A system for the determination of planar force vectors from spontaneously active chicken embryos.

Generally, a combination of kinematic, electromyographic (EMG), and force measurements are used to understand how an organism generates and controls movement. The chicken embryo has been a very useful model system for understanding the early stages of embryonic motility in vertebrates. Unfortunately, the size and delicate nature of embryos makes studies of motility during embryogenesis very challenging. Both kinematic and EMG recordings have been achieved in embryonic chickens, but two-dimensional force vector recordings have not. Here, we describe a dual-axis system for measuring force generated by the leg of embryonic chickens. The system employs two strain gauges to measure planar forces oriented with the plane of motion of the leg. This system responds to forces according to the principles of Pythagorean geometry, which allows a simple computational program to determine the force vector (magnitude and direction) generated during spontaneous motor activity. The system is able to determine force vectors for forces >0.5 mN accurately and allows for simultaneous kinematic and EMG recordings. This sensitivity is sufficient for force vector measurements encompassing most embryonic leg movements in midstage chicken embryos allowing for a more complete understanding of embryonic motility. Variations on this system are discussed to enable nonideal or alternative sensor arrangements and to allow for translation of this approach to other delicate model systems.

Avulsion of the perforating branch of the peroneal artery secondary to an ankle sprain: a cause of acute compartment syndrome in the leg.

In this report, we describe the case of an adult male who developed an acute compartment syndrome localized to the anterior compartment of the leg following an ankle sprain. Compartment syndrome in association with ankle sprain is unusual, and has been previously described in association with avulsion of the perforating peroneal artery. Because of the potential for severe morbidity, we feel that it is important to make foot and ankle surgeons aware of this unusual injury.

Factors associated with the progression of fibrosis on liver biopsy in Alaska Native and American Indian persons with chronic hepatitis C.

BACKGROUND: Various factors influence the development and rate of fibrosis progression in chronic hepatitis C virus (HCV) infection. OBJECTIVES: To examine factors associated with fibrosis in a longterm outcomes study of Alaska Native/American Indian persons who underwent liver biopsy, and to examine the rate of fibrosis progression in persons with subsequent biopsies. METHODS: A cross-sectional analysis of the demographic, inflammatory and viral characteristics of persons undergoing liver biopsy compared individuals with early (Ishak fibrosis score of lower than 3) with those with advanced (Ishak score of 3 or greater) fibrosis. Persons who underwent two or more biopsies were analyzed for factors associated with fibrosis progression. RESULTS: Of 253 HCV RNA-positive persons who underwent at least one liver biopsy, 76 (30%) had advanced fibrosis. On multivariate analysis, a Knodell histological activity index score of 10 to 14 and an alpha-fetoprotein level of 8 ng/mL or higher were found to be independent predictors of advanced liver fibrosis (P<0.0001 for each). When surrogate markers of liver inflammation (alanine aminotransferase, aspartate aminotransferase/alanine aminotransferase ratio and alpha-fetoprotein) were removed from the model, type 2 diabetes mellitus (P=0.001), steatosis (P=0.03) and duration of HCV infection by 10-year intervals (P=0.02) were associated with advanced fibrosis. Among 52 persons who underwent two or more biopsies a mean of 6.2 years apart, the mean Ishak fibrosis score increased between biopsies (P=0.002), with progression associated with older age at initial biopsy and HCV risk factors. CONCLUSIONS: The presence of type 2 diabetes mellitus, steatosis and duration of HCV infection were independent predictors of advanced fibrosis in the present cohort, with significant fibrosis progression demonstrated in persons who underwent serial biopsies.

Persistence of antibody to hepatitis A virus 10 years after vaccination among children and adults.

