Myxoid leiomyosarcoma of the ovary: Analysis of three cases

The first three cases of myxoid leiomyosarcoma occurring in the ovary are reported. Two cases in stage III were found in postmenopausal patients and a further case was found in stage I in a 32-year-old. All masses were large and gelatinous with cystic change, necrosis, and hemorrhage, but both uteri and ligaments and contralateral adnexa appeared normal. Microscopically, the tumors showed a predominantly reticular meshwork of elongated cells surrounded by abundant basophilic material. While electron microscopy proved inconclusive due to nondifferentiation, the use of monoclonal antibodies against smooth muscle actin demonstrated a smooth muscle type of differentiation. The differential diagnosis of this rare ovarian condition includes other myxoid ovarian lesions, such as ovarian edema, myxoma, endodermal sinus tumors, and the sarcomatous component of malignant mixed müllerian tumor and carcinosarcoma, as well as lymphovascular tumors. Since mitotic count due to decreased cellular density is unusually low in myxoid leiomyosarcoma, capsular rupture and clinical stage seem to be more reliable prognostic markers. The highly aggressive behavior of myxoid leiomyosarcoma parallels that of typical ovarian leiomyosarcoma. Two of the three patients in this series died of tumor at 13 and 24 months after diagnosis; the other patient is free of disease at 3 years after diagnosis.     

Lamina propria plasma cells in inflammatory bowel disease: intracellular detection of immunoglobulins using flow cytometry

This is the first application of flow cytometry for the detection of lamina propria plasma cells and their intracellular immunoglobulins in patients with inflammatory bowel disease compared to healthy controls. The study has been focused on the distribution of IgA, IgG, IgM and the four IgG subclasses. Plasma cells were detected as high CD38 positive cells. For fixation and permeabilisation a single step reagent, Ortho Permeafix®, was used.

By flow cytometry, in patients with inflammatory bowel disease compared to healthy controls, a higher percentage of IgG⁺ cells can be observed, in Crohn's disease also a higher percentage of IgM⁺ cells. Regarding the IgG subclass distribution, patients with Crohn's disease show an increase in IgG2⁺ cells, patients with ulcerative colitis an increase in IgG1⁺ and IgG3⁺ cells. These results do agree with and expand the results of earlier immunohistochemical and functional studies, which are favoured today. For the determination of lymphocyte subset proportions and the detection of intracellular antigens, flow cytometry provides a useful alternative to well-established immunohistochemical methods. By analysing a larger number of cells, this method is more reproducible and less prone to interobserver variations than immunohistochemistry, which needs the pre-selection of a mucosal area, the microscopic scoring of a limited number of cells and the circumvention of high background staining. The optimized flow cytometric protocol used in this study might be a promising tool for further investigations of various purposes.     

GLP-1 signalling and effects on glucose metabolism in myocytes from type 2 diabetic patients

Changes in the activity of glycogen synthase a and related kinases (phosphatidylinositol-3-kinase, protein kinase B, p44/42 MAP kinases and p70s6 kinase) evoked by GLP-1 in human myocytes from normal subjects were recently implied in the effect of this hormone upon D-glucose transport and glycogen synthesis in the same cells. The major aims of the present study were i) to investigate the possible extension of this knowledge to myocytes obtained from type 2 diabetic patients, ii) to compare in these patients the response to GLP-1, insulin or the structurally related GLP-1 peptides, exendin (1-39)amide and exendin(9-39)amide, and iii) to explore possible differences in the responsiveness to these agents between normal and diabetic subjects. Apart from the much higher basal PI3K activity and impaired response to insulin of p44/42 MAP kinases in the diabetic patients, the changes in enzyme activity caused by either hormone or peptide, although not identical, were essentially comparable. Nevertheless, significant differences in glucose transport and metabolism parameters were observed in the diabetic patients vs. normal subjects: in the diabetic patients, basal 2-deoxy-glucose uptake and glycogen synthase a activity were lower, accompanied by a similar increasing effect of GLP-1 or insulin; yet, the basal value for glycogen synthesis was higher, coinciding with a lesser relative increment in response to GLP-1 or insulin.     

