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Background The incidence of mucinous appendiceal adenocarcinomas (MAA) has increased over the past three decades. Advanced stage tumor diagnosis is likely attributable to non-specific findings. Here we describe advanced stage appendiceal MAA presenting as inguinal ulcers, scrotal abscesses, and other nonspecific symptoms. To our knowledge, this is the first report of MAA presenting as inguinal pain with inflamed phlegmonous tissue and scrotal abscess. Case presentation A 67-year-old male presented to a rural facility complaining of weight-loss, fatigue, hematuria, dysuria, painful right inguinal ulceration, and right scrotal abscess drainage. Computed tomography of the abdomen and pelvis revealed a distended appendix (> 1.3 cm) and a fistula between the appendix, urinary bladder, right scrotum, and right groin. Laparoscopic appendectomy was performed and diagnosed as MAA. After a right hemicolectomy, the MAA was staged as pT3b pN0 M0 G2. Conclusion This case highlights a unique presentation of late stage appendiceal MAA. Due to the increased incidence of appendiceal MAAs, reports of unique clinical features are needed to facilitate early diagnosis and intervention, especially in rural settings with limited access to specialists.

In terms of managing thrombotic disorders, genotype-based individualized patient care emerged as early as 1994 when the association of factor V Leiden (G1691A), and later, prothrombin (G20210A), with thrombotic phenotypes were discovered. Since then, genetic tests for specific thrombophilic SNPs have been routinely incorporated into daily practices in both thrombotic risk assessment and clinical decision-making with respect to prophylactic anti-thrombotic therapy. Recently, the area of pharmacogenomics in major anti-thrombotic drugs, such as warfarin and clopidogrel, has been the principal driver for personalized therapy based on one’s own individual characteristics.

Personalized healthcare has regained momentum through the unprecedented surge of research in the genomics field and related areas of biology. These new insights compel the optimization of healthcare and therapy for individual subjects. While the potential benefits are large, substantial obstacles need to be overcome for attaining clinical utility in general medial practice. These range from scientific hurdles emerging from biological and genetic complexity, to cultural, economic, legal, regulatory and ethical issues. This article addresses, in broad strokes, these intercalated issues, discussing recent advances, remaining questions and how to move forward. It also highlights recent developments at academic centers devoted to promoting personalized healthcare as an avenue with great potential for improving our healthcare system.

Background: In the early post-transplant period, renal allograft rejection with diffuse peritubular capillary (PTC) C4d deposition predicts poor graft survival. In the late post-transplant setting, that is, one or more yr after transplantation, the implication of diffuse PTC C4d deposition is still a topic of debate. The purpose of our study was to see if diffuse PTC C4d deposition, in late acute rejection (LAR), occurring more than one yr post-transplant, has any impact on graft survival and function. Methods: We selected cases, both cadaveric as well as living donor renal transplant recipients, in whom acute rejection with PTC C4d deposition was first detected after the first year post-transplant. Recipients with multiple acute rejection episodes during the first year post-transplant were excluded from the study. The first biopsy diagnosed with LAR was considered the index biopsy (n = 40). We formed two groups: group 1, C4d-positive LAR (n = 20), and group 2, C4d-negative LAR (n = 20). Groups were matched for maintenance and post-rejection immunosuppressive therapy, baseline serum creatinine levels before the time of the index biopsy, time from transplant to index biopsy, as well as chronic allograft damage index (CADI) score in the index biopsies. We compared the rate of graft loss, and the graft function of the surviving grafts at the end of the study period, as well as histologic parameters in the index biopsy specimens between the two groups. The mean follow-up period was 20 months. Results: No significant differences in the rate of graft loss or graft function were found between groups 1 and 2 at the end of the follow-up period. Histologically, PTC margination and transplant glomerulopathy were more common in the C4d-positive group, and this difference was statistically significant. There was no statistically significant difference in the degree of plasma cell infiltrates. Conclusions: Unlike in the acute setting, the presence or absence of PTC C4d staining in renal allografts with LAR may not have a predictive value regarding graft outcome.

The association of methicillin-resistant Staphylococcus aureus (MRSA) infection with glomerulonephritis (GN) has been well documented in Japan but not in North America. Recently, eight renal biopsies with IgA-predominant or -codominant GN from eight patients with underlying staphylococcal infection, but without endocarditis, were observed at a single institution in a 12-mo period. Renal biopsies were worked up by routinely used methodologies. Eight cases of primary IgA nephropathy were used as controls. Five patients had MRSA infection, one had methicillin-resistant S. epidermidis (MRSE) infection, and two had methicillin-sensitive S. aureus infection. Four patients became infected after surgery; two patients were diabetic and had infected leg ulcers. All patients developed acute renal failure, with active urine sediment and severe proteinuria. Most renal biopsies showed only mild glomerular hypercellularity. Two biopsies had prominent mesangial and intracapillary hypercellularity; one of them (the MRSE-associated case) had large glomerular hyalin thrombi. This patient also had a positive cryoglobulin test. Rare glomerular hyalin thrombi were noted in two other cases. Immunofluorescence showed IgA pre- or codominance in all biopsies. Electron microscopy revealed mesangial deposits in all cases. Five biopsies had rare glomerular capillary deposits as well. In the MRSE-associated GN, large subendothelial electron-dense deposits were present. These cases demonstrate that staphylococcal (especially MRSA) infection-associated GN occurs in the US as well, and a rising incidence is possible. It is important to differentiate a Staphylococcus infection-associated GN from primary IgA nephropathy to avoid erroneous treatment with immunosuppressive medications.

We have previously shown that cytomegalovirus (CMV) can reactivate in lungs of nonimmunosuppressed patients during critical illness. Our recent work has shown that polymicrobial bacterial sepsis can trigger reactivation of latent murine CMV (MCMV). We hypothesize that MCMV reactivation following bacterial sepsis may be caused by inflammatory mediators. To test this hypothesis, BALB/c mice latently infected with Smith strain MCMV received sublethal intraperitoneal doses of lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), or saline. Lung tissue homogenates were evaluated for viral reactivation 3 weeks after mediator injection. Because LPS is known to signal via Toll-like receptor 4 (TLR-4) in mice, further studies blocking this signaling mechanism were performed using monoclonal MTS510. Finally, mice were tested with intravenous TNF-α to determine whether this would cause reactivation. All mice receiving sublethal intraperitoneal doses of LPS, TNF-α, or IL-1β had pulmonary reactivation of latent MCMV 3 weeks following injection, and LPS caused MCMV reactivation with kinetics similar to those for sepsis. When TLR-4 signaling was blocked, exogenous LPS did not reactivate latent MCMV. Intravenous TNF-α administration at near-lethal doses did not reactivate MCMV. Exogenous intraperitoneal LPS, TNF-α, and IL-1β are all capable of reactivating CMV from latency in lungs of previously healthy mice. LPS reactivation of MCMV appears dependent on TLR-4 signaling. Interestingly, intravenous TNF-α did not trigger reactivation, suggesting possible mechanistic differences that are discussed. We conclude that inflammatory disease states besides sepsis may be capable of reactivating CMV from latency.

