Fatty acid composition of serum lipid fractions in type 2 diabetic patients with microalbuminuria

OBJECTIVE: To determine the fatty acid composition of serum phospholipid, triglyceride, and cholesterol ester fractions and to analyze the lipid profile of microalbuminuric type 2 diabetic patients. RESEARCH DESIGN AND METHODS: A case-control study was conducted with 72 patients: 37 were normoalbuminuric (urinary albumin excretion rate [UAER] <20 microg/min), and 35 were microalbuminuric (UAER 20-200 microg/min). After 4 weeks of a standardized diet, the fatty acid composition of phospholipid, triglyceride, and cholesterol ester fractions was determined by gas chromatography. Total cholesterol and triglycerides were measured by enzymatic-colorimetric methods; cholesterol HDL by double precipitation with heparin, MnCl(2), and dextran sulfate; and apolipoprotein B by immunoturbidimetry. RESULTS: Microalbuminuric patients showed a lower proportion of polyunsaturated fatty acids (24.8 +/- 11.0%), especially of the n-6 family (21.7 +/- 10.5%), in triglyceride fraction than normoalbuminuric patients (34.1 +/- 11.3%, P = 0.001 and 31.4 +/- 11.5%, P < 0.001, respectively). Patients with microalbuminuria also presented higher levels of saturated fatty acids in triglyceride fraction (43.4 +/- 18.0% vs. 34.7 +/- 13.1%, P = 0.022). In the logistic regression analysis, only the proportion of polyunsaturated fatty acids in triglyceride fraction remained significantly associated with microalbuminuria (odds ratio [OR] 0.92, 95% CI 0.85-0.98, P = 0.019). Total cholesterol, HDL cholesterol, triglyceride, and apolipoprotein B levels were similar in normo- and microalbuminuric patients. CONCLUSION: Microalbuminuria in type 2 diabetic patients is associated with low polyunsaturated fatty acid contents in serum triglyceride fraction. This association may represent a risk factor for cardiovascular disease and may contribute to the progression of renal disease.     

Mutational effects of retrovirus insertion on the genome of V79 cells by an attenuated retrovirus vector: implications for gene therapy

Attenuated retroviruses are currently the most widely used vectors in clinical gene therapy because of their potential to effect stable and permanent gene transfer. Since gene delivery is accompanied by random insertion of foreign genetic material into the recipient chromosomal DNA, the potential for insertional mutagenesis exists. In this study, we used a defective retrovirus vector containing a selectable marker, the hygromycin phosphotransferase gene, to investigate the mutagenic effects of vector integration on the mammalian genome. V79 Chinese hamster cells were infected with virus supernatants or by coculture with virus producer cells, and provirus insertion events occurred at low and high frequencies, respectively. The frequency of hprt mutagenesis was increased by a factor of 2.3 over the spontaneous hprt mutation frequency only following multiple provirus insertions/cell genome. Multiple provirus insertions (>3/genome) resulted in instability at the hprt locus in 63% of the virally induced hprt mutants, as indicated by rearrangements at the molecular level, whereas no rearrangements were found when the provirus copy number was 1-2/genome. To demonstrate direct proviral involvement in mutagenesis, the defective MLV vector was retrieved along with flanking genomic hprt sequences from one mutant, and localized within intron 5 of the hprt gene. These data suggest that provirus copy number is a key factor when considering the potential hazards of using retrovirus vectors for gene therapy.     

Reduced toxicity of F-deficient Sendai virus vector in the mouse fetus

Current concerns over insertional mutagenesis by retroviral vectors mitigate investigations into alternative, potentially persistent gene therapy vector systems not dependent on genomic integration, such as Sendai virus vectors (SeVV). Prenatal gene therapy requires efficient gene delivery to several tissues, which may not be achievable by somatic gene transfer to the adult. Initially, to test the potential and tropism of the SeVV for gene delivery to fetal tissues, first-generation (replication- and propagation-competent) recombinant SeVV, expressing beta-galactosidase was introduced into late gestation immunocompetent mice via the amniotic and peritoneal cavities and the yolk sac vessels. At 2 days, this resulted in very high levels of expression particularly in the airway epithelium, mesothelium and vascular endothelium, respectively. However, as expected, substantial vector toxicity was observed. The efficiency of gene transfer and the level of gene expression were then examined using a second-generation SeVV. The second generation was developed to be still capable of cytoplasmic RNA replication and therefore high-level gene expression, but incapable of vector spread due to lack of the gene for viral F-protein. Vector was introduced into the fetal amniotic and peritoneal cavities, intravascularly, intramuscularly and intraspinally; at 2 days, expression was observed in the airway epithelia, peritoneal mesothelia, unidentified cells in the gut wall, locally at the site of muscle injection and in the dorsal root ganglia, respectively. Mortality was dramatically diminished compared with the first-generation vector.     

Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice

Inefficient gene transfer, inaccessibility of stem cell compartments, transient gene expression, and adverse immune and inflammatory reactions to vector and transgenic protein are major barriers to successful in vivo application of gene therapy for most genetic diseases. Prenatal gene therapy with integrating vectors may overcome these problems and prevent early irreparable organ damage. To this end, high-dose attenuated VSV-G pseudotyped equine infectious anaemia virus (EIAV) encoding beta-galactosidase under the CMV promoter was injected into the fetal circulation of immuno-competent MF1 mice. We saw prolonged, extensive gene expression in the liver, heart, brain and muscle, and to a lesser extent in the kidney and lung of postnatal mice. Progressive clustered hepatocyte staining suggests clonal expansion of cells stably transduced. We thus provide proof of principle for efficient gene delivery and persistent transgene expression after prenatal application of the EIAV vector and its potential for permanent correction of genetic diseases.     

