Detection of nondisjunction and recombination in meiotic and postmeiotic cells from XYSxr [XY,Tp(Y)1Ct] mice using multicolor fluorescence in situ hybridization.

Current meiotic dogma holds that synapsis is required for recombination and that recombination is required for proper disjunction. The mouse chromosome aberration XYSxr [sex reversal; redesignated XY,Tp(Y)1Ct] appears to challenge this assumption, for although chromosomes X and Y often fail to synapse and recombine, there is no dramatic increase in aneuploid progeny. An explanation of this conundrum might be that X-Y univalent spermatocytes do not survive. The phenotype of sex reversal is generated by the "obligatory" crossover between the X and Y chromosomes, which always occurs proximal to a duplicated copy of the testis-determining gene Sry and transfers one copy from one chromatid of the Y chromosome to one chromatid of the X. Animals that inherit an X chromosome with the Sry gene are chromosomally female but phenotypically male. We have used fluorescence in situ hybridization (FISH) to visualize probes for the X and Y chromosomes and for the Sry sequence and chromosome 8 to track the fate of both recombinant and nonrecombinant chromosomes through metaphases I and II into spermatids and sperm. In the 219 gametes examined by multicolor FISH, the frequency of aneuploid products (XY or "O") was low (3.7%) despite a high frequency (66%) of X-Y separation at metaphase I. In balanced gametes, X and Y recombinant chromosomes slightly exceeded nonrecombinants. Both of these observations support the earlier proposal that asynapsis and nondisjunction in primary spermatocytes lead to their developmental arrest and degeneration.     

Correlates of human herpesvirus 8 seropositivity among heterosexual men in Kenya

BACKGROUND: Several studies have suggested that sexual transmission of human herpesvirus 8 (HHV-8) occurs among homosexual men in developed countries. However, few studies have examined heterosexual HHV-8 transmission, especially among African populations in which HHV-8 is endemic. OBJECTIVES: To determine the seroprevalence and correlates of HHV-8 infection among heterosexual African men. DESIGN: Cross-sectional study. METHODS: Participants were 1061 men enrolled in a prospective cohort study of risk factors for HIV-1 acquisition among trucking company employees in Mombasa, Kenya. Stored frozen sera from the study baseline visit were tested for antibodies to HHV-8 by whole-virus lysate ELISA. RESULTS: HHV-8 seroprevalence was 43%. In multivariate logistic regression analysis, HHV-8 infection was independently associated with older age [for men aged 30-39 years: odds ratio (OR), 1.5; 95% confidence interval (CI), 1.1-2.0; for men aged > or = 40 years: OR, 1.7; 95% CI, 1.1-2.7, compared with men aged < 30 years], Christian religion (OR, 1.6; 95% CI, 1.2-2.1), being uncircumcised (OR, 1.5; 95% CI, 1.0-2.2), and ever having syphilis (OR, 2.2; 95% CI, 1.4-3.5). Ever having used condoms was associated with decreased likelihood of infection (OR, 0.7; 95% CI, 0.6-1.0). Seropositivity was not significantly related to other sexual behaviors characterized or to HIV-1 status. CONCLUSIONS: HHV-8 seropositivity is common in this population and increases with age, suggesting on-going transmission during adulthood. Infection was more common among men who were uncircumcised or who had ever had syphilis and was less common among those who had ever used condoms, suggesting that sexual factors may play a role in HHV-8 transmission. Prospective studies of HHV-8 acquisition in heterosexual African populations are needed to demonstrate whether safer sexual practices can reduce transmission.     

Preliminary report: skeletal muscle cell lipid and oxygen supply

In a group of 12 normal-weight, normotensive, nondiabetic adult females, the intramyocellular lipid (IMCL) to creatine ratio of the soleus muscle was determined using localized (1)H-magnetic resonance spectroscopy ((1)H-MRS) and related to skeletal muscle blood (and oxygen) supply (as assessed by near infrared spectroscopy [NIRS] of the forearm). A significant positive association was found between IMCL content and reoxygenation rate of forearm muscle hemoglobin (Hb) after 1 minute of ischemic exercise (r = .70, P = .01). The relative efficiency of skeletal muscle oxygen supply may be a determining factor of IMCL content in skeletal muscle.     

