Validation and development of MTH1 inhibitors for treatment of cancer.

BACKGROUND: Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. MATERIAL AND METHODS: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8-oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling, proteomics, cellular thermal shift assays, kinase and CEREP panel were used for target engagement, mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E-mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. RESULTS: Here, we demonstrate that recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579, (Karonudib), an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. CONCLUSION: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept.

Preparation of solid drug/cyclodextrin complexes of acidic and basic drugs.

One of the main obstacles in pharmaceutical applications of cyclodextrins is their increase of the formulation bulk. Even at maximum incorporation 500 mg of a solid drug/cyclodextrin complex will only contain between 50 and 125 mg of the drug, assuming a low molecular weight drug (MW 200 to 400 Dalton) and an average molecular weight cyclodextrin (MW about 1500 Dalton). In general, the complexation efficiency is low and consequently the complex powder contains a significant amount of empty cyclodextrin molecules. In the present study the complexation efficiency is increased by ionization of the drug molecule through addition of volatile acid (i.e. acetic acid) or base (i.e. ammonia) to the aqueous complexation media of basic or acidic drugs, respectively. The volatile acid or base was then removed during lyophilization and heating in a vacuum oven resulting in formation of solid cyclodextrin complexes of the unionized drug. Thus, the complexation efficiency was temporary increased by the ionization but then again decreased leading to formation of the thermodynamically unstable solid drug/cyclodextrin complexes. When dissolved the energy of the system was lowered by expelling the drug molecules from the cyclodextrin cavities resulting in formation of supersaturated drug solutions and ultimately precipitation of the drug.

In vitro intestinal permeability of factor Xa inhibitors: influence of chemical structure on passive transport and susceptibility to efflux.

PURPOSE: To study the in vitro intestinal permeability of a number of newly synthesised factor Xa inhibitors to better understand the poor oral absorption of these compounds. METHODS: The bidirectional transport of the fXa inhibitors was studied in the Caco-2 cell model and isolated rat ileal tissue. An attempt was made to characterize efflux mechanisms with the help of commonly used substrates and inhibitors of various transport proteins. In addition, the transport of the fXa inhibitors was studied in MDCK cells transfected with the human MDR1 gene and expressing large amounts of P-glycoprotein (Pgp). RESULTS: The in vitro absorptive permeability was low for all but one of the fXa inhibitors. For compounds with non-substituted amidine, a charge (due to ionisation at neutral pH) may have resulted in poor membrane partitioning. Neutral compounds with substituted amidines were effluxed from the epithelial cells. The significance of the secretion process was illustrated by the results obtained for a neutral analogue showing high absorptive Caco-2 cell permeability that was not obviated by efflux. Transport inhibition studies in Caco-2 and permeability studies in the MDR1-transfected MDCK cells consistently showed that Pgp is not involved in the secretion of fXa inhibitors. Besides efflux, metabolic liability limited the permeation of the neutral lipophilic analogues with a carbamate ester. CONCLUSIONS: Poor intestinal permeability may be an important factor in the incomplete oral absorption of the bisbenzimidazole-type fXa inhibitors. Poor permeability may be related to poor membrane partitioning for hydrophilic analogues, whereas susceptibility to efflux transports and gastro-intestinal enzymatic degradation may limit the permeability of some of the neutral less hydrophilic derivatives.

Chitosans as absorption enhancers of poorly absorbable drugs. 3: Influence of mucus on absorption enhancement.

