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In cynomolgus and rhesus monkeys, the dose-normalized exposure of cyclosporine administered orally as microemulsion preconcentrate (Neoral) was lower than that upon intramuscular administration. For oral administration, mean values ( +/- SD) of Cmax, 24-h area-under-the curve (AUC) and 24-h trough level, all normalized for a 1 mg/kg dose, were 20 +/- 9 ng x kg/mg x ml, 210 +/- 70 ng x h x kg/mg x ml and 2.6 +/- 0.9 ng x kg/mg x ml, respectively. For intramuscular administration, levels were about 5.5-fold, 9-fold and 22-fold higher. Based on pharmacokinetic data, the efficacy of oral cyclosporine treatment (without any other immunosuppressant) was evaluated in life-supporting cynomolgus monkey kidney allotransplantation. Rejection-free kidney allograft survival could be achieved using oral cyclosporine monotherapy with average 24-h trough concentrations above 100 ng/ml during maintenance treatment. Typically, daily oral doses of 100 mg/kg-150 mg/kg during the first two weeks post-transplantation, followed by daily 30 mg/kg-100 mg/kg dose levels during subsequent maintenance can result in long-term allograft survival, with 24-h average trough levels in individual animals during maintenance between 110 ng/ml and 700 ng/ml.

Purpose: To describe the early detection of non-Hodgkin's lymphoma (NHL) with ultrasound in three clinically normal cynomolgus monkeys post-renal transplantation and immunosuppression with cyclosporine. Materials and methods: The monkeys in this report were treated with cyclosporine (Neoral®) after receiving renal transplants. In addition to clinical and laboratory (hematology, serum chemistry) monitoring, renal allografts were monitored every 2 weeks with ultrasound and ultrasound-guided allograft biopsies were performed. Results: Enlarged renal hilar and mesenteric lymph nodes were detected with ultrasound in three monkeys on days 36, 49 and 134 post-transplantation. Sonographically the lymph nodes were inhomogeneous, of low echogenity and rounded. In two animals, the spleen was sonographically enlarged and inhomogeneous. All three monkeys were symptom-free at the time of ultrasound detection and NHL was diagnosed histologically. Conclusion: Ultrasound provides a rapid, non-invasive means of early detection of NHL in animal transplantation models prior to the onset of clinical symptoms of disease.

Xenotransplantation, the process of transplanting live cells, tissues and organs from one species into another, is an emerging novel area in medicine. It is proposed as a treatment for human end-stage organ failure and for other applications such as, for example, the treatment of neurodegenerative and liver disorders, meeting the large demand for donor organs. The pig is considered to be the most suitable donor species. However, the rejection of a pig transplant is much more severe and multifactorial compared with that of a human graft and involves almost all branches of the human immune system. Major rejection mechanisms include complement-mediated rejection, antibody-mediated rejection and cellular rejection. Prevention of rejection requires not only improved immunosuppressive agents, but also innovative approaches including the genetic modification of the pig donor by which organs become more easily acceptable, and the modulation of the human recipient's immune system so that it tolerates the xenograft without the need of extensive immunosuppression. Regarding safety, concerns have been raised on the potential risk of transmission of infectious microorganisms by a pig transplant to a human patient, and subsequent spread of infectious microorganisms into the population by an infected individual. This in particular concerns porcine endogenous retrovirus: to date intensive research has shown no evidence that this virus is transmitted in vivo from pig to humans; also a miniature swine line has been identified which does not give detectable in vitro transmission to human cells. Based on concerns on xenotransplantation, especially the safety of porcine-to-human transplants, many countries in Europe, the USA and Canada have instituted stringent policies for the development of xenotransplantation products, which include guidelines and regulations of clinical trials and additionally include public discussions on the issues associated with xenotransplantation. Based on these initiatives of regulatory authorities, xenotransplantation could advance towards a clinical application by a closely monitored stepwise approach. It is expected that advances in preclinical research during the coming years will give a further basis for such a stepwise development from a promising tool into a new well-accepted clinical entity.

rationale and objectives. To evaluate whether a change in the power Doppler (PD) flow signals produced by the renal cortical interlobular vasculature of allografts in cynomolgus monkey transplant models is useful for the detection of cellular rejection and vasculopathies. methods. Seventy-three monkeys with life-supporting allografts (bilateral native kidney nephrectomy) and 20 monkeys with allografts implanted with only unilateral native kidney nephrectomy were examined with ultrasound that included an examination with PD. Each graft received a PD score of 3 (normal cortical blush), 2 (reduced flow, no blush), or 1 (absence of cortical flow), and the results were compared with histology either from ultrasound-guided biopsy or at necropsy. results. One hundred seventy-one allograft examinations (histological and PD) were compared. Histologically normal grafts were statistically more likely to have normal PD findings than were those with reduced flow or absent flow. Allografts with reduced flow had statistically more severe cellular rejection than those with normal flow. Also, vasculopathies were present in all three PD groups. conclusions. Reduced renal cortical flow in the cynomolgus monkey renal allograft indicates that more severe degrees of cellular rejection are present compared with allografts with normal flow. Overlap in the histological diagnoses of allografts with normal and reduced flow exists, and the finding of reduced flow with PD may be prognostically important and indicates the need for tissue sampling.

