Staphylococcus Research Articles

Effective degradation of various bacterial toxins using ozone ultrafine bubble water.

Infectious and foodborne diseases pose significant global threats, with devastating consequences in low- and middle-income countries. Ozone, derived from atmospheric oxygen, exerts antimicrobial effects against various microorganisms, and degrades fungal toxins, which were initially recognized in the healthcare and food industries. However, highly concentrated ozone gas can be detrimental to human health. In addition, ozonated water is unstable and has a short half-life. Therefore, ultrafine-bubble technology is expected to overcome these issues. Ultrafine bubbles, which are nanoscale entitles that exist in water for considerable durations, have previously demonstrated bactericidal effects against various bacterial species, including antibiotic-resistant strains. This present study investigated the effects of ozone ultrafine bubble water (OUFBW) on various bacterial toxins. This study revealed that OUFBW treatment abolished the toxicity of pneumolysin, a pneumococcal pore-forming toxin, and leukotoxin, a toxin that causes leukocyte injury. Silver staining confirmed the degradation of pneumolysin, leukotoxin, and staphylococcal enterotoxin A, which are potent gastrointestinal toxins, following OUFB treatment. In addition, OUFBW treatment significantly inhibited NF-κB activation by Pam3CSK4, a synthetic triacylated lipopeptide that activates Toll-like receptor 2. Additionally, OUFBW exerted bactericidal activity against Staphylococcus aureus, including an antibiotic-resistant strain, without displaying significant toxicity toward human neutrophils or erythrocytes. These results suggest that OUFBW not only sterilizes bacteria but also degrades bacterial toxins.

Interferon Activating Effect of Staphylococcal Enterotoxins

Interferon Activating Effect of Staphylococcal Enterotoxins

Association between specific IgE to staphylococcal enterotoxin B and the eosinophilic phenotype of asthma.

Sensitization to staphylococcal superantigens (SAgs) could contribute to asthma severity. However, its relevance with eosinophilic phenotype has not yet been clarified. This study aimed to investigate associations between serum specific IgE levels to SAg and eosinophilic airway inflammation in adult asthmatics. The serum specific IgE levels to 3 SAgs, including staphylococcal enterotoxin A (SEA) and B (SEB), and toxic shock syndrome toxin-1 (TSST-1) were measured by ImmunoCAP in 230 adult asthmatic patients and 50 healthy controls (HCs). Clinical characteristics and laboratory parameters, including serum total/free IgE, and 2 eosinophil-activation markers, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN), were analyzed according to blood eosinophil counts (BEC; 150 cells/μL) and serum specific IgE levels to 3 SAgs (0.35 kU/L). Asthmatic patients showed higher serum specific IgE levels to 3 SAgs than HCs (p < 0.05 for all). The serum total/clinfree IgE levels were significantly higher in asthmatics with positive IgE responses to 3 SAgs than those without (p < 0.05 for all). There were no significant differences in clinical parameters including age, asthma severity, comorbidities, or smoking according to IgE responses to 3 SAgs. Patients with positive IgE responses to SEB (not to SEA/TSST-1) had higher serum specific IgE levels to house dust mites and ECP/EDN as well as higher BEC with positive correlations between serum SEB-specific IgE levels and BEC/ECP/EDN (p < 0.05 for all). These findings suggest that serum SEB-specific IgE levels could contribute to eosinophil activation as well as IgE production in adult asthma.

Subinhibitory concentrations of nisin enhance virulence gene expression in Staphylococcus aureus and increase mortality in Galleria mellonella.

Alternative strategies for controlling Staphylococcus aureus and other pathogens have been continuously investigated, with nisin, a bacteriocin widely used in the food industry as a biopreservative, gaining increasing attention. In addition to its antimicrobial properties, bacteriocins have significant effects on genome functionality even at inhibitory concentrations. This study investigated the impact of subinhibitory concentrations of nisin on S. aureus. Culturing in the presence of 0.625 μmol l-1 nisin, led to the increased relative expression of hla, saeR, and sarA, genes associated with virulence while expression of the sea gene, encoding staphylococcal enterotoxin A (SEA), decreased. In an in vivo experiment, Galleria mellonella larvae inoculated with S. aureus cultured in the presence of nisin exhibited 97% mortality at 72h post-infection, compared to over 40% of larvae mortality in larvae infected with S. aureus. A comprehensive understanding of the effect of nisin on the transcriptional response of virulence genes and the impact of these changes on the virulence of S. aureus can contribute to assessing the application of this bacteriocin in food and medical contexts.

