Colon-targeted cell-permeable NFκB inhibitory peptide is orally active against experimental colitis

For the purpose of development of orally active peptide therapeutics targeting NFκB for treatment of inflammatory bowel disease (IBD), two major barriers in oral delivery of therapeutic peptides, metabolic lability and tissue impermeability, were circumvented by introduction of a colon-targeted delivery system and cell permeable peptides (CPP) to NFκB inhibitory peptides (NIP). Suppression of NFκB activation was compared following treatment with various CPP conjugated NIPs (CPP-NIP). The most potent CPP-NIP was loaded in a capsule coated with a colon specific polymer, which was administered orally to colitic rats. The anti-inflammatory activity of the colon-targeted CPP-NIP was evaluated by measuring inflammatory indices in the inflamed colonic tissue. For confirmation of the local action of the CPP-NIP, the same experiment was done after rectal administration. Tissue permeability of the CPP-NIP was examined microscopically and spectrophotometrically using FITC-labeled CPP-NIP (CPP-NIP-FITC). NEMO binding domain peptide (NBD, TALDWSWLQTE) fused with a cell permeable peptide CTP (YGRRARRRARR), CTP-NBD, was most potent in inhibiting NFκB activity in cells. Colon-targeted CTP-NBD, but not colon-targeted NBD and CTP-NBD in an enteric capsule, ameliorated the colonic injury, which was in parallel with decrease in MPO activity and the levels of inflammatory mediators. Intracolonic treatment with CTP-NBD alleviated rat colitis and improved all the inflammatory indicators. CTP-NBD-FITC was detected at much greater level in the inflamed tissue than was NBD-FITC. Taken together, introduction of cell permeability and colon targetability to NIP may be a feasible strategy for an orally active peptide therapy for treatment of IBD.     

Central nervous system dysfunction in a mouse model of FA2H deficiency

Fatty acid 2-hydroxylase (FA2H) is responsible for the synthesis of myelin galactolipids containing hydroxy fatty acid (hFA) as the N-acyl chain. Mutations in the FA2H gene cause leukodystrophy, spastic paraplegia, and neurodegeneration with brain iron accumulation. Using the Cre-lox system, we developed two types of mouse mutants, Fa2h(-/-) mice (Fa2h deleted in all cells by germline deletion) and Fa2h(flox/flox) Cnp1-Cre mice (Fa2h deleted only in oligodendrocytes and Schwann cells). We found significant demyelination, profound axonal loss, and abnormally enlarged axons in the CNS of Fa2h(-/-) mice at 12 months of age, while structure and function of peripheral nerves were largely unaffected. Fa2h(-/-) mice also exhibited histological and functional disruption in the cerebellum at 12 months of age. In a time course study, significant deterioration of cerebellar function was first detected at 7 months of age. Further behavioral assessments in water T-maze and Morris water maze tasks revealed significant deficits in spatial learning and memory at 4 months of age. These data suggest that various regions of the CNS are functionally compromised in young adult Fa2h(-/-) mice. The cerebellar deficits in 12-month-old Fa2h(flox/flox) Cnp1-Cre mice were indistinguishable from Fa2h(-/-) mice, indicating that these phenotypes likely stem from the lack of myelin hFA-galactolipids. In contrast, Fa2h(flox/flox) Cnp1-Cre mice did not show reduced performance in water maze tasks, indicating that oligodendrocytes are not involved in the learning and memory deficits found in Fa2h(-/-) mice. These findings provide the first evidence that FA2H has an important function outside of oligodendrocytes in the CNS.     

Therapeutic hypothermia in infants with hypoxic–ischemic encephalopathy and reversible persistent pulmonary hypertension: short-term hospital outcomes

Aim: Neonatal hypoxic ischemic encephalopathy (HIE) patients are at times accompanied by persistent pulmonary hypertension (PPHN), which is by itself another risk factor of adverse outcomes. We aimed to assess the outcome of therapeutic hypothermia (TH) in these patients whom we managed to reverse the shunt flow, as they are expected to be at much higher risk of adverse neurodevelopmental outcome.

Methods: We reviewed the medical records of 116 HIE infants (13 with PPHN and 103 without PPHN) who underwent TH between 2012 and 2016. We analyzed the short-term hospital outcomes and brain study results (electroencephalogram and magnetic resonance imaging) of TH in these patients.

