A unique antigen (sigma) on sézary cells

Summary Lymphapheresis was performed on a patient with Sézary syndrome. The Sézary cells were purified by removing E-rosette-forming and Fc receptor-bearing cells. Antiserum against these purified Sézary cells was raised in rabbits. This antiserum had cytotoxicity against Sézary cells as well as against normal peripheral blood lymphocytes. Absorption was carried out with chronic lymphocytic leukemia (CLL) and normal lymphocytes. The absorbed antiserum maintained cytotoxicity against Sézary cells but lost cytotoxicity against CLL and normal peripheral blood lymphocytes.Indirect immunofluorescence assay showed that the antiserum reacted against purified Sézary cells and a high percentage (66%) of peripheral blood mononuclear cells from five patients with Sézary syndrome. It also reacted against 5.7% of normal lymphocytes, 8% of CLL cells, 5% of the lymphocytes from a patient who had undergone splenectomy, 2% of lymphocytes from a patient with multiple myeloma, 5% of lymphocytes from a hairy cell leukemia patient, and 1% of acute lymphocytic leukemia cells (T cell). The antiserum did not react against thymocytes but reacted against 34.6% of the bone marrow lymphocytes. This unique marker was designated as sigma () antigen. It was suggested that Sézary syndrome may represent proliferation or malignant transformation of normally present antigen-positive lymphocytes.     

Thymopentin down-regulates both activity and expression of iNOS in blood cells of Sézary syndrome patients

While thymopentin has been used for many years in the experimental treatment of Sézary syndrome (SS), a rare and very aggressive lymphoma, its mechanism of action is still not known. Herein we show that this peptide acts as an inhibitor of isolated iNOS and nNOS isoforms, and reduces iNOS protein/mRNA levels and iNOS activity in blood cells obtained from both healthy donors and SS patients. Similar results were obtained with TPN-2, the N(ω)-nitro analogue of the Arg-Lys motif present in thymopentin. Additional investigations are necessary to confirm the role and the relative importance of the two mechanisms of iNOS down-regulation in the therapeutic action of these peptides against SS.     

Interdigitating reticulum cells in lymph nodes of Sézary syndrome. Freeze-fracture and ultrathin-section morphology

Lymph nodes with extensive leukemic infiltration from three patients with the Sézary syndrome were examined in ultrathin sections and in freeze-fracture replicas. Sézary cells (SC) and interdigitating reticulum cells (IDC) were the predominant cell types in the lymph nodes. Both were closely connected with each other by apparently interdigitating cytoplasmic processes. The projections between these cells were, in the main, processes from the IDC. In freeze-fracture replicas these cellular processes did not appear as interdigitations but were more bubble-like, and for this reason these cells are imprecisely described by the term "interdigitating." The SC were seen to possess only short cytoplasmic processes. The frequent polar grouping of cell organelles in SC in the region of the contact zone with IDC and the high organelle content of IDC ('activated IDC') could be the morphologic expression of intense interaction between IDC and SC. IDC displayed three features in freeze-fracture which are not specific to the Sézary syndrome, but should be applicable to IDC in general: (1) they exhibited an approximately equal density of intramembrane particles in both the E-face and the P-face, (2) some of the intramembrane particles in the P-face were assembled in clusters and (3) the surface showed bubble-like formations of the cytoplasmic processes. On the basis of these properties it was possible to distinguish IDC from macrophages and lymphocytes in freeze-fracture replicas.     

The role of food in the population dynamics of Orchesella cincta (Linné) (Collembola)

Summary The feeding activity of the collembolan species Orchesella cincta (Linné) was studied in the laboratory and in the field. In the laboratory it was shown that at any one time only about 50–60% of the animals were feeding; the remainder were occupied with molting activities. Most of the time similar values were obtained in the field but during winter and during dry summer periods high percentages of empty guts could be demonstrated, indicating an absence or inaccessability of food. When favorable weather returned, a high feeding activity was again observed. Laboratory experiments and field observations indicate that temporary starvation periods are followed by synchronization of the molting and reproductive rhythms and an apparent increase in longevity. These phenomena are important for the restoration of the population after unfavorable periods but reduce the stabilizing force of food scarcity.     

