Nucleoside Combination Research Articles

Nucleoside supplements as treatments for mitochondrial DNA depletion syndrome.

Introduction: In mitochondrial DNA (mtDNA) depletion syndrome (MDS), patients cannot maintain sufficient mtDNA for their energy needs. MDS presentations range from infantile encephalopathy with hepatopathy (Alpers syndrome) to adult chronic progressive external ophthalmoplegia. Most are caused by nucleotide imbalance or by defects in the mtDNA replisome. There is currently no curative treatment available. Nucleoside therapy is a promising experimental treatment for TK2 deficiency, where patients are supplemented with exogenous deoxypyrimidines. We aimed to explore the benefits of nucleoside supplementation in POLG and TWNK deficient fibroblasts. Methods: We used high-content fluorescence microscopy with software-based image analysis to assay mtDNA content and membrane potential quantitatively, using vital dyes PicoGreen and MitoTracker Red CMXRos respectively. We tested the effect of 15 combinations (A, T, G, C, AT, AC, AG, CT, CG, GT, ATC, ATG, AGC, TGC, ATGC) of deoxynucleoside supplements on mtDNA content of fibroblasts derived from four patients with MDS (POLG1, POLG2, DGUOK, TWNK) in both a replicating (10% dialysed FCS) and quiescent (0.1% dialysed FCS) state. We used qPCR to measure mtDNA content of supplemented and non-supplemented fibroblasts following mtDNA depletion using 20µM ddC and after 14- and 21-day recovery in a quiescent state. Results: Nucleoside treatments at 200µM that significantly increased mtDNA content also significantly reduced the number of cells remaining in culture after 7days of treatment, as well as mitochondrial membrane potential. These toxic effects were abolished by reducing the concentration of nucleosides to 50µM. In POLG1 and TWNK cells the combination of ATGC treatment increased mtDNA content the most after 7days in non-replicating cells. ATGC nucleoside combination significantly increased the rate of mtDNA recovery in quiescent POLG1 cells following mtDNA depletion by ddC. Conclusion: High-content imaging enabled us to link mtDNA copy number with key read-outs linked to patient wellbeing. Elevated G increased mtDNA copy number but severely impaired fibroblast growth, potentially by inhibiting purine synthesis and/or causing replication stress. Combinations of nucleosides ATGC, T, or TC, benefited growth of cells harbouring POLG mutations. These combinations, one of which reflects a commercially available preparation, could be explored further for treatment of POLG patients.

TLR7/8 stress response drives histiocytosis in SLC29A3 disorders.

Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs. However, little is known about the mechanism by which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3-/- mice was shown to depend on Toll-like receptor 7 (TLR7), which senses a combination of nucleosides and oligoribonucleotides (ORNs). TLR7 increased phagocyte numbers by driving the proliferation of Ly6Chi immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3-/- mice. Downstream of TLR7, FcRγ and DAP10 were required for monocyte proliferation. Histiocytosis is accompanied by inflammation in SLC29A3 disorders. However, TLR7 in nucleoside-laden splenic monocytes failed to activate inflammatory responses. Enhanced production of proinflammatory cytokines was observed only after stimulation with ssRNAs, which would increase lysosomal ORNs. Patient-derived monocytes harboring the G208R SLC29A3 mutation showed enhanced survival and proliferation in a TLR8-antagonist-sensitive manner. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

Supplementary nucleosides improve the performance through enhanced enzyme activity, intestinal development and transporter genes in broiler chickens

A biological experiment was carried out to evaluate dietary nucleoside supplementation on growth performance, digestive enzymes activities, immune response, and intestinal transporter genes expression in broiler chicken. A total of 720 newly hatched CARIBRO VISHAL broiler chicks were weighed and randomly divided into eight groups with nine replicates. The dietary treatments were as follows: Group I: diet without antibiotic supplement (control), group II: diet supplemented with antibiotic (positive control), groups III, IV and V: diet supplemented with combination of nucleosides at 0.5, 1.0 and 1.5 g/kg feed, respectively, for 14 days, groups VI, VII and VIII: diet supplemented with nucleosides at 0.5, 1.0 and 1.5 g/kg feed, respectively, for 21 days. The combination of nucleosides (equal proportion (1:1:1:1) adenosine, guanosine, cytosine, and uridine with 99% purity) were used in the study. Body weight was significantly higher in the birds fed diets containing antibiotics and 1.5 g/kg nucleosides fed groups. The supplementation had positive effect on the activity of amylase and lipase enzymes and the absorptive surface (villi length). It could be concluded that, the dietary supplementation of nucleosides improved the performance of broilers with better cellular and humoral immunity than control. The study further confirmed that nucleosides supplementation improved gut development and could be an alternative to antibiotic growth promoters in broiler production.

