**4. Conclusions**

We show the measured versus simulated return loss and realized gain of the dual-band antenna at S-band in **Figures 8** and **9** as well as at X-band in **Figures 10** and **11**. We took all realized gain versus frequency measurements at boresight to the antenna. We see general agreement at both bands for all measurements. One

*Advanced Radio Frequency Antennas for Modern Communication and Medical Systems*

**Figure 8.**

**Figure 9.**

**Figure 10.**

**196**

*S-parameters at S-band horizontal polarization port.*

*Realized gain to boresight at S-band horizontal polarization port.*

*S-parameters at X-band horizontal polarization port.*

This chapter describes both the benefits and current challenges facing AM for antennas and RF devices. The forefront of which is a lack of commercially available high-dielectric materials that are compatible with filament fed 3D printers. As a comparison, we fabricated two similar antenna designs utilizing multiple nested dielectrics and an embedded shorting wall within the dielectrics to compare performance between traditional materials and manufacturing methods versus those of AM. Due to restricted access to an AM feedstock with the proper dielectric constant, direct comparisons of the two antennas is not the preferred way of comparison. However, experimental results for both prototypes agree well with the simulation data. There also seems to be no degradation in the performance of the AM prototype over the traditional prototype in terms of the agreement with the respective antenna models. With new methods of extruding higher dielectric filaments for FDM 3D printers, AM seems to be a good fit for future work in antenna design especially as antenna and RF front ends grow increasingly complex and more fully integrated.

**References**

[1] Luneburg R, Herzberger M. Mint: A

*Additive Manufacturing for Antenna Applications DOI: http://dx.doi.org/10.5772/intechopen.92363*

> modeling. Journal of Materials Processing Technology. 2009;**209**

[9] Sood A et al. Improved dimensional accuracy of fused deposition modeling processed part using grey Taguchi method. Journal of Material and Design.

[10] Ang K et al. Investigation of the mechanical properties and porosity relationships in fused deposition modeling-fabricated porous structures. Rapid Prototyping Journal. 2006;**12**(2):

[11] Roberson D, Wicker R, MacDonald E. Ohmic curing of printed silver conductive traces. Journal of Electronic Materials.

Fabrication of low dielectric constant composite filaments for use in fused filament 3D printing. Additive Manufacturing, Science Direct. December 2019;**30**. Available from:

[13] Castles F, Isakov D, Lui A, Lei Q, Dancer CEJ, Wang Y, et al. Microwave dielectric characterization of 3D-printed BaTiO3/ABS polymer composites. Scientific Reports. 2016;**6**. Available from: https://doi.org/10.1038/srep22714

[14] Parsons P, Larimore Z, Mitchell G. Composite materials development for fused filament fabrication of RF systems. In: Proceedings of Applied Computational Electromagnetics Symposium (ACES). Monterey, CA;

[15] Dorsey W, Zaghloul A. Dualpolarized dual-band antenna element for ISM bands. In: Proceedings of IEEE

(15-16):5593-5600

2009;**30**(10):4243-4252

2012;**41**(9):2553-2566

[12] Parsons P, Larimore Z, Muhammed F, Mirotznik M.

https://doi.org/10.1016/j. addma.2019.100888

March 2020

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[3] Moulart A, Marrett C, Colton J. Mint:

ceramics by fused deposition of ceramics.

Polymeric composites for use in electronic and microwave devices. Journal of Polymer Engineering and Science. 2004;**44**:588-597. DOI:

[4] Agarwala MK et al. Structural

In: Proceedings of Solid Freeform Fabrication Symposium. Austin, TX.

[5] Duncan B et al. 3D printing of millimeter wave RF devices. In: Workshop on Additive Manufacturing of Antennas and Electromagnetic Structures. McLean, VA: MITRE; 2017

[6] Castles F, et al. "Microwave Dielectric Characterization of 3D-Printed BaTiO3/ABS Polymer

Composities", US National Library of Medicine, PMC4778131; 2016. Available from: https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC4778131/#b20

[7] Rao Y et al. Novel polymer-ceramic nanocomposite based on high dielectric constant epoxy formula for embedded capacitor application. Journal of Applied Polymer Science. 28 Nov 2001;**83**(5): 1084-1090. Available from: https://doi.

org/10.1002/app.10082

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[8] Ahn D et al. Representation of surface roughness in fused deposition

10.1002/pen.20053

1995. pp. 1-8

mathematical theory of optics. American Journal of Physics. 1996;**34**

(80). DOI: 10.1119/1.1972799

[2] Roper GB, Yarlagadda S, Mirotznik M. Fabrication of a flat Luneburg lens using functional additive manufacturing. In: Proceedings of National Radio Science Meeting (Joint AP-S Symposium), USNC-URSI, Memphis, TN; 6–11 July 2014; DOI: 10.1109/USNC-URSI.2014.6955649


3.1.17. (3*S*,4*S*)-5,8-dihydroxy-7-(hydroxymethyl)-4-methoxy-3-pentylisochroman-1-one (**11**)

To a solution of diMOM hydroxymethyl derivative **11a** (7.2 mg, 24.1 μmol) in MeOH (1.8 mL) was added 6 M aqueous HCl (0.45 mL) at 0 ◦C. After stirring for 4 h at 40 ◦C, the reaction was quenched by adding saturated aqueous NaHCO3 at 0 ◦C. The mixture was extracted with EtOAc (×3) and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified with PTLC (EtOAc:*n*-hexane = 1:1) to give hydroxymethyl derivative **11** (3.9 mg, 52%) as a white solid. m.p. 143–145 ◦C; 1H-NMR (400 MHz, CDCl3) δ 10.99 (1H, s), 7.12 (1H, s), 6.03 (1H, br-s), 4.74 (1H, d, *J* = 2.4 Hz), 4.72 (2H, br-s), 4.48 (1H, ddd, *J* = 2.4, 5.2, 8.0 Hz), 3.38 (3H, s), 2.53 (1H, br-s), 1.96 (1H, m), 1.86 (1H, m), 1.70–1.50 (1H, overlapped), 1.46 (1H, m), 1.40–1.25 (4H, overlapped), 0.91 (3H, t, *J* = 6.8 Hz); 13C-NMR (125 MHz, CDCl3) δ 169.5, 154.2, 145.8, 130.8, 123.8, 121.4, 107.8, 82.1, 69.8, 61.2, 57.2, 31.9, 30.2, 25.2, 22.8, 14.3.; IR (KBr) 2951, 2921, 2854, 1682, 1440, 1302 cm−1; HRMS (ESI) *m*/*z* (M + H)+ calculated for (C16H23O6) + 311.1495, found 311.1498.

3.1.18. ((3*S*,4*S*)-4-methoxy-5,8-bis(methoxymethoxy)-1-oxo-3-pentylisochroman-7-yl) methylmethanesulfonate (**22**)

To a solution of diMOM hydroxymethyl derivative **11a** (7.2 mg, 24.1 μmol) in CH2Cl2 (0.47 mL) were added Et3N (10.8 μL, 77.5 μmol) and MsCl (6.0 μL, 77.5 μmol) at 0 ◦C. After stirring for 40 min at the same temperature, the reaction was quenched by adding water at 0 ◦C. The mixture was extracted with EtOAc (×3) and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified with PTLC (EtOAc:*n*-hexane = 2:3) to give diMOM mesylated derivative **22** (30.4 mg, 91%) as a white wax. 1H-NMR (400 MHz, CDCl3) δ 7.51 (1H, s), 5.45 (1H, d, *J* = 12.0 Hz), 5.37 (1H, d, *J* = 12.2 Hz), 5.25 (2H, s), 5.14 (1H, d, *J* = 6.6 Hz), 5.12 (1H, d, *J* = 6.6 Hz), 4.62 (1H, d, *J* = 1.4 Hz), 4.27 (1H, ddd, *J* = 1.2, 5.6, 7.8 Hz), 3.59 (3H, s), 3.50 (3H, s), 3.33 (3H, s), 3.07 (3H, s), 2.03 (1H, m), 1.82 (1H, m), 1.58 (1H, m), 1.44 (1H, m), 1.40–1.25 (4H, overlapped), 0.91 (3H, t, *J* = 6.8 Hz); 13C-NMR (100 MHz, CDCl3) δ 161.9, 152.2, 150.5, 131.3, 130.5, 120.3, 119.7, 102.8, 95.5, 81.2, 68.6, 66.9, 58.1, 57.4, 56.9, 38.2, 31.9, 30.9, 25.2, 22.8, 14.3.; IR (KBr) 2958, 2930, 2860, 1829, 1681, 1440, 1358, 1175, 933 cm−1; HRMS (ESI) *m*/*z* (M + Na)+ calculated for (C21H32O10SNa)+ 499.1614, found 499.1616.


