Journal of Biomedical Science

BackgroundDengue has been a major health threat globally in recent years. In particular, dengue incidences continue to increase annually and the epidemic area has expanded primarily due to global warming. Therefore, effective case detection and surveillance strategies are crucial to tackle this global health challenge. In clinical practice, the rapid test kit detecting dengue non-structural protein 1 antigen and commonly referred as NS1, is widely employed for early diagnosis. However, real-world studies revealed that the sensitivity of the NS1 test kit ranged from approximately 61% to 95%. Since early diagnosis is really critical for disease surveillance in the early stage of a dengue epidemic, scientists have been working hard to develop novel diagnosis methods that can provide higher sensitivity levels. Methodology/Principal FindingsIn response to this challenge, in this study, we have developed a novel diagnosis procedure that integrates machine learning technologies with the NS1 test kit. Our experimental results revealed that we would be able to raise the sensitivity of the dengue diagnosis procedure to higher than 99% by incorporating machine learning based prediction models to screen the suspected patients with a negative NS1 result. Furthermore, the relative risks between the suspected patients who were predicted to be positive and those who were predicted to be negative exceeded 4.8. Conclusions/SignificanceThese results illustrate that the proposed approach provides an effective and efficient diagnosis procedure to address the global health challenge caused by spread of dengue. Author SummaryThis study has aimed to enhance surveillance of the dengue disease by integrating machine learning technologies with the rapid test kit commonly employed in early diagnosis. In clinical practice, the NS1 rapid test kit is widely employed for early diagnosis. However, real-world studies revealed that a certain percentage of the patients with a negative NS1 test result, ranging from 5% to 39%, were actually infected by dengue. Since early diagnosis is critical for disease control in the early stage of a dengue epidemic, scientists have been working hard to tackle this challenge. Based on this observation, this study was launched to investigate the effects of incorporating machine learning based prediction models to further screen those patients with a negative NS1 test result. The experimental results revealed that the proposed approach was able to identify over 99% of the patients who were infected by the dengue disease. Furthermore, the risk of the suspected patients who were predicted to be positive was 4.8 times higher than the risk of those who were predicted to be negative. The experimental results illustrate that the proposed approach provides an effective and efficient diagnosis procedure to enhance surveillance of the dengue disease.

Administration of ciliary neurotrophic factor (CNTF) reduces food intake and body weight in both humans and experimental animals, where it also ameliorates hyperglycemia, hyperinsulinemia, and dyslipidemia. To exert its anti-obesogenic and anti-diabetogenic effects, CNTF targets brain feeding centers as well as multiple peripheral organs inducing the phosphorylation of the transcription factor signal transducer and activator of transcription 3 (p-STAT3). However, data showing which peripheral cytotypes are specifically targeted by exogenous CNTF in vivo in metabolically relevant organs are currently lacking. Here, we first evaluated the gene expression levels of the subunits of the tripartite CNTF receptor (Cntfr) complex, i.e., the Cntfr, the leukemia inhibitory factor receptor {beta} (Lifr{beta}) and the glycoprotein 130 (gp130), by quantitative real-time PCR in metabolically relevant organs of adult male mice: gastrointestinal (GI) tract, pancreas, liver, visceral and subcutaneous white (WAT) and interscapular brown adipose tissue (iBAT), skeletal muscle and the sciatic nerve. We then quantified p-STAT3 by Western blotting in these organs after intraperitoneal administration of CNTF (0.3 mg/kg) or saline. Finally, we mapped CNTF-responsive cells by immunohistochemistry, followed by morphometric quantification and confocal microscopy in both CNTF- and saline-treated mice. Lifr{beta} and gp130 were ubiquitously detected across all the investigated organs; the Cntfr showed the highest expression levels in the skeletal muscle, sciatic nerve, and iBAT, whereas it was found to be expressed to a lesser extent in the other sites. Administration of CNTF led to a significant increase of p-STAT3/STAT3 protein ratio in all organs examined, except the duodenum, and induced a distinctive pattern of cell nuclear p-STAT3 immunoreactivity. Notably, along the analyzed GI tract CNTF induced nuclear STAT3 phosphorylation in neurons of the submucosal and myenteric plexuses of the enteric nervous system and in contractile cells of the muscularis externa, where the response peaked in the mesenteric gut and colon. In the pancreas, CNTF triggered a higher activation within the endocrine component compared to the exocrine parenchyma. In the liver, CNTF induced STAT3 phosphorylation not only in parenchymal cells but also in sinusoids and resident macrophages. The cytokine activated p-STAT3 in subcutaneous and visceral white adipocytes, but also in brown adipocytes, with a prominent response observed in the beige subcutaneous adipocytes; adipose resident macrophages and endothelial cells of numerous blood vessels were also CNTF-responsive. Lastly, in skeletal muscle, a major site for glucose/lipid utilization, CNTF induced widespread nuclear p-STAT3 immunoreactivity in muscle fibers and in connective and Schwann cells of the peripheral nerves, including the sciatic nerve, supplying the gastrocnemius. In conclusion, our data indicate that CNTF acts across diverse cytotypes within metabolically relevant organs and tissues, likely fostering its peripheral metabolic effects through this cellular heterogeneity.

