Post-immunobiotics increase resistance to primary respiratory syncytial virus infection and secondary pneumococcal pneumonia.

Previously, we demonstrated that post-immunobiotics derived from Lactobacillus gasseri TMT36, TMT39, and TMT40 strains (HK36, HK39 and HK40, respectively) differentially regulated Toll-like receptor 3 (TLR3)-mediated antiviral respiratory immunity in infant mice. In this work, we investigated whether the HK36, HK39 and HK40 nasal treatments were able to improve the resistance against primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. Our results demonstrated that the three treatments increased the resistance to primary viral infection by reducing variations in body weight, RSV titers and lung damage of infected infant mice. Post-immunobiotics significantly enhanced the expressions of interferon (IFN)-λ, IFN-ß, IFN-γ, interleukin(IL) - 1ß, IL-6, IL-27, Mx1, RNAseL and 2'-5'-oligoadenylate synthetase 1 (OAS1) genes and decreased tumour necrosis factor (TNF)-α in alveolar macrophages of RSV-challenged mice. In addition, the studies in the model of RSV-Streptococcus pneumoniae superinfection showed that the HK39 and HK40 treatments were capable of reducing lung damage, lung bacterial cell counts, and the dissemination of S. pneumoniae into the blood of infant mice. The protective effect was associated with increases in IFN-ß, IFN-γ, IL-10, and IL-27 in the respiratory tract. This study demonstrates that the nasal application of the post-immunobiotics HK39 and HK40 stimulates innate respiratory immunity and enhances the defences against primary RSV infection and secondary pneumococcal pneumonia offering an alternative to combat respiratory superinfections in children, which can be fatal.

Soymilk-fermented with Lactobacillus delbrueckii subsp. delbrueckii TUA4408L improves immune-health in pigs.

Lactobacillus delbrueckii subsp. delbrueckii TUA4408L has the ability to grow and ferment soymilk and is able to modulate the innate immune response of intestinal epithelial cells in vitro. These two properties prompt us to evaluate whether the soymilk fermented with the TUA4408L strain can induce beneficial immunomodulatory effects in vivo. For this purpose, pigs were selected as a preclinical model. The studies performed here demonstrated that the L. delbrueckii subsp. delbrueckii TUA4408L-fermented soymilk (TUA4408L FSM) reduced blood markers of inflammation and differentially regulated the expression of inflammatory and regulatory cytokines in the intestinal mucosa. These immunological changes induced by the TUA4408L FSM were associated to an enhanced resistance to pathogenic Escherichia coli and an improved grow performance and meat quality of pigs. The experiments and analysis in our study indicate that the immunobiotic TUA4408L FSM could be an interesting non-dairy functional food to beneficially modulate the intestinal immune system, improve protection against pathogens and reduce inflammatory damage. The preclinical study carried out here in pigs could have a better correlation in humans, compared to a rodent model. However, the clinical relevance of these findings still needs to be confirmed by further research, for example, in controlled human challenge studies.

Effect of chronic kidney disease on excessive daytime sleepiness in Parkinson disease.

BACKGROUND AND PURPOSES: Excessive daytime sleepiness (EDS) is a common sleep disorder in patients with Parkinson disease (PD). Non-ergot dopamine agonists increase the risk of unanticipated sleep episodes. OBJECTIVE: We aimed to assess the influence of renal function on EDS in patients with PD. METHODS: Sixty-two patients treated with ropinirole or pramipexole were recruited for this study. We evaluated the historical and clinical characteristics including the motor symptom rating scales, Epworth Sleepiness Scale (ESS), and estimated glomerular filtration rate (eGFR). An ESS score of 10 or greater was defined as EDS. Participants with eGFR < 60 ml/min/1.73 m(2) were determined to have chronic kidney disease (CKD). Multiple logistic regression analysis was performed to determine the predictive factors of EDS. RESULTS: Chronic kidney disease was found to be a significant predictive factor for EDS in all patients (P = 0.014). We observed a negative correlation between the severity of daytime sleepiness and renal function in patients treated with pramipexole alone (r(s) = -0.637, P < 0.001). CONCLUSIONS: Chronic kidney disease may be a risk factor for EDS, especially in patients treated with pramipexole, which is directly excreted in the urine.

