A simple, rapid, reliable reverse haemolytic plaque assay and positive quality control test for routine use.

A simple, rapid, reliable protein A reverse haemolytic plaque assay is described. Monolayers of protein A-coupled sheep red blood cells, in liquid medium, are formed in shallow 15 mm diameter chambers of the type commercially available for leucocyte migration inhibition assays. No agarose is necessary and the chambers are quick and easy to use, economical of reagents, and of a constant size and volume. Results compare favourably with those obtained using Cunningham chambers. The assay is ideal for clinical studies in which large numbers of samples are assayed daily. A positive quality control test for the protein A reverse hemolytic plaque assays is described. Spleen cells are stimulated with pokeweed mitogen to give high numbers of secreting cells. The cells are harvested on day 6 of culture and stored in aliquots at -70 degrees C. When thawed and tested in the protein A assay, these secreting cells form a sensitive and reproducible monitor of the day-to-day performance of the assay. Variation between operators and between batches of reagents may also be checked if desired, with little additional time, effort or expense.

Specific unresponsiveness to skin allografts in mice. VI. Graft survival in mice pretreated with blood.

Very small amounts (0.5 to 2 microliters) of H-2-incompatible blood given 16 days before skin grafting led to the induction of a long-lived unresponsiveness when the mice were given a short postoperative course of alternating doses of procarbazine hydrochloride and antilymphocyte serum. This unresponsiveness, which was specific for the blood donor strain, was wholly attributable to the white cell moiety, plasma and red blood cells having proved to be ineffective. The optimal dose range of white blood cells was fairly narrow (7 x 10(3) to 1.4 x 10(4)). Of the various attempts made to demonstrate that the blood inoculum modified the response of normal mice, only direct skin grafting gave a positive result in that the survival of allogeneic skin grafts was curtailed. Mixtures of blood from several mouse strains injected into CBA recipients induced long-term unresponsiveness to skin grafts of the blood donor strains. However, although it was possible to create unresponsiveness to DBA/2, C57Bl, or strain A skin grafts in CBA mice pretreated with blood from a closed colony outbred strain (TO), this pretreatment had no beneficial effect to induce unresponsiveness to TO skin grafts in TO recipients. It is argued that these findings are no necessarily inconsistent with the observation that, in inbred animals, the induction of unresponsiveness was strain specific.

Failure to increase the in vivo immunosuppressive activity of antilymphocyte globulin by conjugation with melphalan.

An attempt was made to increase the in vivo immunosuppressive powers of antilymphocyte globulin (ALG) by conjugating it with malphalan (MEL), an alkylating agent, via an inert intermediate carrier (polyglutamic acid). Careful controls to distinguish between increased activity attributable to the conjugate per se, as opposed to synergy between the components of the complex, were included. Conjugation did not destroy the alkylating properties of the drug nor the cytotoxic activity of the antibody. The effect of MEL-ALG complexes on skin allograft survival in both inbred and outbred strains were appraised. In neither system did the immunosuppressive powers of the conjugate exceed those of ALG alone, regardless of the dose used. We conclude that alkylating drugs are not suitable for this particular purpose.

Prospective evaluation of pretransplant blood transfusions in cadaver kidney recipients.

BACKGROUND: A beneficial effect of pretransplant transfusions on graft survival was demonstrated in the early 1970s. In the mid-1980s, however, retrospective studies showed that transfusions had lost their graft-protective effect in the cyclosporine era. During the last 10 years, deliberate transfusion pretreatment of transplant patients has been discontinued. METHODS: Within a collaborative project of 14 transplant centers, prospective recipients of cadaver kidney grafts were randomized to receive either three pretransplant transfusions or transplants without transfusions. RESULTS; The graft survival rate was significantly higher in the 205 transfusion recipients than in the 218 patients who did not receive transfusions (at 1 year: 90+/-2% vs. 82+/-3%, P=0.020; at 5 years: 79+/-3% vs. 70+/-4%, P=0.025). Cox regression analysis showed that this effect was independent of age, gender, underlying disease, prophylaxis with antilymphocyte antibodies, and preformed lymphocytotoxins. CONCLUSIONS; Transfusion pretreatment improves the outcome of cadaver kidney transplants even with the use of modern immunosuppressive regimens.

Evaluation of the flow cytometric crossmatch. Preliminary results of a multicenter study.

