Depleting regulatory T cells with arginine-rich, cell-penetrating, peptide-conjugated morpholino oligomer targeting FOXP3 inhibits regulatory T-cell function.

CD4+CD25+regulatory T cells (T(reg)) impair anti-tumor and anti-viral immunity. As there are higher T(reg) levels in cancer patients compared with healthy individuals, there is considerable interest in eliminating them or altering their function as part of cancer or viral immunotherapy strategies. The scurfin transcriptional regulator encoded by the member of the forkhead winged helix protein family (FOXP3) is critical for maintaining the functions of T(reg). We hypothesized that targeting FOXP3 expression with a novel arginine-rich, cell-penetrating, peptide-conjugated phosphorodiamidate morpholino (PPMO) based antisense would eliminate T(reg) and enhance the induction of effector T-cell responses. We observed that the PPMO was taken up by activated T cells in vitro and could downregulate FOXP3 expression, which otherwise increases during antigen-specific T-cell activation. Generation of antigen-specific T cells in response to peptide stimulation was enhanced by pre-treatment of peripheral blood mononuclear cells with the FOXP3-targeted PPMO. In summary, modulation of T(reg) levels using the FOXP3 PPMO antisense-based genomic strategy has the potential to optimize immunotherapy strategies in cancer and viral immunotherapy.

National estimates of the characteristics of individuals infected with HIV who are likely to report and receive treatment for painful bleeding gums.

Painful bleeding gums may be associated with HIV infection. This study examined the characteristics of persons reporting the symptom "painful bleeding gums" and their likelihood of accessing care. The study population consisted of persons receiving care for HIV as part of the HIV Cost and Services Utilization Study (HCSUS). In this national population, 5.3% reported painful bleeding gums. Significant differences in reporting painful bleeding gums were found between Hispanics/Whites, oral health status, and presence of other symptoms. Compared to younger persons, those in the middle age group were more likely to seek treatment, while persons with the highest CD4 counts were more likely to seek treatment than those with the lowest CD4 counts. This study showed that reporting painful bleeding gums was a function of ethnicity, other symptoms, and perceived oral health, while seeking treatment for painful bleeding gums was related to age and CD4 counts. Dentists and other health care providers can have an active role in improving the quality of life of persons living with HIV by being aware of the relationships that exist between patients with HIV and painful bleeding gums.

Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration.

BACKGROUND: Inflammatory breast cancer (IBC) is an aggressive subtype of breast cancer with distinct molecular profiles. Gene expression profiling previously identified sonic hedgehog (SHH) as part of a gene signature that is differentially regulated in IBC patients. METHODS: The effects of reducing GLI1 levels on protein expression, cell proliferation, apoptosis and migration were determined by immunoblots, MTT assay, Annexin-V/PI assay and conventional and automated cell migration assays. RESULTS: Evaluation of a panel of breast cancer cell lines revealed elevated GLI1 expression, typically a marker for hedgehog-pathway activation, in a triple-negative, highly invasive IBC cell line, SUM149 and its isogenic-derived counterpart rSUM149 that has acquired resistance to ErbB1/2 targeting strategies. Downregulation of GLI1 expression in SUM149 and rSUM149 by small interfering RNA or a small molecule GLI1 inhibitor resulted in decreased proliferation and increased apoptosis. Further, GLI1 suppression in these cell lines significantly inhibited cell migration as assessed by a wound-healing assay compared with MCF-7, a non-invasive cell line with low GLI1 expression. A novel high-content migration assay allowed us to quantify multiple effects of GLI1 silencing including significant decreases in cell distance travelled and linearity of movement. CONCLUSION: Our data reveal a role for GLI1 in IBC cell proliferation, survival and migration, which supports the feasibility of targeting GLI1 as a novel therapeutic strategy for IBC patients.

Seroepidemiology of hepatitis B virus infection: analysis of mass screening in Hawaii.

