Safety evaluation of single-sperm cryopreservation technique applied in intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is a technique that directly injects a single sperm into the cytoplasm of mature oocytes. Here, we explored the safety of single-sperm cryopreservation applied in ICSI. This retrospective study enrolled 186 couples undergoing ICSI-assisted pregnancy. Subjects were allocated to the fresh sperm (group A)/single-sperm cryopreservation (group B) groups based on sperm type, with their clinical baseline/pathological data documented. We used ICSI-compliant sperm for subsequent in vitro fertilization and followed up on all subjects. The recovery rate/cryosurvival rate/sperm motility of both groups, the pregnancy/outcome of women receiving embryo transfer, and the delivery mode/neonatal-related information of women with successful deliveries were recorded. The clinical pregnancy rate, cumulative clinical pregnancy rate, abortion rate, ectopic pregnancy rate, premature delivery rate, live birth delivery rate, neonatal birth defect rate, and average birth weight were analyzed. The two groups showed no significant differences in age, body mass index, ovulation induction regimen, sex hormone [anti-Müllerian hormone (AMH)/follicle-stimulating hormone (FSH)/luteinizing hormone (LH)] levels, or oocyte retrieval cycles. The sperm recovery rate (51.72%-100.00%) and resuscitation rate (62.09% ± 16.67%) in group B were higher; the sperm motility in the two groups demonstrated no significant difference and met the ICSI requirements. Group B exhibited an increased fertilization rate, decreased abortion rate, and increased safety versus group A. Compared with fresh sperm, the application of single-sperm cryopreservation in ICSI sensibly improved the fertilization rate and reduced the abortion rate, showing higher safety.

Single-Cell Profiling Unveils the Inflammatory Heterogeneity within Cutaneous Lesions of Bullous Pemphigoid.

Bullous pemphigoid (BP) is a subepidermal blistering skin disease with a complex pathogenesis involving various immune cells. However, the transcriptional features of these cells remain poorly defined. In this study, we constructed a comprehensive and single-cell resolution atlas of various immune cells within BP skin lesions through integrative single-cell analysis, flow cytometry, and multiplex immunohistochemistry. We observed prominent expansion and transcriptional changes in mast cells, macrophages, basophils, and neutrophils within BP lesions. Mast cells within the lesions adopted an active state and exhibited an elevated capacity for producing proinflammatory mediators. We observed an imbalance of macrophages/dendritic cells within BP lesions. Two macrophage subpopulations (NLRP3+ and C1q+) with distinct transcriptional profiles were identified and upregulated effector programs. T-peripheral helper-like T helper 2 cells were expanded in skin lesions and peripheral blood of patients with BP and were capable of promoting B-cell responses. In addition, we observed clonally expanded granzyme B-positive CD8+ T cells within BP lesions. Chemokine receptor mapping revealed the potential roles of macrophages and mast cells in recruiting pathogenic immune cells and underlying mechanisms within BP lesions. Thus, this study reveals key immune pathogenic features of BP lesions, thereby providing valuable insights for potential therapeutic interventions in this disease.

Integrative single-cell analysis reveals distinct adaptive immune signatures in the cutaneous lesions of pemphigus.

Pemphigus, an autoimmune bullous disease affecting the skin and mucosal membranes, is primarily driven by anti-desmoglein (Dsg) autoantibodies. However, the underlying immune mechanisms of this disease remain largely elusive. Here, we compile an unbiased atlas of immune cells in pemphigus cutaneous lesions at single-cell resolution. We reveal clonally expanded antibody-secreting cells (ASCs) that exhibit variable hypermutation and accumulation of IgG4 class-switching in their immunoglobulin genes. Importantly, pathogenic Dsg-specific ASCs are localized within pemphigus lesions and can evolve from both Dsg-autoreactive and non-binding precursors. We observe an altered distribution of CD4+ T cell subsets within pemphigus lesions, including an imbalance of Th17/Th2 cells. Significantly, we identify a distinct subpopulation of Th17 cells expressing CXCL13 and IL-21 within pemphigus lesions, implying its pivotal role in B cell recruitment and local production of autoantibodies. Furthermore, we characterize multiple clonally expanded CD8+ subpopulations, including effector GMZB+ and GMZK+ subsets with augmented cytotoxic activities, within pemphigus lesions. Chemokine-receptor mapping uncovers cell-type-specific signaling programs involved in the recruitment of T/B cells within pemphigus lesions. Our findings significantly contribute to advancing the understanding of the heterogeneous immune microenvironment and the pathogenesis of pemphigus cutaneous lesions, thereby providing valuable insights for potential therapeutic interventions in this disease.

