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Background. The Decisional Conflict Scale (DCS) measures 5 dimensions of decision making (feeling: uncertain, uninformed, unclear about values, unsupported; ineffective decision making). We examined the use of the DCS over its initial 20 years (1995 to 2015). Methods. We conducted a scoping review with backward citation search in Google Analytics/Web of Science/PubMed, followed by keyword searches in Cochrane Library, PubMed, Ovid MEDLINE, EMBASE, CINAHL, AMED, PsycINFO, PRO-Quest, and Web of Science. Eligible studies were published between 1995 and March 2015, used an original experimental/observational research design, concerned a health-related decision, and provided DCS data (total/subscales). Author dyads independently screened titles, abstracts, full texts, and extracted data. We performed narrative data synthesis. Results. We included 394 articles. DCS use appeared to increase over time. Three hundred nine studies (76%) used the original DCS, and 29 (7%) used subscales only. Most studies used the DCS to evaluate the impact of decision support interventions ( n = 238, 59%). The DCS was translated into 13 languages. Most decisions were made by people for themselves ( n = 353, 87%), about treatment ( n = 225, 55%), or testing ( n = 91, 23%). The most common decision contexts were oncology ( n = 113, 28%) and primary care ( n = 82, 20%). Conclusions. This is the first study to descriptively synthesize characteristics of DCS data. Use of the DCS as an outcome measure for health decision interventions has increased over its 20-year existence, demonstrating its relevance as a decision-making evaluation measure. Most studies failed to report when decisional conflict was measured during the decision-making process, making scores difficult to interpret. Findings from this study will be used to update the DCS user manual.

Background. We explored decisional conflict as measured with the 16-item Decisional Conflict Scale (DCS) and how it varies across clinical situations, decision types, and exposure to decision support interventions (DESIs). Methods. An exhaustive scoping review was conducted using backward citation searches and keyword searches. Eligible studies were published between 1995 and March 2015, used an original experimental/observational research design, concerned a health-related decision, and provided DCS data. Dyads independently screened titles/abstracts and full texts, and extracted data. We performed narrative syntheses and calculated average or median DCS scores. Results. We included 246 articles reporting on 253 studies. DCS scores ranged from 2.4 to 79.7 out of 100. Highest baseline DCS scores were for care planning (44.8 ± 8.9, median = 47.0) and treatment decisions (32.5 ± 12.6, median = 31.9), in contexts of primary care (40.6 ± 18.3), and geriatrics (39.8 ± 11.2). Baseline scores were high among decision makers who were ill (33.2 ± 14.1, median = 30.2) or making decisions for themselves (33.4 ± 13.8, median = 32.0). Total DCS scores 37.4). After DESI use, DCS scores decreased short-term but increased or remained the same long-term (>6 months). Conclusions. DCS scores were highest at baseline and decreased after decision making. DESIs decreased decisional conflict immediately after decision making. The largest improvements after DESIs were in decision makers who were ill or made decisions for themselves. Further meta-analyses are needed for decision type, contexts, and interventions to inform hypotheses about the expected effects of DESIs, the best timing for measurement, and interpretation of DCS scores.

Minimal myelination is proposed to be a contributing factor to the preferential nigral neuronal loss in Parkinson’s disease (PD). Similar to nigral dopaminergic neurons, sympathetic neurons innervating the heart have long, thin axons which are unmyelinated or minimally myelinated. Interestingly, cardiac sympathetic loss in PD is heterogeneous across the heart, yet the spatial relationship between myelination and neurodegeneration is unknown. Here, we report the mapping of myelin basic protein (MBP) expression across the left ventricle of normal rhesus macaques (n = 5) and animals intoxicated with systemic 6-OHDA (50 mg/kg iv) to model parkinsonian cardiac neurodegeneration (n = 10). A subset of 6-OHDA-treated rhesus received daily dosing of pioglitazone (5 mg/kg po; n = 5), a PPARγ agonist with neuroprotective properties. In normal animals, MBP-immunoreactivity (-ir) was identified surrounding approximately 14% of axonal fibers within nerve bundles of the left ventricle, with more myelinated nerve fibers at the base level of the left ventricle than the apex p < 0.014 . Greater MBP-ir at the base was related to a greater number of nerve bundles at that level relative to the apex p < 0.05 , as the percent of myelinated nerve fibers in bundles was not significantly different between levels of the heart. Cardiac sympathetic loss following 6-OHDA was associated with decreased MBP-ir in cardiac nerve bundles, with the percent decrease of MBP-ir greater in the apex (84.5%) than the base (52.0%). Interestingly, cardiac regions and levels with more MBP-ir in normal animals showed attenuated sympathetic loss relative to areas with less MBP-ir in 6-OHDA + placebo (r = −0.7, p < 0.014 ), but not in 6-OHDA + pioglitazone (r = −0.1) subjects. Our results demonstrate that myelination is present around a minority of left ventricle nerve bundle fibers, is heterogeneously distributed in the heart of rhesus macaques, and has a complex relationship with cardiac sympathetic neurodegeneration and neuroprotection.

