Treatment of recurrent rejection in heart transplantation: cytolytic therapy or bolus steroids?

OBJECTIVES: The treatment of recurrent rejection in heart transplant recipients has been a controversial issue for many years. The intent of this retrospective study was to perform a risk-benefit analysis between treatment strategies with bolus steroids only versus anti-thymocyte globulins (RATG; 1.5 mg/kg q 4 days). METHODS: Between 1986 and 1993, 69 of 425 patients (17 male, 52 female; mean age 44 +/- 11 years) who had more than one rejection/patient per month (rej/pt per mo) in the first 3 postoperative months were defined as recurrent rejectors. RESULTS: Repetitive methylprednisolone bolus therapy (70 mg/kg q 3 days) was given in 27 patients (group M; 1.4 +/- 0.2 rej/pt per mo) and RATG therapy for one of the rejection episodes of the 42 remaining patients (group A; 1.5 +/- 0.2 rej/pt per mo). The quality of triple drug immunosuppression in the two study groups was comparable. The rejection-free interval (RFI) following RATG treatment in group A was 21.6 +/- 10 days and 22 +/- 11 in group M. In group M, 3 of 27 patients (11%) had a rejection treatment-related infection (2 bacterial; 1 viral) versus 6 of the 42 patients of group A (14.2%; bacterial 1, viral 5). During postoperative months 3-24, 0.15 +/- 0.12 rej/pat per mo were observed in group M and 0.21 +/- 0.13 rej/pat per mo in group A (n.s.). In this 21-month period cytolytic therapy for rejection was initiated in 8 of the remaining 21 patients of group M (38%) and 15 of the remaining 37 patients of group A (40.5%). The absolute survival and the individual causes of death were not affected by the type of initial treatment of recurrent rejection. The actuarial freedom of graft atherosclerosis is comparable in the two groups with 78% in group A versus 79% in group M free of graft atherosclerosis at 3 years postoperatively. CONCLUSIONS: A comparison of cytolytic therapy versus repeated applications of bolus steroids for treatment of recurrent rejection reveals no significant difference in the long-term patient outcome with respect to the incidence of future rejection episodes and survival.

Generation and functional characterization of ovine bone marrow-derived macrophages.

A method for the culturing and propagation of ovine bone marrow-derived macrophages (BMM) in vitro is described. Bone marrow cells from sterna of freshly slaughtered sheep were cultured in hydrophobic (teflon foil) bags in the presence of high serum concentrations (20% autologous serum and 20% fetal calf serum). During an 18 day culture period in the absence of added conditioned medium, and without medium change, a strong enrichment of mononuclear phagocytes was achieved. Whereas the number of macrophages increased four to fivefold during this time, granulocytes, lymphoid cells, stem cells and undifferentiated progenitor cells were reduced to less than 3% of their numbers at Day 0. This resulted in BMM populations of 94 +/- 3% purity. These cells had morphological and histochemical characteristics of differentiated macrophages, and they performed functions similar to those of non-activated, unprimed human monocyte-derived macrophages. Thus, they avidly ingested erythrocytes coated with IgG of heterologous or homologous origin. They expressed a modest level of procoagulant activity, but upon triggering with lipopolysaccharide (LPS), a marked increase in cell-associated procoagulant activity was observed. LPS triggering promoted the secretion of interleukin-1, as evidenced by measurement of murine thymocyte costimulatory activity, and transforming growth factor-beta. Using the mouse L929 cell cytotoxicity assay as an indication of tumor necrosis factor (TNF) activity, no TNF activity was detected in the same supernatants, a result possibly due to species restriction. BMM generated low levels of O2- upon triggering with phorbol 12-myristate 13-acetate (PMA). On the other hand, no O2- production was observed upon stimulation with zymosan opsonized with ovine or human serum. Using luminol-enhanced chemiluminescence (CL) as a more sensitive indicator of an oxidative burst, both PMA or zymosan were able to trigger CL, but the response was subject to partial inhibition by sodium azide, an inhibitor of myeloperoxidase. This points to non-macrophage cells contributing also to the CL response, and is consistent with the view that unprimed BMM elicit a low oxidative burst upon triggering with strong inducers of a burst. Our functional characterization now allows us to apply priming and activation protocols and to relate their effect to functional alterations.

