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Management of metastatic renal cell carcinoma (mRCC) has evolved considerably in the past 10 years due to better understanding of tumor biology. This development has changed mRCC to a chronic progressive disease with several lines of treatment options. The introduction of several new targeted therapies including immunotherapy has improved median overall survival of approximately 1 year to >2 years in mRCC.

Background: Topotecan is the standard second line agent used in relapsed small cell lung cancer (SCLC). However, the erratic availability and the cost of the drug has been a prohibitive factor for its use in second-line setting in India. Paclitaxel has shown antitumor activity in heavily pretreated patients with SCLC. Hence, this audit was performed to study the efficacy of weekly paclitaxel as a form of metronomic therapy in the second-line setting in SCLC. Materials and Methods: Fifty-seven patients of relapsed SCLC who presented to the thoracic medical oncology unit of Tata Memorial Centre, Mumbai between January 2011 and December 2015 were selected for this analysis. Weekly paclitaxel at a dose of 80 mg/m 2 was administered until progression or development of intolerable side effects or patient refusal. Data regarding baseline demographics, previous treatment history, response rate, progression-free survival, overall survival (OS), and toxicity to weekly paclitaxel was extracted from a prospectively maintained database in the thoracic medical oncology unit and was analyzed using SPSS version 16 (IBM, New York, USA). Kaplan-Meier survival analysis was performed. Results: Median age of the cohort was 58 years (40-77 years). Etoposide with carboplatin was the regimen used in 40 patients (70.2%) whereas the remaining 17 patients received etoposide with cisplatin (29.8%). Eastern Cooperative Oncology Group performance status at relapse was 1 in 3 (5.3%), 2 in 49 (86.0%), and 3 in 5 (8.7%) patients. The response rate and clinical benefit rate were 9.1% (5 patients) and 52.7% (29 patients), respectively. Grade 3-4 toxicities were seen in 10.5% (6 patients). The median PFS was 145 days (95% confidence interval [CI]: 116.6-173.5 days) whereas the median OS was 168 days (95% CI: 112.5-223.5 days). Conclusion: Weekly paclitaxel as a second line agent in relapsed small cell cancer of the lung is a feasible and well-tolerated agent.

Background Studies evaluating neo-adjuvant chemotherapy (NACT) exclusively in gallbladder cancer (GBC) are few and there are no randomized trials on the subject. Locally advanced GBC and indications for NACT in GBC are not yet clearly defined. Methods We analysed 160 consecutive GBC patients who received NACT based on clinico-radiologic criteria suggesting high-risk disease (TMH Criteria) from January 2010 to February 2016. Results On initial assessment, 140 (87.5%) patients had T3/T4 disease and 105 (65%) patients were node positive. Response rate and clinical benefit rate was 52.5% and 70% respectively. Sixty six (41.2%) patients could undergo curative intent resection. With a median follow-up of 33 months, the median OS and EFS of the entire cohort were 13 and 8 months respectively. Patient undergoing curative surgery had a statistically superior OS (49 vs. 7 months; p = 0.0001) and EFS (25 months vs. 5 months; p = 0.0001) compared to those who did not. Conclusion Locally advanced GBC remains a disease with poor prognosis. Chemotherapy with neoadjuvant intent in locally advanced/borderline resectable GBC showed good response rates. This resulted in curative surgical resection or disease stabilisation in significant proportion of patients. Patients who undergo definitive surgery after favourable response to NACT experience good survival.

Surfactant protein D (SP-D), a C-type lectin and pattern-recognition soluble factor, plays an important role in immune surveillance to detect and eliminate human pulmonary pathogens. SP-D has been shown to protect against infections with the most ubiquitous airborne fungal pathogen, Aspergillus fumigatus, but the fungal surface component(s) interacting with SP-D is unknown. Here, we show that SP-D binds to melanin pigment on the surface of A. fumigatus dormant spores (conidia). SP-D also exhibited an affinity to two cell-wall polysaccharides of A. fumigatus, galactomannan (GM) and galactosaminogalactan (GAG). The immunolabeling pattern of SP-D was punctate on the conidial surface and was uniform on germinating conidia, in accordance with the localization of melanin, GM, and GAG. We also found that the collagen-like domain of SP-D is involved in its interaction with melanin, whereas its carbohydrate-recognition domain recognized GM and GAG. Unlike un-opsonized conidia, SP-D–opsonized conidia were phagocytosed more efficiently and stimulated the secretion of proinflammatory cytokines by human monocyte-derived macrophages. Furthermore, SP-D(−/−) mice challenged intranasally with wildtype conidia or melanin ghosts (i.e. hollow melanin spheres) displayed significantly reduced proinflammatory cytokines in the lung compared with wildtype mice. In summary, SP-D binds to melanin present on the dormant A. fumigatus conidial surface, facilitates conidial phagocytosis, and stimulates the host immune response.

Being a major first line of immune defense, the complement system keeps a constant vigil against viruses. Its ability to recognize large panoply of viruses and virus-infected cells, and trigger the effector pathways, results in neutralization of viruses and killing of the infected cells. This selection pressure exerted by complement on viruses has made them evolve a multitude of countermeasures. These include targeting the recognition molecules for the avoidance of detection, targeting key enzymes and complexes of the complement pathways like C3 convertases and C5b-9 formation – either by encoding complement regulators or by recruiting membrane-bound and soluble host complement regulators, cleaving complement proteins by encoding protease, and inhibiting the synthesis of complement proteins. Additionally, viruses also exploit the complement system for their own benefit. For example, they use complement receptors as well as membrane regulators for cellular entry as well as their spread. Here, we provide an overview on the complement subversion mechanisms adopted by the members of various viral families including Poxviridae, Herpesviridae, Adenoviridae, Flaviviridae, Retroviridae, Picornaviridae, Astroviridae, Togaviridae, Orthomyxoviridae and Paramyxoviridae.

Poxviruses display species tropism—variola virus is a human-specific virus, while vaccinia virus causes repeated outbreaks in dairy cattle. Consistent with this, variola virus complement regulator SPICE (smallpox inhibitor of complement enzymes) exhibits selectivity in inhibiting the human alternative complement pathway and vaccinia virus complement regulator VCP (vaccinia virus complement control protein) displays selectivity in inhibiting the bovine alternative complement pathway. In the present study, we examined the species specificity of VCP and SPICE for the classical pathway (CP). We observed that VCP is ∼43-fold superior to SPICE in inhibiting bovine CP. Further, functional assays revealed that increased inhibitory activity of VCP for bovine CP is solely due to its enhanced cofactor activity, with no effect on decay of bovine CP C3-convertase. To probe the structural basis of this specificity, we utilized single- and multi-amino-acid substitution mutants wherein 1 or more of the 11 variant VCP residues were substituted in the SPICE template. Examination of these mutants for their ability to inhibit bovine CP revealed that E108, E120, and E144 are primarily responsible for imparting the specificity and contribute to the enhanced cofactor activity of VCP. Binding and functional assays suggested that these residues interact with bovine factor I but not with bovine C4(H 2 O) (a moiety conformationally similar to C4b). Mapping of these residues onto the modeled structure of bovine C4b-VCP-bovine factor I supported the mutagenesis data. Taken together, our data help explain why the vaccine strain of vaccinia virus was able to gain a foothold in domesticated animals. IMPORTANCE Vaccinia virus was used for smallpox vaccination. The vaccine-derived virus is now circulating and causing outbreaks in dairy cattle in India and Brazil. However, the reason for this tropism is unknown. It is well recognized that the virus is susceptible to neutralization by the complement classical pathway (CP). Because the virus encodes a soluble complement regulator, VCP, we examined whether this protein displays selectivity in targeting bovine CP. Our data show that it does exhibit selectivity in inhibiting the bovine CP and that this is primarily determined by its amino acids E108, E120, and E144, which interact with bovine serine protease factor I to inactivate bovine C4b—one of the two subunits of CP C3-convertase. Of note, the variola complement regulator SPICE contains positively charged residues at these positions. Thus, these variant residues in VCP help enhance its potency against the bovine CP and thereby the fitness of the virus in cattle.

