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1.  Hydrolytic stability of pneumococcal group 6 (type 6A and 6B) capsular polysaccharides. 
Infection and Immunity  1982;37(1):89-103.
The hydrolyses of the immunologically cross-reactive and constitutionally isomeric group 6 pneumococcal polysaccharides, types 6A and 6B, were investigated by 31P nuclear magnetic resonance spectroscopy, gel filtration through Sepharose 4B, reducing-sugar analysis, and rocket immunoelectrophoresis. Phosphorus nuclear magnetic resonance spectroscopy showed that cleavage of the repeating-unit phosphodiester linkages at pH 10, 60 degrees C was considerably faster (greater than 10(3) ) for the type 6A than the type 6B polysaccharide. Under these reaction conditions, 31P nuclear magnetic resonance kinetic measurements showed that the Na+ form of the type 6A polysaccharide underwent phosphodiester-linkage hydrolysis two times slower than the corresponding Ca+2 form; a stoichiometrically excess amount of Ca+2 caused a 30-fold enhancement of the latter hydrolysis rate. The spectroscopic characterization of phosphorus-containing end groups resulting from hydrolysis of the type 6A polymer provided additional mechanistic information. Heating the type 6A and 6B polysaccharides at 56 degrees C for various times led to gel filtration coefficients of distribution (Kd values) which indicated that the type 6A material underwent size reductions considerably faster than did the type 6B antigen; these increased Kd values qualitatively correlated with the loss of immunochemical reactivity measured by rocket immunoelectrophoresis. The application of a statistical theory to the depolymerization of the type 6A and 6B polysaccharides was consistent with random bond cleavage, as evidenced by the calculated versus measured gel filtration patterns. Although the molecular changes causing the size reductions were not fully elaborated, it was established that the acetal linkages of the type 6A and 6B polysaccharides were comparatively resistant to hydrolysis and that depolymerization by hydrolysis of the phosphodiester linkage was a major factor only in the type 6A structure. It was concluded that the hydrolytic stability of the type 6B antigen would favor its use in the polyvalent pneumococcal vaccine rather than the cross-reactive, but comparatively unstable, type 6A polysaccharide, if all other factors are equal.
PMCID: PMC347495  PMID: 7107011
2.  Domain wall motion in magnetic nanowires: an asymptotic approach 
We develop a systematic asymptotic description for domain wall motion in one-dimensional magnetic nanowires under the influence of small applied magnetic fields and currents and small material anisotropy. The magnetization dynamics, as governed by the Landau–Lifshitz–Gilbert equation, is investigated via a perturbation expansion. We compute leading-order behaviour, propagation velocities and first-order corrections of both travelling waves and oscillatory solutions, and find bifurcations between these two types of solutions. This treatment provides a sound mathematical foundation for numerous results in the literature obtained through more ad hoc arguments.
PMCID: PMC3857867  PMID: 24353468
micromagnetics; nanowires; domain wall motion
3.  Altered regional cardiac wall mechanics are associated with differential cardiomyocyte calcium handling due to nebulette mutations in preclinical inherited dilated cardiomyopathy 
Nebulette (NEBL) is a sarcomeric Z-disc protein involved in mechanosensing and force generation via its interaction with actin and tropomyosin-troponin complex. Genetic abnormalities in NEBL lead to dilated cardiomyopathy (DCM) in humans and animal models.
To determine the earliest preclinical mechanical changes in the myocardium and define underlying molecular mechanisms by which NEBL mutations lead to cardiac dysfunction.
Methods and Results
We examined cardiac function in 3-month-old non-transgenic (non-Tg) and transgenic (Tg) mice (WT-Tg, G202R-Tg, A592E-Tg) by cardiac magnetic resonance (CMR) imaging. Contractility and calcium transients were measured in isolated cardiomyocytes. A592E-Tg mice exhibited enhanced in vivo twist and untwisting rate compared to control groups. Ex vivo analysis of A592E-Tg cardiomyocytes showed blunted calcium decay response to isoproterenol. CMR imaging of G202R-Tg mice demonstrated reduced torsion compared to non-Tg and WT-Tg, but conserved twist and untwisting rate after correcting for geometric changes. Ex vivo analysis of G202R-Tg cardiomyocytes showed elevated calcium decay at baseline and a conserved contractile response to isoproterenol stress. Protein analysis showed decreased α-actinin and connexin43, increased cardiac troponin I phosphorylation at baseline in G202R-Tg, providing a molecular mechanism for enhanced ex vivo calcium decay. Ultrastructurally, G202R-Tg cardiomyocytes exhibited increased I-band and sarcomere length, desmosomal separation, and enlarged t-tubules. A592E-Tg cardiomyocytes also showed abnormal ultrastructural changes and desmin downregulation.
