Local injection of HSV-1 viral construct with human proenkephalin gene insert results in targeted tissue expression of opioid protein (Yeomans et al., 2006), whereas systemic administration of some neural agents have undesirable or intolerable side effects (Goss et al., 2002). HSV-1 gene delivery and local opioid expression may potentiate exogenously administered morphine, lowering doses needed and delaying morphine tolerance (Stein et al., 1996). This has been demonstrated in other HSV-based gene therapy models*,* where HSV-viral vector infections potentiated chemotherapy agents in lung cancer (Toyoizumi et al., 1999) and breast cancer (Thomas and Fraser, 2003). Attenuated herpes simplex viruses have already been successfully used in Phase I/II trials for treatment of CNS glioblastoma without apparent nonspecific toxic effects (Papanastassiou et al., 2002). The efficacy and potency of peripheral opioid effects are generally enhanced when drugs are administered during active inflammatory conditions (Antonijevic et al., 1995; Lamigeon et al., 2001; Schafer et al., 1998; Stein, 1995; Walker, 2003).

promoter (hCMV), which **allows expression of inserted gene product at an intracellular locale, in the absence of productive HSV-1 infection and without integration into the host genome** (Wilson et al., 1999). The recombinant viral vectors designed for gene therapy rendered replication conditional or defective do not produce productive viral infection *in vivo,* but can persist for months despite negligible viral protein synthesis. These viral constructs establish a quiescent state similar to natural viral latency but **cannot reactivate to cause active infection in neuronal cells** *in vivo* (Goins et al., 2001; Wilson et al., 1999; Wilson and Yeomans, 2002). Lentiviruses as vectors offer very efficient infection, gene expression in activated cells and genome integration into host DNA for gene replacement therapy. However, current lentiviral constructs result in systemic infection and cannot be contained and the site of lentiviral insertion into the host genome cannot be controlled.They are considered a potential causal agent of cancers, autoimmune diseases and acquired immune deficiency syndrome (AIDS). Potential concerns exist regarding development of helper phenonoma for unwanted expressions and genetic recombination with other lentiviruses, including those already integrated in the host genome. Viral construct design for adenoviruses has also yielded promising results, but adenoviruses may not be

HSV-1 viral constructs may offer unique advantages in peripheral inflammation, as they selectively infect primary sensory neurons but do not integrate into the host genome. Thus, they will be an independent "minipump" source for protein synthesis in the neuronal cytoplasm. As a DNA-based viral construct, (1) the rate of mutation and recombination in these HSV-1 constructs will be minimal to nonexistent since (2) these constructs do not enter into productive infection or latency phases. Further, preliminary data suggest that HSV replication deficient viral constructs do not generate proteins that would induce an amnestic response from the host, activating latent prior HSV-1 infection. These are very important properties of HSV-1 based viral vectors, as 70-90% of the human adult population has evidence of prior HSV-1 infection. This limited potential to generate a host response also improves the potential for using repeated dosings of HSV-based viral constructs. Adenoviruses cause productive infection, induce host inflammation above the inflammation already generated in target tissues and increase potential for extended injury and host innate immune response to the virus making repeated doses problematic (Minter et al.,

Local injection of HSV-1 viral construct with human proenkephalin gene insert results in targeted tissue expression of opioid protein (Yeomans et al., 2006), whereas systemic administration of some neural agents have undesirable or intolerable side effects (Goss et al., 2002). HSV-1 gene delivery and local opioid expression may potentiate exogenously administered morphine, lowering doses needed and delaying morphine tolerance (Stein et al., 1996). This has been demonstrated in other HSV-based gene therapy models*,* where HSV-viral vector infections potentiated chemotherapy agents in lung cancer (Toyoizumi et al., 1999) and breast cancer (Thomas and Fraser, 2003). Attenuated herpes simplex viruses have already been successfully used in Phase I/II trials for treatment of CNS glioblastoma without apparent nonspecific toxic effects (Papanastassiou et al., 2002). The efficacy and potency of peripheral opioid effects are generally enhanced when drugs are administered during active inflammatory conditions (Antonijevic et al., 1995; Lamigeon et al., 2001;

advantageous since they can also involve the central nervous system.

2001).

Schafer et al., 1998; Stein, 1995; Walker, 2003).

#### **6. Anti-inflammatory effects and tissue protection by HSV-Enk treatment**

It is well known that long term administration of morphine reduces immune function through reduced hypothalamic-pituitary-adrenal axis activation. Opioid receptors are constitutively expressed in non-neuronal sites including vascular endothelial cells (Cadet et al., 2000 and Saeed et al., 2000), on immune cells such as macrophages and lymphocytes (Gavériaux et al., 1995), and keratinocytes (Bigliardi et al., 2002). A role for neuronal opiates has also been shown for reduction of inflammation (for review see Machelska and Stein, 2003). While the role/s of specific extra-neuronal peripheral opioid receptors has not been fully established, mu (morphine), kappa (U50488H) and delta-2 (deltorphin II), but not delta-1 (DPDPE) opioid agonists have been shown to produce dose-dependent immunosuppressive effects on a plaque-forming assay, effects that were blocked by respective selective opioid antagonists (Rahim et al., 2001). It is also well established that the spinal cord can regulate peripheral inflammation through a variety of dorsal horn receptor mechanisms and retrograde primary afferent activity (Rees et al., 1994; Ren and Dubner, 1999; Sluka et al., 1993; Sluka et al., 1994; Sluka and Westlund, 1993; Sorkin et al., 2003). This includes glutamate, GABA, substance P and adenosine receptor mechanisms likely influenced by opiates (Boyle et al., 2002). Much less well studied is the report that mu opiates can directly reduce plasma extravasation ((24-36%) Joris et al., 1990; Binder et al., 2001; Green and Levine, 1992; Barber et al., 1993; Taylor et al., 2000). These effects are induced by activation of opioid receptors located in the central and peripheral nervous systems. In the periphery, opioid receptors are expressed on a significant proportion of capsaicin sensitive sensory fibers and sympathetic postganglionic terminals, where they may participate in the modulation of nociceptive information under certain pathological conditions (Zhou et al., 1998). The reversibility of the effects induced by the opioid receptor agonists (edema and extravasation) has been established after the administration of antagonists (i.p.) (Romero et al., 2005). The work of Lei and Rogers (1999) in intact respiratory tissue suggests that opioid receptors located on sensory fibers and immune cells are selectively activated by low (neuronal) or high (non-neuronal) doses of mu- and deltaopioid receptor agonists. The local administration of mu- and delta-opioid receptor agonists, at doses that show no systemic effect, has been shown to decrease plasma extravasation during peripheral inflammation (Hong and Abbott, 1995). This suggests a clinical application for low dose morphine or met-enk as a pre-emptive treatment to avoid endoscopic procedure induced pancreatitis. It is likely that a similar mechanism is reducing inflammatory signs in the HSV-ENK treated animals releasing enkephalin directly to the pancreas.

#### **7. Development of opiate tolerance?**

In our study, hyperalgesia in the control HSV--gal and vehicle treated animals with alcohol and high fat diet induced pancreatitis was maintained through seven weeks. The overexpression of the proenkephalin products in the HSV-ENK treated animals was sufficient to abrogate the effects of the pancreatitis for an extended period of time **without tolerance** demonstrating that this is an adequate model for treatment in clinical studies. The important issue of desensitization and tolerance to the enkephalin generated by the HSV-ENK construct, appears to be a non-issue in the time frame of study. We propose that it is the release of met-enkephalin directly onto receptors at nerve terminals both centrally and

Gene Therapy Approach: HSV-Enkephalin Reduces Fibrosis, Inflammation, and Pain 25

As pancreatitis patient survival is becoming a fortunate reality, consideration of route of administration for viral constructs should be a part of investigative pre-clinical studies. While celiac plexus neurolysis is a currently accepted clinical treatment for severe intractable abdominal pain in abdominal malignancies and chronic pancreatitis, it is usually reserved for patients who have failed other treatment modalities. The reported year 1 success rate for pain relief is reportedly between 57-100%, depending on the approach and ethanol concentration (Okuyama et al., 2002; Vranken et al, 2002; Schmulewitz and Hawes, 2003; Klapman and Chang, 2005). Celiac plexus neurolysis requires surgical or anesthetic specialization and is usually performed at tertiary referral centers. Some procedural approaches require an epidural block for anesthesia. Although generally well-tolerated, reported complications include back pain, hypotension, and diarrhea (Chan, 1996; Kulke, 2002). Celiac plexus neurolysis essentially scleroses the nerve and blocks the afferent and efferent transmission of all neurochemicals. Neural blockade of a significant part of the enteric sympathetic nervous system in an otherwise functioning enteric system may lead to pancreatic or enteric pathologies, as the sympathetic nervous system plays a vital role in modulating intestinal secretory and absorptive processes. This may become more of an issue with the improved cancer treatments and longer lifespan for pancreatic malignancies. Viral vectors could be administered to coeliac ganglia in patients by anesthesiologists. However, it is not entirely clear that HSV-Enk would have sufficient uptake efficiency from the axons themselves as the primary afferents pass through the sympathetic ganglia and do not terminate there. We will compare sympathetic ganglia injections to administration at the pancreatic terminal endings which already appears to be quite robust. Our studies imply that chronic infusions of HSV vectors directly onto the pancreas surface from implantable, transcutaneously refillable pumps would provide relief from chronic pain. This potential treatment would also be nerve plexus sparing and avoid the severe complications of pancreatic duct disruption encountered by endoscopists. The sparing of neural connections

would decrease the potential for pancreatic insufficiency and enteric damage.

Pancreatic cancer and chronic pancreatitis are among those syndromes characterized as causing the most severe pain states. Pancreatic cancer is the 4th leading cause of cancer deaths in the US, and over 70% of pancreatic cancer patients have significant debilitating abdominal pain upon clinical presentation. Over 50% of patients with idiopathic and alcoholic pancreatitis report chronic pain. The current treatment of pain in pancreatic cancer and chronic pancreatitis includes parenteral narcotic agents, surgical intervention at the level of pancreas or neural pathways and complementary therapies. Narcotic agents are not optimal as there are risks of tolerance, addiction and intolerable side effects of sedation and constipation and nausea. Surgical intervention with total pancreatectomy and islet autotransplantation provides pain relief for a considerable number of patients (Blondet et al., 2007; Hildebrand et al., 2011). However, surgery is invasive and can result in transient or suboptimal relief of pain, postoperative diabetes, and maldigestion. Surgery may be therapeutically or fiscally inappropriate based on the clinical status of some patients. Chronic abdominal pain from the pancreas has a significant negative long term impact on patient quality of life and mortality as it profoundly decreases appetite and leads to weight loss. When patients lose >20% of lean body mass, host immunocompetence is profoundly

**9. Clinical significance**

peripherally that provides additive effectiveness for reducing hyperalgesia and tissue protection from inflammatory responses without tolerance. In this site directed manner, enkephalin can affect receptors on neuronal endings that receive information about noxious conditions in the pancreas and have reparative ability as an apparent added benefit. Standard therapies relying on higher and higher levels of circulating opiates, on the other hand, frequently result in intolerable side-effects and development of tolerance. Gene therapy using HSV vectors for gene product delivery may be clinically preferred in patients with prospects of longer life spans and functionality, or in intractable chronic nonmalignant pancreatitis and generally indicated in patients with longer life spans. **HSV-1-based viral vector infections may offer a novel, effective, well-tolerated and advantageous approach for treatment of chronic pancreatic pain in patients. This approach might also be expanded to deliver additional human gene products that could impact the pain, inflammation, structural integrity and repair of the pancreas in patients.**

#### **8. Testing safety and efficacy issues**

Safety and efficacy of replication defective HSV vectors have already been demonstrated for other purposes in Phase 1 clinical trials (Todo, 2002; Shah 2003; Yu et al, 2004; Satoh, 2005; Sawai et al., 2001; Wolfe et al., 2009, a,b). For our studies we examined the spinal cord for evidence of HSV-1 infection after 12 weeks of study. While we expected and have demonstrated HSV-1 in the dorsal root ganglia sensory neurons (Yang et al., 2008), the preferred host cell for HSV, no central nervous system infection was found. This has not been tested in spinal cord previously with these viral vectors. The replication deficient virus has already been used safely when injected directly into the brain as a treatment for glioblastoma (Papanastassiou, et al., 2002). The efficacy and clinical relevance of the direct injection into the pancreas was successfully tested in our pre-clinical studies by examining behavioral, inflammatory and cellular activation responses, such as FOS protein expression in spinal neurons and phospho-p38 in DRG. Effects of met-enkephalin have also been tested in *in vitro* pancreatic cell models. We have infected DRG and pancreas cells in cultures (PANC-1) with HSV vectors to assess the longevity of enkephalin, inflammatory mediator release, reactivation and other safety issues in PANC-1 cells, clonal human pancreatic tumor-derived tubular epithelia equivalent to the cells lining the pancreatic ducts. The PANC-1 cells have been used previously by Eisenberg et al. (2005) to assess increased efficacy of HSV vectors used as an anti-cancer adjuvant to chemotherapeutic agents. In our studies the proenkephalin overexpression significantly reduced cytokine expression (unpublished data).

The phase I clinical trial using a related replication defective viral vector reported successful reduction of pain levels assessed using a numeric rating scale (NRS), the Short Form McGill Pain Questionnaire (SF-MPQ) and concurrent reduction of opiate usage in the terminal cancer patients initially with intractable pain (Wolfe et al., 2009b). The gene therapy studies provide compelling new insights in support of the effectiveness of enkephalin for reduction of tissue injury, fibrosis, inflammation, and pain related behaviors. The HSV viral system offers great potential for simultaneous delivery of multiple other gene products, as the HSV vector cassette is very large. Further improvement of vector system design may provide other anti-inflammatory gene products to protect and restore functional integrity of damaged tissues.

