A Potent Kalihinol Analogue Disrupts Apicoplast Function and Vesicular Trafficking in P. falciparum Malaria.

Here we report the discovery of MED6-189, a new analogue of the kalihinol family of isocyanoterpene (ICT) natural products. MED6-189 is effective against drug-sensitive and -resistant P. falciparum strains blocking both intraerythrocytic asexual replication and sexual differentiation. This compound was also effective against P. knowlesi and P. cynomolgi. In vivo efficacy studies using a humanized mouse model of malaria confirms strong efficacy of the compound in animals with no apparent hemolytic activity or apparent toxicity. Complementary chemical biology, molecular biology, genomics and cell biological analyses revealed that MED6-189 primarily targets the parasite apicoplast and acts by inhibiting lipid biogenesis and cellular trafficking. Genetic analyses in P. falciparum revealed that a mutation in PfSec13, which encodes a component of the parasite secretory machinery, reduced susceptibility to the drug. The high potency of MED6-189 in vitro and in vivo, its broad range of efficacy, excellent therapeutic profile, and unique mode of action make it an excellent addition to the antimalarial drug pipeline.

[Detection of bladder cancer in voided urine samples]. / Détecter le cancer de la vessie à partir d'échantillons d'urine.

Bladder cancer mainly affects patients aged 50 years or more and requires close and repeated surveillance. Flexible cystoscopy associated with urinary cytology are the currently recommended diagnostic and follow-up methods. Because medical imaging techniques remain rather unsatisfying for bladder carcinoma detection, research efforts have focused on urinary markers of the disease. Various approaches were tested with results generally too unconsistant to replace cystoscopy. Recently, the department of Urology at the University of Liège together with the Biotechnology Company OncoMethylome Sciences have been interested in testing whether the detection of hypermethylated genes in voided urine samples would be of value for the detection of bladder cancer. The method is based on the Methylation-Specific PCR technology (MSP). This approach has the theoretical advantage of being non invasive, reproducible and based on DNA, whose stability, in urine, is higher than that of proteins. The results of a large prospective study, recently publised in European Urology, have shown that the identification by MSP of 2 methylated genes, TWIST1 and NID2, in voided urine samples, is a sensitive (+/- 90%) and specific (+/- 93%) test for the detection of bladder cancer. The test is largely more sensitive than cytology while both techniques have similar specificity. Based on these promising results, we are currently evaluating this novel, non invasive MSP approach for the follow-up of patients with non-muscle invasive bladder cancer.

High frequency of RASSF1A and RARb2 gene promoter methylation in morphologically normal endometrium adjacent to endometrioid adenocarcinoma.

AIMS: To identify a DNA methylation signature of endometrioid carcinoma of the endometrium (EEC) in the early stages of endometrial carcinogenesis. METHODS AND RESULTS: Archival biopsy specimens of 39 EECs, 14 cases of atypical hyperplasia (AH), 11 histologically normal endometrial tissues adjacent to EECs and 24 normal control endometrial samples were retrieved. The cases were tested by quantitative methylation-specific polymerase chain reaction with primers hybridizing in the promoter regions of five genes frequently methylated in human cancer (RASSF1A, RARb2, P16, MGMT and GSTPi). Twenty-nine of 39 (74%) EECs and 7/14 (50%) AHs were methylated for the RASSF1A gene, whereas 17/39 (44%) EECs and 6/14 (43%) AHs were positive for the methylation of the RARb2 gene. No significant results were obtained for the other genes (P16, MGMT and GSTPi). Interestingly, 4/11 (36%) and 6/11 (55%) histologically normal endometrial tissues adjacent to EEC showed, respectively, RASSF1A and RARb2 gene methylation. Furthermore, these 11 specimens were microsatellite stable and showed similar proliferative, cell cycle and apoptotic mean labelling indices as the normal endometrial control tissues. CONCLUSIONS: Promoter region methylation of RASSF1A and RARb2 genes is an early event in endometrial carcinogenesis.

