Human leukocyte antigen-DR13 and DR15 are associated with short-term lung transplant outcomes.

BACKGROUND: Lung transplantation outcomes are among the least favorable, with most recipients eventually developing bronchiolitis obliterans syndrome (BOS) and subsequent graft failure. The presence of human leukocyte antigen (HLA)-DR has been implicated in the pathogenesis of BOS and may play a role in these poor outcomes. METHODS: Lung transplant donor and recipient data were retrospectively gathered from the United Network for Organ Sharing database from January 2006 to June 2013. Donor and recipient characteristics, proportion of recipients treated for first year rejection, and 5-y rates of survival and freedom from BOS were determined according to HLA-DR1, -DR7, -DR13, and -DR15 status in both donor and recipient. Each HLA-DR allele was stratified by donor-recipient pair positivity status. RESULTS: A total of 7402 lung transplant recipients met the inclusion and exclusion criteria. There were significant but small differences in donor and recipient characteristics for each HLA-DR group. The recipients in the D(-)R(+) pairing for HLA-DR13 and those in the D(+)R(-) pairing for HLA-DR15 had significantly higher rates of receiving treatment for rejection within the first year after transplant (P = 0.024 and P = 0.001, respectively). There were no differences in 5-y survival or freedom from BOS for any of the four HLA-DR alleles studied. CONCLUSIONS: There are higher rates of patients treated for rejection within the first year who are either negative for the HLA-DR15 allele but received a donor-positive lung or positive for the HLA-DR13 allele but received a donor-negative lung for that allele. However, these differences do not appear to affect long-term outcomes.

Exploring autoimmunity in the pathogenesis of abdominal aortic aneurysms.

The abdominal aortic aneurysm (AAA) is a disease process that carries significant morbidity and mortality in the absence of early identification and treatment. While current management includes surveillance and surgical treatment of low- and high-risk aneurysms, respectively, our narrow understanding of the pathophysiology of AAAs limits our ability to more effectively manage and perhaps even prevent the occurrence of this highly morbid disease. Over the past couple of decades, there has been considerable interest in exploring the role of autoimmunity as an etiological component of AAA. This review covers the current literature pertaining to this immunological process, focusing on research that highlights the local and systemic immune components found in both human patients and murine models. A better understanding of the autoimmune mechanisms in the pathogenesis of AAAs can pave the way to novel and improved treatment strategies in this patient population.

Recipient body mass index and age interact to impact survival after heart transplantation.

Although recipient body mass index (BMI) and age are known risk factors for mortality after heart transplantation, how they interact to influence survival is unknown. Our study utilized the UNOS registry from 1997 to 2012 to define the interaction between BMI and age and its impact on survival after heart transplantation. Recipients were stratified by BMI: underweight (<18.5), normal weight (18.5-24.99), overweight (25-29.99), and either moderate (30-34.99), severe (35-39.99), or very severe (≥40) obesity. Recipients were secondarily stratified based on age: 18-40 (younger recipients), 40-65 (reference group), and ≥65 (advanced age recipients). Among younger recipients, being underweight was associated with improved adjusted survival (HR 0.902; p = 0.010) while higher mortality was seen in younger overweight recipients (HR 1.260; p = 0.005). However, no differences in adjusted survival were appreciated in underweight and overweight advanced age recipients. Obesity (BMI ≥ 30) was associated with increased adjusted mortality in normal age recipients (HR 1.152; p = 0.021) and even more so with young (HR 1.576; p < 0.001) and advanced age recipients (HR 1.292; p = 0.001). These results demonstrate that BMI and age interact to impact survival as age modifies BMI-mortality curves, particularly with younger and advanced age recipients.

Impact of donor age on survival after heart transplantation: an analysis of the United Network for Organ Sharing (UNOS) registry.

