The Transdural Course of Radicular Spinal Cord Veins--A Microangiographical and Microscopical Study.

PURPOSE: This study focuses on the following questions: What are the morphological features at the transdural course of radiculomedullary veins? How are these short transdural segments that may harbour pathological arteriovenous shunts connected to the internal vertebral venous plexus? Is the conception of a reflux-impeding mechanism at the transdural segment indispensable and convincing? METHODS: A total of 102 radiculospinal veins were studied microscopically at various levels of the spinal canal using serial paraffin and semi-thin sections. In addition, 26 vessels were investigated microangiographically following orthograde (12) or attempted retrograde (14) opacification of the intradural venous segment with barium sulphate. After paraplast-embedding, contact-microradiographs were taken using high-resolution spectroscopic plates. RESULTS: At their transdural course, the veins showed narrowing of their lumen accompanied by changes in the vessel wall composition and a tortuous course. Two structurally distinct arrangements of the transdural segment could be identified: A slit type was seen in 60% of the veins studied and a bulge- or nodular type was seen in 35% of the veins. In total, 5% of cases could not be assigned to either one of these types. Reflux to radicular veins from the outside of the dura mater could be produced in 2 out of 14 specimens. The extradural venous plexus, which primarily receives the radicular vein, was composed more frequently of lacunar spaces rather than plexiform blood vessel convolutions. Rare observations were fibrotic, blind ending radiculomedullary veins and continuation of a distinct venous blood vessel after crossing the dura. CONCLUSIONS: Reflux from the epidural plexus to radicular veins is not reliably stopped at the dural level and possibly physiological. Different arrangements of the transdural course of the veins appear to be at least appropriate to modulate flow. The purpose for two different types of radicular vein exit is unclear. The clinical impact of disturbed reflux-control is uncertain, which is in stark contrast to the severe consequences resulting from dural arteriovenous shunts. The functional role of the probably predominant epidural venous plexus for the spinal cord blood circulation remains poorly understood.

Suppression of pulmonary innate host defence in smokers.

BACKGROUND: Smoking increases the susceptibility to pulmonary infection and is a risk factor for the development of chronic obstructive pulmonary disease (COPD). It is postulated that cigarette smoke suppresses the activation of the innate immune system in response to bacterial infection. METHODS: Using sensitive ex vivo analysis, the level of the endogenous antibiotic peptide human beta-defensin-2 (hBD-2) was measured in pharyngeal washing fluid and sputum from patients with community acquired pneumonia. The regulation of antibacterial host defence molecules was studied in vitro. The effect of cigarette smoke on the antibacterial activity of differentiated airway epithelium and the expression of host defence molecules was studied in an in vitro infection model. RESULTS: Current or former smoking was associated with significantly reduced hBD-2 levels in pharyngeal washing fluid and sputum from patients with acute pneumonia. Exposure of airway epithelium to smoke in vitro inhibited the induction of hBD-2 by bacteria. This correlated with decreased antimicrobial activity. This effect was mimicked by hydrogen peroxide, and catalase blunted the smoke-induced inhibition of epithelial host defence. CONCLUSIONS: Smoke exposure suppresses the induction of epithelial antibacterial host defences. These findings link smoking with increased susceptibility to infection. This mechanism may be important in the pathogenesis of pneumonia and COPD.

Neutrophil chemokines in cultured nasal fibroblasts.

BACKGROUND: To gain insight into the mechanisms responsible for tissue neutrophil immigration in sinusitis, primary nasal fibroblasts are analyzed for synthesizing and delivering neutrophil chemokines. METHODS: Primary nasal fibroblast cell culture was treated with tumor necrosis factor (TNF)-alpha concentrations of 20 and 200 ng/ml for 2, 8, 24 and 72 h. Chemokine concentrations in supernatants were determined by enzyme-linked immunoassay (ELISA) and chemokine mRNA expression in fibroblasts was measured by reverse transcriptase polymerase chain reaction (RT-PCR). Biological chemotactic activity was identified by three-step high-performance liquid chromatography (HPLC) and by bioassay measuring neutrophil chemotaxis in a single Boyden chamber system. RESULTS: Interleukin (IL)-8 and growth-related oncogene (GRO)-alpha were induced in nasal fibroblast culture by proinflammatory stimulus. After 24 h of stimulation neutrophil chemotactic activity only was detected for IL-8. Granulocyte chemotactic protein (GCP)-2 mRNA was already significantly up-regulated after 2 h of stimulation. CONCLUSION: Induction of IL-8 protein dominates chemokine synthesis 24 and 72 h after stimulation, whereas induction of GCP-2 mRNA seems to have a role in the early phase after 2 h of exposition with TNF-alpha.