In ovo Administration of Nucleosides Improved the Performance, Apparent Metabolizable Energy and Gut Development in Broiler Chickens.

An in ovo study on the effect of the administration of a combination of nucleosides (25, 50, and 100 mg/egg) on hatchability, growth performance, energy metabolizability, and intestinal morphology in broilers was carried out. Four hundred eighty (480) fertile eggs were divided into four groups (in four replicates each having 30 eggs). On the 18th days of incubation of the eggs, candling was carried out and the fertile eggs were selected and given one of the four in ovo administrations. Group one served as control and was injected with phosphate-buffered saline (PBS). The other groups were given in ovo administration of nucleosides (25, 50, and 100 mg/egg) at 100 μl through the yolk sac route, and chicks of respective groups were hatched out. Among the experimental groups, the hatchability was comparable; however, the hatchability was affected in the group injected with a higher level of nucleosides at 100 mg/egg. The hatched out chicks from higher doses of nucleosides (50 and 100 mg) had higher body weight (BW) (P < 0.05) than the control. Higher energy metabolizability (%) was observed in nucleoside-injected groups. Plasma protein concentration was higher in groups administered with nucleosides (50 and 100 mg). Histologically, the intestinal villi length was maximum in 100 mg-injected group followed by 50 and 25 mg. Relative expression of homeobox (Cdx) in the jejunum was significantly (P < 0.05) upregulated in all the injected groups at 3, 7, and 14 days of age. Nucleoside-administered groups had better performance, energy metabolizability, and intestinal morphology. Among the experimental groups, the administration of nucleosides at 50 mg/egg resulted in higher growth performance, plasma protein, intestinal surface, and villi development in broiler chickens.

Anomeric 5-Aza-7-deaza-2'-deoxyguanosines in Silver-Ion-Mediated Homo and Hybrid Base Pairs: Impact of Mismatch Structure, Helical Environment, and Nucleobase Substituents on DNA Stability.

Nucleoside configuration (α-d vs. β-d), nucleobase substituents, and the helical DNA environment of silver-mediated 5-aza-7-deazaguanine-cytosine base pairs have a strong impact on DNA stability. This has been demonstrated by investigations on oligonucleotide duplexes with silver-mediated base pairs of α-d and β-d anomeric 5-aza-7-deaza-2'-deoxyguanosines and anomeric 2'-deoxycytidines incorporated in 12-mer duplexes. To this end, a new synthetic protocol has been developed to access the pure anomers of 5-aza-7-deaza-2'-deoxyguanosine by glycosylation of either the protected nucleobase or its salt followed by separation of the glycosylation products by crystallization and chromatography. Thermal stability measurements were performed on duplexes with α-d/α-d and β-d/β-d homo base pairs or α-d/β-d and β-d/α-d hybrid pairs within two sequence environments, positions 6 or 7, of oligonucleotide duplexes. The respective Tm stability increases observed after silver ion addition differ significantly. Homo base pairs with β-d/β-d or α-d/α-d nucleoside combinations are more stable than α-d/β-d hybrid base pairs. The positional switch of silver-ion-mediated base pairs has a significant impact on stability. Nucleobase substituents introduced at the 5-position of the dC site of silver-mediated base pairs affect base pair stability to a minor extent. Our investigation might lead to applications in the construction of bioinspired nanodevices, in DNA diagnostics, or metal-DNA hybrid materials.

Improvement of growth rates through nucleoside media supplementation of CHO clones.

Chinese Hamster Ovary (CHO) cells are used for the production of therapeutic proteins. This work examines improving passaging growth rate of two CHO clones. Growth rates were significantly improved for both clones with supplementation of the nucleosides cytidine, hypoxanthine, uridine, and thymidine to the culturing media at the optimal concentration of 100µM of each nucleoside. We investigated supplementing the same combination of nucleosides to seed bioreactors and production fed batch bioreactors. In the seed bioreactors, growth rate and harvest density were improved. However, in the production fed batch bioreactors, no improvements in growth rate or peak viable cell density were observed. Cell cycle analysis of the passaging cells provides evidence that nucleosides can affect the cell cycle. It is not clear from our work how the nucleosides impact the cell cycle regulatory pathways. Overall, nucleoside supplementation in cell culture media is an effective approach for improving growth rate in passaging and seed bioreactors of certain CHO cells.

