Prime-boost-type PspA3 + 2 mucosal vaccine protects cynomolgus macaques from intratracheal challenge with pneumococci.

BACKGROUND: Although vaccination is recommended for protection against invasive pneumococcal disease, the frequency of pneumococcal pneumonia is still high worldwide. In fact, no vaccines are effective for all pneumococcal serotypes. Fusion pneumococcal surface protein A (PspA) has been shown to induce a broad range of cross-reactivity with clinical isolates and afford cross-protection against pneumococcal challenge in mice. Furthermore, we developed prime-boost-type mucosal vaccines that induce both antigen-specific IgG in serum and antigen-specific IgA in targeted mucosal organs in previous studies. We investigated whether our prime-boost-type immunization with a fusion PspA was effective against pneumococcal infection in mice and cynomolgus macaques. METHODS: C57BL/6 mice were intramuscularly injected with fusion PspA combined with CpG oligodeoxynucleotides and/or curdlan. Six weeks later, PspA was administered intranasally. Blood and bronchoalveolar lavage fluid were collected and antigen-specific IgG and IgA titers were measured. Some mice were given intranasal Streptococcus pneumoniae and the severity of infection was analyzed. Macaques were intramuscularly injected with fusion PspA combined with CpG oligodeoxynucleotides and/or curdlan at week 0 and week 4. Then, 13 or 41 weeks later, PspA was administered intratracheally. Blood and bronchoalveolar lavage fluid were collected and antigen-specific IgG and IgA titers were measured. Some macaques were intranasally administered S. pneumoniae and analyzed for the severity of pneumonia. RESULTS: Serum samples from mice and macaques injected with antigens in combination with CpG oligodeoxynucleotides and/or curdlan contained antigen-specific IgG. Bronchial samples contained antigen-specific IgA after the fusion PspA boosting. This immunization regimen effectively prevented S. pneumoniae infection. CONCLUSIONS: Prime-boost-type immunization with a fusion PspA prevented S. pneumoniae infection in mice and macaques.

ggkegg: analysis and visualization of KEGG data utilizing the grammar of graphics.

SUMMARY: The Kyoto Encyclopedia of Genes and Genomes (KEGG) database serves as a valuable systems biology resource and is widely utilized in diverse research fields. However, existing software does not allow flexible visualization and network analyses of the vast and complex KEGG data. We developed ggkegg, an R package that integrates KEGG information with ggplot2 and ggraph. ggkegg enables enhanced visualization and network analyses of KEGG data. We demonstrate the utility of the package by providing examples of its application in single-cell, bulk transcriptome, and microbiome analyses. ggkegg may empower researchers to analyze complex biological networks and present their results effectively. AVAILABILITY AND IMPLEMENTATION: The package and user documentation are available at: https://github.com/noriakis/ggkegg.

Parallel gold enhancement of quantum dots 565/655 for double-labelling correlative light and electron microscopy on human autopsied samples.

Cadmium selenide quantum dots (QDs) are fluorescent and electron-dense nanoparticles. When used as reporter of immunolabeling, this dual visibility is essential for direct comparison of its fluorescent signals on light microscopy (LM) and their ultrastructrual counterparts on electron microscopy (EM) as correlative light and electron microscopy (CLEM). To facilitate EM recognition, QDs on EM grid were gold enhanced, which increased their size and electron density. On histological sections as well, gold-enhanced QDs, used as a reporter of immunolabeling, were easily recognized on EM. Because target structures are visible on bright field microscopy, gold enhancement facilitated trimming the target structures into final EM sections. Furthermore, gold enhancement of rod-shaped QD655 on EM grid was accentuated on their tips while spherical QD565 was gold-enhanced as sphere in contrast. This EM distinction was evident on histological sections where QD565 (green fluorescence) and QD655 (red fluorescence) were used as a reporter pair for double immunolabeling. Double-labeled immuno-fluorescent images, initially captured before EM processing, are now compared with their respective immuno EM counterparts. Specific labeling of each epitope was corroborated by mutual comparison between LM and EM. Although fluoronanogold may be a candidate reporter partner with QDs for gold-enhanced, double-labeling CLEM, its limited penetration into fixed tissue hampers universal use for thick histological sections. Gold-enhancement of QD immunolabeling, now expanded to double-labeling CLEM for human brain samples, will pave the way to translate molecular events into ultrastructural morphopathogenesis in situ.

