Rate and factors associated with surgical site infection following aseptic revision fixation of orthopaedic trauma injuries.

PURPOSE: The primary aim of this study was to define the rate of infection following revision of fixation for aseptic failure. The secondary aims were to identify factors associated with an infection following revision and patient morbidity following deep infection. METHODS: A retrospective study was undertaken to identify patients who underwent aseptic revision surgery during a 3-year period (2017-2019). Regression analysis was used to identify independent factors associated with SSI. RESULTS: Eighty-six patients were identified that met the inclusion criteria, with a mean age of 53 (range 14-95) years and 48 (55.8%) were female. There were 15 (17%) patients with an SSI post revision surgery (n = 15/86). Ten percent (n = 9) of all revisions acquired a 'deep infection', which carried a high morbidity with a total of 23 operations, including initial revision, being undertaken for these patients as salvage procedures and three progressed to an amputation. Alcohol excess (odds ratio (OR) 1.61, 95% CI 1.01-6.36, p = 0.046) and chronic obstructive pulmonary disease (OR 11.1, 95% CI 1.00-133.3, p = 0.050) were independently associated with an increased risk of SSI. CONCLUSION: Aseptic revision surgery had a high rate of SSI (17%) and deep infection (10%). All deep infections occurred in the lower limb with the majority of these seen in ankle fractures. Alcohol excess and COPD were independent risk factors associated with an SSI and patients with a history of these should be counselled accordingly. LEVEL OF EVIDENCE: Retrospective Case Series, Level IV.

Tightly regulated 'all-in-one' lentiviral vectors for protection of human hematopoietic cells from anticancer chemotherapy.

Successful application of gene therapy strategies may require stringently regulated transgene expression. Along this line, we describe a doxycycline (Dox)-inducible 'all-in-one' lentiviral vector design using the pTET-T11 (TII) minimal-promoter and a reverse transactivator protein (rtTA2S-M2) driven by the phosphoglycerate kinase promoter allowing for tight regulation of transgene expression (Lv.TII vectors). Vector design was evaluated in human hematopoietic cells in the context of cytidine deaminase (hCDD)-based myeloprotective gene therapy. Upon Dox administration, a rapid (16-24 h) and dose-dependent (>0.04 µg ml(-1) Dox) onset of transgene expression was detected in Lv.TII.CDD gene-modified K562 cells as well as in primary human CD34(+) hematopoietic cells. Importantly, in both cell models low background transgene expression was observed in the absence of Dox. Functionality of Dox-inducible hCDD expression was demonstrated by >10-fold increase in cytosine arabinoside (1-ß-d-arabinofuranosylcytosine, Ara-C) resistance of Lv.TII.CDD-transduced K562 cells. In addition, Lv.TII.CDD-transduced CD34(+)-derived myeloid cells were protected from up to 300 nm Ara-C (control affected from 50 nm onwards). These data clearly demonstrate the suitability of our self-inactivating lentiviral vector to induce robust, tightly regulated transgene expression in human hematopoietic cells with minimal background activity and highlight the potential of our construct in myeloprotective gene therapy strategies.

Generation of iPSCs from genetically corrected Brca2 hypomorphic cells: implications in cell reprogramming and stem cell therapy.

Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2(Δ) (27/) (Δ27)), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2(Δ) (27/) (Δ27) induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2(Δ) (27/) (Δ27) iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2(Δ) (27/) (Δ27) mouse embryonic fibroblasts. Gene-corrected Brca2(Δ) (27/) (Δ27) iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2(Δ) (27/) (Δ27) recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2(Δ) (27/) (Δ) (27) iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed.

Prospective evaluation of same day versus next day colon manometry results in children with medical refractory constipation.

