Biomedical subjects

M Chakraborty

Publications and source records attributed to M Chakraborty.

At least 37 records · Page 2Linked to original sources

PubMed1995

Induction of human breast cancer-specific antibody responses in cynomolgus monkeys by a murine monoclonal anti-idiotype antibody.

We have generated and characterized a murine monoclonal anti-idiotype (Id) antibody, designated 11D10, which biologically and antigenically mimics a distinct and specific epitope of the high molecular weight human milk fat globule primarily expressed by human breast and some other tumor cells at high density. This epitope is identified by mAb BrE1, which was used as the immunizing antibody or Ab1 to generate the anti-Id (Ab2) 11D10. 11D10 induced antitumor immune responses across species barriers, i.e., in mice and rabbits. In preclinical studies, cynomolgus monkeys were immunized with 2 mg of either 11D10 or the isotype- and allotype-matched control Ab2 3H1 after precipitation with aluminum hydroxide. All monkeys developed high titers of antibodies against the immunizing mouse immunoglobulin. Immunization with 11D10 induced anti-anti-idiotype antibodies (Ab3) which reacted with breast cancer cell lines but not with control T-cell and melanoma cell lines. The Ab3 shared idiotypes with BrE1 (Ab1), as demonstrated by their ability to inhibit 11D10 binding to BrE1. The Ab3 obtained with 11D10 bound specifically to human milk fat globule antigen and competed with BrE1 for binding to breast cancer cell lines, suggesting that Ab1 and Ab3 may bind to the same epitope. In addition, Id-specific cellular immune responses were demonstrated in monkeys immunized with 11D10 by T-cell proliferation assays. These results indicate that aluminum hydroxide-precipitated anti-Id 11D10 can induce breast cancer-specific antibodies in nonhuman primates and can serve as a potential network antigen for breast cancer patients.

Animals↗

PubMed1995

Preclinical evaluation in nonhuman primates of an anti-idiotypic antibody that mimicks the carcinoembryonic antigen.

We have developed and characterized a murine monoclonal antiidiotype (Id) antibody (Ab2), designated 3H1 (IgG1-k) that mimics human carcinoembryonic antigen (CEA). 3H1 was raised against an anti-CEA monoclonal antibody (mAb) 8019 (Ab1) that recognizes a distinct and specific epitope of the 180,000 MW CEA. 3H1 induced specific anti-CEA immune responses in mice and rabbits. In this preclinical study, cynomolgus monkeys (Macaca fascicularis) were immunized with aluminum hydroxide-precipitated 3H1 and tested for the induction of anti-CEA antibodies. Monkeys were injected with 2 mg of 3H1, intracutaneously, four times biweekly. All monkeys developed specific anti-anti-Id (Ab3) responses that were capable of inhibiting binding of the immunizing 3H1 (Ab2) to 8019 (Ab1) and vice versa. Furthermore, immune sera from monkeys contained Ab3 (Abl') antibody that bound to CEA-positive colon carcinoma cell lines but not to CEA-negative MOLT-4 or melanoma cell lines. Also, the Ab3 reacted with purified CEA and competed with Ab1 (8019) for binding to CEA positive LS174-T cells, suggesting that Ab1 and Ab3 may bind to the same epitope. In addition, affinity-purified Ab3 from monkey sera immunoprecipitated the same 180,000 MW CEA as Ab1 8019 and showed an identical pattern as the Ab1 on colon carcinoma specimens by immunoperoxidase staining. The induction of anti-tumor antibodies in monkeys did not cause any apparent side effects. These data suggest that internal image anti-Id can induce tumor-specific humoral immune responses in nonhuman primates and can serve as potential network antigen for triggering active anti-CEA antibodies in colorectal cancer patients.

Animals↗

PubMed1995

Immune response to the carcinoembryonic antigen in patients treated with an anti-idiotype antibody vaccine.