The safety of a technique for ultrasound-guided biopsy of renal allografts was evaluated based on 348 consecutive procedures in cynomolgus monkeys. A spring-loaded biopsy device with an 18G tru-cut biopsy needle was used to biopsy renal allografts in 139 cynomolgus monkeys performed either on clinical indication (n = 95 animals) or as serial protocol biopsies (n = 44 animals) for a total of 348 biopsies. Monkeys having serial biopsies received between 3-9 biopsies per animal. All others received non-protocol biopsies that were performed on clinical indication, and the range was 1-15 biopsies per animal. No life-threatening complications or deaths occurred and there were no clinically detectable minor complications such as macrohematuria. Self-limiting complications such as small arteriovenous fistulas (n = 4, 3-5 mm large) were detected with Doppler ultrasound and resolved hemodynamically after 2-4 weeks. Three animals developed hematomas ranging 4 mm-2 cm in diameter and were no longer sonographically evident 2-4 weeks later. Ultrasound-guided biopsy of renal allografts can be performed with a high degree of safety in small (3-5 kg) laboratory animals such as the cynomolgus monkey and provides a valuable tool for renal transplantation research. Even when cores were taken at two week intervals no major complications occurred and only rarely were clinically irrelevant complications detected. Experience with diagnostic ultrasound, both gray scale and Doppler, is important for both safety and the recognition of complications that may arise.

Mycophenolic acid sodium salt (ERL080) is currently in Phase III clinical trials for the prophylaxis of kidney transplant rejection upon coadministration with Neoral (cyclosporin A microemulsion). To assess the relative side effect profile of ERL080 and MMF as drug substances in Lewis rats, a rat strain commonly used in transplantation experiments, a comparative 4-week tolerability study was performed. Escalating doses of ERL080 and MMF were administered orally at 10–30 mg/kg/d (i.e., doses within or above the immunosuppressive range in rats), either in single compound treatment or in combination with cyclosporine (CsA) at a daily oral dose of 7.5 mg/kg. The compounds were well tolerated as documented by body weight monitoring, hematologic parameters, and weight and histology of organs. Major abnormalities observed were a dose-dependent reduction in thymus weight associated with immunosuppression, in some cases villous atrophy in the jejunum, a reduction in white blood cell counts and lymphocyte counts (mean value in distinct treatment groups not exceeding 40–50%), a decrease in red blood cell counts and hemoglobin concentration (at maximum 25–30%), and an increase in platelet counts (in some groups up to doubling). At a given dose, these adverse effects were slightly more pronounced for MMF than for ERL080, and for groups under CsA coadministration compared to both compounds given alone. No significant potentiation effect of CsA on the changes induced by ERL080 or MMF was observed. Moreover, there were no new toxic entities evident upon CsA microemulsion coadministration.

Objective: FTY720 is a new immunosuppressant active in transplantation models, which modulates lymphocyte recirculation, leading to transient peripheral lymphopenia and increased lymphocytes in lymph nodes and Peyer's patches. Here, we investigated the susceptibility of cynomolgus monkeys to FTY720 given orally either alone or in combination with two other immunosuppressants, Cyclosporin Neoral® or RAD, as an introductory study to transplantation protocols. Methods: Each of the three phases of the study comprised a 3-week treatment period with FTY720 administered daily orally at 0.3, 0.1 or 0.03 mg/kg/day, respectively, followed by a 3-week recovery. FTY720 was given as single compound during the first week and in combination with Neoral® at 20 mg/kg/day p.o. or RAD at 0.5 mg/kg/day p.o. during the subsequent 2 weeks. Main findings: These treatment regimen were well tolerated, except for some body weight loss at high FTY720 dose (0.3 mg/kg/day). FTY720 treatment resulted in a rapid decrease of white blood cell counts which reached a plateau after 3 days. A decrease in both T- and B-lymphocyte counts by up to 80–90% was seen with FTY720 doses of 0.1 and 0.3 mg/kg/day. FTY720 blood levels, both trough levels and AUC0–24 h, showed a linear relationship with FTY720 dose. The reduction in lymphocyte counts was not directly proportional to FTY720 blood levels. The exposure to FTY720 significantly increased upon coadministration of Neoral®. This pharmacokinetic interaction was not observed for coadministration of RAD. However, the peripheral lymphodepletion was slightly increased after coadministration of RAD but not of Neoral®. This may be related to the intrinsic effects of RAD on hematopoietic cells. Conclusions: FTY720 given orally was effective in terms of peripheral T- and B-lymphodepletion and was well tolerated in cynomolgus monkeys even in combination with Cyclosporine Neoral® or RAD, indicating that such combination protocols could be used in allo- and xenotransplantation in this species. However, the data indicate a potentiation of FTY720 exposure by CsA coadministration and additional lymphodepletion by coadministration of FTY720 and RAD which should be carefully monitored.