Onecut transcription factor OC2 is a direct target of T-bet in type-1 T-helper cells.

T-box transcription factor, T-bet, has a central role in the differentiation of T-helper (Th) progenitor cells to Th1 or Th2 effector cells, partly by regulating the expression of genes such as interferon-gamma (IFN-gamma). However, the direct target genes, especially those mediating the transcriptional network initiated by T-bet, are not yet fully understood. By combining chromatin immunoprecipitation from Th1 cells with human cytosine-phosphate-guanine-island array analysis, Onecut 2 (OC2), which encodes a member of the ONECUT class of transcriptional activators, was identified as a direct target gene of T-bet. OC2 is expressed in Th1 but not Th2 cells and reporter assays showed that T-bet transactivates OC2 transcription through putative T-bet half-sites locating -451 to -347 of OC2 promoter region. Moreover, we found that OC2 binds and transactivates human T-bet promoter. These results suggest that not only cell-extrinsic regulation via the IFN-gamma/STAT1 pathway, but also cell-intrinsic transcriptional positive feedback loop between T-bet and OC2 could be involved in Th1 development.

Overexpression, purification and characterization of RecJ protein from Thermus thermophilus HB8 and its core domain.

A recJ homolog was cloned from the extremely thermophilic bacterium Thermus themophilus HB8. It encodes a 527 amino acid protein that has 33% identity to Escherichia coli RecJ protein and includes the characteristic motifs conserved among RecJ homologs. Although T.thermophilus RecJ protein (ttRecJ) was expressed as an inclusion body, it was purified in soluble form through denaturation with urea and subsequent refolding steps. Limited proteolysis showed that ttRecJ has a protease-resistant core domain, which includes all the conserved motifs. We constructed a truncated ttRecJ gene that corresponds to the core domain (cd-ttRecJ). cd-ttRecJ was overexpressed in soluble form and purified. ttRecJ and cd-ttRecJ were stable up to 60 degrees C. Size exclusion chromatography indicated that ttRecJ exists in several oligomeric states, whereas cd-ttRecJ is monomeric in solution. Both proteins have 5'-->3' exonuclease activity, which was enhanced by increasing the temperature to 50 degrees C. Mg(2+), Mn(2+) or Co(2+) ions were required to activate both proteins, whereas Ca(2+) and Zn(2+) had no effects.

Crystal structure of Escherichia coli Fdx, an adrenodoxin-type ferredoxin involved in the assembly of iron-sulfur clusters.

Escherichia coli ferredoxin (Fdx) is an adrenodoxin-type [2Fe-2S] ferredoxin. Recent genetic analyses show that it has an essential role in the maturation of various iron-sulfur (Fe-S) proteins. Fdx probably functions as a component of the complex machinery responsible for the biogenesis of Fe-S clusters. Its crystal structure was determined by the multiple-wavelength anomalous dispersion method using the iron atoms in the [2Fe-2S] cluster of the protein and then refined to R and R(free) values of 0.255 and 0.278, respectively, at 1.7 A resolution. The structure of Fdx is similar to the structures of bovine adrenodoxin (Adx) and Pseudomonas putida putidaredoxin (Pdx) whose respective root-mean-square deviations of the corresponding Calpha atoms are 1.8 and 2.2 A. This analysis also revealed the structure of the C-terminal residues protruding into the solvent, which is missing in Adx and Pdx. The [2Fe-2S] cluster is located at the edge of the molecule and bonds with the Sgamma atoms of Cys42, Cys48, Cys51, and Cys87. Electrostatic potential analysis showed that the surface of Fdx has two negatively charged areas separated by a hydrophobic lane. One is conserved on the surface of Adx which is an area of interaction with adrenodoxin reductase. Cys46 is located on the molecular surface in the vicinity of the [2Fe-2S] cluster, an indication that it may be involved in Fe-S cluster formation.

