Effects of Toll-like receptor 2 agonist Pam(3)CysSK(4) on inflammation and brain damage in experimental pneumococcal meningitis

TLR2 signaling participates in the pathogenesis of pneumococcal meningitis. In infant rats, the TLR2 agonist Pam(3)CysSK(4) was applied intracisternally (0.5 microg in 10 microl saline) alone or after induction of pneumococcal meningitis to investigate the effect of TLR2 activation on cerebrospinal fluid (CSF) inflammation and hippocampal apoptosis. A dose effect of Pam(3)CysSK(4) on apoptosis was investigated by intracisternal application of 0.5 microg in 10 microl saline and 40 microg in 20 microl saline. Pam(3)CysSK(4) neither induced apoptosis in sham-operated mice nor aggravated apoptosis in acute infection. However, Pam(3)CysSK(4) induced pleocytosis, TNF-alpha and MMP-9 in CSF in sham-infection but not during acute meningitis. We conclude that TLR2 signaling triggered by Pam(3)CysSK(4) at a dosage capable to induce a neuroinflammatory response does not induce hippocampal apoptosis in the infant rat model of experimental pneumococcal meningitis.

Distribution of radiodense contrast medium after perineural injection of the palmar and palmar metacarpal nerves (low 4-point nerve block): an in vivo and ex vivo study in horses

REASONS FOR PERFORMING STUDY: Evidence-based information is limited on distribution of local anaesthetic solution following perineural analgesia of the palmar (Pa) and palmar metacarpal (PaM) nerves in the distal aspect of the metacarpal (Mc) region ('low 4-point nerve block'). OBJECTIVES: To demonstrate the potential distribution of local anaesthetic solution after a low 4-point nerve block using a radiographic contrast model. METHODS: A radiodense contrast medium was injected subcutaneously over the medial or the lateral Pa nerve at the junction of the proximal three-quarters and distal quarter of the Mc region (Pa injection) and over the ipsilateral PaM nerve immediately distal to the distal aspect of the second or fourth Mc bones (PaM injection) in both forelimbs of 10 mature horses free from lameness. Radiographs were obtained 0, 10 and 20 min after injection and analysed subjectively and objectively. Methylene blue and a radiodense contrast medium were injected in 20 cadaver limbs using the same techniques. Radiographs were obtained and the limbs dissected. RESULTS: After 31/40 (77.5%) Pa injections, the pattern of the contrast medium suggested distribution in the neurovascular bundle. There was significant proximal diffusion with time, but the main contrast medium patch never progressed proximal to the mid-Mc region. The radiological appearance of 2 limbs suggested that contrast medium was present in the digital flexor tendon sheath (DFTS). After PaM injections, the contrast medium was distributed diffusely around the injection site in the majority of the limbs. In cadaver limbs, after Pa injections, the contrast medium and the dye were distributed in the neurovascular bundle in 8/20 (40%) limbs and in the DFTS in 6/20 (30%) of limbs. After PaM injections, the contrast and dye were distributed diffusely around the injection site in 9/20 (45%) limbs and showed diffuse and tubular distribution in 11/20 (55%) limbs. CONCLUSIONS AND POTENTIAL RELEVANCE: Proximal diffusion of local anaesthetic solution after a low 4-point nerve block is unlikely to be responsible for decreasing lameness caused by pain in the proximal Mc region. The DFTS may be penetrated inadvertently when performing a low 4-point nerve block.

Lead discovery, chemistry optimization, and biological evaluation studies of novel biamide derivatives as CB2 receptor inverse agonists and osteoclast inhibitors

N,N'-((4-(Dimethylamino)phenyl)methylene)bis(2-phenylacetamide) was discovered by using 3D pharmacophore database searches and was biologically confirmed as a new class of CB(2) inverse agonists. Subsequently, 52 derivatives were designed and synthesized through lead chemistry optimization by modifying the rings A-C and the core structure in further SAR studies. Five compounds were developed and also confirmed as CB(2) inverse agonists with the highest CB(2) binding affinity (CB(2)K(i) of 22-85 nM, EC(50) of 4-28 nM) and best selectivity (CB(1)/CB(2) of 235- to 909-fold). Furthermore, osteoclastogenesis bioassay indicated that PAM compounds showed great inhibition of osteoclast formation. Especially, compound 26 showed 72% inhibition activity even at the low concentration of 0.1 μM. The cytotoxicity assay suggested that the inhibition of PAM compounds on osteoclastogenesis did not result from its cytotoxicity. Therefore, these PAM derivatives could be used as potential leads for the development of a new type of antiosteoporosis agent.

