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Background: Carbapenem-resistant Enterobacterales (CRE) are an urgent global public health problem. CRE infections are associated with high mortality and have limited available effective treatment. Carbapenemase enzymes are encoded by genes on mobile genetic elements, such as plasmids, which are highly transmissible between organisms and increase the potential spread of resistance. The study aim was to detect carbapenemase production in Gram negative bacilli by phenotypic (CarbaNP and mCIM only or in conjunction with eCIM) and genotypic (Xpert Carba-R) methods. Material and methods: The various clinical samples were processed as per standard recommended procedures. Identification and antibiotic susceptibility test were done by using GN cards and AST N280 & AST N281 cards of Vitek 2 Compact (bioMérieux) respectively. Phenotypic (CarbaNP and mCIM only or in conjunction with eCIM) and genotypic (Xpert Carba-R) methods were used for detection of carbapenemases. Result: 144 carbapenem-resistant Gram-negative bacilli isolated from various ICUs includes Klebsiella pneumoniae 52.7% (76/144) followed by Escherichia coli 18.05% (26/144) and 15.9% (23/144) Acinetobacter baumannii. Phenotypic test (CarbaNP and mCIM and or in conjunction with eCIM) showed sensitivity of 90%, 100% and 93.75% respectively. Genotypic test of 40 isolates showed predominant expression of NDM in 82.5% (33/40) isolates followed by OXA-48 in 40% (16/40). Conclusion: The study showed mCIM as the most useful diagnostic test with less economic burden to the patients. There is an urgent need for more sensitive, rapid, highly precise and accurate genotypic test which is less expensive and less labor-intensive.

This study is focused on fractionation of insulin-like growth factor I (IGF-I) and transforming growth factor-β2 (TGF-β2) using a new electro-based membrane process calledelectrodialysis with filtration membranes (EDFM). Before EDFM, different pretreatments were tested, and four pH conditions (4.25, 3.85, 3.45, and 3.05) were used during EDFM. It was demonstrated that a 1:1 dilution of defatted colostrum with deionized water to decrease mineral content followed by the preconcentration of GFs by UF is necessary and allow for these compounds to migrate to the recovery compartment during EDFM. MS analyses confirmed the migration, in low quantity, of only α-lactalbumin (α-la) and β-lactoglobulin (β-lg) from serocolostrum to the recovery compartment during EDFM. Consequently, the ratio of GFs to total protein in recovery compartment compared to that of feed serocolostrum solution was 60× higher at pH value 3.05, the optimal pH favoring the migration of IGF-I and TGF-β2. Finally, these optimal conditions were tested on acid whey to also demonstrate the feasibility of the proposed process on one of the main by-products of the cheese industry; the ratio of GFs to total protein was 2.7× higher in recovery compartment than in feed acid whey solution, and only α-la migrated. The technology of GF enrichment for different dairy solutions by combining ultrafiltration and electrodialysis technologies was proposed for the first time.

Invariant natural killer T (iNKT) cells, a unique T cell population, lend themselves for use as adoptive therapy due to diverse roles in orchestrating immune responses. Originally developed for use in cancer, agenT-797 is a donor-unrestricted allogeneic ex vivo expanded iNKT cell therapy. We conducted an open-label study in virally induced acute respiratory distress syndrome (ARDS) caused by the severe acute respiratory syndrome-2 virus (trial registration NCT04582201). Here we show that agenT-797 rescues exhausted T cells and rapidly activates both innate and adaptive immunity. In 21 ventilated patients including 5 individuals receiving veno-venous extracorporeal membrane oxygenation (VV-ECMO), there are no dose-limiting toxicities. We observe an anti-inflammatory systemic cytokine response and infused iNKT cells are persistent during follow-up, inducing only transient donor-specific antibodies. Clinical signals of associated survival and prevention of secondary infections are evident. Cellular therapy using off-the-shelf iNKT cells is safe, can be rapidly scaled and is associated with an anti-inflammatory response. The safety and therapeutic potential of iNKT cells across diseases including infections and cancer, warrants randomized-controlled trials., (© 2024. The Author(s).)

