BACKGROUND:The FOXP3 (forkhead Box p3) transcription factor is a marker for T regulatory cells (Treg). During cellular immune responses, Treg are expected to increase in number to ultimately control and limit this response. In renal transplants massive infiltration by T cells is often seen during rejection crises. This prompted us to examine changes in the numbers of FOXP3 positive T cells accompanying acute cellular rejection events.METHODS:A total of 32 transplant biopsies from 23 patients were studied retrospectively, these 16 protocol biopsies and 16 biopsies taken during rejection episodes included 9 serial pairs (protocol-rejection). To quantify FOXP3 positive T cells, frozen sections were double immunostained with anti-CD3 and anti-FOXP3 antibodies. Areas revealing T cell infiltrates were measured morphometrically and the number of FOXP3 positive cells per 1,000 µm2 of CD3 positive cells was taken as an FOXP3 index.RESULTS:This index was 0.46 (median, range 0.00-1.00) in the 16 protocol biopsies and 0.48 (median, range 0.16-2.31) in rejection episode biopsies. The highest values were seen during rejection crises, exceeding 1.00 in 6/16 biopsies, whereas no protocol biopsies had values greater than 1.00 (0/16) (difference significant p<0.02). In serial biopsies no consistent behavior was observed; the FOXP3 index remained unchanged, fell slightly or rose to a maximum of 13 fold. Expression levels of FOXP3 could vary within weeks. No correlations were found between donor type, initial therapy, therapy at biopsy, serum creatinine at the time of biopsy, at 3 months or 1 year later, and any of the morphometric parameters (CD3 and FOXP3) studied.CONCLUSIONS:During rejection of renal allografts the fraction of FOXP3+ Treg cells within the infiltrating T-cell population can increase transiently. This phenomenon was not consistently seen in acute cellular rejection and the information does not appear to be of value for individual patient management in such cases.

Human immunodeficiency virus infection leads to T-cell exhaustion and involution of lymphoid tissue. Recently, the programmed death-1 pathway was found to be crucial for virus-specific T-cell exhaustion during human immunodeficiency virus infection. Programmed death-1 expression was elevated on human immunodeficiency virus-specific peripheral blood CD8+ and CD4+ T cells and correlated with disease severity. During human immunodeficiency infection, lymphoid tissue acts as a major viral reservoir and is an important site for viral replication, but it is also essential for regulatory processes important for immune recovery. We compared programmed death-1 expression in 2 consecutive inguinal lymph nodes of 14 patients, excised before antiretroviral therapy (antiretroviral therapy as of 1997-1999) and 16 to 20 months under antiretroviral therapy. In analogy to lymph nodes of human immunodeficiency virus-negative individuals, in all treated patients, the germinal center area decreased, whereas the number of germinal centers did not significantly change. Programmed death-1 expression was mostly found in germinal centers. The absolute extent of programmed death 1 expression per section was not significantly altered after antiretroviral therapy resulting in a significant-relative increase of programmed death 1 per shrunken germinal center. In colocalization studies, CD45R0+ cells that include helper/inducer T cells strongly expressed programmed death-1 before and during therapy, whereas CD8+ T cells, fewer in numbers, showed a weak expression for programmed death-1. Thus, although antiretroviral therapy seems to reduce the number of programmed death-1-positive CD8+ T lymphocytes within germinal centers, it does not down-regulate programmed death-1 expression on the helper/inducer T-cell subset that may remain exhausted and therefore unable to trigger immune recovery.

