The immunodominant outer membrane antigen of Actinobacillus actinomycetemcomitans is located in the serotype-specific high-molecular-mass carbohydrate moiety of lipopolysaccharide.

Most patients with juvenile periodontitis manifest serum antibodies, sometimes at very high titers, to antigens of Actinobacillus actinomycetemcomitans, but the antigens inducing the immune response have been only partly characterized. We separated A. actinomycetemcomitans serotype b cells into protein, lipopolysaccharide (LPS), and soluble polysaccharide fractions and characterized them. Coomassie blue- and silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were used to detect protein and LPS components, and gas-liquid chromatography was used to determine their carbohydrate and fatty acid composition. Western blots, dot blots, and enzyme-linked immunosorbent assay inhibition with high-titer sera from juvenile periodontitis patients revealed which components were highest in antibody binding activity. These results showed that the major portion of the immunoglobulin G binding activity resides in the purified mannan-free LPS, with lesser amounts in the total protein fraction. Using Sephacryl S-300 chromatography, we separated LPS into high-molecular-mass components with high carbohydrate contents by gas-liquid chromatography and a low-molecular-mass component consisting mainly of lipid A and the inner core sugar heptulose. The results of quantitative dot blot assays and enzyme-linked immunosorbent assay inhibition show that the serotype-specific antibody binding activity is highly concentrated in the high-molecular-mass carbohydrate-rich LPS fraction and is almost completely absent in the low-molecular-weight lipid-rich fraction. Our observations contrast with previous reports that the predominant serotype antigen of A. actinomycetemcomitans resides in a mannan-rich polysaccharide isolated from spent culture medium. These observations support the conclusion that the immunodominant antigen of the outer membrane is the O antigen of the LPS.     

Long-term haemodynamic effects of amlodipine at rest and during exercise in essential hypertension

The long-term haemodynamic responses to amlodipine, a new long-acting calcium antagonist, were studied both at rest and during exercise in 18 patients (mean age 43 years) with essential hypertension. Blood pressure was measured intra-arterially, cardiac output by dye dilution and heart rate by electrocardiogram. After 11 months of treatment with 5-10 mg amlodipine once daily (mean dose 9 mg/day), mean arterial pressure was reduced by 14% sitting at rest. The reduction in blood pressure was associated with a marked reduction in the total peripheral resistance index (TPRI) of 19% (P less than 0.001). Similar responses were seen supine at rest and during 50W, 100W and 150W bicycle exercise. No significant changes were seen in heart rate. There was a slight increase in stroke index, and cardiac index was preserved at rest and during exercise with a slight trend towards an increase. In 10 of the patients, blood pressure was monitored by a portable blood pressure recorder (Accutracker II, Suntech Medical instruments, Raleigh, North Carolina, USA). Blood pressure was well controlled throughout the full 24 h period after one daily dose. In conclusion, amlodipine exerts a clear antihypertensive effect, both at rest and during exercise, through reduction in the TPRI and without a fall in cardiac pump function. No changes in heart rate were seen and there was no tendency for a reduction in the stroke index during 8 min of exercise at 150 W; on the contrary there was a trend towards an increase. The incidence of side-effects was low (ankle oedema in two patients).     

In vitro characterization of GS-8374, a novel phosphonate-containing inhibitor of HIV-1 protease with a favorable resistance profile

GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (K_i = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4⁺ T cells (50% effective concentration [EC₅₀] = 3.4 to 11.5 nM), and macrophages (EC₅₀ = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC₅₀s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.Fifteen years ago, HIV protease (PR) inhibitors (PIs) were introduced into the clinic as a second class of antiretrovirals, after nucleosides, and launched the era of combination antiretroviral therapy (ART) that brought along a dramatic reduction of the morbidity and mortality among HIV-infected patients (7, 25, 29, 46). PIs evolved to be an important class of agents that are being widely used in combination with other antiretrovirals in both treatment-naïve and -experienced patients (48). On the basis of recent revisions of HIV treatment guidelines, one of several ritonavir-boosted PIs is recommended for use as a third agent of choice in combination with tenofovir and emtricitabine for first-line ART (8, 53). The choice of PIs over other antiretroviral agents is primarily driven by their clinical potency and a higher genetic barrier for resistance development (48). In addition, the clinical use of more recently developed PIs with improved resistance profiles, e.g., darunavir, in combination with new antiretrovirals may represent a promising nucleoside-sparing option for highly treatment-experienced patients (13, 50).Although a total of nine PIs is currently available for the treatment of HIV infection, only a few are widely used. In general, the long-term clinical benefit of PIs across all patient populations can be limited by various factors, including long-term safety and tolerability (3, 27, 38), resistance (36), and drug-drug interactions (18). Among these limitations, the development of viral resistance has been shown to be a major cause of therapy failure (1, 43). Several studies revealed that a significant proportion of patients with detectable viral loads harbor HIV strains resistant to at least one PI (36, 44). Furthermore, transmission of resistant viruses, including strains with reduced susceptibility to approved PIs, has been documented and may limit the choices for the first-line therapy (17, 47). The structural similarity among the multiple PIs used in the clinic increases the possibility of cross-resistance within this therapeutic class (52). Consequently, the mutations conferring resistance are frequently common to multiple PIs (14, 45). Therefore, the design of novel PIs with more favorable resistance profiles and improved pharmacological properties remains an area of high interest.Over more than 2 decades of intense development, HIV therapy became a complex and quickly evolving field of medical research. Novel therapeutic concepts and regimens using both the established and new antiretroviral drug classes are being explored with a primary goal to address limitations of ART in various patient populations. Increasing age of HIV-infected patients brings about additional challenges, such as long-term effects of HIV infection and tolerability of ART (15). In addition, ongoing health care reforms generate pressure to reduce treatment costs, providing an incentive for exploring novel simplified treatment regimens. These evolving aspects of anti-HIV therapy create additional need for potent, durable, and well-tolerated antiretrovirals, including novel PIs.Being aware of both the unique qualities and limitations of PIs, we explored the design of novel compounds through the application of novel modifications to established PI chemotypes. Recently, we reported on inhibitors containing a phosphonate moiety and showed that in comparison with the parent scaffolds, the phosphonate-modified compounds exhibit improved activity against a limited panel of PI-resistant viruses (4). Based on these initial data, we further explored a range of di(alkyl)phosphonate substituents in the structural context of TMC-126, a previously described bis-tetrahydrofuran (bis-THF) peptidomimetic PI (9, 10). Here and in a parallel report (12) we describe the profiling of GS-8374 (Fig. ​(Fig.1),1), a novel diethylphosphonate derivative of TMC-126 that exhibits favorable pharmacological properties, including a resistance profile superior to the profiles of all clinically approved PIs.Open in a separate windowFIG. 1.Structure of GS-8374.     

Contribution of inherited mutations in the BRCA2-interacting protein PALB2 to familial breast cancer

Inherited mutations in the BRCA2-interacting protein PALB2 are known to be associated with increased risks of developing breast cancer. To evaluate the contribution of PALB2 to familial breast cancer in the United States, we sequenced the coding sequences and flanking regulatory regions of the gene from constitutional genomic DNA of 1,144 familial breast cancer patients with wild-type sequences at BRCA1 and BRCA2. Overall, 3.4% (33/972) of patients not selected by ancestry and 0% (0/172) of patients specifically of Ashkenazi Jewish ancestry were heterozygous for a nonsense, frameshift, or frameshift-associated splice mutation in PALB2. Mutations were detected in both male and female breast cancer patients. All mutations were individually rare: the 33 heterozygotes harbored 13 different mutations, 5 previously reported and 8 novel mutations. PALB2 heterozygotes were 4-fold more likely to have a male relative with breast cancer (P = 0.0003), 6-fold more likely to have a relative with pancreatic cancer (P = 0.002), and 1.3-fold more likely to have a relative with ovarian cancer (P = 0.18). Compared with their female relatives without mutations, increased risk of developing breast cancer for female PALB2 heterozygotes was 2.3-fold (95% CI: 1.5-4.2) by age 55 and 3.4-fold (95% CI: 2.4-5.9) by age 85. Loss of the wild-type PALB2 allele was observed in laser-dissected tumor specimens from heterozygous patients. Given this mutation prevalence and risk, consideration might be given to clinical testing of PALB2 by complete genomic sequencing for familial breast cancer patients with wild-type sequences at BRCA1 and BRCA2.     

