Endothelial cell Notch signaling programs cancer-associated fibroblasts to promote tumor immune evasion.

Stromal cells within the tumor tissue promote immune evasion as a critical strategy for cancer development and progression, but the underlying mechanisms remain poorly understood. In this study, we explore the role of endothelial cells (ECs) in the regulation of the immunosuppressive tumor microenvironment. Using mouse pancreatic ductal adenocarcinoma (PDAC) models, we found that canonical Notch signaling in endothelial cells suppresses the recruitment of antitumor T cells and promotes tumor progression by inhibiting the pro-inflammatory functions of cancer-associated fibroblasts (CAFs). Abrogation of endothelial Notch signaling modulates EC-derived angiocrine factors to enhance the pro-inflammatory activities of CAFs, which drive CXCL9/10-CXCR3-mediated T cell recruitment to inhibit tumor growth. Additionally, abrogation of endothelial Notch unleashed interferon gamma responses in the tumor microenvironment, upregulated PDL1 expression on tumor cells, and sensitized PDAC to PD1-based immunotherapy. Collectively, these data uncover a pivotal role of endothelial cells in shaping the immunosuppressive microenvironment, and suggest the potential of targeting EC-CAF interaction as a novel therapeutic modality to boost antitumor immunity.

Exploration of Cell Development Pathways through High-Dimensional Single Cell Analysis in Trajectory Space.

High-dimensional single cell profiling coupled with computational modeling is emerging as a powerful tool to elucidate developmental programs directing cell lineages. We introduce tSpace, an algorithm based on the concept of "trajectory space", in which cells are defined by their distance along nearest neighbor pathways to every other cell in a population. Graphical mapping of cells in trajectory space allows unsupervised reconstruction and exploration of complex developmental sequences. Applied to flow and mass cytometry data, the method faithfully reconstructs thymic T cell development and reveals development and trafficking regulation of tonsillar B cells. Applied to the single cell transcriptome of mouse intestine and C. elegans, the method recapitulates development from intestinal stem cells to specialized epithelial phenotypes more faithfully than existing algorithms and orders C. elegans cells concordantly to the associated embryonic time. tSpace profiling of complex populations is well suited for hypothesis generation in developing cell systems.

Mass Cytometric Analysis of HIV Entry, Replication, and Remodeling in Tissue CD4+ T Cells.

To characterize susceptibility to HIV infection, we phenotyped infected tonsillar T cells by single-cell mass cytometry and created comprehensive maps to identify which subsets of CD4+ T cells support HIV fusion and productive infection. By comparing HIV-fused and HIV-infected cells through dimensionality reduction, clustering, and statistical approaches to account for viral perturbations, we identified a subset of memory CD4+ T cells that support HIV entry but not viral gene expression. These cells express high levels of CD127, the IL-7 receptor, and are believed to be long-lived lymphocytes. In HIV-infected patients, CD127-expressing cells preferentially localize to extrafollicular lymphoid regions with limited viral replication. Thus, CyTOF-based phenotyping, combined with analytical approaches to distinguish between selective infection and receptor modulation by viruses, can be used as a discovery tool.

Trafficking receptor signatures define blood plasmablasts responding to tissue-specific immune challenge.

Antibody-secreting cells are generated in regional lymphoid tissues and traffic as plasmablasts (PBs) via lymph and blood to target sites for local immunity. We used multiparameter flow cytometry to define PB trafficking programs (TPs, combinations of adhesion molecules and chemoattractant receptors) and their imprinting in patients in response to localized infection or immune insults. TPs enriched after infection or autoimmune inflammation of mucosae correlate with sites of immune response or symptoms, with different TPs imprinted during small intestinal, colon, throat, and upper respiratory immune challenge. PBs induced after intramuscular or intradermal influenza vaccination, including flu-specific antibody-secreting cells, display TPs characterized by the lack of mucosal homing receptors. PBs of healthy donors display diverse mucosa-associated TPs, consistent with homeostatic immune activity. Identification of TP signatures of PBs may facilitate noninvasive monitoring of organ-specific immune responses.

Influence of renal and hepatic impairment on the pharmacokinetics of anacetrapib.