Astrocyte-derived thrombospondin-2 is critical for the repair of the blood-brain barrier

Thrombospondin (TSP)-2-null mice have an altered brain foreign body response (FBR) characterized by increases in inflammation, extracellular matrix deposition, and leakage of the blood-brain barrier (BBB). In the present study, we investigated the role of TSP-2 in BBB repair during the brain FBR to mixed cellulose ester filters implanted in the cortex of wild-type (WT) and TSP-2-null mice for 2 days to 8 weeks. Histological and immunohistochemical analysis revealed enhanced and prolonged neuroinflammation in TSP-2-null mice up to 8 weeks after implantation. In addition, recovery of the BBB was compromised and was associated with increased gelatinolytic activity and low levels of collagen type IV in the basement membranes of TSP-2-null blood vessels. An analysis of protein extracts from implantation sites revealed elevated levels of matrix metalloproteinase (MMP)-2 and MMP-9 in TSP-2-null brains. TSP-2-null astrocytes secreted higher levels of both MMPs in vitro compared with their WT counterparts. Furthermore, TSP-2-null astrocytes were deficient in supporting the recovery of barrier function in WT endothelial cells. Finally, Western blot analysis of astrocytes and brain endothelial cells revealed TSP-2 expression only in the former. Taken together, our observations suggest that astrocyte-derived TSP-2 is critical for the maintenance of physiological MMP-2 and MMP-9 levels during the FBR and contributes to the repair of the BBB.     

Nutritional risk and malnutrition determination by anthropometry in cirrhotic children and adolescents

BACKGROUND: The malnutrition is a frequent finding in adults with cirrhosis, but the prevalence of nutritional risk and malnutrition is little known in pediatric patients. AIM: To evaluate through anthropometry the presence of nutritional risk and malnutrition in cirrhotic pediatric patients regularly attended at the Pediatric Gastroenterology Service of "Hospital de Clínicas" of Porto Alegre, RS, Brazil. METHODS: Cross-sectional study with 42 cirrhotic children and adolescents aged between 3 months and 18 years. The nutritional evaluation was made by the determination of the weight/age, height/age, body mass index and triceps skinfold thickness and arm muscle circumference measurements. Patients considered in nutritional risk were < or = -1,28 Z score which corresponds to < or = 10th percentile, and those under -2,0 Z and < or = 3th percentile were in malnutrition status. According to Child-Pugh criteria, 22 patients were classified as A (mild severity), 15 (moderate) B and 5 C (intense). RESULTS: The mean weight/age, height/age and body mass index Z scores were, respectively, - 0,38 +/- 1,4 SD, - 0,83 +/- 1,16 SD and 0,17 +/- 1,3 SD. Patients in nutritional risk were 3/42 (weight/age), 8/42 (height/age), 12/37 (triceps skinfold thickness), 9/37 (arm muscle circumference), 2/38 (body mass index); in malnutrition status were 6/42 (weight/age), 7/42 (height/age), 4/37 (triceps skinfold thickness) and 4/37 (arm muscle circumference) and 3/38 (body mass index). CONCLUSION: The prevalence of nutritional risk was 32.4% and chronic malnutrition was 16.7%. The index which better reflected the nutritional risk in these patients was triceps skinfold thickness. Chronic malnutrition status occurrence was greater in the height/age index.     

Evaluation of tests for microalbuminuria screening in patients with diabetes.

BACKGROUND: The first step in the diagnosis of diabetic nephropathy is to measure albumin in a spot urine sample. The aim of this study was to assess the accuracy of urinary albumin concentration (UAC), urinary albumin-to-creatinine ratio (UACR), and the Micral-Test II in a random urine specimen (RUS) for microalbuminuria screening in diabetes mellitus. METHODS: Two hundred and seventy-eight patients collected 24 h timed urine specimens followed by RUS. Albumin (immunoturbidimetry) and creatinine were measured in protein-negative (Combur-Test) urine samples. Samples were classified as normoalbuminuric [24 h urinary albumin excretion rate (UAER) <20 microg/min; n = 189] and microalbuminuric (UAER =20-199 microg/min; n = 89). Micral-Test II readings were performed in 130 RUS. Receiver operating characteristics (ROC) curves were constructed using UAER as the reference standard. RESULTS: The areas under the ROC curves were similar for UAC (0.934+/-0.032) and UACR (0.920+/-0.035; P = 0.626), but the Micral-Test II had lower accuracy to diagnose microalbuminuria (area = 0.846+/-0.047) than UAC (P = 0.014). The first cutoff point with 100% sensitivity for UAC was 14.4 mg/l (specificity =77.2%), and 15.7 mg/g for UACR (specificity =73.0%). Concerning the Micral-Test II, sensitivity and specificity for the 20 mg/l cutoff point were 90.0 and 46.0%, respectively. The agreement between UAER and the Micral-Test II for microalbuminuria diagnosis was 55.8% (kappa = 0.22; P < 0.001). The cost of diagnosing microalbuminuria was 1.74 dollars(UAC), 2.00 dollars (UACR) and 4.09 dollars (Micral-Test II) per patient. CONCLUSIONS: Measurement of UAC in a RUS was the best choice for the diagnosis screening of microalbuminuria in diabetic patients, considering cost and accuracy.