Lacticaseibacillus casei K11 exerts immunomodulatory effects by enhancing natural killer cell cytotoxicity via the extracellular regulated-protein kinase pathway.

PURPOSE: Probiotics can serve as immunomodulators that regulate the activation of immune cells. This study aimed to screen potential probiotic strains that can enhance NK cell toxicity to improve host immunity. METHODS: In this investigation, we examined three potential probiotic strains, namely Lactiplantibacillus plantarum YZX21 (YZX21), Bifidobacterium bifidum FL-276.1 (FL-276.1) and Lacticaseibacillus casei K11 (K11), to assess their capacity in modulating NK cytotoxicity both in vitro and in vivo, while elucidating the underlying mechanisms involved. RESULTS: The findings demonstrated that K11 exhibited superior efficacy in enhancing NK cytotoxicity. Subsequent analysis revealed that K11 significantly augmented the secretion of perforin and granzyme B by NK cells through activation of receptors NKp30 and NKp46 via the extracellular signal-regulated kinase (ERK) pathway. Furthermore, heat-inactivated K11 also enhanced NK cell activity to an extent comparable to live bacteria, with lipoteichoic acid from K11 identified as a crucial factor mediating the activation of NK cell cytotoxicity. CONCLUSION: Our study suggests that K11 may have potential applications as probiotics or postbiotics for regulating NK cell cytotoxicity to enhance immunity.

Antibody dependent cellular cytotoxicity-inducing anti-EGFR antibodies as effective therapeutic option for cutaneous melanoma resistant to BRAF inhibitors.

Introduction: About 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots. Methods and results: By using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors' and patients' peripheral blood cells. Conclusion: Our data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors.

Soluble MICA in endometriosis pathophysiology: Impairs NK cell degranulation and effector functions.

PROBLEM: Endometriosis exhibits several immune dysfunctions, including deficient natural killer (NK) cell cytotoxicity. MICA (MHC class I chain-related molecule A) is induced by biological stress and soluble MICA (sMICA) negatively modulates the expression of the activating receptor, NKG2D, reducing NK cells activities. We investigated the involvement of soluble MICA in NK cell-deficient activity in endometriosis. METHODS OF STUDY: sMICA levels (serum and peritoneal fluid-PF) were evaluated by ELISA. Circulating NK cell subsets quantification and its NKG2D receptor expression, NK cell cytotoxicity and CD107a, IFN-γ and IL-10 expressions by NK cells stimulated with K562 cells were determined by flow cytometry. RESULTS: We found higher sMICA levels (serum and PF) in endometriosis, especially in advanced and deep endometriosis. Endometriosis presented lower percentages of CD56dim CD16+ cytotoxic cells and impaired NK cell responses upon stimulation, resulting in lower CD107a and IFN-γ expressions, and deficient NK cell cytotoxicity. NK cell stimulation in the MICA-blocked condition (mimicking the effect of sMICA) showed decreased cytotoxicity in initial endometriosis stages and the emergence of a negative correlation between CD107a expression and sMICA levels. CONCLUSIONS: We suggest that soluble MICA is a potential player in endometriosis pathophysiology with involvement in disease progression and severity, contributing to NK cell impaired IFN-γ response and degranulation. NK cell compartment exhibits multiple perturbations, including quantitative deficiency and impaired cytotoxicity, contributing to inadequate elimination of ectopic endometrial tissue.

Clostridioides difficile Infection: Diagnosis and Treatment Challenges.

Clostridioides difficile is the most important cause of healthcare-associated diarrhea in the United States. The high incidence and recurrence rates of C. difficile infection (CDI), associated with high morbidity and mortality, pose a public health challenge. Although antibiotics targeting C. difficile bacteria are the first treatment choice, antibiotics also disrupt the indigenous gut flora and, therefore, create an environment that is favorable for recurrent CDI. The challenge of treating CDI is further exacerbated by the rise of antibiotic-resistant strains of C. difficile, placing it among the top five most urgent antibiotic resistance threats in the USA. The evolution of antibiotic resistance in C. difficile involves the acquisition of new resistance mechanisms, which can be shared among various bacterial species and different C. difficile strains within clinical and community settings. This review provides a summary of commonly used diagnostic tests and antibiotic treatment strategies for CDI. In addition, it discusses antibiotic treatment and its resistance mechanisms. This review aims to enhance our current understanding and pinpoint knowledge gaps in antimicrobial resistance mechanisms in C. difficile, with an emphasis on CDI therapies.

