Roche (Estonia)

HCV, hepatitis C virus; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HIV, human immunodeficiency virus; anti-HCV, HCV antibody; RNA, ribonucleic acid; PCR, polymerase chain reaction; TMA, transcription-mediated amplification; FDA, Food and Drug Administration; HCC, hepatocellular carcinoma; SVR, sustained virologic response; EVR, early virologic response; ETR, end of treatment response; peginterferon, pegylated interferon; G-CSF, granulocyte colony-stimulating factor; HAART, highly active antiretroviral therapy; ddI, didanosine; GM-CSF, granulocyte-macrophage colony-stimulating factor. These recommendations provide a data-supported approach. They are based on the following: (1) a formal review and analysis of the recently published world literature on the topic (Medline search); (2) the American College of Physicians' Manual for Assessing Health Practices and Designing Practice Guidelines1 ; (3) guideline policies, including the American Association for the Study of Liver Diseases' (AASLD) Policy on the Development and Use of Practice Guidelines and the American Gastroenterological Association's Policy Statement on the Use of Medical Practice Guidelines2; the guideline procedures of the Infectious Diseases Society of America3; and (4) the experience of the authors in the specified topic. These recommendations are fully endorsed by the AASLD, the Infectious Diseases Society of America, and the American College of Gastroenterology. Intended for use by physicians, these recommendations suggest preferred approaches to the diagnostic, therapeutic and preventive aspects of care. They are intended to be flexible, in contrast to standards of care, which are inflexible policies to be followed in every case. Specific recommendations are based on relevant published information. In an attempt to characterize the quality of evidence supporting recommendations, the Practice Guidelines Committee of the AASLD requires a category to be assigned and reported with each recommendation (Table 1). The hepatitis C virus (HCV) is a major public health problem and a leading cause of chronic liver disease. In the United States, the Centers for Disease Control and Prevention estimates that there are more than 2.7 million people with ongoing HCV infection.5 HCV is the leading cause of death from liver disease in the United States.6 The purpose of this article is to provide clinicians with approaches to the diagnosis, management, and prevention of HCV infection. The optimal methods of detecting HCV infection are to screen populations for history of risk and to test selected individuals with an identifiable risk factor. With careful questioning, an HCV risk factor can be identified in more than 90% of cases.7 The primary source of HCV transmission is HCV-infected blood or blood products. In the United States, injection drug use is the chief mode of transmission, and anyone who has ever injected illicit drugs should be tested.5, 7 Persons should also be tested if they received a blood or blood component transfusion or organ transplant before 1992, when sensitive tests were first used to screen donors for HCV antibodies. Since that time, HCV infection is rarely transmitted by transfusion.8 Other potential sources of HCV transmission include exposure to an infected sexual partner or multiple sexual partners, frequent exposure to infected blood among health care workers, and perinatal exposure.9-11 Although HCV prevalence rates are consistently higher in persons with multiple sexual partners, sexual transmission of HCV between monogamous partners is rare.7 Thus, while it is common to counsel HCV-infected persons to notify their current partners of their HCV status, they should be told that the risk of sexual transmission is sufficiently low12 that many authorities do not advise use of barrier precautions (i.e., latex condoms). Testing of sexual partners, therefore, is done chiefly for reassurance. There is no need to curtail ordinary household activities except those that might result in blood exposure, such as sharing a razor or toothbrush. HCV is not transmitted by hugging and the sharing of eating utensils. Although a monogamous sexual relationship carries a low risk of transmission of HCV infection, as noted above, the risk is higher in persons involved with multiple sexual partners. Persons with hemophilia should be tested for HCV infection if blood products were received before 1987, when viral inactivation procedures were implemented. It is also advisable to test persons for HCV infection if they have evidence of otherwise unexplained elevations of aminotransferase levels (alanine and/or aspartate aminotransferases; ALT /AST), have ever been on hemodialysis, or have human immunodeficiency virus (HIV) infection.10 Other situations that have been suggested to carry a risk for HCV transmission include certain folk medicine practices (acupuncture, ritual scarification), body piercing, tattooing, and even commercial barbering.13-17 Some studies of HCV infection have reported associations with commercial tattooing, suggesting possible acquisition of HCV infection in this setting.18-20 Most studies of body piercing have not differentiated between ear piercing and piercing of other body parts. As a result of discrepancies in study design, definitive conclusions regarding risks associated with these forms of percutaneous exposures are problematic, although the risk, if present, is likely to be low. Thus, there is no need to routinely test persons who have received tattoos or undergone piercing, particularly if these procedures have taken place in licensed establishments. Table 2, adapted from recommendations published by the Centers for Disease Control, Atlanta, Georgia,10 outlines the list of persons who should be routinely tested for HCV infection. For some of these categories (e.g., injection drug users, persons with hemophilia), the HCV prevalence is high (≈90%); for others (e.g., recipients of blood transfusions prior to 1992), the prevalence is moderate (≈10%). For still others (e.g., persons exposed by needle stick, sexual partners of HCV-infected persons), it is quite low (2%-5%). 1. Persons who should be tested for HCV infection are listed in Table 2(Grade, III). Persons found to be HCV-infected need to be counseled regarding prevention of spread of the virus to others. Good clinical practice dictates that all persons identified as infected with HCV be informed that transmission to others occurs through contact with their blood and that they should therefore take precautions against the possibility of such exposure. Although this advice applies to all HCV-infected persons, it has particular importance for injection drug users who are the leading source of HCV infections. Circumstances requiring counseling are shown in Table 3. 2. Persons infected with HCV should be counseled on how to avoid HCV transmission to others, as indicated in Table 3(Grade, III). Utilizing the tests described in Table 4, several strategies can be employed to detect HCV infection. In clinical practice, the usual approach is to test initially for antibodies to HCV (anti-HCV), then to use HCV ribonucleic acid (RNA) to document viremia. Because most persons with ongoing HCV infection have HCV RNA levels in the range of the quantitative assays and because the quantity of HCV RNA is useful to know before providing and monitoring HCV treatment,21 many experts routinely obtain quantitative rather than qualitative HCV RNA tests to confirm the presence of viremia.22 However, quantitative HCV RNA tests are generally not as sensitive; therefore, some experts prefer a qualitative HCV RNA test either as the primary test or to confirm a positive HCV antibody result in patients with a negative result by quantitative assay.23, 24 A negative sensitive RNA test in a person with HCV antibodies most likely indicates that the HCV infection has resolved. Other interpretations are that the anti-HCV immunoassay is falsely positive, the HCV RNA test is falsely negative, or rarely, that a person has intermittent or low-level viremia. The recombinant immunoblot assay has limited usefulness in clinical practice but may establish the cause of a positive anti-HCV immunoassay in a person with undetectable HCV RNA.24 A negative immunoblot result indicates that a positive anti-HCV immunoassay result represented a false positive result and that no further testing is needed. A positive immunoblot result followed by two or more instances in which HCV RNA cannot be detected using a licensed, qualitative assay suggest that HCV infection has resolved and no further HCV testing is indicated. There are instances in which a negative anti-HCV does not exclude HCV infection in patients with suspected liver disease. These include acute HCV infection or immunosuppressed states. HCV RNA testing can be used to establish acute HCV infection after an exposure because HCV RNA can be detected in 1 to 2 weeks, while antibodies to HCV are detectable an average of 8 weeks later.25-27 HCV RNA testing can also be used to test for HCV infection in persons with negative HCV antibody results who are known to have conditions associated with diminished antibody production, such as HIV infection and chronic hemodialysis.23 HCV RNA can be detected in the blood using amplification techniques such as polymerase chain reaction (PCR) or transcription-mediated amplification (TMA).28 The Food and Drug Administration (FDA) has approved 2 PCR-based tests for qualitative detection of HCV RNA: (1) Amplicor Hepatitis C Virus Test, version 2.0, and (2) Cobas Amplicor Hepatitis C Virus Test, version 2.0 (Roche Molecular Systems, Branchburg, NJ), which have lower limits of detection of approximately 50 IU/mL. Other commercially available nonapproved assays are used by some diagnostic laboratories. Quantitative assays (Table 4) ascertain the quantity of HCV RNA in blood using either target amplification (PCR, TMA) or signal amplification techniques (branched DNA assay). The level of HCV RNA in blood helps in predicting the likelihood of response to treatment, and the change in the level of HCV RNA during treatment can be used to monitor response. The results should be reported in international units to standardize data,29 although the dynamic ranges differ and the results can be difficult to compare between assays, as noted in Table 4. Because a change in the HCV RNA level is used to monitor treatment response, it is important at the outset of treatment to obtain the actual level rather than simply a report indicating that the level exceeds an upper limit of detection, since HCV RNA levels sometimes are above the linear range of currently available assays. In addition, the same quantitative test should be used while on therapy to avoid confusion. The only quantitative test that has currently received FDA approval is Versant HCV RNA version 3.0 (Bayer Diagnostics, Tarrytown, NY) (Table 4). There are 6 major HCV genotypes.30 Although genotype does not predict the outcome of infection, it does predict the likelihood of treatment response, and, in many cases, determines the duration of treatment.31-33 Genotyping can be performed by direct sequence analysis, by reverse hybridization to genotype-specific oligonucleotide probes, or by the use of restriction fragment length polymorphism. Two tests, not yet FDA approved, are currently available for clinical use: (1) the Trugene HCV 5'NC Genotyping Kit (Visible Genetics, Toronto, Canada), which is based on direct sequencing followed by comparison with a reference sequence database, and (2) the line-probe assay (Inno LiPA HCV II, Innogenetics, Ghent, Belgium), which is based on reverse hybridization of PCR amplicons on a nitrocellulose strip coated with genotype-specific oligonucleotide probes.34-36 Once the genotype is identified, the test need not be repeated. Current commercial tests fail to identify the genotype in a small proportion (<3 %) of HCV-positive persons,37 and a similarly low proportion (1%-4%) may display mixed genotypes.37, 38 3. Patients suspected of having chronic HCV infection should be tested for HCV antibodies. (Grade, II-2) 4. HCV RNA testing should be performed in (a) patients with a positive anti-HCV test (Grade, II-2); (b) patients for whom antiviral treatment is being considered, using a quantitative assay (Grade, II-2); (c) patients with unexplained liver disease whose anti-HCV test is negative and who are immune-compromised or suspected of having acute HCV infection (Grade, II-2). 