Flavodoxin overexpression confers tolerance to oxidative stress in beneficial soil bacteria and improves survival in the presence of the herbicides paraquat and atrazine.

AIM: To determine whether expression of a cyanobacterial flavodoxin in soil bacteria of agronomic interest confers protection against the widely used herbicides paraquat and atrazine. METHODS AND RESULTS: The model bacterium Escherichia coli, the symbiotic nitrogen-fixing bacterium Ensifer meliloti and the plant growth-promoting rhizobacterium Pseudomonas fluorescens Aur6 were transformed with expression vectors containing the flavodoxin gene of Anabaena variabilis. Expression of the cyanobacterial protein was confirmed by Western blot. Bacterial tolerance to oxidative stress was tested in solid medium supplemented with hydrogen peroxide, paraquat or atrazine. In all three bacterial strains, flavodoxin expression enhanced tolerance to the oxidative stress provoked by hydrogen peroxide and by the reactive oxygen species-inducing herbicides, witnessed by the enhanced survival of the transformed bacteria in the presence of these oxidizing agents. CONCLUSIONS: Flavodoxin overexpression in beneficial soil bacteria confers tolerance to oxidative stress and improves their survival in the presence of the herbicides paraquat and atrazine. Flavodoxin could be considered as a general antioxidant resource to face oxidative challenges in different micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of plant growth-promoting rhizobacteria or nitrogen-fixing bacteria with enhanced tolerance to oxidative stress in contaminated soils is of significant agronomic interest. The enhanced tolerance of flavodoxin-expressing bacteria to atrazine and paraquat points to potential applications in herbicide-treated soils.

Glycaemic control in patients with type 2 diabetes switching from premixed insulin to long-acting basal insulin analogue plus oral antidiabetic drugs: an observational study.

AIM: To evaluate whether administration of long-acting basal insulin analogue plus oral antidiabetic drugs (OADs) improves glycaemic control in type 2 diabetic patients with glycosylated haemoglobin (HbA1c) > 7% (53 mmol/mol) under premixed insulin therapy. METHODS: This is a multicentre, observational, retrospective study performed in type 2 diabetic patients switching from premixed insulin to long-acting basal insulin analogue plus OADs. Data on patients' medical history and assessments were retrieved from patients' medical charts prior to switching the treatment and 6 months thereafter. RESULTS: A total of 131 evaluable patients were enrolled (mean age, 68.2 ± 9.4 years; female, 65.6%; mean diabetes duration, 12.7 ± 6.9 years; mean time on insulin therapy, 53.2 ± 41.9 months). Patients were receiving premixed insulin (once-daily, 4.7%; twice-daily, 85.0%; thrice-daily, 10.2%), 82.4% of whom in combination with OADs (metformin, 79.4%). After the treatment was switched, only 14.5% required intensification of treatment with additional preprandial insulin. HbA1c decreased -1.4% [mean ± SD, 8.4 ± 1.0% (68.7 ± 11.4 mmol/mol) vs. 7.0 ± 1.0% (53.6 ± 10.9 mmol/mol), p < 0.001] and the proportion of patients achieving HbA1c < 7% (53 mmol/mol) increased to 52.7% (p < 0.001). The percentage of patients with hypoglycaemia decreased (19.2% vs. 10.8%, p < 0.05; symptomatic, 17.6% vs. 4.6%, p < 0.01) and body weight diminished by -1.9 kg (mean ± SD, 78.5 ± 14.7 kg vs. 76.6 ± 13.9 kg, p < 0.05). Basal insulin plus OADs was considered more convenient and flexibly adapted to patients' life in 98.4% and 99.2% of patients, respectively. Additionally, 96.9% of patients reported being more satisfied and 96.9% would recommend it. CONCLUSIONS: Switching the treatment from premixed insulin to long-acting basal insulin analogue plus OADs is a feasible and convenient approach to improve glycaemic control of type 2 diabetic patients poorly controlled with premixed insulin under routine clinical practice conditions.

Strength of association of classical vascular risk factors in young patients with ischaemic stroke: a case-control study.