CURRENT transplantation research has focused on the late loss of graft function and specifically on the vascular alterations, or graft vessel disease (GVD), which contribute to this clinical problem. Mechanisms involved in the multi-factorial GVD process are poorly understood and with most of the available models established in rodents, there are concerns about the predictivity of the mechanisms involved for human grafts. The situation is further complicated by the lack of a proven pharmacologic “gold standard,” to validate the animal models. It is clear that GVD involves both immunological and non-immunological or vascular components, with evidence suggesting that key events at or soon after transplantation have a significant effect upon long-term graft survival. This brief review focuses on the early cascade of events in relation to chronic graft function, concentrating in particular on the involvement of vessel wall cells in the disease process. We conclude that animal GVD models need to include a physiological assessment of graft function, if we are to make progress in the understanding and treatment of the chronic rejection process in man.

Brequinar is an immunosuppressant with the potential to be combined with cyclosporine in synergistic combination therapy. The drug tends to accumulate when given daily per os, and pharmacokinetic interaction with cyclosporine appears to enhance toxicity. Analogues with similar immunosuppressive activity have been identified at Du Pont Merck Pharmaceutical Co., that do not accumulate upon daily oral dosing in rats, and hence could have an improved potential in combination treatment with cyclosporine. We performed a toxicity study with brequinar and two brequinar analogues, administered orally once daily for 4 weeks, either alone or in combination with cyclosporine (Neoral, Novartis Pharma AG). In a first study relatively high doses were evaluated with cyclosporine at non-toxic doses of 5 and 10 mg/kg/d. The maximum tolerated dose of brequinar alone was estimated between 5 and 10 mg/kg/d; that of the analogues was estimated between 10 and 20 mg/kg/d, and above 20 mg/kg/d, respectively. In combination with cyclosporine at 5 and 10 mg/kg/d, approximately a 2-fold reduction in the maximum tolerated dose was observed. In a second study lower doses were evaluated in combination with cyclosporine at 2.5 and 5 mg/kg/d. Also this study revealed increased toxicity of brequinar (analogues) when given in combination with cyclosporine. The side effects observed were typical for drugs in the brequinar class and included leukocytopenia and thrombocytopenia, reduced body weight gain or body weight loss, thymic atrophy, cellular depletion of bone marrow and splenic white pulp, and villous atrophy in jejunum. Concentrations of brequinar (analogues) were determined in blood sampled 4 h after administration at day 1, 14 and 21-28 of the experiment. There was a tendency for drug accumulation in some groups treated with brequinar and cyclosporine. For one of the analogues at a low dose, higher concentrations were measured in groups treated with combinations of this compound and cyclosporine. We conclude that a potential synergism in immunosuppression using combinations of brequinar (analogues) and cyclosporine can be complicated by enhanced toxicity of the compounds. This indicates the need for a careful evaluation of the therapeutic window in a combined treatment together with detailed pharmacokinetics.

OME years ago, Borleffs et al’--? performed a large series of kidney allograft in rhesus monkeys (M~CUCLI nzul~~ttu) to study the effect of pretransplant blood transfusions, cyclosporine, and/or azathioprine on graft survival. In a number of animals, long-term graft survival was obtained after pretransplant blood transfusions and induction treatment with cyclosporinc. A retrospective evaluation showed a beneficial influence of MHC-DR-matched blood transfusions resulting in graft survival without immunosuppression.’ One animal not included in the present study showed permanent graft survival now for more than 13 years after cessation of immunosuppression; a recent biopsy showed normal histology. This animal had received cyclosporine for 12 months, 2 MHC-DR-matched blood transfusions, and no repeated mismatch between blood donors and kidney donor for either MHC class I and II antigens. All other cases rejected their graft during the period they received immunosuppressive drugs or after discontinuation of therapy. The delayed loss of grafts is ascribed not only to the immune reaction of the recipient to the donor organ but also by an atherosclerotic vessel wall response. We therefore revised the histopatholoby of the rcjccted allografts with special emphasis on signs of chronic rejection.

