Immunity, Inflammation and Disease

Background: Gastric carcinoma remains a major cause of cancer related mortality worldwide, with tumor progression increasingly recognized as a consequence of complex interactions within the tumor microenvironment. Hypoxia induced signaling, cancer associated fibroblast (CAF) heterogeneity, and immune checkpoint activation play critical roles in tumor progression and immune evasion. However, their integrated relationship in gastric carcinoma remains insufficiently characterized. Objectives: To evaluate the expression of Hypoxia inducible factor 1 alpha and its association with cancer-associated fibroblast subtypes and Programmed death-ligand 1 expression in gastric carcinoma. Methods: This cross sectional analytical study included 100 histologically confirmed gastric carcinoma cases from Satkhira Medical College. Immunohistochemistry was performed for HIF 1 alpha, smooth muscle actin (SMA), fibroblast activation protein (FAP), and PD L1. CAFs were subclassified into myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs). Associations between biomarkers and clinicopathological variables were analyzed using chi square test, Spearman correlation, and multivariate logistic regression. Receiver operating characteristic (ROC) curve analysis was used to assess model performance. Result: High HIF 1 alpha expression was observed in 55% of cases and demonstrated significant association with poor differentiation (p = 0.001), advanced tumor stage (p = 0.002), and lymph node metastasis (p = 0.001). iCAF predominance was significantly associated with poor differentiation (p = 0.003), advanced stage (p = 0.004), and nodal metastasis (p = 0.004). High PD L1 expression was significantly associated with poor differentiation (p = 0.03), advanced stage (p = 0.001), and lymph node metastasis (p = 0.002). Multivariate logistic regression identified high HIF 1 alpha expression (OR = 3.8, p = 0.001), iCAF dominance (OR = 4.5, p < 0.001), and advanced tumor stage (OR = 2.9, p = 0.004) as independent predictors of high PD L1 expression. Combined high HIF 1 alpha expression and CAF activation demonstrated the highest rate of PD L1 positivity (76.7%, p < 0.001). ROC curve analysis demonstrated good predictive performance of the model with an area under the curve of 0.81. Conclusion: The present study demonstrates a significant interaction between hypoxia, stromal remodeling, and immune checkpoint activation in gastric carcinoma. High HIF 1 alpha expression and inflammatory CAF predominance are strongly associated with aggressive clinicopathological features and increased PD L1 expression, supporting the existence of a coordinated hypoxia stroma immune axis in gastric carcinoma progression. These findings may have potential implications for prognostic stratification and combined targeted therapeutic strategies.

Aim: To investigate whether salivary immune cell profiling can serve as a non-invasive approach to monitor periodontal disease activity and therapeutic response by characterizing innate and adaptive immune cell dynamics in periodontitis. Materials and Methods: This longitudinal study included systemically healthy adults with periodontitis and healthy controls. Periodontal parameters (PPD, BOP, plaque/calculus, and radiographic bone loss) were recorded by calibrated examiners following established criteria. Stimulated saliva and gingival biopsies were collected before and 4-6 weeks after non-surgical periodontal therapy (NSPT), and from healthy controls. Multiparametric flow cytometry was used to characterize myeloid and lymphoid cell populations and polarization markers. Bacterial transcripts and host inflammatory markers were assessed by qRT-PCR. Statistical analyses were performed using one-way ANOVA. Results: Periodontitis subjects exhibited significantly elevated salivary bacterial transcripts, which decreased but did not normalize following NSPT. Both myeloid and lymphoid immune cell populations increased in periodontitis compared with healthy controls and declined after therapy. This was accompanied by a pronounced pro-inflammatory shift with elevated IFN-gamma-producing macrophages, dendritic cells, Th1/Th17 cells, and B cells, including the novel identification of IFN-gamma-producing B cells in saliva and mirrors the gingival immune cell profiles. In contrast, anti-inflammatory populations (IL-10-producing myeloid cells, Tr1 cells, and regulatory B cells) were reduced in disease and partially restored following NSPT. Conclusions: Salivary immunophenotyping non-invasively monitors PD activity and therapeutic response by capturing dynamic immune changes that reflect gingival signatures and track post-therapy resolution.

