Truncating titin mutations are associated with a mild and treatable form of dilated cardiomyopathy.

AIMS: Truncating titin mutations (tTTN) occur in 25% of dilated cardiomyopathy (DCM) cases, but the phenotype and severity of disease they cause have not yet been systematically studied. We studied whether tTTN variants are associated with a clinically distinguishable form of DCM. METHODS AND RESULTS: We compared clinical data on DCM probands and relatives with a tTTN mutation (n = 45, n = 73), LMNA mutation (n = 28, n = 29), and probands who tested negative for both genes [idiopathic DCM (iDCM); n = 60]. Median follow-up was at least 2.5 years in each group. TTN subjects presented with DCM at higher age than LMNA subjects (probands 47.9 vs. 40.4 years, P = 0.004; relatives 59.8 vs. 47.0 years, P = 0.01), less often developed LVEF <35% [probands hazard ratio (HR) 0.38, P = 0.002], had higher age of death (probands 70.4 vs. 59.4 years, P < 0.001; relatives 74.1 vs. 58.4 years, P = 0.008), and had better composite outcome (malignant ventricular arrhythmia, heart transplantation, or death; probands HR 0.09, P < 0.001; relatives HR 0.21, P = 0.02) than LMNA subjects and iDCM subjects (HR 0.36, P = 0.07). An LVEF increase of at least 10% occurred in 46.9% of TTN subjects after initiation of standard heart failure treatment, while this only occurred in 6.5% of LMNA subjects (P < 0.001) and 18.5% of iDCM subjects (P = 0.02). This was confirmed in families with co-segregation, in which the 10% point LVEF increase occurred in 55.6% of subjects (P = 0.003 vs. LMNA, P = 0.079 vs. iDCM). CONCLUSIONS: This study shows that tTTN-associated DCM is less severe at presentation and more amenable to standard therapy than LMNA mutation-induced DCM or iDCM.

Anti-tumor activity of human IgG1 anti-gp75 TA99 mAb against B16F10 melanoma in human FcgammaRI transgenic mice.

Patients suffering from advanced melanoma have a very poor prognosis. Despite recent advances in the understanding of oncogenic mechanisms and therapeutic interventions, the median survival of patients with metastatic disease is less than 12 months. Immunotherapy of melanoma has been intensely investigated and holds great promises. Tyrosinase-related protein-1 or gp75 (TYRP-1/gp75) antigen is a melanosomal polypeptide. It is the most abundant glycoprotein synthesized by pigmented melanocytes and melanomas. It is specific for melanocytes and both primary and metastatic melanomas. In mice, administration of the mouse mAb anti-gp75 TA99 prevents outgrowth of B16F10 melanoma metastases. The activity of TA99 is dependent on the presence and activity of the IgG specific, Fc receptors. TA99 cross-reacts with human gp75, and is currently being used for diagnosis of patients. Here, we sequenced mIgG2a TA99 and found that the locus harboring the endogenous light chain of the fusion partner in the TA99 hybridoma cells is not inactivated, resulting in the production of a mixed pool of mAbs that mitigates binding to gp75. Since human IgG1 (hIgG1) is the most frequently used mAb format in clinical studies, we produced a recombinant hIgG1 TA99 molecule. Whereas it is known that hIgG1 can functionally interact with mouse Fc receptors, we found that hIgG1 TA99 did not exhibit in vivo activity against B16F10 melanoma in wild type C57BL/6 mice. However, results obtained in this study demonstrated anti-tumor activity of hIgG1 TA99 in FcγRIIB knockout mice and in human FcγRI transgenic mice. These results emphasize the need for testing hIgG mAb in mice with functional human FcγRs.

HER2 monoclonal antibodies that do not interfere with receptor heterodimerization-mediated signaling induce effective internalization and represent valuable components for rational antibody-drug conjugate design.

The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (Kadcyla(TM)). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA') fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA', was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA'-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.

Yield of molecular and clinical testing for arrhythmia syndromes: report of 15 years' experience.