Since the initial distribution of the SARS-CoV-19 vaccine, its widespread use has been hypothesized to act as a selective pressure that drives the COVID-19 virus to mutate. This study aims to investigate the correlation between global vaccination rates and the mutation rate of the SARS-CoV-2 Beta variant (B.1.351). From January to July 2021, nucleotide diversity increased in tandem with vaccination rates, demonstrating that the virus evolved more rapidly in response to selective pressure from mass vaccination. Statistical analysis revealed statistically significant positive correlations between both vaccination rates and vaccine doses administered with nucleotide diversity. Thus, our findings indicate a positive correlation between rising vaccination rates and nucleotide diversity, suggesting that increased vaccination coverage acted as a selective pressure that accelerated viral evolution of SARS CoV2.

The KCa2.2 and KCa3.1 channels are fundamental regulator of cellular K+ concentration, and promising target to treat diseases such as spinocerebellar ataxia and cancer. To fully exploit their therapeutic potential, and to continue studying their pathophysiological role, it is crucial to develop selective modulators for each of these two channels. Here we present a computational study to identify the molecular determinants behind the selectivity of two recently reported KCa2.2 modulators. We leveraged a protocol combining in silico mutagenesis, molecular dynamics simulations, and protein-ligand docking to analyse the pockets targeted by these ligands. We identified a Ser353/Pro245 substitution to be the main driver of the distinct pocket shapes in KCa2.2 and KCa3.1 channels, ultimately defining modulator selectivity. This approach provides novel insights into the structural differences of this binding site across potassium channel subtypes, shedding light on the selectivity determinants of modulators targeting this pocket.

Mechanisms that promote organ injury after trauma and hemorrhagic shock (T/HS) remain poorly defined. Endothelial heparan sulfates with a 3-O-sulfate (3-OS) modification, controlled by the HS3ST1 gene, have anticoagulant and anti-inflammatory properties through their interaction with antithrombin. Our objective was to determine whether HS3ST1 deficiency drives organ injury and poor outcomes after T/HS. Hs3st1-/- and wild-type (WT) mice were subjected to T/HS followed by resuscitation with lactated ringers (LR) or fresh frozen plasma (FFP). While no differences were observed between WT and Hs3st1-/- LR resuscitated mice, lung injury and leukocyte infiltrates were significantly increased in FFP resuscitated Hs3st1-/-compared to WT mice. In vitro, leukocyte slow rolling and adherence was increased in HS3ST1 KO compared to WT cells. Among 472 T/HS patients, of which 31 (7%) were homozygous for the rs16881446 variant allele (GG), the number of ventilator free days was lower, and mortality was significantly higher in AG and GG patients. The rs16881446 genotype was independently associated with mortality. In conclusion, HS3ST1 deficiency mitigates organ protection from FFP resuscitation, partly through mediating EC:leukocyte engagement, and predicts mortality after T/HS. These findings identify a novel therapeutic target and prognostic tool that can be leveraged towards improved risk stratification after trauma.

