Allen J. Norin

In vitro and in vivo inhibition of complement activity by a single-chain Fv fragment recognizing human C5

Complement activation has been implicated in the pathogenesis of several human diseases. Recently, a monoclonal antibody, (N19-8) that recognizes the human complement protein C5 has been shown to effectively block the cleavage of C5 into C5a and C5b, thereby blocking terminal complement activation. In this study, a recombinant N19-8 scFv antibody fragment was constructed from the N19-8 variable regions, and produced in both mammalian and bacterial cells. The N19-8 scFv bound human C5 and was as potent as the N19-8 monoclonal antibody at inhibiting human C5b-9-mediated hemolysis of chicken erythrocytes. In contrast, the N19-8 scFv only partially retained the ability of the N19-8 monoclonal antibody to inhibit C5a generation. To investigate the ability of the N19-8 scFv to inhibit complement-mediated tissue damage, complement-dependent myocardial injury was induced in isolated mouse hearts by perfusion with Krebs-Henseleit buffer containing 6% human plasma. The perfused hearts sustained extensive deposition of human C3 and C5b-9, resulting in increased coronary artery perfusion pressure, end-diastolic pressure, and a decrease in heart rate until the hearts ceased beating approximately 10 min after addition of plasma. Hearts treated with human plasma supplemented with either the N19-8 monoclonal antibody or the N19-8 scFv did not show any detectable changes in cardiac performance for at least 1 hr following the addition of plasma. Hearts treated with human plasma alone showed extensive deposition of C3 and C5b-9, while hearts treated with human plasma containing N19-8 scFv showed extensive deposition of C3, but no detectable deposition of C5b-9. Administration of a 100 mg bolus dose of N19-8 scFv to rhesus monkeys inhibited the serum hemolytic activity by at least 50% for up to 2 hr. Pharmacokinetic analysis of N19-8 scFv serum levels suggested a two-compartment model with a T1/2 alpha of 27 min. Together, these data suggest the recombinant N19-8 scFv is a potent inhibitor of the terminal complement cascade and may have potential in vivo applications where short duration inhibition of terminal complement activity is desirable.

Randomized Controlled Trial of Strain-Specific Probiotic Formulation (Renadyl) in Dialysis Patients

Background. Primary goal of this randomized, double-blind, placebo-controlled crossover study of Renadyl in end-stage renal disease patients was to assess the safety and efficacy of Renadyl measured through improvement in quality of life or reduction in levels of known uremic toxins. Secondary goal was to investigate the effects on several biomarkers of inflammation and oxidative stress. Methods. Two 2-month treatment periods separated by 2-month washout and crossover, with physical examinations, venous blood testing, and quality of life questionnaires completed at each visit. Data were analyzed with SAS V9.2. Results. 22 subjects (79%) completed the study. Observed trends were as follows (none reaching statistical significance): decline in WBC count (−0.51 × 109/L, P = 0.057) and reductions in levels of C-reactive protein (−8.61 mg/L, P = 0.071) and total indoxyl glucuronide (−0.11 mg%, P = 0.058). No statistically significant changes were observed in other uremic toxin levels or measures of QOL. Conclusions. Renadyl appeared to be safe to administer to ESRD patients on hemodialysis. Stability in QOL assessment is an encouraging result for a patient cohort in such advanced stage of kidney disease. Efficacy could not be confirmed definitively, primarily due to small sample size and low statistical power—further studies are warranted.

CYCLOSPORIN A IN EXPERIMENTAL LUNG TRANSPLANTATION

Cyclosporin A (Cy A) has been used in combination with low-dose azathioprine (2 mg/kg/day for 14 days) or other immunosuppressives to treat 13 canine lung allograft recipients. Two of five dogs treated with Cy A and azathioprine survive at 13 and 6 months, respectively, with normal lung function and no evident rejection. The other three dogs in this group survived for over 5 months despite evidence of rejection which was reversed with methylprednisolone (50 mg/kg/day for 3 to 5 days). The addition of prophylactic corticosteroids or their substitution for azathioprine resulted in decreased survival without preventing rejection better. The lung allograft rejection that occurred with Cy A was usually later in onset and more easily reversed by corticosteroids than the lung rejection that occurred with standard immunosuppression. Cy A rejection was also sometimes qualitatively different. Perivascular mononuclear cell cuffs and a proportionally greater decrease in allograft perfusion with respect to ventilation were often more prominent than in rejection with standard immunosuppression. In some instances, decreased allograft perfusion evidenced rejection while the plain chest roentgenogram and ventilation remained normal. Except for infection, which only occurred in animals receiving prophylactic corticosteroids, there was no toxicity from Cy A. These findings indicate that this drug is the safest, most effective immunosuppressive agent yet available for use in lung transplantation.

