Assessment of the viability of haemopoietic cells in the livers of mouse fetuses stored at 4 degrees C. Comparison of various methods of testing their usefulness for haemopoietic transplantation

The usefulness of three different tests available in experimental haematology for the assessment of the viability of fetal liver cells obtained from murine fetuses kept in refrigerator at 4 degrees C was compared. The usefulness of these cells as potential transplants for haemopoiesis reconstitution was assessed in the test with trypan blue, in the test based on clonal growth of GM-CFU in agar, and in the test of the splenic colony forming ability of CFU-S. Fetuses kept in refrigerator at 4 degrees C tested 16 hours after circulation arrest contained still about 70% of the initial number of the haemopoietic stem cells (CFU-S). The proportion of committed cells belonging to the granulocyto-monopoiesis line (GM-CFU) decreased at the same time to about 25% of the initial number, and was a sensitive indicator of hypoxia of the studied organ. The test for the viability of the cells based on the use of trypan blue gave results reflecting better the changes in the number of CFU-S than GM-CFU cells.     

Constitutional C-band pattern in patients with adenomatosis of the colon and rectum

The pattern of polymorphism in the C-band-positive constitutive heterochromatin of chromosomes #1, #9, and #16 was studied in fibroblasts from 23 unrelated patients with adenomatosis of the colon and rectum and in peripheral lymphocytes from 78 control persons. The parameters of the heterochromatic regions analyzed were relative size, symmetry-asymmetry within homologous chromosome pairs, and frequency of inversions. The polyposis coli patients had a significantly higher frequency (p less than 0.05) of partial and total heterochromatin inversion on chromosome #9 than the control group (37.0% compared with 21.8%). In the other parameters studied, no significant differences were found between patients and controls.     

Double wavelength measurement of 3,3',5,5'-tetramethylbenzidine (TMB) provides a three-fold enhancement of the ELISA measuring range

A rapid, simple method is described which permits a three-fold enhancement of the working range of ELISA procedures using TMB as a substrate. This consists of measuring absorbance values at a wavelength away from the absorption maximum of TMB and using a predetermined multiplication factor.     

Diagnostische Bewertung des Nachweises von Gliadin-Antikörpern bei Cöliakie

Zusammenfassung Die Methoden der Komplement-bindung und der Präcipitinreaktion in Agar mit Gliadin als Antigen wurden vergleichend auf ihre Anwendbarkeit in der Diagnostik der Cöliakie geprüft.Beim Präcipitinversuch kamen pseudopositive Reaktionen bei 285 Kontrollen nie vor, bei der Komplementbindung zweimal bei 189 Kontrollen.Zwölf sichere Cöliakiefälle ergaben vergleichend geprüft acht bzw. neun positive Reaktionen, wobei die negativen Befunde für die beiden Verfahren nicht das gleiche Serum jeweils betrafen. Wurde die positive Reaktion in nur einer Methode als ausreichend betrachtet, so war das Ergebnis: elf positive Reaktionen auf zwölf Fälle.Während bei klinisch bestätigter Cöliakie die Übereinstimmung der beiden Methoden gut war, war dies bei den bloßen Verdachtsfällen nicht der Fall. Hier gab es bei der Komplementbindung mehr positive Resultate als bei der Präcipitinreaktion.Es wird vorgeschlagen, beide Methoden nebeneinander anzuwenden und sie bei negativem Ausfall zu wiederholen, wobei die gegebenen diätetischen Richtlinien einzuhalten sind. Als Antigen kann ein Glycerinextrakt aus Gluten verwendet werden, wenn gereinigtes Gliadin nicht zur Verfügung steht. Extrakte mit heißem Wasser geben zu viele pseudopositive Reaktionen.

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Summary Complement fixation tests and precipitin reactions in agar with gliadin as antigen were compared in cases of celiac disease.Pseudopositiv reactions did not occur in 285 controll cases when the precipitin test of Ouchterlony was used, but were seen in two cases of 189 controlls with the complement fixation test.If only one of the two reactions was positive and this was considered as a sufficient proof for the formation of gliadin antibodies those were found in 11 of 12 cases of clinically confirmed celiac disease. In those cases the agreement of the two methods was good, but not so in not confirmed cases where complement fixation tests gave more positive results than the precipitin reaction.It is proposed to use both methods for diagnostic help and to repeat the tests if the reactions are negativ.

     

Prevalence of Eikenella corrodens in dental plaque.

The prevalence of Eikenella corrodens in dental plaque and saliva samples of 282 volunteers was determined by a semiquantitative method with a selective medium. E. corrodens was recovered in 58.9% of plaque samples and 0.3% of saliva samples. This prevalence rate was not significantly altered (P greater than 0.05) by variables of sex, race, smoking habits, clinic attended by the patient, general health status, or age; however, patients 7 to 14 years old had a significantly higher prevalence rate (90.5%; P less than 0.05). E. corrodens should be considered as a potential pathogen in infections associated with and wounds inoculated by the human gingival flora.     

β1-Adrenoceptor gene variations: a role in idiopathic dilated cardiomyopathy?