The tight regulation of iron homeostasis is of great importance for cellular health. An increase in intracellular iron levels results in the formation of free radicals, which damages macromolecules and membranes, eventually resulting in cell death by Ferroptosis. Recently, we showed that patients with mutations in VPS41 display a severe neurodegenerative phenotype with iron deposition in the brain. VPS41 is well known as subunit of the HOPS complex required for fusion of late endosomes and autophagosomes with lysosomes. However, VPS41 has also been identified as inhibitor of Ferroptosis and regulator of redox homeostasis. How VPS41 exerts these functions and if these are dependent on the HOPS complex is unknown. Here we show that depletion of VPS41 results in increased intracellular iron levels, ROS formation and mitochondrial fission. Our findings indicate an important role for VPS41 in the regulation of iron homeostasis and mitochondrial fission and suggest Ferroptosis as a possible cause for neurodegeneration in VPS41 patients.

Chaperonins complex into double-ringed octamers to aid peptide folding. Recent evidence implicates dysfunctional chaperonin subunits in cancer and neurodegenerative diseases because their deregulation exacerbates cellular injury. Nevertheless, a gap of knowledge exists regarding the expression and localization of chaperonin subunits in relation to amyloidogenic processes in Alzheimers disease (AD). Here, we show that reduced levels of chaperonin-containing TCP-1 subunits 2 (CCT2) and 3 (CCT3) stratify AD, with the subcellular distribution of their residua being mutually exclusive with both {beta}-amyloid and hyperphosphorylated tau in neurons. We find CCT3 localized to a subset of glial fibrillary acidic protein-positive astrocytes in AD. Increased oxidative stress in vitro upregulated CCT3 expression in astrocyte-like U251 cells. Conversely, CCT3, but not CCT2, loss-of-function in neuron-like SH-SY5Y cells increased intracellular {beta}-amyloid load. These data suggest that CCT2/CCT3 are faithful disease-state indicators and implicate CCT3 in oxidative stress-dependent cellular damage pathways.

Mixed-lineage leukemia translocated to 3 (MLLT3) is vital for maintaining the stemness of hematopoietic stem cells. Loss of MLLT3 in megakaryocyte (MK)-erythrocyte progenitor (MEP) cells leads to its differentiation into MKs. Despite its significance in stemness, the regulatory mechanism of MLLT3 during differentiation remains elusive. In this study, we investigate the regulatory role of histone deacetylase 6 (HDAC6) in modulating MLLT3 levels via heat shock protein 90 (Hsp90) activation during myeloid lineage differentiation into MKs, monocytes, and macrophages. We found that HDAC6 activates Hsp90 through deacetylation, enabling Hsp90 to retain MLLT3 in the cytoplasm where protein kinase C (PKC) phosphorylates MLLT3 at serine residues; leading to loss of MLLT3 during MK and macrophage differentiation but not during monocyte differentiation. This research provides valuable insights into the regulatory mechanisms underlying myeloid lineage commitment and opens new avenues for future investigations into stem cell biology and therapeutic applications.

Traumatic brain injury (TBI) is a condition of high incidence worldwide, but remains mostly undertreated. Previous observations in preclinical studies pointed to a beneficial effect of insulin-like growth factor 1 (IGF-1) in TBI. As brain injury is associated to loss of IGF-1 sensitivity, we tested the therapeutic potential of AIK3a305 (AIK3), a novel IGF-1 sensitizer. Twenty-four hours after mild TBI induced by controlled impact, mice received daily intraperitoneal injections of AIK3 during 4 weeks. We found that TBI-associated sensorimotor disturbances measured with the adhesive-removal test were reverted by AIK3 treatment. In addition, neurological and cognitive disturbances measured by the neurological severity score and Y maze respectively, were also ameliorated by treatment with the IGF-1 sensitizer, whereas increased anxiety after mild TBI was also normalized by AIK3. Circulating levels of IGF-1 were increased after AIK3 treatment in TBI mice, while serum IL-6 levels, a biomarker of inflammation associated to TBI were similar to control mice treated with AIK3. Transcriptomic analysis determined that treatment with AIK3 widely affected gene expression in TBI brains, showing a general reduction in both up- and down-regulated genes. Collectively, these data support the use of IGF-1 sensitizers such as AIK3 for treatment of TBI.

