Influence of Genetic Variants in TPMT and COMT Associated with Cisplatin Induced Hearing Loss in Patients with Cancer: Two New Cohorts and a Meta-Analysis Reveal Significant Heterogeneity between Cohorts

Treatment with cisplatin-containing chemotherapy regimens causes hearing loss in 40–60% of cancer patients. It has been suggested that genetic variants in the genes encoding thiopurine S-methyltransferase (TPMT) and catechol O-methyltransferase (COMT) can predict the development of cisplatin-induced ototoxicity and may explain interindividual variability in sensitivity to cisplatin-induced hearing loss. Two recently published studies however, sought to validate these findings and showed inconsistent results. The aim of this study was to evaluate the role of polymorphisms in the TPMT and COMT genes in cisplatin-induced ototoxicity. Therefore we investigated two independent cohorts of 110 Dutch and 38 Spanish patients with osteosarcoma and performed a meta-analysis including all previously published studies resulting in a total population of 664 patients with cancer. With this largest meta-analysis performed to date, we show that the influence of TPMT and COMT on the development of cisplatin-induced hearing loss may be less important than previously suggested.     

Analysis of the Temporal Relationship between Human Immunodeficiency Virus Type 1 Quasispecies in Sequential Blood Samples and Various Organs Obtained at Autopsy

We studied the temporal relationship between human immunodeficiency type 1 (HIV-1) quasispecies in tissues and in peripheral blood mononuclear cells (PBMC) of infected individuals. Sequential PBMC and tissue samples from various organs obtained at autopsy from three patients who died of AIDS-related complications were available for analysis. Biological HIV-1 clones were isolated from PBMC samples, and cellular tropism and syncytium-inducing (SI) capacity were determined. Genomic DNA was isolated from 1 cm³ of organ tissue, and proviral DNA was amplified by means of PCR and cloned with the PGEM-T vector system. A 185-bp region encompassing the third variable domain of the virus envelope, known to influence HIV-1 biological properties, was sequenced. HIV-1 could be amplified from all PBMC and organ samples, except from liver tissue for two patients. Both SI and non-syncytium-inducing (NSI) genotypes could be detected in the different tissues. Tissue-specific quasispecies were observed in brain, lung, and testis. Lymphoid tissues, such as bone marrow, lymph node, and spleen, harbored several different variants similar to those detected in blood in the last PBMC samples. In general, only tissues in which macrophages are likely to be the main target cell for HIV-1 harbored NSI HIV-1 sequences that clustered separately. Both SI and NSI sequences that clustered with sequences from late-stage PBMC were present in other tissues, which may indicate that the presence of HIV-1 in those tissues is secondary to lymphocyte infiltration rather than to tissue tropism of HIV-1 itself. These data suggest that the viral reservoir may be limited, which will have important implications for the success of HIV-1 eradication.     

Infectious Cellular Load in Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and Susceptibility of Peripheral Blood Mononuclear Cells from Their Exposed Partners to Non-Syncytium-Inducing HIV-1 as Major Determinants for HIV-1 Transmission in Homosexual Couples

To study risk factors for homosexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared 10 monogamous homosexual couples between whom transmission of HIV-1 had occurred with 10 monogamous homosexual couples between whom HIV-1 transmission had not occurred despite high-risk sexual behavior. In the group of individuals who did not transmit virus, peripheral cellular infectious load was lower and the CD4⁺ T-cell counts were higher than in the group of transmitters. HIV-1 RNA levels in serum did not differ between transmitters and nontransmitters. Compared with peripheral blood mononuclear cells (PBMC) from normal healthy blood donors, 8 of 10 nonrecipients and only 3 of 8 recipients had PBMC with reduced susceptibility to in vitro infection with non-syncytium-inducing (NSI) HIV-1 variants isolated from either their respective partners or an unrelated individual. No difference in susceptibility was observed for infection with a syncytium-inducing variant. Among the individuals who had PBMC with reduced susceptibility, five nonrecipients and one recipient had PBMC that were equally or even less susceptible to NSI variants than PBMC that had low susceptibility and that were derived from healthy blood donors that were heterozygous for a 32-bp deletion in the CCR5 gene (CCR5 Δ32). Three of these individuals (all nonrecipients) had a CCR5 Δ32 heterozygous genotype themselves, confirming an association between low susceptibility to NSI variants and CCR5 Δ32 heterozygosity. All three recipients with less susceptible PBMC had partners with a high infectious cellular load; inversely, both nonrecipients with normally susceptible PBMC had partners with a very low infectious cellular load. These results suggest that a combination of susceptibility of target cells and inoculum size upon homosexual exposure largely determines whether HIV-1 infection is established.     

Monocytotropic human immunodeficiency virus type 1 (HIV-1) variants detectable in all stages of HIV-1 infection lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture.

