Hybrid and herd immunity 6 months after SARS-CoV-2 exposure among individuals from a community treatment program.

The death rate from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in 2022 was lower than the death rate in 2021, when the infection rate increased. Hybrid immunity provided by a combination of vaccination and infection, including asymptomatic infection, may confer effective protection against death. We explored the combined effect of asymptomatic infection and hybrid immunity by studying T-cell and antibody responses against SARS-CoV-2 among individuals treated in home health care services 6 months after SARS-CoV-2 exposure. Asymptomatic SARS-CoV-2 infection was demonstrated in 24.4% of close contacts. The levels of immunity were not different between patients and close contacts. Anti-RBD IgG against SARS-CoV-2 increased in a dose-dependent manner with the number of vaccine doses. Interestingly, the T-cell response decreased soon after a booster dose of vaccine. Asymptomatic SARS-CoV-2 infection could not enhance immunity against SARS-CoV-2 among vaccinated close contacts. Full vaccination was crucial to provide hybrid immunity. However, when designing vaccine strategies, T-cell exhaustion after multiple vaccinations should be considered.

Vitamin D3 Supplementation Promotes Regulatory T-Cells to Maintain Immune Homeostasis After Surgery for Early Stages of Colorectal Cancer.

BACKGROUND/AIM: Vitamin D3 (VD3) affects the regulation of the immune system, including the differentiation and function of regulatory T-cells (Tregs). Tregs play an important role in maintaining immune homeostasis in patients with colorectal cancer (CRC). The effects of VD3 on Treg-associated immune function were investigated in Thai patients in the early stages of CRC. MATERIALS AND METHODS: Twenty-eight patients were randomized to one of two groups: Untreated or treatment with VD3 for 3 months. Whole blood samples were collected at baseline, and at 1 and 3 months. Peripheral blood mononuclear cells were isolated and the populations of forkhead box P3-positive Treg cells was analyzed by flow cytometry. The levels of Treg-associated cytokines, interleukin 10 (IL-10) and transforming growth factor beta 1 (TGF-ß1), were measured by enzyme-linked immunosorbent assays. RESULTS: Serum VD3 levels of the VD3-treated group were significantly increased at 1 (p=0.017) and 3 months (p<0.001) compared to the untreated control group. The mean percentage of Tregs was maintained between 1 and 3 months in the VD3-treated group. At 3 months, the untreated group had significantly lower Treg levels than the VD3-treated group (p=0.043). Serum IL-10 levels of the VD3-treated group were statistically increased at 1 month compared to the control group (p=0.032). No significant difference in serum TGF-ß1 levels was observed between the two groups. However, the TGF-ß1 level in the VD3-treated group at 1 month was lower than that of the control. CONCLUSION: Our findings suggest that VD3 supplementation can maintain immune responses in the early stages of CRC, helping to control Treg function. Therefore, VD3 should be supplemented to maintain immune homeostasis, especially in patients with vitamin D deficiency.

Regulatory T cell and cytokine changes in children undergoing 3 days rush venom immunotherapy.

BACKGROUND: Rush venom immunotherapy (VIT) is the recommended treatment for patients with Hymenoptera anaphylaxis. Specific data regarding regulatory T cell and cytokine changes in children receiving rush VIT are sparse. OBJECTIVE: To study the changing of CD4+CD25+FOXP3+ regulatory T cells (Treg) and serum cytokines in children undergoing 3 days rush VIT. METHODS: Children younger than 15 years with systemic reaction to Hymenoptera who had evidence of IgE sensitization to Hymenoptera were enrolled for 3 days rush VIT. Peripheral blood CD4+CD25+FOXP3+ Treg and serum IL-4, IL5, IL-13, IFN-γ, and IL-10 were measured at baseline before rush VIT, achieving maintenance dose, 6 months, and 12 months after reaching maintenance dose. Specific IgE to Hymenoptera was measured at baseline and 12 months after VIT. RESULTS: A total of 15 children (11 boys and 4 girls) aged 6-15 years (mean age, 10 years) were enrolled. Four children were allergic to bee and 11 children were allergic to Vespid. The levels of CD4+CD25+FOXP3+ Treg were significantly increased at 6 months after maintenance dose compared with baseline (6.58% VS 4.01%, p = 0.001). Serum IL-13, IFN-γ, and IL-10 levels did not change significantly from baseline. However, there was a significant reduction of IL-4 in the serum at 12 months after MN when compared to the baseline levels. The systemic reaction requiring epinephrine intramuscular injection occurred only in 1 case who was on Vespid venoms rush VIT. CONCLUSIONS: Three days rush VIT provide acceptable systemic reaction and able to increase the number of CD4+CD25+FOXP3+ Treg in children.

