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PubMed2026

Thyroid-stimulating hormone receptor mediates peripheral-central neuroimmune crosstalk in autoimmune thyroid diseases.

BACKGROUND: Organ-specific autoimmune diseases, particularly Graves' disease (GD) and its extrathyroidal manifestation, Graves' orbitopathy (GO), are characterized by systemic autoimmunity that may extend its impact to the central nervous system (CNS). While thyroid-stimulating hormone receptor (TSHR) is the primary driver of pathological remodeling in the thyroid and orbital tissues, emerging evidence suggests it is also expressed in the brain and may participate in neuroimmune signaling. However, the molecular mechanisms linking peripheral TSHR-driven autoimmunity to these extended systemic features remain unclear. Thus, GD and GO provide a unique window to investigate how peripheral autoantibodies influence CNS involvement as part of its broader pathological spectrum. METHODS: Genome-wide association studies (GWAS) and post-GWAS analyses were integrated with bulk RNA sequencing, single-cell and spatial transcriptomics, and brain imaging phenotypes to comprehensively characterize peripheral and central alterations in GD and GO. Mendelian randomization was applied to test causal relationships between genetic variants and brain signatures. Structural biology analyses were further conducted including protein-protein docking, small-molecule docking, and normal mode dynamics to identify prospective modulators of TSHR. Immunofluorescence staining was performed in a GO mouse model to validate the colocalization of potential interacted proteins in the specific brain region. RESULTS: Brain imaging-derived phenotypes (IDPs) alterations in GO and GO were systematically analyzed to identify neuroanatomical and functional alterations. TSHR was further identified as a shared genetic driver across peripheral and central compartments. TSHR was expressed in spiny projection neurons, microglia, and peripheral T cells, with cell-cell communication analyses highlighting TSHR-mediated interactions among neurons, endothelial cells, and microglia. Immunofluorescence staining in a GO mouse model confirmed the colocalization of TSHR with FN1 and GNAS in the basal ganglia, providing tissue-level validation of the computationally predicted ligand-receptor interactions. Immune profiling further showed immune alterations in GD and GO. Structural modeling supported plausible physical interfaces between TSHR and interacting proteins, and small-molecule screening identified three repurposable compounds - venetoclax, irinotecan, and dutasteride - with predicted favorable docking scores and stable binding poses in our simulations. CONCLUSIONS: These findings demonstrate that TSHR acts as a molecular hub mediating peripheral-central neuroimmune crosstalk in GD and GO. The results support a broader "disease-molecule axis" framework that links genetic susceptibility with multi-level immune and neural mechanisms. This work provides mechanistic insights relevant to the development of TSHR-targeted therapies, with implications for both peripheral immune modulation and central regulation. However, the limited sample size, lack of longitudinal follow-up, and absence of in vivo validation warrant cautious interpretation and further investigation.

Receptors, Thyrotropin↗

PubMed1976

[Rheumatic manifestations in 80 cases of Crohn's disease].

Out of a series of 80 patients suffering from Crohn's disease, 31 presented rheumatic manifestations. In 16 subjects this took the form of synovitis closely dependent on the enteritic evolution, which developed after the alimentary symptoms, and which worsened as they did and sometimes regressed as they did following medical or surgical treatment. In combination with erythema nodosum, aphtosis, and conjunctivitis, synovitis appears to be the expression of an immune response to the enteritic lesion. Three cases of chronic polyarthitis and 6 cases of asymptomatic sacro-ileitis were also observed, and 6 cases of spondylarthritis of a minor radiological type were observed that evolved independently of the Crohn's disease. Typing according to the HLA system using 26 antigens was carried out in 44 subjects; no difference in phenotype frequency was found between a control group (blood donors) and the group of subjects with Crohn's disease alone; however, the antigen W 17 was found significantly more frequently in those subjects with peripheral arthritis and the antigen W 27 was found more frequently in those with spondylarthritis. These findings suggest, although it is not certain, the existence of genetic susceptibility to rheumatic manifestations in certain sites in patients with Crohn's disease.