#### **9. Clinical significance**

24 Chronic Pancreatitis

peripherally that provides additive effectiveness for reducing hyperalgesia and tissue protection from inflammatory responses without tolerance. In this site directed manner, enkephalin can affect receptors on neuronal endings that receive information about noxious conditions in the pancreas and have reparative ability as an apparent added benefit. Standard therapies relying on higher and higher levels of circulating opiates, on the other hand, frequently result in intolerable side-effects and development of tolerance. Gene therapy using HSV vectors for gene product delivery may be clinically preferred in patients with prospects of longer life spans and functionality, or in intractable chronic nonmalignant pancreatitis and generally indicated in patients with longer life spans. **HSV-1-based viral vector infections may offer a novel, effective, well-tolerated and advantageous approach for treatment of chronic pancreatic pain in patients. This approach might also be expanded to deliver additional human gene products that could impact the pain,**

Safety and efficacy of replication defective HSV vectors have already been demonstrated for other purposes in Phase 1 clinical trials (Todo, 2002; Shah 2003; Yu et al, 2004; Satoh, 2005; Sawai et al., 2001; Wolfe et al., 2009, a,b). For our studies we examined the spinal cord for evidence of HSV-1 infection after 12 weeks of study. While we expected and have demonstrated HSV-1 in the dorsal root ganglia sensory neurons (Yang et al., 2008), the preferred host cell for HSV, no central nervous system infection was found. This has not been tested in spinal cord previously with these viral vectors. The replication deficient virus has already been used safely when injected directly into the brain as a treatment for glioblastoma (Papanastassiou, et al., 2002). The efficacy and clinical relevance of the direct injection into the pancreas was successfully tested in our pre-clinical studies by examining behavioral, inflammatory and cellular activation responses, such as FOS protein expression in spinal neurons and phospho-p38 in DRG. Effects of met-enkephalin have also been tested in *in vitro* pancreatic cell models. We have infected DRG and pancreas cells in cultures (PANC-1) with HSV vectors to assess the longevity of enkephalin, inflammatory mediator release, reactivation and other safety issues in PANC-1 cells, clonal human pancreatic tumor-derived tubular epithelia equivalent to the cells lining the pancreatic ducts. The PANC-1 cells have been used previously by Eisenberg et al. (2005) to assess increased efficacy of HSV vectors used as an anti-cancer adjuvant to chemotherapeutic agents. In our studies the proenkephalin

**inflammation, structural integrity and repair of the pancreas in patients.**

overexpression significantly reduced cytokine expression (unpublished data).

The phase I clinical trial using a related replication defective viral vector reported successful reduction of pain levels assessed using a numeric rating scale (NRS), the Short Form McGill Pain Questionnaire (SF-MPQ) and concurrent reduction of opiate usage in the terminal cancer patients initially with intractable pain (Wolfe et al., 2009b). The gene therapy studies provide compelling new insights in support of the effectiveness of enkephalin for reduction of tissue injury, fibrosis, inflammation, and pain related behaviors. The HSV viral system offers great potential for simultaneous delivery of multiple other gene products, as the HSV vector cassette is very large. Further improvement of vector system design may provide other anti-inflammatory gene products to protect and restore functional integrity of

**8. Testing safety and efficacy issues**

damaged tissues.

As pancreatitis patient survival is becoming a fortunate reality, consideration of route of administration for viral constructs should be a part of investigative pre-clinical studies. While celiac plexus neurolysis is a currently accepted clinical treatment for severe intractable abdominal pain in abdominal malignancies and chronic pancreatitis, it is usually reserved for patients who have failed other treatment modalities. The reported year 1 success rate for pain relief is reportedly between 57-100%, depending on the approach and ethanol concentration (Okuyama et al., 2002; Vranken et al, 2002; Schmulewitz and Hawes, 2003; Klapman and Chang, 2005). Celiac plexus neurolysis requires surgical or anesthetic specialization and is usually performed at tertiary referral centers. Some procedural approaches require an epidural block for anesthesia. Although generally well-tolerated, reported complications include back pain, hypotension, and diarrhea (Chan, 1996; Kulke, 2002). Celiac plexus neurolysis essentially scleroses the nerve and blocks the afferent and efferent transmission of all neurochemicals. Neural blockade of a significant part of the enteric sympathetic nervous system in an otherwise functioning enteric system may lead to pancreatic or enteric pathologies, as the sympathetic nervous system plays a vital role in modulating intestinal secretory and absorptive processes. This may become more of an issue with the improved cancer treatments and longer lifespan for pancreatic malignancies. Viral vectors could be administered to coeliac ganglia in patients by anesthesiologists. However, it is not entirely clear that HSV-Enk would have sufficient uptake efficiency from the axons themselves as the primary afferents pass through the sympathetic ganglia and do not terminate there. We will compare sympathetic ganglia injections to administration at the pancreatic terminal endings which already appears to be quite robust. Our studies imply that chronic infusions of HSV vectors directly onto the pancreas surface from implantable, transcutaneously refillable pumps would provide relief from chronic pain. This potential treatment would also be nerve plexus sparing and avoid the severe complications of pancreatic duct disruption encountered by endoscopists. The sparing of neural connections would decrease the potential for pancreatic insufficiency and enteric damage.

Pancreatic cancer and chronic pancreatitis are among those syndromes characterized as causing the most severe pain states. Pancreatic cancer is the 4th leading cause of cancer deaths in the US, and over 70% of pancreatic cancer patients have significant debilitating abdominal pain upon clinical presentation. Over 50% of patients with idiopathic and alcoholic pancreatitis report chronic pain. The current treatment of pain in pancreatic cancer and chronic pancreatitis includes parenteral narcotic agents, surgical intervention at the level of pancreas or neural pathways and complementary therapies. Narcotic agents are not optimal as there are risks of tolerance, addiction and intolerable side effects of sedation and constipation and nausea. Surgical intervention with total pancreatectomy and islet autotransplantation provides pain relief for a considerable number of patients (Blondet et al., 2007; Hildebrand et al., 2011). However, surgery is invasive and can result in transient or suboptimal relief of pain, postoperative diabetes, and maldigestion. Surgery may be therapeutically or fiscally inappropriate based on the clinical status of some patients. Chronic abdominal pain from the pancreas has a significant negative long term impact on patient quality of life and mortality as it profoundly decreases appetite and leads to weight loss. When patients lose >20% of lean body mass, host immunocompetence is profoundly

Gene Therapy Approach: HSV-Enkephalin Reduces Fibrosis, Inflammation, and Pain 27

delivery by the neuronal innervation, may have significant clinical relevance for reducing

These studies were funded by NIH R01 NS039041. Acknowledgement is extended to Drs. Ying Lu and Hong Yang for their original study that is reviewed here through open

Antonijevi, I.; Mousa, S.A.; Schafer, M. & Stein, C. (1995) Perineurial defect and peripheral opioid analgesia in inflammation. *Journal of Neuroscience*, Vol.15, pp.165-172. Apte, M.; Pirola, R. & Wilson, J. (2009) New insights into alcoholic pancreatitis and

Barber, A. (1993) μ and κ-opioid receptor agonists produce peripheral inhibition of

Bigliardi, P.L.; Buchner, S.; Rufli, T. & Bigliardi-Qi, M. (2002) Specific stimulation of

Binder, W.; Machelska, H.; Mousa, S.; Schmitt T, Rivière PJM, Junien JL, Stein C, Schäfer M

Blondet, J.; Carlson, A.; Kobayashi, T.; Jie, T.; Bellin, M.; Hering, B.J.; Freeman, M.L.;

Boyle, D.L., Moore, J., Yang , L., Sorkin, L.S., Firestein, G.S. (2002) Spinal adenosine receptor

Braz, J,; Beaufour, C.; Coutaux, A.; Epstein, A.L.; Cesselin, F.; Hamon, M. & Pohl, M. (2001)

overproduction in sensory neurons. *Journal of Neuroscience* 21: 7881-7888. Burton, E.A.; Wechuck, J.B.; Wendell, S.K.; Goins, W.F.; Fink, D.J. & Glorioso, J.C. (2001)

Cadet P, Bilfinger, T.V.; Fimiani, C,; Peter, D. & Stefano, G.B. (2000) Human vascular and

Chan, V.W. (1996) Chronic diarrhea: an uncommon side effect of celiac plexus block.

Danielson, P.B. & Dores, R.M. (1999) Molecular evolution of the opioid/orphanin gene

family. *General Comparative Endocrinology*, Vol.113, pp. 169-186.

*Receptor Signaling and Transduction Research*, Vol.22, pp.191–199.

arthritis. *Arthritis and Rheumatology, Vol.* 46, pp.3076-3082.

pancreatic cancer. *Journal of Gastroenterology and Hepatology*. Vol.24, Suppl. 3,

neurogenic plasma extravasation in rat skin. *European Journal of Pharmacology*,

migration of human keratinocytes by mu-opiate receptor agonists. *Journal of*

(2001) Analgesic and antiinflammatory effects of two novel κ-opioid peptides*.*

Beilman, G.J. & Sutherland, D.E. (2007) The role of total pancreatectomy and islet autotransplantation for chronic pancreatitis. *Surgical Clinics of North America*,

activation inhibits inflammation and joint destruction in rat adjuvant-induced

Therapeutic efficacy in experimental polyarthritis of viral-driven enkephalin

Multiple applications for replication-defective herpes simplex virus vectors. *Stem*

cardiac endothelia express mu opiate receptor transcripts. *Endothelium,* Vol.7,

access, as well as Drs. Sabrina McIlwrath and Liping Zhang for final edits.

inflammation, pain-related behavior and tissue destruction.

**11. Acknowledgment**

**12. References**

pp.S51-6.

Vol.236, pp.113–120.

Vol.87, pp.1477-1501.

*Cells* 19: 358-377.

pp.185–191.

*Anesthesiology*, Vol. 94, pp.1034–1044.

*Anesthesia and Analgesia*. Ol. 82:205-7.

impaired. **Therefore, effective management of pain is imperative, not just for the legitimate concern for pain's sake, but for improvement in physical functioning, general health, patient survival, quality of life, and functional independence. Effective management of chronic pain would lift the economic burden of pain-induced debilitation on the individual, their support system and public health.**

Pain is a serious public health problem, costing the US about 100 billion dollars/yr (JAHCO report). Pain is the single greatest cause of disability, decreased physical function, decreased work productivity, absenteeism, reactive depression and lower quality of life (QOL, i.e. SF36) scores. In fact, 40-60% of patients with chronic pain report it has significantly and negatively impacted their personal relationships, work productivity and daily routines of living. Considerable improvements have been made in opioid medications, in term of bioavailability, half-life, transdermal delivery, breakthrough opioid combinations and usage with enhancing drugs, such as tricyclic antidepressants. However, side effects and potential for opioid addiction remain a concern to the patient, health care providers and the community. About 60% of patients with chronic pain have expressed fears regarding narcotic medication side effects and fear of addiction to current narcotic regimens. In a Partners Against Pain Survey of 1000 patients in chronic pain, 50% of the patients reported difficulty >1 yr in getting their pain under control and 78% of the patients reported that they would be willing to try new treatments.

#### *Innovative Aspects of the Gene Therapy Studies*

Unique advantages offered by HSV-1 viral vector delivery of proenkephalin expression products by the peripheral nerves include:

#### **10. Conclusion**

In summary, our studies indicate that the proenkephalin gene product delivery to pancreas is reparative and significantly reduce pain related behaviors in rodent pancreatitis models. Gene therapeutic approaches that promote the endogenous opiate enkephalin, particularly delivery by the neuronal innervation, may have significant clinical relevance for reducing inflammation, pain-related behavior and tissue destruction.

#### **11. Acknowledgment**

These studies were funded by NIH R01 NS039041. Acknowledgement is extended to Drs. Ying Lu and Hong Yang for their original study that is reviewed here through open access, as well as Drs. Sabrina McIlwrath and Liping Zhang for final edits.

#### **12. References**

26 Chronic Pancreatitis

impaired. **Therefore, effective management of pain is imperative, not just for the legitimate concern for pain's sake, but for improvement in physical functioning, general health, patient survival, quality of life, and functional independence. Effective management of chronic pain would lift the economic burden of pain-induced debilitation**

Pain is a serious public health problem, costing the US about 100 billion dollars/yr (JAHCO report). Pain is the single greatest cause of disability, decreased physical function, decreased work productivity, absenteeism, reactive depression and lower quality of life (QOL, i.e. SF36) scores. In fact, 40-60% of patients with chronic pain report it has significantly and negatively impacted their personal relationships, work productivity and daily routines of living. Considerable improvements have been made in opioid medications, in term of bioavailability, half-life, transdermal delivery, breakthrough opioid combinations and usage with enhancing drugs, such as tricyclic antidepressants. However, side effects and potential for opioid addiction remain a concern to the patient, health care providers and the community. About 60% of patients with chronic pain have expressed fears regarding narcotic medication side effects and fear of addiction to current narcotic regimens. In a Partners Against Pain Survey of 1000 patients in chronic pain, 50% of the patients reported difficulty >1 yr in getting their pain under control and 78% of the patients reported that they

Unique advantages offered by HSV-1 viral vector delivery of proenkephalin expression

Potential for simultaneous delivery of analgesic peptides to both peripheral and central

Normalization of pain related behavior to near baseline levels as shown in the

Potential for positive impact on pancreatic inflammation as shown in the published

Focused delivery to the target organ allows a much lower viral titer (5-10X lower viral

Novel palliative strategy for alleviating the unremitting pain of pancreatic cancer and

In summary, our studies indicate that the proenkephalin gene product delivery to pancreas is reparative and significantly reduce pain related behaviors in rodent pancreatitis models. Gene therapeutic approaches that promote the endogenous opiate enkephalin, particularly

HSV-1 viral constructs will not incorporate into the host genome or become lytic

sites, neuronal ending sites, optimizing the effects without tolerance More effective and prolonged abrogation of nociceptive responses

HSV-1 viral constructs to be used are replication deficient/ defective

Potential for reduction/elimination of the use of narcotic drugs

**on the individual, their support system and public health.**

would be willing to try new treatments.

*Innovative Aspects of the Gene Therapy Studies*

products by the peripheral nerves include:

published studies

titer than in skin)

chronic pancreatitis

**10. Conclusion**

studies

Gene Therapy Approach: HSV-Enkephalin Reduces Fibrosis, Inflammation, and Pain 29

Merkord J.; Jonas L.; Weber H.; Kröning G.; Nizze H.; Hennighausen G. (1997) Acute

Okuyama, M.; Shibata, T.; Morita, T.; Kitada, M.; Tukahara, Y.; Fukushima, Y.; Ikeda,

Papanastassiou, V.; Rampling R.; Fraser M.; Petty R.; Hadley D.; Nicoll J.; Harland J.;

Pezzilli, R. & Morselli-Labate, A.M. (2009) Alcoholic pancreatitis: pathogenesis,

Rahim, R.T.; Meissler, J.J.J.; Cowan, A., Rogers, T.J.; Geller, E.B.; Gaughan, J.; Adler, M.W. &

Rees, H.; Sluka, K.A.; Westlund, K.N. & Willis, W.D. (1994) Do dorsal root reflexes augment

Ren, K. & Dubner, R. (1999) Central nervous system plasticity and persistent pain. *Journal of*

Romero, A.; Planas, E.; Poveda, R.; Sanchez, S.; Pol, O. & Puig, M.M. (2005) Anti-

Saeed, R.W.; Stefano, D.B.; Murga, J.D.; Short, T.W.; Qi, F.; Bilfinger, T.V. & Magazine, H.I.

Salzet, M. (2001) Neuroimmunology of opioids from invertebrates to human. *Neuro*

Sassani, J.W.; Zagon, I.S. & McLaughlin, P.J. (May 2003). Opioid growth factor modulation

Satoh, T.; Irie, A.; Egawa, S. & Baba, S. (2005) In situ gene therapy for prostate cancer.

exudative effects of opioid receptor agonists in a rat model of carrageenaninduced acute inflammation of the paw. *European Journal of Pharmacology*, Vol.511,

(2000) Expression of functional delta opioid receptors in vascular smooth muscle.

of corneal epithelium: uppers and downers. *Current Eye Research*, Vol.26, No.5, pp.

