Indicators for comparing the incidence of diabetic amputations: a nationwide population-based register study.

OBJECTIVE: To test various indicators for comparing the outcomes of diabetic foot care. DESIGN: All 396,317 patients treated with hypoglycaemic medication in Finland were followed up based on nationwide registers on hospital discharges and causes of death during 1997-2007. MATERIALS AND METHODS: The crude and standardized incidences of lower extremity amputations (LEAs), the minor-major ratio of the first LEA and 2-year survival with a preserved leg after the first minor LEA were used as indicators for regional and temporal variation in diabetic foot care. RESULTS: A total of 13,469 LEAs were recorded in 1997-2007. The standardized population-corrected rate of first major LEA per 100,000 person-years declined from 10.0 (95% CI 9.6-10.5) to 7.3 (6.9-7.6) (p < .001), while the minor-major LEA ratio progressed from 0.86 (0.80-0.92) to 1.35 (1.26-1.46) (p < .001). By using these indicators, variation was observed between the university hospital catchment areas. Nationwide, the 2-year survival with a preserved leg after the first minor LEA increased statistically insignificantly from 50.8% (47.3-54.6%) to 55.4% (51.9-59.0%) (p = .08). CONCLUSIONS: The standardized, population-corrected incidence of major LEA, the minor-major LEA ratio, and major-amputation-free survival proved useful as indicators in comparing the outcomes of diabetic foot care.

Capturing dynamics with Eiger, a fast-framing X-ray detector.

Eiger is the next-generation single-photon-counting pixel detector following the widely used Pilatus detector. Its smaller pixel size of 75 µm × 75 µm, higher frame rate of up to 22 kHz, and practically zero dead-time (~4 µs) between exposures will further various measurement methods at synchrotron sources. In this article Eiger's suitability for X-ray photon correlation spectroscopy (XPCS) is demonstrated. By exploiting its high frame rate, complementary small-angle X-ray scattering (SAXS) and XPCS data are collected in parallel to determine both the structure factor and collective diffusion coefficient of a nano-colloid suspension. For the first time, correlation times on the submillisecond time scale are accessible with a large-area pixel detector.

Influence of non-universal effects on dynamical scaling in driven polymer translocation.

We study the dynamics of driven polymer translocation using both molecular dynamics (MD) simulations and a theoretical model based on the non-equilibrium tension propagation on the cis side subchain. We present theoretical and numerical evidence that the non-universal behavior observed in experiments and simulations are due to finite chain length effects that persist well beyond the relevant experimental and simulation regimes. In particular, we consider the influence of the pore-polymer interactions and show that they give a major contribution to the non-universal effects. In addition, we present comparisons between the theory and MD simulations for several quantities, showing extremely good agreement in the relevant parameter regimes. Finally, we discuss the potential limitations of the present theories.

A pilot study of the implementation of WHO surgical checklist in Finland: improvements in activities and communication.

BACKGROUND: World Health Organisation (WHO) has introduced a surgical safety checklist that has reduced post-operative morbidity and mortality. Prior to national checklist implementation, we assessed its possible impact on the operating room (OR) process, safety-related issues and communication among surgical staff in a high-income country. METHODS: In four university and teaching hospitals, a structured questionnaire was delivered to OR personnel involved in consecutive operations over 4-6 weeks before and after the checklist implementation. The questionnaire resembled the WHO checklist and comprised multiple-choice questions relating to performance of safety checks and communication. Anaesthesiologists (A), surgeons (S) and circulating nurses (CN) answered the questions independently. The WHO checklist was modified for national needs. RESULTS: Questionnaires were returned from 1748 operations, 901 before and 847 after the checklist. Patient's identity was more often confirmed (A: 62.7% vs. 84.0%, S: 71.6% vs. 85.5%, CN: 81.6% vs. 94.2%, P < 0.001) and knowledge of names and roles among team members (A: 65.7% vs. 81.8%, S: 71.1% vs. 83.6%, CN: 87.7% vs. 93.2%, P < 0.01) improved with the checklist. Anaesthesiologists and surgeons discussed critical events pre-operatively (A: 22.0% vs. 42.6%, S: 34.7% vs. 46.2%, P < 0.001) more frequently after the checklist. In addition, fewer communication failures (43 vs. 17, P < 0.05) were reported with checklist. CONCLUSIONS: The checklist increased OR teams' awareness of patient-related issues, the procedure and expected risks. It also enhanced team communication and prevented communication failures. Our findings support use of the WHO checklist in various surgical fields.