BACKGROUND: Data are limited regarding the influence of donor age on outcomes after heart transplantation. We sought to determine if advanced donor age is associated with differences in survival after heart transplantation and how this compares to waitlist survival. METHODS: All adult heart transplants from 2000 to 2012 were identified using the United Network for Organ Sharing database. Donors were stratified into four age groups: 18-39 (reference group), 40-49, 50-54, and 55 and above. Propensity scoring was used to compare status IA waitlist patients who did not undergo transplantation with IA recipients who received hearts from advanced age donors. The primary outcome of interest was recipient survival and this was analyzed with multivariate Cox regression analysis and the Kaplan-Meier method. RESULTS: A total of 22,960 adult heart transplant recipients were identified. Recipients of hearts from all three older donor groups had significantly increased risk of mortality (HR, 1.187-1.426, all p < 0.001) compared to recipients from donors age 18 to 39. Additionally, propensity-matched status IA patients managed medically without transplantation had significantly worse adjusted survival than status IA recipients who received hearts from older donors age ≥55 (HR, 1.362, p < 0.001). CONCLUSIONS: Compared to donors aged 18-39, age 40 and above is associated with worse adjusted recipient survival in heart transplantation. This survival difference becomes more pronounced as age increases to above 55. However, the survival rate among status IA patients who receive hearts from advanced age donors (≥55) is significantly better compared to similar status IA patients who are managed without transplantation.

Transport pathways of macromolecules between the nucleus and the cytoplasm.

Transport between the nucleus and cytoplasm involves both stationary components and mobile factors acting in concert to move macromolecules through the nuclear pore complex. Multiple transport pathways requiring both unique and shared components have been identified. In the past 18 months, new findings have shed light on the nature of some of the mobile components of these pathways. New receptor-cargo pairs for both import and export pathways have been identified extending the breadth of known transport pathways. Surprising findings on the role of Ran and energy in transport have changed our way of thinking about the mechanism of movement through the nuclear pore.

The importance of importin.

The accumulation of karyophilic proteins in the nucleus requires cytoplasmic factors. Cell-free systems that reconstitute nuclear protein import have been used to identify several of these factors and to define the biochemical requirements for the import process. Recently, one factor has been purified and cloned from Xenopus and identified as a homologue of the 'suppressor of RNA polymerase l' (SRP1) gene originally described in yeast. This factor belongs to a closely related group of proteins that may share similar functions in nuclear protein transport.

Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors.

We have developed an in vitro system involving digitonin-permeabilized vertebrate cells to study biochemical events in the transport of macromolecules across the nuclear envelope. While treatment of cultured cells with digitonin permeabilizes the plasma membranes to macromolecules, the nuclear envelopes remain structurally intact and nuclei retain the ability to transport and accumulate proteins containing the SV40 large T antigen nuclear location sequence. Transport requires addition of exogenous cytosol to permeabilized cells, indicating the soluble cytoplasmic factor(s) required for nuclear import are released during digitonin treatment. In this reconstituted import system, a protein containing a nuclear location signal is rapidly accumulated in nuclei, where it reaches a 30-fold concentration compared to the surrounding medium within 30 min. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature dependent. Furthermore, accumulation of the transport substrate within nuclei is completely inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. Together, these results indicate that the permeabilized cell system reproduces authentic nuclear protein import. In a preliminary biochemical dissection of the system, we observe that the sulfhydryl alkylating reagent N-ethylmaleimide inactivates both cytosolic factor(s) and also component(s) in the insoluble permeabilized cell fraction required for nuclear protein import. Because this permeabilized cell model is simple, efficient, and works effectively with cells and cytosol fractions prepared from a variety of different vertebrate sources, it will prove powerful for investigating the biochemical pathway of nuclear transport.

Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope.

Nuclear protein import can be separated into two distinct steps: binding to the nuclear pore complex followed by translocation to the nuclear interior. A previously identified nuclear location sequence (NLS) receptor and a 97-kD protein purified from bovine erythrocytes reconstitute the binding step in a permeabilized cell assay. Binding to the envelope is specific for a functional SV-40 large T antigen NLS and is not ATP or temperature dependent. Modification of p97 with N-ethylmaleimide (NEM) decreases binding to the pore, but interestingly, NEM treatment of the NLS receptor does not. Nuclear envelope binding is inhibited by wheat germ agglutinin suggesting a possible mechanism for the inhibition of transport by the lectin.

Sequence and characterization of cytoplasmic nuclear protein import factor p97.

