Daniel D. Sedmak

Effect of Fixatives and Tissue Processing on the Content and Integrity of Nucleic Acids

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.

Analysis of the Molecular Quality of Human Tissues An Experience From the Cooperative Human Tissue Network

The scientific usefulness of the data obtained from tissue analysis is related to specimen quality, which may be affected by conditions that may contribute to the degradation of the specimen before processing and analysis. We determined the usability of nucleic acids extracted from banked human tissues for further molecular analyses. We assayed 151 tissue specimens, storedfor various times at 4 divisions of the Cooperative Human Tissue Network, National Cancer Institute, Bethesda, MD, for DNA and RNA degradation. Simple electrophoresis, polymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, and Northern blot analysis were compared to determine the optimal quality control procedure. In addition, a time course degradation procedure was performed on human lung tissue. Gel electrophoresis was as informative as PCR, RT-PCR, and Northern blot analysis in determining the molecular usefulness of the human tissues. Overall, 80% of the stored human tissues had good-quality DNA, and 60% had good-quality RNA. Electrophoresis procedures for DNA and RNA offer a quick and valuable measure of the molecular quality of stored human tissues. The DNA and RNA degradation of one tissue type (lung) was stable for both nucleic acids for up to 5 hours after excision.

Novel mechanism of inhibition of cytomegalovirus by the experimental immunosuppressive agent leflunomide.

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.

Inhibition of Cytomegalovirus in vitro and in vivo by the Experimental Immunosuppressive Agent Leflunomide

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.

Lipopolysaccharide, Tumor Necrosis Factor Alpha, or Interleukin-1β Triggers Reactivation of Latent Cytomegalovirus in Immunocompetent Mice

ABSTRACT 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.

Progressive histologic injury in kidneys from heart and liver transplant recipients receiving cyclosporine

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<0.003). The percentage of glomeruli with focal glomerulosclerosis was also increased in transplant recipients followed for more than 80 days. The kidneys of heart and liver recipients who died less than 80 days after transplantation, that is, those patients who received no CsA or low doses of CsA, demonstrated significantly more interstitial fibrosis than the kidneys of control individuals. Furthermore, there was no significant increase in the amount of renal interstitial fibrosis in allograft recipients followed for prolonged periods of time. There were no significant relationships between the severity of renal pathologic changes and serum creatinine or systemic blood pressure levels. In conclusion, non-renal transplant recipients demonstrate renal damage that affects primarily the preglomerular arterioles and results in glomerular obliteration. This renal damage increases in relation to the time of exposure to CsA and in relation to the total dose of CsA that the patient receives. These renal structural changes are not reflected initially in changes in glomerular filtration rate, as determined by serum creatinine.

Morphometric analysis of neointimal formation in murine cardiac allografts.

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.

Prevalence of hepatitis C in patients with idiopathic glomerulopathies in native and transplant kidneys

Previous studies suggest that there is an association between hepatitis C (HCV) infection and glomerular diseases in native and transplanted kidneys. However, the data are controversial. To reexamine this issue, we determined the prevalence of serum anti-HCV antibodies in patients with glomerulopathies of native kidneys (n = 105) and in patients with acute and chronic transplant glomerulopathy (TxGN) (n = 62). Compared with a control group of patients with diabetic nephropathy (n = 37, 0% HCV+), the prevalence of HCV antibodies was significantly higher in patients with focal glomerulosclerosis (FGS) (4 of 32, 13%, P = 0.04 by chi-square), but not in patients with membranous nephropathy (MGN) (1 of 19, 5%) or in patients with membranoproliferative glomerulonephritis (MPGN) (2 of 17, 12%). All of the patients with positive HCV serology had histories of intravenous (IV) drug use. Thus, HCV serology was negative in all of the patients with native glomerulopathies without histories of IV drug use. Compared with a group of 105 transplant patients without TxGN (1.8% HCV+), the prevalence of HCV antibodies was significantly higher in patients with acute (A)TxGN (12 or 41: 29%. P = 0.0004) and in patients with chronic (C)TxGN (9 of 27: 33%. P = 0.0004). Compared with controls, patients with ATxGN also had a significantly higher prevalence of serum immunoglobulin (Ig) M antibodies to cytomegalovirus (CMV) (3% and 26% of patients, respectively, P = 0.0004). However, there were no statistical associations between HCV and CMV serologies. These results do not support the postulate that HCV infection is associated with idiopathic native glomerulopathies; instead, the data suggest that the presence of HCV positivity in these patients can be explained by the inclusion of patients with a history of IV drug use. In contrast, these studies demonstrate for the first time an association between HCV infection and transplant glomerulopathies.

Staphylococcus Infection-Associated Glomerulonephritis Mimicking IgA Nephropathy

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.

The high prevalence of severe early posttransplant renal allograft pathology in hepatitis C positive recipients.

In the USA approximately 10% of candidates for renal transplantation have serum antibodies to hepatitis C (HCV). To assess the possible impact of HCV infection on early posttransplant events we assessed allograft complications during the first 6 months following renal transplantation in three groups of adult renal allograft recipients: (1) HCV antibody positive recipients (R-HCV) (n=32); (2) HCV negative recipients who received kidneys from HCV antibody positive donors (D-HCV) (n=48); and (3) HCV negative recipients of HCV negative allografts who were transplanted during the same time period as R-HCV (Ctrl) (n=204). Allograft biopsies were done for evaluation of allograft dysfunction during the first 6 months posttransplant in 58% of Ctrl, 42% of D-HCV, and 63% of R-HCV (not significantly different). The prevalence of acute tubulointerstitial rejection was similar among the 3 groups of patients. In contrast, compared with Ctrl, both R-HCV and D-HCV had a significantly higher prevalence of acute transplant glomerulopathy (Ctrl, 6%; R-HCV, 55%, P<.0001; D-HCV 40%, P=.0004). Acute vascular rejection was more common in R-HCV (60%) than in Ctrl (28%) (P=.009) and the prevalence of chronic vascular rejection was also higher in R-HCV (60%) than in Ctrl (31%) (P=.01). Furthermore, chronic vascular rejection was diagnosed earlier in R-HCV (64% of cases within one month posttransplantation) than in Ctrl (19% within one month) (P=.01). Death censored renal allograft losses occurred in 14% of Ctrl, 17% of D-HCV, and 26% of R-HCV (not significant). In conclusion, R-HCV patients have a high prevalence of severe acute pathologic findings in renal allograft biopsies obtained early after transplantation and develop chronic vascular rejection more often and earlier than HCV negative recipients. These studies also confirm the previously reported association of HCV with acute transplant glomerulopathy.