Therapeutic Inducers of Natural Killer cell Killing (ThINKK): preclinical assessment of safety and efficacy in allogeneic hematopoietic stem cell transplant settings.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) remains the standard of care for chemotherapy-refractory leukemia patients, but cure rates are still dismal. To prevent leukemia relapse following HSCT, we aim to improve the early graft-versus-leukemia effect mediated by natural killer (NK) cells. Our approach is based on the adoptive transfer of Therapeutic Inducers of Natural Killer cell Killing (ThINKK). ThINKK are expanded and differentiated from HSC, and exhibit blood plasmacytoid dendritic cell (pDC) features. We previously demonstrated that ThINKK stimulate NK cells and control acute lymphoblastic leukemia (ALL) development in a preclinical mouse model of HSCT for ALL. Here, we assessed the cellular identity of ThINKK and investigated their potential to activate allogeneic T cells. We finally evaluated the effect of immunosuppressive drugs on ThINKK-NK cell interaction. METHODS: ThINKK cellular identity was explored using single-cell RNA sequencing and flow cytometry. Their T-cell activating potential was investigated by coculture of allogeneic T cells and antigen-presenting cells in the presence or the absence of ThINKK. A preclinical human-to-mouse xenograft model was used to evaluate the impact of ThINKK injections on graft-versus-host disease (GvHD). Finally, the effect of immunosuppressive drugs on ThINKK-induced NK cell cytotoxicity against ALL cells was tested. RESULTS: The large majority of ThINKK shared the key characteristics of canonical blood pDC, including potent type-I interferon (IFN) production following Toll-like receptor stimulation. A minor subset expressed some, although not all, markers of other dendritic cell populations. Importantly, while ThINKK were not killed by allogeneic T or NK cells, they did not increase T cell proliferation induced by antigen-presenting cells nor worsened GvHD in vivo. Finally, tacrolimus, sirolimus or mycophenolate did not decrease ThINKK-induced NK cell activation and cytotoxicity. CONCLUSION: Our results indicate that ThINKK are type I IFN producing cells with low T cell activation capacity. Therefore, ThINKK adoptive immunotherapy is not expected to increase the risk of GvHD after allogeneic HSCT. Furthermore, our data predict that the use of tacrolimus, sirolimus or mycophenolate as anti-GvHD prophylaxis regimen will not decrease ThINKK therapeutic efficacy. Collectively, these preclinical data support the testing of ThINKK immunotherapy in a phase I clinical trial.

Assessment of new HDAC inhibitors for immunotherapy of malignant pleural mesothelioma.

Background: Malignant pleural mesothelioma (MPM) is a very rare and highly aggressive cancer of the pleura associated in most cases with asbestos exposure. To date, no really efficient treatments are available for this pathology. Recently, it has been shown that epigenetic drugs, particularly DNA methylation or histone acetylation modulating agents, could be very efficient in terms of cytotoxicity for several types of cancer cells. We previously showed that a hypomethylating agent (decitabine) and a histone deacetylase inhibitor (HDACi) (valproic acid (VPA)) combination was immunogenic and led to the induction of an anti-tumor immune response in a mice model of mesothelioma. However, VPA is not very specific, is active at millimolar concentrations and is responsible for side effects in clinic. To improve this approach, we studied four newly synthetized HDACi, two hydroxamates (ODH and NODH) and two benzamides (ODB and NODB), in comparison with VPA and SAHA. We evaluated their toxicity on immune cells and their immunogenicity on MPM cells in combination with decitabine. Results: All the tested HDACi were toxic for immune cells at high concentrations. Combination with decitabine increased toxicity of HDACi only towards T-cell clone. A decrease in the proportion of regulatory T cells and natural killer cells was observed in particular with VPA and ODH. In MPM cells, all HDACi combinations induced NY-ESO-1 cancer testis antigen (CTA) expression and the recognition of the treated cells by a NY-ESO-1 specific T-CD8 clone. However, for MAGE-A1, MAGE-A3 and XAGE-1b mRNA expression, the results obtained depended on the HDACi used and on the CTA studied. Depending on the MPM cell line studied, molecules alone increased moderately PD-L1 expression. When combined, a higher stimulation of this immune check point inhibitor expression was observed. Decitabine-induced anti-viral response seemed to be inhibited in the presence of HDACi. Conclusions: This work shows that the combination of decitabine and HDACi could be of interest for MPM immunotherapy. However, this combination induced PD-L1 expression which suggests that an association with anti-PD-L1 therapy should be performed to induce an efficient anti-tumor immune response.

