Cell-mediated amplification and down regulation of cytotoxic immune response against autologous human cancer

The cytotoxic host immune response toward autologous human cancer may be regulated by the immunoregulatory network. Here we show that helper T cells, cloned from peripheral blood lymphocytes that were sensitized in vitro against an autologous human malignant paraganglioma, proliferated against and made interleukin 2 when cocultured with the tumor-associated antigen in the presence of autologous accessory cells. Furthermore, the helper cell clones amplified cytotoxic immune response by peripheral blood lymphocytes against the paraganglioma cells in coculture with the blood lymphocytes and the paraganglioma cells. An autologous T cell line bearing suppressor phenotype, established from a lymph node that had been infiltrated with the paraganglioma tumor cells, in contrast to the helper cells, selectively suppressed the cytotoxic immune response by the blood lymphocytes against the paraganglioma cells in identical coculture. These results, therefore, demonstrate the existence of cell-mediated immunologic regulations of the cytotoxic immune response (concurrent amplification and suppression in the same host) against an autologous human tumor.     

Regulation of cellular immune response against autologous human melanoma. I. Evidence for cell-mediated suppression of in vitro cytotoxic immune response

The potential existence of down-regulation of cytotoxic immune response against an autologous human melanoma line was investigated as a possible explanation for cytotoxic unresponsiveness against the autologous melanoma cells. The melanoma cell line, PJ-M, was established and lymph node resident lymphocytes (LNL) were isolated from a lymph node which was partially infiltrated with the melanoma cells. Autologous peripheral blood lymphocytes (PBL) were sensitized in in vitro co-culture (IVC) against radiated PJ-M cells in the presence or absence of PJ-M-sensitized LNL and enriched suppressor (OKT8+) or inducer (OKT4+) LNL populations, and were assayed for cytotoxicity in a 4-hr 51Cr-release microcytotoxicity assay. Significant cytotoxic response against PJ-M could be generated in the PBL, but not in the LNL. The addition of sensitized, unfractionated LNL, OKT8+, or OKT4+ LNL populations abrogated cytotoxic response in the PBL against PJ-M. The suppression of cytotoxic response was induced selectively against the PJ-M targets, because IVC of PBL in the presence of the sensitized LNL did not affect the generation of polyclonal cytotoxic alloreactivities, nor did they abrogate the generation of cytotoxic response against allogeneic targets in IVC against the corresponding allogeneic targets. These results suggest the possibility that cytotoxic immune response against the autologous melanoma cells might have been suppressed by the individual's own immunoregulatory circuit.     

UDP-glucose 4-epimerase from Saccharomyces fragilis. Presence of an essential arginine residue at the substrate-binding site of the enzyme

UDP-glucose 4-epimerase from Saccharomyces fragilis was inactivated by the arginine-specific reagents phenylglyoxal, 1,2-cyclohexanedione, and 2,3-butanedione following pseudo first order reaction kinetics. The reaction order with respect to phenylglyoxal was 1.8 and that with respect to the other two diones was close to unity. Protection afforded by substrate and competitive inhibitors against inactivation by phenylglyoxal and the reduced interaction of 1-anilinonaphthalene 8-sulfonic acid, a fluorescent probe for the substrate-binding region after phenylglyoxal modification, suggested the presence of an essential arginine residue at the substrate-binding region. Experiments with [7-14C]phenylglyoxal in the presence of UMP, a ligand known to interact at the substrate-binding region, showed that only the arginine residue at the active site could be modified by phenylglyoxal. The characteristic coenzyme fluorescence of the yeast enzyme was found to be enhanced three times in phenylglyoxal-inactivated enzyme suggesting the incorporation of the phenyl ring near the pyridine moiety of NAD.     

Changes in UsnRNA biosynthesis during rat liver regeneration

Partial hepatectomy (P.H.) induces a partially synchronized growth response of liver under normal regulation of growth. In this phase changes in cellular morphology, radial distribution pattern of cells and other biological as well as major biochemical changes are well documented [24]. Here, we have shown that the cellular content of UsnRNAs altered during this proliferative phase as well. The level of spliceosomal UsnRNAs (U1, U2, U4–U6) gradually decreased by 30–50% upto 48 hrs of P.H. followed by gradual increase to reach the normal level within one month of P.H. The U3 snRNA level on the other hand, was nearly equal to that in normal liver at 48 hrs of P.H. but in 24 and 72 hrs of P.H. its level was high (4 fold) in contrast to that in other UsnRNAs. Thus, it is clear from our data that the level of all the six UsnRNAs decreased during 48 hrs of P.H. compared to that after first 24 hrs. This has been correlated in the kinetics of UsnRNAs' synthesis (in terms of labelling) in isolated hepatocytes, where the rate of labelling of all the six UsnRNAs increased 20–30% in 24 hrs regenerating hepatocytes (R.H.) followed by sharp decrease by 30–50% within next 24 hrs, compared to that in the normal hepatocytes. But from 72 hrs onwards in R.H. the rate of labelling of all the six UsnRNAs again increased by 30–50% (compared to that in normal hepatocytes) followed by decrease of their labelling-rate to reach the normal level in R.H. within one month of P.H. Thus, it may be concluded that the changes in UsnRNAs' level during the proliferative phase of liver regeneration may be either due to the alteration in the rate of synthesis (in terms of labelling) or along with it differential turn over rate; this phenomenon may have some consequences with the regenerative process of liver.This paper was published in Molecular and Cellular Biochemistry131:67–73, 1994. Kluwer Academic Publishers regret the publication of the only partly corrected version.     