Anticarcinogenic Effects of Gold Nanoparticles and Metformin Against MCF-7 and A549 Cells.

Metformin is commonly prescribed to people with diabetes. Metformin has been shown in previous studies to be able to prevent the growth of cancer cells. This study aims to investigate the effects of metformin and gold nanoparticles in MCF7 breast cancer and A549 lung cell lines. The effects of metformin and gold nanoparticles on MCF7 breast cancer and A549 lung cells were determined on cells grown in 24 h cell culture. MCF-7 and A549 cells were incubated for 24 h with the treatment of escalating molar concentrations of ifosfamide. The MTT assay was used to determine the cytotoxicity of metformin toward MCF7 and A549 cell lines. The expression of Bax, BCL2, PI3K, Akt3, mTOR, Hsp60, Hsp70, and TNF-α was measured by RT-PCR. Metformin and gold nanoparticles inhibited the proliferation of MCF-7 and A549 cells in a dose and time-dependent manner with an IC50 value of 5 µM and 10 µg/mL. RT-PCR assays showed ifosfamide + metformin + gold nanoparticles significantly reduced the expression of BCL2, PI3K, Akt3, mTOR, Hsp60 and Hsp70 and increased the expression of TNF-α and Bax. The findings obtained in this study suggest that further studies should be conducted, and metformin and gold nanoparticles can be used in breast cancer and lung cancer treatments.

CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation.

BACKGROUND: The antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric antigen receptors (CARs). Despite antibody or CAR targeting, some tumors remain resistant towards NK cell attack. While the importance of ICAM-1/LFA-1 interaction for natural cytotoxicity of NK cells is known, its impact on ADCC induced by the ErbB2 (HER2)-specific antibody trastuzumab and ErbB2-CAR-mediated NK cell cytotoxicity against breast cancer cells has not been investigated. METHODS: Here we used NK-92 cells expressing high-affinity Fc receptor FcγRIIIa in combination with trastuzumab or ErbB2-CAR engineered NK-92 cells (NK-92/5.28.z) as well as primary human NK cells combined with trastuzumab or modified with the ErbB2-CAR and tested cytotoxicity against cancer cells varying in ICAM-1 expression or alternatively blocked LFA-1 on NK cells. Furthermore, we specifically stimulated Fc receptor, CAR and/or LFA-1 to study their crosstalk at the immunological synapse and their contribution to degranulation and intracellular signaling in antibody-targeted or CAR-targeted NK cells. RESULTS: Blockade of LFA-1 or absence of ICAM-1 significantly reduced cell killing and cytokine release during trastuzumab-mediated ADCC against ErbB2-positive breast cancer cells, but not so in CAR-targeted NK cells. Pretreatment with 5-aza-2'-deoxycytidine induced ICAM-1 upregulation and reversed NK cell resistance in ADCC. Trastuzumab alone did not sufficiently activate NK cells and required additional LFA-1 co-stimulation, while activation of the ErbB2-CAR in CAR-NK cells induced efficient degranulation independent of LFA-1. Total internal reflection fluorescence single molecule imaging revealed that CAR-NK cells formed an irregular immunological synapse with tumor cells that excluded ICAM-1, while trastuzumab formed typical peripheral supramolecular activation cluster (pSMAC) structures. Mechanistically, the absence of ICAM-1 did not affect cell-cell adhesion during ADCC, but rather resulted in decreased signaling via Pyk2 and ERK1/2, which was intrinsically provided by CAR-mediated targeting. Furthermore, while stimulation of the inhibitory NK cell checkpoint molecule NKG2A markedly reduced FcγRIIIa/LFA-1-mediated degranulation, retargeting by CAR was only marginally affected. CONCLUSIONS: Downregulation of ICAM-1 on breast cancer cells is a critical escape mechanism from trastuzumab-triggered ADCC. In contrast, CAR-NK cells are able to overcome cancer cell resistance caused by ICAM-1 reduction, highlighting the potential of CAR-NK cells in cancer immunotherapy.

