Oral immune dysfunction is associated with the expansion of FOXP3+PD-1+Amphiregulin+ T cells during HIV infection.

Residual systemic inflammation and mucosal immune dysfunction persist in people living with HIV, despite treatment with combined anti-retroviral therapy, but the underlying immune mechanisms are poorly understood. Here we report that the altered immune landscape of the oral mucosa of HIV-positive patients on therapy involves increased TLR and inflammasome signaling, localized CD4+ T cell hyperactivation, and, counterintuitively, enrichment of FOXP3+ T cells. HIV infection of oral tonsil cultures in vitro causes an increase in FOXP3+ T cells expressing PD-1, IFN-γ, Amphiregulin and IL-10. These cells persist even in the presence of anti-retroviral drugs, and further expand when stimulated by TLR2 ligands and IL-1ß. Mechanistically, IL-1ß upregulates PD-1 expression via AKT signaling, and PD-1 stabilizes FOXP3 and Amphiregulin through a mechanism involving asparaginyl endopeptidase, resulting in FOXP3+ cells that are incapable of suppressing CD4+ T cells in vitro. The FOXP3+ T cells that are abundant in HIV-positive patients are phenotypically similar to the in vitro cultured, HIV-responsive FOXP3+ T cells, and their presence strongly correlates with CD4+ T cell hyper-activation. This suggests that FOXP3+ T cell dysregulation might play a role in the mucosal immune dysfunction of HIV patients on therapy.

Microbial population dynamics in laboratory-scale solid waste bioreactors in the presence or absence of biosolids.

AIMS: Decomposition of solid waste is microbially mediated, yet little is known about the associated structure and temporal changes in prokaryotic communities. Bioreactors were used to simulate landfill conditions and archaeal and bacterial community development in leachate was examined over 8 months. METHODS AND RESULTS: Municipal solid waste (MSW) was deposited in laboratory bioreactors with or without biosolids and combustion residues (ash). The near-neutral pH fell about half a log by day 25, but recovered to approximately 7.0 by day 50. Cell concentrations in bioreactors containing only MSW were significantly higher than those from co-disposal bioreactors. Archaeal and bacterial community structure was analysed by denaturing gradient gel electrophoresis targeting 16S rRNA genes, showing temporal population shifts for both domains. mcrA sequences retrieved from a co-disposal bioreactor were predominantly affiliated with the orders Methanosarcinales and Methanomicrobiales. CONCLUSION: Regardless of waste composition, microbial communities in bioreactor leachates exhibited high diversity and distinct temporal trends. The solid waste filled bioreactors allowed simulation of solid waste decomposition in landfills while also reducing the variables. SIGNIFICANCE AND IMPACT OF THE STUDY: This study advances the basic understanding of changes in microbial community structure during solid waste decomposition, which may ultimately improve the efficiency of solid waste management.

Mucosal T-cell immunoregulation varies in early and late inflammatory bowel disease.

BACKGROUND AND AIMS: Crohn's disease is a life-long form of inflammatory bowel disease (IBD) mediated by mucosal immune abnormalities. Understanding of the pathogenesis is limited because it is based on data from adults with chronic Crohn's disease. We investigated mucosal T-cell immunoregulatory events in children with early Crohn's disease. METHODS: Mucosal biopsies and T-cell clones were derived from children experiencing the first attack of Crohn's disease, children with long-standing Crohn's disease, infectious colitis, and children without gut inflammation. RESULTS: As in acute infectious colitis, interleukin (IL) 12 induced T cells from early Crohn's disease to acquire a strongly polarised T helper (Th) type 1 response characterised by high IFN-gamma production and IL12Rbeta2 chain expression. Th1 polarisation was not induced in clones from late Crohn's disease. Mucosal levels of IL12p40 and IL12Rbeta2 messenger RNA were significantly higher in children with early than late Crohn's disease. These results demonstrate that susceptibility to IL12-mediated modulation is strongly dependent on the stage of Crohn's disease. CONCLUSIONS: At the onset of Crohn's disease mucosal T cells appear to mount a typical Th1 response that resembles an acute infectious process, and is lost with progression to late Crohn's disease. This suggests that mucosal T-cell immunoregulation varies with the course of human IBD. Patients with the initial manifestations of IBD may represent an ideal population in which immunomodulation may have optimal therapeutic efficacy.