Towards better patient safety: WHO Surgical Safety Checklist in otorhinolaryngology.

OBJECTIVES: The World Health Organisation has developed a Surgical Safety Checklist to improve patient safety during surgery. This checklist has reduced postoperative morbidity and mortality. Prior to checklist implementation, we wanted to evaluate how it would fit into the process of otorhinolaryngology-head and neck surgery and whether it would have an impact on the awareness of safety-related issues. DESIGN: A structured questionnaire was addressed to the operating room team after consecutive operations during a 1-month period before and after checklist implementation. SETTING AND PARTICIPANTS: This study was conducted at the Department of Otorhinolaryngology at the Helsinki University Central Hospital as a part of a multicentre study. Responses were received regarding 288 operations before and 412 after checklist implementation. MAIN OUTCOME MEASURES: The questions concerned patient-related safety checks, teamwork and communication. RESULTS: The checklist improved verification of the patient's identity (P<0.001). Awareness of the patient's medical history, medication and allergies increased (P<0.001). Knowledge of the names and roles among the team members improved. The otolaryngologists and anaesthesiologists discussed possible critical events more often (P<0.001), and postoperative instructions were better recorded after use of the checklist. In addition, the checklist enhanced communication between operation team members. CONCLUSIONS: Our study confirms that the Surgical Safety Checklist fits well into the surgical working process in otorhinolaryngology-head and neck surgery improving the sharing of patient-related medical information between team members. Development of a specific checklist for otolaryngology calls for further study.

Nordic collaborative study of the BARD1 Cys557Ser allele in 3956 patients with cancer: enrichment in familial BRCA1/BRCA2 mutation-negative breast cancer but not in other malignancies.

BACKGROUND: BARD1 was originally identified as a BRCA1-interacting protein but has also been described in tumour-suppressive functions independent of BRCA1. Several studies have indicated that the BARD1 gene is a potential target for germline changes predisposing to breast and ovarian cancer. The C-terminal Cys557Ser change has previously been uncovered to associate with an increased risk of breast cancer and was recently shown to result in defective apoptotic activities. AIM AND METHODS: Conformation-sensitive gel electrophoresis, minisequencing, TaqMan assays, denaturing high-performance liquid chromatography analysis and DNA sequencing were used to investigate the prevalence of the Cys557Ser allele in a large Nordic case-control study cohort consisting of 2906 patients with breast or ovarian cancer, 734 with prostate cancer, 188 with colorectal cancer, 128 men with breast cancer, and 3591 controls from Finland, Iceland, Denmark, Sweden and Norway. RESULTS: The frequency of the BARD1 Cys557Ser variant seemed to increase among patients from families with breast or ovarian cancer lacking BRCA1 or BRCA2 mutations: a significant difference was obtained compared with controls (6.8% v 2.7%; p<0.001; odds ratio (OR) 2.6; 95% confidence interval (CI) 1.7 to 4.0) and with patients from BRCA1/BRCA2 mutation-positive families (6.8% v 2.2%; p = 0.01; OR 3.2; 95% CI 1.2 to 8.3). In contrast, no major association with male breast, ovarian, colorectal or prostate cancer was observed. Additionally, a novel BARD1 allele resulting in Ser558Pro was identified in familial breast cancer cases. CONCLUSION: These results provide further evidence that BARD1 Cys557Ser confers a slightly increased risk of breast cancer in women.

Electrochemically anodized porous silicon: Towards simple and affordable anode material for Li-ion batteries.

