Sequential pretreatment to recover carbohydrates and phosphorus from Desmodesmus sp. cultivated in municipal wastewater.

This study focused on the simultaneous recovery of carbohydrates (CHO) and phosphorus (P) from Desmodesmus sp. biomass cultivated in municipal wastewater, through a sequential pretreatment. The pretreatment consisted first of ultrasound to trigger cell disruption followed by ozonation to recover CHO and P. For ozone pretreatment, three different parameters were considered: ozone concentration (9, 15, 21, 27, 36, and 45 mg O3/L), contact time (15, 25 and 35 min), and pH (8 and 11). The maximum simultaneous release of 84% of CHO and 58% of P was achieved at the experimental parameters of ozone concentration of 45 mg O3/L, contact time of 35 min, and pH of 11. Also, P was concentrated in solution by 8- to 14-fold with respect to municipal wastewater. The sequential pretreatment was conducted at alkaline pH of 11 and atmospheric conditions, which may considerably reduce energy demand and reagents, in comparison to a traditional hydrolysis pretreatment. The results found suggest that the sequential pretreatment could be feasible on a large scale.

Intensified recovery of lipids, proteins, and carbohydrates from wastewater-grown microalgae Desmodesmus sp. by using ultrasound or ozone.

This study evaluates the effect of ultrasound and ozone pretreatments for the subsequent recovery of Desmodesmus sp. biocomponents-lipids, proteins, and carbohydrates-using a response surface methodology. Both pretreatments impact on the recovered lipids quality, solvent waste production and extraction time is analysed for process intensification purposes. For ultrasound pretreatment, independent parameters were energy applied (50-200 kWh/kg dry biomass), biomass concentration (25-75 g/L), and ultrasonic intensity (0.32 and 0.53 W/mL). While for ozone pretreatment, independent parameters were ozone concentration (3-9 mg O3/L), biomass concentration (25-75 g/L), and contact time (5-15 min). In the case of ultrasound pretreatment, recovery yield reached 97 ±â€¯0.4%, 89 ±â€¯3%, and 73 ±â€¯0.6% for proteins, carbohydrates and lipids respectively. Given process required: energy applied of 50 kWh/kg dry biomass, 75 g/L of biomass concentration, 0.32 W/mL of ultrasonic intensity, and 56 min of time process. Ultrasound caused high cell disruption releasing all proteins, thereby obviating downstream processing for its recovery. Ozone pretreatment recovery yield was 85 ±â€¯2%, 48 ±â€¯1.4%, and 25 ±â€¯1.3%, for carbohydrates, lipids and proteins respectively, under the following conditions: 9 mg O3/L of ozone concentration, 25 g/L of biomass concentration, and 5 min of contact time that depicts an energy consumption of 30.64 kWh/kg dry biomass. It was found that ultrasound and ozone pretreatments intensified the lysis and biocomponents recovery process by reducing solvent consumption by at least 92% and extraction time between 80% and 90% compared with extraction of untreated biomass biocomponents. Both pretreatments improve the composition of the recovered lipids. It was noted that the yield of neutral lipids increased from 28% to 67% for ultrasound pretreatment while for ozone pretreatment from 49% to 63%. The method used for lipid extraction may also have an effect but here it was kept constant.

The role of FOXP3+ regulatory T cells in human autoimmune and inflammatory diseases.

CD4+ regulatory T cells (Treg ) expressing the forkhead box protein 3 (FOXP3) transcription factor (Tregs ) are instrumental for the prevention of autoimmune diseases. There is increasing evidence that the human T regulatory population is highly heterogeneous in phenotype and function. Numerous studies conducted in human autoimmune diseases have shown that Treg cells are impaired either in their suppressive function, in number, or both. However, the contribution of the FOXP3+ Treg subpopulations to the development of autoimmunity has not been delineated in detail. Rare genetic disorders that involve deficits in Treg function can be studied to develop a global idea of the impact of partial or complete deficiency in a specific molecular mechanism involved in Treg function. In patients with reduced Treg numbers (but no functional deficiency), the expansion of autologous Treg cells could be a suitable therapeutic approach: either infusion of in-vitro autologous expanded cells, infusion of interleukin (IL)-2/anti-IL-2 complex, or both. Treg biology-based therapies may not be suitable in patients with deficits of Treg function, unless their deficit can be corrected in vivo/in vitro. Finally, it is critical to consider the appropriate stage of autoimmune diseases at which administration of Treg cellular therapy can be most effective. We discuss conflicting data regarding whether Treg cells are more effectual at preventing the initiation of autoimmunity, ameliorating disease progression or curing autoimmunity itself.

