*7.4. Issues and Interpretation of Prompt Emission GRB Correlations*

In the following, attention is given to the above described GRB prompt emission correlations, focusing on possible issues and their physical interpretation. Particular emphasis is given to the functional form of *E*p–*E*iso (or Amati), Ghirlanda and Yonetoku correlations, the most popular and most quoted correlations for prompt emission observables.


# **Quantitative Assessment of Environmental/ Human Health Risks**

Edited by Cheng Yan Printed Edition of the Special Issue Published in *IJERPH*

www.mdpi.com/journal/ijerph

## **Quantitative Assessment of Environmental/Human Health Risks**


*6.2.1. Linear Fresnel reflector*

These collectors can be divided into two types: nonconcentrating (NC) and concentrating

• Heat transfer fluid (HTF) can achieve higher temperatures because the solar beam is fo-

• The thermal efficiency is greater because of the small heat-loss area relative to the receiver area. • With these systems, the HTF reduces exergetic losses through the STT; it means that the interchange between the fluid temperature and environment is less according to the smaller

• For a CC, the cost per unit area of the solar collecting surface is cheaper than of a NC collector.

• In a cloudy day, it may not work; it means, without solar radiation, these systems do not

• Concentrator systems collect little diffuse radiation depending on the concentration ratio. • Some form of tracking system is required to follow the Sun, generating more monetary and

• Solar reflecting surfaces may lose their reflectance with time and require periodic cleaning

Even with these characteristics, not all solar collectors have the same design, depending on different concentrators and receivers; for that reason, they are divided into: linear fresnel reflector (LFR), parabolic trough collector (PTC), parabolic dish reflector (PDR) and central receiver (CR); finally, they are subdivided into solar tower (ST) and solar furnace

The **Figure 6(a)** shows the focus method to achieve the recollected energy of sun and increase the efficiency of collector; the PTC and PD are divided into mobile receivers; it means that the collector and the receiver need a tracking sun technology. On the other hand, LFR, SF, and ST are classified as fixed receivers because the receiver is static. In **Figure 6(b)**, a graph is shown comparing the concentration ratio with the temperature that can be achieved. LFR has a concentration ratio from 10 to 40 units, and a temperature ranges from 60 to 250°C. PTC increases its concentration compared with LFR, between 15 and 45 units; thus, the temperature range can reach from 60 to 300°C. On one hand, ST achieves a concentration ratio of 300–600 and can achieve a temperature above 800°C. On the other hand, PD has a concentration ratio between 100 and 1000 and a temperature range from 100 to 1600°C. Furthermore, SF has the highest concentration range of all STT, reaching 10,000 units; thus, this technology can arrive

(CC). Then, it describes their advantages and disadvantages [48, 49].

Advantages

cused on a point.

54 Sustainable Air Conditioning Systems

area of the receiver.

transform energy.

energetic costs.

and refurbishing.

temperatures above 2000°C [48, 49].

(SF) [48, 49].

Disadvantages

LFR is a type of solar collector that collects sunlight by adapting long, narrow or slightly curved mirrors to reflect the Sun's ray into an absorber tube and concentrate that energy. They usually use water as HTF, which passes through the receiver and change to steam. Considering that the focal line in the LFR can be distorted by astigmatism of the mirror, usually a secondary mirror is placed above the receiver to refocus the Sun´s ray [6]. They have the advantages of easy manufacture and maintenance, and low cost comparing with other solar thermal technologies. However, these systems have the disadvantage of losing some portion of reflector aperture because the arrangement between each other blocks part of the sunlight and affecting their efficiency [50].


**4. Neuroprotection by ASCs — Demyelination, stroke, spinal cord injury**

Early translational studies do suggest that ASCs can be safely administered to nervous tissue injuries and that functional improvement is noted. Transplanted ASCs have been reported to improve functional deficits following middle cerebral occlusion or ischemic stroke [152]-[154], spinal cord contusion injury [155] and peripheral nerve gaps [156], [157]. Histologic analysis of these injury sites has suggested that ASC differentiation into neurons and/or glial cells may play a role in the functional recovery, with transplanted cells staining positively for MAP2 [153], GFAP, Tuj-1 and an oligodendrocyte marker [155]. However, this functional improve‐ ment may be due to paracrine actions on the host more than ASC differentiation, as less then 1% of transplanted ASCs can be found within a spinal contusive injury model, with those remaining appearing to be oligodendrocytes [158]. In addition, extremely low levels ASC differentiation into mature neurons is noted in a model of cerebral cortex injury [159]. However, both of these studies note significant changes in the host tissue with Nakada et al. observing improvements in microvasculature and Zhang et al. measuring increases in host oligodendrocyte formation. Therefore, like wound healing and IR models, ASCs are likely to exert paracrine actions within nervous tissue.

In 2002, Zhao et al. suggested that functional recovery in ischemic brain injury was not due to MSC differentiation but to secreted paracrine factors that act on the host [160]. A similar hypothesis has been put forth by bone marrow MSC groups who have noted increased survival and differentiation of Tuj1+ve neurons and neuroblastoma cells in co-cultures [161] and increased neuronal viability and glial cell differentiation using MSC conditioned media [162]. Consistent with this, ASC/Matrigel constructs implanted into models of mice limb re-inner‐ vation stimulate the regeneration of nerves and induce axon growth, likely through the expression numerous neurotrophins [163]. Moreover, enhanced nerve fiber growth is observed if the ASCs are pre-induced toward the neural lineage thus enhancing their production of brain-derived neurotrophic factor (BNDF). BDNF secretion (together with nerve growth factor/NGF and glial cell-derived neurotrophic factor/GDNF) by ASCs pre-differentiated toward a Schwann Cell (SC) phenotype is thought to be the basis for axonal regeneration in sciatic nerve gap models - although these authors speculate that this regeneration is likely due to the neuroprotective function of these three neurotrophins [164]. In support of this, studies using ASC-conditioned media appeared to further strengthen this theory. Protection against cortical and hippocampal volume loss in rats can be achieved through the infusion of ASCconditioned medium [165]. ASC-conditioned medium containing VEGF, BDNF and NGF is shown to have a protective effect against glutamate excitotoxicity on PC12 cells (a key factor implicated in stroke and neurodegenerative diseases) and increase PC12 viability 94. Condi‐ tioned media from pre-differentiated ASCs infused over one week into a rat model of ischemic stroke 8 days after stroke induction increases the number of CD31+ve cells [166]. Finally, functional deficits in a model of middle cerebral artery occlusion can be dramatically improved using ASC transduced to overexpress BDNF [153].

While these neurotrophic factors may act to protect neurons, ASCs may also play roles in decreasing inflammation and gliosis (i.e. glial cell-mediated scar formation) – two critical events that specifically affect healing in the both the central and peripheral nervous system. Systemic transplantation of human ASCs can attenuate cerebral degeneration in rats, reducing both brain atrophy and glial proliferation [79]. Rats implanted with ASC-derived SCs show significant locomotor function recovery compared with untreated ASCs and also reduction in gliosis [152]. Pre-differentiated canine ASCs in Matrigel scaffolds show better functional recovery and reduced fibrosis and inflammation when implanted into spinal cord injuries [167]. Decreased gliosis is also noted upon intrathecal administration of ASCs in a model of IR neuronal damage in rabbits – an event accompanied by increased expression of BDNF within the first 72 hours following ASCs administration [168]. Finally, a possible anti-inflammatory role for ASCs in sciatic nerve repair might be seen in a recent model describing possible immunosuppression of xenogeneic acellular nerve matrices combined with autologous ASCs [169]. Implantation of this construct does not result in host rejection, making it possible that peripheral nerves repair can be accomplished using commercial nerve matrices combined with the patient's own ASCs.

#### **4.1. Controlled release from ASCs — ASCs as a cellular biopump**

**4. Neuroprotection by ASCs — Demyelination, stroke, spinal cord injury**

Early translational studies do suggest that ASCs can be safely administered to nervous tissue injuries and that functional improvement is noted. Transplanted ASCs have been reported to improve functional deficits following middle cerebral occlusion or ischemic stroke [152]-[154], spinal cord contusion injury [155] and peripheral nerve gaps [156], [157]. Histologic analysis of these injury sites has suggested that ASC differentiation into neurons and/or glial cells may play a role in the functional recovery, with transplanted cells staining positively for MAP2 [153], GFAP, Tuj-1 and an oligodendrocyte marker [155]. However, this functional improve‐ ment may be due to paracrine actions on the host more than ASC differentiation, as less then 1% of transplanted ASCs can be found within a spinal contusive injury model, with those remaining appearing to be oligodendrocytes [158]. In addition, extremely low levels ASC differentiation into mature neurons is noted in a model of cerebral cortex injury [159]. However, both of these studies note significant changes in the host tissue with Nakada et al. observing improvements in microvasculature and Zhang et al. measuring increases in host oligodendrocyte formation. Therefore, like wound healing and IR models, ASCs are likely to

In 2002, Zhao et al. suggested that functional recovery in ischemic brain injury was not due to MSC differentiation but to secreted paracrine factors that act on the host [160]. A similar hypothesis has been put forth by bone marrow MSC groups who have noted increased survival and differentiation of Tuj1+ve neurons and neuroblastoma cells in co-cultures [161] and increased neuronal viability and glial cell differentiation using MSC conditioned media [162]. Consistent with this, ASC/Matrigel constructs implanted into models of mice limb re-inner‐ vation stimulate the regeneration of nerves and induce axon growth, likely through the expression numerous neurotrophins [163]. Moreover, enhanced nerve fiber growth is observed if the ASCs are pre-induced toward the neural lineage thus enhancing their production of brain-derived neurotrophic factor (BNDF). BDNF secretion (together with nerve growth factor/NGF and glial cell-derived neurotrophic factor/GDNF) by ASCs pre-differentiated toward a Schwann Cell (SC) phenotype is thought to be the basis for axonal regeneration in sciatic nerve gap models - although these authors speculate that this regeneration is likely due to the neuroprotective function of these three neurotrophins [164]. In support of this, studies using ASC-conditioned media appeared to further strengthen this theory. Protection against cortical and hippocampal volume loss in rats can be achieved through the infusion of ASCconditioned medium [165]. ASC-conditioned medium containing VEGF, BDNF and NGF is shown to have a protective effect against glutamate excitotoxicity on PC12 cells (a key factor implicated in stroke and neurodegenerative diseases) and increase PC12 viability 94. Condi‐ tioned media from pre-differentiated ASCs infused over one week into a rat model of ischemic stroke 8 days after stroke induction increases the number of CD31+ve cells [166]. Finally, functional deficits in a model of middle cerebral artery occlusion can be dramatically improved

While these neurotrophic factors may act to protect neurons, ASCs may also play roles in decreasing inflammation and gliosis (i.e. glial cell-mediated scar formation) – two critical

exert paracrine actions within nervous tissue.