After cyclosporine treatment, dendritic cells disappear from the rat thymic medulla. The present study was undertaken to examine the ultrastructural alterations in the dendritic cell population during 14-day cyclosporine treatment and subsequent 6-week recovery. Four dendritic cell subtypes were defined ultrastructurally by a newly developed classification system. In addition, the potential effect of cyclosporine on six medullary epithelial cell subtypes was studied. During cyclosporine treatment, a prominent reduction of dendritic cells was seen at the ultrastructural level, whereas the total number of medullary epithelial cells remained largely unchanged. These findings were confirmed by immunohistochemistry. The number of mature dendritic cells declined later than the number of immature ones. A decrease in the antigen-processing capacity of remaining dendritic cells was suggested by the disappearance of Birbeck granules and the reduced number of tubulovesicular complexes. These findings support a disturbance of clonal deletion during cyclosporine treatment. The dendritic cell alterations appeared reversible 4 weeks after the restoration of the original architecture. During recovery, dendritic cells displaying lysosomal elements outnumbered those found in the normal uninvoluted thymus. This phenomenon probably reflects an enhanced turnover of cell organelles. No treatment-related effect on epithelial cell subtypes was seen.

The present studies were carried out to provide further evidence for the hypothesis that the hypotensive response to adenosine A3 receptor activation in the anaesthetized rat involves mediator release from mast cells. Male Sprague-Dawley rats were anaesthetized and given just supramaximal hypotensive doses of either the non-selective A3 receptor agonist, N6(-2)-(4-aminophenyl)ethyladenosine (APNEA: 100 micrograms/kg, preceded by the A1/A2 receptor antagonist, 8-p-(sulphophenyl)theophylline, to "isolate' the A3 receptor-mediated component of the response), the mast cell degranulating agent, compound 48/80 (300 micrograms/kg) or the vehicle for APNEA, intravenously. Blood was withdrawn from a carotid artery between 2 and 10 min after the injection and plasma and serum histamine concentrations measured. Samples of connective tissue (surrounding the abdominal musculature), thymus, mesenteric lymph node, kidney, skin and diaphragm were removed for histological analysis. The plasma and serum histamine concentrations were markedly and significantly higher in the APNEA- or compound 48/80-treated animals compared to vehicle-treated controls. Consistent with this, a substantially greater proportion of mast cells was seen to be undergoing degranulation in all tissues removed from animals treated with APNEA or compound 48/80 compared to those from rats treated with vehicle. Thus, adenosine A3 receptor activation results in rapid mast cell degranulation in the anaesthetized rat. The data provide further evidence of a key role for the mast cell in adenosine A3 receptor-mediated hypotension in this species.

The model of porcine islet cell transplant to the diabetic macaque is well accepted to study efficacy in and immunological interference. This life-supporting model is therefore considered an integral component in translational research, i.e. in efficacy-toxicity studies before the phase transition to clinical trials. Diabetes reversal and graft survival >180 days has been achieved in incidental cases, but not in a consistent way. Especially later after transplantation complications can become manifest that are intrinsic to the model: • There are metabolic incompatibilities in the insulin-glucose homeostasis between species. Fasting glucose levels are lower in monkeys than in pigs or man, and on the other hand C-peptide levels are higher. In most studies efficacy is associated with moderate hyperglycemia. Insulin secretory capacity in vitro and in vivo is much lower in pigs than in human or monkeys. Hence, the amount and quality of porcine islets needed in the pig-to-monkey transplant setting differ from those in the pig-to-human setting. • The efficacy-toxicity window of immunosuppressive drugs is smaller in nonhuman primates than in humans, in particular for small molecular weight xenobiotics. Chronic immunosuppression can induce gastrointestinal toxicity and cachexia, as a result of the high exposure to poorly absorbable drugs necessary to achieve acceptable systemic trough levels can result ingastrointestinal toxicity, with lipid wasting, malabsorption, and inflammation. This affects the diabetes model, namely a reduced challenge of the insulin-producing machinery thanks to protein and carbohydrate malabsorption, wasting and body weight loss. • Husbandry and management of nonhuman primates in chronic survival studies is complicated, in particular under conditions of metabolic perturbations and chronic immunosuppression. In our unit an animal refinement program has been implemented to achieve optimal well-being. Under such conditions long-term survival is facilitated, but also model limitations become more evident. We conclude that there are intrinsic aspects in the pig-to-nonhuman primate model of life-supporting islet transplantation that are not apparent in short-term survival studies, but become manifest later after transplantation. If long-term survival is achieved, it might not unequivocally reflect good graft function, but rather a condition of marginal graft challenge and insulin secreting activity. This phenomenon has consequences for the design and interpretation of pivotal preclinical studies in preparation for the phase transition to clinical trials.

