In vivo properties of attenuated rubella virus, “Cendehill”® strain

Summary Thein vivo properties of the Cendehill strain of attenuated rubella virus were further studied. The absence of seroconversion in rabbits inoculated subcutaneously was confirmed. When the virus was given intravenously most rabbits seroconverted and virus could be recovered from the organs of inoculated animals. Virulent virus given subcutaneously even in minute amounts, regularly evoked antibodies.When given by the combined intrathalamic, intraspinal and intramuscular routes, the Cendehill strain evoked antibodies in about one third ofCercopithecus monkeys in 17 to 19 days. When cortisone was given simultaneously with the virus the seroconversion rate in monkeys inoculated under similar conditions dropped to about 3%. A depressive effect of cortisone on the production of antibodies was also observed when virulent virus or the attenuated RA 27/3 virus were used.Comparative tests with virulent virus and Cendehill virus were also carried out in the following species: rats, mice, ferrets, gerbils and guinea-pigs. They all responded serologically to a single parenteral inoculation of wild virus. Mice, ferrets, gerbils and guinea-pigs failed to seroconvert when inoculated with Cende-hill virus. The rat was the only species in which a seroconversion could be regularly obtained.     

Morphological studies on equine arteritis virus

Summary Electron microscopy of purified preparations of equine arteritis virus (EAV) revealed enveloped, spherical particles with an average diameter of 55 m. The envelope was found to carry tiny projections on the surface, 3 to 5 m in length. No detailed structure of an internal component could be seen.In sections of EAV infected BHK cells 24 hours after inoculation, viral particles were shown to bud from the cytoplasmic matrix into cisternae of the endoplasmic reticulum, the process starting in the vicinity of the Golgi apparatus. The mature particles had an average diameter of 50 m, with an inner core measuring 25m.This study was supported by grant no. B70-16X-744-05-6068-19195 from the Swedish Medical Research Council.     

Comparison of the properties of five pox virus strains isolated from monkeys

Summary Five strains of monkey pox viruses were compared with respect to their cultural characteristics in primary and continuous cell cultures and the lesions developed in embryonated eggs and in rabbit skin as well as to their hemagglutinating activity.Four strains (Copenhagen 65-31 65-32 and 7-61) appeared to be similar in their properties. The cytopathogenic effect (CPE) was identical to that induced by vaccinia virus. There was no detectable virus multiplication in an pig kidney cell line (PEK). All four strains produced small, white, compact, hemorrhagic pock-like lesions on the chorioallantoic membrane.The strain 64–7275, isolated from healthy monkeys kidneys, had all properties of variola virus. It multiplied in the PEK cell line with a CPE. The lesions on the CAM were more compact without hemorrhage. In rabbit skin no detectable reaction occurred after infection with this strain.     

Histochemical staining of clonal mammalian cell lines expressingE. coli β galactosidase indicates heterogeneous expression of the bacterial gene

An evaluation has been made of the E. coli -galactosidase (-gal) gene for use as a reporter gene in mammalian cells in culture. We have adopted a histochemical procedure which enables identification of those cells within a population that express the introduced bacterial gene. Data is presented concerning the sensitivity of the histochemical method relative to an immunological method of detection. It has been found that several clonal cell lines generated after transfection of human 293 cells with a Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter--gal construction are mosaic for expression of the introduced mini-gene. Furthermore, after treatment of these clonal cell lines with the nucleoside analog 5-aza-cytidine (5-aza-C), an increase in production of -gal under control of this promoter element was observed.     

