RESUMO
OBJECTIVE: The management of patients with penetrating neck injuries in the prehospital setting and in the emergency department has evolved with regard to the necessity for spinal immobilisation and the use of multidetector computed tomographic (MDCT) imaging. Questions also arise as to choices of securing a threatened or compromised airway. A structured review of the medical literature was conducted to provide current recommendations for the management of patients with penetrating neck injury. METHODS: Databases for PubMed, MEDLINE, CINAHL and Cochrane EBM Reviews were electronically searched using the subject headings "penetrating neck injury", "penetrating neck trauma", "cervical immobilization", "multi-detector CTA" and "airway management". The results generated by the search were limited to English language articles and reviewed for relevance to the topic. RESULTS: 122 citations were identified that met the criteria for emphasis on emergency department care, cervical spine immobilisation, use of multidetector CT angiography or airway management. After excluding case series, non-peer reviewed articles and editorials, 20 articles were identified and reviewed. CONCLUSIONS: The current literature suggests that prehospital cervical immobilisation may not be necessary unless the patient has focal neurological deficits. Studies show that patients with penetrating neck trauma who are haemodynamically stable and exhibit no "hard signs" of vascular injury may be evaluated initially by MDCT imaging even when platysma violation is present. Airway management is evolving, but traditional laryngoscopy continues to be the mainstay of airway stabilisation.
Assuntos
Serviço Hospitalar de Emergência , Lesões do Pescoço/terapia , Ferimentos Penetrantes/terapia , Humanos , Imobilização/métodos , Intubação Intratraqueal/métodos , Lesões do Pescoço/diagnóstico por imagem , Exame Físico , Tomografia Computadorizada por Raios X , Ferimentos Penetrantes/diagnóstico por imagemRESUMO
Neither GMP malaria antigens nor GMP vaccines have been compared for efficacy in monkeys and humans. It is too risky to base categorical (go/no go) development decisions on results obtained using partially characterized (non-GMP) antigens, adjuvants that are too toxic for human use or unvalidated primate models. Such practices will lead to serious errors (e.g. failure to identify and stop flawed efforts, rejection of effective vaccine strategies) and unjustifiable delays. Successful malaria vaccine development will emphasize definitive field trials in populations at risk of malaria to define and improve vaccine efficacy.
Assuntos
Aotus trivirgatus , Ensaios Clínicos como Assunto , Vacinas Antimaláricas , Malária/prevenção & controle , Saimiri , Animais , Antígenos de Protozoários/imunologia , Modelos Animais de Doenças , Humanos , Plasmodium/imunologiaRESUMO
Merozoite surface protein 1 (MSP-1) is a precursor to major antigens on the surface of Plasmodium spp. merozoites, which are involved in erythrocyte binding and invasion. MSP-1 is initially processed into smaller fragments; and at the time of erythrocyte invasion one of these of 42 kDa (MSP-1(42)) is subjected to a second processing, producing 33 kDa and 19 kDa fragments (MSP-1(33) and MSP-1(19)). Certain MSP-1-specific monoclonal antibodies (mAbs) react with conformational epitopes contained within the two epidermal growth factor domains that comprise MSP-1(19), and are classified as either inhibitory (inhibit processing of MSP-1(42) and erythrocyte invasion), blocking (block the binding and function of the inhibitory mAb), or neutral (neither inhibitory nor blocking). We have mapped the epitopes for inhibitory mAbs 12.8 and 12.10, and blocking mAbs such as 1E1 and 7.5 by using site-directed mutagenesis to change specific amino acid residues in MSP-1(19) and abolish antibody binding, and by using PEPSCAN to measure the reaction of the antibodies with every octapeptide within MSP-1(42). Twenty-six individual amino acid residue changes were made and the effect of each on the binding of mAbs was assessed by Western blotting and BIAcore analysis. Individual changes had either no effect, or reduced, or completely abolished the binding of individual mAbs. No two antibodies had an identical pattern of reactivity with the modified proteins. Using PEPSCAN each mAb reacted with a number of octapeptides, most of which were derived from within the first epidermal growth factor domain, although 1E1 also reacted with peptides spanning the processing site. When the single amino acid changes and the reactive peptides were mapped onto the three-dimensional structure of MSP-1(19), it was apparent that the epitopes for the mAbs could be defined more fully by using a combination of both mutagenesis and PEPSCAN than by either method alone, and differences in the fine specificity of binding for all the different antibodies could be distinguished. The incorporation of several specific amino acid changes enabled the design of proteins that bound inhibitory but not blocking antibodies. These may be suitable for the development of MSP-1-based vaccines against malaria.
