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Biomedical subjects

C F Calvo

Publications and source records attributed to C F Calvo.

At least 19 recordsLinked to original sources
PubMed2001

Antiproliferative properties of sphingosine-1-phosphate in human hepatic myofibroblasts.

Sphingosine-1-phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg-1 and Edg-3. S1P stimulated thymidine incorporation and induced activation of extracellular signal-regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P-evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3-kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin-sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood-brain barrier.

Animals↗
PubMed2001

Sphingosine-1-phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades.

Sphingosine-1-phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg-1 and Edg-3. S1P stimulated thymidine incorporation and induced activation of extracellular signal-regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P-evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3-kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin-sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood-brain barrier.

Animals↗
PubMed2000

Identification of an opioid peptide secreted by rat embryonic mixed brain cells as a promoter of macrophage migration.

Conditioned media from embryonic mixed cells from the rat brain were used in a chemotaxis assay to look for potential chemotactic activity which could account for the infiltration of the developing central nervous system (CNS) by macrophage precursors. The most potent chemotactic activity was found in the conditioned medium from E17 mixed brain cells (E17-CM). Based upon checkerboard analysis, this activity was shown to be chemotactic rather than chemokinetic. This chemoattraction was not restricted to brain macrophages (BM) because it was as pronounced on bone marrow-derived macrophages. The implication of a peptide compound in this activity was suggested by its resistance to heat as well as acid treatments, and by its sensitivity to aminopeptidase M digestion. In agreement with the opioid nature of the peptide, not only naloxone, but also the delta opioid receptor antagonist ICI-174 reduced the migration of BM in response to E17-CM by 60%. This migratory activity was no longer effective when pertussis toxin-treated BM were used. When the chemotactic effects of selective opioid agonists were compared to that of E17-CM, DPDPE, the delta agonist, was the most efficient in attracting BM. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that delta as well as other known opioid receptors were expressed in both BM and E17 mixed brain cells. Finally, a Met-enkephalin-like reactivity was found by RIA in the E17-CM. Altogether, these observations suggest that a delta-like opioid peptide released from embryonic mixed brain cells could be responsible for the infiltration of the developing CNS by macrophages precursors.

Analgesics, Opioid↗
PubMed1998

Synthetic full-length and truncated RANTES inhibit HIV-1 infection of primary macrophages.

OBJECTIVE: To determine the effect of beta-chemokines on HIV-1 infection of primary macrophages, and to search for chemokine derivatives devoid of biological effects but efficient at protecting CD4+ T lymphocytes and macrophages against HIV-1. DESIGN: Use of chemically synthesized molecules devoid of biological contaminants and monocyte-derived macrophages from healthy donors. METHODS: Full-length RANTES was chemically synthesized together with three derivatives, truncated of seven, eight and nine amino acids at the amino-terminus ([8-68]RANTES, [9-68]RANTES and [10-68]RANTES), which were tested for their biological activity and antiviral effects. RESULTS: Whereas full-length and truncated RANTES derivatives bound to beta-chemokine receptor CCR-5 with the same affinity as recombinant RANTES, the truncated forms were not chemotactic and acted as CCR-5 antagonists in this respect, although a partial agonist effect was noted on cell metabolism. Full-length RANTES and [8-68]RANTES protected T lymphocytes and macrophages from infection by HIV-1, although 10-fold higher concentrations of the truncated analogues were necessary to achieve the same effect as full-length RANTES. With regard to the effect of RANTES on HIV-1 infection of primary macrophages, our results contrast with most previously reported data. CONCLUSION: These data indicate that through binding to CCR-5, truncated RANTES derivatives that are devoid of detectable biological effects may represent candidates as drugs to protect both lymphocytes and macrophages from HIV- 1.

Animals↗
PubMed1998

Identification of CSF-1 as a brain macrophage migratory activity produced by astrocytes.

