Quantitative studies of lymphoid organs, blood and lymph in inbred athymic and euthymic LEW rats under germfree and specified-pathogen-free conditions

Four groups of inbred male LEW rats were examined: A, germfree athymic; B, specified pathogen free (SPF) athymic; C, germfree euthymic; D, SPF euthymic. All animals were killed at 18 weeks and compared with respect to body weight, histological appearance and cell density of the lymphoid organs, haematological values and differential counts of bone marrow, peripheral blood and lymph. Athymic rats had a lower body weight, less densely populated lymphoid organs, and fewer lymphocytes in the blood and lymph compared with euthymic animals. No difference was seen between athymic rats under germfree and SPF conditions, and in general the differences between athymic and euthymic animals were less pronounced under germfree conditions.     

Immune suppression and histophysiology of the immune response

Seven daily intramuscular (im) injections of cortisone acetate (25 mg/Kg b.w.) given to rats or rabbits produced, (i) a pronounced reduction in the numbers of small lymphocytes in thymus-independent areas, (ii) atrophy of the thymic cortex, (iii) atrophy of germinal centres and (iv) a consequent depressed production of germinal centre-derived cells. Lymphocyte depletion was not caused by cell lysis. Moreover cell traffic between peripheral lymphoid organs did not seem to be altered. A revival of the depressed germinal centres in cortisone-treated (inbred) rats could be achieved by a transfer of bone-marrow cell suspensions from normal, cortisone-treated or T-cell-deprived animals. It was concluded that cortisone acetate arrests the migration of B-lymphocytes from the bone marrow to germinal centres in peripheral lymphoid organs, and that the accumulations of lymphoid cells in the bone marrow of cortison-treated animals might be composed of immature or mature T- and B-lymphocytes.     

Effect of exposure to stress on the role of T- and B-lymphocytes in the response of the hematopoietic system

Cellular composition of the bone marrow, spleen and peripheral blood was studied after 6-hour immobilization on the back in experiments on 4 groups of (CBAxC57BL)F1 mice with varying degree of T lymphocyte deficiency (thymectomy, sham thymectomy, administration of antilymphocytic serum, B mice). The evidence obtained shows that the "lymphoid peak" recorded in the bone marrow during stress is likely to be formed at the expense of T and B lymphocyte migration from the peripheral lymphoid organs. The data have been also obtained, indicating that T lymphocytes migrating to the bone marrow during the first 6-9 hours after the exposure to stress may participate in granulocytopoiesis activation.     

The stable and permanent expansion of functional T lymphocytes in athymic nude rats after a single injection of mature T cells

Athymic nude rats (PVG.rnu/rnu) were injected at 6 to 10 wk of age with 1 to 200 million thoracic duct lymphocytes (TDL) containing 40 to 60% mature T cells. Thereafter TDL-injected nude recipients were monitored for evidence of T cell function for up to 2 yr. W3/25+ T helper (Th) cells in lymph nodes (LN) increased from 7% at 2 wk to 30% at 8 wk after TDL transfer. The percent of W3/25+ cells remained elevated for the life of the recipient (up to 2 yr), approximating normal levels. The total size of the recirculating pool expanded in TDL-injected nude rats to reach 2/3 the level of euthymic controls by 16 wk, an increase of 10-fold to 15-fold in W3/25+ cells. The expansion of the W3/25+ population was independent of initial TDL dose. With time spleen and LN acquired a normal histological appearance including the development of germinal centres and a marked increase in cellularity in T cell traffic areas. TDL-injected nude rats rejected skin allografts with near normal kinetics. In addition graft vs host (GVH) responsiveness, assessed by the popliteal LN assay, progressively increased reaching a level 9 mo to 1 yr after replacement that resembled the GVH activity in euthymic controls.     

