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BACKGROUND The safety and efficacy of tenofovir disoproxil fumarate (TDF) in HIV-1-infected children have not been evaluated in a randomized controlled trial. METHODS Subjects (2 to

Background & Aims: Treatment with adefovir dipivoxil for 48 weeks resulted in clinical improvement in patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B that was lost when treatment was discontinued. We investigated the efficacy, safety, and resistance profile of adefovir dipivoxil treatment for up to 240 weeks. Methods: HBeAg-negative patients were treated double blind with placebo or adefovir dipivoxil 10 mg once daily for 48 weeks, followed by adefovir dipivoxil from week 49 to 96. At week 97, 125 patients enrolled in a 144-week, open-label phase. Patients received adefovir dipivoxil for up to 192 or 240 weeks. Results: Serum hepatitis B virus (HBV) DNA levels were less than 1000 copies per milliliter in 67% of patients, and alanine aminotransferase (ALT) levels normalized in 69% after 240 weeks. After 192 or 240 weeks of treatment, over 83% of patients had improvement in necroinflammation, and over 73% had improvement in fibrosis. Ishak fibrosis scores improved compared with baseline in 35%, 55%, and 71% of patients after 48, 192, and 240 weeks of adefovir dipivoxil, respectively. After 240 weeks, the cumulative probability of HBV polymerase mutations was 29%, but the cumulative probability of mutations with virologic resistance was 20% and of mutations, virologic resistance, and ALT elevations was 11%. Slight elevations in creatinine were confirmed in 4 (3%) patients. Conclusions: Treatment with adefovir dipivoxil for up to 240 weeks was well tolerated and produced significant, increasing improvement in hepatic fibrosis, durable suppression of HBV replication, normalization of liver enzymes, and delayed development of resistance.

background Durable suppression of replication of the human immunodeficiency virus (HIV) depends on the use of potent, well-tolerated antiretroviral regimens to which patients can easily adhere. methods We conducted an open-label, noninferiority study involving 517 patients with HIV infection who had not previously received antiretroviral therapy and who were randomly assigned to receive either a regimen of tenofovir disoproxil fumarate (DF), emtricitabine, and efavirenz once daily (tenofovir–emtricitabine group) or a regimen of fixed-dose zidovudine and lamivudine twice daily plus efavirenz once daily (zidovudine–lamivudine group). The primary end point was the proportion of patients without baseline resistance to efavirenz in whom the HIV RNA level was less than 400 copies per milliliter at week 48 of the study. results Through week 48, significantly more patients in the tenofovir–emtricitabine group reached and maintained the primary end point of less than 400 copies of HIV RNA per milliliter than did those in the zidovudine–lamivudine group (84 percent vs. 73 percent, respectively; 95 percent confidence interval for the difference, 4 to 19 percent; P = 0.002). This difference excludes the inferiority of the tenofovir DF, emtricitabine, and efavirenz regimen, indicating a significantly greater response with this regimen. Significant differences were also seen in the proportion of patients with HIV RNA levels of less than 50 copies per milliliter (80 percent in the tenofovir– emtricitabine group vs. 70 percent in the zidovudine–lamivudine group; 95 percent confidence interval for the difference, 2 to 17 percent; P = 0.02) and in increases in CD4 cell counts (190 vs. 158 cells per cubic millimeter, respectively; 95 percent confidence interval for the difference, 9 to 55; P = 0.002). More patients in the zidovudine–lamivudine group than in the tenofovir–emtricitabine group had adverse events resulting in discontinuation of the study drugs (9 percent vs. 4 percent, respectively; P = 0.02). In none of the patients did the K65R mutation develop. conclusions Through week 48, the combination of tenofovir DF and emtricitabine plus efavirenz fulfilled the criteria for noninferiority to a fixed dose of zidovudine and lamivudine plus efavirenz and proved superior in terms of virologic suppression, CD4 response, and adverse events resulting in discontinuation of the study drugs. (ClinicalTrials. gov number, NCT00112047.)