RIPK3 promotes islet amyloid-induced ß-cell loss and glucose intolerance in a humanized mouse model of type 2 diabetes.

OBJECTIVE: Aggregation of human islet amyloid polypeptide (hIAPP), a ß-cell secretory product, leads to islet amyloid deposition, islet inflammation and ß-cell loss in type 2 diabetes (T2D), but the mechanisms that underlie this process are incompletely understood. Receptor interacting protein kinase 3 (RIPK3) is a pro-death signaling molecule that has recently been implicated in amyloid-associated brain pathology and ß-cell cytotoxicity. Here, we evaluated the role of RIPK3 in amyloid-induced ß-cell loss using a humanized mouse model of T2D that expresses hIAPP and is prone to islet amyloid formation. METHODS: We quantified amyloid deposition, cell death and caspase 3/7 activity in islets isolated from WT, Ripk3-/-, hIAPP and hIAPP; Ripk3-/- mice in real time, and evaluated hIAPP-stimulated inflammation in WT and Ripk3-/- bone marrow derived macrophages (BMDMs) in vitro. We also characterized the role of RIPK3 in glucose stimulated insulin secretion (GSIS) in vitro and in vivo. Finally, we examined the role of RIPK3 in high fat diet (HFD)-induced islet amyloid deposition, ß-cell loss and glucose homeostasis in vivo. RESULTS: We found that amyloid-prone hIAPP mouse islets exhibited increased cell death and caspase 3/7 activity compared to amyloid-free WT islets in vitro, and this was associated with increased RIPK3 expression. hIAPP; Ripk3-/- islets were protected from amyloid-induced cell death compared to hIAPP islets in vitro, although amyloid deposition and caspase 3/7 activity were not different between genotypes. We observed that macrophages are a source of Ripk3 expression in isolated islets, and that Ripk3-/- BMDMs were protected from hIAPP-stimulated inflammatory gene expression (Tnf, Il1b, Nos2). Following 52 weeks of HFD feeding, islet amyloid-prone hIAPP mice exhibited impaired glucose tolerance and decreased ß-cell area compared to WT mice in vivo, whereas hIAPP; Ripk3-/- mice were protected from these impairments. CONCLUSIONS: In conclusion, loss of RIPK3 protects from amyloid-induced inflammation and islet cell death in vitro and amyloid-induced ß-cell loss and glucose intolerance in vivo. We propose that therapies targeting RIPK3 may reduce islet inflammation and ß-cell loss and improve glucose homeostasis in the pathogenesis of T2D.

Impact of Wooden Breast myopathy on in vitro protein digestibility, metabolomic profile, and cell cytotoxicity of cooked chicken breast meat.

This study investigated the impacts of Wooden Breast (WB) abnormality on in vitro protein digestibility and cytotoxicity of cooked chicken breast meat. Chicken breasts without (non-WB, n = 6) or with severe WB condition (WB, n = 6) were cooked and subjected to static in vitro protein digestion. The results showed no significant differences in free-NH2, degree of hydrolysis and distribution of peptide molecular weight between non-WB and WB samples at late intestinal digestion (P5), suggesting no adverse effects of WB on protein digestibility. Based on peptidomic analysis, P5 fraction of WB showed greater content of peptides with oxidative modification than that of non-WB. Untargeted metabolomics did not find any metabolites with potential toxicity either in non-WB and WB. Hydrolyzed non-WB and WB (1.56-100 µg/mL) did not affect viability of Caco-2 and Vero cells but addition of WB samples reduced Caco-2 cell viability compared with non-WB.

Therapeutic applications of sustainable new chitosan derivatives and its nanocomposites: Fabrication and characterization.

