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T Mertens

Publications and source records attributed to T Mertens.

At least 19 recordsLinked to original sources
PubMed2000

Indolocarbazoles exhibit strong antiviral activity against human cytomegalovirus and are potent inhibitors of the pUL97 protein kinase.

We have analyzed a panel of protein kinase inhibitors (PKIs) and found that some indolocarbazoles (Gö6976, K252a, K252c) proved to be highly effective inhibitors of GCV-sensitive and -resistant human cytomegalovirus (HCMV) strains, but did not show any effect against herpes simplex virus. Antiviral activity was determined by focus reduction assays (IC(50) ranging from 0.009 to 0.4 microM). Other inhibitors of serine/threonine kinases (Gö6850, H-7, roscovitine) were found to be ineffective. Virus yield at 5 days after infection was reduced by three orders of magnitude with nanomolar concentrations of the indolocarbazoles. These compounds were fully effective when added up to 24 h post infection and showed reduced activity up to 72 h post infection. Cytotoxicity assays in proliferating and non-proliferating cells demonstrated that the effective antiviral concentration of these compounds was significantly lower than either antiproliferative (IC(50)/CC(50) ranging from 6.5 to 390) or cytotoxic (IC(50)/CC(50) ranging from 72. 5 to 1000) doses. The effects of PKIs on the virus-encoded protein kinase pUL97 were studied using recombinant vaccinia viruses. Indolocarbazoles strongly inhibited both pUL97 autophosphorylation (IC(50) ranging from 0.0012 to 0.013 microM) and pUL97-dependent ganciclovir phosphorylation (IC(50) ranging from 0.05 to 0.26 microM). Other inhibitors of serine/threonine kinases showed only weak (Gö6850) or no (H-7, roscovitine) effect on these pUL97 functions, while oxoflavone tyrosine kinase inhibitors had no effect at all.

Antiviral Agents↗
PubMed2000

Comparison between human cytomegalovirus pUL97 and murine cytomegalovirus (MCMV) pM97 expressed by MCMV and vaccinia virus: pM97 does not confer ganciclovir sensitivity.

The UL97 protein (pUL97) of human cytomegalovirus (HCMV) is a protein kinase that also phosphorylates ganciclovir (GCV), but its biological function is not yet clear. The M97 protein (pM97) of mouse cytomegalovirus (MCMV) is the homolog of pUL97. First, we studied the consequences of genetic replacement of M97 by UL97. Using the infectious bacterial plasmid clone of the full-length MCMV genome (M. Wagner, S. Jonjic, U. H. Koszinowski, and M. Messerle, J. Virol. 73:7056-7060, 1999), we replaced the M97 gene with the UL97 gene and constructed an MCMV M97 deletion mutant and a revertant virus. In addition, pUL97 and pM97 were expressed by recombinant vaccinia virus to compare both for known functions. Remarkably, pM97 proved not to be the reason for the GCV sensitivity of MCMV. When expressed by the recombinant MCMV, however, pUL97 was phosphorylated and endowed MCMV with the capacity to phosphorylate GCV, thereby rendering MCMV more susceptible to GCV. We found that deletion of pM97, although it is not essential for MCMV replication, severely affected virus growth. This growth deficit was only partially amended by pUL97 expression. When expressed by recombinant vaccinia viruses, both proteins were phosphorylated and supported phosphorylation of GCV, but pUL97 was about 10 times more effective than pM97. One hint of the functional differences between the proteins was provided by the finding that pUL97 accumulates in the nucleus, whereas pM97 is predominantly located in the cytoplasm of infected cells. In vivo testing revealed that the UL97-MCMV recombinant should allow evaluation of novel antiviral drugs targeted to the UL97 protein of HCMV in mice.

Animals↗
PubMed1999

Amino acids of conserved kinase motifs of cytomegalovirus protein UL97 are essential for autophosphorylation.