Silicon is being increasingly studied as the next-generation anode material for Li-ion batteries because of its ten times higher gravimetric capacity compared with the widely-used graphite. While nanoparticles and other nanostructured silicon materials often exhibit good cyclability, their volumetric capacity tends to be worse or similar than that of graphite. Furthermore, these materials are commonly complicated and expensive to produce. An effortless way to produce nanostructured silicon is electrochemical anodization. However, there is no systematic study how various material properties affect its performance in LIBs. In the present study, the effects of particle size, surface passivation and boron doping degree were evaluated for the mesoporous silicon with relatively low porosity of 50%. This porosity value was estimated to be the lowest value for the silicon material that still can accommodate the substantial volume change during the charge/discharge cycling. The optimal particle size was between 10-20 µm, the carbide layer enhanced the rate capability by improving the lithiation kinetics, and higher levels of boron doping were beneficial for obtaining higher specific capacity at lower rates. Comparison of pristine and cycled electrodes revealed the loss of electrical contact and electrolyte decay to be the major contributors to the capacity decay.

Identification of germline MLH1 alterations in familial prostate cancer.

Several linkage and loss of heterozygosity (LOH) analyses suggest that the region 3p21-p26, which is a chromosomal location of MLH1, could harbour a susceptibility gene for prostate cancer (PRCA). Furthermore, in a recent candidate single nucleotide polymorphism (SNP) analysis the I219V variation of the MLH1 gene was associated with PRCA. Microsatellite instability (MSI) and germ-line MLH1 mutations were originally demonstrated in hereditary non-polyposis colorectal cancer (HNPCC) but MSI and loss of MLH1 function have also been detected in PRCA. To assess the contribution of MLH1 germline mutations to the development of PRCA in Finland different approaches were used. First, the samples from 11 PRCA-colon cancer patients were screened for MLH1, MSH2 and MSH6 protein expression by immunohistochemistry (IHC). IHC revealed one patient with a putative MLH1 aberration and sequencing of this sample revealed five sequence variants including two missense variants P434L and I219V. Second, the samples from Finnish hereditary prostate cancer (HPC) families were used for the screening of MLH1 mutations which produced twelve MLH1 sequence variants including two missense mutations, I219V, as in the PRCA-colon cancer patient, and V647M. P434L and V647 were both novel, rare variants. Carrier frequencies of the I219V mutation were compared between hereditary prostate cancer (HPC) patients, unselected PRCA cases, patients with benign prostate hyperplasia and controls, but no differences between the sample groups were found. P434L was not present in this study population and V647M was a very rare variant found only in one HPC family. According to the present results, MLH1 does not have a major role in PRCA causation in Finland.

ELAC2/HPC2 involvement in hereditary and sporadic prostate cancer.

The ELAC2/HPC2 gene at 17p11 is the first candidate gene identified for human prostate cancer (PRCA) based on linkage analysis and positional cloning (S. V. Tavtigian et al. Nat. Genet., 27:172-180, 2001). A truncating mutation was found in one hereditary prostate cancer (HPC) family, whereas two missense variants, Ser217Leu and Ala541Thr, were reported to be associated with increased PRCA risk in the general population. Here, we screened for mutations of the ELAC2/HPC2 gene in 66 Finnish HPC families. Several sequence variants, including a new exonic variant (Glu622Val) were found, but none of the mutations were truncating. We then analyzed the frequency of the three found missense variants in 1365 individuals, including hereditary (n = 107) and unselected (n = 467) PRCA, benign prostatic hyperplasia (n = 223), and population controls (568 healthy male blood donors). Ser217Leu and Ala541Thr variants carried no significantly elevated risk for HPC or PRCA, although the latter variant was associated with benign prostatic hyperplasia. The previously undescribed Glu622Val variant had a 1.0% population prevalence, but a significantly higher frequency in PRCA cases (3.0% odds ratio, 2.94; 95% confidence interval, 1.05-8.23). We conclude that ELAC2/HPC2 truncating mutations are rare in HPC, but that rare variants of the ELAC2/HPC2 require additional study as risk factors for PRCA in the general population.

Feasibility of in vivo intravascular ultrasound tissue characterization in the detection of early vascular transplant rejection.