Chitosans are potent nontoxic absorption enhancers after nasal administration but their effects on the intestinal epithelium in vivo has not been studied in detail. In this study, the effects of chitosans with varying molecular weights and degrees of acetylation on the absorption of a poorly absorbed model drug (atenolol) were studied in intestinal epithelial cell layers with or without a mucus layer and in an in situ perfusion model of rat ileum. The effects of the chitosans on epithelial morphology and release of lactate dehydrogenase (LDH) into the perfusate were investigated in the in situ model. The chitosans had pronounced effects on the permeability of mucus-free Caco-2 layers and enhanced the permeation of atenolol 10- to 15-fold, with different absorption kinetics for different chitosans, in accordance with previous results. In contrast, enhancement of atenolol absorption through rat ileum was modest. LDH release from the tissues perfused with chitosans did not increase, indicating that the chitosans were used at nontoxic concentrations. Morphological examination of the perfused ileal tissues revealed more mucus discharge from the tissues exposed to chitosans than from controls, which suggested that the discharged mucus may inhibit the binding of chitosan to the epithelial surface and hence decrease the absorption-enhancing effect. This hypothesis was supported by studies with intestinal epithelial HT29-H goblet cells covered with a mucus layer. The binding of chitosan to the epithelial cell surface and subsequent absorption-enhancing effects were significantly reduced in mucus-covered HT29-H cultures. When the mucus layer was removed prior to the addition of chitosan, the cell surface binding and absorption-enhancing effects of the chitosans were increased. We conclude that the modest absorption-enhancing effects of unformulated chitosan solutions in the perfused rat ileum are a result of the mucus barrier in this tissue. This effect may be overcome by increasing the local concentrations of both chitosan and drug, i.e,. through formulation of the chitosan into a particulate dosage form.

Automated absorption assessment using Caco-2 cells cultured on both sides of polycarbonate membranes.

PURPOSE: To increase the capacity of in vitro absorption assessment and to decrease the amount of substance needed to perform early mechanistic investigations. METHODS: A liquid handling system, combined with a shaker and heating plates, was used to automate the Caco-2 cell based in vitro absorption assessment assay. In order to decrease the amount of substance needed for early mechanistic studies, a method for culturing Caco-2 cells on the lower side of polycarbonate membranes was also developed. RESULTS: Similar results were obtained with the automated assay as compared to manually performed assays. Data presented suggest that active transport and efflux were decreased in cells cultured on the lower side of the membranes as compared to ordinary seeded cells. CONCLUSIONS: Implementation of a liquid handling system for in vitro absorption assessment as reported here decrease the manual workload and increases the capacity of this in vitro assay substantially. Caco-2 cells cultured on the lower side of polycarbonate membranes, as described in this article, can not be used for analysis of transport mechanisms.

Absorption enhancement in intestinal epithelial Caco-2 monolayers by sodium caprate: assessment of molecular weight dependence and demonstration of transport routes.

Sodium caprate (C10), a medium chain fatty acid, is used clinically to enhance rectal absorption of the low molecular weight (MW) drug ampicillin. The main aim of this study was to investigate whether C10 also enhances the permeability of high MW model drugs in a model of the intestinal epithelium. The second aim was to present visual evidence of the route of enhanced transport across the epithelial cell layer. The studies were performed in Caco-2 monolayers cultured on permeable supports. The effects of non-toxic concentrations (< or = 13 mM) of C10 on drug transport across the monolayers was studied using monodisperse 14C-polyethylene glycols (MW 238-502; 14C-PEGs), 125I-Arg5-vasopressin (MW 1,208), 125I-insulin (MW 6,000) and FITC-labelled dextrans (MW 4,400 and 19,600; FD4 and FD20 respectively) as model drugs. Electron and confocal laser scanning microscopy were used to demonstrate transport routes across the epithelium. 10 mM C10 increased the permeability of all 14C-PEGs to approximately the same extent. 13 mM C10 increased the permeability of 125I-Arg8-vasopressin 10-fold. Only small increases in FD4 and FD20 permeabilities were observed. After C10 exposure, both tight junctions with normal morphology and those with dilatations showed an increased permeability to ruthenium red, indicating that C10 enhanced the paracellular transport of molecules with a MW < 1,000. Confocal microscopy showed that C10 increased the transport of FD4 and FD20 by the paracellular route. In conclusion, non-toxic concentrations of C10 can be used to enhance the permeability of drugs of MW up to approximately 1,200. Enhancement of the absorption of molecules larger than 4,000 is quantitatively insignificant. The enhanced permeability occurred via the paracellular pathway.

