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Biomedical subjects

T Gallart

Publications and source records attributed to T Gallart.

At least 37 records · Page 2Linked to original sources

Desialylation of T lymphocytes overcomes the monocyte dependency of pokeweed mitogen-induced T-cell activation.

Activation of T lymphocytes by pokeweed mitogen (PWM) is strictly monocyte (Mo)-dependent and results in T-cell mitogenesis and interleukin-2 (IL-2) secretion, coupled with an inability to utilize IL-2 due to an impaired expression of functional IL-2 receptor (IL-2R). Such IL-2R impairment could arise in PWM-activated T cells themselves or, alternatively, be the result of Mo-derived influences, as it is known that PWM binds Mo strongly and does not or poorly binds lymphocytes, and Mo becomes rapidly destroyed in PWM-stimulated cultures of blood mononuclear cells or T cells plus Mo. The present study investigated these possibilities. The results show for the first time that desialylation of T lymphocytes strongly increases their PWM-binding capacity and, in addition, overcomes the Mo requirement for PWM to induce T-cell mitogenesis and IL-2 secretion. Such secreted IL-2 levels were even higher that those found in cultures of Mo-dependent PWM-activated T lymphocytes but similarly to the latter, PWM-activated desialylated purified T lymphocytes exhibited negligible high-affinity IL-2 binding capacity and an inability to utilize the IL-2 they produced. These effects were not due to desialylation itself, as indicated by data obtained with peanut agglutinin, a lectin that becomes strongly reactive with desialylated T lymphocytes. The data clearly indicate the existence of PWM-related events capable of impairing the expression of functional IL-2R without affecting IL-2 secretion, and indicate that such events are due to mechanisms arising at the level of PWM-activated T cells themselves.

Adult↗

Immunodetection of mitochondrial glycerophosphate dehydrogenase (mGDH) by a polyclonal antibody raised against a recombinant mGDH fragment product.

The mitochondrial enzyme glycerophosphate dehydrogenase (mGDH) plays an essential role in the B-cell glucose-sensing device and its activity in islet homogenates is impaired in several animal models of type 2 diabetes. We have now developed a polyclonal antibody, raised against a recombinant mGDH fragment product, that could be used for the immunodetection of mGDH. Total RNA was isolated from rat pancreatic islets and used in the synthesis of cDNA. Specific primers were designed that corresponded to the FAD binding domain of mGDH. The PCR product was purified and cloned into an appropriate expression vector used for transformation of Escherichia coli cells. The fusion protein was extracted from the transformed cells, further purified, and used for immunization of rabbits. The antibody recognized a single band of 72 kDa in rat islets and testis. The recombinant mGDH product was also recognized as a single band with the expected 65-kDa reference. An ELISA procedure was designed for detection of antibodies against the recombinant mGDH fragment product. The availability of the mGDH antibody opens the way to a number of further applications such as immunocytochemis- try and mGDH quantification in biological material.

Animals↗

Ovarian dermoid cyst-associated autoimmune hemolytic anemia: a case report with emphasis on pathogenic mechanisms.

Autoimmune hemolytic anemia (AIHA) as a manifestation of ovarian dermoid cyst (ODC) is a rare paraneoplastic syndrome of unknown pathogenesis. Among mechanisms postulated to explain this association, cross-reactivity between cyst and red blood cell (RBC) antigens, and local production of RBC autoantibodies by intracyst B lymphocytes are the most likely. Studies to test these hypothesis were done in a patient diagnosed of AIHA associated with a nonpalpable ODC, in whom the AIHA subsided after tumor excision. The RBC-bound autoantibody was an IgG directed against the Rh complex. The cyst's fluid content lacked detectable RBC autoantibodies or immunoglobulins, the latter being measured by a high-sensitivity assay. It also failed to inhibit the ability of the purified autoantibody to agglutinate RBCs. Ovarian dermoid cyst histology disclosed that (1) the biotin-labelled RBC autoantibody did not bind to ODC structures; (2) scanty amounts of small mature lymphocytes (50% CD45RO+; 50% CD20+) were present only in a few tissue sections; (3) plasma cells producing IgM or IgG were extremely scarce; and (4) deposits of immunoglobulins were not detected into the ODC. These data fail to support any of the aforementioned hypotheses on the pathogenesis of this paraneoplastic syndrome. Other possible mechanisms are discussed, and a wider use of imaging diagnosis to search for ODC in women with AIHA is emphasized.

