Ultrastructural characterization of CD133(+) stem cells bound to superparamagnetic nanoparticles: possible biotechnological applications

CD133 antigen is an integral membrane glycoprotein that can bind with different cells. Originally, however. this cellular surface antigen was expressed in human stem cells and in various cellular progenitors of the haematopoietic system. Human cord blood has been described as an excellent source of CD133(+) haematopoietic progenitor cells with a large application potential. One of the main objectives of the present study is to describe for the first time the ultrastructural characteristics of CD133(+) stem cells using transmission electronic microscopy. Another objective of the manuscript is to demonstrate through transmission electronic microscopy the molecular image of magnetic nanoparticles connected to the stein cells of great biotechnological importance, as well as demonstrating the value of this finding for electronic paramagnetic resonance and its related nanobioscientific value. Ultrastructural results showed the monoclonal antibody anti-CD133 bound to the superparamagnetic nanoparticles by the presence of electrondense granules in cell membrane, as well as in the cytoplasm, revealing the ultrastructural characteristics of CD133(+) cells, exhibiting a round morphology with discrete cytoplasmic projections, having an active nucleus that follows this morphology. The cellular cytoplasm was filled up with mitochondrias, as well as microtubules and vesicles pinocitic. characterizing the process as being related to internalization of the magnetic nanoparticles that were endocyted by the cells in question. Electronic paramagnetic resonance analysis of the CD133(+) stem cells detected that the small (spectrum) generated by the labelled cells comes from the superparamagnetic nanoparticles that are bound to them. These results strongly suggest that these CD133(+) cells can be used in nanobiotechnology applications, with benefits in different biomedical areas.

Substrate-bound carbohydrates stimulate signal transduction and neurite outgrowth in an olfactory neuron cell line

SUBPOPULATIONS of olfactory receptor neurons, which are dispersed throughout the olfactory neuroepithelium, express specific cell surface carbohydrates and project to discrete regions of the olfactory bulb. Cell surface carbohydrates such as N-acetyl-lactosamine have been postulated to mediate sorting and selective fasciculation of discrete axon subpopulations during development of the olfactory pathway. Substrate-bound N-acetyl-lactosamine promotes neurite outgrowth by both clonal olfactory receptor neuron cell lines and olfactory receptor neurons in vitro, indicating that cell surface carbohydrates may be ligands for receptor-mediated stimulation of axon growth in vivo. In the present study, the role of transmembrane signaling in N-acetyl-lactosamine-stimulated neurite outgrowth was examined in the clonal olfactory neuron cell line 4.4.2. Substrate-bound N-acetyl-lactosamine stimulated neurite outgrowth which was specifically inhibited by antagonists to N- and L-type calcium channels and to tyrosine kinase phosphorylation. These results indicate that N-acetyl-lactosamine can evoke transmembrane receptor-mediated responses capable of influencing neurite outgrowth.

Fetal cystoscopy for severe lower urinary tract obstruction-initial experience of a single center

Objective To report the experience with fetal cystoscopy and laser fulguration of posterior urethral values (PUV) for severe lower urinary tract obstruction (LUTO). Methods Between July 2006 and December 2008, fetal cystoscopy was offered to 23 patients whose fetuses presented with severe LUTO. favorable urinary analysis and gestational age <26 weeks. Fetal urinary biochemistry was evaluated before and after cystoscopy. All infants were followed 6-12 months after birth. Abnormal renal function was defined when serum creatinine higher than 50 mu mol/L (2 Standard Deviation) or the necessity of dialysis or renal transplantation. Autopsy was always performed whenever fetal or neonatal deaths occurred. Results Eleven patients decided to undergo fetal therapy and 12 elected to continue with expectant observation. There was no difference between both groups in gestation age at diagnosis and referral examinations. Urethral atresia was diagnosed in 4/11 (36.4%) fetuses by fetal cystoscopy. At 26 weeks, fetuses that were managed expectantly presented with worse urinary biochemistry results (p < 0.05). Survival rates and percentage of infants with normal renal function were significantly higher in the cystoscopic group than in the expectant group (P < 0.05). Conclusions Percutaneous fetal cystoscopy is feasible using a thinner special cannula for prenatal diagnosis and therapy of LUTO. Prenatal laser ablation of the PUV under cystoscopy may prevent renal function deterioration improving postnatal outcome. Copyright (C) 2009 John Wiley & Sons, Ltd.