Chitosan (CS) is a biologically active biopolymer used in different medical applications due to its biodegradability, biocompatibility, and nontoxicity. Nanotechnology is an exciting and quick developing field in medical applications. Nanoparticles have shown great potential in the treatment of cancer and inflammation. In the present work modification of chitosan and its (Ag, Au, or ZnO) nanocomposites by N-aminophthalimide (NAP) occurred through the reaction with epichlorohydrin (ECH) as a crosslinker in the presence or absence of glutaraldehyde (GA) under different reaction conditions using microwave irradiation to give modified chitosan derivatives CS-2, CS-6, and their nanocomposites. Modified chitosan derivatives were characterized using different tools. CS-2 and CS-6 derivatives displayed enhancement of thermal stability and crystallinity compared to chitosan. Additionally, CS-2, CS-6, and their nanocomposites exhibited improvements in antitumor activity against HeLa cancer cells and enzymatic inhibitory against trypsin and α-chymotrypsin enzymes compared to chitosan. However, CS-2 revealed the highest cell growth inhibition% toward HeLa cells (89.02 ± 1.46 %) and the enzymatic inhibitory toward α-chymotrypsin enzyme (17.13 ± 1.59 %). Furthermore, CS-Au-2 showed the highest enzymatic inhibitory against trypsin enzyme (28.14 ± 1.76 %). These results suggested that the new chitosan derivatives CS-2, CS-6, and their nanocomposites could be a platform for medical applications against HeLa cells, trypsin, and α-chymotrypsin enzymes.

Immunohistochemical expression of perforin in adult systemic lupus erythematosus associated macrophage activation syndrome: Clinicohematological correlation and literature review.

OBJECTIVE: To study the bone marrow (BM) immunohistomorphological characteristics in adult systemic lupus erythematosus (SLE) associated macrophage activation syndrome (SLE-MAS). MATERIALS AND METHODS: Immunohistochemical (IHC) expression of CD3, CD8, perforin (PFN), and CD163 was studied on BM trephine biopsies from 30 cytopenic adult SLE cases (male: female = 1:5, age; 24 years, range; 19-32) and compared them with ten age matched controls. Clinicopathological parameters were compared among the cases likely (L) or unlikely (U) to have MAS using probability scoring criteria. The best cut off laboratory parameters to discriminate between the two were obtained through receiver operator curve (ROC) analysis. RESULTS: MAS occurred in 12/30 (40%) cases and was more commonly associated with prior immunosuppressive therapy (p = .07), ≥ 3 system involvement (p = .09), lower fibrinogen (p < .01), increased triglyceride (p = .002), increased BM hemophagocytosis (p = .002), and higher MAS score [185 (176-203) vs. 105 (77-119), p < .01] than MAS-U subgroup. Although PFN+CD8+ T lymphocytes significantly decreased among cases than controls (p < .05), it was comparable between MAS-L and MAS-U subgroups. Fibrinogen (< 2.4 g/L, AUC; 0.93, p < .01), hemophagocytosis score (> 1.5, AUC; 0.71, p = .03), and an MAS probability score of ≥ 164 (AUC; 1, p < .01) discriminated MAS from those without MAS. CONCLUSION: We noted a decrease in perforin mediated CD8 + T cell cytotoxicity in SLE. Immunohistochemical demonstration of the same along with histiocytic hemophagocytosis on BM biopsy may be useful adjunct in early diagnosis and management of MAS in SLE.

The loss of B7-H4 expression in breast cancer cells escaping from T cell cytotoxicity contributes to epithelial-to-mesenchymal transition.

