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Genetic immunization represents a new tool for investigating physiological and pathological immune responses. Here we used genetic immunization with naked DNA to study the immune relevance of aminoacid sequence homologies by evaluating the outcome of immunization to a viral protein homologous to an HLA molecule. The viral protein was Balf2, a protein of Epstein-Barr virus (EBV) that shares aminoacid sequence homology with the HLA allele DRB1*0801. After genetic immunization of BALB/c mice with a construct encoding Balf2, we analyzed T cell responses of immunized mice. We found that cross-reactive proliferative and cytotoxic responses were raised to the homologous sequence as expressed by HLA-DRB1*0801. Furthermore, preferential secretion of Th1-type pro-inflammatory cytokines occurred. This strategy can allow rapid screening of interactive immune networks involving aminoacid sequence homologies between organisms.

Bacterial DNA is enriched in unmethylated CpG motifs that have been shown to activate the innate immune system. These immunostimulatory DNA sequences (ISS) induce inflammation when injected directly into joints. However, the role of bacterial DNA in systemic arthritis is not known. The purpose of the present experiments was to determine whether ISS contributes to the development of adjuvant arthritis in Lewis rats after intradermal injection of heat-killed Mycobacterium tuberculosis (Mtb). The results showed that Mtb DNA was necessary for maximal joint inflammation in adjuvant arthritis but could be replaced by synthetic ISS oligodeoxynucleotides. The arthritis-promoting effect of the Mtb DNA or of the ISS oligodeoxynucleotides correlated with an increased Th1 response to Mtb Ags, as measured by the production of IFN-gamma and increased production of the osteoclast differentiation factor, receptor activator of NF-kappaB ligand (RANKL). The Mtb DNA did not enter the joints but dispersed to the bone marrow and spleen before the onset of systemic joint inflammation. Thus, adjuvant arthritis is a microbial DNA-dependent disease. In this model, we postulate that massive and prolonged activation of macrophages, dendritic cells, and osteoclast precursors in the bone marrow may prime the joints for the induction of inflammatory Th1 immune responses to Mtb Ags.

Rheumatoid arthritis (RA) is an autoimmune/inflammatory disorder with a complex genetic component. We report the first major genomewide screen of multiplex families with RA gathered in the United States. The North American Rheumatoid Arthritis Consortium, using well-defined clinical criteria, has collected 257 families containing 301 affected sibling pairs with RA. A genome screen for allele sharing was performed, using 379 microsatellite markers. A nonparametric analysis using SIBPAL confirmed linkage of the HLA locus to RA (P < .00005), with lambdaHLA = 1.79. However, the analysis also revealed a number of non-HLA loci on chromosomes 1 (D1S235), 4 (D4S1647), 12 (D12S373), 16 (D16S403), and 17 (D17S1301), with evidence for linkage at a significance level of P<.005. Analysis of X-linked markers using the MLOD method from ASPEX also suggests linkage to the telomeric marker DXS6807. Stratifying the families into white or seropositive subgroups revealed some additional markers that showed improvement in significance over the full data set. Several of the regions that showed evidence for nominal significance (P < .05) in our data set had previously been implicated in RA (D16S516 and D17S1301) or in other diseases of an autoimmune nature, including systemic lupus erythematosus (D1S235), inflammatory bowel disease (D4S1647, D5S1462, and D16S516), multiple sclerosis (D12S1052), and ankylosing spondylitis (D16S516). Therefore, genes in the HLA complex play a major role in RA susceptibility, but several other regions also contribute significantly to overall genetic risk.

Recent progress in molecular medicine has provided important tools to identify antigen-specific T cells. In most cases, the approach is based on oligomeric combinations of recombinant major histocompatibility complex-peptide complexes fixed to various rigid supports available for binding by the T-cell receptor. These tools have greatly increased our insight into mechanisms of immune responses mediated by CD8+ T cells. Examples of the diverse fields of application for this technology include immunization, viral infections and oral tolerance induction.

A system that uses genetic immunization for recombinant protein recovery and purification is described. The genetic sequence encoding a target protein is subcloned into both a eukaryotic and a prokaryotic vector. With the eukaryotic construct, a rabbit is genetically immunized and specific polyclonal antibodies to the encoded protein raised. The prokaryotic construct is used for bacterial transformation and expression of recombinant protein. Recovery and purification of target recombinant protein are obtained by passing the lysate of expressing bacteria through an immunoaffinity column prepared with the polyclonal antibodies raised in the genetically immunized animal. This method allows purification of recombinant protein without fusion tails and can be applied to purify any protein whose encoding genetic sequence is known., (Copyright 2000 Academic Press.)

