[Dissertations 25 years after date 43. Temporomandibular joint dysfunction and condylar resorption following orthognathic surgery]. / Proefschriften 25 jaar na dato 43. Disfunctie van het kaakgewricht en condylaire resorptie na orthognatische chirurgie.

A surgical-orthodontic treatment has a direct influence on a patient's skeletal, dental, functional and psychological factors. A variety of surgical and anatomical factors determine the result of this complex treatment. Risk factors are a retrognathy with a steep mandibular angle, and the anatomy of the mandibular condyles and the fossa. The customary surgical techniques have an enhancing influence on the function of the temporomandibular joint. The role of the position of the articular disc remains unclear. Since 1989, more insight has gradually been gained in the aspects having an influence on the function of the temporomandibular joint following orthognathic surgery.

Cystathionine Levels in Patients With Huntington Disease.

BACKGROUND: Recently a profound depletion of cystathionine γ-lyase (CSE), the principal enzyme involved in the generation of cysteine from cystathionine, was shown in Huntington disease (HD) patients and several transgenic HD mouse models. We therefore hypothesized that blood and urine cystathionine levels may be increased in HD patients and that this increase might correlate with disease progression. METHODS: We measured concentrations of cystathionine as well as 22 other amino acids in fasting plasma and 24-h urine samples of nine early-stage HD patients and nine age, sex, and body mass index matched controls. RESULTS: There were no significant differences in the plasma or urine concentrations of cystathionine or any other amino acid between HD patients and controls. CONCLUSION: We found no evidence for changes in plasma or urine concentrations of cystathionine in early-stage HD patients. Therefore, cystathionine levels are unlikely to be useful as a state biomarker in HD.

Development of a porcine small intestinal cDNA micro-array: characterization and functional analysis of the response to enterotoxigenic E. coli.

The intestine is a complex and dynamic ecosystem, in which nutrients, exogenous compounds and micro-flora interact, and its condition is influenced by the complex interaction between these factors and host genetic elements. Furthermore, interactions of immune cells with the other components of the intestinal mucosa are essential in the defense against pathogens. The outcomes of these complex interactions determine resistance to infectious diseases. The development of genomic tools and techniques allows for analysis of multiple and complex host responses. We have constructed a porcine small intestinal micro-array, based on cDNA from jejunal mucosal scrapings. Material from two developmental distinct stages (4- and 12-week-old pigs) was used in order to assure a reasonably broad representation of mucosal transcripts. The micro-array consists of 3468 cDNAs spotted in quadruplicate. Comparison of the 4-week-old versus 12-week-old pigs revealed a differential expression in at least 300 spots. Furthermore, we report the early gene expression response of pig small intestine jejunal mucosa to infection with enterotoxigenic E. coli (ETEC) using the small intestinal segment perfusion (SISP) technique. A response pattern was found in which a marker for innate defense dominated, demonstrating the strength of this applied technology. Further analysis of these response patterns will contribute to a better understanding of enteric health and disease in pigs. The great similarity between pig and human suggest results from these continuing studies should be applicable for both agricultural and human biomedical purposes.

Quantification of tumour vascularity in squamous cell carcinoma of the tongue using CARD amplification, a systematic sampling technique, and true colour image analysis.

The aims of this study of head and neck tissue samples were to develop an immunohistochemical protocol based on the catalysed reporter deposition (CARD) technique to enhance staining results for use in automated true colour image analysis, to assess the reproducibility of systematic tissue sampling in the angiogenic hot spot selection, and quantification of microvessel density (MVD) and other vessel characteristics. The latter data were compared between six metastasised tongue squamous cell carcinomas, vs. four non-metastasised. In comparison to the standard immunohistochemical protocol with anti-CD34 antibodies, CARD amplification resulted in both more intensely stained and larger numbers of vessels. Averaging the 10 most vascularised fields of the 40 to 60 systematically sampled fields in a tissue section resulted in an overall acceptable interobserver reproducibility for most assessed vessel parameters (r> or =0.76 and p< or =0.01). The percentage vessels with diameter <5 microm was significantly higher in the non-metastasised tongue carcinomas (p=0.02). However, for a number of tumours the effect of tissue sampling was significant. We conclude that CARD amplification is needed for reliable segmentation of vessels by image analysis systems, and that tumour heterogeneity is a limiting factor for all procedures in which tumour vascularity is assessed in a single tissue section. Figures on http://www.esacp.org/acp/2001/22-4/hannen.htm.

