Genetic analysis of low V beta 3 expression in humans.

While studying the T cell receptor (TCR) repertoire of normal individuals, we found that more than 20% of adults have low levels of circulating V beta 3.1+ T cells in both CD4 and CD8 populations. A similar frequency was found in fetal cord blood samples, suggesting that in most cases, the V beta 3.1low phenotype is inherited. In support of this conclusion, children expressing low levels were only found in families where one of the parents expressed this phenotype. In two large families, genetic studies showed that low expression was a recessive trait and dependent on inheritance of particular TCR VB gene complexes. Family members with the low phenotype, however, expressed VB3.1 genes with normal sequences and expressed normal levels of receptor per cell. Results from these families suggest that up to 50% of normal individuals may carry a VB3.1 allele that is defective in its ability to rearrange effectively. In another large family, low expression in one individual was shown not to be determined by genes within the TCR VB gene or major histocompatibility complexes, suggesting a different mechanism for low V beta 3.1+ T cells. Overall, our results describe novel mechanisms that result in low levels of V beta 3.1+ T cells in a relatively large subset of the normal human population.

Heterogeneity of autoreactive T cell clones specific for the E2 component of the pyruvate dehydrogenase complex in primary biliary cirrhosis.

The extraordinary specificity of bile duct destruction in primary biliary cirrhosis (PBC) and the presence of T cell infiltrates in the portal tracts have suggested that biliary epithelial cells are the targets of an autoimmune response. The immunodominant antimitochondrial response in patients with PBC is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). Hitherto, there have only been limited reports on the characterization and V beta usage of PDC-E2-specific cloned T cell lines. In this study, we examined peripheral blood mononuclear cells (PBMC) for their reactivity to the entire PDC complex as well as to the E1- and E2-specific components. We also examined the phenotype, lymphokine profile, and V beta usage of PDC-specific T cell clones isolated from cellular infiltrates from the livers of PBC patients. We report that PBMC from 16/19 patients with PBC, but not 12 control patients, respond to the PDC-E2 subunit. Interestingly, this response was directed to the inner and/or the outer lipoyl domains, despite the serologic observation that the autoantibody response is directed predominantly to the inner lipoyl domain. Additionally, lymphokine analysis of interleukin (IL) 2/IL-4/interferon gamma production from individual liver-derived autoantigen-specific T cell clones suggests that both T helper cell Th1- and Th2-like clones are present in the liver. Moreover, there was considerable heterogeneity in the T cell receptor for antigen (TCR) V beta usage of these antigen-specific autoreactive T cell clones. This is in contrast to murine studies in which animals are induced to develop autoimmunity by specific immunization and have an extremely limited T cell V beta repertoire. Thus, our data suggest that in human organ-specific autoimmune diseases, such as PBC, the TCR V beta repertoire is heterogenous.

Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation.

Bone marrow contains a rare population of mesenchymal stem cells (MSCs) capable of giving rise to multiple mesodermal tissues including bone, cartilage, tendon, muscle, and fat. The cell surface antigen recognized by monoclonal antibody SB-10 is expressed on human MSCs but is lost during their developmental progression into differentiated phenotypes. Here we report on the immunopurification of the SB-10 antigen and its identification as activated leukocyte-cell adhesion molecule (ALCAM). Mass spectrometry establishes that the molecular mass of ALCAM is 80,303 +/- 193 Da and that it possesses 17,763 +/- 237 Da of N-linked oligosaccharide substituents. Molecular cloning of a full-length cDNA from a MSC expression library demonstrates nucleotide sequence identity with ALCAM. We also identified ALCAM homologs in rat, rabbit, and canine MSCs, each of which is over 90% identical to human ALCAM in their peptide sequence. The addition of antibody SB-10 Fab fragments to human MSCs undergoing osteogenic differentiation in vitro accelerated the process, thereby implicating a role for ALCAM during bone morphogenesis and adding ALCAM to the group of cell adhesion molecules involved in osteogenesis. Together, these results provide evidence that ALCAM plays a critical role in the differentiation of mesenchymal tissues in multiple species across the phylogenetic tree.