Nuclear location sequence-mediated binding of karyophilic proteins to the nuclear pore complexes is one of the earliest steps in nuclear protein import. We previously identified two cytosolic proteins that reconstitute this step in a permeabilized cell assay: the 54/56-kD NLS receptor and p97. A monoclonal antibody to p97 localizes the protein to the cytoplasm and the nuclear envelope. p97 is extracted from nuclear envelopes under the same conditions as the O-glycosylated nucleoporins indicating a tight association with the pore complex. The antibody inhibits import in a permeabilized cell assay but does not affect binding of karyophiles to the nuclear pore complex. Immunodepletion of p97 renders the cytosol inactive for import and identifies at least three other cytosolic proteins that interact with p97. cDNA cloning of p97 shows that it is a unique protein containing 23 cysteine residues. Recombinant p97 binds zinc and a bound metal ion is required for the nuclear envelope binding activity of the protein.

RanBP1 stabilizes the interaction of Ran with p97 nuclear protein import.

Three factors have been identified that reconstitute nuclear protein import in a permeabilized cell assay: the NLS receptor, p97, and Ran/TC4. Ran/TC4, in turn, interacts with a number of proteins that are involved in the regulation of GTP hydrolysis or are components of the nuclear pore. Two Ran-binding proteins, RanBP1 and RanBP2, form discrete complexes with p97 as demonstrated by immunoadsorption from HeLa cell extracts fractionated by gel filtration chromatography. A > 400-kD complex contains p97, Ran, and RanBP2. Another complex of 150-300 kD was comprised of p97, Ran, and RanBP1. This second trimeric complex could be reconstituted from recombinant proteins. In solution binding assays, Ran-GTP bound p97 with high affinity, but the binding of Ran-GDP to p97 was undetectable. The addition of RanBP1 with Ran-GDP or Ran-GTP increased the affinity of both forms of Ran for p97 to the same level. Binding of Ran-GTP to p97 dissociated p97 from immobilized NLS receptor while the Ran-GDP/RanBP1/p97 complex did not dissociate from the receptor. In a digitonin-permeabilized cell docking assay, RanBP1 stabilizes the receptor complex against temperature-dependent release from the pore. When added to an import assay with recombinant NLS receptor, p97 and Ran-GDP, RanBP1 significantly stimulates transport. These results suggest that RanBP1 promotes both the docking and translocation steps in nuclear protein import by stabilizing the interaction of Ran-GDP with p97.

Role of importin-beta in coupling Ran to downstream targets in microtubule assembly.

The guanosine triphosphatase Ran stimulates assembly of microtubule asters and spindles in mitotic Xenopus egg extracts. A carboxyl-terminal region of the nuclear-mitotic apparatus protein (NuMA), a nuclear protein required for organizing mitotic spindle poles, mimics Ran's ability to induce asters. This NuMA fragment also specifically interacted with the nuclear transport factor, importin-beta. We show that importin-beta is an inhibitor of microtubule aster assembly in Xenopus egg extracts and that Ran regulates the interaction between importin-beta and NuMA. Importin-beta therefore links NuMA to regulation by Ran. This suggests that similar mechanisms regulate nuclear import during interphase and spindle assembly during mitosis.

Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.

Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.

Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription.

Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 (38K) become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared. Immunofluorescence microscopy shows mostly cytoplasmic and some nuclear staining. These observations demonstrate that commonly used inhibitors of transcription affect the physical state of messenger ribonucleoproteins in vivo.

mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence.