OBJECTIVE:We investigated the expression of early and late phase BK virus (BKV) proteins and their interactions with host cell proteins in renal allografts, with ongoing polyomavirus associated nephropathy (PVAN), and correlated this with the nuclear and cell morphology.METHODS:Frozen sections from three patients with renal allografts (two biopsies, one explant) with PVAN were analysed by indirect immunofluorescence using BKV specific anti-polyoma large T-antigen and anti-VP-1 antibodies, as well as anti-p53, anti-Ki67, anti-caspase-3, anti-bcl2 and anti-cytokeratin 22 antibodies. Nuclear morphology and size were estimated by DNA Hoechst staining.RESULTS:In infected tubular cells the early and late phases of infection could be distinguished according to expression of large T-antigen or VP-1. The early phase revealed almost normal nuclear proportions, whereas in later phases nuclear size increased about 2 to 3 fold. Expression of large T-antigen was strongly associated with accumulation of p53 in the nucleus, accompanied by the activation of the cell cycle associated cell protein Ki67. In contrast, expression of BKV VP1 correlated only weakly with p53. Virus dependent cell lysis was due to necrosis, since neither caspase 3 nor nuclear nor cytoskeleton changes indicated apoptosis.CONCLUSION:In our selected patients with PVAN a clear distinction between early and late phases was possible, according to the protein expression patterns of BKV markers. Striking nuclear enlargement is only present in the late phase of infection. In the inflammatory setting of PVAN, BKV dependent effects appear to be mediated by the inhibition of p53, resulting in the activation of the cell cycle. We assume that in PVAN similar BKV mechanisms are operative as in certain in vitro systems.

The human MAGE-3 gene encodes a melanoma antigenic epitope rec ognized by specific cytotoxic T lymphocytes, but its gene product has not been identified thus far. We produced a recombinant MAGE-3 gene product by expression cloning of the entire reading frame in the context of a fusion protein characterized by a 10-histidine tail, allowing purification by metal chelation on a nickel Sepharose column. The semipurified prod uct was used to generate MAGE-3-specific monoclonal antibodies. One reagent could identify by immunoblotting the native MAGE-3 gene prod uct as a ,l/r 48,000 protein in lysates of cell lines showing evidence of MAGE-3 gene expression. No apparent cross-reactivity with recombinant or native MAGE-1 gene product was observed. Immunohistochemistry shows that, closely resembling the MAGE-1 gene product, MAGE-3 is a cytoplasmic protein.

Introduction: We investigated these factors and their relation to 1,25-dihydroxyvitamin D receptor (VDR) expression in freshly removed human muscle tissue.Materials and Methods: We investigated biopsy specimens of the gluteus medius taken at surgery from 20 female patients undergoing total hip arthroplasty (mean age, 71.6 +/- 14.5; 72% > 65 years) and biopsy specimens of the transversospinalis muscle taken at surgery from 12 female patients with spinal operations (mean age, 55.2 +/- 19.6; 28% > 65 years). The specimens were obtained by immunohistological staining of the VDR using a monoclonal rat antibody to the VDR (Clone no. 9A7). Quantitative VDR expression (number of VDR positive nuclei) was assessed by counting 500 nuclei per specimen and person. Serum concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were assessed at day of admission to surgery.Results: All muscle biopsy specimens stained positive for VDR. In the univariate analyses, increased age was associated with decreased VDR expression (r = 0.5: p = 0.004), whereas there were no significant correlations between VDR expression and 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D levels. VDR expression did not differ between patients with hip and spinal surgery. In the multivariate analysis, older age was a significant predictor of decreased VDR expression after controlling biopsy location (gluteus medius or the transversospinalis muscle), and 25-hydroxyvitamin D levels (linear regression analysis: beta-estimate = -2.56; p = 0.047).Conclusions: Intranuclear immunostaining of the VDR was present in muscle biopsy specimens of all orthopedic patients. Older age was significantly associated with decreased VDR expression, independent of biopsy location and serum 25-hydroxyvitamin D levels.

