Detection of trichomonads in patients with lung cancer and transcription analysis on the response of human pulmonary epithelial cells to Trichomonas tenax invasion.

INTRODUCTION: Lung cancer is one of the most prevalent malignancies worldwide. Substantial research has illuminated the intricate interplay between microorganisms and human health, revealing their role in disease regulation. Trichomonads is a flagellated protozoan in the human cavity and have been previously identified as a pathogen associated with pneumonia, contributing to tissue chronic inflammation and carcinogenesis. METHODS: Nested polymerase chain reaction methods were employed to scrutinize the prevalence of trichomonads in the bronchovesicular fluid of patients diagnosed with lung cancer. Subsequently, the influence of Trichomonas tenax invasion on lung cancer cells was elucidated through proliferation assays, migration assays, and transcription analysis. RESULTS: Bronchoalveolar fluid samples from lung cancer patients yielded positive nested PCR results for eight out of twenty-seven samples. Seven of these samples were identified as Trichomonas tenax, while one was identified as Tetratrichomonas spp. Our findings revealed a significant upregulation of pathways associated with carcinogenesis, including cellular proliferation, migration, and drug resistance, in response to T. tenax invasion. CONCLUSIONS: This study underscores the importance of recognizing the presence of trichomonads and the influence of T. tenax invasion on host responses to respiratory diseases. The identified pathways implicated in cancer development may pave the way for developing targeted treatment strategies for pulmonary diseases. These findings hold promise for informing and improving the precision of therapeutic interventions in the context of pulmonary ailments.

Immunization with lytic polysaccharide monooxygenase CbpD induces protective immunity against Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.

Potential role of Acanthamoeba Rab7.

Acanthamoeba castellanii is a free-living protozoan that causes several severe human parasitic diseases such as Acanthamoeba keratitis and granulomatous encephalitis. A. castellanii feeds on bacteria, yeasts, and other organic particles as food sources, but the mechanisms of digestion in acanthamoebal cells are unclear. Rab GTPases participate in endosomal delivery in eukaryotes after phagocytosis. This study aimed to determine the potential functions of A. castellanii Rab7 (AcRab7), which is involved in phagocytosis, and the relationship between AcRab7 and further cellular physiological phenomena. In this study, the inhibitor CID1067700 (CID) was used to specifically inhibit the binding of nucleotides to confirm the potential functions of AcRab7. Cellular proliferation and ATP assays were also used to detect underlying cellular physiological functions after blocking the phagocytosis pathway. We found that AcRab7 expression increased as the co-culture time with Escherichia coli increased. Immunofluorescence staining showed that AcRab7 colocalized with lysosomes in its GTP-activating form. In addition, AcRab7 inhibition resulted in a reduction in cell proliferation and ATP levels. Our results suggest that AcRab7 participates in endosomal delivery and dominates energy production and cell growth.

Siglec-14 Enhances NLRP3-Inflammasome Activation in Macrophages.

Pathogenic microorganisms are sensed by the inflammasome, resulting in the release of the pro-immune and proinflammatory cytokine interleukin-1ß (IL-1ß). In humans, the paired sialic acid-binding Ig-like lectin receptors Siglec-5 (inhibitory) and Siglec-14 (activating) have been shown to have reciprocal roles in regulating macrophage immune responses, but their interaction with IL-1ß signaling and the inflammasome has not been characterized. Here we show that in response to known inflammasome activators (ATP, nigericin) or the sialic acid-expressing human bacterial pathogen group B Streptococcus (GBS), the presence of Siglec-14 enhances, whereas Siglec-5 reduces, inflammasome activation and macrophage IL-1ß release. Human THP-1 macrophages stably transfected with Siglec-14 exhibited increased caspase-1 activation, IL-1ß release and pyroptosis after GBS infection, in a manner blocked by a specific inhibitor of nucleotide-binding domain leucine-rich repeat protein 3 (NLRP3), a protein involved in inflammasome assembly. Another leading pathogen, Streptococcus pneumoniae, lacks sialic acid but rather prominently expresses a sialidase, which cleaves sialic acid from macrophages, eliminating cis- interactions with the lectin receptor, thus attenuating Siglec-14 induced IL-1ß secretion. Vimentin, a cytoskeletal protein released during macrophage inflammatory activation is known to induce the inflammasome. We found that vimentin has increased interaction with Siglec-14 compared to Siglec-5, and this interaction heightened IL-1ß production by Siglec-14-expressing cells. Siglec-14 is absent from some humans because of a SIGLEC5/14 fusion polymorphism, and we found increased IL-1ß expression in primary macrophages from SIGLEC14+/+ individuals compared to those with the SIGLEC14-/+ and SIGLEC14-/- genotypes. Collectively, our results identify a new immunoregulatory role of Siglec-14 as a positive regulator of NLRP3 inflammasome activation.