60 Regenerative Medicine and Tissue Engineering

using ASC transduced to overexpress BDNF [153].

It is possible that the paracrine action of ASCs may be "fine-tuned" so that the ASC secretes a desired factor, hence turning the ASC into a "cellular biopump". This is not a recent concept as the engineering of numerous cell types to secrete a variety of factors has been reported in the literature for over a decade. In the field of stem cell research, bone marrow MSCs have been modified to secrete various factors, including BMP2 [170], [171], bFGF [172], IFN-β [173] and IL12 [174]. Similar to these studies, ASCs have been engineered for the delivery of BMP4 [175], BMP2 [176], [177], and BMP6 [178] in several bone regeneration models. Delivery of TGFβ2 by ASCs for the induction of chondrogenesis has been reported [179]. Adenovirallymediated VEGF secretion by ASCs has been used to induce vascular growth in a bone defect model [180] and adipose tissue grafts [181]. Finally, as described above, BDNF delivery by transduced ASCs into a model of middle cerebral artery occlusion improves functional deficits when compared to control ASCs [153].

However, a more exciting idea might be in the engineering of ASCs in the treatment of disease. In 2007, ASCs engineered to express cytosine deaminase were found to decrease the growth of colon carcinoma cells [182]. ASCs have recently been described in the delivery of an oncolytic myxoma virus that will specifically target gliomas [183]. ASC viability is not impacted with transduction and successful cross-infection of gliomablastoma cells is observed upon 3D coculture with glioblastoma cells, leading to their cell death. More importantly, rat survival is increased with this myxoma virus delivery, with the size of the gliomas significantly decreas‐ ing upon injection of transduced ASCs in comparison to non-transduced ASCs controls. Localization of ASCs and increased apoptosis within tumors has also been reported following intravenous or subcutaneous injection of ASCs engineered to express TRAIL, having no effect on the surrounding healthy tissue [184]. Finally, this approach may have far-reaching effects on autoimmune diseases through the delivery of interleukins and interferons. ASCs engi‐ neered to overexpress IL4 and administered at the time of T cell priming attenuate autoimmune encephalomyelitis and reduce peripheral T cell responses shifting the host pro-inflammatory response to an anti-inflammatory one [71]. With the development of inducible viral systems, there is the possibility that the ASC cellular biopump could be controlled not only at the dose level through the number of cells delivered but at the temporal level, giving clinicians more precise control over their therapeutic regimen.

#### **4.2. ASC uses in the clinic**

In light of their differentiative capacity and paracrine actions, there is great interest in the use of ASCs within the clinic. As source of regenerative stem cells, the ASC may have no equal. Bone marrow aspirates yield on average 6x106 nucleated cells per ml, of which, only 0.001 to 0.01% are thought to be stem cells [185], [186]. In comparison, approximately three-fold more cells can be obtained per gram adipose tissue [187] [188] with 10% of these cells thought to be stem cells [188], [189]. The abundance of ASCs within adipose tissue, combined with the relative ease of its harvest and isolation also makes the ASC a good choice for clinical work. Patient's could conceivably have their adipose tissue harvested relatively painlessly a few weeks prior to their procedure in a simple outpatient procedure, the ASCs isolated and expanded under good manufacturing protocols and then used for regenerative purposes. With the confirmed absence of HLA/MHC class II proteins and continuing xenogeneic animal models, the patient may not even need to use their own stem cells. Donated allogeneic ASC lines could be used in lieu of autologous cells without the fear of immunorejection or inflam‐ matory complications. Such a situation might be perfect in the case of myocardial infarct treatment where a delay in treatment could have serious consequences.

The first published article using ASCs in a clinical setting was in 2004, in which freshly harvested SVF cells were combined with fibrin glue and used in the repair of a traumatic calvarial injury [190]. Three months after reconstruction, CT scans showed new bone formation within the injury. However, it is important to point out that the cells used in this study were not ASCs, purified through plastic adherence and culture time, but the SVF - a heterogenous mixture of ASCs, endothelial cells, pre-adipocytes, pericytes, fibroblasts and red blood cells. Therefore, it is difficult to attribute the observed healing to the action of the ASC itself. Since that time, other clinical studies using the SVF have been attempted [191] and a review by Casteilla et al. does an excellent job of summarizing these works [192]. It is worth noting that with the exception of some cysts and microcalcifications being observed upon breast recon‐ struction [193], the use of SVFs clinically has not resulted in any serious complications.

Because of its heterogeneity, clinical studies using purified ASCs have also been performed for the treatment of such disorders as critical limb ischemia and radiation therapy ([194], [195] – for a more comprehensive review, see [192]). Bone regeneration using ASCs has recently been reported in 2009 with the reconstruction of the maxilla being induced using ASC in combina‐ tion withBMP2 [196].BonyhealingusingBMPshas beendocumentedinnumerous translation‐ al animal models [197]-[201], making this clinical study an exciting addition to the ways bone regenerationandhealingcanbebroughtaboutinthe clinic.However,manyofthese translation‐ al models fail to report the appropriate control – the amount of bone being formed just by the BMPitself.Thefirsttranslational studytocombineASCsandaBMP(i.e.BMP2)failedtomeasure any significant improvement in bone formation when BMP2 and ASC+BMP2 groups were compared [197]. Since this study, others have appeared to suggest that BMP2 may not pro‐ mote the in vivo osteogneic capacity of the ASC [202] but may, in fact, may have a deleterious effect on bone regeneration [203]. Since it is not possible to perform similarly controlled studies clinically, it remains unknown if the addition of ASCs to BMP-treated scaffolds provides any more advantage. However, It is worth noting that, as with the use of SVFs, administration of ASCs into human patients has not been associated with any adverse effects [204].

response to an anti-inflammatory one [71]. With the development of inducible viral systems, there is the possibility that the ASC cellular biopump could be controlled not only at the dose level through the number of cells delivered but at the temporal level, giving clinicians more

In light of their differentiative capacity and paracrine actions, there is great interest in the use of ASCs within the clinic. As source of regenerative stem cells, the ASC may have no equal. Bone marrow aspirates yield on average 6x106 nucleated cells per ml, of which, only 0.001 to 0.01% are thought to be stem cells [185], [186]. In comparison, approximately three-fold more cells can be obtained per gram adipose tissue [187] [188] with 10% of these cells thought to be stem cells [188], [189]. The abundance of ASCs within adipose tissue, combined with the relative ease of its harvest and isolation also makes the ASC a good choice for clinical work. Patient's could conceivably have their adipose tissue harvested relatively painlessly a few weeks prior to their procedure in a simple outpatient procedure, the ASCs isolated and expanded under good manufacturing protocols and then used for regenerative purposes. With the confirmed absence of HLA/MHC class II proteins and continuing xenogeneic animal models, the patient may not even need to use their own stem cells. Donated allogeneic ASC lines could be used in lieu of autologous cells without the fear of immunorejection or inflam‐ matory complications. Such a situation might be perfect in the case of myocardial infarct

The first published article using ASCs in a clinical setting was in 2004, in which freshly harvested SVF cells were combined with fibrin glue and used in the repair of a traumatic calvarial injury [190]. Three months after reconstruction, CT scans showed new bone formation within the injury. However, it is important to point out that the cells used in this study were not ASCs, purified through plastic adherence and culture time, but the SVF - a heterogenous mixture of ASCs, endothelial cells, pre-adipocytes, pericytes, fibroblasts and red blood cells. Therefore, it is difficult to attribute the observed healing to the action of the ASC itself. Since that time, other clinical studies using the SVF have been attempted [191] and a review by Casteilla et al. does an excellent job of summarizing these works [192]. It is worth noting that with the exception of some cysts and microcalcifications being observed upon breast recon‐ struction [193], the use of SVFs clinically has not resulted in any serious complications.

Because of its heterogeneity, clinical studies using purified ASCs have also been performed for the treatment of such disorders as critical limb ischemia and radiation therapy ([194], [195] – for a more comprehensive review, see [192]). Bone regeneration using ASCs has recently been reported in 2009 with the reconstruction of the maxilla being induced using ASC in combina‐ tion withBMP2 [196].BonyhealingusingBMPshas beendocumentedinnumerous translation‐ al animal models [197]-[201], making this clinical study an exciting addition to the ways bone regenerationandhealingcanbebroughtaboutinthe clinic.However,manyofthese translation‐ al models fail to report the appropriate control – the amount of bone being formed just by the BMPitself.Thefirsttranslational studytocombineASCsandaBMP(i.e.BMP2)failedtomeasure any significant improvement in bone formation when BMP2 and ASC+BMP2 groups were

treatment where a delay in treatment could have serious consequences.

precise control over their therapeutic regimen.