Assessment of interactions of efavirenz solid drug nanoparticles with human immunological and haematological systems.

BACKGROUND: Recent work has developed solid drug nanoparticles (SDNs) of efavirenz that have been demonstrated, preclinically, improved oral bioavailability and the potential to enable up to a 50% dose reduction, and is currently being studied in a healthy volunteer clinical trial. Other SDN formulations are being studied for parenteral administration, either as intramuscular long-acting formulations, or for direct administration intravenously. The interaction of nanoparticles with the immunological and haematological systems can be a major barrier to successful translation but has been understudied for SDN formulations. Here we have conducted a preclinical evaluation of efavirenz SDN to assess their potential interaction with these systems. Platelet aggregation and activation, plasma coagulation, haemolysis, complement activation, T cell functionality and phenotype, monocyte derived macrophage functionality, and NK cell function were assessed in primary healthy volunteer samples treated with either aqueous efavirenz or efavirenz SDN. RESULTS: Efavirenz SDNs were shown not to interfere with any of the systems studied in terms of immunostimulation nor immunosuppression. Although efavirenz aqueous solution was shown to cause significant haemolysis ex vivo, efavirenz SDNs did not. No other interaction with haematological systems was observed. Efavirenz SDNs have been demonstrated to be immunologically and haematologically inert in the utilised assays. CONCLUSIONS: Taken collectively, along with the recent observation that lopinavir SDN formulations did not impact immunological responses, these data indicate that this type of nanoformulation does not elicit immunological consequences seen with other types of nanomaterial. The methodologies presented here provide a framework for pre-emptive preclinical characterisation of nanoparticle safety.

Phase I study of OM-174, a lipid A analogue, with assessment of immunological response, in patients with refractory solid tumors.

BACKGROUND: Lipids A, the lipophilic partial structure of lipopolysaccharides, induce regression of several tumor types in animal models. Rather than exerting direct cytotoxic effect, these compounds trigger the immune system which in turn stimulates secretion of cytokines, and activates the inducible nitric oxide synthase, as well as immune cell infiltration of tumors. OM-174 is an analogue of lipid A with dual action on Toll-like receptors 2 and 4. In an experimental model of peritoneal carcinomatosis induced in BDIX rats by intraperitoneal injection of syngeneic PROb colon cancer cells, it induced a complete regression of tumors. The present phase I trial was conducted to determine the maximum tolerated dose, the recommended phase II dose and biological response associated with OM-174 administered as intravenous infusion. METHODS: Patients received OM-174 twice weekly for a total of 5, 10 or 15 injections of either 600, 800 or 1000 µg/m(2). Blood samples for pharmacokinetic analysis and cytokine dosages were collected. NK cells activity and Toll-like receptors 4 polymorphism analysis were also performed. RESULTS: Seventeen patients were included. The highest dose administered was 1000 µg/m(2) repeated in 15 injections. The most common toxicities were a chills, fever, nausea/vomiting, diarrhea, fatigue and headache. No patient experienced haematological side effects. As no dose limiting toxicity was observed, despite a grade 3 respiratory complication, the maximal tolerated dose and recommended dose were not established. Three patients exhibited disease stabilization with a mean duration of 4 months. Pharmacokinetic profile of OM-174 was characterized by a low distribution volume and clearance. Analysis of TLR 4 polymorphysm showed that most (16/17) patients carried the wild type alleles. A progressive increase in NK cell number and activity was observed only in patients receiving 1000 µg/m(2) of OM-174. A peak of IL-8 and IL-10 concentrations were observed after each OM-174 injection. Peaks of TNF-alpha and IL-6 concentrations were detected after the first infusion and decreased progressively suggesting tolerance. CONCLUSION: OM-174 therapy was well tolerated at biologically active concentrations. Whereas the recommended dose was not determined, further studies are planned in combination with chemotherapy as animal models suggest a strong synergistic antitumor effect. TRIAL REGISTRATION: NCT01800812 (ClinicalTrials.gov Identifier).

