Increased frequency of interleukin 2-responsive T cells specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis.

Equal numbers of CD4+ T cells recognizing myelin basic protein (MBP) and proteolipid protein (PLP) are found in the circulation of normal individuals and multiple sclerosis (MS) patients. We hypothesized that if myelin-reactive T cells are critical for the pathogenesis of MS, they would exist in a different state of activation as compared with myelin-reactive T cells cloned from the blood of normal individuals. This was investigated in a total of 62 subjects with definitive MS. While there were no differences in the frequencies of MBP- and PLP-reactive T cells after primary antigen stimulation, the frequency of MBP or PLP but not tetanus toxoid-reactive T cells generated after primary recombinant interleukin (rIL-2) stimulation was significantly higher in MS patients as compared with control individuals. Primary rIL-2-stimulated MBP-reactive T cell lines were CD4+ and recognized MBP epitopes 84-102 and 143-168 similar to MBP-reactive T cell lines generated with primary MBP stimulation. In the cerebrospinal fluid (CSF) of MS patients, MBP-reactive T cells generated with primary rIL-2 stimulation accounted for 7% of the IL-2-responsive cells, greater than 10-fold higher than paired blood samples, and these T cells also selectively recognized MBP peptides 84-102 and 143-168. In striking contrast, MBP-reactive T cells were not detected in CSF obtained from patients with other neurologic diseases. These results provide definitive in vitro evidence of an absolute difference in the activation state of myelin-reactive T cells in the central nervous system of patients with MS and provide evidence of a pathogenic role of autoreactive T cells in the disease.

MHC-restricted depletion of human myelin basic protein-reactive T cells by T cell vaccination.

Activated autoreactive T cells are potentially pathogenic and regulated by clonotypic networks. Experimental autoimmune diseases can be treated by inoculation with autoreactive T cells (T cell vaccination). In the present study, patients with multiple sclerosis were inoculated with irradiated myelin basic protein (MBP)-reactive T cells. T cell responses to the inoculates were induced to deplete circulating MBP-reactive T cells in the recipients. Regulatory T cell lines isolated from the recipients inhibited T cells used for vaccination. The cytotoxicity of the CD8+ T cell lines was restricted by major histocompatibility antigens. Thus, clonotypic interactions regulating autoreactive T cells in humans can be induced by T cell vaccination.

Targeting of tumor necrosis factor to tumor cells: secretion by myeloma cells of a genetically engineered antibody-tumor necrosis factor hybrid molecule.

The construction, synthesis and secretion of a genetically engineered antibody-cytokine fusion molecule is described. To target tumor necrosis factor (TNF) to tumor cells, recombinant antibody techniques were used to produce a Fab-like antibody-TNF conjugate. At the gene level, the heavy chain gene of an antitransferrin receptor antibody was linked to a synthetic TNF gene encoding human TNF. Transfection of the heavy chain-TNF gene into a myeloma derived cell line which was producing the light chain of the same antibody, allowed the isolation of a cell line secreting a fusion protein of the expected molecular weight and composition. The culture supernatant of the cell line contained TNF cytotoxic activity towards murine L929 cells and human MCF-7 cells. Cytotoxicity towards the human cancer cells was inhibited by an excess of the original antitransferrin receptor antibody, indicating that the antibody-TNF molecules are targeted to the transferrin receptor rich tumor cells. Since the antibody genes used are chimeric (i.e. composed of mouse variable and human constant regions) and since DNA encoding human TNF was used, the hybrid protein is an example of a humanized immunotoxin-like molecule. These results illustrate the possibilities of antibody engineering technology to create and produce improved agents for cancer therapy. Furthermore, they demonstrate for the first time the ability of myeloma cells to secrete an antibody-cytokine chimeric molecule.

Autoimmune pathogenesis of multiple sclerosis: role of autoreactive T lymphocytes and new immunotherapeutic strategies.

Accumulating evidence indicates that multiple sclerosis (MS) is an autoimmune disease mediated by autoreactive T lymphocytes with specificity for myelin antigens. Initially, the evidence to support this hypothesis was based mainly on experiments performed in experimental allergic encephalomyelitis (EAE), the animal model of MS. In this model it was demonstrated that T cells reactive to several myelin antigens are encephalitogenic. Many recent immunological and immunohistochemical studies in MS have yielded further data to support this view. For instance, it was demonstrated that activated myelin basic protein (MBP) and proteolipid protein (PLP)-specific T cells accumulate in the central nervous system (CNS), and that clonally expanded MBP-specific T cells persist for several years in the blood of patients with MS. Furthermore, T cells with specificity for MBP were identified in the brain lesions of the patients. It is not yet clear how these autoreactive T cells are activated in the periphery, but several studies have suggested that viral antigens mimicking the myelin epitopes, or superantigens may be involved. Furthermore, we and others have provided evidence showing that the regulatory mechanisms that control autoreactive T cells in healthy subjects are potentially defective in MS patients. In addition to myelin reactive T cells, B cells producing myelin-specific antibodies and gamma delta T cells may also play an important role in the autoimmune cascade. Based on the recent insights in the disease mechanisms, new experimental therapies were developed to target specifically the pathogenic lymphocytes in MS. Some therapies yielded encouraging data in pilot studies, whereas phase III trials of other drugs showed beneficial effects on the disease course. In this article, we overview the most recent data on the role of autoreactive lymphocytes in the pathogenesis of the disease, and discuss some of the recently developed immunotherapeutical strategies in MS.