Tolerance to allografted hearts in human recipients has been observed both in clinical situations and in in vitro experiments. To elucidate whether a quantitative change of alloreactive CTL is one of the mechanisms accounting for this graft tolerance, CTL precursor (CTLp) frequencies in the peripheral blood of 10 heart recipients were measured against spleen cells from donors and HLA nonidentical third-party persons. In this longitudinal follow-up study, we showed that the rejection reaction(s) in the grafted heart correlated with CTLp frequencies in samples taken before transplantation against the donor spleen cells, but not with the CTLp frequencies against the spleen cells from the third-party persons. The CTLp frequencies against the spleen cells from donors decreased 4-6 months after transplantation, and remained at a low level afterward. However, the CTLp frequencies against spleen cells from third-party persons in blood samples obtained 1 year after transplantation were not significantly different from those before transplantation. Therefore, we conclude that donor-reactive CTLs are important in rejecting allografted heart. The decrease in donor-specific CTLp after transplantation could explain the donor-specific tolerance. The decrease may be due to homing of the specific CTLp to the graft, or by clonal deletion of the donor-reactive CTL caused by chronic alloantigen stimulation in the presence of immunosuppressive therapies.

Exposure to sublethal doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Wistar rats results in thymic atrophy and reduced thymic cellularity. In a first experiment, rats were once orally intubated with 0, 1, 5, 25, 50, and 150 micrograms TCDD/kg body wt and sacrificed at Day 10. The dose that produced one-half of the maximal thymic involution was estimated at about 10-20 micrograms/kg. The time course (Days 0-21) of thymic atrophy induced by a single oral intubation of 25 micrograms TCDD/kg body wt revealed four phases. In phase 1 (initiation phase, Days 0-2) a decrease in proliferative activity was seen in cortical thymocytes, whereas no changes occurred in thymic cellularity. Phase 2 (lymphodepletion phase, Days 2-8) was characterized by an initial strong depletion of immature CD4+CD8+ double-positive (DP) cells (Day 4), followed by a more gradual decrease in the number of mature thymocytes (Day 6). On Day 8 the lymphodepletion was maximal. In phase 3 (stationary phase, Days 8-13) no changes occurred in thymic cellularity. Although the first signs of recovery were already seen on Day 6, indicated by a recovery in proliferative activity in the thymus cortex, an increase in thymic cellularity was observed first after Day 13 (phase 4, recovery phase, Day 13 onward). Reversibility of thymic atrophy is therefore observed within the estimated half-life of TCDD in the rat thymus (> 16 days). We conclude that TCDD exerts a rapidly reversible effect on an intrathymic cortical target cell population.

SUMMARYTo evaluate the diagnostic value of serum cytokine levels and cytokine receptor levels in the diagnosis of acute rejection after heart transplantation, we measured soluble CD8 and soluble CD25 in the serum of heart transplant recipients. The results were compared with endomyocardial biopsy (EMB) histopathology, lymphocyte activation by morphologic inspection of peripheral blood cells (cytoimmunologic monitoring), clinically manifested infections, and the maintenance immunosuppressive therapy. Significantly increased levels were observed in eases of lymphocyte activation in cytoimmunologic monitoring indicative of either rejection or infection. In clinically documented cytomegalovirus (CMV), bacterial, and Pneumocystis carinii infections, increased levels of soluble CD25 were observed. Soluble CD8 was only increased in a single ease of P. carinii infection. A statistically significant correlation was calculated between the levels of soluble CDS and whole blood cyetosporin A level. Considering chemotherapy, the levels of soluble CD8 showed an inverse correlation with the daily dosage of azathioprine. In conclusion, the levels of soluble CD8 and CD25 are associated with lymphocyte activation in peripheral blood, but do not differentiate between lymphocyte activation indicative of rejection or infection. No relationship was observed between levels of soluble CD8 and CD25, and EMB histopathology. Therefore, the assessment of these two cell products has no diagnostic potential for monitoring acute rejection after heart transplantation.

Intercellular adhesion molecule 1 (ICAM-1; CD54) is an adhesion molecule, that participates in a variety of cell-cell adhesion phenomena1. It serves as a ligand for the leucocyte integrins LFA-1 and Mac-1 2,3 and as a receptor for different pathogens4,5. ICAM-1 is constitutively expressed in vivo on cells of the high endothelial venules and on germinal centre cells in lymph node6,7, whereas its expression is readily induced on leucocytes and different types of adherent cells. ICAM-1 is especially present in areas of inflammation, where its expression of mRNA and protein has been induced by inflammatory mediators, such as lipopolysaccharide, IFNγ, IL-1 and , TNF8.

