E. Joost Ruitenberg

Infection of a canine macrophage cell line with leishmania infantum: determination of nitric oxide production and anti-leishmanial activity.

We have previously shown that resistance to Leishmania infantum in dogs is associated with a Th1 type of immune response. In this study, we use a canine macrophage cell line (030-D) that can readily be infected with this protozoan parasite. Our aim is to further characterize the effector mechanisms involved in killing of Leishmania parasite in dogs. We observed that activation of 030-D cells by incubation with a supernatant derived from a Leishmania-specific T cell line containing IFN-gamma, TNF-alpha and interleukin-2 (IL-2) resulted in enhanced nitric oxide (NO) production by these cells. In addition, we observed enhanced anti-leishmanial activity of infected 030-cells after activation. Both, NO production and anti-leishmanial activity were abrogated by addition of L-N(G)-nitroargininemethyl ester (L-NAME), an analogue of L-arginine. Thus, NO play an important role in the anti-leishmanial activity of these canine macrophages. We propose the infection of the 030-D cell line as a good in vitro model to further investigate parasite-host cell interactions in dogs, a natural host of Leishmania parasites.

Impaired Immune Response by Isoniazid Treatment During Intravesical BCG Administration in the Guinea Pig

At present, isoniazid (INH) is being used prophylactically to reduce the side effects of intravesical BCG therapy for superficial bladder cancer, although it is not clear whether or not this reduces the antitumor efficacy of BCG. In this study the impact of INH treatment on the immune response after repeated intravesical BCG administration was investigated in guinea pigs. INH was given during 3 days starting on the day prior to the BCG administration. It was found that the administration of INH severely impaired the immunological effects of BCG. The induction of mononuclear cell infiltration in the bladder wall was reduced. Enlargement of the regional lymph nodes (weight and number of cells), and increase of Major Histocompatibility Complex (MHC) class II expression on the lymph node cells, normally observed after intravesical BCG administration, were inhibited by INH. Systemic immunity, measured by the DTH reaction in the skin to PPD, was also diminished due to the combined treatment of BCG with INH. A five-fold increase of the dose of BCG did not overcome the effect of INH. INH probably did not exert a direct suppression of the immune system of the guinea pig as the DNCB skin reactivity was not influenced. Although INH concentrations in the urine were high at the onset of the instillation, in vitro experiments indicated that the effect of INH was probably not caused by killing of the BCG organisms shortly after application in the bladder. In conclusion, our data in guinea pigs suggest that the use of INH may impair the immune response to intravesical BCG. As this response may be important for the antitumor effect of BCG, urologists should be cautious with the prophylactic use of INH. The influence on the antitumor efficacy is now investigated in man.

Molecular cloning and sequencing of the cDNA for dog interleukin-4

T lymphocytes play a crucial role in the development of protective immunity against intracellular pathogens. Studies on mouse CD4 T cell clones showed that helper T (Th) cells can be divided into Th1 and Th2 subsets based on their cytokine production. Interleukin-4 (IL-4) is a cytokine produced predominantly by the Th2 subset. It is involved in stimulation and class switching of B cells, acts as a growth factor for Th2 cells, and inhibits Th1 type of reactivity (Mosmann et al. 1987). In mice resistance and susceptibility toLeishmania infection were highly correlated with predominance of Th1 and Th2 reactivity, respectively (Locksley and Scott 1991). We study the immune responses in dogs infected with Leishmania infantum. Protective immunity in canine visceral leishmaniosis (CVL) is associated with a Th1 type of response (Pinelli et al. 1994, 1995). Our knowledge of CVL would greatly benefit from the development of dog Th2 specific reagents. This paper reports the cloning and sequencing of cDNA containing the coding sequence of dog IL-4 performed by the rapid amplification of cDNA ends (RACE) protocol (Frohman 1995). For this purpose mRNA isolated from concanavalin-A-stimulated dog peripheral blood mononuclear leucocytes isolated from a Leishmania–infected dog was reverse transcribed into

Antibody-dependent mechanisms of immunity against migrating larvae of Trichinella spiralis

Abstract Normal rat peritoneal exudate cells adhere to new-born Trichinella spiralis larvae in the presence of specific antibodies produced during the course of an oral infection. After 24 hours incubation with the peritoneal cells and antibodies most of the larvae were killed. Heated immune serum had less activity. With normal serum no adherence was observed. In order to investigate the influence of antibodies on new-born larvae in vivo, larvae were pre-treated in vitro with immune serum and then inoculated intravenously into normal mice. After 30 days the yield of normal larvae was 50% of the number recovered in a control group infected with non-treated new-born larvae.

