Mechanical Ventilation and Extracorporeal Membrane Oxygenation as a Bridging Strategy to Lung Transplantation: Significant Gains in Survival.

Mechanical ventilation (MV) and extracorporeal membrane oxygenation (ECMO) are increasingly used to bridge patients to lung transplantation. We investigated the impact of using MV, with or without ECMO, before lung transplantation on survival after transplantation by performing a retrospective analysis of 826 patients who underwent transplantation at our high-volume center. Recipient characteristics and posttransplant outcomes were analyzed. Most lung transplant recipients (729 patients) did not require bridging; 194 of these patients were propensity matched with patients who were bridged using MV alone (48 patients) or MV and ECMO (49 patients). There was no difference in overall survival between the MV and MV+ECMO groups (p = 0.07). The MV+ECMO group had significantly higher survival conditioned on surviving to 1 year (median 1,811 days ([MV] vs. not reached ([MV+ECMO], p = 0.01). Recipients in the MV+ECMO group, however, were more likely to require ECMO after lung transplantation (16.7% MV vs. 57.1% MV+ECMO, p < 0.001). There were no differences in duration of postoperative MV, hospital stay, graft survival, or the incidence of acute rejection, renal failure, bleeding requiring reoperation, or airway complications. In this contemporary series, the combination of MV and ECMO was a viable bridging strategy to lung transplantation that led to acceptable patient outcomes.

Proteasome Inhibitor Carfilzomib-Based Therapy for Antibody-Mediated Rejection of the Pulmonary Allograft: Use and Short-Term Findings.

We present this observational study of lung transplant recipients (LTR) treated with carfilzomib (CFZ)-based therapy for antibody-mediated rejection (AMR) of the lung. Patients were considered responders to CFZ if complement-1q (C1q)-fixing ability of their immunodominant (ID) donor-specific anti-human leukocyte antibody (DSA) was suppressed after treatment. Treatment consisted of CFZ plus plasma exchange and immunoglobulins. Fourteen LTRs underwent CFZ for 20 ID DSA AMR. Ten (71.4%) of LTRs responded to CFZ. DSA IgG mean fluorescence intensity (MFI) fell from 7664 (IQR 3230-11 874) to 1878 (653-7791) after therapy (p = 0.001) and to 1400 (850-8287) 2 weeks later (p = 0.001). DSA C1q MFI fell from 3596 (IQR 714-14 405) to <30 after therapy (p = 0.01) and <30 2 weeks later (p = 0.02). Forced expiratory volume in 1s ( FEV1 ) fell from mean 2.11 L pre-AMR to 1.92 L at AMR (p = 0.04). FEV1 was unchanged after CFZ (1.91 L) and subsequently rose to a maximum of 2.13 L (p = 0.01). Mean forced expiratory flow during mid forced vital capacity (25-75) (FEF25-75 ) fell from mean 2.5 L pre-AMR to 1.95 L at AMR (p = 0.01). FEF25-75 rose after CFZ to 2.54 L and reached a maximum of 2.91 L (p = 0.01). Responders had less chronic lung allograft dysfunction or progression versus nonresponders (25% vs. 83%, p = 0.04). No deaths occurred within 120 days and 7 patients died post CFZ therapy of allograft failure. Larger prospective interventional studies are needed to further describe the benefit of CFZ-based therapy for pulmonary AMR.

A novel dual ex vivo lung perfusion technique improves immediate outcomes in an experimental model of lung transplantation.

The lungs are dually perfused by the pulmonary artery and the bronchial arteries. This study aimed to test the feasibility of dual-perfusion techniques with the bronchial artery circulation and pulmonary artery circulation synchronously perfused using ex vivo lung perfusion (EVLP) and evaluate the effects of dual-perfusion on posttransplant lung graft function. Using rat heart-lung blocks, we developed a dual-perfusion EVLP circuit (dual-EVLP), and compared cellular metabolism, expression of inflammatory mediators, and posttransplant graft function in lung allografts maintained with dual-EVLP, standard-EVLP, or cold static preservation. The microvasculature in lung grafts after transplant was objectively evaluated using microcomputed tomography angiography. Lung grafts subjected to dual-EVLP exhibited significantly better lung graft function with reduced proinflammatory profiles and more mitochondrial biogenesis, leading to better posttransplant function and compliance, as compared with standard-EVLP or static cold preservation. Interestingly, lung grafts maintained on dual-EVLP exhibited remarkably increased microvasculature and perfusion as compared with lungs maintained on standard-EVLP. Our results suggest that lung grafts can be perfused and preserved using dual-perfusion EVLP techniques that contribute to better graft function by reducing proinflammatory profiles and activating mitochondrial respiration. Dual-EVLP also yields better posttransplant graft function through increased microvasculature and better perfusion of the lung grafts after transplantation.