BACKGROUND: Unprocessed ultrasound radiofrequency (RF) signal analysis has been shown to distinguish different tissue structures more reliably than gray-scale interpretation of conventional ultrasound images. METHODS AND RESULTS: The objective of this study was to test the feasibility of in vivo intravascular ultrasound (IVUS) RF signal analysis in an animal model of allograft rejection. Six cynomolgus monkeys underwent transplantation of 3-cm aortic allograft segments distal to the renal arteries from immunologically mismatched donors. IVUS imaging with a 30-MHz system was performed 84 to 105 days after the operation. RF signals were acquired from cross sections of the recipient and the allograft aortas in real time with a digitizer at 500 MHz with 8-bit resolution. Sixty-five cross sections and 68 regions of interest (31 in host aorta and 37 in allograft) were analyzed in the adventitial layer with a total number of 8568 vectors processed. For each region of interest, a weighted-average attenuation was calculated on the basis of the attenuation and length for each individual vector. Histological examination was performed at every cross section imaged by IVUS. When the gray-scale images of conventional IVUS scored by an independent observer were compared, no distinction between adventitia of the native aorta and allograft was possible. Analysis of the average RF backscatter power also showed no significant difference (70.32+/-3.55 versus 70.72+/-3.38 dB). However, the average attenuation of allografts was significantly lower than that of the host aortas (2.64+/-1.38 versus 4.02+/-1.16 dB/mm, P<0.001). Histology demonstrated a marked adventitial inflammatory response in all allografts, with no inflammation observed in the host aortas. CONCLUSIONS: In vivo IVUS tissue characterization can be performed during routine imaging. In this model of transplant vasculopathy, RF attenuation measurements were more sensitive than visual or quantitative gray-scale analysis.

Association of E-cadherin germ-line alterations with prostate cancer.

In our recent cancer registry-based study, the incidence of gastric carcinoma was increased up to 5-fold in male relatives of early-onset prostate cancer (PCA) patients. This association may reflect the influence of genetic factors predisposing individuals to both tumor types. Germ-line mutations of the CDH1 gene at 16q have recently been associated with familial gastric cancer. Furthermore, two genome-wide linkage studies of PCA recently reported positivity at 16q. We therefore identified families and individual patients with both gastric and PCA and investigated whether the CDH1 gene mutations were involved in cancer predisposition in these cases. Fifteen of the 180 Finnish hereditary PCA families (8.3%) had one or more gastric cancer cases. No truncating or splice site CDH1 mutations were identified by PCR single-strand conformational polymorphism in these families or in eight individual patients who had both prostate and gastric cancer. However, a novel S270A missense mutation in exon 6 of the CDH1 gene was seen in a single family with four prostate and two gastric cancers. A large-scale population-based survey indicated a higher prevalence of S270A among both familial PCA cases (3.3%; n = 120; P = 0.01) and unselected PCA patients (1.5%; n = 472; P = 0.12) as compared with blood donors serving as population controls (0.5%; n = 923). We conclude that individual rare mutations and polymorphisms in the CDH1 gene, such as S270A, may contribute to the onset of PCA and warrant further investigations in other populations. However, the CDH1 gene does not appear to explain the link between prostate and gastric cancer.

Dominant negative regulation of trans-activation by the rat androgen receptor: roles of the N-terminal domain and heterodimer formation.

A series of deletion mutants was constructed for the rat androgen receptor (AR) to delineate sequences involved in transcriptional activation. Using transient expression conditions in CV-1 cells and in vitro DNA-binding studies, the amino-terminal domain of the receptor was shown to contain a region (residues 147-296) that is mandatory for trans-activation. Receptors with deletions (residues 147-408) in the N-terminal domain, but with intact DNA- and ligand-binding domains, interacted in vitro with androgen-responsive elements albeit with affinities lower than that of the wild type receptor. Coexpression of N-terminal deletion mutants (delta 46-408 and delta 38-296) with the wild type AR blunted trans-activation by the latter protein in a dominant fashion. By contrast, a hormone-binding-deficient receptor (delta 788-902) that had poor intrinsic activity potentiated the trans-activation by the native receptor. Mechanisms by which deletion mutants in the N-terminal region abolish the function of the wild type protein appear to involve heterodimer formation during interaction with DNA and direct competition for available binding sites on DNA, rather than squelching of accessory proteins. In contrast to impaired trans-activation, binding of the ligand to N-terminal deletion mutants brought about conformational changes that were comparable in wild type and mutant forms, as judged by electrophoretic mobility shift assays. Taken together, these data have specified a region in the N-terminal domain of the AR that plays a decisive role in transcriptional regulation.

