Photobiomodulation in the infrared spectrum reverses the expansion of circulating natural killer cells and brain microglial activation in Sanfilippo mice.

Sanfilippo syndrome results from inherited mutations in genes encoding lysosomal enzymes that catabolise heparan sulfate (HS), leading to early childhood-onset neurodegeneration. This study explores the therapeutic potential of photobiomodulation (PBM), which is neuroprotective and anti-inflammatory in several neurodegenerative diseases; it is also safe and PBM devices are readily available. We investigated the effects of 10-14 days transcranial PBM at 670 nm (2 or 4 J/cm2/day) or 904 nm (4 J/cm2/day) in young (3 weeks) and older (15 weeks) Sanfilippo or mucopolysaccharidosis type IIIA (MPS IIIA) mice. Although we found no PBM-induced changes in HS accumulation, astrocyte activation, CD206 (an anti-inflammatory marker) and BDNF expression in the brains of Sanfilippo mice, there was a near-normalisation of microglial activation in older MPS IIIA mice by 904 nm PBM, with decreased IBA1 expression and a return of their morphology towards a resting state. Immune cell immunophenotyping of peripheral blood with mass cytometry revealed increased pro-inflammatory signalling through pSTAT1 and p-p38 in NK and T cells in young but not older MPS IIIA mice (5 weeks of age), and expansion of NK, B and CD8+ T cells in older affected mice (17 weeks of age), highlighting the importance of innate and adaptive lymphocytes in Sanfilippo syndrome. Notably, 670 and 904 nm PBM both reversed the Sanfilippo-induced increase in pSTAT1 and p-p38 expression in multiple leukocyte populations in young mice, while 904 nm reversed the increase in NK cells in older mice. In conclusion, this is the first study to demonstrate the beneficial effects of PBM in Sanfilippo mice. The distinct reduction in microglial activation and NK cell pro-inflammatory signalling and number suggests PBM may alleviate neuroinflammation and lymphocyte activation, encouraging further investigation of PBM as a standalone, or complementary therapy in Sanfilippo syndrome.

A lower motor neuron disease in takahe (Porphyrio hochstetteri) is an endoplasmic reticulum storage disease.

AIMS: To investigate the pathogenesis of a disease in takahe (Porphyrio hochstetteri) with intracytoplasmic inclusion bodies in lower motor neurons. METHODS: Four birds aged between 5 and 12 years, from three different wildlife sanctuaries in New Zealand were examined. Of these, only one had signs of spinal dysfunction in the form of paresis. Stained paraffin sections of tissues were examined by light microscopy and immunostained sections of the ventral horn of the spinal cord by confocal microscopy. Epoxy resin sections of the spinal cord from the bird with spinal dysfunction were examined by electron microscopy. RESULTS: Two types of inclusion bodies were noted, but only in motor neurons of the ventral spinal cord and brain stem. These were large globoid eosinophilic bodies up to 5 µm in diameter, and yellow/brown granular inclusions mostly at the pole of the cell. The globoid bodies stained with Luxol fast blue but not with periodic acid Schiff (PAS), or Sudan black. The granular inclusions stained with Luxol fast blue, PAS and Sudan black. Both bodies were slightly autofluorescent. On electron microscopy the globoid bodies had an even electron-dense texture and were bound by a membrane. Beneath the membrane were large numbers of small intraluminal vesicles. The smaller granular bodies were more heterogeneous, irregularly rounded and membrane-bound accumulations of granular electron-dense material, often with electron-lucent vacuoles. Others were more vesicular but contained varying amounts of electron-dense material. The large globoid bodies did not immunostain for lysosomal markers lysosomal associated protein 1 (LAMP1) or cathepsin D, so were not lysosomal. The small granular bodies stained for cathepsin D by a chromogenic method.A kindred matrix analysis showed two cases to be as closely related as first cousins, and another case was almost as closely related to one of them, but the fourth bird was unrelated to any other. CONCLUSIONS: It was concluded that this was an endoplasmic reticulum storage disease due to a specific protein misfolding within endoplasmic reticulum. It was rationalised that the two types of inclusions reflected the same aetiology, but that misfolded protein in the smaller granular bodies had entered the lysosomal system via endoplasmic reticulum autophagy. Although the cause was unclear, it most likely had a genetic aetiology or predisposition and, as such, has clinical relevance.

Early disease course is unaltered in mucopolysaccharidosis type IIIA (MPS IIIA) mice lacking α-synuclein.

