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Biomedical subjects

S Ramakrishnan

Publications and source records attributed to S Ramakrishnan.

At least 217 records · Page 12Linked to original sources
PubMed1985

Increased efficiency in selective elimination of leukemia cells by a combination of a stable derivative of cyclophosphamide and a human B-cell-specific immunotoxin containing pokeweed antiviral protein.

Leukemia cells were mixed with normal human bone marrow cells to simulate bone marrow from leukemia patients; the mixture was then treated with a combination of stabilized derivative of cyclophosphamide [Mafosfamide (ASTA Z 7557)] and pokeweed antiviral protein-containing immunotoxin. The ability of this protocol for selective elimination of B-ALL cells was evaluated by clonogenic assay. The monoclonal antibody (B43) portion of the immunotoxin was directed against human B-cells and was linked to pokeweed antiviral protein by a disulfide bond. The combination of ASTA Z 7557 and immunotoxin was superior to either ASTA Z 7557 or the immunotoxin alone and produced nearly 7 logs of elimination of leukemia cells from the cell mixtures. About 5 logs of contaminating tumor cells were eliminated from a 200-fold excess of normal marrow under conditions where fewer than 50% of pluripotent stem cells were lost. Moreover, this manipulation did not inhibit subsequent production of pluripotent stem cells in long-term bone marrow cultures, indicating that the more primitive progenitors were not harmed.

Antibodies, Monoclonal↗
PubMed1985

Anti-T cell immunotoxins containing pokeweed anti-viral protein: potential purging agents for human autologous bone marrow transplantation.

The ex vivo anti-leukemic efficacy and stem cell toxicity of two different T cell directed immunotoxins containing pokeweed antiviral protein (PAP) were studied by clonal assays. 5E9-11-PAP, an immunotoxin directed against human transferrin receptors, elicited a maximum leukemic cell kill of 3.9 logs. However, it was also toxic against normal pluripotent stem cells, and therefore is not a clinically useful purgative reagent. PAP conjugated to 3-A1, a monoclonal antibody directed against CD7 (T, p41), was more effective against leukemic T cells than 5E9-11-PAP and eliminated a maximum of 4.8 log of cells. 3A1-PAP was only slightly toxic to pluripotent stem cells: 13% of CFU-GEMM were lost after treatment with 3000 ng of 3A1-PAP/ml, a concentration that eliminated 99.96% of contaminating leukemic T cells from a 200-fold excess of normal bone marrow. Cryopreservation of treated cells by conventional methods did not affect the extreme selectivity and potency of 3A1-PAP. Incubation of 3A1-PAP with peripheral blood mononuclear cells resulted in the complete inhibition of phytohemagglutinin-induced mitogenic response, illustrating the possibility of using this immunotoxin as a potent anti-T cell reagent for prophylaxis against graft vs host disease in allogeneic BMT as well.

Antibodies, Monoclonal↗
PubMed1985

Immunological and biological stability of immunotoxins in vivo as studied by the clearance of disulfide-linked pokeweed antiviral protein-antibody conjugates from blood.

Monoclonal antibodies against human T-cell antigen 3A1, human transferrin receptor, and mouse Thy 1.1 antigen were linked to pokeweed antiviral protein (PAP) by a disulfide bond. Because the ability of the immunotoxin to home on target cells in vivo and the eventual internalization of the hemitoxin polypeptide depends in part on the stability of the conjugate in circulation, the clearance of antibody-PAP conjugates from blood was investigated. Blood samples collected from rabbits at different times after the injection of immunotoxin were analyzed for: (a) total mouse IgG; and (b) intact antibody-PAP conjugate in enzyme-linked immunosorbent assay. Further, antibody-PAP conjugate was separated from PAP by differential precipitation using polyethyleneglycol, and the PAP content of the fractions were analyzed by radioimmunoassay. Free PAP is removed very rapidly from blood, and 95% is cleared within 2 h. Our results showed that the immunotoxin did not dissociate in circulation immediately, and about 90% of the initial concentration of the conjugate was still present for more than 4 h. Analysis by enzyme-linked immunosorbent assay showed a 4- to 8-h lag period in which immunotoxin concentrations were relatively unchanged. This was followed by a steady decline, and the half-life of the conjugate in circulation then ranged between 17 and 24 h. Not only did the immunotoxins remain intact immunologically, but they also retained their biological activity as measured by the ability of blood-borne immunotoxins to efficiently block protein synthesis of target cells in vitro. These data show that the disulfide linkage of toxin to antibody is reasonably stable and that the immunotoxin retains the biological properties of both the antibody and the hemitoxin polypeptide in circulation.