Spontaneous arthritis in nude rats of Rowett hooded strain

Spontaneous arthritis was found in 19 of 55 Rowett hooded strain rats with rnu gene. Most cases were in the male rnu/rnu (15/19) but a few occurred in the male rnu/+ (3/10) and female rnu/rnu (1/8). The lesions were first noted as reddened swelling due to exudative inflammation of periarticular soft tissues including synovial membranes in the tarsal and/or carpal joints. Most of the affected animals cured leaving slight induration but in a few male rnu/rnu ankylosis with pannus formation and exostosis developed. No sign of mycoplasmal or bacterial infection was noticed in the colony.     

Effects of xenogeneic, allogeneic and isogeneic thymus grafts on lymphocyte populations in peripheral lymphoid organs of the nude rat

In order to gain information about the effect of xenografted, allografted and isografted thymic tissue on peripheral lymphoid organs of immune-deficient rats, athymic nude LEW rats of ninth backcross-intercross were grafted with fetal calf and neonatal BDIX and LEW thymus. Adrenalectomy was also performed in some animals in order to obtain a possible enhancement of the immunological reconstitution. Both groups of isogeneic-thymus-grafted animals had more T helper cells than the nude controls. Furthermore, they had more densely populated paracortical areas in the inguinal lymph nodes and higher lymphocyte counts in the thoracic duct lymph. Finally, the inguinal lymph nodes contained germinal centres. Xenogeneic and allogeneic thymus transplants did not induce constant changes in the parameters observed compared with the untreated nudes. No clear difference was observed between the adrenalectomized and non-adrenalectomized thymic-isografted animals. We therefore conclude that of all the experimental animals examined the isografted nude rats show by far the best response and that adrenalectomy seems unnecessary for the success of neonatal isogeneic thymus grafts. We also conclude that the isogeneic-thymus-grafted nude rat is a suitable tool for immunological reconstitution studies.     

A new apparatus and surgical technique for the dual perfusion of human tumor xenografts in situ in nude rats

We present a new perfusion system and surgical technique for simultaneous perfusion of 2 tissue-isolated human cancer xenografts in nude rats by using donor blood that preserves a continuous flow. Adult, athymic nude rats (Hsd:RH-Foxn1(rnu)) were implanted with HeLa human cervical or HT29 colon adenocarcinomas and grown as tissue-isolated xenografts. When tumors reached an estimated weight of 5 to 6 g, rats were prepared for perfusion with donor blood and arteriovenous measurements. The surgical procedure required approximately 20 min to complete for each tumor, and tumors were perfused for a period of 150 min. Results showed that tumor venous blood flow, glucose uptake, lactic acid release, O(2) uptake and CO(2) production, uptake of total fatty acid and linoleic acid and conversion to the mitogen 13-HODE, cAMP levels, and activation of several marker kinases were all well within the normal physiologic, metabolic, and signaling parameters characteristic of individually perfused xenografts. This new perfusion system and technique reduced procedure time by more than 50%. These findings demonstrate that 2 human tumors can be perfused simultaneously in situ or ex vivo by using either rodent or human blood and suggest that the system may also be adapted for use in the dual perfusion of other organs. Advantages of this dual perfusion technique include decreased anesthesia time, decreased surgical manipulation, and increased efficiency, thereby potentially reducing the numbers of laboratory animals required for scientific investigations.     

Quantitative evaluation of the total number and distribution of lymphocytes in young pigs.

In young pigs, the spleen, thymus and all lymph nodes were dissected out and weighed. The relative content of lymphoid cells was determined from histological sections. The number of nucleated cells was evaluated by two different methods: firstly, by measuring the DNA content of samples of lymphoid tissue and dividing by the DNA content of a single nucleus; and, secondly, by counting all lymphoid cells in histological sections of defined volumes of these organs. The number of lymphoid cells in tonsils, gut, bone marrow and lung were determined using histological evaluations and the volumes or weights of these organs. The resulting average number of lymphocytes was 321 times 10 (9) for a pig of 26 kg body weight. The lymphocytes showed the following distribution in lymphoid and non-lymphoid organs: thymus 44%, spleen 9%, mesenteric lymph nodes 17%, cervical lymph nodes 9%, other peripheral lymph nodes 3%, gut-associated lymphocytes 5%, tonsils 2%, bone marrow 5%, blood 3%, lung 0.2% and an estimated figure of 3% for all other tissues.     

