Anti-AIDS agents 81. Design, synthesis, and structure-activity relationship study of betulinic acid and moronic acid derivatives as potent HIV maturation inhibitors.

In our continuing study of triterpene derivatives as potent anti-HIV agents, different C-3 conformationally restricted betulinic acid (BA, 1) derivatives were designed and synthesized in order to explore the conformational space of the C-3 pharmacophore. 3-O-Monomethylsuccinyl-betulinic acid (MSB) analogues were also designed to better understand the contribution of the C-3' dimethyl group of bevirimat (2), the first-in-class HIV maturation inhibitor, which is currently in phase IIb clinical trials. In addition, another triterpene skeleton, moronic acid (MA, 3), was also employed to study the influence of the backbone and the C-3 modification toward the anti-HIV activity of this compound class. This study enabled us to better understand the structure-activity relationships (SAR) of triterpene-derived anti-HIV agents and led to the design and synthesis of compound 12 (EC(50): 0.0006 microM), which displayed slightly better activity than 2 as a HIV-1 maturation inhibitor.

Boosting high-intensity focused ultrasound-induced anti-tumor immunity using a sparse-scan strategy that can more effectively promote dendritic cell maturation.

BACKGROUND: The conventional treatment protocol in high-intensity focused ultrasound (HIFU) therapy utilizes a dense-scan strategy to produce closely packed thermal lesions aiming at eradicating as much tumor mass as possible. However, this strategy is not most effective in terms of inducing a systemic anti-tumor immunity so that it cannot provide efficient micro-metastatic control and long-term tumor resistance. We have previously provided evidence that HIFU may enhance systemic anti-tumor immunity by in situ activation of dendritic cells (DCs) inside HIFU-treated tumor tissue. The present study was conducted to test the feasibility of a sparse-scan strategy to boost HIFU-induced anti-tumor immune response by more effectively promoting DC maturation. METHODS: An experimental HIFU system was set up to perform tumor ablation experiments in subcutaneous implanted MC-38 and B16 tumor with dense- or sparse-scan strategy to produce closely-packed or separated thermal lesions. DCs infiltration into HIFU-treated tumor tissues was detected by immunohistochemistry and flow cytometry. DCs maturation was evaluated by IL-12/IL-10 production and CD80/CD86 expression after co-culture with tumor cells treated with different HIFU. HIFU-induced anti-tumor immune response was evaluated by detecting growth-retarding effects on distant re-challenged tumor and tumor-specific IFN-gamma-secreting cells in HIFU-treated mice. RESULTS: HIFU exposure raised temperature up to 80 degrees centigrade at beam focus within 4 s in experimental tumors and led to formation of a well-defined thermal lesion. The infiltrated DCs were recruited to the periphery of lesion, where the peak temperature was only 55 degrees centigrade during HIFU exposure. Tumor cells heated to 55 degrees centigrade in 4-s HIFU exposure were more effective to stimulate co-cultured DCs to mature. Sparse-scan HIFU, which can reserve 55 degrees-heated tumor cells surrounding the separated lesions, elicited an enhanced anti-tumor immune response than dense-scan HIFU, while their suppressive effects on the treated primary tumor were maintained at the same level. Flow cytometry analysis showed that sparse-scan HIFU was more effective than dense-scan HIFU in enhancing DC infiltration into tumor tissues and promoting their maturation in situ. CONCLUSION: Optimizing scan strategy is a feasible way to boost HIFU-induced anti-tumor immunity by more effectively promoting DC maturation.

High-throughput identification of chemical inhibitors of E. coli Group 2 capsule biogenesis as anti-virulence agents.

Rising antibiotic resistance among Escherichia coli, the leading cause of urinary tract infections (UTIs), has placed a new focus on molecular pathogenesis studies, aiming to identify new therapeutic targets. Anti-virulence agents are attractive as chemotherapeutics to attenuate an organism during disease but not necessarily during benign commensalism, thus decreasing the stress on beneficial microbial communities and lessening the emergence of resistance. We and others have demonstrated that the K antigen capsule of E. coli is a preeminent virulence determinant during UTI and more invasive diseases. Components of assembly and export are highly conserved among the major K antigen capsular types associated with UTI-causing E. coli and are distinct from the capsule biogenesis machinery of many commensal E. coli, making these attractive therapeutic targets. We conducted a screen for anti-capsular small molecules and identified an agent designated "C7" that blocks the production of K1 and K5 capsules, unrelated polysaccharide types among the Group 2-3 capsules. Herein lies proof-of-concept that this screen may be implemented with larger chemical libraries to identify second-generation small-molecule inhibitors of capsule biogenesis. These inhibitors will lead to a better understanding of capsule biogenesis and may represent a new class of therapeutics.

