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While technology-based interventions enhance instruction and improve outcomes for students with disabilities, implementing and integrating technology in authentic learning environments continues to be a challenge. Based on the experiences of a variety of Stepping-Up Technology Implementation projects funded by the U.S. Department of Education, Office of Special Education Programs, this mixed-methods study explored the essential factors for the successful implementation of technology-based interventions in K-12 schools and early childhood programs. Based on the qualitative analysis of projects’ implementation reports and responses to the follow-up questionnaire, four major themes emerged. The barriers and facilitators to technology implementation were reported across such areas as (a) developing and sustaining buy-in, (b) ensuring implementation fidelity to support the intervention, (c) research-to-practice dilemmas, and (d) data serving multiple purposes. The discussion and practical implications for supporting technology implementation are provided.

Don't save your barbecuing for the weekends or company. Your grill can help you get dinner on the table quickly every night this summer. It's an incredibly low-fat cooking method, [...]

Businesses which offer professional services are being challenged for their relevance to marketplace changes and needs. Consumers have reached levels of education which make them more sophisticated and give them the power to select products or services with greater scrutiny. Thus, businesses must strive to inject professionalism, value and a more holistic approach to the services they render. Professionalism must be marked by education beneficial to customers and strong ethical standards., A sure-fire approach to satisfying client expectations Businesses that provide professional services to their clients are increasingly being challenged. Are their services, whether financial, accounting, medical or other really relevant [...]

Byline: Jackson, Terry You don't have to be a master chef to whip up savory, satisfying dishes that are healthy, too. All you really need is a slow cooker, a [...]

Byline: Jackson, Terry We think we have it bad, but a century ago, women toiled 6 hours a day making meals and then cleaning up the mess. Today, we've whittled [...]

I am writing this article after undergoing emergency open-heart surgery. This is not to spotlight my procedure, but to provide the customer's perspective--and my professional viewpoint--on the sterile processing of [...]

Up to 30% of adult patients with sickle cell disease (SCD) will develop pulmonary hypertension (pHTN), a complication associated with significant morbidity and mortality. To identify genetic factors that contribute to risk for pHTN in SCD, we performed association analysis with 297 single nucleotide polymorphisms (SNPs) in 49 candidate genes in patients with sickle cell anemia (Hb SS) who had been screened for pHTN by echocardiography (n = 111). Evidence of association was primarily identified for genes in the TGFβ superfamily, including activin A receptor, type II–like 1 (ACVRL1), bone morphogenetic protein receptor 2 (BMPR2), and bone morphogenetic protein 6 (BMP6). The association of pHTN with ACVRL1and BMPR2corroborates the previous association of these genes with primary pHTN. Moreover, genes in the TGFβ pathway have been independently implicated in risk for several sickle cell complications, suggesting that this gene pathway is important in overall sickle cell pathophysiology. Genetic variation in the β-1 adrenergic receptor (ADRB1) was also associated with pHTN in our dataset. A multiple regression model, which included age and baseline hemoglobin as covariates, retained SNPs in ACVRL1, BMP6, and ADRB1as independently contributing to pHTN risk. These findings may offer new promise for identifying patients at risk for pHTN, developing new therapeutic targets, and reducing the occurrence of this life-threatening SCD complication.

ABSTRACTWe monitored 93 subjects cured of amebic liver abscess (ALA) and 963 close associate controls in Durban, South Africa, and determined by enzyme-linked immunosorbent assay that the intestinal immunoglobulin A (IgA) antibody response to the Entamoeba histolyticagalactose-inhibitable adherence lectin is most accurately represented by a complex pattern of transitory peaks. One or more intestinal anti-lectin IgA antibody peaks occurred in 85.9% of ALA subjects over 36 months compared to 41.6% of controls (P< 0.0001). ALA subjects exhibited a greater number of anti-lectin IgA antibody peaks (P< 0.0001) than controls. In addition, their peak optical density values were higher (peak numbers 1 to 3, P< 0.003), peaks were of longer duration (for peaks 1 and 2, P≤ 0.0054), and there was a shorter time interval between peaks (between 1 and 2 or 2 and 3, P≤ 0.0106) than observed for control subjects. A prior E. histolyticainfection was associated with the occurrence of an anti-lectin IgA antibody peak (79.1%, P< 0.0001) more so than for Entamoeba disparinfection (57.2%, P< 0.001). The annual number of anti-lectin IgA antibody peaks in ALA subjects was 0.71 per year, compared to just 0.22 in controls (P<0.0001), indicating a higher rate of exposure to the parasite than previously appreciated. Anti-lectin IgA antibody peaks were of higher amplitude following a E. histolyticainfection compared to E. dispar(P= 0.01) and, for either, were of greater height in ALA subjects than controls (P< 0.01). ALA subjects demonstrated greater clearance of amebic infection after an anti-lectin IgA antibody peak compared to controls, and only 14.3% remained with a positive culture after the peak, compared to 38.9% in controls (P= 0.035). In summary, this prospective controlled longitudinal study elucidated the dynamic nature of the human intestinal IgA antibody response to E. histolyticaand E. disparinfection and revealed that ALA subjects exhibit heightened intestinal anti-lectin IgA antibody peaks that are associated with clearance of E. histolyticaand E. disparinfection.

