Differential effects of age,cytomegalovirus-seropositivity and end-stage renal disease (ESRD) on circulating T lymphocyte subsets

The age- and cytomegalovirus (CMV)-seropositivity-related changes in subsets and differentiation of circulating T cells were investigated in end-stage renal disease (ESRD) patients (n = 139) and age-matched healthy individuals. The results show that CMV-seropositivity is associated with expansion of both CD4⁺ and CD8⁺ memory T cells which is already observed in young healthy individuals. In addition, CMV-seropositive healthy individuals have a more differentiated memory T cell profile. Only CMV-seropositive healthy individuals showed an age-dependent decrease in CD4⁺ naïve T cells. The age-related decrease in the number of CD8⁺ naïve T cells was CMV-independent. In contrast, all ESRD patients showed a profound naïve T-cell lymphopenia at every decade. CMV-seropositivity aggravated the contraction of CD4⁺ naïve T cells and increased the number of differentiated CD4⁺ and CD8⁺ memory T cells. In conclusion, CMV-seropositivity markedly alters the homeostasis of circulating T cells in healthy individuals and aggravates the T cell dysregulation observed in ESRD patients.     

A complex multilevel attack on Pseudomonas aeruginosa algT/U expression and algT/U activity results in the loss of alginate production

Infection by the opportunistic pathogen Pseudomonas aeruginosa is a leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. This is mainly due to the genotypic and phenotypic changes of the bacteria that cause conversion from a typical nonmucoid to a mucoid form in the CF lung. Mucoid conversion is indicative of overproduction of a capsule-like polysaccharide called alginate. The alginate-overproducing (Alg(+)) mucoid phenotype seen in the CF isolates is extremely unstable. Low oxygen tension growth of mucoid variants readily selects for nonmucoid variants. The switching off mechanism has been mapped to the algT/U locus, and the molecular basis for this conversion was partially attributed to mutations in the algT/U gene itself. To further characterize molecular changes resulting in the unstable phenotype, an isogenic PAO1 derivative that is constitutively Alg(+) due to the replacement of the mucA with mucA22 (PDO300) was used. The mucA22 allele is common in mucoid CF isolates. Thirty-four spontaneous nonmucoid variants, or sap (suppressor of alginate production) mutants, of PDO300 were isolated under low oxygen tension. About 40% of the sap mutants were rescued by a plasmid carrying algT/U (Group A). The remaining sap mutants were not (Group B). The members of Group B fall into two subsets: one similar to PAO1, and another comparable to PDO300. Sequence analysis of the algT/U and mucA genes in Group A shows that mucA22 is intact, whereas algT/U contains mutations. Genetic complementation and sequencing of one Group B sap mutant, sap22, revealed that the nonmucoid phenotype was due to the presence of a mutation in PA3257. PA3257 encodes a putative periplasmic protease. Mutation of PA3257 resulted in decreased algT/U expression. Thus, inhibition of algT/U is a primary mechanism for alginate synthesis suppression.     

Transplantation survival is maintained by granzyme B+ regulatory cells and adaptive regulatory T cells

Granzyme B (GZB) has been implicated as an effector mechanism in regulatory T cells (T(reg)) suppression. In a model of T(reg)-dependent graft tolerance, it is shown that GZB- deficient mice are unable to establish long-term tolerance. Moreover, mice overexpressing the inhibitor of GZB, serine protease inhibitor 6, are also resistant to tolerization to alloantigen. Graft survival was shorter in bone marrow-mixed chimeras reconstituted with GZB-deficient T(reg) as compared with wild-type T(reg). Whereas there was no difference in graft survival in mixed chimeras reconstituted with wild-type, perforin-deficient, or Fas ligand-deficient T(reg). Finally, data also show that if alloreactive effectors cannot express FoxP3 and be induced to convert in the presence of competent T(reg), then graft tolerance is lost. Our data are the first in vivo data to implicate GZB expression by T(reg) in sustaining long-lived graft survival.     

Cerebral perfusion pressure in giraffe: modelling the effects of head-raising and -lowering

Loss of consciousness caused by positional changes of the head results from reduced cerebral blood flow (CBF). CBF is related to cerebral perfusion pressure (CPP). CPP is the difference between mean arterial pressure (MAP) at the head and intracranial pressure (ICP). The positional change of the giraffe head between ground level and standing upright is the largest of all animals yet loss of consciousness does not occur. We have investigated the possibility that an increase in CPP protects giraffe from fainting, using a mechanical model that functioned as an anatomical U-tube. It consisted of a rigid ascending “carotid” limb, a collapsible “brain” tube drained by a rigid, “vertebral venous plexus” (VVP) tube, and a collapsible “head” tube drained by a collapsible tube representing the “jugular vein”. The descending tubes could be rotated relative to the “carotid” tube to be horizontal, or at 30°, 45°, and 60° to the vertical to simulate changes in head position. Pressure at the top of the “carotid” tube was intracranial MAP, at the top of the “VVP” tube was ICP, and the difference CPP. In the simulated “head-up” position and a fluid flow rate of 4 L min⁻¹, CPP was ∼170 mmHg. With the VVP tube horizontal, CPP fell from ∼170 to 45 mmHg, but increased to ∼67 mmHg at 30° “down”, to ∼70 mmHg at 45° “down” and to ∼75 at 60° “down”. The fall in CPP in the head-down positions resulted from a decrease in viscous resistance in, and dissipation of pressure to, the “head” and “jugular” tubes. These data provide an estimate of cranial pressure changes in giraffe during positional changes of the head, and suggest that an increase in CPP plays a significant role in maintaining CBF during head-raising and that it may be an important mechanism for preventing fainting in giraffe.     

