Micronutrient deficits are still public health issues among women and young children in Vietnam

Background

The 2000 Vietnamese National Nutrition Survey showed that the population''s dietary intake had improved since 1987. However, inequalities were found in food consumption between socioeconomic groups. As no national data exist on the prevalence of micronutrient deficiencies, a survey was conducted in 2010 to assess the micronutrient status of randomly selected 1526 women of reproductive age and 586 children aged 6–75 mo.

Principal Findings

In women, according to international thresholds, prevalence of zinc deficiency (ZnD, 67.2±2.6%) and vitamin B12 deficiency (11.7±1.7%) represented public health problems, whereas prevalence of anemia (11.6±1.0%) and iron deficiency (ID, 13.7±1.1%) were considered low, and folate (<3%) and vitamin A (VAD, <2%) deficiencies were considered negligible. However, many women had marginal folate (25.1%) and vitamin A status (13.6%). Moreover, overweight (BMI≥23 kg/m² for Asian population) or underweight occurred in 20% of women respectively highlighting the double burden of malnutrition. In children, a similar pattern was observed for ZnD (51.9±3.5%), anemia (9.1±1.4%) and ID (12.9±1.5%) whereas prevalence of marginal vitamin A status was also high (47.3±2.2%). There was a significant effect of age on anemia and ID prevalence, with the youngest age group (6–17 mo) having the highest risk for anemia, ID, ZnD and marginal vitamin A status as compared to other groups. Moreover, the poorest groups of population had a higher risk for zinc, anemia and ID.

Conclusion

The prevalence of anemia and ID in Vietnam has been markedly reduced over the last decade, but a large part of the population is still at risk for other deficiencies such as zinc, vitamin A, folate and vitamin B₁₂ especially the youngest children aged 6–17 mo. Consequently specific interventions to improve food diversity and quality should be implemented, among them food fortification of staple foods and condiments and improvement of complementary feeding.     

Beneficial effects of resistance exercise on glycemic control are not further improved by protein ingestion

Purpose

To investigate the mechanisms underpinning modifications in glucose homeostasis and insulin sensitivity 24 h after a bout of resistance exercise (RE) with or without protein ingestion.

Methods

Twenty-four healthy males were assigned to a control (CON; n = 8), exercise (EX; n = 8) or exercise plus protein condition (EX+PRO; n = 8). Muscle biopsy and blood samples were obtained at rest for all groups and immediately post-RE (75% 1RM, 8×10 repetitions of leg-press and extension exercise) for EX and EX+PRO only. At 24 h post-RE (or post-resting biopsy for CON), a further muscle biopsy was obtained. Participants then consumed an oral glucose load (OGTT) containing 2 g of [U-¹³C] glucose during an infusion of 6, 6-[²H₂] glucose. Blood samples were obtained every 10 min for 2 h to determine glucose kinetics. EX+PRO ingested an additional 25 g of intact whey protein with the OGTT. A final biopsy sample was obtained at the end of the OGTT.

Results

Fasted plasma glucose and insulin were similar for all groups and were not different immediately post- and 24 h post-RE. Following RE, muscle glycogen was 26±8 and 19±6% lower in EX and EX+PRO, respectively. During OGTT, plasma glucose AUC was lower for EX and EX+PRO (75.1±2.7 and 75.3±2.8 mmol·L⁻¹∶120 min, respectively) compared with CON (90.6±4.1 mmol·L⁻¹∶120 min). Plasma insulin response was 13±2 and 21±4% lower for EX and CON, respectively, compared with EX+PRO. Glucose disappearance from the circulation was ∼12% greater in EX and EX+PRO compared with CON. Basal 24 h post-RE and insulin-stimulated PAS-AS160/TBC1D4 phosphorylation was greater for EX and EX+PRO.

Conclusions

Prior RE improves glycemic control and insulin sensitivity through an increase in the rate at which glucose is disposed from the circulation. However, co-ingesting protein during a high-glucose load does not augment this response at 24 h post-exercise in healthy, insulin-sensitive individuals.     