Prostaglandin E1 testing in heart failure-associated pulmonary hypertension enables transplantation: the PROPHET study

BACKGROUND: Elevated pulmonary vascular resistance (PVR) is relevant to prognosis of congestive heart failure and heart transplantation. Proof of reversibility by pharmacologic testing in potential transplantation candidates is important because it indicates a reduced probability of right ventricular failure or death in the early post-transplant period. This study aimed to clarify the possible extent of acute reversibility of elevated PVR in a large, consecutive cohort of heart transplant candidates. METHODS: This study included 208 consecutive patients (age 52 +/- 10 years, 89% men and 11% women, ejection fraction 21 +/- 9%, Vo2max 12.6 +/- 4.2 ml/kg/min) being evaluated for heart transplantation in 7 transplant centers in Germany and Switzerland. Testing was performed with increasing intravenous doses of prostaglandin E1 (PGE1; average maximum dose 173 +/- 115 ng/kg/min for at least 10 minutes) in 92 patients exhibiting a baseline PVR of > 2.5 Wood units (WU) and/or a transpulmonary gradient (TPG) of > 12 mm Hg. RESULTS: PGE1 testing lowered PVR from 4.1 +/- 2.0 to 2.1 +/- 1.1 WU (p < 0.01), increased cardiac output from 3.8 +/- 1.0 to 5.0 +/- 1.5 liters/min (p < 0.01), and decreased TPG from 14 +/- 4 to 10 +/- 3 mm Hg (p < 0.01), mean pulmonary artery pressure (PAM) from 39 +/- 9 to 29 +/- 9 mm Hg (p < 0.01) and mean pulmonary capillary wedge pressure (PCWP) from 24 +/- 7 to 19 +/- 9 mm Hg (p < 0.01). Mean aortic pressure (MAP) decreased to 85% and systemic vascular resistance (SVR) to 65% of baseline values (p < 0.01). Symptomatic systemic hypotension was not observed. For the whole population the percentage of patients with PVR > 2.5 WU was reduced from 44.2% to 10.5% with PGE1. PVR decreased in each patient; only 2 patients (1%) remained ineligible for listing because of a final PVR of > 4.0 WU. TPG, ejection fraction and male gender were independent predictors of reversibility of PVR. CONCLUSIONS: Elevated PVR in heart transplant candidates is highly reversible and can be normalized during acute pharmacologic testing with PGE1.

TLR expression on neutrophils at the pulmonary site of infection: TLR1/TLR2-mediated up-regulation of TLR5 expression in cystic fibrosis lung disease

Cystic fibrosis (CF) lung disease is characterized by infection with Pseudomonas aeruginosa and a sustained accumulation of neutrophils. In this study, we analyzed 1) the expression of MyD88-dependent TLRs on circulating and airway neutrophils in P. aeruginosa-infected CF patients, P. aeruginosa-infected non-CF bronchiectasis patients, and noninfected healthy control subjects and 2) studied the regulation of TLR expression and functionality on neutrophils in vitro. TLR2, TLR4, TLR5, and TLR9 expression was increased on airway neutrophils compared with circulating neutrophils in CF and bronchiectasis patients. On airway neutrophils, TLR5 was the only TLR that was significantly higher expressed in CF patients compared with bronchiectasis patients and healthy controls. Studies using confocal microscopy and flow cytometry revealed that TLR5 was stored intracellularly in neutrophils and was mobilized to the cell surface in a protein synthesis-independent manner through protein kinase C activation or after stimulation with TLR ligands and cytokines characteristic of the CF airway microenvironment. The most potent stimulator of TLR5 expression was the bacterial lipoprotein Pam(3)CSK(4). Ab-blocking experiments revealed that the effect of Pam(3)CSK(4) was mediated through cooperation of TLR1 and TLR2 signaling. TLR5 activation enhanced the phagocytic capacity and the respiratory burst activity of neutrophils, which was mediated, at least partially, via a stimulation of IL-8 production and CXCR1 signaling. This study demonstrates a novel mechanism of TLR regulation in neutrophils and suggests a critical role for TLR5 in neutrophil-P. aeruginosa interactions in CF lung disease.

Experimental heart transplantation: effect of cyclosporine on expression and activity of metzincins

