Insufficient anti-spike RBD IgA responses after triple vaccination with intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2.

Objectives: This study aims to examine whether the parenterally administered mRNA-based COVID-19 vaccines can induce sufficient mucosal-type IgA responses to prevent SARS-CoV-2 transmission. Methods: We examined the longitudinal kinetics of SARS-CoV-2 spike RBD-specific IgA and IgG responses in sera of Japanese healthcare workers (HCWs) after receiving two doses and the third dose of BNT162b2 mRNA vaccines. During the prospective cohort study, Omicron breakthrough infections occurred in 62 participants among 370 HCWs who had received triple doses of the vaccine. Pre-breakthrough sera of infected HCWs and non-infected HCWs were examined for the levels of anti-RBD IgA and IgG titers. Results: The seropositivity of anti-RBD IgA at 1 M after the second vaccine (2D-1M) and after the third dose (3D-1M) was 65.4% and 87.4%, respectively, and wanes quickly. The boosting effect on anti-RBD Ab titers following breakthrough infections was more notable for anti-RBD IgA than for IgG. There were partial cause-relationships between the lower anti-RBD IgA or IgG at pre-breakthrough sera and the breakthrough infection. Conclusions: Parenterally administered COVID-19 vaccines do not generate sufficient mucosal-type IgA responses despite strong systemic IgG responses to SARS-CoV-2. These results demonstrate the necessity and importance of reevaluating vaccine design and scheduling to efficiently increase oral or respiratory mucosal immunity against SARS-CoV-2.

The appearance of anti-spike receptor binding domain immunoglobulin G4 responses after repetitive immunization with messenger RNA-based COVID-19 vaccines.

OBJECTIVES: It is crucial to analyze the consequences of repeated messenger RNA (mRNA)-based COVID-19 vaccinations on SARS-CoV-2 spike receptor binding domain (RBD)-specific immunoglobulin (Ig)G subclass and the possible causal relationship with breakthrough infection. METHODS: We examined the longitudinal kinetics of RBD-specific IgG subclass antibodies in sera after receiving the second, third, and fourth doses of mRNA-based COVID-19 vaccines in Japanese healthcare workers. Anti-RBD IgG subclass in sera of patients with COVID-19-infected who had not received the COVID-19 vaccine were also examined. We compared anti-RBD IgG subclass antibody titers in the serum of pre-breakthrough-infected vaccinees and non-infected vaccinees. RESULTS: The seropositivity of anti-RBD IgG4 after the vaccination was 6.76% at 1 month after the second dose, gradually increased to 50.5% at 6 months after the second dose, and reached 97.2% at 1 month after the third dose. The seropositivity and titers of anti-RBD IgG1/IgG3 quickly reached the maximum at 1 month after the second dose and declined afterward. The elevated anti-RBD IgG4 Ab levels observed after repeated vaccinations were unlikely to increase the risk of breakthrough infection. CONCLUSIONS: Repeated vaccinations induce delayed but drastic increases in anti-RBD IgG4 responses. Further functional investigations are needed to reveal the magnitude of the high contribution of spike-specific IgG4 subclasses after repeated mRNA-based COVID-19 vaccinations.

Chlamydia pneumoniae Lung Infection in Mice Induces Fatty Acid-Binding Protein 4-Dependent White Adipose Tissue Pathology.

Fatty acid-binding protein 4 (FABP4) is a critical immune-metabolic modulator, mainly expressed in adipocytes and macrophages, secreted from adipocytes in association with lipolysis, and plays essential pathogenic roles in cardiovascular and metabolic diseases. We previously reported Chlamydia pneumoniae infecting murine 3T3-L1 adipocytes and causing lipolysis and FABP4 secretion in vitro. However, it is still unknown whether C. pneumoniae intranasal lung infection targets white adipose tissues (WATs), induces lipolysis, and causes FABP4 secretion in vivo. In this study, we demonstrate that C. pneumoniae lung infection causes robust lipolysis in WAT. Infection-induced WAT lipolysis was diminished in FABP4-/- mice or FABP4 inhibitor-pretreated wild-type mice. Infection by C. pneumoniae in wild-type but not FABP4-/- mice induces the accumulation of TNF-α- and IL-6-producing M1-like adipose tissue macrophages in WAT. Infection-induced WAT pathology is augmented by endoplasmic reticulum (ER) stress/the unfolded protein response (UPR), which is abrogated by treatment with azoramide, a modulator of the UPR. C. pneumoniae lung infection is suggested to target WAT and induce lipolysis and FABP4 secretion in vivo via ER stress/UPR. FABP4 released from infected adipocytes may be taken up by other neighboring intact adipocytes or adipose tissue macrophages. This process can further induce ER stress activation and trigger lipolysis and inflammation, followed by FABP4 secretion, leading to WAT pathology. A better understanding of the role of FABP4 in C. pneumoniae infection-induced WAT pathology will provide the basis for rational intervention measures directed at C. pneumoniae infection and metabolic syndrome, such as atherosclerosis, for which robust epidemiologic evidence exists.

