HIV-1 drug resistance evolution among patients on potent combination antiretroviral therapy with detectable viremia

Many patients infected with HIV do not achieve or maintain virologic suppression below levels of detection while on potent combination antiretroviral therapy. The likelihood of emergence of incident mutations conferring reduced antiretroviral drug susceptibility was estimated among patients maintained on a stable regimen with ongoing detectable plasma HIV RNA levels. Ninety-eight HIV-infected patients were identified who had 2 genotypic antiretroviral resistance tests available. Poisson log-linear regression models were used to identify predictors and estimate incidence rates of number of acquired antiretroviral drug resistance mutations per person-year. At the 1st resistance test, 88% of patients had evidence of at least 1 mutation. Sixty percent of patients acquired at least 1 new mutation during a median of 9.3 months between consecutive resistance tests, with an incidence rate of 1.61 acquired mutations per person-year (95% CI: 1.36-1.90). Predictors of resistance evolution included average plasma HIV RNA level, HIV RNA slope, and number of mutations detected at the 1st resistance test. The likelihood of acquiring drug resistance mutations while remaining on potent combination antiretroviral therapy that does not confer complete suppression of HIV replication is relatively low and depends on the level of viral replication and prior resistance.     

Duplication 18q syndrome

Some variation in the phenotype of patients with dup(18q) is recognized. Our patient has the phenotype described for dup(18qter).     

Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP. Two coding substitutions were found in comparison with the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429]PLAP, into wild-type Chinese hamster ovary cells. We determined the k(cat) and K(m), of the PLAP S, F, and D allozymes using the non-physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples.     

CDK9/CYCLIN T1 expression during normal lymphoid differentiation and malignant transformation

CDK9 is a member of the CDC2-like family of kinases. Its cyclin partners are members of the CYCLIN T family (T1, T2a, and T2b) and CYCLIN K. The CDK9/CYCLIN T1 complex is very important in the differentiation programme of several cell types, controlling specific differentiation pathways. Limited data are available regarding the expression of CDK9/CYCLIN T1 in haematopoietic and lymphoid tissues. The aim of this study was to analyse the expression of the CDK9/CYCLIN T1 complex in lymphoid tissue, in order to assess its role in B- and T-cell differentiation and lymphomagenesis. CDK9/CYCLIN T1 expression was found by immunohistochemistry in precursor B and T cells. In peripheral lymphoid tissues, germinal centre cells and scattered B- and T-cell blasts in interfollicular areas expressed CDK9/CYCLIN T1, while mantle cells, plasma cells, and small resting T-lymphocytes displayed no expression of either molecule. CDK9/CYCLIN T1 expression therefore appears to be related to particular stages of lymphoid differentiation/activation. CDK9 and CYCLIN T1 were highly expressed in lymphomas derived from precursor B and T cells, from germinal centre cells, such as follicular lymphomas, and from activated T cells (ie anaplastic large cell lymphomas). Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma also showed strong nuclear staining. Diffuse large B-cell, Burkitt's lymphomas, and peripheral T-cell lymphomas, among T-cell lymphoproliferative disorders, showed a wide range of values. No expression of CDK9 or CYCLIN T1 was detected in mantle cell and marginal zone lymphomas. However, at the mRNA level, an imbalance in the CDK9/CYCLIN T1 ratio was found in follicular lymphoma and diffuse large B-cell lymphomas with germinal centre phenotype, and in the cell lines of classical Hodgkin's lymphomas, Burkitt's lymphomas, and anaplastic large cell lymphoma, in comparison with reactive lymph nodes. These results suggest that the CDK9/CYCLIN T1 complex may affect the activation and differentiation programme of lymphoid cells. The molecular mechanism through which the CDK9/CYCLIN T1 complex is altered in malignant transformation needs to be elucidated.     

Contraceptive use among South Asian women attending general practices in southwest London.

A cross-sectional survey of contraceptive practices was conducted among 180 South Asian women aged 16 to 50 years, attending inner-city general practices. Overall prevalence of contraceptive use was 63% and ranged from 70% in South Asian teenagers, to only 50% in women over 30 who had completed their family. Thirteen per cent of women had had a termination of pregnancy Although contraceptive use in this group is increasing, it has not yet reached national levels.     

Con A activates an Akt/PKB dependent survival mechanism to modulate TCR induced cell death in double positive thymocytes

While low avidity ligation of the T cell receptor (TCR) leads to positive selection and further maturation of developing thymocytes providing the immune system with mature CD4(+) and CD8(+) (single positive) T cells, high avidity ligation triggers negative selection by apoptotic cell death and therefore the TCR repertoire is purged of autoreactive T cells. On peripheral T cells, however, high avidity ligation of the TCR triggers activation and survival not death. In the present study we used concanavalin A (Con A) and alpha-CD3 epsilon antibody to investigate a possible survival mechanism in connection with TCR ligation. Con A and alpha-CD3 epsilon were used in the study for the following reasons: (1) they both mimic the effects of high avidity TCR ligation by activating peripheral T cells, and (2) they trigger distinctively different physiological changes in developing thymocytes. While Con A supports events associated with cellular survival, alpha-CD3 epsilon induces apoptotic cell death. In our experimental system the TCR was cross-linked by Con A and alpha-CD3 epsilon in thymocytes of major histocompatibility complex (MHC) deficient thymus organ cultures, where signals from the TCR can be triggered on zero background signal level. We have found that TCR cross-linking by Con A and not by alpha-CD3 epsilon decreases the gene and protein expression of the pro-apoptotic molecule, Bad; and that Con A is capable of the activation of the survival signalling pathway including protein kinase B (Akt/PKB) independently of phosphatidyl inositol kinase (PI3K).     

