Platinum(II)-based coordination compounds as nucleic acid labeling reagents: synthesis, reactivity, and applications in hybridization assays.

The synthesis, characterization, and molecular interactions of platinum(II) coordination compounds, which contain a distal nonradioactive reporter molecule, with mono- and polynucleotides are described. A [Pt(II)(en)(NH(2)(CH(2))(6)NH-tBoc)Cl](NO(3)) (en=ethylenediamine) entity has been coupled, after removal of the tBoc group, to a number of hapten and fluorophore molecules through succinimide derivatives. The influence of the various tethered reporter groups within these complexes on the reactivity towards guanosine 5'-monophosphate (5'-GMP), as a model for polynucleotide sequences, was investigated to shed light on the use of these reagents in hybridization assays. Reactivity turned out to be strongly dictated by the chemical nature of the distal reporter molecule present. At pH 7.0 the sequence of reactivity is cationic approximately aromatic (stacking) > neutral > anionic; there is approximately an order of magnitude difference between the fastest reacting complex (k=10.2 x 10(-2) M(-1) s(-1)) and the slowest reacting complex (k=0.93 x 10(-2) M(-1) s(-1)) under these conditions. Platination of an oligodeoxynucleotide (30-mer), dsDNA, or an RNA transcript, shows that a Pt/nucleotide ratio between 1:10 and 1:20 (established by using flameless atomic absorption spectroscopy) results in probes with excellent hybridization characteristics. In terms of applicability and detection limits these platinated nucleic acid probes perform equally well compared to conventionally generated nucleic acid probes, that is, through enzymatic incorporation of covalently labeled nucleotide triphosphates. Applications of these reagents to in situ hybridization assays and gene expression profiling on microarrays illustrate the potential of these monofunctional binding platinum triamine compounds.

Application of a new, universal DNA labeling system in the PCR mediated diagnoses of Chlamydia trachomatis and human papillomavirus type 16 infection in cervical smears.

Non-isotopic DNA labeling procedures are essential for integration of DNA diagnostics into the clinical laboratory. A newly developed reagent was tested for use in reversed hybridisation identification of DNA fragments generated by polymerase chain reaction (PCR) amplification of Chlamydia trachomatis or human papilloma virus type 16 (HPV16) DNA isolated from cervical smears. The platinum-containing chemical compound, equipped with a biotin hapten, enables versatile 'one tube' labeling of amplified DNA. A HPV16-specific probe was immobilised on a nylon strip and reverse hybridisation with the biotin labeled DNA took place. To determine the value of this new, non-isotopic label in combination with clinical material, 98 cervical smears 54 of which contained HPV16, and 51 cervical smears 26 of which contained C. trachomatis, were analysed. The novel type of non-radioactive analysis appeared to be as sensitive as its isotopic counterpart. The DNA isolation and purification method require modification only in samples of poor quality. The labeling procedure is simple, versatile and can be included as a universal linkage system in any PCR test for the detection and identification of DNA molecules.

Further evaluation of quantitative nuclear image features for classification of lung carcinomas.

The usefulness of quantitative nuclear image features (QNI) for the histological classification of lung carcinomas was investigated. As no clear distinction could be established between the distributions of these features for the nuclei of squamous cell, adenocarcinoma, and large cell carcinoma, the attention was restricted to the discrimination between small cell lung carcinoma (SCLC) and non-small cell carcinoma (NSCLC). This discrimination is the crucial one in discussions about the choice of treatment. The differences between SCLC and NSCLC are statistically highly significant for various QNI features. The use of more than one QNI feature hardly raised the discriminatory performance with respect to the distinction between SCLC and NSCLC. Inferences were made about the probability and confidence interval of SCLC for a given QNI feature. It is concluded that in cases of uncertainty or disagreement, nuclear characteristics are useful for the discrimination between SCLC and NSCLC.

The effect of mechanical stress on healing skin wounds: an experimental study in rabbits using tissue expansion.

In rabbits, skin wounds were expanded by inflation of a subcutaneously implanted tissue expander in order to study the effect of mechanical stress on wound healing. Biomechanical and histomorphological properties of both expanded and non-expanded control wounds were evaluated. Expanded wounds demonstrated a significant increase in maximum load (80%) and energy absorption at maximum load (95%), when compared to non-expanded control wounds. Histomorphologically, the expanded wounds were stretched in comparison to the control wounds. The collagen in expanded wounds showed an orientation parallel to the direction of force, and displayed a more organised configuration. It is concluded that the use of tissue expanders permits the standardisation of the mechanical stress applied to experimental skin wounds. It is found that mechanical stress accelerates wound healing by producing stronger and more organised scars, however, at the expense of scar stretching.

