Differential priority effects impact taxonomy and functionality of host-associated microbiomes.

Most multicellular eukaryotes host complex communities of microorganisms, but the factors that govern their assembly are poorly understood. The settlement of specific microorganisms may have a lasting impact on community composition, a phenomenon known as the priority effect. Priority effects of individual bacterial strains on a host's microbiome are, however, rarely studied and their impact on microbiome functionality remains unknown. We experimentally tested the effect of two bacterial strains (Pseudoalteromonas tunicata D2 and Pseudovibrio sp. D323) on the assembly and succession of the microbial communities associated with the green macroalga Ulva australis. Using 16S rRNA gene sequencing and qPCR, we found that both strains exert a priority effect, with strain D2 causing initially strong but temporary taxonomic changes and strain D323 causing weaker but consistent changes. Consistent changes were predominately facilitatory and included taxa that may benefit the algal host. Metagenome analyses revealed that the strains elicited both shared (e.g., depletion of type III secretion system genes) and unique (e.g., enrichment of antibiotic resistance genes) effects on the predicted microbiome functionality. These findings indicate strong idiosyncratic effects of colonizing bacteria on the structure and function of host-associated microbial communities. Understanding the idiosyncrasies in priority effects is key for the development of novel probiotics to improve host condition.

Breast Surgical Oncology Fellowship applicant selection and ranking: A survey of Society of Surgical Oncology programs.

BACKGROUND AND OBJECTIVES: In 2003, the Society of Surgical Oncology (SSO) initiated a breast surgical oncology fellowship, which has now grown to 60 SSO accredited programs as of 2021. Limited knowledge exists on the traits of successful applicants and the factors influencing the rank list. METHODS: A web-based, anonymous survey was sent to all SSO Breast Surgical Oncology Fellowship program directors. The survey consisted of 26 questions. Descriptive statistics were used to analyze survey responses and evaluate impact on applicant interview and rank list. RESULTS: Thirty-four programs (57% response rate) completed the survey. Programs received an average of 70 applications and granted 24 interviews. Most programs reported a minimum ABSITE cut-off score (n = 28, 82%) and a defined publication requirement (n = 22, 65%), including a first-author requirement (n = 18, 53%) to extend an invitation to interview. For postinterview rank, applicant interpersonal skills were highly valued. The interview was the most important aspect for the rank list. CONCLUSIONS: Many programs have ABSITE and publication thresholds before offering an interview. Upon receiving interview invitation, the applicant's interview performance, interpersonal skills, and letters of recommendation were the most important aspect in rank list decision making.

Adjuvant chemotherapy is associated with improved overall survival in select patients with Stage II colon cancer: A National Cancer Database analysis.

BACKGROUND AND OBJECTIVES: Guidelines for Stage II colon cancer recommend adjuvant chemotherapy (AC) only for tumors with high-risk features, but long-term outcomes data are mixed. We aimed to determine if AC was associated with a survival benefit in this population. METHODS: Patients were identified from the National Cancer Database and included if they met the following criteria: diagnosis of Stage II colon cancer, surgery, survival data, and complete data on six high-risk features. The cohort of 57 335 patients was stratified by receipt of AC. Subgroup analysis was performed on patients under the age of 65 years with no comorbidities. Overall survival (OS) was the primary endpoint. RESULTS: An increasing number of high-risk features was associated with significantly decreased median OS. AC was associated with significantly increased OS for patients with 0, 1, 2, and ≥3 high-risk features. On subgroup analysis, receipt of AC was associated with a reduced risk of death (hazard ratio: 0.66; confidence interval: 0.59-0.74). For patients in the subgroup who had a T4 tumor, AC was associated with increased OS (92.7 vs. 83.6 months). CONCLUSIONS: AC should be considered for all younger, healthy patients with Stage II colon cancer and may be associated with a survival benefit for patients with T4 disease.

Central memory CD8+ T cells become CD69+ tissue-residents during viral skin infection independent of CD62L-mediated lymph node surveillance.

Memory CD8+ T cells in the circulation rapidly infiltrate non-lymphoid tissues following infection and provide protective immunity in an antigen-specific manner. However, the subsequent fate of memory CD8+ T cells after entering non-lymphoid tissues such as the skin during a secondary infection is largely unknown. Furthermore, because expression of CD62L is often used to identify the central memory (TCM) CD8+ T cell subset, uncoupling the physical requirement for CD62L-mediated lymph node homing versus other functional attributes of TCM CD8+ T cells remains unresolved. Here, we show that in contrast to naïve CD8+ T cells, memory CD8+ T cells traffic into the skin independent of CD62L-mediated lymph node re-activation and provide robust protective immunity against Vaccinia virus (VacV) infection. TCM, but not effector memory (TEM), CD8+ T cells differentiated into functional CD69+/CD103- tissue residents following viral clearance, which was also dependent on local recognition of antigen in the skin microenvironment. Finally, we found that memory CD8+ T cells expressed granzyme B after trafficking into the skin and utilized cytolysis to provide protective immunity against VacV infection. Collectively, these findings demonstrate that TCM CD8+ T cells become cytolytic following rapid infiltration of the skin to protect against viral infection and subsequently differentiate into functional CD69+ tissue-residents.