Parity-induced changes in global gene expression in the human mammary gland.

The protective effect of an early first full-term pregnancy in relation to breast cancer risk is well established, but the molecular and cell-specific changes in the human mammary gland involved remain unclear. To identify the molecular changes associated with pregnancy-induced differentiation, we analysed the global gene expression profiles of normal mammary tissues from both a parous and a nulliparous woman, using serial analysis of gene expression. This approach allowed us to identify sets of genes, known and unknown, that are differentially expressed in parous versus age-matched nulliparous mammary gland tissues. The normal mammary gland of a multiparous woman is characterized by several known differentiation markers such as casein kappa, casein beta, keratin 14, CCAAT/enhancer binding protein beta and delta and adipsin. Candidate genes involved in cytoarchitectural remodelling and growth inhibition with a potential role in pregnancy-induced protection against breast cancer were also observed. Several genes that are highly expressed in the nulliparous mammary gland and that are lost after pregnancy, encode for growth promoting, cytoskeletal and extracellular matrix proteins. One of these genes, the small breast epithelial mucin, is almost completely downregulated upon a first full-term pregnancy but is known to be expressed in more than 90% of invasive ductal carcinomas.

T cell vaccination: clinical application in autoimmune diseases.

T cell responses to myelin basic protein (MBP) are implicated to play an important role in the pathogenesis of multiple sclerosis (MS). These MBP autoreactive T cells are found to undergo in vivo activation and clonal expansion in patients with MS. They accumulate in the brain compartment and may reside in the brain lesions of patients with MS. As MBP-reactive T cells potentially hold a central position in initiation and perpetuation of the brain inflammation, specific immune therapies designed to deplete them may improve the clinical course of the disease. We review here the recent application of T cell vaccination in patients with MS to deplete circulating MBP-reactive T cells. The results of our phase I clinical trial indicate that T cell vaccination with inactivated MBP autoreactive T cells induces specific regulatory T cell network of the host immune system to deplete circulating MBP-reactive T cells in a clonotype-specific fashion. The immunity induced by T cell vaccination is clonotype specific and long-lasting. Our longitudinal clinical evaluation further suggests a moderately lower rate of clinical exacerbation, disability score, and brain lesions (measured by magnetic resonance imaging) in vaccinated patients than in matched controls. Our study should encourage further investigation on the treatment efficacy of T cell vaccination and further improvement for its clinical administration in other human autoimmune diseases. This review discusses the immune regulation and therapeutic administration of T cell vaccination in human autoimmune diseases, exemplified by our recent T cell vaccination trial in MS.

Construction and expression of antibody-tumor necrosis factor fusion proteins.

The construction, expression and secretion of two genetically engineered antibody-cytokine hybrid fusion proteins is described. To target tumor necrosis factor (TNF) to tumor cells, recombinant antibody techniques were used to generate F(ab')2-like antibody-TNF fusion proteins. At the gene level, an antitransferrin receptor antibody heavy chain gene was linked to a synthetic gene coding for human TNF. The chimeric heavy chain-TNF genes were introduced into a light chain secreting transfectoma cell line, which was producing the light chain of the same antibody. Cell lines were isolated which secreted antibody-TNF fusion proteins of expected size and composition. Culture supernatant of these cell lines contained TNF cytotoxic activity towards murine L929 cells and human MCF-7 cells, indicating that TNF is still active in the fusion protein constructs. These results illustrate the feasibility of the antibody engineering technology to create and produce chimeric mouse-human immunotoxin-like molecules. Furthermore, they demonstrate the ability of mammalian (myeloma) cells to express and secrete antibody-cytokine hybrid molecules with potential use in anticancer therapy.

Development and functional characterization of a murine/human chimeric antibody with specificity for the human interleukin-2 receptor.