Lower N-Acetyl-Aspartate Levels in Prefrontal Cortices in Pediatric Bipolar Disorder: A (1)H Magnetic Resonance Spectroscopy Study

Objective: The few studies applying single-voxel(1)H spectroscopy in children and adolescents with bipolar disorder (BD) have reported low N-acetyl-aspartate (NAA) levels in the dorsolateral prefrontal cortex (DLPFC), and high myo-inositol / phosphocreatine plus creatine (PCr+Cr) ratios in the anterior cingulate. The aim of this study was to evaluate NAA, glycerophosphocholine plus phosphocholine (GPC+PC) and PCr+Cr in various frontal cortical areas in children and adolescents with BD. We hypothesized that NAA levels within the prefrontal cortex are lower in BD patients than in healthy controls, indicating neurodevelopmental alterations in the former. Method: We studied 43 pediatric patients with DSM-IV BD (19 female, mean age 13.2 +/- 2.9 years) and 38 healthy controls (79 female, mean age 13.9 +/- 2.7 years). We conducted multivoxel in vivo (1)H spectroscopy measurements at 1.5 Tesla using a long echo time of 272 ms to obtain bilateral metabolite levels from the medial prefrontal cortex (MPFC), DLPFC (white and gray matter), cingulate (anterior and posterior), and occipital lobes. We used the nonparametric Mann-Whitney U test to compare neurochemical levels between groups. Results: In pediatric BD patients, NAA and GPC+PC levels in the bilateral MPFC, and PCr+Cr levels in the left MPFC were lower than those seen in the controls. In the left DLPFC white matter, levels of NAA and PCr+Cr were also lower in BD patients than in controls. Conclusions: Lower NAA and PCr+Cr levels in the PFC of children and adolescents with BD may be indicative of abnormal dendritic arborization and neuropil, suggesting neurodevelopmental abnormalities. J. Am. Acad. Child Adolesc. Psychiatry, 2011;50(1):85-94.

Lower numbers of circulating natural killer T (NK T) cells in individuals with human T lymphotropic virus type 1 (HTLV-1) associated neurological disease

Human T lymphotropic virus type 1 (HTLV-1) infects 10-20 million people worldwide. The majority of infected individuals are asymptomatic; however, approximately 3% develop the debilitating neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is also currently no cure, vaccine or effective therapy for HTLV-1 infection, and the mechanisms for progression to HAM/TSP remain unclear. NK T cells are an immunoregulatory T cell subset whose frequencies and effector functions are associated critically with immunity against infectious diseases. We hypothesized that NK T cells are associated with HAM/TSP progression. We measured NK T cell frequencies and absolute numbers in individuals with HAM/TSP infection from two cohorts on two continents: Sao Paulo, Brazil and San Francisco, CA, USA, and found significantly lower levels when compared with healthy subjects and/or asymptomatic carriers. Also, the circulating NK T cell compartment in HAM/TSP subjects is comprised of significantly more CD4(+) and fewer CD8(+) cells than healthy controls. These findings suggest that lower numbers of circulating NK T cells and enrichment of the CD4(+) NK T subset are associated with HTLV-1 disease progression.

