Characterisation of the T-cell response to Ebola virus glycoprotein amongst survivors of the 2013-16 West Africa epidemic.

Zaire ebolavirus (EBOV) is a highly pathogenic filovirus which can result in Ebola virus disease (EVD); a serious medical condition that presents as flu like symptoms but then often leads to more serious or fatal outcomes. The 2013-16 West Africa epidemic saw an unparalleled number of cases. Here we show characterisation and identification of T cell epitopes in surviving patients from Guinea to the EBOV glycoprotein. We perform interferon gamma (IFNγ) ELISpot using a glycoprotein peptide library to identify T cell epitopes and determine the CD4+ or CD8+ T cell component response. Additionally, we generate data on the T cell phenotype and measure polyfunctional cytokine secretion by these antigen specific cells. We show candidate peptides able to elicit a T cell response in EBOV survivors and provide inferred human leukocyte antigen (HLA) allele restriction. This data informs on the long-term T cell response to Ebola virus disease and highlights potentially important immunodominant peptides.

Nanogel-based nasal ghrelin vaccine prevents obesity.

Obesity is associated with multiple comorbidities such as cardiovascular diseases and has a huge economic impact on the health-care system. However, the treatment of obesity remains insufficient in terms of efficacy, tolerability, and safety. Here we created a nasal vaccine against obesity for the first time. To avoid the injectable administration-caused pain and skin-related adverse event, we focused on the intranasal route of antigen delivery. We developed a vaccine antigen (ghrelin-PspA (pneumococcal surface protein A)), which is a recombinant fusion protein incorporating ghrelin, a hormone that stimulates food intake and decreases energy expenditure, and PspA, a candidate of pneumococcal vaccine as a carrier protein. Ghrelin-PspA antigen was mixed with cyclic di-GMP adjuvant to enhance the immunogenicity and incorporated within a nanometer-sized hydrogel for the effective antigen delivery. Intranasal immunization with ghrelin-PspA vaccine elicited serum immunoglobulin G antibodies against ghrelin and attenuated body weight gain in diet-induced obesity mice. This obesity-attenuating effect was caused by a decrease in fat accumulation and an increase in energy expenditure that was partially due to an increase in the expression of mitochondrial uncoupling protein 1 in brown adipose tissue. The development of this nasal vaccine provides a new strategy for the prevention and treatment of obesity.

Critical role of TSLP-responsive mucosal dendritic cells in the induction of nasal antigen-specific IgA response.

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine involved in T helper 2 type immune responses. The primary target of TSLP is myeloid dendritic cells (DCs), however, little is known about the mechanism by which TSLP elicits respiratory IgA immune responses upon mucosal immunization. Here, we found that the levels of TSLP and TSLPR were upregulated in the mucosal DCs of mice nasally immunized with pneumococcal surface protein A (PspA) plus cholera toxin (CT) compared with those immunized with PspA alone. PspA-specific IgA responses, but not IgG Ab responses were significantly reduced in both serum and mucosal secretions of TSLPR knockout mice compared with wild-type mice after nasal immunization with PspA plus CT. Furthermore, CD11c+ mucosal DCs isolated from TSLPR knockout mice nasally immunized with PspA plus CT were less activated and exhibited markedly reduced expression of IgA-enhancing cytokines (e.g., APRIL, BAFF, and IL-6) compared with those from equivalently immunized wild-type mice. Finally, exogenous TSLP promoted production of IgAs in an in vitro DC-B cell co-culture system as exhibited by enhanced IL-6 production. These results suggest that TSLP-TSLPR signaling is pivotal in the induction of nasal respiratory immunity against pathogenic pneumococcal infection.

The hnRNP-Htt axis regulates necrotic cell death induced by transcriptional repression through impaired RNA splicing.

In this study, we identify signaling network of necrotic cell death induced by transcriptional repression (TRIAD) by α-amanitin (AMA), the selective RNA polymerase II inhibitor, as a model of neurodegenerative cell death. We performed genetic screen of a knockdown (KD) fly library by measuring the ratio of transformation from pupa to larva (PL ratio) under TRIAD, and selected the cell death-promoting genes. Systems biology analysis of the positive genes mapped on protein-protein interaction databases predicted the signaling network of TRIAD and the core pathway including heterogeneous nuclear ribonucleoproteins (hnRNPs) and huntingtin (Htt). RNA sequencing revealed that AMA impaired transcription and RNA splicing of Htt, which is known as an endoplasmic reticulum (ER)-stabilizing molecule. The impairment in RNA splicing and PL ratio was rescued by overexpresion of hnRNP that had been also affected by transcriptional repression. Fly genetics with suppressor or expresser of Htt and hnRNP worsened or ameliorated the decreased PL ratio by AMA, respectively. Collectively, these results suggested involvement of RNA splicing and a regulatory role of the hnRNP-Htt axis in the process of the transcriptional repression-induced necrosis.

Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNA-associated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques.

We previously established a nanosized nasal vaccine delivery system by using a cationic cholesteryl group-bearing pullulan nanogel (cCHP nanogel), which is a universal protein-based antigen-delivery vehicle for adjuvant-free nasal vaccination. In the present study, we examined the central nervous system safety and efficacy of nasal vaccination with our developed cCHP nanogel containing pneumococcal surface protein A (PspA-nanogel) against pneumococcal infection in nonhuman primates. When [(18)F]-labeled PspA-nanogel was nasally administered to a rhesus macaque (Macaca mulatta), longer-term retention of PspA was noted in the nasal cavity when compared with administration of PspA alone. Of importance, no deposition of [(18)F]-PspA was seen in the olfactory bulbs or brain. Nasal PspA-nanogel vaccination effectively induced PspA-specific serum IgG with protective activity and mucosal secretory IgA (SIgA) Ab responses in cynomolgus macaques (Macaca fascicularis). Nasal PspA-nanogel-induced immune responses were mediated through T-helper (Th) 2 and Th17 cytokine responses concomitantly with marked increases in the levels of miR-181a and miR-326 in the serum and respiratory tract tissues, respectively, of the macaques. These results demonstrate that nasal PspA-nanogel vaccination is a safe and effective strategy for the development of a nasal vaccine for the prevention of pneumonia in humans.

Transcription factor Spi-B-dependent and -independent pathways for the development of Peyer's patch M cells.

Although many of the biological features of microfold cells (M cells) have been known for many years, the molecular mechanisms of M-cell development and antigen recognition have remained unclear. Here, we report that Umod is a novel M-cell-specific gene, the translation products of which might contribute to the uptake function of M cells. Transcription factor Spi-B was also specifically expressed in M cells among non-hematopoietic lineages. Spi-B-deficient mice showed reduced expression of most, but not all, other M-cell-specific genes and M-cell surface markers. Whereas uptake of Salmonella Typhimurium via M cells was obviously reduced in Spi-B-deficient mice, the abundance of intratissue cohabiting bacteria was comparable between wild-type and Spi-B-deficient mice. These data indicate that there is a small M-cell population with developmental regulation that is Spi-B independent; however, Spi-B is probably a candidate master regulator of M-cell functional maturation and development by another pathway.

Intrahepatic venous continuation associated with inferior vena cava hypoplasia: evaluation with sonography, MR imaging and MR angiography.

A 17-year-old Asian girl was admitted to our hospital because of acute onset of abdominal pain. Abdominal imaging work-up revealed an unusual pattern of congenital vascular anomalies, including a hypoplastic IVC and large intrahepatic venous collaterals along with an atypical, accessory hepatic vein. Congenital or acquired abnormalities of the inferior vena cava (IVC) can lead to formation of collateral pathways that help bypass the blood flow obstacle.

MR imaging of uncommon recurrence of fibrolipomatous hamartoma of the ulnar nerve.

We present the MR and histopathologic findings of fibrolipomatous hamartoma (FLH) of the ulnar nerve in a 54-year-old woman, a lipomatous process that rarely affects the ulnar nerve. The case illustrated is further unusual as a local soft tissue recurrent mass developed over a remarkably long course of the disease.

Identification and characterization of human PKNOX2, a novel homeobox-containing gene.

The three-amino-acid loop extension (TALE) homeodomain proteins are highly conserved transcription regulators. Since cooperative function among members of this growing family is critical for regulating transcription, we have tried to explore novel members to understand their regulatory mechanisms in cellular proliferation and differentiation. Here we report identification of PKNOX2, a novel TALE homeodomain protein that shows distinct homology with PKNOX1, a stable partner of PBX proteins. PKNOX2 is composed of 460 amino acids and contains HR1, HR2, and homeodomain, which are highly similar to PKNOX1, suggesting that PKNOX2 may also interact with PBX proteins as well as the same DNA sequence as PKNOX1. Genomic organization of PKNOX2 also showed high similarity to PKNOX1, though PKNOX2 lies on a different chromosomal region, 11q24. Unlike PKNOX1, which was broadly expressed in many tissues, PKNOX2 showed a more restricted pattern of mRNA expression. Nuclear localization of PKNOX2 was confirmed by transfection of epitope-tagged cDNA. Taken together, these data indicate that PKNOX2 is a novel PKNOX-related protein and may interact with PBX proteins and play a tissue-specific regulation of transcription.

