Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection. Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP. c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development. Skin is the natural host tissue for both HPV and AAV. In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift). Addition of anti-Rep78 antibodies inhibited the EMSA supershift. Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays. Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts. Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78. The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process.
Nitric oxide (NO) has diverse effects on immune responses and hepatic functions. In BNL CL.2 cells, the murine embryonic liver cells, inducible nitric oxide synthase (iNOS) mRNA expression appeared after 3 h of treatment with IFN-gamma and LPS. Interestingly, mRNA and protein expression of iNOS was down-regulated by sodium nitroprusside (SNP) and diethylamine dinitric oxide in a time- and dose-dependent manner, but not by H2O2. TNF-alpha gene expression was also dramatically reduced by SNP, but IL-6 gene expression was inhibited much less. IFN-gamma and LPS-induced chloramphenicol acetyltransferase activity of iNOS promoter constructs was inhibited by SNP. Electrophoretic mobility shift assay showed that SNP inhibited IFN-gamma plus LPS-induced Oct-1 binding activity, and the inhibition was reversed by DTT. Mutation in the Oct-1 site completely abolished iNOS promoter activity. In addition, supershift assay and Southwestern analysis demonstrated that the Oct-1 binding activity was inhibited by SNP. Taken together, these results indicate that NO suppresses IFN-gamma plus LPS-induced iNOS expression, and that Oct-1 is an important element in this process.
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The key regulator of the switch from latent to lytic replication of the human herpesvirus 8 (HHV-8; KSHV) is the replication and transcription activator (Rta). The ability of Rta to regulate cellular gene expression was examined by transient transfection into cells that were not infected with HHV-8. Rta induced some, but not all, NF-kappa B-responsive reporters through mechanisms that did not involve activation of classic forms of NF-kappa B. Furthermore, transfection of the NF-kappa B subunit Rel A inhibited the ability of Rta to transactivate some but not all reporters. For example, Rel A inhibited the ability of Rta to transactivate the IL-6 promoter, but only when sequences upstream of the NF-kappa B site were present. The ability of Rel A to inhibit Rta-mediated transactivation was not dependent on a functional NF-kappa B site within the promoter, suggesting an indirect mechanism for inhibition. These studies suggest that Rta expression during lytic reactivation of HHV-8 would lead to expression of some cellular genes, including IL-6, whereas activation of NF-kappa B could inhibit some responses to Rta.
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O-Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNA(Sep)), its cognate Sep-tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNA(Sep) binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep(218), Sep(222)). This system has general utility in protein engineering, molecular biology, and disease research.
Although there are no pathognomonic clinical features of MDRTF at the onset of the illness, high fever ( > 104°F), toxaemia, abdominal distension, abdominal tenderness, hepatomegaly and splenomegaly are often reported. The gold standard for the diagnosis of MDRTF is bacterial isolation of the organism in blood cultures. Ciprofloxacin and ceftriaxone are the drugs most commonly used for treatment of MDRTF and produce good clinical results.
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Seven patients with fulminant hepatitis and one asymptomatic HBV carrier were investigated for screening T1762A1764 mutation by cloning and PCR products direct sequencing. PCR products containing HBV precore/core gene regulatory sequences were directly cloned into chloramphenicol acetyltransferase (CAT) expressing plasmid. CAT assay was done to compare CAT expressing level after transfecting into HepG2 cell line and transient expression.
The relatedness of the isolates was determined by phage typing and XbaI-PFGE. Resistance genes, integrons and transposable elements were identified by PCR amplification and sequencing. Plasmids were characterized by alkaline lysis, S1-PFGE, conjugation, replicon typing and Southern blot hybridization.
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Fowl typhoid caused by Salmonella enterica subsp. enterica serotype Gallinarum biotype Gallinarum is the most important chicken disease in Korea. Due to appearance of new or multiple antibiotics resistances in the recently isolated strains, it was difficult to control the disease using antibiotics in our country. Therefore, the prevalence and genetic contents of class 1 integrons in biotype Gallinarum isolated between 1992 and 2001 were investigated by PCR and direct sequencing, respectively. Out of 90 strains, 35 (39%) carried class 1 integrons. The 1.0, 1.6 and 2.0kbp amplicons were amplified in 32 strains (36%), 2 strains (2%) and 1 strain (1%), respectively. The 1.0, 1.6 and 2.0 kbp amplicons contained one (aadA1a), two (aadB-aadA1b) and three cassettes (dhfrXII-orfF-aadA2), respectively, providing resistances against aminoglycosides (aadA1a, aadA1b, aadB, and aadA2) and trimethoprim (dhfrXII). The integron-carrying strains of biotype Gallinarum appeared in 1996 and acquired additional cassettes in 2000. Although the resistances to ampicillin, tetracycline and chloramphenicol are unrelated to class 1 integrons, relatively high prevalence of integron in biotype Gallinarum may be a dormant threat to the chemotherapy of the disease in the near future because of potency to acquire additional antibiotics resistances.
