The role of extracellular vesicle immune checkpoints in cancer.

Immune checkpoints (ICPs) play a crucial role in regulating the immune response. In the tumor, malignant cells can hijack the immunosuppressive effects of inhibitory ICPs to promote tumor progression. Extracellular vesicles (EVs) are produced by a variety of cells and contain bioactive molecules on their surface or within their lumen. The expression of ICPs has also been detected in EVs. In vitro and in vivo studies have shown that extracellular vesicle immune checkpoints (EV ICPs) have immunomodulatory effects and are involved in tumor immunity. EV ICPs isolated from the peripheral blood of cancer patients are closely associated with the tumor progression and the prognosis of cancer patients. Blocking inhibitory ICPs has been recognized as an effective strategy in cancer treatment. However, the efficacy of immune checkpoint inhibitors (ICIs) in cancer treatment is hindered by the emergence of therapeutic resistance, which limits their widespread use. Researchers have demonstrated that EV ICPs are correlated with clinical response to ICIs therapy and were involved in therapeutic resistance. Therefore, it is essential to investigate the immunomodulatory effects, underlying mechanisms, and clinical significance of EV ICPs in cancer. This review aims to comprehensively explore these aspects. We have provided a comprehensive description of the cellular origins, immunomodulatory effects, and clinical significance of EV ICPs in cancer, based on relevant studies.

SARS-CoV-2 Omicron infection augments the magnitude and durability of systemic and mucosal immunity in triple-dose CoronaVac recipients.

The inactivated whole-virion vaccine, CoronaVac, is one of the most widely used coronavirus disease 2019 (COVID-19) vaccines worldwide. There is a paucity of data indicating the durability of the immune response and the impact of immune imprinting induced by CoronaVac upon Omicron infection. In this prospective cohort study, 41 recipients of triple-dose CoronaVac and 14 unvaccinated individuals were recruited. We comprehensively profiled adaptive immune parameters in both groups, including spike-specific immunoglobulin (Ig) G and IgA titers, neutralizing activity, B cells, circulating follicular helper T (cTfh) cells, CD4+ and CD8+ T cells, and their memory subpopulations at 12 months after the third booster dose and at 4 and 20 weeks after Omicron BA.5 infection. Twelve months after the third CoronaVac vaccination, spike-specific antibodies and cellular responses were detectable in most vaccinated individuals. BA.5 infection significantly augmented the magnitude, cross-reactivity, and durability of serum neutralization activities, Fc-mediated phagocytosis, nasal spike-specific IgA responses, memory B cells, activated cTfh cells, memory CD4+ T cells, and memory CD8+ T cells for both the ancestral strain and Omicron subvariants, compared to unvaccinated individuals. Notably, the increase in BA.5-specific immunity after breakthrough infection was consistently comparable to or higher than that of the ancestral strain, suggesting no evidence of immune imprinting. Immune landscape analyses showed that vaccinated individuals have better synchronization of multiple immune components than unvaccinated individuals upon heterologous infection. Our data provide detailed insight into the protective role of the inactivated COVID-19 vaccine in shaping humoral and cellular immunity to Omicron infection. IMPORTANCE: There is a paucity of data indicating the durability of the immune response and the impact of immune imprinting induced by CoronaVac upon Omicron breakthrough infection. In this prospective cohort study, the anti-severe acute respiratory syndrome coronavirus 2 adaptive responses were analyzed before and after the Omicron BA.5 infection. Our data provide detailed insight into the protective role of the inactivated COVID-19 vaccine in shaping humoral and cellular immune responses to heterologous Omicron infection. CLINICAL TRIAL: This study is registered with ClinicalTrials.gov as NCT05680896.

Effect of serum autoantibodies on the COVID-19 patient's prognosis.

