Improved MAIT cell functions following fecal microbiota transplantation for metastatic renal cell carcinoma.

Strategies to modify the gut microbiome in cancer patients using fecal microbiota transplantation (FMT) have gained momentum as a therapeutic intervention. However, how FMT impacts innate-like, antimicrobial T lymphocytes is unclear. In this study, we assessed peripheral blood (PB) mucosa-associated invariant T (MAIT) cell frequencies and functions in patients with metastatic renal cell carcinoma (mRCC) before and seven days after they received FMT as part of a clinical trial. We found comparable MAIT cell frequencies in healthy controls and mRCC patients. In contrast, γδ T cells exhibited a numerical decline in mRCC, which was partially reversed by FMT. We also found a significant increase in the PB CD4+ MAIT cell compartment of mRCC patients with or without FMT. Paired sample analyses revealed CD69 upregulation on MAIT cells accompanied by decreased PD-1 levels post-FMT. These changes were unique to MAIT cells as non-MAIT T lymphocytes showed either no trend or a trend in the opposite direction. Importantly, FMT did not render MAIT cells exhausted as also judged by their stable expression of TIM-3, LAG-3, BTLA, CTLA-4, TIGIT and VISTA. These findings were corroborated in functional assays in which MAIT cells were stimulated with MR1 ligands or with a combination of IL-12 and IL-18 to produce inflammatory cytokines and granzyme B. Indeed, when stimulated ex vivo with IL-12 and IL-18, MAIT cells mounted a more rigorous TNF-α response post-FMT. In conclusion, FMT improves MAIT cell functions, which should serve patients well in subsequent microbial challenges in the face of cancer-elicited immunosuppression. Trial Registration: https://clinicaltrials.gov/ Identifier: NCT04163289 (registration date: November 14, 2019).

Fexofenadine and Rosuvastatin Pharmacokinetics in Mice with Targeted Disruption of Organic Anion Transporting Polypeptide 2B1.

Organic anion transporting polypeptide 2B1 (OATP2B1) is a widely expressed membrane transporter with diverse substrate specificity. In vitro and clinical studies suggest a role for intestinal OATP2B1 in the oral absorption of medications. Moreover, OATP2B1 is highly expressed in hepatocytes where it is thought to promote liver drug clearance. However, until now, a shortcoming of studies implicating OATP2B1 in drug disposition has been a lack of in vivo models. Here, we report the development of a knockout (KO) mouse model with targeted, global disruption of the Slco2b1 gene to examine the disposition of two confirmed mOATP2B1 substrates, namely, fexofenadine and rosuvastatin. The plasma pharmacokinetics of intravenously administered fexofenadine was not different between KO and wild-type (WT) mice. However, after oral fexofenadine administration, KO mice had 70% and 41% lower maximal plasma concentration (C max) and area under the plasma concentration-time curve (AUC0-last) than WT mice, respectively. In WT mice, coadministration of fexofenadine with grapefruit juice (GFJ) or apple juice (AJ) was associated with reduced C max by 80% and 88%, respectively, while the AUC0-last values were lower by 35% and 70%, respectively. In KO mice, AJ coadministration reduced oral fexofenadine C max and AUC0-last values by 67% and 59%, respectively, while GFJ had no effects. Intravenous and oral rosuvastatin pharmacokinetics were similar among WT and KO mice. We conclude that intestinal OATP2B1 is a determinant of oral fexofenadine absorption, as well as a target for fruit juice interactions. OATP2B1 does not significantly influence rosuvastatin disposition in mice. SIGNIFICANCE STATEMENT: A novel mouse model with targeted disruption of the Slco2b1 gene revealed that OATP2B1 is a determinant of oral absorption but not systemic disposition of fexofenadine, as well as a target of fruit juice interactions. Rosuvastatin oral and intravenous pharmacokinetics were not dependent on OATP2B1. These findings support the utility of the Slco2b1 KO mouse model for defining mechanisms of drug disposition at the intersection of in vitro and clinical pharmacology.

Differences in Warfarin Pharmacodynamics and Predictors of Response Among Three Racial Populations.

BACKGROUND: Population differences in warfarin dosing requirement have been reported; however, unlike the pharmacokinetics (PK) of warfarin, the quantitative influences of pharmacodynamic (PD) factors on the anticoagulation response to warfarin in different ethnic populations are totally unknown. METHODS: Using population PK/PD analysis, we attempted to identify predictors of S-warfarin clearance [CL(S)] and half maximal effective concentration (EC50) to quantify racial differences in both PK and PD parameters, and to assess the contribution of these parameters to the international normalized ratio (INR) and over-anticoagulation response (INR ≥ 4) in a cohort of 309 White, Asian and African American patients. RESULTS: Similar to our previous findings, the median CL(S) was 30% lower in African American patients than Asian and White patients (169 vs. 243 and 234 mL/h, p < 0.01). EC50 showed a greater racial difference than CL(S) [1.03, 1.70 and 2.76 µg/mL for Asian, White and African American patients, respectively, p < 0.01). Significant predictors of INR included demographic/clinical (age, body weight, creatinine clearance and sex) and genotypic (CYP2C9*3,*8 and VKORC1 -1639G>A) factors, as well as African American ethnicity. In all three racial groups, genetic predictors of INR appeared to have greater influence than demographic/clinical predictors. Both CL(S) and EC50 contributed to the over-anticoagulation response to warfarin. Patients having VKORC1 -1639 G>A and/or factors associated with reduced CYP2C9 activity were more likely to have an INR ≥ 4. CONCLUSIONS: Although there were contrasting racial differences in CL(S) and EC50 that impacted on the INR, the racial difference in EC50 was greater than that for CL(S), thus explaining the higher warfarin requirement for African American patients.

