The La protein is a target of autoantibodies in patients suffering from Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Ubiquitous in eukaryotes, La functions as a RNA-binding protein that promotes the maturation of tRNA precursors and other nascent transcripts synthesized by RNA polymerase III as well as other noncoding RNAs. La also associates with a class of mRNAs that encode ribosome subunits and precursors to snoRNAs involved in ribosome biogenesis. Thus, it was surprising that La is dispensable in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the organisms from which it has been characterized most extensively. To determine whether La is essential in mammals and if so, at which developmental stage it is required, mice were created with a disrupted La gene, and the offspring from La ؉/؊ intercrosses were analyzed. La ؊/؊ offspring were detected at the expected frequency among blastocysts prior to implantation, whereas no nullizygotes were detected after implantation, indicating that La is required early in development. Blastocysts derived from La La antigen, also known as Sjögren's syndrome antigen B (SS-B), is a target of autoantibodies in patients suffering from systemic lupus erythematosus, neonatal lupus, and related disorders and exists in cells complexed with various RNAs (20). Homologs of La are present in all of the eukaryote genomes examined, and La proteins have been characterized in ciliates, yeasts, flies, frogs, and mammals (7,22,33). While La has been implicated in many RNA-related pathways, its most established role is protecting the UUU-OH 3Ј ends of precursor tRNAs and other small RNAs from digestion (21,23,29,33).Vertebrate La proteins can modulate the translation of mRNAs that contain internal ribosome entry sites, as well as mRNAs that contain 5Ј-terminal oligopyrimidine motifs that encode ribosome subunits and translation factors (8, 28; reviewed in reference 33). The association of human Mdm2 mRNA with La promotes MDM2 translation with consequent decrease in p53 protein level and leukemia progression (31). La is also found associated with mRNAs in Saccharomyces cerevisiae, including mRNAs that encode ribosome subunits (14). Deletion of La from yeasts leads to alterations in the maturation pathways of pre-tRNAs (2,5,6,16,17,26,35) and pre-snoRNAs involved in rRNA biogenesis (14, 21). Thus, it was surprising that La is nonessential in yeasts, except when tRNAs or RNA-associated factors acquire debilitating mutations (21, 29, 33) and upon a conditional induction of the unfolded protein response (14).The conserved N-terminal domain of La is comprised of a La motif and RNA recognition motif (RRM) that cooperate for high-affinity 3Ј UUU-OH binding (1, 9, 18). However, while these motifs constitute the La proteins of yeasts, metazoan La proteins also contain another, atypical RRM in their C termini (18).In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the organisms in which La has been characterized most extensively, La is dispens...

Staphylococcus aureus causes a wide range of diseases from skin infections to life threatening invasive diseases such as bacteremia, endocarditis, pneumonia, surgical site infections, and osteomyelitis. Skin infections such as furuncles, carbuncles, folliculitis, erysipelas, and cellulitis constitute a large majority of infections caused by S. aureus (SA). These infections cause significant morbidity, healthcare costs, and represent a breeding ground for antimicrobial resistance. Furthermore, skin infection with SA is a major risk factor for invasive disease. Here we describe the pre-clinical efficacy of a multicomponent toxoid vaccine (IBT-V02) for prevention of S. aureus acute skin infections and recurrence. IBT-V02 targets six SA toxins including the pore-forming toxins alpha hemolysin (Hla), Panton-Valentine leukocidin (PVL), leukocidin AB (LukAB), and the superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxins A and B. Immunization of mice and rabbits with IBT-V02 generated antibodies with strong neutralizing activity against toxins included in the vaccine, as well as cross-neutralizing activity against multiple related toxins, and protected against skin infections by several clinically relevant SA strains of USA100, USA300, and USA1000 clones. Efficacy of the vaccine was also shown in non-naïve mice pre-exposed to S. aureus. Furthermore, vaccination with IBT-V02 not only protected mice from a primary infection but also demonstrated lasting efficacy against a secondary infection, while prior challenge with the bacteria alone was unable to protect against recurrence. Serum transfer studies in a primary infection model showed that antibodies are primarily responsible for the protective response.