Structural insights into the binding of nanobodies to the Staphylococcal enterotoxin B

Staphylococcal Enterotoxin Type B (SEB), produced by Staphylococcus aureus bacteria, is notorious for inducing severe food poisoning and toxic shock syndrome. While nanobody-based treatments hold promises for combating SEB-induced diseases, the lack of structural information between SEB and nanobodies has hindered the development of nanobody-based therapeutics. Here, we present crystal structures of SEB-Nb3, SEB-Nb6, SEB-Nb8, SEB-Nb11, and SEB-Nb20 at resolutions ranging from 1.59 Å to 2.33 Å. Crystallographic analysis revealed that Nb3, Nb8, Nb11, and Nb20 bind to SEB at the T-cell receptor (TCR) interface, while Nb6 binds at the major histocompatibility complex (MHC) interface, suggesting their potential to inhibit SEB function by disrupting interactions with TCR or MHC molecules. Molecular biological analyses confirmed the thermodynamic and kinetic parameters of Nb3, Nb5, Nb6, Nb8, Nb11, Nb15, Nb18, and Nb20 to SEB. The competitive inhibition was further confirmed by cell-based experiments demonstrating nanobody neutralization. These findings elucidate the structural basis for developing specific nanobodies to neutralize SEB threats, providing crucial insights into the underlying mechanisms and offering significant assistance for further optimization towards future therapeutic strategies.

Identification and Removal of Pollen Spectral Interference in the Classification of Hazardous Substances Based on Excitation Emission Matrix Fluorescence Spectroscopy.

Sensitively detecting hazardous and suspected bioaerosols is crucial for safeguarding public health. The potential impact of pollen on identifying bacterial species through fluorescence spectra should not be overlooked. Before the analysis, the spectrum underwent preprocessing steps, including normalization, multivariate scattering correction, and Savitzky-Golay smoothing. Additionally, the spectrum was transformed using difference, standard normal variable, and fast Fourier transform techniques. A random forest algorithm was employed for the classification and identification of 31 different types of samples. The fast Fourier transform improved the classification accuracy of the sample excitation-emission matrix fluorescence spectrum data by 9.2%, resulting in an accuracy of 89.24%. The harmful substances, including Staphylococcus aureus, ricin, beta-bungarotoxin, and Staphylococcal enterotoxin B, were clearly distinguished. The spectral data transformation and classification algorithm effectively eliminated the interference of pollen on other components. Furthermore, a classification and recognition model based on spectral feature transformation was established, demonstrating excellent application potential in detecting hazardous substances and protecting public health. This study provided a solid foundation for the application of rapid detection methods for harmful bioaerosols.

Development and validation of an ELISA kit for the detection of Staphylococcus aureus enterotoxin A, B, C1, C2, C3, D, E from food samples

Staphylococcus aureus (S. aureus) is a significant food-borne pathogen that poses a threat to human health. The classical serotypes staphylococcal enterotoxin (SE) A, B, C1, C2, C3, D, E produced by S. aureus are the primary cause of its food-borne pathogenic toxicity. Therefore, establishing a highly sensitive, rapid, robust, high-throughput detection method for the total amount of classical serotypes of SEs is crucial for food safety and public health. This study prepared 10 rabbit polyclonal antibodies and 40 mouse monoclonal antibodies targeting SEA, SEB, SEC1, SEC2, SEC3. Upon identifying the optimal antibody pairs through square array method and optimization, a sandwich ELISA kit for the detection of SEs (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE) was developed, with respective limits of detection at 0.169–0.688 ng/mL. In addition, this sandwich ELISA kit could detect SEs in raw cow milk, pasteurized milk, fermented milk, milk powder, whey powder, rice, raw pork, and canned beef samples with coefficients of variation below 7%. Meanwhile, this sandwich ELISA kit was stable at 4 °C for one-year storage. The study fills a research gap in the detection of the total amount of classical serotypes of SEs.

