Mutation detection in the alpha-1 antitrypsin gene (PI) using denaturing gradient gel electrophoresis.

A method for mutation detection in the alpha-1 antitrypsin gene (protease inhibitor 1; PI) has been developed using denaturing gradient gel electrophoresis of PCR amplified gene fragments. Using this experimental approach, all common phenotypes and mutations could be detected. Denaturing gradient gel electrophoresis (DGGE) was compared with standard isoelectric focusing (IEF) in 20 potential alpha1-antitrypsin deficient patients and their relatives. The genotype determined by DGGE was found to be more reliable in some cases than IEF, which is essential for a proper diagnosis of alpha-1 antitrypsin malfunctioning.

Skewed T-cell receptor variable gene usage in the synovium of early and chronic rheumatoid arthritis patients and persistence of clonally expanded T cells in a chronic patient.

OBJECTIVE: Autoreactive T cells may contribute to the pathogenesis of rheumatoid arthritis (RA). We studied the T-cell receptor (TCR) V-gene repertoire in the blood and synovium of early and chronic RA patients using polymerase chain reaction-enzyme-linked immunosorbent assay to evaluate possible differences between these patient groups. RESULTS: Over-represented TCR V genes were observed in the synovium, but not in the blood of all RA patients (n = 38). The number of over-represented V genes was higher in the synovium of chronic RA patients (n = 31) than in that of early RA patients (n = 7). The V-gene profile was different among patients, and similar in the two knees for patients with bilateral synovitis (n = 5). The clonal composition of over-represented TCR BV genes in a patient with early RA and a patient with chronic RA was further studied by CDR3 region sequence analysis. A high level of clonal diversity was found in the joints and the blood of the early RA patient, suggesting a polyclonal T-cell expansion. In the chronic RA patient, predominant clonal expansions were observed in the blood and synovium, and some expanded clones were still present 2 yr later. CONCLUSIONS: The observation of similar T-cell populations in both joints in patients with bilateral synovitis and the persistence of clonally expanded T cells for more than 2 yr in the joints of a chronic RA patient may indicate a pathogenic role for these cells in the disease process.

Lack of association between estrogen receptor genotypes and bone mineral density, fracture history, or muscle strength in elderly women.

The PvuII polymorphism of the estrogen receptor (ESR) gene and its relation to bone mineral density (BMD), fracture history, and muscle strength was studied in 313 postmenopausal (76 +/- 5 years) women of Caucasian origin, of whom 142 had suffered from a fragility fracture after the age of 50 years (14 with fracture of the hip, 38 of the spine, 45 of the wrist, and 85 of other bones). The ESR genotype distribution was similar in women with and without a history of fragility fracture (PP 21%, Pp 43%, pp 36% compared with PP 18%, Pp 47%, pp 35%). We did not find a correlation between the ESR genotypes and BMD at the lumbar spine, the femoral neck, or the proximal forearm. No association was found with grip or quadriceps strength. We further evaluated the relationship between the vitamin D receptor (VDR) and ESR haplotypes and BMD in a random subgroup of 270 elderly women. No differences were found in women with the BBpp versus the bbPP haplotype in the femoral neck (mean difference +/- SD, in Bbpp compared with bbPP groups: -0.05 +/- 0.15 g/cm2), the spine (0.01 +/- 0.13 g/cm2), or the forearm (0.04 +/- 0.08 g/cm2). The significant association of quadriceps strength with VDR genotypes (25% lower in BB compared with bb genotype, p < 0.05) was not influenced by ESR haplotypes. We conclude that in elderly Caucasian women the PvuII ESR polymorphism is not associated with osteoporosis, fracture history, nor muscle strength and does not influence the association of bone density and muscle strength with polymorphism of the VDR.

Identification of overrepresented T cell receptor genes in blood and tissue biopsies by PCR-ELISA.