This study showed distinct effects of NEBL mutations on sarcomere ultrastructure, cellular contractile function, and calcium homeostasis in preclinical DCM in vivo. We suggest that these abnormalities correlate with detectable myocardial wall motion patterns.
PMCID: PMC3683841  PMID: 23632046
nebulette; dilated cardiomyopathy; sarcomere; cardiac magnetic resonance imaging; troponin; connexin43; desmin
4.  Molecular Mechanics of Cardiac Myosin Binding Protein-C in Native Thick Filaments # 
Science (New York, N.Y.)  2012;337(6099):1215-1218.
The heart's pumping capacity results from highly regulated interactions of actomyosin molecular motors. Mutations in the gene for a potential regulator of these motors, cardiac myosin binding protein-C (cMyBP-C), cause hypertrophic cardiomyopathy. However, cMyBP-C's ability to modulate cardiac contractility is not well understood. Using single particle fluorescence imaging techniques, transgenic protein expression, proteomics, and modeling, we found that cMyBP-C slowed actomyosin motion generation in native cardiac thick filaments. This mechanical effect was localized to where cMyBP-C resides within the thick filament (i.e. the C-zones) and was modulated by phosphorylation and site-specific proteolytic degradation. These results provide molecular insight into why cMyBP-C should be considered a member of a tripartite complex with actin and myosin that allows fine tuning of cardiac muscle contraction.
PMCID: PMC3561468  PMID: 22923435
5.  Concurrent Change in Dehydroepiandrosterone Sulfate and Functional Performance in the Oldest Old: Results From the Cardiovascular Health Study All Stars Study 
The correlation between dehydroepiandrosterone sulfate (DHEAS) decline and age led to the hypothesis that DHEAS might be a marker of primary aging, though conflicting data from observational studies of mortality do not support this. We evaluated concurrent DHEAS and functional decline in a very old cohort to test if DHEAS change tracks with functional change during aging.
DHEAS and functional performance (gait speed, grip strength, Modified Mini-Mental State Examination [3MSE] score, and digit symbol substitution test [DSST] score) were measured in 1996–1997 and 2005–2006 in 989 participants in the Cardiovascular Health Study All Stars study (mean age 85.2 years in 2005–2006, 63.5% women and 16.5% African American). We used multivariable linear regression to test the association of DHEAS decline with functional decline.
After adjustment, each standard deviation decrease in DHEAS was associated with greater declines in gait speed (0.12 m/s, p = .01), grip strength (0.09 kg, p = .03), 3MSE score (0.13 points, p < .001), and DSST score (0.14 points, p = .001) in women only. Additional adjustment for baseline DHEAS attenuated the association with grip strength but did not alter other estimates appreciably, and baseline DHEAS was unassociated with functional decline.
In this cohort of very old individuals, DHEAS decline tracked with declines in gait speed, 3MSE score, and DSST score, but not grip strength, in women independent of baseline DHEAS level. DHEAS decline might be a marker for age-associated performance decline, but its relevance is specific to women.
PMCID: PMC2920580  PMID: 20466773
Aging; Biomarker; Dehydroepiandrosterone sulfate; Function
6.  Mesenteric venous collateral vessels mimicking cystic pancreatic neoplasm 
The British Journal of Radiology  2010;83(992):e175-e178.
We report an unusual case of intrapancreatic mesenteric venous collateral vessels following partial pancreatic surgical resection resembling pancreatic neoplasm upon greyscale sonographic and unenhanced CT examinations.
PMCID: PMC3473515  PMID: 20675462
7.  Differences in Sonographic Conspicuity According to Papillary Thyroid Cancer Subtype. Results from the Ukrainian-American Cohort Study of Thyroid Cancer and Other Thyroid Diseases Following the Chornobyl Accident 
AJR. American journal of roentgenology  2008;191(6):W293-W298.
Over time, the histology of papillary thyroid cancers detected in a population exposed to radiation at Chornobyl has shifted from a more aggressive toward less aggressive subtypes. This change may reflect biologic behavior, but could also be influenced by the detectability of different subtypes. The study objective was to identify whether there is any relationship between the conspicuity of ultrasound-detected papillary cancers and histologic subtype.
Ultrasound images of 84 papillary cancers occurring in young people exposed to radiation at Chornobyl were each given a conspicuity score using a subjective 1 to 5 scale by four independent expert readers blinded to histologic subtype. The effects of tumor subtype, tumor encapsulation, reader, machine type, and size on conspicuity were determined using ANOVA and Spearman correlations.