The pandemic influenza A(H1N1) 2009 virus caused significant morbidity and mortality worldwide thus necessitating the need to understand the host factors that influence its control. Previously, the complement system has been shown to provide protection during the seasonal influenza virus infection, however, the role of individual complement pathways is not yet clear. Here, we have dissected the role of intact complement as well as of its individual activation pathways during the pandemic influenza virus infection using mouse strains deficient in various complement components. We show that the virus infection in C3-/- mice results in increased viral load and 100% mortality, which can be reversed by adoptive transfer of naïve wild-type (WT) splenocytes, purified splenic B cells, or passive transfer of immune sera from WT, but not C3-/- mice. Blocking of C3a and/or C5a receptor signaling in WT mice using receptor antagonists and use of C3aR-/- and C5aR-/- mice showed significant mortality after blocking/ablation of C3aR, with little or no effect after blocking/ablation of C5aR. Intriguingly, deficiency of C4 and FB in mice resulted in only partial mortality (24%-32%) suggesting a necessary cross-talk between the classical/lectin and alternative pathways for providing effective protection. In vitro virus neutralization experiments performed to probe the cross-talk between the various pathways indicated that activation of the classical and alternative pathways in concert, owing to coating of viral surface by antibodies, is needed for its efficient neutralization. Examination of the virus-specific complement-binding antibodies in virus positive subjects showed that their levels vary among individuals. Together these results indicate that cooperation between the classical and alternative pathways not only result in efficient direct neutralization of the pandemic influenza virus, but also lead to the optimum generation of C3a, which when sensed by the immune cells along with the antigen culminates in generation of effective protective immune responses., Author summary The pandemic influenza A(H1N1) 2009 virus is now circulating seasonally and causing a significant disease burden worldwide. Hence, it is important to delineate the immune components required for protection against its infection. Here we demonstrate that presence of intact complement is essential for clearing the pandemic influenza virus infection, wherein complement synthesized by B cells plays a major role. Further, we show that activation of the classical as well as alternative pathways is a requisite for efficient neutralization of the virus as well as the optimum generation of C3a, which is necessary for boosting the protective immune responses. Our results thus reveal that deficiencies of components of the classical and alternative pathways enhance the susceptibility to and severity of the pandemic influenza virus infection.

INTRODUCTION: Sorafenib is an established upfront treatment option for metastatic RCC (mRCC). There is no published literature regarding its performance in Indian Patients. We present an analysis of Sorafenib use in our institute and attempt to validate the Heng criteria as a prognostic score in these patients. MATERIALS AND METHODS: Patients who received Sorafenib as first line treatment for advanced RCC from June 2012 to December 2015 were prognosticated by Heng criteria and retrospectively analysed for baseline demographics, toxicity, response and outcomes. RESULTS: 82 patients (65 males, 17 females) with a median age of 57 years were included for final analysis. Median ECOG PS was 1, 95.2 % of the patients had Stage IV disease and clear cell was the predominant histology (79.4%). 23.2%, 42.7% and 34.1% of patients were classified as low, intermediate and high risk by Heng's criteria, respectively. Dose reduction was required in 24.4% of patients, while 14.6% required permanent cessation of Sorafenib due to intolerable or recurrent side effects. Common adverse events included HFS (68.2%), mucositis (35.3%), fatigue (35.3%), rash (32.9%) and hypertension (25.6%). Response rate observed was 18.2%, while clinical benefit rate was 57.2% in the 57 patients where response was evaluable. Median progression free survival was 7.75 months (5.45-10.05) and median overall survival (OS) was 12.18 months (9.61 – 14.76). Median OS was 19.6, 16.1 and 10.3 months respectively for low, intermediate and high risk patients by Heng criteria and the criteria was statistically discriminatory for the 3 groups for OS (p=0.045, chi-square test). CONCLUSION: Sorafenib is a viable upfront treatment option for metastatic RCC in Indian patients with acceptable PFS, although a high incidence of HFS, mucositis and rash is observed. The Heng score has discriminatory value in mRCC with Sorafenib and can be considered for routine use in the clinic.

Vaccinia virus (VACV), a member of the Poxviridae family, uses cytoplasmic factories for its replication. Recent studies indicated that VACV infection requires a set of nucleoporins. However, how the nucleoporins contribute to viral life cycle remains unclear. Here, we report that the nucleoporins Nup62 and Nup358 localize to the cytoplasmic viral factories (VFs). Nup358 was targeted to the VFs at 6h post-infection (hpi), whereas Nup62, along with the previously reported translation factors such as eIF4E, eIF3η and G3BP1, was recruited to the VFs at 8 hpi. Nup358 depletion led to a decrease in the size and number of viral factories and reduction in viral yield. Further studies showed that Nup358 is involved in recruiting Nup62 and eIF4E to the VFs. Collectively, our results reveal spatio-temporal regulation in the recruitment of nucleoporins and translation factors to VFs, and particularly the importance of Nup358 in VACV infection.

e18260 Background: The therapeutic landscape in medical oncology continues to expand significantly. Newer therapies, especially immunotherapy, offer the hope of profound and durable responses with more tolerable side effect profiles. Integrating this information into the decision making process is challenging for patients and oncologists. Systemic anticancer treatment within the last thirty days of life is a key quality of care indicator and is one parameter used in the assessment of aggressiveness of care. Methods: A retrospective review of medical records of all patients previously treated at Goulburn Valley Health oncology department who died between 1 January 2015 and 30 June 2018 was conducted. Information collected related to patient demographics, diagnosis, treatment, and hospital care within the last 30 days of life. These results were presented to a hospital meeting and a quality improvement intervention program instituted. A second retrospective review of medical records of all patients who died between 1 July 2018 and 31 December 2018 was conducted in order to measure the effect of this intervention. Results: The initial audit period comprised 440 patients. 120 patients (27%) received treatment within the last 30 days of life. The re-audit period comprised 75 patients. 19 patients (25%) received treatment within the last 30 days of life. Treatment rates of chemotherapy reduced after the intervention in contrast to treatment rates of immunotherapy which increased. A separate analysis calculated the rate of mortality within 30 days of chemotherapy from the total number of patients who received chemotherapy was initially 8% and 2% in the re-audit period. Treatment within the last 30 days of life was associated with higher use of aggressive care such as emergency department presentation, hospitalisation, ICU admission and late hospice referral. Palliative care referral rates improved after the intervention. Conclusions: This audit demonstrated that a quality improvement intervention can impact quality of care indicators with reductions in the use of chemotherapy within the last 30 days of life. However, immunotherapy use increased which may be explained by increased access and perceived better tolerability.

