Analysis of PTPN22, ZFAT and MYO9B polymorphisms in Turner Syndrome and risk of autoimmune disease.

Turner syndrome (TS) is one of the most common sexual chromosome abnormalities and is clearly associated with an increased risk of autoimmune diseases, particularly thyroid disease and coeliac disease (CD). Single-nucleotide polymorphism analyses have been shown to provide correlative evidence that specific genes are associated with autoimmune disease. Our aim was to study the functional polymorphic variants of PTPN22 and ZFAT in relation to thyroid disease and those of MYO9B in relation to CD. A cross-sectional comparative analysis was performed on Mexican mestizo patients with TS and age-matched healthy females. Our data showed that PTPN22 C1858T (considered a risk variant) is not associated with TS (X2  = 3.50, p = .61, and OR = 0.33 [95% CI = 0.10-1.10]). Also, ZFAT was not associated with TS (X2  = 1.2, p = .28, and OR = 1.22 [95% CI = 0.84-1.79]). However, for the first time, rs2305767 MYO9B was revealed to have a strong association with TS (X2  = 58.6, p = .0001, and OR = 10.44 [95% C = 5.51-19.80]), supporting a high level of predisposition to CD among TS patients. This report addresses additional data regarding the polymorphic variants associated with autoimmune disease, one of the most common complications in TS.

[Evaluation of visceral adipose tissue and lipid level in premenopausal and postmenopausal women's]. / Evaluación del tejido adiposo visceral y de la concentración de lípidos en mujeres pre y posmenopáusicas.

BACKGROUND: Postmenopausal women present weight gain and intensification of obesity, especially visceral adipose tissue (VAT) increases in postmenopausal women. But it is still not clear whether abdominal fat increases during this stage independently of body weight. OBJECTIVE: compare the VAT and lipid profile between postmenopausal and premenopausal Mexican women. METHODS: A case control study in postmenopausal women matched for BMI with premenopausal women. Anthropometric and laboratory measurements as well as body composition analysis were performed. RESULTS: VAT was increased in postmenopausal women in contrast with premenopausal women (114.8 ± 39.5 vs 97.3 ± 29.0, p<0.05). Compared with premenopausal women, postmenopausal women showed higher total cholesterol (231 .6 ± 56.1 vs 206.8 ± 29.5 p <0.05), and LDL-cholesterol levels (145.9 ± 48.3 vs 121.7 ± 34.1, p < 0.05), whereas H DL-cholesterol remained unchanged. CONCLUSION: The results of the present study have demonstrated that Mexican postmenopausal women had a significant increment in visceral adipose tissue and in other metabolic risk factors, independent of the body mass index.

Lymphocytes and B-cell abnormalities in patients with common variable immunodeficiency (CVID).

BACKGROUND AND AIMS: Common variable immunodeficiency (CVID) is a primary antibody deficiency characterised by decreased antibody production and low or normal B-cell numbers. To elucidate the clinical and immunological heterogeneity of CVID, we studied 16 patients diagnosed with CVID. METHODS: We analysed B, T and NK cell populations. We also assessed CD27 expression to define B-cell subsets and examined the expression of molecules important in B-cell proliferation and differentiation, such as the transmembrane activator and CALM interactor (TACI), inducible costimulator (ICOS), CD154 and CD40. RESULTS: We observed reduced B and T-cell numbers in CVID patients; this reduction was more pronounced in adults. While one group of patients (group I) showed a significant reduction in CD27+ memory B-cells, another group (group II) of patients exhibited numbers of CD27+ memory B-cells similar to the healthy donor. The frequency of B-cells and T-cells expressing CD40 and ICOS, respectively, was significantly lower in all CVID patients compared with healthy donors. Finally, a correlation between the frequency of CD27+ memory B-cells and clinical features was observed in CVID patients. CONCLUSION: These results suggest that in some patients, the combined defects in both T and B-cells may account for CVID. Additionally, patients in group I exhibited an increased frequency of pneumonia and chronic diarrhoea.

C6 knock-out mice are protected from thrombophilia mediated by antiphospholipid antibodies.