Assuntos
Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium falciparum/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Especificidade de Anticorpos/genética , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/genética , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Proteína 1 de Superfície de Merozoito/química , Proteína 1 de Superfície de Merozoito/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Plasmodium falciparum/genética , Conformação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
The C terminus of the circumsporozoite protein (CSP) is anchored to the parasite cell membrane by a glycosylphosphatidylinositol (GPI) glycolipid. This GPI signal sequence functions poorly in heterologous eukaryotic cells, causing CSP retention within internal cell organelles during genetic immunization. Cellular location of antigen has quantitative and qualitative effects on immune responses induced by genetic immunization. Removal of the GPI signal sequence had a profound effect on induction and efficacy of CSP-specific immune response after genetic immunization of BALB/c mice with a gene gun. The CSP produced from the plasmid lacking the GPI anchor signal sequence (CSP-A) was secreted and soluble, but that produced by the CSP+A plasmid was not. The CSP-A plasmid induced a highly polarized Th2 type response, in which the CSP-specific IgG antibody titer was three- to fourfold higher, and the protective effect was significantly greater than that induced by the CSP+A plasmid. Thus, these two physical forms of CSP induced quantitatively and qualitatively different immune responses that also differed in protective efficacy. Engineering plasmid constructs for proper cellular localization of gene products is a primary consideration for the preparation of optimally efficacious DNA vaccines.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Glicosilfosfatidilinositóis/genética , Malária/prevenção & controle , Proteínas de Protozoários/imunologia , Vacinas de DNA/uso terapêutico , Animais , Antígenos de Protozoários/genética , Biolística , Feminino , Imunoglobulina G/biossíntese , Isotipos de Imunoglobulinas/biossíntese , Malária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium berghei/genética , Plasmodium berghei/imunologia , Sinais Direcionadores de Proteínas , Proteínas de Protozoários/genética , Deleção de Sequência , Células Th2/imunologia , Fatores de TempoRESUMO
The circumsporozoite protein (CSP) from the surface of sporozoite stage Plasmodium sp. malaria parasites is among the most important of the malaria vaccine candidates. Gene gun injection of genetic vaccines encoding Plasmodium berghei CSP induces a significant protective effect against sporozoite challenge; however, intramuscular injection does not. In the present study we compared the immune responses and protective effects induced by P. berghei CSP genetic vaccines delivered intradermally with a needle or epidermally with a gene gun. Mice were immunized three times at 4-week intervals and challenged by a single infectious mosquito bite. Although 50 times more DNA was administered by needle than by gene gun, the latter method induced significantly greater protection against infection. Intradermal injection of the CSP genetic vaccine induced a strong Th1-type immune response characterized by a dominant CSP-specific immunoglobulin G2a (IgG2a) humoral response and high levels of gamma interferon produced by splenic T cells. Gene gun injection induced a predominantly Th2-type immune response characterized by a high IgG1/IgG2a ratio and significant IgE production. Neither method generated measurable cytotoxic T lymphocyte activity. The results indicate that a gene gun-mediated CS-specific Th2-type response may be best for protecting against malarial sporozoite infection when the route of parasite entry is via mosquito bite.