Entecavir and Peginterferon Alfa-2a in Adults With Hepatitis B e Antigen-Positive Immune-Tolerant Chronic Hepatitis B Virus Infection.

Monotherapy with interferon or nucleoside analog is generally not recommended during the immune-tolerant (IT) phase of chronic hepatitis B virus (HBV) infection. Recognition that high HBV DNA levels are associated with hepatocellular carcinoma has increased interest in treating HBV in the IT phase. Small pediatric studies reported efficacy with combination nucleoside analog and interferon therapy. The aim of this study was to evaluate the safety and efficacy of the combination of entecavir and peginterferon in adults in the IT phase of chronic HBV infection. Hepatitis B e antigen (HBeAg)-positive adults with HBV DNA > 107 IU/mL and alanine aminotransferase (ALT) ≤ 1.5 times the upper limit of normal (ULN) (male: ≤ 45, female: ≤ 30 U/L) received entecavir 0.5 mg daily for 8 weeks followed by the addition of peginterferon alfa-2a 180 µg/week to entecavir for an additional 40 weeks. The primary endpoint was HBeAg loss and HBV DNA ≤ 1,000 IU/mL 48 weeks after end of treatment (EOT). Among 28 participants from 11 sites, the median age was 37.2 (range: 22-61) years, 54% were male, and 96% were Asian. Nearly all were infected with genotype C (64%) or B (32%). Median baseline HBV DNA was 8.2 log10 IU/mL, and ALT was 0.9 times the ULN. Although one (4%) participant cleared HBeAg, none met the primary endpoint of both HBeAg loss AND HBV DNA ≤ 1,000 IU/mL 48 weeks post-EOT. ALT elevations > 5 times the ULN occurred in eight (29%) participants, and none were associated with icterus. Forty-eight weeks posttreatment, HBV DNA rebounded to baseline levels in all participants, including the participant who lost HBeAg, and ALT values returned to near baseline levels in all but four participants. Conclusion: A lead-in strategy of 8 weeks of entecavir followed by combination peginterferon and entecavir therapy for 40 weeks had limited efficacy in adults in the IT phase of chronic HBV infection and cannot be recommended.

Viral activity and outcome of hepatitis B surface antigen-positive grafts in deceased liver transplantation.

Indications of liver transplantation are extensive, but deceased donation does not meet the demand. Hepatitis B surface antigen (HBsAg)-positive grafts used to be discarded in the past. The aim of this study was to examine viral activity and outcome of HBsAg-positive deceased grafts transplanted to HBsAg-positive recipients. Eleven HBsAg-positive deceased grafts were transplanted to HBsAg-positive patients with acute liver failure (3 patients), hepatocellular carcinoma (6 patients) and repeatedly bleeding varices (2 patients). Postoperatively, hepatitis B virus (HBV) infection was treated by a combination of antiviral nucleoside and nucleotide analogues. HBV DNA and HBsAg were measured periodically. The median (interquartile) model of end-stage liver disease score for the recipients was 19 (16-32) with a range from 11 to 40. HBV DNA was detected in 6 patients with a range from 61 to 1083IU/mL before transplantation. After transplantation, HBV DNA was detected in 4 patients in the first month and 2 patients in the 6th month and became undetectable for all patients at end of the first year. The quantitative HBsAg ranged from 0.86 to 241.1IU/mL at 6months and 0.34 to 238.5IU/mL at 24months (P=.135). Three of the patients died in the early phase, and the other patients were followed up for 40.0±19.2months with normal liver function. In conclusion, HBsAg-positive deceased liver grafts function well with minimal viral activity under treatment of combined antiviral nucleoside and nucleotide analogues. Use of HBsAg-positive deceased grafts is feasible and increases the donor pool to rescue dying patients.