Macrophages are crucial innate immune cells that maintain tissue homeostasis and defend against pathogens; however, their infiltration into tumors has been associated with adverse outcomes. Tumor-associated macrophages (TAMs) represent a significant component of the inflammatory infiltrate in breast tumors, and extensive infiltration of TAMs has been linked to poor prognosis in breast cancer. Here, we detail how TAMs impede a productive tumor immunity cycle by limiting antigen presentation and reducing activation of cytotoxic T lymphocytes (CTLs) while simultaneously supporting tumor cell survival, angiogenesis, and metastasis. There is an urgent need to overcome TAM-mediated immune suppression for durable anti-tumor immunity in breast cancer. To date, failure to fully characterize TAM biology and classify multiple subsets has hindered advancement in therapeutic targeting. In this regard, the complexity of TAMs has recently taken center stage owing to their subset diversity and tightly regulated molecular and metabolic phenotypes. In this review, we reveal major gaps in our knowledge of the functional and phenotypic characterization of TAM subsets associated with breast cancer, before and after treatment. Future work to characterize TAM subsets, location, and crosstalk with neighboring cells will be critical to counteract TAM pro-tumor functions and to identify novel TAM-modulating strategies and combinations that are likely to enhance current therapies and overcome chemo- and immuno-therapy resistance., Competing Interests: JG is a consultant for Glaxo-Smith Kline (GSK), Codagenix, Verseau, Kymera, and Array BioPharma and receives sponsored research support from GSK, Array BioPharma, and Eli Lilly. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mehta, Kadel, Townsend, Oliwa and Guerriero.)

Filtration membranes (FMs) are an integral part of electrodialysis with filtration membranes (EDFM), a green and promising technology for bioactive peptide fractionation. Therefore, it is paramount to understand how physicochemical properties of FMs impact global and selective peptide migration to anionic (A - RC ) peptide recovery compartments during their simultaneous separation by EDFM. In this context, six polyether sulfone (PES) membranes with molecular weight cut-offs (MWCO) of 5, 10, 20, 50, 100 and 300 kDa were characterized and used during EDFM to separate peptides from a complex whey protein hydrolysate. Surface charge, roughness, thickness and surface/pores nature of studied PES membranes were similar with small differences in conductivity, porosity and pore size distribution. Interestingly, global peptides migration to both recovery compartments increased linearly as a function of MWCO. However, peptide selectivity changed according to the recovery compartments and/or the peptide's charge and MW with an increase in MWCO of FMs. Indeed, in A + RC ) peptide recovery compartments during their simultaneous separation by EDFM. In this context, six polyether sulfone (PES) membranes with molecular weight cut-offs (MWCO) of 5, 10, 20, 50, 100 and 300 kDa were characterized and used during EDFM to separate peptides from a complex whey protein hydrolysate. Surface charge, roughness, thickness and surface/pores nature of studied PES membranes were similar with small differences in conductivity, porosity and pore size distribution. Interestingly, global peptides migration to both recovery compartments increased linearly as a function of MWCO. However, peptide selectivity changed according to the recovery compartments and/or the peptide's charge and MW with an increase in MWCO of FMs. Indeed, in A - RC , the relative abundance (RA) of peptides having low negative charge and MW (IDALNENK and VLVLDTDYK) decreased (45% to 19%) with an increase in MWCO, while the opposite for peptides having high negative charge and MW (TPEVDDEALEK, TPEVDDEALEKFDK & VYVEELKPTPEGDLEILLQK) (increased from 16% to 43%). Concurrently, in C + RC , regardless of MWCO used, the highest RA was observed for peptides having low positive charge and MW (IPAVFK & ALPMHIR). It was the first time that the significant impact of charge, MWCO and pore size distribution of PES membranes on a wide range of MWCO was demonstrated on EDFM performances.

