Fetal membranes exhibit selective leukocyte chemotaxic activity during human labor.

One of the characteristics of the labor process in women is leukocyte recruitment into reproductive tissues. These migrating cells may play a role in the induction of functional and biochemical changes associated with the rupture of fetal membranes during labor. This study was undertaken to assess whether human fetal membranes induce leukocyte chemotaxis during labor as well as to identify and characterize leukocyte chemoattractants secreted by these tissues. Leukocyte chemotactic activity of fetal membrane extracts obtained from women with full-term pregnancies and spontaneous active labor was compared with extracts from women without labor. The number and phenotype of attracted leukocytes were analyzed by flow cytometry. Chemokines were quantified using a Multiplex system and were identified by immunofluorescence histochemistry. Although all tested extracts induced chemotaxis of leukocytes, those prepared from women undergoing labor induced higher responses. Polymorphonuclear leukocyte chemotaxis increased approximately three-fold in response to extract from fetal membranes with labor. The same extracts elicited a significant increase in attracted monocytes (36-fold) as well as T and B lymphocytes, and NK cells (all five-fold) when compared to extracts from women without labor. This enhanced chemotactic activity was associated with the presence of IL-8, MCP-1, IP-10 and MIP-1alpha. We conclude that fetal membrane extracts obtained from women during labor exhibit selective chemotaxis for specific leukocyte subpopulations in vitro. This process may contribute to a microenvironment composed of specific leukocytes that promotes and amplifies biochemical changes in the fetal membranes during labor.

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.

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.

Donor lymphocyte infusions for relapse of chronic myeloid leukemia after allogeneic stem cell transplantation: prognostic significance of the dose of CD3(+) and CD4(+) lymphocytes.

Between December 1993 and November 2001, 30 patients with chronic myeloid leukemia who relapsed after stem cell transplantation were studied. Seventeen patients were not treated before donor lymphocyte infusion (DLI), eight patients received interferon-alpha (IFN-alpha), and five underwent chemotherapy. The method of DLI was the bulk dose regimen. The median time between DLIs was 6 weeks. The median number of infusions was three; the median time from transplant to relapse was 17 months and from relapse to DLI 2 months. Eleven patients (37%) were in molecular/cytogenetic relapse, 14 (47%) in chronic phase, and five (16%) in accelerated or blastic phase. Seventeen patients (57%) developed acute graft-versus-host disease (GVHD). Chronic GVHD was observed in 15 of 24 (62%) patients. Four (13%) patients developed cytopenia after a median of 30 days. Nineteen (63%) patients achieved response, 15 of them developed GVHD. The response rate according to the disease phase was molecular or cytogenetic relapse: 91%, chronic phase: 57%, and accelerated or blastic phase: 20%. The median time to response was 6 months. Patients treated with IFN-alpha or no treatment as well as those who were in molecular/cytogenetic relapse and those who received a CD3(+) cell dose <1 x 10(8)/kg and CD4(+) <8 x 10(7)/kg had better survival. We conclude that patients who receive lower doses of lymphocytes have better survival. In some patients IFN-alpha seems to be a good choice to potentiate the graft-versus-leukemia (GVL) effect.

In-vitro secretion of proinflammatory cytokines by human amniochorion carrying hyper-responsive gene polymorphisms of tumour necrosis factor-alpha and interleukin-1beta.

The identification of polymorphisms in genes encoding proinflammatory cytokines that affect transcription or the secretion rate has opened new ways to understand the variation in responses to infection during pregnancy. In this study, human amniochorion carrying hyper-responsive alleles of tumour necrosis factor-alpha (TNF-alpha: TNF*2 at -308) and interleukin-1beta (IL-1beta: IL-1*2 at +3953) were stimulated in vitro with bacterial lipopolysaccharide (LPS) and compared with tissues carrying the common alleles (TNF*1 and IL-1*1). Fetal membranes carrying the TNF*1 allele displayed an identical dose-response pattern to tissues carrying a TNF*2 allele, except at the highest dose of LPS tested (50 ng/ml) there was a significantly greater production of TNF-alpha in the presence of a TNF*2 allele. Membranes carrying the IL-1*2 polymorphism secreted IL-1beta in a dose-response curve that was different from IL-1* tissues when challenged with 5, 10 and 50 ng/ml LPS. These observations support the hypothesis that reproductive tissues carrying hyper-responsive proinflammatory cytokine genes may over-respond to intrauterine infection secreting higher amounts of cytokines, which in turn, may lead to adverse pregnancy outcomes.

IgG antibodies to enterobacteria 60 kDa heat shock proteins in the sera of HLA-B27 positive ankylosing spondylitis patients.

