RESUMO
PfSPZ Vaccine, composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum sporozoites, is administered by direct venous inoculation (DVI) for maximal efficacy against malaria. A critical issue for advancing vaccines that are administered intravenously is the ability to efficiently administer them across multiple age groups. As part of a pediatric safety, immunogenicity, and efficacy trial in western Kenya, we evaluated the feasibility and tolerability of DVI, including ease of venous access, injection time, and crying during the procedure across age groups. Part 1 was an age de-escalation, dose escalation trial in children aged 13 months-5 years and infants aged 5-12 months; part 2 was a vaccine efficacy trial including only infants, using the most skilled injectors from part 1. Injectors could use a vein viewer, if needed. A total of 1222 injections (target 0.5 mL) were initiated by DVI in 511 participants (36 were 5-9-year-olds, 65 were 13-59-month-olds, and 410 infants). The complete volume was injected in 1185/1222 (97.0%) vaccinations, 1083/1185 (91.4%) achieved with the first DVI. 474/511 (92.8%) participants received only complete injections, 27/511 (5.3%) received at least one partial injection (<0.5 mL), and in 10/511 (2.0%) venous access was not obtained. The rate of complete injections by single DVI for infants improved from 77.1% in part 1 to 92.8% in part 2. No crying occurred in 51/59 (86.4%) vaccinations in 5-9-year-olds, 25/86 (29.1%) vaccinations in 13-59-month-olds and 172/1067 (16.1%) vaccinations in infants. Mean administration time ranged from 2.6 to 4.6 minutes and was longer for younger age groups. These data show that vaccination by DVI was feasible and well tolerated in infants and children in this rural hospital in western Kenya, when performed by skilled injectors. We also report that shipping and storage in liquid nitrogen vapor phase was simple and efficient. (Clinicaltrials.gov NCT02687373).
Assuntos
Vacinas Antimaláricas , Malária Falciparum , Adolescente , Animais , Criança , Pré-Escolar , Estudos de Viabilidade , Humanos , Lactente , Quênia , Malária Falciparum/prevenção & controle , Plasmodium falciparum , Esporozoítos , Vacinação , Vacinas AtenuadasRESUMO
BACKGROUND: The presence of miRNAs in human reproductive tissue is intriguing and suggests the possibility that these important regulatory molecules play a role in reproductive function. However, the regulatory role of miRNAs in reproductive tissue remains poorly understood with a significant amount of controversial and contradicting data. OBJECTIVES: To systematically review the high-quality studies published to date investigating miRNAs associated with male human reproduction in order to describe their roles and relations with infertility and update the knowledge in the field. MATERIALS AND METHODS: A comprehensive systematic review of the published literature in MEDLINE-PubMed and EMBASE databases from the earliest available online indexing year until June 2018 (complimentary search until July 2019) was performed, in accordance with the PRISMA guidelines. We have included descriptive, case-control, cross-sectional, and observational prospective and retrospective studies in which fertile/infertile men were well-defined. The primary outcome was the miRNA expression in testis, epididymis, sperm cells, seminal plasma, and extracellular vesicles (i.e., exosomes and microvesicles). RESULTS: We identified 25,204 articles, of which 42 were selected for qualitative analysis. Of the 42 articles included, 15 evaluated the miRNAs in testis, five in epididymis, 13 in spermatozoa, and 11 in seminal plasma and/or extracellular vesicles. Two studies tackled more than one sub-group. As far as miRNA presence and content, the results of this systematic review indicated that every tissue/cell contains a well-defined and stable population of miRNAs that could be potentially related to spermatogenesis and embryogenesis. DISCUSSION AND CONCLUSION: Our systematic review of descriptive and observational studies shows a consistent relationship between aberrant miRNA expression and infertility. Therefore, it seems reasonable that measuring the expression of particular miRNAs might be useful not only as infertility biomarkers, but also for developing therapeutic strategies.