**5. RT and immunotherapy combination modalities**

### **5.1 Pre-clinical models**

202 Prostate Cancer – Diagnostic and Therapeutic Advances

probably due to loss of membrane-bound TGF-β (Cao et al., 2009). Tregs were especially sensitive to low-dose radiation compared to effector T cells (Cao et al., 2011). Another study, in TRAMP mice, had the opposite findings: Treg cell numbers increased in immune organs after local or whole body irradiation without changes to their functional activity (Kachikwu et al., 2010), indicating relative resistance to 0-20 Gy radiation. It remains to be seen what

We showed recently that lymphopenia following prostate and pelvic RT causes preferential death of naïve or unstimulated T-cells (Tabi et al., 2010). Elevated frequencies of Treg cells were observed in the circulation following 44 Gy radiation in 20 fractions to the pelvic nodes and 55 Gy to the prostate (Fig.2). T cell proliferative function was also impaired (Tabi et al., 2010) but it was restored in vitro with exogenous IL-2 without increasing Treg frequencies (Fig. 2). This indicates that IL-2 maybe used to support T cell function after patients

> IL-2 (U/ml) 0 20 100

Fig. 2. IL-2 response of Treg cells from the peripheral blood of PCa patients undergoing standard RT. Frequencies of CD4+CD25+Foxp3+ T cells (see gate in insert) were measured before (RT0), immediately after radical radiation in 20 fractions (RT20) and 4 weeks after the last fraction (pRT4). Means and SD of triplicates are shown from a representative patient. Frequencies of Tregs were elevated at RT20, but returned to pre-radiation (RT0) level at pRT4. Unlike at RT0 or RT20, exogenous IL-2, added to the cells in vitro, did not increase

Most importantly, we identified novel TAA-specific T-cell responses post-RT (Tabi et al., 2010), which were not present before RT. Similar findings were observed by others (Nesslinger et al., 2007; Schaue et al., 2008), indicating the ability of radiation to shift the

balance between tumour-specific regulatory and effector immune mechanisms.

RT0 RT20 pRT4

happens to Tregs in situ during RT of PCa.

completed standard RT.

% CD4+CD25+Foxp3+ cells

0

Treg frequencies at pRT4.

1

2

3

4

5

6

7

The use of IL-2 as a monotherapy in cancer has been extensively researched but due to issues with toxicity its clinical use has been limited. In the murine renal adenocarcinoma model, IR was found to augment the response of pulmonary metastases to IL-2 therapy (Younes et al., 1995). Following IR to one lung, plus systemic IL-2 treatment, a reduction in tumour size was observed in both lungs. The effect is dose-dependent and immunohistological studies show significant infiltration of T cells and macrophages at the tumour site. IL-2 is capable of rescuing T cells from IR-induced apoptosis and restores T cell proliferation after RT (Tabi et al., 2010). Its use in combination with RT may minimise the immunosuppressive effects of RT and enhance tumour cell killing via T cells (Mor & Cohen, 1996). In PCa, the combination of IL-2 and radiation in a mouse bone metastases model demonstrated a ~50% inhibition of tumour growth (Hillman et al., 2003). There was a greater degree of tumour destruction in IL-2-treated irradiated tumours than in irradiated tumours alone and the histology revealed increased fibrosis and elevated numbers of infiltrating inflammatory immune cells.

Antitumour effects were also observed in a model utilising IL-12 and RT. IL-12 is secreted by mature DC and macrophages and required for IFNγ and TNFα production from T cells and mediates a Th1-type immune response. Adenovirus-derived IL-12 plus RT significantly increased local antitumour and systemic antimetastatic effects in a preclinical model of metastatic PCa when compared to either treatment alone (Fujita et al., 2008). This antimetastatic activity is due to the antitumoural activities of natural killer (NK) cells. These results were also observed in the 4T1 mammary carcinoma. The combination of RT and an adenoviral vector encoding IL-12 and the co-stimulatory molecule CD80 resulted in a significant reduction in tumour growth (Lohr et al., 2000). The antitumour effect observed in the combination therapy group was far superior if the IL-12 and CD80-expressing adenovirus was administered after the final fraction of radiation.

Further cytokine studies have evaluated the combined effect of IL-3 and RT. IL-3 differentiates haematopoietic stem cells into myeloid progenitor cells and stimulates the proliferation of myeloid-derived cells such as DC and monocytes. In mouse models of fibrosarcoma and PCa, IL-3 was found to increase the tumour response to radiation. Combining adenoviral-IL-3 and radiation in the TRAMP-C1 mouse prostate model caused significant delays in tumour growth. Further reports indicated that adenoviral-IL-3 plus radiation enhanced IFNγ-producing CD4+ and CD8+ T cell responses in the spleen (Oh et al., 2004). This shifted the immune response to a Th1–type response from a suppressive Th2 type response (Tsai et al., 2006).

The combination of the pro-inflammatory cytokine TNFα and RT in mouse mammary carcinoma delayed tumour growth at a greater extent than either treatment alone (Nishiguchi et al., 1990). Similar synergistic effects have also been observed in mouse melanoma, lung adenocarinoma and brain tumours. The combined effects were attributed to increased recruitment and enhanced activation of lymphocytes and neutrophils (Gridley et al., 1996; Gridley et al., 2002; Gridley et al., 2000; Jin et al., 2005; Li et al., 1998).

Adoptive-cell-transfer (ACT) therapy is the passive transfer of tumour-specific T cells that have been expanded ex vivo. Local tumour irradiation can enhance the therapeutic efficacy of ACT therapy (Teitz-Tennenbaum et al., 2009). Combination of RT with ACT of carcinoembryonic antigen (CEA)-specific CD8+ T cells in a mouse colon carcinoma

Combination of Immunotherapy & Radiotherapy for the Treatment of Prostate Cancer 205

but the hazard ratio for death was 0.56 (95% CI 0.37 to 0.85) in the PSA-TRICOM arm and

The initial results of the phase II trial in combination with radiation were highly encouraging. Thirty patients were randomised in a 2:1 ratio to receive vaccine plus EBRT or EBRT alone. In this trial the vaccine consisted of a priming vaccine with rV-PSA plus rV-B7.1 followed by monthly booster vaccines with rFP-PSA. The immunological adjuvants used were GMCSF and high dose IL-2. PSA-specific T cell responses generated prior to RT were not adversely affected by RT, confirming our observation (Tabi et al., 2010). In total, 13 of the 17 patients in the combination arm had increases in PSA-specific T cells and epitope spreading to 4 other prostate cancer TAA (PSMA, PAP, PSCA and MUC-1) was noted (Gulley et al., 2005), possibly due to cross-presentation of a mix of TAA from dying tumour cells by DC (Obeid et al., 2007). There was some IL-2 toxicity, which was reduced in a singlearm follow-up study using lower doses but longer durations of IL-2; immunological effects

There is one other reported ongoing work of immunotherapy-radiotherapy combination with intraprostatic injections of autologous DC. The first report confirms the safety of this approach in 5 HLA-A2+ patients with high risk, localised disease, also treated with ADT, EBRT to 45 Gy and LDR brachytherapy. Autologous intraprostatic DC injections were given at four timepoints during EBRT. Measurable, induced increases in TAA-specific T cell frequencies in peripheral blood using ELISPOT were observed in some patients. The pattern of distribution of CD8+ cells in tissue was consistent with PCa TAA-targeting, rather than

There are no other published studies in PCa patients of EBRT in combination with cytokines, antibodies, immune modulators or immunologically relevant gene therapy. However, there are a few prostate cancer clinical trials combining RT and immunotherapy currently recruiting according to the ClinicalTrials.gov website, such as anti-CTLA-4 (Ipilimumab) antibody therapy in castration-resistant prostate cancer following RT (Phase III trial, Bristol-Myers Squibb; NCT00861614) and treatment with anti-OX40 and cyclophosphamide in combination with RT in metastatic prostate cancer (Providence Health

Clinical trial design, investigating the benefit of immunotherapy in addition to EBRT is challenging in PCa, as long-term tumour control outcome is already good in both localised (assuming dose escalated image-guided IMRT) and locally advanced disease (assuming combination of long term ADT and EBRT). Phase III trials, adequately powered to show a clinically relevant improvement, would need to address biochemical relapse-free survival or overall survival, both of which require prolonged follow-up of many hundreds and perhaps thousands of patients. Before such trials were undertaken, it is important to optimise the immunotherapy-RT schedules. Reliable biomarkers of treatment-efficacy are needed and this is difficult, especially if neoadjuvant or adjuvant ADT is used, as changes in PSAkinetics become redundant in these patients. Therefore, development of reliable immunological biomarkers is crucial. We believe that the presence of systemic TAA-specific T cell responses is likely to be the most reliable and easily detectable indicator of a sustainable immunological effect. It may also assist patient selection for optimised treatment

of those patients who are most likely to benefit from combination therapies.

the treatment was generally well tolerated.

were equivalent (Gulley et al., 2005; Lechleider et al., 2008).

non-specific organ infiltration (Finkelstein et al., 2011 ).

