The Impact of Implementing a Diabetic Limb-Preservation Program on Amputation Outcomes at an Academic Institution in a Majority-Minority State.

Background. Diabetic foot osteomyelitis may precede major limb amputations and lengthy hospital admission. These complications impact patients' morbidity and mortality. Healthcare institutions with dedicated limb-preservation teams realize reduced amputation rates and improved quality of care. This study evaluates the outcomes following the implementation of a rigorous diabetic limb-preservation program at an academic institution. Methods. Patients with diabetes admitted for osteomyelitis occurring below the knee were identified by ICD-10 codes and included for retrospective review. The number and type of amputations, bone biopsies, revascularizations, and hospital length of stay (LOS) were evaluated. Outcomes were compared using the high-low (Hi-Lo) amputation ratio for the 24 months preceding and the 24 months after the integration of a diabetic limb-preservation service. Results. The authors identified and included 337 patients admitted for diabetic foot osteomyelitis. In the 24-month period prior to program implementation, 140 patients were evaluated. In the 24-month period after program implementation, 197 patients were evaluated. The overall amputation rate decreased from 67.1% (n = 94) to 59.9% (n = 118) (P = .214). Major limb amputation rates significantly decreased from 32.9% (n = 46) to 12.7% (n = 25) (P = .001). Minor amputation rates significantly increased from 34.2% (n = 48) to 47.2% (n = 93) (P = .024). The Hi-Lo amputation ratio decreased from 0.96 to 0.27 (P < .001). The rate of obtaining bone biopsies increased from 32.1% (n = 45) to 72.1% (P < .001). The rate of revascularization increased from 10.7% (n = 15) to 15.2% (n = 30) (P = .299). Average hospital LOS decreased significantly from 11.6 days to 9.8 days (P = .044). Conclusion. After the implementation of a limb-preservation team, there was a precipitous drop in major limb amputations in favor of minor amputations. The average hospital LOS decreased. These findings demonstrated improved clinical care and outcomes in patients with lower extremity osteomyelitis and reinforce the importance of a diabetic foot-preservation service within healthcare institutions.

Investigation Into the Role of ERK in Tyrosine Kinase Inhibitor-Induced Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common and debilitating adverse event that can alter patient treatment options and halt candidate drug development. A case study is presented here describing the preclinical and clinical development of CC-90003, a small molecule extracellular signal-regulated kinase (ERK)1/2 inhibitor investigated as an oncology therapy. In a Phase Ia clinical trial, CC-90003 elicited adverse drug-related neuropathy and neurotoxicity that contributed to discontinued development of CC-90003 for oncology therapy. Preclinical evaluation of CC-90003 in dogs revealed clinical signs and electrophysiological changes consistent with peripheral neuropathy that was reversible. Mice did not exhibit signs of neuropathy upon daily dosing with CC-90003, supporting that rodents generally poorly predict CIPN. We sought to investigate the mechanism of CC-90003-induced peripheral neuropathy using a phenotypic in vitro assay. Translating preclinical neuropathy findings to humans proves challenging as no robust in vitro models of CIPN exist. An approach was taken to examine the influence of CIPN-associated drugs on human-induced pluripotent stem cell-derived peripheral neuron (hiPSC-PN) electrophysiology on multielectrode arrays (MEAs). The MEA assay with hiPSC-PNs was sensitive to CIPN-associated drugs cisplatin, sunitinib, colchicine, and importantly, to CC-90003 in concordance with clinical neuropathy incidence. Biochemical data together with in vitro MEA data for CC-90003 and 12 of its structural analogs, all having similar ERK inhibitory activity, revealed that CC-90003 disrupted in vitro neuronal electrophysiology likely via on-target ERK inhibition combined with off-target kinase inhibition and translocator protein inhibition. This approach could prove useful for assessing CIPN risk and interrogating mechanisms of drug-induced neuropathy.

Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4.