Cancer subtype was related (p<0.01) to conspicuity. The relatively aggressive solid subtype of papillary carcinoma was more conspicuous than the papillary, follicular, and mixed subtypes, p<0.05. The other subtypes did not differ significantly from each other in conspicuity. Conspicuity was not significantly related to nodule size, degree of encapsulation, age and gender of the subject, or machine type. Although the mean conspicuity score for each reader differed significantly, reliability of conspicuity judgments across readers was fair.
In subjects exposed to the Chornobyl accident, the solid subtype of papillary carcinoma appears to be more conspicuous on ultrasound than other subtypes. It is therefore possible that the observed change in subtype over time in this repeatedly screened population is influenced by differences in nodule conspicuity.
PMCID: PMC2841356  PMID: 19020218
8.  Incidence of fractures compared to cardiovascular disease and breast cancer: the Women’s Health Initiative Observational Study 
To compare the absolute risk of fracture to the risk of other conditions by race/ethnicity, we studied 83,724 women, aged 70–79. The projected number of fractures was similar to or exceeded the combined number of cardiovascular events and breast cancers. Osteoporosis prevention efforts should target women of all ethnicities.
The relative risk of fracture is lower in non-white compared to white women but the absolute risk of fracture in comparison to other common chronic conditions is uncertain.
We performed a prospective cohort study of 83,724 women, age 50–79 years. Cardiovascular disease (CVD), invasive breast cancer and all fractures were identified over an average of 7.7±2.6 years.
The incidence of fracture, breast cancer, stroke and CVD varied across ethnicity. The annualized (%) incidence of fracture was greatest in whites (2.4%) and American Indians (2.8%) and lowest among blacks (1.3%). The majority of hip fractures occurred in white women. The projected number of women who will experience a fracture in one year exceeded the combined number of women who would experience invasive breast cancer or a broad category of CVD events in all ethnic groups except blacks. In 10,000 black women, an estimated 153 women would experience CVD, and 35 women, breast cancer compared to 126 women expected to fracture in one year.
The annual risk of suffering a fracture is substantial in women of all ethnicities. Osteoporosis prevention efforts should target all women irrespective of their race/ethnic backgrounds.
PMCID: PMC2663802  PMID: 18629572
Breast cancer; Cardiovascular disease; Fracture; Osteoporosis; Race/ethnicity; Women’s Health Initiative
9.  Excess type 2 diabetes in African-American women and men aged 40-74 and socioeconomic status: evidence from the Third National Health and Nutrition Examination Survey 
OBJECTIVE—To examine whether socioeconomic status (SES) explains differences in the prevalence of type 2 diabetes between African-American and non-Hispanic white women and men.
DESIGN—Cross sectional study of diabetes prevalence, SES, and other risk factors ascertained by physical examination and interview.
SETTING—Interviews were conducted in subjects' homes; physical examinations were conducted in mobile examination centres.
PARTICIPANTS—961 African-American women, 1641 non-Hispanic white women, 839 African-American men and 1537 non-Hispanic white men, aged 40 to 74 years, examined in the Third National Health and Nutrition Examination Survey (NHANES III), a representative sample of the non-institutionalised civilian population of the United States, 1988-1994.
MAIN RESULTS—Among women, African-American race/ethnicity was associated with an age adjusted odds ratio of 1.76 (95% confidence intervals 1.21, 2.57), which was reduced to 1.42 (95% confidence intervals 0.95, 2.13) when poverty income ratio was controlled. Controlling for education or occupational status had minimal effects on this association. When other risk factors were controlled, race/ethnicity was not significantly associated with type 2 diabetes prevalence. Among men, the age adjusted odds ratio associated with African-American race/ethnicity was 1.43 (95% confidence intervals 1.03, 1.99). Controlling for SES variables only modestly affected the odds ratio for African/American race/ethnicity among men, while adjusting for other risk factors increased the racial/ethnic differences.
CONCLUSIONS—Economic disadvantage may explain much of the excess prevalence of type 2 diabetes among African-American women, but not among men.

Keywords: diabetes mellitus; ethnic groups; socioeconomic factors
PMCID: PMC1731578  PMID: 11027198
10.  Response to health insurance by previously uninsured rural children. 
Health Services Research  1999;34(3):761-775.