BACKGROUND: B7 homology 4 (B7-H4), a potential target for cancer therapy, has been demonstrated to inhibit T cell cytotoxicity in the early stages of breast cancer. However, B7-H4 manipulating breast tumor immune microenvironment (TIME) in the tumor progression remains unknown. METHODS: We engineered T cells with B7-H4-specific chimeric antigen receptors (CARs) and performed a T cell co-culture assay to characterize B7-H4 expression level in breast cancer cells escaping from T cell cytotoxicity. We generated B7-H4 knockout (KO) and overexpression (OE) breast cancer cells to determine the epithelial-to-mesenchymal transition (EMT) and stemness characteristics in vitro and in vivo, including tumor proliferation, migration, metastasis and chemoresistance. The Cancer Genome Atlas breast cancer database was accessed to investigate the correlation between B7-H4 expression levels and EMT characteristics in patients with breast cancer. RESULTS: Our result found that B7-H4 expression level was significantly reduced in a subset of breast cancer cells that escaped from the cytotoxicity of B7-H4 CAR-T cells. Compared with wild type cells, B7-H4 KO cells prompt EMT and stemness characteristics, including migration, invasion and metastasis, and OE cells vice versa. The increase in H3K27me3 in KO cells confirmed the epigenetic reprogramming of cancer stem cells. The IC50 of doxorubicin or oxaliplatin significantly increased in KO cells, which was in agreement with a decrease in OE cells. Moreover, a trend of downregulated B7-H4 from stage I to stage II breast cancer patients indicates that the low-expressing B7-H4 breast cancer cells escaping from TIME have spread to nearby breast lymph nodes in the cancer progression. CONCLUSIONS: Our study illuminates the novel role of renouncing B7-H4 in breast cancer cells through immune escape, which contributes to EMT processes and provides new insights for breast cancer treatments.

Scaling-up of carbon dots hydrothermal synthesis from sugars in a continuous flow microreactor system for biomedical application as in vitro antimicrobial drug nanocarrier.

Carbon dots (CDs) are a new class of nanomaterials exhibiting high biocompatibility, water solubility, functionality, and tunable fluorescence (FL) property. Due to the limitations of batch hydrothermal synthesis in terms of low CDs yield and long synthesis duration, this work aimed to increase its production capacity through a continuous flow reactor system. The influence of temperature and time was first studied in a batch reactor for glucose, xylose, sucrose and table sugar precursors. CDs synthesized from sucrose precursor exhibited the highest quantum yield (QY) (175.48%) and the average diameter less than 10 nm (~6.8 ± 1.1 nm) when synthesized at 220°C for 9 h. For a flow reactor system, the best condition for CDs production from sucrose was 1 mL min-1 flow rate at 280°C, and 0.2 MPa pressure yielding 53.03% QY and ~ 6.5 ± 0.6 nm average diameter (6.6 mg min-1 of CDs productivity). CDs were successfully used as ciprofloxacin (CP) nanocarrier for antimicrobial activity study. The cytotoxicity study showed that no effect of CDs on viability of L-929 fibroblast cells was detected until 1000 µg mL-1 CDs concentration. This finding demonstrates that CDs synthesized via a flow reactor system have a high zeta potential and suitable surface properties for nano-theranostic applications.

Endogenous H3.3K27M derived peptide restricted to HLA-A∗02:01 is insufficient for immune-targeting in diffuse midline glioma.

Diffuse midline glioma (DMG) is a childhood brain tumor with an extremely poor prognosis. Chimeric antigen receptor (CAR) T cell therapy has recently demonstrated some success in DMG, but there may a need to target multiple tumor-specific targets to avoid antigen escape. We developed a second-generation CAR targeting an HLA-A∗02:01 restricted histone 3K27M epitope in DMG, the target of previous peptide vaccination and T cell receptor-mimics. These CAR T cells demonstrated specific, titratable, binding to cells pulsed with the H3.3K27M peptide. However, we were unable to observe scFv binding, CAR T cell activation, or cytotoxic function against H3.3K27M+ patient-derived models. Despite using sensitive immunopeptidomics, we could not detect the H3.3K27M26-35-HLA-A∗02:01 peptide on these patient-derived models. Interestingly, other non-mutated peptides from DMG were detected bound to HLA-A∗02:01 and other class I molecules, including a novel HLA-A3-restricted peptide encompassing the K27M mutation and overlapping with the H3 K27M26-35-HLA-A∗02:01 peptide. These results suggest that targeting the H3 K27M26-35 mutation in context of HLA-A∗02:01 may not be a feasible immunotherapy strategy because of its lack of presentation. These findings should inform future investigations and clinical trials in DMG.

A Genetically Encoded Dark-to-Bright Biosensor for Visualisation of Granzyme-Mediated Cytotoxicity.

