Functional expression, monodispersity and conformational changes in the SBMV virus viral VPg on binding TFE.

Southern bean mosaic virus (SBMV) RNA purified from infected plants was used for cloning the viral genome-linked protein (VPg) and was subsequently expressed in Escherichia coli. Circular dichroism (CD), dynamic light scattering (DLS) and saturation transfer difference (STD) by nuclear magnetic resonance (NMR) measurements were employed to determine the degree of monodispersity and to investigate the conformational changes in the absence and presence of trifluoroethanol (TFE) which indicated increased helical content with increasing concentration of TFE. 8-Anilino-1-naphthalenesulfonic acid (ANS) was used as a probe to compare the unfolding regions of the protein before and after addition of TFE. The results indicated that although the TFE concentration influences VPg folding, it does not play a role in nucleotide binding and that the local solvent hydrophobicity causes significant conformational changes.

Contagem de reticulócitos de cães saudáveis ou anêmicos pela citometria de fluxo / Count of reticulocyte counts of healthy or anemic dogs by flow cytometry

Compararam-se os resultados da contagem de reticulócitos pela microscopia de luz e pelo método da citometria de fluxo em 25 cães saudáveis (controle), 60 cães com anemia regenerativa e 40 com anemia arregenerativa. Houve diferença nas contagens absolutas obtidas pela microscopia de luz e pela citometria de fluxo nos três grupos estudados. A contagem de reticulócitos foi mais alta pela citometria de fluxo que a contagem pela microscopia de luz, mostrando ser um método mais sensível, simples e seguro para a quantificação de reticulócitos.

Counts of reticulocytes using both the light microscopy and flow cytometry (FC) methods in 25 healthy control dogs, 60 dogs with regenerative anemia, and 40 dogs with non-regenerative anemia were compared. The absolute counts were submitted to the paired t-Student test, which determined significant differences (P<0.0001) between those methods in the three studied groups. Counts of retyculocytes were higher under flow cytometry, which proved to be a more sensitive method. Flow cytometry is a simple and reliable method for the quantification of reticulocytes.

Quality control of blood irradiation with a teletherapy unit: damage to stored red blood cells after cobalt-60 gamma irradiation.

BACKGROUND: Previous publications have documented the damage caused to red blood cells (RBCs) irradiated with X-rays produced by a linear accelerator and with gamma rays derived from a 137Cs source. The biologic effects on RBCs of gamma rays from a 60Co source, however, have not been characterized. STUDY DESIGN AND METHODS: This study investigated the effect of 3000 and 4000 cGy on the in vitro properties of RBCs preserved with preservative solution and irradiated with a cobalt teletherapy unit. A thermal device equipped with a data acquisition system was used to maintain and monitor the blood temperature during irradiation. The device was rotated at 2 r.p.m. in the irradiation beam by means of an automated system. The spatial distribution of the absorbed dose over the irradiated volume was obtained with phantom and thermoluminescent dosimeters (TLDs). Levels of Hb, K+, and Cl(-) were assessed by spectrophotometric techniques over a period of 45 days. The change in the topology of the RBC membrane was investigated by flow cytometry. RESULTS: Irradiation caused significant changes in the extracellular levels of K+ and Hb and in the organizational structure of the phospholipid bilayer of the RBC membrane. Blood temperature ranged from 2 to 4 degrees C during irradiation. Rotation at 2 r.p.m. distributed the dose homogeneously (92%-104%) and did not damage the RBCs. CONCLUSIONS: The method used to store the blood bags during irradiation guaranteed that all damage caused to the cells was exclusively due to the action of radiation at the doses applied. It was demonstrated that prolonged storage of 60Co-irradiated RBCs results in loss of membrane phospholipids asymmetry, exposing phosphatidylserine (PS) on the cells' surface with a time and dose dependence, which can reduce the in vivo recovery of these cells. A time- and dose-dependence effect on the extracellular K+ and plasma-free Hb levels was also observed. The magnitude of all these effects, however, seems not to be clinically important and can support the storage of irradiated RBC units for at last 28 days.

Quality control of blood irradiation: determination T cells radiosensitivity to cobalt-60 gamma rays.

BACKGROUND: To identify the most appropriate dose for the prevention of transfusion-associated graft-versus-host disease, the radiosensitivity of T cells has been determined in blood bags irradiated with X-rays produced by a linear accelerator and gamma rays derived from the cesium-137 source of a specific irradiator. In this study, the influence of doses ranging from 500 to 2500 cGy was investigated on T cells isolated from red blood cell (RBC) units preserved with ADSOL and irradiated with a cobalt teletherapy unit. STUDY DESIGN AND METHODS: A thermal device consisting of acrylic and foam was constructed to store the blood bags during irradiation. Blood temperature was monitored with an automated data acquisition system. Dose distribution in the blood bags was analyzed based on isodose curves obtained with a polystyrene phantom constructed for this purpose. The influence of cobalt-60 gamma radiation on T cells was determined by limiting-dilution analysis, which measures clonable T cells. T-cell content of the mononuclear cell population plated was assessed by flow cytometry with a monoclonal antibody specific for CD3. RESULTS: Blood temperature ranged from 2 to 4.5 degrees C during irradiation. Dosimetry performed on the phantom showed a homogenous dose distribution when the phantom was irradiated with a parallel-opposite field. A radiation dose of 1500 cGy led to the inactivation of T cells by 4 log, but T-cell growth was observed in all experiments. At 2500 cGy, no T-cell growth was detected in any of the experiments and a greater than 5 log reduction in functional T cells was noted. CONCLUSION: The results showed that a dose of 2500 cGy completely inactivates T cells in RBC units irradiated with cobalt-60 source.