OBJECTIVE: To examine the healthcare utilization and costs of previously uninsured rural children. DATA SOURCES/STUDY SETTING: Four years of claims data from a school-based health insurance program located in the Mississippi Delta. All children who were not Medicaid-eligible or were uninsured, were eligible for limited benefits under the program. The 1987 National Medical Expenditure Survey (NMES) was used to compare utilization of services. STUDY DESIGN: The study represents a natural experiment in the provision of insurance benefits to a previously uninsured population. Premiums for the claims cost were set with little or no information on expected use of services. Claims from the insurer were used to form a panel data set. Mixed model logistic and linear regressions were estimated to determine the response to insurance for several categories of health services. PRINCIPAL FINDINGS: The use of services increased over time and approached the level of utilization in the NMES. Conditional medical expenditures also increased over time. Actuarial estimates of claims cost greatly exceeded actual claims cost. The provision of a limited medical, dental, and optical benefit package cost approximately $20-$24 per member per month in claims paid. CONCLUSIONS: An important uncertainty in providing health insurance to previously uninsured populations is whether a pent-up demand exists for health services. Evidence of a pent-up demand for medical services was not supported in this study of rural school-age children. States considering partnerships with private insurers to implement the State Children's Health Insurance Program could lower premium costs by assembling basic data on previously uninsured children.
PMCID: PMC1089036  PMID: 10445901
11.  A mouse model of myosin binding protein C human familial hypertrophic cardiomyopathy. 
Journal of Clinical Investigation  1998;102(7):1292-1300.
Familial hypertrophic cardiomyopathy can be caused by mutations in genes encoding sarcomeric proteins, including the cardiac isoform of myosin binding protein C (MyBP-C), and multiple mutations which cause truncated forms of the protein to be made are linked to the disease. We have created transgenic mice in which varying amounts of a mutated MyBP-C, lacking the myosin and titin binding domains, are expressed in the heart. The transgenically encoded, truncated protein is stable but is not incorporated efficiently into the sarcomere. The transgenic muscle fibers showed a leftward shift in the pCa2+-force curve and, importantly, their power output was reduced. Additionally, expression of the mutant protein leads to decreased levels of endogenous MyBP-C, resulting in a striking pattern of sarcomere disorganization and dysgenesis.
PMCID: PMC508976  PMID: 9769321
12.  A truncated cardiac troponin T molecule in transgenic mice suggests multiple cellular mechanisms for familial hypertrophic cardiomyopathy. 
Journal of Clinical Investigation  1998;101(12):2800-2811.
Mutations in multiple cardiac sarcomeric proteins including myosin heavy chain (MyHC) and cardiac troponin T (cTnT) cause a dominant genetic heart disease, familial hypertrophic cardiomyopathy (FHC). Patients with mutations in these two genes have quite distinct clinical characteristics. Those with MyHC mutations demonstrate more significant and uniform cardiac hypertrophy and a variable frequency of sudden death. Patients with cTnT mutations generally exhibit mild or no hypertrophy, but a high frequency of sudden death at an early age. To understand the basis for these distinctions and to study the pathogenesis of the disease, we have created transgenic mice expressing a truncated mouse cTnT allele analogous to one found in FHC patients. Mice expressing truncated cTnT at low (< 5%) levels develop cardiomyopathy and their hearts are significantly smaller (18-27%) than wild type. These animals also exhibit significant diastolic dysfunction and milder systolic dysfunction. Animals that express higher levels of transgene protein die within 24 h of birth. Transgenic mouse hearts demonstrate myocellular disarray and have a reduced number of cardiac myocytes that are smaller in size. These studies suggest that multiple cellular mechanisms result in the human disease, which is generally characterized by mild hypertrophy, but, also, frequent sudden death.
PMCID: PMC508871  PMID: 9637714
13.  Functional significance of cardiac myosin essential light chain isoform switching in transgenic mice. 
Journal of Clinical Investigation  1998;101(12):2630-2639.
The different functions of the ventricular- and atrial-specific essential myosin light chains are unknown. Using transgenesis, cardiac-specific overexpression of proteins can be accomplished. The transgenic paradigm is more useful than originally expected, in that the mammalian heart rigorously controls sarcomeric protein stoichiometries. In a clinical subpopulation suffering from heart disease caused by congenital malformations of the outflow tract, an ELC1v-->ELC1a isoform shift correlated with increases in cross-bridge cycling kinetics as measured in skinned fibers derived from the diseased muscle. We have used transgenesis to replace the ventricular isoform of the essential myosin light chain with the atrial isoform. The ELC1v--> ELC1a shift in the ventricle resulted in similar functional alterations. Unloaded velocities as measured by the ability of the myosin to translocate actin filaments in the in vitro motility assay were significantly increased as a result of the isoform substitution. Unloaded shortening velocity was also increased in skinned muscle fibers, and at the whole organ level, both contractility and relaxation were significantly increased. This increase in cardiac function occurred in the absence of a hypertrophic response. Thus, ELC1a expression in the ventricle appears to be advantageous to the heart, resulting in increased cardiac function.