Immune reactivity in cattle with ocular squamous cell carcinoma after intralesional BCG immunotherapy

SummaryLymphocyte stimulation with Con A and specific immune reactivity to BCG (antibody formation to BCG and DTH reaction to PPD) were determined in BCG-treated, surgically treated and untreated cows with ocular squamous cell carcinoma. In tumor-bearing cows the Con A-induced proliferation of lymphocytes was reduced when compared to healthy controls. This suppression consisted of a reduced blastogenic response to Con A of lymphocytes from tumor-bearing cows, and the presence of a factor in the sera of these animals, as these sera suppressed the blastogenic response of lymphocytes from healthy cows. BCG had only a minor influence on the suppressive activity. Antibodies to BCG were demonstrated in 50% of the BCG-treated animals. The formation of antibodies was not influenced by intradermal injection of PPD of Mycobacterium bovis. Absorption of a BCG antibody containing serum with BOSCC tumor extracts did not reveal the existence of cross reacting antigens between BCG and BOSCC. Pretherapeutic and posttherapeutic Con A reactivity could not be correlated with clinical response. Of the 30 BCG treated cows 29 developed a positive DTH reaction to PPD. Correlation between clinical response and immune reactivity was seen only with regard to the DTH reaction to PPD: this reaction remained positive for a longer period after treatment in animals with a favorable clinical outcome than in nonresponding animals.

Control III Surveillance in Swine by Immunodiagnostic Methods

Tests for the detection of Trichinella spiralis infection in pigs at slaughter make use either of direct methods (trichinoscopy, digestion methods) by means of which the parasite itself can be demonstrated (see Chapter 16) or of indirect (serological or skin test) methods. With the latter, the presence of the parasite can be suggested by the demonstration of specific anti-Trichinella humoral or cell-mediated immunity. The reliability of these immunological methods depends largely on the quality of the reagents used. The presence of cell-mediated immunity can be assessed by a skin test, which has been employed only occasionally for Trichinella (Andrews et al., 1976). Much more attention has been paid to the evaluation of a humoral response by a variety of serological techniques.

Histological evaluation of immunologically mediated tumor regression of the line 10 guinea pig hepatocarcinoma

SummaryThe histology of immunologically mediated tumor regression was studied in the syngenic strain 2 guinea pig/line 10 hepatocellular carcinoma tumor system. Tumor regression was induced non-specifically by the intralesional injection of livingBacillus Calmette-Guérin (BCG) in 7-day-old established tumors (diameter 8–10 mm).In untreated line 10 tumors at day 7 a mild to moderate inflammatory reaction was present, which consisted mainly of small mononuclear cells; in addition large mononuclear cells and basophils were present. Intratumoral BCG-treatment induced a prominent increase in the inflammatory reaction due to an influx of small and large mononuclear cells and neutrophils. Small mononuclear cells were identified mainly as lymphocytes whereas large mononuclear cells belonged mainly to the macrophage line. Intratumoral administration of BCG resulted in a granulomatous reaction. A time-related decrease in the number of tumor cells and an increase in inflammation, associated with purulent lysis of the granulomatous tissue, was observed.Specific immune-mediated tumor rejection occurred in animals both after active immunization and after adoptive transfer of immune spleen cells. In actively immunized animals the tumor cells were rapidly rejected and from day 4 onwards no tumor cells could be detected at the injection site. Lymphocytes were the major component of the inflammatory reaction; large mononuclear cells were present to a lesser extent and basophilic granulocytes were regularly observed. After adoptive transfer of immunity with immune spleen cells given simultaneously with an intradermal innoculation of tumor cells, an essentially similar rejection reaction was found, although tumor cell rejection was delayed. Lymphocytes and large mononuclear cells were found in equal proportions, whereas basophilic granulocytes were always present in smaller numbers.After BCG-induced regression and in adoptively transferred immune rejection, a fibroblast component was more prominent than in untreated control tumors. This reaction tended to isolate smaller tumor cell areas into islets of decreasing sizes. In contrast with the fibroblast component of growing tumors, the proliferative pre-existing fibrous tissue in tumors undergoing regression or rejection showed a loosely arranged architecture and contained a marked cellular infiltrate.From the results of the present study it was concluded that the morphological expression of line 10 tumor rejection varies. Without immune cells, BCG is needed for the induction of a local inflammatory reaction, which was granulomatous in type and eventually led to complete tumor cell eradication. In actively immunized animals there was rapid tumor cell rejection associated with a predominantly lymphocytic inflammatory response. After adoptive transfer with immune cells a slower rejection reaction occurred in which both lymphocytes and macrophages were present in equal proportions.

Xeno-Transplantation and Xenozoonoses

Transplantation of organs such as hearts or kidneys from one individual to another has become a highly successful mode of therapy. According to reports of the FDA (US Food and Drug Administration) and CBER (Center for Biologics Evaluation and Research) in the US ten patients die every day in the US, because a needed organ is not available to them. The supply of organs in the US is based on about 5.000 donors annually. The demands, however, rose from about 20.000 in 1990 to more than 60.000 patients waiting for organs in 1998. These huge needs stimulated research on xenografts as donor organs.