We previously demonstrated a correlation between the presence of syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variants showing tropism for cell line H9 and the occurrence of rapid CD4 cell decline and progression to AIDS. In contrast, in stable asymptomatic individuals, we detected only isolates with low replication rates that were non-syncytium-inducing (NSI) and nontropic for the H9 cell line. Here, we investigated the monocytotropism of established HIV-1 isolates with a panel of isolates and with biological HIV-1 clones with distinct phenotypes. Moreover, the prevalence and biological phenotypes of monocytotropic HIV-1 variants in the course of HIV-1 infection were analyzed in comparative primary isolation studies on peripheral blood lymphocytes (PBL) and monocyte-derived macrophages (MDM). In cell-free infection studies with MDM from eight blood donors, 13 of 17 NSI isolates but only 4 of 14 SI isolates were able to infect MDM. NSI isolates also infected significantly more different donors than SI variants (median, 3 of 8 versus 0 of 8). This enhanced monocytotropism of NSI isolates was confirmed in experiments with biological HIV-1 clones with distinct phenotypes recovered from the same donor. To investigate the prevalence and biological phenotypes of monocytotropic variants in different stages of HIV-1 infection, sequential isolates from peripheral blood mononuclear cell samples from nine asymptomatic individuals, five of whom progressed to AIDS and seven of whom had a known time of seroconversion, were recovered by cocultivation with both PBL and MDM. Monocytotropic variants were obtained from 37 of 42 time points. All monocytotropic variants were NSI in PBL culture and non-T-cell-line tropic, even when SI, T-cell-line-tropic HIV-1 variants could be recovered from the same patient sample by cocultivation with PBL. We conclude that monocytotropic HIV-1 variants mostly have an NSI phenotype in PBL and, in contrast to SI variants, are present at all stages of HIV-1 infection. These results suggest an important role for monocytotropic variants in the persistence of HIV-1 infection.     

Role of Ca2+/calmodulin regulated signaling pathways in chemoattractant induced neutrophil effector functions. Comparison with the role of phosphotidylinositol-3 kinase.

In human neutrophils, both changes in intracellular Ca(2+) concentrations, [Ca(2+)]i, and activation of phosphatidylinositol-3 kinase (PtdIns3K) have been proposed to play a role in regulating cellular function induced by chemoattractants. In this study we have investigated the role of [Ca(2+)]i and its effector molecule calmodulin in human neutrophils. Increased [Ca(2+)]i alone was sufficient to induce phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2), p38 mitogen activated kinase (p38 MAPK), protein kinase B (PKB) and glycogen synthase kinase-3alpha (GSK-3alpha). Inhibition of calmodulin using a calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), did not effect N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced ERK, p38 MAPK or GSK-3alpha phosphorylation, but attenuated fMLP induced PKB phosphorylation. PCR analysis of human neutrophil cDNA demonstrated variable expression of members of the Ca(2+)/calmodulin-dependent kinase family. The roles of calmodulin and PtdIns3K in regulating neutrophil effector functions were further compared. Neutrophil migration was abrogated by inhibition of calmodulin, while no effect was observed when PtdIns3K was inhibited. In contrast, production of reactive oxygen species was sensitive to inhibition of both calmodulin and PtdIns3K. Finally, we demonstrated that chemoattractants are unable to modulate neutrophil survival, despite activation of PtdIns3K and elevation [Ca(2+)]i. Taken together, our data indicate critical roles for changes in [Ca(2+)]i and calmodulin activity in regulating neutrophil migration and respiratory burst and suggest that chemoattractant induced PKB phosphorylation may be mediated by a Ca(2+)/calmodulin sensitive pathway in human neutrophils.     

Composition and ecology of the human intestinal flora

Longitudinal quantitative cultures of fecal flora of 20 newborns, 4 older babies and 10 healthy adults were carried out to study the composition and development of the intestinal flora. In all newborns the same sequence of colonization was observed. The numbers of aerobic and anaerobic bacteria fluctuated and reached finally numbers of 10¹⁰/g wet weight. In adults the flora was in balance with 10⁵–10⁷ aerobic and 10¹⁰–10¹¹ anaerobic bacteria/g wet weight. Interaction experiments in vitro showed growth inhibition of Bacteroides fragilis by all intestinal species isolated. Bifidobacteria were not inhibited. The assumption was made that this type of interaction could be one of the mechanisms involved in the intestinal micro-ecology. Three of the Bacteroides fragilis strains tested were able to grow on natural intestinal substrates as gastric mucin, glycogen and a variety of plant polysaccharides. Acetic, lactic, propionic and succinic acids were detected as fermentation products.     