Chitosans as absorption enhancers for poorly absorbable drugs 2: mechanism of absorption enhancement.

PURPOSE: It has recently been shown that the absorption enhancing and toxic effects of chitosans are dependent on their chemical composition. In this study, the mechanisms underlying these effects were investigated at the cellular level. METHODS: The effects on epithelial cells of chitosans with different chemical composition, absorption enhancing properties and toxicities were studied in Caco-2 monolayers. Chitosan C( 1:31) has a low degree of acetylation (DA) (1%) and a low m.w. (31 kD), and displays dose-dependent absorption enhancement and cytotoxicity; chitosan C(35:170) has a higher DA (35%) and a higher m.w. (170 kD), is less dose-dependent in absorption enhancement, and is not cytotoxic. A third non-toxic chitosan C(49:22) with a high DA (49%), a low m.w. (22 kD), and no influence on epithelial permeability was used as control. RESULTS: C(1:31) and C(35:170) bound tightly to the epithelium. Cellular uptake of the chitosans was not observed. Both chitosans increased apical but not basolateral cell membrane permeability and induced a redistribution of cytoskeletal F-actin and the tight junction protein ZO-1. This resulted in increased paracellular permeability of hydrophilic marker molecules of different molecular weights. Addition of negatively charged heparin inhibited the cellular and the absorption enhancing effects of the chitosans, indicating that these effects are mediated via their positive charges. The onset of the effects of C(35:170) on apical membrane permeability and tight junction structure was much faster than that of C(1:31). C(49:22) did not influence any of the properties of the Caco-2 cell monolayers studied. CONCLUSIONS: The binding and absorption enhancing effects of chitosans on epithelial cells are mediated through their positive charges. The interaction of chitosans with the cell membrane results in a structural reorganisation of tight junction-associated proteins which is followed by enhanced transport through the paracellular pathway.

Intestinal safety of water-soluble beta-cyclodextrins in paediatric oral solutions of spironolactone: effects on human intestinal epithelial Caco-2 cells.

Effects of water-soluble beta-cyclodextrins (beta CDs) on intestinal epithelial integrity were investigated, to establish the safe use of these beta CDs as solubilizers of spironolactone in paediatric enteral solutions. Mannitol permeability and transepithelial resistance (TER) of human intestinal epithelial Caco-2 cell monolayers during exposure to dimethyl-beta-cyclodextrin (DM beta CD), hydroxypropyl-beta-cyclodextrin (HP beta CD) and sulphobutyl ether beta-cyclodextrin (SBE beta CD) were followed. Staining methods were used to discern cells with damaged membranes and to study the integrity of cytoskeletal actin and tight junctions. Cytotoxicity of the beta CDs was tested by effects on intracellular dehydrogenase activity. Exposure to HP beta CD and SBE beta CD solutions had only minor effects on the integrity of Caco-2 cell monolayers. In contrast, DM beta CD clearly increased the epithelial permeability for the hydrophilic marker [14C]mannitol across Caco-2 monolayers, decreased TER and showed a dose-dependent cytotoxicity. According to staining, DM beta CD increased the permeability of the apical cell membrane without discernable effects on cytoskeletal actin. HP beta CD and SBE beta CD appear to be safe additives for use in enteral spironolactone preparations with respect to their acute local effects on epithelial integrity.

Chitosans as absorption enhancers for poorly absorbable drugs. 1: Influence of molecular weight and degree of acetylation on drug transport across human intestinal epithelial (Caco-2) cells.