A highly neutralizing human monoclonal antibody targeting a novel linear epitope on staphylococcal enterotoxin B

ABSTRACT Staphylococcal Enterotoxin B (SEB), produced by Staphylococcus aureus (S. aureus), is a powerful superantigen that induces severe immune disruption and toxic shock syndrome (TSS) upon binding to MHC-II and TCR. Despite its significant impact on the pathogenesis of S. aureus, there are currently no specific therapeutic interventions available to counteract the mechanism of action exerted by this toxin. In this study, we have identified a human monoclonal antibody, named Hm0487, that specifically targets SEB by single-cell sequencing using PBMCs isolated from volunteers enrolled in a phase I clinical trial of the five-antigen S. aureus vaccine. X-ray crystallography studies revealed that Hm0487 exhibits high affinity for a linear B cell epitope in SEB (SEB138–147), which is located distantly from the site involved in the formation of the MHC-SEB-TCR ternary complex. Furthermore, in vitro studies demonstrated that Hm0487 significantly impacts the interaction of SEB with both receptors and the binding to immune cells, probably due to an allosteric effect on SEB rather than competing with receptors for binding sites. Moreover, both in vitro and in vivo studies validated that Hm0487 displayed efficient neutralizing efficacy in models of lethal shock and sepsis induced by either SEB or bacterial challenge. Our findings unveil an alternative mechanism for neutralizing the pathogenesis of SEB by Hm0487, and this antibody provides a novel strategy for mitigating both SEB-induced toxicity and S. aureus infection.

Development and validation of a multiplex bead-based immunoassay for the simultaneous detection of fifteen pathogenic biological agents

In this study, a multiplex bead-based capture sandwich immunoassay (MIA) for the simultaneous detection of fifteen plant and bacterial Gram-positive and Gram-negative pathogenic biological agents (PBAs) was developed. This study included the selection of “sandwich” pairs of monoclonal antibodies, the development of an analytical protocol, and an evaluation of the specificity and sensitivity of detection. The working range, linearity, precision, and limits of detection of the developed assay were evaluated. The LLOQ of the multiplex assay was around 104 – 105 CFU/ml for bacteria and 0,4 ng/ml – 6,6 ng/ml for antigens. The assay can be used for the simultaneous detection of 15 PBAs, including Francisella tularensis, Brucella abortus, Bacillus anthracis, Legionella pneumophila, Staphylococcus aureus, Staphylococcus haemolyticus, Pseudomonas aeruginosa, and Salmonella typhimurium, as well as cholera toxin beta-subunit, viscumin, ricin, staphylococcal enterotoxins A and B, and botulinum toxins A and B.

Nasal carriage of Staphylococcus aureus in healthy dairy cows in Algeria: antibiotic resistance, enterotoxin genes and biofilm formation