Purpose To determine if prior exposure to pathogens associated with vascular disease, cytomegalovirus, Chlamydia pneumoniae , and Helicobacter pylori correlates with neovascular age-related macular degeneration (AMD). Design An experimental study. Methods Setting Institutional. Bascom Palmer Eye Institute, October 2001 to December 2002. Patient population 150 patients (47 neovascular amd, 36 dry amd, and 67 non-amd controls) were included in the study. exclusion criteria included hiv infection, malignancy, recent acute illness requiring hospitalization within 6 months, or immunosuppressive illness. Procedure Serum samples were obtained for analysis of cytomegalovirus, chlamydia pneumoniae , and helicobacter pylori igg antibody titers by elisa. Main outcome measure Comparison of the distribution of igg titers between patients with wet amd, dry amd, and controls. Results The average cytomegalovirus IgG titer was higher in patients with wet AMD versus controls (p = 0.02, Student t-test, two-tailed) and patients with dry AMD (p = 0.06). Twenty-six (55%) of 47 subjects with wet AMD had high cytomegalovirus IgG titers compared with 14 (39%) of 36 patients with dry AMD (odds ratio [OR] = 2.23, 95% confidence interval [CI] = 0.77 to 6.44) and 23 (34%) of 67 control patients (OR = 2.49, 95% CI=0.98 to 6.33). There was no major difference in the distribution of titers for Chlamydia pneumoniae IgG and Helicobacter pylori IgG in wet and dry AMD patients. Five of 47 patients with wet AMD (11%) had high antibody titers to all three pathogens, compared with only 1 of 36 patients with dry AMD (3%) (OR = 4.17, 95% CI=0.46 to 37.36). Conclusions There was a significant association of high cytomegalovirus IgG titer with neovascular AMD compared with dry AMD and control patients. Chronic infection with cytomegalovirus may be a novel risk factor for the progression from dry to neovascular AMD.

Clinical and molecular medicines are undergoing a revolution based on the accelerated advances in biotechnology such as DNA microarrays and proteomics. Answers to fundamental questions such as how does the DNA sequence differ between individuals and what makes one individual more prone for a certain disease are eagerly being sought in this postgenomic era. Several government and nonprofit organizations provide the researchers access to human tissues for molecular studies. The tissues procured by the different organizations may differ with respect to fixation and processing parameters that may affect significantly the molecular profile of the tissues. It is imperative that a prospective investigator be aware of the potential contributing factors before designing a project. The purpose of this review is to provide an overview of the methods of human tissue acquisition, fixation, and preservation. In addition, the parameters of procurement and fixation that affect the quality of the tissues at the molecular level are discussed.

Background: Parvovirus B19 has recently been implicated in various vasculitic syndromes including Henoch Schonlein purpura (HSP), Wegener's granulomatosis and microscopic polyarteritis. The association was established through serology, the identification of DNA in the peripheral blood and affected tissues and more recently by RNA localization to cutaneous capillary endothelium. However, direct localization of the viral DNA to the glomerular and cutaneous endothelium in HSP in correlation with the histopathologic findings has not been demonstrated. Methods: Skin and kidney biopsy tissues were processed for hematoxylin and eosin, immunofluorescent, polymerase chain reaction (PCR) and reverse transcriptase in situ PCR studies. Case presentation: A 64-year-old-female presented with palpable purpura and nephrotic range proteinuria. Kidney and skin biopsies showed IgA-associated mesangioproliferative glomerulonephritis and IgA-associated leukocytoclastic vasculitis, respectively. A diagnosis of HSP was rendered. Her clinical course was refractory to prednisone. Parvovirus B19 DNA and tumor necrosis factor α DNA were identified in the dermal and glomerular capillary endothelial cells and surrounding dermal inflammatory cells. Conclusion: This is the first documentation of B19 localization to dermal and glomerular capillary endothelium in HSP. It is important to recognize parvovirus B19-associated adult HSP cases, as the treatment of choice is intravenous gamma globulin in concert with anti-TNFα therapy. In contrast immunosuppressive therapy may lead to a persistent and/or worsening disease course.

We hypothesized that US28, a cytomegalovirus (CMV) CC chemokine receptor homolog, plays a role in modulating the host antiviral defense. Monocyte chemotaxis was induced by supernatants from fibroblasts infected with a US28 deletion mutant of CMV (CMV Delta US28) due to endogenously produced CC chemokines MCP-1 and RANTES. However, these chemokines were sequestered from the supernatants of CMV-infected cells that did express US28. US28 was also capable of sequestering exogenously added RANTES. Surprisingly, cells infected with CMV Delta US28 transcribed and secreted increased levels IL-8, a CXC chemokine, when compared to CMV-infected cells. Finally, because chemokines are potent mediators of immune cell migration through the endothelium, we characterized the CC chemokine binding potential of CMV-infected endothelial cells. We propose that US28 functions as a 'chemokine sink' by sequestering endogenously and exogenously produced chemokines and alters the production of the CXC chemokine IL-8, suggesting that CMV could significantly alter the inflammatory milieu surrounding infected cells.

Critically ill surgery patients are susceptible to pulmonary reactivation of latent cytomegalovirus (CMV), but what triggers this reactivation is unknown. Immunosuppression and bacterial sepsis are thought to stimulate reactivation of CMV, and in this study it was hypothesized that immunosuppressive effects of surgery with or without concomitant bacterial infection may reactivate latent CMV. Mice infected with CMV were allowed to develop latent infections. Latently infected mice underwent a laparotomy with cecal ligation and puncture (CLP; n=30), a laparotomy alone (sham; n=10), or no surgery (control; n=5). Lung tissue homogenates were evaluated for viral activity, and, 2 and 3 weeks after CLP, lungs of 7 of 7 and 5 of 5 mice, respectively, showed reactivation of latent CMV. In contrast, lungs from all sham-operated animals and controls showed no viral reactivation. These findings demonstrate that surgery with subsequent intra-abdominal bacterial infection reactivated CMV in lungs of latently infected mice. The mechanism of this reactivation is unknown but likely involves cytokines induced by sepsis.

CD4 + T lymphocytes are a significant component of the afferent and efferent arms of adaptive immunity and are critical for controlling viral infections. CD4 + T lymphocytes secrete cytokines that augment CD8 + T lymphocyte and B lymphocyte responses and directly inhibit viral replication. The interface between the CD4 + T lymphocyte and virus is the major histocompatibility complex (MHC) class II molecule. Cytomegalovirus, a beta-herpesvirus, has evolved mechanisms for inhibiting MHC class II expression and thus escaping CD4 + T lymphocyte immunosurveillance. Herein, we review cytomegalovirus-mediated down-regulation of inducible and constitutive MHC class II expression, while focusing on lesions that occur at the level of MHC class II transcription.

Microsphere-adenoviral complexes target and transduce the glomerulus in vivo. Background Developing new treatments for glomerulonephritis makes the glomerulus a logical target for gene therapy. Microspheres may lodge in the glomerulus, and replication-deficient recombinant adenoviruses are potent mediators of gene transfer. We postulated that adenoviral-microsphere complexes could result in DNA transfer (transduction) into glomerular cells in vivo. Methods Two adenoviruses, each one containing a luciferase or β-galactosidase (β-gal) transgene expression cassette, were complexed to polystyrene microspheres. To assess in vivo glomerular transduction with this tool, male Sprague-Dawley rats underwent aortic injections with adenovirus linked to 11 or 16 μm diameter microspheres. Results After 48 hours, adenoviral-microsphere complexes resulted in transduction of up to 19% of glomeruli per kidney section. Endothelial and mesangial cells were transduced with this approach, and transprotein expression persisted for 21 days. Transduction efficiency was greater in the 16 μm group. For all rats, there was a strong correlation between kidney luciferase levels and the number of β-gal-positive glomeruli ( r = 0.87), indicating that transgene expression was primarily glomerular in location. This was supported by reverse transcriptase in situ polymerase chain reaction, which demonstrated glomerular localization of the β-gal transgene. Conclusions The aortic injection of adenoviral-microsphere complexes transduces the glomerulus in vivo and may be a useful tool in developing approaches to gene therapy of glomerular disease.