Supplemental material, Appendix_1_included_papers_paper_2.rjf_online_supp for Decisional Conflict Scale Findings among Patients and Surrogates Making Health Decisions: Part II of an Anniversary Review by Mirjam M. Garvelink, Laura Boland, Krystal Klein, Don Vu Nguyen, Matthew Menear, Hilary L. Bekker, Karen B. Eden, Annie LeBlanc, Annette M. O’Connor, Dawn Stacey and France Légaré in Medical Decision Making

Supplemental material, Appendix_1_paper_1.rjf_online_supp for Decisional Conflict Scale Use over 20 Years: The Anniversary Review by Mirjam M. Garvelink, Laura Boland, Krystal Klein, Don Vu Nguyen, Matthew Menear, Hilary L. Bekker, Karen B. Eden, Annie LeBlanc, Annette M. O’Connor, Dawn Stacey and France Légaré in Medical Decision Making

BACKGROUND BK virus nephropathy is one of the most common viral infections that affect up to 10% of renal transplant recipients (RTRs), causing allograft dysfunction and graft loss. Interferon-gamma (IFN-γ) gene polymorphisms have been associated with parvovirus B19, hepatitis C virus, HIV-1/AIDS infection, cytomegalovirus viremia, and disease. IFN-γ is known to have potent inhibitory effects on BK virus gene expression, both at the level of transcription and translation. METHODS It was investigated whether IFN-γ polymorphisms are associated with BKV infection. Genotyping of four single-nucleotide polymorphisms located in the IFN-γ gene were performed on DNA collected from a total of 251 RTRs (71 RTRs with BKV infection and 180 without BKV infection). RESULTS Analysis of the results showed that IFN-γ (rs12369470) CC genotype was significantly associated with susceptibility to BKV infection (OR: 2.9, 95% CI: 1.29-6.44, P=0.007) while the IFN-γ +874 (rs2435061) TT and (rs2406918) CC genotypes appear to be markers for protection against BKV infection (OR: 0.29, 95% CI: 0.1-0.83, P=0.01 for rs245061; OR: 0.61, 95% CI: 0.4-0.94, P=0.02 for rs24069718). A haplotype analysis using the combination of rs2435061-rs2406918-rs2870953 showed that the A-G-T haplotype was associated with a significantly reduced risk for BKV infection (OR: 0.43, 95% CI: 0.25-0.73, P=0.001). CONCLUSION Polymorphisms in the IFN-γ gene may confer certain protection or predisposition for BKV infection.