Abundant nuclear ribonucleoprotein form of CAD RNA.

Transcripts of the CAD gene in Syrian hamster cells are as abundant in the nucleus as in the cytoplasm. This was shown by in situ hybridization of whole cells and by solution and blot hybridization of subcellular fractions. Similar results were obtained both for wild-type cells and for a mutant containing amplified CAD genes in which the level of CAD RNA is 150-fold greater. CAD nuclear RNA is indistinguishable from mature mRNA by gel electrophoresis and blot hybridization. Discrete higher-molecular-weight precursors are undetectable, although the persistence of a short length of intervening sequence in the otherwise fully processed RNA is not excluded. CAD RNA is released from nuclei by sonication in physiological conditions in a ribonucleoprotein form that sediments as a broad peak at about 200S in a sucrose gradient. CAD sequences extracted from nuclei by treatment with EDTA and RNase are found in the 30S particles previously described.

A cell culture system that enhances mononuclear cell IgE synthesis induced by recombinant human interleukin-4.

A new culture system is described in which recombinant human interleukin-4 (rhIL-4) consistently induces the synthesis of large quantities of IgE by human blood mononuclear cells (MNC). Unfractionated MNC were cultured in complete Iscove's modified Dulbecco's medium (C-IMDM), composed of IMDM enriched with human transferrin, bovine insulin, bovine serum albumin, oleic acid, palmitic acid, linoleic acid, and fetal calf serum (FCS). Under these culture conditions, MNC from four donors synthesized mean quantities of IgE of 76 ng/ml at plateau after stimulation with rhIL-4 in concentrations ranging from 0.04 to 80 ng/ml (plateau rhIL-4 concentrations were 5 ng/ml or greater). In contrast, rhIL-4 failed to induce significant IgE synthesis at any of those doses of rhIL-4 in parallel MNC cultures performed in RPMI 1640 supplemented with FCS (RPMI 1640). Additional optimal conditions for the induction of IgE synthesis in this system were a MNC concentration of 1-2 X 10(6)/ml and a culture time of 18 days. Variability was noted in the amount of IgE produced by different donors (CV 0.22) and by the same donor when tested on different occasions (mean CV 0.21), but no donor's MNC failed to produce significant IgE in response to rhIL-4 when cultured in C-IMDM. The geometric mean IgE production induced by optimal IL-4 concentrations for the entire group of 16 subjects was 36.8 ng/ml IgE, with the lowest day 18 mean IgE concentration for any donor being 10.6 ng/ml and the highest 372.2 ng/ml. The enhanced rhIL-4-induced IgE synthesis supported by C-IMDM was due to the combined effects of the added enrichment factors and not to differences in the viabilities of MNC cultured in C-IMDM and RPMI 1640. This culture system will alleviate the problems of inconsistent and low quantities of IgE induced by IL-4 that confound most current culture systems used to examine rhIL-4-induced IgE synthesis. It will, thereby, facilitate further investigation of the regulation of human IgE synthesis.

Cytokine production by T helper cell subpopulations during prolonged in vitro stimulation.

In man, CD4+ T cells can be divided into phenotypically distinguishable subsets with different function whereas CD4+ T cells with the opposite pheno-CD45RO and low levels of CD45RA antigen provide help for mitogen-induced immunoglobulin production whereas CD4+ cells with the opposite phenotype suppress immunoglobulin production. However, studies examining cytokine production by phenotypically defined CD4+ T cell subsets have led to different conclusions. Further, very few studies have examined cytokine production by freshly isolated CD4+ T cell subsets during extended culture periods. Thus, we examined the production of several cytokines (at various time points) by CD4+ T cell subsets that were isolated in several ways, and stimulated with PWM, Con A, and PHA in a well-defined serum-free culture system. We found that CD4+, CD45RA- (or CD45RO+) T cells consistently produced the most IL-2, IFN-gamma, and TNF-alpha after mitogen stimulation for 2 days. PWM induced the largest quantities of each cytokine, although a similar pattern of production was observed in response to Con A and PHA. We were unable to detect IL-4 production by mononuclear cells and CD4+ T cell subsets suggesting that, if it is produced at all, IL-4 is produced in extremely small quantities. When the culture period of initially CD45RO- T cells was extended beyond 2 days, the culture supernatant contained increased quantities of each cytokine and the cells in the culture had an increased number of cells expressing CD45RO antigen. Together, these data indicate that CD4, CD45RA- (or CD45RO+) T cells in peripheral blood are the major producers of IL-2, IFN-gamma, and TNF-alpha following short-term mitogen stimulation, and that phenotypically defined peripheral blood T cell subsets do not maintain a distinct pattern of cytokines during extended culture periods.