Combination of purine and pyrimidine nucleosides influences growth performance, gut morphology, digestive enzymes, serum biochemical indices and immune functions in broiler chickens

The objective of the current study was to investigate the cumulative effects of purine and pyrimidine nucleosides on performance, gut morphology, digestive enzymes and immune system in broilers from 0 to 21days of age. A total of 360day-old male chicks (Cobb 500) were randomly assigned to 4 experimental diets with 6 replications of 15 birds each in a completely randomized design. Experimental diets were 1) control diet based on maize-soybean meal which supplemented with following nucleosides: 2) 0.05% Adenosine + 0.025% Uridine + 0.025% Cytidine (AUC); 3) 0.05% Guanosine + 0.025% Uridine + 0.025% Cytidine (GUC); 4) 0.025% Adenosine + 0.025% Guanosine + 0.025% Uridine + 0.025% Cytidine (AGUC). Body weight and feed intake of birds were measured on days 10 and 21 to calculate the average daily gain, average feed intake and FCR. On days 11 and 21, 2 birds per cage (12 birds/treatment) were selected to collect blood samples and euthanize to obtain jejunal, bursa and spleen samples. Results showed that AUC significantly increased body weight and average daily gain, intestinal villus height and width, activity of brush border enzymes (alkaline phosphatase and aminopeptidase). AUC and AGUC significantly increased high density lipoprotein and uric acid, respectively, while dietary nucleosides did not affect cholesterol and triglyceride concentrations in serum samples. AUC significantly improved immune indices such as the relative weight of the Bursa of Fabricius and the concentration of Ig A. In conclusion, the present study clearly demonstrated that combination of adenosine + uridine + cytidine could be useful feed additive for improving economically important traits in broilers.

5-fluorouracil Toxicity Mechanism Determination in Human Keratinocytes: in vitro Study on HaCaT Cell Line.

5-fluorouracil (5-FU) and capecitabine therapy is often accompanied by palmar-plantar erythrodysesthesia (PPE) which is manifestation of 5-FU toxicity in keratinocytes. The main mechanisms of 5-FU action are thymidylate synthase (TS) inhibition which can be abrogated by thymidine and strengthened by calciumfolinate (CF) and incorporation of fluorouridinetriphosphate into RNA which can be abrogated by uridine. For proper PPE treatment 5-FU mechanism of action in keratinocytes needs to be elucidated. We used the 5-FU toxicity modulators uridine, thymidine and CF to discover the mechanism of 5-FU action in human keratinocyte cell line HaCaT. To measure the cellular viability, we used MTT test and RTCA test. CF did not augment 5-FU toxicity and 5-FU toxicity was weakened by uridine. Therefore, the primary mechanism of 5-FU toxicity in keratinocytes is 5-FU incorporation into RNA. The uridine protective effect cannot fully develop in the presence of CF. Thymidine addition to 5-FU and uridine treated cells not only prevents the toxicity-augmenting CF effect but it also prolongs the 5-FU treated cells survival in comparison to uridine only. Therefore, it can be assumed that in the presence of uridine the 5-FU toxicity mechanism is switched from RNA incorporation to TS inhibition. Although particular 5-FU toxicity mechanisms were previously described in various cell types, this is the first time when various combinations of pyrimidine nucleosides and CF were used for 5-FU toxicity mechanism elucidation in human keratinocytes. We suggest that for PPE treatment ointment containing uridine and thymidine should be further clinically tested.

Flipping the Switch “On” for Aminoglycoside-Resistance Enzymes: The Mechanism Is Finally Revealed!

In a recent issue of Structure, Caldwell etal. (2016) determined crystal structures of APH(2″)-Ia in complex with various combinations of aminoglycosides and nucleosides, which compellingly revealed that the catalytic activity of this resistance enzyme is regulated by a conformational change of the triphosphate of GTP, a mechanism previously unknown for antibiotic kinases.

Effects of Dietary Lecithin, Nucleoside, and Krill Supplementation to a Fishmeal Based Diet on Growth and Feed Utilization of Sharpsnout Sea Bream (Diplodus Puntazzo)