e18000 Background: Febrile neutropenia is a serious complication of chemotherapy. The Multinational Association for Supportive Care in Cancer (MASCC) risk index score can reliably identify patients with febrile neutropenia at low risk of serious complications. Outpatient management programs utilising protocol based risk stratification, daily ambulatory nursing visits, telephone follow up and early outpatient review have been successfully piloted in other Australian cancer treatment centres. Methods: We performed a retrospective review of medical records for all patients admitted at our institution with febrile neutropenia between January 1 2016 and December 31 2018. We collected information regarding patient characteristics, cancer diagnosis and treatment, determined the MASCC risk index score, and if low risk, we determined the potential eligibility for outpatient care and potential reduction in length of stay. Results: A total of 98 hospital admissions with febrile neutropenia were analysed. Of these, 66 were determined to have a MASCC low risk index score. 58 patients met the eligibility criteria for outpatient management. The median age was 67 years. 71% were female. The most common tumour type was breast cancer. 52% were treated with palliative intent. The median length of stay was 3 days. The median potential reduction in length of stay for each admission was 2 days. The total potential reduction in length of stay was 198 days. No admission resulted in serious complications indicating the safety of outpatient care. Conclusions: Febrile neutropenia is a common complication of chemotherapy and a leading cause of hospital admission. This review demonstrates a significant number of hospital admission days can be avoided with outpatient care. We intend to conduct a prospective pilot study at our centre to institute an outpatient febrile neutropenia program for such low risk groups with potential reduction in hospital bed length of stay. This has significant implications on health resource usage, service provision planning and patient quality of life.

Introduction The current treatment of metastatic renal cell carcinoma (mRCC) revolves around targeted agents, which have resulted in a median overall survival of 22 to 26 months in registration trials. However, the outcomes in a non-trial, real-world Indian population have not yet been evaluated. Materials and Methods The present study was a part of a prospective Clinical Trials Registry–India-registered study, the Kidney Cancer Registry, a prospectively maintained kidney cancer registry. The data of patients with a diagnosis of mRCC from February 2007 to August 2015 who were potential candidates for systemic therapy were extracted from the database and analyzed for treatment patterns and outcomes. Results The data from 212 patients were eligible for analysis. Of these 212 patients, 204 (96.2%) received first-line systemic treatment with sunitinib (40.6%), sorafenib (37.7%), pazopanib (2.8%), temsirolimus (2.8%), or everolimus (1.9%). The risk status of 91% of the patients could be stratified using the Heng criteria into favorable (18.9%), intermediate (43.9%), and poor risk (28.3%) categories. The response rate, clinical benefit rate, median progression-free survival, and median overall survival with first-line targeted therapy were 22.5%, 60.7%, 7.09 months, and 12.87 months, respectively. The common adverse events seen included skin rash (31.7%), hypertension (29.4%), grade 3 hand-foot syndrome (27.4%), mucositis (26.4%), dyslipidemia (20%), and hyperglycemia (17.6%). Patients receiving second-line therapy (22.6%) had superior overall survival to patients who had not (16.46 vs. 10.67 months; P = .032). Conclusion The present registry-based study is the first, to the best of our knowledge, of its type from India and showed that the overall outcomes in this real-world cohort appear comparable to non-trial data worldwide. An increased incidence of metabolic adverse events that require monitoring during treatment was also found.

Background: Multimodality treatment of gastrointestinal stromal tumor (GIST) with surgery and adjuvant imatinib mesylate (IM), along with an emerging role for neoadjuvant IM prior to evaluation for resectability has resulted in high survival rates. Methods: We conducted a retrospective analysis of prospectively collected data of patients who underwent surgery for GIST, prior to or followed by IM therapy. A total of 112 patients underwent surgery between January 2009 and March 2015 at our centre. This included 27 patients with upfront resectable disease, 76 patients with locally advanced GIST who received neoadjuvant IM followed by surgery and 9 patients with metastatic disease who had excellent response to IM and were taken for surgery. Results: The primary tumor in the non metastatic patients was in the stomach (53%), duodenum (16%), rectum (12%), jejunum (11%), ileum (7%), and others (2%). Median duration of neoadjuvant IM was 5 months with 4 patients showing disease progression during neoadjuvant IM. Ninety-three percent of all patients had R0 resections, while 7% had R+ resections. The estimated 3- and 5-year DFS in non-metastatic patients was 86.1% and 67% respectively with a 3- and 5-year median OS of 95.4% and 91.7% respectively. Five-year PFS and OS for the metastatic patients was 88.8% and 100% respectively. Lack of adjuvant IM was the only factor related to inferior PFS and OS. Conclusions: Longer duration of neoadjuvant IM should be considered in locally advanced GIST prior to surgery and resection may be considered in responding metastatic patients.

Today image processing and utilizations based on RGB-D data have huge interest in various research areas. Regarding segmentation, colour correction, depth information, comprehensive description of 3-Dimension scenes in RGB-Depth data provides useful hints from another dimension to segregate substance in close colours. Here, super pixel segmentation based a new method is discussed to resolve such mixed, lost, and noise level increase of pixel problems of the RGB-D system and has been simulated using MATLAB software. The advantage of MATLAB is, it is widely available, continuously updated and has wider reach. In our approach, mixed pixel areas are detected using super pixel segmentation of colour and depth, and consolidate them to lost pixel regions. The consolidated regions are replete with neighbouring depth information based on a Robust Edge-Stop (RES) Function; distance transform values of colour edge pixels are used to form this function. In addition, a hybrid filter is used to remove noise level of pixels. Experimental results shows the proposed method gives better performance on Mean, Intensity, Area, Perimeter, Centroid, and Diameter.

Background: Dihydropyrimidine dehydrogenase (DPD) enzyme is the rate limiting step in the metabolism of capecitabine, and its deficiency leads to severe toxicities and rarely, death. Methods: A total of 506 patients were treated in the GI Medical Oncology unit of our institution with capecitabine containing regimens with a dose range of 1,250 to 2,000 mg/m 2 /day during the period from June 2013 to June 2015. Patients with grade (Gr) 3/4 toxicities requiring in-patient care (life threatening complications) were planned for DPD activity testing by peripheral blood PCR sequencing. Results: Thirty-one patients developed Gr 3/4 toxicities during cycle 1 of capecitabine. This included mucositis in 24 (77%), diarrhea in 29 (94%), hand-foot syndrome (HFS) in 13 (42%) and myelosuppression in 5 (16%) patients. Twenty-two (81.4%) were found to be DPD deficient with 6 patients negative for DPD mutation. Three patients did not undergo the DPD analysis as advised. More than one mutation was seen in 11 patients. The relative frequencies of the mutations were IVS14+1G→A in 39%, with 13% having homozygosity, 85 T→C in 36%, 1627 A→G in 32%, 496 A→G in 18% and 2194 G→A in 18%, respectively. After dose reduction in cycle 2 in 17 patients of the DPD mutation positive cohort, statistically significant reduction in the toxicities was seen. Conclusions: Dose reduction in DPD deficient patients, reduces risk of life threatening complications significantly but not completely. Upfront screening for DPD deficiency in Indian patients should be evaluated further in view of potentially high homozygous DPD mutation prevalence.