ABSTRACTImmunity to Entamoebaspecies intestinal infection is associated with the presence of intestinal IgA antibodies against the parasite's galactose-inhibitable adherence lectin. We determined the epitope specificity of serum and intestinal antilectin IgA antibodies by enzyme-linked immunosorbent assay using overlapping fragments of a recombinant portion of the lectin heavy subunit, designated LC3. These findings were correlated with the effects of epitope-specific murine antilectin immunoglobulin A (IgA) monoclonal antibodies (MAbs) on amebic in vitro galactose-specific adherence. LC3 is a highly antigenic and immunogenic cysteine-rich protein (amino acids [aa] 758 to 1150) that includes the lectin's carbohydrate binding domain. The study subjects, from Durban, South Africa, were recently cured of amebic liver abscess (ALA) with or without concurrent Entamoeba histolyticaintestinal infection or were infection free 1 year after cure. We also studied seropositive subjects that were infected with E. histolytica, disease free, and asymptomatic. Serum anti-LC3 IgA antibodies from all study groups exclusively recognized the third (aa 868 to 944) and the seventh (aa 1114 to 1134) LC3 epitopes regardless of clinical status; epitope 6 (aa 1070 to 1114) was also recognized by serum anti-LC3 IgG antibodies. However, IgG antibody recognition of epitope 6 but not 3 or 7 was lost 1 year following cure of ALA. We produced 14 murine anti-LC3 IgA MAbs which collectively recognized five of the seven LC3 epitopes. The majority of the murine MAbs recognized the first epitope (aa 758 to 826), which was not recognized by human IgA antibodies. Interestingly, adherence of E. histolyticatrophozoites to CHO cells was inhibited by MAbs against epitopes 1, 3, 4 (aa 944 to 987), and 6 (P< 0.01). The LC3 epitopes recognized by human IgA antibodies (3 and 7) were further characterized by use of overlapping synthetic peptides. We identified four peptides (aa 891 to 903, 918 to 936, 1114 to 1134, and 1128 to 1150) that in linear or cyclized form were recognized by pooled intestinal IgA antibodies and serum IgG antibodies from subjects with ALA and asymptomatic, seropositive infected subjects. This study identifies the lectin epitopes to be studied in an amebiasis subunit vaccine designed to elicit mucosal immunity mimicking that of humans cured of ALA.

Presents several no-mess recipes such as Greek Lemon Chicken, Orange-Soy Salmon with Vegetables and Jumbo Southwestern Supper Pizza.

We followed 93 subjects with amebic liver abscess (ALA) and 963 close associate controls at 3-month intervals for 36 months to characterize intestinal and humoral antibody responses to the amebic galactose-inhibitable lectin and to determine whether immunity developed to Entamoeba histolytica or Entamoeba dispar infection following cure of ALA. We found that ALA subjects had a higher prevalence and level of intestinal antilectin immunoglobulin A (IgA) and serum anti-LC3 (cysteine-rich recombinant lectin protein) IgA and IgG antibodies, P < 0.01 and P < 0.05, respectively, compared to controls. The intestinal antilectin IgA antibody response was sustained over a longer time period in ALA subjects (71.8% remained positive at 18 months and 52.6% at 36 months, P < 0.001 compared to 17.6% and 10.3% of controls, respectively). ALA subjects were highly immune to E. dispar infection throughout the study (0% infected at 6 and 36 months, compared to 6.5% and 4.9% of control subjects, respectively, P < 0.05). Upon entry into the study, 6.3% of ALA subjects were infected with E. histolytica; the incidence of new E. histolytica infections in controls (as determined by culture) was too low (1.4%) to determine whether ALA subjects exhibited immunity to new infections. We found that stool cultures every 3 months markedly underestimated the occurrence of new E. histolytica infections, as 15.3% of controls seroconverted after 12 months of follow-up. Unfortunately, under the field conditions present in Durban, South Africa, enzyme-linked immunosorbent assay for detection of lectin antigen in stool yielded unreliable results. In summary, subjects cured of ALA exhibited sustained mucosal IgA antibody responses to the amebic galactose-inhibitable lectin and a high level of immunity to E. dispar infection. Determination of immunity to E. histolytica following cure of ALA will require the use of more sensitive and reliable diagnostic methods.

