Do Increasing Rates of Loss to Follow-up in Antiretroviral Treatment Programs Imply Deteriorating Patient Retention?

In several studies of antiretroviral treatment (ART) programs for persons with human immunodeficiency virus infection, investigators have reported that there has been a higher rate of loss to follow-up (LTFU) among patients initiating ART in recent years than among patients who initiated ART during earlier time periods. This finding is frequently interpreted as reflecting deterioration of patient retention in the face of increasing patient loads. However, in this paper we demonstrate by simulation that transient gaps in follow-up could lead to bias when standard survival analysis techniques are applied. We created a simulated cohort of patients with different dates of ART initiation. Rates of ART interruption, ART resumption, and mortality were assumed to remain constant over time, but when we applied a standard definition of LTFU, the simulated probability of being classified LTFU at a particular ART duration was substantially higher in recently enrolled cohorts. This suggests that much of the apparent trend towards increased LTFU may be attributed to bias caused by transient interruptions in care. Alternative statistical techniques need to be used when analyzing predictors of LTFU-for example, using "prospective" definitions of LTFU in place of "retrospective" definitions. Similar considerations may apply when analyzing predictors of LTFU from treatment programs for other chronic diseases.

Identification of Trypanosoma brucei components involved in trypanolysis by normal human serum

Normal human serum (NHS) confers human resistance to infection by the parasite Trypanosoma brucei owing to the trypanolytic activity of apolipoprotein L1 (APOL1), present in two serum complexes termed Trypanolytic Factors (TLF-1 and -2). In order to identify parasite components involved in the intracellular trafficking and activity of TLFs, an inducible RNA interference (RNAi) genomic DNA library constructed in bloodstream form T. brucei was subjected to RNAi induction and selection for resistant parasites under NHS conditions favouring either TLF-1 or TLF-2 uptake. While TLF-1 conditions readily selected the haptoglobin-haemoglobin (HP-HB) surface receptor TbHpHbR as expected, given its known ability to bind TLF-1, under TLF-2 conditions no specific receptor for TLF-2 was identified. Instead, the screen allowed the identification of five distinct factors expected to be involved in the assembly of the vacuolar proton pump V-ATPase and consecutive endosomal acidification. These data confirm that lowering the pH during endocytosis is required for APOL1 toxic activity.

Multibranched acquired periungual fibrokeratomas with confounding histopathologic findings resembling papillomavirus infection: a report of two cases

Acquired periungual fibrokeratomas are benign fibrous tissue tumors and are considered as the topographical variant of acquired digital fibrokeratoma. They usually present as solitary tumors. In some instance, the entity may appear in multibranched fashion. The main histopathologic features consist of acanthosis, thick collagen bundles mainly oriented in a vertical axis forming a central core, and numerous proliferating fibroblasts. In this article, we present two cases of acquired multibranched periungual fibrokeratoma and depict their varying clinical features over time. Binucleation and perinuclear halos of keratinocytes mimicking human papillomavirus (HPV) infection were detected microscopically, but there was no reactivity with HPV immunostaining. In context, anti-HPV immunostaining may be helpful in the differentiation of fibrokeratomas from HPV infection. On the other hand, it should be kept in mind that these histopathologic findings may be found in acral biopsies independent of viral effects.

Genetic epidemiology of gallbladder disease in Mexican Americans and cholesterol 7$\alpha$-hydroxylase gene sequence variation

