RESUMO
BACKGROUND: For patients with cT1 renal lesions, Partial Nephrectomy (PN) is the gold standard treatment. However, 20% of small renal masses are benign, situation in which the PN is an overtreatment. The percutaneous Renal Tumor Biopsy (RTB) may lower the risk of overtreatment as there is a 90% concordance rate on histotype between the RTB and the final pathology. It has been suggested that the RTB could increase the difficulty of the PN and increase the risk of surgical complications. OBJECTIVE: To compare surgical outcomes and complications of PN with or without previous RTB. DESIGN, SETTING, AND PARTICIPANTS: monocentric retrospective review of patients who underwent laparoscopic or robotic-assisted PN between January 2012 and December 2019. MEASUREMENTS: perioperative complications were recorded using Clavien-Dindo classification, peroperative data included operative time, clamping time and blood loss, and histological outcomes of RTB and PN. RESULTS AND LIMITATIONS: In total, 163 patients were included in our study. There were significantly less benign lesions in PN with prior RTB: 7% (4/56) vs. 20% (22/107) without prior RTB (P=0.03). There were no significant differences regarding Clavien-Dindo>2 perioperative complications with respectively 7% (4/56) vs. 10% (11/107) (P=0.57). Same goes for peroperative data such as duration of surgery (P=0.81), warm ischemia (P=0.07) and blood loss (P=0.13). CONCLUSIONS: RTB does not increase the risk of surgical complications of PN and may reduce the risk of small renal masses overtreatment.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Procedimentos Cirúrgicos Robóticos , Biópsia , Carcinoma de Células Renais/patologia , Humanos , Neoplasias Renais/patologia , Neoplasias Renais/cirurgia , Nefrectomia/efeitos adversos , Nefrectomia/métodos , Estudos Retrospectivos , Procedimentos Cirúrgicos Robóticos/efeitos adversos , Procedimentos Cirúrgicos Robóticos/métodos , Resultado do Tratamento , Isquemia QuenteRESUMO
INTRODUCTION: Mitomycin C is the gold standard intravesical adjuvant therapy for intermediate-risk non-muscle-invasive bladder cancer (NMIBC). Tensions in the supply of mitomycin have emerged in France since late 2019. The ANSM in agreement with the AFU proposed to use epirubicin, already available in other European countries in this indication. The objective of our study was to report the initial French experience with the use of epirubicin in adjuvant treatment of NMIBC. MATERIALS AND METHODS: We undertook a French multicenter retrospective descriptive study to collect, from the centers of the members of the CC-AFU bladder, the clinico-pathological data of the patients, the indications, the modalities of use (dose, indication, circuit in the pharmacy) and the tolerance data of epirubicin. The impact of the COVID-19 epidemic on treatment interruptions was also identified. Of the 20 centers contacted, 5 (25%) had implemented the epirubicin administration protocol developed by the CC-AFU bladder subcommittee. A total of 61 patients were treated with endovesical instillations of epirubicin between November 2019 and November 2020 for NMIBC at a single dose of 50mg. RESULTS: A total of 61 patients (mean age 67 years, 64-77 years) were treated with epirubicin, of which 45 (73.8%) were male. The patients had intermediate-risk NMIBC in 88.5%, the rest had high-risk disease. Induction therapy without or with maintenance was planned for 48 (78.7%) and 13 patients (21.3%), respectively. The preparation and administration of epirubicin was similar to that of mitomycin: central pharmacy preparation for same-day dispensing with immediate outpatient instillation. Unlike mitomycin, urinary alkalinization was not required. Of the 498 total instillations scheduled, 345 were performed (69.3%). The COVID-19 epidemic significantly impacted epirubicin delivery: one patient could not start treatment (1.6%), 8 patients (13.1%) had to discontinue it permanently; the rest of the patients underwent delayed instillations (18%). Other causes of discontinuation included infectious complications (9.8%). No major toxicities were reported. CONCLUSION: The implementation of an adjuvant epirubicin treatment protocol presented a good feasibility with low toxicity, without modifying the organization of the patients' care pathway. In the context of unpredictable mitomycin shortage, epirubicin represents a good therapeutic alternative in the endovesical adjuvant treatment of intermediate-risk NMIBC. LEVEL OF PROOF: 3.
