Prevalence of bacterial burden on macroscopic contaminants of orthopaedic surgical instruments following sterilization.

BACKGROUND: Macroscopic contamination of orthopaedic instruments with particulates, including cortical bone and polymethyl methacrylate (PMMA) bone cement, that have previously undergone pre-operative sterilization is frequently encountered peri- or intraoperatively, calling into question the sterility of such instruments. AIM: To determine if macroscopic contaminants of orthopaedic surgical instrumentation maintain a bacterial burden following sterile processing, and to determine the most commonly contaminated instruments and the most common contaminants. METHODS: Macroscopic contaminants in orthopaedic instrument trays were collected prospectively at a single tertiary referral centre over a 6-month period from August 2021 to May 2022. When identified, these specimens were swabbed and plated on sheep blood agar. All specimens were incubated at 37 °C for 14 days, and inspected visually for colony formation. When bacterial colony formation was identified, samples were sent for species identification. RESULTS: In total, 33 contaminants were tested, and only one contaminant was found to be growing bacterial colonies (Corynebacterium sp.). The items most commonly found to have macroscopic contamination were surgical trays (N=9) and cannulated drills (N=7). The identifiable contaminants were bone (N=10), PMMA bone cement (N=4) and hair (N=4). Eleven macroscopic contaminants were not identifiable. CONCLUSION: This study found that 97% of macroscopic orthopaedic surgical instrument contaminants that underwent sterile processing did not possess a bacterial burden. Contaminants discovered during a procedure are likely to be sterile, and do not pose a substantially increased risk of infection to a patient.

An alternatively spliced long form of Fas apoptosis inhibitory molecule (FAIM) with tissue-specific expression in the brain.

The gene encoding Fas apoptosis inhibitory molecule (FAIM) was cloned by differential display using RNA obtained from Fas-resistant and Fas-sensitive primary murine B lymphocytes. FAIM is highly evolutionarily conserved and broadly expressed, suggesting that its gene product plays a key role in cellular physiology. Here we report the identification of a new, longer form of FAIM (FAIM-L) and characterization of the genomic locus that clarifies its origin. The murine FAIM gene is located at chromosome 9f1, a region syntenic to the corresponding location of the human FAIM gene. The gene consists of six exons and contains putative translation initiation sites within exons II and III. The long form of FAIM is generated by all six exons, whereas the originally cloned form of FAIM, now termed FAIM-Short (FAIM-S) is generated from five exons by alternative splicing. FAIM-L is dominantly expressed in the brain whereas FAIM-S is widely expressed in many tissues.

Receptor-specific regulation of B-cell susceptibility to Fas-mediated apoptosis and a novel Fas apoptosis inhibitory molecule.

The susceptibility of primary B cells to Fas (APO-1, CD95)-mediated apoptosis is modulated by signals derived from additional surface receptors: CD40 engagement produces upregulation of Fas expression and marked sensitivity to Fas-induced cell death, whereas antigen receptor engagement, or interleukin-4 receptor (IL-4R) engagement, inhibits Fas killing and thereby produces Fas resistance, even in otherwise susceptible, CD40-stimulated targets. Surface immunoglobulin (sIg) and IL-4R utilize distinct signaling pathways to produce Fas resistance that rely on protein kinase C and signal transducer and activator of transcription 6, respectively sIg signaling for inducible Fas resistance requires nuclear factor-kappaB and depends on new macromolecular synthesis. Proximate mediators for Fas resistance include the known anti-apoptotic gene products Bcl-xL and FLIP (but not Btk), and a novel anti-apoptotic gene that encodes Fas apoptosis inhibitory molecule (FAIM). FAIM was identified by differential display and was cloned as two alternatively spliced forms: FAIM-S is broadly expressed, whereas faim-L expression is tissue specific. faim is highly evolutionarily conserved, suggesting an important function throughout phylogeny. Inducible resistance to Fas-mediated apoptosis is speculated to protect antigen-specific B cells during potentially dangerous interactions with FasL-bearing T cells; the elevated sIg-signaling threshold for inducible Fas resistance in autoreactive, tolerant B cells would insure against autoimmunity. However, aberrant acquisition of Fas resistance may allow autoreactive B cells to escape Fas deletion and malignant lymphocytes to thwart antitumor immunity.