5. HCV genotype should be determined in all HCV-infected persons prior to treatment in order to determine the duration of therapy and likelihood of response (Grade, I). The role of liver biopsy in the management of patients with chronic hepatitis C is currently being debated. In the initial treatment trials of hepatitis C, a liver biopsy was regarded as an important parameter in helping to guide management and treatment, particularly at a time when response to treatment was low. More recently, with the improvement of treatment effectiveness, the value of the liver biopsy has been questioned because of the potential risks of the procedure and the concern of sampling error.39 This has prompted some to challenge the need for biopsy and to suggest that the procedure may not be necessary as a prelude to treatment. However, since current therapy is effective in clearing virus in only about one half of those treated, and since treatment is associated with costs and adverse events, there are likely many individuals in whom therapy can be safely deferred. The liver biopsy furnishes information about the staging of fibrosis and the degree of hepatic inflammation, histopathological features that are helpful to both the patient and the provider for predicting the natural history of disease and thus the relative urgency of therapy.40-42 Three scoring systems for defining the degree of inflammation (grading) and the extent of fibrosis (staging) have been devised, 2 of which—the Metavir scoring system43 and the Ishak grading system44—have received the greatest attention. The components of these systems are shown in Table 5. Using the degree of fibrosis as one component of the basis for therapy, treatment is generally advised if the liver biopsy displays a Metavir score of ≥ 2 or an Ishak score of ≥ 3. Some experts, in considering the need for treatment, also assess the intensity of liver inflammation. However, there are no established guidelines for how to combine the degrees of liver fibrosis and inflammation. Moreover, measurement of liver fibrosis, and especially liver inflammation, can be compromised by sampling error and by difficulties in the histopathologic interpretation. In most studies, the extent of liver fibrosis is an independent predictor of treatment response. Patients with milder degrees of fibrosis generally respond more favorably to treatment than do patients with more advanced fibrosis (bridging fibrosis or cirrhosis).45, 46 However, the need for treatment in such patients is lower than it is for those with advanced fibrosis. The cost-effectiveness of treating patients with no liver fibrosis has been questioned, since the prognosis even without therapy is excellent, further underscoring the importance of accurately staging the severity of liver disease.47 Clinical, laboratory and radiological findings can identify many patients with cirrhosis, but not those with lesser degrees of fibrosis.48 Thus, in persons without strong clinical evidence of cirrhosis, a liver biopsy is useful in providing information about the extent of liver damage associated with chronic infection, the feature that remains the best predictor of prognosis. Although liver fibrosis markers are commercially available, they are currently insufficiently accurate to support their routine use.49 Until sensitive serum markers can be developed that will define all stages of fibrosis and mirror the information derived from liver biopsy, the procedure remains the only means of defining the severity of damage from HCV infection in many patients. After weighing the risks, benefits and costs of existing HCV treatments and of liver biopsy, most experienced clinicians routinely obtain a liver biopsy in patients with HCV genotype-1 infection to guide recommendations for treatment. Patients infected with HCV genotypes 2 and 3, however, have a high likelihood of response and, therefore, some advocate treating all such patients regardless of severity of liver disease without resorting to liver biopsy. For patients with no or little fibrosis (i.e., Metavir score <2 or Ishak score <3), in whom treatment is often deferred, liver biopsy can be used to monitor progression of liver disease. An interval of 4 to 5 years between biopsies may be needed to measure change in such patients.50 Although the spectrum of liver fibrosis tends to be worse in persons with elevated blood levels of aminotransferases than in those with normal aminotransferase levels,51 14% to 24% of persons with persistently normal values have more-than-portal fibrosis on liver biopsy. These persons may have progressive liver disease over time despite persistence of normal aminotransferase values.51, 52 In individuals with normal aminotransferase values and extensive hepatic fibrosis (bridging fibrosis or cirrhosis), treatment should be considered, and liver biopsy is the only available method to obtain the necessary information to guide this decision. In patients with chronic infection and clinical signs of advanced cirrhosis, liver biopsy may add little to the clinical impression and may be riskier than in healthier patients. 6. Regardless of the level of ALT, a liver biopsy should be done when the results will influence whether treatment is recommended, but a biopsy is not mandatory in order to initiate therapy (Grade, III). 7. A liver biopsy may be obtained to provide information on prognosis (Grade, III). Natural history studies indicate that 55% to 85% of persons who develop acute hepatitis C will remain HCV-infected. Among these individuals, 5% to 20% are reported to develop cirrhosis over periods of approximately 20 to 25 years.53, 54 The higher percentage figure of 20% may not reflect the cirrhosis rate in the general population of HCV-infected persons because these data originate largely from studies in tertiary-care settings, and hence may represent referral bias. Persons with HCV-related cirrhosis are at risk for developing end-stage liver disease (a risk of approximately 30% over 10 years) as well as hepatocellular carcinoma (HCC) (a risk of approximately 1% to 2% per year).55 The 15% to 45% of persons with acute hepatitis C who do recover (HCV RNA not detected in their blood) are not subject to long-term complications and do not need treatment. In general clinical practice, however, acute hepatitis C is uncommonly recognized; the majority of patients already have chronic hepatitis C. In persons with persistent infection, evolution to cirrhosis is the primary concern, usually requiring the passage of 2 or more decades, and occurring more often in persons infected at older ages (particularly men), those who drink more than 50 grams of alcohol each day, those who are obese or have substantial hepatic steatosis, and those with HIV coinfection.56-58 More-than-portal fibrosis on liver biopsy (Metavir ≥2 or Ishak ≥3) is an important predictor of future progression of liver disease and the need for HCV treatment.40, 41, 57 Infection with HCV can also be associated with a variety of extra-hepatic manifestations, chief of which is the induction of abnormal circulating proteins called cryoglobulins. The pathologic consequence, termed mixed cryoglobulinemia, is the development of vasculitis, which is associated with certain skin manifestations and internal organ damage that predominantly the The presence of is an for HCV antiviral therapy, regardless of the of liver disease. The of treatment is to complications of HCV this is by of infection. treatment are by the results of HCV RNA Infection is when there is a sustained virologic response as the of HCV RNA in serum by a sensitive test at the end of treatment and 6 Persons who an have a in the HCV RNA level in some studies as a or of HCV RNA weeks therapy, to as an early virologic response of detectable virus at of treatment is to as end of treatment response A patient is to have when HCV RNA undetectable on treatment but is detected after of treatment. Persons in whom HCV RNA levels remain on treatment are while those whose HCV RNA levels (e.g., by but are to as in liver including improvement in fibrosis, has been in patients or pegylated in with particularly in those with an to There have been substantial in the of HCV treatment and there are currently several treatments approved by the FDA (Table In clinical the rates have been with the of of and which the current of care. in therapy of chronic hepatitis C. interferon; pegylated interferon; are by of the to thus and the half of the There are 2 licensed products in the United States, the and the Because of their half they can be by injection In higher rates have been with the of of than with by injection a with or used In these was by was FDA and with of was as a of with a higher of if and if In a of was used with either or the of Since the 2 have not been in a using their relative cannot be However, there were of treatment response and adverse It should be noted that data to be useful for treatment recommendations were not for both forms of For the of the study was the only one of that a treatment duration of 6 is for persons infected with HCV genotypes 2 or recommendations have been to both response rates to and response to genotype and HCV RNA are shown in and 4. The likelihood of an can be by patient as well as by the In all treatment studies, genotype is the predictor of response. In the studies of and rates were higher in patients who or HCV lower HCV RNA levels lower body and of fibrosis and In persons who were with with the independent associated with an genotype than and body than The majority of patients in the first 2 trials who were infected with genotype 2 or 3, but a small were infected with genotypes 4, and 6. In these 2 in patients with genotype-1 were to while the response rates in those with genotype 2 or were to In the study that the data were further by genotype and viral Persons with genotype 1 and a high viral to who received the of and an of the rate among those with genotype 1 and a low viral who were with the same was In in persons with genotypes 2 and and a high viral who were and the rate was while those with genotypes 2 and and a low viral who were similarly an of virologic response rates with and therapy for weeks to genotype and viral virologic response rates with or and to virologic response rates in recipients of and 2 of for 24 or In American patients with genotype-1 infection, rates are lower than in although estimates are not currently available for the of and In the study of with the of an based on the was at as an at from of the HCV RNA of patients with an an among those who not have an EVR, to develop an data were noted in the study that used with Among persons who an EVR, an of those who not have an EVR, developed an The optimal treatment duration and were in a in which all persons received at a of while patients in the 4 received either 24 or weeks of at of either or the of or were not only the HCV but also the viral or 2 of those with genotype 1. In patients with genotype 1 with low-level the was in those who received the higher and who were for weeks This was also optimal for patients with genotype 1 and a high viral an In in patients with genotype 2 or 3, regardless of the viral no were detected with the 4 treatment suggesting that at a of for 24 weeks is associated with such as and and and skin fibrosis and and to with than with associated with such as or Because of the concern of from the use of it is that persons who the drug use methods both during treatment and for a of 6 after treatment. reported in with the use of and include and such as and granulocyte colony-stimulating factor have been used to the adverse of and However, currently there are data to their routine use as a means to avoid or and in clinical to be more in the initial weeks of treatment and often can be with such as or such as and, Current recommendations for treatment of persons with chronic hepatitis C are derived from data in the trials