Psychological morbidity in patients with pemphigus and its clinicodemographic risk factor: A comparative study.

Due to the long disease duration, impact on appearance, social stigmatization, and numerous side effects of treatment, pemphigus, an autoimmune bullous disease, often has a significant psychological impact on patients. On the other hand, mood disorders may exacerbate the disease by affecting the patient's self-management, forming a vicious circle. To investigate anxiety and depressive disorders in patients with pemphigus, a total of 140 patients with pemphigus were recruited for this cross-sectional retrospective study between March 2020 and January 2022. A control group of 118 patients with psoriasis, a commonly known psychosomatic dermatosis, was established. Patients were evaluated at the visiting day with the Beck Anxiety Inventory and Beck Depression Inventory second edition for mood disorders, the Dermatology Life Quality Index and the EuroQol Five Dimensions Questionnaire for disease-related life quality, and the Visual Analogue Scale for pain and itching symptoms. In our cohort, 30.7% of patients with pemphigus suffered from either anxiety disorder (25%) or depressive disorders (14.3%). Propensity score matching was implemented to create a comparable cohort of pemphigus and psoriasis groups considering the baseline discrepancy. Thirty-four comparable pairs of pemphigus and psoriasis patients were extracted. The prevalence and severity of depressive disorder in pemphigus patients were significantly higher than in psoriasis patients, while anxiety disorder levels appeared to be similar in two groups. Multivariate logistic regression analysis further revealed that disease-related hospitalization history, active mucosal damage, and concomitant thyroid disease are independent risk factors for mood disorders in pemphigus patients. Our results showed that pemphigus patients had a high prevalence and severity of mood disorders. Relevant clinicodemographic indicators may be valuable for prediction and early identification of mood disorders in pemphigus. Better disease education from physicians may be important for these patients to achieve overall disease management.

Selenized glucose improves rat semen quality by improving the gut microbiota and serum metabolome.

Selenium (Se), a well-known antioxidant, is important for male fertility and sperm quality. The gut microbiota is involved in vital activities and cross-talk between reproduction and the gut axis. It is still unclear whether the gut microbiota mediates the impact of selenium on semen quality, and what the underlying mechanisms may be. A selenized glucose (SeGlu) derivative is a novel organic Se compound. After 7 days of acclimation, the Sprague-Dawley (SD) male rats (230 g, 6 weeks) were divided into three drinking groups: deionized water group (CK), SeGlu 0.15 group (0.15 mg Se per L), and SeGlu 0.4 group (0.4 mg Se per L). All animals were euthanized 30 days post-treatment. Serum and intratesticular testosterone and semen parameters were measured. Metagenomic and non-targeted metabolomic approaches were used to study the effects of SeGlu on the gut microbiota and serum metabolites of rats. In both the SeGlu 0.15 Group and the SeGlu 0.4 Group, we found a significant increase in seminiferous epithelium thickness. While the SeGlu 0.4 Group had a tendency to increase with insignificant difference, the SeGlu 0.15 Group significantly improved the sperm viability, survival rate, and seminal plasma fructose. SeGlu had no effect on intratesticular testosterone levels, or abnormal sperm counts. Measured serum testosterone levels using ELISA and LC-MS, which showed a decreasing trend. ELISA did not reveal significant differences, but LC-MS indicated a significant decrease in SeGlu 0.4 group. Meanwhile, the SeGlu 0.15 Group reduced the abundance of harmful bacteria such as Rikenella, Barnesiella, Tenacibaculum, and Aeromonas while increasing the abundance of beneficial microbiota such as Intestinimonas, Christensenella, Coprococcus, and Butyrivibrio. Linear discriminant analysis Effect Size (LEfSe) identified the SeGlu 0.15 group's feature genera as Roseburia, Clostridium, Ruminococcus, and Eubacterium. Serum metabolites showed that the SeGlu 0.15 Group increased 5 beta-androstane-3,17-dione while decreasing estrone and 2-methoxyestradiol (2-MeOE2). In conclusion, the SeGlu 0.15 Group can significantly alter the levels of several sex hormones in serum, improve the quality of rats' sperm, and reduce harmful bacterial colonization. SeGlu 0.15 may be used as an effective dietary supplement.

Direct Immunofluorescence of IgG on Formalin-Fixed Paraffin-Embedded Tissue by Heat-Induced Antigen Retrieval as a Sensitive Method for the Diagnosis of Pemphigus.

