A novel adenoviral gutless vector encoding sphingosine kinase promotes arteriogenesis and improves perfusion in a rabbit hindlimb ischemia model

OBJECTIVES: We previously demonstrated that sphingosine kinase (SPK) increases the level of extracellular sphingosine-1-phosphate and promotes neovascularization in a mouse matrigel model. In this study, we tested the hypothesis that SPK gene transfer using a novel adenoviral 'gutless' vector (AGV) can enhance arteriogenesis in a rabbit hindlimb ischemia model. METHODS: Thirty-five male New Zealand white rabbits were randomized to the AGV-SPK group (n=13), AGV-null group (n=13), and control group (n=9). On day 10, after the induction of unilateral hindlimb ischemia, gene vectors or buffer were introduced and the effect examined on day 30, using calf blood pressure, quantitative angiographic analysis, and histology. RESULTS: Calf systolic blood pressure ratios of the ischemic limb to the normal limb on day 30 were 0.77+/-0.13 in control groups, including the AGV-null group, and 0.91+/-0.14 in the AGV-SPK group (P<0.05). Angiographic vessel counts were significantly increased (8.0+/-2.1 at baseline and 11.8+/-3.2 on day 30, P<0.001) in the AGV-SPK group. Histologic analysis showed that microscopic total vessel counts on day 30 were 3.5+/-1.8/field in the control and AGV-null group and 5.4+/-1.0/field in the AGV-SPK group. Arterioles (AGV-SPK; 3.0+/-0.8 versus control and AGV-null; 2.1+/-1.1, P<0.05) were significantly increased in the AGV-SPK group. CONCLUSIONS: This study shows that SPK promotes arteriogenesis, as evidenced by the maximal improvement in the blood pressure restoration and collateral vessel counts. SPK may be an important angiogenic target to improve perfusion in ischemic tissues.     

Deprivation of dietary nucleotides decreases protein synthesis in the liver and small intestine in rats

BACKGROUND & AIMS: Dietary nucleotides are reported to influence the growth and functioning of the liver and small intestine. The aim of this study was to examine the mechanism by which nucleotides exert their effects in these tissues by assessing protein synthesis activity and related parameters in the presence or absence of dietary nucleotides. METHODS: Rats were fed a purified diet with or without nucleotides for 10 days. Fractional protein synthesis rate, RNA and DNA concentrations, polysome size distribution, and number of ribosomes were assessed. RESULTS: Fractional protein synthesis rates of the liver and small intestine were lower in the nucleotide-deprived group than in the control group. In the liver, RNA concentration was also lower in the nucleotide-deprived group, but values in the small intestine were similar in the two groups. In the liver, deprivation of nucleotides resulted in a reduction in the number of ribosomes and in polysome breakdown. Protein and DNA concentrations did not vary in the liver; however, the concentration of DNA was lower in the small intestine of the nucleotide-deprived group than in the control group. CONCLUSIONS: Dietary nucleotides can modulate protein synthesis in the liver and small intestine as a result of tissue-specific nucleic acid changes. (Gastroenterology 1996 Jun;110(6):1760-9)     

The formation of Ca++-dependent complexes of platelet membrane glycoproteins IIb and IIIa in solution as determined by crossed immunoelectrophoresis

