Obesity-Modified CD4+ T-Cells Promote an Epithelial-Mesenchymal Transition Phenotype in Prostate Cancer Cells.

Obesity is associated with low-grade chronic inflammation, and metabolic dysregulation. Evidence shows that chronic inflammation inhibits protective immunity mediated by CD4+ T cells. Additionally, obesity-induced inflammation affects prostate cancer progression. However, the effect of obesity on CD4+ T-cell- response to prostate cancer is not well understood. To investigate whether obesity induces changes in CD4+ T cell cytokine profile, cytokine expression was measured in splenic CD4+ T-cells from 10-week-old male C57Bl/6 mice exposed to conditioned media (CM) from macrophages grown in sera from obese subjects. Additionally, expression levels of key regulators of Epithelial-Mesenchymal Transition (EMT) were measure in prostate cancer epithelial cells exposed to conditioned media from obesity-modified T-cells. Cell migration and invasion was measured in prostate cancer epithelial cells exposed to CM from obesity-modified CD4+ T-cells. Obesity suppressed the expression of IFNγ and IL-2 in CD4+ T-cells but up-regulated the expression of IL-6. Prostate epithelial cancer cells exposed to conditioned media from obesity-modified T cell increased the expression of EMT markers and showed a higher invasive and migratory capacity.

Carbon speciation and flocculation in Neochloris oleoabundans cultures using anaerobically digested stillage.

The effects of bicarbonate loading rate (BLR) and pH on growth kinetics, inorganic carbon speciation, carbon fixation and lipid content in Neochloris oleoabundans cultures using anaerobically digested stillage (ADS) (2% v/v) were investigated. Four different cultures were established: culture A with BLR = 1 g l-1 day-1 and no pH adjustment, culture B with BLR = 0.5 g l-1 day-1 and no pH adjustment, culture C with BLR = 1 g l-1 day-1 and pH adjustment at 7.0, and culture D with BLR = 0.5 g l-1 day-1 and pH adjustment at 7.0. The experiments were carried out in flat plate reactors (4 l) at controlled conditions (light intensity of 134 µmol photon m-1 s-1 and photoperiod 16 light/8 darkness; temperature of 32 ± 1 °C). The effects of pH (7, 10.41, 10.65, and 12), time (15, 30, 60, and 90 min), and concentration of a cationic polyelectrolyte (CP) (10 and 20 mg l-1) on the flocculation efficiency (FE) of N. oleoabundans were also investigated. The results showed that bicarbonate was the predominant carbon species in the media and the main carbon source for microalgae growth in all cultures. The highest productivity (87.70 ± 9.70 mg l-1 day-1) and CO2(aq) fixation rate (0.15 g CO2(aq) l-1 day-1) were found in culture B. The lipid content in N. oleoabundans was affected negatively by the pH adjustment at 7.0 during its growth; higher values were found in cultures with no pH adjustment (37.10% and 38.85% dw for culture A and B, respectively) as compared to those obtained in cultures with pH adjustment (27.35% and 22.20% dw for culture C and D, respectively) (p < 0.05). Regarding flocculation, the addition of 20 mg CP l-1 was required to obtain a FE > 95% in cultures A and B, although a significant FE (40-59%) occurred without CP addition at a high pH (≥ 10.41) in all cultures.

Unique potential of 4-1BB agonist antibody to promote durable regression of HPV+ tumors when combined with an E6/E7 peptide vaccine.

