A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

Background  

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation.     

Autoantibodies to the high-affinity IgE receptor in children with chronic urticaria.

BACKGROUND: Chronic urticaria (CU) in childhood remains a challenge for investigation, and its etiology is largely unknown. Autoantibodies to the high-affinity IgE receptor (FcepsilonRI) are believed to play a role in the pathogenesis of this disease in adults. OBJECTIVE: To determine the prevalence of autoantibodies to FcepsilonRIalpha on basophils in children with CU vs atopic eczema dermatitis syndrome (AEDS). METHODS: Eighty children with CU were compared with 38 children with AEDS. In addition to complete blood cell counts and total IgE measurements, CAP-RASTs to egg, codfish, soy, milk, and peanut were performed. Stool samples were examined for parasites, and autologous serum skin testing and a functional anti-FcepsilonRIalpha assay were conducted to detect autoantibodies. RESULTS: No significant differences were observed between children with CU and controls in mean basophil or eosinophil counts. Twenty (26%) of 77 children with CU and 31 (82%) of 38 with AEDS had positive CAP-RAST results (P < .001). Only 2.5% of the children with CU and 0% with AEDS had stool samples positive for parasites (P = .005). Anti-FcepsilonRIalpha autoantibodies were positive in 37 (47%) of 78 children with CU and in none of 33 with AEDS. Non-IgG histamine-releasing factors were found in 10 (13%) of 78 children with CU. CONCLUSIONS: Children have a similar prevalence of autoantibodies to the FcepsilonRIalpha as has been previously published for adults. Few have type I allergies, and parasite infestation is also uncommon. Further studies are required to investigate the predictive value of the autoantibodies in these children with respect to clinical profile, requirements for medications other than antihistamines, and remission rates.     

A new method to bind allergens for the measurement of specific IgE antibodies

BACKGROUND: Detection of allergen-specific IgE antibodies in patients' sera plays a key role for the diagnosis of IgE-mediated allergy. If no validated test system is available, diagnostic tools must be developed, usually by coupling or binding the allergens to a solid phase. Streptavidin ImmunoCAP is a new solid phase for binding of allergens which can be used in the Pharmacia CAP system. OBJECTIVE: It was the aim of this study to assess the diagnostic validity of Streptavidin ImmunoCAP. METHODS: Biotinylation and allergen concentration for binding to Streptavidin ImmunoCAP were optimized and IgE obtained with natural rubber latex, obeche wood, wheat and rye flour Streptavidin ImmunoCAP were compared with the results of ImmunoCAP and Enzyme Allergo-Sorbent Test (EAST) using sera from patients complaining of workplace-related respiratory symptoms. RESULTS: While the relation of biotin-label and protein was critical (best results were obtained with a 5- fold molar excess), labelled protein for coupling to streptavidin ImmunoCAP was applicable in a wide concentration range. On average, IgE values with streptavidin ImmunoCAP were as high as with ImmunoCAP but considerably higher than values obtained by EAST. CONCLUSION: Streptavidin ImmunoCAP is a valuable tool for sensitive and specific measurement of IgE binding to new allergens superior to cellulose disk-based methods.     

Electron microscopy of the canine corneal basement membranes

The purpose of this study was to characterize the surface topographical features of the epithelial and endothelial (Descemet's) basement membranes of the canine cornea. Corneas were obtained from young, healthy dogs (<2 years old) with no history or evidence of previous ocular disease. The epithelium and endothelium was carefully removed preserving the anterior and posterior basement membranes. The specimens were examined by transmission electron microscopy and scanning electron microscopy. The epithelial and endothelial basement membrane surface topography is an intricate meshwork of pores and fibers measuring in the nanometer size range. The features of the endothelial basement membrane overall are smaller in size than the epithelial basement membrane. These surface topographical features may incite changes in epithelial and endothelial cell behavior.     