Influence of molecular weight on the phase behavior and structure formation of branched side-chain hairy-rod polyfluorene in bulk phase.

We report on an experimental study of the self-organization and phase behavior of hairy-rod pi -conjugated branched side-chain polyfluorene, poly[9,9-bis(2-ethylhexyl)-fluorene-2,7-diyl]-i.e., poly[2,7-(9,9-bis(2-ethylhexyl)fluorene] (PF2/6) -as a function of molecular weight (M(n)) . The results have been compared to those of phenomenological theory. Samples for which M(n) =3-147 kg/mol were used. First, the stiffness of PF2/6 , the assumption of the theory, has been probed by small-angle neutron scattering in solution. Thermogravimetry has been used to show that PF2/6 is thermally stable over the conditions studied. Second, the existence of nematic and hexagonal phases has been phenomenologically identified for lower and higher M(n) (LMW, M(n) < M(*)(n) and HMW, M(n) > M(*)(n) ) regimes, respectively, based on free-energy argument of nematic and hexagonal hairy rods and found to correspond to the experimental x-ray diffraction (XRD) results for PF2/6 . By using the lattice parameters of PF2/6 as an experimental input, the nematic-hexagonal transition has been predicted in the vicinity of glassification temperature (T(g)) of PF2/6 . Then, by taking the orientation parts of the free energies into account the nematic-hexagonal transition has been calculated as a function of temperature and M(n) and a phase diagram has been formed. Below T(g) of 80 degrees C only (frozen) nematic phase is observed for M(n)< M(*)(n) = 10(4) g/mol and crystalline hexagonal phase for M(n) > M(*)(n) . The nematic-hexagonal transition upon heating is observed for the HMW regime depending weakly on M(n) , being at 140-165 degrees C for M(n) > M(*)(n). Third, the phase behavior and structure formation as a function of M(n) have been probed using powder and fiber XRD and differential scanning calorimetry and reasonable semiquantitative agreement with theory has been found for M(n) >or=3 kg/mol. Fourth, structural characteristics are widely discussed. The nematic phase of LMW materials has been observed to be denser than high-temperature nematic phase of HMW compounds. The hexagonal phase has been found to be paracrystalline in the (ab0) plane but a genuine crystal meridionally. We also find that all these materials including the shortest 10-mer possess the formerly observed rigid five-helix hairy-rod molecular structure.

Stimulation of androgen-regulated transactivation by modulators of protein phosphorylation.

The effect of modulators of protein phosphorylation on the transcriptional activity of the androgen receptor (AR) was studied under transient expression conditions. Activators of protein kinase-A [8-bromo-cAMP (8-Br-cAMP)] and protein kinase-C (phorbol 12-myristate 13-acetate) or an inhibitor of protein phosphatase-1 and -2A (okadaic acid) influenced minimally pMMTV-chloramphenicol acetyl-transferase (CAT) activity in CV-1 cells cotransfected with an AR expression plasmid in the absence of androgen. In the presence of testosterone, however, all compounds enhanced AR-mediated transactivation by 2- to 4-fold. A nonsteroidal antiandrogen, Casodex, behaved as a pure antagonist; it blunted the action of testosterone and was not rendered agonistic by activators of protein kinase-A. A reporter plasmid containing two androgen response elements (AREs) in front of the thymidine kinase promoter (pARE2tk-CAT) was also used to examine promoter specificity. It was activated by 8-Br-cAMP, forskolin, or okadaic acid even without AR or androgen. However, when forskolin or okadaic acid was used together with androgen and AR, the resulting AR-dependent transactivation of pARE2tk-CAT was more than additive. Intact DNA- and ligand-binding domains, but not the N-terminal amino acid residues 40-147, of the receptor were mandatory for the synergism between protein kinase-A activators and androgen. Immunoreactive AR content in transfected COS-1 cells was not influenced by exposure to 8-Br-cAMP. Similar results were obtained by ligand binding assays. Quantitative or qualitative differences were not observed in DNA-binding characteristics between receptors extracted from cells treated with testosterone with or without protein kinase-A activator. Collectively, the synergistic stimulation of AR-dependent transactivation by androgen and protein kinase activators is not due to changes in cellular AR content or affinity of the receptor for the cognate DNA element; rather, this phenomenon seems to result from altered interaction of ligand-activated AR with other proteins in the transcription machinery.

