Diagnosis of canine coronavirus infection using nested-PCR

The results of polymerase chain reaction (PCR) and nested polymerase chain reaction (n-PCR) assays for the diagnosis of canine coronavirus (CCV) infection, and the comparison with other diagnostic techniques, such as electron microscopy (EM) and virus isolation using A-72 cell line are reported. The study was carried out on 71 faecal samples of pups with enteritis. Of 71 samples examined 14 were positive in PCR, whereas 30 samples resulted positive in the n-PCR assay. CCV was detected by EM examination in only four out of 45 samples, and by virus isolation in three out of 30 samples n-PCR positive.     

Hepatic lymphoid aggregates in chronic hepatitis C and mixed cryoglobulinemia

Summary We have examined the clinical (virological and immunological), histological and immunohistochemical features of liver lymphoid nodules in hepatitis C virus-positive (HCV⁺)/mixed cryoglobulinemia (type II and III) and chronic hepatitis C. The clinical features of liver disease were found to be similar in all patients. In all these groups, liver lymphoid nodules were observed to a similar extent, being more frequent in earlier phases of liver disease and less in more advanced stages. These data were confirmed by studies in serial biopsy samples taken from individual patients with type II mixed cryoglobulinemia; the loss of lymphoid nodules with progression to more advanced histological stages of disease in these patients was accompanied by a decrease of the serum levels of cryoglobulins (although not statistically significant). By immunohistochemical analysis, the liver lymphoid nodules contained predominantly B cells with a CD5⁺/bcl2⁺/Ki67^– phenotype, which were always polyclonal in type III mixed cryoglobulinemia and chronic hepatitis C, and monoclonal in type II mixed cryoglobulinemia. These immunological features were consistent with an active role of the immune system in HCV-associated liver necro-inflammation. Only in type II mixed cryoglobulinemia was there a clonal restriction of B cells. The immunological profile (autoantibodies) and viral genotypes were examined in some patients, but no significant correlation with clinical and immunohistochemical findings was found; however, the prevalence of genotype 2a was significantly higher in type II mixed cryoglobulinemia than in type III and chronic hepatitis without cryoglobulinemia.     

Vascular pulsations stimulating nitric oxide release during cyclic exercise may benefit health: a molecular approach (review)

It is widely assumed that all exercise, regardless of the degree of difficulty or strenuousness, is good (no pain-no gain). In this speculative review of the literature and our research findings we highlight the fact that strenuous exercise taken to the extreme initiates an immune and vascular proinflammatory situation. However, mild cyclic exercise appears to produce health benefits for an individual. In part, this is due to vascular cyclic pulsations, occurring in mild exercise, stimulating constitutive nitric oxide synthase derived nitric oxide release. This in turn down-regulates vascular endothelial cells and immunocytes, as well as their interaction and inhibits the disassociation of NF-kappaB, preventing the production of proinflammatory cytokines. The nitric oxide so generated may even scavenge excess free radicals, preventing tissue damage. Prolonged strenuous exercise appears to limit these positive phenomena because of the maintained and prolonged high blood pressure that reduces the cyclic pulsations, limiting nitric oxide production. We further note that pathological conditions, i.e., Parkinson's disorder, may benefit from mild exercise, i.e., cyclic nitric oxide production, since the inactivity associated with this disease may lead to compromised nitric oxide production, initiating a progressive deterioration of tissues, including peripheral adrenergic neurons, due to a lack of adequate basal nitric oxide levels required to maintain the vascular microenvironment in a mild state of inhibition. We conclude that mild exercise represents an alternate and economical therapy to preserve health and/or diminish the rate of decline of the normal physiological processes that may even be associated with aging.     