Triton X-100 soluble proteins from 125I-labeled human platelets were studied by crossed immunoelectrophoresis employing a multispecific rabbit antibody raised against whole normal platelets. Emphasis was placed upon an analysis of immunoprecipitates containing 125I-labeled major membrane glycoproteins, and in particular, a prominent immunoprecipitate containing a glycoprotein antigen (s) previously designated as protein 16. SDS-polyacrylamide gel electrophoresis of protein 16 precipitated by a monospecific alloantibody. IgG L . . . , confirmed the presence of both glycoproteins IIb and IIIa. 125I-IgG L . . . , at concentration below that capable of precipitating protein 16 by itself, bound specifically to the precipitate containing protein 16 produced by the multispecific rabbit antibody. No other precipitates formed by the rabbit antibody contained either glycoprotein IIb or IIIa. When platelet proteins, incubated with optimum concentrations of ethylenediamine tetraacetic acid (EDTA) or ethyleneglycol bis (B- aminoethylether) NN1-tetraacetic acid (EGTA), were electrophoresed against the rabbit antibody, previously unobserved immunoprecipitates that contained either free glycoprotein IIb or free glycoprotein IIIa were detected. Upon readdition of excess Ca++, but not Mg++, to the same protein samples, a single immunoprecipitate containing both glycoproteins was once again observed. It is thus demonstrated that glycoproteins IIb and IIIa can form Ca++-dependent complexes (protein 16) in Triton X-100 extracts of normal platelets. The potential significance of the reversible association of these glycoproteins to normal platelet function is discussed.     

The washout rate on the delayed CT image as a diagnostic tool for adrenal adenoma verified by pathology: a multicenter study

Objectives

To establish the undisputed the value of washout rate for adrenal adenoma using delayed enhanced CT, we evaluated diagnostic performance of cut-off value and delayed time of washout rate by final pathologic diagnosis in a multicenter study.

Methods

We reviewed the pathologic and clinical records of 244 patients underwent adrenalectomies at 5 university hospitals between 2005 and 2009. We calculated the mean Housfield units (HU) of adrenal lesion at non-enhancing CT, and early and delayed enhanced CT using the region of interest. We used ROC curves to determine the specificity and sensitivity of non-enhanced CT scans and the washout rate according to the various cut-off for adrenal adenomas.

Results

We divided the patients into adrenal adenoma group (n?=?138) and non-adrenal adenoma group (n?=?106) based on final pathologic report. Using the unenhanced images with a threshold of 10 HU, the sensitivity was 45.7?%, and the specificity was 97.1?%. Using the 15-min-washout rate with a threshold of 55?%, the sensitivity was 93.9?%, and the specificity was 95.8?%.

Conclusions

Regardless of various CT machines and protocols, a washout rate of 15-min-delayed CT was most useful in the diagnosis of adrenal adenomas due to the early inflow and outflow of contrast media in the tissues of adrenal adenomas.     

Guideline for referral of patients with suspected lung cancer by family physicians and other primary care providers

Objective

The aim of this guideline is to assist FPs and other primary care providers with recognizing features that should raise their suspicions about the presence of lung cancer in their patients.

Composition of the committee

Committee members were selected from among the regional primary care leads from the Cancer Care Ontario Provincial Primary Care and Cancer Network and from among the members of the Cancer Care Ontario Lung Cancer Disease Site Group.

Methods

This guideline was developed through systematic review of the evidence base, synthesis of the evidence, and formal external review involving Canadian stakeholders to validate the relevance of recommendations.

Report

Evidence-based guidelines were developed to improve the management of patients presenting with clinical features of lung cancer within the Canadian context.

Conclusion

Earlier identification and referral of patients with lung cancer might ultimately help improve lung cancer morbidity and mortality. These guidelines might also be of value for informing the development of lung cancer diagnostic programs and for helping policy makers to ensure appropriate resources are in place.Lung cancer is the most common cause of cancer death in Canada.¹ Lung cancers are frequently diagnosed at a late stage, and the prognosis is very poor.¹ The chance of surviving lung cancer in Canada is low, with a 5-year survival rate of 13% for men and 19% for women.¹ Delays in the diagnosis of lung cancer are well documented.^2–11 This might in part be owing to patients and clinicians attributing the often common, atypical, or nonspecific symptoms of lung cancer to other, benign diseases.In order to provide guidance for the introduction of lung cancer diagnostic assessment programs (DAPs) in Ontario, the Cancer Care Ontario (CCO) Provincial Primary Care and Cancer Network initiated a collaboration in October 2009 with CCO’s Program in Evidence-based Care (PEBC) to form the Lung Cancer Referral Working Group. The working group was tasked with providing recommendations that would help FPs and other primary care providers recognize and initiate the management of undiagnosed patients presenting with signs or symptoms of lung cancer. The following questions were evaluated in completing this overall objective.

