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R Kandolf

Publications and source records attributed to R Kandolf.

At least 55 records · Page 3Linked to original sources
PubMed1999

Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis.

BACKGROUND: Previous studies dealing with the detection of enteroviral RNA in human endomyocardial biopsies have not differentiated between latent persistence of the enteroviral genome and active viral replication. Enteroviruses that are considered important factors for the development of myocarditis have a single-strand RNA genome of positive polarity that is transcribed by a virus-encoded RNA polymerase into a minus-strand mRNA during active viral replication. The synthesis of multiple copies of minus-strand enteroviral RNA therefore occurs only at sites of active viral replication but not in tissues with mere persistence of the viral genome. METHODS AND RESULTS: We investigated enteroviral RNA replication versus enteroviral RNA persistence in endomyocardial biopsies of 45 patients with left ventricular dysfunction and clinically suspected myocarditis. Using reverse-transcriptase polymerase chain reaction in conjunction with Southern blot hybridization, we established a highly sensitive assay to specifically detect plus-strand versus minus-strand enteroviral RNA in the biopsies. Plus-strand enteroviral RNA was detected in endomyocardial biopsies of 18 (40%) of 45 patients, whereas minus-strand RNA as an indication of active enteroviral RNA replication was detected in only 10 (56%) of these 18 plus-strand-positive patients. Enteroviral RNA was not found in biopsies of the control group (n=26). CONCLUSIONS: These data demonstrate that a significant fraction of patients with left ventricular dysfunction and clinically suspected myocarditis had active enteroviral RNA replication in their myocardium (22%). Differentiation between patients with active viral replication and latent viral persistence should be particularly important in future studies evaluating different therapeutic strategies. In addition, molecular genetic detection of enteroviral genome and differentiation between replicating versus persistent viruses is possible in a single endomyocardial biopsy.

Adult↗
PubMed1999

Hydrocution in a case of Coxsackie virus infection.

An apparently healthy 7-year-old boy attempted to demonstrate his ability to dive into a whirlpool but was retrieved from the water in a state of unconsciousness after several minutes. Resuscitation was unsuccessful. No characteristic signs of drowning were found at the autopsy but examination of the lymph nodes and the cardiac muscle indicated a pre-existent infection. The histological examination revealed a slight degree of predominantly lymphocytic infiltration of the cardiac muscle. IgM antibodies against Coxsackie virus were detected in the serum sample by means of ELISA. The reverse transcriptase polymerase chain reaction (RT-PCR) performed on an extract of formalin-fixed, paraffin-embedded cardiac muscle tissue revealed a DNA sequence specific for Coxsackie B3 virus. Therefore, cardiac failure was due to a myocardial virus infection and the additional strain caused by diving. This case report emphasizes the importance of modern molecular biological methods in cases of sudden death including death by hydrocution.

Antibodies, Viral↗
PubMed1999

h-sgk serine-threonine protein kinase gene as transcriptional target of transforming growth factor beta in human intestine.

BACKGROUND & AIMS: Recently, the immediate early gene h-sgk was cloned as a hypertonicity-induced gene from human hepatoma cells. The aim of this study was to localize h-sgk messenger RNA (mRNA) expression in normal and inflamed intestinal mucosa and to identify potential transcriptional regulators. METHODS: h-sgk mRNA in small intestinal mucosa from healthy persons and patients with Crohn's disease was determined by in situ hybridization. Transcriptional regulation was studied by Northern blot analysis of total RNA isolated from cultured human Intestine 407, U937, and HepG2 cells. RESULTS: In normal ileum, h-sgk mRNA was selectively localized to the apical villus enterocytes, whereas no staining was detected in crypt cells. In Crohn's disease, enterocytes of the crypts expressed h-sgk and abundant h-sgk positive inflammatory cells appeared in the lamina propria. Combined h-sgk in situ hybridization and immunohistochemical analysis of CD68 antigen expression identified a part of these cells as macrophages. In addition to spatial correlation of transforming growth factor (TGF)-beta1 protein and h-sgk mRNA expression, h-sgk transcription in human Intestine 407 and HepG2 cells as well as in U937 monocytes/macrophages was strongly induced by TGF-beta1 in vitro. CONCLUSIONS: h-sgk expression in normal and inflamed intestinal mucosa may be regulated by TGF-beta1 and may contribute to the pleiotropic actions of TGF-beta1 in mucosal cell populations.

