Synthesis and characterization of amino acid substituted sunitinib analogues for the treatment of AML.

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted delivery may reduce such undesired side effects, our goal was to develop novel amino acid substituted derivatives of sunitinib which are potent candidates to be used conjugated with antibodies and peptides. In the current paper we present the synthesis, physicochemical and in vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with peptide-based delivery systems via their carboxyl group. We determined the solubility, pKa, CHI and LogP values of the compounds along with their inhibition potential against FLT3-ITD mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the growth of the MV4-11 leukemia cell line at submicromolar concentration.

Evaluation of carbon dioxide laser therapy for benign tumors of the eyelid margin.

Eyelid margin tumors require special attention based on both anatomical and histological perspectives. Our aim in this study was to evaluate carbon dioxide (CO2) laser therapy for the treatment of eyelid margin tumors. Fifty-two patients with 55 eyelid margin tumors were included in this study. All tumors were removed with a CO2 laser, and histopathological evaluation was obtained in 52 cases. All patients were followed up for a mean period of 8.5 months (range 6 to 14 months). There were no bleedings in the intra- and postoperative period; the wounds were dry and reepithelized after 10-14 days and no recurrence occurred during follow-up period. Compared to the surrounding tissue, the treated area was hypopigmented and maximum five eyelashes (average 2.5) were wasted during the procedure. We achieved complete patient and surgeon satisfaction with cosmetic and therapeutic results. CO2 laser treatment of eyelid margin is a safe and effective procedure; its cosmetic result is beneficial as it does not cause malposition of the eyelid or damage to the lacrimal drainage system if the tumor is located in its proximity.

Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters.

Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca(2+)-dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins and γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the K m and the V max kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild-type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2-driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of crosslinked proteins correlates with the manifestation of degenerative disorders.

[Pyrido[2,3-b]pyrazines inhibiting both erlotinib-sensitive and erlotinib-resistant cell lines, and their preparation via regioselective condensation reaction]. / Erlotinib-érzékeny és erlotinib-rezisztens sejtvonalakat gátló pirido[2,3-b] pirazinok, és eloállításuk régiószelektív kondenzációs reakcióval.

The EGFR inhibitor erlotinib possesses high anti-tumor effect but despite the good clinical responses in most of the cases recrudescence occures. This can be attributed to a secondary, acquired mutation causing resistance to tyrosine kinase inhibitors. In our work we were looking for small-molecule inhibitors, which simultaneously affect on the proliferation of erlotinib-sensitive PC9 cells and PC9-ER erlotinib-resistant cells. A set of molecules were selected from Vichem Chemie Research Ltd.'s kinase inhibitor compound library (Nested Chemical Library™). According to the results of medium throughput screening (MTS) of this set of compounds, novel structures with pyrido[2,3-b]pyrazine core were designed. These compounds were proved to be effective inhibitors of resistant cells in phenotypic screening. Based on these results structure-activity relationships were set up. The pyrido[2,3-b]pyrazine core was synthesized by a condensation reaction, which resulting two asymmetric products. In the reaction two regioisomer intermediates formed, and one of the products is the intermediate of the effective compounds. This condensation reaction was optimized, the regioisomers were identified by NMR analysis and X-ray crystallography. As a result of optimization we found that lower reaction temperature and replacement of dimethylformamide solvent with trifluoroacetic acid provided the undesired isomer in less than 2 % ratio.

Carboxypeptidase-M is regulated by lipids and CSFs in macrophages and dendritic cells and expressed selectively in tissue granulomas and foam cells.

