Hypocretin-2 (orexin-B) modulation of superficial dorsal horn activity in rat

The hypothalamic peptides hypocretin-1 (orexin A) and hypocretin-2 (Hcrt-2; orexin B) are important in modulating behaviours demanding arousal, including sleep and appetite. Fibres containing hypocretin project from the hypothalamus to the superficial dorsal horn (SDH) of the spinal cord (laminae I and II); however, the effects produced by hypocretins on SDH neurones are unknown. To study the action of Hcrt-2 on individual SDH neurones, tight-seal, whole-cell recordings were made with biocytin-filled electrodes from rat lumbar spinal cord slices. In 19 of 63 neurones, Hcrt-2 (30 n m to 1 μ m ) evoked an inward (excitatory) current accompanied by an increase in baseline noise. The inward current and noise were unaffected by TTX but were blocked by the P_2X purinergic receptor antagonist suramin (300–500 μ m ). Hcrt-2 (30 n m to 1 μ m ) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the majority of neurones. The sIPSC increase was blocked by strychnine (1 μ m ) and by TTX (1 μ m ), suggesting that the increased sIPSC frequency was glycine and action potential dependent. Hcrt-2 increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in a few neurones but had no effect on dorsal root-evoked EPSCs in these or in other neurones. Neurones located in outer lamina II, particularly radial and vertical cells, were most likely to respond to Hcrt-2. We conclude that Hcrt-2 has excitatory effects on certain SDH neurones, some of which exert inhibitory influences on other cells of the region, consistent with the perspective that hypocretin has a role in orchestrating reactions related to arousal, including nociception, pain and temperature sense.     

Oestrogen, progesterone, and androgen receptors in ovarian neoplasia: correlation between immunohistochemical and biochemical receptor analyses

AIM: To investigate the correlation between immunohistochemical and biochemical steroid receptor analyses by measurement of oestrogen, progesterone, and androgen receptor status in ovarian neoplasia. METHODS: Tissue samples were obtained from 27 ovarian neoplasms, including two borderline tumours. Immunohistochemical staining of the tissue slides was scored semiquantitatively, incorporating the intensity and percentage of positive staining (histo-score). Tumours with a histo-score of 10 or more were considered steroid receptor positive. The epithelial and stromal fractions of the tumours were analysed separately. To study the uniformity of receptor expression throughout a tumour, up to four samples were analysed. RESULTS: Immunohistochemical histo-scores of the oestrogen receptor in the epithelial fractions were significantly correlated with the biochemical oestrogen receptor values (r = 0.408). Androgen receptor status in the epithelial fraction was correlated with that in the stromal fraction (r = 0.741), while androgen receptor histo-scores in the epithelial fraction correlated with the biochemical assay values (r = 0.463). On biochemical analysis, 17 of the 27 ovarian tumours were oestrogen receptor positive and seven were progesterone receptor positive. On immunohistochemical analysis, eight tumours were oestrogen receptor positive and two were progesterone receptor positive. Biochemical analysis showed that 14 of the 26 tumours were slightly androgen receptor positive (10-50 fmol/mg protein), while all the others were negative. On immunohistochemical analysis, seven of the 26 tumours were androgen receptor positive. When two or more specimens from one tumour were analysed, marked differences in steroid status were found, especially in progesterone receptor and androgen receptor expression. Some parts of a tumour were steroid receptor positive, while other parts were negative owing to heterogeneity of expression. CONCLUSIONS: Immunohistochemical and biochemical analysis of steroid receptors in ovarian tumours correlated weakly or not at all. Heterogeneity of expression within a tumour and the presence of progesterone and androgen receptors in the stromal fraction partly accounted for this observation. Biochemical and immunohistochemical androgen receptor status was much lower than in previous reports.     

Molecular Characterization of Vancomycin-Resistant Enterococci from Hospitalized Patients and Poultry Products in The Netherlands

Vancomycin-resistant enterococci (VRE) pose an emerging health risk, but little is known about the precise epidemiology of the genes coding for vancomycin resistance. To determine whether the bacterial flora of consumer poultry serves as a gene reservoir, the level of contamination of poultry products with VRE was determined. VRE were genotyped by pulsed-field gel electrophoresis (PFGE), and transposon structure mapping was done by PCR. The vanX-vanY intergenic regions of several strains were further analyzed by sequencing. A total of 242 of 305 (79%) poultry products were found to be contaminated with VRE. Of these VRE, 142 (59%) were high-level-vancomycin-resistant Enterococcus faecium strains (VREF). PFGE revealed extensive VREF heterogeneity. Two genotypes were found nationwide on multiple occasions: type A (22 of 142 VREF [15%]) and type B (14 of 142 VREF [10%]). No PFGE-deduced genetic overlap was found when VREF from humans were compared with VREF from poultry. Two vanA transposon types were identified among poultry strains. In 59 of 142 (42%) of the poultry VREF, the size of the intergenic region between vanX and vanY was ~1,300 bp. This transposon type was not found in human VREF. In contrast, all human strains and 83 of 142 (58%) of the poultry VREF contained an intergenic region 543 bp in size. Sequencing of this 543-bp intergenic vanX-vanY region demonstrated full sequence conservation. Though preliminary, these data suggest that dissemination of the resistance genes carried on transposable elements may be of greater importance than clonal dissemination of resistant strains. This observation is important for developing strategies to control the spread of glycopeptide resistance.     