The SCID-hu mouse model offers the possibility to study the human thymus in vivo in an isolated xenogeneic environment. Whilst studying the toxicology of the human thymus using the SCID-hu model, a "control" model to test for differences in toxicokinetics due to the different localization of the thymus is desirable when SCID-hu data have to be extrapolated to the normal human situation. For this reason, SCID-ra mice were constructed by implanting rat fetal or postnatal thymus and liver explants under the renal capsule of severe combined immunodeficient (SCID) mice. Implantation of rat fetal thymus in combination with fetal liver resulted in thymus grafts with a histological appearance that was virtually identical to that of normal age-matched rat thymus. T cells of rat origin were found in the circulating blood and lymphoid organs of the recipient mice. After implantation of postnatal thymus and liver, the thymus grafts showed a dysplastic morphology. These grafts were devoid of thymocytes and consisted mainly of thymic microenvironmental components and large numbers of thymic macrophages. Some SCID-ra mice showed signs of graft-versus-host (GVH) reactions in the skin. The incidence of GVH reactions was higher with increasing developmental stage of the donor material used for implantation.

The tissue distribution of human IgG Fc receptors (Fc gamma Rs) classified in three clusters of differentiation (CD16, using 5 antibodies, CD32, using 2 antibodies; and CD64, using 3 antibodies) was evaluated by immunohistochemistry on lymphoid (lymph node, spleen, thymus, tonsil) and non-lymphoid (heart, jejunum, kidney, liver, lung, muscle, stomach, and skin) tissues. Macrophage-like cells, including Kupffer cells, expressed all three classes of Fc gamma R. Part of the cells coexpressed HLA-DR. Interdigitating dendritic cells that were present in high density in interfollicular areas of a lymph node showing dermatopathic lymphadenopathy were immunoreactive for CD32, but not for CD16 or CD64 antibodies. In lymphoid tissue, mantle zones of secondary follicles were labeled by CD32 and some CD16 antibodies. The immunolabelling of mantle zones was not present after washing the sections at low pH, which suggests that the molecules detected were passively absorbed on the cell surface (i.e. soluble Fc gamma R). The immunolabelling of tonsil sections by various CD16 antibodies showed three patterns. The first (anti-Leu-11b) revealed labelling of solitary macrophage-like cells. The second (BW209/2 and 3G8) revealed, in addition, labelling of germinal centres. The third (CLBgran1 and CLBgran11) revealed labelling of solitary cells and follicle mantles. This labelling on tissue sections was also seen in the analysis of follicular dendritic cells isolated from tonsil. The cells were faintly immunoreactive for 3G8, as well as for CD16 mAb CLBgran1, and both CD32 mAbs. In all tissues investigated there was immunoreactivity for Fc gamma Rs in varying intensity on endothelial cells of blood vessels.

Cyclosporin A (CsA) is well-known for its immunosuppressive characteristics. 1 A peculiar activity ascribed to the compound is the induction of auto-immune phenomena resembling graft-versus-host disease. This emerges in rodents after a short-course CsA treatment during the recovery phase after lethal irradiation and syngeneic bone marrow transplantation. 2 A role for the thymus has been implicated in this so-called syngeneic graft-versus-host disease. 3 Histology of the thymus after CsA treatment shows the almost absence of medullary areas. 4,5 The normal medulla is characterized by the presence of medium-sized lymphocytes with the medullary T-cell immunophenotype, and by the microenvironment consisting of medulla-type epithelium and interdigitating cells (IDC). Besides some small remnants, these components of the medulla are lacking in animals after a two-week period of CsA treatment. The disappearance of medullary epithelium and IDC has been related to a disturbance of negative selection of lymphocytes, resulting in the export of potentially autoreactive cells, that give auto-immune phenomena in the periphery.

Infection with the human immunodeficiency virus type 1 (HIV-1) may result in persistent generalized lymphadenopathy (PGL). Lymph nodes in PGL most commonly show a florid follicular hyperplasia (FH) in which regressive changes, such as loss of mantle zones and fragmentation of the germinal centre (GC) can be observed.1,2 Fragmentation of the framework of follicular dendritic cells (FDC) has been demonstrated by immunohistochemistry.1,3 Intact HIV-1 virus particles are regularly found between the extensions of FDC within GCs of HIV-1 infected individuals.4,5 These observations have been associated with a cytopathic action of the virus on FDC.6,7 A proportion of FDC may be infected and produce virus.8 FDC function as accessory cells in GCs. Impairment of FDC function by cytopathic effects of the virus may influence GC microenvironment. The distinct types of B-cells can be distinguished and quantified at the ultrastructural level.9,10 Ultrastructural analysis of of the light and dark zones in tonsils shows that FDC subtypes can be recognized representing distinct differentiation stages of FDC (Rademakers and Schuurman, this issue). We applied this approach to GCs in lymph nodes of HIV-1 infected patients to analyze alterations in the GC microenvironment.