BackgroundStaphylococcus aureus can colonize and infect a variety of animal species. In dairy herds, it is one of the leading causes of mastitis cases. The objective of this study was to characterize the S. aureus isolates recovered from nasal swabs of 249 healthy cows and 21 breeders of 21 dairy farms located in two provinces of Algeria (Tizi Ouzou and Bouira).MethodsThe detection of enterotoxin genes was investigated by multiplex PCRs. Resistance of recovered isolates to 8 antimicrobial agents was determined by disc-diffusion method. The slime production and biofilm formation of S. aureus isolates were assessed using congo-red agar (CRA) and microtiter-plate assay. Molecular characterization of selected isolates was carried out by spa-typing and Multi-Locus-Sequence-Typing (MLST).ResultsS. aureus was detected in 30/249 (12%) and 6/13 (28.6%) of nasal swabs in cows and breeders, respectively, and a total of 72 isolates were recovered from positive samples (59 isolates from cows and 13 from breeders). Twenty-six of these isolates (36.1%) harbored genes encoding for staphylococcal enterotoxins, including 17/59 (28.8%) isolates from cows and 9/13 (69.2%) from breeders. Moreover, 49.1% and 92.3% of isolates from cows and breeders, respectively, showed penicillin resistance. All isolates were considered as methicillin-susceptible (MSSA). Forty-five (76.3%) of the isolates from cows were slime producers and 52 (88.1%) of them had the ability to form biofilm in microtiter plates. Evidence of a possible zoonotic transmission was observed in two farms, since S. aureus isolates recovered in these farms from cows and breeders belonged to the same clonal lineage (CC15-ST15-t084 or CC30-ST34-t2228).ConclusionsAlthough healthy cows in this study did not harbor methicillin-resistant S. aureus isolates, the nares of healthy cows could be a reservoir of enterotoxigenic and biofilm producing isolates which could have implications in human and animal health.

Assessment of Microbial Contamination and Metabolite Exposure in Cosmetic Products Used in Women's Beauty Salons.

An important route of microbial transmission is the shared use of these products in beauty salons. We aimed to investigate level of contamination with microorganisms and their metabolites in shared-use cosmetics in several women's beauty salons. Bacterial and fungal strains from 320 opened/used cosmetic samples were identified according to the Iranian standards for microbial quality of cosmetic products in Golestan Province (North of Iran) during Jul-Sep 2021. In order to assess production of toxins and protease by the predominant bacterial and fungal isolates, multiplex-polymerase chain reaction and the Lowry protein assay were performed, respectively. Microbial contamination was detected in 180 samples (56.5%), and the highest and lowest rates of microbial contamination were related to skin products (63.88%) and eye beauty products (20%), respectively. The highest level of S. aureus contamination (> 4,000 colony-forming units/g) was found in toner and face wash samples, and the highest level of C. albicans contamination was seen in lipstick samples (>20,000 colony-forming units/g). Only one (2%) S. aureus isolate produced staphylococcal enterotoxin B, while 3 out of 12 (25%) C. albicans isolates were able to produce protease. The shared-used health and beauty products, face products, in the study area are heavily contaminated. Therefore, it is essential to store used cosmetics in dry and cool places, establish strict inspection regulations for cosmetic products before and after entering the market, and increase awareness of beauty salon workers regarding the appropriate use, sanitary control, and maintenance of health and beauty products.

Staphylococcal enterotoxin B exposed to pregnant rats inhibits the hedgehog signaling pathway in thymic T lymphocytes of the offspring

The Hedgehog (Hh) signaling pathway is involved in T cell differentiation and development and plays a major regulatory part in different stages of T cell development. A previous study by us suggested that prenatal exposure to staphylococcal enterotoxin B (SEB) changed the percentages of T cell subpopulation in the offspring thymus. However, it is unclear whether prenatal SEB exposure impacts the Hh signaling pathway in thymic T cells. In the present study, pregnant rats at gestational day 16 were intravenously injected once with 15 μg SEB, and the thymi of both neonatal and adult offspring rats were aseptically acquired to scrutinize the effects of SEB on the Hh signaling pathway. It firstly found that prenatal SEB exposure clearly caused the increased expression of Shh and Dhh ligands of the Hh signaling pathway in thymus tissue of both neonatal and adult offspring rats, but significantly decreased the expression levels of membrane receptors of Ptch1 and Smo, transcription factor Gli1, as well as target genes of CyclinD1, C-myc, and N-myc in Hh signaling pathway of thymic T cells. These data suggest that prenatal SEB exposure inhibits the Hh signaling pathway in thymic T lymphocytes of the neonatal offspring, and this effect can be maintained in adult offspring via the imprinting effect.

