Inter-laboratory validation of PCR-based HPV detection in pathology specimens

The detection and typing of human papilloma virus (HPV) in pathology specimens is gaining increasingly in importance. In the context of the initiative for quality assurance in pathology (QuIP) of the German Society of Pathology and the Professional Association of German Pathologists, four panel laboratories with experience and expertise in polymerase chain reaction (PCR)-based HPV detection were selected to establish an inter-laboratory trial. In a first step, these laboratories performed an internal testing of their own methodologies, which comprised DNA sequencing, multiplex nested PCR and hybridization techniques. Material from 39 samples including paraffin sections and DNA preparations of tissues and plasmids were evaluated by each panel institute according to their own protocols. Despite the different methodologies, a high degree of inter-laboratory reliability was achieved. In this report, we summarise the results. Pretested specimens are available for the external trail and can be ordered from the steering institute via provitro GmbH Berlin (). Supplementary data are online available at (rubric "Forschung"), which includes a web-based photo gallery of HPV-associated lesions and their potential association with specific virus types. The initiative is intended to foster the quality assurance of molecular HPV analysis in pathology and its correlation with morphological changes.     

Transfer of tumor specific immunity with “immune” RNA: prospects for the treatment of human cancer

Summary Ribonucleic acids extracted from specifically sensitized lymphoid cells (I-RNA) have been shown to transfer specific immunoreactivity to normal non-immune lymphoid cells. Evidence for the transfer by I-RNA, of immune responses to tumor-associated antigens of animal and human neoplasms, in vivo and in vitro, is reviewed. Results obtained in our laboratory and in other laboratories indicate that xenogeneic, allogeneic and syngeneic I-RNA extracts mediate specific cytotoxicity to tumor cells, in vitro, and mediate transplantation resistance and tumor rejection responses in vivo. Our results suggest that I-RNA preparations fail to elicit immune responses directed against self antigens. By contrast, I-RNA's directed against non-self tumor-associated antigens appear to induce lymphocytes to effect specific anti-tumor immune responses. The mechanisms responsible for the failure of I-RNA to initiate immune responses against self antigens are not known at present and demand investigation.Preliminary results of a clinical Phase I trial of immunotherapy with xenogeneic I-RNA in selected cancer patients are reviewed. I-RNA might offer promise as a new modality for the immunotherapy of human cancer.Recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft     

Disturbance of delayed match-to-sample in macaques by tetanization of anterior commissure versus limbic system or basal ganglia

Summary Three pig-tailed macaques were trained to select (match) from a pair of colored images that which they had seen (sample) and responded to 5–15 s previously. The anterior commissure (AC) and/or its radiation, various loci in basal ganglia, hippocampal formation and control areas, (splenium of corpus callosum, precentral gyrus, insular cortex), totalling 40 loci, were each tetanized for 4 s during presentation of the sample image, during the delay period, or when the monkey was required to select the matching image. For several loci in the hippocampal formation tetanization at any phase of the task reduced matching to chance levels and gave evidence of electrical after-discharge; but other comparable hippocampal loci had little or no effect. Response to sample or match stimuli were absent during tetanization of basal ganglia or anterior commissure. When finally made, upon cessation of tetanization, responses were equally correct for basal ganglia and control sites, but for AC were at chance levels.     

Effects of sleep deprivation on the activity of selected metabolic enzymes in skeletal muscle

Summary The present study gives the results of a comparison of the recorded and true tibia-calcaneal angles in 17 normal subjects and in 14 patients with abnormally hypoextensible non contracting triceps. 1. For a minimal passive torque, the difference between true and recorded angles varied considerably from one individual to another. The means and ranges for the two groups were respectively: –8 (+7, –21) and –7 (+5, –20). 2. When the passive torque increased as a result of slow passive lengthening of the muscle, the true curve was steeper than the recorded one, owing to differences between the two angle measurements. For each of the two groups the differences in means and ranges were respectively: 6 (0, +13.5) and 8 (3, 12). 3. Subjects made isometric voluntary contractions of the triceps surae at fixed angles which corresponded to step by step muscle lengthening. The resulting true curve was much steeper than the recorded curve. The differences in means and ranges were: 7 (1.5, +15) in children of the two groups and respectively 3 (0, +9) and 12 (10, 14) in adults of the two groups. The present results show that this methodology was the only reliable way of correctly obtaining passive and active torque-angle curves, measuring differences between subjects, appreciating the effects of treatments and these by ascertaining whether or not trophic muscle regulation was defective.     