**4.2. ASC uses in the clinic**

62 Regenerative Medicine and Tissue Engineering

The first phase I clinical trials using ASCs were not conducted on bone formation or even fat grafting but in the healing of chronic fistulae in Crohn's disease [205]-[210]. In 2005, nine rectovaginal fistulae in four patients were treated with ASCs, purified and cultured for up to one month. Of the eight fistulae examined, six showed complete healing in 8 weeks [206]. These fistulae had previously failed to heal using conventional surgical treatments, thus justifying progression to more comprehensive phase II trials. In 2009, a larger phase II trial using patients with and without Crohn's fistulae were treated with ASCs [211]. As seen with their earlier clinical trial, the majority of Crohn's and non-Crohn's fistulae were healed completely using ASCs in comparison to controls. Currently, there are three phase II clinical trials recruiting for the use of ASCs in Crohn's disease fistulae (Clinicaltrials identifiers: NCT01011244, NCT01157650, NCT00999115, http://clinicaltrials.gov/ct2/results?term=adipose+derived +cells), in addition to one phase III trial (NCT00475410) recently completed [212].

One of the reasons ASCs are considered in the treatment of Crohn's disease is their ability to suppress inflammation. This review includes numerous examples of how the ASC may be capable of suppressing the immune system and recent clinical trials have attempted to take direct advantage of this quality. The treatment of multiple sclerosis (MS) with SVFs, containing ASCs, has been described by Riordan and colleagues in 2009, with the 3 enrolled patients showing improvement in numerous functional categories including balance and coordination [213]. The use of culture expanded ASCs in autoimmune diseases like hearing loss, MS and rheumatoid arthritis was recently discussed in 2011 [214]. Prior to this, ASCs have been proposed as a viable therapy for suppression of graft vs. host disease (GVHD) [215]-[218]. Each of these studies report favorable functional outcomes and propose ASCs, or their SVF coun‐ terpart, for the treatment of immune system disorders.

The most obvious application of the ASC clinically should be in breast reconstruction. In the lab, the combination of ASC-containing SVFs with fat grafts through a protocol called cellassisted lipotransfer has enjoyed success [151]. Clinically, treatment of facial lipoatrophy has been reported [219] and two recent trials overseas has suggested that the ASCs within the SVF are capable of increasing breast volume and improving contour 6 months post-surgery [193], [220]. However, the use of ASCs in breast reconstruction is being pursued carefully in light of recent findings that link stem cells to cancer. Bone marrow MSCs have been found to increase proliferation of breast cancer cell lines [221] and subcutaneous injection of MSCs with tumor cells can favor their growth [222]. Similar to this, ASCs can increase tumorigenesis of estab‐ lished breast cancer lines [223]. In this study, ASCs not only promote the growth of metastatic pleural effusion cells both in vitro and in vivo but the ASC also secretes adipsin and leptin – both of which are known to promote breast cancer growth [224]. Additional work in MSCs has documented their ability to secrete large amounts of IL-6 and the corresponding increase in the growth of estrogen receptor alpha-positive cell lines [225]. Increased expression of IL4 and IL10 have also been reported by ASCs isolated from breast cancer tissue [226], leading many to speculate that the ASC may be capable of altering the immune environment within the breast, resulting in the "protection" of the cancerous cells. Such a possibility could have farreaching effects in the development of breast cancer and in its possible reoccurrence if ASCs are used in reconstruction. However, it is encouraging to find that cultured ASCs are resistant to the chemotherapies cisplatin, vincristine or comptothecin and that they still retain their stem cell characteristics [227]. Such findings could make it possible for a more natural reconstruction of the breast if ASCs are found not to contribute to the cancer itself.

#### **4.3. "Paracrines gone wild" — ASCs and adipose disorders**

With the proposed paracrine function of ASCs now well accepted, a re-examination of certain disorders and how the ASC might play a role might now be in order. The most obvious of these disorders would be obesity. However, studying the ASC might allow more information into lesser known dysfunctions such as lipedema and rare adipose disorders (RADs) like Dercum's and Madelung's disease. Normal fat has been described as having an anti-inflam‐ matory milieu with adipocytes storing lipid, regulating energy metabolism, and, together with resident macrophages, secreting anti-inflammatory mediators such as IL-10 and adiponectin to protect against the possible development of inflammation-driven obesity [228]-[230]. However, with chronic nutrient overload, existing adipocytes increase their fat storage to become hypertrophic and resident pre-adipocytes (or ASCs) are thought to undergo increased differentiation to increase adipocyte number (i.e. hyperplasia). The hypertrophic adipocytes increase their secretion of "adipokines" - soluble factors known to affect angiogenesis and inflammation [231], [232]. Specifically, these adipocytes shift their adipokine production from anti-inflammatory to inflammatory, producing a series of feedback cascades that ultimately manifests in obesity [232].

Obesity has been recognized since the 1950s as a chronic state of low-level inflammation associated with excess accumulation of adipose tissue [233]. This inflammation is now thought to be a complex response to cellular events, such as hypoxia and oxidative stresses within the adipocyte. Figure 1 outlines the possible interacting events underlying obesity starting with the creation of hypertrophic adipocytes. These adipocytes become too large to be adequately supplied by the existing vasculature in the adipose depot, resulting in localized areas of hypoxia. This hypoxic state induces the production of numerous pro-inflammatory adipokines (e.g. IL1Rα, IL6, IL8, TNFα, MCP-1, leptin) and decreases the secretion of several key antiinflammatory factors (e.g. IL10, adiponectin). Excellent reviews on these adipokines in obesity can be found in Fain et al. 2010 and Balistreri 2010. In these hypertrophic adipocytes, hypoxia is thought to induce oxidative stress [234], [235]. Oxidative stress is defined as an imbalance in the levels of reaction oxygen species (ROS) relative to the tissue's antioxidant capacity, resulting in the accumulation of oxidative products such as superoxide and hydroxyl radicals, reactive nitrogen species (RNS) and hydrogen peroxide [236]. Excess nutrients and hypertro‐ phic adipocytes can produce ROS through: the nicotinamide dinucleotide phosphate oxidase (NOX) system [237], incomplete mitochondrial respiration due to excess free fatty acids [238] and endoplasmic reticulum (ER) stress due to excess lipid storage [239]. Both mitochondrial and ER dysfunction have been demonstrated to increase the secretion of inflammatory adipokines [239], [240] and numerous studies in obesity models and obese subjects now exist linking hypoxia, oxidative stress and inflammation (reviewed in [236]). Concomitant with the development of hypertrophic adipocytes, there is a shift within the adipose tissue from M2 macrophages, found in normal adipose tissue, to a more pro-inflammatory M1 macrophage subset [241]-[243]. This shift is likely, in part, a consequence of the production of pro-inflam‐ matory adipokines by adipocytes – such as MCP-1, but this infiltration is also likely to be due to the death of these adipocytes [244]. Consistent with this, "crown-like" structures of macro‐ phages are known to be associated with necrotic adipocytes in obese murine adipose tissue [242]. These macrophages may directly contribute to the production of inflammatory agents within obese adipose tissue [245]. However, they may also augment adipokine production by the adipocyte through possible cross-talk mechanisms. While these mechanisms are unclear at this point, there are many who postulate that adipocyte-macrophage interaction is the key factor in inflammation and resulting obesity [230], [246], [247].

**Table 3.** Secretion of Pro-inflammatory Cytokines by ASCs

the growth of estrogen receptor alpha-positive cell lines [225]. Increased expression of IL4 and IL10 have also been reported by ASCs isolated from breast cancer tissue [226], leading many to speculate that the ASC may be capable of altering the immune environment within the breast, resulting in the "protection" of the cancerous cells. Such a possibility could have farreaching effects in the development of breast cancer and in its possible reoccurrence if ASCs are used in reconstruction. However, it is encouraging to find that cultured ASCs are resistant to the chemotherapies cisplatin, vincristine or comptothecin and that they still retain their stem cell characteristics [227]. Such findings could make it possible for a more natural reconstruction

With the proposed paracrine function of ASCs now well accepted, a re-examination of certain disorders and how the ASC might play a role might now be in order. The most obvious of these disorders would be obesity. However, studying the ASC might allow more information into lesser known dysfunctions such as lipedema and rare adipose disorders (RADs) like Dercum's and Madelung's disease. Normal fat has been described as having an anti-inflam‐ matory milieu with adipocytes storing lipid, regulating energy metabolism, and, together with resident macrophages, secreting anti-inflammatory mediators such as IL-10 and adiponectin to protect against the possible development of inflammation-driven obesity [228]-[230]. However, with chronic nutrient overload, existing adipocytes increase their fat storage to become hypertrophic and resident pre-adipocytes (or ASCs) are thought to undergo increased differentiation to increase adipocyte number (i.e. hyperplasia). The hypertrophic adipocytes increase their secretion of "adipokines" - soluble factors known to affect angiogenesis and inflammation [231], [232]. Specifically, these adipocytes shift their adipokine production from anti-inflammatory to inflammatory, producing a series of feedback cascades that ultimately

Obesity has been recognized since the 1950s as a chronic state of low-level inflammation associated with excess accumulation of adipose tissue [233]. This inflammation is now thought to be a complex response to cellular events, such as hypoxia and oxidative stresses within the adipocyte. Figure 1 outlines the possible interacting events underlying obesity starting with the creation of hypertrophic adipocytes. These adipocytes become too large to be adequately supplied by the existing vasculature in the adipose depot, resulting in localized areas of hypoxia. This hypoxic state induces the production of numerous pro-inflammatory adipokines (e.g. IL1Rα, IL6, IL8, TNFα, MCP-1, leptin) and decreases the secretion of several key antiinflammatory factors (e.g. IL10, adiponectin). Excellent reviews on these adipokines in obesity can be found in Fain et al. 2010 and Balistreri 2010. In these hypertrophic adipocytes, hypoxia is thought to induce oxidative stress [234], [235]. Oxidative stress is defined as an imbalance in the levels of reaction oxygen species (ROS) relative to the tissue's antioxidant capacity, resulting in the accumulation of oxidative products such as superoxide and hydroxyl radicals, reactive nitrogen species (RNS) and hydrogen peroxide [236]. Excess nutrients and hypertro‐ phic adipocytes can produce ROS through: the nicotinamide dinucleotide phosphate oxidase (NOX) system [237], incomplete mitochondrial respiration due to excess free fatty acids [238]

of the breast if ASCs are found not to contribute to the cancer itself.