BACKGROUND: Hepatitis A vaccination is effective in preventing disease. However, the duration of protection after vaccination is unknown. METHODS: We enrolled persons who responded to a 3-dose primary series of hepatitis A vaccine. For adults, the first dose was 720 ELISA units (EU) of hepatitis A vaccine, readministered at 1 and 12 months after the first vaccination (hereafter, "0-1-12 months"); for children aged 3-6 years, the first dose was 360 EU, readministered according to 1 of 3 vaccination schedules: 1 and 2 months after the first vaccination ("0-1-2 months"), 1 and 6 months after the first vaccination ("0-1-6 months"), or 1 and 12 months after vaccination ("0-1-12 months"). Specimens collected 1 month and 1-10 years after vaccination were tested for antibody to hepatitis A virus (anti-HAV) by ELISA. Long-term antibody persistence was estimated by using the observed rate of decline in geometric mean concentration (GMC). RESULTS: A total of 144 children and 128 adults were enrolled. Children vaccinated at 0-1-2 months had a significantly lower GMC of antibody than children vaccinated at 0-1-12 months, but this difference was statistically significant only through 4 years of follow-up. All 67 children tested at 10 years and 25 (96%) of 26 adults tested at 8-9 years had detectable anti-HAV. The estimated duration of antibody persistence was 21-27 years, depending on the vaccination schedule. CONCLUSIONS: Anti-HAV persists in adults and children for more than 10 years after the primary vaccination series. Additional studies are needed to evaluate the duration of antibody persistence beyond 10 years and to assess the long-term immunogenicity of the currently recommended 2-dose schedule.

No significant role for angiogenesis in nasal polyposis.

BACKGROUND: Nasal polyposis is a common disease of which little is currently known. Recent studies have shown up-regulation of several proangiogenic factors. The aim of this study was to assess and quantify how much angiogenesis occurs in nasal polyps and therefore whether angiogenesis is involved in the etiology of polyposis. METHODS: Biopsy specimens of polyp tissue and inferior turbinate (IT) were taken from patients undergoing polypectomy and compared with IT samples from control patients. Five patients were used per study group. Biopsy specimens were either stained with a fluorescent lectin for confocal microscopy or snap frozen and sectioned for histology for the examination of multiple measures of angiogenesis. RESULTS: No significant differences in capillary density, capillary-associated proliferation, capillary surface density, or capillary volume density were seen between the three study groups, and the regression of surface density versus volume density described a linear relationship. Polyp samples showed increases in capillary diameter and interstitial proliferation. CONCLUSION: These results show no active angiogenesis occurring in the polyp or changes in capillary bed architecture, although capillaries seem more edematous in the polyp. As the capillary supply increases in line with the physiological needs of the growing polyp, we conclude that angiogenesis is not a driving force in the etiology of nasal polyposis.

Differences in response to a hepatitis B vaccine booster dose among Alaskan children and adolescents vaccinated during infancy.

BACKGROUND: The duration of protection provided by hepatitis B vaccination is unknown, but the presence of immune memory can be evaluated indirectly by measuring the immune response to a booster dose of vaccine. METHODS: Participants included 74 adolescents (aged 11.7-14.9 years) who had received a plasma-derived 3-dose primary vaccine series and 138 adolescents (aged 10.0-14.7 years) and 166 children (aged 5.0-7.0 years) who received a recombinant 3-dose primary vaccine series. All were born to hepatitis B surface antigen-negative mothers and had received the first dose of hepatitis B vaccine within 7 days of birth. The proportion of participants with serologic evidence of protective immunity (antibody to hepatitis B surface antigen > or = 10 mIU/mL) at baseline (prebooster), the proportion who developed an anamnestic response (increase to > or = 10 mIU/mL or at or more than fourfold increase in antibody to hepatitis B surface antigen to > 10 mIU/mL), and the geometric mean concentration by 1, 2, and 4 weeks after a 5-microg recombinant vaccine booster dose were determined. RESULTS: No participant had evidence of chronic hepatitis B virus infection. Overall, 99% of the group of children who received recombinant hepatitis B vaccine, 83% of the group of adolescents who received recombinant hepatitis B vaccine, and 69% of the group of adolescents who received the plasma-derived vaccine had an anamnestic response to a booster dose; among responders, the geometric mean concentration at 2 weeks postbooster was 3360 and 128 mIU/mL among adolescents who received plasma-derived vaccine with antibodies to hepatitis B surface antigen > or = 10 and < 10 mIU/mL at baseline, respectively, compared with 1283 and 369 mIU/mL among adolescents who received recombinant hepatitis B vaccine and 5091 and 696 mIU/mL for children who received recombinant hepatitis B vaccine. The anamnestic response rate at 2 weeks postbooster among participants with antibodies to hepatitis B surface antigen < 10 mIU/mL at baseline was inversely associated with age; 97% of 5-year-olds responded compared with 60% of 14-year-olds. CONCLUSIONS: Although most participants responded to a booster dose of hepatitis B vaccine, the significance of the increased proportion of nonresponses among older adolescents might indicate waning immune memory.

Hepatitis B-associated vasculitis in Alaska Natives: viral genotype, clinical and serologic outcome.

