Development of Lactococcus lactis encoding fluorescent proteins, GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system.

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.

Effects of different surface modifying agents on the cytotoxic and antimicrobial properties of ZnO nanoparticles.

Zinc oxide (ZnO) nanoparticles (NPs) have received considerable attention in the medical field because of their antibacterial properties, primarily for killing and reducing the activity of numerous microorganisms. The purpose of this study was to determine whether surface-modified ZnO NPs exhibit different properties compared with unmodified ZnO. The antimicrobial and cytotoxic properties of modified ZnO NPs as well as their effects on inflammatory cytokine production were evaluated. ZnO NPs were prepared using a wet chemical method. Then, the surfaces of these NPs were modified using 3-aminopropyltriethoxysilane (APTES) and dimethyl sulfoxide (DMSO) as modifying agents via a chemical hydrolysis method. According to infrared spectroscopy analysis (FTIR), the structure of the ZnO remained unchanged after modification. Antibacterial assays demonstrated that APTES modification is more effective at inducing an antimicrobial effect against Gram-negative bacteria than against Gram-positive bacteria. Cytotoxicity studies showed that cell viability was dose-dependent; moreover, pristine and APTES-modified ZnO exhibited low cytotoxicity, whereas DMSO-modified ZnO exhibited toxicity even at a low NP concentration. An investigation of inflammatory cytokine production demonstrated that the extent of stimulation was related to the ZnO NP concentration but not to the surface modification, except for IFN-γ and IL-10, which were not detected even at high NP concentrations.

Recombinant adenovirus delivery of calreticulin-ESAT-6 produces an antigen-specific immune response but no protection against a Mycobacterium tuberculosis challenge.

Bacillus Calmette-Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT-ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT-ESAT-6-CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis.

Efficient secretion of a modified E7 protein from human papilloma virus type-16 by Lactococcus lactis.

AIMS: To create and provide a strain of the food-grade bacterium Lactococcus lactis able to efficiently secrete a modified form of the E7 protein from the human papilloma virus (HPV) type-16. METHODS AND RESULTS: We cloned the coding sequence of a modified E7 (E7m) from the HPV-16 in a plasmid regulated by the strong expression promoter p59. Secretion of the E7m was made by the signal peptide of the usp45 gene. The E7m was detected by Western blot in the cell-free-medium fraction, showing no degradation or aberrant forms. CONCLUSIONS: We constructed a strain of L. lactis able to secrete efficiently a HPV-16 E7 modified protein with diminished transforming activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Human papilloma virus infection is associated with more than 99% of cervical cancers. Immunotherapy targeting E7 to treat HPV-associated cervical malignancies has been demonstrated to be highly efficient. However, native E7 maintains transforming activity. We present this new strain of a food-grade bacterium able to efficiently secrete a HPV-16 E7-modified protein with diminished transforming activity. This new strain could be used as a live vaccine to deliver E7 at a mucosal level and generate antitumour immune responses against HPV-associated tumours.

Targeting and retention of HPV16 E7 to the endoplasmic reticulum enhances immune tumour protection.

The endoplasmic reticulum (ER) is where the major histocompatibility complex (MHC) class I molecules are loaded with epitopes to cause an immune cellular response. Most of the protein antigens are degraded in the cytoplasm to amino acids and few epitopes reach the ER. Antigen targeting of this organelle by Calreticulin (CRT) fusion avoids this degradation and enhances the immune response. We constructed a recombinant adenovirus to express the E7 antigen with an ER-targeting signal peptide (SP) plus an ER retention signal (KDEL sequence). In cell-culture experiments we demonstrated that this new E7 antigen, SP-E7-KDEL, targeted the ER. Infection of mice with this recombinant adenovirus that expresses SP-E7-KDEL showed interferon induction and tumour-protection response, similar to that provided by an adenovirus expressing the E7 antigen fused to CRT. This work demonstrated that just by adding a SP and the KDEL sequence, antigens can be targeted and retained in the ER with a consequent enhancement of immune response and tumour protection. These results will have significant clinical applications.