& Services, Oregon USA, Phase I/II trial; NCT01303705).

**5.3 Designs of combination clinical trials**

demonstrated increased tumour rejection, that could be attributed to the upregulation of the death receptor Fas on the surface of irradiated tumour cells (Chakraborty et al., 2003).

The anti-tumour effect can be further enhanced by intratumoural administration of DC. In a murine metastatic melanoma model reduction in the size of tumour and extent of spontaneous metastasis and prolonged survival were observed after irradiation and intratumoural DC administration. This was associated with an increase in proliferation, accumulation and cytokine production of CD4+ cells. Similar results were observed in DC plus irraditation in melanoma and sarcoma models (J. Huang et al., 2007). Kjaergaard et al. (2005) reported a method of fusing DC with tumour cells via an electric field resulting in a TAA-DC primed vaccine. These DC/tumour cell fusions induce a potent immune response in combination with local cranial RT in mouse glioma. Both CD4+ and CD8+ T cells infiltrate the tumours, leading to complete tumour regression. Tumour rejection was also observed after subsequent tumour challenge, indicating the presence of immunological memory.

Vaccines containing either modified tumour cells that are more immunogenic than the "native" tumour cells or TAA-vaccines have been tested extensively in preclinical models. Combination of cytokine-producing vaccines with local RT in mouse glioma demonstrated that IL-4 and GM-CSF vaccines alone were capable of curing 20-40% of mice but in combination with local RT 80-100% of the mice were cured. The brain tumours were heavily infiltrated with CD4+ T cells (Lumniczky et al., 2002). The increased anti-tumour effect of GM-CSF and RT was also demonstrated in mouse glioma using a vaccine which contained GM-CSF-secreting tumour cells (Newcomb et al., 2006).

Strategies using RT in combination with monoclonal antibodies that are specific for TAA are now commonly used in clinical oncology (reviewed by Drake, 2010). In a mouse lung cancer model, monoclonal antibody to OX40 (a secondary co-stimulatory molecule expressed on activated CD4+ and CD8+ T cells) and RT resulted in a synergistic effect on survival compared to either treatment alone (Yokouchi et al., 2008). The effect was CD8+ T cell dependent. Antibody-based immunotherapy strategies aiming to neutralise molecules implied in immune tolerance have also been examined. Antibodies for the cytotoxic T lymphocyte antigen (CTLA)-4 (a CD28-superfamily molecule causing T cell functional inhibition) have been shown to induce effective anti-tumour responses via lowering the threshold of tumour-specific T cell activation (reviewed by Drake, 2010). Based on the preclinical findings, CTLA-4 inhibition by the antibody Ipilimumab is now an FDA approved treatment of metastatic melanoma (Chambers et al., 2001). Its combination with RT is being tested in animal models (Dewan et al., 2009) and in clinical trials (see next section).

#### **5.2 Clinical trials**

There is huge potential for augmentation of the radiation response with the use of immunotherapy but as yet there are only a few clinical trials published. These were carried out in PCa patients at the National Cancer Institute, using a recombinant viral vaccine consisting of recombinant vaccinia virus (rV) encoding PSA, admixed with rV encoding the co-stimulatory molecule B7.1, followed by booster vaccinations with recombinant fowlpox (rFP) vector expressing PSA prior to RT. The product has been further developed and is presently marketed as Prostvac® which encodes ICAM-1 and LFA-3 in addition to B7.1 (PSA-TRICOM). This agent has been shown to improve median overall survival from 16.6 months to 25.1 months in a phase II multi-centre randomized controlled trial in 125 men with asymptomatic or minimally symptomatic metastatic castrate refractory prostate cancer (Kantoff et al., 2010). There was similar progression-free survival in the two arms of the trial,

demonstrated increased tumour rejection, that could be attributed to the upregulation of the death receptor Fas on the surface of irradiated tumour cells (Chakraborty et al., 2003). The anti-tumour effect can be further enhanced by intratumoural administration of DC. In a murine metastatic melanoma model reduction in the size of tumour and extent of spontaneous metastasis and prolonged survival were observed after irradiation and intratumoural DC administration. This was associated with an increase in proliferation, accumulation and cytokine production of CD4+ cells. Similar results were observed in DC plus irraditation in melanoma and sarcoma models (J. Huang et al., 2007). Kjaergaard et al. (2005) reported a method of fusing DC with tumour cells via an electric field resulting in a TAA-DC primed vaccine. These DC/tumour cell fusions induce a potent immune response in combination with local cranial RT in mouse glioma. Both CD4+ and CD8+ T cells infiltrate the tumours, leading to complete tumour regression. Tumour rejection was also observed after subsequent tumour challenge, indicating the presence of immunological memory. Vaccines containing either modified tumour cells that are more immunogenic than the "native" tumour cells or TAA-vaccines have been tested extensively in preclinical models. Combination of cytokine-producing vaccines with local RT in mouse glioma demonstrated that IL-4 and GM-CSF vaccines alone were capable of curing 20-40% of mice but in combination with local RT 80-100% of the mice were cured. The brain tumours were heavily infiltrated with CD4+ T cells (Lumniczky et al., 2002). The increased anti-tumour effect of GM-CSF and RT was also demonstrated in mouse glioma using a vaccine which contained

Strategies using RT in combination with monoclonal antibodies that are specific for TAA are now commonly used in clinical oncology (reviewed by Drake, 2010). In a mouse lung cancer model, monoclonal antibody to OX40 (a secondary co-stimulatory molecule expressed on activated CD4+ and CD8+ T cells) and RT resulted in a synergistic effect on survival compared to either treatment alone (Yokouchi et al., 2008). The effect was CD8+ T cell dependent. Antibody-based immunotherapy strategies aiming to neutralise molecules implied in immune tolerance have also been examined. Antibodies for the cytotoxic T lymphocyte antigen (CTLA)-4 (a CD28-superfamily molecule causing T cell functional inhibition) have been shown to induce effective anti-tumour responses via lowering the threshold of tumour-specific T cell activation (reviewed by Drake, 2010). Based on the preclinical findings, CTLA-4 inhibition by the antibody Ipilimumab is now an FDA approved treatment of metastatic melanoma (Chambers et al., 2001). Its combination with RT is being tested in animal models

There is huge potential for augmentation of the radiation response with the use of immunotherapy but as yet there are only a few clinical trials published. These were carried out in PCa patients at the National Cancer Institute, using a recombinant viral vaccine consisting of recombinant vaccinia virus (rV) encoding PSA, admixed with rV encoding the co-stimulatory molecule B7.1, followed by booster vaccinations with recombinant fowlpox (rFP) vector expressing PSA prior to RT. The product has been further developed and is presently marketed as Prostvac® which encodes ICAM-1 and LFA-3 in addition to B7.1 (PSA-TRICOM). This agent has been shown to improve median overall survival from 16.6 months to 25.1 months in a phase II multi-centre randomized controlled trial in 125 men with asymptomatic or minimally symptomatic metastatic castrate refractory prostate cancer (Kantoff et al., 2010). There was similar progression-free survival in the two arms of the trial,

GM-CSF-secreting tumour cells (Newcomb et al., 2006).

(Dewan et al., 2009) and in clinical trials (see next section).

**5.2 Clinical trials**

but the hazard ratio for death was 0.56 (95% CI 0.37 to 0.85) in the PSA-TRICOM arm and the treatment was generally well tolerated.

