Zusammenfassung Die Bursabezirke von 49 am 3. Bruttag mit 1,2% und 41 mit 3% bzw. 4% Testosteronpropionat hormonell durch Eintauchen der Eier bursektomierten Hühner wurden im Alter von 1 Tag bis 7 Monaten histologisch untersucht. Bei 24 Tieren ist kein Bursagewebe nachweisbar. Die beobachteten Bursabildungen treten in 5 Typen auf. Typ 1 und Typ 2 entsprechen völlig bzw. nahezu den Bursen von Kontrolltieren. Die Typen 3 und 4 sind gegenüber Kontrollbursen stark in der Größe reduziert, Bursafalten sind nur undeutlich ausgebildet oder fehlen. Bursafollikel kommen in unterschiedlicher Menge, bei jüngeren Tieren häufig ohne Rinde, bei über 2 Wochen alten Hühnern mit gut ausgebildeter Rinde vor. Bei kollektivem Vergleich zwischen den Gruppen sind bei 1,2% Hormonkonzentration mehr und besser differenzierte Bursabildungen vorhanden als bei höherer Konzentration. Trotzdem ist innerhalb beider Gruppen eine weite Variation von Fehlen der Bursa bis zu morphologisch normaler Bursa zu beobachten. Bei älteren Tieren läßt sich ein höherer Anteil von Bursabildungen nachweisen. Es wird diskutiert, ob nach dem Schlupf eventuell eine Erholung der Bursa eintritt. Summary The morphology of the post-embryonic bursa of Fabricius after hormonal bursectomy A histological study was made of the bursal area of 49 chicks between 1 day and 7 months old which were hormonally bursectomized by dipping the eggs at the third day of incubation into 1.2% testosterone propionate and 41 chicks in which 3 or 4% of the hormone was used. In 24 of the birds no bursal tissue was detectable. The bursal structures which did appear were classified into five types. Types 1 and 2 corresponded completely or closely with the bursa of control birds. In types 3 and 4 there was marked reduction in size and the bursal folds were indistinctly formed. The bursal follicles occurred in variable number and in younger birds they were often without a cortex, whereas in pullets more than two weeks old the cortex was well developed. In a comparative study of the groups the best developed bursal structures were in those treated with the hormone at 1.2% level, as compared with higher levels. In spite of this, each group contained birds with pictures varying from no bursa to a bursa which was morphologically normal. A higher proportion with a bursa was found in the older birds. The question whether regeneration of the bursa occurs after hatching is discussed. Résumé Morphologie de la bourse de Fabrice post-embryonnaire, après boursectomie hormonale On examine histologiquement, entre 1 jour et 7 mois, la région de la bourse de poulets boursectomisés hormonalement par immersion des oeufs dans du propionate de testostérone au 3éme jour de couvée (49 poulets, avec du propionate de testostérone 1,2%, 41 avec du propionate de testostérone 3%). Vingt quatre animaux ne présentent plus trace de tissu de la bourse. Les formations de bourse observées appartiennent á 5 types. Les types 1 et 2 correspondent entièrement ou presque aux bourses des animaux témoins. Les bourses des types 3 et 4 ont fortement rétrogradé en grandeur, par rapport aux bourses témoins, les plis de la bourse ne sont que faiblement développés ou font défaut. Les follicules de la bourse sont présents en quantité variable, chez les animaux jeunes, souvent sans cortex, chez les poulets de plus de deux semaines, avec un cortex bien développé. Dans une comparaison collective entre les groupes, on observe des formations de bourse mieux et plus différenciées avec les concentrations hormonales de 1,2%, qu'avec les concentrations plus élevées. Malgré cela, on trouve á l'intérieur de chaque groupe une large variation, depuis l'absence de bourse jusqu'á des bourses morphologiquement normales. Chez les animaux plus âgés, on peut mettre en évidence un pourcentage plus élevé de formations de bourses. On se demande s'il se produirait éventuellement, après éclosion, une régénérescence de la bourse. Resumen Morfología de la bolsa postembrionaria de Fabricio tras bursectomía hormonal En edades comprendidas entre un día y 7 meses se examinaron histológicamente las zonas de la bolsa de 49 gallinas bursectomizadas hormonalmente sumergiendo los huevos el tercer día de incubación en una solución de propionato de testosterona al 1,2% y 41 en otras al 3% y 4%. En 24 animales no se pudo identificar tejido bursátil. Las formaciones observadas de bolsas aparecen en 5 tipos. Los tipos 1 y 2 corresponden por completo o bastante a las bolsas de los animales testigos. Los tipos 3 y 4 están muy reducidos en tamaño frente a las bolsas de los controles, los pliegues solo están formados de modo poco preciso o faltan. En cantidades diversas se encuentran folículos bursátiles, en los animales jóvenes frecuentemente sin corteza, en las gallinas mayores de 2 semanas con corteza bien formada. En la comparación colectiva entre los grupos, en la concentración hormonal al 1,2% están presentes más formaciones bursátiles y mejor diferenciadas que con concentración mayor. A pesar de esto, dentro de ambos grupos se observa una variación amplia desde la falta de bolsa hasta la misma morfológicamente normal. En los animales mayores se identifica una parte más grande de formaciones de bolsas. Se discute si después de la eclosión sobreviene eventualmente la recuperación de la bolsa de Fabricio.