Assuntos
Tratamento Farmacológico da COVID-19 , Neoplasias da Bexiga Urinária , Adjuvantes Imunológicos , Administração Intravesical , Idoso , Antibióticos Antineoplásicos , Vacina BCG/uso terapêutico , Protocolos Clínicos , Epirubicina/uso terapêutico , Feminino , Humanos , Masculino , Mitomicina , Invasividade Neoplásica , Estudos Retrospectivos , Neoplasias da Bexiga Urinária/patologiaRESUMO
PROBLEM: Given the important role of regulatory T cells (Treg) for successful pregnancy, the ability of soluble maternal and fetal pregnancy factors to induce human Treg was investigated. METHOD OF STUDY: Peripheral blood mononuclear cells (PBMCs) or isolated CD4+CD25â cells were cultured in the presence of pooled second or third trimester pregnancy sera, steroid hormones or supernatants from placental explants, and the numbers and function of induced CD4+CD25+FOXP3+ Treg were analysed. RESULTS: Third trimester pregnancy sera and supernatants of early placental explants, but not sex steroid hormones, induced an increase of Tregs from PBMCs. Early placental supernatant containing high levels of tumour necrosis factor-α, interferon-γ, interleukins -1, -6 and -17, soluble human leucocyte antigen-G, and transforming growth factor-ß1, increased the proportion of Treg most effectively and was able to induce interleukin-10-secreting-Treg from CD4+CD25âcells. CONCLUSIONS: Compared with circulating maternal factors, placental- and fetal-derived factors appear to exert a more powerful effect on numerical changes of Treg, thereby supporting fetomaternal tolerance during human pregnancy.
Assuntos
Vilosidades Coriônicas/metabolismo , Citocinas/metabolismo , Linfócitos T Reguladores/metabolismo , Adulto , Linfócitos T CD4-Positivos , Células Cultivadas , Estudos Transversais , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-2 , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-Idade , Gravidez , Segundo Trimestre da Gravidez , Terceiro Trimestre da GravidezRESUMO
The objective of the study was to determine the feasibility of generating a biodegradable, stem cell-loaded osteogenic composite graft from human placenta. Initially, a scaffold from human chorion membrane was produced. Human placenta mesenchymal stem cells (MSCs) derived from either first-trimester chorionic villi or term chorion membrane were differentiated osteogenically on this scaffold. Outgrowth, adherence, and osteogenic differentiation of cells were assessed by immunohistochemistry (IHC), scanning electron microscopy, protein expression, and real-time polymerase chain reaction (RT-PCR). Our results showed that a cell-free extracellular matrix scaffold can be generated from human chorion. Seeded MSCs densely adhered to that scaffold and were osteogenically differentiated. Calcium and alkaline phosphatase were detected in the cell-scaffold constructs as a proof of mineralization and findings were confirmed by IHC and RT-PCR results. This study shows for the first time that generation of an osteogenic composite graft using placental tissue is feasible. It might allow therapeutic application of autologous or allogeneic grafts in congenital skeletal defects by means of a composite graft.