Nasal administration of cholera toxin (CT) suppresses clinical signs of experimental autoimmune encephalomyelitis (EAE).

Cholera toxin (CT), a major enterotoxin produced by Vibrio cholerae, elicits mucosal adjuvant activities by inducing antigen-specific CD4+ T cells secreting T helper type 2 (Th2) cytokines. Experimental autoimmune encephalomyelitis (EAE) is induced by Th1 cells specific for myelin-derived antigens. We induced EAE in C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG) 35-55 and CT was nasally administered as an immunomodulator on day 7 following MOG challenge. Clinical severity in the CT-treated mice was milder when compared to PBS-treated mice, while the levels of expression of interleukin (IL)-12 and interferon (IFN)-gamma in the central nervous system (CNS) of CT-treated mice were lower than PBS-treated mice. Thus, nasal administration of the mucosal immunomodulator CT ameliorated the severity of EAE, which was associated with the suppression of Th1 cell responses.

Role of MOG-stimulated Th1 type "light up" (GFP+) CD4+ T cells for the development of experimental autoimmune encephalomyelitis (EAE).

Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis in humans. EAE can be passively transferred into naive syngeneic animals by administration of MOG-specific T cell clones. Lymphocytes isolated from green fluorescent protein (GFP)-transgenic (Tg) mice can light up by emitting green fluorescence, thus making it feasible to use such animals in a passive transfer model for EAE. When MOG-sensitized splenic lymphocytes from GFP-Tg mice were adoptively transferred to irradiated, syngeneic C57BL/6 and RAG-1(-/-)mice, typical symptoms of EAE developed. Analysis of the reconstituted mice with EAE revealed prominent infiltration of fluorescing (GFP+), CD4+ T cells into the central nervous system (CNS). Real-time confocal imaging revealed these cells in the spinal cords and brains of recipient mice. This infiltration was also confirmed by anti-GFP monoclonal antibodies. Furthermore, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) evaluation indicated that the infiltrating GFP+, CD4+ T cells exclusively produced T helper type 1 (Th1) cytokines, especially interferon-gamma (IFN-gamma). These results clearly show that MOG-specific CD4+ T cells preferentially invade into the CNS and mediate the development of EAE by producing Th1-biased cytokines.

Production of a recombinant hybrid molecule of cholera toxin-B-subunit and proteolipid-protein-peptide for the treatment of experimental encephalomyelitis.

Mucosal administration of experimental autoimmune encephalomyelitis (EAE)-specific autoantigens can reduce the onset of disease. To examine whether cholera toxin-B-subunit (CTB)-conjugated EAE-specific T-cell epitope can reduce development of the autoimmune disease in mice, we produced a recombinant hybrid molecule of CTB fusion protein linked with proteolipid-protein (PLP)-peptide139-151(C140S) at levels up to 0.1 gram per liter culture media in Bacillus brevis as a secretion-expression system. Amino acid sequencing and GM1-receptor binding assay showed that this expression system produced a uniformed recombinant hybrid protein. EAE was induced in SJL/J mice by systemic administration with the PLP-peptide. When nasally immunized 5 times with 70 microg rCTB PLP-peptide hybrid protein, mice showed a significantly suppressed development of ongoing EAE and an inhibition of both the PLP-peptide-specific delayed-type hypersensitivity (DTH) responses and leukocyte infiltration into the spinal cord. In contrast, all mice given the PLP-peptide alone or the PLP-peptide with the free form of CTB did not suppress the development of EAE and DTH responses. These results suggest that nasal treatment with the recombinant B. brevis-derived hybrid protein of CTB and autoantigen peptide could prove useful in the control of multiple sclerosis.

Nasal immunization with E. coli verotoxin 1 (VT1)-B subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies.

Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.

Oral tolerance revisited: prior oral tolerization abrogates cholera toxin-induced mucosal IgA responses.