Objectives: Virus infection closely associated with autoimmune disease. The study aimed to explore the autoantibody profiles and the correlation of autoantibodies with the disease severity and the prognosis of the coronavirus disease 2019 (COVID-19) patients. Methods: Three hundred thirty-seven hospitalized COVID-19 patients from 6th to 23rd January 2023 were enrolled. Logistic and Cox regression analyses were used to analyze the risk factors for the patient's disease severity and outcome. The association between Anti-extractable nuclear antigen antibody (ENA) positivity and the prognosis of COVID-19 patients was analyzed using Kaplan-Meier survival curves. Results: 137 of COVID-19 patients were detected positive for antinuclear antibody (ANA), 61 had positive results for ENA, and 38 were positive for ANA and ENA. ANA positivity rate was higher in non-severe illness group (p = 0.032). COVID-19 patients who died during hospitalization had a high rate of ENA positivity than convalescent patients (p = 0.002). Multivariate logistic regression showed that ANA positivity was a protective factor for the disease severity of COVID-19. Multivariate Cox regression analysis revealed that ENA positivity, white blood cells count (WBC), aspartate aminotransferase (AST), Creatinine (CREA), and CRP were independent risk factors for the outcome of COVID-19 patients, and that COVID-19 patients with ENA positivity had a lower cumulative survival rate (p = 0.002). Conclusion: A spectrum of autoantibodies were expressed in COVID-19 patients, among which ANA and ENA positivity was associated with the severity and prognosis of COVID-19. Therefore, autoantibodies may help to assess the disease severity and prognosis of COVID-19 patients.

Hepatitis B Viral Markers in the Human Milk of HBsAg-Positive Mothers: An Observational Study.

BACKGROUND: Quantification of viral antigens and viral loads in human milk samples from mothers infected with hepatitis B virus is largely unknown. RESEARCH AIM: The aim of the study was to quantitatively measure the levels of viral antigens and deoxyribonucleic acid of hepatitis B virus in human milk from mothers infected with hepatitis B virus. METHODS: Fifty-five pairs of milk and serum samples from mothers with positive hepatitis B surface antigen, including 11 hepatitis B e antigen positive, were quantitatively tested to measure viral antigens by microparticle enzyme immunoassay and viral loads by real-time polymerase chain reaction assay. RESULTS: The median level of hepatitis B surface antigen in the human milk samples of mothers with positive or negative hepatitis B e antigen was each lower than that in the sera, respectively (1.10 vs. 4.32 log10 IU/ml, t = 10.693, p < .001; -0.77 vs. 2.53 log10 IU/ml, t = -25.135, p < .001). The titers of hepatitis B surface antigen or hepatitis B e antigen in the human milk samples were each correlated with that in maternal serum. The detectable level of deoxyribonucleic acid of hepatitis B virus in human milk ranged from 1.42-5.27 log10 IU/ml, whereas that in maternal sera was 1.44-8.66 log10 IU/ml. The viral level in human milk was not correlated with that in maternal circulation. CONCLUSION: The present study data illustrate the relatively low titers of viral markers in the milk of mothers with positive hepatitis B surface antigen.

A Novel Serum tsRNA for Diagnosis and Prediction of Nephritis in SLE.

Objective: Dysregulation of transfer RNA (tRNA)-derived small noncoding RNA (tsRNA) signatures in human serum has been found in various diseases. Here, we determine whether the signatures of tsRNAs in serum can serve as biomarkers for diagnosis or prognosis of systemic lupus erythematosus (SLE). Methods: Initially, small RNA sequencing was employed for the screening serum tsRNAs obtained from SLE patients, followed by validation with TaqMan probe-based quantitative reverse transcription-PCR (RT-PCR) assay. Receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic efficacy. The biological functions of tsRNAs were identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) assay. Results: We first analyzed tsRNA signatures in SLE serum and identified that tRF-His-GTG-1 was significantly upregulated in SLE serum. The combination of tRF-His-GTG-1 and anti-dsDNA could serve as biomarkers for diagnosing SLE with a high area under the curve (AUC) of 0.95 (95% CI = 0.92-0.99), sensitivity (83.72%), and specificity (94.19%). Importantly, the noninvasive serum tRF-His-GTG-1 could also be used to distinguish SLE with LN or SLE without LN with AUC of 0.81 (95% CI, 0.73-0.88) and performance (sensitivity 66.27%, specificity 96.15%). Moreover, the serum tsRNA is mainly secreted via exosome and can directly target signaling molecules that play crucial roles in regulating the immune system. Conclusion: In this study, it has been demonstrated for the first time that serum tsRNAs can be employed as noninvasive biomarkers for the efficient diagnosis and prediction of nephritis in SLE.

