High levels of inflammatory cytokines are associated with poor clinical response to steroid treatment and recurrent episodes of type 1 reactions in leprosy

Levels of leprosy antigen-induced interferon-gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) were measured in 96 leprosy patients with type 1 reactions (T1R) before, during and after a standard 12-week course of steroids. Peripheral blood mononuclear cells (PBMC) from leprosy patients with untreated T1R produced significantly more TNF-alpha than leprosy patients without T1R. Median levels of IFN-gamma and TNF-alpha in T1R patients fell during treatment with steroids; however, TNF-alpha levels increased as the steroid dose was reduced. Median IL-10 levels increased throughout the steroid treatment period and were associated strongly with TNF-alpha levels. Patients with high cytokine levels had a poorer recovery of sensory or voluntary muscle nerve function, a higher risk of reactivation of symptoms during steroid treatment, and a higher risk of another episode of T1R within 2 months of completing the steroid regimen. Rapid and effective reversal of the inflammatory process in T1R is critical to prevent permanent nerve damage from T1R and monitoring cytokine levels during treatment may be useful.     

The glycosphingolipid globoside as a serological marker on cytolytic T lymphocyte precursors and alloantigen-responsive proliferating T lymphocytes in murine spleen

Biochemical analyses of murine lymphocytes have shown that the glycosphingolipid globoside (Glo) is present exclusively on alloantigen-stimulated murine T lymphocytes (Gruner, K. R., Van Eijk, R. V. W. and Mühlradt, P. F., Biochemistry 1981. 20: 4518). An anti-Glo antibody has now been raised in rabbits immunized with purified antigen. Most activity was recovered in the IgM fraction. The specificity of the antibody was ascertained in an enzyme-linked immunosorbent assay with purified glycosphingolipids bound to the solid phase. In antibody-dependent complement lysis experiments the anti-Glo eliminated about 20% of nylon wool-nonadherent splenic T cells of CBA/J mice. To determine the functional identity of these Glo+ cells, the effects of Glo+ cell elimination on mitogen stimulation with concanavalin A and lipopolysaccharide, as well as the effects on the mixed lymphocyte culture (MLC) reaction and cell-mediated lympholysis with mitomycin-treated DBA/2 splenocytes as stimulator cells were studied. Whereas lipopolysaccharide stimulation was not affected by elimination of Glo+ cells, there was a slight inhibitory effect on the concanavalin A stimulation, and a severe inhibition of the MLC reaction and the generation of H-2d-specific cytolytic T lymphocytes. Addition of interleukin 2 increased the MLC reaction, but interleukin 2-saturated cultures were also severely inhibited by anti-Glo and complement treatment. Combined treatment with anti-Glo and anti-Lyt-1 or anti-Lyt-2 antibodies, and determination of cytolytic T lymphocyte precursor frequencies in limiting dilution cultures after Glo+ cell elimination showed that a large proportion of T cells proliferating in a primary MLC are Lyt-1+,2+,3+Glo+, whereas in secondary MLC they are Lyt-1+,2-,3-,Glo+. Fifty % of the cytolytic T lymphocyte precursors in primary as well as secondary MLC are Glo+. The Glo marker is lost upon differentiation to cytolytic T lymphocyte effector cells. It is discussed herein that Glo is a marker for alloantigen-stimulated precursor T lymphocytes of both helper and cytolytic T cells.     

Alteration of the cortisol-cortisone shuttle in leprosy type 1 reactions in leprosy patients in Hyderabad, India

Regulation of inflammation in leprosy may be influenced by local concentrations of active cortisol and inactive cortisone, whose concentrations are regulated by enzymes in the cortisol-cortisone shuttle. We investigated the cortisol-cortisone shuttle enzymes in the skin of leprosy patients with type 1 reactions (T1R), which are characterised by skin and nerve inflammation. Gene expression of the shuttle enzymes were quantified in skin biopsies from 15 leprosy patients with new T1R before and during prednisolone treatment and compared with levels in skin biopsies from 10 borderline leprosy patients without reactions. Gene expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2, which converts cortisol to cortisone, is down-regulated in skin from T1R lesions. However expression levels of 11beta-HSD type 1, which converts cortisone to cortisol, were similar in skin with and without reactions and did not change during anti-leprosy drug treatment. Prednisolone treatment of patients with reactions is associated with an upregulation of 11beta-HSD2 expression in skin. The down regulation of 11beta-HSD2 at the beginning of a reaction may be caused by pro-inflammatory cytokines in the leprosy reactional lesion and may be a local attempt to down-regulate inflammation. However in leprosy reactions this local response is insufficient and exogenous steroids are required to control inflammation.     

