High functional CD70 expression on α-type 1-polarized dendritic cells from patients with chronic lymphocytic leukaemia.

Antigen-loaded dendritic cells (DCs) used as anticancer vaccine holds promise for therapy, but needs to be optimized. The most frequently described DC vaccine is being matured with a cocktail containing prostaglandin E2 (PGE2 DC). However, even though PGE2 DCs express both costimulatory and migratory receptors, their IL-12p70-prodcution is low, leading to an insufficient Th1 immune response. As an alternative, α-type-1 polarized DCs (αDC1s) have shown a superior production of IL-12p70 and subsequent activation of effector cells. From chronic lymphocytic leukaemia (CLL) patients, αDC1s can be generated to induce a functional Th1-immune response. Yet, another costimulatory receptor, CD70, appears to be essential for optimal DC function by promotion of T cell survival and function. So far, PGE2 is suggested as one of the most important factors for the induction of CD70 expression on DCs. Therefore, we wanted to investigate whether αDC1s have the ability to express functional CD70. We found that CD70 expression on αDC1s could be upregulated in the same manner as PGE2 DCs. In an allogeneic mixed leucocyte reaction, we found that antibody-blocking of CD70 on αDC1s from controls reduced effector cell proliferation although this could not be found when using CLL αDC1s. Nevertheless, CD70-blocking of αDC1s from both controls and patients with CLL had a negative influence on the production of both IL-12p70 and the Th1 cytokine IFN-γ, while the production of the Th2 cytokine IL-5 was enhanced. Together, this study further suggests that αDC1s should be considered as a suitable candidate for clinical antitumour vaccine strategies in patients with CLL.

The direct pathway of human T-Cell allorecognition is not tolerized by stimulation with allogeneic peripheral blood mononuclear cells irradiated with high-dose ultraviolet B.

Transfusions of high-dose (> or =10,000 Joule/m(2)) ultraviolet-B (UVB)-irradiated allogeneic leukocytes in rodent models have been shown to induce immunologic tolerance that is mediated by allospecific regulatory CD4(+) T cells. Whether these regulatory T cells recognize alloantigens through the direct or indirect pathway of allorecognition is controversial. Here, we demonstrate that the proliferative response obtained in standard primary mixed leukocyte reactions (MLRs) with human peripheral blood mononuclear cells (PBMCs) reflected a CD4(+) T-cell-dependent direct pathway of allorecognition and that high-dose UVB irradiation of PBMCs totally inhibited their capacity to induce a proliferative alloresponse. Re-stimulation with gamma-irradiated PBMCs from the same allogeneic donor (secondary MLR) elicited a proliferative and Th1-deviated response that was similar to the response induced in unprimed PBMCs. Finally, high-dose UVB was found to induce a rapid and massive apoptosis of irradiated PBMCs. Collectively, these data indicate that leukocytes irradiated with high-dose UVB are unable to prime for unresponsiveness or immune deviation in T cells directly recognizing allogeneic major histocompatibility complex molecules. Because it is well-established that antigens within transfused apoptotic cells are captured by resident tolerogenic spleen dendritic cells, we propose that tolerance induced by transfusions of high-dose UVB-irradiated leukocytes primarily involve T cells indirectly recognizing alloantigens.

Ischemic preconditioning can overcome the effect of moderate to severe cold ischemia on concordant mouse xeno-heart transplants.

PURPOSE: Concordant mouse xeno-heart transplants are relatively sensitive to ischemia-reperfusion injury. We investigated the effect of an ischemic preconditioning (IPC) protocol on the functional and biochemical outcome of mouse xenohearts transplanted to the Lewis rat. MATERIAL AND METHODS: NMRI mice (30 to 40 g) were anesthetized, intubated, and mechanically ventilated. They were subjected either to a IPC protocol leading to an SaO(2) of 70% for 5 minutes followed by normoxia (defined as SaO(2) >90%) for 10 minutes (n = 9) or normoxia only (n = 11). The hearts were then heterotopically transplanted to Lewis rats (220 g). The frequencies of immediate onset and early dysfunction and late dysfunction were registered. The hearts surviving for 6 hours were explanted and the absolute concentrations of phosphocreatine and adenosine triphosphate (ATP) were determined in micromole per gram of heart tissue with high-pressure liquid chromatography. The phosphorylation ratio, PCr/ATP, a known correlate to biochemical and functional outcome, was calculated. RESULTS: Four of 11 (36.4%) of control hearts experienced immediate onset and early dysfunction versus 0% (0/9) in M hearts subjected to IPC (P = .01). Furthermore, the IPC protocol increased the PCr concentration, 15.08 +/- 1.00 versus 9.04 +/- 2.04 micromol/g in controls (P = .01), and the PCr/ATP ratio, 1.80 +/- 0.17 versus 1.27 +/- 0.21 (NS; P = .06). CONCLUSIONS: IPC provides a protective PCr overshoot overcoming the short-term effects of moderate to severe ischemic injury on mouse xeno-heart transplants.