Lectin-dependent cell-mediated cytotoxicity. A new and simple method to quantitate cytotoxic T cell activity in dogs.

Alloimmune T lymphocytes kill a wide spectrum of target cells, even in the absence of alloantigen recognition, providing that a lectin is incorporated into the assay medium. In these studies we have modified the lectin-dependent cell-mediated cytotoxicity (LDCMC) assay to evaluate the cytotoxic activity of canine T cells using a human B lymphoblastoid cell line (Raji) as the target. Canine peripheral blood mononuclear cells activated in vitro in mixed leukocyte cultures or by concan avalin A (Con A) killed 51Cr-labeled Raji target cells (up to 70% in 4 hr) when Con A was included in the assay. T cells expressed cytotoxic activity only when activated and when Con A was present in the assay. Canine T cells activated in vivo also exerted cytotoxic activity in the LDCMC assay. T cells obtained from a rejecting allografted lung by bronchoalveolar lavage displayed considerably higher cytotoxic activity than did T cells obtained from the contralateral normal lung or peripheral blood of the same dog. In contrast, T cells from the lungs and blood of normal dogs and lung-allografted dogs without rejection exerted no cytotoxic activity. We conclude that the LDCMC assay using xenogeneic Raji cells as targets is a valid measure of the cytotoxic activity of canine T cells. Its major advantages include the fact that it is simple to perform and can be used when specific target cells are not available.

Concanavalin A-dependent cell-mediated cytotoxicity in bronchoalveolar lavage fluid. Correlation with lung allograft rejection in mongrel dogs during cyclosporine dose tapering.

Although cyclosporine (CsA) is widely used as the primary agent for inhibiting the rejection of organ allografts in man, the ideal immunosuppressive regimen for utilizing this drug is still uncertain. To investigate this question, a concanavalin A (con A)-dependent cell-mediated cytotoxicity (CDCMC) assay was used to examine the development of intragraft and peripheral blood cytolytic T lymphocyte activity during CsA dose tapering. These studies were conducted in a canine single-lung transplantation model that facilitates serial examination of intragraft effector cells by bronchoalveolar lavage (BAL). A remarkable correlation of increased intragraft CDCMC and clinical evidence of lung allograft rejection was observed during CsA dose tapering in some recipients. In other recipients CDCMC remained low and evidence of rejection was not observed during drug tapering. In contrast, peripheral blood CDCMC did not correlate well with evidence of rejection. Rejection phenomena observed after termination of CsA therapy were reversed by resumption of CsA treatment but were not reversed by administration of methylprednisolone. Furthermore, the increased level of CDCMC was diminished by reinstitution of CsA therapy at the initial dosage. Following termination of CsA therapy, a prolonged period of unresponsiveness was observed in nearly two-thirds of the recipients, and 60% of these latter dogs had unlimited survival of their lung allografts (median greater than 496 days). Intragraft CDCMC remained low during the periods of unresponsiveness and increased upon onset of rejection. We conclude that measurement of intragraft CDCMC is a useful in vitro method of monitoring lung allograft rejection, and therefore provides a technique for adjusting CsA dosage schedules to achieve maximally effective immunosuppression. The use of this assay for monitoring rejection of other organ grafts requires further investigation.

Influence of desmethylimipramine on natural killer cell activity

Mounting evidence suggests that the central nervous system (CNS) and the immune system are extensively interconnected. One question that arises is whether there is cross-reactivity between psychotropic agents, which are active in the CNS, and immune system function. To explore this notion, we examined the in vitro effect of the tricyclic antidepressant, desmethylimipramine (DMI), on human natural killer (NK) cell activity in seven separate experiments. At concentrations greater than or equal to 625 ng/ml, DMI reliably inhibited NK activity. Preincubation of lymphocytes with DMI before assay did not increase the inhibitory effect. Furthermore, removal of the drug from preincubated cells immediately before assay completely eliminated the inhibitory effect. These results demonstrate that DMI reversibly inhibits NK activity at serum concentrations that are not uncommonly found in depressed patients receiving this medication.