Apoptosis: implications for inflammatory bowel disease.

Cells of the intestinal mucosa live in a harsh environment and therefore rely heavily on the highly regulated process of cell death, apoptosis, to maintain tissue integrity. Imbalance in the intracellular events that modulate apoptosis may contribute to the pathogenesis of inflammatory bowel disease.

Crohn's disease and ulcerative colitis mucosal T cells are stimulated by intestinal epithelial cells: implications for immunosuppressive therapy.

BACKGROUND: Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases, and their pathogenesis is attributed, in part, to alterations of the mucosal immune system. This study was designed to define the possible contribution of epithelial cells to the activation of lamina propria T lymphocytes (LPTs) in CD and UC. METHODS: LPTs isolated from CD, UC, and control surgical specimens were cocultured with freshly isolated allogeneic or autologous epithelial cells or epithelial cell lines. Resulting T-cell proliferation was evaluated by tritiated thymidine incorporation on day 5. RESULTS: When intestinal epithelial cells were used to stimulate mucosal T-cell proliferation, CD and UC LPTs were less responsive than control LPTs (p < 0.05 and p < 0.03, respectively). This difference between inflamed and control T cells was consistently observed by using a variety of different intestinal epithelial cell types. CONCLUSIONS: CD and UC mucosal T cells are hyporesponsive to activation by intestinal epithelial cells when compared with control LPTs. Elucidating the mechanism underlying the differential activation of CD and UC LPTs may help to better understand the immunopathogenesis of these conditions.

Selective resistance of mucosal T-cell activation to immunosuppression in Crohn's disease.

BACKGROUND & AIMS: The inappropriately high state of T-cell activation found in Crohn's disease could be due to failure to respond to inhibitory signals. We tested the hypothesis that Crohn's disease mucosal T-cells are resistant to the immunosuppressive action of interleukin4. PATIENTS: Patients with Crohn's disease, ulcerative colitis, and other malignant and non-malignant conditions undergoing bowel resection. METHODS: The effect of interleukin-4 on lamina propria mononuclear cells from Crohn's disease, ulcerative colitis and control mucosa was assessed on various T-cell functions: interleukin-2-induced cytotoxicity, soluble interleukin-2 receptor and interleukin-2 production, and expression of mRNA for interleukin-2R and interferon-gamma. RESULTS: Cytotoxicity of control and ulcerative colitis cells was markedly decreased by interleukin-4, whereas Crohn's disease cells failed to be inhibited. Addition of interleukin-4 to interleukin-2-stimulated cultures decreased soluble interleukin-2R production significantly less in Crohn's disease and ulcerative colitis than control cells. In the same cultures, residual levels of interleukin-2 were significantly increased in control and ulcerative colitis, but not Crohn's disease cultures. Finally, Crohn's disease cells were significantly more resistant to interleukin-4-mediated inhibition of spontaneous and interleukin-2-induced expression of interleukin-2Ralpha and interferon-gamma mRNA compared to control cells. CONCLUSIONS: The effector function, receptor expression and cytokine production of Crohn's disease mucosal T-cells are resistant to interleukin4-mediated inhibition. Failure to respond to down-regulatory signals may contribute to persistent T-cell activation and chronicity of inflammation in Crohn's disease.

Microbial biosorption of copper and lead from aqueous systems.