Diagnóstico diferido de hernia diafragmática congénita derecha tras sepsis por «Streptococcus» del grupo B / Delayed diagnosis of right-sided congenital diaphragmatic hernia following group B streptococcal sepsis

Se presenta el caso de un recién nacido atendido en el Servicio de Pediatría del Hospital Regional Universitario «Carlos Haya» por sepsis y enfermedad respiratoria, que desarrolló coagulopatía y shock en los primeros días, por lo que se le instauró antibioticoterapia y ventilación mecánica. La bronconeumonía inicial cursó con derrame pleural e imágenes persistentemente alteradas del hemidiafragma derecho. Al final y mediante ecografía, se estableció el diagnóstico de hernia diafragmática congénita derecha. Una vez superado el problema infeccioso, fue intervenido quirúrgicamente con éxito

We present the case of a newborn, treated in our unit for sepsis and respiratory disease. In his first few days of life, he developed coagulopathy and shock, requiring antibiotics and mechanical ventilation. The initial bronchopneumonia was associated with pleural effusion, and the imaging studies repeatedly revealed a defect in right hemidiaphragm that proved to be a congenital diaphragmatic hernia on ultrasound. Once the infection resolved, surgical repair was successfully performed

[Continuous glucose monitoring with type 1 diabetes mellitus]. / Monitorización continua de glucosa en la diabetes mellitus tipo 1.

BACKGROUND: Appropriate metabolic control of children with type 1 diabetes mellitus (DM) is based on frequent measurements of capillary glycemia. However, this method offers only partial information on fluctuations in glycemia during the day, while episodes of postprandial hyperglycemia and hypoglycemia, mainly nocturnal, go unnoticed. OBJECTIVES: To analyze pre- and postprandial blood glucose levels, as well as the presence and duration of hypoglycemic episodes in diabetic children aged more than 8 years old with more than one year of disease duration. METHODS: Seventeen patients of both sexes (mean age: 12 years old) with type 1 DM were monitored with the continuous glucose monitoring system (CGMS) during working days. Maximum values of pre- and postprandial glucose (1-3 hours after breakfast, lunch and dinner) were registered. Data were downloaded with a Com-station. RESULTS: The mean duration of sensor-wearing was 2.97 days. Pre- and postprandial values were high: mean preprandial values were between 144.9 and 160.5 mg % and mean postprandial values were between 230.4 and 248.8 mg %. The mean number of hypoglycemic episodes detected with the sensor was 4.9 compared with 1.8 detected with the glucometer (p < 0.05). Episodes of mainly nocturnal asymptomatic hypoglycemia were detected with a mean duration of 145 minutes during the night and 75 minutes during the day. CONCLUSIONS: The use of continuous subcutaneous glucose monitoring demonstrates that glycemic objectives are not achieved by conventional insulin therapy. It also shows that there are a high number of hypoglycemic episodes, most of which are asymptomatic.

Determinación de inmunoglobulina E (IgE) mediante inmunoquimioluminiscencia / Measurement of immunoglobulin E (IgE) by chemiluminiscent imunoassay (Access)

No disponible

Estudio multicéntrico del tratamiento empírico de la neutropenia febril en el enfermo oncohematológico / Multicentric study of empiric treatment of febrile neutiropenia in cancer patients

En el presente trabajo se analiza un estudio multicéntrico de tipo prospectivo, cuyo principal objetivo es conocer el perfil de utilización de antibióticos y antifúngicos empleados en el tratamiento de la neutropenia febril en el paciente oncohematológico y establecer el grado de adhesión a los protocolos instaurados en siete hospitales de la zona Andalucía Oriental. Se incluyeron en el estudio los pacientes que recibieron tratamiento durante el período mayo-junio de 1998 (65 episodios), utilizando para su evaluación unas hojas de recogida de datos consensuadas por los hospitales participantes. En ellas también se recoge la utilización de factores estimulantes de colonias en estos pacientes. Los resultados muestran que la monoterapia genera un interés creciente como alternativa a la terapia combinada, especialmente en pacientes de bajo riesgo. La mayoría de los hospitales participantes poseen protocolos instaurados que recogen el tratamiento antiinfeccioso empírico de la neutropenia febril, variando el grado de adhesión entre un 50 y un 75 por ciento. Destaca la utilización de factores estimulantes de colonias en un alto porcentaje de pacientes (72,3 por ciento) como terapia adyuvante (AU)

Study of omeprazole-gamma-cyclodextrin complexation in the solid state.