Neutralization of human papillomavirus type 11 (HPV-11) by serum from women vaccinated with yeast-derived HPV-11 L1 virus-like particles: correlation with competitive radioimmunoassay titer.

Neutralization of human papillomavirus type 11 (HPV-11) has been demonstrated using serum and cervical secretions from primates vaccinated with virus-like particles (VLPs). Theoretically, neutralizing antibodies could protect women from HPV infection. The immunogenicity of a yeast-derived HPV-11 L1 VLP vaccine was tested in women. Serum specimens were evaluated for HPV-11 titer by competitive radioimmunoassay (cRIA) and for neutralization by use of the athymic mouse xenograft system. Analysis of serum from 104 subjects showed a dose response in HPV-11 cRIA titers and neutralization. Overall, 68 (82.9%) of 82 postimmunization serum specimens from VLP recipients were 100% neutralizing when used in the assay at a 1:50 dilution. Of 69 serum specimens, 63 (91.3%) with cRIA titers >200 milliMerck units per milliliter were neutralizing. Immunization with HPV VLPs elicits a vigorous serum immune response in a high percentage of women. The HPV-11 cRIA titer appears to be a surrogate marker for neutralization.

Detection of multiple human papillomavirus types in the lower genital tract correlates with cervical dysplasia.

Some human papillomavirus (HPV) types, such as HPV 16, are clearly associated with cervical dysplasia; however, the role played by other HPV types occasionally found in dysplasia is less certain. In addition, most methods used to detect HPV in clinical specimens cannot easily distinguish among more than two or three HPV types in a single specimen. Therefore, the significance of infection with multiple HPV types is not known. To address this question, we analyzed cervicovaginal lavage specimens from three cohorts of women for HPV DNA using a PCR/reverse blot assay system that permits the detection and partial quantitation of 26 genital HPV types. As expected, 94.1% of women who had dysplasia (n = 34) and 71.4% of women who had atypical squamous cells of uncertain significance (ASCUS) (n = 21) on cytology had HPV DNA detected compared to 54.5% of age matched women with normal cytology. HPV 16 DNA was detected in 35% of dysplasia patients compared to 9% of cytologic normals (P = 0.0044). Dysplasia patients had a mean of 3.29 (range 0-10) different HPV types detected compared to 1.04 (range 0-7) HPV types among those with normal cytology (P < 0.0001). These data support a possible role for multiple HPV types in the development or progression of cervical dysplasia.

Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge.

This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.

Propagation of human papillomavirus type 59 in the athymic mouse xenograft system.

Studies of human papillomavirus (HPV) infection are hampered by the lack of an adequate culture system. The athymic mouse xenograft system permits propagation of HPV, but only a few HPV types have been grown in this manner. To produce an oncogenic type for studies of HPV pathogenesis, a condylomata acuminata lesion from an immunosuppressed patient was used to prepare an infectious extract. The patient's lesion was shown by PCR analysis to contain abundant HPV 59 (an oncogenic type) and a lesser amount of HPV 11, a nononcogenic type. The extract was used to infect human foreskin tissue which was subsequently implanted into athymic mice. Characterization of implants recovered after 3-4 months of growth revealed the presence of HPV 59 exclusively. A second extract was prepared from one of these implants and used in an additional experiment to demonstrate the passage of HPV 59. Compared to the histopathologic changes induced by the prototypic nononcogenic HPV 11, infection with HPV 59 caused a higher degree of basal cell crowding, less acanthosis, minimal papillomatosis and less pronounced koilocytosis.

Influence of music on ratings of perceived exertion during 20 minutes of moderate intensity exercise.

The effects of different types of music on ratings of perceived exertion (RPE) during 20 min. of moderate intensity exercise were examined. 27 physically active subjects (age 18-30 yr.) performed a graded exercise test on a cycle ergometer to establish peak aerobic power (VO2peak). Four 20-min. exercise sessions were performed at a power output equal to 70% of VO2peak in a soundproof visually sterile room. The sessions were randomly assigned from the conditions of fast upbeat music, classical music, self-selected music, and no music. Heart rate, peripheral RPE, central RPE, and overall RPE were measured every 5 min. during exercise. No significant differences were found in heart rate among the four conditions indicating similar exercise intensity during each condition. Each type of music resulted in a reduced peripheral, central, and overall RPE when compared with the no-music condition. The data indicate that different types of music can act as an effective passive distractor during exercise and are associated with lower ratings of perceived exertion.

Detection of multiple human papillomavirus types in Condylomata acuminata lesions from otherwise healthy and immunosuppressed patients.

