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P Casellas

Publications and source records attributed to P Casellas.

At least 73 records · Page 4Linked to original sources
PubMed1994

Allosteric modulation of peripheral sigma binding sites by a new selective ligand: SR 31747.

The interactions of a new compound SR 31747 with sigma sites were examined in rat spleen membranes and in human peripheral blood leukocytes (PBL). Nanomolar concentrations of SR 31747 selectively inhibited in a non-competitive manner the binding of the prototypic sigma ligands [3H](+)-pentazocine, [3H](+)-3PPP and [3H]DTG on rat spleen membranes. Characterization of SR 31747 binding sites using [3H]SR 31747 as a ligand showed that this compound binds reversibly, with high affinity to one class of sites on rat spleen membranes (Kd 0.66 nM, Bmax 5646 fmol/mg protein). The pharmacological profile of [3H]SR 31747 binding sites was consistent with the presence of specific sites distinct from classical sigma 1 and sigma 2 receptor subtypes strongly suggesting an allosteric modulation of sigma sites by SR 31747. Similarly, [3H]SR 31747 binding sites were demonstrated on human PBL and also on purified subpopulations of human mononuclear cells (granulocytes, NK cells, T4, T8 and B lymphocytes). Administered to mice by i.p. or oral route 30 min before sacrifice, SR 31747 strongly inhibited the binding of [3H](+)-3PPP to mice spleen membranes with ED50 values of 0.18 and 1.43 mg/kg, respectively. Taken together these results could suggest a potential immunological activity of SR 31747 either directly or through allosteric modulation of peripheral sigma sites.

Allosteric Site↗
PubMed1994

Immunopharmacological profile of SR 31747: in vitro and in vivo studies on humoral and cellular responses.

In our preceding paper, we demonstrated that both human and rat lymphocytes possess saturable high-affinity binding sites for the new sigma ligand SR 31747. Here we investigate the potential activity of this ligand on immune responses. In vitro, our study shows that SR 31747 exerts a concentration- and time-dependent inhibition of proliferative response to mitogens on mouse and human lymphocytes without affecting cell viability. This suppressive effect elicited by SR 31747 occurs over a concentration range which correlates with the pharmacological profile of the molecule in binding assays, strongly suggesting that SR 31747 acts through a receptor-mediated process. We showed that the SR 31747 effect, which was observed on purified T lymphocytes, affects a late event in the activation process which occurs after the G1 during the S phase of the cell cycle. Interestingly, no anti-proliferative effect was observed in a variety of tumor cell lines, supporting a specific effect limited to normal immune cells. In vivo, in mice, treatment with SR 31747 prevented both graft-versus-host disease and delayed-type hypersensitivity granuloma formation, while antibody response to sheep red blood cells was not affected. These results strongly suggest that the sigma-related receptor recognized by SR 31747 is very likely coupled to a biological function of lymphocytes.

Animals↗
PubMed1994

Subcellular localization of peripheral benzodiazepine receptors on human leukocytes.

BACKGROUND: The peripheral-type benzodiazepine receptor (PBR) was initially identified in many peripheral tissues and in some blood cells. Drugs that bind with high affinity to PBRs have previously been described as having immunomodulating properties. The number of PBRs varies according to the cell population considered. The aim of this study was to study the localization of PBRs in two human leukocyte populations, T4-lymphocytes, and monocytes. EXPERIMENTAL DESIGN: Both cell populations were purified by negative immunoselection in order to keep only the physiologically accessible sites on the viable cells. Mitochondria were quantified by electron microscopy and flow cytometric analysis. Subcellular localization was then studied after PBR photoaffinity labeling using electron microscopic ultrastructural autoradiography. RESULTS: We have shown that monocytes contain twice as many mitochondria as lymphocytes. We have also shown that the global labeling of monocytes by ultrastructural autoradiography is actually higher than that of lymphocytes and the labeling of monocyte mitochondria is higher than that of lymphocyte mitochondria. In addition, the distribution of subcellular labeling indicates that there are different populations of mitochondria in one cell, i.e., labeled and unlabeled, and that the percentage of labeled mitochondria is greater in monocytes. These results are consistent with those obtained in previous binding studies. Finally, over 50% of receptors are localized in cell compartments devoid of visible mitochondria. CONCLUSIONS: The subcellular distribution of the PBR shows that this receptor could have other physiologic functions towards immune cells than a function associated with mitochondria.

