Biomedical subjects

M H Wilkinson

Publications and source records attributed to M H Wilkinson.

At least 19 recordsLinked to original sources

PubMed1996

Source of respiratory drive during periodic breathing in lambs.

In order to investigate the mechanisms underlying periodic breathing (PB), we studied the initiation of breathing after passive hyperventilation in 14 anaesthetised 10-20 day old lambs. Eight of the lambs exhibited PB following post-hyperventilation apnea (PHA), with an epoch duration of 82.4 +/- 14.2 sec (mean +/- SEM), a cycle duration of 9.7 +/- 0.7 sec and a ratio of ventilatory duration to apnea duration (V-A ratio) of 1.24 +/- 0.32. The remaining lambs showed stable breathing patterns following PHA. The ventilatory response to isocapnic hypoxia was significantly greater in the group that had PB (-7.2 +/- 1.0 ml min-1% Sao2-1 kg-1) than in the animals that did not (-2.5 +/- 1.0 ml min-1%Sao2-1 kg-1). Using experimentally determined ventilatory response curves to O2 and CO2 we calculated that the swings in Sao2 and Paco2 during PB generated chemical drive that accounted for only 16.2% of the ventilatory oscillations observed during PB. Much of the remaining drive appeared to originate in the 'switch-on' characteristics of the respiratory controller, in lambs that exhibited periodic breathing, when breathing began after PHA ventilation jumped abruptly from zero to 55.1% of the eupneic ventilation. The magnitude of this jump in ventilation accounted for 51.9% of the amplitude of ventilatory oscillations that occur during PB. We speculate that this previously unrecognised feature of the respiratory controller, together with an elevated sensitivity to hypoxaemia, play crucial roles in generating PB in the infant.

Animals↗

PubMed1996

Micromorphometrical analysis of rodent related (SPF) and unrelated (human) gut microbial flora in germfree mice by digital image processing.

Digital image processing (DIP) of bacterial smears is a new method of analysing the composition of the gut microbial flora. This method provides the opportunity to compare and evaluate differences in the complex highly concentrated anaerobic fraction of gut microbial flora, based on micromorphological differences. There is ample evidence that this fraction can be characterized as related or unrelated to the host organism by its immunogenicity. In this study germfree ND2 mice were associated with either related (rodent) SPF microflora (SPF-MF) or unrelated human MF (HUM-MF). DIP analysis was performed on original SPF-MF and HUM-MF and on the faeces of ex-germfree mice 4 weeks after association. The micromorphological pattern of highly concentrated anaerobic bacteria in faeces of HUM-MF associated ex-germfree mice was significantly different from SPF-MF associated counterparts with regard to the scores for elongation (P < 0.01) and morphological variety (P < 0.05). Moreover, gross morphological variability was present between individual HUM-MF associated mice but not between individual SPF-MF associated animals. No differences were found between original SPF and HUM-MF. The data are discussed with regard to differences in the presence of (non-)immunogenic bacteria and the ability for related and unrelated flora to colonize the murine gut. This study provides evidence that murine host specificity of microbial flora may not only be reflected in the number of non-immunogenic bacteria but also in the micromorphological pattern of highly concentrated anaerobic bacteria in faeces measured by DIP analysis.

Adult↗

PubMed1995

Effect of venous oxygenation on arterial desaturation rate during repetitive apneas in lambs.

While arterial oxygen desaturation during apnea is a common occurrence in adults and infants, the factors determining the rate of desaturation are poorly understood. We describe a theoretical model which suggests that arterial desaturation during an apneic episode occurs in two stages. In the initial stage (stage 1) the oxygen store in the lung is depleted, while in the second phase of the desaturation process (stage 2) tissue oxygen needs are met predominantly by depletion of the blood store. Our model predicts that preapneic venous oxygenation (SvO2) will strongly influence the rate of the desaturation in stage 1 but not in stage 2. We therefore examined the effect of changing preapneic SvO2 on the rate of arterial oxygen desaturation (SaO2) during stage 1 and stage 2 apnea in anaesthetised 10-20 day-old lambs. Preapneic arterial oxygen saturation was maintained constant. In agreement with the model's prediction there were two stages to the desaturation process and during stage 1 a significant increase in SaO2 was observed when preapneic SvO2 was lowered; SaO2 was -3.1 +/- 0.4%.sec-1 when SvO2 = 47.4 +/- 2.1% increasing to -5.8 +/- 0.7%.sec-1 when SvO2 = 28.3 +/- 1.2%. During stage 2, SaO2 was -1.62 +/- 0.07%.sec-1 and was independent of preapneic SvO2, also in accord with the model's prediction. In order to assess whether the accelerated desaturation rate we observed in stage 1 could have resulted from a decline in lung volume during apnea rather than lower levels of SvO2, we repeated the experiment with CPAP applied. Under these conditions SaO2 continued to be greater at lower preapneic SvO2 levels. In summary, lowered preapneic SvO2 has a potent influence on SaO2 during stage 1 of the desaturation process but not during stage 2.