Metzincins, such as matrix metalloproteases (MMP), and extracellular matrix (ECM) proteins are differentially regulated in inflammation. We hypothesised that metzincins are also dysregulated in experimental acute cardiac allograft rejection. We investigated the Dark Agouti-to-Lewis (DA-to-Lew) rat model of acute cardiac allograft rejection. Cyclosporine (CsA) (7.5 mg/kg/d) was given from transplantation to sacrifice (day +5). At that time, mRNA levels were analysed by Affymetrix genechip and quantitative reverse transcription polymerase chain reaction (qRTPCR). MMP protein and activities were analysed by immunohistology, fluorometry, zymography and Western blots. In untreated rejected DA allografts, mRNA levels of MMP-2/-7/-9/-/12-/14, a disintegrin and metalloprotease (ADAM)-17, tissue inhibitor of metalloprotease (TIMP)-1/-3 were increased, whereas MMP-11/-16/-24 and TIMP-2/-4 were lowered compared to native DA hearts. With respect to these untreated allografts, CsA lowered mRNA levels of MMP-7, TIMP-1/-3 (TIMP-2/-4 remained relatively low) and ADAM17, but augmented mRNA levels of MMP-11/-16/-23 and of many ECM genes. Immunohistology showed increased staining of MMP-2 in acute rejection (AR). Overall MMP activity was augmented in both transplanted groups, but CsA reduced MMP-9 activity and MMP-14 production. Taken together, MMP and TIMP were upregulated during acute AR. CsA ameliorated histology of rejection but showed potential pro-fibrotic effects. Thus, MMP and TIMP may play a role in acute cardiac allograft rejection, and beneficial modification of the MMP-ECM balance requires interventions beyond CsA.

Strengthening the Reporting of Molecular Epidemiology for Infectious Diseases (STROME-ID): an extension of the STROBE statement

Molecular data are now widely used in epidemiological studies to investigate the transmission, distribution, biology, and diversity of pathogens. Our objective was to establish recommendations to support good scientific reporting of molecular epidemiological studies to encourage authors to consider specific threats to valid inference. The statement Strengthening the Reporting of Molecular Epidemiology for Infectious Diseases (STROME-ID) builds upon the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) initiative. The STROME-ID statement was developed by a working group of epidemiologists, statisticians, bioinformaticians, virologists, and microbiologists with expertise in control of infection and communicable diseases. The statement focuses on issues relating to the reporting of epidemiological studies of infectious diseases using molecular data that were not addressed by STROBE. STROME-ID addresses terminology, measures of genetic diversity within pathogen populations, laboratory methods, sample collection, use of molecular markers, molecular clocks, timeframe, multiple-strain infections, non-independence of infectious-disease data, missing data, ascertainment bias, consistency between molecular and epidemiological data, and ethical considerations with respect to infectious-disease research. In total, 20 items were added to the 22 item STROBE checklist. When used, the STROME-ID recommendations should advance the quality and transparency of scientific reporting, with clear benefits for evidence reviews and health-policy decision making.

Organotypic slice cultures from rat brain tissue: a new approach for Naegleria fowleri CNS infection in vitro

The free-living amoeba Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM), a disease leading to death in the vast majority of cases. In patients suffering from PAM, and in corresponding animal models, the brain undergoes a massive inflammatory response, followed by haemorrhage and severe tissue necrosis. Both, in vivo and in vitro models are currently being used to study PAM infection. However, animal models may pose ethical issues, are dependent upon availability of specific infrastructural facilities, and are time-consuming and costly. Conversely, cell cultures lack the complex organ-specific morphology found in vivo, and thus, findings obtained in vitro do not necessarily reflect the situation in vivo. The present study reports infection of organotypic slice cultures from rat brain with N. fowleri and compares the findings in this culture system with in vivo infection in a rat model of PAM, that proved complementary to that of mice. We found that brain morphology, as present in vivo, is well retained in organotypic slice cultures, and that infection time-course including tissue damage parallels the observations in vivo in the rat. Therefore, organotypic slice cultures from rat brain offer a new in vitro approach to study N. fowleri infection in the context of PAM.

Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri

Species in the genus Naegleria are free-living amoebae of the soil and warm fresh water. Although around 30 species have been recognized, Naegleria fowleri is the only one that causes primary amoebic meningoencephalitis (PAM) in humans. PAM is an acute and fast progressing disease affecting the central nervous system. Most of the patients die within 1-2 weeks of exposure to the infectious water source. The fact that N. fowleri causes such fast progressing and highly lethal infections has opened many questions regarding the relevant pathogenicity factors of the amoeba. In order to investigate the pathogenesis of N. fowleri under defined experimental conditions, we developed a novel high- versus low-pathogenicity model for this pathogen. We showed that the composition of the axenic growth media influenced growth behaviour and morphology, as well as in vitro cytotoxicity and in vivo pathogenicity of N. fowleri. Trophozoites maintained in Nelson's medium were highly pathogenic for mice, demonstrated rapid in vitro proliferation, characteristic expression of surface membrane vesicles and a small cell diameter, and killed target mouse fibroblasts by both contact-dependent and -independent destruction. In contrast, N. fowleri cultured in PYNFH medium exhibited a low pathogenicity, slower growth, increased cell size and contact-dependent target cell destruction. However, cultivation of the amoeba in PYNFH medium supplemented with liver hydrolysate (LH) resulted in trophozoites that were highly pathogenic in mice, and demonstrated an intermediate proliferation rate in vitro, diminished cell diameter and contact-dependent target cell destruction. Thus, in this model, the presence of LH resulted in increased proliferation of trophozoites in vitro and enhanced pathogenicity of N. fowleri in mice. However, neither in vitro cytotoxicity mechanisms nor the presence of membrane vesicles on the surface correlated with the pathologic potential of the amoeba. This indicated that the pathogenicity of N. fowleri remains a complex interaction between as-yet-unidentified cellular mechanisms.

