Gene delivery into mouse retinal ganglion cells by in utero electroporation

Background  

The neural retina is a highly structured tissue of the central nervous system that is formed by seven different cell types that are arranged in layers. Despite much effort, the genetic mechanisms that underlie retinal development are still poorly understood. In recent years, large-scale genomic analyses have identified candidate genes that may play a role in retinal neurogenesis, axon guidance and other key processes during the development of the visual system. Thus, new and rapid techniques are now required to carry out high-throughput analyses of all these candidate genes in mammals. Gene delivery techniques have been described to express exogenous proteins in the retina of newborn mice but these approaches do not efficiently introduce genes into the only retinal cell type that transmits visual information to the brain, the retinal ganglion cells (RGCs).     

The impaired pregnancy outcome in murine congenital toxoplasmosis is associated with a pro-inflammatory immune response, but not correlated with decidual inducible nitric oxide synthase expression

Congenital toxoplasmosis is associated with adverse pregnancy outcome. Despite the type 1 immune response, C57BL/6 mice are more susceptible than BALB/c mice to Toxoplasma gondii infection. Additionally, successful pregnancy appears to be correlated with type 2 T helper maternal immunity and regulatory T cells. In order to investigate the mechanisms of susceptibility/resistance to congenital toxoplasmosis in mice with different genetic backgrounds and the influence of inducible nitric oxide synthase in pregnancy outcome, groups of C57BL/6, BALB/c and C57BL/6 iNOS(-/-) females were orally infected with T. gondii ME-49 strain on day 1 of pregnancy and were sacrificed on day 8 p.i. and day 19 p.i. The uterus and placenta were evaluated for the foetal resorption rate, parasite load, immunological and histological changes. C57BL/6 mice presented inflammatory foci in the decidua (endometrium) of the uterus at a higher frequency than BALB/c mice on day 8 p.i., and a large number of pregnant C57BL/6 mice presented necrotic implantation sites. The parasite was seldom found in the uterus or placenta of either lineage of mice. Interestingly, there was no observed difference in inducible nitric oxide synthase expression in the uterus and placenta of infected mice. In addition, higher levels of TNF-α were detected in serum samples from C57BL/6 mice compared with BALB/c mice. Accordingly, C57BL/6 mice presented with levels of 90% abortion compared with 50% in BALB/c mice on day 19 p.i. C57BL/6 iNOS(-/-) mice showed low placental parasite counts and high absorption rates, similar to wild type mice. The data suggest that the impaired pregnancy outcome due to T. gondii infection in C57BL/6 mice could be associated with a higher inflammatory response leading to cell apoptosis and necrosis of implantation sites compared with BALB/c mice, and this phenomenon was not due to inducible nitric oxide synthase expression in the decidua.     

Oxidative Stress,Redox Homeostasis and Cellular Stress Response in Ménière’s Disease: Role of Vitagenes

Ménière’s disease (MD) is characterized by the triad of fluctuating hearing loss, episodic vertigo and tinnitus, and by endolymphatic hydrops found on post-mortem examination. Increasing evidence suggests that oxidative stress is involved in the development of endolymphatic hydrops and that cellular damage and apoptotic cell death might contribute to the sensorineural hearing loss found in later stages of MD. While excess reactive oxygen species (ROS) are toxic, regulated ROS, however, play an important role in cellular signaling. The ability of a cell to counteract stressful conditions, known as cellular stress response, requires the activation of pro-survival pathways and the production of molecules with anti-oxidant, anti-apoptotic or pro-apoptotic activities. Among the cellular pathways conferring protection against oxidative stress, a key role is played by vitagenes, which include heat shock proteins (Hsps) as well as the thioredoxin/thioredoxin reductase system. In this study we tested the hypothesis that in MD patients measurable increases in markers of cellular stress response and oxidative stress in peripheral blood are present. This study also explores the hypothesis that changes in the redox status of glutathione, the major endogenous antioxidant, associated with abnormal expression and activity of carbonic anhydrase can contribute to increase oxidative stress and to disruption of systemic redox homeostasis which can be associated to possible alterations on vulnerable neurons such as spiral ganglion neurons and consequent cellular degeneration. We therefore evaluated systemic oxidative stress and cellular stress response in patients suffering from Meniere’s disease (MD) and in age-matched healthy subjects. Systemic oxidative stress was estimated by measuring protein oxidation, such as protein carbonyls (PC) and 4-hydroxynonenal (HNE) in lymphocytes of MD patients, as well as ultraweak luminescence (UCL) as end-stable products of lipid oxidation in MD plasma and lymphocytes, as compared to age-matched controls, whereas heat shock proteins Hsp70 and thioredoxin (Trx) expression were measured in lymphocytes to evaluate the systemic cellular stress response. Increased levels of PC (P < 0.01) and HNE (P < 0.05) have been found in lymphocytes from MD patients with respect to control group. This was paralleled by a significant induction of Hsp70, and a decreased expression of Trx (P < 0.01), whereas a significant decrease in both plasma and lymphocyte ratio reduced glutathione GSH) vs. oxidized glutathione (GSSG) (P < 0.05) were also observed. In conclusion, patients affected by MD are under condition of systemic oxidative stress and the induction of vitagenes Hsp70 is a maintained response in counteracting the intracellular pro-oxidant status generated by decreased content of GSH as well as expression of Trx. The search for novel and more potent inducers of vitagenes will facilitate the development of pharmacological strategies to increase the intrinsic capacity of vulnerable ganglion cells to maximize antidegenerative mechanisms, such as stress response and thus cytoprotection.     