Adoptive immunotherapy for malignant brain tumors using human peripheral blood mononuclear cells activated by the Streptococcal preparation OK-432.

Adoptive immunotherapy using OK-432-activated mononuclear cells (OK-MCs) offers cell-mediated and cytokine-mediated pathways for antitumor activity. The effectiveness of direct intratumoral administration of OK-MCs via a catheter/reservoir system was studied in patients with malignant brain tumors. Seventeen patients, 12 with malignant glioma, four with metastatic adenocarcinoma, and one with primary sarcoma of the brain, were treated by OK-MC therapy (1.0 to 11.2 x 10(7) cells/person) between June 1989 and April 1999. The OK-MC therapy was given to patients with tumors progressing despite previous cytoreductive surgery, radiation, or chemotherapy. Adverse effects seen after the therapy were fever in 10 patients, seizure in two patients, and hypotension in one patient. Evaluation by computed tomography or magnetic resonance imaging revealed that seven patients showed no change including three with minor response, and 10 showed progressive disease. Adoptive immunotherapy using OK-MC was safe and well tolerated, but the therapeutic potential is limited.

Expression of prostaglandin H synthase-2 in human brain tumors.

Numerous studies have demonstrated that prostaglandin H synthase-2 (PHS-2) is involved in gastrointestinal carcinogenesis, and that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit PHS, can reduce the risk of colon cancer. In brain tumors, elevated prostaglandin production and its correlation to anaplastic grade of gliomas have been demonstrated. To determine whether the increased prostaglandin production is due to enhanced expression of PHS-2 and whether the up-regulation of PHS-2 has any correlation to histopathological findings in brain tumors, we evaluated the profile of PHS expression in several human glioma cell lines and surgical specimens from patients with various types of brain tumors. In glioma cell lines, five out of six cell lines showed constitutive expression of PHS-2, whereas PHS-1 was weakly expressed in all of them. All surgical specimens, except an ependymoma, which expressed both isozymes equally, expressed PHS-2 mRNA predominantly. Immunohistochemistry of various types of brain tumors, including six glioblastomas, nine astrocytomas, six meningiomas, five medulloblastomas, four craniopharyngiomas, three ependymomas, three neurinomas, two oligodendrogliomas, two malignant lymphomas, two dysembryoplastic neuroepitherial tumors and one metastatic brain tumor showed PHS-2 staining in most cases. In gliomas, astrocytomas (grade 2 and 3) were strongly stained, but the staining intensity of glioblastomas was relatively weak. Meningiomas and a metastatic brain tumor were also strongly stained. Our data thus suggest that most brain tumors express PHS-2, which may also play a role in tumorigenesis in the brain.

Detection of simian virus 40 DNA sequence in human primary glioblastomas multiforme.

OBJECT: Deoxyribonucleic acid oncoviruses can induce neoplastic transformation of cells because their viral proteins interfere with antiproliferative cellular proteins. Simian virus 40 (SV40) is a DNA virus that induces the emergence of ependymomas, choroid plexus tumors, mesotheliomas, osteosarcomas, sarcomas, and various tumors when injected into newborn hamsters. Recently, approximately 60% of human ependymomas, choroid plexus tumors, and mesotheliomas were reported to contain and express SV40 DNA sequences. In this study the presence of SV40 DNA sequences was investigated in human brain tumors. METHODS: Three of 32 glioblastomas mutiforme (GBMs), but none of two ependymomas and five medulloblastomas, were found to possess SV40 DNA sequences when examined using polymerase chain reaction (PCR). The DNA sequence analysis of PCR-amplified fragments disclosed that the samples were identical to the regulatory region of SV40. All three GBMs, which arose in elderly patients with wild-type p53, were considered to be primary (de novo) tumors. Although each of the three tumors was immunohistochemically negative for SV40 T antigen, in situ hybridization successfully demonstrated the messenger RNA for SV40 T antigen. CONCLUSIONS: The results of this study indicate that latent infection of SV40 in elderly people may be implicated in the tumorigenesis of certain primary GBMs.

