Contribution of a multiplex serological test for the preoperative diagnosis of prosthetic joint infection: a prospective study.

BACKGROUND: Prosthetic joint infection (PJI) is a severe complication of orthopaedic surgery. Preoperative diagnosis, although sometimes difficult, is key to choose the relevant treatment. METHODS: We conducted a prospective study aimed at evaluating the diagnostic performance of a multiplex serological test for the pre-operative diagnosis of PJI. Blood samples were collected between 1 July 2016 and 31 July 2017 among patients referred for suspected PJI that occurred at least six weeks prior. Infection diagnosis was confirmed using intraoperative bacteriological cultures during prosthetic exchange. RESULTS: Seventy-one patients were included, with a median age of 73 years (interquartile range [IQR]: 66-81) and 40 (56%) were male. Twenty-six patients had aseptic loosening and 45 patients had PJI. Among the latter, median time since the last surgery was 96 weeks (IQR: 20-324). Intraoperative cultures found Staphylococcus spp, Streptococcus spp or both in 39, 5 and 1 patients, respectively. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 81.8, 95.4, 97.3 and 72.4%, respectively, for all patients and 87.5, 93.5, 94.6 and 85.3%, respectively, for staphylococcal infections. Patients with false negative (FN) results had a significantly lower blood lymphocyte count (p = .045). CONCLUSIONS: Multiplex serological test performed well among patients with chronic staphylococcal prosthetic infection. This approach could contribute to PJI diagnosis especially in patients for whom the pre-operative analysis of joint fluid is not informative.

Efficacy of debridement, antibiotic therapy and implant retention within three months during postoperative instrumented spine infections.

BACKGROUND: Postoperative instrumented spine infection (PISI) is a severe complication of invasive spine procedures. METHODS: Retrospective study of patients treated for PISI between 1st January 2008 and 31st December 2012 in a French University Hospital. The objectives of this study were to describe the outcome of patients treated with debridement-irrigation, antibiotic therapy and implant retention (DAIR) within three months after the occurrence of PISI and to identify factors associated with relapse. RESULTS: Among 4290 patients who underwent spinal arthrodesis surgery during the 5-year study period, 129 had PISI treated by debridement-irrigation in the first three months (3.0%, 95% confidence interval [95%CI]: 2.5-3.5). Fifty-two (40%) were female and the median age was 57 years. Fourteen patients (10.8%) had diabetes and 73 (56.6%) had a BMI (Body Mass Index) ≥25 kg/m2. Staphylocccus aureus, enterobacteria or polymicrobial infections were identified in 44.0, 18.0 and 13.0% of cases, respectively. One hundred and six patients (82.2%) and one hundred and twenty-one patients (93.8%) were cured after one DAIR and after two DAIR, respectively. In multivariate logistic analysis, polymicrobial infection was associated with relapse (Odd Ratio [OR] = 3.81; 95%CI: 1.06-13.66; p = .03), while a BMI ≥25 kg/m2 was a protective factor (OR =0.25; 95%CI: 0.07-0.89; p = .03). CONCLUSION: DAIR may be effective for PISI when performed within the first 3 months after onset of infection. Relapses are significantly associated with polymicrobial infection and negatively associated with moderate overweight. These results need to be confirmed in future prospective studies.

Characterization and identification of a chymotryptic hydrolysate of alpha-lactalbumin stimulating cholecystokinin release in STC-1 cells.

Alpha-lactalbumin hydrolysate is of significant interest, due to its potential application as a source of bioactive peptides in nutraceutical and pharmaceutical domains. This study was focused on the cholecystokinin (CCK) family compounds which are small peptides involved in the satiety control. The action of chymotryptic hydrolysate of alpha-lactalbumin on cholecystokinin release from intestinal endocrine STC-1 cells was investigated. We demonstrated for the first time that a chymotryptic hydrolysate of alpha-lactalbumin was able to highly stimulate CCK-releasing activity from STC-1 cells. The peptidic hydrolysate was characterized by LC/MS and MS/MS, thus highlighting the presence of 11 fractions containing 21 peptides, each potentially having the desired activity.

Expression and spatial heterogeneity of dipeptidyl peptidases in endothelial cells of conduct vessels and capillaries.

