Dissecting the complexity of the memory T cell response

Memory immune responses are classically attributed to the reactivation of long-lived, antigen-specific T lymphocytes that persist in a quiescent state. Determining mechanisms for the generation of memory T cells and dissecting the functional nature of the memory T cell pool has been encumbered by an inability to distinguish recently activated effector T cells from memory T cells. We have established new activation and biochemical criteria that distinguish effector and memory T cells and have applied these criteria to follow memory generation from activated cells in vivo. We found that the resultant memory T cell pool is heterogeneous and consists of effector-like and resting memory-like subsets that differ in expression of the homing receptor, CD62L. We discuss these findings in the context of memory T cell heterogeneity identified in human and mouse systems. These results suggest that more than one type of previously activated T cell can mediate recall or memory immune responses and that elucidating the fundamental phenotypic and functional features of memory T cell subsets is therefore critical to deciphering the complex nature of the memory immune response.     

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.     

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.     

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.     

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.     

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.     

Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity

Defects in death receptor-mediated apoptosis have been linked to cancer and autoimmune disease in humans. The in vivo role of caspase 8, a component of this pathway, has eluded analysis in postnatal tissues because of the lack of an appropriate animal model. Targeted disruption of caspase 8 is lethal in utero. We generated mice with a targeted caspase 8 mutation that is restricted to the T-cell lineage. Despite normal thymocyte development in the absence of caspase 8, we observed a marked decrease in the number of peripheral T-cells and impaired T-cell response ex vivo to activation stimuli. caspase 8 ablation protected thymocytes and activated T-cells from CD95 ligand but not anti-CD3-induced apoptosis, or apoptosis activated by agents that are known to act through the mitochondria. caspase 8 mutant mice were unable to mount an immune response to viral infection, indicating that caspase 8 deletion in T-cells leads to immunodeficiency. These findings identify an essential, cell-stage-specific role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. This is consistent with the recent identification of caspase 8 mutations in human immunodeficiency.     

Vascularization and tissue infiltration of a biodegradable polyurethane matrix

Urethanes are frequently used in biomedical applications because of their excellent biocompatibility. However, their use has been limited to bioresistant polyurethanes. The aim of this study was to develop a nontoxic biodegradable polyurethane and to test its potential for tissue compatibility. A matrix was synthesized with pentane diisocyanate (PDI) as a hard segment and sucrose as a hydroxyl group donor to obtain a microtextured spongy urethane matrix. The matrix was biodegradable in an aqueous solution at 37 degrees C in vitro as well as in vivo. The polymer was mechanically stable at body temperatures and exhibited a glass transition temperature (Tg) of 67 degrees C. The porosity of the polymer network was between 10 and 2000 microm, with the majority of pores between 100 and 300 microm in diameter. This porosity was found to be adequate to support the adherence and proliferation of bone-marrow stromal cells (BMSC) and chondrocytes in vitro. The degradation products of the polymer were nontoxic to cells in vitro. Subdermal implants of the PDI-sucrose matrix did not exhibit toxicity in vivo and did not induce an acute inflammatory response in the host. However, some foreign-body giant cells did accumulate around the polymer and in its pores, suggesting its degradation is facilitated by hydrolysis as well as by giant cells. More important, subdermal implants of the polymer allowed marked infiltration of vascular and connective tissue, suggesting the free flow of fluids and nutrients in the implants. Because of the flexibility of the mechanical strength that can be obtained in urethanes and because of the ease with which a porous microtexture can be achieved, this matrix may be useful in many tissue-engineering applications.     

Ultrastructural morphometry of capillary basement membrane thickness in normal and transgenic diabetic mice

Capillary basement membrane (CBM) thickening is an ultrastructural hallmark in diabetic patients and in animal models of diabetes. However, the wide variety of tissues sampled and diverse methods employed have made the interpretation of thickness data difficult. We showed previously that acellular glomerular BMs in OVE26 transgenic diabetic mice were thickened beyond normal age-related thickening, and in the current study we hypothesized that other microvascular BMs likewise would show increased widths relative to age-matched controls. Accordingly, a series of tissues, including skeletal and cardiac muscle, ocular retina and choriod, peripheral nerve, lung, pancreas, and renal glomerulus was collected from 300-350-day-old normal and transgenic mice. Transmission electron micrographs of cross sections through capillary walls were prepared, and CBM thickness (CBMT) was determined by the "orthogonal intercept" method. Morphometric analyses showed highly variable transgene-related BMT increases in the sampled tissues, with glomerular BM showing by far the greatest increase (+87%). Significant thickness increases were also seen in the retina, pulmonary alveolus, and thoracoabdominal diaphragm. BMT increases were not universal; however, most were modestly widened, and those that were thickest in controls generally showed the greatest increase. Although the pathogenesis of diabetes-related increases in CBM is poorly understood, data in the current study showed that in OVE26 transgenic mice increased BMT was a frequent concomitant of hyperglycemia. Accordingly, it seems likely that hyperglycemia-induced microvascular damage may be a contributing factor in diabetic BM disease, and that microvessel cellular and extracellular heterogeneity may limit the extent of CBM thickening in diverse tissues.