Proximal tubular epithelial hyperplasia in patients with chronic glomerular proteinuria. Background Proteinuria is known to affect the proximal tubular epithelial structure and function. The present study tested the hypothesis that chronic proteinuria leads to hyperplasia of proximal tubular epithelium. Methods This hypothesis was tested by morphometric analysis of the renal biopsy specimens in two groups of patients. Group A ( N = 15) was composed of patients with chronic glomerular proteinuria who, for clinical indications, underwent renal biopsy of their native kidneys on two separate occasions. The proteinuria was sustained during the first and second renal biopsies in all but two of the patients with minimal change nephrotic syndrome who experienced transient remission. Group B ( N = 10) was composed of patients with little or no proteinuria who underwent renal biopsy because of unexplained hematuria and whose renal biopsy showed only thin glomerular basement membrane (GBM) disease. Results In Group A, the mean number of epithelial cell nuclei per proximal tubule cross-section increased significantly from the first to the second renal biopsy (11.0 ± 2.7 vs. 13.0 ± 2.2, P = 0.005, paired t -test). Also, those with severe proteinuria showed proximal tubules with reactive epithelium (large pale nuclei with a high nucleus to cytoplasm ratio) and marked hyperplasia (double and triple layers of epithelium). Such changes were not seen in group B renal biopsies. Compared with group A biopsies, group B biopsies showed a lower mean value for proximal tubular epithelial cell nuclei per tubular cross-section ( P = 0.056) and a higher mean proximal tubular volume ( P = 0.049). As a consequence, the mean number of nuclei per relative tubular volume was significantly greater in group A compared with group B (0.55 ± 0.14 vs. 0.40 ± 0.06, P = 0.003, by Wilcoxon rank sum). Conclusions Chronic heavy proteinuria is associated with hyperplasia of proximal tubular epithelium and contraction of proximal tubular volume. These events may impair glomerular filtration and represent another mechanism of progression of renal disease.

BACKGROUND Cytomegalovirus (CMV) is the primary viral cause of complications in transplant recipients. We sought to understand the mechanisms of its dissemination and induction of vascular disease, which may lead to transplant complications. Sialyl Lewis(x) (sLe(x)) and Lewis(x) (Le(x)) are known for their roles in mediating cell adhesion and as tumor-associated carbohydrate antigens. Herein we explore whether CMV induces surface expression of these important molecules in endothelial cells (EC). METHODS Flow cytometry was used to detect surface expression of sLe(x) and Le(x) on CMV-infected human umbilical vein endothelial cells (HUVEC), with or without ultraviolet inactivation of the virus. To elucidate mechanisms of CMV-mediated induction, mRNA coding for predominant HUVEC sialyltransferases (ST) and fucosyltransferases (FT), key enzymes in sLe(x) and Le(x) synthesis, was analyzed by Northern blot. Dual immunohistochemical staining for sLe(x) and Le(x) expression of human colon and placental tissue was performed to investigate in vivo relevance. RESULTS sLe(x) expression on CMV-infected HUVEC was strongly up-regulated by 8 days after inoculation. Le(x) expression was detectable earlier and increased steadily over time. In contrast, ultraviolet-inactivated CMV did not induce expression of these molecules. Northern blot assays demonstrated higher levels of important EC glycosyltransferases ST-IV, FT-III, and FT-IV in CMV-infected EC. Finally, high levels of sLe(x) and Le(x) were expressed in CMV-infected EC in vivo. CONCLUSIONS Given the known biologic functions of sLe(x) and Le(x), we suggest that CMV induction of these molecules may have widespread consequences ranging from CMV dissemination to induction of CMV-associated vascular disease, including thrombosis.

Interferon-gamma stimulates major histocompatibility complex (MHC) class I antigen processing and presentation by inducing the expression of major histocompatibility complex class I heavy chains, beta2-microglobulin, the transporter associated with antigen processing, and components of the proteasome complex. We demonstrate that this effect of interferon-gamma on the major histocompatibility complex class I pathway is inhibited in human cytomegalovirus-infected fibroblasts and endothelial cells. This is the result of a direct human cytomegalovirus/cell interaction leading to a block in interferon-gamma signal transduction beginning at early times after infection and peaking at 72 hr after infection. These observations suggest a novel level of herpesvirus interference with antigen processing: protection of infected cells from the immunoregulatory effects of interferon-gamma. Thus protected, human cytomegalovirus persists and may exacerbate graft rejection or lead to fulminant infection in the immunocompromised transplant recipient.

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) is strongly induced under inflammatory conditions associated with allograft rejection, thereby promoting leukocyte recruitment and activation at the site of inflammation. Enhancement of ICAM-1 expression can also be the result of viral infection, in particular human cytomegalovirus (CMV), a frequent source of complications in the transplant recipient. In vitro studies have shown that CMV infection of endothelial cells (EC) results in the direct enhancement of ICAM-1 expression and consequent leukocyte adhesion/activation suggesting mechanisms by which CMV exacerbates graft vascular disease. Although treatment of EC with ICAM-1-specific antisense oligonucleotides has been shown to attenuate ICAM-1 induction under simulated inflammatory conditions (i.e., TNF-alpha), no studies have addressed their effectiveness on virally-induced ICAM-1 expression. RESULTS: In the current investigation, we show that the progressive increase in endothelial ICAM-1 protein expression that follows inoculation with CMV correlates with a progressive accumulation of ICAM-1 mRNA. Furthermore, we demonstrate that treatment of EC with a partially 2'-O-methoxyethyl modified ICAM-1-specific antisense oligonucleotide before viral inoculation significantly reduces CMV-associated induction of ICAM-1 protein and mRNA expression. Finally, we show that antisense-mediated attenuation in ICAM-1 expression results in a significant reduction of T lymphocyte adhesion to CMV-infected EC monolayers, an interaction that has been implicated in allogeneic T lymphocyte activation, in viral transmission to transiently adherent leukocytes and subsequent hematogenous dissemination. CONCLUSIONS: These findings demonstrate for the first time that antisense oligonucleotides can effectively reverse virally-induced host cellular protein expression, specifically ICAM-1, as well as consequent T lymphocytes adhesion, thus broadening the potential clinical utility of antisense oligonucleotides.

Background Cytomegalovirus (CMV), a betaherpesvirus associated with allograft rejection, infects the endothelium, the cellular interface between allograft tissue and the host immune system. Because of recent appreciation of the phenotypic diversity of endothelial cells (EC) from different vascular compartments, controversy now exists on the universality of CMV-mediated adhesion molecule induction previously described on umbilical vein EC. Therefore, we herein extend these previous studies to arterial and microvascular EC, which represent sites of vascular rejection. Methods Human coronary artery, aortic, umbilical artery, and microvascular EC were mock or CMV infected and/or treated with tumor necrosis factor-a before flow cytometric and immunohistochemical analysis. Results CMV directly enhanced intercellular adhesion molecule-1 on all EC isolates but did not induce E-selectin or vascular cell adhesion molecule-1. Furthermore, CMV-infected EC were refractory to tumor necrosis factor-alpha-mediated induction of these molecules. Conclusion CMV-induced modulations of adhesion molecule expression, which may affect allograft immunogenicity, seem common to all EC regardless of vascular origin.

Renal allograft survival following acute rejection correlates with blood pressure levels and histopathology.BackgroundAcute rejection (AR) is a strong predictor of renal graft survival, but the negative impact of AR on survival is variable, suggesting that other factors modulate this relationship. In this study, we examined the variables that correlate with graft survival after AR, particularly the impact of blood pressure (BP), graft function, and histopathology.MethodsThe study population included patients with no AR (N = 942) and patients with one (N = 407) or two (N = 156) AR during the first year post-transplant. Patients were adults who were recipients of living related (LRD, N = 410) or cadaveric grafts (CAD, N = 1095) and who were transplanted in a single institution and followed for 5.8 ± 4 years.ResultsCompared with patients without AR, those with AR were significantly younger, had more human lymphocyte antigen mismatches, and included more CAD recipients. Graft survival was analyzed beyond six-months post-transplant. In patients with AR, reduced survival correlated (multivariate) with (a) younger recipients (P = 0.01), (b) AR occurring later during the first-year post-transplant (P = 0.0006), (c) elevated serum creatinine (Cr) before (P = 0.05), at the time (P = 0.0001) of, or after AR (P = 0.0004), and (d) average BP levels after AR [systolic BP (P = 0.003 logistic, P < 0.0001 by Cox), diastolic BP (P = 0.007), mean arterial pressure (P < 0.0001)]. This latter correlation was independent of graft function and recipient race. Thus, post-AR BP levels correlated with graft survival in patients with post-AR creatinine ≤2 mg/dl (N = 408, P = 0.0009), in Caucasian recipients (P = 0.001), and in African American recipients (P = 0.01). In contrast, there was no significant correlation between BP levels and graft survival in patients without AR. AR histopathology, analyzed in patients with one AR episode, correlated with graft survival only the first six months after AR but not thereafter.ConclusionsGraft survival after AR can be predicted independently by graft function and BP levels after the event. Patients with elevated BP post-AR have poor graft survival even if they have excellent graft function.