Nitrosative stress in the bronchial mucosa of severe chronic obstructive pulmonary disease

BACKGROUND: Reactive nitrogen species, formed via the reaction of nitric oxide (NO) with superoxide anion and via (myelo)peroxidase-dependent oxidation of NO(2)(-), have potent proinflammatory and oxidizing actions. Reactive nitrogen species formation and nitrosative stress are potentially involved in chronic obstructive pulmonary disease (COPD) pathogenesis. OBJECTIVES: To investigate the expression of markers of nitrosative stress, including nitrotyrosine (NT), inducible NO synthase (iNOS), endothelial NO synthase (eNOS), myeloperoxidase (MPO), and xanthine oxidase (XO) in bronchial biopsies and bronchoalveolar lavage from patients with mild to severe stable COPD compared with control groups (smokers with normal lung function and nonsmokers). METHODS: The expression of NT, iNOS, eNOS, MPO and XO in the bronchial mucosa and bronchoalveolar lavage of patients was measured by using immunohistochemistry, Western blotting, and ELISA and correlated with the inflammatory cell profile. RESULTS: Patients with severe COPD in stable phase had higher numbers of NT(+) and MPO(+) cells in their bronchial submucosa compared with mild/moderate COPD, smokers with normal lung function, and nonsmokers (P < .01). iNOS(+) and eNOS(+) but not XO(+) cells were significantly increased in smokers with COPD or normal lung function compared with nonsmokers (P < .05 and P < .01, respectively). In patients with COPD, the number of MPO(+) cells was significantly correlated with the number of neutrophils (r = +0.61; P < .0025) in the bronchial submucosa. Furthermore, the number of NT(+) and MPO(+) cells was negatively correlated with postbronchodilator FEV(1). CONCLUSION: These data suggest that nitrosative stress, mainly mediated by MPO and neutrophilic inflammation, may contribute to the pathogenesis of severe COPD.     

Spatial and temporal properties of neurons of the lateral suprasylvian cortex of the cat

Neurons in the posteromedial lateral suprasylvian cortex (PMLS) of cats were recorded extracellularly to investigate their response to stimulation by bars and by sinusoidal gratings. Two general types of cells were identified: those that modulated in synchrony with the passage of drifting bars and gratings and those that responded with an unmodulated increase in discharge. Both types responded to contrast reversed gratings with a modulation of activity: the cells that modulated to drifting gratings modulated to the first harmonic of contrast reversed gratings (at appropriate spatial phase and frequency), whereas those that did not modulate to drifting gratings always modulated to the second harmonic of contrast reversed gratings. No cell had a clear null point. Nearly all cells were selective for spatial frequency. The preferred frequency ranged from 0.1 to 1 cycles per degree (cpd), and selectivity bandwidths (full width at half height) were around two octaves. Preferred spatial frequency was not correlated with receptive field size, but bandwidth and receptive field size were positively correlated. Preferred spatial frequency decreased with eccentricity, at about 0.05 octaves/deg. The response of all cells increased as a function of grating contrast up to a saturation level. The contrast threshold for response to a grating of optimal parameters was approximately 1% for most cells and the saturation contrast approximately 10%. The contrast gain was approximately 25 spikes/s per log unit of contrast. All cells were tuned for temporal frequency, preferring frequencies from approximately 3 to 10 Hz, with a selectivity bandwidth approximately 2 octaves. For some cells, the spatial selectivity did not depend on the temporal frequency and vice versa. Others were spatiotemporally coupled, with the preferred temporal frequency being lower at high than at low spatial frequencies, and the preferred spatial frequency lower at high than at low temporal frequencies. Previous results showing broad velocity tuning to a bar were replicated and found to be predictable from the combined spatial and temporal tuning of PMLS cells and the Fourier spectrum of a bar. Preferred temporal frequency steadily decreased with eccentricity, at 0.025 octaves/deg. The results for PMLS cells are compared with those of other visual areas. Acuity and spatial preference and selectivity bandwidth is comparable to all areas except area 17, where they are a factor of about two higher. Temporal selectivity in PMLS is as fine as observed in other areas. The possibility that PMLS cells may be involved with motion detection and detection of motion in depth is discussed.     