Arthritis↗

PubMed2025

High baseline PD-1+ CD8 T Cells and TIGIT+ CD8 T Cells in circulation associated with response to PD-1 blockade in patients with non-small cell lung cancer.

Blockade of PD-1 or its ligand PD-L1 with antibodies revolutionized treatment for stage III and IV non-small cell lung cancer (NSCLC) since FDA approval in 2015. However, resistance to PD-1/PD-L1 blockade remains a challenge, highlighting the need for biomarkers. This study analyzed 36 stage III and IV NSCLC patients, classified as responders or non-responders by iRECIST criteria. Peripheral blood mononuclear cells collected at baseline and post-treatment were examined for surface and intracellular markers via flow cytometry. CITE sequencing of CD8 T cells from three patients and plasma ctDNA analysis from 13 patients was performed using an ultrasensitive barcoding and next-generation sequencing method. Phenotypic analysis of CD8 T cells revealed higher TIGIT and PD-1 expression at baseline in responders compared to non-responders. Long-term responders (> 21 months) exhibited increased TCF-1+PD-1+ CD8 T cell frequencies relative to shorter-term responders (> 15 months) and non-responders. CITE sequencing revealed intrinsic differences in immune regulation pathways between responders and non-responders. Finally, non-responders showed elevated and increasing ctDNA levels post-treatment, correlating with declining TCF-1+PD-1+ CD8 T cells. Our data suggests combining CD8 T cell analysis with ctDNA dynamics could identify promising biomarkers for monitoring clinical response and treatment efficacy to PD-1/PD-L1 blockade in NSCLC.

Humans↗

PubMed2025

Lipid peroxidation and type I interferon coupling fuels pathogenic macrophage activation causing tuberculosis susceptibility.

A quarter of the human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop pulmonary TB. We developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: the development of necrotic lung granulomas and determined that the sst1-susceptible phenotype was driven by the aberrant macrophage activation. This study demonstrates that the aberrant response of the sst1-susceptible macrophages to prolonged stimulation with TNF is primarily driven by conflicting Myc and antioxidant response pathways leading to a coordinated failure (1) to properly sequester intracellular iron and (2) to activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled superinduction of Ifnβ and sustained the type I interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within the granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. The upregulation of the Myc pathway in peripheral blood cells of human TB patients was significantly associated with poor outcomes of TB treatment. Thus, Myc dysregulation in activated macrophages results in an aberrant macrophage activation and represents a novel target for host-directed TB therapies.

Animals↗

PubMed1975

T-cell reconstitution by thymus transplantation and transfer factor in severe combined immunodeficiency.

Reconstitution of cell-mediated immunity was achieved in a 5-month old female infant with severe combined immunodeficiency by fetal thymus transplant given simultaneously with two units of transfer factor. Thymus was obtained from a 15-week gestational age male fetus, and the two units of transfer factor from the lymphocytes of 500 ml of peripheral blood. Three weeks after transplantation, two nel HL-A antigens were detected on the infant's lymphocytes, one of which was present in the mother of the thymus donor; at the same time, some of the infant's own HL-A antigens disappeared. Thereafter, the percent of rosette-forming cells (T-cells) increased and the in vitro response to phytohemagglutinin and allogeneic lymphocytes became normal, and delayed skin tests became positive. Karyotyping of peripheral blood lymphocytes posttransplantation reveals an XY (male) pattern. These results suggest lymphocyte re-population as a result of the thymus-transfer factor therapy. Five months after transplantation, the patient has normal cellular immunity but persistent hypogammaglobulinemia. She is free of infection and growing normally on gammaglobulin injections.

Female↗

PubMed2026

Programmed cell death-1: from a T-cell immune checkpoint to a regulator of Natural Killer cell biology.

