Integrative single-cell RNA-seq analysis of vascularized cerebral organoids.

BACKGROUND: Cerebral organoids are three-dimensional in vitro cultured brains that mimic the function and structure of the human brain. One of the major challenges for cerebral organoids is the lack of functional vasculature. Without perfusable vessels, oxygen and nutrient supplies may be insufficient for long-term culture, hindering the investigation of the neurovascular interactions. Recently, several strategies for the vascularization of human cerebral organoids have been reported. However, the generalizable trends and variability among different strategies are unclear due to the lack of a comprehensive characterization and comparison of these vascularization strategies. In this study, we aimed to explore the effect of different vascularization strategies on the nervous system and vasculature in human cerebral organoids. RESULTS: We integrated single-cell RNA sequencing data of multiple vascularized and vascular organoids and fetal brains from publicly available datasets and assessed the protocol-dependent and culture-day-dependent effects on the cell composition and transcriptomic profiles in neuronal and vascular cells. We revealed the similarities and uniqueness of multiple vascularization strategies and demonstrated the transcriptomic effects of vascular induction on neuronal and mesodermal-like cell populations. Moreover, our data suggested that the interaction between neurons and mesodermal-like cell populations is important for the cerebrovascular-specific profile of endothelial-like cells. CONCLUSIONS: This study highlights the current challenges to vascularization strategies in human cerebral organoids and offers a benchmark for the future fabrication of vascularized organoids.

Covalent Immobilization of Collagen Type I to a Polydimethylsiloxane Surface for Preventing Cell Detachment by Retaining Collagen Molecules under Uniaxial Cyclic Mechanical Stretching Stress.

Surface modification of polydimethylsiloxane (PDMS) with an extracellular matrix (ECM) is useful for enhancing stable cell attachment. However, few studies have investigated the correlation between the stability of deposited ECM and cell behavior on the PDMS surfaces in external stretched cell culture systems. Herein, covalent collagen type I (Col)-immobilized PDMS surfaces were fabricated using 3-aminopropyl-trimethoxysilane, glutaraldehyde, and Col molecules. The immobilized collagen molecules on the PDMS surface were more stable and uniform than the physisorbed collagen. The cells stably adhered to the Col-immobilized surface and proliferated even under uniaxial cyclic mechanical stretching stress (UnCyMSt), whereas the cells gradually detached from the Col-physisorbed PDMS surface, accompanied by a decrease in the number of deposited collagen molecules. Moreover, the immobilization of collagen molecules enhanced cell alignment under the UnCyMSt. This study reveals that cell adhesion, proliferation, and alignment under the UnCyMSt can be attributed to the retention of collagen molecules on the PDMS surface.

Spatial and Sexual Divergence of Gut Bacterial Communities in Field Cricket Teleogryllus occipitalis (Orthoptera: Gryllidae).

The insect gut is colonized by microbes that confer a myriad of beneficial services to the host, including nutritional support, immune enhancement, and even influence behavior. Insect gut microbes show dynamic changes due to the gut compartments, sex, and seasonal and geographic influences. Crickets are omnivorous hemimetabolous insects that have sex-specific roles, such as males producing chirping sounds for communication and exhibiting fighting behavior. However, limited information is available on their gut bacterial communities, hampering studies on functional compartmentalization of the gut and sex-specific roles of the gut microbiota in omnivorous insects. Here, we report a metagenomic analysis of the gut bacteriome of the field cricket Teleogryllus occipitalis using 16S rRNA V3-V4 amplicon sequencing to identify sex- and compartment-dependent influences on its diversity and function. The structure of the gut microbiota is strongly influenced by their gut compartments rather than sex. The species richness and diversity analyses revealed large difference in the bacterial communities between the gut compartments while minor differences were observed between the sexes. Analysis of relative abundance and predicted functions revealed that nitrogen- and oxygen-dependent metabolism and amino acid turnover were subjected to functional compartmentalization in the gut. Comparisons between the sexes revealed differences in the gut microbiota, reflecting efficiency in energy use, including glycolytic and carbohydrate metabolism, suggesting a possible involvement in egg production in females. This study provides insights into the gut compartment dependent and sex-specific roles of host-gut symbiont interactions in crickets and the industrial production of crickets.

