Epidemiological characterization of resistance and PCR typing of Shigella flexneri and Shigella sonnei strains isolated from bacillary dysentery cases in Southeast Brazil

Shigella spp are Gram-negative, anaerobic facultative, non-motile, and non-sporulated bacilli of the Enterobacteriaceae family responsible for "Shigellosis" or bacillary dysentery, an important cause of worldwide morbidity and mortality. However, despite this, there are very few epidemiological studies about this bacterium in Brazil. We studied the antibiotic resistance profiles and the clonal structure of 60 Shigella strains (30 S. flexneri and 30 S. sonnei) isolated from shigellosis cases in different cities within the metropolitan area of Campinas, State of São Paulo, Brazil. We used the following well-characterized molecular techniques: enterobacterial repetitive intergenic consensus, repetitive extragenic palindromic, and double-repetitive element-polymerase chain reaction to characterize the bacteria. Also, the antibiotic resistance of the strains was determined by the diffusion disk method. Many strains of S. flexneri and S. sonnei were found to be multi-resistant. S. flexneri strains were resistant to ampicillin in 83.3% of cases, chloramphenicol in 70.0%, streptomycin in 86.7%, sulfamethoxazole in 80.0%, and tetracycline in 80.0%, while a smaller number of strains were resistant to cephalothin (3.3%) and sulfazotrim (10.0%). S. sonnei strains were mainly resistant to sulfamethoxazole (100.0%) and tetracycline (96.7%) and, to a lesser extent, to ampicillin (6.7%) and streptomycin (26.7%). Polymerase chain reaction-based typing supported the existence of specific clones responsible for the shigellosis cases in the different cities and there was evidence of transmission between cities. This clonal structure would probably be the result of selection for virulence and resistance phenotypes. These data indicate that the human sanitary conditions of the cities investigated should be improved.

Effect of saline infusion for the maintenance of blood volume on pulmonary gas exchange during temporary abdominal aortic occlusion

We analyzed the effects of saline infusion for the maintenance of blood volume on pulmonary gas exchange in ischemia-reperfusion syndrome during temporary abdominal aortic occlusion in dogs. We studied 20 adult mongrel dogs weighing 12 to 23 kg divided into two groups: ischemia-reperfusion group (IRG, N = 10) and IRG submitted to saline infusion for the maintenance of mean pulmonary arterial wedge pressure between 10 and 20 mmHg (IRG-SS, N = 10). All animals were anesthetized and maintained on spontaneous ventilation. After obtaining baseline measurements, occlusion of the supraceliac aorta was performed by the inflation of a Fogarty catheter. After 60 min of ischemia, the balloon was deflated and the animals were observed for another 60 min of reperfusion. The measurements were made at 10 and 45 min of ischemia, and 5, 30, and 60 min of reperfusion. Pulmonary gas exchange was impaired in the IRG-SS group as demonstrated by the increase of the alveolar-arterial oxygen difference (21 ± 14 in IRG-SS vs 11 ± 8 in IRG after 60 min of reperfusion, P = 0.004 in IRG-SS in relation to baseline values) and the decrease of oxygen partial pressure in arterial blood (58 ± 15 in IRG-SS vs 76 ± 15 in IRG after 60 min of reperfusion, P = 0.001 in IRG-SS in relation to baseline values), which was correlated with the highest degree of pulmonary edema in morphometric analysis (0.16 ± 0.06 in IRG-SS vs 0.09 ± 0.04 in IRG, P = 0.03 between groups). There was also a smaller ventilatory compensation of metabolic acidosis after the reperfusion. We conclude that infusion of normal saline worsened the gas exchange induced by pulmonary reperfusion injury in this experimental model.

Antinociceptive properties in mice of a lectin isolated from the marine alga Amansia multifida Lamouroux

The antinociceptive effects of a lectin (LEC) isolated from the marine alga Amansia multifida were determined in Swiss mice. The LEC (1, 5, and 10 mg/kg) inhibited acetic acid-induced abdominal writhings in a dose-dependent manner after intraperitoneal or oral administration. A partial but significant inhibition of writhings was observed after the combination of LEC (10 mg/kg) with avidin (1 mg/kg), a potent inhibitor of the hemmaglutinant activity of the lectin. However, total writhing inhibition was demonstrable in the group of mice treated with LEC plus mannose (1 mg/kg), as compared to LEC alone or to control groups. Furthermore, avidin and mainly mannose also play a role in antinociception, somehow facilitating the interaction of LEC with its active cell sites. In the formalin test, although both phases of the response were significantly inhibited, the effect of LEC was predominant during phase 2, causing inhibition of licking time that ranged from 48 to 88% after oral (5 and 10 mg/kg) and intraperitoneal (1 to 5 mg/kg) administration. As is the case with morphine, the effect of LEC (2 mg/kg) was reversed by naloxone (2 mg/kg), indicating the involvement of the opioid system. LEC was also effective in the hot-plate test, producing inhibitory responses to the thermal stimulus, and its effects were blocked by naloxone. In the pentobarbital-induced sleeping time, although LEC did not alter the onset of sleep significantly, it increased the time of sleep within the same dose range compared to control. These results show that LEC presents antinociceptive effects of both central and peripheral origin, possibly involving the participation of the opioid system.

