The T-locus - inspiration and distraction?

The T/t locus was a major focus of study by mouse geneticists during the 20th century. In the 70s, as the study of cell surface antigens controlling transplantation antigens was taking off, several laboratories hypothesized that alleles of this locus would control cell surface antigens important for embryonic development. One such antigen, the embryonal carcinoma F9 antigen was said to be an example. Other antigens were described on sperm and embryos that were said to be controlled by alleles at the T/t complex. These findings were later found to be false. The history of the findings and their refutation is described.

Germ cell tumors, cell surface markers, and the early search for human pluripotent stem cells.

Many strands of research by different groups, starting from teratocarcinomas in the laboratory mouse, later moving the corresponding human tumors, contributed to the isolation and description of human pluripotent stem cells (PSCs). In this review, I highlight the contributions from my own research, particularly at the Wistar Institute during the 1980s, when with my colleagues we characterized one of the first clonal lines of pluripotent human embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, and identified key features including cell surface antigen markers that have since found a place in the study and exploitation of human PSC. Much of this research depended upon close teamwork with colleagues, many in other laboratories, who contributed different expertise and experience. It was also often driven by circumstance and chance rather than pursuit of a grand design.

Increase of Hepatitis B Surface Antibody Levels After Inactivated COVID-19 Vaccine in Hemodialysis Patients: An Important Single-Center Observation.

BACKGROUND: The effects of COVID-19 vaccines on immunocompromised people such as hemodialysis (HD) patients are an important topic that should be addressed. This study reports an observation of the effect of the third dose of the Sinopharm vaccine (SphV3) on the level of hepatitis B surface antibody (anti-HBs) in HD patients, and the differences between anti-HBs titers before and after SphV3 were analytically evaluated. METHODS: This single-center observational study involved all HD patients presented to Shariati Hospital, Tehran, Iran, from February 2021 to March 2022. All patients received three doses of the Sinopharm vaccine over 8 months. The anti-HBs level is measured every 6 months as the routine evaluation against HBV infection for all HD patients. Three months before (anti-HBs-B3) and 3 months after (anti-HBs-A3) SphV3 were the routine times to measure the anti-HBs titer during this study. RESULTS: Twenty-five HD patients were enrolled. Overall, the anti-HBs-A3 was significantly higher than anti-HBs-B3 (p = 0.001). The anti-HBs levels before and after SphV3 were not statistically remarkable in patients with diabetes and ischemic heart disease. The patients with a history of kidney transplant and those with a history of COVID-19 had significant differences between anti-HBs-B3 and anti-HBs-A3 (p = 0.002, p = 0.003, respectively). CONCLUSION: Our findings revealed that inactivated COVID-19 vaccine may be involved in the humoral immune response to hepatitis B in HD patients. It may be novel and have significant implications for the vaccination protocol for immunocompromised patients, including those undergoing HD and transplant recipients.

Anti-Ri paraneoplastic neurological syndrome presenting with bilateral cranial nerve VI palsy and jaw dystonia-a distinctive syndrome within the anti-Ri spectrum? : Case report and literature review.

OBJECTIVE: Paraneoplastic neurological syndromes (PNS) are rare disorders associated with various onconeuronal antibodies. Anti-Ri antibodies (ANNA-2) are typically found in patients with opsoclonus myoclonus syndrome (OMS) and ataxia. CASE REPORT: We present an anti-Ri antibody-positive 77-year-old woman with subacute progressive bilateral cranial nerve VI palsy, gait disturbance and jaw dystonia. MRI of the brain showed hyperintense signals on T2 bitemporal without contrast enhancement. Cerebrospinal fluid (CSF) examination exhibited mild pleocytosis of 13 cells/µl and positive oligoclonal bands. CSF was overall inconspicuous for a malignant or inflammatory etiology. Immunofluorescence analysis revealed anti-Ri antibodies in both serum and CSF. Subsequent diagnostic work up resulted in a newly diagnosed ductal carcinoma of the right breast. PNS in this case partially responded to the anti-tumor therapy. CONCLUSION: This case shows similarities with recently published anti-Ri syndromes, which might form a distinct triad within the anti-Ri spectrum.

Comparison of HBV DNA and RNA markers in Alaska Native people who did and did not clear hepatitis B surface antigen.

