Tracing of projection neurons from the cervical dorsal horn to the medulla with the anterograde tracer biotinylated dextran amine

In addition to the well-defined role of dorsal horn neurons in pain transmission, neurons in the superficial laminae also provide a rich source of synaptic input to cardiovascular and respiratory centers in the medullary reticular formation. In this study, ascending projection neurons from the superficial laminae of the cervical enlargement were studied in the rat using the anterograde tracer biotinylated dextran amine (BDA). Ipsilateral microinjection of BDA into the cervical spinal cord (C6-C8) resulted in extensive labeling of dorsal horn neurons in laminae I-V. Axons and terminal processes of cervical dorsal horn cells projecting to the medulla were present in the cuneate nucleus (Cu), the nucleus of the solitary tract (NTS), the lateral reticular nucleus, (LRt) as well as the caudal and rostral ventrolateral medulla (VLM). The highest density of BDA labeling was found ipsilaterally in the Cu, LRt, caudal and rostral VLM, while a moderate density of labeling was present in the NTS caudal to the area postrema (AP). Moderate-to-weak labeling was also found in the LRt, the caudal and rostral VLM contralateral to the BDA injection. These results support the existence of a spinomedullary pathway that transmits noxious and innocuous Adelta and C fiber-mediated sensory signals to the medulla. Neurons in this ascending spinal pathway likely participate in the patterning of autonomic responses evoked by pain or during exercise.     

Myocardial Contractility in Premature Neonates With and Without Patent Ductus Arteriosus

Controversy exists as to whether a hemodynamically significant left-to-right shunt due to a patent ductus arteriosus (PDA) affects ventricular contractility. Load-dependent indices such as ejection fraction and shortening fraction have traditionally been used to assess contractility, but the relationship between the rate-corrected velocity of fiber shortening (MVCFc) and wall stress may be more suitable, as it is a preload-independent, afterload-adjusted method of assessing ventricular contractility. Age-related differences have been established for these variables in normal adults and children and it has been recommended for use in the premature neonate. The study was performed to assess left ventricular contractility in premature neonates with a significant left-to-right shunt due to a PDA. Using echocardiography, we measured the relationship of MVCFc to stress at peak systole (SPS) in two groups of premature infants. Group 1 consisted of 15 controls (680–1495 g, 25–32 weeks gestation), and Group 2 of 15 neonates with hemodynamically significant PDA (840–1635 g, 26–33 weeks gestation). In both groups, MVCFc was linearly and inversely related to SPS (p < 0.001). The regression equations were as follows: Group 1, MVCFc = –0.0153SPS + 1.70 (R² = 0.68); and Group 2, MVCF = – 0.019SPS + 1.89 (R² = 0.76). There was no significant difference in the relationship between the two groups, but their slopes were significantly steeper and had a higher Y-intercept than the relationship we previously reported for older children. This preliminary study establishes the normal MVCFc/SPS relationship in the premature neonate (25–33 weeks gestation) and suggests that premature infants function at a higher resting contractile state than older children. A hemodynamically significant PDA has no effect on contractility. These data will be useful in assessing left ventricular contractility in premature neonates with other types of ventricular loading and noncardiac stress.     

Safety and efficacy of olopatadine hydrochloride nasal spray for the treatment of seasonal allergic rhinitis.

BACKGROUND: A nasal spray containing the antiallergy agent olopatadine hydrochloride is being developed for the treatment of seasonal allergic rhinitis (SAR). OBJECTIVE: To evaluate the safety and efficacy of 2 concentrations of olopatadine nasal spray vs placebo in patients with SAR. METHODS: This was a multicenter, randomized, double-blind, placebo-controlled study. After a 3- to 21-day placebo run-in, 565 patients aged 12 to 80 years were randomized to receive 0.4% or 0.6% olopatadine or placebo, 2 sprays per nostril twice daily for 2 weeks. Patients evaluated morning and evening reflective and instantaneous nasal symptoms (sneezing, stuffy nose, runny nose, and itchy nose, which compose the total nasal symptom score [TNSS]) and ocular symptoms and completed the Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ). RESULTS: Olopatadine spray (0.4% and 0.6%) was significantly superior to placebo for percentage change from baseline in overall reflective (P = .004 and P < .001, respectively) and instantaneous (P = .02 and P = .003, respectively) TNSSs. Also, 0.6% olopatadine was significantly superior to placebo for reducing the reflective and instantaneous assessments of sneezing, runny and itchy nose, and itchy eyes; the instantaneous assessments of watery eyes; and the overall and all 7 domain scores of the RQLQ (P < .05). Olopatadine spray exhibited a safety profile comparable with that of placebo. CONCLUSIONS: Olopatadine nasal spray (0.4% and 0.6%) provided statistically significant improvements in allergic rhinitis symptoms compared with placebo regarding TNSSs (reflective and instantaneous) and in quality-of-life variables in patients with SAR. Olopatadine nasal spray administered twice daily was safe and well tolerated in adolescents and adults.     