The immunohistochemical detection of the complement degradation product C4d, a component of the classical complement pathway, offers a new and currently poorly defined tool in the evaluation of renal allograft biopsies. Our retrospective study aims at determining the diagnostic and clinical significance of C4d accumulation in kidney transplants, employing immunofluorescence microscopy. We analyzed 398 diagnostic allograft biopsies (n = 265 patients with 1 to 5 biopsies obtained 7 to 7165 d posttransplantation [tx]) and correlated the detection of C4d with 18 histologic changes, panel-reactive antibody titers, response to treatment, and outcome. One hundred twenty-five native kidney and baseline tx biopsies served as controls. Linear deposition of C4d along peritubular capillaries was only found in a subgroup (30%) of allografts post-tx, mainly during the early time-course (median, 38 d post-tx; range, 7 to 5646 d). There was no significant association with infections. C4d staining could change from negative to positive and vice versa within days to weeks. The accumulation of C4d was most tightly linked to a morphologic subtype of rejection, transplant glomerulitis (P < 0.0001). In addition, tubular MHC class II expression was correlated with C4d deposition (P < 0.0001). Both features are signs of "acute active rejection." In comparison with C4d-negative controls, 43% of C4d-positive patients showed increased (>10%) panel-reactive antibody titers (versus 19% in the negative group; P = 0.001). C4d positivity was frequently associated with higher serum creatinine levels at time of biopsy (compared with C4d-negative group; P < 0.01). More C4d-positive patients were treated with polyclonal antithymocyte globulins (ATG) or monoclonal anti-CD3 antibodies (OKT3) (P < 0.0001). Outcome did not significantly differ between C4d-positive and C4d-negative groups. In conclusion, the detection of C4d identifies a humoral alloresponse in a subgroup of kidney transplants, which is often associated with signs of cellular rejection, i.e. tx glomerulitis. Allograft dysfunction in C4d-positive rejection episodes is often more pronounced. We provide first evidence that C4d-positive rejection might benefit from intensive therapy, potentially preventing the previously reported high graft failure rate. In addition, we show that a subgroup of C4d-positive cases may not require any immediate therapeutic intervention. The presence of C4d is clinically relevant and should be reported in the histologic diagnosis.

ABSTRACT Expression of human herpesvirus 8 viral Bcl-2 protein was demonstrated in spindle cells of late-stage Kaposi's sarcoma lesions but not in primary effusion lymphoma cell lines. In contrast, strong expression of human Bcl-2 was found in stimulated primary effusion lymphoma cells, whereas in Kaposi's sarcoma lesions preferential mononuclear cells, and to a lesser extent spindle cells, stained positive.

Current polymerase chain reaction (PCR) methods for the molecular diagnosis of B- and T-cell lymphomas by determination of clonality of immunoglobulin heavy chain (IgH) and T-cell receptor-gamma rearrangements and by detection of the chromosomal translocations t(14;18) and t(11;14), require several laborious and costly PCR assays for each of these diagnostic tests. We have developed a multiplex PCR assay for the simultaneous determination of B- and T-cell clonality and the detection of the chromosomal translocations t(14;18) and t(11;14) in a single reaction, using four-color fluorescence and automated high-resolution fragment analysis. The 26 primers combined in the multiplex PCR correspond to the sequences of > 90% of the 69 variables and 6 join IgH genes and 100% of the T-cell receptor-gamma variables and join genes that could participate in the respective rearrangements. In addition, they detect the major and the minor breakpoint regions of the t(14;18) and the major breakpoint region of the t(11;14), and amplify the beta -globin gene as an internal control. The specificity of the multiplex PCR was confirmed by analysis of 39 T-cell lymphomas and 58 B-cell lymphomas, including 11 mantle cell lymphomas bearing the t(11;14) and 25 follicular lymphomas bearing the t(14;18), with known rearrangements and/or translocations. Fifteen samples of reactive lymphadenitis remained negative.

Growing evidence suggests that intracellular vitamin D receptors are present in skeletal muscle tissue mediating vitamin D hormone response. The aim of the work reported here was to investigate the in situ expression of 1,25-dihydroxy vitamin D 3 receptor in human skeletal muscle tissue. Intraoperative periarticular muscle biopsies were taken from 20 female orthopaedic patients (17 middle-aged and elderly patients receiving total hip arthroplasty due to osteoarthritis of the hip or an osteoporotic hip fracture and 3 young patients who received back surgery). The immunohistological distribution of the vitamin D 3 receptor was investigated using a monoclonal rat antibody to the receptor (Clone Nr. 9A7). The receptor-positive nuclei were quantified by counting 500 nuclei per biopsy. Strong intranuclear immunostaining of the vitamin D receptor was detected in human muscle cells. Biopsies of hip patients had significantly fewer receptor-positive nuclei compared to those of back surgery patients (Mann–Whitney U -test: p = 0.0025). VDR expression (number of antigen-positive nuclei) was significantly correlated with age (coefficient of correlation = 0.46; p = 0.005), but not with 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D levels. The data clearly demonstrate presence of nuclear 1,25-dihydroxyvitamin D 3 receptor in human skeletal muscle. To our knowledge this is the first in situ detection of the receptor in human skeletal muscle. The difference in the expression of the receptor between hip and spinal muscle biopsies might be explained by age or location. Further research is needed in order to evaluate whether vitamin D 3 receptor expression in human skeletal muscle is age-dependent and varies between different muscles.