PGE2 suppresses NK activity in vivo directly and through adrenal hormones: effects that cannot be reflected by ex vivo assessment of NK cytotoxicity.

Surgery can suppress in vivo levels of NK cell cytotoxicity (NKCC) through various mechanisms, including catecholamine-, glucocorticoid (CORT)-, and prostaglandin (PG)-mediated responses. However, PGs are synthesized locally following tissue damage, driving proinflammatory and CORT responses, while their systemic levels are often unaffected. Thus, we herein studied the role of adrenal factors in mediating in vivo effects of PGs on NKCC, using adrenalectomized and sham-operated F344 rats subjected to surgery or PGE(2) administration. In vivo and ex vivo approaches were employed, based on intravenous administration of the NK-sensitive MADB106 tumor line, and based on ex vivo assessment of YAC-1 and MADB106 target-line lysis. Additionally, in vitro studies assessed the kinetics of the impact of epinephrine, CORT, and PGE(2) on NKCC. The results indicated that suppression of NKCC by epinephrine and PGE(2) are short lasting, and cannot be evident when these compounds are removed from the in vitro assay milieu, or in the context of ex vivo assessment of NKCC. In contrast, the effects of CORT are long-lasting and are reflected in both conditions even after its removal. Marginating-pulmonary NKCC was less susceptible to suppression than circulating NKCC, when tested against the xenogeneic YAC-1 target line, but not against the syngeneic MADB106 line, which seems to involve different cytotoxicity mechanisms. Overall, these findings indicate that elevated systemic PG levels can directly suppress NKCC in vivo, but following laparotomy adrenal hormones mediate most of the effects of endogenously-released PGs. Additionally, the ex vivo approach seems limited in reflecting the short-lasting NK-suppressive effects of catecholamines and PGs.

Assessment of immune function after short-term administration of recombinant human growth hormone in healthy young males.

Growth hormone (GH) is a commonly used drug aimed at improving sport performance. The aim of this study is to evaluate the immunomodulatory effects of short-term administration of recombinant GH (rhGH) in healthy young males. NK cell number, activity and phenotype, T cell number, CD4(+) (Th1/Th2) cytokine production of IL2, IL4, IL6, IL10, TNF-α and IFN-γ and CD4(+)/CD8(+) ratio with particular attention to the possible correlation to IGF-I production were investigated. 30 males (27 ± 9 years) were randomly assigned to placebo (n = 15) or drug (rhGH) 1 mg/day groups (n = 15) with daily injection for 7 days. IGF-I plasma concentration and flow cytometry data were generated at baseline and days 8, 15, 22 and 29 post injection. Data analysis used General Linear Model with repeated measures, Bonferroni correction factor and significance at p ≤ 0.05. Serum IGF-I levels (ng/mL) increased significantly (p ≤ 0.01) on day 8 (0.48 ± 0.78) after injections compared to baseline (0.31 ± 0.07) and days 15 (0.33 ± 0.06), 22 (0.29 ± 0.05) and 29 (0.29 ± 0.06). A significant time effect was noted in IL10 secretion (pg/mL) from day 15 (P = 35.14 ± 19.93, rhGH = 26.63 ± 16.39) to days 22 (P = 61.32 ± 20.41, rhGH = 74.99 ± 46.91) and 29 (P = 101.98 ± 67.25, rhGH = 107.74; ± 122.58). There was no correlation between IGF-I and NK activity, phenotype or number along with T lymphocyte number, CD4(+)/CD8(+) ratio or Th1 and Th2 cytokine production. In conclusion, cytokine secretion spectrum was not affected by short-term rhGH administration in young males.

