Microglia are resident macrophage-like cells in the central nervous system (CNS) and cause innate immune responses via the LPS receptors, Toll-like receptor (TLR) 4 and CD14, in a variety of neuroinflammatory disorders including bacterial infection, Alzheimer’s disease, and amyotrophic lateral sclerosis. Granulocyte macrophage-colony stimulating factor (GM-CSF) activates microglia and induces inflammatory responses via binding to GM-CSF receptor complex composed of two different subunit GM-CSF receptor α (GM-CSFRα) and common β chain (βc). GM-CSF has been shown to be associated with neuroinflammatory responses in multiple sclerosis and Alzheimer’s disease. However, the mechanisms how GM-CSF promotes neuroinflammation still remain unclear.Microglia were stimulated with 20 ng/ml GM-CSF and the levels of TLR4 and CD14 expression were evaluated by RT-PCR and flowcytometry. LPS binding was analyzed by flowcytometry. GM-CSF receptor complex was analyzed by immunocytechemistry. The levels of IL-1β, IL-6 and TNF-α in culture supernatant of GM-CSF-stimulated microglia and NF-κB nuclear translocation were determined by ELISA. Production of nitric oxide (NO) was measured by the Griess method. The levels of p-ERK1/2, ERK1/2, p-p38 and p38 were assessed by Western blotting. Statistically significant differences between experimental groups were determined by one-way ANOVA followed by Tukey test for multiple comparisons.GM-CSF receptor complex was expressed in microglia. GM-CSF enhanced TLR4 and CD14 expressions in microglia and subsequent LPS-binding to the cell surface. In addition, GM-CSF priming increased LPS-induced NF-κB nuclear translocation and production of IL-1β, IL-6, TNF-α and NO by microglia. GM-CSF upregulated the levels of p-ERK1/2 and p-p38, suggesting that induction of TLR4 and CD14 expression by GM-CSF was mediated through ERK1/2 and p38, respectively.These results suggest that GM-CSF upregulates TLR4 and CD14 expression in microglia through ERK1/2 and p38, respectively, and thus promotes the LPS receptor-mediated inflammation in the CNS. Candida albicans, and Cryptococcus neoformans, and initiate an inflammatory response [Microglia are resident macrophage-like cells in the central nervous system (CNS). They become activated in response to bacterial infection including gram-negative bacteria, response ,2. The presponse ,2. TLRs response . TLR4 anresponse ,4 and thresponse , via theresponse .It is reported that TLR4 on microglia recognizes fibrillar Aβ . ActivatGranulocyte macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine secreted by a wide variety of cells including endothelial cells, monocytes, astrocytes, and T cells -17. GM-CGM-CSF has been shown to be upregulated in various neurological disorders like AD, vascular dementia, multiple sclerosis (MS) ,24. IndeHere, we show that the GM-CSF receptor complex, GM-CSFRα and GM-CSFRβ, is strongly expressed in microglia. GM-CSF enhances TLR4 and CD14 expression in microglia, leading to increase of LPS-induced NF-κB nuclear translocation and increased LPS-mediated IL-6, TNF-α, and NO production. GM-CSF-induced expression of TLR4 and CD14 was mediated through ERK1/2 and p38, respectively. These results suggest that GM-CSF promotes the LPS receptor-mediated neuroinflammation via upregulating TLR4 and CD14 expression in microglia.Recombinant mouse GM-CSF was obtained from R&D systems . LPS was obtained from Sigma-Aldrich . Wortmannin (PI3K inhibitor), U0126 (MEK1/2 inhibitor), SB203580 (p38 inhibitor), SP600125  inhibitor), and NF-κB inhibitor SN50 were obtained from Calbiochem . Anti-phospho-p44/42 MAPK (p-ERK1/2), anti-p42/44 MAPK antibodies , anti-phospho-p38 and anti-p38 were obtained from Cell Signaling Technology . The other antibodies used were as follows: anti-TLR4 , anti-CD14 , anti-CD11b , anti-GFAP , anti-Iba1 , anti-MAP2 , anti-GM-CSFRα , and anti-NeuN (Merck Millipore).All animal experiments were conducted under protocols that were approved by the Animal Experiment Committee of Nagoya University. All primary cultures were prepared from C57BL/6 mice .Microglia were isolated from primary mixed glial cell cultures prepared from newborn mice on day 14 using the shaking-off method as previously described . The purPrimary neuronal cultures were prepared from the cortices of embryos at embryonic day 17 (E17) as described previously . BrieflyPrimary astrocytes were isolated as previously described . Briefly5 cells/well. After stimulation with varying concentrations of LPS for 48 h, the total cellular RNA was extracted using the RNeasy Mini Kit . cDNA was synthesized from total cellular RNA that was denatured for 5 minutes at 65°C, followed by reverse transcription reaction using the SuperScript II . The cDNA served as a template to amplify genes in RT-PCRs with TaqMan Gene Expression assays (Applied Biosystems), Universal PCR Master Mix (Applied Biosystems), and Rotor-Gene Q (Qiagen). Expression levels of target genes were calculated using a comparative method and normalized to GAPDH expression levels as previously described [GAPDH, Mm99999915_g1; NOS2, Mm00440502_m1; CD14, Mm00438094_m1; and TLR4, Mm00445273_m1.For quantitative PCR, microglia were treated with GM-CSF (20 ng/ml) for varying times (6 to 72 h). Similarly, microglia were treated with GM-CSF (20 ng/ml) for 48 h or left untreated in RepCell® , in which cells are adherent at 37°C but detach from the plate at 20 to 25°C. Then the cells were harvested, washed twice and seeded on 24-well plates at a density of 1 × 10escribed . The fol′–ACTCACGGGAAATTCAACG–3′; GAPDH antisense, 5′–CCCTGTTGCTGTAGCCGTA–3′; GM-CSFRα sense, 5′–TGGCGAACGACTTGTCACTGCT–3′; GM-CSFRα antisense, 5′–GCACCTTGACCTTGTGACCT–3′; GM-CSFRβ sense, 5′–TGTTCCAGGATGGAGGTAAA–3′; GM-CSFRβ an-tisense, 5′–CCCACACTGCACATCCATAG–3′.For semi-quantitative PCR, the total cellular RNA from astrocytes, neurons and microglia were extracted using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from total cellular RNA as described above with SuperScript II (Life Technologies) and semiquantitative PCR was performed using Ampli Taq DNA polymerase (Applied Biosystems) as previously described  using thFCM was done as previously described . BrieflyImmunocytochemistry was done as previously described . Microgl5 cells/well in 24 well plates. Then the cells were treated with varying concentrations of LPS (0.01 to 1 μg/ml) for 48 h. Supernatants were collected and NO production was determined by the Griess reaction as previously described [Microglia were treated with GM-CSF for 48 h or left untreated in RepCell. Then the cells were harvested, washed twice and seeded at a density of 1 × 10escribed . The lev7 cells/well and treated with LPS (1 μg/ml) for 0 to 30 minutes. Nuclear fractions from the cells were separated and the levels of NF-κB in the nuclear fractions were analyzed using the NF-κB/p65-active ELISA kit  according to the manufacturer’s instructions.Microglia primed with GM-CSF for 48 h or left untreated were seeded on 6-well plates at 1 × 10Microglia were treated with GM-CSF (20 ng/ml) for varying times (15 to 120 minutes). After washing with PBS, cells were lysed with TNES buffer  containing phosphatase (Sigma-Aldrich) and protease inhibitor ; 50 μg of protein from the total lysate was assayed for phosphorylated ERK1/2, phosphorylated p38, total ERK and total p38 by western blotting as described previously .P-values less than 0.05 were considered statistically significant.Statistically significant differences between experimental groups were determined by one-way analysis of variance (ANOVA) followed by Tukey test for multiple comparisons. Statistical analysis was performed using the software program Prism 4.0 . We first assessed the basal expression of GM-CSFRα and GM-CSFRβ mRNA in microglia, astrocytes and neurons. Microglia expressed both GM-CSFRα and GM-CSFRβ mRNA Figure . The expWe next assessed expression of the LPS receptor, TLR4, and CD14 in microglia. GM-CSF time-dependently increased expression of TLR4 and CD14 mRNA, which peaked at 48 h and remained stabilized until 72 h in microglia Figure . FCM als5 cells/well in 24-well plates, and further stimulated with varying doses of LPS (0.01 to 1 μg/ml) for 48 h. GM-CSF priming increased LPS-induced expression of IL-1β, IL-6, TNF-α and NOS2 mRNA as compared to unprimed microglia (Figure We then examined whether GM-CSF affects LPS-mediated function in microglia. We pretreated microglia with GM-CSF for 48 h, then the cells were harvested, washed, seeded at a density of 1 × 10a Figure . Similara Figure .NF-κB plays a critical role in the production of various proinflammatory molecules, including IL-6 and TNF-α, by activation of TLR4 [GM-CSF has been shown to activate JAK2, PI3K, ERK1/2, and p38 in microglia -22. We eHere we have shown that GM-CSFR complex is expressed by microglia but not astrocytes and cortical neurons in the CNS, as no immunoreactivity for GM-CSFRα, which is required for binding GM-CSF, was found in GFAP-positive astrocytes or in MAP2-positive cortical neurons. In contrast to our finding, previous reports suggested expression of GM-CSFRα in astrocytes and neurons -35. HoweHelicobacter pylori LPS upregulate TLR4 expression via ERK1/2 in the gastric epithelial cell line, MKN28, and GM-CSF reportedly upregulate TLR4 expression via ERK1/2 in human blood monocytes respectively [We showed that GM-CSF increased both mRNA and surface expression of TLR4 and CD14 via activation of ERK1/2 and p38, respectively, and pretreatment with U0126, SB203580, or both, indeed inhibit LPS-induced production of proinflammatory mediators in the microglia incubated with GM-CSF. In the peripheral immune system, it has been shown that peritoneal macrophages from GM-CSF knockout mice are hyposensitive to LPS , and GM-ectively ,40. GM-Cectively ,40,41. Sectively . HoweverTLR4 is required for the initiation of innate immune responses to LPS ,6. CD14 GM-CSF increases surface expression of TLR4 and CD14 in microglia, which is mediated by ERK1/2 and p38, respectively. Accordingly, GM-CSF upregulates LPS-induced IL-1β, IL-6, TNF-α, and NO production and may promote neuroinflammation.AD: Alzheimer’s disease; ALS: Amyotrophic lateral sclerosis; ANOVA: One-way analysis of variance; βc: Common β chain; CNS: Central nervous system; ERK: Extracellular signal-regulated kinase; FACS: Fluorescence-activated cell sorter; FCM: Flow cytometry; GFAP: Glial fibrillary acidic protein; GM-CSF: Granulocyte macrophage-colony stimulating factor; IL: Interleukin; NOS2: Nitric oxide synthase 2; JAK: Janus kinase; LPS: Lipopolysaccharide; MAPK: Mitogen-activated protein kinase; mSOD: Mutations in superoxide dismutase; MS: Multiple sclerosis; NF-kB: Nuclear factor-κB; NO: Nitric oxide; PBS: Phosphate-buffered saline; PI3K: Phosphatidylinositol-3 kinase; PCR: Polymerase chain reaction; TLR: Toll-like receptor; TNF: Tumor necrosis factor.The authors declare that they have no competing interests.BP performed the experiments and helped draft the manuscript. YD, JK, and NM performed the cell culture and were involved in the conception of study. TM and TH helped with the preparation of the manuscript. YS was involved in the conception and design of the study and helped draft the manuscript. AS was involved in the conception and design of the study and helped draft the manuscript. All authors read and approved the final manuscript.


