Glycated bovine serum albumin for use in feeding trials with animal models - In vitro methodology and characterization of a glycated substrate for modifying feed pellets.

This study investigated different methods to produce Nε-carboxymethyl-lysine (CML)-enriched bovine serum albumin (BSA) as alternatives to the classical approach using glyoxylic acid (GA) and sodium cyanoborohydride (NaBH3CN) which results in toxic hydrogen cyanide (HCN). The reaction of GA (6 mmol/L) and NaBH3CN (21 mmol/L) to produce CML remained the most effective with CML yields of 24-35%, followed by 13-24% using 300 mmol/L glyoxal (GO). GA promoted specific modification of lysine to CML, and fewer structural modifications of the BSA molecule compared with GO, as evidenced by fluorescence and proteomic analyses. GO promoted greater arginine modification compared with GA (76 vs 23%). Despite structural changes to BSA with GO, murine fecal clearance of CML was similar to literature values. Hence, BSA glycation with 300 mmol/L glyoxal is a suitable alternative to GA and NaBH3CN for generating CML-enriched protein free of HCN, but a CML-only fortification model remains to be described.

An easy, quick and cheap high-throughput method for yeast DNA extraction from microwell plates.

We herein present a high-throughput and cheap method for yeast DNA isolation in a 96 well microplate. About 1500 yeast isolates can be processed within one working day and final DNA concentrations are suitable for direct application in PCR-based molecular typing methods.

Sublingual grass pollen immunotherapy is associated with increases in sublingual Foxp3-expressing cells and elevated allergen-specific immunoglobulin G4, immunoglobulin A and serum inhibitory activity for immunoglobulin E-facilitated allergen binding to B cells.

BACKGROUND: The mechanisms of sublingual immunotherapy (SLIT) are less well understood than those of subcutaneous immunotherapy (SCIT). OBJECTIVES: To determine the effects of grass-pollen SLIT on oral mucosal immune cells, local regulatory cytokines, serum allergen-specific antibody subclasses and B cell IgE-facilitated allergen binding (IgE-FAB). METHODS: Biopsies from the sublingual mucosa of up to 14 SLIT-treated atopics, nine placebo-treated atopics and eight normal controls were examined for myeloid dendritic cells (mDCs) (CD1c), plasmacytoid dendritic cells (CD303), mast cells (AA1), T cells (CD3) and Foxp3 using immunofluorescence microscopy. IL-10 and TGF-beta mRNA expression were identified by in situ hybridization. Allergen-specific IgG and IgA subclasses and serum inhibitory activity for binding of allergen-IgE complexes to B cells (IgE-FAB) were measured before, during and on the completion of SLIT. RESULTS: Foxp3(+) cells were increased in the oral epithelium of SLIT- vs. placebo-treated atopics (P=0.04). Greater numbers of subepithelial mDCs were present in placebo-treated, but not in SLIT-treated, atopics compared with normal controls (P=0.05). There were fewer subepithelial mast cells and greater epithelial T cells in SLIT- compared with placebo-treated atopics (P=0.1 for both). IgG(1) and IgG(4) were increased following SLIT (P<0.001). Peak seasonal IgA(1) and IgA(2) were increased during SLIT (P<0.05). There was a time-dependent increase in serum inhibitory activity for IgE-FAB in SLIT-treated atopics. CONCLUSIONS: SLIT with grass pollen extract is associated with increased Foxp3(+) cells in the sublingual epithelium and systemic humoral changes as observed previously for SCIT.

Grass pollen immunotherapy for seasonal rhinitis and asthma: a randomized, controlled trial.