Carbon stocks and fluxes in tropical lowland dipterocarp rainforests in Sabah, Malaysian Borneo

Deforestation in the tropics is an important source of carbon C release to the atmosphere. To provide a sound scientific base for efforts taken to reduce emissions from deforestation and degradation (REDD+) good estimates of C stocks and fluxes are important. We present components of the C balance for selectively logged lowland tropical dipterocarp rainforest in the Malua Forest Reserve of Sabah, Malaysian Borneo. Total organic C in this area was 167.9 Mg C ha⁻¹±3.8 (SD), including: Total aboveground (TAGC: 55%; 91.9 Mg C ha⁻¹±2.9 SEM) and belowground carbon in trees (TBGC: 10%; 16.5 Mg C ha⁻¹±0.5 SEM), deadwood (8%; 13.2 Mg C ha⁻¹±3.5 SEM) and soil organic matter (SOM: 24%; 39.6 Mg C ha⁻¹±0.9 SEM), understory vegetation (3%; 5.1 Mg C ha⁻¹±1.7 SEM), standing litter (<1%; 0.7 Mg C ha⁻¹±0.1 SEM) and fine root biomass (<1%; 0.9 Mg C ha⁻¹±0.1 SEM). Fluxes included litterfall, a proxy for leaf net primary productivity (4.9 Mg C ha⁻¹ yr⁻¹±0.1 SEM), and soil respiration, a measure for heterotrophic ecosystem respiration (28.6 Mg C ha⁻¹ yr⁻¹±1.2 SEM). The missing estimates necessary to close the C balance are wood net primary productivity and autotrophic respiration.Twenty-two years after logging TAGC stocks were 28% lower compared to unlogged forest (128 Mg C ha⁻¹±13.4 SEM); a combined weighted average mean reduction due to selective logging of −57.8 Mg C ha⁻¹ (with 95% CI −75.5 to −40.2). Based on the findings we conclude that selective logging decreased the dipterocarp stock by 55–66%. Silvicultural treatments may have the potential to accelerate the recovery of dipterocarp C stocks to pre-logging levels.     

Chymase-dependent generation of angiotensin II from angiotensin-(1-12) in human atrial tissue

Since angiotensin-(1-12) [Ang-(1-12)] is a non-renin dependent alternate precursor for the generation of cardiac Ang peptides in rat tissue, we investigated the metabolism of Ang-(1-12) by plasma membranes (PM) isolated from human atrial appendage tissue from nine patients undergoing cardiac surgery for primary control of atrial fibrillation (MAZE surgical procedure). PM was incubated with highly purified ¹²⁵I-Ang-(1-12) at 37°C for 1 h with or without renin-angiotensin system (RAS) inhibitors [lisinopril for angiotensin converting enzyme (ACE), {"type":"entrez-protein","attrs":{"text":"SCH39370","term_id":"1052735772","term_text":"SCH39370"}}SCH39370 for neprilysin (NEP), MLN-4760 for ACE2 and chymostatin for chymase; 50 µM each]. ¹²⁵I-Ang peptide fractions were identified by HPLC coupled to an inline γ-detector. In the absence of all RAS inhibitor, ¹²⁵I-Ang-(1-12) was converted into Ang I (2±2%), Ang II (69±21%), Ang-(1-7) (5±2%), and Ang-(1-4) (2±1%). In the absence of all RAS inhibitor, only 22±10% of ¹²⁵I-Ang-(1-12) was unmetabolized, whereas, in the presence of the all RAS inhibitors, 98±7% of ¹²⁵I-Ang-(1-12) remained intact. The relative contribution of selective inhibition of ACE and chymase enzyme showed that ¹²⁵I-Ang-(1-12) was primarily converted into Ang II (65±18%) by chymase while its hydrolysis into Ang II by ACE was significantly lower or undetectable. The activity of individual enzyme was calculated based on the amount of Ang II formation. These results showed very high chymase-mediated Ang II formation (28±3.1 fmol×min⁻¹×mg⁻¹, n = 9) from ¹²⁵I-Ang-(1-12) and very low or undetectable Ang II formation by ACE (1.1±0.2 fmol×min⁻¹×mg⁻¹). Paralleling these findings, these tissues showed significant content of chymase protein that by immunocytochemistry were primarily localized in atrial cardiac myocytes. In conclusion, we demonstrate for the first time in human cardiac tissue a dominant role of cardiac chymase in the formation of Ang II from Ang-(1-12).     

Spatial differences in the presence of FOXP3+ and GranzymeB+ T cells between the intra- and extravascular compartments in renal allograft vasculopathy

Background

Allograft vasculopathy (AV) and native atherosclerosis (NA) share the presence of a T-cell mediated inflammatory response, but differ in overall plaque morphology and growth rate. We studied the distribution and frequency of regulatory- and cytotoxic T cells in the arterial intima lesions in both conditions.

