P.K. Ray

Antitumor activity with nontoxic doses of protein A

SummaryThis report confirms our previous observation that IV inoculation of purified protein A causes regression of rat mammary adenocarcinomas. In treated tumors, we have obtained histological evidence of changes indicating tumor cell destruction. Protein A treatment does not cause reduction in the body weight or organ weights of rats; nor does it cause any decrease in activity of the enzymes of the microsomal mixed function oxidase system in the liver. Protein A stimulates peripheral white cell counts in normal rats, but not in tumor-bearing rats. We found that protein A infusion reduced (P<0.0005) the level of circulating plasma immune complex concentration. A homing study with 125I-labeled protein A indicated that liver, spleen, and kidney tissues are the major sites of protein A accumulation. Therefore, protein A seemed to exert its antitumor effects without causing any generalized toxicity to the system. It is postulated that the action of protein A may be related to its ability to cause a drastic reduction in circulating plasma immune complex concentration, thus potentiating the immune reactivity of the host observed earlier.

Induction of Immune Rejection of Tumors by Protein A in Mice Bearing Transplantable Solid Tissue Dalton's Lymphoma Tumors

In a transplantable solid tissue Daltons lymphoma tumor model in mice we have studied the mode of antitumor action of protein A, a well known biological response modifier. Protein A (15 ug) was administered intravenously in normal and solid tissue Daltons lymphoma tumor bearing mice on day 3 and 7 after tumor inoculation. Incidence of mortality was more in untreated tumor bearing group than that in PA treated tumor bearers. There was a significant decrease (p less than 0.001) in tumor diameter in PA treated group compared to untreated group. Protein A treatment significantly enhanced the delayed type hypersensitivity (p less than 0.01), T-cell number in spleens (p less than 0.001) and lymph nodes (p less than 0.05) as well as phagocytosis (p less than 0.001) of opsonized SRBC by peritoneal macrophages of tumor bearing animals. Apart from the nonspecific immunopotentiation, Protein A also activates natural Killer (NK) cell activity and also splenic lymphocytes mediated killing of autologous tumor targets in a significant (p less than 0.001) manner. These results suggest that PA treatment activates cellular arc of the immune system in general, and macrophage, T cells and NK cells specifically. In the present communication, we have attempted to provide the information that these immune activations appear to be related to antitumor response induced by Protein A.

Effect of ASTA-Z 7575 (INN Maphosphamide) on Human Lymphokine-Activated Killer Cell Induction

Recent studies combining chemotherapeutic agents with various biological response modifiers for the treatment of cancer have shown promising results. Cyclophosphamide (Cy) is the most widely used alkylating agent and a major constituent of combination chemotherapy regimens for many neoplastic diseases. It has been reported that Cy is a cytotoxic drug, which becomes immunosuppressive at higher doses. A synthetic metabolite of Cy, ASTA-Z, has recently been produced. ASTA-Z is more active and stable by itself and does not need to be metabolically converted to an active compound. The combined effect of Cy and interleukin-2 (IL-2) on the induction of lymphokine-activated killer (LAK) cells is not known. Therefore, we decided to investigate the effect of ASTA-Z on the induction and function of LAK. The coculture of peripheral blood mononuclear cells (PBMC) with various concentrations of ASTA-Z (0, 10(-6), 10(-5), 10(-4), and 10(-3) dilution) and IL-2 (50 U/ml) for 4 days produced significant suppression of cytotoxicity and lytic ability of the LAK cells against NK-sensitive (K562) and NK-resistant (M14) tumor cell lines. The lower doses of ASTA-Z did not affect the generation of LAK cells, its cytotoxicity and lytic ability of ASTA-Z against both NK-sensitive and NK-resistant tumor cell lines. Furthermore, the ASTA-Z produced dose-dependent suppression of the proliferative response of LAK cells. The significant therapeutic benefit in the cancer patient may be achieved by the low dose regimen of Cy and IL-2 because it has no deleterious effect on the induction and function of LAK cells.

Protein A potentiates lymphokine-activated killer cell induction in normal and melanoma patient lymphocytes

The effects of purified protein A from Staphylococcus aureus Cowan I stain on induction of lymphokine (IL-2) activated killer (LAK) activity were studied in normal as well as melanoma patients lymphocyte. The coculture of peripheral blood mononuclear cells (PBMC) with various doses of protein A (0.001, 0.01 and 0.1 microgram/ml) and IL-2 (100 U/ml) for 4 days produced synergistic effect on the LAK cells mediated cytotoxicity. The potentiation of cytotoxicity and lytic ability of LAK cells against NK sensitive (K-562) and NK-resistant (M14) tumor cells were observed. Further there was potentiation of DNA synthesis in PBMC after 4 days culture. Similar results were found when PBMC from melanoma patients were cultured with PA and IL-2. The potentiation of LAK cell induction associated with its cytotoxic and lytic potential by low doses of IL-2/PA regiment may be helpful in the development of LAK immunotherapy of the cancer patients.