BACKGROUND: Grass pollen immunotherapy significantly reduces hay fever symptoms and medication requirements. Effects on seasonal asthma are less clear, and concerns over safety persist. OBJECTIVE: The goal of this study was to assess the effects of grass pollen immunotherapy on symptoms, bronchial hyperresponsiveness, and quality of life in seasonal rhinitis and asthma. METHODS: Forty-four patients with severe summer hay fever (of whom 36 reported seasonal chest symptoms and 28 had seasonal bronchial hyperresponsiveness) participated in a randomized, double-blind, placebo-controlled, parallel group study. After symptom monitoring for one summer, participants received injections of a depot grass pollen vaccine (n = 22) or matched placebo injections (n = 22) in a rapid updosing cluster regimen for 4 weeks, followed by monthly injections for 2 years. Outcome measures included hay fever symptoms and medication use, health-related quality of life, and measurements of nonspecific bronchial responsiveness. RESULTS: Significant reductions were observed in the immunotherapy group compared with the placebo group in hay fever symptoms (49%, 15%; P =.01), medication scores (80%, 18%; P =.007), and seasonal chest symptoms (90%, 11%; P <.05). Impairment of overall quality of life (mean score of 7 domains) during the pollen season was less in the immunotherapy group than in the placebo group (median difference [95% CI], 0.8 [0.18-1.5]; P =.02). During the pollen season there was no change in airway methacholine PC(20) (provocation concentration producing a 20% fall in FEV(1)) in the immunotherapy-treated group (P =.5), compared with an almost 3 doubling-dose decrease in the placebo-treated group (P =.01, between-group difference). There were no significant local or systemic side effects during the study. CONCLUSION: Grass pollen immunotherapy improves quality of life in seasonal allergic rhinitis and reduces seasonal asthma symptoms and bronchial hyperresponsiveness.

Determination of stilbenes (trans-astringin, cis- and trans-piceid, and cis- and trans-resveratrol) in Portuguese wines.

Stilbenes have been shown to have cancer chemopreventive activity and to protect lipoproteins from oxidative damage. A method is described for their direct determination in different types of wine using high-performance liquid chromatography with ultraviolet detection. In a survey of 120 commercial wines from Portugal and France, the highest concentrations of stilbenes were found in red wines. The glucosides of resveratrol were present in higher concentrations than the free isomers. Isolation from wine and characterization of trans-astringin in a large quantity are described for the first time.

Major histocompatibility complex (MHC) class III genetics in two Amerindian tribes from southern Brazil: the Kaingang and the Guarani.

Population genetic studies of the major histocompatibility complex (MHC) class III region, comprising C2, BF and C4 phenotypes, and molecular genetic data are rarely available for populations other than Caucasoids. We have investigated three Amerindian populations from Southern Brazil: 131 Kaingang from Ivaí (KIV), 111 Kaingang (KRC) and 100 Guarani (GRC) from Rio das Cobras. Extended MHC haplotypes were derived after standard C2, BF, C4 phenotyping and restriction fragment length polymorphism (RFLP) analysis with TaqI, together with HLA data published previously by segregation analysis. C2 and BF frequencies corresponded to other Amerindian populations. C4B*Q0 frequency was high in the GRC (0.429) but low in the Kaingang. Unusual C4 alleles were found, viz. C4A*58, A*55 and C4B*22 (presumably non-Amerindian) and aberrant C4A*3 of Amerindian origin occurring with a frequency of 0.223 in the GRC. C4A*3 bands of homo- and heterozygous individuals carrying this variant were Rodgers 1 positive and Chido 1,3 positive, showed a C4A specific lysis type and a C4A like alpha-chain. Polymerase chain reaction studies and sequencing showed that this is based on a C4A*3 duplication with a regular C4A*3 and a partially converted C4A*0304 carrying the C4B specific epitopes Ch 6 and Ch 1,3. Associations of class III haplotypes with particular RFLP patterns were similar to those reported for Caucasoids. The previously described association between combined C4A and CYP21P deletions and the 6.4 kb TaqI fragment was not seen in these Amerindians. This fragment occurred within a regular two locus gene structure in the Kaingang, representing a "short" gene at C4 locus I. C4 and CYP21 duplications were frequently observed. The distribution of extended MHC haplotypes provides evidence for a close relationship between the KIV and KRC and a larger genetic distance between the two Kaingang groups and the GRC.

Trypanosoma cruzi: paninfectivity of CL strain during murine acute infection.

A systematic study of the distribution of intracellular parasites in the organs and tissues of mice acutely infected (15 days) with the CL strain of Trypanosoma cruzi was performed. Almost all tissues and organs were parasitized with different intensities, including several epithelial cell types. In addition to striated, cardiac, and smooth muscles a very high parasitism of fat cells, pancreas, and genital adnexa was observed. A smaller number of parasites was found in all other structures studied except in highly vascularized structures such as in the penile corpora cavernosa, pulmonary and renal parenchyma, islets of Langerhans, hepatic sinusoids, and in atrial endothelium. This paper also shows, for the first time in the literature, the parasitism of milky spots, cornea epithelium, cornea stroma, retroorbital fibroblasts, seminal vesicles, and coagulative, Cowper's, urethral, preputial, sebaceous anal, and clitoris glands. The results indicated that CL strain is highly invasive, being able to infect cells derived from the three embryonic layers (ectoderm, mesoderm, and endoderm), suggesting that the paninfectivity may influence the outcome of immunological and pathological events.