INTRODUCTION: Recent studies have reported an increasing incidence of ischaemic stroke among young adults. However, the strength of the association between traditional vascular risk factors has not been fully established. METHODS: We compared 120 patients with a first ischaemic stroke before the age of 55 years admitted to the stroke unit of our centre with 600 healthy non-stroke controls from a population-based cohort study (HERMEX), matched for sex. Risk factors assessed included: hypertension, obesity, auricular fibrillation, current smoking, estimated glomerular filtration rate (eGFR), total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglycerides, high-density lipoprotein cholesterol (HDL-C) and diabetes mellitus. We used logistic regression analysis and calculated population attributable risk. We performed an overall analysis, by sex and aetiological subgroup. RESULTS: Using logistic regression analysis, we found that overall, the significant risk factors were: hypertension (OR: 1.58; 95%CI: 1.01-2.50), atrial fibrillation (OR: 4.77; 95%CI: 1.20-19.00), low eGFR (OR: 4.74; 95%CI: 1.3-21.94) and low HDL-C (OR: 5.20; 95%CI: 3.29-8.21), as well as smoking for males (OR: 1.86; 95%CI: 1.14-3.03). LDL-C showed an inverse association with stroke. The population attributable risk for HDL-C was 37.8% and for hypertension 21.1%. In terms of aetiological subgroups, only low HDL-C was associated with stroke of undetermined aetiology. CONCLUSIONS: Hypertension, auricular fibrillation, low eGFR, and low HDL-C, plus tobacco use in men, are the main risk factors among patients under 55 years of age with a first ischaemic stroke. We believe that it would be of particular interest to further explore the management of low HDL-C levels as part of preventive strategies in young stroke patients.

[Incidence of ischemic heart disease in the HERMEX study and its association with classical cardiovascular risk factors]. / Incidencia de cardiopatía isquémica en el estudio HERMEX y su asociación con los factores de riesgo cardiovascular clásicos.

OBJECTIVES: Knowledge about the incidence of coronary heart disease in our country is scarce and its sources are not comparable. Our aim was to determine the incidence of ischemic heart disease in a population cohort in a 7 years of follow-up, as well as the risk associated with the different classical cardiovascular risk factors. METHODS: Cohort study with a population sample of 2833 subjects, selected by the simple random method in a population between 25 and 79, from the Don Benito - Villanueva de la Serena (Badajoz) health area, response rate was 80.5%. All episodes of angina pectoris, lethal and non-lethal myocardial infarction were collected in individuals with no previous history of cardiovascular disease. Cumulative incidences and incidence rates adjusted per 100,000 person-years by sex and overall were calculated. RESULTS: From 2833 initial cohort participants, 103 were excluded due to a history of cardiovascular disease and 61 for losses, 2669 completed the follow-up (94.2%). The median follow-up was 6.9 years (IR 6.5-7.5), 56.4% women; 59 events were recorded. The overall incidence rate of ischemic heart disease was 327 cases/100,000 person-years, corresponding to 470 cases/100,000 for men and 211 cases/100,000 people-years for women. The classical cardiovascular risk factors that were associated with a higher risk of presenting events in the follow-up were dyslipidemia and arterial hypertension, in addition to male sex and age. CONCLUSIONS: The incidence of ischemic heart disease in the urban-rural population of Extremadura is high. The classical cardiovascular risk factors most strongly associated with events were dyslipidemia, and arterial hypertension.

Complications related to less-invasive haemodynamic monitoring.

BACKGROUND: The aim of this study was to evaluate the type and incidence of complications during insertion, maintenance, and withdrawal of central arterial catheters used for transpulmonary thermodilution haemodynamic monitoring (PiCCO™). METHODS: We conducted a prospective, observational, multicentre study in 14 European intensive care units (six countries). A total of 514 consecutive patients in whom haemodynamic monitoring by PiCCO™ was indicated were studied. RESULTS: Five hundred and fourteen PiCCO catheters (475 in femoral, 26 in radial, nine in axillary, and four in brachial arteries) were inserted. Arterial access was obtained on the first attempt in 86.4% of the patients. Minor problems such as oozing after insertion (3.3%) or removal of the catheter (3.5%) were observed, but no episodes of serious bleeding (more than 50 ml) were recorded. Small local haematomas were observed after insertion (4.5%) and after removal (1.2%) of the catheter. These complications were not more frequent in patients with coagulation abnormalities. The incidence of site inflammation and catheter-related infection was 2% and 0.78%, respectively. Other complications such as ischaemia (0.4%), pulse loss (0.4%), or femoral artery thrombosis (0.2%) were rare, transient, and all resolved with catheter removal or embolectomy, respectively. CONCLUSIONS: In this series of patients, central arterial catheters used for PiCCO™ monitoring were demonstrated to be a safe alternative for advanced haemodynamic monitoring.