PURPOSE: Chitosan has recently been demonstrated to effectively enhance the absorption of hydrophilic drugs such as peptides and proteins across nasal and intestinal epithelia (1-3). In this study, the effect of the chemical composition and molecular weight of chitosans on epithelial permeability and toxicity was investigated using monolayers of human intestinal epithelial Caco-2 cells as a model epithelium. METHODS: Eight chitosans varying in degree of acetylation (DA) and molecular weight were studied. The incompletely absorbed hydrophilic marker molecule 14C-mannitol was used as a model drug to assess absorption enhancement. Changes in intracellular dehydrogenase activity and cellular morphology were used to assess toxicity. RESULTS: Chitosans with a low DA (1 and 15%) were active as absorption enhancers at low and high molecular weights. However, these chitosans displayed a clear dose-dependent toxicity. Chitosans with DAs of 35 and 49% enhanced the transport of 14C-mannitol at high molecular weights only, with low toxicity. One chitosan (DA = 35%; MW = 170 kD) was found to have especially advantageous properties such as an early onset of action, very low toxicity, and a flat dose-absorption enhancement response relationship. CONCLUSIONS: The structural features of chitosans determining absorption enhancement are not correlated with those determining toxicity, which makes it possible to select chitosans with maximal effect on absorption and minimal toxicity.

Methylated beta-cyclodextrins are able to improve the nasal absorption of salmon calcitonin.

The absorption enhancing effect of methylated beta-cyclodextrins on the nasal absorption of salmon calcitonin (sCT) was studied in rats and rabbits. The nasal absorption of sCT following administration without additives was low in both species. The absorption in rats could be largely improved by coadministration of cyclodextrins as apparent from the effect on serum calcium concentrations. Trimethyl-beta-cyclodextrin (TM beta CD), at a concentration of 5% (w/v), was the least potent enhancer. Randomly methylated-beta-cyclodextrin (RM beta CD) and dimethyl-beta-cyclodextrin (DM beta CD), all at a concentration of 5% (w/v), were almost equally effective in decreasing serum calcium levels, and the hypocalcemic responses were similar to those of i.v. and s.c. injected sCT. Absorption enhancement was already achieved with 1% DM beta CD added to the nasal formulations. In rabbits, only the effect of DM beta CD on the nasal sCT absorption was investigated. A total serum calcium decrement in 4 hours of 9.4 +/- 3.9% (mean +/- SD) was observed following nasal administration of 12.6 IU/kg sCT with 5% DM beta CD, comparable to that of i.v.-injected sCT. In conclusion, the methylated cyclodextrins DM beta CD and RM beta CD are suitable absorption enhancers for nasal sCT administration, which is expected to have a clinical impact on the therapy with calcitonin.

Automated screening for cytomegalovirus infected cells using image analysis. Comparison of two immunoenzymatic staining methods with respect to colour segmentation.

The detection of human cytomegalovirus (HCMV) infected poly-morphonuclear leukocytes (PMNLs) for early finding of the pp65 antigen using automated image analysis has been improved. The routinely used immunoenzyme peroxidase (PO) labelling has been replaced by alkaline phosphatase (AP) using new fuchsin as substrate. The number of automatically detected false positive objects due to incomplete inactivation of endogenous peroxidase and strong variations in counterstain intensity could be reduced by 81% using this AP staining method. The number of detected truly positive cells with both staining methods was not significantly different. Furthermore, a new image analysis system providing processing of colour images was evaluated. Since plain differences in absorption wavelength are required for colour segmentation, the red immuno-staining was combined with a green counterstain using methyl green. Screening of AP- instead of PO-stained slides in combination with colour segmentation resulted in a further reduction of the number of falsely detected alarms from 61% to 11%. Consequently, the sensitivity of the automated detection was improved. For AP staining detection of cells in frequencies of approximately one to one million was demonstrated. Screening for CMV-positive, alkaline phosphatase labelled cells using an image analysis system with colour segmentation resulted in a reduced false alarm rate, a better visual interpretation of the images and subsequently an increase in the sensitivity of the automated screening process.

Detection of immunocytochemically stained rare events using image analysis.

