Primary cilia are critical for tracheoesophageal septation.

INTRODUCTION: Primary cilia play pivotal roles in the patterning and morphogenesis of a wide variety of organs during mammalian development. Here we examined murine foregut septation in the cobblestone mutant, a hypomorphic allele of the gene encoding the intraflagellar transport protein IFT88, a protein essential for normal cilia function. RESULTS: We reveal a crucial role for primary cilia in foregut division, since their dramatic decrease in cilia in both the foregut endoderm and mesenchyme of mutant embryos resulted in a proximal tracheoesophageal septation defects and in the formation of distal tracheo(broncho)esophageal fistulae similar to the most common congenital tracheoesophageal malformations in humans. Interestingly, the dorsoventral patterning determining the dorsal digestive and the ventral respiratory endoderm remained intact, whereas Hedgehog signaling was aberrantly activated. CONCLUSIONS: Our results demonstrate the cobblestone mutant to represent one of the very few mouse models that display both correct endodermal dorsoventral specification but defective compartmentalization of the proximal foregut. It stands exemplary for a tracheoesophageal ciliopathy, offering the possibility to elucidate the molecular mechanisms how primary cilia orchestrate the septation process. The plethora of malformations observed in the cobblestone embryo allow for a deeper insight into a putative link between primary cilia and human VATER/VACTERL syndromes.

Epidemiological investigation of a Legionnaires' disease outbreak in Christchurch, New Zealand: the value of spatial methods for practical public health.

Between April and August 2005 Christchurch, New Zealand experienced an outbreak of Legionnaires' disease. There were 19 laboratory-confirmed case including three deaths. Legionella pneumophila serogroup 1 (Lpsg1) was identified as the causative agent for all cases. A case-control study indicated a geographical association between the cases but no specific common exposures. Rapid spatial epidemiological investigation confirmed the association and identified seven spatially significant case clusters. The clusters were all sourced in the same area and exhibited a clear anisotropic process (noticeable direction) revealing a plume effect consistent with aerosol dispersion from a prevailing southwesterly wind. Four out of five cases tested had indistinguishable allele profiles that also matched environmental isolates from a water cooling tower within the centre of the clusters. This tower was considered the most probable source for these clusters. The conclusion would suggest a maximum dispersal distance in this outbreak of 11·6 km. This work illustrated the value of geostatistical techniques for infectious disease epidemiology and for providing timely information during outbreak investigations.

Multiple essential roles for primary cilia in heart development.

BACKGROUND: The primary cilium is a microtubule-based, plasma membrane-ensheathed protrusion projecting from the basal bodies of almost all cell types in the mammalian body. In the past several years a plethora of papers has indicated a crucial role for primary cilia in the development of a wide variety of organs. We have investigated heart development in cobblestone, a hypomorphic allele of the gene encoding the intraflagellar transport protein Ift88, and uncovered a number of the most common congenital heart defects seen in newborn humans. METHODS: We generated serial sections of mutant cobblestone and wild type embryos in the region encompassing the heart and the cardiac outflow tract. The sections were further processed to generate three-dimensional reconstructions of these structures, and immunofluorescence confocal microscopy, transmission electron microscopy, and in situ hybridization were used to examine signal transduction pathways in the relevant areas. Whole mount in situ hybridization was also employed for certain developmental markers. RESULTS: In addition to an enlarged pericardium and failure of both ventricular and atrial septum formation, the cobblestone mutants displayed manifold defects in outflow tract formation, including persistent truncus arteriosus, an overriding aorta, and abnormal transformation of the aortic arches. To discern the basis of these anomalies we examined both the maintenance of primary cilia as well as endogenous and migratory embryonic cell populations that contribute to the outflow tract and atrioventricular septa. The colonization of the embryonic heart by cardiac neural crest occurred normally in the cobblestone mutant, as did the expression of Sonic hedgehog. However, with the loss of primary cilia in the mutant hearts, there was a loss of both downstream Sonic hedgehog signaling and of Islet 1 expression in the second heart field, a derivative of the pharyngeal mesoderm. In addition, defects were recorded in development of atrial laterality and ventricular myocardiogenesis. Finally, we observed a reduction in expression of Bmp4 in the outflow tract, and complete loss of expression of both Bmp2 and Bmp4 in the atrioventricular endocardial cushions. Loss of BMP2/4 signaling may result in the observed proliferative defect in the endocardial cushions, which give rise to both the atrioventricular septa as well as to the septation of the outflow tract. CONCLUSIONS: Taken together, our results potentially identify a novel link between Sonic hedgehog signaling at the primary cilium and BMP-dependent effects upon cardiogenesis. Our data further point to a potential linkage of atrioventricular septal defects, the most common congenital heart defects, to genes of the transport machinery or basal body of the cilia.

