Endothelial expression of CD40 in renal cell carcinoma.

Recently, the immunoregulative molecule CD40 has also been introduced as a potential surface determinant of endothelial cells that can be induced by various cytokines and thus might be involved in inflammatory vascular reactions. In this study, the ubiquitous endothelial expression of CD40 within the neovascularized areas of renal cell carcinoma is demonstrated. The strong capillary expression of CD40 in 12 tumor samples is contrasted by the absence of endothelial CD40 in the corresponding tumor-free kidney specimens in which only certain tubular segments and few interstitial cells carry CD40. Northern hybridization studies confirmed the presence of CD40 RNA in cytokine-treated endothelial cells and in renal cell carcinoma, whereas no hybridization signal was obtained with normal kidney tissue. That the presence of tumor cells is pertinent to the endothelial expression of CD40 could be substantiated by in vitro experiments, when a renal carcinoma cell line and its supernatant, but not normal kidney cells, could induce CD40 on endothelial cells in culture. According to further experimental results, the carcinoma-derived, CD40-inducing factor(s) is not represented within a variety of pleiotropic cytokines including IFN-gamma, interleukin 1, interleukin 6, and tumor necrosis factor alpha, or common angiogenic factors such as basic fibroblast growth factor, vascular endothelial cell growth factor, angiogenin, and erythropoietin. The immunohistological results showing a widespread, even distribution of CD40 in tumor capillaries suggest that within renal cell carcinoma, the appearance of endothelial CD40 may also be related to angiogenesis in addition to inflammation.

Significance of donor-specific antibodies in acute rejection.

Significant advances in recent years in the diagnosis of antibody-mediated graft rejection have led to the re-evaluation of humoral alloreactivity in organ transplantation. By introducing the "C4d-test" into the work-up of transplant biopsies, donor-specific antibodies were claimed to be directly involved in about 30% of acute rejection episodes. The diagnostic criteria for antibody-mediated rejections of renal grafts are now incorporated in the "Banff classification" as refined at a recent consensus conference. Capillary C4d is not always concordant with circulating anti-HLA-antibodies, even if these are assayed with improved techniques. Antibody absorption within the graft and antigens other than HLA, therefore, have to be considered. Effective therapy of humoral rejection is now available. Serial assessment of humoral alloreactivity also in the posttransplantation period is now mandatory to identify at-risk patients.

Deposition of complement activation products on plastic-adsorbed immunoglobulins. A simple ELISA technique for the detection of defined complement deficiencies.

The activation of complement components in human serum has been studied using immunoglobulins adsorbed to microtiter plates. The sequential deposition of complement fragments was detected by a series of mono- and polyclonal antibodies in an indirect enzyme-linked immunosorbent assay (ELISA). Antibodies against C1q, C1s, C4b/d, C3b/d, factor B, C5b-9 membrane attack complex (MAC), the regulatory complement proteins C4 binding protein (C4bp) and properdin were reactive. Several lines of evidence suggest that complement activation was via the classical pathway: (1) complement activation was highly isotype-restricted with regard to the adsorbed Igs (human IgG1 and IgG3 as well as mouse IgM, IgG2a and IgG2b isotypes are strong activators in contrast to human IgG2, IgG4, IgA and mouse IgG1); (2) Ca2+ depletion, heat treatment (56 degrees C for 45 min), incubation with 0.5 M KSCN or heat-aggregated immunoglobulins (aggIgG) abrogated serum activity; (3) complement deficient sera (C1q def', C2 def', C6 def' human sera; C2 def', C4 def' guinea pig sera) showed impaired deposition of the complement components that follow the missing component in the cascade of activation. In a clinical study sera from patients with systemic lupus erythematosus (SLE) were investigated in order to measure the effect of hypocomplementemia due to complement consumption. The results obtained suggest that this new and simple assay is well suited for (1) the detection of various inherited complement deficiencies, (2) the semiquantitative evaluation of sera with decreased complement levels, (3) a more detailed study of complement components bound to a solid phase.

Efficient separation of human T lymphocytes from venous blood using PVP-coated colloidal silica particles (percoll).

