Sequence capture and next-generation resequencing of multiple tagged nucleic acid samples for mutation screening of urea cycle disorders

Molecular genetic testing is commonly used to confirm clinical diagnoses of inherited urea cycle disorders (UCDs); however, conventional mutation screenings encompassing only the coding regions of genes may not detect disease-causing mutations occurring in regulatory elements and introns. Microarray-based target enrichment and next-generation sequencing now allow more-comprehensive genetic screening. We applied this approach to UCDs and combined it with the use of DNA bar codes for more cost-effective, parallel analyses of multiple samples.

Fractional transepidermal delivery: a histological analysis

In autologous cell therapy, e.g. in melanocyte transplantation for vitiligo, a minimally invasive mode of transepidermal delivery of the isolated cells is of crucial importance to reduce potential side effects such as infections and scarring as well as to minimize the duration of sick leave.

IL28B alleles associated with poor hepatitis C virus (HCV) clearance protect against inflammation and fibrosis in patients infected with non-1 HCV genotypes

Genetic polymorphisms near IL28B are associated with spontaneous and treatment-induced clearance of hepatitis C virus (HCV), two processes that require the appropriate activation of the host immune responses. Intrahepatic inflammation is believed to mirror such activation, but its relationship with IL28B polymorphisms has yet to be fully appreciated. We analyzed the association of IL28B polymorphisms with histological and follow-up features in 2335 chronically HCV-infected Caucasian patients. Assessable phenotypes before any antiviral treatment included necroinflammatory activity (n = 1,098), fibrosis (n = 1,527), fibrosis progression rate (n = 1,312), and hepatocellular carcinoma development (n = 1,915). Associations of alleles with the phenotypes were evaluated by univariate analysis and multivariate logistic regression, accounting for all relevant covariates. The rare G allele at IL28B marker rs8099917-previously shown to be at risk of treatment failure-was associated with lower activity (P = 0.04), lower fibrosis (P = 0.02) with a trend toward lower fibrosis progression rate (P = 0.06). When stratified according to HCV genotype, most significant associations were observed in patients infected with non-1 genotypes (P = 0.003 for activity, P = 0.001 for fibrosis, and P = 0.02 for fibrosis progression rate), where the odds ratio of having necroinflammation or rapid fibrosis progression for patients with IL28B genotypes TG or GG versus TT were 0.48 (95% confidence intervals 0.30-0.78) and 0.56 (0.35-0.92), respectively. IL28B polymorphisms were not predictive of the development of hepatocellular carcinoma.

Systemic and hepatic vein galectin-3 are increased in patients with alcoholic liver cirrhosis and negatively correlate with liver function

Recently we demonstrated higher galectin-3 in portal venous serum (PVS) compared to hepatic venous serum (HVS) in a small cohort of patients with normal liver function suggesting hepatic removal of galectin-3. Here, galectin-3 was measured by ELISA in PVS, HVS and systemic venous blood (SVS) of 33 patients with alcoholic liver cirrhosis and a larger cohort of 11 patients with normal liver function. Galectin-3 was cleared by the healthy but not the cirrhotic liver, and subsequently HVS and SVS galectin-3 levels were significantly increased in the patients with liver cirrhosis compared to controls. In healthy liver galectin-3 was produced by cholangiocytes and synthesis by hepatocytes was only observed in cirrhotic liver. Hepatic venous pressure gradient did not correlate with galectin-3 levels excluding hepatic shunting as the principal cause of higher SVS galectin-3. Galectin-3 was elevated in all blood compartments of patients with CHILD-PUGH stage C compared to patients with CHILD-PUGH stage A, and was higher in patients with ascites than patients without this complication. Galectin-3 was negatively associated with antithrombin-3 whose synthesis is reduced with worse liver function. Galectin-3 positively correlated with urea and creatinine, and PVS galectin-3 showed a negative association with creatinine clearance as an accepted measure of kidney function. To summarize in the current study systemic, portal and hepatic levels of galectin-3 were found to be negatively associated with liver function in patients with alcoholic liver cirrhosis and this may in part be related to impaired hepatic removal and/or increased synthesis in cirrhotic liver.