Serum ferritin has been used widely in clinical medicine chiefly as an indicator of iron stores and inflammation. Circulating ferritin also can have paracrine effects. Despite the clinical significance of serum ferritin, its secretion remains an enigma. The consensus view is that serum ferritin arises from tissue ferritins— principally ferritin light—which can be glycosylated. Ferritin heavy and light chains are cytosolic proteins that form cages of 24 subunits to store intracellular iron. We show that ferritin light is secreted when its expression is increased in stable, transfected HepG2 cells or adenovirus-infected HepG2 cells. Export occurs through the classical secretory pathway and some chains are N-glycosylated. Ferritins do not need to form cages prior to secretion. Secretion is blocked specifically, effectively, and rapidly by a factor in serum. The timing of this inhibition of ferritin secretion suggests that normally cytosolic ferritin L is targeted to the secretory pathway during translation despite the absence of a conventional signal sequence. Thus, secretion of glycosylated and unglycosylated ferritin is a regulated and not a stochastic process.

Thioredoxin (Trx) is a cytosolic, redox-active protein that is secreted from many cells and has several extracellular functions. In activated lymphocytes, the pathway of secretion does not involve the Golgi apparatus. Levels of extracellular Trx are decreased by the antioxidant N-acetylcysteine. Hence, the secretion of Trx could be altered by the redox status of the cell or the protein. To study Trx mutants, we characterized the secretion of human Trx from Chinese hamster ovary cells. Secretion of human Trx is unaffected by brefeldin A, slow but efficient, and sensitive to low temperature and factors in serum. We demonstrate that N-acetylcysteine reduces the cellular level of Trx but not the proportion secreted; thus this chemical does not block the nonclassic pathway for Trx secretion. Furthermore, we find that mutations in either the active site or the dimerization site of Trx do not alter its secretion. Thus the nonclassic secretion of Trx is not dependent on the redox status of either the cell or the protein.

This 96-week, double-blind, active-controlled, phase 3 study, randomized subjects to elvitegravir once daily or raltegravir twice daily with a fully active, ritonavir-boosted protease inhibitor plus a third agent. The proportion of subjects randomized to elvitegravir that achieved and maintained HIV-1 RNA < 50 copies/mL through week 96 was 47.6% (167/351) compared with 45.0% (158/351) for raltegravir with a treatment difference of 2.6% (95% confidence interval: 4.6% to 9.9%). Both regimens were well tolerated, with comparable rates of adverse events and laboratory abnormalities through week 96. Once-daily elvitegravir was noninferior to twice-daily raltegravir, showed durable long-term efficacy, and was well tolerated in HIV+ treatment-experienced patients.

Emtricitabine/rilpivirine/tenofovir disoproxil fumarate (FTC/RPV/TDF) is a next-generation, once-daily complete antiretroviral single-tablet regimen for the treatment of HIV-1 infection in adults. This study evaluated the pharmacokinetics and bioequivalence of 2 distinct investigational coformulations of the FTC/RPV/TDF single-tablet regimen (containing emtricitabine 200 mg, rilpivirine 25 mg, and tenofovir disoproxil fumarate 300 mg) compared with the concurrent administration of a 200-mg strength capsule of FTC, a 25-mg strength tablet of RPV, and a 300-mg strength tablet of tenofovir disoproxil fumarate in healthy subjects. Thirty-six subjects were randomized in a single-dose, open-label, 3-way crossover study design; 34 subjects completed all study treatments. Serial blood samples were obtained over 192 hours following oral administration of each treatment and pharmacokinetic parameters calculated. Formulation bioequivalence was assessed by 90% confidence intervals (CI) for the ratio of geometric least square means (GMR) for Cmax, AUClast and AUCinf for each drug of the FTC/RPV/TDF single-tablet regimen versus the individual components. Emtricitabine, rilpivirine, and tenofovir disoproxil fumarate administered under fed conditions (standardized meal of ~400 kcal) as individual drugs given concurrently or as a fixed‑dose combination tablet were generally well tolerated. Of the 2 single-tablet formulations that were tested, a single coformulation (Test Formulation 1) demonstrated bioequivalence to the reference formulation, with 90% confidence intervals for the ratio of the geometric least-squares means contained within the bounds of 80% to 125% for the AUCinf, AUC0−last, and Cmax values of emtricitabine, rilpivirine, and tenofovir, and was carried forward as the commercial formulation. This tablet is a next-generation, once-daily single-tablet antiretroviral regimen for the treatment of HIV-1 infection and offers an attractive treatment option to efavirenz-containing regimens.