Suppression and prevention of adjuvant arthritis in rats by a monoclonal antibody to the alpha/beta T cell receptor.

Adjuvant arthritis (AA) in rats is an experimentally induced autoimmune disease mediated by T lymphocytes specific for Mycobacterium tuberculosis. We raised the question whether T cells carrying the gamma/delta T cell receptor (TcR), reactive or not to mycobacterial antigens, are involved in the pathogenesis of AA. For this purpose, T cells bearing the TcR alpha/beta were depleted from circulation by treatment with a monoclonal antibody against the rat TcR alpha/beta (R73). This treatment efficiently suppressed existing disease. Even more efficient was pretreatment with R73 from birth, which prevented AA induction completely. In these alpha/beta+ T cell-depleted animals an elevated level of alpha/beta- T cells (about 15% vs. 1% in normal rats) was evident, which was not significantly increased by Mycobacterium tuberculosis injection. We found no positive evidence that gamma/delta + T cells do contribute to AA induction. Moreover, treatment with an anti-TcR alpha/beta monoclonal antibody may be very efficient treatment of T cell-mediated autoimmune diseases.     

Suppression of the local graft-vs.-host reaction in rats by treatment with a monoclonal antibody specific for the interleukin 2 receptor

Local graft-vs.-host reaction (GVHR) was induced in rats by injecting parental cells into young F1 recipients. As a consequence of antigenic stimulation in the course of developing GVHR in the responding lymph nodes, the number of interleukin 2-receptor (IL 2R)-bearing T cells increased from less than 1% up to 10% of the total population. The IL 2R-bearing cells were located mainly in the T cell areas of the reactive lymph nodes. As assessed by the determination of the GVHR indices, treatment of the recipients with anti-T-helper subset-specific mAb (W3/25) or with anti-IL 2R mAb (ART-18) inhibited the GVHR. In parallel, the number of IL 2R-bearing cells was reduced to the normal levels. W3/25 mAb treatment changed the helper/suppressor subset ratio and reduced the number of circulating lymphocytes in the peripheral blood. In contrast, ART-18 mAb treatment did not induce any detectable changes in the subset distribution and it did not affect the number of circulating lymphocytes. The results demonstrate the key role that the IL 2R-positive cells play in the proliferative phase of acute GVHR, and favor the use of anti-IL 2R mAb as selective immunosuppressive agents.     

A DNA vaccine encoding a chimeric allergen derived from major group 1 allergens of dust mite can be used for specific immunotherapy

Immunization with DNA-based constructs has been shown to be against the antigen and the response is skewed in such a way as to ameliorate the symptoms of allergic disease. This approach is particularly useful in the treatment of allergic inflammatory diseases, such as asthma. The major group 1 allergen from house dust mites is one of the triggers of allergic asthma. This study explores whether a chimeric gene R8, derived from the major group 1 allergen of house dust mite species (Dermatophagoides farinae and Dermatophagoides pteronyssinus), can be expressed in Human Embryonic Kidney 293 cells (HEK 293T) and whether such a construct can be used as a DNA vaccine in asthma therapy. The eukaryotic expression vector pcDNA3.1 was used to express the R8 molecule in HEK 293T cells and successful expression of R8 was confirmed using a fluorescence microscope and western blot analysis. The efficacy of R8 as DNA vaccine was also assessed in a mouse asthma model. The in vivo data showed that R8 rectified the TH1/TH2 imbalance typical of allergic inflammation and stimulated the proliferation of regulatory T (Treg) cells. Immunization with the R8 construct also decreased serum allergen-specific IgE production in this mouse asthma model. Our findings suggest that R8 may be a feasible potential DNA vaccine for specific immunotherapy (SIT) in the treatment of allergic asthma.     

