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In the revived interest in crossing ABO barriers in organ transplantation renal A/B antigen expression has been correlated with donor ABO, Lewis, and secretor subtype to predict antigen expression.

Background:For half a century, immunofluorescence (IF) on frozen sections has been the gold standard for immunohistochemical evaluation of renal biopsy specimens. In routine diagnostic immunohistopathologic evaluation, traditional IF has been replaced to a large extent by immunoperoxidase (IP) methods applied to paraffin sections of formaldehyde-fixed tissue. This is caused in part by the practical disadvantages inherent in the IF method, eg, separate tissue specimen and handling, UV microscopy, fading and impermanence of the label-making archiving, and difficult later investigation. Our aim for the present study is to evaluate IP as an alternative to IF in the diagnostic assessment of renal biopsy specimens. Methods:Proteolytic antigen retrieval, antibodies effective on deparaffinized sections, a sensitive detection system (Dako EnVision HRP; Dako, Copenhagen, Denmark), and a standardized and rigorously controlled procedure were applied to a series of renal biopsy specimens (n = 81) previously classified by means of light microscopy (LM) and IF. Staining for immunoglobulin G (IgG), IgA, IgM, C1q, and C3c were recorded as positive or negative for IF and IP in paired proportions, presuming that IF was the test standard. Results:Concordant observations were 71% for all (282 of 398 observations), 82% for IgG (65 of 79 observations), and 89% for IgA (72 of 81 observations). The majority of discordant observations (74 of 116 observations) were positive by means of IP, with mesangial deposits of IgM and C1q that were not found by IF. Statistically, there was no significant difference in outcomes between IF and IP for IgG, IgA, and C3c (P0.2). In addition, IP staining allowed simultaneous evaluation of tissue by LM and therefore correlation between tissue structure and immune deposits not readily attained by IF. Conclusion:In the present study, it is documented that for the detection of IgG, IgA, and C3c, IP applied to protease-digested deparaffinized sections of formaldehyde-fixed renal tissue is, with few exceptions, equal to IF on frozen sections. The EnVision HRP method used here is several times more effective in terms of primary antibody dilution than earlier existing IP methods, and because the avidin-biotin system is not involved, very little nonspecific background staining will occur. Discordant observations (116 of 398 observations; 29%) were in the majority (91 of 116 observations) due to positive IP findings of IgM and C1q, which deserve additional investigation.

Among patients with early severe impairment of renal allograft function we have previously identified a group displaying isolated deposition of complement factor C3 in glomeruli. Here we studied the pattern of complement deposition more extensively in allograft biopsies from five patients using an immunofluorescence technique. We found a prominent deposition of C3c, C3d and C4d antigens in the glomerular capillary walls, and a positive reaction to vitronectin (S-protein), but only trace amounts of the complement factor C9 neoepitope. Clq, C4c, C3a, iC3b, factor B, properdin, immunoglobulins IgG, IgA or IgM were not found in glomeruli or in any other cortical structure. These findings indicate that most of the demonstrated glomerular C3 consists of C3b and/or C3c/C3d molecules. By immunoelectron microscopy the C3 antigen was found within the glomerular basement membrane. Our findings indicate that there is a mechanism of complement activation involving the early steps of the classical pathway, despite the lack of demonstrable immunoglobulins in the tissue. In analogy with similar reactions described recently in heart allografts, we suggest that this may be a manifestation of a humoral rejection, possibly mediated by a low titer of circulating antibodies directed against endothelial surface antigens, presumed to be the initial step leading to complement activation.