BACKGROUND: We evaluated the change in colon manometry (CM) parameters and interpretation comparing results when the study is performed the same day after the motility catheter is placed under anesthesia or the following day. METHODS: CM catheter was placed with colonoscopy under anesthesia and recorded on day 1 and repeated on day 2. Study parameters including motility index during fasting, post-prandial and post-Bisacodyl challenge phase; gastrocolonic response; number, presence and propagation of high amplitude propagating contractions (HAPCs); and, study interpretation were compared between both the days. KEY RESULTS: Motility index (fasting, post-Bisacodyl phase, P<.05), HAPC number (10.1 vs 6.6, P=.01) and the proportion of patients having HAPCs (92% vs 70%, P=.002) was significantly higher on day 2 vs day 1. HAPC propagation improved on day 2 vs day 1 (fully propagated, 49% vs 37%; partially propagated, 43% vs 33%; absent 8% vs 30%). Study interpretation changed from day 1 to day 2. On day 1, 37% had a normal study and 63% had an abnormal study. On day 2, all patients with a normal study on day 1 remained normal, and patients with an abnormal study on day 1, 53% remained abnormal and 47% had a normal study. CONCLUSIONS & INFERENCES: CM parameters are affected the day the catheter is placed with colonoscopy under anesthesia. The number, presence, and propagation of HAPCs were significantly higher/improved on day 2 compared to day 1. Overall, CM interpretation changed from abnormal to normal from day 1 to day 2 in 47% of the patients.

Targeting expression to megakaryocytes and platelets by lineage-specific lentiviral vectors.

Essentials Platelet phenotypes can be modified by lentiviral transduction of hematopoietic stem cells. Megakaryocyte-specific lentiviral vectors were tested in vitro and in vivo for restricted expression. The glycoprotein 6 vector expressed almost exclusively in megakaryocytes. The platelet factor 4 vector was the strongest but with activity in hematopoietic stem cells. SUMMARY: Background Lentiviral transduction and transplantation of hematopoietic stem cells (HSCs) can be utilized to modify the phenotype of megakaryocytes and platelets. As the genetic modification in HSCs is transmitted onto all hematopoietic progenies, transgene expression from the vector should be restricted to megakaryocytes to avoid un-physiologic effects by ectopic transgene expression. This can be achieved by lentiviral vectors that control expression by lineage-specific promoters. Methods In this study, we introduced promoters of megakaryocyte/platelet-specific genes, namely human glycoprotein 6 (hGP6) and hGP9, into third generation lentiviral vectors and analyzed their functionality in vitro and in vivo in bone marrow transplantation assays. Their specificity and efficiency of expression was compared with lentiviral vectors utilizing the promoters of murine platelet factor 4 (mPf4) and hGP1BA, both with strong activity in megakaryocytes (MKs) used in earlier studies, and the ubiquitously expressing phosphoglycerate kinase (hPGK) and spleen focus forming virus (SFFV) enhancer/promoters. Results Expression from the mPf4 vector in MKs and platelets was the strongest similar to expression from the viral SFFV promoter, however, the mPf4 vector, also exhibited considerable off-target expression in hematopoietic stem and progenitor cells. In contrast, the newly generated hGP6 vector was highly specific to megakaryocytes and platelets. The specificity was also retained when reducing the promoter size to 350 bp, making it a valuable new tool for lentiviral expression in MKs/platelets. Conclusion MK-specific vectors express preferentially in the megakaryocyte lineage. These vectors can be applied to develop murine models to study megakaryocyte and platelet function, or for gene therapy targeting proteins to platelets.

Inducible T-cell receptor expression in precursor T cells for leukemia control.

Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T-cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. As expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8(+) T-cell development was required to obtain a mature T-cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T-cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity.

Comparison of the pharmacokinetics of digoxin and dihydrodigoxin in cats in single-dose studies.

Pharmacokinetics of 3H-dihydrodigoxin and 3H-digoxin after single intravenous and intraduodenal administration in cats are compared. Data could be described by an open two compartment body model. The beta half-life in plasma of dihydrodigoxin after initial rapid distribution is 4.6h compared to 10.4 h after digoxin administration. The volume of tissue distribution of dihydrodigoxin is 7 times smaller than that of digoxin (0.311 versus 2.051). The "specific uptake" of dihydrodigoxin into myocardium and some other tissues is very low. Over 5.5h the cumulative biliary and urinary elimination of dihydrodigoxin is definitely higher (45.7% versus 14.4%). An unexpected peak in TLC plates after dihydrodigoxin administration in blood, bile and urine was identified to be the sodium salt of the opened lactone structure: dihydrodigoxin acid.