Control of postnatal apoptosis in the neocortex by RhoA-subfamily GTPases determines neuronal density.

Apoptosis of neurons in the maturing neocortex has been recorded in a wide variety of mammals, but very little is known about its effects on cortical differentiation. Recent research has implicated the RhoA GTPase subfamily in the control of apoptosis in the developing nervous system and in other tissue types. Rho GTPases are important components of the signaling pathways linking extracellular signals to the cytoskeleton. To investigate the role of the RhoA GTPase subfamily in neocortical apoptosis and differentiation, we have engineered a mouse line in which a dominant-negative RhoA mutant (N19-RhoA) is expressed from the Mapt locus, such that all neurons of the developing nervous system are expressing the N19-RhoA inhibitor. Postnatal expression of N19-RhoA led to no major changes in neocortical anatomy. Six layers of the neocortex developed and barrels (whisker-related neural modules) formed in layer IV. However, the density and absolute number of neurons in the somatosensory cortex increased by 12-26% compared with wild-type littermates. This was not explained by a change in the migration of neurons during the formation of cortical layers but rather by a large decrease in the amount of neuronal apoptosis at postnatal day 5, the developmental maximum of cortical apoptosis. In addition, overexpression of RhoA in cortical neurons was seen to cause high levels of apoptosis. These results demonstrate that RhoA-subfamily members play a major role in developmental apoptosis in postnatal neocortex of the mouse but that decreased apoptosis does not alter cortical cytoarchitecture and patterning.

A crucial role for primary cilia in cortical morphogenesis.

Primary cilia are important sites of signal transduction involved in a wide range of developmental and postnatal functions. Proteolytic processing of the transcription factor Gli3, for example, occurs in primary cilia, and defects in intraflagellar transport (IFT), which is crucial for the maintenance of primary cilia, can lead to severe developmental defects and diseases. Here we report an essential role of primary cilia in forebrain development. Uncovered by N-ethyl-N-nitrosourea-mutagenesis, cobblestone is a hypomorphic allele of the IFT gene Ift88, in which Ift88 mRNA and protein levels are reduced by 70-80%. cobblestone mutants are distinguished by subpial heterotopias in the forebrain. Mutants show both severe defects in the formation of dorsomedial telencephalic structures, such as the choroid plexus, cortical hem and hippocampus, and also a relaxation of both dorsal-ventral and rostral-caudal compartmental boundaries. These defects phenocopy many of the abnormalities seen in the Gli3 mutant forebrain, and we show that Gli3 proteolytic processing is reduced, leading to an accumulation of the full-length activator isoform. In addition, we observe an upregulation of canonical Wnt signaling in the neocortex and in the caudal forebrain. Interestingly, the ultrastructure and morphology of ventricular cilia in the cobblestone mutants remains intact. Together, these results indicate a critical role for ciliary function in the developing forebrain.

Prevalence and risk factors for diabetic retinopathy in the multiethnic population of Mauritius.