BACKGROUND: Sudden cardiac death is often caused by inherited arrhythmia syndromes, particularly if it occurs at a young age. In 1996, we started a cardiogenetics clinic aimed at diagnosing such syndromes and providing timely (often presymptomatic) treatment to families in which such syndromes or sudden cardiac death existed. We studied the yield of DNA testing for these syndromes using a candidate-gene approach over our 15 years of experience. METHODS AND RESULTS: We analyzed the yield of DNA testing. In subanalyses, we studied differences in the yield of DNA testing over time, between probands with isolated or familial cases and between probands with or without clear disease-specific clinical characteristics. In cases of sudden unexplained death (antemortem or postmortem analysis of the deceased not performed or providing no diagnosis), we analyzed the yield of cardiological investigations. Among 7021 individuals who were counseled, 6944 from 2298 different families (aged 41 ± 19 years; 49% male) were analyzed. In 702 families (31%), a possible disease-causing mutation was detected. Most mutations were found in families with long-QT syndrome (47%) or hypertrophic cardiomyopathy (46%). Cascade screening revealed 1539 mutation-positive subjects. The mutation detection rate decreased over time, in part because probands with a less severe phenotype were studied, and was significantly higher in familial than in isolated cases. We counseled 372 families after sudden unexplained death; in 29% of them (n=108), an inherited arrhythmia syndrome was diagnosed. CONCLUSIONS: The proportion of disease-causing mutations found decreased over time, in part because probands with a less severe phenotype were studied. Systematic screening of families identified many (often presymptomatic) mutation-positive subjects.

Ebstein anomaly associated with left ventricular noncompaction: an autosomal dominant condition that can be caused by mutations in MYH7.

Left ventricular noncompaction (LVNC) is a relatively common genetic cardiomyopathy, characterized by prominent trabeculations with deep intertrabecular recesses in mainly the left ventricle. Although LVNC often occurs in an isolated entity, it may also be present in various types of congenital heart disease (CHD). The most prevalent CHD in LVNC is Ebstein anomaly, which is a rare form of CHD characterized by apical displacement and partial fusion of the septal and posterior leaflet of the tricuspid valve with the ventricular septum. Several reports of sporadic as well as familial cases of Ebstein anomaly associated with LVNC have been reported. Recent studies identified mutations in the MYH7 gene, encoding the sarcomeric ß-myosin heavy chain protein, in patients harboring this specific phenotype. Here, we will review the association between Ebstein anomaly, LVNC and mutations in MYH7, which seems to represent a subtype of Ebstein anomaly with autosomal dominant inheritance and variable penetrance.

The in vivo mechanism of action of CD20 monoclonal antibodies depends on local tumor burden.

BACKGROUND: CD20 monoclonal antibodies are widely used in clinical practice. Antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and direct cell death have been suggested to be important effector functions for CD20 antibodies. However, their specific contributions to the in vivo mechanism of action of CD20 immunotherapy have not been well defined. DESIGN AND METHODS: Here we studied the in vivo mechanism of action of type I (rituximab and ofatumumab) and type II (HuMab-11B8) CD20 antibodies in a peritoneal, syngeneic, mouse model with EL4-CD20 cells using low and high tumor burden. RESULTS: Interestingly, we observed striking differences in the in vivo mechanism of action of CD20 antibodies dependent on tumor load. In conditions of low tumor burden, complement was sufficient for tumor killing both for type I and type II CD20 antibodies. In contrast, in conditions of high tumor burden, activating FcγR (specifically FcγRIII), active complement and complement receptor 3 were all essential for tumor killing. Our data suggest that complement-enhanced antibody-dependent cellular cytotoxicity may critically affect tumor killing by CD20 antibodies in vivo. The type II CD20 antibody 11B8, which is a poor inducer of complement activation, was ineffective against high tumor burden. CONCLUSIONS: Tumor burden affects the in vivo mechanism of action of CD20 antibodies. Low tumor load can be eliminated by complement alone, whereas elimination of high tumor load requires multiple effector mechanisms.

c-Jun activating binding protein 1 binds to the IgA receptor and modulates protein levels of FcalphaRI and FcRgamma-chain.