Assuntos
Epididimo/metabolismo , MicroRNAs/fisiologia , Reprodução , Espermatozoides/metabolismo , Testículo/metabolismo , Humanos , MasculinoRESUMO
Numerous health consequences of tobacco smoke exposure have been characterized, and the effects of smoking on traditional measures of male fertility are well described. However, a growing body of data indicates that pre-conception paternal smoking also confers increased risk for a number of morbidities on offspring. The mechanism for this increased risk has not been elucidated, but it is likely mediated, at least in part, through epigenetic modifications transmitted through spermatozoa. In this study, we investigated the impact of cigarette smoke exposure on sperm DNA methylation patterns in 78 men who smoke and 78 never-smokers using the Infinium Human Methylation 450 beadchip. We investigated two models of DNA methylation alterations: (i) consistently altered methylation at specific CpGs or within specific genomic regions and (ii) stochastic DNA methylation alterations manifest as increased variability in genome-wide methylation patterns in men who smoke. We identified 141 significantly differentially methylated CpGs associated with smoking. In addition, we identified a trend toward increased variance in methylation patterns genome-wide in sperm DNA from men who smoke compared with never-smokers. These findings of widespread DNA methylation alterations are consistent with the broad range of offspring heath disparities associated with pre-conception paternal smoke exposure and warrant further investigation to identify the specific mechanism by which sperm DNA methylation perturbation confers risk to offspring health and whether these changes can be transmitted to offspring and transgenerationally.
Assuntos
Fumar Cigarros/efeitos adversos , Metilação de DNA , Espermatozoides , Adulto , Ilhas de CpG , Humanos , MasculinoRESUMO
Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8(+) T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8(+), IFN-γ-producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Fígado/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Esporozoítos/imunologia , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Humanos , Injeções Intravenosas , Injeções Subcutâneas , Interferon gama/biossíntese , Interferon gama/imunologia , Macaca mulatta , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/efeitos adversos , Camundongos , Pessoa de Meia-Idade , Coelhos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/imunologia , Adulto JovemRESUMO
In 2004, three wild sea otters were diagnosed with putative Sarcocystis neurona-associated meningoencephalitis by histopathology and immunohistochemistry. Schizonts, free merozoites and tissue cysts were observed in the brains of all three infected animals. Tissue cysts walls from sea otter 1 (SO1) stained positively using anti-S. neurona polyclonal antiserum. However, positive staining does not preclude infection by closely related or cross-reactive tissue cyst-forming coccidian parasites. Two immature tissue cysts in the brain of SO1 were examined using transmission electron microscopy. Ultrastructural features included cyst walls with thin villous projections up to 1 microm long with tapered ends and a distinctive, electron-dense outer lining layer composed of linearly-arranged, semi-circular structures with a "hobnailed" surface contour. Small numbers of microtubules extended down through the villi into the underlying granular layer. Metrocytes were short and plump with an anterior apical complex, 22 sub-pellicular microtubules, numerous free ribosomes and no rhoptries. Some metrocytes appeared to be dividing, with two adjacent nuclear profiles. Collectively these ultrastructural features were compatible with developing protozoal cysts and were similar to prior descriptions of S. neurona tissue cysts. Panspecific 18S rDNA primers were utilized to identify protozoa infecting the brains of these otters and DNA amplification and additional sequencing at the ITS1 locus confirmed that all three otters were infected with S. neurona. No other Sarcocystis spp. were detected in the brains or skeletal muscles of these animals by immunohistochemistry or PCR. We believe this is the first ultrastructural and molecular confirmation of the development of S. neurona tissue cysts in the CNS of any animal.