Serogroups and virulence genotypes of Escherichia coli isolated from patients with sepsis

Sixty strains of Escherichia coli, isolated by hemoculture, from septicemic Brazilian patients were evaluated to determine their serogroup and invasivity to Vero cells. All 60 patients died within 2 days of hospitalization. Furthermore, the molecular study of the following extraintestinal pathogenic E. coli-associated virulence factor (VF) genes was performed by PCR: i) adhesins: type 1 fimbria (fimH), S fimbria (sfaD/E), P fimbria (papC and papG alleles) and afimbrial adhesin (afaB/C); ii) capsule K1/K5 (kpsMTII); iii) siderophores: aerobactin (iucD), yersiniabactin (fyuA) and salmochelin (iroN); iv) toxins hemolysin (hlyA), necrotizing cytotoxic factor type 1 (cnf1) and secreted autotransporter toxin (sat); v) miscellaneous: brain microvascular endothelial cells invasion (ibeA), serum resistance (traT), colicin V (cvaC) and specific uropathogenic protein (usp). Our results showed that isolates are able to invade Vero cells (96.6%), differing from previous research on uropathogenic E. coli (UPEC). The O serogroups associated with UPEC were prevalent in 60% of strains vs 11.7% of other serogroups. The PCR results showed a conserved virulence subgroup profile and a prevalence above 75% for fimH, fyuA, kpsMTII and iucD, and between 35-65% for papC, papG, sat, iroN, usp and traT. The evasion from the immunological system of the host and also iron uptake are essential for the survival of extraintestinal pathogenic E. coli strains. Interestingly, among our isolates, a low prevalence of VF genes appeared. Therefore, the present study contributes to the identification of a bacterial profile for sepsis-associated E. coli.

Hind limb ischemic preconditioning induces an anti-inflammatory response by remote organs in rats

Ischemic preconditioning (IPC), a strategy used to attenuate ischemia-reperfusion injury, consists of brief ischemic periods, each followed by reperfusion, prior to a sustained ischemic insult. The purpose of the present study was to evaluate the local and systemic anti-inflammatory effects of hind limb IPC in male Wistar rat (200-250 g) models of acute inflammation. IPC was induced with right hind limb ischemia for 10 min by placing an elastic rubber band tourniquet on the proximal part of the limb followed by 30 min of reperfusion. Groups (N = 6-8) were submitted to right or left paw edema (PE) with carrageenan (100 µg) or Dextran (200 µg), hemorrhagic cystitis with ifosfamide (200 mg/kg, ip) or gastric injury (GI) with indomethacin (20 mg/kg, vo). Controls received similar treatments, without IPC (Sham-IPC). PE is reported as variation of paw volume (mL), vesical edema (VE) as vesical wet weight (mg), vascular permeability (VP) with Evans blue extravasation (µg), GI with the gastric lesion index (GLI; total length of all erosions, mm), and neutrophil migration (NM) from myeloperoxidase activity. The statistical significance (P < 0.05) was determined by ANOVA, followed by the Tukey test. Carrageenan or Dextran-induced PE and VP in either paw were reduced by IPC (42-58.7%). IPC inhibited VE (38.8%) and VP (54%) in ifosfamide-induced hemorrhagic cystitis. GI and NM induced by indomethacin were inhibited by IPC (GLI: 90.3%; NM: 64%). This study shows for the first time that IPC produces local and systemic anti-inflammatory effects in models of acute inflammation other than ischemia-reperfusion injury.

Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) and protein levels in the right and left heart auricles of naive control and long-term (12 weeks) socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h) was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70%) compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62%) and left (about 81%) auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%), DBH (about 37%) and PNMT (about 60%) only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

Vancomycin-dependent Enterococcus faecium vanA: characterization of the first case isolated in a university hospital in Brazil

In this study, we report the characterization of a strain of Enterococcus faecium vanA, which grows only in the presence of vancomycin (VDEfm-UEL). The bacterium was isolated from the feces of a female patient who had undergone surgical treatment of Reinke’s edema and was receiving intravenous vancomycin therapy for infection with methicillin/oxacillin-resistant Staphylococcus aureus, a postoperative complication. Antimicrobial dependence was further confirmed by the vancomycin E-test. VDEfm-UEL was also shown to be resistant to ampicillin, ciprofloxacin, chloramphenicol, erythromycin, levofloxacin, penicillin, rifampicin, and teicoplanin. The putative virulence genes efaA, gelE and esp were detected by PCR. The ddl gene from VDEfm-UEL was cloned and sequenced. Vancomycin dependence seems to be associated with the insertion of a nucleotide in that sequence, which results in a frame-shift mutation, introducing a premature stop codon. This is the first report of vancomycin-dependent E. faecium isolation in a university hospital in Brazil.

