Novel, selective CDK9 inhibitors for the treatment of HIV infection.

Cyclin Dependent Kinases (CDKs) are important regulators of cell cycle and gene expression. Since an up-to-date review about the pharmacological inhibitors of CDK family (CDK1-10) is not available; therefore in the present paper we briefly summarize the most relevant inhibitors and point out the low number of selective inhibitors. Among CDKs, CDK9 is a validated pathological target in HIV infection, inflammation and cardiac hypertrophy; however selective CDK9 inhibitors are still not available. We present a selective inhibitor family of CDK9 based on the 4-phenylamino-6- phenylpyrimidine nucleus. We show a convenient synthetic method to prepare a useful intermediate and its derivatisation resulting in novel compounds. The CDK9 inhibitory activity of the derivatives was measured in specific kinase assay and the CDK inhibitory profile of the best ones (IC(50) < 100 nM) was determined. The most selective compounds had high selectivity over CDK1, 2, 3, 5, 6, 7 and showed at least one order of magnitude higher inhibitory activity over CDK4 inhibition. The most selective molecules were examined in cytotoxicity assays and their ability to inhibit HIV-1 replication was determined in cellular assays.

Genetic subtypes of HIV type 1 in Hungary.

We examined the diversity of HIV-1 subtypes in 11 adults from Hungary, using the heteroduplex mobility assay (HMA) and DNA sequencing. HMA results showed that HIV-1 gp120 sequences from 10 patients were of subtype B, whereas 1 patient, infected in Africa, carried a subtype C strain. DNA sequencing confirmed the HMA results and revealed a high intrasubtype diversity in the C2V3 region of env in different clade B isolates, which suggests multiple introduction of subtype B to Hungary. This study shows that subtype B is the predominant HIV-1 clade in Hungary.

Strong correlation between the complement-mediated antibody-dependent enhancement of HIV-1 infection and plasma viral load.

OBJECTIVE: We have previously demonstrated that complement-mediated antibody-dependent enhancement (C-ADE) of HIV-1 infection correlates with accelerated immunosuppression and disease progression in HIV-1-infected individuals. In the present work the relationship between C-ADE and plasma HIV-1 RNA concentrations was studied to determine the effect of C-ADE on viral replication. METHODS: Three studies were performed: (a) C-ADE and HIV-1 RNA concentrations were determined in the serum and plasma aliquots taken at the same time from 98 HIV patients, mostly in the advanced stage of the disease; (b) the above two parameters as well as HIV enzyme-linked immunosorbent assay (ELISA)-reactive antibodies (Abbott HIV 1/2 test), and p24 antigen levels (Abbott antigen test; Abbott, Delkenheim, Germany) were determined in four seroconversion panels purchased from the Boston Biomedica firm; (c) changes of HIV-1 RNA concentration and C-ADE during a 17 month follow-up period were determined in 18 HIV-infected patients. C-ADE was measured by the method previously established in our laboratories. The results were expressed by an enhancement/neutralization index (E/NI). HIV-1 RNA levels were determined with the Amplicor monitor kit (Roche, Basel, Switzerland), and in some experiments with the nucleic acid sequence based amplification (Organon Teknika, Turnhout, Belgium) kits. RESULTS: (a) We found a highly significant (P<0.0001) positive correlation between E/NI values reflecting the extent of HIV-1 infection enhancement and plasma HIV-1 RNA levels. Both E/NI and HIV-1 RNA levels negatively correlated to the CD4 cell counts. (b) C-ADE was first detected just before, or concomitantly with, seroconversion in 4/4 seroconversion panels. (c) Both E/NI values and HIV-1 RNA levels significantly (P<0.001) increased during a 17 month observation period in 18 HIV-infected patients. CONCLUSION: We found strong association between the extent of the complement-mediated antibody-dependent enhancement of HIV-1 infection and the plasma viral load in HIV patients. On the basis of these findings, C-ADE correlates with HIV replication in vivo, and potentially contributes to the progression of HIV disease.

S-methylthio-cysteine and cystamine are potent stimulators of thiol production and glutathione synthesis.