IgE receptors on human lymphocytes. II. Detection of cells bearing IgE receptors in unstimulated mononuclear cells by means of a monoclonal antibody

A monoclonal antibody (mAb) specific for lymphocyte IgE receptors (ER) was employed in a rosette assay for the detection of cells bearing IgE receptors (Fc epsilon R). The specificity of the assay was documented by inhibition studies with soluble immunoglobulins (Ig) and anti-Ig antibodies. Moreover, similar results were obtained by employing the F(ab')2 fragment of mAbER instead of intact molecule. Circulating mononuclear cells isolated from normal or allergic adults and from umbilical cord blood contained approximately 8% of Fc epsilon R-bearing cells with values ranging from 0.3 to 17%. Tonsillar lymphocytes contained about 30% of Fc epsilon R+ cells. After the removal of adherent cells, there was a small but significant reduction of the proportion of Fc epsilon R+ cells. When mononuclear cells were separated into T and B cell fractions by two-cycle rosetting with 2-aminoethylisothiouronium bromide hydrobromide-treated sheep red blood cells, most of the Fc epsilon R+ cells were in the B cell fraction; however, a small proportion of Fc epsilon R+ was also found in the enriched T cells and double-labeling experiments confirmed that these cells were indeed T lymphocytes. Fc epsilon R+ cells were purified by rosetting with mAbER-coated erythrocytes and their phenotype was compared to that of Fc epsilon R- cells; Fc epsilon R+ cells contained about 90% of B cells (B1+) together with a small proportion of OKT3+, Leu 7+ and Mo2+ cells. The bulk of T cells, macrophages and natural killer (NK) cells was found in the Fc epsilon R- cells which contained fewer B cells than the fraction of Fc epsilon R+ cells. These data thus indicated that the great majority of Fc epsilon R-bearing cells are B cells but that a small proportion of NK cells, macrophages and T lymphocytes also express Fc epsilon R. Upon incubation at 37 degrees C, B cells lost their Fc epsilon R and this phenomenon was selectively inhibited by IgE; however, purified T cells seemed to express more Fc epsilon R after overnight incubation at 37 degrees C and this was not influenced by IgE. It is finally shown that the expression of Fc epsilon R is cyclic and that Fc epsilon R-bearing B cells do not represent a functionally distinct subpopulation of B lymphocytes.     

Longitudinal Immune Responses and Gene Expression Profiles in Type 1 Leprosy Reactions

Purpose

Leprosy, a chronic disease initiated by Mycobacterium leprae, is often complicated by acute inflammatory reactions. Although such episodes occur in at least 50 % of all leprosy patients and may cause irreversible nerve damage, no laboratory tests are available for early diagnosis or prediction of reactions. Since immune- and genetic host factors are critical in leprosy reactions, we hypothesize that identification of host-derived biomarkers correlated to leprosy reactions can provide the basis for new tests to facilitate timely diagnosis and treatment thereby helping to prevent tissue damage.

Methods

The longitudinal host response of a leprosy patient, who was affected by a type 1 reaction (T1R) after MDT-treatment, was studied in unprecedented detail, measuring cellular and humoral immunity and gene expression profiles to identify biomarkers specific for T1R.

Results

Cytokine analysis in response to M. leprae revealed increased production of IFN-γ, IP-10, CXCL9, IL-17A and VEGF at diagnosis of T1R compared to before T1R, whereas a simultaneous decrease in IL-10 and G-CSF was observed at T1R. Cytokines shifts coincided with a reduction in known regulatory CD39⁺CCL4⁺ and CD25^high T-cell subsets. Moreover, RNA expression profiles revealed that IFN-induced genes, (V)EGF, and genes associated with cytotoxic T-cell responses (GNLY, GZMA/B, PRF1) were upregulated during T1R, whereas expression of T-cell regulation-associated genes were decreased.

Conclusions

These data show that increased inflammation, vasculoneogenesis and cytotoxicity, perturbed T-cell regulation as well as IFN-induced genes play an important role in T1R and provide potential T1R-specific host biomarkers.     

During HIV-1 infection most blood dendritic cells are not productively infected and can induce allogeneic CD4+ T cells clonal expansion.