There are few data on the safety and antiviral activity of tenofovir disoproxil fumarate (TDF) in HIV-1 infected adolescents.A randomized, double-blinded, placebo-controlled study was conducted. Ninety adolescents (12 to18 years) who were viremic while receiving antiretroviral treatment were randomized to receive TDF 300 mg (mean, 216.8 mg/m(2)) or placebo in combination with an optimized background regimen (OBR) for 48 weeks. The primary efficacy endpoint was time-weighted average change in plasma HIV-1 RNA from baseline at week 24Eighty-seven subjects (45 TDF, 42 placebo) received the study drug. Through week 24, the median time-weighted average change in plasma HIV-1 RNA was not different between the TDF and placebo groups (-1.6 versus -1.6 log(10)copies/mL, P = 0.55). The percentages of subjects who achieved HIV-1 RNA400 copies/mL were similar at week 24 (40.9 versus 41.5%). One fourth of subjects in the TDF and placebo groups (24.4 versus 28.6%) had at least 3 active agents in the OBR. Many subjects in both groups had baseline genotypic resistance to TDF (48.9 versus 33.3%). TDF was generally safe and well tolerated. There were no statistically significant differences in changes of renal function and bone mineral density between the 2 groups.This study of TDF in combination with an OBR in antiretroviral-experienced adolescents did not meet its primary or secondary efficacy endpoints. The effectiveness of the OBR and baseline genotypic resistance to TDF in both groups may have confounded the efficacy findings. No clinically relevant TDF-related renal or bone toxicities were observed in this adolescent population.

OBJECTIVE: To assess efficacy and safety of cobicistat versus ritonavir as pharmacoenhancers for atazanavir when administered with emtricitabine/tenofovir df as initial treatment for HIV-1 infection. DESIGN: Randomized, partially placebo-controlled, double-blind, multicenter study. PARTICIPANTS: Antiretroviral treatment-naive adults, screening HIV-1 RNA of at least 5000 copies/ml and CD4 cell count more than 50 cells/μl. INTERVENTION: Randomized 2 : 1 (stratified by HIV RNA ≤ or >100,000 copies/ml) to receive placebo-blinded once-daily cobicistat 150 mg or ritonavir 100 mg with open-label atazanavir and fixed-dose emtricitabine/tenofovir df. MAIN OUTCOME MEASURES: Efficacy and safety at weeks 24 and 48. RESULTS: Eighty-four percent of ATV/co participants and 86% of ATV/r participants suppressed HIV-1 RNA (

To assess the safety and efficacy of two, single-tablet regimens for the initial treatment of HIV infection.Phase 2, randomized, double-blind, double-dummy, multicenter, active-controlled study.Antiretroviral treatment-naive adults with a screening HIV-1 RNA at least 5000 copies/ml and a CD4 cell count more than 50 cells/μl were randomized 2: 1 to receive fixed-dose combination tablets of elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (EVG/COBI/FTC/TDF; N = 48) or efavirenz/emtricitabine/tenofovir disoproxil fumarate (EFV/FTC/TDF; n = 23) for 48 weeks. The primary endpoint was proportion of participants with HIV-1 RNA less than 50 copies/ml at week 24.Participants receiving EVG/COBI/FTC/TDF exhibited a more rapid decline in HIV-1 RNA and a greater proportion suppressed viral load to less than 50 copies/ml than participants receiving EFV/FTC/TDF. Both EVG/COBI/FTC/TDF and EFV/FTC/TDF resulted in high rates of viral suppression and increases in CD4 cell count. Ninety and 83% of participants suppressed HIV-1 RNA to less than 50 copies/ml both at the 24-week and 48-week visits for EVG/COBI/FTC/TDF and EFV/FTC/TDF, respectively. Once-daily administration of EVG/COBI/FTC/TDF provided a mean EVG trough concentration 10-fold over its protein binding-adjusted IC(95) across study visits. EVG/FTC/TDF/GS-9350 was generally well tolerated with a lower rate of drug-related central nervous system (17%) and psychiatric (10%) adverse events versus EFV/FTC/TDF (26 and 44%, respectively). Decreases in estimated glomerular filtration rate occurred within the first few weeks of dosing in participants receiving EVG/COBI/FTC/TDF, remained within the normal range and did not progress at week 24 or 48; no participant experienced a clinical adverse event or discontinued study drug due to changes in serum creatinine or renal function.Once-daily EVG/COBI/FTC/TDF achieved and maintained a high rate of virologic suppression with fewer central nervous system and psychiatric adverse events compared to a current standard-of-care regimen of EFV/FTC/TDF.