Negative tissue parasitism in mice injected with a noninfective clone of Trypanosoma cruzi.

Trypanosoma cruzi is a heterogeneous population of parasites as shown by differences between strains and cloned stock from the same strain. Herein we present evidence of the noninfectivity of CL-14, a clone derived from the CL strain of T. cruzi. In a previous paper we reported the absence of parasitemia and mortality in mice injected with metacyclic trypomastigotes of this clone. To investigate further this lack of infectivity we did and extensive histopathological analysis in mice at different intervals after i.p. (5 and 15 days as well as 1, 4, and 12 months) or i.v. (5 and 30 days) injection of trypomastigotes. In spite of a systematic search in all tissues and organs of the animals, no parasite or significant pathological change was detected in any of the tissue sections. These data suggest the inability of this clone to mediate infection and/or cause pathological alterations in vivo.

BF and C3 genetic polymorphisms in Kaingang Indians from southern Brazil.

The distribution of C3 and BF variants was determined in a sample of 239 Kaingang Indians. The corresponding gene frequencies were as follows: BF*S = 0.9393, BF*F = 0.0356, BF*S05 = 0.0251, C3*S = 0.9769, C3*F = 0.0231. The presence of the BF*S05 allele, which has previously been found only in a Brazilian population, suggests that this allele originated in Amerindians. The comparatively low degree of polymorphism with high frequencies of BF*S and C3*S is in accordance with the relatedness of the Kaingang with other Amerindians, Eskimos and Asian populations.

Trypanosoma cruzi: properties of a clone isolated from CL strain.

BALB/c mice injected i.p. with 2 x 10(6) metacyclic forms of CL-14, a clone isolated from the CL strain of Trypanosoma cruzi, did not show parasitemia as evaluated by direct blood microscopy examination, hemoculture and xenodiagnosis. Moreover, new-born mice (1-2 days old) injected with culture- or insect-derived CL-14 trypomastigotes also displayed negative parasitemia. No mortality was observed in either group of animals. However, despite this apparent non-infectivity, mice injected with clone 14 developed high resistance against a lethal challenge with virulent trypomastigotes. All challenged mice survived and the parasitemia was negative. These results indicate that clone 14 is a very good antigen for the study of acquired immunity in T. cruzi infection and, therefore, a potential candidate for the development of a vaccine against this parasite.

Do self-heart-reactive T cells expand in Trypanosoma cruzi-immune hosts?

Anti-heart T-cell activity was evaluated by a lymph node cell proliferative assay in isogenic strains of mice immunized with several Trypanosoma cruzi epimastigote and trypomastigote antigenic preparations. In addition, chronically infected animals were boosted with trypomastigote antigens and their lymph node cells were tested by in vitro proliferative responses. Our results indicated that (i) use of allogeneic sources of heart antigens may induce alloreactive responses in T. cruzi-immune T cells, (ii) specific autoimmune T-cell reactivity against self-heart constituents could not be demonstrated after immunization of the host with T. cruzi, and (iii) a proportion of chronically infected mice showed a small but detectable level of auto-anti-heart T-cell reactivity. These results argue against the notion that T. cruzi epitopes cross-reactive with self-heart tissue play a role in initiating T-cell-mediated autoimmunity. Anti-heart autoreactive T cells, generated in a proportion of the animals, may result from heart lesions associated with the infection process.

Histopathological study of human placenta in women infected with rubella virus during pregnancy.

A histopathological and virological study is carried out on a group of placentae in the second trimester of pregnancy, obtained during the 1974 rubella epidemic in Cuba. The comparison between the positive and negative cases did not show characteristic lesions of the virus passage through the placenta, which does not coincide with the reports of other authors who generally consider these alterations as a result of this pathology. Eosinophilic intracytoplasmic granules were found in the Hofbauer cells, which should be studied by employing other techniques for accurate identification.