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The temperature-composition phase diagrams of aqueous solutions of gelatin and dextran, which show liquid/liquid phase segregation, were explored at temperatures above the gelation temperature of gelatin. The compositions of the coexisting phases were found to show practically no dependence on temperature between 40 and 80 degrees C. Also, the total polymer concentration at which phase separation occurred was found to be nearly independent of temperature. These observations suggest an entropy-driven phase separation. An explanation in terms of depletion, reversible clustering, and subsequent transient network formation of gelatin at temperatures well above the temperature of gelation is suggested. Phase separation is found to be accompanied by strong fractionation of the molar mass distribution in the two phases.

Many patients with colorectal carcinoma (CRC) mount a cellular as well as a humoral immune response to the tumor. To investigate the nature and specificity of the humoral immune response in a CRC patient, lymphocytes infiltrating the primary colorectal tumor and lymph nodes draining the tumor were used as antibody variable (V)-gene pools for the construction of phage antibody repertoires. These libraries were first validated by selection on the antigen tetanus toxoid and shown to contain antibodies that were probably derived from both naive and memory B cells. The repertoires were then screened for the presence of antibodies directed to CRC by selection on the cell line CaCo2. For comparison, the same selections were performed with a phage antibody repertoire made from B cells of healthy donors. Striking differences were observed in the panel of specificities selected from these different repertoires: although a large panel of antibodies reactive with patient-derived primary tumors was obtained from the immune repertoires, none of these discriminated between normal colonic epithelium and colon cancer and none were reactive with cell-surface antigens. However, selections using the non-immune library did result in numerous antibodies that recognized cell surface markers on CaCo2. These data suggest a bias in the local humoral immune response in this CRC patient, directed primarily toward intracellular epithelial-cell specific target antigens., (Copyright 2001 Wiley-Liss, Inc.)

The aim of this study was to assess the diagnostic performance of breast imaging in the diagnosis of breast cancer in a non-screening population. In a consecutive set of patients referred for mammography in one year, the results of palpation and radiological breast imaging were scored on a 5-point grading scale and linked to pathology as gold standard after a follow up period of one year. The diagnostic performance was studied by logistic regression analysis and ROC-curves. There were 1,944 breast examinations in 1,890 patients and 3,816 breasts. Pathology results reported 118 malignancies in 115 women. With a cut-off point between benign and uncertain benign a sensitivity of 89% and a specificity of 98% was found for radiological imaging. ROC-curves showed a significant increase in diagnostic performance when radiology was added to results of palpation and age (p = 0.007). Radiological imaging tests have a large diagnostic value in the detection of breast cancer in addition to palpation and age. A sensitivity close to 100% could be reached.

In this paper we have derived constitutive equations for an elastic material with anisotropic rigid particles. We have included a dependence on the Finger tensor B and the orientation tensor Q in the expression for the free energy of the system. With this expression for the free energy we have derived an expression for the stress tensor up to second order in both these variables. We have shown that the elastic modulus in this expression depends on Q, and this dependence leads to an effective elastic modulus that depends on the strain. We have calculated the explicit form of the equation for the stress tensor for a deformation in the xy plane with a strain equal to -gamma. For fully isotropic materials with Q=0 this expression reduces to an equation containing only odd powers of gamma. The inclusion of a non-zero value for the orientation tensor leads to an additional set of terms in the equation, all proportional to Qxy (the xy component of the tensor Q), and all proportional to even powers of gamma. We have qualitatively compared these expressions with Fourier transform (FT) rheological measurements of xanthan gels, at concentrations above and below the order-disorder transition. In FT rheometry an oscillatory deformation is applied in the nonlinear regime, and the resulting stress response is analyzed in Fourier space. In the 2% (w/w) xanthan system (disordered state) only odd harmonics were found in the stress response, whereas in the 4% (w/w) xanthan gel (ordered state) even harmonics could be detected. As predicted by our theory, the intensity of these even harmonics first increased with increasing gamma, until a maximum value was reached. Beyond this maximum the intensity decreased continuously with increasing gamma.

Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for therapy of human solid tumours. The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety of human solid tumours and constitutes an attractive target for immunotargeting. We set out to obtain fully human antibodies to this antigen by selecting from a large antibody repertoire displayed on bacteriophages. Two single-chain variable antibody fragments (scFv) were identified that specifically bound recombinant antigen in vitro. One of the selected antibodies (VEL-1) cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2) specifically recognised colon cancer cells. The latter antibody was further characterised with respect to epitope specificity and kinetics of antigen-binding. It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody and had an off-rate of 5 x 10(-2) s-1. To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity, the genes of scFv VEL-2 were re-formatted by fusion to a human (gamma 1) hinge region and CH3 domain. This "minibody" was expressed in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis. These results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules with promising characteristics for in vivo use in tumour targeting.

Immunotargeting of solid tumours using antibodies has become a valuable tool for the detection of cancer metastases and the treatment of minimal residual disease. However, only few tumour antigens useful for targeting have been described to date. To identify cell-surface targets on colorectal carcinoma (CRC), we selected a large, human phage antibody repertoire on freshly isolated colon tumour cells. Two antibodies were identified that reacted with epithelial cell-restricted cell-surface antigens, whereas one clone preferentially reacted with stromal cells. These antigens are tumour-associated antigens, as shown by their uniform expression in tumours of different patients and of different differentiation stages and by their limited expression on normal tissues. The pattern of reactivity in immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) suggests that these antigens are different from previously identified tumour-associated antigens (e.g. Ep-CAM or c-ERB-2). This phage antibody-based method may lead to the cloning of novel tumour antigens that are useful for the immunotargeting of solid tumours.