The long noncoding RNA ROCKI regulates inflammatory gene expression.

Long noncoding RNAs (lncRNAs) can regulate target gene expression by acting in cis (locally) or in trans (non-locally). Here, we performed genome-wide expression analysis of Toll-like receptor (TLR)-stimulated human macrophages to identify pairs of cis-acting lncRNAs and protein-coding genes involved in innate immunity. A total of 229 gene pairs were identified, many of which were commonly regulated by signaling through multiple TLRs and were involved in the cytokine responses to infection by group B Streptococcus We focused on elucidating the function of one lncRNA, named lnc-MARCKS or ROCKI (Regulator of Cytokines and Inflammation), which was induced by multiple TLR stimuli and acted as a master regulator of inflammatory responses. ROCKI interacted with APEX1 (apurinic/apyrimidinic endodeoxyribonuclease 1) to form a ribonucleoprotein complex at the MARCKS promoter. In turn, ROCKI-APEX1 recruited the histone deacetylase HDAC1, which removed the H3K27ac modification from the promoter, thus reducing MARCKS transcription and subsequent Ca2+ signaling and inflammatory gene expression. Finally, genetic variants affecting ROCKI expression were linked to a reduced risk of certain inflammatory and infectious disease in humans, including inflammatory bowel disease and tuberculosis. Collectively, these data highlight the importance of cis-acting lncRNAs in TLR signaling, innate immunity, and pathophysiological inflammation.

Propionibacterium acnes-induced immunopathology correlates with health and disease association.

Genomic studies revealed the existence of health- and acne-associated P. acnes strains and suggested novel approaches for broadening understanding of acne vulgaris. However, clinical association of P. acnes with disease or health has yet to be corroborated experimentally. Current animal models of acne do not closely mimic human disease and have unclear translational value. We have developed a murine model of acne by combining P. acnes inoculation with topical application of a synthetic human sebum. We showed that human sebum promoted persistence of intradermally injected P. acnes with little loss of viability after 1 week and permitted use of more physiologic inoculums. Application of acne-associated P. acnes RT4/5 strains led to development of moderate to severe skin pathology compared with application of health-associated type II P. acnes strains (RT2/6). RT4/5 P. acnes strains uniformly induced higher levels of KC (IL-8), IL-1α, IL-1ß, and IL-6 in vitro and in vivo compared with type II P. acnes strains. Overall, our data provide immunopathologic corroboration of health and disease association of clinical P. acnes strains and inform on a platform to query putative virulence factors uncovered by genomic studies.

Trichomonas vaginalis Induces NLRP3 Inflammasome Activation and Pyroptotic Cell Death in Human Macrophages.

Trichomonas vaginalis is a sexually transmitted, eukaryotic parasite that causes trichomoniasis, the most common nonviral, sexually transmitted disease in the USA and worldwide. Little is known about the molecular mechanisms involved in the host immune response to this widespread parasite. Here we report that T. vaginalis induces NLRP3 inflammasome activation in human macrophages, leading to caspase-1 activation and the processing of pro-IL-1ß to the mature and bioactive form of the cytokine. Using inhibitor-based approaches, we show that NLRP3 activation by T. vaginalis involves host cell detection of extracellular ATP via P2X7 receptors and potassium efflux. In addition, our data reveal that T. vaginalis inflammasome activation induces macrophage inflammatory cell death by pyroptosis, known to occur via caspase-1 cleavage of the gasdermin D protein, which assembles to form pores in the host cell membrane. We found that T. vaginalis-induced cytolysis of macrophages is attenuated in gasdermin D knockout cells. Lastly, in a murine challenge model, we detected IL-1ß production in vaginal fluids in response to T. vaginalis infection in vivo. Together, our findings mechanistically dissect how T. vaginalis contributes to the production of the proinflammatory IL-1ß cytokine and uncover pyroptosis as a mechanism by which the parasite can trigger host macrophage cell death.

Siglec-7 engagement by GBS ß-protein suppresses pyroptotic cell death of natural killer cells.

