Ouabain Protects against Shiga Toxin–Triggered Apoptosis by Reversing the Imbalance between Bax and Bcl-xL

Hemolytic uremic syndrome, a life-threatening disease often accompanied by acute renal failure, usually occurs after gastrointestinal infection with Shiga toxin 2 (Stx2)–producing Escherichia coli. Stx2 binds to the glycosphingolipid globotriaosylceramide receptor, expressed by renal epithelial cells, and triggers apoptosis by activating the apoptotic factor Bax. Signaling via the ouabain/Na,K-ATPase/IP3R/NF-κB pathway increases expression of Bcl-xL, an inhibitor of Bax, suggesting that ouabain might protect renal cells from Stx2-triggered apoptosis. Here, exposing rat proximal tubular cells to Stx2 in vitro resulted in massive apoptosis, upregulation of the apoptotic factor Bax, increased cleaved caspase-3, and downregulation of the survival factor Bcl-xL; co-incubation with ouabain prevented all of these effects. Ouabain activated the NF-κB antiapoptotic subunit p65, and the inhibition of p65 DNA binding abolished the antiapoptotic effect of ouabain in Stx2-exposed tubular cells. Furthermore, in vivo, administration of ouabain reversed the imbalance between Bax and Bcl-xL in Stx2-treated mice. Taken together, these results suggest that ouabain can protect the kidney from the apoptotic effects of Stx2.Hemolytic uremic syndrome (HUS) is a life-threatening disease, with acute renal failure as one of the most prominent symptoms. HUS generally occurs after a gastrointestinal infection with Shiga toxin 2 (Stx2)–producing Escherichia coli.^1,2 Stx2 penetrates the intestine, enters the circulation, and binds with high affinity to glycosphingolipid globotriaosylceramide receptors (Gb3), which are expressed in many tissues but are particularly abundant in renal epithelial cells. The Stx2/Gb3 complex is endocytosed and then translocated via the Golgi apparatus and the endoplasmic reticulum to the cytosol, where the toxic A subunit will exert its effects by acting on ribosomes.³ Apoptosis is a major manifestation of Stx2 toxicity, as shown in biopsy samples from patients with HUS and in kidneys from mice inoculated with Stx2-producing versus Stx-nonproducing E. coli O157:H7, those treated with Stx2, and Stx2-exposed cells.^1,4,5The mechanism by which Stx2 activates apoptotic pathways is not fully understood, but several lines of evidence suggest that it involves activation of caspase-8 and the intrinsic, mitochondrial pathway. It is well documented that Stx2 increases caspase-8 expression.^6,7 Caspase-8 may trigger a signaling cascade that results in caspase-3 cleavage and cell death without involving the mitochondrial pathway (the extrinsic apoptotic pathway), or it may act by stimulating the apoptotic oligomerizing factors Bax and Bak, which bind to the mitochondrial membrane and make it permeable. This leads to a series of events that also result in caspase-3 cleavage and cell death (the intrinsic apoptotic pathway).⁷ Bax belongs to the Bcl-2 family, which includes both pro- and antiapoptotic factors. Bcl-xL is an antiapoptotic member of the Bcl-2 family and a potent inhibitor of Bax.^8,9 Little is known about the effect of Stx2 on Bax and Bcl-xL expression. Two independent studies found that the overexpression of Bcl-xL by transient transfection protected from Stx2 B subunit–mediated apoptosis.^10,11 HUS was previously more common in children, but the recent large outbreak of food-borne Stx2-producing E. coli O104:H4 infection suggested that severe HUS might affect adults as well as children. No available therapy protects patients from acute toxin-mediated cellular injury, including apoptosis.^12,13 Chronic renal affection or failure occurs in up to 10% of patients who survive the acute manifestations of the disease.¹³Our group has identified a novel signaling system that protects against apoptosis.^14,15 The signal is activated by the cardiotonic steroid ouabain. The signaling pathway involves interaction between Na,K-ATPase and the inositol 1,4,5-triphosphate receptor (IP3R), triggering of slow intracellular calcium oscillations, and activation of the NF-kB p65 subunit (or RelA).^16–19 Because NF-κB p65 is known to increase the expression of Bcl-xL,²⁰ we hypothesized that ouabain-triggered Na,K-ATPase signaling may counteract the apoptotic action of Stx2 by upregulation of Bcl-xL and downregulation of Bax. Here we present a series of studies in support of this hypothesis. We show that ouabain in nM concentrations protects Stx2-exposed rat renal epithelial cells from apoptosis by reversing an imbalance between Bax and Bcl-xL. Studies on mice inoculated with Stx2 provide proof of principle and show that treatment with ouabain protects against apoptosis and reverses the imbalance between Bax and Bcl-xL.     

