Assessment of physicochemicalparameters and water quality index of reservoir water

ABSTRACT: The present study was undertaken to know the variation in different seasons in response to physicochemical properties of Lalpari reservoir situated at Rajkot City in Gujarat. The study was carried out over a period of one year. In India there are enormous number of natural andmanmade water bodies used for various purposes, mainly for drinking and agriculture. One of the most severe problems in arid and semi-arid regions is high concentration of salts in soils and water resources. Thus, water quality and its management have received much attention in developing countries.The present study is aimed at assessing the Water Quality Index of reservoir water and assesses the impact of industries and human activities. Physicochemical parameters were monitored for the calculation of Water Quality Index for the monsoon, winter and summerseasons.The analysis reveals that the surface water of the area needs some treatment before consumption; andit also needs to be protected from the perils of contamination

T cell repertoire to citrullinated self-peptides in healthy humans is not confined to the HLA-DR SE alleles; targeting of citrullinated self-peptides presented by HLA-DP4 for tumour therapy

Post-translational modifications are induced in stressed cells which cause them to be recognised by the immune system. One such modification is citrullination where the positive charged arginine is modified to a neutral citrulline. We demonstrate most healthy donors show an oligoclonal CD4 response in vitro to at least one citrullinated vimentin or enolase peptide. Unlike rheumatoid arthritis patients, these T cell responses were not restricted by HLA-DRB1 shared epitope (SE) alleles, suggesting they could be presented by other MHC-II alleles. As HLA-DP is less polymorphic than HLA-DR, we investigated whether the common allele, HLA-DP4 could present citrullinated epitopes. The modification of arginine to citrulline enhanced binding of the peptides to HLA-DP4 and induced high frequency CD4 responses in HLA-DP4 transgenic mouse models. Our previous studies have shown that tumours present citrullinated peptides restricted through HLA-DR4 which are good targets for anti-tumour immunity. In this study we show that citrullinated vimentin and enolase peptides also induced strong anti-tumour immunity (100% survival,

Impaired circulating myeloid CD1c+ dendritic cell function in human glioblastoma is restored by p38 inhibition - implications for the next generation of DC vaccines

Current treatments for glioblastoma (GBM) have limited efficacy and significant morbidity and therefore new strategies are urgently needed. Dendritic cells have the power to create anti-tumour immune responses. The greater potency of circulating dendritic cells (DC) over laboratory-generated monocyte-derived DC makes them exciting new immunotherapeutic candidates. To determine the immune status of GBM patients we initially investigated the frequency and function of circulating DC subsets. Furthermore, we tested the therapeutic potential of inhibiting the p38 mitogen-activated protein kinase pathway (p38i) in circulating DC to overcome DC dysfunction.GBM patients (n=16) had significantly reduced numbers of the major myeloid circulating dendritic cell (cDC2) and plasmacytoid DC vs healthy controls; 1736 vs 4975 (p=0.028) and 893 vs 2287 cells/mL (P

Citrullinated glucose-regulated protein 78 is a candidate target for melanoma immunotherapy