*International Immunopharmacology*, Vol.1, pp.2001–2009.

peripheral inflammation? *Neuroreport,* Vol.5, pp.821-824.

*International Journal of Molecular Medicine*, Vol.6, pp.673–677.

*Orofacial Pain*, Vol.13, pp. 155-163.

*Endocrinology Letters, Vol.*22, pp.467-474.

*Current Gene Therapy*, Vol.5, pp.111-9.

sepsis. *Journal of Immunol*ogy, Vol.167, pp.1053-1059.

5.

398–406.

pp.2763-82.

pp.207-217.

249–62.

interstitial pancreatitis in rats induced by dibutyltin dichloride (DBTC): pathogenesis and natural course of lesions. *Pancreas*, Vol.15, No. 4, pp.392-401. Minter, R.M.; Ferry, M.A.; Murday, M.E.; Tannahill, C.L.; Bahjat, F.R.; Oberholzer, C.;

Oberholzer ,A.; LaFace, D.; Hutchins, B.; Wen, S.; Shinoda, J.; Copeland, E.M., III, & Moldawer LL (2001) Adenoviral delivery of human and viral IL-10 in murine

K.; Fuzita, J. & Shimano T. (2002) A comparison of intraoperative celiac plexus block with pharmacological therapy as a treatment for pain of unresectable pancreatic cancer. *Journal of Hepatobiliary and Pancreatic Surgery*, Vol. 9, pp.372-

Mabbs R.; Brown M (2002) The potential for efficacy of the modified (ICP 34.5(−)) herpes simplex virus HSV1716 following intratumoural injection into human malignant glioma: a proof of principle study, *Gene Therapy, Vol.*9, pp.

incidence and treatment with special reference to the associated pain. *International Journal of Environmental Research and Public Health*, Vol.6, No.11,

Eisenstein, T.K. (2001) Administration of mu-, kappa- or delta2-receptor agonists via osmotic minipumps suppresses murine splenic antibody responses.

Eisenberg, D.P.; Adusumilli, P.S.; Hendershott, K.J.; Yu, Z.; Mullerad, M.; Chan, M.K.; Chou,

Gavériaux, C.; Peluso, J.; Simonin, F.; Laforet, J. & Kieffer, B. (1995) Identification of kappa-

Goins, W.F.; Yoshimura, N.; Phelan, M.W.; Yokoyama, T.; Fraser, M.O.; Ozawa, H.; Bennett,

Goss, J.R.; Goins, W.F.; Lacomis, D.; Mata, M.; Glorioso, J.C. & Fink, D.J. (2002) Herpes

Green, P.G. & Levine, J.D. (1992) δ- and κ-opioid receptor agonists inhibit plasma

Hildebrand, P.; Duderstadt, S.; Jungbluth, T.; Roblick, U.J.; Bruch, H.P. & Czymek, R. (2011)

Hong, Y. & Abbott, F.V. (1995) Peripheral opioid modulation of pain and inflammation in the formalin test. *European Journal of Pharmacology,* Vol.277, pp.21–28. Joris, J.; Costello, A., Dubner, R., Hargreaves, KkM (1990) Opiates suppress carrageenan-

Klapman, J.B. & Chang, K.J. (2005) Endoscopic ultrasound-guided fine-needle injection.

Kulke, M.H. (2002) Metastatic pancreatic cancer. *Current Treatment Options in Oncology*,Vol.

Lamigeon, C.; Bellier, J.P.; Sacchettoni, S.; Rujano, M. & Jacquemont, B. (2001) Enhanced

Lei, Y.H. & Rogers DF (1999) Effects and interactions of opioids on plasma exudation

method in man. *Clinical and Pharmacol ogicalTherapeutics,* Vol.8, pp.521-542. Lu, Y.; McNearney, T.A.; Lin, W.; Wilson, S.P.; Yeomans, D.C. & Westlund, K.N. (2007).

Machelska, H. & Stein, C. (2003) Immune mechanisms of pain and analgesia. Landes

*Gastrointestinal Endoscopy Clinics North America,*Vol.15, pp.169-77.

*Gastrointestinal Surgery*, Vol.9, pp.1068-77.

*Journal of the Pancreas*, Vol.12, No.4, pp.364-71.

276.

pp.1748-1754.

pp.2227-3.

pp.95–103.

3, pp.449-57.

Vol.15, No.10, pp.1812-9.

Bioscience/Eurekah.com. Georgetown, TX.

606.

Vol.49, pp.129–133.

T.C.; & Fong, Y. (2005) 5-fluorouracil and gemcitabine potentiate the efficacy of oncolytic herpes viral gene therapy in the treatment of pancreatic cancer*. Journal of*

and delta-opioid receptor transcripts in immune cells. *FEBS Letters* Vol.369, pp.272–

N., Jr.; de Groat, W.C.; Glorioso, J.C. & Chancellor, M.B. (2001) Herpes simplex virus mediated nerve growth factor expression in bladder and afferent neurons: potential treatment for diabetic bladder dysfunction*. Journal of Urology*, Vol.165,

simplex-mediated gene transfer of nerve growth factor protects against peripheral neuropathy in streptozotocin-induced diabetes in the mouse. *Diabetes*. Vol.51, No.7,

extravasation induced by bradykinin in the knee joint of the rat. *Neuroscience*,

Evaluation of the quality of life after surgical treatment of chronic pancreatitis.

induced oedema and hyperthermia at doses that inhibit hyperalgesia. *Pain, Vol.*43,

neuronal protection from oxidative stress by coculture with glutamic acid decarboxylase-expressing astrocytes. *Journal of Neurochem*istry, Vol. 77, pp.598-

induced by cigarette evaluated by the intraperitoneal bradykinin-evoked pain

Treatment of inflamed pancreas with enkephalin encoding HSV-1 recombinant vector reduces inflammatory damage and behavioral sequelae. *Molecular Therapy*,

Gene Therapy Approach: HSV-Enkephalin Reduces Fibrosis, Inflammation, and Pain 31

Thomas, D.L. & Fraser, N.W. (2003) HSV-1 therapy of primary tumors reduces the number

Todo, T. (2002) Oncolytic virus therapy using genetically engineered herpes simplex viruses.

Toyoizumi, T.; Mick, R.; Abbas, A.E.; Kang, E.H.; Kaiser, L.R. & Molnar-Kimber, K.L. (1999)

Vardanyan, M. & Rilo, H.L. (2010) Pathogenesis of chronic pancreatitis-induced pain.

Vranken, J.H.; Zuurmond, W.W.; Van Kemenade, F.J. & Dzoljic, M. (2002)

Walker, J.S. (2003) Effect of opioids immune mechanisms of pain and analgesia, In: *Immune*

Westlund, K.N. (2000) Visceral nociception, *Current Reviews in Pain*, Vol.4, No.6, pp.478-

Westlund, K.N. (2009) Gene therapy for pancreatitis pain. *Gene Therapy,* Vol.16, No.4,

Westlund, K.N.; Vera-Portocarrero, L.P.; Zhang, L.; Wei ,J.; Quast, M.J. & Cleeland, C.S.

Wilson, S.P. & Yeomans, D.C. (2000) Genetic therapy for pain management. *Current Reviews*

Wilson, S.P. & Yeomans, D.C. (2002) Virally mediated delivery of enkephalin and other

Wilson, S.P.; Yeomans, D.C.; Bender, M.A.; Lu, Y.; Goins, W.F. & Glorioso, J.C. (1999)

Wolfe, D.; Wechuck, J.; Krisky, D.; Mata, M. & Fink, D.J. (2009b) A clinical trial of gene

Yang, H.; McNearney, T.A,; Chu, R.; Lu, Y.; Ren, Y.; Yeomans, D.C.; Wilson, S.P. &

Yeomans, D.C.; Lu, Y.; Laurito, C.E.; Peters, M.C.; Vota-Vellis, G.; Wilson, S.P. & Pappas,

the treatment of chronic pain. *Gene Therapy*, Vol.6, No.4, pp.455-60.

therapy for chronic pain. *Pain Medicine*, Vol.10, No.7, pp.1325-30.

hyperalgesia. *Molecular Therapy*, Vol.13, No.3, pp.589-97.

*Therapy*, Vol.8, pp.543-51.

*Human Cell* Vol.15, pp.151-159.

*Therapy*, Vol.10, pp.3013-3029.

pp.827-30.

pp.483-92a.

*in Pain*, Vol.4, pp.445-450.

*Pain*, Vol.28, No.4, p.8.

*Academy of Science*, Vol.971, pp.515-521.

87.

*Discovery Medicine*, Vol.9, No.47, pp.304-10

Academic/Plenum Publishers, Georgetown, TX.

inflammation in rats. *Neuroimage,* Vol.44, No.1, pp.23-34.

of metastases in an immune-competent model of metastatic breast cancer. *Molecular*

Combined therapy with chemotherapeutic agents and herpes simplex virus type 1 ICP34.5 mutant (HSV-1716) in human non-small cell lung cancer. *Human Gene*

Neurohistopathologic findings after a neurolytic celiac plexus block with alcohol in patients with pancreatic cancer pain. *Acta Anaesthesiology Scandinavia*, Vol.46,

*mechanisms of pain and analgesia,* H. Machelska and C. Stein (Eds.), Kluwer

(2009b) fMRI of supraspinal areas after morphine and one week pancreatic

neuropeptide transgenes in experimental pain models. *Annals of the New York*

Antihyperalgesic effects of infection with a preproenkephalin-encoding herpes virus. *Proceedings of the National Academy of Science USA*, Vol.96, pp.3211-3216. Wolfe, D.; Mata, M. & Fink, D.J. (2009a) A human trial of HSV-mediated gene transfer for

Westlund, K.N. (2008) Enkephalin-encoding herpes simplex virus-1 decreases inflammation and hotplate sensitivity in a chronic pancreatitis model. *Molecular*

G.D. (2006) Recombinant herpes vector-mediated analgesia in a primate model of

Sawai, H.; Yamamoto, M.; Okada, Y.; Sato, M.; Akamo, Y.; Takeyama, H. & Manabe T.

Schmidt, J.; Compton, C.C.; Rattner, D.W.; Lewandrowski, K. & Warshaw, A.L. (1995) Late

Schmidt, J.; Rattner, D.W.; Lewandrowski, K.; Compton, C.C.; Mandavilli, U.; Knoefel, W.T.

Schafer, M.; Zhou, L. & Stein, C. (1998) Cholecystokinin inhibits peripheral opioid analgesia

Schmulewitz, N. & Hawes, R. (2003) EUS-guided celiac plexus neurolysis--technique and

Shah, A.C.; Benos, D.; Gillespie, G.Y. & Markert, J.M. (2003) Oncolytic viruese:cliical

Sluka, K.A.; Jordan, H.H. & Westlund, K.N. (1994) Reduction in joint swelling and

Sluka, K.A. & Westlund, K.N. (1993) Centrally administered non-NMDA but not NMDA

Sluka, K.A.; Willis, W.D. & Westlund, K.N. (1993) Joint inflammation and hyperalgesia are

Sorkin, L.S.; Moore, J.; Boyle, D.L.; Yang, L. & Firestein, G.S. (2003) Regulation of peripheral

Sparmann, G.; Merkord, J.; Jaschke, A.; Nizze, H.; Jonas, L.; Lohr, M.; Liebe, S. & Emmrich, J.

Stein, C. (1995) The control of pain in peripheral tissue by opioids. *New England Journal of*

Stein, C.; Pflüger, M.; Yassouridis, A.; Hoelzl, J.; Lehrberger, K.; Welte, C. & Hassan, A.H.

Steiner, I.; Spivack, J.G.; Deshmane, S.L.; Ace, C.I.; Preston, C.M., & Fraser, N.W. (1990) A

Taylor, B.K.; Peterson, M.A.; Roderick, R.E.; Tate, J.; Green, P.J.; Levine, J.O. & Basbaum, A.I.

reduced by spinal bicuculline. *Neuroreport* Vol.5, pp.109-112.

dichloride. *Gastroenterology,*Vol.112, pp.1664-1672.

local blood flow in rats. *Pain*, Vol.84, pp.263–270.

cells. *Pancreas,Vol* 23, pp.399-405.

*Annals of Surgery*, Vol.215, pp.44-56.

*Neurooncology,*Vol.65, pp.203-226.

antagonist. *Pain*, Vol.59, pp.95-100.

*Neurology*, Vol.184, pp.162-168.

*Medicine*, Vol.332, pp.1685-1690.

225.

9.

1638.

necrotizing pancreatitis. *Digestion* Vol.56, pp.246-252.

in inflamed tissue. *Neuroscience,* Vol.82, pp.603-611.

indication. *Endoscopy*, Vol.35, No.8, pp.S49-53.

(2001) Alteration of integrins by interleukin-1alpha in human pancreatic cancer

histopathologic changes and healing in an improved rodent model of acute

& Warshaw, A.L. (1992) A better model of acute pancreatitis for evaluating therapy.

applications as vectors for the treatment of malignant gliomas. *Journal of*

hyperalgesia following post-treatment with a non-NMDA glutamate receptor

receptor antagonists block peripheral knee joint inflammation. *Pain*, Vol.55, pp.217-

inflammation by spinal adenosine: role of somatic afferent fibers. *Experimental*

(1997) Pancreatic fibrosis in experimental pancreatitis induced by dibutyltin

(1996) No tolerance to peripheral morphine analgesia in presence of opioid expression in inflamed synovia. *Journal of Clinical Investigation*, Vol.98, No.3, pp.793-

herpes simplex virus type 1 mutant containing a nontransinducing Vmw65 protein establishes latent infection in vivo in the absence of viral replication and reactivates efficiently from explanted trigeminal ganglia. *Journal of Virology*, Vol.64, pp.1630-

(2000) Opioid inhibition of formalin-induced changes in plasma extravasation and

**3**

*Romania*

**Pancreatic Acinar and Island Neogenesis**

Garofita-Olivia Mateescu1, Mihaela Hincu2, B. Oprea1,

Maria Comanescu3 and Gabriel Cojocaru1

*2"Ovidius" University, Constanta, Histology Department 3"Victor Babes" Institute, Bucharest, Pathology Department*

*1U.M.F. Craiova, Histology Department*

**Correlated with Vascular and Matrix Dynamics**

The pancreas is an organ that has a remarkable "plasticity" so that the demarcation between normal and pathological is particularly difficult; it is sometimes difficult to determine

In these circumstances of an increasing incidence of chronic pancreatitis, the question of regeneration of acinar structures and islands, in the context of known etiopathological

The prevalence of chronic pancreatitis in the context of different pathologies is difficult to be determined, varying probably between 0.04% and 5%. This signifies a superposition of the etiology of acute and chronic pancreatitis although it is known that the most frequent

Over 40% from the patients with chronic pancreatitis are predisposed to acute pancreatitis. The evaluation of the general medicine consult and of the laboratory data is necessary for the accuracy of the etiology and pathogeny, being known that the metabolic autoimmune pathology, the anatomical anomalies, the abusive consumption of alcohol also induce hepatic lesions, thus it can be spoken from a hepato-pancreatic lesional complex11. In the present paper the etiologic agent prime conductor of the pancreatic lesions is the main concern. That is why we consider indispensable the evaluation of the immunohistochemical markers, of involvement for each matriceal component and pancreatic parenchyma. Thus, the fibrotic replacement of the exocrine pancreatic parenchyma and the appearance of the

In this context, it is compulsory to add immunohistochemical results, knowing the importance of some of them either in the collagen forming or in acino-island tissue and matrix recovery. Therefore we consider extremely important to observe in dynamic the histo-architectural

This is why the approach of island neogenesis and not of the acinar neogenesis must be done like a more complex lesion in a mutual interdependence and vascular-stromal induction.

whether a change in structure is the cause or effect of pathological conditions.

etiology of chronic pancreatitis is alcoholism, in middle-age patients6.

inflammatory infiltrate are the main features of the chronic pancreatitis.

changes of the stroma and parenchyma.

