Effects of consumption of contaminated feed with 2,4-dichlorophenoxyacetic acid (2,4-D) on the rat tibia: analysis by Raman spectroscopy and mechanical properties

Lasers in Medical Science - Studies reported the harmful effects of 2,4-D on body tissues, provoking changes in the anatomy and physiology of the kidneys, liver, and testicles. Thus, the objective...     

Stimulation by mitogens and neuronal membranes of lymphocytes from patients with motor neurone disease

Stimulation of lymphocytes from motor neurone disease patients by either concanavalin A or PHA was shown to be significantly depressed relative to that from normal controls, as assayed by incorporation of [3H]thymidine or [3H]leucine or by glucose uptake. Corresponding significant differences were not shown by assays based upon incorporation of [3H]uridine or of lactate release. Lymphocytes from 4 out of 14 motor neurone disease patients showed a blastogenic response to membranes from rat spinal cord cells, compared with those from 0 out of 9 normal controls. These results not only suggest the possibility of an impaired cellular immune control in MND patients but also indicate the presence of lymphocytes sensitised specifically to neuronal membrane components.     

Anti-inflammatory effects of hydroalcoholic extract and two biflavonoids from Garcinia gardneriana leaves in mouse paw oedema

Garcinia gardneriana (Planch. & Triana) Zappi (Clusiaceae) is widely distributed in Brazil and used in folk medicine to treat inflammation, pain, and urinary tract and other infections. However, very few studies have analyzed these therapeutic effects. In this study, the anti-inflammatory effects of the hydroalcoholic extracts from Garcinia gardneriana (HEGG) and some of its isolated biflavonoids were evaluated. The results showed that HEGG from the leaves, bark and seeds reduced carrageenan-induced mouse paw inflammation, in addition to diminishing the myeloperoxidase activity in the stimulated tissues. The reduction of neutrophil infiltration by treatment with the HEGG from leaves was confirmed by histology. The leaf extract also reduced the paw oedema evoked by bradykinin, histamine, prostaglandin E2 and 12-O-tetradecanoylphorbol acetate. However, it partially decreased substance P and compound 48/80-caused paw oedema, without any influence on the arachidonic acid-induced oedema. Both of the isolated compounds, fukugetin and GB-2a, prevented the carrageenan-induced paw oedema. In conclusion, this study showed important anti-inflammatory effects of HEGG through its interaction with different intracellular signaling pathways, without interfering with the formation of arachidonic acid (AA) metabolites. These characteristics, in addition to the wide distribution and culturing ease of the plant, confirm its popular use and highlight its promise in the development of new anti-inflammatory drugs.     

Release of platelet-activating factor (PAF-acether) and arachidonic acid metabolites from alveolar macrophages

Human, monkey and rat alveolar macrophages (AM) release PAF-acether in a dose-dependent fashion in the presence of 1 to 5 g/ml ionophore A 23187 (2.5 pmol of PAF-acether from 2.5×10⁵ cells) but not in the presence of zymosan. Arachidonic acid (AA) metabolites released from AM from these species were studied. Thromboxane A₂ TxA₂)—detected by its action on rabbit arteries—was released from human, monkey and rat AM upon addition of 0.5 mM AA. This release was inhibited by aspirin and indomethacin. Lipoxygenase and cyclooxygenase AA metabolites from rat AM were identified using high efficiency glass capillary column gas chromatography coupled to mass spectrometry. The cyclooxygenase metabolites PGF₂, E₂ and D₂ and TxB₂ were identified. The lipoxygenase-dependent AA metabolites were explored using aspirin-pretreated AM. Only 12 HETE was found.These data indicate that AM secrete several substances with bronchoconstrictive activity: PGF₂, D₂, TxA₂ and PAF-acether. Therefore an active role of AM in human and experimental bronchoconstriction must be considered.     

Lymphocytic Hypophysitis Presenting as a Pituitary Tumor in a 63-Year-Old Man

This report describes a case of lymphocytic hypophysitis in a 63-year-old man who presented with symptoms of a pituitary mass lesion associated with hypothyroidism and hypogonadism. Postoperative endocrinological testing demonstrated gonadotropic, thyrotropic, and corticotropic hypopituitarism, and the patient was commenced on replacement therapy with hydrocortisone and levothyroxine. Histological examination of the pituitary tissue obtained by transsphenoidal surgery revealed lymphocytic hypophysitis without evidence of a pituitary adenoma. The vast majority of patients with lymphocytic hypophysitis are women particularly during pregnancy and the puerperium. Until recently only four men were reported in the literature. The pathogenesis of lymphocytic hypophysitis is uncertain but autoimmune mechanisms are possibly involved.     