Oral and rectal immunization of adult female volunteers with a recombinant attenuated Salmonella typhi vaccine strain.

An attenuated strain of Salmonella typhi delta(cya) delta(crp-cdt) delta(asd) expressing a gene encoding a hepatitis B virus core-pre-S protein was tested in female adult volunteers for its ability to elicit a systemic and a mucosal immune response. Specifically, our purpose was to evaluate the potential of such a vaccine strain to induce specific secretory immunoglobulin A (sIgA) at genital and rectal surfaces. Oral and rectal routes of immunization were compared: oral immunization induced seroconversion against the bacterial lipopolysaccharide (LPS) in six out of seven volunteers, while after rectal immunization only one out of six volunteers seroconverted against LPS. To our disappointment, the latter volunteer was also the only one who seroconverted against the carried antigen (pre-S1), demonstrating the poor ability of this live vaccine to induce an immune response against the carried antigen. Anti-LPS sIgA was found in both the vaginal and cervical secretions of a volunteer who presented a strong seroconversion after oral immunization (16-fold increase in anti-LPS IgG). Smaller amounts of anti-LPS sIgA were found in the rectal secretions of one orally and one rectally immunized volunteer and in the saliva of three orally and one rectally immunized woman. Our data show for the first time that it is possible to induce specific sIgA in the genital and rectal tracts of women by using an S. typhi vaccine strain.     

The promotion of osteoblastic differentiation of rat bone marrow stromal cells by a polyvalent plant mosaic virus

To investigate the role that the micro/nano-environment plays on the differentiation pathway of bone marrow stromal cells (BMSCs) into osteoblasts, we employed a 2D substrate coated with turnip yellow mosaic virus (TYMV) particles. TYMV is a non-enveloped icosahedral plant virus which has an average diameter 28nm and the protein cage structure consists of 180 identical subunits. The temporal effect of TYMV coated substrate on the adhesion and differentiation capacity of the BMSCs was monitored for selected time periods of 7, 14 and 21 days. We examined the gene expression profile of BMSCs cultured in primary media (undifferentiated cells) and cells induced to osteoblast lineage by real time PCR analysis. To further corroborate our findings, we investigated the expression of osteogenic markers using immunohistochemistry and cytochemical staining. As expected, the genes involved in the process of osteogenic differentiation were activated more during the growth of cells under osteogenic media. In addition, we found that the BMSCs induced to undergo osteogenic differentiation on TYMV coated substrates formed fully mineralized nodules comprising of osteoblast-like cells around day 14. Comparing the gene expression pattern of BMSCs induced to osteogenic differentiation under standard culture conditions with the cells induced on TYMV substrates, we found significant differences in the temporal expression and level of expression of several key genes. Our findings indicate that TYMV, as a biogenic nanoparticle, can be employed as a model to modulate the nano-environment of the substrates in order to gain an insight into the role that the micro/nano-environment has in regulating adhesion, growth and differentiation of BMSCs towards osteogenic lineage, which will be vital for designing compatible biomaterials for tissue engineering purposes.     

The EuroPrevall surveys on the prevalence of food allergies in children and adults: background and study methodology

Background:  The epidemiological surveys in children and adults of the EU-funded multidisciplinary Integrated Project EuroPrevall, launched in June 2005, were designed to estimate the currently unknown prevalence of food allergy and exposure to known or suspected risk factors for food allergy across Europe. We describe the protocol for the epidemiological surveys in children and adults. This protocol provides specific instructions on the sampling strategy, the use of questionnaires, and collection of blood samples for immunological analyses.
Methods:  The surveys were performed as multi-centre, cross-sectional studies in general populations. Case–control studies were nested within these surveys. The studies in children aged 7–10 years and adults aged 20–54 years were undertaken in eight centres representing different social and climatic regions in Europe.
Results:  After a community-based survey collecting basic information on adverse reactions to foods, all those stating they had experienced such reactions, as well as of a random sample of those stating 'no reactions' to foods, completed a detailed questionnaire on potential risks and exposures. Also a blood sample was taken to allow serological analysis to establish patterns of food and aeroallergen sensitization. We also included a questionnaire to schools on their preparedness for dealing with food allergy amongst pupils. Subjects reporting adverse reactions to foods and sensitized to the same food(s) were called in for a full clinical evaluation that included a double blind placebo controlled food challenge (DBPCFC), following a protocol which is described in detail elsewhere.
Conclusions:  The outcome of these studies will help to improve our understanding of several important aspects of food allergies in the European Community, providing for more well-informed policies and effective measures of disease prevention, diagnosis and management.     

