Anti-Thymocyte Globulin Treatment Augments 1,25-Dihydroxyvitamin D3 Serum Levels in Patients Undergoing Hematopoietic Stem Cell Transplantation.

Application of anti-thymocyte globulin (ATG) is a widely used strategy for the prevention of graft-versus-host disease (GvHD). As vitamin D3 serum levels are also discussed to affect hematopoietic stem cell transplantation (HSCT) outcome and GvHD development, we analysed a possible interplay between ATG treatment and serum levels of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 in 4 HSCT cohorts with different vitamin D3 supplementation. ATG is significantly associated with higher serum level of 1,25-dihydroxyvitamin D3 around HSCT (day -2 to 7, peri-transplant), however only in patients with adequate levels of its precursor 25-hydroxyvitamin D3. ATG exposure had no impact on overall survival in patients supplemented with high dose vitamin D3, but was associated with higher risk of one-year treatment-related mortality (log rank test p=0.041) in patients with no/low vitamin D3 supplementation. However, the difference failed to reach significance applying a Cox-model regression without and with adjustment for baseline risk factors (unadjusted P=0,058, adjusted p=0,139). To shed some light on underlying mechanisms, we investigated the impact of ATG on 1,25-Dihydroxyvitamin D3 production by human dendritic cells (DCs) in vitro. ATG increased gene expression of CYP27B1, the enzyme responsible for the conversion of 25-hydroxyvitamin D3 into 1,25-dihydroxyvitamin D3, which was accompanied by higher 1,25-dihydroxyvitamin D3 levels in ATG-treated DC culture supernatants. Our data demonstrate a cooperative effect of 25-hydroxyvitamin D3 and ATG in the regulation of 1,25-dihydroxyvitamin D3 production. This finding may be of importance in the context of HSCT, where early high levels of 1,25-dihydroxyvitamin D3 levels have been shown to be predictive for lower transplant related mortality and suggest that vitamin D3 supplementation may especially be important in patients receiving ATG for GvHD prophylaxis.

Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase.

Lymphocyte depletion with antithymocyte globulin (ATG) can be complicated by systemic coagulation activation. We found that ATG activated tissue factor procoagulant activity (TF PCA) on monocytic cells more potently than other stimuli that decrypt TF, including cell disruption, TF pathway inhibitor inhibition, and calcium ionophore treatment. Induction of TF PCA by ATG was dependent on lipid raft integrity and complement activation. We showed that ATG-mediated TF activation required complement activation until assembly of the C5b-7 membrane insertion complex, but not lytic pore formation by the membrane attack complex C5b-9. Consistently, induction of TF PCA by ATG did not require maximal phosphatidylserine membrane exposure and was not correlated with the magnitude of complement-induced lytic cell injury. Blockade of free thiols, an inhibitory monoclonal antibody to protein disulfide isomerase (PDI), and the small-molecule PDI antagonist quercetin-3-rutinoside prevented ATG-mediated TF activation, and C5 complement activation resulted in oxidation of cell surface PDI. This rapid and potent mechanism of cellular TF activation represents a novel connection between the complement system and cell surface PDI-mediated thiol-disulfide exchange. Delineation of this clinically relevant mechanism of activation of the extrinsic coagulation pathway during immunosuppressive therapy with ATG may have broader implications for vascular thrombosis associated with inflammatory disorders.

Analysis of the prognostic factors of very severe aplastic anemia treated with Chinese Kidney-invigorating drugs in combination with anti-lymphocyte globulin or anti-thymocyte globulin.