The cationic amino acid transporter 2 is induced in inflammatory lung models and regulates lung fibrosis

Background

Cigarette smoke (CS) is known to initiate a cascade of mediator release and accumulation of immune and inflammatory cells in the lower airways. We investigated and compared the effects of CS on upper and lower airways, in a mouse model of subacute and chronic CS exposure.

Methods

C57BL/6 mice were whole-body exposed to mainstream CS or air, for 2, 4 and 24 weeks. Bronchoalveolar lavage fluid (BAL) was obtained and tissue cryosections from nasal turbinates were stained for neutrophils and T cells. Furthermore, we evaluated GCP-2, KC, MCP-1, MIP-3α, RORc, IL-17, FoxP3, and TGF-β1 in nasal turbinates and lungs by RT-PCR.

Results

In both upper and lower airways, subacute CS-exposure induced the expression of GCP-2, MCP-1, MIP-3α and resulted in a neutrophilic influx. However, after chronic CS-exposure, there was a significant downregulation of inflammation in the upper airways, while on the contrary, lower airway inflammation remained present. Whereas nasal FoxP3 mRNA levels already increased after 2 weeks, lung FoxP3 mRNA increased only after 4 weeks, suggesting that mechanisms to suppress inflammation occur earlier and are more efficient in nose than in lungs.

Conclusions

Altogether, these data demonstrate that CS induced inflammation may be differently regulated in the upper versus lower airways in mice. Furthermore, these data may help to identify new therapeutic targets in this disease model.     

Recalcitrant abdominal wall hernias: long-term superiority of autologous tissue repair

Secondary repair of recurrent ventral hernia is difficult, and success depends on re-establishing the functional integrity of the abdominal wall. Current techniques used for closure of these defects have documented recurrence rates as high as 54 percent. The authors' 8-year experience utilizing variations of the components separation technique for autologous tissue repair of recalcitrant hernias emphasizes that recurrent or recalcitrant hernias benefit from the creation of a dynamic abdominal wall. A total of 389 patients were retrospectively identified as having abdominal wall defects, and 284 of these patients met the selection criteria. Study patients were grouped according to the type of surgical repair used. The recurrence rate was 20.7 percent over all study groups and was directly related to the extent of repair required. Group 1 patients (wide tissue undermining) had a recurrence rate of only 15 percent, while in group 2 (complete components separation), the recurrence rate was 22 percent. Group 3 patients (interpositional fascia lata graft) had a 29 percent recurrence rate. Time to recurrence was also significantly different across treatment groups, with study group 3 experiencing earlier hernia recurrence. The most frequent postoperative complication was wound infection, which was directly related to the repair performed. The relative odds of recurrence versus the risk factors of age, sex, perioperative steroid use, wound infection, defect size, and the presence of enterocutaneous fistula were studied with a logistic regression analysis. These factors did not possess statistical significance for predicting hernia recurrence. The preoperative presence of mesh was independently significant for hernia recurrence, increasing the relative odds 2.2 times (p = 0.01). Similarly, when other risk factors were controlled for, increasing the complexity of the treatment group, from study group 1 (wide tissue undermining) to study group 3 (interpositional fascia lata graft), also increased the odds of hernia recurrence 1.5-fold per group (p = 0.04). Average inpatient cost was $24,488. The length of inpatient stay ranged from 2 to 172 days (average, 12.8 days). The length of inpatient stay and costs were directly related to the extent of repair required. Using the analysis of variance test for multiple factors, the presence of an enterocutaneous fistula (p = 0.0014) or a postoperative wound infection (p = 0.008) independently increased the length of inpatient stay and hospital costs. A total of 108 successfully repaired patients were contacted by telephone and agreed to participate in a self-reported satisfaction survey. The patients noticed improvements in the appearance of their abdomen, in their postoperative emotional state, and in their ability to lift objects, arise from a chair or a bed, and exercise. These results suggest that recalcitrant hernia defects should be solved, when possible, by reconstructing a dynamic abdominal wall.     

