Effect of Exchangeable Ions in Natural and Modified Zeolites on Ag Content,Ag Nanoparticle Formation and Their Antibacterial Activity

To broaden the application of silver nanoparticles (AgNPs), which are well-known antibacterial agents, they are supported on different substrates to prevent aggregation, increase their surface area and antibacterial efficiency, and to be separated from the system more effectively at the end of treatment. To produce nanocomposites that consist of silver nanoparticles on natural and modified zeolites, silver ions (Ag⁺) were loaded onto zeolite (natural, Na-modified, H-modified) and then thermally reduced to AgNPs. The effect of the exchangeable cations in zeolite on Ag⁺ uptake, AgNPs formation, size and morphology was investigated by the TEM, SEM, EDX, XPS, UV-vis, XRD and BET methods. The silver amount in the nanocomposites decreased in the following order Na-modified zeolite > natural zeolite > H-modified zeolite. Microscopic techniques showed formation of AgNPs of 1–14 nm on natural and Na-modified zeolite, while the diameter of metal particles on H-modified zeolite was 12–42 nm. Diffuse reflectance UV-vis and XPS methods revealed the presence of both silver ions and AgNPs in the materials indicating that partial reduction of Ag⁺ ions took place upon heating at 400 °C in air. Additionally, antibacterial properties of the nanocomposites were tested against Escherichia coli, and it was found that Ag–containing composites originating from the Na-modified zeolite demonstrated the highest activity.     

Lateral diffusion of phospholipids in the plasma membrane of soybean protoplasts: Evidence for membrane lipid domains

Fluorescent lipid and phospholipid probes were incorporated at 4°C into soybean protoplasts prepared from cultured soybean (SB-1) cells. Fluorescence microscopy showed that the plasma membrane as well as the nucleus were labeled. Fluorescence redistribution after photobleaching (FRAP) analysis was performed on these cells at 18°C to monitor the lateral mobility of the incorporated probes. After labeling at low concentrations (40 μg/ml) of phosphatidyl-N-(4-nitrobenzo-2-oxa-1,3-diazolyl)ethanolamine (NBD-PtdEtn), a single mobile component was observed with a diffusion coefficient (D) of ≈3 × 10^-9 cm²/sec. After labeling at higher probe concentrations (≥100 μg/ml), two diffusing species were observed, with diffusion coefficients of ≈3 × 10^-9 cm²/sec (“fast”) and ≈5 × 10^-10 cm²/sec (“slow”). Similar results were observed with fluorescent derivatives of phosphatidylcholine and fatty acids. In contrast to these results, parallel analysis of 3T3 fibroblasts, using the same probes and conditions, yielded only a single diffusion component. These results suggest that the soybean plasma membrane may contain two distinct lipid domains in terms of lipid mobility. Consistent with this idea, experiments with soybean protoplasts yielded a single diffusion component under the following conditions: (i) labeling with NBD-PtdEtn (100 μg/ml), FRAP analysis at 37°C (D = 1.1 × 10^-8 cm²/sec); (ii) labeling with NBD-PtdEtn (100 μg/ml), FRAP analysis at 18°C in the presence of 2 mM EGTA (D = 4.2 × 10^-9 cm²/sec); (iii) labeling with 5-(N-dodecanoyl)aminofluorescein (a short-chain lipid probe), FRAP analysis at 18°C or 37°C (D = 2.5 × 10^-8 cm²/sec). These results suggest that the plasma membrane of soybean cells may contain stable immiscible domains of fluid and gel-like lipids.     