IntroductionPost translational modification of proteins plays a significant role in immune recognition. In particular the modification of arginine to citrulline which is mediated by PAD enzymes is increased during cellular stress (autophagy) which permits the presentation of modified epitopes upon MHC class II molecules for recognition by CD4 T cells. Citrullination also occurs in tumour cells as a result of continuous environmental stresses and increased autophagy. We have shown in animal models the efficient stimulation of citrullinated epitope specific CD4 T cells resulting in dramatic elimination/regression of tumours. The ER chaperone glucose-regulated protein 78 (GRP78) is known to also be required for stress-induced autophagy and is directly linked to autophagosome formation. GRP78 is known to be highly expressed by many tumour types. In this study we investigate the potential of targeting citrullinated GRP78 for cancer therapy.MethodsA citrullinated GRP78 specific antibody was used to assess citrullinated GRP78 expression in murine and human tumour cells by flow cytometry. Five peptides were selected and used to vaccinate HLA transgenic mice and immune responses were characterised by ex vivo cytokine ELISpot assay. T cell repertoire in humans was assessed through proliferation assays and cytokine ELISpot assay. Citrullinated peptide was identified in murine B16 melanoma by mass spectrometry and the peptide vaccine was assessed for tumour therapy in a mouse melanoma model.ResultsWe show the identification CD4 T cell responses to one citrullinated GRP78 epitope that are restricted through HLA DP*0401 and HLA-DR*0101 alleles. This peptide is detected by mass spectrometry in B16 melanoma grown in vivo and citrulline specific CD4 responses to two peptides spanning this epitope mediate efficient therapy of established B16 melanoma tumours in HHDII/DP4 (p<0.0001) transgenic mouse model. Finally, we demonstrate the existence of a repertoire of responses to the citrullinated GRP78 peptide in healthy individuals (p=0.0023) with 13/17 (76%) individuals showing a response to this peptide.ConclusionWe propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach

T cell recognition of stress-induced post-translational modifications

Background: Citrullination is the post-translational modification of arginine to citrulline mediated by peptidylarginine deiminases (PADs). We have previously shown that vaccination with citrullinated peptides from vimentin and α-enolase which are presented on HLA-DR4 or -DP4 in tumour cells mediate strong anti-tumour responses [1, 2]. This response is dependent upon autophagy and presentation of citrullinated peptides on MHC-II. Cytotoxic CD4 T cells can directly kill tumours expressing MHC-II and cognate peptide. As most tumours do not express MHC-II unless induced to do so by IFNγ we propose that citrullination is a normal stress response but that citrullinated epitopes are only presented on viable cells in the presence of inflammation. Results: Exposure to nutrient starvation induced citrullinated vimentin expression in the murine melanoma cell line B16F1 grown in vitro, measured using the anti-Citrullinated vimentin rabbit polyclonal anti-serum GB17002 (ECV-1), but was also associated with apoptosis. Interestingly, up to 70% of low density plated cells expressed citrullinated vimentin which was mostly cytoplasmic with some cases showing punctate staining, increased phagosome associated LC3-II and increased proliferation marker Ki67 compared to high density cultures, suggesting actively growing cells undergo more autophagy and have greater potential to express citrullinated epitopes. The role the HIPPO pathway and Epithelial-to-Mesenchymal transition (EMT) plays in citrullination was investigated however results were inconclusive and require further work. Approximately 27-55% of human cancers were positive for MHC-II. The expression of MHC-II was associated with survival advantage in breast cancer patients (P=0.004), and positively correlated with citrullinated vimentin (P<0.001), PAD2 (P<0.001), IFNγ receptor (P=0.003) and Ambra1 (P=0.016) in ovarian cancer. In a panel of triple negative breast cancer (TNBC) and ovarian cancer cell lines, 4/8 lines showed expression of MHC-II. This was enhanced following exposure to IFNγ which was dependent on activation of the Class II transactivator (CIITA) and is controlled by the pIV promoter. In addition, a strong correlation between IRF-1 and CIITA mRNA expression was found in 10 cancer types analysed from the TCGA database and in 4 cancer types CIITA expression alone correlated with good prognosis. Interestingly, IFNγ also increased citrullinated vimentin expression in the B16F1, suggesting this cytokine upregulates citrullination and MHC-II. Analysis of the human CD4 T cell repertoire responding to the citrullinated peptides, Vim28-49cit, Vim415-433cit and Eno241-260cit, showed oligoclonal proliferation of mainly T effector memory (Tem) cells but also an increase in the cytotoxic CD45RA expressing Tem population (TEMRA). In total, 28 high frequency TCRs were identified by RNA sequencing of proliferating CD4 T cells in response to Vim28cit (16), Eno241cit (4) and Vim415cit (8). These were transduced using lentiviral technology into Jurkat 76 and/or T cells and tested in antigen presentation assays to confirm their reactivity to citrullinated peptides by upregulation of the activation marker CD69 and the potent T cell stimulating cytokine, IFNγ. Despite numerous improvements to the lentiviral constructs, transfection/transduction methods and recognition assays, no positive responses were detected with any citrullinated peptide transduced TCRs. This suggests that the in vitro assays to detect citrulline specific T cell responses did not sufficiently expand the antigen specific T cells or that the TCR that did expand were of too low affinity for in vitro peptide recognition. Repeat stimulation assays to select for higher affinity more specific TCRs are required. Summary: Lack of cell-to-cell contact induced the greatest levels of citrullination in viable cells which were also highly proliferative and had upregulated autophagy. For stress induced post-translational modifications (siPTMs) to be recognised by T cells, MHC-II expression is also required. This data shows MHC-II is present on human tumour cells at low levels but can be further induced by the inflammatory cytokine IFNγ and is dependent on CIITA expression. Citrullinated peptides presented by MHC-II stimulated oligoclonal proliferation of CD4 T cells that expressed an effector phenotype. IFNγ released from these cells can induce MHC-II expression on tumours thus allowing presentation of siPTM epitopes. Citrullinated peptide vaccines have shown that siPTMs are good targets for immunotherapy but further results are required to determine if specific TCRs can be identified and used for adoptive T cell therapy