Human salivary protein-derived peptides specific-salivary SIgA antibodies enhanced by nasal double DNA adjuvant in mice play an essential role in preventing Porphyromonas gingivalis colonization: an in-vitro study.

BACKGROUND: We previously showed that fimbriae-bore from Poryphyromonas gingivalis (Pg), one of the putative periodontopathogenic bacteria specifically bound to a peptide domain (stat23, prp21) shared on statherin or acidic proline-rich protein 1 (PRP1) molecule of human salivary proteins (HSPs). Here, we investigated whether the nasal administration of DNA plasmid expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotide 1826 as double DNA adjuvant (dDA) with stat23 and prpr21 induces antigen (Ag)-specific salivary secretory IgA (SIgA) antibodies (Abs) in mice. Further, we examined that stat23- and prpr21-specific salivary SIgA Abs induced by dDA have an impact on Pg-binding to human whole saliva-coated hydroxyapatite beads (wsHAPs). MATERIAL AND METHODS: C57BL/6N mice were nasally immunized with dDA plus sta23 or/and prp21 peptide as Ag four times at weekly intervals. Saliva was collected one week after the final immunization and was subjected to Ag-specific ELISA. To examine the functional applicability of Ag-specific SIgA Abs, SIgA-enriched saliva samples were subjected to Pg binding inhibition assay to wsHAPs. RESULTS: Significantly elevated levels of salivary SIgA Ab to stat23 or prp21 were seen in mice given nasal stat23 or prp21 with dDA compared to those in mice given Ag alone. Of interest, mice nasally given the mixture of stat23 and prp21 as double Ags plus dDA, resulted in both stat23- and prp21-specific salivary SIgA Ab responses, which are mediated through significantly increased numbers of CD11c+ dendritic cell populations and markedly elevated Th1 and Th2 cytokines production by CD4+ T cells in the mucosal inductive and effector tissues. The SIgA Ab-enriched saliva showed significantly reduced numbers of live Pg cells binding to wsHAPs as compared with those in mice given double Ags without dDA or naïve mice. Additionally, saliva from IgA-deficient mice given nasal double Ags plus dDA indicated no decrease of live Pg binding to wsHAPs. CONCLUSION: These findings show that HSP-derived peptides-specific salivary SIgA Abs induced by nasal administration of stat23 and prp21 peptides plus dDA, play an essential role in preventing Pg attachment and colonization on the surface of teeth, suggesting a potency that the SIgA may interrupt and mask fimbriae-binding domains in HSPs on the teeth.

Nasal double DNA adjuvant induces salivary FimA-specific secretory IgA antibodies in young and aging mice and blocks Porphyromonas gingivalis binding to a salivary protein.

BACKGROUND: We previously showed that nasal administration of a combination of dendritic cell (DC) targeted DNA plasmid expressing Flt3 ligand and CpG oligodeoxynucleotides 1826 as a mucosal adjuvant (double adjuvant, DA) provoked protective immunity in the upper respiratory tract of young adult and aging mice. Here, we investigated whether the nasal DA system induces secretory (S)IgA antibodies (Abs) toward recombinant fimbrillin (rFimA) of Porphyromonas gingivalis (P. gingivalis) in the saliva of young adult and aging mice. Further, we examined the functional applicability of rFimA-specific salivary SIgA Abs. METHODS: BALB/c mice (8- or 48-week-old) were nasally immunized with rFimA plus DA three times at weekly intervals. Control mice were nasally administered rFimA alone. Saliva samples were collected 1 week after the final immunization, and were subjected to rFimA-specific ELISA. To examine the functional applicability of rFimA-specific SIgA Abs, IgA-enriched saliva samples were subjected to an inhibition assay in order to assess the numbers of P. gingivalis cells bound to the salivary protein statherin. RESULTS: The 8- and 48-week-old mice administered nasal rFimA plus DA showed significantly increased levels of rFimA-specific SIgA Abs in saliva and elevated numbers of CD11c+ DCs in sublingual glands (SLGs), periglandular lymph nodes (PGLNs) and submandibular glands (SMGs) as well as nasopharyngeal-associated lymphoid tissues (NALT) compared to mice administered rFimA alone. Further, rFimA-specific SIgA Abs-containing saliva, in which IgG Abs of 8- and 48-week-old mice administered nasal rFimA plus DA were removed, significantly inhibited binding of P. gingivalis to the salivary protein. CONCLUSIONS: These findings show that this DA system could be an effective nasal vaccine strategy for the enhancement of P. gingivalis-specific protective immunity in the oral cavity of adolescents and older individuals.

