Phase I study targeting newly diagnosed grade 4 astrocytoma with bispecific antibody armed T cells (EGFR BATs) in combination with radiation and temozolomide.

PURPOSE: The purpose of this study was to determine the safety, feasibility, and immunologic responses of treating grade 4 astrocytomas with multiple infusions of anti-CD3 x anti-EGFR bispecific antibody (EGFRBi) armed T cells (EGFR BATs) in combination with radiation and chemotherapy. METHODS: This phase I study used a 3 + 3 dose escalation design to test the safety and feasibility of intravenously infused EGFR BATs in combination with radiation and temozolomide (TMZ) in patients with newly diagnosed grade 4 astrocytomas (AG4). After finding the feasible dose, an expansion cohort with unmethylated O6-methylguanine-DNA methyltransferase (MGMT) tumors received weekly EGFR BATs without TMZ. RESULTS: The highest feasible dose was 80 × 109 EGFR BATs without dose-limiting toxicities (DLTs) in seven patients. We could not escalate the dose because of the limited T-cell expansion. There were no DLTs in the additional cohort of three patients with unmethylated MGMT tumors who received eight weekly infusions of EGFR BATs without TMZ. EGFR BATs infusions induced increases in glioma specific anti-tumor cytotoxicity by peripheral blood mononuclear cells (p < 0.03) and NK cell activity (p < 0.002) ex vivo, and increased serum concentrations of IFN-γ (p < 0.03), IL-2 (p < 0.007), and GM-CSF (p < 0.009). CONCLUSION: Targeting AG4 with EGFR BATs at the maximum feasible dose of 80 × 109, with or without TMZ was safe and induced significant anti-tumor-specific immune responses. These results support further clinical trials to examine the efficacy of this adoptive cell therapy in patients with MGMT-unmethylated GBM. CLINICALTRIALS: gov Identifier: NCT03344250.

Novel PE and APC tandems: Additional near-infrared fluorochromes for use in spectral flow cytometry.

Recent advances in flow cytometry instrumentation and fluorochrome chemistries have greatly increased fluorescent conjugated antibody combinations that can be used reliably and easily in routine experiments. The Cytek Aurora flow cytometer was first released with three excitation lasers (405, 488, and 640 nm) and incorporated the latest Avalanche Photodiode (APD) technology, demonstrating significant improvement in sensitivity for fluorescent emission signals longer than 800 nm. However, there are limited commercially available fluorochromes capable of excitation with peak emission signals beyond 800 nm. To address this gap, we engineered six new fluorochromes: PE-750, PE-800, PE-830 for the 488 nm laser and APC-750, APC-800, APC-830 for the 640 nm laser. Utilizing the principal of fluorescence resonance energy transfer (FRET), these novel structures were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Additionally, each of these fluorochrome conjugates were shown to be compatible with fixation/permeabilization buffer reagents, and demonstrated acceptable brightness and stability when conjugated to antigen-specific monoclonal antibodies. These six novel fluorochrome reagents can increase the numbers of fluorochromes that can be used on a spectral flow cytometer.

Anti-tumor and immune modulating activity of T cell induced tumor-targeting effectors (TITE).

Adoptive transfer of Bispecific antibody Armed activated T cells (BATs) showed promising anti-tumor activity in clinical trials in solid tumors. The cytotoxic activity of BATs occurs upon engagement with tumor cells via the bispecific antibody (BiAb) bridge, which stimulates BATs to release cytotoxic molecules, cytokines, chemokines, and other signaling molecules extracellularly. We hypothesized that the release of BATs Induced Tumor-Targeting Effectors (TITE) by this complex interaction of T cells, bispecific antibody, and tumor cells may serve as a potent anti-tumor and immune-activating immunotherapeutic approach. In a 3D tumorsphere model, TITE showed potent cytotoxic activity against multiple breast cancer cell lines compared to control conditioned media (CM): Tumor-CM (T-CM), BATs-CM (B-CM), BiAb Armed PBMC-CM (BAP-CM) or PBMC-CM (P-CM). Multiplex cytokine analysis showed high levels of Th1 cytokines and chemokines; phospho-protein signaling array data suggest that the prominent JAK1/STAT1 pathway may be responsible for the induction and release of Th1 cytokines/chemokines in TITE. In xenograft breast cancer models, IV injections of 10× concentrated TITE (3×/week for 3 weeks; 150 µl TITE/injection) was able to inhibit tumor growth significantly (ICR/scid, p < 0.003; NSG p < 0.008) compared to the control mice. We tested the key components of the TITE for immune activating and anti-tumor activity individually and in combinations, the combination of IFN-γ, TNF-α and MIP-1ß recapitulates the key activities of the TITE. In summary, master mix of active components of BATs-Tumor complex-derived TITE can provide a clinically controllable cell-free platform to target various tumor types regardless of the heterogeneous nature of the tumor cells and mutational tumor.