Animals↗

PubMed1995

DATAPLOT: a graphical display package for bacterial morphometry and fluorimetry data.

A microbiological image processing system has been developed, which is used to determine both morphometry and fluorimetry data on (mainly faecal) bacteria. A special program for display of these data has been developed for this system. Besides probability density estimates of any one or two parameters and scatter plots of any two or three parameters in the distribution, it can generate scatter plots using the actual bacterial shapes, rather than arbitrary symbols. A particularly powerful feature of the program is the possibility to overlay a contour plot of the probability density distribution of the bacteria in parameter space, with a scatter plot of the actual bacterial shapes involved. In this paper, the program's organization, options, and the algorithms used are discussed.

Algorithms↗

PubMed1995

Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples.

Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and V8 of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide probes. Determination of the genus specificity of the oligonucleotides was performed by whole-cell hybridization with fluorescein isothiocyanate-labelled probes. To this end, cells were fixed on glass slides, hybridized with the probes, and monitored by videomicroscopy. In combination with image analysis, this allowed quantification of the fluorescence per cell and objective evaluation of hybridization experiments. One of the probes developed was used to determine the population of Bifidobacterium spp. in human fecal samples. A comparison was made with results obtained by cultural methods for enumeration. Since both methods gave similar population estimates, it was concluded that all bifidobacteria in feces were culturable. However, since the total culturable counts were only a fraction of the total microscopic counts, the contribution of bifidobacteria to the total intestinal microflora was overestimated by almost 10-fold when cultural methods were used as the sole method for enumeration.

Base Sequence↗

PubMed1995

Fluorescence overlay antigen mapping of the epidermal basement membrane zone: III. Topographic staining and effective resolution.

In this third study on the fluorescence overlay antigen mapping (FOAM) technique, we have addressed the question of which differences of antigen distributions close to the resolving power of the light microscope can be distinguished. An answer to this question should provide clues to future applications of the technique aiming at the topographic differentiation of IgG deposits displayed at the epidermal basement membrane zone (EBMZ) in certain bullous skin disorders. For the present purpose we have developed a topographic staining model in human skin, using structural EBMZ antigens as topographic reference markers. The distribution of these markers relative to one another is visualized in FOAM images obtained by selective double immunofluorescence tracing and videomicroscopic overlay imaging. The theoretical resolution limit of the technique is discussed and suggests an effective lower limit of some 60-65 nm. Although this limit is not reached under present conditions, our results show that it is possible to distinguish topographic differences of antigen distributions with an upper resolution limit of 200 +/- 50 nm. Furthermore, our findings indicate that collagen Type VII and beta 4 integrin are the most suitable molecules to serve as topographic reference markers in future applications of the technique aiming at the differentiation of bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). Preliminary results on this topic are most promising indeed.

Antigens, Surface↗

PubMed1995

Fluorescence overlay antigen mapping of the epidermal basement membrane zone: II. Color fidelity.

In this second report on the fluorescence overlay antigen mapping (FOAM) technique, we highlight some of the errors that may influence faithful color rendition of slide preparations using triple antigen immunofluorescence staining. Reliable interpretation of multicolor fluorescence images requires that the observer can unambiguously assign each color in these images to the presence of a specific combination of the labeled antigens. This is possible only when the image fidelity meets certain standards. The present study concentrates on color fidelity which is easily undermined by spectral matching errors, image contrast errors, and exposure time errors. Evaluation of these errors, using the photomicrographic overlay variant of FOAM, showed the potential unreliability of the simultaneous use of multiple fluorophores for immunofluorescence microscopy. The procedures described here may serve as a solid starting point in formulating technical conditions that allow reliable color rendition in multicolor immunofluorescence microscopy. Furthermore, these procedures can be adapted to studies other than the analysis of basement membrane zone antigens, to which they have been first applied.

Basement Membrane↗

PubMed1995

Changes in the temporal structure of periodic breathing with postnatal development in preterm infants.

