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1.
Scand J Rheumatol ; 50(3): 169-177, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33300420

RESUMO

Objectives: The study aimed to evaluate the feasibility of a blood flow restriction (BFR) training regimen in patients with rheumatoid arthritis (RA); and to compare the effects of 4 weeks of BFR training with low-intensity strength training on muscle strength, muscle endurance, and joint pain in patients with RA.Method: In this non-blinded pilot randomized controlled trial, 18 women with RA aged 18-65 years performed low-intensity strength training for the lower limbs three times a week for 4 weeks, and were randomized to train with or without occlusion bands. The primary outcomes were registration of the recruitment process, compliance with training sessions, side effects, perceived pain, and a satisfaction survey. The secondary outcomes were changes in muscle strength, muscle endurance, and joint pain.Results: The findings of this pilot study included a challenging recruitment process, well tolerated training and test protocols, overall good patient satisfaction, no serious side effects, and high compliance. Both groups achieved significant improvements in knee extensor strength from baseline to follow-up, with a change of 11.5 kg [interquartile range (IQR) 9.8;13.0] in the intervention group and 8.4 kg (IQR 5.5;12.4) in the control group, and a significant between-group difference in favour of the intervention group (p = 0.0342).Conclusions: The feasibility results of this study indicated a challenging recruitment process, general satisfaction with the BFR and exercises, good compliance, and only expected non-serious side effects. BFR training may improve knee extensor strength in women with RA, compared low-intensity strength training without BFR.


Assuntos
Artrite Reumatoide/terapia , Terapia por Exercício/métodos , Articulação do Joelho/fisiopatologia , Força Muscular/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Adulto , Idoso , Artrite Reumatoide/fisiopatologia , Estudos de Viabilidade , Feminino , Humanos , Pessoa de Meia-Idade , Projetos Piloto , Treinamento Resistido/métodos , Resultado do Tratamento
2.
Acta Anaesthesiol Scand ; 61(5): 532-538, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28374467

RESUMO

BACKGROUND: In recent years, large trials have increased the level of evidence for intravenous (IV) fluid therapy, at least in the intensive care setting. It is less clear whether this change in the evidence base has been associated with changes in IV fluid use in different hospital departments. METHODS: We obtained details from the regional pharmacy regarding IV fluids issued to hospital departments in the Danish Capitol Region from January 2012 to May 2015. We used paired Wilcoxon's signed-rank test to analyse changes in the issuing in different departments. RESULTS: Total regional issuing of IV fluids showed increase in crystalloid solutions (9%; P = 0.001) and decrease in colloid solutions (59%; P = 0.005). Subtype analysis showed increased issuing of buffered crystalloids (36%; P = 0.001), human albumin (30%; P < 0.0001) and decreased issuing in synthetic colloid solutions (82%; P < 0.0001) from Q1 2012 to Q2 2015. At the departmental level, the issuing of synthetic colloid solutions decreased markedly to all settings. The issuing of buffered crystalloids increased to orthopaedic (226%; P = 0.03) and to general surgery departments (686%; P = 0.002). Albumin solutions were increasingly issued to anaesthesia departments (63%; P = 0.005) and was rarely issued to general surgery and orthopaedic departments. CONCLUSIONS: The issuing of IV fluid solutions to hospital departments has changed markedly over the last years to less colloid, in particular the synthetic solutions, and relatively more issuing of crystalloids, in particular the buffered solutions.


Assuntos
Albuminas/administração & dosagem , Coloides/administração & dosagem , Hidratação/métodos , Hidratação/estatística & dados numéricos , Departamentos Hospitalares , Soluções Isotônicas/administração & dosagem , Cloreto de Sódio/administração & dosagem , Administração Intravenosa , Soluções Cristaloides , Dinamarca , Humanos
3.
Acta Anaesthesiol Scand ; 61(10): 1384-1397, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28901549