**1. Introduction**

conditions is imperative.

### **Pancreatic Acinar and Island Neogenesis Correlated with Vascular and Matrix Dynamics**

Garofita-Olivia Mateescu1, Mihaela Hincu2, B. Oprea1, Maria Comanescu3 and Gabriel Cojocaru1 *1U.M.F. Craiova, Histology Department 2"Ovidius" University, Constanta, Histology Department 3"Victor Babes" Institute, Bucharest, Pathology Department Romania*

#### **1. Introduction**

32 Chronic Pancreatitis

Yu Z.; Eisenberg D.P.; Singh B.; Shah J.P.; Fong Y.; Wong R.J. (2004) Treatment of aggressive thyroid cancer with an oncolytic herpes virus. *Int J Cancer*, Vol. 112, pp.525–532. Zhou L.; Zhang Q.; Stein C.; Schäfer M. (1998) Contribution of opioid receptors on primary

*Ther*, Vol. 286, No. 2, pp.1000-1006.

afferent versus sympathetic neurons to peripheral opioid analgesia. *J Pharmacol Exp*

The pancreas is an organ that has a remarkable "plasticity" so that the demarcation between normal and pathological is particularly difficult; it is sometimes difficult to determine whether a change in structure is the cause or effect of pathological conditions.

In these circumstances of an increasing incidence of chronic pancreatitis, the question of regeneration of acinar structures and islands, in the context of known etiopathological conditions is imperative.

The prevalence of chronic pancreatitis in the context of different pathologies is difficult to be determined, varying probably between 0.04% and 5%. This signifies a superposition of the etiology of acute and chronic pancreatitis although it is known that the most frequent etiology of chronic pancreatitis is alcoholism, in middle-age patients6.

Over 40% from the patients with chronic pancreatitis are predisposed to acute pancreatitis. The evaluation of the general medicine consult and of the laboratory data is necessary for the accuracy of the etiology and pathogeny, being known that the metabolic autoimmune pathology, the anatomical anomalies, the abusive consumption of alcohol also induce hepatic lesions, thus it can be spoken from a hepato-pancreatic lesional complex11. In the present paper the etiologic agent prime conductor of the pancreatic lesions is the main concern. That is why we consider indispensable the evaluation of the immunohistochemical markers, of involvement for each matriceal component and pancreatic parenchyma. Thus, the fibrotic replacement of the exocrine pancreatic parenchyma and the appearance of the inflammatory infiltrate are the main features of the chronic pancreatitis.

In this context, it is compulsory to add immunohistochemical results, knowing the importance of some of them either in the collagen forming or in acino-island tissue and matrix recovery.

Therefore we consider extremely important to observe in dynamic the histo-architectural changes of the stroma and parenchyma.

This is why the approach of island neogenesis and not of the acinar neogenesis must be done like a more complex lesion in a mutual interdependence and vascular-stromal induction.

Pancreatic Acinar and Island Neogenesis Correlated with Vascular and Matrix Dynamics 35

These cells have long been known to contribute to hepatic fibrosis. Inactive pancreatic stellate cells are triangular, lipid-containing cells predominantly located in the perivascular regions. When activated, they lose their lipid droplets and transform into a fibroblast-like

There is ample evidence to suggest that stellate cells play a central role in the deposition of collagen in the early stages of chronic pancreatitis. Pancreatic specimens from patients with chronic pancreatitis exhibit staining for *α*-smooth muscle actin (present in activated stellate

Our studies showed that the fibrosis begins in extra-lobular spaces by a hyper activity of the stellate cells especially in alcoholic patients which suggest that alcohol and the oxidative stress which it induces form a pathogenic triangle of the necrotic and fibrotic lesions1.

It is known that the cytokine profile within the pancreas in patients with chronic pancreatitis is distinct from normal pancreas. Pancreatic stellate cells are stimulated by a variety of cytokines which are emitted during the inflammatory phase of acute pancreatitis, this

The pancreas is exposed to "oxidative stress" through the systemic circulation or through the reflux of bile into the pancreatic duct, leading to inflammation and tissue damage. Oxidative stress may be exacerbated by increased mixed-function oxidase activity, either from high levels of substrate (*e.g.*, fats, diabetics) or inducers (*e.g.*, alcohol and drugs).

Oxidized products are capable of damaging cellular compounds, possibly inducing

Oxidative stress is involved in the pathway to fibrosis, it has never been proven to *initiate*  the disease. Pancreatic acinar cells are capable of metabolizing alcohol without the involvement of Langerhans island. Oxidized products along with cyto-toxines released in the inflammation represent the main aggression factor for the pancreatic acinar cells,

For our study we used pancreas specimens obtained from patients who died having chronic pancreatitis of various etiology: alcoholism, hepatic cirrhosis, diabetes, etc. For the initial diagnosis classic hematoxylin-eozine and Davenport staining were used to identify pancreatic islands and select the most representative samples for the immunohistochemical

We used 10% formaline solution as a fixative because it is suitable for classical stainings as well as for immunohistochemistry, preserving the antigen expression pattern of cells. The

The samples were fixed 3-4 days depending of their size, and were paraffin-wax processed. The paraffin blocks were cut using the microtome to 5μm thick sections. The sections were

volume of the solution was 10 times higher that the volume of the sample.

suggesting that recurrent acute pancreatitis could lead to chronic pancreatitis.

**2.3 Pancreatic stellate cells**

cells) in fibrotic areas of the pancreas.

**2.4 Oxidative stress**

pancreatic autolysis.

study.

favoring necrosis and fibrosis1.

**3. Materials and methods**

morphologic appearance, migrating to the periacinar areas.

Therefore we consider interesting island neogenesis evaluation in dynamics of various lesions, so the immunohistochemical results are much larger, by a wide use of antibodies, whom target is not only parenchyma neogenesis, but the whole context of lesional chronic pancreatitis. We say this because the study is done on humans and not on experimental models.

We believe that in this case, of the island neogenesis, many factorial etio-pathogenesis, history and family history, therapeutic actions, multiple risk factors (alcohol, smoking, food habits) can create more or less favorable conditions, aspects that can not be extrapolated to the experimental models.

Therefore we can not stop only to identify newly formed islands without assessing the context of lesion, which is why we considered it appropriate to use a wider range of antibodies, covering both parenchyma and vascular-matrix elements.

#### **2. Chronic pancreatitis pathogenic mechanisms**

#### **2.1 Toxic-metabolic mechanism**

Alcohol is directly toxic to acinar cell through a change in cellular metabolism. Alcohol produces cytoplasmic lipid accumulation within the acinar cells, leading to fatty degeneration, cellular necrosis, and eventual widespread fibrosis.

In alcoholics without chronic pancreatitis, cytoplasmic fat droplets were frequently found in the acinar cells. Alcoholic patients with chronic pancreatitis expressed lesions such as atrophy and fibrosis.

Alcohol produced a stepwise progression from fatty accumulation to fibrosis. The morphological changes of the cellular organites may likewise represent a toxic change in cellular metabolism from alcohol.

Alcohol is metabolized in pancreatic acinar cells through oxidative and non-oxidative pathways13.

A criticism of this theory is the lack of proof that pancreatic steatosis is a true precursor to fibrosis, rather than a parallel, reversible, alcohol-related lesion1.

We consider that there is an inter-relation between multiple factors and that alcohol could be the precursor of other pathological events that lead to chronic pancreatic disease.

#### **2.2 Necrosis-fibrosis**

Inflammation and necrosis from episodes of acute pancreatitis produces scarring in the periductular areas. This scarring leads to obstruction of the ductules, leading to stasis within the duct and secondarily, stone formation. Severe obstruction results in atrophy and fibrosis. It was suggested that there is a stepwise progression of fibrosis emerging from recurrent episodes of acute pancreatitis1.

We consider that the necrosis-fibrosis mechanism has to be correlated with the destruction of excreto-secretory elements.

#### **2.3 Pancreatic stellate cells**

34 Chronic Pancreatitis

Therefore we consider interesting island neogenesis evaluation in dynamics of various lesions, so the immunohistochemical results are much larger, by a wide use of antibodies, whom target is not only parenchyma neogenesis, but the whole context of lesional chronic pancreatitis. We say this because the study is done on humans and not on experimental

We believe that in this case, of the island neogenesis, many factorial etio-pathogenesis, history and family history, therapeutic actions, multiple risk factors (alcohol, smoking, food habits) can create more or less favorable conditions, aspects that can not be extrapolated to

Therefore we can not stop only to identify newly formed islands without assessing the context of lesion, which is why we considered it appropriate to use a wider range of

Alcohol is directly toxic to acinar cell through a change in cellular metabolism. Alcohol produces cytoplasmic lipid accumulation within the acinar cells, leading to fatty

In alcoholics without chronic pancreatitis, cytoplasmic fat droplets were frequently found in the acinar cells. Alcoholic patients with chronic pancreatitis expressed lesions such as

Alcohol produced a stepwise progression from fatty accumulation to fibrosis. The morphological changes of the cellular organites may likewise represent a toxic change in

Alcohol is metabolized in pancreatic acinar cells through oxidative and non-oxidative

A criticism of this theory is the lack of proof that pancreatic steatosis is a true precursor to

We consider that there is an inter-relation between multiple factors and that alcohol could

Inflammation and necrosis from episodes of acute pancreatitis produces scarring in the periductular areas. This scarring leads to obstruction of the ductules, leading to stasis within the duct and secondarily, stone formation. Severe obstruction results in atrophy and fibrosis. It was suggested that there is a stepwise progression of fibrosis emerging from recurrent

We consider that the necrosis-fibrosis mechanism has to be correlated with the destruction

be the precursor of other pathological events that lead to chronic pancreatic disease.

antibodies, covering both parenchyma and vascular-matrix elements.

degeneration, cellular necrosis, and eventual widespread fibrosis.

fibrosis, rather than a parallel, reversible, alcohol-related lesion1.

**2. Chronic pancreatitis pathogenic mechanisms**

models.

the experimental models.

atrophy and fibrosis.

**2.2 Necrosis-fibrosis**

episodes of acute pancreatitis1.

of excreto-secretory elements.

pathways13.

**2.1 Toxic-metabolic mechanism**

cellular metabolism from alcohol.

These cells have long been known to contribute to hepatic fibrosis. Inactive pancreatic stellate cells are triangular, lipid-containing cells predominantly located in the perivascular regions. When activated, they lose their lipid droplets and transform into a fibroblast-like morphologic appearance, migrating to the periacinar areas.

There is ample evidence to suggest that stellate cells play a central role in the deposition of collagen in the early stages of chronic pancreatitis. Pancreatic specimens from patients with chronic pancreatitis exhibit staining for *α*-smooth muscle actin (present in activated stellate cells) in fibrotic areas of the pancreas.

Our studies showed that the fibrosis begins in extra-lobular spaces by a hyper activity of the stellate cells especially in alcoholic patients which suggest that alcohol and the oxidative stress which it induces form a pathogenic triangle of the necrotic and fibrotic lesions1.

It is known that the cytokine profile within the pancreas in patients with chronic pancreatitis is distinct from normal pancreas. Pancreatic stellate cells are stimulated by a variety of cytokines which are emitted during the inflammatory phase of acute pancreatitis, this suggesting that recurrent acute pancreatitis could lead to chronic pancreatitis.

#### **2.4 Oxidative stress**

The pancreas is exposed to "oxidative stress" through the systemic circulation or through the reflux of bile into the pancreatic duct, leading to inflammation and tissue damage. Oxidative stress may be exacerbated by increased mixed-function oxidase activity, either from high levels of substrate (*e.g.*, fats, diabetics) or inducers (*e.g.*, alcohol and drugs).

Oxidized products are capable of damaging cellular compounds, possibly inducing pancreatic autolysis.

Oxidative stress is involved in the pathway to fibrosis, it has never been proven to *initiate*  the disease. Pancreatic acinar cells are capable of metabolizing alcohol without the involvement of Langerhans island. Oxidized products along with cyto-toxines released in the inflammation represent the main aggression factor for the pancreatic acinar cells, favoring necrosis and fibrosis1.

#### **3. Materials and methods**

For our study we used pancreas specimens obtained from patients who died having chronic pancreatitis of various etiology: alcoholism, hepatic cirrhosis, diabetes, etc. For the initial diagnosis classic hematoxylin-eozine and Davenport staining were used to identify pancreatic islands and select the most representative samples for the immunohistochemical study.

We used 10% formaline solution as a fixative because it is suitable for classical stainings as well as for immunohistochemistry, preserving the antigen expression pattern of cells. The volume of the solution was 10 times higher that the volume of the sample.

The samples were fixed 3-4 days depending of their size, and were paraffin-wax processed. The paraffin blocks were cut using the microtome to 5μm thick sections. The sections were

Pancreatic Acinar and Island Neogenesis Correlated with Vascular and Matrix Dynamics 37

Since inflammatory elements are preserved in vast areas of fibrosis often peri-lesional, we

Enzyme inductive COX2 normally absent in cells respond to the action of local and general factors in the evaluation using the immune response by pro-inflammatory role in

The main features of chronic pancreatitis are involution of exocrine pancreatic parenchyma and endocrine island elements, accompanied by fibril genetics processes and marked inflammatory infiltrate. Therefore, we can not see unilateral these insular and acinar neogenesis, as long as the accompanying inflammatory process may involve prostaglandins. Inflammatory cells that express COX2, following a specific pancreatic chemo tactics aimed directly the structure, causing necrobiotic damage on one hand, and on the other by

It's important to mention that the changes shown in the figure above appear in areas of island neogenesis which in our opinion could be neo-islands. It is possible that the focal positive Cox2 immunostaining to reflect a local immune reactivity to the neo-islets formation. These aspects have been more obvious in patients consuming alcohol comparing

to diabetes patients, which suggest the low immune capacity of a diabetic person8, 19.