Cucurbitacin E and I target the JAK/STAT pathway and induce apoptosis in Sézary cells

Cutaneous T-cell lymphomas and leukemias (CTCLs) are a heterogeneous group of extranodal non-Hodgkin's lymphomas. These are characterized by an accumulation of malignant CD4⁺ T-lymphocytes in the skin, lymph nodes, and peripheral blood. Novel treatment options are needed for patients who progress to advanced stage disease. Cucurbitacin I has previously shown promising results in Sézary syndrome (Sz). A plethora of cucurbitacins, however, have not yet been tested in CTCL. Herein, we investigated the effect of cucurbitacin E and I in two CTCL cell lines. We show that both cucurbitacins decrease viability and cause apoptosis in these cell lines, although HuT-78 was more affected than SeAx (IC₅₀ of 17.38 versus 22.01 μM for cucurbitacin E and 13.36 versus 24.47 μM for cucurbitacin I). Moreover, both cucurbitacins decrease viability of primary cells of a Sz patient (56.46% for cucurbitacin E and 59.07% for cucurbitacin I). Furthermore, while JAK2 inhibition leads to decreased viability in SeAx cells (IC₅₀ of 9.98 and 29.15 μM for AZD1480 and ruxolitinib respectively), both JAK1 and JAK3 do not. This suggests that JAK2 has a preferential role in promoting survival. Western blotting in SeAx cells revealed that both cucurbitacins inhibit STAT3 activation (P < 0.0001), while only cucurbitacin I inhibits STAT5 activation (P = 0.05). This suggests that STAT3 plays a preferential role in the mechanism of action of these cucurbitacins. Nevertheless, a role of STAT5 and JAK2 cannot be excluded and should be explored further. This knowledge could contribute to the development of effective therapies for CTCL and other malignancies involving dysfunction of the JAK/STAT pathway.     

Expression of TNFα membrane-bound receptors in the peripheral blood mononuclear cells (PMBC) in rheumatoid arthritis patients

ObjectiveTo investigate the expression of TNFα membrane-bound receptors: the percentage of cells expressing these receptors and the number of molecules expressed on different immune cell subsets, and to evaluate serum concentrations of soluble TNFα and its receptors (sTNFRI and sTNFRII) in patients with rheumatoid arthritis in acute stage and after response to treatment compared to healthy donors.MethodsThe objects of the study are peripheral blood mononuclear cells (PBMC) of healthy donors (n = 150) and RA patients (n = 40) subjected to hospital treatment with either biological agents (Rituximab) or glucocorticosteroids (methylprednisolone). To determine PBMC phenotype antibodies anti-hCD3-APC, anti-hCD19 PECy7, anti-hCD14 FITC (eBioscience), as well as anti-hTNFRI-PE and anti-hTNFRII-PE (R&D Systems) were used. To determine receptor number on the cells Quantibrite PE Beads (BD) were used.ResultsCells obtained from patients who responded to therapy and achieved disease remission exhibited either an increase in the percentage of TNFRI+ cells or elevated expression density of this receptor type.ConclusionSubsets of immunocompetent cells from RA patients show variation in the percentage of membrane-bound receptor positive cells and receptor expression density, which influences the development and progression of the pathological processes in RA. Response to therapy and achievement of disease remission are associated with an increase of TNFRI expression.     

Thermal constant of an experimental population of Muscina stabulans (Fallén 1817) (Diptera:Muscidae) in the laboratory

A study was conducted to investigate the thermal constant in an experimental population of Muscina stabulans (Fallén 1817). A natural population was obtained from a livestock and their F1 generations were maintained at four constant temperatures (16 degrees C, 20 degrees C, 26 degrees C and 31 degrees C). The thermal constant was calculated by the hyperbole method using the base temperature, tb = 4.4 degrees C and the thermal constant K = 35.3 GD. Ricker's geometric regression, considering tb = 7.8 degrees C and K = 28.9 GD was also calculated.     

Induction of bacillus-Calmette-Guérin-activated killer cells from human peripheral blood mononuclear cells against human bladder carcinoma cell lines in vitro