BACKGROUND: Complement activation plays a role in pathogenesis of the antiphospholipid syndrome (APS), but the involvement of the C5b-9 membrane attack complex (MAC) is unknown. Here we studied the effects of human polyclonal antiphospholipid (aPL) antibodies on thrombosis and tissue factor (TF) up-regulation in C6 deficient (C6(-/-)) mice. METHODS: C6(-/-) mice or the wild-type C3H/HeJ (C6(+/+)) mice were injected twice with IgG-APS (n = 2) or IgM-APS (n = 1) isolated from APS patients or with the corresponding control immunoglobulins (Igs) of normal human serum, (NHS) (IgG-NHS or IgM-NHS). Then, the sizes of induced thrombi in the femoral vein were determined 72 hours after the first injection. Tissue factor was determined in homogenates of carotid arteries and in peritoneal macrophages. RESULTS: Thrombus sizes were significantly larger in C6(+/+) treated with IgG-APS1 or with IgG-APS2 or with IgM-APS when compared with C6(+/+) mice treated with IgG-NHS or with IgM-NHS, respectively. The sizes of thrombi were significantly smaller in the C6(-/-) mice injected with IgG-APS1, IgG-APS2 or IgM-APS (p < 0.001), compared to their C6(+/+) counterparts showing an important abrogation of thrombus formation in mice lacking C6. The TF expression and activity in the C6(-/-) mice treated with IgG-APS or IgM-APS were diminished when compared to C3H/HeJ (C6(+/+)) mice treated with the same Igs. All mice injected with IgG-APS and IgM-APS had medium-high titers of anticardiolipin (aCL) and anti-ß(2)glycoprotein I (aß(2)GPI) antibodies. CONCLUSIONS: These data indicate that the C6 component of the complement system mediates aPL-thrombogenic effects, underscoring an important pathogenic mechanism and indicating the possibility of inhibiting complement to ameliorate APS-related manifestations.

Antibodies against recombinant heat shock proteins of 60 kDa from enterobacteria in the sera and synovial fluid of HLA-B27 positive ankylosing spondylitis patients.

OBJECTIVE: To study the association of HLA-B27 with IgG antibodies to different enterobacterial HSP60s in patients with ankylosing spondylitis (AS). METHODS: IgG antibodies to 60 kDa enterobacterial HSPs were determined by ELISA in paired samples of sera and synovial fluid from 21 HLA-B27+ ankylosing spondylitis (AS) patients; and in sera from 32 HLA-B27+ AS patients, 35 HLA-B27+ healthy relatives of AS patients, and 60 HLA-B27- healthy individuals with no family members with AS. RESULTS: HLA-B27+ patients and healthy individuals showed significantly higher IgG antibody levels to recombinant enterobacterial HSP60s than HLA-B27- healthy controls. The levels of anti-HSP60Sf and anti-HSP60Ec antibodies correlated with disease activity and anti-HSP60Ec antibodies with male gender. No association between enterobacterial HSP60 antibody levels and disease duration was observed. All groups had lower levels of IgG antibodies to rHSP60 from Streptococcus pyogenes (rHSP60 Spy). In paired samples of sera and synovial fluid from B27+ patients, IgG antibodies to enterobacterial HSP60s were detected, but in significantly higher levels in sera than in synovial fluid. The anti-rHSPSpy IgG response in these samples was lower and similar in the three groups. CONCLUSIONS: A correlation was found between HLA-B27 and the response to recombinat enterobacterial HSP60s. This response could be associated with disease activitir and gender in some proteins and the presence eof IgG antibodies to these proteins in synovial fluid could be associated with the inflammatory process and initiation of AS.

In vivo effects of an inhibitor of nuclear factor-kappa B on thrombogenic properties of antiphospholipid antibodies.