Among Mexican Americans, the second largest minority group in the United States, the prevalence of gallbladder disease is markedly elevated. Previous data from both genetic admixture and family studies indicate that there is a genetic component to the occurrence of gallbladder disease in Mexican Americans. However, prior to this thesis no formal genetic analysis of gallbladder disease had been carried out nor had any contributing genes been identified.^ The results of complex segregation analysis in a sample of 232 Mexican American pedigrees documented the existence of a major gene having two alleles with age- and gender-specific effects influencing the occurrence of gallbladder disease. The estimated frequency of the allele increasing susceptibility was 0.39. The lifetime probabilities that an individual will be affected by gallbladder disease were 1.0, 0.54, and 0.00 for females of genotypes "AA", "Aa", and "aa", respectively, and 0.68, 0.30, and 0.00 for males, respectively. This analysis provided the first conclusive evidence for the existence of a common single gene having a large effect on the occurrence of gallbladder disease.^ Human cholesterol 7$\alpha$-hydroxylase is the rate-limiting enzyme in bile acid synthesis. The results of an association study in both a random sample and a matched case/control sample showed that there is a significant association between cholesterol 7$\alpha$-hydroxylase gene variation and the occurrence of gallbladder disease in Mexican Americans males but not in females. These data have implicated a specific gene, 7$\alpha$-hydroxylase, in the etiology of gallbladder disease in this population.^ Finally, I asked whether the inferred major gene from complex segregation analysis is genetically linked to the cholesterol 7$\alpha$-hydroxylase gene. Three pedigrees predicted to be informative for linkage analysis by virtue of supporting the major gene hypothesis and having parents with informative genotypes and multiple offspring were selected for this linkage analysis. In each of these pedigrees, the recombination fractions maximized at 0 with a positive, albeit low, LOD score. The results of this linkage analysis provide preliminary and suggestive evidence that the cholesterol 7$\alpha$-hydroxylase gene and the inferred gallbladder disease susceptibility gene are genetically linked. ^

A viral gene that activates lytic cycle expression of Kaposi’s sarcoma-associated herpesvirus

Herpesviruses exist in two states, latency and a lytic productive cycle. Here we identify an immediate-early gene encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus eight (HHV8) that activates lytic cycle gene expression from the latent viral genome. The gene is a homologue of Rta, a transcriptional activator encoded by Epstein–Barr virus (EBV). KSHV/Rta activated KSHV early lytic genes, including virus-encoded interleukin 6 and polyadenylated nuclear RNA, and a late gene, small viral capsid antigen. In cells dually infected with Epstein–Barr virus and KSHV, each Rta activated only autologous lytic cycle genes. Expression of viral cytokines under control of the KSHV/Rta gene is likely to contribute to the pathogenesis of KSHV-associated diseases.

CD147 facilitates HIV-1 infection by interacting with virus-associated cyclophilin A

Cyclophilin A (CyPA) is specifically incorporated into the virions of HIV-1 and has been shown to enhance significantly an early step of cellular HIV-1 infection. Our preliminary studies implicated CD147 as a receptor for extracellular CyPA. Here, we demonstrate a role for CyPA–CD147 interaction during the early steps of HIV-1 infection. Expression of human CD147 increased infection by HIV-1 under one-cycle conditions. However, susceptibility to infection by viruses lacking CyPA (simian immunodeficiency virus or HIV-1 produced in the presence of cyclosporin A) was unaffected by CD147. Virus-associated CyPA coimmunoprecipitated with CD147 from infected cells. Antibody to CD147 inhibited HIV-1 entry as evidenced by the delay in translocation of the HIV-1 core proteins from the membrane and inhibition of viral reverse transcription. Viruses whose replication did not require CyPA (SIV or mutant HIV-1) were resistant to the inhibitory effect of anti-CD147 antibody. These results suggest that HIV-1 entry depends on an interaction between virus-associated CyPA and CD147 on a target cell.

Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B

Human cytomegalovirus (HCMV) infection alters the expression of many cellular genes, including IFN-stimulated genes (ISGs) [Zhu, H., Cong, J.-P., Mamtora, G., Gingeras, T. & Shenk, T. (1998) Proc. Natl. Acad. Sci. USA 95, 14470–14475]. By using high-density cDNA microarrays, we show that the HCMV-regulated gene expression profile in fibroblasts does not differ substantially from the response generated by IFN. Furthermore, we identified the specific viral component triggering this response as the envelope glycoprotein B (gB). Cells treated with gB, but not other herpesviral glycoproteins, exhibited the same transcriptional profile as HCMV-infected cells. Thus, the interaction of gB with its as yet unidentified cellular receptor is the principal mechanism by which HCMV alters cellular gene expression early during infection. These findings highlight a pioneering paradigm for the consequences of virus–receptor interactions.