We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.
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Dichloroacetamide (DCAcAm) is an important type of nitrogenous disinfection byproduct. This study is the first to report that DCAcAm can be formed in the absence of chlorinated disinfectants (chlorine and chloramines). This can occur through reduction of three chloramphenicol (CAP) antibiotics by zero valent iron (ZVI). The effects of key experimental parameters, including reaction time, ZVI dose, pH, temperature, water type, and the presence of humic acid (HA) on the formation of DCAcAm were ascertained. The DCAcAm yields from three CAPs all presented the trend of increasing first and then decreasing with time and also increased with increasing ZVI dosage. DCAcAm yields from the ZVI reduction route were higher than those resulting from the chlorination of some previously identified DCAcAm precursors. Acidic conditions favored the formation of DCAcAm by the ZVI route. In addition, lower temperatures increased DCAcAm yields at extended contact times (>12 h). DCAcAm formed from the three CAPs in the presence of HA was lower than in the absence of HA. The formation potential of DCAcAm from the reduction of authentic waters spiked with CAPs by ZVI showed good linear correlations with initial concentrations of the three CAPs. This allows the formation of DCAcAm from the reduction of CAPs by ZVI to be predicted. Given that many wastewater and drinking water distribution networks contain unlined cast iron pipes, reactions between CAPs and ZVI may contribute to the formation of DCAcAm in such systems.
Four schools situated at different locations of Kathmandu valley were included in the study. Throat swabs from 350 students of age group 5-15 years were collected, immediately transported to the laboratory and were processed for S. pyogenes following standard microbiological procedures. Antimicrobial susceptibility testing of the isolates was performed by Kirby Bauer disc diffusion method following CLSI guidelines.
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This study was conducted to estimate the prevalence of Salmonella spp. and their antimicrobial susceptibilities on poultry and swine farms, sampled in 2 regions in Central Vietnam. A total of 67 poultry farms and 46 swine farms were sampled in a period of 5 months (from September 2012 to January 2013). Salmonella spp. was prevalent in 46.3% and 71.7% of poultry and swine farms, respectively. Altogether, 99 non-typhoidal Salmonella were isolated and the most common serovars were Salmonella Weltevreden (19%), followed by Salmonella Typhimurium (12%) and Salmonella 4,,12:i:- (11%). Overall, 71 of 99 (72%) Salmonella isolates were resistant to at least one of the 14 antimicrobial agents tested. Both in poultry and swine farms, high levels of resistance were observed for ampicillin, chloramphenicol, ciprofloxacin, sulphamethoxazole and tetracycline. The presence of Salmonella isolates from poultry and swine farms which were resistant to different classes of antimicrobials suggests that alternative control measures to antimicrobials should be implemented. Moreover, an effective policy should be promoted to encourage a prudent use of these agents in animal farming in Vietnam.
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Tenascin-C (TN-C), an extracellular matrix glycoprotein is expressed during embryonic development, but is present only at low levels in normal adult tissues. TN-C is re-expressed during wound healing, fibrotic diseases and in cancer. To better understand the mechanisms that control TN-C gene expression, we examined the regulation of the human TN-C promoter in human fibroblasts. We demonstrate that a short segment of the TN-C promoter between bp -133 and -27 contains three evolutionarily conserved Ets binding sites (EBS). These three EBSs bind in vitro expressed Fli1 protein and mediate transactivation of the TN-C gene by Fli1. Furthermore, two proximal EBSs contribute significantly to basal activity of the TN-C promoter. GABP, which is present in human fibroblast nuclear extracts, interacts with the two proximal EBSs. In addition, several Sp1 and Sp3 binding sites have been located in close proximity to the EBSs within this promoter region. The studies performed in Drosophila cells demonstrate that either Fli1 or GABPalpha+beta1 functionally interact with Sp1 resulting in a synergistic stimulation of the TN-C promoter activity. In conclusion, this study shows for the first time that the TN-C gene is regulated by Ets proteins, which together with Sp1 act as potent activators of TN-C expression.
