Heterogeneity of the effectors of natural killer activity in CBA mice

The natural cytotoxicity of Percoll fractions of CBA mice splenocytes was tested against cells of human erythroblastosis suspension culture K-562 and murine C3HA hepatoma solid culture MGXXIIa. The 1.076 g/ml dense fraction was shown to have the highest activity in 3H-uridine cytotoxic test against the cell targets. Immunosera prepared by the Golub method against CBA mouse brains and exhausted supplementary by syngeneic thymocytes decreased the natural cytotoxicity of CBA mouse splenocytes against MGXXIIa tumor cells and, on the contrary, increased it against K-562 tumor cells.     

Dynamics of the activity of the normal mouse spleen killers after partial splenectomy

The activity of natural killers from the mouse spleen was determined by 51Cr clearance from labeled target cells (YAC-1) at different times after resection of the two-thirds of the spleen. In the early periods after operation, there was a decrease in the natural cytotoxic activity of splenocytes followed by a relatively rapid rise for 8 days until the maximum exceeding the cytotoxicity of intact spleen cells was reached. However, the total lytic potential of the regenerating spleen did not return to normal during 28 days.     

Effects of cyclosporin on C57BL/6 splenocytes before and after culture with high-dose recombinant interleukin-2: Implications for immunosuppression with cyclosporin

Summary Lymphokine-activated killer cells appear to arise from precursor cells bearing natural killer (NK) cell antigens. Cyclosporin (CsA) is a well-known immunosuppressive agent that can down-regulate NK cell cytotoxicity. Studies were initiated to evaluate the effects of CsA on splenocytes before and after exposure to recombinant interleukin-2 (rIL-2). Normal C57BL/6 mice receiving CsA at a dose of 100 mg/kg demonstrated a decrease in NK cell lysis against the YAC-1 lymphoma target in a 4-h chromium-release assay. When splenocytes obtained from CsA-treated mice were cultured for 3 days in complete medium containing 1000 U rIL-2/ml, they demonstrated a return of NK cell lysis to normal (mean cytotoxicity = 65 LU versus 60 LU for control and CsA-exposed splenocytes respectively;P, NS, five consecutive experiments) but revealed a decrease in the lysis of a NK-resistant target: the MCA-102 sarcoma (mean cytotoxicity = 20 LU vs 12 LU for control and CsA-exposed splenocytes respectively;P <0.02, five consecutive experiments). Fresh splenocytes cultured in media containing rIL-2 and CsA demonstrated a decrease in proliferation, cell-cycle S-phase fraction and cell yields compared to splenocytes cultured in media containing rIL-2 alone. In addition, a decrease in tumor cell lysis for NK-cell sensitive (mean percentage lysis = 98% vs 60%, rIL-2 vs rIL-2 + CsA; effector-to-target ratio 100: 1) and resistant targets (mean percentage lysis = 68% vs 28%, rIL-2 vs rIL-2 + CsA; effector-to-target ratio 100: 1) was also seen. CsA had no effects on the phenotypic antigenic expression of splenocytes cultured with high-dose rIL-2 although activated T cell antigens were down-regulated when fresh splenocytes were evaluated after in vivo exposure to CsA. These studies support the down-regulating effects of CsA on NK cell lysis and suggest that the rIL-2-activated cell population is heterogeneous as demonstrated by the differential down-regulation and recovery of NK-resistant cell lysis versus NK-sensitive cell lysis.     

Sensitization of mouse splenocytes to normal tissue antigens and natural killer activity. I. The effect of immunization with normal syngeneic tissues

Immunization of C3HA mice with homogenized syngeneous liver led to sensibilization of splenocytes towards liver antigens and to the increase in natural killer cell activity towards K-562 cells. The dynamics of sensibilization and cytotoxicity in different time intervals after immunization has led to a conclusion about a correlation between the two above phenomena.     