Methodology/Principal Findings

The study is based on vessels of 15 explanted human renal allografts with AV and 10 carotid artery plaques obtained at surgery. Distribution and frequency of cytotoxic- and regulatory T cells, as identified by the expression of Granzyme B (GrB) and FOXP3 was established in NA and AV. Furthermore, we compared the distribution of these cells in AV with the perivascular, interstitial renal tissue using immunohistochemistry. The total number of T cells was much higher in AV than in NA lesions (711±135 and 37±8 CD3/mm² respectively, p<0.005, mean, ± SEM). Total numbers of FOXP3⁺ regulatory cells were also significantly increased in AV (36±10 and 0.9±0.3 FOXP3⁺/mm² p<0.05), but relative numbers, expressed as a percentage of the total number of CD3⁺ T cells ((FOXP3⁺/CD3⁺) ×100), were not significantly different (4.6%±0.9 and 2.7%±0.6). GrB⁺ cells were rare in NA, but significantly increased numbers of GrB⁺ cells were found in AV lesions (85±24 and 0.2±0.1 GrB⁺/mm², p<0.05). Perivascular tissues in the allografts showed a higher relative frequency of FOXP3⁺ cells than adjacent intimal lesions (14.0%±2.7 and 4.6%±0.9, respectively, p<0.05), but a lower frequency of GrB⁺ cytotoxic T cells (16.1%±2.7 and 22.6%±3.6, p<0.05).

Conclusions

Similar to NA, AV is characterized by a low frequency of intimal FOXP3⁺ regulatory T cells. Moreover, significant spatial differences exist in the distribution of functional T cell subsets between the intra- and extravascular micro-environments of the graft.     

The immune cell composition in Barrett's metaplastic tissue resembles that in normal duodenal tissue

Background and Objective

Barrett''s esophagus (BE) is characterized by the transition of squamous epithelium into columnar epithelium with intestinal metaplasia. The increased number and types of immune cells in BE have been indicated to be due to a Th2-type inflammatory process. We tested the alternative hypothesis that the abundance of T-cells in BE is caused by a homing mechanism that is found in the duodenum.

Patients and Methods

Biopsies from BE and duodenal tissue from 30 BE patients and duodenal tissue from 18 controls were characterized by immmunohistochemistry for the presence of T-cells and eosinophils(eos). Ex vivo expanded T-cells were further phenotyped by multicolor analysis using flowcytometry.

Results

The high percentage of CD4⁺-T cells (69±3% (mean±SEM/n = 17, by flowcytometry)), measured by flowcytometry and immunohistochemistry, and the presence of non-activated eosinophils found in BE by immunohistochemical staining, were not different from that found in duodenal tissue. Expanded lymphocytes from these tissues had a similar phenotype, characterized by a comparable but low percentage of αE(CD103) positive CD4⁺cells (44±5% in BE, 43±4% in duodenum of BE and 34±7% in duodenum of controls) and a similar percentage of granzyme-B⁺CD8⁺ cells(44±5% in BE, 33±6% in duodenum of BE and 36±7% in duodenum of controls). In addition, a similar percentage of α4β7⁺ T-lymphocytes (63±5% in BE, 58±5% in duodenum of BE and 62±8% in duodenum of controls) was found. Finally, mRNA expression of the ligand for α4β7, MAdCAM-1, was also similar in BE and duodenal tissue. No evidence for a Th2-response was found as almost no IL-4⁺-T-cells were seen.

Conclusion

The immune cell composition (lymphocytes and eosinophils) and expression of intestinal adhesion molecule MAdCAM-1 is similar in BE and duodenum. This supports the hypothesis that homing of lymphocytes to BE tissue is mainly caused by intestinal homing signals rather than to an active inflammatory response.     

Temperature-dependence of Weibel-Palade body exocytosis and cell surface dispersal of von Willebrand factor and its propolypeptide

Background

Weibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca²⁺-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca²⁺-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs.

Principal Findings

Ca²⁺-driven WPB exocytosis occurred at all temperatures studied (7–37°C). The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s⁻¹ (37°C) to 0.80±0.14 s⁻¹ (7°C) and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C) to 3.6±1.3% (7°C). A discrepancy was found between the reduction in Ca²⁺-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75–80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion) dispersal from WPBs, although slowed, was complete within 60–120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself.

Conclusions

We report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased.     

Blockade of VEGFR1 and 2 suppresses pathological angiogenesis and vascular leakage in the eye

Objective

VEGFR1 and 2 signaling have both been increasingly shown to mediate complications of ischemic retinopathies, including retinopathy of prematurity (ROP), age-related macular degeneration (AMD), and diabetic retinopathy (DR). This study evaluates the effects of blocking VEGFR1 and 2 on pathological angiogenesis and vascular leakage in ischemic retinopathy in a model of ROP and in choroidal neovascularization (CNV) in a model of AMD.