Bortezomib Eliminates Persistent Chlamydia trachomatis Infection through Rapid and Specific Host Cell Apoptosis.

Chlamydia trachomatis, a parasitic intracellular bacterium, is a major human pathogen that causes millions of trachoma, sexually transmitted infections, and pneumonia cases worldwide. Previously, peptidomimetic inhibitors consisting of a hydrophobic dipeptide derivative exhibited significant inhibitory effects against chlamydial growth. Based on this finding, this study showed that both bortezomib (BTZ) and ixazomib (IXA), anticancer drugs characterized by proteasome inhibitors, have intensive inhibitory activity against Chlamydia. Both BTZ and IXA consisted of hydrophobic dipeptide derivatives and strongly restricted the growth of Chlamydia (BTZ, IC50 = 24 nM). In contrast, no growth inhibitory effect was observed for other nonintracellular parasitic bacteria, such as Escherichia coli. BTZ and IXA appeared to inhibit chlamydial growth bacteriostatically via electron microscopy. Surprisingly, Chlamydia-infected cells that induced a persistent infection state were selectively eliminated by BTZ treatment, whereas uninfected cells survived. These results strongly suggested the potential of boron compounds based on hydrophobic dipeptides for treating chlamydial infections, including persistent infections, which may be useful for future therapeutic use in chlamydial infectious diseases.

Longitudinal Dynamics of SARS-CoV-2 IgG Antibody Responses after the Two-Dose Regimen of BNT162b2 Vaccination and the Effect of a Third Dose on Healthcare Workers in Japan.

Analysis of longitudinal dynamics of humoral immune responses to the BNT162b2 COVID-19 vaccine might provide useful information to predict the effectiveness of BNT162b2 in preventing SARS-CoV-2 infection. Herein, we measure anti-RBD IgG at 1, 3 and 6 months (M) after the second dose of BNT162b2, and at 1 M after a third dose of BNT162b2 vaccination in 431 COVID-19-naïve healthcare workers (HCWs) in Japan. All HCWs mounted high-anti-RBD IgG responses after the two-dose regimen of BNT162b2 vaccinations. Older persons and males presented lower anti-RBD IgG responses than younger adults and females, respectively. The decay in anti-RBD IgG started from 1 M after the second dose of BNT162b2 and anti-RBD IgG titers dropped to nearly one-tenth at 6 M after the second vaccination. Subsequently, the participants received a third dose of BNT162b2 at 8 M after the second dose of BNT162b2 vaccine. Anti-RBD antibody titers 1 M after the third dose of BNT162b2 increased seventeen times that of 6 M after the second dose, and was twice higher than the peak antibody titers at 1 M after the second dose of vaccination. The negative effect of age for the male gender on anti-RBD IgG antibody titers was not observed at 1 M after the third dose of BNT162b2 vaccine. There were no notable adverse events reported, which required hospitalization in these participants. These results suggest that the third dose of BNT162b2 safely improves humoral immunity against SARS-CoV-2 with no major adverse events.

Anti-chlamydial activities of cell-permeable hydrophobic dipeptide-containing derivatives.

The obligate intracellular bacteria chlamydia is major human pathogen that causes millions of trachoma, sexually transmitted infections and pneumonia worldwide. We serendipitously found that both calpain inhibitors z-Val-Phe-CHO and z-Leu-Nle-CHO showed marked inhibitory activity against chlamydial growth in human epithelial HeLa cells, whereas other calpain inhibitors not. These peptidomimetic inhibitors consist of N-benzyloxycarbonyl group and hydrophobic dipeptide derivatives. Both compounds strongly restrict the chlamydial growth even addition at the 12 h post infection. Notably, inhibitors-mediated growth inhibition of chlamydia was independent on host calpain activity. Electron microscopic analysis revealed that z-Val-Phe-CHO inhibited chlamydial growth by arresting bacterial cell division and RB-EB re-transition, but not by changing into persistent state. We searched and found that z-Leu-Leu-CHO and z-Phe-Ala-FMK also inhibited chlamydial growth. Neither biotin-hydrophobic dipeptide nor morpholinoureidyl-hydrophobic dipeptide shows inhibitory effects on chlamydial intracellular growth. Our results suggested the possibility of some chemical derivatives based on z-hydrophobic dipeptide group for future therapeutic usage to the chlamydial infectious disease.