**4. Results and discussions**

pancreatitis.

considered interesting to evaluate the COX23.

phagocytes processes and removing dead cells and cellular debris

Fig. 2. Focal CD68 immunoreaction in a large area of fibrosis, DAB

harvested on poli-L-lisyne slides in order to have better adherence and to exclude the eventual cross reactions. These sections were stained for different antibodies (Table 1) using the Dako's EnVision detection system, and DAB (3,3'-diaminobenzidine) as chromogen substrate. The nuclei were counterstained using Mayer hematoxylin.

Table 1. Antibodies used for immunohistochemistry

Fig. 1. Cox2 positive immunostaining in acinar cells only

### **4. Results and discussions**

36 Chronic Pancreatitis

harvested on poli-L-lisyne slides in order to have better adherence and to exclude the eventual cross reactions. These sections were stained for different antibodies (Table 1) using the Dako's EnVision detection system, and DAB (3,3'-diaminobenzidine) as chromogen

**Antibody Clone Cod Antigen retrieval Dilution**

pH 6 citrate buffer, heat mediated for 11 minutes in the microwave oven

pH 6 citrate buffer, heat mediated for 11 minutes in the microwave oven

pH 6 citrate buffer, heat mediated for 11 minutes in the microwave oven

pH 6 citrate buffer, heat mediated for 11 minutes in the microwave oven

1:100

1:100

1:50

1:50

Dako

Dako

Dako

Cayman Chemical Company

substrate. The nuclei were counterstained using Mayer hematoxylin.

Anti SMA 1A4 M0851

Anti CD68 IgG1k M0814

Anti CD34 QBEnd-10 M 7165

Table 1. Antibodies used for immunohistochemistry

Fig. 1. Cox2 positive immunostaining in acinar cells only

Anti Cox2 CX229

Since inflammatory elements are preserved in vast areas of fibrosis often peri-lesional, we considered interesting to evaluate the COX23.

Enzyme inductive COX2 normally absent in cells respond to the action of local and general factors in the evaluation using the immune response by pro-inflammatory role in pancreatitis.

The main features of chronic pancreatitis are involution of exocrine pancreatic parenchyma and endocrine island elements, accompanied by fibril genetics processes and marked inflammatory infiltrate. Therefore, we can not see unilateral these insular and acinar neogenesis, as long as the accompanying inflammatory process may involve prostaglandins.

Inflammatory cells that express COX2, following a specific pancreatic chemo tactics aimed directly the structure, causing necrobiotic damage on one hand, and on the other by phagocytes processes and removing dead cells and cellular debris

It's important to mention that the changes shown in the figure above appear in areas of island neogenesis which in our opinion could be neo-islands. It is possible that the focal positive Cox2 immunostaining to reflect a local immune reactivity to the neo-islets formation. These aspects have been more obvious in patients consuming alcohol comparing to diabetes patients, which suggest the low immune capacity of a diabetic person8, 19.

Fig. 2. Focal CD68 immunoreaction in a large area of fibrosis, DAB

Pancreatic Acinar and Island Neogenesis Correlated with Vascular and Matrix Dynamics 39

Due to "pseudo-insular" aspect in areas of parenchyma replacement with remaining acinar structures, the presence of activated macrophages is relevant, stimulating the star-shaped cellularity located at the stromal level, responsible for fibrilogenetics processes in areas with

That is why the expression of fibrosis degree with a immunohistochemical response led to

We believe that pancreatic stellate cells (PSC) can act as a central regulator in pancreatic fibrosis in chronic pancreatitis controlling matrix degradation through MMP and TIMP expression, transcriptional regulation process on MMP and TIMP being mediated by

We consider this issue to be important in chronic early pancreatitis. The immunohistochemical response was highly positive while in diabetics and in subjects diagnosed with pancreatitis due to consume alcohol, immunohistochemical response was

In particular we found in subjects with early-stage chronic pancreatitis, alcohol consumers and non-consumers a binucleation cellularity located in the vicinity of Largerhans islands, and acinar structures with proliferative island aspect, which suggested a possibility of transformation of acinar structures and centro-acinar pseudo-cordonal type items in the

In completing this study, the nestin expression in ductal epithelial cells suggests a possible role in island cells neogenesis. It is believed that the birth of new pancreas endocrine cells occur through mature ductal cell differentiation. That is why there are concepts which suggest that we can not use nestin in human pancreas as a marker of endocrine precursor

However, very rarely evidenced in extra-insular cells, with increased nestin expression in neonatal pancreatic islets, we can say, however, it is involved in island neogenesis. This is why it rather indicates a tissue differentiation with diffuse pattern, varying in layout

Immunohistochemical nestin positive response was extinguished met both in pancreatic duct cells in appearance and in isolated and small outbreaks in the structures with pseudoinsular aspect, in mature Langerhans island structures, but necrobiotics damages makes

This reinforces our idea of a possible positivity of tissue remodeling with cell differentiation

As a result of the vast process of fibrosis processes leading to a reversal of the parenchymastroma relationship in favor of the stroma, we were interested in the distribution of collagen

Thus, we observed that in subjects with clinically diagnosed early-stage chronic pancreatitis was present a pattern of continuous distribution of collagen IV in basal membrane around

the use of matrix metalloproteinases (MMP) involved in regulating fibrilogenesis.

weak positive on acinar cells, and even negative in insular cells.

fibrosis12, 14.

cytokines2.

cells21.

drafting of primitive island.

structures with pseudo-insular aspects.

ductal cytolysis lesions.

difficult to differentiate between different structures.

or functionality exocrine or endocrine-related functionality.

IV, a basement membrane major constituent within laminin13.

So, leukocyte infiltration in chronic pancreatitis is represented by a remarkable percentage of mononuclear cells, which suggests that macrophages have great importance in the inflammatory process. With origin in circulating monocytes, macrophages are activated by certain cytokines and a series of endotoxin, so that their activation can induce phagocytosis. That's why COX 2 inhibitors are accepted therapy of chronic inflammatory lesions5, 20.

Immunohistochemical response evaluation was approached differently depending on case. For cases diagnosed with early stage chronic pancreatitis imunoreactivity for COX2 was highly positive (62%), while for subjects with diabetes and those diagnosed with advanced chronic pancreatitis was weakly positive (12.7%) or negative response, both ductal and intra-island.

The immunostaining for macrophages (CD68) was reduced compared to Cox2 staining suggesting that the chronic or sub-acute inflammatory reaction is not that much involved in the acino-island regneration.

In advanced chronic pancreatitis, intra and extra-lobular ductal cells were intensely positive for COX-2 compared with islands with variable staining pattern in areas with insulin-positive cells and clinical diagnosis of diabetes10. Immunoreactivity was low or absent in people with type II diabetes compared with people consuming alcohol and diabetes type I. These aspects were extremely relevant on the studied samples. Therefore, we consider that a positive COX2 immunoreactivity particularly in intralobular ductal cells enables us to corroborate the involvement of these cellular processes into parenchyma and stroma changes17,18.

Fig. 3. Focal CD68 immunoreaction near a optic vide areas possible due to focal necrosis, DAB

So, leukocyte infiltration in chronic pancreatitis is represented by a remarkable percentage of mononuclear cells, which suggests that macrophages have great importance in the inflammatory process. With origin in circulating monocytes, macrophages are activated by certain cytokines and a series of endotoxin, so that their activation can induce phagocytosis. That's why COX 2 inhibitors are accepted therapy of chronic inflammatory lesions5, 20.

Immunohistochemical response evaluation was approached differently depending on case. For cases diagnosed with early stage chronic pancreatitis imunoreactivity for COX2 was highly positive (62%), while for subjects with diabetes and those diagnosed with advanced chronic pancreatitis was weakly positive (12.7%) or negative response, both ductal and

The immunostaining for macrophages (CD68) was reduced compared to Cox2 staining suggesting that the chronic or sub-acute inflammatory reaction is not that much involved in

In advanced chronic pancreatitis, intra and extra-lobular ductal cells were intensely positive for COX-2 compared with islands with variable staining pattern in areas with insulin-positive cells and clinical diagnosis of diabetes10. Immunoreactivity was low or absent in people with type II diabetes compared with people consuming alcohol and diabetes type I. These aspects were extremely relevant on the studied samples. Therefore, we consider that a positive COX2 immunoreactivity particularly in intralobular ductal cells enables us to corroborate the

Fig. 3. Focal CD68 immunoreaction near a optic vide areas possible due to focal necrosis, DAB

involvement of these cellular processes into parenchyma and stroma changes17,18.

intra-island.

the acino-island regneration.

Due to "pseudo-insular" aspect in areas of parenchyma replacement with remaining acinar structures, the presence of activated macrophages is relevant, stimulating the star-shaped cellularity located at the stromal level, responsible for fibrilogenetics processes in areas with fibrosis12, 14.

That is why the expression of fibrosis degree with a immunohistochemical response led to the use of matrix metalloproteinases (MMP) involved in regulating fibrilogenesis.

We believe that pancreatic stellate cells (PSC) can act as a central regulator in pancreatic fibrosis in chronic pancreatitis controlling matrix degradation through MMP and TIMP expression, transcriptional regulation process on MMP and TIMP being mediated by cytokines2.

We consider this issue to be important in chronic early pancreatitis. The immunohistochemical response was highly positive while in diabetics and in subjects diagnosed with pancreatitis due to consume alcohol, immunohistochemical response was weak positive on acinar cells, and even negative in insular cells.

In particular we found in subjects with early-stage chronic pancreatitis, alcohol consumers and non-consumers a binucleation cellularity located in the vicinity of Largerhans islands, and acinar structures with proliferative island aspect, which suggested a possibility of transformation of acinar structures and centro-acinar pseudo-cordonal type items in the drafting of primitive island.

In completing this study, the nestin expression in ductal epithelial cells suggests a possible role in island cells neogenesis. It is believed that the birth of new pancreas endocrine cells occur through mature ductal cell differentiation. That is why there are concepts which suggest that we can not use nestin in human pancreas as a marker of endocrine precursor cells21.

However, very rarely evidenced in extra-insular cells, with increased nestin expression in neonatal pancreatic islets, we can say, however, it is involved in island neogenesis. This is why it rather indicates a tissue differentiation with diffuse pattern, varying in layout structures with pseudo-insular aspects.

Immunohistochemical nestin positive response was extinguished met both in pancreatic duct cells in appearance and in isolated and small outbreaks in the structures with pseudoinsular aspect, in mature Langerhans island structures, but necrobiotics damages makes difficult to differentiate between different structures.

This reinforces our idea of a possible positivity of tissue remodeling with cell differentiation or functionality exocrine or endocrine-related functionality.

As a result of the vast process of fibrosis processes leading to a reversal of the parenchymastroma relationship in favor of the stroma, we were interested in the distribution of collagen IV, a basement membrane major constituent within laminin13.

Thus, we observed that in subjects with clinically diagnosed early-stage chronic pancreatitis was present a pattern of continuous distribution of collagen IV in basal membrane around ductal cytolysis lesions.

Pancreatic Acinar and Island Neogenesis Correlated with Vascular and Matrix Dynamics 41

Importantly, for an overview of the changes occurring in the matrix and the parenchyma elements, exocrine and endocrine aspects with microscopic positive immunohistochemical markers were mentioned. We consider this relevant because in chronic pancreatitis common lesions can be found but differentiated both at the parenchymal and stromal level. Aspects that we found can be considered to be elements of island neogenesis and are certainly induced by the changes occurring in the overall lesion of chronic pancreatitis. Thus, serous acinar cell nucleus were found in different stages of involution, but the vast majority of nucleus had dispersed chromatin lumps in nucleoplasma or condensed chromatin on nuclear membrane, thereby creating a vacuum optical image space, perinucleolar,

Fig. 5. CD 34 positive in vascular endothelial cells and focal in perivascular cells, DAB

has an important role in pancreatic fibrogenesis4, 15, 18.

Chronic pancreatitis is characterized by a large number of reactive miofibroblasts to alpha SMA, chronic pancreatitis stromal remodelation being also caused by miofibroblasts, immunoreactive fibroblast-like cells with contractile properties which are considered to be fibroblasts16, 22. The TGF-beta has a main role in fibrosis development in liver, but also in pancreas, this suggesting a better research of hepatopathies matter. Many evidences suggest that PSC can be also activated by paracrine profibrogenes cytokines, like trombocytes derivated growth factor and TGF-beta derivated by migratory macrophages. In addition, PSC generate EMC components in return for TGF-beta action, suggesting that the cytokine

something known as "owl eye".

Also, large areas of acinar structures were separated by collagen bands while the remaining islands collagen network sketches pseudo-capsular aspect, so insular cytolysis and modified histo-architecture would be possible by the dual mechanisms.

These issues were particularly characteristic in subjects with advanced chronic pancreatitis. Also, this is very useful especially in differential diagnosis of pancreatic adenocarcinoma where the distribution of collagen IV in basal membrane is discontinuous and irregular or absent around individual cells or groups of tumor cells.

It is known that in normal pancreas, CD 34 positive stromal cells are present predominantly in peri-acinar areas. Isolated CD34 positive cells also were observed in stroma of subjects diagnosed chronic pancreatitis in both advanced and early stage7,9.

It seems that the presence of significant amounts of CD34 positive stromal cells in primary lesion is characteristic of chronic inflammatory lesions.

In our study, subjects diagnosed with chronic pancreatitis could see an increase in fibrocites CD34 positive. Also, CD34 was positive in vessels in all cases of chronic pancreatitis studied both in diabetics (type I and type II) and drinkers.

This leads us to the idea of involving fibrotic elements which are CD34 positive in a result of stromal remodeling in chronic pancreatitis. Intensively CD34 positive vascular elements were observed in diabetic subjects compared with the rest of the studied

Fig. 4. CD34 immunoreaction in vascular endothelial cells, DAB

Also, large areas of acinar structures were separated by collagen bands while the remaining islands collagen network sketches pseudo-capsular aspect, so insular cytolysis and modified

These issues were particularly characteristic in subjects with advanced chronic pancreatitis. Also, this is very useful especially in differential diagnosis of pancreatic adenocarcinoma where the distribution of collagen IV in basal membrane is discontinuous and irregular or

It is known that in normal pancreas, CD 34 positive stromal cells are present predominantly in peri-acinar areas. Isolated CD34 positive cells also were observed in stroma of subjects

It seems that the presence of significant amounts of CD34 positive stromal cells in primary

In our study, subjects diagnosed with chronic pancreatitis could see an increase in fibrocites CD34 positive. Also, CD34 was positive in vessels in all cases of chronic pancreatitis studied

This leads us to the idea of involving fibrotic elements which are CD34 positive in a result of stromal remodeling in chronic pancreatitis. Intensively CD34 positive vascular elements

histo-architecture would be possible by the dual mechanisms.

diagnosed chronic pancreatitis in both advanced and early stage7,9.

were observed in diabetic subjects compared with the rest of the studied

Fig. 4. CD34 immunoreaction in vascular endothelial cells, DAB

absent around individual cells or groups of tumor cells.

lesion is characteristic of chronic inflammatory lesions.

both in diabetics (type I and type II) and drinkers.

