Regulation of 25-hydroxyvitamin D3-1 alpha-hydroxylase and production of 1 alpha,25-dihydroxyvitamin D3 by human dendritic cells

25-Hydroxyvitamin D3-1 alpha-hydroxylase (25(OH)D3-1 alpha-hydroxylase), the key enzyme of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) production, is expressed in monocyte-derived macrophages (MACs). Here we show for the first time constitutive expression of 25(OH)D3-1 alpha-hydroxylase in monocyte-derived dendritic cells (DCs), which was increased after stimulation with lipopolysaccharide (LPS). Accordingly, DCs showed low constitutive production of 1,25(OH)2D3, but activation by LPS increased 1,25(OH)2D3 synthesis. In addition, 25(OH)D3-1 alpha-hydroxylase expression was found in blood DCs but not in CD34+-derived DCs. Next we analyzed the functional consequences of these results. Addition of 1,25(OH)2D3 at concentrations comparable with those produced by DCs inhibited the allostimulatory potential of DCs during the early phase of DC differentiation. However, terminal differentiation decreased the responsiveness of DCs to 1,25(OH)2D3. In conclusion, DCs are able to produce 1,25(OH)2D3 especially following stimulation with LPS. Terminal maturation renders DCs unresponsive to the effects of 1,25(OH)2D3, but those cells are able to suppress the differentiation of their own precursor cells in a paracrine way through the production of 1,25(OH)2D3.     

Randomized comparison of reduced fat and reduced carbohydrate hypocaloric diets on intrahepatic fat in overweight and obese human subjects

Obesity-related hepatic steatosis is a major risk factor for metabolic and cardiovascular disease. Fat reduced hypocaloric diets are able to relieve the liver from ectopically stored lipids. We hypothesized that the widely used low carbohydrate hypocaloric diets are similarly effective in this regard. A total of 170 overweight and obese, otherwise healthy subjects were randomized to either reduced carbohydrate (n = 84) or reduced fat (n = 86), total energy restricted diet (-30% of energy intake before diet) for 6 months. Body composition was estimated by bioimpedance analyses and abdominal fat distribution by magnetic resonance tomography. Subjects were also submitted to fat spectroscopy of liver and oral glucose tolerance testing. In all, 102 subjects completed the diet intervention with measurements of intrahepatic lipid content. Both hypocaloric diets decreased body weight, total body fat, visceral fat, and intrahepatic lipid content. Subjects with high baseline intrahepatic lipids (>5.56%) lost ≈7-fold more intrahepatic lipids compared with those with low baseline values (<5.56%) irrespective of diet composition. In contrast, changes in visceral fat mass and insulin sensitivity were similar between subgroups, with low and high baseline intrahepatic lipids. CONCLUSION: A prolonged hypocaloric diet low in carbohydrates and high in fat has the same beneficial effects on intrahepatic lipid accumulation as the traditional low-fat hypocaloric diet. The decrease in intrahepatic lipids appears to be independent of visceral fat loss and is not tightly coupled with changes in whole body insulin sensitivity during 6 months of an energy restricted diet.     

A cell-based screen for resistance of Bcr-Abl-positive leukemia identifies the mutation pattern for PD166326, an alternative Abl kinase inhibitor

In Philadelphia-positive (Ph(+)) leukemia, point mutations within the Bcr-Abl kinase domain emerged as a major mechanism of resistance to imatinib mesylate. We established a cell-based screening strategy for detection of clinically relevant point mutations using Bcr-Abl-transformed Ba/F3 cells. We identified 32 different single-point mutations within the kinase domain of Bcr-Abl. The pattern and frequency of mutations in this cell culture-based screen resembled the pattern and frequency observed in resistant patients. We then applied this screen to an alternative Abl kinase inhibitor. Using PD166326, the frequency of resistant colonies emerging at 5 to 10 times the median growth inhibition (IC50) of PD166326 was significantly lower than with imatinib. In addition, PD166326 produced a distinct pattern of Bcr-Abl mutations. The majority of mutations that came up with both imatinib and PD166326 could effectively be suppressed by increasing the dose of PD166326 to 50 to 500 nM. In contrast, only a few mutations could be suppressed by increasing the imatinib dose to 5 to 10 microM. However, 3 mutations affecting F317 displayed complete resistance to PD166326, but could be effectively inhibited by standard concentrations of imatinib. Thus, this robust and simple screening system provides a rational basis for combinatorial and sequential treatment strategies in targeted cancer therapy.     