Major histocompatibility complex status in breast carcinogenesis and relationship to apoptosis

Major histocompatibility complex (MHC) molecules are of central importance in regulating the immune response against tumors. In this study we used immunohistochemistry to study human leukocyte antigen (HLA) class I and II antigen expression in normal breast tissues and benign, preneoplastic, primary, and metastatic breast lesions using antibodies against beta-2-microglobulin (beta2-m), heavy-chain, and HLA-DR antigens. Whereas all normal tissues and benign lesions were positive for beta2-m and HLA-A, -B, and -C antigens, total loss of HLA class I antigens was found in 37% (11 of 30) of in situ carcinomas, in 43% (56 of 131) of the primary tumors, and in 70% (31 of 45) of the lymph node metastases. HLA-DR was also underexpressed in breast cancer cells; thus 20% (6 of 30) of in situ carcinomas, 15% of invasive carcinomas (20 of 131), and only 1 metastatic case were positive for this antigen. Both HLA class I and II antigen expression were more frequently down-regulated in metastatic lesions than in primary breast lesions (P <0.05), and a tendency toward a simultaneous defective expression of HLA class I and II antigens was observed in primary carcinomas (P = 0.07). However, no correlation was found between the expression of any of the aforementioned molecules and pathological parameters or survival. Interestingly, HLA class I expression was expressed more frequently in tissues with high apoptotic activity and was significantly associated with the expression of the proapoptotic bax gene (P = 0.02), and was inversely associated with expression of the antiapoptotic bcl-2 gene (P = 0.03). We conclude that alterations in HLA class I and II antigen expression are early events in breast carcinogenesis and play significant roles in metastatic progression. In addition, their expression is correlated with apoptosis-regulating proteins, which may influence the cytotoxicity of T cells against HLA class I-specific tumor antigens.     

Antigenic homogeneity among the adenovirus hexon types of subgenus C

Summary Antigenic relationships of hexons of human adenovirus (Ad h) types 1, 2, 5 and 6 of subgenus C were studied with 61 monoclonal antibodies (MAbs) raised against human Ad h1, Ad h35 and bovine adenovirus 2. The reactivity pattern (RP) and the titers of the MAbs were determined in indirect ELISA. In previous experiments with hexons of different subgenera 49 MAbs displayed numerous different intertype specificities besides genus specific and type specific ones. With the four hexon types of subgenus C all MAbs gave identical RPs except the type specific ones. Data reveal the existence of a remarkable homogeneity in the antigenic structure among the hexon types of subgenus C defined by the presence of identical or closely related intertype specific epitopes on the surface of the hexons. The possible significance of the results in the experimental gene therapy is discussed.     

Groups I,II and III extracellular phospholipases A2: Selective inhibition of group II enzymes by indomethacin but not other NSAIDs

The three types (groups I, II and III) of stable extracellular 14 kDa phospholipase A₂ enzymes differ in their primary amino acid sequences and their properties. It may thus be possible to design low-molecular weight inhibitors targeted to the secretory form of mammalian PLA₂. this enzyme has been implicated in inflammatory disorders. We have studied the inhibition of four distinct PLA₂ enzymes by a range of NSAIDs, using³H-oleate release from prelabelled membranes ofE. coli for assay. The enzymes used were cobra venom PLA₂ (Naja naja, a group I enzyme), bee venom PLA₂ (Apis mellifera, group III), recombinant human synovial PLA₂ (group II) and rat peritoneal PLA₂ (group II). Under the conditions of the³H-oleateE. coli assay, 1 mM concentrations of aspirin, sodium salicylate, paracetamol (acetaminophen), oxphenbutazone, ibuprofen, flurbiprofen and nabumetone failed to inhibit significantly any of the four enzymes. However, indomethacin inhibited all four enzymes, although effects were greatest on the two group II enzymes (rat peritoneal and human synovial PLA₂). Approximate IC₅₀ values were 28 and 35 M, respectively. Inhibition by indomethacin was not time dependent and was greater at micromolar rather than millimolar levels of calcium. We conclude that indomethacin but not the other tested classes of NSAID inhibits the group II PLA₂ enzyme in a selective manner and suggest that this may be relevant both to its clinical spectrum and to the design of novel pharmaceutical leads.     

Inhibitory activity of a series of coumarins on leukocyte eicosanoid generation

Sixteen plant-derived or synthetic coumarins with different patterns of substitution were tested for their capacity to modify A23187-induced synthesis of leukotriene B₄ and thromboxane B₂ via the 5-lipoxygenase and cyclooxygenase pathways of arachidonate metabolism in rat peritoneal exudate leukocytes. Five of the 16 coumarins inhibited LTB₄ production: all containortho-dihydroxy substitutions (approximate IC₅₀ values 8–100 M). The mechanism is likely to depend upon a combination of the coumarins' iron-chelating and iron ion-reducing abilities, properties which also confer beneficial activities of these compounds as scavengers of reactive oxygen species (Payá et al., Biochem. Pharmacol.44, 205–214 (1992)). Inhibition of the cyclooxygenase pathway was only demonstrated by one compound, 5,7-dihydroxy-4-methylcoumarin, which did not inhibit 5-lipoxygenase, indicating that the cyclooxygenase inhibitory mechanism is different. Similar effects of the active coumarins were obtained using arachidonic acid as substrate for rat leukocyte eicosanoid generation, confirming that they act at the 5-lipoxygenase/cyclooxygenase level. The same profile of activity was also shown when the coumarins were tested against 5-lipoxygenase in human polymorphonuclear neutrophils. Taken together, these antioxidant and anti-eicosanoid properties of coumarins could be exploited for the design of potentially valuable non-toxic anti-inflammatory agents for treating diseases in which eicosanoid generation and the production of reactive oxygen species are involved.     

