Rapamycin but not FK506 inhibits the proliferation of mononuclear phagocytes induced by colony-stimulating factors

FK506, CsA, and rapamycin are potent inhibitors of T lymphocyte activation; relatively little is known of their effects on cells of the monocyte/macrophage lineage. Studies were undertaken to determine the effects of these drugs on the proliferative response of bone marrow-derived mononuclear phagocytes (BMMP) to CSFs. Rapamycin inhibited the proliferation of BMMP cultured in the presence of 10% L cellconditioned medium, used as a source of macrophage CSF. The inhibition by rapamycin was dose dependent and apparent at concentrations of 0.1 nM or greater. In a similar fashion, rapamycin inhibited the proliferation of BMMP stimulated by the recombinant forms of murine IL-3 and murine granulocyte-macrophage CSF, and human macrophage CSF. In contrast, neither FK506 nor CsA at concentrations as high as 1000 nM diminished the proliferation of BMMP cultured under identical conditions. FK506, but not CsA, blocked the inhibitory effects of rapamycin on the response of BMMP to CSFs. In summary, these data indicate that rapamycin inhibits the proliferation of BMMP in response to CSFs. These results imply that patients receiving rapamycin, but not FK506 or CsA, may have an impaired ability to generate a functional mononuclear phagocyte population. © 1994 by Williams and Wilkins

Adenovirus-mediated gene transfer to liver grafts: An improved method to maximize infectivity

Background. Adenoviral gene therapy in liver transplantation has many potential applications, but current vector delivery methods to grafts lack efficiency and require high titers. In this study, we attempted to improve gene delivery efficacy using three different delivery methods to liver grafts with adenoviral vector encoding the LacZ marker gene (AdLacZ). Methods. AdLacZ was delivered to cold preserved rat liver grafts by: (1) continuous perfusion via the portal vein (portal perfusion), (2) continuous perfusion via both the portal vein and hepatic artery (dual perfusion), and (3) trapping viral perfusate in the liver vasculature by clamping outflow (clamp technique). Results. Using 1x109 plaque-forming units of Ad-LacZ (multiplicity of infection of 0.4), transduction rate in 3-hr preserved liver grafts, determined by 5-bromo-4-chromo-3-indolyl-β-D-galactopyranoside staining and β-galactosidase assay 48 hr after transplantation, was best with clamp technique (21.5±2.7% 5-bromo-4-chromo-3-indolyl-β-D- galactopyranoside-positive cells and 81.1±3.6 U/g β-galactosidase), followed by dual perfusion (18.5±1.8%, 66.6±19.4 U/g) and portal perfusion (8.8±2.5%, 19.7±15.4 U/g). Further studies using clamp technique demonstrated a near-maximal gene transfer rate of 30% at multiplicity of infection of 0.4 with prolonged cold ischemia to 18 hr. Transgene expression was stable for 2 weeks and slowly declined to 7.8±12.1% at day 28. Lack of inflammatory response was confirmed by histopathological examination and liver enzymes. Transduction was selectively induced in hepatocytes with nearly no extrahepatic transgene expression in the lung and spleen. Conclusions. The clamp technique provides a highly efficient viral gene delivery method to cold preserved liver grafts. This method offers maximal infectivity of adenoviral vector with minimal technical manipulation

Changes in life-style after liver transplantation

Sixty-five pediatric patients who received liver transplants between May 1981 and May 1984 were observed for as many as 5 years and examined for changes in life-style. Children were less frequently hospitalized, spent less time hospitalized, required fewer medications, and generally had excellent liver and renal function after hepatic transplantation as compared with their pretransplantation status. Most children were in age-appropriate and standard school classes or were only 1 year behind. Cognitive abilities remained unchanged. Children improved in gross motor function and patients' behavior significantly improved according to parents' perceptions. Enuresis was more prevalent, however, than in the population of children who had not received liver transplants. Parental divorce rates were no greater than those reported for other families with chronically ill children. Overall, objective changes in life-style as well as parents' perceptions of behavior of children appear to be improved after liver transplantation

Ulcerative colitis disease activity as subjectively assessed by patient-completed questionnaires following orthotopic liver transplantation for sclerosing cholangitis

To assess whether or not liver transplantation and subsequent immunosuppression with cyclosporine and prednisone affect ulcerative colitis symptomatology, we surveyed by questionnaire all 23 surviving patients with pretransplant colonoscopy-documented ulcerative colitis who were transplanted for primary sclerosing cholangitis between June 1982 and September 1985. At follow-up [89.8±7.6 weeks (mean±sem], all six patients who had had asymptomatic colonoscopy-documented ulcerative colitis reported continued ulcerative colitis quiescence. Among the 17 patients who had had symptomatic colonoscopydocumented ulcerative colitis at time of liver transplantation, 88.2% reported improvement in overall ulcerative colitis severity (P<0.001), with significant improvement in the frequency of bowel movements reported by 100%, in crampy abdominal pain by 87.5%, in bowel urgency by 75%, in the occurrence of pus or mucus in stool by 87.5%, in the incidence of ulcerative colitis flares by 81.8%, and in the number of days unable to function normally due to ulcerative colitis symptoms by 78.6% (all at least P<0.01). These data demonstrate that ulcerative colitis symptom severity significantly improves following liver transplantation with immunosuppression with cyclosporine and prednisone. © 1991 Plenum Publishing Corporation

Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture

Increasing the intracellular Zn2+ concentration with zinc-ionophores like pyrithione (PT) can efficiently impair the replication of a variety of RNA viruses, including poliovirus and influenza virus. For some viruses this effect has been attributed to interference with viral polyprotein processing. In this study we demonstrate that the combination of Zn2+ and PT at low concentrations (2 µM Zn2+ and 2 µM PT) inhibits the replication of SARS-coronavirus (SARS-CoV) and equine arteritis virus (EAV) in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp), which is the core enzyme of their multiprotein replication and transcription complex (RTC). Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV—thus eliminating the need for PT to transport Zn2+ across the plasma membrane—we show that Zn2+ efficiently inhibits the RNA-synthesizing activity of the RTCs of both viruses. Enzymatic studies using recombinant RdRps (SARS-CoV nsp12 and EAV nsp9) purified from E. coli subsequently revealed that Zn2+ directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn2+ was found to block the initiation step of EAV RNA synthesis, whereas in the case of the SARS-CoV RdRp elongation was inhibited and template binding reduced. By chelating Zn2+ with MgEDTA, the inhibitory effect of the divalent cation could be reversed, which provides a novel experimental tool for in vitro studies of the molecular details of nidovirus replication and transcription

Injury Risk Estimation Expertise Assessing the ACL Injury Risk Estimation Quiz

Background: Available methods for screening anterior cruciate ligament (ACL) injury risk are effective but limited in application as they generally rely on expensive and time-consuming biomechanical movement analysis. A potential efficient alternative to biomechanical screening is skilled movement analysis via visual inspection (ie, having experts estimate injury risk factors based on observations of athletes’ movements). Purpose: To develop a brief, valid psychometric assessment of ACL injury risk factor estimation skill: the ACL Injury Risk Estimation Quiz (ACL-IQ). Study Design: Cohort study (diagnosis); Level of evidence, 3. Methods: A total of 660 individuals participated in various stages of the study, including athletes, physicians, physical therapists, athletic trainers, exercise science researchers/students, and members of the general public in the United States. The ACL-IQ was fully computerized and made available online (www.ACL-IQ.org). Item sampling/reduction, reliability analysis, cross-validation, and convergent/discriminant validity analysis were conducted to optimize the efficiency and validity of the assessment. Results: Psychometric optimization techniques identified a short (mean time, 2 min 24 s), robust, 5-item assessment with high reliability (test-retest: r = 0.90) and consistent discriminability (average difference of exercise science professionals vs general population: Cohen d = 1.98). Exercise science professionals and general population individuals scored 74% and 53% correct, respectively. Convergent and discriminant validity was demonstrated. Scores on the ACL-IQ were most associated with ACL knowledge and various cue utilities and were least associated with domain-general spatial/decision-making ability, personality, or other demographic variables. Overall, 23% of the total sample (40% exercise science professionals; 6% general population) performed better than or equal to the ACL nomogram. Conclusion: This study presents the results of a systematic approach to assess individual differences in ACL injury risk factor estimation skill; the assessment approach is efficient (ie, it can be completed in\3 min) and psychometrically robust. The results provide evidence that some individuals have the ability to visually estimate ACL injury risk factors more accurately than other instrument-based ACL risk estimation methods (ie, ACL nomogram). The ACL-IQ provides the foundation for assessing the efficacy of observational ACL injury risk factor assessment (ie, does simple skilled visual inspection reduce ACL injuries?). It also provides a representative task environment that can be used to increase our understanding of the perceptual-cognitive mechanisms underlying observational movement analysis and to improve injury risk assessment performance

CAD in mammography: lesion-level versus case-level analysis of the effects of prompts on human decisions

Object: To understand decision processes in CAD-supported breast screening by analysing how prompts affect readers’ judgements of individual mammographic features (lesions). To this end we analysed hitherto unexamined details of reports completed by mammogram readers in an earlier evaluation of a CAD tool. Material and methods: Assessments of lesions were extracted from 5,839 reports for 59 cancer cases. Statistical analyses of these data focused on what features readers considered when recalling a cancer case and how readers reacted to CAD prompts. Results: About 13.5% of recall decisions were found to be caused by responses to features other than those indicating actual cancer. Effects of CAD: lesions were more likely to be examined if prompted; the presence of a prompt on a cancer increased the probability of both detection and recall especially for less accurate readers in subtler cases; lack of prompts made cancer features less likely to be detected; false prompts made non-cancer features more likely to be classified as cancer. Conclusion: The apparent lack of impact reported for CAD in some studies is plausibly due to CAD systematically affecting readers’ identification of individual features, in a beneficial way for certain combinations of readers and features and a damaging way for others. Mammogram readers do not ignore prompts. Methodologically, assessing CAD by numbers of recalled cancer cases may be misleading

Production of α1,3-galactosyltransferase-deficient pigs

The enzyme α1,3-galactosyltransferase (α1,3GT or GGTA1) synthesizes α1,3galactose (α1,3Gal) epitopes (Galα1,3Galβ1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of α1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the α1,3GT gene in cloned pigs. A selection procedure based on a bacteria[toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the α1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the α1,3GT protein. Four healthy α1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the α1,3GT gene hold significant value, as they would allow production of α1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use