Synthesis of Seoul virus RNA and structural proteins in cultured cells

Summary. Seoul virus is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). The virion has a tripartite (S, M, and L) negative-stranded RNA genome, which is characteristic of the family Bunyaviridae. However, the molecular basis of virus replication is not well known. We established a Northern blot hybridization (NB) procedure using digoxygenin-labeled RNA probes, to quantitate the hantaviral plus- and minus-strand RNAs separately. Virus RNA replication was analyzed in infected Vero E6 cells. When the Vero E6 cells were infected with Seoul virus strain KI-83-262 (KI) at m.o.i.=0.25, the plus-strand RNA was detected within 1h post-infection (hpi), and the minus-strand RNA was detected subsequently. Using laser confocal microscopy, the nucleocapsid protein (NP) was detected within 2hpi, and accumulated as scattered granules in the cytoplasm until 24hpi. In contrast, the G2 protein first appeared at 8hpi, was immediately transported to the Golgi, and accumulated in the Golgi until 24hpi. Infectious virus particles were released into the medium at 24hhpi. These findings indicate that hantavirus RNA replication starts with the appearance of NP at 2hpi, glycoproteins then accumulate gradually in the Golgi, and virion formation is initiated once the viral RNAs and proteins have accumulated.Received October 10, 2002; accepted April 25, 2003 Published online July 17, 2003     

Virus-specific RNA formed in Newcastle disease virus-infected cells after suppression of protein synthesis by cycloheximide

Summary In Newcastle disease virus-infected cells the accumulation of virus-specific RNA is prevented if protein synthesis is inhibited by cycloheximide at an early stage of infection. At a later stage cycloheximide enhances the synthesis of smaller (18–35 S) virus-specific RNA while the synthesis of 49 S RNA is suppressed. This is accompanied by a corresponding change in the relative amounts of plus and minus RNA strands: the proportion of plus strand is sharply decreased. A possible significance of the phenomenon for the regulation of virus-specific RNA synthesis is discussed.     

Vascular clearance of Venezuelan equine encephalomyelitis viruses as a correlate to virulence for rhesus monkeys

Summary The epizootic Trinidad donkey strain of Venezuelan equine encephalomyelitis virus (VEE) was cleared slowly from the circulation of rhesus monkeys following intravenous inoculation, while the live, attenuated vaccine strain, TC-83, was cleared rapidly. The efficient clearance of TC-83 vaccine may be a factor in the lower viremia and benign course of TC-83 virus infection in rhesus monkeys.With 1 FigureIn conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals, as promulgated by the Committee on Revision of the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, National Research Council. The facilities are fully accredited by the American Association of Accreditation of Laboratory Animal Care.     

Induction of β-family chemokines mRNA in human embryonic astrocytes by inflammatory cytokines

The cellular infiltration found during CNS inflammation consists of monocytes and activated T cells, suggesting the presence of cell-specific chemotactic signals during inflammatory responses. Astrocyte chemokine expression might contribute to site-specific leukocyte infiltration within the CNS. To investigate the factors that regulate astrocyte chemokine expression, we examined the ability of human fetal astrocytes to induce -family chemokine mRNA. Astrocyte-derived monocyte chemoattractant protein-1 (MCP-1), RANTES, macrophage inflammatory protein-1 (MIP-1), and MIP-1 mRNA were easily induced by lipopolysaccharide and/or the proinflammatory cytokines (IFN and/or TNF-), respectively. Addition of both IFN and TNF- together did not lead to an additive effect but resulted in the inhibition of MCP-1 and MIP-1 mRNA expression, indicating that interaction between chemokines and cytokines may play a key role in regulating the local immune response of resident and infiltrating cells at the site of lesion. Interestingly, ultraviolet light-inactivated measles virus, but not cytomegalovirus, strongly induced expression of MCP-1, RANTES, MIP-1, and MIP-1 mRNA in human embryonic astrocytes, especially MCP-1 and MIP-1. An association occurs between the -family chemokine expression in astrocytes and inflammatory factors/virus, suggesting a possible role for -family chemokines in the pathogenesis of CNS inflammatory disease.     

