The ketogenic diet influences taxonomic and functional composition of the gut microbiota in children with severe epilepsy.

The gut microbiota has been linked to various neurological disorders via the gut-brain axis. Diet influences the composition of the gut microbiota. The ketogenic diet (KD) is a high-fat, adequate-protein, low-carbohydrate diet established for treatment of therapy-resistant epilepsy in children. Its efficacy in reducing seizures has been confirmed, but the mechanisms remain elusive. The diet has also shown positive effects in a wide range of other diseases, including Alzheimer's, depression, autism, cancer, and type 2 diabetes. We collected fecal samples from 12 children with therapy-resistant epilepsy before starting KD and after 3 months on the diet. Parents did not start KD and served as diet controls. Applying shotgun metagenomic DNA sequencing, both taxonomic and functional profiles were established. Here we report that alpha diversity is not changed significantly during the diet, but differences in both taxonomic and functional composition are detected. Relative abundance of bifidobacteria as well as E. rectale and Dialister is significantly diminished during the intervention. An increase in relative abundance of E. coli is observed on KD. Functional analysis revealed changes in 29 SEED subsystems including the reduction of seven pathways involved in carbohydrate metabolism. Decomposition of these shifts indicates that bifidobacteria and Escherichia are important contributors to the observed functional shifts. As relative abundance of health-promoting, fiber-consuming bacteria becomes less abundant during KD, we raise concern about the effects of the diet on the gut microbiota and overall health. Further studies need to investigate whether these changes are necessary for the therapeutic effect of KD.

Pivotal advance: inhibition of HMGB1 nuclear translocation as a mechanism for the anti-rheumatic effects of gold sodium thiomalate.

Gold compounds such as gold sodium thiomalate (GST) can reduce the symptoms of rheumatoid arthritis (RA), although their mechanism of action is not well defined. As the proinflammatory mediator high mobility group box chromosomal protein 1 (HMGB1) may play a role in the pathogenesis of RA, we have performed in vitro studies to investigate whether GST inhibits HMGB1 release as the basis of its mode of action. Murine RAW 264.7 or human THP-1 macrophage cells were stimulated in culture with agents causing extracellular HMGB1 release, including LPS, IFN-gamma, polyinosinic:polycytidylic acid, IFN-beta, or NO in the presence of GST, ranging from 0 microM to 250 microM. Secretion and intracellular location of HMGB1 were assessed by Western blotting, HMGB1-specific ELISPOT assay, and immunofluorescent staining. In parallel, TNF and IFN-beta levels were analyzed by ELISPOT and/or ELISA. Supernatant NO production was analyzed by the Griess method. At pharmacologically relevant doses, GST inhibited the extracellular release of HMGB1 from activated macrophages and caused the nuclear retention of this protein; in contrast, no effects were observed on the secretion or production of TNF. Release of the key endogenous mediators of HMGB1 translocation, IFN-beta and NO, was inhibited by GST. This inhibition required gold, as sodium thiomalate did not affect the responses measured. Furthermore, gold chloride also inhibited release of HMGB1. Together, these results suggest a new mechanism for the anti-rheumatic effects of gold salts in RA and the potential of drugs, which interfere with intracellular HMGB1 transport mechanisms, as novel agents to treat RA.

The high mobility group box chromosomal protein 1 is expressed in the human and rat testis where it may function as an antibacterial factor.

BACKGROUND: The high mobility group box chromosomal protein 1 (HMGB1) was originally shown to be a nuclear DNA-binding protein that activates transcription and promotes differentiation. More recently, there have been reports that HMGB1 may also function as a pro-inflammatory and antibacterial factor. The aim of this study was to investigate the testicular expression and antibacterial functions of HMGB1 to elucidate a possible role of HMGB1 in the testicular barrier defence against infections. METHODS AND RESULTS: RT-PCR and in situ hybridization revealed high-level testicular expression of HMGB1 mRNA and localization of this expression to the Sertoli cells and germ cells of the human and rat testis. In addition, immunohistochemical examination demonstrated the presence of the corresponding protein in Sertoli cells and spermatogonia in human and rat testes. Western blotting detected abundant amounts of the HMGB1 protein in the interstitial and intratubular fluids of the intact adult rat testis. Finally, the HMGB1 protein purified from both human and rat testis by reversed-phase high-performance liquid chromatography (HPLC) exerted antibacterial activity towards Bacillus megaterium in an inhibition zone assay. CONCLUSION: HMGB1 is expressed by Sertoli cells and germ cells in the mammalian testis. In addition, purified testicular HMGB1 shows antibacterial activity, indicating that this protein may function as a paracrine host defence factor in the testis.

Interleukin-18 is expressed in rat testis and may promote germ cell growth.