ABSTRACTWe followed 93 subjects with amebic liver abscess (ALA) and 963 close associate controls at 3-month intervals for 36 months to characterize intestinal and humoral antibody responses to the amebic galactose-inhibitable lectin and to determine whether immunity developed to Entamoeba histolyticaor Entamoeba disparinfection following cure of ALA. We found that ALA subjects had a higher prevalence and level of intestinal antilectin immunoglobulin A (IgA) and serum anti-LC3 (cysteine-rich recombinant lectin protein) IgA and IgG antibodies, P< 0.01 and P< 0.05, respectively, compared to controls. The intestinal antilectin IgA antibody response was sustained over a longer time period in ALA subjects (71.8% remained positive at 18 months and 52.6% at 36 months, P< 0.001 compared to 17.6% and 10.3% of controls, respectively). ALA subjects were highly immune to E. disparinfection throughout the study (0% infected at 6 and 36 months, compared to 6.5% and 4.9% of control subjects, respectively, P< 0.05). Upon entry into the study, 6.3% of ALA subjects were infected with E. histolytica;the incidence of new E. histolyticainfections in controls (as determined by culture) was too low (1.4%) to determine whether ALA subjects exhibited immunity to new infections. We found that stool cultures every 3 months markedly underestimated the occurrence of new E. histolyticainfections, as 15.3% of controls seroconverted after 12 months of follow-up. Unfortunately, under the field conditions present in Durban, South Africa, enzyme-linked immunosorbent assay for detection of lectin antigen in stool yielded unreliable results. In summary, subjects cured of ALA exhibited sustained mucosal IgA antibody responses to the amebic galactose-inhibitable lectin and a high level of immunity to E. disparinfection. Determination of immunity to E. histolyticafollowing cure of ALA will require the use of more sensitive and reliable diagnostic methods.

Presents several recipes. Mediterranean Grilled Pita Pizzas; Santa Fe Grilled Shrimp with Bean, Orange and Avocado Salad; BBQ Chicken Burgers; Thai Beef Salad.

ABSTRACTA closed-tube, real-time PCR assay was developed for sensitive and specific detection and differentiation of the two closely related intestinal protozoan parasites Entamoeba histolyticaand Entamoeba dispardirectly from human feces. The assay is performed with the LightCycler system using fluorescence-labeled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, ribosomal DNA-containing ameba episome. The assay was able to detect as little as 0.1 parasite per g of feces. The two pairs of primers used were specific for the respective ameba species, and results were not influenced by the presence of other Entamoebaspecies even when present in exceeding amounts. PCR was evaluated using several hundred stool samples from areas of amebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and ameba culture. PCR was found to be significantly more sensitive than microscopy or culture, as all samples positive by microscopy and 22 out of 25 (88%) samples positive by culture were also positive by PCR, but PCR revealed a considerable number of additional E. histolytica-or E. dispar-positive samples. Compared to culture and subsequent ameba differentiation by isoenzyme analysis, PCR was 100% specific for each of the two Entamoebaspecies. Interestingly, the comparison with PCR revealed that culture, in particular, underestimates E. histolyticainfections. Given the high sensitivity and specificity of the developed PCR assay, the inability of microscopy to distinguish between the two ameba species, and the time it takes to culture and subsequently differentiate entamoebae by isoenzyme analysis, this assay is more suitable than microscopy or culture to correctly diagnose intestinal E. histolyticaor E. disparinfection.