Cyclooxygenase-2 (COX-2) alleles have been associated with allograft outcomes in kidney transplant recipients; however, these alleles may be in linkage with other genes. Human allograft inflammatory factor-1 (AIF-1) is a cytoplasmic protein and is produced by macrophages. Its synthesis is regulated by several cytokines, including interferon gamma. We investigated whether polymorphisms of gene encoding COX-2 and AIF-1 were associated with allograft outcomes among Hispanic renal transplant recipients (RTRs). A total of 527 de novo RTRs of Hispanic ethnicity were included in this study transplanted at St. Vincent Medical Center (SVMC) during 2000–2009. Patients were genotyped for the following: COX-2 (−1195C > T rs689466, intron 6 rs2066826) and AlF1 (rs2269475). Analysis of the results showed that COX-2–1195 CC genotype (OR = 1.92, CI% = 1.00–3.67, p = 0.04) were more frequent, but COX-2–1195 CT genotype was less frequent in kidney allograft acute rejection in comparison with control group (OR = 0.59, CI% = 0.38–0.91, p = 0.017). The genetic variant TT/CT of the AIF-1 gene was associated with a lower risk of rejection (OR = 0.63, CI% = 0.41–0.98, p = 0.038). No association of COX-2 (rs2066826) was observed with allograft rejection. We are unable to find statistically significant association between COX-2 and AIF-1 gene polymorphisms and allograft survival. The −1195C > T in the COX-2 promoter and AIF-1 gene polymorphisms could be a potential predictor of allograft rejection in our Hispanic kidney transplant recipients.

Background The dimeric NF-κB transcription factors play critical roles in diverse cellular processes including adaptive and innate immunity, cell differentiation, proliferation and apoptosis. It regulates the expression of numerous genes that play a key role in the inflammatory response during kidney allograft rejection. This study aims to determine the association of NF-κB gene polymorphisms with allograft outcomes in the Hispanic renal transplant recipients. Methods A total of 607 Hispanic renal transplant recipients at St. Vincent Medical Center between 2001 and 2010 were included in this study. The NF-κB genotypes were studied along with clinical data. In the case of NF-κB genotypes, the following single nucleotide polymorphisms (SNPs) were included: NF-κB1 (rs3774959, rs3774932, rs3774937, rs230526, rs230519), NF-κB2 (rs1056890, rs7897947, rs12769316) and NF-κB inducing kinase (NIK) (rs9908330, rs7222094). The association of each genotype with renal allograft survival and acute rejection was evaluated. Results NF-κB1 (rs3774937) CC genotype showed protective association with allograft rejection (OR = 0.66, 95% CI = 0.44–0.99, p = 0.04). There was a significant increase in allograft survival time associated with the NF-κB1 (rs3774959) A allele (OR = 0.76, 95% CI = 0.60–0.98, p = 0.03) while GG genotype was associated with a higher risk of graft failure (OR = 1.51, 95% CI = 1.02–2.21, p = 0.03). There were no associations between polymorphic markers in NF-κB2 and NIK genes with allograft survival or acute rejection. Among non-genetic factors, we found that the use of tacrolimus, a deceased donor, delayed graft function and acute rejection were associated with allograft failure. Conclusion The result of present study suggests that NF-κB1 gene polymorphisms may determine the incidence of acute rejection or graft survival among Hispanic allograft recipients.

The X-ray structure of the nuclear factor-kappaB (NF-kappaB) p52:RelB:kappaB DNA complex reveals a new recognition feature not previously seen in other NF-kappaB:kappaB DNA complexes. Arg 125 of RelB is in contact with an additional DNA base pair. Surprisingly, the p52:RelB R125A mutant heterodimer shows defects both in DNA binding and in transcriptional activity only to a subclass of kappaB sites. We found that the Arg 125-sensitive kappaB sites contain more contiguous and centrally located A:T base pairs than do the insensitive sites. A protein-induced kink observed in this complex, which used an AT-rich kappaB site, might allow the DNA contact by Arg 125; such a kink might not be possible in complexes with non-AT-rich kappaB sites. Furthermore, we show that the p52:RelB heterodimer binds to a broader spectrum of kappaB sites when compared with the p50:RelA heterodimer. We suggest that the p52:RelB heterodimer is more adaptable to complement sequence and structural variations in kappaB sites when compared with other NF-kappaB dimers.