Assessment of NK cells response to hepatocyte derived chemotactic agents.

This project was aimed to examine the NK92 cells response as the CXC chemokine responder cells in rat model of liver disorder and injuries. Hepatocytes were isolated from Sprague-dawly rats and cultured on collagen type 1. Migration of NK92 cells was assessed using a 48 well micro-chemotaxis technique. Transwell chambers were positioned faced up, blocked Medium supernatant (500 microL) obtained from hepatocytes cultures were placed into the lower compartment of each Transwell. The upper compartment was filled with either 500 microL of NK92 cells. After washing, Membrane-attached cells were fixed; stained and Membrane-attached cells were counted by light microscopy and/or by size gating (9-14 microm) with an automated counter system. Human NK92 cells were attracted to recombinant human IP-10 in a concentration and time-dependent manner. NK92 cells also exhibited a chemotactic response to medium harvested from primary hepatocyte cultures. Isolated and cultured hepatocytes express several different chemokines. Although we identified that medium from hepatocyte cultures contains specific chemokines by immunoblotting, there is potential that migration assays detected yet other chemokines and other factors such as complement components. In this report, we demonstrated that hepatocytes expressed factors that were chemoattractive for human NK92 cells and that the factors must interact with the repertoire of receptors responsible for recruitment of these cells.

Methodological issues in the investigation of ginseng as an intervention for fatigue.

BACKGROUND: Although literature suggests that fatigue is commonly reported by women during and after breast cancer treatment, treatment options are limited. Although ginseng is widely used in Asian countries as a tonic to increase energy, its efficacy for treating cancer-related fatigue has not been carefully studied. We conducted a pilot study to evaluate the feasibility of a larger clinical trial to investigate the efficacy of ginseng for treating breast cancer-related fatigue. PARTICIPANTS/METHODS: Breast cancer survivors seeking treatment for fatigue were recruited to participate in an 8-week randomized, double-blind, placebo-controlled trial. RESULTS: A variety of practical problems were encountered in the study, including large numbers of survivors with conditions that were possible contraindications to ginseng use, inability to achieve blinding for the intervention because of strong odor from the ginseng, and measurement device failure. DISCUSSION: Evaluating ginseng for breast cancer treatment-related fatigue is a great challenge, particularly if a blinded randomized design is desired. This article provides insight into issues related to investigating complementary therapies and the importance of pilot studies for identifying methodological problems.

Immunotoxicological assessment of cyclosporin A by conventional pathological techniques and immune function testing in the rat.

1. Groups of male rats were given different doses of cyclosporin A, ranging from the maximum tolerated dose (20 mg/kg/day) downwards, 7 days a week for 28 days using a protocol derived from OECD test guideline 407. 2. At the end of the test, one set of animals underwent a detailed necropsy and histopathological examination of lymphoid tissues. Immune function was assessed using the lymphoproliferative response and natural killer cell activity of their spleen cells. Another set of animals was immunised with sheep erythrocytes on day 25 and used to evaluate the ability to produce specific anti-sheep red blood cell antibody. 3. Cyclosporin A produced detectable effects on the immune system at all doses and at doses lower than other toxic effects. Both histopathological techniques and one of the immune function tests were able to identify changes at the lowest dose, 1.25 mg/kg/day. 4. The results of this investigation suggest that conventional histopathological techniques, if applied to a range of lymphoid organs, are sufficient to identify potential immunotoxicants without recourse to immune function tests.

Inability to establish ectopic endometrium in a natural killer cell-deficient murine model. Immunologic, histologic and histochemical assessment.