Background. Chronic allograft nephropathy (CAN) is a composite term for various types of damage to a kidney transplant. We wanted to analyse its components in relation to baseline biopsy findings, transplant function, and outcome.Methods. Among renal transplantations performed from 1985 to 1997, 156 were identified where allograft biopsies had been obtained on clinical indication 6 months after transplantation or later, baseline biopsies were available in each case and the patient's original disease was known. Time after transplantation was median 2.2 years (range 0.5–13). The biopsies were reviewed and the Banff 1997 CAN score obtained.Results. All but one late biopsy showed some CAN grade, 48% grade II, and 7.5% grade III. Acute tubulointerstitial rejection was seen in 9% but vascular rejection in only 3%. Arterial wall thickening was present in 66% of the late biopsies, correlated with donor age and its presence at baseline but also with time after transplantation. The Banff CAN score and serum creatinine level were both independent predictors of further graft survival, relative risk 0.35 (confidence interval 0.15–0.82, P=0.015) for CAN grade I vs III and 0.30 (0.14–0.67, P=0.003) for serum creatinine <170 vs >250 μmol/l. Presence of arterial wall thickening had no prognostic impact.Conclusion. The CAN grade is predictive of further graft survival independently of the serum creatinine level. Interstitial fibrosis and tubular atrophy are more prominent features of chronic graft damage than vascular rejection. Unspecific arterial wall thickening is partly dependent on baseline conditions and lacks prognostic impact in this late stage.

The Galα1-3Gal antigen (Galα) is the primary target for human natural anti-pig xenoantibodies. The presence of Galα has been shown in porcine endothelial cells (ECs) using light microscopy, whereas the expression of Galα in other cell structures in the porcine kidney is only partially characterized.

RU 486 is a 19-norsteroid which has a specific high affinity binding to the progesterone and glucocorticoid receptor. It is generally accepted that RU 486 acts as a pure progesterone antagonist almost without agonistic activity. RU 486 acts mainly directly on the target organ, such as the endometrium, but also to some extent indirectly through an effect on the pituitary gonadotrophin secretion. The effect of RU 486 during the menstrual cycle is dependent on time of treatment. Treatment before ovulation will result in a prolongation of the proliferative phase of the menstrual cycle, while treatment during the mid-and late luteal phase will invariably induce bleeding, often followed by a second bleeding episode at the expected time of menstruation. The only treatment period which does not influence the menstrual cycle is treatment immediately following ovulation. Treatment during the proliferative phase has no effect on endometrial morphology but inhibits follicular development and delays oestrogen and LH surge. Treatment on the first days following ovulation has no effect on ovarian steroid concentration, but will significantly delay endometrial development, cause a change in the concentration of oestrogen and progesterone receptor concentration, enzyme activity and production of substances thought to be progesterone dependent. The change in endometrial development is sufficient to prevent implantation. In mid-and late luteal phase, treatment with RU 486 will result in endometrial shedding in spite of normal progesterone levels. Post-ovulatory treatment with RU 486 will also significantly change uterine contractility. In early pregnancy, withdrawal of progesterone inhibition will result in uterine contractility and a significant increase in the sensitivity of the myometrium to prostaglandin. These effects are the scientific background for the combined treatment of RU 486 and prostaglandin for termination of early pregnancy. Clinical results indicate that the effect of RU 486 on the myometrial sensitivity to prostaglandins remains even during the second trimester of pregnancy. Treatment with RU 486 during pregnancy will also induce cervical ripening. The effect does not seem to be mediated through an increased endogeneous prostaglandin production, but inhibition of prostaglandin metabolism still remains a possible mode of action.

Defibrase was used for anticoagulation in dogs during extracorporeal perfusion up to 6 hours with a Travenol membrane oxygenator or a Pall filter. The anticoagulation effect was adequate. The already low fibrinogen concentration became unmeasurable, the platelet count decreased, and the fibrin(ogen) degradation products increased during the perfusion. Histological examination showed platelets and white blood cells on the membranes. Perfusion pressure and blood flow were stable during the experiments, and no bleeding, thromboembolism or other adverse side-effects were noted. Defibrase might be an alternative to heparin for anti-coagulation during extra-corporeal circulation.