Normal reference ranges of antithrombin,protein C and protein S: Effect of sex,age and hormonal status

Introduction

Antithrombin (AT), protein C (PC) and protein S (PS) deficiencies are risk factors for venous thromboembolism. Overlapping values between heterozygous carriers and normal individuals often make a correct classification of a deficiency difficult. The aim of this study was to investigate the effect of sex, age, menopause and hormone therapy on natural anticoagulant plasma levels in a large group of healthy individuals, and to evaluate the need of separate reference ranges.

Materials and Methods

AT and PC were measured with a chromogenic assay, antigenic free PS with an ELISA test. To evaluate the effect of sex, age, oral contraception, hormonal status (and their interaction) on AT, PC and PS levels, linear regression models were used. Biological relevance and the value of the normal deviate z were chosen as rules to decide for separate reference ranges.

Results

The study population consisted of 1837 healthy adult individuals (741 men, 1096 women), aged 18-85 years (median age: 44 years). In men AT levels decreased after the age of 50 years. Men had higher levels of PS than women, particularly at young ages. In women, after correction for menopause, only PC levels increased with age. Menopause affected AT and PS, but not PC levels. Oral contraceptive intake was associated with a decrease of AT and PS, and an increase of PC levels.

Conclusions

For AT, PC and PS, sex- and age-specific normal reference ranges can be useful, in order to better discriminate true carriers of a natural anticoagulant deficiency.     

Evidence for a minimal competence core of consonant sounds in the speech of African American children: A preliminary study

Most studies have stressed those aspects of African American English (AAE) that differ from Standard English (SE) varieties. Therefore word‐ final consonant performance has been investigated most often. In contrast, this intensive study aimed to reveal whether a common core of initial consonants is used by typically developing AAE children at or close to the 3^rd birthday. More than 2000 phonetically transcribed utterances were extracted from the audiovisual archives of natural samples of speech for seven children. All seven children met the productivity criterion for a shared core of 15 sounds in their repertoire of correctly produced word‐initial consonants. The children also met the criterion for using word‐initial consonant clusters or blends that commonly included the stop+sonorant type. The results provided preliminary evidence for a minimal competence core of word‐initial consonants that is consistent with developmental data on typically developing speakers of SE and other languages besides English. Sample sizes of more than 50 spontaneous utterances were required to elicit these core features. Research and clinical implications of these results are discussed.     

Alternative Splicing of the Chromodomain Protein Morf4l1 Pre-mRNA Has Implications on Cell Differentiation in the Developing Chicken Retina

The proliferation, cell cycle exit and differentiation of progenitor cells are controlled by several different factors. The chromodomain protein mortality factor 4-like 1 (Morf4l1) has been ascribed a role in both proliferation and differentiation. Little attention has been given to the existence of alternative splice variants of the Morf4l1 mRNA, which encode two Morf41l isoforms: a short isoform (S-Morf4l1) with an intact chromodomain and a long isoform (L-Morf4l1) with an insertion in or in the vicinity of the chromodomain. The aim of this study was to investigate if this alternative splicing has a function during development. We analysed the temporal and spatial distribution of the two mRNAs and over-expressed both isoforms in the developing retina. The results showed that the S-Morf4l1 mRNA is developmentally regulated. Over-expression of S-Morf4l1 using a retrovirus vector produced a clear phenotype with an increase of early-born neurons: retinal ganglion cells, horizontal cells and cone photoreceptor cells. Over-expression of L-Morf4l1 did not produce any distinguishable phenotype. The over-expression of S-Morf4l1 but not L-Morf4l1 also increased apoptosis in the infected regions. Our results suggest that the two Morf4l1 isoforms have different functions during retinogenesis and that Morf4l1 functions are fine-tuned by developmentally regulated alternative splicing. The data also suggest that Morf4l1 contributes to the regulation of cell genesis in the retina.     

