Knockdown of PIK3R1 by shRNA inhibits the activity of the splenic macrophages associated with hypersplenism due to portal hypertension.

Phosphatidylinositol 3-kinase (PI3K) plays a central role in the metabolic actions of insulin. One 85 kDa regulatory subunit of PIK3 is encoded by phosphoinositide-3-kinase, the regulatory subunit 1 (PIK3R1). Our previous study has demonstrated that PIK3R1 was up-regulated significantly in the splenic macrophage (MΦ) of portal hypertensive spleen. In the present study, RNA interference specific to PIK3R1 was employed to investigate its inhibitive effects on the activity of MΦ associated with hypersplenism due to portal hypertension (HS-PHT). The expression of PIK3R1 in the spleen was detected by immunohistochemical staining. Plasmid vector pGenesil-1 expressing specific small hairpin RNA (shRNA) against PIK3R1 and the scrambled shRNA control was constructed. MΦ were isolated and purified by anchored cultivation from patients with HS-PHT (HS-PHT-MΦ) and traumatic rupture of the spleen (Con-MΦ). After transfection into MΦ, PIK3R1 expression at both the mRNA and the protein level was examined by real-time polymerase chain reaction and Western blot. The activities of MΦ were determined, and the expression and activity of NF-κB were also detected. Immunohistochemistry revealed expression and cellular distribution of PIK3R1 in the spleen. The PIK3R1-shRNA was successfully synthesized and cloned into the plasmid vector pGenesil-1, and specifically suppressed PIK3R1 expression at both the mRNA and the protein level. After transfection into HS-PHT-MΦ and Con-MΦ, PIK3R1 knockdown inhibited the viability of MΦ, reduced the phagocytic rate, the rate of antigen-presenting positive cells, the metabolic rate, and the secretion of IL-1ß and TNF-α (all p<0.05), and decreased the expression and activity of NF-κB. Our data showed that the knocking down of PIK3R1 with shRNA produced by pGenesil-1 led to inhibition of viability and to decreased activity of MΦ associated with HS-PHT in vitro. Therefore, it is tempting to speculate that PIK3R1 might play a considerable role in the pathogenesis of HS-PHT, and inhibition of PIK3R1 expression might be a novel therapeutic strategy for HS-PHT.

MicroRNAome of splenic macrophages in hypersplenism due to portal hypertension in hepatitis B virus-related cirrhosis.

MicroRNAs (miRNAs) are a recently discovered class of post-transcriptional regulators of gene expression with critical functions in health and disease. Their role in the pathogenesis of hypersplenism, however, is completely unknown. To determine whether miRNA expression is altered in splenic macrophages associated with hypersplenism due to portal hypertension in hepatitis-B-virus (HBV)-related cirrhosis, we analyzed the entire miRNAome in macrophages from normal and portal hypertensive spleen samples by microarray and Real-Time PCR. In this study, we identified 99 miRNA differences in expression in splenic macrophages associated with hypersplenism due to portal hypertension in HBV-related cirrhosis. Among the miRNAs identified in this study, hsa-miR-615-3p was significantly up-regulated in hypersplenism. Dynamic changes in miRNA expression occurred during the pathogenesis of portal hypertension-induced hypersplenism in HBV-related cirrhosis. The miRNAs then are novel regulatory RNAs in hypersplenism in patients with HBV-related cirrhosis.

[cDNA microarray-based screening of differentially expressed genes in macrophages in the spleen of patients with portal hypertension and hypersplenism].

OBJECTIVE: To identify the differentially expressed genes associated with hypersplenism in patients with portal hypertension. METHODS: The total RNA were extracted from the macrophages isolated from normal spleen and the spleen of patients with portal hypertension and reversely transcribed to cDNA with the incorporation of fluorescent (cy3 and cy5)-labeled dCTP to prepare the hybridization probes. After hybridization of Biostar-H140s chip containing 14,112 spots of cDNAs with the prepared probes, the gene chip was scanned for fluorescence intensity to screen the differently expressed genes. Three gene chips were used for hybridization and only the genes with differential expression in all the three chips were considered to associate with hypersplenism in patients with portal hypertension. RESULTS: Totaling 896, 1330 and 898 genes were identified to be differentially expressed by the three chips, respectively, and 121 genes (0.86%) showed differential expression in all the three chips, including 21 up-regulated known genes and 73 down-regulated known genes. The differently expressed genes were functionally related with ion channels and transport proteins, cyclins, cytoskeleton, cell receptors, cell signal transduction, metabolism, immunity, and so forth. These genes might be involved in hypersplenism in the condition of portal hypertension. CONCLUSION: cDNA microarray-based screening of differentially expressed genes in the macrophages in the spleen may provide new insights into the pathogenesis of hypersplenism in patients with portal hypertension.