ABSTRACT: The complement system is one of the important factors involved in the hyperacute rejection of xenografts. This report deals with the activation of the complement system in a clinical trial where pig kidneys were extracorporeally connected to two volunteer dialysis patients who were pretreated with plasmapheresis in order to substantially reduce anti‐pig xenoantibodies. The clinical data of the perfusion experiments and the patients humoral immune response to pig xenoantigens have been reported in detail (Xenotransplantation 1996; 3:328–339, 340–353). Three consecutive daily plasmapheresis treatments of the patients reduced the plasma complement protein (C3, C4, and C5) concentrations to 8–27% of the baseline values. The perfusion of the pig kidney connected to patient 1 was terminated at 65 min due to graft rejection and this patient was not hemodynamically affected by the experiment. The second experiment was terminated at 15 min due to an anaphylactic like reaction of the patient. In patient 1 a slight reduction of plasma C3, C4, and C5 and an increase of C5a and SC5b–9 occurred, while C3a decreased during the perfusion. Patient 2 had an increase of all complement parameters, most prominent for C4d and SC5b–9, which occurred concomitant with the appearance of the anaphylactic like side effects. In general, plasma levels of PMN elastase, IL6 and IL8 increased in both patients during the perfusion. Immunohistochemical investigation of the kidney tissues revealed deposition of human complement factors C1q, C4c, and C3c in a congruent pattern with the vasculature of the kidney in patient 1. In kidney 2 only trace amounts of C1q and C3c were found. Both kidneys were negative for properdin. Therefore, in this experimental set up with extracorporeal connection of pig kidneys to the human circulation the human complement cascade is activated mainly through the classical pathway.

The aim of this study was to determine whether specific growth factors, those shown to be involved using PCR and immunohistochemistry, are necessary for the in-vivo mechanisms of normal implantation in mice. The abdomen of pregnant female mice were opened surgically on day 4 to expose the uterine horns, which were microinjected with specific neutralising antibodies against PDGF, CSF-1, TGFb2,3, EGF and EGF-receptor. At autopsy on day 12, the numbers, positions and sizes of all implantation and resorption sites were recorded. Sham-operation controls were utilised to evaluate the implantation model. Normal female mice exhibited a mean of 6.24 implantation sites per uterine horn. Sham-operated mice exhibited a 30.8 reduction in implantation compared with normals, and saline-injected mice exhibited a 45.7 reduction. Antibody-injected horns were compared with horns injected with saline and horns injected with heat deactivated antibody. All neutralising antibodies tested resulted in significant reductions in the implantation rate and the size of the implantation site. These experiments confirm, in vivo, participation of the specific growth factors tested in the mechanisms of murine implantation, as alluded to previously by evidence from PCR, in vitrostimulatory and immunohistochemical work.

To establish an intracytoplasmic sperm injection treatment program for couples with male infertility and to determine those factors important for success.

A variety of cellular interactions is involved in the process of implantation of the mammalian embryo into the uterine tissue. Recent discoveries have demonstrated that intercellular recognition and adhesive events are governed by a class of cell surface molecules known as cell adhesion molecules (CAMs). In the present report, we have investigated the occurrence of the well-characterized cell adhesion molecule cell-CAM 105 on the surface of rat pre- and periimplantation embryos of various stages. This was carried out by indirect immunofluorescence microscopy employing affinity-purified rabbit antibodies against cell-CAM 105. The embryonal stages investigated comprised morulae, normal day-4 blastocysts, and delayed and adhesive blastocysts obtained by using the method of experimentally delayed implantation. Cell-CAM 105 was absent in the early-morula stage, but in normal day-4 blastocysts and delayed blastocysts a specific staining for cell-CAM 105 was seen on the entire surface. However, adhesive-stage blastocysts exhibited a marked polarity with staining of the polar trophoblast cells. Scanning electron microscopy of adhesive-stage blastocysts revealed that the stronger staining of the polar region was not due to a greater number of microvilli on the polar trophoblast cells. Thus, it seems as if cell-CAM 105 is lost or masked from the surface of the mural trophoblast cells of adhesive-stage rat blastocysts. Since the mural trophoblast cells are the first to adhere to the uterine luminal epithelium during the onset of implantation and subsequently invade the uterine stroma, we suggest that the apparent downregulation of cell-CAM 105 in the mural trophoblast cells might be linked to the acquisition of trophoblast invasiveness.