Febrile neutropenia (FN) occurs following myelosuppressive chemotherapy and is associated with morbidity, mortality, costs, and chemotherapy reductions and delays. Granulocyte colony-stimulating factors (G-CSFs) stimulate neutrophil production and may reduce FN incidence when given prophylactically following chemotherapy.A systematic review and meta-analysis assessed the effectiveness of G-CSFs  in reducing FN incidence in adults undergoing chemotherapy for solid tumours or lymphoma. G-CSFs were compared with no primary G-CSF prophylaxis and with one another. Nine databases were searched in December 2009. Meta-analysis used a random effects model due to heterogeneity.Twenty studies compared primary G-CSF prophylaxis with no primary G-CSF prophylaxis: five studies of pegfilgrastim; ten of filgrastim; and five of lenograstim. All three G-CSFs significantly reduced FN incidence, with relative risks of 0.30 (95% CI: 0.14 to 0.65) for pegfilgrastim, 0.57 (95% CI: 0.48 to 0.69) for filgrastim, and 0.62 (95% CI: 0.44 to 0.88) for lenograstim. Overall, the relative risk of FN for any primary G-CSF prophylaxis versus no primary G-CSF prophylaxis was 0.51 (95% CI: 0.41 to 0.62). In terms of comparisons between different G-CSFs, five studies compared pegfilgrastim with filgrastim. FN incidence was significantly lower for pegfilgrastim than filgrastim, with a relative risk of 0.66 (95% CI: 0.44 to 0.98).Primary prophylaxis with G-CSFs significantly reduces FN incidence in adults undergoing chemotherapy for solid tumours or lymphoma. Pegfilgrastim reduces FN incidence to a significantly greater extent than filgrastim. Febrile neutropenia (FN) is defined as neutropenia with fever, usually indicating infection, and is associated with substantial morbidity, mortality, and costs [Neutropenia is the major dose-limiting toxicity of many chemotherapy regimens. Grade 3 and grade 4 neutropenia are defined as a neutrophil count < 1.0 × 10nd costs ,3. In adRecombinant human granulocyte colony-stimulating factors (G-CSFs) stimulate production of mature, functional neutrophils . G-CSFs This paper reports a systematic review and meta-analysis of the effect of primary G-CSF prophylaxis  on incidence of FN. The effect of each G-CSF is assessed in comparison with no primary G-CSF prophylaxis and in comparison with other G-CSFs.The systematic review followed the recommendations in the PRISMA statement ,17. A syStudies were considered suitable for inclusion if they assessed primary G-CSF prophylaxis  administered 1-3 days after the completion of chemotherapy, versus a different G-CSF or versus no primary G-CSF prophylaxis. Studies were only included if they reported incidence of FN. For consistency with the two existing systematic reviews, ,19 only The outcome measure assessed in this review was the incidence of FN over all cycles of chemotherapy within each study. FN was chosen as a key clinical outcome due to its direct bearing on morbidity, mortality and hospitalisation rates, and also because this review was undertaken alongside the development of an economic model which utilised FN rate as a key parameter.Data was extracted by two reviewers using a form developed for this review and any discrepancies were resolved through discussion.2 statistic, which describes the percentage of the variability in effect estimates that is due to heterogeneity rather than sampling error (chance) [Meta-analyses were undertaken to compare the effectiveness of G-CSFs versus no prophylaxis and versus each other for the reduction of FN. Analyses were undertaken using RevMan software . Results for each comparison were presented as a pooled relative risk and 95% CIs. Although clinical and statistical heterogeneity existed between studies, there was insufficient data on individual populations to facilitate separate analyses. Therefore, for consistency with existing reviews, all studies were included in the analysis, and a random effects model was used. Heterogeneity was presented using the I(chance) .The flow chart for study inclusion is shown in Figure A previous systematic review of prophylactic G-CSF use  includedThere was heterogeneity among trials of all three G-CSFs in terms of cancer type, patient age, chemotherapy regimen, number of chemotherapy cycles and cycle length Table . FilgrasThe relative risks of FN incidence are shown in Figure 2 statistic, which ranged from 50-76%; this is likely to reflect the variations between studies in factors such as cancer type, patient age, chemotherapy regimen, number of chemotherapy cycles and cycle length. Individual studies differed on too many variables for formal sub-analyses to be meaningful. However, Figures There was a relatively high level of statistical heterogeneity in the analyses as shown by the IIn terms of comparisons between different G-CSFs, the relative risk of FN for pegfilgrastim versus filgrastim was 0.66 (95% CI: 0.44 to 0.98). There were no head-to-head trials comparing lenograstim to either of the other two G-CSFs.Our systematic review and meta-analyses confirm and strengthen previous evidence that primary prophylaxis with each of the three G-CSFs is effective in reducing the risk of FN following chemotherapy. In particular, our systematic review identified 4 further RCTs of pegfilgrastim vs. no primary G-CSF, -24 whereThis review also strengthens the evidence base regarding the comparative effectiveness of the three G-CSFs; in particular, comparison of the "once-per-cycle" G-CSF pegfilgrastim versus the "once-daily" G-CSF filgrastim. Meta-analysis of five RCTs indicated that FN incidence was significantly lower following primary prophylaxis with pegfilgrastim than with filgrastim. This is consistent with the fact that the reduction in FN risk for pegfilgrastim versus no primary G-CSF was greater than the reduction observed for filgrastim versus no primary G-CSF.As discussed in previous reviews, ,19 thereThis systematic review and meta-analysis demonstrate that primary G-CSF prophylaxis with pegfilgrastim, filgrastim and lenograstim is effective in reducing the risk of FN in adults undergoing chemotherapy for solid tumours or lymphoma. In addition, although heterogeneity existed between studies, a meta-analysis suggests that pegfilgrastim reduces the risk of FN to a greater extent than filgrastim.ANC: Absolute neutrophil count; ASCO: American Society of Clinical Oncology; DARE: Database of Abstracts of Reviews of Effects; EORTC: European Organisation for Research and Treatment of Cancer; FN: Febrile neutropenia; G-CSF: Granulocyte colony-stimulating factor; NCCN: National Comprehensive Cancer Network; NHS-EED: NHS Economic Evaluation Database; RCT: Randomised controlled trial.The research underlying this paper was funded by Amgen Ltd, and a research grant from Amgen (EUROPE) GmbH was provided to support the production of the manuscript. Amgen staff reviewed and made suggested edits to the manuscript, but final content, authorship and right to publication remained with the research team.KC undertook the systematic review and drafted the manuscript. JM undertook the statistical analyses. SW contributed to study selection and interpretation. MS contributed to study selection and interpretation and to the statistical analyses. RA participated in the design and coordination of the study and contributed to the statistical analyses. All authors read and approved the final manuscript.1 Granulocyte colony-stimulating factor/2 Granulocyte colony-stimulating factor, recombinant/3 Colony-stimulating factors, recombinant/4 Filgrastim/5 G-CSF$6 granulocyte colony-stimulating factor$7 filgrastim8 Neupogen9 pegfilgrastim10 Neulasta11 lenograstim12 Granocyte13 Euprotin14 r-metHuG-CSF15 SD-0116 PEG-rmetHuG-CSF17 XM0218 Ratiograstim19 or/1-1820 randomized controlled trial.pt.21 controlled clinical trial.pt.22 randomized controlled trial/23 random allocation/24 double blind method/25 single blind method/26 clinical trial.pt.27 exp clinical trial/28 .ti,ab.29 ((singl$ or doubl$ or trebl$ or tripl$) adj25 (blind$ or mask$)).ti,ab.30 placebos/31 placebos.ti,ab.32 random.ti,ab.33 research design/34 randomised.ti,ab35 randomized.ti,ab36 or/20-3537 19 and 36The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/404/prepub


Azotobacter chroococcum is a widespread free-living soil bacterium within the genus of Azotobacter known for assimilation of atmospheric nitrogen and subsequent conversion into nitrogenous compounds, which henceforth enrich the nitrogen content of soils. A. chroococcum SBUG 1484, isolated from composted earth, exhibits phenol oxidase (PO) activity when growing under nitrogen-fixing conditions. In the present study we provide incipient analysis of the crude PO activity expressed by A. chroococcum SBUG 1484 within comparative analysis to fungal crude PO from the white-rot fungus Pycnoporus cinnabarinus SBUG-M 1044 and tyrosinase (PPO) from the mushroom Agaricus bisporus in an attempt to reveal desirable properties for exploitation with future recombinant expression of this enzyme. Catalytic activity increased with pre-incubation at 35°C; however 70% of activity remained after pre-treatment at 50°C. Native A. chroococcum crude PO exhibited not only strong preference for 2,6-dimethoxyphenol, but also towards related methoxy-activated substrates as well as substituted ortho-benzenediols from over 40 substrates tested. Presence of CuSO4 enhanced crude phenol oxidase activity up to 30%, whereas NaN3 (0.1 mM) was identified as the most inhibiting substance of all inhibitors tested. Lowest inhibition of crude PO activity occurred after 60 minutes of incubation in presence of 15% methanol and ethanol with 63% and 77% remaining activities respectively, and presence of DMSO even led to increasing oxidizing activities. Substrate scope and inhibitor spectrum strongly differentiated A. chroococcum PO activity comprised in crude extracts from those of PPO and confirmed distinct similarities to fungal PO. Azospirillum lipoferum which lives, comparable to several soil fungi, in association with the plant rhizosphere and promotes plant growth. This bacterial PO was determined to be expressed in combination with physiological processes like cell pigmentation and the activation of phenolic plant ingredients. Within our previous studies, nitrogen-fixing cultures of the non-symbiotic Azotobacter chroococcum SBUG 1484, isolated from composted earth, exhibited PO activity when growing with nutritional deficiencies, especially depletion of exogenous nitrogen sources -1-propanesulfonate (CHAPS)) as well as combinations  of each were added to a defined volume of particulate cell debris redissolved in NAc buffer with increasing concentration . When combining detergents, the concentration of CHAPS and guanidine hydrochloride was always set to 5 mM, with increase in molarities of the detergents Triton X-100 and Sarkosyl ranging from 5-40 mM tested. To optimize solubilisation and protein release, lysates were incubated at 30°C for 30 min followed by centrifugation  and subsequently, equal volumes of supernatants tested for remaining 2,6-DMP oxidizing activity.Solubilisation experiments with detergents, chelating agents and chaotropic reagents were used to anticipate interaction of P. cinnabarinus SBUG-M 1044 was cultivated for production of the ligninolytic enzyme laccase (PO) as described previously [P. cinnabarinus SBUG-M 1044 produced an extracellular phenol oxidase with an activity of around 0.5 U mL-1. After several purification steps including removal of secondary metabolites using DEAE-Sephacel , the eluted enzyme fraction contained crude phenol oxidase which was used for following experiments.The fungus eviously . Under tA. chroococcum SBUG 1484 crude phenol oxidase (with a specific activity of 1.18 U mg-1 protein) or P. cinnabarinus SBUG-M 1044 crude PO (1.53 U mL-1). Examinations with tyrosinase (PPO) were equally performed in sodium phosphate buffer  containing 20 μL of a enzyme stock solution , with monitoring enzyme activity after addition of 5 mM catechol  at 25°C. In standard assays, initial rates were determined with individual time-ranges for enzyme preparations . 