Acute respiratory disease caused by influenza viruses is imperfectly mitigated by annual vaccination to select strains. Development of vaccines that elicit lung-resident memory CD8 + T cells (T RM ) would offer more universal protection to seasonal and emerging pandemic viruses. Understanding how lung-resident dendritic cells (DCs) regulate T RM differentiation would be an important step in this process. Here, we used CD11c-cre-Irf4 f/f (KO) mice, which lack lung-resident IRF4-dependent CD11b + CD24 hi DCs and show IRF4 deficiency in other lung cDC subsets, to determine if IRF4-expressing DCs regulate CD8 + memory precursor cells and T RM during influenza A virus (IAV) infection. KO mice showed defective CD8 + T-cell memory, stemming from a deficit of T regulatory cells and memory precursor cells with decreased Foxo1 expression. Transfer of wild-type CD11b + CD24 hi DCs into KO mice restored CD8 + memory precursor cell numbers to wild-type levels. KO mice recovered from a primary infection harbored reduced numbers of CD8 + T RM and showed deficient expansion of IFNγ + CD8 + T cells and increased lung pathology upon challenge with heterosubtypic IAV. Thus, vaccination strategies that harness the function of IRF4-dependent DCs could promote the differentiation of CD8 + T RM during IAV infection.

Sex differences in the incidence and severity of respiratory virus infection are widely documented in humans and murine models and correlate with sex biases in numbers and/or functional responses of innate immune cells in homeostasis and lung infection. Similarly, changes in sex hormone levels upon puberty, pregnancy, and menopause/aging are associated with qualitative and quantitative differences in innate immunity. Immune cells express receptors for estrogens (ERα and ERβ), androgens (AR), and progesterone (PR), and experimental manipulation of sex hormone levels or receptors has revealed that sex hormone receptor activity often underlies sex differences in immune cell numbers and/or functional responses in the respiratory tract. While elegant studies have defined mechanistic roles for sex hormones and receptors in innate immune cells, much remains to be learned about the cellular and molecular mechanisms of action of ER, PR, and AR in myeloid cells and innate lymphocytes to promote the initiation and resolution of antiviral immunity in the lung. Here, we review the literature on sex differences and sex hormone regulation in innate immune cells in the lung in homeostasis and upon respiratory virus infection.

Humans show significant sex differences in the incidence and severity of respiratory diseases, including asthma and virus infection. Sex hormones contribute to the female sex bias in type 2 inflammation associated with respiratory diseases, consistent with recent reports that female lungs harbor greater numbers of GATA-3-dependent group 2 innate lymphoid cells (ILC2s). In this study, we determined whether sex hormone levels govern sex differences in the numbers, phenotype, and function of ILC2s in the murine lung and bone marrow (BM). Our data show that lungs of female mice harbor significantly greater ILC2 numbers in homeostasis, in part due to a major subset of ILC2s lacking killer-cell lectin like receptor G1 (KLRG1), a population largely absent in male lungs. The KLRG1 - ILC2s were capable of type 2 cytokine production and increased with age after sexual maturity, suggesting that a unique functional subset exists in females. Experiments with gonadectomized mice or mice bearing either global or lymphocyte restricted estrogen receptor α ( Esr1 ) deficiency showed that androgens rather than estrogens regulated numbers of the KLRG1 - ILC2 subset and ILC2 functional capacity in the lung and BM, as well as levels of GATA-3 expression in BM ILC2s. Furthermore, the frequency of BM PLZF + ILC precursors was higher in males and increased by excess androgens, suggesting that androgens act to inhibit the transition of ILC precursors to ILC2s. Taken together, these data show that a functional subset of KLRG1 - ILC2s in females contributes to the sex bias in lung ILC2s that is observed after reproductive age., Competing Interests: DISCLOSURES The authors have no financial conflicts of interest.