Adoptive immunotherapy with peripheral blood lymphocytes cocultured in vitro with autologous tumor cells and interleukin-2

A clinical trial of adoptive immunotherapy was carried out with peripheral blood lymphocytes (PBL), cocultured in vitro with autologous tumor cells and interieukin-2 (IL-2), in 14 patients with advanced melanoma. PBL from these patients were cocultured with irradiated autologous tumor cells for 7 days, which was followed by expansion in IL-2-containing medium. These lymphocytes were returned to the patient along with intravenous IL-2 at doses up to 2×10⁶ IU m^–2 day^–1. A dose of 300 mg/m² cyclophosphamide was administered to each patient intravenously 4 days prior to each treatment. Following coculture, the lymphocytes were primarily CD3⁺ T cells and they expressed varied degrees of cytotoxicity against autologous melanoma cells. In 9 patients the activated cells were al least 80% CD4⁺ and in 2 cases they were mostly CD8+. Some of the activated cells exhibited suppressor or helper activity in a functional regulatory coculture assay. No major therapeutic response was observed in this study. Minor responses were observed in 2 patients. Toxicities were those expected from the IL-2 dose administered.This work has been supported by an American Cancer Society Institutional Research Grant (ACS-IRG 91-230), by the University of Connecticut Clinical Research Center (grant 0021), and by the Hartford Hospital Research Fund (grant 1017-20-018). Dr. Sporn is a recipient of American Cancer Society Clinical Oncology Career Development Award 90-230     

Delayed-type cutaneous hypersensitivity to melanoma antigens and its implication in active specific immunotherapy in cancer

Summary In this study, we explored whether soluble tumor-cell surface-associated antigens (TAA) might be derived from autochthonous as well as allogeneic sources as immunogens for active specific immunotherapy. Using two popular cell membrane-bound antigen extraction techniques (3 M KCl and isotonic-hypotonic NaCl), we examined the immunogenic potential of such TAA and the specificity of immunologic host reactivity through a delayed-type cutaneous hypersensitivity reaction (DTH) as a guideline for their immunogenic potential in a human malignant melanoma model system. We found that either extraction technique could provide soluble TAA from both autochthonous and allogeneic sources capable of eliciting DTH. While evidence of positive DTH with autochthonous TAA reaffirms the immunogenicity of such TAA, the specificity of host reactivity against TAA derived from allogeneic sources is extremely difficult to establish, even with TAA partially purified by column chromatography in Sephadex G-200. Patients exhibited reactivity to other TAA derived from tumors of different histologies and often to more than one component isolated by column chromatography. Furthermore, when a group of melanoma patients was tested against a panel of melanoma antigens in any random combination, DTH to allogeneic TAA was seen in an unpredictable order and with inconsistent frequency. We conclude, therefore, that while autochthonous antigen immunizations may be justified, more careful studies will be necessary to define the antigenic profile of a given tumor (individual specificity vs shared specificity), establish specificity of alloantigens, and devise suitable methods for testing immunologic specificity for alloantigens, before rational immunotherapy with allogeneic tumor antigens will be feasible.     

Streptomycin induced changes in growth and metabolism of etiolated seedlings

Effects of various concentrations of streptomycin sulphate either alone or in combination with different cations and hormones on mungbean (Phaseolus aureus L.) seedling growth were studied. The relative inhibition of root growth was stronger than that of hypocotyl growth. Root growth inhibition was completely overcome by calcium, while other cations were ineffective. Inhibition of hypocotyl elongation could not be prevented by cations. IAA and GA₃ were capable of relieving streptomycin inhibition but kinetin was ineffective. In the coleoptiles of streptomycin-treated rice (Oryza sativa L.) seedlings, there were accumulation of nucleic acids and decline in protein content resulting in increased RNA/protein and DNA/protein ratios. High nucleic acid content induced by streptomycin could be correlated with reduced activity of the nucleases.     

Synthesis of azatricyclodiones & octahydro-benzo[f]isoindoles and their antimicrobial evaluation

A series of azatricyclodiones and octahydro-benzo[f]isoindoles have been synthesized by (4+2) Diels-Alder cycloaddition of maleimides with furfuryl amine. Reaction of azatricyclodiones with isocyanates led to the respective ureides. All of the compounds were screened against a number of bacteria and fungi. One of the compounds (2) displayed moderate antitubercular activity while two compounds (2) and (4) inhibited the fungal growth at 25 μg/mL.     

Prophylactic Potential of Synbiotic (Lactobacillus casei and Inulin) in Malnourished Murine Giardiasis: an Immunological and Ultrastructural Study

Probiotics and Antimicrobial Proteins - Giardiasis is a re-emerging infectious disease with outbreaks reported globally specially in children and malnourished individuals leading to malabsorption,...