Produção e extração de vesículas de membrana externa (OMV) de Escherichia coli para o tratamento de infecções nosocomiais

As infecções hospitalares (nosocomiais) são adquiridas por pacientes em ambiente hospitalar. Segundo a OMS, as infecções bacterianas ainda são uma das principais causas de hospitalização e morte em todo o mundo. O uso excessivo inadequado de antibióticos aumenta as infecções nosocomiais em virtude do surgimento de cepas resistentes a estes antimicrobianos causando grande impacto econômico no país. Assim, as vacinas são as estratégias para tratamento destas infecções. Escherichia coli é um dos principais patógenos nosocomiais emergentes e é uma bactéria Gram-negativa em forma de bastonete que coloniza o trato gastrointestinal, responsável por várias doenças, como septicemia, pneumonia, meningite neonatal, peritonite e gastroenterite. A E. coli produz vesículas da membrana externa (OMV) que são pequenas estruturas esféricas e altamente imunogênicas de 20 a 200 nm e pode ser uma vacina alternativa contra infecções nosocomiais. As vesículas são liberadas durante o crescimento celular e a produção pode ser influenciada por fatores como acidez, temperatura, deficiência de nutrientes, etc. O objetivo deste trabalho foi avaliar a produção de OMV por E. coli cepa 17/38 (fornecida pelo HU - Hospital Universitário) em "shaker", utilizando diferentes condições do cultivo (pH, temperatura e tempo) e extração por filtração tangencial ou ultracentrifugação. Para a quantificação e caracterização de OMVs realizamos testes de dosagem de proteína, eletroforese e microscopia eletrônica. Com base nos resultados, concluímos que: o pH, temperatura e tempo afetaram a produção de OMV; extração de OMV pela filtração tangencial mostrou-se uma tecnologia promissora quando comparada com a ultracentrifugação. Assim, apesar do grande conhecimento a respeito de OMVs como mediadores de virulência e da necessidade para a sobrevivência bacteriana, ainda existem muitos aspectos a serem elucidados a respeito do impacto destas OMVs nos microrganismos e no hospedeiro humano.

Integrin α3 is overexpressed in glioma stem-like cells and promotes invasion.

BACKGROUND: Glioma stem-like cell (GSC) properties are responsible for gliomagenesis and recurrence. GSCs are invasive but its mechanism remains to be elucidated. Here, we attempted to identify the molecules that promote invasion in GSCs. METHODS: Neurospheres and CD133⁺ cells were collected from glioblastoma (GBM) specimens and glioma cell lines by sphere-formation method and magnetic affinity cell sorting, respectively. Differential expression of gene candidates, its role in invasion and its signaling pathway were evaluated in glioma cell lines. RESULTS: Neurospheres from surgical specimens attached to fibronectin and laminin, the receptors of which belong to the integrin family. Integrin α3 was overexpressed in CD133⁺ cells compared with CD133⁻ cells in all the glioma cell lines (4 out of 4). Immunohistochemistry demonstrated the localisation of integrin α3 in GBM cells, including invading cells, and in the tumour cells around the vessels, which is believed to be a stem cell niche. The expression of integrin α3 was correlated with migration and invasion. The invasion activity of glioma cells was linked to the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. CONCLUSION: Our results suggest that integrin α3 contributes to the invasive nature of GSCs via ERK1/2, which renders integrin α3 a prime candidate for anti-invasion therapy for GBM.

Silencing of ferrochelatase enhances 5-aminolevulinic acid-based fluorescence and photodynamic therapy efficacy.

