Anti‐PcrV Immunization for Pseudomonas aeruginosa Pneumonia in Cystic Fibrosis

Propagation of multidrug‐resistant Pseudomonas aeruginosa, which causes endemic nosocomial infections, has become a major concern in various parts of the world. In patients with cystic fibrosis, a major cause of death is respiratory tract infections with antibiotic‐resistant P. aeruginosa. This condition has prompted medical research aimed at developing effective prophylaxis and treatments that do not rely on conventional antimicrobial agents. The pathogenesis that results in cytotoxicity and mortality in immunocompromised patients infected with P. aeruginosa is associated with the type III secretion system of this bacterium. Clinical isolates that are cytotoxic and drug‐resistant are involved in acute exacerbation of chronic infectious diseases. The P. aeruginosa V‐antigen PcrV, a Yersinia V‐antigen LcrV homolog, is involved as an indispensable component in the translocational process of type III secretory (TTS) toxins. Vaccination against PcrV ensures survival of infection‐challenged mice and decreases lung inflammation and injury. Furthermore, anti‐PcrV IgG can inhibit translocation of TTS toxins. These observations support the hypothesis that anti‐PcrV strategies have the potential as nonantibiotic immune strategies for preventing aggravation of P. aeruginosa infections in patients with cystic fibrosis

Construction and Characteristics of a Recombinant Single- Chain Antibody Fragment against Bacterial Type III Secretion

Pseudomonas aeruginosa, a Gram-negative pathogen, causes life-threatening infections. Lung injury and the development of sepsis depend largely on expression of the virulence genes associated with the type III secretion system of this bacterium. The type III secretion system functions as a molecular syringe to deliver type III secretory toxins directly into the cytosol of eukaryotic cells and also acts to inhibit innate immune mechanisms, thereby preventing bacterial clearance. Antibodies against PcrV, the cap structure in the translocational needle of type III secretory apparatus of P. aeruginosa, block toxin translocation of the type III secretion system. We have been investigating the therapeutic use of a recombinant anti-PcrV single-chain antibody. In this chapter, as a preliminary step toward an antibody-based immunotherapy against bacterial infections, we summarize our experience of constructing a recombinant single-chain antibody (called scFv166), in which the heavy (VH) and light chain (VL) variable regions of the anti-PcrV monoclonal IgG are joined by a flexible peptide linker. The practical methodologies used to make recombinant scFv166 against a bacterial protein component are described in detail

Basement Membrane beneath Serous Mesothelial Cells Contains α1(IV), α2(IV), α5(IV), and α6(IV) Chains of Type IV Collagen Demonstrated by Chain-specific Monoclonal Antibodies

Serous membrane (SM) covers inner surface of abdominal, thoracic and pericardial cavities, aswell as outer surface of organs inside the cavities. It consists of surface mesothelial cells andloose connective tissue. Between them, a thim layer of basement membrane (BM) is located. Type IVcollagen is major constituent of BM, and consists of 6 different a(IV) chains, a1(IV) through a6(IV).Chain-specific functions are assumed by a chain-specific manner of localization. The a(IV) chaincomposition of skin, covering outer surface of the body, is demonstrated to have a1(IV), a2(IV), a5(IV),and a6(IV) chains, whereas that of SM, covering inner surface of the body, is yet to be analyzed. Abdominal wall, small intestine, thoracic wall, lung, pericardium and epicardium of humanmaterials were used in this study. Chain-specific monoclonal antibodies (mAbs) used were H11(for a1), H22 (for a2), H31 (fo a3), H43 (for a4), H53 (for a5) and H63 (for a6). Fresh frozen sectionswere stained with indirect immunofluorescence staining using the mAbs. Four out of six a(IV) chains, a1, a2, a5 and a6, were demonstrated in BM beneath themesothelial cells of all types of SMs, whereas only capillary BM consisted of a1 and a2. Besides,epicardial SM expressed a3 and a4 moderately as extra components. The a(IV) chain composition was same as those of epidermal skin BM. Therefore, these a(IV)chains are designated to be essential for BM covering inside and outside of the body