PMCID: PMC508853  PMID: 9637696
14.  Detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins confers protection against otitis media in chinchillas. 
Infection and Immunity  1997;65(11):4488-4493.
Detoxified-lipooligosaccharide (dLOS)-protein conjugates from nontypeable Haemophilus influenzae (NTHi) elicited a significant rise of anti-LOS antibodies with bactericidal activity in rabbits (X.-X. Gu, C.-M. Tsai, T. Ueyama, S. J. Barenkamp, J. B. Robbins, and D. J. Lim, Infect. Immun. 64:4047-4053, 1996). In this study, we evaluated whether vaccination with the conjugates would protect against NTHi otitis media in chinchillas. Fifty-eight chinchillas received three subcutaneous or intramuscular injections of dLOS-conjugated tetanus toxoid, dLOS-conjugated high-molecular-weight proteins from NTHi, or saline (control) in Freund's adjuvant and then were challenged by intrabullar inoculation with 140 CFU of NTHi. All vaccinated animals responded with elevated serum titers of anti-LOS antibody, and 49% (19 of 39) demonstrated bactericidal activity against the homologous strain. Otitis media with culture-positive NTHi effusions developed in all 19 controls and 56% (22 of 39) of the vaccinated animals during a period of 21 days (P < 0.001). Bacterial counts of the middle ear effusions were lower in the vaccine groups than in the controls (P < 0.01). The incidences of infection in the unchallenged ear or inner ear were 26 or 28% in the vaccine groups and 53 or 58% in the controls (P < 0.05). The signs of infection observed by otoscopy were less severe in the vaccine groups than in the controls. There was no significant difference between the two vaccine groups. These data indicate that active immunization with LOS-based conjugates reduces the incidence of NTHi-induced otitis media.
PMCID: PMC175645  PMID: 9353024
15.  Cardiac compartment-specific overexpression of a modified retinoic acid receptor produces dilated cardiomyopathy and congestive heart failure in transgenic mice. 
Journal of Clinical Investigation  1997;100(8):1958-1968.
Retinoids play a critical role in cardiac morphogenesis. To examine the effects of excessive retinoid signaling on myocardial development, transgenic mice that overexpress a constitutively active retinoic acid receptor (RAR) controlled by either the alpha- or beta-myosin heavy chain (MyHC) promoter were generated. Animals carrying the alpha-MyHC-RAR transgene expressed RARs in embryonic atria and in adult atria and ventricles, but developed no signs of either malformations or disease. In contrast, beta-MyHC-RAR animals, where expression was activated in fetal ventricles, developed a dilated cardiomyopathy that varied in severity with transgene copy number. Characteristic postmortem lesions included biventricular chamber dilation and left atrial thrombosis; the incidence and severity of these lesions increased with increasing copy number. Transcript analyses showed that molecular markers of hypertrophy, alpha-skeletal actin, atrial natriuretic factor and beta-MyHC, were upregulated. Cardiac performance of transgenic hearts was evaluated using the isolated perfused working heart model as well as in vivo, by transthoracic M-mode echocardiography. Both analyses showed moderate to severe impairment of left ventricular function and reduced cardiac contractility. Thus, expression of a constitutively active RAR in developing atria and/ or in postnatal ventricles is relatively benign, while ventricular expression during gestation can lead to significant cardiac dysfunction.
PMCID: PMC508385  PMID: 9329959
16.  Synthesis and immunological properties of Vi and di-O-acetyl pectin protein conjugates with adipic acid dihydrazide as the linker. 
Infection and Immunity  1997;65(6):2088-2093.
