Examination of gene expression profile of functional human pancreatic islets after 2-week culture.

Islet transplant faces significant challenges, mainly because of the high incidence of primary nonfunction of transplanted islets. Protocol modifications to improve the rate of islet function have included changes in pancreatic preservation and the introduction of short-term culture. Islet culture for 48 to 72 hours has become a standard part of most successful protocols for clinical islet transplantation. We have previously reported gene expression profiles associated with human pancreatic islet function. The aim of this study was to determine the change in gene expression profiles of functional islets after 2 weeks of culture in Memphis-serum free media. Human islets from four isolations were maintained in culture for 14 days in Memphis-serum free media. RNA was extracted from 10000 IEQ for analysis of the gene expression profiles using high-density Affymetrix U133A GeneChips and Genespring software. Islet function was assessed by measurements of human C-peptide at days 7 and 14 posttransplant into NOD-SCID mice. Human C-peptide levels were determined by radioimmunoassay. Our preliminary data showed that genes related to functionality, such as those directed toward insulin processing and secretion, did not vary over 14 days of culture, while genes related to exocrine pancreas and organ architecture and immune-associated genes decreased over time. The ability to maintain islets in culture is an important step toward the development of islet tissue repositories, as well as toward screening human islet preparations for additional pathogens.

OP-142 gene expression profile of nonfunctional human pancreatic islets: predictors of transplant failure?

Islet culture has become a standard part of most successful protocols for clinical islet transplantation. To date, however, islets are transplanted based on crude measures of viability, purity and in vitro insulin production without adequate prior assessment of the potential for in vivo function. The purpose of this study was to define the gene expression profiles of human islets associated with in vivo function using a nonimmune NOD-scid mouse model. Human islets from eight isolations were maintained in culture for 7 to 14 days in Memphis serum-free media until transplanted. The RNA was extracted from 10,000 IEQ using RNASTAT-60. The gene expression profiles were analyzed using high-density Affymetrix U133A GeneChips and Genespring software. An aliquot of 2000 IEQ from each islet preparation was also transplanted into NOD-scid animals (n = 5) for in vivo function assessments. Islet function was assessed by measurements of human C-peptide at days 7 and 14 posttransplant. Human C-peptide levels were determined by radioimmunoassay. Gene analysis of nonfunction islets (4 of 8 islet preparations) showed high relative levels of expression of proinflammatory genes and low relative levels of genes directed toward insulin processing and secretion as well as islet integrity. Overexpression of hypoxia and proinflammatory genes may result in reduced insulin secretion and lead to islet destruction posttransplantation. Identifying and validating those genes could allow the development of a potency assay for human transplantation that would be very useful for screening human islet preparations before clinical transplant.

Respiratory vaccination of mice against influenza virus: dissection of T- and B-cell priming functions.

We find that a single respiratory administration of replicationally inactivated influenza A viral particles most often elicits a waning serum antibody response, as the long-sustained bone marrow antiviral plasma cell populations characteristically induced by viral infection are lacking, though antiviral plasma cells at other sites may occasionally persist for a long time. To determine whether this alteration in the pattern of the B-cell response is a reflection of the nature of T-helper (Th) priming, we simultaneously primed B cells with inactivated influenza A/PR8(H1N1) and Th cells with infectious A/x31(H3N2). We show that Th cells cross-react extensively between these two viruses, although the antibody response to viral envelope glycoproteins is completely non-cross-reactive. Th cells primed by infectious A/x31 have little impact on the antibody response specifically elicted from naïve B cells by inactivated A/PR8 viruses, suggesting that the characteristic vigour of the antibody response to influenza viral infection depends on the direct interaction of antiviral B cells with virally infected dendritic cells. Memory B cells primed by inactivated influenza viral particles however, respond rapidly to secondary challenge with live or inactivated viruses, promptly populating bone marrow with antiviral plasma cells. Moreover, Th cells primed by previous live A/x31 viral challenge alter the pattern of the response of naïve B cells to live A/PR8 challenge by accelerating the appearance of anti-H1/N1 plasma cells in bone marrow, eliminating the early spike of anti-H1/N1 plasma cells in the mediastinal node, and generally diminishing the magnitude of the lymph node response. Inactivated A/PR8 and infectious A/x31 are both effective vaccines against A/PR8 infection, as mice preimmunized with either vaccine exhibit much more rapid viral clearance from the lung after infectious A/PR8 challenge. In fact, even when given during a course of anti-CD8 treatment to preempt cross-reactive cytotoxic T cells, live A/x31 is a more effective vaccine against A/PR8 infection than is inactivated A/PR8 itself.