Assuntos
Vacinas Antimaláricas/administração & dosagem , Malária/prevenção & controle , Plasmodium berghei/imunologia , Proteínas de Protozoários/genética , Vacinas de DNA/administração & dosagem , Animais , Anticorpos Antiprotozoários/sangue , Biolística/métodos , Citocinas/biossíntese , Injeções Intradérmicas , Ativação Linfocitária , Vacinas Antimaláricas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Plasmodium berghei/genética , Proteínas de Protozoários/imunologia , Baço/citologia , Linfócitos T Citotóxicos , Vacinação , Vacinas de DNA/imunologiaRESUMO
A novel human gene, TRPC5, was cloned from the region of Xq23 that contains loci for nonsyndromic mental retardation (MRX47 and MRX35) and two genes, DCX and HPAK3, implicated in two X-linked disorders (LISX and MRX30). Within a single YAC, we have determined the order cen-HPAK3(5'-3')-DCX(3'-5')-DXS7012E-TRPC5(3'-5' )-ter. TRPC5 encodes a 974-residue novel human protein (111.5 kDa predicted mass) and displays 99% homology with mouse TRP5, (MGD-approved symbol Trrp5) a novel member of a family of receptor-activated Ca2+ channels. It contains eight transmembrane domains, including a putative pore region. A transcript larger than 9.5 kb is observed only in fetal and adult human brain, with a relatively higher level in the adult human cerebellum. We devised an efficient method, Incorporation PCR SSCP (IPS), for detection of gene alterations. Five single-nucleotide variations in the TRPC5 gene were identified in males with mental retardation. However, these were found to be polymorphic variants. Exclusive expression of the TRPC5 gene in developing and adult brain suggests a possible role during development and provides a candidate gene for instances of mental retardation and other developmental defects.
Assuntos
Canais de Cálcio/genética , Proteínas de Transporte de Cátions , Alinhamento de Sequência , Sequência de Aminoácidos , Animais , Encéfalo , Canais de Cálcio/isolamento & purificação , Clonagem Molecular , Análise Mutacional de DNA , Proteína Duplacortina , Éxons , Expressão Gênica , Humanos , Deficiência Intelectual/genética , Camundongos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Polimorfismo Conformacional de Fita Simples , Canais de Cátion TRPC , Cromossomo X/genéticaRESUMO
Collections of human sera from malaria-endemic areas would be valuable for identifying and characterizing antigens as malaria vaccine candidates if the contributing serum donors' ability to resist infection were fully characterized. We prepared such a serum collection from 26 apparently immune Nigerian adults who failed to develop patent parasitemia for at least 20 weeks following a documented increase in antibodies to the circumsporozoite protein (CSP) from Plasmodium falciparum. Volunteers were evaluated five times per week for malaria symptoms and bimonthly for parasites by examining thick blood smears. The incidence rate over 13 months for the cohort was 42% (47 malaria-confirmed volunteers) and the risk of infection was 1.3 infections/year. Responses to CSP did not correlate with protection. Because antibody responses to antigens other than CSP may be associated with protection, the sera from these immune individuals may be useful for identifying and characterizing other potential malaria vaccine candidates.
Assuntos
Antígenos de Protozoários/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adulto , Animais , Antígenos de Protozoários/sangue , Antimaláricos/uso terapêutico , Estudos de Coortes , Combinação de Medicamentos , Feminino , Humanos , Incidência , Estudos Longitudinais , Malária Falciparum/sangue , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Masculino , Pessoa de Meia-Idade , Nigéria/epidemiologia , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/sangue , Pirimetamina/uso terapêutico , Chuva , Sulfadoxina/uso terapêuticoRESUMO
Purified rabbit immunoglobulin raised against yeast-expressed recombinant FVO or 3D7 Plasmodium falciparum merozoite surface protein-1 (MSP-1) 19k-D C terminal fragment (MSP-1(19)) was transfused into malaria-naive Aotus nancymai monkeys that were immediately challenged with FVO asexual stage malaria parasites. Control monkeys received rabbit immunoglobulin raised against the sexual stage antigen Pfs25 or Aotus hyperimmune serum obtained from monkeys immunized by P. falciparum infection and drug cure. Passive transfer of rabbit anti-MSP-1(19) failed to protect against homologous or heterologous challenge and, when compared with negative controls, there were no differences in prepatent periods or time to treatment. Interestingly, rabbit anti-MSP-1(19), but not anti-Pfs25, immunoglobulin, and immune monkey serum prevented the development of antibodies directed against MSP-1(19) fragment by infected monkeys, indicating that the antibodies were reactive with native MSP-1(19) antigen in vivo. The prepatent period and time to treatment was greatly delayed in the two monkeys that received Aotus immune serum, both of which developed a chronic intermittent low level infection. In vitro parasite growth inhibition assays (GIAs) confirmed the presence of inhibitory activity (40% maximum inhibition) in concentrated anti-MSP-1(19) immunoglobulin (4.8 mg/ml), but the peak concentrations we achieved in vivo (1 mg/ml) were not inhibitory in vitro. Subinhibitory levels of anti-MSP-1(19) antibodies achieved by passive transfer were not protective against P. falciparum challenge.