Background. This phase 2, randomized, active-controlled, 48-week study assessed the noninferiority of the human immunodeficiency virus (HIV) integrase inhibitor elvitegravir to comparator ritonavir-boosted protease inhibitor (CPI/r) in treatment-experienced subjects. Methods. Subjects had HIV RNA levels ≥1000 copies/mL and ≥1 1 protease resistance mutation. Subjects received nucleoside or nucleotide reverse-transcriptase inhibitors (NRTIs) with or without T-20 and either CPI/r or once-daily elvitegravir at a dose of 20 mg, 50 mg, or 125 mg (blinded to dose) with ritonavir. After week 8, the independent data monitoring committee stopped the elvitegravir 20 mg arm and allowed subjects in the elvitegravir 50 mg and 125 mg arms to add protease inhibitors. The primary end point was the time-weighted average change from baseline in HIV RNA level through week 24 (DAVG 24 ). Results. A total of 278 subjects with a median of 11 protease and 3 thymidine analog mutations were randomized and treated. One-half of subjects received NRTIs without expected antiviral activity. Compared with the DAVG 24 for the CPI/r arm (-1.19 log 10 copies/mL), the elvitegravir 50 mg arm was noninferior (-1.44 log 10 copies/mL), and the elvitegravir 125 mg arm was superior (-1.66 log 10 copies/mL; P = .021). Efficacy was impacted by activity of background agents. There was no relationship between elvitegravir dosage and adverse events. Conclusions. Elvitegravir was well-tolerated and produced rapid virologic suppression that was durable with active background therapy. Trial registration. ClinicalTrials.gov identifier number: NCT00298350.

Apolipoprotein B (apo B) is crucial for the transport of cholesterol in humans. It is a large secretory protein that mediates the uptake of low-density lipoproteins and renders several forms of lipid droplets soluble in the blood. The binding of lipid by apo B also prevents this hydrophobic protein from precipitating in aqueous solution. In the endoplasmic reticulum, nascent secretory proteins must be translocated through an aqueous channel in the membrane into the aqueous lumen, so some novel form of processing may be necessary to maintain the solubility of apo B during its translocation. We have discovered that the biogenesis of apo B in cell-free systems does indeed involve a new variation on protein translocation: unlike typical secretory proteins, apo B is synthesized as a series of transmembrane chains with large cytoplasmic domains and progressively longer amino-terminal regions that are protected against added proteases during the translocation process. In contrast to typical transmembrane proteins, these transmembrane chains are not integrated into the bilayer. Moreover, the transmembrane chains with the shortest protected domains are precursors of forms whose protection is progressively extended to cover the length of the protein. This stepwise conversion occurs post-translationally for the most part. We propose a model on the basis of these findings for the biogenesis of apo B.

Apolipoprotein B is secreted with atherogenic lipids as lipoprotein particles from hepatocytes. Regulation of the secretion of apolipoprotein B is largely post-translational and reflects the balance between processes that leads to particle assembly or to intracellular degradation. Previously, we conducted a proteomic screen to find proteins that bind apolipoprotein B in rat liver microsomes. We identified ferritin heavy and light chains in this screen among other proteins and showed that the two ferritins bind apolipoprotein B directly in vitro. In hepatocytes and other cells, ferritin heavy and light chains form cytosolic cages that store iron. We now show that ferritin heavy or light chains post-translationally inhibit the secretion of apolipoprotein B without altering the export of other hepatic proteins including albumin, factor XIII, and apolipoprotein A–I. This inhibition of apolipoprotein B secretion is not due to diminished lipid synthesis and can be partially overcome by stimulating triglyceride synthesis. The block in apolipoprotein B secretion by ferritins leads to an increase in endoplasmic reticulum-associated degradation of the apolipoprotein. Thus, despite being cytosolic proteins without known chaperone activity, ferritins can specifically regulate the secretion of apolipoprotein B post-translationally. The metabolic pathways for iron storage and intercellular cholesterol and triglyceride transport could intersect.

The green fluorescent protein is a cytosolic protein frequently used as a molecular tag to study protein localization in intact cells. We discovered that this protein is secreted into the medium by several but not all cell lines through a non-classical secretory pathway that is insensitive to brefeldin A. Green fluorescent protein is secreted efficiently by Chinese hamster ovary cells, with 60% of synthesized proteins secreted over 8 hours. This pathway is sensitive to changes in temperature but not to factors in serum or chemicals known to affect other non-classical protein secretion pathways. Fluorescence is observed in cells expressing green fluorescent protein, indicating that some of the protein must be fully folded in the cytosol. However, secreted green fluorescent protein is not fluorescent and therefore not folded properly. Furthermore, cellular fluorescence does not change over 6 hours whereas a significant proportion of green fluorescent protein is secreted. Thus, nascent green fluorescent protein either is folded correctly or incorrectly, and the improperly folded molecules can be exported. Non-classical secretion might be a route by which cells remove an excess of improperly folded, cytosolic proteins.