Antioxidant Activity of Ge-132, a Synthetic Organic Germanium, on Cultured Mammalian Cells.

Ge-132 is a synthetic organic germanium that is used as a dietary supplement. The antioxidant activity of Ge-132 on cultured mammalian cells was investigated in this study. First, Ge-132 cytotoxicity on mammalian cultured cells was determined by measuring lactate dehydrogenase (LDH) levels. Ge-132 had no cytotoxic effect on three different cell lines. Second, the cell proliferative effect of Ge-132 was determined by measuring ATP content of whole cells and counting them. Ge-132 treatment of Chinese hamster ovary (CHO-K1) and SH-SY5Y cells promoted cell proliferation in a dose-dependent manner. Finally, antioxidant activity of Ge-132 against hydrogen peroxide-induced oxidative stress was determined by measuring the levels of intracellular reactive oxygen species (ROS) and carbonylated proteins. Pre-incubation of CHO-K1 and SH-SY5Y cells with Ge-132 suppressed intracellular ROS production and carbonylated protein levels induced by hydrogen peroxide. Our results suggest that Ge-132 has antioxidant activity against hydrogen peroxide-induced oxidative stress.

Dendritic cell-targeting DNA-based nasal adjuvants for protective mucosal immunity to Streptococcus pneumoniae.

To develop safe vaccines for inducing mucosal immunity to major pulmonary bacterial infections, appropriate vaccine antigens (Ags), delivery systems and nontoxic molecular adjuvants must be considered. Such vaccine constructs can induce Ag-specific immune responses that protect against mucosal infections. In particular, it has been shown that simply mixing the adjuvant with the bacterial Ag is a relatively easy means of constructing adjuvant-based mucosal vaccine preparations; the resulting vaccines can elicit protective immunity. DNA-based nasal adjuvants targeting mucosal DCs have been studied in order to induce Ag-specific mucosal and systemic immune responses that provide essential protection against microbial pathogens that invade mucosal surfaces. In this review, initially a plasmid encoding the cDNA of Flt3 ligand (pFL), a molecule that is a growth factor for DCs, as an effective adjuvant for mucosal immunity to pneumococcal infections, is introduced. Next, the potential of adding unmethylated CpG oligodeoxynucleotide and pFL together with a pneumococcal Ag to induce protection from pneumococcal infections is discussed. Pneumococcal surface protein A has been used as vaccine for restoring mucosal immunity in older persons. Further, our nasal pFL adjuvant system with phosphorylcholine-keyhole limpet hemocyanin (PC-KLH) has also been used in pneumococcal vaccine development to induce complete protection from nasal carriage by Streptococcus pneumoniae. Finally, the possibility that anti-PC antibodies induced by nasal delivery of pFL plus PC-KLH may play a protective role in prevention of atherogenesis and thus block subsequent development of cardiovascular disease is discussed.

DeepLabCut-based daily behavioural and posture analysis in a cricket.