The initial results of the phase II trial in combination with radiation were highly encouraging. Thirty patients were randomised in a 2:1 ratio to receive vaccine plus EBRT or EBRT alone. In this trial the vaccine consisted of a priming vaccine with rV-PSA plus rV-B7.1 followed by monthly booster vaccines with rFP-PSA. The immunological adjuvants used were GMCSF and high dose IL-2. PSA-specific T cell responses generated prior to RT were not adversely affected by RT, confirming our observation (Tabi et al., 2010). In total, 13 of the 17 patients in the combination arm had increases in PSA-specific T cells and epitope spreading to 4 other prostate cancer TAA (PSMA, PAP, PSCA and MUC-1) was noted (Gulley et al., 2005), possibly due to cross-presentation of a mix of TAA from dying tumour cells by DC (Obeid et al., 2007). There was some IL-2 toxicity, which was reduced in a singlearm follow-up study using lower doses but longer durations of IL-2; immunological effects were equivalent (Gulley et al., 2005; Lechleider et al., 2008).

There is one other reported ongoing work of immunotherapy-radiotherapy combination with intraprostatic injections of autologous DC. The first report confirms the safety of this approach in 5 HLA-A2+ patients with high risk, localised disease, also treated with ADT, EBRT to 45 Gy and LDR brachytherapy. Autologous intraprostatic DC injections were given at four timepoints during EBRT. Measurable, induced increases in TAA-specific T cell frequencies in peripheral blood using ELISPOT were observed in some patients. The pattern of distribution of CD8+ cells in tissue was consistent with PCa TAA-targeting, rather than non-specific organ infiltration (Finkelstein et al., 2011 ).

There are no other published studies in PCa patients of EBRT in combination with cytokines, antibodies, immune modulators or immunologically relevant gene therapy. However, there are a few prostate cancer clinical trials combining RT and immunotherapy currently recruiting according to the ClinicalTrials.gov website, such as anti-CTLA-4 (Ipilimumab) antibody therapy in castration-resistant prostate cancer following RT (Phase III trial, Bristol-Myers Squibb; NCT00861614) and treatment with anti-OX40 and cyclophosphamide in combination with RT in metastatic prostate cancer (Providence Health & Services, Oregon USA, Phase I/II trial; NCT01303705).

#### **5.3 Designs of combination clinical trials**

Clinical trial design, investigating the benefit of immunotherapy in addition to EBRT is challenging in PCa, as long-term tumour control outcome is already good in both localised (assuming dose escalated image-guided IMRT) and locally advanced disease (assuming combination of long term ADT and EBRT). Phase III trials, adequately powered to show a clinically relevant improvement, would need to address biochemical relapse-free survival or overall survival, both of which require prolonged follow-up of many hundreds and perhaps thousands of patients. Before such trials were undertaken, it is important to optimise the immunotherapy-RT schedules. Reliable biomarkers of treatment-efficacy are needed and this is difficult, especially if neoadjuvant or adjuvant ADT is used, as changes in PSAkinetics become redundant in these patients. Therefore, development of reliable immunological biomarkers is crucial. We believe that the presence of systemic TAA-specific T cell responses is likely to be the most reliable and easily detectable indicator of a sustainable immunological effect. It may also assist patient selection for optimised treatment of those patients who are most likely to benefit from combination therapies.

Combination of Immunotherapy & Radiotherapy for the Treatment of Prostate Cancer 207

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Pfreundschuh, M. & Old, L. (1997). A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening. *Proc Natl Acad Sci USA*

apoptosis in normal human prostate epithelial cells and tumor cell lines. *Cancer Lett*

CD39 and CD73, which suppress T cells through adenosine production. *J Immunol*

S. (2009). Fractionated but not single-dose radiotherapy induces an immunemediated abscopal effect when combined with anti-CTLA-4 antibody. *Clin Cancer*

*Res* 166, 6, (Dec), 819-831.

1, (Jan), 29-44.

2488-2496.

565-571.

in vitro. *Int J Rad Biol* 87, 1, 71-80.

immunotherapy. *Ann Rev Immunol* 19, 565-594.

6801, (Sep 14), 249-257.

94,5,1914-1918.

6, 324-330.

203, 2, (Jan 20), 145-154.

187, 2, (July 15) 676-683.

*Res* 15, 17, 5379-5388.

The successful result of the IMPACT trial showing the survival advantage with Sipuleucel-T is expected to lead to a dramatic increase in the use of systemic immunotherapy for prostate cancer (Sonpavde et al., 2011). Presently there are only production facilities within the USA and the cost is likely to remain prohibitive for many healthcare systems. Radiation has great potential to augment the effect of systemic immunotherapy and we can expect many combination trials in the future. Clinical trial design in this setting will remain challenging as immunotherapy appears to improve overall survival but demonstrable tumour or biochemical responses are often delayed. Augmenting immunotherapy with radiation may improve overall advantage further.


4.1. Degradation of PCBs by Pseudomonas aeruginosa

Samples of wastewater from the Notwane Sewage Treatment Plant were used in this study to find out the degree of biodegradation of PCBs in wastewater un-inoculated and inoculated with the test organism. Spectral changes (a shift in wavelength (λmax) in nm), detected using the UVvisible spectrophotometer, were used as an indication that the compounds were broken down into new products. The results of the wavemax (λmax) nm obtained are presented in Figure 2.

The results shown by chromatogram indicated that there was a shift in λmax from 224 to 270 nm in 0 h of incubation to 96 h of incubation at 30C on a rotary shaker in the dark. These results were obtained using a Cary 300 UV-visible spectrophotometer at a wavelength range of 200–800 nm. The results were an indication that isolates Pseudomonas aeruginosa was able to degrade Aroclor 1260 into chlorobenzoates and derivatives, which have wavelength ranging from 244 to 270 nm, hence the shift in wavelength.

Figure 2. Spectral changes of PCB degradation in water inoculated with isolate Pseudomonas aeruginosa.

PCB standard presented in Figure 3 showed that there were several compounds in the Aroclor; thus, the many picks, which are not found when the same treatment was extended to the

The Role of Bacteria on the Breakdown of Recalcitrant Polychlorinated Biphenyls (PCBs) Compounds in Wastewater

http://dx.doi.org/10.5772/intechopen.75400

147

The chromatogram shows that the components of the standard had retention times ranging from 3.647 to 13.119 min. The components picked by the instrument are indicated by the red line marking the beginning and ending of the peak. The instrument could not detect the actual

The wastewater was sterilized and thereafter inoculated with the test organism and PCBs mixture added. Figure 4 shows that no peaks were picked by the HPLC. This is an indication that the polychlorinated biphenyls added to the wastewater were completely broken down by

Representation of Figure 4 is a clear indication that there were no detectable amounts of compounds after Aroclor was subjected to bacterial treatment. The compounds were broken down in 5 days. There are no peaks shown as compared to the chromatogram shown in Figure 3.

The wavelength maximum (λmax) observed after 96 h ranged from 264 to 269 nm on average for PCBs (Figure 2). These results indicated that the PCBs were broken down forming new products with different wavelength and thus the change. This implies that the bacteria were able to use them as their sole source of carbon; thus, the biodegradation of the PCBs added to wastewater. The shifts are an indication of the presence of initial ring oxidation metabolites and ring fission metabolites [19, 47]. PCBs first get degraded into chlorobenzoates [47] that have been found to have λmax ranging from 210 to 214 nm in the B-band and 244 to 270 nm in the C-band when dissolved in water [48], a range that was observed in the results obtained after 24 h of culturing the organism used in this study in PCBs Aroclor mixture, results of which are shown in Figure 2. The results depicted that the bacteria was able to breakdown the PCBs, which were similar to results in a similar studies by Vrchotova et al. and Seeger et al. [47, 49]. In their studies [47, 49], the product chlorobenzoate was biodegraded into benzoate and eventually pyruvate and

The HPLC run confirmed that the compound was biodegraded by the bacteria isolate Pseudomonas aeruginosa as presented in Figures 3 and 4, which was also proved by Heider and Rabus, Roy et al. and Raja et al. [38, 46, 50] in their studies. No PCB compound was detected after 96 h

Pseudomonas aeruginosa, isolated from wastewater in the Notwane Sewage Treatment Plant was successfully used in biodegradation of recalcitrant polychlorinated biphenyls (PCBs). This having been successfully employed at the micro level, and further tests can be carried out to

isolate Pseudomonas aeruginosa resulting in complete elimination.

acetyladehyde, which are essential in the tricarboxylic cycle (TCA) [47, 49].

of exposure to the bacteria in wastewater.

experiment, resulted in Figure 4.

names of the compounds though.

