Objective Long noncoding RNAs (lncRNA) represent a growing class of noncoding genes with diverse cellular functions. We previously reported on SENCR, a lncRNA that appears to support the vascular smooth muscle cell (VSMC) contractile phenotype. However, information about the vascular smooth muscle cell (VSMC)-specific lncRNAs regulated by myocardin (MYOCD)/SRF, the master switch for VSMC differentiation, is virtually unknown. Approach and Results To define novel lncRNAs with functions related to VSMC differentiation, we performed RNA sequencing in human coronary artery SMCs (HCASMCs) that overexpress MYOCD. A number of novel lncRNAs showed altered expression with MYOCD overexpression and one, named MYOcardin-induced Smooth muscle Long non-coding RNA, Inducer of Differentiation (MYOSLID), was activated by MYOCD and selectively expressed in VSMCs. MYOSLID was a direct transcriptional target of both MYOCD/SRF and TGFβ/SMAD pathways. Functional studies revealed that MYOSLID promotes VSMC differentiation and inhibits VSMC proliferation. MYOSLID showed reduced expression in failed human arteriovenous fistula (AVF) samples compared with healthy veins. While MYOSLID did not affect gene expression of transcription factors such as SRF and MYOCD, its depletion in VSMCs disrupted actin stress fiber formation and blocked nuclear translocation of MYOCD-related transcription factor A (MKL1). Finally, loss of MYOSLID abrogated TGFβ1-induced SMAD2 phosphorylation. Conclusion We have demonstrated that MYOSLID, the first human VSMC-selective and SRF/CArG-dependent lncRNA, is a novel modulator in amplifying the VSMC differentiation program, likely through feed-forward actions of both MKL1 and TGFβ/SMAD pathways.

Antibiotic-resistant S. Typhi secretes typhoid toxin despite antibiotic treatment MAb targeting the receptor-binding or nuclease subunits neutralizes typhoid toxin TyTx11 makes nuclease CdtB inactive by causing catalytic-site conformational change Toxin-neutralizing epitopes identified are conserved across S. Typhi clinical isolates

Non-typhoidal Salmonella enterica strains, including serovar Typhimurium (STm), are an emerging cause of invasive disease among children and the immunocompromised, especially in regions of sub-Saharan Africa. STm invades the intestinal mucosa through Peyer's patch tissues before disseminating systemically. While vaccine development efforts are ongoing, the emergence of multidrug resistant strains of STm affirms the need to seek alternative strategies to protect high-risk individuals from infection. In this report, we investigated the potential of an orally administered O5 serotype-specific IgA monoclonal antibody (mAb), called Sal4, to prevent infection of invasive Salmonella enterica serovar Typhimurium (STm) in mice. Sal4 IgA was delivered to mice prior to or concurrently with STm challenge. Infectivity was measured as bacterial burden in Peyer's patch tissues one day after challenge. Using this model, we defined the minimal amount of Sal4 IgA required to significantly reduce STm uptake into Peyer's patches. The relative efficacy of Sal4 in dimeric and secretory IgA (SIgA) forms was compared. To assess the role of isotype in oral passive immunization, we engineered a recombinant IgG1 mAb carrying the Sal4 variable regions and evaluated its ability to block invasion of STm into epithelial cells in vitro and Peyer's patch tissues. Our results demonstrate the potential of orally administered monoclonal IgA and SIgA, but not IgG, to passively immunize against invasive Salmonella. Nonetheless, the prophylactic window of IgA/SIgA in the mouse was on the order of minutes, underscoring the need to develop formulations to protect mAbs in the gastric environment and to permit sustained release in the small intestine.

As the predominant antibody type in mucosal secretions, human colostrum, and breast milk, secretory IgA (SIgA) plays a central role in safeguarding the intestinal epithelium of newborns from invasive enteric pathogens like the Gram-negative bacterium Salmonella enterica serovar Typhimurium (STm). SIgA is a complex molecule, consisting of an assemblage of two or more IgA monomers, joining (J)-chain, and secretory component (SC), whose exact functions in neutralizing pathogens are only beginning to be elucidated. In this study, we produced and characterized a recombinant human SIgA variant of Sal4, a well-characterized monoclonal antibody (mAb) specific for the O5-antigen of STm lipopolysaccharide (LPS). We demonstrate by flow cytometry, light microscopy, and fluorescence microscopy that Sal4 SIgA promotes the formation of large, densely packed bacterial aggregates in vitro . In a mouse model, passive oral administration of Sal4 SIgA was sufficient to entrap STm within the intestinal lumen and reduce bacterial invasion into gut-associated lymphoid tissues by several orders of magnitude. Bacterial aggregates induced by Sal4 SIgA treatment in the intestinal lumen were recalcitrant to immunohistochemical staining, suggesting the bacteria were encased in a protective capsule. Indeed, a crystal violet staining assay demonstrated that STm secretes an extracellular matrix enriched in cellulose following even short exposures to Sal4 SIgA. Collectively, these results demonstrate that recombinant human SIgA recapitulates key biological activities associated with mucosal immunity and raises the prospect of oral passive immunization to combat enteric diseases.