Drosophila type XV/XVIII collagen, Mp, is involved in Wingless distribution

Multiplexin (Mp) is the Drosophila orthologue of vertebrate collagens XV and XVIII. Like them, Mp is widely distributed in the basement membranes of the developing embryos, including those of neuroblasts in the central and peripheral nervous systems, visceral muscles of the gut, and contractile cardioblasts. Here we report the identification of mutant larvae bearing piggyBac transposon insertions that exhibit decrease Mp production associated with abdominal cuticular and wing margin defects, malformation of sensory organs and impaired sensitivity to physical stimuli. Additional findings include the abnormal ultrastructure of fatbody associated with abnormal collagen IV deposition, and reduced Wingless deposition. Collectively, these findings are consistent with the notion that Mp is required for the proper formation and/or maintenance of basement membrane, and that Mp may be involved in establishing the Wingless signaling gradients in the Drosophila embryo

Id2-, RORγt-, and LTβR-independent initiation of lymphoid organogenesis in ocular immunity

The eye is protected by the ocular immunosurveillance system. We show that tear duct–associated lymphoid tissue (TALT) is located in the mouse lacrimal sac and shares immunological characteristics with mucosa-associated lymphoid tissues (MALTs), including the presence of M cells and immunocompetent cells for antigen uptake and subsequent generation of mucosal immune responses against ocularly encountered antigens and bacteria such as Pseudomonas aeruginosa. Initiation of TALT genesis began postnatally; it occurred even in germ-free conditions and was independent of signaling through organogenesis regulators, including inhibitor of DNA binding/differentiation 2, retinoic acid–related orphan receptor γt, lymphotoxin (LT) α1β2–LTβR, and lymphoid chemokines (CCL19, CCL21, and CXCL13). Thus, TALT shares immunological features with MALT but has a distinct tissue genesis mechanism and plays a key role in ocular immunity

Oral intake of Lactobacillus pentosus strain b240 accelerates salivary immunoglobulin A secretion in the elderly: A randomized, placebo-controlled, double-blind trial

<p>Abstract</p> <p>Background</p> <p>Immunoglobulin A (IgA) secretion in saliva decreases with age and may be the cause of increased vulnerability of the elderly to respiratory infections. The effect of oral intake of lactic acid bacteria on salivary secretory IgA (SIgA) in the elderly has not been reported. The objective of this study was to demonstrate the acceleration of salivary SIgA secretion by oral intake of <it>Lactobacillus pentosus </it>strain b240 (b240) in the elderly.</p> <p>Results</p> <p>A total of 80 healthy elderly individuals were randomly allocated to either an intervention (i.e., b240) or a control (i.e., placebo) group. The elderly individuals in the b240 group were given a sterile water beverage (125 mL) containing heat-killed b240 (4 × 10⁹cells), while those in the placebo group were given only a sterile water beverage (125 mL); both groups received their respective beverages once daily for 12 weeks. Saliva was collected before initiation of the study and every 2 weeks thereafter. Saliva flow rate and SIgA concentration were determined, and the SIgA secretion rate was calculated. The mean salivary SIgA secretion rate in the b240 group steadily increased until week 4 (exhibiting a 20% elevation relative to that at week 0), and then remained stable until week 12. Changes in SIgA secretion rate over the intervention period were significantly greater in the b240 group than in the placebo group. The treatment groups exhibited no significant differences in adverse events.</p> <p>Conclusions</p> <p>Oral intake of <it>L. pentosus </it>strain b240 for 12 weeks significantly accelerated salivary SIgA secretion, thereby indicating its potential utility in the improvement of mucosal immunity and resistance against infection in the elderly.</p

Candida albicansマンナンのマクロファージ傷害作用

Mannan of Candida albicans NIH A-207 strain induced cytotoxic activity in RAW264.7 cells, a murine macrophage cell line, but not in U937 cells, a human monocyte cell line. The mannan greatly increased TNF-α production in RAW264.7 cells. Anti-TNF-α antibody completely inhibited both the TNF-α production from RAW264.7 cells and the cytotoxicity of the cells by mannan. Commercial recombinant TNF-α showed cytotoxic activity in RAW264.7 cells

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection