Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study

Reasons for performing study: Light microscopical studies show that the key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. More precise knowledge of the damage occurring in the lamellar basement membrane zone may result if laminitis affected tissue is examined with the transmission electron microscope. This could lead to better understanding of the pathogenesis of lesions and the means of treatment or prevention. Objectives: To investigate the ultrastructure of acute laminitis as disease of greater severity is induced by increasing oligofructose (OF) dosage. Methods: Three pairs of normal horses, dosed with OF at 7.5, 10 and 12.5 g/kg bwt via nasogastric intubation, developed laminitis 48 h later. Following euthanasia, their forefeet were processed for transmission electron microscopy. Lamellar basal cell hemidesmosome (HD) numbers and the distance between the basal cell plasmalemma and the lamina densa of the basement membrane were estimated and compared to control tissue. Results: Increasing OF dosage caused greater HD loss and more severe laminitis. The characteristic separation of the basement membrane, cytoskeleton failure and rounded basal cell nuclei results from combined HD dysassembly and anchoring filament failure. Conclusions: Without properly assembled HDs, dysadhesion between the lamina densa of the basement membrane (BM) and epidermal basal cells occurs, emphasising the fundamental importance of HDs in maintaining attachment at the lamellar interface. Medical conditions that trigger lamellar matrix metalloproteinase (MMP) activation and/or compromise entry of glucose into lamellar basal cells appear to promote loss and failure of HDs and, therefore, laminitis development. Potential relevance: A correlation between lameness severity and escalating loss of lamellar HDs now exists. Therapy aimed at protecting the lamellar environment from haematogenous delivery of MMP activators or from glucose deprivation may control laminitis development.

On the loss of regularity for a class of weakly hyperbolic operators

In this paper we determine bounds for the optimal loss of regularity in the Sobolev scale for a class of weakly hyperbolic operators. (C) 2009 Elsevier Inc. All rights reserved.

Loss of Ergodicity in the Transition from Annealed to Quenched Disorder in a Finite Kinetic Ising Model

We consider a kinetic Ising model which represents a generic agent-based model for various types of socio-economic systems. We study the case of a finite (and not necessarily large) number of agents N as well as the asymptotic case when the number of agents tends to infinity. The main ingredient are individual decision thresholds which are either fixed over time (corresponding to quenched disorder in the Ising model, leading to nonlinear deterministic dynamics which are generically non-ergodic) or which may change randomly over time (corresponding to annealed disorder, leading to ergodic dynamics). We address the question how increasing the strength of annealed disorder relative to quenched disorder drives the system from non-ergodic behavior to ergodicity. Mathematically rigorous analysis provides an explicit and detailed picture for arbitrary realizations of the quenched initial thresholds, revealing an intriguing ""jumpy"" transition from non-ergodicity with many absorbing sets to ergodicity. For large N we find a critical strength of annealed randomness, above which the system becomes asymptotically ergodic. Our theoretical results suggests how to drive a system from an undesired socio-economic equilibrium (e. g. high level of corruption) to a desirable one (low level of corruption).

Insertion/deletion polymorphism of the angiotensin converting enzyme gene and loss of the insertion allele in aldosterone-producing adenoma

The genetic mechanisms responsible for the formation of adrenocortical adenomas which autonomously produce aldosterone are largely unknown, The adrenal renin-angiotensin system has been implicated in the pathophysiology of these tumours, Angiotensin-converting enzyme (ACE) catalyses the generation of angiotensin II, and the insertion/deletion (I/D) polymorphism of the ACE gene regulates up to 50% of plasma and cellular ACE variability in humans. We therefore examined the genotypic and allelic frequency distributions of the ACE gene I/D polymorphism in 55 patients with aldosterone-producing adenoma, APA, (angiotensin-unresponsive APA n = 28, angiotensin-responsive APA n = 27), and 80 control subjects with no family history of hypertension, We also compared the ACE gene I/D polymorphism allelic pattern in matched tumour and peripheral blood DNA in the 55 patients with APA, The frequency of the D allele was 0.518 and 0.512 and the I allele was 0.482 and 0.488 in the APA and control subjects respectively, Genotypic and allelic frequency analysis found no significant differences between the groups, Examination of the matched tumour and peripheral blood DNA samples revealed the loss of the insertion allele in four of the 25 patients who were heterozygous for the ACE I/D genotype. The I/D polymorphism of the ACE gene does not appear to contribute to the biochemical and phenotypic characteristics of APA, however, the deletion of the insertion allele of the ACE gene I/D polymorphism in 16% of aldosterone-producing adenomas may represent the loss of a tumour suppressor gene/s or other genes on chromosome 17q which may contribute to tumorigenesis in APA.

Loss of heterozygosity of the APC gene in oral squamous cell carcinoma

The aim of this study was to investigate loss of heterozygosity (LOH) of the APC tumor suppressor gene loci, using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) in 40 cases of oral squamous cell carcinoma (OSCC). Observed informativity was 72.5% for APC exon 11 and 82.5% for APC exon 15. LOH at APC exon 11 was observed in 2 (6.9%) of 29 informative cases, and no LOH was observed for APC exon 15. Our results suggest that inactivation of the APC gene plays a minor role in the carcinogenesis of OSCC. (C) 2008 Elsevier GmbH. All rights reserved.

A computerized tomography scan method for calculating the hernia sac and abdominal cavity volume in complex large incisional hernia with loss of domain

Preoperative progressive pneumoperitoneum (PPP) is a safe and effective procedure in the treatment of large incisional hernia (size > 10 cm in width or length) with loss of domain (LIHLD). There is no consensus in the literature on the amount of gas that must be insufflated in a PPP program or even how long it should be maintained. We describe a technique for calculating the hernia sac volume (HSV) and abdominal cavity volume (ACV) based on abdominal computerized tomography (ACT) scanning that eliminates the need for subjective criteria for inclusion in a PPP program and shows the amount of gas that must be insufflated into the abdominal cavity in the PPP program. Our technique is indicated for all patients with large or recurrent incisional hernias evaluated by a senior surgeon with suspected LIHLD. We reviewed our experience from 2001 to 2008 of 23 consecutive hernia surgical procedures of LIHLD undergoing preoperative evaluation with CT scanning and PPP. An ACT was required in all patients with suspected LIHLD in order to determine HSV and ACV. The PPP was performed only if the volume ratio HSV/ACV (VR = HSV/ACV) was a parts per thousand yen25% (VR a parts per thousand yen 25%). We have performed this procedure on 23 patients, with a mean age of 55.6 years (range 31-83). There were 16 women and 7 men with an average age of 55.6 years (range 31-83), and a mean BMI of 38.5 kg/m(2) (range 23-55.2). Almost all patients (21 of 23 patients-91.30%) were overweight; 43.5% (10 patients) were severely obese (obese class III). The mean calculated volumes for ACV and HSV were 9,410 ml (range 6,060-19,230 ml) and 4,500 ml (range 1,850-6,600 ml), respectively. The PPP is performed by permanent catheter placed in a minor surgical procedure. The total amount of CO(2) insufflated ranged from 2,000 to 7,000 ml (mean 4,000 ml). Patients required a mean of 10 PPP sessions (range 4-18) to achieve the desired volume of gas (that is the same volume that was calculated for the hernia sac). Since PPP sessions were performed once a day, 4-18 days were needed for preoperative preparation with PPP. The mean VR was 36% (ranged from 26 to 73%). We conclude that ACT provides objective data for volume calculation of both hernia sac and abdominal cavity and also for estimation of the volume of gas that should be insufflated into the abdominal cavity in PPP.

LOSS OF CD40 ENDOGENOUS S-NITROSYLATION DURING INFLAMMATORY RESPONSE IN ENDOTOXEMIC MICE AND PATIENTS WITH SEPSIS

Signal transduction through the surface molecule CD40 is critical for cellular activation in immunoinflammatory states such as sepsis. The mechanisms regulating this pathway are not completely understood. Because CD40 displays potentially regulatory cysteine residues and CD40 is probably exposed to NO in the inflammatory milieu, we hypothesized that S-nitrosylation, the interaction of NO with cysteines residues, acts as a post-translational modification on CD40, coregulating the signaling activity and, therefore, the level of cellular activation. As assessed by the biotin switch and the reduction/chemiluminescence S-nitrosylation detection techniques, CD40 was found to be S-nitrosylated endogenously and upon exposure to NO donors in both human and murine macrophages. S-nitrosylation of CD40 was associated with milder activation by its ligand (CD40L), leading to reduced in vitro cytokine (IL-1 beta, IL-12, and TNF-alpha) production, which was reversed in the presence of inhibitors of NO synthesis. S-nitrosylated CD40 was found in resting RAW 246.7 macrophages and BALB/c mice peritoneal macrophages, turning into the denitrosylated state upon in vitro or systemic exposure, respectively, to LPS. Moreover, monocytes from patients with sepsis displayed denitrosylated CD40 in contrast to the CD40 S-nitrosylation measured in healthy individuals. Finally, in an attempt to explain how S-nitrosylation regulates CD40 activation, we demonstrate that NO affects the redistribution of CD40 on the cell surface, which is a requirement for optimal signal transduction. Our results support a novel post-translational regulatory mechanism in which the CD40 signal may be, at least in part, dependent on cellular activation-induced receptor denitrosylation.