We report on a 17-year-old patient with severe bronchiolitis due to Mycoplasma pneumoniae infection. Despite an early 10-day course of clarithromycin, she developed progressive dyspnea, cough, fever, and severe obstructive ventilatory impairment. Sixteen days after onset of the disease a severe hemolytic anemia developed with only cold agglutinins positive at serologic screening. Thoracoscopic lung biopsy revealed diffuse bronchiolitis with suppurative intrabronchiolar inflammation, lymphohistiocytic "cuffing" of the bronchioli, and foam cell aggregates within neighboring alveoli. The infiltrate consisted mainly of CD3+, CD8+ lymphocytes and CD68+ macrophages. The diagnosis of Mycoplasma pneumoniae bronchiolitis was based on repeated complement fixation tests, which turned strongly positive only at day 74 after onset of the disease. Pulmonary function improved slowly under long-term prednisone treatment.

Growing evidence suggests that intracellular vitamin D receptors are present in skeletal muscle tissue mediating vitamin D hormone response. The aim of the work reported here was to investigate the in situ expression of 1,25-dihydroxy vitamin D-3 receptor in human skeletal muscle tissue. Intraoperative periarticular muscle biopsies were taken from 20 female orthopaedic patients (17 middle-aged and elderly patients receiving total hip arthroplasty due to osteoarthritis of the hip or an osteoporotic hip fracture and 3 young patients who received back surgery). The immunohistological distribution of the vitamin D-3 receptor was investigated using a monoclonal rat antibody to the receptor (Clone Nr. 9A7). The receptor-positive nuclei were quantified by counting 500 nuclei per biopsy. Strong intranuclear immunostaining of the vitamin D receptor was detected in human muscle cells. Biopsies of hip patients had significantly fewer receptor-positive nuclei compared to those of back surgery patients (Mann-Whitney U-test: p = 0.0025). VDR expression (number of antigen-positive nuclei) was significantly correlated with age (coefficient of correlation = 0.46; p = 0.005), but not with 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D levels. The data clearly demonstrate presence of nuclear 1,25-dihydroxyvitamin D-3 receptor in human skeletal muscle. To our knowledge this is the first in situ detection of the receptor in human skeletal muscle. The difference in the expression of the receptor between hip and spinal muscle biopsies might be explained by age or location. Further research is needed in order to evaluate whether vitamin D-3 receptor expression in human skeletal muscle is age-dependent and varies between different muscles.

Background. Type one complement receptor (CR1) is the only physiological inhibitor of complement on podocytes. CR1 is lost in different glomerulopathies, in particular in lupus nephritis, in which it has been suggested that CR1 is removed by proteolysis from the cell membrane.Methods. To define whether proteolytic cleavage of CR1 on podocytes is a general phenomenon, we analyzed the expression of CR1 in different glomerulopathies using a monoclonal antibody against epitopes present on the extracellular portion of the molecule and a polyclonal antibody directed at the intracellular tail of CR1. The two antibodies were applied on sequential serial histologic sections of renal biopsy.Results. In normal glomeruli, the two antibodies provided similar results, that is, strong staining of podocytes, and both were shown to recognize specifically CR1. Decreased expression of the extracellular portion of CR1 was observed in lupus nephritis (8/8), focal and segmental glomerulosclerosis (FSGS; 7/7), IgA nephritis (6/6), membranous glomerulonephritis (3/3), and minimal change disease (3/3). In each case, the decreased expression was accompanied by a simultaneous decrease of the expression of the intracellular tail of CR1 (Spearman's correlation coefficient r(s) = 0.951, P < 0.001). This observation was confirmed by analyzing focal glomerular lesions on sequential serial sections.Conclusion. These data indicate that there are no CR1 stumps on podocytes, even in lupus nephritis, and suggest that the CR1 loss on podocytes is not due to consumption but to decreased synthesis. A loss of CR1 synthesis might render podocytes highly sensitive to complement attack.