Oral delivery of a large dose or prolonged feeding of protein Ags induce systemic unresponsiveness most often characterized as reduced IgG and IgE Ab- and Ag-specific CD4(+) T cell responses. It remains controversial whether oral tolerance extends to diminished mucosal IgA responses in the gastrointestinal tract. To address this issue, mice were given a high oral dose of OVA or PBS and then orally immunized with OVA and cholera toxin as mucosal adjuvant, and both systemic and mucosal immune responses were assessed. OVA-specific serum IgG and IgA and mucosal IgA Ab levels were markedly reduced in mice given OVA orally compared with mice fed PBS. Furthermore, when OVA-specific Ab-forming cells (AFCs) in both systemic and mucosa-associated tissues were examined, IgG AFCs in the spleen and IgA AFCs in the gastrointestinal tract lamina propria of mice given OVA orally were dramatically decreased. Furthermore, marked reductions in OVA-specific CD4(+) T cell proliferative and cytokine responses in spleen and Peyer's patches were seen in mice given oral OVA but were unaffected in PBS-fed mice. We conclude that high oral doses of protein induce both mucosal and systemic unresponsiveness and that use of mucosal adjuvants that induce both parenteral and mucosal immunity may be a better way to assess oral tolerance.

Cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues.

We tested the notion that the mucosal adjuvant cholera toxin (CT) could target, in addition to nasal-associated lymphoreticular tissues, the olfactory nerves/epithelium (ON/E) and olfactory bulbs (OBs) when given intranasally. Radiolabeled CT ((125)I-CT) or CT-B subunit ((125)I-CT-B), when given intranasally to mice, entered the ON/E and OB and persisted for 6 days; however, neither molecule was present in nasal-associated lymphoreticular tissues beyond 24 h. This uptake into olfactory regions was monosialoganglioside (GM1) dependent. Intranasal vaccination with (125)I-tetanus toxoid together with unlabeled CT as adjuvant resulted in uptake into the ON/E but not the OB, whereas (125)I-tetanus toxoid alone did not penetrate into the CNS. We conclude that GM1-binding molecules like CT target the ON/E and are retrograde transported to the OB and may promote uptake of vaccine proteins into olfactory neurons. This raises concerns about the role of GM1-binding molecules that target neuronal tissues in mucosal immunity.

DNA-DNA reassociation among a bloom-forming cyanobacterial genus, Microcystis.

DNA base composition and DNA-DNA hybridization among the cyanobacterial genus Microcystis were determined using nine axenic Microcystis strains, including the three 'morphological' species of Microcystis aeruginosa, Microcystis viridis and Microcystis wesenbergii. These Microcystis species showed a similar DNA base composition (42.1-42.8 mol% G + C) and demonstrated more than 70% DNA relatedness, confirming their synonymy based on bacterial criteria.

ELISA for a complexed antigen with a monoclonal antibody blocking reaction with the free antigen-assay-specific for complexed prostate-specific antigen.

We have developed an enzyme-linked immunoassay (ELISA) for serum complexed-antigen (prostate-specific antigen; PSA)(c-PSA) with simultaneous blocking of free-antigen (PSA)(f-PSA). The assay utilizes three different monoclonal antibodies (MAbs) recognising three distinct PSA epitopes. The detection limit was established as 0.19 microg/l (n=20, mean of zero standard+2S.D.) and the average recovery of f-PSA was 98-100%. The within-run and between-day coefficients of variation (CV) ranged from 2.1% to 3.2% and 2.8% to 6.3%, respectively. There was a good correlation between serum c-PSA measured by the present ELISA and PSA-alpha(1)-antichymotrypsin complex (PSA-ACT) concentrations (r=0.991). This method should provide a better tool for discriminating between benign and malignant prostatic disease.

[A case of lung cancer (small cell carcinoma) occurring esophago-pericardial fistula and purulent pericarditis].

A 72-year-old woman who had had an endoscopic sclerotherapy for esophageal varices presented with high fever and severe cough. Chest X-ray and CT demonstrated a pneumopericardium and pericardial effusion. Esophagoscopy and esophagography revealed an esophageal perforation into the pericardial cavity and into the lung. Consequently, drainage and irrigation of the pericardial cavity and mediastinum were done for MRSA infection. However, these procedures failed to reduce the inflammation, and she expired because of liver failure soon after placing a covered stent in the esophagus. Postmortem examination revealed small cell carcinoma in the left lung invading into the esophagus and pericardium.