Control of Toxoplasma gondii infection by athymic LEW-Whn rnu rats.

In immunocompetent rats and humans infection with Toxoplasma gondii remains mostly without overt clinical symptoms, but can be fatal, if the T-cell response is impaired. For a better understanding of the lack of control of T. gondii infection under immunosuppressed conditions, congenitally athymic rats were used as the experimental model. Whereas athymic F344-Whn(rnu) (F344 nude) rats die from a generalized infection during the first 3 weeks after peritoneal inoculation with 10(6) tachyzoites of T. gondii strain NTE, LEW-Whn(rnu) (LEW nude) rats and euthymic LEW rats infected with a 10-fold higher number of parasites developed chronic infection. To identify underlying mechanisms of LEW rats resistance to T. gondii infection and to investigate a possible contribution of residual T-cells to LEW-Whn(rnu) rat resistance, we characterized the immune response of LEW rats by determination of cellularity and composition of lymphocyte population, antigen-specific IgG2b response as well as assays of antigen-specific proliferation and production of IL-2, IFN-gamma and TNF-alpha. As only euthymic LEW rats developed production of antigen-specific IgG and cellular in vitro responses, these results strongly suggest that the genetic background of LEW rats permits a control of the infection independent of an adaptive immune response.

Muristerone A-induced nerve growth factor release from genetically engineered human dermal fibroblasts for peripheral nerve tissue engineering.

In this study, human dermal fibroblasts (hDFBs) were genetically modified to release human nerve growth factor (NGF) using an ecdysone-inducible system. NGF cDNA was inserted into the pIND vector and then hDFBs were cotransfected with pIND-NGF and pVgRXR. Muristerone A, an analog of ecdysone, was used as the inducing agent. NGF release from transfected hDFBs was assessed in vitro and in vivo. Transfected hDFBs in the presence of Muristerone A possessed a maximal in vitro release of 8.5 +/- 0.4 pg of NGF/mL per 10(3) cells, demonstrating significantly higher NGF levels compared to control hDFBs. The in vitro release rate curve for transfected hDFBs in the presence of Muristerone A exhibited a maximum of 5.1 +/- 0.2 ng NGF/10(6) cells/day. A PC-12 bioassay demonstrated that the in vitro NGF released is bioactive. When transfected hDFBs in the presence of Muristerone A were placed in vivo in nude rats, NGF levels reach 2074 +/- 257 pg/mL and 1620 +/- 132 pg/mL at 24 and 48 h, respectively. These levels were significantly higher than negative control and wound fluid levels. Results support further in vivo investigation of this molecular "on" switch for peripheral nerve regeneration.

The athymic nude rat: an animal experimental model to reveal novel aspects of innate immune responses?

Athymic nude rats resemble nude mice in their lack of a normal thymus and functionally mature T cells. They have been useful in the study of mechanisms of tumor growth or graft rejection in immunocompromised hosts since they can accept major histocompatibility complex (MHC) mismatched organ allografts or xenografts for several months and because a number of tumor cell lines of human and rodent origin grow well in these rats. Injection of a few helper T (Th) cells from euthymic littermate rats partly restores the pool of mature T cells as well as full immunocompetence to reject organ allografts and has helped to reveal some of the cell interactions necessary for rejection to occur In contrast, immunologically naive athymic nude rats of certain strains, acutely reject allografts consisting of lymphocytes or bone marrow cells, which is due to the presence of alloreactive natural killer cells. These cells can recognize and kill MHC incompatible hematopoietic cells through the recognition of both mismatches within the classical (RT1.A) and nonclassical (RT1.C/E) MHC class I regions with a repertoire of inhibitory and activating killer lectin-like receptors (KLR) for MHC-I molecules, encoded by the Ly-49 portion of the rat natural killer cell gene complex (NKC). Some of these receptors have been identified and molecularly cloned and show similarities with NK receptors identified in the mouse. Other leukocytes in nude rats, such as dendritic cells, may also contribute to specific innate immune responses in the absence of mature T cells. Nude rats develop T-like cells expressing CD3 and T-cell receptor (TCR) with increasing age. Though their phenotype in peripheral lymphatic tissues resembles that of normal T cells, consisting mainly of CD4+ or CD8+ cells, they lack alloreactivity in vivo and their TCR repertoire is more of an oligoclonal nature. Their contribution to allograft rejection in T-cell-reconstituted rats is therefore questionable, and their role in innate immune response in these rats still enigmatic.

Resistance to chemically-induced mammary tumors in Copenhagen X nude-derived F2 athymic rats: evidence that T-cell immunity is not involved in Copenhagen resistance.

