Heat shock protein 60 from Chlamydia pneumoniae elicits an unusual set of inflammatory responses via Toll-like receptor 2 and 4 in vivo

Heat shock protein 60 (HSP60) from Chlamydia pneumoniae was described to trigger in vitro inflammatory and cytokine responses including TNF and IL-12p40. Although it can be found in atherosclerotic plaques of patients, the stimulatory potential of chlamydial and other HSP60 in vivo is unclear. We now report that chlamydial HSP60 fails to induce TNF expression in vivo, and significant serum levels of IL-12p40 are only found upon intraperitoneal injection of high doses of HSP60 or after intravenous application. Upon purification of chlamydial HSP60 with polymyxin B-agarose columns, its ability to induce TNF secretion in vitro is much reduced. However, purified chlamydial HSP60 causes increased serum levels of the CXC chemokines KC and MIP2 in vivo, as well as a strong accumulation of polymorphonuclear neutrophils (PMN) in the peritoneal cavity upon intraperitoneal challenge. With respect to PMN accumulation, chlamydial HSP60 is more potent than endotoxin or the CpG oligonucleotide 1668. The responses observed are completely abolished in Toll-like receptor (TLR)2/4-double-deficient mice, while single-deficient mice respond almost normally. Furthermore, KC induction and PMN accumulation are largely dependent on MyD88. In conclusion, HSP60 from C. pneumoniae triggers inflammatory responses in vivo that differ from responses induced by endotoxin or CpG oligonucleotides and are dependent on TLR2 and 4.     

Suprasternal Doppler ultrasound for assessment of stroke distance

An assessment of a non-invasive technique for measurement of stroke distance was made using a portable Doppler ultrasound machine. The aim was to determine the measurement error of repeated stroke distance measurements (Within-observer variability) and to assess measurement agreement between two operators (between-observer variability). The measurement error (within-observer variability) for both operators was similar at approximately 2 cm. However, the measurements of the two operators (between-observer variability) did not agree well. Using the mean (SD) of three readings by each operator, the mean difference between the operators was -0.21 cm (1.96) giving a 95% confidence interval for the differences of -4.0 to +3.6 cm. There were significant positive and negative correlations between stroke distance and a variety of variables (age, height, weight, heart rate), but the relations were weak. The results indicate that the Doppler ultrasound technique for measurement of stroke distance would best be used to study trend changes in an individual patient, or subject, by a single operator.     

Genetic imbalances detected by multiplex ligation-dependent probe amplification in a cohort of patients with oral squamous cell carcinoma—the first step towards clinical personalized medicine

Oral tumors are a growing health problem worldwide; thus, it is mandatory to establish genetic markers in order to improve diagnosis and early detection of tumors, control relapses and, ultimately, delineate individualized therapies. This study was the first to evaluate and discuss the clinical applicability of a multiplex ligation-dependent probe amplification (MLPA) probe panel directed to head and neck cancer. Thirty primary oral squamous cell tumors were analyzed using the P428 MLPA probe panel. We detected genetic imbalances in 26 patients and observed a consistent pattern of distribution of genetic alterations in terms of losses and gains for some chromosomes, particularly for chromosomes 3, 8, and 11. Regarding the latter, some specific genes were highlighted due to frequent losses of genetic material—RARB, FHIT, CSMD1, GATA4, and MTUS1—and others due to gains—MCCC1, MYC, WISP1, PTK2, CCND1, FGF4, FADD, and CTTN. We also verified that the gains of MYC and WISP1 genes seem to suggest higher propensity of tumors localized in the floor of the mouth. This study proved the value of this MLPA probe panel for a first-tier analysis of oral tumors. The probemix was developed to include target regions that have been already shown to be of diagnostic/prognostic relevance for oral tumors. Furthermore, this study emphasized several of those specific genetic targets, suggesting its importance to oral tumor development, to predict patients’ outcomes, and also to guide the development of novel molecular therapies.     