5. Discussion

6. Conclusion

Figure 3. HPLC chromatogram for PCBs standard using a florescence detector method at 10 μl injection volume.

#### 4.2. Results from high performance liquid chromatography (HPLC)

HPLC chromatogram depicted differences in picks obtained for the experiments, the controls, and the standards. Although concentrations were not determined, no PCBs were detected by HPLC after 96 h of incubation. This was an indication that the bacteria degraded the compounds, hence the chromatogram shown in Figure 4. The chromatogram obtained for the experiments was compared with the chromatogram for the PCB standard using the retention times for the compounds in the standard, which was represented in Figure 3. The results for

Figure 4. Chromatogram for PCB biodegradation experiment after 96 h of incubation.

PCB standard presented in Figure 3 showed that there were several compounds in the Aroclor; thus, the many picks, which are not found when the same treatment was extended to the experiment, resulted in Figure 4.

The chromatogram shows that the components of the standard had retention times ranging from 3.647 to 13.119 min. The components picked by the instrument are indicated by the red line marking the beginning and ending of the peak. The instrument could not detect the actual names of the compounds though.

The wastewater was sterilized and thereafter inoculated with the test organism and PCBs mixture added. Figure 4 shows that no peaks were picked by the HPLC. This is an indication that the polychlorinated biphenyls added to the wastewater were completely broken down by isolate Pseudomonas aeruginosa resulting in complete elimination.

Representation of Figure 4 is a clear indication that there were no detectable amounts of compounds after Aroclor was subjected to bacterial treatment. The compounds were broken down in 5 days. There are no peaks shown as compared to the chromatogram shown in Figure 3.


**1. Introduction**

Multi-agent path planning (MAPP) involves finding the set of least-cost paths for a set of agents co-existing in a given *graph* such that each of the agents is free from collision, where a collision is defined as at least two agents moving to the same location at the same time. MAPP attracts increasing attention due to its practical applications in multi-robot systems for surveillance automation, video gaming, traffic control, and many other domains [1–4]. This problem is, however, difficult to solve because the configuration space grows exponentially with the number of agents in the system, incurring extremely heavy computational efforts. It is an NP-hard problem to find optimal solutions for MAPP in its general form [5].

Approaches to solving MAPP problems fold into three main categories: *coupled, decoupled* and *intermediate* [6]. *Coupled* approaches search the *joint configuration space* of the multi-agent system, which is the *Tensor product* of the free configuration spaces of all the individual agents. A popular coupled planner is the A\* algorithm [7] that directly searches the whole joint configuration space, making such an approach computationally infeasible when the number of agents is large. Enhanced variants of A\*, such as operator decomposition (OD), enhanced partial expansion A\* (EPEA\*), and iterative deepening A\* (IDA\*), can—to some extent—mitigate the exponential growth in the number of neighbors by improving the admissible heuristics [8–11]. Coupled approaches are optimal and complete, but usually at high computational cost. *Decoupled* approaches plan for each agent separately and then adjust the path to avoid collisions. Algorithms in this category are generally faster because they perform a graph search and collision-avoidance adjustment in low-dimensional spaces. However, optimality and completeness are not guaranteed [3,12].

*Intermediate* approaches lie between coupled and decoupled ones because they dynamically couple agents and grow the search space during the planning. In this way, the search space is initially small and grows when necessary. A few intermediate MAPP algorithms can guarantee optimality and completeness. State-of-the-art examples include Conflict-Based Search (CBS) [6,13]. CBS is a two-level algorithm. At the high level, conflicts are added into a *conflict tree* (CT). At the low level, solutions consistent with the constraints given by the CT are found and updated to agents. CBS behaves poorly when a set of agents is strongly coupled. Meta-agent CBS (MA-CBS) is then proposed by merging strongly coupled agents into a meta-agent to handle the strongly coupled scenarios.

The M\* algorithm is a state-of-the-art coupled approach. It starts with decoupled planning and applies a strategy called *sub-dimensional expansion* to dynamically increase the dimensionality of the search space in regions in which agent collisions occur. In this way, an efficient graph search with a strict collision-free constraint can be achieved, while minimizing the explored portion of the joint configuration space. M\* identifies which subsets of agents can be safely decoupled and hence plans for multi-agents in a lower-dimensional space. Compared to CBS and its variant MA-CBS, M\* and its variants, e.g., recursive M\* (rM\*), have much more fine-grained control over some technical details, such as the management of conflict sets for better scalability. The fine-grained nature of M\* allows it to be integrated into MA-CBS to take advantage of both [14]. Recent work extended both M\* and CBS algorithms to handle the imperfect path execution due to unmodeled environments and delays [15,16].

Most fundamental MAPP approaches assume *hard collisions*, which means that solutions in which agents share resources (nodes or edges) are rejected. In many real world scenarios, some degree of resource sharing between agents is acceptable, so the hard-collision constraint needlessly over-constrains the solution space. This paper relaxes the hard collisions constraint by allowing some sharing of resources, including space and various services on edges/nodes, by agents. Such sharing reduces the quality of the path, i.e., the satisfaction level of the agent using it, but as long as the quality reduction for each path is below a settable threshold, the solution is acceptable. We call this concept *soft collisions*. Hard collisions are still supported by having a very strict threshold, i.e., a penalty for sharing is very high. The reduction in satisfaction level experienced by an agent caused by soft collisions on resources in its path is quantified using a *collision score*. In this paper, we develop a generalized version of the M\* algorithm, called *soft-collision M\** (SC-M\*), for solving the MAPP problem in the soft-collision context. Note that we that we are not simply replacing hard with soft collisions, but instead introducing soft collisions as a generalization that allows modeling different types of collisions.

SC-M\* extends M\* by taking the perspective of soft collision on common resources. Specifically, SC-M\* tracks the collision score of each agent and places agents whose collision scores exceed certain thresholds into a *soft-collision set* for *sub-dimensional expansion*, a technique that limits the search space

while maintaining the optimality of the algorithm with respect to the objective. In this way, SC-M\* achieves improved scalability to handle a larger number of agents while limiting the probability of collisions on resources to a bound.

In this paper, we show that SC-M\* has advanced flexibility and scalability for efficiently solving the MAPP problem in the soft-collision context where common resources are considered, and can handle complex environments (e.g., with multiple types of agents requesting multiple types of resources). We theoretically prove that SC-M\* is *complete* and *suboptimal* under the soft-collision constraints on resources. Experimental results demonstrate the advantages/trade-offs of SC-M\* in terms of path cost, success rate and run time against baseline SC-based MAPP planners, such as SC-A\* and SC-CBS.

The rest of the paper is organized as follows. Section 2 discusses the motivation of soft collisions. Section 3 gives technical briefing of the M\* algorithm. Section 4 presents our proposed SC-M\* approach. Section 5 evaluates SC-M\* in a grid public transit network. Finally, Section 6 concludes our work.


**4. Data characteristics**

**Source Technique Factors Suitability**

texture, moisture, organic C,

Residential, extractive industry,

NDVI, LUC, climate data set,

Distance from river, LUC,

crop pattern, distance from residential areas, roads, proximity to interested

EC, electrical conductivity; GDD, growing degree days; NDVI, normalized difference vegetation index.

According to **Table 1**, even if the suitability target is same, there might be small differences between factors. Some of the important essential factors are the same in agricultural suitability studies, such as soil depth, slope, and soil texture. However, there are different factors: erosion rate, soil nutrients, and crop productivity. Data accessibility, database availability, and method can be affected factors despite the same purpose. Also, these factors may be changed with respect to regional differences. If there is flood risk in an area, we have to assess flood risk rate

Van City is located in eastern Turkey, and it is the most crowded city of the Eastern Anatolian Region of Turkey (**Figure 2**). Van Province's population was 1,096,397 in 2015, and almost 472,000 of them lived in Van central city area. Population has increased almost 5.5% during the last 5 years [23]. The main incomes in the region are animal production, agriculture, border trading, and tourism. Mainly continental climate is dominant, but alpine and sub-alpine climate effects are observed in high regions. Van central city's area climate is warmer than in those around it because of Van Lake. This lake is the biggest lake in Turkey and its water

Slope, soil depth,

pH, EC, primary nutrients (N, P, K)

marine industry, recreation subfactors

DEM, economic and

social data

urban areas,

areas

**Table 1.** Sample techniques and factors used in land-use suitability studies.

[37] RS integrated GIS, expertbased weighting using AHP

210 Sustainable Urbanization

[30] GIS-MCE-

[38] RS integrated

AHP

[39] Fuzzy logic, AHP-GIS-WLC

fuzzy AHP

GIS-based MCE,

in land-use suitability analyses.