Thioredoxin is a small protein with a catalytic site for redox reactions that is involved in several physiologic and pathophysiologic processes in humans. A ubiquitous cytosolic protein, thioredoxin also is secreted by some types of cells including cells derived from leukemia, lung, colon, and breast cancers. Extracellular thioredoxin acts as an autocrine growth factor and can potentiate the action of other cytokines on these tumor cell lines. The novel pathway by which thioredoxin is secreted has not been elucidated. Our specific aims are to: identify the amino acid sequence within thioredoxin that directs its secretion from cells, and to identify the cellular gene products that secrete thioredoxin from breast cancer cells. We aim to develop a molecular view of the non-classical process by which thioredoxin is secreted. Armed with a thorough understanding of this export process, it may be possible to slow the growth of breast cancers in humans in an entirely new way by inhibiting the release of thioredoxin.

The biogenesis of apolipoprotein B is quite complex in view of its huge size, hydrophobicity, obligate association with lipids such as cholesterol and triglycerides prior to secretion, and intracellular degradation of a substantial proportion of newly synthesized molecules. Multiple proteins likely serve roles as molecular chaperones to assist in folding, assembly with lipids, and regulation of the secretion of apolipoprotein B. In these studies, we developed a strategy to isolate proteins associated with apolipoprotein B in rat livers. The purification consisted of two stages: first, microsomes were prepared from rat liver and treated with chemical cross-linkers, and second, the solubilized proteins were co-immunoprecipitated with antibody against apolipoprotein B. We found that several proteins were cross-linked to apolipoprotein B. The proteins were digested with trypsin, and the released peptides were sequenced by tandem mass spectrometry. The sequences precisely matched 377 peptides in 99 unique proteins. We show that at least two of the identified proteins, ferritin heavy and light chains, can directly bind apolipoprotein B. These and possibly other proteins identified by this proteomic approach are novel candidates for proteins that affect apolipoprotein B during its biogenesis.

While Kaposi's sarcoma (KS) is extremely common in homosexual men infected with human immunodeficiency virus (HIV), it is also found in several clinical settings in which HIV infection is absent. Recently, sequences from the genome of a novel member of the herpesvirus family have been identified within KS biopsies in the AIDS-associated form of the disease. The presence of these sequences was sought in 6 cases of HIV-negative KS, including 4 cases of endemic KS from Africa, and was found in 5 of the 6. These findings strengthen the association between infection with this virus and the development of KS and argue against the notion that this association is simply the result of opportunistic superinfection of profoundly immunodeficient hosts.

We reviewed the records of 106 patients with cryptococcal infections and the acquired immunodeficiency syndrome (AIDS) treated at San Francisco General Hospital. We examined four issues: the efficacy of treatment with amphotericin plus flucytosine as compared with amphotericin alone, the efficacy of suppressive therapy, the prognostic clinical characteristics, and the course of nonmeningeal cryptococcosis. In 48 of the 106 patients (45 percent), cryptococcosis was the first manifestation of AIDS. Among the 89 patients with cryptococcal meningitis confirmed by culture, survival did not differ significantly between those treated with amphotericin plus flucytosine (n = 49) and those treated with amphotericin alone (n = 40). Flucytosine had to be discontinued in over half the patients because of cytopenia. Long-term suppressive therapy with either ketoconazole or amphotericin was associated with improved survival, as compared with survival in the absence of suppressive therapy (median survival, greater than or equal to 238 vs. 141 days; P less than 0.004). The only clinical features independently associated with a shorter cumulative survival were hyponatremia and a positive culture for cryptococcus from an extrameningeal source. The 14 patients with nonmeningeal cryptococcosis had a median survival (187 days) and rate of relapse (20 percent) similar to those in the patients with meningitis (165 days and 17 percent, respectively). From this retrospective study of cryptococcal infections in patients with AIDS we conclude that the addition of flucytosine to amphotericin neither enhances survival nor prevents relapse, but long-term suppressive therapy appears to benefit these patients.