Monocytosis at the time of diagnosis has a negative prognostic impact in myelodysplastic syndromes with less than 5% bone marrow blasts.

The prognostic impact of monocytosis has not yet been determined in patients with myelodysplastic syndromes (MDS). We examined absolute monocyte counts in the peripheral blood at the time of diagnosis in 1949 patients with a bone marrow blast count < 5%, a condition we call MDS < EB1 (MDS with a blast percentage lower than that of MDS with excess blasts 1, according to the WHO classification). Monocytosis (> 600/µl) was associated with higher median hemoglobin, WBC, and ANC, and more favorable karyotype (p = .001). Nevertheless, monocytosis was associated with shorter overall survival (OS) (108 vs. 126 months, p = .002) and earlier transformation into AML (p < .001). In patients with sideroblastic phenotype, the percentage of ring sideroblasts significantly correlated with the monocyte count (p = .005), and OS was significantly shorter when monocytosis was documented (88 vs. 132 months, p = .004). The survival disadvantage of patients with MDS < EB1 and peripheral blood monocytosis suggests that these patients suffer from a CMML-like disease. Even though they are generally classified as MDS with persistent monocytosis, such patients should be considered candidates for therapeutic options employed in CMML.

Analysis of the impact of adherence to guidelines and expert advice in patients with myelodysplastic syndromes.

The European Leukemia Net (ELN) guidelines for treatment of myelodysplastic syndromes (MDS) connect heterogeneous MDS subgroups with a number of therapeutic options ranging from best supportive care to allogeneic stem cell transplantation (alloSCT). However, it is currently unknown whether adherence to guideline recommendations translates into improved survival. The sizeable database of the Duesseldorf MDS Registry allowed us to address this question. We first performed a retrospective analysis including 1698 patients (cohort 1) to whom we retrospectively applied the ELN guidelines. We compared patients treated according to the guidelines with patients who deviated from it, either because they received a certain treatment though it was not recommended or because they did not receive that treatment despite being eligible. We also performed a prospective study with 381 patients (cohort 2) who were seen in our department and received guideline-based expert advice. Again, we compared the impact of subsequent guideline-adherent versus non-adherent treatment. For the majority of treatment options (best supportive care, lenalidomide, hypomethylating agents, low-dose chemotherapy, and intensive chemotherapy), we found that adherence to the ELN guidelines did not improve survival in cohort 1. The same was true when patient management was prospectively enhanced through guideline-based treatment advice given by MDS experts (cohort 2). The only exceptions were alloSCT and iron chelation (ICT). Patients receiving ICT and alloSCT as recommended fared significantly better than those who were eligible but received other treatment. Our analysis underscores the limited survival impact of most MDS therapies and suggests to pursue alloSCT in all suitable candidates. Graphical abstract.

Analysis of immunohistochemical expression of proinflammatory cytokines (IL-1α, IL-6, and TNF-α) in gallbladder mucosa: comparative study in acute and chronic calculous cholecystitis.

BACKGROUND: Several studies have shown increased serum levels of proinflammatory cytokines (IL-1α, IL-6, and TNF-α) in patients with cholelithiasis. The local expression of the proteins involved in pathogenesis of the disease is poorly recognised. MATERIALS AND METHODS: The authors examined immunohistochemically (IHC) the expression status of IL-1α, IL-6, and TNF-α in gallbladder mucosa of the patients with cholelithiasis as related to acute (ACC) and chronic (CCC) types of cholecystitis. Proinflammatory cytokines were quantitatively evaluated in gallbladder mucosa (epithelium and lamina propria) in ACC (n = 16) and CCC (n = 55) groups using modern spatial visualisation technique. RESULTS: Quantitative analysis of IHC signals showed no significant differences in IL-1α and IL-6, and immunoexpression in patients with ACC and CCC. A significantly greater IHC expression of TNF-α was detected in CCC as compared with ACC group. In either of the patient groups immunoexpression of IL-1α and of TNF-α was significantly higher than that of IL-6. Immunoexpression of TNF-α was significantly higher than that of IL-1α only in CCC group. A positive correlation was disclosed between IHC expression of IL-1α and body mass index in CCC group. IHC expression of TNF-α correlated positively with expression of CD68 molecule (histiocytic marker), number of leukocytes in blood and higher grading of gallbladder wall in ACC group. CONCLUSIONS: A more pronounced IHC expression of TNF-α and IL-1α than IL-6 in both types of cholecystitis may suggest the role of these cytokines in pathogenesis of cholelithiasis. IHC expression of TNF- α shows better correlation with clinical/laboratory data in acute cholecystitis, and its quantitative prevalence over the remaining cytokines points to the role of the TNF-α in maintenance of inflammation in the course of cholelithiasis.