PTCH1 gene mutations in exon 17 and loss of heterozygosity on D9S180 microsatellite in sporadic and inherited human basal cell carcinomas

Background Basal cell carcinomas (BCCs) are the most frequent human cancer that results from malignant transformation of basal cells in the epidermis. Gorlin syndrome is a rare inherited autosomal dominant disease that predisposes with multiple BCCs and other birth defects. Both sporadic and inherited BCCs are associated with mutations in the tumor suppressor gene PTCH1, but there is still uncertainty on the role of its homolog PTCH2. Objectives To search for mutations and genomic instability in sporadic and inherited BCCs. Methods DNA obtained from leukocytes and tumor cells was amplified by polymerase chain reaction regarding five exons of PTCH1 and PTCH2 and neighboring microsatellites. Exons were sequenced and compared with the GenBank database. Results Only D9S180, of six microsatellites, showed loss of heterozygosity in three BCCs (two sporadic and one inherited). One sporadic BCC presented the mutation g. 2885G>C in exon 17 of PTCH1, which predicts the substitution p.R962T in an external domain of the protein. In addition, the leukocytes and tumor cells of one patient with Gorlin syndrome showed the mutation g. 2839T>G in the same exon and gene, which predicts a p.E947stop and truncated protein. All control and tumor samples presented IVS9 + 217T in intron 9 of PTCH1. Conclusion Mutations found in the PTCH1 gene and neighboring repetitive sequences may have contributed to the development of the studied BCCs.

Loss of the Ca2+/calmodulin-dependent protein kinase type IV in dopaminoceptive neurons enhances behavioral effects of cocaine

The persistent nature of addiction has been associated with activity-induced plasticity of neurons within the striatum and nucleus accumbens (NAc). To identify the molecular processes leading to these adaptations, we performed Cre/loxP-mediated genetic ablations of two key regulators of gene expression in response to activity, the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) and its postulated main target, the cAMP-responsive element binding protein (CREB). We found that acute cocaine-induced gene expression in the striatum was largely unaffected by the loss of CaMKIV. On the behavioral level, mice lacking CaMKIV in dopaminoceptive neurons displayed increased sensitivity to cocaine as evidenced by augmented expression of locomotor sensitization and enhanced conditioned place preference and reinstatement after extinction. However, the loss of CREB in the forebrain had no effect on either of these behaviors, even though it robustly blunted acute cocaine-induced transcription. To test the relevance of these observations for addiction in humans, we performed an association study of CAMK4 and CREB promoter polymorphisms with cocaine addiction in a large sample of addicts. We found that a single nucleotide polymorphism in the CAMK4 promoter was significantly associated with cocaine addiction, whereas variations in the CREB promoter regions did not correlate with drug abuse. These findings reveal a critical role for CaMKIV in the development and persistence of cocaine-induced behaviors, through mechanisms dissociated from acute effects on gene expression and CREB-dependent transcription.

E-cadherin and beta-catenin Loss of Expression Related to Bone Metastasis in Prostate Cancer

Objectives: E-cadherin and beta-catenin are adhesion molecules responsible for the maintenance of normal epithelial cell phenotype. A disturbance in epithelial cell adhesion, which leads to a more invasive and metastatic phenotype, is a hallmark of tumor progression. Several immunohistochemical studies have reported a strong correlation between loss of their expression to higher stage and grade in prostate carcinoma, but their influence in metastatic process is not yet known. The aim of this study is to verify the role of adhesion molecules in the progression of prostate cancer (PC), assessing the expression of E-cadherin and beta-catenin in bone metastasis. Materials and Methods: Twenty-eight bone metastases of prostate carcinoma were submitted to immunohistochemistry analysis for E-cadherin and beta-catenin expression. In 6 patients, we were able to assess the expression of the adhesion molecules in the primary tumors and their respective metastases. The definition of normal expression for both antibodies was strong and diffuse expression in more than 70% of tumor cells. Results: In bone metastases, there was loss of expression of E-cadherin and beta-catenin in 86% and 82%, respectively. Among the primary tumors, E-cadherin and beta-catenin expression was normal in 83% and 50% cases, respectively. Considering the 6 patients with paired primary and bone metastasis, we found loss of expression for both E-cadherin and beta-catenin in most of the cases. Conclusions: Comparing primary PC and its metastasis, we showed persistent loss of E-cadherin and beta-catenin expression. This phenomenon may be related to metastatic potential in PC, because we have shown underexpression for E-cadherin and beta-catenin in 86% and 82% of bone metastases.

Non-Human Leukocyte Antigen Antibodies Reactive with Endothelial Cells Could Be Involved in Early Loss of Renal Allografts

Preformed donor-specific human leukocyte antigen (HLA) antibodies have been associated with allograft dysfunction and failure. However, recipients of HLA-identical kidneys can develop acute humoral rejection, implicating putative pathogenic antibodies that are directed against non-HLA antigens. We investigated the presence of endothelial cell reactive antibodies in 11 patients who experienced early loss of their transplanted kidneys owing to humoral rejection and 1 loss from renal venal thrombosis. We examined the potential efficacy of intravenous immunoglobulin to block the binding of these antibodies, as previously suggested for anti-HLA antibodies.