We identified and produced antibodies to the major proteins that interact with poly(A)+ RNAs in the yeast Saccharomyces cerevisiae. The major proteins which were cross-linked by UV light to poly(A)+ RNA in intact yeast cells had apparent molecular weights of 72,000, 60,000, and 50,000. The poly(A) segment of the RNA was selectively cross-linked to the 72,000-molecular-weight protein (72K protein). Mice immunized with purified UV-cross-linked RNA-protein (RNP) complexes produced antibodies to the three major RNP proteins. A yeast genomic DNA library constructed in the lambda gt11 expression vector was screened with the anti-RNP serum, and recombinant bacteriophage clones were isolated. One recombinant phage, lambda YPA72.1, bearing a 2.5-kilobase insert, produced a large beta-galactosidase-RNP fusion protein. Affinity-selected antibodies from the anti-RNP serum on this fusion protein recognized a single 72K protein which was cross-linked to the poly(A) segment of RNA in the intact cell. Furthermore, the fusion protein of lambda YPA72.1 had specific poly(A)-binding activity. Therefore, lambda YPA72.1 encodes the 72K poly(A)-binding protein. Immunofluorescence microscopy showed that this protein was localized in the cytoplasm. Hybrid-selected mRNA translated in vitro produced the 72K poly(A)-binding protein, and mRNA blot analysis detected a single 2.1-kilobase mRNA. DNA blot analysis suggested a single gene for the poly(A)-binding protein. DNA sequence analysis of genomic clones spanning the entire gene revealed a long open reading frame encoding a 64,272-molecular-weight protein with several distinct domains and repeating structural elements. A sequence of 11 to 13 amino acids is repeated three times in this protein. Strikingly, this repeated sequence (RNP consensus sequence) is highly homologous to a sequence that is repeated twice in a major mammalian heterogeneous nuclear RNP protein, A1. The conservation of the repetitive RNP consensus sequence suggests an important function and a common evolutionary origin for messenger RNP and heterogeneous nuclear RNP proteins.

Functional domains in nuclear import factor p97 for binding the nuclear localization sequence receptor and the nuclear pore.

The interaction of the nuclear protein import factor p97 with the nuclear localization sequence (NLS) receptor, the nuclear pore complex, and Ran/TC4 is important for coordinating the events of protein import to the nucleus. We have mapped the binding domains on p97 for the NLS receptor and the nuclear pore. The NLS receptor-binding domain of p97 maps to the C-terminal 60% of the protein between residues 356 and 876. The pore complex-binding domain of p97 maps to residues 152-352. The pore complex-binding domain overlaps the Ran-GTP- and Ran-GDP-binding domains on p97, but only Ran-GTP competes for docking in permeabilized cells. The N-ethylmaleimide sensitivity of the p97 for docking was investigated and found to be due to inhibition of p97 binding to the pore complex and to the NLS receptor. Site-directed mutagenesis of conserved cysteine residues in the pore- and receptor-binding domains identified two cysteines, C223 and C228, that were required for p97 to bind the nuclear pore. Inhibition studies on docking and accumulation of a NLS protein provided additional evidence that the domains identified biochemically are the functional domains involved in protein import. Together, these results suggest that Ran-GTP dissociates the receptor complex and prevents p97 binding to the pore by inducing a conformational change in the structure of p97 rather than simple competition for binding sites.

Institutional Cost of Unplanned 30-Day Readmission Following Open and Endovascular Surgery.

BACKGROUND: Vascular surgical patients have a high rate of readmission, and the cost of readmission for these patients has not been described. Herein, we characterize and compare institutional index hospitalization and 30-day readmission cost following open and endovascular vascular procedures. METHODS: The American College of Surgeons National Surgical Quality Improvement Program database was used to identify inpatient open and endovascular procedures at a single institution, from January 2011 through June 2012. Variable and fixed costs for index hospitalization and unplanned 30-day readmissions were obtained using SAP BusinessObjects. Patient characteristics and outcome variables were analyzed using Student t tests or Wilcoxon rank-sum nonparametric tests for continuous variables and Fisher exact tests for categorical variables. RESULTS: One thousand twenty-six inpatient procedures were included in the analysis. There were 605 (59%) open and 421 (41%) endovascular procedures with a 30-day unplanned readmission rate of 16.9% and 17.8%, respectively (P = .679). The mean index hospitalization costs for open and endovascular procedures were US$27 653 and US$23 999, respectively (P = .146). The mean costs for 30-day unplanned readmission for open and endovascular procedures were US$19 117 and US$17 887, respectively (P = .635). Among open procedures, the mean cost for patients not readmitted was US$28 321 compared to US$31 115 for those readmitted (P = .003). Among endovascular procedures, the mean cost for patients not readmitted was US$26 908 compared to US$32 262 for those readmitted (P = .028). CONCLUSION: The cost of index hospitalization and 30-day unplanned readmission are similar for open and endovascular procedures. Readmitted patients had a higher mean index hospitalization cost irrespective of open or endovascular procedure.

A stimulatory role of cyclic adenosine 3',5'-monophosphate in follicle-stimulating hormone-activated mitogen-activated protein kinase signaling pathway in rat ovarian granulosa cells.