The aim of this 96 day feeding trial was to investigate the effects of the addition of different combinations of dietary lecithin, nucleosides, and krill to a fishmeal-based diet on the growth, feed utilization, feed consumption, and body composition of sharpsnout sea bream (Diplodus puntazzo). Six hundred (600) fish (average weight 21.21 ± 0.06SD g) were divided into 8 groups (triplicate treatments) and fed eight isoenergetic fishmeal-based diets, (C-control, L-lecithin, N-nucleosides, K-krill, L+N-lecithin+nucleosides, N+K-nucleosides+krill, L+K-lecithin+krill, L+N+K-lecithin+nucleosides+krill). The effects of the dietary regimes were evaluated in terms of specific growth rate (SGR), feed conversion ratio (FCR), daily feed intake (DFI), and whole body chemical composition (moisture, crude ash, crude protein and crude lipid). At the end the trial the fish had tripled their initial weight. SGR, FCR and DFI were 1.17-1.24 %, 0.95-1.01, and 1.03-1.14% respectively. Although differences were observed between some groups, none of the tested feed additives improved SGR, FCR, and DFI, compared to the control diet. Analysis of whole body proximate composition showed that moisture, crude ash, crude protein, and crude lipid ranged 61.45-64.00%, 3.96-4.26%, 15.44-17.26% and 14.87-18.82% respectively. Crude lipid concentration was higher in whole body of fish fed the nucleoside supplemented diet compared to control, lecithin, and krill groups. No other effects of the dietary regime on the whole body composition of sharpshout sea bream were observed.

Abacavir-based triple nucleoside regimens for maintenance therapy in patients with HIV.

Regimen simplification can be defined as a change in established effective therapy to reduce pill burden and dosing frequency, to enhance tolerability, or to decrease specific food and fluid requirements. Many patients on suppressive antiretroviral therapy may be considered candidates for a simplification strategy and, among them, those who have achievedvirologic suppression. Several clinical trials have evaluated the efficacy of triple nucleoside combination as a simplification therapy in patients who achieved virologic suppression The aim of this review is to combine randomised, controlled trials to examine whether in patients with undetectable viraemia on a Protease inhibitor (PI) based regimen simplification treatment with abacavir (ABC)-based triple-nucleoside combinations has similar rates of efficacy and tolerability compared with a PI regimen or simplification with a NNRTIs (efavirenz-EFV- or nevirapine-NVP) containing regimen. Studies were included if they had at least two of the three interventions, including one 3NRTI arm. Electronic databases and conference proceedings were searched (1996-2012) with relevant search terms without limits to language. Randomised controlled trials (RCTs) only are included in this review. Patients population is represented by HIV-infected adult patients treated with a PI-containing regimen (PI or boosted PI), with undetectable viral load. Patients on a PI-containing regimen had three possibilities: continue the PI regimen or switch to a simplification maintenance regimen, including switch to a NNRTI (EFV or NVP) containing regimen, or switch to a triple-NRTI regimen (ABC-zidovudine-lamivudine) The primary outcomes were: proportion of patients discontinuing or switching antiretroviral therapy due to virologic failure or to adverse events; death (all cause) and AIDS defining illness; occurrence of myocardial infarction and cardiovascular disease. Secondary outcomes were: proportion of patients maintaining an undetectable viral load (e.g. HIV-RNA <50 or <400 copies/mm(3)); change in mean CD4+ cell count; occurrence of lipodystrophy. We applied Cochrane Collaboration tools to assess each individual study for risk for bias. We included eight RCT, for a total of 1,610 patients. All the studies included HIV-1 infected patients virologically suppressed after a successful treatment with PI containing ART. Articles included in the analysis were published between 2001 and 2010, and could be classified as low risk of bias trials in most of the domains considered. Overall, there was no significant difference between the participants on triple nucleoside combination and controls, either PI-based or NNRTI based in terms of overall failures, death and AIDS related events, and rates of patients with viral load below the detectability cut-off. For the outcomes discontinuation for adverse events and virologic failures, the RRs were not significant , albeit being not far from the alpha level of 0.05, thus suggesting a weak evidence of lower incidence of side effects and an higher incidence of virologic failure in the 3NRTI group compared to controls . Change in lipids and in CD4 cells from baselines were reported in 7 studies, but inconsistency in reporting these data did not allow quantitative analysis. However, all agreed that simplification with ABC had a favourable and significant impact on lipid metabolism compared to control group. An increase in CD4 cells count from baseline was evident in all analysed studies, without significant differences between ABC and controls in individual studies. The strategy of switching to triple nucleoside regimens shows weak evidence of lower incidence of side effects and a higher incidence of virologic failure in the 3NRTI group compared to controls. Simplification with 3NRTI holds the advantages of preserving other classes of antiretroviral drugs, to lower blood lipids, and to be cost effective and simple to administer.Thus, simplification with triple nucleoside regimens AZT + 3TC + ABC should be still considered for individuals who are unable to tolerate or have contraindications to NNRTI or PI based regimens. Additional data are needed on longer-term efficacy of triple NRTI regimens, particularly on the development of antiretroviral resistance. Though studies in the current review were conducted between 2001 and 2010, the largemajority of patients from studies analysed received oldPI regimens (e.g., indinavir, ritonavir, nelfinavir, saquinavir) not longer recommended by International Guidelines. Since current guidelines recommend new "lipid -friendly" PI, future studies should compare regimens containing these news PIs to triple NRTI regimens. More realistically, however, there are opportunities to examine these issues in existing cohorts.