Surgical management of gastroesophageal reflux disease/Barrett's esophagus.

Gastroesophageal reflux disease and its complications, such as Barrett's esophagus, are major health problems. In this review, we highlight the critical components of the disease process and surgical considerations relevant to laparoscopic anti-reflux surgery.

Minimally invasive cancer surgery improves patient survival rates through less perioperative immunosuppression.

The import of the immune system to cancer survival is paramount. Immune effector cells are intimately involved in the patient's response to cancer. People with decreased immune function develop cancer more frequently. In the early stages of solid organ malignancies, surgery can potentially be curative. Surgical intervention, in and of itself, is immunosuppressive. Surgical resections are traditionally performed through large incisions. Technologic advances have allowed minimally invasive surgery (MIS) to evolve to the point it is now being used for cancer treatment. Recent minimally invasive series have reported improved survival and recurrence rates, as compared with historical data. We hypothesized that outcome differences for cancer patients undergoing open surgery vs. MIS are due to differential inhibition of immune effector cell function, in response to the different surgical stimulus. This increased immunosuppression after open surgery could potentially inhibit immune effector cell tumor surveillance as well as inhibit scavenging of any residual or micrometastatic disease or of tumor cells shed at the time of the operation. The less immunosuppressive MIS may leave immune function above a threshold level where remaining tumor is cleared. This difference would lead to less recurrence and to survival advantages. A deeper understanding of the integral components of the immune response to surgery would open the door for immunomodulation strategies and be of great clinical utility in guiding neoadjuvant, surgical, or adjuvant therapeutic decisions.

Multiple transitions to non-B-DNA structures occur in the distal regulatory region of the rat prolactin gene.

The developmentally regulated rat prolactin (rPRL) gene presents a promising model system toward understanding the biological role of non-B-DNA structural elements. Two predominantly alternating purine-pyrimidine (APP) (dA-dC)n.(dG-dT)n repeats of 58 and 178 base-pairs flank the (A + T)-rich distal regulatory region. We have characterized several transitions to non-B-DNA structures within this region in negatively supercoiled plasmids by utilizing high resolution chemical probing. Each repeat undergoes a full-length conversion to a novel left-handed helical structure via the stepwise nucleation and propagation of discrete "segments". These segments are delimited by out-of-alternation bases that are susceptible to attack by potassium permanganate and thus appear to be significantly unstacked within the left-handed helices. Moreover, the spatial order of successive right- to left-handed DNA transitions within each repeat exhibits a clear polarity toward the distal regulatory region of the rPRL gene. An additional transition involving the long-range unpairing of (A + T)-rich sequences establishes a directional propagation toward the regulatory region. These data demonstrate a complex series of quasi-independent transitions to non-B-DNA structures that impinge upon a known regulatory control region.

Production of ISG-15, an interferon-inducible protein, in human corneal cells.

ISG-15, a 15-kDa protein encoded by an interferon (IFN)-stimulated gene (ISG), was produced in human corneal cell cultures prepared from donor corneas in response to each of three major types of IFN, IFN-alpha, IFN-beta, and IFN-gamma. IFN-alpha and IFN-beta induced more ISG-15 in the first 24 h of treatment than did IFN-gamma. ISG-15 was detectable within the first 3 h of treatment with either type I IFN, and production peaked at 24 h, whereas IFN-gamma did not induce detectable ISG-15 until 16 h and did not induce peak production until 48 h. Conjugates of ISG-15 to cellular proteins were detectable by Western blot beginning at 9 h after IFN-alpha or IFN-beta treatment. ISG-15 persisted in IFN-treated cells for as long as 96 h. Free, unconjugated, ISG-15 was secreted by keratocytes into cell culture medium. The extent and kinetics of production, the conjugation of ISG-15 to cell proteins, and the secretion of ISG-15 in human fibroblast derived from corneas were IFN type dependent and dose dependent.