Natural killer (NK) cells are innate immune lymphocytes that recognize and destroy abnormal host cells, such as tumor cells or those infected by viral pathogens. To safely accomplish these functions, NK cells display activating receptors that detect stress molecules or viral ligands displayed at the cell surface, balanced by inhibitory receptors that bind to self-molecules. To date, such activating and inhibitory receptors on NK cells are not known to recognize bacterial determinants. Moreover, NK cell responses to direct interactions with extracellular bacteria are poorly explored. In this study, we observed the human neonatal pathogen group B Streptococcus (GBS) can directly engage human NK cells. The interaction was mediated through the B6N segment of streptococcal ß-protein, binding to the inhibitory receptor Siglec-7 via its amino-terminal V-set domain. Unlike classical Siglec binding, the interaction is also independent of its sialic acid recognition property. In contrast to WT GBS, mutants lacking ß-protein induced efficient pyroptosis of NK cells through the NLRP3 inflammasome, with production and secretion of the proinflammatory cytokine IL-1ß and dissemination of the cytotoxic molecule granzyme B. We postulate that GBS evolved ß-protein engagement of inhibitory human Siglec-7 to suppress the pyroptotic response of NK cells and thereby block recruitment of a broader innate immune response, i.e., by "silencing the sentinel."

Breast cancer survival in Soweto, Johannesburg, South Africa: A receptor-defined cohort of women diagnosed from 2009 to 11.

BACKGROUND: South Africa's public healthcare system is better equipped to manage breast cancer than most other SSA countries, but survival rates are unknown. METHODS: A historical cohort of 602 women newly diagnosed with invasive breast carcinoma during 2009-2011 at Chris Hani Baragwanath Academic Hospital, Soweto, Johannesburg, was followed using health systems data to December 2014. 'Overall survival' time was defined from diagnosis to death or terminal illness. Cox regression was used to estimate hazard ratios (HR) associated with woman and tumour characteristics. RESULTS: During a median 2.1 years follow-up (IQR 0.5-3.8), 149 women died or were classified terminally ill; 287 were lost-to-follow-up. 3-year survival was 84% for early stage (I/II) and 56% for late stage (III/IV) tumours (late v early: HR 2.8 (95% confidence interval (CI): 1.9-4.1), however the 42% cumulative losses to follow-up over this period were greater for late stage, half of which occurred within 6 months of diagnosis. After mutual adjustment for stage, grade, age, receptor subtype and HIV status, lower survival was also associated with triple negative (HR 3.1 (95% CI: 1.9-5.0)) and HER2-enriched (2.5 (95% CI: 1.4-4.5)) compared to ER/PR+ HER2- tumours, but not with age or HIV-infection (1.4 (95% CI: 0.8, 2.3)). CONCLUSION: In this South African cohort, breast cancer survival is suboptimal, but was better for early stage and hormone receptor-positive tumours. Efforts to reduce clinic losses in the immediate post-diagnosis period, in addition to early presentation and accelerated diagnosis and treatment, are needed to prevent breast cancer deaths, and survival improvements need to be monitored using prospective studies with active follow-up.

Engineered proteins with sensing and activating modules for automated reprogramming of cellular functions.

Protein-based biosensors or activators have been engineered to visualize molecular signals or manipulate cellular functions. Here we integrate these two functionalities into one protein molecule, an integrated sensing and activating protein (iSNAP). A prototype that can detect tyrosine phosphorylation and immediately activate auto-inhibited Shp2 phosphatase, Shp2-iSNAP, is designed through modular assembly. When Shp2-iSNAP is fused to the SIRPα receptor which typically transduces anti-phagocytic signals from the 'don't eat me' CD47 ligand through negative Shp1 signaling, the engineered macrophages not only allow visualization of SIRPα phosphorylation upon CD47 engagement but also rewire the CD47-SIRPα axis into the positive Shp2 signaling, which enhances phagocytosis of opsonized tumor cells. A second SIRPα Syk-iSNAP with redesigned sensor and activator modules can likewise rewire the CD47-SIRPα axis to the pro-phagocytic Syk kinase activation. Thus, our approach can be extended to execute a broad range of sensing and automated reprogramming actions for directed therapeutics.Protein-based biosensors have been engineered to interrogate cellular signaling and manipulate function. Here the authors demonstrate iSNAP, a tool to detect tyrosine phosphorylation and activate desired protein enzymes allowing the control of phagocytosis in macrophages.

Examination of the role of galectins in plasma cell differentiation.

Plasma cells are terminally differentiated B cells that develop via the stimulation of mature B cells with various agents such as antigens and mitogens. Recently, we found that plasma cell differentiation can be modulated by galectin-1 and galectin-8; these galectins appear to play additive and redundant roles in promoting the production of antibody. Here, we describe the protocols for how to investigate the roles of galectins in plasma cell differentiation. These methods include the preparation of recombinant galectins from Escherichia coli for exogenously treating primary B cells, generation of galectin_Fc(m) fusion proteins for determining their binding to B cells, introduction of ectopic galectins in primary B cells using retroviral vectors, and inhibition of the binding of galectins to B cells by synthetic disaccharides.