We have generated an IgG1 murine monoclonal anti-idiotype antibody (Ab2) designated 3H1, which mimics a specific epitope on the carcinoembryonic antigen (CEA). Patients with CEA positive tumors are immunologically "tolerant" to CEA. We used 3H1 as a surrogate for CEA for vaccine therapy of 12 patients with advanced colorectal cancer. Each of the patients received a minimum of four intracutaneous injections of aluminum hydroxide precipitated 3H1 at either 1, 2, or 4 mg dosage per injection. 9 of 12 patients demonstrated anti-anti-idiotypic (Ab3) response to 3H1. All nine patients generated specific anti-CEA antibody demonstrated by reactivity with radiolabeled purified CEA; some cases were confirmed by immunoprecipitation of purified CEA. We also demonstrated Ab3 stained both autologous and allogeneic colonic tumors. 7 of 12 patients demonstrated idiotype specific T cell proliferative responses and four also showed T cell proliferation to CEA. Toxicity was limited to local reaction with mild fever and chills. All 12 patients eventually progressed after finishing 4-13 dosages. This is the first report demonstrating that a vaccine therapy is capable of breaking "immune tolerance" to CEA in patients with CEA positive tumors. Future studies will focus on treating patients with minimal residual disease.

Adult↗

PubMed1994

Cell cycle-dependent and kinase-specific regulation of the apical Na/H exchanger and the Na,K-ATPase in the kidney cell line LLC-PK1 by calcitonin.

Calcitonin (CT), which regulates serum calcium through its actions in bone and the kidney tubule, also has a potent natriuretic effect in vivo. Na reabsorption in the proximal kidney tubule is mostly dependent on the activity of the Na,K-ATPase and the apical Na/H exchanger. We have previously shown that CT regulates the activity of the Na,K-ATPase in the proximal kidney tubule cell line LLC-PK1 in a cell cycle-dependent manner. We report here that, in the same cells, CT also regulates the Na/H exchanger through a cell cycle-specific activation of the Ca/calmodulin-dependent protein kinase II. In G2 phase, no changes in ethylisopropyl amiloride-sensitive 22Na uptake is observed, despite an increase in cAMP. In contrast, the hormone inhibits the apical exchanger when the cells are in S phase, resulting in an 80% inhibition of 22Na uptake. These results demonstrate that CT affects the activity of the two major proximal tubule Na transport systems and may help clarify the mechanisms by which CT regulates Na+ reabsorption.

Animals↗

PubMed1993

Accumulation of high level of pp60c-srcN is an early event during GM3-antibody mediated differentiation of neuro-2a neuroblastoma cells.

Neuro-2a neuroblastoma cells, when differentiated via a cAMP-dependent pathway by treatment with anti-GM3 monoclonal antibody, accumulated a high level of pp60c-src protein and pp60c-src kinase activity just before the onset of neurite formation. The specific kinase activity of the accumulated c-src protein was found to be comparable to that of normal cerebellar neurons, but was about 6- to 8-fold higher than that of normal astrocytes. These results, and migrations of peptide fragments in the SDS-polyacrylamide gels after V8 proteolysis, strongly indicate the accumulation of the neuron-specific isoform of the c-src protein (pp60c-srcN) in the GM3 antibody-treated Neuro-2a cells. Similar high levels of pp60c-src protein and pp60c-src kinase activity were observed in the Neuro-2a cells differentiated via a cAMP-dependent pathway by treatment with dibutyryl cAMP, but not in the same cell line when differentiated via a cAMP-independent pathway with 5-bromo-2'-deoxyuridine. These results demonstrate that the accumulation of high levels of the neuron-specific isoform of the pp60c-src protein (pp60c-srcN) in the Neuro-2a neuroblastoma cells depends on the specific signal transduction pathway involved during the differentiation of these cells.

Animals↗

PubMed1993

Sensitivity to nitrate and other oxyanions further distinguishes the vanadate-sensitive osteoclast proton pump from other vacuolar H(+)-ATPases.