Antigens, CD↗
PubMed1999

Mechanisms and consequences of enterovirus persistence in cardiac myocytes and cells of the immune system.

In humans and experimental murine models enteroviruses, and in particular coxsackieviruses of group B (CVB), may induce chronic myocarditis associated with a persistent type of heart muscle infection. Persistent myocardial infection has been characterized by restricted viral replication and gene expression, which is capable of sustaining chronic inflammation. Altered replication and transcription of the virus, in addition to an immune response insufficient to recognize and clear infected cells entirely, are essential mechanisms for initiation and maintenance of persistent heart muscle infection. Viral cytotoxicity was found to be crucial for organ pathology both during acute and persistent infection, indicating that enterovirus myocarditis is a virus-induced rather than an immune-mediated disease. Notably, resistance to the development of persistent heart muscle infection is not linked to the H-2 haplotype of the host. In addition to persistently infected myocytes, detection of the replicative minus-strand RNA intermediate provided evidence for virus replication in lymphoid cells of the spleen, predominantly in splenic B lymphocytes, during the course of the disease. Whereas viral RNA was also detected in certain CD4+ helper T cells and Mac1+ macrophages, no enteroviral genomes were identified in CD8+ T cells. Detection of infected activated B lymphocytes both in heart tissue of CVB3-infected immunocompetent mice and syngenic SCID mice receiving splenocytes from CVB3-infected donors support the concept that B cell traffic may contribute to maintenance of chronic disease. Dissection of the diversity of viral and host-specific determinants in susceptible and resistant hosts will allow us to define the protective mechanisms that mediate resistance to the development of life-threatening acute and chronic enterovirus myocarditis.

Animals↗
PubMed1999

Cleavage of RasGAP and phosphorylation of mitogen-activated protein kinase in the course of coxsackievirus B3 replication.

Recently, we reported on tyrosine phosphorylation of distinct cellular proteins in the course of enterovirus infections (M. Huber, H.-C. Selinka, and R. Kandolf, J. Virol. 71:595-600, 1997). These phosphorylation events were mediated by Src-like kinases and were shown to be necessary for effective virus replication. That study is now extended by examination of the interaction of the adapter protein Sam68, a cellular target of Src-like kinases which has been shown to interact with the poliovirus 3D polypeptide, with cellular signaling proteins as well as the function of the latter during infection. Here, we report that the RNA-binding and protein-binding protein Sam68 associates with the p21(ras) GTPase-activating protein RasGAP. Remarkably, RasGAP is cleaved during infections with different strains of coxsackievirus B3 as well as with echovirus 11 and echovirus 12, yielding a 104-kDa protein fragment. This cleavage event, which cannot be prevented by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, may promote the activation of the Ras pathway, as shown by the activating dual phosphorylation of the mitogen-activated protein kinases Erk-1 and Erk-2 in the late phase of infection. Moreover, downstream targets of the mitogen-activated protein kinases, i.e., the p21(ras) exchange factor Sos-1 and cytoplasmic phospholipase A2, are phosphorylated with parallel time courses during infection. Activation or inhibition of cellular signaling pathways may play a general role in regulating effective enterovirus replication and pathogenesis, and the results of this study begin to unravel the molecular cross talk between enterovirus infection and key cellular signaling networks.

Adaptor Proteins, Signal Transducing↗
PubMed1998

[Syncope in 3rd degree atrioventricular block. Detection of virus genome in the myocardium].