Oxidative DNA damage is important in aging and the degenerative diseases of aging such as cancer. Estimates commonly rely on measurements of 8-oxo-2'-deoxyguanosine (oxo8dG), an adduct that occurs in DNA and is also excreted in urine after DNA repair. Here we examine difficulties inherent in the analysis of oxo8dG, identify sources of artifacts, and provide solutions to some of the common methodological problems. A frequent criticism has been that phenol in DNA extraction solutions artificially increases the measured level of oxo8dG. We found that phenol extraction of DNA contributes a real but minor increase in the level of oxo8dG when compared, under equivalent conditions, with a successful nonphenol method. A more significant reduction in the baseline level was achieved with a modification of the recently introduced chaotropic NaI method, reducing our estimate of the level of steady-state oxidative adducts by an order of magnitude to 24,000 adducts per cell in young rats and 66,000 adducts per cell in old rats. Of several alternative methods tested, the use of this chaotropic technique of DNA isolation by using NaI produced the lowest and least variable oxo8dG values. In further studies we show that human urinary 8-oxo-guanine (oxo8Gua) excretion is not affected by the administration of allopurinol, suggesting that, unlike some methylated adducts, oxo8Gua is not derived enzymatically from xanthine oxidase. Lastly, we discuss remaining uncertainties inherent both in steady-state oxo8dG measurements and in estimates of endogenous oxidation ("hit rates") based on urinary excretion of oxo8dG and oxo8Gua.