Identification and quantitation of cortisol metabolites in rat liver homogenate and -slices

Summary Cortisol-1, 2-H³ was incubated with rat liver homogenate and/or rat liver slices in the presence of a NADPH-generating system. The following metabolites could be identified in adult male rats: -cortol, allo--cortol, 3-allo--cortol, 20-hydroxy-cortisol, 11, 17, 20, 21-tetrahydroxy-5-pregnan-3-one, 3-allotetrahydrocortisol, tetrahydrocortisol, trace amounts of allotetrahydrocortisol and two highly polar metabolites only partly identified. In female rats only tetrahydrocortisol, allotetrahydrocortisol and allodihydrocortisol could be detected in significant amounts.The radioactive metabolites mentioned above were localized and quantitated on paper chromatograms by a 4-radiochromatogram scanner. A nearly perfect correlation was found between these results so obtained and those given by liquid-scintillation counting of each metabolite after its elution from the paper.Part of this work was supported by grant n° 695 of the National Fonds voor Wetenschappelijk Geneeskundig Onderzoek.Stagiair of the Nationaal Fonds voor Wetenschappelijk Onderzoek.     

Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Dissemination in five French hospitals ofKlebsiella pneumoniae serotype K25 harbouring a new transferable enzymatic resistance to third generation cephalosporins and aztreonam

A strain ofKlebsiella pneumoniae K25 resistant to newer-lactam drugs was isolated in clusters in five hospitals in the Paris area. The MICs of ceftazidime and aztreonam (128 mg/l) were higher than that of cefotaxime (16 mg/l) for the strain but when measured in the presence of clavulanic acid, they were 1 mg/l. The donor strains and derivatives produced a-lactamase with a pI of 7.75–7.8 and hydrolysing activity against a wide spectrum of-lactams similar to that of SHV-2 and SHV-3, but with significant hydrolysis of ceftazidime. This new enzyme could be designated SHV-4.     

Immunocytochemical characterization of porcine zona pellucida during follicular development

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 and ZP3. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 and anti-ZP3. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 and ZP3. In secondary follicles, distinct labelling with anti-ZP3 and weak labelling with anti-ZP3 could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 and ZP3. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 and anti-ZP3. Some granulosa cells showed staining for ZP3 and ZP3. The ZP displayed strong immunoreactivity for ZP3 and ZP3. Cells of the corona radiata were strongly immunopositive for ZP3 and ZP3. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 and ZP3 strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.     

Effects of delayed myelination by oligodendrocytes and Schwann cells on the macromolecular structure of axonal membrane in rat spinal cord

Summary The macromolecular structure of axonal membrane from dorsal funiculi of control and irradiated spinal cord of 45-day-old rats was examined with freeze-lracture electron microscopy. In control spinal cords, virtually all myelination is mediated by oligodendrocytes, and the internodal axonal membrane of these fibres displays highly asymmetrical partitioning of intramembranous particles (IMPs). The internodal P-face particle density is 2350 IMPs per m², whereas the E-face IMP density is 150 per m². In control dorsal spinal roots, myelination is mediated by Schwann cells, and the ultrastructure of the internodal axolemma of the myelinated fibres is similar to that displayed by myelinated fibres of dorsal funiculi. On the internodal P-face of Schwann cell-myelinated fibres the IMP density is 2350 per m², whereas on the E-face the density is 175 per m². Irradiation of the lumbosacral spinal cord at 3 days of age results in a glial cell-deficient region within the spinal cord such that myelination in irradiated dorsal funiculi is delayed and subsequent myelination is mediated by both oligodendrocytes and Schwann cells. By 45 days of age, dorsal funiculi of irradiated spinal cords are well populated with fibres myelinated by oligodendrocytes and Schwann cells. However, fibres myelinated by oligodendrocytes display very thin myelin sheaths whereas Schwann cell-myelinated fibres exhibit myelin sheaths with normal thicknesses. Internodal membrane of fibres myelinated by Schwann cells and oligodendrocytes exhibit similar macromolecular structure, with 2400 IMPs per m² on P-faces and 150 IMPs per m² on E-faces. Occasional large (>1.5 m diameter) axons without glial-Schwann cell ensheathment are observed. These axons display a high density of P-face particles (2000 per m²) and a moderate density (350 per m²) of E-face IMPs on their fracture faces. These results demonstrate that CNS fibres exhibit similar axonal membrane ultrastructure irrespective of whether they are myelinated by Schwann cells or oligodendrocytes, or whether myelination is delayed. Moreover, when myelination does not occur, the axolemmal E-face IMP density, which may be related to the density of voltage-sensitive sodium channels, is not reduced.     