BACKGROUND: Recurrence of glioma frequently occurs within the marginal area of the surgical cavity due to invading residual cells. 5-Aminolevulinic acid (5-ALA) fluorescence-guided resection has been used as effective therapeutic modalities to improve discrimination of brain tumour margins and patient prognosis. However, the marginal areas of glioma usually show vague fluorescence, which makes tumour identification difficult, and the applicability of 5-ALA-based photodynamic therapy (PDT) is hampered by insufficient therapeutic efficacy in glioma tissues. METHODS: To overcome these issues, we assessed the expression of ferrochelatase (FECH) gene, which encodes a key enzyme that catalyses the conversion of protoporphyrin IX (PpIX) to heme, in glioma surgical specimens and manipulated FECH in human glioma cell lines. RESULTS: Prominent downregulation of FECH mRNA expression was found in glioblastoma tissues compared with normal brain tissues, suggesting that FECH is responsible for PpIX accumulation in glioblastoma cells. Depletion of FECH by small interference RNA enhanced PpIX fluorescence after exposure to 5-ALA concomitant with increased intracellular PpIX accumulation in glioma cells. Silencing of FECH caused marked growth inhibition and apoptosis induction by PDT in glioma cells. CONCLUSION: These results suggest that knockdown of FECH is a potential approach to enhance PpIX fluorescent quality for optimising the subjective discrimination of vague fluorescence and improving the effect of 5-ALA-PDT.

Vitamin A as adjuvant to the mouse immune response to pertussis, tetanus and diphtheria vaccines

Vitamin A was used as adjuvant, comparatively with Al(OH)3, in pertussis, tetanus and diphtheria vaccines. Both groups induced a primary immune response in mice, and one single booster dose elevated the antibodies titers in average 554 times to vitamin A groups and 104 times to Al(OH)3. These antibodies titers correlate with sera IL-4 in immunized animals, suggesting a Th2 response. Other cytokines detected in the sera and/or lymphocytes culture supernatants (IL-2 and IFN- ) indicated that vitamin A could also modulate a Th1 response in DPT and acellular pertussis vaccines.

Use of 5-FU plus hyperbaric oxygen for treating malignant tumors: evaluation of antitumor effect and measurement of 5-FU in individual organs.

PURPOSE: Hyperbaric oxygen (HBO) has been shown to increase tumor radiosensitivity. Several reports indicate that it also increases sensitivity to alkylating agents, but other reports suggest that it may speed angiogenesis and tumor growth. To throw light on these questions, we investigated the effects of HBO and 5-fluorouracil (5-FU), individually and in combination, on Sarcoma 180 implants in mice. METHODS: We administered 5-FU at a dose of 0.75 mg/mouse six times per week and HBO at 2 atm absolute pressure for 90 min six times per week, both 17 times in total. In combination treatment, HBO was administered immediately after 5-FU injection. RESULTS: Over the treatment period, tumor diameter increased 277.8% in the untreated control group, 244.1% in the group receiving HBO monotherapy, 182.7% in the group receiving 5-FU monotherapy, and 138.5% in the group receiving combination therapy. Concomitant HBO increased accumulation of 5-FU in the tumors, liver, and kidneys, but not in the brain, of recipient animals. CONCLUSIONS: Based on the above results, we conclude that concomitant HBO enhances the effects of 5-FU.

Distinct roles of cathepsin K and cathepsin L in osteoclastic bone resorption.

The role of cathepsin K (CAK), cloned as a novel collagenolytic cysteine protease (CCP), cathepsin L (CAL) and cathepsin B (CAB) in bone resorption was investigated. In mouse calvarial organ culture medium, CAL, detected in trace amounts in control conditions, and CCP activity were increased by stimulants of bone resorption: 1alpha,25-(OH)2D3 parathyroid hormone (PTH), IL-1alpha, IL-6 or TNF-alpha. CAK was the most abundantly detected CCP and was not increased by these stimulants. In the absence of the stimulants, only antisense oligodeoxynucleotide (AS-ODN) for CAK suppressed collagenolysis and CCP activity. On the other hand, in the presence of the stimulants, AS-ODN for both CAK and CAL suppressed collagenolysis and CCP activity, and these activities were additive. AS-ODN for CAB did not suppress collagenolysis. These results suggested CAK was constitutively synthesized as the main CCP, and CAL was synthesized as an inducible CCP in osteoclasts to degrade type-I collagen in combination with CAK.

Regulation of collagenolytic cysteine protease synthesis by estrogen in osteoclasts.