A novel, non-apoptotic role for Scythe/BAT3: a functional switch between the pro- and anti-proliferative roles of p21 during the cell cycle.

BACKGROUND: Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. METHODS/PRINCIPAL FINDINGS: Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. CONCLUSION: Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle.

Anti-beta(1)-adrenergic receptor antibodies and heart failure: causation, not just correlation.

Antibodies specific for the beta(1)-adrenergic receptor are found in patients with chronic heart failure of various etiologies. From work presented in this issue of the JCI, we can now infer that these antibodies actually contribute to the pathogenesis of chronic heart failure. This commentary discusses mechanisms by which these antibodies may engender cardiomyopathy.

Accessibility, availability and affordability of anti-malarials in a rural district in Kenya after implementation of a national subsidy scheme.

BACKGROUND: Poor access to prompt and effective treatment for malaria contributes to high mortality and severe morbidity. In Kenya, it is estimated that only 12% of children receive anti-malarials for their fever within 24 hours. The first point of care for many fevers is a local medicine retailer, such as a pharmacy or chemist. The role of the medicine retailer as an important distribution point for malaria medicines has been recognized and several different strategies have been used to improve the services that these retailers provide. Despite these efforts, many mothers still purchase ineffective drugs because they are less expensive than effective artemisinin combination therapy (ACT). One strategy that is being piloted in several countries is an international subsidy targeted at anti-malarials supplied through the retail sector. The goal of this strategy is to make ACT as affordable as ineffective alternatives. The programme, called the Affordable Medicines Facility - malaria was rolled out in Kenya in August 2010. METHODS: In December 2010, the affordability and accessibility of malaria medicines in a rural district in Kenya were evaluated using a complete census of all public and private facilities, chemists, pharmacists, and other malaria medicine retailers within the Webuye Demographic Surveillance Area. Availability, types, and prices of anti-malarials were assessed. There are 13 public or mission facilities and 97 medicine retailers (registered and unregistered). RESULTS: The average distance from a home to the nearest public health facility is 2 km, but the average distance to the nearest medicine retailer is half that. Quinine is the most frequently stocked anti-malarial (61% of retailers). More medicine retailers stocked sulphadoxine-pyramethamine (SP; 57%) than ACT (44%). Eleven percent of retailers stocked AMFm subsidized artemether-lumefantrine (AL). No retailers had chloroquine in stock and only five were selling artemisinin monotherapy. The mean price of any brand of AL, the recommended first-line drug in Kenya, was $2.7 USD. Brands purchased under the AMFm programme cost 40% less than non-AMFm brands. Artemisinin monotherapies cost on average more than twice as much as AMFm-brand AL. SP cost only $0.5, a fraction of the price of ACT. CONCLUSIONS: AMFm-subsidized anti-malarials are considerably less expensive than unsubsidized AL, but the price difference between effective and ineffective therapies is still large.

Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.

Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. A novel method for monitoring caspase activity in living Xenopus oocytes and early embryos is described here. The approach, using microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we show that brief caspase activity (10 min) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing the inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that the eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggest that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators.

Development and characterization of anti-nitr9 antibodies.

The novel immune-type receptors (NITRs), which have been described in numerous bony fish species, are encoded by multigene families of inhibitory and activating receptors and are predicted to be functional orthologs to the mammalian natural killer cell receptors (NKRs). Within the zebrafish NITR family, nitr9 is the only gene predicted to encode an activating receptor. However, alternative RNA splicing generates three distinct nitr9 transcripts, each of which encodes a different isoform. Although nitr9 transcripts have been detected in zebrafish lymphocytes, the specific hematopoietic lineage(s) that expresses Nitr9 remains to be determined. In an effort to better understand the role of NITRs in zebrafish immunity, anti-Nitr9 monoclonal antibodies were generated and evaluated for the ability to recognize the three Nitr9 isoforms. The application of these antibodies to flow cytometry should prove to be useful for identifying the specific lymphocyte lineages that express Nitr9 and may permit the isolation of Nitr9-expressing cells that can be directly assessed for cytotoxic (e.g. NK) function.

Anti-hypotensive treatment and endothelin blockade synergistically antagonize exercise fatigue in rats under simulated high altitude.