OBJECTIVE: To explore the prognostic factors for very severe aplastic anemia (VSAA) patients treated mainly with Chinese Kidney (Shen)-invigorating drugs (CKID) combined with anti-lymphocyte globulin (ALG) or anti-thymocyte globulin (ATG). METHODS: Twenty-seven VSAA patients were treated with CSID+ALG/ATG therapy in conjunction with cyclosporine A, androgen, hemopoietic growth factor, etc. The relationship of the effectiveness and some factors (age of patients, course of illness, blood and bone marrow figures, etc.) were analyzed. RESULTS: In the 25 evaluated VSAA patients who had been followed up for over 1 year, 9 patients (36.0%) were basically cured, 5 (20.0%) remitted, 6 (24.0%) were markedly improved, and 5 (20.0%) were treated in vain, with the total effective rate of treatment being 80.0% (20/25). Better clinical therapeutic effects were shown in patients newly diagnosed with VSAA, of male sex (P=0.037), >20 years old (P=0.045), with an illness course [Symbol: see text] month (P=0.048), with peripheral neutrophil count >0.1 × 10(9)/L (P=0.023), and with reticulocyte count >10 × 10(9)/L (P=0.002). Platelet count (P=0.620) and bone marrow lymphocyte percentage (P=0.736) showed no correlation with the therapeutic effectiveness. Multi-factor analysis by the Kaplan-Meier procedure on the factors influencing survival showed that rather longer survival times occurred in patients > 20 years old, with peripheral neutrophil count [Symbol: see text] 0.1 × 10(9)/L, reticulocyte count [Symbol: see text]10 × 10(9)/L, and platelet count > 10 × 10(9)/L (all P=0.0001). Bone marrow lymphocyte percentage and the initiation time of ALG/ATG application (from onset of the illness) showed no significant influence on patients' survival time (P=0.085 and P=0.935, respectively). CONCLUSIONS: CSKD+ALG/ATG therapy for treatment of VSAA could enhance the current clinical therapeutic effects and elevate patients' survival rate. Conditions including male sex, age >20 years, illness course [Symbol: see text]1 month, neutrophil count >0.1 × 10(9)/L, and reticulocyte count >10 × 10(9)/L are the likely effective indices for predicting favorable therapeutic effectiveness in newly diagnosed VSAA patients.

T-cell-dependent immunobiological activity of a desmuramyl analog of muramyl dipeptide, adamantylamide dipeptide (AdDP), and D-isoglutamine.

The present experiments demonstrate that, similar to immunomodulatory muramyl dipeptide, its desmuramyl analog adamantylamide dipeptide is able to induce mild and fully reversible paw edema in mice. This effect is an immune-related phenomenon depending on the activation of T-cell/macrophage interactions and on production of prostaglandins. Possible involvement of certain immunoregulatory/inflammatory cytokines (e.g. IL-1, IL-2) has been suggested. The most probable intrinsic moiety of the adamantylamide dipeptide molecule responsible for triggering the edema formation is obviously D-isoglutamine.

The importance of non-human primates for preclinical testing of immunosuppressive monoclonal antibodies.

Monoclonal antibodies (MAb) specific for lymphocyte markers can be considered as very specific immunomodulating drugs for treatment of allograft rejection and autoimmune diseases. Although the selection of potentially useful specificities of MAb can be made in rodents, human specific MAb can only be evaluated in man or a closely related species in which these human specific MAb are equally reactive. Because of the restricted reactivity of human specific MAb, non-human primates are the only available species for efficacy and safety studies. This article illustrates the usefulness of such studies in rhesus monkeys and chimpanzees for the testing of T cell specific MAb and other MAb interfering with the immune response in transplantation and autoimmunity.

Propagation and characterization of lymphocytes from rejecting human cardiac allografts.

In recent years heart transplantation has become an increasingly common therapeutic measure for cases of heart failure not amenable to other forms of management. Despite major advances, graft rejection continues to be the main cause of transplant failure. To expand our knowledge of the rejection process, we have grown lymphocytes from biopsy specimens obtained from potentially rejecting allografts. The growth of the lymphocytes requires the presence of interleukin-2 and was shown to correlate well with histologic criteria in identifying rejection (p less than 0.003, chi-square analysis). Of 125 biopsy specimens, 61 (49%) became positive for lymphocyte growth within 30 days. Of the biopsy specimens that were positive on culture, 54 (88%) were histologically diagnosed as rejecting, and seven (11%) were histologically nonrejecting. Of the 64 specimens that were negative on culture, 28 (44%) were histologically negative for rejection, and 36 (56%) were positive for some grades of rejection. Of these culture negative, microscopically positive specimens, however, 31 (86%) were associated with mild (grade 3) or equivocal (grade 2) rejection. Discrepancies between culture and histology appeared to be related to degree of rejection, quantity of sample, and sampling variation. Phenotypic identification of cultured cells showed that the suppressor/cytotoxic phenotype was the predominant cell type in all cultures (mean +/- standard error = 83% +/- 3% of cells). Antihuman mature inducer/helper T cell (OKT4+) T-lymphocytes in culture were associated with biopsy specimens obtained immediately before onset or at the beginning of rejection. Preliminary studies testing in vitro sensitivities of lymphocytes to antithymocyte globulin (ATG) and other control immunosuppressants not believed to function by direct cytolysis (methylprednisolone, cyclosporine, azathioprine) were undertaken. These yielded reproducible dose-response curves, and the drug concentration producing 50% cytotoxicity (LD50) was calculated for each drug-culture combination. The mean LD50 for ATG was in the range of in vivo therapeutic concentrations, whereas LD50 values for control drugs were threefold to twelvefold higher than therapeutic concentrations. Resistance to the effects of ATG showed a tendency to correlate with prolongation of rejection; however, a similar tendency was noted for control drugs, suggesting the possibility of a nonspecific mechanism of resistance to the effects of ATG. The technique of interleukin-2 lymphocyte culture from rejecting allografts promises to further our understanding of mechanisms of graft rejection.