Granzyme B (GZMB) is a key enzyme released by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells to induce apoptosis in target cells. We designed a novel fluorogenic biosensor which is able to assess GZMB activity in a specific and sensitive manner. This cleavage-responsive sensor for T cell activity level (CRSTAL) is based on a fluorescent protein that is only activated upon cleavage by GZMB or caspase-8. CRSTAL was tested in stable cell lines and demonstrated a strong and long-lasting fluorescence signal upon induction with GZMB. It can detect GZMB activity not only by overexpression of GZMB in target cells but also following transfer of GZMB and perforin from effector cells during cytotoxicity. This feature has significant implications for cancer immunotherapy, particularly in monitoring the efficacy of chimeric antigen receptor (CAR)-T cells. CAR-T cells are a promising therapy option for various cancer types, but monitoring their activity in vivo is challenging. The development of biosensors like CRSTAL provides a valuable tool for monitoring of CAR-T cell activity. In summary, CRSTAL is a highly sensitive biosensor that can detect GZMB activity in target cells, providing a means for evaluating the cytotoxic activity of immune cells and monitoring T cell activity in real time.

Simultaneous modulation of CHO cell cytotoxicity, turbidity, and DOC by coagulation with or without pre-oxidation in water from the Pearl River Delta region, China.

Coagulation with or without pre-oxidation are important drinking water treatment processes. However, the efficacy of these processes in mitigating water toxicity remains unknown. To further improve drinking water safety, we employed water from the Pearl River Delta region of southern China to investigate a treatment approach consisting of coagulation with or without pre-oxidation to simultaneously modulate health-relevant cytotoxicity to CHO cells, on top of the conventional foci of turbidity and dissolved organic carbon (DOC) during water treatment. Three coagulants (two aluminum-based and one iron-based salts) and three pre-oxidants (ozone, permanganate, and peroxymonosulfate) were studied. For coagulation without pre-oxidation, intermediate coagulant doses and pH reached optimum cytotoxicity to CHO cells, turbidity, and DOC control simultaneously. Introducing oxidants reduced cytotoxicity to CHO cells significantly, enhanced by increasing oxidant concentrations and pre-oxidation duration. The cytotoxicity to CHO cells mitigation capabilities of three pre-oxidants were: ozone > peroxymonosulfate > potassium permanganate. Modulation of water cytotoxicity to CHO cells was mostly attributable to controlling DOC (specifically humic-acid like substances, tyrosine, tryptophan). However, the addition of pre-oxidants led to significant shifts in water cytotoxicity to CHO cells forcing drivers, rendering humic-acid like substances the sole decisive cytotoxicity-inducing fluorophores. For the first time, 'sweet spots' were identified to simultaneously monitor cytotoxicity to CHO cells alongside turbidity and DOC. These methods better modulate water cytotoxicity to CHO cells without sacrificing conventional water treatment goals while shedding light onto the mechanisms behind.

rWTC-MBTA: autologous vaccine prevents metastases via antitumor immune responses.