A substantial body of evidence suggests involvement of the human beta1-adrenoceptor (beta1-AR) gene in the pathophysiology of dilated cardiomyopathy (DCM), a severe heart disease of significant public health impact. Beta1-AR-mediated signal transduction is dramatically altered due to downregulation, resulting in an impairment of myocardial response. The important role of genetic factors in idiopathic dilated cardiomyopathy (IDCM) recently recognized, we analyzed this prime candidate gene for genetic variation in carefully selected patients and controls. In this preliminary study, 18 single nucleotide polymorphisms were observed, 17 of which were located in the N-terminal and C-terminal region of the coding exon, resulting in 7 amino acid exchanges: Ser-49-Gly, Ala-59-Ser, Gly-389-Arg, Arg-399-Cys, His-402-Arg, Thr-404-Ala, and Pro-418-Ala. These mutations resulted in 11 different beta1-AR genotypes. Importantly, the genotypes carrying the Ser-49-Gly mutation in the N-terminus of the molecule in a heterozygous or homozygous form were observed significantly more frequently in the group of IDCM patients. The present results may provide a clue on the molecular mechanisms involved in IDCM, and add moreover interesting information on nature, distribution, and evolutionary aspects of sequence variation in human adrenergic receptor genes.     

The bingo model of survivorship. V. The problems of conformation to the empirical evidence

We discuss the statistical and biological problems of adapting the theoretical bingo model to the analysis of empirical data. A distinction is made between an idealized pathogenetic model, which aims to represent the disease in as much authentic detail as the present state of knowledge allows and in components that have literal interpretation, and an empirical model, which deals with those effects of the pathogenetic model that one may hope to observe clinically. We review a variety of empirical models distinguishable by the amount of data available on intermediate degrees of damage short of total destruction. The relationship of damage to time is explored, and we consider the criteria and usefulness of linearization of this relationship where the diachronic ("longitudinal") data are few and extend over a comparatively short time. Every time a patient is examined, the degree of cumulative damage is assessed in each of the body systems of interest. Thus the examination will furnish a set of measurements, which is obtained on each of several examinations, taken over a period that for preference is long relative to the survival of the system. Specific disorders discussed include dentition and enlargement of the aorta with age in the Marfan syndrome.     

Location of immunodominant antigenic determinants on fragments of the tick-borne encephalitis virus glycoprotein: Evidence for two different mechanisms by which antibodies mediate neutralization and hemagglutination inhibition

A model showing the topological distribution, functions, and serological specifities of eight distinct, monoclonal antibody-defined epitopes on the tick-borne encephalitis (TBE) virus glycoprotein has been presented in a previous publication [Heinz et al., 1983]. Virology 126, 525–537.) In the present report the influence of conformational change, chemical modification, and fragmentation on the antigenic reactivity of each epitope has been analyzed by the use of blocking enzyme immunoassays and “Western blotting.” One of the two major antigenic domains (A), composed of three different epitopes, completely lost its antigenicity upon incubation at pH 5.0 or by treatment with guanidine-HCl/urea, SDS, reduction and carboxymethylation, as well as by proteolytic (trypsin, α-chymotrypsin, thermolysin) and chemical (CNBr) fragmentation. The second major antigenic domain (B), however, defined by four distinct monoclonal antibodies, three of which are hemagglutination (HA)-inhibiting, neutralizing, and protective, was shown to be resistant to low pH, guanidine-HCl/urea treatment, and proteolytic cleavage of the native protein. Also, polyclonal immune sera from mice and rabbits contained antibody populations reactive with antigenic determinants which are resistant and others which are sensitive to conformational change and fragmentation. Glycoprotein fragments with molecular weights of about 9000, generated by proteolysis of the native protein, were immunoreactive with neutralizing and protective monoclonal antibodies (defining domain B) as well as with a polyclonal mouse immune serum. Thus, these fragments appear to contain antigenic determinants which are immunodominant on the native protein and play an important role in the induction of a protective immune response against TBE virus. In addition, these results show that antibody binding to antigenic domains which are topologically and structurally completely unrelated may result in neutralization and/or HA inhibition. As the presence of two receptor-binding sites is unlikely, different effector mechanisms may account for the effects of these antibodies. The antigenic reactivity of domain A is sensitive to the same treatments which also inactivate HA activity of TBE virus, whereas domain B is resistant. These treatments include a change of domain A induced by incubation at slightly acidic pH which also results in inactivation of virus infectivity. Antibodies to domain A therefore presumably block viral activities by direct binding at or near the putative receptor-binding site whereas antibodies to domain B may cause loss of biological activities by inducing a conformational change of the receptor-binding site.     

Dopamine and methionine-enkephalin in human brain

The contents of dopamine (determined radioenzymatically) and methionine-enkephalin (assayed by a radioimmunoassay) were measured in several areas of the human brain. The peptide was principally localized in dopamine-rich structures. In patients with Parkinson's disease, in contrast to the general dopamine deficiency, the reduction in methionine-enkephalin was restricted to the mesencephalon, putamen and lateral pallidum.