The purpose of this study was to compare the vascular response seen with intravascular ultrasound (IVUS) at 1-year follow-up between statin-treated and non-statin-treated patients. Patients (n = 10) undergoing percutaneous transluminal angioplasty (PTA) of the femoropopliteal artery were studied with IVUS immediately after PTA and at 1-year follow-up. In nondilated matched vascular segments, the change in lumen, vessel, and plaque volume was assessed. In balloon-dilated matched vascular segments, the change in lumen, vessel, and plaque area was assessed. A comparison was made between statintreated (n = 5) and non-statin-treated patients (n = 5) in lumen, vessel, and plaque changes. At follow-up, both statin-treated and non-statin-treated patients showed a similar increase in plaque volume at the nondilated segment (+4% and +2%, respectively). In statin-treated patients the plaque volume increase was compensated by an increase in vessel volume (+2%), resulting in an increase in lumen volume (+1%). In non-statin-treated patients, on the other hand, the increase in plaque volume was associated with a decrease in vessel volume (-2%), resulting in a decrease in lumen volume (-4%). At the balloon-dilated segment a similar trend in changes of lumen, vessel, and plaque was encountered. Differences between both groups of patients were not statistically significant. Despite the nonsignificant nature of the observation, this small retrospective IVUS study may generate the hypothesis that statin therapy may contribute to superior long-term lumen dimensions by inducing positive vascular remodeling both in nondilated and balloon-dilated vascular segments.

In view of a prospective intravascular ultrasound (IVUS) study, the reproducibility of the extent of the calcified lesion in IVUS images derived from separate pull-back maneuvers was assessed. Patients (n = 34) were imaged with IVUS before and after percutaneous transluminal angioplasty (PTA) and at 1-y follow-up. In the presence of a calcified lesion, the largest arc and the length of the matched calcified lesions was assessed. Interobserver differences in arc measurements were low (< or = 0.7%), with low coefficients of variation (< or = 5.8%). Similarly, interexamination differences in arc and length measurements were small (< or = 1.1%), with low coefficients of variation (< or = 3.2%). At follow-up, a nonsignificant increase in both the arc (1.9%) and length (1.7%) of the calcified lesion was observed. This study showed that measurements of the calcified lesion are highly reproducible; changes seen at 1-y follow-up were not significant. We conclude that IVUS may be used to monitor the effect of medical intervention on the extent of the calcified lesion in a longitudinal study.

Rationale and Objectives: To develop a new automated calibration method for vessel measurements in vascular x-ray images., Methods: Radiopaque marker bands mounted equidistantly on a small catheter were acquired in vitro at five image intensifier (II) sizes in x-ray projection images. The positions of the marker centers were detected by using a Hough transform and were computed at subpixel precision by using either a novel, iterative center-of-gravity approach (CGA) or a symmetry filter. Curve-fitting procedures were used to reject false-positive marker detections and to calculate intermarker distances. The calibration factor was calculated from the true marker distance and the average of the measured distances in pixels. Results were compared statistically with a grid calibration method, which was taken as the gold standard. A simulation study was performed to assess the influence of image noise on the CGA method., Results: The iterative CGA method was convergent and faster than the symmetry-based technique. For four II sizes (17, 20, 25, and 31 cm), the results from the CGA method were not significantly different from the results obtained with grid calibration. For the II size of 38 cm, a significant difference (0.3% of the grid calibration factor) was found; however, this was caused by the quantification error in the image data and was not clinically relevant. In general, the performance of the CGA method improved with increasing signal-to-noise ratio., Conclusions: A practical new calibration method for small catheter sizes was developed and validated for quantitative vascular arteriography.

In this study, the possibilities for quantification of vessel diameters of peripheral arteries in gadolinium contrast-enhanced magnetic resonance angiography (Gd CE MRA) were evaluated. Absolute vessel diameter measurements were assessed objectively and semi-automatically in maximum intensity projections (MIPs) of contrast-enhanced T1-weighted 3D spoiled gradient-echo datasets, studied with digital subtraction techniques. In vivo, the complete peripheral arterial bed of six patients was studied, from the aorto-iliac bifurcation down to the distal run-off. By measuring the signal intensity (SI) over the lumen of a vessel in the MIP, an SI-plot was obtained. Next, the vessel boundaries were determined using a threshold algorithm; from these boundary points individual diameter values could be obtained along the trajectory of the vessel. In an in vitro study, an optimal threshold value of 30% of the range of SI-values between the background and the maximal SI in the vessel was obtained for accurate diameter measurement in Gd CE MRA (i.e., full-width 30%-maximum). Furthermore, the relationship between the accuracy of these measurements and the scan resolution was investigated. Accuracy was found to be acceptable (i.e., less than 10% over/underestimation) for vessel sizes covering at least 3 pixels. In six patients, diameters were measured in MIPs of the total datasets (i.e., D(T)) as well as in selective MIPs of the clipped datasets (i.e., D(S)) (n = 209). D(T) and D(S) were statistically significantly correlated (p < 0.01) with a Pearson correlation coefficient rP = 0.98. Measurements in the total MIPs yielded statistically significant (p < 0.01) smaller diameter values compared with measurements in selective MIPs, with a mean difference of 0.15 mm. Diameter values from the selective MIPs of the aorto-iliac arteries were also compared with diameter values measured at corresponding anatomic positions in X-ray angiograms of these patients (i.e., D(x)) (n = 70). D(X) and D(S) were statistically significantly correlated (p < 0.01) with a Pearson correlation coefficient rP = 0.92. Diameters measured in the selective MIPs were smaller than those measured in the X-ray angiograms (mean difference 0.49 mm) and this difference was statistically significant (p < 0.01). In conclusion, diameter values can be evaluated accurately in MIPs of vessels with at least 3 pixels in diameter, using the full-width 30%-maximum criterion.