BACKGROUND: Autologous tumor cell-based vaccines (ATVs) aim to prevent and treat tumor metastasis by activating patient-specific tumor antigens to induce immune memory. However, their clinical efficacy is limited. Mannan-BAM (MB), a pathogen-associated molecular pattern (PAMP), can coordinate an innate immune response that recognizes and eliminates mannan-BAM-labeled tumor cells. TLR agonists and anti-CD40 antibodies (TA) can enhance the immune response by activating antigen-presenting cells (APCs) to present tumor antigens to the adaptive immune system. In this study, we investigated the efficacy and mechanism of action of rWTC-MBTA, an autologous whole tumor cell vaccine consisting of irradiated tumor cells (rWTC) pulsed with mannan-BAM, TLR agonists, and anti-CD40 antibody (MBTA), in preventing tumor metastasis in multiple animal models. METHODS: The efficacy of the rWTC-MBTA vaccine was evaluated in mice using breast (4T1) and melanoma (B16-F10) tumor models via subcutaneous and intravenous injection of tumor cells to induce metastasis. The vaccine's effect was also assessed in a postoperative breast tumor model (4T1) and tested in autologous and allogeneic syngeneic breast tumor models (4T1 and EMT6). Mechanistic investigations included immunohistochemistry, immunophenotyping analysis, ELISA, tumor-specific cytotoxicity testing, and T-cell depletion experiments. Biochemistry testing and histopathology of major tissues in vaccinated mice were also evaluated for potential systemic toxicity of the vaccine. RESULTS: The rWTC-MBTA vaccine effectively prevented metastasis and inhibited tumor growth in breast tumor and melanoma metastatic animal models. It also prevented tumor metastasis and prolonged survival in the postoperative breast tumor animal model. Cross-vaccination experiments revealed that the rWTC-MBTA vaccine prevented autologous tumor growth, but not allogeneic tumor growth. Mechanistic data demonstrated that the vaccine increased the percentage of antigen-presenting cells, induced effector and central memory cells, and enhanced CD4+ and CD8+ T-cell responses. T-cells obtained from mice that were vaccinated displayed tumor-specific cytotoxicity, as shown by enhanced tumor cell killing in co-culture experiments, accompanied by increased levels of Granzyme B, TNF-α, IFN-γ, and CD107a in T-cells. T-cell depletion experiments showed that the vaccine's antitumor efficacy depended on T-cells, especially CD4+ T-cells. Biochemistry testing and histopathology of major tissues in vaccinated mice revealed negligible systemic toxicity of the vaccine. CONCLUSION: The rWTC-MBTA vaccine demonstrated efficacy in multiple animal models through T-cell mediated cytotoxicity and has potential as a therapeutic option for preventing and treating tumor metastasis with minimal systemic toxicity.

IL-17A and Th17 Cells Contribute to Endometrial Cell Survival by Inhibiting Apoptosis and NK Cell Mediated Cytotoxicity of Endometrial Cells via ERK1/2 Pathway.

Immune status including the immune cells and cytokine profiles has been implicated in the development of endometriosis. In this study, we analyzed Th17 cells and IL-17A in peritoneal fluid (PF) and endometrial tissues of patients with (n=10) and without (n=26) endometriosis. Our study has shown increased Th17 cell population and IL-17A level in PF with endometriosis patients. To determine the roles of IL-17A and Th17 cells in the development of endometriosis, the effect of IL-17A, major cytokine of Th17, on endometrial cells isolated from endometriotic tissues was examined. Recombinant IL-17A promoted survival of endometrial cells accompanied by increased expression of anti-apoptotic genes, including Bcl-2 and MCL1, and the activation of ERK1/2 signaling. In addition, treatment of IL-17A to endometrial cells inhibited NK cell mediated cytotoxicity and induced HLA-G expression on endometrial cells. IL-17A also promoted migration of endometrial cells. Our data suggest that Th17 cells and IL-17A play critical roles in the development of endometriosis by promoting endometrial cell survival and conferring a resistance to NK cell cytotoxicity through the activation of ERK1/2 signaling. Targeting IL-17A has potential as a new strategy for the treatment of endometriosis.

Release Assays and Potency Assays for CAR T-Cell Interventions.

Chimeric antigen receptor (CAR) T-cells are considered "living drugs" and offer a compelling alternative to conventional anticancer therapies. Briefly, T-cells are redirected, using gene engineering technology, toward a specific cancer cell surface target antigen via a synthetic chimeric antigen receptor (CAR) protein. CARs have a modular design comprising four main structures: an antigen-binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains for T-cell activation. A major challenge in the CAR T-cell manufacturing field is balancing product quality with scalability and cost-effectiveness, especially when transitioning from an academic clinical trial into a marketed product, to be implemented across many collection, manufacturing, and treatment sites. Achieving product consistency while circumnavigating the intrinsic variability associated with autologous products is an additional barrier. To overcome these limitations, a robust understanding of the product and its biological actions is crucial to establish a target product profile with a defined list of critical quality attributes to be assessed for each batch prior to product certification. Additional challenges arise as the field progresses, such as new safety considerations associated with the use of allogenic T-cells and genome editing tools. In this chapter, we will discuss the release and potency assays required for CAR T-cell manufacturing, covering their relevance, current challenges, and future perspectives.