An automated instrument for the performance of enzymatic DNA sequencing reactions.

An instrument has been developed for the automation of enzymatic DNA sequencing reactions. Up to 96 DNA templates contained in a microtiter plate can be processed for either radioactive or fluorescence-based sequence analysis in a three-hour period. The quality of the resultant data is comparable to that obtained manually. The system is simple, flexible and is readily adapted to the use of new polymerases or modified experimental protocols.

The GenDev Curriculum Development Workshop.

PIP: This article describes the second Curriculum Development Workshop held in May 1997 at the Asian Institute of Technology (AIT) in Bangkok, Thailand. The workshop aimed to review critically and restructure the Gender and Development Studies (GenDev) curriculum and to assess AIT's role in training gender experts for the region. Participants included 22 people from 16 countries in Asia, Europe, and the US who were teaching graduate students about gender issues and who were activists with nongovernmental organizations working on gender issues. It was determined that the following were required courses: Culture, Knowledge and Gender Relations; Gender, Technology, and Development; Principles of Gender Research and Methodology in Science and Technology; and Gender Analysis and Field Methods. Other suggested core courses included: Gender and Natural Resource Management; Enterprise Management, Technology, and Gender; Gender and Agrarian Reform; Urbanization: A Gender Perspective; Gender-Responsive Development Planning; and Gender and Economic Change: Past and Present Concerns. Participants distinguished between GenDev courses offered to anyone attending AIT and training courses designed to produce gender experts in the region. The aim of training courses for AIT graduate students was to sensitize potential managers, technologists, and others on gender issues and to create awareness of the importance of including gender perspectives within decision-making, policy formation, and implementation. Training courses to produce gender experts should be directed to those with a prior background in gender studies and include gender analysis in field methods. Participants agreed that there should be an independent and autonomous field of gender and development studies. Participants made six recommendations for such a field of study.^ieng

Immunoregulatory properties of ISG15, an interferon-induced cytokine.

ISG15 is a 15-kDa protein of unique primary amino acid sequence, which is transcriptionally regulated by interferon (IFN) alpha and IFN-beta. Because it is synthesized in many cell types and secreted from human monocytes and lymphocytes, we postulated that ISG15 might act to modulate immune cell function. ISG15 stimulated B-depleted lymphocyte proliferation in a dose-dependent manner with significant proliferation induced by amounts of ISG15 as low as 1 ng/ml (58 pM). Maximal stimulation of [3H]thymidine incorporation by B-depleted lymphocytes occurred at 6-7 days. Immunophenotyping of ISG15-treated B-depleted lymphocyte cultures indicated a 26-fold expansion of natural killer (NK) cells (CD56+). In cytotoxicity assays, ISG15 was a potent inducer of cytolytic activity directed against both K562 (100 lytic units per 10(6) cells) and Daudi (80 lytic units per 10(6) cells) tumor cell targets, indicating that ISG15 enhanced lymphokine-activated killer-like activity. ISG15-induced NK cell proliferation required coculturing of T and NK cells, suggesting that soluble factor(s) were required. Measurement of ISG15-treated cell culture supernatants for cytokines indicated production of IFN-gamma (> 700 units/ml). No interleukin 2 or interleukin 12 was detected. IFN-gamma itself failed to stimulate lymphocyte proliferation and lymphokine-activated killer cell activation. Further, induced expression of IFN-gamma mRNA was detected by reverse transcription-PCR in T lymphocytes after ISG15 treatment but not in NK cells. Enhancement of NK cell proliferation, augmentation of non-major histocompatibility complex-restricted cytotoxicity, and induction of IFN-gamma from T cells identify ISG15 as a member of the cytokine cascade and suggest that it may be responsible for amplifying and directing some of the immunomodulatory effects of IFN-alpha or IFN-beta.

High-speed separations of DNA sequencing reactions by capillary electrophoresis.

Fluorescently labeled DNA fragments generated in enzymatic sequencing reactions are rapidly separated by capillary gel electrophoresis and detected at attomole levels within the gel-filled capillary. The application of this technology to automated DNA sequence analysis may permit the development of a second generation automated sequencer capable of efficient and cost-effective sequence analysis on the genomic scale.

