Association of leptin and insulin with childhood obesity and retinal vessel diameters.

OBJECTIVE: Childhood obesity is associated with an impaired retinal microcirculation. The aim of the study was to investigate the association between specific obesity-related biomarkers, physical fitness and retinal vessel diameters in school children. DESIGN AND SUBJECTS: We studied 381 children aged 10-11 years (body mass index (BMI): 19.3±3.7 kg m(-2)) in a school-based setting. MEASUREMENTS: Anthropometric measurements and blood sampling were conducted using standard protocols for children. The serum biomarkers leptin, adiponectin, insulin as well as interleukin-6 (IL-6) were analyzed. Physical fitness was determined by a six-item-test battery and physical activity by use of a questionnaire. Central retinal arteriolar equivalent (CRAE), central retinal venular equivalent (CRVE) and the arteriolar-to-venular diameter ratio (AVR) were assessed with a non-mydriatic vessel analyzer (SVA-T) using a computer-based program. RESULTS: Compared with normal weight children (n=254), obese children (n=39) showed higher leptin (P<0.001), higher insulin (P<0.001), higher IL-6 (P<0.001) and lower adiponectin levels (P=0.013). Obese children demonstrated wider CRVE (P=0.041) and lower AVR (P<0.001). Higher leptin levels were associated with wider CRVE (P=0.032) and lower AVR (P=0.010), that was BMI dependent. Insulin levels were associated with arteriolar (P=0.045) and venular dilatation (P=0.034) after adjustment for BMI. No significant associations between adiponectin levels, IL-6 levels, physical fitness or physical activity and retinal vessel diameter were observed. Lower leptin levels were independently correlated with higher physical fitness (r=-0.33; P<0.001). CONCLUSION: Leptin and insulin levels are associated with changes of the retinal microcirculation. Especially insulin seems to be a good target marker for the cardiometabolic risk assessment in children since elevated insulin levels are independently associated with microvascular end-organ alterations at an early stage. Lifestyle intervention studies are warranted to examine whether improvement of physical fitness or weight reduction can affect cardiometabolic risk markers and reverse alterations of the retinal microcirculation.

Effects of a physical education program on physical activity, fitness, and health in children: the JuvenTUM project.

The purpose of the study was to investigate the effects of a school-based prevention program on physical activity, fitness, and obesity. We performed a prospective study in eight Bavarian primary schools (n = 724 children, 8.4 ± 0.7 years) randomized one to one to either an intervention school (IS, n = 427) or a control school (CS, n = 297). Children in IS attended 10 health-related lessons at school over a period of 1 year. Parents and teachers attended two and three educational health-related lessons, respectively, and also received 10 newsletters on health issues. Daily physical activity (≥ 60 min/day), physical fitness (six-item test battery), and anthropometric data were obtained at baseline and after 1 year. Physical activity and physical fitness increased in IS, but it failed to reach significant intervention effects. Nevertheless, a reduction in waist circumference was observed for all children [mean change 1.7 cm; 95% confidence interval (CI) 1.2-2.3; P < 0.001). This effect was more pronounced in overweight children (> 90th percentile, n = 99, mean change 3.2 cm; 95% CI 1.5-4.8; P < 0.001). This easily administered preventative program involving children, parents, and teachers revealed that a generalized approach increasing physical activity will even be favorable in a subgroup of obese children.

Comparative genomes of Chlamydia pneumoniae and C. trachomatis.

Chlamydia are obligate intracellular eubacteria that are phylogenetically separated from other bacterial divisions. C. trachomatis and C. pneumoniae are both pathogens of humans but differ in their tissue tropism and spectrum of diseases. C. pneumoniae is a newly recognized species of Chlamydia that is a natural pathogen of humans, and causes pneumonia and bronchitis. In the United States, approximately 10% of pneumonia cases and 5% of bronchitis cases are attributed to C. pneumoniae infection. Chronic disease may result following respiratory-acquired infection, such as reactive airway disease, adult-onset asthma and potentially lung cancer. In addition, C. pneumoniae infection has been associated with atherosclerosis. C. trachomatis infection causes trachoma, an ocular infection that leads to blindness, and sexually transmitted diseases such as pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy and epididymitis. Although relatively little is known about C. trachomatis biology, even less is known concerning C. pneumoniae. Comparison of the C. pneumoniae genome with the C. trachomatis genome will provide an understanding of the common biological processes required for infection and survival in mammalian cells. Genomic differences are implicated in the unique properties that differentiate the two species in disease spectrum. Analysis of the 1,230,230-nt C. pneumoniae genome revealed 214 protein-coding sequences not found in C. trachomatis, most without homologues to other known sequences. Prominent comparative findings include expansion of a novel family of 21 sequence-variant outer-membrane proteins, conservation of a type-III secretion virulence system, three serine/threonine protein kinases and a pair of parologous phospholipase-D-like proteins, additional purine and biotin biosynthetic capability, a homologue for aromatic amino acid (tryptophan) hydroxylase and the loss of tryptophan biosynthesis genes.

Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis.

Analysis of the 1,042,519-base pair Chlamydia trachomatis genome revealed unexpected features related to the complex biology of chlamydiae. Although chlamydiae lack many biosynthetic capabilities, they retain functions for performing key steps and interconversions of metabolites obtained from their mammalian host cells. Numerous potential virulence-associated proteins also were characterized. Several eukaryotic chromatin-associated domain proteins were identified, suggesting a eukaryotic-like mechanism for chlamydial nucleoid condensation and decondensation. The phylogenetic mosaic of chlamydial genes, including a large number of genes with phylogenetic origins from eukaryotes, implies a complex evolution for adaptation to obligate intracellular parasitism.

Human seminal plasma inhibition of antibody complement-mediated killing and opsonization of Neisseria gonorrhoeae and other gram-negative organisms.

Seminal plasma diluted 1:5-1:1,000 gave marked inhibition of serum antibody complement-mediated bactericidal and opsonic effects against Neisseria gonorrhoeae and other gram-negative organisms. Serum that was bactericidal at a dilution of 1:5,120 was not bactericidal at a dilution of 1:10 when seminal plasma was added. Bactericidal action of immune human or rabbit sera, or purified immunoglobulin (Ig)G or IgM plus complement for six strains of N. gonorrhoeae, serogroups A, B, C, and Y of Neisseria meningitidis, Escherichia coli and other gram-negative rods was inhibited by seminal plasma. Using C8- or C7-deficient sera as antibody and complement sources, opsonization, phagocytosis, and killing of N. gonorrhoeae and E. coli 014-K7 were inhibited by seminal plasma. Opsonization, phagocytosis, and killing of Staphylococcus aureus 502A was not inhibited. For the gram-negative organisms, the early phase of the opsonization process, probably complement activation, appeared to be inhibited rather than the ingestion or polymorphonuclear leukocyte killing steps; addition of seminal plasma yielded a significant reduction in the percentage of polymorphonuclear cells with associated bacteria. Seminal plasma did not prevent attachment of IgG, IgM, or IgA antibodies to gonococci. It reduced serum hemolytic whole complement activity by 25%. The seminal plasma inhibitor was of low molecular weight and was stable at 56 degrees C for 30 min, but inhibitory activity was lost after heating to 100 degrees C for 10 min. It is likely that the inhibitory factor(s) is a low-molecular weight protease or protease inhibitor. Seminal plasma probably has an important role in inhibition of complement and antibody functions in the genital tract. It may enhance pathogenesis of agents of sexually transmitted diseases.

Chlamydia outer membrane protein discovery using genomics.

Outer membrane proteins of microbial pathogens serve essential roles in engaging the host environment and can be important immunotherapeutic targets. Because of the difficulty of growing large quantities of chlamydiae suitable for biochemical fractionation, little was known about their outer membrane protein composition prior to the recent sequencing of the C. trachomatis and C. pneumoniae genomes. Using bioinformatic approaches to characterize chlamydial open reading frames, novel outer membrane proteins were predicted. Several of the predicted outer membrane proteins recently have been shown to be translated and localized to the surface of the chlamydial outer membrane.

Bactericidal action of nafcillin, vancomycin, and three cephalosporins against nafcillin-susceptible and nafcillin-resistant coagulase-negative staphylococci.

Coagulase-negative staphylococci (S. epidermidis, 43 strains; S. warneri, 16 strains; S. haemolyticus, five strains; and others, four strains) were tested by the agar dilution method for nafcillin susceptibility: 53 were susceptible with a minimal inhibitory concentration (MIC) of less than or equal to 2 micrograms/ml; four were of indeterminate susceptibility, MIC = 4-16 micrograms/ml; and 11 were resistant, MIC greater than or equal to 32 micrograms/ml. The bactericidal activities from 0 to 24 hr for nafcillin, vancomycin, cephalothin, cefazolin, and cefamandole, each at 16 micrograms/ml in broth, were determined for all the isolates. The data indicate that a nafcillin agar dilution susceptibility test result of resistance does not consistently predict lack of killing activity by the cephalosporins. It is likely that each cephalosporin would have to be tested against individual coagulase-negative staphylococci in order to determine a suitable therapeutic or prophylactic cephalosporin, if a cephalosporin were to be used. Vancomycin was bactericidal for all the nafcillin-resistant coagulase-negative organisms tested.