Photocatalytic reduction of Cr(VI) from agricultural soil column leachates using zinc oxide under UV light irradiation.

The photocatalytic reduction of Cr(VI) from agricultural soil leachates irrigated with Cr(VI)-containing waste hydroponic solution was evaluated in this work. For this purpose, zinc oxide was used as a catalyst under UV irradiation (lambda = 365 nm). The reduction of Cr(VI) was preliminarily evaluated on synthetic solutions with a concentration of 15 mg L(-1) to optimize the catalyst loading and the solution pH and to determine the effect of organic matter. Greater removal of Cr(VI) was observed at pH 7, and the optimum catalyst loading was found to be 2 g L(-1), which achieved an 84% Cr(VI) reduction in 6 h. The influence of dissolved organic matter on the reduction of Cr(VI) was evaluated through the addition of different concentrations of humic acid (HA) to the chromium solution. The removal of Cr(VI) was continuously enhanced as the HA concentration gradually increased from 0 to 14 mg L(-1). The percentage of hexavalent chromium reduction from soil leachates was in the range of 13-99%, and the rate constant was significantly enhanced by the presence of organic compounds in the soil pore water. Thus, a marked synergistic effect between the photocatalytic reduction of Cr(VI) and the organic matter in soil (e.g. humic substances) was observed in real samples and was similar to that observed in the Cr(VI) synthetic solution that contained HA.

Relation of serum cholesterol, lipid, serotonin and tryptophan levels to severity of depression and to suicide attempts.

OBJECTIVE: To determine if there is a relation to low serum cholesterol, lipoprotein, serotonin or tryptophan levels in patients with depression who have recently attempted suicide. DESIGN: Biochemical and behavioural study. SETTING: Inpatient and outpatient treatment at the Instituto Mexicano de Psiquiatría. PARTICIPANTS: Thirty-three patients with a diagnosis of major depressive episode. Eighteen of these patients had attempted suicide in the month before the start of the study; 15 patients had never attempted suicide. OUTCOME MEASURES: Serum levels of total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, serotonin (5-HT) and tryptophan. Scores on Hamilton Depression Rating Scale, Carroll Depression Rating Scale, Beck Hopelessness Scale and Beck Suicide Attempt Severity Scale. RESULTS: There were no significant differences between patients who had attempted suicide and those who had not in terms of serum cholesterol, HDL, LDL and triglyceride levels. Serum levels of 5-HT and tryptophan were significantly lower in patients with depression who had a recent suicide attempt than in those patients who had never attempted suicide. A comparison of patients not taking antidepressant medication found serum 5-HT levels to be more than 3 times lower in those patients with a recent suicide attempt than in patients with no history of suicide attempt. CONCLUSIONS: The study found no difference in lipid profiles between patients who had attempted suicide and those who had not. Low serum levels of 5-HT may increase the risk of suicide attempt in patients who are depressed.

Incomplete T cell receptor V beta allelic exclusion and dual V beta-expressing cells.

Recent studies have documented incomplete TCR V alpha-chain allelic exclusion and dual V alpha-bearing T cells. Herein, we show that V beta allelic exclusion is also incomplete, since a significant proportion of peripheral T cells express dual V beta in both TCR transgenic and normal mice. Studies in TCR transgenic mice indicated that although a small proportion of T cells escaped allelic exclusion in the thymus, dual V beta-expressing cells expanded dramatically in the periphery with age, and such expanded cells had an activated phenotype. Although not as pronounced, age-related increases in dual V beta-bearing cells were also observed in normal mice. These findings may have important implications for TCR selection and specificity, age-related repertoire changes, and autoimmune disease pathogenesis.