The analysis of T cell receptor variable (TCR V) gene repertoires in blood or tissues may provide important information when studying immunopathological mechanisms. The overexpression of a TCR gene may indicate the expansion of the corresponding T cell subset. In autoimmune diseases, clonally expanded T cell subsets in the affected organs may represent pathogenic lymphocytes. We describe a simple, rapid and sensitive method to determine the TCR AV and BV gene repertoire using a PCR-ELISA method. RNA is extracted from lymphocytes, transcribed to cDNA, which is then used as a template for PCR with 19 different TCR AV gene and 20 BV gene specific primers as the forward primer, and a digoxigenin (DIG) labeled AC/BC primer as the reverse primer. The DIG labeled PCR amplicons are hybridized with a fluorescein isothiocyanate (FITC) labeled TCR C region specific probe. Finally, the amplicons are quantified by ELISA using anti-FITC coated microtiter plates, and an anti-DIG conjugated peroxidase. Although PCR-ELISA cannot accurately quantify the expression level of a given TCR gene, overrepresented TCR V genes are easily identified by comparing the relative expression levels of each individual V gene in the total V gene repertoire. We demonstrate that this technique can be used to determine TCR profiles in blood and tissue samples containing as few as 50,000 T cells. In combination with CDR3 fragment size analysis, this method is an efficient tool to identify clonally expanded T cell subsets in the synovial biopsies of rheumatoid arthritis patients.

Cytokine mRNA profile of myelin basic protein reactive T-cell clones in patients with multiple sclerosis.

Autoimmune mechanisms involving T-cell responses to (a) myelin autoantigen(s), such as myelin basic protein (MBP), are thought to contribute to the pathogenesis of multiple sclerosis (MS). Cytokines may play a central role in the regulation of the pathogenic autoimmune responses in MS and the mediation of tissue damage in the disease. To study the cytokine expression of myelin reactive T-cells in MS, we determined the cytokine mRNA levels in a panel of blood derived MBP-specific T-cell clones derived from MS patients (33 clones) and normal controls (21 clones), using a novel quantitative RT-PCR method. Our results demonstrate that MBP-specific T-cells, both from MS patients and control subjects, predominantly display a Th1- or Th0-like cytokine pattern. Although MS clones express higher levels of TNFalpha and IL-10 mRNA, these differences do not reach statistical significance. Interestingly, significantly increased TNFalpha and IFNgamma mRNA levels were observed among clones derived from HLA-DR2 positive versus HLA-DR2 negative MS patients. This HLA halpotype is known to be associated with MS. The high levels of TNFalpha and IFNgamma mRNA observed in MBP-reactive T-cell clones from MS patients indicate an important role of these cytokines in the disease process. Our data lend further support to the pathogenic role of MBP-reactive T-cells in MS.

Gammadelta T cell responses to activated T cells in multiple sclerosis patients induced by T cell vaccination.

To explore the hypothesis that gammadelta T cells may regulate activated alphabeta T cells, we studied gammadelta T cell responses to alphabeta T cell clones in Multiple Sclerosis (MS) patients who received attenuated autologous autoreactive T cells. We recently conducted a pilot study of T cell vaccination with myelin basic protein reactive T cells in MS. Since T cell vaccination upregulates the anti-vaccine T cell responses, we evaluated gammadelta T cell reactivity towards the vaccine in the vaccinated patients. Lymphocytes were stimulated in vitro with irradiated vaccine cells and the responding lines were checked for the presence of gammadelta T cells. Our data demonstrate that in the majority of vaccinated MS patients gammadelta T cells expand upon stimulation with the vaccine cells. The responding gammadelta T cells were predominantly Vdelta1+/Vgamma1+, and represented diverse clonal origins. The gammadelta T cells could not inhibit in vitro proliferation of the vaccine T cells and displayed low cytotoxic reactivity towards the vaccine clones. However, they produced high levels of IL2, TNFalpha and IL10. These results indicate that gammadelta T cells can be stimulated by activated alphabeta T cells, and that these gammadelta T cell responses are upregulated after T cell vaccination. These findings suggest that gammadelta T cells are involved in peripheral mechanisms to control activated autoreactive T cells.