BACKGROUND: Sanfilippo syndrome (mucopolysaccharidosis type IIIA; MPS IIIA) is an inherited paediatric-onset neurodegenerative disorder caused by the lysosomal deficiency of sulphamidase with subsequent accumulation of heparan sulphate. The pathological mechanisms responsible for clinical disease are unknown; however, intraneuronal accumulation of aggregation-prone proteins such as α-synuclein, phosphorylated tau and amyloid precursor protein suggests inefficient intracellular trafficking and lysosomal degradation. AIM: To investigate the contribution the accumulating α-synuclein plays in early symptom emergence that is, impaired cognition, reduced anxiety and motor deficits, first detectable between 3-5 months of age. METHODS: We have crossed congenic MPS IIIA mice with α-synuclein-deficient (Sncatm1Rosl /J) mice and evaluated phenotype and brain disease lesions. RESULTS: In a battery of behavioural tests performed on mice aged 12-22 weeks, we were unable to differentiate α-synuclein-deficient MPS IIIA mice from those with one or both copies of the α-synuclein gene; all three affected genotypes were significantly impaired in test performance when compared to wild-type littermates. Histological studies revealed that the rate, location and nature of deposition of other proteinaceous lesions, the disruption to endolysosomal protein expression and the inflammatory response seen in the brain of α-synuclein-deficient MPS IIIA mice reflected that seen in MPS IIIA mice homo- or heterozygous for α-synuclein. CONCLUSION: Deletion and/or deficiency of α-synuclein does not influence clinical and neuropathological disease progression in murine MPS IIIA, demonstrating that in and of itself, this protein does not initiate the cognitive and motor symptoms that occur in the first 5 months of life in MPS IIIA mice.

REpeated AutoLogous Infusions of STem cells In Cirrhosis (REALISTIC): a multicentre, phase II, open-label, randomised controlled trial of repeated autologous infusions of granulocyte colony-stimulating factor (GCSF) mobilised CD133+ bone marrow stem cells in patients with cirrhosis. A study protocol for a randomised controlled trial.

INTRODUCTION: Liver disease mortality and morbidity are rapidly rising and liver transplantation is limited by organ availability. Small scale human studies have shown that stem cell therapy is safe and feasible and has suggested clinical benefit. No published studies have yet examined the effect of stem cell therapy in a randomised controlled trial and evaluated the effect of repeated therapy. METHODS AND ANALYSIS: Patients with liver cirrhosis will be randomised to one of three trial groups: group 1: Control group, Standard conservative management; group 2 treatment: granulocyte colony-stimulating factor (G-CSF; lenograstim) 15 µg/kg body weight daily on days 1-5; group 3 treatment: G-CSF 15 µg/kg body weight daily on days 1-5 followed by leukapheresis, isolation and aliquoting of CD133+ cells. Patients will receive an infusion of freshly isolated CD133+ cells immediately and frozen doses at days 30 and 60 via peripheral vein (0.2×10(6) cells/kg for each of the three doses). Primary objective is to demonstrate an improvement in the severity of liver disease over 3 months using either G-CSF alone or G-CSF followed by repeated infusions of haematopoietic stem cells compared with standard conservative management. The trial is powered to answer two hypotheses of each treatment compared to control but not powered to detect smaller expected differences between the two treatment groups. As such, the overall α=0.05 for the trial is split equally between the two hypotheses. Conventionally, to detect a relevant standardised effect size of 0.8 point reduction in Model for End-stage Liver Disease score using two-sided α=0.05(overall α=0.1 split equally between the two hypotheses) and 80% power requires 27 participants to be randomised per group (81 participants in total). ETHICS AND DISSEMINATION: The trial is registered at Current Controlled Trials on 18 November 2009 (ISRCTN number 91288089, EuDRACT number 2009-010335-41). The findings of this trial will be disseminated to patients and through peer-reviewed publications and international presentations.

Worth a thousand words: Integrating clinical photographs into an electronic medical record.

Technological advances have made clinical photographs ubiquitous but the tremendous value of images is often burdened by concerns that are both legitimate and unsubstantiated. Compliant photos dramatically improve patient value and should be encouraged after adequate institutional preparation. As healthcare networks continue to transition to electronic medical records, clinicians and administrators should outline reasonable policies to integrate photos safely.

Does Acute Physiology and Chronic Health Evaluation II provide a valid metric to directly compare disease severity in trauma versus surgical intensive care unit patients?