Assuntos
Encéfalo/parasitologia , Sistema Nervoso Central/parasitologia , Cistos/parasitologia , Lontras/parasitologia , Sarcocystis/isolamento & purificação , Sarcocistose/transmissão , Animais , Cistos/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico 18S/genética , Sarcocystis/genética , Sarcocystis/ultraestrutura , Sarcocistose/genética , Sarcocistose/veterinária , Água do MarRESUMO
Sea otters in California are commonly infected with Toxoplasma gondii. A unique Type X strain is responsible for 72% of otter infections, but its prevalence in terrestrial animals and marine invertebrates inhabiting the same area was unknown. Between 2000 and 2005, 45 terrestrial carnivores (lions, bobcats, domestic cats and foxes) and 1396 invertebrates (mussels, clams and worms) were screened for T. gondii using PCR and DNA sequencing to determine the phylogeographic distribution of T. gondii archetypal I, II, III and Type X genotypes. Marine bivalves have been shown to concentrate T. gondii oocysts in the laboratory, but a comprehensive survey of wild invertebrates has not been reported. A California mussel from an estuary draining into Monterey Bay was confirmed positive for Type X T. gondii by multilocus PCR and DNA sequencing at the B1 and SAG1 loci. This mussel was collected from nearshore marine waters just after the first significant rainfall event in the fall of 2002. Of 45 carnivores tested at the B1, SAG1, and GRA6 typing loci, 15 had PCR-confirmed T. gondii infection; 11 possessed alleles consistent with infection by archetypal Type I, II or III strains and 4 possessed alleles consistent with Type X T. gondii infection. No non-canonical alleles were identified. The four T. gondii strains with Type X alleles were identified from two mountain lions, a bobcat and a fox residing in coastal watersheds adjacent to sea otter habitat near Monterey Bay and Estero Bay. Confirmation of Type X T. gondii in coastal-dwelling felids, canids, a marine bivalve and nearshore-dwelling sea otters supports the hypotheses that feline faecal contamination is flowing from land to sea through surface runoff, and that otters can be infected with T. gondii via consumption of filter-feeding marine invertebrates.
Assuntos
Bivalves/parasitologia , DNA de Protozoário/genética , Felidae/parasitologia , Lontras/parasitologia , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/transmissão , Animais , California , DNA de Protozoário/análise , Monitoramento Ambiental/métodos , Fezes/parasitologia , Oceanos e Mares , Oocistos , Reação em Cadeia da Polimerase , Toxoplasma/genéticaRESUMO
In September 2004, a neonatal sea otter pup was found alive on the beach in northern Monterey Bay, CA. Efforts to locate the mother were unsuccessful. Due to a poor prognosis for successful rehabilitation, the pup was euthanized. Postmortem examination revealed emaciation, systemic lymphadenopathy and a malformation of the left cerebral temporal lobe. On histopathology, free tachyzoites and tissue cysts compatible with Toxoplasma gondii were observed in the brain, heart, thymus, liver, lymph nodes and peri-umbilical adipose. The presence of T. gondii within host tissues was associated with lymphoplasmacytic inflammation and tissue necrosis. Immunofluorescent antibody tests using postmortem serum were positive for anti-T. gondii IgM and IgG (at 1:320 and 1:1280 serum dilution, respectively), but were negative for IgG directed against Sarcocystis neurona and Neospora caninum (<1:40 each). Brain immunohistochemistry revealed positive staining for tachyzoites and tissue cysts using antiserum raised to T. gondii, but not S. neurona or N. caninum. T. gondii parasite DNA was obtained from extracts of brain and muscle by PCR amplification using the diagnostic B1 locus. Restriction enzyme digestion followed by gel electrophoresis and DNA sequencing confirmed the presence of Type X T. gondii, the strain identified in the majority of southern sea otter infections.
Assuntos
Transmissão Vertical de Doenças Infecciosas/veterinária , Lontras , Toxoplasmose Animal/transmissão , Animais , Animais Recém-Nascidos , Animais Selvagens , Encéfalo/parasitologia , Encéfalo/patologia , Feminino , Miocárdio/patologia , Gravidez , Toxoplasmose Animal/patologiaRESUMO
Toxoplasma gondii affects a wide variety of hosts including threatened southern sea otters (Enhydra lutris nereis) which serve as sentinels for the detection of the parasite's transmission into marine ecosystems. Toxoplasmosis is a major cause of mortality and contributor to the slow rate of population recovery for southern sea otters in California. An updated seroprevalence analysis showed that 52% of 305 freshly dead, beachcast sea otters and 38% of 257 live sea otters sampled along the California coast from 1998 to 2004 were infected with T. gondii. Areas with high T. gondii exposure were predominantly sandy bays near urban centres with freshwater runoff. Genotypic characterisation of 15 new T. gondii isolates obtained from otters in 2004 identified only X alleles at B1 and SAG1. A total of 38/50 or 72% of all otter isolates so far examined have been infected with a Type X strain. Type X isolates were also obtained from a Pacific harbor seal (Phoca vitulina) and California sea lion (Zalophus californianus). Molecular analysis using the C8 RAPD marker showed that the X isolates were more genetically heterogeneous than archetypal Type I, II and III genotypes of T. gondii. The origin and transmission of the Type X T. gondii genotype are not yet clear. Sea otters do not prey on known intermediate hosts for T. gondii and vertical transmission appears to play a minor role in maintaining infection in the populations. Therefore, the most likely source of infection is by infectious, environmentally resistant oocysts that are shed in the feces of felids and transported via freshwater runoff into the marine ecosystem. As nearshore predators, otters serve as sentinels of protozoal pathogen flow into the marine environment since they share the same environment and consume some of the same foods as humans. Investigation into the processes promoting T. gondii infections in sea otters will provide a better understanding of terrestrial parasite flow and the emergence of disease at the interface between wildlife, domestic animals and humans.