Rapid Detection of Listeria by Bacteriophage Amplification and SERS-Lateral Flow Immunochromatography

A rapid Listeria detection method was developed utilizing A511 bacteriophage amplification combined with surface-enhanced Raman spectroscopy (SERS) and lateral flow immunochromatography (LFI). Anti-A511 antibodies were covalently linked to SERS nanoparticles and printed onto nitrocellulose membranes. Antibody-conjugated SERS nanoparticles were used as quantifiable reporters. In the presence of A511, phage-SERS nanoparticle complexes were arrested and concentrated as a visible test line, which was interrogated quantitatively by Raman spectroscopy. An increase in SERS intensity correlated to an increase in captured phage-reporter complexes. SERS limit of detection was 6 × 10⁶ pfu·mL⁻¹, offering detection below that obtainable by the naked eye (LOD 6 × 10⁷ pfu·mL⁻¹). Phage amplification experiments were carried out at a multiplicity of infection (MOI) of 0.1 with 4 different starting phage concentrations monitored over time using SERS-LFI and validated by spot titer assay. Detection of L. monocytogenes concentrations of 1 × 10⁷ colony forming units (cfu)·mL⁻¹, 5 × 10⁶ cfu·mL⁻¹, 5 × 10⁵ cfu·mL⁻¹ and 5 × 10⁴ cfu·mL⁻¹ was achieved in 2, 2, 6, and 8 h, respectively. Similar experiments were conducted at a constant starting phage concentration (5 × 10⁵ pfu·mL⁻¹) with MOIs of 1, 2.5, and 5 and were detected in 2, 4, and 5 h, respectively.     

Quantitative X-ray Elemental Imaging in Plant Materials at the Subcellular Level with a Transmission Electron Microscope: Applications and Limitations

Energy-dispersive X-ray microanalysis (EDX) is a technique for determining the distribution of elements in various materials. Here, we report a protocol for high-spatial-resolution X-ray elemental imaging and quantification in plant tissues at subcellular levels with a scanning transmission electron microscope (STEM). Calibration standards were established by producing agar blocks loaded with increasing KCl or NaCl concentrations. TEM-EDX images showed that the salts were evenly distributed in the agar matrix, but tended to aggregate at high concentrations. The mean intensities of K⁺, Cl⁻, and Na⁺ derived from elemental images were linearly correlated to the concentrations of these elements in the agar, over the entire concentration range tested (R > 0.916). We applied this method to plant root tissues. X-ray images were acquired at an actual resolution of 50 nm × 50 nm to 100 nm × 100 nm. We found that cell walls exhibited higher elemental concentrations than vacuoles. Plants exposed to salt stress showed dramatic accumulation of Na⁺ and Cl⁻ in the transport tissues, and reached levels similar to those applied in the external solution (300 mM). The advantage of TEM-EDX mapping was the high-spatial-resolution achieved for imaging elemental distributions in a particular area with simultaneous quantitative analyses of multiple target elements.     

Genome-wide association identifies OBFC1 as a locus involved in human leukocyte telomere biology

Telomeres are engaged in a host of cellular functions, and their length is regulated by multiple genes. Telomere shortening, in the course of somatic cell replication, ultimately leads to replicative senescence. In humans, rare mutations in genes that regulate telomere length have been identified in monogenic diseases such as dyskeratosis congenita and idiopathic pulmonary fibrosis, which are associated with shortened leukocyte telomere length (LTL) and increased risk for aplastic anemia. Shortened LTL is observed in a host of aging-related complex genetic diseases and is associated with diminished survival in the elderly. We report results of a genome-wide association study of LTL in a consortium of four observational studies (n = 3,417 participants with LTL and genome-wide genotyping). SNPs in the regions of the oligonucleotide/oligosaccharide-binding folds containing one gene (OBFC1; rs4387287; P = 3.9 × 10⁻⁹) and chemokine (C-X-C motif) receptor 4 gene (CXCR4; rs4452212; P = 2.9 × 10⁻⁸) were associated with LTL at a genome-wide significance level (P < 5 × 10⁻⁸). We attempted replication of the top SNPs at these loci through de novo genotyping of 1,893 additional individuals and in silico lookup in another observational study (n = 2,876), and we confirmed the association findings for OBFC1 but not CXCR4. In addition, we confirmed the telomerase RNA component (TERC) as a gene associated with LTL (P = 1.1 × 10⁻⁵). The identification of OBFC1 through genome-wide association as a locus for interindividual variation in LTL in the general population advances the understanding of telomere biology in humans and may provide insights into aging-related disorders linked to altered LTL dynamics.     