Metabolic activation of 3-hydroxyanisole by isolated rat hepatocytes

A tyrosinase-directed therapeutic approach for malignant melanoma therapy uses the depigmenting phenolic agents such as 4-hydroxyanisole (4-HA) to form cytotoxic o-quinones. However, renal and hepatic toxicity was reported as side effects in a recent 4-HA clinical trial. In search of novel therapeutics, the cytotoxicity of the isomers 4-HA, 3-HA and 2-HA were investigated. In the following, the order of the HAs induced hepatotoxicity in mice, as measured by increased in vivo plasma transaminase activity, or in isolated rat hepatocytes, as measured by trypan blue exclusion, was 3-HA > 2-HA > 4-HA. Hepatocyte GSH depletion preceded HA induced cytotoxicity and a 4-MC-SG conjugate was identified by LC/MS/MS mass spectrometry analysis when 3-HA was incubated with NADPH/microsomes/GSH. 3-HA induced hepatocyte GSH depletion or GSH depletion when 3-HA was incubated with NADPH/microsomes was prevented by CYP 2E1 inhibitors. Dicumarol (an NAD(P)H: quinone oxidoreductase inhibitor) potentiated 3-HA- or 4-methoxycatechol (4-MC) induced toxicity whereas sorbitol (an NADH generating nutrient) greatly prevented cytotoxicity indicating a quinone-mediated cytotoxic mechanism. Ethylendiamine (an o-quinone trap) largely prevented 3-HA and 4-MC-induced cytotoxicity indicating that o-quinone was involved in cytotoxicity. Dithiothreitol (DTT) greatly reduced 3-HA and 4-MC induced toxicity. The ferric chelator deferoxamine slightly decreased 3-HA and 4-MC induced cytotoxicity whereas the antioxidants pyrogallol or TEMPOL greatly prevented the toxicity suggesting that oxidative stress contributed to 3-HA induced cytotoxicity. In summary, ring hydroxylation but not O-demethylation/epoxidation seems to be the bioactivation pathway for 3-HA in rat liver. The cytotoxic mechanism for 3-HA and its metabolite 4-MC likely consists cellular protein alkylation and oxidative stress. These results suggest that 3-HA is not suitable for treatment of melanoma.     

Expression and characterization of <Emphasis Type="Italic">Momordica Chanrantia</Emphasis> anti-hyperglycaemic peptide in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A nucleic acid sequence MC, encoding Momordica Chanrantia anti-hyperglycaemic peptide MC6 (accession: AAX06814) synthesized according to Escherichia coli preferred codons, was cloned and expressed in E. coli. Recombinant protein pQE8-MC (about 3.5 kDa) was purified and analyzed by 20% SDS–PAGE and western blot. It revealed that the expressed pQE8-MC had good solubility in aqueous media. An HPLC assay was used to confirm the expression of pQE8-MC. Subsequent pharmacological activity assay revealed a significant hypoglycemic effect of low dose treatments of pQE8-MC on male kunming mice. Four hours after an intravenous tail injection, the blood sugar levels of mice treated with pQE8-MC saline solution A3 (1 mg/kg BW) decreased greatly (P < 0.01) relative to the levels of a control group. This suggests that pQE8-MC, expressed in bioengineered E. coli, has a similar hypoglycemic function to the natural protein MC6 from M. Chanrantia. These results reveal the possibility of using bio-engineered bacteria as an anti-diabetic agent.     

Induction of cytotoxicity from fresh splenocytes after in vivo administration of cyclophosphamide

Summary Cyclophosphamide, combined with lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2), is known to mediate regression of tumors, but the effects of cyclophosphamide on the subsequent generation of LAK cells are unclear. It was the aim of the experiments in this paper to determine whether fresh splenocytes cultured with rIL-2 would maintain or regain their cytotoxicity in vitro after being exposed to the cytotoxic agent cyclophosphamide in vivo. Functional monitoring of splenocytes after in vitro incubation with rIL-2 was performed at various times through chromium-release assays, thymidine assays and cell-cycle analysis. Chromium-release assays determined that the cytotoxicity of cultured splenocytes returned to normal after 12 days of in vitro culture with rIL-2. The thymidine assays indicated a normal rate of uptake of thymidine after 7 days in culture, while the cell cycle was still abnormal by day 12 of culture. The growth and expansion of rIL-2-activated splenocytes after different times of in vitro culture indicated a return to normal compared to control animals after 7 days of continuous in vitro exposure to rIL-2. It is concluded that murine splenocytes can demonstrate cytotoxicity after exposure to cyclophosphamide, through prolonged continuous in vitro culture with rIL-2. Since cyclophosphamide did not jeopardize the production of splenocyte cytotoxic effectors generated with rIL-2, it appears to be a strong contender for use in chemoimmunotherapy protocols.Supported in part by grants from The Alberta Heritage Foundation for Medical Research and the National Cancer Institute of Canada     

Natural killer activity of the splenocytes in C3HA strain mice during 20-methylcholanthrene-induced carcinogenesis

The induction of tumor in C3HA mice after intramuscular injection with 20-methylcholanthrene is accompanied by a decrease in natural antitumor resistance. This conclusion is based on the observation of the decreased natural killer activity per total number of splenocytes, from the time of carcinogen application till the appearance of tumor nodes.     