In a comparison between 50 Alaska Native persons with chronic hepatitis B who cleared HBV surface antigen (HBsAg) and 50 Alaska Native age-, sex-, and HBV genotype-matched controls, we found differences in changes in HBV DNA and HBV RNA levels over time but no difference in hepatitis B core-related antigen. These findings suggest that serial HBV DNA and HBV RNA may be associated with HBV functional cure defined by HBsAg clearance.

Paraneoplastic Ataxia: Antibodies at the Forefront Have Become Routine Biomarkers.

Paraneoplastic cerebellopathies are immune-mediated disorders targeting primarily the cerebellar circuitry, often presenting in a subacute course. The syndrome often develops before the cancer. Therefore, its identification often leads secondarily to a diagnosis of cancer, a critical step to stabilize symptoms. Two categories of antibodies have been identified these last 30 years: (a) onconeuronal antibodies which are directed against intracellular antigens, and (b) antibodies which are directed against synaptic and cell surface proteins. These latter impact on the location and function of the antigens, causing a genuine neuronal dysfunction. Appropriate and fast tumor screening has emerged as a recommendation facing a subacute cerebellar syndrome suspected to be paraneoplastic. Search for antibodies is now a milestone for the diagnosis.

An interesting case of isolated false-negative hepatitis B surface antigen in a blood donor.

BACKGROUND AND OBJECTIVES: An important requirement for a hepatitis B surface antigen (HBsAg) screening assay is reliable detection of HBsAg mutant forms, especially in blood donation. Here we investigate and describe the case of an isolated false-negative result of commercial serology HBsAg screening assay of a blood donor. MATERIALS AND METHODS: The current donation was routinely tested for HBsAg and hepatitis B virus (HBV) DNA in the mini-pool mode nucleic acid testing (MP-NAT of six samples), and further evaluated by individual donation ID-NAT. Finally, it was quantified and sequenced. All previous donations were found to have negative HBsAg and HBV DNA, as also the subsequent sample taken 3 months after the marked donation. RESULTS: The current donation of the 53-year-old unvaccinated female with 14 previous donations was initially HBsAg negative and HBV DNA (MP-NAT) positive. Further testing showed HBsAg positive using other HBV serological assays, antibodies to HBV core antigen immunoglobulin M positive and HBV DNA ID-NAT positive, and contained 200 IU/mL of HBV DNA. The implicated donation carried genotype D strains, subtype ayw2 (F83S, V96A, V190A, L193S, I195T, L213S, F220L). The mutations in three positions, namely amino acids T118A, P120T, and P127T, were proven subsequently. CONCLUSION: This unique mutation combination near the target epitope of one of the immunoassay monoclonals is a possible cause of the reduced analytical sensitivity of the serology assay.

Comprehensive characterization of Cysteine-rich protein-coding genes of Giardia lamblia and their role during antigenic variation.

Giardia lamblia encodes several families of cysteine-rich proteins, including the Variant-specific Surface Proteins (VSPs) involved in the process of antigenic variation. Their characteristics, definition and relationships are still controversial. An exhaustive analysis of the Cys-rich families including organization, features, evolution and levels of expression was performed, by combining pattern searches and predictions with massive sequencing techniques. Thus, a new classification for Cys-rich proteins, genes and pseudogenes that better describes their involvement in Giardia's biology is presented. Moreover, three novel characteristics exclusive to the VSP genes, comprising an Initiator element/Kozak-like sequence, an extended polyadenylation signal and a unique pattern of mutually exclusive transcript accumulation are presented, as well as the finding that High Cysteine Membrane Proteins, upregulated under stress, may protect the parasite during VSP switching. These results allow better interpretation of previous reports providing the basis for further studies of the biology of this early-branching eukaryote.

[Analysis of the therapeutic efficacy and factors influencing sequential combination of nucleos(t)ide analogues with pegylated interferon alpha for 48~96 weeks in the treatment of patients with chronic hepatitis B].