Thirteen point mutations targeting predicted domains conserved in homologous protein kinases were introduced into the UL97 coding region of the human cytomegalovirus. All mutagenized proteins were expressed in cells infected with recombinant vaccinia viruses (rVV). Several mutations drastically reduced ganciclovir (GCV) phosphorylation. Mutations at amino acids G340, A442, L446, and F523 resulted in a complete loss of pUL97 phosphorylation, which was strictly associated with a loss of GCV phosphorylation. Our results confirm that in rVV-infected cells pUL97 phosphorylation is due to autophosphorylation and show that several amino acids conserved within domains of protein kinases are essential for this pUL97 phosphorylation. GCV phosphorylation is dependent on pUL97 phosphorylation.

Amino Acid Sequence↗
PubMed1999

Altered expression of extracellular matrix in human-cytomegalovirus-infected cells and a human artery organ culture model to study its biological relevance.

The influence of human cytomegalovirus (HCMV) on the transcription of 11 selected, representative extracellular matrix genes was investigated in cell culture. Northern blot hybridization indicated the downregulation of all mRNAs investigated. Based on our results and the known repression of other extracellular matrix transcripts and the beta-actin transcription during HCMV infection, we suggest that one molecular mechanism contributing to the cytopathic effect may be the transcriptional downregulation of genes encoding proteins involved in cell structure and intercellular connection. To further study the biological relevance of this and other pathogenetic mechanisms, we established a human renal artery organ culture system and characterized this new infection model for HCMV. Our model is a new suitable system for the investigation of molecular as well as functional consequences of HCMV infection in a more physiological microenvironment.

Cells, Cultured↗
PubMed1999

[All around the deathbed of Lubbert ten Busch. The Modern Devotion and the plague in Deventer in 1398].

The seriousness of plague epidemics can be expressed in numbers and medical terms, but we get closer to the past, and it becomes our own history more than in any other way, when we can empathise with the story of a single individual. In the summer months of 1398, the plague raged in the city of Deventer, which lies on the river IJssel, in the east of the present-day Netherlands. This plague epidemic also threatened the small community of priests and minor clerics which gave rise to the new spiritual movement of the Modern Devotion. This new community was concerned about its survival. Their vocation required that the brethren should help the citizens who remained behind. However, this could prove to be their undoing, and therefore it would be better to leave the city. They resolved the dilemma by dividing into two groups. Half of the brethren left to ensure the continuity of the community; the other half stayed in Deventer to help the people. The two groups stayed in contact by means of letters, the text of which has survived in several sources. The fears and forebodings became reality. The plague also affected the new community of brethren. The deputy rector, Lubbert ten Busch, also died of the plague. There is a letter from him which he wrote just before he died. This farewell letter was sent together with a letter from one of the brethren, saying that Lubbert had meanwhile died, and describing the scene of his death. Because of the personal tone and the tragic of content, the letters about the death of Lubbert ten Busch are unique medieval documents. They give a good insight into the way in which the plague could personally affect someone and his immediate companions in the late Middle Ages. This paper focuses attention on these letters, and reconstructs the events around this death, so that the letters speak to us once again.

Catholicism↗
PubMed1998

Higher detection rate of hepatitis G and C virus RNA in liver tissue than in serum of deceased injection drug users.

To examine the prevalence of hepatitis G virus (HGV) and hepatitis C virus (HCV) infections in deceased injection drug users and for comparison of the detection rates of HGV and HCV RNA in liver tissue with detection rates in postmortem serum samples, RT-PCR was performed in 50 drug abuse-related fatalities. HGV RNA was detectable in liver tissue samples from 17/50 suddenly deceased drug abusers (34%). In 16 of these 17 positive cases, serum samples were also available but HGV RNA was detected in only 10. From 29/50 anti-HCV positive individuals, HCV RNA was detected in 23/50 liver tissue samples (46%), but HCV RNA was detectable in only 6/22 of the corresponding serum samples. In 12 anti-HCV positive cases (10 being also positive for HCV RNA in the liver), the examinations revealed a coinfection with HGV by detection of HGV RNA in the liver tissue samples. A significant association between the detection of HCV RNA in the liver and the occurrence of antibodies against the HCV NS4 protein, but not against HCV core antigen or NS3 protein was observed. The probability of anti-HCV and HCV RNA positivity increased with the age of the individuals. No HGV or HCV infection was detected in a control group of 50 persons who died suddenly by violent impact. The prevalence of active HCV and HGV infections in injection drug users detected by RT-PCR in liver tissue is in good accordance with data obtained from sera from living injection drug users. In contrast, the detection rate in postmortem serum samples was clearly lower. Possible reasons for this observation are discussed and the use of liver tissue for postmortem detection of hepatitis virus RNA is recommended.