Importantly, for an overview of the changes occurring in the matrix and the parenchyma elements, exocrine and endocrine aspects with microscopic positive immunohistochemical markers were mentioned. We consider this relevant because in chronic pancreatitis common lesions can be found but differentiated both at the parenchymal and stromal level. Aspects that we found can be considered to be elements of island neogenesis and are certainly induced by the changes occurring in the overall lesion of chronic pancreatitis. Thus, serous acinar cell nucleus were found in different stages of involution, but the vast majority of nucleus had dispersed chromatin lumps in nucleoplasma or condensed chromatin on nuclear membrane, thereby creating a vacuum optical image space, perinucleolar, something known as "owl eye".

Fig. 5. CD 34 positive in vascular endothelial cells and focal in perivascular cells, DAB

Chronic pancreatitis is characterized by a large number of reactive miofibroblasts to alpha SMA, chronic pancreatitis stromal remodelation being also caused by miofibroblasts, immunoreactive fibroblast-like cells with contractile properties which are considered to be fibroblasts16, 22. The TGF-beta has a main role in fibrosis development in liver, but also in pancreas, this suggesting a better research of hepatopathies matter. Many evidences suggest that PSC can be also activated by paracrine profibrogenes cytokines, like trombocytes derivated growth factor and TGF-beta derivated by migratory macrophages. In addition, PSC generate EMC components in return for TGF-beta action, suggesting that the cytokine has an important role in pancreatic fibrogenesis4, 15, 18.

Pancreatic Acinar and Island Neogenesis Correlated with Vascular and Matrix Dynamics 43

Instead, binucleate cellularity was observed in areas of parenchyma completely missing. We noticed the oval binucleate cells with nucleus whose chromatin is cloggy or condensed on the nuclear membrane. Cytoplasm of these cells may present some kind of vacuolization or

Fig. 7. Anti alpha-SMA staining identifies positive star-shaped cells in parenchyma

Perhaps these cells are involved in pancreatic parenchymal cytological regeneration.

As a result of Davenport staining, we noticed groups of cells containing finely granular and hypo-chromic nuclei, located in the interlobular spaces near the excretory channels. Their appearance is the type of "pseudo-insular" irregular looking with hypo-chromic nuclei and

In the immediate vicinity of these islands we have noted the presence of monoclonal cells

These aspects observed in the conjunctive-vascular septas, suggested the hypothesis that, although completely modified, the cyto-architecture of pancreatic parenchyma can

peri-nuclear argirofile areas in particular.

replacement areas, DAB

intra-insular argentafine areas.

with hypo-chromic nucleus fine granular cytoplasm aspect.

regenerate, but without a full morpho-functional reconstruction.

Therefore, we believe that these cell formations are a "primitive form islands."

The apoptosis research, inflammatory injuries, tumoral genesis are controlled by a nuclear transcription factor, NF-kB factor or Kappa B Nuclear Factor. In chronic pancreatitis NF-kB is positive, with intranuclear distribution. Chronic pancreatitis associated hepatic injuries are characterized by hepatocelular apoptosis and necrosis, NF-kB being dependent of liver releasing cytokines and chemakines. Chronic pancreatitis activation can induce hepatic damage through derived Kupfer cells cytokines activation and Fas/Fasl releasing.

Fig. 6. Anti alpha-SMA staining identifies large vessels with hyaline perivascular deposits, DAB

One aspect we found is that the presence of binucleate cells in the exocrine pancreatic parenchyma. Binucleate cells of polygonal or oval shape present nucleus with condensed chromatin on the nuclear membrane, nucleols and nucleoplasma, obvious tendency to create optically empty spaces. These issues lead us to the possibility of parenchyma neogenesis that we can not exclude taking into account the common embryonic origin of the pancreas with the liver.

Close to capillaries responsible for nutrition of the exocrine parenchyma areas we found the presence of two cell types, some of which were binucleate cells with condensed chromatin on nuclear membrane, with or without obvious nucleoli, and some globular, uninucleate and with nucleoli.

The presence of these cells enables us to believe that they are involved in a possible recovery of pancreatic parenchyma, with the existence of metabolic processes.

The apoptosis research, inflammatory injuries, tumoral genesis are controlled by a nuclear transcription factor, NF-kB factor or Kappa B Nuclear Factor. In chronic pancreatitis NF-kB is positive, with intranuclear distribution. Chronic pancreatitis associated hepatic injuries are characterized by hepatocelular apoptosis and necrosis, NF-kB being dependent of liver releasing cytokines and chemakines. Chronic pancreatitis activation can induce hepatic

damage through derived Kupfer cells cytokines activation and Fas/Fasl releasing.

Fig. 6. Anti alpha-SMA staining identifies large vessels with hyaline perivascular deposits,

One aspect we found is that the presence of binucleate cells in the exocrine pancreatic parenchyma. Binucleate cells of polygonal or oval shape present nucleus with condensed chromatin on the nuclear membrane, nucleols and nucleoplasma, obvious tendency to create optically empty spaces. These issues lead us to the possibility of parenchyma neogenesis that we can not exclude taking into account the common embryonic origin of the pancreas

Close to capillaries responsible for nutrition of the exocrine parenchyma areas we found the presence of two cell types, some of which were binucleate cells with condensed chromatin on nuclear membrane, with or without obvious nucleoli, and some globular, uninucleate

The presence of these cells enables us to believe that they are involved in a possible recovery

of pancreatic parenchyma, with the existence of metabolic processes.

DAB

with the liver.

and with nucleoli.

Instead, binucleate cellularity was observed in areas of parenchyma completely missing. We noticed the oval binucleate cells with nucleus whose chromatin is cloggy or condensed on the nuclear membrane. Cytoplasm of these cells may present some kind of vacuolization or peri-nuclear argirofile areas in particular.

Fig. 7. Anti alpha-SMA staining identifies positive star-shaped cells in parenchyma replacement areas, DAB

Perhaps these cells are involved in pancreatic parenchymal cytological regeneration.

As a result of Davenport staining, we noticed groups of cells containing finely granular and hypo-chromic nuclei, located in the interlobular spaces near the excretory channels. Their appearance is the type of "pseudo-insular" irregular looking with hypo-chromic nuclei and intra-insular argentafine areas.

In the immediate vicinity of these islands we have noted the presence of monoclonal cells with hypo-chromic nucleus fine granular cytoplasm aspect.

These aspects observed in the conjunctive-vascular septas, suggested the hypothesis that, although completely modified, the cyto-architecture of pancreatic parenchyma can regenerate, but without a full morpho-functional reconstruction.

Therefore, we believe that these cell formations are a "primitive form islands."

Pancreatic Acinar and Island Neogenesis Correlated with Vascular and Matrix Dynamics 45

As a result of our study we agreed that pancreatic stellar stroma cells mediates fibrosis in chronic pancreatitis with matrix metalloproteinases (MMPS) and tissue inhibitors of

We consider that in the pathogenesis of chronic pancreatic disease, multiple factors and theories are involved leading to parenchyma destruction and fibrosis with elements of

Immunohistochemical results in the dynamic vascular-stromal and island neogenesis does not clarify the subject. We believe that on the human models we can not have so spectacular results as those experimental models, because the subjects with chronic pancreatitis are

[1] Tyler Stevens, Darwin L. Conwell, Gregory Zuccaro, Pathogenesis of chronic

[2] Adam J. Ottaviano, Limin Sun, Vijayalakshmi Ananthanarayanan, Hidayatullah G.

[3] Anirban Maitra, Md, Raheela Ashfaq, Md, Carla R. Gunn, Ayman Rahman, Charles J.

[4] Apte M.V., P S Haber, S J Darby, S C Rodgers, G W Mccaughan,M A Korsten, R C Pirola,

[5] Ardeleanu Carmen, Comănescu Violeta, Zaharia B. - Imunohistochimie, ed. Sitech, 1999;

[6] Banciu T., Susan L., Jovin G. şi col. – Prevalence of chronic (latent) pancreatitis in

[7] Barth P. J., Ebrahimsade S., Hellinger A., Moll R., Ramaswamy Annette - CD34[+]

[8] Gray Kd, Simovic Mo, Blackwell Ts, Christman Jw, May Ak, Parman Ks, Chapman Wc,

[9] Kuroda N, Toi M, Nakayama H, Miyazaki E, Yamamoto M, Hayashi Y, Hiroi M, Enzan

American Journal of Gastroenterology, 2004; 99: 2256-2270.

Transforming Growth Factor- βß1 Cancer Res 2006; 66: 7032-7040;

Automated Cellular ImagingAm J Clin Pathol 118(2):194-201, 2002.

implications for pancreatic fibrogenesis – Gut., 1999;44:534–541;

pancreatitis: An Evience Based Review of past theories and recent developments,

Munshi - Extracellular Matrix-Mediated Membrane-Type 1 Matrix Metalloproteinase Expression in Pancreatic Ductal Cells Is Regulated by

Yeo, Md, Taylor A. Sohn, Md, John L. Cameron, Md, Ralph H. Hruban, Md, Robb E. Wilentz, Md - Cyclooxygenase 2 Expression in Pancreatic Adenocarcinoma and Pancreatic Intraepithelial Neoplasia: An Immunohistochemical Analysis With

J S Wilson - Pancreatic stellate cells are activated by proinflammatory cytokines:

hospitalized chronic consumers of alcohol, Rom. J. Med., 1991, Jan – Jun; 29 (1 -2):

fibrocytes in neoplastic and inflammatory pancreatic lesions European Society of Pathology Virchows Archiv (Virchows Arch.) ISSN 0945-6317

Stain Sc. Activation of nuclear factor kappa B and severe hepatic necrosis may mediate systemic inflammation in choline-deficient/ethionine-supplemented diet-

H. - The distribution and role of myofibroblasts and CD34 - positive stromal cells in

metalloproteinases (LONG) -1 and -2 as a modulator of fibrosis

exposed to a lot of factors in different evolutionary stages.

2002, vol. 440, no2, pp. 128-133 (24 ref.);

induced pancreatitis. Pancreas. 2006 Oct;33(3):260-7.

**5. Conclusions**

**6. References**

acino-insular neogenesis.

140-146;

49 – 53;

Fig. 8. Alpha-SMA positive staining in middle caliber vessels and intra parenchymatous, DAB

Endocrine parenchymal regeneration issues we noticed in other samples studied, where we found the presence of large cells compared with the rest of the cell population, on the outskirts of Largenhans located islands.

These cells are clearly distinguishable from other island cells, their cell boundaries, appearing not to keep. Cytoplasm of these cells has a fine granular and the nucleus, binucleolat, presents condensed chromatin on the nuclear membrane, giving an embattled appearance.

Besides these large cells, with localization throughout the periphery of the island, we noted the presence of binucleate cells, irregularly shaped. Monoclonal cells with bulky appearance and with binucleate cells were observed mainly in the periphery of Langerhans islands.

That is why the presence of mono-and binucleate cells, large in size compared with the rest of the islet, suggests partial restoration of island celularity.

Pancreatic fibrosis, a characteristic histopathologic appearance of chronic pancreatitis in our study was an active process that, according to some authors, may be reversible in the early stages13.

Identification and characterization of cells that have fibrilogenesis capacity greatly helped us in the study of pancreatic stroma in chronic pancreatitis. COX-2 played an important proinflammatory role in pancreatitis by regulating prostaglandin synthesis.

As a result of our study we agreed that pancreatic stellar stroma cells mediates fibrosis in chronic pancreatitis with matrix metalloproteinases (MMPS) and tissue inhibitors of metalloproteinases (LONG) -1 and -2 as a modulator of fibrosis

#### **5. Conclusions**

44 Chronic Pancreatitis

Fig. 8. Alpha-SMA positive staining in middle caliber vessels and intra parenchymatous, DAB

Endocrine parenchymal regeneration issues we noticed in other samples studied, where we found the presence of large cells compared with the rest of the cell population, on the

These cells are clearly distinguishable from other island cells, their cell boundaries, appearing not to keep. Cytoplasm of these cells has a fine granular and the nucleus, binucleolat, presents condensed chromatin on the nuclear membrane, giving an embattled

Besides these large cells, with localization throughout the periphery of the island, we noted the presence of binucleate cells, irregularly shaped. Monoclonal cells with bulky appearance and with binucleate cells were observed mainly in the periphery of Langerhans islands.

That is why the presence of mono-and binucleate cells, large in size compared with the rest

Pancreatic fibrosis, a characteristic histopathologic appearance of chronic pancreatitis in our study was an active process that, according to some authors, may be reversible in the early

Identification and characterization of cells that have fibrilogenesis capacity greatly helped us in the study of pancreatic stroma in chronic pancreatitis. COX-2 played an important

proinflammatory role in pancreatitis by regulating prostaglandin synthesis.

outskirts of Largenhans located islands.

of the islet, suggests partial restoration of island celularity.

appearance.

stages13.

We consider that in the pathogenesis of chronic pancreatic disease, multiple factors and theories are involved leading to parenchyma destruction and fibrosis with elements of acino-insular neogenesis.

Immunohistochemical results in the dynamic vascular-stromal and island neogenesis does not clarify the subject. We believe that on the human models we can not have so spectacular results as those experimental models, because the subjects with chronic pancreatitis are exposed to a lot of factors in different evolutionary stages.