Polyadenylylated nuclear RNA encoded by Kaposi sarcoma-associated herpesvirus.

A newly recognized gamma herpesvirus known as Kaposi sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is present in Kaposi sarcomas and body-cavity-based lymphomas. Here we identify a novel abundant 1.2-kb RNA, polyadenylated nuclear RNA (PAN RNA), encoded by the virus. The majority of cDNAs produced from poly(A)-selected RNA isolated from a human body cavity lymphoma cell line 48 hr after butyrate induction of KSHV lytic replication represented PAN RNA. Within PAN RNA were two 9 and 16 nt stretches with 89% and 94% identity to U1 RNA. A third stretch of 14 nt was 93% complementary to U1. The 5' upstream region of PAN RNA contained both proximal and distal sequence elements characteristic of regulatory regions of U snRNAs, whereas the 3' end was polyadenylylated. PAN RNA was transcribed by RNA polymerase II, lacked a trimethylguanosine cap, and did not associate with polyribosomes. PAN RNA formed a speckled pattern in the nucleus typical of U snRNAs and colocalized with Sm protein. Therefore, PAN represents a new type of RNA, possessing features of both U snRNA and mRNA.

Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism.

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, is a human herpesvirus associated with epithelial cell malignancies (nasopharyngeal carcinoma) as well as B-cell malignancies. Understanding how viral latency is disrupted is a central issue in herpesvirus biology. Epithelial cells are the major site of lytic EBV replication within the human host, and viral reactivation occurs in EBV-associated nasopharyngeal carcinomas. It is known that expression of a single viral immediate-early protein, BZLF1, is sufficient to initiate the switch from latent to lytic infection in B cells. Cellular regulation of BZLF1 transcription is therefore thought to play a key role in regulating the stringency of viral latency. Here we show that, unexpectedly, expression of another viral immediate-early protein, BRLF1, can disrupt viral latency in an epithelial cell-specific fashion. Therefore, the mechanisms leading to disruption of EBV latency appear to be cell-type specific.

CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells.

Dendritic cells are potent antigen-presenting cells that initiate primary immune responses. Although dendritic cells derive from bone marrow stem cells, the intermediate stages in their development remain unknown. In this study, plastic-adherent blood monocytes (CD14+, CD1a-) cultured for 7 days with granulocyte-monocyte colony-stimulating factor, interleukin 4, and tumor necrosis factor alpha were shown to differentiate into CD1a+ CD83+ dendritic cells. These cells displayed all phenotypic and morphologic characteristics of mature dendritic cells and were the most potent stimulatory cells in allogeneic mixed leukocyte reactions. The identification of specific culture conditions that generate large numbers of dendritic cells from purified monocytes uncovers an important step in dendritic cell maturation that will allow the further characterization of their role in autoimmune diseases, graft rejection, and human immunodeficiency virus infection.

Dissecting the signaling mechanisms of IL-7-mediated autophagy regulation in T-cell acute lymphoblastic leukemia

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Effects of chronic exposure to low-level cadmium on renal tubular function and CYP2A6-mediated coumarin metabolism in healthy human subjects

Relationships between cadmium (Cd) body burden, kidney function and coumarin metabolism were investigated using two groups of 197 and 200 healthy Thais with men and women in nearly equal numbers. A mean age of one group was 30.5 years and it was 39.3 years for the other group. Of 397, 20 subjects (5%) excreted urine Cd between 1.4 mug/g and 3.8 mug/g creatinine and these subjects faced 10-15% increase in the probability of having abnormal urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG-uria). The prevalence of NAG-uria varied with Cd body burden in a dose-dependent manner (chi(2) = 22, P < 0.008). Also NAG-nuria was one of the three kidney effect markers tested that showed the greatest strength of correlation with urine Cd in both men and women (r = 0.48 P < 0.001). In addition, urine Cd excretion of men and women showed a positive correlation (r = 0.46 to 0.54. P < 0.001) with urine 7-hydroxycoumarin (7-OHC) excretion which was used as a marker of liver cytochrome P450 2A6 (CYP2A6) enzyme activity. Urinary CA excretion accounted for 25% of the total variation in urine 7-OHC excretion (P < 0.001). These data suggest that Cd may increase the expression of CYP2A6 in liver, resulting in enhanced coumarin metabolism in subjects with high Cd body burden. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