Correlation between inhibition of (Na+, K+)-membrane-ATPase and positive inotropic activity of cardenolides in isolated papillary muscles of guinea pig.

Concentrations of 17 cardenolides, cardenolide glucuronides and sulfates producing half-maximal inhibition of (Na+, K+)-membrane-ATPase from different organs and animal species were determined in vitro. In addition the concentrations that increased the contractility of guinea pig isolated papillary muscles to a particular level were investigated. Comparisons between ATPase-inhibiting and positive inotropic cardiac activities showed extensive parallelism: the correlation coefficients after log/log transformation were between 0.92 and 0.97. The same close correlations are found if dissociation constants of cardenolide receptor complexes and concentrations causing 86Rb-uptake inhibition in human erythrocytes are examined. The concentrations necessary for inhibition of (Na+, K+)-membrane-ATPase of the guinea pig heart and the concentrations required to achieve a defined positive inotropic effect in guinea pig papillary muscle showed a log/log correlation coefficient of 0.97 (P less than 0.001). In both tests the potencies covered more than three orders of magnitude. The results support Repke's hypothesis on the digitalis receptor.

The sensitivity of acoustic cough recording relative to intraesophageal pressure recording and patient report during reflux testing.

BACKGROUND: One of the primary indications for reflux testing with multichannel intraluminal impedance with pH (pH-MII) is to correlate reflux events with symptoms such as cough. Adult and pediatric studies have shown, using cough as a model, that patient report of symptoms is inaccurate. Unfortunately, intraesophageal pressure recording (IEPR) to record coughs is more invasive which limits its utility in children. The primary aim of this study was to validate the use of acoustic cough recording (ACR) during pH-MII testing. METHODS: We recruited children undergoing pH-MII testing for the evaluation of cough. We simultaneously placed IEPR and pH-MII catheters and an ACR device in each patient. Each 24 h ACR, pH-MII, and IEPR tracing was scored by blinded investigators. Sensitivities for each method of symptom recording were calculated. KEY RESULTS: A total of 2698 coughs were detected; 1140 were patient reported PR, 2425 were IEPR detected, and 2400 were ACR detected. The sensitivity of PR relative to ACR was 45.9% and the sensitivity of IEPR relative to ACR was 93.6%. There was strong inter-rater reliability (κ = 0.78) for the identification of cough by ACR. CONCLUSIONS & INFERENCES: Acoustic recording is a non-invasive, sensitive method of recording cough during pH-MII testing that is well suited for the pediatric population.

Relationship between postprandial metabolomics and colon motility in children with constipation.

BACKGROUND: The metabolic pathways associated with colonic motility are unknown. To identify potential metabolic targets for treatment of constipation, we examined the metabolic profile before and after a meal challenge in a cohort of children with constipation and determined its relationship with postprandial colon motility patterns. METHODS: In this prospective study, 187 metabolites were measured by liquid chromatography-mass spectrometry at multiple time points before and after a standardized meal in constipated children undergoing a colon manometry. Postprandial metabolite levels were compared with baseline and also correlated with multiple manometric measurements, including the number, frequency, and amplitude of pressure peaks as well as the motility index (MI). KEY RESULTS: A total of 20 subjects were included (mean age 13.1 ± 3.4 years). No significant metabolite changes were observed at 10 min after the meal, whereas 16 amino acid and 22 lipid metabolites had significant (P < 0.005) postprandial changes, including decreases in methylhistamine, histamine, and GABA, by 60 min. Correlations were observed between normal and abnormal postprandial motility patterns and changes in specific metabolites, including glycerol, carnosine, alanine, asparagine, cytosine, choline, phosphocholine, thyroxine, and triiodothyronine. Interestingly, subjects without the normal postprandial increase in area under the curve (AUC), had markedly increased levels of kynurenic acid and adenosyl-homocysteine. CONCLUSIONS & INFERENCES: This is the first study to examine postprandial metabolic changes in children and also to correlate changes in specific metabolites with colonic motility. The results suggest possible metabolic pathways associated with motility and identify potential targets for the treatment of constipation.

Activation of Evi1 inhibits cell cycle progression and differentiation of hematopoietic progenitor cells.