The detection of rare-event cells circulating in peripheral blood using automated image analysis was evaluated using a model system consisting of cells from a breast cancer cell line (SKBR3) seeded in a mononuclear cell suspension. Slides of cells with optimal morphology were prepared according to an optimized preparation procedure based on centrifugal cytology in combination with formalin fixation. SKBR3 cells were immunocytochemically stained for cytokeratin using the cam 5.2 monoclonal antibody and labelled with alkaline phosphatase using CAS-red as substrate. Because, for optimal segmentation of cell images, plain differences in absorption wavelength are required, the red immunostaining was combined with a green nuclear counter-staining based on ethyl green. Slides were automatically screened for cytokeratin-positive SKBR3 cells resulting in a lowest detectable frequency of one positive cell per 1.87 x 10(6) negative cells. A comparison between manual screening and automated screening for cytokeratin-positive cells showed a high level of correlation (0.9998). For the definition of the total number of objects per slide, two counting procedures were evaluated. Results were close to the visual score with a coefficient of variation of 0.47% for the counting procedure used in this study. It is concluded that optimization of preparation and staining procedures for the detection of rare-event cells using automated image analysis results in optimal image contrast and, consequently, in an increase in sensitivity for detecting rare events.

Scoring of radiation-induced micronuclei in cytokinesis-blocked human lymphocytes by automated image analysis.

The micronucleus assay in human lymphocytes is, at present, frequently used to assess chromosomal damage caused by ionizing radiation or mutagens. Manual scoring of micronuclei (MN) by trained personnel is very time-consuming, tiring work, and the results depend on subjective interpretation of scoring criteria. More objective scoring can be accomplished only if the test can be automated. Furthermore, an automated system allows scoring of large numbers of cells, thereby increasing the statistical significance of the results. This is of special importance for screening programs for low doses of chromosome-damaging agents. In this paper, the first results of our effort to automate the micronucleus assay with an image-analysis system are represented. The method we used is described in detail, and the results are compared to those of other groups. Our system is able to detect 88% of the binucleated lymphocytes on the slides. The procedure consists of a fully automated localization of binucleated cells and counting of the MN within these cells, followed by a simple and fast manual operation in which the false positives are removed. Preliminary measurements for blood samples irradiated with a dose of 1 Gy X-rays indicate that the automated system can find 89% +/- 12% of the micronuclei within the binucleated cells compared to a manual screening.

Hypocalcemic effect of salmon calcitonin following single and repeated nasal and intravenous administration in young rabbits.

The effect of the polypeptide salmon calcitonin (sCT) on serum calcium concentrations following intranasal and intravenous administration was studied in young rabbits. A small, hypocalcemic effect was observed after nasal administration of sCT without additives, indicating that the nasal sCT absorption was low. The absorption could be improved by addition of an absorption-enhancing adjuvant to the nasal preparation. The absorption, however, was still far from complete as was apparent from the much stronger effect of intravenously injected sCT. When a number of sCT doses were given during a 10-week period, the hypocalcemic effect per sCT dose in the young rabbits decreased after intravenous and, although less pronounced, after nasal administration. The decreased response to sCT is probably not related to the induction of neutralizing antibodies or desensitization of sCT receptors, but is more likely associated with the age-dependent level of bone activity.

Nasal administration of an ACTH(4-9) peptide analogue with dimethyl-beta-cyclodextrin as an absorption enhancer: pharmacokinetics and dynamics.

1. The systemic absorption and the neurotrophic effect of the metabolically stabilized ACTH (4-9) analogue, Org2766, were investigated following intranasal (i.n.) administration. 2. Without additives the nasal bioavailability of the peptide was in the order of 15 and 10% in rats and rabbits, respectively. The absorption could be improved by addition of a variety of absorption enhancers to the nasal preparation. The beta-cyclodextrin derivative, dimethyl-beta-cyclodextrin (DM beta CD) at a concentration of 5% (w/v) improved the absorption in rats about 5 fold from 13 +/- 4% (mean +/- s.d.) for administration of the peptide alone to 65 +/- 21%, and in rabbits 1 to 2 fold, from 10 +/- 6% to 17 +/- 8%. 3. The increased permeability of the rat nasal mucosa for Org2766 caused by DM beta CD in rats reversed substantially within 1 h. However, the nasal absorption had not yet completely returned to the level without enhancer. 4. S.c. administered Org2766 accelerated the functional recovery from peripheral nerve damage in rats. However, the peptide did not facilitate nerve repair following i.n. administration with DM beta CD, in spite of the fact that Org2766 was well absorbed. I.v. injection of Org2766 was also ineffective.

