N-acetylcysteine alleviated tris(2-chloroisopropyl) phosphate-induced sperm motility decline and functional dysfunction in mice through reversing oxidative stress and DNA damage.

The decline in male fertility caused by environmental pollutants has attracted worldwide attention nowadays. Tris(2-chloroisopropyl) phosphate (TCPP) is a chlorine-containing organophosphorus flame retardant applied in many consumer products and has multiple side effects on health. However, whether TCPP impairs spermatogenesis remains unclear. In this study, we found that TCPP reduced the sperm motility and blastocyst formation, inhibited proliferation and induced apoptosis in mice testes and spermatocyte cell line GC-2. Moreover, TCPP induced imbalance of oxidant and anti-oxidant, DNA damage and mitochondrial dysfunction, thus induced abnormal spermatogenesis. In this process, p53 signaling pathway was activated and N-acetylcysteine treatment partially alleviated the side effects of TCPP, including decrease of sperm motility, activation of p53 signaling pathway and DNA damage. Finally, our study verified that TCPP elevated reactive oxygen species (ROS), decreased mitochondrial membrane potential and induced apoptosis in human semen samples. Overall, ROS mediated TCPP-induced germ cell proliferation inhibition and apoptosis, which finally led to the decline of sperm motility.

Cresyl Diphenyl Phosphate exposure induces reproductive functional defects in men and male mice.

Cresyl Diphenyl Phosphate (CDP), as a novel organophosphate esters (OPEs), achieves widely used and exposed in multiple industries. However, its male reproductive toxicity and underlying mechanism remains unclear. In vivo, male mice were gavaged with CDP (0, 4, 20, or 100 mg/kg/d) for 8 weeks. And we treated TM3, TM4 and GC-2 cells with 0, 10, 25, and 50 µM CDP for 24 h to detect its reproductive toxicity effect in vitro. In our study, we revealed that CDP inhibited proliferation and induced apoptosis in mice testis and GC-2 cells, thereby leading to the decreased sperm quality. In mechanism, CDP trigger the oxidative stress and ROS production, thus partially causing DNA damage and cell apoptosis. Moreover, CDP exposure causes injury to Ledyig cells and Sertoli cells, thus disturbing the testicular microenvironment and inhibiting spermatogonia proliferation. In conclusion, this research reveals multiple adverse impacts of CDP on the male reproductive system and calls for further study of the toxicological effects of CDP on human health.

Decreased granzyme-B expression in CD11c+CD8+ T cells associated with disease progression in patients with HBV-related hepatocellular carcinoma.

Introduction: CD11c+CD8+ T cells are an unconventional CD8+ T cell subset that exerts antiviral activity in infectious diseases. However, its characteristics in hepatocellular carcinoma (HCC) have not been elucidated. Methods: Twenty-six patients with hepatitis B virus (HBV)-related HCC and 25 healthy controls (HC) were enrolled. The frequency and phenotype of CD11c+CD8+ T cells in peripheral blood and tumors in situ were detected by flow cytometry and immunohistochemistry. Results: Both the HCC group and HC group had similar frequency and phenotype characteristics of CD11c+CD8+ T cells in the periphery. CD11c+CD8+ T cells were mainly composed of effector T cells, most of which were CD45RA+CCR7-. Compared with CD11c-CD8+ T cells, CD11c+CD8+ T cells had a higher proportion of CD38 and HLA-DR double positive, and expressed high levels of granzyme-B (GB) and degranulation marker CD107a, and produced high levels of interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ). However, the ability of degranulation and TNF-α production of CD11c+CD8+ T cells in patients with HCC were significantly lower than that in healthy controls. The GB expression level of peripheral CD11c+CD8+ T cells in patients with advanced stage of HCC was significantly lower than that in patients with early stage of HCC, and the GB expression level of liver-infiltrating CD11c+CD8+ T cells in tumor tissues was lower than that in non-tumor tissues. More importantly, the GB expression level of peripheral CD11c+CD8+ T cells was negatively correlated with tumor volume. Conclusions: These findings indicate that CD11c+CD8+ T cells may have potential anti-tumor activity and that GB+CD11c+CD8+ T cells are associated with disease progression in patients with HBV-related HCC.