Immunological effects of donor lymphocyte infusion in patients with chronic myelogenous leukemia relapsing after bone marrow transplantation.

Allogeneic bone marrow transplantation (alloBMT) is the only curative therapy for chronic myelogenous leukemia (CML). This success is explained by the delivery of high doses of antineoplastic agents followed by the rescue of marrow function and the induction of graft-versus-leukemia reaction mediated by allogeneic lymphocytes against host tumor cells. This reaction can also be induced by donor lymphocyte infusion (DLI) producing remission in most patients with CML who relapse after alloBMT. The immunological mechanisms involved in DLI therapy are poorly understood. We studied five CML patients in the chronic phase, who received DLI after relapsing from an HLA-identical BMT. Using flow cytometry we evaluated cellular activation and apoptosis, NK cytotoxicity, lymphocytes producing cytokines (IL-2, IL-4 and IFN-gamma), and unstimulated (in vivo) lymphocyte proliferation. In three CML patients who achieved hematological and/or cytogenetic remission after DLI we observed an increase of the percent of activation markers on T and NK cells (CD3/DR, CD3/CD25 and CD56/DR), of lymphocytes producing IL-2 and IFN-gamma, of NK activity, and of in vivo lymphocyte proliferation. These changes were not observed consistently in two of the five patients who did not achieve complete remission with DLI. The percent of apoptotic markers (Fas, FasL and Bcl-2) on lymphocytes and CD34-positive cells did not change after DLI throughout the different study periods. Taken together, these preliminary results suggest that the therapeutic effect of DLI in the chronic phase of CML is mediated by classic cytotoxic and proliferative events involving T and NK cells but not by the Fas pathway of apoptosis.

Immunological effects of donor lymphocyte infusion in patients with chronic myelogenous leukemia relapsing after bone marrow transplantation

Allogeneic bone marrow transplantation (alloBMT) is the only curative therapy for chronic myelogenous leukemia (CML). This success is explained by the delivery of high doses of antineoplastic agents followed by the rescue of marrow function and the induction of graft-versus-leukemia reaction mediated by allogeneic lymphocytes against host tumor cells. This reaction can also be induced by donor lymphocyte infusion (DLI) producing remission in most patients with CML who relapse after alloBMT. The immunological mechanisms involved in DLI therapy are poorly understood. We studied five CML patients in the chronic phase, who received DLI after relapsing from an HLA-identical BMT. Using flow cytometry we evaluated cellular activation and apoptosis, NK cytotoxicity, lymphocytes producing cytokines (IL-2, IL-4 and IFN-gamma), and unstimulated (in vivo) lymphocyte proliferation. In three CML patients who achieved hematological and/or cytogenetic remission after DLI we observed an increase of the percent of activation markers on T and NK cells (CD3/DR, CD3/CD25 and CD56/DR), of lymphocytes producing IL-2 and IFN-gamma, of NK activity, and of in vivo lymphocyte proliferation. These changes were not observed consistently in two of the five patients who did not achieve complete remission with DLI. The percent of apoptotic markers (Fas, FasL and Bcl-2) on lymphocytes and CD34-positive cells did not change after DLI throughout the different study periods. Taken together, these preliminary results suggest that the therapeutic effect of DLI in the chronic phase of CML is mediated by classic cytotoxic and proliferative events involving T and NK cells but not by the Fas pathway of apoptosis.

Influence of menstrual cycle on NK activity.

Natural killer (NK) cells are CD3- CD56+ and/or CD16+ cytotoxic lymphocytes that mediate first-line defense against various types of target cells without prior immunization. To assess the effect of the menstrual cycle and gender on NK activity we evaluated 30 healthy women (mean age 28.1 years, range 21-39) in follicular and luteal phases, 29 postmenopausal women (mean age 58.8 years, range 42-72) and 48 healthy men (mean age 31.6 years, range 21-40). In a flow cytometric test of NK activity, peripheral blood mononuclear effector cells were mixed with K562 targets cells labeled with DiO (3,3'-dioctadecyloxacarbocyanine perchlorate) at effector:target cell ratios of 40, 20, 10 and 5:1. Dead cells were stained with propidium iodide and results were expressed as lytic units per 10(7) cells. In addition, progesterone levels were determined in the luteal phase of the menstrual cycle of healthy women by a chemiluminescence assay. Our results showed that (1) NK cytotoxicity was higher in the follicular than in the luteal phase of the menstrual cycle (P < 0.0001); (2) postmenopausal women and men showed NK activity similar to women in the follicular phase but higher than women in the luteal phase of the menstrual cycle (P < 0.05); and (3) there was no correlation between NK activity and levels of progesterone. The data suggest that progesterone does not influence NK activity directly and that other factors may explain the reduction of NK activity in the luteal phase of the menstrual cycle.