Air-liquid interface (ALI) cultures consisting of well-differentiated primary respiratory epithelial cells (PRECs) provide a versatile in vitro model for pharmacological studies and to investigate host-pathogen interactions. Proliferation and differentiation of PRECs require complex media containing several growth factors, hormones, and nutrients. Usually, some of these essential components are provided by the addition of fetal calf serum (FCS). However, several disadvantages of FCS and, most importantly, ethical concerns regarding the method of serum collection have encouraged researchers to find alternatives. Human platelet lysate (hPL) has emerged as a promising alternative to FCS for supporting cell expansion in vitro. In the present study, we investigated the effects of different concentrations of hPL on the proliferation of porcine PRECs and their subsequent differentiation under ALI conditions. Cell morphology was assessed by phase-contrast microscopy, while cell proliferation was evaluated using the ClickTech EdU Cell Proliferation Kit and visualization of proliferating cells by fluorescence microscopy. Differentiation under ALI conditions was monitored by immunofluorescence staining of ciliated cells and the establishment of an intact epithelial barrier was confirmed by measuring transepithelial electrical resistance (TEER). We found that 5% hPL supported efficient cell growth and the subsequent formation of a functional, well-differentiated airway epithelium comparable to or even better than 10% FCS. Thus, hPL offers a reproducible, ethically sound, and scalable alternative to FCS for complex cell culture models in respiratory research, drug development, and host-pathogen interaction studies. LO_SCPLOWAYC_SCPLOW SO_SCPLOWUMMARYC_SCPLOWRespiratory epithelial cells from the lungs of slaughtered animals, such as pigs, can be used for cell culture models to study respiratory diseases and drug development. Air-liquid interface (ALI) cultures closely mimic the natural environment of the airways by exposing the cells to air, making them a valuable alternative to animal experiments. To grow and mature properly, these cells require nutrients and growth factors that are commonly supplied by serum from unborn calves (FCS). However, for ethical and scientific reasons, the use of FCS should be avoided. Therefore, we evaluated whether human platelet lysate (hPL) derived from expired blood donations could replace FCS in ALI cultures. We found that adding 5% hPL to the medium supported efficient cell growth and the development of a well-differentiated airway epithelium. This approach enables the use of an improved and ethically superior model of the (porcine) respiratory tract in accordance with the 3Rs principle.

Background: Subepithelial connective tissue grafting remains a reference treatment for predictable root coverage. Although short- and medium-term outcomes of tunnel-based procedures are well documented, evidence regarding stability beyond 10 years remains limited. This study evaluated the long-term clinical performance of a minimally invasive tunnel technique combined with subepithelial connective tissue grafting (SCTG) under routine clinical conditions. Methods: This retrospective longitudinal cohort study included 74 patients (57 women and 17 men) contributing 710 gingival recession sites treated between 2009 and 2025. All sites were treated with a tunnel approach and SCTG, with enamel matrix derivative (EMD) used in selected cases. The mean follow-up was 6.0 for 4.0 years, with a maximum observation period of 16 years. The primary outcome was recession depth reduction. Secondary outcomes included complete root coverage (CRC), mean root coverage, and long-term marginal stability. Clinically relevant relapse was defined as a 1 mm increase in recession after initial healing. Results: Mean recession reduction was 2.72 mm. Complete root coverage was achieved at 83.4% of treated sites. At the final available follow-up, no treated site showed a clinically relevant relapse of 1 mm after initial healing, and no site deteriorated beyond its baseline recession level. Treatment effects were observed across anterior and posterior regions. Conclusions: Within the limitations of a retrospective cohort design, tunnel surgery combined with SCTG was associated with high root-coverage predictability and durable marginal soft-tissue stability for observation periods extending to 16 years. These real-world data support phenotype-enhancing, minimally invasive soft-tissue augmentation as a durable therapeutic strategy for localized and multiple gingival recessions. Keywords: gingival recession; tunnel technique; subepithelial connective tissue graft; root coverage; periodontal plastic surgery; long-term stability

Background: Oral dysbiosis has been associated with oral squamous cell carcinoma (OSCC); however, most microbiome studies rely on 16S ribosomal RNA (rRNA) gene sequencing, limiting species-level taxonomic resolution. Methods: Dental plaque, saliva, and tumor tissue samples from 10 patients with OSCC and dental plaque and saliva samples from 10 healthy controls were analyzed in this exploratory cross-sectional study. DNA was extracted and subjected to shotgun metagenomic sequencing using the Illumina MiSeq platform. Sequence reads were quality filtered with fastp, taxonomically classified using Kraken2 v2.1.3, and species-level abundances were re-estimated with Bracken v2.9 following the removal of human reads and low abundance taxa. Relative abundances were compared using the Mann Whitney U test with the Benjamini Hochberg false discovery rate correction, while the Bray Curtis principal coordinate analysis was used as an exploratory approach to visualize microbial community patterns. Results: Shotgun metagenomic sequencing revealed distinct bacterial community profiles across the oral microenvironment. Dental plaque exhibited the highest taxonomic diversity and relative abundance. The control plaque was enriched in Streptococcus koreensis, Capnocytophaga sp. oral taxon 878, Treponema sp. Marseille Q4132, and Leptotrichia sp. oral taxon 498, whereas the plaque from patients with OSCC showed a higher relative abundance of Pyramidobacter piscolens, Parvimonas parva, and Gemella sanguinis. Salivary samples displayed lower diversity and a more homogeneous composition, predominantly comprising Capnocytophaga endodontalis, Prevotella jejuni, Aggregatibacter aphrophilus, and Gemella sanguinis. The tumor tissue showed relatively higher abundance of Sellimonas catena, Escherichia coli, Solobacterium moorei, and Lacrimispora sp. HJ 01. Conclusions: This exploratory study provides species-level characterization of the oral microbiome across multiple oral microenvironments in OSCC and generates hypotheses for future integrative metagenomic and functional studies investigating the potential contribution of oral bacterial communities to OSCC pathogenesis.

