Search for the genome of bovine herpesvirus types 1, 4 and 5 in bovine semen.

Bovine herpesvirus type 1 (BoHV-1) causes respiratory and reproductive disorders in cattle. Recently, bovine herpesvirus type 5 (BoHV-5) and bovine herpesvirus type 4 (BoHV-4) have been identified to be associated with genital disease. In this study, the presence of the genome of BoHV-1, BoHV-4 and BoHV-5 in bovine semen of Argentinean and international origin was analyzed by PCR assays. The most important finding of this study is the detection of the genome of BoHV-1 and BoHV-4 in semen of bulls maintained at artificial insemination centers. It is particularly relevant that BoHV-1 DNA was also identified in one sample of international origin suggesting the need for extensive quality control measures on international transport of bovine semen.

Detection of bovine leukemia virus specific antibodies using recombinant p24-ELISA.

We developed an indirect ELISA test (rp24-ELISA) for the detection of antibodies against the p24 capsid protein of bovine leukemia virus (BLV) in bovine serum samples. A recombinant p24 (rp24) was expressed in the Escherichia coli expression system, purified in a nickel charged resin and used as antigen in the ELISA test. A cut-off point was established considering the distribution of reactivity values obtained by rp24-ELISA on a set of 555 field serum samples that were stated as double positive (DP) or double negative (DN) by the combination of commercial agar gel immunodiffusion (AGID) assay and gp51-ELISA tests results. The rp24-ELISA showed good concordance with the agar gel immunodiffusion assay, when 710 serum samples were analyzed. In addition, rp24-ELISA demonstrated to be a precise assay with good repeatability and reproducibility and a better analytical sensitivity than AGID. From 67 discordant rp24-ELISA-AGID sera, 4 negative reactors were from the DP group, 28 positive samples were from the DN group and 35 positive samples were stated as negative by AGID but positive by the gp51-ELISA. Samples from this last subgroup were sent to an OIE reference laboratory where 28 samples were stated as negative, 5 samples were stated as positive and 2 as inconclusive. Complete concordance with blind previous results from an international proficiency test panel confirmed the capability of the assay to discriminate between infected and non-infected animals. In conclusion, the rp24-ELISA developed and standardized demonstrated to have good analytical characteristics to be considered for screening of BLV.

Severe infantile hyperkalaemic periodic paralysis and paramyotonia congenita: broadening the clinical spectrum associated with the T704M mutation in SCN4A.

The authors describe an Italian kindred with nine individuals affected by hyperkalaemic periodic paralysis associated with paramyotonia congenita (hyperPP/PMC). Periodic paralysis was particularly severe, with several episodes a day lasting for hours. The onset of episodes was unusually early, beginning in the first year of life and persisting into adult life. The paralytic episodes were refractory to treatment. Patients described minimal paramyotonia, mainly of the eyelids and hands. All affected family members carried the threonine to methionine substitution at codon 704 (T704M) in exon 13 of the skeletal muscle voltage gated sodium channel gene (SCN4A). The association between T704M and the hyperPP/PMC phenotype has been only recently revealed. Nevertheless, such a severe phenotype has never been reported so far in families with either hyperPP or hyperPP/PMC. These data further broaden the clinical spectrum of T704M and support the evidence that this mutation is a common cause of hyperPP/PMC.

Effects of hyperoxia on tumor necrosis factor alpha and Grobeta expression in newborn rabbit lungs.

Chemokines have been implicated in the pathogenesis of many inflammatory processes, including bronchopulmonary dysplasia in mechanically ventilated premature infants. We hypothesized that early expression of the proinflammatory cytokine, tumor necrosis factor alpha (TNFalpha), would be followed by later expression of the downstream chemokine, Grobeta, in the oxygen-injured newborn lung. Reverse transcriptase-polymerase chain reaction (RT-PCR) and ribonuclease protection assay (RPA) were used to assess TNFalpha and Grobeta mRNA expression in lung RNA samples from newborn rabbits exposed to > 95% O2 for 8-9 days, followed by 60% O2 for a further 2-4 weeks or from control rabbits exposed to air. Four lung samples per condition were collected every 2 days from day 0 to day 14, and at days 22 and 36. Rabbit alveolar macrophages (AM) stimulated in vitro with bacterial lipopolysaccharide served as positive controls ( n = 8). Grobeta mRNA expression in rabbit lung samples increased with oxygen exposure until day 8, then returned toward baseline levels. This corresponded to previously described elevations in neutrophil number in the lungs. TNFalpha mRNA expression in lung samples was below the limit of detection by RPA and showed no upregulation in hyperoxic lung samples by RT-PCR. TNFalpha activity was assessed in lung lavage ( n = 2 samples per condition per time) using an L929 cell line bioassay and was not increased in hyperoxic animals. The expression of Grobeta mRNA without antecedent or concurrent TNFalpha mRNA expression or activity makes it unlikely that Grobeta in the hyperoxic newborn rabbit lung is elaborated in response to a stimulus by TNFalpha.

