Molecular mechanisms underlying responses of the Antarctic coral Malacobelemnon daytoni to ocean acidification.

Benthic organisms of the Southern Ocean are particularly vulnerable to ocean acidification (OA), as they inhabit cold waters where calcite-aragonite saturation states are naturally low. OA most strongly affects animals with calcium carbonate skeletons or shells, such as corals and mollusks. We exposed the abundant cold-water coral Malacobelemnon daytoni from an Antarctic fjord to low pH seawater (LpH) (7.68 ± 0.17) to test its physiological responses to OA, at the level of gene expression (RT-PCR) and enzyme activity. Corals were exposed in short- (3 days) and long-term (54 days) experiments to two pCO2 conditions (ambient and elevated pCO2 equaling RCP 8.5, IPCC 2019, approximately 372.53 and 956.78 µatm, respectively). Of the eleven genes studied through RT-PCR, six were significantly upregulated compared with control in the short-term in the LpH condition, including the antioxidant enzyme superoxide dismutase (SOD), Heat Shock Protein 70 (HSP70), Toll-like receptor (TLR), galaxin and ferritin. After long-term exposure to low pH conditions, RT-PCR analysis showed seven genes were upregulated. These include the mannose-binding C-Lectin and HSP90. Also, the expression of TLR and galaxin, among others, continued to be upregulated after long-term exposure to LpH. Expression of carbonic anhydrase (CA), a key enzyme involved in calcification, was also significantly upregulated after long-term exposure. Our results indicated that, after two months, M. daytoni is not acclimatized to this experimental LpH condition. Gene expression profiles revealed molecular impacts that were not evident at the enzyme activity level. Consequently, understanding the molecular mechanisms behind the physiological processes in the response of a coral to LpH is critical to understanding the ability of polar species to cope with future environmental changes. Approaches integrating molecular tools into Antarctic ecological and/or conservation research make an essential contribution given the current ongoing OA processes.

Ocean acidification effects on the stress response in a calcifying antarctic coastal organism: The case of Nacella concinna ecotypes.

Ocean acidification (OA) could become a serious threat for the Antarctic marine ecosystem over coming years, as the solubility of atmospheric CO2 and CaCO3 minerals increases at lower temperatures. We evaluated the effect of OA on the stress response of the limpet Nacella concinna by measuring gene expression levels. The experiment was performed with the two ecotypes (Littoral and Sublittoral) of the species during 54 days (IPCC, 2019 scenario RCP8.5; control, ~375 ppm; low-pH treatment, ~923 ppm). Exposure to low-pH treatment during 15 days triggered the down-regulation of two heat-shock protein genes (HSP70A, HSP70B) only in sublittoral individuals. Little variation in the relative expression values of all genes in both ecotypes was observed probably, due to a historical exposure to the substantial daily natural pH fluctuations recorded in the study area during the experiment. This study provides relevant baseline data for future OA experiments on coastal species in Antarctica.

Assessment of treatment response in non-alcoholic steatohepatitis using advanced magnetic resonance imaging.

BACKGROUND: Magnetic resonance imaging-derived measures of liver fat and volume are emerging as accurate, non-invasive imaging biomarkers in non-alcoholic steatohepatitis (NASH). Little is known about these measures in relation to histology longitudinally. AIM: To examine any relationship between MRI-derived proton-density fat-fraction (PDFF), total liver volume (TLV), total liver fat index (TLFI), vs. histology in a NASH trial. METHODS: This is a secondary analysis of a 24-week randomised, double-blind, placebo-controlled trial of 50 patients with biopsy-proven NASH randomised to oral ezetimibe 10 mg daily (n = 25) vs. placebo (n = 25). Baseline and post-treatment anthropometrics, biochemical profiling, MRI and biopsies were obtained. RESULTS: Baseline mean PDFF correlated strongly with TLFI (Spearman's ρ = 0.94, n = 45, P < 0.0001) and had good correlation with TLV (ρ = 0.57, n = 45, P < 0.0001). Mean TLV correlated strongly with TLFI (ρ = 0.78, n = 45, P < 0.0001). After 24 weeks, PDFF remained strongly correlated with TLFI (ρ = 0.94, n = 45, P < 0.0001), maintaining good correlation with TLV (ρ = 0.51, n = 45, P = 0.0004). TLV remained strongly correlated with TLFI (ρ = 0.74, n = 45, P < 0.0001). Patients with Grade 1 vs. 3 steatosis had lower PDFF, TLV, and TLFI (P < 0.0001, P = 0.0003, P < 0.0001 respectively). Regression analysis of changes in MRI-PDFF vs. TLV indicates that 10% reduction in MRI-PDFF predicts 257 mL reduction in TLV. CONCLUSIONS: The MRI-PDFF and TLV strongly correlated with TLFI. Decreases in steatosis were associated with an improvement in hepatomegaly. Lower values of these measures reflect lower histologic steatosis grades. MRI-derived measures of liver fat and volume may be used as dynamic and more responsive imaging biomarkers in a NASH trial, than histology.