Antibody modulation of T-cell coinhibitory (e.g., CTLA-4) or costimulatory (e.g., 4-1BB) receptors promotes clinical responses to a variety of cancers. Therapeutic cancer vaccination, in contrast, has produced limited clinical benefit and no curative therapies. The E6 and E7 oncoproteins of human papilloma virus (HPV) drive the majority of genital cancers, and many oropharyngeal tumors. We discovered 15-19 amino acid peptides from HPV-16 E6/E7 for which induction of T-cell immunity correlates with disease-free survival in patients treated for high-grade cervical neoplasia. We report here that intranasal vaccination with these peptides and the adjuvant alpha-galactosylceramide elicits systemic and mucosal T-cell responses leading to reduced HPV(+) TC-1 tumor growth and prolonged survival in mice. We hypothesized that the inability of these T cells to fully reject established tumors resulted from suppression in the tumor microenvironment which could be ameliorated through checkpoint modulation. Combining this E6/E7 peptide vaccine with checkpoint blockade produced only modest benefit; however, coadministration with a 4-1BB agonist antibody promoted durable regression of established genital TC-1 tumors. Relative to other therapies tested, this combination of vaccine and α4-1BB promoted the highest CD8(+) versus regulatory FoxP3(+) T-cell ratios, elicited 2- to 5-fold higher infiltration by E7-specific CTL, and evoked higher densities of highly cytotoxic TcEO (T cytotoxic Eomesodermin) CD8 (>70-fold) and ThEO (T helper Eomesodermin) CD4 (>17-fold) T cells. These findings have immediate clinical relevance both in terms of the direct clinical utility of the vaccine studied and in illustrating the potential of 4-1BB antibody to convert therapeutic E6/E7 vaccines already in clinical trials into curative therapies.

Phytofiltration of anaerobically digested sugarcane ethanol stillage using a macrophyte with high potential for biofuel production.

Anaerobically digested stillage (ADS) requires treatment before being discharged into water bodies or soils to avoid adverse effects. Phytofiltration systems are eco-friendly technologies for wastewater treatment, and they simultaneously serve as a source of biomass for biofuel production. The aim of the present study was to investigate the phytofiltration of ADS using Azolla sp. The effects of the ADS strength (dilutions 1: 50 and 1 : 25 v/v) and initial biomass density (IBD) [15.44 (IBD1) and 23.16 (IBD2) g dry weight (dw) m-2] on plant growth and pollutant removal were assessed. Productivities obtained at ADS 1: 50 (2.93 and 3.04 g m-2 d-1 for IBD1 and IBD2, respectively) were not significantly different from those of a synthetic medium (2.56 and 3.15 g m-2 for IBD1 and IBD2, respectively). Higher organic matter removal was found using ADS at 1: 25 than that obtained using ADS 1: 50 (52.16-53.34 vs 32.29-38.16%), while no IBD effect was observed. The nutrient concentrations in ADS were reduced significantly, especially the concentrations of NH4-N (75.11-82.54%), PO4-P (88.72-92.90%) and SO4-S (55.95-66.61%). The conversion of nutrients from ADS into Azolla biomass may result in an effective way to produce an attractive feedstock for biofuel production.

Prostate cancers with distinct transcriptional programs in Black and White men.

BACKGROUND: Black men are at a higher risk of prostate cancer (PC) diagnosis and present with more high-grade PC than White men in an equal access setting. This study aimed to identify differential transcriptional regulation between Black and White men with PC. METHODS: We performed microarray of radical prostatectomy tissue blocks from 305 Black and 238 White men treated at the Durham Veterans Affairs Medical Center. Differential expression, gene set enrichment analysis, master regulator analysis, and network modeling were conducted to compare gene expression by race. Findings were validated using external datasets that are available in the Gene Expression Omnibus (GEO) database. The first was a multi-institutional cohort of 1152 prostate cancer patients (596 Black, 556 White) with microarray data (GEO ID: GSE169038). The second was an Emory cohort of 106 patients (22 Black, 48 White, 36 men of unknown race) with RNA-seq data (GEO ID: GSE54460). Additionally, we analyzed androgen receptor (AR) chromatin binding profiles using paired AR ChIP-Seq datasets from Black and White men (GEO IDs: GSE18440 and GSE18441). RESULTS: We identified 871 differentially expressed genes between Black and White men. White men had higher activity of MYC-related pathways, while Black men showed increased activity of inflammation, steroid hormone responses, and cancer progression-related pathways. We further identified the top 10 transcription factors (TFs) in Black patients, which formed a transcriptional regulatory network centered on the AR. The activities of this network and the pathways were significantly different in Black vs. White men across multiple cohorts and PC molecular subtypes. CONCLUSIONS: These findings suggest PC in Black and White men have distinct tumor transcriptional profiles. Furthermore, a highly interactive TF network centered on AR drives differential gene expression in Black men. Additional study is needed to understand the degree to which these differences in transcriptional regulatory elements contribute to PC health disparities.