The progressive decrease in the periodic cycle duration (PCD) of periodic breathing with postnatal age in term infants has been previously reported by a number of authors and is thought to be associated with peripheral chemoreceptor maturation. We hypothesized that a similar decrease should be observed in preterm infants. Therefore, in this study we measured the changes in PCD with postnatal age in a small group of preterm (n = 4) infants followed longitudinally (36 afternoon nap studies) over the first 6 mo postnatally. PCD declined in these infants from 17.1 +/- 3.3 s (mean +/- 2 SD) at 9 d to 9.8 +/- 3.2 s (mean +/- 2 SD) at 105 d. The regression slope was -0.072 s/d. Beyond 105 d there was no change in PCD up to 6 mo postnatally. We found no significant difference between active and quiet sleep. These results are similar to results previously published in term infants but apparently contradict recent data on a group of preterm infants. Possible reasons for this discrepancy are discussed. By examining long epochs of periodic breathing in these infants we also identified characteristic changes in PCD and V/A ratio, defined as the duration of the ventilatory period divided by the duration of the apneic interval. V/A ratio fell from the start of an epoch from 1.21 +/- 0.08 (mean +/- SEM) to a minimum of 0.62 +/- 0.03 and then increased again to 0.8 +/- 0.05 at the end of the epoch.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

PubMed1994

Shading correction and calibration in bacterial fluorescence measurement by image processing system.

An image processing system with applications in bacterial (immuno-)fluorescence measurement has been developed. To reach quantitative results, correction for non-uniformities in system sensitivity, both as a function of time (calibration for drifts) and as a function of image coordinates (shading correction), is essential. Both problems can be handled simultaneously by acquiring images of a uniformly fluorescent, solid standard as a reference image. To measure bacterial fluorescence, the average fluorescence intensity of isolated areas of interest (the bacteria) is computed, and corrected using the reference images. Two shading correction methods are theoretically and experimentally compared: direct averaging in the corrected image, and (weighted and unweighted) averaging using the raw image and a separate shading image to determine the weights and correct for shading during the averaging. The latter method proved computationally 3.5-6.5 times faster on average and reduced propagation of truncation errors during computation, resulting in 40% less noise, for 8-bits/pixel images.

Bacteria↗

PubMed1994

A simple data compression scheme for binary images of bacteria compared with commonly used image data compression schemes.

A run length code compression scheme of extreme simplicity, used for image storage in an automated bacterial morphometry system, is compared with more common compression schemes, such as are used in the tag image file format. These schemes are Lempel-Ziv and Welch (LZW), Macintosh Packbits, and CCITT Group 3 Facsimile 1-dimensional modified Huffman run length code. In a set of 25 images consisting of full microscopic fields of view of bacterial slides, the method gave a 10.3-fold compression: 1.074 times better than LZW. In a second set of images of single areas of interest within each field of view, compression ratios of over 600 were obtained, 12.8 times that of LZW. The drawback of the system is its bad worst case performance. The method could be used in any application requiring storage of binary images of relatively small objects with fairly large spaces in between.

Bacteria↗

PubMed1994

Computer processing of microscopic images of bacteria: morphometry and fluorimetry.

Several techniques that use computer analysis of microscopic images have been developed to study the complicated microbial flora in the human intestine, including measuring the shape and fluorescence intensity of bacteria. These techniques allow rapid assessment of changes in the intestinal flora and could apply equally to other complex microbial ecosystems.

Bacteria↗

PubMed1994

Fluorescence overlay antigen mapping of the epidermal basement membrane zone: I. Geometric errors.

To identify in tissue sections the relative positions of antigen distributions close to the resolving power of the microscope, we have developed the fluorescence overlay antigen mapping (FOAM) procedure. As this technique makes high demands on the geometric fidelity of the overlay image, it is essential to recognize geometric errors resulting from optical imperfections. This applies in particular to the image shift difference (ISD) that may routinely occur during fluorescence overlay. We describe here procedures for assessment and mechanical correction of the ISD in tissue sections. Furthermore, we describe an alignment verification test to assess the accuracy of the ISD correction procedure, using collagen Type VII as the geometric verification marker. These procedures should enable reliable evaluation of relative antigen distributions in tissue sections using photomicrographic multicolor fluorescence overlay. Further details of the FOAM technique, such as color fidelity and its utility for diagnostic and research purposes, will be published separately.

Basement Membrane↗

PubMed1993

Origin of tonic electrical activity patterns in the diaphragm of the fetal lamb.