Development of a high resolution x-ray spectrometer for the National Ignition Facility (NIF).

A high resolution (E/ΔE = 1200-1800) Bragg crystal x-ray spectrometer is being developed to measure plasma parameters in National Ignition Facility experiments. The instrument will be a diagnostic instrument manipulator positioned cassette designed mainly to infer electron density in compressed capsules from Stark broadening of the helium-ß (1s2-1s3p) lines of krypton and electron temperature from the relative intensities of dielectronic satellites. Two conically shaped crystals will diffract and focus (1) the Kr Heß complex and (2) the Heα (1s2-1s2p) and Lyα (1s-2p) complexes onto a streak camera photocathode for time resolved measurement, and a third cylindrical or conical crystal will focus the full Heα to Heß spectral range onto an image plate to provide a time integrated calibration spectrum. Calculations of source x-ray intensity, spectrometer throughput, and spectral resolution are presented. Details of the conical-crystal focusing properties as well as the status of the instrumental design are also presented.

Magnetic resonance elastography identifies fibrosis in adults with alpha-1 antitrypsin deficiency liver disease: a prospective study.

BACKGROUND: Limited data exist on the clinical presentation and non-invasive detection of liver fibrosis in adults with homozygous Z genotype alpha-1 antitrypsin (AAT) deficiency. AIMS: To compare demographic, biochemical, histological and imaging data of AAT deficient patients to normal-control and biopsy-proven non-alcoholic fatty liver disease (NAFLD) patients, and to assess the diagnostic accuracy of magnetic resonance elastography (MRE) in detecting fibrosis in AAT deficiency. METHODS: Study includes 33 participants, 11 per group, who underwent clinical research evaluation, liver biopsy (AAT and NAFLD groups), and MRE. Histological fibrosis was quantified using a modified Ishak 6-point scale and liver stiffness by MRE. Diagnostic performance of MRE in detecting fibrosis was assessed by receiver operating characteristic (ROC) analysis. RESULTS: Mean (±s.d.) of age and BMI of normal-control, AAT and NAFLD groups was 57 (±19), 57 (±18), and 57 (±13) years, and 22.7 (±2.5), 24.8 (±4.0) and 31.0 (±5.1) kg/m(2) respectively. Serum ALT [mean ± s.d.] was similar within normal-control [16.4 ± 4.0] and AAT groups [23.5 ± 10.8], but was significantly lower in AAT than NAFLD even after adjustment for stage of fibrosis (P < 0.05, P = 0.0172). For fibrosis detection, MRE-estimated stiffness had an area under the ROC curve of 0.90 (P < 0.0001); an MRE threshold of ≥3.0 kPa provided 88.9% accuracy, with 80% sensitivity and 100% specificity to detect presence of any fibrosis (stage ≥1). CONCLUSIONS: This pilot prospective study suggests magnetic resonance elastography may be accurate for identifying fibrosis in patients with alpha-1 antitrypsin deficiency. Larger validation studies are warranted.

Non-invasive screening of diabetics in primary care for NAFLD and advanced fibrosis by MRI and MRE.