Follicular dendritic cells (FDC) are among the first cells involved in tissue damage after infection with Human Immunodeficiency Virus type-1 (HIV-1). These cells, being of mesenchymal origin, contribute to the stroma of germinal centers in lymphoid organs. In the first phase after HIV-1 infection, these germinal centers are hyperplastic, with distinct fragmentations and indentations. At these locations, FDC give the impression of being damaged by the infection. An active role of HIV-1 components has been proposed in this process. HIV-1 proteins, complete virions and HIV-1 RNA have been observed in the germinal center, in higher density than at other locations of the lymphoid tissue. 1,2 Apparently, protein and virus particles are concentrated, presumably in the form of immune complex, in the labyrinth of cytoplasmic protusions of FDC in the germinal center. Studies on FDC purified from a lymph node of an HIV-1-infected patient, 3 and in vitro infection of FDC from normal tissue, 4 have provided support for the suggestion that the cells can be infected and actively produce virus.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known for inducing cortical atrophy in the rat thymus. The present study was conducted to provide ultrastructural evidence for the cortical epithelium to be a target for TCDD in vivo. Juvenile male Wistar rats were orally intubated once with either 50 or 150 micrograms/kg TCDD and killed 4 or 10 days thereafter. Major changes were found in the cortical thymic epithelium. First, a relative shift occurred from "pale" to darker cortical epithelial cell types, as judged by their nuclear and cytoplasmic electron density. This effect was most prominent at 10 days after exposure to 150 micrograms/kg TCDD. The increased electron density of the cortical epithelium indicates an altered state of cellular differentiation. Secondly, at the 150 micrograms/kg dose level focal epithelial cell aggregates were seen both at day 4 and day 10 after administration. This aggregation may either be compound induced or represent a secondary event to the collapse of the thymic stroma. Thirdly, increased vacuolation of cortical epithelial cells was apparent. This effect is interpreted as a consequence rather than a cause of thymocyte depletion from the cortex. This study indicates that TCDD exposure affects the cortical epithelium of the rat thymus at a high dose level. Electron microscopy reveals that the differentiation of epithelial cells is altered. In addition, epithelial cell aggregates are formed.

In the first phase of infection by the Human Immunodeficiency Virus type 1 (HIV-1), persistent generalised lymphadenopathy can be the only feature of the disease, with or without associated clinical symptoms. The main histologic abnormality observed in the swollen lymph node is hyperplasia of follicles. The network comprising of follicular dendritic cells (FDC) appears to be damaged. Immunohistochemistry shows signs of fragmentations and indentations. HIV-1 components have been suggested to play a role in this process, as proteins, 1,2 virions3 and mRNA2,4 of the virus are concentrated in the germinal centers. We5 and others6 have provided evidence that FDC can be infected by HIV-1. Some authors have proposed a role for the immune response towards the virus. This was supported by the demonstration of cytotoxic cells, documented by the presence of mRNA encoding serine esterase Granzyme B, which occurs in granules of cytotoxic cells.7

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces thymic atrophy in rats. The present study was initiated to provide (immuno)histological data on the mechanism of action. Juvenile male Wistar rats were orally intubated once with 50 or 150 micrograms/kg TCDD. They were euthanized 4 or 10 days thereafter, or were allowed to stay alive until Day 20 or 26. Growth retardation occurred rapidly in all TCDD-treated animals. Lethality was demonstrated within 20-21 days after administration. At Days 4 and 10 after intubation, thymic atrophy was shown by reduction of thymic weight and cortex/medulla ratio. Staining patterns for T-cell markers in the atrophic thymuses coincided with the reduction of cortical areas. There was no evidence indicating that the effects were indirectly caused by stress. TCDD-induced thymic atrophy persisted until Day 26 after administration. Immunohistochemical analysis revealed prominent changes in the cortical thymic epithelium at the 150-micrograms/kg dose level. First, in the cortex epithelial cell aggregates were observed both at Day 4 and at Day 10 after administration. Apparently, the architecture of the epithelium had changed in these animals. Second, at 10 days after administration epithelial cells were found with the simultaneous expression of markers that in the normal uninvoluted thymus only occur either in the subcapsular/medullary area or in the cortex. This phenotype points to an unusual stage of differentiation. We conclude that TCDD exposure affects the cortical epithelium of the rat thymus at a high dose level. Apparently, it disturbs the epithelial network and interfers with the differentiation of epithelial cells.