It has been shown that endothelial cell (EC) activation and tissue factor (TF) upregulation in EC and monocytes by antiphospholipid antibodies (aPL Abs) leads to a prothrombotic state and involves translocation of nuclear factor-kappa B (NF-kappaB). Here we examined the effects of an NF-kappaB inhibitor on aPL-induced thrombosis, TF activity, and EC in vivo. We treated CD1 mice with IgG from a patient with antiphospholipid syndrome (IgG-APS) or with control IgG (IgG-NHS). The adhesion of leukocytes (number of white blood cells) to EC in cremaster muscle (as an indication of EC activation) as well as the size of an induced thrombus in the femoral vein of the mice were examined. Some mice in each group were infused with 10 microM MG132 (an inhibitor of NF-kappaB). TF activity was determined using a chromogenic assay in homogenates of carotid arteries and in peritoneal cells of mice. In vivo, IgG-APS increased significantly the number of white blood cells adhering to ECs (4.7 +/- 2.2) when compared to control mice (1.5 +/- 0.8), and these effects were significantly reduced when mice were pretreated with MG132 (0.8 +/- 0.2). IgG-APS increased significantly the thrombus size and MG132 inhibited that effect (93%). Treatment of the mice with IgG-APS also induced significantly increased TF function in peritoneal cells and in homogenates of carotid arteries. Pretreatment of the mice with MG132 abrogated those effects significantly. Mice injected with IgG-APS or with IgM-APS with or without the inhibitor had medium-high titers of anticardiolipin antibodies in serum at the time of the surgical procedures. The data show that prothrombotic and proinflammatory properties of IgG-APS and IgM-APS are downregulated in vivo by an NF-kappaB inhibitor. These findings may be important in designing new modalities of targeted therapies to treat thrombosis in patients with APS.

CD4+ CD25+ lymphocyte and dendritic cell mobilization with intermediate doses of recombinant human granulocyte colony-stimulating factor in healthy donors.

We prospectively conducted a quantitative and phenotypic analysis of T, B, natural killer (NK), NKT, type 1 and 2 dendritic cells (DC), and regulatory T cells, before and after mobilization with intermediate doses of granulocyte colony-stimulating factor (G-CSF) (16 microg/kg per day). Between November, 2003, and December, 2004, we collected stem cells from 25 HLA identical sibling donors for allogeneic hematopoietic stem cell transplantation. Before mobilization and 3 h after the fourth and fifth doses of G-CSF, blood samples were taken for blood counts and flow cytometry. The median number of regulatory T cells before and after G-CSF was statistically different (69 +/- 41 x 10(6)/L versus 161 +/- 159 x 10(6)/L, p < 0.01). We observed a 1.7-fold increase in NK and NKT cells (p < 0.009 and p < 0.02, respectively). DC were mobilized with a 11.5-fold increase in type 2 (p < 0.004) and a 8.5-fold increase in type 1 DC (p < 0.003). The patients received a mean of: 2.2 x 10(7)/kg +/- 1.4 x 10(7)/kg of NK cells, 0.95 x 10(7)/kg +/- 0.81 x 107/kg of NKT cells, 0.43 x 107/kg +/- 0.53 x 10(7)/kg of type 1 DC, 0.3 v 10(7)/kg +/- 0.45 x 10(7)/kg of type 2 DC and 1.4 x 10(7)/kg +/- 1.2 x 10(7)/kg of regulatory T cells. Using intermediate doses of G-CSF, we have demonstrated the mobilization of different lymphocyte subsets, in particular regulatory T cells and DC, which can be expanded later and used in the treatment of cancer and autoimmune diseases.

TLRs and NODs mRNA expression pattern in healthy mouse eye.

AIMS: To look for TLR and NOD mRNA expression in the healthy eye and in other immune privileged and non-immune privileged mouse organs. METHODS: Semiquantitative RT-PCR was performed to look for TLR1-9 and NOD1 and NOD2 mRNA expressions in the whole eye, in the anterior (AP) and posterior (PP) portions of the eye, in corneal fibroblasts (CF) and in ovary, brain, testis, heart, lung, and spleen. RESULTS: All the TLR mRNAs were expressed in the whole eye of Balb/c mice. NIH and C57BL/6 did not express TLR9 and TLR8, respectively. NIH expressed higher levels of TLR1, 2, 3, and 6 than the other strains. C57BL/6 expressed the lowest levels of all TLRs. TLR9, 5, and 4 were the less expressed in all strains. All TLRs were expressed in Balb/c PP and TLR1 was not expressed in AP. In NIH and Balb/c CF the majority of TLRs were overexpressed with LPS. In testis, expression of most TLRs was absent. Non-immune privileged organs expressed most of the TLRs. All the organs expressed NOD1 and NOD2. In PP NOD2 was not expressed. CONCLUSION: TLRs and NODs are expressed in the eye, and could have an important role in the innate immunity.

Klebsiella pneumoniae 35 and 36 kDa porins are common antigens in different serotypes and induce opsonizing antibodies.