Adult↗

Anti-islet cell and anti-insulin antibody production by CD5+ and CD5- B lymphocytes in IDDM.

Although CD5 + B lymphocytes are mostly committed to the production of polyreactive natural autoantibodies, CD5 + B lymphocytes committed to the production of somatically mutated and monoreactive high-affinity IgM autoantibodies have been also shown. Increased proportions of CD5 + B lymphocytes in some autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM), have been noticed. The present study was undertaken to analyse the differences between CD5 + and CD5- B lymphocyte subsets for production of IDDM-related autoantibodies, i.e. anti-human insulin antibodies (IA) and anti-human islet cell antibodies (ICA). For this purpose, Epstein-Barr Virus (EBV)-transformation of FACS cell-sorted CD5 + and CD5- B lymphocytes and unfractionated enriched B lymphocytes from nine IDDM patients treated exclusively with recombinant human insulin, and from four healthy control subjects was performed; a mean of 102-216 microcultures with a mean of 1,000-2,333 cells/microculture for each B-lymphocyte fraction and individual was established. Data show that both CD5 + and CD5- B-lymphocyte subsets from either normal subjects or from IDDM patients receiving recombinant human insulin, contain B lymphocytes committed to the production of IA-IgM as a common element of their repertoire. In contrast, cells committed to the production of IA-IgG were only detected among the CD5- B lymphocyte subset from some IDDM patients. Only one microculture, out of a total of 6,211 screened (from control subjects and patients), in the CD5- B-cell subset from a recently-diagnosed IDDM patient, was found to produce ICA-IgM lambda. This might suggest that the frequency of circulating B lymphocytes committed to the production of ICA is very low even in IDDM patients bearing serum ICA. EBV-transformed B cells producing the ICA-IgM lambda were stabilized and cloned by somatic hybridization technique. This ICA-IgM lambda human monoclonal antibody, designated HY1-MB91, is not polyreactive, but shows a restricted reactivity with human pancreatic islets, failing to react with other human tissues including cerebellar cortex, and lacking rheumatoid factor and anti-DNA antibody activities. It also lacks reactivity with pancreatic islets from other mammalian species (rat, mouse and monkey) as well as with other rat tissues, including cerebellar cortex. The antigen recognized by HY1-MB91 antibody in human islet cells is a cytoplasmic component mostly found in beta cells.

Adolescent↗

[CD4+ lymphocytes and opportunistic infections and neoplasms in patients with human immunodeficiency virus infection].

BACKGROUND: The CD4+ lymphocytes are the principal target cell for the human immunodeficiency virus (HIV). Their depletion originates a very severe cell immunosuppression, which conditions the appearance of opportunistic infections and neoplasms characteristic of AIDS. The aim of this study was to evaluate whether there is a relation between the degree of cell immunosuppression and the type of opportunistic infections and neoplasms which these patients develop in Spain. METHODS: The CD4+ lymphocyte counts in 400 adults with HIV infection who developed opportunistic infections or neoplasms were retrospectively reviewed (1987-1991). This determination was carried out during between two months prior to diagnosis of AIDS (CDC, 1987) to one month after such diagnosis. RESULTS: The results allowed opportunistic infections to be classified into three groups according to the grade of immunosuppression: 1) opportunistic infections with more than 0.2 x 10(9) CD4+ lymphocytes/l (45-60% of cases of tuberculosis, esophageal candidiasis and enteritis by Isospora belli); 2) opportunistic infections with 0-0.2 x 10(9) CD4/l (87-100% of the cases of pneumonia by Pneumocystis carinii, encephalic toxoplasmosis, visceral leishmaniasis and enteritis by Cryptosporidium); 3) opportunistic infections with 0-0.1 x 10(9) CD4 lymphocytes/l (70-100% of the cases of systemic cryptococcosis, retinitis by cytomegalovirus, progressive multifocal leukoencephalopathy and infection by Mycobacterium avium-intracellulare). With respect to the neoplasms, Kaposi's sarcoma was observed in patients with different degrees of immunosuppression. Seventy-five and 80% of the patients with non Hodgkin's lymphoma and primary cerebral lymphoma had less than 0.2 x 10(9)/l and less than 0.1 x 10(9)/l CD4+ lymphocytes, respectively. CONCLUSIONS: The CD4 lymphocyte counts may predict the type of opportunistic infections which patients with the human immunodeficiency virus infection may develop.

