Diminished transmission of drug resistant HIV-1 variants with reduced replication capacity in a human transmission model

Background

Different patterns of drug resistance are observed in treated and therapy naïve HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naïve population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns.As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals.

Results

In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5⁺ Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5⁺ Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs.

Conclusions

Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.

     

Generation of “Virtual” Control Groups for Single Arm Prostate Cancer Adjuvant Trials

It is difficult to construct a control group for trials of adjuvant therapy (Rx) of prostate cancer after radical prostatectomy (RP) due to ethical issues and patient acceptance. We utilized 8 curve-fitting models to estimate the time to 60%, 65%, … 95% chance of progression free survival (PFS) based on the data derived from Kattan post-RP nomogram. The 8 models were systematically applied to a training set of 153 post-RP cases without adjuvant Rx to develop 8 subsets of cases (reference case sets) whose observed PFS times were most accurately predicted by each model. To prepare a virtual control group for a single-arm adjuvant Rx trial, we first select the optimal model for the trial cases based on the minimum weighted Euclidean distance between the trial case set and the reference case set in terms of clinical features, and then compare the virtual PFS times calculated by the optimum model with the observed PFSs of the trial cases by the logrank test. The method was validated using an independent dataset of 155 post-RP patients without adjuvant Rx. We then applied the method to patients on a Phase II trial of adjuvant chemo-hormonal Rx post RP, which indicated that the adjuvant Rx is highly effective in prolonging PFS after RP in patients at high risk for prostate cancer recurrence. The method can accurately generate control groups for single-arm, post-RP adjuvant Rx trials for prostate cancer, facilitating development of new therapeutic strategies.     

Testosterone suppresses the expression of regulatory enzymes of fatty acid synthesis and protects against hepatic steatosis in cholesterol-fed androgen deficient mice

Aims

Non-alcoholic fatty liver disease and its precursor hepatic steatosis is common in obesity and type-2 diabetes and is associated with cardiovascular disease (CVD). Men with type-2 diabetes and/or CVD have a high prevalence of testosterone deficiency. Testosterone replacement improves key cardiovascular risk factors. The effects of testosterone on hepatic steatosis are not fully understood.

Main methods

Testicular feminised (Tfm) mice, which have a non-functional androgen receptor (AR) and very low serum testosterone levels, were used to investigate testosterone effects on high-cholesterol diet-induced hepatic steatosis.

Key findings

Hepatic lipid deposition was increased in Tfm mice and orchidectomised wild-type littermates versus intact wild-type littermate controls with normal androgen physiology. Lipid deposition was reduced in Tfm mice receiving testosterone treatment compared to placebo. Oestrogen receptor blockade significantly, but only partially, reduced the beneficial effects of testosterone treatment on hepatic lipid accumulation. Expression of key regulatory enzymes of fatty acid synthesis, acetyl-CoA carboxylase alpha (ACACA) and fatty acid synthase (FASN) were elevated in placebo-treated Tfm mice versus placebo-treated littermates and Tfm mice receiving testosterone treatment. Tfm mice on normal diet had increased lipid accumulation compared to littermates but significantly less than cholesterol-fed Tfm mice and demonstrated increased gene expression of hormone sensitive lipase, stearyl-CoA desaturase-1 and peroxisome proliferator-activated receptor-gamma but FASN and ACACA were not altered.

Significance

An action of testosterone on hepatic lipid deposition which is independent of the classic AR is implicated. Testosterone may act in part via an effect on the key regulatory lipogenic enzymes to protect against hepatic steatosis.     

Copepod development rates in relation to genome size and 18S rDNA copy number.

It is known that body sizes and temperature-independent developmental durations within two genera of calanoid Copepoda (Crustacea) are positively related to nuclear DNA contents of their somatic nuclei. Evidently because of the constraint of similar cell numbers among the species, (nucleotypic) effects of nucleus size on cell size and on cell-level processes are expressed at the whole-organism level. Here, we show that developmental durations of eight species of five genera are also negatively related to their greatly differing numbers of 18S rRNA genes per unit DNA. We propose that levels of rDNA iteration among copepods have been controlled by natural selection to regulate ribosome concentrations, therefore protein production and development rates, independently of the large variations in genome sizes, which are in turn adapted to regulating cell and therefore body sizes.     