BACKGROUND: Klebsiella pneumoniae is a major cause of neonatal sepsis and nosocomial infections in Mexico. Antibiotic therapy is the first choice for treatment but the increase in multiple resistance strains has forced scientists to look for alternative treatments, such as immunotherapy. In this work, we propose that porins could be a common antigen among four different capsular serotypes of Klebsiella pneumoniae for the production of immune sera with opsonizing capacity. METHODS: The 35 and 36 kDa porins from four different serotypes of the bacteria were isolated by the Nikaido method followed by purification in Sephacryl column chromatography. The 36 kDa of serotype K8 was further purified by electroelution. The 35 and 36 kDa porins were used to obtain rabbit polyclonal antibodies (PolyAb) to the four serotypes and the 36 kDa from K8 for the production of monoclonal antibodies (MoAb). Antigenic reactivity of PolyAb and MoAb were analyzed by ELISA and WB and their opsonizing capacity for human PMN was measured by chemiluminescence (CL) using capsulated and non-capsulated bacteria. RESULTS: Porins from the four strains showe electrophoretic homology and cross reaction by ELISA and WB. CL assays indicated that PolyAb opsonized heterologous strains and that MoAb perform this in the absence of capsule. CONCLUSIONS: K.pneumoniae 35 and 36 kDa porins are common antigens for the four serotypes studied and induce opsonizing antibodies.

Opsonic capacity of a hyperimmune serum against outer membrane proteins of Klebsiella pneumoniae.

The opsonic capacity of a hyperimmune rabbit serum against a porin-rich outer membrane protein preparation of a strain of K. pneumoniae was evaluated. By immunoblot, the antiserum recognized mainly the porins from an outer membrane protein preparation. Using an ELISA, the titer of anti-porin antibodies was determined. Through a chemiluminescence assay, the increase in the respiratory burst of murine hyperimmune serum was recorded. These data correlate with the results of the microbicidal assays and with the electron microscopy preparations obtained where a great number of bacteria were seen within the macrophages. The in vitro data show that there is a greater bacterial killing when the macrophage is infected with bacteria opsonized with the hyperimmune serum.

Recognition of Streptococcus pyogenes and skin autoantigens in guttate psoriasis.

BACKGROUND: Guttate psoriasis is associated with infections by Streptococcus pyogenes and cross-reactions between skin and streptococcal antigens have been reported, suggesting an autoimmune component in the disease. METHODS: In this work, the authors looked for antibodies against S. pyogenes M-5 antigens by immunoblot in 52 sera of psoriasis patients and in 52 sera of normal individuals. Histological and immunohistochemical analysis in skin biopsies from lesions of another group of 16 clinically diagnosed guttate psoriasis patients and four healthy controls were also carried out. RESULTS: All guttate psoriasis patients studied (11) had IgG antibodies that intensively recognized three different proteins of 70, 60 and 14 kDa, as compared to sera from patients with other forms of psoriasis or from healthy controls. The diagnosis of psoriasis was confirmed in 14 of the patients by hematoxylineosin staining. Of the other two patients, one was diagnosed as parapsoriasis and the other as liquen. By indirect immunofluorescence (IFI), all 14 psoriatic patients had autoantibodies against their own lesional skin that did not recognize normal skin from control subjects or from the two non-psoriatic patients. The parapsoriatic and the liquen patients did not have autoantibodies. A rabbit immune serum against S. pyogenes antigens reacted with lesional skin from the 14 guttate psoriatic patients, but not with normal skin from controls or with lesional skin from the 2 non-psoriatic patients. CONCLUSIONS: The recognition by immunoblot of streptococcal antigens by serum of guttate psoriasis patients, the presence of autoantibodies against their own skin, and recognition of the same skin antigens by anti-streptococcal rabbit antibodies confirm the participation of the immune system and of streptococcal infections in guttate psoriasis.

Peripheral blood mobilization of different lymphocyte and dendritic cell subsets with the use of intermediate doses of G-CSF in patients with non-Hodgkin's lymphoma and multiple myeloma.