Cytotoxicity against two human bladder carcinoma cell lines (BT-A and BT-B) was investigated using human peripheral blood mononuclear cells (PBMC) stimulated with viable bacillus Calmette-Guérin (BCG) or sonicated BCG (s-BCG). We applied a cytotoxicity assay based on radioactive labelling of tumour cells by incorporation ofl[³H]methionine. The results were compared with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interleukin-2 (IL-2) and interferon (IFN). BCG-stimulated PBMC showed a cytotoxic potential against BT-A and BT-B comparable to that of IFN-generated LAK cells, but this did not reach the level of IL-2-generated LAK cells. We termed these cytotoxic effectors BCG-activated killer (BAK) cells. In contrast to their cytotoxicity against bladder tumour cells. BAK cells did not differ from unstimulated PBMC in the killing of K562 cells. Only viable but not sonicated BCG was able to induce cytotoxicity against BT-A and BT-B. We could demonstrate the presence of the cytokines IFN, IL-2, tumour necrosis factor (TNF) and TNFß in the supernatants harvested during the generation of BAK cells. Monoclonal antibodies neutralizing IFN were able to inhibit BCG-mediated cytotoxicity, giving evidence of the involvement of IFN in the induction of BAK cells. Furthermore, we performed experiments to investigate the cytotoxic potential of distinct cell populations. The cells effective in BCG-activated killing of bladder tumour cells could be localized within the CD8+/CD56+ lymphocyte subset. CD4+ cells and macrophages did not exhibit cytolytic activity. Our findings imply that the activation by BCG of CD8+/CD56+ killer cells might be an important antitumoral mechanism during BCG therapy against superficial urothelial bladder cancer.     

Diagnostic utility of α-methylacyl CoA racemase (P504S) & HMWCK in morphologically difficult prostate cancer

The epidermal growth factor receptor (EGFR) family, consisting of four tyrosine kinase receptors, c-erbB1-4, seems to be influential in gliomagenesis. The aim of this study was to investigate EGFR gene amplification and expression of c-erbB1-4 receptor proteins in human anaplastic astrocytomas. Formalin-fixed and paraffin-embedded sections from 31 cases were investigated by standard immunohistochemical procedures for expression of c-erbB1-4 receptor proteins using commercial antibodies. EGFR gene amplification was studied by fluorescence in situ hybridization using paraffin-embedded tissues. Two monoclonal antibodies, NCL-EGFR-384 and NCL-EGFR, were used for EGFR detection and they displayed positive immunoreactivity in 97% and 71%, respectively. For c-erbB2 detection three monoclonal antibodies, CB11, 3B5, and 5A2, were applied and they displayed positive immunoreactivity in 45%, 100%, and 52%, respectively. Positive immunostaining for c-erbB3 and c-erbB4 was encountered in 97% and 74%, respectively. The EGFR gene was amplified in 9 out of 31 tumors (29%). After adjusting for age, Karnofsky performance status, and extent of surgical resection, Cox multiple regression analysis with overall survival as the dependent variable revealed that c-erbB2 overexpression detected by the monoclonal antibody clone CB11 was a statistically significant poor prognostic factor (P = 0.004). This study shows the convenience and feasibility of immunohistochemistry when determining the expression of receptor proteins in tissue sections of human astrocytomas. The synchronous overexpression of c-erbB1-4 proteins in anaplastic astrocytomas supports their role in the pathogenesis of these tumors. Further, c-erbB2 overexpression seems to predict aggressive behaviour.     

Distribution, abundance and population parameters of Diapterus rhombeus (Pisces: Gerreidae) in Laguna de Términos, Campeche, Mexico

Diapterus rhombeus dominates the nektonic community of Terminos Lagoon and is associated to zones with submerged vegetation, temperatures between 27 and 33 degrees C and salinity between 25 and 36.5 UPS. A total of 3,024 individuals were collected with a trawl net in 23 sites between September 1997 and September 1998. The length-weight model parameters are a = 0.0098; b = 3.155 with a 96% correlation. The growth parameters are L infinity = 20.4 cm, k = 0.74 and t(o) = -4.207 and the total mortality coefficient is 3.526. Fish length is 2.5-18.0 cm with a mode of 7.5 cm. Recruitment is continuous with a peak from May to August. The species uses several habitats of the lagoon mainly for feeding and growth, reproduction probably occurs in the adjacent continental shelf. Movements between continental shelf and the estuarine system represent a continuous energy flow.     

Subpopulations of human T lymphocytes. IV. Quantitation and distribution in patients with mycosis fungoides and Sézary syndrome.

T cell subpopulations (Tμ and Tγ cells) were examined in the peripheral blood from fourteen patients with mycosis fungoides and Sézary syndrome. One patient with Sézary syndrome having low lymphocyte count had higher proportions of Tγ cells when compared to controls while the other with high lymphocyte count (75% Sézary cells) lacked Tγ cells and had normal proportions of Tμ cells. T cells from a third patient with Sézary syndrome having high lymphocyte count (95% Sézary cells) lacked almost completely both Tμ and Tγ cells. Three of eleven patients with mycosis fungoides had a high proportion of Tγ cells and one had a high proportion of Tμ cells. Study of T cells in the peripheral blood, lymph nodes, and bone marrow from two patients with mycosis fungoides demonstrated that the quantitative abnormality of tμ and Tγ cells is shared by the peripheral blood and bone marrow and not by the lymph nodes. Heterogeneity of T cells subsets in mycosis fungoides appears to be in non-malignant T cells. However, in Sézary syndrome malignant Sézary T cells demonstrate heterogeneity with regard to receptors for IgM (Tμ) and IgG (Tγ).     