Adult↗
PubMed1998

HCV and HGV in B-cell non-Hodgkin's lymphoma.

BACKGROUND/AIMS: A causative role of hepatitis C virus infection (HCV) has been discussed in the pathogenesis of mixed cryoglobulinaemia and in B-cell non-Hodgkin's lymphoma. No data are available concerning the newly discovered hepatitis G virus (HGV) and extrahepatic manifestations such as haematological malignancies. But, HCV and HGV most probably belong to the same family of Flavivirus. Consequently, we looked for the prevalence of HCV, HGV and cryoglobulins in patients with B-cell non-Hodgkin's lymphoma. METHODS: Serum samples from 69 patients with non-Hodgkin's lymphoma were studied. Diagnosis of non-Hodgkin's lymphoma was established according to the Kiel classification. Active HCV- and HGV infections were investigated using polymerase chain reaction for detection of viral RNA. Cryoglobulins were detected from serum and monoclonal immunoglobulin components were analysed with immunofixation electrophoresis. In addition, we assessed the clinical course of HCV- and HGV-infected patients under chemotherapy. RESULTS: Three of 69 (4.3%) patients with B-cell non-Hodgkin's lymphoma were HCV-infected and nine non-Hodgkin's lymphoma patients (13.0%) were positive for hepatitis G virus RNA. All HGV infected patients were suffering from low-grade non-Hodgkin's lymphoma. No HGV-infected patient was co-infected by HCV and neither HCV- nor HGV-infected patients showed clinical signs of chronic liver disease before, during or after chemotherapy. Serum samples from all patients were devoid of cryoglobulins. CONCLUSIONS: HCV seems to have no significance for the pathogenesis of non-Hodgkin's lymphoma in Germany. The increased prevalence of hepatitis G (16.3%) in patients with low-grade non-Hodgkin's lymphoma could suggest a pathological consequence of HGV infection outside of the liver. Evidence of clinically relevant hepatic disease in HGV infected patients was not obtained. Further, chemotherapy does not seem to affect the subsequent clinical course of HGV infection.

Adult↗
PubMed1998

Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself.

In order to identify functional regions of the human cytomegalovirus protein pUL97 (i) different 5' fragments of the UL97 open reading frame (ORF) were fused to the coding region of the green fluorescent protein and (ii) recombinant vaccinia viruses (rVV) were generated carrying two full-length and 11 mutated UL97 ORFs. The results indicated the presence of an N-terminal region within pUL97 which changed the intracellular distribution of the fusion proteins. pUL97 was localized in the nucleus, but not in the nucleoli, and was detected in the nuclear matrix fraction. Expression of all pUL97 mutants could be confirmed by Western blot analysis. pUL97-associated ganciclovir (GCV) phosphorylation in rVV-infected cells, determined quantitatively by HPLC analysis, was abolished completely using individual UL97 deletion mutants. Phosphorylation of full-length and some of the mutated pUL97 was detected in cells infected with the rVVs. The UL97 constructs carrying point mutations from GCV-resistant HCMV isolates at positions 460M, 520H, 594V, and the 4 aa deletion 590AACR593, also resulted in decreased but not abolished phosphorylation of GCV in the rVV system, whereas the phosphorylation of pUL97 itself was not influenced. The rVV system is a suitable method for quantitatively testing the functional relevance of pUL97 mutations.