IkappaB Kinase (IKK)alpha is required for activation of an alternative NF-kappaB signaling pathway based on processing of the NF-kappaB2/p100 precursor protein, which associates with RelB in the cytoplasm. This pathway, which activates RelB:p52 dimers, is required for induction of several chemokine genes needed for organization of secondary lymphoid organs. We investigated the basis for the IKKalpha dependence of the induction of these genes in response to engagement of the lymphotoxin beta receptor (LTbetaR). Using chromatin immunoprecipitation, we found that the promoters of organogenic chemokine genes are recognized by RelB:p52 dimers and not by RelA:p50 dimers, the ubiquitous target for the classical NF-kappaB signaling pathway. We identified in the IKKalpha-dependent promoters a novel type of NF-kappaB-binding site that is preferentially recognized by RelB:p52 dimers. This site links induction of organogenic chemokines and other important regulatory molecules to activation of the alternative pathway.

IκBβ, one of the major IκB proteins, is only partially degraded in response to most extracellular signals. However, the molecular mechanism of this event is unknown. We show here that IκBβ exists in at least two different forms: one that is bound to the NF-κB dimer and the other bound to both NF-κB and κB-Ras, a Ras-like small G protein. Removal of cellular κB-Ras enhances whereas excess κB-Ras blocks induced IκBβ degradation. Remarkably, κB-Ras functions in both GDP- and GTP-bound states, and mutations of the conserved guanine-binding residues of κB-Ras abrogate its ability to block degradation of IκBβ. κB-Ras also directly blocks the in vitro phosphorylation of IκBβ by IKKβ. These observations suggest that IκBβ in the ternary complex is resistant to degradation by most signals. We suggest that specific signals, in addition to those that activate only IKK, are essential for the complete degradation of IκBβ.

We have recently identified an RNA aptamer for the transcription factor NF-κB p50 homodimer [(p50) 2 ], which exhibits little sequence resemblance to the consensus DNA target for (p50) 2 , but binds (p50) 2 with an affinity similar to that of the optimal DNA target. We describe here the 2.45-Å resolution x-ray crystal structure of the p50 RHR/RNA aptamer complex. The structure reveals that two RNA molecules bind independent of each other to the p50 N-terminal Ig-like domains. The RNA secondary structure is comprised of a stem and a stem–loop separated by an internal loop folded into a kinked helix because of the cross-strand stacking of three internal loop guanines. These guanines, placed at the edge of the 3′ helix, together with the major groove of the irregular 3′ helix, form the binding surface for p50. Each p50 monomer uses the same surface to recognize the distorted RNA major groove as observed in the κB DNA/p50 RHR complex, resulting in strikingly similar interfaces. The structure reveals how the aptamer specifically selects p50 and discriminates against p65. We also discuss the physiological implications of RNA binding by (p50) 2 .

The SRPK family is distinguished from typical eukaryotic protein kinases by several unique structural features recently elucidated by X-ray diffraction methods [Nolen et al. (2001) Nat. Struct. Biol. 8, 176-183]. To determine whether these features impart unique catalytic function, the phosphorylation of the physiological Sky1p substrate, Npl3p, was monitored using steady-state and pre-steady-state kinetic techniques. While Sky1p has a low apparent affinity for ATP compared to other protein kinases, it binds Npl3p with very high affinity. The latter is achieved through a combination of local and distal factors in the protein substrate. The phosphoryl donor ATP has access to the nucleotide pocket in the absence or presence of Npl3p, indicating that a large protein substrate does not enforce an ordered addition of ligands. Sky1p binds two Mg(2+)-the first is essential whereas the second further enhances catalysis. While the turnover number is low (0.5 s(-1)), Npl3p is rapidly phosphorylated in the active site (40 s(-1)) based on single turnover experiments. These results indicate that Sky1p employs a catalytic pathway involving fast phosphoryl transfer followed by slow net release of products. These studies represent the first kinetic investigation of a member of the SRPK family and the first pre-steady-state kinetic study of a protein kinase using a natural protein substrate.

The NFκB family of dimeric transcription factors regulate inflammatory and immune responses. While the dynamic control of NFκB dimer activity via the IκB-NFκB signaling module is well understood, there is little information on how specific dimer repertoires are generated from Rel family polypeptides. Here we report the iterative construction – guided by in vitro and in vivo experimentation – of a mathematical model of the Rel-NFκB generation module. Our study reveals that IκBβ has essential functions within the Rel-NFκB generation module, specifically for the RelA:RelA homodimer, which controls a subset of NFκB target genes. Our findings revise the current dogma of the three classical, functionally-related IκB proteins by distinguishing between a positive ‘licensing’ factor (IκBβ) that contributes to determining the available NFκB dimer repertoire in a cell’s steady state, and negative feedback regulators (IκBα and -ε) that determine the duration and dynamics of the cellular response to an inflammatory stimulus.