Clonal expansion of mycobacterial heat-shock protein-reactive T lymphocytes in the synovial fluid and blood of rheumatoid arthritis patients.

OBJECTIVE: To examine the reactivity pattern and T cell receptor (TCR) characteristics of mycobacterial heat-shock protein 65 (hsp65)-reactive T cells generated from paired synovial fluid (SF) and peripheral blood (PB) samples obtained from rheumatoid arthritis (RA) patients and from healthy subjects. METHODS: The reactivity pattern of hsp65-reactive T cell clones generated under limiting-dilution conditions was analyzed in 3H-thymidine incorporation assays. The TCR variable regions of these hsp65-reactive T cells were characterized by polymerase chain reaction with TCR AV- and BV-specific primers and by DNA sequence analysis of the third complementarity-determining region (CDR3). RESULTS: The hsp65-reactive T cells derived both from RA patients and controls preferentially recognized the 1-170 and 303-540 regions of hsp65 and did not cross-react with human hsp60. The hsp65-reactive T cell clones derived from RA patients displayed a restricted TCR AV and BV gene usage, which can be attributed to the limited clonal origin(s) of the independent T cell clones, as evidenced by CDR3 sequence analysis. These clonally expanded T cells were found in both PB and SF and in different inflamed joints of RA patients. CONCLUSION: Our study suggests that there is in vivo clonal activation and expansion of mycobacterial hsp65-reactive T cells in patients with RA.

Influence of the vitamin D receptor gene alleles on bone mineral density in postmenopausal and osteoporotic women.

It is well established that genetic factors contribute to bone turnover and bone density. Evidence exists suggesting that a major part of this genetic influence may be due to polymorphisms in the vitamin D receptor (VDR) gene. However, it is not clear whether the VDR genotype effect persists in elderly women. In the present study, the relationship between the BsmI, ApaI, and TaqI polymorphisms in the VDR gene, and the bone mineral density (BMD) at the lumbar spine, the femoral neck (FN), and the proximal radius was investigated in a large group of elderly women (75.5 +/- 5.0 years) of Caucasian origin and in 84 Type I osteoporotic women (66.6 +/- 8.4 years). We did not find a correlation between the VDR genotypes and BMD in elderly women. However, a significantly higher FN-BMD was observed in obese (body mass index [BMI] > 30 kg/m2) versus nonobese (BMI < 30 kg/m2) women (p < 0.01). This relationship was observed for all BsmI genotypes. Furthermore, the FN-BMD of nonobese women with bb BsmI genotype was 5% higher than that of women with the BB genotype (p = 0.04). We conclude that the VDR gene polymorphisms influence the FN-BMD in nonobese postmenopausal women. In a second part of the study, possible correlations between the VDR gene polymorphisms and osteoporosis Type I were analyzed. Our data could not reveal any association between these parameters.

Quadriceps and grip strength are related to vitamin D receptor genotype in elderly nonobese women.

Osteoporotic fragility fractures are related to bone density and injury, which are both related to muscle strength. The influence of genetic factors, such as the vitamin D receptor (VDR) polymorphism on bone mineral density (BMD), is documented but still controversial, and is not known for muscle strength. In the present study, we investigated the association between the VDR BsmI polymorphism and BMD (femoral neck [FN], lumbar spine [LS], and proximal forearm [FA]) and muscle strength (quadriceps and grip strength) in 501 healthy women older than 70 years. No association was found between the VDR genotypes and BMD in elderly women. However, in nonobese women (body mass index <30 kg/cm2), the BMD in the FN was 5% higher in women with the bb BsmI genotype than in women with the BB genotype (p < 0.05). After correction for muscle strength, no association was found. A significant association between the VDR genotypes and quadriceps and grip strength was observed. In nonobese women, a 23% difference in quadriceps strength (p < 0.01) and 7% in grip strength (NS) was observed between the bb and BB genotype of the VDR. After correction for confounding factors and BMD, this association was significant for quadriceps and grip strength. These results indicate a major association of an allelic variant at the VDR locus with muscle strength in elderly nonobese women, which could explain a small association between VDR polymorphism with BMD in the femoral neck in nonobese women. No such associations were found in obese women, suggesting that factors related to obesity obscure such an association.