Amino Acid Sequence↗
PubMed1998

Intracellular metabolism of the N7-substituted acyclic nucleoside analog 2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine, a potent inhibitor of herpesvirus replication.

We investigated the intracellular metabolism of S2242 (2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine), the only known antivirally active acyclic nucleoside analogue with the side chain substituted at the N7 position of the purine ring. Uptake of S2242 by CEM cells increased linearly with increasing extracellular concentrations of the compound and was blocked by inhibitors of nucleoside transport. S2242 was phosphorylated in a time- and concentration-dependent manner to its monophosphates, diphosphates, and triphosphates. Intracellular half-life of the diphosphates and triphosphates in CEM cells was approximately 3-6 hr. A strong correlation was found between the cytostatic action of the compound and its phosphorylation in different cell lines. In accord with the findings that (1) the cytostatic potential of S2242 is reversed by deoxycytidine (dCyd) and (2) the growth of deoxycytidine kinase-deficient (dCK-) cells is refractory to the inhibitory effect of S2242, the amount of metabolites formed from S2242 in the dCK- cell line was approximately one hundredth of that in the wild-type cells. The observation that purified dCK phosphorylates S2242 to its monophosphate further corroborates these results. The activity of S2242 against herpes simplex virus, varicella-zoster virus, and human herpesvirus type 6 was reversed by 50-100-fold on the addition of exogenous dCyd. Compound S2242 was not preferentially phosphorylated in herpes simplex virus 1-, varicella-zoster virus-, or human herpesvirus type 6-infected cells (Vero, human embryonic lung, and HSB-2 cells, respectively), and exogenously added dCyd reduced substantially the formation of S2242 metabolites in these cells. In human cytomegalovirus (HCMV)-infected human embryonic lung cells, a 5-25-fold increase in S2242 metabolite formation was observed compared with the noninfected cells, suggesting that an HCMV-encoded or -induced enzyme causes the specific phosphorylation of S2242. Exogenously added dCyd had little effect on the activity of S2242 against HCMV and on the phosphorylation of the compound in HCMV-infected cells. S2242 was not specifically phosphorylated by the HCMV-encoded UL-97 kinase in cells infected with a vaccinia/UL-97 recombinant. S2242 was found to be a substrate (K(m) = 90 microM) for purified human deoxyguanosine kinase; the latter enzyme was stimulated 3-4-fold in HCMV-infected cells.

Alkaline Phosphatase↗
PubMed1997

Phosphorylation of aciclovir, ganciclovir, penciclovir and S2242 by the cytomegalovirus UL97 protein: a quantitative analysis using recombinant vaccinia viruses.

We used recombinant vaccinia viruses (rVV) containing the UL97 open reading frame (ORF) of the human cytomegalovirus (HCMV) to investigate the UL97-dependent phosphorylation of different nucleoside analogs. The rVV T1 expressed the wild-type UL97 protein whereas rVV A5 contained a 12 bp deletion in the UL97 which had been known to be responsible for resistance of HCMV to ganciclovir (GCV). The rVV T1opal was generated which contained a stop codon at position 1089 of the UL97 ORF and which expressed a truncated UL97 protein. We quantitatively analyzed the capability of these rVVs to phosphorylate GCV, penciclovir (PCV), aciclovir (ACV) and 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl] purine (S2242) as well as the natural nucleosides deoxycytidine and deoxythymidine. Moreover, we compared their phosphorylating capability with that of herpes simplex virus type 1 strains. In thymidine kinase (TK)-deficient 143B cells infected with rVV T1, the three compounds GCV, ACV and PCV were phosphorylated with different efficiency whereas in cells infected with the rVV A5 a markedly reduced but not completely abolished phosphorylation of these compounds was observed. In rVV T1opal-infected cells no specific phosphorylation of the compounds was detectable at all. Neither S2242 nor the natural substrates of TKs were phosphorylated by any of the vaccinia recombinants. The rVVs proved to be a suitable tool for analysis of UL97-dependent phosphorylation of nucleoside analogs and also allowed to quantitatively study the influence of UL97 mutations on drug phosphorylation.