RESUMO

BACKGROUND: Patients in the intensive care unit (ICU) are often anaemic due to blood loss, impaired red blood cell (RBC) production and increased RBC destruction. In some studies, more than half of the patients were treated with RBC transfusion. During storage, the RBC and the storage medium undergo changes, which lead to impaired transportation and delivery of oxygen and may also promote an inflammatory response. Divergent results on the clinical consequences of storage have been reported in both observational studies and randomised trials. Therefore, we aim to gather and review the present evidence to assess the effects of shorter vs. longer storage time of transfused RBCs for ICU patients. METHODS: We will conduct a systematic review with meta-analyses and trial sequential analyses of randomised clinical trials, and also include results of severe adverse events from large observational studies. Participants will be adult patients admitted to an ICU and treated with shorter vs. longer stored RBC units. We will systematically search the Cochrane Library, MEDLINE, Embase, BIOSIS, CINAHL and Science Citation Index for relevant literature, and we will follow the recommendation by the Cochrane Collaboration and the Preferred Reporting Items for Systemtic Review and Meta-Analysis (PRISMA)-statement. We will assess the risk of bias and random errors, and we will use the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach to evaluate the overall quality of evidence. CONCLUSION: We need a high-quality systematic review to summarise the clinical consequences of RBC storage time among ICU patients.


Assuntos
Preservação de Sangue , Transfusão de Eritrócitos , Unidades de Terapia Intensiva , Preservação de Sangue/efeitos adversos , Protocolos Clínicos , Humanos , Avaliação de Resultados em Cuidados de Saúde , Revisões Sistemáticas como Assunto , Fatores de Tempo
4.
BMC Prim Care ; 24(1): 206, 2023 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-37798651

RESUMO

BACKGROUND: People with a severe mental illness (SMI) have shorter life expectancy and poorer quality of life compared to the general population. Most years lost are due to cardiovascular disease, respiratory disease, and various types of cancer. We co-designed an intervention to mitigate this health problem with key stakeholders in the area, which centred on an extended consultations for people with SMI in general practice. This study aimed to1) investigate general practitioners' (GPs) experience of the feasibility of introducing extended consultations for patients with SMI, 2) assess the clinical content of extended consultations and how these were experienced by patients, and 3) investigate the feasibility of identification, eligibility screening, and recruitment of patients with SMI. METHODS: The study was a one-armed feasibility study. We planned that seven general practices in northern Denmark would introduce extended consultations with their patients with SMI for 6 months. Patients with SMI were identified using practice medical records and screened for eligibility by the patients' GP. Data were collected using case report forms filled out by practice personnel and via qualitative methods, including observations of consultations, individual semi-structured interviews, a focus group with GPs, and informal conversations with patients and general practice staff. RESULTS: Five general practices employing seven GPs participated in the study, which was terminated 3 ½ month ahead of schedule due to the COVID-19 pandemic. General practices attempted to contact 57 patients with SMI. Of these, 38 patients (67%) attended an extended consultation, which led to changes in the somatic health care plan for 82% of patients. Conduct of the extended consultations varied between GPs and diverged from the intended conduct. Nonetheless, GPs found the extended consultations feasible and, in most cases, beneficial for the patient group. In interviews, most patients recounted the extended consultation as beneficial. DISCUSSION: Our findings suggest that it is feasible to introduce extended consultations for patients with SMI in general practice, which were also found to be well-suited for eliciting patients' values and preferences. Larger studies with a longer follow-up period could help to assess the long-term effects and the best implementation strategies of these consultations.


Assuntos
COVID-19 , Medicina Geral , Transtornos Mentais , Humanos , Estudos de Viabilidade , Pandemias , Qualidade de Vida , COVID-19/epidemiologia , Transtornos Mentais/diagnóstico , Transtornos Mentais/epidemiologia , Transtornos Mentais/terapia , Encaminhamento e Consulta
5.
Curr Opin Microbiol ; 1(1): 75-81, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10066469

RESUMO

Recent studies show that the coupling of fimbrial adhesins of uropathogenic Escherichia coli and pathogenic Neisseria species to their complementary receptors on host cells is a dynamic event, involving specific signaling to the bacteria as well as to the host cell. These studies have unveiled intriguing and novel mechanisms by which bacteria utilize their fimbriae to promote virulence at the mucosal surface and in deeper tissue.