Purpose: What is perceived to be a disability is both culturally specific and related to levels of development and modernity. This paper explores knowledge and attitudes towards people with disabilities among rural women in Nepal, one of the poorer countries in South Asia., Method: Four hundred and twelve married women of reproductive age (aged 15-49 years), from four villages in two different parts of Nepal, who had delivered a child within the last 24 months preceding the study, completed a standard questionnaire., Results: The majority of the participants only considered physical conditions that limit function of an individual and are visible to naked eyes, such as missing a leg or arm, to be disability. Attitudes towards people with disability were generally positive, for example most women believed that disabled people should have equal rights and should be allowed to sit on committees or get married. Most respondents thought that disability could result from: (i) accidents; (ii) medical conditions; or (iii) genetic inheritance. Fewer women thought that disability was caused by fate or bad spirits., Conclusions: There is need to educate the general population on disability, especially the invisible disabilities. There is also a need for further research on disability and its social impact., Implications for Rehabilitation: • There is need to educate the general population on disability, especially the invisible disabilities and its rehabilitation. There is also a need for further research on disability and its social impact.

Cutaneous melanoma occurs in both familial and sporadic forms. We investigated a melanoma-prone family through linkage analysis and high-throughput sequencing and identified a disease-segregating germline mutation in the promoter of the telomerase reverse transcriptase (TERT) gene, which encodes the catalytic subunit of telomerase. The mutation creates a new binding motif for Ets transcription factors and ternary complex factors (TCFs) near the transcription start and, in reporter gene assays, caused up to twofold increase in transcription. We then screened the TERT promoter in sporadic melanoma and observed recurrent ultraviolet signature somatic mutations in 125 of 168 (74%) of human cell lines derived from metastatic melanomas, 45 of 53 corresponding metastatic tumor tissues (85%), and 25 of 77 (33%) primary melanomas. The majority of those mutations occurred at two positions in the TERT promoter and also generated binding motifs for Ets/TCF transcription factors.

Cartilage acidic protein 1 (CRTAC1), a novel human marker which allowed discrimination of human chondrocytes from osteoblasts and mesenchymal stem cells in culture was so far studied only on the RNA-level. We here describe its genomic organisation and detect a new brain expressed (CRTAC1-B) isoform resulting from alternate last exon usage which is highly conserved in vertebrates. In humans, we identify an exon sharing process with the neighbouring tail-to-tail orientated gene leading to CRTAC1-A. This isoform is produced by cultured human chondrocytes, localized in the extracellular matrix of articular cartilage and its secretion can be stimulated by BMP4. Of five putative O-glycosylation motifs in the last exon of CRTAC1-A, the most C-terminal one is modified according to exposure of serial C-terminal deletion mutants to the O-glycosylation inhibitor Benzyl-alpha-GalNAc. Both isoforms contain four FG-GAP repeat domains and an RGD integrin binding motif, suggesting cell-cell or cell-matrix interaction potential. In summary, CRTAC1 acquired an alternate last exon from the tail-to-tail oriented neighbouring gene in humans resulting in the glycosylated isoform CRTAC1-A which represents a new extracellular matrix molecule of articular cartilage.

Leukocyte recruitment to lymph nodes or inflammatory sites is regulated by adhesion and activation. L-selectin (CD62L) is expressed on leukocytes and mediates tethering and rolling of leukocytes on endothelial cells. Upon stimulation L-selectin is down-regulated by proteolytic cleavage but the molecular mechanisms regulating this shedding step are poorly defined. To study intracellular mechanisms, we induced shedding of L-selectin by cross-linking with an immobilized L-selectin antibody (Dreg56) in Jurkat cells. The loss of surface expression was quantitated by flow cytometry and the increase of soluble L-selectin was determined by Western blot analysis. We find that Jurkat and p56(lck)-deficient JCaM1.6 cells released L-selectin to similar extent (18+/-4% and 17+/-3%, respectively) and revealed comparable inhibition with the src-tyrosine kinase inhibitor PP2. Glutathione (GSH), an inhibitor of the neutral sphingomyelinase, PD98059, a MAP-kinase (MAP-K) inhibitor and metalloprotease inhibitors (MPI) (TAPI, Ro 31-9790, and BB-3103) reduced significantly L-selectin-induced shedding by 60-80%. In Jurkat cells, L-selectin was present in Triton X-100 insoluble membrane rafts and was constitutively tyr-phosphorylated. Dreg56 cross-linking enhanced phosphorylation and recruitment of L-selectin into rafts which was significantly decreased by pretreatment of cells with PD98059. We conclude, that the metalloproteinase-mediated cleavage of L-selectin from cell surface is triggered by intracellular signaling pathways that are independent of p56(lck) tyrosine kinase activity, but require other tyrosine kinases and the neutral sphingomyelinase. The cleavage of L-selectin might involve membrane rafts as signaling platform.