BACKGROUND: The highest incidence of hepatitis B virus (HBV)-associated vasculitis in the world has been reported in Alaska Natives. We examined the incidence of HBV-associated vasculitis before and after mass HBV vaccine immunization and the association between HBV genotype and vasculitis in a population-based cohort study in Alaska natives chronically infected with HBV. METHODS: Genotyping was performed in vasculitis cases and 644 hepatitis B-positive controls without vasculitis using polymerase chain reaction and sequencing of the S gene. Occurrence of HBV vasculitis from 1974 to 2004 was calculated. HBV vasculitis patients and controls were also tested for basal core promoter and precore mutations. RESULTS: Fifteen cases of HBV-associated vasculitis were identified: 13 (86%) had genotype D and one each genotype A and F. Genotype D was more commonly found in patients with vasculitis than controls [odd ratio (OR)=5.9, confidence interval (95% CI) 1.2, 21.8; P<0.015). CONCLUSIONS: HBV-associated vasculitis was associated with genotype D.

Results of a general hepatitis C lookback program for persons who received blood transfusions in a neonatal intensive care unit between January 1975 and July 1992.

OBJECTIVE: To notify persons who received a blood transfusion in a neonatal intensive care unit between January 1975 and July 1992 of their risk for hepatitis C infection and to encourage them to seek hepatitis C antibody testing. DESIGN: Neonatal intensive care unit, blood bank, and public access records were queried to identify current mailing addresses and persons deceased. All persons were notified by letter. SETTING: Anchorage, Alaska. PARTICIPANTS: Persons who received health care in an integrated health care system, the Alaska Native Medical Center, or in the private sector. Main Exposure Transfusion in the neonatal period. MAIN OUTCOME MEASURES: Prevalence of test results positive for the hepatitis C virus antibody and RNA and awareness of having received a blood transfusion in a neonatal intensive care unit. RESULTS: Alaska Native Medical Center (n = 401) and private sector (n = 1396) persons were targeted for notification. Letters were mailed to 277 Alaska Native Medical Center (69%) and 374 private sector (27%) persons, with 151 (55%) and 65 (17%) screened for hepatitis C, respectively. Among those screened (n = 216), 7 (3%) were hepatitis C antibody positive, with 6 (<3%) also hepatitis C virus-RNA positive. Among 147 persons who responded, 75 (51%) were unaware they had received a transfusion. CONCLUSIONS: Compared with the private sector, a higher proportion of persons were identified and tested from the integrated health care system and more than half of respondents were unaware of their transfusion history. It would be prudent to screen neonatal intensive care unit patients who received transfusions before July 1992 for hepatitis C virus infection.

Hepatitis B virus genotypes in Alaska Native people with hepatocellular carcinoma: preponderance of genotype F.

BACKGROUND: The development of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B virus (HBV) infection has been associated with specific HBV genotypes and the presence of specific mutations. METHODS: From a cohort of Alaska Native people with chronic HBV infection, we genotyped 47 patients with HCC and 1129 patients without HCC, and we tested patients with HCC and control patients for mutations in the basal core promoter and precore regions. RESULTS: Genotype F was found in 68% of patients with HCC, versus 18% of those without HCC (P<.001). For patients with genotype F, the median age at diagnosis of HCC was lower than that for patients with other genotypes (22.5 vs. 60 years, respectively; P=.002). Overall, there were no significant differences in the number of basal core promoter and precore region mutations between patients with HCC and control patients. CONCLUSIONS: We found a significant association between genotype F and the development of HCC among Alaska Native people with chronic HBV infection but no significant association between HCC and basal core promoter or precore mutations in genotype F.

VEGF trap abolishes shear stress- and overload-dependent angiogenesis in skeletal muscle.

OBJECTIVE: To determine which elements of the angiogenic process are controlled by VEGF under physiological conditions. METHODS: VEGF Trap was administered at 10 mg x kg(-1) by subcutaneous injection twice weekly to mice, which were subject to one of two established angiogenic stimuli: (1) increasing blood flow by administration of prazosin (50 mg L(-1)); (2) muscle overload by extirpation of a synergist. Angiogenesis was determined by calculating capillary to fiber ratio (C:F), and proliferating cell nuclear antigen (PCNA) staining localized to capillaries or the interstitium used to measure cell proliferation. Characteristic ultrastructural changes were quantified using electron microscopy. RESULTS: Administration of VEGF Trap abolished the increases in C:F seen in both models, and prevented growth of luminal filopodia and large vacuole formation. Overload-induced proliferation associated with capillaries was reduced, along with endothelial cell number abluminal sprouts and basement membrane breakage. However, interstitial proliferation remained high, along with the increased capillary association of pericytes and fibroblasts. CONCLUSIONS: VEGF is required for both models of angiogenesis, although some morphological changes lie upstream, or are independent of, VEGF involvement. The abolition of angiogenesis in a model unaffected by NO inhibition shows that NO is not required for all VEGF-dependent angiogenesis in vivo.