Background. Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) remains a major cause of morbidity and mortality among pharmacologically immunosuppressed organ transplant recipients, frequently engaging the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective against acute and chronic allograft rejection in animal models. Because a number of CMV proteins are known to be phosphorylated, we tested the hypothesis that this agent might exert inhibitory activity against CMV. Methods and results. Plaque assays demonstrated dramatic dose-dependent attenuation of production of multiple clinical CMV isolates in leflunomide-treated human fibroblasts and endothelial cells, common targets for CMV infection in vivo. As shown by Northern blot analysis and immunohistochemical staining, leflunomide neither interferes with transcription of immediate early or late viral genes, nor with expression of corresponding proteins. CMV-specific DNA dot blots and biochemical enzyme assays indicated that, in contrast to currently approved anti-CMV drugs, leflunomide exerts no inhibitory effect on the accumulation of viral DNA in infected cells, or on viral DNA polymerase activity. Rather, as visualized by transmission electron microscopy, this agent appears to act at a late stage in virion assembly by preventing tegument acquisition by viral nucleocapsids. Finally we have demonstrated equivalent inhibitory activity of leflunomide against multi-drug-resistant CMV isolates. Conclusions. These findings imply that leflunomide, an effective immunosuppressive agent, shows potential to concurrently attenuate a major complication of immunosuppression, CMV disease, by a novel mechanism of antiviral activity.

There are clinical situations in which it may be advantageous to monitor delayed type hypersensitivity (DTH) responses, an index of cell-mediated immunity, without exposing patients directly to the challenge antigens. For example, transplant patients may be at risk for becoming sensitized to donor antigens if injected with donor antigen during traditional skin tests. We describe an alternative method for human DTH testing, which involves the transfer of human peripheral blood mononuclear cells plus antigen into the pinnae or footpads of naive mice. This induces a measurable DTH-like swelling response, which we refer to as the "trans vivo DTH response." As proof of principle, we provide data obtained during trans vivo DTH studies with tetanus toxoid, cytomegalovirus (CMV) and alloantigens. In general, human T cells must be co-localized with antigen and human macrophages to produce swelling responses, and such responses are antigen-specific and require prior antigen sensitization. Not only does this assay offer a simple, reliable clinical monitoring device, but it also provides a model with which to study the in vivo mechanisms of human DTH responses.

The type I IFNs represent a primordial, tightly regulated defense system against acute viral infection. IFN-α confers resistance to viral infection by activating a conserved signal transduction pathway that up-regulates direct antiviral effectors and induces immunomodulatory activities. Given the critical role of IFN-α in anti-human cytomegalovirus (HCMV) immunity and the profound ability of HCMV to escape the host immune response, we hypothesized that HCMV blocks IFN-α-stimulated responses by disrupting multiple levels of the IFN-α signal transduction pathway. We demonstrate that HCMV inhibits IFN-α-stimulated MHC class I, IFN regulatory factor-1, MxA and 2′,5-oligoadenylate synthetase gene expression, transcription factor activation, and signaling in infected fibroblasts and endothelial cells by decreasing the expression of Janus kinase 1 and p48, two essential components of the IFN-α signal transduction pathway. This investigation is the first to report inhibition of type I IFN signaling by a herpesvirus. We propose that this novel immune escape mechanism is a major means by which HCMV is capable of escaping host immunity and establishing persistence.

Background. After transplantation renal allografts frequently develop interstitial fibrosis and tubular atrophy, and these pathologic changes are the hallmarks of chronic allograft nephropathy (CN). However, the diagnosis of CN has no specific pathogenic implications. In this study we sought to determined whether a subclassification of CN according to vascular pathology correlates with posttransplant events, particularly acute rejection, and graft survival. Methods. A total of 419 patients with moderate to severe CN were subdivided into: (1) transplant arteriopathy (TA, n=233, 56%); (2) arteriolar hyalinosis (AH, n=89, 21%); and (3) no characteristic vascular pathology (IFb, n=97, 23%). Results. Patients with AH differed significantly from patients with TA or IFb in the following parameters: (1) AH was diagnosed later after transplantation (P=0.001); (2) fewer patients with AH had acute rejection (AR) before the diagnosis of CN (P

Viruses have evolved numerous mechanisms that modulate MHC-mediated antigen presentation, which in turn protect infected cells from T-lymphocyte-mediated immunosurveillance. Recent studies of previously identified viral immunomodulatory proteins reveal the allelic specificity of these proteins, their ability to function in xenogeneic systems and the difficulty in translating in vitro data to in vivo models; moreover, new mechanisms of viral modulation of MHC expression have emerged.

Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) remains a major cause of morbidity and mortality among pharmacologically immunosuppressed transplant recipients, frequently engaging the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective against acute and chronic rejection in animal models. Herein we summarize our recent studies demonstrating that leflunomide inhibits the production of multiple clinical CMV isolates (including multi-drug-resistant virus) in both human fibroblasts and endothelial cells. In contrast to all other anti-CMV drugs currently in use, leflunomide does not inhibit viral DNA synthesis, but rather appears to interfere with virion assembly. Finally, preliminary studies in a rat model suggest that this agent reduces viral load in vivo. These findings imply that leflunomide, an effective immunosuppressive agent, shows potential to concurrently attenuate a major complication of immunosuppression, CMV disease, by a novel mechanism of antiviral activity.

BACKGROUND: Herein we investigated the relationships between acute rejection (AR), infection, and renal allograft infarcts, particularly those infarcts that occur beyond the immediate posttransplant period and that affect functioning grafts. METHODS: Infarcts (n=59) were classified as: (1) early (EI; 2 months; n=27). Controls included patients with severe AR but without infarction (n=84). RESULTS: There were not significant differences in donor or recipient characteristics between infarcts and controls. At diagnosis, patients with infarcts were more likely to be infected (30%) than controls (14%, P=0.01); 15% of infarcts and 1% of controls had disseminated cytomegalovirus (P=0.04). Infarct and AR coexisted in the biopsy specimens of 66% of patients with EI and 62% of patients with LI, but the AR severity ranged from borderline to severe. Furthermore, 30% of patients with EI/LI had a history of severe AR. Graft survival was 47% in patients with EI, 22% in patients with LI (NS), and 71% in controls (P

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to persist for decades in its host. HCMV has evolved protean countermeasures for anti-HCMV cellular immunity that facilitate establishment of persistence. Recently it has been shown that HCMV inhibits interferon gamma (IFN-gamma)-stimulated MHC class II expression, but the mechanism for this effect is unknown. IFN-gamma signal transduction (Jak/Stat pathway) and class II transactivator (CIITA) are required components for IFN-gamma-stimulated MHC class II expression. In this study, we demonstrate that both a clinical isolate and a laboratory strain of HCMV inhibit inducible MHC class II expression at the cell surface and at RNA level in human endothelial cells and fibroblasts. Moreover, reverse transcriptase polymerase chain reaction and Northern blot analyses demonstrate that neither CIITA nor interferon regulatory factor 1 are upregulated in infected cells. Electrophoretic mobility shift assays reveal a defect in IFN-gamma signal transduction, which was shown by immunoprecipitation to be associated with a striking decrease in Janus kinase 1 (Jak1) levels. Proteasome inhibitor studies with carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone suggest an HCMV-associated enhancement of Jak1 protein degradation. This is the first report of a mechanism for the HCMV-mediated disruption of inducible MHC class II expression and a direct virus-associated alteration in Janus kinase levels. These findings are yet another example of the diverse mechanisms by which HCMV avoids immunosurveillance and establishes persistence.