Granulomatous inflammations, characterized by the presence of activated macrophages (MAs) forming epithelioid cell (EPC) clusters, are usually easy to recognize. However, in ambiguous cases the use of a MA marker that expresses selectively in EPCs may be needed. Here, we report that carboxypeptidase-M (CPM), a MA-differentiation marker, is preferentially induced in EPCs of all granuloma types studied, but not in resting MAs. As CPM is not expressed constitutively in MAs, this allows utilization of CPM-immunohistochemistry in diagnostics of minute granuloma detection when dense non-granulomatous MAs are also present. Despite this rule, hardly any detectable CPM was found in advanced/active tubercle caseous disease, albeit in early tuberculosis granuloma, MAs still expressed CPM. Indeed, in vitro both the CPM-protein and -mRNA became downregulated when MAs were infected with live mycobacteria. In vitro, MA-CPM transcript is neither induced remarkably by interferon-γ, known to cause classical MA activation, nor by IL-4, an alternative MA activator. Instead, CPM is selectively expressed in lipid-laden MAs, including the foam cells of atherosclerotic plaques, xanthomatous lesions and lipid pneumonias. By using serum, rich in lipids, and low-density lipoprotein (LDL) or VLDL, CPM upregulation could be reproduced in vitro in monocyte-derived MAs both at transcriptional and protein levels, and the increase is repressed under lipid-depleted conditions. The microarray analyses support the notion that CPM induction correlates with a robust progressive increase in CPM gene expression during monocyte to MA maturation and dendritic cell (DC) differentiation mediated by granulocyte-MA-colony-stimulating factor+IL-4. M-CSF alone also induced CPM. These results collectively indicate that CPM upregulation in MAs is preferentially associated with increased lipid uptake, and exposure to CSF, features of EPCs, also. Therefore, CPM-immunohistochemistry is useful for granuloma and foam MA detections in tissue sections. Furthermore, the present data offer CPM for the first time to be a novel marker and cellular player in lipid uptake and/or metabolism of MAs by promoting foam cell formation.

Development of a cell-selective and intrinsically active multikinase inhibitor bioconjugate.

Multikinase inhibitors are potent anticancer drugs that simultaneously intervene in multiple related signaling cascades, thus being capable of blocking salvage pathways that may play a role in the development of drug resistance. Multikinase inhibitors are increasingly evaluated for indications other than cancer, but long-term safety risks dictated by off-organ toxicities of these agents may prevent their safe and effective use. Here, we describe a new approach in which platinum coordination chemistry is applied for the development of a cell-selective multikinase inhibitor bioconjugate. The platinum(II) kinase inhibitor bioconjugate was designed to be active with the linker attached to the inhibitor and displayed improved activity by enhanced cell specificity as well as enhanced intracellular retention, thereby prolonging its pharmacological activity. In addition, the utilized platinum-based linkage technology potentiated the inhibitory activity of the multikinase inhibitor. These features in combination with carrier-mediated uptake in the target cells may revolutionize dosing regimens and safety profiles of (multi)kinase inhibitors.

[Malignant insulinoma]. / Malignus insulinoma.

Authors present the history of a 56-year-old man who was evaluated for recurrent epigastric pain. Clinical investigation revealed a 4-cm tumor in the head of the pancreas and a solitary liver metastasis. Pathological examination of the surgically excised pancreatic tumor indicated a moderately differentiated neuroendocrine carcinoma and Ki-67 labeling index revealed moderate proliferative activity. Despite short-term chemotherapy combined with interferon and somatostatin analogue administration, the metastatic disease rapidly progressed. Octreotide scintigraphy disclosed abundant expression of somatostatin receptors both on primary tumor and hepatic metastases. 9°Yttrium-DOTATOC treatment was performed in three sessions within 9 months (3 x 200 mCi) with a mixed therapeutic response. Endocrine symptoms were not observed during the first 33 months of the disease. 34 months after the initial diagnosis of the neoplastic disease, imaging studies and chromogranin A measurement revealed rapidly progressing disease and the patient developed frequent episodes of hypoglycemic attacks. Serum insulin and C-peptide measurements confirmed insulin oversecretion. Continuous administration of somatostatin analogue was supplemented with diazoxide, but the latter therapy was not tolerated because of severe water retention. The high dose oral carbohydrate intake was supplemented with continuous glucose infusion. As a rescue procedure, repeated liver chemoembolization was performed, which resulted only in a short-term effect. The autopsy and the immunohistochemical investigations confirmed the diagnosis of insulin-producing, metastatic neuroendocrine carcinoma.

Transglutaminase-mediated intramolecular cross-linking of membrane-bound alpha-synuclein promotes amyloid formation in Lewy bodies.