In ovariectomized (Ovx) mice, collagenolytic cysteine protease (CCP) activity in calvaria significantly increased 7 days after ovariectomy and was about 50% of that observed in sham-operated (Sham) mice 3 weeks later. In Ovx mice, subcutaneously (s.c.) administered estradiol-17beta (E2) (10 microg/kg) for 2 weeks led to a decrease in CCP activity in calvaria to the level observed in Sham mice. In Ovx mice, though the amount of cathepsin L increased more than that of cathepsin K, cathepsin K and cathepsin L content increased by 200-400% compared with the Sham mice; cathepsin K was detected in larger amounts than cathepsin L in calvaria from both Sham and Ovx mice. The amounts of cathepsin K and cathepsin L in Ovx mice were reduced to the values seen with Sham mice after administration (s.c.) of E2 (10 microg/kg) for 2 weeks. In mouse calvarial organ culture, the increase of CCP activity and release of hydroxyproline, an indicator of degradation of type-I collagen, in the presence of 1alpha,25-(OH)(2)D(3), parathyroid hormone, interleukin (IL)-1alpha, IL-6, or tumor necrosis factor-alpha was suppressed by E2 (10(-9)-10(-7) M). In all cases, secretion of both cathepsin K and cathepsin L were suppressed by E2. In osteoclasts, expression of cathepsin K and cathepsin L was suppressed by E2 at the mRNA level. Cathepsin B was detected faintly or not at all. These results suggest that synthesis of cathepsin K and cathepsin L was negatively regulated by E2 at the mRNA level. In Ovx mice, deficiency of E2 resulted in an augmentation of cathepsin K and cathepsin L synthesis, and the cathepsins might share roles in bone resorption in vivo.

Regulation of collagenolytic protease secretion through c-Src in osteoclasts.

The role of pp60c-src activity in the synthesis and secretion of the collagenolytic cysteine proteases (CCPs), cathepsin K (CAK), cathepsin L (CAL), and cathepsin B (CAB), by osteoclasts was investigated. Synthesis and secretion of CAL were up-regulated by 1alpha,25-(OH)2D3, but neither those of CAK, dominant relative to CAL, nor CAB, barely detectable, levels changed in the experiments. Though PP1, a pp60c-src inhibitor, had no effect on CCPs synthesis, suppressed the CAK and CAL secretion. Wortmannin, a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor that works as a second messenger for pp60c-src, and cytochalasin B, an inhibitor of actin polymerization, suppressed the secretion of both CAK and CAL without suppressing synthesis. Hydroxyproline release, an indicator of degradation of type-I collagen, and F-actin ring formation, a structure linked to osteoclastic bone resorption, were suppressed by PP1, cytochalasin B or wortmannin. These results suggested inhibition of pp60c-src activity affected the osteoclastic cytoskeleton, which in turn reflected the suppression of bone resorption.

Long-term follow-up study of serum immunoglobulin G and immunoglobulin A antibodies after Helicobacter pylori eradication.