Materials and Methods

Neutralizing antibodies specific for mouse VEGFR1 (MF1) and VEGFR2 (DC101) were administrated systemically. CNV was induced by laser photocoagulation and assessed 14d after laser treatment. Retinal NV was generated in oxygen-induced ischemic retinopathy (OIR) and assessed at p17. NV quantification was determined by measuring NV tufts and vascular leakage was quantified by measuring [³H]-mannitol leakage from blood vessels into the retina. Gene expression was measured by real-time quantitative (Q)PCR.

Results

VEGFR1 and VEGFR2 expressions were up-regulated during CNV pathogenesis. Both MF1 and DC101 significantly suppressed CNV at 50 mg/kg: DC101 suppressed CNV by 73±5% (p<0.0001) and MF1 by 64±6% (p = 0.0002) in a dosage-dependent manner. The combination of MF1 and DC101 enhanced the inhibitory efficacy and resulted in an accumulation of retinal microglia at the CNV lesion. Similarly, both MF1 and DC101 significantly suppressed retinal NV in OIR at 50 mg/kg: DC101 suppressed retinal NV by 54±8% (p = 0.013) and MF1 by 50±7% (p<0.0002). MF1 was even more effective at inhibiting ischemia-induced BRB breakdown than DC101: the retina/lung leakage ratio for MF1 was reduced by 73±24%, p = 0.001 and for DC101 by 12±4%, p = 0.003. The retina/renal leakage ratio for MF1 was reduced by 52±28%, p = 0.009 and for DC101 by 13±4%, p = 0.001.

Conclusion

Our study provides further evidence that both VEGFR1 and 2 mediate pathological angiogenesis and vascular leakage in these models of ocular disease and suggests that antagonist antibodies to these receptor tyrosine kinases (RTKs) are potential therapeutic agents.     

Peroral Amphotericin B Polymer Nanoparticles Lead to Comparable or Superior In Vivo Antifungal Activity to That of Intravenous Ambisome? or Fungizone?

Background

Despite advances in the treatment, the morbidity and mortality rate associated with invasive aspergillosis remains unacceptably high (70–90%) in immunocompromised patients. Amphotericin B (AMB), a polyene antibiotic with broad spectrum antifungal activity appears to be a choice of treatment but is available only as an intravenous formulation; development of an oral formulation would be beneficial as well as economical.

Methodology

Poly(lactide-co-glycolode) (PLGA) nanoparticles encapsulating AMB (AMB-NPs) were developed for oral administration. The AMB-NPs were 113±20 nm in size with ∼70% entrapment efficiency at 30% AMB w/w of polymer. The in vivo therapeutic efficacy of oral AMB-NPs was evaluated in neutropenic murine models of disseminated and invasive pulmonary aspergillosis. AMB-NPs exhibited comparable or superior efficacy to that of Ambisome® or Fungizone™ administered parenterally indicating potential of NPs as carrier for oral delivery.

Conclusions

The present investigation describes an efficient way of producing AMB-NPs with higher AMB pay-load and entrapment efficiency employing DMSO as solvent and ethanol as non-solvent. The developed oral formulation was highly efficacious in murine models of disseminated aspergillosis as well as an invasive pulmonary aspergillosis, which is refractory to treatment with IP Fungizone™and responds only modestly to AmBisome®.     

Larvicidal activity of novel anthraquinone analogues and their molecular docking studies

To investigate the larvicidal activities of novel anthraquinones (1a-1k) against Culex quinquefasciatus mosquito larvae. Novel anthraquinones (1a-1k) derivatives were synthesis via condensation method. The compounds were confirmed through FT-IR spectroscopy, ¹H & ¹³C NMR spectrum, and mass spectral studies. The larvicidal activity of compound 1c was highly active LD₅₀ 20.92 µg/mL against Culex quinquefasciatus compared standard permethrin with LD₅₀ 25.49 µg/mL. Molecular docking studies were carried out for compound 1c against Odorant-binding protein of Culex quinquefasciatus. The compound 1c (−9.8 Kcal/mol) was a potent larvicide with more binding energy than control permethrin (−9.7 Kcal/mol). Therefore, compound (1c) may be more significant inhibitors of mosquito larvicidal.     