Assuntos
Escherichia coli/patogenicidade , Fímbrias Bacterianas/fisiologia , Neisseria/patogenicidade , Adesinas Bacterianas/metabolismo , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Humanos , Neisseria meningitidis/patogenicidade , Infecções por Neisseriaceae/imunologia , Infecções por Neisseriaceae/microbiologia , Fagocitose , Transdução de Sinais , Sistema Urinário/microbiologia
6.
Biochim Biophys Acta ; 1397(2): 137-40, 1998 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-9565669

RESUMO

The nucleotide sequence of a 3 kb region downstream of pilC1 in Neisseria gonorrhoeae MS11 was analyzed. This region contains two open reading frames, ORF1 and ORF2, and several repetitive DNA elements. ORF1 encodes an outer membrane protein that shows homology to orf98 of Pediococcus acidilactici. PCR with primers specific for ORF1 revealed that the gene is present in all gonococcal strains tested. The other open reading frame, ORF2, is highly homologous to the putative integral membrane protein HI1680 of Haemophilus influenzae.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Fímbrias , Neisseria gonorrhoeae/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Fases de Leitura Aberta , Sequências Repetitivas de Ácido Nucleico
7.
FEMS Microbiol Lett ; 162(1): 25-30, 1998 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-9595660

RESUMO

Neisseria gonorrhoeae and Neisseria meningitidis are exclusively human pathogens. A crucial property of the pathogenicity of neisserial infection is the ability to adhere to human epithelial cells. Pili mediate adherence of these bacteria to target cells and thereby promote colonization and infection of mucosal surfaces. In order to identify and to learn more about the initial event during infection, a cDNA clone from a human cervical epithelial cell line was identified in a panning experiment using purified gonococcal pili as probe. Upon transfection of the cloned cDNA into COS-7 cells, both gonococci and meningococci adhered to these otherwise non-binding cells. The deduced amino acid sequence of the cDNA clone showed homology to a recently reported human cDNA, called WWP2, that encodes an N-terminal C2-like domain. The C2 domain has been shown to bind membrane phospholipids in a calcium-dependent manner and is thought to function in the intracellular compartmentalization of proteins. Antiserum raised against the product encoded by the cDNA did not inhibit bacterial adherence, indicating that the cloned gene is most likely involved in up-regulation of a surface receptor for pathogenic Neisseria.


Assuntos
Aderência Bacteriana/fisiologia , Proteínas de Transporte/fisiologia , DNA Complementar/genética , Células Epiteliais , Neisseria gonorrhoeae/patogenicidade , Neisseria meningitidis/patogenicidade , Animais , Células COS , Linhagem Celular , Colo do Útero/citologia , Clonagem Molecular , Células Epiteliais/microbiologia , Feminino , Fímbrias Bacterianas , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
8.
Vaccine ; 23(17-18): 2214-7, 2005 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-15755598

RESUMO

Animal infection models are valuable for the development and preclinical assessment of meningococcal vaccines in the absence of clear in vitro correlates of protection for protein-based serogroup B vaccines. It is only in animal models that interactions of the organism with the innate, humoral and cellular immune systems can be assessed. However, humans are the only natural host for Neisseria meningitidis and there is no ideal disease model using laboratory animals that mimics the course of human disease. The two most widely used models are intraperitoneal (i.p.) infection of adult mice or infant rats. The mouse i.p. infection model requires an exogenous iron source (e.g. human transferrin) to obtain a lethal bacteraemic infection and can be used to assess both active and passive immunisation. The virulence of wild-type and knockout mutants can also be compared. i.p. infection of infant rats has been used to assess passive protection provided by sera raised against vaccine candidates or human vaccine sera. However, the duration of bacteraemia is short, mortality is low and active protection cannot be assessed. Recent developments using transgenic mice expressing human CD46 give hope that improved models will be developed.