The alpha-synuclein immunopositive and chaotrope-insoluble material from human brains with Lewy body pathology was analyzed by mass spectrometry. From the proteinase K-cleavable peripheral fraction of Lewy bodies, which was densely cross-linked by gamma-glutamyl-epsilon-lysine bonds between HspB1 and ubiquitin in a pattern similar to neurofibrillary tangles (Nemes, Z., Devreese, B., Steinert, P. M., Van Beeumen, J., and Fésüs, L. (2004) FASEB J. 18, 1135-1137), 53 proteins were identified. In the core of Lewy bodies only alpha-synuclein was found, and it contained a low amount of intramolecular cross-links between Gln-99 and Lys-58. In vitro cross-linking of alpha-synuclein by transglutaminases 1-3 and 5 produced a heterogeneous population of variably cross-linked alpha-synucleins in solution, which inhibited the aggregation of the protein into amyloid. However, in the presence of phosphatidylserine-rich membranes and micromolar calcium concentrations, the cross-linking by transglutaminases 1, 2, and 5 showed specificity toward the utilization of Gln-99 and Lys-58. As shown by thioflavin T fluorescence monitoring, the formation of this cross-link accelerated the aggregation of native alpha-synuclein. Chemical cross-linking of residues 58-99 triggered amyloid formation, whereas such bonding of residues 99 to 10 was inhibitory. Our findings reveal the pivotal role of membrane attachment and transglutaminase-mediated intermolecular cross-linking for the propagative misfolding and aggregation of alpha-synuclein.

Clinical decision making and outcome in routine care for people with severe mental illness (CEDAR): study protocol.

BACKGROUND: A considerable amount of research has been conducted on clinical decision making (CDM) in short-term physical conditions. However, there is a lack of knowledge on CDM and its outcome in long-term illnesses, especially in care for people with severe mental illness. METHODS/DESIGN: The study entitled "Clinical decision making and outcome in routine care for people with severe mental illness" (CEDAR) is carried out in six European countries (Denmark, Germany, Hungary, Italy, Switzerland and UK). First, CEDAR establishes a methodology to assess CDM in people with severe mental illness. Specific instruments are developed (and psychometric properties established) to measure CDM style, key elements of CDM in routine care, as well as CDM involvement and satisfaction from patient and therapist perspectives. Second, these instruments are being put to use in a multi-national prospective observational study (bimonthly assessments during a one-year observation period; N = 560). This study investigates the immediate, short- and long-term effect of CDM on crucial dimensions of clinical outcome (symptom level, quality of life, needs) by taking into account significant variables moderating the relationship between CDM and outcome. DISCUSSION: The results of this study will make possible to delineate quality indicators of CDM, as well as to specify prime areas for further improvement. Ingredients of best practice in CDM in the routine care for people with severe mental illness will be extracted and recommendations formulated. With its explicit focus on the patient role in CDM, CEDAR will also contribute to strengthening the service user perspective. This project will substantially add to improving the practice of CDM in mental health care across Europe. TRIAL REGISTER: ISRCTN75841675.

Methods to analyze transglutamination of proteins involved in apoptosis.

Enhanced expression of transglutaminases is a frequent, though not obligatory phenomenon in apoptosis, which is associated with cells dying in steady interaction with their tissue environment. Modification of cellular proteins by transglutamination is a tightly controlled procedure. If transglutamination is dysregulated, it has profound and potentially detrimental consequences on cellular functioning. Under conditions normally occurring in living cells transglutaminase activity is usually undetectably low (latent) and can only be tested by careful preselection of proteins of interest. In late stages of apoptosis, transglutaminases can become rampant in dying cells and a minuscule fraction of dead cells may overshadow many more living ones, which may cause inherent and severe methodological and interpretation bias. Therefore, in this chapter, we describe the analysis of dead cell remnants for protein-bound transglutaminase-mediated cross-link content. In the techniques described below, we rely on the increasing availability and user-friendliness of mass spectrometric equipments.

Analysis of EGFR gene amplification, protein over-expression and tyrosine kinase domain mutation in recurrent glioblastoma.

Gefitinib and erlotinib are both selective EGFR tyrosine kinase inhibitors (EGFR-TKIs) that have produced responses in a small subgroup of lung cancer patients. The strongest evidence for a role of EGFR in the biology of glioblastoma stems from clinical trials in which 15-20% of recurrent glioblastoma patients experienced significant tumour regression in response to these small-molecule EGFR kinase inhibitors. We examined the protein-kinase domain of the EGFR gene, EGFR protein expression and EGFR gene amplification in 20 cases of recurrent GBMs. EGFR protein over-expression was found in 65% of cases. EGFR protein over-expression was associated with EGFR gene amplification in 35% of cases, and with high polysomy in 15% of cases. No mutations were found in the TK domain of the EGFR gene. Our results confirm that mutations in the kinase domain are absent in recurrent GBM, and this might be a preponderant factor in the lack of major clinical responses of TKIs in GBM, recent studies have suggested that responsiveness to EGFR kinase inhibitors was strongly associated with coexpression of EGFRvIII and PTEN. Further prospective validation of EGFRvIII and PTEN as predictors of the clinical response to EGFR kinase inhibitors in recurrent GBM is strongly anticipated.