Phage antibody display technology offers a powerful tool for the isolation of specific antibodies to defined target antigens. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well-defined antigen source. However, when the target antigen cannot be purified (e.g., an integral membrane protein), or if the antigen is unknown (e.g., when searching for novel markers on cells or tissues), panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental challenges. One focus of our research is to select antibodies directed to novel cancer-induced antigens expressed by tumours and by the tumour vasculature. To understand the parameters governing selection on complex antigen sources and to assess the efficiency of these phage library selections, we have set up two model selection systems in which both tumour cells and vascular endothelial cells serve as target "antigen". We describe a model based on phage antibodies directed to the tumour antigen epithelial glycoprotein-2, to compare phage antibody selections on a range of different antigen sources including purified and recombinant antigen, whole live cells, tissue cryosections and in vivo grown solid tumours. Secondly, we describe a model based on a phage antibody directed against the endothelial cell inducible adhesion molecule E-selectin. We compare selections on cultured cell monolayers with selections on cell suspensions immobilised on columns, to determine which selection approach is most suitable for the identification of novel tumour endothelial cell markers. Our data provide insight into the efficiency and thus potency of different selection strategies and show that there are very large differences in the recovery and enrichment of binding phage between the different methods tested. Our results further demonstrate the feasibility of phage antibody selections on whole, intact cells and show that these may sometimes compare favourably to selections on purified antigen. Selections on endothelial cells immobilised on columns compare favourably with selections on cell-monolayers; the most favourable conditions for both selection procedures are described. The implications of our data for phage antibody selections on these different complex antigen sources using either non-immune or immune phage antibody repertoires are discussed. The use of model systems such as the ones described here will help to determine optimal experimental conditions for phage library selections on complex antigens and aid in developing more powerful selection procedures for target discovery.

In patients with rheumatoid arthritis (RA) synovitis activity is the dominant clinical variable that determines the therapeutic approach. At present, the amount of painful and swollen joints assessed by physical examination, is generally used to measure the degree of synovitis activity. A gold standard for the assessment of synovitis activity is not available. The availability of an objective and reproducible method to evaluate synovitis activity in RA would be of great value in patient management and in examination of therapeutic effects. An advantage of the use of radiopharmaceuticals in detection of arthritis activity, compared with other imaging techniques, is the possibility to depict all joints in a single image. Furthermore the technique may image joints which are difficult to assess clinically or radiographically and may also detect joint inflammation in an early phase. In this overview different scintigraphic techniques are compared with each other and with other diagnostic imaging modalities.

Acute urinary retention in women is not a common problem. The incidence is 0.07 per 1000 inhabitants per year. The causes of acute urinary retention can be divided into four groups: obstructive, neurological, pharmacological and psychogenic. More than half of acute urinary retentions occur after surgery or parturition. The most common obstructive cause is a gynaecologic tumor. A psychogenic cause is a diagnosis per exclusionem. The treatment of the acute urinary retention has to be catheterisation before further investigation is done.

Patients suffering from rheumatoid arthritis (RA) may experience a temporary reduction of disease symptoms during pregnancy. As indicated by the occurrence of RA-disease symptoms during pregnancy, three categories of patients were defined, namely, remission, relapse and unchanged. In all three categories changes in the plasma level and glycosylation of alpha1-acid glycoprotein (AGP) were determined longitudinally in comparison to those occurring in pregnancy of healthy women. In healthy pregnancy, we observed: (i) a peak in the plasma concentration at week 18 and a minimum at week 30; (ii) a continuous increase in the degree of branching of the glycans during the entire pregnancy period, and (iii) a decrease in the degree of alpha3-fucosylation of AGP-glycans with a minimum occurring at week 25. Comparable pregnancy-induced changes in glycosylation were found for two other acute-phase proteins alpha1-protease inhibitor (PI) and alpha1-antichymotrypsin (ACT). Increased oestrogen levels, known to occur during pregnancy, may be one of the factors that induce these changes, because the increased branching and decreased alpha3-fucosylation is in agreement with our earlier findings regarding an involvement of this hormone in the regulation of acute phase protein glycosylation in oestrogen-treated males as well as females. In all three clinical categories in RA, pregnancy also induced a continuous increase in the degree of branching of the glycans of AGP. However, similar changes in concentration and fucosylation were only found during remission of the disease symptoms. In the relapse and unchanged categories in RA, the degree of fucosylation and the plasma concentration of AGP remained constant throughout pregnancy. This indicates a relationship between changes in alpha3-fucosylation of AGP and RA disease activity.

Acute and chronic inflammation-induced expression of sialyl LewisX has already been shown to occur on alpha1-acid glycoprotein. We now demonstrate that this phenomenon is not restricted to alpha1-acid glycoprotein but also occurs on two other acute-phase proteins. ie on alpha1-antichymotrypsin and on haptoglobin. The level of expression of sialyl LewisX on these proteins was lower than on alpha1-acid glycoprotein, in all likelihood because alpha1-acid glycoprotein is the only acute-phase protein containing tetraantennary glycans. No expression of sialyl LewisX was detectable on alpha1-protease inhibitor, a protein with a high diantennary glycan content. Non-sialylated LewisX was not detectable on these major acute-phase proteins in any of the conditions studied. This indicates that the majority of the a3-linked fucose residues are present as sialyl LewisX on alpha1-acid glycoprotein, alpha1-antichymotrypsin and haptoglobin. The absolute contribution to the total phenotype in plasma of protein containing this determinant in a multivalent form was highest for alpha1-acid glycoprotein. This leads us to propose that alpha1-acid glycoprotein is, among the acute-phase proteins studied, the one with the highest potential for interference with the extravasation of leukocytes by binding to the selectins.

Lethal hypophosphatasia, spur type: case report and fetopathological study: Hypophosphatasia (HP) is characterised by severe undermineralisation of the skeleton owing to deficiency of tissue nonspecific alkaline phosphatase. Clinically a perinatal, infantile, childhood and adult type is distinguished. Clinical signs in the perinatal type of HP show considerable overlap with other skeletal dysplasias such as osteogenesis imperfecta type IIA and type IIC, and achondrogenesis type IA. If present, "spurs" of the limbs are diagnostic for HP. We present a prenatally diagnosed case of HP and discuss the differential diagnosis based on clinical, radiological and pathological findings. Our findings indicate that two types of spurs can be distinguished in hypophosphatasia: midshaft type and joint type.