Cervical secretory immunoglobulin A in adolescent girls.

PURPOSE: To determine whether there are differences in levels of cervical secretory immunoglobulin A (sIgA) between adolescent girls in the secretory and proliferative phases of their menstrual cycle. METHODS: Sexually active adolescent girls (n = 117) at health maintenance organization (HMO) based adolescent medical clinic were recruited into the study. In addition to demographic and clinical data, cervical specimens were collected for sIgA measurement and gonorrhea culture, urine for chlamydia ligase chain reaction, and blood for progesterone levels. Subjects were classified as being in the proliferative phase or secretory phase of the menstrual cycle on the basis of their progesterone levels. RESULTS: The mean age of the subjects was 17.2 years old. There was no difference in the sIgA levels between those in the proliferative phase of their cycle (n = 45; mean sIgA level, 0.0055 mg/mL) and those in the secretory phase (n = 40; mean sIgA level, 0.0032 mg/mL) (p > .10). CONCLUSIONS: The secretory phase of the menstrual cycle does not appear to be associated with higher levels of sIgA in adolescent girls. These results suggest that adolescents with anovulatory cycles, i.e., those who lack a secretory phase, may not be at increased risk for genital tract infections such as chlamydia or gonorrhea.

Retinal vessel diameter, obesity and metabolic risk factors in school children (JuvenTUM 3).

BACKGROUND: The prevalence of childhood obesity is high and its association with future cardiovascular disease in adulthood is well established. The cross-sectional data presented analyze the prevalence of obesity and the association between metabolic risk factors, physical inactivity and retinal vessel diameter in young school children. METHODS: The examination included 578 school children aged 11.1±0.6 years from secondary schools in the District of Munich, Germany. Anthropometric measurements and blood sampling were conducted using standard protocols for children. Physical activity was evaluated by use of a questionnaire. Retinal microvascular diameters and the arteriolar to venular ratio (AVR) were assessed with a non-mydriatic vessel analyser (SVA-T) using a computer-based program. RESULTS: In our population, 128 (22.2%) children were overweight (ow) or obese (ob). The mean retinal arteriolar and venular calibres were 208.0±15.6 µm and 236.2±16.2 µm, respectively, with a mean AVR of 0.88±0.01. Girls had significantly wider arteriolar and venular diameters compared to boys (p<0.001). ow and ob children had a lower AVR compared to normal weight (nw) children (mean(95% CI); nw: 0.89(0.88-0.89); ow: 0.87(0.86-0.88); ob: 0.85(0.83-0.87); p≤0.05). Wider venular diameters were independently associated with higher BMI and higher hsCRP. Blood pressure was associated with retinal vessel constriction. Higher physical inactivity and BMI were independently associated with a reduced AVR (p=0.032 and p<0.001, respectively). CONCLUSIONS: Cardiometabolic risk factors and physical inactivity are associated with retinal microvascular alterations in young children, comparable to associations in adults. Retinal vessel imaging seems to be a feasible assessment for the detection of microvascular impairments in children at risk of developing cardiovascular disease in adulthood.

A unique adenosine diphosphoglucose pyrophosphorylase associated with maize embryo tissue.

A comparison of heat stabilities and various kinetic properties between the adenosine diphosphoglucose pyrophosphorylases isolated from endosperm and embryo tissues from starchy maize seeds indicates that the adenosine diphosphoglucose pyrophosphorylase associated with the embryo is distinct from the enzyme isolated from the endosperm. The embryo enzyme is more stable to incubation for 5 minutes at 60 C while the endosperm enzyme is labile to this treatment. Both enzymes are activated by glycerate-3-P. The embryo enzyme is more sensitive to inhibition by phosphate than is the endosperm enzyme. Glycerate-3-P, which reverses the inhibition of the endosperm enzyme by phosphate, has little effect on the phosphate inhibition of the embryo enzyme. Other kinetic studies distinguish the two enzymes.

Scanning electron microscopy of piliated Neisseria gonorrhoeae processed with hexamethyldisilazane.