Pressão arterial periférica por Dinamap e pressão arterial central por tonometria planar de absorção no ambulatório de Medicina Geral e Familiar

IntroductionCentral blood pressure (CBP) is the pressure exerted by the blood column at any given moment on the aortic and carotid artery walls, which is a close proxy for the blood pressure inside the brain and the heart, and is thus a better marker of cardiovascular morbidity and mortality than peripheral blood pressure (PBP).ObjectiveTo assess how the augmentation index (AI), peripheral pulse pressure (pPP), central pulse pressure (cPP) and subendocardial viability ratio (SEVR) vary in hypertensive patients according to level of control of CBP and PBP.MethodsWe performed an observational, cross-sectional study in a convenience sample from a general practice in Central Portugal over a period of four days in May 2010. Measurements were taken after a four-minute resting period. The following values were considered to reflect controlled pressures: PBP <140/90 mmHg, CBP <130/80 mmHg, pPP <55 mmHg and cPP <45 mmHg.ResultsThe sample included 92 patients, 38 male (41.3%), mean age 62.3±11.1 years, with no significant difference in gender distribution. PBP was controlled in 55 (59.8%), and CBP in 53 (57.6%). Both PBP and CBP were controlled in 50 patients (54.3%) and neither was controlled in 34 (37.9%). pPP and cPP were significantly lower in those with controlled PBP (p<0.001) and CBP (p<0.001). AI was non-significantly lower in those with controlled PBP (78±9 vs. 80.7) and those with controlled CBP (78±9 vs.81±7) (p=0.02). SEVR was within the desirable range in 92 patients (92.2%). 78.4% of individuals were taking drugs acting on the renin angiotensin aldosterone system (RAAS).ConclusionsIn a convenience sample of 92 patients, PBP and CBP were controlled in 59.8% and 57.6%, respectively. Those with controlled PBP had significantly better peripheral systolic and diastolic blood pressure, CBP, pPP and cPP; the same was true of those with controlled CBP, who also had a significantly better AI. The percentage of the cardiac cycle in diastole had a desirable value for 92,2% of the subjects.     

Enzymatic characterization of Vibrio alginolyticus strains isolated from bivalves harvested at Venice Lagoon (Italy) and Guanabara Bay (Brazil)

The aquatic ecosystem is the natural habitat of microorganisms including Vibrio and Aeromonas genus which are pathogenic to human and animals. In the present investigation the frequency of these bacteria and the enzymatic characteristics of 34 Vibrio alginolyticus strains isolated from bivalves harvested in Venice Lagoon (Italy) and Guanabara Bay (Brazil) were carried out from November 2003 to February 2004. The mussels' samples were submitted to enrichment in Alkaline Peptone Water (APW) added with 1% of sodium chloride (NaCl) and APW plus 3% NaCl incubated at 37 degrees C for 18-24 h. Following the samples were streaked onto TCBS Agar (Thiossulfate Citrate Bile Sucrose Agar) and the suspected colonies were submitted to biochemical characterization. Also, the Vibrio alginolyticus strains were evaluated to collagenase, elastase and chondroitinase production. The results showed the isolation of 127 microorganisms distributed as follows: 105 Vibrio strains such as V. alginolyticus (32.4%), V. harveyi (19%) and V. parahaemolyticus (7.6%), 20 Aeromonas strains and two Plesiomonas shigelloides were the main pathogens isolated. We observed the production of the three enzymes from V. alginolyticus strains considered as the main virulence factors of the bacteria, especially in cases of human dermatological infection.     

Functional relevance of NLRP3 inflammasome-mediated interleukin (IL)-1β during acute allergic airway inflammation