Immunomodulating effect of cyclophosphamide on cytotoxic activity of rats and mice splenocytes

This study examines the immunomodulating effect of cytostatic drug--cyclophosphamide (Cy)--on natural cytotoxic activity of rats and mice splenocytes. The cytotoxicity of the effector cells against the confluent monolayer cell lines of rat hepatoma Zajdela and HTC and mice hepatoma MH-22a was estimated by means the morphometric analysis. It was shown, that 48 h after single intraperitoneal injection of Cy produced a immunomodulating effect on the activity of splenocytes--suppressor action on cells of Zajdela hepatoma and immunopotentiating action on the target cells of HTC and MH-22a hepatomas. The possible mechanisms of immunopotentiating effect of Cy are discussed.     

Characteristics of natural killer cells (NK cells) and features of the cytotoxic test in Syrian hamsters

The cytotoxic test in vitro with the use of xenogeneic target cells of human myeloma, strain K-562, labeled with 51Cr has demonstrated natural cytotoxicity of lymphoid cells from noninbred Syrian hamsters. This cytotoxicity occurs at the cost of non-adherent splenocytes. NK may be isolated over the gradient density of ficoll (1.078), selective for large granular lymphocytes. To detect the maximal lytic activity of NK from Syrian hamsters in the cytotoxic test in vitro, they should be brought into 10-12 hour contact with sensitive target cells K-562. In Syrian hamsters, the highest natural cytotoxicity is shown by the cells of the blood and spleen. In the bone marrow and thymus, it is little pronounced and is virtually absent from the peripheral lymph nodes.     

Immunomodulating effect of cyclophosphamide on cytotoxic activity of rat and mouse splenocytes

The goal of this work consisted in study of the immunomodulating action of the cytostatic drug cyclophosphamide (CP) on the natural cytotoxic activity of rat and mice splenocytes. The cytotoxicity of effector cells (EC) with respect to monolayer cell lines of the Zajdela rat hepatoma and the HTC rat hepatoma and of the MH-22a mouse hepatoma was determined with the aid of morphometric analysis. CP at a dose of 100 mg/kg 48 h after administration to animals has been shown to produce an immunomodulating effect on cytotoxicity of splenocytes—a suppressive one with respect to cell-targets (CT) of Zajdela hepatoma and an immunopotentiating one with respect to CT of HTC and MH-22 hepatomas. Possible mechanisms of the CP immunopotentiating action are discussed.     

Effect of 3-methylcholanthrene on the biliary excretion of bromsulphthalein and eosine in newborn rats.

The biliary excretion rates of bromsulphthalein (BSP), bromsulphthalein-glutathione conjugate (BSP-GSH) and eosine have been studied in 3-methylcholanthrene (3-MC)-pretreated (100 mg/kg i.p.) and control rats aged 10 days. Liver weight was invariably increased after 3-MC treatment, associated with enhanced biliary excretion of total BSP. The increase in the biliary excretion of total BSP was due solely to the increased excretion of BSP-GSH. Following 3-MC pretreatment, BSP-GSH and eosine appeared in the bile in the same amount as in the control rats after i.v. administration of BSP-GSH and eosine. Pretreatment with 3-MC increased the ratio of BSP-GSH to BSP in the liver and bile. Our results suggest that the increased biliary excretion of total BSP following 3-MC treatment was due to an enhanced conjugation of BSP with GSH.     

The influence of tumor growth on natural cytotoxicity and activity of some lysosomal enzymes of human effector cells and the C3HA mouse splenocytes

In the course of malignant growth processes in patients with lung cancer, a decrease of natural cytotoxic activity of peripheral blood lymphocytes was observed. This process was accompanied by changes of activities of two lysosomal enzymes, arylsulfatase and acid phosphatase, suggesting participation of these enzymes in manifestation of effector functions of lymphocytes in cancer patients. The level of activity of granular enzyme, beta-glucuronidase, remained unchanged at all stages of disease. A study of natural killer activity of C3HA mice splenocytes after inoculation of transplantable hepatoma 22-a cells revealed a relative stability of the level of their cytotoxicity, and of the activities of lysosomal enzymes--arylsulfatase, acid phosphatase, alpha-mannosidase, acid lipase, N-acetyl-beta-D-glucosidase, and beta-galactosidase, beginning from the 3rd day after hepatoma implantation.     