Ultrastructural alterations induced in quail ciliary neurons by postganglionic nerve crush and by Ricinus toxin administration, separately and in combination

The response to postganglionic nerve crush and Ricinus toxin administration by the ciliary neurons of the quail ciliary ganglion was investigated at the ultrastructural level. The toxin was either applied at the crush site on the postganglionic nerves or injected into the anterior eye chamber without any other operative intervention. Crush of postganglionic nerves without toxin administration and saline injection into the anterior eye chamber served as controls for the two toxin administration procedures. Postganglionic nerve crush caused a distinct chromatolytic reaction, accompanied by massive detachment of the preganglionic axon terminals from the ciliary neurons and loss of most of the synapses, both chemical and electrical. This process does not induce cell death and is reversible. Saline injection in the anterior eye chamber caused a moderate retrograde reaction in some of the ciliary neurons, presumably as a consequence of paracentesis. The changes consisted mainly of an increase of perikaryal neurofilaments with, at most, a minor detachment of the preganglionic boutons from a small portion of the cell body at the nuclear pole. Ricinus toxin administration induced neuronal degeneration following a pattern common to both delivery modes. The degenerative process consisted of disruption and detachment of polyribosomes from the rough endoplasmic reticulum, an increase of smooth cisterns and tubules, a dramatic increase of neurofilament bundles, compartmentalization of the cytoplasmic organelles and, finally, karyorrhexis and cell lysis. The final stages of Ricinus toxin degeneration involve a progressive accumulation of extracellular flocculo-filamentous material and cell lysis. After administration of Ricinus toxin to the crush site, ricin-affected neurons showed withdrawal of the preganglionic boutons from a portion of the ciliary neuron, especially at the nuclear pole. After Ricinus toxin injection into the anterior eye chamber, however, the bouton shell surrounding the affected ciliary neurons remained intact in the early stages of degeneration. Detachment of the preganglionic terminals and disruption of the cell junctions, therefore, is the consequence of nerve crush and not of the toxin itself.

This study demonstrates that quail ciliary neurons are a suitable model for experimental neuropathology and neurotoxicology.     

Distribution of acid alpha-naphthyl acetate esterase among human T lymphocyte subsets

Human T lymphocyte subsets, identified by means of OKT3, 4 and 8 monoclonal antibodies, were isolated by a fluorescence activated cell sorter (FACS IV) and analyzed for distribution of alpha-naphthyl acetate esterase (ANAE) activity. As compared to OKT8⁺ lymphocytes a higher proportion of OKT4⁺ lymphocytes was ANAE-positive exibiting a spot or dot-like pattern in the cytoplasm. OKT8 and 4 positive subsets showed a similar ANAE distribution in diffuse granular form. Although OKT4 and OKT8 populations presented a different ANAE dot-like reactivity, this marker did not allow as clear a distinction between them as that reported for T_G and T_M lymphocytes.     

Congenital afibrinogenaemia caused by uniparental isodisomy of chromosome 4 containing a novel 15-kb deletion involving fibrinogen Aalpha-chain gene

Among rare inherited deficiencies of coagulation factors, congenital afibrinogenaemia is characterised by the lack of fibrinogen in plasma. In the last few years, several genetic defects underlying afibrinogenaemia (mostly point mutations) have been described in the fibrinogen gene cluster. In this study, the molecular basis responsible for afibrinogenaemia in a Thai proband was defined. Point mutation screening was accomplished by directly sequencing the three fibrinogen genes. The impossibility to amplify fibrinogen Aalpha-chain gene (FGA) exons 5 and 6 suggested the presence of a homozygous deletion. A specific long-range PCR assay enabled the identification of a novel 15-kb deletion, representing the largest afibrinogenaemia-causing deletion described so far. Direct sequencing of the deletion junction allowed mapping of the breakpoints in FGA intron 4 and in the intergenic region between Aalpha- and Bbeta-chain genes. Since the mutation was inherited only from the mother and nonpaternity was ruled out, a maternal uniparental disomy (UPD) was hypothesised. UPD test, carried out with markers covering the whole chromosome 4, revealed that maternal isodisomy was responsible for homozygosity of the 15-kb deletion in the proband. The apparently normal phenotype of the proband, except for afibrinogenaemia, suggests that UPD for chromosome 4 is clinically silent. This represents the first case of a documented complete isodisomy of chromosome 4 causing the phenotypic expression of a recessive disorder. In silico analyses of the regions surrounding the breakpoints suggested that the 15-kb deletion might have originated from an inappropriate repair of a double-strand break by the nonhomologous end joining mechanism.