Role of vacuolating cytotoxin in gastritis due to Helicobacter pylori in gnotobiotic piglets.

To investigate the role of the Helicobacter pylori cytotoxin in the pathogenesis of gastritis, gnotobiotic piglets were colonized with either toxigenic H. pylori or a nontoxigenic isogenic mutant. Only piglets given the toxigenic strain developed toxin-neutralizing antibodies (indicating that toxin is expressed in vivo), but there was no difference in bacterial colonization, epithelial vacuolation, or gastritis between the two groups of piglets.     

Helicobacter pylori gastric infection in gnotobiotic beagle dogs.

Establishment of infection with Helicobacter pylori and gastritis in nonhuman species is currently only successful in gnotobiotic piglets. This study was designed to determine whether H. pylori will colonize the gastrointestinal tract of gnotobiotic dogs. Gnotobiotic beagle pups were derived by standard methods. Group A (five dogs) was orally challenged with 3 x 10(8) H. pylori at 7 days of age. Group B (two dogs) received only peptone water but was contact-exposed beginning on day 23 postinfection (p.i.). Necropsy was performed on dogs on day 30 p.i. H. pylori colonized the stomach of all dogs (groups A and B). Urease map analysis correlated with the microbiologic findings and indicated that the density of colonization was less than that observed in human tissue. Organisms were also recovered from the pharynx, esophagus, duodenum, and rectum of 1, 2, 2, and 1 dog, respectively. All group A and one group B dog developed serum immunoglobulin G specific for H. pylori by day 30 p.i. Gross lesions were restricted to the stomach and consisted of small (less than 1 mm) lymphoid follicles. Microscopically, there were focal to diffuse lymphoplasmacytic infiltrates with follicle formation and mild to moderate infiltration of neutrophils and eosinophils in the gastric lamina propria. With the Warthin-Starry silver stain, organisms were seen on the surface of the gastric epithelial cells, beneath the mucus layer. We conclude that H. pylori colonizes the stomachs of gnotobiotic dogs for at least 1 month and the lesions resemble those seen in humans. H. pylori is transmissible by contact from infected to noninfected dogs.     

Serotonergic neural precursor cell grafts attenuate bilateral hyperexcitability of dorsal horn neurons after spinal hemisection in rat

Hemisection of the rat spinal cord at thoracic level 13 provides a model of spinal cord injury that is characterized by chronic pain attributable to hyperexcitability of dorsal horn neurons. Presuming that this hyperexcitability can be explained in part by interruption of descending inhibitory modulation by serotonin, we hypothesized that intrathecal transplantation of RN46A-B14 serotonergic precursor cells, which secrete serotonin and brain-derived neurotrophic factor, would reduce this hyperexcitability by normalizing the responses of low-threshold mechanoreceptive, nociceptive-specific, and multireceptive dorsal horn neurons. Three groups (n=45 total) of 30-day-old male Sprague-Dawley rats underwent thoracic level 13 spinal hemisection, after which four weeks were allowed for development of allodynia and hyperalgesia. The three groups of animals received transplants of no cells, 10(6) RN46A-V1 (vector-only) or 10(6) RN46A-B14 cells at lumbar segments 2-3. Electrophysiological experiments were done two weeks later. Low-threshold mechanoreceptive, nociceptive-specific, and multireceptive cells (n=394 total) were isolated at depths of 1-300 and 301-1000 micro in the lumbar enlargement. Responses to innocuous and noxious peripheral stimuli were characterized, and analyses of population responses were performed. Compared with normal animals, dorsal horn neurons of all types in hemisected animals showed increased responsiveness to peripheral stimuli. This was true for neurons on both sides of the spinal cord. After hemisection, the proportion of neurons classified as multireceptive cells increased, and interspike intervals of spontaneous discharges became less uniform after hemisection. Transplantation of RN46A-B14 cells restored evoked responses to near-control levels, normalized background activity, and returned the proportion of multireceptive cells to the control level. Restoration of normal activity was reversed with methysergide.These electrophysiological results corroborate anatomical and behavioral studies showing the effectiveness of serotonergic neural precursors in correcting phenomena associated with chronic central pain following spinal cord injury, and provide mechanistic insights regarding mode of action.     

1141610241Altered expression of HuR, an mRNA stabilizing protein, mediates aberrant Placenta Growth Factor (PlGF) expression in preeclampsia

Background:  Control of mRNA stability is an essential regulatory process in eukaryotic gene expression. HuR, a 3'UTR mRNA binding protein, can protect AU-rich mRNA from degradation in response to stresses. PlGF, an angiogenic growth factor, contains two consensus AU-rich sites suggesting that under normal conditions HuR may protect PlGF mRNA from degradation. Trophoblast expression of PlGF is significantly decreased in preeclampsia and by hypoxia in vitro . We hypothesize that decreased levels of cytoplasmic HuR may contribute to decreased PlGF expression in hypoxic and preeclamptic trophoblast.
Methods:  Western blots were used to determine relative effects of in vitro hypoxia on HuR protein expression and subcellular localization in trophoblast. Immunohistochemistry was used to compare HuR expression patterns in trophoblast of preeclamptic and normal placentae.
Results:  Cytoplasmic expression of HuR was decreased 1.4 fold in the cytoplasm and 1.2 fold in the nucleus of JEG3 cells. A shift in HuR was more apparent in primary trophoblast with a greater than 2-fold decrease in the cytoplasm and a 1.4 fold decrease in the nucleus following 24 hr of hypoxia. Immunohistochemical analyses detected HuR expression in near term trophoblast in situ . However, this technical approach did not detect a significant change in HuR expression between normal and preeclamptic trophoblast.
Conclusions:  HuR expression is decreased in hypoxic trophoblast, at least in vitro , which may provide a causal link to decreased PlGF mRNA expression. Down regulation of trophoblast PlGF expression is thought to contribute to the pathophysiology associated with preeclampsia including the relative lack of perfusion of the placenta and systemic renal effects.     