Oral feeding with pig peripheral lymphocytes decreases the xenogeneic delayed type hypersensitivity reaction in galactosyltransferase knockout mice.

PURPOSE: Oral tolerance induction has shown promising results in experimental allotransplantation models but is not well investigated in xenotransplantation. We investigated the possibility to induce tolerance against pig peripheral lymphocytes (pPBL) in galactosyltransferase knockout mice (gal -/-), which produce antibodies against Galalpha1-3Gal. MATERIAL AND METHODS: Female (gal -/-) mice 6 to 8 weeks old weighing 35 to 40 g (n = 10) were fed orally every third day five times with 2 x 10(7) isolated, viable pPBL, or with phosphate-buffered saline (PBS) only (n = 7). They were then immunized subcutaneously on day 0 with a subcellular lysate from 4 x 10(7) isolated, viable pPBL. On day 13, 25 microL of a subcellular lysate corresponding to 1 x 10(7) isolated, viable pPBL was injected in the right dorsal foot pad, and the delayed type hypersensitivity (DTH) reaction was calculated after 24 hours by subtracting the swelling response from 25 microL PBS in the left footpad. Anti-Galalpha1-3Gal immunoglobulin IgG and IgM antibody titers were measured in the serum before oral feeding and at day 14. RESULTS: The DTH reaction of the pPBL fed mice was 0.07 +/- 0.05 mm vs 0.57 +/- 0.23 mm for the controls (P < .001). No significant differences in anti Gal alpha1-3 Gal IgG and IgM antibody titers were seen. CONCLUSIONS: This study demonstrates for the first time that oral delivery of pPBL can counteract the indirect T-cell reaction against xenogeneic subcellular antigens from pPBL. These observations warrant further investigation in immunologically modified mice and perhaps in primate models of xenotransplantation.

Direct allorecognition promotes activation of bystander dendritic cells and licenses them for Th1 priming: a functional link between direct and indirect allosensitization.

T-cell sensitization to indirectly presented alloantigens (indirect pathway of allorecognition) plays a critical role in chronic rejection. The usual very efficient priming of such self-restricted, T helper type 1 (Th1)-deviated CD4+ T cells obviously conflicts with the fact that allogeneic MHC molecules are poorly immunogenic per se. The aim of the present study is to elucidate whether direct allosensitization induces production of inflammatory mediators that may affect recruitment and activation of immature bystander (host) dendritic cells (DC). These potential mechanisms were studied in vitro by conducting primary allogeneic mixed leucocyte reactions (MLR), mimicking the priming phase in secondary lymphoid organs, and secondary MLR, mimicking the effector phase within the graft. Primary, and particularly secondary, MLR supernatants were found to contain high levels of monocyte/immature DC-recruiting CC chemokines and pro-inflammatory cytokines. Exposure of immature DC to primary or secondary MLR supernatants was found to upregulate CD40 expression and further enhanced lipopolysaccharide-induced interleukin-12 (IL-12) p70 production. Secondary MLR supernatants additionally induced upregulation of CD86 and deviated allogeneic T-cell responses towards Th1 (enhanced interferon-gamma production without concomitant induction of detectable IL-4 or IL-10 production). These findings indicate that direct allorecognition may act as a Th1-deviating adjuvant for indirect allosensitization.

Intragraft heat shock protein-60 expression after small bowel transplantation in the mouse.