HISTORY AND CLINICAL FINDINGS: A 28-year-old woman was admitted after syncope which had been preceded by several flulike episodes. There was no history of any other serious disease. Physical examination was unremarkable. Heart sounds were regular and normal, there were no murmurs. INVESTIGATIONS: White cell count was 9400/microliter, with a normal differential count. Erythrocyte sedimentation rate and C-reactive protein were also normal. Virus serology revealed no abnormality. The electrocardiogram (ECG) showed complete (third degree) atrioventricular (AV) block with an idioventricular rhythm of 38 beats/min and right bundle branch block pattern. TREATMENT AND COURSE: A temporary transvenous pacemaker was inserted on the first hospital day. As myocarditis was suspected a right ventricular endomyocardial biopsy was obtained. Histological and immunohistological examinations demonstrated no unequivocal findings. But molecular-biological tests revealed. Coxsackie-B3 virus genome. The pacemaker was removed on the 6th day, when the ECG had shown intermittent second degree AV block. Regular sinus rhythm with a PR interval of 0.18 s was recorded on day 12, and 24-hour ECG monitoring for several days until her discharge on the 18th day confirmed this rhythm throughout. CONCLUSION: In aetiologically undetermined disease molecular-biological techniques can be indispensable for the exact diagnosis and may be decisive for administering specific treatment.

Adult↗
PubMed1998

Transgenic expression of replication-restricted enteroviral genomes in heart muscle induces defective excitation-contraction coupling and dilated cardiomyopathy.

Numerous studies have implicated Coxsackievirus in acute and chronic heart failure. Although enteroviral nucleic acids have been detected in selected patients with dilated cardiomyopathy, the significance of such persistent nucleic acids is unknown. To investigate the mechanisms by which restricted viral replication with low level expression of Coxsackieviral proteins may be able to induce cardiomyopathy, we generated transgenic mice which express a replication-restricted full-length Coxsackievirus B3 (CVB3) cDNA mutant (CVB3DeltaVP0) in the heart driven by the cardiac myocyte-specific myosin light chain-2v (MLC-2v) promoter. CVB3DeltaVP0 was generated by mutating infectious CVB3 cDNA at the VP4/VP2 autocatalytic cleavage site from Asn-Ser to Lys-Ala. Cardiac-specific expression of this cDNA leads to synthesis of positive- and negative-strand viral RNA in the heart without formation of infectious viral progeny. Histopathologic analysis of transgenic hearts revealed typical morphologic features of myocardial interstitial fibrosis and in some cases degeneration of myocytes, thus resembling dilated cardiomyopathy in humans. There was also an increase in ventricular atrial natriuretic factor mRNA levels, demonstrating activation of the embryonic program of gene expression typical of ventricular hypertrophy and failure. Echocardiographic analysis demonstrated the presence of left ventricular dilation and decreased systolic function in the transgenic mice compared with wild-type littermates, evidenced by increased ventricular end-diastolic and end-systolic dimensions and decreased fractional shortening. Analysis of isolated myocytes from transgenic mice demonstrate that there is defective excitation-contraction coupling and a decrease in the magnitude of isolated cell shortening. These data demonstrate that restricted replication of enteroviral genomes in the heart can induce dilated cardiomyopathy with excitation-contraction coupling abnormalities similar to pressure overload models of dilated cardiomyopathy.

Animals↗
PubMed1998

Low-level expression of a mutant coxsackieviral cDNA induces a myocytopathic effect in culture: an approach to the study of enteroviral persistence in cardiac myocytes.

BACKGROUND: Enteroviral ribonucleic acids have been identified in heart muscle of a subset of patients with myocarditis and dilated cardiomyopathy as well as in a mouse model of persistent coxsackievirus B3 (CVB3) infection, suggesting that persistent viral infection along with activation of an immune response may contribute to the pathogenesis of ongoing cardiac disease and dilated cardiomyopathy in certain patients. It is still not known whether persistence of the viral genome contributes to the pathogenesis of dilated cardiomyopathy. METHODS AND RESULTS: To determine whether low-level enteroviral gene expression similar to that observed with viral persistence can induce myocytopathic effects without formation of infectious virus progeny, the full-length infectious cDNA copy of CVB3 was mutated at the VP0 maturation cleavage site. This prevented formation of infectious virus progeny. In myocytes transfected with this mutated cDNA copy of the viral genome, both positive- and negative-strand viral RNAs were detected, demonstrating that there was replication of the viral genome by the RNA-dependent RNA polymerase. The level of viral protein expression was found to be below limits of detection by conventional methods of protein detection, thus resembling restricted virus replication. Nonetheless, the CVB3 mutant was found to induce a cytopathic effect in transfected myocytes, which was demonstrated by inhibition of cotransfected MLC-2v luciferase reporter activity and an increase in release of lactate dehydrogenase from transfected cells. CONCLUSIONS: This study demonstrates that restricted replication of enteroviral genomes in myocytes in a pattern similar to that observed in hearts with persistent viral infection can induce myocytopathic effects without generation of infectious virus progeny.