Assuntos
Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Placenta/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Córion/citologia , Estudos de Viabilidade , Feminino , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , GravidezRESUMO
OBJECTIVE: Preeclampsia is associated with perinatal brain injury. Autologous placenta stem cell transplantation represents a promising future treatment option for neuroregeneration. The aim of this study was to compare the neuroregenerative capacity of preeclampsia-placenta stem cells to previously characterized placentas from uncomplicated pregnancies. STUDY DESIGN: Placenta stem cells from amnion (epithelium, mesenchyme) and chorion were assessed for cell surface markers and the formation of neuronal-like cells, oligodendrocytes and their progenitors in culture. RESULTS: Markers of preeclampsia-placenta stem cells were different from uncomplicated pregnancies-placenta stem cells in amnion epithelium and chorion, but not in amnion mesenchyme. Similarly to uncomplicated pregnancies-placenta stem cells, preeclampsia-placenta stem cells derived from amnion and chorion differentiated preferably into nestin-positive stem/progenitor cells and Tuj-1-positive neurons. However, other important markers were varying after neurogenic differentiation of uncomplicated pregnancies- and preeclampsia-placenta stem cells. CONCLUSION: Surface marker expression patterns of preeclampsia-placenta stem cell's and uncomplicated pregnancies-placenta stem cell's differ. In vitro differentiation assays, however, provide evidence that both preeclampsia-placenta stem cells and uncomplicated pregnancies-placenta stem cells are comparably suitable for neuroregeneration purposes.
Assuntos
Neurônios/citologia , Placenta/citologia , Pré-Eclâmpsia/patologia , Células-Tronco/citologia , Âmnio/citologia , Diferenciação Celular , Células Cultivadas , Córion/citologia , Feminino , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/metabolismo , Gravidez , Tubulina (Proteína)/metabolismoRESUMO
OBJECTIVE: We aimed to induce neural stem (NSC) and progenitor cells (NPC) from human placental tissues. STUDY DESIGN: Placental stem cells from first-trimester placental chorionic villi and term chorion were isolated. Neural differentiation was initiated with plating on collagen, retinoic acid, and/or human brain-derived neurotrophic factor and epidermal and fibroblast growth factor. Differentiation into neurons, oligodendrocytes, and astrocytes was monitored by immunohistochemistry. Two-dimensional polyacrylamide gel electrophoresis, high-performance liquid chromatography, and tandem mass spectrometry were used to identify proteins involved in the differentiation. RESULTS: Differentiated cells were mostly immediately postmitotic with some more but not fully mature postmitotic neurons. Neurons had dopaminergic or serotonergic character. Some cells differentiated into predominantly immature oligodendrocytes. Upon differentiation, neuron-specific proteins were up-regulated, whereas placental proteins were reduced. CONCLUSION: Stem cells derived from human placenta can be differentiated into neural progenitors.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Neurônios/citologia , Oligodendroglia/citologia , Placenta/citologia , Feminino , Humanos , Imuno-Histoquímica , Mitose , Proteínas Musculares/metabolismo , Neurônios/metabolismo , Gravidez , Tubulina (Proteína)/metabolismo , Regulação para CimaRESUMO
Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy-based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23-24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications.
Assuntos
Separação Celular/métodos , Células-Tronco Embrionárias/citologia , Placenta/citologia , Âmnio/citologia , Âmnio/imunologia , Animais , Antígenos de Superfície/metabolismo , Adesão Celular , Diferenciação Celular , Córion/citologia , Córion/imunologia , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Embrionárias/imunologia , Células-Tronco Embrionárias/transplante , Células Epiteliais/citologia , Células Epiteliais/imunologia , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Tolerância Imunológica , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Placenta/imunologia , Gravidez , Transplante de Células-Tronco , Células Estromais/citologia , Células Estromais/imunologia , Bancos de Tecidos , Trofoblastos/citologia , Trofoblastos/imunologiaRESUMO
We manufactured a cell-free extracellular matrix scaffolds in order to obtain a support material for amnion cell outgrowth, eventually being used for repair of prematurely ruptured fetal membrane. Human preterm or term amnion tissue was separated into its collagenous extracellular matrix and cell components. The acellular scaffold was explored for its capacity to support regrowth of isolated human amnion epithelial or mesenchymal cells in vitro. The outgrowth of amnion cells on and in the scaffold was investigated by scanning and transmission electron microscopy, and confocal laser scanning microscopy. Cell-free amnion matrix scaffolds demonstrated a porous collagen fiber network similar as in native amnion. Inoculation of acellular amnion scaffolds with human amnion cells revealed that its property to support amnion cell outgrowth was retained. Amnion epithelial and mesenchymal cells were found to grow into dense layers on the surface of the scaffold within 3-4 days and 7-8 days, respectively, and to some extent, invaded the scaffold during the culture period. Manufactured acellular amnion matrix retains structural and functional properties required for cell outgrowth in vitro. It may become useful to repair prematurely ruptured fetal membranes.