Measuring the diaspora for virus-specific CD8+ T cells.

The CD8(+) T cell diaspora has been analyzed after secondary challenge with an influenza A virus that replicates only in the respiratory tract. Numbers of D(b)NP(366)- and D(b)PA(224)-specific CD8(+) T cells were measured by tetramer staining at the end of the recall response, then followed sequentially in the lung, lymph nodes, spleen, blood, and other organs. The extent of clonal expansion did not reflect the sizes of the preexisting memory T cell pools. Although the high-frequency CD8(+) tetramer(+) populations in the pneumonic lung and mediastinal lymph nodes fell rapidly from peak values, the "whole mouse" virus-specific CD8(+) T cell counts decreased only 2-fold over the 4 weeks after infection, then subsided at a fairly steady rate to reach a plateau at about 2 months. The largest numbers were found throughout in the spleen, then the bone marrow. The CD8(+)D(b)NP(366)+ and CD8(+)D(b)PA(224)+ sets remained significantly enlarged for at least 4 months, declining at equivalent rates while retaining the nucleoprotein > acid polymerase immunodominance hierarchy characteristic of the earlier antigen-driven phase. Lowest levels of the CD69 "activation marker" were detected consistently on virus-specific CD8(+) T cells in the blood, then the spleen. Those in the bone marrow and liver were intermediate, and CD69(hi) T cells were very prominent in the regional lymph nodes and the nasal-associated lymphoid tissue. Any population of "resting" CD8(+) memory T cells is thus phenotypically heterogeneous, widely dispersed, and subject to broad homeostatic and local environmental effects irrespective of epitope specificity or magnitude.

A conserved domain of the Epstein-Barr virus nuclear antigens 3A and 3C binds to a discrete domain of Jkappa.

EBNA-3C can affect the LMP-1 promoter in both a positive and a negative manner through distinct DNA sequence elements. The viral transactivator EBNA-2 normally binds DNA indirectly via Jkappa to activate transcription, but this activation is prevented in the presence of EBNA-3C. The DNA element recognized by Jkappa is both required and sufficient for this inhibition. Jkappa clones isolated in a yeast two-hybrid screen using EBNA-3C as bait allowed us to delineate the sequences of both proteins mediating the interaction. Two isoforms of Jkappa that differ in exon 1, Jkappa-1 and RBP-2N, interact with EBNA-3C, suggesting that exon 1 is not required for this interaction; indeed, clones with deletion of the N-terminal third of Jkappa interacted as efficiently with EBNA-3C as full-length Jkappa clones. A Jkappa domain as small as 56 amino acids was sufficient to bind to EBNA-3C. A 74-amino-acid domain of EBNA-3C, conserved in all three EBNA-3 family members, was sufficient to interact with Jkappa. A specific mutation in this conserved domain suppressed the ability of EBNA-3C to downregulate transcription. Accordingly, EBNA-3A was also able to interact with Jkappa and downregulate Jkappa-mediated transcription as efficiently as EBNA-3C. The ability of the EBNA-3 proteins to prevent Jkappa from binding to DNA in vitro and suppress transactivation via Jkappa DNA elements suggests that the EBNA-3 proteins act analogously to the Drosophila protein Hairless.