The transcription factor Evi1 has an outstanding role in the formation and transformation of hematopoietic cells. Its activation by chromosomal rearrangement induces a myelodysplastic syndrome with progression to acute myeloid leukemia of poor prognosis. Similarly, retroviral insertion-mediated upregulation confers a competitive advantage to transplanted hematopoietic cells, triggering clonal dominance or even leukemia. To study the molecular and functional response of primary murine hematopoietic progenitor cells to the activation of Evi1, we established an inducible lentiviral expression system. EVI1 had a biphasic effect with initial growth inhibition and retarded myeloid differentiation linked to enhanced survival of myeloblasts in long-term cultures. Gene expression microarray analysis revealed that within 24 h EVI1 upregulated 'stemness' genes characteristic for long-term hematopoietic stem cells (Aldh1a1, Abca1, Cdkn1b, Cdkn1c, Epcam, among others) but downregulated genes involved in DNA replication (Cyclins and their kinases, among others) and DNA repair (including Brca1, Brca2, Rad51). Cell cycle analysis demonstrated EVI1's anti-proliferative effect to be strictly dose-dependent with accumulation of cells in G0/G1, but preservation of a small fraction of long-term proliferating cells. Although confined to cultured cells, our study contributes to new hypotheses addressing the mechanisms and molecular targets involved in preleukemic clonal dominance or leukemic transformation by Evi1.

[Relationship between digoxin plasma concentrations and frequency of digoxin intoxication (author's transl)]. / Beziehung zwischen Digoxin-Plasma-Konzentrationen und Digoxin-Intoxikationshäufigkeit.

Using appropriate transformations the relationship between the frequency of digoxin intoxication and the corresponding digoxin plasma levels can be shown to be linear. Therefore the relative frequencies have to be transformed into probits and the digoxin plasma levels have to be replaced by their logarithms. With intoxication frequencies from published data the IC50, i.e., the plasma level leading to an intoxication in 50% of cases, is 2.9 ng/ml. At 1.0 ng/ml the intoxication frequency is estimated below 1%, at 4.0 ng/ml the estimated frequency is just below 80%. In the intoxication diagnosis of a single patient the plasma level is of only poor significance.

[Drug release in vitro from digoxin formulations with high bioavailability (author's transl)]. / Wirkstoffliberation in vitro bei Digoxin-Präparaten mit hoher Bioverfügbarkeit.

The drug release of four brands of digoxin tablets (A, B, C, D) with known bioavailability was examined using three liberation systems. The paddle method (1) could only ascertain a uniformly good release of all brands. A flow-cell (II) and a special method testing the supersaturation (III) indicated significant differences in drug release. Both II and III pointed to the brand having the best bioavailability. Beyond it no satisfactory correlation was found between the values of bioavailability and drug release.

[On the pharmacology of the alpha-receptor blocker BE 2254 (HEAT) (author's transl)]. / Zur Pharmakologie des alpha-Rezeptoren-Blockers BE 2254 (HEAT).

The alpha-adrenolytic activity of BE 2254 was investigated in in vitro as well as in vivo assays. On the isolated rat anococcygeus muscle, 2-[beta-(4-hydroxyphenyl)-ethyl-amino-methyl]tetralone(1) (BE 2254) shows a high affinity for postsynaptic alpha-adrenoceptors (pA2 = 8.9), in contrast to its much weaker potency (pA2 = 6.7) in inhibiting clonidine on the electrically driven rat vas deferens, thus suggesting a relative preference for postsynaptic alpha-adrenoceptors. BE 2254 effects on other catecholamine receptors are either negligible or not detectable. The hypotensive action of BE 2254 is shown to be solely due to alpha-blockade. All alpha-adrenolytic actions studied were of competitive nature.

Relationship between dose and plasma level of digoxin and patient characteristics.