**3. Study area**

**field**

Urban land suitability

Eco-city evaluation

Waste water disposal area suitability

Agriculture 2015

**Year**

2015

2015

2016

The research data set contains (i) satellite images and LUC data, (ii) digital elevation model (DEM) data, (iii) LUA data, (iv) distance from road and urban built-up area data, and (v) restrictive areas or constrained areas (**Figure 3**).

#### **4.1. Satellite images**

Landsat 5 TM and Landsat 8 OLI data sets were used to detect urban area change between 2002 and 2015 in Van central city area. Landsat imageries have a great potential for monitoring the land-use/cover change because of large time series database and available spatial (30 m) and spectral (VIS, NIR, SWIR, and TIR) resolutions [24]. Two Landsat images were used for LUC and change detection. An earlier image was taken on August 2002, and the later one was recorded on August 2015.

**Figure 3.** Input data set (criteria or factors) used in the urban sprawl suitability of Van City.

#### **4.2. DEM data**

Elevation data were obtained from ASTER Global DEM data set in 30 m spatial resolution. Slope and hillshade images were produced from DEM data in GIS interface. Additionally, DEM data were used as ancillary data in LUC classification stage to detect the LUC change based on elevation. Also, such data sets may be improved for the accuracy of the LUC classi‐ fication [2].

#### **4.3. LUA data**

The LUA of Turkey was mapped by Soil Survey Staff of Turkey. This map shows capable lands and capability degrees in eight categories (1 refers to very capable and 8 refers to not capable) based on slope, soil depth, soil type, geological type, etc. Highly available lands can be used for agriculture, urban built-up, and industrial facilities. However, land-use suitabili‐ ty is variable according to the scenarios. For example, agricultural areas are important be‐ cause available lands are limited so, in ecological or sustainable scenarios, highly available lands must be protected for agriculture.

### **4.4. Distance images**

Distance from roads and urban built-up areas are produced from road maps and urban area maps, which are produced from classified LUC map. Road and settlement distance is signif‐ icant to evaluate the infrastructure and superstructure availability of a land for urban sprawl.

#### **4.5. Restrictive areas**

Urban cannot sprawl to regions, such as water bodies, existing built-up areas, security areas, and historical protection areas. Also, restrictive areas are modified according to the scenarios. Some wetlands are closed for urban sprawl in the ecological scenario.


**Matrix Restructuring During Liver Regeneration is Regulated by Glycosylation of the Matrix Glycoprotein Vitronectin**

Haruko Ogawa1, Kotone Sano2,

Naomi Sobukawa1 and Kimie Asanuma-Date1 *1Graduate School of Advanced Sciences and Humanities, and Glycoscience Institute, Ochanomizu University, 2Faculty of World Heritage, Department of Liberal Arts, Cyber University, Tokyo, Japan*

#### **1. Introduction**

78 Liver Regeneration

York, J.D. & Majerus, P.W. (1994). *Nuclear phosphatidylinositols decrease during S-phase of the*

There are three major approaches for regenerative medicine. The most innovative approach among them is: induction of target cells from various stem cells such as induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells) and implantation of them to regenerate the organ. The second approach is: in vitro tissue regeneration that involves preparation of artificial tissue by combining human cells with scaffolding biomaterials and growth factors. The third is: promotion of self-regeneration through controlling the repair activity of each tissue, which most organisms do naturally, is a more fundamental approach, but it will also be important in cell therapy to regulate tissue organization after induction of differentiation.

Because tissue homeostasis depends on spatially and temporally controlled expression of multifunctional adhesive glycoproteins and receptors, many studies have examined the changes of expression of extracellular matrix (ECM) molecules during tissue remodeling, inflammation and invasion by cancer cells (DeClerck, Y.A., et al. 2004; Seiffert, D. 1997; Kato, S., et al. 1992; Hughes, R.C. 1997) on the one hand. On the other hand, there is increasing evidence that glycosylations post-translationally modulate various biological phenomena by altering the activity and specificity or the stability of glycoproteins through the biosignaling functions of oligosaccharides (Varki, A. 1993; Varki, A., et al., 2009). During tissue remodeling, the glycosylated ECM molecules are different from those of normal tissue owing to the changes in the expression of many proteins that are responsible for glycan synthesis (Dalziel, M., et al. 1999). However, the glycan modulation of most glycoproteins that are involved in tissue remodeling has remained unknown.

When the three big lobes of a liver are excised, the remaining liver recovers its former mass and function within about two weeks in humans or 7 to 10 days in rats (Diehl, A.M. and Rai, R.M. 1996). ECM degradation occurs in the early stage of this process, followed by biosynthesis of the matrix, cell proliferation, and cell differentiation. During this process, many glycosyl transferases (Bauer, C.H., et al. 1976; Serafini-Cessi, F. 1977; Okamoto, Y., et

Matrix Restructuring During Liver Regeneration

to play a central role in matrix remodeling.

K., et al. 2010).

is Regulated by Glycosylation of the Matrix Glycoprotein Vitronectin 81

The ligand-binding sites on the domain structure of vitronectins are shown in Fig. 1. Vitronectin regulates the proteolytic degradation of matrix and fibrinolysis through binding with urokinase-type plasminogen activator, PAI-1, and urokinase receptor (Seiffert, D. 1997; Schvartz, I., et al. 1999; Preissner, K.T. 1991; Pretzlaff, R.K., et al. 2000). Besides this function, vitronectin plays a key role in cell adhesion and cellular motility during tissue remodeling through binding to major ECM receptors, integrins such as αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and other ECM components like collagen and proteoglycans (Schvartz, I., et al. 1999). In plasma, vitronectin has been shown to regulate coagulation and thrombolytic and complement systems (Preissner, K.T. 1991). By providing a link between plasmin-regulated matrix proteolysis and integrin-mediated cell migration, activated vitronectin is considered

Fig. 1 also shows the glycosylation structures of vitronectins and ligand-binding sites. *N*glycosylation sites of vitronectins are well conserved among mammals, and vitronectin contains almost fully sialylated complex-type *N*-linked glycans at these sites. Most vitronectins, except normal human, additionally contain various amounts of *O*-linked glycans, which differ by species: bovine, rabbit, and especially chicken vitronectins possess more *O*-glycans than *N*-glycans. Human vitronectin does not contain *O*-glycans in the normal state, while rat vitronectin contain several *O*-glycans in the connecting region (Sano,

Fig. 1. Glycosylation and domain structure models of vitronectins. Human, rat/mouse, and porcine vitronectin contain three, four, and two *N*-glycosylation sites, respectively (Ogawa, H., Yoneda, A., et al. 1995) (Yoneda, A., Ogawa, H., et al. 1993) (Sano, K., Miyamoto, Y., et al. 2010). The numbers of *O*-glycosylation sites differ by animal species. The major ligandbinding sites that have been identified are indicated at the top of the figure (Yoneda, A., Ogawa, H., et al. 1998) (Preissner, K.T. 1991) (Preissner, K.T. and Seiffert, D. 1998).

al. 1978; Ip, C. 1979; Oda-Tamai, S., et al. 1985; Miyoshi, E., et al. 1995) and total glycoconjugates in the liver have been reported to change (Okamoto, Y. and Akamatsu, N. 1977; Kato, S. and Akamatsu, N. 1984; Kato, S. and Akamatsu, N. 1985; Ishii, I., et al. 1985). However, the nature of the links between such glycans changes, and the process of tissue remodeling has remained unclear. We consider it important to identify which molecules play important roles in the tissue remodeling during liver regeneration, and we will discuss the glycan modulation of one extracellular molecule, vitronectin, in this chapter. Vitronectin is a multifunctional adhesive glycoprotein that plays a central role in tissue remodeling by connecting pericellular tissue lysis with cell adhesion and motility.