T cell recognition of stress-induced post-translational modifications

Background: Citrullination is the post-translational modification of arginine to citrulline mediated by peptidylarginine deiminases (PADs). We have previously shown that vaccination with citrullinated peptides from vimentin and α-enolase which are presented on HLA-DR4 or -DP4 in tumour cells mediate strong anti-tumour responses [1, 2]. This response is dependent upon autophagy and presentation of citrullinated peptides on MHC-II. Cytotoxic CD4 T cells can directly kill tumours expressing MHC-II and cognate peptide. As most tumours do not express MHC-II unless induced to do so by IFNγ we propose that citrullination is a normal stress response but that citrullinated epitopes are only presented on viable cells in the presence of inflammation. Results: Exposure to nutrient starvation induced citrullinated vimentin expression in the murine melanoma cell line B16F1 grown in vitro, measured using the anti-Citrullinated vimentin rabbit polyclonal anti-serum GB17002 (ECV-1), but was also associated with apoptosis. Interestingly, up to 70% of low density plated cells expressed citrullinated vimentin which was mostly cytoplasmic with some cases showing punctate staining, increased phagosome associated LC3-II and increased proliferation marker Ki67 compared to high density cultures, suggesting actively growing cells undergo more autophagy and have greater potential to express citrullinated epitopes. The role the HIPPO pathway and Epithelial-to-Mesenchymal transition (EMT) plays in citrullination was investigated however results were inconclusive and require further work. Approximately 27-55% of human cancers were positive for MHC-II. The expression of MHC-II was associated with survival advantage in breast cancer patients (P=0.004), and positively correlated with citrullinated vimentin (P<0.001), PAD2 (P<0.001), IFNγ receptor (P=0.003) and Ambra1 (P=0.016) in ovarian cancer. In a panel of triple negative breast cancer (TNBC) and ovarian cancer cell lines, 4/8 lines showed expression of MHC-II. This was enhanced following exposure to IFNγ which was dependent on activation of the Class II transactivator (CIITA) and is controlled by the pIV promoter. In addition, a strong correlation between IRF-1 and CIITA mRNA expression was found in 10 cancer types analysed from the TCGA database and in 4 cancer types CIITA expression alone correlated with good prognosis. Interestingly, IFNγ also increased citrullinated vimentin expression in the B16F1, suggesting this cytokine upregulates citrullination and MHC-II. Analysis of the human CD4 T cell repertoire responding to the citrullinated peptides, Vim28-49cit, Vim415-433cit and Eno241-260cit, showed oligoclonal proliferation of mainly T effector memory (Tem) cells but also an increase in the cytotoxic CD45RA expressing Tem population (TEMRA). In total, 28 high frequency TCRs were identified by RNA sequencing of proliferating CD4 T cells in response to Vim28cit (16), Eno241cit (4) and Vim415cit (8). These were transduced using lentiviral technology into Jurkat 76 and/or T cells and tested in antigen presentation assays to confirm their reactivity to citrullinated peptides by upregulation of the activation marker CD69 and the potent T cell stimulating cytokine, IFNγ. Despite numerous improvements to the lentiviral constructs, transfection/transduction methods and recognition assays, no positive responses were detected with any citrullinated peptide transduced TCRs. This suggests that the in vitro assays to detect citrulline specific T cell responses did not sufficiently expand the antigen specific T cells or that the TCR that did expand were of too low affinity for in vitro peptide recognition. Repeat stimulation assays to select for higher affinity more specific TCRs are required. Summary: Lack of cell-to-cell contact induced the greatest levels of citrullination in viable cells which were also highly proliferative and had upregulated autophagy. For stress induced post-translational modifications (siPTMs) to be recognised by T cells, MHC-II expression is also required. This data shows MHC-II is present on human tumour cells at low levels but can be further induced by the inflammatory cytokine IFNγ and is dependent on CIITA expression. Citrullinated peptides presented by MHC-II stimulated oligoclonal proliferation of CD4 T cells that expressed an effector phenotype. IFNγ released from these cells can induce MHC-II expression on tumours thus allowing presentation of siPTM epitopes. Citrullinated peptide vaccines have shown that siPTMs are good targets for immunotherapy but further results are required to determine if specific TCRs can be identified and used for adoptive T cell therapy