Dose effects of oxaliplatin on persistent and transient Na+ conductances and the development of neurotoxicity

Background

Oxaliplatin, a platinum-based chemotherapy utilised in the treatment of colorectal cancer, produces two forms of neurotoxicity- acute sensorimotor neuropathic symptoms and a dose-limiting chronic sensory neuropathy. Given that a Na⁺ channelopathy has been proposed as the mechanism underlying acute oxaliplatin-induced neuropathy, the present study aimed to determine specific mechanisms of Na⁺ channel dysfunction.

Methodology/Principal Findings

Specifically the function of transient and persistent Na⁺ currents were followed during treatment and were investigated in relation to oxaliplatin dose level. Eighteen patients were assessed before and after a single oxaliplatin infusion with motor and sensory axonal excitability studies performed on the median nerve at the wrist. While refractoriness (associated with Na⁺ channel inactivation) was significantly altered post-oxaliplatin infusion in both motor (Pre: 31.7±6.4%; Post: 68.8±14.5%; P≤.001) and sensory axons (Pre: 31.4±5.4%; Post: 21.4±5.5%; P<.05), strength-duration time constant (marker of persistent Na⁺ conductances) was not significantly altered post-infusion (Motor Pre: 0.395±0.01 ms; Post: 0.394±0.02 ms; NS; Sensory Pre:0.544±0.03 ms; Post: 0.535±0.05 ms; NS). However, changes in strength-duration time constant were significantly correlated with changes in refractoriness in motor and sensory axons (Motor correlation coefficient = −.65; P<.05; Sensory correlation coefficient = .67; P<.05).

Conclusions/Significance

It is concluded that the predominant effect of acute oxaliplatin exposure in human motor and sensory axons is mediated through changes in transient rather than persistent Na⁺ conductances. These findings are likely to have implications for the design and trial of neuroprotective strategies.     

Burkholderia dabaoshanensis sp. nov., a Heavy-Metal-Tolerant Bacteria Isolated from Dabaoshan Mining Area Soil in China

Heavy-metal-tolerant bacteria, GIMN1.004^T, was isolated from mine soils of Dabaoshan in South China, which were acidic (pH 2–4) and polluted with heavy metals. The isolation was Gram-negative, aerobic, non-spore-forming, and rod-shaped bacteria having a cellular width of 0.5−0.6 µm and a length of 1.3−1.8 µm. They showed a normal growth pattern at pH 4.0–9.0 in a temperature ranging from 5°C to 40°C.The organism contained ubiquinone Q-8 as the predominant isoprenoid quinine, and C_16∶0, summed feature 8 (C_18∶1 ω7c and C_18∶1 ω6c), C_18∶0, summed feature 3 (C_16∶1 ω7c or iso-C_15∶0 2-OH), C_17∶0 cyclo, C_18∶1 ω9c, C_19∶0 cyclo ω8c, C_14∶0 as major fatty acid. These profiles were similar to those reported for Burkholderia species. The DNA G+C % of this strain was 61.6%. Based on the similarity to 16S rRNA gene sequence, GIMN1.004^T was considered to be in the genus Burkholderia. The similarities of 16S rRNA gene sequence between strain GIMN1.004^T and members of the genus Burkholderia were 96−99.4%, indicating that this novel strain was phylogenetically related to members of that genus. The novel strain showed the highest sequence similarities to Burkholderia soli DSM 18235^T (99.4%); Levels of DNA-DNA hybridization with DSM 18235^T was 25%. Physiological and biochemical tests including cell wall composition analysis, differentiated phenotype of this strain from that closely related Burkholderia species. The isolation had great tolerance to cadmium with MIC of 22 mmol/L, and adsorbability of 144.94 mg/g cadmium,and it was found to exhibit antibiotic resistance characteristics. The adsorptive mechanism of GIMN1.004^T for cadmium depended on the action of the amide,carboxy and phosphate of cell surface and producing low-molecular-weight (LMW ) organic acids to complex or chelated Cd²⁺.Therefore, the strain GIMN1.004^T represented a new cadmium resistance species, which was tentatively named as Burkholderia dabaoshanensis sp. nov. The strain type is GIMN1.004^T ( = CCTCC M 209109^T =  NRRL B-59553^T ).     

Measuring the Pharmacodynamic Effects of a Novel Hsp90 Inhibitor on HER2/neu Expression in Mice Using 89Zr-DFO-Trastuzumab

Background

The positron-emitting radionuclide ⁸⁹Zr (t _1/2 = 3.17 days) was used to prepare ⁸⁹Zr-radiolabeled trastuzumab for use as a radiotracer for characterizing HER2/neu-positive breast tumors. In addition, pharmacodynamic studies on HER2/neu expression levels in response to therapeutic doses of PU-H71 (a specific inhibitor of heat-shock protein 90 [Hsp90]) were conducted.