Acyclovir↗
PubMed1997

Dirt and diarrhoea: formative research in hygiene promotion programmes.

Investment in the promotion of better hygiene for the prevention of diarrhoeal diseases and as a component of water and sanitation programmes is increasing. Before designing programmes capable of sustainably modifying hygiene behaviour in large populations, valid answers to a number of basic questions concerning the site and the intended beneficiaries have to be obtained. Such questions include 'what practices favour the transmission of enteric pathogens?', 'what advantages will be perceived by those who adopt safe practices?' and 'what channels of communication are currently employed by the target population?' A study of hygiene and diarrhoea in Bobo-Dioulasso, Burkina Faso, used a mixture of methods to address such questions. This paper draws on that experience to propose a plan of preliminary research using a variety of techniques which could be implemented over a period of a few months by planners of hygiene promotion programmes. The techniques discussed include structured observation, focus group discussions and behavioural trials. Modest investment in such systematic formative research with clear and limited goals is likely to be repaid many times over in the increased effectiveness of hygiene promotion programmes.

Adult↗
PubMed1997

Flow cytometric analysis of herpes simplex virus type 1 susceptibility to acyclovir, ganciclovir, and foscarnet.

We established a quantitative flow cytometric method for determination of herpes simplex virus type 1 (HSV-1) susceptibility to acyclovir (ACV), ganciclovir, and foscarnet in vitro. Susceptibility was defined in terms of the drug concentration which reduced the number of cells expressing HSV-1 glycoprotein C (gpC) with a fluorescence intensity of > or =10(2) by 50% (IC50). Flow cytometry allowed us to use a high (1.0) as well as a low (0.005) multiplicity of infection, and determination of the IC50 was possible after one or more viral replicative cycles. IC50s were dependent on virus input and on time postinfection. In mixture experiments, 1 to 2% resistant viruses added to a sensitive strain could be detected. The results obtained by flow cytometry showed a good qualitative correlation with those achieved by cytopathic effect inhibitory assay. However, flow cytometry might detect more quantitative differences in drug susceptibility, especially among resistant strains, as confirmed also by determination of intracellular drug phosphorylation. The mean IC50s for ACV-sensitive strains were 0.45 to 1.47 microM, and those for ACV-resistant strains were between 140 and 3,134 microM. Flow cytometric analysis was fast and accurate, automatizable, and highly reproducible. Flow cytometry may be a more powerful tool than standard cytopathic effect-based assays and could have advantages for the detection of low levels of drug resistance or mixtures of sensitive and resistant virus strains.

Acyclovir↗
PubMed1996

DNA vector constructs that prime hepatitis B surface antigen-specific cytotoxic T lymphocyte and antibody responses in mice after intramuscular injection.

We tested the efficiency of induction of immune responses to the small hepatitis B surface antigen (HBsAg) in mice by intramuscular DNA immunization using different vector constructs that allow high levels of HBsAg expression in mouse cells. The HBsAg-specific responses of class I-restricted cytotoxic T lymphocytes (CTL) and of B cells (serum antibody titers) were measured. Following the intramuscular inoculation of 'naked' DNA, five different vector constructs of 4-8 kb, that contained or did not contain an intron and/or the neo gene, in which HBsAg expression was driven by promoter sequences derived from the immediate early region of HCMV, the SV40 enhancer/promoter region, or a retroviral 3' LTR efficiently primed responses of class I-restricted CD8+ CTL precursors. In contrast, the constructs in which HBsAg expression was driven by HCMV-derived promoter sequences stimulated significantly higher levels of HBsAg-specific serum antibody titers after intramuscular DNA injection than the SV40 or MPSV vector constructs. Large (15 kb) episomal vector constructs did not stimulate CTL or antibody responses. The data demonstrate that: (i) intramuscular DNA immunization represents an efficient technique for priming CTL and antibody responses to HBsAg; (ii) many vectors can be constructed that express an immunogenic product after intramuscular inoculation of 'naked' DNA; (iii) the efficiency of the tested vector constructs to prime after DNA immunization, either a CTL response, or an antibody response, differs.