[Development of animal experimental model for bacterial peritonitis]. / Desarrollo de modelo experimental animal de peritonitis bacteriana.

OBJECTIVE: The aim of the study was to develop a model of abdominal sepsis in the experimental animal. METHODS: Sprague-Dawley male rats of 5 weeks (N=39) were used. Initially, a pilot study (N = 9) was performed and distributed in 3 groups with 1cc inoculum of Escherichia coli ATCC 25922 intraperitoneally at concentrations of 10E8, 10E9 and 10E10 CFU. Subsequently, concentrations of 10E10 CFU are used in two groups of 3 rats with dilutions of 10 cc and 15 cc of distilled water respectively. Finally, a randomized trial of 24 rats was started in three treatment groups after intraperitoneal infection: Group I with physiological serum (N = 6), Group II with ceftriaxone (N = 9), Group III with ceftriaxone plus allicin (N = 9). Microbiological samples of blood and peritoneal fluid were made, as well as histopathological study of intraperitoneal organs (liver, diaphragm and peritoneum). RESULTS: Death of 100% of the rats infected with 10E10 E. coli UFC concentration with the dilution of 15 ml of distilled water and without antibiotic was oberved. The blood culture and peritoneal fluid culture was positive for the same strain in all of them. The formation of abscesses on the liver surface and polymorphonuclear infiltration in tissues were observed. CONCLUSIONS: The lethal dose of E. coli is 10E10 CFU diluted in 15 cc distilled water by intraperitoneal injection.

Patient characteristics, clinical course and factors associated to ICU mortality in critically ill patients infected with SARS-CoV-2 in Spain: A prospective, cohort, multicentre study. / Características, evolución clínica y factores asociados a la mortalidad en UCI de los pacientes críticos infectados por SARS-CoV-2 en España: estudio prospectivo, de cohorte y multicéntrico.

BACKGROUND: The clinical course of COVID-19 critically ill patients, during their admission in the intensive care unit (UCI), including medical and infectious complications and support therapies, as well as their association with in-ICU mortality has not been fully reported. OBJECTIVE: This study aimed to describe clinical characteristics and clinical course of ICU COVID-19 patients, and to determine risk factors for ICU mortality of COVID-19 patients. METHODS: Prospective, multicentre, cohort study that enrolled critically ill COVID-19 patients admitted into 30 ICUs from Spain and Andorra. Consecutive patients from March 12th to May 26th, 2020 were enrolled if they had died or were discharged from ICU during the study period. Demographics, symptoms, vital signs, laboratory markers, supportive therapies, pharmacological treatments, medical and infectious complications were reported and compared between deceased and discharged patients. RESULTS: A total of 663 patients were included. Overall ICU mortality was 31% (203 patients). At ICU admission non-survivors were more hypoxemic [SpO2 with non-rebreather mask, 90 (IQR 83 to 93) vs. 91 (IQR 87 to 94); P<.001] and with higher sequential organ failure assessment score [SOFA, 7 (IQR 5 to 9) vs. 4 (IQR 3 to 7); P<.001]. Complications were more frequent in non-survivors: acute respiratory distress syndrome (ARDS) (95% vs. 89%; P=.009), acute kidney injury (AKI) (58% vs. 24%; P<10-16), shock (42% vs. 14%; P<10-13), and arrhythmias (24% vs. 11%; P<10-4). Respiratory super-infection, bloodstream infection and septic shock were higher in non-survivors (33% vs. 25%; P=.03, 33% vs. 23%; P=.01 and 15% vs. 3%, P=10-7), respectively. The multivariable regression model showed that age was associated with mortality, with every year increasing risk-of-death by 1% (95%CI: 1 to 10, P=.014). Each 5-point increase in APACHE II independently predicted mortality [OR: 1.508 (1.081, 2.104), P=.015]. Patients with AKI [OR: 2.468 (1.628, 3.741), P<10-4)], cardiac arrest [OR: 11.099 (3.389, 36.353), P=.0001], and septic shock [OR: 3.224 (1.486, 6.994), P=.002] had an increased risk-of-death. CONCLUSIONS: Older COVID-19 patients with higher APACHE II scores on admission, those who developed AKI grades ii or iii and/or septic shock during ICU stay had an increased risk-of-death. ICU mortality was 31%.