Discovery and development of extreme selective inhibitors of the ITD and D835Y mutant FLT3 kinases.

Aberrant activation of FMS-like tyrosine receptor kinase 3 (FLT3) is implicated in the pathogenesis of acute myeloid leukemia (AML) in 20-30% of patients. In this study we identified a highly selective (phenylethenyl)quinazoline compound family as novel potent inhibitors of the FLT3-ITD and FLT3-D835Y kinases. Their prominent effects were confirmed by biochemical and cellular proliferation assays followed by mice xenograft studies. Our modelling experiments and the chemical structures of the compounds predict the possibility of covalent inhibition. The most effective compounds triggered apoptosis in FLT3-ITD AML cells but had either weak or no effect in FLT3-independent leukemic and non-leukemic cell lines. Our results strongly suggest that our compounds may become therapeutics in relapsing and refractory AML disease harboring various ITD and tyrosine kinase domain mutations, by their ability to overcome drug resistance.

Immunohistochemical antibodies in breast cancer HER2 diagnostics. A comparative immunohistochemical and fluorescence in situ hybridization study.

Overexpression and/or gene amplification of the HER2 oncogene predicts worse prognosis and altered sensitivity to chemotherapy. Trastuzumab is capable of improving prognosis of HER2-positive breast cancer, but for the success of treatment appropriate HER2 testing is essential. Our aim was to determine the value of immunohistochemical (IHC) screening prior to fluorescence in situ hybridization (FISH). We assessed five conventional IHC assays (NCL-CB11, Pathway CB11, CBE356, CBE1, HercepTest) and the novel rabbit monoclonal antibody, RM-4B5, combined with FISH on 199 invasive breast cancer cases. Taking FISH as the endpoint, we calculated sensitivity, specificity, positive and negative predictive values (PPV, NPV) and accuracy for all IHC assays with either taking both 2+/3+ cases or only 3+ cases as IHC positives. With 2+/3+ cases HercepTest showed 100% sensitivity and NPV, while the highest specificity, PPV and accuracy was associated with RM-4B5 (97.36, 80 and 95.34%, respectively). The second highest values belonged to either NCL-CB11 or Pathway CB11. When calculating only with 3+ cases, the results were reversed with increased specificity, PPV and accuracy. Our findings suggest that improving sensitivity by using two parallel IHC reactions might be beneficial; we recommend primarily HercepTest and Pathway CB11. Nevertheless, we may consider performing FISH analysis without prior IHC screening.

The presence of carboxypeptidase-M in tumour cells signifies epidermal growth factor receptor expression in lung adenocarcinomas: the coexistence predicts a poor prognosis regardless of EGFR levels.

PURPOSE: Carboxypeptidase-M (CPM) is a membrane-bound peptidase that metabolizes peptides, and is present in pneumocytes. CPM hydrolyses the C-terminal arginine of epidermal growth factor (EGF) resulting in des-Arg53-EGF which binds to the EGF receptor (EGFR) with an equal or greater affinity than native EGF. Therefore, this study focused on the possible presence of CPM in human lung adenocarcinomas (ADC) and evaluated the relationship between CPM and EGFR by assessing the impact of expressions on patient clinical outcome. METHODS: This is a retrospective study of 110 patients who underwent resection of the primary tumour (92) or metastatic tissues (18) for treatment or diagnosis. Immunohistochemistry (IHC) for CPM and EGFR was made in serial sections using standard methods. RESULTS: This study demonstrates for the first time that 23.6% of ADCs express carboxypeptidase-M (26/110), mainly in membrane-bound forms. The amounts and the extent of CPM within tumours vary from low levels to obviously overexpressed forms. The immunohistochemical positivity (+) for CPM in ADCs negatively correlated with disease survival. In addition, 80% of CPM+ adenocarcinomas (21/26) showed a coexpression with EGFR suggesting a high prevalence for coexistence. The follow up data indicated a significantly shorter 5-year survival time for patients with CPM+-EGFR+ (double-positive) tumours compared to those harbouring neoplasias negative for both proteins (9.5 vs. 60.4% survivals, P < 0.001). CONCLUSION: The fact that CPM+ ADCs often co-express with EGFR suggests a functional-regulatory link between these proteins which might have therapeutical consequences. The present novel data could lead to improved IHC tests in lung adenocarcinomas for EGFR expression.