Functional Restoration of Bacteriomes and Viromes by Fecal Microbiota Transplantation.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI). However, the overall mechanisms underlying FMT success await comprehensive elucidation, and the safety of FMT has recently become a serious concern because of the occurrence of drug-resistant bacteremia transmitted by FMT. We investigated whether functional restoration of the bacteriomes and viromes by FMT could be an indicator of successful FMT. METHODS: The human intestinal bacteriomes and viromes from 9 patients with rCDI who had undergone successful FMT and their donors were analyzed. Prophage-based and CRISPR spacer-based host bacteria-phage associations in samples from recipients before and after FMT and in donor samples were examined. The gene functions of intestinal microorganisms affected by FMT were evaluated. RESULTS: Metagenomic sequencing of both the viromes and bacteriomes revealed that FMT does change the characteristics of intestinal bacteriomes and viromes in recipients after FMT compared with those before FMT. In particular, many Proteobacteria, the fecal abundance of which was high before FMT, were eliminated, and the proportion of Microviridae increased in recipients. Most temperate phages also behaved in parallel with the host bacteria that were altered by FMT. Furthermore, the identification of bacterial and viral gene functions before and after FMT revealed that some distinctive pathways, including fluorobenzoate degradation and secondary bile acid biosynthesis, were significantly represented. CONCLUSIONS: The coordinated action of phages and their host bacteria restored the recipients' intestinal flora. These findings show that the restoration of intestinal microflora functions reflects the success of FMT.

Metagenome Data on Intestinal Phage-Bacteria Associations Aids the Development of Phage Therapy against Pathobionts.

The application of bacteriophages (phages) is proposed as a highly specific therapy for intestinal pathobiont elimination. However, the infectious associations between phages and bacteria in the human intestine, which is essential information for the development of phage therapies, have yet to be fully elucidated. Here, we report the intestinal viral microbiomes (viromes), together with bacterial microbiomes (bacteriomes), in 101 healthy Japanese individuals. Based on the genomic sequences of bacteriomes and viromes from the same fecal samples, the host bacteria-phage associations are illustrated for both temperate and virulent phages. To verify the usefulness of the comprehensive host bacteria-phage information, we screened Clostridioides difficile-specific phages and identified antibacterial enzymes whose activity is confirmed both in vitro and in vivo. These comprehensive metagenome analyses reveal not only host bacteria-phage associations in the human intestine but also provide vital information for the development of phage therapies against intestinal pathobionts.

Antigen-Specific Mucosal Immunity Regulates Development of Intestinal Bacteria-Mediated Diseases.