Placental transfer of the thromboxane synthetase inhibitor ridogrel in the late-pregnant ewe.

OBJECTIVES: To assess the occurrence of placental transfer of the thromboxane synthetase inhibitor ridogrel in the pregnant ewe and to determine its effect on prostanoid levels in the ewe and fetal lamb, on uterine contractility and on maternal and fetal hemodynamics. STUDY DESIGN: Five chronically instrumented pregnant ewes at 122 days of gestation received intravenous infusions of 5 mg/kg/3 h ridogrel and solvent. Maternal and fetal arterial samples were obtained at predetermined intervals to determine concentrations of ridogrel and prostaglandin metabolites TXB2, 6-keto-PGF1alpha, PGF2alpha, and PGE2. Maternal and fetal responses of blood flow and pressures were determined. RESULTS: Fetal ridogrel levels were 25% of maternal concentrations. Ridogrel showed rapid and marked thromboxane synthetase inhibition and augmentation of levels of prostaglandin metabolites. There was no evidence of change in amniotic pressure, uterine blood flow, maternal and fetal blood pressure and heart rate. CONCLUSION: Ridogrel is a potent thromboxane synthetase inhibitor which passes the sheep placenta, does not influence maternal and fetal hemodynamics and uterine contractility, and shows similar antiplatelet activity in the ewe and the fetal lamb.

A novel gene coding for a Fas apoptosis inhibitory molecule (FAIM) isolated from inducibly Fas-resistant B lymphocytes.

The sensitivity of primary splenic B cells to Fas-mediated apoptosis is modulated in a receptor-specific fashion. Here we used a differential display strategy to detect cDNAs present in B cells rendered Fas resistant but absent in those rendered Fas sensitive. This led to the cloning and characterization of a novel 1.2-kb gene that encodes a Fas apoptosis inhibitory molecule (FAIM). faim-transfected BAL-17 B lymphoma cells were less sensitive by half or more to Fas-mediated apoptosis than were vector-transfected controls, using Fas ligand-bearing T cells or a cytotoxic anti-Fas antibody to trigger Fas, and this was associated with inhibition of Fas- induced poly-ADP ribose polymerase (PARP) cleavage. In primary B cells, the time course of faim mRNA and FAIM protein expression correlated with the induction of Fas resistance by surface (s)Ig engagement. Thus, FAIM is an inducible effector molecule that mediates Fas resistance produced by sIg engagement in B cells. However, faim is broadly expressed in various tissues and the faim sequence is highly conserved evolutionarily, suggesting that its role extends beyond lymphocyte homeostasis. As FAIM has no significant regions of homology to other gene products that modulate Fas killing, it appears to represent a distinct, new class of antiapoptotic protein.

Bcl-x protects primary B cells against Fas-mediated apoptosis.

Primary murine splenic B cells are rendered sensitive or resistant to Fas-mediated apoptosis in a receptor-specific fashion. B cells stimulated though CD40 are Fas sensitive unless they also receive a signal though surface Ig that produces a state of resistance to Fas killing. Protection from Fas-mediated apoptosis takes time to develop and requires ongoing macromolecular synthesis; therefore, it appears to involve the induction and accumulation of one or more gene products. The role of Bcl-x was evaluated by examining the expression and function of this gene in primary B cells. bcl-x mRNA was induced by anti-IgM treatment of otherwise sensitive (CD40 ligand-treated) B cells. Bcl-x protein expression was induced by anti-IgM and appeared in a time frame that correlates well with the onset of anti-IgM-induced Fas resistance. Further, B cells from Bcl-x Tg mice were found to be resistant to Fas-mediated apoptosis. These results strongly suggest that the protection against Fas killing afforded by cross-linking surface Ig is mediated, at least in part, by an increase in Bcl-x.