Animals↗
PubMed1998

[Enteroviral myocarditis and dilated cardiomyopathy].

In situ hybridization and PCR studies have demonstrated that enteroviruses of the human picornavividae, and in particular coxsackieviruses of group B (CVB), are detectable in endomyocardial biopsies of patients with acute and chronic myocarditis, indicating the possibility of enterovirus persistence in the human heart. As well, such infections are observed in patients with end-stage dilated cardiomyopathy, suggesting an etiologic link between myocarditis and dilated cardiomyopathy. The molecular diagnosis of persistent heart muscle infection allows to differentiate myocarditis and dilated cardiomyopathy, sustained by virus persistence, from postviral immune-mediated cardiac disease. Apart from providing an etiologic diagnosis, there are therapeutic implications from in situ demonstration of myocardial enterovirus infection. As to whether antiviral therapy with interferon is capable of providing protection against enterovirus myocarditis must be determined by controlled prospective clinical studies. Immunosuppressive therapy of myocarditis appears to be justified only after exclusion of persistent heart muscle infection. Experimental studies indicate that altered viral replication strategies, the incompetence of effector mediators of local immunity to eliminate persistently infected myocardial cells as well as infection of cellular constituents of the immune system itself, are major pathogenic determinants for development and maintenance of chronic myocarditis and cardiomyopathy.

Antiviral Agents↗
PubMed1998

Detection of human papillomavirus type 16 E6/E7 oncogene transcripts in dysplastic and nondysplastic cervical scrapes by nested RT-PCR.

Infections with high-risk human papillomaviruses (e.g., HPV-16) play an important role in the development of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (IC). Continued expression of the viral E6 and E7 genes is believed to be a key factor for oncogenic transformation of infected cells. Two spliced transcripts of the E6/E7 oncogenes, termed E6*I and E6*II, can be detected by reverse transcriptase polymerase chain reaction (RT-PCR). To increase the sensitivity of detecting E6/E7 transcripts in cervical scrapes we took advantage of a nested RT-PCR (nRT-PCR) protocol. In a series of 30 HPV-16-positive patients with histologic diagnoses ranging from nondysplastic epithelium to IC, the application of nRT-PCR significantly improved the detection of E6/E7 transcripts compared to conventional RT-PCR. The prevalence of E6/E7 spliced transcripts correlated with lesion severity and the nRT-PCR protocol allowed detection of these transcripts even in nondysplastic epithelium and CIN I lesions. Therefore, in epidemiologic follow-up studies, detection of E6/E7 transcripts by nRT-PCR should prove to be a useful diagnostic tool for risk evaluations regarding the development of CIN and its progression to cervical cancer, especially in high-risk HPV-type-infected patients with CIN 0 and CIN I.

Blotting, Southern↗
PubMed1998

[Detection of enteroviral RNA in endomyocardial biopsies in inflammatory cardiomyopathy and idiopathic dilated cardiomyopathy].