Autoradiography↗
PubMed1993

Peripheral benzodiazepine receptor modulation with phagocyte differentiation.

Peripheral benzodiazepine receptor (PBR) was found to be less expressed in the immature phagocytic HL-60 and U-937 cell lines than in the more mature monocytic THP-1 cell line. Cell differentiation by several agents induced a strong enhancement of PBR density on these three phagocytic cell lines but not on the lymphocytic CEM cell line. Detailed analysis of phorbol 12-myristate 13-acetate-treated THP-1 cells showed an increased PBR expression and the rise came along with an increase of CD11a and CD11b antigens and a secretion of macrophagic cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-8. Quantitation of mRNA using polymerase chain reaction (PCR)-based technique showed that overexpression of PBR did not parallel mRNA expression, indicating a gene-independent regulation. These results suggest that PBR predominance on phagocytic cells could be related to maturation process.

Antigens, CD↗
PubMed1993

Up-regulation of 5-HT2 receptors in the rat brain by repeated administration of SR 46349B, a selective 5-HT2 receptor antagonist.

Chronic administration (twice a day for three days and on the morning of the fourth day) of SR 46349B (trans-4-[(3Z)3-(2-dimethylaminoethyl)oxyimino-3-(2-fluoroph enyl)propen-1- yl]phenol hemifumarate) (10 mg/kg, orally), a selective 5-HT2 receptor antagonist, caused 24 h later a marked increase (+42%) of the maximum binding capacity of [3H]ketanserin in rat brain cortical membranes without change in its affinity constant. Further, administration of the 5-HT2 receptor agonist, (+/-)-DOI((+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) (1 mg/kg, i.p.), produced in chronic SR 46349B treated rats a significant increase in the amount of [3H]-inositol phosphate compared to corresponding controls. In addition, subacute administration of SR 46349B caused a 2-fold increase in the head-twitch response to (+/-)-DOI (0.5 mg/kg, i.p.). This enhanced response was blocked by an acute administration of ritanserin (6-(2-[4-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-7- methyl-5H-thiazolo[3,2-a]pyrimidin-5-one) (10 mg/kg). Finally, a significant enhancement (+29%) of 5-HT2 receptor mRNA levels was observed in the cortex. Taken together, these data showed that an up-regulation of 5-HT2 receptors occurred in rats following repeated treatment with a selective 5-HT2 receptor antagonist. The effects of SR 46349B on 5-HT2 receptors might implicate pre-translational regulation.

Amphetamines↗
PubMed1993

Cannabinoid-receptor expression in human leukocytes.

Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS), probably through the cannabinoid receptor, which has recently been cloned in rat and human. While numerous reports have also described effects of cannabinoids on the immune system, the observation of both mRNA and cannabinoid receptor has hitherto been exclusively confined to the brain, a reported detection in the testis being the sole example of its presence at the periphery. Here we report the expression of the cannabinoid receptor on human immune tissues using a highly sensitive polymerase-chain-reaction-based method for mRNA quantification. We show that, although present in a much lower abundance than in brain, cannabinoid receptor transcripts are found in human spleen, tonsils and peripheral blood leukocytes. The distribution pattern displays important variations of the mRNA level for the cannabinoid receptor among the main human blood cell subpopulations. The rank order of mRNA levels in these cells is B cells > natural killer cells > or = polymorphonuclear neutrophils > or = T8 cells > monocytes > T4 cells. Cannabinoid-receptor mRNA, which is also found in monocytic, as well as T and B leukemia cell lines but not in Jurkat cells, presents a great diversity of expression on these cells as well, B-cell lines expressing a much higher level than T-cell lines. The cannabinoid receptor PCR products from leukocytes and brain are identical both in size and sequence suggesting a strong similarity between central and peripheral cannabinoid receptors. The expression of this receptor was demonstrated on membranes of the myelomonocytic U937 cells using the synthetic cannabinoid [3H]CP-55940 as ligand. The Kd determined from Scatchard analysis was 0.1 nM and the Bmax for membranes was 525 fmol/mg protein. The demonstration of cannabinoid-receptor expression at both mRNA and protein levels on human leukocytes provides a molecular basis for cannabinoid action on these cells.

Animals↗
PubMed1993

Distribution profile and properties of peripheral-type benzodiazepine receptors on human hemopoietic cells.