Expression of phenotypic markers of mast cells, macrophages and dendritic cells in gallbladder mucosa with calculous cholecystitis.

The study aimed at quantitative analysis of expression involving markers of mast cells (tryptase), monocytes/macrophages (CD68 molecule) and dendritic cells (S100 protein) in gallbladder mucosa with acute and chronic calculous cholecystitis. Routinely prepared tissue material from the patients with acute (ACC) (n = 16) and chronic calculous cholecystitis (CCC) (n = 55) was evaluated. Three cellular markers were localized by immunocytochemistry. Their expression was quantified using spatial visualization technique. The expression of tryptase was similar in acute and chronic cholecystitis. CD68 expression in ACC was significantly higher than in the CCC group. Expression of S100 protein was significantly higher in CCC as compared to the ACC group. No significant correlations were disclosed between expression of studied markers and grading in the gallbladder wall. A weak negative correlation was noted between expression of CD68 and number of gallstones in the CCC group. The spatial visualization technique allowed for a credible quantitative evaluation of expression involving markers of mast cells (MCs), monocytes/macrophages (Mo/Ma) and dendritic cells (DCs) in gallbladder mucosa with ACC and CCC. For the first time mucosal expression of S100 protein-positive DCs was evaluated in calculous cholecystitis. The results point to distinct functions of studied cell types in the non-specific immune response in calculous cholecystitis.

Role of high endothelial postcapillary venules and selected adhesion molecules in periodontal diseases: a review.

Periodontitis is accompanied by the proliferation of small blood vessels in the gingival lamina propria. Specialized postcapillary venules, termed periodontal high endothelial-like venules, are also present, and demonstrate morphological and functional traits similar to those of high endothelial venules (HEVs) in lymphatic organs. The suggested role of HEVs in the pathogenesis of chronic periodontitis involves participation in leukocyte transendothelial migration and therefore proinflammatory effects appear. Recent observations suggest that chronic periodontitis is an independent risk factor for systemic vascular disease and may result in stimulation of the synthesis of acute phase protein by cytokines released by periodontal high endothelial cells (HECs). However, tissue expression of HEV-linked adhesion molecules has not been evaluated in the gingiva of patients with chronic periodontitis. This is significant in relation to potential therapy targeting expression of the adhesion molecules. In this review, current knowledge of HEV structure and the related expression of four surface adhesion molecules of HECs [CD34, platelet endothelial cell adhesion molecule 1, endoglin and intercellular adhesion molecule 1 (ICAM-1)], involved in the key steps of the adhesion cascade in periodontal diseases, are discussed. Most studies on the expression of adhesion molecules in the development and progression of periodontal diseases pertain to ICAM-1 (CD54). Studies by the authors demonstrated quantitatively similar expression of three of four selected surface markers in gingival HEVs of patients with chronic periodontitis and in HEVs of reactive lymph nodes, confirming morphological and functional similarity of HEVs in pathologically altered tissues with those in lymphoid tissues.

Cellular expression of interleukin 2 (IL-2) and its receptor (IL-2R, CD25) in lung tumours.

In view of the unclear prognostic and diagnostic role of interleukin 2 (IL-2) and its receptor in human tumours, we examined the cellular expression of IL-2 and of the subunit alpha of its receptor (IL-2Ralpha, CD25) in relation to the proliferative activity of various subtypes of lung tumours. The immunocytochemical ABC technique was applied to archival tissue material of neuroendocrine lung tumours: lung carcinoids, including typical carcinoids (TC), atypical carcinoids (AC) and small-cell lung cancers (SCLC) and squamous cell lung cancers (non-small cell lung cancers, NSCLC). Expression of IL-2 was detected in all types of lung tumours. The highest frequency of IL-2 expression (93%) was noted and the most pronounced semi-quantitatively evaluated expression of IL-2 was detected in AC tumour cells. The expression was more pronounced as compared to neoplastic SCLC (p = 0.01) and NSCLC cells (p = 0.005). The results suggest a negative correlation between IL-2 expression and the proliferative activity of tumour cells (evaluated by expression of Ki-67) in AC. The frequency of detection of IL-2 receptor (IL-Ralpha, CD25) was the highest in NSCLC (94%). Semi-quantitative expression of IL-2R, like that of IL-2, also dominated in the group of atypical lung carcinoids but manifested a significant difference only as compared to typical carcinoids (p = 0.014). Within the groups of tumours studied no correlation could be detected between cellular expressions of IL-2 and IL-2R. Our results demonstrate variable expression of IL-2 and its receptor in various types of lung tumours, but no simple relationship could be detected between tissue expression of the markers and proliferative activity. Appraisal of the diagnostic and/or prognostic significance of the results requires further study.