Circadian rhythms are indispensable intrinsic programs that regulate the daily rhythmicity of physiological processes, such as feeding and sleep. The cricket has been employed as a model organism for understanding the neural mechanisms underlying circadian rhythms in insects. However, previous studies measuring rhythm-controlled behaviours only analysed locomotive activity using seesaw-type and infrared sensor-based actometers. Meanwhile, advances in deep learning techniques have made it possible to analyse animal behaviour and posture using software that is devoid of human bias and does not require physical tagging of individual animals. Here, we present a system that can simultaneously quantify multiple behaviours in individual crickets - such as locomotor activity, feeding, and sleep-like states - in the long-term, using DeepLabCut, a supervised machine learning-based software for body keypoints labelling. Our system successfully labelled the six body parts of a single cricket with a high level of confidence and produced reliable data showing the diurnal rhythms of multiple behaviours. Our system also enabled the estimation of sleep-like states by focusing on posture, instead of immobility time, which is a conventional parameter. We anticipate that this system will provide an opportunity for simultaneous and automatic prediction of cricket behaviour and posture, facilitating the study of circadian rhythms.

Oral clarithromycin enhances airway immunoglobulin A (IgA) immunity through induction of IgA class switching recombination and B-cell-activating factor of the tumor necrosis factor family molecule on mucosal dendritic cells in mice infected with influenza A virus.

We previously reported that the macrolide antibiotic clarithromycin (CAM) enhanced the mucosal immune response in pediatric influenza, particularly in children treated with the antiviral neuraminidase inhibitor oseltamivir (OSV) with low production of mucosal antiviral secretory IgA (S-IgA). The aims of the present study were to confirm the effects of CAM on S-IgA immune responses, by using influenza A virus (IAV) H1N1-infected mice treated with or without OSV, and to determine the molecular mechanisms responsible for the induction of mucosal IgA class switching recombination in IAV-infected CAM-treated mice. The anti-IAV S-IgA responses and expression levels of IgA class switching recombination-associated molecules were examined in bronchus-lymphoid tissues and spleens of infected mice. We also assessed neutralization activities of S-IgA against IAV. Data show that CAM enhanced anti-IAV S-IgA induction in the airway of infected mice and restored the attenuated antiviral S-IgA levels in OSV-treated mice to the levels in the vehicle-treated mice. The expression levels of B-cell-activating factor of the tumor necrosis factor family (BAFF) molecule on mucosal dendritic cells as well as those of activation-induced cytidine deaminase and Iµ-Cα transcripts on B cells were enhanced by CAM, compared with the levels without CAM treatment, but CAM had no effect on the expression of the BAFF receptor on B cells. Enhancement by CAM of neutralization activities of airway S-IgA against IAV in vitro and reinfected mice was observed. This study identifies that CAM enhances S-IgA production and neutralizing activities through the induction of IgA class switching recombination and upregulation of BAFF molecules in mucosal dendritic cells in IAV-infected mice.

A combination of Flt3 ligand cDNA and CpG oligodeoxynucleotide as nasal adjuvant elicits protective secretory-IgA immunity to Streptococcus pneumoniae in aged mice.

Our previous study showed that a combination of a plasmid-expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotides (CpG ODN) as a combined nasal adjuvant elicited mucosal immune responses in aged (2-y-old) mice. In this study, we investigated whether a combination of pFL and CpG ODN as a nasal adjuvant for a pneumococcal surface protein A (PspA) would enhance PspA-specific secretory-IgA Ab responses, which could provide protective mucosal immunity against Streptococcus pneumoniae infection in aged mice. Nasal immunization with PspA plus a combination of pFL and CpG ODN elicited elevated levels of PspA-specific secretory-IgA Ab responses in external secretions and plasma in both young adult and aged mice. Significant levels of PspA-specific CD4(+) T cell proliferative and PspA-induced Th1- and Th2- type cytokine responses were noted in nasopharyngeal-associated lymphoreticular tissue, cervical lymph nodes, and spleen of aged mice, which were equivalent to those in young adult mice. Additionally, increased numbers of mature-type CD8, CD11b-expressing dendritic cells were detected in mucosal inductive and effector lymphoid tissues of aged mice. Importantly, aged mice given PspA plus a combination of pFL and CpG ODN showed protective immunity against nasal S. pneumoniae colonization. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for protection against S. pneumoniae in the elderly.

Complete mitochondrial genome sequences of two ground crickets, Dianemobius fascipes nigrofasciatus and Polionemobius taprobanensis (Orthoptera: Grylloidea: trigonidiidae).