Ischemic post-conditioning attenuates the intestinal injury induced by limb ischemia/reperfusion in rats

The purpose of this study was to investigate the protective effects of ischemic post-conditioning on damage to the barrier function of the small intestine caused by limb ischemia-reperfusion injury. Male Wistar rats were randomly divided into 3 groups (N = 36 each): sham operated (group S), lower limb ischemia-reperfusion (group LIR), and post-conditioning (group PC). Each group was divided into subgroups (N = 6) according to reperfusion time: immediate (0 h; T1), 1 h (T2), 3 h (T3), 6 h (T4), 12 h (T5), and 24 h (T6). In the PC group, 3 cycles of reperfusion followed by ischemia (each lasting 30 s) were applied immediately. At all reperfusion times (T1-T6), diamine oxidase (DAO), superoxide dismutase (SOD), and myeloperoxidase (MPO) activity, malondialdehyde (MDA) intestinal tissue concentrations, plasma endotoxin concentrations, and serum DAO, tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) concentrations were measured in sacrificed rats. Chiu’s pathology scores for small intestinal mucosa were determined under a light microscope and showed that damage to the small intestinal mucosa was lower in group PC than in group LIR. In group PC, tissue DAO and SOD concentrations at T2 to T6, and IL-10 concentrations at T2 to T5 were higher than in group LIR (P < 0.05); however, tissue MPO and MDA concentrations, and serum DAO and plasma endotoxin concentrations at T2 to T6, as well as TNF-α at T2 and T4 decreased significantly (P < 0.05). These results show that ischemic post-conditioning attenuated the permeability of the small intestines after limb ischemia-reperfusion injury. The protective mechanism of ischemic post-conditioning may be related to inhibition of oxygen free radicals and inflammatory cytokines that cause organ damage.

Cardiac autonomic modulation during progressive upper limb exercise by patients with coronary artery disease

The purpose of this study was to investigate the behavior of heart rate (HR) and HR variability (HRV) during different loads of resistance exercise (incline bench press) in patients with coronary artery disease (CAD) and healthy sedentary controls. Ten healthy men (65 ± 1.2 years, control group, CG) and 10 men with clinically stable CAD (66 ± 2.4 years, CADG) were recruited. A discontinuous progressive protocol was applied with an initial load of 10% of the maximum load achieved in the 1RM (1 repetition maximum) with increases of 10% until 30% 1RM was reached, which was followed by subsequent increases of 5% 1RM until exhaustion. HRV was analyzed by linear and non-linear methods. There was a significant reduction in rMSSD (CG: 20 ± 2 to 11 ± 3 ms; CADG: 19 ± 3 to 9 ± 1 ms) and SD1 indexes (CG: 14 ± 2 to 8 ± 1 ms; CADG: 14 ± 2 to 7 ± 1 ms). An increase in HR (CG: 69 ± 5 to 90 ± 5 bpm; CADG: 62 ± 4 to 75 ± 4 bpm) and in systolic blood pressure (CG: 124 ± 3 to 138 ± 3 mmHg; CADG: 122 ± 6 to 126 ± 9 bpm) were observed (P < 0.05) when comparing pre-effort rest and 40% 1RM in both groups. Furthermore, an increase in RMSM index was also observed (CG: 28 ± 3 to 45 ± 9 ms; CADG: 22 ± 2 to 79 ± 33 ms), with higher values in CADG. We conclude that loads up to 30% 1RM during incline bench press result in depressed vagal modulation in both groups, although only stable CAD patients presented sympathetic overactivity at 20% 1RM upper limb exercise.