Peripheral blood natural killer cell cytotoxicity in recurrent miscarriage: a systematic review and meta-analysis.

Dysregulated natural killer (NK) cells have been associated with recurrent miscarriages (RM). Studies have suggested that high peripheral blood NK cell cytotoxicities (pNKCs) are associated with an increased risk of RM. The aim of this systematic review (SR) and meta-analysis (MAs) is to investigate the difference in pNKC between nonpregnant and pregnant women with RM and controls and determine whether pNKC is reduced by immunotherapy. We searched the PubMed/Medline, Embase, and Web of Science databases. The MAs were conducted to compare pNKCs between women with and without RM before and during pregnancy as well as pre- and post-immunotherapy. Risk of bias in nonrandomized studies was assessed by the Newcastle-Ottawa Scale. Statistical analysis was performed using the Review Manager software. A total of 19 studies were included in the SR and 14 studies in the MAs. The MAs revealed higher pNKCs among nonpregnant women with RM compared to controls (MD, 7.99 95 %CI 6.40-9.58; p < 0.00001). pNKCs were also higher in pregnant women with RM than in pregnant controls (MD, 8.21 95 %CI 6.08-10.34; p < 0.00001). Women with RM showed significantly decreased pNKCs after the immunotherapy compared to before (MD, -8.20 95 %CI -10.20 - -6.19; p < 0, 00001). Additionally, there is an association between high pNKCs and the risk of pregnancy loss in women with RM. However, included studies showed substantial heterogeneities regarding the inclusion criteria of patients, techniques measuring pNKC, and types of immunotherapies. More studies are needed to evaluate the clinical efficiency of pNKCs in managing RM.

Higher proinflammatory responses possibly contributing to suppressed cytotoxicity in patients with COVID-19 associated mucormycosis.

INTRODUCTION: COVID-19 Associated Mucormycosis (CAM), an opportunistic fungal infection, surged during the second wave of SARS Cov-2 pandemic. Since immune responses play an important role in controlling this infection in immunocompetent hosts, it is required to understand immune perturbations associated with this condition for devising immunotherapeutic strategies for its control. We conducted a study to determine different immune parameters altered in CAM cases as compared to COVID-19 patients without CAM. METHODOLOGY: Cytokine levels in serum samples of CAM cases (n = 29) and COVID-19 patients without CAM (n = 20) were determined using luminex assay. Flow cytometric assays were carried out in 20 CAM cases and 10 controls for determination of frequency of NK cells, DCs, phagocytes, T cells and their functionalities. The cytokine levels were analyzed for their association with each other as well as with T cell functionality. The immune parameters were also analyzed with respect to the known risk factors such as diabetes mellitus and steroid treatment. RESULTS: Significant reduction in frequencies of total and CD56 + CD16 + NK cells (cytotoxic subset) was noted in CAM cases. Degranulation responses indicative of cytotoxicity of T cell were significantly hampered in CAM cases as compared to the controls. Conversely, phagocytic functions showed no difference in CAM cases versus their controls except for migratory potential which was found to be enhanced in CAM cases. Levels of proinflammatory cytokines such as IFN-γ, IL-2, TNF-α, IL-17, IL-1ß, IL-18 and MCP-1 were significantly elevated in cases as compared to the control with IFN-γ and IL-18 levels correlating negatively with CD4 T cell cytotoxicity. Steroid administration was associated with higher frequency of CD56 + CD16- NK cells (cytokine producing subset) and higher MCP-1 levels. Whereas diabetic participants had higher phagocytic and chemotactic potential and had higher levels of IL-6, IL-17 and MCP-1. CONCLUSION: CAM cases differed from the controls in terms of higher titers of proinflammatory cytokines, reduced frequency of total and cytotoxic CD56 + CD16 + NK cell. They also had reduced T cell cytotoxicity correlating inversely with IFN-γ and IL-18 levels, possibly indicating induction of negative feedback mechanisms while diabetes mellitus or steroid administration did not affect the responses negatively.