Overall asthmatic symptoms can be controlled with diverse therapeutic agents. However, certain symptomatic individuals remain at risk for serious morbidity and mortality, which prompts the identification of novel therapeutic targets and treatment strategies. Thus, using an adjuvant-free T helper type 2 (Th2) murine model, we have deciphered the role of interleukin (IL)-1 signalling during allergic airway inflammation (AAI). Because functional IL-1β depends on inflammasome activation we first studied asthmatic manifestations in specific inflammasome-deficient [NACHT, LRR and PYD domains-containing protein 3 (NLRP3^−/−) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC^−/−)] and IL-1 receptor type 1^−/− (IL-1R1^−/−) mice on the BALB/c background. To verify the onset of disease we assessed cellular infiltration in the bronchial regions, lung pathology, airway hyperresponsiveness and ovalbumin (OVA)-specific immune responses. In the absence of NLRP3 inflammasome-mediated IL-1β release all symptoms of AAI were reduced, except OVA-specific immunoglobulin levels. To address whether manipulating IL-1 signalling reduced asthmatic development, we administered the IL-1R antagonist anakinra (Kineret®) during critical immunological time-points: sensitization or challenge. Amelioration of asthmatic symptoms was only observed when anakinra was administered during OVA challenge. Our findings indicate that blocking IL-1 signalling could be a potential complementary therapy for allergic airway inflammation.     

Engineering of Escherichia coli strains for plasmid biopharmaceutical production: Scale-up challenges

Plasmid-based vaccines and therapeutics have been making their way into the clinic in the last years. The existence of cost-effective manufacturing processes capable of delivering high amounts of high-quality plasmid DNA (pDNA) is essential to generate enough material for trials and support future commercialization. However, the development of pDNA manufacturing processes is often hampered by difficulties in predicting process scale performance of Escherichia coli cultivation on the basis of results obtained at lab scale. This paper reports on the differences observed in pDNA production when using shake flask and bench-scale bioreactor cultivation of E. coli strains MG1655ΔendAΔrecA and DH5α in complex media with 20 g/L of glucose. MG1655ΔendAΔrecA produced 5-fold more pDNA (9.8 mg/g DCW) in bioreactor than in shake flask (1.9 mg/g DCW) and DH5α produced 4-fold more pDNA (8 mg/g DCW) in bioreactor than in shake flask (2 mg/g DCW). Accumulation of acetate was also significant in shake flasks but not in bioreactors, a fact that was attributed to a lack of control of pH.     

Contrasting effects of recombinant human granulocyte-macrophage colony- stimulating factor (CSF) and granulocyte CSF treatment on the cycling of blood elements in childhood-onset cyclic neutropenia

Recombinant human granulocyte colony-stimulating factor (G-CSF) treatment has been shown to increase average neutrophil counts substantially in patients with childhood-onset cyclic neutropenia (or "cyclic hematopoiesis"), but not to eliminate the cyclic oscillations of neutrophil counts or those of other blood elements (monocytes, platelets, eosinophils, and reticulocytes) that are characteristic of this hematopoietic disorder. Indeed, oscillations of neutrophil counts are amplified during G-CSF treatment. We have compared the effects of recombinant granulocyte-macrophage-CSF (GM-CSF) with those of G-CSF in three patients with this disease (2 men and 1 woman, 17, 30, and 32 years of age). These patients were treated with GM-CSF (2.1 micrograms/kg/day, subcutaneously) for 6 weeks, preceded and followed by 6 to 13 weeks of detailed observation to document changes in the cyclic oscillations of blood neutrophils and other blood elements; two of the patients were subsequently treated with G-CSF (5.0 micrograms/kg/d, subcutaneously) and observed for comparable periods of time. Unlike G-CSF treatment, which increased average neutrophil counts more than 20-fold, GM-CSF increased neutrophil counts only modestly, from 1.6- to 3.9-fold, although eosinophilia of varying prominence was induced in each patient. However, at the same time, GM-CSF treatment dampened or eliminated the multilineage oscillations of circulating blood elements (neutrophils, monocytes, platelets, and/or reticulocytes) in each of the patients. In contrast, G-CSF treatment of the same patients markedly amplified the oscillations of neutrophil counts and caused the cycling of other blood elements (monocytes in particular) to become more distinct. These findings support the conclusion that the distinctive cycling of blood cell production in childhood-onset cyclic neutropenia results from abnormalities in the coordinate regulation of both GM-CSF-responsive, multipotential progenitor cells and G-CSF-responsive, lineage-restricted, neutrophil progenitors.