OBJECTIVE: To investigate what effect natural killer (NK) cells have on the implantation of heterologous endometrial scrapings. STUDY DESIGN: Anti-asialo GM1 (AA-GM1) anti-sera have been shown to eliminate NK cell activity in various strains of rats and mice. Either AA-GM1 antibodies (+) or rabbit antiglobulin (-) was administered to beige mice (NK cell deficient) or beige control mice (not NK cell deficient of the same strain). The heterologous endometrial scrapings were prepared by scraping seven pairs of uterine horns from normal mice of the same strain. Beige and normal mice were then injected intraperitoneally every 3 days with the heterologous endometrial scraping and antibodies for a period of 50 days. The four experimental groups (n = 10 per group) can be summarized as being beige (+), beige (-), normal (+) and normal (-). RESULTS: There was no evidence of ectopic endometrial tissue in any of the four test groups by histologic examination or by using immunohistochemical staining techniques. Histologic evidence of an impaired immune response was clearly demonstrated in the beige mice receiving AA-GM1 antibodies. CONCLUSION: Using this model, a deficiency of NK cell activity did not appear to enhance the implantation of endometrial tissue on the abdominal peritoneum of mice.

Assessment of the involvement of central nervous system and peripheral opioid receptors in the immunomodulatory effects of acute morphine treatment in rats.

The present study assessed the involvement of opioid receptors both in the central nervous system (CNS) and in the periphery (i.e., on immunocytes) in the immune alterations produced by acute morphine treatment in rats. The first experiment showed that the in vitro suppressive effects of morphine on the mitogen-stimulated proliferation of splenic and blood lymphocytes are produced only by a very high concentration of morphine and are not naltrexone-reversible. These results suggest that the in vitro immunomodulatory effects of morphine are not mediated by classical opioid receptors on immunocytes. A second experiment showed that s.c. doses of N-methylnaltrexone that do not gain access to the CNS, as determined by the tail-withdrawal assay, do not antagonize the suppressive effects of a single, s.c. injection of morphine on the mitogen-stimulated proliferation of splenic and blood lymphocytes, splenic natural killer cell activity and the production of interferon-gamma by stimulated splenocytes. Only a high s.c. dose of N-methylnaltrexone that does gain access to the CNS, as determined by the tail-withdrawal assay, blocks morphine's immunomodulatory effects. A third experiment demonstrated that N-methylnaltrexone is 4 to 5 log units more potent in antagonizing most of the immune alterations produced by a single, s.c. injection of morphine when administered i.c.v. than s.c. Taken together, the results of the present study strongly suggest that CNS opioid receptors play an important role in the immune alterations produced by acute morphine treatment in rats.

Increase in CD57 + CD16-lymphocytes in workers exposed to benzidine and beta-naphthylamine: assessment of natural killer cell subpopulations.

Previously, we found a decrease in CD4 + CD45RA + T lymphocytes in workers exposed to the aromatic amines (AAs) [benzidine (BZ) and beta naphthylamine (BNA)]. For further investigation of the effects of AAs on lymphocyte subpopulations, we measured natural killer (NK) cell subpopulations using two-color staining with anti-Leu7 (CD57) and anti-Leu11 (CD16) monoclonal antibodies in peripheral blood in 78 male dyestuff workers. The workers had been exposed to AAs before 1972 at a chemical plant, either in the production of AAs (40 workers, high-exposure group) or in other work that involved handling dyestuffs (38 workers, low-exposure group). The controls were 30 "healthy" male volunteers without a history of occupational exposure to AAs or hazardous chemicals. The number of CD57 + CD16- cells in the high-exposure group was significantly higher than that in the controls (P < 0.01, analysis of covariance with age as a covariate). No significant differences were found in CD57 + CD16-, CD57 + CD16+ and CD57- CD16 + NK cells between the low-exposure group and the controls. It is suggested that a decrease in the number of CD4+ T lymphocytes following exposure to AAs might be compensated by the increase in CD57 + CD16- cells, i.e. circulating peripheral lymphocytes with poor NK cell activity.

Assessment of immunotoxicity of buprenorphine.