**4.3. "Paracrines gone wild" — ASCs and adipose disorders**

manifests in obesity [232].

64 Regenerative Medicine and Tissue Engineering

So obesity results from a complex series of cellular events that ultimately increases the production of inflammatory adipokines within the tissue. These adipokines are known to further increase adipocyte hypertrophy producing a positive feedback system. This feedback system could be augmented further by the secretory activity of non-fat cells – i.e. the preadipocyte and even the ASC. Pre-adipocytes and adipocytes secrete many of the same proinflammatory factors listed above - with the exception of leptin and adiponectin, factors secreted by the adipocyte (reviewed in [235]). Furthermore, a review of the current literature

**Figure 1.** Possible interactions in obesity. Excess energy leads to development of hypertophic adipocytes. Hypertro‐ phic adipocytes lead to the development of cellular stresses and hypoxia, via HIFI1α signaling, which can induce the adipocyte to release numerous pro-inflammatory cytokines. Hypoxia can also result in the death of adipocytes, induc‐ ing infiltration by pro-inflamatory/M1 macrophages into the adipose tissue. Paracrine activity by macrophages could affect the release of inflammatory cytokines from the adipocytes. In addition the macrophage may also release these cytokines directly. The resulting inflammation is likely to set up a feedback loop to enhance hypertrophic adipocyte development. The role of the ASC remains unknown in obesity but possible points of interaction could be the differ‐ entiation of ASCs, leading to adipocyte hyperplasia and the release of similar pro-inflammatory cytokines. Paracrine activity is shown as solid arrows.

turns up many studies that document the secretion of similar pro-inflammatory factors by ASCs (Table 3). It is possible that the secretion of inflammatory factors, like IL6 or TNFα, by ASCs may play a crucial role in inflammation and the development of obesity. Alternatively, it is possible that inflammation and obesity may result from "defective" ASCs that fail to secrete key anti-inflammatory factors such as IL-10 or have lost their ability to ameliorate oxidative stresses, thus allowing inflammation to go on unchecked. Unfortunately, the effect of inflam‐ mation and the ASC is under-represented in today's literature. Those studies that do exist document the inhibition of ASC adipogenesis under inflammatory conditions [248]. This is an interesting finding, as the ASC might be thought of as the logical source for adipocyte hyperplasia observed in obesity. However, if it is the paracrine activity of the ASC that plays a crucial role in the development or maintenance of obesity, then ASC differentiation capacity might be sacrificed in the name of maintaining this function. In light of what we know about adipocytes and pre-adipocytes in obesity, more in-depth studies on the ASC are certainly warranted.

A similar argument for ASCs could be made for other adipose disorders such lipedema and rare adipose disorders (RADs) such as Dercum's (aka Adiposa Dolorosa) [249] and Madelung's disease or Multiple Symmetric Lipomatosis (MSL) [250]. Lipedema (LD), or edema of the fat, is defined as the symmetrical accumulation of adipose tissue in the lower extremities [251]. Because the fat may also be painful as the disorder progresses, LD is often described in the same spectrum as Dercum's [252]. While lipedema and obesity share many similarities – leading to the misdiagnosis of lipedema in up to 15% of the population as obesity, there are some significant differences between LD and obesity. Specifically, excess fat accumulates almost exclusively in the lower limbs in LD and this adipose tissue is stubbornly resistant to loss through dieting [253]. LD is almost exclusively seen in women in their 30s or older, suggesting a hormonal component [251]. Despite these differences, the etiology of obesity and LD may share some commonalities, in that LD is thought to be mediated, in part, through hypoxia and the production of inflammatory cytokines (Figure 2). Like obesity, LD is initially characterized by adipocyte hypertrophy and hyperplasia [254], although the reason for this hypertrophy cannot be attributed to nutrient overload and currently remains unknown. This hypertrophy results in hypoxia, which is thought to result in inflammatory adipokine secretion and a putative positive feedback cascade as seen in obesity. Like obesity, LD fat is characterized by macrophage "crowns" in close association with hypertrophic and/or necrotic adipocytes [132]. These macrophages will almost certainly contribute to the inflammatory reactions occurring in LD fat. Furthermore, when examining adipose tissues isolated from Dercum's, similar immune infiltrations in association with perivascular cells and hypertrophic adipocytes are also seen, again, suggesting that LD and Dercum's may be points along the same spectrum [252]. In light of these commonalities with obesity, it would be logical to assume that the ASC would also play some critical role in mediating inflammation in LD or RADs through its production of paracrine factors. Unfortunately, these studies do not exist at this point.

Despite sharing many of the same characteristics, there are some important distinctions between obesity and LD that may also be at work. These distinctions are also likely to be found in RADs like Dercum's and Madelung's disease. Specifically, LD (and possibly Dercum's and Madelung's) is associated with defects in the microvasculature, together with lymphatic dysfunction [252]. Current theories propose that adipocyte hypertrophy leads to hypoxia, which results in increased angiogenesis. However, this angiogenesis is pathologic and the resulting capillaries are said to be "fragile" or "leaky" [255]. In support of this, perivascular cells, indicative of vascular damage, can be found in LD adipose tissue [254] and pathologic angiogenesis producing fragile capillaries have been found in many eye diseases [256], [257]. What produces this pathology is unknown but studies have shown that leptin can increase the number of fenestrations in capillaries [258] and increased plasma VEGF levels can be found in LD patients [259]. Increased plasma VEGF levels can also be found in LD patients [259], so it is possible that paracrine secretion from hypertrophic and hypoxic adipocytes could disrupt angiogenesis within LD adipose tissue. With studies showing ASCs capable of secreting

turns up many studies that document the secretion of similar pro-inflammatory factors by ASCs (Table 3). It is possible that the secretion of inflammatory factors, like IL6 or TNFα, by ASCs may play a crucial role in inflammation and the development of obesity. Alternatively, it is possible that inflammation and obesity may result from "defective" ASCs that fail to secrete key anti-inflammatory factors such as IL-10 or have lost their ability to ameliorate oxidative stresses, thus allowing inflammation to go on unchecked. Unfortunately, the effect of inflam‐ mation and the ASC is under-represented in today's literature. Those studies that do exist document the inhibition of ASC adipogenesis under inflammatory conditions [248]. This is an interesting finding, as the ASC might be thought of as the logical source for adipocyte hyperplasia observed in obesity. However, if it is the paracrine activity of the ASC that plays a crucial role in the development or maintenance of obesity, then ASC differentiation capacity

activity is shown as solid arrows.

66 Regenerative Medicine and Tissue Engineering

**Figure 1.** Possible interactions in obesity. Excess energy leads to development of hypertophic adipocytes. Hypertro‐ phic adipocytes lead to the development of cellular stresses and hypoxia, via HIFI1α signaling, which can induce the adipocyte to release numerous pro-inflammatory cytokines. Hypoxia can also result in the death of adipocytes, induc‐ ing infiltration by pro-inflamatory/M1 macrophages into the adipose tissue. Paracrine activity by macrophages could affect the release of inflammatory cytokines from the adipocytes. In addition the macrophage may also release these cytokines directly. The resulting inflammation is likely to set up a feedback loop to enhance hypertrophic adipocyte development. The role of the ASC remains unknown in obesity but possible points of interaction could be the differ‐ entiation of ASCs, leading to adipocyte hyperplasia and the release of similar pro-inflammatory cytokines. Paracrine

**Figure 2.** Lipedema. Development of lipedema may have numerous commonalities with obesity starting with the de‐ velopment of hypertrophic adipocytes. Howerver, causation for this is unknown at this time may involve the ASC. As with obesity, adipocyte hypertrophy can lead to the development of hypoxia and the release of inflammatory cyto‐ kines from the adipocyte. Possible release of these factors from the ASC due to hypoxia is also shown. In addition, adipocyte hypertrophy is also accompanied by the development of "leaky" capillaries and lymphatics. While the cause of pathologic angiogenesis remains unclear, a role for the gene Prox-1 is though to be involved in lymphatic patholo‐ gy. Increased filtration from capillaries, combined with poor lypmphatic drainage (due to hypertrophic adipocytes and the the leaking of lymph back from the lymphatic vessel) leads to an accumulation of protein rich fluids within the tissue. Fluid accumulation and hypoxia may induce pro-inflammatory cytokine release. Other mechanisms of obesity (e.g. macrophage infiltration) are also likely to be involved. Paracrine activity is shown as solid arrows.

numerous paracrine factors, including VEGF, and inducing endothelial differentiation and vessel formation, the question of whether the ASC plays a role in this vascular pathology should be asked. The fragile capillaries allow the filtration of protein-rich plasma into the interstitial space, driving the formation of edema [255]. In the early stages of LD, lymphatic drainage can keep up [260]. However with progression of the disorder, lymphatic drainage does decrease as the patient ages [253]. Added to this, the hypertrophic adipocytes are thought to physically restrict fluid drainage and the smaller lymphatic vessels themselves are thought to become "leaky", possibly through the appearance of microaneurysms in these vessels [253]. All of this results in the accumulation of lymph within the adipose tissue. Recent studies now suggest that "lymph can make you fat" [261]. In support of this, adipogenesis in vitro increases when cells are cultured in the presence of lymph [262], [263].  More recently, mice heterozygous for a mutation in the Prox1 gene not only exhibit leaky lymphatics, but develop obesity as they age [264]-[266]. What it is in the lymph that enhances adipogenesis is unclear. It simply could be the result of edema causing hypoxia, inflammation and adipocyte hypertrophy – not unlike obesity. Alternatively, factors in the lymph could directly induce the ASC to differentiate or the mature adipocyte to store more fat. Since lymph is interstitial fluid combined with emulsified fats, non-reabsorbed proteins and immunocompetent leukocytes, any of these factors could conceivably alter the behavior of the ASC. As it stands, more studies investigating the exact consequences of lymph accumulation on ASC and adipocyte behavior are needed.