Primary structure and phylogenetic analysis of the coat protein of a Toyama isolate of tobacco necrosis virus.

The amino acid sequence of the coat protein (CP) of a tobacco necrosis virus (TNV) strain, Toyama isolate, was determined by a combination of peptide and cDNA sequencing. The deduced sequence of 276 residues was compared with CPs of other TNV isolates and other plant virus isolates of Tombusviridae. It showed the highest similarity to the TNV Nebraska isolate with 92% identity and moderate similarity to the TNV strain A with 51% identity, confirming the previous serological analysis. It also showed overall similarity with CPs of mostly genera Necrovirus and Sobemovirus, and partial similarity with CPs of genera Tombusvirus and Carmovirus. Among 13 CPs that showed overall similarity, there were 10 completely conserved residues. These included three residues that participate in Ca2+ ligation at the interfaces of virion subunits in TNV crystal structure, suggesting that similar metal binding occur in the viruses of genera Necrovirus and Sobemovirus.

Apoptosis induced by ischemia-reperfusion and fasting in gastric mucosa compared to small intestinal mucosa in rats.

The effect of ischemia-reperfusion and 48-hr fasting on apoptosis was characterized in rat gastric mucosa and compared to small intestinal mucosa. Under halothane anesthesia, the celiac artery or superior mesenteric artery in the rat was occluded for 60 min followed by reperfusion. Occlusion of the celiac artery reduced blood flow in the stomach and occlusion of the mesenteric artery reduced blood flow in the small intestine. Additional rats were fasted for 48 hr to evaluate the effect of fasting on mucosal apoptosis. The ratios of fragmented DNA to total DNA, electrophoresis, and immunohistochemical staining were examined after ischemia-reperfusion or fasting. Apoptosis was not induced significantly in the gastric mucosa after ischemia-reperfusion, although it increased dramatically in the intestinal mucosa after ischemia-reperfusion. Further, after 48 fasting, apoptosis was induced in the small intestine, but not in the stomach. These results indicate that rat gastric mucosa is not as sensitive as small intestinal mucosa to ischemia-reperfusion or fasting-induced apoptosis.

[Simple antireflux measures for management of gastroesophageal reflux disease].

It is widely accepted that proton pump inhibitors are the most effective therapy for management of gastroesophageal reflux disease. H2-receptor antagonists, prokinetic agents, and antacids are used in treatment of gastroesophageal reflux disease. In addition to these medical treatments, beneficial effects of simple anti-reflux measures on management of gastroesophageal disease are evaluated. Simple anti-reflux measures include elevation of the head of the bed, weight loss, reduced food intake, and the avoidance of precipitating foods and drugs. In this chapter, we summarized the utility of these simple anti-reflux measures for management of gastroesophageal reflux disease.

[Clinical study of cerebral blood flow in bilateral chronic subdural hematoma measured by 99mTc-HMPAO SPECT].

Cerebral blood flow(CBF) in 34 patients with bilateral chronic subdural hematoma was measured by 99mTc-HMPAO SPECT before operation. The regional CBF was measured in 26 regions of the 10 cortical regions, putamen, thalamus and cerebellar hemisphere on both sides. According to the thickness of subdural hematoma, the thicker hematoma side was measured and examined as the thick hematoma side, and the other side as the thin hematoma side. Thirty four cases with bilateral chronic subdural hematoma were classified into four groups on the basis of clinical symptoms: 13 cases with headache(headache group), 10 cases with hemiparesis(hemiparesis group), 5 cases with tetraparesis(tetraparesis group) and 6 cases with consciousness disturbance or dementia(consciousness disturbance group), and into two groups according to the degree of midline brain shift on MRI: 14 cases of non-shifted group and 20 cases of shifted group. The average CBF of 34 patients in each region indicated a regional CBF reduction in the frontal, parietal and occipital cortices on the thin hematoma side, and in the putamen on the thick hematoma side. In the headache group, the regional CBF reduction on the thin hematoma side was found in the frontal, parietal and occipital cortices compared with the corresponding regions on the thick hematoma side, and in thalamus on the thick hematoma side. In the hemiparesis and tetraparesis groups, there was no statistically significant CBF reduction between the thick and thin hematoma sides. In the consciousness disturbance group, the CBF reduction in whole brain was remarkably significant. By the degree of the midline brain shift, the CBF reductions between the thick and thin hematoma sides were observed. Namely, in the shifted group, the CBF reductions were noted in the frontal, parietal and occipital cortices in the thin hematoma side, and in the putamen in the thick hematoma side. We concluded that the CBF reduction of bilateral chronic subdural hematoma was bilaterally found in the hemiparesis and tetraparesis groups, and which was finally observed in whole brain in the consciousness disturbance group.

