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.

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.

Identification of peptides presented by HLA class I molecules on cervical cancer cells with HPV-18 infection.

In this work we eluted peptides from purified class I MHC molecules, isolated from a novel human cervical carcinoma cell line (INBL), generated in our laboratory and positive for HPV-18 infection. A fraction of these peptides was capable of stimulating T lymphocytes obtained from a donor matched for HLA-Cw4 and who was also HPV-18+. Direct N-terminal Edman degradation of these peptides, revealed the sequence (XQFPIFLQF) that matched 85% with the sequence NVFPIFLQM localized in between the 54 and 62 residues of the HPV-18 L1 protein. After stimulation with the synthetic peptide NVFPIFLQM, T lymphocytes from the donor were capable to lyse INBL cells. Our results provide evidence of the existence of naturally occurring viral epitopes presented on cervical cancer cells by the HLA-Cw4 allele, that could be useful for immunotherapy on this type of patient.

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.

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.