Is multiple SNP testing in BRCA2 and BRCA1 female carriers ready for use in clinical practice? Results from a large Genetic Centre in the UK

BRCA1 and BRCA2 are major breast cancer susceptibility genes. Nineteen single nucleotide polymorphisms (SNPs) at 18 loci have been associated with breast cancer. We aimed to determine whether these predict breast cancer incidence in women with BRCA1/BRCA2 mutations. BRCA1/2 mutation carriers identified through the Manchester genetics centre between 1996 and 2011 were included. Using published odds ratios (OR) and risk allele frequencies, we calculated an overall breast cancer risk SNP score (OBRS) for each woman. The relationship between OBRS and age at breast cancer onset was investigated using the Cox proportional hazards model, and predictive ability assessed using Harrell's C concordance statistic. In BRCA1 mutation carriers we found no association between OBRS and age at breast cancer onset: OR for the lowest risk quintile compared to the highest was 1.20 (95% CI 0.82–1.75, Harrell's C = 0.54), but in BRCA2 mutation carriers the association was significant (OR for the lowest risk quintile relative to the highest was 0.47 (95% CI 0.33–0.69, Harrell's C = 0.59). The 18 validated breast cancer SNPs differentiate breast cancer risks between women with BRCA2 mutations, but not BRCA1. It may now be appropriate to use these SNPs to help women with BRCA2 mutations make maximally informed decisions about management options.     

Hoechst staining and exposure to UV laser during flow cytometric sorting does not affect the frequency of detected endogenous DNA nicks in abnormal and normal human spermatozoa

Controlling the sex of offspring by the separation of X and Y chromosome-bearing spermatozoa using flow cytometry has been reported as a clinical technique aiding prevention of X-linked diseases. Although this technique has resulted in several hundred normal births in animals and at least one human birth, there is still concern over its genetic safety due to the involvement of two potentially mutagenic agents: UV light and the fluorochrome dye, Hoechst 33342 (H33342). Human spermatozoa, particularly those considered abnormal, may be more likely to suffer DNA damage following exposure to mutagenic agents, compared with other mammalian species. The stability of normal fresh and decondensed human spermatozoa were examined after exposure to a range of levels of UV and H33342 staining, using an assay that detects endogenous nicks in the DNA of spermatozoa. The stability of abnormal and normal, fresh and frozen-thawed human spermatozoa was examined following UV laser, H33342 staining and flow cytometry treatments utilizing the same assay. There was an increase in the presence of endogenous nicks when spermatozoa were decondensed compared with fresh spermatozoa. There was no increase in the incidence of nicks in any group of spermatozoa after UV and fluorochrome exposure compared with controls without exposure.      

Modulation of disease severity of dystrophic epidermolysis bullosa by a splice site mutation in combination with a missense mutation in the COL7A1 gene

Dystrophic epidermolysis bullosa (EBD) is a clinically heterogeneous skin disorder, characterized by abnormal anchoring fibrils (AF) and loss of dermal-epidermal adherence. EBD has been linked to the COL7A1 gene at chromosome 3p21 which encodes collagen VII, the major component of the AF. Here we investigated two unrelated EBD families with different clinical phenotypes and novel combinations of recessive and dominant COL7A1 mutations. Both families shared the same recessive heterozygous 14 bp deletion at the exon-intron 115 boundary of the COL7A1 gene. The deletion caused in-frame skipping of exon 115 and the elimination of 29 amino acid residues from the pro-alpha1(VII) polypeptide chain. As a result, procollagen VII was not converted to collagen VII and the C-terminal NC-2 propeptide which is normally removed from the procollagen VII prior to formation of the anchoring fibrils was retained in the skin. All affected individuals also carried missense mutations in exon 73 of COL7A1 which lead to different glycine- to-arginine substitutions in the triple-helical domain of collagen VII. Combination of the deletion mutation with a G2009R substitution resulted in a mild phenotype. In contrast, combination of the deletion with a G2043R substitution led to a severe phenotype. The G2043R substitution was a de novo mutation which alone caused a mild phenotype. Thus, different combinations of dominant and recessive COL7A1 mutations can modulate disease activity of EBD and alter the clinical presentation of the patients.