Monoclonal antibody against macrophage colony-stimulating factor suppresses circulating monocytes and tissue macrophage function but does not alter cell infiltration/activation in cutaneous lesions or clinical outcomes in patients with cutaneous lupus erythematosus.

This study's objective was to assess the effects of PD-0360324, a fully human immunoglobulin G2 monoclonal antibody against macrophage colony-stimulating factor in cutaneous lupus erythematosus (CLE). Patients with active subacute CLE or discoid lupus erythematosus were randomized to receive 100 or 150 mg PD-0360324 or placebo via intravenous infusion every 2 weeks for 3 months. Blood and urine samples were obtained pre- and post-treatment to analyse pharmacokinetics and pharmacodynamic changes in CD14(+) CD16(+) monocytes, urinary N-terminal telopeptide (uNTX), alanine/aspartate aminotransferases (ALT/AST) and creatine kinase (CK); tissue biopsy samples were taken to evaluate macrophage populations and T cells using immunohistochemistry. Clinical efficacy assessments included the Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI). Among 28 randomized/analysed patients, peak/trough plasma concentrations increased in a greater-than-dose-proportional manner with dose increases from 100 to 150 mg. Statistically significant differences were observed between active treatment and placebo groups in changes from baseline in CD14(+) CD16(+) cells, uNTX, ALT, AST and CK levels at most time-points. The numbers, density and activation states of tissue macrophages and T cells did not change from baseline to treatment end. No between-group differences were seen in CLASI. Patients receiving PD-0360324 reported significantly more adverse events than those receiving placebo, but no serious adverse events. In patients with CLE, 100 and 150 mg PD-0360324 every 2 weeks for 3 months suppressed a subset of circulating monocytes and altered activity of some tissue macrophages without affecting cell populations in CLE skin lesions or improving clinical end-points.

Abnormal connexin expression in human chronic wounds.

BACKGROUND: Regulated alteration of connexin expression has been shown to be integral to acute wound repair. Downregulation of the gap-junction protein connexin 43 at the wound edge has been correlated with keratinocyte and fibroblast migration, while abnormal overexpression of connexin 43 significantly perturbs healing, as shown in the streptozotocin diabetic rodent impaired healing model. OBJECTIVES: To examine the protein expression levels of connexin 43, in addition to connexins 26 and 30, in a variety of human chronic wounds. METHODS: Wound-edge punch biopsies and a matched control from the arm were taken from a cohort of patients with venous leg, diabetic foot or pressure ulcers. Wound connexin expression in each patient was compared with that in a matched, nonwounded arm punch. Tissue was sectioned, stained and imaged by confocal microscopy using identical parameters per patient to permit quantification. RESULTS: Epidermal connexin 43, connexin 26 and connexin 30, and dermal connexin 43 were discovered to be strikingly upregulated in every ulcer from all three wound types, pointing to connexin upregulation as a common feature between chronic wounds. CONCLUSIONS: This result supports efforts to target connexin 43 to promote cell migration and wound healing in chronic ulcers.

Kaleidoscope model of diabetes care: time for a rethink?

National Audit Data highlight persistent sub-optimum control among increasing numbers of people living with diabetes, with severe consequences for the individual and the NHS. The aim of the present review was to introduce a new cohesive, holistic model of care, tailored to individual needs to support optimum diabetes outcomes. This model of diabetes is necessary in order to understand the driving forces behind behaviour and their impact on diabetes management. Feelings (an emotional state or reaction) and beliefs (an acceptance that something is true or real) are fundamental behavioural drivers and influence diabetes self-management choices. Individually, these explain some of the complexities of behaviour and, collectively, they impact on personal motivation (rationale/desire to act) to achieve a specific outcome. Inevitably, they independently affect diabetes self-management and the environment in which individuals live. A model of care that proposes the encompassing of environment, intrinsic thought and therapy regimens to provide tailored, personalized healthcare should support enhanced diabetes self-management and outcomes from diagnosis. The Kaleidoscope model of care could be deliverable in routine care, incorporating each of the influences on diabetes self-management, and should benefit both individuals with diabetes and healthcare professionals.