Acute Kidney Injury in Hospitalized Children with COVID19.

BACKGROUND: Acute kidney injury (AKI) has been recognized as a significant risk factor for mortality among adults with severe acute respiratory syndrome coronavirus infection. AIM: The aim of this study is to assess the prevalence and risk factors for AKI and mortality in children with coronavirus disease 2019 (COVID19) from a resource-limited setting. METHODS: Cross-sectional analysis of laboratory confirmed COVID19 children admitted from 1 March to 30 November 2020 in a tertiary care hospital in New Delhi, India was done. Clinical features and associated comorbidities of COVID19 were noted. Baseline serum creatinine (height-independent Hoste's equation) and peak serum creatinine were used for staging of AKI by the 2012 Kidney Disease Improving Global Outcomes serum creatinine criteria. Univariate analysis and Kaplan-Meier survival analysis were used to compare the overall outcome in the AKI vs. the non-AKI group. RESULTS: A total of 64 810 children between 1 month and 18 years visited the hospital; 3412 were tested for suspected COVID19, 295 tested positive and 105 (54% boys) were hospitalized. Twenty-four hospitalized children (22.8%) developed AKI; 8 in Stage 1 (33.3%), 7 in Stage 2 (29.2%) and 9 in Stage 3 (37.5%) respectively. Overall, three patients received KRT. Highest reported mortality was (66.6%) in AKI Stage 3. Risk factors for AKI included associated sepsis (OR 95% CI, 1.22-9.43, p < 0.01), nephrotic syndrome (OR 95% CI, 1.13-115.5, p < 0.01), vasopressor support (OR 3.59, 95% CI, 1.37-9.40, p value< 0.007), shock at presentation (OR 2.98, 95% CI, 1.16-7.60, p value 0.01) and mechanical ventilation (OR 2.64, 95% CI, 1.04-6.71, p value< 0.03). Mortality (25.71%) was higher in the AKI group (OR 95% CI, 1.14-8.35, p < 0.023) with shock (OR 45.92; 95% CI, 3.44-612.0, p value <0.004) and ventilation (OR 46.24; 95% CI, 1.6-1333.0 p value< 0.02) as significant risk factors for mortality. CONCLUSION: AKI is an important modifiable risk factor for mortality in children with COVID19 in a resource-limited setting. Our study supports the strengthening of kidney replacement therapy and its timely initiation to reduce the progression of AKI and thus mortality in children.

Granzyme B Is a Biomarker for Suspicion of Malignant Seromas Around Breast Implants.

