Inhibition of naive Th1 CD4+ T cells by glatiramer acetate in multiple sclerosis.

We investigated whether glatiramer acetate (GA) treatment may affect Th1 differentiation at various T-cell maturation stages. Specifically, we analyzed the effect of in vivo GA treatment on intracellular synthesis of IL-2 and TNF-alpha by naive, memory and effector CD4(+) and CD8(+) T cells by five-colour flow cytometry. Our data indicate that GA treatment downregulates/normalizes an accelerated Th1 differentiation of CD4(+) T cells in RRMS patients at all stages of T-cell maturation. Most notably, we conclude that, by altering naive, unprimed CD4(+) T cells, GA treatment appears to affect T-cell differentiation, at least in part, in an antigen-independent manner.

Calretinin and calbindin D-28k delay the onset of cell death after excitotoxic stimulation in transfected P19 cells.

In some neurological diseases, injury to neurones reflects an over-stimulation of their receptors for excitatory amino acids. This response may disturb the Ca(2+)-homeostasis and lead to a pronounced and sustained increase in the intracellular concentration of this ion. On the basis of data derived from correlative studies, calcium-binding proteins have been postulated to play a protective role in these pathologies. We tested, directly, the capacity of the three calcium-binding proteins calretinin (CR), calbindin D-28k (CB) and parvalbumin (PV) to buffer [Ca(2+)], and to protect cells against excitotoxic death. We used P19 murine embryonic carcinoma cells, which can be specifically induced (by retinoic acid) to transform into nerve-like ones. The differentiated cells express functional glutamate-receptors and are susceptible to excitotoxic shock. Undifferentiated P19-cells were stably transfected with the cDNA for CR, CB or PV, induced to differentiate, and then exposed to NMDA, a glutamate-receptor agonist. The survival rates of clones expressing CR, CB or PV were compared with those of untransfected P19-cells using the lactate-dehydrogenase assay. CR- and CB-expressing cells were protected from death during the first 2 h of exposure to NMDA. This protection was, however, transient, and did not suffice to rescue P19-cells after prolonged stimulation. Two of the three PV-transfected clones raised were vulnerable to NMDA-induced excitotoxicity; the third, which expressed the lowest level of PV, was protected to a similar degree as that found for the CR- and CB-transfected clones. Our results indicate that in the P19-cell model, CR and CB can help to delay the onset of cell death after excitotoxic stimulation.

Beneficial effect of co-polymer 1 on cytokine production by CD4 T cells in multiple sclerosis.

Multiple sclerosis (MS) has been associated with an imbalance in the T helper type 1 (Th1) and Th2 subsets. We investigated, at the single-cell level, the synthesis of pro-inflammatory cytokines by CD4 and CD8 T cells from MS patients. We report the relationship between priming of CD4 and CD8 T cells for interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) and disease evolution in MS patients, clinically subdivided into relapsing-remitting MS (RRMS) in remission, RRMS in relapse, or chronic progressive MS (CPMS). Moreover, we report the in vivo influence of co-polymer 1 (COP) treatment on the pattern of cytokine producers in RRMS patients. We show that the frequency of CD4 T cells primed for TNF-alpha synthesis increased in all stages of MS, including RRMS remitting, and was normalized to control values in COP-treated patients (43.2 +/- 11.8% in treated patients versus 47 +/- 7.3% in RRMS remitting versus 40.3 +/- 8% in controls). In addition, a significant decrease in the frequency of CD4 T cells primed for IL-2 was found in COP-treated patients as compared to the other groups of patients, reaching values below that of controls (59.1 +/- 9.9% in treated patients versus 70 +/- 11.6% in RRMS remitting versus 67.1 +/- 7.4% in controls). Unexpectedly, COP-treated patients also showed a significantly decreased priming for IFN-gamma at the CD4 T-cell level (9.1 +/- 3.4% in treated patients versus 18.8 +/- 0.6.4% in RRMS remitting versus 15.4 +/- 4.7% in controls), but not at the CD8 T-cell level. This bystander suppression on the inflammatory cells should be considered in the monitoring of MS patients submitted to COP treatment, in order to evaluate better its clinical efficacy.

Inhibition of the proliferative cycle and apoptotic events in WiDr cells after down-regulation of the calcium-binding protein calretinin using antisense oligodeoxynucleotides.

The colon adenocarcinoma cell line WiDr expresses the calcium-binding protein calretinin (CR). In order to deduce possible functions of calretinin in these cells we decreased its concentration by antisense techniques. Treatment of WiDr cells with phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) led to a drop in calretinin expression, as evidenced by immunohistochemical staining of WiDr cells and Western blot analysis of cytosolic cell extracts. The morphology of these epithelial cells changed from polygonal to spherical and they formed dense cell clusters. Cells displaying morphological alterations typical for apoptotic cells were observed after incubation with AS-ODNs, as evidenced by phase-contrast and electron microscopy. The mitotic rate of AS-ODN-treated cells dropped significantly, as demonstrated by mitotic labeling and time-lapse microcinematography. Furthermore, an accumulation of cells in phase G1 and a reduction of [3H]thymidine-labeled cells was observed in antisense-treated cells. The basal level of [Ca2+]i was not influenced by the down-regulation of calretinin. WiDr cells incubated with the nonsense, reverse-sense, or with an oligodeoxynucleotide with a totally unrelated sequence did not show any significant differences when compared to control cells. We conclude that calretinin levels have an impact on the progression of the cell cycle of WiDr cells.

Changes in shape and motility of cells transfected with parvalbumin cDNA.

Parvalbumin is thought to act as a Ca2+ buffer in skeletal muscle fibers, but its physiological role in brain, kidney, and testis remains unclear. We have transfected parvalbumin cDNA into a human ovarian adenocarcinoma cell line, which normally does not express this protein. The induced expression of parvalbumin under the control of three different promoters causes: (1) changes in the morphology of the cells from epitheloid to fusiform, (2) an increase in motility of whole cell clusters, and (3) a decrease in the mitotic rate. Transfection with a mutated cDNA of rat parvalbumin incapable of binding Ca2+ had no effect on these three parameters. Our results indicate that ectopic expression of parvalbumin influences not only cell division [Rasmussen and Means (1989) Mol. Endocrinol. 3, 588-596], but also cell shape and motility by modulating intracellular Ca2+ handling. This may be a basic function of parvalbumin when it is intrinsically expressed in differentiated nonmuscle cells.