AIDS-Related Opportunistic Infections↗

Characterization of a monoclonal IgMK (IgMGAS) anti-Gd cold agglutinin (CA). Its coexistence with a monoclonal IgG3K (IgGGAS) without CA activity that might be clonally related to IgMGAS.

A monoclonal IgMK (IgMGAS) cold agglutinin (CA) of the infrequent anti-Gd specificity, found in a patient with Waldenström macroglobulinemia, has been characterized. IgMGAS uses a VH gene homologous (93.7%) to the reported VH251 germ-line, one of the two functional genes of the VH5 family, with differences in both framework regions and complementary determining regions (CDR). The VL gene is homologous to the reported 15AVK1 germ-line gene, recently described in an anti-i CA, with differences mostly clustered in CDR. In the patient's serum, IgMGAS coexisted with a monoclonal IgG3K (IgGGAS) that lacked CA activity but expressed the private idiotopes found on IgMGAS. Both Ig lacked reactivity with antibodies detecting VH1 or VHIII or VKIII subgroup regions or the VH4-21 gene product that is expressed by anti-I/i CAs. The K chains from both Igs showed the same isoelectrical mobility. Moreover, the k chains from both serum Igs showed the same N-terminal amino acid sequence. This sequence was identical to that predicted by the nucleotide sequence of VK1GAS gene segment, including one discrepancy at position 15 (Ile for Val) with respect to the consensus VK1 subgroup regions. Although these data do not exclude a possible independent clonal origin, they are consistent with the notion that IgGGAS might be clonally related to IgMGAS.

Aged↗

Delayed appearance of anti-myelin-associated glycoprotein antibodies in a patient with chronic demyelinating polyneuropathy.

A patient who had a polyneuropathy compatible with a chronic inflammatory demyelinating polyneuropathy and was initially negative for anti-myelin-associated glycoprotein (MAG) antibodies developed a double monoclonal gammopathy, IgM kappa and IgM lambda, two years after the diagnosis. The IgM kappa, but not the IgM lambda, exhibited strong anti-MAG antibody activity. The late appearance of the anti-MAG immunoreactivity suggests that in patients with an initial diagnosis of chronic inflammatory demyelinating polyneuropathy, the search for anti-MAG antibodies should be repeated during the course of the neuropathy.

Autoantibodies↗

Impaired post-transcriptional expression of interleukin-2 receptor in pokeweed mitogen-activated T cells.

The expression and role of interleukin-2/interleukin-2 receptor (IL-2/IL-2R) system in the pokeweed mitogen (PWM)-induced T cell mitogenesis was studied. In the absence of monocytes (Mo), both soluble and Sepharose-bound PWM fail to induce T cell mitogenesis even when exogenous IL-2 or IL-1 or IL-1 + IL-2 or IL-4 are also present. In the presence of Mo, PWM stimulation of T lymphocytes (highly depleted of B lymphocytes) induces as much IL-2 mRNA as phytohemagglutinin (PHA), but results in higher and persistent IL-2 levels in culture supernatants despite the concomitant T cell mitogenesis, suggesting that PWM-activated T cells do not utilize the IL-2 they produce. Confirming this notion, Mo-dependent PWM-preactivated T cells, as compared to PHA-preactivated ones: (a) failed to consume exogenous IL-2 and their mitogenic response did not increase upon exposure to exogenous IL-2; (b) exhibited very low numbers of high-affinity IL-2R; and (c) showed lower expression of IL-2R p55 and undetectable expression of IL-2R p75 on their surface. Moreover, the PWM-induced T cell mitogenesis was not inhibited by anti-IL-2 or CD25 antibodies and only partially (50%-60%) inhibited by cyclosporin A, while these treatments abrogated the PHA-induced one. PWM-activated T cells, as compared to the PHA-activated ones, exhibited as high (p55) or even higher (p75) mRNA expression of both IL-2R p55 and p75 subunits. The possibility that PWM interferes with IL-2R subunits once expressed on the T cell surface was excluded. Thus, intracellular PWM-related events are likely to impair IL-2R expression post-transcriptionally. Possible explanations for this effect and its relation with the capacity of PWM to induce T cell-dependent B cell differentiation are discussed.