BACKGROUND: Current guidelines do not recommend screening for non-alcoholic fatty liver disease (NAFLD) or advanced fibrosis. Patients with type 2 diabetes mellitus (T2DM) are known to be at increased risk for NAFLD and advanced fibrosis. AIM: To assess the feasibility in diabetics in a primary care setting of screening for NAFLD and advanced fibrosis, by using non-invasive magnetic resonance imaging (MRI) to estimate the hepatic proton density fat fraction (MRI-PDFF) and magnetic resonance elastography (MRE) to estimate hepatic stiffness. METHODS: We performed a cross-sectional analysis of a prospective study that included 100 (53% men) consecutively enrolled diabetics who did not have any other aetiology of liver disease. All patients underwent a standardised research visit, laboratory tests, MRI-PDFF, and MRE. RESULTS: Mean (±s.d.) age and body mass index (BMI) was 59.7 (±11.2) years and 30.8 (±6.5) kg/m(2) , respectively. The prevalence of NAFLD (defined as MRI-PDFF ≥5%) and advanced fibrosis (defined as MRE ≥3.6 kPa) was 65% and 7.1%, respectively. One patient with advanced fibrosis had definite hepatocellular carcinoma. When compared to those without NAFLD, patients with NAFLD were younger (P = 0.028) and had higher mean BMI (P = 0.0008), waist circumference (P < 0.0001) and prevalence of metabolic syndrome (84.6% vs. 40.0%, P < 0.0001). Only 26% of those with NAFLD had elevated alanine aminotransferase. CONCLUSIONS: This proof-of-concept study demonstrates that T2DM has significant rates of both NAFLD and advanced fibrosis. Concomitant screening for NAFLD and advanced fibrosis by using MRI-proton density fat fraction and magnetic resonance elastography in T2DM is feasible and may be considered after validation in a larger cohort.

Novel association between serum pentraxin-2 levels and advanced fibrosis in well-characterised patients with non-alcoholic fatty liver disease.

BACKGROUND: Pentraxin-2 (PTX-2), a serum protein, inhibits inflammation and fibrosis, and recombinant PTX-2 is being tested as an anti-fibrotic agent. AIM: To evaluate the association between serum PTX-2 levels and fibrosis stage in patients with non-alcoholic fatty liver disease (NAFLD). METHODS: Serum pentraxin-2 levels were compared between four groups of well-characterised patients including NAFLD with no fibrosis, NAFLD with mild-moderate fibrosis (stage 1-2), NAFLD with advanced fibrosis (stage 3-4), and age-sex matched non-NAFLD controls. RESULTS: Sixty subjects were included in the study. The mean age was 58.9 years, 68% were male and 58% were Caucasian. In univariate analysis, serum PTX-2 levels significantly decreased from non-NAFLD controls to mild NAFLD with no fibrosis, to NAFLD with mild-moderate fibrosis and were lowest in patients with NAFLD and advanced fibrosis, in a dose-dependent manner (P < 0.0001). In multivariable-adjusted analyses controlling for age, sex, albumin, and CRP, the results remained consistent and statistically significant. Serum PTX-2 level had an AUROC of 0.84 (95% CI: 0.71-0.97) for the diagnosis of NAFLD, and an AUROC of 0.77 (95% CI: 0.65-0.90) for the diagnosis of advanced fibrosis in NAFLD. Serum PTX-2 levels also decreased with increasing liver stiffness as estimated by magnetic resonance elastography (r = -0.31, P = 0.02). CONCLUSIONS: PTX-2 levels are significantly lower in patients with NAFLD compared to non-NAFLD controls, and decline further in patients with advanced fibrosis. PTX-2 may therefore be both a biomarker of disease and a potential target for anti-fibrotic therapy with the recombinant pentraxin-2.

Clinical, biochemical and histological differences between HIV-associated NAFLD and primary NAFLD: a case-control study.