Between June 2003 and November 2004, we collected mobilized peripheral blood units from 29 patients with non-Hodgkin's lymphoma and multiple myeloma for autologous peripheral blood stem cell transplantation. They received granulocyte colony-stimulating factor (G-CSF) (16 micro g/kg/day) for a total of 5 days. Immediately before and 3 h after the fourth and fifth dose of G-CSF, we performed flow cytometry analysis to quantify: T cells (CD3+CD4+, CD3+CD8+), B cells (CD19+), NK cells (CD3-CD16+CD56+), NKT cells (CD3+CD16+CD56+), type 1 dendritic cells (DC1) (lin-HLA-DR+CD11c+), type 2 dendritic cells (DC2) (lin-HLA-DR+CD123+), regulatory T cells (Tregs) (CD4+CD25+), and activated T cells (CD3+HLA-DR+). All cell subsets were mobilized after G-CSF treatment with the exception of B, NK, and NKT lymphocytes. The median number of Treg cells before and after G-CSF was statistically different (29+/-14.9x10(6)/l vs 70.1+/-46.1x10(6)/l, P<0.02). DCs were mobilized significantly with a 5.9-fold increase in DC2 (15.1+/-30.3x10(6)/l vs 89.8+/-81.0x10(6)/l, P<0.02) and a 2.6-fold increase for DC1 (41+/-42.5x10(6)/l vs 109.5+/-58.0x10(6)/l, P<0.04). Patients received a mean of 3.1+/-1.2x10(7)/kg NK cells, 1.3+/-0.9x10(7)/kg NKT cells, 0.41+/-0.29x10(7)/kg DC1, 0.2+/-0.22x10(7)/kg DC2, and 1.8+/-1.9x10(7)/kg Tregs. In conclusion, intermediate doses of G-CSF induce mobilization of different lymphocyte subsets, with the exception of B, NK, and NKT cells. The mobilization of certain suppressive populations (DC2 and Treg) could be in theory deleterious, at least in patients with cancer.

Clinical relevance of NK, NKT, and dendritic cell dose in patients receiving G-CSF-mobilized peripheral blood allogeneic stem cell transplantation.

To analyze the relationship between the cellular composition of peripheral blood allografts and clinical outcome, we performed a prospective study in 45 adult patients who underwent allogeneic peripheral blood hematopoietic stem cell transplantation (HSCT) from a histocompatibility leukocyte antigen identical sibling donor for different hematological malignancies. The dose of CD34+, CD3+, CD4+, CD8+, and CD19+ lymphocytes, natural killer (NK) cells, natural killer T (NKT) cells, type 1 and type 2 dendritic cells (DC1 and DC2), as well as regulatory T (Treg) lymphocytes was analyzed. All patients were conditioned with busulphan and cyclophosphamide (BuCy2) +/- VP-16 and received a short course of methotrexate and cyclosporin-A as graft-versus-host disease (GVHD) prophylaxis. Acute GVHD (aGVHD) was present in 9 of 43 (21%) patients, and chronic GVHD (cGVHD) developed in 18 of 39 (46%) patients. There was a significantly higher incidence of aGVHD in patients receiving more than 6x10(6)/kg CD34+ cells. In univariate analysis, variables associated with better survival were as follows: a dose of less than 1.5x10(7)/kg NKT cells and less than 1.7x10(6)/kg DC2 for disease-free survival (DFS), and a dose of less than 3x10(7)/kg NK cells, less than 1.5x10(7)/kg NKT cells, less than 3x10(6)/kg DC1, and less than 1.7x10(6)/kg DC2 for overall survival (OS). In the Cox regression analysis, the dose of NKT cells was the only variable associated with better DFS, while the doses of NK, NKT, and CD34+ cells (less than 8x10(6)/kg) were associated with better OS. In conclusion, different circulating cell populations, other than CD34+ cells, are also of relevance in predicting the clinical outcome after allogeneic peripheral blood HSCT.