The effect of splenectomy for hypersplenism on whole body protein turnover, resting metabolic rate and growth in sickle cell disease.

OBJECTIVE: To determine whether, in the same individual, an observed fall in whole body protein turnover following splenectomy in children with hypersplenism and homozygous sickle cell (SS) disease is associated with a measurable fall in resting metabolic rate (RMR) and an increase in rate of growth. SUBJECTS: Six children (5 SS disease, 1 S beta degree thalassaemia), aged 68 to 126 months, were studied before and after splenectomy for hypersplenism. DESIGN: Protein turnover was measured by the end product method using prime/intermittent oral doses of 15N-glycine and RMR by indirect calorimetry before preoperative transfusion and repeated at least eight weeks after splenectomy. Height and weight velocities were measured over six month periods before and after splenectomy. SETTING: University Hospital of the West Indies in Jamaica and the Medical Research Laboratories (Jamaica). RESULTS: After splenectomy protein turnover fell significantly by 30% and RMR by 34 kJ/kg/d. Mean weight velocity which was below normal before surgery, z = -2.3, improved significantly after surgery, z = 0.7, (P = 0.03). Height velocity increased in two children but the mean height velocity did not change following splenectomy. The reduction in protein turnover was estimated to account for 62% of the fall in RMR. CONCLUSION: This study confirms that there is a significant reduction in energy expenditure following splenectomy for hypersplenism in SS disease. A reduction in protein turnover was a major contributor to the saving in energy, although it is not clear whether it accounted for all. In the present group of children the energy saved was associated with an improvement in the wasting present before splenectomy.

The effect of splenectomy for hypersplenism on whole body protein turnover, resting metabolic rate and growth in sickle cell disease

OBJECTIVE: To determine whether, in the same individual, an observed fall in whole body protein turnover following splenectomy in children with hypersplenism and homozygous sickle cell (SS) disease is associated with a measurable fall in resting metabolic rate (RMR) and an increase in rate of growth. SUBJECTS: Six children (5 SS disease, 1 S beta degree thalassaemia), aged 68 to 126 months, were studied before and after splenectomy for hypersplenism. DESIGN: Protein turnover was measured by the end product method using prime/intermittent oral doses of 15N-glycine and RMR by indirect calorimetry before preoperative transfusion and repeated at least eight weeks after splenectomy. Height and weight velocities were measured over six month periods before and after splenectomy. SETTING: University Hospital of the West Indies in Jamaica and the Medical Research Laboratories (Jamaica). RESULTS: After splenectomy protein turnover fell significantly by 30 percent and RMR by 34 kJ/kg/d. Mean weight velocity which was below normal before surgery, z = -2.3, improved significantly after surgery, z = 0.7, (p = 0.03). Height velocity increase in two children but the mean height velocity did not change following splenectomy. The reduction in protein turnover was estimated to account for 62 percent of the fall in RMR. CONCLUSION: This study confirms that there is a significant reduction in energy expenditure following splenectomy for hypersplenism in SS disease. A reduction in protein turnover was a major contributor to the saving in energy, although it is not clear whether it accounted for all. In the present group of children the energy saved was associated with an improvement in the wasting present before splenectomy.(AU)

[Hereditary thrombophilia as a cause of recurrent transplant thromboses]. / Hereditäre Thrombophilie als Ursache rezidivierender Transplantatthrombosen.

We report a case of hereditary protein S and protein C deficiency which are a rare defects of the anti-coagulation-system. Protein S is a vitamin K dependent glycoprotein that functions as a cofactor to activated Protein C in the inactivation of coagulation factors V and VIIIa. A deficiency of these proteins caused by a genetic defect increase the risk of recurrent thrombosis at a younger age. Acquired decreases in protein S and C concentration have been reported in connection with age, sex, pregnancy and with oral anticonception. The higher risk for thrombotic diseases of patients with thrombophilia requires a sufficient treatment and prophylaxis, e.g. with fresh frozen plasma or a protein C concentrate.