Generation of protection against Francisella novicida in mice depends on the pathogenicity protein PdpA, but not PdpC or PdpD.

Previous results suggest that mutations in most genes in the Francisella pathogenicity island (FPI) attenuate the bacterium. Using a mouse model, here we determined the impact of mutations in pdpA, pdpC, and pdpD in Francisella novicida on in vitro replication in macrophages, and in vivo immunogenicity. In contrast to most FPI genes, deletion of pdpC (FnΔpdpC) and pdpD (FnΔpdpD) from F. novicida did not impact growth in mouse bone-marrow derived macrophages. Nonetheless, both FnΔpdpC and FnΔpdpD were highly attenuated when administered intradermally. Infected mice produced relatively normal anti-F. novicida serum antibodies. Further, splenocytes from infected mice controlled intramacrophage Francisella replication, indicating T cell priming, and mice immunized by infection with FnΔpdpC or FnΔpdpD survived secondary lethal parenteral challenge with either F. novicida or Francisella tularensis LVS. In contrast, deletion of pdpA (FnΔpdpA) ablated growth in macrophages in vitro. FnΔpdpA disseminated and replicated poorly in infected mice, accompanied by development of some anti-F. novicida serum antibodies. However, primed Th1 cells were not detected, and vaccinated mice did not survive even low dose challenge with either F. novicida or LVS. Taken together, these results suggest that successful priming of Th1 cells, and protection against lethal challenge, depends on expression of PdpA.

Burkholderia multivorans survival and trafficking within macrophages.

Cystic fibrosis (CF) patients are at great risk of opportunistic lung infection, particularly by members of the Burkholderia cepacia complex (Bcc). This group of bacteria can cause damage to the lung tissue of infected patients and are difficult to eradicate due to their high levels of antibiotic resistance. Although the highly virulent Burkholderia cenocepacia has been the focus of virulence research for the past decade, Burkholderia multivorans is emerging as the most prevalent Bcc species infecting CF patients in North America. Despite several studies detailing the intramacrophage trafficking and survival of B. cenocepacia, no such data exist for B. multivorans. The results of this study demonstrated that the clinical CF isolates C5568 and C0514 and an environmental B. multivorans isolate, ATCC 17616, were able to replicate and survive within murine macrophages in a manner similar to that of B. cenocepacia strain K56-2. These strains were also able to survive but were unable to replicate within human THP-1 macrophages. Differences in macrophage uptake were observed among all three B. multivorans strains; these variances were attributed to major differences in O-antigen production. Unlike B. cenocepacia-containing vacuoles, which delay phagosomal maturation in murine macrophages by 6 h, all B. multivorans-containing vacuoles co-localized with lysosome-associated membrane protein-1, a late endosome/lysosomal marker, and the lysosomal marker dextran within 2 h of uptake. Together, these results indicated that, whilst both Bcc species were able to survive and replicate within macrophages, they utilized different intramacrophage survival strategies. To observe differences in virulence, the strains were compared using the Galleria mellonella (wax worm) model. When compared with the B. multivorans strains tested, B. cenocepacia K56-2 was highly virulent in this model and killed all worms within 24 h when injected at 10(7) c.f.u. B. multivorans clinical isolates C5568 and C0514 were significantly more virulent than the soil isolate ATCC 17616, which was avirulent even when worms were injected with 10(7) c.f.u. These results suggest strain differences in the virulence of B. multivorans isolates.

Essential genes from Arctic bacteria used to construct stable, temperature-sensitive bacterial vaccines.

All bacteria share a set of evolutionarily conserved essential genes that encode products that are required for viability. The great diversity of environments that bacteria inhabit, including environments at extreme temperatures, place adaptive pressure on essential genes. We sought to use this evolutionary diversity of essential genes to engineer bacterial pathogens to be stably temperature-sensitive, and thus useful as live vaccines. We isolated essential genes from bacteria found in the Arctic and substituted them for their counterparts into pathogens of mammals. We found that substitution of nine different essential genes from psychrophilic (cold-loving) bacteria into mammalian pathogenic bacteria resulted in strains that died below their normal-temperature growth limits. Substitution of three different psychrophilic gene orthologs of ligA, which encode NAD-dependent DNA ligase, resulted in bacterial strains that died at 33, 35, and 37 degrees C. One ligA gene was shown to render Francisella tularensis, Salmonella enterica, and Mycobacterium smegmatis temperature-sensitive, demonstrating that this gene functions in both Gram-negative and Gram-positive lineage bacteria. Three temperature-sensitive F. tularensis strains were shown to induce protective immunity after vaccination at a cool body site. About half of the genes that could be tested were unable to mutate to temperature-resistant forms at detectable levels. These results show that psychrophilic essential genes can be used to create a unique class of bacterial temperature-sensitive vaccines for important human pathogens, such as S. enterica and Mycobacterium tuberculosis.