FSH promotes the differentiation of ovarian follicular granulosa cells via a cAMP-dependent mechanism. Based upon the presence of a prominent phospholipid/diolein/Ca2+-independent myelin basic protein kinase activity in soluble extracts of proliferating immature rat granulosa cells, we determine whether this activity was attributable to the mitogen-activated protein kinases (MAPKs), one of the ubiquitous families of myelin basic protein kinases, and whether FSH acutely regulated the MAPKs in rat granulosa cells. Granulosa cells were obtained from large preantral follicles in ovaries of immature rats treated with 17beta-estradiol to promote granulosa cell proliferation. Exposure of granulosa cells, cultured overnight in serum-free medium containing 10 nM 17beta- estradiol, to 50 ng/ml FSH for 10 min promoted a 2- to 5- fold increase in MAPK activity. The effects of FSH were mimicked by forskolin and inhibited by the inhibitor of cAMP- dependent protein kinase H89, but were not inhibited by the tyrosine kinase inhibitor Ag-18. FSH also promoted increased phosphorylation of the 90-kDa ribosomal S6 protein kinase and phosphorylation of exogenous S6 protein. These results suggest that the cAMP-directed pathway by which FSH initiates granulosa cell differentiation includes activation of MAPKs.

Trends in induced abortion in England and Wales.

Analysis of routinely published abortion and fertility data for England and Wales between 1968 and 1980 shows that the age-specific abortion rates increased from 1968 until about 1973 when the rates peaked for all ages; rates then declined until 1977 but have subsequently returned to higher levels. Two factors are implicated: (1) the recent changes are related to parallel changes in the fertility rate; but (2) there is also a tendency for recent cohorts of women to resort to abortion more readily. These relationships are derived from analyses of fertility rates and abortion ratios, the proportion of conceptions that result in abortions. The results are discussed in terms of attitudes and practices related to birth control.

Lung transplantation: chronic allograft dysfunction and establishing immune tolerance.

Despite significant medical advances since the advent of lung transplantation, improvements in long-term survival have been largely unrealized. Chronic lung allograft dysfunction, in particular obliterative bronchiolitis, is the primary limiting factor. The predominant etiology of obliterative bronchiolitis involves the recipient's innate and adaptive immune response to the transplanted allograft. Current therapeutic strategies have failed to provide a definitive treatment paradigm to improve long-term outcomes. Inducing immune tolerance is an emerging therapeutic strategy that abrogates allograft rejection, avoids immunosuppression, and improves long-term graft function. The aim of this review is to discuss the key immunologic components of obliterative bronchiolitis, describe the state of establishing immune tolerance in transplantation, and highlight those strategies being evaluated in lung transplantation.

The HMGB1-RAGE axis mediates traumatic brain injury-induced pulmonary dysfunction in lung transplantation.

Traumatic brain injury (TBI) results in systemic inflammatory responses that affect the lung. This is especially critical in the setting of lung transplantation, where more than half of donor allografts are obtained postmortem from individuals with TBI. The mechanism by which TBI causes pulmonary dysfunction remains unclear but may involve the interaction of high-mobility group box-1 (HMGB1) protein with the receptor for advanced glycation end products (RAGE). To investigate the role of HMGB1 and RAGE in TBI-induced lung dysfunction, RAGE-sufficient (wild-type) or RAGE-deficient (RAGE(-/-)) C57BL/6 mice were subjected to TBI through controlled cortical impact and studied for cardiopulmonary injury. Compared to control animals, TBI induced systemic hypoxia, acute lung injury, pulmonary neutrophilia, and decreased compliance (a measure of the lungs' ability to expand), all of which were attenuated in RAGE(-/-) mice. Neutralizing systemic HMGB1 induced by TBI reversed hypoxia and improved lung compliance. Compared to wild-type donors, lungs from RAGE(-/-) TBI donors did not develop acute lung injury after transplantation. In a study of clinical transplantation, elevated systemic HMGB1 in donors correlated with impaired systemic oxygenation of the donor lung before transplantation and predicted impaired oxygenation after transplantation. These data suggest that the HMGB1-RAGE axis plays a role in the mechanism by which TBI induces lung dysfunction and that targeting this pathway before transplant may improve recipient outcomes after lung transplantation.