SFEMG in ocular myasthenia gravis diagnosis.

BACKGROUND AND OBJECTIVE: In typical cases, the patient's history and clinical examination make it possible to diagnose ocular myasthenia gravis (OMG). But, in many cases a clear clinical picture is not present and OMG diagnosis is very difficult because gold diagnostic standard tests are not available. The diagnostic tests for OMG are usually unable to display a good sensitivity and specificity simultaneously. In this paper, we studied 86 cases submitted for suspected OMG. METHODS: The patients were studied clinically and with various other tests used in OMG diagnosis (SFEMG, repetitive nerve stimulation, Ab anti AChR titration, tensilon test). RESULTS AND CONCLUSION: SFEMG showed the highest sensitivity (100%) while Ab anti AChR showed the highest specificity (100%). To our knowledge this is the largest population of suspected OMG studied using most of the diagnostic parameters, reported in the literature.

Bcl-2 family gene expression during severe hyperoxia induced lung injury.

Exposure of the lung to severe hyperoxia induces terminal transferase dUTP end-labeling (TUNEL) indicative of DNA damage or apoptosis and increases expression of the tumor suppressor p53 and of members of the Bcl-2 gene family. Because cell survival and apoptosis are regulated, in part, by the relative abundance of proteins of the Bcl-2 family, we hypothesized that lung cells dying during exposure would show increased expression of pro-apoptotic members, such as Bax, whereas surviving cells would have increased expression of anti-apoptotic members, such as Bcl-X(L). The hypothesis is tested in the current study by determining which Bcl-2 genes are regulated by hyperoxia, with specific focus on correlating expression of Bax and Bcl-X(L) with morphologic evidence of apoptosis or necrosis. Adult mice exposed to greater than 95% oxygen concentrations for 48 to 88 hours had increased whole-lung mRNA levels of Bax and Bcl-X(L), no change in Bak, Bad, or Bcl-2, and decreased levels of Bcl-w and Bfl-1. In situ hybridization revealed that hyperoxia induced Bax and Bcl-X(L) mRNA in uniform and overlapping patterns of expression throughout terminal bronchioles and parenchyma, coinciding with TUNEL staining. Electron microscopy and DNA electrophoresis, however, suggested relatively little classical apoptosis. Unexpectedly, Western analysis demonstrated increased Bcl-X(L), but not Bax, protein in response to hyperoxia. Bax and Bfl-1 were not altered by hyperoxia in p53 null mice; however, oxygen toxicity was not lessened by p53 deficiency. These findings suggest that oxygen-induced lung injury does not depend on the relative expression of these Bcl-2 members.

Discordant pulmonary proinflammatory cytokine expression during acute hyperoxia in the newborn rabbit.

Newborn animals are resistant to oxygen toxicity. To investigate this phenomenon, the proinflammatory cytokines interleukin (IL)-1 beta, IL-8, and monocyte chemoattractant protein-1 (MCP-1) were measured during newborn rabbit hyperoxic lung injury. Pups were exposed to > 95% O2 for 8-9 days, followed by 60% O2 until 36 days of age. Lung lavage fluid, RNA, and tissue sections were collected at 0, 2, 4, 6, 8, 10, 12, 14, 22, and 36 days. Acute inflammation occurred by 6-10 days of hyperoxia, and fibrosis by 22 days. Northern hybridization of lung homogenates from hyperoxia-exposed pups showed elevated MCP-1 and IL-8 mRNA expression at 6 and 10 days, respectively, compared to age-matched, air-exposed controls. Lavage fluid IL-8 protein also peaked at 10 days, and was strongly correlated to neutrophil numbers in lavage. In situ hybridization revealed elevated IL-1 beta mRNA in macrophages, alveolar epithelial and interstitial cells at 2-10 days, elevated MCP-1 mRNA in similar cell types at 4-8 days, and elevated IL-8 mRNA in these cells and neutrophils at 4-10 days. IL-1 beta and IL-8 expression peaked during peak inflammation, whereas peak MCP-1 expression preceded macrophage influx. Comparing newborn and adult animals' chemokine response may help explain their differences in hyperoxia susceptibility.

Hyperoxia increases keratinocyte growth factor mRNA expression in neonatal rabbit lung.