Biotin labeling of red cells was tested in dogs as a preclinical study for cell survival. Red cells were labeled with either spacered Biotin-X-NHS (BxNHS) or water-soluble biotin compounds. After reinfusion, biotinylated red cells were detected in small blood samples (5 μl) with flow cytometry. Improved BxNHS labeling allows an easy detection of positive red cells for almost 100 days, whereas labeling with watersoluble compounds – despite strong labeling during the first days – results in a decrease of label, which prevented a discrimation between labeled and negative cells after about 4 weeks. When biotin labeling of red cells was compared with 51Cr labeling, slopes of red cell survival were quite similar after the latter were corrected for elution. Survival slopes were linear, and the mean survival time was t=93d. In two blood-donor dogs the slopes of red cell survival where log linear and the mean survival time was t=45d. In conclusion, BxNHS, but not the water-soluble biotin compounds, is a good nonradioactive, nontoxic alternative for red cell survival studies. No health hazards are to be expected from the very low dose of Dimethylformamide, which is used as a solvent for biotin-x-NHS.

T-cell subsets were studied by flow cytometry in 58 feline leukaemia virus (FeLV)-positive cats with naturally acquired FeLV infection to determine whether the changes in CD4+ or CD8+ T cell populations differed from those observed in 55 feline immunodeficiency virus (FIV)-positive cats with naturally acquired FIV infection. The sole criterion for inclusion into the study was seropositivity. Mean (SD) CD4+ T cell values of FeLV positive cats were decreased to 31.1 (8.0) per cent and their CD8+ T cell values were increased to 22.8 (6.3) per cent in comparison with uninfected control cats (37.9 [9.5] per cent CD4+; 15.2 [6.3] per cent CD8+). The CD4+/CD8+ ratio was reduced to 1.5 (0.7), compared with 3.0 (1.5) in 39 FeLV-and FIV-negative control cats. Differences from control values were significant, but there was no significant difference between CD4+ and CD8+ lymphocytes of FeLV-versus FIV-infected cats. These findings indicate that FeLV and FIV have similar effects on T lymphocyte subsets. Both retrovirus infections can induce immunodeficiency, both viruses infect a broad range of lymphohaemopoietic cells, despite having different primary target cells, and can induce the killing of lymphocytic cells in vitro. It is concluded that a decreased CD4+/CD8+ ratio is not restricted to FIV infections but may also occur in FeLV infection.

Untreated control OS chickens develop spontaneous autoimmune thyroiditis (SAT). In contrast, OS chickens treated with a monoclonal anti-CD4 antibody failed to develop SAT. The preventive effect of anti-CD4 antibody on SAT was associated with the marked depletion of CD4+ T-cells by anti-CD4 treatment. These results indicate that CD4+ T-cells play a crucial role in SAT of OS chickens.