Background: There is a need for markers in colorectal cancer which will allow subclassification of stage groups into subgroups with high versus low risk of recurrent disease., Aims: To develop monoclonal antibodies that recognise antigens or immature crypt base cells, on the assumption that in a neoplasm undifferentiated but not the terminally differentiated cells will be responsible for tumour progression., Methods: Colon crypt cells which were isolated from human colonic mucosa by EDTA/EGTA incubation were studied. By stepwise harvesting, crypt base cell enriched fractions were obtained, and after incubation with antibodies against dominant antigens, used as immunogens., Results: Of one crypt base cell specific antibody (5E9), the reactive epitope appeared to be a non-terminal carbohydrate in the mucin O-glycans of the colon. The epitope did not seem to be colon specific, but was expressed in a variety of other tissues. In colorectal carcinomas, 5E9 immunoreactivity identified a subgroup of patients with a tendency for worse prognosis., Conclusion: A mucin associated maturation epitope was identified in colonic crypt base cells, the expression of which in Dukes' stage B3 colorectal carcinoma may be associated with poor prognosis.

A follow-up study was performed to assess the functional and radiological outcome after operative treatment of tibial plateau fractures. Of 62 patients operated upon from 1985 to 1993, 46 patients were traced and re-examined. Follow-up averaged 60 months. Three functional scores were applied: the HSS knee score, the Lysholm score and the Tegner score. Radiographs obtained at follow-up were rated for deformity and signs of arthrosis, using a score described by Reswick and Niwayama. Patients reported most functional complaints during the first 3 years and after 6 years. Patients seen between 3 and 6 years after injury showed better functional scores. Arthritic changes were seen from 5 years after the fracture. Patients over 60 years of age seemed to suffer less functional loss than younger patients, indicating that operative treatment should not be withheld on grounds of age alone.

Previous in vitro and in vivo model studies have shown that when E-cadherin expression in carcinoma cells is reduced, invasive behaviour ensues. The situation in human cancer in vivo, however, appears to be more complex, as immunohistochemically determined E-cadherin expression in various carcinomas, including colorectal cancer, does not always correlate with invasive growth. Loss of cell adhesion during invasion in spite of E-cadherin expression might be associated with a defective cadherin-catenin complex. The expression of alpha- and beta-catenin in comparison with E-cadherin was therefore examined in colorectal adenomas and carcinomas and in lymph node and liver metastases. In normal colonic mucosa, alpha- and beta-catenin immunoreactivity occurred along the lateral plasma membranes of the epithelial cells, in a pattern identical to E-cadherin staining. A similar pattern was found in colorectal adenomas and in most malignancies. In general, in neoplastic epithelia, the majority of the cancer cells displayed a normal (matching) pattern of E-cadherin and catenin expression. It is concluded that the patterns of expression of E-cadherin and alpha- and beta-catenin are very similar in colorectal neoplasms. This observation indicates that invasion in colorectal cancer is not paralleled by consistent loss of expression of the components of the cadherin-catenin complex.

The occurrence of differences in acute-phase response, with respect to concentration and glycosylation of alpha 1-acid glycoprotein (AGP) was studied in the sera of patients, surviving or not from septic shock. Crossed affino-immunoelectrophoresis was used with concanavalin A and Aleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of sialyl Lewisx (SLeX) groups on AGP. Septic shock apparently induced an acute-phase response as indicated by the increased serum levels and changed glycosylation of AGP. In the survivor group a transient increase in diantennary glycan content was accompanied by a gradually increasing fucosylation and SLeX expression, comparable to those observed in the early phase of an acute-inflammatory response. Remarkably, in the non-survivor group a modest increase in diantennary glycan content was accompanied by a strong elevation of the fucosylation of AGP and the expression of SLeX groups on AGP, typical for the late phase of an acute-phase response. Our results suggest that these changes in glycosylation of AGP can have a prognostic value for the outcome of septic shock.

Background: Point mutations within the family of the ras genes are detected in approximately 50% of human colorectal adenomas and carcinomas. Therefore, it is generally accepted that the occurrence of ras-point mutations constitute an important step in colorectal carcinogenesis. In addition, many studies have demonstrated that the tumorigenicity of the human colorectal carcinoma cell line, CaCo 2, strongly increases after transfection with the c-Ha-ras oncogene. This cell line is suitable for gaining more insight into the mechanism of c-Ha-ras induced tumorigenesis., Material and Methods: Proliferation, differentiation, and proteolytic capacity of c-Ha-ras oncogene transfected CaCo 2 cells were studied in vitro., Results: It was found that gelatinolytic capacity and production of urokinasetype plasminogen activator increased, whereas the production of tissue-type plasminogen activator was similar. Proliferative activity, as measured by the potential doubling time, did not alter. The expression of the differentiation markers sucraseiso-maltase, mucin, and chromogranin A was not different from that of the parental CaCo 2 cell line, which indicates that an increased tumorigenic capacity of c Ha-ras oncogene transfected CaCo 2 cells is not accompanied by loss of differentiation., Conclusion: These data demonstrate that the highly increased tumorigenic capacity of c Ha-ras oncogene-transfected CaCo 2 cells is associated with an enchanced proteolytic capacity.

We describe a 20-week-old fetus with multiple congenital anomalies exposed to acitretin in the first trimester of pregnancy. Acitretin and its ethylester etretinate are both vitamin A congeners; drugs of this group are well-known teratogenic agents. Since the marketing of acitretin only one report on human teratogenicity associated with acitretin has been published. The present male fetus showed severe symmetric anomalies of upper and lower limbs, craniofacial anomalies, ear anomalies, and an atrioventricular septal defect (ASD). Although the craniofacial anomalies resemble the abnormalities described in classical "retinoic acid embryopathy," limb anomalies were seldomly reported after maternal use of vitamin A congeners. However, in laboratory animals limb defects were frequently observed after retinoid exposure in utero. This case emphasizes again that extreme care and precaution are needed before prescribing a potentially teratogenic drug to a fertile woman.