The BCL-6 proto-oncogene is expressed in germinal center B lymphocytes, in their neoplastic counterparts, and in a subpopulation of germinal center and perifollicular T lymphocytes. Rearrangements and/or mutations of the 5′ noncoding region of the bcl-6 gene have been demonstrated in a large majority of diffuse large B cell lymphomas. Some, but not all, of these genetic alterations lead to dysregulation of the protein. Recently, anaplastic large cell lymphomas with T and null cell phenotypes, as well as T lymphoblastic lymphomas, have also been reported to exhibit immunoreactivity to the anti-BCL-6 antibody. We collected 33 T cell non-Hodgkin lymphomas (T-NHLs) and analyzed their expression of the BCL-6 protein by immunohistochemistry and investigated the organization of the bcl-6 gene by Southern blot and single strand conformation polymorphism (SSCP). The expression of BCL-6 was demonstrated in 37.5% of lymphoblastic (LBL), 40% of anaplastic large cell (ALCL), and 33% of peripheral T cell lymphomas (PTCL). BCL-6–positive malignant cells exhibited the CD4+ or CD4+/CD8+ phenotype. The bcl-6 gene was in a germline configuration in all T-NHLs examined, and a mutation at the first exon-intron boundary region structure of the wild-type bcl-6 gene was detected in 3 of 12 PTCL. One case of PTCL with mutations of the 5′ noncoding region expressed BCL-6. In conclusion, expression of the BCL-6 protein is demonstrable independently of bcl-6 alterations in T-NHLs. This further suggests that molecular mechanisms other than rearrangements and/or mutations of the 5′ noncoding region of the bcl-6 gene can result in expression of the protein. Whether these lymphomas arose from T cells expressing BCL-6 or expressed BCL-6 as part of the malignant transformation process needs to be determined. Finally, structural alterations of bcl-6 are rare in T-NHLs, but mutations do occur in the 5′ noncoding region. We suggest that expression of BCL-6 in T cells may facilitate lymphomagenesis by repressing critical cytokines and cell cycle regulators.

NY-ESO-1 gene encodes a novel member of the cancer/testis (CT) family of human tumour-associated antigens (TAA). Specific monoclonal antibodies (mAb) have identified the corresponding gene product in lysates of tumour cell lines as a 22 kDa protein but no data are available concerning its intracellular location or distribution within neoplastic tissues. We have generated NY-ESO-1 specific mAbs recognizing the target molecule in cytospin preparations and in sections from clinical tumour specimens. These reagents identify NY-ESO-1 TAA in melanoma cell lines expressing the specific gene as a cytoplasmic protein, sharing the intracellular location of most MAGE TAA. In a series of 12 melanoma specimens, specific staining, limited to neoplastic cells, was detectable in the five cases where NY-ESO-1 gene expression was observed. In two of them over 90% of tumour cells showed evidence of positive staining. Lower percentages of positive neoplastic cells ranging between single cells and 50% were observed in the remaining tumours. These data suggest that active specific immunotherapies targeting NY-ESO-1, alone or in combination with other TAA could be of high clinical relevance in sizeable subgroups of melanoma patients. © 2000 Cancer Research Campaign