Implantation of cultured thymic fragments in congenitally athymic nude rats: ignorance of thymic epithelial haplotype in generation of alloreactivity

We studied the development of thymus-dependent immunity in congenitally athymic nude rats after implantation of cultured thymic fragments (CTF), particularly the development of in vitro alloreactivity in allogeneic combinations. CTF of DA (RT1a), PVG (RT1c), and RP (RT1p(u,1] origin were implanted in nude rats of WAG (RT1u) origin. In analysis 14 to 18 wk later, all recipients exhibited thymus-dependent immunocompetence assessed by (immuno)-histology of lymphoid organs and responsiveness to in vitro concanavalin A stimulation and in vivo ovalbumin immunization. Control nude animals were unresponsive. Also, in vitro alloreactivity was observed, measured by mixed lymphocyte reaction and cell-mediated lympholysis. The alloresponse to the allogeneic CTF donor haplotype was as to a third party, but that to the recipient was negative. The CTF before implantation were devoid of lymphoid elements and revealed epithelial-like cells as the major component. Cells in CTF showed expression of RT1 class I and class II antigens. CTF at autopsy had the architecture of a normal thymus. In immunohistochemistry using haplotype-specific antibodies, lymphocytes showed RT1u class I expression as in the normal WAG thymus. In the cortex-like area of CTF, stromal cells revealed class I and class II haplotype expression of the donor thymus, but in the medulla-like area, class II haplotype expression was that of the recipient WAG rat. These data indicate that after implantation in nude rats, CTF become populated not only with lymphoid elements, but also with stromal components from the recipient. In induction of thymus-dependent immunity, these acceptor-derived stromal (dendritic) cells may be involved in generation of allospecificity; class I and class II haplotype expression by the donor cortex (epithelial) compartment is ignored in this process.     

Detection of heterogenicity of the lymphoid populations by measuring the diameter of the cells and cell nuclei]

Some parameters of distribution according to the lymphoid cell and its nuclei size in the peripheral blood, bone marrow, thymus and spleen of healthy rats were studied. A comparative assay revealed population homogeneity for the thymus and bone marrow lymphocytes as well as their mean diameter differences. Mixing of these cell types markedly changed the distribution parameters of newly formed population, as shown on the model of the bone marrow lymphoid reaction caused by migration of thymic lymphocytes after 5-fluorouracil use. Preliminary thymectomy excluded migration and homogeneity of the bone marrow lymphocyte size remained unchanged.     

Continuous intravenous infusion in athymic (nude) rats: an animal model for evaluating the efficacy of anti-cancer agents

The athymic (nude) rat (rnu/rnu) has been used for a number of years in research into various human tumours involving xenotransplantation. We now report the validation of a continuous intravenous infusion method in nude rats using a tail cuff tether, which enables the study of the efficacy of novel anti-cancer materials in this mutant strain, using intravenous infusion and with no restriction of the animals or of the tumour implantation sites by jackets. Ten animals each had a cannula surgically implanted into the vena cava via the femoral vein and exteriorized via a tail cuff. Animals were housed singly in conventional cages following surgery. Following a recovery period of 5 days all animals were continuously infused with physiological saline at an infusion rate of 0.5 ml/h for a further 37 days. Body weights and food consumption were recorded weekly. Blood samples were taken approximately 14 days post-surgery and analysed for haematology and clinical chemistry parameters. All animals were successfully cannulated, and no unexpected adverse clinical signs were noted during the recovery period and the 37 days of infusion. The results demonstrate that it is possible to surgically cannulate the femoral vein of athymic (nude) rats and infuse them in conventional cages for a period of up to 37 days with minimal adverse effects. The minimal restraint required provides benefits both to the animal and to the conduct of studies such as assessment of tumour growth in the absence of a jacket. Recent work has demonstrated that the same techniques can be successfully applied to the nude mouse.     