Mutagenicity,antioxidant potential,and antimutagenic activity against hydrogen peroxide of cashew (Anacardium occidentale) apple juice and cajuina

Fresh and processed cashew (Anacardium occidentale) apple juice (CAJ) are among the most popular drinks in Brazil. Besides their nutritional benefits, these juices have antibacterial and antitumor potential. The chemical constituents of both the fresh juice and the processed juice (cajuina) were analyzed and characterized as complex mixtures containing high concentrations of vitamin C, various carotenoids, phenolic compounds, and metals. In the present study, these beverages exhibited direct and rat liver S9-mediated mutagenicity in the Salmonella/microsome assay with strains TA97a, TA98, and TA100, which detect frameshifts and base pair substitution. No mutagenicity was observed with strain TA102, which detects oxidative and alkylating mutagens and active forms of oxygen. Both CAJ and cajuina showed antioxidant activity as determined by a total radical-trapping potential assay. To test whether this antioxidant potential might result in antimutagenesis, we used a variation of the Salmonella/microsome assay that included pre-, co-, and posttreatment of hydrogen peroxide-exposed Salmonella typhimurium strain TA102 with the juices. CAJ and cajuina protected strain TA102 against mutation by oxidative damage in co- and posttreatments. The antimutagenic effects during cotreatment with hydrogen peroxide may be due to scavenging free radicals and complexing extracellular mutagenic compounds. The protective effects in posttreatment may be due to stimulation of repair and/or reversion of DNA damage. The results indicate that CAJ and cajuina have mutagenic, radical-trapping, antimutagenic, and comutagenic activity and that these properties can be related to the chemical constituents of the juices.     

Immunization of Aotus Monkeys with Recombinant Plasmodium falciparum Hybrid Proteins Does Not Reproducibly Result in Protection from Malaria Infection