Investigation of the “valency” of antibodies by means of azoproteins

Summary The valency of antibodies was studied by the method of exhaustion of antisera against mono-and diazoproteins, and subsequent cross reactions both with the antibodies left over in the supernatant fluid of the serum and with the precipitating and nonprecipitating antibodies isolated from the precipitate.It was proved that the antibodies interact with the antigens as multivalent compounds.The valency determined with regard to the azoproteins is dependent upon the number of groups introduced.Thus, bivalent antibodies correspond to monoazoproteins and trivalent ones to diazoproteins.The valency of antibodies is, evidently, determined by the structural similarity of the heterologous and the immunizing antigens as well as by the less complete specific conformity between the individual structural peculiarities of the antigen and its antibody.From the Tashkent Pharmaceutical Institute (Director-Docent M. A. Azizov)Presented by Active Member AMN SSSR N. N. Zhukov-Verezhnikov     

Heterotypic and homotypic re-inoculation of mice already latently infected with herpes simplex virus type 1

Summary Mice were infected at 4 weeks of age with a type 1 strain of herpes simplex virus (HSV) and re-infected 4 weeks later with either a type 1 or a type 2 strain of HSV. The virus used for first infection could be distinguished from that used later since it was resistant to phosphonoformic acid and formed syncytial plaques. Sites used for the second inoculation were as follows: at the site of primary infection, at a different site within the same dermatome or in the equivalent dermatome on the opposite side (also called remote site).Re-infection caused no detectable reactivation of the latent PFA resistant virus. After re-infection with a homotypic virus replication of the re-infecting virus was limited to the inoculation site. However after heterotypic re-infection the type 2 strain was occasionally isolated from the ganglia. Previous infection with the PFA resistant type 1 strain clearly reduced the ability of the homotypic or heterotypic strains to establish a latent infection. However, in a few animals ganglia were found to be latently infected with virus from both the first and second inoculations. Analysis of the results suggests that resistance to the establishment of a second latent infection in a ganglion is determined by the general immunity of the animal rather than immunity of the latently infected ganglion itself.     

Facilitation by 5-hydroxytryptamine of ATP-activated current in rat pheochromocytoma cells

The effects of 5-hydroxytryptamine (5-HT) on an inward current activated by extracellular ATP were investigated in rat pheochromocytoma PC12 cells. Under whole-cell voltage-clamp conditions 5-HT (10 M) reversibly enhanced the amplitude of the current activated by 30 M ATP. The enhancement may not be due to an increase in the number of functional channels because the current activated by 300 M ATP was not remarkably augmented compared with the current activated by 30 M ATP. The current enhancement by 100 M 5-HT was less obvious than that by 10 M 5-HT. When the current kinetics were compared, activation of the ATP-evoked current was accelerated to the same extent by either 10 or 100 M 5-HT, whereas deactivation was largely more accelerated by 100 M 5-HT. Propranolol (10 M), a 5-HT₁ receptor antagonist, or LY53857 (10 M), a 5-HT₂ receptor antagonist, exerted an agonistic effect: the ATP-activated current was facilitated by these compounds. Metoclopramide (10 M), a 5-HT₃ receptor antagonist, neither facilitated the ATP-activated current, nor blocked the current facilitation by 5-HT. Guanosine 5-O-(2-thiodiphosphate) (GDP[S]) (2 mM), the non-hydrolysable analog of guanosine 5-triphosphate (GTP), or K-252a (2 M), a protein kinase inhibitor, did not affect the facilitation by 5-HT of the ATP-activated current when they were included in the intracellular solution. The ATP-activated current pre-facilitated by 10 M dopamine was not enhanced by 10 M 5-HT. Similarly, the pre-facilitation by 5-HT attenuated the current enhancement by dopamine. The results suggest that 5-HT facilitates the ATP-activated channels through receptors that are not readily classified into conventional subclasses of 5-HT receptors. The reciprocal masking between the current facilitation by 5-HT and that by dopamine, combined with their sensitivities to the compounds involved in the intracellular solution, indicates that the facilitation by 5-HT may share not all, but some, common cellular mechanism with that by dopamine.     