The relationship between genetic aberrations as detected by comparative genomic hybridization and vascularization in glioblastoma xenografts.

Angiogenesis is of vital importance for the growth of solid tumors and constitutes a target for anti-cancer therapy. Glioblastomas (GBMs) are histologically characterized by striking microvascular proliferation. The identification of the mechanism of angiogenesis is of major importance for the further development of anti-angiogenic therapy. Tumor angiogenesis might be the result of a combination of local tissue conditions (especially hypoxia) and specific genetic alterations acquired during oncogenesis. In order to investigate the relationship between genetic aberrations and tumor angiogenesis in GBM xenograft lines, the genetic alterations were examined by Comparative Genomic Hybridization (CGH). Two vascular phenotypes of GBM xenografts could be identified: a well vascularized and a poorly vascularized type. In this model, the poorly vascularized type had a larger number of genetic alterations. However, there was no unequivocal correlation between angiogenesis, growth rate and patterns of genetic alterations as detected by CGH.

A highly specific and sensitive sandwich blocking ELISA based on baculovirus expressed pseudorabies virus glycoprotein B.

A direct sandwich blocking enzyme-linked immunosorbent assay (BacgB ELISA) based on the reaction between a monoclonal antibody (MAb) and a recombinant glycoprotein B (gB) of pseudorabies virus (PRV) was developed. This protein was obtained in large quantities from insect cells infected with a PRV gB recombinant baculovirus. Expression of the gB was confirmed by immunoperoxidase monolayer assay (IPMA) with gB specific MAbs. The specificity and sensitivity of the developed BacgB ELISA were evaluated and compared with two commercially available tests by using sets of sera of known PRV infection or vaccination history. For validation, 347 serum samples have been tested. The BacgB ELISA had a high sensitivity and specificity, which were comparable with those of the two commercial tests. In addition, the BacgB ELISA allows detecting anti-gB antibodies in pig serum as early as 7 days following infection. Also maternal antibodies in uninfected pig sera were detected. We conclude that the BacgB ELISA is a useful tool for the detection of as well vaccinated as infected pigs (including derivatives from gE negative vaccine strains), with the added advantage that it uses an antigen that can be produced safely and in large quantities.

AgarCyto: a novel cell-processing method for multiple molecular diagnostic analyses of the uterine cervix.

In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer.

Development and validation of a computerized cytomorphometric method to assess the maturation of vaginal epithelial cells.

BACKGROUND: After menopause, declining levels of estrogens may cause vaginal discomfort, or so-called "vaginal atrophy." Evaluation of therapies for vaginal atrophy may be performed using the so-called "maturation index." The maturation index is expressed as the percentage of (para)-basal, intermediate, and superficial epithelial cells in a vaginal smear. Manual assessment of the maturation index is subject to inter- and intraobserver variations. In this study, assessment of the maturation of cells in vaginal smears using automated image analysis was investigated. MATERIALS AND METHODS: Automated assessment, using a commercially available image analysis system, was performed on hematoxylin-eosin-stained cytospin specimens. A training set was constructed by an experienced cytotechnologist, based upon visual classification of stored grey value images. From this, two discriminant functions (DFs) were calculated capable of classifying cells in one of the three types. These cell classifiers were capable of classifying 97% of the cells correctly. Data from automated assessment were compared with those of classical manual counting. Specimens of 13 mature and 6 atrophic vaginal specimens were assessed in duplicate, both manually and by image analysis, using the DFs. RESULTS: No significant interobserver effect was found for image analysis, whereas a significant effect was found for manual counting. Both methods were able to distinguish between matured and atrophic specimens. CONCLUSIONS: It was concluded that for assessment of vaginal maturation, the use of automated image analysis systems is recommended. Besides increased reproducibility, image analysis systems yield additional data describing the size and shape of the cytoplasm and nucleus of cells, which might increase discriminating power.

Human papilloma virus detection by in situ hybridisation signal amplification based on biotinylated tyramine deposition.