The time course of heat shock protein 60 (hsp 60) expression after intestinal transplantation in syngeneic and allogeneic combination was correlated with the degree of rejection. Hsp 60 expression was assessed by immunostaining; rejection degree was established by histologic examination on posttransplantation days 1, 3, 6, and 8. No signs of rejection occurred in syngeneic grafts at any time. In the allogeneic setting, rejection was absent in all but 1 case on postoperative day 3. Three days later moderate rejection was evident based on focal crypt destruction and focal mucosal ulceration, whereas at postoperative day 8 extensive mucosal sloughing was the dominant feature, consistent with advanced rejection. Hsp 60 remained undetectable in the syngeneic setting at all times. In allografts, hsp 60 was initially expressed on posttransplant day 3, increasing synchronously with the progression of rejection at days 6 and 8. Hsp 60 expression was localized almost exclusively to the crypt area and the lower third of the villi. In conclusion, the rejection of murine allogeneic intestinal grafts is characterized by a progressive expression of hsp 60 in the epithelium.

Anti-SSA/Ro antibody determination by enzyme-linked immunosorbent assay as a supplement to standard immunofluorescence in antinuclear antibody screening.

The aim of this study was to investigate the frequency and possible clinical relevance of SSA/Ro antibodies, as determined by enzyme-linked immunosorbent assay (ELISA), in patient sera not exhibiting a concomitant positive reaction by the standard immunofluorescence (IF) test using HEP-2 cells as substrate. SSA/Ro reactivity, as shown by ELISA, was found in 285 (7%) of 4025 serum samples consecutively remitted for antinuclear antibody (ANA) screening. Seventy-five of these serum samples (26%), derived from 64 patients, were negative by the IF-ANA screening test. Serum samples from all 64 patients exhibiting SSA/Ro reactivity by ELISA without concomitant positivity by IF-ANA were further investigated by IF using transfected HEP-2 cells hyperexpressing the 60,000 MW SSA/Ro antigen (HEP-2000(R)) and by immunodiffusion (ID) and Western blot. In 55 of these 64 patients, SSA/Ro reactivity could be verified by one or more of the other techniques investigated. Twelve of these patients fulfilled four or more American College of Rheumatology (ACR) criteria for systemic lupus erythematosus (SLE) and another five patients exhibited a histologically confirmed cutaneous lupus erythematosus (LE). In four of the 12 IF-ANA-negative patients with a diagnosis of SLE, the SSA/Ro reactivity was only detectable by ELISA and Western blot. In conclusion, the use of a sensitive ELISA assay could provide a clinically important supplement to the routine ANA screening by IF, which does not detect certain anti-SSA/Ro-containing sera among patients with relevant autoimmune diagnoses. Detection of anti-SSA/Ro antibodies, however, does not alone signify cutaneous LE or SLE but adds weight to these diagnoses that should rely heavily on other clinical information.

Ro 52kD autoantibodies are detected in a subset of ANA-negative sera.

To define Ro 52kD, Ro 60kD, and La specificities of autoantibodies within ANA-negative sera, samples from 64 ANA-negative but SSA positive patients undergoing investigation due to suspected CTD were analysed, using recombinant antigens and synthetic peptides by immunoblotting and ELISA. The sera were selected from 4025 sera submitted for routine ANA analysis. Antibodies to Ro or La were detected in 42/64 sera (65%). Anti-Ro 52kD antibodies occurred most frequently and were present in 42/64 sera (65%). This was the only specificity of autoantibody detected in 18 sera. No patient had only anti-La or anti-Ro 60 antibodies. In total 18.64 patients (28%) had Ro 60 antibodies and 14/64 had anti-La antibodies (21%). Eight patients had antibodies reacting with all three antigens. We used the same set of sera to test the antigenicity of different regions of Ro 52kD represented by deletion clones and peptides derived from the Ro 52kD sequence. Out of 30 sera reacting with a recombinant deletion clone encompassing as residues 136-227, 12 sera reacted with a peptide corresponding to a 200-239. Some sera gave a low positive OD value with a peptide of a 176-196. Based on the results of this study in which we demonstrate Ro 52kD autoantibodies in 65% of selected ANA negative sera and define an autocephitope within the Ro 52kD protein composed of the leucine zipper domain, we suggest that testing for Ro 52kD antibodies could be included in an extended investigation of ANA negative patients with suspected connective tissue disease.

Canine antinuclear antibodies: comparison of immunofluorescence staining patterns and precipitin reactivity.