Janus plasmonic-aggregation induced emission nanobeads as high-performance colorimetric–fluorescent probe of immunochromatographic assay for the ultrasensitive detection of staphylococcal enterotoxin B in milk

Herein, a colorimetric–fluorescent hybrid bifunctional nanobead with Janus structure (J-cf-HBN) was synthesized via one-pot microemulsification. Oleylamine-coated AuNPs and aggregation-induced emission luminogens (AIEgens) were suggested as building blocks to obtain high-performance colorimetric–fluorescent signals. The as-prepared J-cf-HBNs were used as a signal amplification probe to construct an immunochromatographic assay (J-cf-HBNs-ICA) platform for the ultrasensitive detection of staphylococcal enterotoxin B (SEB) in milk samples. Owing to the rational spatial distribution of AuNPs and AIEgens, the J-cf-HBNs present a highly retained photoluminescence and enhanced colorimetric signals. Combined with a pair of highly affinitive anti-SEB antibodies, the J-cf-HBN-ICA platform enabled the fast naked-eye visualization and fluorescent quantitative detection of SEB in various milk matrices. Given the advantages of the dual-mode high-performance J-cf-HBNs, the proposed strip achieved a high sensitivity for SEB qualitative determination with a visual limit of detection (LOD) of 1.56 ng mL−1 and exhibited ultrasensitivity for SEB quantitative detection with a LOD of 0.09 ng mL−1, which is 139-fold lower than that of ELISA using same antibodies. In conclusion, this work provides new insights into the construction of multimode immunochromatographic methods for food safety detection in the field.

Investigation of activation-induced markers (AIM) in porcine T cells by flow cytometry.

Activation-induced markers (AIMs) are frequently analyzed to identify re-activated human memory T cells. However, in pigs the analysis of AIMs is still not very common. Based on available antibodies, we designed a multi-color flow cytometry panel comprising pig-specific or cross-reactive antibodies against CD25, CD69, CD40L (CD154), and ICOS (CD278) combined with lineage/surface markers against CD3, CD4, and CD8α. In addition, we included an antibody against tumor necrosis factor alpha (TNF-α), to study the correlation of AIM expression with the production of this abundant T cell cytokine. The panel was tested on peripheral blood mononuclear cells (PBMCs) stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, Staphylococcus enterotoxin B (SEB) or PBMCs from African swine fever virus (ASFV) convalescent pigs, restimulated with homologous virus. PMA/ionomycin resulted in a massive increase of CD25/CD69 co-expressing T cells of which only a subset produced TNF-α, whereas CD40L expression was largely associated with TNF-α production. SEB stimulation triggered substantially less AIM expression than PMA/ionomycin but also here CD25/CD69 expressing T cells were identified which did not produce TNF-α. In addition, CD40L-single positive and CD25+CD69+CD40L+TNF-α- T cells were identified. In ASFV restimulated T cells TNF-α production was associated with a substantial proportion of AIM expressing T cells but also here ASFV-reactive CD25+CD69+TNF-α- T cells were identified. Within CD8α+ CD4 T cells, several CD25/CD40L/CD69/ICOS defined phenotypes expanded significantly after ASFV restimulation. Hence, the combination of AIMs tested will allow the identification of primed T cells beyond the commonly used cytokine panels, improving capabilities to identify the full breadth of antigen-specific T cells in pigs.

In vitro selection of DNA aptamers against staphylococcal enterotoxin A

Staphylococcal enterotoxin A (SEA) is the most frequently reported in staphylococcal food poisoning (SFP) outbreaks. Aptamers are single-stranded nucleic acids that are seen as promising alternatives to antibodies in several areas, including diagnostics. In this work, systematic evolution of ligands by exponential enrichment (SELEX) was used to select DNA aptamers against SEA. The SELEX protocol employed magnetic beads as an immobilization matrix for the target molecule and real-time quantitative PCR (qPCR) for monitoring and optimizing sequence enrichment. After 10 selection cycles, the ssDNA pool with the highest affinity was sequenced by next generation sequencing (NGS). Approximately 3 million aptamer candidates were identified, and the most representative cluster sequences were selected for further characterization. The aptamer with the highest affinity showed an experimental dissociation constant (KD) of 13.36 ± 18.62 nM. Increased temperature negatively affected the affinity of the aptamer for the target. Application of the selected aptamers in a lateral flow assay demonstrated their functionality in detecting samples containing 100 ng SEA, the minimum amount capable of causing food poisoning. Overall, the applicability of DNA aptamers in SEA recognition was demonstrated and characterized under different conditions, paving the way for the development of diagnostic tools.