Assuntos
Vacinas Bacterianas/farmacologia , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/prevenção & controle , Neisseria meningitidis Sorogrupo B/imunologia , Administração Intranasal , Animais , Antígenos CD/genética , Modelos Animais de Doenças , Humanos , Imunização Passiva , Injeções Intraperitoneais , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/genética , Infecções Meningocócicas/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neisseria meningitidis Sorogrupo B/patogenicidade , Ratos , Vacinação
9.
J Bacteriol ; 167(1): 231-7, 1986 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-3087954

RESUMO

Cryptic plasmid DNA of Neisseria gonorrhoeae was found integrated into the gonococcal chromosome in both plasmid-bearing strains and plasmid-free strains. At several chromosomal locations only segments of the plasmid were found. However, in at least two strains an intact copy of the plasmid seemed to be present with the joints between the plasmid and the chromosomal DNA being located within the cppB gene of the cryptic plasmid. The cppB gene was shown to undergo a sequence-specific intragenic deletion. The deletion removed 54 base pairs, representing 18 amino acids, and did not affect the reading frame. It is proposed that the cryptic plasmid integrates into the chromosome and other gonococcal plasmids within this site-specific deletion region. Models for the site-specific recombination are presented.


Assuntos
Neisseria gonorrhoeae/genética , Plasmídeos , Recombinação Genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Deleção Cromossômica , Cromossomos Bacterianos , DNA Bacteriano/genética , Genes , Modelos Genéticos
10.
Proc Natl Acad Sci U S A ; 89(8): 3204-8, 1992 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-1348857

RESUMO

Pili of Neisseria gonorrhoeae undergo both phase and structural variation. Phase variation of gonococcal pili can be caused by a RecA-independent on/off switch in PilC, a protein involved in pilus biogenesis. We show here that spontaneous nonpiliated PilC- derivatives as well as PilC- insertional mutants have also acquired sequence alterations in pilE relative to the pilE gene of the piliated MS11mk(P+)-u parent, so that the pilin produced is processed to soluble S-pilin and can be released into the medium. It is proposed that pilin alterations are selected for in PilC- bacteria if the parental nonassembled pilin is toxic to the cells--i.e., is not degradable to S-pilin at rates sufficient to allow viability of the cells. Toxicity is indicated by the extreme instability of certain unassembled pilin sequences and by the low frequency of nonpiliated, pilin+, PilC- variants that emerge from piliated recA- cells. The presence of a point mutation changing leucine-39 to phenylalanine at the cleavage site for S-pilin in one nonpiliated, PilC-, recA- variant relative to its piliated parent is a further argument for a selective mechanism of structural diversity of the gonococcal pilin.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Fímbrias Bacterianas/fisiologia , Genes Bacterianos , Variação Genética , Neisseria gonorrhoeae/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Proteínas de Fímbrias , Fímbrias Bacterianas/ultraestrutura , Dados de Sequência Molecular , Neisseria gonorrhoeae/fisiologia , Neisseria gonorrhoeae/ultraestrutura , Sondas de Oligonucleotídeos , Recombinases Rec A/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência do Ácido Nucleico
11.
Cell Microbiol ; 2(4): 341-51, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11207590

RESUMO

In this study, we characterize the interaction between non-piliated (P-) Neisseria gonorrhoeae and human epithelial cells. P- mutants lacking the pilus subunit protein PilE attach at low levels to cells. Although the binding may not lead to heavy inflammatory responses, the interaction between P- Neisseria and host cells most probably play a role in colonization and asymptomatic carriage of the pathogen. Here we show that the adherence of P N. gonorrhoeae is blocked by GDP-beta-S [guanosine 5'-O(thio)diphosphate], a non-hydrolyzable GTP analogue, and by C3 exotoxin, an inhibitor of the small G-protein Rho. G-protein activators such as cholera toxin, that activates Gs, and fluoroaluminate, a general G-protein activator, induced bacterial adherence. Furthermore, increase of the extracellular free [Ca2+] dramatically enhanced adherence of non-piliated Neisseria. The pharynx and the urogenital tract are natural entry sites of the pathogenic Neisseria species, and at both sites the epithelial cells can be exposed to wide variations in Ca2+ concentration. Taken together, these data show the importance of extracellular Ca2+ in the pathogenic Neisseria-host interaction, and reveal a novel function of cholera toxin, namely induction of bacterial adherence.