Objective: To explore the therapeutic efficacy and factors influencing the sequential combination of nucleos(t)ide analogues (NAs) with pegylated interferon alpha (Peg-IFN-α) in the treatment of patients with chronic hepatitis B (CHB). Methods: 144 CHB cases with NAs treatment for more than 1 year, HBV DNA < 20 IU/ml, hepatitis B surface antigen (HBsAg) quantification < 3 000 IU/ml, treated with a sequential combination of Peg-IFN-α treatment for 48 to 96 weeks, and followed up were selected from the Fifth Medical Center of the PLA General Hospital between May 2018 and May 2020. Intention-to-treat analysis was used to measure the HBsAg clearance rate at 96 weeks. The Kaplan-Meier method was used to compute the cumulative HBsAg clearance rate at 96 weeks. Univariate and multivariate logistic regression were used to analyze the factors influencing HBsAg clearance at 48 weeks of sequential combination therapy. Univariate and multifactorial COX proportional hazard models were used to analyze the factors influencing HBsAg clearance following 96 weeks of prolonged PEG-IFN-α treatment. The receiver operating characteristic curve was used to assess the predictive value of factors influencing HBsAg clearance. A Mann-Whitney U test was used to compare the measurement data between groups. The count data was compared using the χ(2) test between groups. Results: 41 (28.47%) cases achieved HBsAg clearance at 48 weeks of sequential combination therapy. The HBsAg clearance rate at 96 weeks was 40.28% (58/144) by intention-to-treat analysis. The Kaplan-Meier method computed that the cumulative HBsAg clearance rate at 96 weeks was 68.90%. Multivariate logistic regression analysis showed that HBsAg quantification at baseline (OR = 0.090, 95%CI: 0.034-0.240, P < 0.001) and a 24-week drop in HBsAg level (OR = 7.788, 95%CI: 3.408-17.798, P < 0.001) were independent predictors of HBsAg clearance in CHB patients treated sequentially in combination with NAs and Peg-IFN-α for 48 weeks. Receiver operating characteristic curve analysis showed that the baseline HBsAg quantification [area under the receiver operating characteristic curve (AUC), 0.911, 95% CI: 0.852-0.952)] and 24-week drop in HBsAg level (AUC = 0.881, 95%CI: 0.814-0.930) had equally good predictive value for 48-week HBsAg clearance, but there was no statistically significant difference between the two (Z = 0.638, P = 0.523). The value of the combination of baseline HBsAg quantification and 24-week drop in HBsAg level (AUC = 0.981, 95%CI: 0.941-0.997) was superior to that of single baseline HBsAg quantification (Z = 3.017, P = 0.003) and 24-week drop in HBsAg level (Z = 3.214, P = 0.001) in predicting HBsAg clearance rate at 48 weeks. Multivariate COX proportional hazards model analysis showed that HBsAg quantification at 48 weeks (HR = 0.364, 95%CI: 0.176-0.752, P = 0.006) was an independent predictor of HBsAg clearance with a prolonged course to 96 weeks of Peg-IFN-α treatment. Conclusion: The HBsAg clearance rate can be accurately predicted with baseline HBsAg quantification combined with a 24-week drop in HBsAg level in patients with CHB who are treated with a sequential combination of NAs and Peg-IFN-α therapy for 48 weeks. Prolonging the course of Peg-IFN-α treatment can enhance the HBsAg clearance rate's capability. An independent predictor of HBsAg clearance is HBsAg quantification at 48 weeks of sequential combination therapy with a prolonged course of 96 weeks of Peg-IFN-α treatment.

Detection of the Hepatitis B Surface Antigen in Patients with Occult Hepatitis B by Use of an Assay with Enhanced Sensitivity.