ABSTRACTInfection by field strains of Foot-and-mouth disease virus(FMDV) is initiated by binding to certain species of arginine-glycine-aspartic acid (RGD)-dependent integrin including avß3 and the epithelial integrin avß6. In this report we show that the integrin avß1, when expressed as a human/hamster heterodimer on transfected CHOB2 cells, is a receptor for FMDV. Virus binding and infection mediated by avß1 was inefficient in the presence of physiological concentrations of calcium and magnesium but were significantly enhanced by reagents that activate the integrin and promote ligand binding. The ability of chimeric a5/av integrin subunits, in association with the ß1 chain, to bind FMDV and mediate infection matched the ligand binding specificity of avß1, not a5ß1, thus providing further evidence for the receptor role of avß1. In addition, data are presented suggesting that amino acid residues near the RGD motif may be important for differentiating between the binding specificities of avß1 and avß6.

ABSTRACTField isolates of foot-and-mouth disease virus (FMDV) are believed to use RGD-dependent integrins as cellular receptors in vivo. Using SW480 cell transfectants, we have recently established that one such integrin, avß6, functions as a receptor for FMDV. This integrin was shown to function as a receptor for virus attachment. However, it was not known if the avß6 receptor itself participated in the events that follow virus binding to the host cell. In the present study, we investigated the effects of various deletion mutations in the ß6 cytoplasmic domain on infection. Our results show that although loss of the ß6 cytoplasmic domain has little effect on virus binding, this domain is essential for infection, indicating a critical role in postattachment events. The importance of endosomal acidification in avß6-mediated infection was confirmed by experiments showing that infection could be blocked by concanamycin A, a specific inhibitor of the vacuolar ATPase.

ABSTRACTField isolates of foot-and-mouth disease virus (FMDV) have been shown to use the RGD-dependent integrin avß3 as a cellular receptor on cultured cells. However, several other RGD-dependent integrins may have the potential to act as receptors for FMDV in vivo. Of these, avß6 is a likely candidate for use as a receptor by FMDV as it is expressed on epithelial cells, which correlates with the tissue tropism of the virus. In this report, we show that human colon carcinoma cells (SW480) that are normally nonpermissive for FMDV become susceptible to infection as a result of transfection with the integrin ß6 subunit and expression of avß6 at the cell surface. Integrin avß6 is the major site for virus attachment on the ß6-transfected cells, and binding to avß6 serves to increase the rate of virus entry into these cells. In addition, we show that virus binding and infection of the ß6-transfected cells is mediated through an RGD-dependent interaction that is specifically inhibited by a monoclonal antibody (10D5) that recognizes avß6. These studies establish a role for avß6 as a cellular receptor for FMDV.

ABSTRACTSaliva from subjects with amebic liver abscess (ALA), acute amebic colitis, asymptomatic infection with Entamoeba histolyticaor Entamoeba dispar, and uninfected controls was tested by enzyme-linked immunosorbent assay (ELISA) for the presence of E. histolyticagalactose-inhibitable lectin antigen and salivary immunoglobulin (IgG) antibodies to a recombinant cysteine-rich lectin-derived protein (LC3). Salivary lectin antigen was found in 65.8% of subjects with acute colitis, compared to 22.2% of those convalescent from ALA, 10.0% with asymptomatic E. histolyticainfection, 9.8% with E. disparinfection, and 2.6% of controls (subjects from the United States and study patients with nonamebic diarrhea) (P< 0.001 for each compared to values for subjects with colitis). Salivary anti-LC3 IgG antibodies were found in 92% of ALA patients regardless of duration of illness and in 83.3% of colitis patients who were symptomatic for at least 7 days (P< 0.001 compared to other study groups). Serum anti-LC3 IgG antibodies were detected in 56.3% of subjects with acute colitis, 100% of subjects with ALA or prolonged colitis, 45% of subjects with asymptomatic E. histolyticainfection, 32.3% of subjects with E. disparinfection, and 23.4% of diarrhea controls. In comparison to ELISA for serum anti-LC3 IgG antibodies, the salivary lectin antigen assay is a more sensitive and specific test for acute amebic colitis. Detection of salivary anti-LC3 IgG antibodies by ELISA is an effective means for the diagnosis of ALA and prolonged cases of amebic colitis.