Assuntos
Anticorpos Antiprotozoários/imunologia , Imunização Passiva , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium falciparum/imunologia , Animais , Aotus trivirgatus , Malária Falciparum/prevenção & controle , Plasmodium falciparum/crescimento & desenvolvimento , Coelhos , Proteínas Recombinantes/imunologiaRESUMO
The circumsporozoite protein (CSP) is a target for effector Ab and cell mediated immunity against malaria parasites; DNA vaccination can induce both types of effector response. The immunogenicity and efficacy of two DNA plasmids expressing different amounts of Plasmodium berghei CSP were evaluated by immunizing BALB/c mice i.m. or epidermally and by varying the number of immunizations (one to three doses) and the interval between immunizations. Expanding the interval gave the strongest effect, increasing efficacy and antibody boosting, and, in the case of epidermal vaccination, promoting a switch in CSP-specific IgG isotypes from IgG1 to a balance with IgG2a. The strongest humoral immune response and the greatest level of protection were induced by vaccinating epidermally with high expresser plasmid, using a gene gun to administer three doses at 6-wk intervals. For this group, the mean, repeat-specific, prechallenge antibody titer among mice not infected after challenge was significantly higher than that in infected mice, but the mean prechallenge titers for antibody reactive with whole sporozoites were not significantly different. The interval-dependent induction of IgG2a antibodies by epidermal vaccination contradicts the widely held belief that antibody responses induced by this method are restricted to those that are Th2 dependent.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Plasmodium berghei/imunologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Animais , Bovinos , Epiderme , Feminino , Humanos , Esquemas de Imunização , Imunização Secundária , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Injeções Intradérmicas , Injeções Intramusculares , Malária/imunologia , Malária/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/biossíntese , Plasmídeos/síntese química , Plasmídeos/genética , Plasmodium berghei/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Análise de Sequência de DNARESUMO
Serum antibodies from malaria immune donors can inhibit merozoite dispersal by forming immune complexes through surface-accessible regions of membrane associated antigens. Such merozoite forms are referred to as immune clusters of merozoites (ICM). Antibodies dissociated from ICM of Plasmodium falciparum identify a restricted subset of antigens, including merozoite surface protein-1 (MSP-1). We performed epitope mapping by comparing the reactivity of whole immune sera and ICM-derived antibodies in immunoblotting assays, using fourteen overlapping recombinant MSP-1 fragments, and by ELISA, using each of the 1720 octapeptides encoded within MSP-1. Antibodies in immune sera reacted with thirteen recombinant fragments and hundreds of octapeptides, but antibodies derived from ICM reacted with only six recombinant fragments and twenty octapeptides. Recombinant fragment recognition by ICM-derived antibodies was delimited to three regions 150-200 residues long, with seven of the octapeptide epitopes also mapping to these regions. The octapeptides recognized most strongly by antibodies in whole serum corresponded to the degenerate repeats near the N-terminus of MSP-1, however, neither recombinant fragments, nor octapeptides containing these degenerate repeats, were recognized by ICM-derived antibodies. Compared to reactions with recombinant fragments, the reactions observed with octapeptides were weak and may represent low-affinity mimetopes or cross-reactions. Alternatively, they may represent reactions with a portion of an epitope assembled from more than one non-contiguous peptide. These results suggest that ICM-derived antibodies can be used to map surface-accessible epitopes on MSP-1 and that the recombinant fragments with which they react are appropriate candidates for further evaluation as components of a malaria vaccine.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Mapeamento de Epitopos , Plasmodium falciparum/imunologia , Precursores de Proteínas/imunologia , Proteínas de Protozoários/imunologia , Animais , Antígenos de Protozoários/química , Clonagem Molecular , Epitopos/imunologia , Escherichia coli/genética , Soros Imunes/imunologia , Immunoblotting , Proteína 1 de Superfície de Merozoito , Oligopeptídeos/imunologia , Fragmentos de Peptídeos , Plasmodium falciparum/fisiologia , Precursores de Proteínas/química , Proteínas de Protozoários/química , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Two clinical lots of alum-adsorbed SPf66 vaccine produced in the United States were evaluated in separate, open-label, Phase I clinical trials involving 15 healthy, malaria-naive, 18-45-year old men and women. Subjects received 2 mg doses subcutaneously in alternate arms at 0, one, and six months. Safety was assessed by monitoring local and systemic reactions and laboratory parameters. The most common side effects were erythema and local tenderness at the site of injection, which increased in frequency with subsequent doses of vaccine. These local reactions were considered mild and resolved within 24-48 hr. Eleven of 14 volunteers who received all three doses of vaccine seroconverted by enzyme-linked immunosorbent assay. The distribution of high, medium, and low nonresponders was comparable with that seen in trials of Colombian-produced vaccine. One high responder developed antibodies reactive with asexual stage parasite antigens by immunofluorescence and immunoblot. The results indicated that at full adult doses, SPf66 of U.S. origin is mildly reactogenic and induces immune responses similar to those reported for SPf66 of Colombian origin.