Cloning and sequencing of the gene that codes for the Klebsiella pneumoniae GroEL-like protein associated with ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease of the sacroiliac joints and vertebral column of unknown aetiology, but strongly related to the presence of the HLA-B27 antigen. The participation of bacterial infections as triggering factors have also been suggested. We have associated the 60 kDa heat shock protein of Klebsiella pneumoniae (HSP60Kp) with AS since we have previously demonstrated that most of the patients have IgG antibodies and active T cells that recognize preferentially this protein, but we have not yet identified the epitopes involved in the recognition. In order to know the amino acid sequence of HSP60Kp, and to be able to analyse in the future the relevant epitopes; we amplified by PCR and cloned the gene coding for this protein into the SmaI site of pUC19. The nucleotide sequence of the gene was obtained by the Sanger method using both manual and automatic techniques. Amino acid sequence of the HSP60Kp was deduced by translating the nucleotide sequence of the gene. The antigenic analysis of this sequence was compared to the antigenic analysis of the reported sequences of Escherichia coli GroEL and Yersinia enterocolitica HSP60. Using a software to predict HLA class I motifs, the nonapeptide (KRGIDKAVL) residues 117-125 of HSP60Kp showed a much higher affinity for HLA-B27 than the similar nonapeptide of E. coli GroEL and Y. enterocolitica HSP60. The only difference between the three peptides was in position nine. This finding could explain the association of AS only with the HSP60 of Klebsiella pneumoniae. On the other hand, hydrophilicity analysis, which indicates B cell epitopes, showed three similar strongly antigenic regions in the three proteins.

Contributions to the elucidation of S XIV pneumococcal polysaccharide structure.

Rabbits were immunized with a hemocyanin (Hcy)-p-amino phenyl beta lactoside (PAFBLO) conjugate and the gamma globulin fraction from immune sera was purified. Antibodies were demonstrated by means of the quantitative precipitin reaction using an egg albumin (EA)-PAFBLO conjugate as antigen. PAFBLO, D-lactose, D-galactose, cellobiose and D-galacturonic acid were used as inhibitors of the precipitin reaction and their relative affinity constants were calculated. The best inhibitors were the homologous hapten, D-lactose and D-galactose. Anti PAFBLO antibodies precipitated with types 6, 14 and 18 pneumococcal polysaccharides (SVI, SXIV and SXVIII) probably due to the presence of either D-lactose or D-galactose in similar linkage as in PAFBLO in these polymers. Antibodies also reacted with human red blood cells type B.

[Capsular polysaccharide of Klebsiella pneumoniae. II. Immunogenic properties]. / Polisacárido capsular de Klebsiella pneumoniae. II. Propiedades inmunogénicas.

It has been considered that the polysaccharide capsular material from microorganisms such as Klebsiella pneumoniae induces mainly thymus-independent humoral immunity, nevertheless studies done with Bacteroides fragilis have shown the participation of cellular immune effector mechanisms. Works done to define the immunogenic role of the capsular polysaccharide of Klebsiella pneumoniae indicate that both active and passive immunization with these antigens confer protection against infections with this bacteria. Several studies propose the use of vaccines prepared with one or various capsular serotypes of Klebsiella pneumoniae in immunoprophylaxis or immunotherapy.

Autoantibodies to autologous skin in guttate and plaque forms of psoriasis and cross-reaction of skin antigens with streptococcal antigens.

BACKGROUND: Psoriasis is a chronic disease of the skin that appears to be of autoimmune nature. It has a strong association with throat streptococcal infections, as well as with stressful events. Although many groups consider psoriasis to be a T-cell-mediated autoimmune disease, autoantibodies could also play a role in the development of this process. METHODS: In this work, we looked for autoantibodies to psoriatic skin in 21 psoriatic patients and four healthy donors (controls). The immunoperoxidase technique was used to look for autoantibodies in autologous sera in skin sections obtained from lesions or from healthy areas of the same patient, before and after immunoadsorption with a Streptococcus pyogenes extract. The skin biopsies were also analyzed with a pool of sera from mice immunized with the streptococcal extract. RESULTS: We found that all psoriatic patients had autoantibodies to antigens present in keratinocytes, whereas healthy subjects did not. These antibodies did not recognize epitopes on healthy skin from the same psoriatic patients or controls. Immunoadsorption of autologous sera removed the reactivity to antigens in skin lesions in all cases. Mouse anti-streptococcal sera recognized epidermal antigens present in lesional psoriatic skin, but not in healthy skin from psoriatic patients or controls. Deposits of immunoglobulin G (IgG) were not detected in the lesions. CONCLUSIONS: It seems that autoantibodies, although they do not appear to participate in the pathogenesis of psoriasis, are an important feature, and that skin antigens, which appear in lesional immature keratinocytes, cross-react with S. pyogenes and contribute to the autoimmune process in psoriasis.