We found that the glycans of vitronectin drastically change during liver regeneration after partial hepatectomy. In our studies to determine the glycan structures during the initial stage of the liver regeneration after partial hepatectomy of rats, we found that alterations in glycosylation, especially decreased sialylation of vitronectin, modulate the biological activities of vitronectin during tissue-remodeling processes by multiple steps (Uchibori-Iwaki, H., et al. 2000; Sano, K., et al. 2010). Liver regeneration is a normal repair process, while fibrosis and cirrhosis are considered to be excessive and abnormal repair processes that often give rise to cancer. In this context, elucidating how alterations of glycans occur and understanding how glycans modulate the glycans on vitronectin is useful in order to develop a strategy to regulate matrix remodeling in regeneration and deposition in liver cirrhosis. Therefore, we aimed to elucidate glycan modulation during liver regeneration after partial hepatectomy. We focused on the changes in vitronectin during liver regeneration, especially the changes of the glycan moiety, which plays a crucial role in controlling survival of hepatic stellate cells.

#### **1.1 Structure and function of vitronectin**

Vitronectin is a multifunctional adhesive glycoprotein that originates mainly in hepatocytes and circulates in the blood at high concentrations (0.2 mg/ml in human and no more than 0.1 mg/ml in rats). Vitronectin was first isolated from human serum by Holmes in 1967 as an 'α-1 protein' (Holmes, R. 1967), and has been referred to as 'serum spreading factor', 'epibolin', or 'S-protein'. It induces cell growth in vitro and is known as a major celladhesive component in cell culture mediums (Hayman, E.G., et al. 1983). Vitronectin is present as an ECM component in the liver, as well as various other organs, including skeletal muscle, kidney, and brain (Seiffert, D. 1997; Seiffert, D., et al. 1991). Most vitronectin in normal plasma is present as an inactive monomer form that does not bind to various ligands in the plasma (Gebb, C., et al. 1986; Izumi, M., et al. 1989). *In vivo*, vitronectin is activated in the presence of certain ligands such as heparin, type-1 plasminogen activator inhibitor (PAI-1), thrombin-AT-III, membrane attack complex of complements, and through a partial conformational change and multimerization process (Preissner, K.T. and Muller-Berghaus, G. 1987). Tissue vitronectin is considered to be present as an active multimeric form. Conformation-dependent binding of vitronectin was also observed for sulfatide (Gal(3-SO4)β1-1ceramide), cholesterol 3-sulfate, and various phospholipids including phosphatidylserine, but not gangliosides, while vitronectin bound to cholesterol 3-sulfate regardless of its conformational state (Yoneda, A., et al. 1998). The binding of vitronectin to the membrane lipids and β-endorphin-binding activities were found to be attributable to hemopexin domain 2 and hemopexin domain 1, as well as type I collagen and heparin (Yoneda, A., et al. 1998).

al. 1978; Ip, C. 1979; Oda-Tamai, S., et al. 1985; Miyoshi, E., et al. 1995) and total glycoconjugates in the liver have been reported to change (Okamoto, Y. and Akamatsu, N. 1977; Kato, S. and Akamatsu, N. 1984; Kato, S. and Akamatsu, N. 1985; Ishii, I., et al. 1985). However, the nature of the links between such glycans changes, and the process of tissue remodeling has remained unclear. We consider it important to identify which molecules play important roles in the tissue remodeling during liver regeneration, and we will discuss the glycan modulation of one extracellular molecule, vitronectin, in this chapter. Vitronectin is a multifunctional adhesive glycoprotein that plays a central role in tissue remodeling by

We found that the glycans of vitronectin drastically change during liver regeneration after partial hepatectomy. In our studies to determine the glycan structures during the initial stage of the liver regeneration after partial hepatectomy of rats, we found that alterations in glycosylation, especially decreased sialylation of vitronectin, modulate the biological activities of vitronectin during tissue-remodeling processes by multiple steps (Uchibori-Iwaki, H., et al. 2000; Sano, K., et al. 2010). Liver regeneration is a normal repair process, while fibrosis and cirrhosis are considered to be excessive and abnormal repair processes that often give rise to cancer. In this context, elucidating how alterations of glycans occur and understanding how glycans modulate the glycans on vitronectin is useful in order to develop a strategy to regulate matrix remodeling in regeneration and deposition in liver cirrhosis. Therefore, we aimed to elucidate glycan modulation during liver regeneration after partial hepatectomy. We focused on the changes in vitronectin during liver regeneration, especially the changes of the glycan moiety, which plays a crucial role in

Vitronectin is a multifunctional adhesive glycoprotein that originates mainly in hepatocytes and circulates in the blood at high concentrations (0.2 mg/ml in human and no more than 0.1 mg/ml in rats). Vitronectin was first isolated from human serum by Holmes in 1967 as an 'α-1 protein' (Holmes, R. 1967), and has been referred to as 'serum spreading factor', 'epibolin', or 'S-protein'. It induces cell growth in vitro and is known as a major celladhesive component in cell culture mediums (Hayman, E.G., et al. 1983). Vitronectin is present as an ECM component in the liver, as well as various other organs, including skeletal muscle, kidney, and brain (Seiffert, D. 1997; Seiffert, D., et al. 1991). Most vitronectin in normal plasma is present as an inactive monomer form that does not bind to various ligands in the plasma (Gebb, C., et al. 1986; Izumi, M., et al. 1989). *In vivo*, vitronectin is activated in the presence of certain ligands such as heparin, type-1 plasminogen activator inhibitor (PAI-1), thrombin-AT-III, membrane attack complex of complements, and through a partial conformational change and multimerization process (Preissner, K.T. and Muller-Berghaus, G. 1987). Tissue vitronectin is considered to be present as an active multimeric form. Conformation-dependent binding of vitronectin was also observed for sulfatide (Gal(3-SO4)β1-1ceramide), cholesterol 3-sulfate, and various phospholipids including phosphatidylserine, but not gangliosides, while vitronectin bound to cholesterol 3-sulfate regardless of its conformational state (Yoneda, A., et al. 1998). The binding of vitronectin to the membrane lipids and β-endorphin-binding activities were found to be attributable to hemopexin domain 2 and hemopexin domain 1, as well as type I collagen and heparin

connecting pericellular tissue lysis with cell adhesion and motility.

controlling survival of hepatic stellate cells.

**1.1 Structure and function of vitronectin**

(Yoneda, A., et al. 1998).

The ligand-binding sites on the domain structure of vitronectins are shown in Fig. 1. Vitronectin regulates the proteolytic degradation of matrix and fibrinolysis through binding with urokinase-type plasminogen activator, PAI-1, and urokinase receptor (Seiffert, D. 1997; Schvartz, I., et al. 1999; Preissner, K.T. 1991; Pretzlaff, R.K., et al. 2000). Besides this function, vitronectin plays a key role in cell adhesion and cellular motility during tissue remodeling through binding to major ECM receptors, integrins such as αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and other ECM components like collagen and proteoglycans (Schvartz, I., et al. 1999). In plasma, vitronectin has been shown to regulate coagulation and thrombolytic and complement systems (Preissner, K.T. 1991). By providing a link between plasmin-regulated matrix proteolysis and integrin-mediated cell migration, activated vitronectin is considered to play a central role in matrix remodeling.

Fig. 1 also shows the glycosylation structures of vitronectins and ligand-binding sites. *N*glycosylation sites of vitronectins are well conserved among mammals, and vitronectin contains almost fully sialylated complex-type *N*-linked glycans at these sites. Most vitronectins, except normal human, additionally contain various amounts of *O*-linked glycans, which differ by species: bovine, rabbit, and especially chicken vitronectins possess more *O*-glycans than *N*-glycans. Human vitronectin does not contain *O*-glycans in the normal state, while rat vitronectin contain several *O*-glycans in the connecting region (Sano, K., et al. 2010).

Fig. 1. Glycosylation and domain structure models of vitronectins. Human, rat/mouse, and porcine vitronectin contain three, four, and two *N*-glycosylation sites, respectively (Ogawa, H., Yoneda, A., et al. 1995) (Yoneda, A., Ogawa, H., et al. 1993) (Sano, K., Miyamoto, Y., et al. 2010). The numbers of *O*-glycosylation sites differ by animal species. The major ligandbinding sites that have been identified are indicated at the top of the figure (Yoneda, A., Ogawa, H., et al. 1998) (Preissner, K.T. 1991) (Preissner, K.T. and Seiffert, D. 1998).