The cellular localization of peripheral-type benzodiazepine receptors (PBRs) was characterized in several human blood cell subpopulations including erythrocytes, platelets, monocytes and polymorphonuclear neutrophils (PMN), B, NK, T8 and T4-cells. Pharmacological properties of the PBR were established by binding studies and PBR mRNA expression was measured by quantitative polymerase chain reaction based method. These data clearly indicate 1) the PBR is pharmacologically homogeneous in the various types of blood cells, 2) the rank order of PBR cell density is monocytes = PMN > lymphocytes >> platelets > erythrocytes, 3) the PBR appears to be transcriptionally regulated since mRNA levels are roughly correlated with PBR density.

Antibodies, Monoclonal↗
PubMed1992

Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control.

We describe a simplified and reliable polymerase chain reaction-based method for assaying RNAs of low abundancy. The technique involves the co-amplification of cellular RNA-derived cDNA with a multispecific cDNA of synthetic origin added as an internal standard, using primer pairs common to both templates. We show that the co-amplified templates accumulate in a parallel manner throughout both the exponential and nonexponential phases of amplification, even when the starting amounts of the templates differ by up to 2 orders of magnitude. This finding means that preliminary experiments designed to determine either the late exponential region or the amplification efficiency for each pair of primers are unnecessary. This has enabled us to develop a greatly simplified quantitation protocol. We illustrate our approach by quantifying the effect of the immunosuppressor cyclosporin A on the accumulation of interleukin-4, interferon-gamma, and interleukin-2 receptor mRNAs in phytohemagglutinin-stimulated human peripheral blood mononuclear cells.

Base Sequence↗
PubMed1992

Internalization and intracellular fate of anti-CD5 monoclonal antibody and anti-CD5 ricin A-chain immunotoxin in human leukemic T cells.

We have investigated the entry and subsequent intracellular fate of T101 monoclonal antibody (MoAb) and T101-ricin A-chain (RTA) immunotoxin (IT) directed against the CD5 antigen (Ag) expressed on human leukemic CEM cells. We provide direct evidence for the internalization of T101 MoAb and the corresponding IT. Both the MoAb and IT were internalized at a relatively low rate. This slow internalization process could be related to the partial recycling of the MoAb/Ag or IT/Ag complexes. Analysis of the internalized molecules showed that their molecular weight was only partially altered after internalization and that no free A-chain could be found inside the cells, indicating that lysosomal degradation and cleavage of disulfide-linked conjugates is a quantitatively minor phenomenon compared with the localization of internalized anti-CD5 ITs in an endosomo-Golgi compartment, followed by their recycling to the cell surface. We believe that this is the major factor explaining the low efficacy of anti-CD5 IT when assayed in the absence of potentiating substances. Finally, we showed that the presence of ammonium chloride and monensin, which both dramatically enhance the kinetics of IT activity, did not affect the rate of internalization or the intracellular localization of the conjugate, suggesting that these activators could act at the postendocytotic level on a limited number of IT molecules.

Ammonium Chloride↗
PubMed1992

Contrasting effects of the protein kinase C inhibitor staurosporine on the interleukin-1 and phorbol ester activation pathways in the EL4-6.1 thymoma cell line.

EL 4-6.1 cells, variants of the murine EL4 thymoma cell line, can be activated by interleukin 1 (IL-1) or phorbol 12-myristate-13-acetate (PMA), or PMA+IL-1 to secrete interleukin 2 (IL-2) and interleukin 4 (IL-4) and to express the IL-2 receptor (IL-2R). To compare the different activation pathways, we examined the effects of staurosporine (STAR) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), two protein kinase C (PKC) inhibitors, on the induction of interleukin secretion and IL-2R expression in these cells. We report here that nanomolar concentrations of STAR strongly potentiated (20- to 30-fold) the production of IL-2 or IL-4, when EL 4-6.1 cells were induced by IL-1 alpha (or IL-1 beta) alone. By contrast, at identical concentrations, STAR dose-dependently inhibited the production of IL-2 and IL-4 resulting from PMA or PMA+IL-1 cell treatment. STAR also negatively affected the expression of IL-2R, which was dependent on PMA-sensitive PKC with either IL-1, PMA, or PMA+IL-1 stimulation. The changes in interleukin production and IL-2R expression in EL 4-6.1 activated cells were correlated with changes at the mRNA level measured by quantitative polymerase chain reaction (PCR). This finding suggests a pretranslational effect of the drug. At micromolar concentrations, H7 showed the same effects as STAR, but only increased IL-1-triggered interleukin secretions twofold. We observed that the action of PKC inhibitors did not result from modification of IL-1 receptor (IL-1R) expression in EL 4-6.1 cells. Thus, our data show that PKC inhibitors clearly distinguish between IL-1 and PMA stimulatory pathways. In addition, they suggest that the IL-1 stimulatory pathway involves PKC(s) [or other undefined kinase(s)] which regulate this pathway and differ from PKC(s) activated by PMA.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

In vivo cytotoxic efficacy of immunotoxins prepared from anti-CD5 antibody linked to ricin A-chain.