So while the mechanisms may differ at points, at the basis of obesity, LD and RADs is inflammation. How the ASC participates in this inflammation remains to be seen, but the ASC could be used in the treatment of these disorders. If inflammation results in adipocyte hypertrophy, then ameliorating this process could decrease the size and number of these cells. In this regard, the anti-inflammatory, anti-oxidant properties of ASCs could be taken advant‐ age of and enhanced in the hopes of mitigating the damaging effects of inflammation in these adipose disorders. However, before this could be attempted, more information is definitely required on the exact roles the ASC plays in adipose tissue formation and how these roles can go wrong when adipose disorders develop.


*Case Report* **Myoepithelial Carcinoma Arising in a Salivary Duct Cyst of the Parotid Gland: Case Presentation**

**Michał Gontarz 1,\*, Jolanta Orłowska-Heitzman 2, Krzysztof G ˛asiorowski 1, Jakub Bargiel 1, Tomasz Marecik 1, Paweł Szczurowski 1, Jan Zapała 1 and Grazyna Wyszy ´ ˙ nska-Pawelec 1**

**Abstract:** Cystic lesions observed in parotid glands are relatively rare and comprise 2–5% of all parotid primaries. A salivary duct cyst (SDC) is a true cyst representing 10% of all salivary gland cysts. The risk of malignant transformation of SDC's epithelium is extremely rare. In the literature, only three cases of carcinoma ex SDC of the parotid gland are described. This report presents the first in the literature case of myoepithelial carcinoma (MECA) arising from a parotid SDC. A 75-year-old male patient was referred to the Department of Cranio-Maxillofacial Surgery of the Jagiellonian University in Cracow, Poland due to a cystic tumor arising from the right parotid gland. Superficial parotidectomy with facial nerve preservation was performed. Histological examination confirmed a rare case of MECA emerging from the SDC. The immunohistochemical profile of MECA ex SDC was presented. During 6 months of the follow-up, local recurrence or distant metastasis was not observed.

**Keywords:** salivary duct cyst; parotid gland; myoepithelial carcinoma; parotid cyst; parotid cancer; malignant parotid cyst

#### **1. Introduction**

The World Health Organization (WHO) 2022 Classification of Head and Neck Tumours distinguishes tumors and tumor-like lesions, which include a variety of cysts [1]. Cystic lesions observed in parotid glands are relatively rare and comprise 2–5% of parotid primaries [2]. According to Takita et al., cystic lesions in the parotid gland can be all divided into non-neoplastic cysts, benign tumors with cystic formation (Warthin tumor, cystadenoma), and malignant tumors with macrocystic change (mucoepidermoid carcinoma, acinic cell carcinoma) [2]. Non-neoplastic cysts include salivary duct cysts (SDC), lymphoepithelial cysts, HIV-associated salivary gland disease, dermoid cysts, and lymphangioma [2]. SDC is a true cyst representing 10% of all salivary gland cysts [3]. SDCs develop from the cystic dilatation of the salivary gland duct and are observed in the superficial lobe of the parotid gland and minor salivary glands [4]. A SDC is an acquired cyst, which occurs following obstruction of the salivary duct. The main causes of salivary duct obstruction are sialoliths and mucous plaques, as well as postoperative, posttraumatic, or postinflammatory stenosis [5]. However, in most cases, the specific cause of the salivary duct obstruction is unknown. A SDC is lined with double- or multi-layered columnar and/or cuboid cells: sometimes squamous or oncocytic metaplasia might be observed [3,5]. The risk of malignant transformation of the SDC's epithelium is extremely rare. In the literature, only three cases of carcinoma ex SDC of the parotid gland are presented: undifferentiated carcinoma [6], adenocarcinoma [7], and mucoepidermoid carcinoma [8].

The current report describes the first in the literature case of myoepithelial carcinoma (MECA) arising from the parotid SDC.

**Citation:** Gontarz, M.; Orłowska-Heitzman, J.; G ˛asiorowski, K.; Bargiel, J.; Marecik, T.; Szczurowski, P.; Zapała, J.; Wyszy ´nska-Pawelec, G. Myoepithelial Carcinoma Arising in a Salivary Duct Cyst of the Parotid Gland: Case Presentation. *Medicina* **2023**, *59*, 184. https://doi.org/ 10.3390/medicina59020184

Academic Editors: Daniela Vrinceanu, Adriana Neagos, Codrut Sarafoleanu and Mahmut Tayyar Kalciogu

Received: 14 December 2022 Revised: 13 January 2023 Accepted: 16 January 2023 Published: 17 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **2. Case Report**

In September 2021, a 75-year-old male patient was admitted to the Department of Cranio-Maxillofacial Surgery of the Jagiellonian University in Cracow, Poland, with a tenyear history of a slowly growing, painless cystic tumor of the left parotid gland. Progression in the last period was not observed. The patient suffered from arterial hypertension, and mitral and tricuspid valve regurgitation. In 2018, the patient had a stroke of the right hemisphere of the brain, with left-sided paresis and balance disorders. Physical examination revealed a soft, movable, and fluctuating cystic tumor of the parotid gland with unchanged skin above it. In fine needle aspiration cytology (FNAC), 15 mL of brown fluid content was obtained. Any signs of peripheral facial nerve paresis were not observed. Computed tomography (CT) showed a unilateral, solitary, well-defined, thin-walled, multilocular cystic lesion with dimensions of 49 mm (ax.) × 29 mm (cor.) × 37 mm (sag.) located in the superficial lobe of the parotid gland. In the distal part of the cystic lesion, a contrastenhancing solid tumor 9 mm in diameter was revealed (Figure 1A). Imaging of the neck, chest, and abdomen excluded regional and distant metastases.

**Figure 1.** (**A**) Computed tomography (CT) scan at diagnosis showed a unilateral, solitary, welldefined, thin-walled, multilocular cystic lesion located in the superficial lobe of the right parotid gland. In the distal part of the cystic lesion, a contrast-enhancing solid tumor 9 mm in diameter was revealed (red arrow). (**B**) The thin-walled multilocular cyst (white arrow) with the superficial lobe of the parotid gland as a surgical specimen. The cyst was located directly on the lateral aspect of the temporal, zygomatic, and buccal branches of the facial nerve (black arrows). (**C**) The tumor was composed of mixed small cells, partially epithelioid, partially spindle without marked cytological atypia, with a tendency to form trabecular structures. HE, obj. magnification 200×.

#### *2.1. Surgery*

The patient was qualified for surgical treatment. A modified Blair incision was used to perform superficial parotidectomy with facial nerve preservation. During the surgery, intraoperative neuromonitoring was used to identify branches of the facial nerve (Inomed C2 Nerve Monitor). As an antibiotic prophylaxis, 1 g of Cefazolin was administrated. The superficial lobe of the parotid gland with the thin-walled cyst was excised. The tumor was located in the lumen of the cyst, which was situated in the superficial lobe, directly on the lateral aspect of the temporal, zygomatic, and buccal branches of the facial nerve (Figure 1B). The inferior pole of the parotid gland was intact. The great auricular nerve and parotid fascia were preserved. Active drainage was removed on the second day after surgery. Four days following surgery, the patient was discharged home. The healing process was uneventful with proper facial nerve function.

#### *2.2. Pathology*

The histopathological examination of the surgical specimen revealed a salivary gland with lobules separated by adipose tissue and preserved histological structure. Adjacent to the salivary gland a multilocular cyst with a diameter of 40 mm was found. The cyst was lined with mainly single-layered cuboid glandular cells, in some places with double-layered, and focal formation of small papillary outgrowths up to 0.1 mm in size.

One of the cyst's cavities included a tumor with an 8-mm diameter growing from the cyst's wall. The tumor was composed of mixed small cells, partially epithelioid, partially spindle without marked cytological atypia, with a tendency to form trabecular structures (Figure 1C). The tumor mass surrounded the glandular structures of the salivary gland with positive staining for cytokeratin 19 (CK19) (Figure 2A). The immunohistochemical profile of MECA ex SDC characterized positive staining for keratins AE1:AE3, S-100 protein, smooth muscle actin (SMA), p40, DOG1, and SOX10 (Figures 2B, 3 and 4A). Only limited staining for epithelial membrane antigen (EMA) was found. Immunoreactivity for chromogranin and thyroglobulin was negative (Figure 4B). The Ki-67 labeling index (LI) in the tumor was about 10% (Figure 4C).

**Figure 2.** (**A**) The tumor mass surrounded the glandular structures of the salivary gland with positive staining for cytokeratin 19 (CK19). Obj. magnification 200× (**B**) The immunohistochemical profile of the tumor characterized positive staining for keratins AE1:AE3 and (**C**) S-100 protein. Obj. magnification 200×.

**Figure 3.** (**A**) Positive immunohistochemical staining for smooth muscle actin (SMA), (**B**) p40 and (**C**) DOG1. Obj. magnification 200×.

**Figure 4.** (**A**) Positive immunohistochemical staining for SOX10. (**B**) Negative immunoreactivity for chromogranin. (**C**) The Ki-67 labeling index (LI) in the tumor was about 10%. Obj. magnification 200×.

The entire histological picture supported the diagnosis of a low-grade myoepithelial carcinoma ex-salivary duct cyst. A cluster of cancer cells was found in the cyst wall except for the well-limited remains of the tumor tissue thrust into the lumen of the cyst. Minimally infiltrative growth through the cystic wall without surrounding tissue involvement was observed. Perineural and angiolymphatic invasion was not present. MECA ex SDC was resected with adequate surgical margins (>5 mm, R0 resection). Moreover, in the surgical specimen of the superficial lobe of the parotid gland, ten small lymph nodes with features of reactive changes without metastases were also found.