Estimating the net contribution of interleukin-28B variation to spontaneous hepatitis C virus clearance

The identification of associations between interleukin-28B (IL-28B) variants and the spontaneous clearance of hepatitis C virus (HCV) raises the issues of causality and the net contribution of host genetics to the trait. To estimate more precisely the net effect of IL-28B genetic variation on HCV clearance, we optimized genotyping and compared the host contributions in multiple- and single-source cohorts to control for viral and demographic effects. The analysis included individuals with chronic or spontaneously cleared HCV infections from a multiple-source cohort (n = 389) and a single-source cohort (n = 71). We performed detailed genotyping in the coding region of IL-28B and searched for copy number variations to identify the genetic variant or haplotype carrying the strongest association with viral clearance. This analysis was used to compare the effects of IL-28B variation in the two cohorts. Haplotypes characterized by carriage of the major alleles at IL-28B single-nucleotide polymorphisms (SNPs) were highly overrepresented in individuals with spontaneous clearance versus those with chronic HCV infections (66.1% versus 38.6%, P = 6 × 10(-9) ). The odds ratios for clearance were 2.1 [95% confidence interval (CI) = 1.6-3.0] and 3.9 (95% CI = 1.5-10.2) in the multiple- and single-source cohorts, respectively. Protective haplotypes were in perfect linkage (r(2) = 1.0) with a nonsynonymous coding variant (rs8103142). Copy number variants were not detected. CONCLUSION: We identified IL-28B haplotypes highly predictive of spontaneous HCV clearance. The high linkage disequilibrium between IL-28B SNPs indicates that association studies need to be complemented by functional experiments to identify single causal variants. The point estimate for the genetic effect was higher in the single-source cohort, which was used to effectively control for viral diversity, sex, and coinfections and, therefore, offered a precise estimate of the net host genetic contribution.

Long-term course of lung clearance index between infancy and school-age in cystic fibrosis subjects

Multiple breath washout (MBW) measurements have recently been shown to be sensitive for detection of early cystic fibrosis (CF) lung disease, with the lung clearance index (LCI) being the most common measure for ventilation inhomogeneity. The aim of this observational study was to describe the longitudinal course of LCI from time of clinical diagnosis during infancy to school-age in eleven children with CF. Elevated LCI during infancy was present in seven subjects, especially in those with later clinical diagnosis. Tracking of LCI at follow-up was evident only in the four most severe cases. We provide the first longitudinal data describing the long-term course of LCI in a small group of infants with CF. Our findings support the clinical usefulness of MBW measurements to detect and monitor early lung disease in children with CF already present shortly after clinical diagnosis.

Clearance of p-cresol sulfate and β-2-microglobulin from dialysate by commercially available sorbent technology

Dialysate regeneration by sorbents is an alternative to conventional single-pass dialysis. Little is known about the capacity of sorbents to clear dialysate of “middle molecules” and protein-bound uremic toxins. We studied p-cresol sulfate (PCS) and β-2-microglobulin (β2M) removal from dialysate by a sorbent: 1. PCS (40 mg PCS dissolved in 4 L of fresh dialysate) was recirculated through a sorbent cartridge (SORB Technology, Inc.) for analysis of PCS removal. 2. Spent peritoneal dialysate was recirculated on the “blood” side of a high-flux dialyzer. On the “dialysate” side of the membrane, bicarbonate dialysate was recirculated through a sorbent cartridge. β2M was measured in both streams. Two results are of particular importance for the use of regenerated fluid in chronic dialysis: 1. PCS was virtually completely removed from the dialysate. On average, PCS concentration was reduced to 1.4% of the starting concentration after 60 minutes. PCS extraction across the sorbent was nearly complete at any time. 2. β2M was on average reduced to 14.3% of the starting concentration after 60 minutes. Postsorbent concentrations were consistently below the validated range of the test method. We conclude that PCS and β2M are efficiently removed from the dialysate by commercially available sorbent technology. Spent peritoneal dialysis fluid can be cleared of β2M when circulated against sorbent-regenerated dialysate using a high-flux membrane.

Strain-specific spleen remodelling in Plasmodium yoelii infections in Balb/c mice facilitates adherence and spleen macrophage-clearance escape

Knowledge of the dynamic features of the processes driven by malaria parasites in the spleen is lacking. To gain insight into the function and structure of the spleen in malaria, we have implemented intravital microscopy and magnetic resonance imaging of the mouse spleen in experimental infections with non-lethal (17X) and lethal (17XL) Plasmodium yoelii strains. Noticeably, there was higher parasite accumulation, reduced motility, loss of directionality, increased residence time and altered magnetic resonance only in the spleens of mice infected with 17X. Moreover, these differences were associated with the formation of a strain-specific induced spleen tissue barrier of fibroblastic origin, with red pulp macrophage-clearance evasion and with adherence of infected red blood cells to this barrier. Our data suggest that in this reticulocyte-prone non-lethal rodent malaria model, passage through the spleen is different from what is known in other Plasmodium species and open new avenues for functional/structural studies of this lymphoid organ in malaria.