Immunologic changes after house dust mite modified rush subcutaneous immunotherapy in allergic rhinitis children.

BACKGROUND: House dust mites (HDM) are the major causative allergen for allergic rhinitis. The sole disease-modifying therapy for allergic rhinitis is allergen immunotherapy (AIT). Rush immunotherapy is the accelerated build-up schedules to reach the target maintenance dose. OBJECTIVE: To evaluate the kinetic changes of peripheral blood CD4+CD25+FOXP3+ regulatory T cells (Treg) and serum cytokines in children undergoing 2-day modified rush HDM AIT. METHODS: Children aged 5-15 years with allergic rhinitis were enrolled for a 2-day modified rush HDM AIT. Peripheral blood CD4+CD25+FOXP3+ Treg, serum interleukin (IL)-4, IL-13, interferon-γ, and IL-10 were measured at baseline, finishing rush, achieving maintenance dose, 6 months, and 12 months after reaching maintenance dose. Specific IgE (sIgE) to HDM was evaluated at baseline and 12 months after getting the maintenance dose. Rhinitis symptoms were assessed daily using a daily card. RESULTS: A total of 12 children with a mean age of 13 years were enrolled. Rhinitis symptom-free days per month increased significantly after reaching the maintenance dose compared to baseline (from 9.5 days to 19.5 days, p = 0.002), and the maximum improvement was seen at 1 year. The levels of Treg were significantly increased at 6 months after maintenance dose compared to baseline level (6.27%±1.63% vs. 3.83%±1.80%, p < 0.001). After treatment, there were significantly decreased serum IL-13 at 1 year after maintenance but no significant changes in sIgE to HDM. The systemic reaction during AIT occurred 7 episodes from 119 shots (5.9%). CONCLUSION: Two-day modified rush HDM AIT provides acceptable systemic reactions and increases the number of CD4+CD25+FOXP3+ Treg in children.

Application of QuantiFERON ELISA for Detection of Interferon-Gamma Autoantibodies in Adult-Onset Immunodeficiency Syndrome.

OBJECTIVE: Patients who develop interferon-gamma autoantibodies (IFN-ɤ autoAbs) in adult-onset immunodeficiency (AOID) syndrome are more likely to develop opportunistic and recurrent intracellular infections. The assay to detect IFN-ɤ autoAbs is essential for the diagnosis and therapeutic monitoring of AOID syndrome. Therefore, this study applied the QuantiFERON assay for the detection of IFN-ɤ autoAbs. METHODS: Serum from patients with AOID syndrome (n = 19) and serum from healthy patients (n = 20) was collected and applied using 2 neutralizing platforms of enzyme-linked immunosorbent assay (ELISA) kits (the BD ELISA and the QuantiFERON ELISA) for IFN-ɤ autoAbs detection. RESULTS: The pooled serum from patients with AOID syndrome showed >50% inhibition at 1:5000 dilution (positive), whereas the pooled serum from healthy patients showed <50% inhibition at 1:5000 dilution (negative) according to the neutralizing QuantiFERON ELISA. Each specimen showed the same result according to both the neutralizing BD ELISA and the neutralizing QuantiFERON ELISA. Moreover, the patient serum showed a variation in titer ranging from 1:5000 to >1:5,000,000 according to the neutralizing QuantiFERON ELISA. CONCLUSION: The QuantiFERON ELISA kit could be applied for the detection of IFN-ɤ autoAbs for the diagnosis and therapeutic monitoring of AOID syndrome.

Inhibitory activity of FOXP3+ regulatory T cells reveals high specificity for displaying immune tolerance in remission state rheumatoid arthritis.

Immune regulation status may indicate immunological remission in rheumatoid arthritis (RA). This cross-sectional study aimed to determine the Regulatory T cell (Treg) properties, together with 14 plasma cytokines levels between active RA and clinical remission patients. Peripheral blood (PB) Foxp3+ Treg was collected from RA patients for determination of Treg inhibitory activity using a co-culture system. Other PB T cell types and plasma cytokines were determined by flow-cytometry. The Treg results were analyzed according to the disease activity score-28 (DAS28). Then sensitivity and specificity were calculated for the indication of the remission status. The number and inhibitory activity of Treg are higher in the clinical remission as compared to the active RA (p value < 0.0001). Also, Treg: CD4+CD25+CD127+ cell ratio demonstrates the similar result (p value < 0.05). Treg inhibitory activity is inversely correlated with the DAS28. Specificity and positive likelihood ratio of inhibitory activity for indicating remission status are 92.31% (95% CI 63.97-99.81) and 11.14 (95% CI 1.67-74.14), respectively. Treg inhibitory activity is a promising prognostic marker and probably represents the immunological remission status in RA.