Differential gene and protein expression in abluminal sprouting and intraluminal splitting forms of angiogenesis.

In adult skeletal muscle, abluminal sprouting or longitudinal splitting of capillaries can be initiated separately by muscle overload and elevated microcirculation shear stress respectively. In the present study, gene and protein expression patterns associated with the different forms of angiogenesis were examined using a targeted gene array (Superarray), validated by quantitative RT (reverse transcription)-PCR and immunoblots. Sprouting angiogenesis induced large changes in expression levels in genes associated with extracellular matrix remodelling, such as MMP-2 (matrix metalloproteinase-2), TIMP (tissue inhibitor of metalloproteinases), SPARC (secreted protein, acidic and rich in cysteine) and thrombospondin. Changes in neuropilin, midkine and restin levels, which may underpin changes in endothelial morphology, were seen during splitting angiogenesis. Up-regulation of VEGF (vascular endothelial growth factor), Flk-1, angiopoietin-2 and PECAM-1 (platelet/endothelial cell adhesion molecule-1) was seen in both forms of angiogenesis, representing a common angiogenic response of endothelial cells. In conclusion, the present study demonstrates that general angiogenic signals from growth factors can be influenced by the local microenvironment resulting in differing forms of capillary growth to produce a co-ordinated expansion of the vascular bed.

A differential role for nitric oxide in two forms of physiological angiogenesis in mouse.

NO plays a role in a variety of in vitro models of angiogenesis, although confounding effects of NO on non-endothelial tissues make its role during in vivo angiogenesis unclear. We therefore examined the effects of NO on two physiological models of angiogenesis in mouse skeletal muscle: (1) administration of prazosin (50 mg l-1) thereby increasing blood flow; and (2) muscle overload from surgical ablation of a functional synergist. These models induce angiogenesis via longitudinal splitting and capillary sprouting, respectively. Administration of NG-nitro-L-arginine (L-NNA) abolished the increase in capillary to fibre ratio (C:F) in response to prazosin administration, along with the increases in luminal filopodia and large endothelial vacuoles. L-NNA prevented luminal filopodia and vacuolisation in response to extirpation, but had no effect on abluminal sprouting, and little effect on C:F. Comparison of mice lacking endothelial (eNOS-/-) and neuronal NO synthase (nNOS-/-) showed that longitudinal splitting is eNOS-dependent, and Western blotting demonstrated an increase in eNOS but not inducible NOS (iNOS) expression. These data show that there are two pathways of physiological angiogenesis in skeletal muscle characterised by longitudinal splitting and capillary sprouting, respectively. NO generated by eNOS plays an essential role in splitting but not in sprouting angiogenesis, which has important implications for angiogenic therapies that target NO.

Results of a hepatitis C general transfusion lookback program for patients who received blood products before July 1992.

BACKGROUND: The Centers for Disease Control and Prevention recommend hepatitis C virus (HCV) antibody (anti-HCV) screening for persons who received blood products before July 1992. A general transfusion lookback program was implemented to identify, counsel, and screen persons who received transfusions at the Alaska Native Medical Center between January 1980 and July 1992. STUDY DESIGN AND METHODS: Hard-copy transfusion records data were entered, and available databases were queried to identify deceased patients and the mailing address of those living. Patients were notified by letter of their HCV risk and encouraged to seek counseling and testing. Serum samples were screened for anti-HCV and HCV RNA, and program costs were estimated. RESULTS: Overall, 3169 transfusion recipients were identified, with 1356 (43%) living and targeted for notification. Of 764 patients notified and screened by this program, 41 (5%) were anti-HCV-positive and 19 (2%) were HCV RNA-positive. There was a higher probability of detecting anti-HCV with each subsequent increase of a transfusion event. Among 298 lookback patients, 33 percent were unaware of having received a blood transfusion. The estimated cost per person sent notification was US$57 and to detect an anti-HCV-positive case it was US$3146. CONCLUSION: This general transfusion lookback program successfully notified and screened patients at a reasonable cost. Further investigation would be helpful in determining the role these programs or other measures could play in promoting HCV screening in persons receiving transfusions before July 1992, especially among those who are unaware of their transfusion history.