Assuntos
Aderência Bacteriana/efeitos dos fármacos , Cálcio/farmacologia , Toxina da Cólera/farmacologia , Células Epiteliais/microbiologia , Proteínas de Ligação ao GTP/fisiologia , Guanosina Difosfato/análogos & derivados , Neisseria/patogenicidade , Proteínas rho de Ligação ao GTP/fisiologia , Alumínio/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Toxinas Botulínicas/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Fímbrias Bacterianas/genética , Flúor/farmacologia , Guanosina Difosfato/farmacologia , Humanos , Mutação , Neisseria/genética , Tionucleotídeos/farmacologia , Ativação Transcricional , Proteínas rho de Ligação ao GTP/antagonistas & inibidores
12.
EMBO J ; 10(2): 477-88, 1991 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1671354

RESUMO

Pili prepared from Neisseria gonorrhoeae contain minor amounts of a 110 kd outer membrane protein denoted PilC. The corresponding gene exists in two copies, pilC1 and pilC2, in most strains of N.gonorrhoeae. In the piliated strain MS11(P+), only one of the genes, pilC2, was expressed. Inactivation of pilC2 by a mTnCm insertion resulted in a nonpiliated phenotype, while a mTnCm insertion in pilC1 had no effect on piliation. Expression of pilC was found to be controlled at the translational level by frameshift mutations in a run of G residues positioned in the region encoding the signal peptide. Nonpilated (P-), pilin expressing colony variants that did not express detectable levels of PilC were selected; all P+ backswitchers from these P-, PilC- clones were found to be PilC+. The structural gene for pilin, pilE, was sequenced and found to be identical in one P-, PilC- and P+, PilC+ pair. Most PilC- cells were completely bald whereas the PilC+ backswitcher had 10-40 pili per cell. Thus, a turn ON and turn OFF in the expression of PilC results in gonococcal pili phase variation. These results suggest that PilC is required for pilus assembly and/or translocation across the gonococcal outer membrane.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Fímbrias Bacterianas/fisiologia , Mutação da Fase de Leitura , Genes Bacterianos , Variação Genética , Neisseria gonorrhoeae/genética , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Proteínas de Fímbrias , Fímbrias Bacterianas/ultraestrutura , Dados de Sequência Molecular , Peso Molecular , Mutagênese Insercional , Neisseria gonorrhoeae/fisiologia , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico
13.
Mol Microbiol ; 25(1): 11-25, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11902714

RESUMO

Adherence of pathogenic Neisseria to target host cells is mediated by pili. PilC1 and PilC2 are two high-molecular-weight proteins involved in pilus assembly and cellular adherence functions of the pili. Inactivation of pilC1 or pilC2 in N. meningitidis resulted in clones that expressed the same number of pili as the parent, contained no alterations in pilE and showed no detectable differences in PilE glycosylation. However, the PilC2+ pilC1- mutant showed much reduced adherence to target cells, indicating that production of PilC1 is essential for pilus-mediated adherence. To study further the functional differences between the meningococcal pilC genes, we determined the complete nucleotide sequence of pilC1 and pilC2 of N. meningitidis. Alignment of six PilC sequences demonstrated that PilC is composed of both conserved and variable regions. By immunogold labelling of bacterial sections we showed that PilC is present in the membranes of both piliated and non-piliated bacteria. Further, we demonstrated that PilC is associated with the bacterial cell surface.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Fímbrias , Neisseria meningitidis/genética , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Sequência de Bases , Membrana Celular/metabolismo , Clonagem Molecular , Sequência Conservada , DNA Bacteriano/análise , Glicosilação , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Neisseria meningitidis/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência , Células Tumorais Cultivadas
14.
Microbiology (Reading) ; 141 ( Pt 10): 2367-77, 1995 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7581997