Alterations of Cytokine Profiles in Patients With Recurrent Implantation Failure.

Serum cytokine profile and T helper (Th)1/Th2 cell balance are related to the success of embryo implantation, although not yet firmly linked to recurrent implantation failure (RIF), a repeated failure to achieve clinical pregnancy following multiple high-quality embryo transfer. In this prospective study, comprehensive bioinfomatic analysis and logistic regression analysis were used to compare the serum cytokine profiles of 41 RIF patients with those of 29 subjects with first-cycle successful pregnancy in the mid-luteal phase and to assess the alterations of cytokine profiles in patients with clinical pregnancy at five weeks post-transplantation. We found several elevated pro-inflammatory cytokines, decreased anti-inflammatory cytokines, and increased Th1/Th2 cytokine ratios in RIF patients compared to control subjects. Specifically, the receiver operating characteristic (ROC) curve generated using multiple indicators provides a high predictive value for diagnosing RIF (area under the curve [AUC] = 0.94, 95% confidence interval [CI] 0.87-1.00, P < 0.0001), with a sensitivity of 96.55% and a specificity of 87.50%. Meanwhile, at five weeks post-transplantation, patients in both groups diagnosed with clinical pregnancy exhibited increased levels of several cytokines compared with pre-pregnancy levels, and a gradual shift in Th1/Th2 balance toward Th2. These findings suggest that inflammatory serum cytokines and the predominance of Th1 cells likely contribute to RIF and possibly reflect the immune environment at the maternal-fetal interface, suggesting their value as outcome indicators in assisted reproductive therapy.

Characterization and distribution of HIV-infected cells in semen.

Semen is a known vector for both human immunodeficiency virus (HIV) infection and transmission. However, the distribution and characteristics of HIV-infected cells in semen remain unclear. Investigating the possibility of transmission through the spermatozoon in semen is of great clinical significance to improve the strategies for exposure prevention and assisted reproduction for HIV-infected partners. Twenty-six HIV-infected patients, including twelve treatment-naïve (TN) patients and fourteen antiretroviral treated (ART) patients, were enrolled in this study. HIV p24 protein in spermatozoa was detected using imaging flow cytometry and immunohistochemistry, and HIV RNA was identified using next-generation RNAscope in situ hybridization. Additionally, we described the rates of HIV-positive spermatozoon and CD4+ T lymphocytes in semen, and found that p24+ spermatozoon were mainly CD4 negative regardless of whether the patients received ART. Of note, p24-positive cells in semen are predominantly spermatozoa, and we confirmed that motile spermatozoa carried HIV into peripheral blood mononuclear cells of healthy men in vitro. Our findings provide evidence regarding the risk of HIV-infected spermatozoa.

Implications of the accumulation of CXCR5+ NK cells in lymph nodes of HIV-1 infected patients.

BACKGROUND: B cell follicles are immune-privileged sites where intensive HIV-1 replication and latency occur, preventing a permanent cure. Recent study showed that CXCR5+ NK cells in B cell follicles can inhibit SIV replication in African green monkeys, but this has not been reported in HIV-1 infected patients. METHODS: Lymphocytes and tissue sections of lymph node were collected from 11 HIV-1 positive antiretroviral therapy (ART)-naive and 19 HIV-1 negative donors. We performed immunofluorescence and RNA-scope to detect the location of CXCR5+ NK cells and its relationship with HIV-1 RNA, and performed flow cytometry and RNA-seq to analyze the frequency, phenotypic and functional characteristics of CXCR5+ NK cells. The CXCL13 expression were detected by immunohistochemistry. FINDINGS: CXCR5+ NK cells, which accumulated in LNs from HIV-1 infected individuals, expressed high levels of activating receptors such as NKG2D and NKp44. CXCR5+ NK cells had upregulated expression of CD107a and ß-chemokines, which were partially impaired in HIV-1 infection. Importantly, the frequency of CXCR5+NK cells was inversely related to the HIV-1 viral burden in LNs. In addition, CXCL13-the ligand of CXCR5-was upregulated in HIV-1 infected individuals and positively correlated with the frequency of CXCR5+ NK cells. INTERPRETATION: During chronic HIV-1 infection, CXCR5+ NK cells accumulated in lymph node, exhibit altered immune characteristics and underlying anti-HIV-1 effect, which may be an effective target for a functional cure of HIV-1.