In vitro susceptibility ofHaemophilus influenzae to cefaclor,cefixime, cefetamet and loracarbef

The susceptibility of 2,212Haemophilus influenzae isolates cultured in UK clinical laboratories in 1991 was determined for four orally-administered -lactam drugs. These isolates included 1,893 ampicillin-susceptible, 191 -lactamase-positive and 128 ampicillin-resistant, -lactamase-negativeHaemophilus influenzae. While 150 (6.8 %) isolates were resistant to cefaclor (MIC 16 mg/l) and 85 (3.8 %) to loracarbef, all were inhibited by 2 mg/l cefetamet and 1 mg/l cefixime and were therefore susceptible to these agents. Ranges and modes of inhibition zone diameters and MICs indicated that the susceptibility of a variable proportion of the 191 -lactamase-positive isolates to cefaclor, loracarbef and cefetamet was reduced compared with the fully susceptible population. In contrast, a major reduction in susceptibility to all four antimicrobial agents was seen among the 128 ampicillin-resistant (MIC 1–64 mg/l) -lactamase-negative isolates such that these accounted for 53 % and 67 % of the total number of organisms resistant to cefaclor and loracarbef respectively. In addition, 23 of 25 isolates inhibited only by 1 mg/l cefetamet and all eight inhibited only by 0.5 mg/l cefixime showed this type of resistance to ampicillin. Results indicate the importance of detecting non--lactamase-mediated resistance to ampicillin and any concomitant diminished susceptibility to other -lactam drugs.     

Recognition by peptide mapping of three different structural groups of outer membrane protein YOP-1 of Yersinia enterocolitica and Yersinia pseudotuberculosis

The structural relation of YOP-1 of european and american Yersinia enterocolitica serotypes O3, O9, O5, 27, and O8 and O20, respectively, and Y. pseudotuberculosis serotypes I, II, and III was compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping using Staphylococcus aureus protease V8. Apparent molecular weights of YOP-1 ranged from 206,000 (O3) to approx. 180,000 (O8). According to their respective peptide maps YOP-1 of the european and american Y. enterocolitica serotypes and Y. pseudotuberculosis serotypes could be assigned to three different groups. Evaluation of several isolates of Y. enterocolitica serotypes O3, O9, and O8 by peptide mapping indicated that YOP-1 is conserved within a serotype. However, one serotype O8 isolate differed from the consensus peptide pattern of the other serotype O8 and O20 isolates. The similarity of the peptide patterns of Yersinia serotypes which predominate in certain geographical locations, i. e., european and american Y. enterocolitica serotypes, suggest common evolution of YOP-1 of these serotypes independent of the evolution of the other serotypes.     

S-100 protein immunostaining in cells of dendritic morphology within reactive germinal centers by ABC immunoperoxidase method

Summary The dendritic and the interdigitating (IRC) reticulum cells are commonly reported to have different topographical distribution in the human lymphoid tissue and some peculiar cytochemical and immunohistochemical features. These include the detection of S-100 protein by PAP (peroxidase anti-peroxidase) method only in IRC located in the thymus-dependent areas. In 15 reactive lymphoid tissue specimens (11 lymph nodes, 3 tonsils, and 1 adenoid) the presence of S-100 protein was tested by ABC (avidin-biotin complex) immunoperoxidase method. IRC were constantly positive. Other positive cells were located within the follicular germinal centers; these immunostained cells appeared as a striking network composed of dendritic-shaped processes displaying a finely granular positivity for S-100 protein. It is suggested that by using this very sensitive technique, S-100 protein can also be detected in intrafollicular cells of dendritic morphology.This study was in part supported by Grant n.84.00525.44 from Consiglio Nazionale delle Ricerche Progetto Finalizzato Oncologia Roma     

Fluorescence histochemical and electron-microscopical observations on the innervation of the atrial myocardium of the adult human heart