BK virus-associated nephropathy (BKVN) can cause clinically significant viral infection in renal transplant recipients, leading to allograft dysfunction and loss. The usual management of BKVN involves the reduction of immunosuppression and the addition of leflunomide, quinolones, and cidofovir, but the rate of graft loss remains high. The aim of this study was to assess the impact of treatment with intravenous human immunoglobulin (IVIG) on the outcome of BKVN in renal transplant recipients. Upon diagnosis of BKVN, patients remained on anti-polyomavirus treatment, consisting of the reduction of immunosuppression and the use of leflunomide therapy. Treatment with IVIG was given only to patients who did not respond to 8 weeks of the adjustment of immunosuppression and leflunomide. All 30 patients had persistent BKV viremia and BKVN with their mean BK viral loads higher than the baseline (range, 15,000-2 million copies/mL). Mean peak BK load was 205,314 copies/mL compared with 697 copies/mL after 1 year of follow-up. Twenty-seven patients (90%) had a positive response in clearing viremia. The actuarial patient and graft survival rates after 12 months were 100% and 96.7%, respectively. IVIG administration appeared to be safe and effective in treating BKV viremia and BKVN and preventing graft loss in patients who had inadequate response to immunosuppression reduction and leflunomide therapy.

Mycophenolic acid (MPA), a widely used immunosuppressant, has a complex metabolism that involves a number of enzymes. Some of its metabolites are thought to be the cause of gastrointestinal (GI) side effects. In this study, we investigated whether polymorphisms of UDP-glucuronosyltransferases (UGT1) A8, 1A9, and hepatocyte nuclear factor (HNF1α) genes or pharmacokinetic parameters of mycophenolic acid (MPA) were associated with the severity of GI symptoms in patients receiving MPA therapy. A total of 109 kidney transplant patients taking mycophenolic acid (MPA) derivatives were genotyped for UGT1A8, 1A9 and HNF1α genes. Among these, a total of 15 patients were participants in the pharmacokinetic study. Severity of GI symptoms was assessed using a validated Gastrointestinal Symptom Rating Scale (GSRS). The overall and subscale GSRS scores were measured at 1 week (baseline), 2 weeks, 3 months and 6 months post-transplantation. In the case of the pharmacokinetic study, EC-MPS was administered and a total of nine blood samples were obtained at − 1, 0, 0.5, 1, 2, 4, 6, 8, and 12 h. Genotypes of UGT1A8 were significantly associated with the overall GSRS scores at week 1 (p = 0.02) and week 2 (p = 0.036). Subscales were only statistically significant for constipation at week 1 (p = 0.002) and indigestion at week 2 (p = 0.02), while UGT1A9 was only significant for the constipation at week 1 (p = 0.04). HNF1α genotypes were significantly different at week 1 in the overall GSRS (p = 0.004), and for abdominal pain (p = 0.04), acid reflux (p = 0.036) and constipation subscales (p = 0.04). In addition, abdominal pain was statistically significantly different at 3 months and 6 months after transplantation (p = 0.03 and 0.02, respectively). In the case of the pharmacokinetic study, we have found some correlations between MPAC0 and constipation (p = 0.02) where MPAAUC was correlated with acid reflux (p = 0.02) and constipation (p = 0.012), MPAGCL/F was correlated to acid reflux, indigestion, constipation and the sum of the GSRS scores (p = 0.037, p = 0.032, p = 0.033 and p = 0.04, respectively). Multinomial regression analysis for MPAGCL/F showed a statistical significance for the subscale indigestion and the sum of the GSRS (p = 0.033 and p = 0.037, respectively). Our data suggests that among patients receiving MPA the UGT1A9 alleles might play a role in determining the severity of early GI side effects, while the HNF1α allele appears to be associated with a later effect as well as early side effects. Our data also showed that some kinetic parameters might predict MPA side effects.