BACKGROUND The aim of this study was to assess whether kidney-pancreas transplantation (KPT) compromises the prognosis of kidney transplantation (KT). METHODS This study included 368 paired recipients who received grafts from the same donor (184 KPT/184 KT), i.e., renal grafts with the same pretransplant functional and pathologic characteristics. RESULTS KPT recipients (KPR) were significantly younger and included fewer African-Americans (22% vs. 6%, P=0.0005) than recipients of kidney alone (KR). During year 1 after transplant surgery, KPR were re-admitted more often than KR (4.2+/-2 vs. 2.8+/-2, P < 0.0001). The number of acute rejections (AR) and the serum creatinine were not significantly different in KR and KPR up to 3 years after transplant. After 44+/-29 months, 13% of KR and 17% of KPR died (NS), and 17% of KR and 16% of KPR lost their kidneys (NS). In KPR, reduced renal graft survival did not correlate with AR (P=0.44), but it correlated with: older donors, younger recipients, elevated serum creatinine at 6 months, pancreas loss, and the number of episodes of acute graft dysfunction evaluated by biopsy (multivariate analysis). By Cox, graft and patient survival were not significantly different in KR and KPR. However, the patient survival of KPR < 40 years of age was lower than that of KR (P=0.02). Renal biopsies (n=165) in 40 paired recipients showed no significant differences in AR, interstitial fibrosis, or vascular pathology. CONCLUSIONS Renal graft function, structure, and survival are not different in KPR and KR, but the correlates of renal graft survival are different in these two groups of recipients.

An adult man presented severely ill with vasculitis of his lower extremities and with impaired kidney function. After detailed evaluation at a local hospital, a diagnosis of essential type III cryoglobulinemia was made. High-dose steroid and cyclophosphamide therapy was begun. The patient improved dramatically. However, 6 wk later when his steroid dose was reduced to 30 mg daily, vasculitis recurred. Intensifying his immunosuppressive therapy only worsened his condition. He was than transferred to the Ohio State University Medical Center for consideration for plasmapheresis for the presumed essential type III cryoglobulinemia. However, our evaluation showed that he had bacterial endocarditis causing his type III cryoglobulinemia. When immunosuppressive drugs were stopped and antibiotics were begun, his condition resolved completely. This case illustrates the difficulty of identifying infectious causes of cryoglobulinemia and emphasizes that an initial, highly favorable response of vasculitis to immunosuppressive therapy does not exclude an infectious cause for the vasculitis.

Background. This study examined the relationship between transplant vascular sclerosis (TVS) and tissue fibrosis, features of chronic rejection that can develop rapidly in accepted heterotopic murine cardiac allografts. Methods. The rate of development of interstitial fibrosis or TVS development was determined by computerized analysis of tissue sections from DBA/2→C57BL/6 heterotopic cardiac allografts after immunosuppression with gallium nitrate. Results. In accepted cardiac allografts, neointimal fibrosis developed by 30 days after transplant, whereas TVS was minimal by day 30, and maximal by day 60. Variable levels of fibrosis were found throughout the allografts. DBA/2→DBA/2 cardiac isografts never displayed TVS in this time period, but displayed allograft-like fibrosis within 60 days of transplantation. Conclusions. Interstitial fibrosis can be dissociated from the TVS development in this experimental model of chronic cardiac allograft rejection. Apparently, it is caused, at least in part, by alloantigen-independent factors other than TVS-related tissue ischemia.

Recent understanding of the mechanism of immunoglobulin G (IgG) catabolism has yielded new insight into antibody-mediated diseases. We proposed that beta2-microglobulin (beta2m)-deficient mice have been protected from systemic lupus erythematosis (SLE)-like syndromes because they lack the beta2m-associated IgG protection receptor (FcRn) and therefore catabolize IgG, including pathogenic IgG autoantibodies, considerably more rapidly than normal mice. Such an hypothesis would predict that beta2m-deficient mice would also be resistant to experimental bullous pemphigoid, a disease with a pathogenesis thought to be much simpler than SLE, being the result of antibody directed toward a pathogenic epitope on the epidermal hemidesmosome that anchors basal keratinocytes to the basement membrane. To test this hypothesis, we administered pathogenic rabbit antibody directed toward the hemidesmosome to beta2m-deficient mice and to normal control mice, both intraperitoneally and intradermally, and assessed the mice clinically, histologically, and immunologically for manifestations of skin disease. We found that the beta2m-deficient mice were protected when the antibody was given intraperitoneally whereas intradermal administration resulted in blisters only slightly less severe than those seen in normal mice. These data would indicate that autoantibody-mediated inflammation might be prevented or controlled by appropriate modulation of FcRn function.

The present study is a prospective, controlled, blinded trial of enalapril therapy in experimental immune complex (IC)-mediated glomerulonephritis (GN) in the nonhuman primate (cynomolgus monkey [CYN]). Two groups of CYNs were studied: those with established GN (study A) and those in which GN was being induced (study B). In study A, 12 CYNs had GN established by 8 or 10 weeks of daily intravenous infusion of bovine gamma-globulin (BGG). These CYNs were then assigned to either 4 weeks of daily oral enalapril therapy (n = 6) or daily oral placebo therapy (n = 6). The daily BGG infusions were continued during the 4 weeks of enalapril or placebo therapy. At the start of the enalapril/placebo protocol, the two groups were similar with respect to proteinuria and level of precipitating antibody to BGG, which determined the daily BGG dose. Renal biopsy was performed in each CYN at the start and end of the 4-week period of enalapril/placebo protocol. In study B, 15 normal CYNs were immunized to BGG over a period of 4 weeks. The CYNs were then assigned to daily oral enalapril therapy (n = 8) or placebo therapy (n = 7) based on level of precipitating antibody to BGG. At this point, daily intravenous BGG was begun along with daily enalapril or placebo for 8 weeks. Renal biopsy was performed in each CYN before and at the end of this 8-week period. In study A, enalapril therapy was associated with a significant decrease in mesangial matrix volume (mean change, -27.7%; P = 0.031) and a trend toward decreased mesangial matrix deposits (mean change, -34.1%; P = 0.188). By contrast, in CYNs receiving placebo therapy, mesangial matrix volume increased compared with the enalapril group (P = 0.002) and mesangial deposits were unchanged. In study B, both the enalapril and placebo groups showed significant increases in mesangial matrix volume, mesangial deposits, mesangial cell volume, and capillary wall deposits during the 8 weeks of daily BGG infusion. However, none of the differences between the groups achieved statistical significance. Changes in mesangial cell volume and capillary wall deposits were also evaluated in study A and study B, but were not found to be different between the enalapril and placebo groups. In both study A and study B, blood pressure was lower in the enalapril groups. In conclusion, in the initial phase of IC-GN induction (0 to 8 weeks), enalapril therapy does not significantly influence the glomerular accumulation of mesangial matrix or immune deposits. However, in established IC-GN (after 8 weeks of GN induction), enalapril therapy significantly decreases the further accumulation of mesangial matrix and may decrease the further accumulation of mesangial deposits. Whether this benefit of enalapril therapy was related to lower blood pressure or to other effects of angiotensin-converting enzyme (ACE) inhibition was not determined in this study.