The Composites of Graphene Oxide with Metal or Semimetal Nanoparticles and Their Effect on Pathogenic Microorganisms

The present experiment describes a synthesis process of composites based on graphene oxide, which was tested as a carrier for composites of metal- or metalloid-based nanoparticles (Cu, Zn, Mn, Ag, AgP, Se) and subsequently examined as an antimicrobial agent for some bacterial strains (Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). The composites were first applied at a concentration of 300 µM on all types of model organisms and their effect was observed by spectrophotometric analysis, which showed a decrease in absorbance values in comparison with the control, untreated strain. The most pronounced inhibition (87.4%) of S. aureus growth was observed after the application of graphene oxide composite with selenium nanoparticles compared to control. Moreover, the application of the composite with silver and silver phosphate nanoparticles showed the decrease of 68.8% and 56.8%, respectively. For all the tested composites, the observed antimicrobial effect was found in the range of 26% to 87.4%. Interestingly, the effects of the composites with selenium nanoparticles significantly differed in Gram-positive (G⁺) and Gram-negative (G⁻) bacteria. The effects of composites on bacterial cultures of S. aureus and MRSA, the representatives of G⁺ bacteria, increased with increasing concentrations. On the other hand, the effects of the same composites on G⁻ bacteria E. coli was observed only in the highest applied concentration.     

Green Synthesis of Stable Spherical Monodisperse Silver Nanoparticles Using a Cell-Free Extract of Trichoderma reesei

In the current study, a green method for the preparation of silver nanoparticles (AgNPs) is presented as an alternative to conventional chemical and physical approaches. A biomass of Trichoderma reesei (T. reesei) fungus was used as a green and renewable source of reductase enzymes and metabolites, which are capable of transforming Ag⁺ ions into AgNPs with a small size (mainly 2–6 nm) and narrow size distribution (2–25 nm). Moreover, extracellular biosynthesis was carried out with a cell-free water extract (CFE) of T. reesei, which allows for facile monitoring of the bioreduction process using UV–Vis spectroscopy and investigation of the effect of experimental conditions on the transformation of Ag⁺ ions into AgNPs, as well as the simple isolation of as-prepared AgNPs for the study of their size, morphology and antibacterial properties. In continuation to our previous results about the influence of media on T. reesei cultivation, the amount of biomass used for CFE preparation and the concentration of Ag⁺ ion solution, herein, we present the impact of temperature (4, 20, 30 and 40 °C), agitation and time duration on the biosynthesis of AgNPs and their properties. A high stability of AgNPs in aqueous colloids was observed and attributed to the capping effect of the biomolecules as shown by the zeta potential (−49.0/−51.4 mV) and confirmed by the hydrodynamic size of 190.8/116.8 nm of AgNPs.     

Abundance and significance of neuroligin-1 and glutamate in Hirschsprung’s disease