BACKGROUND: Granzyme B (GrB) is a serine protease secreted, along with pore-forming perforin, by cytotoxic lymphocytes to mediate apoptosis in target cells. GrB has been detected in tumor cells associated with systemic and breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) but its potential use for detection of early BIA-ALCL has not been fully investigated. OBJECTIVES: Prompted by the increased incidence of BIA-ALCL, the aim of this study was to assess GrB as a new biomarker to detect early disease in malignant seromas and to better understand the nature of the neoplastic cell. METHODS: A Human XL Cytokine Discovery Magnetic Luminex 45-plex Fixed Panel Performance Assay was used to compare cytokine levels in cell culture supernatants of BIA-ALCL and other T-cell lymphomas, as well as malignant and benign seromas surrounding breast implants. Immunohistochemistry was employed to localize GrB to cells in seromas and capsular infiltrates. RESULTS: Differences in GrB concentrations between malignant and benign seromas were significant (P < 0.001). GrB was found in and around apoptotic tumor cells, suggesting that the protease may be involved in tumor cell death. CONCLUSIONS: GrB is a useful marker for early detection of malignant seromas and to identify tumor cells in seromas and capsular infiltrates. Because there is an overlap between the lowest concentrations of soluble GrB in malignant seromas and the highest concentrations of GrB in benign seromas, it is recommended that GrB be used only as part of a panel of biomarkers for the screening and early detection of BIA-ALCL.

Determinants of Expenditure on Diabetes Care: A Community Based Longitudinal Study in aResettlement Colony of Delhi.

INTRODUCTION: Diabetes is one of the expensive diseases due to its chronic nature and gradual involvement of multiple organs, Moreover loss of economic productivity further enhances the cost of care. Several factors were reported to have impact on overall economic burden in diabetic patients. So, the present study aims to determine influence of various socio-demographic and clinical factors on expenditure of diabetes care among patients residing in resettlement colony of East Delhi. METHODOLOGY: A community based one year longitudinal study was conducted in Kalyanpuri area of East Delhi. All the diabetes patients aged 25 years and who were the permanent residents of Kalyanpuri, attending the Diabetic Clinic of a government hospital in November-December 2014 were selected for the study. A pre-tested semi-structured interview schedule was used as study tool. Each subject was followed up 3 monthly from January to December 2015. RESULTS: Data of 150 study subjects was analyzed. Out of 150 subjects 45(30 %) were male and 105 (70%) female. Overall mean age of study subjects was 53 ± 10 years Among socio-demographic factors, Expenditure on diabetes care showed significant association with male gender and among clinical factors, longer duration since diagnosis, use of Insulin with Oral Hypoglycemic drugs, hospitalization and utilization of private care has shown positive association with expenditure on diabetes care. CONCLUSION: The present study concludes that there is need of better provisioning of services for diabetes care in government health facilities to cater needs of growing diabetic population..

Riboswitch regulation in cyanobacteria is independent of their habitat adaptations.

Cyanobacteria are one of the ancient bacterial species occupying a variety of habitats with diverse metabolic preferences. RNA regulators like riboswitches play significant role in controlling the gene expression in prokaryotes. The taxonomic distribution of riboswitches suggests that they might be one of the oldest mechanisms of gene control system. In this paper, we analyzed the distribution of different riboswitch families in various cyanobacterial genomes. It was observed that only four riboswitch classes were abundant in cyanobacteria, B12-element (Cob)/AdoCbl/AdoCbl-variant riboswitch being the most abundant. The analysis suggests that riboswitch mode of regulation is present in cyanobacterial species irrespective of their habitat types. A large number of unidentified genes regulated by riboswitches listed in this analysis indicate the wide range of targets for these riboswitch families. The analysis revealed a large number of genes regulated by riboswitches which may assist in elaborating the diversity among the cyanobacterial species.

Targeting CD138-/CD20+ Clonogenic Myeloma Precursor Cells Decreases These Cells and Induces Transferable Antimyeloma Immunity.

This phase Ib clinical trial evaluated whether pretargeting of CD20(+) clonogenic myeloma precursor cells (CMPCs) with anti-CD3 × anti-CD20 bispecific antibody-armed T cells (BATs) before autologous stem cell transplantation (SCT) in patients with standard-risk and high-risk multiple myeloma would induce antimyeloma immunity that could be detected and boosted after SCT. All 12 patients enrolled in this study received 2 BATs infusions before SCT, and 4 patients received a booster infusion of BATs after SCT. Pretargeting CD138(-)/CD20(+) CMPCs with BATs before SCT was safe and reduced levels of CMPCs by up to 58% in the postinfusion bone marrow in patients who remained in remission. Four of 5 patients who remained in remission had a >5-fold increase in IFN-γ enzyme-linked immunospot responses. SOX2 antibody increased after BATs infusions and persisted after SCT. The median anti-SOX2 level at 3 months after SCT was 28.1 ng/mL (range, 4.6 to 256 ng/mL) in patients who relapsed and 46 ng/mL (range, 28.3 to 73.3 ng/mL) in patients who remained in remission. The immune correlates suggest that infusions of targeted T cells given before SCT were able to reduce CMPC levels and induced cellular and humoral antimyeloma immunity that could be transferred and boosted after SCT.