The authors sequenced the complete mitochondrial (mt) genomes of the band-legged ground cricket (Dianemobius fascipes nigrofasciatus Matsumura, 1904) and a temperate form of the lawn ground cricket (Polionemobius taprobanensis Walker, 1869), collected in Japan. The length of the mt genome sequences was 15,354 bp in D. fascipes nigrofasciatus and 16,063 bp in P. taprobanensis. Annotation of the mt genome sequences revealed 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. The orientation of the genes was the same as in other Grylloidea species, and the order was the same as in other Trigonidiidae species. In our phylogenetic analysis, D. fascipes nigrofasciatus formed a clade with D. fascipes collected in China, and the temperate form of P. taprobanensis formed a clade with P. taprobanensis collected in China. Comparison of the numbers of positions with different amino acid residues encoded by the protein-coding genes implied the separate species status of each member of each of the two pairs of ground crickets. The mt genome sequences of D. fascipes nigrofasciatus and P. taprobanensis will contribute to phylogenetic and taxonomic studies of the Trigonidiidae.

Genome and Genetic Engineering of the House Cricket (Acheta domesticus): A Resource for Sustainable Agriculture.

Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics.

Notch-ligand expression by NALT dendritic cells regulates mucosal Th1- and Th2-type responses.

Our previous studies showed that an adenovirus (Ad) serotype 5 vector expressing Flt3 ligand (Ad-FL) as nasal adjuvant activates CD11c(+) dendritic cells (DCs) for the enhancement of antigen (Ag)-specific IgA antibody (Ab) responses. In this study, we examined the molecular mechanism for activation of CD11c(+) DCs and their roles in induction of Ag-specific Th1- and Th2-cell responses. Ad-FL activated CD11c(+) DCs expressed increased levels of the Notch ligand (L)-expression and specific mRNA. When CD11c(+) DCs from various mucosal and systemic lymphoid tissues of mice given nasal OVA plus Ad-FL were cultured with CD4(+) T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice, significantly increased levels of T cell proliferative responses were noted. Furthermore, Ad-FL activated DCs induced IFN-γ, IL-2 and IL-4 producing CD4(+) T cells. Of importance, these APC functions by Ad-FL activated DCs were down-regulated by blocking Notch-Notch-L pathway. These results show that Ad-FL induces CD11c(+) DCs to the express Notch-ligands and these activated DCs regulate the induction of Ag-specific Th1- and Th2-type cytokine responses.

Study on the subgel-phase formation using an asymmetric phospholipid bilayer membrane by high-pressure fluorometry.

The myristoylpalmitoylphosphatidylcholine (MPPC) bilayer membrane shows a complicated temperature-pressure phase diagram. The large portion of the lamellar gel (L(ß)'), ripple gel (P(ß)'), and pressure-induced gel (L(ß)I) phases exist as metastable phases due to the extremely stable subgel (L(c)) phase. The stable L(c) phase enables us to examine the properties of the L(c) phase. The phases of the MPPC bilayers under atmospheric and high pressures were studied by small-angle neutron scattering (SANS) and fluorescence spectroscopy using a polarity-sensitive fluorescent probe Prodan. The SANS measurements clearly demonstrated the existence of the metastable L(ß)I phase with the smallest lamellar repeat distance. From a second-derivative analysis of the fluorescence data, the line shape for the L(c) phase under high pressure was characterized by a broad peak with a minimum of ca. 460 nm. The line shapes and the minimum intensity wavelength (λ″(min)) values changed with pressure, indicating that the L(c) phase has highly pressure-sensible structure. The λ″(min) values of the L(c) phase spectra were split into ca. 430 and 500 nm in the L(ß)I phase region, which corresponds to the formation of a interdigitated subgel L(c) (L(c)I) phase. Moreover, the phase transitions related to the L(c) phase were reversible transitions under high pressure. Taking into account the fluorescence behavior of Prodan for the L(c) phase, we concluded that the structure of the L(c) phase is highly probably a staggered structure, which can transform into the L(c)I phase easily.

Secretory-IgA antibodies play an important role in the immunity to Streptococcus pneumoniae.