Longer duration of neoadjuvant (NA) imatinib (IM) used for locally advanced (LA) gastrointestinal stromal tumours (GIST) is not based on biology of the tumour reflected by kit mutation analysis.LA or locally recurrent (LR) GIST treated with NA IM from May 2008 to March 2015 from a prospective database were included in the analysis. Archived formalin-fixed paraffin-embedded tissues (FFPE) were used for testing KIT exons 9, 11, 13 and 17 by PCR.One hundred twenty-five patients with LA or LR GIST were treated with NA IM. Forty-five patients (36 %) had undergone c-kit mutation testing. Exon 11 was seen in 25 patients (55.5 %), 3 with exon 9 (6.7 %) and 2 with exon 13 (4.4 %). Twelve were wild type (26.6 %) and 3 (6.7 %) were declared uninterpretable. Response rate (RR) for the exon 11 mutants was higher than the non-exon 11 mutant group (84 vs. 40 %, p = 0.01). Disease stabilization rate (DSR) rates were also higher in the exon 11 subgroup than non-exon 11 group (92 vs. 75 %). Eighty-four per cent exon 11 and 75 % non-exon 11 mutants were surgical candidates. Patients undergoing surgery had significantly improved event free survival (EFS) (p 0.001) compared to patients not undergoing surgery, with the same trend seen in OS (p = 0.021). Patients with a SD on response to NA IM had a lower EFS (p = 0.076) and OS compared to patients achieving CR/PR. There were no differences between the various exon variants in terms of outcomes and responses CONCLUSION: Upfront evaluation of kit mutation status may help us in delineating separate treatment strategies for potentially biologically different tumours and assessing the correct timing of surgery for this subset of GIST.

In this paper, theoretical investigation of single feed, single layer, double band, Microstrip patch antenna is presented that resonate at 3.105GHz cover the frequency range into s-band of electromagnetic spectrum. Main purpose of this project for enhancement of the Return Loss by cut/made out slots within the patch as well as Bandwidth increased by increasing the height between the dielectric substrate and finite ground plane. The proposed antenna consists of rectangular radiating patch with T and U shaped slots, truncated corner with meshed finite ground plane. The proposed antenna is simulated on IE3D simulation software achieve maximum return loss value −42.57 at resonant frequency 3.105 GHz and bandwidth increase upto 21% that cover range 2.81–3.47 GHz into s-band. Good radiation pattern and efficiency of about 92% has achieved.

Variola and vaccinia viruses, the two most important members of the family Poxviridae, are known to encode homologs of the human complement regulators named smallpox inhibitor of complement enzymes (SPICE) and vaccinia virus complement control protein (VCP), respectively, to subvert the host complement system. Intriguingly, consistent with the host tropism of these viruses, SPICE has been shown to be more human complement-specific than VCP, and in this study we show that VCP is more bovine complement-specific than SPICE. Based on mutagenesis and mechanistic studies, we suggest that the major determinant for the switch in species selectivity of SPICE and VCP is the presence of oppositely charged residues in the central complement control modules, which help enhance their interaction with factor I and C3b, the proteolytically cleaved form of C3. Thus, our results provide a molecular basis for the species selectivity in poxviral complement regulators.

The complement system is a major component of the innate immune system that recognizes invading pathogens and eliminates them by means of an array of effector mechanisms, in addition to using direct lytic destruction. Viruses, in spite of their small size and simple composition, are also deftly recognized and neutralized by the complement system. In turn, as a result of years of coevolution with the host, viruses have developed multiple mechanisms to evade the host complement. These complex interactions between the complement system and viruses have been an area of focus for over three decades. In this article, we provide a broad overview of the field using key examples and up-to-date information on the complement-evasion strategies of viruses.

e16159Background: The CAPTEM regimen has significant activity in well differentiated pancreatic NETs. There is limited data regarding CAPTEM in patients (pts) with intermediate to high grade NETs. ...

The web applications and websites of the enterprises are accessed by a huge number of users with the expectation of reliability and high availability. Social networking sites are generating the data exponentially large amount of data. It is a challenging task to store data efficiently. SQL and NoSQL are mostly used to store data. As RDBMS cannot handle the unstructured data and huge volume of data, so NoSQL is better choice for web applications. Graph database is one of the efficient ways to store data in NoSQL. Graph database allows us to store data in the form of relation. In Graph representation each tuple is represented by node and the relationship is represented by edge. But, to handle the exponentially growth of data into a single server might decrease the performance and increases the response time. Data partitioning is a good choice to maintain a moderate performance even the workload increases. There are many data partitioning techniques like Range, Hash and Round robin but they are not efficient for the small transactions that access a less number of tuples. NoSQL data stores provide scalability and availability by using various partitioning methods. To access the Scalability, Graph partitioning is an efficient way that can be easily represent and process that data. To balance the load data are partitioned horizontally and allocate data across the geographical available data stores. If the partitions are not formed properly result becomes expensive distributed transactions in terms of response time. So the partitioning of the tuple should be based on relation. In proposed system, Schism technique is used for partitioning the Graph. Schism is a workload aware graph partitioning technique. After partitioning the related tuples should come into a single partition. The individual node from the graph is mapped to the unique partition. The overall aim of Graph partitioning is to maintain nodes onto different distributed partition so that related data come onto the same cluster.

Introduction: Docetaxel/oxaliplatin/capecitabine (TEX) is a commonly used combination chemotherapeutic regimen in advanced gastric cancer (AGC). Application strategies in routine clinical practice are reported in this study. Materials and Methods: Patients diagnosed with AGC, receiving biweekly TEX (docetaxel - 60 mg/m (2)-D1; oxaliplatin - 85 mg/m (2)-D1, and capecitabine 500–625 mg/m (2) orally twice daily for 14 days) between July 2012 and May 2016 were retrospectively analyzed for tolerance, prognostic factors, event-free survival (EFS), and overall survival (OS). The proportion of patients continuing and terminating chemotherapy at various time-points was enumerated. Results: Overall, 208 patients were started on TEX. Median EFS was 6.34 months (95% confidence interval [CI] 5.80–6.87), and median OS was 15.31 (95% CI 12.65–17.96). Post 8 cycles of TEX, further 30 patients (14.4%) were continued on chemotherapy (docetaxel, capecitabine, or TEX) whereas 47 patients (22.6%) were on observation only, and there was a statistically significant difference in the median OS of these two groups (22.55 months vs. 14.89 months; P = 0.028). Raised serum alkaline phosphatase (SAP) levels (>100 U/L) predicted inferior survival (P = 0.006). Conclusion: TEX chemotherapy is a feasible, efficacious triplet regimen that can be used in clinical practice. SAP levels >100 U/L is a poor prognostic factor, as observed in this study. An initial “induction” such as combination chemotherapy regimen followed by monotherapy as continuation requires further evaluation.