Heterodimerization is mainly responsible for the dominant negative activity of amino-terminally truncated rat androgen receptor forms.

Rat androgen receptor (rAR) mutants devoid of the amino-terminal transactivation domain are able to behave as dominant negative regulators of wild-type rAR. To address the underlying mechanisms of the trans-dominant negative action, we have examined the roles of the DNA-binding domain (DBD) and the ligand-binding domain (LBD) in this process. Transactivation experiments in CV-1 cells complemented by electrophoretic mobility shift assays revealed that the dominant negative receptor forms repress the function of wild-type rAR mainly through heterodimer formation, rather than through competition for binding to cognate DNA elements. Heterodimerization of receptor forms containing LBDs may take place even in the absence of specific DNA binding.

The presence of a transcription activation function in the hormone-binding domain of androgen receptor is revealed by studies in yeast cells.

To assess the importance of various regions of the androgen receptor (AR) in transcriptional regulation, we have compared its activation functions (AFs) in yeast and mammalian cells. The receptor's amino-terminal region contains a major transcriptional activator (AF-1) in both cell types, whereas AF-2 in the ligand-binding domain (LBD) is very weak in mammalian cells but clearly functional in the yeast. Hormone-binding ability of LBD is mandatory for AF-2 to operate, as illustrated by mutated LBD constructs. The activity of AF-2 in yeast is severely attenuated when the hinge region is attached to LBD, suggesting that the former region modulates AF-2 in vivo, probably by presenting an interface for interacting proteins.

Epithelial re-growth is associated with inhibition of obliterative airway disease in orthotopic tracheal allografts in non-immunosuppressed rats.

BACKGROUND: Because epithelial cells are targets of alloimmune injury leading ultimately to airway obliteration, we tested whether epithelial re-growth could prevent obliterative airway disease (OAD) in orthotopic tracheal allografts. METHODS: Brown Norway tracheal segments were orthotopically transplanted into nonimmunosuppressed Lewis rats. Allografts were removed on days 2-10 (n=13), 30 (n=4), and 60 (n=5) for histology, computerized morphometry (obliteration), and immunohistochemical detection of mononuclear cells, smooth muscle alpha-actin, and tissue phenotype. Normal tracheas, host tracheas, and heterotopically transplanted allografts served as controls. RESULTS: Orthotopic allografts removed on days 2-10 exhibited epithelial damage and re-growth and mononuclear cell infiltration. On days 30 and 60, partially ciliated cuboidal or attenuated epithelium completely covered the lumen. Although mononuclear cells declined, numerous T cells with a high CD4/CD8 ratio were found in the epithelium till day 60. Orthotopic allograft epithelium expressed donor phenotype on day 7, but recipient phenotype on days 30 and 60. Despite subepithelial alpha-actin positive myofibroblast proliferation, obliteration did not progress from day 7 to 30 and 60 (35, 30, and 33%, respectively). Although more than in normal or host tracheas, the obliteration in orthotopic allografts on days 30 and 60 was significantly less (P<0.001) than in heterotopic allografts. CONCLUSIONS: We describe, for the first time, longterm patency of fully histoincompatible orthotopic tracheal allografts in nonimmunosuppressed rats. Despite acute alloimmune injury and induction of myofibroblast proliferation, epithelial re-growth from the host limited the progression of OAD, thus emphasizing the role of epithelium in the control of airway obliteration.

Prevention of acute allograft rejection in nonhuman primate lung transplant recipients: induction with chimeric anti-interleukin-2 receptor monoclonal antibody improves the tolerability and potentiates the immunosuppressive activity of a regimen using low doses of both microemulsion cyclosporine and 40-O-(2-hydroxyethyl)-rapamycin.