Human T helper cells specific for HIV reverse transcriptase: possible role in intrastructural help for HIV envelope-specific antibodies.

Cooperation between B cells specific for an antigen exposed on a viral structure and T helper (Th) cells specific for an internal antigen, as demonstrated with influenza, hepatitis B and rabies viruses, has been termed intrastructural help. Th cells specific for internal proteins of HIV, which are much less mutated than its exposed antigens, may be valuable in vaccine design against this virus. We investigated the human Th repertoire specific for the core HIV antigen reverse transcriptase (p66), and determined whether these cells could be candidate intrastructural T helpers. CD4+ T lines and clones were generated from non-immune individuals by stimulation with p66-pulsed antigen-presenting cells (APC). Specific lines were obtained with p66 from 19 out of 21 (90%) of these individuals, vs. 7 out of 29 (24%) with gp120. Diverse epitopes were recognized by different individuals, and various V beta genes were used by these clones. Clones using the same V beta genes were of diverse origin, according to VDJ region sequence. Of these lines 45% responded to p66 in the context of HIV virions. Moreover, p66-specific clones could respond to APC that had internalized HIV complexed with envelope-specific monoclonal antibodies, suggesting that p66-specific Th cells may participate in intrastructural help. These studies indicate that p66-specific Th cells are detectable in vitro in most naive individuals and exhibit clonal heterogeneity, and that the majority recognize an HIV conserved antigen. They respond to p66 following processing of whole virions and are clearly candidates for intrastructural help. If confirmed in vivo, p66 should be included among vaccine candidates investigated to optimize the anti-HIV Th response.

Recognition of human T-leukemia virus (HTLV-1) envelope by human CD4+ T-cell lines from HTLV-1 seronegative individuals: specificity and clonal heterogeneity.

Because T-helper cells are critical for immune responses in retroviral infections, CD4+ T-cell lines specific for the human T-leukemia virus type 1 (HTLV-1) envelope have been generated from peripheral T lymphocytes of nonimmune donors to study their naive repertoire. Recombinant fragments (RE1, amino acids [aa] 26-200; RE3, aa 165-307; RE5, aa 308-401; and RE6, aa 165-401) of HTLV-1 envelope, whole envelope glycoprotein, and synthetic peptides were used to induce T-cell lines. CD4+ T-cell lines specific for one or more fragments were obtained from seven of eight individuals tested. T-cell lines generated against envelope glycoprotein from five of five donors did not cross-react with the RE fragments and vice versa. The lines specific for RE and env were mapped with overlapping peptides. The lines with single peptide (narrow) specificity contained a variety of clones that used different T-cell receptor V beta genes. These data (1) suggest that most of the normal individuals carry T-helper precursors specific for epitopes on HTLV-1 envelope; (2) indicate that heterogeneity of HTLV-1 envelope-specific T cells can be detected in the naive repertoire; and (3) define optimal antigenic preparations to be used to assess cellular immunity in HTLV-1-infected individuals.

V beta gene repertoire in the aging mouse: a developmental perspective.

To define age-associated alterations in the immune system at the molecular level, we have analyzed TCR V beta gene expression patterns at the fetal, neonatal, adult, and advanced ages of mice. In contrast to V gamma and VH genes, V beta genes rearranged without any preference related to their chromosomal organization. Endogenous superantigen-mediated clonal deletions were registered for the first time at the neonatal stage, presumably reflecting the late developmental appearance of these molecules. Such deletions, once established, were maintained throughout life with little, if any, leakage in this process. Furthermore, bone marrow transplantation and other studies indicated that an involuted thymus maintained its capacity to perform both its functions, i.e. positive and negative selection. Although overall V beta repertoires showed remarkable stability with advanced age, modifications in expression levels for some V beta, particularly those associated with the CD8 subset and presumably reflecting antigenic stimulation, were recorded. Mice with lupus and early-life thymic involution were fully capable of deleting endogenous superantigen-reactive V beta clones, and even lupus mice with a genetic defect in the apoptosis-promoting Fas gene were normal in this regard. The results indicate that, aside from some anticipated clonal expansions induced by antigenic stimulation, age-associated alterations in immune functions are not caused by any profound changes in the overall TCR repertoire.