Nasal insulin delivery with dimethyl-beta-cyclodextrin as an absorption enhancer in rabbits: powder more effective than liquid formulations.

The nasal absorption of insulin using dimethyl-beta-cyclodextrin (DM beta CD) as an absorption enhancer in rabbits was studied. The nasal administration of insulin/DM beta CD liquid formulations did not result in significant changes in serum insulin and blood glucose concentrations. In contrast, previous experiments in rats showed that the addition of DM beta CD to the liquid nasal formulation resulted in an almost-complete insulin absorption, with a concomitant strong hypoglycaemic response. Apparently, the effect of the cyclodextrin derivative on insulin absorption differs between animal species following nasal delivery of insulin/DM beta CD solutions. On the other hand, nasal administration of the lyophilized insulin/DM beta CD powder dosage form in rabbits resulted in increased serum insulin concentrations, and a maximum decrease in blood glucose of about 50%. The absolute bioavailability of the nasally administered insulin/DM beta CD powder was 13 +/- 4%, compared to 1 +/- 1% for both an insulin/DM beta CD liquid and an insulin/lactose powder formulation. It is concluded that insulin powder formulations with DM beta CD as an absorption enhancer are much more effective than liquid formulations.

DNA damage in UV-irradiated human skin in vivo: automated direct measurement by image analysis (thymine dimers) compared with indirect measurement (unscheduled DNA synthesis) and protection by 5-methoxypsoralen.

The incidence of the various types of skin cancer in the general population has been increasing at an annual rate of 2-8% over the past 2 decades. In spite of considerable media coverage on the risk of skin cancer the acquisition of a suntan is still very popular. Thus the UV exposures required for tanning pose a serious carcinogenic risk, particularly to individuals who tan poorly. On the other hand, the presence of natural skin pigment, or the ability to tan easily can protect the skin against some of the harmful effects of subsequent UV exposures (Kollias et al. 1991). We have recently shown (Young et al. 1991) in human volunteers, using an indirect measurement of DNA damage (unscheduled DNA synthesis (UDS) detected by autoradiography), that a tan induced by UV in the presence of a UVB sunscreen (Parsol MCX) preparation containing 5-methoxypsoralen (5-MOP) is more effective at protecting the skin against a subsequent DNA-damaging challenge dose of UV than a tan induced by UV alone, particularly in individuals who tan poorly. No such protection was seen with the same sunscreen lacking 5-MOP. 5-MOP is an ingredient in natural citrus oils and in many other plants. Here we show the same pattern of protective action when measuring, for the first time, DNA damage directly using a monoclonal antibody to thymine dimers (a major category of DNA lesion induced by UV radiation) on fixed human skin sections and automated image analysis. There is a good correlation between UV exposure dose and the levels of thymine dimers in epidermal nuclei. The levels of thymine dimers (measured as absorption by the mean integrated optical density (IOD)) also correlated well with the levels of UDS (grains per nucleus). These findings are of importance in the comparative risk-benefit assessment of sunscreens with and without 5-MOP. The techniques described have applications for measuring other DNA lesions following UV and other exposures.

Value of counting colonic mucosal Ig-containing cells in the differential diagnosis of chronic inflammatory bowel disease.