Dipeptidyl peptidase IV (DPPIV)/CD26 is by far the most extensively studied member of the prolyl oligopeptidase family of serine proteases. The discovery of the related enzymes DPP8 and DPP9 necessitates a (re-)evaluation of the DPPIV-like enzymatic activity in cells and organs. In this study, we aimed (1) to investigate the expression of the individual dipeptidyl peptidases in different types of endothelial cells (ECs) and (2) to reconsider published data in relation to our findings. Examination of DPP expression in rat primary ECs of aortic, endocardial and cardiac microvascular origin revealed the presence of DPPIV-like activity in all cell lysates. More than half of this activity could be attributed to DPP8/9. Western blot analysis revealed an abundance of the DPP8 protein as compared to DPP9. The expression of DPPIV and DPP8 was significantly higher in the cardiac microvascular endothelium than in the other ECs, suggesting a more pronounced role of these DPPs in the microvasculature. In situ, DPP activity in ventricular microvasculature was completely inhibited by sitagliptin, indicating that DPPIV is the predominant DPPIV-like enzyme in this organ. By contrast, immunohistochemical studies indicated DPP9 as the predominant DPP in human carotid artery ECs. In conclusion, our results support a highly regulated expression of individual DPPs in ECs, with a spatial heterogeneity in the cardiovascular tree.

Minimal antimicrobial peptidic sequence from hemoglobin alpha-chain: KYR.

Hemoglobin is an animal protein described as a source of biologically active peptides. Peptic digestion of bovine hemoglobin alpha-chain allowed obtaining peptide fractions with antimicrobial activity. These peptides were purified by reverse-phase High-Performance Liquid Chromatography (HPLC) and characterized by mass spectrometry. The minimal inhibitory concentration and mode of action of these peptides were studied against five bacterial strains including Escherichia coli and Salmonella enteritidis as Gram-negative bacteria and Listeria innocua, Micrococcus luteus and Staphylococcus aureus as Gram-positive bacteria. The action aforementioned peptides were studied on artificial membranes as well. The most active peptides resulted to be the short ones. Consequently, the minimal peptidic sequence necessary for the antibacterial activity was clearly determined: KYR.

Enzyme activity and immunohistochemical localization of dipeptidyl peptidase 8 and 9 in male reproductive tissues.

The mRNA expression pattern of dipeptidyl peptidase (DPP) 8 and DPP9, two DPP4 homologs, was studied previously and showed a broad tissue distribution. In this study, protein expression and activity of DPP8 and DPP9 were investigated in male reproductive tissues of different mammals. Based on specific DPP activities and inhibition profiles, the proline-selective DPP activity in the bovine and rat testis could predominantly be attributed to DPP8/9 and not to DPP4. This is in contrast to the epididymis, where most of the activity was caused by DPP4. Bovine sperm preparations had very low or undetectable DPP8/9 activity. After characterization of polyclonal antibodies specific for DPP8 or DPP9, we could localize both enzymes in seminiferous tubules of the testis. A specific staining for DPP9 was found associated with spermatozoids embedded in the epithelium, just before their release into the lumen, and in spermatids. DPP8 was localized in spermatozoids in an earlier stage of maturation. These findings help to provide insight into the physiological role of DPP4-like enzymes in the male reproductive system. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 2: isoindoline containing inhibitors.

To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors' affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II.

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 1: identification of dipeptide derived leads.

Dipeptide derivatives bearing various P2 residues and pyrrolidine derivatives as P1 mimics were evaluated in order to identify lead structures for the development of DPP8 and DPP9 inhibitors. Structure-activity-relationship data obtained in this way led to the preparation of a series of alpha-aminoacyl ((2S, 4S)-4-azido-2-cyanopyrrolidines). These compounds were shown to be nanomolar DPP8/9 inhibitors with modest overall selectivity toward DPP IV and DPP II.

Contrasting effects of human THP-1 cell differentiation on levofloxacin and moxifloxacin intracellular accumulation and activity against Staphylococcus aureus and Listeria monocytogenes.

BACKGROUND AND AIMS: Listeria monocytogenes and Staphylococcus aureus invade and multiply in THP-1 monocytes. Fluoroquinolones accumulate in these cells, but are less active against intracellular than extracellular forms of L. monocytogenes and S. aureus. We examined whether differentiation of THP-1 monocytes into adherent, macrophage-like cells increases fluoroquinolone uptake and activity. METHODS: THP-1 monocytes were differentiated with phorbol myristate acetate (PMA) and compared with unstimulated cells for: (i) moxifloxacin and levofloxacin accumulation; and (ii) activity against phagocytosed L. monocytogenes and S. aureus (5 h contact). RESULTS: The differentiation of THP-1 monocytes caused: (i) a 3- to 4-fold increase in moxifloxacin uptake and a significant increase in its activity against intracellular L. monocytogenes (from 1.3 log(10) to 2.1 log(10) cfu decrease compared with the post-phagocytosis inoculum), but not against S. aureus (1.0-1.2 log(10) cfu decrease throughout); and (ii) no change in levofloxacin accumulation and intracellular activity against either L. monocytogenes or S. aureus. CONCLUSIONS: Although differentiation of monocytes enhances the uptake and activity of moxifloxacin against L. monocytogenes, this cannot be extended to other intracellular bacteria and to levofloxacin. These results further demonstrate that antibiotic intracellular accumulation and activity are not necessarily linked and suggest that intracellular drug and pathogen combinations must be studied individually.