Resistance to chemically-induced mammary tumors in the Copenhagen rat is well defined, but the mechanism of resistance has yet to be determined. We have tested whether or not Copenhagen rat resistance is dependent on T-cells, since several lines of evidence supported an involvement of the immune system. We crossed Copenhagen rats with an athymic nude rat to produce F1s, that were interbred to produce F2 animals, some of which were athymic with partial Copenhagen rat background. A comparison of the mammary tumor incidences between the nude athymic F2 animals and their non-nude littermates allowed us to determine what role, if any, T-cells played in resistance. Following treatment with N-methyl-N-nitrosourea, we observed no difference in the tumor incidences between the two groups. Furthermore, the mammary tumor incidences in the F2 nude and non-nude animals was almost zero. These results indicate that T-cells are not involved in Cop resistance, and that nude rats are resistant to N-methyl-N-nitrosourea-induced mammary tumorigenesis.

Characterization of the T cells in aged rat bone marrow.

In this study, we determined the characteristics of CD3-positive (CD3+) T cells existing in rat bone marrow (BM). In contrast to splenic T cells, BM CD3+ T cells are composed of a higher proportion of CD8+ T cells, and the number of both cell types increased with age. Such CD3+ T cells in aged rats showed a similar usage of TCR V beta as splenic T cells, suggesting that BM CD3+ T cells are thymus-dependent and composed of an ordinary population in view of the expression of the TCR beta-chain. Purified T cells obtained from aged rat BM showed a markedly proliferative response by stimulation with immobilized anti-CD3 mAb, as did splenic T cells. However, the addition of BM non-T cells completely inhibited the response of both BM and splenic T cells in vitro. These results suggest that T cells in rat BM are negatively regulated by BM non-T cells in their response to the TCR-mediated signal not to disrupt the microenvironment of the BM.

Factores inmunológicos y no inmunológicos involucrados en el control del crecimiento tumoral / Involved immunological and nonimmunological factors in the control of the tumorlike growth

El propósito de este trabajo es determinar si existe un patrón común a ambos grupos de tumores: los inmunogénicos y los que no inducen respuesta inmune. Dado que el estudio de la resistencia concomitante ha recibido distintos enfoques según la inmunogenicidad del tumor, el objetivo es ver si en el caso de los tumores inmunogénicos se genera algún mecanismo análogo al observado en los no inmunogénicos. En tal caso se sobreagregaría a este factor, el componente inmunológico. (AU)

Factores inmunológicos y no inmunológicos involucrados en el control del crecimiento tumoral / Involved immunological and nonimmunological factors in the control of the tumorlike growth

El propósito de este trabajo es determinar si existe un patrón común a ambos grupos de tumores: los inmunogénicos y los que no inducen respuesta inmune. Dado que el estudio de la resistencia concomitante ha recibido distintos enfoques según la inmunogenicidad del tumor, el objetivo es ver si en el caso de los tumores inmunogénicos se genera algún mecanismo análogo al observado en los no inmunogénicos. En tal caso se sobreagregaría a este factor, el componente inmunológico.

The extrathymic T-cell development pathway.

In normal mice, not all T-lineage cells are generated and selected in the thymus; an alternative, extrathymic, development pathway exists. Extrathymic T cells are rare in the spleen and lymph nodes, but are abundant in some tissues, such as the gut. Here, Benedita Rocha, Pierre Vassalli and Delphine Guy-Grand discuss the rules of selection of extrathymic T cells, assess the possible role of these cells in the defence of epithelial integrity and their potential role in autoimmune disease.

The appearance and role of gamma delta T cells in the peritoneal cavity and liver during primary infection with Listeria monocytogenes in rats.

We have previously reported that gamma delta T cells play important roles in protection during the early stage of infection with Listeria monocytogenes in mice. To generalize the protective roles of gamma delta T cells in listerial infection to different species, we examined the appearance of gamma delta T cells during infection with L. monocytogenes in Fisher F344 rats. The numbers of bacteria in the peritoneal cavity and liver increased to a maximum level on day 3 and then decreased to an undetectable level by day 10 after an intraperitoneal infection with a sublethal dose (1 x 10(8)) of viable L. monocytogenes in rats. CD3+ alpha beta- T cells in the peritoneal cavity and liver began to increase on day 3, reached a maximum level on day 6, and thereafter decreased gradually by day 10 after infection. Northern blot analysis confirmed that the CD3+ alpha beta- T cells expressed TCR delta and gamma gene messages. In vivo treatment with anti-TCR alpha beta mAb, which suppressed most of the alpha beta T cells in the periphery and impaired resistance during the late stage of listerial infection, did not affect the host defense by day 6 after infection. A significantly increased number of gamma delta T cells was detected in the peritoneal cavity of the TCR alpha beta-suppressed rats on day 6 after infection. These results suggest that the early appearing gamma delta T cells may contribute to the host defense at a relatively early stage during listeriosis in rats.