The occurrence of antinuclear antibodies (ANAs) against several specific nuclear antigens is clearly associated with certain systemic rheumatic disorders in human patients. Determination of ANAs on a routine basis, usually by indirect immunofluorescence (IIF) technique, has therefore become an important diagnostic tool. The subdividing of positive ANA-sera into different nuclear IIF staining patterns often gives clues to antibody specificity. The present investigation aimed at studying whether such subgroups of staining patterns in IIF ANA positive canine sera may represent certain specific ANAs that can be verified by standard methods used for specificity determination in humans. The presence of precipitating antibodies, determined by the Ouchterlony immunodiffusion (ID) technique, was found to be strictly associated with a positive IIF ANA, exhibiting a speckled staining pattern without any chromosomal reactivity. None of the sera with chromosomal reactivity contained precipitating antibodies. Among the ID positive serum samples, different antigenic reactivities were detected, represented by different ID subgroups. Only 1 of the 4 main subgroups obtained by ID showed identity with any of the common and disease-associated human ANA specificities, exhibiting anti-RNP reactivity. One of these serum samples concomitantly exhibited precipitating antibodies against the Sm antigen.

Xenograft rejection of fetal porcine islet-like cell clusters in the rat: effects of active and passive immunization.

The process of islet xenograft rejection is still poorly understood. To elucidate further possible mechanism(s) involved in xenograft rejection, the effect of different immunization protocols was investigated. Fetal porcine islet-like cell clusters (ICCs) were transplanted under the kidney capsule in otherwise untreated rats, rats pre-immunized by s.c. injections of ICCs and in rats passively immunized with immune serum. The rejection process was evaluated with regard to antibody and complement deposition in the graft, as well as to morphology and phenotype of the infiltrating cells. In otherwise untreated animals, a moderate perigraft mononuclear cell infiltrate was seen after 3 days. Graft destruction became evident on day 6 with marked intragraft infiltration by macrophages (ED1 positive), whereas T cells were in the minority and mainly located in the perigraft area. In contrast to the findings in non-immunized rats, the rejection process in pre-immunized rats was characterized by marked intragraft infiltration by macrophages 3 days after transplantation. Moreover, both T cells and macrophages heavily infiltrated the adjacent kidney parenchyma, and major histocompatibility complex (MHC) class II expression in surrounding kidney tubular cells was concomitantly enhanced. Syngeneic rat islets mixed with porcine ICCs escaped the rejection process in non-immunized rats but were affected in pre-immunized animals. Thus, the specificity of the rejection process in non-immunized animals seems to be lost in pre-immunized animals. The early macrophage infiltration was also accelerated in rats passively immunized with immune serum, but no early switch from perigraft to intragraft infiltration or subsequent cellular infiltration in the adjacent kidney parenchyma was seen. Circulating xenoreactive antibodies of the IgG isotype increased after transplantation in normal and otherwise untreated rats. No distinct IgG deposition in the ICC xenografts was observed until day 12 after transplantation in untreated rats, whereas perigraft deposition of IgG was found 1 day after transplantation in pre-immunized rats and in rats given immune serum. No deposition of complement was observed within the ICC xenograft in any of the groups during the observation period. The dependence on T cells, the massive infiltration of macrophages with a unique phenotype, the cellular distribution, and the loss of specificity (bystander killing) of the rejection process in immunized rats suggest that ICC xenograft rejection shares some of its main characteristics with a delayed type hypersensitivity-like (DTH) immune response.

Antibodies against double-stranded DNA and development of polymyositis during treatment with interferon.

Alpha interferons have become effective palliative treatments for patients with neuroendocrine tumours such as carcinoids and endocrine pancreatic tumours. However, several reports indicate an increased incidence of both autoantibodies and autoimmune diseases in patients treated with interferon-alpha (IFN-alpha). We studied the development of antibodies against double-stranded DNA (dsDNA) and clinical signs of autoimmune disease in 214 patients with malignant carcinoids or endocrine pancreatic tumours consecutively admitted for treatment with IFN-alpha. Seventeen patients (8%) developed antibodies against dsDNA, predominantly females (12 females and 5 males). One patient had clinical and laboratory signs of polymyositis. Among the other 16 patients, three developed hypothyroidism and in six patients the anti-dsDNA autoantibodies normalized despite continuing therapy. Although a significant number of patients developed autoantibodies against dsDNA, overt autoimmune disease related to these antibodies is a rare event and many patients spontaneously normalize these titres despite continuing IFN-alpha treatment.