Results: While infants with PPHN were significantly more likely to be outborn or have meconium aspiration syndrome, and required a longer duration of inotrope and intensive care support, electroencephalographic and brain magnetic resonance findings did not significantly differ according to PPHN status.

Conclusion: Based on our study, the hospital outcomes of infants with HIE accompanied by reversible PPHN who underwent TH were in general not significantly graver than those not accompanied by PPHN. Our results suggest that undergoing TH may be more beneficial in HIE infants with PPHN and the risks for possible adverse effects may not be as so high.     

Prenatal stress promotes development of spasms in infant rats

We have developed a new model of cryptogenic infantile spasms with prenatal betamethasone brain priming to increase susceptibility to development-specific spasms triggered by N-methyl-d-aspartate (NMDA). A recent clinical study linked severe prenatal stress to increased risk for development of infantile spasms. Here, we determined whether prenatal restraint stress (2 × 45 min) in rats on gestational day 15 would increase susceptibility to develop spasms on postnatal day 15. Prenatal stress significantly accelerated onset and increased number of NMDA-triggered spasms compared to handled controls. A single adrenocorticotropic hormone (ACTH or corticotropin) dose delivered acutely had no effects, whereas long-term (3 day) ACTH pretreatment significantly increased latency to onset and decreased number of spasms (an effect similar to that in the human condition). Our data support the notion that extra care should be provided during pregnancy to minimize stress.     

Comparative In Vitro Activities of Torezolid (DA-7157) against Clinical Isolates of Aerobic and Anaerobic Bacteria in South Korea

