Quantification noise in single cell experiments.

In quantitative single-cell studies, the critical part is the low amount of nucleic acids present and the resulting experimental variations. In addition biological data obtained from heterogeneous tissue are not reflecting the expression behaviour of every single-cell. These variations can be derived from natural biological variance or can be introduced externally. Both have negative effects on the quantification result. The aim of this study is to make quantitative single-cell studies more transparent and reliable in order to fulfil the MIQE guidelines at the single-cell level. The technical variability introduced by RT, pre-amplification, evaporation, biological material and qPCR itself was evaluated by using RNA or DNA standards. Secondly, the biological expression variances of GAPDH, TNFα, IL-1ß, TLR4 were measured by mRNA profiling experiment in single lymphocytes. The used quantification setup was sensitive enough to detect single standard copies and transcripts out of one solitary cell. Most variability was introduced by RT, followed by evaporation, and pre-amplification. The qPCR analysis and the biological matrix introduced only minor variability. Both conducted studies impressively demonstrate the heterogeneity of expression patterns in individual cells and showed clearly today's limitation in quantitative single-cell expression analysis.

Detection of chromosomal aneuploidies in fetal cells isolated from maternal blood using single-chromosome dual-probe FISH analysis.

Detection of chromosomal aneuploidies using fetal cells isolated from maternal blood, for prenatal non-invasive genetic investigation, has been a long-sought goal of clinical genetics to replace amniocentesis and chorionic villous sampling to avoid any risk to the fetus. The purpose of this study was to develop a sensitive and specific new assay for diagnosing aneuploidy with circulating fetal cells isolated from maternal blood as previously reported using two novel approaches: (i) simultaneous immunocytochemistry (ICC) evaluation using a monoclonal antibody for i-antigen, followed by fluorescence in situ hybridization (FISH); (ii) dual-probe FISH analysis of interphase nuclei using two differently labeled probes, specific for different loci of chromosomes 21 and 18; in addition, short tandem repeats (STR) analysis on single cells isolated by micromanipulation was applied to confirm the presence of fetal cells in the cell sample enriched from maternal blood. Blood samples were obtained from women carrying trisomic fetuses, and from non-pregnant women and men as controls. Using ICC-FISH approach, a large heterogeneity in immunostaining pattern was observed, which is a source of very subjective signal interpretation. Differently, dual-probe FISH analysis provided for a correct diagnosis of all pregnancies: the mean percentage of trisomic cells was 0.5% (range, 0.36-0.76%), while the mean percentage of trisomic cells in the control group (normal pregnancies or non-pregnant women) was ≤0.20%. The application of the dual-probe FISH protocol on fetal cells isolated from maternal blood enables accurate molecular detection of fetal aneuploidy, thus providing a foundation for development of non-invasive prenatal diagnostic testing.

Conjunctivitis, red nose and skin hypersensitivity as signs of food allergy in veal calves.

The severity of diarrhoea and the degree of hyperaemia of the conjunctiva and nose were recorded in veal calves being fattened on either cows milk or milk-replacer. A skin-prick test using the milk-replacer as antigen was also performed. Control calves received only their mother's milk. Among these control calves there were no abnormalities, and in the calves fed other cows milk only slight abnormalities were seen. In contrast, in the calves fattened on milk-replacer, and especially in the calves showing the 'cachexia' syndrome, there were moderate to severe abnormalities and clear correlations were found between the severity of the diarrhoea and the hyperaemia of the conjunctiva and nose, and the scores recorded in the skin-prick test.

Gastrointestinal food allergy and its role in large domestic animals.

The significance of food allergy as a primary cause for gastrointestinal disturbances in domestic animals, especially calves and piglets, is discussed. The immunological backgrounds and pathogenesis are described in some detail. The clinical and pathological manifestations in animals are related to those in man. Diagnostic possibilities, therapy and prevention, as far as known in animals, are mentioned and, based on human experiences, further extensions are proposed.

[Findings on infectious bovine rhinotracheitis (author's transl)]. / Enkele ervaringen met IBR.

Findings on infectious bovine rhinotracheitis (IBR) since this disease was diagnosed for the first time in the province of Gelderland in 1973, are reported. The clinical picture varies markedly, unmistakable symptoms are followed by a period of impaired health in a number of cases. There is no evidence to suggest that this impaired state of health is due to the causative virus of IBR. The number of infected herds is constantly increasing and is currently estimated at approximately 20 per cent. The disease also shows an increasing incidence in veal-calf herds. Intranasal vaccination will afford adequate protection of other members of the herd in the event of IBR but the immunity produced is short-lived.

[Interpretation of results of serologic analyses in large animal practices]. / De interpretatie van serologische onderzoekresultaten in de grote huisdierenpraktijk.

The backgrounds to and possibilities of serological studies for the purpose of establishing are reviewed, all this with regard to bovine disease. The serology of the virus infections infectious bovine rhinotracheitis (IBR), bovine viral diarrhoea (BVD), para-influenza virus 3 infection (PI3), bovine respiratory syncytial virus (BRS) and Chlamydia is discussed in greater detail. Likewise, the interpretation of serological findings in cases of brucellosis, leptospirosis and Johne's disease is dealt with. In addition, attention is paid to the results of serological testing in cases of lung-worm disease and fascioliasis.

[Lameness in Scottish Highlanders in the Imbos area]. / Kreupelheden bij Schotse Hooglanders in de Imbos.

Lameness occurred in Scottish Highlanders which were pastured in a part of the National Park Veluwezoom. This was followed by a general clinical picture, cachexia and death in a number of cases. Osteodystrophy resulting from calcium and phosphorus deficiencies in the local soil is regarded as the main cause.

[A field experiment using E. coli-K99 vaccine]. / Een praktijkproef met een E. coli-K99 vaccin.

