Adipose tissue: a new target for electroporation-enhanced DNA vaccines.

DNA vaccines delivered using electroporation (EP) have had clinical success, but these EP methods generally utilize invasive needle electrodes. Here, we demonstrate the delivery and immunogenicity of a DNA vaccine into subcutaneous adipose tissue cells using noninvasive EP. Using finite element analysis, we predicted that plate electrodes, when oriented properly, could effectively concentrate the electric field within adipose tissue. In practice, these electrodes generated widespread gene expression persisting for at least 60 days in vivo within interscapular subcutaneous fat pads of guinea pigs. We then applied this adipose-EP protocol to deliver a DNA vaccine coding for an influenza antigen into guinea pigs. The resulting host immune responses elicited were of a similar magnitude to those achieved by skin delivery with EP. The onset of the humoral immune response was more rapid when the DNA dose was spread over multiple injection sites, and increasing the voltage of the EP device increased the magnitude of the immune response. This study supports further development of EP protocols delivering gene-based therapies to subcutaneous fat.

Prototype development and preclinical immunogenicity analysis of a novel minimally invasive electroporation device.

The magnitude of the immune response to a DNA vaccine depends on three criteria--the optimized vector design, the use of a suitable adjuvant and the successful delivery and subsequent expression of the plasmid in the target tissue. In vivo electroporation (EP) has proved to be particularly effective in efficiently delivering DNA immunogens to the muscle and the skin, and indeed several devices have entered into human clinical trials. Here, we report on a novel concept of DNA delivery to the dermal tissue using a minimally invasive EP device, which is powered using low-voltage parameters. We show that this prototype device containing a novel 4 × 4-electrode array results in robust and reproducible transfection of dermal tissue and subsequent antigen expression at the injection site. Using DNA encoding for NP and M2e influenza antigens, we further show induction of potent cellular responses in a mouse model as measured by antigen-specific T-cell ELISpot assays. Importantly, 100% of the immunized animals were protected when challenged with VN/1203/04 (H5N1) strain of influenza. We have also extended our findings to a guinea-pig model and demonstrated induction of HI titers greater than 1:40 against a pandemic novel H1N1 virus showing proof of concept efficacy for DNA delivery with the prototype device in a broad spectrum of species and using multiple antigens. Finally, we were able to generate protective HI titers in macaques against the same novel H1N1 strain. Our results suggest that the minimally invasive dermal device may offer a safe, tolerable and efficient method to administer DNA vaccinations in a prophylactic setting, and thus potentially represents an important new option for improved DNA vaccine delivery in vivo.

Specimen Collection for Translational Studies in Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory disorder characterized by painful nodules, sinus tracts, and scars occurring predominantly in intertriginous regions. The prevalence of HS is currently 0.053-4%, with a predominance in African-American women and has been linked to low socioeconomic status. The majority of the reported literature is  retrospective, population based, epidemiologic studies. In this regard, there is a need to establish a repository of biospecimens, which represent appropriate gender and racial demographics amongst HS patients. These efforts will diminish knowledge gaps in understanding the disease pathophysiology. Hence, we sought to outline a step-by-step protocol detailing how we established our HS biobank to facilitate the formation of other HS tissue banks. Equipping researchers with carefully detailed processes for collection of HS specimens would accelerate the accumulation of well-organized human biological material. Over time, the scientific community will have access to a broad range of HS tissue biospecimens, ultimately leading to more rigorous basic and translational research. Moreover, an improved understanding of the pathophysiology is necessary for the discovery of novel therapies for this debilitating disease. We aim to provide high impact translational research methodology for cutaneous biology research and foster multidisciplinary collaboration and advancement of our understanding of cutaneous diseases.

Insect renal tubules constitute a cell-autonomous immune system that protects the organism against bacterial infection.