Synthesis and characterization of halogen-containing polyisophthalamides

Two series of aromatic polyamides were prepared from m-phenylenediamine (MPD) and 4,4′-diaminodiphenyl sulfone (DDS) in combination with a set of new halogenated diacyl chlorides, namely, 5-fluoroisophthaloyl chloride, 5-chloroisophthaloyl chloride, 5-bromoisophthaloyl chloride and 5-iodoisophthaloyl chloride. The polymers were synthesized in high yields and in high molecular weight (as determined by gel permeation chromatography) by means of the low temperature condensation method. Noticeable changes in the spectral characteristics of the halogen-modified polyisophthalamides were observed compared to those of the corresponding unmodified polyisophthalamides. Slight improvements were observed for the glass transition temperatures, which increased 5–30°C relative to those of the conventional polyisophthalamides. Moreover, a correlation was found between the size of the halogen substituents and the increase in T_g values. Thermogravimetric analysis showed that the new polymers are almost as thermally resistant as the unmodified ones, while solubility in organic solvents was some-what poorer.     

Nanoarchitectures for efficient IgE cross-linking on effector cells to study amoxicillin allergy

Background

Amoxicillin (AX) is nowadays the β-lactam that more frequently induces immediate allergic reactions. Nevertheless, diagnosis of AX allergy is occasionally challenging due to risky in vivo tests and non-optimal sensitivity of in vitro tests. AX requires protein haptenation to form multivalent conjugates with increased size to be immunogenic. Knowing adduct structural features for promoting effector cell activation would help to improve in vitro tests. We aimed to identify the optimal structural requirement in specific cellular degranulation to AX using well-precised nanoarchitectures of different lengths.

Method

We constructed eight Bidendron Antigens (BiAns) based on polyethylene glycol (PEG) linkers of different lengths (600–12,000 Da), end-coupled with polyamidoamine dendrons that were terminally multi-functionalized with amoxicilloyl (AXO). In vitro IgE recognition was studied by competitive radioallergosorbent test (RAST) and antibody–nanoarchitecture complexes by transmission electron microscopy (TEM). Their allergenic activity was evaluated using bone marrow-derived mast cells (MCs) passively sensitized with mouse monoclonal IgE against AX and humanized RBL-2H3 cells sensitized with polyclonal antibodies from sera of AX-allergic patients.

Results

All BiAns were recognized by AX-sIgE. Dose-dependent activation responses were observed in both cellular assays, only with longer structures, containing spacers in the range of PEG 6000–12,000 Da. Consistently, greater proportion of immunocomplexes and number of antibodies per complex for longer BiAns were visualized by TEM.

Conclusions

BiAns are valuable platforms to study the mechanism of effector cell activation. These nanomolecular tools have demonstrated the importance of the adduct size to promote effector cell activation in AX allergy, which will impact for improving in vitro diagnostics.

     

Immunohistochemical characterization of fibroblast subpopulations in normal peritoneal tissue and in peritoneal dialysis-induced fibrosis

Peritoneal fibrosis is one of the most common morphological changes observed in continuous ambulatory peritoneal dialysis (CAPD) patients. Both resident fibroblasts and new fibroblast-like cells derived from the mesothelium by epithelial-to-mesenchymal transition are the main cells involved fibrogenesis. In order to establish markers of peritoneal impairment and pathogenic clues to explain the fibrogenic process, we conducted an immunohistochemical study focused on peritoneal fibroblasts. Parietal peritoneal biopsies were collected from four patient groups: normal controls (n=15), non-CAPD uremic patients (n=17), uremic patients on CAPD (n=27) and non-renal patients with inguinal hernia (n=12). To study myofibroblastic conversion of mesothelial cells, -smooth muscle actin (SMA), desmin, cytokeratins and E-cadherin were analyzed. The expression of CD34 by fibroblasts was also analyzed. Fibroblasts from controls and non-CAPD uremic patients showed expression of CD34, but no myofibroblastic or mesothelial markers. The opposite pattern was present during CAPD-related fibrosis. Expression of cytokeratins and E-cadherin by fibroblast-like cells and -SMA by mesothelial and stromal cells supports that mesothelial-to-myofibroblast transition occurs during CAPD. Loss of CD34 expression correlated with the degree of peritoneal fibrosis. The immunophenotype of fibroblasts varies during the progression of fibrosis. Myofibroblasts seem to derive from both activation of resident fibroblasts and local conversion of mesothelial cells.Manuel López-Cabrera and Rafael Selgas contributed equally to the article.