The aim of this study was to establish a preclinical lit vivo model to evaluate the suppressive effect of unconjugated anti-human T (CD3, 5, 7)-cell monoclonal antibodies (mAb) of mouse IgG2a or rat IgG2b isotype, Therefore, severe combined immunodeficient (SCID) mice were transplanted with human peripheral blood lymphocytes (PBL) of healthy donors (hu-PBL-SCID) or with neoplastic T cells of the human T-ALL cell line Jurkat. In preselected hu-PBL-SCID mice with substantial T cell chimerism single antibody injection caused prompt suppression of circulating human T lymphocytes within 2 days followed by occasional T cell recovery during the following weeks, Furthermore, antibody-mediated T cell suppression was measured by prolonged survival of SCID mice that had been injected with Jurkat cells preadapted to cause 100% mortality within 40 days, Injection of preadapted Jurkat cells caused fatal metastasis in lymphoid as well as non-lymphoid organs, The progression of leukemic cells was successfully suppressed when cells and anti-T cell mAb were given i.p. In contrast to control mice, tumor mortality of antibody-treated animals was delayed or completely suppressed. We conclude that SCID mice with reproducible human T cell chimerism are a relevant animal model to test the suppressive effect of anti-human T cell mAb in preclinical studies.

We studied T cell receptor variable β (TCR Vβ) gene usage by autoreactive T cells in spontaneous autoimmune thyroiditis (SAT) of obese strain (OS) chickens. Chicken αβ T cells may express either Vβ1 or Vβ2 genes, the products of which can be recognized by TCR2 and TCR3 monoclonal antibodies, respectively. Selective depletion of Vβ1+ or Vβ2+ T cells in OS chickens was accomplished by repeated injections of TCR2 or TCR3 antibodies into embryonic and 1–3-week-old chickens. The birds were killed at 20 days of age and their spleens and thyroid glands evaluated by immunohistochemistry. We found that Vβ1+ T cells preferentially infiltrated OS chicken thyroid glands. Antibody treatments resulted in a 41% reduction in frequency of Vβ1+, and a 87% reduction of the frequency of Vβ2+ cells in the circulation, and in a profound decrease of the respective T cells in spleens and thyroid glands. Selective suppression of Vβ1+ T cells partially inhibited SAT development in that thyroid-infiltrating cells and destruction of thyroid follicles were reduced by more than 50%. Thyroglobulin autoantibody serum levels were also reduced in Vβ1+ T cell-depleted OS chickens, whereas selective depletion of Vβ2+ T cells did not inhibit SAT development. These findings indicate preferential TCR Vβ1 gene usage by autoreactive T cells in SAT of OS chickens.

Rat monoclonal antibodies were produced against the C-terminus of Epstein-Barr virus nuclear antigens 2A (EBNA2A) and 28 (EBNA2B) expressed as bacterial trpE fusion proteins The initial screening was performed using a soluble bacterial extract containing the fusion proteins. Positive hybridomas were confirmed by immunofluorescence on SF158 (Spodoptera frugiperda) insect cells infected with recombinant baculovirus (Autographa californica nuclear polyhedrosis virus) and expressing the complete EBNA2A or EBNA2B genes. We selected a panel of antibodies which reacted either with both antigens or specifically with EBNA2A or with EBNA2B. The antibodies were extensively characterized using immunoprecipitation. Western blotting, epitope mapping on synthesized peptide segments of EBNA2A, immunocytology, and immunohistology on both cryostat sections and paraffin sections of AIDS-associated primary central nervous system lymphomas.

