Susceptibility of diverse primary HIV isolates with varying co-receptor specificity's to CXCR4 antagonistic compounds

The chemokine receptors CCR5 and CXCR4 are an obvious target for HIV therapies. Two compounds, T-22 and AMD-3100, have been shown to inhibit infection of CXCR4-using HIV-1 isolates. The specificity of T-22 and AMD-3100 was further confirmed by their ability to block entry of HIV-1 in GHOST-CXCR4 transfected cells with no effect on viral entry in the GHOST-CCR5 cells. The ability of T-22 to block replication of diverse HIV-1 isolates (group M, subtypes A, B, D, E, and F as well as group O) and HIV-2 primary isolates with varying coreceptor specificities ranging from exclusive CCR5 usage to multiple coreceptor usage was examined in detail. T-22 was found to be highly effective (>90%) at blocking infection of diverse HIV-1 (subtypes A-F, and group O) and HIV-2 isolates that use multiple coreceptors in human PBMCs homozygous for a 32-bp deletion in CCR5 (CCR5-/-), but less effective in CCR5 +/+ PBMCs. Additionally, sequential primary HIV-1 isolates obtained from a longitudinal cohort who had switched from single coreceptor usage to a broad range of multiple receptors could be blocked effectively by both T-22 and AMD-3100 in CCR5-/- PBMCs. Our data suggest that CXCR4 antagonistic compounds are highly effective in blocking the entry of X4-tropic HIV-1, and that these compounds could be a useful additive to current anti-retroviral therapy for clinical management of HIV disease.     

Enhancing a primary care environment: a case study of effects on patients and staff in a single general practice

BACKGROUND: Few studies have examined the effect on patients and staff of the physical environment in primary care facilities. AIM: To explore changes in patient and staff satisfaction, patient anxiety, and patient-doctor communication when a GP surgery moves from old premises to enhanced purpose-built accommodation. DESIGN OF STUDY: Questionnaire surveys, interviews, and focus groups pre- and post move. SETTING: An urban general practice in Bristol. METHOD: Patient questionnaires assessed anxiety (Spielberger State-Trait Anxiety Inventory; STAI), satisfaction with the environment, and communication during the consultation. Staff questionnaires assessed satisfaction with the environment and job satisfaction. Qualitative methods explored patient and staff views in more depth. RESULTS: A total of 1118 pre-move and 954 post-move patient questionnaires showed significant increases in satisfaction scores for reception/waiting areas (mean 6.46, 95% confidence interval [CI]=5.97 to 6.95) and consulting rooms (mean 3.80, 95% CI=3.44 to 4.15) in the new premises. Patients' satisfaction with patient-doctor communication also increased (mean 0.88, 95% CI=0.30 to 1.46) and anxiety scores were significantly reduced before and after the consultation in the new premises compared with the old (STAI mean difference before consultation 0.72, 95% CI=0.37 to 1.08; mean after consultation 0.37, 95% CI=0.03 to 0.72). Patients highlighted the increased space and light, more modern appearance, greater comfort, and novel works of art in the new surgery. Staff workplace satisfaction increased significantly after moving and remained higher than in the old building. CONCLUSION: This large-scale study examining the effects of a UK primary care environment on patients and staff shows that an enhanced environment is associated with improvements in patients' perception of patient-doctor communication, reduction in anxiety, and increases in patient and staff satisfaction.     

Pattern and predictors of weight gain during pregnancy among HIV-1-infected women from Tanzania