Combination vaccine based on citrullinated vimentin and enolase peptides induces potent CD4-mediated anti-tumor responses

Background Stress-induced post-translational modifications occur during autophagy and can result in generation of new epitopes and immune recognition. One such modification is the conversion of arginine to citrulline by peptidylarginine deiminase enzymes.Methods We used Human leukocyte antigen (HLA) transgenic mouse models to assess the immunogenicity of citrullinated peptide vaccine by cytokine Enzyme linked immunosorbant spot (ELISpot) assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma and ID8 ovarian models expressing either constitutive or interferon-gamma (IFNγ) inducible Major Histocompatibility Complex (MHC) class II (MHC-II) as represented by most human tumors. To determine the importance of CD4 T cells in tumor therapy, we analyzed the immune cell infiltrate into murine tumors using flow cytometry and performed therapy studies in the presence of CD4 and CD8 T cell depletion. We assessed the T cell repertoire to citrullinated peptides in ovarian cancer patients and healthy donors using flow cytometry.Results The combination of citrullinated vimentin and enolase peptides (Modi-1) stimulated strong CD4 T cell responses in mice. Responses resulted in a potent anti-tumor therapy against established tumors and generated immunological memory which protected against tumor rechallenge. Depletion of CD4, but not CD8 T cells, abrogated the primary anti-tumor response as well as the memory response to tumor rechallenge. This was further reinforced by successful tumor regression being associated with an increase in tumor-infiltrating CD4 T cells and a reduction in tumor-associated myeloid suppressor cells. The anti-tumor response also relied on direct CD4 T cell recognition as only tumors expressing MHC-II were rejected. A comparison of different Toll-like receptor (TLR)-stimulating adjuvants showed that Modi-1 induced strong Th1 responses when combined with granulocyte-macrophage colony-stimulating factor (GMCSF), TLR9/TLR4, TLR9, TLR3, TLR1/2 and TLR7 agonists. Direct linkage of the TLR1/2 agonist to the peptides allowed the vaccine dose to be reduced by 10-fold to 100-fold without loss of anti-tumor activity. Furthermore, a CD4 Th1 response to the citrullinated peptides was seen in ovarian cancer patients.Conclusions Modi-1 citrullinated peptide vaccine induces potent CD4-mediated anti-tumor responses in mouse models and a CD4 T cell repertoire is present in ovarian cancer patients to the citrullinated peptides suggesting that Modi-1 could be an effective vaccine for ovarian cancer patients