The role of enteroviral myocardial infection in the development of dilated cardiomyopathy could only be substantiated after the introduction of molecular biological techniques (polymerase chain reaction, in-situ hybridization) in virological diagnostics of dilated cardiomyopathy. By using histological and especially immunohistological techniques for the detection of myocardial inflammation in patients with the tentative clinical diagnosis of dilated cardiomyopathy, a differentiation between inflammatory cardiomyopathy and idiopathic dilated cardiomyopathy on the basis of the WHO classification 1995 (31) was made. Inflammatory cardiomyopathy is defined by myocarditis in association with cardiac dysfunction and is diagnosed by established histological and especially immunohistological techniques. The combination of histological, immunohistological, and molecularbiological techniques enabled a subgroup analysis of the incidence of enteroviral myocardial RNA in patients with inflammatory cardiomyopathy in comparison to patients with idiopathic dilated cardiomyopathy. The study involved a total of 75 patients with impaired left ventricular function (EF < 50%) and the tentative clinical diagnosis of dilated cardiomyopathy. Right ventricular endomyocardial biopsies were obtained from all patients for further clarification of the cause of left ventricular functional disorder. All biopsies were analyzed for the presence of acute and chronic inflammatory myocardial alterations by histological ("Dallas" criteria) and immunohistological techniques (lymphocytic infiltrates, MHC antigen expression). Furthermore, each biopsy was examined by reverse transcriptase polymerase chain reaction (RT-PCR) in combination with Southern blot hybridization for the presence of enteroviral RNA. Active myocarditis was excluded in all patients by histological examination according to the "Dallas" criteria. Using immunohistological techniques, 26/75 patients (35%) had evidence for chronic inflammatory myocardial alterations in the sense of lymphocytic infiltrates (> or = 2,0 CD3 T-lymphocytes/ visual field at 400 magnification (HPF); > or = 7 CD3 T-lymphocytes/mm2). These patients were diagnosed as having inflammatory cardiomyopathy. To differentiate between patients with and without myocardial inflammation, cases with focal cellular infiltration and an average cell number between 2.5 and 2.0 CD3 T-lymphocytes/HPF and an increased expression of additional immune markers, i.e., MHC antigens, were not addressed in the group of patients with inflammatory cardiomyopathy. This is in contrast to Kühl et al (19). Consequently these patients were classified as patients with idiopathic dilated cardiomyopathy. These criteria of diagnosing myocardial inflammation were based on published results (20, 23, 26, 27, 49) and on our own control group (n = 85) (19) in which mean CD3 T-lymphocyte count/HPF in normal myocardial tissue were 0.7 (range 0.0-1.4). In addition, a subgroup analysis was performed of patients with a CD3 T-lymphocyte count > or = 3 CD3 T-lymphocytes/HPF (> or + 11 CD3 T-lymphocytes/mm2). The other 49/75 patients without myocardial inflammation (< 2.0 CD3 T-lymphocytes /HPF) were diagnosed as having idiopathic dilated cardiomyopathy. In 27/75 patients (36%), RT-PCR in combination with Southern blot hybridization revealed enteroviral RNA in the endomyocardial biopsies. The detection rate of enteroviral RNA did not differ between inflammatory cardiomyopathy (8/26 (31%)) and idiopathic dilated cardiomyopathy (19/49 39%)). In the subgroups of patients with a CD3 T-lymphocyte cell count > or = 3 CD3 T-lymphocytes/HPF (> or = 11 CD3 T-lymphocytes/mm2) (mean 4.4 +/-2.1 CD3 T-lymphocytes/HPF), three of the ten patients were enteroviral RNA positive (30%). In summary, the introduction of histological and immunohistological techniques in the extended diagnostics of dilated cardiomyopathy enables a subgroup analysis of the incidence of enteroviral myocardial RNA in

Adult↗
PubMed1998

Coxsackie B virus and its interaction with permissive host cells.

BACKGROUND: Observations in humans and the results of experiments on laboratory animals have provided evidence that coxsackieviruses of group B (CVB) are major etiologic agents of acute and chronic enterovirus myocarditis and various other virus-induced diseases. OBJECTIVE: This minireview briefly summarizes the investigations to elucidate various molecular mechanisms for the induction and maintenance of persistent CVB infections. With regard to the recent findings that CVB may use several different receptor proteins, this article focuses on virus-host cell interactions and the potential impact of these interactions for enteroviral replication. STUDY DESIGN: The interaction of CVB with specific cell surface proteins was analyzed in cultured cell lines and murine tissues at the level of virus attachment and virus internalization. As example for the interaction of CVB with intracellular proteins, the state of p21rasGTPase-activating protein (RasGAP) was investigated in mock-infected and CVB3-infected HeLa cells. RESULTS AND CONCLUSIONS: The experiments to elucidate the virus receptor interactions revealed the necessity to differentiate between CVB attachment proteins and proteins involved in virus internalization. Since more than one protein may be required to initiate the uptake of CVB into permissive host cells, a model of the putative interaction of these proteins within a multimeric receptor complex is proposed. It is further tempting to speculate that the presence of multiple attachment proteins may influence the tissue tropism of CVB as well as pathogenicity.