The clinical-grade 42-kilodalton fragment of merozoite surface protein 1 of Plasmodium falciparum strain FVO expressed in Escherichia coli protects Aotus nancymai against challenge with homologous erythrocytic-stage parasites

A 42-kDa fragment from the C terminus of major merozoite surface protein 1 (MSP1) is among the leading malaria vaccine candidates that target infection by asexual erythrocytic-stage malaria parasites. The MSP1(42) gene fragment from the Vietnam-Oak Knoll (FVO) strain of Plasmodium falciparum was expressed as a soluble protein in Escherichia coli and purified according to good manufacturing practices. This clinical-grade recombinant protein retained some important elements of correct structure, as it was reactive with several functional, conformation-dependent monoclonal antibodies raised against P. falciparum malaria parasites, it induced antibodies (Abs) that were reactive to parasites in immunofluorescent Ab tests, and it induced strong growth and invasion inhibitory antisera in New Zealand White rabbits. The antigen quality was further evaluated by vaccinating Aotus nancymai monkeys and challenging them with homologous P. falciparum FVO erythrocytic-stage malaria parasites. The trial included two control groups, one vaccinated with the sexual-stage-specific antigen of Plasmodium vivax, Pvs25, as a negative control, and the other vaccinated with baculovirus-expressed MSP1(42) (FVO) as a positive control. Enzyme-linked immunosorbent assay (ELISA) Ab titers induced by E. coli MSP1(42) were significantly higher than those induced by the baculovirus-expressed antigen. None of the six monkeys that were vaccinated with the E. coli MSP1(42) antigen required treatment for uncontrolled parasitemia, but two required treatment for anemia. Protective immunity in these monkeys correlated with the ELISA Ab titer against the p19 fragment and the epidermal growth factor (EGF)-like domain 2 fragment of MSP1(42), but not the MSP1(42) protein itself or the EGF-like domain 1 fragment. Soluble MSP1(42) (FVO) expressed in E. coli offers excellent promise as a component of a vaccine against erythrocytic-stage falciparum malaria.     

The effect of stable macromolecular complexes of ionic polyphosphazene on HIV Gag antigen and on activation of human dendritic cells and presentation to T-cells

Neonates and infants are susceptible to infection due to distinct immune responses in early life. Therefore, development of vaccine formulation and delivery systems capable of activating human newborn leukocytes is of global health importance. Poly[di(carboxylatophenoxy)phosphazene] (PCPP) belongs to a family of ionic synthetic polyphosphazene polyelectrolyte compounds that can form non-covalent interactions with protein antigens and demonstrate adjuvant activity in animals and in human clinical trials. However, little is known about their ability to activate human immune cells. In this study, we characterized the effects of PCPP alone or in combination with a model antigen (recombinant HIV-Gag (Gag)), on the maturation, activation and antigen presentation by human adult and newborn dendritic cells (DCs) in vitro. PCPP treatment induced DC activation as assessed by upregulation of co-stimulatory molecules and cytokine production. Studies benchmarking PCPP to Alum, the most commonly used vaccine adjuvant, demonstrated that both triggered cell death and release of danger signals in adult and newborn DCs. When complexed with Gag antigen, PCPP maintained its immunostimulatory characteristics while permitting internalization and presentation of Gag by DCs to HIV-Gag-specific CD4⁺ T cell clones. The PCPP vaccine formulation outlined here has intrinsic adjuvant activity, can facilitate effective delivery of antigen to DCs, and may be advantageous for induction of beneficial T cell-mediated immunity. Moreover, polyphosphazenes can further reduce cost of vaccine production and distribution through their dose-sparing and antigen-stabilizing properties, thus potentially eliminating the need for cold chain distribution.     

