Impaired affinity maturation in Cr2-/- mice is rescued by adjuvants without improvement in germinal center development

Cr2-/- mice have an impairment in humoral immunity, as shown by the decrease in the Ab titers against T cell-dependent Ags and abnormalities in germinal center formation. Germinal centers are present, but they are decreased in size and number, indicating problems in their development. In this study, we investigated whether this abnormality in germinal center development is associated with problems in the establishment of optimal affinity maturation and the generation of memory B cells, processes closely related to the germinal center reaction. We immunized the Cr2-/- animals with different Ags with or without adjuvants. We showed that, when immunized without adjuvants, complement receptors are absolutely required for optimal affinity maturation. Although limited affinity maturation is elicited in the Cr2-/- Ab response, it is decreased as compared with normal animals. Memory B cell generation is also impaired. In the presence of adjuvants, germinal center development in the Cr2-/- mice is still abnormal, as demonstrated by their decreased size and number. Surprisingly, adjuvants establish optimal affinity maturation and partially restore the amount of Ab produced during the primary response and memory B cell generation. However, adjuvants cannot improve the ability of follicular dendritic cells to retain Ags in the form of immune complexes. These observations indicate that immunization with inflammatory Ags offset some of the immunological abnormalities found in the Cr2-/- mice and show that optimal affinity maturation in the Cr2-/- mice can be achieved in the absence of normal germinal centers.     

Genetic diversity among isolates of Trichinella spiralis from the Province of Buenos Aires, Argentina

Random Amplified Polymorphic DNAs, (RAPDs) are used to study the occurrence of Trichinella britovi and T5 among domestic animals in the Province of Buenos Aires, Argentina and to assess the genetic diversity among isolates of T. spiralisfrom this area in a number of infected hosts. All the local isolates proved to be T. spiralis. Six of the eight primers used indicate that the Buenos Aires isolates are distinct from each other as they produce a considerable number of polymorphic bands. Our overall estimates are relatively higher than other intraspecific distances previously estimated within species of this genus and among T. spiralis isolates. Such high degrees of variability observed among local isolates and between isolates from Buenos Aires and Spain should be taken into account when defining isolates within this species, and considering differences in the epidemiology of T. spiralis.     

Regulatory mechanisms for modulation of signaling through the cell integrity Slt2-mediated pathway in Saccharomyces cerevisiae

Signal transduction mediated by the mitogen-activated protein kinase (MAPK) Slt2 pathway is essential to maintain the cell wall integrity in Saccharomyces cerevisiae. Stimulation of MAPK pathways results in activation by phosphorylation of conserved threonine and tyrosine residues of MAPKs. We have used an antibody that specifically recognizes dually phosphorylated Slt2 to gain insight into the activation and modulation of signaling through the cell integrity pathway. We show that caffeine and vanadate activate this pathway in the absence of osmotic stabilization. The lack of the putative cell surface sensor Mid2 prevents vanadate- but not caffeine-induced Slt2 phosphorylation. Disruption of the Rho1-GTPase-activating protein genes SAC7 and BEM2 leads to constitutive Slt2 activation, indicating their involvement as negative regulators of the pathway. MAPK kinases also seem to participate in signaling regulation, Mkk1 playing a greater role than Mkk2 in signal transmission to Slt2. Additionally, one of the phosphatases involved in Slt2 dephosphorylation is likely to be the dual specificity phosphatase Msg5, since overexpression of MSG5 in a sac7Delta mutant eliminates the high Slt2 phosphorylation, and disruption of MSG5 in wild type cells results in increased phospho-Slt2 levels. These data present the first evidence for a negative regulation of the cell integrity pathway.     