Investigating trends in interest for benign prostatic hyperplasia surgery options using Google Trends.

Understanding patient interest among surgical options is challenging. We used Google Trends to analyze interest in benign prostatic hyperplasia (BPH) surgeries recommended for prostate volumes <80 cc. Google Trends was queried with five BPH surgeries. Final rank of search terms was TURP, UroLift, Rezum, Aquablation, and Greenlight. Google Trends can be an effective tool for evaluating public interest trends in BPH surgery.

Identification of Coxiella burnetii type IV secretion substrates required for intracellular replication and Coxiella-containing vacuole formation.

Coxiella burnetii, the etiological agent of acute and chronic Q fever in humans, is a naturally intracellular pathogen that directs the formation of an acidic Coxiella-containing vacuole (CCV) derived from the host lysosomal network. Central to its pathogenesis is a specialized type IVB secretion system (T4SS) that delivers effectors essential for intracellular replication and CCV formation. Using a bioinformatics-guided approach, 234 T4SS candidate substrates were identified. Expression of each candidate as a TEM-1 ß-lactamase fusion protein led to the identification of 53 substrates that were translocated in a Dot/Icm-dependent manner. Ectopic expression in HeLa cells revealed that these substrates trafficked to distinct subcellular sites, including the endoplasmic reticulum, mitochondrion, and nucleus. Expression in Saccharomyces cerevisiae identified several substrates that were capable of interfering with yeast growth, suggesting that these substrates target crucial host processes. To determine if any of these T4SS substrates are necessary for intracellular replication, we isolated 20 clonal T4SS substrate mutants using the Himar1 transposon and transposase. Among these, 10 mutants exhibited defects in intracellular growth and CCV formation in HeLa and J774A.1 cells but displayed normal growth in bacteriological medium. Collectively, these results indicate that C. burnetii encodes a large repertoire of T4SS substrates that play integral roles in host cell subversion and CCV formation and suggest less redundancy in effector function than has been found in the comparative Legionella Dot/Icm model.

Avocados: A Recipe for Good Guacamole or Lower Cancer Risk?

Diet plays a role in modifying cancer risk. This study from Ericsson and colleagues provides new evidence suggesting that avocado intake could aid in cancer prevention. However, these effects were observed only in men, pointing to intriguing key differences by sex. Also, associations were only seen for some (colorectal, lung, and bladder cancers), but not all cancer types. Still, the number of avocado servings and ways to eat avocado to obtain these benefits remain to be determined. This brief commentary discusses the study and provides an opinion on the proposed role of avocados in reducing cancer risk. See related article by Ericsson et al., p. 211.

The effects of glycemic index on prostate cancer progression in a xenograft mouse model.

