Successful conversion from conventional immunosuppression to anti-CD154 monoclonal antibody costimulatory molecule blockade in rhesus renal allograft recipients.

BACKGROUND: Several conventional forms of immunosuppression have been shown to antagonize the efficacy of anti-CD154 monoclonal antibody- (mAb) based costimulatory molecule blockade immunotherapy. Our objective was to determine if allograft recipients treated with a conventional immunosuppressive regimen could be sequentially converted to anti-CD154 mAb monotherapy without compromising graft survival. METHODS: Outbred juvenile rhesus monkeys underwent renal allotransplantation from MHC-disparate donors. After a 60-day course of triple therapy immunosuppression with steroids, cyclosporine, and mycophenolate mofetil, monkeys were treated with: (1) cessation of all immunosuppression (control); (2) seven monthly doses of 20 mg/kg hu5C8 (maintenance), or; (3) 20 mg/kg hu5C8 on posttransplant days 60, 61, 64, 71, 79, and 88 followed by five monthly doses (induction+maintenance). Graft rejection was defined by elevation in serum creatinine>1.5 mg/dl combined with histologic evidence of rejection. RESULTS: Graft survival for the three groups were as follows: group 1 (control): 70, 75, >279 days; group 2 (maintenance): 83, 349, >293 days, and; group 3 (induction+maintenance): 355, >377, >314 days. Acute rejection developing in two of four monkeys after treatment with conventional immunosuppression was successfully reversed with intensive hu5C8 monotherapy. CONCLUSIONS: Renal allograft recipients can be successfully converted to CD154 blockade monotherapy after 60 days of conventional immunosuppression. An induction phase of anti-CD154 mAb appears to be necessary for optimal conversion. Therefore, although concurrent administration of conventional immunosuppressive agents including steroids and calcineurin inhibitors has been shown to inhibit the efficacy of CD154 blockade, sequential conversion from these agents to CD154 blockade appears to be effective.     

Renal transplantation in sensitized recipients with positive luminex and negative CDC (complement‐dependent cytotoxicity) crossmatches

Recently, Luminex‐crossmatch (LumXm) was introduced. The aim of this study was to evaluate clinical outcomes in sensitized recipients with a positive Luminex‐crossmatch (LumXm (+)) and a negative complement‐dependent cytotoxicity crossmatch (CDCXm (?)) after renal transplantation. Fifty‐five renal transplant recipients with a CDCXm (?) and PRA class I or II ≥20% were enrolled in this study between February 2008 and December 2010 at Severance Hospital. Eighteen patients displayed LumXm (+) defined as LumXm positive class I or II and 37 patients displayed LumXm (?). Mean duration of follow‐up was 18.9 ± 8.3 months. During this period, no patient death or graft loss occurred. The incidence of biopsy‐proven or clinically presumed rejection was higher in the LumXm (+) group (n = 12, 66.7%) than in the LumXm (?) group (n = 6, 18.2%) (P = 0.001). All biopsy‐proven acute rejections (n = 12) were diagnosed as acute cellular rejection. No significant difference in mean serum creatinine level or eGFR was observed between the groups at 18 months post‐transplantation. The short‐term outcome of renal transplantation in sensitized patients with a LumXm (+) and a CDCXm (?) may be considered to be acceptable. However, patients with a LumXm (+) have a substantially higher immunological risk for the development of acute cellular rejection.     

The molecular mechanism of apoptosis induced by xenogeneic cytotoxicity

Abstract: In order to clarify the role of natural killer (NK) cells in delayed xenograft rejection (DXR) of discordant xenotransplantation, we used in vitro xenogeneic combination of human NK cells and pig kidney target cells (PK13, and investigated the mechanism of xenogeneic cytotoxicity caused by human NK cells. In the presence of decomplemented human serum or human IgG, freshly isolated human peripheral blood lymphocytes (PBLs) caused both membrane (⁵¹Cr release) and DNA (³H release) damage on PK15. In contrast, only membrane damage was detected in the presence of normal human serum. To clarify the participation of perforin/granzymes-cell mediated cytotoxicity (P/G-CMC), when EGTA or concanamycin B (CMB) was added to the cytotoxicity assays, both cytotoxicities were completely inhibited by these drugs in a dose-dependent manner. In terms of the involvement of Fas/FasL-based cytotoxicity (F-CMC), while the cytotoxicity assays were performed in the presence of antagonistic anti-human FasL mAb, this antibody was not able to block the cytotoxicity. From these results, it is concluded that xenogeneic cytotoxicity is due to NK cell dependent ADCC (antibody-dependent cell-mediated cytotoxicity), and their effector mechanism can cause apoptosis on target cells via P/G-CMC.     