Cerebral small vessel disease (cSVD) is a major contributor to stroke and cognitive decline, ultimately leading to vascular dementia (VaD). Genetic factors play a key role in the disease susceptibility and progression, and variants in COL4A1 cause one of the most common genetic cSVD. COL4A1 encodes the 1 subunit of type IV collagen, the principle extracellular matrix (ECM) protein in the basement membrane of vasculature. In the central nervous system (CNS), the neurovascular unit (NVU) has the unique astrocyte-derived parenchymal basement membrane (pBM), in addition to the vascular basement membrane (vBM), which together contributing to the regulation of the blood-brain barrier (BBB) function. However, the role of pBM in cSVD remains under investigated and poorly understood. The lack of relevant human models has limited our ability to dissect specific cell-cell and cell-matrix interactions, hindering the identification of effective therapeutic targets. In this study, we hypothesised that astrocyte-mediated ECM remodelling contributes to BBB dysfunction in COL4A1-associated cSVD. To investigate this, human induced pluripotent stem cells (hiPSCs) derived from a patient carrying the COL4A1G755R variant and its isogenic control line were differentiated into astrocytes and brain microvascular endothelial cells (BMECs). Comparing to isogenic controls, the COL4A1G755R astrocytes significantly reduced the expression of ECM-related genes and abnormally increased glutamate uptake. ECM preparations from COL4A1G755R astrocytes significantly damaged the tight junction (TJ) structure formed by control iPSC-derived BMECs and failed to rescue the compromised TJ integrity in COL4A1G755R BMECs. The secretome from COL4A1G755R astrocytes exaggerated the ECM abnormality in COL4A1G755R BMECs. Most importantly, reduced expression of HTRA1, a crucial serine protease known to regulate both ECM turnover and homeostasis, and increased TGF-{beta} signalling was observed in COL4A1G755R astrocytes. Functional rescue by recombinant human HTRA1 protein restored the disrupted TJ continuity in COL4A1G755R BMECs and normalized TGF-{beta} signalling and glutamate uptake in astrocytes. Together, these findings defined a previously unrecognised astrocyte-driven pBM mechanism in COL4A1-associated cSVD and highlight HTRA1 in ECM remodelling as a therapeutic target.

Regulator of G protein Signaling 6 (RGS6), heavily implicated in neurological and neuropsychiatric disorders, is enriched in mouse and human brain. Our initial cloning effort identified 36 RGS6 mRNAs in human brain. However, we recently identified an additional RGS6 protein isoform that is larger ([~]69kDa) than the ubiquitously expressed [~]56kDa RGS6L(+GGL) isoforms. Notably, this isoform, named "RGS6B" for "brain-specific", is selectively expressed in the nervous system of mice and humans. Here, we report the cloning of a new RGS6-encoding mRNA, which resembles the RGS6L1(+GGL) transcript identified in our initial cloning effort but includes a highly conserved novel exon (Alternative 3, A3) that alters the reading frame of terminal exon resulting in an extension of the protein C-terminus. When expressed in cells, RGS6LA31(+GGL) co-migrates with RGS6B, and, importantly, interfering RNA targeting exon A3 results in selective depletion of RGS6B in isolated primary cortical astrocytes. RGS6B is capable of stabilizing RGS6 binding partners R7BP and G{beta}5 and, in fact, exhibits an increased protein half-life relative to RGS6L. Both RGS6L and RGS6B are downregulated in human gliomas and share the ability to kill U87MG glioblastoma cells when overexpressed indicating conservation of non-canonical cytotoxic activity between RGS6L and RGS6B species. However, RGS6B lacks the ability to counteract Gi/o-dependent suppression of cAMP signaling, indicating a lack of functional GTPase activating protein (GAP) activity. Instead, RGS6B functions in a dominant negative manner to block Gi/o regulation by RGS6L. RGSB is the first identified RGS protein member that functions to promote, rather than inhibit, G protein signaling. The discovery of the molecular identity of RGS6B will now allow for delineation of unique functions for RGS6 protein isoforms in both physiological and pathophysiological brain states.