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Recent studies in our laboratory indicated that arsenic trioxide has the ability to cause significant cytotoxicity, and induction of a significant number of stress genes in human liver carcinoma cells, HepG2. However, similar investigations with atrazine did not show any significant effects of this chemical on HepG2 cells, even at its maximum solubility of 100 microg/mL in 1% dimethyl sulfoxide (DMSO). Further cytogenetic studies were therefore carried out to investigate the combined effects of arsenic trioxide and atrazine on cell viability and gene expression in immortalized human hepatocytes. Cytotoxicity was evaluated using the MTT-assay for cell viability, while the CAT-Tox (L) assay was performed to measure the induction of stress genes in thirteen different recombinant cell lines generated from human liver carcinoma cells (HepG2), by creating stable transfectants of different mammalian promoter-chloramphenicol acetyltransferase (CAT) gene fusions. Cytotoxicity experiments yielded LC50 values of 11.9 +/- 2.6 microg/mL for arsenic trioxide in de-ionized water, and 3.6 +/- 0.4 microg/mL for arsenic trioxide in 100 microg/mL atrazine; indicating a 3 fold increase in arsenic toxicity associated with the atrazine exposure. Co-exposure of HepG2 cells to atrazine also resulted in a significant increase in the potency of arsenic trioxide to upregulate a number of stress genes including those of the glutathione-S-transferase Ya subunit--GST Ya, metallothioneinIIa--HMTIIA, 70-kDa heat shock protein--HSP70, c-fos, 153-kDa growth arrest and DNA damage (GADD153), 45-kDa growth arrest and DNA damage (GADD45), and 78-kDa glucose regulated protein--GRP78 promoters, as well as the xenobiotic response element--XRE, tumor suppressor protein response element--p53RE, cyclic adenosine monophosphate response element--CRE, and retinoic acid response element--RARE. No significant changes were observed with respect to the influence of atrazine on the modulation of cytochrome P450 1A1-CYP 1A1, and nuclear factor kappa (B site) response element--NFkappaBRE by arsenic trioxide. These results indicate that co-exposure to atrazine strongly potentiates arsenic trioxide-induced cytotoxicity and transcriptional activation of stress genes in transformed human hepatocytes.
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The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) modulates production of proinflammatory cytokines in brain tissue and in peripheral inflammatory cells. Transcription of the genes for these proinflammatory cytokines is regulated by the nuclear factor kappaB (NF-kappaB). NF-kappaB is also activated by proinflammatory cytokines. Degradation of the cytoplasmic inhibitor IkappaBalpha protein results in activation of NF-kappaB. Because of increasing evidence that NF-kappaB is involved in brain injury and inflammation and neurodegenerative disease, we examined whether alpha-MSH inhibits activation of NF-kappaB and limits degradation of IkappaBalpha protein induced by lipopolysaccharide (LPS) in human glioma cells (A-172) and in mouse brain. Electrophoretic mobility shift assays of nuclear extracts from A-172 cells and whole mouse brains stimulated with LPS revealed that alpha-MSH does suppress NF-kappaB activation. Western blot analysis demonstrated that alpha-MSH preserved expression of IkappaBalpha protein in vitro (glioma cells) and in vivo (brain tissue). Chloramphenicol acetyltransferase assay indicated that alpha-MSH suppresses NF-kappaB-dependent reporter gene expression induced by LPS in A-172 cells. The findings are consistent with the possibility that the anti-inflammatory action of alpha-MSH in CNS inflammation occurs via modulation of NF-kappaB activation by peptide-induced inhibition of degradation of IkappaBalpha protein.
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Each genome segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), encodes two genes in ambisense orientation, separated by an intergenic region (IGR). The 3' ends of subgenomic viral mRNAs have been mapped to a stem-loop structure within the IGR, suggesting structure-dependent transcription termination. We have studied the role of the LCMV IGR by using a minigenome (MG) rescue system based on RNA analogues of the short genome segment. An ambisense MG coding for chloramphenicol acetyltransferase (CAT) and green fluorescent protein reporter genes instead of the nucleoprotein and glycoprotein open reading frames, respectively, served as a template for synthesis of full-length anti-MG (aMG) replicate and subgenomic size mRNA for reporter gene expression. An analogous MG without IGR was amplified by the virus polymerase with equal efficiency, but subgenomic mRNA was undetectable. Reporter gene expression from IGR-deficient aMG CAT-sense RNA of genomic length was approximately 5-fold less efficient than that from subgenomic CAT mRNA derived from an IGR-containing MG, but at least 100-fold more efficient than that from a T7 RNA polymerase transcript with the same sequence. Therefore, in the absence of IGR-mediated transcription termination, a fraction of full-length aMG RNA appears to behave as bona fide mRNA. Unexpectedly, MGs without IGR were dramatically impaired in their ability to passage reporter gene activity via infectious virus-like particles. These data suggest that the LCMV IGR serves individual functions in transcription termination for enhanced gene expression and in the virus assembly and/or budding, which are required for the efficient propagation of LCMV infectivity.