DNA surveillance: web-based molecular identification of whales,dolphins, and porpoises

DNA Surveillance is a Web-based application that assists in the identification of the species and population of unknown specimens by aligning user-submitted DNA sequences with a validated and curated data set of reference sequences. Phylogenetic analyses are performed and results are returned in tree and table format summarizing the evolutionary distances between the query and reference sequences. DNA Surveillance is implemented with mitochondrial DNA (mtDNA) control region sequences representing the majority of recognized cetacean species. Extensions of the system to include other gene loci and taxa are planned. The service, including instructions and sample data, is available at http://www.dna-surveillance.auckland.ac.nz.     

Demonstration of diet-induced decoupling of fatty acid and cholesterol synthesis by combining gene expression array and 2H2O quantification

The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate-free diet. At the end of the dietary intervention, the two groups received (2)H(2)O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a custom-designed pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydrate-free diet. In support of these findings, (2)H(2)O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered.     

Schistosoma mansoni: migration potential of normal and radiation attenuated parasites in naive guinea pigs

Compressed tissue autoradiography using [75Se]selenomethionine labelled parasites has been used to investigate the migration potential of normal and radiation attenuated cercariae of Schistosoma mansoni in naive guinea pigs. By Day 14 after infection. 44% of normal parasites were detected as reduced silver foci in the liver; this value corresponded well with the number of liver parasites recovered by retrograde perfusion of the hepatic portal system on Day 42 (42% of the challenge). In contrast, cercariae subjected to 50 krad of gamma irradiation failed to migrate out of the skin. The migration capacity of 20 krad irradiated parasites was less severely affected in that about half of the challenge parasites reached the lungs, but virtually none moved to the liver. These data are discussed in relation to the kinetics of immunity induced in guinea pigs by infection or vaccination with normal or radiation attenuated parasites.     

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae. International Journal for Parasitology16: 491–510. Naive guinea pigs and guinea pigs vaccinated 4 weeks previously with highly irradiated cercariae were challenged percutaneously with normal cercariae. Skin samples from the challenge site were then harvested at varying times to provide histological, quantitative and ultrastructural data on the respective cellular responses to cercarial invasion. The primary cutaneous reaction was characterised by neutrophils; these cells reached peak numbers (16% of total cells) by 18 h. Eosinophils and basophils made only a small contribution to the infiltrate (2.9 and 5.7% respectively). Some basophils showed evidence of anaphylactic degranulation, others seemed to be damaged, but most appeared normal. Mononuclear cells of varied morphology were present at all times, but activated fibroblasts were prominent, and collagen deposition increased with time. Degranulating mast cells were recognised at 24 and 48 h. Dead schistosomula were never seen in naive-challenged skin, although one or two of the observed larvae showed minor tegumental abnormalities. In vaccinated guinea pigs, the cutaneous cellular response to challenge was significantly enhanced, with basophils dominating the reaction (33% of total cells at 24 h). Many basophils were already degranulating by the anaphylactic mechanism at 3 h post challenge, and free basophil granules were seen frequently. Both intact cells and free granules congregated in close proximity to invading larvae. Eosinophils were also present in greater numbers at secondary reaction sites, but they never exceeded 6% of the total infiltrate. Mononuclear cells believed to be immature eosinophils were prominent from 3 h. For the first time, the mechanism of eosinophil attachment and degranulation onto a multicellular target, described previously only from in vitro investigations, was recognized in vivo. Neutrophil numbers matched those recorded in naive-challenged skin at 3 and 6 h, but declined thereafter, while mast cells were seen degranulating at these early times. Mononuclear cells again presented a variety of morphological appearances; of particular note were large cells that had phagocytosed debris and were presumed to be macrophages, and small rounded cells with scant cytoplasm and few organelles, that may have been lymphocytes. By 12 h, large areas of the dermis had become severely disorganised and numerous, free basophil granules were distributed amongst the other cellular constituents. Dead schistosomula, denuded of tegument, were clearly recognised at 6 h, and such individuals invariably had neutrophils attached to their exposed muscle layers. Since dead schistosomula were not identified in naive-challenged guinea pig skin, it is concluded that a percentage of the challenge larvae, however small, is preferentially killed in the skin of the vaccinated animals.