BackgroundKATNIP (Katanin-interacting protein), also known as KIAA0556, is one of the human genes with pathogenic variants linked to Joubert syndrome, an archetypal neurodevelopmental ciliopathy. KATNIP is a scaffolding protein with a critical role in ciliogenesis. In this study, we characterized the ciliopathy phenotypes due to KATNIP gene deletion. ResultsWe produced a Katnip null mouse model using CRISPR-Cas12a (Cpf1). The null heterozygotes appeared normal while the homozygotes died around postnatal day 9, showing severe hydrocephalus and deficiency in neuroprogenitor cell proliferation. Katnip-deficient cells in the brain have a higher rate of cilia formation and longer cilia than wild type cells. ConclusionKATNIP loss of function gives rise to hydrocephalus found in Joubert syndrome. The results indicate that KATNIP restricts ciliogenesis and cilia extension and supports proliferation of neuroprogenitor cells in the brain.

AimsPercutaneous coronary intervention has improved survival following myocardial infarction, yet strategies to further reduce infarct size are limited. This study investigates the role of cytotoxic {gamma}{delta}-T cells in ischemic cardiomyocyte death and potential therapeutic interventions to reduce infarct size. MethodsGenetic and pharmacological approaches were used to delete {gamma}{delta}-T cells and their specific proteins to assess their involvement in cardiomyocyte death using mouse models of permanent ligation (PL) and ischemia/reperfusion (IR). Results{gamma}{delta}-T cells accumulated in infarct zones within 6h post-PL, expressing IFN-{gamma}, TNF-, granzyme B, and perforin. Their deletion reduced infarct size by 73% (PL) and 64% (IR). They induced cardiomyocyte death via apoptosis, gasdermin E-dependent pyroptosis, and MLKL-dependent necroptosis; {gamma}{delta}-T cell depletion reduced apoptosis by 80% and pyroptosis by 38%, with perforin deletion yielding similar effects. Necroptosis, attributed to combined IFN-{gamma}/TNF- cytotoxicity, decreased by 67%. Cytoplasmic DNA (cDNA) in stressed cardiomyocytes activated the cGAS/STING pathway, inducing expression of chemoattractant MCP-1 and death signal RAE-1. These signals recruited and activated {gamma}{delta}-T cells, which then triggered the death of the stressed cardiomyocytes. STING inhibition suppressed these expressions, reducing {gamma}{delta}-T cell accumulation and infarct size. NKG2D-deficient {gamma}{delta}-T cells prevented activation and reduced infarct size. Administration of an anti-IFNAR antibody at PL onset markedly reduced infarct size. ConclusionEarly activation of cytotoxic {gamma}{delta}-T cells via cardiomyocyte stress signals contributes significantly to immunogenic cardiomyocyte death. Targeting the STING pathway and type I interferon signalling presents a promising therapeutic avenue to mitigate infarct size and improve outcomes.

Sympathetic neuronal (SN) activity critically regulates the development and function of peripheral organs and tissues. Activity-dependent plasticity has been shown to modulate SN output, suggesting that compensatory forms of plasticity could contribute to maintaining stability of sympathetic circuits. Early SN hyperactivity drives the development of hypertension in humans and in the spontaneously hypertensive rat (SHR). In this study we used chemogenetic and pharmacological approaches, and took advantage of the enhanced activity of SHR SNs, to examine how long-term changes in activity impact synaptic properties in neonatal SN cultures. We showed that bidirectional changes in SN activity result in compensatory shifts in synaptic density that counteract long-term activity manipulations. These changes were mediated by satellite glial cells (SGCs), a non-neuronal cell in the sympathetic ganglia that has been shown to influence cholinergic synaptic sites during development. In the absence of SGCs there was no induction of homeostatic plasticity. Further, direct chemogenetic activation of SGCs was sufficient to drive compensatory plasticity, while glial inhibition blocked SN plasticity. We found that SGCs respond to cholinergic signaling by downregulating the expression of the synaptic regulators NGF and TNF, suggesting that neurons and glia interact to stabilize sympathetic output during long-term changes in circuit activity. Finally, we investigated whether these plasticity mechanisms are present in neonatal SHR SNs. We demonstrated that SHR SNs have an attenuated response to glia, both during synapse formation and activity-dependent plasticity. Taken together, this work outlines a novel homeostatic activity-dependent plasticity mechanism in the peripheral nervous system.