Tirilazad Mesylate—Effects of the 21-Aminosteroid on the Lymphoid System of Laboratory Animals: A Comparison with the Glucocorticoid Methylprednisolone. Jackson, T. A., Ochoa, R., and Kakuk, T. J. (1995). Fundam. Appl. Toxicol. 26, 246-257. Four-week toxicity studies with the 21-aminosteroid tirilazad mesylate were conducted in Sprague-Dawley rats, beagle dogs, and cynomolgus monkeys to support development of the drug for use in various clinical syndromes of injury to the central nervous system of humans. As the immune system is involved in many of the obvious side effects of glucocorticoids used currently for this indication, particular attention was directed to the lymphoid system; results were contrasted with similar data from studies with methylprednisolone, a classical glucocorticoid. Administration of tirilazad mesylate to rats, dogs and monkeys for 4 weeks had no effects at the highest doses tested on parameters assessed, including absolute peripheral blood lymphocyte counts, thymus or adrenal weights, circulating levels of cortisol, or lymphocyte proliferation response to phytohemagglutinin-P. Germinal centers in lymphoid tissues from dogs given high doses of tirilazad contained small numbers of macrophages with vacuolated cytoplasm but no other changes; lymphoid tissues in rats and monkeys given tirilazad were morphologically normal. Administration of methylprednisolone for a similar duration in rats and dogs at high dose levels was associated with increased death rates due to bacterial infections, markedly decreased peripheral blood lymphocyte counts and weights of thymus and adrenal glands, and prominent lymphoid atrophy as well as decreased circulating levels of cortisol. Female dogs infused for 10 days with a high dose of methylprednisolone had depression of the in vitro proliferation response of peripheral blood lymphocytes to phytohemagglutinin-P. Copyright 1995, 1999 Academic Press

Several letters to the editor are presented in response to articles in previous issues including "International Buys Jeep!," in the April 2009 issue, "Jeep Brand Sold to International," in the April 2009 issue, and an article on April Fool's joke in the April 2009 issue.

Technological advances in telemarketing will give operators more time to focus on complicated sales calls and allow computers to handle simple orders, provide information, route calls, and take messages. Five independent technologies are identified that will influence the future of telemarketing: (1) digitized voice response, (2) digitized voice storage, (3) voice recognition, (4) text-to-speech conversion, and (5) advanced network technology.

Cindy Vaughn, front-end manager, Shop 'N Save, Collinsville, III.: People never understand this business until they work in it. My husband will ask me why I spend so many hours [...]

Several letters to the editor are presented in response to articles published in the October 2006 issue of the journal including "The Next Disruptors," "Can This Super Car Rev Up Nissan?" and "Why Free Agents Don't Feel Free."

The article reflects on the importance and value of the Meeting Professionals International's 2007 Professional Education Conference-North America (PEC-NA) to the city of New Orleans in Louisiana. He comments on the personal and professional effect of hosting the PEC-NA on members in the city. He also explores the financial implications surrounding the conference.

The life expectancy of many patients with sickle cell disease (SCD) is well into the 5th and 6th decade, but this remains extremely variable. Little is known about the biological factors that protect certain SCD patients from early demise while others never reach mid-adulthood. Recently, McKerrell and colleagues (2004) compared the clinical and laboratory profiles of SCD patients aged 40 years and over with SCD patients who were between 21 and 30 years of age. Similarly, we have compared clinical and genetic correlates of older SCD patients (50 years and over) with those of younger patients (18–30 years). Among 514 patients in our total study population, 49 (10%) were categorized as “older” and 194 (38%) were categorized as “younger.” Older SCD patients had lower hemoglobin (older: 7.8 ± 1.1 vs. younger: 8.5 ± 1.2, p=0.004), platelet count (older: 372 ± 126 vs. younger: 460 ± 225, p=0.02), MCV (older: 92 ± 12 vs. younger: 89 ± 9, p=0.08), MCHC (older: 33.6 ± 1.4 vs. younger: 34.3 ± 1.8, p=0.05), and WBC (older: 10.2 ± 2.7 vs. younger: 13.1 ± 4.1, p<0.001). Older patients also had lower total bilirubin (p=0.01), and increased alkaline phosphatase (p=0.0002) and creatinine (p=0.0002), which was associated with poorer creatinine clearance (p<0.0001). The older SCD patients also had increased systolic (p<0.0001) and diastolic (p=0.008) blood pressure, decreased O2 saturation (p=0.03), and a history of fewer pain episodes per year requiring medical treatment (p<0.0001). Many of our findings are consistent with those of McKerrell et al. (2004). In order to identify genetic factors associated with longevity in SCD, we examined 155 SNPs in a total of 41 genes, primarily involved in red blood cell adhesion and inflammation pathways. Chi Square tests of association were constructed for the genotypes of each SNP with the two clinical categories: “older” and “younger.” When the number of rare homozygotes was less than 5 individuals, we combined those individuals with the heterozygote individuals for analysis. All p-values are uncorrected for multiple testing. We found putative associations with 5 SNPs in 3 genes. Three non-coding SNPs in Klotho, not in linkage disequilibrium, exhibited different genotype frequencies in the older versus younger SCD patients (p=0.007, p=0.01 and p=0.01). Similarly, a single non-coding SNP in NOS2A (p=0.02) and TGFBR2 (p=0.02) also exhibited significantly different genotype frequencies in the older versus younger patients. These data support the clinical findings in aging SCD patients reported by McKerrell and colleagues (2004), and they also suggest that genetic factors contribute to variability in longevity in SCD. Interestingly, multiple SNPs in Klotho exhibited differing genotype frequencies in older versus younger patients. Mutations in Klotho have been previously associated with aging-related phenotypes in mice. A better understanding of the biological mechanisms associated with longevity in SCD may help identify those at risk for early demise and in need of more specialized medical care.