In a comparative study, human peripheral T lymphocytes were separated as E rosettes by density centrifugation through various gradient media. Sheep red blood cells (SRBC) were removed by dissociation of the E rosettes at 37 degrees C with subsequent centrifugation on a similar density gradient prewarmed to 37 degrees C. In particular, gradients made of Ficoll Urovison were compared with Percoll gradients with regard to both separation steps. Using Percoll gradients, a maximal T cell recovery of 75% was obtained, whereas Ficoll separation yielded only 46%. T lymphocytes separated with Percoll exhibited equal viability compared to Ficoll isolated cells and consisted of 98% EAET-RFC. No inhibition of cellular function by Percoll treatment was detected, whereas Ficoll treatment led to an impaired mitogenic response. An inherent mitogenicity of Percoll was not observed. The method described results in considerably shortened centrifugation times due to the low viscosity of the Percoll medium and simultaneously seems to be less harmful to the rather fragile rosettes. Reproducibility was found to depend on careful control of density and osmolarity of the Percoll medium.

Assessment of soluble adhesion molecules (sICAM-1, sVCAM-1, sELAM-1) and complement cleavage products (sC4d, sC5b-9) in urine. Clinical monitoring of renal allograft recipients.

Increasing evidence exists that inducible adhesion molecules are involved in cell-mediated allograft rejection. In addition, complement activation during rejection has been described. This study investigated, whether specific molecules derived from either pathway are excreted into urine during rejection and whether they can provide useful diagnostic tools for the monitoring of renal transplant recipients. Urinary concentrations of soluble adhesion molecules (sICAM-1, sVCAM-1, sE-selectin) and of complement cleavage products (sC4d and sC5b-9), were determined by standardized ELISA in 30 normal controls and 80 samples from 49 recipients of renal allografts. In contrast to the low amounts of adhesion molecules and complement components uniformly excreted by healthy persons (group 0), marked differences were observed among allograft recipients. To prove the clinical relevance of these differences in excretion, patient samples were assigned to 5 categories according to clinical and histopathological criteria: group I--acute steroid-resistant rejection (n = 10); group II--acute steroid-sensitive rejection (n = 10); group III--chronic rejection (n = 23); group IV--stable graft function (n = 27); and group V--miscellaneous disorders (n = 10), including infections, CsA overdoses, and glomerulonephritis. Urinary levels of sICAM-1, sVCAM-1, and sC4d were significantly higher in group I compared with all other groups (P < 0.01). The difference in sICAM-1 excretion between groups III and IV also reached statistical significance (P < 0.05). Urinary concentrations of sICAM-1, sVCAM-1, and sC4d were reflective of their histological distribution in corresponding graft biopsies. None of the patients excreted E-selectin in detectable amounts. Excretion of the terminal membrane attack complex C5b-9 was not significantly associated with any diagnosis. It is concluded that for clinical purposes the combined evaluation of sICAM-1, sVCAM-1, and sC4d is most useful and can provide valuable information with regard to the severity and the type of allograft rejection.

Early renal graft dysfunction. The role of preformed antibodies to DR-typed lymphoblastoid cell lines.

Diverse pathogenetic factors may lead to the complex syndrome of early graft dysfunction, an important determinant of later renal graft outcome. That humoral factors could play a prominent role in the development of the syndrome was suggested by the capillary deposition of complement fragment C4d in about 50% of graft biopsies. This study investigates whether the presumed classical activation of complement is derived from preformed antibodies that would possibly react against endothelial HLA-class II molecules. Such antibodies were detectable by flow cytometry using a representative collection of 11 DR-typed lymphoblastoid cell lines (LCL) as targets. Simultaneous discrimination between complement-activating and -nonactivating antibodies was achieved by two-color FACS analysis. Using this method, 44 out of 86 pretransplant serum samples from recipients with early dysfunction showed reactivity against LCL (18 complement-activating, 14 nonactivating, 12 complement-activating non-IgG). Conventional panel-reactivity was observed in 20 sera only (14 also LCL-reactive). Evaluation of corresponding graft biopsies revealed that capillary C4d was associated with LCL (P = 0.018) and panel reactivity (P = 0.015) alone and in combination (P = 0.001; Pearson's chi-square test). Thirteen subsequent graft losses within one year were observed in the LCL-reactive group as compared with seven losses in the nonreactive group (panel-reactive: 7; nonreactive: 13). Thus, measurement of LCL-reactive antibodies in prospective transplant recipients improves the assessment of an individual immunological risk. The results further demonstrate that performed antibodies do not simply reflect the enhanced overall immune reactivity of certain recipients but rather act locally in vivo, thus emphasizing the role of humoral factors in the development of early graft dysfunction.

Distribution of cell adhesion molecules (ICAM-1, VCAM-1, ELAM-1) in renal tissue during allograft rejection.