HIV-1-Specific CD11c+ CD8+ T Cells Display Low PD-1 Expression and Strong Anti-HIV-1 Activity.

Exhaustion of HIV-1-specific CD8+ T cells prevents optimal control of HIV-1 infection. Identifying unconventional CD8+ T cell subsets to effectively control HIV-1 replication is vital. In this study, the role of CD11c+ CD8+ T cells during HIV-1 infection was evaluated. The frequencies of CD11c+ CD8+ T cells significantly increased and were negatively correlated with viral load in HIV-1-infected treatment-naïve patients. HIV-1-specific cells were enriched more in CD11c+ CD8+ T cells than in CD11c- CD8+ T cells, which could be induced by HIV-1-derived overlapping peptides, marking an HIV-1-specific CD8+ T cell population. This subset expressed higher levels of activating markers (CD38 and HLA-DR), cytotoxic markers (granzyme B, perforin, and CD107a), and cytokines (IL-2 and TNF-α), with lower levels of PD-1 compared to the CD11c- CD8+ T cell subset. In vitro analysis verified that CD11c+ CD8+ T cells displayed a stronger HIV-1-specific killing capacity than the CD11c- counterparts. These findings indicate that CD11c+ CD8+ T cells have potent immunotherapeutic efficacy in controlling HIV-1 infection.

Immune Dysfunctions of CD56neg NK Cells Are Associated With HIV-1 Disease Progression.

Background: Populations of natural killer cells lacking CD56 expression [CD56neg natural killer (NK) cells] have been demonstrated to expand during human immunodeficiency virus (HIV)-1 infection. However, their phenotypic and functional characteristics have not been systematically analyzed, and their roles during disease progression remain poorly understood. Methods: In this study, 84 donors, namely 34 treatment-naïve HIV-1-infected patients (TNs), 29 HIV-1-infected patients with successful antiretroviral therapy (ARTs), and 21 healthy controls (HCs), were enrolled. The phenotypic and functional characteristics of CD56neg NK cells were analyzed using single-cell RNA-sequencing (scRNA-seq) and flow cytometry. A potential link between the characteristics of CD56neg NK cells and the clinical parameters associated with HIV-1 disease progression was examined. Results: The frequency of the CD56neg NK cell population was significantly increased in TNs, which could be partially rescued by ART. Flow cytometry analyses revealed that CD56neg NK cells were characterized by high expression of CD39, TIGIT, CD95, and Ki67 compared to CD56dim NK cells. In vitro assays revealed reduced IFN-γ and TNF-α secretion, as well as decreased expression of granzyme B and perforin in CD56neg NK cells. In line with the data obtained by flow cytometry, scRNA-seq analysis further demonstrated impaired cytotoxic activities of CD56neg NK cells. Notably, a negative correlation was observed between CD39, CD95, and Ki67 expression levels in CD56neg NK cells and CD4+ T cell counts. Conclusions: The results presented in this study indicate that the CD56neg NK cell population expanded in HIV-1-infected individuals is dysfunctional and closely correlates with HIV-1 disease progression.

[Study on the vertical transmission of Y chromosome microdeletions from father to son].

OBJECTIVE: To analyze the Y chromosome microdeletions in the family of the infertile male and to study on the vertical transmission of Y chromosome microdeletions from father to son. METHODS: The peripheral blood of infertile patients' family male members was extracted and analyzed with modified multiplex PCR. The infertile family tree was drawn according to the results. RESULTS: Two cases in twelve investigated families had azoospermia factor (AZFc) microdeletion heredity. The others had no heredity. CONCLUSION: AZFc microdeletion of the Y chromosome can be transmitted to the male offspring naturally,and the same deletion can result in different phenotypes in different individuals.

[Application of suspension array assay to detect marker genes expression of circulating tumor cells for early prediction of breast cancer metastasis].