Metal-Mediated DNA Base Pairing: Alternatives to Hydrogen-Bonded Watson–Crick Base Pairs

With its capacity to store and transfer the genetic information within a sequence of monomers, DNA forms its central role in chemical evolution through replication and amplification. This elegant behavior is largely based on highly specific molecular recognition between nucleobases through the specific hydrogen bonds in the Watson-Crick base pairing system. While the native base pairs have been amazingly sophisticated through the long history of evolution, synthetic chemists have devoted considerable efforts to create alternative base pairing systems in recent decades. Most of these new systems were designed based on the shape complementarity of the pairs or the rearrangement of hydrogen-bonding patterns. We wondered whether metal coordination could serve as an alternative driving force for DNA base pairing and why hydrogen bonding was selected on Earth in the course of molecular evolution. Therefore, we envisioned an alternative design strategy: we replaced hydrogen bonding with another important scheme in biological systems, metal-coordination bonding. In this Account, we provide an overview of the chemistry of metal-mediated base pairing including basic concepts, molecular design, characteristic structures and properties, and possible applications of DNA-based molecular systems. We describe several examples of artificial metal-mediated base pairs, such as Cu(2+)-mediated hydroxypyridone base pair, H-Cu(2+)-H (where H denotes a hydroxypyridone-bearing nucleoside), developed by us and other researchers. To design the metallo-base pairs we carefully chose appropriate combinations of ligand-bearing nucleosides and metal ions. As expected from their stronger bonding through metal coordination, DNA duplexes possessing metallo-base pairs exhibited higher thermal stability than natural hydrogen-bonded DNAs. Furthermore, we could also use metal-mediated base pairs to construct or induce other high-order structures. These features could lead to metal-responsive functional DNA molecules such as artificial DNAzymes and DNA machines. In addition, the metallo-base pairing system is a powerful tool for the construction of homogeneous and heterogeneous metal arrays, which can lead to DNA-based nanomaterials such as electronic wires and magnetic devices. Recently researchers have investigated these systems as enzyme replacements, which may offer an additional contribution to chemical biology and synthetic biology through the expansion of the genetic alphabet.

Abacavir Use and Risk of Acute Myocardial Infarction and Cerebrovascular Events in the Highly Active Antiretroviral Therapy Era

Some studies have suggested that exposure to antiretroviral therapy (ART) with abacavir is associated with an increased risk of acute myocardial infarction (AMI). Using the Veterans Health Administration's Clinical Case Registry we calculated the risk of AMI and cerebrovascular events (CVA) associated with the cumulative use of abacavir and other nucleoside combinations. We also evaluated the impact of pre-existing chronic kidney disease on the selection of abacavir versus tenofovir in the last recorded ART regimen, and on highly active antiretroviral therapy-associated AMI and CVA risks. A total of 19,424 human immunodeficiency virus-infected patients contributed 76,376 patient-years of follow. After adjusting for age, hypercholesterolemia, hypertension, type 2 diabetes, and smoking, the hazard ratio (HR) for each year of abacavir use was 1.18 (95% confidence interval [CI], .92-1.50; P=.191) for AMI and 1.16 (95% CI, .98-1.37; P=.096) for CVA. Abacavir use was more common among patients with prior chronic kidney disease than was tenofovir use (12.46% versus 7.15%; P=.0001), and chronic kidney disease was associated with a significantly higher risk of AMI (HR, 2.41; 95% CI, 1.73-3.36), and CVA (HR, 1.80; 95% CI, 1.44-2.24). Compared with patients who received neither tenofovir nor abacavir, patients who received tenofovir had lower risk of AMI (HR, 0.16; 95% CI, .08-.33; P=.0001) and CVA (HR, 0.22; 95% CI, .15-.32; P=.001). Use of abacavir was associated with lower risk of CVA (HR, 0.60; 95% CI, .45-.79). We observed no association between cumulative or current abacavir use and AMI or CVA. Abacavir use was more common than was tenofovir use among patients with prior chronic kidney disease, and chronic kidney disease independently predicted higher rates of AMI and CVA.