#### *2.3. Follow Up*

After tumor board consultation, due to the radical resection and early stage of lowgrade cancer pT1N0, the patient was qualified for systematic oncological follow-up in accordance with the NCCN guidelines. A follow-up ultrasound examination 3 months after the procedure was performed without signs of locoregional recurrence. During 6 months of the follow-up, locoregional recurrence was not observed and the facial nerve function was preserved. Unfortunately, in February 2022, the patient died due to acute pancreatitis with peritonitis.

#### **3. Discussion**

Eighty percent of SDC occurs in the parotid gland and comprises about 1–2% of parotid lesions and 10% of all salivary gland cysts [2,3]. SDC is frequently observed in patients older than 30 years old without sex predilection [2,3]. Usually, SDC is a thin-walled, unilateral, solitary cyst situated in the superficial lobe of the parotid gland. Wall thickening might occur due to hemorrhage or infection as a result of FNAC. The differential diagnosis for SDC includes cystadenoma and low-grade mucoepidermoid carcinoma with cystic formation [4]. Surgical resection is the treatment of choice. However, after FNAC, the SDC can disappear completely and the surgery should be postponed until the recurrence of the cyst.

MECAs, also known as malignant myoepitheliomas, are rare entities and are composed of neoplastic myoepithelial cells with infiltrative growth. Myoepithelial cells are observed around the acinus and intercalated ducts of the saliva [9]. MECAs account for less than 2% of all salivary gland cancers and 0.1–0.45% of all salivary gland tumors [10–13]. The most common site of occurrence is the parotid gland (76.6%) followed by the submandibular gland [11]. This was also confirmed by our previous research, in which MECA was found in three cases localized in the parotid gland, which accounted for 1.3% of epithelial salivary gland cancers and 0.37% of all epithelial salivary gland tumors [14]. Etiological factors of MECAs origin are unknown and males and females are affected equally with an average age of around 60 years [13,15,16]. MECAs can either develop de novo or can arise in a pleomorphic adenoma and myoepithelioma [13,16]. Nevertheless, to the best of our knowledge, this case report describes the first case of MECA arising in the SDC of the parotid gland.

The pathological definition of myoepithelioma remains under discussion due to the morphological diversity of the neoplastic myoepithelial cells [16]. Differential diagnoses between benign and malignant myoepitheliomas can also be problematic. In some cases, MECA might be misclassified as a benign salivary gland tumor [10]. According to Nagao et al., MECAs are distinguished by >7 mitotic figures/10 high power fields (HPFs) or Ki-67 LI > 10%. According to Kane and Bagwan, MECAs of the parotid gland might have microscopically partial or complete encapsulation [17]. Histologically, MECAs are characterized by a wide range of morphologic tumor cell types: epithelioid, clear, hyaline, spindle, and mixed [15,17]. About 60% of MECAs are low-grade cancers with quite uniform small- to intermediate-size nuclei with gently distributed chromatin and inconspicuous nucleoli [15]. The immunohistochemical profile of MECA is characterized by the expression of Vimentin, S-100 protein, and the three antibodies against keratins (CAM5.2, AE1:AE3, and 34βE12) [15–19]. In addition, SMA and calponin are immunoreactive in 50% and 75% of tumors, respectively [15].

According to NCCN guidelines from 2022, surgery is the MECAs treatment of choice [20]. Low-grade MECAs with a clinically negative neck do not require elective neck dissection, unlike high-grade MECAs [20]. Xiao et al.'s study presented that occult metastases after elective neck dissection are observed in 15.2% of patients with cN0 neck. This study also confirmed that high-grade MECAs are independent, significant predictors of regional nodal disease in multivariate analysis (OR = 4.42) [21]. Clinically and/or in diagnostic imaging, enlarged lymph nodes should be treated with therapeutic neck dissection with primary MECA resection [20]. Postoperative radiotherapy or radiochemotherapy should be considered in cases of high-grade T3–4a MECAs with close or positive margins, perineural/angiolymphatic invasion, and lymph node metastases found in the surgical specimen [20].

Savera et al.'s study revealed that 29% of MECAs patients die from the disease after an average of 32 months. Only high-grade MECAs are strongly correlated with poor prognosis [15,21]. On the other hand, Luo in the cohort of 290 patients, reported 5-, 10-, and 15-year overall survival (OS) of 68.9%, 53%, and 38.1%, respectively. The independent prognostic factors influencing OS were TNM stage, grade, and postoperative radiotherapy [11]. The presented case of a low-grade MECA ex SDC consisted of mixed (epithelioid and spindle) tumor cell types. Radical resection of MECA ex SDC (R0 resection) and early stage of the disease indicated only observation without adjuvant treatment. Unfortunately, the followup period was only 6 months (with proper facial nerve function, without locoregional recurrence) due to the patient's death from another cause.

#### **4. Conclusions**

In conclusion, carcinoma ex SDC of the parotid gland is an extremely rare entity. To our knowledge, this is the first described case of the MECA arising from the parotid SDC. Surgery is the standard of care for both SDC and MECA. MECA emerging from SDC can be successfully treated with superficial parotidectomy with facial nerve preservation without recurrence. MECA, depending on the grade and stage, might require additional neck dissection as well as adjuvant treatment.

**Author Contributions:** Conceptualization, M.G., J.B., J.O.-H. and G.W.-P.; methodology, M.G., J.B., J.O.-H. and K.G.; software, M.G., T.M. and P.S.; validation, M.G., J.B. and K.G.; formal analysis, M.G., T.M., J.O.-H. and J.Z.; investigation, M.G., J.O.-H., J.B., K.G. and P.S.; resources, M.G., J.O.-H., J.Z. and G.W.-P.; data curation, M.G., T.M. and P.S.; writing—original draft preparation, M.G., T.M., J.O.-H. and G.W.-P.; writing—review and editing, M.G., J.O.-H. and G.W.-P.; visualization, M.G. and J.B.; supervision, J.Z. and G.W.-P.; project administration, M.G. and J.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Jagiellonian University (No: 122.6120.287.2016).

**Informed Consent Statement:** Patient consent was waived so long as all personal information remained confidential.

**Data Availability Statement:** Restrictions apply to the availability of these data. Data were obtained from patients treated at the Department of Cranio-Maxillofacial Surgery, Cracow, Poland, and cannot be shared, in accordance with the General Data Protection Regulation (EU) 2016/679.

**Acknowledgments:** We thank Krzysztof Sliwi ´ ´ nski for technical assistance in clinical imaging.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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*1.3. Objectives and the Paper Content*

The main subject of this research was the impact of the occurrence of power limitation on the production system. The production system is a deliberately designed and organized system, within which the available resources are used to produce specific products that meet the consumer needs [5]. This system is based on the execution of the production process, using four key resources. The resources include material resources, human resources, information resources, and capital resources. In the last few years, more and more attention has been paid to including the energy component in the definition of the production system. This component is one of the inputs to the manufacturing process. Electricity makes it possible to perform technological operations on machines as well as to ensure the functioning of the entire production environment.

It is likely that soon, both the increase in electricity prices and the reduction in the availability of electricity may constitute one of the main challenges for manufacturing companies. In this context, this paper aims to propose a method that would enable manufacturing companies to analyze electricity consumption in rapidly changing production conditions. In particular, the method should answer the question of how the constrained availability of power or large differences in electricity prices between different times can be compensated by changing the production plan. A change in the production plan is understood as an extension of the execution time for a given production batch, moving the execution to another time, or determining which part of the production batch will not be completed on the previously planned date. This type of approach has not been considered in the literature thus far. The main conclusion of this paper is that with the help of available simulation modeling programs, it is possible to analyze electricity consumption in production processes and draw practical benefits for the company. An additional conclusion is the possibility of developing and applying the proposed method to various production processes, which requires further research.


**3. Etiopathogenesis**

Lupus anticoagulants and anticardiolipin antibodies are immunoglobulins that were originally thought to react only with phospholipid. However, it is now well established that these antibodies react directly with epitopes on β2-GPI(McNeil HP 1990, Galli M 1990) or prothrombin(Rao LVM 1996, Bevers EM 1991 ), that subsequently bind to anionic phospholipid. Anticardiolipin antibodies are low-affinity monovalent antibodies to β2-GPI when in solution, and the monovalent complexes bind weakly to anionic phospholipids. However, when the antigen density is high, bivalent complexes are formed that have a high affinity for phospholipid surfaces. The fact that β2-GPI antibodies are polyclonal reacting

event for which no underlying cause may be apparent; (4) patients receiving certain drugs, including procainamide and phenothiazines(a high prevalence of the lupus anticoagulant and a positive antinuclear antibody test are observed in psychotic patients receiving longterm chlorpromazine therapy); other drugs or biologics that can induce the lupus anticoagulant include hydralazine, quinidine, and possibly α-interferon; (5) patients with a recent acute viral infection, in whom the antibody is usually transient; (6) patients with human immunodeficiency virus infection; (7) women with recurrent fetal wastage;(8) occasionally in older patients with malignancies and (9) patients seeking medical attention for a variety of disorders in whom the lupus anticoagulant is discovered as an incidental finding, usually discovered because of a prolonged partial thromboplastin time (PTT)

