Reinhard Kurth

Inhibition of HIV-1 replication and NF-kappa B activity by cysteine and cysteine derivatives.

HIV-1 proviral DNA contains two binding sites for the transcription factor NF-x B. HIV-1-infected individuals have, on average, abnormally high levels of tumour necrosis factor α (TNFα) and abnormally low plasma cysteine levels. We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and NF-x B expression. The experiments in this report show that cysteine or N-acetylcysteine (NAC) raise the intracellular glutathione (GSH) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells. However, inhibition of HIV-1 replication appears not to be directly correlated with CSH levels. Cysteine and NAC also inhibit NF-x B activity as determined by electrophoretic mobility shift assays and chloramphenicol acetyl-transferase (CAT) gene expression under control of NF-x B binding sites in uninfected cells. This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of NF-x B-dependent genes and enhance HIV-1 replication. NAC may be considered for the treatment of HIV-1-infected individuals.

Lack of evidence for xenotropic murine leukemia virus-related virus(XMRV) in German prostate cancer patients

BackgroundA novel gammaretrovirus named xenotropic murine leukemia virus-related virus (XMRV) has been recently identified and found to have a prevalence of 40% in prostate tumor samples from American patients carrying a homozygous R462Q mutation in the RNaseL gene. This mutation impairs the function of the innate antiviral type I interferon pathway and is a known susceptibility factor for prostate cancer. Here, we attempt to measure the prevalence of XMRV in prostate cancer cases in Germany and determine whether an analogous association with the R462Q polymorphism exists.Results589 prostate tumor samples were genotyped by real-time PCR with regard to the RNaseL mutation. DNA and RNA samples from these patients were screened for the presence of XMRV-specific gag sequences using a highly sensitive nested PCR and RT-PCR approach. Furthermore, 146 sera samples from prostate tumor patients were tested for XMRV Gag and Env antibodies using a newly developed ELISA assay. In agreement with earlier data, 12.9% (76 samples) were shown to be of the QQ genotype. However, XMRV specific sequences were detected at neither the DNA nor the RNA level. Consistent with this result, none of the sera analyzed from prostate cancer patients contained XMRV-specific antibodies.ConclusionOur results indicate a much lower prevalence (or even complete absence) of XMRV in prostate tumor patients in Germany. One possible reason for this could be a geographically restricted incidence of XMRV infections.

Expression of Human Endogenous Retrovirus K in Melanomas and Melanoma Cell Lines

The human endogenous retrovirus K family (HERV-K) comprises 30 to 50 closely related proviruses, most of which are defective. In contrast to all other human endogenous retroviruses, some HERV-K proviruses have maintained open reading frames for all viral proteins. In addition to the structural proteins Gag and Env and the reverse transcriptase, two regulatory proteins (Rec and Np9) have been described. Malignant melanoma has the highest mortality among skin cancers and is particularly aggressive. To study the expression of HERV-K, a set of seven primers was developed that allows discrimination between full-length and spliced mRNA and mRNA from deleted and undeleted proviruses. Expression of full-length mRNA from deleted and undeleted proviruses was detected in all human cells investigated. Expression of spliced env and rec was detected in a teratocarcinoma cell line, in 45% of the metastatic melanoma biopsies, and in 44% of the melanoma cell lines. In normal neonatal melanocytes, spliced rec was detected but not spliced env. Viral proteins were shown to be expressed in primary melanomas, metastases, and melanoma cell lines by immunohistochemistry, immunofluorescence, and Western blot analyses using specific antisera. For the first time, antibodies against HERV-K were found in melanoma patients. Melanomas are, in addition to teratocarcinomas and human breast cancer, the third tumor type with enhanced expression of HERV-K.

Interleukin-16 stimulates the expression and production of pro-inflammatory cytokines by human monocytes.

Interleukin‐16 (IL‐16) acts as a chemoattractant for CD4+ cells, as a modulator of T‐cell activation, and plays a key role in asthma. This report describes the cytokine‐inducing effects of IL‐16 on total peripheral blood mononuclear cells (PBMC) and PBMC subpopulations. While CD4+ T lymphocytes did not secrete cytokines in response to rhIL‐16, CD14+ CD4+ monocytes and maturing macrophages secrete IL‐1β, IL‐6, IL‐15 and tumour necrosis factor‐α (TNF‐α) upon rhIL‐16 stimulation. The mRNA species for these four cytokines were detected as early as 4 hr post‐stimulation, with protein being secreted by 24 hr. Secretion of IL‐1β and IL‐6 by total PBMC was dose dependent, with maximal secretion being observed using 50 ng/ml rhIL‐16. However, for IL‐15 or TNF‐α maximal secretion by total PBMC occurred with all concentrations between 5 ng/ml to 500 ng/ml rhIL‐16. Purified monocytes/macrophages secreted maximal concentrations of all four cytokines in the presence of 500 ng/ml rhIL‐16, except for monocytes where maximal secretion of IL‐15 was, interestingly, observed with only 50 ng/ml rhIL‐16. The use of higher concentrations of rhIL‐16 (1000 ng/ml) inhibited secretion of all four cytokines. While these IL‐16‐induced cytokines are likely to be involved in the immune systems response to antigen, the data suggest that IL‐16 may play a key role in initiating and/or sustaining an inflammatory response.

Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs

Abstract:  Background:  Xenotransplantation using porcine cells, tissues or organs may be associated with the transmission of porcine endogenous retroviruses (PERVs). More than 50 viral copies have been identified in the pig genome and three different subtypes of PERV were released from pig cells, two of them were able to infect human cells in vitro. RNA interference is a promising option to inhibit PERV transmission.