Incidental granular parakeratosis associated with molluscum contagiosum

A patient presented with a 4-month history of slowly progressive pruritic papules on her trunk and extremities. Biopsies from 2 of these lesions revealed molluscum contagiosum. One of the biopsies also showed several small foci of granular parakeratosis. Based on the clinical features and course of this patient, the granular parakeratosis seems to be an incidental finding.     

Topical anti-itch therapy

Topical anti-itch therapy could be causative (antiviral, antimycotic, and/or antiparasitic preparations) or symptomatic. Symptomatic therapy includes substituting some other sensation by cooling, heating, and/or counterirritation, by anesthesia of sensory nerve endings with local anesthetics, blocking mediators of pruritus (to deplete substance P or to block acetylcholine release), and reducing inflammation of the skin with corticosteroids or topical immunomodulators (pimecrolimus and tacrolimus). In addition to drugs, the patient should be taught to use emollients and to avoid skin dryness and vasodilatation by contact with irritants. Topical anti-itch preparations can be recommended not only for treatment of localized pruritus, but also for therapy of generalized pruritus when general measures are not effective, systemic drugs are contraindicated, and/or as addition to causative or systemic therapy. Topical anti-itch preparations should be prescribed after the diagnosis is made and used as the first-choice treatment together with general measures.     

Structural and sequence variants in patients with Silver‐Russell syndrome or similar features—Curation of a disease database

Silver‐Russell syndrome (SRS) is a clinically and molecularly heterogeneous disorder involving prenatal and postnatal growth retardation, and the term SRS‐like is broadly used to describe individuals with clinical features resembling SRS. The main molecular subgroups are loss of methylation of the distal imprinting control region (H19/IGF2:IG‐DMR) on 11p15.5 (50%) and maternal uniparental disomy of chromosome 7 (5%–10%). Through a comprehensive literature search, we identified 91 patients/families with various structural and small sequence variants, which were suggested as additional molecular defects leading to SRS/SRS‐like phenotypes. However, the molecular and phenotypic data of these patients were not standardized and therefore not comparable, rendering difficulties in phenotype–genotype comparisons. To overcome this challenge, we curated a disease database including (epi)genetic phenotypic data of these patients. The clinical features are scored according to the Netchine‐Harbison clinical scoring system (NH‐CSS), which has recently been accepted as standard by consensus. The structural and sequence variations are reviewed and where necessary redescribed according to recent recommendations. Our study provides a framework for both research and diagnostic purposes through facilitating a standardized comparison of (epi)genotypes with phenotypes of patients with structural/sequence variants.     

Anti‐biofilm Effects of Honey Against Wound Pathogens Proteus mirabilis and Enterobacter cloacae

Biofilm growth and its persistence within wounds have recently been suggested as contributing factors to impaired healing. The goal of this study was to investigate the anti‐biofilm effects of several honey samples of different botanical origin, including manuka honey against Proteus mirabilis and Enterobacter cloacae wound isolates. Quantification of biofilm formation was carried out using a microtiter plate assay. All honeys at a sub‐inhibitory concentration of 10% (w/v) significantly reduced the biofilm development of both isolates. Similarly, at a concentration of 50% (w/v), each of the honeys caused significant partial detachment of Pr. mirabilis biofilm after 24 h. On the other hand, no honey was able to significantly detach Ent. cloacae biofilm. In addition, treatment of Ent. cloacae and Pr. mirabilis biofilms with all honeys resulted in a significant decrease in colony‐forming units per well values in a range of 0.35–1.16 and 1.2–7.5 log units, respectively. Of the tested honeys, manuka honey possessed the most potent anti‐biofilm properties. Furthermore, methylglyoxal, an antibacterial compound of manuka honey, was shown to be responsible for killing biofilm‐embedded wound bacteria. These findings suggest that manuka honey could be used as a potential therapy for the treatment of wounds containing Pr. mirabilis or Ent. cloacae. Copyright © 2013 John Wiley & Sons, Ltd.