Autoantibody profiles in canine ANA-positive sera investigated by immunoblot and ELISA.

Canine systemic lupus erythematosus (SLE) has a similar disease expression as human SLE, but the serological characterisation of the canine disease is as yet incomplete. In the present study, we examined the specificity of antinuclear antibodies (ANA) in indirect immunofluorescence (IIF) positive canine sera. Sixty-four canine IIF ANA positive sera were characterised using HeLa cell nuclear extract immunoblots and recombinant U1-70K ELISA. We compared these results with a previously shown concordance between indirect immunofluorescence and immunodiffusion in canine SLE serological diagnosis. One canine serum reacting with Sm proteins was observed, and five canine sera presented anti-RNP autoantibodies against the antigens 70K, A, C, and/or B/B'. The autoantigen most frequently recognised was a 43 kDa nuclear protein, previously described as hnRNP G. This prominent canine autoantigen was missing in the commercially available extract designed for immunodiffusion testing of human sera. Other prominent canine autoantigens were found not to be identical with the principal human ones, thus making present human test systems deficient for the use in canine systemic connective disease diagnosis. The development of antigenic extract designed for canine autoimmune autoantigens is necessary in order to make immunodiffusion a useful method in canine diagnosis. The anti-RNP positive canine sera were examined in more detail and we found that the human major antigenic region of the most prominent RNP antigen, the U1-70K protein, also is targeted by canine autoantibodies. Thus, the response against the RNP antigen seems to be conserved between man and dog.

Efficacy of immunosuppressive drugs in islet xenotransplantation: leflunomide in combination with cyclosporine and mycophenolate mofetil prevents islet xenograft rejection in the pig-to-rat model.

BACKGROUND: Morphological characteristics of islet xenograft rejection differ from those of islet allograft rejection. Therefore, prevention of islet xenograft rejection probably requires a different type of immunosuppression from that used in allogeneic transplantation. METHODS: Fetal porcine islet-like cell clusters (ICC) were transplanted into the renal subcapsular space of rats treated with different immunosuppressive protocols. The existence of a cellular infiltrate or deposits of antibodies and complement in the grafts was evaluated at different times after transplantation using immunohistochemistry. RESULTS: Treatment with leflunomide (LEF), cyclosporine (CsA), mycophenolate mofetil (MMF), 15-deoxyspergualin, and rapamycin alone or in combination had an insufficient inhibitory effect on ICC xenograft rejection. However, in animals treated with LEF+CsA, the rejection process was markedly inhibited. However, some macrophages and T cells were still present, and at 24 days, the xenografts were destroyed. In LEF+CsA-treated animals that were given sera containing an excessive amount of rat anti-porcine xenoreactive antibodies, marked deposits of IgG, and to some extent C3 as well, were detected along the border between intact ICC, and the xenografts were surrounded by macrophages. However, almost no cells infiltrated the grafts, and there were many intact ICC. In animals treated with LEF+CsA+MMF, only occasional infiltrating cells were seen at 12 and 24 days after transplantation, and the endocrine tissue was completely intact. CONCLUSIONS: LEF+CsA+MMF prevented rejection of porcine ICC xenografts in the rat for up to 24 days after transplantation.

Successful retransplantation of mouse-to-rat cardiac xenografts under immunosuppressive monotherapy with cyclosporine.

The present study was undertaken to investigate whether retransplantation with a second xenograft, from the same species as the primary graft, is possible to achieve using only moderate immunosuppression. Heterotopic mouse-to-rat cardiac transplantations were performed, and the recipients were treated with 15-deoxyspergualin (DSG) and cyclosporine (CsA) at high doses for days -1 to 4 and at moderate doses for days 5 to 28. From day 29 and onward, the immunosuppressive protocol consisted of daily oral administration of CsA 10 mg/kg as monotherapy. Animals that had beating grafts when DSG treatment was stopped were retransplanted 56-154 days after the primary transplantation, either with a vascularized graft (heart) or with nonvascularized graft (pancreatic islets), under continued therapy with CsA. Six of 10 secondary cardiac xenografts functioned for more than 50 days and were harvested beating after 60-100 days. In contrast, nonimmunosuppressed or DSG-treated rats are known to reject a second cardiac mouse graft hyperacutely. The unresponsiveness was confined to cardiac tissue, as the pancreatic islets, transplanted under the kidney capsule, were totally rejected after 14 days. Long-term functioning cardiac xenografts, primary and secondary, had a well-preserved morphology, and infiltrating mononuclear cells were found just in the periphery of the grafts. A majority of these cells were macrophages expressing the ED1, but not the ED2, antigen. No deposition of IgG or complement was seen in any of the graft vessels, whereas a slight deposition of IgM was observed in some vessels of both primary and secondary grafts. In conclusion, we have demonstrated that unresponsiveness can be induced by effective immunosuppression of the recipient at the time of the initial transplantation, so that retransplantation with a second xenograft can be performed successfully under single-drug immunosuppressive therapy with CsA.