Antibodies against T cells are widely used as immunosuppressive agents in clinical therapy. As effector functions of chimeric or humanized anti-T cell antibodies cannot be predicted in vitro, we compared T cell-depleting effects of human isotypes in vivo with their immunosuppressive consequences in a mouse BMT model. This system is based on chimeric antibodies with a mouse pan T cell specificity and human constant regions. To secure optimal immunosuppression, the specificity for Thy-1.2—one of the best-characterized T cell antigens—was selected, as Thy-1.2-specific antibodies prevent graft-versus-host disease in fully mismatched mice. Chimeric mouse anti-Thy-1.2 antibody with the human IgG1 Fc part was found to be equally effective in preventing graft-versus-host disease mortality as the highly protective anti-Thy-1.2 mouse IgG2a isotype, while human IgG3 was far less effective. This was not predictable by measuring the degree of T cell depletion in peripheral blood. T cell depletion in lymph nodes, however, exactly reflected the results obtained in the BMT system. In addition, this system offers the advantage of assessing the influence of reduced antigen density by using heterozygous Thy-1.2 mice.

Biotin labeling of red cells was studied using different approaches to see if biotinylation is a useful label for determination of erythrocyte survival. Mouse red cells were labeled with biotin, either in vivo by injection or in vitro. In vivo labeled red cells were followed up in some mice without transfusing the labeled erythrocytes. Furthermore, in vivo labeled as well as in vitro labeled red cells were transfused into syngeneic mice. The biotin label allows an easy discrimination between labeled and unlabeled red cells during FACS analysis, and it is relatively stable for at least 50 days. All the three different approaches give similar results. Mean red cell life spans of in vivo or in vitro labeled red cells either transfused or followed up in vivo were between 44 and 52 days (T50 mean 23.9 days) when red cell destruction was assumed to be only a result of senescence. Mean red cell life spans were between 8 and 18 days (T50 mean 9.5 days) when a random destruction independent of red cell age was suggested. All the survival slopes are neither simple linear functions of time nor logarithmic functions, but they show an overlay of linear function by a logarithmic function where the components of both are unknown.

Surprisingly little graft-versus-host disease (GVHD) has been observed in severe combined immunodeficient (SCID) mice injected intraperitoneally (IP) with human blood lymphocytes (hu-PBL-SCID), which raised the question as to whether GVHD in such a distant species is sporadic or suppressed because of immunologic reasons. After screening for blood T-cell chimerism, we hereby describe generalized lethal xenogeneic human GVHD in unconditioned SCID chimeras, which resembles GVHD in SCID mice injected with allogeneic lymphocytes. We adapted an immunocytochemical slide method for minute cell numbers, which allowed us to follow, by multimarker phenotyping of weekly mouse-tail bleeds, the chimeric status of 100 hu-PBL-SCID injected with 10(7) or 10(8) hu-PBL of Epstein-Barr virus- (EBV-) donors. More than half of the mice showed no or less than 2% T cells. However, 13% to 21% developed substantial blood T-lymphocyte chimerism (10% to 80% human CD+ cells) and high mortality. Immunohistology showed more human CD8+ than CD4+ T cells in the splenic white pulp. The cells developed HLA-DR activation markers and infiltrated the red pulp where human B cells also appeared. Expression of activation and proliferation markers increased within 5 to 6 weeks. Many human CD3+ cells were also found in the portal triads of the liver and in the lung, pancreas, and kidney. The thymus also became heavily infiltrated. The intestines and skin of hu-PBL-SCID were less infiltrated by donor cells than in SCID with allogeneic GVHD. The tongue contained almost no human T cells. Our data show that a relatively low overall incidence of human xenogeneic GVHD, even when high numbers of human PBL are injected, is the consequence of a dichotomy between mice with no or transient T-cell chimerism and a minority of mice with high-blood T-lymphocyte chimerism and GVHD mortality.