Regulatory T Cell Suppressive Activity Predicts Disease Relapse During Disease-Modifying Anti-rheumatic Drug Dose Reduction in Rheumatoid Arthritis: A Prospective Cohort Study.

When the dose of conventional disease-modifying anti-rheumatic drugs (cDMARDs) is tapered in rheumatoid arthritis (RA) patients who achieve sustained remission, biomarkers for predicting disease relapse may be needed. A prospective, unblinded cohort study was conducted in nine RA patients with remission. Peripheral blood samples were collected at baseline and at 6, 12, and 24 weeks after cDMARD dose reduction (dose of combination regimens reduced to 50%) to determine the number of regulatory Foxp3+T cells (Tregs) and other T cell subpopulations as well as Treg suppressive activity. Additionally, plasma levels of 14 cytokines at each time-point were measured via flow cytometry. Univariate and multivariate analyses were performed to identify the factor(s) associated with RA relapse during the observational period. In univariate analysis, Treg suppression and DAS28 and VAS scores were associated with RA relapse after cDMARD dose tapering. However, in multivariate analysis, only Treg suppressive activity (<42%) was found to be an independent factor associated with RA relapse after cDMARD dose reduction to 50%. Of all patients who had ≥42% Treg suppressive activity during cDMAD reduction, three-fourth patients remained in the remission stage for 24 weeks. Treg suppressive activity (<42%) in RA patients with remission could be a potential biomarker for predicting RA relapse after cDMARD dose reduction, especially over a short-term period (24 weeks).

Efficient short-term expansion of human peripheral blood regulatory T cells for co-culture suppression assay.

Regulatory T cells (Tregs) are a small population of CD4+ lymphocytes and play a key role as suppressors of the immune system, a role that can be identified by employing a co-culture suppression assay. Conventional protocol requires a long period of in vitro expansion of Treg numbers; hence, this study describes an establishment of a co-culture suppression assay using a short-term expansion of peripheral blood (PB) Tregs and autologous T cells (Tconvs) IL-2-pre-cultured in parallel for the same length of time, thereby obviating the need of freeze/thawed autologous Tconvs. Tregs and Tconvs were isolated from PB mononuclear cells employing magnetic bead-aided depletion of CD8+ cells followed by cell sorting of CD4+ CD25high+CD127low- (Treg) and CD4+ CD25-CD127+ (Tconv) cell populations. Following a 3-day co-cultivation period under optimized conditions, Treg suppression activity was monitored by comparing using flow cytometry the number of carboxyfluorescein succinimidyl ester-labeled Tconvs to that of Treg-minus control. The assay allowed significant differentiation between Treg suppression activity of patients with active rheumatoid arthritis and those in remission. This method should be more convenient and time-saving than the conventional Treg suppression assay in current use.

Quorum sensing triggers the stochastic escape of individual cells from Pseudomonas putida biofilms.

The term 'quorum sensing' (QS) is generally used to describe the phenomenon that bacteria release and perceive signal molecules to coordinate cooperative behaviour in response to their population size. QS-based communication has therefore been considered a social trait. Here we show that QS signals (N-acyl-homoserine lactones, AHLs) are stochastically produced in young biofilms of Pseudomonas putida and act mainly as self-regulatory signals rather than inducing neighbouring cells. We demonstrate that QS induces the expression of putisolvin biosurfactants that are not public goods, thereby triggering asocial motility of induced cells out of microcolonies. Phenotypic heterogeneity is most prominent in the early stages of biofilm development, whereas at later stages behaviour patterns across cells become more synchronized. Our findings broaden our perspective on QS by showing that AHLs can control the expression of asocial (self-directed) traits, and that heterogeneity in QS can serve as a mechanism to drive phenotypic heterogeneity in self-directed behaviour.

The genetic basis of cadmium resistance of Burkholderia cenocepacia.

Burkholderia species are highly resistant to heavy metals (HMs), yet their resistance mechanisms are largely unknown. In this study we screened 5000 mini-Tn5 transposon insertion mutants of Burkholderia cenocepacia H111 for loss of cadmium tolerance. Of the four genes identified three affected outer membrane biogenesis and integrity or DNA repair. The fourth gene, BCAE0587, encoded a P1-type ATPase belonging to the CadA family of HM exporters. CadA-deficient strains lost the ability to grow in the presence of cadmium, zinc and lead, whereas resistance to nickel, copper and cobalt was not affected. Expression studies using a transcriptional fusion of the cadA promoter to gfp confirmed this specificity, as induction was only observed in presence of cadmium, zinc and lead. The promoter activity was found to be highest at neutral pH with an activation threshold of 30 nM cadmium. Inoculation of the HM-hyperaccumulating plant Arabidopsis halleri with a RFP-marked derivative of B. cenocepacia H111 containing the PcadA -gfp fusion demonstrated the applicability of this biosensor for monitoring cadmium at the single cell level in a natural environment.