Methodology/Principal Findings

Trastuzumab was functionalized with desferrioxamine B (DFO) and radiolabeled with [⁸⁹Zr]Zr-oxalate at room temperature using modified literature methods. ImmunoPET and biodistribution experiments in female, athymic nu/nu mice bearing sub-cutaneous BT-474 (HER2/neu positive) and/or MDA-MB-468 (HER2/neu negative) tumor xenografts were conducted. The change in ⁸⁹Zr-DFO-trastuzumab tissue uptake in response to high- and low-specific-activity formulations and co-administration of PU-H71 was evaluated by biodistribution studies, Western blot analysis and immunoPET. ⁸⁹Zr-DFO-trastuzumab radiolabeling proceeded in high radiochemical yield and specific-activity 104.3±2.1 MBq/mg (2.82±0.05 mCi/mg of mAb). In vitro assays demonstrated >99% radiochemical purity with an immunoreactive fraction of 0.87±0.07. In vivo biodistribution experiments revealed high specific BT-474 uptake after 24, 48 and 72 h (64.68±13.06%ID/g; 71.71±10.35%ID/g and 85.18±11.10%ID/g, respectively) with retention of activity for over 120 h. Pre-treatment with PU-H71 was followed by biodistribution studies and immunoPET of ⁸⁹Zr-DFO-trastuzumab. Expression levels of HER2/neu were modulated during the first 24 and 48 h post-administration (29.75±4.43%ID/g and 41.42±3.64%ID/g, respectively). By 72 h radiotracer uptake (73.64±12.17%ID/g) and Western blot analysis demonstrated that HER2/neu expression recovered to baseline levels.

Conclusions/Significance

The results indicate that ⁸⁹Zr-DFO-trastuzumab provides quantitative and highly-specific delineation of HER2/neu positive tumors, and has potential to be used to measure the efficacy of long-term treatment with Hsp90 inhibitors, like PU-H71, which display extended pharmacodynamic profiles.     

Intentional weight loss and dose reductions of anti-diabetic medications--a retrospective cohort study

Background and Aim

Intentional weight loss, primarily by improving insulin resistance, is known to decrease the need for anti-diabetic medications. In this study, we assess the magnitude of weight loss that resulted in dose reductions or discontinuation of anti-diabetic medications in overweight or obese patients with type 2 diabetes (DM) undergoing weight loss treatment.

Methods

Case records of 50 overweight or obese patients with DM who successfully decreased dosage or discontinued diabetes medications after losing weight via attendance at two University-based, outpatient weight management centers were analyzed. Follow-up visits, weight reduction interventions, and decisions for dose reductions or discontinuation of medications were individualized to patient needs by the treating physician.

Results

Mean starting BMI was 35 kg/m², mean age 53.4 years, and 58% were male. All 50 used at least one anti-diabetic medication (30 metformin, 39 sulfonylureas, 31 insulin, 21 sitagliptin) to manage blood sugar. Mean duration of follow-up was 30.2 months. Mean weight loss was 10.8±4.1 kgs (11.1% of initial body weight ±4.7%). 22/50 patients (44%) discontinued anti-diabetes medications (14 sulfonylureas [36%], 7 insulin [23%], 4 sitagliptin [19%]). The mean percentage weight loss achieved at the point of successful discontinuation of medication was 11.2%±3.5% (14% for sulphonylureas, 11% for insulin, and 7.1% for sitagliptin). Mean percentage weight loss of 5.6%±2.8% (5.1% for sulphonylureas, 4.3% for insulin, and 7.1% for sitagliptin) was required for initial dose reduction. For every 5% weight loss, predicted dose reductions were sulphonylureas, 39%; insulin, 42%; and any anti-diabetic medications, 49%.

Conclusion

Among overweight or obese patients with type 2 diabetes, intentional weight loss of 7–14% was typically required for full discontinuation of at least one anti-diabetic medication. Discontinuation of insulin was achieved at a mean weight reduction of 11% of initial body weight.     

Mycobacterium tuberculosis complex mycobacteria as amoeba-resistant organisms

Background

Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii.

Methodology/Principal Findings

Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03), correlating with the significantly larger size of M. canetti organisms (P = 0.035). The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57). M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10⁻⁶) higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02) M. avium organisms (34×10⁴ CFU/mL) than M. tuberculosis (42×10¹ CFU/mL) and M. bovis (35×10¹ CFU/mL) in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde.