Lupus susceptibility loci in New Zealand mice.

Susceptibility to systemic lupus erythematosus has been unequivocally established to be an inherited trait, but the exact genes and how they confer susceptibility remain largely unknown. In this study of (NZB x NZW)F2 intercross mice, we used linkage analysis of markers covering > 90% of the autosomal genome and identified eight susceptibility loci (Lbw1 to -8, chromosomes 17, 4-7, 18, 1, 11, respectively) associated with antichromatin autoantibody production, glomerulonephritis, and/or mortality. Only one locus, the major histocompatibility complex, was linked to all three traits. Two other loci were associated with both glomerulonephritis and mortality, whereas the remaining loci were linked to one of the above traits. Two additional loci (Sbw1 and -2) that contributed to splenomegaly were also identified. The Sbw2 locus mapped to the identical region as Lbw2, a locus on chromosome 4 linked to glomerulonephritis and mortality, suggesting a single locus with pleiotropic effects. The results indicate that the immunopathologic features of lupus are affected by distinct, but additive, genetic contributions. Studies to determine the nature of the genes associated with these loci should help define the genetic mechanisms involved in this systemic autoimmune disease.

Crystal structure of a peptide complex of anti-influenza peptide antibody Fab 26/9. Comparison of two different antibodies bound to the same peptide antigen.

The three-dimensional structure of the complex of a second anti-peptide antibody (Fab 26/9) that recognizes the same six-residue epitope of an immunogenic peptide from influenza virus hemagglutinin (HA1; 75-110) as Fab 17/9 with the peptide has been determined at 2.8 A resolution. The amino acid sequence of the variable region of the 26/9 antibody differs in 24 positions from that of 17/9, the first antibody in this series for which several ligand-bound and free structures have been determined and refined. Comparison of the 26/9-peptide with the 17/9-peptide complex structures shows that the two Fabs are very similar (r.m.s.d. 0.5 to 0.8 A) and that the peptide antigen (101-107) has virtually the same conformation (r.m.s.d. 0.3 to 0.8 A) when bound to both antibodies. A sequence difference in the 26/9 binding pocket (L94; His in 26/9, Asn in 17/9) results in an interaction with a bound water molecule that is not seen in the 17/9 structures. Epitope mapping shows that the relative specificity of 26/9 and 17/9 antibodies for individual positions of the peptide antigen are slightly different. Amino acid substitutions in the peptide, particularly at position SerP107, are tolerated to different extents by 17/9 and 26/9. Structural and sequence analysis suggests that amino acid differences near the peptide-binding site are responsible for altering slightly the specificity of 26/9 for three peptide residues and illustrates how amino acid substitutions can modify antibody-antigen interactions and thereby modulate antibody specificity.

V beta repertoire of murine hepatic T cells. Implication for selection of double negative alpha beta + T cells.

To further define the origin, selection, and diversity of hepatic T cells, we have determined V beta gene expression profiles in double negative (DN, CD4-8-) and single positive (SP, CD4+8- or CD8+4-) alpha beta + liver T cells of DBA/2 mice. These I-E+ mice express mouse mammary tumor (Mtv) provirus-encoded endogenous superantigens of the Mlsa,c type, and thus display deletions/depletions of several V beta-bearing SP cells. Total liver alpha beta + T cells of these mice exhibited an overall V beta expression profile similar to splenic T cells, with the notable exception of high V beta 7 and V beta 8.1 expression. As previously reported, DN alpha beta + T cells were enriched highly in the liver. This subset exhibited a V beta expression profile similar to thymic DN alpha beta + cells with deletions/depletions in several V beta s, but high V beta 7 expression in both populations. Surprisingly, hepatic CD4+ cells also displayed high V beta 7 expression compared with splenic T cells, suggesting that hepatic DN alpha beta + and CD4+ T cells are selected via a common pathway. The V beta 7-expressing DN alpha beta + and CD4+ liver T cell populations were polyclonal, as evidenced by cloning and sequencing. High V beta 7 expression in these cells was undiminished with age. On the basis of V beta repertoire and surface phenotype, DN alpha beta + and/or certain CD4+ T cells seem to constitute a distinct population primarily found in the liver, thymus, and bone marrow. These cells may originate from SP T cells that have down-regulated their accessory molecules under certain activation conditions and, because of the accompanying expression of particular adhesion molecules, they accumulate in tissues such as the liver and thymus.