BACKGROUND: In previous studies of cynomolgus monkey lung allograft recipients, we demonstrated significant immunosuppressive efficacy but reduced tolerability after combined treatment with high doses of microemulsion cyclosporine (CsA) and SDZ RAD (40-O-(2-hydroxyethyl)-rapamycin). The current study was designed to compare efficacy and tolerability of a combination of low-dose CsA and high-dose SDZ RAD (CTL group) to triple therapy using the chimeric anti-interleukin-2 (IL-2) receptor (CD25) monoclonal antibody (mAb) basiliximab (anti-IL-2 receptor mAb) for induction therapy (basiliximab: 5 mg intravenously on days 0 and 4) plus low-dose CsA and low-dose SDZ RAD for maintenance immunosuppression (CD25 group). CsA and anti-IL-2 receptor mAb are drugs that reduce cytokine synthesis and block IL-2-mediated lymphocyte stimulation, respectively. SDZ RAD blocks lymphocyte stimulation by other cytokines (e.g., IL-15) that are not inhibited by anti-IL-2 receptor mAb. METHODS: Twelve unilateral lung transplants were performed. Recipients were observed for 49 days by daily weight assessment, hemograms, blood chemistries, radiographs, and lung biopsies. Monkeys were euthanized before day 49 in the event of excessive weight loss (>25%) or organ failure. Target CsA trough levels were 100-200 ng/ml. Target SDZ RAD trough levels in the CTL group (no mAb) were 20-40 ng/ml, and 10-20 ng/ml in the CD25 group. RESULTS: None of the monkeys in the CD25 group needed to be euthanized early due to signs of drug toxicity. In contrast, four monkeys in the CTL group were sacrificed on days 28-35 as a result of excessive weight loss (n=3) and renal functional impairment (n=1). Three recipients in the CD25 group were euthanized on days 36, 38, and 46 as a result of persistent high fever associated with severe rejection. The median animal survival in the CTL group was 32 vs. 46 days in the CD25 group (P<0.04). The only two long-term survivors in the CTL group showed moderate rejection at day 49. The median rejection scores at day 14 (A0) and day 28 (A2) were identical in the two groups, despite the fact that the mean SDZ RAD trough level was significantly lower in the CD25 group (CTL: 38+/-3 ng/ml, CD25: 18+/-2 ng/ml, P<0.0001). After basiliximab levels fell below the minimum therapeutic level (1 mg/ml) on day 28, the median rejection score at day 49 increased to A4 in the CD25 group. CONCLUSION: This is the first study to combine an anti-IL-2 receptor mAb with a drug from the rapamycin class plus CsA. Our study shows that induction therapy with basiliximab enabled SDZ RAD blood levels to be significantly reduced, which led to improved tolerability without the penalty of increased rejection.

Sirolimus (rapamycin) halts and reverses progression of allograft vascular disease in non-human primates.

BACKGROUND: Current immunosuppressive protocols fail to prevent chronic rejection often manifested as graft vascular disease (GVD) in solid organ transplant recipients. Several new immunosuppressants including sirolimus, a dual function growth factor antagonist, have been discovered, but studies of drug efficacy have been hampered by the lack of a model of GVD in primates, as a prelude to clinical trials. As described earlier, we have developed a novel non-human primate model of GVD where progression of GVD is quantified by intravascular ultrasound (IVUS). METHODS: Twelve cynomolgus monkeys underwent aortic transplantation from blood group compatible but mixed lymphocyte reaction-mismatched donors. To allow the development of GVD in the allograft, no treatment was administered for the first 6 weeks. Six monkeys were treated orally with sirolimus from day 45 after transplantation to day 105. RESULTS: Progression of GVD measured as change in intimal area from day 42 to 105 was halted in sirolimus-treated monkeys compared to untreated monkeys (P<0.001, general linear model). On day 105, the intimal area +/- SEM was 3.7+/-1.0 and 6.4+/-0.5 mm2, respectively (P<0.05, t test). The magnitude of allograft intimal area on day 105 correlated inversely with sirolimus trough levels (R2=0.67, P<0.05). Regression of the intimal area was seen in four of six sirolimus-treated monkeys, which was significantly different from the untreated monkeys (P<0.05). CONCLUSIONS: Our results in the first non-human primate model of GVD showed that treatment with sirolimus not only halted the progression of preexisting GVD but also was associated with partial regression. Sirolimus trough blood levels were correlated with efficacy. Therefore, sirolimus has the potential to control clinical chronic allograft rejection.