Transformed dermatofibrosarcoma protuberans: real time polymerase chain reaction detection of COL1A1-PDGFB fusion transcripts in sarcomatous areas.

BACKGROUND: Recent cytogenetic studies have shown that reciprocal translocation t (17;22)(q22;q13) and a supernumerary ring chromosome derived from the translocation r(17;22) are highly characteristic of dermatofibrosarcoma protuberans (DFSP). The chromosomal rearrangements fuse the collagen type Ialpha1 (COL1A1) and the platelet-derived growth factor B-chain (PDGFB) genes. The COL1A1-PDGFB fusion transcript has been shown not only in conventional DFSP but also in a small series of DFSP with fibrosarcomatons areas (DFSP-FS) using reverse transcriptase-based conventional polymerase chain reaction. Nothing is known about the status of the COL1A1-PDGFB chimaeric gene in the pleomorphic areas of DFSP-PleoSarc (formerly known as DFSP-malignant fibrous sarcoma). AIMS: To show the COL1A1-PDGFB fusion transcript in transformed malignant fibrous histiocytoma. METHOD: A real-time polymerase chain reaction assay for the COL1A1-PDGFB fusion transcript in a series of DFSP containing sarcoma was conducted to determine whether the chimaeric gene could be identified in both components of DFSP-FS and DFSP-PleoSarc. Eight cases were analysed. RESULTS: In seven cases, transcriptable RNA was detected, and in these cases, translocations were found between COL1A1 and PDGFB genes involving exons 27, 32, 34, 40 and 47 of the COL1A1 gene and exon 2 of the PDGFB gene. CONCLUSIONS: From a diagnostic aspect, this assay can be particularly useful in confirming the diagnosis of sarcomatous DFSP. On the other hand, the COL1A1-PDGFB fusion gene was shown in three cases of DFSP containing pleomorphic sarcoma, which supports the theory of the common histogenesis.

The role of microglia/macrophage system in the tissue response to I-125 interstitial brachytherapy of cerebral gliomas.

OBJECTIVE: To study histopathologic changes and the role of the microglia/macrophage cell in the therapeutic effect of I-125 interstitial brachytherapy on the cerebral gliomas. METHODS: Out of a series of 60 cases with cerebral astrocytomas and other brain tumors treated with I-125 interstitial brachytherapy, autopsy materials were available in ten cases 0.75 and 60 months after irradiation. The patients were treated with the maximum dosage (60 Gy) on the tumor periphery. Besides the routine hematoxylin-eosine and Mallory's PTAH trichrome staining, immunohistochemical reactions were carried out for CD15, CD31, CD34, CD45, CD68, CPM, HAM56 and HLR-DR antigens on paraffin sections to study immunologic phenotypic characteristics of the reaction cell population around gliomas after I-125 treatment. RESULT: One month after irradiation, a necrotic zone developed around the I-125 seeds within the 72 Gy isodose curve. Histologically, there was a fresh coagulation necrosis in the center of the lesion. Reactive zone has not yet developed but scattered interstitial and perivascular CD68 positive macrophages were present in the surrounding brain tissues. Six months after the I-125 isotope treatment, a reactive zone developed: a microglial rim around the necrosis tissue, and a broad area of proliferating vessels and glial fibrillary acidic protein (GFAP) positive astroglial cells which contained CD68 positive activated microglial and macrophage cells. Fifty-four months after I-125 interstitial irradiation, the necrotic center became colliquative and cystic. The microglial rim was replaced by round end stage (HLR-DR and CD31 positive) macrophages. The reactive zone was characterized by astrocytic gliosis but vascular proliferation and macrophages were lacking. CONCLUSION: Results of the present immunohistochemical study suggest that the early lesions are characterized by migrating macrophages apparently concerned with the removal of necrotic debris. The established phase of reactive zone around the necrotic center is characterized by a narrow inner rim of microglial accumulation and a broad outer area characterized by astrocytic gliosis, vascular proliferation, activated microglia and infiltration by macrophages. In the burned-out phases of I-125 interstitial brachytherapy of gliomas, the necrosis undergoes liquefaction and the microglial rim is replaced by astrocytic gliosis which can be considered as equivalent to the scar tissue formed around necrosis outside the central nervous system.

Retrospective analysis of the prognostic role of tissue eosinophil and mast cells in Hodgkin's lymphoma.