Thymosin {beta}4 (T{beta}4) is a conserved acidic polypeptide with 43-amino acids participating in multiple pathophysiological processes. In this study in vivo effects of T{beta}4 on liver regeneration are investigated in carbon-tetrachloride (CCL4) induced rodent animal liver jury models. Results illustrate that exogenous T{beta}4 treatment significantly reduced CCL4-rendered liver necrosis around central vein. At 48 hours after CCL4 insults hepatocytes proliferation occur mainly around the periportal area, while hepatocytes proliferation around the necrosis area is prominently increased by exogenous T{beta}4 treatment. The holistic proliferation level of liver tissues are also enhanced by exogenous T{beta}4. Hepatocyte proliferation activities negatively correlate with the necrosis extent of the liver tissue. These results suggested firstly exogenous T{beta}4 treatment could enhance liver regeneration and exhibit prosperous potential for application in clinical conditions such as liver transplantation.

BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) is an oncogenic retrovirus which is transmitted via cell-containing blood fluids or from mother to child via breastfeeding, leading to severe diseases such as adult T-cell leukemia/lymphoma (ATL) and neuroinflammation. Most studies focus on virus detection in peripheral blood due to limited access of tissue material, especially in infants. Thus, our understanding of viral distribution in organs, in particular along the oral route of transmission, is still a critical gap in HTLV-1 research. Methodology/Principal FindingsHere, we present an analysis of tissues from a non-human primate (NHP) colony (olive baboon: Papio anubis) naturally infected with the closely related counterpart of HTLV-1, simian T-lymphotropic virus type 1 (STLV-1). Various organs and tissues of the oropharyngeal and gastrointestinal tract including tonsils, stomach, small intestine and colon were analyzed for the presence or absence of STLV-1. Beside TaqMan qPCR measuring relative copy numbers, we established a highly sensitive and precise droplet digital (dd) PCR protocol to measure absolute copy numbers of viral Tax DNA. Tax DNA was detectable in the tonsils in two NHPs, but to a greater extent in stomach in four NHPs. We also found Tax in parts of the small intestine, i.e. duodenum and Peyers patches, in a NHP with high blood proviral load. Conclusion/SignificanceThese data provide a quantitative analysis of STLV-1 Tax in the gastrointestinal tract and are, to our knowledge, the first indication of STLV-1 detection in stomach tissue of naturally STLV-1-infected asymptomatic NHPs. Although it is unclear how infection occurred - from mother-to-child, sexual or via animal bites - our study suggests that these parts of the gastrointestinal tract might either serve as site of virus transmission or as viral reservoir. Author summaryHuman T-lymphotropic virus type 1 (HTLV-1) is a human oncogenic retrovirus being transmitted via cell-containing body fluids such as breast milk, blood, or semen. The estimated number of infected people is around 10 to 20 million. To study the viral distribution and persistence of HTLV-1 in different organs in the oropharyngeal and gastrointestinal tract, a suitable in vivo model is essential. In this study, tissues of non-human primates (NHPs, baboon: Papio anubis) naturally infected with the closely related simian counterpart of HTLV-1, simian T-lymphotropic virus type 1 (STLV-1), were analyzed for the presence or absence of the viral gene Tax. We identified the presence of STLV-1 Tax in the stomach, duodenum and Peyers patches by using two different detection methods: TaqMan-based qPCR and the more sensitive droplet digital PCR (ddPCR). Tax could be detected in tonsils in two NHPs only, but in stomach in four NHPs. Together, this is the first time that STLV-1 Tax was detected in stomach tissue of STLV-1-infected asymptomatic NHPs, highlighting the importance of investigating viral persistence and/or viral reservoirs of primate T-lymphotropic viruses independently of the entry route.