The receptor for IgA, FcalphaRI or CD89, is expressed on myeloid cells and can trigger phagocytosis, tumor cell lysis, and release of inflammatory mediators. These functions critically depend on the associated FcR gamma-chain; however, some biological functions, like receptor internalization, are solely mediated by FcalphaRI alpha-chain. Little is known as to how FcalphaRI regulates these processes and the FcalphaRI intracellular domain does not contain recognized signalling motifs. We searched for associating proteins and identified c-Jun activating binding protein 1 (JAB1) as a binding partner specifically for FcalphaRI. We found increased FcalphaRI surface expression after ectopic expression of JAB1 as well as diminished protein levels of total FcR gamma-chain levels after JAB1 knock-down. These data functionally link JAB1 with controlling protein expression levels of FcalphaRI-FcR gamma-chain protein complex.

In vivo cytotoxicity of type I CD20 antibodies critically depends on Fc receptor ITAM signaling.

Antibody-Fc receptor (FcR) interactions play an important role in the mechanism of action of most therapeutic antibodies against cancer. Effector cell activation through FcR triggering may induce tumor cell killing via antibody-dependent cellular cytotoxicity (ADCC). Reciprocally, FcR cross-linking of antibody may lead to the induction of apoptotic signaling in tumor cells. The relative importance of these bisecting pathways to in vivo antibody activity is unknown. To unravel these roles, we developed a novel mouse model with normal FcR expression but in which FcR signaling was inactivated by mutation of the associated gamma-chain. Transgenic mice showed similar immune complex binding compared with wild-type mice. In contrast, ADCC of cells expressing frequently used cancer targets, such as CD20, epidermal growth factor receptor, Her2, and gp75, was abrogated. Using the therapeutic CD20 antibodies ofatumumab and rituximab, we show that FcR cross-linking of antibody-antigen immune complexes in the absence of gamma-chain signaling is insufficient for their therapeutic activity in vivo. ADCC therefore represents an essential mechanism of action for immunotherapy of lymphoid tumors.

Inside-out regulation of Fc alpha RI (CD89) depends on PP2A.

To achieve a correct cellular immune response toward pathogens, interaction between FcR and their ligands must be regulated. The Fc receptor for IgA, FcalphaRI, is pivotal for the inflammatory responses against IgA-opsonized pathogens. Cytokine-induced inside-out signaling through the intracellular FcalphaRI tail is important for FcalphaRI-IgA binding. However, the underlying molecular mechanism governing this process is not well understood. In this study, we report that PP2A can act as a molecular switch in FcalphaRI activation. PP2A binds to the intracellular tail of FcalphaRI and, upon cytokine stimulation, PP2A becomes activated. Subsequently, FcalphaRI is dephosphorylated on intracellular Serine 263, which we could link to receptor activation. PP2A inhibition, in contrast, decreased FcalphaRI ligand binding capacity in transfected cells but also in eosinophils and monocytes. Interestingly, PP2A activity was found crucial for IgA-mediated binding and phagocytosis of Neisseria meningitidis. The present findings demonstrate PP2A involvement as a molecular mechanism for FcalphaRI ligand binding regulation, a key step in initiating an immune response.

Signaling through mutants of the IgA receptor CD89 and consequences for Fc receptor gamma-chain interaction.