The autoimmune response to vimentin after renal transplantation in nonhuman primates is immunosuppression dependent

BACKGROUND: Chronic allograft nephropathy (CAN) is a common late complication of kidney transplantation. Antibodies to both human leukocyte antigen and nonhuman leukocyte antigen antigens have been implicated in the development of this condition. Here we investigated the presence of antivimentin antibodies in nonhuman primate recipients of kidney allografts as a possible predictor of CAN and the effects of immunosuppression. METHODS: Thirty seven rhesus monkeys received a kidney allograft to study the potency of several different immunosuppressive regimens (conventional immunosuppression, n=19, vs. costimulatory blockade, n=18). Monkeys were tested for antivimentin antibody by enzyme-linked immunosorbent assay and for anti-donor antibody by staining donor spleen cells with recipient serum. The appearance of antibodies was correlated with the graft pathology in biopsy and necropsy material. RESULTS: Antivimentin antibodies were found in 31 of 37 animals, whereas only 15 of 32 animals made anti-donor antibodies. Conventional immunosuppression did not prevent antivimentin antibody formation. Costimulation blockade, in particular blocking CD40 and CD86, significantly delayed or prevented antivimentin antibody formation, but did not prevent CAN. Antivimentin antibodies were not significantly associated with development of CAN. CONCLUSIONS: We postulate that vimentin acts as an autoantigen after renal transplantation; it elicits an autoimmune response that is not regulated by cyclosporine. This autoimmune response may be part of the complex immunologic events occurring posttransplantation and may contribute to the development of CAN, but cannot be considered as a major cause of CAN because this condition also develops without antivimentin antibodies.     

Sequential actions of immune effector cells induced by viral activation of dendritic cells to eliminate murine neuroblastoma

Purpose

In preclinical trails, we reported the antitumor effect of dendritic cells activated with Sendai virus (rSeV/DC) combined with γ-irradiation against neuroblastoma. However, what kind of effector cells for the combined therapy were used to show the antitumor effect was unclear. In this study, we performed radiation and rSeV/DC therapy in vivo and examined the effector cells involved.

Myeloid-derived suppressor cell (MDSC) key genes analysis in rat anti-CD28-induced immune tolerance kidney transplantation

BackgroundIn the field of transplantation, inducing immune tolerance in recipients is of great importance. Blocking co-stimulatory molecule using anti-CD28 antibody could induce tolerance in a rat kidney transplantation model. Myeloid-derived suppressor cells (MDSCs) reveals strong immune suppressive abilities in kidney transplantation. Here we analyzed key genes of MDSCs leading to transplant tolerance in this model.MethodsMicroarray data of rat gene expression profiles under accession number {"type":"entrez-geo","attrs":{"text":"GSE28545","term_id":"28545"}}GSE28545 in the Gene Expression Omnibus (GEO) database were analyzed. Running the LIMMA package in R language, the differentially expressed genes (DEGs) were found. Enrichment analysis of the DEGs was conducted in the Database for Annotation, Visualization and Integrated Discovery (DAVID) database to explore gene ontology (GO) annotation and their Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Their protein-protein interactions (PPIs) were provided by STRING database and was visualized in Cytoscape. Hub genes were carried out by CytoHubba.ResultsThree hundred and thirty-eight DEGs were exported, including 27 upregulated and 311 downregulated genes. The functions and KEGG pathways of the DEGs were assessed and the PPI network was constructed based on the string interactions of the DEGs. The network was visualized in Cytoscape; the entire PPI network consisted of 192 nodes and 469 edges. Zap70, Cdc42, Stat1, Stat4, Ccl5 and Cxcr3 were among the hub genes.ConclusionsThese key genes, corresponding proteins and their functions may provide valuable background for both basic and clinical research and could be the direction of future studies in immune tolerance, especially those examining immunocyte-induced tolerance.     

Drug immunosuppression therapy for adult heart transplantation. Part 1: immune response to allograft and mechanism of action of immunosuppressants

In the early days of transplantation, immunosuppression therapy was rather broad and nonspecific, mainly using high-dose corticosteroids and azathioprine. Thereafter we progressively narrowed the target of immunosuppressive strategy starting with polyclonal antibodies. The introduction of cyclosporine, OKT3, and tacrolimus further narrowed the target on the T-cell pathways. More recently mycophenolate mofetil progressively took the place of azathioprine with its higher lymphocyte specificity and sirolimus and interleukin-2 receptor antibodies were introduced. In this field in constant movement the aim is to find a drug or a regimen that provides optimal immunosuppression therapy with minimal side effects, in other words to find the right balance between overimmunosuppression and underimmunosuppression therapy. This review is divided into two parts. The first part will provide a basic understanding of the immunologic response to allograft and explain how conventional and recently introduced immunosuppressive agents work. The second part will describe the clinical application of immunosuppressive drugs to provide practical information for those in charge of heart transplant recipients.     