Function of CD4 T cell subsets in vivo: expression of CD45R isoforms.

Currently high (hi) and low (lo) molecular weight isoforms of CD45R are used to identify naive and memory T cells. However, CD45R isoforms were found to be expressed cyclically on CD4 T cells in vivo. A switch in isoform paralleled a change of function. During early post-thymic development, a switch from CD45Rlo to CD45Rhi occurred prior to antigen encounter. Naive CD45Rhi T cells when stimulated by antigen became CD45Rlo but reverted to CD45Rhi with time. It is argued that CD45Rlo T cells functionally resemble effector T cells and are at a pivotal stage of differentiation. Many are equipped to migrate into sites of inflammation. Removed from antigen, antigen-primed CD45Rlo T cells become CD45Rhi (memory) and thus adopt the same phenotype as naive T cells.

Allograft rejection in CD4+ T cell-reconstituted athymic nude rats--the nonessential role of host-derived CD8+ cells.

PVG-rnu/rnu nude rats reject fully allogenic renal (DA) and skin (BN, AO) allografts after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many nude-derived CD8+ leukocytes within the graft. In addition, mononuclear cells infiltrating the rejecting renal grafts in these animals display cytotoxic activity in vitro against specific and third-party alloantigens. In this investigation we have treated CD4+ T cell-restored nude rats bearing renal or skin allografts with the mAb MRC OX8 to deplete the host of CD8+ cells. In vivo treatment with OX8 completely eliminated CD8+ cells from rejecting grafts of both kidney and skin, but it did not prevent graft rejection, nor did OX8 treatment abolish the cytotoxic effector cells found in nude rat spleen or in graft-infiltrating cells (GIC) of rejecting renal allografts. The nature of the cytotoxic activity was examined with anti-CD3 mAb 1F4, which was shown to block conventional CD8+ Tc killing in vitro but did not inhibit allogeneic target cell lysis by spleen cells from nude rats. The cytotoxic activity found in GIC of rejecting allografts was not inhibited by anti-CD3 mAb, suggesting that these cytotoxic effector cells were CD3-CD8- and were of extrathymic origin. We conclude that non-thymus-derived CD8+ GIC are not essential for allograft rejection in CD4+ T cell-restored nude rats.

Xenogeneic osteogenin, a bone morphogenetic protein, and demineralized bone matrices, including human, induce bone differentiation in athymic rats and baboons.

Subcutaneous implantation of xenogeneic demineralized bone matrix does not initiate endochondral bone differentiation. Dissociative extraction in 4 M guanidine-HCl or 6 M urea has shown that the apparent species-specificity of intact bone matrix resides in its insoluble immunogenic component, since there is homology in solubilized osteogenic proteins amongst mammals. To further investigate the species-specificity and cross-species reactivity of bone matrix components, baboon and human demineralized bone matrix (DBM) and bovine osteogenin, purified greater than 50,000-fold and with an apparent molecular mass of 28-42 kilodaltons, were implanted in the subcutaneous space of athymic and euthymic rats and into the rectus abdominis of 16 baboons (Papio ursinus). Baboon DBM was also implanted in athymic and euthymic mice. Alkaline phosphatase activity and histology of implants harvested at day 11 and 30 showed that baboon and human DBM induced endochondral bone differentiation both in athymic rats and baboons. Bovine osteogenin in conjunction with baboon insoluble collagenous matrix induced extensive bone differentiation in athymic rats and baboons. Baboon and human DBM did not induce bone differentiation in euthymic rats and, in athymic mice, baboon DBM failed to induce bone differentiation, determining instead the recruitment of multinucleated giant cells. The results indicate that in rodents bone differentiation induced by intact bone matrix is species specific and that T-cell functions are not a requirement for bone induction, although immunologically competent rats block bone differentiation from xenogeneic matrix. Bone differentiation induced by human DBM in baboons suggests that intact bone matrices may not be species-specific amongst primates.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucosal immune activation and maturation of the small intestine at weaning in the hypothymic (nude) rat.