Acrylamtoe Induced Immunosuppression in Rats and Its Modulan by 6-MFA, An Interferon Inducer

In the present communication, we describe acrylamide (ACR) induced immunotoxicity and its modulation by an interferon inducer, the 6th mycelial fraction acetone (6-MFA) of Aspergillus ochraceus ATCC 28706. ACR administration to rats produced a significant decrease in the weight of spleen (p < 0.001), thymus (p < 0.001) and mesenteric lymph nodes (p < 0.05). A decrease in cellularity of spleen (p < 0.001), thymus (p < 0.001), bone marrow (p < 0.001) and circulating blood lymphocyte population (p < 0.001) was also recorded. ACR suppressed the humoral as well as cell mediated immunity as assessed by erythrocyte antibody complement (EAC)-rosettes (p < 0.001), hemagglutination titre (p < 0.001), PFC (p < 0.001) and the delayed type hypersensitivity response against sheep red blood cells (SRBC, p < 0.001). ACR treated immunosuppressed rats when treated with 6-MFA restored the circulating lymphocyte number to the normal level and a partial recovery in the weight of spleen and thymus. Potentiation of EAC-rosettes, hemagglutination titre, IgM-PFC and DTH response against SRBC was observed. It is concluded that 6-MFA ameliorate the ACR induced toxicity. This study may be of significance in prevention of ACR toxicity.

Antitumour activity of protein A in a mouse skin model of two-stage carcinogenesis

Protein A (PA) is an immunostimulating glycoprotein (mol. wt. 43,000 kDa) obtained from Staphylococcus aureus cowan I. The antitumour property of PA is well documented in the literature in various transplantable tumours of rats and mice. In the present set of investigations, the antitumour property of PA was tested in Swiss albino mice in a two-stage initiation-promotion mouse skin carcinogenesis model. The animals were initiated topically with a single subcarcinogenic dose (52 microgram) of 7,12-dimethylbenzanthracene (DMBA). PA was administered intraperitoneally (1 microgram/animal), twice weekly for 2 weeks. Promotion was performed by twice weekly applications of 12-O- tetradecanoyl phorbol-13-acetate (TPA) at a dose of 5 microgram/animal for 32 weeks. The result showed that the treatment schedule can effectively check the onset of tumorigenesis, the cumulative number of tumours and the average number of tumours per mouse. In the PA administered group, 30% of the animals remained tumour free until the termination of the experiments (i.e. 32 weeks of promotion). Thus the present study proves that protein A can effectively inhibit DMBA initiated and TPA promoted mouse skin carcinogenesis.

Protection against 7,12-dimethylbenzanthracene-induced tumour initiation by protein A in mouse skin

Protein A is an immunostimulating glycoprotein obtained from Staphylococcus aureus Cowan I. Its antitumour activity is proven in various tumour models. Its ability to provide protection against tumour initiation by the chemical carcinogen 7,12-dimethylbenzanthracene (DMBA) has been investigated in the present study using a mouse skin model of two-stage carcinogenesis. Protein A was administered intraperitoneally (1 microgram/animal 20 g body wt.) twice a week for 2 weeks, prior to initiation by DMBA. The promotion was performed by twice weekly applications of 12-O-tetradecanoyl phorbol-13-acetate (TPA) (3 or 5 micrograms/animal in 100 microliters acetone). Protein A provided significant protection to animals from DMBA-induced tumour initiation as was observed by the decrease in cumulative number of tumours, percent of animals developing tumours, number of tumours per animal and rate of tumour growth. Our data indicate that protein A has anticarcinogenic properties.

Biomethanation of spent wash: Heavy metal inhibition of methanogenesis in synthetic medium

Abstract Five heavy metals detected in distillery waste were lead (1.0–8.8 μg/ml), copper (1.7–15.7 μg/ml), zinc (3.1–11.8 μg/ml), iron (36.0–43.5 μg/ml), and manganese (3.0–5.1 μg/ml). Their toxicity to biomethanogenesis in a synthetic medium containing 1% sodium acetate, propionate, or butyrate was measured by batch fermentation, after cultivating the bacterial biomass semicontinuously. Lead, copper, and zinc in decreasing order were found to be toxic to biomethanogenesis. Lead at the concentration of 10 μg/ml completely stopped methane production. Iron did not produce any notable change in the process while manganese stimulated the rate of methane production. The toxicity of lead, copper, and zinc to methanogenic bacteria and methane production was dose-dependent but the growth of acetogenic bacteria was impaired at higher concentrations (2.5–10.0 μg/ml) of lead, copper, and zinc. Manganese stimulated the growth of only methanogenic bacteria, but not that of non-methanogenic bacteria or acetic acid production. The reduction in the synthesis of acetic acid via butyrate was more in the presence of these three metals than the synthesis of this acid via propionate.

Modulation of benzene induced toxicity by protein A

Administration of benzene (i.p. 1.0 mL/kg body weight) for 3 consecutive days produced leucopenia and lymphocytopenia in female albino rats. In addition, the total iron content, lipid peroxidation and superoxide dismutase activity of the liver and bone marrow were significantly (P < 0.001) increased. Low molecular weight (LMW) bleomycin-detectable iron accumulated only in bone marrow. Prior administration of Protein A (PA), a multipotent immunostimulant and interferon inducer (60 micrograms/kg body weight, i.v. twice weekly for 2 weeks), ameliorated most of the adverse effects of benzene. PA restored the changes in hepatic histological architecture, reversed leucopenia and superoxide dismutase activity, lipid peroxidation, total iron content and LMW iron content of bone marrow were normalized. Isozymes of glutathione-S-transferase (alpha, pi, mu) which decreased following benzene exposure increased in PA pretreated benzene exposed rats. This study suggests that pretreatment with PA modulates the toxicity of benzene.

Induction of glutathione-s-transferase isoenzymes by Protein A in rat liver

The administration of Protein A, a cell wall protein of Staphylococcus aureus Cowan I cells, causes an induction of glutathione-s-transferase in rat liver. Proteins, cross reactive with anti human glutathione-s-transferase, acidic (pi), basic (alpha, and neutral (mu) isoenzymes, are induced by 5.8, 2.2 and 6.15 fold respectively. The induction of glutathione -s-transferases, at least in part, might play a role in manifestation of therapeutic properties of Protein A.