Patients with occult hepatitis B infection (OBI) have undetectable hepatitis B surface antigen (HBsAg) by conventional assays but detectable hepatitis B virus (HBV) DNA in blood/liver. We evaluated the key performance characteristics of a sensitive HBsAg assay (Architect HBsAg Next qualitative assay, referred to as NEXT) with respect to HBsAg detection. Assay precision, sample carryover, and seroconversion sensitivity of NEXT were evaluated. HBsAg was measured by NEXT in 1,138 individuals, including 1,038 patients who attended liver clinics in a tertiary hospital (200 HBV DNA-positive blood donors whose HBsAg was undetectable by conventional assays, 38 patients receiving immunosuppressive therapy, and 800 chronic hepatitis B patients with HBsAg seroclearance) and 100 HBsAg-negative subjects recruited from a community project. The within-run and within-laboratory coefficients of variation were <6% for the positive sample pools. In 9 seroconversion panels tested, NEXT allowed an earlier HBsAg detection than conventional assays. NEXT detected HBsAg in 10/200 (5%) HBsAg-negative blood donors, 1/20 (5%) and 0/18 HBsAg-negative patients with and without HBV reactivation, respectively, and 59/800 (7.3%) patients with HBsAg seroclearance. HBsAg was detectable by NEXT in 27.8%, 8.2%, 6.9%, 3.8%, and 1.9% samples at <3, 3 to 5, >5 to 8, >8 to 11, and >11 years after HBsAg seroclearance, respectively. Seven out of 100 HBsAg-negative community-identified subjects were tested positive by NEXT. Compared with conventional HBsAg assays, NEXT demonstrated a higher sensitivity and conferred an increment of 5 to 7% detection rate in patients with OBI, thereby helping in identifying HBV carriers and prevention of OBI-associated HBV transmission and reactivation.

Randomised controlled trial: effect of metformin add-on therapy on functional cure in entecavir-treated patients with chronic hepatitis B.

INTRODUCTION AND OBJECTIVES: Hepatitis B surface antigen (HBsAg) clearance, indicating functional cure or resolved chronic hepatitis B (CHB), remains difficult to achieve via nucleos(t)ide analogue monotherapy. We investigated whether metformin add-on therapy could help achieve this goal in entecavir-treated patients with hepatitis B e antigen (HBeAg)-negative CHB. PATIENTS AND METHODS: Patients with HBeAg-negative CHB who met eligibility criteria (entecavir treatment for > 12 months, HBsAg < 1000 IU/mL) were randomly assigned (1:1) to receive 24 weeks of either metformin (1000 mg, oral, once a day) or placebo (oral, once a day) add-on therapy. The group allocation was blinded for both patients and investigators. Efficacy and safety analyses were based on the intention-to-treat set. The primary outcome, serum HBsAg level (IU/mL) at weeks 24 and 36, was analysed using mixed models. RESULTS: Sixty eligible patients were randomly assigned to the metformin (n = 29) and placebo (n = 31) groups. There was no substantial between-group difference in the HBsAg level at week 24 (adjusted mean difference 0.05, 95% confidence interval -0.04 to 0.13, p = 0.278) or week 36 (0.06, -0.03 to 0.15, p = 0.187), and no significant effect of group-by-time interaction on the HBsAg level throughout the trial (p = 0.814). The occurrence of total adverse events between the two groups was comparable (9 [31.0%] of 29 vs. 5 [16.1%] of 31, p = 0.227) and no patient experienced serious adverse events during the study. CONCLUSION: Although it was safe, metformin add-on therapy did not accelerate HBsAg clearance in entecavir-treated patients with HBeAg-negative CHB.

Incidences and Determinants of Functional Cure During Entecavir or Tenofovir Disoproxil Fumarate for Chronic Hepatitis B.

BACKGROUND: Long-term incidences and baseline determinants of functional cure (hepatitis B surface antigen [HBsAg] seroclearance) during entecavir (ETV) or tenofovir disoproxil fumarate (TDF) treatment are incompletely understood. METHODS: This is an international multicenter cohort study of treatment-naive patients with chronic hepatitis B who started ETV or TDF treatment without baseline cancer. Patients were observed for HBsAg seroclearance until death or loss to follow-up. We calculated the incidences and explored the baseline determinants of HBsAg seroclearance using competing risk regression. RESULTS: The analysis included 4769 patients (median age, 50 years; 69.05% male), with a median follow-up of 5.16 years (26 614.47 person-years). HBsAg clearance occurred in 58 patients, yielding a 10-year cumulative incidence of 2.11% (95% confidence interval, 1.54%-2.88%) and an annual rate of 0.22% (.17%-.28%). Baseline predictors included low-level viremia with hepatitis B virus DNA <2000 IU/mL (adjusted subdistribution hazard ratio, 3.14 [95% confidence interval, 1.80-5.49]), elevated serum alanine aminotransferase >200 U/L (3.68 [2.07-6.53]), serum bilirubin (1.11 per mg/dL; [1.06-1.17 mg/dL]), and fatty liver (1.84 [1.03-3.29]). CONCLUSION: HBsAg seroclearance rarely occurs in patients with chronic hepatitis B treated with ETV or TDF and is associated with low-level viremia, alanine aminotransferase flare, bilirubin level, and fatty liver.Functional cure of hepatitis B virus infection rarely occurred at an average annual rate of 0.22% during first-line oral antiviral treatment, with higher chances observed in patients with low-level viremia, high-level aminotransferase flare, elevation of serum bilirubin, and fatty liver.