This study was designed to investigate whether secretory-IgA (S-IgA) Abs induced by a pneumococcal surface protein A (PspA)-based nasal vaccine are necessary for prevention of streptococcal colonization. Mice nasally immunized with PspA plus a plasmid expressing Flt3 ligand (pFL) cDNA as a mucosal adjuvant showed significantly higher levels of PspA-specific S-IgA and IgG Ab responses in both plasma and nasal washes when compared with naive mice. Although IgA(-/-) mice given nasal PspA plus pFL had significantly high levels of PspA-specific IgG Abs, high numbers of CFUs were detected in nasal washes and nasal passages. In contrast, vaccinated wild-type mice showed essentially no bacteria in the nasal cavity. Further, a nasal vaccine consisting of PspA plus pFL effectively reduced pre-existing Streptococcus pneumoniae in the nasal cavity. These results show that PspA-based vaccine-induced specific S-IgA Abs play a necessary role in the regulation of S. pneumoniae colonization in the nasal cavity.

Dissecting cricket genomes for the advancement of entomology and entomophagy.

Significant advances in biophysical methods such as next-generation sequencing technologies have now opened the way to conduct evolutionary and applied research based on the genomic information of greatly diverse insects. Crickets belonging to Orthoptera (Insecta: Polyneoptera), one of the most flourishing groups of insects, have contributed to the development of multiple scientific fields including developmental biology and neuroscience and have been attractive targets in evolutionary ecology for their diverse ecological niches. In addition, crickets have recently gained recognition as food and feed. However, the genomic information underlying their biological basis and application research toward breeding is currently underrepresented. In this review, we summarize the progress of genomics of crickets. First, we outline the phylogenetic position of crickets in insects and then introduce recent studies on cricket genomics and transcriptomics in a variety of fields. Furthermore, we present findings from our analysis of polyneopteran genomes, with a particular focus on their large genome sizes, chromosome number, and repetitive sequences. Finally, how the cricket genome can be beneficial to the food industry is discussed. This review is expected to enhance greater recognition of how important the cricket genomes are to the multiple biological fields and how basic research based on cricket genome information can contribute to tackling global food security. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-021-00924-4.

A nasal double DNA adjuvant system induces atheroprotective IgM antibodies via dendritic cell-B-1a B cell interactions.

We previously demonstrated that the dendritic cell (DC)-targeting nasal double DNA adjuvant system, which consists of a DNA plasmid expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotide 1826 (CpG ODN), elicits specific immune responses to various antigens in the mucosal and systemic compartments. Here, we investigated, using phosphorylcholine (PC)-conjugated keyhole limpet hemocyanin (PC-KLH) as an antigen, whether the nasal double DNA adjuvant system induces protective immunity to atherosclerosis in apolipoprotein E-deficient (ApoE KO) mice. Further, we assessed the molecular and cellular mechanisms in the induction of anti-PC-specific immune responses. Nasal immunization with PC-KLH plus pFL and CpG ODN enhanced induction of PC-specific IgM in plasma, peritoneal fluids, and nasal washes when compared with mice administered PC-KLH alone. Of importance, these antibodies exhibited highly specific binding to the PC molecule, and dose-dependent binding to anti-T15 idiotype (AB1-2). Twelve weeks after the last immunization, the nasal double DNA adjuvant system with PC-KLH resulted in a reduction of atherogenesis in the aortic arch of ApoE KO mice. Therefore, we next assessed immunocytological mechanism to induce these antibodies. The nasal double DNA adjuvant system with PC-KLH resulted not only in significantly increased frequencies of CD11c+ DCs in the spleen, peritoneal cavity (PEC), and nasopharyngeal-associated lymphoid tissues (NALT), but also significantly increased expression of a proliferation-inducing ligand and B-cell-activating factor by CD11c+ DCs. In addition, the double DNA adjuvant system induced significantly increased numbers of B-1 B cells in the spleen, PEC, and NALT, and increased expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor on CD5+ B220+ (B-1a) B cells. These findings demonstrated that the nasal double DNA adjuvant system with PC-KLH resulted in the induction of T15-like antibodies in the mucosal and systemic lymphoid tissues through interaction between DCs and B-1a B cells, and inhibited the progression of atherogenesis.