The tissue distribution of cellular adhesion molecules (CAMs) was studied in specimens from 10 normal human kidneys and in 52 biopsies from kidney allografts with cell-mediated rejection. In addition to the vascular presence of ICAM-1, a common finding in normal kidneys, expression of ICAM-1 on tubular cells was observed in 22 graft biopsies. Compared with normal kidneys, where VCAM-1 was present on Bowman's capsules and few proximal tubular cells, a markedly enhanced expression of VCAM-1 in numerous tubuli (including distal tubular segments) was observed in 51 graft biopsies. In 41 graft specimens VCAM-1 appeared also in variable numbers of peritubular capillaries. Infiltrating leukocytes carrying VCAM-1 were observed in 7 grafts. ELAM-1 could not be found in normal kidneys but was restricted to some peritubular capillaries in 29 grafts. Comparable results were obtained with cultured renal tubular cells when stimulated by TNF-alpha. That the induced appearance of adhesion molecules was in fact related to actual cellular synthesis was demonstrated by Northern blot analysis. Thus, little ICAM-1 specific mRNA of 3.4-kb length could be detected in unstimulated cultured renal tubular cells, but hybridization was markedly increased after stimulation with TNF-alpha. A substantial amount of VCAM-1 specific mRNA of 3.2-kb length was present already in unstimulated renal tubular cells. Likewise, synthesis of VCAM-1 mRNA was enhanced by stimulation with TNF-alpha. TNF-stimulated endothelial cells also showed weak synthesis of VCAM-1 mRNA. The results provide further evidence that constitutive and inducible expression of cell adhesion molecules contributes to the process of allograft rejection.

Detection of humoral rejection in human cardiac allografts by assessing the capillary deposition of complement fragment C4d in endomyocardial biopsies.

BACKGROUND: There are no well-established diagnostic criteria to detect humoral rejection in organ transplantation. The value of commonly used markers in immunohistochemistry, such as C1q, C3c, IgG, IgM and fibrinogen, is questioned by some groups. Complement fragment C4d is a more stable marker of complement activation as it is covalently bound to graft capillaries. C4d has been shown to identify clinically relevant, but otherwise undetectable humoral anti-graft reactions in human kidney transplants. METHODS: Immunohistochemical techniques were used to evaluate 155 endomyocardial biopsies from 56 heart transplant recipients less than 3 months post transplantation for the presence of capillary C4d staining. In a subset of patients, C4d staining was compared with C1q, C3c, IgM and fibrin staining and was correlated with clinical outcome. RESULTS: Within 3 months 9 of 56 patients died. Five of these nonsurvivors had prominent C4d staining (p < .05), whereas C1q, C3c and IgM showed no correlation with clinical outcome. Presence of fibrin correlated with clinical outcome and C4d staining (p < .05). CONCLUSIONS: The capillary deposition of complement split product C4d in human endomyocardial biopsies was significantly associated with graft loss. Determination of fibrin deposition may yield additional information to establish a diagnosis of humoral rejection. The immunohistochemical assessment of capillary deposition of C4d and fibrin appears to be an appropriate tool for the identification of patients, who may require additional or alternative immunosuppressive therapy targeted against the humoral immune system.

Role of complement in glomerular injury.

The alternative and classical pathway of activation enable the complement system to operate in various phases of infection. Both pathways are tightly controlled by membrane-bound and circulating regulatory proteins. The immediate effects of complement activation comprise the direct lysis of target structures, the generation of proinflammatory molecules and the recruitment of circulating leukocytes. In addition, complement is involved in antigen processing and in the regulation of appropriate immune responses. All functions of complement contribute to the development of glomerular injury. The terminal membrane attack complex acts directly on resident glomerular cells, whereas the earlier components are effective via the recruitment of leukocytes. That the early components are critically involved also in the metabolism and clearance of immune complexes is of special relevance to the pathogenesis of certain glomerular diseases. The exact role of complement components produced locally by resident glomerular cells has yet to be determined.

Complement activation in human lymphoid germinal centres.

The presence of complement activation products has been studied in morphologically normal human lymphatic tissue from tonsil, spleen and lymph node. Newly established monoclonal antibodies (mAbs) with reactivity against the C4 cleavage fragments C4a, C4b, C4c and C4d were applied on cyrostat sections in the indirect immunoperoxidase staining technique. Irrespective of organ type, C4d activation product could be detected in germinal centres of all secondary lymphoid follicles. To substantiate this finding, the complete sequence of complement activation products was investigated by a series of mono- and polyclonal antibodies to the complement proteins C1, C2, C3, factor B, C5, C9 to C5b-9 neoantigens and to the regulatory complement proteins C4 binding protein (C4bp), factor I, factor H and properdin. Similar to C4d, all secondary follicles exhibited a strong staining reaction for C3d antigens restricted to germinal centres. At the same site, albeit with distinctly weaker intensity, components of the membrane attack complex (MAC) C5b-9 were found. The simultaneous deposition of C1, C4b and C4bp in certain germinal centres indicates that complement activation is induced via the classical pathway. Concomitant deposition of IgM suggests IgM-antigen complexes that have been trapped on follicular dendritic cells (FDC) during normal immune response as the most likely candidates for activators of the classical pathway. Our data demonstrate that human lymphoid germinal centres as important sites of immune regulation closely interrelate with the complete cascade of complement-activation products, including the membrane attack complex (MAC).