The characterization of the Phlebotomus papatasi transcriptome.

As important vectors of human disease, phlebotomine sand flies are of global significance to human health, transmitting several emerging and re-emerging infectious diseases. The most devastating of the sand fly transmitted infections are the leishmaniases, causing significant mortality and morbidity in both the Old and New World. Here we present the first global transcriptome analysis of the Old World vector of cutaneous leishmaniasis, Phlebotomus papatasi (Scopoli) and compare this transcriptome to that of the New World vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed using pooled mRNA from Phlebotomus papatasi larvae, pupae, adult males and females fed sugar, blood, or blood infected with Leishmania major. A total of 47 615 generated sequences was cleaned and assembled into 17 120 unique transcripts. Of the assembled sequences, 50% (8837 sequences) were classified using Gene Ontology (GO) terms. This collection of transcripts is comprehensive, as demonstrated by the high number of different GO categories. An in-depth analysis revealed 245 sequences with putative homology to proteins involved in blood and sugar digestion, immune response and peritrophic matrix formation. Twelve of the novel genes, including one trypsin, two peptidoglycan recognition proteins (PGRP) and nine chymotrypsins, have a higher expression level during larval stages. Two novel chymotrypsins and one novel PGRP are abundantly expressed upon blood feeding. This study will greatly improve the available genomic resources for P. papatasi and will provide essential information for annotation of the full genome.

Two newly identified cat allergens: the von Ebner gland protein Fel d 7 and the latherin-like protein Fel d 8.

INTRODUCTION: Characterization of the complete IgE binding spectrum of cat allergens is important for the development of improved diagnosis and effective immunotherapeutics. While Fel d 1 remains unchallenged as the major cat allergen, we now report the isolation of two new allergens capable of binding similar concentrations of IgE in the allergic sera of some individuals. MATERIALS AND METHODS: Cat tongue and submandibular salivary gland cDNA libraries were screened by DNA hybridisation and IgE immunoassay. The isolated DNA fragments were sub-cloned into an E. coli expression system and the IgE reactivity was examined with human cat-allergic sera using a DELFIA IgE quantitation assay. RESULTS: Fel d 7, an 18 kDa von Ebner gland protein Can f 1 homologue, was isolated from the tongue library. Fel d 8, a 24-kDa latherin-like protein with homology to Equ c 5, was isolated from the submandibular library. The frequency of IgE binding of cat-allergic sera to recombinant Fel d 1, 7 and 8 was 60.5, 37.6 and 19.3%, respectively. Inhibition studies indicated some IgE binding cross-reactivity between Fel d 7 and dog dander extracts. DISCUSSION: The study reports the isolation and characterization of two new cat allergens. The isolation of these allergens provides the opportunity to determine the role that IgE binding proteins other than Fel d 1 play in cat-allergic disease. For cat-allergic individuals with moderate to mild rhinoconjunctivitis these allergens may play a more important role in the manifestation of their allergic disease.

CD8+ lymphocyte depletion without SIV infection does not produce metabolic changes or pathological abnormalities in the rhesus macaque brain.

BACKGROUND: Simian immunodeficiency virus (SIV) infection and persistent CD8(+) lymphocyte depletion rapidly leads to encephalitis and neuronal injury. The objective of this study is to confirm that CD8 depletion alone does not induce brain lesions in the absence of SIV infection. METHODS: Four rhesus macaques were monitored by proton magnetic resonance spectroscopy ((1) H-MRS) before and biweekly after anti-CD8 antibody treatment for 8 weeks and compared with four SIV-infected animals. Post-mortem immunohistochemistry was performed on these eight animals and compared with six uninfected, non-CD8-depleted controls. RESULTS: CD8-depleted animals showed stable metabolite levels and revealed no neuronal injury, astrogliosis or microglial activation in contrast to SIV-infected animals. CONCLUSIONS: Alterations observed in MRS and lesions in this accelerated model of neuroAIDS result from unrestricted viral expansion in the setting of immunodeficiency rather than from CD8(+) lymphocyte depletion alone.

Neuropsychological differences between survivors of supratentorial and infratentorial brain tumours.