RegPhos 2.0: an updated resource to explore protein kinase-substrate phosphorylation networks in mammals.

Protein phosphorylation catalyzed by kinases plays crucial roles in regulating a variety of intracellular processes. Owing to an increasing number of in vivo phosphorylation sites that have been identified by mass spectrometry (MS)-based proteomics, the RegPhos, available online at http://csb.cse.yzu.edu.tw/RegPhos2/, was developed to explore protein phosphorylation networks in human. In this update, we not only enhance the data content in human but also investigate kinase-substrate phosphorylation networks in mouse and rat. The experimentally validated phosphorylation sites as well as their catalytic kinases were extracted from public resources, and MS/MS phosphopeptides were manually curated from research articles. RegPhos 2.0 aims to provide a more comprehensive view of intracellular signaling networks by integrating the information of metabolic pathways and protein-protein interactions. A case study shows that analyzing the phosphoproteome profile of time-dependent cell activation obtained from Liquid chromatography-mass spectrometry (LC-MS/MS) analysis, the RegPhos deciphered not only the consistent scheme in B cell receptor (BCR) signaling pathway but also novel regulatory molecules that may involve in it. With an attempt to help users efficiently identify the candidate biomarkers in cancers, 30 microarray experiments, including 39 cancerous versus normal cells, were analyzed for detecting cancer-specific expressed genes coding for kinases and their substrates. Furthermore, this update features an improved web interface to facilitate convenient access to the exploration of phosphorylation networks for a group of genes/proteins. Database URL: http://csb.cse.yzu.edu.tw/RegPhos2/

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes.

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galß1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.

Advanced mass spectrometry and chemical analyses reveal the presence of terminal disialyl motif on mouse B-cell glycoproteins.

The occurrence of a terminal disialyl motif on mammalian O-glycans is increasingly being identified through recent mass spectrometry (MS)-based glycomic profiling. In most cases, it is carried on simple core 1 structures in which both the galactose and N-acetyl galactosamine can be disialylated. In contrast, a disialyl motif on N-glycans is less readily revealed by MS mapping, since additional MS/MS analysis is required to determine the distribution of the various sialic acids on typically multisialylated complex type N-glycans. In our MS-based glycomic screening, we found that a mouse B lymphoma cell line, BCL1, ranks among those that have the highest amount of disialyl motif on its O-glycans, including those carried on CD45. More intriguingly, detailed chemical and MS/MS analyses unambiguously showed that the Neu5Gcα2-8Neu5Gc disialyl motif is also present on the N-glycans and that it can be carried on the termini of polylactosaminoglycan chains, which can be further sulfated on the proximal GlcNAc, occurring alongside other monosialylated sulfated LacNAc termini. Upon silencing the expression of mouse α2,8-sialyltransferase VI (ST8Sia VI), the overall disialyl content decreases significantly, but more so for that on the N-glycans than the O-glycans. ST8Sia VI was further shown to be the most significantly upregulated ST8Sia during plasma cell differentiation, which coincides with increasing content of the disialyl motif. Increasing terminal disialylation without leading to polysialylation may thus have important biological consequences awaiting further investigation. Likewise, the expression of mono- and disialylated sulfated LacNAc may constitute novel recognition codes modulating B-cell activation and differentiation.

Galectin-1 promotes immunoglobulin production during plasma cell differentiation.

Galectin-1, a beta-galactoside-binding soluble lectin, has been implicated in regulating immune system homeostasis. We investigated the function of galectin-1 in plasma cell differentiation and found that it is induced in primary murine and human differentiating B cells. B lymphocyte-induced maturation protein-1 (Blimp-1), a master regulator for plasma cell differentiation, was necessary and sufficient to induce galectin-1 expression. Notably, ectopic expression of galectin-1 in mature B cells increased Ig mu-chain transcript levels as well as the overall level of Ig production. This function of galectin-1 was dependent on binding to cell surface glycosylated counter receptors, as a galectin-1 mutant deficient in beta-galactoside binding showed diminished ability to promote Ig production. Extracellular galectin-1 bound more significantly to mature B cells than to plasma cells. Lastly, we found that the sugar compound N-acetyllactosamine blocked the binding of galectin-1 to murine splenic B cells and inhibited their differentiation. Taken together, these data are the first to demonstrate a role for galectin-1 in promoting Ig production during plasma cell differentiation.