1 Unit of activity is defined as the amount of enzyme that catalyzes the conversion of 1 μmol mL-1 min-1 substrate at 25°C.Standard assays were conducted spectrophotometrically  in a total volume of 1 mL containing NAc buffer  supplemented with 2,6-DMP , and reactions initiated by adding either A. chroococcum SBUG 1484 PO, PO-containing crude extracts were pre-incubated for 30 min in 100 mM NAc buffer (pH 5) at temperatures ranging from 25-55°C, and the remaining activity monitored with ABTS (5 mM) as well as 2,6-DMP (5 mM) at 25°C.For determination of optimum pH for catalytic activity of the bacterial crude PO, oxidation of ABTS  due to change in absorbance at various pH (pH 1 - 7.5) and in different buffers  was monitored over a time range of 3 min. For examination of thermal stability of A. chroococcum and P. cinnabarinus crude PO, whereas tyrosinase (PPO) from A. bisporus was monitored using 5 mM catechol.Influence of metal ions and sensitivity to specific organic and inorganic enzyme inhibitors were examined with concentrations from 0.1 to 6 mM. The substrate used for testing of enzyme activity in the presence of repressors and activators was 2,6-DMP (5 mM) for A. chroococcum SBUG 1484 PO comprised in crude extracts, P. cinnabarinus crude PO and A. bisporus PPO was accomplished using seven different groups of model compounds applied for determining laccase and tyrosinase activity. All tested compounds were prepared as 50 mM stock solutions in bidestilled water and examined in 5 mM concentration for enzymatic conversion at wavelengths obtained from literature or deduced from related structures. Substrates were added to initiate the reaction, and the ΔA values of enzymatic oxidation determined spectrophotometrically at various time ranges that were equal for each of the analyzed enzymes with the same substrate and reveal the greatest initial rate of enzymatic oxidation. All experiments were performed with a four-fold repetition to ensure the validity of the results.A comparative analysis of substrate scope for A. chroococcum SBUG 1484, native PAGE using a non-denatured protein molecular weight marker kit was conducted. After electrophoresis, proteins were immediately fixed using Coomassie brilliant blue R-25 (5 min), and then shortly washed with distilled water before transferring into 100 mM NAc buffer (pH 5) containing 2,6-DMP for activity staining. Procedures described for characterization of molecular weights in polyacrylamide gels were accomplished according to [A. chroococcum SBUG 1484 PO was assessed on the basis of its electrophoretic mobility Rf in comparison to that of the used marker proteins.For non-denaturing poylacrylamide gel electrophoresis (native PAGE), resolving gels with acrylamide concentrations ranging from 10, 9, 8, 7, 6, 5.5, 5 and 4.5% were used in combination with a 4.5% stacking gel. Samples were mixed in a 2:1 ratio (v/v) with sample buffer (20% glycerol (w/v), 0.0025% bromophenol blue in destilled water) and electrophoresis performed at 20 mA for 2 h in 0.05 M Tris-HCL/0.38 M glycine electrode buffer (pH 8.3). For determination of the relative molecular weight of the herein described bacterial PO from the strain rding to  and the para-phenylenediamine. Gels were incubated at 37°C until active proteins became visible. Activity stained protein bands were cut into small slices and added to 100 μL of 100 mM NAc buffer (pH 5). Release of proteins from gel slices was performed by sonication for 2 min. Afterwards, 50 μL of a 2-mercaptoethanol-containing sample buffer was transferred to these samples with heating at 95°C for 15 min. Samples were then analysed by standard SDS-PAGE. Staining of these gels was conducted using the PageSilver™ silver-staining kit .Semi-denatured SDS-PAGE was performed as described by  with mod®-Mark BICOLOR marker  and a low molecular weight range marker .The molecular masses of proteins from semi-denatured and SDS-PAGE were compared using a pre-stained Rotiortho-vanillin were obtained from Acros Organics ; dopamine hydrochloride was purchased from Alfa Aesar® . Eugenol was acquired from ABCR  and 3-isopropylcatechol from ChemService, Inc. . Lyophilized tyrosinase (EC 1.14.18.1) from mushroom (A. bisporus) and a non-denatured protein molecular weight marker kit were purchased from Sigma-Aldrich .All chemicals were purchased from Sigma-Aldrich . Vanillin azine and A. chroococcum SBUG 1484 which undergo encystment and a melanogenic change in appearance [A. chroococcum crude extracts were not successful due to irreversible absorption of this enzyme to a wide range of columns and membranes under a variety of conditions. Therefore, instead of using mandatory purified enzyme preparation for comprehensive enzyme characterization studies, experiments were conducted with crude phenol oxidase aiming to provide an initial understanding of general characteristics of this particular PO.Phenol oxidase (PO) activity had previously been determined in nitrogen-fixing cells of the strain pearance . Examinapearance  and therWith regard to this, solubilisation experiments with different detergents were conducted in this current study to monitor the release of cell-associated bacterial phenol oxidase from particulate cell debris for determining effects on activity. For enzyme extraction, non-ionic, anionic and zwitterionic reagents as well as combinations of each were added at various concentrations (5 mM up to 40 mM). With reference to the nature and target location of the detergents used for solubilisation of proteins, integration of laccase in particulate cell fractions could be estimated and elevated release into crude extracts realized.g) was monitored by using the anionic detergent EDTA (5 mM)  Figure . A 20-30) Figure . In the The effect of pH on crude phenol oxidase activity was monitored with the canonical substrate ABTS in different buffers ranging in 0.5 steps from pH 1 to 7.5, with the highest activity observed around pH 4.5-5.0 in NAc buffer (100 mM) 2Pb were conducted with the substrate ABTS as they were observed to stimulate strong autoxidation of 2,6-DMP. A. chroococcum SBUG 1484 crude PO showed strongest sensitivity towards iron sulfate, where at a concentration of 0.1 mM, a 75% reduction in activity was observed. In contrast, trivalent iron ions (FeCl3) did not show the same inhibiting effects at the tested concentrations. Addition of 1 mM ZnSO4, H3BO3 and (CH3COO)2Pb revealed crude PO inhibition with 60% remaining activity, whereas the presence of MnSO4, CoCl2 and CaCl2 showed lower inhibitory effects with relative activities around 80%. In the presence of 0.1 mM Mg2+ and Cu2+ salts, positive effects on oxidizing activity could be monitored with increases in recovered activity of up to 10%. Remarkable enhancement of activity was achieved with the addition of 1 mM CuSO4 with an increased relative activity of 130%. Slight stimulation of crude PO activity was also determined with the addition of NiCl3 and NaMoO4.The effects of metal ions  on 2,6-DMP oxidizing capacity of the herein examined bacterial crude phenol oxidase are summarised in Table n-hexane, tetrahydrofuran (THF)), aprotic polar solvents (dimethylsulfoxide (DMSO), acteonitrile) and the protic solvents ethanol and methanol. All solvents were tested in 5 percent steps from 5% up to 35% solvent concentrations. Generally, when incubating crude enzyme preparations in presence of solvents, the inhibitory effects determined after 20 min were found to be compensated over incubation time of 60 min. Incubation of crude phenol oxidase in the presence of 15% solvent concentration revealed 69% (n-hexane) and 38% (THF) relative activities after 60 min  with aprotic unpolar solvents (3) was identified as the most inhibiting substance of all tested inhibitors towards A. chroococcum SBUG 1484 as well as P. cinnabarinus SBUG-M 1044 crude PO activity, whereas tyrosinase of A. bisporus still showed 65% relative activity in the presence of 2 mM NaN3 , whereby crude PO of P. cinnabarinus revealed a two-fold higher remaining activity. Addition of organic inhibitors such as the disulfide-bond cleaving agent dithiothreitol (DTE) and copper chelator diethyldithiocarbamic acid (DDC) revealed strong decreases in 2,6-DMP oxidizing activities of both PO preparations and gave complete inactivation of fungal PPO. The metal chelator tropolone also gave a strong negative influence on bacterial and fungal PO activity (12% and 31% remaining activity respectively), and was found to completely inhibit A. bisporus PPO  gave only negligible inhibitory effects on A. chroococcum PO (3% inhibition) as well as fungal PO (1% inhibition), whereas catechol oxidizing activity of A. bisporus PPO was reduced to 50% relative activity. The addition of SDS showed remarkable enhancement of 2,6-DMP oxidizing activity of A. chroococcum crude extracts and A. bisporus PPO up to 135%, whereas activity of P. cinnabarinus PO preparation was relative unaffected by the addition of 2 mM SDS  and tyrosinase (PPO) of mushroom . Within this, seven different groups of potential substrates were tested with group 1-substances structurally derived from guaiacol, including mono-methoxylated (and methylated) monophenols. Group 2 (dimethoxylated monophenols) consisted of substrates related to 2,6-DMP, which was examined as the most preferred aromatic substrate of A. chroococcum crude PO preparations. Groups 3 and 4 included ortho- and para-dihydroxylated compounds that were defined as typical catecholase substrates (3) and laccase substrates (4) respectively. In addition to melanin-precursors (group 5), meta-dihydroxylated alkylresorcinols (group 6) as well as exclusive PO and PPO substrates (group 7) were examined in this analysis. Comparison of the results obtained with crude bacterial and fungal phenol oxidase as well as fungal polyphenol oxidase are summarised in Table As the crude PO derived from entation , experimA. chroococcum crude PO towards methoxy-substituted monophenols was confirmed. Generally, model compounds that reveal a methoxy-substituent in position 2 could be described as A. chroococcum crude PO preferred substrates. Activity in crude extract preparations was enhanced when at position 6 of the aromatic ring a further methyl substituent was present (e.g. 2-methoxy-6-methyl phenol). With tyrosinase, none of the tested substrates except guaiacol were found to be oxidized. However, P. cinnabarinus PO crude preparation showed significant activity towards guaiacol