APA can be detected in the absence of thrombosis or pregnancy morbidity or other systemic autoimmune diseases. During ongoing infectious disease, during treatments with a variety of drugs and even in healthy individuals, APA positivity may occur. The prevalence of APA ranges from 1% to 10% in the general population, 16% in patients with rheumatoid arthritis, and 30% to 40% in patients with SLE(Petri M 2000, Lim W et al 2006). The prevalence of positive tests for lupus anticoagulant and anticardiolipin antibody in a normal population has been reported in several studies. Because of the non-Gaussian distribution of anticardiolipin antibody levels in normal subjects, the cut-off points between normal and abnormal results is difficult to determine. One study reported IgG and IgM anticardiolipin antibodies in approximately 5% of normal individuals, although only 2% had persistently elevated levels on repeat testing. Shi and colleagues detected anticardiolipin antibodies in 6% of normal blood donors, respectively, and detected lupus anticoagulant activity by kaolin clotting time in 4%Shi W 1993). The prevalence of anticardiolipin antibody appears to increase with age. The prevalences of elevated levels of IgG and IgM anticardiolipin antibody in healthy pregnant women were 2% to 3% and 4%, respectively(Harris EN 1991, Aoki K 1994, Lockshin MD 1997). Most of these were low titer; only 0.2% were high titer. In other studies, the incidence of anticardiolipin antibodies in pregnant individuals ranged from 1% to 2%

When the patient does not exhibit any other symptom that would allow the diagnosis of another associated autoimmune disease, the antiphospholipid syndrome is considered primary, or isolated. The term 'secondary' APLS is sometimes used for patients suffering

Lupus anticoagulants and anticardiolipin antibodies are immunoglobulins that were originally thought to react only with phospholipid. However, it is now well established that these antibodies react directly with epitopes on β2-GPI(McNeil HP 1990, Galli M 1990) or prothrombin(Rao LVM 1996, Bevers EM 1991 ), that subsequently bind to anionic phospholipid. Anticardiolipin antibodies are low-affinity monovalent antibodies to β2-GPI when in solution, and the monovalent complexes bind weakly to anionic phospholipids. However, when the antigen density is high, bivalent complexes are formed that have a high affinity for phospholipid surfaces. The fact that β2-GPI antibodies are polyclonal reacting

performed as a routine preoperative evaluation.

and lupus anticoagulant 1% to 4%(Petri M 2000).

from another autoimmune or inflammatory disease.

**3. Etiopathogenesis**

**2. Epidemiology**

with different epitopes on the β2-GPI molecule and the increased affinity of the divalent antigen-antibody complexes for phospholipid surfaces explains why some anticardiolipin antibodies have anticoagulant activity and some do not(Arnout J 2003). This anticoagulant activity correlates best with the incidence of thrombosis(Galli M 2003), and a subset of lupus anticoagulants caused by anti–β2-GPI antibodies with specificity for an epitope on domain I. In some patients the anticardiolipin antibody will react with immobilized cardiolipin in vitro but not prolong phospholipid-dependent coagulation tests. Similarly, some of the antiprothrombin antibodies can prolong coagulation tests and some will not.

As with most autoimmune conditions, the etiology of APLS is not understood. It has been demonstrated that normal healthy individuals without APS have memory B cells that produce aPL antibodies; in a study of patients with infectious mononucleosis, 10 to 60 percent of immunoglobulin M aCL-producing cells expressed CD 27, the marker of memory B cells(Lieby P 2003).

Although antibodies against anionic phospholipid moieties arise during the course of infections such as syphilis and lyme disease, those are distinct from antibodies generated by patients with the syndrome because they generally recognize phospholipid epitopes directly and are not associated with the clinical manifestations of the syndrome.

Reports of familial clustering of raised aPL antibody levels indicate that genetic susceptibility can play a role in their development(Donald I. Feinstein 2007). In one study of 84 APLS patients, more than 35% had at least one relative, and more than 20% had two or more relatives, with evidence of at least one clinical feature of APS, such as thrombosis or recurrent fetal loss(Weber M 2000).

Many different mechanisms have been described for thrombosis during APLS, mainly after in-vitro experiments: (1) activation of endothelial cells by complexes of β2 GPI and anti-β2 GPI, these complexes could bind to annexin 2 or even Toll-like receptors on the surface of endothelial cells(Zhang J 2005, Fischetti F 2005); (2) platelet activation after direct binding of the β2 GPI, which targets the autoantibodies on the surface of these cells, the β2 GPI is selectively bound by the activator receptor apo ER 2(Lutters BC 2003); (3) functional dysregulation of hemostasis by the presence of autoantibodies against natural anticoagulant proteins like annexin 5 and activated protein C; (4) abnormal fibrinolysis directly linked to the presence of APL(Cesarman-Maus G 2006).

APL can stimulate platelet aggregation(Lin YL 1992), an effect that might be promoted via signalling through apolipoprotein E receptor 2(apoER2) receptors; the beta2GPI binding site for apo ER2 on platelets was localized to its domain V. Beta2GPI also has a dampening effect on platelet adhesion by interfering with the platelet-von Willebrand factor interaction, and consequently aPL antibodies, by interfering with this dampening, can increase platelet adhesion in flow systems(Hullstein JJ 2007).

Normal endothelial function includes control over thrombosis and thrombolysis, platelets and leukocyte interaction with the vessel wall, and regulation of vascular tone and smooth muscle proliferation. Several in vitro studies and studies on animal models have shown that incubation of endothelial cells with aPL from APLS patients generates different effects on endothelial function via β2 GPI. As a whole this might cooperate in sustaining endothelial perturbation that has been suggested to have a pivotal pathogenetic role in APS associated thrombosis(Stalc M 2006).

Because high-level aPLs may persist for years in asymptomatic persons, it is likely that vascular injury, endothelial cell activation, or both immediately precede the occurrence of thrombosis in those bearing the antibody (second-hit hypothesis). Of note, at least 50% of

Antiphospholipd Syndrome and Venous Thrombosis 59

The first clinical aspect of the APLS is thrombosis, which can affect arterial or venous vessels, as well as small vessels, and must be confirmed by means of imaging studies and/or histopathology. Arterial thrombosis mainly occurs in the central nervous system. But all arteries can be effected and myocardial infarction, peripheral gangrene, aseptic osteonecrosis and adrenal insufficency can develop with respect to effected arterial site. The venous thrombosis commonly localizes to the deep veins of the limbs and can be complicated by pulmonary embolism. As in arterial thrombosis, any segment of the venous

A definitive diagnosis of APLS is based on fulfilling at least one of the Updated Sapporo Clinical criteria(vascular thrombotic event or pregnancy morbidity) and at least one of the laboratory criteria(Table 1). In general, medium titer aCL is considered 40 U or more and high titer, more than 80 U; titers between 20 and 40 U should be evaluated cautiously. Transient APL positivity is common during infections; thus documentation of the persistence(at least 12 weeks apart) of autoimmune APL is crucial for both diagnostic and

The choice of initial APL tests remains a subject of debate. In general, the LA test is more specific for APL-related clinical events. The specificity of aCL for APL-related clinical events increases with higher titers. The IgG isotype is more strongly associated with APL-related clinical events than the IgM isotype. In a patient with suspected APS, testing for LA and IgG/IgM aCL should be ordered initially. If these tests are negative or low-titer and there is still a high level of suspicion for APS, then testing for antiβ2GPI antibodies and IgA aCL/antiβ2GPI can be pursued(George D 2009). Antiphospholipid antibody tests developed based on other phospholipids such as phosphatidylserine, phosphatidylinositol, or phosphatidylethanolamine or phospholipid-binding plasma proteins(such as prothrombin)

Although any vasculature can be affected by thrombosis, stroke and transient ischemic attack are the most common presentations of arterial thrombosis, whereas deep vein thrombosis with or without pulmonary embolism is the most common presentation of venous thrombosis in APLS(George D 2009). Antiphospholipid antibodies can cause both arterial and venous thrombosis in the same patient. Reccurent thromboses tend to occur in the same vascular distribution(venous followed by venous and arterial followed by arterial). in some studies the incidence of venous thrombosis (70%) is greater than the incidence of

Superficial thrombophlebitis, superior vena cava syndrome, renal vein thrombosis, Budd Chiari syndrome, central retinal vein occlusion, pulmonary hypertension due to recurrent pulmonary embolism, and diffuse pulmonary hemorrhage due to microthrombosis are

Of unselected patients with antiphospholipid antibody, 1% to 2.5% per year will develop thromboembolism(Galli M 2003, Finazzi G 1996),and 10% to 25% of patients with deep venous thrombosis will be found to have antiphospholipid antibodies(Ginsburg KS 1992).However, in a prospective population based study of 66140 individuals in Norway(Naess IA 2005), elevated anticardiolipin antibody levels were not a risk factor for

vasculature can be effected, which will induce different manifestations.

therapeutic purposes.

**5. Clinical features**

are not yet well standardized and accepted.

arterial thrombosis.(Galli M 1997, Triplett DA 1995).

some of the thrombotic manifestations of APLS.

APLS patients with vascular factors possess other acquired thrombosis risk factors at the time of their events(Kaul M 2006, Erkan D 2002).

Both persons congenitally lacking ß2GPI39 and ß2GPI knockout mice appear normal(Sheng Y 2001). ß2GPI polymorphisms influence the generation of aPLs in individuals, but they have only a weak relationship to the occurrence of APLS. A cluster of 50 upregulated genes may have an effect on the occurrence of thrombosis in aPL-positive individuals(Potti A 2006).


*Edited by Yuri V. Kim*

The purpose of this book is to present some uses of the Kalman filter (KF) in engineering activities that can produce a robust and technically acceptable result while keeping as close as possible to the optimal (most accurate) solution. KF suboptimization is often required, due to the realities of implementation and real-life operational conditions. The book brings together the experiences of specialists from different engineering areas using the KF in their practice.

ISBN 978-1-80356-575-0

Kalman Filter - Engineering Applications

Published in London, UK © 2023 IntechOpen © watchara\_tongnoi / iStock

## Kalman Filter Engineering Applications

*Edited by Yuri V. Kim*


**4. Current situation: environmental conflicts and an ecosystem in danger of extinction**

The dynamics of the expansion in the occupation of spaces during the last 80 years in the equatorial high mountain areas of Colombia have brought as a consequence and dramatic decrease of forests in these regions and taking into account the figure of less than 5% of existence of the original Andean forests we could affirm that this ecosystem vital for the regulation of biogeochemical cycles and ecological corridors of fauna and the flora that it lends to the whole of the Main Ecological Structure of the nation, is today in danger of extinction [8].