Purpose: Direct immunofluorescence (DIF) on frozen sections (DIF-F) plays a key role in the identification and differential diagnosis of bullous dermatoses, which are a group of critical autoimmune diseases that include pemphigus, bullous pemphigoid (BP), and epidermolysis bullosa acquisita (EBA). However, this technique requires specialized laboratory equipment conditions, sample acquisition and sample preservation. In this study, the application value of DIF on paraffin-embedded tissue sections (DIF-P) detecting IgG using heat-induced antigen retrieval (HIAR) in the diagnosis of bullous dermatosis was explored. Patients and Methods: Samples from 12 patients with pemphigus vulgaris (PV), 10 patients with pemphigus foliaceus (PF), 17 patients with BP, and 4 patients with EBA were retrospectively studied for DIF-P IgG detection. Formalin-fixed, paraffin-embedded tissue (FFPE) was used, and the antigen retrieval method used in the experiment was HIAR. All patients were diagnosed with the autoimmune bullous disease (AIBD) based on clinical presentation, histopathology, DIF-F, and enzyme-linked immunosorbent assay (ELISA). Results: Intercellular staining for IgG in the epidermis was successful in paraffin-embedded tissue sections in 11 of 12 PV samples and in all 10 PF samples. IgG at the basement membrane zone (BMZ) was not detected by immunofluorescent staining in 17 BP samples and 4 EBA samples. Conclusion: The detection of IgG by DIF-P using HIAR can be used for the diagnosis of pemphigus as an alternative method to DIF-F.

Transcriptomic profiling of pemphigus lesion infiltrating mononuclear cells reveals a distinct local immune microenvironment and novel lncRNA regulators.

Pemphigus is an autoimmune skin disease. Ectopic lymphoid-like structures (ELSs) were found to be commonly present in the pemphigus lesions, presumably supporting in situ desmoglein (Dsg)-specific antibody production. Yet functional phenotypes and the regulators of Lymphoid aggregates in pemphigus lesions remain largely unknown. Herein, we used microarray technology to profile the gene expression in skin lesion infiltrating mononuclear cells (SIMC) from pemphigus patients. On top of that, we compared SIMC dataset to peripheral blood mononuclear cells (PBMC) dataset to characterize the unique role of SIMC. Functional enrichment results showed that mononuclear cells in skin lesions and peripheral blood both had over-represented IL-17 signaling pathways while neither was characterized by an activation of type I Interferon signaling pathways. Cell-type identification with relative subsets of known RNA transcripts (CIBERSORT) results showed that naïve natural killer cells (NK cells) were significantly more abundant in pemphigus lesions, and their relative abundance positively correlated with B cells abundance. Meanwhile, plasma cells population highly correlated with type 1 macrophages (M1) abundance. In addition, we also identified a lncRNA LINC01588 which might epigenetically regulate T helper 17 cells (Th17)/regulatory T cells (Treg) balance via the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Here, we provide the first transcriptomic characterization of lesion infiltrating immune cells which illustrates a distinct interplay network between adaptive and innate immune cells. It helps discover new regulators of local immune response, which potentially will provide a novel path forward to further uncover pemphigus pathological mechanisms and develop targeted therapy.

Differential Expression of Long Noncoding RNAs Reveals a Potential Biomarker for Intractable Pemphigus.

BACKGROUND: Long noncoding RNAs (lncRNAs) are involved in autoimmune diseases. However, the role of lncRNAs in pemphigus remains elusive. OBJECTIVE: The study is aimed at investigating the expression profile in pemphigus patients to identify a circulating lncRNA as a novel biomarker for pemphigus. METHOD: A global lncRNA expression profile in peripheral blood mononuclear cells (PBMCs) was measured by lncRNA microarray. Differentially expressed lncRNAs were validated by quantitative reverse transcriptase-PCR (qRT-PCR). The functional and biological processes of the differentially expressed lncRNAs were analyzed by bioinformatics. RESULTS: lncRNA ENST00000585297 in the PBMCs of pemphigus patients was highly expressed compared with those of HCs and BP patients. The area under the receiver operating characteristic (ROC) curve was 0.846 (95%confidence interval (CI) = 0.7526 to 0.9397). Intriguingly, we found that the expression of ENST00000585297 was upregulated in pemphigus patients whose symptoms could not be managed within four weeks compared to other patients whose symptoms could be managed in four weeks or less (P < 0.05). In addition, ENST00000585297 expression in pemphigus patients was positively correlated with the dosage of prednisone needed to manage the disorder (r = 0.4905, P = 0.0094). A competing endogenous RNA (ceRNA) regulatory network was constructed based on the ceRNA theory. Further verification demonstrated that silencing of ENST00000585297 increased the expression of miR-584-3p. CONCLUSIONS: Our study revealed for the first time the expression profile of lncRNAs in pemphigus patients. In addition, our study identified ENST00000585297 as a biomarker and indicator for the intractable course of pemphigus.