Knowledge and attitude of doctors and nurses regarding indication for catheterization and prevention of catheter-associated urinary tract infection in a tertiary care hospital.

BACKGROUND AND AIMS: Catheter-associated urinary tract infection (CAUTI) is one of the most common health care acquired infection encountered in clinical practice. The present study was planned to assess the knowledge and attitude of health care providers regarding the indications for catheterization and methods of preventing CAUTI. METHODS: A prospective questionnaire-based survey was done from March 2011 to August 2011. A structured questionnaire comprising of 41 items related to demographic details of the respondents, their knowledge regarding indications for catheterization and methods of preventing CAUTI was given to 54 doctors and 105 nurses. The response was evaluated for statistical correlation using a computer software. RESULTS: The mean years of experience of the respondents in the health care setup was 6.8 years. Only 57% of the respondents could identify all the measures for prevention of CAUTI. The knowledge regarding the indication for catheterization though suboptimal was significantly better amongst the doctors as compared to nurses. CONCLUSION: The knowledge regarding indication and preventive measures was suboptimal in our study group. There is a tremendous scope of improvement in catheterization practices in our hospital and education induced intervention would be the most appropriate effort toward reducing the incidence of CAUTI.

Ipilimumab augments antitumor activity of bispecific antibody-armed T cells.

BACKGROUND: Ipilimumab is an antagonistic monoclonal antibody against cytotoxic T-lymphocyte antigen-4 (CTLA-4) that enhances antitumor immunity by inhibiting immunosuppressive activity of regulatory T cells (Treg). In this study, we investigated whether inhibiting Treg activity with ipilimumab during ex vivo T cell expansion could augment anti-CD3-driven T cell proliferation and enhance bispecific antibody (BiAb)-redirected antitumor cytotoxicity of activated T cells (ATC). METHODS: PBMC from healthy individuals were stimulated with anti-CD3 monoclonal antibody with or without ipilimumab and expanded for 10-14 days. ATC were harvested and armed with anti-CD3 x anti-EGFR BiAb (EGFRBi) or anti-CD3 x anti-CD20 BiAb (CD20Bi) to test for redirected cytotoxicity against COLO356/FG pancreatic cancer cell line or Burkitt's lymphoma cell line (Daudi). RESULTS: In PBMC from healthy individuals, the addition of ipilimumab at the initiation of culture significantly enhanced T cell proliferation (p = 0.0029). ATC grown in the presence of ipilimumab showed significantly increased mean tumor-specific cytotoxicity at effector:target (E:T) ratio of 25:1 directed at COLO356/FG and Daudi by 37.71% (p < 0.0004) and 27.5% (p < 0.0004), respectively, and increased the secretion of chemokines (CCL2, CCL3, CCL4,CCL5, CXCL9, and granulocyte-macrophage colony stimulating factor(GM-CSF)) and cytokines (IFN-γ, IL-2R, IL-12, and IL-13), while reducing IL-10 secretion. CONCLUSIONS: Expansion of ATC in the presence of ipilimumab significantly improves not only the T cell proliferation but it also enhances cytokine secretion and the specific cytotoxicity of T cells armed with bispecific antibodies.

Targeted cancer immunotherapy via combination of designer bispecific antibody and novel gene-engineered T cells.