Assuntos
Lontras/parasitologia , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/transmissão , Microbiologia da Água , Animais , Sequência de Bases , California , DNA de Protozoário/análise , Monitoramento Ambiental/métodos , Dados de Sequência Molecular , Oocistos , Técnica de Amplificação ao Acaso de DNA Polimórfico , Água do Mar , Toxoplasma/genética , Toxoplasmose Animal/diagnóstico , ZoonosesRESUMO
EAT-2 is an adaptor expressed in innate immune cells, including natural killer (NK) cells. It is closely related to the adaptor SAP, which regulates signaling lymphocyte activation molecule (SLAM)-related receptors by recruiting the kinase FynT to the receptors. Here we have studied the function of EAT-2 in NK cells by creating mice lacking or overexpressing EAT-2. Like SAP, EAT-2 was associated with the SLAM-related receptor 2B4 in NK cells. However, unlike SAP, EAT-2 was an inhibitor of NK cell function. EAT-2 repressed natural cytotoxicity and interferon-gamma secretion by a mechanism involving tyrosine phosphorylation of its C terminus. We have demonstrated a similar function for the adaptor ERT, a newly identified SAP family member expressed in mouse NK cells. These data identify a previously unknown mechanism of NK cell inhibition. Moreover, they indicate that EAT-2 and SAP have distinct and at times opposing functions in natural immunity.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Células Matadoras Naturais/imunologia , Fatores de Transcrição/fisiologia , Domínios de Homologia de src/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Antígenos CD/genética , Antígenos CD/fisiologia , Células CHO , Linhagem Celular , Cricetinae , Citotoxicidade Imunológica , Regulação para Baixo , Interferon gama/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Matadoras Naturais/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Dados de Sequência Molecular , Fosforilação , Receptores Imunológicos/genética , Receptores Imunológicos/fisiologia , Alinhamento de Sequência , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Família de Moléculas de Sinalização da Ativação Linfocitária , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Tirosina , Domínios de Homologia de src/genéticaRESUMO
Toxoplasma gondii-associated meningoencephalitis is a significant disease of California sea otters (Enhydra lutris nereis), responsible for 16% of total mortality in fresh, beachcast carcasses. Toxoplasma gondii isolates were obtained from 35 California otters necropsied between 1998 and 2002. Based on multi-locus PCR-restriction fragment length polymorphism and DNA sequencing at conserved genes (18S rDNA, ITS-1) and polymorphic genes (B1, SAG1, SAG3 and GRA6), two distinct genotypes were identified: type II and a novel genotype, here called type x, that possessed distinct alleles at three of the four polymorphic loci sequenced. The majority (60%) of sea otter T. gondii infections were of genotype x, with the remaining 40% being of genotype II. No type I or III genotypes were identified. Epidemiological methods were used to examine the relationship between isolated T. gondii genotype(s) and spatial and demographic risk factors, such as otter stranding location and sex, as well as specific outcomes related to pathogenicity, such as severity of brain inflammation on histopathology and T. gondii-associated mortality. Differences were identified with respect to T. gondii genotype and sea otter sex and stranding location along the California coast. Localised spatial clustering was detected for both type II (centred within Monterey Bay) and x (centred near Morro Bay)-infected otters. The Morro Bay cluster of type x-infected otters overlaps previously reported high-risk areas for sea otter infection and mortality due to T. gondii. Nine of the 12 otters that had T. gondii-associated meningoencephalitis as a primary cause of death were infected with type x parasites.