AIMS: To investigate whether counting cells containing immunoglobulin (Ig) subclass in colonic biopsy specimens of patients with chronic inflammatory bowel disease, in addition to conventional histological evaluation, can improve the differentiation of patients with Crohn's disease from those with ulcerative colitis. METHODS: The colonic and rectal biopsy specimens of 40 patients with chronic inflammatory bowel disease, comprising 20 patients with Crohn's disease and 20 with ulcerative colitis, were used and sections were stained specifically for IgA, IgM, and IgG heavy chains using an indirect immune peroxidase method. The immunoglobulin subclass containing cells were counted using an ocular grid counting method in a light microscope. A linear stepwise discriminant analysis was performed on Ig subclass containing cell counts in combination with 16 reproducible histological features. The results of this discriminant analysis were compared with the results of the discriminant analyses in which only the histological features were used. RESULTS: Applying stepwise discriminant analysis, two histological features (an excess of histiocytes in the lamina propria and the villous or irregular aspect of the mucosal surface) in combination with IgMax were selected as the most discriminatory parameters that distinguish Crohn's disease from ulcerative colitis. IgMmax was defined as the maximum value of the mean percentage of IgM containing cells over all the biopsy locations. The use of this combination resulted in a better classification in 20% of the patients with Crohn's disease and in 9% of the patients with ulcerative colitis compared with the use of histological features alone. CONCLUSIONS: Morphometric enumeration of Ig subclass containing cells in colonic mucosal biopsy specimens has diagnostic value as a means of differentiating individual patients with Crohn's disease from those with ulcerative colitis.

Quantification by laser scan microscopy of intracellular doxorubicin distribution.

Changes in intracellular drug localization accompany doxorubicin resistance in multidrug resistant tumor cells. The purpose of this study was to develop a method to quantify these changes and so detect different levels of resistance. Tumor cells were incubated with the fluorescent anthracycline doxorubicin (excitation at 480 nm; emission maximum at 560-590 nm) and were quantified using laser scanning microscopy. The fluorescent mode was used to record the intracellular drug distribution, whereas the absorption mode was used to define the nuclear and cytoplasmic boundaries. The cell compartments were delineated interactively on an image processing system and the ratio nuclear fluorescence/cytoplasmic fluorescence (N/C ratio) was determined. N/C ratios were: 1.8 in the Chinese hamster ovarian cell line AUXB1 and 0.1 in its MDR subline CHRC5; 3.8 in the human squamous lung cancer cell line SW-1573 and 1.8 and 0.4 in its MDR sublines SW-1573/2R120 and SW-1573/2R160, respectively; and 3.6 in the human myeloma cell line 8226/S and 2.1 and 1.0 in its MDR sublines 8226/Dox4 and 8226/Dox40, respectively. The doxorubicin distribution was independent of the doxorubicin concentration within a range from 1-32 microM. Furthermore, the progressive mean of the nuclear/cytoplasmic doxorubicin fluorescence ratio showed that a minimal sample size of 30 cells is necessary for reliable results. The results of two independent assessments showed a high reproducibility (r = 0.97). Thus, with the method described in this paper, it is possible to detect relatively low levels of doxorubicin resistance (factor 8).

Histopathological evaluation of colonic mucosal biopsy specimens in chronic inflammatory bowel disease: diagnostic implications.

In a prospective blind evaluation of multiple colonic mucosal biopsy specimens, 45 clinically well defined patients with chronic inflammatory bowel disease (21 Crohn's disease and 24 ulcerative colitis) and 16 control subjects (seven normal subjects and nine patients with diverticular disease) were studied to identify reproducible histopathological features which could distinguish chronic inflammatory bowel disease (CIBD) from non-CIBD and Crohn's disease from ulcerative colitis. Using kappa statistics 16 of 41 histological features were sufficiently reproducible for further stepwise discriminant analysis to differentiate between CIBD and non-CIBD, and between Crohn's disease and ulcerative colitis. Using the combination of three features (an increase of lymphocytes and plasma cells in the lamina propria, the presence of branching of crypts, and neutrophils in the crypt epithelium) we were able to distinguish CIBD from non-CIBD in 89% of the cases with high probability (p greater than 0.85). To separate Crohn's disease from ulcerative colitis three features (an excess of histiocytes in combination with a villous or irregular aspect of the mucosal surface and granulomas) had a high predictive value. Using these features 70% of Crohn's disease patients and 75% of ulcerative colitis patients were correctly classified with a high probability (p greater than 0.85). These findings indicate that the pathologist is dependent on the presence of only a few histological features for a reliable classification of Crohn's disease and ulcerative colitis.