and was also found to oxidize ortho-vanillin, 2-hydroxy-5-methoxy benzaldehyde and eugenol. This fungal crude PO was also active in vanillin azine oxidation, whereby PO-containing crude extracts of A. chroococcum failed in the oxidation of this particular substrate.With group 1 compounds, preference of P. cinnabarinus crude PO substrate spectrum as they were marginally converted by fungal tyrosinase with exception of 4-methyl-2,6-dimethoxyphenol. As with the group 1-compound vanillin azine, syringaldazine which is structurally deduced from 2,6-DMP was only oxidized by P. cinnabarinus PO preparations.With 2,6-DMP belonging to group 2 compounds (dimethoxylated monophenols) bacterial crude PO revealed the highest activity towards all tested substrates of this class followed by oxidation of 2,3-dimethoxyphenol. All tested substances were also found to belong to the Ortho-dihydroxylated substrates (group 3) were converted with highest rates using tyrosinase from mushroom, with activities typically between 2-8 fold higher than those of bacterial and fungal crude PO preparations. This confirms the distinctive feature of PPOs, especially tyrosinase, in exhibition of catecholase activity, and enhances the link to POs that were described for their ability in oxidizing ortho-dihydroxylated compounds. When comparing A. chroococcum PO-comprising crude extracts and P. cinnabarinus PO preparations, similar activities towards methyl-, methoxy- and alkyl substituted catechols were determined. Hydroquinoid substrates belonging to group 4 that were designated as substrates for explicit differentiation of PO activity from PPO activity were found to be oxidized by A. chroococcum as well as P. cinnabarinus crude PO. In this respect, tyrosinase was interestingly determined to be active in the oxidation of 1,4-dihydroxylated compounds such as methylhydroquinone, methoxyhydroquinone and tert-butylhydroquinone. Despite the aforementioned differences of POs and PPOs on the basis of activity towards 1,2- and 1,4-dihydroxylated substrates, with the examined model compounds of group 4 in conjunction with bacterial and fungal crude PO; we observed an extended time range in which measurements must be conducted due to lower activities when compared to ortho-dihydroxylated substrates. Generally, 2,6-dimethoxy-hydroquinone was the most preferred compound of A. chroococcum crude PO as well as P. cinnabarinus PO preparations, whereby other tested hydroquinoid compounds revealed two to seven-fold diminished activity for PO-containing extracts of A. chroococcum. With melanin-precursors and related compounds (group 5) A. chroococcum crude PO was able to activate the monophenolic compound 4-hydroxyindole as well as 3--L-alanine (L-DOPA) and dopamine with comparable activity, whereby fungal PO preparations revealed slightly lower values. In comparison to both POs, tyrosinase from mushroom exhibits up to seven times increased activity rates when using L-DOPA and dopamine as substrates. This observation confirms that these ortho-dihydroxylated compounds are appropriate substances for determining catecholase activity expressed by tyrosinases, whereby tyrosine is the sole substrate that was converted by mushroom tyrosinase during the herein described spectrophotometric examinations. As expected, activity towards tyrosine could not be monitored with A. chroococcum and P. cinnabarinus enzyme preparations.meta-dihydroxylated alkylresorcinols (group 6) were not found to be oxidized by any of the tested members belonging to the enzymatic classes of POs and PPOs.Resorcinol and related A. chroococcum PO were examined to show highest preference towards the non-phenolic substrate ABTS, with activity values comparable to those gained with 2,6-DMP. With P. cinnabarinus PO, slightly higher oxidation rates could be determined whereby this substrate gave the greatest activity values of all tested compound groups. With ABTS oxidation, tyrosinase from mushroom showed in comparison to the researched POs a three-fold reduced rate of conversion; however using pyrogallole it was found to express a two-fold greater oxidation rate than A. chroococcum crude PO. The aminated non-phenolic compound para-phenylenediamine was oxidized equally by A. chroococcum and P. cinnabarinus PO preparations. Activity towards para-cresol was only observable in assay mixtures with tyrosinase, confirming that PO of A. chroococcum exhibits no PPO characteristics by means of a cresolase activity.Referring to group 7 compounds, extracts containing A. chroococcum PO present in crude extracts could be constructed based upon activity observations: 2,6-DMP ≥ ABTS >ortho-dihydroxylated compounds ≥ mono methoxylated monophenols > dimethoxylated monophenols >para-dihydroxylated compounds. Lowest levels of activity with crude PO preparations from P. cinnabarinus SBUG-M 1044 were also observed when using para-dihydroxylated compounds as substrates.Based upon the screening of potential PO substrates in comparison to known PPO substrates, the following range of appropriate substrates for Azotobacter PO was accomplished indirectly in a non-denaturing system according to [f) facilitated creation of linear plots, whereby the molecular weight of the PO was calculated as 142 kDa (data not shown). This indicates that A. chroococcum PO acts as a homotrimer under non-denaturing conditions as already postulated previously [para-phenylenediamine), results were revealed to be much more complex than in turbid supernatant samples gained from crude extracts  mediated by metal ions, particularly calcium. Through addition of EDTA to cyst or membrane fractions, lipopolysaccharides which are usually stabilised by divalent cations such as Ca2+, Mn2+ and Mg2+ become more fluid, resulting in permeabilization and subsequent release of proteins due to the formation of EDTA-metal complexes [A. chroococcum crude PO to exist as a native active homotrimer (142 kDa), which is also active in monomeric and dimeric states. Accordingly, PO of Streptomyces psammoticus was found to act as a monomer with an apparent molecular weight of 45 kDa [Streptomyces griseus PO showed comparable results to A. chroococcum SBUG 1484 with an active native homotrimer comprised of 38 kDa monomers [P. cinnabarinus, Aspergillus niger and Aspergillus oryzae PO [A. chroococcum SBUG 1484 in cell-free extracts revealed a pH optimum of 4.5 when oxidizing the substrate ABTS. Together with a beneficial pre-incubation at 35°C and 25-30°C  respectively, parallels can be drawn to phenol oxidases and laccase-like enzymes especially expressed by other soil bacteria known for establishment of dormant stages during their life-cycle [E. coli [Daedalea quercina laccase [Solubilisation experiments with particulate cell debris were only effective with anionic EDTA and the non-ionic agent Triton X-100. EDTA was identified as being the most effectual agent in protein release, indicating that omplexes . Furtheromplexes . Herein f 45 kDa , wherebymonomers . Smallerryzae PO  up to 10fe-cycle . Crude Pfe-cycle . Copper [E. coli . This ca laccase .A. chroococcum SBUG 1484 was tested for activity in various organic solvents often used in biotransformation and biocatalysis. Generally, DMSO was determined as the most activity enhancing solvent presumably allowing the PO to retain its active confirmation [Azotobacter PO activity recovered in crude extracts was monitored through addition of 15% acetonitrile, even though it is an aprotic polar solvent like DMSO. The noticeable degree of solvent stability, which is displayed by A. chroococcum crude PO, raises potential future application of this enzyme in bioremedial and biotechnological processes of which organic solvents can often be found as a co-solvent.The application of enzyme-mediated synthesis of fine chemicals is accepted as an effective and preferred alternative towards chemical synthetic routes. Non-aqueous enzymology by means of medium-engineering has been proposed as alternative to common biotransformation reactions in industry , and witirmation , whereasP. cinnabarinus SBUG-M 1044 and PPO (tyrosinase) of A. bisporus. A clear preference of A. chroococcum SBUG 1484 PO-containing crude extracts was determined towards ortho-dihydroxylated compounds followed by mono-methoxylated monophenols, and dimethoxylated compounds . Diminished activity towards para-dihydroxylated compounds was somewhat surprising as POs are strictly differentiated from catecholases and tyrosinases due to an exclusive and pronounced oxidation of 1,4-dihydroxylated compounds [P. cinnabarinus SBUG-M 1044 showed comparable results to those obtained for A. chroocoocum crude phenol oxidase preparations. According to [para-dihydroxylated substances could be reasoned with low molar adsorption coefficients at visible regions and instability of the formed products, suggesting para-diphenols as non-appropriate substrates in spectrophotometric standard assays. Substrates belonging to seven different compound classes were equally tested in a 5 mM concentration , which in some instances may lead to substrate inhibition of the tested enzymes. The pronounced oxidative activity of A. chroococcum SBUG 1484 crude PO towards 2,6-DMP and ABTS, a feature shared with P. cinnabarinus SBUG-M 1044 preparations, clearly confirms laccase-type characteristics of the herein described Azotobacter phenol oxidase. Furthermore, the studied bacterial PO comprised in cell-free crude extracts exhibited no PPO activity towards oxidation of tyrosine and para-cresol, which was however clearly determined with A. bisporus PPO. Crude PO of A. chroococcum SBUG 1484 possesses 128% relative activity towards 2,6-DMP when compared to ABTS [S. psammoticus PO with 214% activity relative to that with ABTS. Despite the similarity between A. chroococcum SBUG 1484 and P. cinnabarinus SBUG-M 1044 crude PO preparations in 2,6-DMP oxidation, the examined prokaryotic PO differentiates from fungal PO due to an inability to oxidize vanillin azine and the typical laccase substrate syringaldazine. Both of these substrates reveal methoxy-substituents with an azine bridge at the C4-carbon atom of the aromatic ring. Structurally similar compounds, with methyl-, methylen or hydroxy-groups at the C4-position did not reduce activity of A. chroococcum crude PO, whereas substrates containing an acid or aldehyde residue as the terminal group were not oxidized. A. chroococcum SBUG 1484 as well as P. cinnabarinus SBUG-M 1044 PO preparations revealed no activity towards meta-dihydroxylated compounds, which has also been observed in many other POs with resorcinol derived substrates [ortho-diphenols the herein examined crude POs can be distinctly distinguished from PPO activity on the basis of decreased ΔA values within an extended time-range in which highest initial rates were obtained. However, comparing activity of A. chroococcum SBUG 1484 crude PO towards catechol-derivatives with other tested compounds, a high affinity towards substituted catechols was estimated. Laccase activity towards both, ortho- and para-dihydroxylated compounds has also been previously reported [In order to estimate the native ecological role of this prokaryotic enzyme, primary biochemical investigations were conducted in an attempt to explore substrate scope in comparison to fungal