Placental transfer and maternal and fetal hemodynamic effects of ketanserin in the pregnant ewe.

OBJECTIVE: To determine placental transfer of ketanserin and to assess the effect of serotonin-2 receptor blockade by ketanserin on serotonin- and phenylephrine-induced vasoconstriction. STUDY DESIGN: Five chronically instrumented pregnant ewes at 120 days gestation were injected with 20 mg ketanserin i.v., and fetal and maternal arterial samples were obtained at predetermined intervals to assess placental transfer. Maternal and fetal responses of blood flows and pressures were determined after injected of serotonin (20 micrograms/kg) or phenylephrine (10 micrograms/kg) before and after ketanserin (0.75 mg/kg). RESULTS: In the ewe, ketanserin is transferred across the placenta and reaches measurable levels in the fetal lamb. Ketanserin blocks the maternal and fetal serotonin-induced rise in arterial pressure, but not the serotonin-induced reduction in uterine blood flow. CONCLUSION: In the pregnant ewe, the serotonin-induced rise in maternal and fetal blood pressure is effectively antagonized by ketanserin, whereas the serotonin-induced reduction in uterine blood flow is not.

Intracellular signaling for inducible antigen receptor-mediated Fas resistance in B cells.

CD40 ligand-activated B cells are sensitive targets for CD4+ Th1 effector cells that kill in a Fas-dependent fashion. Susceptibility to apoptosis is counteracted by Ag receptor binding that produces a state of resistance to Fas engagement in otherwise sensitive targets. In the present study, protection from Th1-mediated apoptosis was found to be induced by protein kinase C and calcium signals, which in combination mimicked the level of Fas resistance produced by surface Ig engagement. Signaling for Fas resistance did not alter Fas expression. Furthermore, B cells that were protected against Th1-mediated apoptosis were also resistant to apoptosis mediated by soluble, rFas ligand. Taken together, these results indicate that signaling for protection against Fas-mediated apoptosis does not depend on alteration of the interaction between B cell target and Th1 effector populations. Instead, surface IgM-derived protein kinase C and calcium signals appear to produce an intracellular change in the Fas signaling pathway that develops over a period of hours and interferes with the apoptotic process through a mechanism that depends on protein synthesis.

Effects of subcutaneous recombinant human erythropoietin in normal subjects: development of decreased reticulocyte hemoglobin content and iron-deficient erythropoiesis.

The present study evaluates the properties of the reticulocytes produced in healthy volunteers after treatment with different regimens of recombinant human erythropoietin (r-HuEPO). Twenty-four subjects were randomly assigned to one of three different subcutaneous (SC) r-HuEPO (Protcrit; Ortho Biotech) administration protocols (I: 300 U/kg on days 1, 4, 7, 10; II: 400 U/kg on days 1, 5, 9; III: 600 U/kg on days 1, 10) with oral iron supplementation (Niferex; 150 mg, twice a day). The characteristics of the reticulocytes produced were examined with a flow cytometry method that allows measurements of individual reticulocyte cell volume, hemoglobin concentration, and hemoglobin content. Administration of SC r-HuEPO was associated with a significant increase in the production of reticulocytes. The hemoglobin content of reticulocytes (CHr, in picograms of hemoglobin per cell) in the three groups was 28.5 +/- 1.0, 28.2 +/- 0.5, and 28.5 +/- 1.3, respectively, at baseline, decreased to 24.6 +/- 1.6 (p < 0.001), 24.5 +/- 2.3 (p < 0.001), and 27.5 +/- 1.8 (not significant) at day 10, and returned to baseline after r-HuEPO was discontinued (28.8 +/- 0.9, 28 +/- 0.8, and 28.8 +/- 1.4, respectively, at day 22). The percentage of reticulocytes with cell hemoglobin content less than 23 pg was taken as an indicator of iron-deficient erythropoiesis. At baseline, 5.6% +/- 2.7%, 6.9% +/- 3.4%, and 8.3% +/- 3.8% of reticulocytes had less than 23 pg hemoglobin in groups I, II, and III, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of different dosing regimens for recombinant human erythropoietin in a simulated perisurgical setting: the importance of iron availability in optimizing response.