BACKGROUND: Previously, we found low-carbohydrate diets slowed prostate cancer (PC) growth and increased survival vs. a Western diet in mice, by inhibiting the insulin/IGF-1 axis. Thus, we tested whether modifying carbohydrate quality to lower glycemic index (GI) without changing quantity results in similar benefits as with reduced quantity. METHODS: Male SCID mice injected with LAPC-4 cells were single-housed and randomized when their tumors reached 200 mm3 on average to a LoGI (48% carbohydrate kcal, from Hylon-VII) or HiGI Western diet (48% carbohydrate kcal, from sucrose). Body weight and tumor volume were measured weekly. Body composition was assessed 35 days after randomization. Blood glucose and serum insulin, IGF-1 and IGFBP3 were measured at study end when tumor volumes reached 800 mm3. We analyzed gene expression of mice tumors by RNA-sequencing and human tumors using the Prostate Cancer Transcriptome Atlas. RESULTS: There were no significant differences in tumor volume (P > 0.05), tumor proliferation (P = 0.29), and overall survival (P = 0.15) between groups. At 35 days after randomization, the LoGI group had 30% lower body fat (P = 0.007) despite similar body weight (P = 0.58). At sacrifice, LoGI mice had smaller livers (P < 0.001) and lower glucose (P = 0.15), insulin (P = 0.11), IGF-1 (P = 0.07) and IGF-1:IGFBP3 ratio (P = 0.05), and higher IGFBP3 (P = 0.09) vs. HiGI, although none of these metabolic differences reached statistical significance. We observed differential gene expression and pathway enrichment in mice tumors by diet. The most upregulated and downregulated gene in the LoGI group showed expression patterns more closely resembling expression in human benign prostate tissue vs. PC. CONCLUSIONS: In this single mouse xenograft model, consuming a low GI diet did not delay PC growth or survival vs. a high GI diet despite suggestions of decreased activation of the insulin/IGF-1 pathway. These data suggest that improving carbohydrate quality alone while consuming a high carbohydrate diet may not effectively slow PC growth.

27-hydroxycholesterol and DNA damage repair: implication in prostate cancer.

Introduction: We previously reported that cholesterol homeostasis in prostate cancer (PC) is regulated by 27-hydroxycholesterol (27HC) and that CYP27A1, the enzyme that converts cholesterol to 27HC, is frequently lost in PCs. We observed that restoring the CYP27A1/27HC axis inhibited PC growth. In this study, we investigated the mechanism of 27HC-mediated anti-PC effects. Methods: We employed in vitro models and human transcriptomics data to investigate 27HC mechanism of action in PC. LNCaP (AR+) and DU145 (AR-) cells were treated with 27HC or vehicle. Transcriptome profiling was performed using the Affymetrix GeneChip™ microarray system. Differential expression was determined, and gene set enrichment analysis was done using the GSEA software with hallmark gene sets from MSigDB. Key changes were validated at mRNA and protein levels. Human PC transcriptomes from six datasets were analyzed to determine the correlation between CYP27A1 and DNA repair gene expression signatures. DNA damage was assessed via comet assays. Results: Transcriptome analysis revealed 27HC treatment downregulated Hallmark pathways related to DNA damage repair, decreased expression of FEN1 and RAD51, and induced "BRCAness" by downregulating genes involved in homologous recombination regulation in LNCaP cells. Consistently, we found a correlation between higher CYP27A1 expression (i.e., higher intracellular 27HC) and decreased expression of DNA repair gene signatures in castration-sensitive PC (CSPC) in human PC datasets. However, such correlation was less clear in metastatic castration-resistant PC (mCRPC). 27HC increased expression of DNA damage repair markers in PC cells, notably in AR+ cells, but no consistent effects in AR- cells and decreased expression in non-neoplastic prostate epithelial cells. While testing the clinical implications of this, we noted that 27HC treatment increased DNA damage in LNCaP cells via comet assays. Effects were reversible by adding back cholesterol, but not androgens. Finally, in combination with olaparib, a PARP inhibitor, we showed additive DNA damage effects. Discussion: These results suggest 27HC induces "BRCAness", a functional state thought to increase sensitivity to PARP inhibitors, and leads to increased DNA damage, especially in CSPC. Given the emerging appreciation that defective DNA damage repair can drive PC growth, future studies are needed to test whether 27HC creates a synthetic lethality to PARP inhibitors and DNA damaging agents in CSPC.