In the chicken three types of T-cell receptors can be defined by monoclonal antibodies TCR1, TCR2 and TCR3, which recognize gammadelta T cells, and Vbeta1- and Vbeta2-expressing alphabeta T cells, respectively. In the present report we have analysed means of selectively depleting the gammadelta T cells and the Vbeta1+ alphabeta T cells.Gammadelta T cells, which represent up to 66% of all T cells in blood of a 6-month-old chicken, can be effectively depleted by neonatal thymectomy (Tx) to levels as low as 1%. Immunohistology demonstrates a similar depletion in lymphoid organs while intestinal epithelium-associated gammadelta T cells are affected by Tx to a lesser extent.Vbeta1-bearing alphabeta T cells, which comprise about 80% of the alphabeta T cells, were depleted by embryonic and neonatal injection of the TCR2 antibody. In the thymus such treatment depleted only the Vbeta1+ alphabeta T cells with high density expression of T-cell receptor. Therefore, we thymectomized TCR2-treated animals in order to prevent development of mature Vbeta1+ alphabeta T cells from the low density immature thymocytes. Treatment of chickens with a total of 22 mg of TCR2 antibody plus Tx reduced Vbeta1+ alphabeta T cells from an average of 65% to 10% of all T cells. In these TCR2 antibody-treated animals the Vbeta2-expressing alphabeta T cells become the predominant type of T cell (average 85%).

Monoclonal antibodies (mAb) were produced by immunizing BALB/c mice with non-adherent dog lymphocytes. M10 was specific for a subset of dog lymphocytes. M10 belonged to the IgG1 subclass and reacted with 26% of dog peripheral blood lymphocytes, 24% of spleen lymphocytes, 81% of thymus cells, 1.2% of bone marrow cells (5.8% of bone marrow lymphocytes) and 23% of PHA-stimulated lymphocytes. Immunohistology of snap-frozen thymus and spleen showed that the spleen B-cell area stained negative, whereas the spleen T-cell area and the thymus medulla exhibited positive reaction in 20-30%. The thymus cortex was strongly positive. M10 diminished cell lysis by 58% in cell mediated lysis assays (CML). Immunoblot assays revealed that M10 recognized an antigen with a molecular weight of 76 kD under non-reducing and 33 kD under reducing conditions. Finally, M10 bound to a canine CD8alpha transfected rat T-cell line (NB2). These findings characterize M10 as an antibody directed against the dog CD8 antigen.

In the present study the therapeutic efficacy and the side effects of two antiretroviral compounds used in human acquired immunodeficiency syndrome (AIDS) research, 3'-azido-2',3'-dideoxy-thymidine (AZT, zidovudine, Retrovir) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), were investigated in the treatment of cats naturally infected with feline immunodeficiency virus (FIV) and cats naturally infected with feline leukemia virus (FeLV). AZT was administered subcutaneously at a dose of 5 mg kg-1 body weight every 12 h and PMEA was administered subcutaneously at a dose of 2.5 mg kg-1 body weight every 12 h during a 3 week hospitalization. The therapeutic efficacy of both compounds was investigated. There was a stronger potency of PMEA than of AZT on the regression of stomatitis in FIV and in FeLV infected cats. In addition, in FIV infection PMEA had a stronger effect on the improvement of the general clinical status. Both antiretroviral compounds were potent agents to improve the immunologic status of FIV infected cats by raising the CD4/CD8 ratio. In FeLV infection PMEA and AZT appeared to reduce antigenemia. The hematological side effects caused by PMEA were severe and stronger than those of AZT. Therefore the advantage of PMEA in clinical and immunologic improvement was diminished by the hematologic disorders, which do not allow long term treatment with this drug in the dose used.

T-cell subsets were studied by fluorescence-activated cell sorter analysis in 57 feline immunodeficiency virus (FIV)-seropositive cats with naturally acquired FIV infection to see whether CD4(+)-CD8+ alterations were comparable to those observed in human immunodeficiency virus-infected patients. CD4+ values were decreased and CD8+ values were increased. The CD4+/CD8+ ratio was reduced to 1.6, compared with 3.3 in 33 FIV-seronegative control cats. Variance analysis of data showed a significant influence of FIV seropositivity, sex, and spaying of female cats on CD4+ values. CD8+ values were significantly influenced by FIV seropositivity, age, and breed. These findings indicate a similarity between FIV and human immunodeficiency virus infections, as far as alterations of T-cell subsets are concerned.