A human T lymphoid cell variant resistant to the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine shows a unique combination of a phosphorylation defect and increased efflux of the agent.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral agent with activity against herpes viruses and retroviruses, including human immunodeficiency virus, but its metabolism and mechanism of action remain unclear. We have isolated a human T lymphoid cell line (CEMr-1) that is resistant to the antiproliferative effects of PMEA. The antiviral effects of PMEA against human immunodeficiency virus-1 infection were also greatly reduced in CEMr-1 cells, compared with the parental cells. This mutant showed cross-resistance to the related acyclic nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)diaminopurine and 9-(2-phosphonylmethoxyethyl)guanine and the lipophilic prodrug bis(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine-( bispome-PMEA), as well as partial resistance to the purine nucleosides 2-chlorodeoxyadenosine, 2-fluro-9-beta-D-arabinosylfuranosyladenine, and adenosine, but did not show resistance to 2'-deoxyadenosine or 9-beta-D-arabinosylfuranosyladenine. We compared the uptake and metabolism of [3H]PMEA and [3H]-bispom-PMEA in the mutant and parental cells. The analysis of radioactive products by high pressure liquid chromatography revealed marked alterations in the ability of the mutant cell line to accumulate PMEA and its anabolites, compared with the parental cells. Accumulation of PMEA, PMEA monophosphate, and PMEA bisphosphate (major metabolites formed with either PMEA or bispom-PMEA) decreased by 50, 95, and 97%, respectively. Compared with the parental cells, the variant cells showed a approximately 7-fold increase in the rate of efflux of PMEA and a 2-fold decrease in the activity of adenylate kinase. In contrast, other enzymes of nucleotide metabolism, such as adenosine kinase, deoxycytidine kinase, and 5-phosphoribosyl-1-pyrophosphate synthetase, showed no significant change in the two cell lines. Overall, these results suggest that the mutation in this resistant cell line is of a novel type, involving an alteration in the cellular efflux of PMEA as the major basis for the resistant phenotype.

A Fleur de Lys modification of the TRAM flap for breast reconstruction.

A major problem of the standard TRAM flap is uncertain vascularity. Many modifications have been made to overcome this problem, but they usually lead to an increase in both the duration and complexity of the operative procedure. The modification described here uses a single muscle pedicle flap, but adds a vertical component comprising the well vascularised tissue above the umbilicus in continuity with the usual horizontal skin flap. This has the advantage of greater volume of available tissue, much improved blood supply, shorter operative time, greater exposure for dissection, a technically more simple procedure, and quicker recovery. The vertical and horizontal skin flaps are approximated as a cone to provide excellent breast projection. All this can be achieved at the cost of a vertical abdominal scar, which is well accepted when explained to patients.

Rate of glycolysis during ischemia determines extent of ischemic injury and functional recovery after reperfusion.

The efficacy of increasing glycolysis during ischemia for enhancing the salutary effects of reperfusion was evaluated in isolated perfused rabbit hearts subjected to low-flow ischemia followed by reperfusion. Control hearts were perfused with buffer containing 0.4 mM palmitate, 5 mM glucose, and 70 mU/l insulin. Additional groups of hearts were perfused with double glucose/insulin and 1 mM dichloroacetate or were subjected to substrate priming to increase preischemic glycogen content. Ischemic contracture was completely prevented in hearts perfused with high glucose/insulin and was delayed markedly by either dichloroacetate or enhanced preischemic glycogen [45 +/- 14 and 31 +/- 20 min, respectively; P < 0.01 each vs. control (11 +/- 10 min)] and inversely related to the rate of lactate production. With reperfusion, recovery of developed pressure was 56 +/- 23% of baseline in control hearts, 90 +/- 8% in hearts receiving high glucose/insulin, 92 +/- 5% in hearts receiving dichloroacetate, and 79 +/- 19% in hearts with increased glycogen (P < 0.05 each vs. control hearts). Creatine kinase release was reduced by > 55% in treated hearts. Thus enhancement of glycolysis by diverse mechanisms during ischemia decreased ischemic damage and improved the recovery of contractile function with reperfusion.