Animals↗
PubMed1996

Reactivated fulminant hepatitis B virus replication after bone marrow transplantation: clinical course and possible treatment with ganciclovir.

A female chronic hepatitis B virus carrier (HBV-DNA negative) suffered from simultaneous hepatitis B virus and cytomegalovirus reactivation after in vivo T cell depletion preceding transplantation of an in vitro T cell depleted marrow graft for treatment of acute leukaemia. Interstitial pneumonia developing after bone marrow transplantation was successfully treated with ganciclovir (day 13 until day 46). The initially unnoticed extensive hepatitis B virus replication finally led to clinical hepatitis (day 85) and liver failure (day 96). Liver transplantation was performed, but the patient died from septicaemia. Retrospective analysis of hepatitis B virus DNA revealed that the HBV replication started immediately after T cell depletion and was completely suppressed during ganciclovir administration. Screening for HBV-DNA seems to be mandatory in comparable cases, and antiviral chemotherapy should be seriously considered.

Adult↗
PubMed1996

The UL97 gene product of human cytomegalovirus is an early-late protein with a nuclear localization but is not a nucleoside kinase.

The temporal expression of the UL97 gene product during human cytomegalovirus (HCMV) infection of human foreskin fibroblasts (HFF) and subcellular localization of this protein were analyzed by using a polyclonal antiserum raised against a truncated UL97 protein of 47 kDa. The UL97 protein was detectable 16 h after infection by Western blot (immunoblot) analysis. Since only reduced UL97 expression occurred in the presence of two inhibitors of DNA replication, phosphonoacetic acid and ganciclovir, we conclude that UL97 is an early-late gene, requiring DNA replication for maximum expression. By indirect immunofluorescence, the protein could be visualized in the nuclei of virus-infected HFF 22 h after infection. Nuclear localization of the UL97 protein was also detected in thymidine kinase-deficient 143B cells infected with a recombinant vaccinia virus containing the entire UL97 open reading frame (ORF), as well as in HFF transiently expressing the entire UL97 ORF under the control of HCMV major immediate-early promoter. However, transiently expressed 5'-terminal deletion mutants of the UL97 ORF in addition showed a cytoplasmic localization of the UL97 protein, confirming the presence of a nuclear localization site in the N-terminal region of the protein. Our high-pressure liquid chromatography analyses confirmed the ganciclovir phosphorylation by the UL97 protein, but no specific phosphorylation of natural nucleosides was observed, indicating that the UL97 protein is not a nucleoside kinase. During plaque purification of recombinant UL97-deficient HCMV, this virus was growth defective; hence, we presume that UL97 may be essential for the viral life cycle.

Amino Acid Sequence↗
PubMed1996

Myocarditis--cardiomyopathy. Consensus Report of the German Association for Internal Medicine, presented at the 100th annual meeting, Wiesbaden, 13 April 1994.

On the basis of several, partially divergent definitions, myocarditis-unlike the often aetiologically unexplained (idiopathic) cardiomyopathies (dilated, hypertrophic, restrictive)-can be classified as c specific disease of the myocardium. This disease often has a known cause and can be due to a large variety of aetiological agents (viral, autoreactive, toxic, infectious processes, etc.) (1). A question still to be answered is whether there is a connection between dilated cardiomyopathy and myocarditis, i.e. whether (virus-induced) myocarditis can progress via a subacute or chronic stage to dilated cardiomyopathy. This question is of considerable diagnostic and therapeutic relevance. The following consensus report thus deals with the clinically important problems related to the diagnosis of myocarditis and dilated cardiomyopathy, examines the aetiological role of immunological mechanisms and discusses the treatment of the two diseases and prognostic factors.

Cardiomyopathy, Dilated↗