Activation of the mucosal immune system peaks at weaning on days 21 and 22 of life in the rat. We have investigated activation in the gut associated lymphoid tissue and maturation of intestinal mucosa in hypothymic (nude) and in phenotypically normal heterozygous CBH rats at 22 days of life. Intestinal maturation, as assessed by villus area, crypt length, crypt cell production rate and disaccharidase activity, was similar in hypothymic and normal rats, and indices of mucosal immune function were elevated in both groups at this time. The proportion of mononuclear cells from the mesenteric lymph node expressing IL-2R was 11% in heterozygous and 14% in hypothymic rats as determined by flow cytometry. Immunoperoxidase staining of MLN sections confirmed the presence of IL-2R+ cells in the T-dependent interfollicular areas. However, the number of T-cells was considerably depleted in hypothymic rats. Intraepithelial lymphocyte counts and serum rat mucosal mast cell protease II concentrations were similar in the two groups, while counts of jejunal mucosal mast cells and eosinophils were paradoxically increased in hypothymic animals. As T lymphocyte function is thought to be impaired in hypothymic rats, the intact mucosal immune activity in hypothymic rats could be due to activation of intrinsic "thymic independent" T lymphocytes in hypothymic rats, or to engraftment with extrinsic maternal milk-derived lymphocytes and their activation in the infant rat gut.

Effect of rIFN-gamma and IL-2 treatments in mouse and nude rat infections with Toxoplasma gondii.

Mice and nude rats lethally infected with T. gondii and treated with recombinant rat interferon-gamma (rIFN-gamma) or recombinant human interleukin-2 (rIL-2) were protected against death, when compared with untreated infected controls. In mice rIFN-gamma and rIL-2 played an important role in "prophylactic treatment", but not in "curative therapy". The survival rate was 42% in mice treated with 3 doses of 20,000 U of rIFN-gamma at days -2, -1, 0 before challenge and up to 66% in mice treated with 3 doses of 10,000 U of rIFN-gamma at days -2, 0, +2 before and after infection. Whereas the survival rate was 33% in mice that received 3 doses of 500 U rIL-2 at days -2, -1, 0 before infection, or -2, 0, +2 before and after infection respectively, up to 50% of the mice treated with 3 doses of 1,000 U rIL-2 at days -2, -1, 0 survived. In nude rats rIFN-gamma had a slight effect in "prophylactic treatment", whereas rIL-2 was active only in "curative treatment". The survival rate was 25% both in nude rats treated with doses of 400,000 U of rIFN-gamma at days -3, 0 before challenge, or with doses of 5,000 U of rIL-2 at days +2, +6, +9 after infection. These results lead us to hypothesise that the mechanism by which the lymphokine treatment exerts a protective effect on Toxoplasma infected mice is different from that on nu/nu rats. We conclude that these cytokines may play a notable role in modulating the host's immune defence against T. gondii infection.

The athymic nude rat. Immunobiological characteristics with special reference to establishment of non-antigen-specific T-cell reactivity and induction of antigen-specific immunity.

The aim of the present review has been to describe some immunobiological characteristics of the athymic nude rat and on the basis of these to describe the possibilities of establishing non-antigen-specific T-cell reactivity and inducing antigen-specific immunity. After a brief introduction, some general characteristics of the autosomal recessive rnu/rnu rat are outlined. This athymic mutant normally has a longer lifespan than athymic nude mice and is easier to breed. Inbred nude rats are now available with several rat strain backgrounds. Since the rnu/rnu rat is athymic, the morphology and function of the thymus are briefly described. The thymus has two main functions: production of T lymphocytes and production of thymic hormones. The intrathymic T-cell ontogeny is described along with the two thymocyte selection mechanisms, positive and negative selection. Different thymic hormones are mentioned and their possible function outlined. However, thymic hormones still do not play an important role in immunotherapy. There are some striking differences in the morphology of lymphatic tissues of athymic and euthymic rats. The thymic area in nude rats consists only of fat and clusters of epithelial cells. Little research has been performed on bone marrow of athymic nude rats but morphologically there seems to be no difference from findings in normal animals. The thymus-dependent areas of peripheral lymphoid organs, i.e. the paracortical area of lymph nodes, the periarteriolar sheet of the splenic white pulp, and the interfollicular areas of Peyer's patches, are severely cell-depleted in the athymic nude rat. The lower lymphocyte content of these organs is not reflected in the lymphocyte counts of peripheral blood, but the lymphocyte counts of thoracic duct lymph are much lower than those found in euthymic animals. Few T-lymphocyte marker-positive cells are found in the athymic nude rat, and in the epithelium of the intestine there are also lymphocytes bearing neither T nor B markers. These cells probably represent the tau delta receptor-bearing cells. Immunologically the athymic nude rat reflects the thymic aplasia. Normal B-lymphocyte function has been found in both in vitro and in vivo tests, whereas the T-cell function is virtually absent both in vitro and in vivo. Non-MHC-restricted cells with killer activity like NK cells are present in the nude rat, and these cells function normally both in vitro and in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Alloreactive lymphokine-activated killer cells from athymic nude rats do not express CD3-associated alpha/beta or gamma/delta T cell receptors.