Cyclosporine↗

Analysis of islet cell antibodies reactivity to a human islet cell line.

A human pancreatic beta cell line (HP62) was tested for reactivity with islet cell antibodies (ICA) as compared with previously-established methods. Using indirect immunofluorescence test, we found that HP62 cell line failed to react in a specific way with ICA from type 1 (insulin-dependent) diabetic patients since sera from normal controls showed a reactivity similar to that found in the patients. So, the usefulness of this human beta cell line as a tool of immunological purpose is questioned when indirect immunofluorescence procedures are used.

Autoantibodies↗

Stimulation through the TCR/CD3 complex up-regulates the CD2 surface expression on human T lymphocytes.

Despite the well known interrelationship between the CD2- and CD3-mediated signal transduction pathways, it is not well established whether the CD2 surface expression can be regulated by triggering of TCR/CD3 complex. In this study we show that the stimulation of human PBMC with the Cris-7 (CD3) mAb, both in soluble and particulate form, results in hyperexpression of the CD2 surface Ag, as assessed by immunofluorescence and semi-quantitative immunoprecipitation assays. Similar effects on CD2 surface expression were obtained when different CD3 mAb (OKT3, RW2-8C8 and Leu-4) were tested. The CD3-mediated CD2 up-regulation was suppressed by cycloheximide and actinomycin D, indicating that it requires de novo protein and RNA synthesis. In agreement with this, increased CD2 RNA levels were observed after 3 h of stimulation, reaching a plateau at 24 h that was maintained for 72 h. The CD2 up-regulation was concomitant to other CD3-induced activation-related events such as induction of surface CD25 and CD71 and high RNA levels for c-myc, IL-2R alpha- and beta-chains, CD71, and IFN-gamma. CD2 up-regulation appeared to be elicited by a protein kinase C-dependent mechanism because it was abrogated by staurosporine, a potent protein kinase C inhibitor. Moreover, IL-2-dependent events may also help in enhancing CD2 hyper-expression because it was only partially inhibitable by cyclosporine, dexamethasone, or Mar-108 (CD25) mAb. In conclusion, our data suggest that CD2 up-regulation can be a relevant event in T cell activation triggered by the physiologic engagement of the TCR/CD3 complex.

Antibodies, Monoclonal↗

Increased CD5-positive B lymphocytes in type I diabetes.

CD5+ B lymphocytes have been implicated in the production of polyspecific and monospecific antibodies that bind self-antigens, and increased proportions of this B cell subset occur in patients with some autoimmune diseases. We investigated the proportion of peripheral blood CD5+ B lymphocytes in type I diabetic patients. Compared with 18 age-matched healthy subjects, 11 out of 28 (39.2%) type I diabetic patients had increased proportions of circulating CD5. B lymphocytes with no alterations in the numbers of circulating B and T lymphocytes. Although all patients with increased CD5 B lymphocytes also had serum islet cell antibodies and/or insulin autoantibodies, the occurrence of increased proportions of CD5+ B lymphocytes and serum autoantibodies was not significantly correlated. Increased proportions of CD5+ B lymphocytes was not related to the time elapsed since the clinical onset of diabetes. In addition, regardless of being increased or normal, the proportion of CD5+ B lymphocytes appeared as a relatively constant phenotype after 1 year of follow-up studies at 3-month intervals in eight patients. Although the significance of these findings remains to be established, the possibility exists that CD5+ cells play a role in the pathogenesis of type I diabetes.