Transplanted interleukin-4--secreting mesenchymal stromal cells show extended survival and increased bone mineral density in the murine femur.

BACKGROUND: Mesenchymal stromal cell (MSC)-based therapy has great potential to modulate chronic inflammation and enhance tissue regeneration. Crosstalk between MSC-lineage cells and polarized macrophages is critical for bone formation and remodeling in inflammatory bone diseases. However, the translational application of this interaction is limited by the short-term viability of MSCs after cell transplantation. METHODS: Three types of genetically modified (GM) MSCs were created: (1) luciferase-expressing reporter MSCs; (2) MSCs that secrete interleukin (IL)-4 either constitutively; and (3) MSCs that secrete IL-4 as a response to nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) activation. Cells were injected into the murine distal femoral bone marrow cavity. MSC viability and bone formation were examined in vivo. Cytokine secretion was determined in a femoral explant organ culture model. RESULTS: The reporter MSCs survived up to 4 weeks post-implantation. No difference in the number of viable cells was found between high (2.5 × 106) and low (0.5 × 106) cell-injected groups. Injection of 2.5 × 106 reporter MSCs increased local bone mineral density at 4 weeks post-implantation. Injection of 0.5 × 106 constitutive IL-4 or NFκB-sensing IL-4-secreting MSCs increased bone mineral density at 2 weeks post-implantation. In the femoral explant organ culture model, LPS treatment induced IL-4 secretion in the NFκB-sensing IL-4-secreting MSC group and IL-10 secretion in all the femur samples. No significant differences in tumor necrosis factor (TNF)α and IL-1ß secretion were observed between the MSC-transplanted and control groups in the explant culture. DISCUSSION: Transplanted GM MSCs demonstrated prolonged cell viability when transplanted to a compatible niche within the bone marrow cavity. GM IL-4-secreting MSCs may have great potential to enhance bone regeneration in disorders associated with chronic inflammation.

B cell-intrinsic CD84 and Ly108 maintain germinal center B cell tolerance.

Signaling lymphocyte activation molecules (SLAMs) play an integral role in immune regulation. Polymorphisms in the SLAM family receptors are implicated in human and mouse model of lupus disease. The lupus-associated, somatically mutated, and class-switched pathogenic autoantibodies are generated in spontaneously developed germinal centers (GCs) in secondary lymphoid organs. The role and mechanism of B cell-intrinsic expression of polymorphic SLAM receptors that affect B cell tolerance at the GC checkpoint are not clear. In this study, we generated several bacterial artificial chromosome-transgenic mice that overexpress C57BL/6 (B6) alleles of different SLAM family genes on an autoimmune-prone B6.Sle1b background. B6.Sle1b mice overexpressing B6-derived Ly108 and CD84 exhibit a significant reduction in the spontaneously developed GC response and autoantibody production compared with B6.Sle1b mice. These data suggest a prominent role for Sle1b-derived Ly108 and CD84 in altering the GC checkpoint. We further confirm that expression of lupus-associated CD84 and Ly108 specifically on GC B cells in B6.Sle1b mice is sufficient to break B cell tolerance, leading to an increase in autoantibody production. In addition, we observe that B6.Sle1b B cells have reduced BCR signaling and a lower frequency of B cell-T cell conjugates; the reverse is seen in B6.Sle1b mice overexpressing B6 alleles of CD84 and Ly108. Finally, we find a significant decrease in apoptotic GC B cells in B6.Sle1b mice compared with B6 controls. Our study establishes a central role for GC B cell-specific CD84 and Ly108 expression in maintaining B cell tolerance in GCs and in preventing autoimmunity.

Genetic testing for Lynch syndrome in the province of Ontario.

BACKGROUND: In November 2001, genetic testing for Lynch syndrome (LS) was introduced by the Ministry of Health and Long-Term Care (MOH) in Ontario for individuals at high risk for LS cancers according to either tumor immunohistochemistry staining or their family history. This article describes the outcomes of the program and makes recommendations for improving it and informing other public health care programs. METHODS: Subjects were referred for molecular testing of the mismatch repair (MMR) genes MutL homolog 1, MutS homolog 2, and MutS homolog 6 if they met 1 of 7 MOH criteria. Testing was conducted from January 2001 to March 2015 at the Molecular Diagnostic Laboratory of Mount Sinai Hospital in Toronto. RESULTS: A total of 1452 subjects were tested. Of the 662 subjects referred for testing because their tumor was immunodeficient for 1 or more of the MMR genes, 251 (37.9%) carried a germline mutation. In addition, 597 subjects were tested for a known family mutation, and 298 (49.9%) were positive; 189 of these 298 subjects (63.4%) were affected with cancer at the time of testing. An additional 193 subjects were referred because of a family history of LS, and 34 of these (17.6%) had a mutation identified. CONCLUSIONS: These results indicate that the provincial criteria are useful in identifying LS carriers after an MMR-deficient tumor is identified. Placing greater emphasis on testing unaffected relatives in families with a known mutation may identify more unaffected carriers and facilitate primary prevention in those individuals. Cancer 2016;122:1672-9. © 2016 American Cancer Society.