The osteoclast proton pump (OC H(+)-ATPase) differs from other vacuolar H(+)-ATPases (V-ATPases) in its sensitivity to vanadate and in the subunit composition of its catalytic domain, where isoforms of subunits A and B are expressed [Chatterjee et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6257-6261]. In the present study, the sensitivity of the osteoclast H(+)-ATPase to various oxyanions was tested. The results indicate that H+ transport by microsomal preparations isolated from chicken osteoclasts is 20-100-fold more sensitive to nitrate that any other animal and fungal V-ATPases and 10-20-fold more sensitive than plant V-ATPases, as is the ATPase activity of the affinity-purified enzyme. This inhibition by nitrate is not due to a chaotropic effect of the oxyanion and is complete at 1 mM concentrations with an IC50 of 100 microM. In contrast, proton transport by the OC H(+)-ATPase was insensitive to other oxyanions (phosphate, sulfate, and acetate) which inhibit other V-ATPases. These results further demonstrate that the proton pump present in osteoclast membranes differs from other vacuolar ATPases. It is speculated that, since cells of the macrophage lineage can generate high intracellular concentrations of nitrate, it may be possible to physiologically or therapeutically regulate the activity of the OC H(+)-ATPase in the osteoclast without affecting the other V-ATPases in the same or in other cells.

Animals↗

PubMed1993

Immuno-suppressive effect of human alphafetoprotein: a cross species study.

Alphafetoprotein (AFP) is the major serum protein of fetal life in human and other mammalian species. The phylogenetical conservatism of AFP demonstrated by extensive immunological cross reaction between human AFP and AFP of a number of species, suggest that AFP plays a general role in the successful pregnancy of all mammalian species. The present work clearly demonstrates the antiproliferative effect of human AFP on lymphocytes, harvested from normal human donors. The inhibitory effect of human AFP is quite significant in the same dose during blastic transformations of the lymphocytes. In this study, peripheral blood mononuclear cells were induced to blastic transformations with phytohaemagglutinin (PHA-M) and the effect of AFP was quantified by the incorporation of [3H]-thymidine into newly synthesized DNA during 24 hrs pulse. Moreover, human AFP shows similar immuno-suppressive effect to other species of lymphocytes also. In all the three species (mouse, rat and hamster) studied, a parallelism was noted in their respective percentage of thymidine incorporation values at the comparable doses. These results establish a cross species inhibitory effect of human AFP and it may be stated that this effect is directly targeted on T-helper cells and has no interaction with interleukin-2 (IL-2).

Animals↗

PubMed1992

Sensitivity to vanadate and isoforms of subunits A and B distinguish the osteoclast proton pump from other vacuolar H+ ATPases.

Analysis of proton (H+) transport by inside-out vesicles derived from highly purified chicken osteoclast (OC) membranes has revealed the presence of a newly discovered type of vacuolar H+ ATPase (V-ATPase). Unlike vesicles derived from any other cell type or organelle, H+ transport in OC-derived vesicles is sensitive to V-ATPase inhibitors (N-ethylmaleimide and Bafilomycin A1) and vanadate (IC50, 100 microM), an inhibitor previously found to affect only P-type ATPases. The OC H+ ATPase contains several V-like subunits (115, 39, and 16 kDa) but subunits A and B of the catalytic domain of the enzyme differ from that of other V-ATPases. In OCs, subunit A has a mass of 63 kDa instead of the 67-70 kDa expressed in monocytes, macrophages, and kidney microsomes, which contain a vanadate-insensitive H+ ATPase. Moreover, two types of 57- to 60-kDa B subunits are also found: one is expressed predominantly in OCs and the other is expressed in kidney microsomes. The OC H+ pump may therefore constitute a class of H+ ATPase with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme. This H+ ATPase is involved in resorption of bone and may be expressed in a cell-specific manner, thereby opening possibilities for therapeutic intervention.

Animals↗

PubMed1992

Differentiation of Neuro-2a neuroblastoma cells by an antibody to GM3 ganglioside.