The increasing use of nanotechnology in consumer products and medical applications underlies the importance of understanding its potential toxic effects to people and the environment. Although both fullerene and carbon nanotubes have been demonstrated to accumulate to cytotoxic levels within organs of various animal models and cell types and carbon nanomaterials have been exploited for cancer therapies, the molecular and cellular mechanisms for cytotoxicity of this class of nanomaterial are not yet fully apparent. To address this question, we have performed whole genome expression array analysis and high content image analysis based phenotypic measurements on human skin fibroblast cell populations exposed to multiwall carbon nano-onions (MWCNOs) and multiwall carbon nanotubes (MWCNTs). Here we demonstrate that exposing cells to MWCNOs and MWCNTs at cytotoxic doses induces cell cycle arrest and increases apoptosis/necrosis. Expression array analysis indicates that multiple cellular pathways are perturbed after exposure to these nanomaterials at these doses, with material-specific toxigenomic profiles observed. Moreover, there are also distinct qualitative and quantitative differences in gene expression profiles, with each material at different dosage levels (6 and 0.6 microg/mL for MWCNO and 0.6 and 0.06 microg/mL for MWCNT). MWCNO and MWCNT exposure activates genes involved in cellular transport, metabolism, cell cycle regulation, and stress response. MWCNTs induce genes indicative of a strong immune and inflammatory response within skin fibroblasts, while MWCNO changes are concentrated in genes induced in response to external stimuli. Promoter analysis of the microarray results demonstrate that interferon and p38/ERK-MAPK cascades are critical pathway components in the induced signal transduction contributing to the more adverse effects observed upon exposure to MWCNTs as compared to MWCNOs.