Limitations of pulse oral calcitriol therapy in continuous ambulatory peritoneal dialysis patients.

Calcitriol is increasingly used for therapy of secondary hyperparathyroidism in patients with end-stage renal disease. Its therapeutic efficacy, however, often has been limited by the associated increase in intestinal calcium and phosphorus absorption. Previous studies reported that these side effects could be avoided by intermittent administration of calcitriol in high doses, subsequently referred to as pulse therapy. The present study was designed to investigate pulse oral calcitriol therapy in a patient subgroup especially susceptible to the development of hypercalcemia and hyperphosphatemia under standard continuous calcitriol treatment. We examined 15 peritoneal dialysis patients with moderate degrees of hyperparathyroidism (intact parathyroid hormone [iPTH] levels, 150 to 903 pg/mL) ingesting between 1.5 and 6 g of calcium salts as the sole phosphate binders. Treatment consisted of 0.5 microgram calcitriol twice weekly. Eight of these patients had been previously converted to low calcium dialysate to tolerate the necessary doses of phosphate-binding calcium salts. During the study period, comprising 8 pretreatment weeks and 8 weeks of therapy, dialysates and doses of calcium salts were not changed, so that only calcitriol influenced the determined parameters. As expected, iPTH levels decreased rapidly in all patients (P < 0.0001). However, within 4 weeks of treatment a marked increase in calcium phosphorus products was observed (P < 0.0001). Overt hypercalcemia developed in five patients. We concluded that pulse oral calcitriol has to be carefully monitored in peritoneal dialysis patients receiving high doses of calcium salts because of the increased risk for hypercalcemia and hyperphosphatemia.

Classical pathway of complement activation in normal and diseased human glomeruli.

Monoclonal antibodies (mAb) reactive against complement components involved in the classical activation pathway were applied in an indirect immunoperoxidase technique for the histological study of normal and diseased human renal tissues. Prominent staining with antibodies against the C4d fragment was seen in all glomeruli and some renal arteriolar walls. The C4d staining was mesangial with light microscopy, whereas the subendothelial site of the glomerular basement membrane (GBM) also appeared to be positive in immunoelectron microscopy. In similar localization, albeit with distinctly weaker intensity, IgM and C4 binding protein (C4bp) were detected. In kidney biopsies from patients with various types of glomerulonephritis, C4d reactive antibodies stained the glomerular structures in a strong, diffuse or granular pattern in contrast to the more segmental distribution and weaker staining intensity in normal kidney specimens. Increased amounts of C4d, occasionally also of C4b, were paralleled in diseased kidney tissues by distinct deposits of IgM and/or IgG in the presence of C4bp. This study suggests that the C4d fragment in normal human glomeruli is indicative of a continuous, local complement activation via the classical pathway induced by the physiological deposition of IgM-containing immune complexes.

[Fasciola hepatic infection in a family: diagnosis and therapy]. / Fasciola hepatica--Infektion einer Familie. Diagnostik und Therapie.

A 39-year-old man was hospitalized because of a 5-week history of feeling very ill, with fever up to 39 degrees C and nonspecific upper abdominal pain. He looked very pale and his spleen was painful on palpation. There was a blood eosinophilia of over 50% and computed tomography demonstrated hypodense areas in the liver, suggesting a parasitic infection with liver involvement. An ELISA factor of over 100 and the finding of liver fluke eggs in bile confirmed the diagnosis of Fasciola hepatica infection, which was probably acquired by eating wild watercress when visiting in the Allgäu. A fasciola infection was also proven in his 37-year-old sister who for some time had complained of colicky right-sided upper abdominal pain, her 40-year-old husband with similar symptoms and their 10-year-old daughter. All four were successfully treated for two days with 10 mg/kg triclabendazole daily by mouth. Persons eating raw vegetables and salads of wild-growing plants are at risk of being infected with Fasciola hepatica.