Intracellular expression of pro-inflammatory cytokines (IL-1alpha, TNF-alpha, and IL-6) in chronic hepatitis C.

The study aimed at localizing TNF-alpha, IL-1alpha, IL-6 at light and electron microscope levels in patients with chronic hepatitis C, using the immunocytochemical techniques in biopsy material from patients with chronic hepatitis C and at comparing the expression of the cytokines with histopathological changes. Our studies demonstrated an augmented expression of all cytokines in liver biopsies in chronic hepatitis C, in comparison with respective values, obtained in control biopsy material. The highest expression of the cytokines was observed in hepatocytes. That was confirmed by electron microscopy, which demonstrated the cytokines mainly in altered ER cisterns and in the cytoplasm. In children, the expression of IL-1alpha was negatively correlated with staging, while in adult patients; the staging was positively correlated with the expression of TNF-alpha. The new element involves demonstration of cellular and subcellular expression of TNF-alpha, IL-1alpha and IL-6 in hepatocytes in in vivo infection.

Hybridocytochemical detection of mRNA for calcitonin, CGRP, NPY and somatostatin in thyroid parafollicular (C) cells in three rodent species.

The present study was aimed at hybridocytochemical (HCC) detection and interspecies comparison of mRNA for calcitonin (CT), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and somatostatin (SS) in thyroid C cells of two rodent families of wild Microtidae: pine voles and common voles and also of laboratory Muridae, Wistar rats. Studies were performed on adult males. The HCC method in situ and immunomax technique were used to detect mRNA. DNA oligonucleotide probes labeled with digoxigenin were used in the HCC method. The obtained results were compared to the results of immunocytochemical (ICC) examinations, where rabbit or mouse antibodies against human CT, SS, NPY and rat CGRP, as well as chromogranin A were performed. In the present study, HCC reaction has demonstrated the presence of mRNA for CT and CGRP in all thyroid C cells in all the species examined. However, mRNA for NPY and SS was observed in very few C cells in rat and in many more C cells in the two species of wild rodents. The distribution of the positive cells corresponded with that of ICC detected cells.

PTHrP and cytokeratins in human epidermis.

We have demonstrated the presence of parathyroid hormone-related peptide (PTHrP) in cells of human epidermis, employing immunocytochemical techniques. Cells of human epidermal layers demonstrated variable intensity of the reaction. The least pronounced reaction was detected in cells of the basal and the most pronounced reaction in cells of the granular layer. Ultrastructural studies demonstrated that gold particles labeled bundles of keratin filaments. Therefore, at the subsequent stage of the studies we examined the type of filaments to which PTHrP was bound, using immunocytochemical reactions with antibodies against cytokeratins 10, 14, 16 and 19. Positive reaction was obtained for cytokeratins 10, 14 and 16. The reaction pattern obtained for cytokeratins 10 and 16 most closely resembled that of PTHrP. Double labeling with colloidal gold was performed at the ultrastructural level. The results obtained in this way demonstrated that PTHrP most probably binds to filaments built of cytokeratin 16. By binding to the cytokeratin, PTHrP may possibly affect growth and differentiation of keratinocytes.

Expression of mRNA for cytokines (TNF-alpha and IL-1alpha) in human cytomegalovirus (HCMV) and hepatitis B virus (HBV) infections.

