IL-13 production by regulatory T cells protects against experimental autoimmune encephalomyelitis independently of autoantigen

Treatment with an anti-inflammatory Salmonella vaccine expressing enterotoxigenic Escherichia coli colonization factor Ag 1 (CFA/I) proved effective in stimulating protective, potent CD25(+)CD4(+) regulatory T (T(reg)) cells in susceptible mice challenged with experimental autoimmune encephalomyelitis (EAE). Because the Salmonella vector was considerably less protective, we questioned whether altering fimbrial subunit expression to resemble conventional Salmonella expression may impact T(reg) cell potency. The Salmonella-CFA/I vaccine was modified to limit fimbrial subunit expression to the intracellular compartment (Salmonella-CFA/I(IC)). SJL mice were challenged with proteolipid protein peptide 139-151 to induce EAE and orally treated with one of three Salmonella vaccines 6 days postchallenge. Treatment with Salmonella-CFA/I(IC) greatly reduced clinical disease, similarly as Salmonella-CFA/I, by subduing IL-17 and IL-21; however, mechanisms of protection differed as evident by increased IL-13 and IFN-gamma but diminished TGF-beta production by T(reg) cells from Salmonella-CFA/I(IC)-treated mice. Adoptive transfer of T(reg) cells from both CFA/I-expressing constructs was equivalent in protecting against EAE, showing minimal disease. Although not as potent in its protection, CD25(-)CD4(+) T cells from Salmonella-CFA/I(IC) showed minimal Th2 cells, but vaccination did prime these Th2 cells rendering partial protection against EAE challenge. In vivo IL-13 but not IFN-gamma neutralization compromised protection conferred by adoptive transfer with Salmonella-CFA/I(IC)-induced T(reg) cells. Thus, the Salmonella-CFA/I(IC) vaccine elicits T(reg) cells with attributes from both the Salmonella vector and Salmonella-CFA/I vaccines. Importantly, these T(reg) cells can be induced to high potency by simply vaccinating against irrelevant Ags, offering a novel approach to treat autoimmune diseases independently of the autoantigen.     

Low-dose tolerance is mediated by the microfold cell ligand, reovirus protein sigma1

Mucosal tolerance induction generally requires multiple or large Ag doses. Because microfold (M) cells have been implicated as being important for mucosal tolerance induction and because reovirus attachment protein sigma1 (psigma1) is capable of binding M cells, we postulated that targeting a model Ag to M cells via psigma1 could induce a state of unresponsiveness. Accordingly, a genetic fusion between OVA and the M cell ligand, reovirus psigma1, termed OVA-psigma1, was developed to enhance tolerogen uptake. When applied nasally, not parenterally, as little as a single dose of OVA-psigma1 failed to induce OVA-specific Abs even in the presence of adjuvant. Moreover, the mice remained unresponsive to peripheral OVA challenge, unlike mice given multiple nasal OVA doses that rendered them responsive to OVA. The observed unresponsiveness to OVA-psigma1 could be adoptively transferred using cervical lymph node CD4(+) T cells, which failed to undergo proliferative or delayed-type hypersensitivity responses in recipients. To discern the cytokines responsible as a mechanism for this unresponsiveness, restimulation assays revealed increased production of regulatory cytokines, IL-4, IL-10, and TGF-beta1, with greatly reduced IL-17 and IFN-gamma. The induced IL-10 was derived predominantly from FoxP3(+)CD25(+)CD4(+) T cells. No FoxP3(+)CD25(+)CD4(+) T cells were induced in OVA-psigma1-dosed IL-10-deficient (IL-10(-/-)) mice, and despite showing increased TGF-beta1 synthesis, these mice were responsive to OVA. These data demonstrate the feasibility of using psigma1 as a mucosal delivery platform specifically for low-dose tolerance induction.     

Seasonal changes in cold tolerance,water relations and accumulation of cations and compatible solutes in Atriplex halimus L.

We determined the cold (freezing) tolerance for field-grown plants of Atriplex halimus L. (Chenopodiaceae) in relation to plant ploidy level, leaf water relations and accumulation of osmolytes. Plants were grown at two sites in Murcia (Spain), having average minimum temperatures in the coldest month of 0.6 and 12.1 °C, respectively. LT₅₀ values derived from laboratory freezing tests, using leaves taken from the plants in early winter and in spring, showed greater tolerance for winter-harvested leaves; the acclimation was more pronounced at the cold-winter site. Cold tolerance was related positively with leaf K and/or Na accumulation. Analysis of compatible organic solutes (soluble sugars, total amino acids and quaternary ammonium compounds) showed that cold tolerance (measured both as LT₅₀ and as winter freezing damage in situ) was related most closely with leaf concentrations of soluble sugars. The leaf percentage dry matter content was related to both in vitro and in vivo tolerance, while tolerance in vitro was correlated also with the osmotic (potential ψ_s) and the relative water content. The two diploid (2n = 2x = 18) populations, from Spain, showed greater cold tolerance than the three tetraploid (2n = 4x = 36) populations, from North Africa and Syria, which may be related to the latter's greater cell size and consequent dilution of osmolytes. In this halophytic species, cold tolerance, like salinity and drought tolerance, seems to depend on osmotic adjustment, driven by vacuolar accumulation of K and Na and cytoplasmic accumulation of compatible solutes.     

