Effect of the viable-yellow (A(vy)) agouti allele on skin tumorigenesis and humoral hypercalcemia in v-Ha-ras transgenic TGxAC mice

We previously reported that papillomas can arise from the follicular epithelium of v-Ha-ras transgenic TGxAC mice. Since the viable-yellow mutation (A(vy)) of the mouse agouti gene which regulates coat color pigmentation by acting within the micro-environment of the hair follicle has been shown to function as a tumor promoter in the liver, we hypothesized that it may also play a role in TGxAC skin tumorigenesis. Endogenous agouti protein product was detected in the outer root sheath of anagen hair follicles following plucking of the hair shaft, but not in the interfollicular epithelium, in TGxAC mice on an FVB/N genetic background. It was also detected in papillomas from these mice produced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment or plucking. Expression of the A(vy) allele in the v-Ha-ras transgenic TGxAC mouse line results in an approximately 2-fold increase in papilloma development compared with controls which did not carry the A(vy) allele following twice-weekly treatment with 1.25, 2.5 or 5.0 microg TPA. In addition, TPA-treated, papilloma-bearing F1 mice which carried the A(vy) allele, but not F1 mice which did not carry the A(vy) allele, exhibited a syndrome of humoral hypercalcemia mediated by parathyroid hormone-related protein (PTHrP) that led to weight loss, hypercalcemia and hypophosphatemia. Thus, we conclude that the A(vy) allele can influence the development of skin tumors and PTHrP-mediated humoral hypercalcemia in v-Ha-ras transgenic TGxAC mice.      

The evolution of ductus arteriosus treatment

The treatment of patent ductus arteriosus (PDA) has evolved over the years. We reviewed 231 non-premature children (group 1) undergoing surgical closure of a PDA between January 1985 and December 1997, and 30 children (group 2) undergoing transcatheter closure from May 1995 to December 1998. The median age and weight at operation in group 1 were 13 months (range, 0.5-174 months) and 9.5 kg (range, 1.9-49.7 kg), respectively. There was one intra-operative death (0.4%) secondary to hemorrhage. Immediate extubation was performed in 208 patients (90%). Intra-operative chest tube use decreased from 73.3% to 10% between the 1985-88 and 1996-97 periods (P < 0.001). Postoperative pneumothoraces occurred in 33/131 (25%) patients with only one patient (0.7%) requiring drainage. Eleven patients had complications including wound infection in four, vocal cord paralysis in three, and left pulmonary artery stenosis in one. The median length of stay (LOS) was 5 days (range, 2-43 days). Follow-up echocardiogram was performed in 146/230 patients (63%) and revealed a residual PDA in six (4%); two being re-ligated, two remaining clinically insignificant, and two spontaneously resolved at 7 and 28 months follow-up. The remaining 84 patients had no clinical signs of a residual PDA. In group 2, where a transcatheter coil occlusion technique was used, the median age and weight at procedure were 31 months (range, 9-320 months) and 14.9 kg (range, 9-69.7 kg), respectively. Vascular complications occurred in four patients (13.3%). One patient developed hemolysis and hemoglobinuria requiring hospital admission. Four patients required a second intervention. At the most recent echocardiographic assessment, four patients (13.3%) had a residual PDA.     

The preterm gut microbiota: changes associated with necrotizing enterocolitis and infection

Aim: To describe gut colonization in preterm infants using standard culture and 16S gene rRNA profiling, exploring differences in healthy infants and those who developed NEC/late onset sepsis (LOS). Methods: Ninety‐nine stools from 38 infants of median 27‐week gestation were cultured; 44 stools from 27 infants had their microbial profiles determined by 16S. Ordination analyses explored effects of patient variables on gut communities. Results: Standard microbiological culture identified a mean of two organisms (range 0–7), DGGE 12 (range 3–18) per patient. Enterococcus faecalis and coagulase negative staphylococci (CONS) were most common by culture (40% and 39% of specimens). Meconium was not sterile. No fungi were cultured. Bacterial community structures in infants with NEC and LOS differed from healthy infants. Infants who developed NEC carried more CONS (45% vs 30%) and less Enterococcus faecalis (31% vs 57%). 16S identified Enterobacter and Staphylococcus presence associated with NEC/LOS, respectively. Conclusions: Important differences were found in the gut microbiota of preterm infants who develop NEC/LOS. The relationship of these changes to current practices in neonatal intensive care requires further exploration.     

