The microvasculature in the equine distal phalanx: implications for fracture healing.

OBJECTIVES: To describe the intra-osseous microvasculature of the distal phalanx of the equine forelimb with regard to its potential clinical relevance. METHODS: Eleven clinically normal equine forelimbs were used from six adult horses (range: 4 to 18 years old) euthanatized for reasons unrelated to lameness. In each limb the median artery was catheterized at the level of the carpus and India ink was injected under constant manual pressure. The limbs were frozen and 5 mm thick sections of the foot were cut in the sagittal, coronal, or transverse planes on a band saw. The sections were fixed in 10% formalin and cleared using a modified Spalteholz technique. Once cleared, the sections were photographed and the microvascular anatomy identified. RESULTS: The vascular injections revealed a rich intra-osseous microvascular supply of the distal phalanx originating from the medial and lateral palmar digital arteries. In addition, numerous smaller vessels from the terminal arch, formed by anastomosis of the medial and lateral palmar digital arteries, could be seen branching into the distal aspects of the distal phalanx. This distal portion of the distal phalanx appeared more densely vascularized than the proximal part in all specimens examined. CLINICAL SIGNIFICANCE: The increased vascularity demonstrated in the distal portion of the distal phalanx appears to correlate with improved fracture healing reported in this area. This may also explain why healing fractures which involve both the distal and proximal portions of the distal phalanx have been described as progressing from distal-to-proximal.

Expression of voltage-gated chloride channels in human glioma cells.

Voltage-gated chloride channels have recently been implicated as being important for cell proliferation and invasive cell migration of primary brain tumors cells. In the present study we provide several lines of evidence that glioma Cl- currents are primarily mediated by ClC-2 and ClC-3, two genes that belong to the ClC superfamily. Transcripts for ClC-2 thru ClC-7 were detected in a human glioma cell line by PCR, whereas only ClC-2, ClC-3, and ClC-5 protein could be identified by Western blot. Prominent ClC-2, -3, and -5 channel expression was also detected in acute patient biopsies from low- and high-grade malignant gliomas. Immunogold electron microscopic studies as well as digital confocal imaging localized a portion of these ClC channels to the plasma membrane. Whole-cell patch-clamp recordings show the presence of two pharmacologically and biophysically distinct Cl- currents that could be specifically reduced by 48 hr exposure of cells to channel-specific antisense oligonucleotides. ClC-3 antisense selectively and significantly reduced the expression of outwardly rectifying current with pronounced voltage-dependent inactivation. Such currents were sensitive to DIDS (200-500 microm) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (165 microm). ClC-2 antisense significantly reduced expression of inwardly rectifying currents, which were potentiated by hyperpolarizing prepulses and inhibited by Cd2+ (200-500 microm). Currents that were mediated by ClC-5 could not be demonstrated. We suggest that ClC-2 and ClC-3 channels are specifically upregulated in glioma membranes and endow glioma cells with an enhanced ability to transport Cl-. This may in turn facilitate rapid changes in cell size and shape as cells divide or invade through tortuous extracellular brain spaces.

[Three infants with epidural hematoma]. / Drie zuigelingen met een epiduraal hematoom.

Three infants, two boys aged 10 months and one girl aged 5 months, developed drowsiness and emesis within a few hours of a fall (after which they had not lost consciousness). Radiological examination revealed an epidural haematoma which was treated with emergency decompression. The children subsequently made a good recovery. An epidural haematoma is a potentially life-threatening event. More than 20% of all cases of epidural haematoma occur in childhood. In contrast with distinct symptoms in most adults and older children, an epidural haematoma in infancy can have a minimum of symptoms. The open fontanelle means compression occurs less rapidly, but consequently blood loss can be greater, leading to acute anaemia, hypovolaemic shock and consumptive coagulopathy.

Electronic communication in ethics committees: experience and challenges.

Experience with electronic communication in ethics committees at two hospitals is reviewed and discussed. A listserver of ethics committee members transmitted a synopsis of the ethics consultation shortly after the consultation was initiated. Committee comments were sometimes incorporated into the recommendations. This input proved to be most useful in unusual cases where additional, diverse inputs were informative. Efforts to ensure confidentiality are vital to this approach. They include not naming the patient in the e-mail, requiring a password for access to the listserver, and possibly encryption. How this electronic communication process alters group interactions in ethics committees is a fruitful area for future investigation.

Measurement of proteases in human subgingival dental plaque by fluorescence polarization.