Susceptibilities of zidovudine-resistant variants of human immunodeficiency virus type 1 to inhibition by acyclic nucleoside phosphonates.

The acyclic purine nucleoside phosphonates, a newly described class of broad-spectrum antiviral agents, effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro and in animal AIDS models. 9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is currently being evaluated in clinical trials in patients with AIDS. In this study, we investigated the efficacy of PMEA and a related analog, 9-(2-phosphonylmethoxypropyl)diaminopurine (PMPDAP), against HIV-1 isolates exhibiting various degrees of resistance to zidovudine (azidothymidine [AZT]). HIV isolates highly (approximately 50 to 200-fold) resistant to AZT were found to be about two- to eightfold less susceptible to PMEA. A comparable degree of cross-resistance to PMPDAP, a structurally related analog of PMEA, was also observed. However, the 50% effective dose values of PMEA or PMPDAP against a panel of HIV isolates showing intermediate levels (approximately 8 to 25-fold) of AZT resistance was indistinguishable from the 50% effective dose values of PMEA (0.7 to 1.7 versus 2 microM) or PMPDAP (0.4 to 1.4 versus 0.8 to 1 microM) against HIV isolates from patients who had not previously used AZT. In addition, we were unable to generate PMEA- (or PMPDAP)-resistant HIV-1 variants by > 30 serial passages of the virus in the presence of increasing concentrations of PMEA. Careful analysis of HIV-1 isolates from patients previously treated with AZT for cross-resistance to PMEA are needed to evaluate the significance of these observations.

Breastfeeding after reduction mammaplasty.

A retrospective study was performed of 30 women who had undergone breast reduction and subsequently wished to breastfeed. Breastfeeding capabilities were assessed by a trained lactation consultant. Findings indicate that in women who have a physiological type of operation then breastfeeding is usually possible (18 patients out of 19), although complementary feeds may be required. We strongly suggest that all functioning breast tissue that remains after reduction mammaplasty be left attached to the nipple in a physiological manner to allow subsequent breastfeeding.

The contralateral breast flap in reconstruction of the breast and chest wall.

The contralateral breast flap is a useful method of reconstruction of the breast and chest wall after the treatment of carcinoma of the breast by surgery and x-ray therapy. Breast tissue is an excellent donor tissue to solve these difficult problems and the only disadvantage is the risk of a second primary breast cancer. Young patients with an identifiable high risk of a second primary tumor are not suitable for this technique. If, however, we confine the operation to the group in whom we are prepared to preserve the breast, with or without a breast reduction, then there is no logical reason why the risk should be any greater in having the residual breast tissue on two sides rather than one. The operation involves the transfer of breast tissue, normally discarded in a breast reduction, to the other side in a two-stage procedure; this is a useful method, both for reconstruction of the breast and also for repair of the chest wall after irradiation damage, particularly in elderly patients. I have found this technique to be suitable in approximately 10% of breast reconstructions I have performed; there has been no patient with a second primary tumor over a 15-year period in 60 carefully selected patients.

Selection of Marek's disease virus recombinants expressing the Escherichia coli gpt gene.

We developed a positive selection method for recovering Marek's disease virus (MDV) recombinants. The Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt), under the control of the major immediate-early promoter from cytomegalovirus, was inserted into the inverted repeats flanking the unique long (UL) region of a non-pathogenic serotype 2 MDV strain 281MI/1. In a second demonstration of the usefulness of the positive selection system, the gpt gene was inserted into the inverted repeats flanking the unique short (US) region of the turkey herpesvirus (HVT) strain FC126. The targeted insertion site in 281MI/1 was in a previously established nonessential site for virus replication. The targeted insertion site for FC126, at the junction of the UL and US regions, is a nonessential site for in vitro replication of herpes simplex virus. Recombinant viruses were easily selected by incubating the transfected cells in mycophenolic acid (MPA)-containing medium. Purification of recombinants resulted from a series of trypsinization and sonication steps combined with the culturing of virus in MPA-containing medium to inhibit wild-type virus replication. This simple technique for recovering MDV and HVT recombinants should increase the efficiency of identifying nonessential sites and gene function analysis by insertional mutagenesis.