In vitro and in vivo secretion of human ISG15, an IFN-induced immunomodulatory cytokine.

ISG15, a 15-kDa protein of unique primary amino acid sequence, functions intracellularly as a ubiquitin homologue and a cytokine that induces production of IFN-gamma and augments NK/lymphokine-activated killer cell proliferation and function. ISG15 is secreted from monocytes and lymphocytes, and in this study we have characterized in vitro and in vivo production of ISG15 in response to IFN-alphabeta. Low levels of ISG15 were present constitutively in PBMCs; dose-dependent ISG15 synthesis was observed in response to IFN-alpha or IFN-beta, but not IFN-gamma. High m.w. conjugates, present in PBMC extracts constitutively, were enhanced after IFN-alpha or IFN-beta treatment. Metabolic labeling experiments demonstrated that IFN-beta-induced ISG15 was released from primary cultures of peripheral blood CD3+ (including both CD4+ and CD8+ subpopulations). Furthermore, ISG15 was released from viable cell lines of monocyte, T lymphocyte, B lymphocyte, and epithelial origins. Since ISG15 was secreted in response to IFN treatment in vitro, its levels in the serum of healthy human volunteers treated with IFN-beta(ser) were quantitated by asymmetric sandwich ELISA. Both single and multiple doses of IFN-beta(ser) increased serum ISG15 levels significantly (p < 0.01) over baseline. A maximum 7.3-fold enhancement of serum ISG15 was obtained after multiple injections of 8 million units of IFN-beta(ser). Significant change was observed at 24 and 48 h of multiple 0.02-million-unit injections, yielding 1.2- and 1.7-fold increases over basal levels, respectively. These studies suggest that ISG15 is a novel member of the cytokine cascade that is synthesized and released in response to IFN-beta both in vitro and in vivo.

Regulation of rat AMP deaminase 3 (isoform C) by development and skeletal muscle fibre type.

AMP deaminase (AMPD) is characterized by a multigene family in rodents and man. Highly conserved rat and human AMPD1 and AMPD2 genes produce protein products that exhibit cross-species immunoreactivities (AMPD1, rat isoform A and human isoform M; AMPD2, rat isoform B and human isoform L). A third gene, AMPD3, has been described in humans, but antisera raised against its purified protein product (isoform E) reportedly does not cross-react with a third activity purified from rat tissues (isoform C). This study was designed to address this latter issue by cloning, sequencing and expressing rat AMPD3 cDNA species. Similarly to the human AMPD3 gene, the rat AMPD3 gene produces multiple transcripts that differ at or near their 5' ends. The boundary at which these alternative sequences diverge is precisely conserved in both species. Across the region that is common to all rat and human AMPD3 cDNA species, nucleotide and predicted amino acid sequences are 89% and 93% identical respectively, although the rat open reading frame is lacking two separate in-frame codons in the 5' end. Extreme 5' regions between the two species are entirely divergent, and one alternative rat sequence is predicted to confer at least 36 additional N-terminal residues to its encoded AMPD3 polypeptide. A comparison of 3' untranslated regions indicates that the rat sequence is 250 bp longer and contains multiple consensus polyadenylation signals. Examination of relative rat AMPD3 gene expression shows (1) variable patterns of alternative mRNA abundance across adult tissues, (2) developmental regulation in skeletal muscle and liver, and (3) greater mRNA abundance in adult red (soleus) than in mixed (plantaris) and white (outer gastrocnemius) skeletal muscle. Finally, baculoviral expression of rat and human AMPD3 proteins produces enzymes that are chromatographically and kinetically similar. Moreover, both recombinant activities immunoreact with anti-C and anti-E serum. These combined results demonstrate that rat isoform C and human isoform E are homologous cross-species AMPD3 proteins.

High speed DNA sequencing by capillary electrophoresis.

A major challenge of the Human Genome Initiative is the development of a rapid, accurate, and efficient DNA sequencing technology. A major limitation of current technology is the relatively long time required to perform the gel electrophoretic separations of DNA fragments produced in the sequencing reactions. We demonstrate here that it is possible to increase the speed of sequence analysis by over an order of magnitude by performing the electrophoresis and detection in ultra thin capillary gels. An instrument which utilizes these high speed separations to simultaneously analyze many samples will constitute a second generation automated DNA sequencer suitable for large-scale sequence analysis.