Horseradish peroxidase binding to intestinal brush-border membranes of Cyprinus carpio. Identification of a putative receptor

Morphologic studies have shown that the classic endocytosis tracer horseradish peroxidase (HRP) is actively internalized by vesicular transport in the carp intestine, suggesting the existence of specific binding sites in the apical membrane of enterocytes. The aim of the present study was to develop an in vitro binding assay using isolated carp intestinal brush-border membranes (BBM) to demonstrate and characterize these specific HRP binding sites. The results obtained show that HRP binding to BBM exhibits a saturable mode and high affinity (K(d) = 22 nM). In addition, HRP binding sites are highly enriched in BBM compared to basolateral membranes. On the other hand, HRP interaction with these sites is apparently of an ionic character because binding increased concomitantly with decreasing NaCl concentrations in the assay, reaching a maximum in the absence of NaCl. Other proteins that are also internalized in carp intestine did not significantly inhibit HRP binding to BBM. A lectin-type of interaction was discarded because neither manan nor ovoalbumin inhibited HRP binding. Proteinase K treatment of BBM reduced HRP binding by 70%, suggesting a proteic nature for this binding site. Finally, ligand blotting assays showed that HRP binds specifically to a 15.3-kDa protein. Taken together, these results are consistent with the existence of a functional receptor for HRP in carp intestinal mucosa that could mediate its internalization.     

Carbon dioxide uptake efficiency by outdoor microalgal cultures in tubular airlift photobioreactors

The influence of solar irradiance and carbon dioxide molar fraction of injected CO(2)-air mixtures on the behavior of outdoor continuous cultures of the microalga Phaeodactylum tricornutum in tubular airlift photobioreactors was analyzed. Instantaneous solar irradiance, pH, dissolved oxygen, temperature, biomass concentration, and the mass flow rates of both the inlet and outlet oxygen and carbon with both the liquid and gas phases were measured. In addition, elemental analysis of the biomass and the cell-free culture medium was performed. The oxygen production rate and carbon dioxide consumption rate increased hyperbolically with the incident solar irradiance on the reactor surface. Carbon losses showed a negative correlation with the daily variation of the carbon dioxide consumption rate. The maximum CO(2) uptake efficiency was 63% of the CO(2) supplied when the CO(2) concentration in the gas supplied was 60% v/v. Carbon losses were >100% during the night, due to CO(2) production by respiration, and hyperbolically decreased to values of 10% to 20% in the midday hours. An increase in the carbon fixed in the biomass with the solar cycle was observed. A slight daily decrease of carbon content of the cell-free culture medium indicated the existence of carbon accumulation in the culture. A decrease in CO(2) molar fraction in the injected gas had a double benefit: first, the biomass productivity of the system was enhanced from 2.05 to 2.47 g L(-1) day(-1) by reduction of CO(2) inhibition and/or pH gradients; and second, the carbon losses during the daylight period were reduced by 60%. The fluid dynamics in the reactor also influenced the carbon losses: the higher the liquid flow rate the higher the carbon losses. By using a previous mass transfer model the experimental results were simulated and the usefulness of this method in the evaluation and scale-up of tubular photobioreactors was established.     

Craniofacial shortening by contraction osteogenesis: an experimental model

Application of gradual external forces to correct craniofacial deformities challenges many procedures in conventional craniomaxillofacial surgery. Distraction osteogenesis is replacing traditional osteotomies for correction of patients with craniomaxillofacial deficiencies. However, the reverse concept, contraction osteogenesis, has yet to be established for patients with craniomaxillofacial excesses. The purpose of this investigation is to demonstrate the contraction osteogenesis phenomenon applied in a controlled animal model during the craniofacial growth period. Twenty-six 26-day-old rabbits were assigned to one of four groups: 0, control; 1, pin control (pin insertion); 2, no contraction (pins and contraction device application, without active contraction); and 3, contraction (pin insertion, contraction device application, and active contraction). An external fixator was placed across the incisive-maxillary suture, and the effects after 4.5 weeks of contraction at a rate of 0.5 mm twice a week were compared with control groups. The results were assessed by craniometric and cephalometric measurements and by histologic examination. Gross alterations were evident in the contraction group, characterized by midface anteroposterior shortening, maxillary regression, snout deviation, and anterior crossbite. Histologic examination of the contraction group demonstrated a significant increase in osteoblastic activity. Contraction osteogenesis is a new treatment concept in craniofacial development and may offer therapeutic opportunities for shortening skeletal structures without the need of osteotomies, thus taking advantage of the potential of craniofacial growth and remodeling.     