Mono- and bispecific single-chain antibody fragments for cancer therapy.

Especially when dealing with solid cancers, single-chain antibody fragments (scFvs) have a lot of advantages. Due to their small size (27 kDa), these proteins clear more rapidly from the blood, and penetrate faster and deeper into tissues, than whole antibodies. Furthermore, the lack of constant regions ensures that they are not retained in tissues such as the liver and kidney. This reduces possible toxic side-effects. Single-chain construction is normally done by polymerase chain reaction (PCR). To decrease the overall cost of oligonucleotide primer synthesis, time-consuming primer design, multiple PCR reactions and individual PCR optimization, we designed a universal single-step overlap extension PCR protocol using hybridoma cDNA as a template. To overcome the lack of effector function, bispecific scFvs, consisting of an scFv produced against a tumour-associated antigen fused to a T cell marker-specific scFv, are being created, starting from already assembled scFv, by means of two additional PCR reactions. In this paper we describe both PCR methods that were successfully used to create scFvs against the human transferrin receptor, the human interleukin-2 receptor, the human CD3 molecule, a breast tumour-associated antigen and an anti-transferrin-anti-CD3 bispecific scFv.

Immunotherapy for cancer: construction, expression and functional characterization of chimeric antibodies.

Monoclonal antibodies (Mabs) are a potential key component for the treatment of cancer, because of their specificity and multiple effector functions. Hybridoma technology and progress in genetic engineering made it possible to customize antibody molecules, rendering them more suitable for selective application. A widely used technique is the construction of mouse-human hybrid molecules by recombinant DNA techniques. These so-called chimeric antibodies contain the murine variable (V) regions fused to the human constant (C) regions. In this report, a general approach is described for the production of chimeric antibodies. The gene segments encoding the murine variable heavy and light chain are isolated by the polymerase chain reaction and cloned into expression vectors containing the human gamma 1 heavy chain gene and the human K light chain gene, respectively. Subsequently, these constructs are transfected into a non-Ig-producing murine hybridoma, eg SP2/0 cells. The in vitro study of the functional characteristics and biological properties of the thus obtained chimeric antibodies are discussed.

Vaccination with autoreactive T cell clones in multiple sclerosis: overview of immunological and clinical data.

Although the etiology and pathogenesis of Multiple Sclerosis (MS) remain elusive, accumulating evidence indicates that MS is a chronic inflammatory disease with an autoimmune component, mediated by autoreactive T lymphocytes specific for myelin antigens. The triggering T cell autoantigen has not been identified yet, but recent immunological studies in MS and parallel experiments in experimental allergic encephalomyelitis (EAE), the animal model of MS, have indicated that myelin basic protein (MBP) can be considered as one of the major candidate autoantigens in the pathogenesis of the disease. Based on these observations, several therapeutic strategies have been developed aimed at the specific elimination or inactivation of MBP reactive T cells in MS. One of these approaches involves the immunization of MS patients with autologous attenuated autoreactive T cells to induce an immune response specifically targeted at these autoreactive T cells. This method, termed T cell vaccination, has been shown to prevent and treat EAE. We have recently conducted a pilot trial of T cell vaccination in a limited group of MS patients to evaluate the immunological responses to the injected cells. The data obtained indicate that this type of vaccination induces an effective anti-clonotypic T cell response leading to a specific depletion of circulating MBP reactive T cells. Preliminary data on the clinical effects are promising, encouraging further clinical trials.

Quantification of cytokine messenger RNA in transfected human T cells by RT-PCR and an automated electrochemiluminescence-based post-PCR detection system.