Exposure to thalidomide during a critical window of development results in limb defects in humans and non-human primates while mice and rats are refractory to these effects. Thalidomide-induced teratogenicity is dependent on its binding to cereblon (CRBN), the substrate receptor of the Cul4A-DDB1-CRBN-RBX1 E3 ubiquitin ligase complex. Thalidomide binding to CRBN elicits subsequent ubiquitination and proteasomal degradation of CRBN neosubstrates including SALL4, a transcription factor of which polymorphisms phenocopy thalidomide-induced limb defects in humans. Herein, thalidomide-induced degradation of SALL4 was examined in human induced pluripotent stem cells (hiPSCs) that were differentiated either to lateral plate mesoderm (LPM)-like cells, the developmental ontology of the limb bud, or definitive endoderm. Thalidomide and its immunomodulatory drug (IMiD) analogs, lenalidomide, and pomalidomide, dose-dependently inhibited hiPSC mesendoderm differentiation. Thalidomide- and IMiD-induced SALL4 degradation can be abrogated by CRBN V388I mutation or SALL4 G416A mutation in hiPSCs. Genetically modified hiPSCs expressing CRBN E377V/V388I mutant or SALL4 G416A mutant were insensitive to the inhibitory effects of thalidomide, lenalidomide, and pomalidomide on LPM differentiation while retaining sensitivity to another known limb teratogen, all-trans retinoic acid (atRA). Finally, disruption of LPM differentiation by atRA or thalidomide perturbed subsequent chondrogenic differentiation in vitro. The data here show that thalidomide, lenalidomide, and pomalidomide affect stem cell mesendoderm differentiation through CRBN-mediated degradation of SALL4 and highlight the utility of the LPM differentiation model for studying the teratogenicity of new CRBN modulating agents.

Hyperhemolysis Syndrome without Underlying Hematologic Disease.

Introduction. Hyperhemolysis is characterized by a life-threatening hemolytic transfusion reaction, with hemoglobin (Hb) and hematocrit (Hct) dropping markedly lower than before transfusion. This phenomenon, commonly described in sickle cell disease, is a rare occurrence in patients without hemoglobinopathies. Case Report. A 55-year-old male presented to the hospital after a motorcycle crash and received 10 units of cross-matched blood for active bleeding. The patient was blood group O, with a negative antibody screen. Ten days later, he represented complaining of dyspnea and was found to have a hematocrit of 12%. The direct antiglobulin test was positive for anti-immunoglobin G and complement. Indirect antiglobulin test was positive for anti-Jka alloantibodies. The presence of Jka antigen was revealed in one unit of previously transfused blood; patient's RBCs were negative for the Jka antigen. Laboratory data demonstrated findings consistent with DHTR, as well as reticulopenia and elevated ferritin levels. He continued to show signs of active hemolysis, requiring a total of 4 subsequent units of pRBCs. Each transfusion precipitated a drop in Hb and Hct to levels lower than before transfusion; once transfusions were held, the patient slowly recovered. Discussion. Hyperhemolysis in the setting of a DHTR can occur in patients without hematologic disease.

An Unusual Etiology for Elevation of Activated Partial Thromboplastin Time (aPTT) in SLE: Acquired Hemophilia and Lupus Anticoagulant.

A 60-year-old female who has a history significant for diabetes, depression, and rheumatoid arthritis presented with a progressively enlarging hematoma of the left upper extremity. She was found to have an enlarging hematoma and an isolated elevation of activated partial thromboplastin time (aPTT). Lab work-up revealed low factor VIII activity levels and inhibitor titers at 13.38 Bethesda units (BU). Dilute Russell's viper venom time (dRVVT) revealed a lupus anticoagulant. Hemostasis was achieved with factor VIII inhibitor bypassing activity (FEIBA) and inhibitor eradication with-rituxan after the failure of first-line treatment with cyclophosphamide and prednisone.

Drug-induced hemolytic anemia and thrombocytopenia associated with alterations of cell membrane lipids and acanthocyte formation.

CXCR3 is a chemokine receptor, upregulated upon activation of T cells and expressed on nearly 100% of T cells in sites of inflammation. SCH 900875 is a selective CXCR3 receptor antagonist. Thrombocytopenia and severe hemolytic anemia with acanthocytosis occurred in rats at doses of 75, 100, and 150 mg/kg/day. Massively enlarged spleens corresponded histologically to extramedullary hematopoiesis, macrophages, and hemosiderin pigment and sinus congestion. Phagocytosed erythrocytes and platelets were within splenic macrophages. IgG and/or IgM were not detected on erythrocyte and platelet membranes. Ex vivo increased osmotic fragility of RBCs was observed. Lipid analysis of the RBC membrane revealed modifications in phosphatidylcholine, overall cholesterol, and/or sphingomyelin. Platelets exhibited slender filiform processes on their plasma membranes, analogous to those of acanthocytes. The presence of similar morphological abnormalities in acanthocytes and platelets suggests that possibly similar alterations in the lipid composition of the plasma membrane have taken place in both cell types. This phenotype correlated with alterations in plasma lipids (hypercholesterolemia and low triglycerides) that occurred after SCH 900875 administration, although other factors cannot be excluded. The increased cell destruction was considered triggered by alterations in the lipid profile of the plasma membranes of erythrocytes and platelets, as reflected morphologically.