Western blotting in evaluating Lyme seropositivity and the utility of a gel densitometric approach.

The antibody response to Borrelia burgdorferi is widely used in the diagnosis of Lyme neuroborreliosis and other manifestations of Lyme disease. However, a problem with immunoassays has been a significant number of false positives. The Western blotting technique is a useful adjunct in the serodiagnosis of other infections, but its use in Lyme borreliosis has been limited because of a lack of definition of what constitutes a positive assay. Using a gel densitometric analysis, we devised quantitative criteria for positivity and tested our criteria by matching blot results with clinical characteristics in a retrospectively studied group of 20 patients with Lyme disease, 23 healthy controls, and 18 patients with other neurologic and rheumatologic diseases. We then evaluated these criteria prospectively in serum from 35 ELISA-positive patients, and found that the serum from the majority of patients with positive serologies by ELISA were negative by Western blot. The Western blot-negative seropositive patients usually had other inflammatory or infectious diseases. We conclude that quantitative Western blotting is a helpful test in the serodiagnosis of Lyme neuroborreliosis and other manifestations of Lyme disease.

Anti-ligand antibodies as potential autoantigens: in vitro and in vivo characteristics of anti-bungarotoxin antibodies in the idiotype network.

Some antibodies to ligands of a receptor will have combining sites that structurally resemble the receptor's binding site for that ligand. The network hypothesis predicts that anti-idiotypic antibodies to these anti-ligand antibodies will also bind to the receptor. We pursued these hypotheses in the well-defined ligand-receptor system, alpha-bungarotoxin(BTX)-acetylcholine receptor (AChR). Monoclonal antibodies (mAbs) to BTX were generated; native BTX was used as the immunogen to optimize the probability of obtaining mAbs to the AChR binding site. These mAbs were then characterized for their ability to "mimic" AChR in the following in vitro assays: neutralization of BTX binding to native AChR on the surface of the cell line TE671, formation of a ternary complex with 125BTX-AChR, and ability of cholinergic ligands to interfere with binding to BTX. Three aBTX mAbs which had in vitro attributes of the AChR on the basis of these assays, were injected into C3H mice and serial sera tested for antibodies to Torpedo and murine AChR. Anti-AChR antibodies directed primarily to the gamma and delta subunits of the Torpedo AChR were detected, as well as low amounts of anti-mouse AChR antibody. The generation of anti-AChR antibodies by immunization with aBTX antibodies supports the network hypothesis and provides a theoretical basis for initiation of autoimmunity to cell receptors.

Prevalence of CD8+ T-cell expansions in relation to age in healthy individuals.

Clonal CD8+ T-cell expansions have been identified in the peripheral blood of healthy adults and occasionally in children. These expansions are often large, yet their etiology is unknown. This study evaluated the relationship between age and the prevalence of these expansions in a healthy population (n = 147) aged 9 months to 85 years. Expansions were determined using immunofluorescence staining with monoclonal antibodies to different T-cell receptor (TCR)-variable regions. The overall prevalence was 13.6% and increased linearly with age as follows: 0% for 9-month-olds, 2.7% for 4- to 12-year-olds, 13.3% for 20- to 30-year-olds, 20.7% for 35- to 50-year-olds, and 33.4% for 65- to 85-year-olds. Multiple expansions were observed only in the oldest group. Certain TCR-variable regions appeared to be preferentially utilized by these expansions, which suggests a response to a particular antigenic stimulus. Childhood illness and vaccination histories did not provide insight into the etiology of these expansions. Age was the only measured factor that was associated with these expansions.

In vitro neutralization by monoclonal antibodies of alpha-bungarotoxin binding to acetylcholine receptor.

In order to develop monoclonal antibodies that would neutralize binding of alpha-bungarotoxin to acetylcholine receptor in vitro, mice were hyperimmunized with native toxin. Frequent small doses of toxin were used. Hybridoma supernatants were screened by ELISA and six monoclonal antibodies isolated and tested. The anti-alpha-bungarotoxin monoclonal antibodies consisted of IgM, IgG1 or IgG2a antibodies. In an in vitro neutralization assay measuring the effect of the antibodies on the binding of iodinated alpha-bungarotoxin to BC3H1 and TE671 (mouse and human cell lines bearing acetylcholine receptor), three of the six monoclonal antibodies were able to neutralize toxin binding. These studies demonstrate the feasibility of using native toxin for the generation of hybridomas, and the potential of using in vitro neutralization assays to screen hybridomas for in vivo neutralization.