BACKGROUND: Redirection of T lymphocytes against tumor antigens can induce dramatic regression of advanced stage malignancy. The use of bispecific antibodies (BsAbs) that bind both the T-cell receptor (TCR) and a target antigen is one promising approach to T-cell redirection. However, BsAbs indiscriminately bind all CD3+ T-cells and trigger TCR activation in the absence of parallel costimulatory signals required to overcome T-cell unresponsiveness or anergy. METHODS: To address these limitations, a combination platform was designed wherein a unique BsAb referred to as frBsAb exclusively engages T-cells engineered to express a novel chimeric receptor comprised of extracellular folate receptor fused to intracellular TCR and CD28 costimulatory signaling domains in tandem; a BsAb-binding immune receptor (BsAb-IR). As a surrogate TCR, the BsAb-IR allows for concomitant TCR and costimulatory signaling exclusively in transduced T-cells upon engagement with specific frBsAbs, and can therefore redirect T-cells on command to desired antigen. Human primary T-cells were transduced with lentiviral vector and expanded for 14-18 days. BsAb-IRs were harvested and armed with frBsAbs to test for redirected cytotoxicity against CD20 positive cancer cell lines. RESULTS: Using frBsAbs specific for CD20 or HER2, the lytic activity of primary human T-cells expressing the BsAb-IR was specifically redirected against CD20+ leukemic cells or HER2+ epithelial cancer cells, respectively, while non-engineered T-cells were not activated. Notably, elimination of the CD28 costimulatory domain from the BsAb-IR construct significantly reduced frBsAb-redirected antitumor responses, confirming that frBsAbs are capable of delivering simultaneous TCR activation and costimulatory signals to BsAb-IR T-cells. CONCLUSION: In summary, our results establish the proof of concept that the combination of BsAbs with optimized gene-engineered T-cells provides the opportunity to specify and augment tumor antigen-specific T-cell activation and may improve upon the early success of conventional BsAbs in cancer immunotherapy.

Targeting refractory/recurrent neuroblastoma and osteosarcoma with anti-CD3×anti-GD2 bispecific antibody armed T cells.

BACKGROUND: The survival benefit observed in children with neuroblastoma (NB) and minimal residual disease who received treatment with anti-GD2 monoclonal antibodies prompted our investigation into the safety and potential clinical benefits of anti-CD3×anti-GD2 bispecific antibody (GD2Bi) armed T cells (GD2BATs). Preclinical studies demonstrated the high cytotoxicity of GD2BATs against GD2+cell lines, leading to the initiation of a phase I/II study in recurrent/refractory patients. METHODS: The 3+3 dose escalation phase I study (NCT02173093) encompassed nine evaluable patients with NB (n=5), osteosarcoma (n=3), and desmoplastic small round cell tumors (n=1). Patients received twice-weekly infusions of GD2BATs at 40, 80, or 160×106 GD2BATs/kg/infusion complemented by daily interleukin-2 (300,000 IU/m2) and twice-weekly granulocyte macrophage colony-stimulating factor (250 µg/m2). The phase II segment focused on patients with NB at the dose 3 level of 160×106 GD2BATs/kg/infusion. RESULTS: Of the 12 patients enrolled, 9 completed therapy in phase I with no dose-limiting toxicities. Mild and manageable cytokine release syndrome occurred in all patients, presenting as grade 2-3 fevers/chills, headaches, and occasional hypotension up to 72 hours after GD2BAT infusions. GD2-antibody-associated pain was minimal. Median overall survival (OS) for phase I and the limited phase II was 18.0 and 31.2 months, respectively, with a combined OS of 21.1 months. A phase I NB patient had a complete bone marrow response with overall stable disease. In phase II, 10 of 12 patients were evaluable: 1 achieved partial response, and 3 showed clinical benefit with prolonged stable disease. Over 50% of evaluable patients exhibited augmented immune responses to GD2+targets post-GD2BATs, as indicated by interferon-gamma (IFN-γ) EliSpots, Th1 cytokines, and/or chemokines. CONCLUSIONS: This study demonstrated the safety of GD2BATs up to 160×106 cells/kg/infusion. Coupled with evidence of post-treatment endogenous immune responses, our findings support further investigation of GD2BATs in larger phase II clinical trials.

Race-related Differences in Sipuleucel-T Response Among Men with Metastatic Castrate-Resistant Prostate Cancer.