After intradermal genetic immunization, naked DNA is transported from the site of injection to regional lymph nodes. Little is known on how inflammation influences this process and whether DNA is transported beyond local lymph nodes. In the experiments herein reported, we injected naked DNA in the presence of adjuvant to address questions related to 1) the fate of naked DNA in the presence of inflammation; 2) the generation of immune responses to the encoded protein during inflammation; and, more in general, 3) the fate of ingested molecules beyond regional lymph nodes during inflammation. Two sites of inflammation were induced in vivo in mice. Naked DNA was injected in the nape together with adjuvant, and adjuvant only was injected at a distant peritoneal site. Injected DNA, uptaken at the primary dermal site of inflammation, was transported beyond regional lymph nodes to distant organs such as the spleen and to the distant peritoneal site of inflammation. This transport, mediated by CD11b+ cells, was cumulative during chronic inflammation. These results indicate a novel route of transport of DNA beyond regional lymph nodes and may have specific implications for DNA-based immune modulation.

By means of a novel technique for identification and isolation of MHC class II-restricted antigen-specific T cells, we describe here in non-transgenic BALB / c mice physiological positive selection of an oligoclonal population of T cells which recognizes both a self MHC-derived peptide (Ialpha52) and a bacterial homologoue (Hi15). The results support a model for self peptide-mediated generation of T cells which have specificity for microbial antigens through molecular mimicry. This mechanism may be a model for the ontogeny of a physiological T cell response to infectious agents. Loss of control of these circuits may be part of the inciting factors of autoimmunity.

Objective: Previously, we showed that rheumatoid arthritis (RA) had both antibodies and T cells specific for the QKRAA-encompassing Escherichia coli dnaJ protein. These findings suggest that the bacteria induced anti-dnaJ responses may cross react with the human homolog of bacterial dnaJ in the joint, resulting in tissue damage., Methods: We used the combinatorial library technique to isolate and characterize an IgG monoclonal anti-dnaJ antibody (designated CG1) from the blood of a patient with RA., Results: Sequence analysis of CG1 revealed that its heavy and light chain V regions were respectively most homologous to the 3d279d VH4 and the O18 Vk1 genes. Interestingly, 3d279d is frequently expressed by B cells stimulated with staphylococcal enterotoxin; and O18 is the main gene employed by the Vk1 IgG antibodies against Haemophilus influenzae., Conclusion: The combinatorial immunoglobulin library method represents an interesting model of how to approach the isolation and characterization of antibody-like reagents in the elucidation of autoantigens in RA.

Induction of oral tolerance to antigens that are targets of self-reactive immune responses is an attractive approach to antigen-specific immune therapy of autoimmune diseases. Oral tolerization has indeed proven to be safe and effective in amelioration of autoimmune diseases in animal models. In humans, results have been somewhat controversial. The emphasis given to clinical outcome rather than to immunomodulation, and the difficulty in identifying appropriate candidate antigens contribute to the controversy. Heat shock proteins are promising targets for immune intervention. Immune reactivity to heat shock proteins has indeed been correlated with autoimmune arthritis in animal models, and abnormal immune responses to heat shock proteins have been described in human arthritis as well. Despite significant recent progress, little is known at a molecular level regarding the mechanisms which are responsible for a switch from autoimmunity to tolerance in humans. This is particularly true with respect to sequential analysis of several molecular and immunologic markers during both the course and treatment of disease. Novel approaches are currently under way to fill the gaps. We will briefly detail here the experience gained to date, and identify some of the avenues which future research will explore.

A novel 'multistep molecular mimicry' mechanism for induction of rheumatoid arthritis (RA) by bacterial antigens that activate T lymphocytes previously 'educated' by peptides derived from a class of human histocompatibility antigens is reported here. These antigens have the amino acid sequence QKRAA, which is also present on the Escherichia coli heat-shock protein dnaJ. Synovial fluid cells of early RA patients have strong immune responses to the bacterial antigen, but cells from normal subjects or controls with other autoimmune diseases do not. The activated T cells may cross-react with autologous dnaJ heat-shock proteins that are expressed at synovial sites of inflammation. Our findings may have direct relevance to new strategies for the immune therapy of RA.