BackgroundStrategies to define human monocytes by flow cytometry vary considerably across studies. Recently, toll-like receptor 2 (TLR2) has been proposed as a marker to identify "all monocytes" in human peripheral blood. However, the TLR2-defined monocytes also contained a previously ignored TLR2posCD14dim/negCD16neg population, which we termed the unclassified subset (UCS). MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors and patients with multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS) were analyzed by multiparameter flow cytometry using TLR2pos gating. PBMCs from additional healthy donors were analyzed to characterize the UCS population, including the impact of using either TLR2pos or a negative selection-based gating strategy. ResultsThe TLR2pos CD14dim/neg CD16neg UCS population was present in healthy controls, MGUS, and MM patients. The UCS expressed the monocyte-macrophage scavenger receptor CD163 and was significantly reduced in MM patients compared to healthy donors (P<0.002). Further phenotypic characterization in healthy blood donors revealed that approximately 80% of UCS cells expressed CD163 at levels comparable to classical monocytes, yet phenotypically resembled CD163pos dendritic cells (DCs). Importantly, gating strategies influenced the composition of the UCS: negative selection-based gating captured all DC subsets, whereas TLR2pos gating primarily included CD1cpos DCs that were highly CD163pos. ConclusionsThese findings demonstrate that circulating CD163pos CD1cpos DCs are included in the TLR2pos cell population previously described as exclusively monocytes, highlighting the impact of gating strategy on monocyte subset identification. Further, the lower level of TLR2pos CD14dim/neg CD16neg CD163pos cells in MM patients may represent decreased levels of circulating DCs that may contribute to the immune dysregulation in this disease.

RationaleNeurogenic inflammation is recognised as an important contributor to the pathophysiology of Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). Substance P (Sub P), a neuropeptide released from sensory nerves, is a potent inducer of mast cell degranulation through the Mas-related G protein-coupled receptor member X2 (MRGPRX2), resulting in the release of pro-inflammatory mediators that perpetuate chronic bladder inflammation. Medihoney, a medical-grade M[a]nuka honey, possesses well-established antimicrobial and anti-inflammatory properties, and we have recently demonstrated its ability to stabilise mast cells through inhibition of histamine release. However, its effects on Sub P-induced mast cell activation and MRGPRX2-mediated neurogenic inflammation have not previously been investigated. Aim of the studyWe aimed to investigate the inhibitory effects of Medihoney and a sugar-free M[a]nuka honey extract on Substance P-induced mast cell degranulation and MRGPRX2 activation as potential therapeutic approaches for chronic neurogenic inflammation associated with IC/BPS. In addition, we examined the expression of MRGPRX2 in bladder biopsies from patients with IC/BPS. Materials and methodsHuman LAD2 mast cells were stimulated with Substance P (1 M) for 40 minutes following 20-minute pre-incubation with Medihoney or a sugar-free M[a]nuka honey extract. Mast cell degranulation was quantified by measuring {beta}-hexosaminidase release. MRGPRX2 activation was assessed by intracellular calcium imaging using Fluo-4 in MRGPRX2-expressing HEK-293 cells. Bladder biopsies obtained from patients with IC/BPS and healthy controls were immunostained for mast cell tryptase, chymase and MRGPRX2. ResultsMedihoney at 2% and 4% markedly inhibited Substance P-induced mast cell degranulation in LAD2 cells by approximately 90%, an effect that was similarly observed with the sugar-free M[a]nuka honey extract. Both preparations produced a dose-dependent inhibition of Substance P-induced intracellular signalling in MRGPRX2-expressing HEK-293 cells, demonstrating suppression of MRGPRX2 activation. Furthermore, immunohistochemical analysis of bladder biopsies revealed that approximately 66% of tryptase-positive mast cells expressed MRGPRX2 in patients with IC/BPS, which was significantly higher than that observed in healthy control tissues (25%). ConclusionThe present study demonstrates that mast cells within IC/BPS bladder tissue express increased levels of MRGPRX2, suggesting enhanced responsiveness to Substance P and supporting a role for neurogenic inflammation in the pathophysiology of IC/BPS. Medihoney and the sugar-free M[a]nuka honey extract significantly inhibit Substance P-induced mast cell degranulation through modulation of MRGPRX2-mediated intracellular signalling, highlighting their potential as novel therapeutic agents for reducing neurogenic bladder inflammation associated with IC/BPS. ImpactThis study provides evidence that MRGPRX2-mediated neurogenic mast cell activation is enhanced in IC/BPS and demonstrates, for the first time, that Medihoney and a sugar-free M[a]nuka honey extract effectively inhibit Substance P-induced mast cell degranulation through modulation of MRGPRX2 signalling. These findings provide new mechanistic insight into the anti-inflammatory actions of M[a]nuka honey-derived preparations and identify MRGPRX2 as a potential therapeutic target in IC/BPS. The observed inhibition of neurogenic mast cell activation suggests that these naturally derived preparations may offer a novel strategy for limiting chronic bladder inflammation. Overall, this work provides a foundation for future preclinical and clinical studies evaluating the safety and therapeutic efficacy of Medihoney and M[a]nuka honey-derived compounds in patients with IC/BPS.