Conclusions/Significance

These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.     

Formulation and Optimization of Nonionic Surfactants Emulsified Nimesulide-Loaded PLGA-Based Nanoparticles by Design of Experiments

This investigation aimed to develop nimesulide (NMS)-loaded poly(lactic-co-glycolic acid) (PLGA)-based nanoparticulate formulations as a biodegradable polymeric drug carrier to treat rheumatoid arthritis. Polymeric nanoparticles (NPs) were prepared with two different nonionic surfactants, vitamin E d-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) and poly(vinyl alcohol) (PVA), using an ultrasonication solvent evaporation technique. Nine batches were formulated for each surfactant using a 3² factorial design for optimal concentration of the emulsifying agents, 0.03–0.09% for vitamin E TPGS and 2–4% for PVA. The surfactant percentage and the drug/polymer ratio (1:10, 1:15, 1:20) of the NMS-loaded NPs were investigated based on four responses: encapsulation efficiency, particle size, the polydispersity index, and the surface charge. The response surface plots and linearity curves indicated a relationship between the experiment’s responses and a set of independent variables. The NPs produced with both surfactants exhibited a negative surface charge, and scanning electron micrographs revealed that all of the NPs were spherical in shape. A narrower size distribution and higher drug loadings were achieved in PVA-emulsified PLGA NPs than in the vitamin E TPGS emulsified. Decreasing amounts of both nonionic surfactants resulted in a reduction in the emulsion’s viscosity, which led to a decrease in the particle size of NPs. According to the ANOVA results obtained in this present research, vitamin E TPGS exhibited the best correlation between the independent variables, namely drug/polymer ratio and the surfactant percentage, and the dependent variables (encapsulation efficiency R² = 0.9603, particle size R² = 0.9965, size distribution R² = 0.9899, and surface charge R² = 0.8969) compared with PVA.KEY WORDS: ANOVA, factorial design, nanoparticles, nimesulide, PLGA, PVA, vitamin E TPGS     

Influence of short-term glucocorticoid therapy on regulatory T cells in vivo

Background

Pre- and early clinical studies on patients with autoimmune diseases suggested that induction of regulatory T(T_reg) cells may contribute to the immunosuppressive effects of glucocorticoids(GCs).

Objective

We readdressed the influence of GC therapy on T_reg cells in immunocompetent human subjects and naïve mice.

Methods

Mice were treated with increasing doses of intravenous dexamethasone followed by oral taper, and T_reg cells in spleen and blood were analyzed by FACS. Sixteen patients with sudden hearing loss but without an inflammatory disease received high-dose intravenous prednisolone followed by stepwise dose reduction to low oral prednisolone. Peripheral blood T_reg cells were analyzed prior and after a 14 day GC therapy based on different markers.

Results

Repeated GC administration to mice for three days dose-dependently decreased the absolute numbers of T_reg cells in blood (100 mg dexamethasone/kg body weight: 2.8±1.8×10⁴ cells/ml vs. 33±11×10⁴ in control mice) and spleen (dexamethasone: 2.8±1.9×10⁵/spleen vs. 95±22×10⁵/spleen in control mice), which slowly recovered after 14 days taper in spleen but not in blood. The relative frequency of FOXP3⁺ T_reg cells amongst the CD4⁺ T cells also decreased in a dose dependent manner with the effect being more pronounced in blood than in spleen. The suppressive capacity of T_reg cells was unaltered by GC treatment in vitro. In immunocompetent humans, GCs induced mild T cell lymphocytosis. However, it did not change the relative frequency of circulating T_reg cells in a relevant manner, although there was some variation depending on the definition of the T_reg cells (FOXP3⁺: 4.0±1.5% vs 3.4±1.5%*; AITR⁺: 0.6±0.4 vs 0.5±0.3%, CD127^low: 4.0±1.3 vs 5.0±3.0%* and CTLA4+: 13.8±11.5 vs 15.6±12.5%; * p<0.05).

Conclusion

Short-term GC therapy does not induce the hitherto supposed increase in circulating T_reg cell frequency, neither in immunocompetent humans nor in mice. Thus, it is questionable that the clinical efficacy of GCs is achieved by modulating T_reg cell numbers.     

Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle

Background

Within the animal kingdom, horses are among the most powerful aerobic athletic mammals. Determination of muscle respiratory capacity and control improves our knowledge of mitochondrial physiology in horses and high aerobic performance in general.