Key takeaways for knowledge expansion of early-career scientists conducting Transdisciplinary Research in Energetics and Cancer (TREC): a report from the TREC Training Workshop 2022.

The overall goal of the annual Transdisciplinary Research in Energetics and Cancer (TREC) Training Workshop is to provide transdisciplinary training for scientists in energetics and cancer and clinical care. The 2022 Workshop included 27 early-to-mid career investigators (trainees) pursuing diverse TREC research areas in basic, clinical, and population sciences. The 2022 trainees participated in a gallery walk, an interactive qualitative program evaluation method, to summarize key takeaways related to program objectives. Writing groups were formed and collaborated on this summary of the 5 key takeaways from the TREC Workshop. The 2022 TREC Workshop provided a targeted and unique networking opportunity that facilitated meaningful collaborative work addressing research and clinical needs in energetics and cancer. This report summarizes the 2022 TREC Workshop's key takeaways and future directions for innovative transdisciplinary energetics and cancer research.

Pontederia sagittata and Cyperus papyrus contribution to carbon storage in floating treatment wetlands established in subtropical urban ponds.

Carbon sequestration is considered an ecosystem service of regulation provided by diverse ecosystems, including wetlands. It has been widely evaluated in the soil of natural wetlands while in constructed wetlands, there is scanty information. In Floating Treatment Wetlands (FTW) there is none. Previously, our research group reported the efficient performance of FTW in an urban polluted pond for two years. As a follow up, the aim of this work was to investigate the contribution of Cyperus papyrus and Pontederia sagittata to carbon storage (CS) in four FTW established in eutrophic urban ponds in a subtropical region. Plant growth, productivity, and CS were assessed in the aboveground biomass of C. papyrus and P. sagittata and the belowground biomass (root mix from C. papyrus and P. sagittata), throughout 26 months in 2 FTW with an area of 17.5 m2 (FTW1) and 33 m2 (FTW2) and throughout 19 months in 2 FTW with an area of 25 m2 (FTW3) and 33 m2 (FTW4), respectively. The macrophyte growth depended on various factors, such as the season, the plant species, and the location of the FTW. High relative growth rate values were found for both species (0.125 and 0.142 d-1 for P. sagittata and C. papyrus, respectively), especially during summer and early autumn. The highest values of productivity were 337 ± 125 gdw m-2d-1 for the aboveground biomass of C. papyrus in FTW2, 311 ± 96.90 gdwm-2d-1 for the aboveground of P. sagittata in FTW1, and 270 ± 107 gdw m-2d-1 for the belowground biomass in FTW2. The mean values of CS for P. sagittata found in FTW1 were 1.90 ± 0.94 kg m-2, while for C. papyrus in FTW2 they were 4.09 ± 0.73 kg m-2. The contribution of the belowground biomass to CS was also significant in FTW2 (4.58 ± 0.59 kg m-2).

Characterizing molecular subtypes of high-risk non-muscle-invasive bladder cancer in African American patients.

BACKGROUND: We sought to determine whether differences in subtype distribution and differentially expressed genes exist between African Americans (AAs) and European Americans (EAs) in patients with high-risk nonmuscle-invasive bladder cancer (NMIBC). METHODS: We performed a retrospective cohort study including 26 patients (14 AAs and 12 EAs) from the University of Texas Medical Branch and the Durham Veterans Affair Health Care System from 2010 to 2020 among treatment naïve, high-risk NMIBC. Profiled gene expressions were performed using the UROMOL classification system. RESULTS: UROMOL racial subtype distributions were similar with class 2a being most common with 10 genes commonly upregulated in AAs compared to EAs including EFEMP1, S100A16, and MCL1 which are associated with progression to muscle-invasive bladder cancer, mitomycin C resistance, and bacillus Calmette-Guérin durability, respectively. We used single nuclei analysis to map the malignant cell heterogeneity in urothelial cancer which 5 distinct malignant epithelial subtypes whose presence has been associated with different therapeutic response prediction abilities. We mapped the expression of the 10 genes commonly upregulated by race as a function of the 5 malignant subtypes. This showed borderline (P = 0.056) difference among the subtypes suggesting AAs and EAs may be expected to have different therapeutic responses to treatments for bladder cancer. AAs were enriched with immune-related, inflammatory, and cellular regulation pathways compared to EAs, yet appeared to have reduced levels of the aggressive C3/CDH12 bladder tumor cell population. CONCLUSIONS: While premature, gene expression differed between AAs and EAs, supporting potential race-based etiologies for muscle-invasion, response to treatments, and transcriptome pathway regulations.