The organotypic culture of HPV-transformed keratinocytes: an effective in vitro model for the development of new immunotherapeutic approaches for mucosal (pre)neoplastic lesions.

The purpose of this study is to develop a reliable in vitro human model to test new immunotherapeutic approaches for squamous cell carcinoma that develop on mucosal surfaces. The organotypic (raft) culture permits cells to proliferate and differentiate at an air-liquid interface on a dermal equivalent support. Normal keratinocytes stratify and fully differentiate in a manner similar to the normal squamous epithelial tissues, while human papillomavirus-immortalized and established squamous carcinoma cell lines exhibit dysplastic morphologies similar to (pre)neoplastic lesions seen in vivo. We have demonstrated the ability of these organotypic cultures to be manipulated by altering the epithelial stratification with cytokines (interferon-gamma and tumor necrosis factor-alpha) and by integrating activated lymphocytes or dendritic cells into the in vitro formed epithelial sheet. This model may provide a useful tool to investigate the factors contributing to the presence and function of immunocompetent cells within a neoplastic epithelium that develops on a mucosal surface.

Safety and efficacy of ritonavir and saquinavir in combination with zidovudine and lamivudine.

BACKGROUND: Ritonavir is a potent inhibitor of cytochrome P4503A4 that strongly increases saquinavir bioavailability. In this study we assessed the safety and antiretroviral efficacy of the combination of these two compounds in patients pretreated and receiving continued treatment with zidovudine and lamivudine who were protease inhibitor naive and who had a CD4 cell counts below 200/mm3. METHODS: In this 48-week pilot study, all patients received 600 mg ritonavir and 400 mg saquinavir twice daily. Administration of zidovudine and lamivudine was continued without a change in previous doses. Viral load, CD4 cell count, and the emergence of resistance to the two protease inhibitors were evaluated repeatedly up to week 48. RESULTS: Sixteen patients were included in the study. Previous nucleoside analog treatment duration was 48+/-22 months (mean +/- SD). Two patients quit taking both protease inhibitors within 2 weeks. The ritonavir dose had to be reduced in 10 other patients because of side effects. Between inclusion and week 48, plasma viremia varied from 4.87+/-0.43 to 3.00+/-1.29 log10 copies/mL and CD4 cell counts ranged from 98+/-61 to 250+/-139/mm3. Ten patients (63%) had viral loads below 200 copies/mL and 7 (44%) had viral loads below 50 copies/mL. A single key mutation that conferred ritonavir resistance I84V and V82A/V developed in two patients. A mutation at codon 54 developed in another patient. These mutations were associated with repeated cessations of antiretroviral treatment. No lipodystrophy was observed. CONCLUSION: Ritonavir and saquinavir in combination are quite well tolerated and induce a high and sustained antiretroviral efficacy. A four-drug combination that includes these two protease inhibitors should be considered as a first line of treatment in patients with low CD4 cell counts.

HLA-G transcription studies during the different stages of normal and malignant hematopoiesis.

Specific expression of the non classical class I HLA-G gene on trophoblasts, the only fetal tissue in contact with maternal cells which lack MHC class I antigens, may indicate a role of this gene in fetal-maternal tolerance. We recently reported HLA-G transcription in peripheral blood leukocytes. In this work, we have investigated HLA-G transcription in hematopoietic stem cells, in different hematopoietic lineages and in malignant cells by using a RT-PCR technique. PCR amplification with primers specific to the exon 2 and the 3' untranslated region has enabled to detect HLA-G transcription in B and T cell populations. No transcription was found in CD34+ cells, in thymocytes, in polynuclear cells, in monocytes and in natural killer cells. Among the malignancies analyzed, HLA-G is transcribed in 2 of 13 cases of acute leukemia characterized by a monocytic contingent, in 3 of 6 CLL and in all the cases of B-NHL (n = 6). No HLA-G transcription was detected in myeloma (n = 2). The splicing type does not seem to be linked to a lymphocyte subpopulation nor to a malignant proliferation stage. These results suggest that HLA-G is a marker of mature lymphoid cells and may play an immunological function as a peptide presenting molecule. HLA-G transcription in some cases of malignancy might indicate a contribution to the tumoral progression by blocking natural killing reaction.