Combination effects of chronic cadmium exposure and gamma-irradiation on the genotoxicity and cytotoxicity of peripheral blood lymphocytes and bone marrow cells in rats

This work investigated the effects of chronic cadmium (Cd) exposure combined with γ-ray irradiation on the cytotoxicity and genotoxicity of peripheral blood cells and bone marrow cells in rats. Results showed that when the rats were exposed to low dose (LD) Cd of 0.1mg CdCl?/(kgd) for 8 and 12 weeks, the Cd concentration in blood reached to 135-140 μg/L and no toxic effects on peripheral blood lymphocytes, white blood cells (WBC) and granulocyte-monocyte (GM) progenitor cells were observed except polychromatic erythrocytes (PCE) of bone marrow. Moreover, this chronic LD Cd exposure significantly decreased irradiation-induced micronucleus (MN) formation and hypoxanthine-guanine phosphoribosyl transferase (hprt) mutation in lymphocytes and PCE, while the combination of LD Cd exposure and irradiation induced the additive metallothionein (MT) protein expression in bone marrow cells. When the rats were exposed to a high dose (HD) Cd of 0.5mg CdCl/?(kgd) for 8 and 12 weeks, the blood Cd level approached to 458-613 μg/L and an inflammatory response was induced, meanwhile, MN formation and hprt mutation were markedly increased, and the ratio of PCE/NCE (normochromatic erythrocyte) was significantly decreased. Furthermore, when the rats were exposed to HD Cd plus 2 Gy irradiation, additive toxic effects on MN formation, hprt mutation, PCE damage and GM progenitor cell proliferation were observed, while this combination treatment resulted in an obvious reduction of MT protein compared to HD Cd group. In conclusion, chronic exposure to LD Cd induced the adaptive response to irradiation in the genotoxicity of peripheral blood lymphocytes and PCE of bone marrow by the up-regulation of Cd-induced MT protein, but the combination of HD Cd exposure and irradiation generated the additive effects on the cytotoxicity and genotoxicity in peripheral blood lymphocytes and bone marrow cells.     

Lymphocyte subpopulations in sensitization and specific immunotherapy in an experiment. Lymphocyte subpopulations in sensitization to staphylococci, Artemisia pollen and their combinations

The distribution of T- and B-lymphocytes in the body of guinea pigs was studied in different groups of the animals. As shown in this study, in delayed hypersensitivity to staphylococci the number of PE- and E-rosette-forming cells increased in the blood, the spleen, and the lymph nodes and decreased in the thymus; the number of EA- and EAC-rosette-forming cells decreased in the bone marrow and the spleen, the number of T gamma-suppressors decreased in the bone marrow and the distant lymph node. Immediate hypersensitivity to tarragon pollen induced the general increase of the content of T- and B-lymphocytes; the number of T gamma-cells decreased in the thymus, the bone marrow, and the lymph nodes and increased in the spleen. The characteristic features of combined microbial-pollen sensitization were the high content of B-cells in all lymphoid organs (except the thymus), a low level of T-lymphocytes in the blood and the peripheral lymphoid organs, the decreased number of T gamma-cells in most of the immunogenetic organs.     

State of several rat hematopoietic organs following total and subtotal irradiation and after bone marrow autotransplantation]

Hemopoiesis was studied in rats after x-ray irradiation. Lethal doses of 800--820 R were applied totally, with screening the shin and with subsequent autotransplantation of bone marrow taken from noninjured hemopoietic tissue. Survival of the animals and status of hemopoietic organs (quantitative indices of the peripheral blood, bone marrow and the spleen, as well as morphological changes in hemopoietic organs) served as tests. All totally irradiated animals died by the 20th day, the 30th day in the group of screened animals 32% survived, in the group with autotransplantation of bone marrow--62%. According to the indices studied restoration of hemopoiesis proceeded more quickly and completely in the group with autotransplantation of bone marrow and somewhat slower in the group with screening the shin (but without autotransplantation); this was accompanied by repopulation of bone marrow comparing with the totally irradiated animals. Restoration of the hemopoietic organs was followed by a comparatively rapid increase in the number of myeloid cells, while the number of lymphoid cells increased more slowly.     