A fast method is reported for the precise and accurate quantification of cytokine mRNA, based on a quantitative polymerase chain reaction (PCR) assay. Post-PCR detection of the amplification products is achieved using an automated electrochemiluminescent (ECL) detection system. The target is amplified using a biotinylated forward and a tris(2,2'-bipyridine)ruthenium (II) (TBR)-labeled reverse primer. The amplification products are then captured on streptavidin coated paramagnetic beads and quantified by measuring the ECL signal of the TBR label. The results obtained are reproducible and accurate over a wide range (3 orders of magnitude) of concentrations. Quantitative results can be obtained using a standard curve which is generated with a synthetic external standard. This technique was applied to quantify tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin-2(IL-2) mRNA levels in human T cells transfected with the corresponding genes.

In vivo clonotypic regulation of human myelin basic protein-reactive T cells by T cell vaccination.

Autoreactive T cells specific for myelin basic protein (MBP) are implicated in the pathogenesis of multiple sclerosis. The mechanism by which MBP-reactive T cells are regulated in vivo remains unknown, but is thought to involve the clonotypic regulatory network that can be induced by immunization with attenuated T cells and TCR peptides. We reported previously that immunization of multiple sclerosis patients with irradiated MBP-reactive T cells (T cell vaccination) induced T cell responses to the immunizing clones, resulting in a clonal depletion of circulating MBP-reactive T cells. In this study, we demonstrated that in the majority of the recipients, MBP-reactive T cells remained undetectable in circulation over a period of 1 to 3 yr after vaccination, while they reappeared in some individuals (three of nine), coinciding with clinical exacerbation. The reappearing MBP-reactive T cells were found to originate from clonal origins different from those of T cells persisting before immunization, suggesting a shift of the T cell repertoire to other determinants of MBP. The immunization induces predominantly CD8+ regulatory T cells capable of lysing the immunizing clones in a clonotype-specific manner. The T cell responses induced by immunization were restricted to the immunizing clones and did not affect MBP-reactive clones not used for immunization. Our data further suggest that different hypervariable regions of the TCR may be involved in the observed clonotypic interaction. This study provides useful information for designing future clinical trials using T cell vaccination and other TCR-based therapeutic strategies.

Superantigen reactivity of gamma delta T cell clones isolated from patients with multiple sclerosis and controls.

gamma delta T cells have been implicated as playing a role in the demyelinating processes of multiple sclerosis (MS). However, the nature of the ligands which lead to activation and accumulation of gamma delta T cells in the brain lesions remains unknown. This study was undertaken to examine whether gamma delta T cells derived from cerebrospinal fluid and blood of MS patients could be stimulated by bacterial superantigens: staphylococcal enterotoxins A and B and toxic shock syndrome toxin-1. A panel of 16 gamma delta T cell clones isolated from MS patients and controls was found to react with the superantigens used at a nanogram range and displayed specific cytotoxic activity toward target cells pulsed with the corresponding superantigens. The responses of the gamma delta T cell clones did not require MHC-matched accessory cells and were not blocked by antibodies to the MHC molecules, suggesting a non-MHC restricted interaction. The superantigen reactivity was associated with both V delta 2+/V gamma 2+ and V delta 1+/V gamma 1+ subsets, reportedly found in the MS lesions. Our data suggest an alternative pathway which may account for gamma delta T cell activation in MS.

Quantification of cytokine mRNA expression by RT-PCR and electrochemiluminescence.

Variable gene expression constitutes a major mechanism for controlling cell development and cell function. To investigate these changed mRNA levels, a sensitive and quantitative assay is required. We describe a quick and easy method to quantify specific mRNAs by a combination of PCR and an electrochemiluminescent (ECL) detection of the amplified products. Total cellular RNA is reverse-transcribed and amplified with a biotinylated forward primer and a Tris (2,2'-bipyridine) ruthenium (II) (TBR)-labeled reverse primer. The amplification product is captured on streptavidin-coated paramagnetic beads and quantified by ECL detection using the QPCR system 5000. The results can be converted to quantitative values with an external standard curve. This method permits accurate and reliable quantitation of cytokine mRNA expression.