A Two-to-Five Year Follow-Up of a Pediatric Acute-Onset Neuropsychiatric Syndrome Cohort

Child Psychiatry & Human Development - Little is known about the long-term prognosis of children with pediatric acute-onset neuropsychiatric syndrome (PANS). Out of the 46 eligible patients...     

Surgical Apgar Score Predicted Postoperative Morbidity After Esophagectomy for Esophageal Cancer

Background

Recently, a simple and easy complication prediction system, the Surgical Apgar Sore (SAS) calculated by three intraoperative parameters (estimated blood loss, lowest mean arterial pressure, and lowest heart rate), has been proposed for general surgery. This study aimed to determine if the SAS could accurately predict perioperative morbidity in patients undergoing esophagectomy for esophageal cancer.

Methods

We investigated 399 patients who underwent esophagectomy at the Kumamoto University Hospital between April 2007 and March 2015. Clinical data, including intraoperative parameters, were collected retrospectively. Patients had postoperative morbidities classified as Clavien–Dindo grade III or more. Univariate and multivariate analyses were performed to elucidate factors that affected the development of complications.

Results

The mean age of the study population was 65.7 years, 357 patients (89.5 %) were male. The frequency of any morbidity was 32.3 %. Univariate analyses showed that the SAS as well as preoperative chemotherapy, volume of bleeding, and reconstruction of organs were associated with morbidities. Multivariate analysis showed that a SAS < 5 was found to be an independent risk factor for morbidities.

Conclusion

The SAS is considered to be useful for predicting the development of postoperative morbidities after esophagectomy for esophageal cancer.

     

Non‐surgical treatment of deep wounds triggered by harmful physical and chemical agents: a successful combined use of collagenase and hyaluronic acid

Some chronic ulcers often occur with slough, not progressing through the normal stages of wound healing. Treatment is long and other therapies need to be performed in addition to surgery. Patients not eligible for surgery because of ASA class (American Society of Anesthesiologists class) appear to benefit from chemical therapy with collagenase or hydrocolloids in order to prepare the wound bed, promoting the healing process. We describe four cases of traumatic, upper limb deep wounds caused by different physical and chemical agents, emphasising the effectiveness of treatment based on topical application of collagenase and hyaluronic acid (HA) before standardised surgical procedures. We performed careful disinfection of lesions combined with application of topical cream containing hyaluronic acid, bacterial fermented sodium hyaluronate (0·2%w/w) salt, and bacterial collagenase obtained from non‐pathogenic Vibrio alginolyticus (>2·0 nkat1/g). In one patient a dermo‐epidermal graft was used to cover the wide loss of substance. In two patients application of a HA‐based dermal substitute was done. We obtained successful results in terms of wound healing, with satisfactory aesthetic result and optimal recovery of the affected limb functionality. Topical application of collagenase and HA, alone or before standardised surgical procedures allows faster wound healing.     

Exosomes in human atherosclerosis: An ultrastructural analysis study

Cell-to-cell communication, or signaling, is absolutely essential in orchestrating the activities of cells in multicellular organisms, to grow, develop, detect environmental changes and compensate for them in an internal, coordinated fashion. In the last few years, a considerable amount of new data have demonstrated the occurrence of a sophisticated intercellular signaling pathway based on the release of specialized vesicular structures, called exosomes, whose secretion appears to be regulated by various natural and experimental stimuli, physiological states, and disease processes. In the cardiovascular system, the study of exosomes is still in its infancy. Here, we aim to provide the first ultrastructural evidence for the presence of exosomes in human atherosclerotic plaque. We demonstrate by means of transmission electron microscopy that both lesional smooth muscle cells and endothelial cells are able to generate these membraneous microvesicles within specific compartments of the cell, called multivesicular bodies. Notably, in our series no signs of apoptosis have been detected in vascular cells secreting exosomes and no evidence of calcification has been observed associated with these structures in the extracellular space. Our results suggest the possible existence of a new mechanism of intercellular communication in the plaque milieu.