RESUMO

Pili of Neisseria gonorrhoeae mediate attachment of the bacteria to target cells and undergo both phase and antigenic variation. PilC is a 110 kDa minor pilus-associated protein involved in pilus biogenesis and attachment. The expression of PilC is turned on and off at high frequency and is controlled by frameshift mutations in a run of G residues positioned in the region encoding the signal peptide. Most strains of N. gonorrhoeae carry two copies of pilC. The DNA sequence of pilC1 of strain MS11 is presented and compared to the sequence of the 3' end of pilC2. These two genes are highly homologous, but not identical. The putative transcriptional terminator of pilC1 contains a pair of inverted uptake sequences for gonococcal DNA (5'-GCCGTCTGAA-3'). An 88 bp sequence located upstream of the pilC1 gene has also been reported to precede several opa genes of N. gonorrhoeae. Shorter regions positioned both downstream and upstream of pilC1 can also be found in silent pil loci as well as close to opa genes. The pilC genes are part of a duplication of a larger DNA region extending more than 2 kb downstream of the coding region.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Fímbrias , Genes Bacterianos , Família Multigênica , Neisseria gonorrhoeae/genética , Pili Sexual/genética , Sequência de Aminoácidos , Aderência Bacteriana/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Dados de Sequência Molecular , Pili Sexual/metabolismo , Regiões Promotoras Genéticas , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico
15.
Infect Immun ; 66(2): 469-73, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9453597

RESUMO

Piliated and nonpiliated Neisseria gonorrhoeae organisms were added on top of confluent layers of HEC-1-B cells, each maintained on a microporous Transwell-COL membrane. The bacteria released into the lower chamber were characterized with respect to the following virulence determinants: pili, which mediate adherence to target host cells; PilE, the major pilus subunit protein; and PilC, which is involved in pilus biogenesis and adherence. Even if >99% of the added bacteria of N. gonorrhoeae MS11 were piliated, bacteria recovered on the other side of the cell layer were predominantly nonpiliated. The recovered clones still expressed unassembled PilE protein, but 50% had lost PilC production. Nonpiliated gonococci, in which the 5' end of pilE had been deleted, were released in reduced numbers, and piliated recA bacteria added to the cell layer were not released at all, at time points when piliated recA+ clones were found at high numbers in the lower chamber. Our data indicate that bacteria producing unassembled PilE protein are selected for during passage through an epithelial cell layer. The finding that the pilE gene sequence was altered in the transmigrants suggests that pilin sequence variation is involved in the transcellular passage of N. gonorrhoeae.


Assuntos
Fímbrias Bacterianas/fisiologia , Neisseria gonorrhoeae/fisiologia , Sequência de Aminoácidos , Células Epiteliais/microbiologia , Feminino , Humanos , Dados de Sequência Molecular , Recombinases Rec A/genética
16.
Microb Pathog ; 24(5): 299-308, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9600862

RESUMO

Murine monoclonal antibodies (MAbs) against the A, B and C LPS serotypes of M. catarrhalis were generated and their binding specificity was examined in an enzyme-linked immunosorbent assay (ELISA). Two broadly cross-reactive monoclonal antibodies (MCA1 and MCC2) against the outer core region of LPS were further characterized. A panel of synthetic glycoproteins and glycolipids was used to determine the binding specificity of the MAbs. MCA1 and MCC2 bound specifically to alpha-Gal-(1-4)-beta-Gal of galabiose and globotriose glycoconjugates. The reactivity of the MAbs with galabiose was higher than that with globotriose. The MAbs could recognize the alpha-Gal-(1-4)-beta-Gal epitope only when it was in a terminal position. MCA1 was further shown to react with a similar epitope in the glycosylated type IV pili of N. meningitidis, which has been shown to contain a 1-4 linked digalactose at the terminal part of the saccharide present in the pili. MCA1 could efficiently recognize this epitope indicating that it was exposed on the surface of the pili.