Assuntos
Lontras/parasitologia , Toxoplasma/patogenicidade , Toxoplasmose Animal/parasitologia , Animais , Sequência de Bases , California/epidemiologia , DNA de Protozoário/genética , Suscetibilidade a Doenças , Genótipo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Fatores de Risco , Fatores Sexuais , Toxoplasma/genética , Toxoplasma/isolamento & purificação , Toxoplasmose Animal/mortalidade , Toxoplasmose Animal/patologiaRESUMO
Antigens that are not normally seen by the host but that are nevertheless, accessible to host immune effector molecules and cells such as the native endoantigens associated with the intestinal epithelium of haematophagous tissue-dwelling parasites, could be potentially useful vaccine antigens. In this study, intestines were dissected from adult Dirofilaria immitis, homogenised, and a 105,000 x g pellet obtained and extracted with Triton X-100. The soluble 105,000 x g supernatant from this extract induced partial protection (51%) against a challenge infection of third stage larvae (L3) implanted in micropore chambers. Sera from mice immunised with this soluble detergent extract reacted with proteins ranging in size from 38 to 130 kDa. Immunolocalisation studies indicated the mouse sera reacted primarily to the lumenal surface of the intestines of adult D. immitis, though reactivity to the lateral nerve/epithelial chords, hypodermis and reproductive tracts was also noted, indicating the presence of shared antigens. Tissues of L3s were also recognised by the immunised mouse sera. These mouse sera did not react to a dog blood fraction prepared identically to the D. immitis fraction. Only those sera from D. immitis-infected dogs with heavy or long-term infections were reactive to a single 42 kDa protein. After 24 h incubation in fluorescein isothiocyanate-conjugated serum the intestinal tract of Onchocerca volvulus and D. immitis L3 and L4 fluoresced, indicating the serum had been ingested. These data suggest that filarial gut-associated antigens (apart from the single 42 kDa antigen) are not seen by normally infected hosts, that they can be accessible to antibodies and that they can induce an immune response which is partially protective.
Assuntos
Antígenos de Helmintos/imunologia , Dirofilaria immitis/imunologia , Dirofilariose/imunologia , Imunização , Animais , Anticorpos Anti-Helmínticos/biossíntese , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/isolamento & purificação , Western Blotting , Dirofilaria immitis/metabolismo , Dirofilariose/prevenção & controle , Cães , Feminino , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Onchocerca volvulus/imunologia , Onchocerca volvulus/metabolismoRESUMO
By using BAC transgenic mice, we have shown that increased human ABCA1 protein expression results in a significant increase in cholesterol efflux in different tissues and marked elevation in high density lipoprotein (HDL)-cholesterol levels associated with increases in apoAI and apoAII. Three novel ABCA1 transcripts containing three different transcription initiation sites that utilize sequences in intron 1 have been identified. In BAC transgenic mice there is an increased expression of ABCA1 protein, but the distribution of the ABCA1 product in different cells remains similar to wild type mice. An internal promoter in human intron 1 containing liver X response elements is functional in vivo and directly contributes to regulation of the human ABCA1 gene in multiple tissues and to raised HDL cholesterol, apoAI, and apoAII levels. A highly significant relationship between raised protein levels, increased efflux, and level of HDL elevation is evident. These data provide proof of the principle that increased human ABCA1 efflux activity is associated with an increase in HDL levels in vivo.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Apolipoproteína A-I/metabolismo , HDL-Colesterol/metabolismo , Camundongos Transgênicos , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Elementos de Resposta , Transportador 1 de Cassete de Ligação de ATP , Animais , Sequência de Bases , Células COS , Linhagem Celular , Células Cultivadas , Colesterol/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA , Humanos , Imuno-Histoquímica , Íntrons , Lipídeos/sangue , Fígado/metabolismo , Receptores X do Fígado , Macrófagos/metabolismo , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Receptores Nucleares Órfãos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Transfecção , Células Tumorais CultivadasRESUMO
Screening a DNA bank from 50 patients with enzymatic confirmation of their diagnosis of nonketotic hyperglycinemia gave allele frequencies of 5% for R515S of P-protein (glycine decarboxylase) and 7% for R320H of T-protein (aminomethyltransferase). In a previous report we found that 3% of the same patient alleles were positive for T-protein IVS7-1G>A. In total, testing for these three mutations identified 15% of alleles and positive results (one or two mutations) were found in 11 of the 50 patients. In addition, a novel point mutation in T-protein, N145I, was found in a single case and a PCR/restriction enzyme assay was developed for its detection.