RationaleMast degranulation is a driver of several pathologies. Several endogenous peptides have been established as stimulators of mast cell degranulation. Compounds that stabilise mast cells have in part demonstrated inhibitory effect on the inflammatory cascade. Manuka honey has been widely used for the treatments of various inflammatory diseases. However, its effect on A23187 (Calcium ionophore) stimulated mast cell degranulation, cytokine release and cellular signalling pathways has not yet been investigated. Aim of the studyWe aim to investigate the effect of Medical Grade Manuka Honey (MGMH) on A23187 induced mast cell degranulation, cytokine release and downstream signalling pathways in a human mast cell lines LAD2 model. Materials and methodsThe cytotoxic effect of MGMH on LAD2 cells was assessed using LDH assay. LAD2 cells were pre-incubated with MGMH and challenged with A23187.Mast cell degranulation was measured by {beta}-hexosaminidase release whilst histamine and cytokines released were measured by ELISA. The effect of MGMH on downstream signalling of Mitogen Activated Protein Kinase (MAPK) pathways was determined and quantified by SDS-PAGE western blotting. ResultsMGMH at 2% and 4% was well tolerated by LAD2 cells. MGMH at all concentrations tested significantly inhibited the A23187 triggered release of {beta}-hexosaminidase but failed to inhibit the release of histamine. MGMH 4% significantly inhibited the release of GM-CSF and IL-8. MGMH (2% and 4%) significantly down regulated the expression of both ERK I and ERK II. However, MGMH at all the doses tested had no effect on the expressions of JNK and p38. On the contrary, an increase expression of these p38 and JNK were noted with MGMH pre-incubation. ConclusionOur present study provides evidence that MGMH inhibition of A23187 stimulated degranulation of mast cells and cytokine release through down regulation of ERK I and II signalling. The results suggest potential use as a mast cell stabiliser and effective treatments of mast cell mediated inflammatory diseases. ImpactThis study provides the first evidence that Medical Grade Manuka Honey (MGMH) can attenuate A23187-induced mast cell activation and pro-inflammatory cytokine release in human LAD2 mast cells through modulation of ERK1/2 signalling pathways. The findings advance our understanding of the cellular and molecular mechanisms underlying the anti-inflammatory properties of Manuka honey. The demonstration that MGMH inhibits {beta}-hexosaminidase release suggests a mast cell-stabilising effect, highlighting its potential as a novel natural therapeutic agent for mast cell-mediated disorders, including allergic diseases, interstitial cystitis, asthma, chronic inflammatory skin conditions, and mast cell activation syndromes. The observed reduction in GM-CSF and IL-8 production further indicates that MGMH may help limit the amplification and persistence of inflammatory responses. Mechanistically, the selective downregulation of ERK1/2 signalling provides new insight into how MGMH exerts its biological effects and identifies a potential molecular target through which its anti-inflammatory activity is mediated. These findings contribute to the growing evidence base supporting the therapeutic value of Manuka honey beyond its established antimicrobial and wound-healing properties. Overall, this work lays the foundation for future preclinical and clinical studies investigating MGMH as a safe, naturally derived mast cell stabiliser and anti-inflammatory intervention, with potential applications across a broad spectrum of allergic and inflammatory diseases.

Background A persistent gap between motivation and action threatens voluntary blood supply. This study examined the publics knowledge, attitudes, and practices (KAP) regarding blood donation, with a particular focus on identifying the different determinants of past blood donation behavior and future willingness to donate. Methods Convenience sampling was used to conduct a cross-sectional survey among 1,058 eligible people in Qingdao, China, between July and November 2025. Data were collected via a self-designed KAP questionnaire. To find independent characteristics linked to previous behavior and future intention, respectively, multivariable binary logistic regression was used. Results Overall, 37.0% of participants (n=391) had a lifetime donation history, while 39.2% (n=415) intended to donate in the next 12 months. Past behavior was positively associated with older age (36-45 years: OR=6.84; 95% CI: 3.21-14.58), higher education (OR=2.06; 95% CI: 1.33-3.17), and interpersonal interaction channels (OR=1.45; 95% CI: 1.01-2.09) but hindered by safety concerns (OR=0.23; 95% CI: 0.16-0.34). Conversely, future intention was positively correlated with male sex (OR=1.69; 95% CI: 1.24-2.29), prior donation history (OR=2.69; 95% CI: 1.87-3.86), having family members or friends in need of blood (OR=2.75; 95% CI: 1.96-3.85), and traditional media exposure (OR=3.33; 95% CI: 2.18-5.10). Higher education was adversely correlated with future intention (OR=0.55; 95% CI: 0.38-0.79). Conclusion There is a substantial disparity between donation motivation and action. The determinants of past behavior and future intention are asymmetric, suggesting that stage-specific interventions are required, using social mobilization for initiating first-time donations, while employing family reciprocity and authoritative communication to sustain long-term engagement.