Purification and characterization of dipeptidyl peptidase IV-like enzymes from bovine testes.

Until now, only recombinant forms of dipeptidyl peptidase (DPP) 8 and 9 have been characterized. We purified non DPPII-non DPPIV enzymes from a natural source. A first DPP8/9-like enzyme was enriched 1160-fold from bovine testes and identified as 'DPP9-like enzyme' by using an anti-DPP9 antibody. A second 576-fold enriched preparation ('DPP enriched peak 3') also showed DPP8/9-like activity. SDS-PAGE analysis showed that the DPP9-like enzyme had a monomeric molecular mass of approx. 100 kDa. Size exclusion chromatography generated a native molecular mass of 164 kDa for the DPP9-like enzyme and one of 234 kDa for the DPP enriched peak 3, suggesting that both proteins appeared to be dimeric. Both enriched preparations and rDPP8 showed roughly similar substrate specificity and inhibitor profiles. The DPP9-like enzyme and the DPP enriched peak 3 possessed a neutral pH optimum and were stable at -80 degrees C. We can conclude that the natural DPP9-like enzyme and the DPP enriched peak 3 are closely related to the recombinant forms of human DPP9 and DPP8.

Dipeptidyl peptidase 8/9-like activity in human leukocytes.

The proline-specific dipeptidyl peptidases (DPPs) are emerging as a protease family with important roles in the regulation of signaling by peptide hormones. Inhibitors of DPPs have an intriguing, therapeutic potential, with clinical efficacy seen in patients with diabetes. Until now, only recombinant forms of DPP8 and DPP9 have been characterized. Their enzymatic activities have not been demonstrated in or purified from any natural source. Using several selective DPP inhibitors, we show that DPP activity, attributable to DPP8/9 is present in human PBMC. All leukocyte types tested (lymphocytes, monocytes, Jurkat, and U937 cells) were shown to contain similar DPP8/9-specific activities, and DPPII- and DPPIV-specific activities varied considerably. The results were confirmed by DPPIV/CD26 immunocapture experiments. Subcellular fractionation localized the preponderance of DPP8/9 activity to the cytosol and DPPIV in the membrane fractions. Using Jurkat cell cytosol as a source, a 30-fold, enriched DPP preparation was obtained, which had enzymatic characteristics closely related to the ones of DPP8 and/or -9, including inhibition by allo-Ile-isoindoline and affinity for immobilized Lys-isoindoline.

Inhibition of lethal endotoxin shock with an L-pyridylalanine containing metalloproteinase inhibitor selected by high-throughput screening of a new peptide library.

Gelatinase B/MMP-9 fulfills crucial regulator or effector functions in disease states and may be pharmacologically targeted by specific inhibitors. The characteristics of cleavages of a natural gelatinase B substrate were simulated and amino acids with zinc-ion chelating side-groups were employed to design a library of peptide-based inhibitors. Here, we extend previous findings of combinatorial chemical synthesis and subsequent library deconvolution with a recently established high-throughput technology. This enabled us to study MMP inhibitors with two zinc-binding groups and to identify a new L-pyridylalanine-containing gelatinase B inhibitor. The peptide analog was found to inhibit, almost to the same degree, the neutrophil enzymes collagenase 2/ MMP-8 and MMP-9 and the monocytic tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE/ADAM-17) in vitro and to protect mice against lethal endotoxinemia in vivo. These data illustrate the usefulness of the screening platform for protective inhibitor discovery and complement recent insights in the pathogenesis and treatment of shock syndromes.

New antibacterial peptide derived from bovine hemoglobin.

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration.

Cross-infection due to imipenem-resistant Bacteroides fragilis associated with a totally implantable venous port.

Four patients in an oncology ward developed Bacteroides fragilis bacteremia over a 12-day period. Cross-infection between two of them, due to an imipenem-resistant strain, was demonstrated by epidemiological investigation and genotypic typing methods (arbitrarily primed PCR fingerprinting and nucleotide sequencing of the cfiA genes and upstream IS1186/IS1168 elements).