PURPOSE: Sipuleucel-T is an autologous cellular immunotherapy that targets prostatic acid phosphatase (PAP) and is available for treatment of men with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). In this single-arm, two-cohort, multicenter clinical study, potential racial differences in immune responses to sipuleucel-T in men with mCRPC were explored. PATIENTS AND METHODS: Patients' blood samples were obtained to assess serum cytokines, humoral responses, and cellular immunity markers pre- and post-treatment. Baseline cumulative product parameters (total nucleated and CD54+ cell counts, and CD54 upregulation) were evaluated. IgM titers against the immunogen PA2024, the target antigen PAP, prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA) were quantified by ELISA. Cytotoxic T lymphocyte activity was determined by ELISpots, and cytokine and chemokine concentrations by Luminex. RESULTS: Twenty-nine African Americans (AA) and 28 non-African Americans (non-AA) with mCRPC received sipuleucel-T. Baseline total nucleated cell count, CD54+ cell count, CD54 expression, and cumulative product parameters were higher in non-AA. Although PSA baseline levels were higher in AA, there were no racial differences in IgM antibody and IFN- ELISpots responses against PA2024, PAP, PSA and PSMA pre- and post-treatment. Expression of co-stimulatory receptor ICOS on CD4+ and CD8+ T cells, and the levels of Th1 cytokine granulocyte-macrophage colony-stimulating factor and chemokines CCL4 and CCL5, were significantly higher in AA pre- and/or post-treatment. Despite no difference in the overall survival, PSA changes from baseline were significantly different between the two races. CONCLUSIONS: The data suggest that immune correlates in blood differ in AA and non-AA with mCRPC pre- and post-sipuleucel-T.

The immunological contribution of NF-κB within the tumor microenvironment: a potential protective role of zinc as an anti-tumor agent.

Over decades, cancer treatment has been mainly focused on targeting cancer cells and not much attention to host tumor microenvironment. Recent advances suggest that the tumor microenvironment requires in-depth investigation for understanding the interactions between tumor cell biology and immunobiology in order to optimize therapeutic approaches. Tumor microenvironment consists of cancer cells and tumor associated reactive fibroblasts, infiltrating non-cancer cells, secreted soluble factors or molecules, and non-cellular support materials. Tumor associated host immune cells such as Th(1), Th(2), Th17, regulatory cells, dendritic cells, macrophages, and myeloid-derived suppressor cells are major components of the tumor microenvironment. Accumulating evidence suggests that these tumor associated immune cells may play important roles in cancer development and progression. However, the exact functions of these cells in the tumor microenvironment are poorly understood. In the tumor microenvironment, NF-κB plays an important role in cancer development and progression because this is a major transcription factor which regulates immune functions within the tumor microenvironment. In this review, we will focus our discussion on the immunological contribution of NF-κB in tumor associated host immune cells within the tumor microenvironment. We will also discuss the potential protective role of zinc, a well-known immune response mediator, in the regulation of these immune cells and cancer cells in the tumor microenvironment especially because zinc could be useful for conditioning the tumor microenvironment toward innovative cancer therapy.

CD20-targeted T cells after stem cell transplantation for high risk and refractory non-Hodgkin's lymphoma.