Acute hyperoxic lung injury remains a major factor in the development of chronic lung disease in neonates. A critical step in the repair of acute lung injury is the proliferation of type II alveolar epithelial cells. Type II cell proliferation is stimulated by keratinocyte growth factor (KGF), an epithelial cell-specific mitogen. We sought to investigate KGF mRNA expression in relation to type II cell proliferation during hyperoxic lung injury. We studied a previously described newborn (NB) rabbit model of acute and chronic hyperoxic injury [C. T. D'Angio, J. N. Finkelstein, M. B. LoMonaco, A. Paxhia, S. A. Wright, R. B. Baggs, R. H. Notter, and R. M. Ryan. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L720-L730, 1997]. NB rabbits were placed in 100% O2 for 9 days and then recovered in 60% O2. RT-PCR was used to synthesize and amplify a 267-bp fragment of rabbit KGF cDNA from whole lung RNA. KGF mRNA expression was analyzed by ribonuclease protection assay, and mRNA abundance was quantified by phosphorimaging. Proliferating cell nuclear antigen immunohistochemistry was used on lung sections to identify proliferating cells. The rabbit partial cDNA sequenced was >95% homologous to human cDNA, and all amino acids were conserved. Whole lung KGF mRNA expression was increased 12-fold after 6 days of hyperoxia compared with control lungs, and remained increased throughout the 100% O2 exposure period. Proliferating cell nuclear antigen immunohistochemistry showed an increase in type II cell proliferation after 8-12 days of hyperoxia. NB rabbits exposed to hyperoxic injury exhibit increased whole lung KGF mRNA expression preceding type II cell proliferation. KGF may be an important mitogen in the regulation of alveolar epithelial repair after hyperoxic lung injury.

Fibronectin expression in bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic fibrotic lung disease of neonates. Fibronectin (FN), a component of the extracellular matrix, is increased in the tracheobronchial effluent of neonates destined to develop BPD. Pulmonary FN is derived from plasma and local cellular synthesis. In order to identify which pulmonary cells synthesize FN and to test the hypothesis that FN is more abundant in lungs with BPD, we examined the distribution of pulmonary FN by in situ hybridization (for mRNA) and immunohistochemistry (for protein) in neonatal autopsy lung specimens, comparing lungs with BPD to those without. We used a staging system in which BPD is characterized by disruption of alveolar architecture, severe vascular changes, airway epithelial necrosis, smooth muscle hypertrophy, and peribronchial fibrosis. FN mRNA and protein were found in vascular endothelium, macrophages, fibroblasts, vascular and airway smooth muscle, and chondrocytes as well as in the pulmonary parenchyma in neonates with and without BPD. Hyaline membranes, when present, immunostained intensely for FN protein. FN mRNA was not seen in airway epithelial cells of either group. FN mRNA and protein were first increased in early acute BPD with their levels appearing greatest during the chronic reparative stage of BPD. In long-standing "healed" BPD, lower levels of FN mRNA and protein were seen. These findings are consistent with the association of increased FN with adult fibrotic lung disease and the previously reported increase in FN tracheal effluent levels in infants with BPD. Our results suggest an important role for pulmonary cell-derived FN in the early inflammatory and later proliferative stages of BPD.

A case of Guyon syndrome with neuroapraxic block resolved after surgical decompression.

Guyon syndrome is the well-known ulnar entrapment at the wrist; usually surgical decompression improves the symptoms. Neurophysiological studies are essential to demonstrate the abnormality of ulnar nerve conduction at the wrist. We report a case of Guyon syndrome onset after 6 h of cycling. Neurophysiological study revealed ulnar neuroapraxic block at the wrist, and axonal impairment. Post-operative clinical and neurophysiological follow-up showed marked clinical improvement and neurophysiological resolution of abnormalities. To our knowledge, no cases of Guyon syndrome with neuroapraxic block are reported in the literature.

Italian multicentre study of carpal tunnel syndrome: study design. Italian CTS Study Group.

In September 1996, during a carpal tunnel syndrome (CTS) symposium, the Italian CTS Study Group was funded. The group designed a strict clinical and neurophysiological protocol for performing a wide multicentre study on idiopathic CTS in hands. In addition to the traditional evaluations, the group also adopted a validated patient-oriented measurement in order to obtain comprehensive and reliable data for the clinical picture. The study was designed to: (1) better assess the clinical picture of the CTS population, especially using new measurement tools (patient-oriented); (2) evaluate the sensitivity of an electrodiagnostic protocol; (3) re-evaluate the validity of a neurophysiological classification; and (4) evaluate the influence of social status and quantify some of the aspects regarding economic and social costs. The study design is described.

Neurophysiological classification and sensitivity in 500 carpal tunnel syndrome hands.