Plasmodium falciparum antigens SERP, HRPII, MSAI, and 41-3 have shown promise as vaccine components. This study aimed at reproducing and extending previous results using three hybrid molecules. Antibody responses were reproduced in Aotus monkeys, but solid protection from a P. falciparum blood-stage challenge that showed an unintendedly enhanced pathogenicity was not observed.The increasing drug resistance of Plasmodium falciparum, the most pathogenic human malaria parasite, underlines the need for an effective malaria vaccine. Identification, testing, and optimization of candidate molecules originating from all developmental stages of the parasite are under way. Previously, a successful trial in Aotus monkeys employed the Escherichia coli-expressed hybrid proteins MS2/SERP/HRPII and SERP/MSAI/HRPII (11). Both hybrid proteins contain a region of the serine repeat protein SERP (1, 9), including two putative T-cell epitopes (13) and previously shown to induce a partial protective response in Aotus monkeys (5), and the C-terminal half of the histidine-rich protein HRPII (14), which has also been shown to induce a partially protective response (5, 8). SERP/MSAI/HRPII contains in addition a conserved N-terminal region of the merozoite surface antigen MSAI (7) that includes at least four T-cell epitopes (3, 6). Here we report on further analysis of three hybrid proteins of this type in a vaccination trial with Aotus monkeys. Two of the proteins, SERP/HRPII and SERP/MSAI/HRPII, are improved versions of the hybrid proteins mentioned above, obtained by deleting nonmalaria protein regions and changing an internal restart residue (methionine-729 of SERP) into alanine. Thus, the SERP/HRPII hybrid protein comprises residues 630 to 893 of SERP fused to the 189 C-terminal residues of HRPII, and SERP/MSAI/ HRPII comprises residues 630 to 764 of SERP fused to residues 146 to 259 of MSAI, which is fused to the 189 C-terminal residues of HRPII. SERP/41-3/HRPII contains the same components as SERP/HRPII, and additionally includes residues 77 to 188 of antigen 41-3 (10), which was previously shown to confer protection against a P. falciparum challenge (5). The internal restart residue (methionine-100) was also mutagenized into alanine and another residue, arginine-319, was changed into leucine to prevent proteolytic degradation. SERP/MSAI/HRPII was partially purified to a final purity of about 30%, as described previously (8), in order to match the quality of the proteins used in the successful previous trials (5, 11). The other two hybrid proteins were purified from bacterial lysates to over 90% purity by size exclusion chromatography (SERP/41-3/HRPII) or by sequential cation and anion exchange and then size exclusion chromatography (SERP/ HRPII) (data not shown). The final products were dialyzed against phosphate-buffered saline–3 M urea and adjusted to 100 μg of protein per ml. Efficacy was tested following an experimental protocol identical to the one used in the previous successful trial (11).Fifteen laboratory-raised Aotus azarae boliviensis karyotype VI monkeys were randomly assigned to one of four experimental groups (three groups of four and one group of three monkeys) and immunized with 1 ml of antigen or with the diluent alone (control group), both mixed with 100 μl of polyalphaolefin (4) as an adjuvant, on days 0, 21, and 42. Each vaccine dose was administered subcutaneously at two separate sites in the right and left flank and was well tolerated. The seroconversion results, as measured by enzyme-linked immunosorbent assay with SERP/HRPII as the solid-phase antigen and peroxidase-labelled rabbit anti-human immunoglobulin G (1:10,000 dilution; Pierce) as the secondary antibody, are shown in Fig. ​Fig.1.1. All experimental monkeys developed comparable antibody responses to SERP/HRPII, irrespective of the immunogen. Control monkey sera did not react significantly (not shown). A boosting effect is obvious after the second injection in all three groups (Fig. ​(Fig.1),1), as well as after the third SERP/41-3/HRPII injection (Fig. ​(Fig.1C).1C). This is similar to the seroconversion pattern observed previously (11). Prechallenge sera were also tested by immunofluorescence (IFA) for reactivity with P. falciparum schizonts. All preimmune sera and control group immune sera were negative (1:100 dilution). IFA titers from the experimental animals were all 1:1,600, except for animals A381 and A462 (titer, 1:800) and A452 and A292 (titer, 1:3,200). Thus, antibodies specific for native parasite determinants were induced. The relatively low IFA titers were comparable to those obtained in previous successful trials (5, 11). Open in a separate windowFIG. 1Development of antibody responses in Aotus monkeys during the immunization period as determined by enzyme-linked immunosorbent assay. Monkeys in different immunization groups were immunized at weeks 0, 3, and 6 (indicated by arrows) and challenged at week 8 (indicated by an asterisk). All sera were tested for reactivity with SERP/HRPII in a 1:100 dilution. The hybrid antigens used for immunization were SERP/MSAI/HRPII (A), SERP/HRPII (B), and SERP/41-3/HRPII (C). Sera of the three control monkeys remained negative in this assay (not shown). OD, optical density.At week 7 all monkeys were splenectomized, and at week 8 they received intravenously 2 × 10⁶ parasitized erythrocytes, which had been isolated from an Aotus monkey infected with an in vivo-passaged FUP-Cayenne isolate of P. falciparum (a kind gift of W. E. Collins) (Fig. ​(Fig.1).1). Monkey A293 appeared to have no spleen, although there was no prior history of splenectomy. The immunoglobulin G response of monkey A293 was nevertheless comparable to that of the other animals (Fig. ​(Fig.1B).1B). Figure ​Figure22 shows the course of parasitemia after challenge. Two of three control animals rapidly developed a parasitemia which required mefloquine therapy (20 mg/kg of body weight orally) when parasitemia reached 10% (day 8 for A371 and day 10 for A320). In A432, parasitemia developed to 8.6% (day 10) and then fluctuated until rapidly reaching 19% (day 21), at which point mefloquine was administered (Fig. ​(Fig.2A).2A). None of the three immunized groups showed a solid protective response (Fig. ​(Fig.2B2B to D). A340 (SERP/HRPII group) (Fig. ​(Fig.2C)2C) and A292 (SERP/41-3/HRPII group) (Fig. ​(Fig.2D)2D) showed low fluctuating parasitemias with a peak around 2.5% at the end of the observation period (day 25). Otherwise, parasitemias of the experimental animals did not significantly differ from those of the controls. No obvious correlation between prechallenge antibody levels and protection was evident. Open in a separate windowFIG. 2Course of infection with the FUP-Cayenne isolate of P. falciparum in control Aotus monkeys (A) and in Aotus monkeys immunized with SERP/MSAI/HRPII (B), SERP/HRPII (C), and SERP/41-3/HRPII (D). Parasitemias of ≥10% were cured with mefloquine.It seems unlikely that small conservative changes designed to improve SERP/HRPII and SERP/MSAI/HRPII expression in E. coli and to remove nonrelevant sequences adversely affected immune response development. After challenge, parasitemia developed markedly faster than in the previous trial, which had shown protection. Challenge with 2 × 10⁶ parasitized erythrocytes now resulted in high parasitemias on days 7 to 9 in 2 of the 3 controls and in 6 of the 12 experimental animals, whereas previously controls were untreated until day 14 (11). Also, one control and three experimental animals suffered recrudescence, which was not seen previously (11) or with later infections with the same parasite stock (5). It is remarkable that this apparent enhanced pathogenicity developed after a single passage in A. nancymai just before the present trial started. It is likely that this unintended pathogenicity influenced the experimental outcome. The protection of two monkeys in the SERP/HRPII and SERP/41-3/HRPII group may, however, reveal some protective effect of these vaccine candidates.For demonstration of the protective potential of antigens in the primate model the pathogenicity of the challenge strain in the respective primate (sub)species, i.e., the equilibrium of immune response and pathogenicity, seems to be crucial (2, 12). The disturbance of this equilibrium may explain the discrepancy between previous successful trials (5, 11) and the present study. Recombinant proteins shown to be protective in the Aotus model (5, 11) failed to protect Saimiri monkeys, in which the course of parasitemia is quite different from that observed in Aotus monkeys. Similarly, no protection could be demonstrated in A. nancymai against the same challenge strain as was used in the successful trials with A. azarae boliviensis and A. lemurinus griseimembra (7a). The poor standardization of these models due to the scarcity of monkeys susceptible to human malaria remains an obstacle for the evaluation of human malaria vaccine candidates.