Priapism is a complication of sickle cell disease (SCD) that occurs due to obstruction of the corpora cavernosa of the penis. We have studied priapism in relation to several clinical and genetic factors in 249 adult male patients with SCD, 92 (37%) of whom reported a positive history of priapism. The mean age of male patients without a history of priapism was 35.2 years (± 10.8 years) compared with a mean age of 36.4 years (± 11.3 years) in male patients with a positive history of priapism. Because of the possible relationship with nitric oxide biology, we examined the co-occurrence of priapism with proteinuria, leg ulcers and stroke. Of the males with a positive history of priapism, 20% also had a history of 2+ or greater proteinuria, compared to a presence of 2+ or greater proteinuria in only 10% of males without a history of priapism (p=0.03). Similarly, 34% of males with a positive history of priapism also had a history of leg ulcers, compared to the presence of leg ulcers in 22% of males without priapism (p=0.03). No statistically significant association between the occurrence of priapism and stroke was observed. In an effort to identify genetic risk factors for priapism, we examined 262 single nucleotide polymorphisms (SNPs) in a total of 56 genes, primarily involved in red blood cell adhesion and inflammation pathways. Chi Square tests of association were constructed for the genotypes of each SNP with two clinical categories: patients with a positive history of priapism and patients without a history of priapism. When the frequency of rare homozygotes was less than five individuals, we combined these rare homozygote individuals with heterozygote individuals for analysis. All p-values were uncorrected for multiple testing. We found associations with 12 SNPs in 8 genes: SLC4A2 (p=0.003); ITGAV (p=0.004 and p=0.02 for two different SNPs); F13A1 (p=0.004 and p=0.02 for two different SNPs); AQP1 (p=0.01 and p=0.04 for two different SNPs); TGFBR2 (p=0.01 and p=0.02 for two different SNPs); ADRB2 (p=0.03); MGC (p=0.04); and ARG2 (p<0.05). These genes are involved in a variety of functions, including adhesion, coagulation, signal transduction, NO biology and immune response. We examined 21 non-coding SNPs in the Klotho gene, but we did not find an association between priapism and Klotho, as was recently reported by Nolan and colleagues (2005). The only possible trend for association we observed in Klotho was at marker rs1888057 (p=0.07); we did not observe association with the SNP (rs2249358) (p=0.82) Nolan and colleagues found associated with priapism. These data support our over-arching hypothesis that genetic factors mediate the variability and risk of developing organ-specific complications of SCD. A better understanding of the genetic factors that contribute to the occurrence of complications such as priapism should ultimately lead to a better understanding of SCD pathophysiology as well as to improved treatment for patients with SCD.