The etiopathogenesis of childhood chronic autoimmune disease is, in most cases, unknown. Most likely, several factors overlap in determining the loss of tolerance toward certain autoantigens that become the target of the disease and the main cause of its perpetuation. Infectious agents have often been implicated in the pathogenesis of these diseases, but, to date, compelling evidence for a horizontal transmission or for localized epidemics is lacking. Human pathogens may nevertheless play a role in determining the loss of tolerance toward certain self-antigens by means of mechanisms other than classic infection. It is common knowledge that human pathogens often express proteins with high antigenic potential with important homologies with human proteins. Evolutionary pressures based upon the necessity of escaping the host's specific immune responses may have determined this phenomenon, called "molecular mimicry". It is reasonable to assert that certain individuals can develop abnormal immune responses upon contact with an antigen that mimics a self-protein. These responses may ultimately lead to self-reactivity and autoimmune disease. In this model of molecular mimicry, self-reactivity is triggered by cross-recognition of a self and an exogenous protein that bear the same sequence. A disease triggered by such a mechanism should present with: i) some form of an acute or chronic autoimmune clinical manifestation; ii) a documented clinical correlation between contact with a human pathogen and the autoimmune disease; iii) immune cross-reaction between a protein from a pathogen and a homologous human protein. Acute rheumatic fever, Reiter's syndrome and the other reactive arthritides fulfill the above conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

We describe two children with clinical and laboratory features of hypocomplementemic urticarial vasculitis syndrome. Both patients had severe, life-threatening manifestations: rapidly progressive glomerulonephritis (patient 1) and pulmonary hemorrhage (patient 2). We conclude that this syndrome may be a potentially severe multisystem disease.

By using a sandwich ELISA, soluble human IL-6 receptor (sIL-6 R) levels were measured in the sera of 20 healthy children and of 25 patients with systemic juvenile rheumatoid arthritis (JRA). In patients with systemic JRA, serum sIL-6 R levels (114.6 +/- 37.7 ng/ml) were significantly lower (P < 0.01) than those of healthy children (161.2 +/- 45.5 ng/ml). Serum sIL-6 R levels were negatively correlated (r = -0.610, P < 0.001) with serum IL-6 levels measured with the B9 cells. The serum IL-6/sIL-6 R complex was detected using an ELISA based on a monoclonal antibody to IL-6 for capture and on a monoclonal antibody to human sIL-6 R for detection. Healthy controls had little, if any, detectable serum IL-6/sIL-6 R complex (OD 0.024 +/- 0.027), while the majority of patients with systemic JRA presented measurable serum IL-6/sIL-6 R complex (OD 0.492 +/- 0.546). IL-6 levels estimated in the circulating IL-6/sIL-6 R complexes were in the range of nanograms per milliliter and approximately 20-fold higher than those measured by the B9 cells. Since serum C-reactive protein concentrations were much more correlated with serum levels of IL-6/sIL-6 R complexes (r = 0.713, r2 = 0.51, P < 0.0001) than with the serum IL-6 levels measured with the B9 cells (r = 0.435, r2 = 0.19, P = 0.05), the large quantities of serum IL-6 present in IL-6/sIL-6 R complexes appear to be biologically relevant in vivo, at least as far as the induction by IL-6 of acute phase protein production.

Patients with juvenile rheumatoid arthritis frequently have abnormal immune responses to the hsp65 class of bacterial heat shock proteins. However, lymphocytes from children with other inflammatory diseases may also recognize hsp65, and the role of these antigens in juvenile rheumatoid arthritis remains controversial. We have studied humoral and cellular immune responses to a distinct, recently described bacterial heat shock protein, designated dnaJ. The Escherichia coli dnaJ gene was cloned and expressed, and the purified recombinant protein was used as an antigen. Neither normal children nor children with various chronic inflammatory diseases had lymphocyte proliferative responses to recombinant dnaJ. However, lymphocytes from patients with polyarticular, pauciarticular, and systemic manifestations of juvenile rheumatoid arthritis responded strongly to the antigen. Cellular immune responses to dnaJ were higher in synovial fluid than in blood and higher in children with active disease than in children in remission. These data show that increased immune reactivity to dnaJ is characteristic of juvenile rheumatoid arthritis and that the magnitude of the immune response is linked to disease activity. The results suggest that an abnormal immune response to antigens on commensal gut bacteria may contribute to the generation of chronic inflammation in juvenile rheumatoid arthritis.