Comparison of Guidelines for Management of Pemphigus: a Review of Systemic Corticosteroids, Rituximab, and Other Immunosuppressive Therapies.

Pemphigus is a severe autoimmune bullous dermatosis that affects the skin and/or mucosa, and it may be life-threatening without proper treatment. The guidelines and/or consensus statements for treatment vary widely between groups. We selected 6 guidelines and consensus statements established by different associations about the management of pemphigus vulgaris (PV) and/or pemphigus foliaceus (PF) to review, compare, and contrast the similarities and differences of these recommendations and provide optimal management suggestions to physicians. Corticosteroids remain a first-line therapy for pemphigus, but there are many differences in initial dose, tapering schedule, and management of relapse between different guidelines. Rituximab is a monoclonal antibody targeting CD20-positive B lymphocytes that is approved as a first-line therapy in moderate-to-severe pemphigus. Immunosuppressive agents, such as azathioprine (AZA) and mycophenolate mofetil (MMF), are also widely used as corticosteroid-sparing drugs, but the adjuvant applications and dosage regimens of different recommendations are not standardized. We attribute these differences to the clinical scoring adopted, the standards for disease severity evaluation, the publication year of each guideline, and local and regional healthcare differences.

The Pathogenic Role of CD4+ Tissue-Resident Memory T Cells Bearing T Follicular Helper-Like Phenotype in Pemphigus Lesions.

In skin lesions caused by pemphigus, a group of life-threatening autoimmune bullous diseases, an over-representation of CD4+ tissue-resident memory T (TRM) cells was found. We sought to investigate the contributions of CD4+ TRM cells to the severity and refractoriness of pemphigus and their role in local immunological pathogenesis. Our data showed that CD4+ TRM cells accumulated significantly in pemphigus skin lesions. These CD4+ TRM cells expressed a specific set of T follicular helper cell‒related costimulatory molecules. We also found that CD4+ TRM cells remaining in the lesions produced IL-17A and IL-21. In vitro, CD4+ TRM cells exhibited strong support and assistance to autoantibody production. Through transcriptomic sequencing and bioinformatics analysis, we identified that the transcription factor IRF4 was responsible for IL-21 overexpression and autoantibody production. Our results showed that T follicular helper-like CD4+ TRM cells in pemphigus lesions promoted local autoantibody production, resulting in the formation and recurrence of lesions, which supports targeting this cell subset in pemphigus treatment. IRF4 might serve as a potential therapeutic target.

Nitidine chloride efficiently induces autophagy and apoptosis in melanoma cells via AMPK-mTOR signaling pathway.

Nitidine chloride (NC) has displayed anti-tumor effects in various types of cancer. However, the role of NC in human melanoma is largely unknown. This study aimed to investigate the effects of NC on melanoma cancer cells A375 and WM35 by MTT assay. Apoptosis was measured by detecting caspase-3 protein expression level and its activity. Autophagy was measured by TEM image, immunostaining and immunoblotting assays. MTT assays showed that NC significantly blocks melanoma cells proliferation. Immunoblotting and caspase-3 activity assays showed that NC inhibited melanoma cells proliferation by inducing cell apoptosis. TEM, immunostaining and immunoblotting assays showed that NC also triggers melanoma cells autophagy and activation of the AMPK-mTOR pathway, which plays an important role in autophagy initiation. Finally, we found that blocking autophagy by 3-MA or AMPK pathway inhibitor greatly enhanced NC-induced apoptosis and cell death, indicating that NC-induced autophagy may have a cytoprotective effect in melanoma cells. Together, these results suggested that NC has strong anti-tumor effects on melanoma cells.

Pivotal Role of Lesional and Perilesional T/B Lymphocytes in Pemphigus Pathogenesis.