Chlamydia trachomatis targets mitochondrial dynamics to promote intracellular survival and proliferation.

Chlamydia trachomatis is an obligate intracellular bacterium that scavenges host metabolic products for its replication. Mitochondria are the power plants of eukaryotic cells and provide most of the cellular ATP via oxidative phosphorylation. Several intracellular pathogens target mitochondria as part of their obligatory cellular reprogramming. This study was designed to analyse the mitochondrial morphological changes in response to C. trachomatis infection in HeLa cells. Mitochondrial elongation and fragmentation were found at the early stages and late stages of C. trachomatis infection, respectively. C. trachomatis infection-induced mitochondrial elongation was associated with the increase of mitochondrial respiratory activity, ATP production, and intracellular growth of C. trachomatis. Silencing mitochondrial fusion mediator proteins abrogated the C. trachomatis infection-induced elevation in the oxygen consumption rate and attenuated chlamydial proliferation. Mechanistically, C. trachomatis induced the elevation of intracellular cAMP at the early phase of infection, followed by the phosphorylation of fission-inactive serine residue 637 (S637) of Drp1, resulting in mitochondrial elongation. Accordingly, treatment with adenylate cyclase inhibitor diminished mitochondrial elongation and bacterial growth in infected cells. Collectively, these results strongly indicate that C. trachomatis promotes its intracellular growth by targeting mitochondrial dynamics to regulate ATP synthesis via inhibition of the fission mediator Drp1.

Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum.

Peroxisome biogenesis disorders (PBDs) manifest as neurological deficits in the central nervous system, including neuronal migration defects and abnormal cerebellum development. However, the mechanisms underlying pathogenesis remain enigmatic. Here, to investigate how peroxisome deficiency causes neurological defects of PBDs, we established a new PBD model mouse defective in peroxisome assembly factor Pex14p, termed Pex14 ΔC/ΔC mouse. Pex14 ΔC/ΔC mouse manifests a severe symptom such as disorganization of cortical laminar structure and dies shortly after birth, although peroxisomal biogenesis and metabolism are partially defective. The Pex14 ΔC/ΔC mouse also shows malformation of the cerebellum including the impaired dendritic development of Purkinje cells. Moreover, extracellular signal-regulated kinase and AKT signaling are attenuated in this mutant mouse by an elevated level of brain-derived neurotrophic factor (BDNF) together with the enhanced expression of TrkB-T1, a dominant-negative isoform of the BDNF receptor. Our results suggest that dysregulation of the BDNF-TrkB pathway, an essential signaling for cerebellar morphogenesis, gives rise to the pathogenesis of the cerebellum in PBDs.

Chlamydia pneumoniae exploits adipocyte lipid chaperone FABP4 to facilitate fat mobilization and intracellular growth in murine adipocytes.

Fatty acid-binding protein 4 (FABP4), a cytosolic lipid chaperone predominantly expressed in adipocytes and macrophages, modulates lipid fluxes, trafficking, signaling, and metabolism. Recent studies have demonstrated that FABP4 regulates metabolic and inflammatory pathways, and in mouse models its inhibition can improve type 2 diabetes mellitus and atherosclerosis. However, the role of FABP4 in bacterial infection, metabolic crosstalk between host and pathogen, and bacterial pathogenesis have not been studied. As an obligate intracellular pathogen, Chlamydia pneumoniae needs to obtain nutrients such as ATP and lipids from host cells. Here, we show that C. pneumoniae successfully infects and proliferates in murine adipocytes by inducing hormone sensitive lipase (HSL)-mediated lipolysis. Chemical inhibition or genetic manipulation of HSL significantly abrogated the intracellular growth of C. pneumoniae in adipocytes. Liberated free fatty acids were utilized to generate ATP via ß-oxidation, which C. pneumoniae usurped for its replication. Strikingly, chemical inhibition or genetic silencing of FABP4 significantly abrogated C. pneumoniae infection-induced lipolysis and mobilization of liberated FFAs, resulting in reduced bacterial growth in adipocytes. Collectively, these results demonstrate that C. pneumoniae exploits host FABP4 to facilitate fat mobilization and intracellular replication in adipocytes. This work uncovers a novel strategy used by intracellular pathogens for acquiring energy via hijacking of the host lipid metabolism pathway.

Maoto, a Traditional Japanese Herbal Medicine, Inhibits Uncoating of Influenza Virus.