Staphylococcus aureus (SA) infections cause high mortality and morbidity in humans. Being central to its pathogenesis, S. aureus thwarts the host defense by secreting a myriad of virulence factors, including bicomponent, pore-forming leukotoxins. While all vaccine development efforts that aimed at achieving opsonophagocytic killing have failed, targeting virulence by toxoid vaccines represents a novel approach to preventing mortality and morbidity that are caused by SA. The recently discovered leukotoxin LukAB kills human phagocytes and monocytes and it is present in all known S. aureus clinical isolates. While using a structure-guided approach, we generated a library of mutations that targeted functional domains within the LukAB heterodimer to identify attenuated toxoids as potential vaccine candidates. The mutants were evaluated based on expression, solubility, yield, biophysical properties, cytotoxicity, and immunogenicity, and several fully attenuated LukAB toxoids that were capable of eliciting high neutralizing antibody titers were identified. Rabbit polyclonal antibodies against the lead toxoid candidate provided potent neutralization of LukAB. While the neutralization of LukAB alone was not sufficient to fully suppress leukotoxicity in supernatants of S. aureus USA300 isolates, a combination of antibodies against LukAB, α-toxin, and Panton-Valentine leukocidin completely neutralized the cytotoxicity of these strains. These data strongly support the inclusion of LukAB toxoids in a multivalent toxoid vaccine for the prevention of S. aureus disease.

Cytolytic pore-forming toxins including alpha hemolysin (Hla) and bicomponent leukotoxins play an important role in the pathogenesis of Staphylococcus aureus. These toxins kill the polymorphonuclear phagocytes (PMNs), disrupt epithelial and endothelial barriers, and lyse erythrocytes to provide iron for bacterial growth. The expression of these toxins is regulated by the two-component sensing systems Sae and Agr. Here, we report that a point mutation (L18P) in SaeS, the histidine kinase sensor of the Sae system, renders the S. aureus Newman hemolytic activity fully independent of Hla and drastically increases the PMN lytic activity. Furthermore, this Hla-independent activity, unlike Hla itself, can lyse human erythrocytes. The Hla-independent activity towards human erythrocytes was also evident in USA300, however, under strict agr control. Gene knockout studies revealed that this Hla-independent Sae-regulated activity was entirely dependent on gamma hemolysin A subunit (HlgA). In contrast, hemolytic activity of Newman towards human erythrocytes from HlgAB resistant donors was completely dependent on agr. The culture supernatant from Newman S. aureus could be neutralized by antisera against two vaccine candidates based on LukS and LukF subunits of Panton-Valentine leukocidin but not by an anti-Hla neutralizing antibody. These findings display the complex involvement of Sae and Agr systems in regulating the virulence of S. aureus and have important implications for vaccine and immunotherapeutics development for S. aureus disease in humans.

Staphylococcus aureus has been acquiring multiple drug resistance and has evolved into superbugs such as Methicillin/Vancomycin-resistant S. aureus (MRSA/VRSA) and, consequently, is a major cause of nosocomial and community infections associated with high morbidity and mortality for which no FDA-approved vaccines or biotherapeutics are available. Previous efforts targeting the surface-associated antigens have failed in clinical testing. Here, we generated hyperimmune products from sera in rabbits against six major S. aureus toxins targeted by an experimental vaccine (IBT-V02) and demonstrated significant efficacy for an anti-virulence passive immunization strategy. Extensive in vitro binding and neutralizing titers were analyzed against six extracellular toxins from individual animal sera. All IBT-V02 immunized animals elicited the maximum immune response upon the first boost dose against all pore-forming vaccine components, while for superantigen (SAgs) components of the vaccine, second and third doses of a boost were needed to reach a plateau in binding and toxin neutralizing titers. Importantly, both anti-staphylococcus hyperimmune products consisting of full-length IgG (IBT-V02-IgG) purified from the pooled sera and de-speciated F(ab’)2 (IBT-V02-F(ab’)2) retained the binding and neutralizing titers against IBT-V02 target toxins. F(ab’)2 also exhibited cross-neutralization titers against three leukotoxins (HlgAB, HlgCB, and LukED) and four SAgs (SEC1, SED, SEK, and SEQ) which were not part of IBT-V02. F(ab’)2 also neutralized toxins in bacterial culture supernatant from major clinical strains of S. aureus. In vivo efficacy data generated in bacteremia and pneumonia models using USA300 S. aureus strain demonstrated dose-dependent protection by F(ab’)2. These efficacy data confirmed the staphylococcal toxins as viable targets and support the further development effort of hyperimmune products as a potential adjunctive therapy for emergency uses against life-threatening S. aureus infections.