The effects of methylthio-cysteine disulfide (MT-Cy) and cystamine (CAM) on the thiol production and glutathione content of a human T cell line (CEM-SS) have been investigated. MT-Cy per se and CAM in the presence of cystine greatly enhanced thiol production and glutathione content of cells while cystine alone exerted no or slight influence in the first hours. The MT-Cy- or CAM-induced extracellular SH-generation was observed both in a complete nutrient medium and even more in SH-free D-PBS. The acid-soluble thiol level and glutathione content of cells elevated markedly (up to 5-6 fold in two hours) when incubating cells in complete medium. Inhibition of glutathione synthesis by DL-buthionine (S,R)-sulfoximine did not alter the MT-Cy- or CAM-induced extracellular thiol production indicating that glutathione synthesis is not involved in this effect. The results suggest that MT-Cy easily enters the cells thus accelerating the thiol cycle in SH-poor medium while CAM promotes cystine uptake into the cells. Phenylalanine and leucine inhibited both MT-Cy- and CAM-dependent thiol production in D-PBS most effectively suggesting the involvement of the L membrane transport system in these effects.

[Nervous system lymphoma in HIV infection]. / Les lymphomes du système nerveux au cours de l'infection par le VIH.

Primary CNS lymphoma is recognized as one of the criteria of AIDS. The incidence is 0.4 to 0.56% of AIDS complications. Non-Hodgkin's lymphomas appear as multicentric tumours, mostly located in the hemispheres; they have a B phenotype and have a high grade of malignancy. They are sometimes associated with opportunistic infections. Survival is around 4 to 5 months. The presence of EBV in these tumours is an argument to propose a synergistic role of this virus in the pathogenesis of these tumours. Secondary lymphomas are observed in 40 to 47% of these tumours and must be systematically looked for. Hodgkin's disease is rare. It is mostly diagnosed as secondary compressive epidural tumors.

Virological, Neurological and Histological Investigations of a Child Born to a Mother with AIDS.

HIV-1 was isolated from a child at 6 and 9 months of age, proving the vertical transmission of infection from the mother with AIDS. The p24 antigen test of the plasma at 9 months of age was positive as well. A positive PCR reaction was detected in J34 cells, infected with the supernatant of the peripheral blood lymphocytes of the child. According to phenotypic characterization, the virus proved to be a SI (syncytium inducing) isolate, growing in PBL, MT2, J34 and other T and monocytic cell lines. The isolate was AZT sensitive. Two methods were applied for genotypic characterization: 1. Heteroduplex mobility assay (HMA), 2. Sequence analysis of a part of the env gene. On the basis of both of these methods, this virus belongs to the B subtype of HIV-1, which is prevalent mainly in Europe and in the USA. The neurological status of the child was followed regularly. At autopsy the presence of p24 antigen was detected in glial cells of the frontal cortex, proving the presence of the virus in the brain. A retardation of the development of the central nervous system could be observed as well.

Three cases of HIV-1 seroreversion.

Three patients were enrolled, two as hemophiliacs, and one with acute EBV infection. Serial serum samples of each patient were tested with at least 3 different HIV antibody EIA tests, an immunofluo-rescent test and two western blots (WB). In the third case, PCR and reverse transcriptase enzyme activity measurement were also done. One of the regularly checked serum samples of hemophiliac patients was reactive with different HIV screening and confirmatory assays. Their next blood samples, two weeks and one month later, respectively, were negative with the same tests. In Case 3. two and a half years after the first examination, the EIA tests results changed to negative, but the WB was still indeterminate. In the case of the two hemophiliac patients, the patients may have been exposed to HIV containing blood products (before 1985), but were not infected. Regular treatment with factor VIII concentrate, in which HIV antigens may be present, can boost the immune response and results in transient seropositivity. In the case of the EBV infected patient, the transient HIV seropositivity may be the consequence of EBV induced proliferation of anti-HIV-antibody producing B cell clones. During our ten year HIV confirmatory practice we tested more than 40000 samples, from which transient seropositivity were observed only in the three cases summarized in this paper.

Serological survey for type D retroviruses and simian immunodeficiency virus in monkeys living in Hungary.