The Acute Physiology and Chronic Health Evaluation II (APACHE II) score has never been validated to risk-adjust between critically ill trauma (TICU) and general surgical (SICU) intensive care unit patients, yet it is commonly used for such a purpose. To study this, we evaluated risk of death in TICU and SICU patients with pneumonia. We hypothesized that mortality for a given APACHE II would be significantly different and that using APACHE II to directly compare TICU and SICU patients would not be appropriate. We conducted a retrospective review of patients admitted to the TICU or SICU at a tertiary medical center over an 18-month period with pneumonia. Admission APACHE II scores, in-hospital mortality, demographics, and illness characteristics were recorded. One hundred eighty patients met inclusion criteria, 116 in the TICU and 64 in the SICU. Average APACHE II scores were not significantly different in the TICU versus SICU (25 vs 24; P = 0.4607), indicating similar disease severity; overall mortality rates, however, were significantly different (24 vs 50%; P = 0.0004). Components of APACHE II, which contributed to this mortality differential, were Glasgow Coma Score, age, presence of chronic health problems, and operative intervention. APACHE II fails to provide a valid metric to directly compare the severity of disease between TICU and SICU patients with pneumonia. These groups represent distinct populations and should be separated when benchmarking outcomes or creating performance metrics in ICU patients. Improved severity scoring systems are needed to conduct clinically relevant and methodologically valid comparisons between these unique groups.

Creation and implementation of an emergency general surgery registry modeled after the National Trauma Data Bank.

BACKGROUND: As emergency general surgery (EGS) evolves, an EGS patient-tracking database (EGS registry [EGSR]) similar to the National Trauma Data Bank (NTDB) will be essential to study outcomes and improve care. The goal of this study was to establish diagnostic ICD-9 codes to define EGS patients. The hypothesis was that creating standardized ICD-9-based inclusion criteria would facilitate patient identification for an EGSR and aid in its ongoing development. STUDY DESIGN: We conducted a retrospective review of EGS admissions over a 9-month period to define ICD-9 diagnostic codes of patients admitted to our EGS service. Subsequently, prospective data were collected into the EGSR by testing ICD-9-based inclusion criteria over 1 month. Patient, hospital, and severity scoring variables, as well as quality assurance information, were identified. RESULTS: We identified 959 admissions to the EGS service over 9 months with 306 ICD-9 diagnosis codes that define EGS patients; the prospective population of the EGSR confirmed feasibility of ICD-9-based inclusion criteria. The EGSR captures 107 data points and 33 comorbidities per patient over 11 categories, akin to the 10 NTDB categories. CONCLUSIONS: Following the model of the NTDB, we have successfully completed creation and initial implementation of an EGSR by using ICD-9-based inclusion criteria. Our comprehensive EGSR creates a prospective data-collection modality to capture and define EGS patients. A uniform patient-tracking EGSR, akin to the NTDB, will advance the science of acute care surgery, improve EGS patient outcomes, and facilitate multi-institutional collaboration.

Intracisternal enzyme replacement therapy in lysosomal storage diseases: routes of absorption into brain.

AIMS: The research concerns enzyme replacement therapy in lysosomal storage diseases with central nervous system involvement. The principle aim was to understand the routes of entry of enzyme into the brain when delivered directly into the cerebrospinal fluid (CSF) via the cerebellomedullary cistern. METHODS: Pathways for absorption of replacement enzyme were investigated in dogs with mucopolysaccharidosis IIIA (MPSIIIA) following intracisternal injections of human recombinant N-sulphoglucosamine sulphohydrolase (rhSGSH, EC3.10.1.1) by light and confocal microscopy using chromogenic and fluorescent immune probes. RESULTS: Enzyme entered the brain superficially by penetration of the pia/glia limitans interface, but the main route was perivascular along large veins, arteries and arterioles extending onto capillaries. It further dispersed into surrounding neuropil to be taken up by neurones, macrophages, astrocytes and oligodendroglia. Enzyme also entered the lateral ventricles adjacent to the choroid plexus, probably also by the tela choroidea and medullary velum, with further spread throughout the ventricular system and spinal canal. There was secondary spread back across the ependyma into nervous tissue of brain and spinal cord. CONCLUSIONS: Enzyme mainly enters the brain by a perivascular route involving both arteries and veins with subsequent spread within the neuropil from where it is taken up by a proportion of neurones and other cells. Penetration of enzyme through the pia/glia limitans is minor and superficial.

Outcome after mastectomy for ipsilateral breast tumor recurrence after breast conserving surgery.