The Vi capsular polysaccharide of Salmonella typhi, a licensed vaccine for typhoid fever in individuals > or = 5 years old, induces low and short-lived antibodies in children, and reinjection does not elicit booster responses at any age. Its immunogenicity was improved by binding Vi to proteins by using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) as a linker. Similar findings were observed with the structurally related, di-O-acetyl derivative of pectin [poly-alpha(1-->4)-D-GalpA] designated OAcP. Protein conjugates of Vi and OAcP were synthesized by carbodiimide-mediated synthesis with adipic acid dihydrazide (ADH) as the linker. Hydrazide groups were introduced into proteins (bovine serum albumin or recombinant Pseudomonas aeruginosa exoprotein A) by treatment with ADH and 1-ethyl-3(3-dimethylaminopropyl carbodiimide (EDC). The resultant adipic acid hydrazide derivatives (AH-proteins), containing 2.3 to 3.4% AH, had antigenic and physicochemical properties similar to those of the native proteins. The AH-proteins were bound to Vi and OAcP by treatment with EDC. The immunogenicity of Vi or OAcP, alone or as protein conjugates, was evaluated in young outbred mice and guinea pigs by subcutaneous injection of 2.5 and 5.0 microg, respectively, of polysaccharide, and antibodies were measured by enzyme-linked immunosorbent assay. All conjugates were significantly more immunogenic than Vi or OAcP alone and induced booster responses with 5- to 25-fold increases of antibodies. Vi conjugates were significantly more immunogenic than their OAcP analogs. A carboxymethyl derivative of yeast beta-glucan enhanced the anti-Vi response elicited by an OAcP conjugate but had no effect on the immunogenicity of Vi or of OAcP alone. Vi and OAcP conjugates synthesized by this scheme will be evaluated clinically.
PMCID: PMC175288  PMID: 9169736
17.  Synthesis, characterization, and immunological properties in mice of conjugates composed of detoxified lipopolysaccharide of Salmonella paratyphi A bound to tetanus toxoid with emphasis on the role of O acetyls. 
Infection and Immunity  1996;64(7):2709-2715.
Salmonella paratyphi A, the second most common cause of enteric fever in Southeast Asia, is a habitant of and a pathogen for humans only. Lipopolysaccharides (LPS) are both essential virulence factors and protective antigens for systemic infections caused by groups A, B, C, and D nontyphoidal salmonellae. The O-specific polysaccharide of S. paratyphi A is composed of a trisaccharide, -->2-alpha-D)-Manp-(1-->4)-alpha-L-Rhap-(1-->3)-alpha-D-Galp -(1-->, with a branch of D-paratose from the C-3 of alpha-D-mannose, and the C-3 of beta-L-rhamnose is partially O acetylated (C. G. Hellerqvist, B. Lindberg, K. Samuelsson, and A. A. Lindberg, Acta Chem. Scand. 25:955-961, 1971). On the basis of data from our investigational vaccines for enteric bacterial pathogens, including group B salmonellae (D. C. Watson, J. B. Robbins, and S. C. Szu, Infect. Immun. 60:4679-4686, 1992), conjugates composed of the detoxified LPS of S. paratyphi A bound to tetanus toxoid (TT) were prepared by several schemes. LPS was detoxified with acetic acid or with hydrazine; the latter removed O acetyls from the O-specific polysaccharide. The detoxified polysaccharides were activated with cyanogen bromide (CNBr) or with 1-cyano-4-dimethylaminopyridinium tetratfluoroborate (CDAP) and bound to TT with or without a spacer. Solutions of 2.5 microgram of saccharide, alone or as a conjugate, were injected subcutaneously into young mice, and LPS and TT antibodies were measured by enzyme-linked immunosorbent assaying. A conjugate synthesized with higher-molecular-weight O-SP elicited the highest anti-LPS levels. Only conjugates with O acetyls elicited serum immunoglobulin G anti-LPS with bactericidal activity. There were no statistically significant differences between LPS antibody levels elicited by conjugates synthesized with or without a spacer. The conjugate with O-specific polysaccharide activated by CDAP and bound to TT without a spacer elicited the highest level of TT antibodies. Clinical evaluation (if S. paratyphi A conjugates is planned.
PMCID: PMC174130  PMID: 8698499
19.  Ablation of the murine alpha myosin heavy chain gene leads to dosage effects and functional deficits in the heart. 
Journal of Clinical Investigation  1996;98(8):1906-1917.
The alpha-myosin heavy chain (alpha-MyHC) is the major contractile protein expressed in the myocardium of adult mice. We have produced mice carrying a null mutation of alpha-MyHC by homologous recombination in murine ES cells. Homozygous null animals die between 11 and 12 d in utero of gross heart defects, while alpha-MyHC+/- heterozygotes survive and appear externally normal. The presence of a single functional alpha-MyHC+ allele in heterozygous animals results in reduced levels of the transcript and protein as well as fibrosis and alterations in sarcomeric structure. Examination of heart function using a working heart preparation revealed severe impairment of both contractility and relaxation in a subset of the alpha-MyHC+/- animals. Thus, two alpha-MyHC+ alleles are necessary for normal cardiac development, and hemizygosity for the normal allele can result in altered cardiac function.
PMCID: PMC507631  PMID: 8878443
20.  Safety and immunogenicity of investigational Shigella conjugate vaccines in Israeli volunteers. 
Infection and Immunity  1996;64(10):4074-4077.