In order to identify genetic variants that modify the clinical severity of sickle cell disease (SCD), 118 patients with Hb SS or Hb Sβ0-thalassemia at our centers have undergone echocardiography (echo). Of these, 45 have results consistent with pulmonary hypertension (PHT), defined as a peak tricuspid jet velocity of at least 2.5 m/s. Analysis by De Castro et al (ASH 2004) of the Duke subset of these patients showed that ≥ 1+ proteinuria is highly associated with risk for PHT. Thus, we examined our candidate gene data to identify genetic polymorphisms associated with risk for PHT and proteinuria in this larger dataset. In all, we genotyped these patients for 101 SNPs in 31 candidate genes primarily involved in adhesion, coagulation, inflammation and cell signaling. For analyses of PHT, we compared patients with PHT, versus patients with other echo abnormalities and normal echos. Two levels of proteinuria were examined in our analyses: ≥1+ or ≥2+. For each SNP, contingency tables and tests of association were constructed for the genotypes by each clinical variable. The β2 adrenergic receptor gene (ADRB2) is associated with risk for asthma, diabetes, and obesity, as well as with the incidence of stroke in SCD. Thus, we examined 5 SNPs in ADRB2, including two in the leader cistron (RS1042711 and RS1801704), arg16gly (RS1042713), glu27gly (RS1042714), and HCV2084766 in exon 1. We found a trend for association with PHT and RS1042713 (p=0.06). SNPs associated with ≥1+ proteinuria were RS1042711 (p=0.03), RS1801704 (p=0.03), and RS1042714 (p=0.03). RS1042711 and RS1042714 were in high linkage disequilibrium (LD). ADCY6 is the major cardiac adenylyl cyclase isoform activated downstream of signaling though ADRB2, and is associated with cardiac hypertrophy. Thus we also examined four intronic SNPs in ADCY6 (HCV1244841, RS3730070, HCV1244851, and HCV1244859). Two SNPs in high LD were associated with PHT (HCV1244841, p=0.02, and RS3730070, p=0.01). Shores et al (2003) suggested that low cholesterol in both children and adults with SCD may exacerbate SCD complications. The LCAT gene converts cholesterol to cholesterol esters, and LCAT variants are associated with proteinuria and/or renal failure in other settings. We examined a SNP in exon 6 of LCAT (HCV11441833), as well as three other SNPs within genes close to LCAT (CTRL: RS2301246; SLC12A4: HCV2846928; and DPEP3: RS2271296). HCV11441833 was associated with PHT (p=0.05), ≥1+ proteinuria (p=0.01) and ≥2+ proteinuria (p=0.03). HCV2846928 was in high LD with the LCAT SNP and was similarly associated. Because the bone morphogenetic protein receptor II gene (BMRP2) is associated with primary PHT, we also investigated 5 SNPs (HCV1711012, HCV11510297, HCV2915656, HCV2915585, HCV2915597) in BMRP2. Two SNPs (HCV2915587 and HCV2915656) in high LD were associated with PHT (p=0.03). These preliminary data suggest the presence of genetic modifiers that significantly affect risk for both PHT and proteinuria in SCD. These data also support a common etiology of PHT and proteinuria, which are clinically associated, as noted by De Castro et al.

Single nucleotide polymorphisms (SNPs) of the β2adrenergic receptor (β2AR) gene (ADRB2) on chromosome 5 have been implicated in clinical variability of several cardiopulmonary disorders. Also, Hoppe et al. [2004] have recently found evidence that polymorphisms of ADRB2 correlate with the incidence of stroke in sickle cell disease (SCD). Stimulation of sickle red cell (SS RBC) adrenergic receptors by epinephrine, as well as stimulation of adenylate cyclase by forskolin, activates the B-CAM/LU laminin receptor [Hines et al. 2003]. There is also evidence that activation of B-CAM/LU on SS RBCs occurs in vivo [Zen et al. 2004]. However, SS RBCs from different individuals show variable baseline adhesion to laminin, as well as variable responsiveness to stimulation by epinephrine. We have found that butoxamine, which specifically inhibits the β2AR, blocked epinephrine-induced SS RBC adhesion by 91%, thus implicating β2ARs in activation of adhesion. Thus, we explored whether SNPs of the genes encoding β2AR (the mediator of epinephrine effects) and adenylate cyclase (a downstream effector of β2AR) affect the degree of baseline adhesion to laminin, as well as the degree to which RBCs upregulate adhesion in response to epinephrine. All samples studied were obtained from non-transfused Hb SS patients in steady state. SS RBCs from 14 of 20 patients studied showed increased adhesion in response to epinephrine, with a mean fold increase of 1.98±0.9. These results are similar to those previously described by Hines. Four SNPs of ADRB2 were examined by direct sequencing; two of these reside in the leader sequence, while the other two encode amino acid substitutions. Four intronic SNPs in ADCY6 were studied by Taqman assays. We found that the arg16gly substitution in β2AR is associated with elevation of baseline SS RBC adhesion measured at 1 dyne/cm2: arg/arg=122, arg/gly=149, gly/gly=262 (mean adherent cells/mm2, n=3, 11, 6 respectively, p=0.03 for gly/gly vs arg/arg + arg/gly). However, the arg16gly substitution was not found to be associated with a statistically significant difference either in the maximal adhesion observed after stimulation by epinephrine: arg/arg=116, arg/gly=263, gly/gly=286 (mean adherent cells/mm2), or in the fold increase observed after stimulation. In addition, two linked SNPs (HCV1244841 and RS3730070, r2=0.8) of ADCY6, which is encoded by chromosome 12, also had a statistically significant effect on baseline but not on epinephrine-stimulated SS RBC adhesion to laminin. For HCV1244841, mean baseline adhesion to laminin at 1 dyne/cm2was 118 and 303 cells/mm2for AA+AG and GG, respectively (n=13 and 6, p=0.007), while for RS3730070 mean baseline adhesion was 130 and 327 cells/mm2for CC+CG and GG, respectively (n=15 and 5, p=0.01). However, these ADCY6 SNPs also showed no statistically significant effect on epinephrine-stimulated adhesion. Overall, both the β2AR and ADCY6 polymorphisms appeared to significantly affect baseline adhesion but not epinephrine-stimulated adhesion in this study. We conclude that β2AR and adenylate cyclase polymorphisms affect red cell adhesive function. We theorize that signaling pathway polymorphisms, by affecting baseline activation of SS RBC adhesion, may influence the severity of SCD.