Microalgae-Based Biorefineries: Challenges and Future Trends to Produce Carbohydrate Enriched Biomass, High-Added Value Products and Bioactive Compounds.

Microalgae have demonstrated a large potential in biotechnology as a source of various macromolecules (proteins, carbohydrates, and lipids) and high-added value products (pigments, poly-unsaturated fatty acids, peptides, exo-polysaccharides, etc.). The production of biomass at a large scale becomes more economically feasible when it is part of a biorefinery designed within the circular economy concept. Thus, the aim of this critical review is to highlight and discuss challenges and future trends related to the multi-product microalgae-based biorefineries, including both phototrophic and mixotrophic cultures treating wastewater and the recovery of biomass as a source of valuable macromolecules and high-added and low-value products (biofertilizers and biostimulants). The therapeutic properties of some microalgae-bioactive compounds are also discussed. Novel trends such as the screening of species for antimicrobial compounds, the production of bioplastics using wastewater, the circular economy strategy, and the need for more Life Cycle Assessment studies (LCA) are suggested as some of the future research lines.

27-Hydroxycholesterol Impairs Plasma Membrane Lipid Raft Signaling as Evidenced by Inhibition of IL6-JAK-STAT3 Signaling in Prostate Cancer Cells.

We recently reported that restoring the CYP27A1-27hydroxycholesterol axis had antitumor properties. Thus, we sought to determine the mechanism by which 27HC exerts its anti-prostate cancer effects. As cholesterol is a major component of membrane microdomains known as lipid rafts, which localize receptors and facilitate cellular signaling, we hypothesized 27HC would impair lipid rafts, using the IL6-JAK-STAT3 axis as a model given its prominent role in prostate cancer. As revealed by single molecule imaging of DU145 prostate cancer cells, 27HC treatment significantly reduced detected cholesterol density on the plasma membranes. Further, 27HC treatment of constitutively active STAT3 DU145 prostate cancer cells reduced STAT3 activation and slowed tumor growth in vitro and in vivo. 27HC also blocked IL6-mediated STAT3 phosphorylation in nonconstitutively active STAT3 cells. Mechanistically, 27HC reduced STAT3 homodimerization, nuclear translocation, and decreased STAT3 DNA occupancy at target gene promoters. Combined treatment with 27HC and STAT3 targeting molecules had additive and synergistic effects on proliferation and migration, respectively. Hallmark IL6-JAK-STAT gene signatures positively correlated with CYP27A1 gene expression in a large set of human metastatic castrate-resistant prostate cancers and in an aggressive prostate cancer subtype. This suggests STAT3 activation may be a resistance mechanism for aggressive prostate cancers that retain CYP27A1 expression. In summary, our study establishes a key mechanism by which 27HC inhibits prostate cancer by disrupting lipid rafts and blocking STAT3 activation. IMPLICATIONS: Collectively, these data show that modulation of intracellular cholesterol by 27HC can inhibit IL6-JAK-STAT signaling and may synergize with STAT3-targeted compounds.

Continuous dye adsorption and desorption on an invasive macrophyte (Salvinia minima).