DataSheet_1_Citrullinated glucose-regulated protein 78 is a candidate target for melanoma immunotherapy.docx

IntroductionPost translational modification of proteins plays a significant role in immune recognition. In particular the modification of arginine to citrulline which is mediated by PAD enzymes is increased during cellular stress (autophagy) which permits the presentation of modified epitopes upon MHC class II molecules for recognition by CD4 T cells. Citrullination also occurs in tumour cells as a result of continuous environmental stresses and increased autophagy. We have shown in animal models the efficient stimulation of citrullinated epitope specific CD4 T cells resulting in dramatic elimination/regression of tumours. The ER chaperone glucose-regulated protein 78 (GRP78) is known to also be required for stress-induced autophagy and is directly linked to autophagosome formation. GRP78 is known to be highly expressed by many tumour types. In this study we investigate the potential of targeting citrullinated GRP78 for cancer therapy.MethodsA citrullinated GRP78 specific antibody was used to assess citrullinated GRP78 expression in murine and human tumour cells by flow cytometry. Five peptides were selected and used to vaccinate HLA transgenic mice and immune responses were characterised by ex vivo cytokine ELISpot assay. T cell repertoire in humans was assessed through proliferation assays and cytokine ELISpot assay. Citrullinated peptide was identified in murine B16 melanoma by mass spectrometry and the peptide vaccine was assessed for tumour therapy in a mouse melanoma model.ResultsWe show the identification CD4 T cell responses to one citrullinated GRP78 epitope that are restricted through HLA DP*0401 and HLA-DR*0101 alleles. This peptide is detected by mass spectrometry in B16 melanoma grown in vivo and citrulline specific CD4 responses to two peptides spanning this epitope mediate efficient therapy of established B16 melanoma tumours in HHDII/DP4 (pConclusionWe propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach.</p

Weight-bearing in ankle fractures: An audit of UK practice.

INTRODUCTION: The purpose of this national study was to audit the weight-bearing practice of orthopaedic services in the National Health Service (NHS) in the treatment of operatively and non-operatively treated ankle fractures. METHODS: A multicentre prospective two-week audit of all adult ankle fractures was conducted between July 3rd 2017 and July 17th 2017. Fractures were classified using the AO/OTA classification. Fractures fixed with syndesmosis screws or unstable fractures (>1 malleolus fractured or talar shift present) treated conservatively were excluded. No outcome data were collected. In line with NICE (The National Institute for Health and Care Excellence) criteria, "early" weight-bearing was defined as unrestricted weight-bearing on the affected leg within 3 weeks of injury or surgery and "delayed" weight-bearing as unrestricted weight-bearing permitted after 3 weeks. RESULTS: 251 collaborators from 81 NHS hospitals collected data: 531 patients were managed non-operatively and 276 operatively. The mean age was 52.6 years and 50.5 respectively. 81% of non-operatively managed patients were instructed for early weight-bearing as recommended by NICE. In contrast, only 21% of operatively managed patients were instructed for early weight-bearing. DISCUSSION: The majority of patients with uni-malleolar ankle fractures which are managed non-operatively are treated in accordance with NICE guidance. There is notable variability amongst and within NHS hospitals in the weight-bearing instructions given to patients with operatively managed ankle fractures. CONCLUSION: This study demonstrates community equipoise and suggests that the randomized study to determine the most effective strategy for postoperative weight-bearing in ankle fractures described in the NICE research recommendation is feasible