B lymphocyte stimulator (BLyS) is a member of the TNF superfamily of cytokines. The biological activity of BLyS is mediated by three cell surface receptors: BR3/BAFF-R, TACI and BCMA. The expression of these receptors is highly restricted to B cells, both normal and malignant. A BLyS-gelonin fusion toxin (BLyS-gel) was generated consisting of the recombinant plant-derived toxin gelonin fused to the N-terminus of BLyS and tested against a large and diverse panel of B-NHL cell lines. Interestingly, B-NHL subtypes mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL) and B cell precursor-acute lymphocytic leukemia (BCP-ALL) were preferentially sensitive to BLyS-gel mediated cytotoxicity, with low picomolar EC50 values. BLyS receptor expression did not guarantee sensitivity to BLyS-gel, even though the construct was internalized by both sensitive and resistant cells. Resistance to BLyS-gel could be overcome by treatment with the endosomotropic drug chloroquine, suggesting BLyS-gel may become trapped within endosomal/lysosomal compartments in resistant cells. BLyS-gel induced cell death was caspase-independent and shown to be at least partially mediated by the “ribotoxic stress response.” This response involves activation of p38 MAPK and JNK/SAPK, and BLyS-gel mediated cytotoxicity was inhibited by the p38/JNK inhibitor SB203580. Finally, BLyS-gel treatment was shown to localize to sites of disease, rapidly reduce tumor burden, and significantly prolong survival in xenograft mouse models of disseminated BCP-ALL, DLBCL, and MCL. Together, these findings suggest BLyS has significant potential as a targeting ligand for the delivery of cytotoxic “payloads” to malignant B cells.

BackgroundCigarette smoking (CS) remains the primary cause of chronic lung diseases. After a steady decline, smoking rates have recently increased especially with the introduction of newer electronic nicotine delivery devices, and it is also emerging that dual or poly product usage is on the rise. Additionally, with the introduction of IQOS (a heated tobacco product) globally, its impact on human health needs to be investigated. In this study we tested if dual exposure (CS+IQOS) is detrimental to lung epithelial cells when compared to CS or IQOS exposure alone.MethodsHuman airway epithelial cells (BEAS-2B) cells were exposed to either CS or IQOS or their dual combinations at concentrations 0.1, 1.0, 2.5 and 5.0%. Cytotoxicity, oxidative stress, mitochondrial homeostasis, mitophagy, and effects on epithelial mesenchymal transition (EMT) signaling were assessed.ResultsBoth CS and IQOS alone significantly induced loss of cell viability in a concentration-dependent manner which was further enhanced by dual exposure compared to IQOS alone (p<0.01). Dual exposure significantly increased oxidative stress and perturbed mitochondrial homeostasis when compared to CS or IQOS alone (p<0.05). Additionally, dual exposure induced EMT signaling as shown by increased in mesenchymal (α-SMA and N-cadherin) and decreased epithelial (E-cadherin) markers when compared to CS or IQOS alone (p<0.05).ConclusionCollectively, our study demonstrates that dual exposure enhances pathogenic signaling mediated by oxidative stress and mitochondrial dysfunction leading to EMT activation which is an important regulator of small airway fibrosis in obstructive lung diseases.

B lymphocyte stimulator (BLyS) is a member of the TNF superfamily of cytokines. BLyS is expressed by cells of myeloid origin and is one of many soluble factors that regulate human B-cell activation. The biological activity of BLyS is mediated by three cell surface receptors: B-cell-activating factor-receptor (BAFF-R/BR3), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA). These receptors are expressed by normal and malignant B-cells at various stages of development. To target malignant B cells expressing BLyS receptors, the recombinant plant-derived toxin gelonin was fused to the N-terminus of BLyS to generate the fusion toxin BLyS-gel. Gelonin is an N-glycosidase that removes a critical adenine from eukaryotic 28S rRNA, disrupting ribosome function and inhibiting protein synthesis. Importantly, gelonin is not toxic to cells unless coupled with a targeting moiety that can enter the cell. BLyS-gel was used to treat a panel of nearly 50 malignant non-Hodgkin lymphoma (NHL) cell lines expressing BLyS receptors. NHL subtypes mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL) and B cell precursor-acute lymphocytic leukemia (BCP-ALL) were preferentially sensitive to BLyS-gel mediated cytotoxicity, with EC50 values in the low picomolar range. BLyS-gel cytotoxicity was mediated primarily by BR3 and TACI in sensitive cell lines; however, cell surface expression of these BLyS receptors did not necessarily confer sensitivity to BLyS-gel. The internalization of BLyS-gel was confirmed in both sensitive and resistant cell lines, indicating resistance was not due to a lack of internalization. As expected, BLyS-gel treatment inhibited protein synthesis in sensitive, but not resistant cell lines. In most cell lines, cell death seemed to be mediated by the “ribotoxic stress response.” This response involves activation of p38 MAPK and JNK/SAPK, and BLyS-gel mediated cytotoxicity was inhibited by the p38/JNK inhibitor SB203580. Finally, BLyS-gel treatment significantly prolonged survival in three distinct disseminated xenograft models (BCP-ALL, DLBCL & MCL) in SCID mice, and BLyS-gel was shown to specifically localize to sites of disease. Together, these findings suggest BLyS has potential as a targeting ligand for the therapeutic delivery of toxins and/or drugs directly to malignant B cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3861. doi:1538-7445.AM2012-3861