Genome-wide identification of pathogenicity factors of the free-living amoeba Naegleria fowleri.

BACKGROUND The free-living amoeba Naegleria fowleri is the causative agent of the rapidly progressing and typically fatal primary amoebic meningoencephalitis (PAM) in humans. Despite the devastating nature of this disease, which results in > 97% mortality, knowledge of the pathogenic mechanisms of the amoeba is incomplete. This work presents a comparative proteomic approach based on an experimental model in which the pathogenic potential of N. fowleri trophozoites is influenced by the compositions of different media. RESULTS As a scaffold for proteomic analysis, we sequenced the genome and transcriptome of N. fowleri. Since the sequence similarity of the recently published genome of Naegleria gruberi was far lower than the close taxonomic relationship of these species would suggest, a de novo sequencing approach was chosen. After excluding cell regulatory mechanisms originating from different media compositions, we identified 22 proteins with a potential role in the pathogenesis of PAM. Functional annotation of these proteins revealed, that the membrane is the major location where the amoeba exerts its pathogenic potential, possibly involving actin-dependent processes such as intracellular trafficking via vesicles. CONCLUSION This study describes for the first time the 30 Mb-genome and the transcriptome sequence of N. fowleri and provides the basis for the further definition of effective intervention strategies against the rare but highly fatal form of amoebic meningoencephalitis.

A conserved role for p48 homologs in protecting dopaminergic neurons from oxidative stress.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by the progressive loss of dopaminergic (DA) neurons. Both environmental and genetic factors are thought to contribute to the pathogenesis of PD. Although several genes linked to rare familial PD have been identified, endogenous risk factors for sporadic PD, which account for the majority of PD cases, remain largely unknown. Genome-wide association studies have identified many single nucleotide polymorphisms associated with sporadic PD in neurodevelopmental genes including the transcription factor p48/ptf1a. Here we investigate whether p48 plays a role in the survival of DA neurons in Drosophila melanogaster and Caenorhabditis elegans. We show that a Drosophila p48 homolog, 48-related-2 (Fer2), is expressed in and required for the development and survival of DA neurons in the protocerebral anterior medial (PAM) cluster. Loss of Fer2 expression in adulthood causes progressive PAM neuron degeneration in aging flies along with mitochondrial dysfunction and elevated reactive oxygen species (ROS) production, leading to the progressive locomotor deficits. The oxidative stress challenge upregulates Fer2 expression and exacerbates the PAM neuron degeneration in Fer2 loss-of-function mutants. hlh-13, the worm homolog of p48, is also expressed in DA neurons. Unlike the fly counterpart, hlh-13 loss-of-function does not impair development or survival of DA neurons under normal growth conditions. Yet, similar to Fer2, hlh-13 expression is upregulated upon an acute oxidative challenge and is required for the survival of DA neurons under oxidative stress in adult worms. Taken together, our results indicate that p48 homologs share a role in protecting DA neurons from oxidative stress and degeneration, and suggest that loss-of-function of p48 homologs in flies and worms provides novel tools to study gene-environmental interactions affecting DA neuron survival.

New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli.

Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

Targeting the PI3K/AKT/mTOR signaling pathway in medulloblastoma

Medulloblastoma is the most common malignant childhood brain tumor and is associated with a poor outcome. There is an urgent need to develop novel targeted therapeutic approaches for medulloblastoma, which will arise from an enhanced understanding of the disease at the molecular level. Medulloblastoma has been recognized to be a heterogeneous disease, and no recurrent cancer gene mutations have been found, although many of the mutations described so far affect key intracellular signaling pathways, such as sonic hedgehog (SHH) and Wnt/β-catenin. The PI3K/AKT/mTOR (PAM) signaling pathway controls key cellular responses, such as cell growth and proliferation, survival, migration and metabolism. Over the last decades, it has been recognized that this intracellular signaling pathway is frequently activated by genetic and epigenetic alterations in malignant brain tumors, including medulloblastoma. Clinical trials have started to evaluate the safety and efficacy of agents targeting this pathway in malignant brain tumors. Due to the complexity of the PAM signaling pathway, there remain significant difficulties in the development of novel therapeutic approaches. The future challenges in developing effective treatments for cancer patients include the development of predictive biomarkers and combinatorial approaches to effectively target multiple signal transduction pathways. In this review article, we will summarize the current knowledge about the role of PAM signaling in medulloblastoma and discuss the strategies that are currently being evaluated with targeted agents against this pathway.