The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.     

Cytomegalovirus Infection in Inflammatory Bowel Disease Is Not Associated with Worsening of Intestinal Inflammatory Activity

Background

Cytomegalovirus is highly prevalent virus and usually occurs in immunocompromised patients. The pathophysiology and treatment of inflammatory bowel disease often induce a state of immunosuppression. Because this, there are still doubts and controversies about the relationship between inflammatory bowel disease and cytomegalovirus.

Aim

Evaluate the frequency of cytomegalovirus in patients with inflammatory bowel disease and identify correlations.

Methods

Patients with inflammatory bowel disease underwent an interview, review of records and collection of blood and fecal samples. The search for cytomegalovirus was performed by IgG and IgM blood serology, by real-time PCR in the blood and by qualitative PCR in feces. Results were correlated with red blood cell levels, C-reactive protein levels, erythrocyte sedimentation rates and fecal calprotectin levels for each patient.

Results

Among the 400 eligible patients, 249 had Crohn''s disease, and 151 had ulcerative colitis. In the group of Crohn''s disease, 67 of the patients had moderate or severe disease, but 126 patients presented with active disease, based on the evaluation of the fecal calprotectin. In patients with ulcerative colitis, only 21 patients had moderate disease, but 76 patients presented with active disease, based on the evaluation of the fecal calprotectin. A large majority of patients had positive CMV IgG. Overall, 10 patients had positive CMV IgM, and 9 patients had a positive qualitative detection of CMV DNA by PCR in the feces. All 400 patients returned negative results after the quantitative detection of CMV DNA in blood by real-time PCR. Analyzing the 19 patients with active infections, we only found that such an association occurred with the use of combined therapy (anti-TNF-alpha + azathioprine)

Conclusion

The findings show that latent cytomegalovirus infections are frequent and active cytomegalovirus infection is rare. We did not find any association between an active infection of CMV and inflammatory bowel disease activity.     

Nodulation ofTrifolium subterraneum byRhizobium leguminosarum

Summary Four cultivars ofTrifolium subterraneum were nodulated by five strains ofRhizobium leguminosarum; all combinations except one gave 100% nodulation. Rates of nodule formation and total nodule numbers were similar to those with an effectiveR. trifolii strain. The nodules were more commonly associated with lateral roots and were ineffective in nitrogen fixation.     

Phylogenetic analysis of the north‐east Atlantic and Mediterranean species of the genus Stenosoma (Isopoda,Valvifera, Idoteidae)

Xavier, R., Santos, A. M., Harris, D. J., Sezgin, M., Machado, M., Branco, M. (2012). Phylogenetic analysis of the north‐east Atlantic and Mediterranean species of the genus Stenosoma (Isopoda, Valvifera, Idoteidae). —Zoologica Scripta, 41, 386–399. The marine isopod genus Stenosoma is widespread in the northern hemisphere. However, 12 of its 14 known species are found within the Mediterranean basin and adjacent regions of the north‐east Atlantic and the Black Sea. Such a high level of diversity confined to a limited region of a much larger circumglobal distribution suggests that the Mediterranean region may have played a crucial role in the evolutionary history of this genus. In the present work, the phylogeny of the genus Stenosoma was investigated on the basis of DNA sequencing data from one nuclear (28SrRNA) and two mitochondrial (COI, ND4) gene fragments obtained for nine of 12 Atlantic–Mediterranean species. Divergence time estimates point to a Tethyan origin of Stenosoma and suggest that the speciation events from which stem most of the extant species took place well before the Messinian Salinity Crisis. Stenosoma spinosum and Stenosoma appendiculatum are the only exceptions, as they apparently arose within the Mediterranean during the Pleistocene. Phylogenetic reconstruction agrees with current taxonomic status of most species. However, Stenosoma capito clustered in two distinct and well‐supported clades, one composed of eastern Mediterranean and Black Sea specimens and the other by western Mediterranean and Atlantic ones. Such polyphyly suggests the existence of a previously unrecognized species, Stenosoma sp., which so far has been confounded with S. capito.     

Characteristics of Environmental <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Isolates

The ecological niche or exact habitat of the fungus Paracoccidioides brasiliensis is not known, and few isolates have been obtained from the environment. In this study, ten isolates were analyzed with respect to antigenic composition, serology, pathogenicity, and molecular aspects. Gp43 is considered to be the molecular basis for the serodiagnosis of paracoccidioidomycosis; however, in this study only six of the environmental isolates secreted this molecule (four in great amounts and two in small amounts). Other molecules were also produced. When exoantigens from these isolates were tested using immunodiffusion, only four preparations were positive by ID tests. However, when these exoantigens were tested by ELISA, all of them except one were able to detect anti-P. brasiliensis antibodies. In Western blot assays, these exoantigens showed different reactivities. Isolates that secreted gp43 presented positive reactions for this molecule, and isolates that did not secrete gp43 gave positive reactions for other minor molecules. RAPD analysis revealed that there is great genetic variation between these environmental isolates. These isolates were non-pathogenic: no mortality was observed among the inoculated mice during an 18-month follow-up period.     

Mitochondrial dysfunction in platelets and hippocampi of senescence-accelerated mice

Senescence-accelerated mice (SAM) strains are useful models to understand the mechanisms of age-dependent degeneration. In this study, measurements of the mitochondrial membrane potential (Δψ_m) of platelets and the Adenosine 5^′-triphosphate (ATP) content of hippocampi and platelets were made, and platelet mitochondria were observed in SAMP8 (faster aging mice) and SAMR1 (aging resistant control mice) at 2, 6 and 9 months of age. In addition, an Aβ-induced (Amyloid beta-protein) damage model of platelets was established. After the addition of Aβ, the Δψ_m of platelets of SAMP8 at 1and 6 months of age were measured. We found that platelet Δψ_m, and hippocampal and platelet ATP content of SAMP8 mice decreased at a relatively early age compared with SAMR1. The platelets of 6 month-old SAMP8 showed a tolerance to Aβ-induced damages. These results suggest that mitochondrial dysfunction might be one of the mechanisms leading to age-associated degeneration in SAMP mice at an early age and the platelets could serve as a biomarker for detection of mitochondrial function and age related disease.     

Reassembly of a large multisubunit protein promoted by nonprotein factors. Effects of calcium and glycerol on the association of extracellular hemoglobin

The effects of cations and glycerol on the dissociation induced by pressure and on the reassembly of Glossoscolex paulistus hemoglobin were examined by light scattering, gel filtration, and electron microscopy. Calcium stabilized the quaternary structure of the hemoglobin against pressure dissociation. In the presence of 50 mM Ca2+, the half-dissociation pressure (p 1/2) increased by 400 bar, which corresponds to an average stabilization of -0.62 kcal/mol of dissociating subunit. Calcium also promoted a large increase in the yield of recovery of fully assembled hemoglobin at the expense of the partially dissociated (one-twelfth subunit) and fully dissociated forms. Glycerol protected the hemoglobin from pressure dissociation, increasing the half-dissociation pressure (p 1/2) and promoted an increase in the yield of recovery of fully assembled hemoglobin by about 40%. Addition of calcium after return to atmospheric pressure increased recovery of the fully associated form only in a long time scale (many days). The existence of time-dependent changes in the conformation of the dissociated subunits is suggested to explain the partial association to one-twelfth subaggregates (drifted forms) that lack the ability to reassemble to native hemoglobin. The promotion of reassembly by nonprotein factors (calcium and glycerol) is suggested to occur by preventing the formation of wrong intermediate forms (drifted one-twelfth subunits).