• What main known risk factors are predictive of lung cancer?
• What signs, symptoms, and other clinical features are predictive of lung cancer?
• What is the diagnostic accuracy of investigations for lung cancer?
• Which patient and provider factors are associated with delayed referral?
• Does a delay in the time to consultation affect patient outcomes?

The aim of this guideline is to assist primary care clinicians in recognizing and managing clinical features that should raise their suspicion of lung cancer and ultimately lead to more timely and appropriate referrals. The recommendations are targeted to managing patients presenting in primary care settings. They are also intended to help policy makers ensure that resources such as lung cancer DAPs are in place so that target wait times are achieved.     

Colonic mucosal tears in collagenous colitis

In general, the colonic mucosa is macroscopically normal in collagenous colitis, although minor, non-specific abnormalities may be found. Significant endoscopic abnormalities, “mucosal tears” representing longitudinal mucosal lacerations, have been reported in a few patients with collagenous colitis. We report the cases of three women with collagenous colitis and mucosal tears detected at the index colonoscopy in order to illustrate the endoscopic characteristics and review the literature. Including the present cases, a total of 12 patients with mucosal tears and collagenous colitis have been reported. In 10 patients, the mucosal lacerations involved the ascending or the transverse colon. Three of the 12 patients had a colonic perforation immediately after the colonoscopy. The colonoscopist should be aware that the risk of perforation is likely to be increased when mucosal tears are present.     

β‐catenin regulates parathyroid hormone/parathyroid hormone–related protein receptor signals and chondrocyte hypertrophy through binding to the intracellular C‐terminal region of the receptor

Objective

To investigate the underlying mechanisms of action and functional relevance of β‐catenin in chondrocytes, by examining the role of β‐catenin as a novel protein that interacts with the intracellular C‐terminal portion of the parathyroid hormone (PTH)/PTH‐related protein (PTHrP) receptor type 1 (PTHR‐1).

Methods

The β‐catenin–PTHR‐1 binding region was determined with deletion and mutagenesis analyses of the PTHR1 C‐terminus, using a mammalian two‐hybrid assay. Physical interactions between these 2 molecules were examined with an in situ proximity ligation assay and immunostaining. To assess the effects of gain‐ and loss‐of‐function of β‐catenin, transfection experiments were performed to induce overexpression of the constitutively active form of β‐catenin (ca‐β‐catenin) and to block β‐catenin activity with small interfering RNA, in cells cotransfected with either wild‐type PTHR1 or mutant forms (lacking binding to β‐catenin). Activation of the G protein α subunits G_αs and G_αq in the cells was determined by measurement of the intracellular cAMP accumulation and intracellular Ca²⁺ concentration, while activation of canonical Wnt pathways was assessed using a TOPflash reporter assay.

Results

In differentiated chondrocytes, β‐catenin physically interacted and colocalized with the cell membrane–specific region of PTHR‐1 (584–589). Binding of β‐catenin to PTHR‐1 caused suppression of the G_αs/cAMP pathway and enhancement of the G_αq/Ca²⁺ pathway, without affecting the canonical Wnt pathway. Inhibition of Col10a1 messenger RNA (mRNA) expression by PTH was restored by overexpression of ca‐β‐catenin, even after blockade of the canonical Wnt pathway, and Col10a1 mRNA expression was further decreased by knockout of β‐catenin (via the Cre recombinase) in chondrocytes from β‐catenin–floxed mice. Mutagenesis analyses to block the binding of β‐catenin to PTHR1 caused an inhibition of chondrocyte hypertrophy markers.

Conclusion

β‐catenin binds to the PTHR‐1 C‐tail and switches the downstream signaling pathway from G_αs/cAMP to G_αq/Ca²⁺, which is a possible mechanism by which chondrocyte hypertrophy may be regulated through the PTH/PTHrP signal independent of the canonical Wnt pathway.