A monoclonal antibody against GM3 ganglioside (GM3Ab) was found to trigger differentiation of Neuro-2a cells in culture. The differentiation of Neuro-2a cells by GM3Ab was accompanied by increased levels of intracellular serotonin and amino acid neurotransmitters viz. aspartate, glutamate, glutamine, glycine and taurine. Further study indicated that the increase in the serotonin level was not due to a higher rate of serotonin synthesis but rather to a higher rate of active transport of serotonin from the medium. Studies on the cell surface gangliosides revealed that unlike the proliferating cells, the GM3Ab-mediated differentiated cells contained higher gangliosides in addition to GM3 and GM2 gangliosides. Analysis of total cellular proteins indicated the appearance of a 25 kDa protein, pI 5.4, in the GM3Ab-treated cells--a small amount of this protein was observed in dibutyryl cAMP (Bt2cAMP)-treated cells, however, the protein was totally absent in the 5-bromo-2'-deoxyuridine (BrdU)-treated cells. Investigation of the mode of action of GM3Ab indicated that the cellular differentiation was due to increased cAMP accumulation resulting from an increase in the adenylate cyclase activity. Further studies with different agents affecting protein kinase C (PKC) activity and direct assay of PKC ruled out the possibility that GM3Ab mediated its effect via PKC. This GM3Ab-induced differentiation could be inhibited by protein kinase A (PKA) inhibitor, H8, but could not be inhibited by sphingosine, an inhibitor of PKC. Pertussis toxin could mimic the effect of GM3Ab, suggesting that GM3Ab caused the elevation in the adenylate cyclase activity by reducing the Gi-protein inhibition of the adenylate cyclase. The data suggests that GM3Ab, after interaction with cell surface GM3, elevated intracellular cAMP level by withdrawing the inhibitory effect of some undefined factor(s) present in culture medium which normally keeps adenylate cyclase activity low through activation of Gi-protein.

Amino Acids↗

PubMed1992

Thyroidal influence on the cell surface GM1 of granule cells: its significance in cell migration during rat brain development.

1. No difference was observed in the in vitro growing ability of granule cells isolated from hypothyroid or normal rat brain. When granule cells were taken from hypothyroid rat brain and grown in normal culture medium containing 10% fetal calf serum, they behaved similarly to the granule cells obtained from normal rat brain. 2. In both cases there were progressive losses of in vitro growing ability of the granule cells with the age of the animal and it became impossible to grow them when derived from 21 days or older animals. 3. A marked decrease in cell surface GM1 was observed when the cells were maintained under thyroid hormone-deficient conditions in culture. 4. Anti-GM1 antibody was found to inhibit significantly the migration of granule cells along the astrocyte fibers. 5. These results indicate that GM1 has an important role in thyroid hormone-dependent postnatal brain maturation in rat.

Age Factors↗

PubMed1992

Differential effects of the 3',5'-cyclic adenosine monophosphate and protein kinase C pathways on the response of isolated rat osteoclasts to calcitonin.

Calcitonin (CT) activates both the cAMP and the protein kinase C (PKC) pathways in the kidney cell line LLC-PK1. Although CT also activates cAMP in osteoclasts, its effects on PKC in this cell type are unknown. In order to determine whether the response of osteoclasts to CT also involves the PKC pathway, the effects of activators and inhibitors of PKC on bone resorption and cell surface area were analyzed in isolated rat osteoclasts. As expected, CT inhibited in a dose-dependent manner bone resorption by rat osteoclasts cultured for 24 h on devitalized bovine bone slices and this effect could be mimicked by cAMP. The inhibitory effect of CT could however also be mimicked by phorbol-12,13-dibutyrate (PDBu) and blocked by the PKC inhibitor sphingosine, as well as by the less specific inhibitors H7 and H8, none of which had detectable effects in the absence of CT. No changes in the number of attached osteoclasts were observed under any of these conditions. These results indicate that CT activates PKC in osteoclasts and that this activation, like the activation of cAMP-dependent protein kinase, leads to an inhibition of bone resorption. Quantitative time-lapse videomicroscopy showed that the CT-induced retraction of osteoclasts also involved activation of the PKC pathway and could therefore be induced by phorbol esters. In contrast, (Bu)2 cAMP (1-200 microM) failed to induce rapid cell retraction. It is concluded that, in osteoclasts, CT receptors are coupled to both the cAMP-dependent protein kinase and the PKC pathways. Although these two second messengers can have additive inhibitory effects on bone resorption, only activation of the PKC pathway induces rapid cell retraction. These two effects of calcitonin on osteoclasts are therefore independent and may be functionally unrelated.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

The osteoclast proton pump differs in its pharmacology and catalytic subunits from other vacuolar H(+)-ATPases.