Carbon monoxide (co) downregulates hepatic inducible nitric oxide synthase (inos) by a mitogen-activated protein kinase (mapk) dependent mechanism

Background: Low dose CO is non-toxic and has been shown to possess potent anti-inflammatory properties. We hypothesize that CO would decrease cytokine-stimulated iNOS induction in rat hepatocytes. Methods: Primary hepatocytes from Sprague-Dawley rats were exposed to combinations of cytokines (TNFα, IL-1, IFγ) in the presence or absence of CO pre- and co- treatment at 250 ppm. Protein was harvested for Western blotting at 0, 1 and 6 hours after treatment. Greiss reaction was used to quantify NO production and crystal violet staining was used to measure cell viability. Experiments were performed 2-3 times each and t-test was used to assess significance between groups. Results: CO exposure at 250 ppm did not decrease hepatocyte viability measured by crystal violet staining at 24 hours. Pretreatment with CO resulted in a statistically significant decrease in cytokine mixture (TNFα 500 U/ml, IL-1 200 U/ml, IFγ 100 U/ml) stimulated hepatocyte NO production measured by Greiss reaction (37.8 ± 7.4 to 20.8 ± 5.7 μM, p < 0.05). Western blot 6 hours after stimulation resulted in a similar decrease in iNOS protein in the CO exposed hepatocytes. Pretreatment with CO up-regulated mitogen activated protein kinase phosphatase-1 (MKP-1) protein and decreased TNFα induced c-Jun N-terminal kinase (JNK) activation measured by Western blot. Conclusions: CO pretreatment decreased cytokine stimulated hepatocyte iNOS protein synthesis and NO production. A possible mechanism for this effect is the up-regulation of MKP-1 protein and subsequent decrease in cytokine stimulated JNK activation. The study provides a new mechanism for the anti-inflammatory effects of CO and validates the importance of JNK activation in pro-inflammatory signaling.     

The effect of the immunomodulator RU 41,740 (biostim) on the specific and nonspecific immunosuppression induced by thermal injury or protein deprivation

We studied the effect of RU 41,740 (biostim), a primary macrophage stimulator, in the following two immunosuppressive conditions in rats: (1) a 30% full-thickness burn that leads to significant decreases in cell-mediated (delayed-type hypersensitivity [DTH]), humoral (anti-tetanus toxoid antibody production), and nonspecific immunity (Staphylococcus aureus 502a skin abscess) and (2) protein malnutrition using a 2% protein diet for eight weeks. Biostim administered by gastric intubation at dosages of 10 and 50 mg/kg of body weight for five days following thermal trauma did not prevent the DTH suppression induced by the thermal injury, but resulted in a significant dose-related increase in the amount of anti-tetanus toxoid antibody produced. In the malnourished rats given biostim at dosages of 10 and 50 mg/kg of body weight for seven days, there was a significant dose-related increase in the DTH response in the presence of continued protein depletion in these animals, with a modest but significant reduction in the S aureus 502a skin abscess at three days. Antibody production was not affected with this model. Biostim partially overcomes the suppression in humoral immunity following thermal injury, but not cell-mediated or nonspecific immunity. On the other hand, biostim augments both the cell-mediated and nonspecific immune suppression induced by prolonged protein deprivation but does not affect humoral immunity.     

The use of immune RNA (i-RNA) to prevent and treat burn infection

The immune RNA was extracted from the spleens and livers of immunizing sheep with Ps. aeruginosa, E. coli and S. aureus. After animal trial, the i-RNA activity and its antibacterial protective effect has been demonstrated. 95 cases of burn infection treated with i-RNA plus antibiotics were compared with a concurrent control group treated with antibiotics alone. Clinical observation confirmed that an obvious effect on the prevention and treatment of burn infection with gram negative bacteria was manifested. Among them, 59 cases with burn area 15-49% II degrees, III degrees of total body surface, the ratio of incidence of gram negative septicemia and wound pyemia between i-RNA and control group was 1.69%: 22%, p less than 0.01. In another 36 cases with burn area over 50% body surface II degrees, III degrees treated with i-RNA the gram negative septicemia or wound sepsis were found in 3 cases (morbidity 8.3%). And one died overall mortality was 2.8%. Whereas, the morbidity and mortality were 51.4% and 45.9% respectively in 37 control cases. The ratio of morbidity between i-RNA and control group was 8.3%: 51.4%, p less than 0.01, and that of mortality was 2.8%: 45.9%, p less than 0.01.     