Assuntos
Aminoácido Oxirredutases/genética , Glicina/sangue , Hidroximetil e Formil Transferases/deficiência , Hidroximetil e Formil Transferases/genética , Hiperglicinemia não Cetótica/genética , Mutação , Aminometiltransferase , Primers do DNA/química , Éxons , Frequência do Gene , Glicina/metabolismo , Glicina Desidrogenase (Descarboxilante) , Heterozigoto , Humanos , Hiperglicinemia não Cetótica/enzimologia , Iminas , Recém-Nascido , Cetose , Fígado/enzimologia , Reação em Cadeia da Polimerase , Diagnóstico Pré-NatalRESUMO
PURPOSE: beta-D(+) glucose-glucose oxidase (E.C.# 1.1.3.4)-catalase (E.C.# 1.11.1.6) (GO-CAT) is being investigated as a new antioxidant system for use in pharmaceutical solutions. This study reports the results of tests for pyrogenicity and antigenicity of GO-CAT derived from Aspergillus niger when used parenterally in autoclaved preparations. METHODS: The Limulus amebocyte lysate (LAL) method was used to test the pyrogenicity of native GO-CAT. Pyrogenicity/antigenicity was evaluated in vivo by injecting autoclaved GO-CAT into New Zealand white rabbits. Antigenicity was also evaluated by Ouchterlony and Western blotting. RESULTS: None of the native GO-CAT concentrations tested (up to 30.83 u/ml) produced a positive gel clot in the LAL test, thereby suggesting its non-pyrogenicity. The rabbits, which received seven injections of autoclaved GO-CAT over a period of eleven weeks, remained healthy during and after the GO-CAT injections. All Ouchterlony and Western blot assays using sera from rabbits injected with autoclaved GO-CAT were negative. Furthermore, autoclaved GO-CAT could not be detected in Ouchterlony assays using a mouse monoclonal antibody (GO40 mAb) to native A. niger glucose oxidase. Control samples containing native GO-CAT produced an antigen-antibody complex reaction in Ouchterlony assays against the GO40 mAb. Antigen-antibody complexes could be detected by non-denaturing PAGE in samples containing native GO-CAT/GO40 and boiled GO-CAT/GO40, but not in samples containing autoclaved GO-CAT/GO40. These results indicate autoclaved GO-CAT is neither pyrogenic nor antigenic. CONCLUSIONS: Based on these results, there is potential for the use of beta-D(+) glucose-glucose oxidase-catalase as an antioxidant system in pharmaceutical solutions, particularly in terminally autoclaved aqueous formulations for parenteral use.
Assuntos
Antioxidantes , Catalase/farmacologia , Febre/induzido quimicamente , Glucose Oxidase/farmacologia , Animais , Formação de Anticorpos , Antígenos , Western Blotting , Catalase/imunologia , Feminino , Glucose Oxidase/imunologia , Caranguejos Ferradura , CoelhosRESUMO
A novel splice site mutation (IVS7-1G-->A) in the T-protein gene (aminomethyltransferase, or AMT) of the glycine cleavage enzyme complex was found in a patient with nonketotic hyperglycinemia (NKH). A PCR/restriction enzyme method to detect this mutation was used to screen 100 NKH alleles and identified the mutation in three unrelated families.