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Summary A simplified procedure to isolate-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate-connectin is introduced. Separation of-connectin from-connectin is introduced. Separation of-connectin from-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that both- and-connectin consist of 60%-sheet and 30%-turn. It thus appears that the whole elastic filament of connectin has a folded-strand structure. Proteolysis of-connectin by calpain resulted in formation of-connectin and smaller peptides. The-connectin interacted with both myosin and actin filaments similarly to-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with-connectin (titin 1, TI) but only weakly with-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears that-connectin extends from the edge of M-line to the above epitope region in the I-band.     

Kombinationsformen der Arteriitis

Zusammenfassung Ausgehend von den UntersuchungenRössles zum Formenkreis der rheumatischen Gewebsveränderungen, wird an Hand von 4 eigenen Fällen die Problematik der morphologischen Differentialdiagnose zwischen rheumatischer Arteriitis, Periarteriitis (Panangiitis) nodosa und Thrombangitis obliterans unter Berücksichtigung der Literatur aufgezeigt. Mit der morphologischen Definition der verwendeten Begriffe fibrinöse Entzündung und proliferierende und granulierende Angiitis wird versucht, die drei Formen der Gefäßwandentzündung morphologisch abzugrenzen. Die überschneidungen der morphologischen Symptome und die Kombinationsformen werden aus Beschreibung und Abbildung einschlägiger veröffentlichter Fälle aufgezeigt. Aus dem Nachweis dieses Schrifttumsvergleichs und dem der Differentialdiagnose der 4 eigenen Fälle wird gefolgert, daß bei solchen nicht-klassischen Gefäßentzündungen die Annahme von Kombinationsformen richtiger ist als die immer stärkere Erweiterung des sog. rheumatischen Formenkreises.

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Summary With reference to the investigations ofRössle On the Classification of the Rheumatic Tissue Changes, the problems that are associated with the differential diagnosis of rheumatic arteritis, periarteritis (panangiitis) nodosa, and thromboangiitis obliterans are discussed, exemplified by the report of four cases, and by a review of the literature. By applying the morphological criteria of fibrinous inflammation, proliferative and granulomatous angiitis, an attempt is made histologically to differentiate these three forms of inflammation of the vascular wall. The overlapping of the morphological criteria and the combined forms that mav occur are illustrated in the four cases reported. As shown by comparing the cases in the literature, and as exemplified by the differential diagnosis of the four cases, the inference is made, that with such types of non-classical vascular inflammation, it is better to refer to combined forms than to the more diffuse so-called rheumatic classification.

     

Der gegenwärtige Stand der Mastzell-Forschung

Zusammenfassung Seit der Erstbeschreibung der MZ durchEhrlich (1877) ist die Kenntnis über die biologische Rolle dieser Zellen wesentlich erweitert worden; diese, mit metachromatischen Granula erfüllten Zellen haben nicht nur die Eigenschaft, Substanzen zu speichern (Mastzellen), sie haben auch die Fähigkeit, biologisch aktive Substanzen zu bilden, diese an das Erfolgsgewebe abzugeben (sekretorische Zellen, Heparinocyten, Histaminocyten) und sind sowohl in morphologischer als auch in funktioneller Hinsicht in den Bauplan der vegetativen nervösen Peripherie mit eingeschlossen (neurohormonale Zellen).