BACKGROUND: The purpose of this study is to evaluate the relationship between brain tumour location and core areas of cognitive and behavioural functioning for paediatric brain tumour survivors. The extant literature both supports and refutes an association between paediatric brain tumour location and neurocognitive outcomes. We examined neuropsychological test data to identify any differences in neurocognitive and behavioural profile associated with supratentorial versus infratentorial tumour location. METHODS: Following Institutional Review Board approval, the medical records and neuropsychological test data collected between 1997 and 2002 for 70 children treated for brain tumour at Children's Hospital Los Angeles were reviewed. Fifty-one per cent of the participants had tumours located in the supratentorial regions of the brain, whereas 49% had infratentorial tumours. Primary medical treatments involved tumour resection (90%), cranial radiation therapy (76%), chemotherapy (71%), and 59% all three medical procedures. The two tumour location groups did not differ significantly in the cumulative treatment dose of irradiation to the tumour bed or in the dose delivered to the whole brain. Neuropsychological test data included measures of verbal and non-verbal intellectual functioning, attention/working memory, processing speed, verbal and visual memory, fine motor skills, visual-motor integration, academic achievement, and social-emotional functioning. Differences between the two groups were evaluated using anova, t-tests and chi-squared statistical tests. RESULTS: The supratentorial and infratentorial tumour location groups did not differ on measures of intellectual functioning. However, survivors of infratentorial tumours performed more poorly on selected measures of more specific cognitive functions and on parent-report of social-emotional functioning relative to survivors of supratentorial tumours, even when age at diagnosis was held as a covariate. Higher frequency of auditory deficits was noted in the infratentorial tumour group and was associated with lowered academic achievement scores. CONCLUSIONS: The differences by location found in more specific neurocognitive and social-emotional variables, after controlling for age at diagnosis, may possibly reflect tumour location-specific effects. However, this interpretation remains tentative given the limitations in our study and inability to control for the range of medical and treatment-related factors that may have contributed towards the outcomes observed in our sample. At the same time, most of our findings appear consistent with reports from recent studies in this area.

Brain dopamine and serotonin receptor sites revealed by digital subtraction autoradiography.

Autoradiography combined with image analysis permitted quantitative visualization of dopamine (D2) and serotonin (S2) binding sites in rat brain. Forebrain sections were incubated with tritiated spiroperidol alone or with tritiated spiroperidol plus unlabeled compounds that saturated the D2 or S2 sites. By subtracting the digitized image of an autoradiograph derived from the latter sections from that of the former, the D2 or S2 sites were specifically revealed. The resulting quantitative images demonstrate the differing anatomical distributions of these sites. The D2 site is largely restricted to the striatal complex (caudate-putamen, nucleus accumbens septi, and olfactory tubercle), whereas the S2 site is enriched in layer 5 of motor cortex, the perirhinal and cingulate cortices, and the claustrum.

Potential Energy Surface Including Electron Correlation for F + H2 rarr FH + H: Refined Linear Surface.

A priori quantum mechanical calculations have been carried out at about 150 linear geometries for the fluorine plus hydrogen molecule system. An extended basis set of Gaussian functions was used, and electron correlation was treated explicitly by configuration interaction. Comparison with the experimental activation energy and exothermicity suggests that the theoretical potential surface is quite realistic.

Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine.

Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster controlled a highly pathogenic immunodeficiency virus challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the acquired immune deficiency syndrome epidemic.

Synchronous Aberrant Cerebellar and Opercular Development in Fetuses and Neonates with Congenital Heart Disease: Correlation with Early Communicative Neurodevelopmental Outcomes, Initial Experience.