Establishment and Characterization of Molecular Clones of Porcine Endogenous Retroviruses Replicating on Human Cells

ABSTRACT The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming immunological and physiological barriers, the existence of numerous porcine microorganisms poses the risk of initiating a xenozoonosis. Recently, different classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We therefore examined whether completely intact proviruses exist that produce infectious and replication-competent virions. Several proviral PERV sequences were cloned and characterized. One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A second clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-A mutation in the first start codon (Met to Ile) of the env gene, preventing this provirus from replicating. However, a genetic recombinant, PERV-B(33)/ATG, carrying a restoredenv start codon, became infectious and could be serially passaged on 293 cells similar to virus clone PERV-B(43). PERV protein expression was detected 24 to 48 h posttransfection (p.t.) using cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomic env transcript of 3.1 kb. PERV-B(33) and PERV-B(43) differ in the number of copies of a 39-bp segment in the U3 region of the long terminal repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-free animals.

Neutralizing antibodies against conserved domains of p15E of porcine endogenous retroviruses: basis for a vaccine for xenotransplantation?

Porcine xenotransplants may offer a potential solution to the problem posed by the limited supply of allotransplants. However, xenotransplantation may be associated with the risk of transmission of microorganisms, in particular of porcine endogenous retroviruses (PERVs) that are an integral part of the porcine genome and able to infect human cells in vitro. Possible strategies to prevent virus transmission include the development of PERV knockout animals or of effective vaccines. When antisera prepared against the main structural proteins of PERV were screened, a goat antiserum against the recombinant ectodomain of the transmembrane envelope protein p15E was found to neutralize PERV infectivity. Epitope mapping using overlapping peptides revealed two epitopes, E1 (GPQQLEK) and E2 (FEGWFN). These sequences are identical for all PERVs and are highly conserved among all gammaretroviruses. Interestingly, antibodies isolated from AIDS patients and specific for sequences of HIV-1 partially homologous with E2 (Mab4E10, LWNWFN) or located in close proximity to E2 (Mab2F5, ELDKWA) are known to neutralize several strains of HIV-1. It is the first report showing epitope mapping of gammaretrovirus-specific neutralizing antibodies and demonstrating similarity to corresponding epitopes in HIV. These domains of the transmembrane proteins of different retroviruses are an effective target for neutralizing antibodies and may be a useful antigen to create an antiretroviral vaccine.

Simian immunodeficiency virus from African green monkeys.

When virologists talk about African green monkeys (AGM), they usually mean species belonging to the subgenus Cercopithecus aethiops of the genus Cercopithecus. This genus can be subdivided into eight subgenera (C. mona, C. cephus, C. mitis, C. i’hoesti, C. hamlyni, C. neglectus, C. diana, C. aethiops). A subgenus comprises several species and is defined by the common ecological specialization (“life-style”) of its member species. Therefore, species belonging to the same subgenus are geographically separated, as they would otherwise compete for resources. Some of the species can be further subdivided into altogether over 70 subspecies (for the entire genus) distinguished by subtle morphological differences. Members of similar subspecies usually do not mate in the wild. There are a number of excellent books delineating monkey species (GRZIMEK 1988; HILL 1972; NAPIER and NAPIER 1985).

Transspecies Transmission of the Endogenous Koala Retrovirus

ABSTRACT The koala retrovirus (KoRV) is a gammaretrovirus closely related to the gibbon ape leukemia virus and induces leukemias and immune deficiencies associated with opportunistic infections, such as chlamydiosis. Here we characterize a KoRV newly isolated from an animal in a German zoo and show infection of human and rat cell lines in vitro and of rats in vivo, using immunological and PCR methods for virus detection. The KoRV transmembrane envelope protein (p15E) was cloned and expressed, and p15E-specific neutralizing antibodies able to prevent virus infection in vitro were developed. Finally, evidence for immunosuppressive properties of the KoRV was obtained.

Porcine endogenous retroviruses: no infection in patients treated with a bioreactor based on porcine liver cells

BACKGROUND Acute liver failure (ALF) remains a disease with high mortality. Bioartificial liver support systems, which combine living cells of the liver in an extracorporeal circuit, have been successfully used in first clinical trials. The shortage of human organs to be used for bioreactors and the lack of safe and effective human liver cell lines have resulted in pigs becoming an important hepatic cell source. However, using these cells may be associated with the risk of transmission of porcine endogenous retroviruses (PERVs). PERVs are present in the genome of all pigs and are able to infect human cells in vitro. However, it remains unclear whether PERVs infect transplant recipients in vivo and, if so, whether they are pathogenic. OBJECTIVES To detect antibodies directed against specific epitopes from PERVs in seven individuals who were treated with porcine liver cell bioreactor therapy prior to liver transplantation. METHODS Sera from seven patients treated with a hybrid liver support system based on porcine liver cells for ALF who survived the treatment and were discharged from hospital were investigated for antibodies against PERV. For this in addition to methods already reported (Xenotransplantation (2001) 125), new immunological detection methods were developed. RESULTS PERV-specific antibodies were found in none of the patients using Western blot assays based on purified virus or recombinant viral core and envelope proteins or ELISA based on synthetic diagnostic peptides. CONCLUSION The assays used are specific and sensitive, and correlated in their diagnostic value. The data indicate that no PERV infection had occurred in none of the patients treated with the CellModule bioreactor containing porcine cells.