Innate immunity is a widespread and important defence against microbial attack, which in insects is thought to originate mainly in the fat body. Here we demonstrate that the fluid-transporting Malpighian (renal) tubule of Drosophila melanogaster constitutes an autonomous immune-sensing tissue utilising the nitric oxide (NO) signalling pathway. Reverse transcriptase PCR (RT-PCR) shows that tubules express those genes encoding components of the Imd pathway. Furthermore, isolated tubules bind and respond to lipopolysaccharide (LPS), by upregulating anti-microbial peptide (diptericin) gene expression and increased bacterial killing. Excised, LPS-challenged tubules, as well as tubules from LPS-infected flies, display increased NO synthase (NOS) activity upon immune challenge. Targetted expression of a Drosophila NOS (dNOS) transgene to only principal cells of the tubule main segment using the GAL4/UAS system increases diptericin expression. In live flies, such targetted over-expression of dNOS to tubule principal cells confers increased survival of the whole animal upon E. coli challenge. Thus, we describe a novel role of Malpighian tubules in immune sensing and insect survival.

Potential role of interleukin-1 in the development of bronchopulmonary dysplasia.

Increased activities of inflammatory mediators unopposed by their inhibitors contribute to chronic lung injury and impaired healing in BPD. The deleterious effects of IL-1 beta, a cytokine involved in inflammation and host defense, are blocked by IL-1 receptor antagonist (IL-1Ra). We proposed that an imbalance of IL-1 beta and its inhibitors may contribute to the development of BPD. To determine the relative antigen concentrations of IL-1 beta and IL-1Ra and functional IL-1 activity in lung lavage of infants at risk for BPD, lung lavage was serially obtained from 1 to 28 days from 17 infants with evolving BPD, 13 infants with self-limited RDS, and 6 controls ventilated for nonpulmonary reasons. Overall, there was a high correlation between IL-1 beta antigen concentration and IL-1 activity (r = 0.82, p = 0.0001). There were no significant differences among the groups for lung lavage variables on day 1. However, in infants who developed BPD, IL-1 beta antigen concentration and IL-1 activity increased 16- and 61-fold, respectively, during the first week. IL-1Ra remained relatively unchanged during the first month. IL-1 beta/IL-1Ra antigen ratio was significantly higher on days 5 (median 0.024) and 7 (median 0.025) compared with day 1 (median 0.004), p < 0.05. These results suggest that a relative imbalance of IL-1 beta and IL-1Ra may contribute to prolonged inflammation in BPD.

Fast MR imaging of fetal CNS anomalies in utero.

BACKGROUND AND PURPOSE: Although sonography is the primary imaging technique for evaluating the developing fetus, significant limitations exist in the sonographic prenatal diagnosis of many brain disorders. Fast MR imaging is increasingly being used to determine the underlying cause of nonspecific fetal CNS abnormalities detected sonographically and to confirm or provide further support for such anomalies. Our goal was to determine the value of MR imaging in establishing the diagnosis of fetal CNS anomalies, to ascertain how this information might be used for patient counseling, and to assess its impact on pregnancy management. METHODS: We prospectively performed MR examinations of 73 fetuses (66 pregnancies) with suspected CNS abnormalities and compared these with available fetal sonograms, postnatal images, and clinical examinations. Retrospectively, the impact on patient counseling and pregnancy management was analyzed. RESULTS: Images of diagnostic quality were routinely obtained with in utero MR imaging, which was particularly valuable in detecting heterotopia, callosal anomalies, and posterior fossa malformations, and for providing excellent anatomic information. We believe that 24 (46%) of 52 clinical cases were managed differently from the way they would have been on the basis of sonographic findings alone. In every case, the referring physicians thought that MR imaging provided a measure of confidence that was not previously available and that was valuable for counseling patients and for making more informed decisions. CONCLUSION: Sonography is the leading technique for fetal assessment and provides reliable, inexpensive diagnostic images. Fast MR imaging is an important adjunctive tool for prenatal imaging in those instances in which a complex anomaly is suspected by sonography, when fetal surgery is contemplated, or when a definitive diagnosis cannot be determined.

Neu-Laxova syndrome: a case report.

Neu-Laxova syndrome is a rare form of congenital malformation characterized by intrauterine growth retardation, microcephaly with bizarre facial features, short neck, apparent edema, scaly skin, and perinatal death. Chromosomal analysis in reported cases has revealed a normal karyotype, and an autosomal recessive transmission has been postulated. We present a case of Neu-Laxova syndrome. The pathologic features and the prenatal radiographic appearance are described.