A murine/human chimeric antibody, with specificity for the human interleukin-2 receptor, was developed by genetic engineering. For this purpose, the light and heavy chain variable region exons encoding the murine monoclonal antibody 2C8 were isolated and inserted into expression vectors containing the human kappa and gamma-1 constant regions. After transfection by electroporation of the chimeric genes into murine Sp 2/0 hybridoma cells, transfectomas secreting the complete chimeric antibody were selected. The chimeric antibody has similar binding properties as the original murine antibody. The in vitro cytotoxic effects of the murine and the chimeric antibodies were compared.

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.

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.

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.

Superfusion of normal and neoplastic mouse mammary tissue with estrogens.

Slices of normal mammary tissue from pregnant and lactating mice and slices of neoplastic (MXT) mouse mammary tissue were superfused with estradiol (E2) and estrone (E1) each labeled with a different isotope. Isotope concentrations in tissue and perfusate at the steady state were used to calculate fractions and rates of uptake, metabolism and release of estrogens by the tissue perfused. Both E2 and E1 entered equally well and were concentrated to the same extent by normal and neoplastic mammary tissue. However, much smaller tissue to medium ratios and larger diffusible fractions of estrogens were found in tumor slices as compared to mammary tissue from both pregnant and lactating mice, the uptake being the highest in pregnancy mammary glands. The interconversion between E1 and E2 was found to favor the formation of E2 in normal mammary tissue, the metabolic activity being the highest in lactating glands. In the MXT tumor slices the conversion of E1 into E2 was predominant as well but in contrast to the almost negligible metabolism of E2 in normal mammary tissue, a large fraction of E2 was converted into E1. The observed differences in estrogen uptake and metabolism between pregnant and lactating mammary glands were in concordance with the functional characteristics of the tissues. Furthermore, E1 was shown to play an important role in the mouse mammary gland as a metabolic precursor of E2. A different metabolic pattern was found in neoplastic mammary tissue.

Prediction of the DST results in depressives by means of urinary-free cortisol excretion, dexamethasone levels, and age.

This study investigates the relationships between cortisol escape from suppression by dexamethasone during a depressive episode, and the baseline activity of the hypothalamic-pituitary-adrenal (HPA) axis, circulating dexamethasone levels, and age. To this end, we measured urinary-free cortisol (UFC) excretion in 24-hr urine samples and the 8 AM cortisol and dexamethasone levels after administration of 1 mg dexamethasone in 50 depressive patients. We found that up to 54% of the variance in the postdexamethasone cortisol values could be explained by the multiple regression on UFC, age, and dexamethasone levels. By utilizing these three parameters, the dexamethasone suppression test (DST) nonsuppressor/suppressor state was correctly identified in 92% of the subjects. It was shown that an important part of the variance in postdexamethasone cortisol is actually background variance, irrelevant to depression and produced by the cumulative effects of the three aforementioned parameters. Only a small part (less than 20%) of the variance in postdexamethasone cortisol is determined by the actual depressive state. It was concluded that (1) baseline hypersecretion of cortisol, (2) decrements in the bioavailability of the test substance, (3) increasing age, and (4) the depressive state per se--all of which are cumulative--contribute independently to cortisol escape from suppression by 1 mg dexamethasone.

Effects of dexamethasone on the availability of L-tryptophan and on the insulin and FFA concentrations in unipolar depressed patients.

Several authors have shown that the availability of L-tryptophan (L-TRP) in the serum is lower in patients with major depression than in controls. It has recently been reported that the administration of a dose of dexamethasone sufficient to cause cortisol suppression also caused significant decrements in the availability of L-TRP. In order to elucidate the putative pathophysiological mechanisms underlying this decreased L-TRP disposition, the authors examined 37 depressed women categorized according to the DSM-III. L-TRP, the sum of five competing amino acids (CAA), the L-TRP/CAA ratio, and insulin and free fatty acid (FFA) levels were determined both before and after oral administration of 1 mg dexamethasone. The availability of L-TRP was significantly lower in depressed women with melancholia compared with simple major and minor depressives. The baseline disposal of L-TRP was related neither to insulin nor to FFA concentrations Dexamethasone administration significantly reduced the L-TRP and L-TRP/CAA values and increased FFA and insulin levels. Postdexamethasone L-TRP and FFA levels were significantly and positively correlated.

Pituitary and adrenal hormone responsiveness to Synacthen in melancholic subjects versus subjects with minor depression.

Increased adrenal cortex responsiveness to adrenocorticotropic hormone (ACTH) has been suggested to contribute to increased cortisol secretion in dexamethasone nonsuppression and melancholia. To further examine this hypothesis, the following variables were examined in 68 patients with unipolar depression (minor, n = 24; simple major, n = 25; melancholic, n = 19): basal or post-Synacthen [ACTH(1-24), 250 micrograms IV] intact ACTH(1-39), beta-endorphin/beta-lipotropin, cortisol, and androstenedione concentrations, as well as the postdexamethasone (DST) plasma ACTH(1-39) and cortisol values. Melancholic subjects showed significantly higher baseline ACTH(1-39), beta-endorphin/beta-lipotropin, and androstenedione values compared with subjects with minor depression. No significant differences in post-Synacthen cortisol or androstenedione secretion between any of the groups or between [ACTH(1-39) or cortisol] DST nonsuppressors and suppressors were found. No significant relationships between DST and ACTH test results were observed. Abnormally increased post-DST cortisol values in melancholic subjects were highly predicted (> 68% of the variance) by post-DST intact ACTH levels. ACTH(1-39) values were significantly lower after Synacthen administration in melancholic subjects than in subjects with minor depression. These results are not consistent with the hypothesis that melancholia is characterized by an increased adrenocortical responsivity to exogenous ACTH compared with minor depression or that DST nonsuppression is due to adrenal hyperresponsiveness.

Decreased serum dipeptidyl peptidase IV activity in major depression.

It has been recently shown that severe depression is characterized by immune dysfunctions such as blunted mitogen-induced blast transformation, which is linked to interleukin-2 (IL-2) mechanisms, and to autoimmune responses. In order to explore one of the putative pathophysiological mechanisms underlying both factors, we have measured the predexamethasone and postdexamethasone serum dipeptidyl-peptidase IV (DPP IV) activity in depressed inpatients and normal controls. This enzyme is an important mediator of IL-2-related blast proliferation, and it may play a role in autoimmunity. We found significantly lower DPP IV levels in major depressives as compared with healthy controls, and melancholics exhibited significantly lower enzyme activity than minor depressives. There was a significant negative correlation between serum DPP IV activity and the severity of illness. However, we were unable to detect any significant relationships between DPP IV on the one hand, and mitogen-induced blast transformation, soluble IL-2 receptor accumulation in PHA culture supernatant, total number of leukocytes and lymphocytes, T lymphocytes, CD4+ and CD25+ cells, on the other. Men exhibited significantly higher serum DPP IV levels than women.

Fine analysis of cytolytic and natural killer T lymphocytes in the CSF in multiple sclerosis and other neurologic diseases.

Cytolytic T lymphocyte precursors (CTL-p) and natural killer precursors (NK-p) in the CSF of 15 MS patients and 11 patients with other neurologic disorders (OND) were quantitatively assessed, using a T-lymphocyte microculture system that allows clonal expansion of all human T cells. CSF CTL-p and NK-p frequencies were higher in patients with OND of inflammatory nature than in patients with noninflammatory OND. In all MS patients, these frequencies were higher in the CSF than in their peripheral blood. Surprisingly, in all patients studied, the CSF contained a substantial number of CTL-p with a helper (CD4+) phenotype.

Encephalomyelitis-associated antimyelin autoreactivity induced by streptococcal exotoxins.

OBJECTIVE: After implicating Streptococcus pyogenes as causing acute disseminated encephalomyelitis (ADEM) in a child, we wanted to prove that in vivo activation of autoreactive T lymphocytes by superantigens of this Streptococcus contributed to the dramatic demyelination. BACKGROUND: ADEM is a demyelinating disorder of the CNS sharing many similarities with MS. Demyelination in MS is considered to be the result of an autoimmune process mediated by autoreactive T lymphocytes with specificity for myelin antigens. METHODS: Phenotypic analysis and proliferation assays on blood monocytes, as well as isolation of myelin basic protein (MBP)-reactive T-cell lines/clones; and TCR repertorium analysis by PCR-ELISA and cytokine production. RESULTS: 1) The blood T-cell receptor (TCR) repertoire was compatible with in vivo expansion induced by S. pyogenes exotoxins. 2) TCR expression analysis indicated clonal expansion of CD8+ MBP-reactive T cells, suggesting in vivo activation. MBP-reactive T cells showed crossreactivity to S. pyogenes supernatant and exotoxins. 3) Cytokine mRNA quantification of the mononuclear cells revealed a Th2-biased profile. CONCLUSION: In vivo exposure to S. pyogenes may have induced activation of pathogenic myelin reactive T cells, contributing to the dramatic inflammatory demyelination.

A murine monoclonal antibody to human breast cancer cells associated with DNA ploidy status.

A murine monoclonal antibody, 5D10, raised against the human breast cancer cell line MCF7 reacted preferably with mammary carcinomas and weakly with normal epithelial cells. The antigens recognised by the antibody had molecular weights of about 28 and 90 kD. The reactivity of the antibody to human breast carcinomas correlated with the DNA ploidy status of the tumour cells. Upon analysis of 54 breast carcinoma specimens, the percentage of antibody positive cells was significantly higher in tumours with an aneuploid stemline than in those with a diploid DNA content (P less than 0.001). This antibody therefore could be a useful tool in evaluating the prognosis of breast carcinomas.