Surveillance of SARS-CoV-2 infection among frontline health care workers in Wuhan during COVID-19 outbreak.

INTRODUCTION: As an emerging infectious disease, coronavirus disease 2019 (COVID-19) has rapidly spread throughout worldwide. Health care workers (HCWs) on frontline directly participated in the diagnosis, treatment, and care of COVID-19 patients are at high risk of getting infected with the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus that causes COVID-19. In Nanjing Drum Tower Hospital, a total of 222 medical staff went to Wuhan city for support. In this study, we aimed to determine any nosocomial infection among our cohort of HCWs who worked in Wuhan. METHODS: Throat swab samples were obtained for RNA testing on day 1 and 14 of their quarantine upon their return to Nanjing. Radiological assessments were performed by chest computed tomography (CT) on day 14 of their quarantine. The blood was collected from 191 HCWs between May 12 and May 15. Anti-SARS-CoV-2 immunoglobulin M (IgM) and IgG antibody responses were determined by a chemiluminescence immunoassay. RESULTS: All the throat swab specimens were found negative for SARS-CoV-2. The radiological analysis revealed that there was no typical chest CT scan of COVID-19 among 222 HCWs. Consistently, anti-SARS-CoV-2 IgM or IgG was also found to be negative among 191 HCWs. CONCLUSIONS: There was no nosocomial infection of SARS-CoV-2 among our cohort of the frontline HCWs, suggesting that zero occupational infection is an achievable goal with appropriate training, strict compliance, and psychological support for the frontline HCWs.

High mutation prevalence of precore and basal core promoter in pregnant women who underwent spontaneous HBeAg seroconversion within one year postpartum.

BACKGROUND: Seroconversion of hepatitis B e antigen (HBeAg) is a critical event in the natural course of hepatitis B virus (HBV) infection. AIM: We herein characterize the virological factors associated with postpartum spontaneous HBeAg seroconversion. METHODS: A total of 214 pregnant women positive for both hepatitis B surface antigen (HBsAg) and HBeAg were followed up at 7-12 months postpartum. RESULTS: Of the subjects, 26 (12.1%) achieved spontaneous HBeAg seroconversion. Receiver operating curve analysis indicated that HBV DNA level <1.0 × 107 IU/mL, HBsAg <1.0 × 104 IU/mL and HBeAg <7.36 × 102 S/CO each independently predicted HBeAg seroconversion within 12 months postpartum. At delivery, 73.1% (19/26) women with postpartum HBeAg seroconversion had precore (PC) and/or basal core promoter (BCP) mutations, higher than that (5/36, 13.9%) in the women without postpartum seroconversion. Binary logistic regression analysis indicated that the presence of mutations in PC, BCP, and both PC and BCP at delivery was associated with an increased likelihood (OR = 13.286, 16. 238, and 22.143 respectively, all P < 0.05) to undergo postpartum spontaneous HBeAg seroconversion. CONCLUSION: These results suggest that quantitative determination of virological markers and sequencing PC and BCP can predict spontaneous HBeAg seroconversion, which could be valuable in deciding antiviral therapy against HBV.

Is hepatitis B antigen in cord blood an immunotolerogen playing a critical role in the pathogenesis of chronic hepatitis B?

