Inflammatory immune responses induced by lipopolysaccharides (LPS) of gram-negative bacteria play an important role in the pathogenesis of preterm labor and delivery, and in neonatal disorders. To ...better characterize LPS-induced inflammatory response, we determined the cytokine profile of umbilical cord blood mononuclear cells (UBMC) stimulated with LPS of seven vaginal gram-negative bacteria commonly found in pregnant women with preterm labor and preterm rupture of membrane. UBMC from ten newborns of healthy volunteer mothers were stimulated with purified LPS of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter calcoaceticus, Citrobacter freundii, and Pseudomonas aeruginosa. UBMC supernatants were tested for the presence of secreted pro-inflammatory cytokines (IL-6, IL-1β, TNF), anti-inflammatory cytokine (IL-10), TH1-type cytokines (IL-12, IFN-γ), and chemokines (IL-8, MIP-1α, MIP-1β, MCP-1) by Luminex technology. The ten cytokines were differentially induced by the LPS variants. LPS of E. coli and E. aerogenes showed the strongest stimulatory activity and P. aeruginosa the lowest. Interestingly, the ability of UBMC to respond to LPS varied greatly among donors, suggesting a strong individual heterogeneity in LPS-triggered inflammatory response.
This study describes the comparative expression and purification of hepatitis B surface antigen (HBsAg) particles produced upon infection of human primary hepatocytes and human hepatoma cell lines ...(HuH-7 and HepG2) with recombinant vaccinia viruses. The highest levels of HBsAg expression were found in HuH-7 hepatoma cells following infection with recombinant vaccinia viruses, which contain the S gene under control of a 7.5 k-promoter. Four different methods for purification of the HBsAg particles were examined: isopycnic ultracentrifugation, sucrose cushion sedimentation, isocratic column gel filtration, and binding to anti-HBs-coated microparticles. The highest degree of purity of HBsAg particles was reached by the method based on anti-HBs-coated microparticles. The resulting product was >98% pure. Biochemical analysis and characterization of purified HBsAg particles were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and electron microscopy. The HBsAg, purified from human hepatoma cell lines and from human primary hepatocytes, consisted of both the non-glycosylated (p25) and the glycosylated (gp27) form and assembled into typical 22-nm particles, and thus may be of great interest and importance for research, diagnostics, and medical treatments.
Recurrence of cytomegalovirus reactivation remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Monitoring cytomegalovirus-specific cellular ...immunity using a standardized assay might improve the risk stratification of patients. A prospective multicenter study was conducted in 175 intermediate- and high-risk allogeneic hematopoietic stem cell transplant recipients under preemptive antiviral therapy. Cytomegalovirus-specific cellular immunity was measured using a standardized IFN-γ ELISpot assay (T-Track® CMV). Primary aim was to evaluate the suitability of measuring cytomegalovirus-specific immunity after end of treatment for a first cytomegalovirus reactivation to predict recurrent reactivation. 40/101 (39.6%) patients with a first cytomegalovirus reactivation experienced recurrent reactivations, mainly in the high-risk group (cytomegalovirus-seronegative donor/cytomegalovirus-seropositive recipient). The positive predictive value of T-Track® CMV (patients with a negative test after the first reactivation experienced at least one recurrent reactivation) was 84.2% in high-risk patients. Kaplan-Meier analysis revealed a higher probability of recurrent cytomegalovirus reactivation in high-risk patients with a negative test after the first reactivation (hazard ratio 2.73; p=0.007). Interestingly, a post-hoc analysis considering T-Track® CMV measurements at day 100 post-transplantation, a time point highly relevant for outpatient care, showed a positive predictive value of 90.0% in high-risk patients. Our results indicate that standardized cytomegalovirus-specific cellular immunity monitoring may allow improved risk stratification and management of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation. This study was registered at www.clinicaltrials.gov as #NCT02156479.
During pregnancy, infections caused by the gram-positive bacteria
(
),
(
), and
(
) are major reasons for preterm labor, neonatal prematurity, meningitis, or sepsis. Here, we propose cytokine ...responses to bacterial infections by the immature perinatal immune system as central players in the pathogenesis of preterm birth and neonatal sepsis. We aimed to close the gap in knowledge about such cytokine responses by stimulating freshly isolated umbilical blood mononuclear cells (UBMC) with lysates of
,
, and
collected from pregnant women in preterm labor. Bacterial lysates and, principally,
and
distinctly triggered most of the eleven inflammatory, anti-inflammatory, TH
/TH
cytokines, and chemokines quantified in UBMC culture media. Chemokines depicted the most robust induction. Among them, MIP-1β was further enhanced in UBMC from female compered to male newborn infants. Due to its stability and high levels, we investigated the diagnostic value of IL-8. IL-8 was critically upregulated in cord blood of preterm neonates suffering from infections compared to gestational age-matched controls. Our results provide novel clues about perinatal immunity, underscoring a potential value of IL-8 for the timely detection of infections and suggesting that MIP-1β constitutes an early determinant of sex-specific immunity, which may contribute, e.g., to male's vulnerability to preterm birth.