In this study we evaluated the presence of anti-nucleosome reactivity in children with systemic lupus erythematosus (SLE), and assessed its clinical correlations in comparison with anti-dsDNA and anti-histone IgG levels. Reactivities to nuclear substrates were determined by enzymatic immunoassays in 80 sera from 22 children with SLE, and solid phase adsorption experiments were performed. In children with active SLE, the anti-dsDNA, anti-histone, and anti-nucleosome IgG levels were elevated, and were significantly correlated with disease severity; during remission anti-histone (but not anti-dsDNA and anti-nucleosome) antibodies, although reduced, were still significantly elevated. The results of adsorption studies showed that anti-dsDNA antibodies contributed to anti-nucleosome reactivity, while anti-histone antibodies did not. The recognition of the H3H4-DNA nucleosome subparticle, rather than H2AH2B-DNA, may be related to the nucleosome reactivity. In children with SLE anti-nucleosome reactivity is present; it is in part due to anti-dsDNA antibodies and in part to antibodies recognizing conformational epitopes that may be related to the H3H4-DNA nucleosome subparticle. Its clinical significance for diagnosis is not greater than that of the anti-dsDNA antibodies.

Objective: It has been suggested that double negative (CD4-CD8-) (DN) and gamma/delta T cells may be involved in some autoimmune diseases. We investigated peripheral blood DN and gamma/delta T cell levels in patients with active juvenile rheumatoid arthritis (JRA)., Methods: DN and gamma/delta T cell levels were measured in 42 patients with active JRA and in 10 healthy controls comparable for age by an immunofluorescence double staining procedure., Results: All 3 JRA onset types had DN and gamma/delta T cell levels not significantly different from those of controls, although a wide scattering of data was present. No correlation was found between DN or gamma/delta T cell levels and erythrocyte sedimentation rate values or the number of active joints. When patients were divided according to treatment, we found that DN and gamma/delta T cell levels were significantly lower (p = 0.001, p = 0.02, respectively) in patients receiving methotrexate (MTX) than in patients not receiving MTX. The association of MTX treatment with a decrease in DN and gamma/delta T cell levels was also confirmed in a followup study of individual patients. Among patients not receiving MTX, patients with systemic JRA presented DN T cell levels significantly higher than those of controls. In 5 patients with pauciarticular JRA DN and gamma/delta T cell levels were higher in synovial fluid than in the peripheral blood., Conclusions: We found an increase in peripheral blood DN T cell levels in systemic JRA; treatment with MTX appears to be associated with a decrease in DN and gamma/delta T cell levels.

An adult patient had a syndrome associating the features of juvenile dermatomyositis and cutaneous polyarteritis nodosa that followed a cyclic course from childhood; recurrences were always associated with a rise of serum antistreptococcal antibodies. Regions of homology between streptococcal type 5 M protein and skeletal myosin were found. These findings suggest that streptococcal infection, possibly through a molecular mimicry mechanism, played a role in the pathogenesis of the disease in our patient.

Our experiments have led us to conclude that the rheumatoid arthritis shared epitope may act as a peptide that is important for positive and negative selection of T lymphocytes, that T lymphocytes are skewed by positive selection to recognize epitopes that are similar but not identical to self, and that peptide sequences that are similar to the RA-shared epitope are abundantly expressed by microorganisms that chronically infect most people. This combination of events could partly explain the association of the shared epitope with the severe forms of RA. The hypothesis cannot be tested directly, because we do not postulate that any unique population of autoreactive T cells is expanded in RA; however, the role of positive selection in molding the human T-cell repertoire to exogenous antigens can be tested by mapping T-cell antigenic determinants on the E. coli dnaJ protein or the gp110 protein of EBV in people with different HLA-DR types. Moreover, positive selection models imply that maternal antigens that cross the placenta can influence the T-cell repertoire. Thus, one might expect to find that the frequency of HLA-DR4 in the mothers of patients with RA who themselves lack the DR4 antigen, would be more frequent than predicted by chance alone. As the principles of positive selection are more precisely delineated in animal systems, it should become possible to ascertain more clearly how the shared epitope on HLA-DR molecules enhances the severity of autoimmune reactions; however, RA only occurs in humans; possibly because of the unique inability of human macrophages to replicate. Thus, only the direct analysis of patients can directly reveal the mechanisms of disease pathogenesis.