Germinal centers in lymph nodes with follicular hyperplasia from 15 patients with HIV-1 infection were analyzed by qualitative and quantitative electron microscopical methods and compared with control follicular hyperplasia (FH). Using a pattern recognition method, two main clusters were recognized within the germinal centers of HIV and FH lymph nodes on the basis of the relative frequencies of small centroblast and centrocytes. All FH lymph nodes and 6 HIV-1 lymph nodes (HIV-Clu-1) were placed in cluster 1; 9 HIV-1 lymph nodes (HIV-Clu-2) formed cluster 2. Germinal centers in the HIV-Clu-2 lymph nodes were characterized by a cell composition of predominantly lymphoid blasts and decreased numbers of centrocytes, but without altered numbers of mitotic figures. The frequency distribution of ultrastructurally distinct FDC subtypes differed between these clusters. In HIV-Clu-2 the frequencies of FDC types with an undifferentiated and regressive morphology occurred at a higher frequency, whereas FDC types with a highly differentiated morphology had a lower frequency. We conclude that 9 out of 15 lymph nodes with HIV-1 associated follicular hyperplasia show changes in FDC morphology indicative of a less differentiated functional stage of FDC. The changes in FDC morphology are closely associated with changes in the germinal center B-cell population resulting in an inverted blast to the centrocyte ratio.

T-cell receptor (TCR)ß-chain usage and expression of the CD3, CD4, and CD8 differentiation antigens were analyzed in 14 spontaneous AKR lymphomas. Lymphoma cells massively infiltrated and/or proliferated in the organs analyzed (thymus, spleen, and mesenteric lymph nodes), giving rise to a loss of organ structure. One lymphoma occurred only in the thymus, and failed to express CD3, CD4, and CD8. All other lymphomas expressed the CD3/TCR complex. With respect to CD4 and CD8 expression, the lymphomas were either double-negative (DN), double-positive (DP), or single-positive (SP). The frequency of DP (CD4+8+) lymphomas was low compared to the frequency of DP thymocytes in a normal AKR thymus. A substantial heterogeneity was seen in the intensity of CD4 and CD8 expression among various lymphomas, which was independent of the level of CD3 expression. Considering TCR Vßgene family usage, 2 out of 14 lymphomas expressed Vß6. Normally, Vß6+thymocytes are deleted from the thymocyte pool at the immature DP stage of T-cell development in AKR mice. These data support the hypothesis that the lymphocytes in the immature DP stage of T-cell development are susceptible to the induction of AKR lymphomagenesis. The presence of Vß6+lymphoma cells indicates that the lymphomagenesis is accompanied by a defective clonal deletion of cells expressing a possible autoreactive TCR.

Patients with alcoholic liver disease frequently reveal an increase in IgA serum concentration and IgA deposits in a continuous pattern along hepatic sinusoids. We investigated whether the hepatic IgA deposits are a passive reflection of changes in concentration or composition of IgA in the circulation, or represent a distinct effect of alcohol on the liver. Forty-one patients with alcoholic liver disease (daily alcohol intake at least 50 gm for more than five consecutive years) were compared with 41 patients with nonalcoholic liver disease. Patients in both groups were matched for serum IgA and histopathological changes in the liver biopsy. IgA deposits in the liver were found in 78% of the alcoholic patients and in 12% of the nonalcoholic patients. The presence of deposits was not related to histopathological changes in the liver or to the serum IgA concentration. In serum IgA subclass distribution, alcoholic patients differed from nonalcoholic patients by a slight but significant shift to IgA2; in contrast, the hepatic IgA deposits in alcoholic patients were almost of the IgA1 subclass. Serum secretory component (which is an equivalent of serum secretory IgA) was elevated in both alcoholic and nonalcoholic patients; patients with a liver biopsy revealing hepatitis showed the highest level. In contrast, the hepatic deposits did not contain secretory component. We conclude that the continuous deposits of IgA along liver sinusoids are not a passive reflection of changes in concentration or composition of circulating IgA, but may represent a distinct effect of alcohol on the liver related to the role of this organ in IgA metabolism.

The diagnostic relevance of the serum immunoglobulin A (IgA) concentration and liver deposition of IgA for chronic excessive alcohol consumption was evaluated in 164 patients with biochemical liver abnormalities. A relationship was demonstrated between the amount of daily alcohol consumption and the two IgA parameters and serum gamma-glutamyl transpeptidase. A continuous pattern of IgA deposition along hepatic sinusoids proved to be the best diagnostic feature, combining a specificity of 0.91 with a sensitivity of 0.75. Although serum IgA has a specificity of 0.78, its diagnostic value is restricted by a sensitivity of 0.50, making it not superior to serum gamma-glutamyl transpeptidase. Furthermore, serum IgA proved to be related to liver histopathology. High levels of serum IgA are found in hepatitis and cirrhosis, without significant differences between alcoholic and nonalcoholic patients. However, in the case of mild histopathologic changes in the liver, such as steatosis and fibrosis, significantly higher serum IgA concentrations are found in alcoholic than in nonalcoholic liver disease.