Selectins are mediating transient contacts of leukocytes with endothelium during inflammatory processes and in the development of the immune system. L-selectin expressed on almost all leukocytes also functions as a signaling receptor. Recently, we have identified different signaling pathways in T lymphocytes by L-selectin. One signaling cascade leads via the tyrosine kinase p56lck to the small G-proteins Ras and Rac and to MAP-kinases. A second independent pathway results in ceramide release. In this study, an L-selectin-induced translocation of the transcription factor NFAT to the nucleus was identified. Using genetically modified JCaM1.6 cells, pharmacological inhibitors, and antisense molecules, it was shown that L-selectin-induced NFAT activation depends on src-tyrosine kinases, calcineurin and small G-proteins. MAP-kinases and actin filaments were identified as Ras effectors involved in NFAT translocation. We conclude that L-selectin cross-linking results in activation of NFAT by different signaling pathways. The activation of NFAT might modulate the immune response of leukocytes interacting with endothelial cells., (©2002 Elsevier Science (USA).)

L-Selectin-mediated rolling of leukocytes on endothelial cells is an important step for lymphocyte homing and an early event in the immune response to pathogens or inflammatory stimuli. We have previously elucidated intracellular signaling cascades upon L-selectin engagement resulting in activation of Ras, Rac and JNK as well as cytoskeletal changes, oxygen release, ceramide synthesis and receptor capping. Activation of the src-tyrosine kinase p56lck is followed by phosphorylation of the L-selectin molecule and MAP-K. Here we show a tyrosine kinase dependent phosphorylation of the Cbl adapter protein after L-selectin engagement in lymphocytes. Phosphorylation of Cbl was absent in Jurkat cells that are pharmacologically treated with tyrosine kinase inhibitors and in lck-deficient JCaM cells. There is an activation induced association of tyrosine phosphorylated Cbl with Grb2 and CrkL, respectively, but not CrkII. Therefore, the adapter protein Cbl plays a role in L-selectin signaling and might modulate immune function by the specific recruitment of signaling molecules to multiprotein complexes., (Copyright 2001 Academic Press.)

Intraalveolar leukocyte accumulation is one of the hallmarks during respiratory distress. In the intraalveolar space, leukocyte activation is mediated by pathogens, cytokines, and different ligands binding to adhesion molecules. Leukocyte stimulation via the adhesion molecule L-selectin is specifically induced by ligands expressed on leukocytes, platelets, endothelial cells, or lipopolysaccharide. Recently, we have demonstrated that leukocyte activation by L-selectin transmits several intracellular signaling cascades resulting in capping and cytoskeletal changes, the activation of kinases and neutral sphingomyelinase, the recruitment of adaptor proteins to the cell membrane, the activation of the small G-proteins Ras and Rac, and the release of oxygen. In the present study, we examined the effects of surfactant on L-selectin-induced signal transduction in leukocytes. Using fluorescence microscopy, we provide evidence that preincubation of leukocytes with surfactant significantly inhibits receptor capping; 28+/-7% of cells show capping after L-selectin stimulation versus 8+/-5% and 3+/-1% of cells after preincubation with Exosurf and Curosurf, respectively (p < 0.05). The activity of the neutral sphingomyelinase in cell lysates is also modulated by surfactant. In addition, we show that the activation of the tyrosine kinase p56lck is diminished by approximately 50% after surfactant treatment. This results in inhibition in tyrosine phosphorylation of certain intracellular proteins. The interaction of the L-selectin molecule with its antibody was not influenced by surfactant as shown by flow cytometry. Surfactant inhibits intracellular signaling events of the L-selectin receptor in leukocytes and might therefore contribute to the modulatory effects of surfactant on immune function.