Osteoclasts are multinucleated cells derived from the mononuclear phagocyte system in the hematopoietic bone marrow. Their function is to resorb bone during skeletal growth and remodeling. They perform this function by acidifying an enclosed extracellular space, the bone resorbing compartment. Analysis of proton transport by inside-out vesicles derived from highly purified chicken osteoclast membranes has revealed the presence of a novel type of multisubunit vacuolar-like H(+)-ATPase. Unlike H(+)-ATPases derived from any other cell type or organelle, proton transport and ATPase activity in osteoclast vesicles are sensitive to two classes of inhibitors, namely V-ATPase inhibitors [N-ethyl-maleimide (NEM) and bafilomycin A1] and vanadate (IC50 100 mumol l-1), an inhibitor previously found to affect only P-ATPases. The osteoclast V-ATPase morphologically resembles vacuolar proton pumps and contains several vacuolar-like subunits (115 x 10(3), 39 x 10(3) and 16 x 10(3)M(r)), demonstrated by Western blot analysis. Subunits A and B of the catalytic domain of the enzyme, however, differ from that of other V-ATPases. In osteoclasts, subunit A has an M(r) of 63 x 10(3) instead of 67 x 10(3)-70 x 10(3); in contrast, monocytes, macrophages and kidney microsomes, which contain a vanadate-insensitive H(+)-ATPase, express the classical subunit A (70 x 10(3)M(r)). Moreover, two types of 57 x 10(3)-60 x 10(3)M(r) B subunits are also found: they are differentially recognized by antibodies and one is expressed predominantly in osteoclasts and the other in bone marrow cells and in kidney microsomes. Preliminary cloning data have indicated that the B subunit expressed in osteoclasts may be similar to the brain isoform. The osteoclast proton pump may, therefore, constitute a novel class of V-ATPase, with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme.

Adenosine Triphosphatases↗

PubMed1992

Anemia in experimental visceral leishmaniasis in hamsters.

Experimental infection of hamsters with Leishmania donovani caused visceral leishmaniasis in which hematological changes occurred. The infected hamsters were anemic and reticulocyte counts were high. No significant change in the serum erythropoietin level was noted. Red cell membrane Na(+)-K(+)-ATPase and acetylcholinesterase activities increased. Osmotic fragility of the erythrocytes from infected animals increased. The level of 2,3-diphosphoglycerate of the red cells increased with the degree of anemia.

2,3-Diphosphoglycerate↗

PubMed1991

Use of high-performance liquid chromatography for assay of glutamic acid decarboxylase. Its limitation in use for post-mortem brain.

Rat brain, obtained 10 min after death, contained high levels of endogenous gamma-aminobutyric acid (GABA) and glutamic acid. Incubation of this brain homogenate at 37 degrees C indicated decrease of GABA with time due to degradation by GABA-transaminase. Reported high-performance liquid chromatographic (HPLC) methods for glutamic acid decarboxylase (GAD) assay depend on the difference between the GABA content of the reaction mixture after and before the incubation period. None of the methods considered the degradation of GABA during incubation. Furthermore, during determination of the Michaelis constant (KM) for the reaction none of them considered the endogenous substrate. Here we have focused on these factors which seriously affect the maximum velocity (Vmax) and KM values during GAD assay by the HPLC technique. By a simple and rapid HPLC technique we have measured GAD activity in post-mortem rat brain after removing endogenous glutamic acid by charcoal treatment and using gabaquline to prevent GABA degradation during incubation period. By this method a Vmax value of 46 +/- 4 nmol/h/mg protein and a KM value of 7.5 +/- 0.6 mM were observed for GAD activity of crude brain homogenate. For a comparative study, we have carried out radiometric assay of GAD activity from the same sample and observed a Vmax of 48 +/- 6 nmol/h/mg protein and KM of 6.9 +/- 0.4 mM.

Animals↗

PubMed1991

Effect of erythropoietin on the interaction of concanavalin A with rat erythrocytes.