Granzyme B (GZMB) and perforin 1 gene (PRF1) are key effector molecules of cytotoxic T lymphocytes, in causing acute and chronic solid organ transplant rejection. In this study, we analyzed the impact of GZMB and PRF1 polymorphism on kidney allograft outcomes. In all, 527 de novo kidney Hispanic allograft recipients were genotyped for PRF1 (rs10999426, rs35947132) and GZMB (rs8192917, rs7144366). PRF1 (rs10999426, rs35947132) G alleles and GG genotypes were negatively associated with allograft rejection, demonstrating protection against allograft rejection (OR = 0.61, p = 0.005 for rs1099946; OR = 0.4, p = 0.01 for rs 35947132). On the other hand, the GA heterozygosity of PRF1 was found marginally associated with the rejection group (OR = 1.53, p = 0.05 for rs10999426; OR = 2.24, p = 0.07 for rs35947132). There was a significant increase in allograft survival in time period studied for the PRF1 (rs10999426) GG genotype, while the GA heterozygosity was associated with graft failure. We found no association for polymorphic markers in GZMB gene with allograft rejection. Survival was significantly improved for patients who were homozygous TT for the GZMB (rs8192917) (TT vs. CC/TT, p = 0.041). The result suggests that PRF1 and GZMB gene polymorphisms may determine the incidence of acute rejection or graft survival among Hispanic allograft recipients.

Transcription factors of the nuclear factor kappaB (NF-κB) family arise through the combinatorial association of five distinct Rel subunits into functional dimers. However, not every dimer combination is observed in cells. The RelB subunit, for example, does not appear as a homodimer and forms heterodimers exclusively in combination with p50 or p52 subunits. We previously reported that the RelB homodimer could be forced to assemble through domain swapping in vitro. In order to understand the mechanism of selective dimerization among Rel subunits, we have determined the x-ray crystal structures of five RelB dimers. We find that RelB forms canonical side-by-side heterodimers with p50 and p52. We observe that, although mutation of four surface hydrophobic residues that are unique to RelB does not affect its propensity to form homodimers via domain swapping, alteration of two interfacial residues converts RelB to a side-by-side homodimer. Surprisingly, these mutant RelB homodimers remain distinct from canonical side-by-side NF-κB dimers in that the two monomers move away from one another along the 2-fold axis to avoid non-complementary interactions at the interface. The presence of distinct residues buried within the hydrophobic core of the RelB dimerization domain appears to influence the conformations of the surface residues that mediate the dimer interface. This conclusion is consistent with prior observations that alterations of domain core residues change dimerization propensity in the NF-κB family transcription factors. We suggest that RelB has evolved into a specialized NF-κB subunit with unique amino acids optimized for selective formation of heterodimers with p50 and p52.

Polymorphism of genes encoding components of the vitamin D pathway including vitamin D receptor (VDR) and vitamin D binding protein (VDBP), have been widely explored due to the complex role played by vitamin D in renal transplant outcomes. In this study, we investigated whether polymorphisms of genes encoding VDR and VDBP were associated with allograft survival or acute rejection (AR) among a Hispanic kidney transplant population. A total of 502 Hispanic renal allograft recipients at the St. Vincent Medical Center between 2001 and 2010 were genotyped for four different single nucleotide polymorphisms of VDR: FokI C>T (rs2228570), BsmI G>A (rs1544410), ApaI T>G (rs7975232), and TaqI T>C (rs731236). We also performed genotyping for one common polymorphism in the VDBP gene (rs4588). Survival was significantly improved for patients who were homozygous GG for the rs4588 G>T allele in the VDBP gene (GG vs. GT + TT, OR = 0.63, p = 0.02) while GT genotype was associated with a higher risk of graft loss (GT vs. GG + TT, OR = 1.67, p = 0.01). We found no association for polymorphic markers in VDR with allograft survival and AR. The frequency of the haplotype GTCG (in the order of VDR FokI C>T, BsmI G>A, ApaI T>G, and TaqI T>C), was significantly different in the patients with graft rejection compared to the control (p = 0.007) while ACCA haplotype was found to be associated with graft loss (p = 0.02). Hence, the VDBP G>T polymorphism (rs4588) and two haplotypes (GTCG and ACCA) of VDR appear to be associated with renal allograft outcomes among Hispanic allograft recipients.