EEG asymmetries may be affected by cranial and Brain parenchymal asymmetries

Summary The plagiocephaly index, an index that reflects an underlying anatomic asymmetry of the brain, was assessed in ten schizophrenic patients and its values were correlated with the lateral distribution of quantitatively evaluated EEG. The correlations between the index and alpha power at F7 were significant, positive for frontal asymmetry (frontal bulging) and negative for occipital flattening. We then studied ten normal subjects in an attempt to illuminate the contribution of several cephalic and cranial variables to the imbalance of alpha-afterdischarges (AD) of VEP recorded at O1–O2. The asymmetry index of AD was computed and correlated with asymmetries of CT-derived measures of occipital bone thickness, occipital lobe width, mastoid area, and sulcal asymmetry (the asymmetry of intraparietal sulcus location from the longitudinal fissure). With the exception of the sulcal variable all measures significantly covaried with alpha AD. These findings caution that it may be important to determine cranial and brain parenchymal asymmetries where brain laterality is pertinent to studies of EEG.     

Morphometric study of histological changes in sublabial salivary glands due to aging process.

The sublabial salivary glands were studied by morphometric methods in 68 healthy volunteers to establish possible changes related to age in those tissue components that are affected in Sjögren''s syndrome and connective tissue diseases (and which might stimulate Sjögren''s syndrome). There was an increase in the amount of connective tissue and intralobular ducts with age and a corresponding decrease in acinar tissue. During the aging process changes in the intralobular ducts occurred: the outer and inner diameters of these ducts and the thickness of the epithelium decreased, but the ratio of the outer and inner diameters of the ducts remained constant. The amount of diffuse lymphoplasmacytic infiltrate and the vascularity of the tissue remains constant with age. In 15 of the subjects, however, discrete lymphocytic foci were seen and in six of these more than one focus/4 mm2 of salivary tissue was found, which has been described as suggestive of Sjögren''s syndrome. The volume percentage of lymphocytic foci is constant during the aging process. The histological features commonly used to diagnose Sjögren''s syndrome may occur in normal people, and false positive diagnoses will occur if these criteria are rigidly adhered to. Morphometry may provide more reliable criteria for distinguishing changes induced by inflammation and related to age which occur in salivary tissue.     

Chest radiography in general practice: indications, diagnostic yield and consequences for patient management

BACKGROUND: Chest radiography (CXR) is frequently performed in Western societies. There is insufficient knowledge of its diagnostic value in terms of changes in patient management decisions in primary care. AIM: To assess the influence of CXR on patient management in general practice. DESIGN OF STUDY: Prospective cohort study. SETTING: Seventy-eight GPs and three general hospitals in the Netherlands. METHOD: Patients (n = 792) aged > or =18 years referred by their GPs for CXR were included. The main outcome was change in patient management assessed by means of questionnaires filled in by GPs before and after CXR. RESULTS: Mean age of the patients was 57.3+/-16.2 years and 53% were male. Clinically relevant abnormalities were found in 24% of the CXRs. Patient management changed in 60% of the patients following CXR. Main changes included: fewer referrals to a medical specialist (from 26 to 12%); reduction in initiation or change in therapy (from 24 to 15%); and more frequent reassurance (from 25 to 46%). However, this reassurance was not perceived as such in a quarter of these patients. A change in patient management occurred significantly more frequently in patients with complaints of cough (67%), those who exhibited abnormalities during physical examination (69%), or those with a suspected diagnosis of pneumonia (68%). CONCLUSION: Patient management by the GP changed in 60% of patients following CXR. CXR substantially reduced the number of referrals and initiation or change in therapy, and more patients were reassured by their GP. Thus, CXR is an important diagnostic tool for GPs and seems a cost-effective diagnostic test.     

Public health impact of isoniazid-resistant Mycobacterium tuberculosis strains with a mutation at amino-acid position 315 of katG: a decade of experience in The Netherlands

A previous limited study demonstrated that Mycobacterium tuberculosis isolates with a mutation at amino-acid position 315 of katG (Delta315) exhibited high-level resistance to isoniazid and were more frequently resistant to streptomycin. In the present study, isoniazid-resistant M. tuberculosis isolates from 8,332 patients in The Netherlands (1993-2002) were screened for the Delta315 mutation. Isoniazid resistance was found in 592 (7%) isolates, of which 323 (55%) carried Delta315. IS6110 restriction fragment length polymorphism analysis showed that Delta315 isolates occurred in clusters, suggesting recent transmission, at the same frequency as isoniazid-susceptible isolates. In contrast, other isoniazid-resistant isolates clustered significantly less frequently. Delta315 isolates were high-level isoniazid-resistant, streptomycin-resistant and multidrug-resistant significantly more often, and may have a greater impact on public health, than other isoniazid-resistant isolates.     