Condylomata acuminata, or genital warts, are proliferative lesions of genital epithelium caused by human papillomavirus (HPV) infection. HPV types 6 and 11 are most often detected in these lesions. Genital lesions consistent with exophytic condylomata acuminata were removed by excision biopsy from 65 patients, 41 of whom were otherwise healthy individuals (control group) and 24 of whom had conditions known to cause immunosuppression. Histologically, the majority of the lesions were typical condylomata acuminata. Three lesions removed from immunosuppressed individuals also contained foci of moderate to severe dysplasia (intraepithelial neoplasia grade II/III). A recently developed PCR and reverse blot strip assay was used to determine the specific HPV types present in the genital lesions. With a set of oligonucleotide primers based on the same primer binding regions used for the MY09 and MY11 primer pair, this PCR assay detects the presence of 27 HPV types known to infect the genital tract. All but two condylomata acuminata contained either HPV type 6 or 11. The predominant type in the lesions from control patients was HPV 6, while lesions from immunosuppressed types most often contained HPV 11. Condylomata acuminata from immunosuppressed patients contained significantly more overall HPV types than lesions from the control group. HPV types associated with an increased risk of dysplasia (high-risk types) were detected in 42 (64.6%) of the total of 65 specimens; 18 (43.9%) specimens were detected in the 41 otherwise healthy individuals, and 24 (100%) specimens were detected in the 24 immunosuppressed patients. HPV 16 was the most common high-risk type detected, found in 21 of 65 (32.3%) specimens. After HPV types 6 and 11, HPV types 53 and 54 were the most frequently detected low-risk HPV types. This study demonstrates that a high percentage of condylomata acuminata lesions contain multiple HPV types, including types associated with a high risk of dysplastic abnormalities. Further studies are needed to determine the influence these additional HPV types have on the epidemiology of genital tract HPV infections and the natural history of condylomata acuminata, especially in immunosuppressed patients.

Sodium bicarbonate ingestion does not attenuate the VO2 slow component during constant-load exercise.

We examined the effects of sodium bicarbonate ingestion on the VO2 slow component during constant-load exercise. Twelve physically active males performed two 30-min cycling trials at an intensity above the lactate threshold. Subjects ingested either sodium bicarbonate (BIC) or placebo (PLC) in a randomized, counterbalanced order. Arterialized capillary blood samples were analyzed for pH, bicarbonate concentration ([HCO3-]), and lactate concentration ([La]). Expired gas samples were analyzed for oxygen consumption (VO2). The VO2 slow component was defined as the change in VO2 from Minutes 3 and 4 to Minutes 28 and 29. Values for pH and [HCO3-] were significantly higher for BIC compared to PLC. There was no significant difference in [La] between conditions. For both conditions there was a significant time effect for VO2 during exercise; however, no significant difference was observed between BIC and PLC. While extracellular acid-base measures were altered during the BIC trial, sodium bicarbonate ingestion did not attenuate the VO2 slow component during constant-load exercise.

Characterisation of macrophage inflammatory protein-5/human CC cytokine-2, a member of the macrophage-inflammatory-protein family of chemokines.

A human monocyte-activating CC chemokine has been identified based on sequences in an expressed sequence tag (EST) cDNA database. The protein shows highest sequence identity to the macrophage inflammatory protein (MIP) group of chemokines, particularly MIP-3 (76.7%) and MIP-1alpha (75.4%), and has been named MIP-5. Model building confirms that the protein has a similar three dimensional structure to other chemokines, but has an additional third disulphide bond. Northern blot analysis and reverse-transcriptase PCR show that the mRNA for MIP-5 is expressed at a high levels in liver, intestine and in lung leukocytes. MIP-5 induces chemotaxis of human monocytes, T-lymphocytes and, to a lesser degree, eosinophils at nanomolar concentrations; it has no effect on neutrophil migration. In receptor-binding assays, MIP-5 shows IC50 values of 12 nM for competition with 125I-MIP-1alpha for binding to CC-chemokine receptor (CCR)1, and 2.5 nM for competition with 125I-MCP-3 for binding to CCR3. It shows no ability to compete with ligand for binding to the two interleukin (IL)-8 receptors (CXC-chemokine receptors 1 and 2) or to CCR2, CCR4 or CCR5. Consistent with this binding data, MIP-5 was only able to induce calcium fluxes in CHO cells stably transfected with CCR1 or CCR3.

A comparison of three fluid replacement strategies for maintaining euhydration during prolonged exercise.