Piliated Neisseria gonorrhoeae are virulent and attach readily to some human mucosal cells. The study of interactions between piliated Neisseria gonorrhoeae and surface structures of eukaryotic cells in tissue culture requires consistent high resolution imaging in scanning electron microscopy (SEM). The combination of the fixatives glutaraldehyde, osmium, tannic acid, and uranyl acetate improves preservation of pili and other delicate structures. Following the critical point drying (CPD) process, pili bundles remained intact, but charging produced image distortion in most of the specimens. The use of hexamethyldisilazane (HMDS) with air drying substantially reduced charging and image distortion. Less contrast and greater resolution of pili bundles and surface structures of bacteria or tissue culture cells were obtained at magnifications of 10,000 or higher. As an alternative to CPD, HMDS processing of cell culture monolayers was simple and was more efficient when a large number of samples was processed.

Antibodies against IgA1 protease are stimulated both by clinical disease and asymptomatic carriage of serogroup A Neisseria meningitidis.

IgA1 protease was purified from a strain of serogroup A Neisseria meningitidis subgroup IV-1, representative of bacteria that caused an epidemic of meningococcal meningitis in The Gambia in 1982-1983. ELISAs and immunoblot assays were done using this protease as antigen with paired acute- and convalescent-phase sera from patients from that epidemic and from one in Finland caused by other serogroup A meningococci. Paired sera were also tested from healthy Gambians who were persistent nasopharyngeal carriers, persistent noncarriers, or persons who became carriers after the first serum sample was taken. The results correlated well between the two methods: Antibodies were stimulated by disease or acquisition of carriage, and they remained at a constant level upon continued carriage.

Recall of original serologic response after challenge with homologous and heterologous Chlamydia trachomatis serovars.

Chlamydia trachomatis serovar-specific major outer membrane protein (MOMP) antigens are important targets of immune neutralization in vitro, and natural immunity to infection is associated with serovar specificity. Reinfection, often by different serovars, plays an essential role in chlamydial disease pathogenesis. By use of a murine model, the anamnestic serologic response was characterized following priming and challenge inoculations using 6 different serovars. The serologic response was evaluated using synthetic peptides representing MOMP variant segments (VS) 1,2,3, and 4 antigenic same serovar resulted in serologic responses to homologous VS1 peptides. After challenge with a different serovar, anti-VS1 serologic responses were often elicited with specificity to both the priming serovar and the challenge serovar. The recall of serologic response to the original serovar was typically dependent upon the antigenic relationship of the 2 serovars.

Opa (protein II) influences gonococcal organization in colonies, surface appearance, size and attachment to human fallopian tube tissues.

Opa-expressing variants of Neisseria gonorrhoeae strain F62-SF and an Opa- variant, all non-piliated, were examined for differences in the interaction of the bacteria within colonies and in attachment to and damage of human fallopian tube mucosa. Expression of certain Opas was associated with the formation of transparent colonies where the bacteria were tightly packed and evenly spaced within the colonies. Expression of other Opas was associated with the formation of opaque colonies where the gonococci were less tightly packed and were unevenly spaced. Distinct differences in the size of the gonococci and in their surface characteristics were dependent upon the Opa being expressed. Certain Opas were associated with gonococci that had significantly larger cross-sectional areas and bigger perimeters. Scanning electron microscopy showed that OpaC- and OpaD-containing variants yielded greater mucosal damage than OpaB-containing and Opa- variants with the least damage caused by the OpaA-containing variant (clumped bacteria from dark opaque friable colonies). The mucosal damage after 60 min incubation included shortening and decreased numbers of microvilli on non-ciliated cells and invagination and sloughing of ciliated cells. Differences in the interactions of gonococci within colonies and in attachment to fallopian tube mucosa and damage to the mucosal cells occurred with different Opa-expressing variants of N. gonorrhoeae strain F62-SF.

Neisseria gonorrhoeae coordinately uses Pili and Opa to activate HEC-1-B cell microvilli, which causes engulfment of the gonococci.