Although host-defence mechanisms, designed to preserve the integrity of the developing germ cells are operative in the testis, the components of this protective system have yet to be characterised in detail. Here, we report that the cytokine interleukin-18 (IL-18) is expressed in the rat testis and may contribute to these defences. Thus, analysis by RT-PCR and Western blotting revealed pronounced testicular expression of pro-IL-18 from postnatal day 5 and onwards. Expression of both IL-18 mRNA and protein was found to be localised to meiotic and post-meiotic germ cells as evaluated by in situ hybridisation and immunohistochemistry, respectively. The mRNA species coding for the IL-18 receptor and IL-1beta converting enzyme, which activates pro-IL-18, were also shown to be expressed by the seminiferous tubules. Recombinant IL-18 was seen to stimulate spermatogonial DNA synthesis in cultures of staged segments of rat seminiferous tubules, without influencing germ cell apoptosis. These results suggest that IL-18 may have host-protective and growth-promoting functions in the testis, but further investigations need to be done to confirm this.

Single subcutaneous administration of chorionic gonadotropin to rats induces a rapid and transient increase in testicular expression of pro-inflammatory cytokines.

hCG has been reported to cause an inflammation-like effect in the testis, although the background and consequences of this phenomenon remain to be understood. This investigation reveals that a single injection of hCG (100 U) induces a transient surge in pro-inflammatory cytokine expression in the adult rat testis. Reverse transcriptase PCR analysis demonstrated onset of testicular expression of IL-1beta and IL-6 mRNA and increases in the levels of mRNA encoding the constitutively expressed cytokines IL-1alpha, IL-1 receptor antagonist, and tumor necrosis factor-alpha 4 h after hCG injection and a maximal response after 8-12 h. These increases were accompanied by a transient increase in testicular IL-1 bioactive protein. Twenty-four hours after administration of hCG, the levels of all cytokine mRNA had decreased, although most were still elevated above control. Immunohistochemical staining revealed that the IL-1beta protein was undetectable in normal testes but was seen to be localized to interstitial macrophages but not Leydig cells after hCG treatment. Testes devoid of Leydig cells after pretreatment with ethane dimethane sulphonate exhibited normal staining for interstitial macrophages but failed to respond to hCG with increases in IL-1beta mRNA and protein expression. We conclude that hCG induces testicular inflammation via local activation by Leydig cells of the production of pro-inflammatory cytokines by resident macrophages. It remains to be investigated whether the high-dose hCG regimens used for treatment of boys with cryptorchidism could induce similar increases of pro-inflammatory cytokines in the human testis and if such treatments could adversely affect future testicular function.

Production and secretion of interleukin-1alpha proteins by rat testis.

The present study characterizes constitutively expressed rat testicular interleukin-1alpha (IL-1alpha) proteins. IL-1 bioactivity of crude testis protein was completely neutralized by IL-1alpha antiserum, IL-1 receptor antagonist, and soluble type I IL-1 receptor. Upon non-denaturating gel permeation chromatography, bioactive IL-1 eluted at molecular sizes of 45, 31, and 17kDa and at charges of pH 5.7 and 6.0 after chromatofocusing. SDS-PAGE/Western blot analysis of proteins extracted from whole testis, seminiferous tubules, interstitial, and seminiferous tubule fluids all demonstrated IL-1alpha immunoreactivity at 45, 24, and 19kDa. Activated macrophages and tissue proteins from endotoxin treated rats showed immunoreactive 31 and 19kDa IL-1alpha. The results indicate that the testis produces three isoforms of IL-1alpha proteins that are secreted into the interstitial compartment and tubular lumen where they may exert paracrine functions. The testicular IL-1alpha isoforms may represent posttranslationally modified precursor, mature IL-1alpha, and a 24-kDa alternate splice form.

High mobility group box chromosomal protein 1 (HMGB1) is an antibacterial factor produced by the human adenoid.

Antibacterial factors were purified from human adenoid glands by tissue extraction and consecutive steps of reversed-phase chromatography and assayed for bactericidal activity against the airway pathogen Moraxella catarrhalis and also Escherichia coli and Bacillus megaterium. One of the most active components isolated from adenoids was identified by N-terminal sequence analysis and mass spectrometry as high mobility group box chromosomal protein 1 (HMGB1). This novel finding was further substantiated by Western blot analysis, demonstrating a protein of expected size reactive with HMGB1 antiserum. Local synthesis was confirmed by reverse-transcriptase PCR and in situ hybridization. Adenoid-derived HMGB1 and recombinant HMGB1 revealed comparable antibacterial activity at high rate. More than 95% of bacteria were eradicated within 5 min by HMGB1 in the cultures. Secretion from the adenoid gland surface was also demonstrated to contain antibacterial activity, mainly mediated by alpha-defensins, but not by HMGB1. We conclude that HMGB1, produced and stored intracellularly in the adenoid gland, contributes to the local antibacterial barrier defense system in the upper respiratory tract.