Rapid ascent to high altitude causes illness and fatigue, and there is a demand for effective acute treatments to alleviate such effects. We hypothesized that increased oxygen delivery to the tissue using a combination of a hypertensive agent and an endothelin receptor A antagonist drugs would limit exercise-induced fatigue at simulated high altitude. Our data showed that the combination of 0.1 mg/kg ambrisentan with either 20 mg/kg ephedrine or 10 mg/kg methylphenidate significantly improved exercise duration in rats at simulated altitude of 4,267 m, whereas the individual compounds did not. In normoxic, anesthetized rats, ephedrine alone and in combination with ambrisentan increased heart rate, peripheral blood flow, carotid and pulmonary arterial pressures, breathing rate, and vastus lateralis muscle oxygenation, but under inspired hypoxia, only the combination treatment significantly enhanced muscle oxygenation. Our results suggest that sympathomimetic agents combined with endothelin-A receptor blockers offset altitude-induced fatigue in rats by synergistically increasing the delivery rate of oxygen to hypoxic muscle by concomitantly augmenting perfusion pressure and improving capillary conductance in the skeletal muscle. Our findings might therefore serve as a basis to develop an effective treatment to prevent high-altitude illness and fatigue in humans.

Le rôle de l'hormone anti-müillerienne dans la régulation du fonctionnement de l'ovaire. Revue de la littérature

Anti-mullerian hormone, also called AMH, belongs to the large family of transforming growth factor P. Its role in the sexual differentiation of male fetus is now well known. Recently, AMH has been demonstrated to play an important role in the ovarian function. In fact, AMH seems to regulate the kinetics of follicular development, inhibiting the follicular recruitment and the follicular growth. Thus, this intra-gonadic cybernin could be a decisive determinant of the rapidity of follicular pool exhaustion. Today, some experimental data from the literature suggest that AMH could be a reliable marker of ovarian reserve. This review summarizes the present knowledge about AMH and its role in physiology but also in ovarian pathology.

The island-scale internal wave climate of Moorea, French Polynesia

Analysis of five-year records of temperatures and currents collected at Moorea reveal strong internal wave activity at predominantly semi-diurnal frequencies impacting reef slopes at depths 30m around the entire island. Temperature changes of 1.5C to 3C are accompanied by surges of upward and onshore flow and vertical shear in onshore currents. Superimposed on annual temperature changes of approximately 3C, internal wave activity is high from Oct-May and markedly lower from Jun-Sep. The offshore pycnocline is broadly distributed with continuous stratification to at least 500m depth, and a subsurface fluorescence maximum above the strong nutricline at approximately 200m. Minimum buoyancy periods range from 4.8 to 6min, with the maximum density gradient occurring at 50 to 60m depth in summer and deepening to approximately 150 to 200m in winter. The bottom slope angle around all of Moorea is super-critical relative to the vertical stratification angle suggesting that energy propagating into shallow water is only a portion of total incident internal wave energy. Vertical gradient Richardson numbers indicate dominance by density stability relative to current shear with relatively limited diapycnal mixing. Coherence and lagged cross-correlation of semi-diurnal temperature variation indicate complex patterns of inter-site arrival of internal waves and no clear coherence or lagged correlation relationships among island sides. Semi-diurnal and high frequency internal wave packets likely arrive on Moorea from a combination of local and distant sources and may have important impacts for nutrient and particle fluxes in deep reef environments. © 2012 American Geophysical Union. All Rights Reserved.

Time-Resolved Synchronous Fluorescence for Biomedical Diagnosis.

This article presents our most recent advances in synchronous fluorescence (SF) methodology for biomedical diagnostics. The SF method is characterized by simultaneously scanning both the excitation and emission wavelengths while keeping a constant wavelength interval between them. Compared to conventional fluorescence spectroscopy, the SF method simplifies the emission spectrum while enabling greater selectivity, and has been successfully used to detect subtle differences in the fluorescence emission signatures of biochemical species in cells and tissues. The SF method can be used in imaging to analyze dysplastic cells in vitro and tissue in vivo. Based on the SF method, here we demonstrate the feasibility of a time-resolved synchronous fluorescence (TRSF) method, which incorporates the intrinsic fluorescent decay characteristics of the fluorophores. Our prototype TRSF system has clearly shown its advantage in spectro-temporal separation of the fluorophores that were otherwise difficult to spectrally separate in SF spectroscopy. We envision that our previously-tested SF imaging and the newly-developed TRSF methods will combine their proven diagnostic potentials in cancer diagnosis to further improve the efficacy of SF-based biomedical diagnostics.