Aim-To describe a method for amplifying human papilloma virus (HPV) in situ hybridisation (ISH) signals.Methods-Three human cervical cell lines, namely CaSKi, HeLa and SiHa, containing different copy numbers of integrated HPV DNA were studied. Following ISH, catalysed reporter deposition (CARD), based on the deposition of biotinylated tyramine at the location of the DNA probe, was used to amplify the ISH signal.Results-Using CARD-ISH, one to three HPV type 16 copies were detected in situ both in cell suspensions and paraffin wax sections of SiHa cells. CARD-ISH can also be used to detect oncogenic HPV DNA sequences, such as HPV types 16 and 18, in routinely processed formalin fixed, paraffin wax embedded cervical specimens.Conclusions-CARD-ISH is a fast and highly sensitive ISH method for the routine detection of low copy number HPV DNA sequences in cervical cell lines and routinely processed tissue sections. Application of this technology also enables the routine detection and cellular localisation of other viral DNA sequences present at copy numbers below the detection limit of conventional ISH methods.

Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretion receptor superfamily with an unusual extracellular domain.

CD97 is a monomeric glycoprotein of 75 to 85 kDa that is induced rapidly on the surface of most leukocytes upon activation. We herein report the isolation of a cDNA encoding human CD97 by expression cloning in COS cells. The 3-kb cDNA clone encodes a mature polypeptide chain of 722 amino acids with a predicted molecular mass of 79 kDa. Within the C-terminal part of the protein, a region with seven hydrophobic segments was identified, suggesting that CD97 is a seven-span transmembrane molecule. Sequence comparison indicates that CD97 is the first leukocyte Ag in a recently described superfamily that includes the receptors for secretin, calcitonin, and other mammalian and insect peptide hormones. Different from these receptors, CD97 has an extended extracellular region of 433 amino acids that possesses three N-terminal epidermal growth factor-like domains, two of them with a calcium-binding site, and a single Arg-Gly-Asp (RGD) motif. The existence of structural elements characteristic for extracellular matrix proteins in a seven-span transmembrane molecule makes CD97 a receptor potentially involved in both adhesion and signaling processes early after leukocyte activation. The gene encoding CD97 is localized on chromosome 19 (19p13.12-13.2).

A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine.

For amplification of in situ hybridization (ISH) signals, we describe a method using catalyzed reporter deposition (CARD). This amplification method is based on the deposition of biotinylated tyramine (BT) at the location of the DNA probe. The BT precipitate can then visualized with fluorochrome- or enzyme-labeled avidin. Both for bright-field ISH (BRISH) and for fluorescence ISH (FISH), the detection limit was highly increased. This method is especially suitable for visualization of very weak ISH signals, such as those obtained by ISH using locus-specific DNA probes. Furthermore, CARD amplification of ISH signals (CARD-ISH) is highly sensitive, rapid, flexible, and easy to implement. Successful application of CARD-ISH with locus-specific DNA probes on histological and cytological samples may improve the determination of structural chromosomal aberrations in archival material.

Double-target in situ hybridization in brightfield microscopy.

For brightfield detection of two different DNA target sequences in one sample, we developed a double-target in situ hybridization (ISH) technique, using biotin- and digoxigenin-labeled chromosome-specific DNA probes. First, several immunochemical detection systems were optimized and compared for sensitivity and simultaneous applicability. Two non-interfering immunochemical systems were chosen for simultaneous detection of the DNA probe labels. This resulted in combination of an alkaline phosphatase (AP)-conjugated avidin-biotin system with a horseradish peroxidase (HRP)-conjugated antibody system for detection of biotin- and digoxigenin-labeled DNA probes, respectively. Development of AP with New Fuchsin-naphthol phosphate and HRP with diaminobenzidine-H2O2 resulted in stable, well-contrasting (red and black, respectively) color precipitates visible by conventional light microscopy. The double-target ISH technique was successfully applied on a wide variety of biological materials, such as metaphase spreads, cytospin, and Thin-prep samples of cytological specimens, frozen tissue sections, and formalin-fixed, paraffin-embedded tissue sections. In particular, on tissue sections, where quantitative interpretation of ISH data can be hampered by truncation of nuclei, the double-target ISH technique appeared to be a valuable tool for demonstration of chromosome aberrations and chromosome imbalances.

Condylar resorption in orthognathic surgery. The role of intermaxillary fixation.