Survey of savanna chimpanzees (Pan troglodytes verus) in Southeastern Sénégal

A survey of the western subspecies of chimpanzee (Pan troglodytes verus) was conducted from 1 February to 9 April 2000 in Sénégal, West Africa, by the Miami Assirik Pan Project (MAPP). In addition to the Assirik area of the Parc National du Niokolo Koba (PNNK), areas south and east of the park were surveyed. Nests made by chimpanzees were used to estimate chimpanzee distribution and densities. Within the PNNK, chimpanzees were estimated to occur at an average of 0.13 individuals/km(2). Chimpanzee nests were recorded in nine of 10 locales surveyed outside of the PNNK, as well as within the park. Data on 994 nests made by chimpanzees were recorded outside the PNNK, while 736 nests were recorded inside the park. Nest density in areas surveyed outside the PNNK, such as Bandafassi, Tomboronkoto, and Segou, was comparable to that of Assirik in habitats where nests were concentrated (i.e., evergreen gallery forest). The purpose of MAPP was to initiate long-term research of chimpanzees in southeastern Sénégal, as a follow-up to the Stirling African Primate Project (SAPP) of the 1970s. We sought to replicate the standards set by the SAPP project, except when technological innovations allowed improvement in data collection procedures (e.g., the global positioning system (GPS)).     

Characterization of renal interstitial fibroblast-specific protein 1/S100A4-positive cells in healthy and inflamed rodent kidneys

Fibrosis is considered as a central factor in the loss of renal function in chronic kidney diseases. The origin of fibroblasts and myofibroblasts that accumulate in the interstitium of the diseased kidney is still a matter of debate. It has been shown that accumulation of myofibroblasts in inflamed and fibrotic kidneys is associated with upregulation of fibroblast-specific protein 1 (FSP1, S100A4), not only in the renal interstitium but also in the injured renal epithelia. The tubular expression of FSP1 has been taken as evidence of myofibroblast formation by epithelial–mesenchymal transition (EMT). The identity of FSP1/S100A4 cells has not been defined in detail. We originally intended to use FSP1/S100A4 as a marker of putative EMT in a model of distal tubular injury. However, since the immunoreactivity of FSP1 did not seem to fit with the distribution and shape of fibroblasts or myofibroblasts, we undertook the characterization of FSP1/S100A4-expressing cells in the interstitium of rodent kidneys. We performed immunolabeling for FSP1/S100A4 on thin cryostat sections of perfusion-fixed rat and mouse kidneys with peritubular inflammation, induced by thiazides and glomerulonephritis, respectively, in combination with ecto-5-nucleotidase (5NT), recognizing local cortical peritubular fibroblasts, with CD45, MHC class II, CD3, CD4 and Thy 1, recognizing mononuclear cells, with alpha smooth muscle actin (SMA), as marker for myofibroblasts, and vimentin for intracellular intermediate filaments in cells of mesenchymal origin. In the healthy interstitium of rodents the rare FSP1/S100A4+ cells consistently co-expressed CD45 or lymphocyte surface molecules. Around the injured distal tubules of rats treated for 3–4 days with thiazides, FSP1+/S100A4+, 5NT+, SMA+, CD45+ and MHC class II+ cells accumulated. FSP1+/S100A4+ cells consistently co-expressed CD45. In the inflamed regions, SMA was co-expressed by 5NT+ cells. In glomerulonephritic mice, FSP1+/S100A4+ cells co-expressed Thy 1, CD4 or CD3. Thus, in the inflamed interstitium around distal tubules of rats and of glomerulonephritic mice, the majority of FSP1+ cells express markers of mononuclear cells. Consequently, the usefulness of FSP1/S100A4 as a tool for detection of (myo)fibroblasts in inflamed kidneys and of EMT in vivo is put into question. In the given rat model the consistent co-expression of SMA and 5NT suggests that myofibroblasts originate from resident peritubular fibroblasts.Ivan Hegyi and Michel Le Hir contributed equally to the study     

Chocolate: (un)healthy source of polyphenols?