Highlights d Antibodies targeting glycan-receptor binding B subunits can be split into two classes d The two classes are grouped by their epitope locations on the B subunit homopentamer d B homopentamers act similar to heteropentamers due to their skewed A subunit positioning d The two antibody classes exhibit significantly different neutralizing efficacies

AbstractBackgroundNon-typhoidal Salmonella enterica strains, including serovar Typhimurium (STm), are an emerging cause of invasive disease among children and the immunocompromised, especially in regions of sub-Saharan Africa. STm invades the intestinal mucosa through Peyer’s patch tissues before disseminating systemically. While vaccine development efforts are ongoing, the emergence of multidrug resistant strains of STm affirms the need to seek alternative strategies to protect high-risk individuals from infection.Methodology/Principal FindingsIn this report we investigated the potential of an orally administered O5 serotype-specific IgA monoclonal antibody (mAb), called Sal4, to protect mice against invasive Salmonella enterica serovar Typhimurium (STm) infection. Sal4 IgA was delivered to mice prior to or concurrently with STm challenge. Infectivity was measured as bacterial burden in Peyer’s patch tissues one day after challenge. Using this model, we defined the minimal amount of Sal4 IgA required to significantly reduce STm uptake into Peyer’s patches. The relative efficacy of Sal4 in dimeric and secretory IgA (SIgA) forms was compared, as was a second lower avidity O5-specific IgA mAb that we produced from STm immunized mice. To assess the role of isotype in oral passive immunization, we engineered a recombinant IgG1 mAb carrying the Sal4 variable regions and evaluated its ability to block invasion of STm into epithelial cells in vitro and Peyer’s patch tissues.Conclusions/SignificanceOur results demonstrate the potential of orally administered monoclonal IgA and SIgA, but not IgG, to passively immunize against invasive Salmonella. Nonethless, the prophylactic window of IgA/SIgA in the mouse was on the order of minutes, underscoring the need to develop formulations to protect mAbs in the gastric environment and to permit sustained release in the small intestine.Author SummaryThe bacterium Salmonella enterica is responsible for significant morbidity and mortality in the developed and developing worlds. While the pathogen is most renowed as the causative agent of typhoid fever, the emergence of invasive non-typhoid strains like S. enterica serovar Typhimurium (STm) are of great concern because of their propensity to cause severe disease in children under the age of five. In this report, we demonstrate in a mouse model that oral administration of a monoclonal antibody targeting the surface of STm is able to prevent the bacterium from infecting gastrointestinal tissues, the first step in the dissemination process. We show that IgA antibodies (which are normally found in the gut) were far superior than an equivalent IgG antibody (normally found in blood) at protecting the intestine from STm infection. These results lay the foundation for future studies aimed at the development of an orally administered antibody cocktail capable of providing temporary immunity to pathogens like S. enterica.

Secretory IgA (SIgA) is the most abundant antibody type in intestinal secretions where it contributes to safeguarding the epithelium from invasive pathogens like the Gram-negative bacterium, Salmonella enterica serovar Typhimurium (STm). For example, we recently reported that passive oral administration of the recombinant monoclonal SIgA antibody, Sal4, to mice promotes STm agglutination in the intestinal lumen and restricts bacterial invasion of Peyer’s patch tissues.

Eliminating diarrheal diseases as a leading cause of childhood morbidity and mortality in low-and middleincome countries (LMICs) will require multiple intervention strategies. In this review, we spotlight a series of preclinical studies investigating the potential of orally administered monoclonal secretory IgA (SIgA) antibodies (MAbs) to reduce disease associated with three enteric bacterial pathogens: Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC), and invasive Salmonella enterica serovar Typhimurium. IgA MAbs targeting bacterial surface antigens (flagella, adhesins, and lipopolysaccharide) were generated from mice, humanized mice, and human tonsillar B cells. Recombinant SIgA1 and/or SIgA2 derivates of those MAbs were purified from supernatants following transient transfection of 293 cells with plasmids encoding antibody heavy and light chains, J-chain, and secretory component (SC). When administered to mice by gavage immediately prior to (or admixed with) the bacterial challenge, SIgA MAbs reduced infection C. jejuni, ETEC, and S. Typhimurium infections. Fv-matched IgG1 MAbs by comparison were largely ineffective against C. jejuni and S. Typhimurium under the same conditions, although they were partially effective against ETEC. While these findings highlight future applications of orally administered SIgA, the studies also underscored the fundamental challenges associated with using MAbs as prophylactic tools against enteric bacterial diseases.