Lymphokine activated killer (LAK) cells from athymic nude rats, previously shown to selectively kill MHC-incompatible small lymphocytes in vitro, were examined for rearrangement and expression of genes for the T cell antigen receptors. Southern blots showed no rearrangement of the TCR beta-chain genes, and Northern blots showed transcription only of truncated 1.0 kb beta-chain messages, but not of full-length, 1.3 kb beta-chain mRNA. Transcription of the alpha-chain of the TCR could not be detected, and surface staining with the mAb R73 showed no expression of the rat TCR alpha/beta heterodimer. Transcripts hybridizing with a rat TCR C gamma probe were detected on Northern blots, but probes for all presently characterized mouse V gamma genes failed to hybridize to the same filters, indicating that the C gamma-containing transcripts probably were from non-rearrangement genes. CD3 delta- and epsilon-chain transcripts could not be detected by Northern blot analysis. Less than 2% of the cells stained with the anti-rat CD3 monoclonal antibody 1F4, and incubation with 1F4 had no effect on the alloreactivity of nude rat LAK cells. We have previously shown that immunoglobulin is not involved in the killing of the allogeneic lymphocytes. The most likely interpretation of these results is therefore that nude rat LAK cells express a novel receptor structure involved in allorecognition.

Identification of the mononuclear cell infiltrate in the superior cervical ganglion of athymic nude and euthymic rats after guanethidine-induced sympathectomy.

Guanethidine sulphate 40 mg/kg intraperitoneally for 14 days induced chromatolysis and nerve cell death in the superior cervical ganglia of athymic nude (rnu/rnu) LEW/Mol rats and their euthymic (+/rnu) LEW/Mol heterozygous littermates. Histologically the sympathetic ganglia were dominated by an infiltration of small inflammatory cells. By means of monoclonal antibodies these cells were identified. The number of B-lymphocytes increased following guanethidine in both athymic and euthymic rats. The number of T-lymphocytes increased to a great extent in euthymic rats, but was virtually missing in athymic rats. The number of NK-cells and monocytes/macrophages increased in both athymic and euthymic rats. The conclusion is, that guanethidine exerts a direct effect on sympathetic ganglion cells followed by a thymus-independent immune response.

The origin of T cells in permanently reconstituted old athymic nude rats. Analysis using chromosome or allotype markers.

Athymic PVG-rnu/rnu rats receiving a single intravenous injection of syngeneic euthymic thoracic duct lymphocytes (TDL) develop normal levels of CD4+ T lymphocytes and survive for more than 2 years in a conventional animal house. We investigated the origin of the T cells (and B cells) in reconstituted nude recipients by transferring TDL carrying either the 3T chromosome marker or the RT6b + Igk-1b allotype or the RT7b (leucocyte-common) allotype markers. Karyotype analysis of spleen and lymph node (LN) cells from 1- to 2-year-old PVG-3T/3T-reconstituted nude recipients, stimulated in vitro with phytohaemagglutinin (PHA), unexpectedly revealed that a majority (79-97%) of dividing cells were of nude origin. However, extensive nude cell division was also recorded in PHA-stimulated cultures using mixtures of euthymic (PVG-3T/3T) and unreconstituted nude spleen cells; the assumption that only T cells divide in PHA-stimulated cultures thus appears to be erroneous. In contrast to the karyotype analysis, sIg- RT6b+ LN cells obtained from nude recipients reconstituted 2 years earlier with PVG-RT6b allotype-marked TDL, were all of donor origin with no indication of a nude-derived sIg- RT6a+ population. Igk-1b+ donor B cells were not found in these same recipients. Dual fluorescence analysis of TDL from 18- to 20-month RT7b-reconstituted nudes showed that 91-100% of CD4+ cells were donor-derived. When tested functionally, sIg- RT7b+ (donor) cells, but not sIg- RT7b- (nude-derived) cells, were able to reject skin allografts and induce local graft-versus-host (GVH) responses. Donor T cells, in contrast to CD4+ cells of nude origin, divided extensively in nude recipients; FACS-purified RT7b+ (donor) TDL retransferred from 17-month primary reconstituted nude rats, expanded further (60-100-fold) in secondary nude recipients. In conclusion, only the donor-derived CD4+ cells in reconstituted nude rats displayed T-cell function; evidence to the contrary from karyotype analysis was flawed. At no stage in their life did uninjected or T-cell reconstituted nude rats develop endogenous cells that in any way resembled CD4+ products of the thymus.