#### **6. References**

**Part 2**

**Diagnosis and Treatment**

**of Chronic Pancreatitis**

normal pancreas and various pancreatic lesions. Histol Histopathol. 2004 Jan;19(1):59-67;

## **Part 2**

**Diagnosis and Treatment of Chronic Pancreatitis**

46 Chronic Pancreatitis

[10] Mateescu Garofita, Bold Adriana, Mogoanta L., Busuioc Cristina, PANDURU

[11] O'reilly Da, Roberts Jr, Cartmell Mt, Demaine Ag, Kingsnorth An. - Heat shock factor-1

[12] Palm K, Salin-Nordstrom T, Levesque Mf & Neuman T - Fetal and adult human CNS

[13] Panduru Laurenţia, Bogdan Fl., Mărgăritescu C., Mateescu Garofiţa, – Dinamica

[14] Phillips Pa, Mccarroll Ja, Park S, Wu Mj, Pirola R, Korsten M, et al. Rat pancreatic

[15] Phillips Pa, Wu Mj, Kumar Rk, Doherty E, Mccarroll Ja, Park S, et al. Cell migration: a

[16] Rietze Rl, Valcanis H, Brooker Gf, Thomas T, Voss Ak & Bartlett Pf - Purification of a

[17] Schlosser W, Schlosser S, Ramadani M, Gansauge F, Gansauge S, Beger Hg. -

[18] Seiya Yoshida, Michael Ujiki, Xian-Zhong Ding, Carolyn Pelham, Mark S Talamonti,

[19] Shi C, Zhao X, Lagergren A, Sigvardsson M, Wang X, Andersson R. - Immune status

[20] Shimizu K, Kobayashi M, Tahara J, Shiratori K. Cytokines and peroxisome proliferator-

[21] Street C N, J R T Lakey, K Seeberger, L Helms, R V Rajotte, A M J Shapiro, Korbutt G S -

an islet precursor marker. Journal of Endocrinology (2004) 180, 213–225. [22] Suda K, Fukumura Y, Takase M, Kashiwagi S, Izumi M, Kumasaka T, Suzuki F.

fibrogenesis in chronic alcoholic pancreatitis. Pathol Int 2007; 57:21-5.

pancreatitis. Scand J Gastroenterol. 2006 Apr;41(4):472-8.

novel aspect of pancreatic stellate cell biology. Gut 2003; 52:677-82.

Jan;19(1):59-67;

JOP. 2006 Mar 9;7(2):174-84.

Ştiinţifice, Craiova, 2004;

739.

Jul;25(1):26-30.

turnover. Gut 2003; 52:275-82.

in PSCs. Mol Cancer. 2005; 4: 27.

Gastroenterology 2005; 128:2105-18.

Developmental Brain Research, 2000, 78 192–195.

normal pancreas and various pancreatic lesions. Histol Histopathol. 2004

LAURENTIA – Studiul histologic parenchimo-stromal al pancreasului la persoanele cu diabet zaharat tip 2, Sesiune de Comunicări Stiinţifice, Craiova, 2004;

and nuclear factor-kappaB are systemically activated in human acute pancreatitis.

stem cells have similar molecular characteristics and developmental potential.

stromei pancreatice cronice alcoolice şi nonalcoolice, Sesiune de Comunicări

stellate cells secrete matrix metalloproteinases: implications for extracellular matrix

pluripotent neural stem cell from the adult mouse brain. Nature, 1994. 412 736–

Cyclooxygenase-2 is overexpressed in chronic pancreatitis: Pancreas. 2002

Richard H Bell, Jr, Woody Denham, Thomas E Adrian - Pancreatic Stellate Cells (PSCs) express Cyclooxygenase-2 (COX-2) and pancreatic cancer stimulates COX-2

and inflammatory response differ locally and systemically in severe acute

activated receptor gamma ligand regulate phagocytosis by pancreatic stellate cells.

Heterogenous expression of nestin in human pancreatic tissue precludes its use as

Activated perilobular, not periacinar, pancreatic stellate cells contribute to

**4**

*Brazil*

**The Role of Endoscopic Ultrasound**

**to Diagnose, Exclude or Stablish the**

José Celso Ardengh1 and Eder Rios Lima-Filho2

*1Endoscopy Unit, Hospital 9 de Julho, São Paulo, 2Endoscopy Unit, Division of Surgery and Anatomy, Ribeirão Preto School of Medicine, University of São Paulo,*

**Parenchimal Changes in Chronic Pancreatitis**

Chronic pancreatitis (CP) can be defined as an inflammatory disease with progressive and irreversible morphological changes (1). There is loss of endocrine and / or exocrine function, with or without pain (2-6). The structure is altered by inflammation, necrosis, fibrosis and

Alcoholism accounts for 70 to 80% of CP ; 10 to 20% are idiopathic and the remaining 5-10% are caused by hypercalcemia, trauma, hereditary diseases, hyperlipidemia (types I, IV and

CP is associated with a mortality rate of approximately 50% within 20 to 25 years after its onset (2,6). About 15 to 20% of patients die due to complications associated with attacks of CP, and most of the remaining deaths are due to trauma, malnutrition, infection or smoking (often associated with alcoholism) (2,6). Very little is known about the actual prevalence or incidence of CP, although estimates indicate an incidence of 3.5 to 4 cases per 100.000 (2,6).

Clinical presentation of CP is characterized by attacks of pain (1). This is intense, localized in the epigastrium, radiating to the back and may also be present in the right or left hypochondria and be associated with nausea and vomiting (1). Usually lasts for hours, although some patients experience continuous pain for days or weeks (3, 5, 6). Along with the destruction of pancreatic tissue there are signs of endocrine pancreatic insufficiency (impaired glucose tolerance, diabetes mellitus) and / or exocrine (steatorrhea) (3, 5, 6).

The diagnosis is based on morphological (abnormalities of pancreatic channels) and functional criteria (pancreatic insufficiency). Although it is easy in advanced forms (Figure 1), in early stages, diagnosis is difficult (7). Ideal diagnostic criteria would be histological (8). However, pancreatic biopsy is susceptible to serious complications, especially in normal or

**1. Introduction**

**2. Symptomatology**

**3. Diagnosis**

loss of exocrine and endocrine elements (5,6).

V) or nutritional causes (tropical pancreatitis) (3, 5, 6).

## **The Role of Endoscopic Ultrasound to Diagnose, Exclude or Stablish the Parenchimal Changes in Chronic Pancreatitis**

José Celso Ardengh1 and Eder Rios Lima-Filho2 *1Endoscopy Unit, Hospital 9 de Julho, São Paulo, 2Endoscopy Unit, Division of Surgery and Anatomy, Ribeirão Preto School of Medicine, University of São Paulo, Brazil*

#### **1. Introduction**

Chronic pancreatitis (CP) can be defined as an inflammatory disease with progressive and irreversible morphological changes (1). There is loss of endocrine and / or exocrine function, with or without pain (2-6). The structure is altered by inflammation, necrosis, fibrosis and loss of exocrine and endocrine elements (5,6).

Alcoholism accounts for 70 to 80% of CP ; 10 to 20% are idiopathic and the remaining 5-10% are caused by hypercalcemia, trauma, hereditary diseases, hyperlipidemia (types I, IV and V) or nutritional causes (tropical pancreatitis) (3, 5, 6).

CP is associated with a mortality rate of approximately 50% within 20 to 25 years after its onset (2,6). About 15 to 20% of patients die due to complications associated with attacks of CP, and most of the remaining deaths are due to trauma, malnutrition, infection or smoking (often associated with alcoholism) (2,6). Very little is known about the actual prevalence or incidence of CP, although estimates indicate an incidence of 3.5 to 4 cases per 100.000 (2,6).

#### **2. Symptomatology**

Clinical presentation of CP is characterized by attacks of pain (1). This is intense, localized in the epigastrium, radiating to the back and may also be present in the right or left hypochondria and be associated with nausea and vomiting (1). Usually lasts for hours, although some patients experience continuous pain for days or weeks (3, 5, 6). Along with the destruction of pancreatic tissue there are signs of endocrine pancreatic insufficiency (impaired glucose tolerance, diabetes mellitus) and / or exocrine (steatorrhea) (3, 5, 6).

#### **3. Diagnosis**

The diagnosis is based on morphological (abnormalities of pancreatic channels) and functional criteria (pancreatic insufficiency). Although it is easy in advanced forms (Figure 1), in early stages, diagnosis is difficult (7). Ideal diagnostic criteria would be histological (8). However, pancreatic biopsy is susceptible to serious complications, especially in normal or

The Role of Endoscopic Ultrasound to Diagnose,

shadow in a patient with calcifying CP.

dilation and intracanalicular lesions (16-18).

Exclude or Stablish the Parenchimal Changes in Chronic Pancreatitis 51

calcifications (Figure 2) (2,9-11). Intravenous injection of secretin may be useful in early forms of CP, enhancing discrete changes in MPD caliber (12). Sensitivity and specificity of ultrasound to diagnose CP ranges from 50 to 70% and 80 to 90%, respectively (6, 10, 13).

Fig. 2. Echoendoscopic image showing multiple hyperechoic areas with posterior acoustic

A study comparing US, CT and endoscopic retrograde cholangiopancreatography (ERCP) demonstrated that US has a sensitivity of 58% and specificity of 75% to diagnose CP (14). CT is more sensitive and accurate than plain films or US. The suggestive findings of CP include: glandular atrophy, irregularities in pancreatic contours, dilation and irregularities of pancreatic channels with pancreatic calcifications (15). CT is the most sensitive test to detect calcifications and it is still important to search for complications such as pseudocysts. CT sensitivity varies from 74 to 90% and its specificity is over 85% to diagnose CP (2,6). In

Magnetic resonance imaging (MRI) and magnetic resonance cholangiopancreatography (MRCP) are new non-invasive methods, (no sedation, no contrast, no endoscope insertion). They can obtain images of both parenchyma (MRI) and hepatic and billiary channels (MRCP). They can identify pancreatic atrophy, MPD stenosis or dilation, collateral branches

Our experience is according to literature, and shows that the concordance between MRCP and ERCP varies from 83% to 92% in canalicular dilation, 70% to 92% in canalicular stenosis and 92% to 100% when intracanalicular lesions are present (16, 18, 19). Often, minor abnormalities detectable by ERCP are often undetectable by MRCP and, the rate of falsepositive results in canalicular stenosis is high (16,18). Although MRCP is useful to detect

Intravenous administration of secretin during MRCP is an alternative to enhance image of MPD in the early stages of CP, as it improves the diagnostic yield (20,21). Image interpretation should be cautious, keeping in mind that artifacts may occur during its reconstruction, leading to a misconception of obstruction, stenosis and stones (21, 22).

moderate and advanced forms of CP, it has a limited value in the early stages (9,18).

aforementioned study, CT had a sensitivity of 75% and specificity of 95% (14).

slightly impaired pancreas. In addition, irregular distribution of lesions can lead to diagnostic errors (false-negative) (8,9).

(a)

(b)

Fig. 1. Image obtained by wirsungography (a): dilation of MPD and secondary branches. It suggests CP. Echoendoscopic image (b) of the same patient. Besides dilatation of main pancreatic duct (MPD), there are hyperechoic streaks, and hypoechoic areas permeating normal parenchyma.

The development of a technique that could be able to detect early morphological abnormalities and besides that could search for specific cellular or CP biochemical markers in pancreatic juice would represent a significant advance in this field (6,9).

#### **4. Imaging**

Similar to AP, US is considered the first exam for patients with suspected CP (7). It shows localized or diffuse increase in pancreatic volume, irregularities and dilations of main pancreatic duct (MPD), or cystic collections adjacent to the gland, and pancreatic

slightly impaired pancreas. In addition, irregular distribution of lesions can lead to

(a)

(b) Fig. 1. Image obtained by wirsungography (a): dilation of MPD and secondary branches. It suggests CP. Echoendoscopic image (b) of the same patient. Besides dilatation of main pancreatic duct (MPD), there are hyperechoic streaks, and hypoechoic areas permeating

The development of a technique that could be able to detect early morphological abnormalities and besides that could search for specific cellular or CP biochemical markers

Similar to AP, US is considered the first exam for patients with suspected CP (7). It shows localized or diffuse increase in pancreatic volume, irregularities and dilations of main pancreatic duct (MPD), or cystic collections adjacent to the gland, and pancreatic

in pancreatic juice would represent a significant advance in this field (6,9).

diagnostic errors (false-negative) (8,9).

normal parenchyma.

**4. Imaging**

calcifications (Figure 2) (2,9-11). Intravenous injection of secretin may be useful in early forms of CP, enhancing discrete changes in MPD caliber (12). Sensitivity and specificity of ultrasound to diagnose CP ranges from 50 to 70% and 80 to 90%, respectively (6, 10, 13).

Fig. 2. Echoendoscopic image showing multiple hyperechoic areas with posterior acoustic shadow in a patient with calcifying CP.

A study comparing US, CT and endoscopic retrograde cholangiopancreatography (ERCP) demonstrated that US has a sensitivity of 58% and specificity of 75% to diagnose CP (14). CT is more sensitive and accurate than plain films or US. The suggestive findings of CP include: glandular atrophy, irregularities in pancreatic contours, dilation and irregularities of pancreatic channels with pancreatic calcifications (15). CT is the most sensitive test to detect calcifications and it is still important to search for complications such as pseudocysts. CT sensitivity varies from 74 to 90% and its specificity is over 85% to diagnose CP (2,6). In aforementioned study, CT had a sensitivity of 75% and specificity of 95% (14).

Magnetic resonance imaging (MRI) and magnetic resonance cholangiopancreatography (MRCP) are new non-invasive methods, (no sedation, no contrast, no endoscope insertion). They can obtain images of both parenchyma (MRI) and hepatic and billiary channels (MRCP). They can identify pancreatic atrophy, MPD stenosis or dilation, collateral branches dilation and intracanalicular lesions (16-18).

Our experience is according to literature, and shows that the concordance between MRCP and ERCP varies from 83% to 92% in canalicular dilation, 70% to 92% in canalicular stenosis and 92% to 100% when intracanalicular lesions are present (16, 18, 19). Often, minor abnormalities detectable by ERCP are often undetectable by MRCP and, the rate of falsepositive results in canalicular stenosis is high (16,18). Although MRCP is useful to detect moderate and advanced forms of CP, it has a limited value in the early stages (9,18).

Intravenous administration of secretin during MRCP is an alternative to enhance image of MPD in the early stages of CP, as it improves the diagnostic yield (20,21). Image interpretation should be cautious, keeping in mind that artifacts may occur during its reconstruction, leading to a misconception of obstruction, stenosis and stones (21, 22).

The Role of Endoscopic Ultrasound to Diagnose,

Exclude or Stablish the Parenchimal Changes in Chronic Pancreatitis 53

(a)

(b)

Fig. 3. Echoendoscopic images in patients with abdominal pain. (a) Hypoechoic areas, intermingled with normal parenchyma and hyperechoic streaks. The aspect remembers a "honeycomb". This aspect suggests early-stage chronic pancreatitis. (b) The same aspect, but

more pronounced.

ERCP is considered the "gold standard" for diagnosis and treatment of CP (6, 9). Its sensitivity ranges from 74% to 95% and specificity from 90% to 100% (14). A recent study including 202 patients with suspected CP tried to compare the results of ERCP and pancreatic stimulation by secretin- pancreozymin test (TSP), as it is a more sensitive method to assess pancreatic function. The results showed significant correlation between ERCP and TSP, although in 21% of patients, they were discordant and in 15% the results have been contradictory (normal ERCP and abnormal TSP, or vice- versa) (23).

ERCP has some limitations to diagnose CP. Failure in pancreatic channels opacification occurs in 7.5% of cases, particularly when there is an obstruction in the papillary region caused by a stone (9). Invasive nature of ERCP leads to a risk of acute pancreatitis in 5% to 10% of cases, especially in normal or slightly altered pancreas (24). Pancreatography can be normal in 15% of CP and diagnosis is based on clinical course, pancreatic function tests or other imaging methods (6). This occurs more frequently in non-calcified forms of CP (2, 5, 6, 9).