Background 10–30% of dialysis population awaiting renal transplantation is sensitized. Mycophenolic acid (MPA) has been shown to reduce panel reactive antibody (PRA) formation in kidney transplant recipients. Our aim was to investigate whether MPA could effectively reduce anti-HLA antibody levels and allow successful transplantation. Methods A total of 40 highly sensitized patients were treated orally with MPA. All patients had T-cell PRA values greater than 30% (73% of patients were ≥ 75%). The PRAs, T-cell/B-cell flow cytometry crossmatch (FCXM) mean channel shift (MCS), patient/graft survival, acute rejection, and serum creatinine (SCr) were recorded. Results All 40 patients showed a decrease in PRA levels. Eighteen of the 40 patients (40%) received a transplant. All four living donor recipients converted to a negative crossmatch. There was a significant decrease in FCXM MCS in all 18 transplanted patients. The mean SCr at 24 months was 1.00 ± 0.25 mg/dL. Five patients (28%) experienced acute rejection. The overall one year actuarial patient and graft survival were 94% and 88%, respectively. Conclusions These findings suggest that MPA therapy is effective in reducing PRAs and increases the likelihood of successful transplantation in sensitized recipients in a potentially simpler and more cost effective manner than the current regimens employed.

We describe here the X-ray crystal structure of NF-kappaB p50/RelB heterodimer bound to a kappaB DNA. Although the global modes of subunit association and kappaB DNA recognition are similar to other NF-kappaB/DNA complexes, this complex reveals distinctive features not observed for non-RelB complexes. For example, Lys274 of RelB is removed from the protein-DNA interface whereas the corresponding residues in all other subunits make base-specific contacts. This mode of binding suggests that RelB may allow the recognition of more diverse kappaB sequences. Complementary surfaces on RelB and p50, as revealed by the crystal contacts, are highly suggestive of assembly of multiple p50/RelB heterodimers on tandem kappaB sites in solution. Consistent with this model our in vitro binding experiments reveal optimal assembly of two wild-type p50/RelB heterodimers on tandem HIV kappaB DNA with 2 bp spacing but not by a mutant heterodimer where one of the RelB packing surface is altered. We suggest that multiple NF-kappaB dimers assemble at diverse kappaB promoters through direct interactions utilizing unique protein-protein interaction surfaces.

The NF-kappaB p50 is the N-terminal processed product of the precursor, p105. It has been suggested that p50 is generated not from full-length p105 but cotranslationally from incompletely synthesized molecules by the proteasome. We show that the 20S proteasome endoproteolytically cleaves the fully synthesized p105 and selectively degrades the C-terminus of p105, leading to p50 generation in a ubiquitin-independent manner. As small as 25 residues C-terminus to the site of processing are sufficient to promote processing in vivo. However, any p105 mutant that lacks complete ankyrin repeat domain (ARD) is processed aberrantly, suggesting that native processing must occur from a precursor, which extends beyond the ARD. Remarkably, the mutant p105 that lacks the internal region including the glycine-rich region (GRR) is completely degraded by 20S proteasome in vitro. This suggests that the GRR impedes the complete degradation of the p105 precursor, thus contributing to p50 generation.

IkappaBbeta, one of the major IkappaB proteins, is only partially degraded in response to most extracellular signals. However, the molecular mechanism of this event is unknown. We show here that IkappaBbeta exists in at least two different forms: one that is bound to the NF-kappaB dimer and the other bound to both NF-kappaB and kappaB-Ras, a Ras-like small G protein. Removal of cellular kappaB-Ras enhances whereas excess kappaB-Ras blocks induced IkappaBbeta degradation. Remarkably, kappaB-Ras functions in both GDP- and GTP-bound states, and mutations of the conserved guanine-binding residues of kappaB-Ras abrogate its ability to block degradation of IkappaBbeta. kappaB-Ras also directly blocks the in vitro phosphorylation of IkappaBbeta by IKKbeta. These observations suggest that IkappaBbeta in the ternary complex is resistant to degradation by most signals. We suggest that specific signals, in addition to those that activate only IKK, are essential for the complete degradation of IkappaBbeta.