In a field experiment in which vaccine containing E. coli-K99 antigen were administered to approximately thirty per cent of the pregnant dairy cows in herds in which the calves were affected with scours, during the first five days of life (the other animals were treated with a placebo), following results were obtained: The colostrum of vaccinated cows contained significantly higher concentrations of immunoglobulins to E. coli-K99 than did that of controls (Table 1). From the start of the experiment, severe scouring disappeared in the calves of vaccinated cows, and from those of the controls. E. coli-K99 could no longer be isolated. Zoötechnical measures such as adequate supplementation colostrum, hygienic procedures, etc., probably played an important role in the disappearance of the problems of scouring in these herds.

[Role of bovine respiratory syncytial virus in the aetiology of viruspneumonia in calves and yearlings ("pinkengriep") (author's transl)]. / De Etiologische Rol Van Het Bovine Respiratory Syncytial Virus Bij Pinkengriep. Voorlopige mededeling

The clinical picture of "pinkengriep", an enzootic form of bronchopneumonia in young cattle, is described. In addition to cough, conjunctivitis and a soporous state, accelerated respiration initially is an outstanding clinical symptom. In some cases, symptoms of fog fever appear during the second stage of the disease. In the autumn of 1973 and that of 1974, a total number of 292 animals with "pinkengriep" were serologically examined for known respiratory virus infections and Chlamydia. Complement fixation tests showed that there was significant increase in antibodies to respiratory syncytial virus in 76 per cent of the animals studied. In the case of para-influenza virus 3, virus diarrhoea virus, the adenoviruses of the antigen groups 1, 2 and 3, those of the antigen groups 4 to 10 inclusive and Chlamydia, these proportions were 48, 13, 12, 11 en 10 per cent respectively. On the other hand, symptoms of infection with infectious bovine rhinotracheitis virus were absent in the herds studied. These findings suggest a possible role of bovine respiratory syncytial virus in "pinkengriep".

[Results obtained using an attenuated bovine syncytial virus vaccine against bronchopneumonia of yearlings (author's transl)]. / Ervaringen met een geattenueerd RS-virusvaccin tegen pinkengriep. Praktijkmededeling.

Bronchopneumonia of yearlings is a respiratory infection with bovine respiratory syncytial virus (BRS virus) occurring as a herd disease in young cattle. In severe cases, the clinical picture is identifiable; characteristic features are laboured respiration and a high morbidity rate. Following an acute onset, atypical interstitial pneumonia is observed on postmorten examination and bacteriological studies are negative. An attenuated live BRS virus vaccine was used against bronchopneumonia of yearlings in 1976, 1977 and 1978. This vaccine was inoculated in twenty-six herds supervised by the present author and numbering approximately 1,200 animals in the sensitive period of life. Moreover, the results obtained by sixty practitioners on vaccinating approximately 13,000 animals in 1978 were collected. Conclusive evidence of the effectiveness of the vaccine was not provided by these field trials. However, it is concluded from the findings that the satisfactory results obtained in the majority of cases were not accidental and that these results suggest that BRS virus vaccine is very likely to have a protective action against bronchopneumonia of yearlings.

[Winter diarrhea]. / Winterdiarree.

Winter dysentery is a highly contagious, herd-enzootic disease of cattle, most frequently observed during the winter season. The disease is characterized by a sudden onset of severe diarrhoea, which normally disappears spontaneously within two or three days. The aetiology continues to be obscure, though a virus is a probable causative agent.

A new PAT/QbD approach for the determination of blend homogeneity: combination of on-line NIRS analysis with PC Scores Distance Analysis (PC-SDA).

A novel and straightforward multivariate analytical tool for the qualitative determination of powder blend uniformity using on-line Near-Infrared Spectroscopy (NIRS) is presented. The approach combines current chemometric methods, e.g. spectral pre-processing and Principal Component Analysis (PCA), with (1) a new approach of data analysis to determine the end-point of the blending process, (2) building a design space (DS) for blend homogeneity and (3) developing a solid statistical rationale to stop blending according to Quality-by-Design (QbD) principles of FDA's Process Analytical Technology (PAT) initiative. The new approach comprises calculation of Euclidean distances between PCA scores in a multidimensional space and determination of Moving Block Standard Deviations (MBSDs) of successive Principal Component (PC) scores distances to estimate a time-window during blending where spectral variability decreases to a preset minimum. Hotelling's T(2) statistics is then used to monitor and report blend homogeneity. This technique is called "Principal Component Scores Distance Analysis" (PC-SDA). A Central Composite Design resulting in 10 batches mixed in a bin-blender (same composition, different blender fill level, different number of revolutions) was executed. NIR Chemical Imaging (NIR-CI) in combination with Symmetry Parameter Image Analysis (SPIA) was used to verify the NIRS analyzer response and assess homogeneity of all NIR-active components.

p53-mediated apoptosis prevents the accumulation of progenitor B cells and B-cell tumors.

We propose that the apoptotic function of p53 has an important role in B-cell homeostasis, which is important for the prevention of B-cell lymphomas. We created a mouse model (mDeltapro) that lacked residues 58-88 of the proline-rich domain of p53. mDeltapro is defective for apoptosis, but is able to arrest cell-cycle progression in hematopoietic tissues. mDeltapro develops late-onset B-cell lymphoma, but not the thymic T-cell tumors found in p53-null mice. Interestingly, mDeltapro lymphomas comprised incorrectly differentiated B cells. B-cell irregularities were also detected in mDeltapro before tumor onset, in which aged mice showed an increased population of inappropriately differentiated B cells in the bone marrow and spleen. We predict that by keeping B-cell populations in check, p53-dependent apoptosis prevents irregular B cells from eventuating in lymphomas.