This study examines the prevalence of, and risk factors for, diabetic retinopathy in Asian Indian, Chinese, and Creole Mauritians in whom there is an increasing prevalence of non-insulin-dependent diabetes mellitus (NIDDM). As part of a population-based survey on the Indian Ocean island of Mauritius in 1992, glucose tolerance was classified using a 75-g oral glucose tolerance test on 6,553 persons. Subjects with newly diagnosed (n = 358) or known diabetes (n = 388), and a random sample of one in four subjects with impaired glucose tolerance (n = 165), had stereoscopic 45 degrees retinal photographs taken of three fields in the right eye after mydriasis. Photographs were graded according to a modified version of the Airlie House criteria. The prevalence of nonproliferative and proliferative retinopathy was: 14.5% and 0.3%, respectively, in newly diagnosed diabetic subjects; 42.0% and 2.3%, respectively, in known diabetic subjects; and 9.1% and 0%, respectively, in persons with impaired glucose tolerance. Muslim Indians had the lowest prevalence of retinopathy (10.8% and 34.0% for new and known diabetes, respectively), but after adjusting for other factors, this was significantly different only to Creoles (18.8% and 53.8%, respectively). Univariate analysis revealed significant differences between diabetic subjects with and without retinopathy in mean age, body mass index, fasting and 2-hour plasma glucose levels, systolic and diastolic blood pressure, fasting triglycerides, serum creatinine, and urinary albumin levels. For known diabetes, mean duration of diabetes and the proportion using insulin were also greater in those with retinopathy. Multivariate analysis using logistic regression confirmed that increasing duration of diabetes, fasting plasma glucose, systolic blood pressure, and urinary albumin concentration, and decreasing body mass index, were independently associated with retinopathy. The high prevalence of diabetic retinopathy observed in all major ethnic groups in Mauritius portends a serious public health problem, given the relative recency of the NIDDM epidemic in that country and the limited resources for laser photocoagulation. Strategies to minimize this problem among those already known to have diabetes should include strict control of plasma glucose and blood pressure.

Autoantibodies to glutamic acid decarboxylase and phenotypic features associated with early insulin treatment in individuals with adult-onset diabetes mellitus.

We investigated the association of serum antibodies to glutamic acid decarboxylase (GADab) with early start of insulin treatment (< or = 1 year from diagnosis, or < or = 2 years from diagnosis) using data from a representative sample of 374 adult-onset insulin-treated individuals from the Tasmanian Diabetes Register. Furthermore, we examined whether this association was stronger than the phenotypic characteristics (age at diagnosis, sex, family history of diabetes, level of obesity, duration of diabetes) often used for diabetes classification. In this cohort, 35.9% of males and 38.5% of females were GADab positive. Within the first year from diagnosis, 78.4% of GADab positive people compared to 44.0% of GADab negative people (p < 0.001) had started insulin treatment. Univariate associations with insulin treatment < or = 1 year from diagnosis included GADab positivity, no family history of diabetes, lower BMI for men, and GADab positivity and lower BMI for women. In multivariate models, significant associations with insulin treatment < or = 1 year from diagnosis included a family history of diabetes (OR = 0.47, 95% CI = 0.23-0.95) and GADab positivity (OR = 2.19, 95% CI = 1.01-4.73) for men, but only GADab positivity (OR = 7.53, 95% CI = 3.09-18.30) for women. Age at diagnosis was not associated with insulin treatment < or = 1 year or < or = 2 years from diagnosis for either sex. These findings indicate that a positive GADab test result is strongly associated with start of insulin treatment within 1 or 2 years from diagnosis, more so than characteristics such as level of obesity and age at diagnosis.

Diabetes and nontraumatic lower extremity amputations. Incidence, risk factors, and prevention--a 12-year follow-up study in Nauru.