OBJECTIVE: There have been few studies concerning serum titers of anti-Helicobacter pylori immunoglobulin G (IgG) antibody >12 months after eradication of the original infection. Moreover, clinical usefulness of immunoglobulin A (IgA) antibody levels remains to be established. The purpose of this study was to investigate long-term responses of serum IgG-specific and IgA-specific antibodies to H pylori in children after eradication therapy. STUDY DESIGN: A total of 34 children, 2 to 17 years of age (mean: 11.7 years) with H pylori-associated gastroduodenal disease received eradication therapy (proton pump inhibitor-based dual or triple regimens). Diagnoses included nodular gastritis (n = 8), gastric ulcer (n = 7), and duodenal ulcer (n = 19). Upper gastrointestinal endoscopy and biopsy were performed before the therapy and at 1 to 2 months' posttreatment. H pylori infection and eradication were defined by biopsy-based tests; eradication was successful in 28 patients and unsuccessful in 6. Pretreatment IgG was positive in 30 patients (88. 2%), and the IgA was positive in 31 (91.2%), who were entered into this study (duration /=30% decrease in titer of the IgA antibody at 6 months indicated eradication with sensitivity of 90.5% and specificity of 100%. For the IgG antibody, a 30% decrease at 12 months showed equal sensitivity and specificity. Seroreversion rates of IgG and IgA antibodies were 53% and 48% at 12 months and were 86% and 81% at 24 months, respectively. The mean periods from the completion of eradication therapy to seroreversion of IgG and IgA antibodies were 11.2 +/- 7.0 and 11.6 +/- 7.8 months, respectively (not significantly different). A higher pretreatment titer of IgG antibody was related to a longer period of seroreversion (r = 0.44). In one patient, (13)C-urea breath test-confirmed reinfection was accompanied by reappearance of significant titers of the IgG and IgA antibodies. CONCLUSIONS: A serology test is useful for evaluating eradication in children. Approximately half of patients with successful eradication remained to be IgG-seropositive and IgA-seropositive at 12 months' posttreatment. When a decrease titer in antibody is used for assessing eradication, an endpoint of >/=6 months is required. The IgA antibody may be a more convenient indicator of H pylori status than is the IgG antibody.

Helicobacter pylori reinfection rates in children after eradication therapy.

BACKGROUND: There are few studies of Helicobacter pylori reinfection in childhood. In the current study the reinfection rate of H. pylori and ulcer recurrence were investigated during a follow-up period of 12 months or more in children who had undergone eradication therapy. METHODS: Twenty-seven patients aged 5 to 16 years (6 with gastric ulcer, 13 with duodenal ulcer, and 8 with nodular gastritis) were studied. Biopsy-based H. pylori tests performed 1 to 2 months after eradication therapy demonstrated that eradication was successful in 23 patients (5 with gastric ulcer, 11 with duodenal ulcer, and 7 with nodular gastritis) and unsuccessful in 4 (1 with gastric ulcer, 2 with duodenal ulcer, and 1 with nodular gastritis). Twenty-three successfully treated patients were observed for a mean of 22 months (a total of 42.2 patient years of follow-up). To assess H. pylori status, all 23 patients underwent a 13C-urea breath test 1 year after eradication therapy. If the test result was negative, the patients underwent the follow-up test once every year thereafter. In successfully and unsuccessfully treated patients, endoscopy was performed if a patient reported symptoms suggesting ulcer recurrence. RESULTS: The initial follow-up 13C-urea breath tests showed that all 23 patients remained free of infection at 12 to 19 months. Among 17 patients, the second test confirmed reinfection in 1 at 28 months. In two patients studied, the third test showed a negative result. The reinfection rate was 2.4% per patient year. Over the follow-up period, ulcer recurrence was found in 2 of 3 ulcer patients with eradication failure but in none of the 16 ulcer patients with successful eradication. The recurrence rate was significantly lower in successfully treated patients than in unsuccessfully treated patients (p < 0.05). CONCLUSIONS: Reinfection with H. pylori is rare in children aged more than 5 years, and successful eradication significantly reduces ulcer recurrence. This study supports the benefit of eradication therapy in older children.

Synthesis of peptide aldehyde derivatives as selective inhibitors of human cathepsin L and their inhibitory effect on bone resorption.

Cathepsin L, a lysosomal cysteine protease, is secreted by osteoclasts and participates in bone collagen degradation. In a search for cathepsin L inhibitors as antiosteoporotic agents, a series of peptide aldehyde derivatives were prepared by two synthetic approaches, DMSO oxidation of the corresponding alcohol derivatives and DIBAL-H reduction of the corresponding N, O-dimethylhydroxylamide derivatives, and evaluated for inhibitory activity against human cathepsin L and for inhibitory effects on bone resorption. Some of the peptide aldehyde derivatives including alpha-acylamino aldehyde derivatives showed potent activities. Among these compounds, N-(1-naphthalenylsulfonyl-L-isoleucyl-L-tryptophanal (12) was selected as a candidate for further investigation. Compound 12, a potent, selective, and reversible inhibitor of human cathepsin L with an IC50 of 1.9 nM, inhibited the release of Ca2+ and hydroxyproline from bone in in vitro bone culture system and also prevented bone loss in ovariectomized mice at an oral dose of 50 mg/kg.