Glomerular deposition of mannose-binding lectin (MBL) indicates a novel mechanism of complement activation in IgA nephropathy

Background. IgA nephropathy (IgA-N) is considered the most common glomerular disease in the world and leads to renal failure in a substantial number of patients. Although many studies have looked at the pathogenesis of the disease, many points need to be clarified, including the mechanism of complement activation. Recent studies have shown that mannose-binding lectin (MBL or mannose binding protein, MBP) initiates activation of the complement cascade (lectin pathway) utilizing two types of MBP-associated serine protease, namely MASP-1 and MASP-2. The present study was undertaken to elucidate whether the lectin pathway was involved in the pathogenic mechanism of IgA-N. Methods. Forty-five renal biopsy cases with IgA-N, 35 cases with other forms of glomerulonephritis (GN), and normal kidney tissues were collected and an immunohistochemical study was performed using monoclonal antibodies against MBL and MASP-1. Furthermore, clinicopathological and serological features were also analysed in the patients with IgA-N. Results. Glomerular deposition of MBL, which was accompanied by MASP-1, was detected in 11 of 45 (24.4%) cases with IgA-N, while it was detected in only one case with other forms of GN. The deposited MBL/MASP-1 was observed to associate with C3b/C3c and C5b-9 but not with IgG, IgM, Clq, C4c, or properdin. Compared with MBL/MASP-1 negative cases with IgA-N, the positive cases with IgA-N were young and the renal biopsies had been performed at an early stage of the disease. No significant correlation was found between glomerular deposition of MBL/MASP-1 and proteinuria, haematuria, creatinine clearance, and serum levels of IgA, C3, or C4 at the time of renal biopsy. There were also no significant differences between MBL/MASP-1 positive cases and negative cases in the plasma levels of circulating immune complexes or soluble C5b-9. Conclusion. The lectin pathway of complement activation, which is initiated by the MBL/MASPs complex, evidently contributes to the development of glomerular injury in a significant number of cases with IgA-N. In addition, these findings will add insight to the pathogenesis of IgA-N, including its relation to infection, since MBL plays a crucial role in the host defence against various pathogens.     

Dexamethasone increases protein degradation in cultured myotubes through a Calcium-calmodulin Kinase II (CaMK-II) dependent mechanism

Introduction. Previous reports suggest that glucocorticoids are important mediators of muscle protein breakdown in various muscle-wasting conditions, including sepsis. The mechanisms by which glucocorticoids induce muscle proteolysis are poorly understood. Previous studies provided evidence that glucocorticoids may influence cellular calcium homeostasis, and, in other experiments, calcium increased muscle protein breakdown. We tested the hypothesis that glucocorticoid-induced protein degradation in muscle cells is at least in part mediated by calcium and the calcium-activated enzyme CaMK-II. Methods. Cultured L6 myotubes, a rat skeletal muscle cell line, were treated with 1 μM dexamethasone for 24 h. Protein degradation was assessed by measuring the release of TCA-soluble ³H-tyrosine from proteins that had been pre-labeled with the amino acid. The role of calcium in dexamethasone-induced protein degradation was tested by treating the cells with the intracellular calcium chelator BAPTA-AM (20 μM). The involvement of CaMK-II was tested by using the CaMK-II inhibitor KN-93 (20 μM). Results. Treatment of L6 myotubes with dexamethasone resulted in a 12% increase in protein degradation, confirming previous reports from this laboratory. This effect of dexamethasone was blocked by BAPTA-AM and KN-93. KN-93 also inhibited basal protein degradation, in the absence of dexamethasone (P < 0.001 by ANOVA). Conclusions. This study provides the first evidence that calcium and the calcium-activated enzyme CaMK-II may be involved in glucocorticoid-induced muscle protein degradation.

TABLE—ABSTRACT 15.

	− DEX	+ DEX	% Change with DEX
Control	14.2 ± 0.2	15.9 ± 0.2	+ 12.0%∗
BAPTA-AM	13.6 ± 0.2	13.5 ± 0.1	Not significant
KN-93	10.0 ± 0.2	10.4 ± 0.2	Not significant

Note. Protein degradation (% 24 h) in myotubes treated with dexamethasone, BAPTA-AM,and/or KN-93. Results are means ± SEM with n = 6 per group.