Summary The existence of both adrenergic and cholinergic innervation of the atrial myocardium of the adult human heart was demonstrated by means of fluorescence induced by formaldehyde or glyoxylic acid and by electron microscopy.The adrenergic fluorescing axons (1) followed the course of blood vessels as typical perivascular nerve plexuses, and (2) formed a three-dimensional fairly dense nerve net obviously not related to the blood vessels. The varicosities frequently came into close apposition on myocardial cells.Several types of nerve terminals were differentiated at electron microscopy: (1) an adrenergic type containing small (diameter 450–700 Å) dense-cored vesicles and usually (in various proportions) small empty and/or large (900–1500 Å) dense-cored vesicles, (2) a cholinergic type containing small (ca. 500 Å) empty vesicles and occasionally also some large (mean diameter ca. 1200 Å) dense-cored vesicles, (3) a pale type containing only a few or no vesicles, (4) a disintegrated type containing degenerated mitochondria, autophagic vacuoles, and occasional normal-looking mitochondria, (5) nerve terminals containing a large number of mitochondria in addition to varying vesicle populations, and (6) a (possibly baroreceptive type of) nerve terminal containing myelinlike lamellated structures. The disintegrated and the pale types of nerve terminals possibly represent different stages of axonal degeneration, or may correspond to diminution in the transmitter substance concentration under certain pathophysiologic conditions, respectively. Nerve terminals crowded with mitochondria may be sensory and involved in mechano-or chemoreceptive functions.In preliminary experiments convincing evidence was obtained that the glyoxylic acid-induced fluorescence histochemical method will be suitable for comparative studies on (human) clinical specimens, e.g., for analyzing the degree of the functional activity of the intrinsic adrenergic innervation of the myocardium under various pathophysiologic conditions. The modification which appeared most appropriate for such studies is described in detail, and is proposed for use as a standard method in other similar or related studies on human clinical series. The essential criteria for analyzing the specimens at fluorescence microscopy are suggested as well.     

In situ expression of β1, β3 and β4 integrin subunits in non-neoplastic endothelium and vascular tumours

Endothelial cells play an important role in adhesive interactions between circulating cells and extracellular matrix proteins. In vitro studies have shown that many of these processes are mediated by a superfamily of heterodimeric transmembrane glycoproteins called integrins. The distribution patterns of 1, 3 and 4 integrin subunits in endothelial cells (EC) in situ were examined immunohistochemically on serial forzen sections of a wide range of non-neoplastic tissues and of vascular tumours, both benign and malignant. Expression of the 1 subunit was a constitutive feature of EC. Among the 1-associated subunits, 5 and 6 were broadly distributed in EC, irrespective of vessel size and microenvironment. The 3 subunit displayed intermediate levels of expression with a slight preference for small vessel EC. Presence of 1 was confined to EC of capillaries and venules/small veins. Expression of 2 in EC was inconsistent. With rare exceptions, the 4 chain was absent in EC. The 3 and v subunits were expressed in most EC, though not always concomitantly. In contrast to the 1 chain, however, these integrin subunits were absent in EC of glomerular capillaries and were expressed variably in sinusoidal EC. The 4 chain was evenly present in the great majority of EC, except for those of large vessels. In vascular tumours, the patterns of 1 and 1 to 6 subunit expression generally corresponded to those found in their non-neoplastic counterparts. Expression of 3, v and 4 chains, however, decreased in neoplasia, especially in angiosarcomas. These data show that EC dispose of broad and at the same time differential repertoires of integrin subunits that presumably reflect vessel-type associated functional differences among these cells. In vascular tumours, the orthologous distribution patterns of 1 and 1 to 6 chains are conserved in most instances while the amounts of 3, v and 4 subunits expressed in EC tend to decrease in the course of malignant transformation.Dedicated to Prof. Dr. med. Dres. h.c. Wilhelm Doerr on the occasion of his 80th birthday     

Transforming growth factor β gene maps to human chromosome 19 long arm and to mouse chromosome 7

Transforming growth factors (TGF) are defined as biologically active polypeptides which reversibly confer the transformed phenotype onto untransformed cultured cells. They have been subdivided into two classes: type and type TGFs. TGF- acts synergistically with TGF- in inducing phenotypic transformation. TGF- can also act as negative autocrine growth factor. A human 1050-bp EcoRI cDNA fragment was used to map the human locus for TGF- by Southern blotting of DNA prepared from 17 human × Chinese hamster somatic cell hybrids. The humanspecific restriction fragments segregated with human chromosome 19 in all of 14 informative hybrids. All other human chromosomes were discordant with the TGF- bands in at least four hybrids. After in situ hybridization of the tritiated TGF- probe to normal human metaphase spreads, 151 silver grains were scored in 54 cells. Of 24 grains over chromosome 19, 16 grains (11%) lay over region 19q13.1 q13.3. Of the 54 cells analyzed, 16 (30%) had label over region 19q13.1 q13.3. Thus,TGFB is assigned to chromosome 19, subbands q13.1 q13.3. TheTgf- locus in the mouse was mapped to chromosome 7 by hybridizing a murine cDNA probe to a Chinese hamster × mouse hybrid panel. Human chromosome 19 and proximal mouse chromosome 7 share another four homologous loci.     