Lower cytokine secretion ex vivo by natural killer T cells in HIV-infected individuals is associated with higher CD161 expression

Objective: Natural killer T (NKT) cells are efficiently targeted by HIV and severely reduced in numbers in the circulation of infected individuals. The functional capacity of the remaining NKT cells in HIV-infected individuals is poorly characterized. This study measured NKT cell cytokine production directly ex vivo and compared these responses with both the disease status and NKT subset distribution of individual patients. Methods: NKT cell frequencies, subsets, and ex-vivo effector functions were measured in the peripheral blood mononuclear cells of HIV-infected patients and healthy controls by flow cytometry. We measured cytokines from NKT cells after stimulation with either a-galactosyl ceramide-loaded CD1d dimers (DimerX-alpha GalCer) or phorbol myristate acetate and ionomycin. Results: The frequencies of NKT cells secreting interferon-gamma and tumor necrosis factor-alpha were significantly lower in HIV-infected patients than healthy controls after DimerX-alpha GalCer treatment, but responses were similar after treatment with phorbol myristate acetate and ionomycin. The magnitude of the interferon-gamma response to DimerX-alpha GalCer correlated inversely with the number of years of infection. Both interferon-gamma and tumor necrosis factor-alpha production in response to DimerX-alpha GalCer correlated inversely with CD161 expression. Conclusion: The ex-vivo Th1 responses of circulating NKT cells to CD1d-glycolipid complexes are impaired in HIV-infected patients. NKT cell functions may be progressively lost over time in HIV infection, and CD161 is implicated in the regulation of NKT cell responsiveness. (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

A method for better positioning bipolar electrodes for lower limb EMG recordings during dynamic contractions

To obtain a high quality EMG acquisition, the signal must be recorded as far away as possible from muscle innervations and tendon zones, which are known to shift during dynamic contractions. This study describes a methodology, using commercial bipolar electrodes, to identify better electrode positions for superficial EMG of lower limb muscles during dynamic contractions. Eight female volunteers participated in this study. Myoelectric signals of the vastus lateralis, gastrocnemius medialis, peroneus longus and tibialis anterior muscles were acquired during maximum isometric contractions using bipolar electrodes. The electrode positions of each muscle were selected assessing SENIAM and then, other positions were located along the length of muscle up and down the SENIAM site. The raw signal (density), the linear envelopes, the RMS value, the motor point site, the position of the IZ and its shift during dynamic contractions were taken into account to select and compare electrode positions. For vastus lateralis and peroneus longus, the best sites were 66% and 25% of muscle length, respectively (similar to SENIAM location). The position of the tibialis anterior electrodes presented the best signal at 47.5% of its length (different from SENIAM location). The position of the gastrocnemius medialis electrodes was at 38% of its length and SENIAM does not specify a precise location for signal acquisition. The proposed method should be considered as another methodological step in every EMG study to guarantee the quality of the signal and subsequent human movement interpretations. (C) 2009 Elsevier B.V. All rights reserved.

Incidence of ipsilateral postoperative deep venous thrombosis in the amputated lower extremity of patients with peripheral obstructive arterial disease

Objective: Patients undergoing amputation of the lower limb due to peripheral arterial disease (PAD) are at risk of developing deep venous thrombosis (DVT). Few studies in the research literature report the incidence of DVT during the early postoperative period or the risk factors for the development of DVT in the amputation stump. This prospective study evaluated the incidence of DVT during the first 35 postoperative days in patients who had undergone amputation of the lower extremity due to PAD and its relation to comorbidities and death. Methods: Between September 2004 and March 2006, 56 patients (29 men), with a mean age of 67.25 years, underwent 62 amputations, comprising 36 below knee amputations (BKA) and 26 above knee amputations (AKA). Echo-Doppler scanning was performed preoperatively and on postoperative days 7 and 31 (approximately). All patients received acetylsalicylic acid (100 mg daily) preoperatively and postoperatively, but none received prophylactic anticoagulation. Results: DVT occurred in 25.8% of extremities with amputations (10 ARA and 6 BKA). The cumulative incidence in the 35-day postoperative period was 28% (Kaplan-Meier). There was a significant difference (P = .04) in the incidence of DVT between AKA (37.5%) and BKA (21.2%). Age >= 70 years (48.9% vs 16.8%, P = .021) was also a risk factor for DVT in the univariate analysis. Of the 16 cases, 14 (87.5%) were diagnosed during outpatient care. The time to discharge after amputation was averaged 6.11 days in-hospital stay (range, 1-56 days). One symptomatic nonfatal pulmonary embolism occurred in a patient already diagnosed with DVT. There was no relation between other comorbidities and DVT. The multivariate analysis showed no association between risk factors and the occurrence of DVT in the amputated extremity. DVT ipsilateral to the amputation did not influence the mortality rate (9.7%). Conclusion: The incidence of DVT in the early postoperative period (<= 35 days) was elevated principally in patients aged >= 70 years and for AKA. Patients with PAD who have recently undergone major amputations should be considered at high risk for DVT, even after hospital discharge. Given the high rate of postoperative DVT observed in this study, we now recommend prophylactic anticoagulation for these patients, but further study is needed to determine the optimal duration and efficacy of this treatment. (J Vasc Surg 2008;48:1514-9.)