Assuntos
Âmnio/citologia , Matriz Extracelular/química , Engenharia Tecidual/métodos , Processos de Crescimento Celular/fisiologia , Sistema Livre de Células , Células Cultivadas , Células Epiteliais/citologia , Matriz Extracelular/fisiologia , Feminino , Ruptura Prematura de Membranas Fetais/terapia , Humanos , Modelos Biológicos , Gravidez , TransplantesRESUMO
OBJECTIVE: Mesenchymal stem cells (MSCs) have a broad differentiation potential. We aimed to determine if MSCs are present in fetal membranes and placental tissue and to assess their potential to differentiate into neurogenic and mesodermal lineages. STUDY DESIGN: MSCs isolated from first and third trimester chorion and amnion and first trimester chorionic villi and characterized morphologically and by flourescence-activated cell sorting analysis. Their ability to mature under different culture conditions into various cells of mesodermal and neuroectodermal cell lines was assessed by immuno- and cytochemical staining. RESULTS: Independent of gestational age, cells isolated from fetal membranes and placenta showed typical MSC phenotype (positive for CD166, CD105, CD90, CD73, CD49e, CD44, CD29, CD13, MHC I; negative for CD14, CD34, CD45, MHC II) and were able to differentiate into mesodermal cells expressing cell markers/cytologic staining consistent with mature chondroblasts, osteoblasts, adipocytes, or myocytes and into neuronal cells presenting markers of various stages of maturation. The differentiation pattern was mainly dependent on cell type. CONCLUSION: Mesenchymal cells from chorion, amnion, and villous stroma can be differentiated into neurogenic, chondrogenic, osteogenic, adipogenic, and myogenic lineage. Placental tissue obtained during prenatal chorionic villous sampling or at delivery might be an ideal source for autologous stem cell graft for peripartum neuroregeneration and other clinical issues.
Assuntos
Diferenciação Celular , Ectoderma/citologia , Membranas Extraembrionárias/citologia , Células-Tronco Mesenquimais/citologia , Mesoderma/citologia , Placenta/citologia , Feminino , Doenças Fetais/cirurgia , Humanos , Transplante de Células-Tronco Mesenquimais , Regeneração Nervosa , Sistema Nervoso/citologia , Gravidez , Primeiro Trimestre da Gravidez , Terceiro Trimestre da GravidezRESUMO
Cat scratch disease (CSD) is a frequent clinical outcome of Bartonella henselae infection in humans. Recently, two case reports indicated Bartonella clarridgeiae as an additional causative agent of CSD. Both pathogens have been isolated from domestic cats, which are considered to be their natural reservoir. B. clarridgeiae and B. henselae can be distinguished phenotypically by the presence or absence of flagella, respectively. Separation of the protein content of purified flagella of B. clarridgeiae by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis indicated that the flagellar filament is mainly composed of a polypeptide with a mass of 41 kDa. N-terminal sequencing of 20 amino acids of this protein revealed a perfect match to the N-terminal sequence of flagellin (FlaA) as deduced from the sequence of the flaA gene cloned from B. clarridgeiae. The flagellin of B. clarridgeiae is closely related to flagellins of Bartonella bacilliformis and several Bartonella-related bacteria. Since flagellar proteins are often immunodominant antigens, we investigated whether antibodies specific for the FlaA protein of B. clarridgeiae are found in patients with CSD or lymphadenopathy. Immunoblotting with 724 sera of patients suffering from lymphadenopathy and 100 healthy controls indicated specific FlaA antibodies in 3.9% of the patients' sera but in none of the controls. B. clarridgeiae FlaA is thus antigenic and expressed in vivo, providing a valuable tool for serological testing. Our results further indicate that B. clarridgeiae might be a possible etiologic agent of CSD or lymphadenopathy. However, it remains to be clarified whether antibodies to the FlaA protein of B. clarridgeiae are a useful indicator of acute infection.