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The two most common pathogens for AOM with tympanic perforation were H. influenzae and Staphylococcus aureus. Both pathogens were mostly sensitive to antibiotics.
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Mediterranean herring gulls (Larus cachinnans) were investigated as a possible reservoir of antibiotic resistant bacteria and of cassette-borne resistance genes located in class 1 integrons. Two hundred and fourteen isolates of the family Enterobacteriaceae were collected from cloacal swabs of 92 chicks captured in a natural reserve in the North East of Italy. They showed high percentages of resistance to ampicillin and streptomycin. High percentages of resistance to trimethoprim/sulfamethoxazole were found in Proteus and Citrobacter and to chloramphenicol in Proteus. Twenty-two (10%) isolates carried the intI1 gene. Molecular characterization of the integron variable regions showed a great diversity, with the presence of 11 different cassette arrays and of one integron without integrated cassettes. The dfrA1-aadA1a and aadB-aadA2 cassette arrays were the most frequently detected. Also the estX cassette, alone or in combination with other cassettes, was detected in many isolates. From this study it is concluded that the enteric flora of Mediterranean herring gulls may act as a reservoir of resistant bacteria and of resistance genes. Due to their feeding habits and their ability to fly over long distances, these free-living birds may facilitate the circulation of resistant strains between waste-handling facilities, crops, waters, and urban areas.
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This was a cross-sectional study performed in psychology and ENT department of Moradi Hospital of Rafsanjan University of Medical Sciences in 2008 (Kerman, Iran). Nasopharyngeal cultures were taken from 50 opium smokers before and 2 to 3 months after cessation of opium smoking. Potential pathogens were identified.
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The availability of genes coding for monoclonal Fab fragments of a desired specificity permits their expression in bacteria and provides a simple method for the generation of good quality reagents. In this paper we describe a new phagemid vector for the production of recombinant Fabs from genes obtained from phage display combinatorial libraries. The phagemid features an antibiotic resistance cassette which, once inserted between the heavy chain fragment and the light chain genes, avoids unwanted recombination and preserves useful restriction sites not affecting the Fab production rate.
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Death due to sepsis by S. aureus is rapidly increasing because of their potent weaponries against macrophage mediated killing. Macrophages serve as intracellular reservoirs of S. aureus. Although significant resources have been invested during the last decade in new treatments for sepsis, only antibiotic therapy has failed to improve outcomes. Moreover the host pathogen interaction resulted in host cell death triggering inflammation. So, successful therapy requires amalgamation of therapies to delineate pathogen along with providing protection to host cell. With this idea, LNMMA, the iNOS inhibitor is used along with antibiotics Ofloxacin or Chloramphenicol on S. aureus infected mouse peritoneal macrophage. ROS like H2O2, O2(-) production has been measured. NO inhibition by iNOS inhibitor and antioxidant levels has been analysed. COX2, TLR2 and iNOS expression along with proinflammatory cytokine level was studied. It was found that the use of iNOS inhibitor LNMMA along with antibiotics not only enhances bacterial clearance but also decreases proinflammatory responses in Staphylococcus aureus infected macrophages. Inhibition of TLR2 as well as COX2 has also been found in combined treatment groups. The use of iNOS inhibitor LNMMA plus Ofloxacin or Chloramphenicol pretreatment enhanced bacterial clearance by increasing ROS. Decreases in NO protect the cell from harmful peroxynitril as well as inflammatory damage by changes in iNOS, COX2 activity along with reduced proinflammatory cytokines like TNFα, IFNγ, IL1-β etc. Changes in antioxidant level has been found. This in-vitro realm of augmented bacterial clearance and regulated inflammation may be considered as a novel and important therapeutic intervention.
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The strains were susceptible to aminoglycosides (MIC(90) values: gentamicin, 0.75 mg/L; and streptomycin, 6.0 mg/L), tetracyclines (MIC(90) values: tetracycline, 0.5 mg/L; and doxycycline, 1.0 mg/L), quinolones (MIC(90) values: ciprofloxacin, 0.047 mg/L; and levofloxacin, 0.023 mg/L) and chloramphenicol (MIC(90) value: 1.5 mg/L), i.e. antibiotics commonly used in therapy. Tigecycline (MIC(90) value: 0.19 mg/L) and rifampicin (MIC(90) value: 1.0 mg/L) were also active against F. tularensis strains, while resistance to erythromycin (MIC(90) value: >256 mg/L) and linezolid (MIC(90) value: 32 mg/L) was observed in all strains.