Pemphigus is a skin and mucosal membrane-targeting autoimmune bullous disease. Previous studies have shown that circulating anti-desmoglein1/3 antibodies are pathogenic and mediate blister formation. However, the role of infiltrating immune cells in lesional skin has not been fully investigated. In this study we showed that there existed a large number of B and T lymphocytes and plasma cells in the skin lesions by immunohistochemistry and immunofluorescence staining. In addition, a significantly increased number of Dsg1- and Dsg3-specific B cells could be identified by flow cytometric analysis or enzyme-linked immunospot technique (i.e., ELISPOT) assay. Furthermore, anti-Dsg1 and Dsg3 antibodies could be detected from the supernatant of in vitro cultures with isolated lymphocytes from lesional skin. We found that most T lymphocytes infiltrating pemphigus vulgaris lesions were CD4+ T helper cells expressing IL-21 and IL-17a but not typical T follicular helper cells expressing CXCR5. Additionally, our microarray assay showed that the level of chemokine CCL19 was significantly elevated, suggesting active T-/B-lymphocyte trafficking and aggregation in the pemphigus vulgaris lesions. Collectively, our results suggest a critical role of locally infiltrating lymphocytes in pemphigus pathogenesis.

Immune cellular regulation on autoantibody production in pemphigus.

Pemphigus is an autoantibody-mediated blistering disease in the skin and mucous membranes. The autoantibodies primarily target desmoglein (Dsg)1 or/and Dsg3, transmembrane glycoproteins of skin epidermal cells, leading to loss of desmosome adhesion and acantholysis through signaling dependent and independent pathways. Thus, the pemphigus autoantibodies themselves and immune processes, particularly cellular regulation of antibody production, remain as interesting topics in probing pemphigus pathogenesis. In this review, we focus on current advances regarding how the pemphigus antibody production is highly regulated by the key immune effectors including T cells, B cells and their secreted cytokines. Specifically targeting these immune effectors involved in the pemphigus autoantibody production should provide better therapeutic options for disease treatment of pemphigus.

The imbalance of Th17 and regulatory T cells in pemphigus patients.

UNLABELLED: Pemphigus is a complex dermatologic autoimmune bullous disease whose pathogenic mechanism is not fully understood. Anti-desmoglein-1 (Dsg1) and anti- Dsg3 autoantibodies play an important role in the pathogenesis of pemphigus. OBJECTIVES: To investigate the role of T-helper17 (Th17) and regulatory T (Treg) cells in the pathogenesis of pemphigus in fifty-one patients and twenty-six healthy individuals (control group). METHODS: Levels of CD3(+)CD8(-) IL-17 expressing Th cells and CD4(+)CD25(hi)Foxp3(+) Treg cells were determined by FACS in both groups, along with anti-Dsg1 and Dsg3 antibody titers. An analysis of the correlation between Th17 and Treg cells was performed. RESULTS: Th17 cell numbers were significantly higher in pemphigus patients than in normal controls (P = 0.014), especially in the acute onset and chronic active stages (P = 0.004 and 0.022). Conversely, Treg cells in pemphigus patients were significantly fewer than in the control group (P<0.001). The same trend was observed between the acute onset and the remittent stage patients (P = 0.006). We found a negative correlation between Th17 and Treg cell populations (r = -0.532, P<0.001). Anti-Dsg1 and Dsg3 antibody titers were higher in patients in the active stage than in the remittent stage, with an increased IgG4/IgG1 subclass ratio. There was no statistically significant correlation between Th17/Treg ratios and anti-Dsg1 or Dsg3 antibody titers. CONCLUSION: These findings show an imbalance of Th17 and Treg cell populations in pemphigus patients, which might result in the activation and proliferation of effector T cells, further up-regulating B cell activity and antibody production.

Cognate Th2-B cell interaction is essential for the autoantibody production in pemphigus vulgaris.

Pemphigus vulgaris (PV) is a Th2-dominant autoimmune skin disease. We showed that indeed active PV patients had a biased Th2 response and specific IgG4 autoantibodies were dominant. To further investigate the role of antigen-specific Th2 cells in the regulation of pathogenic Dsg3-IgG antibody production, we used recombined Dsg3 protein to immunize wild-type C57BL/6 mice with aluminum hydroxide or complete Freund's adjuvant as adjuvant. CD4(+) T cells from Dsg3-immunized mice were adoptively transferred into TCR-ß chain deficient mice. The transferred CD4(+) T cells were readily seen in the peripheral blood and spleen, and interacted with B cells, resulting in B-cell activation. Furthermore, transferred CD4(+) T cells from mice immunized with Dsg3 plus Alum with Th2 phenotype were able to render unprimed B cells to secrete Dsg3-specific IgG1 antibody in vivo. Taken together, these results provide the first demonstration of direct role of Dsg3-reactive CD4(+) T (Th2) cells in the regulation of pathologic anti-Dsg3 antibody production.