Single nucleotide polymorphisms (SNPs) of the β2 adrenergic receptor (β2AR) gene (ADRB2) on chromosome 5 have been implicated in clinical variability of several cardiopulmonary disorders. Also, Hoppe et al. [2004] have recently found evidence that polymorphisms of ADRB2 correlate with the incidence of stroke in sickle cell disease (SCD). Stimulation of sickle red cell (SS RBC) adrenergic receptors by epinephrine, as well as stimulation of adenylate cyclase by forskolin, activates the B-CAM/LU laminin receptor [Hines et al. 2003]. There is also evidence that activation of B-CAM/LU on SS RBCs occurs in vivo [Zen et al. 2004]. However, SS RBCs from different individuals show variable baseline adhesion to laminin, as well as variable responsiveness to stimulation by epinephrine. We have found that butoxamine, which specifically inhibits the β2AR, blocked epinephrine-induced SS RBC adhesion by 91%, thus implicating β2ARs in activation of adhesion. Thus, we explored whether SNPs of the genes encoding β2AR (the mediator of epinephrine effects) and adenylate cyclase (a downstream effector of β2AR) affect the degree of baseline adhesion to laminin, as well as the degree to which RBCs upregulate adhesion in response to epinephrine. All samples studied were obtained from non-transfused Hb SS patients in steady state. SS RBCs from 14 of 20 patients studied showed increased adhesion in response to epinephrine, with a mean fold increase of 1.98±0.9. These results are similar to those previously described by Hines. Four SNPs of ADRB2 were examined by direct sequencing; two of these reside in the leader sequence, while the other two encode amino acid substitutions. Four intronic SNPs in ADCY6 were studied by Taqman assays. We found that the arg16gly substitution in β2AR is associated with elevation of baseline SS RBC adhesion measured at 1 dyne/cm2: arg/arg=122, arg/gly=149, gly/gly=262 (mean adherent cells/mm2, n=3, 11, 6 respectively, p=0.03 for gly/gly vs arg/arg + arg/gly). However, the arg16gly substitution was not found to be associated with a statistically significant difference either in the maximal adhesion observed after stimulation by epinephrine: arg/arg=116, arg/gly=263, gly/gly=286 (mean adherent cells/mm2), or in the fold increase observed after stimulation. In addition, two linked SNPs (HCV1244841 and RS3730070, r2=0.8) of ADCY6, which is encoded by chromosome 12, also had a statistically significant effect on baseline but not on epinephrine-stimulated SS RBC adhesion to laminin. For HCV1244841, mean baseline adhesion to laminin at 1 dyne/cm2 was 118 and 303 cells/mm2 for AA+AG and GG, respectively (n=13 and 6, p=0.007), while for RS3730070 mean baseline adhesion was 130 and 327 cells/mm2 for CC+CG and GG, respectively (n=15 and 5, p=0.01). However, these ADCY6 SNPs also showed no statistically significant effect on epinephrine-stimulated adhesion. Overall, both the β2AR and ADCY6 polymorphisms appeared to significantly affect baseline adhesion but not epinephrine-stimulated adhesion in this study. We conclude that β2AR and adenylate cyclase polymorphisms affect red cell adhesive function. We theorize that signaling pathway polymorphisms, by affecting baseline activation of SS RBC adhesion, may influence the severity of SCD.

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