British HIV Association guidelines for antiretroviral treatment of HIV‐2‐positive individuals 2010

British HIV Association guidelines for antiretroviral treatment of HIV‐2‐positive individuals 2010

The non-nucleoside reverse transcriptase inhibitor efavirenz stimulates replication of human immunodeficiency virus type 1 harboring certain non-nucleoside resistance mutations

We measured the effects of non-nucleoside reverse transcriptase (RT) inhibitor-resistant mutations K101E+G190S, on replication fitness and EFV-resistance of HIV(NL4-3). K101E+G190S reduced fitness in the absence of EFV and increased EFV resistance, compared to either single mutant. Unexpectedly, K101E+G190S also replicated more efficiently in the presence of EFV than in its absence. Addition of the nucleoside resistance mutations L74V or M41L+T215Y to K101E+G190S improved fitness and abolished EFV-dependent stimulation of replication. D10, a clinical RT backbone containing M41L+T215Y and K101E+G190S, also demonstrated EFV-dependent stimulation that was dependent on the presence of K101E. These studies demonstrate that non-nucleoside reverse transcriptase inhibitors can stimulate replication of NNRTI-resistant HIV-1 and that nucleoside-resistant mutants can abolish this stimulation. The ability of EFV to stimulate NNRTI-resistant mutants may contribute to the selection of HIV-1 mutants in vivo. These studies have important implications regarding the treatment of HIV-1 with combination nucleoside and non-nucleoside therapies.

Dissecting interactions between nucleosides and germination receptors in Bacillus cereus 569 spores

Bacillus cereus 569 spores germinate either with inosine as a sole germinant or with a combination of nucleosides and L-alanine. Whereas the inosine-only germination pathway requires the presence of two different germination receptors (GerI and GerQ) to be activated, the nucleoside/alanine germination pathway only needs one of the two receptors. To differentiate how nucleoside recognition varies between the inosine-only germination pathway and the nucleoside/alanine germination pathway, we tested 61 purine analogues as agonists and antagonists of the two pathways in wild-type, DeltagerI and DeltagerQ spores. The structure-activity relationships of germination agonists and antagonists suggest that the inosine-only germination pathway is restricted to recognize a single germinant (inosine), but can be inhibited in predictable patterns by structurally distinct purine nucleosides. B. cereus spores encoding GerI as the only nucleoside receptor (DeltagerQ mutant) showed a germination inhibition profile similar to wild-type spores treated with inosine only. Thus, GerI seems to have a well-organized binding site that recognizes inosine and inhibitors through specific substrate-protein interactions. Structure-activity analysis also showed that the nucleoside/alanine germination pathway is more promiscuous toward purine nucleoside agonists, and is only inhibited by hydrophobic analogues. B. cereus spores encoding GerQ as the only nucleoside receptor (DeltagerI mutant) behaved like wild-type spores treated with inosine and L-alanine. Thus, the GerQ receptor seems to recognize substrates in a more flexible binding site through non-specific interactions. We propose that the GerI receptor is responsible for germinant detection in the inosine-only germination pathway. On the other hand, supplementing inosine with l-alanine allows bypassing of the GerI receptor to activate the more flexible GerQ receptor.

Antiretroviral Exposure and Lymphocyte mtDNA Content Among Uninfected Infants of HIV-1-Infected Women