The nasal dendritic cell-targeting Flt3 ligand as a safe adjuvant elicits effective protection against fatal pneumococcal pneumonia.

We have previously shown that a pneumococcal surface protein A (PspA)-based vaccine containing DNA plasmid encoding the Flt3 ligand (FL) gene (pFL) as a nasal adjuvant prevented nasal carriage of Streptococcus pneumoniae. In this study, we further investigated the safety and efficacy of this nasal vaccine for the induction of PspA-specific antibody (Ab) responses against lung infection with S. pneumoniae. C57BL/6 mice were nasally immunized with recombinant PspA/Rx1 (rPspA) plus pFL three times at weekly intervals. When dynamic translocation of pFL was initially examined, nasal pFL was taken up by nasal dendritic cells (DCs) and epithelial cells (nECs) but not in the central nervous systems, including olfactory nerve and epithelium. Of importance, nasal pFL induced FL protein synthesis with minimum levels of inflammatory cytokines in the nasal washes (NWs) and bronchoalveolar lavage fluid (BALF). NWs and BALF as well as plasma of mice given nasal rPspA plus pFL contained increased levels of rPspA-specific secretory IgA and IgG Ab responses that were correlated with elevated numbers of CD8(+) and CD11b(+) DCs and interleukin 2 (IL-2)- and IL-4-producing CD4(+) T cells in the nasal mucosa-associated lymphoid tissues (NALT) and cervical lymph nodes (CLNs). The in vivo protection by rPspA-specific Abs was evident in markedly reduced numbers of CFU in the lungs, airway secretions, and blood when mice were nasally challenged with Streptococcus pneumoniae WU2. Our findings show that nasal pFL is a safe and effective mucosal adjuvant for the enhancement of bacterial antigen (Ag) (rPspA)-specific protective immunity through DC-induced Th2-type and IL-2 cytokine responses.

Respiratory FimA-Specific Secretory IgA Antibodies Upregulated by DC-Targeting Nasal Double DNA Adjuvant Are Essential for Elimination of Porphyromonas gingivalis.

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis (P. gingivalis) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis, a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA (rFimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal rFimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal rFimA plus FL/CpG resulted in increased levels of rFimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given rFimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by rFimA-stimulated CD4+ T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with rFimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal rFimA plus FL/CpG were challenged with nasal P. gingivalis, the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated rFimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with rFimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients.

Comparative Analysis of Mitochondrial Genomes in Gryllidea (Insecta: Orthoptera): Implications for Adaptive Evolution in Ant-Loving Crickets.

Species of infraorder Gryllidea, or crickets, are useful invertebrate models for studying developmental biology and neuroscience. They have also attracted attention as alternative protein sources for human food and animal feed. Mitochondrial genomic information on related invertebrates, such as katydids, and locusts, has recently become available in attempt to clarify the controversial classification schemes, although robust phylogenetic relationships with emphasis on crickets remain elusive. Here, we report newly sequenced complete mitochondrial genomes of crickets to study their phylogeny, genomic rearrangements, and adaptive evolution. First, we conducted de novo assembly of mitochondrial genomes from eight cricket species and annotated protein-coding genes and transfer and ribosomal RNAs using automatic annotations and manual curation. Next, by combining newly described protein-coding genes with public data of the complete Gryllidea genomes and gene annotations, we performed phylogenetic analysis and found gene order rearrangements in several branches. We further analyzed genetic signatures of selection in ant-loving crickets (Myrmecophilidae), which are small wingless crickets that inhabit ant nests. Three distinct approaches revealed two positively selected sites in the cox1 gene in these crickets. Protein 3D structural analyses suggested that these selected sites could influence the interaction of respiratory complex proteins, conferring benefits to ant-loving crickets with a unique ecological niche and morphology. These findings enhance our understanding of the genetic basis of cricket evolution without relying on estimates based on a limited number of molecular markers.