In order to use buprenorphine as an analgesic in immunological experiments, we have studied the potential immunotoxicity of buprenorphine. Three-week-old male Wistar Riv:TOX rats were subcutaneously treated with buprenorphine by injection of 0.1, 0.4, or 1.6 mg/kg body weight per day over a period of 4 weeks. Concentrations used were within the range for analgesia in rats. A slight decrease of body weight gain was observed at the highest dose in one but not in a duplicate study. Decreased liver weights were observed in all dose groups. Histopathologically glycogen storage was decreased and fatty vacuolation was found to be increased starting from the lowest dose group. The relative but not absolute weight of the lungs was slightly increased at the lowest dose, this phenomenon was therefore not dose-dependent. Histopathologically, a dose-dependent increase in interstitial pneumonia in the lung was found. At the 2 higher dose levels the weight of the adrenal glands was increased. No haematological changes were found, nor were there effects on bone marrow. In one of 2 studies indications of potential immunotoxicity noted were: an increased weight of the thymus, as well as an increased weight of popliteal and mesenteric lymph nodes. No effects on the weight of the spleen were found. Histologically, there were no changes in the lymphoid organs tested. Total immunoglobulin A concentrations in serum were significantly decreased in the highest dose group, whereas IgG concentrations were increased, albeit not statistically significantly. IgM and IgE concentrations showed no alterations. Two types of immune function assays were carried out: determination of natural killer cell activity and of mitogen responsiveness of spleen cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional and phenotypic assessment of neonatal human leucocytes expressing natural killer cell-associated antigens.

A subpopulation of mononuclear leucocytes was prepared from umbilical cord venous blood by immunomagnetic depletion of lymphocytes and monocytes using monoclonal antibodies to CD2, CD3, CD14 and CD19 antigens, and examined for NK cell-associated phenotypic and functional properties. The depleted population was enriched for the NK markers CD16 (mean 53.6% positive) and CD56 (mean 42.7% positive). While there was considerable overlap of these two markers, approximately one-third of CD16+ cells were CD56-; in contrast, few CD56+ CD16- cells were found. CD16+/CD56+ cells also co-expressed CD7 and CD45RA antigens, while a minority weakly expressed CD8. Another marker of adult NK cells, CD57, was virtually absent from CD16+/CD56+ cells, as was MHC Class 2. Freshly depleted cord cells had virtually absent natural cytotoxicity to K562 targets in a chromium release assay, but NK activity could be induced after 18 h exposure to recombinant human IL-2, without significant change in phenotype. These findings confirm the phenotypic differences and functional defects of NK cells in cord blood as compared to adult blood, and identify a subset of cells with unique phenotype (CD2- CD3- CD7+ CD16+ CD56- CD57-). The precise relationship of this subset of cells to NK lineage remains to be defined.

A sequential multi-assay protocol for the preclinical assessment of natural product complex carbohydrate immunomodulators.

A major barrier to the understanding, development and utilization of natural product complex carbohydrate immunomodulators has been the lack of standardization during pre-clinical efficacy and safety testing. In addition, it has been our experience that no single assay system or model is adequate for assessing preclinical efficacy and safety of these agents. To address these important issues, our laboratory group has developed a sequential multi-assay protocol for the preclinical evaluation of natural product complex carbohydrate immunomodulators. This sequential multi-assay screening protocol is divided into four phases: 1) physiochemical characterization of the carbohydrate polymer; 2) evaluation of immune stimulatory activity; 3) assessing in vivo anti-microbial activity and anti-tumor efficacy and 4) preclinical safety evaluation. This sequential protocol provides an effective, reproducible and rational approach to the preclinical assessment of complex carbohydrate immunomodulators that, in our experience, is predictive of clinical safety and efficacy.

Immunotoxicologic assessment of subacute exposure of rats to carbon tetrachloride with comparison to hepatotoxicity and nephrotoxicity.