Assessment of ileal pouch inflammation by single-stool calprotectin assay

PURPOSE: Assessment of inflammation within the ileal pouch to establish a diagnosis of pouchitis requires both pouch endoscopy and biopsy because there can be a poor correlation between macroscopic and histologic assessments of inflammation. A simplified diagnostic test would be of clinical advantage. Calprotectin is a stable myelomonocytic protein, measurable in feces. It quantitatively relates to inflammation within the gastrointestinal tract. This study was designed to compare single and 24-hour stool measurements of calprotectin in patients with and without evidence of ileal pouch inflammation with endoscopic, histologic, and immunohistochemical indices. METHODS: Twenty-four-hour stool collections were made in ileal pouch patients, 9 with and 15 without (7 with ulcerative colitis and 8 with familial polyposis coli) evidence of pouch inflammation. First-morning stool concentration and total 24-hour calprotectin were quantified by use of a single step enzyme-linked immunosorbent assay. Biopsies from the reservoir were taken for conventional histology and scoring of intraepithelial neutrophil infiltrate. Cells positive for CD3, CD45RO, CD14, and CD15 within the lamina propria were quantified by use of immunohistochemistry. RESULTS: The mean first-morning stool calprotectin concentration correlated with the 24-hour level (r=0.91;P=<0.0001). The median single-stool calprotectin concentrations were 39 mg/l, 4 mg/l, and 8.5 mg/l (normal range, 0.2–10 mg/l) in patients with inflamed, noninflamed ulcerative colitis, and familial adenomatous polyposis, respectively. All nine patients with endoscopic and histologic evidence of pouch inflammation had raised stool calprotectin. Two of 15 patients without evidence of pouch inflammation had abnormal stool calprotectin. Single-stool calprotectin concentration correlated with the percentage of mature granulocytes (CD15;r=0.46;P=0.04) and activated macrophages (CD14;r=0.65;P=0.006), but not memory T cells (CD45RO;r=–0.05;P=0.4) within the lamina propria. CONCLUSION: Single first-morning stool calprotectin levels provide a quantitative measure of pouch inflammation, which may be helpful in the diagnosis and assessment of pouchitis.Supported by a grant from the Henry Smith Foundation.Presented at the Faulk symposium on the pelvic ileal reservoir in ulcerative colitis, Oxford, United Kingdom, April 17 to 19, 1997.     

Early life environmental control: effect on symptoms, sensitization, and lung function at age 3 years

We investigated whether environmental control during pregnancy and early life affects sensitization and lung function at the age of 3 years. High-risk children (n = 251) were prenatally randomized to stringent environmental control (active) or no intervention (control). Questionnaires, skin testing, IgE, and specific airway resistance (sRaw) measurement were completed at the age of 3 years. Children in the active group were significantly more frequently sensitized compared with control subjects (at least one allergen by skin tests: risk ratio, 1.61; 95% confidence interval [CI], 1.02-2.55; p = 0.04; mite by IgE: risk ratio, 2.85; 95% CI, 1.02-7.97; p = 0.05). However, sRaw was significantly better in the active group (kiloPascal/second, geometric mean [95% CI]: 1.05 [1.01-1.10] vs. 1.19 [1.13-1.25], p < 0.0001, active vs. control). Maximal flow at functional residual capacity was measured using rapid thoracic compression at the age of 4 weeks in a subgroup. Prospective lung function data (at infancy and 3 years) were obtained in 32 children (14 active and 18 control). There was no difference in infant lung function between the groups, but at 3 years, sRaw was significantly lower in the active compared with control children (p = 0.003). Stringent environmental control was associated with increased risk of mite sensitization but better results for some measurements of lung function in high-risk children at the age of 3 years.     