PURPOSE: The goal of this study was to develop a short-term, practical, yet effective regimen for the perioperative use of recombinant human erythropoietin (r-HuEPO) as an alternative to autologous blood donation and/or homologous transfusion. In addition, changes in iron kinetics during accelerated erythropoiesis were examined. PATIENTS AND METHODS: A randomized trial was performed on 24 healthy, iron-replete men. Subjects were given r-HuEPO in one of three dosage schedules, receiving a total dose of 1200 U/kg r-HuEPO subcutaneously: Group I--300 U/kg on Days 1, 4, 7, and 10; Group II--400 U/kg on Days 1, 5, and 9; Group III--600 U/kg on Days 1 and 10. All subjects received 300 mg of elemental iron orally each day for 10 days beginning on Day 1. Complete blood counts (CBC), absolute reticulocyte counts, serum ferritin, serum iron, serum total iron-binding capacity (TIBC), and serum transferrin receptor protein concentrations were measured periodically during the 24-day study period. RESULTS: All groups showed a statistically significant increase in hematocrit, hemoglobin, and absolute reticulocyte count. There was no significant difference in response among the three groups with respect to hemoglobin and hematocrit. The mean maximum increases in hematocrit were 5.4 +/- 1.7, 6.0 +/- 2.1, and 7.2 +/- 2.6 in groups I, II, and III, respectively. The increase in hematocrit positively correlated with log baseline ferritin (r = 0.682, p < 0.001). Administration of r-HuEPO was associated with a highly significant (p < or = 0.0005) 74% decrease in serum ferritin, as well as a marked decrease in percent saturation of TIBC from 39% +/- 14% to 14% +/- 4% (p < or = 0.0005). This was despite the fact that subjects lost less than 250 mL of blood as a result of venipunctures during the entire course of the study. CONCLUSION: Each of these r-HuEPO dose schedules provides an effective, convenient regimen for perisurgical use. However, "normal" iron stores for basal erythropoiesis may not always be sufficient to supply optimal amounts of iron for the accelerated erythropoiesis associated with acute r-HuEPO administration, even with oral iron supplementation. Nonetheless, these findings provide support for further study of the use of r-HuEPO as an alternative to autologous blood donation in the perisurgical setting.

Similarities between the oxygen-sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin.

The ability to adapt successfully to periods of relative hypoxia is crucial to the survival of all higher life forms. Several genes have previously been identified which are up-regulated in response to hypoxia; these include the genes encoding erythropoietin (Epo), platelet-derived growth factor B chain, endothelin, interleukin-1 alpha, ornithine decarboxylase, and vascular endothelial growth factor (VEGF). However, the molecular mechanisms by which hypoxia is sensed remain enigmatic. In addition, it is unknown whether the genes mentioned share a common oxygen-sensing signal transduction pathway. In this report we demonstrate multiple similarities between the oxygen-sensing mechanisms regulating the expression of VEGF and Epo. The expression of both mRNAs is significantly up-regulated by hypoxia and cobalt chloride (CoCl2), and the half-life of both mRNAs is markedly prolonged by cycloheximide. In addition, hypoxic induction of both Epo and VEGF is inhibited by carbon monoxide. As part of our investigation into the signal transduction pathway responsible for the hypoxia and cobalt induction of these genes, we discovered that the expression of members of the jun and fos protooncogene families is also up-regulated early after exposure to either of these stimuli. These findings provide support for the hypothesis that the mechanism(s) by which hypoxia is sensed at a molecular level may be highly conserved and tightly regulated.

Clinical validation of an RIA for natural and recombinant erythropoietin in serum and plasma.