Programmed cell death protein 1 (PD-1, CD279) is a pivotal inhibitory immune checkpoint receptor that plays a central role in maintaining immune homeostasis and peripheral tolerance. Originally characterized as a negative regulator of T-cell activation, PD-1 limits excessive immune responses and prevents autoimmunity, while its sustained expression under conditions of chronic antigen stimulation contributes to T-cell dysfunction and exhaustion. The discovery that blockade of the PD-1 pathway can restore anti-tumor immunity has revolutionized cancer therapy and established immune checkpoint inhibition as a cornerstone of modern oncology. Although PD-1 has traditionally been viewed as a key regulator of adaptive immunity, accumulating evidence indicates that its biological functions extend beyond T cells. In recent years, PD-1 expression has been identified in several innate immune cell populations, particularly Natural Killer (NK) cells, where it has emerged as an important modulator of effector functions, cytokine production, metabolic fitness, and antitumor activity. These findings have challenged the classical view of PD-1 biology and revealed unexpected similarities between NK-cell dysfunction and the exhausted phenotype described in chronically stimulated T cells. In the tumor microenvironment, PD-1 expression on NK cells has been associated with impaired cytotoxicity and reduced immune surveillance, suggesting that NK cells may also represent relevant targets of PD-1-mediated immunosuppression. At the same time, the mechanisms regulating PD-1 expression and signaling in NK cells appear to differ, at least in part, from those operating in T lymphocytes, highlighting the complexity of this pathway across distinct immune cell subsets. In this review, we summarize the current knowledge of PD-1 biology, from its established role in T-cell regulation to its emerging functions in NK cells. We discuss the molecular mechanisms governing PD-1 expression and signaling, its contribution to immune dysfunction in cancer and chronic diseases, and the potential implications of targeting the PD-1 axis to enhance both adaptive and innate antitumor immunity.

Natural Killer (NK) cells↗

PubMed2025

Genetic haplotypes in VWA8, OSBPL6, and ADAMTS9-AS2 are associated with immune-related adverse effects in ICI-treated patients with cancer.

BACKGROUND: Immune-related adverse events (irAEs) remain largely unpredictable, potentially affecting multiple organ systems and occurring at almost any point during and even occasionally after immune checkpoint inhibitor (ICI) treatment. To identify populations at risk for these immune-mediated toxicities, we analyzed genetic characteristics and immune markers associated with clinically significant irAEs. METHODS: We carried out a genome-wide association study on 373 white patients receiving ICI treatment. We identified single nucleotide polymorphisms associated with irAEs. Blood cytokine profiling and peripheral blood mononuclear cell RNA sequencing were performed at pretreatment baseline and 6-8 weeks after ICI initiation. Findings were validated in two external cohorts. RESULTS: We identified genetic haplotypes in VWA8 (Von Willebrand Factor A Domain Containing 8), OSBPL6 (Oxysterol Binding Protein Like 6), and ADAMTS9-AS2 (ADAM Metallopeptidase With Thrombospondin Type 1 Motif 9 Antisense RNA 2) associated with grade ≥2 irAEs. Patients carrying risk haplotypes for one or more genes exhibited significantly greater rates of grade ≥2 (OR 3.02; 95% CI 1.83 to 5.02; p<0.001), grade ≥3 (OR 3.59; 95% CI 1.93 to 6.64; p<0.001), and multiple type irAE (OR 2.60; 95% CI 1.53 to 4.39; p<0.001). Serum CCL3 levels were significantly elevated in individuals carrying risk haplotypes (p=0.03). Gene expression analysis demonstrated activated autoimmune and inflammatory pathways in the genetic risk group. CONCLUSIONS: Novel polymorphisms in VWA8, OSBPL6, and ADAMTS9-AS2 may impact immune pathways, promote inflammation, potentiate autoimmune phenotypes, and convey risk of irAE in ICI-treated patients.

Humans↗

PubMed2025

High MGMT expression identifies aggressive colorectal cancer with distinct genomic features and immune evasion properties.