Animals↗
PubMed1998

Multiple gene expression analysis reveals distinct differences between G2 and G3 stage breast cancers, and correlations of PKC eta with MDR1, MRP and LRP gene expression.

A possible link between protein kinase C (PKC) and P-glycoprotein (P-gp)-mediated-multidrug resistance (MDR) was assumed from studies on MDR cell lines selected in vitro. The functional relevance of PKC for the MDR phenotype remains unclear, and the involvement of a particular PKC isozyme in clinically occurring drug resistance is not known. Recently, we have demonstrated significant correlations between the expression levels of the PKC eta isozyme and the MDR1 or MRP (multidrug resistance-associated protein) genes in blasts from patients with acute myelogenous leukaemia (AML) and in ascites cell aspirates from ovarian cancer patients. To extend these findings to further types of human tumours we analysed specimens from 64 patients with primary breast cancer for their individual expression levels of several MDR-associated genes (MDR1, MRP, LRP (lung cancer resistance-related protein), topoisomerase (Topo) II alpha/IIbeta, cyclin A and the PKC isozyme genes (alpha, beta1, beta2, eta, theta, and mu) by a cDNA-PCR approach. We found significantly enhanced mean values for MRP, LRP and PKC eta gene expression, but significantly decreased Topo II alpha and cyclin A gene expression levels in G2 tumours compared with G3. Remarkably, significant positive correlations between the MDR1, MRP or LRP gene expression levels and PKC eta were determined: MDR1/PKC eta (rs = +0.6451, P < 0.0001) n = 62; MRP/PKC eta (rs = +0.5454, P < 0.0001) n = 63; LRP/PKC eta (rs = +0.5436, P < 0.0001) n = 62; MRP/LRP (rs = +0.7703, P < 0.0001) and n = 62, MDR1/MRP (rs = +0.5042, P < 0.0001) n = 62. Our findings point to the occurrence of a multifactorial MDR in the clinics and to PKC eta as a possible key regulatory factor for up-regulation of a series of MDR-associated genes in different types of tumours.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Molecular typing of enteroviruses: current status and future requirements. The European Union Concerted Action on Virus Meningitis and Encephalitis.

Human enteroviruses have traditionally been typed according to neutralization serotype. This procedure is limited by the difficulty in culturing some enteroviruses, the availability of antisera for serotyping, and the cost and technical complexity of serotyping procedures. Furthermore, the impact of information derived from enterovirus serotyping is generally perceived to be low. Enteroviruses are now increasingly being detected by PCR rather than by culture. Classical typing methods will therefore no longer be possible in most instances. An alternative means of enterovirus typing, employing PCR in conjunction with molecular genetic techniques such as nucleotide sequencing or nucleic acid hybridization, would complement molecular diagnosis, may overcome some of the problems associated with serotyping, and would provide additional information regarding the epidemiology and biological properties of enteroviruses. We argue the case for developing a molecular typing system, discuss the genetic basis of such a system, review the literature describing attempts to identify or classify enteroviruses by molecular methods, and suggest ways in which the goal of molecular typing may be realized.

Encephalitis, Viral↗
PubMed1998

Apoptosis in restenosis versus stable-angina atherosclerosis: implications for the pathogenesis of restenosis.