Objective: Our purpose was to examine the immunohistochemical properties of epithelial cells in peritoneal fluid and to compare the staining characteristics with cells of endometrium, menstrual effluent, peritoneum, and endometriotic lesions., Study Design: Samples of menstrual effluent, endometrium, and peritoneal fluid and biopsy specimens of endometriotic lesions and peritoneum from 16 patients were examined. Monoclonal antibodies against vimentin, cytokeratin 18 and 19, and the monoclonal antibody BW495/36, staining an epithelial marker present in endometrium and absent in peritoneal epithelium, were used., Results: All but one sample of menstrual effluent and peritoneal fluid cells stained positively with antibodies against vimentin and cytokeratin 18 and 19. BW495/36 stained 14 of 16 menstrual effluent samples and nine of 16 peritoneal fluid cell samples. Endometriotic specimens showed staining with all markers. No major differences in staining properties were observed in menstrual effluent, endometrium, and peritoneal fluid cells between patients with or without endometriosis., Conclusion: These results support the contention of transport of menstrual detritus to the peritoneal cavity in women with patent fallopian tubes.

Objective: To analyze the cause of infertility in a patient with polycystic kidney disease., Setting: General hospital., Patient: A 32-year-old man known with adult polycystic kidney disease, complaining of infertility and testicular pain., Interventions: Testicular biopsy; imaging., Results: Examination revealed bilateral severe congestion of the epididymis. The semen contained no spermatozoa and practically no fructose. Testicular biopsy, however, showed normal spermatogenesis. Transrectal ultrasonography and computer tomography revealed severe congestion and extensive cystic changes in the seminal vesicles., Conclusion: The dilated cysts in the seminal vesicles had led to obstructive azoospermia, probably through compression of the ductal system.

The aim of this review is to summarize the research efforts of the last two decades with respect to (i) the determination and characterization of the changes in glycosylation of AGP under various physiological and pathological states; and (ii) the effects of such changes on its possible anti-inflammatory functions. It will become clear that the heterogeneity observed in the glycosylation of AGP in serum, represents various so-called glycoforms of AGP, of which the relative amounts are strictly determined by the (patho) physiological conditions.

Fracture of the sesamoid bone of the thumb is a rare injury. In this report three additional cases with fracture of an ulnar sesamoid bone of the thumb are reported. The anatomic considerations, the mechanisms of injury, the differential diagnoses, and management are discussed with the English-language literature.

In this study we investigated the tumorigenicity, growth pattern and spontaneous metastatic ability of a series of nine human colorectal carcinoma cell lines after subcutaneous and intracaecal xenografting in nude mice. CaCo2 cells were found to be poorly tumorigenic to non-tumorigenic in either site; the other cell lines were tumorigenic in both sites. SW1116, SW480 and SW620 did not show local invasive in the NCI-H716 and LS174T cells were both invasive in the caecum, but only NCI-H716 was invasive in the subcutis. HT29 and 5583 (S and E) cells were invasive in the caecum and from that site metastatic to the lungs and/or the liver. HT29 and 5583S cells were both invasive in the subcutis, but 5583E cells were not. Of each category of in vivo behaviour in the caecum, one cell line was further investigated with regard to invasion in vitro (into embryonic chick heart fragments), E-cadherin expression in vivo and in vitro and in vitro production of u-PA and t-PA. These parameters were chosen in view of their purported role in extracellular matrix degradation and intercellular adhesion, which are all involved in the invasive and metastatic cascade. Invasion in vitro was not predictive for invasion or metastasis in vivo. In the cell line which showed invasion in embryonic chick heart tissue, heterogeneous E-cadherin expression was observed in vitro together with a relatively high production of u-PA. The non-invasive cell lines showed in vitro homogeneous expression of E-cadherin with a relatively low production of u-PA. In vivo expression of E-cadherin was either absent or heterogeneous. We conclude that: (1) colorectal carcinoma xenografts show site-specific modification of in vivo invasive and metastatic behaviour; (2) invasion in vitro does not correlate with invasion and metastasis in vivo; (3) in vitro non-invasion might be associated with homogeneous E-cadherin expression and low production of u-PA; (4) E-cadherin expression in vitro differs from E-cadherin expression in vivo. The results support the notion that the microenvironment in which cancer cells grow is one of the factors involved in the regulation of invasive and metastatic behaviour.

In 67 patients with longstanding anterior knee pain intra-osseous phlebography of the patella and arthroscopy of the knee were performed. No statistical correlation could be demonstrated between the delayed drainage of the contrast medium from the patellar veins and the degree of damage to the patellar cartilage.

L-CAM, also known as E-cadherin, is a cell adhesion molecule expressed on the plasma membranes of epithelial cells at the intercellular interface. From in vitro gene transfection experiments the idea has been conceived that loss of L-CAM expression might be related to the invasive capacity as well as metastatic potential of tumour cells. In several tumours a relation between the grade of differentiation and L-CAM expression has been noticed: loss of differentiation appears to be associated with loss of L-CAM immunoreactivity. Also, in lymph node metastases of poorly differentiated carcinomas loss of L-CAM expression was demonstrated. In this study we describe L-CAM expression in lymphogenous and haematogenous metastases of large bowel adenocarcinomas, using an indirect immunoperoxidase method with the monoclonal anti-L-CAM antibody 6F9. All the metastases studied--lymphogenous as well as haematogenous--demonstrated L-CAM immunoreactivity in a pattern comparable to that of primary tumours. Intratumour heterogeneity in expression was noted, with normal intercellular, apical (non-functional), and focally negative areas in the same tumour. The data indicate that primary tumours and their metastases do not differ strikingly in their pattern of L-CAM expression. This would be consistent with transient rather than constitutive down-regulation of L-CAM in invasive and metastatic cancer cells.