crude PO preparations of ompounds . Howeverrding to , results to ABTS . studiedbstrates . In casereported .A. chroococcum SBUG 1484 crude PO we have been able to describe remarkable differences in substrate specificity of prokaryotic POs, and also between prokaryotic and eukaryotic POs; and which may also enable reconsideration of previously designated bacterial and fungal PO activities. Studies on the substrate spectrum of A. chroococcums crude PO indicate that this enzyme could be useful in biotransformations involving mono- and dimethoxy-substituted phenols as well as ortho-diphenolic compounds.Through examining activity of Azotobacter is a well-studied soil bacterium interacting with plant rhizosphere and evoking improved plant growth due to cells nitrogen-fixing activity and production of plant growth regulators, there may exist possible physiological advantages when expressing PO activity, which is closely related in terms of the determined substrate preferences related to (methoxylated) plant phenolic compounds. As enzyme preparations of A. chroococcum revealed activity towards selected melanin-precursors, PO expression can be linked to synthesis of melanin and the concurrent consumption of oxygen during pigmentation of this prokaryote. Therefore, besides its assumed relevance in interaction with plants and compounds which can be derived there from; oxygen-requiring PO of A. chroococcum SBUG 1484 may play a contributory role in protection of oxygen-sensitive nitrogenase, besides a high inner respiratory rate, and which evokes an enhanced rate of oxygen consumption. A detailed kinetic analysis of this enzyme will be facilitated by future investigation into cloning and recombinant expression. Together with the knowledge gained herein as to the preferred substrates of this PO and the effects of various solvents and reagents, the application of this enzyme as a potential valuable biocatalyst is one step nearer.As The authors declare that they have no competing interests.A. chroococcum crude PO preparation towards oxidation of ABTSpH optimum of . Relative activities of A. chroococcum crude PO preparation in different buffers ranging from pH 1 to 7.5 monitored due to the oxidation of 1 mM ABTS. Substrate oxidation in pH ranging from 1 to 3 was examined using maleate buffer, whereas activities from pH 3.5 to 6 were monitored in sodium acetate buffer (100 mM) and pH 6 to 7.5 in 100 mM phosphate-citrate buffer.Click here for fileA. chroococcum PO comprised in crude extracts monitored with ABTS and 2,6-DMPThermal stability of . Thermal stability determined with ABTS (filled square) and 2,6-DMP (filled triangle), with samples pre-incubated for 30 minutes in NAc buffer  at temperatures ranging from 25-50°C. Error bars refer to standard deviation by means of four replicates.Click here for file


Anorexia nervosa commonly arises during adolescence and is associated with more than one medical morbidity. Abnormalities in brain structure (defined as “pseudoatrophy”) are common in adolescents with anorexia nervosa; however, their correlations with endocrinological profiles and clinical parameters are still unclear. In particular, no study has described the impact of BMI (body mass index) variations (speed and magnitude of weight loss) on cerebral trophism changes.Eleven adolescents with anorexia nervosa and 8 healthy controls underwent cerebral MRI (magnetic resonance imaging) examination to obtain global and partial volumes  and clinical evaluation. The Mann-Whitney U test was used to compare partial volumes and clinical variables between cases and controls. The Spearman non-parametric test was performed in order to explore correlations between the variables studied.The patients diagnosed with AN showed significantly increased cerebrospinal fluid (CSF) volumes and decreased total gray (GM) and white matter (WM) volumes. The degree of weight loss (deltaBMI) correlated inversely with the GM volume; the increase of CSF compartment correlated directly with the rapidity of weight loss (DeltaBMI/disease duration).This study suggests a correlation between cerebral alterations in AN and the speed and magnitude of weight loss, and outlines its importance for the therapeutic treatment. Anorexia nervosa (AN) is a psychiatric illness that commonly arises during adolescence. Among all the psychiatric disorders anorexia has the highest mortality rate and it is associated with severe medical morbidity . Regard 3 (triiodothyronine), whereas T4 (thyroxine) and TSH (thyroid stimulating hormone) are normal or slightly reduced [The typical hypothalamic amenorrhea is associated with low serum levels of gonadotropins and sexual hormones. Plasmatic levels of leptin, a peptide produced by fat store and involved in appetite-regulating mechanisms, are also reduced. GH (growth hormone) levels are often increased accompanied by low levels of IGF-1 (insulin-like growth factor) which suggests an acquired peripheral resistance to GH. An hypercortisolemia is common to almost all women with AN, but not associated to the typical Cushingoid features and often not suppressible after dexamethasone administration. Abnormalities of the thyroid axis are also relevant and characterized by low levels of T reduced .Previous neuroimaging studies on AN showed global gray (GM) and white matter (WM) reduction and an increase in cerebrospinal fluid volumes (CSF) ,7, while3[In literature, associations between brain alterations and hormonal profile changes as hypercortisolemia ,11,12 an3 are descWeight restoration tends to improve brain abnormalities in AN, but it is still not clear whether reversibility is complete ,10,15,16In literature associations between brain volumes and BMI in AN are known , anyway Eleven girls, aged 11–17, who fulfilled the DSM-IV-TR diagnostic criteria for AN were enrolled. They were all new patients of the Eating Disorders Unit of the Department of Child and Adolescent Neuropsychiatry, at the S. Gerardo Hospital in Monza, University of Milano Bicocca  from August 2008 to January 2010. Clinical variables such as age, body-mass-index (BMI), age of AN onset, deltaBMI , deltaBMI/disease duration (rapidity of BMI variation), the presence of primary/secondary amenorrhea were evaluated by means of a clinical interview. No patient was taking any medication. The Hollingshead 4-factor index was carried out as a measure of the socio-economical status (SES). A normal academic performance was reported for all the girls enrolled. Patients did not have any concomitant medical diseases (except for one girl affected by celiac disease) or psychiatric comorbidity and no prenatal/perinatal/postnatal cerebral suffering was reported.Eight age-matched girls without psychiatric disorders were also enrolled as controls.http://www.fmrib.ox.ac.uk/fsl/). For each MRI scan a binary mask of cerebral volume was obtained with the BET extraction tool (threshold fixed was 0.5) and then manually outlined.Each subject underwent a high-resolution T1-weighted volume MRI scan Acquisition consists of a set of adjacent axial images with a slice thickness of 1 mm and pixel size 0.94 _ 0.94 mm, using spoiled gradient echo sequence . All MRI data was acquired on the same scanner (1.5 T Achieva Philips) using the same parameters according to strictly standardized procedures. Total cerebral and intracranial volumes  were calculated using the FAST and BET extraction tools from the FSL package . The Mann-Whitney U test was used to compare partial volumes , and clinical variables between cases and controls. In the group of adolescents with anorexia nervosa, the Spearman non-parametric test was performed in order to explore correlations between the variables studied. The level of significance was set at p < 0.05. Statistical analysis was performed using the SPSS 19 package.The socio-demographic features and BMI of participants with AN and controls are described in Table 3, SD = 60592 in the AN group vs 689829 mm3, SD = 64033 in the control group; p < 0.005) and WM volumes , and higher CSF volumes than controls .Differences in WM, GM and CSF volumes, when comparing the two groups Table  were fouThe DeltaBMI was inversely correlated with the fraction of GM volume, which means that girls with AN with the greatest weight loss between the onset of the disease and the time at diagnosis had the greatest reduction of GM volume. Delta BMI also directly correlated with the enlargement of CSF volume. Moreover, a direct correlation was also observed between the CSF compartment volume and the fraction DeltaBMI/disease duration Table .To our knowledge, this is the first study conducted on a sample of young adolescents with AN in which correlations between cerebral volumes and speed and magnitude of weight loss are explored. A negative correlation between deltaBMI and the fraction of gray matter volume and a positive correlation between deltaBMI and the enlargement of CSF volume were observed. To the contrary, no correlations between cerebral volume parameters and BMI value were found , which mGirls with AN displayed significant reduced GM and WM volumes and increased CSF volumes, confirming data from the literature . DiffereOur study results outline the importance of an early diagnosis of the illness in order to limit also cerebral alterations. In fact, a precocious intervention and screening programs might be necessary in order to prevent major cerebral alterations of GM, WM and CSF. Concerning this problem, our sample of anorexic adolescents get the first access to our specialized unit with a mean disease duration of more than one year. More awareness is needed among pediatricians and neuropsychiatrists so that adolescents at risk can be promptly recommended a consult with a specialist. The main limitation of the present work was sample size that should be increased in future studies. However, our sample is comparable with other published studies on adolescents with AN. Furthermore our intent was to investigate a group of patients homogeneous for age, type of anorexia and illness severity .Anorexia nervosa is a psychiatric illness with a high prevalence and origin in adolescence.In adolescents with AN, the speed and magnitude of weight loss, and not only BMI, represent a possible indicator of disease severity. If confirmed by other studies, our results could provide additional support concerning the benefits of paediatricians’ and neuropsichiatrists’ increased awareness, early detection and treatment of patients with anorexia nervosa.3: Triiodothyronine; T4: Thyroxine; TSH: Thyroid stimulating hormone; DSM-IV-TR: Diagnostic and statistical manual of mental disorders, Fourth Edition, Text Revision; SES: Socio-economical status; SD: Standard deviation; SPSS: Statistical Package for Social Science.BMI: Body mass index; MRI: Magnetic resonance; CSF: Cerebrospinal fluid; GM: Gray matter; WM: White matter; DeltaBMI: Delta body mass index; AN: Anorexia nervosa; GH: Growth hormone; IGF-1: Insuline-like growth factor; TAuthors declare that they have no competing interests.MB and RN planned the study. MB, AR, VF and RN conducted patients' clinical evaluation. MG and SM conducted radiological evaluation. MB and SM conducted statistical analysis. All authors contributed to draft the manuscript, which they read and approved in the final version.