Progression of HIV disease is often accompanied by weight loss and wasting. Gestational weight gain is a strong determinant of maternal and neonatal outcomes; however, the pattern and predictors of weight gain during pregnancy among HIV-positive women are unknown. We obtained monthly anthropometric measurements in a cohort of 957 pregnant women from Tanzania who were HIV infected. We estimated the weekly rate of weight gain at various points during the second and third trimesters of pregnancy and computed rate differences between levels of sociodemographic, nutritional, immunologic, and parasitic variables at the first prenatal visit. The change in mid-upper arm circumference (MUAC) from baseline to delivery was also examined. The rate of weight gain decreased progressively during pregnancy. There was an average decline of 1 cm in MUAC between weeks 12 and 38. Lower level of education and helminthic infections at first visit were associated with decreased adjusted rates of weight gain during the third trimester. High baseline MUAC, not contributing to household income, lower serum retinol and selenium concentrations, advanced clinical stage of HIV disease, and malaria infection were related to decreased rates of weight gain during the second trimester. Low baseline CD4 T-cell counts were related to a poorer pattern of weight gain throughout pregnancy. Prevention and treatment of parasitic infections and improvement of nutritional status are likely to enhance the pattern of gestational weight gain among HIV-infected women.     

Construction and characterization of affibody-Fc chimeras produced in Escherichia coli

Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human IgG. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture. Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of "artificial antibodies" was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such "artificial antibodies" should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.     

Quantitative detection of Borrelia burgdorferi in 2-millimeter skin samples of erythema migrans lesions: correlation of results with clinical and laboratory findings

Variability of disease manifestations has been noted in patients with Lyme disease. A contributing factor to this variation may be the number of spirochetes present in infected patients. We evaluated clinical and laboratory findings for patients with erythema migrans with regard to the number of Borrelia burgdorferi organisms detected by quantitative PCR (qPCR) in 2-mm skin biopsy specimens. B. burgdorferi was detected in 80% (40 of 50) of the specimens tested; the mean number of spirochetes in these specimens ranged over 3 orders of magnitude (10 to 11,000 spirochetes per 2-mm biopsy specimen). Larger numbers of spirochetes were significantly associated with a shorter duration of the erythema migrans skin lesion (P = 0.020), smaller skin lesions (P = 0.020), and infection with a specific genotype of B. burgdorferi (P = 0.008) but not with the number or severity of symptoms. Skin culture positivity was significantly associated with skin lesions containing larger numbers of spirochetes (P = 0.019).     

Standardized BACTEC Method To Measure Clarithromycin Susceptibility of Mycobacterium genavense

A standardized clarithromycin susceptibility test for Mycobacterium genavense is reported. The BACTEC radiometric broth dilution test method recommended for Mycobacterium avium complex was modified to develop a reliable and reproducible procedure. Test development involved optimization of medium pH and inoculum densities for antibiotic vials as well as growth control vials. MIC control organisms included Mycobacterium simiae, Mycobacterium avium, and Mycobacterium xenopi. Growth control vials required two to three inoculum dilutions, which varied for each species. Clarithromycin MICs and MBCs for 12 isolates and 1 colonial variant of M. genavense ranged from ≤0.06 to 0.25 μg/ml.     

Study of discharge communications from hospital doctors to an inner London general practice

In inner London patients are now being discharged from hospital earlier to be cared for by the community services. In this study the general practitioners in one inner London practice were asked to evaluate discharge communications from hospital doctors. The general practitioners were dissatisfied with the delay in receiving over one third of the letters and with the content of almost a fifth. They also felt that delay and lack of detail affected their management in 24% of cases. They would have liked more information in the letters, particularly about drug regimens. Some suggestions for improvement in written discharge communications are made.     

Neuroimaging of NREM sleep in primary insomnia: a Tc-99-HMPAO single photon emission computed tomography study

STUDY OBJECTIVES: The objectives of this study were to: 1) demonstrate the feasibility of combining polysomnography and SPECT neuroimaging to study NREM sleep in primary insomnia and 2) evaluate possible functional CNS abnormalities associated with insomnia. DESIGN: Patients with insomnia and good sleeper controls were studied polysomnographically for three nights with a whole brain SPECT Scan of NREM sleep on Night 3. Groups were screened for medical/psychiatric history, substance use, and matched on age, body mass index, and education. SETTING: Sleep Research Laboratory and Nuclear Medicine Center PARTICIPANTS: Nine females, 5 patients with chronic psychophysiologic insomnia and 4 healthy good sleepers (mean age 36 years, SD 12, range 27-55). INTERVENTIONS: N/A MEASUREMENTS AND RESULTS: Tomographs of regional cerebral blood flow during the 1st NREM sleep cycle were successfully obtained. Contrary to our expectations, patients with insomnia showed a consistent pattern of hypoperfusion across all 8 pre-selected regions of interest, with particular deactivation in the basal ganglia (p=.006). The frontal medial, occipital, and parietal cortices also showed significant decreases in blood flow compared to good sleepers (p<.05). Subjects with insomnia had decreased activity in the basal ganglia relative to the frontal lateral cortex, frontal medial cortex, thalamus, occipital and parietal cortices (p<.05). CONCLUSIONS: This study demonstrated the feasibility of combining neuroimaging and polysomnography to study cerebral activity in chronic insomnia. These preliminary results suggest that primary insomnia may be associated with abnormal central nervous system activity during NREM sleep that is particularly linked to basal ganglia dysfunction.     