Effect of Erythropoietin (Ep) on the interaction of Concanavalin A (Con A) with rat erythrocytes was studied using 125I-labelled Con A. Binding of Con A to erythrocytes was dependent on time and cell concentration. Starvation caused an elevation of the lectin binding capacity of red cells which again came down towards the normal level on Ep administration to starved rats. Binding of Con A to erythrocytes decreased linearly with increasing concentration of Ep. Specificity of binding was confirmed by inhibition studies with alpha-methyl-D-mannopyranoside (Me Man) Cells from the starved rats compared to those from normal and Ep treated animals were less prone to inhibition by this sugar analog. Positive cooperative binding of Con A to rat erythrocyte was observed at low concentration of Con A but was absent at higher lectin concentrations. Starvation caused an increase in the number of binding sites per cell which returned to normal level after Ep treatment. Under identical conditions, binding affinities were not much changed in these cells. Cells from the starved animals were more susceptible to agglutination compared to those from normal and Ep-treated rats. Microviscosity and cholesterol/phospholipid ratio of red cell membrane decreased in the starved animals which retraced its way back towards the normal level after Ep treatment.

Animals↗

PubMed1991

Cell cycle-dependent coupling of the calcitonin receptor to different G proteins.

Calcitonin is a calcium regulating peptide hormone with binding sites in kidney and bone as well as in the central nervous system. The mechanisms of signal transduction by calcitonin receptors were studied in a pig kidney cell line where the hormone was found to regulate sodium pumps. Calcitonin receptors activated the cyclic adenosine monophosphate (cAMP) or the protein kinase C (PKC) pathways. The two transduction pathways required guanosine triphosphate (GTP)-binding proteins (G proteins) (the choleratoxin sensitive Gs and the pertussis toxin sensitive Gi, respectively) and led to opposite biological responses. Moreover, selective activation of one or the other pathway was cell cycle-dependent. Therefore, calcitonin may induce different biological responses in target cells depending on their positions in the cell cycle. Such a modulation of ligand-induced responses could be of importance in rapidly growing cell populations such as during embryogenesis, growth, and tumor formation.

Adenylyl Cyclases↗

PubMed1991

Epitope analysis of the oncofetal antigen alphafetoprotein using monoclonal antibodies.

Alphafetoprotein (AFP), an oncofetal antigen, plays very important roles in the early embryonic life and oncogenesis. Under various physiological and pathological conditions AFP exhibits microheterogeneity, probably as a result of differential expression of its epitopes. To analyse the epitopes we have developed a panel of monoclonal antibodies against human AFP purified by a new and efficient method using an immunoadsorbent consisting of polyclonal antibodies immobilized on cyanogen bromide activated Sepharose. Clones producing antibodies of various isotypes, e.g. IgG1, IgG2a, IgG2b, IgA and IgM have been subcloned and characterized. The antibodies showed high avidity for AFP (with half-maximal binding concentrations between 0.012 and 3.87 nM). Mutual inhibition efficiencies of a panel of 14 monoclonal antibodies were determined by RIA. Based on these inhibition data a computer program was used to group these antibodies with respect to their "epitope specificity distance". As a result of this grouping, clones have been identified which can recognize at least five different epitopes on AFP. This panel of antibodies may be very useful for analysis of the epitopic variation of AFP under various physiological and pathological conditions.

Adult↗

PubMed1991

Neurosurgical experience with carotid endarterectomy: a 12-year study.

In the 13-year period from 1975 to 1988, 91 carotid endarterectomies were performed on 83 patients in a neurosurgical unit. Sixty-seven of these patients had continued to have symptoms after the best medical treatment. Seventy-one presented with transient ischaemic attacks (TIAs), nine with TIA and minor completed stroke (MCS), and three with MCS alone. Follow-up ranged from 8 months to 12 years with a mean of 5.5 years. Within the follow-up period, including operative complications, four deaths of cerebral origin (4.8%) and three major cerebral events (3.6%) occurred--an annual stroke morbidity and mortality rate of less than 1.5%, which compares favourably with a minimum stroke risk of 5% per annum for the first 3 years following a TIA and 3% for subsequent years. The annual stroke and/or vascular death rate including myocardial infarction was 3.5% compared to an expected stroke and/or vascular death rate of 7.4%. It appears that carotid endarterectomy is a useful adjunct to medical therapy. Myocardial ischaemia is the major cause of death in the follow-up period in this group of patients. It is suggested that patients with TIAs and MCS should be investigated, and those who do not respond to medical therapy should be identified for carotid endarterectomy.

Adult↗