Assuntos
Aminoácido Oxirredutases/genética , Proteínas de Transporte/genética , Hidroximetil e Formil Transferases/genética , Hiperglicinemia não Cetótica/genética , Complexos Multienzimáticos/genética , Mutação/genética , Sítios de Splice de RNA/genética , Transferases/genética , Aminometiltransferase , Triagem de Portadores Genéticos , Humanos , Hiperglicinemia não Cetótica/enzimologia , Íntrons/genética , Reação em Cadeia da PolimeraseRESUMO
As a model of the cell proliferation occurring in posterior capsule opacification (PCO), lens epithelial cells (LEC) from human and rabbit capsulotomies, and a rabbit LEC line (N/N1003A) were grown in Dulbecco's Minimal Essential Media (MEM) with 10% fetal calf serum. LEC were exposed to the calcium ionophore, calcimycin, and viability was assessed by trypan blue staining, growth by 3H-thymidine incorporation and apoptosis by annexin/propidium iodide staining, calcein AM/ethidium bromide staining and DNA laddering. Human capsulotomy samples were similarly exposed to calcimycin, and apoptosis assayed by calcein AM/ethidium bromide staining. Calcimycin exposure induced apoptosis in both rabbit LEC cultures and human LEC, and changes leading to apoptosis could be detected within 30 minutes of calcimycin treatment. The decrease in viability and growth in human and rabbit LEC was dose-dependent. These data support the further evaluation of apoptosis induction as a possible treatment mechanism to prevent development of PCO following primary cataract surgery in humans.
Assuntos
Apoptose/efeitos dos fármacos , Calcimicina/farmacologia , Catarata/prevenção & controle , Ionóforos/farmacologia , Cristalino/citologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Cristalino/efeitos dos fármacos , Masculino , CoelhosRESUMO
The investigation of 14 unrelated patients with nonketotic hyperglycinemia led to the identification of mutations in 4 cases. Patients were initially categorized into probable P- or T-protein defects of the glycine cleavage enzyme complex, by the use of the glycine exchange assay without supplemental H-protein, then screened for mutations in the P-protein and T-protein genes, respectively.
Assuntos
Análise Mutacional de DNA , Hiperglicinemia não Cetótica/genética , Metiltransferases/genética , Sequência de Aminoácidos , Primers do DNA/química , Éxons , Feminino , Glicina/metabolismo , Homocisteína S-Metiltransferase , Humanos , Hiperglicinemia não Cetótica/enzimologia , Recém-Nascido , Íntrons , Fígado/enzimologia , Masculino , Metiltransferases/metabolismo , Dados de Sequência Molecular , LinhagemRESUMO
Peroxidoxins are a recently described family of antioxidants. They have an ancient origin, being present in organisms as primitive as the archaea, and they appear to be ubiquitous in living cells. Here, Sharon McGonigle, John Dalton and Eric James review the present understanding of the functions and mechanism of action of these enzymes and suggest that these antioxidants may represent the ;missing link' in the metabolism of reactive oxygen species by some protozoan and helminth parasites. Also, by performing sequence comparisons of homologues entered in the public databases, they have classified the parasite peroxidoxins as 1-cys or 2-cys enzymes. The discovery of these antioxidants may change our understanding of how reactive oxygen species, of parasite or host origin, are managed by parasites.
RESUMO
Toxoplasma gondii is a protozoan parasite that infects birds and mammals, including humans. T. gondii T-263 is an attenuated mutant strain that is being developed as a live vaccine to protect cats from shedding oocysts. A cryopreservation procedure for T. gondii T-263 bradyzoites has been developed to meet the requirement for product stability. A Me2SO-based procedure for the cryopreservation of tachyzoites was used as a basis for process optimization. A modified cell culture plaque assay was used to determine the effects of selected cryobiological parameters on bradyzoite viability. The major parameters evaluated were: (i) cooling rates; (ii) intermediate plunge temperature; and (iii) thawing and dilution rates and temperatures. The optimized cryopreservation protocol comprised incubation in 12.5% Me2SO and 4% BSA for 30 min at room temperature, cooling at 1 degree C min-1 to -40 degrees C, followed by direct transfer into liquid nitrogen. Rapid thawing (approximately 120 degrees C min-1) followed by slow dilution of cryoprotectant over 15 min resulted in the highest survival. The optimized procedure increased survival 10,000-fold over that obtained using an established tachyzoite protocol. This procedure is to be adapted for the large-scale cryopreservation of T. gondii T-263 bradyzoites in individual vaccine doses.