Assuntos
Anticorpos Antibacterianos/sangue , Infecções por Bartonella/microbiologia , Bartonella/imunologia , Flagelina/genética , Flagelina/imunologia , Doenças Linfáticas/microbiologia , Sequência de Aminoácidos , Animais , Infecções por Bartonella/diagnóstico , Sequência de Bases , Western Blotting , Doença da Arranhadura de Gato/diagnóstico , Doença da Arranhadura de Gato/microbiologia , Gatos , Clonagem Molecular , Flagelos/química , Flagelina/química , Humanos , Doenças Linfáticas/diagnóstico , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF-E. VEGF-E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF-E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat-stable, secreted dimer. VEGF-E and VEGF-A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF-A, VEGF-E was found to bind with high affinity to VEGF receptor-2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF-A, VEGF-E did not bind to VEGF receptor-1 (Flt-1). VEGF-E is thus a potent angiogenic factor selectively binding to VEGF receptor-2. These data strongly indicate that activation of VEGF receptor-2 alone can efficiently stimulate angiogenesis.
Assuntos
Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/fisiologia , Linfocinas/genética , Linfocinas/fisiologia , Vírus do Orf/genética , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Proteínas Virais/genética , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Linhagem Celular , Primers do DNA/genética , Escherichia coli/genética , Expressão Gênica , Genes Virais , Humanos , Dados de Sequência Molecular , Neovascularização Fisiológica/genética , Coelhos , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Tromboplastina/metabolismo , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Binding of a particular opacity outer membrane protein (Opa) of Neisseria gonorrhoeae to cell surface heparan sulfate proteoglycans (HSPGs) of epithelial cells results in tight bacterial adherence; however, the role of this ligand-receptor interaction in triggering the subsequent bacterial internalization step is uncertain. Here we have used latex beads coated with HSPG-ligating antibodies as an in vitro model to study the role of HSPGs in gonococcal uptake into epithelial cells. Beads and gonococci showed the same cell line-specified adherence patterns and increase in phagocytic uptake mediated by serum or purified vitronectin (Vn). Heparitinase digestion as well as antibody competition experiments indicate that a critical level of HSPG ligation is necessary and sufficient to trigger phagocytic uptake into epithelial cells. Vn was found to specifically enhance HSPG-dependent phagocytic uptake while phagocytosis resulting from the ligation of other cell surface receptors was unaffected in the presence of Vn. Pharmacological studies with PKC inhibitors suggest a role for PKC in phagocytic uptake of HSPG-ligating beads. The use of drugs impairing cytoskeletal functions indicates that HSPG-dependent phagocytosis requires actin polymerization by a process distinct from receptor-mediated endocytosis.
Assuntos
Anticorpos Antibacterianos/metabolismo , Anticorpos Monoclonais/metabolismo , Células Epiteliais/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Proteoglicanas de Heparan Sulfato/farmacocinética , Actinas/metabolismo , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Aderência Bacteriana/fisiologia , Biopolímeros/metabolismo , Reagentes de Ligações Cruzadas , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/microbiologia , Células HeLa , Proteoglicanas de Heparan Sulfato/imunologia , Humanos , Ligantes , Microesferas , Neisseria gonorrhoeae/imunologia , Neisseria gonorrhoeae/metabolismo , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Sensibilidade e Especificidade , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/microbiologia , Vitronectina/farmacologiaRESUMO
In this article, we describe our experience with oral morphine for the treatment of patients with cancer pain who were referred to a Palliative Care Unit. Morphine was used in 3,399 days treatment with an average of 65 days per patient. In 50 patients (96%), there was a decreased intensity of pain from severe (Visual Analogue Scale average (VAS) 8.7) to mild (VAS average 1.9). Daily average dose was 95.6 mg. Only 26 patients (50%) required an increase in dosage. The side effects were minimal and controllable. Even though most authors recommend morphine q4h, in our population q6h was enough to achieve pain control in 83% of patients. It should be known by the medical population that morphine is essential therapy for pain in cancer patients. This study confirms that morphine is an excellent drug for the control of pain in cancer patients, with minimal dosages and controllable side effects.