The antitumoral efficacy of various anti-CD5 immunotoxins, prepared with whole monoclonal antibody (mAb), F(ab')2 or Fab fragment linked to native ricin A-chain (RTA) or partially deglycosylated ricin A-chain (dRTA), was examined in vivo in ascitic nude mice bearing a large burden of Ichikawa human tumour cells. We first demonstrated that after systemic administration of IgG-RTA or F(ab')2-dRTA, the cytotoxic activity of immunotoxin molecules specifically bound to tumour cells was preserved. Secondly we showed, by using different immunotoxins with various targeting capacities, that their cytotoxic effect in vivo was related to the number of immunotoxin molecules bound per cell. However, even when antigen saturation was achieved after i.p. injection, the cytotoxic effect did not exceed 53% of the tumour burden. By contrast, when the immunotoxin was administered i.p. or i.v. with the enhancer monensin conjugated to human serum albumin and injected i.p., 90% of the tumour cells were killed. This potentiating effect was demonstrated even when the tumour localisation was as low as 5% of the saturation level. Such an effect could be completely prevented by addition of unconjugated monoclonal antibody, demonstrating the specificity of the immunotoxin-induced cytotoxicity in the presence of the enhancer. However this enhancement was demonstrated whatever the route of immunotoxin administration, i.p. or i.v., but was only observed when the enhancer was injected i.p. and not i.v.. These results emphasize the importance of optimizing the therapeutic course to improve the antitumoral efficacy of immunotoxins.

Animals↗
PubMed1990

Internalization of the cytotoxic molecules of T101 F(ab')2-(ricin-A-chain) immunotoxin into human T-leukemic cells.

We have investigated the internalization step of an immunotoxin and its relationship with cytotoxicity, with the F(ab')2-T101(ricin-A-chain) immunotoxin, directed against the CD5 antigen expressed on leukemic CEM cells. We first demonstrated that the biological action of the conjugate was related to its entry into the cell by an energy-dependent endocytotic process. We also found that during the first hours of cell intoxication, internalization is not the rate-limiting step of immunotoxin cytotoxicity. Internalization becomes limiting in cell intoxication only when the entry rate is low. Lastly we show that ammonium chloride, which strongly enhances immunotoxin potency, acts on internalized molecules for a very short time, suggesting that this enhancer affects an early intracellular step.

Ammonium Chloride↗
PubMed1990

Parameters affecting tumor-specific delivery of anti-CD5 antibody-ricin A chain immunotoxins in vivo.

The tumor localization of various anti-CD5 immunotoxins (ITs) was studied in vivo in BALB/c nude mice bearing human ascitic Ichikawa tumor cells. Plasma and ascitic clearance of IT as well as the number of intact IT molecules taken up per tumor cell was measured with a highly sensitive immunoenzymometric assay. This assay allowed the quantification of as few as 400 molecules per cell, either bound to cell surface antigens or internalized. Tumor cell localization of an i.v.-administered IT composed of a whole IgG linked by a disulfide bond to native ricin A chain (RTA) was extremely small. The highest binding level of this IT to ascitic cells did not exceed 1,600 molecules per cell, whereas up to 25,000 molecules per cell were quantified after i.p. injection. To improve IT localization after i.v. injection, both the size of the monoclonal antibody moiety and glycosylation of the ricin A chain were examined. Two major additive improvements were obtained by (a) Fc removal and (b) partial deglycosylation of RTA. The localization of F(ab')2-deglycosylated RTA IT was 5.5 times greater than that of IgG-RTA IT, resulting from reduced plasma clearance and better extravasation of the conjugate. However, the IT targeting observed in vivo after i.v. administration was unexpectedly lower than the theoretical number of bound IT molecules, calculated from the concentration of soluble IT molecules in the ascitic fluid. This discrepancy was found to be related to a dramatic decrease in the antigen binding capacity of the antibody moiety in ascitic fluid.