Direct binding of hydroxylamine to the heme iron of Arthromyces ramosus peroxidase. Substrate analogue that inhibits compound I formation in a competetive manner.

The interaction of hydroxylamine (HA) with Arthromyces ramosus peroxidase (ARP) was investigated by kinetic, spectroscopic, and x-ray crystallographic techniques. HA inhibited the reaction of native ARP with H(2)O(2) in a competitive manner. Electron absorption and resonance Raman spectroscopic studies indicated that pentacoordinate high spin species of native ARP are converted to hexacoordinate low spin species upon the addition of HA, strongly suggesting the occurrence of a direct interaction of HA with ARP heme iron. Kinetic analysis exhibited that the apparent dissociation constant is 6.2 mm at pH 7.0 and that only one HA molecule likely binds to the vicinity of the heme. pH dependence of HA binding suggested that the nitrogen atom of HA could be involved in the interaction with the heme iron. X-ray crystallographic analysis of ARP in complex with HA at 2.0 A resolution revealed that the electron density ascribed to HA is located in the distal pocket between the heme iron and the distal His(56). HA seems to directly interact with the heme iron but is too far away to interact with Arg(52). In HA, it is likely that the nitrogen atom is coordinated to the heme iron and that hydroxyl group is hydrogen bonded to the distal His(56).

Crystal structure of a repair enzyme of oxidatively damaged DNA, MutM (Fpg), from an extreme thermophile, Thermus thermophilus HB8.

The MutM [formamidopyrimidine DNA glycosylase (Fpg)] protein is a trifunctional DNA base excision repair enzyme that removes a wide range of oxidatively damaged bases (N-glycosylase activity) and cleaves both the 3'- and 5'-phosphodiester bonds of the resulting apurinic/apyrimidinic site (AP lyase activity). The crystal structure of MutM from an extreme thermophile, Thermus thermophilus HB8, was determined at 1.9 A resolution with multiwavelength anomalous diffraction phasing using the intrinsic Zn(2+) ion of the zinc finger. MutM is composed of two distinct and novel domains connected by a flexible hinge. There is a large, electrostatically positive cleft lined by highly conserved residues between the domains. On the basis of the three-dimensional structure and taking account of previous biochemical experiments, we propose a DNA-binding mode and reaction mechanism for MutM. The locations of the putative catalytic residues and the two DNA-binding motifs (the zinc finger and the helix-two-turns-helix motifs) suggest that the oxidized base is flipped out from double-stranded DNA in the binding mode and excised by a catalytic mechanism similar to that of bifunctional base excision repair enzymes.

Histaminergic effect on apoptosis of rat small intestinal mucosa after ischemia-reperfusion.