Relationship between sick leave, unemployment, disability, and health-related quality of life in patients with inflammatory bowel disease

BACKGROUND: The goal of this study was to determine the rate of work disability, unemployment, and sick leave in an unselected inflammatory bowel disease (IBD) cohort and to measure the effect of working status and disability on the patient's health-related quality of life (HRQOL). MATERIALS AND METHODS: All eligible patients were clinically examined and interviewed at the 5-year follow-up visit. In addition, they completed the 2 HRQOL questionnaires, the Short Form-36 Health Survey (SF-36) and the Inflammatory Bowel Disease Questionnaire validated for use in Norway (N-IBDQ). Data regarding sick leave, unemployment, and disability pension (DP) also were collected. RESULTS: All together, 495 patients were or had been in the workforce during the 5-year follow-up period since diagnosis. Forty-two patients (8.5%) were on DP compared with 8.8% in the background population. Women with Crohn's disease (CD) had the highest probability of receiving DP (24.6%). A total of 58 patients (11.7%) reported they were unemployed at 5 years. This was equally distributed between men and women but was more frequent in patients with ulcerative colitis. Sick leave for all causes was reported in 47% with ulcerative colitis and 53% with CD, whereas IBD-related sick leave was reported in 18% and 23%, respectively. A majority (75%) had been sick <4 weeks, and a relatively small number of patients (25%) contributed to a large number of the total sick leave days. Both unemployment and DP reduced HRQOL scores, but the most pronounced effect on HRQOL was found in patients reporting IBD-related sick leave, measured with SF-36 and N-IBDQ. The observed differences also were highly clinically significant. Multiple regression analysis confirmed that IBD-related sick leave was the independent variable with the strongest association to the observed reduction in HRQOL scores. CONCLUSIONS: Unemployment or sick leave is more common in IBD patients than in the Norwegian background population. The number of patients receiving DP is significantly increased in women with CD but not in the other patient groups. Unemployment, sick leave, and DP are related to the patient's HRQOL in a negative way, but this effect is most pronounced in patients reporting IBD-related sick leave.     

A prospective study of same day bi-directional endoscopy in the evaluation of patients with occult gastrointestinal bleeding

OBJECTIVE: Previous studies show conflicting results in the diagnostic yield of oesophagogastroduodenoscopy (OGD) and colonoscopy (bi-directional) in identifying potential bleeding sources (PBS) in patients investigated for occult gastrointestinal bleeding (OGIB). The aims of this study were to evaluate the diagnostic yield of bi-directional endoscopy in patients presenting with OGIB and to assess the factors predictive of a positive yield. MATERIAL AND METHODS: Patients with OGIB referred to the gastroenterology unit were prospectively included in the study. Colonoscopy was immediately followed by OGD. Predetermined criteria for the diagnosis of a PBS were used. Potential clinical factors predictive of positive yield were assessed. RESULTS: Of the 219 patients (mean age 65 years, 34% M), 110 (50%) had at least one PBS. Colonoscopy revealed 87 PBS in 73 patients (33%), the most common being adenoma. OGD detected 49 PBS in 48 patients (22%), gastric ulcer being the most common. Of the patients with PBS at OGD, 23% also had a PBS at colonoscopy, including 3 patients with colonic cancers. Patients presenting with either a positive faecal occult blood test (FOBT) or iron-deficiency anaemia (IDA) only had a significantly lower yield for PBS than patients with combined positive FOBT and IDA. The percentages of patients with a PBS increased substantially with age. CONCLUSIONS: A PBS was found in only 50% of the patients. Colonoscopy had a slightly higher diagnostic yield, and as expected, resulted in a significantly higher cancer detection rate than OGD. In older patients, colonoscopy should be done irrespective of the findings at OGD. Gastrointestinal-specific symptoms and the use of ASA/NSAIDs were not predictive in finding or localizing PBS.