Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test.

The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

Mycobacterium tuberculosis epitope-specific interferon-g production in healthy Brazilians reactive and non-reactive to tuberculin skin test

The interferon (IFN)-γ response to peptides can be a useful diagnostic marker of Mycobacterium tuberculosis (MTB) latent infection. We identified promiscuous and potentially protective CD4+ T-cell epitopes from the most conserved regions of MTB antigenic proteins by scanning the MTB antigenic proteins GroEL2, phosphate-binding protein 1 precursor and 19 kDa antigen with the TEPITOPE algorithm. Seven peptide sequences predicted to bind to multiple human leukocyte antigen (HLA)-DR molecules were synthesised and tested with IFN-γ enzyme-linked immunospot (ELISPOT) assays using peripheral blood mononuclear cells (PBMCs) from 16 Mantoux tuberculin skin test (TST)-positive and 16 TST-negative healthy donors. Eighty-eight percent of TST-positive donors responded to at least one of the peptides, compared to 25% of TST-negative donors. Each individual peptide induced IFN-γ production by PBMCs from at least 31% of the TST-positive donors. The magnitude of the response against all peptides was 182 ± 230 x 106 IFN-γ spot forming cells (SFC) among TST-positive donors and 36 ± 62 x 106 SFC among TST-negative donors (p = 0.007). The response to GroEL2 (463-477) was only observed in the TST-positive group. This combination of novel MTB CD4 T-cell epitopes should be tested in a larger cohort of individuals with latent tuberculosis (TB) to evaluate its potential to diagnose latent TB and it may be included in ELISPOT-based IFN-γ assays to identify individuals with this condition.

Low levels of heat shock proteins-60 and -65 autoantibodies in Sjögren's syndrome.

BACKGROUND: Heat shock proteins are highly conserved immunodominant antigens found in various species. Humoral immune responses to mycobacterial HSP65 and human HSP60 have been established in a number of human autoimmune diseases. OBJECTIVE: To assess the prevalence of antibodies to HSP60 kDa and HSP65 kDa in patients with Sjögren's syndrome as compared to normal subjects. METHODS: Thirty-seven patients with SS were compared with normal controls. The antibodies against human HSP60 were measured by the Anti-Human (IgG/IgM) HSP60 ELISA kit. IgGs and IgMs to mycobacterial HSP65 were determined using an enzyme-linked immunosorbent assay with mycobacterial recombinant HSP65 antigens. RESULTS: The levels of both anti-HSP60 and -HSP65 were lower in patients compared with controls. IgG autoantibodies to HSP60 were significantly different between groups: 162 +/- 55.1 ng/ml in controls versus 112.3 +/- 30.6 ng/ml in SS patients (P < 0.001). The levels among controls of anti-HSP65 IgM isotype were also significantly higher than among the SS patients: 111.6 +/- 33.4 U/ml versus 96.1 +/- 8.9 U/ml (P= 0.01). CONCLUSIONS: The results of the present study show that the levels of different isotypes of anti- HSP60 and HSP65 antibodies were lower in patients with SS than in normal subjects. Additional studies in larger patient populations are required to evaluate the prevalence of these autoantibodies in SS patients.

The magnitude of the immune response to heat shock protein-65 following BCG immunisation is associated with the extent of experimental atherosclerosis.

Several studies have reported associations between coronary heart disease (CHD) and infection. Recent studies have implicated immune responses to heat shock protein(s) (HSP) as a contributary factor. Using an immunisation model, we have assessed the relationship between the immune responses to HSP and subsequent atherosclerosis. Rabbits were immunised with bacillus Calmette-Guerin (BCG) vaccine (n=10) or saline (n=10) and subsequently fed a 0.25-1.0% cholesterol diet for 10 weeks. Plasma levels of IgG specific for mycobacterial antigen A60 and human HSP-60, but not for human HSP-70, rose following BCG immunisation, reaching a peak after 8 weeks. The percentage aortic area covered by atherosclerotic plaque was greater in animals immunised with BCG (30.5+/-3.8) compared to saline treated animals (16.4+/-2.6) (P<0.05). Furthermore, the individual titres of anti-HSP-60 in the BCG-immunised animal antibodies at week 8 (prior to starting the cholesterol diet) correlated with the percentage aortic area covered by plaque after 18 weeks (R2=0.72; P<0.05). No correlation was found between anti-A60 antibody titres and plaque area. Antiserum from BCG-immunised, but not control, animals stained heat-shocked endothelial cells. These data suggest that immune responses to HSP may be implicated in the relationship between specific infections and CHD.