T cell receptor V beta 15 dominates the antiacetylcholine receptor response in Lewis rat T cell lines.

Ten Lewis rat T cell lines responsive to Torpedo californica acetylcholine receptor (AChR) were assayed for TCR V beta usage. All lines were CD4+, OX-22-, and exhibited reactivity to one or more AChR chains. Several different V beta s were expressed by these lines, but V beta 15 was dominant in 5 of 10 lines. Unique CDR3 sequences were observed among the 10 lines, although three of the V beta 15 rearrangements used J beta 1.4. These data suggest that V beta 15+ T cells are selected in the in vitro response to the antigenically complex AChR in the Lewis rat.

V beta gene repertoires in T cells expanded in local self-healing and lethal systemic murine cutaneous leishmaniasis.

Inbred mice infected with Leishmania major promastigotes display two different courses of leishmaniasis: resistant strains develop self-healing local sores, while susceptible strains show progressive systemic disease with lethal outcome. Resistance predominantly correlates with the production of T helper type 1 (TH1) lymphokines and susceptibility with production of TH2-type lymphokines. Here, we analyzed whether this TH phenotype difference correlates with expression of particular T cell receptor V beta chains. Our results show that T cells expand strongly during infection in all groups of mice and invariantly express the same V beta gene families as prior to infection. Our data indicate that TH1 and TH2 cells use similar V beta gene families, and argue against the engagement of a restricted set of V beta by dominant determinants associated with L. major.

T-cell receptor genes in autoimmunity.

T cells are primary participants in the pathogenesis of the MHC-dependent autoimmune diseases, and therefore, evidence for association of TCR V-gene repertoires with such disorders has been actively sought. With very few exceptions, no clear-cut evidence for correlation of particular RFLP-defined V-C-region genomic polymorphisms with autoimmune disease predisposition has thus far been demonstrated. With regard to TCR V-gene repertoires engaged in responses to autoantigens, restricted use of certain V beta and V alpha genes in response to myelin basic protein has been documented in animal models. In many spontaneous and experimentally induced animal and human autoimmune diseases, however, the picture is far from clear. Although dominance of certain TCR V genes has been noted, the clonal restrictions are not absolute; they differ from one study to another and from one patient to another. Such variations may be caused by MHC allele-dependent determinant selection mechanisms, secondary T-cell infiltrates in inflammatory sites, different patient populations and stages of disease, or the involvement of different pathogens that, nevertheless, lead to the same clinical entity. Overall, the results indicate that efforts to intervene therapeutically in autoimmune diseases by vaccination with modified T-cell clones, V region-synthetic peptides, or TCR blocking analogues may not be easily applicable. Further studies on the characterization of the specific antigens involved in autoimmune disease pathogenesis is required in order to accurately address the issue of TCR utilization in autoimmune diseases.

Genomic composition and allelic polymorphisms influence V beta usage by the Mycoplasma arthritidis superantigen.