Adolescent↗

Serum levels of secretory IgA in ankylosing spondylitis.

An ELISA test was performed in order to measure the serum levels of secretory IgA (sIgA) in 51 patients with ankylosing spondylitis (AS) and 30 healthy controls. Raised values of sIgA were found in AS patients compared to controls. Patients with active disease, defined by clinical criteria, showed a significant elevation of serum sIgA compared to healthy controls (p = 0.03) or to patients with inactive disease (p = 0.02). A positive correlation between total serum IgA and sIgA was found (p = 0.001). Our findings further support the role of the mucosal stimulation in the pathogenesis of AS.

C-Reactive Protein↗

Phosphorylation-mediated changes in the electrophoretic mobility of CD5 molecules.

This work shows that tumor promoter agents (TPA) induce the post-translational modification of the human lymphocyte surface CD5 antigen (Tp67) in several cellular types. Treatment of [32P]orthophosphate- and [35S]cysteine-labeled normal and lymphoblastoid T and B cells with active tumor promoters induced the rapid, transitory and dose-dependent appearance of hyperphosphorylated CD5 forms with higher apparent molecular masses. These changes in the electrophoretic mobility of CD5 molecules were independent of RNA and protein synthesis, as well as of differences in neuraminic acid content. The inhibition of the TPA-mediated changes by protein kinase C inhibitors (staurosporine and 1-(5-isoquinolylsulfonyl)-2-methylpiperazine) indicated its protein-kinase-C-mediated nature. Phosphatase digestion of CD5 immunoprecipitates reverted the TPA-mediated mobility changes showing its dependence on phosphorylation. Neuraminidase digestion of intact cells revealed that the target of the TPA effects are surface-expressed CD5 molecules. In conclusion, we suggest that the heterogeneity in the electrophoretic mobility induced by TPA could reflect some structural and/or functional differences within CD5 molecules.

Antigens, Differentiation↗

Protein kinase C-dependent up-regulation of CD5 surface expression on normal and lymphoblastoid T cells.

As an approach to study the mechanisms regulating the surface expression of CD5 antigen on T cells, the effects of agents that activate different lymphocyte functions were examined. Active tumour promoter agents (TPA), such as phorbol ester analogues [phorbol 12-myristate 13-acetate (PMA), phorbol 12, 13-dibutyrate (PBu2)] and mezerein, were able to increase the expression of CD5 on the cell surface of all T-cell lines), as deduced from immunofluorescence and immunoprecipitation analysis. The TPA-induced CD5 up-regulation occurred in a dose-and time-dependent manner and was dependent on protein and RNA synthesis. From the other stimuli tested, the T-cell mitogens [phytohaemagglutinin (PHA) and concanavalin A (Con A)], as well as monoclonal antibodies (mAb) against the CD3 complex, also increased CD5 expression, although to a lesser degree. In all cases the increments were shown to be dependent on protein kinase C (PKC), as evidenced by their inhibition with staurosporine, a potent inhibitor of PKC activation. These data suggest that CD5 up-regulation on T cells can be a physiological event that depends on PKC activation.

Antigens, Differentiation↗

Cellular activation without proliferation to B cell growth factor and interleukin 2 in chronic lymphocytic leukaemia B cells stimulated with phorbol ester plus calcium ionophore.