Classification of the underlying renal disorder in kidney transplant patients involves some uncertainties. To allow evaluation of the risk of recurrence of renal disease in the transplanted kidney and other characteristics and risks inherent to the specific renal disorder we have investigated the basis for and reevaluated the diagnosis of 1000 consecutive patients who received transplants in Göteborg 1985-1993. In the original registry, 36 of patients had been given the diagnosis chronic glomerulonephritis but the diagnosis was confirmed by biopsy in only half of them, 18 of the total population. Systemic vasculitides and hereditary disorders other than adult polycystic kidney disease which constituted 3 and 6, respectively, had frequently been overlooked. The term chronic pyelonephritis (15) covered a variety of conditions including toxic tubulointerstitial disease, but was dominated by those caused by congenital urinary tract formations. Diabetic nephropathy (21) was the consequence of Type 1 diabetes in 18 and Type 2 or other forms of diabetes in 3. The proportion of patients with unknown cause of renal failure was 20. The registry allows identification of small, distinct entities, which may be characterised as regards prerequisites for and consequences of kidney transplantation.

A pig kidney perfusion model aimed for use in immunological and physiological xenotransplantation research has been developed. Organ viability was characterised by clearance studies, functional response to hormones/diureticum and by light microscopical examination. The pig kidney was perfused in a specially designed plexiglass chamber, using a roller pump and a small membrane oxygenator (O2/CO2. 95/5). The recirculating perfusate used was autologous pig blood diluted by Tyrodes solution to a hematocrit of 30, at a total starting volume of 600-650 ml. The temperature was 37°C. It was crucial for good organ function that the nephrectomy operating time, as well as the warm (1-2 min) and cold ischemia (average 43 min) times were minimized. The average total perfusion time was 151 minutes. Physiological parameters were measured during 10-15 minute periods at average times of 40, 63, 88 and 142 minutes. The clearance values of inulin in these periods were 54 ± 13, 59 ± 15, 48 ± 23, 27 ± 5 and for PAH; 103 ± 14, 121 ± 14, 106 ± 30, 114 ± 34 ml/min/100 g tissue weight. The plasma flows were 123 ± 12, 155 ± 17, 136 ± 36 and 206 ± 57 ml/min/100 g. The injection of 0.5 μg of αANP to the perfusate resulted in a significant decrease in vascular resistance, and increase in urine production (107), as well as sodium (112) and potassium (46) excretion. Ten mg furosemide doubled the urine production and sodium excretion, while potassium excretion increased marginally. The number of leucocytes decreased by 39 during the perfusion, while the platelet count was unaffected. Light microscopy of the renal tissue after termination of the experiments revealed endothelial damage to variable extent. Loss of endothelial cells was most obvious at the level of arcuate and interlobular arteries, while the endothelium was intact in larger arteries and veins. Accumulation of polymorphonuclear granulocytes was found predominantly in the peritubular vessels, and to a lesser degree in the cortical venules. In the tubular cells, only minimal epithelial swelling and irregular cytoplasmic vacuolisation was found. Thus, a good functional viability can be maintained during 2 hours in vitroperfusion, although a decline in function as well as structural damage can be seen at the end of the experiment.

A 40-year-old man with renal failure due to membranous glomerulonephritis received a cadaveric renal transplant and immunosuppressive therapy with cyclosporine, azathioprine and steroids. Initially the transplantation was successful. 12 days after the transplantation, however, serous secretion appeared in the wound. Later, black necrosis was seen. Fungal culture showed growth of a zygomycete species, Rhizopus rhizopodiformis, with high in-vitro resistance to amphotericin B, flucytosine, fluconazole, ketoconazole and itraconazole. The MIC value for the allylamine derivative SF86-327 (Exoderil®) was 1.6 μg/ml. Microscopic examination of sections from a surgical revision showed necrosis of the fat tissue and massive hyphal invasion of the perirenal fat, which contained semi-crystalline material anisotropic as seen in polarized light and characteristically staining with rubeanic acid. These histological data indicate a lipase-induced in-vivo splitting of lipids into fatty acids. In-vitro R. rhizopodiformis showed very high extracellular lipase production. 11 days after initiation of amphotericin B therapy cultures and sections remained positive for rhizopus. Amphotericin B was therefore supplemented with Exoderil® orally, cyclosporine and steroids were maintained, and azathioprine was discontinued. The wound granulated, shrank, and healed completely in 10 weeks.