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating immune responses and have emerged as potential biomarkers and therapeutic targets in complex diseases. Leishmaniasis is a neglected disease that compromises host immunity and is associated with challenging treatments regimens. Leishmania amazonensis (L. amazonensis), an intracellular protozoan parasite, causes cutaneous leishmaniasis by replicating inside mammalian macrophages to establish infection. In this context, miRNAs have emerged as vital post-transcriptional factors that regulate the inflammatory landscape during infection. In this study, we aimed to analyze the function of miR-721 in macrophages during L. amazonensis infection by integrating in silico miR-721 target prediction with RNAseq data from macrophages of two distinct mouse genotypes, resistant C57BL/6 and susceptible BALB/c. We found that miR-721 is induced in macrophages infected with L. amazonensis, but is not in LPS-stimulated macrophages, suggesting a TLR4-independent activation. Integrating miR-721 target prediction with comparative transcriptomic analyses in resistant C57BL/6 and susceptible BALB/c models revealed the TNF-IRF1 axis as a primary miR-721-associated regulatory network. Specifically, miR-721 is predicted to target the 3UTRs of Tnf and Irf1 to suppress the inflammatory response. Functional inhibition of miR-721 successfully restored Tnf and Irf1 expression and reduced the amastigote burden over 24 hours. Furthermore, we showed that the miR-721/TNF-IRF1 axis regulates downstream genes associated with macrophage response, such as Serpine1, Csf1, Cd69 and Maf. Our work demonstrated that Leishmania induces miR-721, which negatively modulates the TNF-IRF1 axis, thereby suppressing the immune response and favoring parasite persistence. While C57BL/6 macrophages exhibit a robust activation of the TNF-IRF1 network, promoting inflammatory response, BALB/c macrophage showed a breakdown of this network. This was associated with post-transcriptional suppression of inflammatory responses, thereby favoring parasite persistence. These findings link miR-721 to the establishment of macrophage polarization, providing relevant insights into the mechanisms of parasite subversion of the host immune response.

Spinal cord injury (SCI) is a devastating neurological condition with limited regenerative capacity. Stem cell-based approaches have emerged as promising strategies due to their neuroprotective and immunomodulatory properties, largely mediated by small extracellular vesicles (sEVs) and their molecular cargo, including miRNAs. In this study, we aimed to evaluate the neuroprotective and anti-apoptotic potential of sEVs derived from SPC-01 and iMR-90 neural stem cell sources using an in vitro rat model of SCI. sEVs were isolated from conditioned media and characterized by multi-angle dynamic light scattering and Western blot analysis. Organotypic spinal cord slices (SCS) were used as an in vitro SCI model, with injury induced at 18-20 days, followed by immediate sEV application. After 72 h, tissue samples were collected and tissue was analyzed for markers of apoptosis, cytoskeletal integrity, and survival-related signaling pathways. Results show that SCI induced cytoskeletal disruption and increased apoptotic markers. Treatment with sEVs mitigated these changes, reducing injury-associated protein levels toward baseline. Both SPC-01- and iMR-90-derived sEVs exerted comparable neuroprotective effects, accompanied by decreased PTEN expression, enhanced STAT3 phosphorylation, and increased levels of the anti-apoptotic protein Bcl-xL. In parallel, reduced Nogo-A expression and normalization of RhoA suggested improved cytoskeletal stability and attenuation of inhibitory signaling. Together, these findings demonstrate that neural stem cell-derived sEVs promote early neuroprotective responses in vitro by modulating key signaling pathways, reducing apoptosis, and stabilizing cytoskeletal dynamics, supporting their potential as a cell-free therapeutic strategy for SCI.