We have considered the possibility that antigen-presenting cells of the dendritic cell lineage may be infected in vivo and spread HIV-1 at the time dendritic cells initiate the clonal expansion of antigen-specific T cells. Dendritic cells were isolated from 25 HIV-1-infected subjects (CDC stages II-IV). Fewer dendritic cells were recovered from most infected subjects. Reduced numbers of total non-T cells were also found in these patients, so that preferential loss of dendritic cells did not occur. Dendritic cell function was assessed by stimulatory capacity for allogeneic CD4+ T cells in the mixed leucocyte reaction (MLR). Potent MLR stimulator activity was retained in the dendritic cell-enriched populations from HIV-infected patients. Seven out of nine patients without AIDS (asymptomatic, lymphadenopathy or ARC) and three out of six patients with AIDS had proliferative responses equivalent to those induced by dendritic cells from controls. Dendritic cells from HIV+ subjects were able to initiate the expansion of allogeneic CD4+ T cell clones with cloning efficiency not different from controls and without evidence of cytopathic effect in the expanding CD4+ clones. In situ hybridization of the different mononuclear cell populations with a gag-specific riboprobe demonstrated positive cells in the T cell fractions of 12 of the 15 patients tested. None of the asymptomatic or ARC patients had riboprobe-positive cells in the dendritic cell-enriched populations. Four out of nine patients with AIDS had cells positive for HIV-1 expression in the dendritic cell-enriched fraction. However, the positive cells had the nuclear profile of lymphocytes, and by cytofluorography some residual low-density T cells were present. By limiting dilution and polymerase chain reaction (PCR), CD4+ lymphocytes carried HIV provirus in inocula of 500-5000 cells, while provirus could only be detected in 50,000 cells from the dendritic cell-enriched fraction. The latter signal may be due to the demonstrated levels of T cell contamination. Our data indicate that productive or latent HIV-1 infection of blood dendritic cells in vivo is rare, certainly no greater than in T lymphocytes, and that in vitro dendritic cell preparations from patients can expand CD4+ T cells efficiently and therefore may be able to expand T cells with immunotherapeutic activity.     

Effects of prednisolone treatment on cytokine expression in patients with leprosy type 1 reactions

Leprosy type 1 reactions (T1R) are due to increased cell-mediated immunity and result in localized tissue damage. The anti-inflammatory drug prednisolone is used for treatment, but there is little good in vivo data on the molecular actions of prednisolone. We investigated the effect of prednisolone treatment on tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-10, and transforming growth factor beta1 (TGF-beta1) mRNA and protein expression in blood and skin biopsies from 30 patients with T1R in India. After 1 month of prednisolone treatment the sizes of the skin granulomas were reduced, as were the grades of cells positive for TNF-alpha and IL-10 in skin lesions. Increased production of TGF-beta1 was seen in skin lesions after 6 months of prednisolone treatment. Expression of mRNA for TNF-alpha, IL-1beta, and TGF-beta1 was reduced, whereas no change in IL-10 mRNA expression was detected during treatment. The circulating cytokine profiles were similar in patients with and without T1R, and prednisolone treatment had no detectable effects on cytokine expression in the blood. The data emphasize the compartmentalization of pathology in T1R and the importance of the immune response in the skin. Clinical improvement and cytokine expression were compared. Surprisingly, patients with improved skin and nerve function and patients with nonimproved skin and nerve function had similar cytokine profiles, suggesting that clinical improvement is not directly mediated by the cytokines studied here. This in vivo well-controlled study of the immunosuppressive effects of prednisolone showed that the drug does not switch off cytokine responses effectively.     

Interleukin 2 induces a transient downregulation of protein phosphatase 1 and 2A activity in human T cells

Stimulation of human CD4* T cell lines with interleukin 2 (IL-2) induces tyrosine, serine and threonine phosphorylation of a series of proteins involved in the IL-2 receptor (IL-2R) signaling pathway. Here, we examined whether IL-2 induces changes in the activity of protein serine/threonine phosphatases in antigen specific, CD4* human T cell lines. Using inhibitors of protein phosphatases 1 (PP1), PP2A, and PP2B, we provide evidence, that IL-2 induces a downregulation of PP activity in the cytoplasmic/membrane fraction. Thus, IL-2R ligation for 30 min triggers a 16 percent decrease in total PP2A activity (p<0.00005, n =17) and a seven percent decrease in PP1 activity (p<0.00005, n =17). Cytokine-induced downregulation of PP2A activity reaches a maximum 60 min after IL-2R ligation, and returns to baseline levels within two hours. Downregulation of PP1 activity reaches a maximum after 30 min and is largely reversed one hour after IL-2 stimulation. As determined from immunoblotting experiments using a specific anti-PP1 or anti-PP2A antibody, the amount of PP1 and PP2A recovered from cytosolic/membrane fraction remains unchanged after IL-2 treatment suggesting that the drop in PP1/PP2A activity might be due to a regulatory change rather than to a change in the amount of PP1 and PP2A. In conclusion, we provide evidence, for the first time, that IL-2 induces a transient downregulation of PP2A activity in T cells. In addition, our findings indicate that cytoplasmic PPl activity is transiently downregulated following IL-2R ligation in antigen-specific, human CD4* T cells.     