Conformational changes are thought to play a key role in the function of active protein kinases, although little is known about how these changes relate to the mechanism of phosphorylation. Here we present four high-resolution structures of a single crystal form of Sky1p, a constitutively active serine kinase implicated in yeast RNA processing, each in a different state of nucleotide binding. By comparing the apoenzyme structure to the ADP- and ATP-bound Sky1p structures, we have revealed conformational changes caused by ATP binding or conversion from nucleotide reactant to product. Rotation of the small lobe of the kinase closes the cleft upon binding, allowing the nucleotide to interact with residues from both lobes of the kinase, although some interactions thought to be important for phosphotransfer are missing in the ATP-containing structure. In the apoenzyme, a kinase-conserved phosphate-anchoring loop is in a twisted conformation that is incompatible with ADP and ATP binding, providing a potential mechanism for facilitating ADP release in Sky1p. The nonhydrolyzable ATP analogue AMP-PNP binds in a unique mode that fails to induce lobe closure. This observation, along with comparisons between the two independent molecules in the asymmetric unit of each structure, has provided new molecular details about how the nucleotide binds and induces closure. Finally, we have used mutational analysis to establish the importance of a glycine within the linker that connects the two lobes of Sky1p.

Among prospective kidney transplant recipients, antibodies to HLA-DP are most frequently identified in re-transplant candidates. The significance of these antibodies and their ability to cause rejection is subject to an ongoing debate. The following is a case study of a kidney re-transplant recipient with many HLA-DP antibodies. The patient is a 27 year-old female who had received one prior transplant. Anti-HLA antibodies were determined by Luminex® single antigen beads. Crossmatching was performed by complement-dependent cytotoxicity (CDC) with and without Dithiothreitol (DTT), and flow cytometry (FXM) with and without Pronase®. The patient exhibited only Class II antibodies, including 16 HLA-DP specificities with MFI >10,000. The recipient had a single donor-specific antibody to HLA-DP11 (21,000 MFI) to the potential living donor. The preliminary crossmatch was negative by both CDC and FXM and was reconfirmed with subsequent crossmatches. Post-transplant, the recipient demonstrated good kidney function and a steady decrease in serum creatinine (sCr) to 1.1 mg/dL until developing pyelonephritis two weeks later. The infection was resolved with antibiotics, but the patient’s sCr increased a week later to 3.1 mg/dL, and a kidney biopsy was performed. The biopsy was positive for vasculitis and cellular rejection, with equivocal C4d staining. Following treatment with steroids, IVIG, and thymoglobulin, the patient stabilized with a sCr of 1.6 mg/dL, and the single HLA-DP11 donor-specific antibody persists. Crossmatches performed with current serum and freshly drawn cells from the living donor remained negative. In the presence of negative crossmatches, the recipient experienced cellular and antibody-mediated rejection associated with a donor specific antibody against HLA-DP. The findings suggest that donor-specific HLA-DP antibodies present a risk to kidney transplantation.

The X-ray structure of the RelB dimerization domain (DD) reveals that the RelBDD assumes an unexpected intertwined fold topology atypical of other NF-kappaB dimers. All typical NF-kappaB dimers are formed by the association of two independently folded immunoglobulin (Ig) domains. In RelBDD, two polypeptides reconstruct both Ig domains in the dimer with an extra beta sheet connecting the two domains. Residues most critical to NF-kappaB dimer formation are invariant in RelB, and Y300 plays a positive role in RelBDD dimer formation. The presence of RelB-specific nonpolar residues at the surface removes several intradomain surface hydrogen bonds that may render the domain fold unstable. Intertwining may stabilize the RelBDD homodimer by forming the extra beta sheet. We show that, as in the crystal, RelB forms an intertwined homodimer in solution. We suggest that the transiently stable RelB homodimer might prevent its rapid degradation, allowing for heterodimer formation with p50 and p52.