This study aimed to examine the relationship between a harmful effect of histamine and apoptosis following ischemia-reperfusion in the rat intestine. The superior mesenteric artery was occluded for 60 min followed by reperfusion for 60 min. Rats were infused with H1-receptor antagonist (chlorpheniramine maleate) or H2-receptor antagonist (cimetidine). Additional rats were pretreated with aminoguanidine (100 mg/kg). Percent apoptosis in the intestinal mucosa increased after reperfusion, but neither H1 nor H2 antagonists had any effect on apoptosis. Aminoguanidine pretreatment inhibited activity of diamine oxidase and increased the plasma histamine concentration. Aminoguanidine attenuated the increase in mucosal apoptosis following reperfusion. Apoptosis induced by an ischemic insult to the intestinal mucosa was not related to an undesirable effect of histamine. Attenuation of increased intestinal apoptosis might be due to increased plasma histamine level and/or other pharmacological action of aminoguanidine, including inhibition of inducible nitric oxide synthase.

Crystal structure of tobacco necrosis virus at 2.25 A resolution.

The crystal structure of tobacco necrosis virus (TNV) has been determined by real-space averaging with 5-fold non-crystallographic symmetry, and refined to R=25.3 % for diffraction data to 2.25 A resolution. A total of 180 subunits form a T=3 virus shell with a diameter of about 280 A and a small protrusion at the 5-fold axis. In 276 amino acid residues, the respective amino terminal 86, 87 and 56 residues of the A, B and C subunits are disordered. No density for the RNA was found. The subunits have a "jelly roll" beta-barrel structure, as have the structures of the subunits of other spherical viruses. The tertiary and quaternary structures of TNV are, in particular, similar to those of southern bean mosaic virus, although they are classified in different groups. Invisible residues 1 to 56 with a high level of basic residues are considered to be located inside the particle. Sequence comparison of the coat proteins of several TNV strains showed that the sequences of the disordered segment diverge considerably as compared with those of the ordered segment, consistent with a small tertiary structural constraint being imposed on the N-terminal segment. Basic residues are localized on the subunit interfaces or inner surface of the capsid. Positive charges of the basic residues facing the interior, as well as those of the N-terminal segment, may neutralize the negative charge of the RNA inside. Five calcium ions per icosahedral asymmetric unit are located at the subunit interfaces; three are close to the exterior surface, the other two away from it. The environments of the first three are similar, and those of the other two sites are similar. These calcium ions are assumed to be responsible for the stabilization/transition of the quaternary structure of the shell. Three peptide segments ordered only in the C subunits are clustered around each 3-fold (quasi-6-fold) axis forming a beta-annulus, and may lead to quasi-equivalent interactions for the organization of the T=3 shell.

Interaction of UvrA and UvrB proteins with a fluorescent single-stranded DNA. Implication for slow conformational change upon interaction of UvrB with DNA.

UvrA and UvrB proteins play key roles in the damage recognition step in the nucleotide excision repair. However, the molecular mechanism of damage recognition by these proteins is still not well understood. In this work we analyzed the interaction between single-stranded DNA (ssDNA) labeled with a fluorophore tetramethylrhodamine (TMR) and Thermus thermophilus HB8 UvrA (ttUvrA) and UvrB (ttUvrB) proteins. TMR-labeled ssDNA (TMR-ssDNA) as well as UV-irradiated ssDNA stimulated ATPase activity of ttUvrB more strongly than did normal ssDNA, indicating that this fluorescent ssDNA was recognized as damaged ssDNA. The addition of ttUvrA or ttUvrB enhanced the fluorescence intensity of TMR-ssDNA, and the intensity was much greater in the presence of ATP. Fluorescence titration indicated that ttUvrA has higher specificity for TMR-ssDNA than for normal ssDNA in the absence of ATP. The ttUvrB showed no specificity for TMR-ssDNA, but it took over 200 min for the fluorescence intensity of the ttUvrB-TMR-ssDNA complex to reach saturation in the presence of ATP. This time-dependent change could be separated into two phases. The first phase was rapid, whereas the second phase was slow and dependent on ATP hydrolysis. Time dependence of ATPase activity and fluorescence polarization suggested that changes other than the binding reaction occurred during the second phase. These results strongly suggest that ttUvrB binds ssDNA quickly and that a conformational change in ttUrvB-ssDNA complex occurs slowly. We also found that DNA containing a fluorophore as a lesion is useful for directly investigating the damage recognition by UvrA and UvrB.