ABSTRACT
Polymerase chain reaction (PCR) has been used as a substitute for conventional serological methods in order to provide blood or blood products free from contaminating viruses and recently attempts have focused to detect 2 or 3 viruses by a single multiplex PCR (M-PCR) reaction. We were able to detect human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV) and human cytomegalovirus (HCMV) simultaneously by a single M-PCR. However detection by gel electrophoresis of the products from M-PCR suffers from drawbacks such as low sensitivity and product sizes. Here we report enhanced detection systems of M-PCR based on nucleic acid hybridization with arrays built on membrane. Membrane array was manufactured by spotting appropriate probe DNAs on nylon membrane. Single or multiplex PCR was performed and the PCR products were labeled with DIG and allowed to hybridize with the membrane array. Results indicate that nonspecific hybridization was not observed for membrane DNA array. Additionally, membrane array method could detect small amount of viruses that were not detectable by conventional gel electrophoresis. At least 25-fold, and in some cases more than 125-fold increases in sensitivity was obtained with DNA array method. Thus, the nucleic acid hybridization with membrane array could be applied for the detection of M-PCR of viruses in blood or blood products.
Subject(s)
Female , Humans , Cytomegalovirus , DNA , Electrophoresis , Hepacivirus , Hepatitis B virus , HIV-1 , Membranes , Metrorrhagia , Multiplex Polymerase Chain Reaction , Nucleic Acid Hybridization , Nylons , Oligonucleotide Array Sequence Analysis , Polymerase Chain ReactionABSTRACT
Purification of islets in pancreas islet cell transplantation has some potential advantages ,such as more safety, improved islet cell implantation, and reduced immunogenicity, compared with an unpurified pancreas islet cell transplantation. We evaluated the effect of islet cell purification on islet yields, hemodynamics, and graft outcome following intraportal canine pancreas islet cell transplantation. The baseline characteristics, including body weight, pancreas weight and collagenase recirculation time for the unpurified group(n=12) and the purified group(n=17) did not show any significant difference(P>0.05). The mean cell pellet volume before intraportal injection was 25.4 ml in the unpurified group and 7.2ml in the purified group(P0.05). Mean recovery rate of islet after purification was 66.8%. The portal pressure change after intraportal islet injection was significantly less in the purified group(16.2+/-11.3 cmH2O vs 6.2+/-2.0 cmH2O, P OR =70%, n=4 vs low purity <70%, n=13) in the purified group, showed significant hemodynamic stability in the high purity group, but no significant difference in the islet recovery rate between the low and high purity group(58.6% vs 61.7%). Glucose was controlled in 3 cases(25.0%) in the unpurified group and 7 cases(41.2%) in the purified group. Death due to portal hypertension occurred in 2 cases(16.7%) in unpurified group and 2 cases(11.7%) in the purified group. Interestingly, in the highly purified group, all the animals were alive with normoglycemia during the follow up period. We conclude that purified pancreas islet cell transplantation, especially in a highly purified group, has distinct hemodynamic advantages compared with unpurified islet transplantation following intraportal islet injection. However further research to develop the methods that will minimize the loss of islet yields during purification and enhance the purity is neccessary to achieve a successful islet transplantation with a long-term good result.
Subject(s)
Animals , Body Weight , Collagenases , Follow-Up Studies , Glucose , Heart Rate , Hemodynamics , Hypertension, Portal , Islets of Langerhans Transplantation , Islets of Langerhans , Pancreas , Portal Pressure , TransplantsABSTRACT
Organ transplantation has become a' widely accepted treatment modality for end-stage organ disease. The shortage of allogenic donors for organ transplantation has brought about the necessity of xenotransplantation as an unlimited source of organ donation. However, organ transplantation between different species have never been successful because of hyperacute rejection. Although the mechanism of this phenomenon is not fully understood, many researchers believe that the natural antibodies present in the recipient's serum may bind to the graft and induce the activation of complement cascade triggering the process of hyperacute rejection. ...continue...