INTRODUCTION: The epigenetic silencing of O6-methylguanine DNA methyltransferase (MGMT) is associated with reduced DNA repair capacity, carcinogenesis and increased sensitivity to alkylating chemotherapy. However, the biological role and clinical significance of MGMT overexpression in cancer remains poorly understood. METHODS: Using multiplexed quantitative immunofluorescence we measured the localized levels of MGMT protein, γH2AX and CD8+ T cells in multiple retrospective colorectal cancer (CRC) cohorts. Genomic and transcriptomic features of selected cases were also studied with whole exome DNA sequencing and genome-wide methylation analysis. MGMT-methylated human CRC cells SW620 were transfected with an MGMT-containing plasmid and co-cultured with allogeneic peripheral blood mononuclear cells. RESULTS: A subset of CRCs showed MGMT protein upregulation associated with lower γH2AX, reduced CD8+ tumor infiltrating lymphocytes (TILs), mismatch repair proficient (pMMR) status and shorter survival. CD8+ TILs were more distant from MGMT-expressing cells than MGMT-negative cells and the MGMT promoter methylation status did not highly correlate with MGMT protein levels in CRC. In genomic/transcriptomic analysis, high MGMT expression was associated with a lower nonsynonymous somatic mutational burden, higher transition-to-transversion mutation ratio, increased deleterious TP53 variants and distinct transcriptomic profiles. The exogenous expression of MGMT in SW620 CRC cells reduced the number of spontaneous nonsynonymous mutations, reproduced mutational features of MGMT-high CRC and limited the in vitro T-cell-mediated killing of malignant cells induced by proinflammatory cytokines in tumor/immune cell co-cultures. CONCLUSIONS: MGMT overexpression identifies a previously undescribed subset of CRCs with distinct biological and clinical properties including reduced mutagenesis, adaptive immune evasion, predominantly pMMR phenotype and aggressive clinical course. Direct, quantitative assessment of MGMT protein expression using spatially resolved analysis is more reliable than inference of MGMT expression by promoter methylation status in CRC.

Humans↗

PubMed1975

B lymphocytes in primary and secondary deficiencies of humoral immunity.

The quantitative studies of B lymphocytes in peripheral blood have been performed in various forms of primary and secondary immunodeficiency disease in man. X-linked agammaglobulinemia was found to comprise two sub-types, one lacking B-cell population, the other showing low numbers of B lymphocytes. The absence of B cells in severe combined immunodeficiency was corrected by marrow transplants in 3 children. Cases of DiGeorge syndrome and lepromatous leprosy showed an absolute increase in numbers of B lymphocytes in peripheral blood, probably a compensatory mechanism in the market deficit of T-cell population and function. The reconstitution of DiGeorge syndrome by fetal thymus transplant reversed the abnormally high percentage of B lymphocytes.

Agammaglobulinemia↗

PubMed2026

Epigenome-wide analysis of DNA-methylation signatures following climate-related disasters.

BACKGROUND: Floods and tropical cyclones (TCs), two of the most frequent and costliest climate-related disasters worldwide, have been linked to sustained health risks extending beyond acute hazards. However, evidence on the underlying epigenetic mechanisms remains scarce. We aimed to characterize DNA methylation patterns associated with exposure to floods and TCs of varying intensities. METHODS: We collected peripheral blood samples from 479 women (132 twin pairs and 215 of their sisters) across Australia. Blood-derived DNA methylation profiles were assessed using the Illumina HumanMethylation450 BeadChip array. Daily flood and TC exposure data for the 6 years preceding each blood draw were obtained from the Dartmouth Flood Observatory and the International Best Track Archive for Climate Stewardship, respectively, and linked to participants based on residential addresses. Using a within-sibship analytical framework that accounted for shared familial factors and other relevant covariates, we examined associations between flood and TC exposures of varying intensities and site-specific methylation at each cytosine-guanine dinucleotide (CpG). Differentially methylated regions (DMRs) were identified using a combination of the comb-p and DMRcate algorithms. RESULTS: There were 164 CpGs and 219 DMRs associated with flood and TC exposures (Bonferroni-adjusted p value < 0.05), mapping to 242 genes enriched in pathways related to inflammation and immune regulation. These genes have been implicated in a wide range of human diseases or phenotypes. The number of differentially methylated CpGs increased with more recent and higher-intensity exposures. Intensity-dependent gene regulation was observed, with genes such as AMT and C22orf45 consistently implicated across various exposure levels, whereas RNF39 and ACY3 emerged only at higher intensities. CONCLUSIONS: Exposures to floods and TCs were associated with differentially DNA methylated signals across the human genome, exhibiting intensity-dependent patterns. The identified signals and related gene pathways may shed light on the biological mechanism underlying the profound health effects of climate-related disasters.