Comparison of polymerase chain reaction with culture and serology for diagnosis of murine experimental Lyme borreliosis.

After the intradermal inoculation of mice with Borrelia burgdorferi, the antibody response, culture, and histology of blood and target organs were assessed and compared with results of a nested polymerase chain reaction (PCR) assay. Of 247 specimens of heart, brain, bladder, and blood, the tested concordance between the PCR and culture was 72%. In the 69 instances of discordance, the PCR was positive in 51 and the culture was positive in 18; thus, the PCR was concordant or more sensitive in 93% of the tested organs. In mice infected with 10 spirochetes, serology confirmed by Western blotting (immunoblotting) was more sensitive than either culture or PCR of brain, bladder, or heart specimens. The organs most commonly culture or PCR positive were the heart and bladder; the brain was infected in only 26% of the animals. DNA hybridization was helpful in confirming the PCR product as being specific and, in some cases, in demonstrating a positive product in the face of negative agarose gels. PCR was less sensitive than culture in detecting the presence of spirochetes in blood specimens, possibly because of the presence of blood inhibitors. We thus found a nested PCR assay, using primers from a genomic sequence, to be a valuable adjunct to serology and culture in the study of murine Lyme borreliosis. The assay confirmed that, after small numbers of spirochetes are injected intradermally, the heart and bladder, and less frequently the brain, are sites of persistence of the spirochetes.

Chronic murine experimental myasthenia gravis: strength testing and serology.

CB6 (Balb/c x C57Bl/6 F1) and C57Bl/6 (B6) mice were hyperimmunized with Torpedo acetylcholine receptor (AChR) for 7 months. Control groups were hyperimmunized with bovine serum albumin. Antibody titers against Torpedo AChR rose quickly, reaching plateau levels by 3-4 months, while antibody to mouse AChR lagged by a few months, reaching plateau levels in 5 months. After the last immunization the mice maintained a state of stable autoimmunity for 9 months with high levels of antibodies against Torpedo and mouse AChR. Fatigability was measured on a programmable treadmill and remained present through the 9 months after the last immunization. CB6 mice had less weakness than the B6 mice, but the latter strain when immunized with BSA had more "false-positive" weakness. Titers of antibodies did not correlate with the degree of weakness measured on the treadmill. Despite the weakness and the high titers of anti-AChR antibodies, sera from myasthenic mice, in contrast to sera from myasthenic humans, were not able to block bungarotoxin binding to native AChR on the surface of BC3H1 cells.

Murine Lyme borreliosis: route of inoculation determines immune response and infectivity.

Outer surface protein A OspA is the major outer surface protein of B. burgdorferi, the causative agent of Lyme disease, and has been advocated as a vaccine candidate. It is recognized late or not at all in the course of human Lyme disease, but has been identified as a major antigenic epitope for the anti-spirochetal immune response in a number of experimental models of B. burgdorferi infection. We injected B.burgdorferi into mice and tested the appearance of immunoreactivity to OspA by Western blotting. Three routes of infection were studied; other variables investigated were inoculum size and isolate of spirochete and strain of mouse. OspA immunoreactivity, as determined by Western blotting, was readily elicited by injection of sonicates under almost any condition. Intraperitoneal or intravenous injection of infectious spirochetes, especially at infective inoculum sizes, or injection of the noninfectious B31 isolate by any route, resulted in OspA immunoreactivity. However, mice from the three strains tested infected intradermally did not develop significant OspA immunoreactivity, but instead developed strong responses to B.burgdorferi proteins of different molecular weights. These data suggest that during infection within the skin after intradermal inoculation, the OspA protein may be altered in some way to make it less immunogenic than when it is presented to the immune system under other circumstances.(ABSTRACT TRUNCATED AT 250 WORDS)

Mesenchymal stem cells in osteobiology and applied bone regeneration.