OBJECTIVE: To study the association of HLA-B27 and the IgG response to the 60 kDa HSPs of Klebsiella pneumoniae, Yersinia enterocolitica, Shigella flexneri, Escherichia coli and Salmonella typhi. METHODS: IgG against the 60 kDa HSPs of enterobacteria was determined by ELISA in the sera from 49 HLA-B27+ ankylosing spondylitis (AS) patients; 41 HLA-B27+ healthy relatives of AS patients and 101 HLA-B27-unrelated healthy individuals. RESULTS: HLA-B27+ patients and healthy individuals, showed significantly higher IgG antibody levels to the Klebsiella, Yersinia and Salmonella HSPs than HLA-B27- healthy controls. B27+ patients had a significantly higher response to E. coli HSP than the two other groups. IgG response anti-Shigella HSP was similar in the three groups. CONCLUSIONS: There is a relationship between HLA-B27 and the response to HSPs 60 from Klebsiella, Yersinia, Escherichia and Salmonella, that may be important in the initiation of AS.

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.

[Tumor necrosis factor-alpha and interleukin-1 beta in maternal, fetal and retroplacental intravascular compartments at term and preterm labor]. / Factor de necrosis tumoral-alpha e interleucina-1 beta en los compartimentos intravascular materno, fetal y retroplacentario en parto a término y pretérmino.

Neonato preterm birth (before 37 pregnancy weeks) account more than 80% perinatal deaths not attributable to congenital malformations. Preterm and term labor full mechanisms are unknown at present. Proinflammatory cytokinesis direct participation have been involved in the phenomena by several experimental evidence. The study's aim was to determine TNF-alpha and IL-1 beta concentration at maternal, fetal and fetal-maternal vascular compartments in women with term and preterm delivery and in women at term childbirth without labor. TNF-alpha and IL-1 beta concentration were determinated by commercial immunoassay. TNF-alpha concentration showed a tendency to be in more proportion at fetal and fetal-maternal compartments in preterm and term childbirth groups versus TNF-alpha concentration in term group without labor at same places. IL-1 beta concentration showed same tendency of increase than TNF-alpha in preterm and term childbirth groups, but alone at fetal-maternal compartment. Statistical difference were not documented at any compartment or group compared. Data allow to identify fetal-maternal compartments as target places where TNF-alpha and IL-1 beta were synthesized. Gradient concentration synthesis of cytokinesis allows to intend fetus as TNF-alpha initial producer.

Recognition of B cells epitopes of the Klebsiella pneumoniae GroEL-like protein by HLA-B27 positive subjects.

The presence of antibodies against antigens of K. pneumoniae in HLA-B27 positive patients with ankylosing spondylitis (AS), has been well documented. We have previously reported that sera from HLA-B27 positive subjects react with the K. pneumoniae GroEL-like protein (HSP60Kp) and have higher titers than HLA-B27 negative individuals. We cloned the gene that codes for this protein, determined hydrophilic regions by computer analysis of the predicted amino acid sequence and found that residues 389-397, 360-368 and 282-290, were possible B cell epitopes. To test this prediction, and to determine if the HLA-B27 positive and negative AS patients recognize the same or different epitopes, we truncated the hsp60Kp gene, from the 3; terminal nucleotide, to obtain fragments having or not the predicted epitopes. Four polypeptides of 40, 37, 30 and 18 kDa were obtained and analysed, by ELISA and inhibition of ELISA, for their reactivity with IgG antibodies from three high responders HLA-B27 positive AS patients and three HLA-B27 negative subjects who recognized the rHSP60Kp. Sera from both HLA-B27 positive and negative subjects reacted equally well with rHSP60Kp or with the 40 and 37 kDa peptides, which do not have residues 389-397 and 360-368, respectively, but reactivity was lost with the 30 kDa peptide, which also lacks residues 282-290. Contrary to what we expected, antibodies from HLA-B27 negative and positive individuals recognized the same epitope of the HSP60Kp. Our results indicate that the important epitope for B cells could be the 282-290 region and that the contribution of the two other predicted regions is minimal. We also conclude that the differences in response to the HSP60Kp in HLA-B27 positive AS patients and HLA-B27 negative individuals is not qualitative, but only quantitative.

Antibody response to Klebsiella pneumoniae 60 kDa protein in familial and sporadic ankylosing spondylitis: role of HLA-B27 and characterization as a GroEL-like protein.

OBJECTIVE: To study the antibody response of HLA-B27+ patients with ankylosing spondylitis (AS) and their first degree relatives to the 60 kDa protein of Klebsiella pneumoniae and to characterize this protein. METHODS: Sera from 84 individuals were analyzed by ELISA to determine the titer of antibodies against the 60 kDa protein of K. pneumoniae. Subjects were divided into 3 categories: Group 1: 44 HLA-B27+ AS related individuals (35 patients, 9 healthy controls); Group 2: 28 healthy B27- AS related individuals; and Group 3: 12 healthy B27- non-AS related subjects. The 60 kDa protein of K. pneumoniae was induced at 45 degrees C and purified by electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was characterized as a GroEL-like heat shock protein (HSP). The recognition of GroEL-like protein was confirmed by immunoblot of 2 dimension electrophoresis. The response to GroEL-like protein from other bacteria and the response to lipopolysaccharide (LPS) was also analyzed by immunoblot. RESULTS: HLA-B27+ individuals (Group 1), independent of their disease status, showed a significant higher response to the 60 kDa protein of K. pneumoniae than HLA-B27- subjects from Groups 2 and 3 (p < 0.0001). This protein was characterized as a HSP of the GroEL family and designated HSP60Kp. The GroEL of other enterobacteria as well as that of Mycobacterium leprae were recognized by HLA-B27+ individuals by immunoblot, whereas HLA-B27- individuals did not. LPS was not recognized by HLA-B27 positive or negative subjects. CONCLUSION: These findings suggest a relationship between HLA-B27 and the response to a GroEL-like protein that could have implications in AS.