Patients with congenital heart disease (CHD) demonstrate multidomain cognitive delays. Cingulo-opercular and cerebellar brain networks are critical to language functions. This is a description of our initial experience aiming to identify an anatomic correlate for CHD patients with expressive language delays. Fetal CHD patients, prospectively enrolled, underwent serial fetal (1.5T), postnatal pre- and postoperative (3T) MRI. Non-CHD patients were enrolled retrospectively from the same epoch. Comparable fetal and neonatal T2 contrast was used for manual linear cross-sectional measurement. Multivariable analysis was used for adjustments and curve fitting. Neurodevelopment was assessed with Battelle Developmental Inventory, 2nd ed. between 9 and 36 months of age. This interim analysis included patients from our longitudinal CHD study who had fetal, postnatal imaging and neurodevelopmental data-yielding a total of 62 mothers (11 CHD fetuses and 51 non-CHD fetuses). Altered brain trajectories were seen in selected cerebellar and opercular measurements in CHD patients compared with the non-CHD group. Smaller inferior cerebellar vermis measurements were associated with multiple communication-related abnormalities. Altered early structural development of the cerebellum and operculum is present in patients with CHD, which correlates with specific neurodevelopmental abnormalities.

Modulation of isocitrate dehydrogenase activity in Acinetobacter calcoaceticus by acetate.

The addition of acetate to a culture of Acinetobacter calcoaceticus grown in medium containing limiting succinate as the sole carbon and energy source leads to an increase in the specific activity of isocitrate dehydrogenase. This is in contrast to similar studies with several other microorganisms in which acetate induces an ATP-dependent phosphorylation and concomitant decrease in the specific activity of this enzyme.

Separation of isoenzymes of citrate synthase and isocitrate dehydrogenase by fast protein liquid chromatography.

Fast protein liquid chromatography (FPLC) has been shown to be a rapid and effective method of separating isoenzymes of citrate synthase and isocitrate dehydrogenase in extracts of Pseudomonas aeruginosa and Acinetobacter calcoaceticus. The advantages of FPLC over conventional methods of fractionation are discussed and it is suggested that this may be a valuable and more general technique for isoenzyme resolution.

Synaptic organization of columnar elements in the lamina of the wild type in Drosophila melanogaster.

The synaptic connections within the lamina, the first of the optic neuropiles underlying the insect's compound eye, have been little studied in Drosophila melanogaster until now, despite the genetic advantages of this animal. Here we report the reconstruction through its entire depth of one of the lamina modules, or cartridges, of a female wild-type Drosophila, for which a series of EM cross sections was analysed at levels extending from the retinal basement membrane to the first optic chiasma. A complete, comprehensive catalogue of the synaptic connections of all columnar elements has been compiled from this single series, confirmed from comparisons with less completely photographed cartridges. Combinations of the 12 types of cartridge neurons form divergent multiple-contact synapses (dyads, triads, and tetrads) throughout the lamina's depth. These 12 neuron types include 11 narrow-field elements (one class of receptor terminal, R1-R6, providing input to the cartridge; two types of long visual fiber from the ommatidium, R7 and R8; five types of monopolar cell, L1-5; and three types of medulla cell--two centrifugal neurons C2 and C3, and a third, T1) as well as a wide-field intrinsic or amacrine cell. Connections within the lamina formed by L4 from two adjacent cartridges (posterodorsal and posteroventral) contribute to the matrix of connections. In addition, connections of at least one other wide-field element have also been incorporated.

Sensorimotor impairment and elevated levels of dopamine metabolites in the neostriatum occur rapidly after intranigral injection of 6-hydroxydopamine or gamma-hydroxybutyrate in awake rats.

The unilateral injection of 6-hydroxydopamine (8 micrograms) into the ventral tegmental area of awake rats produced a rapidly developing and irreversible sensory neglect to contralateral tactile stimuli. This neglect developed in a caudal to rostral direction on the affected body surface and coincided with significant elevation in the concentrations of dopamine and two of its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the ipsilateral neostriatum. The unilateral injection of procaine or gamma-hydroxybutyric acid (GHB) into the substantia nigra of awake animals also produced a contralateral neglect that developed in a caudal to rostral direction, but the behavioral effect of these drugs diminished within 1 hr. Concentrations of dopamine, dihydroxyphenylacetic acid and homovanillic acid in the neostriatum were markedly elevated during continuous infusions of procaine or gamma-hydroxybutyric acid. The extent of sensory neglect and changes in dopamine metabolism in the neostriatum varied according to the amount of gamma-hydroxybutyric acid injected into the nigra and according to the proximity of injections of gamma-hydroxybutyric acid to the pars compacta. The rapid onset of sensory neglect following microinjections of 6-hydroxydopamine, procaine or gamma-hydroxybutyric acid is consistent with the ability of each of these drugs to block the conduction of impulses in mesostriatal neurons and suggests that concomitant increases in levels of dopamine, dihydroxyphenylacetic acid and homovanillic acid in the neostriatum resulted from decreases in the release of dopamine coupled with increased synthesis of dopamine. These findings also indicate that the catabolism of dopamine to dihydroxyphenylacetic acid or homovanillic acid may originate intraneuronally, without prior release of dopamine and its recapture by mesostriatal terminals, if the flow of impulses in this pathway has been blocked.

Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences.

Wolbachia are a group of intracellular inherited bacteria that infect a wide range of arthropods. They are associated with a number of different reproductive phenotypes in their hosts, such as cytoplasmic incompatibility, parthenogenesis and feminization. While it is known that the bacterial strains responsible for these different host phenotypes form a single clade within the alpha-Proteobacteria, until now it has not been possible to resolve the evolutionary relationships between different Wolbachia strains. To address this issue we have cloned and sequenced a gene encoding a surface protein of Wolbachia (wsp) from a representative sample of 28 Wolbachia strains. The sequences from this gene were highly variable and could be used to resolve the phylogenetic relationships of different Wolbachia strains. Based on the sequence of the wsp gene from different Wolbachia isolates we propose that the Wolbachia pipientis clade be initially divided into 12 groups. As more sequence information becomes available we expect the number of such groups to increase. In addition, we present a method of Wolbachia classification based on the use of group-specific wsp polymerase chain reaction (PGR) primers which will allow Wolbachia isolates to be typed without the need to clone and sequence individual Wolbachia genes. This system should facilitate future studies investigating the distribution and biology of Wolbachia strains from large samples of different host species.

Virus threshold determines disease in SIVsmmPBj14-infected macaques.

Simian immunodeficiency virus (SIV) variant SIVsmmPBj14 is unique in producing an acutely lethal enteropathic syndrome in pigtail macaques. To determine whether the nature of the PBj14 disease would be attenuated by decreasing virus input and to relate tissue virus burden to the severity of disease, we infected pigtail macaques with serial 10-fold doses of SIVsmmPBj14 clone bcl.3 spanning 10(-2) through 10(4)TCID50. The results revealed a strikingly narrow difference between minimum infectious and fatal disease-inducing doses and a close association between enteric lymphoid tissue virus burden and disease. All animals infected with as much as 10(4) TCID50 through as little as 100 TCID50 of virus died of the lethal PBj14 syndrome between 7 and 13 days postinfection. Animals receiving 10(-1) TCID50 became infected (PCR+) but did not develop clinical disease. Animals receiving 10(-2) TCID50 did not become infected. The clinical syndrome was surprisingly similar in all affected macaques, although the time to disease onset and total survival time increased slightly as virus input decreased from 10(4) to 10 degrees TCID50. Highest terminal virus loads in plasma, gut-associated lymphoid tissue (GALT), and lymph nodes and greatest lesion severity were attained at intermediate levels of virus input (10(1) to 10(2) TCID50), probably owing to optimal time for virus amplification in target tissues. The present study reinforces others on the PBj14 system, suggesting that once a threshold level of virus replication is attained in intestinal lymphoid tissues, the cascade of events precipitating the lethal PBj14 syndrome is triggered irreversibly.

In vivo cell and tissue tropism of SIVsmmPBj14-bcl.3.