The continuous adsorption-desorption of methylene blue (MB) on an invasive macrophyte, Salvinia minima, was investigated in fixed-bed columns. The effects of bed depth (h) (9.30, 18.70, and 28 cm), inlet dye concentration (C0) (51 ± 1.20, 154 ± 2.00, and 250 ± 1.50 mg L-1), and flow rate (Q) (7 and 14 mL min-1) on dye removal and breakthrough curves were assessed. Thomas, modified dose-response (MDR) and bed depth service time (BDST) models were fitted to the experimental data. Desorption and regeneration studies were also performed. The breakthrough time was affected by h, C0, and Q. The dynamic bed capacity at the breakthrough point (qb) increased with increasing h but decreased with increasing C0 and Q. Dynamic bed capacities (qe) from 318 to 322 mg g-1 were achieved at h = 28 cm, C0 = 154 ± 2.0, or 250 ± 1.50 mg L-1, independently of the Q value. High MB removals were also observed (75-78%). FTIR analysis revealed that hydroxyl and carboxyl groups could be involved in dye adsorption. MDR and BDST models were both successfully used to predict the breakthrough curves of MB adsorption onto S. minima. A high regeneration efficiency (> 87%) was obtained after three adsorption-desorption cycles. These results confirm that the use of S. minima biomass could be a very efficient and eco-friendly alternative for MB adsorption in continuous mode.

Constructed wetland mesocosms for the treatment of diluted sugarcane molasses stillage from ethanol production using Pontederia sagittata.

Sugarcane molasses stillage contains a very high concentration of organic matter and toxic/recalcitrant compounds. Its improper disposal has become a global problem and there is very scanty information about its treatment using phytotechnologies. This work aimed at evaluating the performance of subsurface flow constructed wetlands (SSF CWs) mesocosms planted with Pontederia sagittata and operating at two hydraulic retention times (HRTs), compared to an unplanted SSF CWs, for the treatment of diluted stillage subjected to no pre-treatment apart from an adjustment to pH 6.0. CWs were fed with very high surface COD loading rates (i.e. 47.26 and 94.83gCOD/m(2)d). The planted CWs were able to remove COD in the range of 80.24-80.62%, BOD(5) in the range of 82.20-87.31%, TKN in the range of 73.42-76.07%, nitrates from 56-58.74% and sulfates from 68.58-69.45%, depending on the HRT. Phosphate and potassium were not removed. It was concluded that this type of CWs is a feasible option for the treatment of diluted stillage.

Prophylactic Sublingual Immunization with Mycobacterium tuberculosis Subunit Vaccine Incorporating the Natural Killer T Cell Agonist Alpha-Galactosylceramide Enhances Protective Immunity to Limit Pulmonary and Extra-Pulmonary Bacterial Burden in Mice.

Infection by Mycobacterium tuberculosis (Mtb) remains a major global concern and the available Bacillus Calmette-Guerin (BCG) vaccine is poorly efficacious in adults. Therefore, alternative vaccines and delivery strategies focusing on Mtb antigens and appropriate immune stimulating adjuvants are needed to induce protective immunity targeted to the lungs, the primary sites of infections and pathology. We present here evidence in support of mucosal vaccination by the sublingual route in mice using the subunit Mtb antigens Ag85B and ESAT-6 adjuvanted with the glycolipid alpha-galactosylceramide (α-GalCer), a potent natural killer T (NKT) cell agonist. Vaccinated animals exhibited strong antigen-specific CD4 and CD8 T cells responses in the spleen, cervical lymph nodes and lungs. In general, inclusion of the α-GalCer adjuvant significantly enhanced these responses that persisted over 50 days. Furthermore, aerosolized Mtb infection of vaccinated mice resulted in a significant reduction of bacterial load of the lungs and spleens as compared to levels seen in naïve controls or those vaccinated with subunit proteins, adjuvant , or BCG alone. The protection induced by the Mtb antigens and-GalCer vaccine through sublingual route correlated with a TH1-type immunity mediated by antigen-specific IFN-γ and IL-2 producing T cells.