Comparison of Two Culture Methods for Detection of Tobramycin-Resistant Gram-Negative Organisms in the Sputum of Patients with Cystic Fibrosis

A culture method utilizing quantitative plating on antibiotic-containing media has been proposed as a technique for the detection of tobramycin-resistant organisms that is more sensitive than standard methods. Typical sputum culture methods quantitate the relative amounts of each distinct morphotype, followed by antibiotic susceptibility testing of a single colony of each morphotype. Sputum specimens from 240 cystic fibrosis patients were homogenized, serially diluted, and processed in parallel by the standard method (MacConkey agar and OF basal medium with agar, polymyxin, bacitracin, and lactose) and by plating on antibiotic-containing media (MacConkey agar with tobramycin added at 25 microg/ml [MAC-25] and 100 microg/ml [MAC-100]). MICs of tobramycin were determined for all Pseudomonas aeruginosa isolates by broth microdilution. Growth of P. aeruginosa on MAC-25 was considered to be equivalent to a tobramycin MIC of > or = 16 microg/ml, and growth on MAC-100 was considered to be equivalent to a tobramycin MIC of > or = 128 microg/ml. Analysis of method-specific detection rates showed that tobramycin-containing medium was more sensitive than the standard method for the detection of tobramycin-resistant P. aeruginosa, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans but was less sensitive for the detection of Burkholderia cepacia than the standard method. When MICs for P. aeruginosa that grew on tobramycin-containing medium were tested by broth microdilution, the MICs for 28 of 121 strains (23%) growing on MAC-25 and 22 of 56 strains (39%) growing on MAC-100 were MICs < 16 and < 128 microg/ml, respectively. Addition of a tobramycin-containing MacConkey plate to the routine media for sputum culture may provide additional, clinically relevant microbiologic data.     

Physicians' weight loss counseling in two public hospital primary care clinics.

PURPOSE: Primary care physicians are an important source of information on weight management. Nevertheless, weight loss counseling by these physicians remains inadequate. This study sought to determine physicians' barriers to providing weight loss counseling in a public hospital, patients' recall of physicians' weight loss recommendations, and the influence of physicians' counseling on patients' understanding, motivation, and behavior regarding weight loss. METHOD: In 2001, four focus groups of faculty and residents were held at two primary care clinics affiliated with the Louisiana State University Health Sciences Center-Shreveport to determine the barriers to providing weight loss counseling. Scripted probes were used to uncover consensus norms. In 2001-02, structured exit interviews were conducted with 210 overweight or obese patients recruited from the clinics to determine patients' recall of physicians' weight loss recommendations, and patients' understanding of the relationship between weight and health, and their stages of readiness for weight loss. RESULTS: Physicians identified major barriers to providing weight loss counseling, including insufficient confidence, knowledge, and skills. Obesity was underdocumented as a distinct clinical diagnosis. Only 5% of the patients recalled being given the combined weight loss strategy of diet and exercise. However, patients who recalled being counseled to lose weight were more likely to understand the risks of obesity, the benefits of weight loss, and were at a higher stage of readiness for weight loss. CONCLUSIONS: Physicians' weight loss counseling had a significant effect on patients' understanding of and motivation for weight loss. However, physicians provided insufficient guidance on weight management strategies, possibly because of inadequate counseling skills and confidence.