AIM: To investigate the abundance and potential diagnostic significance of neuroligin-1 and glutamate(Glu) in Hirschsprung's disease(HSCR).METHODS: Ninety children with HSCR and 50 children without HSCR matched for similar nutritional status, age and basal metabolic index were studied. The expression and localization of neuroligin-1 and Glu were assessed using double-labeling immunofluorescence staining of longitudinal muscles with adherent myenteric plexus from the surgically excised colon of children with HSCR. Western blot analysis, quantitative real-time PCR(q RT-PCR) and immunohistochemistry were performed to evaluate the abundance of neuroligin-1 and Glu in different HSCR-affected segments(ganglionic, transitional, and aganglionic segments). Enzyme-linked immunosorbent assay(ELISA) was used to detect and compare serum Glu levels in the long-segment HSCR, short-segment HSCR and non-HSCR samples.RESULTS: Neuroligin-1 and Glu were co-expressed highest to lowest in the ganglionic, transi tional and aganglionic segments based on Western blot(neuroligin-1: 0.177 ± 0.008 vs 0.101 ± 0.006, 0.177 ± 0.008 vs 0.035 ± 0.005, and 0.101 ± 0.006 vs 0.035 ±0.005, P 0.005; Glu: 0.198 ± 0.006 vs 0.115 ± 0.008, 0.198 ± 0.006 vs 0.040 ± 0.003, and 0.115 ± 0.008 vs 0.040 ± 0.003, P 0.005) and q RT-PCR(neuroligin-1: 9.58 × 10-5 ± 9.94 × 10-6 vs 2.49 × 10-5 ± 1.38 × 10-6, 9.58 × 10-5 ± 9.94 × 10-6 vs 7.17 × 10-6 ± 1.12 × 10-6, and 2.49 × 10-5 ± 1.38 × 10-6 vs 7.17 × 10-6 ± 1.12 × 10-6, P 0.005). Serum Glu level was the highest to lowest in the non-HSCR, short-type HSCR and long-type HSCR samples based on ELISA(in nmol/μL, 0.93 ± 0.31 vs 0.57 ± 0.25, 0.93 ± 0.31 vs 0.23 ± 0.16, and 0.57 ± 0.25 vs 0.23 ± 0.16, P 0.005).CONCLUSION: Neuroligin-1 and Glu may represent new markers of ganglion cells, whose expression may correlate with the pathogenesis, diagnosis, differential diagnosis or classification of HSCR.     

The toxin-binding protein of sugarcane, its role in the plant and in disease development

The toxin-binding protein of sugarcane susceptible to eyespot disease also possesses raffinose-binding activity. The K_m's for binding are: helminthosporoside (toxin) 6.8 × 10^-5 M, raffinose 2.5 × 10^-5 M, and melibiose 2.6 × 10^-5 M. Evidence obtained by administering [¹⁴C]raffinose to sugarcane protoplasts suggests that this protein participates in α-galactoside transport. Cells from a resistant clone of sugarcane do not possess an active-binding protein, and likewise, do not actively take up raffinose. Interestingly, the K⁺, Mg⁺⁺ ATPase (ATP phosphohydrolase, EC 3.6.1.3.) on the plasma membrane of the susceptible sugarcane is 30% activated by the toxin at 3 mM. In addition, toxin-treated tissue slices show a rapid uptake of ⁸⁶Rb⁺-K⁺ which is in agreement with the toxin activation of the membrane K⁺, Mg⁺⁺ ATPase. Since the ATPase does not directly interact with the toxin, the activation effect occurs by means of the toxin-binding protein. Membrane proteins may be influenced by the toxin-binding protein acting by one of several different mechanisms.     

Helicobacter pylori infection in Mongolian gerbils does not initiate hematological diseases

AIM: To investigate whether Helicobacter pylori (H. pylori) infection contributes to idiopathic thrombocytopenic purpura (ITP) or iron-deficiency anemia (IDA) onset in gerbils.METHODS: A total of 135 Mongolian gerbils were randomly divided into two groups: an H. pylori infection group and a control group. Both groups were fed the same diet and the same amount of food. Each group was then divided into three subgroups, which were sacrificed at 6, 12, or 18 mo for analysis. At each time point, arterial blood was collected from the abdominal aorta and a complete blood cell count was analyzed in the clinical laboratory in the First Affiliated Hospital of Nanchang University.RESULTS: There were no significant differences in platelet counts (938.00 ± 270.27/L vs 962.95 ± 162.56 × 10⁹/L), red blood cell counts (8.11 ± 1.25/L vs 8.44 ± 1.48 × 10¹²/L), or hemoglobin levels (136.9 ± 8.76 g/L vs 123.21 ± 18.42 g/L) between the control and the H. pylori groups, respectively, at 18 mo. With the exception of the mean corpuscular volume (MCV), all other indicators, including white blood cell counts, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red blood cell distribution width, mean platelet volume, platelet distribution width, lymphocyte count, and lymphocyte count percentage, showed no significant differences between the control and H. pylori infection groups at each time point. The MCV in the H. pylori infection group (52.32 f/L ± 2.86 f/L) was significantly lower than the control group (55.63 ± 1.89 f/L) at 18 mo (P = 0.005), though no significant differences were observed at 6 (54.40 ± 2.44 f/L vs 53.30 ± 1.86 f/L) or 12 mo (53.73 ± 2.31 f/L vs 54.80 ± 3.34 f/L).CONCLUSION: A single H. pylori infection is insufficient to cause onset of ITP or IDA and other factors may be required for disease onset.     