Propylene glycol ingestion causes D-lactic acidosis

Propylene glycol (1,2-propanediol) is a solvent in numerous pharmaceuticals and a major preservative and source of carbohydrates in processed foods. In mammals, propylene glycol is metabolized similar to ethanol, proceeding via hepatic alcohol and aldehyde dehydrogenases to lactate, which can then enter gluconeogenesis. We observed that cats ingesting 1.6 gm of propylene glycol/kg body weight/day developed increased anion gap. To investigate this further, we measured D- and L-lactate concentrations in these cats; we also measured D-lactate in cats ingesting high doses of propylene glycol (8.0 gm/kg). While L-lactate actually decreased throughout the 35-day course of propylene glycol feeding, D-lactate levels were significantly increased on a dose-dependent basis and correlated positively with anion gap. In cats ingesting the high dose of propylene glycol, D-lactate concentrations were as high as 7 mmol/liter, levels associated with encephalopathy in humans. Indeed, this group of cats developed depression and ataxia, consistent with intoxication by D-lactate. These findings are significant not only for animals ingesting diets which contain propylene glycol, but for humans who receive propylene glycol-containing medications.     

A model of corrective gene transfer in X-linked ichthyosis

Single gene recessive genetic skin disorders offer attractive prototypes for the development of therapeutic cutaneous gene delivery. We have utilized X-linked ichthyosis (XLI), characterized by loss of function of the steroid sulfatase arylsulfatase C (STS), to develop a model of corrective gene delivery to human skin in vivo. A new retroviral expression vector was produced and utilized to effect STS gene transfer to primary keratinocytes from XLI patients. Transduction was associated with restoration of full-length STS protein expression as well as steroid sulfatase enzymatic activity in proportion to the number of proviral integrations in XLI cells. Transduced and uncorrected XLI keratinocytes, along with normal controls, were then grafted onto immunodeficient mice to regenerate full thickness human epidermis. Unmodified XLI keratinocytes regenerated a hyperkeratotic epidermis lacking STS expression with defective skin barrier function, effectively recapitulating the human disease in vivo. Transduced XLI keratinocytes from the same patients, however, regenerated epidermis histologically indistinguishable from that formed by keratinocytes from patients with normal skin. Transduced XLI epidermis demonstrated STS expression in vivo by immunostaining as well as a normalization of histologic appearance at 5 weeks post-grafting. In addition, transduced XLI epidermis demonstrated a return of barrier function parameters to normal. These findings demonstrate corrective gene delivery in human XLI patient skin tissue at both molecular and functional levels and provide a model of human cutaneous gene therapy.      

Induction of B cell apoptosis by co-cross-linking CD23 and sIg involves aberrant regulation of c-myc and is inhibited by bcl-2

A novel system to study the effects of co-cross-linking CD23/FceRII and sIg on murine B lymphocytes utilizes a highly multivalent form of anti- Ig prepared by covalently linking anti-Ig antibodies to a DNP-dextran backbone. CD23-sIg co-cross-linking is accomplished by the addition of DNP-specific monoclonal IgE. Previous studies demonstrated that co- cross-linking CD23 and sIg significantly inhibited mouse B cell proliferation, especially at high doses of the multivalent anti-Ig. Interestingly, examination of early activation signals reveals no difference in B cells subjected to co-cross-linking conditions as compared to B cells activated with anti-Ig alone. Total cellular protein tyrosine phosphorylation levels are unchanged by co-cross- linking. Analysis of B cell mRNA reveals that co-cross-linking the receptors does not alter the expression levels of ornithine decarboxylase 8 h after stimulation as compared to the controls. In contrast, levels of the proto-oncogene c-myc were significantly elevated 1 h after inducing B cell activation under co-cross-linking conditions. However, it remains unclear whether this aberrant c-myc regulation plays any role in inducing apoptosis. In addition, on day 3 after stimulation, the co-cross-linking of CD23 and sIg resulted in the formation of apoptotic B cells, determined by both photomicroscopy of the B cell cultures and FACS analysis of B cell nuclei. B cells obtained from bcl-2 transgenic mice proliferated as well as controls, and failed to undergo apoptosis when CD23 and sIg were co-cross-linked on their surface. These studies indicate that co-cross-linking of CD23 with B cell sIg inhibits B cell proliferation by a mechanism that is distinct from that seen by co-cross-linking of the Fc gamma RII and sIg. In addition, these results suggest a means by which antigen- specific IgE can down-regulate additional B cell activation and IgE synthesis.      

An investigation of minimisation criteria

Background  

Minimisation can be used within treatment trials to ensure that prognostic factors are evenly distributed between treatment groups. The technique is relatively straightforward to apply but does require running tallies of patient recruitments to be made and some simple calculations to be performed prior to each allocation. As computing facilities have become more widely available, minimisation has become a more feasible option for many. Although the technique has increased in popularity, the mode of application is often poorly reported and the choice of input parameters not justified in any logical way.