Objective: To detect the expression of integrins and E-cadherin in cells from peritoneal fluid (PF), endometrium, menstrual effluent, peritoneum, and endometriotic lesions during the early follicular phase of the menstrual cycle., Design: An immunohistochemical study., Setting: Tertiary care university medical center., Patients: Sixteen patients undergoing a diagnostic laparoscopy as part of a subfertility work-up. All patients had regular and ovulatory cycles., Interventions: A laparoscopy was performed in the early follicular phase (days 2 to 5). Simultaneously, samples were taken from endometrium, menstrual effluent, and PF, and a representative biopsy of an endometriotic lesion was obtained. If endometriosis was not noted, a peritoneal biopsy was obtained instead., Main Outcome Measures: The expression of cell adhesion molecules, including the integrin alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1 and E-cadherin, as determined by immunohistochemistry on frozen sections., Results: All integrins tested could be detected in the endometrium samples and in endometriotic lesions. In menstrual effluent samples, positive staining for the integrins alpha 2 beta 1 and alpha 3 beta 1 was found in epithelial cells in 13 of 16 cases. Integrin alpha 5 beta 1 was detected in 11 of 16 samples, and integrins alpha 4 beta 1 and alpha 6 beta 1 were detected in 5 of 16 samples. In PF, integrin alpha 3 beta 1 was found in epithelial cells in 12 of 16 samples, integrin alpha 5 beta 1 in 5 of 16, and integrins alpha 4 beta 1 in 2 of 16. The antibody for E-cadherin showed positive staining of epithelial cells in 6 of 16 menstrual effluent samples. All endometrial tissue samples showed positive staining for E-cadherin. In PF, E-cadherin was detected in the epithelial cells of one sample. One peritoneum biopsy revealed positive staining for E-cadherin., Conclusion: Integrins alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, and E-cadherin, important cell adhesion molecules, are expressed in endometriotic lesions and in cells and tissues that are potentially involved in the development of endometriosis. These cell adhesion molecules could be involved in the shedding of endometrial tissue during menstruation and the attachment of endometrial tissue fragments to the peritoneum.

Endocrine cells occur in approximately 30% of all colorectal adenocarcinomas, and this feature appears to correlate with a relatively poor prognosis. To study the factors regulating endocrine differentiation in colorectal cancer, which may bear resemblance to the regulation of endocrine differentiation in normal intestinal mucosa, models in which differentiation can be manipulated are essential. However, endocrine features in colorectal cancer cell lines are scarce and are almost exclusively observed in xenografts, presumably as a result of differentiation induction by stromal components. We attempted to demonstrate endocrine differentiation in the colonic adenocarcinoma cell line Caco-2, which is frequently used as a model for enterocytic differentiation. In vitro endocrine tumor cells were not encountered. In vivo studies were cumbersome, because of the low take rate of Caco-2 cells. We did manage to establish nude mouse xenografts of Caco-2 cells by inoculating cells in collagen gel and by suppressing natural killer cell activity. In an attempt to induce a better take rate and to investigate the effect of Ras oncoprotein overexpression on endocrine differentiation, Caco-2 cells were transfected with a point-mutated c-Ha-Ras gene. The cell line Caco-2 EJ6, generated from these experiments, could be xenografted in nude mice with a high take rate, yielding a moderately well differentiated adenocarcinoma, morphologically identical to the tumors derived from untransfected Caco-2 cells. The xenografts displayed goblet cell, enterocytic, Paneth cell and endocrine differentiation. In vitro endocrine differentiation was observed neither under standard conditions nor with extracellular matrix components as differentiation inducers. We conclude that the Caco-2 cell line and its c-Ha-Ras transfected subline Caco-2 EJ6 in vivo display endocrine differentiation. Ras overexpression does not enhance endocrine differentiation. Due to its favorable growth properties in vivo, Caco-2 EJ6 is a suitable model for studies on endocrine differentiation in colorectal cancer.

Endocrine cells occur in +/- 30% of colorectal adenocarcinomas. The significance of this phenomenon in terms of tumor behavior is still controversial. Endocrine differentiation in colorectal cancer cell lines is almost confined to tumor xenografts in vivo, suggesting that endocrine differentiation might be regulated by epithelial-stromal interactions. This hypothesis was studied in the cecal adenocarcinoma-derived cell line NCI-H716 by comparing the expression of chromogranin A protein and messenger RNA in vivo and in vitro and by attempts to induce differentiation in vitro. We found that chromogranin A expression, which was strongest in vivo, could be significantly enhanced in vitro by culturing tumor cells in the presence of native extracellular matrix, on fibroblast feeder layers, and in a defined medium with basic fibroblast growth factor. The results suggest that the extracellular matrix induces endocrine differentiation through factors (e.g., basic fibroblast-growth factor) that may be produced by stromal cells and after secretion bind to the extracellular matrix.

L-CAM is a cell adhesion molecule which is expressed at the intercellular borders of most epithelial cells. L-CAM has been demonstrated to act as an invasion suppressor in carcinoma cell lines. In order to determine whether or not L-CAM expression might distinguish between invasive and non-invasive or metastatic and non-metastatic colon neoplasms, we studied L-CAM expression in normal colon mucosa, colon adenomas with various degrees of dysplasia, and colon carcinomas by immunohistochemistry, using the 6F9 monoclonal anti-L-CAM antibody. Normal mucosa showed evenly distributed distinct L-CAM immunoreactivity along intercellular borders. In adenomas and carcinomas, a similar though weaker expression was observed. This pattern showed a tendency to decrease in parallel with decreasing differentiation. However, no correlation was found with Dukes stage or area within the tumour. In some carcinomas, L-CAM was expressed at the luminal surface of the cells. In others, L-CAM expression was not found. These results suggest that L-CAM expression is disregulated or lost as an early event in the development of colon neoplasia and indicate that L-CAM expression does not correlate with invasive or metastatic potential.