Assuntos
Anticorpos Monoclonais , Epitopos/análise , Fímbrias Bacterianas/química , Lipopolissacarídeos/análise , Moraxella catarrhalis/química , Neisseria meningitidis/química , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Bactérias , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Fímbrias Bacterianas/imunologia , Humanos , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Moraxella catarrhalis/imunologia , Moraxella catarrhalis/patogenicidade , Neisseria meningitidis/imunologia
17.
Infect Immun ; 69(10): 6419-26, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11553586

RESUMO

Pili of Neisseria gonorrhoeae are phase-variable surface structures that mediate adherence to host target cells. Each pilus is composed of thousands of major pilus subunits, pilins, pilus-associated protein PilC, and possibly other components. Piliated and nonpiliated gonococcal clones may secrete a soluble smaller pilin (S-pilin) that is cleaved after amino acid 39 of the mature pilin protein. Here, purified S-pilin was found to migrate as a 61- to 64-kDa double band on nondenaturing gels, suggesting the formation of tetrameric S-pilin proteins with two isomeric forms. In situ studies of binding to formalin-fixed tissue sections demonstrated the binding of S-pilin to human tissue but not to tissue from mouse or rat organs, showing the presence of a human-specific receptor-binding domain within the pilin polypeptide. Pretreatment of the target tissues with proteinase K decreased gonococcal binding dramatically, whereas pretreatment with neuraminidase and meta-periodate, which cleave carbon-carbon linkages between vicinal hydroxyl groups in carbohydrates, did not affect gonococcal binding. In overlay assays, purified S-pilin bound to a band with a migration pattern and size similar to those of CD46, a cellular pilus receptor. Further, binding of N. gonorrhoeae to target cells and tissues could be blocked by both CD46 antibodies and purified S-pilin. These data argue that S-pilin interacts with a protein domain(s) of the CD46 receptor on human cells.


Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Membrana/metabolismo , Neisseria gonorrhoeae/metabolismo , Pili Sexual/metabolismo , Resinas Acrílicas , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias , Humanos , Masculino , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Desnaturação Proteica , Ratos , Ratos Sprague-Dawley , Solubilidade , Células Tumorais Cultivadas
18.
Mol Microbiol ; 25(4): 639-47, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9379894

RESUMO

Pili of Neisseria gonorrhoeae and Neisseria meningitidis mediate binding of the bacteria to human cell-surface receptors. We found that purified pili bound to a 55- to 60-kDa doublet band on SDS-PAGE of separated human epithelial cell extracts. This is a migration pattern typical of membrane cofactor protein (MCP or CD46). MCP is a widely distributed human complement regulatory protein. Attachment of the bacteria to epithelial cells was blocked by polyclonal and monoclonal antibodies directed against MCP, suggesting that this complement regulator is a receptor for piliated Neisseria. We proved this hypothesis by demonstrating that piliated, but not non-piliated, gonococci bound to CHO cells transfected with human MCP-cDNA. We also demonstrated a direct interaction between purified recombinant MCP and piliated Neisseria. Finally, recombinant MCP protein produced in E. coli inhibited attachment of the bacteria to target cells. Taken together, our data show that MCP is a human cell-surface receptor for piliated pathogenic Neisseria.


Assuntos
Antígenos CD/metabolismo , Fímbrias Bacterianas/metabolismo , Glicoproteínas de Membrana/metabolismo , Neisseria/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Antígenos CD/imunologia , Antígenos CD/farmacologia , Aderência Bacteriana , Western Blotting , Células CHO , Cricetinae , Citometria de Fluxo , Expressão Gênica , Humanos , Imuno-Histoquímica , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/farmacologia , Neisseria/patogenicidade , Receptores de Superfície Celular/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/metabolismo , Células Tumorais Cultivadas
19.
J Immunol ; 166(11): 6764-70, 2001 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-11359834