Humans↗

PubMed1978

Secretory immunoglobulin deficiency in a family with inflammatory bowel disease.

A family with 4 of 10 first-degree relatives affected with inflammatory bowel disease (IBD) was studied to determine whether any distinct immunological abnormalities occur in the affected members, as compared with unaffected members of the family, normal controls, and other unrelated patients with IBD. Red cell blood type and HL-A phenotypes did not distinguish between healthy and affected members, although HL-A2, 32, B27, and B12 were the predominant haplotypes in members with IBD. There was no significant difference between the two groups in the lymphocyte subpopulation counts of T cells, B cells, and cells carrying Fc or complement receptors. The in vitro mitogen response, however, to phytohemagglutinin and pokeweed mitogen were depressed in the affected members. Serum IgA and C3 levels were significantly elevated in members with IBD compared to healthy subjects with values of 232 +/- 69 (mean +/- SD) versus 148 +/- 29 mg per dl for IgA (P less than 0.05) and 173 +/- 32 versus 115 +/- 22 mg per dl for C3 (P less than 0.025), respectively. Plasma and, to a lesser extent, peripheral lymphocytes from 2 affected members who were tested were cytotoxic to allogeneic colonic epithelial cells. Salivary IgA was normal in the affected family members and unrelated patients with IBD. However, the free secretory component of salivary IgA was absent or markedly depressed in family members, as well as in unrelated patients with ulcerative colitis. This deficiency of the secretory immune system appears to characterize more frequently ulcerative colitis than Crohn's disease and may compromise mucosal host defenses in IBD.

Adolescent↗

PubMed1977

Functional heterogeneity among the T-derived lymphocytes of the mouse. VII. Conversion of T1 cells to T2 cells by antigen.

The T1 subpopulation of peripheral T cells was defined in mice by its short half life, insensitivity to anti-thymocyte sera (ATS) in vivo, and slow kinetics of response to antigen. The T2 subpopulation was defined by its long life time, elimination by ATS in vivo, and rapid response to antigen. Mice containing only T1-type T cells were constructed by adult thymectomy (ATx) followed immediately by the elimination of T2 cells by ATS treatment. Immunization of these mice with SRBC led to the production of memory helper cells in the T2 subpopulation. This process depended on the presence of T1 cells and for the most part required SRBC immunization, although a few SRBC-specific T2 cells reappeared in the mice in the absence of antigen. We conclude that T1 cells can give rise to T2 cells in an antigen-driven step and that the two populations correspond to virgin and memory T cells, respectively.

Animals↗

PubMed1976

Alloantigens defined by cytotoxic isoantisera prepared against peripheral rabbit lymphocytes.

Seven lymphocytotoxic antisera were produced in rabbits by means of isoimmunization with purified peripheral lymphocytes. The phenotypic profile of the defined lymphocyte antigens was determined in a panel of 97 rabbits. The results indicated the existence of at least two major antigenic determinants. Further typing and cross-absorptions were performed with several of the lymphocytotoxic antisera in order to demonstrate monospecificity. The anti-3 antiserum was used to determine the distribution of the respective antigens in various rabbit organs. The highest concentration of the alloantigen was contained in the spleen, followed in descending order by the lymph nodes, lung, skin, kidney, heart and liver. Fat and red blood cells (rbc) contained no detectable antigen. A two-stage physical adherence column method was used to separate T-derived from B-derived lymphocytes and each fraction was tested for the alloantigen. Alloantigen was detected on both cell types. The fact that the anti-3 antiserum never killed more than 30% of the cells suggests that it could define a subpopulation of lymphocytes.