Despite the great morbidity and mortality that childhood bacterial meningitis (BM) is experiencing in Africa, diagnosis of BM in resource-limited contexts is still a challenge. Several algorithms and clinical predictors have been proposed to help physicians in decision-making but a lot of these markers used variables that are calculable only in well-equipped laboratories. Predictors or algorithm based on parameters that can be easily performed in basic laboratories can help significantly in BM diagnosis, even in resource-limited settings, rural hospitals or health centers.This retrospective study examined 145 cerebral-spinal fluid (CSF) specimens from children from 2 months to 14 years. CSF specimens were divided into two groups, according to the presence or not of a clinical diagnosis of BM. For each specimen, CSF aspect, CSF white blood cells (WBC) count, CSF glucose and protein concentration were analyzed and statistical analysis were performed. CSF WBC count ≥10/µl is no more a valuable predictor of BM. CSF protein concentration ≥50 mg/dl has a better sensitivity for BM diagnosis and when used with CSF glucose concentration ≤40 mg/dl, can help to diagnose correctly almost all the BM cases. An algorithm including CSF protein concentration, glucose concentration and WBC count has been proposed to rule out BM and to correctly diagnose it.In resource-limited health centers, the availability of a combination of easy-to-obtain parameters can significantly help physicians in BM diagnosis. The prompt identification of a BM case can be rapid treated or transferred to adequate structures and can modify the outcome in the patient. Haemophilus influenzae type b (Hib) vaccine, childhood bacterial meningitis (BM) remains one of the most important diseases responsible for infant mortality in developing countries Streptococcus pneumoniae and Neisseria meningitidisDespite the introduction of a In scientific literature several algorithms have been published: they use clinical and laboratory features to differentiate various forms of meningitis and to quickly diagnostic a BM. Various parameters have been investigated: haemoglobin, temperature, Glasgow coma score, Blantyre coma score, focal neurological signs, additional focus of infection, peripheral white blood cells (WBC) count, glycaemia, cerebral-spinal fluid (CSF) opening pressure, CSF WBC count, CSF polymorphs %, CSF protein concentration, CSF glucose, CSF-serum glucose ratio, positive bacterial culture and positive CSF latex agglutination. Unlucky none of these parameters has revealed an absolute clinical prediction of BM and the algorithms authors found have to been evaluated separately due to the context where studies were performed In this study we aim to assess how basic laboratory parameters of CSF analysis can aid BM diagnostic even in the absence of a bacterial culture. The parameters we evaluated included CSF WBC count, CSF glucose concentration, CSF protein concentration and Gram staining.The study was conducted at Divina Providencia Hospital, Luanda (Angola), settled within the poorest suburbs of the capital. The hospital is a 110-bed municipal hospital; the emergency unit receives daily 200 patients. Patients may arrive from other health centres or other hospitals but most children arrive spontaneously from the great capital area.The study was a nested case-control study of patients who underwent a lumbar puncture in the emergency unit to diagnose or rule out BM between January 1, 2009 and December 31, 2010. Lumbar puncture was performed for patients presenting impaired consciousness, convulsions, prostration, meningismus or bulging fontanel. Exclusion criteria included age <2 months or >14 years, second lumbar puncture within 14 days, clotted samples, CSF red blood cell count >10000/µl and positive malaria thick smear. Medical records of patients admitted in the study were reviewed to extract additional information to ensure that diagnosis or exclusion of BM was correct and to verify that exclusion and inclusion criteria were met. Written informed consent was obtained from all patients or from their relatives if patient was unable to provide consent. Ethical approval for this study was obtained by the Institutional Review Board at our institution.CSF from the lumbar puncture at the time of admission was analysed. Processing of CSF specimens followed standard hospital procedures Data recorded manually were entered in duplicate into secure databases created in Microsoft Excel and Access and analysed using SPSS version 15. Confidence intervals (CI) were calculated using standard normal distribution; sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and odds ratio (OR) were calculated for each predictive variable A total of 179 CSF specimens were collected from January 1, 2009 and December 31, 2010. 34 CSF specimens were excluded: 32 were from patients >14 years, one had a red blood cell count >10000/µl and one patient was admitted for a second lumbar puncture within 14 days. The remaining 145 CSF specimens were divided into two groups: control group (n = 87) and BM group (n = 58). Diagnosis of BM was confirmed according to medical chart information. Patients' characteristics are represented in All the children who underwent a lumbar puncture at Divina Providencia Hospital belonged to a low socioeconomic group, which represents 90% of Luanda population There were no significant differences in mean age between control group and BM group and the distribution of males and females into the two groups is almost equal. Case fatality ratio among children diagnosed with BM was 15.52% . When compared, the difference in CSF WBC count mean between control and BM group is statistically significant ; also the difference in CSF glucose and protein concentration means are statistically significant . For each parameter we examined, Using standard cut-off values Using a combined analysis we calculated the OR of a combination of two parameters: the OR of CSF WBC count ≥10/µl and glucose concentration ≤40 mg/dl is 5.83 ; the OR of CSF WBC count ≥10/µl and protein concentration ≥50 mg/dl is 11.59 ; the OR of CSF glucose concentration ≤40 mg/dl and protein concentration ≥50 mg/dl is 16.41 .The OR of all the three parameters for BM is 9.79 .We also calculated sensitivity, specificity, PPV and NPV for these parameters and their combinations . The NPVGram staining results were analyzed separately. Gram staining was only performed for 62 CSF specimens due to limitations in personnel. Gram-staining revealed a different percentage of Gram positive and Gram negative bacteria. In total 27 specimens resulted positive for Gram staining: 70.37% were identified as diplococcos Gram-positive while 26.63% resulted in coccos Gram-negative. Using a clinical diagnostic of BM as gold-standard, the sensitivity and specificity of Gram staining were respectively 69.44% and 92.31%; PPV and NPV were respectively 92.59% and 68.57%.Physicians are likely to suspect a BM in children when CSF WBC count is far from normal values. When no other clinical or biochemical factors consistent with BM are identified, these children are often hospitalized and treated empirically with antibiotics CSF protein concentration has a better OR (12.52) than CSF WBC count has. It also has the highest NPV within the examined parameters (88.71%). These results can demonstrate that CSF protein concentration lower than 50 mg/dl is likely to exclude a BM. Even with a sensitivity of 87.93%, the PPV of CSF protein concentration alone is poor (61.45%), so we discourage the use of CSF protein concentration as single marker to predict BM.When the parameters are examined together as predictors of BM, the best OR belongs to CSF glucose and protein concentration (16.41). This couple has also the best PPV of all the combinations we examined and it seems to be a good marker of BM in our cases. The sensitivity and the specificity of this couple are respectively 72.21% and 86.41%: when used together these two BM markers can aid to correctly identify BM in 72.21 of 100 cases. Its NPV (82.42) also demonstrates that in resource-limited settings the evaluation of only two parameters can aid significantly to rule out BM. When other BM-associated factors are also present, the co-presence of high protein concentration and low glucose concentration is a strong indicator of BM.In this study CSF protein concentration has the best sensitivity (87.93%), so we can start from here to construct an algorithm to be used in resource-limited contexts. The presence of CSF protein concentration higher than 50 mg/dl indicates a high probability of BM. Notwithstanding, 12.07% of BM cases occurred in CSF samples with protein concentration lower than 50 mg/dl. 86% of these cases had CSF WBC count higher than 10/µl whilst 50% of them had CSF glucose concentration lower than 40 mg/dl. CSF glucose concentration has a better sensitivity than CSF WBC count so we can add CSF glucose concentration to our algorithm to aid to identify possible CSF specimens with BM.In resource-limited settings we advice to use CSF protein concentration as initial predictor of BM, followed by CSF WBC count and CSF glucose concentration. This is in fact the sorting order of parameters that demonstrate the better OR to diagnose BM. If CSF protein concentration is lower than 50 mg/dl, we suggest examining CSF WBC count: if it is higher than 10/µl and/or if CSF glucose concentration is lower than 40 mg/dl, there is a strong suspect of BM. To evaluate the efficacy of the algorithm we proposed, we calculated its specificity and sensitivity for the 145 CSF specimens collected. The application of our algorithm in diagnosing BM resulted in the identification of 57 out of 58 CSF specimens of BM group. This means that our algorithm has a 98.28% sensibility. Our algorithm revealed also 96.55% specificity because it identified 3 out of 87 CSF specimens positive to BM diagnosis although they belong to the control group. These low percentages of false-negative and false-positive results demonstrate the fine accuracy our algorithm has when it is used to support the BM clinical diagnosis.We have also evaluated the possibility to include other parameters in our algorithm. The only marker that is easy-to-perform in resource-limited settings is CSF aspect. Due to the fact that CSF aspect evaluation is prone to inter-observer variability, we did not include this parameter in our algorithm. We are aware that other more accurate parameters as CSF-serum glucose ratio, positive bacterial culture and positive CSF latex agglutination could notably improve the accuracy of our algorithm, but these markers required a well-equipped laboratory and, consequently, are not applicable to rural African health centres.The limit of our study is also represented by the fact that CSF glucose and protein concentration can be altered by other factors other than BM. The most accurate way physicians have to exclude a BM is a negative bacterial culture; due to the fact that resource-limited settings do not possess facilities to perform a bacterial culture, our algorithm can not reach 100% specificity if compared to bacterial culture. CSF glucose level is lower than normal in BM, meningealcarcinoma and sometimes in intracranial haemorrhage. CSF protein level can increase in BM, brain tumors, diabetes, multiple sclerosis, syphilis and guillian-Barre syndrome. In African contexts little is known about epidemiology of these diseases and even littler is known about the correlation between these diseases and CSF parameters. For that reason we strongly suggest that BM diagnosis has always to be accompanied by clinical evaluation in order to exclude diseases that can be responsible for altered CSF parameters and to assess the reliability of BM.Streptococcus pneumoniae –a Gram-positive bacteria- is likely the most important pathogen responsible for BM in our sample population as it appeared in 70.37% within positive Gram-staining specimens; Neisseria meningitidis –a Gram-negative bacteria- is less present (26.63%). These findings are consistent with other results reported in Angola after the introduction of the Hib vaccine When a Gram staining is available it can help physicians optimize their decision making with respect to possible hospitalization and antibiotic treatment. In our study only 62 CSF specimens were analyzed for Gram staining. Gram staining resulted in a good specificity (92.31%) and a poor sensitivity (69.44%), reflected also by a low NPV (68.75%) and a good PPV (92.59%). These values reflect the fact that Gram staining could aid to diagnose BM, but the use of Gram staining alone can lead to the lost of positive BM specimens due to the high proportion of false-negative results. Our positive Gram staining specimens revealed a higher percentage of Gram-positive bacteria compared to Gram-negative bacteria. As it was not possible to perform a bacterial culture, we can only speculate on the identity of possible pathogens responsible for BM. In resource-limited settings, the availability of CSF parameters that can be calculated even in basic laboratories can aid physicians to diagnose or rule out BM. As previously suggested Accurate diagnosis and prompt treatment in African hospitals remain a challenge. We attempted to produce a simple diagnostic rule using basic laboratory tests, available in most health care facilities in Angola. Because it was retrospective in nature and because of the low number of CSF specimens we analyzed, our study has certain limitations that can be overwhelmed only if our findings and our algorithm are accompanied by clinical features. Our findings regard some easily measurable variables and they can be of value only in resource-limited countries. When interpretation of Gram staining results is doubtful and when bacterial culture is no available, we suggest using the easy algorithm we provide in order to diagnose correctly BM. Meningitis caused by other pathogens, as fungi or viruses, present chemical values that differ from the ones here described. Viral meningitis use to have a normal value of CSF glucose concentration We insert our study in a more comprehensive effort that other scientists are doing in Africa with the aim to improve quality and rapidity of BM diagnosis. Above the use of a valid diagnostic test and a predictive combination of CSF characteristics, encouraging parents to seek medical cares for their children promptly, transferring patients without delay and ruling out cerebral malaria are some of the tools at hand today to improve the prognosis of childhood bacterial meningitis in sub-Saharan Africa.