The possibility of obtaining inclusion complexes between omeprazole (OME) and gamma-cyclodextrin (gamma-CD) by kneading, spray-drying, coprecipitation, and freeze-drying was evaluated. All these methods lead to the isolation of a true inclusion compound, as evidenced by differential scanning calorimetry (DSC), infrared spectroscopy, and X-ray diffractometry on powder (PXRD). Moreover, PXRD and scanning electron microscopy (SEM) afforded data concerning crystallinity and surface characteristics of the solid phases obtained. In all cases, a significant increase of the release rate with respect to the drug alone was found, and it was attributed to the formation of an inclusion compound. Among the solid phases obtained, the comprecipitated product presented the highest dissolution rate.

Effects of the host cavity size and the preparation method on the physicochemical properties of ibuproxam-cyclodextrin systems.

The effect of cyclodextrin (Cd) complexation on ibuproxam (IBUX) dissolution properties was studied by evaluating both the influence of Cd cavity size and the preparation method used for obtaining solid inclusion complexes. Binary systems of IBUX with natural Cds, prepared using different techniques (kneading, sealed-heating, spray-drying), were studied by differential scanning calorimetry (DSC), hot-stage microscopy (HSM), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and their dissolution behavior was evaluated according to the dispersed amount method. The nature and the dissolution performance of the end product appeared to be related to both steric factors of host molecule and preparation method of the solid system. The alpha Cd cavity size was less suitable for accommodating the IBUX molecule, whereas spray-drying and sealed-heating methods led to a true inclusion complex of IBUX in the beta Cd and gamma Cd cavity. In contrast, the kneading method did not lead in any case to a real inclusion complex. Spray-dried systems with beta Cd and gamma Cd were the most effective in achieving the enhancement of the IBUX dissolution rate.

[Chylous ascites in cirrhotic and non-cirrhotic patients]. / Ascitis quilosa en pacientes cirróticos y no cirróticos.

Chylous ascites is an accumulation of lymph in the abdominal cavity. The diagnosis is established when the concentration of triglycerides in plasma is greater than in ascitic fluid over a level of 200 mg/dl. The clinical and biochemical characteristics of 22 patients with chylous ascites (11 cirrhotics and 11 non cirrhotics) were studied in order to assess differences between patients with and without hepatic cirrhosis. The cirrhotic patients with chylous ascites showed lower protein (1.3 +/- 0.74 mg/dl, p = 0.002) and cholesterol concentration (46.0 +/- 45.2 mg/dl, p = 0.02) in ascitic fluid than non cirrhotic patients (3.1 +/- 1.09 mg/dl, and 100.85 +/- 41.7 mg/dl, respectively). In addition, the cellularity in the ascitic fluid was also lower in cirrhotic patients (209.09 +/- 113.96 cel/mm3) versus (831.8 +/- 945.08 cel/mm3; p < 0.05). Four patients (18.18%) presented high adenosine deaminase levels (ADA) in the ascitic fluid in the absence of tuberculous peritonitis. The authors conclude that the biochemical differences observed in the ascitic fluid of the cirrhotic patients with chylous ascites may be explained by a dilutional mechanism due to the combination of "clear" ascites secondary to portal hypertension and chylous ascites. Furthermore, chylous ascites could be the cause of an elevation in ADA in the absence of tuberculous peritonitis.

Dissolution properties and in vivo behaviour of triamterene in solid dispersions with polyethylene glycols.

