Achievement of insulin independence via pancreatic islet transplantation using a remote isolation center: a first-year review.

BACKGROUND: Owing to advances in both immunosuppressive protocols and pancreatic islet isolation techniques, insulin independence has recently been achieved in type 1 insulin-dependent diabetics (IDDM) via pancreatic islet transplantation (PIT). Although the dissemination of immunosuppressive protocols is relatively easy, transferring the knowledge and expertise required to isolate a large number of quality human islets for transplantation is a far greater challenge. Therefore, in an attempt to centralize the critical islet processing needed for islet transplantation and to avoid the development of another islet processing center, we have established a collaborative islet transplant program between two geographically distant transplant centers. PATIENTS AND METHODS: Eleven consecutive type 1 IDDM patients with a history of severe hypoglycemia and metabolic instability underwent PIT at the Methodist Hospital (TMH) in Houston, Texas, utilizing pancreatic islets isolated at the Diabetes Research Institute (DRI) at the University of Miami in Miami, Florida between January 1, 2002 and June 31, 2003. Forty-one pancreata have been procured in the Houston area and have subsequently been transported for isolation at the DRI following enzymatic ductal perfusion by the automated method (Ricordi chamber). Following purification the islets were immediately transported back to TMH in Houston and transplanted via percutaneous transhepatic portal infusion. Immunosuppression regimen consisted of sirolimus, tacrolimus, and daclizumab. RESULTS: Following harvesting, donor pancreata arrived at the DRI for initiation of the isolation process within 6.5 hours of cross-clamping (median time 5.4 hours; range 4.8 to 6.5 hours). The islets were immediately transported back to TMH for final sterility and viability tests and transplanted via percutaneous transhepatic portal vein infusion. The harvesting of 41 pancreata has yielded a number of pancreatic islets sufficient for transplantation (>5000 IEQ/kg recipient body weight) 26 times (63% of harvested pancreata). Thus far, three patients have received three PITs and eight patients have received two PITs. Six remain insulin independent. All have experienced a decrease in serum hemoglobin A(1c) levels, and both basal and stimulated C-peptide levels have increased. There have been no major complications related to the procedure or the immunosuppressive regimen used. CONCLUSIONS: Our series demonstrates that pancreatic islets isolated at a remote isolation center can successfully and safely be used for PIT and the achievement of insulin independence.

Interventional uroradiology.

The development of interventional uroradiologic techniques has had a major impact on the care of the urologic patient by allowing nonoperative treatment of many disease processes. This article will review percutaneous nephrostomy with emphasis on urologic calculi, interventional therapy for neoplasms and trauma of the urinary tract, diagnosis and treatment of renovascular hypertension, and the management of complications following renal transplantation.

Cloning and analysis of a gene from Streptomyces lividans 66 encoding a novel secreted protease exhibiting homology to subtilisin BPN'.

Amino-terminal degradation has been observed for many of the secreted heterologous proteins produced by S. lividans 66. We, therefore, set out to characterize the relevant proteinases and their genes. A tripeptide chromogenic substrate was used to identify a gene that was shown to encode a secreted protein which removed tripeptides from the amino terminus of extracellular proteins (tripeptidyl aminopeptidase, Tap; Butler et al. 1995). This activity was removed by a homologous gene deletion replacement and the ability of the S. lividans strain to remove N-terminal tripeptides was greatly reduced, but still significant. When the tap-deleted strain was used as a host for the rescreening of a S. lividans 66 genomic DNA library, a number of other genes encoding proteases with aminopeptidase activities were discovered. One clone (P5-4) produced a 45-kDa secreted protein (Ssp), which showed activity against Ala-Pro-Ala-beta-naphthylamide (APA-beta NH-Nap) substrate. Further analysis of the cloned DNA showed an open-reading frame encoding a protein larger than 45 kDa. Direct Edman degradation of the secreted protein confirmed that it was encoded within the cloned DNA and probably processed from a larger precursor. Protein sequence analysis revealed a striking homology to subtilisin BPN' in three regions around the active-site residues suggesting that the protein is a serine protease. As expected, the protease activity was inhibited by phenylmethylsulphonyl fluoride. Mutant strains with most of the ssp gene deleted exhibited reduced activity against APA-beta NH-Nap substrate compared to their non-deleted parental strains.

Use of a Wallstent in successful treatment of IVC obstruction following liver transplantation.

Obstruction of the IVC occurs in only 1-2% of patients after liver transplantation. The mortality of this complication can be as high as 66%. This case report describes the use of a Wallstent for an IVC obstruction that was unresponsive to conventional balloon angioplasty.

Cloning and characterization of a gene encoding a secreted tripeptidyl aminopeptidase from Streptomyces lividans 66.

The gene encoding a tripeptidyl aminopeptidase (Tap) from Streptomyces lividans was cloned by using a simple agar plate activity assay. Overexpression of the cloned gene results in the production of a secreted protein which has an apparent subunit molecular weight of 55,000 and is responsible for the major amino-terminal degradative activity in culture broths of S. lividans strains. A DNA sequence analysis revealed a potential protein-encoding region of the size expected to encode the observed protein, which contained a sequence that exhibited significant homology around a putative active site serine residue observed for lipases, esterases, and acyl transferases. Preceding the amino terminus of the secreted protein was a predicted signal peptide of 36 amino acids followed by a tripeptide, which could be autocatalytically removed from a secreted Tap precursor. The transcriptional start site for the gene was mapped by primer extension. Mutant strains of S. lividans lacking detectable Tap activity were able to grow and sporulate normally. Cross-species hybridization experiments showed that DNA homologs of the tap gene are present in most of the Streptomyces strains tested.