Intraparenchymal migration of macrophages occurs in the CNS during development or as a consequence of tissue injuries. In the present study, we have shown, by using an in vitro chemotaxis assay, that cultured rat astrocytes obtained from the developing cerebral cortex and striatum produce soluble factors, which attract purified brain macrophages. The effect of astrocyte-derived factors on macrophages was strongly reduced in the presence of antibodies neutralizing colony-stimulating factor 1 (CSF-1, also called M-CSF), and recombinant CSF-1 was found to act as a chemotactic agent on brain macrophages. Synthesis of CSF-1 by cultured astrocytes was confirmed by northern detection of CSF-1 transcripts. In contrast, the CSF-1 gene was not expressed by cultured neurons from the cerebral cortex and striatum or by the brain macrophage population responsive to CSF-1 gradient. ELISA detection of CSF-1 in tissue extracts revealed the occurrence of this cytokine in the rat cerebral cortex during postnatal development and in adults. Altogether, our results demonstrate that astrocytes, through CSF-1 secretion, can trigger the polarized migration of brain macrophages and suggest a new mechanism which could regulate the locomotion of these cells in the cerebral cortex during ontogenesis or following lesions.

Animals↗
PubMed1996

Production of monocyte chemotactic protein-1 by rat brain macrophages.

In the present study, we show that cultured rat brain macrophages release a soluble factor that stimulates the migration of bone marrow-derived macrophages, as determined by an in vitro chemotaxis assay. A checkerboard analysis indicated that most of this effect resulted from a polarized migration of the cells (chemotactic phenomenon), rather than in an increase in cell motility (chemokinesis). This activity was significantly decreased by an immune serum directed against the rat monocyte chemoattractant protein-1 (chemokine MCP-1). Northern blot analysis demonstrated expression of the MCP-1 gene in cultured brain macrophages, but its absence in unstimulated bone marrow-derived macrophages. Up-regulation of MCP-1 expression was observed when lipopolysaccharide was added to cultured brain macrophages, a peak occurring after a 6 h period of stimulation. Also, inflammatory cytokines such as interleukin (IL)-1 beta, colony stimulating factor-1, tumour necrosis factor-alpha and IL-6 individually increased the basal level of MCP-1 mRNA. Subsequently, we demonstrated the in vivo production of MCP-1 in the adult rat brain following injury induced by a local injection of kainic acid. MCP-1 synthesis was localized in both astrocytes and brain macrophages. These results suggest that the activation of resting microglial cells into brain macrophages and their subsequent secretion of chemokines could contribute to the mechanism(s), leading to the infiltration of the CNS by blood-derived monocytes, as observed in several pathologies.

Animals↗
PubMed1996

Recruitment of brain macrophages: roles of cytokines and extracellular matrix proteins produced by glial or neuronal cells.

Brain macrophages are a subpopulation of microglial cells which occur in the developing or in the injured CNS. These cells actively contribute to CNS tissue remodeling by acting on neuronal and macroglial lineages. Recruitment of brain macrophages is promoted by transformation of resting microglial phenotypes, infiltration of the tissue by exogenous macrophage precursors and local proliferation of phagocytes. These events are regulated by extracellular signals produced by glial cells or neurons. Studies based on in vitro cell cultures or experimental tissue lesions suggest that the infiltration of phagocytes involves intracerebral production of chemotactic factors acting on monocytes such as chemokines or extracellular matrix proteins. Proliferation of brain macrophages in stimulated by colony-stimulating factors which seem to be primarily secreted by glial cells.

Astrocytes↗
PubMed1994

Substance P stabilizes interleukin-2 mRNA in activated Jurkat cells.

We investigated here the mechanism leading to the enhancement of interleukin (IL)-2 mRNA that we described in a previous work when Jurkat cells were co-stimulated with PHA+PMA and 10(-12) M of the Substance P (SP) neuropeptide. We show that the SP-augmented IL-2 mRNA signal is totally abrogated by an early addition of cyclosporin A, actinomycin D or cycloheximide. SP does not affect the IL-2 gene transcription, as evidenced by nuclear run on assays. In contrast, a posttranscriptional alteration of the IL-2 mRNA is shown, by demonstrating that the degradation rate of IL-2 mRNA following the addition of actinomycin D, at 4 h, was delayed in the (PHA+PMA)-activated cell cultures containing 10(-12) M of SP. Thus, the SP-induced augmentation of secreted IL-2 in activated T cells we demonstrated previously must result from an SP increase of the IL-2 mRNA stability.