Eine neue Bestimmung der Neutralfette im Blutserum und Gewebe

Zusammenfassung Die Neutralfette im Blutserum und Gewebe lassen sich über Glycerin exakt ermitteln. Freies und nach alkoholischer Verseifung nachweisbares Gesamt-Glycerin werden mit Glycerokinase in einer dreistufigen enzymatischen Nachweisreaktion im Mikroverfahren bestimmt. Die Differenz wird als Glycerid-Glycerin angesprochen. Reaktionsprinzip, Durchführung der Methode und Berechungsweise werden ausführlich besprochen.Für freies Glycerin werden 0,2 ml Serum, für Gesamtglycerin etwa 0,007–0,015 ml Serum äquivalente Hydrolysatmengen in den Test eingesetzt. Bei einer Empfindlichkeit von 0,0008 µMol (wenn bei 340 nm, bzw. 0,0015 µMol Glycerin, wenn bei 366 nm gemessen wird), können noch 1/20 bis 1/10 des im Normalserum vorhandenen freien bzw. Gesamtglycerins mit den Standardansätzen erfaßt werden. Die obere Nachweisgrenze reicht dann bis 0,15 bzw. 0,3 µMol Glycerin, das entspricht der 8- bzw. 15fachen Normalmenge für freies und Gesamtglycerin. In den angegebenen Grenzen liegen die über Glyceridglycerin nachweisbaren Triglyceridkonzentrationen. Empfindlichkeit und obere Nachweisgrenze können durch andere Serum- bzw. Hydrolysatmengen in weiten Grenzen variiert und der jeweiligen Fragestellung angepaßt werden.

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Summary Triglycerides in blood serum and tissue can be identified exactly by means of glyceride-glycerol. Free and total glycerol, measurable after alcoholic saponification, are determined by glycerolkinase in a three-step enzymatic proof-reaction by means of micro-methods. The difference between total and free glycerol is called glycerideglycerol. Reaction principles, working instructions and the calculation will be discussed in detail.The sensitivity of the standard test runs to 0,0008 respeccively 0,0015 µMol glycerol or 1/20 resp. 1/10 of the normal contentrations. The upper proof limit is at 0,15 respectively 0,3 µMol glycerol per test. This is equal to 8–15 times more than the normal of free and total glycerol. Sensitivity and upper proof limit can easily by varied and adapted to the formulation of the question.

     

Predicting metabolic cost of level walking

Summary Energy expenditure in walking is usually expressed as a function of walking speed. However, this relationship applies only to freely adopted step length-step rate patterns. Both the step length and the step rate must be used to predict the energy expenditure for any combination of step length and step rate. Evidence on seven subjects indicates that the energy demand for such a combination can be determined by conducting two experiments. In the first, the subject is allowed to freely choose his own walking pattern to achieve a set of prescribed speeds. In the second, the speed is kept constant but the subject is forced to adopt a range of prescribed step rates. The results of the two experiments combined yield enough data to make possible the determination of the energy equation of the pattern, encompassing both free and forced gaits. Results show that the freely chosen step rate requires the least oxygen consumption at any given speed. Any other forced step rate at the same speed increases the oxygen cost over that required for the free step rate.     

The Involvement of Dopamine in Strengthening Cortical Signals Activating NMDA Receptors in the Striatum (a hypothetical mechanism)

A possible mechanism is proposed for the enhancement/weakening of those cortical signals in the cortex-basal ganglia-thalamus-cortex neural network which induce/do not induce opening of NMDA channels in the spiny neurons of the striatum and which can be regarded as strong/weak in terms of this measure. The mechanism is based on the modulatory influences of dopamine on changes in the efficiency of corticostriatal inputs. In the absence of dopamine, relative increases in the intensity of strong (weak) cortical signals can lead to the induction of long-term potentiation (depression) of corticostriatal synapses. In this case, because of the differently directed influences on thalamic cells of signals passing via strionigral and striopallidal cells, strong signals at the output of the thalamus are weakened, while weak signals are strengthened. Activation of dopamine D₁ (D₂) receptors on strionigral (striopallidal) neurons may facilitate increases in the extent of long-term potentiation/depression (decreases in the extent of long-term potentiation/depression or induction of long-term potentiation/depression). The consequence of this is that strong signals at the output of the thalamus can be strengthened synergistically, while weak signals cab be weakened synergistically. Background cortical signals evoking tonic release of dopamine in the striatum can decrease strengthening because of weakening of the modulatory influence of dopamine on the modification of corticostriatal synapses.     