Differential binding of Haemophilus influenzae to human tissues by fimbriae.

The hypothesis was investigated that tissue tropism of Haemophilus influenzae during colonisation and infection is associated with the ability of fimbriate bacteria to bind to the organs and cell types involved. H. influenzae type b with fimbriae (strain 770235f+) bound to several cell types, including ciliated columnar epithelial cells, pneumocytes, ependymal cells, glial cells, connective tissue fibroblasts, synovial cells, antigen-presenting cells, lymphocytes, erythrocytes and endothelial cells. Binding of H. influenzae to kidney, liver and conjunctiva cells was poor. Fimbriae-specific monoclonal antibody (MAb 6HE8) inhibited this binding. Some binding to endothelial cells and macrophages was also observed with non-fimbriate strains. This binding was not inhibited by MAb 6HE8. We conclude that in-vitro binding of fimbriate H. influenzae is mainly to those tissues and cells where H. influenzae is found during colonisation and infection. The data suggest that a shift to the non-fimbriate form is required for bacteria in the bloodstream to escape clearance mechanisms mediated by blood cells.

Microwave irradiation in label-detection for diagnostic DNA-in situ hybridization.

A DNA-in situ hybridization protocol was adapted for application to sections of routinely processed paraffin embedded material. This protocol was developed previously for detecting DNA-virus infected cells in whole cell preparations and employs biotinylated DNA as probe. Three different biotin detection methods were optimized and applied. The first uses streptavidin and a biotinylated complex of alkaline phosphatase, the second consists of an immunogold-silver staining, and the third of a peroxidase technique using a silver amplification. The alkaline phosphatase method was the most rapid, and as sensitive as the immunogold-silver staining. The peroxidase method was the most sensitive. Microwave irradiation was applied to the different incubation steps of these three detection methods. Short incubations with microwave irradiation gave results comparable to those obtained with conventional incubations, when streptavidin, antibiotin, complexed alkaline phosphatase, or gold labelled goat antirabbit were used. It was thus shown that microwave irradiation creates the possibility of a very rapid label-detection for nonradioactive DNA-in situ hybridization.

Ampullopancreatic carcinoma: preoperative TNM classification with endosonography.

Endosonography (ES) was used for the preoperative TNM (1987) staging of tumors in 43 patients with pancreatic cancer and 24 patients with ampullary carcinomas. These results were correlated with the histologic findings of resected specimens. Early-stage tumors could be distinguished from advanced stages of cancer with ES. Detailed images of ductular and parenchymal abnormalities allowed distinction between pancreatic and ampullary carcinomas based on anatomic location. The overall accuracy of ES in the assessment of tumor classification in pancreatic and ampullary carcinoma was 92% and 88%, respectively. In diagnosing regional lymph nodes in pancreatic and ampullary tumors the accuracy of ES was 74% and 54%, respectively. For diagnosing metastatic lymph nodes in pancreatic and ampullary carcinoma the accuracy of ES was 91% and 80%, respectively. The prevalence of lymph node metastases in T1 pancreatic cancers and T1 ampullary carcinomas was 40% and 0%, respectively. Discrimination between inflammation and metastases was difficult with ES. ES was not accurate in assessing distant metastases because of the limited penetration depth of ultrasound.

Combined beta-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization.

A combination of beta-galactosidase enzyme and the immunogold/silver staining method was studied for evaluation of double-staining experiments. Applications are shown for immunohistochemical double staining using two monoclonal antibodies and for combined immunohistochemistry and DNA in situ hybridization in one tissue section. The following advantages for the present double-staining method were evaluated: superior sensitivity of the immunogold/silver staining method for at least one epitope, which also allows detection of biotinylated DNA probes. The structure of the indolyl precipitate after revelation of beta-galactosidase activity did not show a concealing effect during a sequential double-staining method, as compared with the visualization of peroxidase with diaminobenzidine. These factors, and the sharply contrasting colored reaction products of beta-galactosidase (blue-green) and the immunogold/silver staining method including silver enhancement (brown-black), allow clear distinction of mixed-stained cell constituents.