The respiratory pattern generator in fetal and postnatal life activates the phrenic nucleus and diaphragm muscle with phasic bursts of activity. In the fetus, diaphragmatic activity is also characterized by tonic activity patterns of unknown origin. We have examined whether such activity is diaphragmatic, or radiated from nearby ribcage muscles, by placing two sets of electrodes side-by-side in the costal portion of the diaphragm in five fetuses. The rationale for this approach is that if tonic activity, radiates to the diaphragm it should be recorded by both sets of electrodes and there should be no delay between the action potentials from each set of electrodes. Of 24 single tonic units identified, 15 were recorded from only one of the two sets of electrodes in the diaphragm. In the 9 tonic units recorded from both sets of electrodes, there was a time delay between the appearance of the action potentials in the two recordings (mean +/- S.E.M. 1.6 +/- 0.2 ms). This is the expected conduction delay along the muscle fibres separating the two electrodes. Since tonic diaphragmatic activity persisted in fetuses with the spinal cord transected rostral or caudal to the phrenic nucleus, we conclude that the spinal cord alone is sufficient to produce the tonic activity recorded from the fetal diaphragm but that the brain may also generate such activity.

Animals↗

PubMed1993

Characterization of human faecal flora by means of an improved fluoro-morphometrical method.

The use of polyclonal antibodies in differentiating between samples of faecal microflora derived from ten healthy volunteers was assessed. The distribution of FITC-labelled antibodies (of the IgA, IgG and IgM isotype) over 144 morphologically distinct subsets of faecal bacteria ('morphotypes') was quantified by means of digital morphometry and quantitative immunofluorescence. Furthermore, a new dataprocessing algorithm was developed which makes objective quantitation of the antibody distributions over the faecal morphotypes possible. The results of this study imply that the antibody binding capacity of faecal morphotypes is a unique characteristic of faecal microflora.

Adult↗

PubMed1992

Circulating antibodies against faecal bacteria assessed by immunomorphometry: combining quantitative immunofluorescence and image analysis.

A new technique to study the prevalence of circulating antibodies directed against different morphological groups ('morphotypes') of bacteria in fresh faeces is presented. The technique combines quantitative indirect immunofluorescence with digital image analysis. Plasma antibody titres and patterns of IgA, IgG and IgM isotype against morphotypes of faecal bacteria were determined in ten healthy individuals.

Adult↗

PubMed1991

Objective quantitation of serum antibody titres against Enterobacteriaceae using indirect immunofluorescence, read by videocamera and image processing system.

A new way of measuring indirect immunofluorescence (IIF) of microscopic bacterial slide preparations by videocamera and an image processing system is presented. This method is compared with the conventional method of reading the slides by eye. The advantages of this new approach are objective reading, greater accuracy and easier performance. We have applied the method to measure serum antibody titres against endogenous Enterobacteriaceae. The method offers the opportunity to combine IIF with automatic morphological analysis, thereby maximally exploiting the possibilities of the immunofluorescence technique.

Antibodies, Bacterial↗

PubMed1991

Electromyographic changes in the isolated rat diaphragm during the development of fatigue.

To investigate whether the power spectrum of the electromyogram of a fatiguing muscle can be used to infer the degree to which the muscle is fatigued, we recorded isometric tension and two monopolar electromyograms from eight isolated rat diaphragm preparations suspended in an organ bath containing a balanced salt solution. Each preparation was excited with a fixed phrenic nerve impulse pattern made up of a 70-Hz train of impulses of supramaximal voltage delivered for 170 ms with a 500-ms recovery period. Tension fell rapidly over the first 60 s of the fatigue run and more slowly for the remaining 60 s analysed. The duration of extracellular action potentials increased and their amplitude decreased as the tension developed by the diaphragm decreased; conduction velocity along muscle fibres also decreased. The centroid frequency (fcen) of the power spectrum of the first action potential elicited by each train of stimuli decreased rapidly until tension fell to approximately 70% of the initial value; thereafter little change in fcen occurred, although tension continued to fall to 33% of its initial value. Our results demonstrated that under controlled conditions, fcen provided a sensitive index of fatigue in its early stages, but provided no information once fatigue was pronounced.

Animals↗

PubMed1991

Morphometrical parameters of gut microflora in human volunteers.

The morphology of faecal microflora of nine healthy human volunteers was studied by digital image analysis of microscopic slides. Weekly specimens were collected during an 8-week period. Seven morphometrical parameters were derived: the means and medians of components 1, 2 and 3, and morphometrical entropy. Statistically significant differences among subjects were found for means of components 1 and 2, medians of components 1, 2 and 3, and entropy. The stability in normal circumstances provides an excellent basis for the detection of pathological change in gut flora balance.

Analysis of Variance↗