Long-acting forms of Sonic hedgehog with improved pharmacokinetic and pharmacodynamic properties are efficacious in a nerve injury model

The therapeutic effects of the Sonic hedgehog (Shh) have been difficult to evaluate because of its relatively short serum half-life. To address this issue polyethylene glycol modification (PEGylation) was investigated as an approach to improve systemic exposure. Shh was PEGylated by a targeted approach using cysteines that were engineered into the protein by site-directed mutagenesis as the sites of attachment. Sixteen different versions of the protein containing one, two, three, or four sites of attachment were characterized. Two forms were selected for extensive testing in animals, Shh A192C, which provided a single site for PEGylation, and Shh A192C/N91C, which provided two sites. The PEGylated proteins were evaluated for reaction specificity by SDS-PAGE and peptide mapping, in vitro potency, pharmacokinetic and pharmacodynamic properties, and efficacy in a sciatic nerve injury model. Targeted PEGylation was highly selective for the engineered cysteines and had no deleterious effect on Shh function in vitro. Systemic clearance values in rats decreased from 117.4 mL/h/kg for unmodified Shh to 29.4 mL/h/kg for mono-PEGylated Shh A192C that was modified with 20 kDa PEG-maleimide and to 2.5 mL/h/kg for di-PEGylated Shh A192C/N91C modified with 2, 20 kDa PEG vinylsulfone adducts. Serum half-life increased from 1 h for unmodified Shh to 7.0 and 12.6 h for the mono- and di-PEGylated products. These changes in clearance and half-life resulted in higher serum levels of Shh in the PEG-Shh-treated animals. In Ptc-LacZ knock-in mice expressing lacZ under regulation of the Shh receptor Patched, about a 10-fold lower dose of PEG-Shh was needed to induce beta-galactosidase than for the unmodified protein. Therapeutic treatment of mice with PEG-Shh enhanced the regeneration of injured sciatic nerves. These studies demonstrate that targeted PEGylation greatly alters the pharmacokinetic and pharmacodynamic properties of Shh, resulting in a form with improved pharmaceutical properties.     

Immunoelectron microscopic localization of the opioid growth factor receptor (OGFr) and OGF in the cornea

This study was conducted to determine the cellular and subcellular location(s) of the opioid growth factor receptor (OGFr), and the opioid growth factor (OGF), [Met(5)]-enkephalin, in the corneal epithelium. Laser scanning confocal microscopy analysis revealed that both OGFr and OGF were colocalized in the paranuclear cytoplasm and cell nuclei in basal, as well as suprabasal, cells of adult rat corneal epithelium. Using a postembedding immunogold procedure for immunoelectron microscopy that included embedding in Unicryl, both single- and double-face labeling studies were performed. Immunogold labeling of OGFr was detected on the outer nuclear envelope, in the paranuclear cytoplasm proximal to the nuclear envelope, perpendicular to the nuclear envelope in a putative nuclear pore complex, and within the nucleus adjacent to heterochromatin. Immunoreactivity for OGF was noted in locations similar to that for OGFr. In addition, aggregates of staining for OGF were found throughout the cytoplasm, including subjacent to the plasma membrane. Double labeling experiments revealed that complexes of OGF-OGFr were colocalized on the outer nuclear envelope, in the paranuclear cytoplasm, extending across the nuclear pore complex, and in the nucleus. Anti-OGFr IgG by itself, but not anti-OGF IgG alone, was associated with the outer nuclear envelope, and uncomplexed OGF immunoreactivity was detected in the cytoplasm in dual labeling experiments. These results based on complementary approaches of confocal microscopy and immunoelectron microscopy, suggest that: (i) OGFr resides on the outer nuclear envelope, (ii) OGF interacts with OGFr at the outer nuclear envelope, (iii) the colocalized receptor and peptide translocates between the cytoplasm and the nucleus at the nuclear pore, and (iv) signal transduction for modulation of cell proliferation necessitates a peptide-receptor complex that interfaces with chromatin in the nucleus.     

Frontotemporal lobar degeneration – tau as a pied piper?