We previously reported in randomized controlled trials that maoto, a traditional herbal medicine, showed clinical and virological efficacy for seasonal influenza. In this study, a culturing system for influenza was used to test the effect of maoto. A549 cells in the culture were infected with influenza virus A (PR8) and followed after treatment with maoto; the virus titers in the culture supernatant, intracellular viral proteins, and viral RNA were determined. When infected cells were cultured with maoto for 24 hr, the virus titer and protein were significantly reduced compared with medium only. Other subtypes, A/H3N2, H1N1pdm, and B, were also inhibited by maoto. Proliferation of viral RNA in a 6 hr culture was inhibited by maoto in the early phase, especially in the first 30 min. Focusing on the entry step of the influenza virus, we found that endosomal pH, regulated by vacuolar-type H+ ATPase (V-ATPase) located in the membrane, was increased when treated with maoto. We also found that uncoating of influenza viruses was also inhibited by maoto, resulting in the increase of the number of virus particles in endosomes. These results strongly suggest that the inhibition of endosomal acidification by maoto results in blocking influenza virus entry to cytoplasm, probably through the inhibition of V-ATPase. The present study provides evidence that supports the clinical use of maoto for the treatment of influenza.

Pancreatic fistulae secondary to trypsinogen activation by Pseudomonas aeruginosa infection after pancreatoduodenectomy.

BACKGROUND: Pancreatic fistula after pancreatoduodenectomy (PD) is associated with high mortality and morbidity. Trypsinogen activation and bacteria, although hypothesized to be interrelated etiopathogenetically, have not had their relationship and pathogenic mechanisms elucidated. This study investigated bacterial involvement in pancreatic juice activation perioperatively after PD at sites of pancreatic fistula formation. METHODS: Fifty patients underwent PD; postoperative pancreatic fistulae were graded based on the International Study Group for Pancreatic Fistula grading criteria. Bacteria were isolated from cultures of drainage fluid. Digested peptides from trypsinogen and bacterial culture supernatants underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and mass spectrometric analysis. Zymography was used to detect the trypsinogen activator. RESULTS: Pseudomonas aeruginosa and Enterobacter cloacae isolated from drainage fluid in patients with grades B and C pancreatic fistulae could cause trypsinogen activation. Trypsinogen activation by P. aeruginosa and E. cloacae were preventable by the use of a serine protease inhibitor in vitro. A protease in the supernatant from P. aeruginosa-positive cultures acted as the trypsinogen activator. CONCLUSIONS: Infection with P. aeruginosa perioperatively to PD entails secretion of a protease activator of trypsinogen to trypsin. Bacterial infection control in the perioperative PD period could be crucial to prevent development of pancreatic fistula.

Chlamydia pneumoniae harness host NLRP3 inflammasome-mediated caspase-1 activation for optimal intracellular growth in murine macrophages.

Chlamydia pneumoniae is an obligate intracellular pathogen that replicates within a vacuole and acquires host cell nutrients. We show that C. pneumoniae utilizes host innate immune signaling NLRP3/ASC/caspase-1 inflammasome for intracellular growth. Bone marrow-derived macrophages (BMMs) secreted mature interleukin-1ß upon infection with C. pneumoniae depending on the NLRP3 inflammasome activation. Intracellular growth of C. pneumoniae was severely impaired in BMMs from Nlrp3(-/-), Asc(-/-), and Casp1(-/-) mice but not wild type or Nlrc4(-/-) mice. Furthermore defective NLRP3 inflammasome components led to accumulation of lipid droplets inside the infected BMMs, suggesting that uptake and/or utilization of lipids is disturbed in the absence of NLRP3 inflammasome activation. These results suggest C. pneumoniae has evolved to harness both host innate immune response and NLRP3 inflammasome activation, for the acquisition of essential nutrients necessary for intracellular growth. This unique property of C. pneumoniae may shed a new light on how C. pneumoniae increase the risk of atherosclerosis and metabolic syndrome.

Vitamin D-dependent cathelicidin inhibits Mycobacterium marinum infection in human monocytic cells.