Studies on mouse auto-reactive cells. II. Cytotoxicity exerted by mouse lymphocytes against syngeneic erythrocytes.

In mice, an in vitro cell-mediated cytotoxicity is directed towards syngeneic erythrocytes used as targets. This phenomenon occurs in lymphoid organs of normal, non-immunized mice. Its intensity is greater in the thymus than in spleen or lymph nodes. The study of the nature of the cell responsible for this spontaneous syngeneic cytotoxicity ruled out the role of macrophages. The involvement of T cells in this phenomenon is assessed by its disappearance after the lymphoid cells had been treated with anti-theta serum plus complement, by its disappearance from lymphoid organs of mice previously treated by hydrocortisone, and by its decrease in the presence of synthetic thymic factor. Moreover, spontaneous syngeneic cytotoxicity is lost when lymphoid cells are depleted at autologous rosettes by centrifugation on a Ficoll-Hypaque gradient after rosette formation. Cytotoxic lymphocytes might belong to the population of auto-rosettes previously characterized as a population of immature T cells.

Lipopolysaccharide-induced autoantibody response. II. Age-related change in plaque-forming cell response to bromelain-treated syngeneic erythrocytes.

In spleen of normal mice, there are relatively large numbers of plaque-forming cells (PFC) against syngeneic or allogeneic red blood cells treated with bromelain (Br. MRBC) and the numbers of PFC remarkably increase by the injection of bacterial lipopolysaccharide (LPS). In this report, age-related change in the anti-Br. MRBC PFC response was studied in vivo and in vitro. It was revealed that mean numbers of splenic anti-Br. MRBC PFC increased in both untreated and LPS-injected mice with age (3--14 months). The increase in anti-Br.MRBC PFC detected in LPS-stimulated spleen was dependent on the numbers of PFC in untreated spleen, but not related to the numbers of anti-TNP PFC nor the degree of 3H-thymidine incorporation assessed in in vitro culture. These results imply that enhanced anti-Br.MRBC PFC response was not merely due to polyclonal activation of B lymphocytes. Reasons for increased PFC numbers were analyzed in special references to suppressor activities. The experimental results were obtained suggesting that suppressors were not involved in anti-Br.MRBC PFC response. There were also no marked differences in the acceptability of suppressive signals between young and older spleen cells. It was concluded that hightened anti-Br.MRBC PFC response in older mice might be due to spontaneous increase in the size of Br.MRBC-reactive clone.

Regulatory mechanism of autoantibody production in mice to bromelin-treated isologous red blood cells.

In the spleen of mice immunized with bromelin treated rat red blood cells (RBC), the number of PFC against bromelin treated isologous RBC increased, although immunization with non-treated rat RBC or bromelin treated isologous RBC gave no increase in number of these PFC. This immune response was found to be T-independent and the PFC developed are exclusively of direct or Ig-M type. In the secondary immune response, production of these PFC was depressed rather than increased. This can be thought of as one of the defence mechanisms against overproduction of autoantibodies in confrontation to foreign antigens cross-reactive with self antigenic determinants. This depressed secondary immune response can be adoptively transferred by primed spleen cells but no active suppressor effect was found. We concluded that clonal elimination by exhaustive differentiation may be operative in this depressed secondary immune response.

The in vitro suppression of spontaneous erythrocyte autoimmune responses with lymphocytes activated with concanavalin A.

When normal mouse spleen cells are cultured in vitro, large numbers of cells develop that produce antibody toward antigens found on bromelain-treated mouse erythrocytes (BrMRBC). The in vitro culture also generates T cells that mediate DTH toward these antigens. We have suggested that under in vivo conditions, suppressor T cells maintain these immune responses at a low level but that this suppression wanes when the cells are cultured in vitro. The present study examines the effect of concanavalin A (Con A) on the in vitro development of humoral and cell-mediated immunity to Br-MRBC. Mitogenic concentrations of Con A prevented the development of both the PFC and TDTH responses toward BrMRBC. The Con A-induced suppression was due to the induction of suppressor T cells; thus the addition of Con A-activated cells to fresh spleen cell cultures prevented the development of both the PFC and TDTH response against BrMRBC.

Induction of hapten-specific cell-mediated immunity in rhesus monkeys treated with adsorbent purified antilymphocyte antibody.