BACKGROUND Transplant vascular sclerosis (TVS) is manifested in transplanted human and murine hearts as a concentric, intimal lesion. The purpose of this study was to characterize the rate, location, and intensity of developing TVS lesions in murine cardiac allografts using quantitative morphometric analysis. METHODS Murine cardiac allografts, treated with the immunosuppressant gallium nitrate, were explanted at 30, 60, and 90 days after transplant. The grafts were histologically stained and evaluated for intimal thickening by deriving a neointimal index (NI) using a computerized image-analysis system. RESULTS In cardiac allografts, mild vascular lesions of varying NI were detectable by day 30 and lesion severity increased significantly by day 60. Thereafter, average lesion severity stabilized, although the percentage of affected vessels continued to increase from day 30 to day 90. In contrast, day-90 cardiac isografts showed little to no TVS development. Vascular lesions developed randomly without regard for vessel location or size. TVS developed more regularly in vessels of the interventricular septum than in the right or left ventricular walls. The degree of TVS development fluctuated along the length of individual vessels, even as late as 90 days after transplant. The smaller vessels (

Human cytomegalovirus (CMV) has been associated with allograft rejection and, in particular, with transplant-associated arteriosclerosis. However, the role CMV plays in the development of transplant-associated arteriosclerosis remains unclear. CMV can infect the endothelium, the interface between allograft tissue and the host immune cells, but the direct induction of endothelial human leukocyte antigen (HLA) class II by CMV remains controversial. Our previous studies with venous endothelial cells (EC) have shown that CMV does not directly induce this antigen on infected EC and, furthermore, renders these cells refractory to interferon (IFN)-gamma induction. However, questions have arisen regarding the relevance of these findings to arterial endothelia. Thus, we have extended these studies to determine whether similar interactions occur in arterial EC. EC derived from human coronary artery, aorta, and umbilical artery were assayed by immunofluorescence flow cytometry and dual immunohistochemical staining following IFN-gamma treatment and/or inoculation with CMV. Data generated by these experiments demonstrate that regardless of vascular origin: (1) CMV does not directly induce endothelial surface or cytoplasmic HLA class II, and (2) although uninfected arterial EC are HLA class II inducible by IFN-gamma, infected cells are completely refractory to this effect. These results suggest that CMV-mediated inhibition of HLA class II expression is a phenomenon shared by human arterial and venous endothelia of both fetal and adult origin.

Background. Transplant vascular sclerosis is expressed in transplanted human and murine hearts as a concentric intimal thickening. The purpose of this study was to characterize the location, distribution, and intensity of transplant vascular sclerosis in murine cardiac allografts using computerized morphometric analysis. Methods. Murine cardiac allograft recipients were treated with the immunosuppressant gallium nitrate to promote graft survival. The grafts were removed at 60 days after transplantation and histologically stained. The coronary arteries were analyzed for intimal thickening using a neointimal index (NI) derived with a computer imaging system. Results. A cross-section taken from the middle of a cardiac allograft showed four major coronary arteries, each with widely different NI values (65, 0, 92, and 0). The same four vessels in two other grafts also showed highly variable NI values, but different patterns of vessel involvement. Next, NI values were determined along the length of a single vessel from aorta to apex. This revealed variable, fluctuating intimal thickening along the length of the vessel. In general, arteries from the aortic versus apical regions of the grafted hearts expressed similar amounts of intimal thickening (analysis of variance, P=0.4826). Finally, a method was devised to quantitate intimal thickening from a sampling of three tissue cross-sections taken from the middle of each cardiac allograft. This value was statistically indistinguishable from values obtained by analysis of intimal thickening in multiple sections covering the entire heart (P=0.6734, 0.9021, and 0.1474). Conclusions. Intimal thickening in the coronary arteries of murine cardiac allografts appears to be variable in terms of location, distribution, and intensity. This is true for different regions of the same vessel, different vessels in the same heart region, and the same vessels in different cardiac allografts.

We have treated DBA/2-->C57BL/6 murine cardiac allograft recipients with anti-CD4 monoclonal antibody or with gallium nitrate to promote long-term (>60 days) allograft survival. Within this period, all grafts developed histologic evidence of ongoing vascular and parenchymal tissue remodeling, including interstitial fibrosis and neointimal hyperplasia, which are characteristic of chronic allograft rejection. To evaluate residual alloimmunity associated with the pharmacologic avoidance of acute graft rejection and the development of chronic tissue remodeling, we subjected these graft recipients to a battery of histologic and immunologic tests. Similar test results were obtained for graft recipients treated with either of the two immunosuppressive agents. All long-surviving allografts displayed histologic evidence of ongoing microvascular endothelial activation and interstitial leukocytic infiltration. Reverse transcriptase-polymerase chain reaction analyses demonstrated intragraft expression of mRNAs for interleukin (IL)-1, IL-2, IL-4, IL-6, tumor necrosis factor, interferon-gamma, and transforming growth factor-beta. All recipients had limiting dilution analysis-detectable, graft-reactive cytolytic T lymphocytes and helper T lymphocytes in their spleens and grafts, and all produced high titers of graft-reactive alloantibodies. In general, these observations indicate that (1) a similar immune status is achieved in long-surviving allografts and their recipients when either anti-CD4 monoclonal antibody or gallium nitrate was used for antirejection therapy, (2) this immune status is characterized by continuous, long-term inflammatory and immune processes that are qualitatively similar to those observed during acute allograft rejection, and (3) no specific immune responses developed selectively in long-term graft recipients to account for the avoidance of acute graft rejection or the development of chronic tissue remodeling in the graft.

We report a case of rapidly progressive diabetic nephropathy, from little diabetic change on renal biopsy to severe, nodular, diabetic nephrosclerosis over 32 months. The patient was taking an angiotensin converting enzyme inhibitor and had a mean arterial pressure of 95 mm Hg over this time period. Her dietary protein intake was low, at least upon presentation. She had three additional mechanisms or potential mechanisms of injury: monoclonal kappa light chains; IgA immune deposits on the first, but not the second biopsy; and longstanding hypertension. Her renal histology was typical for diabetic nephropathy but was not characteristic of kappa light chain disease. We suggest that diabetic nephropathy may develop more rapidly than previously assumed, especially when additional mechanisms of injury, or additional promoters of mesangial matrix accumulation are present.

Apoptosis, or the induction of programmed cell death, is a mechanism commonly used by cytotoxic T cells to cause target cell lysis. We evaluated the frequency and distribution of apoptotic cells in DBA/2-->DBA/2 heterotopic cardiac isografts, acutely rejecting DBA/2-->C57BL/6 cardiac allografts, and accepted, 60 day DBA/2-->C57BL/6 allografts from mice treated with anti-CD4 Mab (GK1.5) or gallium nitrate (GN). Apoptosis was identified in histologic sections via TUNEL analysis of nuclear DNA fragmentation. We observed the following. (1) Cardiac isografts display no detectable TUNEL+ cells. (2) Rejecting cardiac allografts display rare (

Cyclosporine (CsA) causes both acute and chronic nephrotoxicity. The goal of the present studies was to examine the nature of the chronic renal pathologic lesions of CsA nephrotoxicity and to determine whether these lesions progress with prolonged exposure to the drug. With this purpose, we examined large sections of kidneys obtained at autopsy from 15 heart transplant recipients, 7 liver transplant recipients, and 10 nontransplanted controls. Tissue sections were examined blindly by light microscopy, histomorphometry, and color computer image analysis. With increasing time following transplantation, there was a progressive increase in renal arteriolar hyalinosis and in the percentage of glomeruli demonstrating global sclerosis. In addition, there was a statistically significant relationship between arteriolar hyalinosis and global glomerulosclerosis (r=0.61, P