The applicability of the solid dispersion technique as a method for enhancing the GI absorption of a drug has been explored in order to procure better dissolution characteristics and better bioavailability for triamterene. The physicochemical characterization of the systems has shown the absence of chemical reaction between the drug and the polymers during the solid dispersion elaboration process (melting carrier method). In vitro release profiles have been studied and quantified in terms of dissolution efficiency over the first 30 min (DE30) and dissolution percentage over the first 30 min (DP30). The results have shown that there were no significant differences between the three polyethylene glycols (PEGs) under test. The in vivo effectiveness of the different preparations was also investigated by means of the urinary volumetric excretion (UVE)--pharmacologic effect--and by the estimation of Ke, tmax, and MRT-pharmacokinetic parameters. At end, an analysis of the relative bioavailabilities between formulations has been performed.

Small bowel bacterial overgrowth in patients with alcoholic cirrhosis.

A total of 89 patients with alcoholic cirrhosis and 40 healthy subjects were included in a study to assess the prevalence of intestinal bacterial overgrowth and to analyze its relationship with the severity of liver dysfunction, presence of ascites, and development of spontaneous bacterial peritonitis (SBP). Bacterial overgrowth was measured by means of a breath test after ingestion of glucose. Intestinal bacterial overgrowth was documented in 27 (30.3%) of the 89 patients with alcoholic cirrhosis and in none of the healthy subjects. The prevalence of intestinal bacterial overgrowth was significantly higher in cirrhotics with ascites (37.1%) than in those with no evidence of ascites (5.3%) and among patients with Pugh-Child class C (48.3%) than in patients with a class A (13.1%) or B (27%). Twelve (17.1%) of the 70 patients with ascites developed an episode of SBP. The prevalence of spontaneous bacterial peritonitis was significantly higher in patients who had intestinal bacterial overgrowth (30.7%) than in patients who did not (9.09%). We conclude that intestinal bacterial overgrowth occurs in approximately one third of patients with cirrhosis secondary to alcohol, particularly in patients with ascites and advanced liver dysfunction. Moreover, bacterial overgrowth may be a condition favoring infection of the ascitic fluid.

Small bowel bacterial overgrowth in patients with alcoholic cirrhosis.

A total of 89 patients with alcoholic cirrhosis and 40 healthy subjects were included in a study to assess the prevalence of intestinal bacterial overgrowth and to analyze its relationship with the severity of liver dysfunction, presence of ascites, and development of spontaneous bacterial peritonitis (SBP). Bacterial overgrowth was measured by means of a breath test after ingestion of glucose. Intestinal bacterial overgrowth was documented in 27 (30.3%) of the 89 patients with alcoholic cirrhosis and in none of the healthy subjects. The prevalence of intestinal bacterial overgrowth was significantly higher in cirrhotics with ascites (37.1%) than in those with no evidence of ascites (5.3%) and among patients with Pugh-Child class C (48.3%) than in patients with class A (13.1%) or B (27%). Twelve (17.1%) of the 70 patients with ascites developed an episode of SBP. The prevalence of spontaneous bacterial peritonitis was significantly higher in patients who had intestinal bacterial overgrowth (30.7%) than in patients who did not (9.09%). We conclude that intestinal bacterial overgrowth occurs in approximately one third of patients with cirrhosis secondary to alcohol, particularly in patients with ascites and advanced liver dysfunction. Moreover, bacterial overgrowth may be a condition favoring infection of the ascitic fluid.

Drinking habits and pain in chronic pancreatitis.

To study the role that continuous drinking plays in the pain of chronic pancreatitis, we have examined 67 patients with alcoholic chronic pancreatitis with pain and 29 patients without pain, and we report on their alcoholic habits. Drinking habits played a part 92 (67.6%) of 136 times in patients with pain; in 185 without pain, 86 (46.5%) had continued their drinking habit (p < 0.001). Advanced pancreatic exocrine insufficiency was seen in 27 patients; 11 of them had pain during follow-up, whereas 16 did not. The nondrinker rate was similar in patients with and without pain, whereas among 69 patients with better pancreatic exocrine function, 56 had pain episodes and 13 did not. Alcoholic consumers were significantly more in number in the pain group: 70.4% versus 35% of the no pain group (p < 0.002). Our study shows that drinking alcohol in patients with chronic pancreatitis increased the frequency of painful episodes when there was relatively good pancreatic function, whereas in severe pancreatic insufficiency drinking had less influence on the development of pain.