Rhesus monkeys were treated with sufficient amounts of rabbit antilymphocyte sera and immunoadsorbent purified antilymphocyte antibody to retain full-thickness skin grafts 23 and 29 days respectively. During this immunosuppressed period hapten-specific cell-mediated immunity was induced in six of seven rhesus monkeys immunized with p-azophenylarsono-N-acetyltyrosine (R-tyr) in complete Freund's adjuvant. These observations suggest that in rhesus monkeys different populations of T cells may be responsible for cutaneous sensitization to antigen and rejection of skin grafts.

Life-span investigations for carcinogenicity of some immune-stimulating, immunodepressive and neurotropic substances in Sprague-Dawley-rats.

In a long-term trial in a total of 858 Sprague-Dawley-rats of both sexes the possible carcinogenicity of 12 substances was investigated. Complete Freunds' adjuvant which is among the immune-stimulating substances was found to decrease significantly the lifetime of the animals; whereas an increase in tumor incidences was not observed. Bacille Calmette-Guérin (BCG), human albumin, and vitamin-A-acid were neither carcinogenic, nor had they an influence on the survival times. Rabbit-antirat lymphocytic serum which is a well-known immunodepressive agent caused a significant decrease in survival times, it was not found to be carcinogenic. The known carcinogenic properties of cyclophosphamide were verified. The applied dose of this compound significantly reduced the mean survival times of the animals. Hydrocortisone and amethopterin were found to have no carcinogenic effects. Atropine, pilocarpine, nicotine, and phenyl-ethyl-barbituric acid (phenobarbital) which are known to have neurotropic effects were tested. Nicotine and phenobarbital were found to diminish the mean survival times. None of these neurotropic substances was found to be carcinogenic.

In vitro evaluation of inhibitors of platelet release and aggregation.

In order to determine which drugs would be most effective as inhibitors of platelet release and aggregation, in vitro release reactions and platelet aggregometry were used to evaluate aspirin, dipyridamole, sulfinpyrazone, flurbiprofen, low molecular weight dextran (dextran 40), prostaglandin E1 (PGE1), apyrase, and adenosine. Adenosine diphosphate-induced aggregation was most effectively inhibited by PGF1, sulfinpyrazone, and dipyridamole. The latter had to be used in large doses. Collagen and epinephrine-induced release and aggregation were inhibited by the same drugs as well as by aspirin and apyrase. Antihynocyte globulin (ATG)-induced release and aggregation could only be partially blocked by these agents. In vitro studies suggest that sulfinpyrazone is one of the most effective of platelet inhibitors currently available for clinical testing.

Enhancement and retardation of spontaneous reticulum cell neoplasm development in SJL/J mice.

The enhancement or delay in the appearance of spontaneous reticulum cell neoplasms (RCN-B) in SJL/J mice was tested. Immunosuppressive treatment with antithymocyte serum and prolonged antigenic stimulation by repeated injections of sheep erythrocytes, leukemogenic cell-free centrifugates, and multichain synthetic polypeptides had no effect on the incidence or latency of spontaneous tumor appearance. Treatment with mineral oil or incomplete Freund adjuvant markedly enhanced tumor appearance (though the abundance of plasma cells induced by these treatments did not evoke the development of plasmacytomas, nor did it affect the incidence of serum paraproteinemia among mice with spontaneous tumors). Treatment with cortisone acetate, however, markedly retarded spontaneous tumor development. Tumors occurring early due to mineral oil and adjuvant treatment all had the characteristics of "RCN-B plasma cell type," and tumors occurring late due to cortisone treatment were all of the "RCN-B reticulum cell type." The possible involvement of the stem cell pool size in the enhancement or delay of spontaneous tumor development was discussed.

The allografted parathyroid gland: evaluation of function in the immunosuppressed host.

Parathyroid autografts were performed in 19 random bred mongrels and parathyroid allografts were exchanged between seven random bred mongrels. Three pairs of siblings from a beagle colony were immunosuppressed and parathyroid allografts exchanged between them. The parathyroid autografts were successful, while parathyroid allografts failed on all occasions in the non-immunosuppressed host. In immunosuppressed animals, however, the allografts were successful in five of six dogs. The success of parathyroid transplantation was determined by the following observations: 1) The experimental animal, having grafted parathyroid tissue as the only source of hormone, maintained a normal or near normal serum calcium concentration. 2) Following removal of the parathyroid graft, there was an immediate fall in the serum calcium concentration associated with tetany and/or death. 3) Histological study of the grafted gland revealed normal architecture, and 4) Radioimmunoassay of extracted grafts revealed moderate to large quantities of parathyroid hormone.