Acquired resistance to crizotinib from a mutation in CD74-ROS1.

Crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK), has also recently shown efficacy in the treatment of lung cancers with ROS1 translocations. Resistance to crizotinib developed in a patient with metastatic lung adenocarcinoma harboring a CD74-ROS1 rearrangement who had initially shown a dramatic response to treatment. We performed a biopsy of a resistant tumor and identified an acquired mutation leading to a glycine-to-arginine substitution at codon 2032 in the ROS1 kinase domain. Although this mutation does not lie at the gatekeeper residue, it confers resistance to ROS1 kinase inhibition through steric interference with drug binding. The same resistance mutation was observed at all the metastatic sites that were examined at autopsy, suggesting that this mutation was an early event in the clonal evolution of resistance. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

B cell-intrinsic TLR7 signaling is essential for the development of spontaneous germinal centers.

Spontaneous germinal center (Spt-GC) B cells and follicular helper T cells generate high-affinity autoantibodies that are involved in the development of systemic lupus erythematosus. TLRs play a pivotal role in systemic lupus erythematosus pathogenesis. Although previous studies focused on the B cell-intrinsic role of TLR-MyD88 signaling on immune activation, autoantibody repertoire, and systemic inflammation, the mechanisms by which TLRs control the formation of Spt-GCs remain unclear. Using nonautoimmune C57BL/6 (B6) mice deficient in MyD88, TLR2, TLR3, TLR4, TLR7, or TLR9, we identified B cell-intrinsic TLR7 signaling as a prerequisite to Spt-GC formation without the confounding effects of autoimmune susceptibility genes and the overexpression of TLRs. TLR7 deficiency also rendered autoimmune B6.Sle1b mice unable to form Spt-GCs, leading to markedly decreased autoantibodies. Conversely, B6.yaa and B6.Sle1b.yaa mice expressing an extra copy of TLR7 and B6.Sle1b mice treated with a TLR7 agonist had increased Spt-GCs and follicular helper T cells. Further, TLR7/MyD88 deficiency led to compromised B cell proliferation and survival after B cell stimulation both in vitro and in vivo. In contrast, TLR9 inhibited Spt-GC development. Our findings demonstrate an absolute requirement for TLR7 and a negative regulatory function for TLR9 in Spt-GC formation under nonautoimmune and autoimmune conditions. Our data suggest that, under nonautoimmune conditions, Spt-GCs initiated by TLR7 produce protective Abs. However, in the presence of autoimmune susceptibility genes, TLR7-dependent Spt-GCs produce pathogenic autoantibodies. Thus, a single copy of TLR7 in B cells is the minimal requirement for breaking the GC-tolerance checkpoint.

Distinct and synergistic roles of FcγRIIB deficiency and 129 strain-derived SLAM family proteins in the development of spontaneous germinal centers and autoimmunity.

The inhibitory IgG Fc receptor (FcγRIIB) deficiency and 129 strain-derived signaling lymphocyte activation molecules (129-SLAMs) are proposed to contribute to the lupus phenotype in FcγRIIB-deficient mice generated using 129 ES cells and backcrossed to C57BL/6 mice (B6.129.RIIBKO). In this study, we examine the individual contributions and the cellular mechanisms by which FcγRIIB deficiency and 129-derived SLAM family genes promote dysregulated spontaneous germinal center (Spt-GC) B cell and follicular helper T cell (Tfh) responses in B6.129.RIIBKO mice. We find that B6 mice congenic for the 129-derived SLAM locus (B6.129-SLAM) and B6 mice deficient in FcγRIIB (B6.RIIBKO) have increased Spt-GC B cell responses compared to B6 controls but significantly lower than B6.129.RIIBKO mice. These data indicate that both FcγRIIB deficiency and 129-SLAMs contribute to elevated Spt-GC B cell responses in B6.129.RIIBKO mice. However, only 129-SLAMs contribute significantly to augmented Tfh responses in B6.129.RIIBKO mice, and do so by a combination of T cell-dependent effects and enhanced B cell and DC-dependent antigen presentation to T cells. Elevated Spt-GC B cell responses in mice with FcγRIIB deficiency and polymorphic 129-SLAMs were associated with elevated metabolic activity, improved GC B cell survival and increased differentiation of naïve B cells into GC B cell phenotype. Our data suggest that the interplay between 129-SLAM expression on B cells, T cells and DCs is central to the alteration of the GC tolerance checkpoint, and that deficiency of FcγRIIB on B cells is necessary to augment Spt-GC responses, pathogenic autoantibodies, and lupus disease.