Amine-reactive pyridylhydrazone-based PEG reagents for pH-reversible PEI polyplex shielding

PEGylation which is reversed after the therapeutic agent reaches the target cell presents an attractive feature for drug, protein or nucleic acid delivery. Amine-reactive, endosomal pH cleavable polyethylene glycol aldehyde-carboxypyridylhydrazone, N-hydroxysuccinimide esters (PEG-HZN-NHS) were synthesized and applied for bioreversible surface shielding of DNA polyplexes. Monofunctional mPEG-HZN-NHS was synthesized by reacting succinimidyl hydraziniumnicotinate with mPEG-butyraldehyde (20 kDa). Bifunctional OPSS-PEG-HZN-NHS was synthesized analogously via a omega-2-pyridyldithio-PEG (10 kDa) propionaldehyde intermediate. Polyethylenimine (PEI) polyplexes were reacted with the pH-sensitive (mPEG-HZN-NHS) or the corresponding stable (mPEG-NHS) reagent. Both types of polyplexes remained shielded at pH 7.4 as demonstrated by particle size and zeta potential measurements after 4h of incubation at 37 degrees C. Polyplex deshielding at endosomal pH 5 was observed only with the mPEG-HZN-NHS shielded particles. This was confirmed by fluorescence correlation spectroscopy using the analogous Alexa-488 fluorescently labeled bifunctional PEGylation reagents. Luciferase gene transfections with epidermal growth factor (EGF) containing polyplexes using EGF-receptor overexpressing hepatoma HUH7 cells showed an up to 16-fold enhancement in gene expression with the reversibly shielded polyplexes as compared to stably shielded polyplexes. Consistently, the reversibly shielded polyplexes mediated also an enhanced tumor specific in vivo transgene expression after intravenous administration in a subcutaneous HUH7 tumor model in SCID mice.     

A role for endocannabinoids in indomethacin-induced spinal antinociception

Inhibition of prostaglandins synthesis does not completely explain non-steroidal anti-inflammatory drug-induced spinal antinociception. Among other mediators, endocannabinoids are involved in pain modulation. Indomethacin-induced antinociception, in the formalin test performed in spinally microdialysed mice, was reversed by co-administration of the cannabinoid 1 (CB(1)) antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1-H-pyrazole-3-carboxamide (AM-251), but not by co-infusion of prostaglandin E(2). Indomethacin was ineffective in CB(1) knockout mice. AM-251 also reversed the indomethacin-induced antinociception in a test of inflammatory hyperalgesia to heat. Furthermore, during the formalin test, indomethacin lowered the levels of spinal nitric oxide (NO), which activates cellular reuptake and thus breakdown of endocannabinoids. The pronociceptive effect of an NO donor, 3-methyl-N-nitroso-sydnone-5-imine (RE-2047), was abolished by co-administration of the endocannabinoid transporter blocker N-(4-hydroxyphenyl) arachidonoyl amide (AM-404). Moreover, the antinociceptive activity of the NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME), was reversed by AM-251. Thus we propose that at the spinal level, indomethacin induces a shift of arachidonic acid metabolism towards endocannabinoids synthesis secondary to cyclooxygenase inhibition. In addition, it lowers NO levels with subsequent higher levels of endocannabinoids.     

Detection of MDM2 alterations in cultured human hepatocytes treated with 17beta-estradiol or 17alpha-ethinylestradiol