Binding properties of fibrinogen receptor GPIIb-IIIa purified from human erythroleukemia cells.

Large amounts (2.3 mg) of an inactive form of the glycoprotein GPIIb-IIIa were obtained in highly purified form from 7-liter cultures of human erythroleukemia (HEL) cells. The purified GPIIb-IIIa was converted to an activated form after its immobilization to 96-well plastic plates. The binding of fibrinogen to the activated GPIIb-IIIa was investigated using 24 kinds of RGD peptides and showed that the IC50 values of these peptides correlated with those obtained with activated platelets. These findings indicated that the binding properties of the activated GPIIb-IIIa from HEL cells are quite similar to those of the platelets.

Two distinct placental lactogen-like substances in serum during mid-pregnancy in the rat.

Two forms of placental lactogen (PL), designated PL-I and PL-II, have been reported in the serum of pregnant rats; PL-I was secreted during mid-pregnancy and PL-II was secreted near term. In the present study, we found that two distinct forms with PL-like activity were secreted into the blood during mid-pregnancy. Serum from day-12 pregnant rats was subjected to high-performance liquid chromatography (HPLC) gel-filtration (TSK-G3000SswXL column), and PL activity was assayed by radioreceptor assay using 125I-labeled rat PRL and hepatocyte plasma membrane from pregnant rats. HPLC-gel filtration separated the PL activity into two peaks with mol wt of 55-60 K and 45-50 K, as estimated by reference to the elution volume of standard proteins. We tentatively designated these peaks PL-alpha and PL-beta, respectively. Considering these mol wt, PL-beta seemed to be identical to PL-I. mRNA was extracted from samples of placenta obtained each day from day 8 to day 12 of pregnancy and analyzed by means of a translation system involving micro-injection into Xenopus oocytes. The time of appearance of the mRNAs corresponding to PL-alpha and PL-beta did not correspond and differed according to the day of pregnancy, suggesting that there are individual mRNAs for each PL in rat placenta. Treatment of PL-alpha and PL-beta with peptide: N-glycosidase F completely abolished the binding activity to their receptors. Also since they were sensitive to glycosidases such as endoglycosidase H, endoglycosidase F and neuraminidase, these PLs were considered to possess N-linked glycoresidue(s) and to be sialylated.(ABSTRACT TRUNCATED AT 250 WORDS)

Placental lactogen secretion during prolonged-pregnancy in the rat: the ovary plays a pivotal role in the control of placental function.

The serum of rats at mid-pregnancy contains at least 2 distinct placental lactogen (PL)-like substances tentatively termed placental lactogen-alpha (PL-alpha) and placental lactogen-beta (PL-beta) (Endocrinol Japon 38: 533-540, 1991). We have investigated the secretory patterns of three placental lactogens (PL-alpha, PL-beta and placental lactogen-II) during normal pregnancy and in two prolonged-pregnancy models. Pregnancy was prolonged by the introduction of new corpora lutea by inducing ovulation on day 15 of pregnancy by successive treatments with PMSG (30 IU/rat, sc on day 12) and hCG (10 IU/rat, iv on day 14), and in the second model by progesterone implants on day 15 of pregnancy. During normal pregnancy, each of the 3 PLs exhibited only one secretory peak in the serum; PL-alpha and PL-beta on day 12 and placental lactogen II (PL-II) on day 20. Interestingly, in the rats with new sets of corpora lutea, serum PL-alpha and PL-beta levels began to increase again on day 18 and showed peaks on day 20 for PL-alpha and on day 22 for PL-beta. In this model, the initiation of PL-II secretion was not affected, but high levels were maintained until day 26, when parturition occurred. In rats receiving either PMSG or hCG, the secretory patterns of the PLs were similar to as those during normal pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)