Structure-Function Characterization of Streptococcus intermedius Surface Antigen Pas.

Streptococcus intermedius, an oral commensal bacterium, is found at various sites, including subgingival dental plaque, purulent infections, and cystic fibrosis lungs. Oral streptococci utilize proteins on their surface to adhere to tissues and/or surfaces localizing the bacteria, which subsequently leads to the development of biofilms, colonization, and infection. Among the 19 genomically annotated cell wall-attached surface proteins on S. intermedius, Pas is an adhesin that belongs to the antigen I/II (AgI/II) family. Here, we have structurally and functionally characterized Pas, particularly focusing on its microbial-host as well as microbial-microbial interactions. The crystal structures of VPas and C123Pas show high similarity with AgI/II of Streptococcus mutans. VPas hosts a conserved metal binding site, and likewise, the C123Pas structure retains its conserved metal binding sites and isopeptide bonds within its three DEv-IgG domains. Pas interacts with nanomolar affinity to lung alveolar glycoprotein 340 (Gp340), its scavenger receptor cysteine-rich domains (SRCRs), and with fibrinogen. Both Candida albicans and Pseudomonas aeruginosa, the opportunistic pathogens that cohabitate with S. intermedius in the lungs of CFTR patients were studied in dual-species biofilm studies. The Pas-deficient mutant (Δpas) displayed significant reduction in dual-biofilm formation with C. albicans. In similar studies with P. aeruginosa, Pas did not mediate the biofilm formation with either the acute isolate (PAO1) or the chronic isolate (FRD1). However, the sortase A-deficient mutant (ΔsrtA) displayed reduced biofilm formation with both C. albicans and P. aeruginosa FRD1. Taken together, our findings highlight the role of Pas in both microbial-host and interkingdom interactions and expose its potential role in disease outcomes. IMPORTANCE Streptococcus intermedius, an oral commensal bacterium, has been clinically observed in subgingival dental plaque, purulent infections, and cystic fibrosis lungs. In this study, we have (i) determined the crystal structure of the V and C regions of Pas; (ii) shown that its surface protein Pas adheres to fibrinogen, which could potentially ferry the microbe through the bloodstream from the oral cavity; (iii) characterized Pas's high-affinity adherence to lung alveolar protein Gp340 that could fixate the microbe on lung epithelial cells; and (iv) most importantly, shown that these surface proteins on the oral commensal S. intermedius enhance biofilms of known pathogens Candida albicans and Pseudomonas aeruginosa.

Contribution of Noncanonical Antigens to Virulence and Adaptive Immunity in Human Infection with Enterotoxigenic E. coli.

Enterotoxigenic Escherichia coli (ETEC) contributes significantly to the substantial burden of infectious diarrhea among children living in low- and middle-income countries. In the absence of a vaccine for ETEC, children succumb to acute dehydration as well as nondiarrheal sequelae related to these infections, including malnutrition. The considerable diversity of ETEC genomes has complicated canonical vaccine development approaches defined by a subset of ETEC pathovar-specific antigens known as colonization factors (CFs). To identify additional conserved immunogens unique to this pathovar, we employed an "open-aperture" approach to capture all potential conserved ETEC surface antigens, in which we mined the genomic sequences of 89 ETEC isolates, bioinformatically selected potential surface-exposed pathovar-specific antigens conserved in more than 40% of the genomes (n = 118), and assembled the representative proteins onto microarrays, complemented with known or putative colonization factor subunit molecules (n = 52) and toxin subunits. These arrays were then used to interrogate samples from individuals with acute symptomatic ETEC infections. Surprisingly, in this approach, we found that immune responses were largely constrained to a small number of antigens, including individual colonization factor antigens and EtpA, an extracellular adhesin. In a Bangladeshi cohort of naturally infected children <2 years of age, both EtpA and a second antigen, EatA, elicited significant serologic responses that were associated with protection from symptomatic illness. In addition, children infected with ETEC isolates bearing either etpA or eatA genes were significantly more likely to develop symptomatic disease. These studies support a role for antigens not presently targeted by vaccines (noncanonical) in virulence and the development of adaptive immune responses during ETEC infections. These findings may inform vaccine design efforts to complement existing approaches.