BACKGROUND: Although hepatitis B seroprevalence has been studied extensively in California and New York, detailed information for other high-risk areas in the United States is lacking. To study current prevalence and risk for hepatitis B virus (HBV) infection in Hawaii, we analyzed cross-sectional data from Hawaii residents screened between July 2003 and April 2006. METHODS: We retrospectively reviewed the screening records of 3,989 participants recruited at health fairs and clinics. Prevalence and risk factors for HBV infection were estimated using univariate and multivariate logistic regression models. RESULTS: Total prevalence of hepatitis B surface antigen (HBsAg) was 3.6%. Gender, age, and ethnicity were independently associated with HBsAg seropositivity. In a multivariate logistic regression model, males were at increased risk for HBsAg compared with females (odds ratio [OR] = 1.53, 95% confidence interval [CI]: 1.09-2.16) and persons aged 70 years or older were less likely to test positive than those younger than 30 (OR = 0.25, 95% CI: 0.11-0.61). In addition, multivariate ORs of HBsAg seropositivity were 3.24 (95% CI: 1.04-10.09), 4.13 (95% CI: 1.66-10.29), and 7.47 (95% CI: 2.52-22.11) for Vietnamese, Chinese, and Pacific Islanders, respectively, compared with Whites. CONCLUSIONS: This study furthers current knowledge of HBV epidemiology in areas with large populations of high-risk immigrants and demonstrates the relevance of screening programs for hepatitis B.

The distribution of scrapie-associated fibrils in neural and non-neural tissues of advanced clinical cases of natural scrapie in sheep.

The distribution of scrapie-associated fibrils (SAFs) throughout four brain regions, the pituitary gland, along the whole length of the spinal cord and in the sciatic nerve was assessed in 10 sheep terminally affected by scrapie and in four control sheep. Tonsils, retropharyngeal, broncho-mediastinal and mesenteric lymph nodes, the distal ileum, proximal colon and spleen were also examined for fibrils in all 14 sheep. Fibrils were detected in all four brain regions and throughout the length of the spinal cord in nine of the scrapie affected sheep. SAFs were not detectable in any of the sciatic nerve samples tested. In one of the 10 clinically affected sheep only minimal lesions were found by histopathology and fibrils were detected only from the cerebrum and one spinal cord region (taken at the C1 C2 vertebrae). Fibrils were not detected in the tonsils or retropharyngeal lymph nodes but were detected in other non-neural tissues of some of the scrapie-affected sheep. These tissues included pituitary gland, broncho-mediastinal and mesenteric portal lymph nodes, distal ileum, proximal colon and spleen. Fibrils could not be detected in any of the tissues taken from the four control sheep.

Scrapie associated fibril detection from formaldehyde fixed brain tissue in natural cases of ovine scrapie.

The medulla oblongata of the brains of 71 scrapie-suspect cases were routinely fixed in 10 per cent formal saline and assessed for vacuolation on HE-stained sections. A pool of fresh brain material was also dissected from each animal and extracts prepared for the routine detection of scrapie-associated fibrils by negative stain transmission electron microscopy. The remaining formaldehyde fixed medulla samples, which were not used for the histological examination, were coded and subjected to a pretreatment with sodium borohydride and then processed using the routine fibril detection procedure. Of the 71 samples tested 46 were considered positive by all three test procedures. Sixteen samples were negative for all three tests. Four samples were positive by histopathological examination and positive for fibrils using fresh tissue, but fibrils could not be detected in the fixed tissue preparations. Conversely, there were five fixed samples in which fibrils could be detected which were negative for the other two tests. The fibrils observed in fixed preparations were indistinguishable from those observed in fresh tissue extracts. The sensitivity of the test for fibril detection using fixed tissue was 92 per cent and the specificity 76 per cent. It is concluded that scrapie-associated fibrils can be recovered from formaldehyde fixed tissue, as presented for routine histopathological examination, and therefore the method has potential in the retrospective analysis of archived brain tissue where only fixed material was stored.

Comparison of detergent and protease enzyme combinations for the detection of scrapie-associated fibrils from the central nervous system of sheep naturally affected with scrapie.

Standardized samples of tissue from the central nervous system of four sheep naturally affected with scrapie and from four healthy control sheep were subjected to a centrifugal extraction technique used to obtain scrapie-associated fibrils; the latter were then demonstrated by negative-contrast transmission electron microscopy. This regime was used to evaluate the fibril yield obtained from the 25 possible combinations of five different detergents and five different proteolytic enzymes. N-lauroylsarcosine detergent was found to be the most efficient detergent for all five enzymes, followed by sulphabetaine 3-14. Sodium dodecyl sulphate detergent was successful only in combination with a subtilisin Carlsberg enzyme. Octylglucoside and nonidet P40 detergents did not produce fibrils with any of the enzymes. Proteinase K was the least efficient of the five enzymes when used in combination with N-lauroylsarcosine; subtilisin Carlsberg, clostripain, pronase and trypsin enzymes all gave higher fibril yields. A combination of N-lauroylsarcosine detergent and subtilisin Carlsberg proteolytic enzyme gave the highest fibril yield.