The study was aimed at detecting cellular sources of transcripts for two cytokines, TNF-alpha and IL-1alpha in infection with human cytomegalovirus (HCMV) or hepatitis B virus (HBV). The studies were performed on paraffin sections of organs (liver, pancreas, spleen, lungs) obtained upon autopsy from a child deceased due to acute inborn HCMV infection, on paraffin sections of liver biopsy, obtained from a child with HCMV-induced chronic hepatitis, and of liver biopsies obtained from children with chronic type B hepatitis (n = 13). The classical in situ hybridization was applied with digoxygenin-labeled probes and amplification by the ImmunoMax technique. In HCMV infection, the most pronounced expression of mRNA for TNF-alpha and Il-1alpha was detected in pancreatic islets (mainly in beta cells) and, then, in a decreasing sequence, in liver (in macrophages and sinusoidal endothelial cells) and in lungs (in alveolar macrophages). No expression of the two cytokines was detected in the spleen. In HBV infection, weak expression of TNF-alpha and more intense expression of IL-1alpha in the liver were observed, mainly in sinusoidal endothelial cells and in macrophages as well as in hepatocytes. These results were confirmed by immunocytochemical experiments.

A rapid method for the identification and differentiation of Helicoverpa nucleopolyhedroviruses (NPV Baculoviridae) isolated from the environment.

A diagnostic method is described for the identification and differentiation of nucleopolyhedrovirus (NPV) pathogens of Helicoverpa species (Lepidoptera: Noctuidae) isolated from the environment. The method is based on the polymerase chain reaction (PCR) used in conjunction with restriction fragment length polymorphism (RFLP) analysis and comprises three parts. The first part describes procedures for obtaining PCR quality viral DNA from individual diseased H. armigera cadavers recovered during bioassay analyses of soil and other types of environmental sample. These procedures were modified from standard techniques used for the routine purification and dissolution of NPV polyhedra and provided an overall PCR success rate of 95% (n=60). The second part describes the design of several sets of PCR primers for generating DNA amplification products from closely and distantly related NPVs. These PCR primers were designed from published DNA sequence data and from randomly cloned genomic DNA fragments isolated from a reference H. armigera SNPV (HaSNPV) isolate. The final part of the method describes how specific PCR products when digested with specific restriction endonuclease enzymes, can be used to generate diagnostic DNA profiles (haplotypes) that can be used both to identify heterologous NPVs e.g. Autographa californica MNPV and related viruses, and to differentiate genotypic variants of Helicoverpa SNPV. In the latter case, only two PCR products and four restriction digests were required to differentiate a reference set of 10 Helicoverpa SNPV isolates known to differ 0.1--3.5% at the nucleotide level. The diagnostic method described below marks the second part of a two-phase quantitative-diagnostic protocol that is now being applied to a variety of ecological investigations. In particular, its application should lead to a significant improvement in our understanding of the distribution and population genetics of Helicoverpa SNPVs in the Australian environment, as well as providing a sound basis for the design of pre- and post-release monitoring systems for genetically enhanced bioinsecticides. It is also likely that this method can be adapted readily to the study of other insect pathogen associations important economically.

Cellular expression of TNF-alpha in human cytomegalovirus (HCMV) infection.

Human cytomegalovirus (HCMV) belongs to the most frequent human pathogens. Even if the respective pathomorphological patterns are known in detail, the mechanisms which lead to persistence of the virus in its latent form, its reactivation as well as mechanisms of cell death in the symptomatic infection remain to be clarified. It is postulated that HCMV controls expression of TNF-alpha gene and the associated secondary inflammatory response. On the other hand, TNF-alpha has been shown in in vitro studies to represent a potential stimulator of HCMV major IE promoter. The present studies have been aimed at evaluation of TNF-alpha expression in HCMV-infected brain, liver, kidney and pancreas, obtained upon autopsy from children deceased due to an inborn HCMV infection. In situ hybridisation using digoxigenin-labelled oligonucleotide probe demonstrated the expression of TNF-alpha transcript in the liver (in macrophages and endothelial cells) and in pancreatic islets of Langerhans (in beta cells). Immunocytochemical studies aimed at detection of TNF-alpha protein in the material yielded negative results.

Immunocytochemical localization of PTHrP in human and rat salivary glands.

Parathyroid hormone-related protein (PTHrP) was isolated from tumours and is thought to represent the main factor responsible for humoral hypercalcaemia, which accompanies neoplastic diseases. At present, the protein is known to reside in multiple tissues and organs of both humans and animals. Our study was aimed at demonstrating the presence of PTHrP in normal salivary glands (parotid and submandibular) of rats and humans. Application of immunocytochemical techniques permitted to document the presence of PTHrP in the human and in the rat salivary glands. In all cases, an intense reaction was observed in intra- and interlobular ducts. In rat salivary glands, PTHrP was also present in cells of mucous acini. In our opinion, the presence of PTHrP in the ducts indicates participation of the protein in electrolyte transport across the epithelial cells. The positive reaction noted in mucous acini of rat salivary glands may indicate accessory role of PTHrP in the secretory processes in the glands.