BACKGROUND: There are limited data regarding the clinical, biochemical and liver histological characteristics of patients with HIV-associated nonalcoholic fatty liver disease (NAFLD), and whether this entity differs in presentation and severity from primary NAFLD AIM: To examine the clinical and histological differences between HIV-associated NAFLD and primary NAFLD. METHODS: This is a cross-sectional, case-control study comparing patients with HIV-associated NAFLD vs. patients with primary NAFLD. HIV-infected patients were identified from a database of consecutive liver biopsies performed at the University of California at San Diego, over a 13-year period. HIV-infected patients with biopsy-proven NAFLD were selected as cases, after exclusion of other causes of liver disease and hepatic steatosis. Age-sex-matched controls with biopsy-proven primary NAFLD were randomly identified from the same pathology database. All biopsies underwent a standardised, detailed, histological research evaluation by a liver pathologist who was blinded to clinical and case-control status. RESULTS: Compared to age-sex-matched patients with primary NAFLD (n = 33), patients with HIV-associated NAFLD (n = 33) had significantly higher mean aspartate aminotransferase (P < 0.001), alanine aminotransferase (P < 0.001), alkaline phosphatase (P = 0.003) and serum triglycerides (P = 0.024). Similarly, compared to age-sex-matched primary NAFLD, patients with HIV-associated NAFLD had significantly higher rates of definite steatohepatitis (37% vs. 63%, P = 0.04), and more features of liver injury, including lobular inflammation (<0.001) and acidophil bodies (<0.001). CONCLUSION: Compared to age-sex-matched primary NAFLD, HIV-associated NAFLD has increased severity of liver disease and a higher prevalence of NASH.

Risk assessment for coronary heart disease in patients with haemophilia: a single centre study in the United States.

In haemophilia, coronary heart disease (CHD) occurs at a similar frequency as in the general population, but the contributing risk factors in haemophilia are incompletely understood. To investigate risk factors and 10-year CHD risk in a single centre cohort of patients with haemophilia (PWH) ≥20 years old (n = 89). We retrospectively applied the modified Framingham National Cholesterol Education Program/Adult Treatment Panel (NCEP/ATP) III risk prediction equation. Three risk levels were defined: <10% (low), 10-20% (intermediate) and >20% (high). Results were compared to the National Health and Nutrition Examination Survey (NHANES). Mean age in both cohorts was similar. Compared to NHANES, systolic blood pressures were significantly higher in PWH, but current smoking and cholesterol were lower. CHD risk differed significantly between PWH and NHANES (P = 0.005) with a higher proportion of PWH classified at low risk (77.5% vs. 61.0%). The proportion of low risk patients was also significantly higher for severe haemophilia patients compared to non-severe haemophilia patients (88.6% vs. 66.7%, P = 0.02). Among PWH, and compared to PWH who were hepatitis C (HepC) negative, HepC positive patients had significantly lower cholesterol, LDL and triglycerides. The CHD risk of HepC positive patients differed significantly from NHANES (P = 0.03) with a lower proportion of HepC positives being classified as high risk (5.7% vs. 17.3%). Favourable CHD risk classification in PWH may be influenced by low cholesterol associated with HepC infection. Estimates of CHD risk in PWH by composite scoring may not be accurate and will require studies correlating risk factors with incident CHD.

Arthrocentesis and therapeutic joint injection: an overview for the primary care physician.

Athrocentesis and therapeutic joint injection is a safe and useful primary care procedure. Fluid collection and analysis from effused joints is important to establish a cause and therefore inform appropriate management. Therapeutic joint injection can give patients significant, rapid, localized pain relief.

Synergistic association between alcohol intake and body mass index with serum alanine and aspartate aminotransferase levels in older adults: the Rancho Bernardo Study.

BACKGROUND: The association between body-mass-index (BMI), alcohol consumption and their joint effect in increasing the risk of elevated serum alanine (ALT) and aspartate (AST) is unclear in older community-dwelling adults. AIM: To determine the association between alcohol, BMI, and their combined effect with serum ALT and AST in older community-dwelling adults in the United States. METHODS: A cross-sectional, population-based study in participants (n = 2364) from the Rancho Bernardo Study (54% women; mean age: 70 years, BMI: 25 kg/m(2), alcohol users: 63%) who attended a research visit in 1984-87. BMI was recorded by a trained nurse and alcohol use ascertained by a validated questionnaire. Odds-ratio (OR) and 95% confidence intervals (CI) of elevated serum ALT and AST (defined as > or =30 U/L in men and > or =19 U/L in women) were calculated for alcohol and BMI separately and their joint exposure using logistic regression models. RESULTS: In multivariate logistic regression models adjusted for age, alcohol use, total cholesterol, serum triglycerides, fasting plasma glucose, systolic blood pressure, and diabetes mellitus, obesity independently increased the odds of elevated ALT in this cohort of older men and women by 3.0 (95% CI, 1.7-5.3) and 1.8 (95% CI, 1.1-2.7) respectively. Joint effects of consuming >3 alcoholic drinks/day and obesity raised the odds of elevated ALT by 8.9 (95% CI, 2.4-33.1) and AST by 21-fold (95% CI, 2.6-170.1), demonstrating synergism. Obese participants had higher odds of elevated ALT even at 0 < or = 1 drink/day. CONCLUSIONS: In older men and women, the combination of obesity with alcohol is synergistic in increasing the risk of liver injury.