The prototypic receptor for IgA (FcalphaRI, CD89) is expressed on myeloid cells and can trigger phagocytosis, tumor cell lysis, and release of inflammatory mediators. The functions of FcalphaRI and activating receptors for IgG (FcgammaRI and FcgammaRIII) are dependent on the FcR gamma-chain dimer. This study increases our understanding of the molecular basis of the FcalphaRI-FcR gamma-chain transmembrane interaction, which is distinct from that of other activatory FcRs. FcalphaRI is unique in its interaction with the common FcR gamma-chain, because it is based on a positively charged residue at position 209, which associates with a negatively charged amino acid of FcR gamma-chain. We explored the importance of the position of this positive charge within human FcalphaRI for FcR gamma-chain association and FcalphaRI functioning with the use of site-directed mutagenesis. In an FcalphaRI R209L/A213H mutant, which represents a vertical relocation of the positive charge, proximal and distal FcR gamma-chain-dependent functions, such as calcium flux, MAPK phosphorylation, and IL-2 release, were similar to those of wild-type FcalphaRI. A lateral transfer of the positive charge, however, completely abrogated FcR gamma-chain-dependent functions in an FcalphaRI R209L/M210R mutant. By coimmunoprecipitation, we have demonstrated the loss of a physical interaction between FcR gamma-chain and FcalphaRI M210R mutant, thus explaining the loss of FcR gamma-chain-dependent functions. In conclusion, not only the presence of a basic residue in the transmembrane region of FcalphaRI, but also the orientation of FcalphaRI toward the FcR gamma-chain dimer is essential for FcR gamma-chain association. This suggests the involvement of additional amino acids in the FcalphaRI-FcR gamma-chain interaction.

Renal tubular epithelial cells modulate T-cell responses via ICOS-L and B7-H1.

BACKGROUND: Renal tubular epithelial cells (TECs) play an active role in renal inflammation. Previous studies have demonstrated the capacity of TECs to modulate T-cell responses both positively and negatively. Recently, new costimulatory molecules [inducible T cell costimulator-L (ICOS-L) and B7-H1] have been described, which appear to be involved in peripheral T-cell activation. METHODS: We characterized expression and regulation of costimulatory molecules on primary human TECs and the TEC line human kidney-2 (HK-2) with reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. Immunohistochemistry was performed on human kidney biopsies. The capacity of TECs to modulate T-cell activation was studied in TEC/T-cell cultures. RESULTS: We demonstrate that TECs express ICOS-L and B7-H1 in vitro and in vivo. Stimulation with interferon-gamma (IFN-gamma) resulted in increased expression of B7-H1, whereas ICOS-L expression was marginally increased upon stimulation with CD40L, with no effect of interleukin (IL-1), IL-17, or tumor necrosis factor-alpha (TNF-alpha). Furthermore, we show that TECs are able to costimulate T cells that have received signal-1 using alphaCD3 antibodies, inducing strong IL-10 production, which was partially mediated by ICOS-L. In contrast, B7-H1 appeared to be involved in inhibition of proliferation and cytokine synthesis. In addition, TECs were able to alter the cytokine profile of fully activated T cells, which were incubated with alphaCD3 and alphaCD28 antibodies, resulting in low IFN-gamma and high IL-10 production. This activity appeared to be independent of ICOS-L and B7-H1. CONCLUSION: Interaction of tubular epithelial cells and kidney infiltrating T cells via ICOS-L and B7-H1 may change the balance of positive and negative signals to the T cells, leading to IL-10 production and limitation of local immune responses.

NF-kappaB mediated IL-6 production by renal epithelial cells is regulated by c-jun NH2-terminal kinase.

Tubular epithelial cells (TEC) play an important role in tubulointerstitial inflammation, a hallmark of most renal diseases, via production of cytokines and chemokines. In this study, the role of mitogen-activated protein kinases (MAPK) in regulation of the proinflammatory cytokine IL-6 in cultured human TEC in response to the leukocyte-derived factors IL-1, TNF-alpha, IL-17, and CD40L was investigated. IL-6 production induced by IL-1, TNF-alpha, and IL-17 was specifically inhibited by the c-jun NH(2)-terminal kinase (JNK) inhibitor SP600125, but not by a selective inhibitor of p38 MAPK, and was moderately increased when the ERK1/2 pathway was inhibited. Also for CD40L stimulation, inhibition of JNK resulted in a pronounced inhibition of IL-6 production. Although stimulation of TEC induced activation of activator protein-1 (AP-1), the down-stream target of JNK, reporter assays demonstrated that mutation of the AP-1 binding site in the IL-6 promoter did not affect gene transcription. Furthermore, IL-1-induced transcriptional activation of the IL-6 promotor was repressed by SP600125 or by co-transfection of a dominant-negative expression plasmid of c-jun even in the absence of a functional AP-1 binding site. This suggests that IL-6 production by renal epithelial cells is regulated by JNK, via a mechanism, however, independent of the AP-1 binding site. The data rather suggest that the JNK pathway may interfere with other signaling pathways, involving NF-kappaB and possibly ERK.