Loin pain-hematuria syndrome associated with thin glomerular basement membrane disease and hemorrhage into renal tubules. Loin painhematuria (LPH) syndrome is a poorly understood disorder in which the patients, mainly young women, experience unexplained severe chronic unilateral or bilateral flank pain associated with gross and/or microscopic hematuria. By contrast, thin glomerular basement membrane (GBM) disease is generally thought to be a benign disorder, affecting males and females equally, in which the major manifestation is asymptomatic microscopic hematuria. Herein we describe seven patients (6 females, 1 male) in whom thin GBM appeared to be the cause of the LPH syndrome. The gross hematuria in these patients could be attributed to thin GBM disease because the renal biopsy demonstrated red cells in renal tubules (indicating glomerular hematuria) and the only glomerular abnormality present was thin GBM. In addition, the other causes of gross hematuria were excluded by appropriate testing. The flank pain in these patients might also have been the result of their thin GBM disease. This is suggested by renal biopsy findings of multiple renal tubules filled with red cells, apparently occluding the tubules. We suggest that occlusion of a relatively small fraction of renal tubules could cause renal pain if back-leak of glomerular filtrate occurred that was of sufficient magnitude to expand renal parenchymal volume and stretch the renal capsule. Preliminary observations suggest that treatment with the angiotensin converting enzyme (ACE) inhibitor enalapril importantly reduces the frequency and severity of the episodes of gross hematuria and flank pain in most patients. ACE inhibition might decrease glomerular hemorrhage in patients with thin GBM by decreasing glomerular hydrostatic pressure. We conclude that (1) Thin GBM disease can be the cause of gross hematuria, apparently as a result of rupture of thin GBM. (2) Rupture of thin GBM resulting in hemorrhage into renal tubules may be the cause of the flank pain and gross hematuria in some patients with the LPH syndrome.

We have investigated the vascular endothelial phenotypes found at various times posttransplant in murine B10-->C3H liver grafts. In this model, liver allografts are spontaneously accepted, and survive indefinitely unless the recipient is first allosensitized with a skin allograft, in which case the liver allografts are rejected within five days. In our previous studies, allograft inflammation was associated with the development of vascular endothelial reactivity with the mAbs MECA-32 and M/K-2 (anti-VCAM-1). We observed that vascular endothelia in both liver isografts and allografts develop reactivity with MECA-32 mAb within two days of transplantation, indicating endothelial activation in both situations. In contrast, only the endothelia in liver allografts develop VCAM-1 expression, as detected with M/K-2 mAb. VCAM-1 was expressed in both rejecting and accepting liver allografts, demonstrating that endothelial VCAM-1 expression is indicative of ongoing graft inflammation but not necessarily graft rejection. Liver parenchymal cells did not appear to develop reactivity with either antibody under any of the conditions tested. In contrast, bile duct epithelia developed M/K-2 reactivity (VCAM-1 expression), but not MECA-32 reactivity in liver allografts, but not isografts. These data demonstrate alloantigen-dependent and alloantigen-independent patterns of endothelial behavior in murine liver grafts that are quite similar to those found in murine cardiac grafts. Further, they demonstrate that the expression of VCAM-1 by graft endothelia is not diagnostic for acute rejection of liver allografts.

Association of thin glomerular basement membrane with other glomerulopathies. In the present study we assessed the prevalence of thin glomerular basement membrane (TGBM) in a large group of native kidney biopsies done for evaluation of renal disease. TGBM was present in 54 of 1078 biopsies (5%). In 12 of 54 biopsies (24%), TGBM was the only abnormality present. In the remaining biopsies TGBM was associated with other pathologic diagnoses. The overall prevalence of TGBM in this series is comparable to previous population studies. TGBM is significantly more common in patients with IgA nephropathy and mesangial proliferative glomerulonephritis. Compared to control patients, individuals with TGBM were more likely to have a history of familial hematuria (14% vs. 43%, P = 0.02). Furthermore, examination of urinary sediments in first degree relatives of patients with TGBM demonstrated microscopic hematuria in 92% of families and, in those families, hematuria was present in 47 ± 6% of relatives. In contrast, hematuria was discovered in 38% of families of control patients, affecting 25 ± 5% of relatives. In conclusion, the presence of TGBM in a kidney biopsy is highly predictable for the presence of familial microscopic hematuria, even in patients in whom TGBM is associated with another glomerulopathy. The present data also indicate that patients with TGBM nephropathy often have concomitant IgA nephropathy and mesangial proliferative glomerulonephritis.

We have investigated the contribution of monocytes (Mo) to the activation of purified human T cells by allogeneic human vascular endothelial cells (HUVEC). We have previously demonstrated that allogeneic HUVEC stimulate IL-2 production by CD8+ helper T lymphocytes (HTL), but not CD4+ HTL, in the absence of accessory Mo. We now show that addition of responder-autologous Mo to such cultures stimulates a high frequency of CD4+ HTL (1/6500), but no additional CD8+ HTL (1/35,000), as detected by limiting dilution analysis (LDA). The CD4+ HTL production of IL-2 increased with increasing numbers of Mo. Monoclonal antibodies to MHC class II interfered with HTL responses to allogeneic HUVEC in the presence, but not in the absence of autologous Mo. In contrast, IFN-gamma-treated HUVEC stimulated a high frequency of CD4+ HTL in the absence of autologous Mo. However, deletion experiments revealed that the population of HTL responsive to IFN-gamma-treated HUVEC is distinct from the population that responds to HUVEC in the presence of autologous Mo. These data suggest that Mo promote IL-2 production by presenting HUVEC-derived alloantigens via MHC class II molecules to CD4+ HTL, rather than by providing cytokines that promote more efficient IL-2 production by CD8+ HTL, or by inducing MHC class II expression on the HUVEC. In general, these data demonstrate that autologous Mo can play a significant role in the response of T cells to allogeneic HUVEC. Further, they demonstrate that the activation of human HTL by allogeneic HUVEC is complex, and can occur by at least three pathways: (1) direct stimulation of a small number of CD8+ HTL, but no CD4+ HTL, by quiescent HUVEC, (2) direct stimulation of a large number of CD4+ HTL by IFN-gamma-treated HUVEC, and (3) indirect stimulation of a different subset of CD4+ HTL by HUVEC in the presence of autologous monocytes. These three pathways of alloactivation are not unique to allogeneic HUVEC, but this experimental system provides a convenient and relevant model with which each pathway can be easily and independently investigated.

Previous studies of the in vitro effects of recombinant erythropoietin (rEPO) on T and B cells and studies of lymphocyte subsets in dialysis patients receiving rEPO therapy suggest that rEPO might augment immune responses. In the present study indices of autoimmunity (antinuclear antibody, anti-double-stranded DNA, and antiphospholipid antibody [immunoglobulins G and M]) were measured before, during, and after rEPO therapy in five systemic lupus erythematosus patients without renal failure (mean serum creatinine, 1.5 +/- 0.3 mg/dL). The rEPO therapy was self-administered by subcutaneous injection in doses ranging from 4,000 units once weekly to 3,000 units three times weekly for 3 to 7 months. On rEPO therapy, each patient experienced an increase in hematocrit. The mean baseline hematocrit increased from 32 +/- 2.0 to a peak of 42.2 +/- 3 (P0.001) at 3 to 7 months and then decreased to baseline values 1 to 2 months after rEPO was discontinued. During this time the indices of autoimmunity were not significantly changed by rEPO therapy. Systemic lupus erythematosus activity, assessed by serum C3, serum creatinine, urinalysis, and 24-hour proteinuria, also was unchanged by rEPO therapy. The rEPO therapy was generally well tolerated. However, one patient, who was also receiving replacement estrogen therapy and had high-titer antiphospholipid antibody, experienced episodes of thrombophlebitis while on rEPO therapy. In conclusion, we found no evidence that rEPO increases autoimmunity in systemic lupus erythematosus. However, we observed a temporal relationship between episodes of thrombophlebitis and rEPO therapy in a single patient with high-titer anticardiolipin antibody who was also receiving replacement estrogen therapy. These associations require further investigation.