Toxicoepigenetic alteration of the kidney injury molecule 1 gene in gentamicin-exposed rat kidney.

Chromatin modifications are now widely accepted as being essential steps involved in activation, repression, and poising of the expression of a large number of genes within the genome. Not only does understanding the role of such changes provide an opportunity to elucidate mechanisms controlling gene expression but in parallel offers the ability to develop novel indicative and predictive biomarkers of disease and toxicity. In the current study, we have applied the chromatin immunoprecipitation assay to investigate putative changes in the chromatin environment associated with the kidney injury molecule 1 (Kim1) gene upon exposure of rats to the nephrotoxicant, gentamicin. Chromatin was isolated from the kidneys of both control and gentamicin-treated animals and interrogated using specific antibodies recognizing two modifications of histone H3, acetylation of lysine 9, and trimethylation of lysine 4, along with RNA polymerase II. Enriched chromatin fractions were analyzed by quantitative PCR using tiled primer pairs covering 4 kb of the Kim1 gene (spanning -2 kb to + 2 kb, relative to the transcription start site). The results demonstrate a substantial increase in the presence of RNA polymerase II and both histone modifications following gentamicin treatment in regions downstream but not upstream of the transcriptional start site of the Kim1 gene. These changes were associated with a marked increase in messenger RNA coding for the Kim1 protein. Together these data suggest, for the first time, that the Kim1 gene is regulated in an epigenetic fashion under conditions of nephrotoxicity.

Towards consensus practices to qualify safety biomarkers for use in early drug development.

Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.

Comparative effects of interferon alpha-2b and pegylated interferon alpha-2b on menstrual cycles and ovarian hormones in cynomolgus monkeys.

BACKGROUND: The covalent modification of interferon (IFN) alpha2b with monomethyoxy polyethylene glycol (PEG) reduces its clearance rate and increases its half-life. High doses of interferon (IFN) alpha2b have previously been shown to affect maintenance of pregnancy in rhesus monkeys. Given the role of ovarian hormones in reproductive function and pregnancy, this study was conducted to assess the effects of PEG-IFNalpha2b or IFNalpha2b (comparative control) on ovarian hormones and menstrual cyclicity in cynomolgus monkeys. In addition, the potential for reversibility of PEG-IFNalpha2b or IFNalpha2b-related observations was assessed. METHODS: Monkeys were administered 3,105 microg/m(2) human recombinant (hr) IFNalpha2b or 52, 262, or 4,239 microg/m(2) PEG-hr-IFNalpha2b every other day for one menstrual cycle, followed by a post-dose period of up to two menstrual cycles. RESULTS: Monkeys administered 3,105 microg/m(2) hr-IFNalpha2b or 52, 262, or 4,239 microg/m(2) PEG-hr-IFNalpha2b exhibited transient decreases in food consumption, leukocyte and erythrocyte parameters. Monkeys administered 3,105 microg/m(2) hr-IFNalpha2b exhibited lengthened menstrual cycles that were associated with a delay in reaching peak ovarian hormone levels and lower respective peak concentrations. Similarly, monkeys administered 4,239 microg/m(2) PEG-hr-IFNalpha2b exhibited lengthened menstrual cycles and a delay in reaching peak ovarian hormone levels and slightly lower respective peak concentrations. Post-dosing menstrual cycle length, estradiol and progesterone profiles exhibited evidence of recovery in both the hr-IFNalpha2b and the high-dose PEG-hr-IFNalpha2b groups. CONCLUSIONS: Administration of hr-IFNalpha2b or PEG-hr-IFNalpha2b at high doses to cynomolgus monkeys resulted in similar effects on menstrual cycles, estradiol and progesterone profiles, and exhibited evidence of reversibility upon cessation of dosing. These results suggest that the previously observed high-dose IFNalpha-related effects on the maintenance of pregnancy in monkeys are likely the result of altered ovarian function.

Quantitative gene expression analysis in a nonhuman primate model of antibiotic-induced nephrotoxicity.