Ipsilateral breast tumor recurrence (IBTR) is a risk after breast conserving surgery, and is traditionally treated with mastectomy. Given the limited literature on outcome after mastectomy for IBTR, we evaluated our long-term data for this group. A retrospective review was conducted using a database of 2101 breast cancer patients at a single institution. Fifty-nine patients underwent breast conserving surgery and experienced an IBTR. Exclusion criteria included repeat lumpectomy or metastatic disease before mastectomy. Patients presented with invasive ductal (58%), invasive lobular (7%), other invasive (11%), or ductal carcinoma in situ (24%). Initial tumors were Tis (24%), T1 (42%), T2 (20%), T3 (2%), or not recorded (12%). IBTR lesions were Tis (20%), T1 (46%), T2 (25%), or T3 (9%). Median follow-up after mastectomy was 4.6 years. Thirteen patients (22%) had postmastectomy recurrence (PMR), which decreased overall survival (P = 0.002). PMR was more common with larger IBTR tumors (P = 0.03), specifically IBTR > or = T2 (P = 0.003). Eighty-five per cent of PMR occurred within 2 years of mastectomy. Mastectomy for IBTR remains effective treatment for most patients, but the risk of PMR remains. Patients with IBTR tumors >2 cm have an increased risk of PMR. Strict follow-up should be routine, especially during the first 24 months.

Validation of the sheep as a large animal model for the study of vertebral osteoporosis.

Rats have long been the animal of choice for research in the field of osteoporosis. In the search for a complementary large animal model the sheep appears useful but hitherto the extent of bone loss from the spine has failed to reach a level that is generally accepted as osteoporotic in humans. Osteoporosis was induced in ten sheep using ovariectomy, low calcium diet and steroid injection for 6 months. Bone samples of iliac crest (IC), lumbar spine (LS), and proximal femur (PF) from the osteoporotic sheep were compared with those from four normal sheep using densitometry, histomorphometry, biochemistry and basic mechanical testing. The differences were examined using an analysis of variance with Tukey-Kramer test. Overall, the bone mineral density at LS and PF decreased more than 25% after treatment. Trabecular bone volume decreased by 29.2, 33.4 and 42.6% in IC, LS and PF, respectively. The failure load of the LS in axial compression was reduced to 2,003 from 6,140 N. The extent of bone loss was sufficient to categorise these sheep as osteoporotic although the pattern of bone loss varied between sites. Reduced mechanical competence in LS confirmed the suitability of this model for evaluation of potential treatments for osteoporosis.

Effect of high dose, repeated intra-cerebrospinal fluid injection of sulphamidase on neuropathology in mucopolysaccharidosis type IIIA mice.

Mucopolysaccharidosis type IIIA (MPS IIIA) is an inherited neurodegenerative lysosomal storage disorder characterised by progressive loss of learned skills, sleep disturbance and behavioural problems. Reduced activity of sulphamidase (SGSH; EC 3.10.1.1) results in intracellular accumulation of heparan sulphate (HS), with the brain the primary site of pathology. We have used a naturally-occurring MPS IIIA mouse model to determine the effectiveness of SGSH replacement via the cerebrospinal fluid (CSF) to decrease neuropathology. This is a potential therapeutic option for patients with this disorder. Mice received intra-CSF injections of recombinant human SGSH (30, 50 or 70 mug) fortnightly from six-18 weeks of age, and the cumulative effect on neuropathology was examined and quantified. Anti-SGSH antibodies detected in plasma at euthanasia did not appear to impact upon the health of the mice or the experimental outcome, with significant, but region- and dose-dependent reductions in an HS-derived oligosaccharide observed in the brain and spinal cord using tandem mass spectrometry. SGSH infusion reduced the number of storage inclusions observed in the brain when visualised using electron microscopy and this correlated with a significant decrease in the immunohistochemical staining of a lysosomal membrane marker (LIMP-II). Reduced numbers of activated isolectin-B4-positive microglia and GFAP-positive astrocytes were seen in many, but not all, brain regions. Significant reductions in the number of ubiquitin-positive intracellular inclusions were also observed. These outcomes demonstrate the effectiveness of this method of enzyme delivery in reducing the spectrum of neuropathological changes in murine MPS IIIA brain.

Quantitative backscattered electron imaging of bone in proximal femur fragility fracture and medical illness.

Bone quality consists of a number of factors including the amount of bone, bone architecture and the degree of bone mineralization. Quantitative backscattered electron imaging is a technique that allows the degree of mineralization of trabeculae to be assessed and in this study is applied to inter-trochanteric bone biopsies of the proximal femur. Biopsy cores from 22 controls, nine individuals with acute and chronic medical conditions and 22 fragility fracture individuals undergoing total hip replacement were processed into methyl methacrylate, polished and analysed in a Philips XL20 scanning electron microscope. A mean and distribution of weight percent calcium were determined for each individual, and for the control, medically ill and fragility fracture groups. All individuals and groups of individuals showed normal distributions of percent calcium with both the ill and fragility fracture groups being under mineralized relative to the control group. The shape and position of the mineralization distributions suggest that the fragility group resulted from increased bone turnover with a slow progression to under mineralization. In contrast, the ill group appears to have had a more rapid change in the mineralization dynamic. Clear distinctions between the control, fragility fracture and medically ill groups could be seen when the mineralization data were plotted as a scatter graph against age. Graphing the data in this way showed an age-related increase in the degree of mineralization in control individuals with the under-mineralized, fragility fracture group scattered below this normal trend. The medically ill group was similarly less mineralized which highlighted the degree to which medical conditions and treatments can alter bone matrix mineralization.