Kaposica, the complement regulator of Kaposi’s sarcoma-associated herpesvirus, inhibits complement by supporting factor I-mediated inactivation of the proteolytically activated form of C3 (C3b) and C4 (C4b) (cofactor activity [CFA]) and by accelerating the decay of classical and alternative pathway C3-convertases (decay-accelerating activity [DAA]). Previous data suggested that electrostatic interactions play a critical role in the binding of viral complement regulators to their targets, C3b and C4b. We therefore investigated how electrostatic potential on Kaposica influences its activities. We built a homology structure of Kaposica and calculated the electrostatic potential of the molecule, using the Poisson–Boltzmann equation. Mutants were then designed to alter the overall positive potential of the molecule or of each of its domains and linkers by mutating Lys/Arg to Glu/Gln, and the functional activities of the expressed mutants were analyzed. Our data indicate that 1) positive potential at specific sites and not the overall positive potential on the molecule guides the CFAs and classical pathway DAA; 2) positive potential around the linkers between complement control protein domains (CCPs) 1–2 and 2–3 is more important for DAAs than for CFAs; 3) positive potential in CCP1 is crucial for binding to C3b and C4b, and thereby its functional activities; 4) conversion to negative or enhancement of negative potential for CCPs 2–4 has a marked effect on C3b-linked activities as opposed to C4b-linked activities; and 5) reversal of the electrostatic potential of CCP4 to negative has a differential effect on classical and alternative pathway DAAs. Together, our data provide functional relevance to conservation of positive potential in CCPs 1 and 4 and the linkers of viral complement regulators.

Herpesvirus saimiri encodes a functional homolog of human regulator-of-complement-activation proteins named CCPH that inactivates complement by accelerating the decay of C3 convertases and by serving as a cofactor in factor I-mediated inactivation of their subunits C3b and C4b. Here, we map the functional domains of CCPH. We demonstrate that short consensus repeat 2 (SCR2) is the minimum domain essential for classical/lectin pathway C3 convertase decay-accelerating activity as well as for factor I cofactor activity for C3b and C4b. Thus, CCPH is the first example wherein a single SCR domain has been shown to display complement regulatory functions.

Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is ∼90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement.

The complement system has evolved to annul pathogens, but its improper regulation is linked with diseases. Efficient regulation of the system is primarily provided by a family of proteins termed regulators of complement activation (RCA). The knowledge of precise structural determinants of RCA proteins critical for imparting the regulatory activities and the molecular events underlying the regulatory processes, nonetheless, is still limited. Here, we have dissected the structural requirements of RCA proteins that are crucial for one of their two regulatory activities, the cofactor activity (CFA), by using the Kaposi’s sarcoma-associated herpesvirus RCA homolog Kaposica as a model protein. We have scanned the entire Kaposica molecule by sequential mutagenesis using swapping and site-directed mutagenesis, which identified residues critical for its interaction with C3b and factor I. Mapping of these residues onto the modeled structure of C3b–Kaposica–factor I complex supported the mutagenesis data. Furthermore, the model suggested that the C3b-interacting residues bridge the CUB (complement C1r-C1s, Uegf, Bmp1) and MG2 (macroglobulin-2) domains of C3b. Thus, it seems that stabilization of the CUB domain with respect to the core of the C3b molecule is central for its CFA. Identification of CFA-critical regions in Kaposica guided experiments in which the equivalent regions of membrane cofactor protein were swapped into decay-accelerating factor. This strategy allowed CFA to be introduced into decay-accelerating factor, suggesting that viral and human regulators use a common mechanism for CFA.

Herpesvirus saimiri (HVS) is a lymphotropic virus that causes T-cell lymphomas in New World primates. It encodes a structural homolog of complement control proteins named complement control protein homolog (CCPH). Previously, CCPH has been shown to inhibit C3d deposition on target cells exposed to complement. Here we have studied the mechanism by which it inactivates complement. We have expressed the soluble form of CCPH in Escherichia coli, purified to homogeneity and compared its activity to vaccinia virus complement control protein (VCP) and human complement regulators factor H and soluble complement receptor 1. The expressed soluble form of CCPH bound to C3b (KD = 19.2 microm) as well as to C4b (KD = 0.8 microm) and accelerated the decay of the classical/lectin as well as alternative pathway C3-convertases. In addition, it also served as factor I cofactor and supported factor I-mediated inactivation of both C3b and C4b. Time course analysis indicated that although its rate of inactivation of C4b is comparable with VCP, it is 14-fold more potent than VCP in inactivating C3b. Site-directed mutagenesis revealed that Arg-118, which corresponds to Lys-120 of variola virus complement regulator SPICE (a residue critical for its enhanced C3b cofactor activity), contributes significantly in enhancing this activity. Thus, our data indicate that HVS encodes a potent complement inhibitor that allows HVS to evade the host complement attack.

Compstatin is a peptidic complement inhibitor that prevents the cleavage of complement factor 3 (C3) by C3 convertase. Compstatin differs from other C3-regulatory proteins, such as complement receptor (CR) 1 and decay-accelerating factor (DAF), in that it binds native as well as activated C3 fragments and acts through mechanisms that do not involve the destabilization of the C3 convertase or the accelerated degradation of C3b. Compstatin's activity most likely relies on its affinity for native C3 and the conformational change that results upon binding with C3. Although the intermolecular interactions between compstatin and C3 have been studied, the identity of the targeted region on C3 is still elusive. To address this issue, we synthesized a photo-crosslinking compstatin analog and used it to probe C3 for sites of interaction. We identified a 40-kDa region at the C-terminus of the beta-chain of C3 that included the binding site of the compstatin analog. The specificity of the binding was confirmed by inhibition studies, which showed reduced crosslinking signal after pre-incubation of C3 with compstatin but not with various inactive analogs. Binding studies performed with a recombinant homolog of the 40-kDa region confirmed these findings. Five smaller recombinant proteins corresponding to various overlapping regions of the 40-kDa fragment did not bind compstatin, suggesting that a proper protein conformation, only found in larger fragments, is required for compstatin binding. The identified region on the beta-chain has, thus far, not been implicated in C3 cleavage or interactions with other proteins. Therefore, further research on this part of the C3 molecule may have implications for studies on the regulation of C3 cleavage, as well as for complement-based drug design.

The complement system is an integral participant in the innate mechanisms of immunity and thus has the burden of performing surveillance in the host. The complement system achieves this effectively by covalently attaching C3b, the activated form of complement protein C3, to the surface of foreign pathogens and labeling them as non-self. Although viruses are small and have relatively simple structure, both acute and latent viruses can be efficiently recognized and neutralized by the complement system. However, to combat the complement system of the host and succeed as pathogens, viruses have developed principles to avoid being ‘labeled' as non-self by complement and thus are considered as self by the complement system.