Human renal epithelial cells produce the long pentraxin PTX3.

BACKGROUND: Pentraxin 3 (PTX3) is a prototypic long pentraxin with structural similarities in the C-terminal domain to the classical short pentraxins C-reactive protein (CRP) and serum amyloid P component. PTX3 is suggested to play an important role in the innate resistance against pathogens, regulation of inflammatory reactions, and clearance of apoptotic cells. Unlike the classic pentraxins, PTX3 is mainly expressed extrahepatically. The present study was designed to investigate the expression of PTX3 by human proximal renal tubular epithelial cells (PTECs). METHODS: PTECs were cultured in the presence or absence of inflammatory cytokines. PTX3 mRNA expression was measured by reverse transcription-polymerase chain reaction (RT-PCR) in human kidney and PTECs. PTX3 protein levels in PTEC cultures were quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: PTX3 mRNA was shown to be constitutively expressed in human kidney. Constitutive expression and production of PTX3 was shown in primary mesangial cells, in primary PTECs, and in renal fibroblasts. Further analysis showed that interleukin (IL)-1 and tumor necrosis factor-alpha (TNF-alpha) stimulation strongly enhanced the expression and production of PTX3 in PTECs in a dose- and time-dependent manner. In addition, activation of PTECs with IL-17 and CD40L, respectively, but not with IL-6 or IL-4, resulted in strongly increased production of PTX3, whereas granulocyte macrophage-colony-stimulating factor (GM-CSF) inhibited IL-1-induced PTX3 production. PTX3 produced by PTEC is functionally active in binding C1q. CONCLUSION: These results indicate that PTX3 is expressed and released by PTECs and that in proinflammatory conditions PTX3 production is up-regulated. Local expression of PTX3 may play a role in the innate immune response and inflammatory reactions in the kidney.

Mechanism of steroid action in renal epithelial cells.

Renal tubular epithelial cells (TEC) are thought to play an active role in tubulointerstitial inflammation. Various immune and non-immune factors activate TEC to produce a variety of cytokines and chemokines, contributing to attraction of inflammatory cells to the kidney. The proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) appears to be a key player in these responses and tubular expression of NF-kappaB has been demonstrated in vitro and in vivo. Although glucocorticoids are known to inhibit NF-kappaB activation at different levels, the proinflammatory capacity of TEC was not inhibited. In contrast, glucocorticoids seemed to enhance the profibrotic response of TEC, emphasizing the cell-type specific characteristics of glucocorticoid action. We propose that specific inhibition of NF-kappaB activation in TEC might be an attractive strategy for therapeutic intervention in renal inflammation.

Steroid responsiveness of renal epithelial cells. Dissociation of transrepression and transactivation.

Glucocorticoids modulate cellular and inflammatory responses via stimulation or inhibition of gene transcription. Inhibition of cytokine gene expression is mediated via repression of transcription factors, including NF-kappaB. Previously we have shown that cytokine production by renal epithelial cells is insensitive to the inhibitory action of dexamethasone. In this study we demonstrate that dexamethasone is unable to inhibit NF-kappaB activation in the renal epithelial cell line HK-2, as measured by IkappaB-alpha degradation and DNA binding activity. Transfection of an NF-kappaB-inducible reporter gene demonstrated that non-stimulated HK-2 cells contain a high level of constitutively active NF-kappaB compared with the steroid-sensitive airway epithelial cell line A549, which was not blocked by dexamethasone. Expression and nuclear translocation of the glucocorticoid receptor (GR) was comparable in both cell types. In HK-2 cells, dexamethasone stimulated expression of two glucocorticoid-responsive genes, beta(2)-adrenoreceptors and angiotensinogen. The capacity of GR to transactivate the native angiotensinogen glucocorticoid-responsive element (GRE) using chromatin-IP was not impaired. Moreover, dexamethasone activation of a GRE-driven reporter construct appeared to be equally effective, although less sensitive compared with A549 cells. In conclusion, we provide evidence that glucocorticoids are unable to repress the activity of NF-kappaB in renal epithelial cells in the presence of an intact stimulatory pathway.

