Improved bioassay for the detection of porcine tumor necrosis factor using a homologous cell line: PK(15)

Close similarities of various physiological parameters makes the pig one of the preferred animal models for the study of human diseases, especially those involving the cardiovascular system. Unfortunately, the use of pig models to study diseases such as viral hemorrhagic fevers and endotoxic shock syndrome have been hampered by the lack of the necessary immunological tools to measure important immunoregulatory cytokines such as tumor necrosis factor (TNF). Here we describe a TNF-bioassay which is based on the porcine kidney cell line PK(15). Compared to the widely used murine fibroblastoid cell line L929, the PK(15) cell line displays a 100–1000-fold higher sensitivity for porcine TNF-, a higher sensitivity for human TNF-, and a slightly lower sensitivity for murine TNF-. Using a PK(15) bioassay we can detect recombinant TNF- as well as cytotoxic activity in the supernatants of lipopolysaccharide (LPS)-activated porcine monocytes at high dilutions. This suggests that the sensitivity of the test should permit the detection of TNF in biological specimens such as pig serum.     

Follow-up results of osteosynthesis of medial femoral neck fractures with the dynamic hip screw

The operative treatment of subcapital femoral neck fractures of stages Garden III and IV in the young patient is still a problem. The current methods of osteosynthesis show a high rate of avascular necrosis of the femoral head. We controlled 24 patients with subcapital femoral neck fracture, who were treated with a dynamic hip screw (DHS). The average age of these patients is 55 years. They were examined after 30-89 months from the operation. All patients were assessed regarding clinical and radiological parameters. All of the six patients with femoral neck fractures of stages Garden I and II had no pain, their clinical results were good. In one of these patients we found a partial avascular necrosis of the femoral head. Four out of the 18 patients with femoral neck fractures of stages Garden III and IV had painful complications, 3 of them needed a reoperation. Fourteen patients of the group with femoral neck fractures of stages Garden III and IV had no pain and wer satisfied with the result. But in this group we found 7 patients with partial avascular necrosis of the femoral head. These results are discussed and compared with data published elsewhere.     

The MHC-haplotype influences primary, but not memory, immune responses to an immunodominant peptide containing T- and B-cell epitopes of the caprine arthritis encephalitis virus Gag protein

In this report, we describe a short peptide, containing a T helper- and a B-cell epitope, located in the Gag protein of the caprine arthritis encephalitis virus (CAEV). This T-cell epitope is capable of inducing a robust T-cell proliferative response in vaccinated goats with different genetic backgrounds and to provide help for a strong antibody response to the B-cell epitope, indicating that it may function as a universal antigen-carrier for goat vaccines. The primary immune response of goats homozygous for MHC class I and II genes showed an MHC-dependent partitioning in rapid-high and slow-low responses, whereas the memory immune response was strong in both groups, demonstrating that a vaccine based on this immunodominant T helper epitope is capable to overcome genetic differences.     

Expression in Escherichia coli and sequencing of the coding region for the capsid protein of Dutch maedi-visna virus strain ZZV 1050: application of recombinant protein in enzyme-linked immunosorbent assay for the detection of caprine and ovine lentiviruses.

Maedi-visna in sheep and caprine arthritis-encephalitis in goats are caused by two closely related and widespread lentiviruses. The infections are characterized by life-long virus persistence and slow induction of antiviral antibodies. The diagnosis is based on the detection of antiviral antibodies. We have used the polymerase chain reaction (PCR) to amplify a part of the gag gene coding for the entire capsid protein and for parts of the matrix and nucleocapsid proteins. Sequencing of the PCR fragment of the Dutch maedi-visna virus strain ZZV 1050 revealed 85 and 92% homology to the DNA and deduced amino acid sequences, respectively, of the distantly related Icelandic visna virus strain 1514. The respective homologies with caprine arthritis-encephalitis virus strain CO were 76 and 80%. The PCR fragment was cloned into pGEX-2T and expressed as a glutathione S-transferase fusion protein. The recombinant protein could be detected on immunoblots by using a monoclonal antibody and polyclonal antisera and was further purified by glutathione-based affinity chromatography. Enzyme-linked immunosorbent assay with purified recombinant fusion protein is shown to be a sensitive and specific diagnostic tool for the detection of lentiviral infection in goats and sheep.     