Weekly prophylactic urokinase instillation in tunneled central venous access devices.

PURPOSE: To determine the safety and efficacy of weekly prophylactic urokinase therapy in tunneled central venous access devices (VADs). MATERIALS AND METHODS: A prospective, randomized study was performed in 105 patients who underwent tunneled VAD placement between March 1997 and April 1998. The patients were randomized to receive either twice-daily heparin flushes (14 heparin flushes per week; group A, n = 52) or twice-daily heparin flushes with once-weekly urokinase (UK) instillation (13 heparin flushes, one UK flush per week; group B, n = 53). Patients were followed up by examination and/or interview at 1, 3, and 6 months for signs and symptoms of delayed catheter-related complications. RESULTS: The total number of indwelling catheter-days was similar between groups (5,450 in group A, 5,276 in group B). The total number of infectious complications and fibrin sheaths formed was greater for group A (n = 11; 21.1%) than group B (n = 3; 5.7%) (P = .02). There were no side effects noted from the prophylactic UK administrations. CONCLUSION: Prophylactic UK is advantageous in preventing delayed catheter-related complications.

Model organisms: new insights into ion channel and transporter function. L-type calcium channels regulate epithelial fluid transport in Drosophila melanogaster.

The neuropeptide CAP2b stimulates fluid transport obligatorily via calcium entry, nitric oxide, and cGMP in Drosophila melanogaster Malpighian (renal) tubules. We have shown by RT-PCR that the Drosophila L-type calcium channel alpha1-subunit genes Dmca1D and Dmca1A (nbA) are both expressed in tubules. CAP2b-stimulated fluid transport and cytosolic calcium concentration ([Ca2+]i) increases are inhibited by the L-type calcium channel blockers verapamil and nifedipine. cGMP-stimulated fluid transport is verapamil and nifedipine sensitive. Furthermore, cGMP induces a slow [Ca2+]i increase in tubule principal cells via verapamil- and nifedipine-sensitive calcium entry; RT-PCR shows that tubules express Drosophila cyclic nucleotide-gated channel (cng). Additionally, thapsigargin-induced [Ca2+]i increase is verapamil sensitive. Phenylalkylamines bind with differing affinities to the basolateral and apical surfaces of principal cells in the main segment; however, dihydropyridine binds apically in the tubule initial segment. Immunocytochemical evidence suggests localization of alpha1-subunits to both basolateral and apical surfaces of principal cells in the tubule main segment. We suggest roles for L-type calcium channels and cGMP-mediated calcium influx in both calcium signaling and fluid transport mechanisms in Drosophila.

Disordered pathways of fibrin turnover in lung lavage of premature infants with respiratory distress syndrome.

Premature infants who have self-limited respiratory distress syndrome (RDS) rapidly improve, whereas infants with a complicated respiratory course are more likely to develop bronchopulmonary dysplasia (BPD), a chronic lung disorder that is the result of prolonged lung injury and impaired healing. The balance of competing activities of coagulation and fibrinolysis may contribute to the premature lung's response to acute injury and determine, in part, whether there is early resolution or protracted alveolar inflammation. To determine the relative activities of the coagulation and fibrinolytic pathways in neonatal lung injury, procoagulant (PC) and plasminogen activator (PA) activities were measured in undiluted cell-free lung lavage samples obtained serially over the first 28 days of life from 11 infants with self-limited RDS, 11 infants with evolving BPD, and 5 mechanically ventilated control infants without lung disease. Lung lavage from all three groups contained readily detectable procoagulant activity due mainly to the tissue factor-Factor VII complex. Plasminogen activator activity was relatively high in control lavage samples but depressed on the first day of life in the two groups of infants with lung disease: median, 0.3814 IU/ml (control); 0.0541 IU/ml (RDS); and 0.0454 IU/ml (BPD), p < 0.05 in each case compared with control. Two infants with severe lung disease had no detectable plasminogen activator activity in lung lavage on the first day of life. Depressed fibrinolytic activity correlated with severity of lung disease assessed radiographically and by pulmonary function measurements. Plasminogen activator activity was due to both tissue plasminogen activator and urokinase.(ABSTRACT TRUNCATED AT 250 WORDS)