Plasmid-associated effects on test gene expression and Marek's disease virus plaque formation during recombination trials.

Marek's disease virus (MDV), a herpesvirus of avian origin, is being examined for suitability as a vector for expressing foreign genes. We observed that plasmids encoding the LacZ gene of E. coli under the control of either the herpes simplex virus alpha 4 immediate-early promoter or the cytomegalovirus major immediate-early promoter inhibited MDV plaque formation. Plaque numbers were decreased by one-third, and transient expression of the beta-galactosidase reporter gene was increased by up to 6-fold, when the plasmids were linearized. Sequences associated with the heterologous promoter were identified as being responsible for inhibiting MDV replication.

Nasal augmentation in chondrodysplasia punctata using tissue expansion.

Chondrodysplasia punctata is an uncommon group of congenital bone dysplasias. A common feature of this disease is a characteristic facial appearance including nasal hypoplasia. This paper describes the management of a case presenting with this disease in which soft tissue augmentation was achieved with tissue expansion of the nasal skin. Follow-up of more than 4 years demonstrates that a satisfactory result has been achieved, with no facial scars. The relevant literature is discussed.

Regional macrodontia and regional bony enlargement associated with congenital infiltrating lipomatosis of the face presenting as unilateral facial hyperplasia. Brief review and case report.

Congenital infiltrating lipomatosis of the face (CIL-F) is a rare lesion. Presented here is a review of the literature concerning this condition, with the report of a case with clinical signs indistinguishable from those of unilateral facial hyperplasia. Previously unreported clinical and radiographic features of regional bony hypertrophy and macrodontia associated with this case of CIL-F are described and the proposition raised that previously reported cases of unilateral facial hyperplasia may have been due to CIL-F. It is suggested that the clinical findings in this case of facial asymmetry associated with CIL-F, regional macrodontia and regional bony enlargement may constitute a previously undescribed syndrome.

Serum antibodies to periodontal bacteria.

The purpose of this study was to determine how serum antibodies reactive with periodontitis-associated bacteria with relates to the diagnosis of periodontitis subjects. Study groups included localized juvenile periodontitis (LJP) subjects, severe periodontitis (SP) subjects, chronic adult periodontitis (AP) subjects, and age matched controls. Twenty-two bacterial strains, representing 18 different species most commonly found in early onset periodontitis were evaluated using serum from LJP, SP, and age matched controls. Serum IgG reactive with these organisms was determined using a radioimmunoassay (RIA). Serum antibody reactive with 13 bacterial strains differed significantly (P less than 0.01) between the three clinical groups. Discriminate analysis revealed that antibodies reactive with 5 bacterial strains of the 13 were able to identify the clinical group to which subjects belonged 79% of the time with control subjects being correctly identified 100% of the time, LJP subjects 78% of the time, and SP subjects 60% of the time. These strains included two strains of Actinobacillus actinomycetemcomitans (Y4 and N27), Fusobacterium nucleatum (E1D1), Eubacterium brachy, and Bacteroides gingivalis. The low classification rate of SP subjects suggested heterogeneity. The SP group could be divided into three subgroups using the serological data. One subgroup, with "super" severe attachment loss, generally lacked antibody reactive with these five organisms, another subgroup was serologically similar to LJP subjects, while the third subgroup had antibodies to additional organisms. This suggests that some SP subjects may represent a more advanced form of LJP. Comparison of antibody reactivity of AP subjects with age matched controls to 23 bacterial types revealed that mean serum antibody reactivity to only Bacteroides gingivalis was higher in AP subjects.(ABSTRACT TRUNCATED AT 250 WORDS)