Recently, we showed that infants with or without fetal hepatitis B e antigen (HBeAg) exposure had comparable antibody response to hepatitis B vaccination and proposed that fetal HBeAg exposure appears to not induce immunotolerance to HBV. Here, we summarized the different results on the topic of fetal HBeAg exposure in inducing immunotolerance to HBV nucleocapsid protein, and the evidence to back up that the tendency of high infection rate in infants of HBeAg-positive mothers is more likely associated with higher maternal viral loads and is less likely associated with the fetal HBeAg exposure. We consider that whether fetal HBeAg exposure plays an important role in the pathogenesis of chronic HBV infection remains to be an open question.

Comparison of antibody response to hepatitis B vaccination in infants with positive or negative maternal hepatitis B e antigen (HBeAg) in cord blood: implication for the role of HBeAg as an immunotolerogen.

Hepatitis B e antigen (HBeAg) has been considered to cause immunotolerance to hepatitis B virus (HBV) in newborn infants after fetal HBeAg exposure. This study compared anti-HBs responses to hepatitis B vaccination in infants who were born to HBeAg-positive and -negative mothers respectively, to investigate whether fetal HBeAg exposure may induce immunotolerance to HBV. Totally 265 infants who received recommended neonatal immunoprophylaxis against hepatitis B and had no HBV infection were included. Anti-HBs levels were compared between 124 infants with cord blood positive HBeAg and 141 infants with cord blood negative HBeAg at 7-12 months of age. The infants in two groups had similar age at the follow-up (10.0 ± 2.3 vs 10.1 ± 2.3 months, P = 0.590). Overall, 259 (97.7%) of 265 infants achieved anti-HBs levels (mIU/ml) ≥10 and 6 (2.3%) others had anti-HBs <10. Of 124 HBeAg-positive infants at birth, 46.0%, 39.5%, 12.1%, and 2.4% had anti-HBs levels (mIU/ml) ≥1000, 100-999.9, 10-99.9, and <10, respectively. Of 141 HBeAg-negative infants at birth, 35.5%, 48.9%, 13.5%, and 2.1% showed ≥1000, 100-999.9, 10-99.9, and <10, respectively. The proportions of each anti-HBs level between the two groups were comparable (all P > 0.05). Additionally, the distribution of anti-HBs response levels were also comparable in infants with high and low HBeAg levels (P = 0.818). In conclusions, the fetal HBeAg exposure does not inhibit the antibody response to neonatal hepatitis B vaccination. The data suggest that HBeAg appears not inducing immunotolerance to HBV.

Insignificant interference of Elevit in pregnant women serum samples with HBsAg immunoassay on Sysmex.

BACKGROUND: The pregnant women often take Elevit as the multivitamin supplement which has a substantial amount of biotin that might potentially interfere with the HBsAg immunoassay performed by the prevalent Sysmex system in clinical laboratories. We therefore wanted to determine this, so that the therapeutic intervention on the hepatitis B virus infection during pregnancy and birth would not be missed. METHODS: Elevit was both serially diluted in vitro and orally taken by healthy volunteers whose blood samples were then taken at different time points. All samples were added to a serum sample with a known result of HBsAg and then measured by Sysmex. The Abbott immunoassay system was used as the control as it involves no streptavidin-biotin binding in the reagent set. Besides, the HBsAg results were compared between the pregnant women taking or not taking Elevit. RESULTS: Biotin at 25 ng/mL in the Elevit started to suppress the HBsAg and reached about 50% suppression at 100 ng/mL on Sysmex. In the volunteers, biotin reached the peak concentration at 2 hours. However, their blood samples showed no suppression on the HBsAg detection by Sysmex. In samples from pregnant women who took Elevit, the HBsAg results by Sysmex were highly correlated with those by Abbott (R2  = 0.96). Comparison of the results from Sysmex between the age- and pregnancy-matched females with and without Elevit intake showed no difference. CONCLUSION: Elevit intake in pregnant women shows no significant interference with HBsAg immunoassay on Sysmex.

Stable Protein Gel Storage in Acetonitrile for Mass Spectrometric Analysis.