Cell Line↗
PubMed1993

DNA regions flanking the mouse Ig 3' alpha enhancer are differentially methylated and DNAase I hypersensitive during B cell differentiation.

Two B cell-specific enhancer elements are associated with the IgH gene cluster. One enhancer is located within the J-C mu intron (E mu), whereas a second enhancer (3' alpha E) is approximately 12.5 kb 3' of the C alpha membrane exon. In an attempt to understand the function of 3' alpha E, we have characterized its surrounding structural milieu during various stages of B cell differentiation through analysis of methylation patterns and the identification of DNAse I-hypersensitive sites. We observed a correlation between the chromatin structure of this region and the differentiation state of the cell. Compared to liver and brain, the region 3' of alpha was hypermethylated in pre-B and T cell lines and became progressively demethylated as B cell differentiation continued. A DNAse I-hypersensitive site was present in pre-B cell lines about 17 kb 3' of 3' alpha E. In fully differentiated myeloma cell lines, a second cluster of DNAse I-hypersensitive sites was present immediately 5' of 3' alpha E. Our data indicate that the 3' alpha enhancer is relatively sequestered during early stages of B cell differentiation and becomes increasingly accessible at later stages.

Animals↗
PubMed1993

A versatile ELISA-PCR assay for mRNA quantitation from a few cells.

Gene expression studies require a sensitive and quantitative assay of mRNA amounts present in small samples. We describe a general method of quantifying specific mRNA quickly and easily from purified RNA or directly from a few cells by PCR and enzyme-linked immunosorbent assay (ELISA) revelation of the resulting products (sensitivity of the last step: < 0.1 fmol). Cells are digested and the total cellular RNA is reverse-transcribed and then amplified with 5' and 3' primers; the former being 5' biotinylated. The amplification product is captured on avidin-coated microplates and quantified by hybridization with a digoxigenin-labeled internal oligonucleotide probe. After revelation with an anti-digoxigenin alkaline phosphatase coupled antibody (anti-DIG-AP1), the amount of hybridized probe is determined by optical reading. The results can be easily converted to absolute values by comparison with an external DNA standard curve. An internal DNA or RNA standard can also be used. The method we describe can be adapted to any cellular or viral gene of known sequence in a matter of days. Since it uses nonradioactive probes, commercially available reagents and standard microplate readers, it is inexpensive and could be automated easily. In this study, interleukin-2 mRNA expression could be studied in as few as 40 Jurkat cells. It was also possible to quantify human immunodeficiency virus (HIV) DNA from 1500 to 1.5 copies out of 1.5 x 10(5) human genomic DNA copies.

Avidin↗
PubMed1992

Substance P enhances IL-2 expression in activated human T cells.

Jurkat and HUT 78 T cell lines, as well as peripheral blood human T cells activated with PHA plus PMA were used to investigate the capacity of substance P (SP) neuropeptide to regulate IL-2 production. By using Northern blot analysis and dosage of the IL-2 release in cell supernatants, we show that SP can act as cosignal with PHA + PMA to enhance the expression of specific IL-2 mRNA and IL-2 secretion in T cells. By using the N-terminal SP(1-4) or the C-terminal SP(4-11) fragments of the entire molecule, we show that the cosignal activity is carried by the C-terminal portion of SP. The SP and SP(4-11) optimal effects were observed at 10(-12) M and 10(-10) M when a broad range of concentrations from 10(-6) M to 10(-13) M was tested. The increase of IL-2 mRNA obtained with 10(-12) M of SP in the activated Jurkat cells was reduced by adding 10(-10) or 10(-9) M of the SP antagonist (D-Pro2,-D-Phe7,-D-Trp9)SP to the culture, indicating the specificity of SP action. The up-regulation observed when 10(-12) M of SP was applied together with the mitogens on Jurkat cells, persisted after a 16-h culture period, time at which the IL-2 mRNA signal is normally back to a minimum level when the mitogens are used alone. Furthermore, an induction of IL-2 mRNA accumulation, in a 2-h pulse, was obtained with 10(-12) M of SP on Jurkat cells previously activated with mitogens for 16 h.