The composition of reactive cell populations, which constitute the majority of tumor load in Hodgkin's lymphoma (HL), can influence the prognosis of the disease. Besides widely accepted and applied prognostic scores, the authors evaluate biological factors that may have a prognostic impact. Previous data indicate that the rate of eosinophils and mast cells in the reactive cell population, determined already at diagnosis, can be used for this purpose. Histological samples from 104 patients with HL with an average follow-up period of 110 (24-214) months were retrospectively analyzed. Mast cell positivity was associated with better overall survival, although this difference was only of borderline statistical significance (p=0.092). No significant difference was found in parameters like overall survival (OS, p=0.906) or event-free survival (EFS, p=0.307) of eosinophil-positive vs. -negative cases or in EFS (p=0.742) of mast cell-positive vs. -negative individuals (criterion for a positive specimen was more than 5% of appropriate cells in the reactive cell population). Looking at the effect of eosinophilia and mastocytosis together, there was no significant difference between the subgroups categorized according to the combined presence of the two cell types. It seems that tissue eosinophil and mast cell predominance have no prognostic value that could be used in clinical practice, although a tendency for correlation of mast cell positivity with overall survival could be seen. For a definitive statement, multicenter studies should be performed involving a higher number of patients suffering from HL.

Tissue microarray technology in breast cancer HER2 diagnostics.

Tissue microarrays (TMAs) as current medical research tools significantly lower the costs of immunohistochemical examinations (IHC) and fluorescence in situ hybridization (FISH) while enabling high levels of standardization and reliability. Taking HER2 testing of breast cancer into consideration, we assessed the routine applicability of TMAs. A hundred and seventy-four consecutive samples of invasive breast cancer cases were selected. TMAs were constructed in order to conduct double HER2 immunohistochemical analysis and FISH abreast using the conventional slide by slide method. Comparing the immunohistochemical data obtained from TMAs with the routinely processed large sections, we found a 94.5%/92.7%, 85.7%/88.9% and 91.2%/90% concordance at immunohistochemically HER2-negative, HER2 2+ and 3+ cases using the CB11/HercepTest, respectively. FISH performed on TMAs helped to determine Herceptin therapy suitability in all cases, and when discordance was found, we controlled FISH on "large sections". Being able to conduct FISH examinations at a reasonable price with or without prior immunohistochemical analysis, departments confronted with a certain frequency of breast cancer cases might extensively use the type of TMAs applied in our study. This is a relieve not only with regard to diagnostic work using microarrays, but this also allows to take new directions in research by shedding light on certain unusual cases.

Pathology of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease is one of the leading causes of death and morbidity worldwide. Despite intensive investigation, its pathology and pathophysiology are not well understood. The hallmarks of the disease are irreversible airflow limitation and chronic inflammation. Small airway obstruction due to progressive inflammation and fibrosis, and the loss of elastic recoil mediated by elastolysis and apoptosis equally contribute to pathologic changes. However, it is debated to what extent the obstruction of large airways leads to altered lung function. Three morphologic entities are described in the literature under one disease; chronic bronchitis, obstructive bronchiolitis and emphysema may appear in the same patient at the same time. The authors review pathologic changes observed in chronic obstructive pulmonary disease, including acute exacerbations and secondary pulmonary hypertension as severe but common complications of the disease. Furthermore, we detail recent scientific evidences for major cellular and molecular inflammatory pathway activation. These mechanisms result in accelerated apoptosis, remodeling and increased proinflammatory cytokine release. Targeting intracellular pathological changes may lead to the discovery of a new generation of drugs that could reduce chronic obstruction before airway irreversibility is established.

Effect of IBD sera on expression of inducible and endothelial nitric oxide synthase in human umbilical vein endothelial cells.

AIM: To study the expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) and their role in inflammatory bowel disease (IBD). METHODS: We examined the effect of sera obtained from patients with active Crohn's disease (CD) and ulcerative colitis (UC) on the function and viability of human umbilical vein endothelial cells (HUVEC). HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofluorescence, and quantified by the densitometry of Western blots. Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10(-4) mol/L L-NAME. Apoptosis and necrosis was examined by the annexin-V-biotin method and by propidium iodide staining, respectively. RESULTS: In HUVEC immediately after exposure to UC, serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control (18%+/-16% vs 23%+/-15% P<0.01). UC or CD serum caused a significant increase in iNOS compared to control (UC: 300%+/-21%; CD: 275%+/-27% vs 108%+/-14%, P<0.01). Apoptosis/necrosis characteristics did not differ significantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum. CONCLUSION: IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced anti-inflammatory protection of endothelial cells.