N-acetylcysteine reduces transformed 3T3-SV40 fibroblast sensitivity to lysis by natural killer cells

We have shown that 18-20 h cultivation of transformed mouse fibroblasts 3T3-SV40 in the presence of antioxidant, N-acetylcysteine (NAC, 10 mM), did not change their sensitivity to lysis by natural killer (NK) mouse splenocytes. However, in 18-20 h after NAC removal 3T3-SV40 cells demonstrated resistance to NK cell activity. The cytotoxicity index (CI) was reduced up to 4.6 +/- 2.4 % (in comparison with the control value 31.8 +/- 2.4 %) approximating to the value in non-transformed 3T3 mouse fibroblasts. Normal 3T3 cells were resistant to NK action in all experimental conditions (CI varied within 0.7-5.3 %). These results show that NAC can induce partial reversion of transformed phenotype. We suggest that this effect may be due to the NAC-induced modifications of the cell surface and extracellular matrix proteins.     

Cancare-a herbal formulation inhibits chemically induced tumours in experimental animals

Cancer chemopreventive potential of Cancare, a multi-herbal formulation on chemically induced tumours was studied by N-nitrosodiethylamine (NDEA) induced hepatocarcinogenesis in rats and 20-methylcholanthrene (20-MC) induced sarcoma development in mice. Oral administration of Cancare was found to inhibit the liver tumour development induced by N-nitrosodiethylamine. Animals administered with NDEA had visible liver tumours by the end of 30th weeks and the liver weight was raised to 6.1 +/- 1.4 g/ 100 g body wt. None of the animals treated with Cancare (150 mg/ kg) developed any visible liver tumours by this period and the liver weight was 3.0 +/- 0.6 g/ 100 g body wt. Gamma-Glutamyl transpeptidase, a marker of hepatocellularcarcinoma, which was raised to 83.7 +/- 8. 9 U/l in serum of NDEA treated group was reduced to 35.2 +/- 6.1 U/l by simultaneous administration of Cancare. Elevated levels of serum alkaline phosphatase, glutamate pyruvate transaminase, bilirubin, liver glutathione S-transferase, glutathione and gamma-Glutamyl transpeptidase in the NDEA administered group was significantly reduced by Cancare administration. Cancare administration inhibited the sarcoma development and increased the life span of mice administered with 20-MC dose dependently. All animals in the control group developed sarcomas by 150th day and dead by 174th day after 20-MC administration. Cancare administration (30 mg and 150 mg/kg) inhibited the sarcoma development (46.7 and 60%) as well as increased the life span (53.3 and 66.7%) as estimated on 240th day after 20-MC administration. The results are indicative of the chemopreventive potential of Cancare against chemically induced neoplasmas.     

Natural killer cell activity in mice infected with Acanthamoeba culbertsoni]

The natural killer cell activity of splenocytes and TBC, active NK cells, recycling capacity of natural killer cells were observed by means of both the 51Cr-release cytotoxicity assay and single cell cytotoxicity assay against YAC-1. C3H/HeJ mice were infected intranasally with 1 x 10(4) or 1 x 10(5) trophozoites of pathogenic Acanthamoeba culbertsoni. The infected mice showed mortality rate of 34% in 1 x 10(4) group and 65% in 1 x 10(5) group, and mean survival time was 16.40 +/- 3.50 and 13.20 +/- 4.09 days respectively. The cytotoxic activity of natural killer cells of the 2 groups was significantly higher than that of non-infected mice from the 12th hour to the 2nd day after infection, showing the highest on the first day. On the 10th day after infection, the cytotoxic activity of natural killer cells was significantly suppressed as compared with that of the control. There was no significant difference in NK cell cytotoxicity between two infected groups. The target-binding capacity and active NK cells of natural killer cells in 1 x 10(5) trophozoite infected mice was significantly increased on the 12th hour and the first day after infection as compared with the control group. Maximal recycling capacity (MRC) was not changed during the observation period. The present results indicated that the elevation of natural killer cell activity in the mice infected with A. culbertsoni was due to elevation of target-binding capacity and increased active NK cells of natural killer cells, and not due to the maximal recycling capacity of the individual NK cell, and there was no difference between two experimental dose groups.     