A phase I trial of infusing anti-CD3 × anti-CD20 bispecific antibody (CD20Bi) armed activated T cells (aATC) was conducted in high-risk/refractory non-Hodgkin's lymphoma patients to determine whether aATC infusions are safe, affect immune recovery, and induce an antilymphoma effect. Ex vivo expanded ATC from 12 patients were armed with anti-CD20 bispecific antibody, cryopreserved, and infused after autologous stem cell transplantation (SCT). Patients underwent SCT after high-dose chemotherapy, and aATC infusions were started on day +4. The patients received 1 infusion of aATC per week for 4 weeks after SCT with doses of 5, 10, 15, and 20 × 10(9). aATC infusions were safe and did not impair engraftment. The major side effects were chills, fever, hypotension, and fatigue. The mean number of IFN-γ Enzyme-linked Immunosorbent Spots (ElSpots) directed at CD20 positive lymphoma cells (DAUDI, P = .0098) and natural killer cell targets (K562, P < .0051) and the mean specific cytotoxicity directed at DAUDI (P = .037) and K562 (P = .002) from pre-SCT to post-SCT were significantly higher. The increase in IFN-γ EliSpots from pre-SCT to post-SCT in patients who received armed ATC after SCT were significantly higher than those in patients who received SCT alone (P = .02). Serum IL-7, IL-15, Macrophage inflammatory protein (MIP)-1 beta, IP-10, MIP-1α, and Monokine induced by gamma interferone increased within hours after infusion. Polyclonal and specific antibodies were near normal 3 months after SCT. aATC infusions were safe and increased innate and specific antilymphoma cell immunity without impairing antibody recovery after SCT.

Generation and immunologic functions of Th17 cells in malignant gliomas.

Th17 cells, a recently discovered inflammatory T cell subtype, have been implicated with autoimmune disorders. However, mechanism of generation or functions of intratumoral Th17 cells are still unclear. We have been investigating the mechanism of induction and role of Th17 cells in malignant gliomas using primary tumor as well as cell lines. We report here that: (1) a higher frequency of Th17 cells in gliomas were associated with higher number of myeloid (CD11b) cells as well as the expression of TGF-ß1 or IL-6; (2) conditioned medium from glioma cells (Gl CM) induced Th17 cell differentiation, which was inhibited by anti-TGF-ß1 and anti-IL-6; (3) glioma-associated monocytes secreted Th17-promoting cytokines IL-1ß and IL-23; (4) CM from glioma and monocyte co-culture (Gl+Mo CM) induced high frequency of Th17 cells in naïve T cell culture, which was abrogated by anti-IL-1ß and anti-IL-23 antibodies; (5) In vitro Gl+Mo CM-mediated Th17 generation was associated with a decrease in IFN-γ and a concomitant increase in IL-10 secretion. Anti-TGF-ß1, but not anti-IL-6, significantly reversed this cytokine profile. These results demonstrate prevalence of Th17 cells in gliomas and implicate the cytokines derived from the tumor as well as infiltrating myeloid cells in the induction of Th17 cells in glioma microenvironment. Moreover, the data also suggest that glioma-associated Th17 cells may contribute to immune-suppression via TGF-ß1-induced IL-10 secretion. Further studies on the mechanism of tumor-infiltration, developmental pathways, and pro-/anti-tumor functions of Th17 cells will provide rationale for developing novel adjuvant immunotherapeutic strategies for malignant gliomas.

Microenvironment generated during EGFR targeted killing of pancreatic tumor cells by ATC inhibits myeloid-derived suppressor cells through COX2 and PGE2 dependent pathway.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are one of the major components of the immune-suppressive network, play key roles in tumor progression and limit therapeutic responses. Recently, we reported that tumor spheres formed by breast cancer cell lines were visibly smaller in a Th1 enriched microenvironment with significantly reduced differentiation of MDSC populations in 3D culture. In this study, we investigated the mechanism(s) of bispecific antibody armed ATC mediated inhibition of MDSC in the presence or absence of Th1 microenvironment. METHODS: We used 3D co-culture model of peripheral blood mononuclear cells (PBMC) with pancreatic cancer cells MiaPaCa-2 [MiaE] and gemcitabine resistant MiaPaCa-GR [MiaM] cells to generate MDSC in the presence or absence of Th1 cytokines and EGFRBi armed ATC (aATC). RESULTS: We show significantly decreased differentiation of MDSC (MiaE, p<0.005; MiaM, p<0.05) in the presence of aATC with or without Th1 cytokines. MDSC recovered from control cultures (without aATC) showed potent ability to suppress T cell functions compared to those recovered from aATC containing co-cultures. Reduced accumulation of MDSC was accompanied by significantly lower levels of COX2 (p<0.0048), PGE2 (p<0.03), and their downstream effector molecule Arginase-1 (p<0.01), and significantly higher levels of TNF-α, IL-12 and chemokines CCL3, CCL4, CCL5, CXCL9 and CXCL10 under aATC induced Th1 cytokine enriched microenvironment. CONCLUSIONS: These data suggest aATC can suppress MDSC differentiation and attenuation of their suppressive activity through down regulation of COX2, PGE2 and ARG1 pathway that is potentiated in presence of Th1 cytokines, suggesting that Th1 enriching immunotherapy may be beneficial in pancreatic cancer treatment.