Although bone marrow transplantation (BMT) from an HLA identical sibling is considered as treatment of choice in pediatric patients with severe aplastic anemia (SAA), a significant number of them experience graft failure (GF) after BMT. We report a case of an 8-year-old male patient with SAA who presented with a complicated posttransplant course due to parvovirus B19 infection and GF. A subsequent attempt to support the graft by antithymocyte globulin (ATG) and a peripheral stem cell boost resulted in transitory autologous recovery of hematopoiesis followed by mixed chimerism, supported by donor lymphocyte infusions (DLIs) and finally graft rejection with relapse of SAA. Permanent complete chimerism was achieved by a second BMT. Dynamics of graft function, measured by a single nucleotide polymorphism (SNPs) analysis, are discussed. The diagnostic criteria for SAA include bone marrow hypocellularity to less than 25% and two of the following: absolute neutrophil count (ANC) <0.5 × 10First-line treatment of SAA in pediatric patients is bone marrow transplantation (BMT) from an HLA identical sibling. Alternatively, for patients lacking an HLA-matched related donor, immunosuppressive therapy with an ATG-based regimen is recommended. Although BMT is considered as treatment of choice, a significant number of patients experience early or late graft failure (GF) after BMT. Analysis of chimerism is an important tool for evaluation of graft function, early detection of GF, and identification of its possible causes in the posttransplant period. Single nucleotide polymorphism- (SNP-) based assays represent a novel rapid method for assessment of hematopoietic chimerism after allogeneic BMT .Quantitative analysis of donor chimerism from peripheral blood was performed by an RT-PCR, based on single nucleotide polymorphisms (SNPs) assessment. Informative SNPs are identified and used as a tool to distinguish between donor/recipient cells after BMT. All SNPs from the peripheral blood samples of donor and recipient were compared before BMT. In the following analysis at least 2 informative SNPs were selected. After BMT cell subpopulations were isolated from the recipient's peripheral blood sample, using Miltenyi immune magnetic method (MACS). The purity of isolated cell subtypes was assessed by a fluorescence-activated cell sorting (FACS) analysis. DNA was extracted from the basic material and the selected subpopulations and RT-PCR was performed for measuring the percentage of chimerism .A previously healthy 8-year-old boy was transferred to the Division of Pediatric Hematology and Oncology in Graz in February 2011 with a one-month history of pancytopenia preceded by a febrile episode. Clinical course, laboratory findings, and bone marrow aspiration were highly suggestive of idiopathic aplastic anemia. Prior to transfer the patient had received 3 erythrocytes and 7 platelet transfusions.9/L; reticulocytes, 18 × 109/L; platelets, 17 × 109/L. Parvovirus B 19 (PVB19) PCR was tested positive above detection limit in bone marrow. Bone marrow aspiration and biopsy confirmed marked hypocellularity without signs of malignant infiltration, fibrosis, or dysplastic changes. There was no evidence of chromosomal abnormalities associated with MDS. The patient fulfilled all criteria for SAA and was prepared for allogeneic bone marrow transplantation from his HLA-identical brother (BMT1).Physical examination revealed pallor and hematomas on the lower extremities. Blood cell counts were ANC, 0.24 × 108/kg nucleated cells with 4.1 × 106 CD34+/kg and 0.29 × 108 CD3+/kg was administered. After transplantation immunosuppression was started with cyclosporine A. WBC engraftment followed on day +25 with counts exceeding 1.0 × 109/L. Platelets were above 50 × 109/L after day +23. The last red blood cell transfusion was administered on day +26. The immunosuppressive therapy was discontinued 4 months after BMT1. In the follow-up period SNP analysis revealed mixed chimerism (The conditioning regimen consisted of cyclophosphamide (4 × 50 mg/kg/d) and rabbit ATG (3 × 20 mg/kg/d). A bone marrow suspension of 1100 mL containing 3.53 × 10himerism , phase 1himerism , phase 26/kg CD34+ cells. Prophylactic immunosuppressive therapy with mycophenolate mofetil was given for 17 days. No signs of GVHD were observed.Due to possible reactivation of parvovirus B19-infection and the presence of circular exanthema on the lower extremities, therapy with immunoglobulin was administered and an attempt to enhance the graft by a stem cell boost from peripheral blood of the donor was undertaken. The preparative regimen with horse ATG (40 mg/kg/d) for 4 days was well tolerated by the patient. The mononuclear cell suspension administered was T-cell depleted by MACS cell separation and consisted of a pure fraction of 10 × 109/L on day +14. Platelet counts continued to increase progressively. Chimerism analysis of donor granulocytes demonstrated slowly increasing donor DNA to a maximum of 93.98% in the immediate period after the PBSC boost and a subsequent decrease in the percentage of both donor-derived granulocytes and lymphocytes while peripheral blood counts were increasing. These findings were interpreted as an imminent loss of graft function and partial recovery of autologous hematopoiesis  [The presence of parvovirus B 19 viraemia, confirmed by PCR, both at diagnosis and relapse, suggests PVB19 as a possible causative agent of SAA and GF 18 months after BMT1 in our case. Virus monitoring showed no evidence of parvovirus infection in the interval between the diagnosis and GF. There are very few published data reporting parvovirus B 19 as a pathogen, associated with SAA in immunocompetent and otherwise healthy individuals –14, whilIn our patient parvovirus B19-PCR was positive at diagnosis without any preceding history of immune deficiency or chronic hemolytic anaemia. At the time of reactivation, the patient was also considered to be immunocompetent as immunologic recovery was already achieved. A previous report with parvovirus B 19 associated SAA encompasses another 3 patients who underwent either matched sibling donor BMT (2 patients) or matched unrelated donor PSCT (1 patient) . No compTreatment alternatives after graft failure in patients with SAA include administration of rhGM-CSF, second BMT (BMT2) from the same or alternative donor, and allogeneic peripheral blood stem cell (PBSC) rescue , 21, 22.The rationale for rhGM-CSF use after GF is possible acceleration of trilineage hematopoiesis and/or prevention of lethal infectious complications, thus increasing the time span for hematopoietic recovery . This opAlthough our patient fulfilled all criteria for favorable outcome after BMT2, a T-cell depleted PBSC boost from the same donor following treatment with ATG was the preferred therapeutic approach aiming at enhancement of graft function and rapid hematologic recovery while minimizing the risk of serious complications such as GVHD . However


The beneficial effects of human milk on decreasing rates of paediatric infections such as necrotizing enterocolitis (NEC) and sepsis have been clearly demonstrated. Donor breastmilk has been encouraged as the milk of choice when a mother's own breastmilk is not available. The objectives of this study were to assess feasibility of providing donor breastmilk to infants in a resource limited Neonatal Prem Unit (NPU). In addition we sought to determine whether donor breastmilk could be safely pasteurized and administered to infants without any adverse events.Low birth weight infants < 1800 g and under 32 weeks gestational age were followed up in the NPU over a 3 week period; feeding data and morbidity data was collected in order to determine if there were any adverse events associated with donor breastmilk. Samples of pasteurized breastmilk were cultured to check for any bacterial contamination.191 infants met the inclusion criteria of whom 96 received their mother's own breastmilk. Of the 95 infants who were potentially eligible to receive donor milk, only 40 did in fact receive donor milk. There was no evidence of bacterial contamination in the samples analyzed, and no evidence of adverse events from feeding with donor breastmilk.It is feasible to supply donor breastmilk to infants in an NPU in a resource limited setting, however staff needs to be sensitized to the importance of donor breastmilk to improve uptake rates. Secondly we showed that it is possible to supply donor breastmilk according to established guidelines with no adverse events therefore making it possible to prevent NEC and other side effects often associated with formula feeding of premature infants. The particular benefits of human breastmilk for preterm and term infants have been well described in medical literature . Human mBecause of the well documented benefits of donor breastmilk, a donor breastmilk bank was set up in the Neonatal Prem unit (NPU) of King Edward VIII Hospital (KEH) in Durban, South Africa at the beginning of 2009. KEH is a public hospital that serves a disadvantaged community and its NPU has an average intake of 180 infants per month. As part of the routine protocol in the NPU all mothers of HIV unexposed infants are encouraged to provide breastmilk to their infants. HIV infected mothers are routinely counseled on appropriate infant feeding for their situation according to WHO guidelines. HIV infected women who opt to breastfeed are taught how to heat treat their own expressed breastmilk (HTEBM) before they feed it to their infants. The new breastmilk bank that was opened a few months prior to the commencement of this evaluation study operates under the guidelines of the Human Milk Banking Association of South Africa (HMBASA) . As it iImplementing a milk bank in this resource-poor setting presented multiple challenges particularly because of inadequate staffing and lack of funds for maintenance of equipment. Therefore a decision was made to use a method of pasteurization that would be more appropriate within these limitations. This pasteurization method, known as flash-heat, is a simple method which involves heating individual jars of breastmilk in a water bath. Flash-heat mimics commercial high-temperature short-time (HTST) pasteurization. This 'low-tech' method of pasteurization, has been documented to inactivate cell-free  and cellst World Health Assembly Meeting in Geneva (May 2008) calling on the World Health Organization to support countries to conduct research on the feasibility of implementing breastmilk banks. There have been no prospective studies in South Africa examining the feasibility or safety of donor breastmilk supplied by breastmilk banks in NPUs. However there has been a report of anecdotal data suggesting that it was feasible and safe to provide donor breastmilk to infants during an outbreak of rotavirus [There is skepticism among health care workers and policy makers in South Africa around feasibility and safety of human milk banks in this area mainly because of the fear of HIV transmission and the cultural acceptability. These concerns have severely hampered the use of donor breastmilk and there is therefore an urgent need for this information from South Africa to clarify these issues. This study hopes to provide some of this information which is in keeping with a resolution passed during the 61otavirus .The objectives of this study were twofold. First, we sought to address feasibility by documenting whether the medical staff would prescribe donor milk for infants; and whether eligible mothers in the unit would be prepared to donate breastmilk to the bank. We also investigated the feasibility of adhering to the HMBASA quality assurance guidelines for microbial testing of breastmilk. Secondly we sought to evaluate an aspect of the safety of providing donor milk by documenting clinical adverse events