Resistance of Gram-positive pathogens to first-line antimicrobial agents has been increasing in many parts of the world. We compared the in vitro activities of torezolid with those of other antimicrobial agents, including linezolid, against clinical isolates of major aerobic and anaerobic bacteria. Torezolid had an MIC₉₀ of ≤0.5 μg/ml for the Gram-positive bacterial isolates tested and was more potent than either linezolid or vancomycin.Antimicrobial resistance in Gram-positive cocci has become a major problem in recent years. Oxazolidinones, a new therapeutic class of synthetic drugs, are active against Gram-positive pathogens. Linezolid, the only marketed oxazolidinone, inhibits the initiation of bacterial protein translation by binding to the 23S rRNA peptidyl transferase region (15). The widely used drug linezolid is effective against most Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus spp., and penicillin-resistant Streptococcus pneumoniae (1, 2). However, several recent studies have reported the emergence of linezolid-resistant staphylococci and enterococci in Brazil, China, France, Germany, Italy, and Sweden. The dominant resistance mechanisms are mutations of the 23S rRNA gene and the recently described mobile chloramphenicol-florfenicol resistance (cfr) methyltransferase gene (9).The antibacterial activity of oxazolidinones depends on their affinity for the site of action on the ribosome. Therefore, by modifying their chemical structure, novel oxazolidinones with improved antimicrobial activity can be obtained. Accordingly, it is important to find more useful and less toxic oxazolidinones. Torezolid [TR-700, DA-7157; R-3-(4-(2-(2-methyltetrazol-5-yl)pyridine-5-yl)-3-fluorophenyl)-5-hydroxymethyl oxazolidin-2-on] is the active moiety of the prodrug torezolid phosphate (TR-701, DA-7218) (Fig. ​(Fig.1).1). In a recent study, torezolid was 4- to 8-fold more active than linezolid against Gram-positive bacteria collected from the United States (3). In another study, torezolid demonstrated an 8- to 16-fold increase in potency against all of the linezolid-resistant isolates tested, including MRSA, MRSA carrying the mobile cfr methyltransferase gene, and vancomycin-resistant enterococci (14). However, as far as we know, the activities of torezolid against anaerobic bacteria have not been reported.Open in a separate windowFIG. 1.Chemical structure of torezolid.Human plasma protein binding of torezolid was about 80% (data not shown), and the MIC was unaffected by the presence of 20% human plasma (4). Torezolid has a better pharmacokinetic profile than linezolid. After oral administration of torezolid at 200 mg once a day, the maximum concentration of the drug in serum, half-life, and area under the curve were 2.0 μg/ml, 11.2 h, and 25.4 μg·h/ml, respectively (13). In another study, torezolid phosphate was safe and effective with once-daily 200-mg dosing over 5 to 7 days of treatment for severe complicated skin and skin structure infections caused by Gram-positive bacteria (16). In this study, we compared the in vitro activities of torezolid with those of other antimicrobial agents, including linezolid, against clinical isolates of major aerobic and anaerobic Gram-positive and Gram-negative bacteria.(Part of this study was presented at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2004 [12]).Five hundred ten nonduplicate aerobic and anaerobic bacterial isolates were collected between 2002 and 2004 from patients at a South Korean tertiary-care hospital. The species were identified by conventional methods or by using either the ID 32 GN or the ATB 32A system (bioMérieux, Marcy-l''Etoile, France). Antimicrobial susceptibility was tested by the CLSI agar dilution method (5, 6, 7). The media used were Mueller-Hinton agar (Becton Dickinson, Sparks, MD) for testing of Staphylococcus spp., Enterococcus spp., and Moraxella catarrhalis; Mueller-Hinton agar supplemented with 5% sheep blood for Streptococcus spp.; Haemophilus test medium for Haemophilus influenzae; and brucella agar (Becton Dickinson) supplemented with 5 μg hemin, 1 μg vitamin K₁ per ml, and 5% laked sheep blood for anaerobic bacteria.The antimicrobial agents used were torezolid and linezolid (Dong-A, Seoul, South Korea); erythromycin, tetracycline, oxacillin, penicillin G, and cefuroxime (Sigma Chemical, St. Louis, MO); piperacillin and tazobactam (Yuhan, Seoul, South Korea); azithromycin and sulbactam (Pfizer Korea, Seoul, South Korea); clindamycin (Korea Upjohn, Seoul, South Korea); levofloxacin (Daiichi, Tokyo, Japan); ampicillin, gentamicin, and chloramphenicol (Chong Kun Dang, Seoul, South Korea); cefotaxime (Han-Dok, Seoul, South Korea); cefoxitin and imipenem (Merck Sharp & Dohme, Rahway, NJ); cefotetan (Je Il, Seoul, South Korea); metronidazole (Choong Wae, Seoul, South Korea); trimethoprim and sulfamethoxazole (Dong Wha, Seoul, South Korea); cefaclor and vancomycin (Daewoong, Seoul, South Korea); and teicoplanin (Sanofi Aventis, Bridgewater, NJ).American Type Culture Collection strains of S. aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), S. pneumoniae (ATCC 49619), H. influenzae (ATCC 49247), Bacteroides fragilis (ATCC 25285), and Bacteroides thetaiotaomicron (ATCC 29741) were used as reference strains. The meningeal breakpoints of penicillin G and cefotaxime were used for S. pneumoniae.MRSA continues to be prevalent in South Korea, accounting for 64% of the S. aureus strains in one study (10). In this study, all of the isolates of staphylococci tested were inhibited by torezolid at ≤1 μg/ml and the MIC for 90% of the strains tested (MIC₉₀) was 4- to 8-fold lower than that of linezolid (Table ​(Table1).1). The majority of the MRSA isolates was resistant to erythromycin, clindamycin, gentamicin, levofloxacin, and tetracycline.

TABLE 1.

Comparative antimicrobial activities of torezolid and other antimicrobial agents against aerobic and anaerobic bacteria

Role of p38 MAP kinase in the development of acute lung injury.

Acute lung injury (ALI) is characterized by an intense pulmonary inflammatory response, in which neutrophils play a central role. The p38 mitogen-activated protein kinase pathway is involved in the regulation of stress-induced cellular functions and appears to be important in modulating neutrophil activation, particularly in response to endotoxin. Although p38 has potent effects on neutrophil functions under in vitro conditions, there is relatively little information concerning the role of p38 in affecting neutrophil-driven inflammatory responses in vivo. To examine this issue, we treated mice with the p38 inhibitor SB203580 and then examined parameters of neutrophil activation and acute lung injury after hemorrhage or endotoxemia. Although p38 was activated in lung neutrophils after hemorrhage or endotoxemia, inhibition of p38 did not decrease neutrophil accumulation in the lungs or the development of lung edema under these conditions. Similarly, the increased production of proinflammatory cytokines and activation of NF-kappaB in lung neutrophils induced by hemorrhage or endotoxemia was not diminished by p38 inhibition. These results indicate that p38 does not have a central role in the development of ALI after either hemorrhage or endotoxemia.