To gain insight into the unique pathogenicity of simian immunodeficiency virus (SIV) variant PBj14, which produces an acutely lethal enteropathic syndrome in infected pigtail macaques, we investigated the cell and tissue tropisms of a highly pathogenic biologic clone (bcl.3) of SIVsmmPBj14. To compare the relative amount of viral antigen in lymphoid organs of infected macaques we used an objective semiquantitative immunohistochemistry (sQIHC) assay. We found that in all animals viral antigen load was greater in alimentary-associated lymphoid tissues (gut-associated lymphoid tissue [GALT], tonsil, mesenteric and retropharyngeal lymph nodes) than in non-alimentary-associated lymphoid tissues (spleen, thymus, inguinal and axillary lymph nodes). Moreover, in six of nine animals examined, virus load in GALT was greater than that in any other lymphoid tissue. To determine whether the acute pathogenicity and prolific replication of SIVsmmPBj14 might be explained by a broader in vivo cell tropism than is typical of SIVs, we used cell subset separation and nested PCR. We found that the primary target cells in mesenteric lymph node for SIVsmmPBj14 were CD4+ T lymphocytes. However, the virus also infected macrophages, as well as CD8+ T cells and B cells, albeit at low frequencies. These results suggest that alimentary lymphoid tissue localization rather than unusual cell phenotype tropism distinguishes the singular pathogenesis of SIVsmmPBj14.

Early pathogenesis of disease caused by SIVsmmPBj14 molecular clone 1.9 in macaques.

We have studied the early pathogenesis of infection by molecular clone 1.9 of SIVsmmPBj14 in pig-tailed and cynomolgus macaques. Like the uncloned PBj14 parent, SIVsmmPBj14-1.9 consistently induced an acute clinical syndrome characterized by behavioral depression, fever, profuse diarrhea, dehydration, lymphadenopathy, splenomegaly, and mucocutaneous exanthema that began at 7 days postinfection (DPI). The acute clinical disease coincided with a marked cell-associated and cell-free viremia, during which SIV p27 was demonstrated in 4 to 68% of circulating mononuclear leukocytes between 4 and 17 DPI. Also characteristic were monocytosis and reductions in CD4+ and CD8+ T lymphocytes, as well as CD20+ B lymphocytes. The most profound depletion occurred in the CD44hi subset of CD4+ T cells. Unlike animals infected previously with uncloned or biologically cloned PBj14, however, all SIVsmmPBj14-1.9-infected macaques survived the acute-phase disease to progress to a chronic, largely asymptomatic phase of infection. Recovery from the acute-phase disease correlated with down modulation of virus replication and the appearance of antibodies to SIV Env and Gag proteins. Similar to the PBj14 parent, PBj14-1.9 targeted to intestine, spleen, bone marrow, lymph node, and cerebellum. Saliva contained substantial quantities of infectious virus and no viral antibodies during the early phase of infection. By contrast, saliva from chronically infected animals usually contained antibodies but no virus. This study extends previous work demonstrating that the acute clinical syndrome produced by SIVsmmPBj14 in pig-tailed macaques represents a unique model of lentiviral pathogenesis.

Obstructive jaundice in rats results in exaggerated hepatic production of tumor necrosis factor-alpha and systemic and tissue tumor necrosis factor-alpha levels after endotoxin.

BACKGROUND: Obstructive jaundice (OJ) predisposes patients to postoperative sepsis. We determined whether OJ led to an increased endotoxin stimulated tumor necrosis factor-alpha (TNF-alpha) production by macrophage-rich organs and whether a lack of intraluminal gut bile contributed to this increased sensitivity. METHODS: Rats underwent laparotomy and common bile duct ligation and division (CBDL) or sham operation after they were given low-dose endotoxin or saline solution (NS). TNF-alpha levels in plasma, perfusate from the isolated perfused rat liver, and tissue from lung, spleen, and liver were measured 90 minutes later. An additional group underwent creation of a choledochal-vesical fistula and endotoxin administration. RESULTS: The plasma TNF-alpha, liver perfusate TNF-alpha, and the tissue TNF-alpha levels in liver, lung, and spleen were significantly elevated in the CBDL + endotoxin (CBDL + ETX) group compared with the SHAM + ETX and CBDL + NS groups (p < 0.05). The choledochal-vesical fistula group after endotoxin had plasma TNF-alpha levels only 27% that of the CBDL + ETX group (p < 0.05). CONCLUSIONS: OJ sensitizes macrophage-rich organs to produce larger amounts of TNF-alpha in response to endotoxin. This sensitization is not solely due to decreased intraluminal gut bile.