Gene expression patterns using microarrays have been described for rodent models of nephrotoxicity. To determine if significant gene expression changes previously identified have application across multiple species, we studied quantitative gene expression changes in the kidneys of female cynomolgus monkeys after exposure to two nephrotoxicants. Animals were dosed with the aminoglycoside gentamicin (10 mg/kg), the experimental oligosaccharide antibiotic everninomicin (30 or 60 mg/kg), or a combination of gentamicin (10 mg/kg) and everninomicin (30 mg/kg) for 7 days. Monkeys receiving these drugs in combination developed renal lesions as early as Day 1. By Day 7, monkeys dosed with 60 mg/kg everninomicin alone also developed renal lesions, while the group exposed to both compounds had more extensive renal damage. The modulation of several genes previously reported to be associated with nephrotoxicity in rodent models was confirmed using quantitative real-time PCR. Among these, waf-1, matrix metalloproteinase-9, and vimentin exhibited changes consistent with the definition of a genomic indicator of toxicity. In addition, we identified three early gene biomarkers that may be predictive of drug-induced nephrotoxicity: clusterin, osteopontin, and hepatitis A virus cellular receptor-1. Logistic regression demonstrated a high degree of correlation between changes in gene expression and the probability of the development of histopathologic lesions. These results are the first confirming rodent gene expression changes associated with nephrotoxicity in a nonhuman primate model and provide preliminary evidence for identifying early gene expression changes predicting the onset of drug-induced renal tubular damage in cynomolgus monkeys.

Porcine toxicology studies of SCH 58500, an adenoviral vector for the p53 gene.

Adenoviral vectors are being actively investigated for their potential utility in gene therapy. SCH 58500, a replication-deficient adenoviral vector, carries the normal p53 tumor suppressor gene, which is frequently mutated or absent in several human cancers. To assess the potential toxicity associated with adenoviral use, Yorkshire pigs were dosed by intravenous, intrahepatic, or local routes (subcutaneous and intradermal) to support a variety of potential clinical indications. Porcine cells were shown to support replication of wild-type human adenovirus. The nonlethal and asymptomatic dose in pigs following dosing via the intrahepatic route was greater than 3 x 10(8) plaque-forming units (pfu)/kg (2.2 x 10(11) particles/kg), but less than 2.1 x 10(9) pfu/kg (1.5 x 10(12) particles/kg). By the intravenous route it was 1 x 10(8) pfu/kg, and by the ip route it was greater than or equal to 3 x 10(8) pfu/kg. In a multicycle intraperitoneal study in pigs, the high dose of 3 x 10(8) pfu/kg caused an increased antibody and/or an inflammatory response. By the intravenous route, plaque-forming units were present in most pigs at 5 min postdose, but only in a few at 10 min postdose. No expression was found in gonadal tissue approximately 3 weeks after a single intravenous injection of 3 x 10(8) pfu/kg. At high intrahepatic doses (about 1.5 x 10(12) particles/kg), acute cardiovascular and hemodynamic effects were found, which in subsequent studies were also present at high doses by intravenous administration. Based on these findings, careful evaluation of hemodynamic parameters in patients receiving systemic doses of SCH 58500 is warranted.

Artifactually increased serum bicarbonate values in two horses and a calf with severe rhabdomyolysis.

Extremely high bicarbonate (HCO3-) and anion gap values were measured in two horses and a calf using the Hitachi 911 automated serum biochemistry analyzer. All three animals had severe muscle disease as evidenced by markedly increased aspartate aminotransferase and creatine kinase activities. Laboratory error was suspected as the source of the increased HCO3- because values calculated from blood gas analysis were normal. It was hypothesized that increased serum lactate dehydrogenase (LDH) activity and pyruvate concentration overwhelmed the oxamate LDH inhibitor in the enzymatic HCO3- assay, resulting in consumption of NADH and falsely elevated spectrophotometric reading. Serum LDH activity was markedly increased in all three patients. In an attempt to reproduce this interference in vitro, LDH and pyruvate were added to normal bovine serum. Bicarbonate concentration was artifactually increased in a linear, dose-response relationship proportional to the amount of LDH activity in the sample; addition of pyruvate augmented this increase. It was concluded that increased serum LDH activity and pyruvate concentration secondary to severe muscle disease can result in artifactual increases in serum HCO3- values obtained by routine enzymatic assay.