Microarray analysis of gene expression in soybean roots susceptible to the soybean cyst nematode two days post invasion.

Soybean root cells undergo dramatic morphological and biochemical changes during the establishment of a feeding site in a compatible interaction with the soybean cyst nematode (SCN). We constructed a cDNA microarray with approximately 1,300 cDNA inserts targeted to identify differentially expressed genes during the compatible interaction of SCN with soybean roots 2 days after infection. Three independent biological replicates were grown and inoculated with SCN, and 2 days later RNA was extracted for hybridization to microarrays and compared to noninoculated controls. Statistical analysis indicated that approximately 8% of the genes monitored were induced and more than 50% of these were genes of unknown function. Notable genes that were more highly expressed 2 days after inoculation with SCN as compared to noninoculated roots included the repetitive proline-rich glycoprotein, the stress-induced gene SAM22, ss-1,3-endoglucanase, peroxidase, and those involved in carbohydrate metabolism, plant defense, and signaling.

Assisted reproductive technologies in europe encompass diverse and complex ethical viewpoints: issues to be considered in reporting research in human reproduction.

The many dimensions of the field of reproduction (clinical, scientific, social, ethical, legal) must be addressed by those with editorial responsibility for the major journals in the field. As the editorial team of the leading reproductive journal Human Reproduction, we have evolved processes that attempt to ensure a transparent but robust approach to complexities whose impact varies across international boundaries.

Profiling local gene expression changes associated with Eimeria maxima and Eimeria acervulina using cDNA microarray.

Eimeria parasites show preferential sites of invasion in the avian intestine and produce a species-specific host immune response. Two economically important species, Eimeria acervulina and Eimeria maxima, preferentially invade and develop in the avian duodenum and jejunum/ileum, respectively. To investigate local host immune responses induced by parasite infection, global transcriptional changes in intestinal intraepithelial lymphocytes (IELs) induced by oral inoculation of chickens with E. acervulina or E. maxima were monitored using cDNA microarrays containing 400 unique chicken genes. Multiple gene transcripts were significantly up- or down-regulated following primary or secondary infection with E. acervulina or E. maxima. In general, infection by either parasite resulted in the expression changes of more genes following primary infection than following secondary infection, and E. acervulina caused more changes than did E. maxima. Although different regions of the small intestine were infected, similar changes in the levels of several cytokine mRNAs were observed in both Eimeria species following primary infection. Also identified was a set of transcripts whose expression was commonly enhanced or repressed in intestinal IELs of chickens infected with either parasite. Microarray analysis of chicken genes induced or repressed following Eimeria infection offers a powerful tool to enhance our understanding of host-parasite interactions leading to protective immunity.

A physical map of a gene-dense region in soybean linkage group A2 near the black seed coat and Rhg (4) loci.

Soybean ( Glycine max L. Merrill) linkage group A2 contains a major resistance gene to the soybean cyst nematode ( Heterodera glycines Ichinohe) at the Rhg (4) locus near a gene encoding aspartokinase homoserine dehydrogenase (AK-HSDH) and also near the I locus affecting seed coat color. To identify clones related to this region of the genome, we used a PCR assay using primers designed from a gene encoding AK-HSDH to screen approximately 40,000 clones from a bacterial artificial chromosome (BAC) library constructed from genomic DNA of the susceptible cv. Williams 82. The identified BACs were screened with a second PCR assay using primers designed from DNA sequence associated with the I locus to confirm the location of the BACs. Only BAC Gm_ISb001_056_G02 (56G2) was positive for both assays. BAC 56G2 contains several genes previously associated with stress or defense response including genes with high sequence similarity to those encoding chalcone synthase, glucosyl-transferase, a heat-shock transcription factor, a membrane-associated salt-inducible protein, adenosyl homocysteinase, a protein kinase, and a G10-like protein. The map contributes to our understanding of the organization of the soybean genome and to the completion of a physical map of the soybean genome. In addition, the genes identified provide landmarks to identify BAC clones near the Rhg (4) locus in resistant soybean genomic libraries and provide a foundation for comparison of soybean cyst nematode resistant and -susceptible DNA sequences in this region.