Decreases in programmed cell death (apoptosis) may contribute to restenotic hyperplasia by prolonging the life span of intimal cells. Apoptotic events were compared in restenotic versus primary lesions, by using atherectomy samples from 16 restenotic and 30 primary human peripheral and coronary lesions from patients presenting with stable angina. We used transmission electron microscopy to identify apoptosis, quantify its frequency, distinguish apoptosis from necrosis, and relate these events to cellular composition. Smooth muscle cell (SMC) density was higher in restenotic versus primary lesions (P<0.0001), whereas the number of macrophages was significantly reduced (P<0.01) and the number of lymphocytes was lower, but not significantly (P=0.06). As the main finding, restenotic lesions contained fewer apoptotic cells compared with primary lesions (3% versus 13%, P=0.002), whereas no differences were found for cellular necrosis. With regard to cell type, the lower frequency of apoptotic cells observed in restenotic tissue was attributable to both SMCs and macrophages. The key finding of less apoptosis in restenotic versus primary lesions was in agreement with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis (2% versus 9%, P<0.001). For all lesions analyzed, significant inverse correlations were observed between the density of SMCs and the frequency of apoptotic cell death (r=-0.60, P<0.001) as well as the density of SMCs and that of macrophages (r=-0.74, P<0.001). No relationship was seen between the frequency of apoptosis and the density of macrophages. In conclusion, the data of the present study indicate that a low level of apoptosis may be an important mechanism leading to restenotic intimal lesion development after interventional procedures.

Adult↗
PubMed1998

Detection of human papillomavirus 16 transcriptional activity in cervical intraepithelial neoplasia grade III lesions and cervical carcinomas by nested reverse transcription-polymerase chain reaction and in situ hybridization.

Continued expression of the oncogenes E6 and E7 of human papilloma virus "high risk" type 16 (HPV16) initiates neoplastic transformation and maintenance of the malignant phenotype in cervical carcinoma cells. The transcriptional activity of the HPV16 E6/E7 oncogenes was investigated in HPV16-containing cervical cell lines, cervical carcinomas, and cervical intraepithelial neoplasia grade III lesions using the techniques of reverse transcription-polymerase chain reaction (RT-PCR), Southern blotting, and in situ hybridization. To facilitate detection of the full-length HPV16 E6/E7 oncogene transcript and its characteristic splice products E6*I and E6*II in cervical tissues, a nested RT-PCR (nRT-PCR) assay was designed. Specific detection of HPV E6/E7 oncogene transcripts in clinical specimens was found to be improved by nRT-PCR, being as sensitive as the combination of conventional RT-PCR and subsequent Southern blot hybridization. Regarding the progression of premalignant lesions to cervical cancer, detection of the HPV transcriptional activity by nRT-PCR may provide additional information for risk evaluations. Moreover, improvements in the amplification of HPV oncogene transcripts may also be advantageous for monitoring the activity of HPV before and after transcript-targeted gene therapy of cervical cancer.

Carcinoma↗
PubMed1998

Visualization of enteroviral replication in myocardial tissue by ultrastructural in situ hybridization: identification of target cells and cytopathic effects.

In humans as well as in various murine models, enteroviruses are capable of inducing a severe acute and chronic myocarditis, which is characterized by myocytotoxic alterations and interstitial mononuclear infiltrates. With regard to the pathogenesis of enteroviral myocarditis, coxsackievirus B3 (CVB3)-infected immunocompetent A.CA/SnJ (H-2f) mice were used as a model to trace viral plus- and minus-strand RNA during acute and chronic organ infection by ultrastructural in situ hybridization techniques. For electron microscopic detection of enteroviral RNA in myocardial tissue, a pre-embedding hybridization technique was developed and optimized for excellent conservation of structural integrity and RNA retention. Herein, we demonstrate how the virus gains access to the myocardium during viremia involving infection of the capillary endothelial cells. In myocytes, viral replication was found to be closely associated with the generation of vesicular regions and lysis of myofibrils, resulting in complete destruction of the internal architecture of the cell. In the course of acute infection, the direct cell-to-cell spread of the virus from one myocyte to the other was found to be related with filaments of the cytoskeleton. The observation of prominent cytopathic alterations in close spatial association with viral replication before the development of the reactive cellular immune response strongly implies that the loss of host cell integrity is a direct consequence of acute viral replication. In addition to myocytes, non-heart muscle cells were found to be infected during acute as well as chronic disease. Viral replication observed in myocardial fibroblasts and immune cells such as B lymphocytes proved to be associated with minor cytopathic effects. The technique of electron microscopic in situ hybridization established for the detection of viral RNA within myocardial tissue provides a powerful tool for the elucidation of molecular and structural interrelationships in organ pathology.

Animals↗