Gene Expression↗
PubMed1991

DNA sequences 3' of the Ig H chain cluster rearrange in mouse B cell lines.

A mouse myeloma cell line MPC11 (IgG2b, kappa) and variants derived from it have been used to study DNA rearrangements that occur at the Ig H chain locus. One variant, F5.5, has acquired both VH gene and C epsilon gene rearrangements. Through genomic Southern blot analysis initially directed to mapping the C epsilon gene rearrangement, we observed that the VH region rearrangement was linked, through an inversion event, to sequences that originate 3' of the CH cluster, i.e., 3' of the C alpha gene. Subsequent studies have shown that DNA rearrangements within the region 3' of the C alpha gene are detected in several other mouse myeloma and hybridoma cell lines and are not associated with the expression of specific isotypes.

Animals↗
PubMed1990

CD1 stimulation of human T cell lines induces a rapid increase in the intracellular free Ca2+ concentration and the production of IL-2.

The effect of a battery of CD1 mAb on intracellular free Ca2+ concentration and IL-2 production has been examined on different T cell lines in this study. Both 0249F and NU-T2 two CD1b specific mAb tested, induced a rapid increase in the intracellular Ca2+ concentration on HPBALL T cells whereas only one (L161) among three different CD1c mAb (L161, 10C3, and M241) produced a similar effect. In contrast the addition of four different CD1a mAb directed against two different epitopic groups of this molecule were uneffective in modifying the intracellular Ca2+. Both L161 and 0249F also induced a comparable increase in the intracellular Ca2+ concentration on MOLT 4 and JURKAT, two other T cell lines of similar phenotype. The effect of L161 mAb on the IL-2 production of the IL-2 producing T cell line JURKAT was also examined. The association of the latter with PMA strongly induced the production of IL-2 on this cell model while either L161 or PMA alone had no effect. Although the natural ligand and the function of CD1 molecules are still unknown, the accumulation of these data strongly suggest that CD1b and CD1c might represent two activatory pathways for immature T cells operating before the classical CD2 and CD3 activation pathways.

Antibodies, Monoclonal↗
PubMed1990

Role of interleukin 2 receptor beta chain in initiating anti-CD3 and interleukin 2-induced proliferation of human resting T cells.

We have examined the role of isolated interleukin 2 receptor (IL2R) beta chains expressed by human resting T cells in the early period of primary T cell activation induced by soluble OKT3 monoclonal antibody (mAb) and exogenous IL2. In the initial 3-day-stimulation phase, high IL2 concentrations were required, in association with soluble OKT3 mAb, to induce the formation of IL2R alpha/beta heterodimers, while later, low IL2 concentrations were sufficient to promote cell growth. When added during the initial phase, TU27 mAb directed at the IL2 binding site of IL2R beta chain substantially inhibited the appearance of functional high-affinity IL2R. Lo-Tact-1 mAb directed at the IL2 binding site of the IL2R alpha chain had only a marginal effect. Strong induction of IL2R alpha mRNA occurred within 3 days upon OKT3 and IL2 stimulation even in the presence of Lo-Tact-1 mAb, but not in the presence of TU27 mAb. OKT3 alone failed to induce significant IL2R alpha gene transcription and that induced by IL2 alone was very weak. The constitutive expression of IL2R beta mRNA was visualized in resting T cells. It remained at a rather stable level, at least during the initial stimulation period which was examined herein. Given the fact that OKT3 alone was ineffective in up-regulating IL2R beta mRNA expression and that pre-incubation of the cells with OKT3 alone did not allow them to respond to high concentrations of IL2, it is highly probable that isolated IL2R beta chains constitutively expressed by CD8+ T cells (the main reactive cells in this system) are primarily responsible for the initial interaction of IL2 with these cells. Such an interaction will result in the formation of high-affinity IL2R and in the initiation of cell proliferation provided that a CD3-derived co-signal is simultaneously delivered to the cells.