Absorption↗

PubMed1979

Aberrations of suppressor T cells in human graft-versus-host disease.

To determine whether imbalances in immunoregulatory T-cell subsets exist in patients with graft-versus-host disease, we analyzed T cells in three patients with acute and in six patients with chronic graft-versus-host disease after bone-marrow transplantation. The normal human peripheral-blood T-cell compartment is composed of 80 per cent TH2-and 20 per cent TH2+ T cells, and defined by reactivity with subset-specific heteroantiserums. Human suppressor cells are TH2+, whereas helper cells are TH2-. Patients with acute and chronic graft-versus-host disease had abnormalities in these populations, and their T cells frequently bore la-like antigens. Patients with acute disease lacked TH2+ cells, and the reappearance of this subset preceded the cessation of disease activity. Chronic disease, in contrast, was more heterogeneous. Suppressor cells were lacking in two patients but increased in the other four. Two of these four patients had TH2+, la+ T cells, suggesting in vivo activation of suppressor cells. Studies showing that these TH2+, la+ cells actively suppressed the in vitro immune response support this hypothesis and suggest that the immunoregulatory cells may profoundly affect the overall immune response.

Acute Disease↗

PubMed1979

Expression of Ly-6 alloantigen during differentiation of cytotoxic T cells.

We have prepared anti-Ly-6.2 by immunization of (CBA/Ca X A-Thy-1a)F1 with AKR/Crc lymphocytes and used the antiserum to study heterogencity of peripheral T lymphocytes. The Ly-6 alloantigen is found on all activated cytotoxic T cells studied, directed to a variety of different target antigens. The antiserum reveals heterogeneity among the precursors of T killer cells, i.e. allogeneic precursors are Ly-6-, whereas the generation of cytotoxic cells to hapten-modified syngeneic cells and xenogeneic cells is partially abrogated by serum pretreatment. Precursors of some in vivo primed responses are also Ly-6+. These findings indicate that the expression of Ly-6 antigen is, at least, partially related to the stage of T cell activation. The in vitro proliferative response of T cells to allogeneic Ia antigens is substantially reduced by pretreatment with anti-Ly-6.2 serum suggesting that this antiserum may be a useful tool for the study of heterogeneity in the Ly-1+ pool of T cells.

Animals↗

PubMed1978

The lymphoreticular system in triggering virus plus self-specific cytotoxic T cells: evidence for T help.

The thymus determines the spectrum of the receptor specificities of differentiating T cells for self-H-2; however, the phenotypic expression of T cell's specificity for self plus virus is determined predominantly by the H-2 type of the antigen presenting cells of the peripheral lymphoreticular system. Furthermore, virus specific helper T cells are essential for the generation of virus-specific cytotoxic T cells. For cooperation between mature T cells and other lymphocytes to be functional in chimeras, thymic epithelial cells and lymphohemopoietic stem cells must share the I region; killer T-cell generation also requires in addition compatibility for at least one K or D region. These conclusions derive from the following experiments: A leads to (A X B)F1 chimeric lymphocytes do produce virus-specific cytotoxic T-cell activity for infected A but not for infected B cells; when sensitized in an acutely irradiated and infected recipient (A X B)F1 these chimeric lymphocytes respond to both infected A and B. Therefore the predominantly immunogenically infected cells of chimeras the radiosensitive and by donor stem cells replaced lymphoreticular cells. In this adoptive priming model (KAIA/DB leads to KAIA/DC) chimeric lymphocytes could be sensitized in irradiated and infected F1 against KA and DC but not against infected DB targets. In contrast KBIB/DA leads to KCIC/DA chimeras' lymphocytes could not be sensitized at all in appropriately irradiated and infected F1 recipients. Thus these latter chimeras probably lack functional I-specific T helper cells that are essential for the generation of T killer cells against infected D compatible targets. If T cells learn in the thymus to recognize H-21 or K, D markers that are not at least partially carried themselves in other cells of the lymphoreticular system immunological interactions will be impossible and this paradox situation results in phenotypic immune incompetence in vivo.