Concern for potential adverse effects of antiretroviral (ARV) chemotherapy used to prevent mother-to-child HIV transmission has led the US Public Health Service to recommend long-term follow-up of ARV-exposed children. Nucleoside reverse transcriptase inhibitor ARV agents can inhibit DNA polymerase gamma, impairing mitochondrial DNA (mtDNA) synthesis and resulting in depletion or dysfunction. We measured the mtDNA content of stored peripheral blood mononuclear cells (PBMCs) of 411 healthy children who were born to HIV-uninfected women and 213 uninfected infants who were born to HIV-infected women with or without in utero and neonatal ARV exposure. Cryopreserved PBMC mtDNA was quantified by using the Primagen Retina Mitox assay. Geometric mean PBMC mtDNA levels were lower at birth in infants who were born to HIV-infected women. Among HIV-exposed children, mtDNA levels were lowest in those who were not exposed to ARVs, higher in those with exposure to zidovudine alone, and higher still in those with combination nucleoside reverse transcriptase inhibitor exposure. A similar pattern was observed in the corresponding women. Levels of mtDNA increased during the first 5 years of life in all HIV-exposed children but achieved normal levels only in those with ARV exposure. Levels of mtDNA are lower than normal in HIV-exposed children. Contrary to expectation, PBMC mtDNA levels are significantly higher in ARV-exposed, HIV-uninfected infants and their infected mothers compared with ARV-unexposed infants and women. By 5 years, levels of PBMC mtDNA rise to normal concentrations in ARV-exposed children but remain depressed in ARV-unexposed children.

Chemoimmuno-Therapy with Fludarabine, Cyclophosphamide and Alemtuzumab (FC-Cam) in Patients with Relapsed or Genetic High-Risk CLL: Final Analysis of the CLL2L Trial of the German CLL Study Group.

Abstract 209 Introduction:Purine nucleoside combination therapy has become the standard treatment approach in B-CLL. In order to enhance efficacy, various combinations with monoclonal antibodies are under investigation. Alemtuzumab has been proven to be the most effective antibody in CLL treatment. Since the combination of Fludarabin and Campath (FluCam) resulted in impressive responses, we developed a multicenter phase II trial combining fludarabine, cyclophosphamide and alemtuzumab in a 4-weekly schedule Methods:Fludarabine 25mg/m2 iv, cyclophosphamide 200mg/m2 iv and alemtuzumab 30 mg sc were given on days 1 – 3 and repeated on day 29 for up to six cycles. A 2-day escalation of alemtuzumab was administered prior to the first cycle. Minimal residual disease (MRD) was measured by 4-color flow cytometry. Antiinfective prophylaxis consisted of TMP-SMZ DS twice daily 3d/wk and valacyclovir during and at least two months after completion of treatment. CMV antigen in blood was tested in 14-days intervals. Results:Target recruitment of 61 patients has been reached, 5 patients were excluded due to protocol violation (withdrawn consent, secondary malignancies, death prior to therapy). Data for all 56 patients are available for response and adverse event analysis. Median age is 63 years (range 36–78) with a medium number of 1 prior regimen (range 0–4); 38/56 patients completed at least four cycles with a median number of 5 given cycles. Pretreatment included fludarabine (15/56), fludarabine and cyclophosphamide (24/56) or rituximab combinations (16/56). CTC grade III/IV thrombocytopenia and neutropenia were the most common serious side effects. 12/56 patients died during or within 6 months after last chemoimmunotherapy, of which 5 were related to therapy, 3 were related to concomitant disease and 4 patients died due to progressive disease. Serious adverse events (SAE) included CMV-reactivation (5 patients), herpes-zoster-reactivation (1 patient), pneumonia (5 patients, 2 of which had Aspergillosis-pneumonias), AIHA (1 patient) and fever of unknown origin (12 patients). Four of 56 treated patients were excluded from response analysis: 1 patient stopped therapy because of secondary malignancy, 3 patients died within therapy. The overall response rate for the remaining 52 patients was 68% with 11 CR (22%), 6 CRu (11%), 18 PR (35%), 8 SD (15%) and 9 PD (17%), response was independent of FISH status. A correlation of response with prior treatment was observed, with 81% ORR for fludarabine pre-treatment versus only 63% for those patients pretreated with fludarabine and cyclophosphamide. Finalized data of all patients will be presented. Conclusions:The concomitant application of fludarabine, cyclophosphamide and alemtuzumab is as an effective approach for patients with relapsed CLL. There is some increased toxicity due to the addition of cyclophosphamide compared to the original FluCam regimen. Disclosures:Elter:BayerSchering AG: Honoraria, Research Funding. Off Label Use: The anti-CD52 antibody Alemtuzumab (Campath, MabCampath) is normally administered three-weekly as monotherapy. We are using a four-weekly schedule in combination with fludarabin for the same indication.. Stilgenbauer:BayerScheringAG: Honoraria, Research Funding. Hallek:BayerScheringAG: Honoraria, Research Funding. Engert:BayerScheringAG: Honoraria, Research Funding.