[Needs of schoolchildren with type 1 diabetes in Extremadura: Family perceptions]. / Necesidades del alumnado con diabetes tipo 1 en Extremadura: percepción de las familias.

INTRODUCTION: School-aged children with type 1 diabetes (DM1) require access to appropriate and safe care for their disease during their stay in the educational centre. OBJECTIVE: To identify the needs perceived by families of schoolchildren with DM1 that affect their educational integration, safety, and well-being during the school day. METHODOLOGY: A descriptive and cross-sectional study was conducted using a questionnaire based on information and opinions provided by families of 362 schoolchildren between 3 and 16 years old with DM1 registered in their health history in the Public Health System of Extremadura. RESULTS: The response rate was 56.9% (206). It was shown that 35% of schoolchildren with DM1 were treated with continuous subcutaneous insulin infusion therapy. Almost all of them (95.1%) required glucose monitoring, and 57.8% required insulin administration during the school day. Most (88%) children had adjusted well to school and did not describe any type of discriminatory treatment (87.4%). Glucagon is available in 82% of educational centres, in which 43.7% had a trained adult person to administer it. That teachers could recognise a hypoglycaemia was expressed by 21.4% of the families, and 29.1% were unaware of the existence of coordination protocols in the school. More than half (58.7%) claimed that the information available in schools about diabetes was low, and 77.2% stated that the control of the disease would improve if more training was provided to teachers. CONCLUSIONS: There are aspects optimally covered in the care of schoolchildren with DM1 in the schools of Extremadura. Among situations identified with potential room for improvement were adherence to the coordination protocol, information about diabetes, and training of adults to deal with emergency situations.

Trends in stroke hospitalisation rates in Extremadura between 2002 and 2014: Changing the notion of stroke as a disease of the elderly. / Tendencias en las tasas de hospitalización por ictus en Extremadura en el periodo 2002-2014. Cambiando la idea del ictus como una enfermedad propia de la senectud.

BACKGROUND: The incidence of stroke in Spain has been evaluated in several studies, whose results are highly variable and not comparable. No studies of stroke have analysed epidemiological changes in younger patients. METHOD: We conducted a retrospective observational study using the Spanish health system's Minimum Data Set and included all patients older than 19 hospitalised due to stroke (ICD-9-CM codes 434.01, 434.11, 434.91, 430, 431, 432.9, 436, and 435) between 2002 and 2013. The analysis was performed using joinpoint regression. RESULTS: A total of 39,321 patients were identified (47.25% were women); 3.73% were aged 20-44, 6.29% were 45-54, 11.49% were 55-64, 23.89% were 65-74, and 54.60% were > 74 years. The hospitalisation rate due to ischaemic stroke has increased significantly in men aged 45-54 (+6.7%; 95% CI, 3.3-10.2) and in women aged 20-44 and 45-54 (+6.1%; 95% CI, 0.8-11.7 and +5.7%; 95% CI, 3.0-8.4, respectively). We also observed a significant increase in the rate of hospitalisation due to ischaemic stroke in men aged over 74 (+4.2%; 95% CI, 1.3-7.2). The rate of hospitalisations due to transient ischaemic attack has also increased significantly whereas the rate of hospitalisations due to brain haemorrhage has stabilised over time. CONCLUSIONS: Our results provide indirect evidence that the epidemiological profile of stroke is changing based on the increase in hospitalisation rates in young adults.

[Utility of distinct urinalysis parameters in the diagnosis of urinary tract infections]. / Utilidad de los distintos parámetros del perfil urinario en el diagnóstico de infección urinaria.