Biosorption of metal ions from aqueous systems was evaluated using a culture of acidic soil isolates grown in a completely mixed, aerobic, semi-batch culture reactor. The laboratory scale system was used to test single and bimetallic solutions of copper and lead with sulfates, chlorides, or nitrates. To elucidate the key factors influencing biosorption and to characterize metal uptake by cellular and extra cellular components of the microbial system, a dialysis testing procedure was developed. A direct contact technique was used to determine the rate of metal sorption on cellular surfaces. The effectiveness of biosorption was influenced by pH, initial metal concentrations, and anionic composition. Respirometric tests were carried out to identify potential inhibitory effects of metal accumulation on microbial oxygen uptake rates.

Biogeochemical assessment of natural attenuation of JP-4-contaminated ground water in the presence of fluorinated surfactants.

The biogeochemistry of the natural attenuation of petroleum-contaminated ground water was investigated in a field study. The focus of the study was a fire training site located on Tyndall Air Force Base in Florida. The site has been used by the Air Force for approximately 11 years in fire fighting exercises. An on-site above-ground tank of JP-4 provided fuel for setting controlled fires for the exercises. Various amounts of water and aqueous film forming foams (AFFF) were applied to extinguish the fires. The sources of contamination included leaks from pipelines transporting the fuel, leaks from an oil/water separator and runoff and percolation from the fire fighting activities. Previous investigations had identified jet fuel contamination at the site, however, no active remediation efforts have been conducted to date. The goal of this study was to use biogeochemical monitoring data to delineate redox zones within the site and to identify evidence of natural attenuation of JP-4 contamination. In addition to identifying several hydrocarbon metabolites, fluorinated surfactants (AFFF) were detected down-gradient of the hydrocarbon plume.

TGF-ß conditions intestinal T cells to express increased levels of miR-155, associated with down-regulation of IL-2 and itk mRNA.

Transforming growth factor (TGF)-ß, is an immunosuppressive cytokine that inhibits T-cell activation. We hypothesized that TGF-ß mediates its immunoinhibitory effects by modulation of micro RNA (miRNA)-155 (miR-155). Interleukin (IL)-2 and interferon-γ are down-regulated by TGF-ß in activated CD4 peripheral blood T cells and lamina propria T cells (LPT), but miR-155 is upregulated ninefold specifically in LPT. Consequently, this study focuses on the role of TGF-ß-enhanced miR-155 on LPT immune responses. TGF-ß induces miR-155 in both freshly isolated and LPT lymphoblasts, whereas other inducible miRNAs are not regulated by TGF-ß. Using MAMI bioinformatics database, we determined that inducible T-cell kinase (itk) is a functional target of miR-155 that exhibits an inverse mRNA response to that of miR-155. To determine experimentally that miR-155 regulates itk, transfection experiments were performed that demonstrated miR-155 overexpression decreased itk and IL-2 mRNA, whereas antagonism of miR-155 restored both mRNAs in activated cells. These findings describe a TGF-ß-dependent function for miR-155 in modulating cytokine and T-cell immune responses in the gut.

Compromised gastrointestinal integrity in pigtail macaques is associated with increased microbial translocation, immune activation, and IL-17 production in the absence of SIV infection.

Pigtail macaques (PTMs) rapidly progress to AIDS after simian immunodeficiency virus (SIV) infection. Given the strong association between human immunodeficiency virus (HIV) and SIV disease progression and microbial translocation and immune activation, we assessed whether high basal levels of immune activation and microbial translocation exist in PTMs. We found that before SIV infection, PTMs had high levels of microbial translocation that correlated with significant damage to the structural barrier of the gastrointestinal tract. Moreover, this increased microbial translocation correlated with high levels of immune activation and was associated with high frequencies of interleukin-17-producing T cells. These data highlight the relationship among mucosal damage, microbial translocation and systemic immune activation in the absence of SIV replication, and underscore the importance of microbial translocation in the rapid course of disease progression in SIV-infected PTMs. Furthermore, these data suggest that PTM may be an ideal model to study therapeutic interventions aimed at decreasing microbial translocation-induced immune activation.

Regulation of life and death in lamina propria T cells.