Matrix Restructuring During Liver Regeneration

value on the BSA-immobilized reference cell.

**early stage of liver regeneration**

is Regulated by Glycosylation of the Matrix Glycoprotein Vitronectin 83

Fig. 2. Changes in electrophoretic mobility and collagen-binding of rat vitronectin at 24 h after partial hepatectomy. (A) SDS-PAGE of vitronectins from non-operated rats (N), partially hepatectomized (PH) rats at 6-240h after operation, and sham-operated (SH) rats at 24 h after operation. (B) Type I collagen (1 μg/100 μL) was coated onto wells of microtiter plates. After blocking with 5% BSA, various concentrations of purified vitronectins were added to each well. The bound vitronectin was measured using HRP-conjugated rabbit antihuman vitronectin IgGs and ELISA. The absorbance of collagen-bound vitronectin was corrected for the antibody reactivity of each vitronectin. (C) Collagen was immobilized on a CM5 sensor chip, and each vitronectin in PBS was injected onto the sensor chip at a flow rate of 20 μL/min at 20°C. The change of resonance units (RU) was corrected by subtracting the

**2.1.2 Changes in glycosylation and carbohydrate concentration of vitronectin during**

As shown in Fig. 3, the carbohydrate analyses of the three vitronectins indicated that total carbohydrate contents of PH-VN and SH-VN decreased to one-third and one-half of that of NO-VN, respectively, and that a remarkable decrease in sialic acids and amounts of glycans occurred due to partial hepatectomy. The lectin reactivity of the three vitronectins indicated that these vitronectins contain complex-type *N*-linked oligosaccharides. The reactivity toward *Phaseolus vulgaris* lectin L4 (L-PHA) varied remarkably among vitronectins, and PH-VN showed marked reactivity with L-PHA, but SH- and NO-VNs reacted only slightly, suggesting that tri- or tetraantennary lactosamine-branched structures multiplied dramatically after partial hepatectomy. The specificity of PVL toward clustered sialyl residues (Ueda, H., Kojima, K., et al. 1999) (Ueda, H., Matsumoto, H., et al. 2002), the


**5. Nano-Antioxidants**

Antioxidants have been accorded as effective therapeutic and prophylactic agents for various diseases. However, these antioxidants have received very limited success until now, as most of the antioxidants show low permeability, and are poorly soluble in water, demonstrate instability during storage and gastrointestinal degradation, which are some of their limitations [61]. The amalgamation of material sciences with nanotechnology has substantially improved and reduced the free radical synthesis during nanoparticle production in different areas and the nanoparticles synthesized for this purpose are regarded as nano-antioxidants [62,63]. Carbon nanotubes, metal and metal oxide nanoparticles and various types of polymer-loaded antioxidant nanoparticles, have been reported to exhibit antioxidant properties [63]. In the past few decades, various preparation protocols, such as emulsion/solvent evaporation, supercritical fluid technology, solvent displacement method, templating method and nanoprecipitation techniques, have been used for synthesizing nano-oxidants [63]. Some oxide nanoparticles can scavenge the reactive nitrogen and reactive oxygen species (RNS/ROS) and mimic the antioxidant molecule, due to their intrinsic physicochemical properties [64]. In the biomedical field, cerium oxide nanoparticles (CONPs) have gathered special attention for their multi-enzymatic scavenging of ROS and their regenerative abilities [65]. These CONPs have unique properties, like the coexistence in both oxidation states i.e., Ce3+ and Ce4+, the ability to reversibly switch between both oxidation states and the reduction potential of ~1.52 V [66]. Cerium dioxide as a bulk crystal primarily contains Ce4+, but during its reduction to nano-size, substantially enhances the relative amount of Ce3+, therefore, leading to higher catalytic activity, in contrast to various biological processes and biological antioxidants [67,68]. Hirst et al. (2013) conducted an in vivo test on mice to assess the antioxidant potential of nanoceria, which were injected intravenously in the subject, and the result of the study revealed that nanoceria significantly decreased the lipoperoxidation after the three weeks, which indicates that CONPs are effective in treating oxidative stress [69]. Caputo et al. (2015) conducted a comparative study to assess the antioxidant potential between CONPs and NAC (N-acetyl-cysteine) and Trolox (soluble analogues of vitamin E) [70]. The results of this study revealed that NAC and Trolox reduced the oxidative 2- -7- -Dichlorofluorescein (DCF) signal triggered by irradiated TiO2 nanoparticles, but the antioxidant potential was significantly lower in comparison to CONPs. This result also highlights the stability of CONPs because of their auto-regenerative redox cycle, which allows them to surpass the challenges related to the stability of the antioxidants molecules.

On the other hand, synthetic polymeric NPs have emerged as the promising nano-drug delivery system, as they can encapsulate the therapeutic agent and progressively release the therapeutic compound at the target site. Poly-D, L-lactide (PLA) and poly (lactic-co-glycolic acid) (PLGA) are some examples of synthetic biodegradable polymers that have been approved safe by the European Medicine Agency (EMA) and U.S. Food and Drug Administration (FDA) for administration (Table 2).

Liposomes are also used for delivering the antioxidant agents to the target site. The amphiphilic and biocompatible nature of these liposomes allows them to load both hydrophilic and lipophilic compounds and favor the encapsulation of the water-soluble and water-insoluble antioxidant enzymes [79].

Furthermore, chitosan is the material predominantly used for synthesizing nanoparticles as a sole material or in amalgamation with another [80]. Chitosan shows mucoadhesive properties, which improves the targeted delivery in mucosal surfaces such as intestinal and nasal epithelium [81]. Curcumin encapsulated in nanocarrier and covered and stabilized with chitosan has also been developed and evaluated for free radical scavenging in comparison with free curcumin, and showed the protective effect of chitosan on the antioxidant activity of curcumin [82]. Pu et al. (2014) reported the encapsulation of curcumin antioxidant compounds within the nanocarrier and regulation of release of antioxidant compounds, by changing the pH and oxidative stress of inflamed tissues to increase the overproduction of RNS/ROS synthesized by lipopolysaccharide (LPS)-stimulated macrophage [83].


**1. Introduction**

This is an opportune time for Dr. Duncan's IntechOpen book on "Enhanced" Liposuction. Liposuction and body contouring is the world #1 surgical procedure. For the modern Liposuctions surgeon, the simple removal of fat has been replaced with adipose contouring in concert with the soft tissue contraction and skin remodeling to deliver body shaping enhancement with an optimized skin

envelope. We might even call this book "Enhanced Lipo-contraction", as many times, in smaller areas we are not aspirating fat. Our current Lipo-contouring patients include older patients with laxity, younger patient with atrophic striatum and encompasses soft tissue contouring and remodeling of the periocular, perioral, jawline, neck and all body areas, including formally "too risky" zones such as the lax upper arm, braline, inner thighs and post weight loss abdomens and mommy tummy's. In the current body contouring market, the modern liposuction surgeon must invest in energy based internal devices and technology that offers "enhanced liposuction' to address the diverse and challenges needs of suboptimal skin elasticity and contours.

As a result of InMode's innovations and development of RFAL technology, there has been the creation of a whole new market segment they have call the GAP patients [1]. RFAL or BodyTite treatments can often provide results that are reasonably comparable to a modest tummy tuck, arm lift or thigh lift procedures, but without the extensive scars [2–8]. This RFAL procedure can bridge the gap between non-invasive treatments with very minimal/modest results, minimally invasive treatments that lack the soft tissue contraction, or safety of RFAL and the standard open plastic surgery excisional procedures (**Figure 1**).

The FaceTite, AccuTite RFAL applicators and the Morpheus8 percutaneous OUTSIDE-IN RFAL remodeling tips are all available on the BodyTite workstation, as well as the Embrace RF and EmpowerRF workstations. Each applicator and Hand piece was specifically designed, engineered, produced and sold to serve a very large and growing market segment in the esthetic plastic surgery space; that is, the non-excisional Body contouring, face and neck lift and *GAP skin tightening market*. Non-excisional skin tightening technology and procedure growth has shown 30–40% year-over-year growth rates over the past five to eight years.

#### **Figure 1.**

*BodyTite RFAL procedures can serve as a GAP procedure between non-invasive, non-thermally invasive and standard excisional procedures.*