Techniques of molecular biology in morphological diagnosis of DNA and RNA viruses.

Recognition of virus structure and biology as well as the increasingly more complete understanding of pathogenesis in infectious diseases have been possible due to the rapid development of the molecular biology techniques. In the recent few years, most of the studies employing those techniques in diagnosis of infectious diseases concerned the detection of novel viruses, clarification of the virus role in diseases of unknown aetiology and determination of the effect of virus mutants on the course of the infection. The pathogenetic mechanisms of chronic infections, oncogenesis and fibrogenesis are continued to be studied. This paper presents the advantages of using in situ hybridisation in the microscopical diagnosis of viruses. Moreover, principal techniques of amplifying the level of virus detection (in situ PCR and its variants, Immunomax) have been described. Direct application of the Immunomax technique in combination with the in situ hybridisation and with immunocytochemistry have been illustrated with our own studies on tissue expression of selected DNA viruses (HBV and HCMV).

Correlation of morphological alterations of light and electron microscopy in chronic type B and C hepatitis.

Chronic type B and C hepatitis involves inflammatory lesions of a variable intensity and variably advanced fibrosis. Considering current, progressively growing requirements for correct evaluation of lesions in liver biopsies, an attempt was made to appraise suitability of selected techniques for a broadened histopathological diagnosis. The lesions were evaluated at the level of light and electron microscopy. Material for the study consisted of liver biopsies obtained from adults and children (n = 60) with serological markers of chronic type B or type C hepatitis. Routine techniques of staining for light and electron microscopy, as well as the techniques of Brachet and Feulgen, were applied. HBcAg expression and HBV-DNA detection in children with chronic type B hepatitis were studied employing the avidin-biotin peroxidase complex (ABC) technique and in situ hybridisation with the ImmunoMax signal amplification. Slight or moderately intense inflammatory lesions (grading of 1 to 2 points) and a low level of fibrosis advancement (staging of 1 to 2 points) prevailed in the material, independently of the etiologic agent involved and age of the patient. Both in children and in adults, extensive lesions in the nuclear chromatin represented the common trait of chronic type B and type C hepatitis examined by light microscopy. Ultrastructural patterns confirmed the lesions and demonstrated virus-resembling particles in the cell nuclei. In HCV infection, hepatocyte cytoplasm contained tubular and horseshoe-shaped structures with lesions of mitochondria, while in HBV infection Dane's particles and tubular forms of HBsAg were detected. For cognitive reasons and due to frequently equivocal literature data, our data on ultrastructural lesions in chronic type C hepatitis seem to be of particular interest. Using the ImmunoMax signal amplification, we were able to diagnose HBV infection under light microscope and to define stage of the infection. Their sensitivity, specificity and relatively short time required for performing the tests makes them advisable in the routine diagnosis of the two infections.

Detection of DNA, mRNA and early antigen of the human cytomegalovirus using the immunomax technique in autopsy material of children with intrauterine infection.

The present study focuses on the immunomax technique in association with the avidin-biotin-peroxidase complex (ABC) technique and a non-isotopic variation of in situ hybridisation (ISH) for optimal microscopical detection of human cytomegalovirus (HCMV). The studies were performed on an archival paraffin material originating from five children deceased due to intrauterine infection. The results of immunocytochemical and hybridocytochemical studies, with or without amplification using biotinylated tyramine, were compared with the routine histopathological results and results obtained using the polymerase chain reaction (PCR). Early antigen (EA)-HCMV was demonstrated in approximately twice as many cells as detected in the routine staining and also in cells that seemed morphologically intact. The hybridocytochemical studies confirmed the presence of HCMV DNA in cells that were positive in the immunocytochemical tests and, in addition (using the ISH-immunomax technique), in cell nuclei of intact myocardial myocytes. In general, fewer cells manifested the presence of HMCV mRNA than the presence of HCMV DNA. The immunomax technique was found to be more sensitive than the techniques of classical immunocytochemistry or of ISH. The former technique permitted the documentation of a higher number of HCMV replication sites than could be detected using the latter techniques. However, the clinical course of HCMV infection or the cause of death of the children was not directly related to the intensity of HCMV expression in tissues.