BackgroundEpidemiological studies have consistently documented an inverse association between atopic dermatitis (AD) and glioblastoma (GBM), yet the immunogenetic mechanisms underlying this paradox remain elusive. We hypothesized that distinct immune subsets driven by shared genetic variants exhibiting antagonistic pleiotropy may explain this relationship. ObjectiveTo dissect the immunogenetic basis underlying the inverse association between AD and GBM by integrating single-cell transcriptomics and clustered Mendelian randomization, and to identify shared immune subsets and genetic variants exhibiting antagonistic pleiotropy that may explain this epidemiological paradox. MethodsWe integrated single-cell RNA sequencing (scRNA-seq) of publicly available datasets from AD skin (GSE153760) and GBM tumors (GSE256490) with genome-wide association study (GWAS) summary statistics. Disease-specific immune cell subsets were identified, and pathway enrichment was conducted on marker genes. Clustered Mendelian randomization (MR-Clust) was applied to detect heterogeneous causal effects, followed by drug target enrichment analysis using the DGIdb database. ResultsscRNA-seq revealed that Th2A cells were the predominant pathogenic subset in AD lesions, whereas S100A9+HLA-low suppressive monocytes were enriched in the GBM microenvironment. Both subsets shared enrichment in the NF-{kappa}B and Fc{varepsilon}RI signaling pathways, revealing a common immunological framework linking peripheral Type 2 inflammation to central nervous system immunosuppression. MR-Clust identified a distinct genetic cluster (Cluster 2) comprising 32 genes (e.g., IL4R, JAK1, SYK, FCER1G) significantly overexpressed in these cell types. This cluster exhibited antagonistic pleiotropy: it was directionally associated with reduced AD risk (OR = 0.930, 95% CI 0.846-1.023, p = 0.137) but a non-significant risk trend for GBM (OR = 1.447, 95% CI 0.737-2.841, p = 0.283). Drug target analysis indicated that Cluster 2 genes are primary targets of approved AD therapies, including dupilumab (IL4R) and JAK inhibitors (JAK1). ConclusionOur integrative analysis uncovers an immune-genetic axis linking Th2A cells in AD to suppressive monocytes in GBM, providing a mechanistic basis for their inverse comorbidity. These findings highlight a potential therapeutic paradox, underscoring the need for pharmacovigilance regarding long-term cancer risk in AD patients receiving targeted immunomodulators.

Behcets disease (BD) is a systemic inflammatory disease. It is considered as an autoinflammatory disease triggered by innate immunity rather than adaptive immunity. Human leukocyte antigen-B51 (HLA-B51) is the strongest genetic factor associated with BD. This study investigated how HLA class 1 molecules interact with innate immune cells and induce cytokine secretion. For this purpose, 293T cells transfected with a plasmid encoding HLA-B51 were cultured with natural killer (NK) cells obtained from healthy human donors. Within 24 h, the concentrations of interleukin-4 (IL-4), IL-8, and interferon-{gamma} (IFN-{gamma}) in the medium increased, indicating that NK cells secreted cytokines without undergoing cellular expansion for cytolysis. NK cells stimulated by nonself HLA-B51 produced IFN-{gamma} levels comparable to those produced by NK cells stimulated by self HLA-B51. NK cells carrying HLA-B51 were accurately recognized by overexpressing HLA-B51 on 293T cells. Moreover, ample intracellular IFN-{gamma} levels were detected in NK cells after stimulation with phorbol 12-myristate-13-acetate (PMA) plus ionomycin. KLRK1 (CD314)-positive cells mainly primarily accounted for IFN-{gamma}-producing cells, whereas KLRK1-negative cells did not. In contrast, both NCR1 (CD335)-positive and -negative cells contributed to IFN-{gamma} production. We next investigated whether HLA-B51 on the surface of 293T cells stimulates KLRK1 as a ligand causing IFN-{gamma} secretion. In masking experiments using anti-KLRK1 antibodies, NK cells with high levels of cell surface KLRK1 decreased the production of IFN-{gamma}. Conversely, human NK cell line KHYG1 cells also produced IFN-{gamma} in culture with 293T cells, but did not increase IFN-{gamma} through HLA-B51 stimulation. The mRNA expression of the signal adaptor protein HCST (DAP10) in KHYG1 cells was lower than that in NK cells, whereas the relative expression of IL-2RA in KHYG1 cells was higher than that in NK cells. These findings suggest that HLA-B51 can interact with KLRK1 on the NK cells inducing IFN-{gamma} secretion, whereas IL-2 signals outweigh HLA-51 stimulation in KHYG1 cells.