Acute interstitial nephritis: a clinical and morphological study in 27 patients.

The clinical presentation, the morphological findings, and the clinical outcome of 27 patients with biopsy-proven acute interstitial nephritis were studied. All patients except one presented with acute renal failure. Typical clinical findings were often absent. Only four patients showed the classical triad of pyrexia, rash and arthralgia. In more than half of the patients an increased blood eosinophil count was not present. Kidney biopsy is therefore needed to establish the diagnosis of acute interstitial nephritis. In many patients acute interstitial nephritis was diagnosed in the biopsy when clinically this type of kidney disease was not expected. In 17 patients renal function improved spontaneously after withdrawal of the drug responsible or treatment of the infection. In ten patients who showed further deterioration of renal function in the first 2 weeks after admission, prednisone therapy was instituted. In all of them improvement of renal function was observed, with six returning to normal.

Comparison of beclomethasone dipropionate (2 and 3 mg) and prednisolone sodium phosphate enemas (30 mg) in the treatment of ulcerative proctitis. An adrenocortical approach.

Twenty-three patients with attacks of distal ulcerative colitis were treated randomly with either 2 or 3 mg of topically administered beclomethasone dipropionate (BDP) or 30 mg of prednisolone sodium phosphate (PP). The effect of the steroid enemas on adrenocortical function was assessed by ACTH tests, which were performed before and after treatment. Endoscopic, clinical and histological scores were comparable in the three treatment groups in this pilot trial. Fasting cortisol in the PP group decreased significantly from 0.47 +/- 0.12 mumol/l before to 0.22 +/- 0.14 mumol/l (P less than 0.05) after therapy; in the BDP group no significant changes were found. Urinary cortisol excretion in the PP group was not detectable after therapy; in the BDP group no changes were found. It is concluded that in the topical treatment of ulcerative colitis, BDP may be preferable to PP because it exerts a promising anti-inflammatory action without affecting adrenocortical function.

Subtotal duodenopancreatectomy for pancreatic duct, distal bile duct and periampullary carcinoma: short- and long-term results.

Ninety patients with pancreatic duct, distal bile duct, and ampullary carcinoma underwent pancreatic resection. Following a standard policy of resection based on surgical findings, all the patients who had resection first underwent subtotal extended pancreatectomy (n = 68) and if they were considered not to fulfill the criteria for this operation, total pancreatectomy (n = 22). Thus, 68 of the 90 patients (72%) were managed with subtotal pancreatic resection irrespective whether they had ampullary, pancreatic duct, or distal common bile duct carcinoma. On the basis of our results, subtotal duodenopancreatectomy is regarded as the method of choice for many patients with pancreatic duct, distal bile duct, or ampullary carcinoma.

Simultaneous application of in situ DNA hybridization and immunohistochemistry on one tissue section.

Combined application of a non-radioactive in situ DNA hybridization procedure and the immunoperoxidase technique on one tissue section is described. Of six potential protocols, only one proved to be successful. First, the immunohistochemical procedure including visualization of enzyme activity is performed; the in situ DNA hybridization protocol is then applied. Using this protocol, several antigens, detected with monoclonal antibodies, and target DNAs, detected by using biotinylated human cytomegalovirus or human papilloma virus type 16 DNA probes, could be distinguished by their peroxidase activity (brown precipitate) and alkaline phosphatase activity (purple-blue precipitate) respectively. The method allows immunophenotyping of virus-infected cells as well as simultaneous visualization of two viral parameters. This technique has important implications for research and diagnostic purposes.

Barrett's esophagus: development of dysplasia and adenocarcinoma.