RNA interference against human papillomavirus oncogenes in cervical cancer cells results in increased sensitivity to cisplatin

Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAi-based cancer therapies because the oncogenes that cause cervical cancer, E6 and E7, are expressed only in cancerous cells. We investigated whether targeting HPV E6 and E7 oncogenes yields cancer cells more sensitive to chemotherapy by cisplatin, the chemotherapeutic agent currently used for the treatment of advanced cervical cancer. We have designed siRNAs directed against the HPV E6 oncogene that simultaneously targets both E6 and E7, which results in an 80% reduction in E7 protein and reactivation of the p53 pathway. The loss of E6 and E7 resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. Interference was specific in that no effect on HPV-negative cells was observed. We demonstrate that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells. The IC50 for HeLa cells treated with cisplatin was 9.4 mu M, but after the addition of a lentivirus-delivered shRNA against E6, the IC50 was reduced almost 4-fold to 2.4 mu M. We also observed a decrease in E7 expression with a concurrent increase in p53 protein levels upon cotreatment with shRNA and cisplatin over that seen with individual treatment alone. Our results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, probably because of increased p53 levels.

Genetic diversity of human metapneumovirus over 4 consecutive years in Australia

The molecular epidemiologic profile of human metapneumovirus (hMPV) infection has likely been skewed toward certain genetic subtypes because of assay-design issues, and no comprehensive studies have been conducted to date. Here, reverse-transcription polymerase chain reaction was used to screen 10,319 specimens from patients presenting to hospitals with suspected respiratory tract infections during 2001 - 2004. After analysis of 727 Australian hMPV strains, 640 were assigned to 1 of 4 previously described subtypes. hMPV was the most common pathogen detected, and subtype B1 was the most common lineage. Concurrent, annual circulation of all 4 hMPV subtypes in our study population was common, with a single, usually different hMPV subtype predominating in each year.

A multimodal perspective on the composition of cortical oscillations:frontiers in human neuroscience

An expanding corpus of research details the relationship between functional magnetic resonance imaging (fMRI) measures and neuronal network oscillations. Typically, integratedelectroencephalography(EEG) and fMRI,orparallel magnetoencephalography (MEG) and fMRI are used to draw inference about the consanguinity of BOLD and electrical measurements. However, there is a relative dearth of information about the relationship between E/MEG and the focal networks from which these signals emanate. Consequently, the genesis and composition of E/MEG oscillations requires further clarification. Here we aim to contribute to understanding through a series of parallel measurements of primary motor cortex (M1) oscillations, using human MEG and in-vitro rodent local field potentials. We compare spontaneous activity in the ~10Hz mu and 15-30Hz beta frequency ranges and compare MEG signals with independent and integrated layers III and V(LIII/LV) from in vitro recordings. We explore the mechanisms of oscillatory generation, using specific pharmacological modulation with the GABA-A alpha-1 subunit modulator zolpidem. Finally, to determine the contribution of cortico-cortical connectivity, we recorded in-vitro M1, during an incision to sever lateral connections between M1 and S1 cortices. We demonstrate that frequency distribution of MEG signals appear have closer statistically similarity with signals from integrated rather than independent LIII/LV laminae. GABAergic modulation in both modalities elicited comparable changes in the power of the beta band. Finally, cortico-cortical connectivity in sensorimotor cortex (SMC) appears to directly influence the power of the mu rhythm in LIII. These findings suggest that the MEG signal is an amalgam of outputs from LIII and LV, that multiple frequencies can arise from the same cortical area and that in vitro and MEG M1 oscillations are driven by comparable mechanisms. Finally, corticocortical connectivity is reflected in the power of the SMC mu rhythm. © 2013 Ronnqvist, Mcallister, Woodhall, Stanford and Hall.