The flow cytometric crossmatch is a technique that is increasingly being used by clinical transplant laboratories. In this multicenter study by the British Society for Histocompatibility and Immunogenetics Flow Cytometry Group, a series of crossmatches were carried out to determine whether different centers obtained same results when performing the same crossmatch. There was greater than 80% agreement among participating laboratories on the results of 35/54 tests. There was no clear agreement in the remaining 20 cases. Quantitative analysis, estimating the number of cell-bound fluorescein molecules, demonstrated that differences in the criteria used by each center to define a positive crossmatch were responsible for some discordant results. When applied, definition of positivity based on the molecules of fluorescein increased concordance from 57.5% to 81.4%.l. These results suggest that a criterion for the interpretation of results based on quantitative analysis of bound antibody may be more reliable than methods in current routine use.

The use of paramagnetic beads for the detection of major histocompatibility complex class I and class II antigens.

Specific immunoadsorbents were prepared using paramagnetic particles (Dynabeads), and their ability to immunoprecipitate major histocompatibility complex (MHC) Class I and Class II antigens compared with conventional protein A Sepharose immunoadsorbents. Lysates of lymphoblastoid cells provided the antigen source which were visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Dynabeads were found to be as effective as protein A Sepharose immunoadsorbents at immunoprecipitating MHC Class I and Class II antigens, but had a much lower nonspecific binding capacity resulting in fewer interference bands and lower backgrounds.

Combined interleukin-2 and okt3 therapy in the treatment of metastatic melanoma.

Objective tumour responses have been documented in patients with malignant melanoma following therapy with recombinant interleukin-2 (rIL-2). Recent attempts to reduce toxicity and retain therapeutic efficacy of rIL-2 have involved low-dose subcutaneous rIL-2 administration and the exploration of synergy with other agents. The anti-CD3 monoclonal antibody OKT3 (Cilag), is immunosuppressive when given at high doses. However, at low doses anti-CD3 mAb are vigorously immunostimulatory in vitro and have been associated with tumour regression in animal models. We have explored the immunomodulatory properties and toxicity of combining intravenous OKT3 with subcutaneous low-dose rIL-2 in a patient with malignant melanoma.

The effect of low-dose okt3 monoclonal-antibody therapy on k562 cytotoxicity in human cancer-patients.

Low concentrations of monoclonal antibodies (mAb) specific for the CD3 component of the T cell receptor complex (TcR) may be both immunosuppressive or potent activators of T cell function in vitro depending upon the experimental situation. The anti-CD3 mAb OKT3 is imnmunosuppressive at high doses (5 mg/day for 10 to 14 days) in transplant patients. We undertook a preliminary study to investigate the effect of a single intravenous injection of 50 mu g to 0.5 mu g of OKT3 mAb in cancer patients. OKT3 modulated cytotoxicity with minimal clinical toxicity at low doses and may be of use in cancer immunotherapy protocols.

The treatment of colorectal hepatic metastases by continuous local infusion of interleukin-2.

Patients with hepatic metastases from colorectal cancer appear to be resistant to most forms of therapy. Recent reports suggest synergism between systemically administered recombinant interleukin-2 (rIL-2) and 5-fluorouracil (5-FU) with responses documented in the treatment of advanced colonic tumours albeit with significant toxicity. Local continuous infusion of rIL-2 into selected sites may reduce toxicity and increase efficacy. We have assessed the feasibility of continuously infusing rIL-2 into the region of a hepatic metastasis in 3 patients via a catheter placed within the liver under ultrasound guidance in a regimen including systemic rIL-2 and 5FU.

Anti-CD3 and anti-cd16 monoclonal-antibody in-vitro enhancement of NK activity in pbl from gastrointestinal-tract cancer-patients.

Natural killer (NK) cell cytotoxicity was assessed using peripheral blood mononuclear cells from 103 healthy volunteers and 51 cancer patients. Peripheral blood cells were assessed by flow cytometry, and cytotoxicity in a standard 4-hour Cr-51-release assay using K562 cells as targets. Anti-CD3 and anti-CD16 mAb significantly enhanced cytoxicity in vitro. NK cell numbers correlated with levels of cytotoxicity. Patients with liver metastases had significantly more CD3+ lymphocytes coexpressing NK markers than patients without liver involvement. These CD3+ NK cells may also mediate cytotoxicity. Enhancement of cellular cytotoxicity by anti-CD3 and anti-CD16 may be of use in cancer immunotherapy protocols.

The application of flow cytometry to histocompatibility testing.