Barrett's esophagus is considered to be a premalignant condition, and long-term surveillance seems mandatory with a careful search for dysplasia and carcinoma by means of multiple and repeated sets of biopsies. Reliable nonhistologic markers indicative of dysplasia or developing carcinoma are not yet available. To investigate development of dysplasia and carcinoma a prospective follow-up study was performed on 50 patients with Barrett's esophagus, without carcinoma at entrance to the study, for a period of 1.5-14 yr (mean, 5.2 yr). Barrett's epithelium was classified as fundic type, junctional or cardia type, or specialized columnar type. When classification in one of these three types was not possible because of lack of the characteristic features of the epithelia, the epithelium was classified as intermediate type. At entrance to the study, low-grade dysplasia was found in 6 patients, high-grade in 1 patient. During follow-up, dysplasia increased in frequency as well as in severity and was found almost exclusively in the specialized columnar- and intermediate-type epithelium. At the end of the observation period dysplasia had been found in 13 patients, in 10 scored as low-grade and in 3 as high-grade, and adenocarcinoma had developed in another 5 patients. This prospective study shows an incidence of carcinoma in Barrett's esophagus of 1 in 52 patient-years, a 125-fold increase compared with the general Dutch population. A sequence of worsening of dysplasia with development of carcinoma was observed in specialized columnar and intermediate-type epithelium. The results of this study support the need for a long-term clinical, endoscopic, and histologic follow-up program in patients with Barrett's esophagus.

Multiple immunoenzyme staining techniques. Use of fluoresceinated, biotinylated and unlabelled monoclonal antibodies.

Simultaneous detection of multiple tissue epitopes with an overlapping distribution pattern by monoclonal antibodies is sometimes needed for routine immunohistological evaluations. Therefore, multistep double and triple immunoenzymatic methods using antibodies from the same species or Ig (sub)class have been developed. Since only commercially available monoclonal antibodies (either unlabelled, biotinylated or as fluorescein conjugate) have been used, the techniques may be regarded as generally applicable. The staining protocol for double staining consists of six incubation steps: (1) unlabelled monoclonal antibody 1; (2) enzyme I-conjugated anti-mouse Ig; (3) normal mouse serum--for blocking; (4) fluoresceinated monoclonal antibody 2; (5) rabbit anti-fluorescein isothiocyanate--employing the fluorochrome as hapten; (6) enzyme II-conjugated anti-rabbit Ig. For enzymes I and II, peroxidase, alkaline phosphatase and beta-galactosidase can be applied; excellent results were obtained with the following colour combinations: peroxidase activity in red/alkaline phosphatase in blue and beta-galactosidase in green/alkaline phosphatase in violet. Moreover, this double staining method can be extended to provide an immunoenzyme triple staining technique by mixing biotinylated monoclonal antibody 3 and avidin-biotin enzyme III complex with the steps 4 and 5 reagents, respectively. In this way three tissue epitopes can simultaneously be detected clearly and selectively in green (beta-galactosidase), blue (alkaline phosphatase) and red (peroxidase).

Resectional surgical procedures for carcinoma of the head of the pancreas.

Of a total series of 103 patients with preoperatively diagnosed carcinoma of the head of the pancreas (including ampullary carcinoma, carcinoma of the distal part of the common bile duct and pancreatic duct and acinar cell carcinoma), 78 underwent pancreatic resection. The remaining 25 had palliative surgical treatment, either a gastric or biliary bypass, and are not included in the present study. Three of the 78 patients who underwent pancreatic resection died, and ten patients required early reoperation. Predictive criteria could be formulated for the prognosis and outcome of the patients with carcinoma of the head of the pancreas. The most reliable index for survival time of the patients proved to be the radicality of the resection, which was directly related to the differentiation of the primary tumor. Forty-three of 48 patients who underwent radical resection are alive, with a survival time ranging from three to 49 months. Eleven of 23 patients who underwent palliative resection are alive, with a survival time ranging from two to 29 months. Of 44 patients with well or moderately differentiated adenocarcinoma who underwent radical resection, 38 are alive, with a survival time ranging from six to 41 months (mean of 29 months).

Human gut wall reactivity to monoclonal antibodies against M. avium glycolipid in relation to Crohn's disease (preliminary results).

In order to evaluate the role of mycobacteria in the pathogenesis of Crohn's disease (CD), 4 monoclonal antibodies (McAb's) raised against M. avium specific glycolipid were tested on bowel resection specimens of CD patients and relevant controls. Two of the McAb's had shown a positive reaction to a CD-Mycobacteria isolate (CD-Myc). The same two McAb's showed a positive reaction within the gut wall, not only in CD patients, but also in the controls. In non-CD controls, however, the positive cells were limited to the lamina propria, while in CD patients positivity was also found in the submucosa and subserosa. Furthermore the mean number of positive cells in CD patients tends to be higher than in the control groups. Using double staining techniques the positive cells appeared to be B-cells of IgA isotype. These preliminary results indicate that mycobacteria might play a role in the pathogenesis of Crohn's disease.