Anti-CMV-IgG Positivity of Donors Is Beneficial for alloHSCT Recipients with Respect to the Better Short-Term Immunological Recovery and High Level of CD4+CD25high Lymphocytes

Hematopoietic stem cell transplantation from anti-cytomegalovirus immunoglobulin G (anti-CMV-IgG) positive donors facilitated immunological recovery post-transplant, which may indicate that chronic CMV infection has an effect on the immune system. This can be seen in the recipients after reconstitution with donor lymphocytes. We evaluated the composition of lymphocytes at hematologic recovery in 99 patients with hematologic malignancies post hematopoietic stem cell transplantation (HSCT). Anti-CMV-IgG seropositivity of the donor was associated with higher proportions of CD4+ (227.963 ± 304.858 × 10⁶ vs. 102.050 ± 17.247 × 10⁶ cells/L, p = 0.009) and CD4+CD25high (3.456 ± 0.436 × 10⁶ vs. 1.589 ± 0.218 × 10⁶ cells/L, p = 0.003) lymphocytes in the blood at hematologic recovery. The latter parameter exerted a diverse influence on the risk of acute graft-versus-host disease (GvHD) if low (1.483 ± 0.360 × 10⁶ vs. 3.778 ± 0.484 × 10⁶ cells/L, p < 0.001) and de novo chronic GvHD (cGvHD) if high (3.778 ± 0.780 × 10⁶ vs. 2.042 ± 0.261 × 10⁶ cells/L, p = 0.041). Higher values of CD4+ lymphocytes in patients who received transplants from anti-CMV-IgG-positive donors translated into a reduced demand for IgG support (23/63 vs. 19/33, p = 0.048), and these patients also exhibited reduced susceptibility to cytomegalovirus (CMV), Epstein–Barr virus (EBV) and/or human herpes 6 virus (HHV6) infection/reactivation (12/50 vs. 21/47, p = 0.032). Finally, high levels (≥0.4%) of CD4+CD25high lymphocytes were significantly associated with better post-transplant survival (56% vs. 38%, four-year survival, p = 0.040). Donors who experience CMV infection/reactivation provide the recipients with lymphocytes, which readily reinforce the recovery of the transplanted patients’ immune system.     

Impaired suppressive activities of human MUTYH variant proteins against oxidative mutagenesis

AIM: To investigate the suppressive activity of MUTYH variant proteins against mutations caused by oxidative lesion, 8-hydroxyguanine (8OHG), in human cells.METHODS: p.R154H, p.M255V, p.L360P, and p.P377L MUTYH variants, which were previously found in patients with colorectal polyposis and cancer, were selected for use in this study. Human H1299 cancer cell lines inducibly expressing wild-type (WT) MUTYH (type 2) or one of the 4 above-mentioned MUTYH variants were established using the piggyBac transposon vector system, enabling the genomic integration of the transposon sequence for MUTYH expression. MUTYH expression was examined after cumate induction using Western blotting analysis and immunofluorescence analysis. The intracellular localization of MUTYH variants tagged with FLAG was also immunofluorescently examined. Next, the mutation frequency in the supF of the shuttle plasmid pMY189 containing a single 8OHG residue at position 159 of the supF was compared between empty vector cells and cells expressing WT MUTYH or one of the 4 MUTYH variants using a supF forward mutation assay.RESULTS: The successful establishment of human cell lines inducibly expressing WT MUTYH or one of the 4 MUTYH variants was concluded based on the detection of MUTYH expression in these cell lines after treatment with cumate. All of the MUTYH variants and WT MUTYH were localized in the nucleus, and nuclear localization was also observed for FLAG-tagged MUTYH. The mutation frequency of supF was 2.2 × 10^-2 in the 8OHG-containing pMY189 plasmid and 2.5 × 10^-4 in WT pMY189 in empty vector cells, which was an 86-fold increase with the introduction of 8OHG. The mutation frequency (4.7 × 10^-3) of supF in the 8OHG-containing pMY189 plasmid in cells overexpressing WT MUTYH was significantly lower than in the empty vector cells (P < 0.01). However, the mutation frequencies of the supF in the 8OHG-containing pMY189 plasmid in cells overexpressing the p.R154H, p.M255V, p.L360P, or p.P377L MUTYH variant were 1.84 × 10^-2, 1.55 × 10^-2, 1.91 × 10^-2, and 1.96 × 10^-2, respectively, meaning that no significant difference was observed in the mutation frequency between the empty vector cells and cells overexpressing MUTYH mutants.CONCLUSION: The suppressive activities of p.R154H, p.M255V, p.L360P, and p.P377L MUTYH variants against mutations caused by 8OHG are thought to be severely impaired in human cells.     