The following environmental problems, which have become real socio-ecological conflicts, which are at the root of this worrying situation are:

i.Expansion of agricultural frontiers to affirm the post-Thispánicos settlements in the mountain ranges, which were constituted during the time of La Colonia (1530–1819) as a basis for the creation of the Republic of Colombia from the War of Independence, which although it brought contributions from European Modernity and gave birth to a mestizo people with the peasantry, It was carried ahead with its demographic catastrophe product of violence and diseases and until the near extinction of indigenous cultures such as the Muisca, the Tairona, and others cornered them in the depths of the jungles and mountains. During this period and for the construction of nascent cities such as the cities of Popayán, Santafé de Antioquia, Cali, Popayán, Bogotá, Tunja and Pamplona, Villa de Leyva, and Guaduas among the main colonial settlements of the Andean mountain, the wood of the equatorial high Andean forests was used and use of the fauna was made that was intense until the second half of the twentieth century and still continues hunting and Illicit trade in the fauna and flora of these ecosystems. The Conquest and the Colony with its disastrous traces of destruction of nature and ancestral peoples and the peasant people, the main victim of all the violence of the nineteenth and twentieth centuries, after the birth of the Republic, has not yet ended in these territories. However, despite the destruction of important forest masses during this period, until the late 1950s when the Agustín Codazzi Geographic Institute generated the cartography of Colombia

to support Law 2 of 1959 that created the Zones of Forest Reserve of the Nation, large areas of high Andean forests existed throughout the country.

municipalities to the northeast of this as are corrales, Beteitiva, Paz del Rio, and others. This has led to strong and dynamic socioenvironmental conflicts between the peasantry and foreign oil and coal companies favored by the prevailing extractivist policies [14, 15].

	- ix.This currently constitutes the basis of socio-environmental conflicts over water and in defense of forests and moors in the department of Boyacá which is one of the departments with the greatest presence of high Andean forests in Colombia, along with the departments of Cundinamarca, Cauca, Tolima, Santander, and the Coffee Axis.


**Yao-Lin Kao 1, Kuan-Hsun Huang 2, Hann-Chorng Kuo 3 and Yin-Chien Ou 1,4,\***

Received: 23 November 2019; Accepted: 11 December 2019; Published: 13 December 2019

**Abstract:** Neurogenic and non-neurogenic urethral sphincter dysfunction are common causes of voiding dysfunction. Injections of botulinum toxin A (BoNT-A) into the urethral sphincter have been used to treat urethral sphincter dysfunction (USD) refractory to conventional treatment. Since its first use for patients with detrusor sphincter dyssynergia in 1988, BoNT-A has been applied to various causes of USD, including dysfunctional voiding, Fowler's syndrome, and poor relaxation of the external urethral sphincter. BoNT-A is believed to decrease urethral resistance via paralysis of the striated sphincter muscle through inhibition of acetylcholine release in the neuromuscular junction. Recovery of detrusor function in patients with detrusor underactivity combined with a hyperactive sphincter also suggested the potential neuromodulation effect of sphincteric BoNT-A injection. A large proportion of patients with different causes of USD report significant improvement in voiding after sphincteric BoNT-A injections. However, patient satisfaction might not increase with an improvement in the symptoms because of concomitant side effects including exacerbated incontinence, urinary urgency, and over-expectation. Nonetheless, in terms of efficacy and safety, BoNT-A is still a reasonable option for refractory voiding function. To date, studies focusing on urethral sphincter BoNT-A injections have been limited to the heterogeneous etiologies of USD. Further well-designed studies are thus needed.

**Keywords:** botulinum toxin; urethral sphincter; urethral sphincter dysfunction; lower urinary tract symptoms; urodynamics

**Key Contribution:** This article summarized the effects of sphincteric BoNT-A injections among various USDs in view of pathophysiology, urodynamic outcome, and subjective patient reports.


**1. Introduction**

Mg alloys are increasing in demand due to their high specific strength (strength to density ratio) and they have attractive applications in automotive industries, aerospace, medical implants, electronic appliances, and sport equipment [1,2]. Different methods are available for the joining of magnesium alloys. The primary methods include tungsten arc welding (TIG), laser beam welding (LBW), resistance spot welding (RSW), electromagnetic welding (EMW), electron beam welding (EBW), diffusion bonding, soldering, brazing, adhesive bonding, and mechanical fastening. Fusion based welding methods, like TIG, LBW, RSW, etc., have major problems that are mostly related to low boiling temperature of magnesium alloys that leads to porosities and hot cracking in the weld line and heat affected zone (HAZ). The other main limitations include high energy consumption and the need of consumables [3,4]. Therefore, the development of solid-state processes, like friction stir welding (FSW), friction stir spot welding (FSSW), and ultrasonic spot welding (USW) helped to avoid problems that are related to melting and provide opportunities for dissimilar material welding [5–7]. However, FSW and FSSW have the limitations of making "keyhole" and the need to fixture and a long process time [5]. USW is considered to be a solid-state welding process, with low energy consumption and time saving

process and no need for consumables or atmosphere control. It is using relatively high frequency ultrasonic vibrations (20–40 KHz) that induce the workpieces to be held together with simultaneous pressure, which leads to frictional shear forces parallel to the interface. Under the effect of frictional shear forces, surface oxide layers break down and two joining parts approach bare contact and the temperature increases due to friction, and then local welding occurs between the asperities of two faying surfaces. Finally, the spreading of local micro-welds and coalescence results in the creation of a metallurgical bond in the weld spot [5,7–11].

High-power ultrasonic spot welding (HP-USW) is an effective way of achieving sound strong joints among different USW methods. This method uses higher power and lower energy and time when compared to other spot-welding processes [12]. The use of high energy levels (or equally longer welding times) may cause extra-thinning of the joint and induce cracks in the weld nugget that limits access to maximum joint strength [9]. Furthermore, the use of interlayer in USW mostly related to weldability improvement, especially in dissimilar joining. Typically, use of metallic foil interlayer is more convenient and cost effective than other methods, like sputtering, plating, etc. [8]. The first trials of current research also performed using Cu foil in different thicknesses and USW energies. However, those experiments resulted in relatively strong joints, but the use of Cu coating was more effective in achieving better weldability and mechanical properties.

According to authors knowledge and a review on previous research studies, the use of Cu as an interlayer in the AZ31B Mg alloy "similar USW" has not been recorded. Previous efforts mostly were related to processes other than USW, the same as TLP (transient liquid phase), eutectic, or diffusion bonding. HP-USW of AZ31 Mg alloy classifies similar joining, but, when Cu used as an interlayer in contact with Mg, it forms a dissimilar welding combination at the interface. Hence, it is beneficial to review the Mg-Cu bonding processes and diffusion behavior, as well as AZ31 that is interlayered with Cu through other processes to find out the possible interactions in this system.

Diffusion kinetics and the identification of interfacial layers between Mg and Cu (pure metals) were studied by Nonaka et al. [13] and they reported the formation of Mg2Cu as major interfacial layer near Mg and a thin layer of MgCu2 adjacent to Cu (410–475 ◦C). Additionally, they suggested that the diffusion of Cu in Mg is faster than the inverse. However, in a nearly same recent study by Dai et al. [14], there was agreement about formation of two intermetallic layers between Mg and Cu, but they reported a different interdiffusion priority based on measured diffusion coefficients and activation energy data. These are basic studies regarding the Mg-Cu reaction diffusion couple, but, in the field of ultrasonic welding, Macwan and Chen [15] applied HP-USW for joining AZ31 to Cu (2000 W, 1000–2500 J) and observed the formation of a diffusion layer around the interface containing Mg + Mg2Cu that spread just towards the Mg side. They proposed a mechanism that is mainly based on the preferable diffusion of Cu in Mg to form Mg2Cu intermetallic compound besides liquid phase formation through the eutectic reaction. This behavior was mostly attributed to the differences in the physical properties between Cu and Mg, such as melting point, thermal conductivity, heat capacity, and atomic radius.

One of trials to use the Cu interlayer was related to the field of eutectic bonding, where Elthalabawy et al. [16,17] studied joining AZ31 to 316L steel using a Cu insert (at 530 ◦C), and found that the eutectic reaction between Mg and Cu after 1 min. resulted to formation of 4 distinct interfacial layers in Mg part. Middle interfacial layers included Mg2Cu and a layer of ternary Mg + Cu + Al composition wherein Al content depleted from AZ31 base metal to participate in the Al-containing product.

The participation of AZ31 alloying elements in interfacial phenomena makes it more complicated, which can lead to non- stoichiometric compositions. Anyway, the formation of intermetallic compound (IMC) reaction products in dissimilar joining is prevalent and, in most cases, it has negative effect on mechanical properties of the joint, because they can embrittle the interface, especially when a continuous layer formed. Panteli et al. [18–20], during HP-USW of AZ31 to Al-6111, observed a 5 μm thickness continuous IMC layer along the interface, which resulted in low tensile shear strength with interfacial fracture. Additionally, they found that, under dynamic high strain rates of USW, the rate

of IMC product formation was twice when compared to normal static conditions [18–20]. Patel et al. [21,22] also used HP-USW to join AZ31-Al 5754 and AZ31-HSLA steel combinations while applying a 50 μm thickness Sn interlayer and, finally, the Sn interlayer improved the weld strength in lower energies. The improvement was attributed to the formation of a composite-like Sn + Mg2Sn eutectic structure instead of forming a brittle IMC product without an interlayer.

The objective of the current research is to study the effects of using a Cu coating interlayer in HP-USW of AZ31B Mg alloy on the weldability, mechanical strength, and process optimization from the point of view of energy, time, and interface characterization.