BACKGROUND: 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) up-regulates the production of human cathelicidin antimicrobial peptide (CAMP) from monocytes/macrophages infected with Mycobacterium tuberculosis (M. tbc). CAMP facilitates the co-localization of autophagolysosomes with M. tbc, promoting the antimicrobial activity of monocytes. Mycobacterium marinum (M. marinum) is an acid-fast bacillus that causes less severe granulomatous skin lesions compared with M. tbc. OBJECTIVE: We investigated whether autophagic antimicrobial activity is promoted by 1,25(OH)2D3 or C-terminal of cathelicidin LL-37 in human monocytes upon infection with M. marinum. METHODS: Human monocytes (THP-1) were infected with M. marinum. Effects of simultaneous treatments of 1,25(OH)2D3, exogenous LL-37 peptide, autophagolysosome inhibitors, 3-methyladenine or chloroquine, were examined. RESULTS: CAMP was strongly induced by adding 1,25(OH)2D3 to the culture of THP-1 cells. In the absence of 1,25(OH)2D3 M. marinum infection alone did not induce CAMP, however, simultaneous addition of 1,25(OH)2D3 to M. marinum infection accelerated CAMP production more than 1,25(OH)2D3 alone. Proliferation of M. marinum was markedly decreased in the presence of 1,25(OH)2D3 or exogenous LL-37 in THP-1 cells. Co-localization of CAMP with autophagolysosome was evident in 1,25(OH)2D3 and LL-37 treated THP-1 cells after M. marinum infection. Autophagolysosome inhibitors abrogated the antimicrobial effects of 1,25(OH)2D3 and exogenous LL-37 against M. marinum infection in THP-1 cells. CONCLUSIONS: Human monocytic cells, whose CAMP production is up-regulated by 1,25(OH)2D3-vitamin D receptor pathway, accelerate antimicrobial function of autophagolysosome in M. marinum infection.

Osteoplastic ameloblastoma: a case report and literature review.

Ameloblastoma with bone formation is rare. We report a case of a 55-year-old woman with ameloblastoma accompanied by prominent osteoplasia. Histopathological examination exhibited an abundant stromal component between tumor nests. Therefore, she was diagnosed as the desmoplastic variant, except for the numerous bone trabeculae. The distinction between new bone formation and invasion of the bone marrow poses a problem. A thin rim of fibrous bone that can be accentuated by Masson-trichrome staining suggests the former.

Stimulatory actions of lysophosphatidic acid on mouse ATDC5 chondroprogenitor cells.

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive lysophospholipids that affect various cellular processes through G protein-coupled receptors. In our current study, we found by in situ hybridization that E11.5 mouse embryos strongly expressed the LPA receptor subtype LPA(1) in cartilaginous bone primordia and the surrounding mesenchymal cells. However, despite their wide-ranging actions, the roles of lysophospholipids in chondrogenesis remain poorly understood. The mouse clonal cell line ATDC5 undergoes a sequential differentiation of chondroprogenitor cells in vitro. Undifferentiated and differentiated ATDC5 cells express LPA(1) and other lysophospholipid receptors including S1P receptor S1P(1) and S1P(2). Taking advantage of this cell model, we studied the effects of LPA on the activities of chondroprogenitor cells. LPA markedly stimulates both DNA synthesis and the migration of ATDC5 chondroprogenitor cells in culture, whereas S1P suppresses the migration of these cells. Treatment with Ki16425, an LPA(1)- and LPA(3)-specific receptor antagonist, suppressed the fetal bovine serum-stimulated migration of ATDC5 cells by almost 80%. These results indicate that LPA plays an important role in the activation of chondroprogenitor cells.

Functional domains and dynamic assembly of the peroxin Pex14p, the entry site of matrix proteins.

The 41-kDa membrane-anchored peroxin Pex14p functions as the peroxisome targeting signal (PTS) receptor-mediated, initial import site for matrix proteins. We here identify the functional domains of Pex14p involved in the assembly of import site subcomplexes. The minimal region of Pex14p required for restoring impaired protein import in pex14 Chinese hamster ovary cell mutant lies at residues 21-260 in the primary sequence. A highly conserved N-terminal region, encompassing residues 21-70, interacts with the PTS1 receptor Pex5p, Pex13p, and Pex19p that is essential for membrane biogenesis. N-terminal residues 21-140, including a hydrophobic segment at 110-138, function as a topogenic sequence. Site-directed mutagenesis, size fractionation, and chemical cross-linking analyses demonstrate that the coiled-coil domain at residues 156-197 regulates homodimerization of Pex14p. Moreover, AXXXA and GXXXG motifs in the transmembrane segment mediate homomeric oligomerization of Pex14p, giving rise to assembly of high molecular mass complexes and thereby assuring Pex13p-dependent localization of Pex14p to peroxisomes. Pex5p, Pex13p, and Pex19p bind to Pex14p homo-oligomers with different molecular masses, whereas cargo-unloaded Pex5p apparently disassembles Pex14p homo-oligomers. Thus, Pex14p most likely forms several distinct peroxin complexes involved in peroxisomal matrix protein import.