Loss-of-function mutations in the genes encoding prokineticin-2 or prokineticin receptor-2 cause autosomal recessive Kallmann syndrome

Context: Physiological activation of the prokineticin pathway has a critical role in olfactory bulb morphogenesis and GnRH secretion in mice. Objective: To investigate PROK2 and PROKR2 mutations in patients with hypogonadotropic hypogonadism (HH) associated or not with olfactory abnormalities. Design: We studied 107 Brazilian patients with HH (63 with Kallmann syndrome and 44 with normosmic HH) and 100 control individuals. The coding regions of PROK2 and PROKR2 were amplified by PCR followed by direct automatic sequencing. Results: In PROK2, two known frameshift mutations were identified. Two brothers with Kallmann syndrome harbored the homozygous p. G100fsX121 mutation, whereas one male with normosmic HH harbored the heterozygous p. I55fsX56 mutation. In PROKR2, four distinct mutations (p. R80C, p. Y140X, p. L173R, and p. R268C) were identified in five patients with Kallmann syndrome and in one patient with normosmic HH. These mutations were not found in the control group. The p. R80C, p. L173R, and p. R268C missense mutations were identified in the heterozygous state in the HH patients and in their asymptomatic first-degree relatives. In addition, nomutations of FGFR1, KAL1, GnRHR, KiSS-1, or GPR54 were identified in these patients. Notably, the new nonsense mutation (p. Y140X) was identified in the homozygous state in an anosmic boy with micropenis, bilateral cryptorchidism, and high-arched palate. His asymptomatic parents were heterozygous for this severe defect. Conclusion: We expanded the repertoire of PROK2 and PROKR2 mutations in patients with HH. In addition, we show that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome or normosmic HH, whereas homozygous loss-of-function mutations either in PROKR2 or PROK2 are sufficient to cause disease phenotype, in accordance with the Prokr2 and Prok2 knockout mouse models.

Down-regulation of expression of osteoblast and osteocyte markers in periodontal tissues associated with the spontaneous alveolar bone loss of interleukin-10 knockout mice

The aim of this study was to unravel the mechanisms by which interleukin (IL)-10, a potent pleiotropic cytokine, modulates alveolar bone homeostasis in C57BL/6 wild-type (WT) and IL-10 knockout (IL-10 KO) mice, evaluated at 8, 24, and 48 wk of age. Interleukin-10 KO mice presented significant alveolar bone loss when compared with WT mice, and this was not associated with changes in leukocyte counts or bacterial load. The levels of expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, IL-6, transforming growth factor-beta (TGF-beta), receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), and matrix metalloproteinase 13 (MMP13) were similar between both strains, whereas a significant decrease of tissue inhibitor of metalloproteinase 1 (TIMP1) mRNA expression was found at 48 wk in IL-10 KO mice. The osteoblast markers core binding factor alpha1 (CBFA1) and type I collagen (COL-I) were expressed at similar levels in both strains, whereas the levels of alkaline phosphatase (ALP) and osteocalcin (OCN), and those of the osteocyte markers phosphate-regulating gene endopeptidases (PHEX) and dentin matrix protein 1 (DMP1) were significantly lower in IL-10 KO mice. Our results demonstrate that the alveolar bone loss in the absence of IL-10 was associated with a reduced expression of osteoblast and osteocyte markers, an effect independent of microbial, inflammatory or bone-resorptive pathways.

Allelic status of 1p and 19q in oligodendrogliomas and glioblastomas: multiplex ligation-dependent probe amplification versus loss of heterozygosity

Identification of the 1p/19q allelic status in gliomas, primarily those with a major oligodendroglial component, has become an excellent molecular complement to tumor histology in order to identify those cases sensitive to chemotherapy. In addition to loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), or comparative genomic hybridization (CGH), multiplex ligation-dependent probe amplification (MLPA) has been shown to be an alternative methodology to identify deletions of those chromosome arms. We used MLPA to explore the 1p and 19q glioblastomas, and a series of 76 gliomas: 41 tumors with a major oligodendroglial component, 34 glioblastomas, and one low-grade astrocytoma. We compared the MLPA findings of the oligodendroglial cases with those previously obtained using LOH in the same samples. Thirty-eight of 41 oligodendrogliomas displayed identical findings by both LOH and MLPA, and losses at either 1p and/or 19q were identified in 12 of 35 (34%) astrocytic tumors. These findings agree with data previously reported comparing MLPA versus FISH or CGH in gliomas and suggest that MLPA can be used in the identification 1p/19q allelic deletions on these brain neoplams. (c) 2009 Elsevier Inc. All rights reserved. reserved.