HLA-G transcription studies during the different stages of normal and malignant hematopoiesis.

Specific expression of the non classical class I HLA-G gene on trophoblasts, the only fetal tissue in contact with maternal cells which lack MHC class I antigens, may indicate a role of this gene in fetal-maternal tolerance. We recently reported HLA-G transcription in peripheral blood leukocytes. In this work, we have investigated HLA-G transcription in hematopoietic stem cells, in different hematopoietic lineages and in malignant cells by using a RT-PCR technique. PCR amplification with primers specific to the exon 2 and the 3' untranslated region has enabled to detect HLA-G transcription in B and T cell populations. No transcription was found in CD34+ cells, in thymocytes, in polynuclear cells, in monocytes and in natural killer cells. Among the malignancies analyzed, HLA-G is transcribed in 2 of 13 cases of acute leukemia characterized by a monocytic contingent, in 3 of 6 CLL and in all the cases of B-NHL (n = 6). No HLA-G transcription was detected in myeloma (n = 2). The splicing type does not seem to be linked to a lymphocyte subpopulation nor to a malignant proliferation stage. These results suggest that HLA-G is a marker of mature lymphoid cells and may play an immunological function as a peptide presenting molecule. HLA-G transcription in some cases of malignancy might indicate a contribution to the tumoral progression by blocking natural killing reaction.

The HLA-G gene is expressed at a low mRNA level in different human cells and tissues.

Recently, HLA-G transgenic mice were shown to exhibit transgene transcription in several extraembryonic tissues. To determine whether HLA-G mRNAs are also expressed in other human tissues, we have undertaken Northern blot and RT-PCR assays using HLA-G locus-specific probe and primers. These studies demonstrate that the HLA-G gene is transcribed in a variety of cells and adult tissues obtained from different individuals (peripheral blood leukocytes, placenta, skin, spleen, thymus, prostate, testicle, ovary, small intestine, colon, heart, brain, lung, liver, and kidney), as well as in fetal tissues (heart, lung, liver, and kidney). The HLA-G mRNA level observed in most tissues is orders of magnitude lower than the level of classic class I genes in the same tissues. RT-PCR studies have demonstrated that alternative splicing of the HLA-G primary transcript is different from tissue to tissue and could be regulated in a tissue-specific fashion. Sequencing of keratinocyte transcripts has confirmed previous observations: (a) three different alternative splicing transcripts are produced (a full-length transcript, an mRNA lacking exon 3, and a transcript devoid of exon 3 and 4) and (b) HLA-G polymorphism is limited in the coding regions. In view of this wide HLA-G tissue distribution, a new hypothesis dealing with possible HLA-G function is proposed.

HIV-associated thrombocytopenia: in vitro megakaryocyte colony formation in 10 patients.

In vitro bone-marrow megakaryocyte colony formation was studied in 10 patients with HIV-associated thrombocytopenia to investigate the mechanism of thrombocytopenia. Increased colony formation was observed in 3 patients and decreased growth in 7 patients. No relationship was noted between the growth potential of megakaryocyte progenitors and platelet count, number of CD4+ celts, platelet response to azidothymidine, and platelet count 7 days after culture. In all patients, megakaryocyte morphology was abnormal: blebbing of the membrane and abnormal chromatin with separated lobes of nuclei. Further studies are needed to determine if growth potential of megakaryocyte progenitors is useful in understanding the mechanism of thrombocytopenia in HIV-infected individuals.

In vitro megakaryocyte colony formation in patients with idiopathic thrombocytopenic purpura: differences between acute and chronic ITP.