Diminished cytokines gene expression in lymphoid organs of healthy aged rats

Immunuosenescence-related dysregulation of cytokine production is not fully understood and the roles of cytokines in organ aging have been insufficiently studied. This work aimed to compare the expression of interleukin-2 (IL-2), IL-4, IL-6, interferon-gamma (IFNγ), and transforming growth factor-beta (TGFβ) in lymphoid organs of young (3 months; n = 10), adult (1 year; n = 10), and aged (2 years; n = 7) rats under healthy, steady-state conditions. Cytokine mRNA levels were determined with TaqMan real-time PCR. In the spleen, all cytokine expression gradually declined with age (for representative cytokine IL-2 averages dCt in spleens of 3 months, 1 year and 2 years rats were 13,645, 13,19 and 15,470, respectively). In lymph nodes, all cytokines except TGF-β showed markedly upregulated expression in adult compared to young rats, but all were expressed at very low levels in aged rats. In bone marrow, adult animals showed enhanced IL-2, IFNγ, and TGFβ expression, but similar IL-4 and IL-6 expression, compared to young rats. Bone marrow of aged rats showed very low expression of all the measured cytokines. Our results demonstrated that changes occurred unevenly with aging in different immune system compartments.     

Effect of feed restriction on the peripheral blood and bone marrow cell counts of Wistar rats

The effects of 33% and 66% restricted feeding on body and organ weights, and hematological and bone marrow cellular findings in rats were investigated. The body weight gains were suppressed by restriction of feed amount and the body weights of 66% restricted-feeding groups were almost unchanged for three months (110 g in males and 80 g in females). Marked organ weight reduction (both absolute and relative) was found in the liver and thymus of rats of both sexes. Neutrophils and lymphocytes in the peripheral blood were reduced. Reticulocytes in the 66% restricted groups were decreased to 1/4 of the control values at one month, but recovered slightly thereafter. Nucleated cell counts in bone marrow in the 66% restricted groups were decreased to 1/3 of the control values after three months. Thus, the most important effect of feed restriction seemed to be on bone marrow cells rather than on peripheral blood cells except for the reticulocytes. There was no significant difference between males and females.     

Hymenolepis nana: worm recovery from congenitally athymic nude and phenotypically normal rats and mice

When eggs or mouse-derived cysticercoids of Hymenolepis nana were inoculated into previously uninfected congenitally athymic nude (rnu/rnu) rats of an outbred Rowett strain, they failed to mature in the intestinal lumen. They also failed to mature in phenotypically normal (rnu/+) littermates, except when these hosts were treated with cortisone acetate from the beginning of the lumen phase. The Rowett rat, either thymus-deficient or not, was susceptible to tissue cysticercoids but resistant to luminal adults. It is therefore considered to be an unnatural host, at least for mouse-derived H. nana. There was little or no difference in susceptibility to initial tissue cysticercoids between these nude rats and phenotypically normal ones. The normal rats became completely resistant to reinfection with eggs and no secondary cysticercoids developed in their intestinal tissue, whereas the nude rats showed unaltered susceptibility to secondary tissue cysticercoids. Thus, acquired resistance to egg challenge, assessed by the failure of tissue cysticercoid recovery, was thymus-dependent. However, innate resistance to both a primary egg dose, assessed by the low recovery rates of tissue cysticercoids, and to a primary cysticercoid dose, assessed by the failure of luminal adult recovery, were thymus-independent. The effect of cortisone acetate to initiate maturation of H. nana appeared to be unrelated to thymus function. In contrast, all mice, either thymus-deficient or not, were highly susceptible to both phases. The number of worms recovered was more than 10 times greater than that of cysticercoids established in the rat's intestinal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peyer's patches export lymphocytes throughout the lymphoid system in sheep