BACKGROUND & AIMS: Dysregulation of the microbiome has been associated with development of complex diseases, such as obesity and diabetes. However, no method has been developed to control disease-associated commensal microbes. We investigated whether immunization with microbial antigens, using CpG oligodeoxynucleotides and/or curdlan as adjuvants, induces systemic antigen-specific IgA and IgG production and affects development of diseases in mice. METHODS: C57BL/6 mice were given intramuscular injections of antigens (ovalbumin, cholera toxin B-subunit, or pneumococcal surface protein A) combined with CpG oligodeoxynucleotides and/or curdlan. Blood and fecal samples were collected weekly and antigen-specific IgG and IgA titers were measured. Lymph nodes and spleens were collected and analyzed by enzyme-linked immunosorbent assay for antigen-specific splenic T-helper 1 cells, T-helper 17 cells, and memory B cells. Six weeks after primary immunization, mice were given a oral, nasal, or vaginal boost of ovalbumin; intestinal lamina propria, bronchial lavage, and vaginal swab samples were collected and antibodies and cytokines were measured. Some mice were also given oral cholera toxin or intranasal Streptococcus pneumoniae and the severity of diarrhea or pneumonia was analyzed. Gnotobiotic mice were gavaged with fecal material from obese individuals, which had a high abundance of Clostridium ramosum (a commensal microbe associated with obesity and diabetes), and were placed on a high-fat diet 2 weeks after immunization with C ramosum. Intestinal tissues were collected and analyzed by quantitative real-time polymerase chain reaction. RESULTS: Serum and fecal samples from mice given injections of antigens in combination with CpG oligodeoxynucleotides and curdlan for 3 weeks contained antigen-specific IgA and IgG, and splenocytes produced interferon-gamma and interleukin 17A. Lamina propria, bronchial, and vaginal samples contained antigen-specific IgA after the ovalbumin boost. This immunization regimen prevented development of diarrhea after injection of cholera toxin, and inhibited lung colonization by S pneumoniae. In gnotobiotic mice colonized with C ramosum and placed on a high-fat diet, the mice that had been immunized with C ramosum became less obese than the nonimmunized mice. CONCLUSIONS: Injection of mice with microbial antigens and adjuvant induces antigen-specific mucosal and systemic immune responses. Immunization with S pneumoniae antigen prevented lung infection by this bacteria, and immunization with C ramosum reduced obesity in mice colonized with this microbe and placed on a high-fat diet. This immunization approach might be used to protect against microbe-associated disorders of intestine.

How to demix Alzheimer-type and PSP-type tau lesions out of their mixture -hybrid approach to dissect comorbidity.

Neurofibrillary tangles (NFTs), are shared between progressive supranuclear palsy (PSP) and Alzheimer disease (AD). Histological distinction of PSP and AD is possible based on the distribution of NFTs. However, neuropathologists may encounter diagnostic difficulty with comorbidity of PSP and AD. In this study, we tried to circumvent this difficulty by analyzing five autopsied brains harboring both PSP and AD pathology. Tau-positive lesions were sorted based on their cell type (neuron versus glia), and tau isoforms: three-repeat (3R) versus four-repeat (4R) tau. 16 regions were selected to map these lesions throughout the brain. 4R-tau lesions were present in all areas examined. Among them, 3R-tau lesions were absent in some areas. These 4R selective (4R+/3R-) areas dictate prototypic distribution of PSP, not usually found in AD, such as pontine nucleus, red nucleus, inferior olivary nucleus, dentate nucleus, globus pallidus and putamen, each contained both glial and neuronal lesions. In contrast, additional 3R-tau lesions were found in hippocampal formation to neocortex, where 3R immunoreactivity (IR) was predominant over the 4R counterpart mainly in neurons as found in AD but not in PSP. Although tau lesions in central grey matter, substantia nigra and locus coeruleus are found in both AD and PSP, 4R-selectivity with glial component suggests PSP origin. Even if the presence of 3 R IR in these areas suggests AD pathology, it does not exclude the involvement of PSP-type lesion because distinction of 4R IR into PSP or AD is not yet possible. Further demixing may be possible if biochemical difference of 4R tau between PSP and AD is identified.

Brainstem tau pathology in Alzheimer's disease is characterized by increase of three repeat tau and independent of amyloid ß.