Recently, Tamura et al. (18) compared ERCP to MRI in patients with CP. The authors studied the results of both methods to measure the diameter and characteristics of MPD. The study showed that ERCP tends to overestimate MPD caliber and that MRI is more accurate to define discrete changes in its caliber (18)

#### **5. Echoendoscopy**

Echoendoscopy can evaluate in detail all the pancreatic parenchyma as well MPD with no fluoroscopy or contrast (25). Moreover, it is a less invasive method and so, unlike ERCP, the patient has no risk of acute pancreatitis. Echoendoscopic criteria for CP are based on parenchyma and canalicular abnormalities (Table 1) (26,27).

Table 1. Echoendoscopic criteria for chronic pancreatitis.

US establishes criteria only in severe CP. There are no criteria for mild and moderate CP (28). Figures 3, 4, 5 and 6 show different echoendoscopic degrees of CP. EE has described new imaging criteria to diagnose CP (29).

ERCP is considered the "gold standard" for diagnosis and treatment of CP (6, 9). Its sensitivity ranges from 74% to 95% and specificity from 90% to 100% (14). A recent study including 202 patients with suspected CP tried to compare the results of ERCP and pancreatic stimulation by secretin- pancreozymin test (TSP), as it is a more sensitive method to assess pancreatic function. The results showed significant correlation between ERCP and TSP, although in 21% of patients, they were discordant and in 15% the results have been

ERCP has some limitations to diagnose CP. Failure in pancreatic channels opacification occurs in 7.5% of cases, particularly when there is an obstruction in the papillary region caused by a stone (9). Invasive nature of ERCP leads to a risk of acute pancreatitis in 5% to 10% of cases, especially in normal or slightly altered pancreas (24). Pancreatography can be normal in 15% of CP and diagnosis is based on clinical course, pancreatic function tests or other imaging methods (6). This occurs more frequently in non-calcified forms of CP (2, 5, 6, 9). Recently, Tamura et al. (18) compared ERCP to MRI in patients with CP. The authors studied the results of both methods to measure the diameter and characteristics of MPD. The study showed that ERCP tends to overestimate MPD caliber and that MRI is more

Echoendoscopy can evaluate in detail all the pancreatic parenchyma as well MPD with no fluoroscopy or contrast (25). Moreover, it is a less invasive method and so, unlike ERCP, the patient has no risk of acute pancreatitis. Echoendoscopic criteria for CP are based on

Type of change Catalano et al. Sahai et al. Parenchyma Parenchyma Ductal Parenchyma Ductal Echotexture Heterogeneous --- --- ---

Streaks (interlobular septa) Hyperechoic --- Hyperechoic --- Lobulation Present --- Present ---

Calcifications and cysts > 5mm --- Present

Diameter --- > 3 mm --- Dilated Appearence --- Tortuous --- Irregular

Hyperechoic wall --- --- --- Present Secondary ducts --- Ectasia --- Visible

US establishes criteria only in severe CP. There are no criteria for mild and moderate CP (28). Figures 3, 4, 5 and 6 show different echoendoscopic degrees of CP. EE has described

contradictory (normal ERCP and abnormal TSP, or vice- versa) (23).

accurate to define discrete changes in its caliber (18)

parenchyma and canalicular abnormalities (Table 1) (26,27).

3 mm)

Focus Echogenic (1 to

Table 1. Echoendoscopic criteria for chronic pancreatitis.

**5. Echoendoscopy**

Pancreatic duct

inside

Hyperechoic focus

new imaging criteria to diagnose CP (29).

(a)

(b)

Fig. 3. Echoendoscopic images in patients with abdominal pain. (a) Hypoechoic areas, intermingled with normal parenchyma and hyperechoic streaks. The aspect remembers a "honeycomb". This aspect suggests early-stage chronic pancreatitis. (b) The same aspect, but more pronounced.

The Role of Endoscopic Ultrasound to Diagnose,

Exclude or Stablish the Parenchimal Changes in Chronic Pancreatitis 55

(a)

(b)

Fig. 5. Echoendoscopic aspect of calcifying CP. Hyperechoic areas with acoustic shadow and

lobular aspect of the gland. (a) dilation of MPD and secondary ducts.

(a)

(b)

Fig. 4. Echoendoscopic image of a lobulated pancreatic gland, with hyperechoic striations interspersed with oval hypoechoic areas, and discrete posterior hyperechoic enhancement. This aspect suggests moderate CP.

(a)

(b)

Fig. 4. Echoendoscopic image of a lobulated pancreatic gland, with hyperechoic striations interspersed with oval hypoechoic areas, and discrete posterior hyperechoic enhancement.

This aspect suggests moderate CP.

(a)

(b)

Fig. 5. Echoendoscopic aspect of calcifying CP. Hyperechoic areas with acoustic shadow and

lobular aspect of the gland. (a) dilation of MPD and secondary ducts.

The Role of Endoscopic Ultrasound to Diagnose,

there is no gold standard (Figure 7).

hypoechogenicity of the entire gland.

histological tests.

Exclude or Stablish the Parenchimal Changes in Chronic Pancreatitis 57

Diagnosis of early stage is a huge challenge. The inability to obtain biopsies makes this presumptive diagnosis difficult. The avaiable diagnostic imaging methods do not offer greater benefits. EE is a promising diagnostic modality and unlike ERCP does not have the same complication rate. Minimal changes in echotexture are difficult to interpret because

**6. Echoendoscopy to diagnose early stage chronic pancreatitis**

Fig. 7. Echoendoscopic image of pancreas showing parenchymal changes such as: longitudinal hyperechoic streaks, hypoechoic areas, secondary duct dilation and

can only be obtained from studies with more rigorous methodology.

There is some evidence in the literature suggesting that these early changes may progress to a more advanced disease (30). It is generally accepted that in the absence of all criteria, CP is unlikely, whereas when there is 5 or more of these criteria CP is likely, even when ERCP and pancreatic function tests are normal. Significance of 1-4 criteria is still uncertain, particularly if other diagnostic methods such as ERCP and functional tests are normal (30). In these cases, there is strong evidence of CP, even if these changes detected by EE could not be confirmed by other diagnostic modalities. A question still remains : How can we improve and understand the abnormalities detected by EE when other tests are normal? The answer

Yusoff & Sahai (31) prospectively studied 1157 patients. The most important predictive factor for parenchymal abnormalities was alcohol intake, male, clinical suspicion of pancreatic disease and smoking. The authors conclude that many variables can change echoendoscopic aspects and severe abnormalities may be found in asymptomatic individuals. The diagnosis of CP by EE needs to be assessed by clinical, functional and

In our department we investigated the meaning of echoendoscopic criteria for MPD and parenchyma abnormalities in patients without suspicion of CP compared to chronic alcoholics (alcohol intake exceeding 80g/ day). Two hundred and twenty-eight patients underwent EE. One hundred and eighty-nine were prospectively studied, using criteria of

(a)

(b)

Fig. 6. Echoendoscopic images showing MPD with a single stone.

(a)

(b)

Fig. 6. Echoendoscopic images showing MPD with a single stone.

### **6. Echoendoscopy to diagnose early stage chronic pancreatitis**

Diagnosis of early stage is a huge challenge. The inability to obtain biopsies makes this presumptive diagnosis difficult. The avaiable diagnostic imaging methods do not offer greater benefits. EE is a promising diagnostic modality and unlike ERCP does not have the same complication rate. Minimal changes in echotexture are difficult to interpret because there is no gold standard (Figure 7).

Fig. 7. Echoendoscopic image of pancreas showing parenchymal changes such as: longitudinal hyperechoic streaks, hypoechoic areas, secondary duct dilation and hypoechogenicity of the entire gland.

There is some evidence in the literature suggesting that these early changes may progress to a more advanced disease (30). It is generally accepted that in the absence of all criteria, CP is unlikely, whereas when there is 5 or more of these criteria CP is likely, even when ERCP and pancreatic function tests are normal. Significance of 1-4 criteria is still uncertain, particularly if other diagnostic methods such as ERCP and functional tests are normal (30). In these cases, there is strong evidence of CP, even if these changes detected by EE could not be confirmed by other diagnostic modalities. A question still remains : How can we improve and understand the abnormalities detected by EE when other tests are normal? The answer can only be obtained from studies with more rigorous methodology.

Yusoff & Sahai (31) prospectively studied 1157 patients. The most important predictive factor for parenchymal abnormalities was alcohol intake, male, clinical suspicion of pancreatic disease and smoking. The authors conclude that many variables can change echoendoscopic aspects and severe abnormalities may be found in asymptomatic individuals. The diagnosis of CP by EE needs to be assessed by clinical, functional and histological tests.

In our department we investigated the meaning of echoendoscopic criteria for MPD and parenchyma abnormalities in patients without suspicion of CP compared to chronic alcoholics (alcohol intake exceeding 80g/ day). Two hundred and twenty-eight patients underwent EE. One hundred and eighty-nine were prospectively studied, using criteria of

The Role of Endoscopic Ultrasound to Diagnose,

and need further confirmation (35).

inflammatory mass and CP (37).

useful to diagnose CP and its complications (38).

Clin North Am 1999;79(4):815-27, x.

Surg Clin North Am 1999;79(4):829-45.

We agree with it.

**9. Conclusion**

**10. References**

90.

497.

2004;93(1):29-37.

Exclude or Stablish the Parenchimal Changes in Chronic Pancreatitis 59

Patients with CP show another diagnostic difficulty : differencial diagnosis between a real pancreatic mass and a pseudotumour. This diagnosis is relatively difficult to make and an accurate diagnosis could avoid unnecessary surgical treatment (35). Several techniques associated with EE have been described for this purpose. The use of Echo-guided Power Doppler for differential diagnosis showed sensitivity and specificity of 93% and 77% respectively (35). The use of contrast agent (Sonovue) seems to increase sensitivity and specificity rates to 91% compared to EE alone versus 73% and 93% vs. 83%, respectively (36). Echo-guided elastography also contributes for diagnosis, but studies are still preliminary

EE-FNA has a sensitivity, specificity, positive and negative predictive value and accuracy of 87.5%, 100%, 100%, 98.1% and 98.3% respectively, for differencial diagnosis between

EE has continuously gained importance and has proven to be of clinical value in patients with CP, as it has a low complication rate when compared to ERCP. Some authors indicate EE for examining CP, as it is the imaging method of choice to asses MPD and parenchyma criteria, but there are some limitations. EE has two main limitations that prevent it being the gold standard: the lack of standard criteria for appropriate education and learning (38).

EE is difficult to learn and therefore, teaching has to be standardized. Moreover, a general platform to compare Cambridge criteria is necessary, in order to be accepted as the gold standard to diagnose CP. Except calcifications, the difficulties in assessing some parenchymal criteria depend on differentiation of the natural aging process, the sequelae of pancreatic fibrosis, acute ingestion of alcohol and the advanced stage of CP. Another important point to mention is that the differentiation of hypoechoic (inflammatory x cancer) and cystic lesions (inflammatory x neoplastic) is difficult. In this area, complementary imaging methods have also low sensitivity. Thus there is no doubt that EE has proven to be

[1] Kataoka K, Kanemitsu D, Sakagami J, Mitsufuji S, Okanoue T. [Clinical symptoms and

[2] Steer ML, Waxman I, Freedman S. Chronic pancreatitis. N Engl J Med 1995;332(22):1482-

[4] Lankisch PG. Progression from acute to chronic pancreatitis: a physician's view. Surg

[5] Clain JE, Pearson RK. Diagnosis of chronic pancreatitis. Is a gold standard necessary?

[6] Dimagno MJ, Dimagno EP. Chronic pancreatitis. Curr Opin Gastroenterol 2006;22(5):487-

[3] Mergener K, Baillie J. Chronic pancreatitis. Lancet 1997;350(9088):1379-85.

diagnostic standards in chronic pancreatitis]. Nippon Naika Gakkai Zasshi

Catalano et al. (26) and Sahai et al. (27). Alcoholic patients (p <0.001) showed more abnormalities than nonalcoholic for both scores (parenchyma and MPD). Comparison of ROC curve between the two groups showed a better specificity and sensitivity when the two scores were combined (29). Our results demonstrate a correlation between these signals and the disease, but it is important to emphasize that, in our opinion, echo-guided fine needle aspiration can aid diagnosis and, beyond a shadow of doubts, will be the gold standard for diagnosis of CP, particularly in early phase.

#### **7. Comparison between EE and ERCP**

Three studies compared EE to ERCP in order to correlate ultrasonographic and wirsungography signs with severity of CP. In the first, 35 patients with CP were analyzed. Sixty per cent of patients had a history of alcohol intake. There was clear correlation between alcohol abuse and CP (p <0.05) as well with abnormalities of MPD by EE with ERCP (p <0.01). These authors conclude that EE should be the first line method for diagnosis of parenchymal and MPD abnormalities (32).

In the second study, the sensitivity and specificity of EE were 85%. CP is likely because it has a positive predictive value of 85% when more than two criteria (for all CP) or more than six criteria (for moderate and severe forms) are present. Moderate or severe CP is unlikely when less than three criteria are present (negative predictive value> 85%). Independent predictive factors of CP were: calcifications (p = 0.000001), history of alcoholism (p = 0.002) and total number of criteria (p = 0.008) (27).

In the third study, EE and ERCP were compared with a functional (secretin test) and showed a sensitivity and specificity of 84% and 98% to diagnose CP. Although correlation between EE and ERCP is excellent in normal pancreas or in moderate or advanced forms of CP, in early forms it is poor (26). Another study reported sensitivity and specificity of 87% and 89% to diagnose CP (33).

Another study compared the agreement among 11 experienced echoendoscopists to diagnose CP. There was agreement for a definitive diagnosis of CP (Kappa index = 0.45). Agreement was higher for criteria such as ductal dilatation (kappa = 0.6) and lobular aspect (kappa = 0.51). All other parameters showed poor agreement (kappa <0.4). The authors conclude that EE is reliable to diagnose CP, with good correlation among experienced observers (34).

#### **8. Echoendoscopy (EE) alone and associated with fine needle aspiration (FNA-EE)**

Hollerbach et al. (8) has recently reported the value of EE with fine needle aspiration puncture (FNAB) to diagnose CP. They concluded that EE is so sensitive and effective as ERCP to detect CP, particularly in early cases. However, echoendoscopic aspects are poor, especially in patients with early disease. EE-FNA is safe and increases negative predictive value of EE. A negative puncture and the absence of echoendoscopic findings of CP could exclude it. It is noteworthy that cytology alone does not increase the specificity of the method, suggesting that tissue collection could impose the use of EE-FNA as a routine to diagnose CP at any stage.

We agree with it.

58 Chronic Pancreatitis