OBJECTIVE: To measure the 12-year incidence (1982-1994) of nontraumatic lower extremity amputations (LEAs) in Nauruans, a population at high risk for NIDDM, and to determine the risk factors for amputation in Nauruans with diabetes. RESEARCH DESIGN AND METHODS: Amputation data were abstracted from operating theater records in Nauru, hospital databases in Australia, and Nauru government records. Baseline characteristics of a cohort of 1,564 Nauruans aged > or = 20 years examined during a population-based survey in 1982 were used to determine risk factors for first LEAs. RESULTS: Over this 12-year period, 46 first LEAs were performed on people with NIDDM, of whom 30 were members of the 1982 study cohort. The incidence of first LEAs in Nauruans aged > or = 25 years with NIDDM was 8.1 per 1,000 person-years in the study cohort and an estimated 7.6 per 1,000 person-years nationally. Amputations were associated significantly with lower BMI, lower blood pressure, higher fasting plasma glucose (FPG) level, and longer mean duration of diabetes at baseline, but levels of other risk factors, including cigarette smoking, plasma triglycerides, and plasma cholesterol, were also elevated in amputees. There were no amputations among individuals with baseline FPG levels < 7.8 mmol/l, irrespective of diabetes duration. FPG, baseline diabetes duration, and male sex were independent risk factors for first amputation using the Cox proportional hazards model. There was a decrease in the incidence of amputations after the commencement of a national foot care health education and prevention campaign in June 1992. CONCLUSIONS: The incidence of LEAs in diabetic Nauruans was higher than in other populations after adjusting for age and duration. Given the apparent success of the Nauruan footcare program in reducing amputation rates, other populations with high rates of NIDDM and LEAs should consider population-wide prevention strategies.

The 1984 Tasmanian insulin treated diabetes mellitus prevalence cohort: an eight and a half year mortality follow-up investigation.

Total mortality and underlying cause of death were examined in a population-based prevalence cohort (n = 1232) of Tasmanians with insulin-treated diabetes mellitus. Eight and a half years after the establishment of the registry, the cause of death based on death certificate information was determined for the overall cohort and for three classification groups of insulin-treated diabetes: Group A--childhood-onset IDDM cases; Group B--adult-onset IDDM cases; and Group C--adult-onset insulin-treated NIDDM cases. A total of 378 deaths occurred, providing an overall SMR of 2.2 (95% CI 2.0-2.4) compared to the Tasmanian population. Diabetic females experienced a higher SMR (2.6, 95% CI 2.3-3.0) than diabetic males (1.9, 95% CI 1.6-2.2). The all-cause SMRs for the diabetic classification groups were 4.6 (95% CI 3.4-6.1) in Group A, 1.8 (95% CI 1.5-2.1) in Group B, and 2.2 (95% CI 1.9-2.6) in Group C. After adjusting for age, gender and duration of diabetes, the mortality in Group C was significantly higher compared to Group B (odds ratio 1.6, 95% CI 1.2-2.3). This study indicates that people with childhood-onset IDDM experience 4.6 times the death rate compared to the Tasmanian population and that the excess mortality is most pronounced in females.

Differing frequency of autoantibodies to glutamic acid decarboxylase among Koreans, Thais, and Australians with diabetes mellitus.

The wide racial-geographic differences in the incidence and prevalence of insulin-dependent diabetes mellitus (IDDM) between Europids and Asian populations prompted us to compare frequencies of positivity of autoantibody to glutamic acid decarboxylase (GAD). The patients with IDDM included 41 Koreans, 30 Thais, and 45 Australian Europids; the Koreans included 14 cases regarded as atypical IDDM by reason of a delayed requirement for insulin treatment. Autoantibodies were measured by radioimmunoprecipitation using iodinated purified porcine brain GAD. The frequency of positive tests for anti-GAD of 30% (8/27) for Koreans and 51% (20/39) for Thais was significantly lower than the 84% (38/45) for Australian Europids, even after stratifying by age of onset. Correspondingly, the mean levels of anti-GAD among seropositive cases were significantly lower for Koreans than for Australian Europids. In contrast to Thais and Australians, more than half the Koreans were diagnosed at age > 20 years, but there was no significant difference in positivity for anti-GAD between those over or under the age of 20 at diagnosis. The different frequency of positivity in tests for anti-GAD among Koreans, Thais, and Australian Europids with IDDM suggests that there is a greater etiologic heterogeneity of IDDM among Asian than Europid populations, in whom autoimmune destruction of pancreatic islets predominates.