T cells are essential to initiation, amplification, and regulation of an immune response. This response is terminated when T cells undergo apoptosis, a physiological process of cell death triggered by various mechanisms and regulated by signaling pathways leading to enzymatic degradation of chromatin. An effective immune response depends on the proper balance between proliferation and death of activated T cells. This is particularly important in the intestine, where mucosal T cells are subjected to the high antigenic pressure of lumenal antigens and apoptosis is required to induce tolerance and maintain a state of 'physiological' inflammation. Insufficient apoptosis may result in excessive T cell retention and chronic intestinal inflammation, as seen in conditions associated with defective apoptosis of lamina propria T cells.

Immunology of inflammatory bowel disease.

The immunology of inflammatory bowel disease continues to be an intense area of investigation for clues to the pathogenesis of Crohn disease and ulcerative colitis. As typical with complex diseases, inflammatory bowel disease research is continuously evolving. Without abandoning traditional areas of study, such as humoral and cellular immunity and cytokines, investigation is broadening to explore new molecules and biologic phenomena. Novel cytokines and cell adhesion molecules appear to be involved in inflammation, while the role of nitric oxide is being clarified. Leukocyte resistance to apoptosis appears to be a major contributing factor to Crohn disease. Epithelial cell-derived defensins and receptors are arising as key molecules mediating the interaction of innate and acquired mucosal immunity with the enteric flora, and explaining how the latter participates in gut inflammation. The results of these combined studies are opening novel therapeutic horizons whose implementation offers better forms of treatment.

Mechanism of pokeweed mitogen inhibition of rhIL-4-induced human IgE synthesis.

Pokeweed mitogen (PWM) suppressed rhIL-4-induced IgE synthesis in a concentration-dependent manner. When rhIL-4 was present from Day 0, PWM added to cultures on Day 0 or 3 inhibited MNC IgE synthesis but not when it was added on Day 6 or later. The concentration of interferon-gamma (IFN-gamma) in MNC culture supernatants varied directly with the quantity of PWM added. Conversely, rhIL-4-stimulated MNC culture IgE concentrations varied inversely with the dose of PWM added and the IFN-gamma concentrations induced. The addition of a rabbit polyclonal neutralizing anti-human IFN-gamma antibody to rhIL-4 plus PWM-stimulated cultures partially or completely reversed PWM-induced inhibition of rhIL-4-induced IgE synthesis. PWM failed to inhibit rhIL-4-induced IgE synthesis by isolated B cells cocultured with monocytes and T cells from a clone unable to produce IFN-gamma message or protein. These findings are consistent with the postulate that PWM inhibits rhIL-4-induced IgE synthesis by inducing the production of IFN-gamma.

Recombination and postreplication repair.

The available data concerning postreplication repair are summarized. In Escherichia coli, recombination is implicated in this repair because the recA+ gene is necessary and because strand exchanges occur that extend over long regions. Other experiments involving phage-induced resistance also point to an interrelation between recombination and repair. In this phenomenon, gene products of lambda bacteriophage are introduced into bacteria, resulting in an increased resistance of the cells when they are subsequently exposed to X rays.

Recombinant human IL-4 induces IgE and IgG synthesis by normal and atopic donor mononuclear cells. Similar dose response, time course, requirement for T cells, and effect of pokeweed mitogen.

Unfractionated human blood mononuclear cells (MNC) from normal and atopic donors cultured in enriched Iscove's modified Dulbecco's medium supplemented with 10% FCS responded similarly to stimulation with purified human rIL-4 (rhIL-4) with respect to the concentration required to induce IgE synthesis and the magnitude and kinetics of the IgE response. The IgE response of MNC was IL-4 dose-dependent, increasing linearly with IL-4 concentrations between 0.2 and 2.5 ng/ml and plateauing at concentrations of 5 ng/ml or more. rhIL-4-induced IgE synthesis was first detected at 9 days after stimulation and supernatant IgE concentrations reached a maximum on day 18. rhIL-4 stimulated IgE synthesis by MNC from all donors tested, with peak supernatant IgE concentrations ranging from 3 to 372 ng/ml. The nonatopic group (n = 15) geometric mean peak concentration was 24.0 ng/ml and that of the atopic group (n = 19) was 20.0 ng/ml (p = NS). rhIL-4 also stimulated IgG synthesis by MNC from some (but not all) donors in quantities comparable to those induced by PWM. Maximum supernatant IgG concentrations in responders were found 18 days after stimulation. When PWM was added to the IL-4-stimulated cultures, it completely inhibited rhIL-4-induced IgE and IgG synthesis. rhIL-4 also completely inhibited PWM-induced IgG synthesis. Stimulation of IgE synthesis by rhIL-4 required the presence of T cells. T cell clone supernatants did not support rhIL-4-induced IgE synthesis by B cells. T cells from atopic and nonatopic donors restored rhIL-4-stimulated IgE synthesis by B cells from either source to a similar extent.