Expression and function of a CD4-like molecule in parathyroid tissue.

BACKGROUND: Parathyroid tissue expresses the T-lymphocyte antigens CD3 and CD4, and parathyroid CD3 has earlier been proposed to interact in the regulation of parathyroid hormone (PTH) release. METHODS: Anti-Leu3a, a monoclonal antibody recognizing CD4, was used to stain parathyroid tissue immunohistochemically, to influence PTH secretion from enzymatically dispersed parathyroid cells, and to immunoprecipitate parathyroid CD4. Northern blot and polymerase chain reaction were used to clarify the similarity between parathyroid and lymphocytic CD4. Serum PTH level was measured with an immunoradiometric assay in healthy control subjects and individuals with human immunodeficiency virus type 1. RESULTS: The parenchyma of normal and abnormal parathyroid tissue displayed strikingly variable CD4 expression. Immunoprecipitation showed a 56 kd molecule, and Northern blot and polymerase chain reaction confirmed the similarity with lymphocyte CD4. Anti-Leu3a inhibited preferentially low calcium-stimulated secretion of PTH from dispersed parathyroid cells, without discernible influences on the cytoplasmic calcium concentration of these cells. Individuals with human immunodeficiency virus type 1 displayed significantly lower serum PTH levels than healthy control subjects. CONCLUSIONS: The results suggest that the human parathyroid chief cell expresses a CD4 moiety, which seems to interact in the PTH release in vitro and in vivo and which seems to use another second messenger system than the structurally similar T-cell equivalent.

Tumor-specific deposition of immunoglobulin G and complement in papillary thyroid carcinoma.

Despite its predilection for multifocal growth and regional metastasis, papillary thyroid carcinoma (PTC) is a clinically indolent malignancy with an exceptionally favorable long-term prognosis. Together with the often striking inflammatory reaction present in PTC, its quiescent behavior has been suggested to reflect the activation of a tumor-induced immune response. To examine this possibility, we have studied the deposition of immunoglobulins and complement in PTC tissue. Samples from 70 cases of neoplastic and autoimmune thyroid diseases, including PTC (n = 41), follicular, anaplastic, and medullary carcinomas (n = 12), follicular adenoma (n = 6), Graves' disease (n = 8), and Hashimoto's thyroiditis (n = 3) were analyzed immunohistochemically. Cellular deposits of immunoglobulin G (IgG), particularly subclasses IgG1 and IgG4, and complement factors C3d, C4d, and C5 were shown in up to 80% of the PTC cases, whereas the other thyroid diseases studied showed little or no cellular deposition. Nonneoplastic tissue of PTC-containing thyroid glands (n = 22) lacked staining for IgG in 50% of the cases, and 82% were devoid of complement. The results suggest a tumor-specific immune response in PTC with activation of the classical complement cascade.

Xenograft rejection of porcine islet-like cell clusters in immunoglobulin- or Fc-receptor gamma-deficient mice.

The aim of the present study was to evaluate the role of xenoreactive antibodies in islet-like cell cluster (ICC) xenograft rejection. For this purpose, normal mice, mice with a targeted disruption of the Fc-receptor (FcR) gamma-chain, or the membrane exon of the immunoglobulin mu-chain gene, were transplanted with fetal porcine ICC under the kidney capsule. Mice lacking the FcR gamma have no functional FcR for IgG or IgE. Mice with disruption of the immunoglobulin mu-chain cannot produce antibodies, because B cell development is arrested at the stage of preB cells. All animals, irrespective of recipient group, readily rejected the ICC xenograft. Analyses of the pattern of cellular infiltration revealed only minor dissimilarities between the different experimental groups. Xenograft destruction was evident on day 6 after transplantation, and a large number of mononuclear cells were found to be evenly distributed throughout the ICC graft. The majority of the infiltrating cells were large, macrophage-like cells expressing the macrophage-specific phenotype marker F4/80. CD3-positive T lymphocytes were found to be mainly accumulated in the peripheral parts of the ICC xenograft. This study has demonstrated that xenoreactive antibodies are not crucial to ICC xenograft rejection in the pig-to-mouse model.