Prevalence of hepatitis B virus infection among women at reproductive age at a German university hospital

BACKGROUND: Mother to infant transmission of hepatitis B virus (HBV) represents a major factor in maintaining chronic infection and depends on the degree of maternal infectivity status. OBJECTIVES: To examine the seroprevalence of hepatitis B virus surface antigen (HBsAg) in women at reproductive age admitted to the Department of Gynaecology at a German university hospital. STUDY DESIGN: The seroprevalence of hepatitis B surface antigen (HBsAg) in 5518 women at reproductive age was examined, HBsAg-positive samples were tested for additional HBV markers to verify the infection status. RESULTS: Out of 5518 samples from women at reproductive age, 88 women (1.59%) were positive for HBsAg and 7 of these HBV-positive women (7.95%) were additionally positive for HBeAg. The majority of the study population were German citizens, however most HBV infected persons originated from countries with a high HBV prevalence. The HBV seroprevalence in our study group is about two times higher compared to the average seroprevalence in the German citizen adult population, thus probably resulting in an underestimation of the infection rate in a multinational setting. CONCLUSIONS: Screening for HBsAg during pregnancy is still necessary and important for reduction of perinatal HBV transmission even in countries with low HBV prevalence.     

PCR-based DNA fingerprinting of Staphylococcus haemolyticus to investigate nosocomial infections

Objective: To apply PCR-based DNA fingerprinting in a clinical microbiology laboratory to investigate nosocomial infections with Staphylococcus haemolyticus.
Method: DNA fingerprints were generated by PCR on 99 S. haemolyticus isolates using different primer combinations based on ERIC, REP or arbitrarily chosen simple repeat sequences.
Results: Primer combinations REP1+(GTC)₆ and ERIC1+ERIC2 had sufficient discrimatory power and were chosen to analyze the clinical isolates. DNA fingerprint patterns from strains isolated from the patients nursed in the same hospital ward in the period 1991–94 were approximately 90% similar to each other. One staff member, sampled in 1991, carried a strain with a similar fingerprint.
Conclusions: PCR based DNA fingerprinting is a suitable method to perform in a clinical laboratory. An S. haemolyticus strain appeared to be endemic in the hospital ward and had most probably been transmitted from patient to patient. S. haemolyticus may carry glycopeptide resistance and needs attention as a causative agent of nosocomial infections.     

Influence of Adipose Tissue Blood Flow on the Lipolytic Response to Circulating Noradrenaline at Normal and Reduced pH

Hypercapnic acidosis (pH 7.0) inhibits the lipolytic response of canine subcutaneous adipose tissue to i.v. infused noradrenaline (NA) by 80 per cent or more. The response to sympathetic nerve stimulation, on the other hand, is only reduced by 1040 per cent during acidosis. The fate of intravenously infused ³H-labelled NA (0.35 ug × kg^-1× min^-1 for 30 min) was not significantly altered by acidosis. The rate of disappearance of unmetabolized NA from the arterial plasma after an infusion was the same at pH 7.4 and 7.0 and the calculated increase in circulating NA during infusions was 4 ng/ml at both pH:s. I.v. infusion of Na increases adipose tissue blood flow, an effect which is attenuated by acidosis. There was a significant correlation (p< 0.001) between adipose tissue blood flow and the lipolytic response at normal pH. Preventing the NA-induced increase in blood flow by constant flow perfusion reduced the lipolytic response at normal pH. The degree of inhibition by acidosis of the lipolytic response to i.v. NA was significantly reduced (from 79 to 56 per cent, p < 0.05) when the adipose tissue was perfused at constant flow. These data suggest that adipose tissue blood flow is important in determining the lipolytic response to i.v. NA, probably by influencing the delivery of NA to the tissue. The marked inhibition by acidosis of lipolysis due to i.v. infused NA therefore appears to be the combined effect of a direct antilipolytic effect of acidosis and a decreased delivery of N A to the adipose tissue due to the attenuated blood flow response.     

Enzymatic activities induced by interferon in human fibroblast cell lines differing in their sensitivity to the anticellular activity of interferon

IF^r, a subline of transformed human fibroblast cells, which is sensitive to the antiviral but resistant to the anticellular activity of interferon, was found to be equally well inducible as its parental cell line RSa for the two major interferon-mediated double-stranded RNA-dependent enzymatic activities, 2–5A synthetase and 73K phosphoprotein kinase. The induction of 2-5A synthetase as a function of interferon dose, the specific activity of the 2-5A synthetase, the nature of the 2-5A oligonucleotide products, and the activity of the 2-5A-activated endonuclease were essentially the same for both cell lines. The 73K phosphoprotein kinase was induced at a similar rate of activity, whether detected in solution or after immobilization on poly(I)·poly(C)-Sepharose. Our observations thus suggest that the induction of these two enzymatic activities are not sufficient for the anticellular activity of interferon.