肋横突关节关节面的形态特点及其意义

目的　通过对成人肋横突关节骨性关节面的形态学测量,探讨其形态特征规律为临床诊疗、置钉设计及相关生物力学研究提供理论依据。 方法 选取40套（80侧）成人胸廓干骨标本,进行相关指标观测,并按不同节段统计分析。 结果　横突肋凹横槽形仅见于T1和T2,凹面形T1~4渐增,平面形T5~9渐增,T11、T12基本无肋凹;横突肋凹上位T4~8渐增,中位T1~4渐增,T5~9由63.7%渐减到11.3%,下位T1~7渐减;肋结节关节面平面型呈正态分布,凸面型除R7外,均占23.0%~50.0%,凹面型R1、R2分别为27.5%、33.8%,其余分布极少,R11、R12基本无关节面;肋结节关节面仅R1出现上位,占10.0%,中位R1占80.0%,R3~11占3.8%~26.3%,下位R2~10占60%~96.2%。 结论　椎骨横突肋凹由凹到平、位置逐渐上移;肋结节关节面由凸变平位置逐渐下移;临床应用应根据1~12肋的相关参数选取适合的治疗方案。     

A Single-tube multiplex system for the simultaneous detection of 10 common cystic fibrosis mutations

We describe a nonisotopic single-tube polymerase chain reaction (PCR) multiplex system that detects 10 of the more common cystic fibrosis (CF) mutations (ΔF508, ΔI507, V520F, G551D, G542X, R553X, R117H, 621 + lG→T, N1303K, A455E). The use of this method detects approximately 90% of the CF alleles in the British population. Five exons of the CF gene are amplified simultaneously by PCR, followed by an overnight triple enzyme restriction digest, and then resolved by high-resolution gel acrylamide electrophoresis. © 1995 Wiley-Liss, Inc.     

Immunoregulatory Factor Released From a Cell Line Derived From Human Decidual Tissue

ABSTRACT: The culture supernatant of the TTK-1 cell line, established from human decidual tissue, was found to contain a factor that strongly suppressed the mixed lymphocyte reaction (MLR). The mechanism of the MLR-suppressive activity as well as the biochemical characterization of this factor was analyzed. The TTK-1 supernatant suppressed the MLR much more strongly than the culture supernatants of the three other malignant cell lines examined. The molecular weight of this factor was estimated to be between 43 kilodaltons (kd) and 67 kd by gel filtration chromatography. The TTK-1 supernatant also suppressed the proliferation of the interleukin 2 (IL-2)-dependent T cell lines, but did not suppress that of the IL-2-independent T cell lines, suggesting that the TTK-1 supernatant inhibited the action of IL-2 and subsequently suppressed the MLR. The fact that the TTK-1 cell line originated from human decidual tissue might imply the important role of this factor in immunological fetomaternal balance.     

Fel d 1-derived T cell peptide therapy induces recruitment of CD4+CD25+; CD4+ interferon-γ+ T helper type 1 cells to sites of allergen-induced late-phase skin reactions in cat-allergic subjects

BACKGROUND: Specific immunotherapy with whole allergen extracts is associated with local accumulation of IFN-gamma+ and CD25+ cells indicating recruitment of activated T-helper type 1 (Th1) and/or T regulatory cells. We have studied allergen-induced, late-phase skin biopsies before and after T cell peptide therapy for evidence of alterations in the pattern of local recruitment of Th1, T-helper type 2 (Th2) and T regulatory cells. OBJECTIVE: To evaluate the effect of T cell peptide therapy on the allergen-induced cutaneous late-phase reaction. METHODS: Increasing doses of synthetic Fel d 1-derived peptides were administered (by intradermal injection) to eight cat-allergic asthmatics at 14-day intervals. Twenty-four-hour skin biopsies were taken from whole cat allergen- and diluent-injected sites, before and after treatment and studied by immunohistochemistry and in situ hybridization. RESULTS: Fel-d 1 peptides decreased airway hyper-responsiveness (P = 0.02) and inhibited the late-phase cutaneous reaction (LPCR) to whole cat allergen (P = 0.03). This was associated with significant increases (post- vs. pre-treatment) in the number of cutaneous CD4+/IFN-gamma+ (P = 0.03) and CD4+/CD25+ cells (P = 0.04), but not in CD4+/IL-10+ or CD4+/CTLA-4+ cells. CONCLUSIONS: Treatment with allergen-derived T cell peptides results in allergen-dependent recruitment to the skin of Th1, rather than T regulatory cells, to cutaneous late-phase reaction sites.