The safety and immunogenicity of investigational conjugates, composed of the O-specific polysaccharides of Shigella sonnei and Shigella flexneri type 2a covalently bound to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), were evaluated in 192 Israeli soldiers. None had significant local reactions or fever. Fourteen days after injection, 90% of S. sonnei-rEPA recipients and 73 to 77% of S. flexneri-rEPA recipients had a fourfold or greater increase in serum immunoglobulin G (IgG) and IgA anti-lipopolysaccharide (anti-LPS) levels; at 2 years, these remained higher than at prevaccination (P < 0.01). There was a fourfold or greater increase in IgM anti-LPS in 20% of vaccinees at 2 weeks, but levels returned to prevaccination values at 6 to 12 months. IgG was the highest and most sustained class of LPS antibodies. Reinjection at day 42 did not boost antibody levels. Eighteen of 23 (78%) who received S. sonnei-rEPA and 13 of 19 (68%) who received S. flexneri-rEPA. had significant IgA-secreting cell responses. Significant IgG antibody-secreting cell responses were detected in 19 of 23 (83%) and 11 of 19 (58%) volunteers following vaccination with S. sonnei-rEPA and S. flexneri 2a-rEPA, respectively. On the basis of these data, further evaluation of the Shigella conjugates for protective efficacy in field trials in Israel was started.
PMCID: PMC174339  PMID: 8926071
21.  Synthesis, characterization, and immunologic properties of detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins. 
Infection and Immunity  1996;64(10):4047-4053.
Nontypeable Haemophilus influenzae (NTHi) is an important cause of otitis media in children and of pneumonitis in adults with depressed resistance. Lipooligosaccharide (LOS) is a major surface antigen of NTHi and elicits bactericidal and opsonic antibodies. We prepared detoxified LOS (dLOS) protein conjugates from NTHi for use as experimental vaccines. LOS from NTHi 9274 was treated with anhydrous hydrazine and had its toxicity reduced to clinically acceptable levels. dLOS was bound to tetanus toxoid (TT) or high- molecular-weight proteins (HMPs) from NTHi through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratio of the dLOS to protein carriers ranged from 26:1 to 50:1. The antigenicity of the conjugates was similar to that of the LOS alone as determined by double immunodiffusion. Subcutaneous or intramuscular injection of the conjugates elicited a 28- to 486-fold rise in the level of immunoglobulin G antibodies in mice to the homologous LOS after two or three injections and a 169- to 243-fold rise in the level of immunoglobulin G antibodies in rabbits after two injections. The immunogenicity of the conjugates in mice and rabbits was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced LOS antibodies induced complement-mediated bactericidal activity against the homologous strain 9274 and prototype strain 3189. These results indicate that a detoxified LOS-protein conjugate is a candidate vaccine for otitis media and pneumonitis caused by NTHi.
PMCID: PMC174335  PMID: 8926067
22.  Characterization of a human monoclonal immunoglobulin M (IgM) antibody (IgMBEN) specific for Vi capsular polysaccharide of Salmonella typhi. 
Infection and Immunity  1995;63(11):4429-4432.
A search for human monoclonal antibodies to protective antigens of bacteria revealed an immunoglobulin M lambda chain [IgM(lambda); designated IgMBEN] reactive with the Vi capsular polysaccharide of Salmonella typhi. Vi, a linear homopolymer of alpha(1-->4)GalApNAc that is O acetylated at C-3, is a licensed vaccine for typhoid fever. Immunologic properties of IgMBEN were compared to those of burro globulin prepared by intravenous injections of S. typhi (B339-340). IgMBEN and B339-340 yielded identical precipitin lines with Vi by double immunodiffusion. IgMBEN and B339-340 produced similar precipitation results with Vi and its derivatives prepared by de-O-acetylation, carboxyl reduction, and removal or replacement of the N-acetyl at C-2 with O-acetyl. B339-340 yielded maximal precipitation with Vi (0.41 mg of antibody per ml with 1.4 micrograms of Vi); next was carboxyl-reduced, O-acetylated Vi, which precipitated 0.325 mg of antibody per ml with 2.5 micrograms of Vi. IgMBEN yielded maximal precipitation with de-O-acetylated, carboxyl-reduced Vi (approximately 11.0 mg of antibody per ml with approximately 1.3 micrograms of antigen); next were de-O-acetylated Vi (9.89 mg/ml) and Vi (9.19 mg/ml). The precipitin curves and equivalence points of these three antigens were similar. Pneumococcus type 1, which contains GalApNAc, did not precipitate with Vi or its derivatives. These slight differences in specificity between IgMBEN and B339-340 were related to our proposed structure of Vi. We plan to use IgMBEN as a reference for measurement of vaccine-induced Vi antibodies.