The immunotoxicity, hepatotoxicity, and nephrotoxicity of subacute exposure to carbon tetrachloride (CCl4) were evaluated in young adult (8-9 weeks old) male Fischer 344 rats dosed by gavage with CCl4 for 10 consecutive days at 0, 5, 10, 20 or 40 mg/kg/day. Two days following the last treatment rats were evaluated for alterations in immune function by monitoring the following: body and lymphoid organ weights; mitogen and mixed leukocyte reaction lymphoproliferative responses; natural killer cell activity; and cytotoxic T lymphocyte responses. A separate group of similarly dosed rats was immunized with sheep red blood cells (SRBC) on Day 9 of dosing, and the primary antibody response was assessed 4 days later. Hepatic and renal toxicity were assessed 2 days after the last treatment by monitoring organ weights, serum indicators of hepatic and renal damage, and hepatic cytochrome P450 levels, as well as by histological evaluation. Significant increases in relative liver weights were observed in rats dosed at 40 mg/kg/day. Histologically, these livers displayed mild to moderate vacuolar degeneration and minimal to mild hepatocellular necrosis. In addition, serum levels of aspartate aminotransferase and alanine aminotransferase were elevated at this dosage, as well as at 20 mg/kg/day. There were no renal effects observed at these dosages of CCl4. In addition, no consistent alterations were observed in the immune parameters examined in these same animals nor in the rats immunized with SRBC. Furthermore, there was no difference in the antibody response to SRBC in another set of rats dosed at 40, 80 or 160 mg/kg/day CCl4. These results indicate that CCl4 is not immunotoxic in the rat at dosages that produce overt hepatotoxicity.

Suppression of murine natural killer cell activity by tributyltin: in vivo and in vitro assessment.

The effects of tributyltin chloride (TBTCl) and inorganic tin (IT) on murine natural killer (NK) cell activity were tested in vivo and in vitro. In vivo studies demonstrated that mice fed with TBT (10 and 100 ppm) daily for 1 week exhibited suppression in NK activity 38-46% at effector:target (E:T) ratio = 50:1 compared to control mice. On the other hand, animals treated with inorganic tin showed no change in activity of NK cells. In vitro studies showed leukocytes, preincubated with TBTCl (0.01-0.1 ppm) at room temperature for 1 h and then washed three times, demonstrated significant suppression in NK activity (41 and 85%) at concentrations 0.01 and 0.05 ppm, respectively. Increasing the dose to 0.1 ppm, resulted in complete inhibition of the activity of NK cells. In contrast, IT had no effect on NK activity in vitro at the same concentrations of TBTCl. The effect of TBTCl appears to be due to interference with the binding capacity of effector cells, a necessary prerequisite for target cell lysis. In conclusion, TBTCl proved to be a very potent inhibitor of NK activity; this inhibition may predispose animals to malignancy, which is a characteristic feature reported recently for some TBT compounds.

Immunotoxicity assessment of gentamycin and liquamycin.

Rats were injected with gentamycin (GEN) or the oxytetracycline, liquamycin 200 (LA200), and multiple immune responses were measured to assess the immunotoxicity of these antibiotics. Animals were treated by sc or im injection for 12 days with either GEN or LA200 given at a therapeutic dose or at 10-, 50-, or 100-fold greater concentrations. The following immune parameters were assessed in each animal: delayed-type hypersensitivity (DTH), natural killer (NK) cell cytotoxicity, antibody production, and synthesis of interleukin 2 (IL2) and gamma interferon (IFN). The DTH response and IFN production in GEN- and LA200-injected animals were suppressed in a dose-dependent manner. Production of IFN was significantly suppressed in all groups treated with LA200. Suppression of DTH was evident when GEN or LA200 were given at the recommended therapeutic dose. Both LA200- and GEN-treated rats had suppressed NK cell cytotoxicity, but only at the highest dosages. Antibody production was suppressed in a dose-dependent manner in LA200-treated rats. Synthesis of IL2 was suppressed in rats treated with the high dose of GEN. Body weight loss occurred, and hepatic and renal toxicity were apparent at the 2 highest dosages of GEN or LA200. It was apparent that relatively high doses of GEN and LA200 can suppress specific and non-specific cell-mediated immune responses. Also, the data suggest the certain immune responses (eg, DTH reaction and possibly IFN production) may be suppressed at the recommended therapeutic doses in the absence of other signs of toxicity. These results indicate that these antibiotics should not be administered at doses or exposure periods in excess of those recommended.(ABSTRACT TRUNCATED AT 250 WORDS)