The complement system is a potent innate immune mechanism consisting of cascades of proteins which are designed to fight against and annul intrusion of all the foreign pathogens. Although viruses are smaller in size and have relatively simple structure, they are not immune to complement attack. Thus, activation of the complement system can lead to neutralization of cell-free viruses, phagocytosis of C3b-coated viral particles, lysis of virus-infected cells, and generation of inflammatory and specific immune responses. However, to combat host responses and succeed as pathogens, viruses not only have developed/adopted mechanisms to control complement, but also have turned these interactions to their own advantage. Important examples include poxviruses, herpesviruses, retroviruses, paramyxoviruses and picornaviruses. In this review, we provide information on the various complement evasion strategies that viruses have developed to thwart the complement attack of the host. A special emphasis is given on the interactions between the viral proteins that are involved in molecular mimicry and the complement system.

The genome analysis of Kaposi's sarcoma-associated herpesvirus (KSHV) has revealed the presence of an open reading frame (ORF 4) with sequence homology to complement control proteins. To assign a function to this protein, we have now expressed this ORF using thePichiaexpression system and shown that the purified protein inhibited human complement-mediated lysis of erythrocytes, blocked cell surface deposition of C3b (the proteolytically activated form of C3), and served as a cofactor for factor I-mediated inactivation of complement proteins C3b and C4b (the subunits of C3 convertases). Thus, our data indicate that this KSHV inhibitor of complement activation (kaposica) provides a mechanism by which KSHV can subvert complement attack by the host.

We have previously identified compstatin, a 13-residue cyclic peptide, that inhibits complement activation by binding to C3 and preventing C3 cleavage to C3a and C3b. The structure of compstatin consists of a disulfide bridge and a type I beta-turn located at opposite sides to each other. The disulfide bridge is part of a hydrophobic cluster, and the beta-turn is part of a polar surface. We present the design of compstatin analogs in which we have introduced a series of perturbations in key structural elements of their parent peptide, compstatin. We have examined the consistency of the structures of the designed analogs compared with compstatin using NMR, and we have used the resulting structural information to make structure-complement inhibitory activity correlations. We propose the following. 1) Even in the absence of the disulfide bridge, a linear analog has a propensity for structure formation consistent with a turn of a 3(10)-helix or a beta-turn. 2) The type I beta-turn is a necessary but not a sufficient condition for activity. 3) Our substitutions outside the type I beta-turn of compstatin have altered the turn population but not the turn structure. 4) Flexibility of the beta-turn is essential for activity. 5) The type I beta-turn introduces reversibility and sufficiently separates the two sides of the peptide, whereas the disulfide bridge prevents the termini from drifting apart, thus aiding in the formation of the hydrophobic cluster. 6) The hydrophobic cluster at the linked termini is involved in binding to C3 and activity but alone is not sufficient for activity. 7) beta-Turn residues Gln(5) (Asn(5))-Asp(6)-Trp(7)(Phe(7))-Gly(8) are specific for the turn formation, but only Gln(5)(Asn(5))-Asp(6)-Trp(7)-Gly(8) residues are specific for activity. 8) Trp(7) is likely to be involved in direct interaction with C3, possibly through the formation of a hydrogen bond. Finally we propose a binding model for the C3-compstatin complex.

Purpose Perioperative chemotherapy improves survival outcomes in locally advanced (LA) gastric cancer. Materials and methods We retrospectively analyzed patients with LA gastric cancer who were offered perioperative chemotherapy consisting of epirubicin, oxaliplatin, and capecitabine (EOX) from May 2013 to December 2015 at Tata Memorial Hospital in Mumbai. Results Among the 268 consecutive patients in our study, 260 patients (97.0%) completed neoadjuvant chemotherapy, 200 patients (74.6%) underwent D2 lymphadenectomy, and 178 patients (66.4%) completed adjuvant chemotherapy. The median follow-up period was 17 months. For the entire cohort, the median overall survival (OS), 3-year OS rate, median progression-free survival (PFS), and 3-year PFS rate were 37 months, 64.4%, 31 months, and 40%, respectively. PFS and OS were significantly inferior in patients who presented with features of obstruction than in those who did not (P=0.0001). There was no difference in survival with respect to tumor histology (well to moderately differentiated vs. poorly differentiated, signet ring vs. non-signet ring histology) or location (proximal vs. distal). Survival was prolonged in patients with an early pathological T stage and a pathological node-negative status. In a multivariate analysis, postoperative pathological nodal status and gastric outlet obstruction on presentation significantly correlated with survival. Conclusions EOX chemotherapy with curative resection and D2 lymphadenectomy is a suggested alternative to the existing perioperative regimens. The acceptable postoperative complication rate and relatively high resection, chemotherapy completion, and survival rates obtained in this study require further evaluation and validation in a clinical trial.

Background: Poor-risk advanced Renal cell carcinoma (RCC) are an under-evaluated and difficult to treat subset of patients with poor prognosis. While Temsirolimus is the approved first line therapy for this category, Tyrosine kinase inhibitors (TKIs) are also commonly uses as initial treatment. We present an analysis of poor-risk advanced RCC treated in our institute. Materials and Methods: Patients diagnosed as poor-risk (as per Heng criteria) advanced RCC from June 2008 to December 2015 were analysed for baseline demographics, treatment received, toxicity (primarily Grade 3 and Grade 4), response rates (RR) and survival. Results: 60 patients (43 males, 17 females) with a median age of 53 years were included for final analysis. Median ECOG PS was 1, clear cell was the predominant histology (63.3%), and 46.7% of patients had greater than 2 sites of metastases. Sorafenib, Sunitinib, Temsirolimus and Pazopanib were used to treat 43.3%, 36.7%, 8.3% and 6.7% of patients respectively, while 3 patients were offered upfront best supportive care. Common adverse events included skin rash (31.5%), HFS (Grade 2 and 3 – 30.8%), mucositis (26.3%), hypertension (24.5%), and dyslipidaemias (22.8%). 41 patients were available for response – overall response rate observed was 15%, while clinical benefit rate was 50%. Median progression free survival was 5.78 months (4.67-6.89) and median overall survival (OS) was 10.05 months (7.31-12.79). Conclusion: A majority of poor-risk metastatic RCC patients in our study were treated with TKIs and the survival outcomes appear to suggest that this strategy is a feasible alternative to Temsirolimus in the Indian setting.

The complement system surveillance in the host is effective in controlling viral propagation. Consequently, to subvert this effector mechanism, viruses have developed a series of adaptations. One among these is encoding mimics of host regulators of complement activation (RCA) which help viruses to avoid being labeled as ‘foreign’ and protect them from complement-mediated neutralization and complement-enhanced antiviral adaptive immunity. In this review, we provide an overview on the structure, function and evolution of viral RCA proteins.