Rapid conversion of effector mechanisms from NK to T cells during virus-induced lysis of allogeneic implants in vivo

Viral infections can strongly stimulate both NK cell and allospecific CD8 T cell responses, and these same effector cells can lyse allogeneic cell lines in vitro. However, the impact of viral infections on the effector systems mediating rejection of allogeneic tissues in vivo has not been fully explored. Using in vivo cytotoxicity assays, we evaluated the effector systems mediating the rejection of CFSE-labeled allogeneic splenocytes after an infection of C57BL/6 (B6) mice with lymphocytic choriomeningitis virus. Naive B6 mice predominantly used a NK cell-effector mechanism to reject allogeneic splenocytes because they rejected BALB/C (H2(d)) splenocytes but not CBA (H2(k)) splenocytes, and the rejection was prevented by immunodepletion of NK1.1(+) or Ly49D(+) NK cells. This rapid and efficient in vivo cytotoxicity assay recapitulated the specificity of NK cell-mediated rejection seen in longer duration in vivo assays. However, as early as 1 day after infection with lymphocytic choriomeningitis virus, a CD8 T cell-dependent mechanism participated in the rejection process and a broader range of tissue haplotypes (e.g., H2(k)) was susceptible. The CD8 T cell-mediated in vivo rejection process was vigorous at a time postinfection (day 3) when NK cell effector functions are peaking, indicating that the effector systems used in vivo differed from those observed with in vitro assays measuring the killing of allogeneic cells. This rapid generation of allospecific CTL activity during a viral infection preceded the peak of viral epitope-specific T cell responses, as detected by in vivo or in vitro cytotoxicity assays.     

Differential contribution of Fas- and perforin-mediated mechanisms to the cell-mediated cytotoxic activity of naive and in vivo-primed intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IELs) are known to exert strong constitutive cytotoxic activity. In the present study we compared the Ag-specific cytotoxic activity and the effector mechanisms involved in non-Ag-primed, naive and in in vivo-primed IELs and splenic CD8 T cells. Ex vivo isolated naive CD8alphaalpha TCRalphabeta IELs, CD8alphabeta IELs, and splenocytes from lymphocytic choriomeningitis virus (LCMV)-specific TCR transgenic mice exert Ag-specific cytotoxic activity in a long-term, but not in a short-term, cytotoxicity assay. This cytotoxic activity is mainly Fas-Fas ligand mediated and is significantly reduced in the presence of 20 microg/ml Fas-Fcgamma1 fusion protein. Both CD8alphabeta IELs and CD8alphabeta splenocytes isolated from LCMV-infected C57BL/6 mice exert potent perforin-dependent cell-mediated cytotoxicity. CD8alphaalpha TCRalphabeta IELs from LCMV-infected animals, however, show only minimal Ag-specific cytotoxicity. The potent cytotoxic activity of in vivo activated CD8alphabeta IELs is not affected by the addition of Fas-Fcgamma1. Nevertheless CD8alphabeta IELs from LCMV-infected perforin-deficient mice exert Ag-specific cytotoxicity in a short-term cytotoxicity assay, and this cytotoxicity is almost completely blocked by the addition of Fas-Fcgamma1. These results demonstrate that naive CD8alphabeta IELs exert Ag-specific, Fas-Fas ligand-mediated, constitutive cytotoxic activity in a long-term cytotoxicity assay, whereas primed CD8alphabeta IELs primarily use the perforin-dependent exocytosis pathway to exert their potent cytotoxic activity. Furthermore, these results clearly illustrate the requirement for Ag-specific determination of IEL-mediated cytotoxicity, because the elevated, but variable, frequencies of memory-type T cells in this compartment may lead to ambiguous results when polyclonal activation or redirected assays are used.     

Effect of immunosuppressors cyclophosphane and 5-fluorouracil on natural cytotoxicity and activity of lysosomal enzymes of splenocytes in C3HA mice

Effects of two immunosuppressors, cyclophosphane abd 5-fluorouracyl, used in clinical practice for treatment of oncological diseases, were assessed in respect to cytotoxicity and activity of several lysosomal enzymes located in splenocyte granules of C3HA mice. 48 h after a single intraperitoneal injection, both preparations produced a marked decrease in their cytotoxic activity, which was accompanied by a pronounced splenopathy. Both preparations were shown to decrease activity of arylsulfatase. Administration of cyclophosphane brought about the rise of activity of acid lipase as compared to control. Activities of acid phosphatase, alpha-mannosidase, beta-galactosidase, and N-acetyl-beta-D-glucosidase did not change after administration of the used immunosuppressors. It may be suggested that only arylsulfatase and acid lipase are involved in performance and(or) manifestation of the natural killer activity in splenocytes of the C3HA mice after their administration with cyclophosphane or 5-fluorouracyl.