The lymphocyte output from small intestine containing either the long continuous ileal Peyer's patch (PP) or several smaller jejunal PP was examined in young lambs. Most studies were done in 2-mo-old lambs, 1 mo after removal of mesenteric lymph nodes (MLN). Extracorporeal perfusion of part of the intestine and addition of fluorescein isothiocyanate to the perfusate led to the labeling, in their normal microenvironment, of a regionally defined population of cells. One day later considerable numbers of emigrant lymphocytes were identified by fluorescence microscopy in the spleen, MLN and peripheral lymph nodes, jejunal PP, and bone marrow. In nonperfused ileal PP and thymus the labeling indexes were low. The highest labeling index was in the blood where 3.7% of the lymphocytes were labeled. A similar organ distribution of emigrant cells was found on day 3. When MLN were included in the perfused region more emigrants were identified. In some animals the intestinal lymphatic draining the perfused ileum was cannulated. Continual lymph drainage caused a dramatic decrease in the labeling indexes in other lymphoid organs. A substantial number of lymphocytes leave both ileal PP and jejunal PP via lymphatics and travel to all other lymphoid organs. However, the number of emigrant lymphocytes compared with the total number of labeled lymphocytes in the perfused tissue was about 10 times greater after perfusing gut with the jejunal PP than after ileal PP perfusion. We conclude that relatively more lymphocytes emigrate from the jejunal PP than from the ileal PP.     

B-lymphocyte differentiation in lethally irradiated and reconstituted mice. III. The influence of splenectomy on the recovery of the B-cell populations.

The recovery of the B-cell population was studied in irradiated and fetal liver-reconstituted mice. Since in irradiated and reconstituted mice the B-cell population in the spleen recovers much more rapidly than in the other lymphoid organs, we assessed the role of the spleen in the recovery of the B-cell compartment in the other organs. It was found that the absence of the spleen did not delay or diminish the recovery of the immunoglobulin (Ig)-bearing (B)-cell population in the bone marrow, lymph nodes, Peyer's patches, and peripheral blood. Throughout the recovery period the number of B lymphocytes in the lymphoid organs of splenectomized mice was even greater than in the same organs of sham-operated mice. B cells obtained from the bone marrow of splenectomized, irradiated, and reconstituted mice appeared to be fully immunocompetent, as shown by their ability to cooperate with thymocytes in an adoptive plaque-forming cell response to sheep red blood cells. The compensatory effect of the increased numbers of B cells in the bone marrow and peripheral lymphoid organs of splenectomized mice was reflected in the level of the serum immunoglobulins. Apart from a lower IgM concentration in the serum of splenectomized mice, no significant differences were found in IgG₁, IgG_2b, and IgA levels between splenectomized and sham-splenectomized mice. It is concluded that the spleen is not essential for both normal B-lymphocyte differentiation and maturation after irradiation and reconstitution.     

Toxoplasma gondii: Effect of maternal infection in the development of lymphoid organs of BALB/c neonates

Toxoplasmosis is one of the worldwide parasitic zoonoses. Alterations in the lymphopoietic system are still poorly studied. We analyzed lymphoid organs of BALB/c mice neonates from Toxoplasma gondii-intraperitoneally-infected mothers on 19th day of gestation, with 30 tachyzoites of strain RH. Normal non-infected pregnant females were used as controls. At 7 days after birth, animals were classified as neonates from infected (NIM) and neonates from non-infected mothers (NNIM). Weight of the thymus and number of thymic cells in NIM were decreased, percentage of apoptosis was significantly increased. Decrease in lymphocytes and monocytes and an increase of plasma cells were observed in bone marrow of NIM. Peripheral blood of NIM showed an increase of monocytes and neutrophils and a decrease in lymphocytes. Infection of the mother during the last day of gestation provokes in the neonates changes in the lymphoid organs that could explain survival of 75% of them.