Animals↗
PubMed1984

Inhibition of human acute lymphoblastic leukemia cells by immunotoxins: potentiation by chloroquine.

Immunotoxins containing pokeweed antiviral protein and monoclonal antibodies against human T cells or human transferrin receptor efficiently killed acute lymphoblastic leukemia cells. Chloroquine specifically enhanced the rate of protein synthesis inhibition by immunotoxin. Depending on its concentration, chloroquine (10 to 100 micromolar) reduced by up to 65-fold the amount of immunotoxin required to inhibit protein synthesis in the target cells 50 percent.

Antibodies, Monoclonal↗
PubMed1984

Comparison of the selective cytotoxic effects of immunotoxins containing ricin A chain or pokeweed antiviral protein and anti-Thy 1.1 monoclonal antibodies.

Ricin A chain and pokeweed antiviral protein (PAP), two enzymes that inhibit the action of eukaryotic ribosomes, were coupled by cleavable, N-succinimidyl-3-(2-pyridyldithio)propionate, and noncleavable m-maleimidobenzoyl-N-hydroxysuccinimide ester, cross-linking reagents to monoclonal antibodies directed against Thy 1.1 antigen. Leukemia cells that contained Thy 1.1 antigen were selectively killed compared to Thy 1.2-containing cells. The composition of the conjugates was determined by radioimmunoassay, and most of the immunotoxins contained about equal molar quantities of antibody and ribosomal inhibitor. Ricin A chain linked to antibody by a noncleavable m-maleimidobenzoyl-N-hydroxysuccinimide ester cross-link was not cytotoxic, but PAP coupled to the same antibody was. Both immunotoxins linked by a cleavable disulfide bond were cytotoxic. Disulfide-linked F(ab')2-PAP was cytotoxic, but it was about 45 times less efficient than disulfide-linked IgG-PAP. There was only a 3.2-fold difference in their ability to inhibit ribosomes in vitro. The relative difference between in vitro action and cytotoxicity could be accounted for by differences in the affinity of the immunotoxins to the cell surface. Neither PAP or ricin A chain disulfide linked to a monoclonal antibody against Mr 15,000 envelope protein, a murine leukemia virus coat protein, were not cytotoxic, although both conjugates bound to the cell surface. Because of its stability, ease of purification, and lack of an analogue of ricin B chain, PAP may be more useful than ricin A for immunotoxin synthesis.

Animals↗
PubMed1984

Prevention of growth of leukemia cells in mice by monoclonal antibodies directed against Thy 1.1 antigen disulfide linked to two ribosomal inhibitors:pokeweed antiviral protein or ricin A chain.

Pokeweed antiviral protein (PAP) and ricin A chain are potent inhibitors of protein synthesis that inactivate eukaryotic 60S ribosomal subunits. Immunotoxins were prepared by linking monoclonal anti-Thy 1.1 antibodies to PAP and ricin A chain through a disulfide bond. Both the conjugates were shown earlier to specifically inhibit protein synthesis of Thy 1.1-positive target leukemic cells (AKR SL3). In the present study, the efficacy of the immunotoxins to prevent the growth of AKR SL3 cell-induced tumor was checked in vivo in a model system. Injection of AKR SL3 cells s.c. into AKR/Cum (Thy 1.2-positive) mice developed into a solid tumor which was fatal. Administration of 31-E6:PAP and 31-E6:ricin A chain suppressed tumor growth. Suppression was specific, as similar treatment could not prevent the growth of a nontarget Thy 1.2-positive leukemia cell line (AKR SL1) derived from a congenic mouse. Unconjugated anti-Thy 1.1 immunoglobulin antibodies also showed significant tumor protection; however, administration of F(ab')2 fragment could not prevent the tumor growth. Injection of F(ab')2:PAP efficiently protected mice from AKR SL3-induced tumor. All the conjugate-treated mice showed antibody response against the toxin polypeptide. Anti-toxin antibody response was found as early as 26 days after the initiation of therapy and lasted as long as 179 days of observation. Further studies indicate that the presence of anti-toxin antibodies blocked completely the inhibitory ability of the respective immunotoxin in vitro. Anti-ricin antibodies neutralized the activity of 31-E6:ricin A chain conjugate but not OX-7:PAP immunotoxin, and similarly, anti-PAP antibodies inhibited the activity of the latter and not the activity of 31-E6:ricin A chain conjugate. These observations indicate that the use of alternate immunotoxins having an immunologically distinct toxin polypeptide may be necessary for tumor therapy during relapse, as exposure to the conjugates results in the formation of specific neutralizing anti-toxin antibodies. The anti-toxin antibodies did not prevent the binding of immunotoxin to target cells. Nevertheless, preincubation of conjugate with anti-toxin antibodies specifically blocked the respective conjugate-induced inhibition of polyuridylic acid translation in a cell-free assay system.