Clinical utility of quantifying hepatitis B surface antigen in African patients with chronic hepatitis B.

The clinical utility of quantifying hepatitis B surface antigen (qHBsAg) levels in African subjects with chronic hepatitis B virus (HBV) infection has been poorly documented. From a multicentre cohort of 944 HBV-infected African patients, we aimed to assess whether qHBsAg alone can accurately identify i) those in a HBeAg-negative chronic HBV infection phase at low risk of liver disease progression and ii) those in need of antiviral therapy according to the 2017 EASL guidelines. We analysed 770 HBV mono-infected treatment-naïve patients, mainly males (61%) from West Africa (92%), median age 35 years (IQR: 30-44), median HBV DNA: 95.6 IU/ml (10.0-1,300.0), median qHBsAg 5,498 IU/ml (1,171-13,000) and HBeAg-pos 38 (5%). A total of 464/770 (60.2%) patients were classified as HBeAg-negative chronic infection (median age 36 years (31-46), median ALT 23 IU/l (18-28), median HBV-DNA 33.5 IU/ml (3.8-154.1), median LSM 4.8 kPa (4.1-5.8)) and qHBsAg levels had poor accuracy to identify these subjects with an AUROC at 0.58 (95%CI: 0.54-0.62), sensitivity 55.0% and specificity 55.6%; 118/770 (15.3%) patients were eligible for treatment according to the 2017 EASL criteria. qHBsAg correlated poorly with HBV DNA and had poor accuracy to select patients for antiviral therapy with an AUROC at 0.54 (0.49-0.60), sensitivity 46.6% and specificity 46.9%. In African treatment-naïve HBV-infected subjects, the clinical utility of qHBsAg to identify subjects in HBeAg-negative infection phase or subjects eligible for antiviral therapy seems futile. Whether qHBsAg levels can be used as a predictor of long-term liver complications in Africa needs to be further investigated.

Host-pathogen interaction in the tissue environment during Plasmodium blood-stage infection.

Human malarial infection occurs after an infectious Anopheles mosquito bites. Following the initial liver-stage infection, parasites transform into merozoites, infecting red blood cells (RBCs). Repeated RBC infection then occurs during the blood-stage infection, while patients experience various malarial symptoms. Protective immune responses are elicited by this systemic infection, but excessive responses are sometimes harmful for hosts. As parasites infect only RBCs and their immediate precursors during this stage, direct parasite-host interactions occur primarily in the environment surrounded by endothelial lining of blood vessels. The spleen is the major organ where the immune system encounters infected RBCs, causing immunological responses. Its tissue structure is markedly altered during malarial infection in mice and humans. Plasmodium falciparum parasites inside RBCs express proteins, such as PfEMP-1 and RIFIN, transported to the RBC surfaces in order to evade immunological attack by sequestering themselves in the peripheral vasculature avoiding spleen or by direct immune cell inhibition through inhibitory receptors. Host cell production of regulatory cytokines IL-10 and IL-27 limits excessive immune responses, avoiding tissue damage. The regulation of the protective and inhibitory immune responses through host-parasite interactions allows chronic Plasmodium infection. In this review, we discuss underlying interaction mechanisms relevant for developing effective strategies against malaria.

Genetic polymorphism of the extracellular region in surface associated interspersed 1.1 gene of Plasmodium falciparum field isolates from Thailand.