Assuntos
Anticorpos Antiprotozoários/sangue , Imunoglobulina G/sangue , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/efeitos adversos , Proteínas Recombinantes , Adulto , Animais , Feminino , Humanos , Injeções Subcutâneas , Malária Falciparum/imunologia , Masculino , Proteínas de Protozoários/administração & dosagem , Estados UnidosRESUMO
The model of protective immunity induced by immunization with irradiated plasmodia sporozoites (SPZ) has become the prototype for a promising vaccine strategy based on Ab and CTL responses directed against pre-erythrocytic stage Ags, in particular the circumsporozoite protein (CSP) and sporozoite surface protein 2 (SSP2). However, results from recently conducted vaccine studies suggest that T cell responses directed against additional specificities might also be required for protection. We have tested this hypothesis by examining human T lymphocytes from irradiated Plasmodium falciparum SPZ-immune volunteers for proliferative reactivities to parasitized red blood cells (pRBC) and recombinant proteins and synthetic peptides representing certain liver and blood stage Ags. In this work, we report that although SPZ-induced protective immunity is stage-specific, SPZ-immune lymphocytes recognized determinants associated with erythrocytic and liver stage parasites. Thus, protective immunity induced by irradiated SPZ may depend upon responses against pre-erythrocytic Ags in addition to CSP and SSP2.
Assuntos
Antígenos de Protozoários/sangue , Fígado/parasitologia , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Linfócitos T/imunologia , Adulto , Sequência de Aminoácidos , Animais , Humanos , Fígado/imunologia , Ativação Linfocitária , Masculino , Dados de Sequência MolecularRESUMO
The virulent avian influenza virus A/Ty/Ont/7732/66 (H5N9) (Ty/Ont) causes a rapid destruction of lymphoid cells in infected birds. Avian macrophage cell lines, HD11 and MQ-NCSU, support productive replication of Ty/Ont and other influenza viruses. Therefore, the ability of these cell lines to produce nitric oxide (NO), a potentially cytotoxic mediator, in response to infection with Ty/Ont was examined. Although treatment with bacterial lipopolysaccharides (LPS) resulted in high NO levels, infection of macrophages with Ty/Ont resulted in NO levels lower than NO levels in untreated cells. Furthermore, Ty/Ont was able to inhibit the positive response to LPS in cultures simultaneously treated with LPS and virus. However, inactivated influenza virus did not exhibit this inhibitory effect. Different strains of influenza virus varied in their ability to inhibit NO production by the macrophages; this may be related to the level of virus replication in these cells. These data suggest that the ability of the avian macrophage to activate the NO synthesis pathway is seriously impaired by infection with virulent influenza viruses such as Ty/Ont.