Double-Enhanced Photothermal Lateral Flow Biosensor Based on Dual Gold Nanoparticle Conjugates.

Bacterial toxins emerge as the primary triggers of foodborne illnesses, posing a significant threat to human health. To ensure food safety, it is imperative to implement point-of-care testing methods. Lateral flow biosensors (LFBs) based on gold nanoparticles (GNPs) have been commonly used for rapid detection, but their applicationis limited by low sensitivity. Based on the localized surface plasmon resonance and photothermal effect of dual gold nanoparticle conjugates (DGNPs), we developed a smartphone-integrated photothermal LFB (PLFB) with double-enhanced colorimetric and photothermal sensitivity. Through numerical simulations, we verified that DGNPs have significantly enhanced photothermal performance compared to single 15 nm GNPs (SGNPs), and applied DGNPs in PLFB for the detection of staphylococcus enterotoxin A (SEA). The colorimetric and photothermal limits of detection of DGNPs-based PLFB for SEA were 0.091 and 0.0038 ng mL-1, respectively. Compared with the colorimetric detection of the SGNPs-based LFB, the colorimetric detection sensitivity of the DGNPs-based PLFB was increased by 10.7 times, and the photothermal detection sensitivity was further improved by 255.3 times. Moreover, the PLFB exhibits robust reproducibility and exceptional specificity and is applicable for detecting SEA in milk samples. This smartphone-integrated PLFB based on DGNPs allows users to detect toxins simply, conveniently, and quickly and has huge application potential in the field of food safety.

Phenotypic and Genotypic Characterization of Staphylococcus aureus Isolated from Nasal Samples of Healthy Dairy Goats in Algeria.

The present study aimed to determine the phenotypic and genotypic characteristics of S. aureus isolates from the nasal swabs of goats. A total of 232 nasal samples (one per animal) were collected from goats on 13 farms located in two regions of Algeria and were analyzed for the presence of S. aureus. The detection of virulence factors was carried out using PCR. The antibiotic susceptibility of the recovered isolates was assessed using the disc diffusion method. The biofilm formation ability was assessed by the Congo red agar method and a microtiter plate assay, and the molecular characterization of isolates was carried out by spa-typing, and for selected isolates also by multilocus sequence typing (MLST). Overall, 36 out of 232 nasal swabs (15.5%) contained S. aureus, and 62 isolates were recovered. Regarding the virulence factors, at least one staphylococcal enterotoxin gene was detected in 30 (48.4%) isolates. The gene tst encoding the toxic shock syndrome toxin was detected in fifteen isolates (24.2%), but none of the isolates harbored the gene of Panton-Valentine leukocidin (lukF/S-PV). Nine different spa-types were identified, including the detection of a new one (t21230). The recovered isolates were assigned to three clonal complexes, with CC5 (51.8%) being the most common lineage. Two isolates were methicillin-resistant (MRSA) and belonged to ST5 (CC5) and to spa-types t450 and t688. Moreover, 27 (43.5%) of the S. aureus isolates were found to be slime producers in Congo red agar, and all of the recovered isolates could produce biofilms in the microtiter plate assay. Our study showed that the nares of healthy goats could be a reservoir of toxigenic and antibiotic-resistant strains of S. aureus isolates, including MRSA, which could have implications for public health.

Phage K exposure generates phage-neutralizing activity in a rabbit model-humoral receptiveness may impact efficacy of phage therapy.