Immunohistology of the early course of lentivirus-induced arthritis

Caprine arthritis encephalitis (CAE) is a lentiviral infection of goats characterized by mononuclear cell infiltration of various tissues, most prominently the joints, mammary glands and, in young animals, the brain. We have investigated the early stages of arthritis induced by intracarpal and intravenous infection with molecularly cloned CAE virus. Analysis of the synovial membranes by immunohistological methods showed that the proportion of CD8⁺T cells peaked around day 12 post-infection. CD4⁺ T cells increased to a lesser degree. The relative proportion of B cells rose steadily post-infection. At 33 days post-infection, plasma cells accounted for over one third of all inflammatory cells in the inflamed synovium. Histopathologically, the arthritic lesions in the synovial membranes closely resembled those in membranes of animals with a 2-year history of chronic arthritis. Our observations indicate that this type of short-term experimental infection is particularly suitable for studying the pathogenesis of goat lentiviral infection. In addition, our observations support the view that a predominantly humoral (type 2) immune response may contribute to the pathogenesis of CAE.     

Direction selectivity of simple cells in cat striate cortex to moving light bars

Summary Quantitative estimates of the direction selectivities of 118 simple cells in response to moving light bars were expressed as a percentage calculated from the ratio of the response peaks: (preferred minus nonpreferred)/preferred. Virtually all simple cells were direction selective to some degree (mean direction selectivity 73.6%). Static-field plots to a stationary flashing bar were prepared from 74 of the 118 cells. Particular attention was given to the 42 cells with only two subregions in their static-field plot, one subregion responding at light on and the other at light off. It was concluded that interactive effects between subregions, whether synergistic or antagonistic, have little if any influence on the direction selective mechanism when the stimulus is a narrow light bar. Eighty two of the 118 cells were also tested with moving light and dark edges and of these 53 had response profiles with only two response peaks, one to the light edge and the other to the dark edge. Forty one of the 53 cells were each not only direction selective for both a light edge and a dark edge but also had a preferred direction for both edges that was the same as that for a light bar. Only two cells had preferred directions for both light and dark edges that were opposite to the direction preferred by the light bar. With one possible exception, every cell with two response peaks to moving edges and two subregions in the static-field plot showed a one-to-one correspondence between the ordinal sequence of the response peaks and the ordinal sequence of the subregions. Depending upon the polarity of the moving edge and the ordinal sequence of the subregions, the mean level of the direction selectivity to a moving edge was significantly below that to a narrow moving light bar. This reduction in the degree of the direction selectivity appears to be due to an interaction between the subregions leading to a reduction in the amplitude of the response in the preferred direction rather than a suppression of the direction selective mechanism that operates in the nonpreferred direction. Moving edges cause a weak interactive effect between the subregions that seems always to reduce the degree of the direction selectivity, never increasing it.     

Mechanisms of contour perception in monkey visual cortex. II. Contours bridging gaps

We have studied the mechanism of contour perception by recording from neurons in the visual cortex of alert rhesus monkeys. We used stimuli in which human observers perceive anomalous contours: A moving pair of notches in 2 bright rectangles mimicked an overlaying dark bar. For control, the notches were closed by thin lines so that the anomalous contours disappeared or half of the figure was blanked, with a similar effect. Orientation-selective neurons were studied. With the receptive fields centered in the gap, 23 of 72 (32%) neurons tested in area V2 responded to the moving "bar" even though the stimulus spared their response fields, and when the notches were closed, their responses were reduced or abolished. Likewise, when half of the figure was removed, the neurons usually failed to respond. Neurons with receptive fields within 4 degrees of the fovea signaled anomalous contours bridging gaps of 1 degree-3.5 degrees. The anomalous-contour responses were compared quantitatively with response field profiles and length-summation curves and found to exceed the predictions by linear-summation and summation-to-threshold models. Summation models also fail to explain the effect of closing lines which add only negligible amounts of light. In V1, only one of 26 neurons tested showed comparable responses, and only with a narrow gap. The others responded only when the stimulus invaded the response field and did not show the effect of closing lines, or failed to respond at all. The contour responses in V2, the nonadditivity, and the effect of closure can be explained by the previously proposed model (Peterhans et al., 1986), assuming that the corners excite end-stopped fields orthogonal to the contour whose signals are pooled in the contour neurons.     