Fluorescence polarization (FP) was examined as a rapid quantitative method to assay the proteases in subgingival plaque. Protease activity was measured by a decrease in FP at 0.5-min intervals over 5 min, using BODIPY-alpha-casein, a protein substrate. To quantitate activity, the least absolute deviation (LAD) slope for each assay was determined. Protease activity increased with the quantity of plaque (r=0.416, P<0.001). Of the 208 subgingival plaque samples, 87 contained detectable protease activity, with a mean of about 4 microg trypsin equivalents above a general background of 1 microg per site. The mean plaque protease activity of 89 paired samples from 15 individuals had decreased by 1.1 microg trypsin equivalents per site when measured at 8 months after tooth scaling and root planing (P<0.01). Most isolates of Porphyromonas gingivalis, Treponema denticola, Prevotella nigrescens, and Prevotella intermedia implicated in the pathogenesis of adult periodontitis exhibited high activity in the FP assay. The assay is rapid, quantitative and requires only one-tenth of the plaque sampled using a single pass with a Gracey curette at a single tooth site.

Simultaneous attainment of high electron and ion temperatures in discharges with internal transport barriers in ASDEX upgrade

Internal transport barriers have been demonstrated to exist also under conditions with T(e) approximately T(i) approximately 10 keV and predominant electron heating of the tokamak core region. Central electron cyclotron heating was added to neutral beam injection-heated ASDEX Upgrade discharges with a preexisting internal transport barrier, established through programmed current ramping leading to shear reversal. Compared to a reference internal transport barrier discharge without electron cyclotron resonance heating, the electron heat conductivity in the barrier region was found not to increase, in spite of a fivefold increase in electron heat flux, and also angular momentum and ion energy transport did not deteriorate.

Identifying the promoter region of the human brain sodium channel subtype II gene (SCN2A).

Sodium channel genes are highly regulated. To begin analyzing the human brain sodium channel subtype II gene, SCN2A, at the transcriptional level, we mapped multiple transcriptional start sites within a 397 bp stretch of the 5'-UTR and -flanking region. When inserted into a basic luciferase reporter vector, this 397 bp region can promote luciferase expression in transiently transfected neuroblastoma cells, but not in non-neuronal cells. Thus, this study provides the initial description of a functional promoter in a human voltage-gated sodium channel gene.

Mitogenic activity of the outer membrane of Treponema denticola.

The outer membrane (OM) was isolated by detergent extraction from Treponema denticola ATCC 35405, ATCC 33521 and ATCC 35404, representing serovars a, b and c, respectively, as well as from two fresh isolates of T. denticola. Strict precautions were undertaken against the introduction of contaminant lipopolysaccharide when the OM was isolated. The OM was active in mitogenic stimulation of C3H/HeOuJ mouse spleen cultures, but to a somewhat lesser extent than purified lipopolysaccharide (LPS) from Escherichia coli 055:B5. Polymyxin B only partially inhibited the response. Unheated OM abrogated mitogenic activity of E. coli LPS, but heated preparations enhanced the mitogenic activity of E. coli LPS, suggesting the presence of a heat-labile cytolytic factor associated with T. denticola OM in addition to a putative lipopolysaccharide and/or heat-stable lipoprotein.

Reducing medium for the cultivation of Porphyromonas gingivalis.

Porphyromonas gingivalis, a micro-organism frequently associated with human periodontal diseases, is commonly cultured under a reducing atmosphere in enclosed cabinets or glove boxes. This paper reports a modified Wilkins-Chalgren (MWC) medium for the culture of P. gingivalis under atmospheric conditions at 37 degrees C. On the basis of preliminary tests, WC broth was supplemented as follows: 500 mg/l cysteine hydrochloride; 250 mg/l sodium thioglycolate; and 1,000 mg/l sodium bicarbonate. Three P. gingivalis isolates (JKG-I, 33277 and A7436) showed very similar growth over 24 and 48 h periods when cultured both in WC medium in an anaerobic chamber and in the MWC medium. Culture of these isolates both in the anaerobic chamber and in the MWC medium yielded very similar data with respect to trypsin-like activity, total protease activity, and reactivity to monoclonal antibodies specific for P. gingivalis. Growth in the MWC medium varied over a 3- to 4-fold range for seven additional isolates (JKG-7, D86B6, D13B11, D84D2, JKG9, D67D9, D82F5) over 24 and 48 h periods.

BODIPY-alpha-casein, a pH-independent protein substrate for protease assays using fluorescence polarization.