There is recent epidemiological evidence that chocolate consumption may improve vascular health. Furthermore, several small-scale human intervention studies indicate that habitual chocolate intake enhances the production of vasodilative nitric oxide and may lower blood pressure. It is hypothesized that potential beneficial effects of chocolate on vascular health are at least partly mediated by cocoa polyphenols including procyanidins. Based on cell culture studies, molecular targets of chocolate polyphenols are endothelial nitric oxide synthetase as well as arginase. However, human bioavailability studies suggest that the plasma concentrations of cocoa polyphenols are manifold lower than those concentrations used in cultured cells in vitro. The experimental evidence for beneficial vascular effects of chocolate in human interventions studies is yet not fully convincing. Some human intervention studies on chocolate and its polyphenols lack a stringent study design. They are sometimes underpowered and not always placebo controlled. Dietary chocolate intake in many of these human studies was up to 100 g per day. Since chocolate is a rich source of sugar and saturated fat, it is questionable whether chocolate could be recommended as part of a nutrition strategy to promote vascular health.     

Dissemination of the entomopathogenic fungus verticillium lecanii (Zimmermann) Viégas (Hyphomycetales: Moniliaceae) in a population of Frankliniella occidentalis (Pergande, 1895) (Thysanoptera: Thripidae)

Tests were carried out to investigate the dissemination of the entomopathogenic fungus Verticillium lecanii (Zimmermann) Viégas in a population of Frankliniella occidentalis. The tested factors, which influence the efficacy of the fungus against the pest insect, have been the population density of the thrips at the application moment as well as the temperature. The population density influenced the dissemination of the fungal spores in the population. The higher the density has been, the higher the insetting control effect has been as well. The temperature influenced the speed of the fungal effect, too. The higher the temperature has been, the earlier the control effect started. However, an increase of the natural mortality was found as well.     

Induction of apoptosis in bacillus Calmette-Guérin-activated T cells by transforming growth factor-beta

In view of the critical role played by bacillus Calmette-Guérin (BCG) in the development and functional activation of protective T cells against tuberculosis, it has become important to understand the mechanisms by which cytokines regulate BCG-mediated immune responses. There is evidence that cytokine-mediated suppression of T cell function by mechanisms, including apoptosis, may reduce host resistance in tuberculosis. However, it is unclear whether cytokine-mediated suppression of antigen-responsive T cells through apoptotic mechanisms may be operating during human cellular activation induced by BCG. Here we present evidence, for the first time, that treatment of BCG-activated T cells with transforming growth factor-beta (TGF-beta) induces cellular apoptosis. These results were further supported by the fact that treatment of cells with a blocking mAb directed to TGF-beta significantly inhibited the percentage of apoptosis induced by TGF-beta. Interestingly, TGF-beta-mediated death of BCG-activated T cells in cultures containing interleukin (IL)-12 was observed. Moreover, our results demonstrated the induction of apoptosis by TGF-beta in BCG-activated T cells cultured in the presence of exogenous IL-12. In addition, our data indicated that TGF-beta significantly inhibited both BCG-induced cell growth determined by thymidine uptake and BCG-induced IFN-gamma secretion. Finally, TGF-beta-induced apoptosis in BCG-activated T cells correlated inversely with BCG-induced IFN-gamma secretion. Taken together, these findings indicate that TGF-beta induces apoptosis in human T cells activated with BCG and at the same time suggest that loss of BCG-reactive T cells through apoptotic mechanisms could contribute to an increased susceptibility to Mycobacterium tuberculosis infection.     

Induction of T → G and T → A transversions by 5-formyluracil in mammalian cells

Oxidatively damaged thymine, 5-formyluracil (5-fU), was incorporated into a predetermined site of double-stranded shuttle vectors. The nucleotide sequences in which the modified base was incorporated were 5′-CFTAAG-3′ and 5′-CTFAAG-3′ (F represents 5-fU), the recognition site for the restriction enzyme AflII (5′-CTTAAG-3′). The 5-fU was incorporated into a template strand of either the leading or lagging strand of DNA replication. The modified DNAs were transfected into simian COS-7 cells, and the DNAs replicated in the cells were recovered and were analyzed after the second transfection into Escherichia coli. The 5-fU did not block DNA replication in mammalian cells. The 5-fU residues were weakly mutagenic, and their mutation frequencies in double-stranded vectors were 0.01–0.04%. The T → G and T → A transversions were the mutations found most frequently, suggesting the formation of 5-fU·C and 5-fU·T base pairs, respectively. This is the first report that clearly shows the induction of transversion mutations by an oxidized pyrimidine base in DNA in mammalian cells.