Anaplastic lymphoma kinase ( ALK ) gene rearrangements are present in a small subset of non-small-cell lung cancers. ALK -positivity confers sensitivity to small-molecule ALK kinase inhibitors, such as crizotinib. The integration of crizotinib into standard treatment practice in NSCLC will rest on the widespread implementation of an effective screening system for newly diagnosed patients with NSCLC which is flexible enough to incorporate new targets as treatments are developed for them. Phase I and II studies of crizotinib in ALK -positive lung cancer have demonstrated significant activity and impressive clinical benefit, which led to its early approval by USFDA in 2011. Although crizotinib induces remissions and extends the lives of patients, there have been reports of emerging resistance to Crizotinib therapy. In this review, we discuss the history, mechanism of action, uses, adverse effects, dose modifications and future challenges and opportunities for patients with ALK -positive lung cancers.

The molecular mechanisms involved in the interaction of complement receptor 2 (CR2) with its natural ligands iC3b and C3d are still not well understood. In addition, studies regarding the binding site(s) of the receptor on C3 as well as the affinities of the C3 fragments for CR2 have produced contradictory results. In the present study, we have used surface plasmon resonance technology to study the interaction of CR2 with its ligands C3d, iC3b, and the EBV surface glycoprotein gp350/220. We measured the kinetics of binding of the receptor to its ligands, examined the influence of ionic contacts on these interactions, and assessed whether immobilized and soluble iC3b bound with similar kinetics to CR2. Our results indicate that 1) gp350 binding to CR2 follows a simple 1:1 interaction, whereas that of the C3 fragments is more complex and involves more than one intramolecular component; 2) kinetic differences exist between the binding of C3d and iC3b to CR2, which may be due to an additional binding site found on the C3c region of iC3b; and 3) iC3b binds to CR2 with different kinetics, depending on whether the iC3b is in solution or immobilized on the surface. These findings suggest that binding of CR2 to iC3b and C3d is more complex than previously thought.

Complement protein C3 is a central molecule in the complement system whose activation is essential for all the important functions performed by this system. After four decades of research it is now well established that C3 functions like a double-edged sword: on the one hand it promotes phagocytosis, supports local inflammatory responses against pathogens, and instructs the adaptive immune response to select the appropriate antigens for a humoral response; on the other hand its unregulated activation leads to host cell damage. In addition, its interactions with the proteins of foreign pathogens may provide a mechanism by which these microorganisms evade complement attack. Therefore, a clear knowledge of the molecule and its interactions at the molecular level not only may allow the rational design of molecular adjuvants but may also lead to the development of complement inhibitors and new therapeutic agents against infectious diseases.

Complement activation products are major components of the inflammatory response induced by cardiac surgery and cardiopulmonary bypass which contribute to postoperative organ dysfunction, fluid accumulation, and morbidity. Activation of the complement system occurs during extracorporeal circulation, during reperfusion of ischemic tissue, and after the formation of heparin-protamine complexes. In this study we examine the efficacy of Compstatin, a recently discovered peptide inhibitor of complement, in preventing heparin/protamine-induced complement activation in baboons. The study was performed in baboons because Compstatin binds to baboon C3 and is resistant to proteolytic cleavage in baboon blood (similar to humans); Compstatin inhibits only the activation of primates' complement system. After testing various doses and administration regimens, Compstatin produced complete inhibition at a total dose of 21 mg/kg when given as a combination of bolus injection and infusion. Compstatin completely inhibited in vivo heparin/protamine-induced complement activation without adverse effects on heart rate or systemic arterial, central venous, and pulmonary arterial pressures. This study indicates that Compstatin is a safe and effective complement inhibitor that has the potential to prevent complement activation during and after clinical cardiac surgery. Furthermore, Compstatin can serve as the prototype for designing an orally administrated drug.

Immune evasion is critical for survival of viruses that establish persistent or recurrent infections. However, at the molecular level, little is known about how viruses evade immune attack in vivo. Herpes simplex virus (HSV)-1 glycoprotein gC has two domains that are involved in modulating complement activation; one binds C3, and the other is required for blocking C5 and properdin (P) binding to C3. To evaluate the importance of these regions in vivo, HSV-1 gC mutant viruses were constructed that lacked one or both gC domains and studied in a murine model of infection. Each gC region of complement regulation contributed to virulence; however, the C3 binding domain was far more important, as virus lacking this domain was much less virulent than virus lacking the C5/P inhibitory domain and was as attenuated as virus lacking both domains. Studies in C3 knockout mice and mice reconstituted with C3 confirmed that the gC domains are inhibitors of complement activation, accounting for a 50-fold difference in virulence between mutant and wild-type viruses. We conclude that the C3 binding domain on gC is a major contributor to immune evasion and that this site explains at a molecular level why wild-type virus resists complement attack.

Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that inhibits both the classical and the alternative pathways of complement activation. DAF has been studied extensively in humans under two clinical settings: when absent from the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) patients, who suffer from complement-mediated hemolytic anemia, and in transgenic pigs expressing human DAF, which have been developed to help overcome complement-mediated hyperacute rejection in xenotransplantation. Nevertheless, the exact role of DAF in regulating complement activationin vivoon the cell surface and the species specificity of this molecule remain to be fully characterized. To address these issues, we have used gene targeting to produce mice lacking GPI-anchored DAF. We found that erythrocytes from mice deficient in GPI-anchored DAF showed no increase in spontaneous complement activationin vivobut exhibited impaired regulation of zymosan-initiated bystander and antibody-triggered classical pathway complement activationin vitro, resulting in enhanced complement deposition. Despite a high level of C3 fixation, no homologous hemolysis occurred. It is noteworthy that GPI-linked DAF knockout erythrocytes, when tested with human and guinea pig sera, were more susceptible to heterologous complement lysis than were normal erythrocytes. These results suggest that DAF is capable of regulating homologous as well as heterologous complement activation via the alternative or the classical pathway. They also indicate that DAF deficiency alone is not sufficient to cause homologous hemolysis. In contrast, when the assembly of the membrane-attack complex is not properly regulated, as in the case of heterologous complement activation or in PNH patients, impaired erythrocyte DAF activity and enhanced C3 deposition could lead to increased hemolytic reaction.

Compstatin, a newly described C3-binding peptide, inhibits complement activation by blocking C3 convertase-mediated cleavage of C3. As the complement activation is an essential part of the rejection reaction, we evaluated the ability of Compstatin to delay or prevent hyperacute rejection in an ex vivo xenograft model. Pig kidneys were perfused with fresh human blood containing either Compstatin (n=6) or a control agent (n=6). Graft survival and activation of complement, leukocytes and platelets both in the fluid-phase and in the tissue were examined. The survival of the Compstatin-perfused kidneys (median, 380 min) was significantly (P=0.0036) longer than that of the controls (median, 90 min). The classical complement pathway (C1rs-C1inhibitor and C4bc) was significantly and equally activated in both groups during the first 60 min. C3 activation products increased fivefold and terminal complement complex eightfold in the control group, but no increase occurred in the Compstatin group during this period. Immunohistochemistry showed less C3 and fibrin deposition and immune electron microscopy showed less terminal SC5b-9 complement complex deposition in the Compstatin group. A significant change in total white cells, neutrophils, myeloperoxidase, and expression of the surface activation markers CD11b (CR3) and CD35 (CR1) and CD62L (L-selectin) was observed in both groups. Leukocyte activation was lower in the Compstatin group but the difference was not statistically significant. There were no differences in platelet counts, thrombospondin, soluble P-selectin or beta-thromboglobulin between the groups. We conclude that Compstatin prolongs graft survival and suggest that it may be a useful agent for attenuating hyperacute rejection by inhibiting C3 and thus terminal complement pathway activation.