INTRODUCTION: Alzheimer-type neuropil threads (NTs) and neurofibrillary tangles (NFTs) are comprised of either 4 repeat (4R)-tau, 3 repeat (3R)-tau, or a mixture of both. In the hippocampus, the number of NFTs, and the proportion of 3R tau progressively increases. If this preferential accumulation of 3R tau also occurs in the brainstem, it may be fundamentally related to progression of Alzheimer pathology. METHODS: Midbrain and pontine sections of brainstems from 23 cases (Braak-NFT stages I/II: 8, III/IV: 8, and V/VI: 7) were double immunofluorolabeled for 4R and 3R tau. High-resolution (0.645 µm/pixel), in-focus snapshots were tiled to cover entire brain sections using a virtual slide system. Each lesion was classified by size (NT < 200 µm2 < NFT) and staining profile (3R/4R). In addition, the localization and quantity of amyloid ß (Aß) deposits were examined in adjacent sections for comparison with tau. RESULTS: The data sets obtained from approximately 286 gigabytes of image files consisted of 847,763 NTs and 7859 NFTs. The proportion of 3R tau-positive NTs and NFTs in the midbrain, and 3R tau-positive NTs in the pons gradually increased with advancing NFT stages, while the proportion of 3R tau-positive NFTs in the pons was already elevated at early stages. Aß deposits were absent at NFT stages I/II, and when present at later stages, their regional distribution was different from that of tau. These observations suggest that a progressive increase in the proportion of 3R tau occurs independently of Aß deposits. CONCLUSIONS: This is the first quantitative analysis of NFTs and NTs in the human brainstem. We demonstrate that the proportion of 3R tau in the brainstem neurofibrillary changes increases with disease progression. Because this phenomenon is shared between the brainstem and the hippocampus, this increase may be fundamental to the pathogenesis of Alzheimer disease.

[Headache as a manifestation of SAPHO syndrome with a lesion extending to the dura mater, parietal bone, and temporal muscle].

A 50-year-old woman with a history of palmoplantar pustulosis, femur osteomyelitis, and sterno-costo-clavicular hyperostosis presented with a chronic severe left temporal headache that had progressed during the previous year. Her CRP level was elevated. Cranial images showed Gadolinium-enhancement of the left temporal muscle, left parietal bone and dura mater. (99m)Tc-HMDP scintigram showed increased uptake in the left parietal bone, left sterno-costo-clavicular joint, right femoral head and intervertebral joints. Biopsy of the lesion demonstrated 1) proliferation of connective tissue in both perimysium and endomysium of the temporal muscle with mild inflammatory cell infiltration within the interstitium, 2) marked infiltration of granulocytes to the bone marrow of the parietal bone, 3) necrosis and moderate fibrosis in the interstitium with inflammatory cell infiltration in the parietal bone, and 4) moderate fibrosis and slight infiltration of inflammatory cells in the dura mater. The patient was diagnosed with a cranial lesion of synovitis-acne-pustulosis-hyperostosis-osteitis (SAPHO) syndrome. There was a moderate response to treatment with intravenous steroid pulse therapy and subsequent methotrexate. In a case of headache accompanied by inflammatory response, palmoplantar pustulosis and joint lesions such as hyperostosis, the possibility of a rare cranial manifestation of SAPHO syndrome should be considered.

Atomic identification of fluorescent Q-dots on tau-positive fibrils in 3D-reconstructed pick bodies.

Pick body disease, characterized by the presence of Pick bodies, is distinguished from neurofibrillary tangles in Alzheimer disease on the basis of their smooth, spherical shape. Quantum dots (QDs) are nanometer-scale, water-soluble fluorophores that are detectable both as a fluorescent signal by light microscopy and as electron-dense particles under electron microscopy. In this study, tau-positive Pick bodies were immunofluorescently labeled with QD nanocrystals composed of cadmium selenide for three-dimensional (3D) reconstruction and subsequently subjected to electron microscopic observation to identify QD immunolabeling on the same Pick body for comparison in detail. The identity of the QD nanocrystals, which label the tau-positive fibrils, was confirmed by the presence of both cadmium and selenium on these nanocrystals, demonstrated as parallel peaks corresponding to these atoms on energy-dispersive X-ray spot analysis under super-resolution scanning transmission electron microscopy. This confirmation of the specificity of the QD labeling through both its fluorescence and energy-dispersive X-ray spectra reinforces the reliability of the labeling. In addition, this exact comparison of the same structure by electron microscopy and 3D light microscopy demonstrates how its ultrastructural details are related to its surrounding structures on a 3D basis, providing further insights into how molecules woven into specific pathological ultrastructures are at work in situ.