Assuntos
Analgésicos Opioides/administração & dosagem , Morfina/administração & dosagem , Neoplasias/fisiopatologia , Dor/tratamento farmacológico , Administração Oral , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Analgésicos Opioides/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Morfina/efeitos adversos , Dor/etiologia , Medição da Dor , Fatores de TempoRESUMO
Vascular colonisation by Bartonella henselae may cause vaso-proliferative tumour growth with clumps of bacteria found in close association with proliferating endothelial cells. By using B. henselae-infected human umbilical vein endothelial cells as an in vitro model for endothelial colonisation, we report here on a novel mechanism of cellular invasion by bacteria. First, the leading lamella of endothelial cells establishes cellular contact to sedimented bacteria and mediates bacterial aggregation by rearward transport on the cell surface. Subsequently, the formed bacterial aggregate is engulfed and internalised by a unique host cellular structure, the invasome. Completion of this sequence of events requires 24 hours. Cortical F-actin, intercellular adhesion molecule-1 and phosphotyrosine are highly enriched in the membrane protrusions entrapping the bacterial aggregate. Actin stress fibres, which are anchored to the numerous focal adhesion plaques associated with the invasome structure, are typically found to be twisted around its basal part. The formation of invasomes was found to be inhibited by cytochalasin D but virtually unaffected by nocodazole, colchicine or taxol, indicating that invasome-mediated invasion is an actin-dependent and microtubuli-independent process. Bacterial internalisation via the invasome was consistently observed with several clinical isolates of B. henselae, while a spontaneous mutant obtained from one of these isolates was impaired in invasome-mediated invasion. Instead, this mutant showed increased uptake of bacteria into perinuclear localising phagosomes, suggesting that invasome-formation may interfere with this alternative mechanism of bacterial internalisation. Internalisation via the invasome represents a novel paradigm for the invasion of bacteria into host cells which may serve as a cellular colonisation mechanism in vivo, e.g. on proliferating and migrating endothelial cells during Bartonella-induced vaso-proliferative tumour growth.
Assuntos
Angiomatose Bacilar/microbiologia , Bartonella henselae/fisiologia , Bartonella henselae/ultraestrutura , Endocitose/fisiologia , Endotélio Vascular/microbiologia , Actinas/análise , Actinas/metabolismo , Aderência Bacteriana/fisiologia , Bartonella henselae/patogenicidade , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Membrana Celular/química , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Molécula 1 de Adesão Intercelular/análise , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microtúbulos/metabolismo , Organelas/química , Organelas/microbiologia , Organelas/ultraestrutura , Fosfotirosina/análise , Pseudópodes/microbiologia , Pseudópodes/fisiologia , Estresse Mecânico , Veias Umbilicais/citologia , VirulênciaRESUMO
The cysteine synthase gene (cysK) from Flavobacterium K3-15 was cloned and sequenced. The gene exhibits 30-50% identity to known cysteine synthases on both the DNA and the amino acid levels. The pyridoxal phosphate binding site of the enzyme is part of a conserved motif comprising seven amino acids (SIKDRIA). The lys31 residue of the flavobacterial enzyme is conserved in all known cysteine synthases. The cysK gene from Flavobacterium K3-15 was heterologously expressed and the gene product identified by immunoblotting and determination of the enzyme activity.