Most previous studies to evaluate endothelial cell-T lymphocyte interactions have used human peripheral blood as a source of T lymphocytes and human umbilical vein endothelial cells as a source of endothelia. Implicit in this experimental system are allogeneic lymphocyte-endothelial interactions, which are largely ignored. To overcome this problem, we isolated gonadal vein endothelial cells (GVEC) along with matched splenic macrophages and T lymphocytes from cadaveric donors, thus providing a completely autologous series of cells for experimentation. First, GVEC were analyzed for morphology, surface phenotype, and cytokine mRNA expression, and found to be indistinguishable from human umbilical vein endothelial cells. Using this system, we observed that irradiated GVEC were able to promote a 2- to 3-fold increase in the proliferation of matched autologous splenic T cells after PHA stimulation. This indicates that the costimulator activity of endothelial cells reported by others is an intrinsic property of endothelial cells, and is not a consequence of endothelial alloantigens. We also used this system to assess the relative abilities of GVEC and macrophages obtained from the same donor to stimulate the proliferation of purified allogeneic CD3+ PBL. We found the following hierarchy of alloantigenicity in this experimental system: splenic macrophages > IFN-gamma-treated GVEC >> untreated GVEC = TNF alpha-treated GVEC. These studies demonstrate that allogeneic macrophages are intrinsically more antigenic than endothelial cells derived from the same donor. Furthermore, they illustrate the utility of this experimental system to obtain data regarding lymphocyte-endothelial interactions that are otherwise unobtainable.

Damage to the vasculature contributes significantly to the overall pathologic consequences of acute radiation exposure. Although at least part of this damage may be accountable to inflammatory reactions involving leukocyte/endothelial interactions, little is known of post-irradiation endothelial expression of the immunologically relevant surface molecules which are intimately involved in these processes. To address this issue, we have employed immunofluorescence flow cytometry to examine the effects of γ-radiation on the constitutive and cytokine-induced expression of HLA class I, HLA class II, and intercellular adhesion molecule-1 (ICAM-1) by cultured human endothelial cells (EC). EC were -γ-irradiated (137Cesium, 1.2 Gray [GyJ/min) in confluent monolayer at doses up to 100 Gy, treated with interferon7 (IFNγ, 200 IU/ml, 72 hr), tumor necrosis factor α (TNFα, 40 IU/ml, 24 hr), or incubated in cytokine-free medium, then harvested and reacted with fluorescein-labelled monoclonal antibodies with specificitie...

CMV has been associated with allograft rejection and transplantation-associated arteriosclerosis. CMV infects endothelium, the interface between allograft tissue and the host immune system. Although endothelial HLA class II expression is a hallmark of vascular rejection, CMV does not directly induce these antigens on infected endothelial cells (EC) and, indeed, renders them refractory to HLA DR induction by IFN-gamma. Our earlier studies have demonstrated, however, that CMV-infected EC are capable of eliciting vigorous activation responses by allogeneic, CMV-seropositive donor-derived CD4+ T cells. We now show that T cells thus activated can induce HLA DR expression on noninfected EC. Human umbilical vein endothelial cells (HUVEC) were inoculated at low titer with CMV strain VHL/E, cocultured with allogeneic, CMV-seropositive or CMV-seronegative donor-derived CD4+ T cells, then dual immunohistochemically stained for CMV antigen and HLA DR, or assayed for HLA DR expression by fluorescence flow cytometry. Results demonstrated that HLA DR was induced in 20-70% of HUVEC in monolayers containing less than 0.5% CMV-infected EC after coculture with CMV-seropositive donor-derived T cells. No such induction was observed in experiments employing T cells isolated from CMV-seronegative individuals. Expression of this antigen was strictly limited to noninfected cells. Rare HLA DR induction was observed in virus-free cocultures. To determine whether endothelial HLA DR was induced by a soluble factor(s) elaborated by activated T cells, noninfected HUVEC monolayers were treated for 72 hr with cell-free supernatant from CMV-infected or noninfected HUVEC/T cell cocultures and assayed as above. Again, HLA DR expression was induced by supernatant from CMV-positive cocultures (only when cocultured T cells were isolated from CMV-seropositive donors), but not by supernatant from CMV-negative cocultures or from T cells cultured alone. The soluble factor was identified as IFN-gamma by the complete attenuation of HLA DR induction by anti-IFN-gamma mAb. These findings suggest that allograft endothelium harboring low level CMV may be capable of eliciting host CD4+ T cell activation, and that consequent release of IFN-gamma is capable of inducing endothelial HLA class II expression, as observed in vascular rejection and transplantation-associated arteriosclerosis. Results of these studies thus provide insight into mechanisms that may help elucidate the association between CMV and rejection-related immune events in the allograft.

We have employed a murine model of cardiac transplantation and two monoclonal antibodies, M/K-2 and MECA-32, to study the responses of graft endothelia during allograft rejection. Using immunohistologic techniques, we demonstrate that the monoclonal antibody M/K-2, which binds to the murine cellular adhesion molecule VCAM-1, reacts with an inducible endothelial epitope found in rejecting cardiac allografts, but not in cardiac isografts, normal cardiac tissues, or extracardiac vasculature from allografted mice. Similar, but focal, M/K-2 reactivity is also found in nontransplanted hearts undergoing virally induced myocarditis. M/K-2 reactivity does not develop in the nonrejecting cardiac allografts from nu/nu mice, and M/K-2 reactivity is found only in grafts that develop CD25+ graft-infiltrating cells--i.e., allografts but not isografts. PCR analyses of grafts during development of VCAM-1 expression indicate that allografts, but not isografts, contain mRNA for the cytokines IL-2 and IFN-gamma, and either of these cytokines may be associated with the expression of M/K-2 reactivity in rejecting allografts. Unlike M/K-2, MECA-32 identifies an inducible epitope that is observed on myocardial endothelia of both isografts and allografts, but not normal cardiac tissues. Further, expression of the MECA-32 epitope can occur in grafts that do not develop CD25+ infiltrating lymphocytes, since it is observed in isografts and the native hearts of transplanted or sham-operated mice. Indeed, MECA-32 reactivity may be T cell independent, since it is also found in nonrejecting allografts of nu/nu mice. PCR analyses of grafts during development of MECA-32 reactivity indicate that cardiac isografts contain mRNA for IL-1, IL-6, TNF, and lymphotoxin. One or more of these might be associated with induction of MECA-32 reactivity.

Cytomegalovirus, a source of serious complications among immunosuppressed individuals, infects endothelial cells in vivo, and has been epidemiologically associated with allograft rejection and transplantation-associated accelerated arteriosclerosis (TxAA). As the interface between the host immune system and allograft tissues, the endothelium may be of primary importance in mediating pathogenic immune responses to CMV. We have therefore investigated T lymphocyte responses to CMV-infected allogeneic human umbilical vein endothelial cells (HUVE) in vitro. Proliferation assays demonstrated dramatically enhanced responses by CMV-seropositive donor-derived PBMC or purified T cells cocultured with CMV-infected HUVE, as compared with those elicited by noninfected stimulator cells. As determined by limiting dilution analysis of IL-2-producing cells, the frequency of T cells responding to infected HUVE was generally found to exceed by approximately one order of magnitude those responding to uninfected cells. Similar analyses of isolated T cell subsets revealed that these responses (proliferation and IL-2 production) were nearly entirely accountable to the CD4+ fraction. Responses of CD8+ populations, however, varied among donors. The marked activation of CD4+ cells is particularly intriguing since we have shown that CMV-infected HUVE do not express HLA class II antigens. Responses of lymphocytes derived from CMV-seronegative donors were minimal in all assays. These studies show that purified T cells can respond to CMV in the exclusive context of allogeneic endothelial cells. Furthermore, we demonstrate the heretofore undescribed phenomenon of CD4+ T cell activation in the absence of serologically detectable HLA class II antigen. As vascular rejection and TxAA are characterized by subendothelial T cell infiltration, these findings support a role for CMV/endothelial interaction in their pathogenesis.