BACKGROUND: Antiretroviral therapy has dramatically reduced the morbidity and mortality of infection due to HIV. The emergence of drug-resistant virus has limited the usefulness of many drugs. OBJECTIVE: To determine the prevalence of HIV drug resistance in the population of adults receiving care in the United States. DESIGN AND METHODS: HIV drug susceptibility assays were performed on plasma virus from a random sample representative of the 132500 HIV-infected American adults who had received medical care in early 1996 yet were viremic with > 500 copies/ml of HIV RNA in late 1998. A blood sample was obtained from 1797 patients who comprised a representative sample of the 208900 adults receiving urban care for HIV infection in early 1996 who survived to late 1998. The sampling procedure permitted weighting each evaluated patient to reflect demographic and other characteristics of the target population. RESULTS: We estimated that 132500 (63%) of the target population had HIV viremia of > 500 copies/ml. Among viremic patients, an estimated 76% had resistance to one or more antiretroviral drugs. The odds of resistance were significantly higher in patients with a history of antiretroviral drug use, advanced HIV disease, higher plasma HIV viral load and lowest CD4 cell count by self-report. CONCLUSIONS: The frequent selection for drug-resistant virus among viremic patients during the first 3 years of widespread use of potent antiretroviral combination therapy has significant implications for HIV treatment and transmission.

The periodic, seven-stripe pattern of the primary pair-rule gene even-skipped (eve) is initiated by crude, overlapping gradients of maternal and gap gene proteins in the early Drosophila embryo. Previous genetic studies suggest that one of the stripes, stripe 2, is initiated by the maternal morphogen bicoid (bcd) and the gap protein hunchback (hb), while the borders of the stripe are formed by selective repression, involving the gap protein giant (gt) in anterior regions and the Krüppel (Kr) protein in posterior regions. Here, we present several lines of evidence that are consistent with this model for stripe 2 expression, including in vitro DNA-binding experiments and transient cotransfection assays in cultured cells. These experiments suggest that repression involves a competition or short-range quenching mechanism, whereby the binding of gt and Kr interferes with the binding or activity of bcd and hb activators at overlapping or neighboring sites within the eve stripe 2 promoter element. Such short-range repression could reflect a general property of promoters composed of multiple, but autonomous regulatory elements.