BODIPY-alpha-casein is a new fluorescent protein substrate designed for fluorescence polarization studies to measure proteolytic activity at any pH over the range from pH 2 to 11. Kinetic protease assays in real-time were performed in 1 to 5 min using an FPM-1 fluorescence polarization instrument. A purified enzyme or bacterial culture was mixed with the BODIPY-alpha-casein in a buffer of an appropriate pH and the decrease in fluorescence polarization was automatically recorded at 0.5-min intervals. The initial decrease in fluorescence polarization with time was dependent on protease concentration. In 3-min assays at 37 degrees C, the sensitivity of detection was 8 mU for pepsin at pH 2.0, 1 mU for papain at pH 6.0, 0.6 mU for proteinase K at pH 7.4, and 2 mU for Streptomyces griseus alkaline protease at pH 11. Only 1-10 microliters of a growing culture was necessary to assay the protease activity of Porphyromonas gingivalis or Treponema denticola, oral bacteria that possess certain proteases on their surfaces. These assays have clinical applications, since certain pathogens use proteolytic activity as a virulence mechanism and differ from their nonpathogenic counterparts in this characteristic. Fluorescence polarization assays are simple, rapid, and reproducible.

Molecular epidemiology of an outbreak of hepatitis A in Italy.

The relationship of hepatitis A virus (HAV) isolates associated with an outbreak in Genoa, Italy, in 1993 was examined using direct sequencing of amplicons derived by antigen capture PCR (AC/PCR) from faecal samples of the infected persons. Forty samples recovered from 38 primary and two secondary cases were examined. The latter were household contacts of the primary cases. In addition, faecal material of 2 unrelated persons infected simultaneously with hepatitis A in Genoa were tested. The PCR products derived from the P1/P2 junction of the HAV genome were analysed. A 100% nucleotide identity was detected between the viral isolates originating from the primary as well as the secondary cases. The viral isolates recovered from the faecal samples of the two unrelated cases differed from the virus causing the outbreak as well as from each other. These results indicate that a single HAV strain caused the outbreak. The virus might have been transmitted by ingestion of contaminated food or water since all hepatitis A infected employees of the factory had eaten in the same canteen. Definitions of HAV genotypes are based on numerous genetic comparisons of different strains. The sequence comparison of the investigated isolates with published HAV sequences of the P1/P2 genome region revealed that the virus associated with the outbreak belongs to HAV subgenotype IA, whereas the strains recovered from the viral isolates of the unrelated cases belong to subgenotype IB.

Effect of outer membrane of Treponema denticola on bone resorption.

The effect of the outer membrane (outer sheath) of Treponema denticola on bone resorption was studied. Bone resorption was measured by the release of previously incorporated 45Ca from the shafts of the radii and ulnae of 19-day fetal rats. A treated-over-control ratio (T/C ratio) significantly greater than 1 indicated the stimulation of bone resorption by the test substance. The addition of outer membrane of T. denticola increased the release of 45Ca from the assay bones. The minimum concentrations required to yield significant 45Ca release from the assay bones were 15, 22 and 75 micrograms protein/ml for serovars a, b and c, respectively. These protein values corresponded to estimated lipopolysaccharide contents of 0.6, 0.8 and 2.8 micrograms/ml, based on 3-deoxy-2-manno-octulosonate analysis. Heat treatment of outer membrane (60 degrees for 30 min) did not change the effect on 45Ca release. Parathyroid hormone or prostaglandin E2, known to act synergistically with lipopolysaccharides in bone resorption, was also added to the assay system. Neither prostaglandin E2 at 10(-7) M nor parathyroid hormone at 40 ng/ml, by itself, increased 45Ca release. However, in the presence of 10 micrograms protein/ml of outer membrane of serovar b at 120 h, the T/C ratio was increased to 1.31 +/- 0.07 and 1.58 +/- 0.118, respectively. These results suggest that a lipopolysaccharide-like material is present in the outer membrane of T. denticola that may be responsible for bone resorption in the in vitro system.

Detection of HIV-1 provirus in bronchoalveolar lavage cells by polymerase chain reaction.