Flow cytometry is a powerful technique that enables the sensitive and quantitative detection of both cellular antigens and bound biological moieties. This article reviews how flow cytometry is increasingly being used as histocompatibility laboratories for the analysis of antibody specificity and HLA antigen expression. A basic description of flow cytometry principles and standardisation is given, together with an outline of clinical application in the areas of pre-transplant cross-matching, antibody screening, post-transplant antibody monitoring and HLA-B27 detection. It is concluded that flow cytometry is a useful multi-parametric analytical tool, yielding clinical benefit especially in the identification of patients at risk of early transplant rejection.

The relevance of anti-A549 antibody in adult renal transplantation.

Sera from 228 individuals were examined for cytotoxic antibodies against the cultured lung epithelial cell line A549. All samples were screened by the conventional complement mediated cytotoxicity assay. The pattern of the anti-A549 reactivity in normal volunteers (n = 12) was 42% negative, 30% weak positive and 25% strong positive. The overall incidence of anti-A549 antibody in chronic renal failure patients (n = 84) was 7% weak positive and 7% strong positive. In 132 patients with a functioning renal allograft the frequency of anti-A549 reactivity increased (19% weak positive, 27% strong positive) and was comparable to that found in normal controls. The presence of anti-A549 antibody did not correlate with panel antibody allosensitization, cytomegalovirus status, age, mode of dialysis, rejection episodes, or with subsequent graft survival. We conclude that in adult renal transplantation the presence of anti-A549 antibody is not a contra-indication to transplantation.

The importance of E-selectin as a marker for renal transplant rejection.

Vascular endothelial cells express membrane bound adhesion molecules which play a direct role in the localization and subsequent movement of leucocytes from the blood into sites of inflammation. E-Selectin is a cytokine induced adhesion molecule, known to be expressed by endothelial cells in inflammatory conditions, which binds to various leucocyte subpopulations. In a prospective study we have investigated the expression and distribution of E-selectin on renal allograft needle biopsies taken from 16 pretransplant kidneys and 119 post-transplant kidneys. Post-transplant biopsies were taken at times of graft dysfunction and at times of normal graft function. Formal histology was also performed and assessed independently. E-Selectin was found predominantly on the intertubular endothelium and on the endothelium of larger vessels. E-Selectin was present, at low intensity, in some pretransplant biopsies and also some post-transplant biopsies which were reported histologically as normal. In post-transplant biopsies taken for dysfunction E-selectin was present in the majority of cases. Expression was strong in biopsies showing acute cellular rejection and this was associated with a CD4 positive cellular infiltrate. Biopsies showing other causes of dysfunction, in particular acute tubular necrosis, also were E-selectin and CD4 positive with lower intensity than those with acute cellular rejection. These results suggest that E-selectin is a good marker for endothelial activation in renal transplant biopsies. Its presence in histologically apparently normal biopsies suggests that its in vivo kinetics may differ from previously reported in vitro kinetics. E-Selectin may be a potential target for therapeutic intervention.

Lack of correlation of soluble E-selectin level with renal transplant rejection.

E-Selectin is a 115-kDa cell surface glycoprotein transiently expressed on vascular endothelium in response to interleukin-1 and tumour necrosis factor-alpha with a peak in expression at four hours. Its distribution in transplant biopsies has been associated with inflammatory events such as allograft rejection. Recently, a soluble isoform of E-selectin has been detected in the culture medium of cytokine activated endothelial cells by an ELISA method. In this study soluble E-selectin levels in renal allograft recipients were compared with the incidence of rejection, acute tubular necrosis (ATN), cyclosporin A (CyA) toxicity, and use of orthoclone OKT3 (muromonab-CD3) to establish whether early endothelial activation and inflammatory damage could be detected. The mean soluble E-selectin level in normal volunteers was 89 ng/ml serum compared to 120 ng/ml for a group of chronic renal failure patients. Soluble E-selectin levels declined upon transplantation but this was not significant, nor was the difference in samples from patients experiencing rejection, ATN or CyA toxicity. A dramatic and sustained rise in soluble E-selectin levels was found within 24 hours of the first dose of OKT3 treatment. This study shows that soluble E-selectin does not provide early unequivocal indication of pathological sequelae in renal transplantation, although extensive endothelial activation can be demonstrated with OKT3 treatment.

A protein-binding assay for measurement of biotin in physiological fluids.

A sensitive and convenient protein-binding assay for biotin in physiological fluids is described. The method is based upon the binding of an iodinated biotin conjugate by avidin followed by separation of bound and free conjugate by charcoal absorption. Adult plasma biotin levels averaged 1.26 pmol/ml, a value comparable to that determined by microbiological assays for biotin.