Induction of anti-myelin antibodies in EAE and their possible role in demyelination.

Experimental allergic encephalomyelitis is characterized by invasion of lymphocytes and macrophages into the central nervous system resulting in inflammation, edema, and demyelination. Sera from Lewis rats from 7-95 days after immunization with purified guinea pig CNS myelin were examined with respect to their ability to opsonize myelin. This was correlated with the appearance of antibody components and the relative amounts of antibody to myelin basic protein (MBP) and proteolipid protein (PLP). Sera from rats 10-95 days after immunization preincubated with purified myelin induced phagocytosis of myelin by cultured macrophages with the resulting production of cholesterol ester. This opsonization activity as measured by the percentage of cholesterol esterified reached a peak at 26-27 days after immunization but remained significantly elevated up to 95 days post-immunization compared to the activity of serum from the Freund's adjuvant-injected controls. Immunoblots of the sera revealed a gradual increase in antibody activity against myelin components. ELISA assays for MBP and PLP antibody showed a similar pattern. Antibody to galactocerebroside (GC) was not detected by immunostains nor by the ELISA assay. Areas of demyelination were observed histologically by luxol-fast blue stained spinal cords up to 60 days post-immunization. These results indicate that antibodies to myelin protein when given access to myelin through or within the blood brain barrier could initiate or enhance the phagocytic response by peripheral or resident macrophages.

Veterans health administration hepatitis B testing and treatment with anti-CD20 antibody administration.

AIM: To evaluate pretreatment hepatitis B virus (HBV) testing, vaccination, and antiviral treatment rates in Veterans Affairs patients receiving anti-CD20 Ab for quality improvement. METHODS: We performed a retrospective cohort study using a national repository of Veterans Health Administration (VHA) electronic health record data. We identified all patients receiving anti-CD20 Ab treatment (2002-2014). We ascertained patient demographics, laboratory results, HBV vaccination status (from vaccination records), pharmacy data, and vital status. The high risk period for HBV reactivation is during anti-CD20 Ab treatment and 12 mo follow up. Therefore, we analyzed those who were followed to death or for at least 12 mo after completing anti-CD20 Ab. Pretreatment serologic tests were used to categorize chronic HBV (hepatitis B surface antigen positive or HBsAg+), past HBV (HBsAg-, hepatitis B core antibody positive or HBcAb+), resolved HBV (HBsAg-, HBcAb+, hepatitis B surface antibody positive or HBsAb+), likely prior vaccination (isolated HBsAb+), HBV negative (HBsAg-, HBcAb-), or unknown. Acute hepatitis B was defined by the appearance of HBsAg+ in the high risk period in patients who were pretreatment HBV negative. We assessed HBV antiviral treatment and the incidence of hepatitis, liver failure, and death during the high risk period. Cumulative hepatitis, liver failure, and death after anti-CD20 Ab initiation were compared by HBV disease categories and differences compared using the χ(2) test. Mean time to hepatitis peak alanine aminotransferase, liver failure, and death relative to anti-CD20 Ab administration and follow-up were also compared by HBV disease group. RESULTS: Among 19304 VHA patients who received anti-CD20 Ab, 10224 (53%) had pretreatment HBsAg testing during the study period, with 49% and 43% tested for HBsAg and HBcAb, respectively within 6 mo pretreatment in 2014. Of those tested, 2% (167/10224) had chronic HBV, 4% (326/7903) past HBV, 5% (427/8110) resolved HBV, 8% (628/8110) likely prior HBV vaccination, and 76% (6022/7903) were HBV negative. In those with chronic HBV infection, ≤ 37% received HBV antiviral treatment during the high risk period while 21% to 23% of those with past or resolved HBV, respectively, received HBV antiviral treatment. During and 12 mo after anti-CD20 Ab, the rate of hepatitis was significantly greater in those HBV positive vs negative (P = 0.001). The mortality rate was 35%-40% in chronic or past hepatitis B and 26%-31% in hepatitis B negative. In those pretreatment HBV negative, 16 (0.3%) developed acute hepatitis B of 4947 tested during anti-CD20Ab treatment and follow-up. CONCLUSION: While HBV testing of Veterans has increased prior to anti-CD20 Ab, few HBV+ patients received HBV antivirals, suggesting electronic health record algorithms may enhance health outcomes.