Animals↗

PubMed1977

Cellular presensitization to alloantigens in haemodialyzed patients.

The state of cellular alloimmunity in haemodialyzed patients has been assessed by a microcytotoxicity assay and compared with that in healthy donors. Some of the patients were also studied during the weeks following a first kidney transplant rejection. For comparison, lymphocytes of multiparous women have been studied. Skin fibroblasts from a panel of normal donors were used as targets, and effector cells were cryopreserved peripheral blood lymphocytes. Lymphocyte-mediated cytotoxicity (LMC) demonstrated restricted reactivity against target cells, an indication of specificity. It detected presensitization more often than a standard test for cytotoxic antibodies. This difference is not attributable to differences in sensitivity of the two tests, since some patients were at least once LMC negative and cytotoxic antibody positive, and the antigens detected by both tests were different in patients who had positive LMC and cytotoxic antibodies simultaneously. In some experiments, LMC target determinants were not HLA serologically detectable antigens. Furthermore, lymphocytes obtained after transplant rejection were cytotoxic for targets which did not share the donors' serological mismatches. These results suggest that antigens other than HLA-A and B may be the determinants recognized in the microcytotoxicity assay.

Antibody Specificity↗

PubMed2026

Simulation of CRISPR/Cas9-mediated gene editing for the Vitellogenin gene in Apis mellifera.

CRISPR/Cas9 genome editing provides a powerful framework for interrogating gene function in Apis mellifera. Yet, empirical application remains challenging due to biological constraints, including haplodiploid genetics, narrow embryonic injection window, and the social rearing requirements that complicate functional validation. These constraints necessitate in silico pre-screening to maximize editing success before resource-intensive wet-lab implementation. Within the omnigenic framework, which distinguishes core regulatory genes from peripheral loci buffered by network effects, vitellogenin (Vg) represents an optimal target which is ancestrally dedicated to yolk provisioning; it has been co-opted to orchestrate diverse non-reproductive functions including longevity, stress resistance, immunity, and social behavior. We developed a computational pipeline to design a list of 57 and 56 candidate guide RNAs (gRNA) for targeted Vg knockout, evaluating candidate sites in both functional exons 2 and 3 based on structural accessibility and frameshift efficiency. Comparative analysis revealed complementary strengths in two top-best candidates from initial target pool of predicted gRNAs. The gRNA targeting exon 2 exhibits weaker secondary structure (ΔG = -0.25 kcal/mol versus -2.10 kcal/mol for exon 3), aligning with empirical evidence that sites with ΔG > -1.0 kcal/mol achieve 2-5 × higher Cas9 binding efficiency. This site yielded moderate frameshift frequency (77.8%; 61.9 percentile). Conversely, the predicted editing outcome for the gRNA targeting exon 3, despite stronger structural constraints, demonstrated superior functional disruption metrics demonstrating very high frameshift frequency (88.3%; 95.2 percentile), high in silico editing precision, minimal microhomology-mediated repair bias, and reproducible outcomes wherein nearly all predicted indels disrupt the coding sequence. Protein structure and domain analyses further predict that frameshift edits will generate a truncated protein missing all downstream functional domains. We recommend parallel empirical validation of both exon 2 and exon 3 targets to resolve the trade-off between structural accessibility (favoring higher editing rates) and frameshift efficacy (favoring complete loss-of-function). This dual-target strategy accommodates uncertainty in in vivo performance while maximizing the probability of generating informative phenotypes. Our in silico framework enables rational CRISPR design in non-model organisms by computationally balancing biophysical accessibility with functional impact, accelerating functional genomics in species where empirical optimization faces substantial biological constraints.

Animals↗