Effectiveness of Endoclips for the Treatment of Stigmata of Recent Hemorrhage in the Colon of Patients with Acute Lower Gastrointestinal Tract Bleeding

Coaptive thermo coagulation (CTC) for the treatment of stigmata of recent hemorrhage (SRH) in the colon is risky. We evaluated the safety and effectiveness of endoclips in 13 patients with acute lower gastrointestinal bleeding (GIB). Thirty-day re-bleeding and complication rates were comparable to a historical cohort of 41 patients (group 2) who underwent CTC/IE (injection epinephrine) for the management of acute lower GIB. There was no difference in the 30-day re-bleeding rates in the two groups. In group I, immediate hemostasis was successful in all patients. Three of 13 patients (23.1%) developed re-bleeding. In group II, 41 patients from six prior studies underwent CTC and/or IE for the treatment of HRS where 12 (29.3%) developed re-bleeding. There were no immediate complications. Endoclip deployment is as effective as CTC and/or IE for treatment of SRH in the colon.

The role of BPH, lower urinary tract symptoms, and PSA levels on erectile function of Brazilian men who undergo prostate cancer screening

Introduction. Lower urinary tract symptoms (LUTS) and erectile dysfunction (ED) are common problems in middle-aged and older men. Recently, epidemiologic studies have shown significant associations between severity of LUTS and male sexual dysfunction. Aim. We analyzed the role of prostate enlargement, LUTS, and prostate specific antigen (PSA) levels in the erectile function of Brazilian men who underwent prostate cancer (PCa) screening. Method. We analyzed data from 1,008 consecutive patients enrolled in a PCa screening program. Benign prostatic hyperplasia (BPH) was defined as a prostate weight greater than 30 g as defined by digital rectal examination. For statistical analysis, we used the chi-squared and analysis of variance tests. The odds ratios (OR) for correlation of ED with prostate volume LUTS and PSA were estimated using logistic regression models. Main Outcome Measure. The American Urological Association (AUA) symptom score for LUTS and the International Index of Erectile Function. Results. Mean patient age was 61.2 years (45-87) and median PSA value was 1.9 ng/mL. BPH was identified in 48.5% of patients. Mild, moderate, and severe LUTS were found in 52.3%, 30.9%, and 16.8% of cases, respectively. ED was classified as absent, mild, mild to moderate, moderate, and severe in 18.6%, 23.1%, 18.6%, 15.2%, and 24.5%, respectively. While only 5.4% of the patients with no ED presented severe LUTS, this finding was observed in 27.1% of patients with severe ED (P<0.001). Univariate logistic regression analysis demonstrated that age, prostate volume, AUA symptom score, and PSA levels were significant predictors of ED. However, when controlled for patient age, only LUTS remained as an independent predictor of ED. Conclusions. Controlling for patient age, LUTS are independent risk factors for the development of ED among Brazilian men who undergo PCa screening.

High voltage electrical stimulation as an alternative treatment for chronic ulcers of the lower limbs

High voltage electrical stimulation has been recommended as a means of accelerating the wound healing process. The effects of high voltage electrical stimulation were evaluated in the treatment of three volunteers with chronic ulcers of the lower limbs. After fifteen weeks of treatment, a reduction was found in the area of all the ulcers, suggesting that high voltage electric stimulation is an effective therapeutic option for chronic ulcers.

The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination. (C) 2009 Elsevier B.V. All rights reserved.