Frontotemporal lobar degeneration (FTLD) is the second most-common form of cortical dementia in the presenium after Alzheimer disease. Clinically three disease entities can be distinguished: frontotemporal dementia, semantic dementia, and primary progressive aphasia. The underlying neuropathology can be classified into disorders with tau pathology (including Pick disease, corticobasal degeneration, progressive supranuclear palsy, and familial frontotemporal dementia with parkinsonism linked to chromosome 17 – FTDP-17), and into disorders that lack tau abnormalities (including dementia lacking distinctive histology and motor neuron disease inclusion dementia). The recent discovery of tau gene mutations in FTDP-17 brought tau to the center stage, but led to the erroneous trend of collectively grouping all forms of FTLD as tauopathies. However, clinicopathological and genetic studies strongly suggest that the majority of sporadic and familial FTLD cases are not associated with tau pathology and/or tau gene mutations. Furthermore, recent studies have linked several autosomal dominantly inherited familial frontotemporal dementia cases to a variety of gene loci on different chromosomes. Thus, this review is intended to summarize our current knowledge about the sporadic and familial FTLD disorders that lack tau pathology, and shall further strengthen the view that FTLD is heterogeneous, both in terms of clinicopathological phenotypes as well as genetic backgrounds. Electronic Publication     

Risk factors for premature birth in French Guiana: the importance of reducing health inequalities

Objectives: French Guiana has the highest birth rate in South America. This French territory also has the highest premature birth rate and perinatal mortality rate of all French territories. The objective was to determine the premature birth rate and to identify the prevalence of risk factors of premature birth in French Guiana.

Methods: A retrospective study of all births in French Guiana was conducted between January 2013 and December 2014 using the computerized registry compiling all live births over 22 weeks of gestation on the territory.

Results: During this period 12 983 live births were reported on the territory. 13.5% of newborns were born before 37 (1755/12 983). The study of the registry revealed that common sociodemographic risk factors of prematurity were present. In addition, past obstetrical history was also important: a scarred uterus increased the risk of prematurity adjusted odds ratio =1.4, 95%CI (1.2–1.6). Similarly, obstetrical surveillance, the absence of preparation for birth or of prenatal interview increased the risk of prematurity by 2.4 and 2.3, the excess fraction in the population was 69% and 72.2%, respectively.

Conclusions: Known classical risk factors are important. In the present study excess fractions were calculated in order to prioritize interventions to reduce the prematurity rate.     

Effects of acute inorganic lead administration on in vivo drug metabolism and cytochrome P-450 in the rat

An acute dose of inorganic lead acetate (4.0 $\text{mg}\text{kg}$ i.p.) prolonged rat methohexital-induced narcosis while doses of 1.5 and 2.0 $\text{mg}\text{kg}$ i.p. did not. The in vivo metabolism of methohexital was tested 24 h after the administration of the lead. Hepatic cytochrome P-450 content was decreased 12 to 24 h following the administration of 4.0 $\text{mg}\text{kg}$ i.p. lead acetate. Hepatic microsomal protein and cytochrome B₅ content did not change 12 or 24 h following lead treatment.     

Cholangiocarcinoma: molecular targeting strategies for chemoprevention and therapy

Cholangiocarcinomas are devastating cancers that are increasing in both their worldwide incidence and mortality rates. The challenges posed by these often lethal biliary tract cancers are daunting, with conventional treatment options being limited and the only hope for long-term survival being that of complete surgical resection of the tumor. Unfortunately, the vast majority of patients with cholangiocarcinoma typically seek treatment with advanced disease, and often these patients are deemed poor candidates for curative surgery. Moreover, conventional chemotherapy and radiation therapy have not been shown to be effective in prolonging long-term survival, and although photodynamic therapy combined with stenting has been reported to be effective as a palliative treatment, it is not curative. Thus, there is a real need to develop novel chemopreventive and adjuvant therapeutic strategies for cholangiocarcinoma based on exploiting select molecular targets that would impact in a significant way on clinical outcome. This review focuses on potential preventive targets in cholangiocarcinogenesis, such as inducible nitric oxide synthase, cyclooxygenase-2, and altered bile acid signaling pathways. In addition, molecular alterations related to dysregulation of cholangiocarcinoma cell growth and survival, aberrant gene expression, invasion and metastasis, and tumor microenvironment are described in the context of various clinical and pathological presentations. Moreover, an emphasis is placed on the importance of critical signaling pathways and postulated interactions, including those of ErbB-2, hepatocyte growth factor/Met, interleukin-6/glycoprotein130, cyclooxygenase-2, vascular endothelial growth factor, transforming growth factor-beta, MUC1 and MUC4, beta-catenin, telomerase, and Fas pathways as potential molecular therapeutic targets in cholangiocarcinoma.     