In vitro megakaryocyte colony formation from the bone marrow of patients with acute idiopathic thrombocytopenic purpura (ITP) or chronic ITP was compared using a plasma clot system. The number of megakaryocyte colony-forming units (CFU-Meg) was significantly higher (p less than 0.05) in acute ITP compared to chronic ITP (54.3 +/- 68.4 vs. 12.9 +/- 15.3/10(5) nonadherent mononuclear cells, mean +/- SD), and significantly lower (p less than 0.05) in chronic ITP compared to controls (12.9 +/- 15.3 vs. 22.8 +/- 15.9). A significant correlation was observed between platelet recovery 7 and 30 days after culture, and the number of CFU-Meg (r = 0.49 and 0.45, respectively, p less than 0.05). An inverse correlation was observed between platelet count at the time of culture and the number of Megs per colony (r = -0.48, p less than 0.05). These results indicated a difference between acute and chronic ITP in the ability to promote in vitro Meg colony formation and may suppose a different immune mechanism for thrombocytopenia in these two disorders.

Spontaneous in vitro megakaryocyte colony formation in primary thrombocythemia: relation to platelet factor 4 plasma level and beta-thromboglobulin/platelet factor 4 ratio.

Seventeen patients with primary thrombocythemia (PT) were evaluated for in vitro bone marrow megakaryocyte progenitors (CFU-MK) using a plasma clot system. The aim of this study was to find out whether spontaneous growth of CFU-MK could be used in the diagnosis of PT. The number of CFU-MK was normal in 7 patients and reduced in 10 patients. In the absence of stimulating factor, CFU-MK grew spontaneously in 12 patients, while in 5 patients no spontaneous CFU-MK were observed. The mean plasma level of platelet factor 4 (PF4) was significantly higher (p less than 0.05) in patients without spontaneous CFU-MK (59.8 +/- 59.6 IU/ml; mean +/- SD) compared to patients with (18.1 +/- 20.7 UI/ml). The mean plasma level of beta-thromboglobulin did not differ between patients with or without spontaneous CFU-MK. The beta-thromboglobulin/PF4 ratio was significantly higher (p less than 0.01) in patients with spontaneous CFU-MK (9.9 +/- 7.1) compared to patients without (3.1 +/- 1.4). These results suggest that PF4 could inhibit in vitro spontaneous growth of CFU-MK.

Carbohydrate composition of human megakaryocyte membranes in culture: identification using binding of seven lectins.

Seven tetramethylrhodamine B isothiocyanate- (TRITC) labeled lectins: lens culinaris (LCH), ulex europeus-1 (UEA-1), lycopersicon esculentum (LEA), wheat germ agglutinin (WGA), dolichos biflorus (DBA), soybean agglutinin (SBA) and erythrina cristagalli (ECA) were applied on cultured human megakaryocytes (Megs) detected by immunofluorescence. All stages of Megs (from lymphocyte-like Megs to mature Megs) and platelets were labeled by LCH, LEA, UEA-1 and WGA. ECA binds to platelets but only to some Megs. DBA did not bind to platelets but did bind to some Megs, irrespective of stage. SBA binds to all stages of Megs, but did not bind to platelets. These results indicate the presence of mannose, glucose (LCH), sialic acid (WGA), and glucosamine (UEA-1, LEA, WGA) on the surface of all cells of the Meg lineage, a variable presence of galactosamine (DBA, SBA, ECA), and a discrepancy in the presence of some galactosamine compounds between platelets and Megs (DBA, SBA).

Inhibitory effect of highly purified human platelet beta-thromboglobulin on in vitro human megakaryocyte colony formation.

The effect of highly purified human beta-thromboglobulin (beta TG), a glycoprotein present in platelet alpha granules, was tested on human bone marrow in vitro megakaryocyte (MK) colony formation. A concentration of 5 micrograms/ml of beta TG induced a 50% reduction in the number of MK colonies, and concentrations of 10 and 20 micrograms/ml completely inhibited MK growth. This inhibition was of importance for MKs because a higher concentration of beta TG (10 micrograms/ml) was needed to obtain a nonsignificant decrease in erythroblastic progenitors (erythroid burst-forming units, BFU-E), and no inhibition of granulocyte-macrophage colony-forming units (CFU-GM) was observed. beta TG acts mainly on maturation of MKs. These results indicate that beta TG could play a role in the physiological regulation of platelet production by MKs.