Assuntos
Vírus da Influenza A/patogenicidade , Macrófagos/metabolismo , Macrófagos/microbiologia , Óxido Nítrico/biossíntese , Aminoácido Oxirredutases/antagonistas & inibidores , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Linhagem Celular , Galinhas , Vírus da Influenza A/fisiologia , Influenza Aviária/etiologia , Macrófagos/imunologia , Camundongos , Óxido Nítrico Sintase , Orthomyxoviridae/patogenicidade , Orthomyxoviridae/fisiologia , Especificidade da Espécie , Replicação Viral , ômega-N-MetilargininaRESUMO
Antibody responses to the glycoprotein precursor of the major merozoite surface antigens of Plasmodium falciparum (gp195) were investigated in acutely infected Thai adults. Specific IgG antibody was assayed by enzyme-linked immunosorbent assay using a recombinant fragment derived from the N-terminal region of gp195 as the capture antigen. Two control groups were found to be without significant cross-reacting antibody. Among occupationally exposed soldiers, 84 of 85 men developed positive antibody responses during acute falciparum malaria. Mean antibody levels began to increase at the time of diagnosis, peaking, often at high titers, within two weeks, and then decreased with an initial serum half-life of less than one month. The high frequency of gp195 antibody responses underscores a potential role in serodiagnosis, whereas the dynamic nature of the response suggests that a rigorous schedule of prospective serum sampling will be required to accurately assess the relationship between these antibodies and protection.
Assuntos
Antígenos de Protozoários/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Adolescente , Animais , Antígenos de Protozoários/uso terapêutico , Criança , Humanos , Imunidade Inata , Malária Falciparum/terapia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , TailândiaRESUMO
The virulent avian influenza virus A/Ty/Ont/7732/66 (H5N9) (Ty/Ont) causes severe destruction of the lymphoid cells in infected birds. Previous studies have suggested that viral infection of macrophages may be involved. However, Ty/Ont failed to replicate productively in primary cultures of chicken macrophages. Therefore, in an effort to develop an in vitro system for our studies, we examined the susceptibility of an avian macrophage cell line, HD11, to Ty/Ont. We found that Ty/Ont replicated in the HD11 cells to high titres, as measured by haemagglutination (HA) assays and infectivity yields. To determine whether this property was unique to Ty/Ont, we also examined the replication of influenza viruses representative of all 13 HA subtypes and an attenuated variant of Ty/Ont. All of the tested viruses replicated in HD11 cells; the avirulent strains required the presence of trypsin in the culture medium whereas virulent viruses and the attenuated variant of Ty/Ont did not. These results suggest that the HD11 cells can support the replication of a wide variety of influenza viruses and that this continuous avian cell line may prove useful for in vitro studies on these viruses.
Assuntos
Vírus da Influenza A/fisiologia , Macrófagos/microbiologia , Replicação Viral , Animais , Linhagem Celular , GalinhasAssuntos
Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Malária/imunologia , Glicoproteínas de Membrana/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários , Sequência de Aminoácidos , Animais , Deleção Cromossômica , Humanos , Immunoblotting , Glicoproteínas de Membrana/genética , Homologia de Sequência do Ácido NucleicoRESUMO
MSA2 is a strain variable blood-stage merozoite surface antigen of Plasmodium falciparum. We have derived the MSA2 nucleotide sequence for four cloned parasite isolates. Comparison with three other published sequences suggests that variation may be limited, and that the architecture of the gene can be conveniently described by segregation into four distinct regions. The N and C terminal regions (Regions 1 and 4) are highly conserved in all seven genes. Six of these seven MSA2 genes can be grouped in a single family, within which variation is largely limited to a region characterized by the presence of tandem repeats (Region 2). We have observed two new forms of repeat in a Gly, Ser, Ala-rich block, and noted the absence of repeat in this block of the CAMP strain. The region downstream of the repeat region (Region 3) is highly conserved within this family. Immunochemical analysis reveals that MSA2 is one of the antigens recognized by immune antibodies eluted from intact merozoites. Regions 2 and 3, expressed as recombinant proteins, are recognized by these antibodies, suggesting that these regions are exposed at the surface of the intact merozoite.