Structural analysis of the glycosylation of gene-activated erythropoietin (epoetin delta, Dynepo)

Recently, a novel recombinant human erythropoietin (epoetin delta, Dynepo) has been marketed in the European Union for the treatment of chronic kidney disease, cancer patients receiving chemotherapy, and so forth. Epoetin delta is engineered in cultures of the human fibrosarcoma cell line HT-1080 by homologous recombination and “gene activation.” Unlike recombinant erythropoietins produced in other mammalian cells, epoetin delta is supposed to have a human-type glycosylation profile. However, the isoelectric focusing profile of epoetin delta differs from that of endogenous erythropoietin (both urinary and plasmatic). In this work, structural and quantitative analysis of the O- and N-glycans of epoetin delta was performed and compared with glycosylation from recombinant erythropoietin produced in Chinese hamster ovary (CHO) cells. From the comparison, significant differences in the sialylation of O-glycans were found. Furthermore, the N-glycan analysis indicated a lower heterogeneity from epoetin delta when compared with its CHO homologue, being predominantly tetraantennary without N-acetyllactosamine repeats in the former. The sialic acid characterization revealed the absence of N-glycolylneuraminic acid. The overall sugar profiles of both glycoproteins appeared to be significantly different and could be useful for maintaining pharmaceutical quality control, detecting the misuse of erythropoietin in sports, and establishing new avenues to link glycosylation with biological activity of glycoproteins.     

Seasonal and interannual cycles of endemic cholera in Bengal 1891–1940 in relation to climate and geography

The temporal distribution of chlorophyll a (chl a), suspended matter (SM), and vertical flux of chl a and organic carbon (OC) has been investigated at three sites along the north-eastern coast of the Gulf of Aqaba during the period from January 1991 through December 1992. Highly significant temporal and interannual variations were found in chl a, and in the vertical flux of chl a and OC. The SM and its OC content did not show any significant temporal or interannual variations. The recorded levels of the forementioned variables were generally low. The temporal distribution of chl a in the water column was polymodal in 1991 with peaks in March, June, August and December, and bimodal in 1992 with peaks in May–June, and October. Maximum values of SM and its OC content occurred in July of 1991 and September of 1992. The temporal variations in the vertical flux of total particles, chl a, and OC followed those of chl a in the water column. The bulk of the sediment material was of inorganic nature, derived from desert sand carried from Wadi Araba by the prevailing northerly winds, and dust of exported raw phosphate. The temporal changes described appear to be related to the temporal variations in water stability, horizontal advection, and winds.     

Variation in Resistance to High Hydrostatic Pressure and rpoS Heterogeneity in Natural Isolates of Escherichia coli O157:H7

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.     

Solubilization of immune precipitates by complement in the absence of properdin or factor D

Various experiments have demonstrated that immune precipitates (IPs) are not solubilized by complement in the absence of alternative pathway function. To determine whether the characteristics of the IPs were responsible for these observations, we studied the solubilization (Sol) of IPs formed by bovine serum albumin (BSA)-rabbit antiBSA and tetanus toxoid (TT)-human antiTT. Sera deficient in properdin solubilized a fraction of BSA-antiBSA precipitates, although only when the IPs were formed in antibody excess. The same sera solubilized TT-antiTT precipitates with some delay but almost as efficiently as normal serum. Factor D-depleted serum solubilized a fraction of TT-antiTT precipitates too, indicating that Sol may proceed through activation of the classical pathway only. Thus, the requirements for complement-mediated Sol depend on the characteristics of the IPs and do not necessarily include alternative pathway function.     

Composition of citrus pulp

Eight groups of citrus varieties grown in Spain were included in this study. Citrus pulp composition was (dry matter basis) 60–65% peel, 30–35% segment pulp and 0–10% seeds. Citrus seeds had a high percentage of protein, ether extract and crude fibre. Segment pulp contained slightly less fibre than citrus pulp. The proximate composition of citrus pulp was similar to that of the peel fraction and did not differ from previous results. Significant differences in fibre and acid detergent lignin of the dried citrus pulp samples were found between varieties.The drying process was studied. Temperatures over 130°C and the addition of calcium hydroxide affected some fractions of the pulp, mainly by increasing ash and fibre contents.