Acquired immunodeficiency in non-human primates has been associated with two types of retroviruses: (i) Simian Retrovirus (SRV), a type D virus and (ii) a lentivirus, Simian Immunodeficiency Virus (SIV). Both types represent a serious health threat to animals living in different primate research centres, zoos, medical institutions and in private homes. The sera of 175 monkeys belonging to different species were tested for the presence of antibodies against type D retroviruses and SIV. The Mason-Pfizer monkey virus, the prototype of D retroviruses, and Simian Immunodeficiency Virus, designated SIVmac251, were used. None of the sera tested proved to be positive for the presence of type D retroviruses and only one serum of an African green monkey was positive for the presence of antibodies to SIV.

Establishment of an MT4 cell line persistently producing infective HIV-1 particles.

Human T cell Lymphotropic Virus-I (HTLV-I) carrying human T cell line MT4 is highly sensitive to Human Immunodeficiency Virus-1 (HIV-1). After HIV-1 infection cell clusters characteristic of intact MT4 rapidly disintegrate, syncytia appear and the cells die. Surviving MT4 cells were subcultured following HIV-1 infection of high multiplicity. We succeeded to establish an MT4 cell line continuously producing infective HIV (MT4/HIV-1). The original and the HIV-1 infected MT4 cells were morphologically similar. The MT4/HIV-1 cells proved to be nearly 100% positive in indirect immunofluorescence assay using the serum of an HIV-1 antibody positive individual. OKT4 surface antigen could not be demonstrated on MT4/HIV-1 cells. On electron microscopic pictures typical and atypical virus particles could be seen near the surface of the cell membrane. The persistently produced virus particles were infective for H9 and MT4 cells. The antigenic structure of the virus produced by MT4 cells was similar to that produced by H9 cells.

A solid phase reverse transcriptase micro-assay for the detection of human immunodeficiency virus and other retroviruses in cell culture supernatants.

A simple and rapid solid-phase reverse transcriptase assay was developed based on the use of poly(rA):oligo(dT)12-18 as template primer immobilized on DEAE cellulose paper squares to detect human immunodeficiency virus (HIV) and/or other retroviruses in cell culture supernatants. It was found that PEG (per se) -up to 4% concentrations (w/v)--did not inhibit reverse transcriptase activity. Optimal conditions of the assay were determined. This solid-phase technique is much faster and more convenient than the methods described previously.

Take and growth of the transplantable MC29 hepatoma in allogeneic and xenogeneic hosts.

We report here the unexpected biological behaviour of the transplantable MC29 virus-induced hepatoma. This neoplasm, originating from an inbred white Leghorn (Duke) chicken, is maintained in our laboratory by serial in vivo passages in Hunnia hybrid chickens allogeneic to the original host. More than 80% of the tumours developing after subcutaneous inoculation of 3 x 10(6) hepatoma cells into newly hatched chickens grew progressively, while after injection of the same number of cells into 7 days old birds regressive tumour growth was observed. Transplantation from the allogeneic hosts into 7 days old inbred white Leghorn (Duke) chickens also resulted in regression of tumours in the great majority of cases. After inoculation to xenogeneic Japanese quails, progressor tumours developed in both two weeks old and adult birds with a dramatic increase of the frequency of liver metastases. Transplantation to another xenogeneic host, the turkey, revealed an age-related resistance similar to that of Hunnia hybrid chickens.

Tyrosine aminotransferase induction in normal and tumor-bearing chickens.

Tyrosine aminotransferase (TAT) induction by glucagon and dexamethasone in the liver of tumor-bearing chickens was studied and compared with induction in healthy animals. The transplantable tumor was caused by inoculation of cells from a cell line induced by MC29 avian leukosis virus. TAT was hardly detectable in tumor tissue of control and dexamethasone-treated chickens, but it was induced by glucagon to levels which were significant although very low when compared to those in host liver or the liver of non-tumor-bearing controls after glucagon treatment. Dexamethasone failed to induce TAT in host liver at 8 A.M. while it significantly indiced TAT in the normal liver at the same time of the day. Similar failure of TAT induction was not detectable when glucagon was used instead of dexamethasone. Furthermore, it was found that diurnal variations in basal and dexamethasone or glucagon-induced TAT levels are considerably mitigated in host liver as compared to those observed in the liver of healthy animals. The possible reasons for these findings are discussed.