Animals↗
PubMed1983

Seasonal variations in different forms of pokeweed antiviral protein, a potent inactivator of ribosomes.

Pokeweed antiviral proteins enzymatically inactivate the 60 S subunit of eucaryotic ribosomes in cell-free preparations. Three different species of the enzyme can be isolated from spring leaves, summer leaves, and seeds of pokeweed. Sequence analyses of the NH2-terminal residues show that pokeweed antiviral protein, isolated from spring leaves and seeds, are homologous and differ in 11 of the 28 residues compared. Ricin contains a polypeptide (ricin A chain) that has functional similarities to pokeweed antiviral protein, yet the sequences of the pokeweed proteins show little similarity with ricin A chain. Ricin B chain is responsible for helping ricin A chain across the plasma membrane; since pokeweed antiviral has no counterpart to ricin B chain, it is not nearly as cytotoxic as ricin. However, when pokeweed antiviral protein was covalently coupled to ricin B chain, a cytotoxic species was formed. Pokeweed antiviral protein fails to interact noncovalently with the ricin B chain to produce a cytotoxic species equivalent in function to ricin.

Amino Acid Sequence↗
PubMed1983

Glucose concentration and insulin release in 5-thio-D-glucose-treated mice.

Male albino mice were given a single dose of various concentrations (25, 50, and 100 mg/kg body weight) of 5-thio-D-glucose or daily infusions (33 mg/kg body weight) of 5-thio-D-glucose for 21 days. Elevated blood glucose and immunoreactive insulin (IRI) levels were observed in the mice treated with 5-thio-D-glucose. Fasting glucose levels reached a maximum in 30 minutes and IRI levels reached a maximum in 60 to 90 minutes in the single-dose treated animals compared to preintubation levels. In the mice treated for 21 days, the fasting and fed glucose and IRI levels were significantly increased. Single dose of glucose (1 g/kg body weight) given to fasting and fed mice did not alter the glucose and IRI levels in the treated animals. However, a single dose of 5-thio-D-glucose (33 mg/kg body weight) given to fasting and fed treated animals increased the IRI levels significantly but not the glucose concentration. These data show that both single-dose and 3-week treatment with 5-thio-D-glucose produced a hyperinsulinemic diabetes in male albino mice.

Animals↗
PubMed1982

Effect of 5-thio-D-glucose on testicular lipids of mice.

Spermatogenesis is reported to be completely inhibited by 5-thio-D-glucose in mice. In an investigation of this inhibition, testicular lipid constituents, namely, total lipids, phospholipids, triacylglycerol, free and total cholesterol, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and NADPH generators like glucose-6-phosphate dehydrogenase, isocitrate dehyrogenase and malic enzyme were estimated in mice fed with 5-thio-D-glucose (33 mg/kg) by gastric intubation for 21 days. Significant increase in cholesteryl ester, glucose-6-phosphate dehydrogenase activity and malic enzyme and a decrease in free cholesterol and phospholipids were observed.

Animals↗
PubMed1982

Radioimmunoassay of ricin A- and B-chains applied to samples of ricin A-chain prepared by chromatofocusing and by DEAE Bio-Gel A chromatography.

A radioimmunoassay for ricin and ricin A- and B-chains was developed. Amounts as low as 100 pg of A-chain and 500 pg of B-chain could easily be quantitated. We showed, however, that the free chains were more reactive in the radioimmunoassay than the equivalent quantity of the individual chains when combined in intact ricin. The usefulness of the assay was demonstrated by determining the concentration of contaminating A- or B-chains in preparations of the separate polypeptides purified by DEAE Bio-Gel A chromatography and by chromatofocusing.

Chromatography, Gel↗