Condylar resorption that occurs after orthognathic surgery was investigated in a large sample of patients treated in the Department of Oral and Maxillofacial Surgery of the Free University in Amsterdam, the Netherlands. The findings correspond with previous publications on this subject. In a 1-year follow-up study the role of intermaxillary fixation was investigated radiologically. In a group of 158 patients prone to show occurrence of condylar resorption, 24 (26.4%) of the 91 patients treated with intermaxillary fixation showed signs of condylar resorption. In the group of 67 patients treated without intermaxillary fixation only eight (11.9%) of the patients showed signs of reduced volume of the condyle. Avoidance of intermaxillary fixation seems to reduce the incidence of condylar resorption after orthognathic surgery in patients with a mandibular deficiency with high mandibular plane angle.

DNA in situ hybridization as a diagnostic tool in the discrimination of melanoma and Spitz naevus.

As the clinical and histological differential diagnosis between Spitz naevus and cutaneous melanoma may be very difficult, we have investigated whether DNA in situ hybridization maybe helpful in resolving this problem. To this end, routinely-processed paraffin sections of 15 typical Spitz naevi, 15 typical nodular melanomas, and five cases originally misdiagnosed as Spitz naevi but which later metastasized and were reclassified as melanoma were analysed using a method previously described (De Wit et al., J Invest Dermatol 1992; 98: 450-458). Microscopical semi-quantitative evaluation revealed that the number of nuclei with supernumerary aberrations of the centromere region of chromosome 1, suggestive of aneuploidy, was significantly different in Spitz naevi and nodular melanoma. The mean number of aberrant nuclei per high power field was 0.41 and 4.01, respectively (P = 0.0001). On applying the results of the typical lesions to the equivocal, originally misdiagnosed lesions, three out of five could be identified as melanoma. These results suggest that the application of DNA in situ hybridization may contribute to the positive identification of histologically equivocal pigmented lesions. The advantages of this technique are that it is cheap, requires little tissue, and can be applied on routinely-processed paraffin sections.

Identification of numerical chromosome aberrations in archival tumours by in situ hybridization to routine paraffin sections: evaluation of 23 phaeochromocytomas.

We have applied non-isotopic in situ hybridization (ISH) to interphase cell nuclei of 23 phaeochromocytomas (18 primary and 5 metastatic tumours) within routine paraffin-embedded tissue sections. Each tumour was screened for numerical aberrations with a defined alphoid repetitive DNA probe set containing DNA probes specific for chromosomes 1, 7, 15, and Y. Normal adrenal medullas and other normal human cell types served as cytogenetic controls. Preservation of tissue morphology enabled targeted analysis of tumour cells. The presence of numerical chromosome changes in the tumour cells could easily be evaluated by comparing the ISH results of the DNA probes. Numerical abnormalities not previously reported in this neoplasm included overrepresentation of chromosomes 1 and 7, loss of chromosome 15, and both gain and loss of chromosome Y (P values < 0.01). The percentage of aneuploid cell nuclei in a tumour correlated well with the percentage of cells in the 4C peak of flow cytometric DNA histograms from these neoplasms. We conclude that interphase ISH can be used for the identification of new and reported cytogenetic changes in tumour cell nuclei within archival tissue sections. This novel procedure also allows for retrospective analysis of previously not karyotyped material.

Histological preservation after in situ hybridization to archival solid tumour sections allows discrimination of cells bearing numerical chromosome changes.

In this study, non-isotopic in situ hybridization (ISH) was used for the cytogenetic and histological examination of urological (prostatic adenocarcinoma) and endocrine (phaeochromocytoma) tumour cell nuclei in 4 microns paraffin-embedded tissue sections. In order to investigate preservation of tissue morphology, standard heat denaturation was compared with a mild enzymatic treatment for the production of single-stranded (ss)-DNA for ISH. Numerical analysis by ISH with chromosome-specific repetitive DNA probes for chromosomes 1, 7, and 11 revealed overrepresentation of chromosome 7 in the phaeochromocytoma (P < 0.01). The constitutional underrepresentation of the Y chromosome was easily detected in the prostate tumour (P << 0.01) when probed for chromosomes 7, 16, and Y. The enzymatic treatment appeared superior to heat denaturation with respect to tissue architecture in the phaeochromocytoma, while no clear difference was observed in the prostatic cancer. ISH probe patterns were similar for the two types of denaturation in both tumours (P > or = 0.20). We conclude that (1) ISH can be used for the identification of numerical cytogenetic changes in solid tumour cell nuclei within archival tissue sections; and (2) mild 'denaturation' protocols, replacing heat, are preference in retaining tissue architecture in fragile tumour specimens.