Fractional flow reserve-guided PCI versus medical therapy in stable coronary disease

The preferred initial treatment for patients with stable coronary artery disease is the best available medical therapy. We hypothesized that in patients with functionally significant stenoses, as determined by measurement of fractional flow reserve (FFR), percutaneous coronary intervention (PCI) plus the best available medical therapy would be superior to the best available medical therapy alone.

Increased surfactant protein D fails to improve bacterial clearance and inflammation in serpinB1-/- mice

Previously, we described the protective role of the neutrophil serine protease inhibitor serpinB1 in preventing early mortality of Pseudomonas aeruginosa lung infection by fostering bacterial clearance and limiting inflammatory cytokines and proteolytic damage. Surfactant protein D (SP-D), which maintains the antiinflammatory pulmonary environment and mediates bacterial removal, was degraded in infected serpinB1-deficient mice. Based on the hypothesis that increased SP-D would rescue or mitigate the pathological effects of serpinB1 deletion, we generated two serpinB1(-/-) lines overexpressing lung-specific rat SP-D and inoculated the mice with P. aeruginosa. Contrary to predictions, bacterial counts in the lungs of SP-D(low)serpinB1(-/-) and SP-D(high) serpinB1(-/-) mice were 4 logs higher than wild-type and not different from serpinB1(-/-) mice. SP-D overexpression also failed to mitigate inflammation (TNF-α), lung injury (free protein, albumin), or excess neutrophil death (free myeloperoxidase, elastase). These pathological markers were higher for infected SP-D(high)serpinB1(-/-) mice than for serpinB1(-/-) mice, although the differences were not significant after controlling for multiple comparisons. The failure of transgenic SP-D to rescue antibacterial defense of serpinB1-deficient mice occurred despite 5-fold or 20-fold increased expression levels, largely normal structure, and dose-dependent bacteria-aggregating activity. SP-D of infected wild-type mice was intact in 43-kD monomers by reducing SDS-PAGE. By contrast, proteolytic fragments of 35, 17, and 8 kD were found in infected SP-D(low)serpinB1(-/-), SP-D(high) serpinB1(-/-) mice, and serpinB1(-/-) mice. Thus, although therapies to increase lung concentration of SP-D may have beneficial applications, the findings suggest that therapy with SP-D may not be beneficial for lung inflammation or infection if the underlying clinical condition includes excess proteolysis.

Electrolyte-free water clearance versus modified electrolyte-free water clearance: do the results justify the effort?

Calculation of electrolyte-free water clearance (EFWC) allows for quantification of renal losses of free water and was shown to be helpful in the differential diagnosis of dysnatremias and might help in the correction of the electrolyte disorders. A modified EFWC formula (MEFWC) was described to be more accurate than the conventional one; however, it has never been evaluated clinically.

Treatment response of airway clearance assessed by single-breath washout in children with cystic fibrosis

We studied the ability of 4 single-breath gas washout (SBW) tests to measure immediate effects of airway clearance in children with CF.

Cross-sectional observational study of 208 patients with non-classical urea cycle disorders

Urea cycle disorders (UCDs) are inherited disorders of ammonia detoxification often regarded as mainly of relevance to pediatricians. Based on an increasing number of case studies it has become obvious that a significant number of UCD patients are affected by their disease in a non-classical way: presenting outside the newborn period, following a mild course, presenting with unusual clinical features, or asymptomatic patients with only biochemical signs of a UCD. These patients are surviving into adolescence and adulthood, rendering this group of diseases clinically relevant to adult physicians as well as pediatricians. In preparation for an international workshop we collected data on all patients with non-classical UCDs treated by the participants in 20 European metabolic centres. Information was collected on a cohort of 208 patients 50% of which were ≥ 16 years old. The largest subgroup (121 patients) had X-linked ornithine transcarbamylase deficiency (OTCD) of whom 83 were female and 29% of these were asymptomatic. In index patients, there was a mean delay from first symptoms to diagnosis of 1.6 years. Cognitive impairment was present in 36% of all patients including female OTCD patients (in 31%) and those 41 patients identified presymptomatically following positive newborn screening (in 12%). In conclusion, UCD patients with non-classical clinical presentations require the interest and care of adult physicians and have a high risk of neurological complications. To improve the outcome of UCDs, a greater awareness by health professionals of the importance of hyperammonemia and UCDs, and ultimately avoidance of the still long delay to correctly diagnose the patients, is crucial.