Comparison of biochemical extraction techniques for the detection of scrapie-associated fibrils in the central nervous system of sheep naturally affected with scrapie.

Standardized samples of brain material from four sheep naturally affected with scrapie and from four healthy control sheep were subjected to six different extraction techniques used for the detection of scrapie-associated fibrils by negative-contrast transmission electron microscopy. The six methods were compared in respect of fibril yield and clarity of ultrastructure. The simplest method consisting of a single N-lauroylsarcosine detergent extraction and differential centrifugation, followed by proteinase K enzyme digestion, gave the best overall results. The use of proteinase and nuclease inhibitors made no apparent difference to the yield or ultrastructural clarity of fibrils. Density gradient centrifugation appeared to reduce tungstate stain penetration and often obscured the ultrastructural clarity. The results suggested that the preferred technique could be improved by the use of a double homogenization stage at the beginning of the procedure and by adding an ultrasonic disintegration step to resuspend the final pellet prior to tungstate staining.

Comparative study of electron microscopical techniques for the detection of scrapie-associated fibrils.

Samples of cervical spinal cord and four anatomical regions of the brains of 12 sheep with natural scrapie and six control sheep were examined by electron microscopy, after the tissues had been stored at 4 degrees C and -20 degrees C. The tissues were tested for the presence of scrapie-associated fibrils by a centrifugal extraction technique and by a touch-grid technique. The touch-grid technique was no better than the centrifugal extraction technique for the detection of fibrils. Structures which could have been classified as tubulofilaments were detected in touch-grid preparations without detergent treatment. With the centrifugal extraction technique there was a significant reduction of the fibril scores in some of the tissue extracts stored at -20 degrees C, but not in any of the extracts stored at 4 degrees C. There was, however, a reduction in the fibril scores when the final extracted pellets were stored at 4 degrees C. The stability of the fibrils on the test grids was unaffected by six months storage at room temperature but the clarity of their ultrastructure did deteriorate. Poor hydrophilic spread of the sample on the test grids did not have a significant effect on the fibril scores.

Effect of interferon-gamma on the 5-lipoxygenase pathway of rat lung macrophages.

In view of conflicting reports concerning the effect of macrophage activation on arachidonic acid metabolism, we examined the effect of the macrophage activator, interferon-gamma (IFN-gamma), on the 5-lipoxygenase pathway in rat lung macrophages. Rat lung macrophages were conditioned in the presence or absence of 10(2) U/ml IFN-gamma for 4 h before stimulation with 1 microM A23187 for 15 min or 100 micrograms/ml opsonized zymosan for 60 min at 37 degrees C as well as other stimuli. Lipoxygenase products in extracted cell supernatants were identified and analyzed by high-pressure liquid chromatography and ultraviolet spectroscopy. The predominant lipoxygenase products included leukotriene (LT) B4, LTC4, and 5-hydroxyeicosatetraenoic acid (5-HETE). These products were not qualitatively altered by conditioning with IFN-gamma. However, 5-lipoxygenase pathway activity, as measured by LTB4 release, was maximally increased 2-fold after conditioning with IFN-gamma and stimulating with either A23187 or opsonized zymosan. IFN-gamma-conditioned macrophages, stimulated with A23187, released greater quantities of lipoxygenase products in comparison with control cells (307.6 +/- 13.3 versus 167.6 +/- 3.9 pmol LTB4/10(6) cells) (mean +/- SEM) (P less than 0.05). Similar results were obtained with the less potent stimulus, opsonized zymosan. IFN-gamma had no direct stimulatory effect on the 5-lipoxygenase pathway. No effect was observed with a variety of other stimuli with or without IFN-gamma conditioning.(ABSTRACT TRUNCATED AT 250 WORDS)

Community care for late adolescents.

This paper describes supportive accommodation for late adolescents in Cambridge provided by the Castle Project in conjunction with the Young People's Psychiatric Service. It outlines the characteristics of young people referred to the Project over the first years of its existence, their lengths of stay and next moves, and suggests that there are members of two groups, those with schizophrenic-type disorders and those with pronounced antisocial behaviour, who present long-term problems of care and accommodation and whose needs are not yet catered for appropriately.