Effect of dehydroepiandrosterone supplementation on bone mineral density, bone markers, and body composition in older adults: the DAWN trial.

UNLABELLED: We present results of a randomized, placebo-controlled trial to examine the effect of 50 mg daily oral DHEA supplementation for one year on bone mineral density (BMD), bone metabolism and body composition in 225 healthy adults aged 55 to 85 years. INTRODUCTION: Dehydroepiandrosterone (DHEA) levels decline dramatically with age, concurrent with the onset of osteoporosis, suggesting a role for DHEA supplementation in preventing age-related bone loss. METHODS: We conducted a randomized, placebo-controlled trial to examine the effect of 50 mg daily oral DHEA supplementation for one year on bone mineral density (BMD), bone metabolism and body composition in 225 healthy adults aged 55 to 85 years. RESULTS: DHEA treatment increased serum DHEA and DHEA sulfate levels to concentrations seen in young adults. Testosterone, estradiol and insulin-like growth factor (IGF-1) levels increased in women (all p < 0.001), but not men, receiving DHEA. Serum C-terminal telopeptide of type-1 collagen levels decreased in women (p = 0.03), but not men, whereas bone-specific alkaline phosphatase levels were not significantly altered in either sex. After 12 months, there was a positive effect of DHEA on lumbar spine BMD in women (p = 0.03), but no effect was observed for hip, femoral neck or total body BMD, and no significant changes were observed at any site among men. Body composition was not affected by DHEA treatment in either sex. CONCLUSION: Among older healthy adults, daily administration of 50 mg of DHEA has a modest and selective beneficial effect on BMD and bone resorption in women, but provides no bone benefit for men.

Hemolin gene silencing by ds-RNA injected into Cecropia pupae is lethal to next generation embryos.

There is increasing evidence of an intimate connection between participants in the innate immune system and in development. Molecules involved in the determination of dorso-ventral polarity in Drosophila have related counterparts in the signalling pathways for immune gene activation in both insects and mammals. Hemolin from the Giant silkmoth, Hyalophora cecropia, identified as a bacteria-inducible molecule and a member of the immunoglobulin superfamily, is present as protein and transcripts in oocytes and embryos. We used RNA interference (RNAi) to investigate H. cecropia gene function in vivo and demonstrated that Hemolin is crucial for the normal development of embryos. When RNAi-females were mated, no larvae emerged from their eggs and when dissected, the eggs revealed malformed embryos. Western blot analysis confirmed the lack of Hemolin gene products. We conclude that Hemolin is necessary for development, since the silencing of Hemolin gene expression leads to embryonic lethality.

The insect immune protein hemolin is expressed during oogenesis and embryogenesis.

Hemolin is the most abundant bacteria-induced proteins in Hyalophora cecropia hemolymph. Its structural features, both at the protein and gene level, ascribe this molecule to the immunoglobulin gene superfamily (IgSF) with particular homology to neural cell adhesion molecules. An increasing number of evidence suggest a role in immune recognition and in cell adhesion events. Hemolin is also developmentally regulated as suggested by changes in its concentration during larval and pupal ecdysis (Trenczek, T., 1998. Endogenous defense mechanisms of insects. Zoology 101, 298-315; Lanz-Mendoza, H., Faye, I., 1999. Physiological aspects of the immunoglobulin superfamily in invertebrates. Dev. Comp. Immunol. 23, 359-374). In the present study the expression of hemolin was investigated in oogenesis and in early embryogenesis. Our results reveal that hemolin is expressed in follicles and in epidermal and neural tissues of embryos.