Clinical applications of three-dimensional photography in breast surgery

Three-dimensional imaging in breast surgery has several uses clinically. The most practical applications are for the evaluation of breast asymmetries, both congenital and acquired, and for the evaluation of factors affecting breast shape in augmentation mammaplasty. Other uses of three-dimensional imaging that we have found clinically helpful are for evaluation of patients desiring reduction mammaplasty and for evaluation of patients undergoing unilateral breast reconstruction to determine the expander and permanent implant size that gives the best symmetry with the contralateral breast. We present five cases in which we investigate the use of three-dimensional imaging clinically by using the images to determine quantitative information about the breast, such as volume or projection. Overall, three-dimensional imaging is very helpful in providing objective information about the breast for use in preoperative planning. In addition, by analyzing clinical cases, it can provide objective data about the breast and surgical mammaplasty (especially augmentation mammaplasty) that may help surgeons better understand those factors that contribute to breast shape and influence surgical outcomes. There are currently some limitations of this system, influenced by patients with significant ptosis or obesity, which may introduce errors into the three-dimensional data, making them unreliable. However, we believe three-dimensional imaging has great clinical potential in surgical mammaplasty.     

Synthesis of nucleotide analogues that potently and selectively inhibit human DNA primase

DNA primase synthesizes short RNA oligonucleotides that DNA polymerase alpha further elongates in order to initiate the synthesis of all new DNA strands during eukaryotic DNA replication. To develop potent and specific primase inhibitors, we combined 2'-modified sugars with bases incapable of normal Watson-Crick hydrogen bonding. The presence of a 2'-hydroxyl in either the ara or ribo configuration greatly enhances the ability of primase to polymerize a nucleotide. Further modifying the 2'-position by including both a hydroxyl and methyl group at this position greatly reduced the ability of primase to polymerize the resulting nucleotides. Replacing the base of the NTP with analogues incapable of normal Watson-Crick hydrogen bonding (benzimidazole, nitrobenzimidazole, and dichlorobenzimidazole) resulted in compounds that inhibited primase quite well and with similar potency. We synthesized arabinofuranosylbenzimidazole triphosphate (araBTP) and found that this sugar change increased inhibition by 2-4-fold relative to the ribofuranosyl analogue. AraBTP inhibited polymerization of both purines and pyrimidines, although primase polymerized only small amounts of the compound. Interestingly, even though araBTP was not readily polymerized by primase, it inhibited primase almost as potently as araATP, a compound that primase polymerizes extremely rapidly and that results in very strong chain termination. Importantly, this compound was a very weak inhibitor of and only slowly polymerized by DNA polymerase alpha, indicating that it is a specific primase inhibitor. The potential utility and mechanistic implications of these inhibitors are discussed.     

Reproductive biology of Psammobatis extenta

Psammobatis extenta collected from the commercial bottom trawl fishery operating in Puerto Quequén, Argentina, between 2000 and 2001, had a sex ratio of 1:1 (535 specimens). Both the largest male and female measured 313 mm total length ( L _T). The relationship between total body mass and L _T was significantly different between sexes; mature females weighed more than mature males for a given L _T. The size at which 50% of males were sexually mature was 262 mm L _T (83·6% of the maximum size), whereas 50% of females were mature at 249 mm L _T (80·0% of the maximum size). The gonadosomatic index in mature males and females was slightly higher during summer, but did not show significant variation over the sampling period. The hepatosomatic index of males and females was higher during the autumn and winter and followed a distinct seasonal pattern. Evidence suggested a continuous reproductive cycle during the year, with a maximum number of females carrying egg-cases in summer.