Surgical approaches to the management of primary biliary cholangiocarcinoma of the porta hepatis: the decision-making dilemma.

Fifty patients with proximal malignant biliary obstruction confined to or above the junction of the main hepatic ducts underwent surgical treatment. Group A patients (n = 30) underwent complete or partial removal of the tumor with no supplementary procedure, group B patients (n = 20) complete removal of the tumor and a supplementary procedure. Additional procedures were liver resection alone (11/20), and liver resection plus resection and reconstruction of regional vascular structures (9/20). Reconstruction of the intrahepatic biliary tree was carried out in all patients using intrahepatic cholangiojejunostomies between common segmental hepatic stomata and a Roux-en-Y jejunal loop. In each common segmental hepatic stoma, two or three segmental hepatic ducts were drained. Transanastomotic tubes were used only temporarily. Eight patients died, three from group A (3/30) and five from group B (5/20). Survivors were relieved of jaundice and had no subsidiary cholangitis or problems associated with the anastomotic tubes. Seventeen patients of group A and 12 of group B are alive, with a mean survival of 29 and 31 months, respectively. Both alternatives offer good results. The choice of the surgical approach should be based on a precise evaluation of each patient's anatomical and individual clinical peculiarities.

The influence of variations in the measuring procedure on quantitative nuclear image features in histologic sections of lung tissue.

The qualitative and quantitative features of cell nuclei in tissue sections play an important role in diagnostic histopathology; variations in staining intensity and measuring procedures may interfere with their proper evaluation. To identify nuclear features that are relatively insensitive to these technical variables, the influence of critical steps in a scanning-stage densitometer measuring system was studied on 87 quantitative nuclear image (QNI) features in histologic sections of lung tissue. The influences of the following measuring variations were evaluated: interactive segmentation (with and without median filtering; with and without 5% uniform distributed noise added); scanning (with and without median filtering); calibration of the photomultiplier (different background localizations and different intensity levels); and time. In addition, the influence of artificially changed intensity variations was investigated. The results showed that, while the coefficient of variation (CV) induced by variations in the measuring system was usually low (below 10%), for some QNI features the CV can be high (up to 216%). The influence of artificial variations in intensity was restricted: only a minority of the QNI features showed a significant difference. Of the 87 QNI features, 35 had a CV of less than 10%, and 25 of these were significantly correlated with each other. Thus, only ten uncorrelated, low-CV QNI features remained; these belonged to all of the different QNI feature categories used. These features may be diagnostically important since they may best describe the morphologic properties of the nuclei. The results of this study should help in selecting quantitative nuclear image features that are less sensitive to variations in the measuring procedure and staining intensity.

Comparison of 5-aminosalicylic acid (3 g) and prednisolone phosphate sodium enemas (30 mg) in the treatment of distal ulcerative colitis. A prospective, randomized, double-blind trial.

Twenty-nine patients with attacks of distal ulcerative colitis were treated randomly with 3 g 5-aminosalicylic acid (5-ASA) or 30 mg of prednisolone phosphate sodium (PP) enemas (40 ml). Endoscopic, clinical, and histologic improvement were comparable in the two treatment groups. Our study showed that topical treatment with 5-ASA is as efficacious as PP in improving distal ulcerative colitis.

The effect of sampling on quantitative nuclear image features in histologic sections of lung carcinomas.

A feasibility study showed that quantitative nuclear image (QNI) analysis, in which the morphology of the nucleus is described by a number of mathematical parameters, can be used to make the therapeutically and prognostically important distinction between small cell lung carcinoma (SCLC) and non-SCLC, which can be difficult to make with subjective histologic typing. In the present study, the effects of sample size and sample site on the QNI features were investigated. For all sample sites in a given tumor, comparison was made between the histologic classification, the ultrastructural findings and the classification based on the QNI features. Using a running mean, it was found that a sample size of 25 nuclei is sufficiently large. Histologic and quantitative classifications of samples from different sites of the same tumors were in agreement with regard to the separation of SCLC and non-SCLC in 19 of 20 sections. In the case in which disagreement occurred in one section, the ultrastructural findings supported the quantitative classification. These data indicate that sampling from different sites has no essential influence on the QNI classification of lung carcinomas.