We review BK-virus nephropathy (BKN) as a new complication that increasingly affects renal allografts and causes dysfunction. Since starting in 1996, we have seen 11 cases. Currently, the prevalence of BKN is 3% in our graft biopsies. The diagnosis can only be made histologically. The virus affects tubular epithelial cells that show characteristic intranuclear inclusion bodies. The major reason for impaired graft function and a possible way for viral particles to gain access to the blood via peritubular capillaries is necrosis of infected epithelial cells. BK-virus DNA in the plasma, which can be detected by PCR, is closely associated with nephropathy. BK-virus does not stimulate tubular MHC-class II expression as judged by immunofluorescence double labelling. The inflammatory response is inconsistent and the frequency of rejection episodes is not increased during disease. Clinical manifestation of viral nephropathy evolves in several stages. (i) Initial, asymptomatic and reversible activation of the virus, judged by the presence of inclusion bearing cells in the urine. (ii) High dose immunosuppressive drug regimens, often including tacrolimus. (iii) Tubular injury and viraemia as additional promoting conditions. BKN nephropathy was associated with graft loss in 45% of our patients. The remaining patients with persistent viral nephropathy showed renal dysfunction (serum creatinine levels on average 150% above baseline readings). Currently, no established antiviral therapy is available. We discuss attempts to lower immunosuppression as a means to control viral replication. We propose a diagnostic algorithm for screening and monitoring the disease.

Hintergrund: Bei Melanomen können Tumor-assoziierte Antigene (TAA) Ziele einer spezifischen zytotoxischen T-Zellantwort sein. Das NY-ESO-1-Gen kodiert ein neues TAA sogenanntes „tumorspezifisches Antigen“. Bislang wurde die Antigenexpression nur auf der Transkriptionsebene nachgewiesen, und es war nicht bekannt, ob das entsprechende Protein in Tumorzellen produziert wird. Wir haben deshalb NY-ESO-1-spezifische monoklonale Antikörper (mAk) hergestellt, die den Nachweis des NY-ESO-1-TAA auf Proteinebene ermöglichen.

Chronic transplant glomerulopathy (TG) is one of the leading causes of severe posttransplantation proteinuria and graft loss. Our current knowledge about risk factors for the development of TG, as well as factors that affect its dynamics and prognosis, is poor. We sought to describe the pathological and clinical risk factors and correlations of TG as well as parameters that influenced the survival of grafts with that pathology.We retrospectively reevaluated 86 kidney transplant cases with TG that have been recognized on the basis of an indication biopsy since 1997. All TG as well as all pre-TG (previous) biopsies were characterized for the presence of C4d deposits in the graft.Younger recipient age and minimal immunosuppression due to drug withdrawal or suboptimal drug doses/blood levels within 3 to 6 months preceding the biopsy were associated with C4d deposition in peritubular capillaries (PTC; P = .0053 and P = .0365, respectively). Diffuse PTC-itis (P = .029, RR [95% confidence interval] = 3.349 [1.131–9.919]) and total interstitial inflammation score (P = .015, RR [95% confidence interval] = 9.662 [1.784–52.329]) were observed to show a negative impact on graft survival. C4d deposition in PTC and glomeruli, the level of pretransplantation sensitization, episodes of acute rejection, and C4d in previous (pre-TG) biopsies did not influence the survival of grafts with TG.Younger recipient age and minimal immunosuppression were associated with C4d positivity in grafts with TG. The survival of kidney grafts with TG was significantly affected by the magnitude of inflammation in the interstitium and PTC, but not by C4d positivity in PTC and glomeruli.

Complement factor I deficiency is known to be associated with recurrent pyogenic infections. The patient described here had recurrent attacks of otitis, sinusitis, and bronchopneumonia since childhood. At the age of 24 years, he had an acute episode of systemic vasculitis with purpura, but no nephritis. A factor I deficiency was diagnosed when he was 36 years old. Because of the uncontrolled activation of the alternative pathway of complement, several other components were depleted, in particular C3, which explained the predisposition for pyogenic infections. A progressive loss of renal function accompanied by proteinuria and hematuria started after the age of 40 years. Renal biopsy showed a focal segmental glomerulonephritis (GN) with glomerular deposits of immunoglobulins and complement C3 and C4 fragments. The glomerular podocytes showed an almost complete loss of complement receptor 1 (CR1; CD35). The expression of CR1 was very low on erythrocytes, as well. Thus, CR1, the most efficient cell-bound cofactor for the inactivation of C4b/C3b by factor I, appears to be consumed when factor I is missing. Although this is the first report of factor I deficiency associated with GN, it is unlikely that the development of the nephritis was fortuitous because GN has been found in many other diseases characterized by uncontrolled activation of the alternative pathway.