The third component of the complement system, C3, is a common denominator in the activation of the classical, alternative, and lectin pathways. The ability of C3 molecule to interact with at least 20 different proteins makes it the most versatile component of this system. Since these interactions are important for phagocytic, immunoregulatory, and immune evasion mechanisms, the analysis of its structure and functions has been a subject of intense research. Here we review our current work on the C3-ligand interactions, C3-related viral molecular mimicry, evolution of the complement system, and identification of C3-based complement inhibitors.

An intramolecular thioester bond in complement protein C3 is vital for covalent attachment of C3b (the proteolytically activated form of C3) to biological surfaces and for activation of the complement system. Proteolytic removal of C3a from C3 activates the thioester in the C3b fragment. Activated C3b primarily forms ester bonds with hydroxyl groups of carbohydrates on complement activating surfaces, but it has also been shown to react with the hydroxyl group of tyrosine and with specific Ser and Thr residues on IgG and on complement protein C4b. To examine the reactivity of the thioester, several families of hydroxylated compounds were examined. Reactivity of a series of substituted phenols varied over two orders of magnitude and demonstrated a linear correlation between reactivity and the Hammett substituent constants. Hydroxylated drugs including members of the L-DOPA/epinephrine family and hydroxamic acids also were examined. Compounds were identified that were 20,000 times more reactive than carbohydrates. These compounds were found to inhibit both the classical and alternative pathways of complement activation. Although the specificity of the thioester for its natural biological targets appears to be determined by many structural features, the data presented here demonstrate that increasing the nucleophilic character of the target hydroxyl group can increase the potency of a synthetic inhibitor many orders of magnitude.

Activation of the complement system on host cells is regulated primarily by a family of proteins termed as regulators of complement activation (RCA). A characteristic feature of the RCA family of proteins is the presence of repeating complement control protein (CCP) domains. Recent advances in genome sequencing resulted in availability of a large pool of sequences encoding CCP domain-containing proteins. However, their functional annotation could not be performed as CCP domains are not exclusive to RCA family (e.g., also present in proteins involved in complement activation, cell adhesion, coagulation, neurotransmission, cytokine signalling and blood clotting) and not all RCA family members are capable of regulating complement (e.g., Complement Receptor 2). In the present study, we therefore, sought to identify whether RCA proteins with complement regulatory function contain any sequence motif indicative of their function. In silico analysis of three N-terminal CCP domains of mammalian and viral RCA proteins resulted in identification of five motifs. These motifs are present in a specific order and are conserved spatially. Phylum-wide analysis of genomes for the presence of proteins with these motifs showed that they are present in CCP domain-containing proteins of a wide variety of organisms ranging from mammalia to nematoda, which included annotated and unannotated proteins for complement regulatory function. Thus, this approach seems to have broad-range sensitivity. To experimentally validate whether these motifs are indeed critical for complement regulatory activities, we performed functional characterization of human SVEP1 (Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1)/Polydom expressed in placenta, which contains these motifs, but has not been characterised to have complement regulatory function. The protein displayed the complement regulatory activities. We therefore propose that the sequence motifs we have identified are capable of distinguishing between regulatory and non-regulatory CCP domains and thus are apt for identifying new complement regulatory proteins in a continually expanding dataset of CCP containing proteins.

Objectives To evaluate the performance and treatment profile of advanced EML4—ALK positive Non-small cell lung cancer (NSCLC) patients in a developing country with potentially restricted access to Crizotinib. Materials and Methods A retrospective analysis of advanced ALK positive NSCLC patients who were treated from June 2012 to September 2015 was conducted. The primary goal was to evaluate outcomes of advanced ALK positive NSCLC in our practice and examine the logistic constraints in procuring Crizotinib. Results 94 patients were available for analysis. 21 (22.3%) patients were started on Crizotinib upfront, 60 (63.8%) on chemotherapy, 10 (10.6%) on Tyrosine kinase inhibitors (in view of poor PS) and 3 (3.2%) patients were offered best supportive care. Reasons for not starting Crizotinib upfront included symptomatic patients needing early initiation of therapy (23.3%), ALK not tested upfront (23.3%) and financial constraints (21.9%). 69 patients (73.4%) received Crizotinib at some stage during treatment. Dose interruptions (> 1 week) with Crizotinib were seen in 20 patients (29%), with drug toxicity being the commonest reason (85%). Median Progression free survival (PFS) on first line therapy for the entire cohort was 10 months, with a significant difference between patients receiving Crizotinib and those who did not ever receive Crizotinib (10 months vs. 2 months, p = 0.028). Median Overall Survival (OS) was not reached for the entire cohort, with 1 year survival being 81.2%. Patients with an ECOG Performance Status (PS) of >2 had a significantly reduced PFS compared to patients with PS < = 2 (1.5 months vs. 11 months, p< 0.001). 47 patients with financial constraints (68.1%) received Crizotinib completely free via various extramural support schemes. Conclusion A majority of our ALK positive NSCLC patients were exposed to Crizotinib through the help of various support mechanisms and these patients had similar outcomes to that reported from previously published literature.

Activation of C3 results in the generation of metastable C3b, which has been shown to preferentially react with the hydroxyl groups of carbohydrates and with specific serine and threonine residues in proteins. In this study we have examined the reactivity of metastable C3b with the third type of hydroxyl group present in proteins, tyrosine (Tyr). The results demonstrated that Tyr reacts with the thioester of metastable C3b and that this reactivity was 11-fold better than that of threonine, 47-fold better than serine and 50-fold better than the reactivity of carbohydrates. Model peptides containing Tyr showed even higher reactivity than free Tyr, demonstrating that incorporation into peptide structures does not block C3b attachment. The site of attachment was found to be in the alpha'-chain of C3b and the bond was hydroxylamine sensitive, indicating an ester linkage. The stability of the C3b-Tyr complex was measured under physiological conditions (pH 7.4, 37 degrees C) and compared to the stability of other C3b complexes. C3b-Tyr decayed 50% in 19 hr at 37 degrees C, but C3b bound to a Tyr-containing peptide was more stable, exhibiting a t1/2 of 53 hr. The ester linked complexes C3b-IgG and C3b-glycerol were less stable each exhibiting a t1/2 of approximately 8 hr. As yet, only two specific C3b attachment sites on proteins have been identified, Ser1217 in C4b and Thr144 in IgG1. The present evidence demonstrates that Tyr residues are highly reactive and that the C3b-Tyr linkage is stable. The findings suggest that complexes involving tyrosine residues as the site of attachment will also be found.