OBJECTIVES: To evaluate the following points about carpal tunnel syndrome (CTS): 1) characterization of a wide population; 2) sensitivity of electrodiagnostic tests, and particularly the contribution of disto-proximal ratio test; 3) validity of a neurophysiological classification developed by us. MATERIAL AND METHODS: Prospective study in 500 hands with CTS symptoms. Neurophysiological "standard" tests were always performed: sensory nerve conduction velocity (SNCV) first- and third digit-wrist and distal motor latency (DML). In "standard negative" hands disto-proximal ratio technique (R) was performed. Neurophysiological classification: Extreme CTS (absence of median motor, sensory responses), Severe (absence of sensory response, abnormal DML), Moderate (abnormal SNCV, abnormal DML), Mild (abnormal SNCV, normal DML), Minimal (abnormal R or other segmental/comparative test, normal standard tests). RESULTS: Sensibility of standard tests: 77%. R increased the diagnostic yield by 20%. CTS classification appeared reliable with significant differences between groups. CONCLUSION: R is a useful test, the classification may be useful in clinical/therapeutical decisions.

Changes in surfactant protein gene expression in a neonatal rabbit model of hyperoxia-induced fibrosis.

Lung injuries, including bronchopulmonary dysplasia, alter the surfactant system. We developed a newborn rabbit model of acute, followed by chronic, hyperoxic injury to study surfactant protein (SP) gene expression. Initial litters were exposed to >95% O2 until 50% died (LD50; 7-11 days old). Subsequent litters were exposed to >95% O2 for 8 days, followed by 60% O2 until 22-36 days. Controls were exposed to room air. LD50 animals displayed acute pulmonary inflammation, edema, protein leak, and surfactant dysfunction. These changes resolved, and fibrosis developed by 22 days. Whole lung SP-A mRNA expression (measured by membrane hybridization) was twice control levels at 4 days of >95% O2, with specific elevations in terminal bronchioles and type II cells at 4 days and the LD50 by in situ hybridization. Whole lung SP-B and SP-C mRNA were unchanged from control throughout exposure. However, in situ hybridization showed elevations in SP-B and SP-C mRNA in type II cells in inflamed areas at the LD50. SP mRNA alterations resolved by 22-36 days. The surfactant system recovers from acute hyperoxic injury, despite continued 60% O2 exposure.

Mononeuropathy of a distal branch of the femoral nerve in a body building champion.

A unique case of a body building champion with localised atrophy of the distal portion of the vastus lateralis muscle is reported; neurophysiological evaluation suggests a selective lesion of a distal branch of the vastus lateralis nerve (a motor branch of the femoral nerve). A necroscopic study in four cases was performed to better clarify the site and mechanism of nerve lesion. The data suggest that stretching and compression of the nerve has probably occurred during strenous exercise.

Low-rate nerve stimulation during regional ischemia in the diagnosis of muscle glycogenosis.

Five patients with muscle glycogenoses (Gly), 30 normal subjects (NS), and 52 disease controls received 3 Hz repetitive stimulation of the ulnar nerve for 4 min (P-LRNS) during regional ischemia and during normal circulation. During regional ischemia, the compound muscle action potential (CMAP) of NS showed 8.5% (SE: 1.0) facilitation after 1 min of P-LRNS and 4.8 +/- 1.5% facilitation after 4 min. Gly showed 8.1 +/- 1.0% facilitation after 1 min of P-LRNS but -58.6 +/- 6.9% depression after 4 min of P-LRNS. During normal circulation, in 18 NS and 4 Gly tested, the facilitation detected after 1 min of P-LRNS was unchanged until the end of stimulation. Compared with the mean +/- 2 SD of NS, individual values of CMAP depression were abnormal in all Gly patients already by the 3rd min of ischemic P-LRNS. Of disease controls, myasthenia gravis patients only showed a CMAP depression during ischemic P-LRNS which was distinguishable from that detected in Gly, being mainly induced by neuromuscular transmission block.

Surgical prognosis in carpal tunnel syndrome: usefulness of a preoperative neurophysiological assessment.

In this study 37 CTS hands underwent pre- and post-operative (15 days 2 and 6 months) evaluation of median nerve distal motor latency (DML) and sensory nerve conduction velocities (SNCV: I digit and III digit-wrist). Pre-operatively, CTS hands were classified as mild (decreased SNCV, normal DML), moderate (decreased SNCV, increased DML) or severe (absent sensory nerve action potentials, increased DML). Post-operatively, all hands presented clinical and neurophysiological improvement. The three groups of patient showed different clinical and neurophysiological responses to nerve decompression: we observed a rapid restitutio ad integrum in mild group, an improvement with normalisation in about 50% of the hands in moderate group, and a high percentage of restore of the sensory responses with no normalisation in severe group. A marked improvement of sensory symptoms was observed in all cases, but some degree of motor and/or sensory deficit was still present six months after surgery in more advanced cases. Preoperative electrophysiological assessment of median nerve function in CTS hands have an important role in predicting the outcome of surgical decompression.

Suprascapular nerve entrapment. Neurophysiological localization in 6 cases.

We report 6 cases of a suprascapular nerve lesion: 3 of them presented a nerve entrapment at the suprascapular notch and 3 at the spinoglenoid notch. Electrophysiological investigation localized the site of entrapment in all cases.