A review of the scientific evidence available on the validity of urinalysis in the diagnosis of urinary tract infection in childhood is presented. Numerous studies evaluating the validity of distinct urinalysis parameters have been published. Although many of these studies have methodological deficiencies, the indicators of validity evaluated in the various systematic reviews can be assumed to be applicable to clinical practice; however, the grade of evidence supporting these studies is less than optimal. There are some parameters or combinations of parameters that have better sensitivity or specificity, but few present sufficiently high positive and negative predictive values simultaneously to form the exclusive basis for diagnosis. Bacteriuria presents certain advantages over other parameters, although this test is not routinely available and loses validity in non-sterile samples. Nevertheless, there are several circumstances that can modify the applicability of these tests in clinical practice: the technique used in urine collection, the patient's age and clinical status, and especially the degree of urgency with which the diagnosis is required.

[Acute myocardial infarction complicating radiofrequency catheter ablation in a girl]. / Infarto agudo de miocardio tras ablación por radiofrecuencia de vía cardiaca anómala en un niño.

Paroxysmal supraventricular tachycardia is the most common tachyarrhythmia in childhood and adolescence. The current treatment of choice for managing and preventing frequent recurrences is radiofrequency catheter ablation, which provides a definitive solution in most cases. This technique is very effective but not devoid of potential complications, particularly in children and adolescents. We report the case of a 3-year-old girl who suffered acute myocardial infarction after occlusion of the right coronary artery during this procedure. We describe intraoperative and postoperative management. Progress was good and the patient was discharged 14 days after ablation.

Myocardial infarction in older than 75 years: An increasing population. CASTUO Study. / Infarto de miocardio en mayores de 75 años: una población en aumento. Estudio CASTUO.

OBJECTIVES: Elderly patients with acute myocardial infarction constitute a population that is not adequately represented in clinical trials or medical registries. Our objective was to compare the clinical characteristics, treatments administered and mortality among patients younger and older than 75 years. MATERIAL AND METHODS: Observational retrospective study of patients hospitalized for acute myocardial infarction in the decade 2000-2009. Multivariate models were constructed to determine hospital and late mortality (median, 4.6 years; IQR 25-75: 2.1-7.3). RESULTS: We included 2,177 patients (995 men [79%]), with a mean age of 70.8 years (SD, 12.6). A total of 917 (42.0%) of the patients were 75 years of age or older. When compared with the patients younger than 75 years, the older patients had a greater prevalence of diabetes (38.3% vs. 32.5%; P<.002), chronic obstructive pulmonary disease (15.6% vs. 11.2%; P<.002), stroke (14.3% vs. 7.3%; P<.001), chronic renal failure (11.0% vs. 3.9%; P<.001), atrial fibrillation (15.9% vs. 6.9%; P<.001), heart failure (28.0% vs. 23.4%; P<.008). The older patients were treated with fewer beta-blockers (55.9% vs. 71.2%; P<.001), statins (44.3% vs. 62.3%; P<.001), coronary angiographies (17.9% vs. 48.5%; P<.001) and angioplasties (10.8% vs. 29.1%; P<.001). The patients older than 75 years had lower survival (mortality, 44.5% vs. 18.9%; HR 1.89; 95% CI 1.57-2.29). The use of beta-blockers (HR, 0.74; 95% CI 0.62-0.89), statins (HR 0.73; 95% CI 0.58-0.91) and angioplasty (HR, 0.42; 95% CI 0.30-0.57) was inversely correlated with mortality. CONCLUSIONS: Patients older than 75 years with acute myocardial infarction had lower survival and were treated with fewer beta-blockers, statins and angioplasty, indications that are associated with lower mortality.

Effects of cannabinoid receptor ligands on electrophysiological properties of myenteric neurones of the guinea-pig ileum.