Production of inflammatory mediators by renal epithelial cells is insensitive to glucocorticoids.

1. In the present study we investigated the effect of glucocorticoids on the activation of renal tubular epithelial cells, which are thought to play an important role in inflammatory processes in the kidney. 2. Activation of renal epithelial cells by IL-1, TNF-alpha or CD40L resulted in increased production of cytokines and chemokines. Both in the renal epithelial cell line HK-2 and in primary cultures of human proximal tubular epithelial cells (PTEC) production of IL-6, IL-8 and monocyte chemotactic protein 1 (MCP-1) was not inhibited by glucocorticoids, independent of the stimulus. 3. In contrast, dexamethasone strongly inhibited cytokine production by immortalized renal fibroblasts and an airway epithelial cell line (A549). 4. Stimulation of renal epithelial cells resulted in activation of NF-kappaB, a pivotal transcription factor in the regulation of cytokine genes, as was shown by IkappaB-alpha degradation and increased DNA-binding activity. In contrast to dexamethasone, addition of the NF-kappaB inhibitors pyrrolidine dithiocarbamate (PDTC) and n-tosyl-l-phenylalanine chloromethyl ketone (TPCK) completely abolished cytokine and chemokine production. 5. Renal epithelial cells express abundant levels of the functional glucocorticoid receptor alpha (GRalpha) isoform and low levels of the inhibitory beta isoform (GRbeta). 6. In conclusion, cytokine production by renal epithelial cells is insensitive to the inhibitory effects of glucocorticoids. The lack of dexamethasone-mediated inhibition was specific for renal epithelial cells and could not be explained by an increased expression of the glucocorticoid receptor beta isoform.

Interleukin-17 and CD40-ligand synergistically enhance cytokine and chemokine production by renal epithelial cells.

Renal allograft rejection is characterized by an influx of inflammatory cells. Interaction between infiltrating T cells and resident parenchymal cells might play an important role in the ongoing inflammatory response. The present study demonstrates that CD40L, a product of activated T cells, is locally expressed in kidneys undergoing rejection. Furthermore, during rejection, CD40 expression not only is present on most graft infiltrating cells but also is increased on resident tubular epithelial cells (TEC). To obtain more detailed insight in the consequences of T cell/TEC interaction, we analyzed the production of chemokines, including interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and regulated upon activation, normal T cell expressed and secreted (RANTES), and the production of IL-6 by cultured human primary TEC in response to activation with CD40L in vitro. In addition, we studied the interaction with IL-17, a T-cell-specific cytokine previously demonstrated to be present during renal allograft rejection. The results, obtained by enzyme-linked immunosorbent assay, indicate that simultaneous activation of TEC with IL-17 and CD40L synergistically enhances production of IL-6 (2.1-fold higher than sum of single stimulations) and the chemokines IL-8 (15-fold) and RANTES (5.8-fold) as demonstrated by statistical analysis (P: < 0.05), whereas effects on MCP-1 (1.4-fold) are additive. Part of the synergy can be explained by increased CD40 expression on TEC upon IL-17 stimulation. The synergy is not unique for TEC, because similar responses were found with human synoviocytes and a foreskin fibroblast cell line (FS4). Stimulation of TEC with CD40L results in activation of NF-kappaB and induction of cytokine production by IL-17 and CD40L is prevented by addition of the NF-kappaB inhibitor pyrrolidine dithiocarbamate. These data suggest an important role for T cells in renal allograft rejection by acting on parenchymal cells via both soluble mediators and direct cellular contact.