Implications of hemolin glycosylation and Ca2+-binding on homophilic and cellular interactions.

Insects are useful models for the study of innate immune mechanisms because of their lack of antibodies and receptors involved in adaptive immune response. Nevertheless, hemolin cloned from moths is a soluble and membrane associated Ig-related molecule that is up-regulated during immune response [Lanz-Mendoza, H. & Faye, I. (1999) Dev. Comp. Immunol. 23, 359-374]. The hemolin monomeric form has four, pair-wise, interacting Ig-domains, forming a strongly bent horseshoe structure [Su, X.-D., Gastinel, L.N., Vaughn, D.E., Faye, I., Poon, P. & Bjorkman, P. (1998) Science 281, 991-995]. To elucidate the nature of its homophilic and cellular interactions, the glycosylation and Ca2+-binding properties of hemolin were investigated. We used Hyalophora cecropia hemolin isolated from hemolymph of bacteria-injected pupae, or produced as a recombinant protein in a baculovirus/insect cell system. Both types of hemolin contain N-acetylglucosamine and probably sialic acid, as indicated by peptide:N-glycosidase F and neuraminidase digestion and glycosylation detection by Western-blotting analysis. The N-acetylglucosamine residues on hemolin were confirmed with the use of specific lectins. In addition, hemolin was shown to specifically bind calcium when spotted onto nitrocellulose and treated as for 45Ca2+ autoradiography. Earlier studies demonstrated that hemolin can bind to hemocytes and this was tested for its dependence on calcium and carbohydrates, using hemolin-coated fluorescent microspheres. A greater level of attachment of microspheres occurred in the presence of calcium than if calcium was absent. Furthermore, this binding was inhibited by EGTA and N-acetylglucosamine or N-acetylneuraminic acid, implying that carbohydrates and calcium are crucial factors in homophilic binding and cell-adhesion events mediated by this Ig-superfamily molecule.

Evaluation of the simple calculated osteoporosis risk estimation (SCORE) in older Caucasian women: the Rancho Bernardo study.

Osteoporosis is a major health problem in older women. A risk assessment tool, the Simple Calculated Osteoporosis Risk Estimation (SCORE), has been developed to identify postmenopausal women likely to have low bone mass who should be referred for bone densitometry. The objective of this study was to calculate the sensitivity, specificity and predictive values of SCORE in a community-dwelling sample of older women. A total of 1013 postmenopausal Caucasian women aged 44-98 years provided a standard medical history including history of osteoporotic fractures and medication use. Bone mineral density (BMD) was measured at the femoral neck using dual-energy X-ray absorptiometry. In accordance with the SCORE protocol, low BMD was defined as 2 or more standard deviations below the mean BMD in healthy young women. Among these older women (mean age = 72.5 years), 67% had low BMD. Using the recommended SCORE cutpoint of 6, the sensitivity of SCORE was 98% but the specificity was only 12.5%. The positive predictive value (PPV) and negative predictive value (NPV) were 69% and 75%, respectively, meaning that all but 5.5% of the women would be recommended for bone densitometry. Increasing the cutpoint of 11, based on ethnicity and the receiver operating characteristic (ROC) curve, reduced sensitivity to 80% but improved specificity to 46%. The PPV and NPV were 75% and 53%, respectively, meaning that bone scans would not be recommended for 28% of the women. However, 13% of the women with low BMD would be missed. Analyses restricted to women <74 years of age reduced the rate of recommended bone densitometry but increased the number of women with low BMD who would be missed. We conclude that SCORE has limited value as a method for appropriately referring older ambulatory women for bone densitometry.

Hormone replacement therapy and stroke risk in older women.