Assuntos
Antígenos de Protozoários/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Alelos , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Sequência de Bases , Western Blotting , DNA de Protozoário/genética , Dados de Sequência Molecular , Proteínas de Protozoários/imunologia , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
When malaria schizont-infected erythrocytes are cultured with immune serum, antibodies prevent dispersal of merozoites, resulting in the formation of immune clusters of merozoites (ICM) and inhibition of parasite growth. Antigens recognized by these antibodies were identified by probing two dimensional immunoblots of Plasmodium falciparum antigens with antibodies dissociated from immune complexes present at the surface of merozoites in ICM. Total immune serum recognized 88 of the 135 protein spots detected by colloidal gold staining, but antibodies dissociated from immune complexes recognized only 15 protein spots attributable to no more than eight distinct antigens. Antigens recognized by antibodies that inhibit merozoite dispersal include the precursor to the major merozoite surface antigens (gp195), a 126-kDa serine-repeat antigen (SERA), the 130-kDa protein that appears to bind to glycophorin (GBP130), and the approx. 45-kDa merozoite surface antigen. One other antigen (230/215-kDa doublet) was identified by using antibodies affinity purified from recombinant expression proteins. The identities of the other three antigens (150 kDa, 127 kDa and less than 30 kDa) were not determined. This approach provides a strategy for identifying epitopes accessible at the merozoite surface which may be important components of a multivalent vaccine against blood stages of P. falciparum.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/análise , Eritrócitos/imunologia , Plasmodium falciparum/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/isolamento & purificação , Especificidade de Anticorpos , Antígenos de Protozoários/imunologia , Eletroforese em Gel de Poliacrilamida , Eritrócitos/parasitologia , Haplorrinos , ImmunoblottingRESUMO
DNA encoding an antigen of 101,000 apparent molecular weight from the human malaria parasite Plasmodium falciparum was cloned and sequenced. Genomic DNA from the Camp strain covering the complete coding region along with cDNA from the FCR3 strain covering 81% of the coding region were obtained. The cloned DNA specified a full-length protein of 743 amino acids which included two tandemly repeated regions, one near the amino terminus containing eight hexapeptide repeats of sequence TVNDEDED, and the second near the carboxyl terminus containing primarily KE and KEE repeats. The latter repeated region is encoded by a 174-base stretch of mRNA containing only a single pyrimidine. Except for a putative leader sequence located at the amino terminus of the protein, the protein is hydrophilic and highly charged with a calculated isoelectric point of 5.6. Sequences from the Camp and FCR3 strains are very close and are also nearly identical to the partial cDNA sequence of the acidic basic repeated antigen (ABRA) protein from the FC27 strain (Stahl, H.D., Bianco, A.E., Crewther, R.F., Anders, R.F., Kyne, A.P., Coppel, R. L., Mitchell, G.F., Kemp, D.J., and Brown, G.V. (1986) Mol. Biol. Med. 3, 351-368). ABRA was previously shown to be located at the merozoite surface and in the parasitophorous vacuole. Because of its location and because it becomes complexed to merozoites when schizonts rupture in the presence of immune serum, ABRA is a candidate component of a malaria vaccine.
Assuntos
Antígenos de Protozoários/análise , Plasmodium falciparum , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Antígenos de Protozoários/genética , Sequência de Bases , Clonagem Molecular , Técnicas de Imunoadsorção , Ponto Isoelétrico , Dados de Sequência Molecular , Plasmodium falciparum/genética , Vacúolos/análiseRESUMO
The liver merozoites of malaria parasites are of paramount importance, as they initiate the parasite invasion of red blood cells and start the cycle associated with the clinical features of malaria. Investigating liver merozoite antigen is difficult because of the lack of a rodent model of human malaria. In addition, only a low proportion of cells are obtained in vivo, the parasites from Cebus and Aotus monkeys are immature, and in-vitro experiments with liver cells are often confounded by contamination with the natural mosquito flora copurified with the sporozoites used for seeding the liver cultures. In our study, mature liver schizonts were shown to possess many of the antigenic determinants recognized by MoAbs and sera specific for defined sporozoite and blood-stage antigens. We employed an immunofluorescence procedure based on evaluating parasites in cryosections prepared from infected chimpanzee liver. Sufficient numbers of sectioned parasites were evaluated with each antibody to assure the reproducibility of the results, and the fixation procedure used was sufficiently non-destructive to parasite antigens so that clear differences between reactions of specific antibodies and negative controls were observed. Our evidence for sharing of epitopes by liver merozoites and sporozoites or by liver merozoites and asexual blood-stage parasites raises the possibility that immune responses elicited against sporozoites or asexual stage antigens being considered as vaccine candidates may also act against this important, little-studied stage of the parasite.