IL-4 induction of IgE class switching by lipopolysaccharide-activated murine B cells occurs predominantly through sequential switching.

Resting murine B cells activated with bacterial LPS co-express membrane (m)IgG1 and mIgE upon stimulation with IL-4. In this report, we combine both cellular and molecular approaches to elucidate the mechanism underlying this co-expression. We demonstrate that an anti-IgG1 antibody specifically and selectively inhibits IgE secretion (approximately 70%) by LPS + IL-4-stimulated B cells, which provides functional evidence for mIgG1 expression by precursors of IgE-secreting cells. The IgG1 and IgE secretory responses are separated temporally by approximately 16 h, with IgE production developing later than IgG1. A similar delay is observed in the appearance of mIgE+ cells suggesting that class switching to IgG1 precedes that to IgE. In the sort-purified, mIgG1+mIgE+ B cell population approximately 25% of cells expressed cytoplasmic (c) (secretory) IgG1 and approximately 15% expressed cIgE at the time of their isolation. However, only a small percent of the mIgG1+mIgE+ cells co-expressed cIgG1 and cIgE, further suggesting a temporal separation in IgG1 and IgE secretion within individual cells, but indicating that single cells can co-secrete these two Ig isotypes. Furthermore, the absolute level and rate of increase of IgG1 secretion by mIgG1+mIgE+ cells, upon their isolation and reculture, is lower than that for mIgG1+mIgE- cells suggesting a loss of CH gamma 1 expression in the former population. Analysis of total, unselected circular DNA excision products in LPS + IL-4-activated B cells demonstrates that most, if not all, of the DNA encoding the IgG1 constant heavy gene (CH gamma 1) (i.e., products of a class switch to IgE) have been rearranged. Collectively this data provides strong evidence at both the cellular and molecular level that the predominant mode of switching to IgE in response to in vitro stimulation by LPS + IL-4 is from IgM to IgG1 to IgE.

Mononuclear cells from patients with the hyper-IgE syndrome produce little IgE when they are stimulated with recombinant human interleukin-4.

To investigate whether B cells from patients with the hyper-IgE syndrome are more sensitive to the effects of interleukin-4 in vitro than B cells of normal or atopic individuals, we stimulated blood mononuclear cells (MNC) with varying doses of recombinant human interleukin 4 (rhIL-4) and measured supernatant IgE concentrations after 18 days of culture. Geometric mean spontaneous IgE synthesis after 18 days of culture without rhIL-4 was low (less than 3 ng/ml) and similar for MNCs from nine patients with the hyper-IgE syndrome, nine atopic and nine normal subjects. As found in our previous studies, MNCs from the nine atopic and the nine normal donors produced significant and similar quantities of IgE (geometric mean maximum IgE, 25.2 and 18.7 ng/ml, respectively) when MNCs were stimulated with rhIL-4. MNCs from both donor groups had similar sensitivity to the concentration of IL-4 eliciting the IgE response. In striking contrast, MNCs from the nine patients with the hyper-IgE syndrome failed to produce significant IgE over that produced spontaneously when MNCs were stimulated by a wide range of rhIL-4 concentrations. Coculture of B cell-enriched subpopulations from patients with the hyper-IgE syndrome with T cell-enriched subpopulations from nonatopic and atopic donors failed to restore responsiveness to rhIL-4. The addition of anti-CD40 monoclonal antibody to MNC cultures did result in enhancement of rhIL-4 IgE synthesis by MNCs from patients with the hyper-IgE syndrome, but the concentration of anti-CD40 required to elicit this enhancement was tenfold higher than for control MNCs.(ABSTRACT TRUNCATED AT 250 WORDS)