Quantitative immunohistologic study of lip biopsies. Evaluation of diagnostic and prognostic value in Sjögren's syndrome.

In a group of 45 patients with Sjögren's syndrome (SS) and 80 controls the high specificity (95%) and sensitivity (100%) of a recently proposed bivariate quantitative immunohistologic (QIH) criterion for SS, based on percentages of IgA and IgG-containing plasma cells in labial salivary gland (LSG) tissue, was confirmed. The best univariate QIH criterion for discrimination between LSG biopsies of SS patients and controls appeared to be based on the percentage of IgA containing plasma cells, and had a specificity of 99% and a sensitivity of 96%. A criterion based only on the percentages of IgM-containing plasma cells, proposed in another recent study, resulted in a high number (31%) of false negatives. Interobserver reproducibility of QIH diagnoses was excellent. Moreover it was demonstrated that accuracy, precision and the interobserver reproducibility of plasma cell counting depends on the choice of tissue fixation and immunohistologic staining procedure. The combination of formol sublimate fixation and peroxidase anti-peroxidase procedure appeared to be the best combination for QIH examination. Furthermore, in 2 SS patients systemic monoclonal IgM/kappa gammopathy was preceded by high predominance of IgM and kappa containing plasma cells in the plasma cellular infiltrate of the LSG tissue.

Gas chromatographic determination of the pesticide dodemorph for assessment of occupational exposure.

As part of a health hazard survey of occupational exposure to pesticides in greenhouse growing of ornamentals, analytical methods are developed and validated for measurement of exposure of workers to the fungicide dodemorph. A gas chromatographic method is developed using on-column injection and nitrogen-phosphorus detection for quantification. Methods for the determination of (potential) dermal exposure by the analysis of foliar dislodgeable residues and cotton gloves are validated with respect to background, analytical recovery, stability, limit of detection, and between-day coefficients of variation. Analytical recovery from 'foliar dislodgeable residue solutions' and cotton gloves is more than 95%. Dodemorph in 'foliar dislodgeable residue solutions' and on cotton gloves is stable for at least five and six months, respectively, when stored in the refrigerator. Between-day coefficients of variation are 6% for both matrices. The limit of detection is 3 micrograms per leaf sample and 150 micrograms per pair of gloves. Institute of Occupational Medicine (IOM) samplers, designed for the collection of a defined inspirable fraction of aerosols, are tested for sampling air-borne dodemorph. IOM samplers equipped with glass-fiber or cellulose filters appear unsuitable for reliable sampling of the fungicide because of breakthrough or breakdown during sampling.

Influence of fixation and immunohistological technique on accuracy, precision and inter-observer reproducibility of plasma cell counting.

Recently two highly sensitive and specific diagnostic criteria for Sjögren's syndrome based on percentages of IgA-, IgG-, and IgM-containing plasma cells measured in immunohistologically stained labial salivary gland tissue have been described. The reliability of such a criterion is dependent on the accuracy, precision and inter-observer reproducibility in plasma cell counting. The present study evaluates the effect of tissue fixation and immunohistological procedures on the aforementioned factors. Immunoglobulin (Ig)-containing plasma cells in sections of lamellated submandibular salivary gland tissue, alternately fixed in a 4% buffered formol solution or formol-sublimate solution and stained with an indirect immunoperoxidase and unlabelled peroxidase anti-peroxidase (PAP) method respectively, were enumerated by three independent observers. Relative numbers of Ig-containing plasma cells appeared to be less sensitive for systematic errors due to tissue fixation and immunohistological procedure than absolute numbers of Ig-containing plasma cells. The best inter-observer reproducibility of plasma cell counts was obtained in sections from formol sublimate-fixed specimens stained according to the PAP procedure.

Condylar atrophy and osteoarthrosis after bimaxillary surgery.

Radiographic evidence of condylar atrophy was seen in 12 patients out of 206 patients who underwent surgical orthodontic treatment. All 12 patients had the same dentofacial deformity, high-angle mandibular retrognathia (Class II open bite), and all but one had bimaxillary surgery. The etiologic factors are discussed. The dentofacial deformity is considered to be the main reason for condylar resorption, but orthognathic surgery is supposed to stimulate the progress of the disease by increased loading, disk displacement, and immobilization.