BACKGROUND: Preneoplastic and neoplastic lesions of the liver are suspected to arise as a result of estrogen treatment. Here we present the first report on the modulational effects of the steroids 17beta-estradiol (E2) and 17alpha-ethinylestradiol (EE2) on oncogene MDM2 in human hepatocytes. MATERIALS AND METHODS: Collagen-embedded cultures of hepatocytes stimulated with different E2/EE2 concentrations were analyzed by immunocytochemistry, RT-PCR and sequencing for MDM2 protein/mRNA expression, MDM2 mRNA splicing and MDM2 gene mutation. RESULTS: The hepatocytes responded to stimulation with steroid E2/EE2 concentrations from 1-100 nmol/l with the overexpression of MDM2 protein while non-stimulated cells were negative. Stimulation with 1 nmol/l E2 and 10-100 nmol/l EE2 induced MDM2 splicing variants. Hepatocytes treated with 100 nmol/l E2 contained full-length MDM2 mRNA carrying a new type of MDM2 gene mutation. Unstimulated hepatocytes revealed neither mRNA splicing nor alteration of the MDM2 genes. CONCLUSION: The data show that steroid hormones are involved in the induction of MDM2 alterations in benign human hepatocytes. We speculate that some of the alterations may influence MDM2 function, thus possibly favouring genesis of liver changes.     

Office-based ultrasound-guided cryoablation of breast fibroadenomas

BACKGROUND: Fibroadenomas commonly found by palpation and routine mammography account for approximately 20% of open surgical breast biopsies. Alternatives to open surgery include tumor removal using an automated coring device and tumor ablation using heating or cooling elements. We report our initial experience with cryoablation of biopsy-proven benign fibroadenomas. METHODS: A table-top cryoablation system employing a 2.4-mm cryoprobe was used to treat biopsy-proven benign fibroadenomas up to 4 cm in maximum diameter in a prospective nonrandomized fashion. The cryoprobe was placed under ultrasound guidance. Using a treatment algorithm based on fibroadenoma size, all tumors were subjected to two freeze cycles with an interposing thaw. Skin appearance and temperature, probe temperature, iceball size, and patient comfort were closely monitored during the procedure. Follow-up examinations including ultrasonography and photographs were scheduled for up to 12 months postablation. RESULTS: Fifty patients with 57 core biopsy-proven benign fibroadenomas were treated. Seven early cases were treated in an ambulatory surgery center setting. The remaining procedures were completely office-based using only local anesthetic. Tumor diameter varied from 7 mm to 42 mm (mean 21 mm). The iceball engulfed the target lesion in each case. Transient postoperative side effects were local swelling and ecchymosis. Postoperative discomfort rarely required medication beyond acetaminophen or ibuprofen. Lesions showed progressive shrinkage and disappearance over 3 to 12 months. No skin injury was noted and appearance remained excellent. Patient satisfaction was excellent. CONCLUSIONS: With office-based use of ultrasound-guided cryoablation for fibroadenomas there was little or no pain, target lesions were reduced in size or eliminated, scarring was minimal, cosmesis outstanding, and patient satisfaction was excellent. Cryoablation offers a useful office-based alternative to surgical excision of benign fibroadenomas.     

The localizing and lateralizing value of ictal/postictal coughing in patients with focal epilepsies.

Postictal coughing has so far been reported to indicate a temporal origin of focal epilepsy. A trend towards non-dominant hemisphere lateralization and mesial temporal localization has been suggested. However, postictal coughing has also been reported in a few patients with extratemporal epilepsies. We have retrospectively evaluated the localizing and lateralizing value of ictal/postictal coughing in 197 patients with temporal and extratemporal epilepsy who received presurgical video-EEG long-term recordings from 1999 to 2001. There was no statistical significant difference in percentage of coughing patients in both groups. However, only patients belonging to the temporal group presented with coughing as a regular element of seizure semiology (simple partial and complex partial seizures) whereas in the extratemporal group coughing occurred more sporadically. Within the temporal group a statistically significant tendency to left-sided seizure onset and a statistically not significant preponderance of mesial seizure onset was observed. Additional vegetative signs were observed only in about half of the patients. These results suggest that coughing occurs in both temporal and extratemporal lobe epilepsy and may only be indicative of temporal lobe seizure onset if representing a regular semiologic element. Coughing may be due to two different mechanisms, one dependent and the other independent from additional vegetative symptoms.     