Animals↗
PubMed1989

An anti-CD5 immunotoxin for chronic lymphocytic leukemia: enhancement of cytotoxicity with human serum albumin-monensin.

Five patients with B-cell chronic lymphocytic leukemia (B-CLL) were treated with 6 courses of the anti-CD5 immunotoxin T101-ricin A chain (T101-RTA). Each course consisted of 8 bi-weekly infusions of T101-RTA (7 or 14 mg/m2). The immunotoxin was well tolerated in all cases with no major toxicities. Though saturation of circulating leukemic cell-associated target antigen was demonstrated by FACS analysis in all patients, no intact immunotoxin was detected in bone-marrow or lymph-node aspirates. Pharmacokinetic studies revealed rapid clearance of T101-RTA, with a half-life of 43 min. None of the patients developed detectable titers of antibody against either T101 murine antibody or ricin A chain. Clinical response was limited to a rapid and transient fall in WBC count lasting less than 24 hr, most likely secondary to the antibody portion of the conjugate. In vitro, fresh B-CLL cells were resistant to T101-RTA at concentrations up to 10(-8)M, while fresh malignant T-cells with a 10-fold increase in expression of CD5 antigen were sensitive. In the presence of the enhancing agent human serum albumin-monensin, fresh B-CLL cells were sensitive to T101-RTA, with an ID50 more than 2 logs below the maximal concentration of immunotoxin achieved in vivo. We conclude that T101-RTA is a potentially useful agent in the treatment of T-cell leukemias. In the presence of HSA-monensin, this spectrum of activity may be extended to B-CLL.

Aged↗
PubMed1988

Comparison of the cytotoxic potency of T101 Fab, F(ab')2 and whole IgG immunotoxins.

The in vitro killing of the human CEM cell line was studied by using ricin A-chain immunotoxins constructed with either the whole IgG or the Fab and F(ab')2 fragments of the same T101 (anti-CD5) antibody. In the presence of ammonium chloride as an activator, the "whole" immunotoxin as well as the "fragment" immunotoxins did not show any significant difference in the cell killing efficacy. In contrast, without the activator, the efficacy of the T101 immunotoxin was greatly improved when fragments were used. Indeed, at a saturating dose, a cytoreduction of three orders of magnitude was obtained with the fragment immunotoxins vs less than one order of magnitude for the whole immunotoxin, as assessed in a clonogenic assay. This enhancing effect was related to better cell killing kinetics, because with a similar amount of A-chain molecules bound per cell, T101 fragment immunotoxins achieved a twofold faster protein synthesis inactivation rate than the corresponding whole IgG immunotoxin. No significant difference in activity was shown between monovalent (Fab) and divalent (F(ab')2) forms of fragment immunotoxins. The observation that T101 fragment immunotoxins were more potent than intact immunotoxins was extended to another fragment immunotoxin constructed with an antibody (F111.98) directed against a different epitope of the CD5 Ag. In another model (anti-CD22 1G11 antibody on Raji cells), the fragment immunotoxin did not show any superiority over the IgG immunotoxin which was by itself very potent, strongly suggesting an Ag-dependent phenomenon.

Animals↗
PubMed1988

Trichokirin, a ribosome-inactivating protein from the seeds of Trichosanthes kirilowii Maximowicz. Purification, partial characterization and use for preparation of immunotoxins.

A protein, here named trichokirin, was extracted from the seeds of Trichosanthes kirilowii and purified by ion-exchange and gel-filtration chromatography. Trichokirin is a basic glycoprotein of apparent relative molecular mass of 27,000 with a strong ribosome-inactivating activity. Alignment of the trichokirin, trichosanthin and momordin N-terminal sequences shows a substantial degree of homology. Trichokirin was conjugated to a monoclonal antibody directed against the Thy 1.2 antigen with the cleavable dimethyl 3,3'-dithiobispropionimidate cross-linking reagent. This immunotoxin selectively killed leukemia cells expressing the Thy 1.2 antigen. The addition of ammonium chloride, which increases the cytotoxicity of ricin A-chain immunotoxins, blocks that of the trichokirin immunotoxin, suggesting that they enter cells by different mechanisms. In vivo studies showed that the pharmacokinetic properties of the trichokirin immunotoxin could be more advantageous than those of the ricin A-chain immunotoxins for in vivo applications.

Amino Acid Sequence↗