Regulation of gene expression by the HlyX protein of Actinobacillus pleuropneumoniae.

The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae and the fnr gene of Escherichia coli encode very similar proteins. The hlyX gene is able to complement delta fnr mutations and will permit the growth of E. coli fnr strains in nitrate minimal salts medium under anoxic conditions. In addition, the hlyX gene product appears to induce the expression of a latent haemolytic activity as evidenced by the presence of a strong zone of haemolysis around E. coli (hlyX+) colonies grown on bovine or ovine blood. In this study, the ability of the hlyX gene product to induce haemolytic activity and regulate expression of frdA and its own gene was examined in the presence of various carbon sources and in the presence and absence of iron; fnr was included for comparison. The HlyX protein was able to induce the synthesis of the latent E. coli haemolytic activity only under anoxic conditions. Haemolytic activity was highest during the late exponential phase and then levelled off in the stationary phase. The hlyX gene product was able to activate the expression of a phi (frdA'-lacZ) in E. coli JRG1787 (delta fnr); however, the level of expression depended on carbon source, growth phase and copy number. Like fnr, the hlyX gene product appeared to affect its own synthesis but the nature and extent of regulation depended not only on the presence of oxygen but also on growth conditions.

The aminopeptidase N-encoding pepN gene of Streptomyces lividans 66.

The gene (pepN) encoding an aminopeptidase N (PepN) has been cloned from Streptomyces lividans. This was done using either leucine-beta-naphthylamide or arginine-beta-naphthylamide in a liquid overlayer on colonies growing on agar medium to screen for overproduction of the ability to hydrolyse the substrates. The nucleotide sequence of pepN was determined and shown to encode a 95-kDa protein, which displayed significant homology to PepN proteins from other organisms. Analysis of the overproduced proteinase confirmed that this protein was located intracellularly as a monomeric active species. PepN is a metallo-exopeptidase cleaving next to Leu, Arg and Lys in peptide-bond-containing substrates.

Cloning and characterization of btr, a Bordetella pertussis gene encoding an FNR-like transcriptional regulator.

To determine whether hemolytic factors other than the bifunctional hemolysin-adenylate cyclase toxin (cyclolysin) are expressed by Bordetella pertussis, a gene library was constructed from a virulent strain of B. pertussis, BP504, transformed into nonhemolytic Escherichia coli, and screened on blood agar plates. A strongly hemolytic colony which contained the plasmid pHLY1A was isolated. Nucleotide sequencing of pHLY1A revealed an open reading frame that could encode a 27-kDa protein. No similarity was detected between the deduced amino acid sequence of this open reading frame and those of any known bacterial cytolysins. However, significant homology was detected with FNR of E. coli and several other transcriptional regulators including HylX from Actinobacillus pleuropneumoniae, which can also confer a hemolytic phenotype on E. coli. An fnr mutant of E. coli, JRG1728, could be complemented by pHLY1A. Thus, the B. pertussis transcriptional regulator-like gene and the protein which it encoded were named btr and BTR, respectively. A BTR-deficient B. pertussis strain, BJB1, was constructed. The btr::kan mutation had no effect on the expression of hemolytic activity or on phase variation. Northern (RNA) blotting revealed that btr expression was not regulated by the BvgAS two-component sensor-regulator. On the basis of sequence similarity to FNR-like transcriptional regulators and the ability to complement an anaerobically deficient E. coli strain (JRG1728) in growing anaerobically, BTR may regulate B. pertussis gene expression in response to changes in oxygen levels or to changes in the redox potential of the bacterial environment. Its role in virulence remains to be determined.

Placement of dual bird's nest filters in an unusual case of duplicated inferior vena cava.

The authors describe an unusual variant of inferior vena cava duplication, with azygos continuation of the right vena cava and hemiazygos continuation of the left vena cava, discovered at cavography in a patient with pulmonary embolism. Following unsuccessful attempts to advance titanium Greenfield filters through tortuous iliac veins, bilateral Bird's Nest filters were placed successfully.

Actinobacillus pleuropneumoniae hlyX gene homology with the fnr gene of Escherichia coli.

The hlyX gene from Actinobacillus pleuropneumoniae, which confers a hemolytic phenotype on Escherichia coli, was sequenced, and its role in regulation of gene expression was investigated. No similarity was found between the hlyX sequence and sequences of known hemolysin or cytotoxin genes. However, the hlyX sequence was very similar to that of the fnr gene of Escherichia coli which encodes the global regulatory protein, FNR. Comparison of the deduced amino acid sequence of the hlyX gene product (HlyX) with that of FNR revealed a high degree of well-aligned sequence correlation throughout the polypeptide chain. For example, 23 of 24 amino acids in the DNA-binding region of FNR are identical in the corresponding region of HlyX. Four cysteine residues in the amino-terminal region are also conserved. The promoter region of hlyX is very similar to that of fnr. It has a putative -10 sequence which closely resembles the E. coli -10 consensus sequence. This sequence is overlapped by a potential operator which is very similar to the FNR-binding-site consensus sequence. Functional homology between HlyX and FNR was also demonstrated. Plasmids carrying hlyX complemented the nutritional lesion of an fnr deletion strain of E. coli. These data suggest that HlyX may regulate, rather than mediate, hemolytic activity in E. coli, but the possibility that HlyX is both a regulator of gene expression and a hemolysin cannot be excluded.