The ex vivo labelling of DNA-synthesizing epithelial cells in colonic and vaginal mucosa was compared with in vivo labelling. For this purpose, in vivo S-phase cells were labelled with [3H]thymidine (Tdr) and ex vivo labelling was continued by culturing tissue specimens in bromodeoxyuridine (BrdU). Various methods of tissue culture were employed in order to improve diffusion of medium (and BrdU) in the tissue. BrdU and 3H-TdR labelling were evaluated by immunohistochemistry and autoradiography respectively. Ex vivo labelling resulted in a patchy distribution of labelled cells, which did not correspond with the 3H-TdR labelling pattern obtained in vivo. Under the described conditions ex vivo labelling does not appear to be a reliable for estimation of the proliferative activities in vivo.

A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37 degrees C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.

Groups of male Wistar rats were fed semi-synthetic diets containing 0, 200 or 500 mg indole-3-carbinol (13C)/kg for 2, 7, 14 or 28 days. After 2 days, P-450 activities were already induced, but the isoenzyme pattern induced was different in the liver and the small intestine. Hepatic P4501A1, P4501A2 and P4502B1 apoprotein levels were dose-relatedly enhanced, whereas in the small intestine induced levels of P4502B1 and P4501A1 were detected but P4501A2 was not induced. Pentoxy- and ethoxyresorufin dealkylation (PROD and EROD) were dose-relatedly enhanced in the liver (5- and 7-fold, respectively, in the higher dose group) as well as in the small intestine (8- and 13-fold, respectively, at 500 mg 13C/kg diet). Testosterone 16 alpha- and 16 beta-hydroxylation in the small intestine were enhanced (6-9-fold) from day 2 onwards, but in the liver these activities were only slightly enhanced from day 7 onwards. Thus, the major forms induced in the liver appear to be P4501A1, P4501A2, P4502B1 and, to a lesser extent, P4503A, whereas in the small intestine all of the effects that were found are associated with only one cytochrome P-450, P4502B1. After 2 days I3C (500 mg/kg) induced glutathione S-transferase in the liver (1.3-fold) and small intestine (1.5-fold). Hepatic glucuronyl transferase (GT1) was induced (about 1.6-fold) after 7, 14 and 28 days. DT-diaphorase was induced in the liver (2.7-fold) and small intestine (1.5-fold) after 14 days of exposure to 500 mg I3C/kg diet. Treatment of rat hepatocytes with indole-3-acetonitrile and 3,3'-diindolylmethane, but not I3C and indole-3-carboxaldehyde, enhanced EROD activity and halved testosterone 16 alpha- and 2 alpha-hydroxylation. All four indoles slightly induced glutathione S-transferase in cultured hepatocytes. Thus, the in vitro studies suggest that the in vivo effects of I3C have to be attributed to indole-condensation products, such as 3,3'-diindolylmethane, but not to I3C itself.

The rotary electric toothbrush, Rota-dent, is often recommended and used as an alternative to the conventional toothbrush. The purpose of this short-term study was to evaluate the effectiveness of the Rota-dent on plaque removal and gingival health in comparison to a conventional toothbrush. Twenty-four subjects were divided into two groups. For 6 weeks one group used the Rota-dent, while the other group used a conventional (Oral-B 40) toothbrush. Plaque and gingival indexes were evaluated each week. Results indicated that the Löe and Silness Gingival Index scores improved significantly for both groups. There was a significant improvement in plaque removal as measured by Turesky modification of the Quigley-Hein Plaque Index for the Rota-dent group, while there were no significant differences in the Silness and Löe Plaque Index for either group. Thus, both devices had a positive effect on gingival health, and the Rota-dent removed plaque better than a conventional toothbrush as measured by one but not another plaque index.

The expression of the adenosine deaminase complexing protein (ADCP) was investigated by immunohistochemistry in the normal and hyperplastic human prostate, in 30 prostatic adenocarcinomas, and in seven human prostatic adenocarcinoma cell lines grown as xenografts in athymic nude mice. In the normal and hyperplastic prostate, ADCP was localized exclusively in the apical membrane and the apical cytoplasm of the glandular epithelial cells. In prostatic adenocarcinomas, four distinct ADCP expression patterns were observed: diffuse cytoplasmic, membranous, both cytoplasmic and membranous, and no ADCP expression. The expression patterns were compared with the presence of metastases. We found an inverse correlation between membranous ADCP immunoreactivity and metastatic propensity. Exclusively membranous ADCP immunoreactivity occurred only in non-metastatic tumours. In contrast, the metastatic tumours showed no or diffuse cytoplasmic ADCP immunoreactivity. This suggests that immunohistochemical detection of ADCP might predict the biological behaviour of prostatic cancer. However, the occurrence of membranous ADCP immunoreactivity in the xenograft of a cell line (PC-EW), derived from a prostatic carcinoma metastasis, indicates that not only the tendency to metastasize modulates ADCP expression.

A study has been made of the efficacy of different immunization protocols using low antigen levels for the generation of monoclonal antibodies capable of detecting antigens (ADCP) in processed tissues. Protocols using unprocessed native antigen immobilized on nitrocellulose were more efficient than soluble antigen in generating serum antibodies reactive with both native antigen and processed tissues. The derived monoclonal antibodies reacted with native but not processed antigen. The use of antigen immobilized on polyvinylidene (PVDF) and subsequently processed as for histochemistry was successful in generating monoclonal antibodies reactive with processed antigen.