Long‐term outcomes of dual kidney transplantation—a single center experience

Abstract:  The shortage of kidney donors has led to broadening of the acceptance criteria for deceased donor organs beyond the traditional use of young donors. We determined long‐term post‐transplant outcomes in recipients of dual expanded criteria donor kidneys (dECD, n = 44) and compared them to recipients of standard criteria donor kidneys (SCD, n = 194) and single expanded criteria donor kidneys (sECD, n = 62). We retrospectively reviewed these 300 deceased donor kidney transplants without primary non‐function (PNF) or death in the first two wk, at our center from 1996 to 2003. The three groups were similar in baseline characteristics. Kidney allograft survival and patient survival (nine yr) were similar in the three respective donor groups, SCD, sECD and dECD (60% vs. 59% vs. 64% and 82% vs. 73% vs. 73%). Acute rejection in the first three months was 23.2%, 16.1%, and 22.7% in SCD, sECD and dECD, respectively (p = 0.49) and delayed graft function was 25.2%, 31.9% and 17.1% in the three groups, respectively (p = 0.28). When PNF and death within the first two wk was included, there was no significant difference in graft survival between the three groups. In our population, recipients of dECD transplants have acceptable patient and graft survival with kidneys that would have usually been discarded.

Immunologic, morphologic, and functional evaluation of long-term-surviving beagle lung allograft recipients treated with lethal total-body irradiation, autologous bone marrow, and methotrexate

Immunologic, morphologic, and functional evaluations were performed in beagle dogs with single lung allografts surviving 3–13 years after transplantation. Immunosuppressive treatment included lethal totalbody irradiation, autologous bone marrow reconstitution, and three doses of methotrexate. Three beagle recipients with full DLA-haplotype-matched grafts and five recipients with one-haplotype-mismatched grafts were studied. Evidence of rejection—i.e., infiltrates on chest roentgenograms, hypoperfusion on radionuclide lung scans, and histopathologic changes—were absent in the matched recipients and in three of the five mismatched recipients. Two of the mismatched recipients had decreased perfusion to their allografted lungs, and open-lung biopsy specimens revealed diffuse fibrotic blood vessels with narrowed lumina but no other abnormalities. Decreased fractional blood flow to the lung allograft of the five one-haplotype-mismatched recipients was correlated (r=-0.92) with the level of donor-specific cytolytic lymphocyte activity generated in mixed lymphocyte cultures (MLC). In contrast, the level of proliferative activity in donor-specific MLC did not correlate well with graft function. These findings suggest that the mechanism of tolerance to these lung allografts (with particular regard to vascular integrity) involves attenuation of the response against major histocompatibility complex (MHC) class I alloantigen since the induction of cytolytic T lymphocytes in MLC is directed primarily against these molecules. Though all of the mismatched recipients had the ability to react against MHC class II alloantigens in vitro (as demonstrated by proliferative responses in MLC), in vivo responses to class II gene products may not occur because of the lack of expression of these molecules on long-term surviving grafts.

Bronchial anastomotic healing in canine lung allotransplants treated with cyclosporine

Bronchial anastomotic healing was evaluated in 22 long-term-surviving canine lung allotransplant recipients treated with cyclosporine as the major immunosuppressive agent. Mean survival in these dogs was over 155 days, and 4 animals survived 1-3 years. Bronchial anastomotic complications were limited to 5 cases of minimal (less than 15%) bronchostenosis. The bronchial anastomoses became somewhat edematous and friable during rejection episodes, but no clinically serious sequelae--such as hemorrhage, peribronchial abscess, or bronchial dehiscence--were observed. Gross and microscopic evaluation of the recipient and donor segments of the anastomoses revealed excellent healing, with only scattered areas of inflammatory cells. The decreased frequency and severity of rejection episodes in animals treated with cyclosporine permits early revascularization of the bronchus to take place and reduces the need for other immunosuppressive agents that may interfere with bronchial healing. Cyclosporine is an effective immunosuppressive agent for canine lung allotransplantation and allows normal bronchial anastomotic healing to occur.

Laboratory Studies in Cross-Species Lung Transplantation

Abstract. The lack of sufficient suitable human donor lungs for the many patients requiring pulmonary transplantation as life-saving therapy for end-stage lung diseases has generated extensive interest in cross-species lung transplantation. Ethical concerns and those of animal rights advocates have prompted studies of nonprimate species as potential solid organ donors for humans. This paper provides an overview of some of the laboratory studies of cross-species pulmonary transplantation performed over the past 20 years and focuses, in particular, on more recent work (from our laboratory and others) in the area of porcine-to-primate pulmonary xenotransplantation.