The correlation between the daily dose of digoxin, its plasma level and clinical characteristics of 213 patients receiving beta-acetyldigoxin treatment has been evaluated. After logarithmic transformation of lognormally distributed variables multiple linear regression analysis was performed. Eight predictor variables were chosen: sex, age, height, weight, glycoside dose, creatinine and potassium level in serum and dose of spironolactone. The resulting correlation coefficient was 0.46, i.e. only 100 x r2 = 21.4% of the variance of the steady state digoxin plasma level could be interpreted with the aid of these variables. For only 4 of the 8 variables (age, glycoside dose, serum level of creatinine, and spironolactone dose) were partial coefficients of regression and of correlation significantly different from zero. Almost 80% of the variance could not be accounted for. This finding is in accordance with conclusions in the literature.

[Concentration of cardiac glycosides in the heart and brain (author's transl)]. / Konzentration von Herzglykosiden im Myokard und im Gehirn

In cats the concentration of cardio-active glycosides in the heart and brain were investigated with trititum-labelled substances. Steady-state conditions were achieved by repeated i.v. injections of ouabain, digoxin, beta-methyldigoxin, digitoxin, and oleandrin over 5 days. 5 h after the last application glycoside concentrations were measured in plasma, urine, heart, cerebrum and cerebellum. Furthermore the metabolic pattern in these compartments was determined. 1. The glycoside concentration in the heart per g wet weight ranges only from 0.93 (oleandrin) to 1.88% (ouabain) of the daily administered dose per kg. 2. The concentrations in the brain show much higher differences between the diverse glycosides: ouabain with a concentration of 0.02%/g wet weight in the cerebrum shows the lowest and oleandrin with 1.60% the highest value. 3. By calculating the mean relative weights for the hearts (3.9 g/kg) and the brains (11.1 g/kg) 3.7% of the daily administered ouabain activity were found in the whole heart and only 0.18% in the whole brain. In contrast to these data the content of heart and brain after giving the more lipophilic oleandrin was 3.6% and 17.7% (!), respectively. 4. Under steady-state conditions the glycosides ouabain, digoxin, beta-methyldigoxin and digitoxin in heart and brain are mainly unchanged whereas oleandrin is transformed at a higher rate to polar metabolites.

[Digitoxin plasma concentrations during oral treatment (author's transl)]. / Digitoxin-Plasmakonzentrationen während oraler Behandlung.

In 198 patients, among them 153 with a creatinine clearance of less than 20 ml/min, the relationship between the digitoxin plasma level and retrospective data on daily digitoxin dose, age, body weight and renal function has been evaluated. A multiple regression analysis yielded only a very weak correlation (100 r2 = 13.2%, n = 186), with the digitoxin dose having by far the highest partial coefficient of determination (100 r2 = 11.5%). The partial correlation for the renal function was too small as to be relevant (100 r2 = 0.6%). Owing to the weakness of correlation it is impossible to predict the digitoxin plasma level on the basis of standard clinical data. A single dose in the range of 0.07 to 0.1 mg/day seems to be an appropriate treatment for most patients. Corresponding to a median dose of 0.082 mg digitoxin daily during steady state a median plasma level of 14.6 ng/ml has been calculated.

[Saluretic and diuretic effects of xipamide-triamterene combinations in varying dose ratios in rats]. / Untersuchung der saluretischen und diuretischen Wirkung von Xipamid-Triamteren-Kombinationen in unterschiedlichen Dosisverhältnissen an Ratten.

The saluretic and diuretic properties of 4-chloro-5-sulfamoyl-2',6'-salicyloxylidide (xipamide) and 2,4,7-triamino-6-phenyl-pteridine (triamterene) were determined in rats following sole and combined application in various dosages and dose ratios. Xipamide dosages ranged from 0.01-30 mg/kg body weight. Xipamide, when given alone, revealed a significant dose-dependent increase in sodium excretion and urine volume compared to control animals even in the smallest dose to be tested (0.01 mg/kg). Triamterene as sole agent led to an increased sodium and water excretion when given in a natriuretic threshold dose of approximately 1.0 mg/kg. Potassium excretion was slightly enhanced following xipamide application and decreased significantly with triamterene treatment. The combined application of xipamide and triamterene in dose ratios of 1:1-1:4 (xipamide/triamterene) resulted in an increased sodium excretion which was almost additive following high triamterene dosages. Potassium elimination decreased significantly when threshold triamterene dosages were added. High triamterene dosages in all dose ratios of the combined application resulted in potassium levels which only could be registered following sole triamterene application.