Bio-Control of Salmonella Enteritidis in Foods Using Bacteriophages

Two lytic phages, vB_SenM-PA13076 (PA13076) and vB_SenM-PC2184 (PC2184), were isolated from chicken sewage and characterized with host strains Salmonella Enteritidis (SE) ATCC13076 and CVCC2184, respectively. Transmission electron microscopy revealed that they belonged to the family Myoviridae. The lytic abilities of these two phages in liquid culture showed 10⁴ multiplicity of infection (MOI) was the best in inhibiting bacteria, with PC2184 exhibiting more activity than PA13076. The two phages exhibited broad host range within the genus Salmonella. Phage PA13076 and PC2184 had a lytic effect on 222 (71.4%) and 298 (95.8%) of the 311 epidemic Salmonella isolates, respectively. We tested the effectiveness of phage PA13076 and PC2184 as well as a cocktail combination of both in three different foods (chicken breast, pasteurized whole milk and Chinese cabbage) contaminated with SE. Samples were spiked with 1 × 10⁴ CFU individual SE or a mixture of strains (ATCC13076 and CVCC2184), then treated with 1 × 10⁸ PFU individual phage or a two phage cocktail, and incubated at 4 °C or 25 °C for 5 h. In general, the inhibitory effect of phage and phage cocktail was better at 4 °C than that at 25 °C, whereas the opposite result was observed in Chinese cabbage, and phage cocktail was better than either single phage. A significant reduction in bacterial numbers (1.5–4 log CFU/sample, p < 0.05) was observed in all tested foods. The two phages on the three food samples were relatively stable, especially at 4 °C, with the phages exhibiting the greatest stability in milk. Our research shows that our phages have potential effectiveness as a bio-control agent of Salmonella in foods.     

Changes of Host Immunity Mediated by IFN-γ+ CD8+ T Cells in Children with Adenovirus Pneumonia in Different Severity of Illness

The host immunity of patients with adenovirus pneumonia in different severity of illness is unclear. This study compared the routine laboratory tests and the host immunity of human adenovirus (HAdV) patients with different severity of illness. A co-cultured cell model in vitro was established to verify the T cell response in vitro. Among 140 patients with confirmed HAdV of varying severity, the number of lymphocytes in the severe patients was significantly reduced to 1.91 × 10⁹/L compared with the healthy control (3.92 × 10⁹/L) and the mild patients (4.27 × 10⁹/L). The levels of IL-6, IL-10, and IFN-γ in patients with adenovirus pneumonia were significantly elevated with the severity of the disease. Compared with the healthy control (20.82%) and the stable patients (33.96%), the percentage of CD8⁺ T cells that produced IFN-γ increased to 56.27% in the progressing patients. Adenovirus infection increased the percentage of CD8⁺ T and CD4⁺ T cells that produce IFN-γ in the co-culture system. The hyperfunction of IFN-γ⁺ CD8⁺ T cells might be related to the severity of adenovirus infection. The in vitro co-culture cell model could also provide a usable cellular model for subsequent experiments.     