In view of the outstanding progress of machine learning (ML) and growing cost of health systems, it is a current challenge to incorporate artificial intelligence tools into actual medical practice. Here we explored the feasibility and reliability of using machine learning to perform an important immunological investigation that currently requires experienced biologists : Anti-nuclear cytoplasmic antibodies (ANCAs) are important markers for vasculitis and they may be evidenced by microscopic examination of cells labeled with patients' sera. The use of a reliable ML classifier to discriminate between positive and negative samples would increase the rapidity and decrease the cost of immunofluorescence-based ANCA detection. Here, we tested seven well-documented ML algorithms, ranging from simple models such as k nearest neighbors to more complex convolutional neural networks involving millions of adjustable parameter. We studied the feasibility and reliability of classifying 1114 serum samples that had been collected for about 3 years and assayed with conventional procedure. We compared four strategies consisting of assaying either whole microscope fields or individual cell images, and natural images or histograms. The following conclusions were obtained : (i) Several different strategies allowed us to build models stable enough to discriminate between positive and negative samples collected during about 27 months, with a comparison to human classification yielding a kappa index of about 0.7, that may be considered as fairly good and intermediate between the performance of junior and senior biologists. (ii) Simpler ML models combined with theoretical thinking might provide the most rapid and efficient way of developing a reliable test within the framework of a single institution. (iii) In addition, the interpretability of the simplest model provided some theoretical insight into important classification parameters. (iv) An important point and caveat is that the multiplicity and versatility of currently available tools make it an essential requirement to test repeatedly a given model, that must be chosen as simple as possible, to achieve a reliability compatible with medical use. It is concluded that our study provides a strong incentive to incorporate ML tools in well defined medical tests, which might reduce the risk of human errors and pave the way to fully automatic procedures.

Efficient cerebrovasculature is vital to neuronal health and cognition and evidence shows most dementia patients have cerebrovascular abnormalities. Brain vasculature is regulated by Vascular Endothelial Growth Factors (VEGFs) binding VEGF receptor2 (VEGFR2) and stimulating angiogenesis, and neuroprotection. Presenilin1 (PS1) is the main proteolytic component of {gamma}-secretase and PS1 mutants are the most common cause of Familial Alzheimer Disease (FAD). Here we show that an ADAM17 cleavage of extracellular VEGFR2 produces the membrane-bound {gamma}-secretase substrate VEGFR2/CTF1 (called VCTF1), comprising the transmembrane and intracellular domains of VEGFR2. PS1 FAD mutants and {gamma}-secretase inhibitors both accumulate VCTF1 and suppress VEGF-A-induced brain angiogenesis. Moreover, PS1 FAD mutants, {gamma}-secretase inhibitors, and PS1 downregulation, all decrease {gamma} secretase processing of VCTF1, thereby increasing its accumulation and impairing VEGF-A-induced VEGFR2 dimerization/activation, signaling, and endothelial cell (EC) functions. Importantly, VCTF1 binds fulllength VEGFR2 monomers suppressing VEGFR2 dimerization/activation, signaling, and EC functions. These data show that VCTF1 suppresses VEGFR2 dimerization and downstream signaling and functions of the brain VEGF-A-/VEGFR2 system. PS1 FAD mutants increase vulnerability of brain neurons to ischemic stress and exert antimorphic effects on {gamma}-secretase cleavage of VCTF1, increasing its concentration and abolishing VEGF-A-induced VEGFR2 dimerization/activation, signaling, neuroprotection and cognition. Importantly, we detected molecular markers of decreased VEGFR2 dimerization and angiogenic dysfunction in human brain tissue from PS1 FAD mutant genotypes. Together, our data suggest a pathway through which FAD mutants promote dementia by increasing VCTF1 and decreasing brain angiogenesis and neuroprotection, suggesting that PS1 FAD patients may benefit from therapeutic methods that decrease brain VCTF1.