Background: Molecular markers and mediators of adverse post-hospital outcomes following critical illness remain incompletely characterized. Anemia and red blood cell (RBC) indices integrate inflammation, nutritional status, and hematopoietic function, and may represent biological processes that influence long-term outcomes. We therefore examined whether pre-discharge anemia and hematologic clusters derived from correlated RBC indices were associated with one-year mortality among critical illness survivors. Methods: We conducted an exploratory, retrospective cohort study of adult ICU patients discharged alive using MIMICIV. Pre-hospital discharge RBC indices (hemoglobin, red cell distribution width (RDW), mean corpuscular volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), and RBC count) were analyzed. Hemoglobin and anemia were first evaluated, followed by Gaussian mixture modeling of standardized RBC indices to identify patient clusters in a derivation cohort (70%) and validated findings in a held-out cohort (30%). Associations with one-year mortality were assessed using multivariable-adjusted Cox models. Results: Among 20,233 ICU survivors with available pre-discharge hemoglobin (median age, 67 years), based on the WHO definition of anemia, 17,247 (85.2%) critical illness survivors were anemic at hospital discharge. Anemia was associated with higher one-year mortality (adjusted HR 1.42, p<0.001). Mortality was 20% overall and decreased across increasing hemoglobin quartiles, from 28.8% to 12.5% (log-rank P<0.005); lower hemoglobin was associated with progressively higher adjusted one-year mortality risk. Six hematologic clusters derived from RBC indices were identified with one-year mortality ranging from 10.1% to 31.4% (log-rank p<0.005). Compared with the lowest-risk group, the cluster characterized by elevated RDW and MCV had a twofold higher adjusted mortality risk (HR 2.14; 95% CI, 1.81-2.53). Conclusion: Both anemia and hematologic clusters derived from standard RBC indices at hospital discharge are associated with one-year mortality following critical illness. These hypothesis-generating findings support further investigation of RBC indices as potential biomarkers for post-discharge risk stratification and of the underlying biological pathways as possible therapeutic targets.

BackgroundiPSCs generated from healthy individuals constitute an important control resource for disease modelling applications but existing biobanks are highly skewed towards populations of European ancestry while well characterized control lines from Indian populations remain limited. Given the extensive genetic diversity of the Indian subcontinent, the availability of ethnically relevant healthy control lines is important for developing accurate disease models and reducing population specific confounding effects. MethodologyWe used peripheral blood mononuclear cells (PBMNCs) of a healthy female donor of Eastern Indian origin for the generation a wild type iPSC line using non-integrating episomal reprogramming vectors. Established colonies were expanded and characterized through morphological assessment, expression of pluripotency and trilineage markers, episomal vector clearance analysis, and chromosomal stability evaluation and mycoplasma contamination analysis. ResultsThe line generated exhibited characteristic pluripotent stem cell morphology and also showed strong expression of pluripotency markers, was free from any contamination and free from the reprogramming vectors confirming an integration free system. The cells maintained a normal diploidy number during characterization. Expression of lineage specific markers associated with ectoderm, mesoderm and endoderm confirmed the developed iPSCs functional capacity to undergo trilineage differentiation. ConclusionWe have developed and validated an iPSC line from an underrepresented Indian population. This well characterized, ethnicity specific iPSC line provides a valuable cell line for establishing a high quality, well characterized control baseline, which is a major missing element in South Asian stem cell repositories and thus will provide a solid foundation for future disease specific modelling and screening.

Severe dengue can damage the liver through unestablished mechanisms. We investigated the role of myeloperoxidase (MPO), a neutrophil enzyme, in dengue through patients, fatal liver samples, and mouse infection models. Observations from two independent clinical cohorts revealed elevated plasma MPO levels in dengue and, in one cohort, MPO was further linked to liver injury markers during the critical phase of disease, whereas livers from dengue fatal cases revealed MPO build-up in the vicinity of CD177+ activated neutrophils. In mice, dengue led to MPO overexpression, oxidative damage, and broad activation of innate and systemic inflammatory pathways in livers. Blocking MPO activity alleviated these and improved survival in one model and delayed disease progression without preventing death in another. These findings establish MPO as a functional mediator of severe dengue-associated liver injury and inflammation, which warrants further preclinical investigation into its hepatic pathogenic mechanism and its validity as target for therapeutic intervention.