Phytosulfokine-α, a sulfated pentapeptide, stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites

Peptide growth factors were isolated from conditioned medium derived from rice (Oryza sativa L.) suspension cultures and identified to be a sulfated pentapeptide [H-Tyr(SO₃H)-Ile-Tyr(SO₃H)-Thr-Gln-OH] and its C-terminal-truncated tetrapeptide [H-Tyr(SO₃H)-Ile-Tyr(SO₃H)-Thr-OH]. These structures were identical to the phytosulfokines originally found in asparagus (Asparagus officinalis L.) mesophyll cultures. The pentapeptide [phytosulfokine-α (PSK-α)] very strongly stimulated colony formation of rice protoplasts at concentrations above 10⁻⁸ M, indicating a similar mode of action in rice of phytosulfokines. Binding assays using ³⁵S-labeled PSK-α demonstrated the existence of both high- and low-affinity specific saturable binding sites on the surface of rice cells in suspension. Analysis of [³⁵S]PSK-α binding in differential centrifugation fractions suggested association of the binding with a plasma membrane-enriched fraction. The apparent K_d values for [³⁵S]PSK-α binding were found to be 1 × 10⁻⁹ M for the high-affinity type and 1 × 10⁻⁷ M for the low-affinity type, with maximal numbers of binding sites of 1 × 10⁴ sites per cell and 1 × 10⁵ sites per cell, respectively. Competition studies with [³⁵S]PSK-α and several synthetic PSK-α analogs demonstrated that only peptides that possesses mitogenic activity can effectively displace the radioligand. These results suggest that a signal transduction pathway mediated by peptide factors is involved in plant cell proliferation.     

The role of 9-O-acetylated ganglioside D3 (CD60) and ��4��1 (CD49d) expression in predicting the survival of patients with S��zary syndrome

Background

Sézary syndrome is a rare and very aggressive leukemic variant of cutaneous T-cell lymphoma characterized by extensive skin involvement and a malignant circulating CD4⁺ T-cell clone which homes to the skin, over-expresses CD60, and lacks CD7, CD26 and CD49d. So far prognostic markers in this disease are limited to treatment with systemic steroids, age, serum lactate dehydrogenase, and a white blood cell count of 20×10⁹/L or higher: no other biological marker with prognostic value, especially related to malignant cells, has been described.

Design and Methods

We used flow activated cell sorting analysis to compare the distribution of the T-cell receptor-Vβ repertoire and several surface molecules (CD7, CD26, CD49d and CD60) within the circulating CD4⁺ T-cell population in 62 patients with Sézary syndrome, 180 with mycosis fungoides, 6 with B-cell lymphomas, and 19 with chronic eczema. We calculated the 5-year overall survival of patients with Sézary syndrome after first hospital admission using Kaplan–Meier product–limit estimates and hazard ratios from the Cox proportional hazards model.

Results

We found that both higher number of CD60⁺ and lower number of CD49d⁺ cells within circulating CD4⁺ T cells at disease presentation were significantly associated with a lower probability of survival. An exceedingly high risk of death was observed for patients with a combination of a high proportion of CD4⁺CD60⁺ cells (≥ 0.5×10⁹/L) and low proportion of CD4⁺CD49d⁺ cells (<0.5×10⁹/L) (hazard ratio = 12.303, 95% confidence interval 1.5–95.9; P<0.02). In addition, a skewed usage of T-cell receptor-Vβ subfamilies was observed in the circulating T-cell clone for 61.9% of all patients with Sézary syndrome, T-cell receptor-Vβ 2 and 5.1 subfamilies being the most frequently represented (42.8%), followed by T-cell receptor-Vβ 12 and 13.1.

Conclusions

In this study we showed that up-regulation of CD60 and down-regulation of CD49d on circulating CD4⁺ T cells are two useful markers for predicting a very poor outcome in patients with Sézary syndrome.