BACKGROUND: A novel variable surface antigens (VSAs), Surface-associated interspersed proteins (SUFRINs), is a protein that is modified on the surface of infected red blood cell (iRBC). Modified proteins on the iRBC surface cause severe malaria, which can lead to death throughout the life cycle of a malaria parasite. Previous study suggested that SURFIN1.1 is an immunogenic membrane-associated protein which was encoded by using the surf1.1 gene expressed during the trophozoite and schizont stages. This study aimed to identify the regions of SURFIN1.1 and investigate the genetic diversity of the extracellular region of the surf1.1 gene. METHODS: A total of 32 blood samples from falciparum malaria cases that were diagnosed in Si Sa Ket Province, Thailand were collected. Plasmodium genomic DNA was extracted, and the extracellular region of surf1.1 gene was amplified using the polymerase chain reaction (PCR). A sequence analysis was then performed to obtain the number of haplotypes (H), the haplotype diversity (Hd), and the segregating sites (S), while the average number of nucleotide differences between two sequences (Pi); in addition, neutrality testing, Tajima's D test, Fu and Li's D* and F* statistics was also performed. RESULTS: From a total of 32 patient-isolated samples, 31 DNA sequences were obtained and analysed for surf1.1 gene extracellular region polymorphism. Researchers observed six distinct haplotypes in the current research area. Haplotype frequencies were 61.3%, 16.2%, and 12.9% for H1, H2, and H3, respectively. The remaining haplotype (H4-H6) frequency was 3.2% for each haplotype. Hd was 0.598 ± 0.089 with the Pi of 0.00381, and S was 15. The most common amino acid polymorphic site was E251Q; other sites included N48D, I49V, E228D, E235S, L265F, K267T, E276Q, and S288F. Fu and Li's D* test value was - 1.24255, Fu and Li's F* test value was - 1.10175, indicating a tendency toward negative balancing selection acting on the surf1.1 N-terminal region. The most polymorphic region was variable 2 (Var2) while cysteine-rich domain (CRD) was conserved in both the amino acid and nucleotide extracellular region of surf1.1 gene. CONCLUSIONS: The Thai surf1.1 N-terminal region was well-conserved with only a few polymorphic sites remaining. In this study, the data regarding current bearing on the polymorphism of extracellular region of surf1.1 gene were reported, which might impact the biological roles of P. falciparum. In addition, may possibly serve as a suitable candidate for future development of SURFIN-based vaccines regarding malaria control.

Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer.

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting.

Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays.

BACKGROUND: Few bead-based multiplex assays have been described that detect antibodies against the protozoan parasite Toxoplasma gondii in large-scale seroepidemiological surveys. Moreover, each multiplex assay has specific variations or limitations, such as the use of truncated or fusion proteins as antigens, potentially masking important epitopes. Consequently, such an assay must be developed by interested groups as none is commercially available. RESULTS: We report the bacterial expression and use of N-terminal fusion-free, soluble, in vivo biotinylated recombinant surface antigens SAG1 and SAG2A for the detection of anti-T. gondii IgG antibodies. The expression system relies on three compatible plasmids. An expression construct produces a fusion of maltose-binding protein with SAG1 (or SAG2A), separated by a TEV protease cleavage site, followed by a peptide sequence recognized by E. coli biotin ligase BirA (AviTag), and a terminal six histidine tag for affinity purification. TEV protease and BirA are encoded on a second plasmid, and their expression leads to proteolytic cleavage of the fusion protein and a single biotinylated lysine within the AviTag by BirA. Correct folding of the parasite proteins is dependent on proper disulfide bonding, which is facilitated by a sulfhydryl oxidase and a protein disulfide isomerase, encoded on the third plasmid. The C-terminal biotinylation allowed the oriented, reproducible coupling of the purified surface antigens to magnetic Luminex beads, requiring only minute amounts of protein per determination. We showed that an N-terminal fusion partner such as maltose-binding protein negatively influenced antibody binding, confirming that access to SAG1's N-terminal epitopes is important for antibody recognition. We validated our bead-based multiplex assay with human sera previously tested with commercial diagnostic assays and found concordance of 98-100% regarding both, sensitivity and specificity, even when only biotinylated SAG1 was used as antigen. CONCLUSIONS: Our recombinant in vivo-biotinylated T. gondii antigens offer distinct advantages compared to previously described proteins used in multiplex serological assays for T. gondii. They offer a cheap, specific and sensitive alternative to either parasite lysates or eukaryotic-cell expressed SAG1/SAG2A for BBMA and other formats. The described general expression strategy can also be used for other antigens where oriented immobilization is key for sensitive recognition by antibodies and ligands.

Cell culture media notably influence properties of human mesenchymal stroma/stem-like cells from different tissues.

BACKGROUND AIMS: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues. METHODS: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium. RESULTS: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected. CONCLUSIONS: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.