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.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Evidence for a direct relationship between mitochondrial genome organization and regeneration ability in hexaploid wheat somatic tissue cultures

Summary Embryogenic and non-embryogenic long-term callus cultures of hexaploid wheat exhibit differences in the organization of their mitochondrial genome. Embryogenic and non-embryogenic fractions of callus cultures initiated from immature embryos of the wheat cultivar Chinese Spring have been isolated and subsequently subcultured. DNA-DNA hybridization experiments using labelled cloned wheat mitochondrial DNA fragments have shown that the mitochondrial DNA organization of embryogenic subcultures derived from embryogenic parts of Chinese Spring calli is closely related to that of the initial Chinese Spring calli, while non-embryogenic subcultures derived from non-embryogenic fragments of Chinese Spring calli exhibit a mitochondrial DNA organization similar to that found in non-embryogenic calli derived from cultivar Aquila. In addition, somatic tissue cultures initiated from three other non-embryogenic wheat cultivars (Talent, Thésée and Capitole) display mitochondrial DNA arrangements similar to those found in cultivar Aquila. These results strongly suggest that, in wheat callus cultures, a particular mitochondrial genome organization is correlated with the ability of cultured cells to regenerate whole plants.Abbreviations mtDNA mitochondrial DNA - ctDNA chloroplast DNA - rRNA ribosomal RNA - kb kilobase pair - cv cultivar - 2,4-D 2,4-dichlorophenoxyacetic acid     

Modelling of electrolyte transport in renal and intestinal epithelia

Summary Epithelia can be classified as leaky and tight epithelia due to their conductive properties and their modes of solute transport. Both the proximal segment of the nephron and the intestinal tract are leaky whereas the distal nephron and the colon are tight. Consequently, inborn errors and exogenous disorders of solute transport often involve both the proximal tubule and the small intestine. In addition, effects on ion and water transport in the distal nephron closely resemble those in the large intestine. Models of solute transport in leaky and tight epithelia are presented employing porter systems known in mammalian tissues. These porter systems are discussed as possible sites of transport defects and as targets for pharmacological agents.     

On the terms “reticulosis” and “reticulum cell sarcoma” with regard to the modern concept of the monocyte macrophage system

Summary The terms reticulosis and reticulum cell sarcoma (= malignant lymphoma, histiocytic type) are discussed regarding the modern concept of the monocyte macrophage system which today has replaced the ancient theory of the reticuloendothelial system. The monocyte macrophage system is not independent, but closely related to the myeloid system. Thus, a third blood forming system as was believed in the case of RES does not exist. Phagocytic reticulum cells of the various hematopoietic organs are highly activated monocyte-derived macrophages. All those conditions formerly termed reticuloses have been found to belong either to the myeloid or to the lymphatic system. Considering the reticulum cell sarcomas or malignant histiocytic lymphomas, most of them seem to be of lymphatic rather than of macrophage origin, representing highgrade malignant lymphomas, possibly immunoblastic sarcomas. No relationship between these tumours and the monocyte macrophage system has been established, so far. Therefore, the terms reticulosis and reticulum cell sarcoma should be no longer used in order to avoid confusion, in order to stimulate sufficient diagnostic efforts which will really clarify such cases, and in order to give full credit to modern results of hematopathology.     

A Proposed Heuristic for Communicating Heritability Estimates to the General Public, with Obesity as an Example

It is well established that many continuously distributed traits have a heritable component. However, it is often difficult to communicate to the general public the meaning of quantitative estimates of heritability. To address this problem, the present paper introduces a heuristic for communicating heritability to nonscientific audiences. This heuristic involves adopting an extremely simplified model of inheritance and artificially (and somewhat arbitrarily) defining a cutoffs of low environmental risk and affectation status. Using body weight and obesity as an example, we present a table which gives estimates of the proportion of obese persons who are genetically obese assuming varying levels of environmental risk for obesity and relative body weight scores for defining obesity. The resulting statistic may prove useful for lay audiences in understanding a heritability estimate.     

Three-dimensional reconstruction of transmitter-identified central neurons by “en bloc” immunofluorescence histochemistry and confocal scanning microscopy

Summary A new method for three-dimensional reconstruction of transmitter-identified neurons is presented which involves en bloc immunofluorescence histochemistry and confocal scanning microscopy. The technique was applied to different types of neurons in the rat brain and lamprey spinal cord. Thick sections or tissue blocs (50–200 m thick) were incubated with antisera against neuropeptides or monoaminergic markers, followed by fluorescent secondary antibodies. Three-dimensional reconstructions were obtained by scanning the preparations in sequential focal planes with a thin laser beam, while sampling the emitted light in each focal plane. The method is convenient and can be applied to a wide variety of neuron types. The reconstructions obtained are accurate since the optical serial sections of the specimen are perfectly aligned, and optic disturbances such as halo phenomena do not occur.