TLR2 activation in corneal stromal cells by Staphylococcus aureus‐induced keratitis

The Toll‐Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus‐induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air‐interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub‐groups exposed to UV‐killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL‐8 mRNA expressions were analyzed by quantitative real‐time RT‐PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL‐8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial‐corneal tissue interactions and their inflammatory consequences.     

Risk factors associated with delayed methotrexate clearance and increased toxicity in pediatric patients with osteosarcoma

High‐dose methotrexate (HD‐MTX; 12 g/m²) is part of standard therapy for pediatric osteosarcoma (OS). Risk factors associated with MTX toxicity in children with OS are not well defined. We investigated the association between peak MTX levels (four‐hour) and delayed MTX clearance or treatment toxicity. Information was retrieved from electronic medical records of 33 OS patients treated with HD‐MTX at Texas Children's Hospital from 2008 to 2015. We found that the four‐hour MTX level did not contribute to toxicity or delayed MTX clearance. We demonstrated that certain demographic characteristics are associated with delayed clearance and increased toxicity.     

Consensus Meeting on Microdialysis in Neurointensive Care

Background Microdialysis is used in many European neurointensive care units to monitor brain chemistry in patients suffering subarachnoid hemorrhage (SAH) or traumatic brain injury (TBI).Discussion We present a consensus agreement achieved at a meeting in Stockholm by a group of experienced users of microdialysis in neurointensive care, defining the use of microdialysis, placement of catheters, unreliable values, chemical markers, and clinical use in SAH and in TBI.Conclusions As microdialysis is maturing into a clinically useful technique for early detection of cerebral ischemia and secondary brain damage, there is a need to following such definition regarding when and how to use microdialysis after SAH and TBI.     

Influence of hyperthermal regimes on experimental teratoma development in vitro

We screened for the impact of hyperthermal regimes varying in the cumulative equivalent minutes at 43°C (CEM43°C) and media composition on tumour development using an original teratoma in vitro model. Rat embryos (three germ layers) were microsurgically isolated and cultivated at the air‐liquid interface. During a two week period, ectodermal, mesodermal and endodermal derivatives developed within trilaminar teratomas. Controls were grown at 37°C. Overall growth was measured, and teratoma survival and differentiation were histologically assessed. Cell proliferation was stereologically quantified by the volume density of Proliferating Cell Nuclear Antigen. Hyperthermia of 42°C, applied for 15 minutes after plating (CEM43°C 3.75 minutes), diminished cell proliferation (P ? .0001) and enhanced differentiation of both myotubes (P ? .01) and cylindrical epithelium (P ? .05). Hyperthermia of 43°C applied each day for 30 minutes during the first week (CEM43°C 210 minutes) impaired overall growth (P ? .01) and diminished cell proliferation (P ? .0001). Long‐term hyperthermia of 40.5°C applied for two weeks (CEM43°C 630 minutes) significantly impaired survival (P ? .005). Long‐term hyperthermia of 40.5°C applied from the second day when differentiation of tissues begins (CEM43°C 585 minutes) impaired survival (P ? .0001), overall growth (P ? .01) and cartilage differentiation (P ? .05). No teratomas survived extreme regimes: 43°C for 24 hours (CEM43°C 1440 minutes), hyperthermia in the scant serum‐free medium (CEM43°C 630 minutes) or treatment with an anti‐HSP70 antibody before long‐term hyperthermia 40.5°C from the second day (CEM43°C 585 minutes). This in vitro research provided novel insights into the impact of hyperthermia on the development of experimental teratomas from their undifferentiated sources and are thus of potential interest for future therapeutic strategies in corresponding in vivo models.     

High Level of Cross-Resistance between Kanamycin,Amikacin, and Capreomycin among Mycobacterium tuberculosis Isolates from Georgia and a Close Relation with Mutations in the rrs Gene

The aminoglycosides kanamycin and amikacin and the macrocyclic peptide capreomycin are key drugs for the treatment of multidrug-resistant tuberculosis (MDR-TB). The increasing rates of resistance to these drugs and the possible cross-resistance between them are concerns for MDR-TB therapy. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs, and mutations of the tlyA gene, which encodes a putative rRNA methyltransferase, are thought to confer capreomycin resistance in Mycobacterium tuberculosis bacteria. Studies of possible cross-resistance have shown variable results. In this study, the MICs of these drugs for 145 clinical isolates from Georgia and the sequences of the rrs and tlyA genes of the isolates were determined. Of 78 kanamycin-resistant strains, 9 (11.5%) were susceptible to amikacin and 16 (20.5%) were susceptible to capreomycin. Four strains were resistant to capreomycin but were susceptible to the other drugs, whereas all amikacin-resistant isolates were resistant to kanamycin. Sequencing revealed six types of mutations in the rrs gene (A514C, C517T, A1401G, C1402T, C1443G, T1521C) but no mutations in the tlyA gene. The A514C, C517T, C1443G, and T1521C mutations showed no association with resistance to any of the drugs. The A1401G and C1402T mutations were observed in 65 kanamycin-resistant isolates and the 4 capreomycin-resistant isolates, respectively, whereas none of the susceptible isolates showed either of those mutations. The four mutants with the C1402T mutations showed high levels of resistance to capreomycin but no resistance to kanamycin and amikacin. Detection of the A1401G mutation appeared to be 100% specific for the detection of resistance to kanamycin and amikacin, while the sensitivities reached 85.9% and 94.2%, respectively.Although the first-line anti-tuberculosis (anti-TB) drugs rifampin (RMP; rifampicin), isoniazid (INH), ethambutol (EMB), pyrazinamide (PZA), and streptomycin (SM) were discovered several decades ago, they are still used today in standard short-course regimens for the treatment of TB. These regimens are, however, ineffective for the treatment of multidrug-resistant (MDR) TB (defined as resistance to at least the two most powerful anti-TB drugs, RMP and INH), leading to the use of less effective and more toxic second-line drugs (SLDs). Injectable drugs such as kanamycin (KAN), amikacin (AMK), and capreomycin (CAP) are the key SLDs for the treatment of MDR-TB (17). The emergence of extensively drug-resistant TB, defined as MDR-TB with additional resistance to any fluoroquinolone and at least one of the injectable drugs (10), once again underlines the importance of fast and reliable testing for susceptibility to these antibiotics.Mutations in the 3′ part of the 16S rRNA gene (rrs), particularly at positions 1401, 1402, and 1484 (1, 7, 11, 12), have been associated with resistance to each of the drugs. It has also been suggested that mutations in the tlyA gene are responsible for resistance to CAP (8). Additionally, reports of cross-resistance among various aminoglycosides and CAP have been variable (1, 4, 6, 16). Most of the previous investigations were done with laboratory-generated mutants and with only a limited number of clinical isolates. In this work, we investigated the correlation between mutations in the rrs and tlyA genes and the in vitro resistance to the three injectable drugs of clinical Mycobacterium tuberculosis isolates.