We measured serum and synovial fluid interleukin 6 (IL-6) levels in patients with polyarticular-onset and pauciarticular-onset juvenile chronic arthritis (JCA), using the hybridoma cell line B9. During active disease, but not during remission, serum IL-6 levels were significantly elevated in patients with polyarticular (p < 0.0001 vs controls) and in patients with pauciarticular JCA (p < 0.01 vs controls). In patients with active polyarticular disease (polyarticular and extended pauciarticular) serum IL-6 levels correlated with the extent and severity of joint involvement (p < 0.05), with erythrocyte sedimentation rate values (p < 0.005) and with C reactive protein concentrations (p < 0.05). In patients with persistent pauciarticular JCA, serum IL-6 levels correlated with the joint swelling score (p < 0.05). Synovial fluid IL-6 levels, measured in 5 patients with pauciarticular JCA, were markedly elevated (range 600-24,000 HGF U/ml). In conclusion, our data suggest that IL-6 is an important pathogenic mediator in polyarticular and pauciarticular JCA.

Susceptibility to rheumatoid arthritis (RA) maps to residues QKRAA/QRRAA in the third hypervariable region of the HLA DR beta 1 chain. Peptides from the same area of MHC class II molecules are able to modulate the T-cell repertoire by deleting self-reactive T-cells. The Epstein Barr virus glycoprotein gp110 and the dna J heat-shock protein from E. coli mimic the third hypervariable region of HLA-Dw4DR beta 1. Thus, the same area of HLA DR beta 1 carries susceptibility to RA, modulates the T-cell repertoire and is mimicked by human pathogens. RA may originate from a particular shape imposed on the T-cell repertoire by the QKRAA/QRRAA sequence in the third hypervariable region of HLA DR beta 1.

Immunological responses to bacterial heat shock proteins have been implicated in the pathogenesis of arthritis in animals and humans. The predicted amino acid sequence of dnaJ, a heat shock protein from Escherichia coli, contains an 11-amino acid segment that is homologous to the third hypervariable region of the human histocompatibility antigen (HLA) DRB10401 (formerly known as HLA Dw4), the part of the molecule that carries susceptibility to rheumatoid arthritis. To test the biological significance of this finding, we expressed and purified recombinant dnaJ (rdnaJ), and determined its immunologic cross-reactivity with HLA DRB10401. A rabbit antipeptide antiserum raised against the sequence of the third hypervariable region of HLA DRB10401 specifically bound to 'dnaJ, thus confirming that a similar sequence is expressed on the bacterial protein. Of greater consequence, an antiserum to the 'dnaJ protein recognized not only a peptide from the third hypervariable region of HLA DRB10401, but also the intact HLA DRB10401 polypeptide. Furthermore, the antibody to 'dnaJ reacted with HLA DRB10401 homozygous B lymphoblasts, but not with HLA DRB11501, DRB10101, DRB10301, and DRB10701 (formerly known as HLA Dw2, DR 1, DR 3, and DR 7, in the same order) homozygous cells. These results demonstrate that exposure to a bacterial heat shock protein can elicit antibodies against the rheumatoid arthritis susceptibility sequence in the third hypervariable region of HLA DRB10401.

To understand the biologic significance of amino acid sequence sharing between proteins from pathogens and hypervariable regions of HLA DR molecules, we studied the immunological status of a peptide from the third hypervariable region of IEb, the mouse equivalent of HLA DRb. We found that allo MHC peptides are recognized and self MHC peptide is tolerated. This suggests that MHC class II molecules may modulate the T cell repertoire not only by selective binding of antigenic peptides (determinant selection) but also by deleting or inactivating T cells specific for self MHC peptides. In the human, such a mechanism may explain why some HLA DR4 subjects have a deficient control of EBV infection.

As a first step in the analysis of a molecular mimicry model of rheumatoid arthritis, we addressed the question of whether tolerance to self-major histocompatibility complex (MHC) class II molecules includes tolerance to peptides from the third hypervariable region of their beta chain. We studied T cell responses to a peptide from the third hypervariable region of the Es beta chain, Es beta peptide (PEFLEQRRAAVDTYC), in different mouse strains after footpad priming with peptide in complete Freund's adjuvant. Strains of mice of the k or d haplotype (B10D2; H-2d, B10BR; H-2k) mounted a vigorous T cell response to the Es beta peptide. In mice expressing the Es beta chain either on the cell surface (B10S9R) or in the cytoplasm as free unassociated chain (B10S), no response could be detected. Binding studies using purified MHC class II molecules and competition for antigen presentation showed that the Es beta peptide binds Ak, Ad and As but not Ek. Thus, the nonresponder status of B10S and B10S9R mice appears to reflect self tolerance. Tolerance was also suggested by the observation that responder x nonresponder F1 crosses such as (B10D2 x B10S9R) and (B10BR x B10S9R) did not respond to Es beta peptide. Interestingly, mice derived from the (B10BR x B10S) cross responded to the Es beta peptide, suggesting that the immune system may not always tolerate peptides from the third hypervariable region of self-MHC class II molecules.