UNLABELLED: Once-monthly (50/50, 100, and 150 mg) and daily (2.5 mg; 3-year vertebral fracture risk reduction: 52%) oral ibandronate regimens were compared in 1609 women with postmenopausal osteoporosis. At least equivalent efficacy and similar safety and tolerability were shown after 1 year. INTRODUCTION: Suboptimal adherence to daily and weekly oral bisphosphonates can potentially compromise therapeutic outcomes in postmenopausal osteoporosis. Although yet to be prospectively shown in osteoporosis, evidence from randomized clinical trials in several other chronic conditions shows that reducing dosing frequency enhances therapeutic adherence. Ibandronate is a new and potent bisphosphonate with antifracture efficacy proven for daily administration and also intermittent administration with a dose-free interval of >2 months. This report presents comparative data on the efficacy and safety of monthly and daily oral ibandronate regimens. MATERIALS AND METHODS: MOBILE is a 2-year, randomized, double-blind, phase III, noninferiority trial. A total of 1609 women with postmenopausal osteoporosis were assigned to one of four oral ibandronate regimens: 2.5 mg daily, 50 mg/50 mg monthly (single doses, consecutive days), 100 mg monthly, or 150 mg monthly. RESULTS: After 1 year, lumbar spine BMD increased by 3.9%, 4.3%, 4.1%, and 4.9% in the 2.5, 50 /50, 100, and 150 mg arms, respectively. All monthly regimens were proven noninferior, and the 150 mg regimen superior, to the daily regimen. All monthly regimens produced similar hip BMD gains, which were larger than those with the daily regimen. All regimens similarly decreased serum levels of C-telopeptide, a biochemical marker of bone resorption. Compared with the daily regimen, a significantly larger proportion of women receiving the 100 and 150 mg monthly regimens achieved predefined threshold levels for percent change from baseline in lumbar spine (6%) or total hip BMD (3%). All regimens were similarly well tolerated. CONCLUSIONS: Monthly ibandronate is at least as effective and well tolerated as the currently approved daily ibandronate regimen in postmenopausal osteoporosis.

The past decade has seen a significant increase in the number of potentially tolerogenic therapies for treatment of new-onset diabetes. However, most treatments are antigen nonspecific, and the mechanism for the maintenance of long-term tolerance remains unclear. In this study, we developed an antigen-specific therapy, insulin-coupled antigen-presenting cells, to treat diabetes in nonobese diabetic mice after disease onset. Using this approach, we demonstrate disease remission, inhibition of pathogenic T cell proliferation, decreased cytokine production, and induction of anergy. Moreover, we show that robust long-term tolerance depends on the programmed death 1 (PD-1)-programmed death ligand (PD-L)1 pathway, not the distinct cytotoxic T lymphocyte-associated antigen 4 pathway. Anti-PD-1 and anti-PD-L1, but not anti-PD-L2, reversed tolerance weeks after tolerogenic therapy by promoting antigen-specific T cell proliferation and inflammatory cytokine production directly in infiltrated tissues. PD-1-PD-L1 blockade did not limit T regulatory cell activity, suggesting direct effects on pathogenic T cells. Finally, we describe a critical role for PD-1-PD-L1 in another powerful immunotherapy model using anti-CD3, suggesting that PD-1-PD-L1 interactions form part of a common pathway to selectively maintain tolerance within the target tissues.

We have previously proposed the hypothesis that asymmetric membranes behave like bilayer couples: the two layers of the bilayer membrane can respond differently to a particular perturbation. Such a perturbation, for example, can result in the expansion of one layer relative to the other, thereby producing a curvature of that membrane. In experiments with erythrocytes and lymphocytes, we now demonstrate that different membrane perturbations which have opposite effects on membrane curvature can compensate and neutralize one another, as expected from the bilayer couple hypothesis. This provides a rational basis, for example, for understanding the effects of amphipathic drugs on a variety of cellular phenomena which involve shape changes of membranes.

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.

T follicular helper (Tfh) cells represent a Th subset engaged in the help of B-cell responses in germinal centers (GCs). Tfh cells abundantly express the transcription repressor B-cell lymphoma 6 (Bcl6), a factor that is necessary and sufficient for their development in vivo. Whether Tfh or Tfh-committed cells are involved in the help of B cells outside GCs remains unclear, particularly in humans. In this study, we identified a previously undefined BCL6-expressing CD4(+) T-cell subset in human tonsils. This subset expressed IL-7 receptor and chemokine receptor 5 (CXCR5) and inducible costimulator (ICOS) at low levels (CXCR5(lo)ICOS(lo)), and it was found exclusively outside GCs. CXCR5(lo)ICOS(lo) CD4(+) T cells secreted larger amounts of IL-21 and IL-10 than CXCR5(hi)ICOS(hi) GC-Tfh cells upon activation, and they induced proliferation and differentiation of naïve B cells into Ig-producing cells more efficiently than GC-Tfh cells. However, this subset lacked the capacity to help GC-B cells because of the induction of apoptosis of GC-B cells through the FAS/FAS-ligand interaction. CXCR5(lo)ICOS(lo) CD4(+) T cells expressed equivalent amounts of BCL6 transcript with CXCR5(hi)ICOS(hi) GC-Tfh cells, but they expressed less Bcl6 protein. Collectively, our study indicates that CXCR5(lo)ICOS(lo) CD4(+) T cells in human tonsils represent Tfh lineage-committed cells that provide help to naïve and memory B cells outside GCs.

Many scientific investigations generate both longitudinal data and sur- vival data. Methods for the combined analysis of both kinds of data have been developed in recent years, with the main emphasis being on modeling and estima- tion. In cancer research it is common for there to be long term survivors or cured patients and methods have been developed to analyze such data. In this article, we review both joint models for the analysis of longitudinal and survival data and cure models. We then present a joint longitudinal-survival-cure model to analyze data from a study of prostate cancer patients treated with radiation therapy. In this model each patient is assumed to be either cured or susceptible to clinical recur- rence. The cured fraction is modeled as a logistic function of baseline covariates. The longitudinal PSA data is modeled as a non-linear hierarchical mixed model, with different models for the cured and susceptible groups. The clinical recurrences are modeled as a time-dependent proportional hazards model for those in the sus- ceptible group. The baseline variables are covariates in both the failure time and longitudinal models. We use both a Monte Carlo EM algorithm and Markov chain Monte Carlo techniques to fit the model. The results from the two estimation methods are compared. We focus on both selected parameters of the model and derived interpretable quantities.