High responsiveness of HLA-B57-restricted Gag-specific CD8+ T cells in vitro may contribute to the protective effect of HLA-B57 in HIV-infection

HLA-B57 has been shown to be associated with long-term asymptomatic HIV-1 infection. To investigate the biological mechanism by which the HLA-B57 allele could protect from HIV-1 disease, we studied both the number of CD8(+) T cells as well as CD8(+) T cell responsiveness directed to different HIV-1 Gag peptides presented by HLA-A2, -B8 or -B57. T cells specific for the HLA-B57 peptide KAFSPEVIPMF responded more readily and to a higher extend to antigenic stimulation in vitro than T cells specific for the HLA-A2 peptide SLYNTVATL or the HLA-B8 peptide EIYKRWII. This phenomenon was reproducible with T cells from individuals expressing HLA-B57 in combination with one or both of the other alleles and was persistent during long-term follow-up. Lower reactivity of A2- and B8-restricted T cells was not explained by mutations in the B8- or A2-restricted Gag-peptides. Moreover, no correlation between peptide mutation frequency and IFN-gamma production by the corresponding Gag-specific T cells was observed. In conclusion, functional differences were observed between T cells specific for HIV epitopes derived from the same protein presented by different HLA molecules. B57-restricted KAFSPEVIPMF-specific CD8(+) T cells have relatively high responsiveness, which could contribute to the protective effect of HLA-B57 in HIV infection.     

Three-dimensional fiber-deposited PEOT/PBT copolymer scaffolds for tissue engineering: influence of porosity, molecular network mesh size, and swelling in aqueous media on dynamic mechanical properties

Among novel scaffold fabrication techniques, 3D fiber deposition (3DF) has recently emerged as a means to fabricate well-defined and custom-made scaffolds for tissue regeneration, with 100% interconnected pores. The mechanical behavior of these constructs is dependent not only on different three-dimensional architectural and geometric features, but also on the intrinsic chemical properties of the material used. These affect the mechanics of the solid material and eventually of 3D porous constructs derived from them. For instance, poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) block copolymers are known to have mechanical properties, depending on the PEOT/PBT weight ratio in block form and on the molecular weight of the initial poly(ethylene glycol) (PEG) blocks. These differences are enhanced even more by their different swelling properties in aqueous media. Therefore, this article examines the influence of copolymer compositions in terms of their swelling on dynamic mechanical properties of solid material and porous 3DF scaffolds. The molecular weight of the starting PEG blocks used in the copolymer synthesis varied from 300 to 1000 g/mol. The PEOT/PBT weight ratio in the blocks used varied from 55/45 to 80/20. This corresponded to an increase of the swelling ratio Q from 1.06 to 2.46, and of the mesh size xi from approximately 9 Angstrom to approximately 47 Angstrom. With increased swelling, dynamic mechanical analysis (DMA) revealed a decrease in elastic response and an increase of viscoelasticity. Thus, by coupling structural and chemical characteristics, the viscoelastic properties of PEOT/PBT 3DF scaffolds may be fine tuned to achieve mechanical requirements for a variety of engineered tissues. Ultimately, the combination of 3DF and DMA may be useful to validate the hypothesis that mimicking the biomechanical behavior of a specific tissue for its optimal replacement is an important issue for at least some tissue-engineering applications.     

Kinetics of gene expression in murine cutaneous graft-versus-host disease

The kinetics of gene expression associated with the development of cutaneous graft-versus-host disease (GVHD) were examined in a mouse model of MHC-matched allogeneic hematopoietic stem cell transplantation. Ear skin was obtained from recipient mice with or without GVHD between 7 and 40 days after transplantation for histopathological analysis and gene expression profiling. Gene expression patterns were consistent with early infiltration and activation of CD8(+) T and mast cells, followed by CD4(+) T, natural killer, and myeloid cells. The sequential infiltration and activation of effector cells correlated with the histopathological development of cutaneous GVHD and was accompanied by up-regulated expression of many chemokines and their receptors (CXCL-1, -2, -9, and -10; CCL-2, -5, -6, -7, -8, -9, -11, and -19; CCR-1 and CCR-5), adhesion molecules (ICAM-1, CD18, Ly69, PSGL-1, VCAM-1), molecules involved in antigen processing and presentation (TAP1 and TAP2, MHC class I and II, CD80), regulators of apoptosis (granzyme B, caspase 7, Bak1, Bax, and BclII), interferon-inducible genes (STAT1, IRF-1, IIGP, GTPI, IGTP, Ifi202A), stimulators of fibroblast proliferation and matrix synthesis (interleukin-1beta, transforming growth factor-beta1), and markers of keratinocyte proliferation (keratins 5 and 6), and differentiation (small proline-rich proteins 2E and 1B). Many acute-phase proteins were up-regulated early in murine cutaneous GVHD including serum amyloid A2 (SAA2), SAA3, serpins a3g and a3n, secretory leukocyte protease inhibitor, and metallothioneins 1 and 2. The kinetics of gene expression were consistent with the evolution of cutaneous pathology as well as with current models of disease progression during cutaneous GVHD.