Phage therapy has not been established in the clinical routine, in part due to uncertainties concerning efficacy and immunogenicity. Here, three rabbits were immunized against staphylococcal phage K to assess viral potency in the presence of immunized serum. Three rabbits received weekly intramuscular injections of ~1010±1 pfu/mL phage K. Phage K-specific IgG formation was measured by an enzyme-linked immunosorbent assay (ELISA); phage inactivation was assessed by calculating K-rates. Using transmission electron microscopy (TEM) and immunogold labeling, antibody binding to phage K was visualized. This was numerically assessed by objective imaging analysis comparing the relative distances of each gold particle to the nearest phage head and tail structure. Immunization led to a strong IgG response, plateauing 7 days after the last phage injection. There was no significant correlation between K-rate and antibody titer over time. TEM showed IgG binding to the head structure of phage K. Image analysis showed a significant reduction in relative distances between antibodies and phage head structures when comparing samples from day 0 and day 28 (P < 0.0001). These results suggest that while individual serum analysis for antibodies against therapeutic phage bears consideration prior to and with prolonged therapy, during phage application, the formation of specific antibodies against phage may only partially explain decreased phage potency in the presence of immunized serum. Instead, other factors may contribute to an individual's "humoral receptiveness" to phage therapy. Future investigations should be directed toward the identification of the humoral factors that have the most significant predictive value on phage potency in vivo.

Keratinocytes Present Staphylococcus aureus Enterotoxins and Promote Malignant and Nonmalignant T Cell Proliferation in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma is characterized by malignant T cells proliferating in a unique tumor microenvironment dominated by keratinocytes (KCs). Skin colonization and infection by Staphylococcus aureus are a common cause of morbidity and are suspected of fueling disease activity. In this study, we show that expression of HLA-DRs, high-affinity receptors for staphylococcal enterotoxins (SEs), by KCs correlates with IFN-γ expression in the tumor microenvironment. Importantly, IFN-γ induces HLA-DR, SE binding, and SE presentation by KCs to malignant T cells from patients with Sézary syndrome and malignant and nonmalignant T-cell lines derived from patients with Sézary syndrome and mycosis fungoides. Likewise, preincubation of KCs with supernatant from patient-derived SE-producing S aureus triggers proliferation in malignant T cells and cytokine release (including IL-2), when cultured with nonmalignant T cells. This is inhibited by pretreatment with engineered bacteriophage S aureus-specific endolysins. Furthermore, alteration in the HLA-DR-binding sites of SE type A and small interfering RNA-mediated knockdown of Jak3 and IL-2Rγ block induction of malignant T-cell proliferation. In conclusion, we show that upon exposure to patient-derived S aureus and SE, KCs stimulate IL-2Rγ/Jak3-dependent proliferation of malignant and nonmalignant T cells in an environment with nonmalignant T cells. These findings suggest that KCs in the tumor microenvironment play a key role in S aureus-mediated disease activity in cutaneous T-cell lymphoma.

Perforin production by T cells and Natural Killer cells in CMV-reactivated allogeneic cell transplant patients is associated with early post-transplant death

Abstract Introduction: Cytomegalovirus (CMV) is a prevalent herpesvirus that poses a serious risk to immunocompromised patients, particularly hematopoietic stem cell transplant (HSCT) recipients. CMV reactivation increases the risk of mortality due to secondary infections. CMV infection has been shown to epigenetically induce immune exhaustion and decrease non-specific immunity. Methods: We reviewed flow cytometry data from CMV seropositive HSCT recipients to determine if CMV reactivation predicted mortality via modulation of lymphocyte function. Peripheral blood mononuclear cells (PBMCs) were collected from recipients 30 days following transplant and stimulated with CMV-specific peptide pools of pp65 and staphylococcal enterotoxin B (SEB) to assess CMV-specific and non-specific immune function. Results: Non-specific, SEB-induced, polyfunctionality of CD4 and CD8 T cells was diminished in deceased CMV seropositive HSCT recipients. In deceased individuals, T-bet and perforin expression were elevated, with decreased IFNγ, TNFα, or IL2 SEB-induced production. Discussion: Mortality in CMV reactivated HSCT recipients was associated with non-specific immune exhaustion phenotypes, but it remains unclear whether this was a result of a higher pathogenic burden, recurrent CMV reactivation, or chemotherapy and myeloablation.