Non-comparative evaluation of the safety of aerosolized amphotericin B lipid complex in patients undergoing allogeneic hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplant (HSCT) recipients are at increased risk for invasive fungal infections (IFIs) over prolonged periods of time. Aerosolized amphotericin B lipid complex (ABLC) has shown promise in lung transplant recipients as a convenient means of delivering protective drug to the upper airways avoiding systemic toxicities. The safety and tolerability of aerosolized ABLC in 40 subjects undergoing allogeneic HSCT was prospectively investigated in an open-labeled, non-comparative study. Subjects received aerosolized ABLC treatment once daily for 4 days, then once weekly for 13 weeks; fluconazole was administered daily as standard of care through post-transplant day 100. Pulmonary mechanics were measured before and after each dose of inhaled ABLC; adverse events (AEs) and the development of IFI were also monitored. Cough, nausea, taste disturbance, or vomiting followed 2.2% of 458 total inhaled ABLC administrations; 5.2% of inhaled ABLC administrations were associated with >or=20% decrease in pulmonary function measurements (forced expiratory volume in 1 second or forced vital capacity) and none required treatment with bronchodilators or withdrawal from study. Four mild AEs were considered possibly or probably related to study treatment; no deaths or withdrawals from treatment were attributed to study drug. Of 3 proven IFIs occurring during the study period, only 1, a catheter-related case of disseminated fusariosis, occurred while the subject was receiving study medication. Aerosolized ABLC was well tolerated in allogeneic HSCT recipients. With only 1 of 40 subjects developing IFI while receiving treatment, the combination of fluconazole and inhaled ABLC warrants further study as antifungal prophylaxis following allogeneic HSCT.     

Early somatic mosaicism is a rare cause of long-QT syndrome

Somatic mosaicism, the occurrence and propagation of genetic variation in cell lineages after fertilization, is increasingly recognized to play a causal role in a variety of human diseases. We investigated the case of life-threatening arrhythmia in a 10-day-old infant with long QT syndrome (LQTS). Rapid genome sequencing suggested a variant in the sodium channel Na_V1.5 encoded by SCN5A, {"type":"entrez-nucleotide","attrs":{"text":"NM_000335","term_id":"124518660","term_text":"NM_000335"}}NM_000335:c.5284G > T predicting p.(V1762L), but read depth was insufficient to be diagnostic. Exome sequencing of the trio confirmed read ratios inconsistent with Mendelian inheritance only in the proband. Genotyping of single circulating leukocytes demonstrated the mutation in the genomes of 8% of patient cells, and RNA sequencing of cardiac tissue from the infant confirmed the expression of the mutant allele at mosaic ratios. Heterologous expression of the mutant channel revealed significantly delayed sodium current with a dominant negative effect. To investigate the mechanism by which mosaicism might cause arrhythmia, we built a finite element simulation model incorporating Purkinje fiber activation. This model confirmed the pathogenic consequences of cardiac cellular mosaicism and, under the presenting conditions of this case, recapitulated 2:1 AV block and arrhythmia. To investigate the extent to which mosaicism might explain undiagnosed arrhythmia, we studied 7,500 affected probands undergoing commercial gene-panel testing. Four individuals with pathogenic variants arising from early somatic mutation events were found. Here we establish cardiac mosaicism as a causal mechanism for LQTS and present methods by which the general phenomenon, likely to be relevant for all genetic diseases, can be detected through single-cell analysis and next-generation sequencing.There is growing recognition that somatic mosaicism, i.e., genetic variation within an individual that arises from errors in DNA replication during early development, may play a role in a variety of human diseases other than cancer (1). However, the extent to which cellular heterogeneity contributes to disease is minimally understood. One report suggests that 6.5% of de novo mutations presumed to be germline in origin may instead have arisen from postzygotic mosaic mutation events (2), and recent genetic investigations directly interrogating diseased tissues in brain malformations, breast cancer, and atrial fibrillation have revealed postzygotic causal mutations absent from germline DNA (3–6). Pathogenic mosaic structural variation is also detectable in children with neurodevelopmental disorders (7). However, a consequential category of genetic variation has not been surveyed systematically in clinical or research studies of other human diseases.The pathophysiological basis of long-QT syndrome (LQTS) is prolongation of cardiac ventricular repolarization by acquired factors such as drug exposure or genetic variation in the proteins controlling transmembrane ion-concentration gradients (8, 9); parental gonadal mosaicism is an infrequently described phenomenon in LQTS (10–12). Knowledge of the molecular subtyping of disease in LQTS has provided a foundation for genotype-specific risk stratification and treatment strategies (8, 9, 13, 14). However, nearly 30% of probands remain undiagnosed using standard commercial gene-panel testing, suggesting there is unrecognized genetic variation (genic, regulatory, or otherwise) yet to be associated with disease (15).In its most severe form, LQTS may occur in the neonatal period with bradycardia and functional 2:1 AV block that occur secondary to a severely prolonged ventricular refractory period greater than the short R–R interval characteristic of a normal heart rate in infancy (16). Patients of all ages presenting with LQTS are predisposed to torsades de pointes (TdP), a life-threatening cardiac arrhythmia, with patients presenting in infancy showing particularly poor outcomes (13, 16–18). In this study, we applied rapid-turnaround whole-genome sequencing (WGS) on day of life (DOL) 3 in a premature infant with perinatal LQTS and life-threatening arrhythmia and investigated the contribution of a discovered mosaic variant to abnormal cardiac electrophysiology at the molecular and tissue levels. Additionally we surveyed 7,500 individuals already tested for genetic arrhythmias, allowing us to estimate the prevalence of mosaicism in an unbiased population sample.     