Anti-CD3 enhancement of cellular cytotoxicity in cancer patients treated with interleukin-2.

Anti-CD3 monoclonal antibody was found to enhance non-MHC restricted cellular cytotoxicity in vitro in the peripheral blood mononuclear cells from normal healthy individuals. This effect was dose dependent (maximal at 0.11 micrograms/ml) and was complete within 30 minutes. Peripheral blood mononuclear cells from 4 out of 8 cancer patients treated with Interleukin-2 were deficient in their ability to respond to anti-CD3 in vitro. In contrast, peripheral blood mononuclear cells from cancer patients who subsequently displayed a tumour response to Interleukin-2, were able to respond to anti-CD3 by enhanced non-MHC restricted cellular cytotoxicity. This study demonstrates the importance of CD3+ lymphocytes in the generation of antitumour activity following in vivo Interleukin-2 administration and the ability of CD3+ve lymphocytes to respond to anti-CD3 in vitro may indicate those patients who would benefit from Interleukin-2 therapy.

Changes in biochemical laboratory investigation in patients treated with constant infusion recombinant interleukin-2.

Eight patients with metastatic hypernephroma were treated with constant infusion recombinant Interleukin-2 (rIL-2), changes in renal and hepatic function and protein levels were monitored during 2 cycles of treatment. The rIL-2 infusion caused a reversible fall in ures and a non-reversible rise in creatinine. Liver function tests (bilirubin, ALT, ALP and GGT) rose during rIL-2 treatment and had returned to pretreatment levels 3 weeks after the last day of rIL-2. There was also a reversible fall in serum protein levels during rIL-2 infusion. Although constant infusion rIL-2 ameliorated much of the severe toxicities usually seen with high-dose bolus rIL-2, the non-reversible rise in serum creatinine levels is not a previously reported feature of rIL-2 therapy.

Evaluation of the safety of recombinant interleukin-2 (rIL-2) and rIL-2 plus 5-fluorouracil (5-FU) in the adjuvant treatment of gastric cancer patients.

Eight patients received either recombinant Interleukin-2 (rIL-2) alone or rIL-2 plus 5-Fluorouracil (5-FU) by constant infusion after undergoing potentially curative surgery for gastric cancer. rIL-2, given at a dose of 18 x 10(6) IU/m2/24 hours, was safely tolerated and only two episodes of WHO grade 3 toxicities occurred, both of which promptly responded to treatment and temporary interruptions of rIL-2 infusions. 5-FU infusions given at 12.5 mg/kg/24 hours did not alter the rebound lymphocytosis seen after completion of rIL-2 infusions. We conclude that the administration of rIL-2 and rIL-2 plus 5-FU to cancer patients recovering from major surgery is safe and well tolerated.

Role of human leukocyte antigen, donor-specific antibodies, and their impact in renal transplantation.

INTRODUCTION: The clinical significance of the presence of antibody against human leukocyte antigen (HLAb) and donor-specific antibodies (DSAb) prior to renal transplantation remains unclear. This study was done to assess the impact of HLAb and DSAb on graft function, rejection episodes, and graft survival in renal transplantation. METHODS: The Luminex (Luminex, Austin, Texas, United States) is a solid-phase assay using micro-spheres and it is more sensitive at detecting human leukocyte antigen (HLA) antibodies than conventional tests. This retrospective analysis involved 141 consecutive renal transplant recipients between May 2007 and 2009 and with a minimum of 2 years of follow-up. RESULTS: Luminex was positive for HLA class I in 35 and negative in 106; similarly class II positivity was noted in 23 and negative in 118. The DSAb were positive in 33 and negative in 108 recipients. The HLA class I, class II, and DSA-positive groups showed no difference in renal function assessed by estimated glomerular filtration rate (eGFR) at 2 years (52 ± 29 vs 52 ± 22; 56 ± 29 vs 51 ± 29; 48 ± 18 vs 53 ± 19; P = not significant [NS]). But rejection episodes at 1 year were significantly high in HLA class I and DSAb-positive group (17/35 vs 27/106; P = .019 and 16/33 vs 29/108; P = .035). The rejection episodes in the HLA class II-positive group did not show any difference when compared with the negative group (9/23 vs 40/118; P = .63). Graft survival was not affected by positivity to any of these antibodies at 2 years. CONCLUSION: Having HLA class I, class II, and DSAb does not have any influence on early and intermediate graft function. The HLA class I and DSAb positivity increases rejection episodes within 1 year in renal transplantation. Graft survival was not affected by class I, class II, and DSAb at 2 years.