Antibodies, Monoclonal↗
PubMed1988

Mechanism of class switching.

We have documented the action of unequal sister chromatid exchange on antibody genes, and our studies provide the first molecular description of an unequal sister chromatid exchange event. We delineated the sequences that mediate class switch recombination in normal B cells through genomic Southern analysis of five IgG2-producing hybridomas. Successive subclass switching was documented in two of eight maps and may possibly account for interposed DNA in three additional maps, implying that subclass switching may be a relatively frequent occurrence in normal B cells. As in most other myelomas and hybridomas, the 5' breakpoint of switch recombination in our panel of hybridomas falls 5' to Cmu-associated tandem repeats, whereas the 3' breakpoint falls within C gamma-associated tandem repeats. We consider various hypotheses in the text, the most interesting of which is that deletion of Cmu-associated tandem repeats is a necessary first step before class switch recombination in mature B cells. This hypothesis suggests that Cmu-associated tandem repeats may be a separately regulated DNA element.

Animals↗
PubMed1986

Persistence of T8+/HNK-1+ suppressor lymphocytes in the blood of long-term surviving patients after allogeneic bone marrow transplantation.

Fifteen patients and their respective bone marrow donors were entered in this study 1 to 5 yr after allogeneic bone marrow transplantation. Peripheral blood E rosetting (T) cells were analyzed for their phenotypic characteristics as well as for their ability to regulate Ig synthesis in the in vitro PWM system. A close relationship was found between a high proportion of T8+/HNK-1+ cells and/or T8+/HLA-DR+ cells and a strong (greater than or equal to 50%) inhibition of the antibody response. It was noteworthy that even the patients without suppressor activity had high proportions of such cells when compared with normal marrow donors. Moreover, the suppression occurred irrespective of the presence or absence of chronic GVHD. Through negative selection experiments (with MAb and complement) and through immunofluorescence cell sorting, it was shown that the suppressor cells expressed the T8+, HNK-1+, HLA-DR- phenotype. They did not carry the Leu-11, NKH1A, or NKH2 determinants, which are expressed on mature functional NK cells. When examined by electron microscopy, they exhibited a morphology of resting agranular lymphocytes. The significant increase of these suppressor cells among the BMT patients was not correlated with clinical syndromes such as chronic GVHD or opportunistic viral infections, which argues against the notion of in vivo profound immunodeficiency coexisting with these cells.

Antibody Formation↗
PubMed1986

Regulation of immunoglobulin synthesis by human T cell subsets as defined by anti-D44 monoclonal antibody within the CD4+ and CD8+ subpopulations.

In our previous paper, we demonstrated that anti-D44 MAb can, in the presence of complement, eliminate all the allocytotoxicity generated during a mixed lymphocyte reaction without affecting the alloproliferative response. As approximately 70% of CD4+ cells and 30% of CD8+ will be stained with anti-D44 MAb, we researched the functional role of the D44+ and D44- cells in each of these T cell subsets in the PWM-induced antibody response. We found that most of the helper activity for immunoglobulin (Ig) synthesis was mediated by CD4+ D44+ lymphocytes and that virtually all the suppressive activity was mediated by CD8+ D44- lymphocytes. Surprisingly enough, we noticed that the low level of Ig synthesis induced in B cells by CD4+ D44- lymphocytes could be strongly amplified by the addition of radiosensitive CD8+ lymphocytes, suggesting coexisting opposite immunoregulatory functions within the CD8+ T cell subset. These results, together with previous data, indicate that anti-D44 MAb subdivides T cells into subpopulations with distinct functional repertoires: a CD4+ D44+ helper subpopulation, a CD8+ D44+ cytotoxic subpopulation, and a CD8+ D44- suppressor subpopulation.

Antibodies, Monoclonal↗