Long-term storage of protein samples for transportation is a challenge in the field of mass spectrometric analysis because the low temperature condition is not easy to be maintained. Here we introduce a simple method to preserve proteins at room temperature for at least one month. In this method, the protein sample is run shortly into a polyacrylamide gel which is then excised after Coomassie staining. The protein gel band is then dehydrated by 100% acetonitrile three times and kept in 100% acetonitrile for storage at room temperature. By the TMT 10-plex based quantitative proteomic analyses, we have found that these proteins are stable in their levels and modifications (phosphorylation, oxidation, and ubiquitination) for 30 days. Further analysis has revealed this storage method also well preserves proteins even at 45 °C. We therefore recommend to use acetonitrile to dehydrate and store protein gel pieces as an effective alternative for sample shipping over days. Therefore, it might facilitate worldwide collaborations in the mass spectrometry-based proteomic research.

Survey of Clinical Extended-Spectrum Beta-Lactamase-Producing Enterobacter cloacae Isolates in a Chinese Tertiary Hospital, 2012-2014.

A total of 72 consecutive and nonduplicate clinical extended-spectrum ß-lactamase (ESBL)-producing Enterobacter cloacae isolates were collected from our hospital from 2012 to 2014 for analyzing the prevalence of plasmid-mediated quinolone resistance (PMQR) genes, 16S rRNA methyltransferase (16S-RMTase) encoding genes, and carbapenem-hydrolyzing ß-lactamase (CHßL) genes, as well as integrons. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were carried out to characterize the genetic relatedness. The isolates mainly harbored blaCTX-M (n = 51, 70.8%) and blaSHV (n = 46, 63.9%) genes. High prevalence of PMQR genes qnrA1 (n = 24, 33.3%), qnrB (n = 4, 5.6%), qnrS1 (n = 2, 2.8%), and aac(6')-Ib-cr (n = 21, 29.2%) was observed. Furthermore, CHßLs IMP-4 (n = 6, 8.3%) and IMP-8 (n = 4, 5.6%), as well as class I integrons (n = 29, 40.3%), were found in the ESBL-producing E. cloacae isolates. PFGE revealed 69 pulsotypes. MLST distinguished 44 sequence types (STs) with ST124 (n = 7, 9.7%), ST50 (n = 3, 4.2%), ST45 (n = 3, 4.2%), and ST93 (n = 3, 4.2%) being the predominant STs. The results indicate a possible clonal transmission of ST124 isolates in the hospital that needs further surveillance. The genetic diversity of the other numerous distinctive STs indicates that most of the ESBL-producing E. cloacae in our hospital might arise through stepwise accumulations of multiple drug-resistance determinants in different clones.

Complete Genome Sequence of Aerococcus urinaeequi Strain AV208.

Aerococcus urinaeequi strain AV208 was isolated from an ascites sample from a patient with chronic kidney disease. The assembled genome contained 2,227,638 bp with a 39.1% G+C content. The genome harbors a Tn1546 transposon-like structure with a vanA gene causing vancomycin resistance phenotypes of strain AV208.

Clonal dissemination of KPC-2 producing Klebsiella pneumoniae ST11 clone with high prevalence of oqxAB and rmtB in a tertiary hospital in China: results from a 3-year period.