Outcomes of unrelated umbilical cord blood transplantation for X-linked adrenoleukodystrophy.

Adrenoleukodystrophy (ALD) is an X-linked disorder caused by a defect in the metabolism of long chain fatty acids leading to demyelination, neurodegeneration, and death. The disease typically presents in young boys and adolescent boys. Allogeneic bone marrow transplantation has been used to halt progression of the disease. However, many patients lack suitable HLA- matched related donors and must rely on unmatched donors for a source of stem cells. The purpose of this study was to evaluate outcomes of unrelated donor umbilical cord blood transplantation after chemotherapy-based myeloablative conditioning and retrospectively determine if baseline studies correlate and help predict outcome. Between November 22, 1996, and November 3, 2005, 12 boys with X-linked ALD who lacked HL- matched related donors were referred to Duke University Medical Center for transplantation. These children were conditioned with myeloablative therapy including busulfan, cyclophosphamide, and antithymocyte globulin before receiving umbilical cord-blood transplants from unrelated donors. Baseline studies of neurophysiologic, neuroimaging, and neurodevelopmental status were performed and patients were subsequently evaluated for survival, engraftment, graft-versus-host disease, and neurodevelopmental outcomes. A substudy evaluated whether baseline neuroimaging and neurophysiologic studies correlated with cognitive and motor function and if these studies were predictive of posttransplantation outcomes. The umbilical cord blood grafts had normal levels of very long chain fatty acids. They delivered a median of 6.98 x 10(7) nucleated cells per kilogram of recipient body weight and were discordant for up to 4 of 6 HLA markers. Neutrophil engraftment occurred at a median of 22.9 days after transplantation. Three patients had grade II-IV acute graft-versus-host disease; 2 had extensive chronic graft-versus-host disease. Cumulative incidence of overall survival of the group at 6 months is 66.7% (95% confidence interval 39.9-93.3%). Median follow-up was 3.3 years (range 12 days to 6.3 years). As previously reported with bone marrow transplantation, symptomatic patients faired poorly with lower survival and rapid deterioration of neurologic function. This study included 3 patients transplanted at a very young age (2.6-3.5 years) before the onset of clinical symptoms who continue to develop at a normal rate for 3-5 years posttransplant. Although baseline Loes scores correlated with cognitive and motor outcome, neurophysiologic studies failed to show statistically significant differences. Transplantation of boys with X-linked ALD using partial HLA-matched umbilical cord blood yields similar results to those previously reported after bone marrow transplantation. Superior outcomes were seen in neurologically asymptomatic boys less than 3.5 years of age at the time of transplantation. Baseline Loes scores were a strong predictor of cognitive and motor outcome.     

Contribution of cellular HIV-1 DNA quantification to the efficacy analysis of antiretroviral therapy: a randomized comparison of 2 regimens, including 3 drugs from 2 or 3 classes (TRIANON, ANRS 081)

Cellular HIV-1 DNA level was sequentially measured by quantitative polymerase chain reaction in 141 patients not previously treated with highly active antiretroviral therapy (HAART), who were enrolled in a 72-week randomized trial (ANRS 081 "Trianon") comparing 2 regimens, including 3 drugs from 2 classes (indinavir + stavudine + lamivudine, group 1) or 3 classes (indinavir + stavudine + nevirapine, group 2). The median decrease from baseline to week 72 in cellular HIV-1 DNA level was not significantly different between the 2 groups (0.54 and 0.45 log10 copies/10 peripheral blood mononuclear cells [PBMCs] in groups 1 and 2, respectively), whereas a higher proportion of patients maintained a plasma HIV-1 RNA level less than 20 copies/mL at week 72 in group 1 than in group 2 (79% and 52%; P = 0.0009). Furthermore, the difference in cellular HIV-1 DNA decrease from baseline to week 72 between patients who achieved a plasma HIV-1 RNA level less than 20 copies/mL at week 72 and those who did not was not statistically significant (0.54 and 0.45 log10 copies/10 PBMCs, respectively; P = 0.14). The decay in cellular HIV-1 DNA from baseline to week 72 was higher in antiretroviral-naive patients than in pretreated patients (0.55 and 0.23 log10 copies/10 PBMCs, respectively; P = 0.0008). The cellular HIV-1 DNA level change under therapy was best fitted to a 2-phase decay model with a junction point at week 16, from which its half-life was estimated at 18 weeks during the initial phase and at 104 weeks thereafter. In conclusion, the changes under therapy in cellular HIV-1 DNA level, which were mostly coincident to those of plasma HIV-1 RNA, did not add significant information to the comparison of the viral efficacy of the 2 studied regimens.