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There is an urgent need for the development of novel antimicrobial agents against the highly resistant E. faecalis. The present study shows that the peptide PsVP-10 might make a contribution to the solution of this serious problem.
Among its other biological activities, pifithrin-alpha is an inhibitor of firefly luciferase activity. Caution must therefore be taken when using this compound, which has been characterised as an inhibitor of p53 transcriptional activity, to investigate effects on gene expression using transiently transfected reporter plasmids. Furthermore, these results demonstrate that when using novel compounds, the choice of vectors used in the experimental procedures might be of great importance for the correct conclusions to be made.
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The adequate management of central nervous system (CNS) infections requires that antimicrobial agents penetrate the blood-brain barrier (BBB) and achieve concentrations in the CNS adequate for eradication of the infecting pathogen. This review details the currently available literature on the pharmacokinetics (PK) of antibacterials in the CNS of children. Clinical trials affirm that the physicochemical properties of a drug remain one of the most important factors dictating penetration of antimicrobial agents into the CNS, irrespective of the population being treated (i.e. small, lipophilic drugs with low protein binding exhibit the best translocation across the BBB). These same physicochemical characteristics determine the primary disposition pathways of the drug, and by extension the magnitude and duration of circulating drug concentrations in the plasma, a second major driving force behind achievable CNS drug concentrations. Notably, these disposition pathways can be expected to change during the normal process of growth and development. Finally, CNS drug penetration is influenced by the nature and extent of the infection (i.e. the presence of meningeal inflammation). Aminoglycosides have poor CNS penetration when administered intravenously. Intrathecal gentamicin has been studied in children with more promising results, often exceeding the minimum inhibitory concentration. There are very limited data with intrathecal tobramycin in children. However, in the few patients that have been studied, the CSF concentrations were highly variable. Penicillins generally have good CNS penetration. Aqueous penicillin G reaches greater concentrations than procaine or benzathine penicillin. Concentrations remain detectable for ≥ 12 h. Of the aminopenicillins, both ampicillin and parenteral amoxicillin reach adequate CNS concentrations; however, orally administered amoxicillin resulted in much lower concentrations. Nafcillin and piperacillin are the final two penicillins with pediatric data: their penetration is erratic at best. Cephalosporins vary greatly in regard to their CSF penetration. Few first- and second-generation cephalosporins are able to reach higher CSF concentrations. Cefuroxime is the only exception and is usually avoided due to its adverse effects and slower sterilization of the CSF than third-generation agents. Ceftriaxone, cefotaxime, ceftazidime, cefixime and cefepime have been studied in children and are all able to adequately penetrate the CSF. As with penicillins, concentrations are greatest in the presence of meningeal inflammation. Meropenem and imipenem are the only carbapenems with pediatric data. Imipenem reaches higher CSF concentrations; however, meropenem is preferred due to its lower incidence of seizures. Aztreonam has also demonstrated favorable penetration but only one study has been completed in children. Both chloramphenicol and sulfamethoxazole/trimethoprim (cotrimoxazole) penetrate into the CNS well; however, significant toxicities limit their use. The small size and minimal protein binding of fosfomycin contribute to its favorable CNS PK. Although rarely used, it achieves higher concentrations in the presence of inflammation and accumulation is possible. Linezolid reaches high CSF concentrations; however, more frequent dosing might be required in infants due to their increased elimination. Metronidazole also has very limited information but it demonstrated favorable results similar to adult data; CSF concentrations even exceeded plasma concentrations at certain time points. Rifampin (rifampicin) demonstrated good CNS penetration after oral administration. Vancomycin demonstrates poor CNS penetration after intravenous administration. When combined with intraventricular therapy, CNS concentrations are much greater. Of the antituberculosis agents, isoniazid, pyrazinamide and streptomycin have been studied in children. Isoniazid and pyrazinamide have favorable CSF penetration. Streptomycin appears to produce unpredictable CSF levels. No pediatric-specific data are available for clindamycin, daptomycin, macrolides, tetracyclines, and fluoroquinolones. Daptomycin, fluoroquinolones, and tetracyclines have demonstrated favorable CNS penetration in adults; however, data are limited due to their potential pediatric-specific toxicities and newness within the marketplace. Macrolides and clindamycin have demonstrated poor CNS penetration in adults and thus have not been studied in pediatrics.