BACKGROUND: Carbapenemase-producing Klebsiella pneumoniae (CPKP) strains have emerged as a major problem for healthcare systems. The aim of this study was to determine the circulating clones and analyze the clinical and molecular characteristics of CPKP in our hospital. METHODS: A total of 74 carbapenemase producers collected from our hospital from 2012 to 2014 were analyzed for the prevalence of extended-spectrum ß-lactamase (ESBLs), plasmid-mediated quinolone resistance genes (PMQRs), exogenously acquired 16S rRNA methyltransferase (16S-RMTase), and plasmid-mediated AmpC enzyme (pAmpCs) by PCR and DNA sequencing. The sequence types (STs) of the carbapenemase producers were analyzed by multi-locus sequence typing (MLST). And Pulsed-field gel electrophoresis (PFGE) was performed to investigate the genetic relationship of KPC-2 producing strains. Clinical data were retrieved from the medical records. RESULTS: KPC-2 (n = 72) was the predominant enzyme followed by NDM-1 (n = 2); The genes blaCTX-M, blaSHV, blaTEM-1, blaDHA-1, rmtB, armA, oqxA, oqxB, and qnrB were present in 29 (39.2 %), 27 (36.5 %), 46 (62.2 %), 2 (2.7 %), 25 (33.8 %), 1 (1.4 %), 60 (81.1 %) and 56 (75.7 %), 6 (8.1 %) isolates, respectively. MLST analysis revealed 10 different STs. The most dominant ST was ST11 (78.4 %, 58/74), followed by ST15 (8.1 %, 6/74). PFGE patterns of the KPC-2 producing K. pneumoniae isolates exhibited clonal dissemination of ST11 and ST15 clones as well as a genetic diversity of the remaining strains. CONCLUSION: The intra- and inter-hospital cross-transmission of KPC-2-producing K. pneumoniae ST11 co-carrying oqxAB and rmtB in our hospital strongly suggested that rapid identification of colonized or infected patients and screening of carriers is quite necessary to prevent a scenario of endemicity.

Clonal dissemination of linezolid-resistant Staphylococcus capitis with G2603T mutation in domain V of the 23S rRNA and the cfr gene at a tertiary care hospital in China.

BACKGROUND: The present study aims to investigate the potential mechanism of linezolid-resistant Staphylococcus capitis (LRSC) isolates collected from our hospital. METHODS: The susceptibilities of 5 Staphylococcus capitis isolates displaying resistance towards linezolid were determined by E-test. Polymerase chain reactions (PCRs) and DNA sequencing were used to investigate the potential molecular mechanism. Clonal relatedness between these strains was analyzed by pulsed-field gel electrophoresis (PFGE). RESULTS: The MICs of linezolid on these 5 isolates were >256 µg/mL. The G2603T mutation was observed in the domain V of the 23S rRNA with cfr gene being also widely detected among these 5 strains. PFGE analysis displayed close genetic relatedness between these linezolid-resistant isolates. CONCLUSIONS: The emergence of LRSC isolates carrying G2603T mutation in the domain V of the 23S rRNA and harboring cfr gene in our hospital may pose a potential challenge to the public health.

Genotypic characteristics of multidrug-resistant Escherichia coli isolates associated with urinary tract infections.

Escherichia coli is an important pathogen involved in community-acquired urinary tract infections (CA-UTIs). In this study, we analyzed the prevalence of frequently occurring genes and the distribution of integrons in 51 multidrug-resistant (MDR) E. coli isolates associated with CA-UTIs. The clonality of these strains was investigated by phylogrouping, multi-locus sequence typing, and pulsed-field gel electrophoresis (PFGE). All these strains were found to produce two or more resistance determinants, ceftazidime-hydrolyzing CTX-M-type extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated quinolone resistance determinants were the most prevalent (92.2% and 51.0%, respectively). A sulfhydryl variable-61-producing E. coli strain was identified for the first time in China. The prevalence of class 1 integrons was 54.9%, class 2 integrons were detected in three isolates but no isolate contained a class 3 integron. Phylogenetic group D was the dominant, observed in 70.6% of the isolates. PFGE analysis revealed a high level of diversity. Twenty-four distinctive sequence types (STs) including four major STs (ST648, ST224, ST38, and ST405) were identified. To our knowledge, this is the first report on the characterization of MDR E. coli isolates associated with CA-UTIs in China; our results suggest that an MDR D-ST648 clone producing CTX-M-ESBLs has emerged as a major clone in the community setting.

Prevalence and characterization of class I integrons among Pseudomonas aeruginosa and Acinetobacter baumannii isolates from patients in Nanjing, China.

Class I integrons were detected in 40.8% (40/98) of Pseudomonas aeruginosa strains and 52.8% (56/106) of Acinetobacter baumannii strains in the Nanjing area of China, including several cassette arrays not previously reported.