Cross-sectional study of availability and pharmaceutical quality of antibiotics requested with or without prescription (Over The Counter) in Surabaya,Indonesia

Background  

Antimicrobial resistance is an increasing problem in developing countries and antibiotic use is widespread. Our previous surveys in Java, Indonesia, revealed that most antibiotic use was probably unnecessary or ineffective. The aim of this study was to explore a potential connection between resistance and substandard antibiotics sold in the area.     

Adherence of Acinetobacter baumannii strains to human bronchial epithelial cells

Acinetobacter baumannii is an important nosocomial pathogen, but the mechanisms contributing to its epidemicity and virulence are largely unknown. The organism is able to colonize skin and mucosal surfaces of the human host. Adherence of microorganisms to host cells is an important virulence factor as it is the initial step of the colonization process. In the present study, adherence of A. baumannii to human bronchial epithelial NCI-H(292) cells was examined by light and scanning electron microscopy. Thirty-seven strains were investigated including 18 from outbreaks, 16 not associated with outbreaks, and three for which an epidemic implication was unknown. Eight and 11 isolates belonged to European clone I and II, respectively. Two types of adherence were observed, dispersed adherence of bacteria to the cell, and adherence of clusters of bacteria at localized areas of the cells. Bacteria with dispersed adherence interacted with the epithelial cells through fimbriae, but were also entrapped by protrusions extending from the epithelial cells. Quantitative adherence varied considerably among strains but there was no significant correlation of the outbreak-associated strains with the percentage of infected cells. There was, however, a correlation between the clonal lineage and the percent of infected cells, with clone II being more adherent than clone I (P<0.05). Ten consecutive isolates from one outbreak were investigated to test whether adherence increased during passage among patients, but this appeared not to be the case. This study showed that A. baumannii adheres to human bronchial epithelial cells in vitro and that A. baumannii strains of clone II had a relatively high capacity for adhering to these cells.     

The viral RNase E prevents IFN type-I triggering by pestiviral single- and double-stranded RNAs

Interferon (IFN) type-I is of utmost importance in the innate antiviral defence of eukaryotic cells. The cells express intra- and extracellular receptors that monitor their surroundings for the presence of viral genomes. Bovine viral diarrhoea virus (BVDV), a Pestivirus of the family Flaviviridae, is able to prevent IFN synthesis induced by poly(IC), a synthetic dsRNA. The evasion of innate immunity might be a decisive ability of BVDV to establish persistent infection in its host. We report that ds- as well as ssRNA fragments of viral origin are able to trigger IFN synthesis, and that the viral envelope glycoprotein E^rns, that is also secreted from infected cells, is able to inhibit IFN expression induced by these extracellular viral RNAs. The RNase activity of E^rns is required for this inhibition, and E^rns degrades ds- and ssRNA at neutral pH. In addition, cells infected with a cytopathogenic strain of BVDV contain more dsRNA than cells infected with the homologous non-cytopathogenic strain, and the intracellular viral RNA was able to excite the IFN system in a 5′-triphosphate-, i.e. RIG-I-, independent manner. Functionally, E^rns might represent a decoy receptor that binds and enzymatically degrades viral RNA that otherwise might activate the IFN defence by binding to Toll-like receptors of uninfected cells. Thus, the pestiviral RNase efficiently manipulates the host's self–nonself discrimination to successfully establish and maintain persistence and immunotolerance.