This study was prompted by the renewed clinical interests in understanding the natural history or early morphogenesis of the human temporomandibular joint. Using histologic preparations of 52 representative human embryos and fetuses, each of the major components of the joint was systematically assessed for its changing structure and related to an approximate time scale. The emergence and continued morphogenesis of the joint articular fossa, mandibular condyle, disk, capsule, lateral pterygoid muscle fibers, and both joint cavities occur in regular temporal and spatial pattern. A key observation from this study of embryos and fetuses ranging in age from 32 days to 22 weeks is that each of the component parts of the TMJ progressively emerge with some kind of continuity from a common mass of embryonic mesenchyme interposed between the future temporal bone and mandibular regions. The observations of this study lead to the suggestion that significant developmental disturbances to this common tissue mass or "developmental field" can lead to anomalous morphogenesis of those structures expected to emerge over time from the "developmental field" of the temporomandibular joint. Timing and the identification of a critical time period for the joint are important variables. This study identifies the critical period in the early morphogenesis of TMJ structures as generally falling between the early 7th and 11th prenatal weeks.

In order to study the interaction between tumors and host environmental factors, we xenografted cells from the human colonic carcinoma cell lines HT29 and 5583-S in the subcutis, cecum, spleen and liver of nude mice and compared growth characteristics, metastatic potential and some phenotypic features of the xenografts in these sites. No remarkable differences were observed between the tumors at different inoculation sites in regard of their expression of carcinoembryonic antigen and secretory component or type of mucin produced. Also the proportion of DNA synthesizing cells as determined by bromodeoxyuridine incorporation appeared to be comparable in the studied implantation sites. Local invasive growth characteristics and metastatic potential, however, showed marked differences. Subcutaneous and cecal xenografts frequently showed tumor cell invasion into the surrounding tissue, vasoinvasive growth and discontinuous basement membrane deposition, whereas splenic and hepatic implants demonstrated more encapsulation, no invasion of blood vessels and more continuous basement membrane deposition. Subcutaneous xenografts produced no metastasis. With HT29 cells liver and lymph node metastases occurred frequently from the splenic as well as the cecal xenografts. 5583 cells regularly produced liver metastases from the splenic xenografts, whereas no metastasis from cecal xenografts were observed. We conclude that although the patterns of invasive growth and the metastatic potential differ for various implantation sites, antigen expression and cell kinetic features of tumor implants are hardly influenced by the site of inoculation.

Unlabelled: Between 1977 and 1980, 118 breast cancer patients with locally advanced disease, T3B-4, any N, M0 or T1-3, tumor positive axillary apex biopsy, were randomized to one of three arms: I: radiotherapy (RT) to the breast and adjacent lymph node areas; II: RT followed by 12 cycles of cyclophosphamide, methotrexate, 5 fluorouracil (CMF) and tamoxifen during the chemotherapy period; III: 2 cycles of adriamycin and vincristine (AV), alternated with 2 cycles of CMF, then RT, followed by another 4 cycles of AV, alternated with 4 CMF; tamoxifen during the entire treatment period. The median follow-up period was 5 1/2 years. The adjuvant chemo- and hormonal therapy did not improve the overall survival; the 5-year survival was 37% for all three treatment arms. There was no statistically significant difference in RFS between the three modalities, nor when arm I was compared to arm II and III together, p = 0.11. Local recurrence (LR) was observed in 24 of the 86 patients (28%) who had reached complete remission (CR). LR was not statistically different over the three treatment arms. In 18 of the 24 patients with LR, distant metastases appeared within a few months from the local recurrence. In arm III, the CR rate after 4 cycles AV plus CMF and RT hardly changed after another 8 cycles of chemotherapy. The menopausal status did not influence the treatment results. Dose reduction in more than 4 cycles of chemotherapy was accompanied by better results, p = 0.04., In Conclusion: adjuvant chemo- and hormonal therapy did not improve RFS and overall survival. These findings do not support the routine use of adjuvant chemo- and endocrine therapy for inoperable breast cancer.

This literature review presents an inventory of the nature and incidence of side-effects that arise from the clinical application of monoclonal antibodies (MoAb) for the diagnosis and treatment of cancer. Most side-effects occurred during therapy. Toxic reactions, such as fever, sweating and chills, were more common than immunological skin reactions; they were observed predominantly in association with the elimination of circulating target cells. Dosage and rate of administration of the MoAb appeared to have little influence on the reactions, which disappeared quickly and did not necessitate discontinuation of treatment. Serum sickness, anaphylactic reactions and bronchospasms were not common; the patients reacted quickly to the indicated therapy. Prevention of the side-effects described here, especially during diagnostic applications, was such that they need not form a barrier to the clinical use of MoAb.

In a series of 61 primary colorectal carcinomas, we attempted to determine which primary tumor characteristics correlated with the possibility to continuously maintain tumor cells in vitro or in vivo, and to what extent the characteristics of a primary tumor were maintained in vitro or as xenograft. Four continuous cell lines and 10 serially transplantable tumors were obtained. Only one cell line could be maintained both in vitro and in vivo. Tumors that had metastized and tumors in the proximal colon showed a higher success for in vivo and in vitro growth. DNA analysis showed that in most xenografts the DNA index was identical to that the primary tumor. However, in some cases tumor cell subpopulations were lost or genetically variant new subpopulations were generated. In general, the degree of differentiation in the xenografts corresponded with the least differentiated areas in the primary tumor. Xenografts appeared to display comparable of antigen expression.

Growth curves recorded by the MS-2 (high performance photometer) provide a new means for studying PBPs. Indeed, the increase in prelytic OD (observed during the first two hours of contact between beta- lactams and cultures) is concentration- independent for antibiotics bound to PBPs3 ( Yourassowsky et al., 1981). Oxacillin has high affinity for PBP3 of E. coli (Curtis et al., 1979). Growth curves confirm biochemical studies: prelytic increase of OD of E. coli cultures is concentration- independent for oxacillin (100-2000 micrograms/ml), conversely to results observed with ampicillin and amoxycillin.