[A 35-year old male patient with fever and painful swelling of the pectoral glands following a short visit to Sri Lanka]. / Ein 35jähriger Patient mit Fieber und schmerzhafter Brustdrüsenschwellung nach Aufenthalt in Sri Lanka.

HISTORY AND CLINICAL FINDINGS: One week after returning from a two-week holiday in Sri Lanka a 35-year-old man started to have recurrent bouts of fever, up to 39.2 degrees C, as well as pain over the left upper abdomen, the back of the right thorax and bilateral pain on pressure with swelling of both breasts. He went to the Tropical Institute in Munich to have malaria excluded. There signs of cholestasis were noted and sonography revealed multiple round foci in the liver. As he had lost 10 kg in 3 weeks he was admitted to a medical unit for further tests. Physical examination now showed bilateral gynaecomastia and marked pressure resistance in the upper abdomen. Proprioceptor reflexes were greatly increased but equal bilaterally. INVESTIGATIONS: Inflammatory parameters were raised (C-reactive protein 22.6 mg/dl, ferritin level 2674 micrograms/l, erythrocyte sedimentation rate 50/82 mm), there also were a leucocytosis (20,600 WBC/mm3) and a raised lactate dehydrogenase level of 613 U/l. In addition, thyroid stimulating hormone was reduced to < 0.03 microU/ml, while free thyroxine was raised to 2.7 ng/dl. The pregnancy test was positive. On quantitative analysis the human beta-chorionic gonadotropin (hCG) level was markedly raised to 193,200 mIU/ml. Abdominal and thoracic computed tomography revealed multiple round metastasis-like masses in the liver and in the lung, and a thickened cardia. Serology for malaria, amoebiasis and echinococciasis was negative, sonography of the testes and thyroid was unremarkable. Endoscopy revealed a polypoid tumour at the gastro-oesophageal junction which histologically was an undifferentiated hCG-positive choriocarcinoma. TREATMENT AND COURSE: The neoplasm at first responded with partial remission (hCG minimally 39 mIU/ml) to chemotherapy (PEI schema: cisplatin, etoposide, ifosfamide) but then progressed, also under treatment of recurrences with paclitaxel, ifosfamide and cisplatin. The patient has since received high-dosage chemotherapy with autologous stem-cell transplantation.

Increased antibody titers against mycobacterial heat-shock protein 65 in patients with vasculitis and arteriosclerosis.

Heat shock proteins (HSPs) are a group of highly conserved proteins that show extensive homology at the DNA and protein level among bacterial and mammalian species. Furthermore, bacterial HSPs induce specific cellular and humoral immune responses in mammals. Cross-reacting antibodies may therefore be induced in chronic infections. Recently, it has been claimed that patients with arteriosclerosis (AS) of the carotid arteries have significantly elevated antibody titers to mycobacterial HSPs. In this study, we extended the spectrum of vascular diseases and analyzed sera from patients with systemic vasculitis and systemic lupus erythematosus (SLE) for the presence of anti-HSP antibodies. Anti-HSP antibodies, tested in an ELISA with recombinant mycobacterial HSP 65, were significantly elevated in patients with vasculitis (n = 56; p < 0.01) and AS (n = 29; p < 0.0001), but only marginally in patients with SLE (n = 22; p > 0.05) compared to healthy controls (n = 90). These findings further support the concept of infection-induced immune reactions playing a pathogenic role in the development of both AS and vasculitis.

Two distinct Fc receptors for IgG on human peripheral T lymphocytes.

The proportion of human peripheral T lymphocytes forming rosettes with IgG-coated ox erythrocytes (ORBC) is increased after controlled hypotonic treatment. This increment may be as high as 40% of total T cells, depending on the lymphocyte donor. Such treatment is shown not to result in selective cell loss. Induced rosetting is mediated by a receptor specific for the Fc portion of human IgG (Fc gamma R). Inhibition of induced Fc gamma R activity is equally well accomplished by monomeric and by aggregated IgG of defined size. This is in contrast to the Fc gamma R detected before hypotonic treatment, which is not significantly inhibited by monomeric IgG. Capping studies established the structural independence of these two types of Fc gamma R in the lymphocyte membrane by virtue of selective cross-linking of either receptor while leaving the respective counterpart unaffected. The biochemical basis of the hypotonic effect is not yet resolved. However, the data presented suggest that hypotonicity results in removal of Fc gamma R-bound cytophilic IgG. Operationally, we propose the term induced Fc gamma R (Fc gamma R-I) for the here-described new type of receptor with high affinity for monomeric IgG.Fc gamma R that are directly assayable without hypotonic induction and not inhibited by monomeric IgG are termed free Fc gamma R (Fc gamma R-F).