The evolution of extracranial approaches to the pituitary and anterior skull base

The development of extracranial approaches to the anterior skull base has been a process in development for over a 100 years. Many neurosurgical (and non-neurosurgical) pioneers have contributed to its evolution. In this paper, we will review the major contributors and contributions to this evolving field.     

Oxidative stress in humans following the Pringle manoeuvre

BACKGROUND: Oxidative stress is induced in the liver by application of the Pringle manoeuvre. Malondialdehyde is a carbonyl compound formed during lipid peroxidation and prostaglandin biosynthesis, which combines with DNA to form a number of adducts. Among them is the DNA ad-duct; 3-(2-deoxybeta-dierythropentafuranosyl) pyr [1,2-alpha]-purin-10(3H) one or M1G. This study was undertaken to determine the suitability of M1G as a novel marker of ischemia-reperfusion injury in the liver and its correlation with both the length of Pringle clamp application and the overall length of the operation. METHODS: Normal and colorectal liver metastatic tissues were obtained in 12 patients before and after application of the Pringle manoeuvre. All samples were snap-frozen in liquid nitrogen at -80 ℃. DNA was extracted and M1G quantification was performed by immunoslotblot analysis. RESULTS: M1G levels in normal liver tissue were 4.0 + 1.0 per 107 nucleotides before the Pringle manoeuvre and 7.4 ± 1.0 per 10 nucleotides after the Pringle manoeuvre (mean ± standard deviation) (P<0.05 by ANOVA). M1G levels in malignant liver tissue were 2.5 ±1.4 per 107 nucleotides before the Pringle manoeuvre and 6. 5 ±1.9 adducts per 10 nucleotides after the Pringle manoeuvre (P <0. 05). Ad-duct levels in normal liver tissue showed a significant correlation with cumulative period of Pringle application. CONCLUSIONS: This is the first time that the tissue levels of M1G before and after application of the Pringle manoeuvre have been studied. The results show that the Pringle manoeuvre exerts significant oxidative stress in human hepa-tocytes, which is Pringle-time dependent. The results highlight the potential for oxidative DNA adducts levels as a tool for measuring the severity of ischemia-reperfusion injury.