Studies of the effect of hormone replacement therapy (HRT) on the risk of stroke in postmenopausal women have yielded divergent results. Many of the studies had small numbers of estrogen-using women and relatively young women. All were confounded because women who use estrogen are healthier than the general population. Case-control studies, which included a larger number of cases, usually were limited to stroke survivors and had problems of recall bias regarding exposure. We describe the independent risk of fatal and nonfatal stroke in a socioeconomically homogeneous cohort of older, postmenopausal, community-dwelling women in Rancho Bernardo, California, who had a high rate of past or current HRT use. During 1984-1987, estrogen use and stroke risk factors were ascertained for 1031 women over age 60 without a history of stroke in the Rancho Bernardo cohort. Stroke deaths were monitored by obtaining 100% of death certificates, and nonfatal strokes were determined through questionnaires. At baseline, the average age was 73.1 years, 27% were current estrogen users, and 45% were past estrogen users. Over 8.8 follow-up years, 263 deaths occurred, and 37 of the death certificates listed stroke as an underlying or contributing cause of death. Only 7 of current HRT users had a stroke compared with 16 former users and 14 never users. The age-adjusted odds ratio (OR) was 0.92 (95% CI = 0.56-1.50) in HRT users versus never users. Results were not materially changed in multiply adjusted analyses. Similar analyses of nonfatal strokes and transient ischemic attacks (n = 20) showed an increased risk (OR = 3.02, 95% CI = 0.70-13.08). This study provides no evidence that HRT prevents stroke in older women, but the confidence intervals are wide, suggesting that more studies are needed.

Cell adhesion properties of hemolin, an insect immune protein in the Ig superfamily.

The isolation of antibacterial peptides from the giant silkmoth Hyalophora cecropia has opened the area of animal antibiotics [Boman, H. G. (1991) Cell 65, 205-207] and the study of insect immune genes has revealed striking similarities to many immune response genes in mammals [Hultmark, D. (1994) Nature 267, 116-117]. However, the molecules and mechanisms behind primordial immune recognition are not understood. One candidate for one such recognition molecule is hemolin, a 48-kDa immunoglobulin-related protein first isolated from H. cecropia, where it is up-regulated upon infection and secreted into the hemolymph. Hemolin was shown to bind to bacteria and to hemocytes, giving rise to changes in hemocyte adhesiveness and intracellular phosphorylation patterns [Faye, I. & Kanost, M. (1997) in Molecular mechanisms of immune responses in insects (Brey, P. T. & Hultmark, D., eds) Chapman and Hall, London]. In the present publication, we give evidence for the presence of a 52-kDa membrane form of hemolin on hemocytes, based on flow-activated cell sorting and membrane protein extractions. In addition we reveal calcium-dependent homophilic binding properties of hemolin, using hemolin-coated microspheres. When biotinylated recombinant hemolin was allowed to bind to hemocyte membranes, higher molecular-mass complexes were formed. Furthermore, we used immunological methods and Northern-blot analysis to demonstrate the presence of hemolin in embryos and retinal discs, suggesting that hemolin is expressed in several tissues at different developmental stages. These results show novel cell adhesion features of hemolin, corroborating its multifunctional character with putative roles in cellular and humoral immunity and in development.

Regulation of the insect immune response: the effect of hemolin on cellular immune mechanisms.

Hemolin is a bacteria-inducible protein of the immunoglobulin superfamily identified in the silk moth Hyalophora cecropia. The role of this protein, in hemocyte aggregation and phagocytosis, was studied in vitro. Hemocyte aggregation, stimulated by phorbol myristate acetate or lipopolysaccharide (LPS), was prevented by hemolin in a dose-dependent fashion, but hemolin did not disrupt aggregates once they had been formed. Furthermore, hemolin was able to stimulate phagocytic activity in both hemocytes and hemocytic mbn-2 cells and this activity was enhanced by LPS. The enhanced phagocytosis produced by a combination of hemolin and LPS was prevented by the protein kinase C (PKC) inhibitors staurosporine and H-7, and PKC activity in hemocyte crude extracts was enhanced by hemolin and LPS, with the highest activity observed in the presence of both. Hemolin affected tyrosine phosphorylation of hemocyte proteins, enhancing the phosphorylation of two proteins of 20 and 30 kDa and preventing tyrosine phosphorylation of two proteins of 35 and 40 kDa. These results suggest that hemolin is involved in the regulation of the cellular immune responses via a pathway that includes PKC activation and protein tyrosine phosphorylation.