When pBR322 plasmid-harboring Escherichia coli strains RR1 or chi1776 were grown in the presence of 1 mg of uridine or cytidine per ml and later treated with chloramphenicol, as much as three times more plasmid deoxyribonucleic acid was recovered than would normally be obtained by routine plasmid amplification procedures.

Removal of 80% of the 5'-terminal 7-methyl-guanosine (m7G) from methylated reovirus mRNA by beta elimination results in a concomitant loss of the ability to bind to wheat germ ribosomes. The mRNA molecules that retain the m7G account for most of the residual binding. Removal of the m7G from all molecules in preparations of methylated reovirus and vesicular stomatitis virus mRNA reduces the extent of binding to wheat germ ribosomes from 80% to 5-7%. In the reticulocyte lysate, however, significant binding (17-34%) of the beta-eliminated viral RNAs occurs. This m7G-independent binding appears to be due to recognition by ribosomes of other structural features of the 5'-proximal sequences. Initiation complexes involving beta-eliminated animal virus mRNAs and rabbit reticulocyte ribosomes appear to be more stable than the more heterologous combination of the same viral mRNAs and wheat germ ribosomes. In addition, evidence is presented to show that the 2'-O-methylated nucleoside of the 5'-terminal cap has a positive influence on the ribosome binding of viral mRNA and of capped synthetic ribopolymers. A model involving recognition of multiple structural features of the 5'-terminal region of mRNA by ribosomes during initiation of protein synthesis is presented.

OBJECTIVE: In order to investigate potential regulatory mechanisms for the increased production of prostaglandin E2 (PGE2) in interleukin-1 beta (IL-1 beta)-stimulated rheumatoid synovial fibroblasts (RSF), this study examined the induction of phospholipase A2 (PLA2) and prostaglandin H synthase (PGHS) enzymes and the correlation of these events with PGE2 production in IL-1 beta-stimulated RSF. METHODS: Protein and messenger RNA (mRNA) levels of cytosolic PLA2 (cPLA2) and PGHS-2 enzymes in IL-1 beta-stimulated RSF were measured by Western and Northern blotting, respectively, using specific antisera and complementary DNA probes. Enzymatic activity of cPLA2 was determined in cell-free reaction mixtures utilizing mixed micelles of 14C-phosphatidylcholine and Triton X-100 as the substrate. PGE2 levels were quantitated using a commercial enzyme immunoassay kit. RESULTS: Incubation of RSF with IL-1 beta increased the mRNA and protein levels for the high molecular weight cPLA2 as well as for the mitogen/growth factor-responsive PGHS (PGHS-2). The IL-1 receptor antagonist completely abolished the induction of these two enzymes and the stimulation of PGE2 production by IL-1 beta in RSF. In contrast, levels of the other known forms of these enzymes, i.e., the 14-kd secretory group II PLA2 (sPLA2) and the constitutive form of PGHS (PGHS-1), were unaffected by IL-1 beta treatment. CONCLUSION: These are the first data to demonstrate the coordinate induction by IL-1 of cPLA2 and PGHS-2 in RSF. The time-course for the induction of these enzymes suggests that their increase contributes to the increased production of PGE2 in IL-1-treated RSF, and may help explain the capacity of RSF to produce large amounts of PGE2.

The protease activated receptor-2 (PAR-2) belongs to a family of G-protein-coupled receptors that are activated by proteolysis. Trypsin cleaves PAR-2, exposing an N-terminal tethered ligand (SLIGRL) that activates the receptor. Messenger RNA (mRNA) for PAR-2 was found in guinea pig airway tissue by reverse transcription-polymerase chain reaction, and PAR-2 was found by immunohistochemistry in airway epithelial and smooth-muscle cells. In anesthetized guinea pigs, trypsin and SLIGRL-NH(2) (given intratracheally or intravenously) caused a bronchoconstriction that was inhibited by the combination of tachykinin-NK(1) and -NK(2) receptor antagonists and was potentiated by inhibition of nitric oxide synthase (NOS). Trypsin and SLIGRL-NH(2) relaxed isolated trachea and main bronchi, and contracted intrapulmonary bronchi. Relaxation of main bronchi was abolished or reversed to contraction by removal of epithelium, administration of indomethacin, and NOS inhibition. PAR-1, PAR-3, and PAR-4 were not involved in the bronchomotor action of either trypsin or SLIGRL-NH(2), because ligands of these receptors were inactive either in vitro or in vivo, and because thrombin (a PAR-1 and PAR-3 agonist) did not show cross-desensitization with PAR-2 agonists in vivo. Thus, we have localized PAR-2 to the guinea-pig airways, and have shown that activation of PAR-2 causes multiple motor effects in these airways, including in vivo bronchoconstriction, which is in part mediated by a neural mechanism.