The effectiveness of a new water delivery system (the Water-Del) was examined for maintaining euhydration compared to other fluid replacement strategies. Subjects (N = 10) performed three 60-min cycling trials (/50% of VO2max) in an environmental chamber (27 degrees C; RH = 50%). Trials were randomly assigned from Water-Del (metered: 200 ml water every 15 min), ad libitum every 15 min (ad-lib-15), and ad libitum (ad-lib). Total water intake (TWI), changes in plasma volume (delta PV), body weight (delta BW), thirst, skin temperature (Tsk), and heart rate (HR) were measured. A significant difference (p < or = .05) among trials was observed for TWI, with metered (1,200 +/- 0 ml) being greater than ad-lib-15 (358 +/- 48 ml) and ad-lib (522 +/- 106 ml). No significant difference was found for delta PV. A significant difference (p < or = .05) for delta BW was observed with metered (0.28 +/- 0.16 kg) being different than ad-lib-15 (-0.63 +/- 0.12 kg) and ad-lib (-0.34 +/- 0.14 kg). No significant differences (p > .05) were found for thirst, Tsk, or HR. The water-Del provides for greater fluid intake during exercise compared to other replacement strategies.

Totally automated analysis by robotized PrepStation and liquid chromatography: direct-sample analysis of felbamate.

A totally automated analysis of felbamate was developed by using a robotized PrepStation for extraction, followed by automated liquid chromatographic (LC) analysis and data reduction. This is one of the newer direct-sample analysis approaches by LC. Felbamate was a previously approved antiepileptic agent used to treat partial seizures with and without generalization and to treat Lennox-Gastaut syndrome in pediatric patients. However, due to the reported incidences of aplastic anemia, its clinical application was recently restricted to the treatment of the latter syndrome. The automated assay using Bench Supervisor, PrepStation, and LC, based on a previously developed manual method, used 200 microliters of serum standards, quality control, or patients' plasma. These were mixed with 600 microliters of internal standard (IS) W509 dissolved in acetonitrile for protein precipitation. After axial centrifugation and standing, aliquots of the clear supernatant were transferred and washed with hexane. Aliquots of the supernatant were transferred and injected into a high-performance liquid chromatograph (HPLC). HPLC parameters included an mu Bondapak C-18 column, phosphate/acetonitrile (8:2) as mobile phase, and detection at 214 nm. Retention times were 2.9 and 4.2 min for felbamate and IS, respectively. Calibration was linear for concentrations from 10 to 200 mg/L with r > 0.994. Precision studies showed coefficients of variation ranging from 2.7% to 8.8%. Correlation with the manual method showed that r = 0.934, slope = 1.048, intercept = -2.642, and n = 21. Phenobarbital coeluted with the IS. This study demonstrated the feasibility of using a robotized, automated method for monitoring felbamate, readily extended to monitoring other antiepileptic drugs with minimal modification.

A new variant of type IV glycogenosis with primary cardiac manifestation and complete branching enzyme deficiency. In vivo detection by heart muscle biopsy.

Type IV glycogenosis (polyglucosan body disease) is a rare congenital autosomal recessive inherited disorder, caused by lack of the branching enzyme (amylo-1,4-1,6 transglucosidase). This deficiency leads to storage of abnormal glycogen (polyglucosan bodies) in the liver and other tissues. The clinical onset of the disease is insidious with non-specific gastrointestinal symptoms followed by progressive hepatic failure. Usually patients die due to hepatic cirrhosis within 4 years. Sometimes myopathy of the heart and skeletal muscle is also present. In these cases, the clinical onset is often later than in typical cases. We report on two brothers with primarily cardiac manifestation and late onset of the disease. The older one started to suffer from progressive dilated cardiomyopathy at the age of 18 years, presenting with severe heart failure, hepatosplenomegaly, ascites and peripheral oedema. He also demonstrated myopathy and muscular atrophy especially of the shoulder and lower limbs. Initially he improved on medical therapy, but one year later severe heart failure recurred followed shortly afterwards by sudden cardiac death. Right heart and skeletal muscle biopsies were performed while he was alive. These, as well as the autopsy, revealed massive accumulation of polyglucosan bodies. In both heart and skeletal muscle, complete branching enzyme deficiency could be proven. His 14-year-old brother showed similar clinical findings of mild dilated cardiomyopathy. His muscle biopsy also revealed polyglucosan body myopathy. Thus, in young patients presenting with congestive cardiomyopathy, type IV glycogenosis has to be considered in the differential diagnosis.

Resistance to the Bacillus sphaericus toxin in cultured mosquito cells.

Increasing doses of Bacillus sphaericus toxin were used to select a toxin-resistant cell line from the Culex quinquefasciatus line. This resistant cell line was proven to be C. quinquefasciatus in origin by isozyme analysis and karyotype. The resistant line bound fluorescent-labeled toxin as did the unselected susceptible line. A high level of resistance was quickly achieved, and this level was maintained after 4 mo. culture in the absence of toxin.