OBJECTIVE: To develop a suspension array assay to detect the expression of multiple genes in the circulating breast cancer cells simultaneously so as to identify the marker genes for human breast cancer metastasis. METHODS: Peripheral blood samples were obtained from 73 breast cancer patients, including 31 breast cancer metastasis patients, 30 patients with benign breast diseases, and 40 healthy women, and peripheral blood mononuclear cells (PBMCs) were isolated. Total RNA was extracted and cDNA was synthesized. PCR was used to amplify 8 breast cancer-related genes: hMAM, HER2, CK19, SBEM, EPG2, hTERT, beta-HGG, and B305D. Suspension array of the PCR products was developed and underwent Luminex 100 laser Flow-type analysis to read the fluorescence signal. COX proportional hazard model was used to find the independent prognostic predictors of breast cancer metastasis. RESULTS: hMAM expression was detected in 57.5%, HER2 in 57.5%, CK19 in 53.4%, SBEM in 52.1%, EPG2 in 31.5%, hTERT in 26.1%, beta-HCG in 21.9%, and B305D in 15.1% of the blood samples respectively. Compared with serum CA15-3 detection, the multigene detection has higher sensitivity (P < 0.05). The expression of SBEM-mRNA in the peripheral blood was correlated with the stage of breast cancer (P < 0.05); and hMAM, SBEM, HER2, and ER could be considered as the independent prognostic predictors of breast cancer metastasis (all P < 0.05). CONCLUSION: The suspension array assay thus developed is practical in diagnosis of the prognosis of breast cancer. The expression of hMAM, SBEM, and HER2 in peripheral blood can be considered as the independent prognostic predictors of breast cancer metastasis.

Evaluation of a combinational use of serum microRNAs as biomarkers for liver diseases.

Circulating microRNAs (miRNA) have been widely recognized as a novel noninvasive biomarker in a variety of physiological and pathological conditions. In order to assess the sensitivity and reliability of potential miRNAs as diagnostic markers for liver disease related to viral infection, alcohol abuse, or chemical exposure, we collected serum samples from 326 participants and evaluated the single and combination diagnostic values of three serum miRNAs (miR-122, miR-125b, miR-192) compared with a conventional marker ALT. We found that serum miR-122 is significantly elevated in patients with active HBV. MiR-125b increased in HCV positive patients, whereas miR-192 and miR-122 were associated with chemical-induced liver injury. None of the aforementioned miRNAs were shown to increase significantly in alcohol-related liver injuries. Furthermore, we analyzed different combinations and found that a set of miR-122 and miR-125b enhanced the sensitivity of detecting liver injury. Among the 58 ALI/ALF patients, miR-122 responded more rapidly than ALT in successful treatments. Patients with spontaneous recovery from ALI/ALF showed significantly higher serum levels of miR-122 and miR-125b compared to non-recovered patients. In conclusion, our results suggest that the combination of miR-122 and miR-125b may serve as efficient biomarkers for liver injury and may be of a prognostic value in predicting ALI/ALF outcome.

[Screening for Y chromosomal microdeletions in AZF region with modified multiplex PCR].

OBJECTIVE: Screening for Y chromosomal microdeletions in azoospermia factor (AZF) region with modified multiplex PCR. METHODS: 160 cases with spermatogenetic failure were recruited in the experimental group, while 90 cases of donors in controls. According to the laboratory guidelines supported by European Academy of Andrology (EAA) and European Molecular Genetics Quality Network (EMQN), Y chromosomal microdeletions in AZFa, b, c regions were screened with multiplex PCR. The primers of sequence targeted sites (STSs) and conditions of PCR were modified. RESULTS: Using modified multiplex PCR, 14 (8.75%) cases with Y chromosomal microdeletions were found in the experimental group, while no case in controls. There were 12 cases in AZFc, 1 case in AZFa + b + c, 1 case in AZFb + c. According to statistics, the difference between two groups was significant (P <0.001). Reaction products could be clearly separated with agarose gel and finished in 1 h. CONCLUSION: Modified multiplex PCR protocols supported by EAA and EMQN proved to be very accurate, sensitive and quick, which could be put into screening practice for Y chromosomal microdeletions in AZF region.