ABSTRACT Angiotensin (Ang)‐II induces vascular wall inflammation by activating NF‐κB. Aspirin inhibits IKKβ in vitro; however, the in vivo relevance of the phenomenon is unclear. We tested the hypothesis that aspirin protects from Ang II‐induced endorgan damage by inhibiting NF‐κB activation in vivo. Rats harboring human renin and angiotensinogen genes received high‐ (600 mg/kg/day) or low‐ (25 mg/kg/day) dose aspirin. High‐dose aspirin reduced mortality, cardiac hypertrophy, fibrosis, and albuminuria independent of blood pressure, whereas both doses reduced cyclooxygenase activity. High‐dose aspirin inhibited NF‐κB and AP‐1 activation and inflammation in heart and kidney. These in vivo results serve to explain the clinical utility of high‐dose aspirin in inflammatory disorders and suggest additional therapeutic avenues that may be relevant to cardiovascular disease.

A total clone bank of cDNAs synthesized from mouse liver poly(A)-RNA was constructed in Escherichia coli K-12 RR1 using the plasmid pBR322. Sequences of cDNAs were inserted into the PST-I site of pBR322 by the "G-C-tailing" method. Bulk preparations of these cDNA sequences were obtained by treatment of the resultant total hybrid plasmid population with PST-I. Aliquots of this cDNA material were then labeled with 32P by "nick translation" using E. coli DNA polymerase I for the preparation of hybridization probes. Experiments involving the back-hybridization of these probes to the total hybrid-plasmid clone bank population revealed that virtually all of the liver poly(A)-RNA sequences were represented in the total clone bank. The long term stability of these sequences residing in RR1 host cells was then examined through extensive serial passage of the initial total clone cell population culture. The results showed: 1) that the small percentage of natural pBR322 molecules (containing no cDNA inserts) usually present in such total clone bank preparations do not outgrow the respectively hybrid specie of plasmids in these populations: 2) few, if any, cDNA sequences are completely lost; and 3) some redistribution of the abundant and more unique DNA sequences probably does occur. The use of total clone hybrid-plasmid populations (constructed from poly(A)-RNA isolated from different tissues) described here should allow the identification of tissue-specific RNA sequences through the use of cross-hybridizaton techniques.

The uptake of norepinephrine (NE) by the human platelet resembles that of the adrenergic neuron. Platelets incubated with d1‐ 14 C‐NE (2.1 x 10 −6 M) for one hour at 37° C. concentrated the amine to a distribution ratio of 5.1 ± 0.1. Uptake was markedly reduced by cold and metabolic inhibitors such as cyanide, fluoride, dinitrophenol, and PCMB. NE uptake was also diminished by ouabain, quinidine, and substitution of lithium for sodium in the medium, suggesting the uptake mechanism is related to a sodium pump. Drugs which interfere with the uptake of NE by the neuron have a similar effect on the platelet. Uptake of 14 C‐NE was impaired by serotonin (5HT), tryptamine, tyramine, amphetamine, bretylium, debrisoquin, guanethidine, desmethylimipramine, and various antihistamines. 14 C‐NE accumulated in the platelet by a preliminary incubation was released by reserpine, tyramine, phenylephrine, and debrisoquin, less by guanethidine, but not by ephedrine or mephentermine. 14 C‐5HT was released by reserpine, tyramine, debrisoquin, and mephentermine, less by ephedrine, but not by guanethidine or phenylephrine. Thus, release or failure of release of NE or 5HT from platelets will not necessarily reflect the action of a drug on sympathetic neurons.

Allelic deletions involving chromosomes 18q occur in a significant number of colorectal cancers. Recently, a highly conserved gene called 'deleted in colorectal cancer' (DCC) has been identified on chromosome 18q. DCC has been postulated to be a colorectal tumor-suppressor gene. In order to understand the role of DCC in cell transformation, we have established a stable Rat-1 cell line expressing dexamethasone-inducible DCC antisense RNA. High levels of dexamethasone-inducible DCC antisense RNA were detected in the Rat-1 transfectants. The antisense DCC-expressing Rat-1 cells showed a faster growth rate, anchorage independence and tumorigenicity in nude mice. Exposure of the parental Rat-1 cells to antisense oligodeoxyribonucleotides to DCC resulted in inhibition of cell adhesion to the substratum which could be abrogated by various extracellular matrices. On the other hand, a bone marrow-derived stromal cell line which does not express DCC showed no detachment from the substratum when treated with the antisense oligo to DCC. These results suggest that the DCC gene is involved in cell adhesion and provide the first direct biological evidence for the possible role of DCC as a tumor-suppressor gene.

Orlistat, a lipase inhibitor, acts locally in the gastrointestinal tract; its systemic exposure is not required for its efficacy. However, knowledge of the extent of its systemic exposure is important for its safe use in obese patients, the intended target population. Pharmacokinetic screening in obese patients was carried out by monitoring plasma concentrations of unchanged orlistat and its metabolites in five key double-blind, placebo-controlled phase II/III studies. Results of these studies involving the monitoring of plasma samples indicate that detection of intact orlistat in plasma was sporadic, and measurable concentrations were low (< 10 ng/mL or 0.02 microM) without evidence of accumulation, which is consistent with minimal absorption. It is concluded that systemic exposure of orlistat is negligible; at a clinically efficacious dose level, orlistat is unlikely to produce systemic lipase inhibition.