This study was undertaken to examine concomitant roles of pili and colony opacity-associated proteins (Opa) in promoting Neisseria gonorrhoeae adherence to and invasion of human endometrial HEC-1-B cells. Adherence of N. gonorrhoeae to cultured HEC-1-B cells was saturable, even though organisms adhered to <50% of the cells. During 4 to 6 h of incubation, adherent mono- and diplococci formed microcolonies on the surfaces of the cells. Microvilli of the HEC-1-B cells adhered by their distal ends to individual cocci within the microcolonies. When the microcolonies grew from isogenic pilus-negative (P-) Opa-, P- Opa+, or P+ Opa- gonococci, microvilli did not elongate, and the colonies were not engulfed. In contrast, the microvilli markedly elongated during exposure to P+ Opa+ gonococci. The microvilli adhered to the organisms along their full lengths and appeared to actively participate in the engulfment of the microcolonies. Internalized microcolonies, with P+ Opa+ gonococci, contained dividing cocci and appeared to be surrounded by cell membrane but were not clearly within vacuoles. In contrast, degenerate individual organisms were within vacuoles. Low doses of chloramphenicol, which inhibits protein synthesis by both prokaryotes and eukaryotes, prevented the microvillar response to and internalization of the P+ Opa+ gonococci; higher doses caused internalization without microvillus activation. Cycloheximide and anisomycin, which inhibit only eukaryotic protein synthesis, caused dose-dependent enhancement of uptake. Cytochalasins reduced engulfment; colchicine had no effect. These results show that gonococci must express both pili and Opa to be engulfed efficiently by HEC-1-B cells.

Starch Synthetase, Phosphorylase, ADPglucose Pyrophosphorylase, and UDPglucose Pyrophosphorylase in Developing Maize Kernels.

Soluble ADPglucose-alpha-glucan 4-alpha-glucosyltransferase (starch synthetase), ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and phosphorylase were assayed in extracts from developing kernels of maize (Zea mays). Normal, waxy and amylose-extender maize at stages of development ranging from 8 days to 28 days after pollination were studied. Shrunken-4 maize at the 22-day stage was also studied. There is adequate activity of both ADPglucose pyrophosphorylase and starch synthetase at all stages of development to account for the synthesis of starch. Thus all starch could be synthesized via the ADPglucose pathway. High levels of UDPglucose pyrophosphorylase and of phosphorylase activities were also found at all stages of development. The possible role of phosphorylase in starch synthesis could not be discounted. The levels of phosphorylase, ADPglucose pyrophosphorylase, starch synthetase, and UDPglucose pyrophosphorylase activities in shrunken-4 kernels were about 20 to 40% of that found in normal maize kernels. It appears that the mutation in shrunken-4 affects the activities of more than one enzyme. The defective starch synthesis seen in this mutant could be due to the low activities of ADPglucose pyrophosphorylase and starch synthetase rather than the low activity of phosphorylase.

In vitro model of Haemophilus ducreyi adherence to and entry into eukaryotic cells of genital origin.

Electron microscopy was used to examine Haemophilus ducreyi adherence to and entry into eukaryotic cells of genital origin. A clinical H. ducreyi isolate (90-244) adhered in snake-like whorls to the surfaces of cervical carcinoma cells (HeLa 229), endometrial adenocarcinoma cells (HEC-1-B), and human neonatal foreskin fibroblast (HFF) cells. A prototype strain of H. ducreyi (CIP542) adhered in randomly organized clumps on the surfaces of HFF. Strain 90-244 entered HFF and HEC-1-B cells but did not enter HeLa cells. The H. ducreyi in the HFF cells at 2 h were partly surrounded by a membrane consistent with that of a phagocytic vacuole. At 2 h, strain CIP542 was found in interstitial spaces between the HFF cells and also in the cytoplasm of the cells. After 7 and 24 h, both strains of H. ducreyi were found in the large interstitial spaces between the HFF cells, in the cytoplasm, and extracellularly. This model of in vitro H. ducreyi infection of eukaryotic cells will allow for more specific study of factors that determine the virulence of H. ducreyi.

Relation of protein I and colony opacity to serum killing of Neisseria gonorrhoeae.

Serum bactericidal tests were done for isogenic Neisseria gonorrhoeae that formed transparent or opaque colonies. Analysis of the data from individual strains showed that the molecular weight of protein I was a highly significant, but not universal, determinant of serum sensitivity or resistance. N. gonorrhoeae organisms with high-molecular-weight protein I were more serum-sensitive. Multivariate analysis of the data from 43 strains indicated that N. gonorrhoeae organisms from transparent colonies were more serum-resistant than isogenic organisms from opaque colonies (P = 0.01). Survivors of bactericidal reactions in which the initial inoculum was from opaque colonies tended to form transparent colonies. Bactericidal action by sera from men was highly associated (P less than 0.02) with the molecular weight of protein I and with the presence of protein II bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas bactericidal action by sera from women was associated only with the molecular weight of protein I.