This study was undertaken to evaluate whether HIV-seropositive individuals harbor HIV provirus in cells obtained by bronchoalveolar lavage (BAL). BAL cells were obtained from 14 HIV-positive patients undergoing bronchoscopy for evaluation of acute pulmonary symptoms. Cells were fractionated into macrophage-enriched and lymphocyte-enriched populations. The quantity of HIV-1 proviral DNA in the unfractionated BAL cells and in each population of fractionated cells was determined following polymerase chain reaction (PCR) amplification. Detectable quantities (3-90 copies/100,000 cells) of HIV-1 proviral DNA were found in unfractionated BAL cells in 12 of 14 patients. In the other two patients, provirus was detected after a sevenfold enrichment of lymphocytes. Provirus was also detected in BAL macrophages from 8/14 patients although proviral content was significantly higher in the lymphocyte fraction (133 +/- 72 vs. 35 +/- 22 proviral copies, p = 0.03). No correlation was seen with the ability to detect provirus in lymphocyte- or macrophage-enriched fractions and clinical diagnosis (e.g., Pneumocystis carinii pneumonia). The data suggest that lymphocytes are the predominant cells that contain provirus found in the lungs, although macrophages may be infected in some patients.

Complement and antibody mediate enhancement of HIV infection by increasing virus binding and provirus formation.

Previous studies have shown that infection of complement receptor (CR2)-bearing cells with HIV pretreated with antibody (Ab) plus complement (C) resulted in increased virus expression. The current study was designed to determine whether C-mediated 'enhancement' of HIV-1 production was the result of increased virus infection of cells as assessed by provirus formation and virus binding. Virus was incubated with anti-HIV Ab and/or C and added to CR2-positive MT-2 cells. Increased virus expression by MT-2 cells correlated with increased numbers of HIV-immunofluorescent-positive cells at 24 and 48 h and higher levels of provirus detected 8-28 h after infection. MT-2 cells also bound threefold more Ab-plus-C-treated virus than untreated virus. Serial dilutions of C showed that high levels of C with Ab did not enhance but rather suppressed virus expression. Studies were also performed which showed that terminal C components C5 and C8 were not necessary for the enhancing effect. The increased binding of C-coated HIV to CR-positive cells has important implications for the fate of virus in vivo.

Sequence analysis of bovine lens aldose reductase.

The covalent structure of bovine lens aldose reductase (alditol-NADP+ oxidoreductase, EC 1.1.1.21) was determined by sequence analysis of peptides generated by specific and chemical cleavage of the homogeneous apoenzyme. Peptides, purified by reverse-phase high performance liquid chromatography were subjected to compositional analysis and sequencing by gas-phase automated Edman degradation. Aldose reductase was found to contain 315 amino acid residues. The enzyme is blocked at the amino terminus, and mass spectrometry was employed to identify the blocking acetyl group and to sequence the amino-terminal tryptic peptide. The aldose reductase was shown to contain no carbohydrate despite the fact that the enzyme contains the consensus sequence -Asn-Lys-Thr- for N-linked glycosylation. Comparative sequence analysis and application of algorithms for prediction of secondary structure and nucleotide binding domains are consistent with the view that aldose reductase is a double-domain protein with a beta-alpha-beta secondary structural organization. The NADPH binding site appears to be associated with the amino-terminal half of the enzyme. Modeling studies based on the tertiary structures of dihydrofolate and glutathione reductases indicate that the NADPH binding site begins at Lys-11 and continues with a beta-alpha-beta fold characteristic of nucleotide binding proteins.

The spectrum of vitamin B12 deficiency.

Low serum vitamin B12 levels are not uncommon in the elderly. Patients with vitamin B12 deficiency manifest a spectrum of clinical findings. Pernicious anemia and malabsorption syndrome are the usual causes of vitamin B12 deficiency. Pernicious anemia is confirmed by the presence of intrinsic factor blocking antibody or abnormal results on the Schilling test. Patients with neuropsychiatric symptoms of vitamin B12 deficiency may have a normal Schilling test and no evidence of macrocytic anemia. In such patients, vitamin B12 deficiency is confirmed by determining serum levels of homocysteine and methylmalonic acid.

Do not resuscitate decisions: discussions with patients.

The problem of psychological pain caused by discussions of do not resuscitate status with patients is addressed. Case histories of patients with such distress are given. We propose that not all patients should be informed of their do not resuscitate status, that the information about such status be given incrementally, and that the giving of further information be guided by the patient's reaction to earlier information. While some affirm the duty of the physician always to inform the patient about his or her do not resuscitate status, we affirm the duty of the physician to determine whether the patient wishes to enter into this discussion.

Retinal vein thrombosis in a patient with pernicious anemia and anticardiolipin antibodies.

A 45-year-old man with pernicious anemia presented with sudden loss of vision due to central retinal vein thrombosis, and was found to have anticardiolipin antibodies and a clotting disorder consistent with the presence of a lupus anticoagulant. He was treated with low dose aspirin and has remained free of recurrent thrombosis over a period of one year. The association between lupus anticoagulant and pernicious anemia is rare, having been reported in only one prior case.