∗

P < 0.001 versus no dex by ANOVA.

Full-size table

     

Reactive oxygen species independent cytotoxicity induced by radiocontrast agents in tubular cells (LLC-PK1 and MDCK)

PURPOSE: Radiocontrast agents (RAs) cause renal tubular damage by hemodynamic imbalance, which could cause hypoxic stimulus and direct cytotoxicity. However, reactive oxygen species (ROS) could be an important factor in RAs' direct cytotoxicity. This study investigated the involvement of ROS in deleterious effects produced by RAs on normoxic and hypoxic renal tubular cells. MATERIALS AND METHODS: LLC-PK1 and MDCK were exposed to diatrizoate and ioxaglate in normoxic and hypoxic conditions. Apoptotic and necrotic cell death were assessed by acridine orange/ethidium bromide and annexin V methods. Hydrogen peroxide, superoxide anion, and malondialdehyde levels were analyzed by, respectively, 2',7'-dichlorofluorescein, luminal, and thiobarbituric acid. Antioxidant agents were used to prevent cellular RAs damage. RESULTS: Diatrizoate and ioxaglate decreased cellular viability in both cells, and this effect was enhanced by hypoxic conditions. Diatrizoate induced more injury than ioxaglate to both cell lines. LLC-PK1 underwent necrosis, while MDCK cells underwent apoptosis when exposed to diatrizoate. These results could not be attributed to an increase in osmolality. RAs did not increase hydrogen peroxide, superoxide anion or malondialdehyde levels in both cells. Additionally, N-acetyl-L-cysteine (NAC), ascorbic acid, alpha-tocopherol, glutathione, beta-carotene, allopurinol, cimetidine, and citric acid did not protect cells against RAs damage. Surprising, NAC increased the cellular damage induced by ioxaglate in the both cell lines. CONCLUSION: The present study shows that RAs induce damage in cultured tubular cells, especially in hypoxic conditions. ROS were not involved in the observed RAs' cytotoxicity, and NAC increased ioxaglate-induced tubular damage.     

The roles of macrophage (M phi) and PGE-2 in postburn immunosuppression.

Postburn immunosuppression in mice with full skin thickness burns was assessed after Con A stimulation of splenic lymphocytes. The immunosuppression was detected within 24 h of injury and was maximal 3-7 days later. Such lymphocyte proliferation was enhanced by depletion of adherent cells (M phi) in burned mice and could be depressed in normal mice by injection of these adherent cells from burned mice. These results suggest the presence of M phi s with a suppressor function. No difference in spontaneous blastogenic transformation (SBT) of spleen cells between the burned and normal mice was found, but after removal of the M phi s the SBT of spleen cells of burned mice became significantly elevated. Hence both M phi and lymphocytes in mouse spleen cells were activated after severe thermal injury.     

Effects of serum immunosuppressive factors on the cytotoxicity of lymphokine-activated killer (LAK) cells induced by recombinant interleukin 2(R-IL2)

Fundamental studies were performed on adoptive immunotherapy, especially on effects on lymphocytic cytotoxic activity, of nonspecific immunosuppressive factors (ferritin, IAP, AFP) and of serum factors obtained from gastric cancer patients, and possible intervention of suppressor T cells in serum immunosuppressive activity on the cytotoxicity was also examined. The following results were obtained. 1) Cytotoxicity of LAK cells induced by culturing normal peripheral blood lymphocytes (PBL) with R-IL2 in the medium containing normal AB-type sera, was higher than that of PBL. 2) Effect of nonspecific immunosuppressive factors on cytotoxicity of LAK cells was lower than that on cytotoxicity of PBL. 3) Cytotoxicity of PBL was inhibited in a relatively specific fashion by sera from patients of the cancers which were of identical histological types with the target tumor cells, while that of LAK cells was hardly inhibited by patients' sera. 4) Cytotoxicity of Leu 15-PBL was inhibited by nonspecific immunosuppressive factors and also by cancer patients' sera in a relatively specific fashion in relation to histology. 5) Cytotoxicity of Leu 15-LAK cells was hardly inhibited by serum nonspecific and specific immunosuppressive factors. The above results showed that serum immunosuppressive factors might act on PBL cytotoxicity without intervention of suppressor T cells, and that LAK cells were hardly inhibited by such immunosuppressive factors. All these results suggested usefulness of adoptive immunotherapy with LAK.