Antibody response to Mycobacterium tuberculosis antigens in children with pulmonary tuberculosis and in BCG vaccinated non tuberculous pediatric patients.

In this work we look for the antibody response to M. tuberculosis antigens in 38 children with pulmonary tuberculosis: 18 bacteriologically and 20 clinically diagnosed as compared to the response of 52 children with other respiratory illnesses in which tuberculosis was definitively ruled out. In this control group 24 had been BCG vaccinated and 28 had not. Antibodies were detected by ELISA and immunoblot using a crude extract of M. tuberculosis as antigen. All 38 tuberculous diagnosed children tested positive, by ELISA, to the bacterial extract as did previously vaccinated controls (18/24), while all 28 non vaccinated controls gave negative results (sensitivity = 1.0, specificity = 0.68). By Western blot, tuberculous patients recognized a 12 kDa protein that was not recognized by the control vaccinated group. This partially purified protein which was used in an ELISA resulted positive in all 18 bacteriologically, in 17/20 clinically diagnosed patients, and in only 6/24 vaccinated controls. Specificity increased to 0.86, with a sensitivity of 0.93.

Antibody response to nitrogenase-positive and -negative Klebsiella pneumoniae strains in juvenile-onset ankylosing spondylitis patients and their first degree relatives: lack of differential recognition of the bacterial nitrogenase.

In the search for the pathogenic consequences of the molecular mimicry between the Klebsiella pneumoniae nitrogenase and the HLA-B27 antigen, sera from individuals belonging to 16 kindreds with juvenile-onset ankylosing spondylitis cases, were analyzed for antibodies against nitrogenase-positive and -negative K. pneumoniae whole bacterial extracts. An initial screening for nitrogenase producing K. pneumoniae strains was performed in 31 clinical isolates. The best nitrogenase producing strain was selected as well as a non producing one for immunoblot analysis using sera from 82 subjects, 55 HLA-B27 positive, of which 26 had some clinical manifestations. Even though electrophoretic patterns were different in both strains, there was no distinctive differential recognition of the 30-40 kDa proteins where the nitrogenase subcomponent which shares the sequence QTDRED with the HLA-B27 molecule is located. On the other hand, strong recognition of a protein of 60 kDa (p60Kp) was detected in 75% of HLA-B27 positive tested subjects independently of their clinical status. Studies on the nature of this protein and its participation in the pathogenesis of ankylosing spondylitis are now in progress.

Cellular immune response to Klebsiella pneumoniae antigens in patients with HLA-B27+ ankylosing spondylitis.

OBJECTIVE: To study the reactivity of peripheral blood mononuclear cells (PBMC) of patients with ankylosing spondylitis (AS) and rheumatoid arthritis (RA) and healthy controls to Klebsiella pneumoniae antigens and to the GroEL-like proteins from K. pneumoniae (HSP60Kp) and Mycobacterium leprae recombinant heat shock protein 65 (rHSP65Ml). METHODS: PBMC of 13 patients with AS and 9 with RA and 10 controls were stimulated in vitro by heat shock induced K. pneumoniae antigens in a cell blot assay, by insolubilized HSP60Kp, by cytosolic proteins (CP) from K. pneumoniae cultivated at 37 degrees C or 45 degrees C, by soluble HSP60Kp, or by rHSP65Ml. RESULTS: In the cell blot assay 7/13 AS and 3/9 RA patients responded to fraction 4, which contains mainly HSP60Kp, and no controls responded (AS vs. controls: p = 0.007). The response to the insolubilized HSP60Kp was positive in 6/13 AS patients but negative in RA patients and controls (p = 0.004). The response to CP45 degrees C was positive in 7/13 AS, in 2/9 RA, and no controls (AS vs controls: p<0.015). Response to the soluble HSP60Kp was found in 7/13 AS and 5/9 RA patients, but no controls (AS vs. controls: p = 0.0075). Response to rHSP65Ml was positive in 3/13 AS, 7/9 RA patients, and 1/10 controls (AS vs RA: p = 0.027; RA vs. controls: p = 0.005; AS vs. controls: nonsignificant). CONCLUSION: In PBMC of the majority of patients with AS and in some with RA, but not in healthy controls, there are cells that proliferate in the presence of HSP60 of K. pneumoniae.