RESUMO

C4b-binding protein (C4BP) is an important plasma inhibitor of the classical pathway of complement activation. Several bacterial pathogens bind C4BP, which may contribute to their virulence. In the present report we demonstrate that isolated type IV pili from Neisseria gonorrhoeae bind human C4BP in a dose-dependent and saturable manner. C4BP consists of seven identical alpha-chains and one beta-chain linked together with disulfide bridges. We found that pili bind to the alpha-chain of C4BP, which is composed of eight homologous complement control protein (CCP) domains. From the results of an inhibition assay with C4b and a competition assay in which we tested mutants of C4BP lacking individual CCPs, we concluded that the binding area for pili is localized to CCP1 and CCP2 of the alpha-chain. The binding between pili and C4BP was abolished at 0.25 M NaCl, implying that it is based mostly on ionic interactions, similarly to what have been observed for C4b-C4BP binding. Furthermore, the N-terminal part of PilC, a structural component of pili, appeared to be responsible for binding of C4BP. Membrane cofactor protein, previously shown to be a receptor for pathogenic N. gonorrhoeae on the surface of epithelial cells, competed with C4BP for binding to pili only at high concentrations, suggesting that different parts of pili are involved in these two interactions. Accordingly, high concentrations of C4BP were required to inhibit binding of N. gonorrhoeae to Chang conjunctiva cells, and no inhibition of binding was observed with cervical epithelial cells.


Assuntos
Complemento C4b/metabolismo , Proteínas Inativadoras do Complemento , Proteínas de Fímbrias , Fímbrias Bacterianas/metabolismo , Glicoproteínas , Neisseria gonorrhoeae/metabolismo , Receptores de Complemento/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas de Bactérias/metabolismo , Ligação Competitiva/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Fímbrias Bacterianas/classificação , HEPES/farmacologia , Humanos , Proteínas Ligantes de Maltose , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Polissorbatos/farmacologia , Ligação Proteica/efeitos dos fármacos , Receptores de Complemento/sangue , Receptores de Complemento/isolamento & purificação , Receptores de Complemento/fisiologia , Soroalbumina Bovina/farmacologia , Cloreto de Sódio/farmacologia , Hidróxido de Sódio/farmacologia , Trometamina , Células Tumorais Cultivadas
20.
Mol Microbiol ; 13(3): 403-16, 1994 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-7997158

RESUMO

Pili of Neisseria gonorrhoeae are correlated with increased bacterial attachment to epithelial cells and undergo both phase and antigenic variation. Phase variation of gonococcal pili can be brought about by recombination events in the pilin structural gene, pilE, or by the on/off switch in expression of PilC, a pilus biogenesis protein for which two loci exist. We have studied the binding to epithelial cell lines and to fixed tissue sections of N. gonorrhoeae MS11 derivatives and mutants carrying structurally defined PilE and PilC proteins. In situ binding studies of N. gonorrhoeae to formalin-fixed tissue sections resulted in a binding pattern similar to that obtained using viable epithelial cell lines of different origin. Piliated gonococcal clones, containing different pilE sequences, varied dramatically from one another in their efficiencies at binding to corneal and conjunctival tissue, but bound equally well to cervical and endometrial tissues. Further, the binding data suggested that PilC expression by itself, i.e. without pili, cannot confer bacterial binding and that expression of either PilC1 or PilC2 does not confer different binding properties to the bacterial cells. Possible receptors for piliated gonococci were expressed in human tissues, such as cervix, endometrium, cornea, intestine, stomach, mid-brain and meninges, but not in human kidney. Pretreatment of the target tissues with Proteinase K decreased the gonococcal binding dramatically, whereas pretreatment with neuraminidase and meta-periodate, which cleave carbon-carbon linkages between vicinal hydroxyl groups in carbohydrates, did not affect attachment of gonococci. These data argue that pilus-dependent attachment of N. gonorrhoeae to human tissue may be mediated by a eukaryotic receptor having protein characteristics, and that the pilus subunit sequence may play an important role in the interaction with human cornea.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Fímbrias Bacterianas/genética , Neisseria gonorrhoeae/genética , Sequência de Aminoácidos , Animais , Aderência Bacteriana/genética , Proteínas de Bactérias/fisiologia , Sequência de Bases , Linhagem Celular , Epitélio , Proteínas de Fímbrias , Genes Bacterianos , Variação Genética , Humanos , Camundongos , Dados de Sequência Molecular , Neisseria gonorrhoeae/patogenicidade , Especificidade de Órgãos , Ratos , Recombinação Genética , Especificidade da Espécie
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