Bone marrow contains a population of rare progenitor cells capable of differentiating into bone, cartilage, muscle, tendon, and other connective tissues. These cells, referred to as MSCs, can be purified and culture expanded from animals and humans. This review summarizes recent experimentation focused on characterizing the cellular aspects of osteogenic differentiation, and exploration of the potential for using autologous stem cell therapy to augment bone repair and regeneration. The authors have completed an array of preclinical studies showing the feasibility and efficacy of MSC based implants to heal large osseous defects. After confirming that syngeneic rat MSCs could heal a critical size segmental defect in the femur, it was established that human MSCs form bone of considerable mechanical integrity when implanted in an osseous defect in an immunocompromised animal. Furthermore, bone repair studies in dogs verify that the technology is transferable to large animals, and that the application of this technology to patients at geographically remote sites is feasible. These studies suggest that by combining MSCs with an appropriate delivery vehicle, it may be possible to offer patients new therapeutic options.

Analysis of clonal CD8+ T cell expansions in normal individuals and patients with rheumatoid arthritis.

In the course of studying the circulating TCR repertoire in humans, we noted several individuals with an increase in the percentage of CD8+ T cells expressing a particular V region. In some cases, these CD8 expansions were dramatic, occupying over 40% of the total CD8 repertoire. Using a panel of mAbs to different TCR V regions, we found that over 30% of healthy adults (> 35 years of age) harbor an expansion that alters the peripheral blood CD8 TCR repertoire. A wide range of V regions were expressed by these expansions. Considering that the mAbs used cover only a portion of the V beta repertoire, the data suggest that over 70% of adults are likely to harbor such expansions. Junctional region sequencing showed that the CD8 subset expansions were clonal, and serial studies as long as 4 years showed that they persisted indefinitely. Expansions were not identified in the CD4 population. Discordant expression of one large V beta 6.7+ clone was found in one identical twin set, suggesting the possibility that an environmental exposure is involved in their generation and/or expansion. In one large family, we found five family members with a large CD8 subset expansion. Remarkably similar usage of J beta regions was noted, and two individuals demonstrated V beta 3-expressing clones with homologous CDR3 regions, differing by only one major substitution. The repertoire data from this family suggest that the T cell clones have arisen in response to a common Ag. Studies of patients with rheumatoid arthritis found a significantly increased frequency of circulating CD8 subset expansions that expressed a different V region repertoire compared with the healthy individuals studied. Overall, our results emphasize a frequent alteration in the human CD8 TCR repertoire, most likely related to an environmental exposure, in both healthy individuals and patients with rheumatoid arthritis. The presence of these expansions will be important to consider in any study of human TCR repertoire, and their implication for health and disease will be important to understand.

TCR expression of activated T cell clones in the lungs of patients with pulmonary sarcoidosis.

Sarcoidosis is a systemic granulomatous disease of unknown etiology in which CD4+ T cells seem to be critically involved. In the lungs of patients with pulmonary disease, CD4+ T cells accumulate in large numbers, and a subset of these cells is activated. By using both quantitative PCR and anti-V beta mAbs, we analyzed the TCR repertoire of total and activated bronchoalveolar lavage T cells, the latter subset being defined by the ability to proliferate in short-term culture supplemented with IL-2. Overall, there was little difference when TCR V beta expression of freshly isolated lung and peripheral blood cells was compared in individual patients. Some individuals did demonstrate a modest increase in a few V beta-expressing subsets. However, after 1 to 2 wk of in vitro growth in IL-2-supplemented media, bronchoalveolar lavage cells from most patients, but not from any healthy individuals, demonstrated a selective expansion of particular V beta-expressing subsets. Interestingly, different V beta-bearing subsets were expanded in different patients. Junctional region sequencing indicated that the proliferating T cells in culture were strikingly oligoclonal and were derived from T cell clones already selectively expanded in vivo. These results provide evidence for a disease process that involves recognition of local Ag(s) by specific subsets of CD4+ T cells. Analysis of the Ag specificity of these IL-2-expanded populations is likely to provide insight into the pathogenesis of this disease.