Weight concerns and dieting among 8 to 12-year-old children

According to a recent study it could be shown that more than one third of female high school students and more than 20 % of male students in the age of 14 to 18 years indicated an impaired eating behaviour, expressed as a medium or high risk to develop eating disorders based upon the standardized Eating Attitudes Test (EAT) . The current study tested the prevalence of weight concerns and dieting as an indicator of problematic eating behaviour among younger children. 230 Thuringian female and male students in the 3 (rd) and 4 (th) year (between 8 and 12-years-old) as well as their parents were investigated using standardized and modified instruments. Weight and height were measured directly to determine the Body Mass Index (BMI) that was put into relation to some factors of potential influence such as parents, peers, media and eating behaviours. It could be shown that 42 % of the boys and 53 % of the girls preferred a thin ideal body image; 32 % of the children revealing normal weight expressed the wish to be thinner. 18 % of the boys and 19 % of the girls tried to lose weight at the time of the investigation. Dieting was influenced by the perceived (not actual) dieting behaviour of the parents. The BMI significantly predicted weight concerns: In total, 85 % of the overweight children but also 17 % of the children with normal weight were convinced to be overweight. The girls' eating behaviour was mostly influenced by the peers, the boys' behaviour by criticism of the parents. Generally, a significant influence of the media could be demonstrated.     

Sympathetic and parasympathetic neuropathy are frequent in both type 1 and type 2 diabetic patients

OBJECTIVE: The aim of this study was to evaluate the frequency of sympathetic versus parasympathetic neuropathy among type 1 and type 2 diabetic patients. RESEARCH DESIGN AND METHODS: There were 43 patients with type 1 and 17 with type 2 diabetes who were investigated. Sympathetic nerve function was assessed by measurement of the vasoconstriction (VAC) index by laser Doppler perfusion imaging of a locally heated finger followed by indirect cooling. Parasympathetic nerve function was assessed by R-R interval variation during deep breathing as measured by the expiration/inspiration (E/I) ratio. Results were expressed as age-corrected z scores in SD; VAC index >1.64 SD and E/I ratio <-1.64 SD were considered abnormal. RESULTS: VAC index was abnormal in 40% with type 1 and 41% with type 2 diabetes, whereas the E/I ratio was abnormal in 42% with type 1 and 65% with type 2 diabetes. There was a clear association between VAC index and E/I ratio among type 1 (rs=0.525; P=0.0002) but not among type 2 (rs=0.10) diabetic patients. Among type 2 diabetic patients, the degree of dysfunction was most severe regarding parasympathetic function (P=0.0167). CONCLUSIONS: Sympathetic and parasympathetic neuropathy were frequent in both type 1 and type 2 diabetic patients. However, there was a difference between the two types of diabetes. Sympathetic and parasympathetic nerve functions correlated in type 1 but not in type 2 diabetic patients. The explanation for this discrepancy might be that parasympathetic nerve function was most severely affected among type 2 diabetic patients.     

Pattern motion integration in infants

To investigate the development of motion integration in infants, we used an eye movement technique to measure subjects' ability to track leftward versus rightward pattern motion in a stimulus consisting of a field of spatially segregated moving gratings. Each grating moved in one of two oblique directions, with the two directions interleaved across the display. When spatially integrated, pattern motion for these paired component motions was either rightward or leftward. To control for the possibility that horizontal eye movements elicited by this stimulus were due to the horizontal motion vector present in each obliquely moving grating, we also measured responses to a field where every grating moved in the same oblique direction. The difference in performance between the integration stimulus and this control stimulus was taken as a measure of integration. Data from 2-, 3-, 4-, and 5-month-old infants revealed significant motion integration, suggesting that higher order motion areas, such as the middle temporal area (MT) may develop at a relatively early age. In addition, the integration effect decreased consistently and significantly with age (p <.005), suggesting a reduction in the spatial extent of motion integration over the course of development.