Individual leukaemic B cells of chronic lymphocytic leukaemia (CLL) do not proliferate to B cell growth factor (BCGF) or interleukin 2 (IL-2) when co-stimulated with immunoglobulin (Ig) ligands. To exclude possible defective signalling via surface Ig (sIg), phorbol myristate acetate (PMA) plus calcium ionophore (A23187) were used to activate purified CLL B cells and compared with staphylococcal protein A coupled to sepharose beads (Seph-PA). RNA synthesis and phenotypic changes after PMA plus A23187 stimulation indicate that CLL B cells from (10) different individuals are similarly able to undergo the G0 to the G1 phase transition and express surface activation antigens. In contrast, they are variable in the capacity to show DNA synthesis, which occurred in only six out of 10 cases. Even in the presence of BCGF (10%, v/v) or IL-2 (50 U/ml) four out of nine CLL B cells activated with PMA plus A23187 or PMA alone were still unable to proliferate although they were induced to express CD23, 4F2, CD25 and OKT9 antigens by PMA plus A23187. However, PMA plus A23187 induced IgM secretion which increased further in response to IL-2 even in the absence of DNA synthesis. Moreover, in other CLL B cell populations, the unresponsiveness to growth factors upon co-stimulation with Ig ligands (Seph-PA) may simply reflect a defective signalling via sIg cross-linking which can be circumvented by PMA plus A23187 stimulation. Recombinant Interferon-gamma (50 U/ml) failed to affect DNA synthesis and IgM secretion.

B-Lymphocytes↗

Differential responsiveness of human B lymphocytes to phorbol ester and calcium ionophore based on their state of activation.

For tonsil B cells of a particular high density (below 65% Percoll), both phorbol myristate acetate (PMA) (5 ng/ml) and calcium ionophore A23187 (500 nM) were required to induce RNA synthesis, significant DNA synthesis also occurring in the presence of 12,000 MW B-cell growth factor (BCGF). In contrast, PMA alone, even at 1 ng/ml, was a sufficient stimulus to induce strong DNA synthesis in low-density B cells (45-50% Percoll) and strong proliferative responsiveness to BCGF in intermediate-density B cells (55-65% Percoll). In these latter B-cell populations, A23187 (500 nM), acted synergistically with non-mitogenic PMA doses to induce strong DNA synthesis, the PMA dose required being 5-50 times lower in low-density B cells (0.1-1 ng/ml) than in intermediate-density B cells (5 ng/ml). Preactivation for 30 hr with anti-Ig antibodies plus BCGF, known to drive B cells into late G1, rendered high-density B cells responsive to PMA (1-10 ng/ml) with high, dose-related DNA synthesis. These data indicate that the B-cell mitogenicity of a given nanomolar dose of PMA depends on the more advanced state of activation of B cells. It was also found that the above optimal dose of A23187 (500 nM) paradoxically inhibited the PMA-induced DNA synthesis of low-density B cells and in vitro preactivated high-density B cells. Data obtained with low-density B cells suggest that a calcium influx during the PMA-induced proliferative phase of B cells may provide a negative signal for the DNA synthesis.

B-Lymphocytes↗

Combined assay of adenosine deaminase, purine nucleoside phosphorylase, and lactate dehydrogenase in the early clinical evaluation of B-chronic lymphocytic leukemia.

The levels of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), lactic dehydrogenase (LDH), and LDH isoenzyme patterns (LD1 to LD5) have been measured in lymphocyte extract from 28 patients with B-chronic lymphocytic leukemia (B-CLL). The activities of ADA, PNP, and LDH have been correlated with two morphological groups of B-CLL classified according to the percentage of large, nongranular, atypical lymphocytes (AL) in peripheral blood: "typical" B-CLL (less than 10% of AL, 21 cases) and "atypical" B-CLL (10-25% of AL, seven cases). Patients with atypical B-CLL had significantly (P less than 0.001) higher activities of ADA (0.46 +/- 0.17 U/10(9) cells), PNP (1.74 +/- 1.0 U/10(9) cells), and LDH (48.3 +/- 9.7 U/10(9) cells) than patients with typical B-CLL (ADA, 0.29 +/- 0.1 U/10(9) cells; PNP, 0.58 +/- 0.23 U/10(9) cells; and LDH, 29 +/- 10 U/10(9) cells). In addition, the "treatment-free period" was also significantly (P less than 0.025) shorter in the group of atypical B-CLL compared with the typical B-CLL group. No clear-cut statistical differences in lymphocyte surface markers or in several other prognostic factors between the two subgroups of B-CLL were found. The present study supports the idea that in B-CLL the simultaneous determination of ADA, PNP, and LDH might be helpful in better understanding the pathophysiology, prognosis, and natural history of the disease.

Adenosine Deaminase↗