Targeting and killing of glioblastoma with activated T cells armed with bispecific antibodies.

BACKGROUND: Since most glioblastomas express both wild-type EGFR and EGFRvIII as well as HER2/neu, they are excellent targets for activated T cells (ATC) armed with bispecific antibodies (BiAbs) that target EGFR and HER2. METHODS: ATC were generated from PBMC activated for 14 days with anti-CD3 monoclonal antibody in the presence of interleukin-2 and armed with chemically heteroconjugated anti-CD3 × anti-HER2/neu (HER2Bi) and/or anti-CD3 × anti-EGFR (EGFRBi). HER2Bi- and/or EGFRBi-armed ATC were examined for in vitro cytotoxicity using MTT and 51Cr-release assays against malignant glioma lines (U87MG, U118MG, and U251MG) and primary glioblastoma lines. RESULTS: EGFRBi-armed ATC killed up to 85% of U87, U118, and U251 targets at effector:target ratios (E:T) ranging from 1:1 to 25:1. Engagement of tumor by EGFRBi-armed ATC induced Th1 and Th2 cytokine secretion by armed ATC. HER2Bi-armed ATC exhibited comparable cytotoxicity against U118 and U251, but did not kill HER2-negative U87 cells. HER2Bi- or EGFRBi-armed ATC exhibited 50--80% cytotoxicity against four primary glioblastoma lines as well as a temozolomide (TMZ)-resistant variant of U251. Both CD133- and CD133+ subpopulations were killed by armed ATC. Targeting both HER2Bi and EGFRBi simultaneously showed enhanced efficacy than arming with a single BiAb. Armed ATC maintained effectiveness after irradiation and in the presence of TMZ at a therapeutic concentration and were capable of killing multiple targets. CONCLUSION: High-grade gliomas are suitable for specific targeting by armed ATC. These data, together with additional animal studies, may provide the preclinical support for the use of armed ATC as a valuable addition to current treatment regimens.

Assessment of knowledge, attitude and practices among healthcare workers in a tertiary care hospital on needle stick injury.

PURPOSE: The study aims to assess healthcare workers' needle-stick injury (NSI) knowledge, attitudes and practices (KAP). DESIGN/METHODOLOGY/APPROACH: A cross-sectional study was conducted in a 600-bedded hospital throughout six months. The data were collected using an anonymous, self-reporting questionnaire. Participants were various healthcare workers (HCW) drawn through stratified random sampling and their knowledge, attitude and practice regarding NSI were assessed. FINDINGS: There is significant difference in the mean knowledge, attitude and practice scores among healthcare workers. Even though scores are better for doctors and nurses, practice scores were better for technical staff. Healthcare workers, who had better practice scores, had suffered fewer NSIs. Since this study is a cross-sectional, the population's NSI incidence could not be calculated. PRACTICAL IMPLICATIONS: This study emphasizes that applying knowledge to practice is required to prevent NSIs. Various recommendations to help prevent and deal with NSIs are made. ORIGINALITY/VALUE: This study analyses healthcare workers' NSI knowledge, attitude and practices, and also assesses their correlation with NSI incidence, which has not been done previously.

SARS-CoV-2 Infection in Children with Idiopathic Nephrotic Syndrome: A Multicentric Study.

A multicenter retrospective study was conducted to assess the clinical spectrum of 30 severe acute respiratory syndrome coronavirus (SARS-CoV-2)-positive children with idiopathic nephrotic syndrome. Difficult to treat nephrotic syndrome was found to be a high-risk group with a high incidence of acute kidney injury and mortality.