1. The effect of cannabinoid receptor agonists was studied in guinea-pig myenteric neurones in vitro by use of conventional intracellular recording techniques. 2. Exposure of myenteric neurones of the S-cell type to the cannabinoid receptor agonists WIN 55,212-2 (100 nM) and CP 55,940 (100 nM) reversibly and significantly depressed the amplitude of fast excitatory synaptic potentials (fast e.p.s.ps) by 46% and 37%, respectively. 3. The depressant effect of WIN 55,212-2 and CP 55,940 on fast e.p.s.p. amplitude (expressed as the area above the amplitude-time curve (mVs)) was significantly greater than that of the vehicle, Tween 80, which had no detectable effect. 4. The inhibitory effect of WIN 55,212-2 appeared to be concentration-dependent over the range 1-100 nM. WIN 55,212-3, its (-)-enantiomer (100 nM), was inactive. 5. The cannabinoid CB1 receptor antagonist, SR141716A (1 microM), reversed the inhibitory effects of WIN 55,212-2 on fast e.p.s.ps in 38% of neurones tested (3/8) and acetylcholine (ACh)-induced depolarizations in 42% of neurones tested (5/12). 6. When tested on its own, SR141716A (1 microM) caused a 40-50% reduction in the amplitude of fast e.p.s.ps (n = 9). 7. WIN 55,212-2 reversibly depressed the amplitude of the slow e.p.s.p. and, in 2 out of 7 neurones, this effect was reversed by SR141716A (1 microM). 8. It is concluded that cannabinoid-induced inhibition of fast cholinergic synaptic transmission occurred by reversible activation of both presynaptic and postsynaptic CB1 receptors and that slow excitatory synaptic transmission can also be reversibly depressed by cannabinoids. Furthermore, it would seem that subpopulations of myenteric S-neurones and their synapsing cholinergic and non-cholinergic, non-adrenergic terminals are not endowed with cannabinoid receptors.

Differences in heart transplant physiology according to surgical technique.

A new cardiac transplantation technique that preserves the shape of the left atrium and leaves the right atrium intact has been introduced. To compare the new and the standard techniques, we studied cardiac physiology with Doppler echocardiography and catheterization in 26 patients who underwent operation with the standard technique (group A) and I1 who underwent operation with the new technique (group B). Right atrial dimensions were significantly lower in group B (right atrial area index 8.4 +/- 1.5 vs 14.5 +/- 1.9 cm2/m2, p < 0.001), whereas left atrial dimensions were slightly lower (left atrial area index 10.8 +/- 2.0 vs 16.4 +/- 7.0 cm2/m2, p = 0.07). Right atrial contraction, as reflected by peak late tricuspid velocity, was greater in group B (37 +/- 15 vs 30 +/- 10 cm/sec, p < 0.05). The subsequent systolic vena caval flow-velocity integral was also greater in group B at all respiratory phases (inspiration 10.0 +/- 4.0 vs 5.2 +/- 4.0 cm, p < 0.001; expiration 4.8 +/- 1.9 vs 2.9 +/- 1.4 cm, p < 0.001; apnea 5.3 +/- 2.0 vs 2.9 +/- 1.9 cm, p < 0.001) suggesting better atrial relaxation. Filling pressures on the right side of the heart were lower in group B (mean right atrial pressure 5.5 +/- 2.4 vs 6.6 +/- 2.8 mm Hg, p = 0.1; right atrial A wave 6.0 +/- 3.1 vs 8.3 +/- 3.2 mm Hg, p < 0.01; right atrial V wave 6.8 +/- 3.1 vs 9.2 +/- 3.2 mm Hg, p < 0.01; right ventricular end-diastolic pressure 5.6 +/- 3.2 vs 7.3 +/- 2.9 mm Hg, p < 0.05); however, no significant differences were found in left ventricular end-diastolic pressure or cardiac index. We conclude that patients undergoing the new technique exhibit cardiac physiologic improvements. Follow-up study is indicated to ascertain whether this finding implies improved long-term prognosis.

Glutamate transporter GLT-1 is highly expressed in activated microglia following facial nerve axotomy.

Glutamate transporters play an important role in the re-uptake of glutamate after its release from glutamatergic synapses. So far five of such transporters subtypes have been cloned from rodent and human brains. The densities of glutamate transporters are recognised to be developmentally regulated, but the role of glutamate transporters in the mechanisms underlying the occurrence of neuronal traumatic injury has not been widely studied. In the present study quantitative Western blotting and immunohistochemical technique were employed to study the expression of GLT-1/EAAT2 in the facial nuclei of adult rats following unilateral facial nerve axotomy. The total content of GLT-1 protein decreased in the ipsilateral axotomised rat facial nucleus. However, activated microglia surrounding motoneurons showed high expression of GLT-1 after facial nerve axotomy. Parallel studies revealed that primary cultured microglial cells also showed GLT-1-immunoreactivity. To our knowledge, this is the first direct demonstration of the expression of GLT-1 protein in activated microglial cells, suggesting a neuroprotective role of microglia against glutamate excitotoxicity following nerve axotomy.