The Mycoplasma arthritidis superantigen (MAM) is produced by an organism that causes systemic disease in rodents leading to chronic proliferative arthritis. MAM is a typical superantigen that requires presentation to T cells by MHC molecules without processing and T cell recognition of MAM occurs through the V beta chains of the TCR. Several major findings are presented here. First, different MAM-MHC class II isotype complexes may engage different sets of V beta TCR. Thus, activation of V beta 6- and V beta 8.3-bearing T cells is more dependent upon the I-E molecule of the murine H-2 MHC than is activation of cells bearing the V beta 5.1, 8.1, and 8.2 TCR. Secondly, both genomic composition and allelic polymorphisms at the V beta chain segment of the TCR exert profound effects upon the pattern of V beta that are used by MAM. Thus, in V beta b haplotype mice, MAM engages V beta 5.1, 6, and the V beta 8 family of TCR whereas in V beta a (C57BR) and V beta c (RIIIS) haplotype mice that lack various combinations of these V beta, activation of cells bearing V beta 1, 3.1, 7, and 16 can be demonstrated. These differences in V beta usage by MAM appear to be caused by both differences in the avidity of MAM for the various V beta s and to structural allelic polymorphisms in these V beta. Clonal expansion of specific V beta in vivo after injection of MAM is also dependent upon the genomic composition of the mice, because expansion of the V beta 8 TCR seen in V beta b haplotype mice (B10.RIII) whereas marked expansion of V beta 6 is seen in V beta a mice (C57BR). In as much as these TCR have been implicated in a number of experimental autoimmune diseases, MAM may represent an ideal model to evaluate the role of superantigens in the triggering of autoimmune disease.

Genesis and evolution of antichromatin autoantibodies in murine lupus implicates T-dependent immunization with self antigen.

Autoantibodies reacting with chromatin and its components, histones and DNA, are characteristic of the human autoimmune disease SLE and drug-induced lupus, but the mechanisms of their induction remain unknown. Serial serum samples collected over short intervals from lupus-prone MRL/MP-lpr/lpr and BXSB mice were tested by ELISA on chromatin and its substructures to characterize the initial autoimmune response to these antigens. Direct binding studies demonstrated that the early autoantibodies recognized discontinuous epitopes on native chromatin and the (H2A-H2B)-DNA subnucleosome. As the immune response progressed, native DNA and other chromatin constituents generally became antigenic. Based on adsorption studies and IgG subclass restriction, antibodies to native DNA were more related to chromatin than to denatured DNA. The kinetics of autoantibody appearance and the Ig class distribution were similar to the kinetics and distribution seen in antibodies induced by immunization with an exogenous T-dependent antigen. These results are most consistent with the view that autoantibodies reacting with chromatin are generated by autoimmunization with chromatin, and antibodies to native DNA are a subset of the wide spectrum of antichromatin autoantibodies.

Application of a multiprobe RNase protection assay and junctional sequences to define V beta gene diversity in Sezary syndrome.

Nineteen patients with mycosis fungoides/Sezary syndrome (MF/SZ), a malignancy of the mature helper T-cell phenotype (CD4+TCR alpha beta+), were screened for clonotypic V beta expansions in peripheral blood with a multiprobe RNase protection assay. A different predominant V beta gene was identified in 9 of 14 patients with high peripheral blood CD4/CD8 ratios, whereas 4 of these patients showed T-cell expansions expressing V beta genes other than those included in the assay. In contrast, five patients with few, if any, malignant cells in the circulation had V beta expression levels similar to that in normal peripheral blood. A unique V-D-J sequence was found for each highly expressed V beta gene, thereby documenting monoclonality of the expanded T-cell populations. Polymerase chain reaction (PCR) primers specific for the D-J beta junction accurately identified the corresponding malignant clonotype in peripheral blood. The diverse TCR V beta gene usage found in these MF/SZ patients suggests that T-cell receptor (TCR) specificity has no bearing on this disease.

V beta repertoire in rats and implications for endogenous superantigens.

Endogenous superantigens of mice, encoded by mammary tumor virus proviral integrants, induce intrathymic deletion of entire T cell populations that express specific V beta gene products, a phenomenon proposed to be important in self-tolerance and prevention of toxic responses to exogenous microbial superantigens. Evidence for the presence of V beta selecting/deleting endogenous superantigens in other species is lacking. We report here that rats do not exhibit endogenous superantigen-induced V beta clonal deletions despite their strong response to bacterial superantigens. These findings indicate that endogenous superantigens are not obligatory in V beta repertoire shaping.