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

In vitro anti-inflammatory, cytotoxic and antioxidant activities of boesenbergin A, a chalcone isolated from Boesenbergia rotunda (L.) (fingerroot)

The current in vitro study was designed to investigate the anti-inflammatory, cytotoxic and antioxidant activities of boesenbergin A (BA), a chalcone derivative of known structure isolated from Boesenbergia rotunda. Human hepatocellular carcinoma (HepG2), colon adenocarcinoma (HT-29), non-small cell lung cancer (A549), prostate adenocarcinoma (PC3), and normal hepatic cells (WRL-68) were used to evaluate the cytotoxicity of BA using the MTT assay. The antioxidant activity of BA was assessed by the ORAC assay and compared to quercetin as a standard reference antioxidant. ORAC results are reported as the equivalent concentration of Trolox that produces the same level of antioxidant activity as the sample tested at 20 µg/mL. The toxic effect of BA on different cell types, reported as IC50, yielded 20.22 ± 3.15, 10.69 ± 2.64, 20.31 ± 1.34, 94.10 ± 1.19, and 9.324 ± 0.24 µg/mL for A549, PC3, HepG2, HT-29, and WRL-68, respectively. BA displayed considerable antioxidant activity, when the results of ORAC assay were reported as Trolox equivalents. BA (20 µg/mL) and quercetin (5 µg/mL) were equivalent to a Trolox concentration of 11.91 ± 0.23 and 160.32 ± 2.75 µM, respectively. Moreover, the anti-inflammatory activity of BA was significant at 12.5 to 50 µM and without any significant cytotoxicity for the murine macrophage cell line RAW 264.7 at 50 µM. The significant biological activities observed in this study indicated that BA may be one of the agents responsible for the reported biological activities of B. rotunda crude extract.

Three-dimensional kinematic analysis of upper and lower limb motion during gait of post-stroke patients

The aim of this study was to analyze the alterations of arm and leg movements of patients during stroke gait. Joint angles of upper and lower limbs and spatiotemporal variables were evaluated in two groups: hemiparetic group (HG, 14 hemiparetic men, 53 ± 10 years) and control group (CG, 7 able-bodied men, 50 ± 4 years). The statistical analysis was based on the following comparisons (P ≤ 0.05): 1) right versus left sides of CG; 2) affected (AF) versus unaffected (UF) sides of HG; 3) CG versus both the affected and unaffected sides of HG, and 4) an intracycle comparison of the kinematic continuous angular variables between HG and CG. This study showed that the affected upper limb motion in stroke gait was characterized by a decreased range of motion of the glenohumeral (HG: 6.3 ± 4.5, CG: 20.1 ± 8.2) and elbow joints (AF: 8.4 ± 4.4, UF: 15.6 ± 7.6) on the sagittal plane and elbow joint flexion throughout the cycle (AF: 68.2 ± 0.4, CG: 46.8 ± 2.7). The glenohumeral joint presented a higher abduction angle (AF: 14.2 ± 1.6, CG: 11.5 ± 4.0) and a lower external rotation throughout the cycle (AF: 4.6 ± 1.2, CG: 22.0 ± 3.0). The lower limbs showed typical alterations of the stroke gait patterns. Thus, the changes in upper and lower limb motion of stroke gait were identified. The description of upper limb motion in stroke gait is new and complements gait analysis.

Hydrogen sulfide postconditioning protects isolated rat hearts against ischemia and reperfusion injury mediated by the JAK2/STAT3 survival pathway

The JAK2/STAT3 signal pathway is an important component of survivor activating factor enhancement (SAFE) pathway. The objective of the present study was to determine whether the JAK2/STAT3 signaling pathway participates in hydrogen sulfide (H2S) postconditioning, protecting isolated rat hearts from ischemic-reperfusion injury. Male Sprague-Dawley rats (230-270 g) were divided into 6 groups (N = 14 per group): time-matched perfusion (Sham) group, ischemia/reperfusion (I/R) group, NaHS postconditioning group, NaHS with AG-490 group, AG-490 (5 µM) group, and dimethyl sulfoxide (DMSO; <0.2%) group. Langendorff-perfused rat hearts, with the exception of the Sham group, were subjected to 30 min of ischemia followed by 90 min of reperfusion after 20 min of equilibrium. Heart rate, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and the maximum rate of increase or decrease of left ventricular pressure (± dp/dt max) were recorded. Infarct size was determined using triphenyltetrazolium chloride (TTC) staining. Myocardial TUNEL staining was used as the in situ cell death detection method and the percentage of TUNEL-positive nuclei to all nuclei counted was used as the apoptotic index. The expression of STAT3, bcl-2 and bax was determined by Western blotting. After reperfusion, compared to the I/R group, H2S significantly improved functional recovery and decreased infarct size (23.3 ± 3.8 vs 41.2 ± 4.7%, P < 0.05) and apoptotic index (22.1 ± 3.6 vs 43.0 ± 4.8%, P < 0.05). However, H2S-mediated protection was abolished by AG-490, the JAK2 inhibitor. In conclusion, H2S postconditioning effectively protects isolated I/R rat hearts via activation of the JAK2/STAT3 signaling pathway.

Arginine induces GH gene expression by activating NOS/NO signaling in rat isolated hemi-pituitaries

The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (~250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (a NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.