Differential effects of hemorrhage and LPS on tissue TNF-alpha, IL-1 and associate neuro-hormonal and opioid alterations

LPS administration and hemorrhage are frequently used models for the in vivo study of the stress response. Both challenges stimulate cytokine production as well as activate opiate and neuro-endocrine pathways; which in turn modulate the inflammatory process. Differences in the magnitude and tissue specificity of the proinflammatory cytokine and neuro-hormonal responses to these stressors are not well established. We contrasted the tissue specificity and magnitude of the increase in circulating and tissue cytokine (TNF-alpha, IL-1alpha and IL-1beta) content in response to either fixed-pressure hemorrhage (approximately 40 mm Hg) followed by fluid resuscitation (HEM) or lipopolysaccharide (LPS; 100 microg/100 g BW) administration. LPS and HEM elevated circulating levels of TNF-alpha, while neither stress altered circulating IL-1-alpha and IL-beta. LPS-induced increases in TNF-alpha content were greater than those elicited by HEM in all tissues studied except for the lung, where both stressors produced similar increases. Tissue (lung, spleen and heart) content of IL-1alpha was increased by HEM but was not affected by LPS. Tissue (lung, spleen, and heart) content of IL-1beta was increased by LPS but was not affected by HEM. HEM produced greater increases than LPS in epinephrine (16- vs. 4-fold) and norepinephrine (4-fold vs. 60%) levels and similar elevations in beta-endorphin. LPS produced greater elevation in corticosterone levels (2-fold) than HEM (50%). These results suggest differential tissue cytokine modulation to HEM and LPS, both with respect to target tissue and cytokine type. The hormonal milieu to HEM is characterized by marked catecholaminergic and moderate glucocorticoid while that of LPS is characterized by marked glucocorticoid with moderate catecholaminergic influence.     

A process for high yield and scaleable recovery of high purity eicosapentaenoic acid esters from microalgae and fish oil

A low expense process is developed for recovering esterified eicosapentaenoic acid (EPA) from microalgae and fish oil. Over 70% of the EPA content in the esterified crude extract of microalgae were recovered at purities exceeding 90%. The recovery scheme utilizes either wet or freeze-dried algal biomass. The process consists of only three main steps: 1) simultaneous extraction and transesterification of the algal biomass; 2) argentated silica gel column chromatography of the crude extract; and 3) removal of pigments by a second column chromatographic step. Argentated silica gel chromatography recovered about 70% of the EPA ester present in the crude fatty ester mixture of fish oil, but at a reduced purity ( approximately 83% pure) compared to the microalgal derived EPA. The optimal loading of the fatty ester mixture on the chromatographic support was about 3% (w/w) but loadings up to 4% did not affect the resolution significantly. The process was scaled up by a factor of nearly 320 by increasing the diameter of the chromatography columns. The elution velocity remained constant. Compared to the green alga Monodus subterraneus, the diatom Phaeodactylum tricornutum had important advantages as a potential commercial producer of EPA. For a microalgal EPA process to be competitive with fish oil derived EPA, P. tricornutum biomass (2.5% w/w EPA) needs to be obtained at less than $4/kg. If the EPA content in the alga are increased to 3.5%, the biomass may command a somewhat higher price. The quality of microalgal EPA compares favorably with that of the fish oil product. Compared to free fatty acid, EPA ester is more stable in storage. Shelf-life is extended by storing in hexane. The silver contamination in the final purified EPA was negligibly small (<210 ppb).     

A study of factors that may influence the determination of copper, iron, and zinc in human milk during sampling and in sample individuals

The aim of this study was to establish the possible effects of the sampling protocol (between-breast, within-feed, and diurnal differences) and the mother’s personal factors (age, parity, iron supple-mentation, smoking habits, and lactation period) on the copper, iron, and zinc contents in human milk. One hundred thirty-six human milk samples identified by their origin and sampling conditions were analyzed. The samples were obtained from the 2nd to 15th d postpartum from 62 women. The data on the individuals required for the study were available. Mineral determinations were analyzed by flame atomic absorption spectrometry following a standarized protocol. The results showed that iron contents were higher in hind-milk samples and at the nighttime feeding and depended on the breast from which the sample was taken. The copper and zinc concentrations showed no significant variations. There was no significant relationship among the mothers’ age, parity, smoking habits, iron supplementation, and copper content. Milk from older women had lower zinc contents than that of younger women. Increased amounts of iron were found in multiparous women. Between colostrum and transitional milk, a sharp decrease in zinc content was observed, whereas copper and iron contents remained constant. All of these results make it clear that standardized sampling protocols are needed in order to obtain comparable values.