PMCID: PMC173630  PMID: 7591081
23.  Physicians' beliefs and behaviour during a randomized controlled trial of episiotomy: consequences for women in their care. 
OBJECTIVE: To evaluate whether physicians' beliefs concerning episiotomy are related to their use of procedures and to differential outcomes in childbirth. DESIGN: Post-hoc cohort analysis of physicians and patients involved in a randomized controlled trial of episiotomy. SETTING: Two tertiary care hospitals and one community hospital in Montreal. PARTICIPANTS: Of the 703 women at low risk of medical or obstetric problems enrolled in the trial we studied 447 women (226 primiparous and 221 multiparous) attended by 43 physicians. Subjects attended by residents or nurses were excluded. MAIN OUTCOME MEASURES: Patients: intact perineum v. perineal trauma, length of labour, procedures used (instrumental delivery, oxytocin augmentation of labour, cesarean section and episiotomy), position for birth, rate of and reasons for not assigning women to a study arm, postpartum perineal pain and satisfaction with the birth experience, physicians: beliefs concerning episiotomy. RESULTS: Women attended by physicians who viewed episiotomy very unfavorably were more likely than women attended by the other physicians to have an intact perineum (23% v. 11% to 13%, p < 0.05) and to experience less perineal trauma. The first stage of labour was 2.3 to 3.5 hours shorter for women attended by physicians who viewed episiotomy favourably than for women attended by physicians who viewed episiotomy very unfavorably (p < 0.05 to < 0.01), and the former physicians were more likely to use oxytocin augmentation of labour. Physicians who viewed episiotomy more favourably failed more often than those who viewed the procedure very unfavourably to assign patients to a study arm late in labour (odds ratio [OR] 1.88, p < 0.05), both overall and because they felt that "fetal distress" or cesarean section necessitated exclusion of the subject. They used the lithotomy position for birth more often (OR 3.94 to 4.55, p < 0.001), had difficulty limiting episiotomy in the restricted-use arm of the trial and diagnosed fetal distress and perineal inadequacy more often than the comparison groups. The patients of physicians who viewed episiotomy very favourably experienced more perineal pain (p < 0.01), and of those who viewed episiotomy favourably and very favourably experienced less satisfaction with the birth experience (p < 0.01) than the patients of physicians who viewed the procedure very unfavourably. CONCLUSIONS: Physicians with favourably views of episiotomy were more likely to use techniques to expedite labour, and their patients were more likely to have perineal trauma and to be less satisfied with the birth experience. This evidence that physician beliefs can influence patient outcomes has both clinical and research implications.
PMCID: PMC1487268  PMID: 7664230
24.  Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice. 
Nucleic Acids Research  1995;23(16):3301-3309.
Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms.
PMCID: PMC307192  PMID: 7667107
25.  Comparative immunogenicity of conjugates composed of Escherichia coli O111 O-specific polysaccharide, prepared by treatment with acetic acid or hydrazine, bound to tetanus toxoid by two synthetic schemes. 
Infection and Immunity  1995;63(8):2805-2810.
Escherichia coli O111, of various H types and virulence factors, causes enteritis throughout the world, especially in young children. This O type is found rarely in healthy individuals. Serum antibodies to the O-specific polysaccharide of O111 lipopolysaccharide (LPS) protect mice and dogs against infection with this E. coli serotype. The O111 O-specific polysaccharide is composed of a pentasaccharide repeat unit with two colitoses bound to the C-3 and C-6 of glucose in a trisaccharide backbone; this structure is identical to that of Salmonella adelaide (O35), another enteric pathogen. Nonpyrogenic O111 O-specific polysaccharide was prepared by treatment of its LPS with acetic acid (O-SP) or the organic base hydrazine (DeA-LPS). The O-SP had a reduced concentration of colitose. These products were derivatized with adipic acid dihydrazide (ADH) or thiolated with N-succinimidyl-3(2-pyridyldithio) propionate (SPDP). The four derivatives were covalently bound to tetanus toxoid (TT) by carbodiimide-mediated condensation or with SPDP to form conjugates. Immunization of BALB/c and general-purpose mice by a clinically acceptable route showed that DeA-LPS-TTADH, of the four conjugates, elicited the highest level of LPS antibodies. Possible reasons to explain this differential immunogenicity between the four conjugates are discussed.
PMCID: PMC173380  PMID: 7542631

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