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.

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.

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.

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.

[Sequential activation of cellular and humoral immunity in leprosy: considerations based on recent findings]. / Activación secuencial de la inmunidad celular y la inmunidad humoral en la lepra: consideraciones con base en hallazgos recientes.

Lepromatous leprosy in the human being evolves showing a progressive loss of cell mediated immunity (CMI) to the antigens of Mycobacterium leprae (ML). This does not prevent the host to respond with antibodies to the same microorganism. On the other hand, the production of antibodies to the great majority of exogenous antigens results from cell-to-cell interactions that involve the participation of helper T cells. On this ground, a satisfactory explanation for the loss of CMI to M. leprae (which indicates either the loss or inactivation of specific helper T cells), with no effect on the humoral response to the same microorganism (this implying the participation of functional specific helper T cells), was difficult to found. It was not until Mosmann established, in the mouse, the existence of two subpopulations of helper T cells, that a feasible explanation for the apparent immunological paradox observed in leprosy was possible to offer. The work described here, based to a great extent in our experience on murine leprosy, refers to recent concepts concerning this issue.

Comparison of the immune response against Mycobacterium tuberculosis antigens between a group of patients with active pulmonary tuberculosis and healthy household contacts.

The mycobacterial antigens and the factors related to protection for the development of active tuberculosis are not known. In a natural model of tuberculosis, we studied 10 patients with active pulmonary tuberculosis (non-protective immune response) and 38 healthy household contacts (protective immune response). We tested the lymphocyte proliferative response by T cell Western blotting to eight different antigen fractions and to two purified mycobacterial antigens of 30 and 64 kD. Patients with active tuberculosis recognized fractions with molecular weights of 80-114, 60-80, 28-41 and 14-19 kD. Household contacts recognized the same fractions except the 14-19 kD. The response to the 64-kD antigen was not significantly different between groups. In contrast, 10% of the patients with active tuberculosis and 73% of the household contacts responded to the 30-kD antigen. The humoral response against the 30-kD antigen by ELISA showed a significantly higher production of antibodies in tuberculosis patients compared with household contacts. We conclude that patients with active pulmonary tuberculosis develop an immune response characterized by poor proliferative response to the 30-kD antigen with a strong humoral response, whereas the opposite occurs in healthy subjects infected by Mycobacterium tuberculosis.

Activation of T cells to peptides that span the 19 kDa protein of Mycobacterium tuberculosis.

It has been suggested that the cellular immune response to mycobacterial antigens, is implicated in the pathogenicity of inflammatory joint diseases such as rheumatoid arthritis. Therefore, the aim of this study was to identify T-cell epitopes in a series of 20-mer peptides spanning the entire 19-kDa protein of M. tuberculosis. Mononuclear cells obtained from six rheumatoid arthritis (RA) patients, were analyzed for their proliferation to both the 19-kDa containing immunoblot fraction and to the synthetic peptides. Mononuclear cells from three rheumatoid arthritis patients responded in a dose-dependent manner to peptides 1-20, 60-79, 71-90, 82-101, 90-109 and 121-140, whereas the other eight peptides: 11-30, 21-40, 41-60, 50-69, 100-119, 112-131, 131-150 and 140-159 did not stimulate significant proliferative responses in any of the patients tested. These results indicate, for the first time, the presence of dominant epitopes in the cellular immune response to the 19-kDa M. tuberculosis antigen by rheumatoid T cells.

Peptide competition at the level of MHC-binding sites using T cell clones from a rheumatoid arthritis patient.

The inhibition of antigen presentation in rheumatoid arthritis by blocking peptide binding to MHC at the antigen presenting cell (APC) level was investigated using various synthetic peptides derived from the 65 kDa mycobacterial protein. Human T cell clones from tuberculosis and rheumatoid arthritis patients were stimulated with peptides in the presence of irradiated APCs (autologous or DR homozygous EBV-B cell lines). Two peptides (residues 65-85 and 412-426) were found to be able to bind to the HLA-DR1 protein. Cross-competition was observed between these peptides when APCs were cultured with a suboptimal concentration of stimulator peptide in the presence of various concentrations of competitor peptides and T cell clones from tuberculosis patients as responder cells. These T cell clones responded not only to the peptides but also to the native protein. In other experiments, we used T cell clones from a rheumatoid arthritis patient to demonstrate the blocking of MHC-binding sites by adding the p412-426 in the recognition of DR1 restricted T cell clone specific to p65-85; MHC binding was not observed using a control peptide (residues 198-217). This approach has permitted the identification of MHC-specific blockers. Further experimentation is required to determine the particular amino acids involved in MHC binding. Our data support the idea that modulation of antigen presentation by peptide competition could be a useful tool for immunotherapy in autoimmune diseases.