Molecular differences between ductal carcinoma in situ and adjacent invasive breast carcinoma: a multiplex ligation-dependent probe amplification study

Ductal carcinoma in situ (DCIS) accounts for approximately 20% of mammographically detected breast cancers. Although DCIS is generally highly curable, some women with DCIS will develop life-threatening invasive breast cancer, but the determinants of progression to infiltrating ductal cancer (IDC) are largely unknown. In the current study, we used multiplex ligation-dependent probe amplification (MLPA), a multiplex PCR-based test, to compare copy numbers of 21 breast cancer related genes between laser-microdissected DCIS and adjacent IDC lesions in 39 patients. Genes included in this study were ESR1, EGFR, FGFR1, ADAM9, IKBKB, PRDM14, MTDH, MYC, CCND1, EMSY, CDH1, TRAF4, CPD, MED1, HER2, CDC6, TOP2A, MAPT, BIRC5, CCNE1 and AURKA.There were no significant differences in copy number for the 21 genes between DCIS and adjacent IDC. Low/intermediate-grade DCIS showed on average 6 gains/amplifications versus 8 in high-grade DCIS (p=0.158). Furthermore, alterations of AURKA and CCNE1 were exclusively found in high-grade DCIS, and HER2, PRDM14 and EMSY amplification was more frequent in high-grade DCIS than in low/intermediate-grade DCIS. In contrast, the average number of alterations in low/intermediate and high-grade IDC was similar, and although EGFR alterations were exclusively found in high-grade IDC compared to low/intermediate-grade IDC, there were generally fewer differences between low/intermediate-grade and high-grade IDC than between low/intermediate-grade and high-grade DCIS.In conclusion, there were no significant differences in copy number for 21 breast cancer related genes between DCIS and adjacent IDC, indicating that DCIS is genetically as advanced as its invasive counterpart. However, high-grade DCIS showed more copy number changes than low/intermediate-grade DCIS with specifically involved genes, supporting a model in which different histological grades of DCIS are associated with distinct genomic changes that progress to IDC in different routes. These high-grade DCIS specific genes may be potential targets for treatment and/or predict progression.     

Influence of seasonal variation on bioassessment of streams using macroinvertebrates

The EU Water Framework Directive requires assessment of the ecological quality of running waters using macroinvertebrates. One of the problems of obtaining representative samples of organisms from streams is the choice of sampling date, as the scores obtained from macroinvertebrate indices vary naturally between seasons, confounding the detection of anthropogenic environmental change. We investigated this problem in a 4th order calcareous stream in the western Carpathian Mountains of central Europe, the Stupavsky potok brook. We divided our 100 m study site into two stretches and took two replicate samples every other month alternately from each stretch for a period of 1 year, sampling in the months of February, April, June, August, October and December. Multivariate analysis of the macroinvertebrate communities (PCA) clearly separated the samples into three groups: (1) April samples (2) June and August samples (3) October, December and February samples. Metric scores were classified into two groups those that were stable with respect to sampling month, and those that varied. Of the metrics whose values increase with amount of allochthonous organic material (ALPHA_MESO, hyporhithral, littoral, PASF, GSI new, DSI, CSI), the highest scores occurred in February, April, October and December, while for metrics whose values decrease with content of organic material (DSII, DIS, GFI D05, PORI, RETI, hypocrenal, metarhithral, RP, AKA, LITHAL, SHRED, HAI) the highest values occurred in February, April, June and December. We conclude that sampling twice a year, in early spring and late autumn, is appropriate for this type of metarhithral mountain stream. Sampling in summer is less reliable due to strong seasonal influences on many of the metrics examined while sampling in winter is inappropriate for logistical reasons. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

A phosphatidylinositol transfer protein alpha-dependent survival factor protects cultured primary neurons against serum deprivation-induced cell death

Selective neuronal loss is a prominent feature in both acute and chronic neurological disorders. Recently, a link between neurodegeneration and a deficiency in the lipid transport protein phosphatidylinositol transfer protein alpha (PI-TPalpha) has been demonstrated. In this context it may be of importance that fibroblasts overexpressing PI-TPalpha are known to produce and secrete bioactive survival factors that protect fibroblasts against UV-induced apoptosis. In the present study it was investigated whether the conditioned medium of cells overexpressing PI-TPalpha (CMalpha) has neuroprotective effects on primary neurons in culture. We show that CMalpha is capable of protecting primary, spinal cord-derived motor neurons from serum deprivation-induced cell death. Since the conditioned medium of wild-type cells was much less effective, we infer that the neuroprotective effect of CMalpha is linked (in part) to the PI-TPalpha-dependent production of arachidonic acid metabolites. The neuroprotective activity of CMalpha is partly inhibited by suramin, a broad-spectrum antagonist of G-protein coupled receptors. Western blot analysis shows that brain cortex and spinal cord express relatively high levels of PI-TPalpha, suggesting that the survival factor may be produced in neuronal tissue. We propose that the bioactive survival factor is implicated in neuronal survival. If so, PI-TPalpha could be a promising target to be evaluated in studies on the prevention and treatment of neurological disorders.