Novel non-viral method for transfection of primary leukemia cells and cell lines

BACKGROUND: Tumor cells such as leukemia and lymphoma cells are possible targets for gene therapy. However, previously leukemia and lymphoma cells have been demonstrated to be resistant to most of non-viral gene transfer methods. METHODS: The aim of this study was to analyze various methods for transfection of primary leukemia cells and leukemia cell lines and to improve the efficiency of gene delivery. Here, we evaluated a novel electroporation based technique called nucleofection. This novel technique uses a combination of special electrical parameters and specific solutions to deliver the DNA directly to the cell nucleus under mild conditions. RESULTS: Using this technique for gene transfer up to 75% of primary cells derived from three acute myeloid leukemia (AML) patients and K562 cells were transfected with the green flourescent protein (GFP) reporter gene with low cytotoxicity. In addition, 49(+/- 9.7%) of HL60 leukemia cells showed expression of GFP. CONCLUSION: The non-viral transfection method described here may have an impact on the use of primary leukemia cells and leukemia cell lines in cancer gene therapy.     

IMCL area density, but not IMCL utilization, is higher in women during moderate-intensity endurance exercise, compared with men

Women use more fat during endurance exercise as evidenced by a lower respiratory exchange ratio (RER). The contribution of intramyocellular lipid (IMCL) to lipid oxidation during endurance exercise is controversial, and studies investigating sex differences in IMCL utilization have found conflicting results. We determined the effect of sex on net IMCL use during an endurance exercise bout using an ultrastructural evaluation. Men (n = 17) and women (n = 19) completed 90-min cycling at 63% Vo(2peak). Biopsies were taken before and after exercise and fixed for electron microscopy to determine IMCL size, # IMCL/area, IMCL area density, and the % IMCL touching mitochondria. Women had a lower RER and carbohydrate oxidation rate and a higher lipid oxidation rate during exercise (P < 0.05), compared with men. Women had a higher # IMCL/area and IMCL area density (P < 0.05), compared with men. Women, but not men, had a higher % IMCL touching mitochondria postexercise (P = 0.03). Exercise decreased IMCL area density (P = 0.01), due to a decrease in the # IMCL/area (P = 0.02). There was no sex difference in IMCL size or net use. In conclusion, women have higher IMCL area density compared with men, due to an increased # IMCL and not an increased IMCL size, as well as an increased % IMCL touching mitochondria postexercise. Endurance exercise resulted in a net decrease in IMCL density due to decreased number of IMCL, not decreased IMCL size, in both sexes.     

Comparative analysis of crystallins and lipids from the lens of Antarctic toothfish and cow

Animal model systems of senile cataract and lens crystallin stability are essential to understand the complex nature of lens transparency. Our aim in this study was to assess the long-lived Antarctic toothfish Dissostichus mawsoni (Norman) as a model system to understand long-term lens clarity in terms of solubility changes that occur to crystallins. We compared the toothfish with the mammalian model cow lens, dissecting each species’ lens into a cortex and nuclear region. In addition to crystallin distribution, we also assayed fatty acid (FA) composition by negative ion electrospray ionization mass spectrometry (ESI-MS). The majority of toothfish lens crystallins from cortex (90.4%) were soluble, whereas only a third (31.8%) from the nucleus was soluble. Crystallin solubility analysis by SDS-PAGE and immunoblots revealed that relative proportions of crystallins in both soluble and urea-soluble fractions were similar within each species examined and in agreement with previous reports for bovine lens. From our data, we found that both toothfish and cow crystallins follow patterns of insolubility that mirror each animals lens composition with more γ crystallin aggregation seen in the toothfish lens nucleus than in cow. Toothfish lens lipids had a large amount of polyunsaturated fatty acids that were absent in cow resulting in an unsaturation index (I _U) four-fold higher than that of cow. We identified a novel FA with a molecular mass of 267 mass units in the lens epithelial layer of the toothfish that accounted for well over 50% of the FA abundance. The unidentified lipid in the toothfish lens epithelia corresponds to either an odd-chain (17 carbons) FA or a furanoid. We conclude that long-lived fishes are likely good animal models of lens crystallin solubility and may model post-translational modifications and solubility changes better than short-lived animal models.