Rationale: Infections due to Extended-spectrum {beta}-lactamases-producing Enterobacterales (ESBL-PE) require empirical treatment with carbapenems. ESBL-PE carriage is considered as a risk factor for ESBL-PE involvement during ICU infection. Our aim was to determine factors that may predict the actual involvement of ESBL-PE. Methods: A two-periods bicentric ambispective study including ICU ESBL-PE carriers patients from April 2011 to January 2019. All ESBL-PE carriers who developed an infection were analyzed. Results: 6112 patients and 4902 patients were screened during the two periods. 384 and 232 ESBL-PE carriers were identified. Total number of infectious episodes were 146 and 114, respectively. A total of 144 pneumonias, 42 urinary tract infection and 45 digestive infections were studied. An ESBL-PE was involved in 35 (24.3%) episodes of pneumonia, and 44 (37.9%) of extra-pulmonary infections. The most frequent ESBL-PE involved were K. pneumoniae, E. cloacae and E. coli. Similar species and phenotypes were present in colonisation and infection in 29 (82.8%) of pneumonia and in 40 (90.9%) of extra-respiratory infection. Multivariate analysis identified Klebsiella pneumonia or Enterobacter cloacae carriage as risk factor for ESBL-PE involvement in pneumonia and E. coli carriage and detection of ESBL-PE carriage before ICU admission as protective factors. Conclusion: In our study an ESBL-PE involvement is infrequent in pneumonia. A known carriage before ICU admission and E. coli carriage are factors associated with the absence of ESBL-PE un the episode of respiratory infection. A confirmation of our findings could lead to a reduction in the empirical use of carbapenems in this population.

Adipose-derived mesenchymal stem cells (gAD-MSCs) are promising candidates for veterinary regenerative medicine, yet the characterization of gAD-MSCs from locally adapted Indonesian goat breeds remains limited. This study aimed to isolate and characterize gAD-MSCs from Peranakan Ettawa (PE) goats using tissue explant culture. Subcutaneous adipose tissue was collected from the base of the tail of healthy PE goats (n=3). Primary cell outgrowth from explants was observed by Day 5, displaying characteristic fibroblast-like, spindle-shaped morphology and strong plastic adherence. Serial passaging to Passage 3 (P3) yielded a morphologically stable, homogeneous cell population. Assessment of cellular metabolic activity via the resazurin assay demonstrated sustained cell viability and a statistically significant increase in metabolic activity between Day 3 and Day 5 (p < 0.05). Furthermore, functional clonogenic capacity, evaluated using the colony-forming unit (CFU) assay, showed continuous temporal expansion of colonies over 14 days, yielding an average of 52.0 + - 4.1 colonies per dish. These findings confirm that expanded gAD-MSCs P3from PE goats maintain characteristic mesenchymal morphology, sustained metabolic activity, and clonogenic capacity. This work provides a baseline cellular profile of PE goat gAD-MSCs, supporting their potential use in veterinary regenerative medicine and tissue engineering.

Periodontitis, a chronic inflammatory disease affecting approximately 40% of U.S. adults aged 30 years and older, is characterized by dysbiosis of the dental plaque microbiome. However, although scaling and root planing (SRP) is the cornerstone of periodontal treatment, its effects on the taxonomic composition and functional potential of the dental plaque microbiome remain incompletely understood. In this study, we used whole-metagenome shotgun sequencing to characterize taxonomic composition and functional potential in dental plaque microbiomes collected from 39 patients with Stage II or III generalized periodontitis before and 3-4 months after SRP. Consistent with clinical improvement, periodontal therapy significantly reduced bleeding on probing and plaque index. Whole-metagenome shotgun sequencing identified 3.18 million non-redundant genes and 12,353 microbial species across 78 samples, revealing increased gene and species richness after treatment, along with a significant restructuring of microbial community. Established periodontal pathogens, including Porphyromonas gingivalis and Tannerella forsythia, as well as the emerging pathogen Escherichia coli, decreased following treatment, whereas health-associated early colonizers, including multiple Actinomyces species and Streptococcus cristatus, increased. Functional annotation using the Carbohydrate-Active Enzymes (CAZy) database identified treatment-associated differences in several carbohydrate-active enzymes, including multiple glycosyltransferases, indicating remodeling of the predicted functional potential of the dental plaque microbiome. These findings demonstrate that successful SRP promotes coordinated taxonomic and predicted functional remodeling of the dental plaque microbiome and highlight the value of shotgun metagenomic sequencing for characterizing both taxonomic and functional recovery following periodontal therapy.