Diagnosing liver fibrosis: a narrative review of current literature for dermatologists

Chronic liver disease is a growing problem worldwide due to obesity, alcohol‐related liver disease and viral hepatitis. Liver fibrosis is generally asymptomatic and patients may not present until they have advanced cirrhosis, when the scope for reversibility is limited. Identification of asymptomatic individuals at an early stage is fundamental to reversing the rising toll of liver‐related morbidity and mortality. Awareness of liver disease and the techniques for diagnosis is important for dermatologists, not only due to the burden of disease in the general population but also because some dermatology cohorts may have an elevated risk. For example, there is an increased prevalence of metabolic syndrome and excess alcohol use in those with psoriasis and hidradenitis suppurativa. In isolation, standard liver function tests lack sensitivity to detect advanced fibrosis and cirrhosis and are of limited value. Traditionally diagnosis has relied on liver biopsy, which remains the gold standard but is both costly and invasive. There have been several recent advances in the development of noninvasive alternatives. These include scoring systems combining clinical and conventional laboratory parameters for use as screening tools, direct serum biomarkers of fibrogenesis and tissue elastography using both ultrasound (Fibroscan) and magnetic resonance. This review summarizes current and future noninvasive diagnostic techniques for evaluation of liver fibrosis.     

Compromised first permanent molars: an orthodontic perspective

The first permanent molar (FPM) is commonly subject to significant compromise which may arise due to caries or endodontic complication, or from developmental anomalies such as hypoplasia. Compromised teeth with questionable prognosis may result in short and long-term clinical dilemmas. This review article highlights the factors that require careful consideration when a compromised FPM is detected and the importance of timely FPM extraction. Several clinical cases are described in detail to discuss possible treatment options from the orthodontic perspective.     

A robust protocol for regional evaluation of methacholine challenge in mouse models of allergic asthma using hyperpolarized 3He MRI

Hyperpolarized (HP) ³He magnetic resonance imaging has been recently used to produce high‐resolution images of pulmonary ventilation after methacholine (MCh) challenge in mouse models of allergic inflammation. This capability presents an opportunity to gain new insights about these models and to more sensitively evaluate new drug treatments in the pre‐clinical setting. In the current study, we present our initial experience using two‐dimensional (2D), time‐resolved ³He MRI of MCh challenge‐induced airways hyperreactivity (AHR) to compare ovalbumin‐sensitized and challenged (N = 8) mice to controls (N = 8). Imaging demonstrated that ovalbumin‐sensitized and challenged animals exhibited many large ventilation defects even prior to MCh challenge (four out of eight) compared to no defects in the control animals. Additionally, the ovalbumin‐sensitized and challenged animals experienced a greater number of ventilation defects (4.5 ± 0.4) following MCh infusion than did controls (3.3 ± 0.6). However, due to variability in MCh delivery that was specific to the small animal MRI environment, the difference in mean defect number was not statistically significant. These findings are reviewed in detail and a comprehensive solution to the variability problem is presented that has greatly enhanced the magnitude and reproducibility of the MCh response. This has permitted us to develop a new imaging protocol consisting of a baseline 3D image, a time‐resolved 2D series during MCh challenge, and a post‐MCh 3D image that reveals persistent ventilation defects. Copyright © 2009 John Wiley & Sons, Ltd.     

Deleterious effects of irradiation and bone marrow transplantation therapy in the genetically anemic an/an mouse

The efficacy and outcome of bone marrow transplantation therapy following lethal irradiation were examined in syngeneic mice that had a hereditary macrocytic anemia (an/an) or were genotypically normal (+/+). Successful RBC and WBC replacement, based on blood cell parameters and donor genetic markers, were observed in all combinations of transplant therapy. Nevertheless, the an/an mice died prematurely several months after treatment, whether they received +/+ or an/an marrow cells. In contrast, the +/+ recipients of either +/+ or an/an marrow cells survived for at least 1 year after transplantation. Premature death of the an/an mice was associated with lymphopenia, anemia, kidney lesions, and severe pathogen-free pneumonitis. On the basis of our results, we hypothesize that the premature deaths of an/an mice are caused by a kind of chronic irradiation damage to which an/an mice are especially susceptible.     