T-cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a central role in the control of the virus. In this study, we evaluated the performance of T-Track® ...SARS-CoV-2, a novel CE-marked quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay, which relies on the combined evaluation of IFNG and CXCL10 mRNA levels in response to the S1 and NP SARS-CoV-2 antigens, in 335 participants with or without a history of SARS-CoV-2 infection and vaccination, respectively. Of the 62 convalescent donors, 100% responded to S1 and 88.7% to NP antigens. In comparison, of the 68 naïve donors, 4.4% were reactive to S1 and 19.1% to NP. Convalescent donors <50 and ≥50 years of age demonstrated a 100% S1 reactivity and an 89.1% and 87.5% NP reactivity, respectively. T-cell responses by T-Track® SARS-CoV-2 and IgG serology by recomLine SARS-CoV-2 IgG according to the time from the last immunisation (by vaccination or viral infection) were comparable. Both assays showed a persistent cellular and humoral response for at least 36 weeks post immunisation in vaccinated and convalescent donors. Our results demonstrate the very good performance of the T-Track® SARS-CoV-2 molecular assay and suggest that it might be suitable to monitor the SARS-CoV-2-specific T-cell response in COVID-19 vaccinations trials and cross-reactivity studies.
Cytomegalovirus (CMV) immunoglobulin (CMVIG) is used for the prophylaxis of CMV infection after transplantation. Beyond providing passive CMV-specific immunity, CMVIG exerts enhancing and suppressive ...immunomodulatory functions. Although the anti-inflammatory activities of CMVIG have been extensively documented, its immunostimulatory activities remain poorly characterized.
This exploratory study analyzed the capacity of CMVIG to modulate cell-mediated innate and adaptive immunities in vitro on freshly isolated peripheral blood mononuclear cells (PBMCs) of CMV-seropositive and -seronegative healthy individuals, using interferon-γ (IFN-γ) enzyme-linked immunospot and intracellular cytokine staining assays.
We showed that CMVIG treatment increases the number of IFN-γ-secreting PBMCs of both CMV-seronegative and -seropositive individuals, indicating a global stimulatory effect on innate immune cells. Indeed, CMVIG significantly increased the frequency of natural killer cells producing the T helper cell 1-type cytokines tumor necrosis factor and IFN-γ. This was associated with the induction of interleukin-12-expressing monocytes and the activation of cluster of differentiation (CD) 4
and CD8
T cells, as measured by the expression of tumor necrosis factor and IFN-γ. Interestingly, stimulation of PBMCs from CMV-seropositive subjects with CMVIG-opsonized CMV antigens (phosphoprotein 65, CMV lysate) enhanced CD4
and CD8
T-cell activation, suggesting that CMVIG promotes the immunogenicity of CMV antigens.
Our data demonstrate that CMVIG can stimulate effector cells of both innate and adaptive immunities and promote the immunogenicity of CMV antigens. These immunostimulatory properties might contribute to the protective effect against CMV infection mediated by CMVIG.
Tuberculosis (TB) is one of the leading causes of death by an infectious disease. It remains a major health burden worldwide, in part due to misdiagnosis. Therefore, improved diagnostic tests ...allowing the faster and more reliable diagnosis of patients with active TB are urgently needed. This prospective study examined the performance of the new molecular whole-blood test T-Track
TB, which relies on the combined evaluation of
and
mRNA levels, and compared it to that of the QuantiFERON
-TB Gold Plus (QFT-Plus) enzyme-linked immunosorbent assay (ELISA). Diagnostic accuracy and agreement analyses were conducted on the whole blood of 181 active TB patients and 163 non-TB controls. T-Track
TB presented sensitivity of 94.9% and specificity of 93.8% for the detection of active TB vs. non-TB controls. In comparison, the QFT-Plus ELISA showed sensitivity of 84.3%. The sensitivity of T-Track
TB was significantly higher (
< 0.001) than that of QFT-Plus. The overall agreement of T-Track
TB with QFT-Plus to diagnose active TB was 87.9%. Out of 21 samples with discordant results, 19 were correctly classified by T-Track
TB while misclassified by QFT-Plus (T-Track
TB-positive/QFT-Plus-negative), and two samples were misclassified by T-Track
TB while correctly classified by QFT-Plus (T-Track
TB-negative/QFT-Plus-positive). Our results demonstrate the excellent performance of the T-Track
TB molecular assay and its suitability to accurately detect TB infection and discriminate active TB patients from non-infected controls.
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