Construction of self-transmissible green fluorescent protein-based biosensor plasmids and their use for identification of N-acyl homoserine-producing bacteria in lake sediments.

Many bacteria utilize quorum sensing (QS) systems to communicate with each other by means of the production, release, and response to signal molecules. N-Acyl homoserine lactone (AHL)-based QS systems are particularly widespread among the Proteobacteria, in which they regulate various functions. It has become evident that AHLs can also serve as signals for interspecies communication. However, knowledge on the impact of AHLs for the ecology of bacteria in their natural habitat is scarce, due mainly to the lack of tools that allow the study of QS in bacterial communities in situ. Here, we describe the construction of self-mobilizable green fluorescent protein (GFP)-based AHL sensors that utilize the conjugation and replication properties of the broad-host-range plasmid RP4. We show that these novel AHL sensor plasmids can be easily transferred to different bacterial species by biparental mating and that they give rise to green fluorescent cells in case the recipient is an AHL producer. We also demonstrate that these sensor plasmids are capable of self-spreading within mixed biofilms and are a suitable tool for the identification of AHL-producing bacteria in lake sediment.

Quorum sensing regulates dpsA and the oxidative stress response in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal human tropical disease. The non-specific DNA-binding protein DpsA plays a key role in protecting B. pseudomallei from oxidative stress mediated, for example, by organic hydroperoxides. The regulation of dpsA expression is poorly understood but one possibility is that it is regulated in a cell population density-dependent manner via N-acylhomoserine lactone (AHL)-dependent quorum sensing (QS) since a lux-box motif has been located within the dpsA promoter region. Using liquid chromatography and tandem mass spectrometry, it was first established that B. pseudomallei strain PP844 synthesizes AHLs. These were identified as N-octanoylhomoserine lactone (C8-HSL), N-(3-oxooctanoyl)homoserine lactone (3-oxo-C8-HSL), N-(3-hydroxyoctanoyl)-homoserine lactone (3-hydroxy-C8-HSL), N-decanoylhomoserine lactone (C10-HSL), N-(3-hydroxydecanoyl) homoserine lactone (3-hydroxy-C10-HSL) and N-(3-hydroxydodecanoyl)homoserine lactone (3-hydroxy-C12-HSL). Mutation of the genes encoding the LuxI homologue BpsI or the LuxR homologue BpsR resulted in the loss of C8-HSL and 3-oxo-C8-HSL synthesis, demonstrating that BpsI was responsible for directing the synthesis of these AHLs only and that bpsI expression and hence C8-HSL and 3-oxo-C8-HSL production depends on BpsR. In bpsI, bpsR and bpsIR mutants, dpsA expression was substantially down-regulated. Furthermore, dpsA expression in Escherichia coli required both BpsR and C8-HSL. bpsIR-deficient mutants exhibited hypersensitivity to the organic hydroperoxide tert-butyl hydroperoxide by displaying a reduction in cell viability which was restored by provision of exogenous C8-HSL (bpsI mutant only), by complementation with the bpsIR genes or by overexpression of dpsA. These data indicate that in B. pseudomallei, QS regulates the response to oxidative stress at least in part via the BpsR/C8-HSL-dependent regulation of DpsA.

Functional genetic analysis reveals a 2-Alkyl-4-quinolone signaling system in the human pathogen Burkholderia pseudomallei and related bacteria.

Pseudomonas aeruginosa synthesizes diverse 2-alkyl-4(1H)-quinolones (AHQs), including the signaling molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), via the pqsABCDE locus. By examining the genome databases, homologs of the pqs genes were identified in other bacteria. However, apart from P. aeruginosa, only Burkholderia pseudomallei and B. thailandensis contained a complete pqsA-E operon (termed hhqA-E). By introducing the B. pseudomallei hhqA and hhqE genes into P. aeruginosa pqsA and pqsE mutants, we show that they are functionally conserved and restore virulence factor and PQS production. B. pseudomallei, B. thailandensis, B. cenocepacia, and P. putida each produced 2-heptyl-4(1H)-quinolone (HHQ), but not PQS. Mutation of hhqA in B. pseudomallei resulted in the loss of AHQ production, altered colony morphology, and enhanced elastase production, which was reduced to parental levels by exogenous HHQ. These data reveal a role for AHQs in bacterial cell-to-cell communication beyond that seen in P. aeruginosa.