Abstract Objective Commercially available next-generation sequencing (NGS) platforms in China routinely adopt a lung cancer-derived tumor microenvironment (TME) subtyping signature from a European cohort to classify colorectal cancer (CRC), yet its diagnostic performance in Chinese CRC patients remains unvalidated. This study aimed to evaluate the subtyping efficiency of the lung cancer TME signature in a Chinese CRC cohort, screen CRC-specific immune mRNA biomarkers for TME subtyping, and explore the clinical utility of IRF1, CD8A and LAG3 for distinguishing immune-enriched (IE) and immune-desert plus fibrotic (D+F) subtypes. Methods A total of 87 FFPE CRC specimens with complete NGS and clinicopathological data were retrospectively enrolled, including 15 IE subtype and 72 D+F subtype patients. Thirty-one mRNA transcripts covering 13 immune-metabolic homeostasis genes and 18 immune checkpoint/infiltration-related genes were divided into two functional modules. Spearman correlation analysis was performed to assess co-expression patterns among candidate genes. Receiver operating characteristic (ROC) curves combined with five-fold cross-validation were used to compare the discriminatory efficacy of single-gene markers and the three-gene combined panel. Results Strong positive co-expression was observed between IRF1, CD8A and LAG3 (IRF1-CD8A: r=0.93; IRF1-LAG3: r=0.84; CD8A-LAG3: r=0.73). Nominal P-values indicated elevated expression of IRF1, CD8A and LAG3 in IE subtype, though no intergroup significance remained after Benjamini-Hochberg FDR correction, largely attributed to the limited sample size of IE cases. Single-gene ROC analysis showed AUC values of 0.763 (IRF1), 0.752 (CD8A) and 0.771 (LAG3), with LAG3 exhibiting the best individual discriminatory capacity. The three-gene combined panel yielded a cross-validated AUC of 0.717, inferior to single LAG3, due to severe collinearity that generated redundant predictive information. Conclusions The lung cancer-originated TME subtyping system cannot be directly extrapolated to Chinese CRC patients. LAG3 serves as a promising independent transcriptomic candidate marker for distinguishing CRC TME subtypes. The robust collinearity among IRF1, CD8A and LAG3 eliminates additional predictive benefits of the combined signature. Large independent multi-center Chinese CRC cohorts are required to construct population-specific immune transcriptomic biomarkers for standardized clinical NGS TME stratification.

Hematopoietic stem and progenitor cells (HSPCs) sustain the production of hundreds of billions of new cells per day to maintain our blood and immune system. In this process, HSPCs regulate the hematopoietic output by sensing and integrating diverse physiological cues. Thus, HSPCs express many receptors traditionally studied for their functions in the immune system, and this allows HSPCs to directly detect microbial compounds, endogenous danger signals, cytokines, and other inflammatory mediators. However, how the expression levels of such receptors on HSPCs change under chronic inflammation and how such changes alter HSPC functions and immune cell production remains unexplored. Working in a murine model of rheumatoid arthritis, we demonstrate the induction of microbial sensors TLR2 and CD14, orphan inflammatory receptor TREM1, and checkpoint receptor PD-L1 on HSPCs and particularly the myeloid progenitor cells in the arthritis-afflicted mice. Furthermore, we demonstrate that the stimulation of HSPCs through these receptors in culture can significantly alter the dynamics of cell expansion and differentiation, with distinct responses from HSPCs of arthritis-afflicted versus healthy control mice. We hypothesize that the induction and stimulation of HSPCs through these immune receptors under chronic inflammation may impact the output and functional properties of their immune cell progeny, positing HSPCs as central players in the pathogenic inflammatory responses of rheumatoid arthritis and potentially other chronic inflammatory diseases. HIGHLIGHTSO_LIHematopoietic progenitor cells in murine models of rheumatoid arthritis show an upregulation of immune receptors TREM1, PD-L1, TLR2, and CD14. C_LIO_LIStimulation of murine hematopoietic stem and progenitor cells through these receptors in culture alters the dynamics of their expansion and differentiation. C_LIO_LIIn such cultures, hematopoietic stem and progenitor cells from mice afflicted with rheumatoid arthritis show altered responses to stimulation as compared to healthy controls. C_LI

Hematopoietic stem cells (HSCs) give rise to all blood cell lineages and possess long-term self-renewal potential. HSCs undergo symmetric division for their expansion and asymmetric division to generate one HSCs and one progenitor cells which contribute to production of mature blood cells. The midbody is a structure which is formed in the center of the intercellular bridge during cytokinesis. However, the midbody is either asymmetrically inherited by one daughter cell or symmetrically released after cell division, whether these distinct patterns of midbody inheritance influence HSC fate remain poorly understood. In this study, we designed a fusion protein hmKO2 and MgcRacGAP which is a component of midbody. We then traced the midbody inheritance during cell division and the future cell fates of HSC daughters after division by time-lapse imaging. As a result, we found that the midbody release correlated with the delay of the time to the next division but not to the lineage potential of HSCs, indicating the possibility that midbody remnant plays some roles in cell cycle progression. HighlightHematopoietic stem cells exhibit a low frequency of midbody inheritance. Midbody inheritance does not affect the lineage potential of daughter cells. Midbody loss is associated with delayed entry into the next cell cycle.