Neonatal resuscitation by laryngeal mask airway after elective cesarean section

The aim of this case-control study was to determine whether unplanned resuscitation using a laryngeal mask airway (LMA) is suitable for neonates delivered by elective cesarean section, a procedure known to carry a risk of inadequate physiological response to birth with a consequent adverse respiratory outcome. During a 3-year period, from January 1998 to December 2000, all newborns delivered by elective cesarean section at term were compared with the next infant born vaginally in the same maternity unit (level III center). The two groups were matched for gestational age >37 weeks. The requirement for resuscitation with positive-pressure ventilation (PPV) using either the LMA or an endotracheal tube (ETT), together with the occurrence of an adverse neonatal outcome, was recorded and analyzed in the cesarean section and vaginal delivery groups. During this time 1,284 at-term elective cesarean sections were performed. 3% (n = 43) of the cesarean section deliveries and 1.4% (n = 18) of the vaginal controls required PPV resuscitation by LMA or ETT, a significant difference (OR 1.26; 95% CI 2.38-5.1; p < 0.01). Of the cesarean section group requiring resuscitation, 30 neonates were managed with the LMA and 13 with the ETT, while in the control vaginal delivery group the numbers were 13 and 5, respectively. LMA use accounted of about 70% of the overall PPV resuscitations and was associated with a successful outcome in 42 of 43 cases. One case was unsuccessfully managed with the LMA, and the ETT subsequently used was effective. Moreover, the probability for the LMA-resuscitated newborns of both cesarean and vaginal groups to have a <5 Apgar score at 1 and 5 min, neonatal intensive care unit admission, and respiratory insufficiency requiring oxygen and intermittent mandatory ventilation was statistically lower than for the ETT group (p < 0.01). In conclusion, infants born by elective cesarean section at term are at increased risk of requiring PPV resuscitation as compared with those born by vaginal delivery. We have shown that about 70% of the neonates who required PPV resuscitation after elective cesarean section and vaginal delivery were arbitrarily treated with LMA by the attending anesthesiologist, without adverse negative respiratory outcome.     

Overcoming resistance to gamma-rays in squamous carcinoma cells by poly-drug elevation of ceramide levels

Recent strategies to sensitize radioresistant tumours are based on combining gamma-irradiation with inducers of apoptosis. We report that the combination of three inhibitors of sphingolipid metabolism, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCl(DL-PDMP)+imipramine +/- D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-MAPP), with 10-Gy irradiation triggers both mitotic and apoptotic killing in radioresistant SQ20B squamous carcinoma cells. In these cells, apoptosis is defective due to a lack of ceramide generation upstream, which cannot be explained by sphingomyelinase (neutral and acidic) deficiency or rapid derivation to the sphingolipid pathway. We present evidence of a functional transduction death pathway when ceramide generation is restored, which involves the mitochondrial-mediated pathway coupled to alterations in redox status and to executive caspases activation. The poly-drug treatment restored apoptosis to levels similar to those observed in radiosensitive SCC61 squamous carcinoma cells. Simultaneous exposure to gamma-irradiation and poly-drug treatment acted synergistically in SQ20B cells to produce a marked increase in both mitochondrial dysfunction and caspase cleavage, which led to a 7.8-fold increase in apoptosis within 48 h, relative to irradiated cells. Moreover, the results suggest that the ceramide released by irradiation or poly-drug treatment converges upon common cellular targets. Modulation of endogenous ceramide levels by inhibitors of sphingolipid metabolism may represent a new cellular target for the sensitization of radioresistant tumours to gamma-ray therapy.