Cytokine induction in human mononuclear cells stimulated by IgG-coated culture surfaces and by IgG for infusion

The effect of IgG on cytokine production by human mononuclear cells (MNC) was studied. Tumor necrosis factor-alpha (TNF) was determined both by bioassay and by immunoassay. Interleukin-1 (IL1) was measured by a thymocyte costimulator assay, which was shown to be completely inhibitable by polyclonal anti-IL1. Precautions were taken to avoid inadvertent exposure of the studied cells to endotoxin. In a first model, TNF and IL1 production by adherent MNC in IgG-coated cluster plates were determined. IgG induced a strong TNF response, usually leveling off after 6 hr, and was comparable in kinetics and magnitude with an LPS-induced response. The thymocyte co-stimulatory activity response was relatively weak and peaked at 6 hr. In contrast, LPS-induced co-stimulatory activity production steadily increased over 24 hr. In a second model, MNC in suspension cultures containing autologous serum were exposed to IgG for intravenous use (IgG-IV). Cells exposed to IgG-IV produced higher amounts of cytokines than control counterparts and were primed for enhanced production of cytokines upon a second, unrelated stimulus. This implies that the effect of IgG-IV on suspended MNC resembles that of surface-adsorbed IgG and raises the possibility that cytokine release is an integral part of the mechanism of action of infused IgG. Evidence is presented suggesting that both surface IgG and IgG-IV act directly on monocytes, in a Fc-dependent manner.

T-cell reprogramming through targeted CD4-coreceptor and T-cell receptor expression on maturing thymocytes by latent Circoviridae family member porcine circovirus type 2 cell infections in the thymus

Although porcine circovirus type 2 (PCV2)-associated diseases have been evaluated for known immune evasion strategies, the pathogenicity of these viruses remained concealed for decades. Surprisingly, the same viruses that cause panzootics in livestock are widespread in young, unaffected animals. Recently, evidence has emerged that circovirus-like viruses are also linked to complex diseases in humans, including children. We detected PCV2 genome-carrying cells in fetal pig thymi. To elucidate virus pathogenicity, we developed a new pig infection model by in vivo transfection of recombinant PCV2 and the immunosuppressant cofactor cyclosporine A. Using flow cytometry, immunofluorescence and fluorescence in situ hybridization, we found evidence that PCV2 dictates positive and negative selection of maturing T cells in the thymus. We show for the first time that PCV2-infected cells reside at the corticomedullary junction of the thymus. In diseased animals, we found polyclonal deletion of single positive cells (SPs) that may result from a loss of major histocompatibility complex class-II expression at the corticomedullary junction. The percentage of PCV2 antigen-presenting cells correlated with the degree of viremia and, in turn, the severity of the defect in thymocyte maturation. Moreover, the reversed T-cell receptor/CD4-coreceptor expression dichotomy on thymocytes at the CD4(+)CD8(interm) and CD4SP cell stage is viremia-dependent, resulting in a specific hypo-responsiveness of T-helper cells. We compare our results with the only other better-studied member of Circoviridae, chicken anemia virus. Our data show that PCV2 infection leads to thymocyte selection dysregulation, adding a valuable dimension to our understanding of virus pathogenicity.

The kinases NDR1/2 act downstream of the Hippo homolog MST1 to mediate both egress of thymocytes from the thymus and lymphocyte motility

The serine and threonine kinase MST1 is the mammalian homolog of Hippo. MST1 is a critical mediator of the migration, adhesion, and survival of T cells; however, these functions of MST1 are independent of signaling by its typical effectors, the kinase LATS and the transcriptional coactivator YAP. The kinase NDR1, a member of the same family of kinases as LATS, functions as a tumor suppressor by preventing T cell lymphomagenesis, which suggests that it may play a role in T cell homeostasis. We generated and characterized mice with a T cell-specific double knockout of Ndr1 and Ndr2 (Ndr DKO). Compared with control mice, Ndr DKO mice exhibited a substantial reduction in the number of naïve T cells in their secondary lymphoid organs. Mature single-positive thymocytes accumulated in the thymus in Ndr DKO mice. We also found that NDRs acted downstream of MST1 to mediate the egress of mature thymocytes from the thymus, as well as the interstitial migration of naïve T cells within popliteal lymph nodes. Together, our findings indicate that the kinases NDR1 and NDR2 function as downstream effectors of MST1 to mediate thymocyte egress and T cell migration.