Methodology/Principal Findings

We applied high-resolution respirometry and multiple substrate-uncoupler-inhibitor titration protocols to study mitochondrial physiology in small (1.0–2.5 mg) permeabilized muscle fibres sampled from triceps brachii of healthy horses.Oxidative phosphorylation (OXPHOS) capacity (pmol O₂•s⁻¹•mg⁻¹ wet weight) with combined Complex I and II (CI+II) substrate supply (malate+glutamate+succinate) increased from 77±18 in overweight horses to 103±18, 122±15, and 129±12 in untrained, trained and competitive horses (N = 3, 8, 16, and 5, respectively). Similar to human muscle mitochondria, equine OXPHOS capacity was limited by the phosphorylation system to 0.85±0.10 (N = 32) of electron transfer capacity, independent of fitness level. In 15 trained horses, OXPHOS capacity increased from 119±12 to 134±37 when pyruvate was included in the CI+II substrate cocktail. Relative to this maximum OXPHOS capacity, Complex I (CI)-linked OXPHOS capacities were only 50% with glutamate+malate, 64% with pyruvate+malate, and 68% with pyruvate+malate+glutamate, and ∼78% with CII-linked succinate+rotenone. OXPHOS capacity with glutamate+malate increased with fitness relative to CI+II-supported ETS capacity from a flux control ratio of 0.38 to 0.40, 0.41 and 0.46 in overweight to competitive horses, whereas the CII/CI+II substrate control ratio remained constant at 0.70. Therefore, the apparent deficit of the CI- over CII-linked pathway capacity was reduced with physical fitness.

Conclusions/Significance

The scope of mitochondrial density-dependent OXPHOS capacity and the density-independent (qualitative) increase of CI-linked respiratory capacity with increased fitness open up new perspectives of integrative and comparative mitochondrial respiratory physiology.     

High fat diet-induced changes in mouse muscle mitochondrial phospholipids do not impair mitochondrial respiration despite insulin resistance

Background

Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD) increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance.

Methodology

C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD) or HFD (45 kcal%). Skeletal muscle mitochondria were isolated and fatty acid (FA) composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR.

Principal Findings

At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9) were decreased (−4.0%, p<0.001), whereas saturated FA (16∶0) were increased (+3.2%, p<0.001) in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6) showed a pronounced increase (+4.0%, p<0.001). Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002) and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks.

Conclusions/Interpretation

Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in mitochondrial fat oxidative capacity and (muscle) insulin resistance.     

Randomized clinical trial on ivermectin versus thiabendazole for the treatment of strongyloidiasis

Background

Strongyloidiasis may cause a life-threatening disease in immunosuppressed patients. This can only be prevented by effective cure of chronic infections. Direct parasitologic exams are not sensitive enough to prove cure if negative. We used an indirect immune fluorescent antibody test (IFAT) along with direct methods for patient inclusion and efficacy assessment.

Methodology/Principal Findings

Prospective, randomized, open label, phase III trial conducted at the Centre for Tropical Diseases (Verona, Italy) to compare efficacy and safety of ivermectin (single dose, 200 µg/kg) and thiabendazole (two daily doses of 25 mg/Kg for two days) to cure strongyloidiasis. The first patient was recruited on 6^th December, 2004. Follow-up visit of the last patient was on 11^th January, 2007. Consenting patients responding to inclusion criteria were randomly assigned to one of the treatment arms. Primary outcome was: negative direct and indirect (IFAT) tests at follow-up (4 to 6 months after treatment) or subjects with negative direct test and drop of two or more IFAT titers. Considering 198 patients who concluded follow-up, efficacy was 56.6% for ivermectin and 52.2% for thiabendazole (p = 0.53). If the analysis is restricted to 92 patients with IFAT titer 80 or more before treatment (virtually 100% specific), efficacy would be 68.1% for ivermectin and 68.9% for thiabendazole (p = 0.93). Considering direct parasitological diagnosis only, efficacy would be 85.7% for ivermectin and 94.6% for thiabendazole (p = 0.21). In ivermectin arm, mild to moderate side effects were observed in 24/115 patients (20.9%), versus 79/108 (73.1%) in thiabendazole arm (p = 0.00).

Conclusion

No significant difference in efficacy was observed, while side effects were far more frequent in thiabendazole arm. Ivermectin is the drug of choice, but efficacy of single dose is suboptimal. Different dose schedules should be assessed by future, larger studies.

Trial Registration

Portal of Clinical Research with Medicines in Italy 2004–004693–87