A sensitive radioimmunoassay (RIA) for the detection of erythropoietin (EPO) was developed using antibody directed against EPO from human urine. With 100 microL of sample, the assay is sensitive to 7 U/L, well below the mean EPO level in normal males (15.1 +/- 3.5 U/L) or females (15.4 +/- 4.8 U/L). Dilutions of a variety of human serum samples show a parallel relationship with the standard EPO. Clinical validation of the RIA was confirmed by appropriate increases or decreases of EPO levels in various types of anemia and polycythemia. Serum EPO levels were also measured in volunteers participating in an autologous blood donation study. The RIA proved to be quite sensitive, detecting small increases even after a single unit phlebotomy. This RIA of human EPO meets all the requirements of a routine clinical assay in terms of specificity and clinical sensitivity and can be easily conducted in routine clinical laboratories.

Modulators of protein kinase C inhibit hypoxia-induced erythropoietin production.

The human hepatoma cell line, Hep 3B, produces biologically active erythropoietin (Epo) in response to normal physiologic stimuli and thus provides a model system for the study of Epo regulation. The addition of phorbol 12-myristate 13-acetate (PMA) to Hep 3B cells subsequently grown under hypoxic conditions resulted in a dose-dependent inhibition of hypoxia-induced Epo production by as much as 95 +/- 1% with half-maximal inhibition at 8 ng/mL. By Northern blot analysis, Epo mRNA levels were correspondingly decreased after treatment with PMA. Direct measurement of both membrane and cytosolic protein kinase C activity in Hep 3B cells following treatment with PMA demonstrated a biphasic response as a function of time. Membrane-associated protein kinase C activity initially increased but subsequently decreased to baseline levels by 12 hours. The PMA-induced inhibition of hypoxia-induced Epo production was shown to occur as early as 3 hours after PMA addition, suggesting that the initial activation, rather than the subsequent decrease in protein kinase C activity, is of primary importance. The relative specificity of the PMA-induced inhibition of Epo production was demonstrated by 1) the finding that overall protein and RNA synthesis were not similarly decreased as measured by 3H-leucine and 3H-uridine pulse labeling studies and 2) the observation that the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, failed to have any effect on hypoxia-induced Epo production. In addition, the synthetic analog of diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore, A23187, inhibited hypoxia-induced Epo production up to 85 +/- 3% and 82 +/- 4%, respectively, in a dose-dependent manner. Taken together, these findings suggest that hypoxia-induced Epo production may be negatively regulated by activators of a protein kinase C-mediated pathway.

Effect of inflammatory cytokines on hypoxia-induced erythropoietin production.

The effects of the inflammatory cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha) on erythropoietin (Epo) production in Hep3B cells were examined. The addition of IL-1 alpha, IL-1 beta, or TNF-alpha resulted in a dose-dependent inhibition of hypoxia-induced Epo production by as much as 89%. IL-1 beta was the most effective cytokine tested, demonstrating half-maximal inhibition at 0.4 U/mL compared with 1.0 and 10.0 U/mL for IL-1 alpha and TNF-alpha, respectively. TGF-beta also inhibited hypoxia-induced Epo production, but only by as much as 56%. In contrast to IL-1 alpha, IL-1 beta, TNF-alpha, and TGF-beta, the addition of IL-6 to hypoxic Hep3B cells resulted in a dose-dependent stimulation of hypoxia-induced Epo production by as much as 81%. However, IL-6 did not stimulate Epo synthesis in the absence of hypoxia, and was thus synergistic with hypoxia in inducing Epo production. Combinations of IL-1 alpha, TNF-alpha, and IL-6 were found to be additive in their effects on hypoxia-induced Epo production. By Northern blot analysis, Epo messenger RNA levels in Hep3B cells grown in 1% O2 were decreased when concurrently exposed to either IL-1 alpha or TNF-alpha. The effects that IL-1 alpha, IL-1 beta, TGF-beta, TNF-alpha, and IL-6 have on hypoxia-induced Epo production may provide new insights into the signal transduction pathway by which hypoxia leads to changes in gene expression. In addition, the effects of these inflammatory cytokines on hypoxia-induced Epo production in vitro suggest that in various inflammatory disorders these cytokines may affect Epo production in vivo and may play a significant role in the pathogenesis of the anemia of chronic disease.