of infants fed on donor milk compared to formula milk. We did not seek to investigate whether infants fed on donor milk had a better outcome than those fed on formula milk as this has already been established as concluded in a Cochrane Review .The study was an observational longitudinal cohort study. Mothers with low birth weight infants were recruited to participate in this study. The study population consisted of Low birth weight infants < 1800 g and under 32 weeks gestational age who were admitted to the NPU at KEH during the 6 month period June 2009 to December 2009. We selected 1800 g as an upper weight limit since larger babies were more likely to have abbreviated hospital stays and thus insufficient days for observation. We also particularly focused on infants < 1800 g and < than 32 weeks gestational age as they are at particular risk of developing NEC and other problems associated with formula feeds. Exclusion criteria included infants whose hospital stay was less than 3 days; infants with congenital abnormalities and infants who were only admitted to the unit > 24 hours after birth. Infants whose mothers were not able or willing to sign written informed consent were also not eligible for the study.Enrolment data was collected from the peripartum history and maternal medical history recorded in the hospital charts. During the infant's hospital stay morbidity and feeding data was collected. Frequency of neonatal complications during a 3 week observation period were recorded after clinical examination by the study doctor (IC) and from perusal of the clinical records. Infection was documented by the presence of clinical and laboratory signs of a systemic inflammatory response and/or positive cultures for pathogenic organisms at one or more of the following sites: blood, spinal fluid, urine, stool, respiratory secretions, umbilicus, or surgical wound. The morbidity was analyzed according to receipt of any donor milk vs receipt of formula milk only.An aliquot of 5 mls was removed from the first sample of pasteurized expressed breastmilk from each donor and was kept frozen at -20°C until it was analyzed.Frozen samples were transported in batches to the National Health Laboratory Services at Inkosi Albert Luthuli Hospital, Durban. The methodology involved plating a 100 μl of the pasteurized milk sample onto MacConkey agar which was incubated for 24 hrs at 37°C. The following day a semi-quantitative count was performed. If any suspicious colonies were observed they were identified further.© SPSS Inc.). Continuous variables were checked for normality of distribution and means/standard deviations (SD) are reported for the normally distributed variables and medians/inter-quartile ranges for the skewed variables. Counts and observed percentages are reported for the categorical data. Chi squared tests were used to compare the characteristics and complications present in the infants who received donor breastmilk and/or formula feeds.The study was analyzed using SPSS 15.0  and the King Edward V111Written informed consent was obtained from mothers for their infants' clinical records to be used in the study.During the study period June to November 2009, a total of 191 infants met the inclusion criteria and were enrolled into the study. The baseline characteristics of the 191 mother/infant pairs who were enrolled in the study are shown in table In terms of adverse events/clinical outcome, as can be seen in table Infant A was born at 32 weeks gestation (birth weight: 1.7 kg) to a 33 year old HIV negative, para 3 mother following a caesarian section for severe pre-eclampsia. Mother had left arm paralysis since adolescence following a head injury. She had chosen to breastfeed antenatally, however the nurses decided as she had a paralytic arm, she would be unable to hold her baby and started her baby on formula. NEC developed on day 2 following formula feeds . Feeds were discontinued for 2 days, following which breastfeeding was initiated by the mother supported by the study doctor. Donor milk was given for four days while the mother's milk was coming down. NEC resolved after 6 days on treatment. Baby was doing well and discharged on breastfeeds and expressed breastmilk top-ups, 4 days after resolution of NEC.Infant B was born at 29 weeks gestation (birth weight 1.1 kg) to a 41 yr old HIV negative, para 4 mother, with previous breast cancer and therefore not planning to breastfeed. Infant received formula milk feeds for the first 3 weeks of life and developed NEC . He was taken off feeds for 5 days and then commenced on donor breastmilk for a further 3 weeks until discharge. Did extremely well on donor breastmilk and showed marked clinical improvement. In fact during the period of receiving donor breast milk the nursing staff attempted re-introducing formula feeds but these were not tolerated at all and were accompanied by projectile vomiting so were discontinued and donor breastmilk re-introduced.We were successful in obtaining donors from amongst the mothers who were in hospital usually waiting for their infants to gain weight or get better. During the 6 month period of study we were able to recruit 35 donors. Because the concept of donation of breastmilk was new in the hospital when there were periods of insufficient supplies the breastmilk bank also sourced breastmilk from a nearby, NGO run, community based breastmilk bank. It was estimated that during the first 12 months of operation approximately 20% of the supplies were externally sourced whereas in the last 12 months (Jan 2010 to December 2010) the bank is operating completely on in-house donations of breastmilk.Of the 35 in-house donors, 30 of the samples were analyzed for presence of bacterial contamination, post-pasteurization (flash heating). No bacterial contamination was found in any of the 30 samples analyzed.This evaluation has shown that donor milk was in fact prescribed for 40 of the potentially 95 eligible infants. There were obvious missed opportunities which were due to medical staff not discussing with mothers that there was donor breastmilk available and that this was an option. We did not evaluate in each case why donor breastmilk was not prescribed when it was indicated but anecdotal evidence suggests that there were 2 main reasons. First, at the time of the study, where there was no routine nevirapine prophylaxis during breastfeeding, the standard NPU protocol was to prescribe preterm formula for HIV exposed infants whose mothers had either chosen to formula feed or who had chosen to breastfeed but were unable to provide the necessary milk. Second, because donor breastmilk was a very new option in the unit and because of the rotation of medical staff through the unit it takes a while to train all the staff and gain acceptance for donor breastmilk. Additionally it is easier and less time consuming for nurses and doctors to prescribe formula feeds compared to donor milk and the formula feeds are easier to access compared to the donor milk. In effect 42% of the eligible infants had donor breastmilk prescribed for them which we believe is an acceptable percentage for such a new concept. While this evaluation study was being conducted a separate acceptability study was also conducted in the unit. Results of this study showed that there were indeed concerns and lack of understanding around donor breastmilk which participants felt could be overcome with education and dissemination of information . In respThis evaluation has also shown that it is possible to supply donor breastmilk in neonatal prem units in resource poor settings with no adverse events experienced by the infants, therefore making it possible to prevent the NEC and other side effects often associated with formula feeding of premature infants. This also has important implications in terms of protecting and promoting breastfeeding and has the potential to play a role in decreasing infant mortality rate as has been shown in a country like Brazil which has reported dramatic decreases in its under-five mortality rate following the growth in donor breastmilk banking . There aBacillus sp. [Bacillus sp. may survive pasteurization, unlike cow's milk this is thought to be a rare contaminant of human breast milk [Bacillus species that are present from growing. Some of these safeguards include, pasteurizing the milk as soon as possible after expression and then storing the sample frozen which will eliminate the majority of bacteria and thus avoid extensive growth of Bacillus during storage. Additionally pasteurized samples should remain covered and the lids should not be removed until feeding. It is also important to note that although Bacillus sp. may be a contaminant in food and dairy sources when it is present in breastmilk this danger is considerably attenuated because the medium of breastmilk unlike that of other food and dairy sources contains important factors that minimize bacterial growth. Furthermore it is important to balance the benefits of providing donor milk compared to the alternative of infant formula which has also been shown to be prone to contamination even with Bacillus sp [Finally, although the small number of samples that were assayed showed no bacteriological contamination it is possible that with a much larger number of samples we would have detected some samples with bacterial contamination as was shown recently in an analysis from breastmilk banks in the US . In partllus sp. . Howeverast milk . Regardlillus sp .Bacillus sp., especially for low-cost settings desiring to utilize a simple method of pasteurization such as Flash-heat described here. However the current process of culture methods would be cost-prohibitive in resource poor settings. A methodology that may be more applicable in resource poor settings may be the system employed in the Brazilian banks in which a pH test is utilized to identify potential bacterial contamination [We acknowledge that further investigation into cost-effective and simplified donor milk screening is warranted to allow for testing of each sample specifically for mination . We recoThis study has shown firstly that it is feasible to access mothers who are prepared to dnate breastmilk and it is feasible for infants to receive donated breastmilk. Secondly we have shown that it is possible to supply donor breastmilk according to established guidelines in NPUs in resource poor settings with no adverse events, therefore making it possible to prevent NEC and other side effects often associated with formula feeding of premature infants. We believe this low-tech system of breastmilk banking can be reproduced in similar settings and would recommend that for successful scale-up, the endorsement and involvement of the Ministry of Health is essential as has been seen in the Brazilian system of breastmilk banking. Furthermore we recommend that low income countries should follow the Brazilian model of having a comprehensive, integrated breastfeeding promotion strategy including donor breastmilk banking with the ultimate aim of improving child survival. In South Africa especially, scale up of donor breastmilk banks would have a profound impact on breastfeeding promotion which is vital in order to improve infant survival rates. Furthermore this would assist to reverse current predictions that South Africa is unlikely to meet the millennium development goal 4 .The authors declare that they have no competing interests.IC conceived the study and all authors contributed towards planning and design of the study. IC together with a Zulu speaking assistant enrolled all the study participants and IC collected all the clinical data which was supervised by MA and NN. IC wrote the first draft of the paper and the other authors contributed to editing and finalizing the document. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/356/prepub