Expression of heat shock proteins 65, 72 and 90 in mercuric chloride-treated human peripheral blood mononuclear cells.

Peripheral blood mononuclear cells (PBMC) were challenged with different concentrations of mercuric chloride and the expression of heat shock protein (hsp) 65, 72 and 90, were investigated by an indirect peroxidase method as well as the proliferation was measured by uptake of tritiated thymidine into DNA. Hsp 65 immunoreactivity was peripherally located in the cells at a mercuric chloride concentration of 5.5-2.8x10(-5) mol/L, which also gave a decreased DNA synthesis. Hsp 72 positive cells, with a nuclear and cytoplasmic immunoreactivity, were found at a concentration of 5.5-2.2x10(-5) mol/L. Hsp 90 positive cells also with an immunoreactivity at a nuclear and cytoplasmic level, were found at 1.1x10(-5)-5.5x10(-6) mol/L. A stimulated DNA synthesis was obtained already at 2.2x10(-5) mol/L and was found down to 5.5x10(-6) mol/L. High concentrations of mercuric chloride might induce expression of hsp 65 and 72 as a protective mechanism. Hsp 90 as well as hsp 72 might be involved in the proliferation of human PBMC to mercuric chloride. Thus, this expression of hsp 72 may have a dual role.

Serum and salivary antibodies to a mycobacterial 65-kDa stress protein are elevated in HIV-positive patients and modified by oral candidiasis.

Serum immunoglobulin G (IgG) and IgA, and salivary IgA antibodies to a mycobacterial stress protein (mSP65) were determined in human immunodeficiency virus (HIV)-positive patients, acquired immunodeficiency syndrome (AIDS) patients and HIV-negative controls with or without oral candidiasis. Serum IgG antibodies were elevated in patients with HIV infection and AIDS and especially in subjects with candidiasis compared with controls (P < 0.02, P < 0.005). This was not apparent with serum IgA. In the absence of candidiasis, salivary IgA antibodies were elevated in HIV-positive patients compared with AIDS (P < 0.005) patients and healthy controls (P = 0.001). The relative avidity of serum IgG antibodies to mSP65 in controls with candidiasis was lower than healthy controls (P < 0.0001). In saliva there was a decrease in the relative avidity of IgA antibodies in AIDS patients with candidiasis compared with HIV patients (P < 0.03). In patients without candidiasis, the relative avidity was higher in HIV patients than healthy controls (P = 0.02). The results suggest that HIV infection leads to raised serum and salivary antibodies to heat shock proteins. Concurrent Candida infection may modify both the titer and relative avidity differently for serum and saliva.

Treatment of severe Raynaud's syndrome by injection of autologous blood pretreated by heating, ozonation and exposure to ultraviolet light (H-O-U) therapy.

OBJECTIVE: To determine the effect of re-injection of small samples of autologous blood, pretreated with heat, ozone and ultraviolet light (H-O-U therapy) in patients with severe Raynaud's syndrome. EXPERIMENTAL DESIGN: Open trial in 4 patients. SETTING: Temperature/humidity controlled vascular laboratory. PATIENTS: Severe Raynaud's syndrome of more than 5 years duration and defined as more than 5 attacks daily or 10 attacks in one week, at least half of which were painful and lasting for more than 30 minutes. Three patients were refractory to infusions of Iloprost. INTERVENTIONS: Patients were treated daily or on alternate days for a two to three weeks period by re-injection of citrated autologous blood pre-treated with heat, ozone and ultraviolet light (H-O-U therapy). MEASURES: Clinical observations; mean equilibrated hand temperature (infrared thermography); distributive and microcirculatory blood-flow (venous occlusion strain-gauge plethysmography, infrared photoplethysmography, laser Doppler flowmetry) iontophoresis of acetylcholine and sodium nitroprusside; estimations: serum levels of 6-keto-PGF1alpha and serum levels of anti-hsp65 antibody. RESULTS: Reduction or abolition of Raynaud's attacks for at least three months after treatment. Mean equilibrated hand temperature increased but did not normalise. Blood flow parameters improved but did not reach statistical significance. Iontophoresis of acetylcholine showed an increase in laser Doppler flowmetry which was statistically significant. Serum levels of 6-keto-PGF1alpha, fell significantly in three patients. Serum levels of anti-hsp65 antibody fell in the one patient which was followed sequentially. CONCLUSIONS: H-O-U therapy may prove useful in patients with severe Raynaud's syndrome.