Detection of antinuclear antibodies by indirect immunofluorescence in dog sera: comparison of rat liver tissue and human epithelial-2 cells as antigenic substrate.

Rat liver sections and a human epithelial cell line (HEp-2) were compared as substrates for the detection of antinuclear antibodies (ANA) in the serum of normal dogs and dogs with suspected autoimmune disease, using a standard indirect immunofluorescence (IIF) technique. Antibody reactivity against rat hepatocyte nuclei was frequently found at low serum dilutions in normal dog sera. Using rat liver sections, a minimum significant positive titer, allowing negativity in more than 95% of normal dog sera, was found to be 1/100. With this titer, ANA positivity could be verified in 64 of 112 (57%) reanalysed serum samples from dogs with suspected autoimmune disease, earlier determined as ANA-positive. No reactivity against nuclei of HEp-2 cells was observed in any of the normal dog sera analyzed at a screening dilution of 1/25. Using this dilution as a minimum significant positive titer, 63 of the 112 (56%) re-evaluated serum samples were positive. These 63 samples were from the same dogs as the 64 samples that were positive on rat liver sections. Thus, the 2 methods of ANA-IIF detected a nearly identical population of dogs with suspected autoimmune disease once the level of significance of a positive titer was adjusted to > 95% specificity for each method. HEp-2 cells were found to be superior to rat liver cryostate sections as ANA substrate because of their low reactivity with normal sera, and the ease of discernment of the ANA fluorescence pattern. The recognition and documentation of specific pattern types may give clues to ANA subspecificities, which could prove useful if they are found to correlate with well-defined subgroups of immune mediated clinical diseases in dogs.

Xenograft rejection of porcine islet-like cell clusters in normal and natural killer cell-depleted mice.

Fetal porcine islet-like cell clusters (ICC) were transplanted under the renal capsule of normoglycemic normal or athymic (nu/nu) C57BL/6 mice. Control animals were implanted with allogeneic minced kidney tissue from C57BL/Ks mice. The animals were killed 6 or 14 days after transplantation and the grafts were processed for flow cytometric analyses or immunohistochemistry. Xenograft destruction was evident in normal mice on day 6 after transplantation. The majority of infiltrating cells were macrophage-like cells expressing the F4/80 antigen. Lymphocytes expressing the CD3 antigen were in minority and mainly located in the peripheral parts of the ICC xenograft. The frequency and distribution of CD4+ cells were found to resemble those of the CD3+ cells. A large number of infiltrating cells, including several macrophage-like cells, expressed the Thy 1.2 antigen. Flow cytometry of infiltrating cells in the ICC xenograft revealed that approximately half of the cells expressing the F4/80 antigen also expressed Thy 1.2 and/or CD4. No cells were found expressing both the F4/80 and CD8 antigens. Both the F4/80 single-positive and the F4/80, CD4 double-positive cells were found to be larger and more granular than the CD4 single-positive cells. No co-expression of CD4 or Thy 1.2 with the F4/80 antigen was detected on cells infiltrating allogeneic tissue grafts. Moreover, a relative large number of cells (approximately 15%) in the xenograft expressed the NK 1.1 antigen as determined by flow cytometry. The role of natural killer (NK) cells in islet xenograft rejection was further evaluated in mice depleted of NK cells, using intraperitoneal injections of the monoclonal antibody NK 1.1. The simultaneous inoculation and subsequent growth of the NK cell-sensitive beta 2-microglobulin-deficient mutant, C4.4-25-, lymphoma cell line EL-4 served as an in vivo control of NK cell depletion. However, all NK cell-depleted mice rejected the ICC xenograft. In contrast, athymic mice permanently accepted the porcine ICC xenograft but, readily rejected the NK cell-sensitive lymphoma cell line. Taken together, ICC xenograft rejection in mice seems to be T cell dependent, as evidenced in the nude mice model, while the main effector cell appears to be a macrophage with a unique phenotype.