Intact microglia are cultured and non-invasively harvested without pathological activation using a novel cultured cell recovery method.

Because spontaneous host regeneration of damaged tissues is limited, novel therapeutics utilizing cultured cells with the aid of tissue engineering methods are promising alternatives for tissue replacement. One critical shortcoming is current requirement for invasive cell harvest from culture to fabricate cell-based devices. Although microglia that secrete neurotrophic factors are attractive candidates for novel cell transplantation therapy for damaged central nervous system tissue, the intact harvest of cultured microglia is presently not achievable. Therefore, primary microglia were plated onto culture surfaces grafted with the temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). This surface undergoes rapid, reversible temperature-dependent changes in its hydration state and surface hydrophilicity. Microglia attached and proliferated on PIPAAm-grafted dishes at 37 degrees C. By reducing culture temperature, more than 90% of the cells spontaneously detached from the dishes within several minutes without trypsin or EDTA treatment. Recovered and replated microglia exhibited phenotypic properties comparable to those of primary microglia freshly isolated from brain. By contrast, less than 60% of the cells were harvested by trypsin digestion, and exhibited significant alteration of characteristic cellular properties as monitored by pathological states in vivo. This new technology exhibits utility for the preparation of cell sources required for cell transplantation as well as microglial function analysis.

The microglia/macrophage response in the neonatal rat facial nucleus following axotomy.

Microglia represent a population of brain macrophage precursor cells which are intrinsic to the CNS parenchyma. Transection of the facial nerve in the newborn rat causes death of the affected motor neurons which is accompanied by massive activation of local microglia. Many of these cells develop into macrophages as can be shown by immunocytochemistry for OX-42 and ED1. Using the new polyclonal microglial marker ionized calcium binding adapter molecule 1, iba1, in combination with immunocytochemical double-labeling for the proliferating cell nuclear antigen (PCNA), or [3H]thymidine autoradiography, and confocal microscopy, qualitative as well as quantitative differences can be demonstrated between the newborn and the adult axotomized rat facial nucleus. While microglial cells are the only cell population which responds to axotomy by cell division in the adult facial nucleus, GFAP positive reactive astrocytes can be shown to undergo mitosis following axotomy in the newborn rat. Furthermore, ED1 immunoreactivity, early expression of MHC class II molecules and morphological transformation of microglia into macrophages can only be observed under conditions of neuronal degeneration, i.e., in the neonatal rat facial nucleus. Thus, the combination of cellular markers described here should be useful for studies employing the neonatal rat facial nucleus as an in vivo assay system to test the efficacy of neurotrophic factors.

Urinary enzyme activities in patients treated with gold and other antirheumatic drugs.

We have studied 48 rheumatoid arthritis (RA) patients treated with nonsteroidal antiinflammatory drugs (NSAIDs), except salicylates, in 31 of whom parenteral gold was associated as therapeutic agent. In order to assess initial tubular involvement, the activities of some urinary enzymes were measured: N-acetylglucosaminidase (NAG, EC 3.2.1.30), microsomal amino-peptidase (MAP, EC 3.4.11.2) and gamma-glutamyltransferase (GGT; EC 2.3.2.2). Results were compared with a control group of 51 subjects of similar age, with no rheumatic symptoms and normal renal function. Both groups of patients (31 with gold therapy and 17 without) showed a significantly increased activity of NAG in urine, but the increase was greater in those treated with gold. MAP and GGT were not elevated significantly in either group. There was no correlation, however, between the increase of NAG and the cumulative dose of gold. NAG, MAP and GGT activities in serum yielded no relevant information. All the usual tests of renal function were also normal. Determination of NAG in urine may be regarded as a sensitive test, capable of detecting selective involvement of renal tubular cells, whose final diagnostic and prognostic significance merits further evaluation.