Periscope Proteins are variable-length regulators of bacterial cell surface interactions

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal “Periscope Proteins” as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this “rule,” suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.

Bacteria encounter complex and dynamic environments, including within human hosts, and have thus evolved various mechanisms that enable a rapid response for survival within, and exploitation of, new conditions. In addition to classical control by regulation of gene expression, bacteria exploit mechanisms that give rise to random variation to facilitate adaptation [e.g., phase and antigenic variation (1)]. In Gram-positive and Gram-negative human pathogens, DNA inversions (2, 3), homologous recombination (4), DNA methylation (1), and promoter sequence polymorphisms (5) govern changes in bacterial surface components, including capsular polysaccharide and protein adhesins, which can impact bacterial survival and virulence in the host (1, 6). Many of these mechanisms are very well studied and widespread across bacteria.A less well-studied mechanism is length variation in bacterial surface proteins. Variability in the number of sequence repeats in the Rib domain (7)–containing proteins on the surface of Group B streptococci has been linked to pathogenicity and immune evasion (8). The repetitive regions of the Staphylococcus aureus surface protein G (SasG) (9) and Staphylococcus epidermidis SasG homolog, Aap (10), also demonstrate sequence repeat number variability. In SasG, this variability regulates ligand binding by other bacterial proteins in vitro (11) in a process that has been proposed to enable bacterial dissemination in the host. Variations in repeat number have also been noted in the biofilm forming proteins Esp from Enterococcus faecalis (12) and, more recently, CdrA from Pseudomonas aeruiginosa (13). High DNA sequence identity in the genes that encode these proteins is likely to facilitate intragenic recombination events that would lead to repeat number variation (14) and, in turn, to protein sequence repetition. However, such sequence repetition is usually highly disfavored in large multidomain proteins (15), so its existence in these bacterial surface proteins suggests that protein length variation provides an evolutionary benefit. SasG, Aap, and Rib contain N-terminal host ligand binding domains and C-terminal wall attachment motifs; thus our recent demonstration that the repetitive regions of both SasG (16) and Rib (17) form unusual highly elongated rods suggests that host-colonization domains will be projected differing distances from the bacterial surface.Here, we show that repeat number variation in predicted bacterial surface proteins is more widespread and we characterize a third rod-like repetitive region in the Streptococcus gordonii protein (Sgo_0707) formed by tandem array of Streptococcal High Identity Repeats in Tandem (SHIRT) domains. Thus, we propose a growing class of “Periscope Proteins,” in which long, highly similar DNA repeats facilitate expression of surface protein stalks of variable length. This mechanism could enable changes in response to selection pressures and confer key advantages to the organism that include evasion of the host immune system (8) and regulation of surface interactions (11) involved in biofilm formation and host colonization.     

Expression of specific granule markers on the cell surface of neutrophil cytoplasts

The widespread assumption that cytoplasts generated from human polymorphonuclear leukocytes (PMNs) are vesicles consisting solely of cytoplasm surrounded by plasma membrane and devoid of granule activity remains to be tested. PMN cytoplasts were prepared by centrifugation of intact cells on a Ficoll step gradient in the presence of cytochalasin B. Two granule membrane markers, Mol, a fluorometrically detectable antigen, and cytochrome b, both of which have been shown to translocate to the plasma membrane during granule release, were compared for their activity in cytoplasts and intact PMNs. We found that the amount of Mol detected on the plasma membrane of intact PMNs, as compared with other membrane markers (such as antigens LFA-1 and beta 2m), increased 1.6- fold upon exposure of PMNs to Ficoll plus cytochalasin B prior to centrifugation. Another twofold increase in Mol expression occurred upon cytoplast preparation. Release of the granule enzymes, vitamin B12- binding protein, and lysozyme were also followed and correlated well (r = .78 and .92) with the amount of Mol antigen present on the cell surface. Cytochrome b was also found to be higher (1.4-fold) on plasma membranes isolated from cytoplasts than on plasma membranes isolated from intact control cells. These results indicate that some fusion of granule membranes and plasma membranes occurred during treatment of PMNs with Ficoll plus cytochalasin b and during cytoplast preparation.