Estimating the date of hepatitis C virus infection from patient interviews and antibody tests on stored sera.

OBJECTIVES: Studies on the natural history and outcome of chronic hepatitis C virus (HCV) infection differ regarding the proportion of persons who develop serious sequelae over time. Most of these studies use an estimated date of HCV infection based on risk factor data obtained from patient interviews. The date of HCV infection is often estimated using the year of a pre-1992 blood transfusion (BT), or the first year of injecting drug use (IDU). We sought to determine the accuracy of these dates obtained by interview. METHODS: We compared BT dates reported by patients in a long-term HCV outcome study to dates confirmed in a BT-Lookback project, and also compared the reported first year of IDU to seroconversion dates estimated from HCV tests on historical sera. RESULTS: Of 28 BT recipients who were interviewed in the HCV outcome study and identified in the Lookback project, 14 (50%; 95% CI: 31-69%) were unaware they had received a BT. Of 25 persons identified in the BT-Lookback project with historical sera available, 9 (36%; 95% CI: 19-57%) had anti-HCV results that did not correlate with their confirmed BT date. Of 216 persons with a history of IDU and historical serum samples available, 66 (31%; 95% CI: 25-37%) had anti-HCV results that did not correlate with their reported first year of IDU. CONCLUSIONS: Inaccuracies in the length of HCV could occur in outcome studies that rely on patient recall of risk-factor history. Statistical methods that incorporate the uncertainty in assigning infection date are needed.

Hepatitis A vaccine: immunogenicity following administration of a delayed immunization schedule in infants, children and adults.

Current immunization schedules for hepatitis A vaccine specify administration of a booster within 6-12 or 6-18 months of the primary dose. However, there may be circumstances that disrupt this schedule and the efficacy of administering a booster beyond the recommended time is a practical concern for healthcare providers. In this study, a booster was administered to 268 participants (137: <18 years old), an average of 27 months (range 20-31) after the primary dose. In those tested after the booster, the median anti-HAV GMT was 1544 milli-international units per milliliter (mIU/ml). Response to a delayed booster was strong in children over 2 years old (GMT 1500-1960 mIU/ml) and adults (GMT 1622 mIU/ml), but was significantly lower in children under 2 years old (GMT 1109 mIU/ml). Findings suggest a booster administered 20-31 months after the primary dose is immunogenic and GMT in persons >2 years of age were comparable to those seen in adults and children who receive hepatitis A vaccine per schedule.

Prevalence of autoimmune liver disease in Alaska Natives.

OBJECTIVE: There is limited information on the prevalence of autoimmune liver disease in nonwhite populations. We conducted a population-based study on the prevalence of autoimmune liver diseases in Alaska natives. METHODS: Clinical records from 1984 to July, 2000 were reviewed to identify Alaska natives with autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis, autoimmune cholangitis, and overlap syndromes of two of the above. AIH was defined as definite or probable, based on criteria established by the International Autoimmune Hepatitis Group. The diagnosis of PBC was based on a positive antimitochondrial antibody of > or = 1: 40, biochemical evidence of cholestasis, and compatible liver biopsy. Autoimmune cholangitis was defined as PBC but without a positive antimitochondrial antibody. Primary sclerosing cholangitis was diagnosed on the basis of cholangiogram. RESULTS: Seventy-seven patients with possible autoimmune liver disease were identified. Of these, 42 had definite and seven probable AIH. At presentation, 34.7% of patients with AIH presented with acute icteric hepatitis, and 65.3% were asymptomatic. Persons presenting with mild or no symptoms were more likely to have moderate to severe fibrosis on liver biopsy than those presenting with jaundice. Eighteen persons were diagnosed with PBC, five with autoimmune cholangitis, five with overlap syndrome, and none with primary sclerosing cholangitis. The combined point prevalence of AIH Alaska natives was 42.9/100,000 (95% CI = 31-57.7). The prevalence of PBC was 16/100,000 (95% CI = 12.9-25.4). CONCLUSIONS: This population-based study demonstrates that the prevalence rates of AIH and PBC in Alaska natives are comparable with reported rates in other populations.