The binding of antinuclear antibody-positive juvenile arthritis (JA) sera to bovine thymus histones H1, H2A, H2B, H3, and H4 was studied by an enzyme-linked immunosorbent assay. Seventy-five percent of the JA patients tested positive for at least 1 antibody specificity. Antihistone antibodies were predominantly IgM, while IgG antibodies were less common and were restricted to histones H1 or H3. In the group of patients with JA of pauciarticular onset, antihistone antibodies were significantly more elevated in patients with past or present uveitis than in patients without a history of uveitis. Anti-H1 antibodies in JA patients were found to react mostly with determinants located in the carboxyl-terminal domain of the H1 molecule. Sera were also reactive with human histone H1(0) or chicken histone H5, which are H1 variants found only in nondividing cells.

Unstimulated and pokeweed mitogen (PWM)-stimulated "in vitro" IgM synthesis by peripheral blood lymphocytes has been evaluated by an ELISA method in 33 patients with juvenile rheumatoid arthritis (JRA). Lymphocyte PWM responses were significantly reduced in patients with JRA compared with healthy controls. When patients were divided according to disease activity, a significant increase of stimulation index (ratio of IgM synthesis with over that without PWM) has been observed in patients with disease remission compared with those with active disease. These findings confirm the defect in PWM-induced IgM synthesis by peripheral blood lymphocytes of patients with JRA and suggest that these abnormalities may be related to disease activity.

Fourteen patients with juvenile dermatomyositis (JDM) have been investigated for the presence of several serum autoantibodies: antinuclear (ANA), anti-single-stranded and double-stranded DNA, anti-histones, anti-Sm, anti-ribonucleoprotein, anti-SSA/SSB, anti-PM-1, anti-Jo-1, anti-mitochondrial, anti-smooth muscle, anti-gastric parietal cells, anti-cardiolipin (ACA) antibodies and rheumatoid factor. Patients were negative for all autoantibodies except for ANA and ACA. ANA were detected in 50% of the patients when tested on rat liver, but the percentage of positivity rose to 86% when HEp-2 cells were used as substrate. This finding suggests that HEp-2 cells represent a more sensitive substrate than rat liver for the detection of ANA in JDM. Three patients were positive for ACA; two of these presented vascular complications, thus suggesting a possible relationship between ACA and vascular involvement in JDM.

The aim of this work was to study possible differences in the humoral response against autologous and heterologous (bacterial and mammalian) ssDNA in children with diffuse connective tissue diseases (DCTD) compared with age matched controls. We found that IgM anti ssDNA were significantly increased in systemic lupus eritematosus (SLE) and in juvenile arthritis (JA), but not in juvenile dermatomyositis (JDM). IgG anti ssDNA were significantly elevated only in children with SLE. We next evaluated the binding specificity to human and bacterial ssDNA by inhibition assays. We found that SLE and JA sera recognised epitopes shared in common to endogenous and bacterial ssDNA. In contrast, in normal subjects IgG binding to bacterial DNA was not inhibited by human DNA, while IgG anti human ssDNA were cross reactive with the bacterial antigen. These data suggest that natural antibodies (IgM) producing cells are activated in some but not all DCTD, and that normal children have different reactivity against autologous and heterologous ssDNA with respect to SLE and JA patients.

In order to evaluate the diagnostic value of a lymphocyte stimulation test with casein and beta lactoglobulin in cow milk intolerance (CMI), we studied 26 patients with a diagnosis of CMI and 25 age-matched controls with intestinal diseases other than CMI. Lymphocyte stimulation test to casein (P less than .001) and beta lactoglobulin (P less than .001) was significantly higher among CMI patients. Seventeen patients were RAST positive and nine negative for whole milk proteins. No differences were found in the lymphocyte stimulation test response between RAST-positive and RAST-negative subjects. These results suggest that lymphocyte stimulation test with highly purified antigens in optimal concentrations is specific and could be useful in the diagnosis of CMI.