Two open-label, parallel-group studies evaluated the influence of renal and hepatic insufficiency on the pharmacokinetics of a single-dose anacetrapib 100 mg. Eligible participants included adult men and women with moderate hepatic impairment (assessed by Child-Pugh criteria) or severe renal impairment (CrCl <30 mL/min/1.73 m(2)). In both studies, patients were matched (race, age, sex, BMI) with healthy control subjects. Twenty-four subjects were randomized in each study (12 with either moderate hepatic or severe renal impairment and 12 matched healthy controls). In the hepatic insufficiency study, the geometric mean ratio (GMR; mean value for the group with moderate hepatic insufficiency/mean value for the healthy controls) and 90% CIs for the area under the concentration-time curve from time zero to infinity (AUC(0-∞)) and the maximum concentration of drug in plasma (C(max)) were 1.16 (0.84, 1.60) and 1.02 (0.71, 1.49), respectively. In the renal insufficiency study, the GMRs (mean value for the group with severe renal insufficiency/mean value for the healthy controls) and 90% CIs for AUC(0-∞) and Cmax were 1.14 (0.80, 1.63) and 1.31 (0.93, 1.83), respectively. Anacetrapib was generally well tolerated and there was no clinically meaningful effect of moderate hepatic or severe renal insufficiency on the pharmacokinetics of anacetrapib.

Diary of hot flashes reported upon occurrence: results of a randomized double-blind study of raloxifene, placebo, and paroxetine.

OBJECTIVE: This trial examined diaries of hot flash events reported upon occurrence to assess the test/retest reliability of the diaries and their ability to measure treatment effects on hot flash frequency and severity. METHODS: Forty-two postmenopausal women (aged ≥40 y; 5-50 hot flashes/wk) were randomized (3:3:1) to placebo, raloxifene 60 mg, or paroxetine 20 mg daily for 12 weeks. Diaries of hot flash frequency and severity were evaluated at 1-week intervals (twice before study treatment and thrice during study treatment). RESULTS: Forty-one women were evaluated. Baseline characteristics were similar between groups (eg, mean, 29.8 hot flashes/wk). Concordance correlation coefficients between screening (week -2) and baseline (week -1) measures of hot flash frequency and severity were 0.73 and 0.71, respectively. After 12 weeks, the mean (95% CI) percent changes from baseline in weekly hot flash frequency were as follows: placebo, -37.4% (-60.9 to -14.0); raloxifene, -14.2% (-37.7 to 9.3); paroxetine, -49.8% (-88.6 to -11.0); the mean (95% CI) percent changes in hot flash severity were as follows: placebo, -39.9% (-69.1 to -10.8); raloxifene, -9.6% (-38.8 to 19.6); paroxetine, -36.6% (-84.7 to 11.5). There were no significant differences in hot flash diary results between treatment groups. CONCLUSIONS: Measures of hot flash frequency and severity show acceptable test/retest reliability between screening and baseline. Reductions in vasomotor symptoms by raloxifene are numerically less than those seen with placebo, but no statistically significant treatment differences have been documented in this small study. The large effect of placebo and the significant reduction in vasomotor symptoms by paroxetine are consistent with other studies. The diary seems to be suitable for use in hot flash clinical trials.

MK-0448, a specific Kv1.5 inhibitor: safety, pharmacokinetics, and pharmacodynamic electrophysiology in experimental animal models and humans.

BACKGROUND: We evaluated the viability of I(Kur) as a target for maintenance of sinus rhythm in patients with a history of atrial fibrillation through the testing of MK-0448, a novel I(Kur) inhibitor. METHODS AND RESULTS: In vitro MK-0448 studies demonstrated strong inhibition of I(Kur) with minimal off-target activity. In vivo MK-0448 studies in normal anesthetized dogs demonstrated significant prolongation of the atrial refractory period compared with vehicle controls without affecting the ventricular refractory period. In studies of a conscious dog heart failure model, sustained atrial fibrillation was terminated with bolus intravenous MK-0448 doses of 0.03 and 0.1 mg/kg. These data led to a 2-part first-in-human study: Part I evaluated safety and pharmacokinetics, and part II was an invasive electrophysiological study in healthy subjects. MK-0448 was well-tolerated with mild adverse experiences, most commonly irritation at the injection site. During the electrophysiological study, ascending doses of MK-0448 were administered, but no increases in atrial or ventricular refractoriness were detected, despite achieving plasma concentrations in excess of 2 µmol/L. Follow-up studies in normal anesthetized dogs designed to assess the influence of autonomic tone demonstrated that prolongation of atrial refractoriness with MK-0448 was markedly attenuated in the presence of vagal nerve simulation, suggesting that the effects of I(Kur) blockade on atrial repolarization may be negated by enhanced parasympathetic neural tone. CONCLUSIONS: The contribution of I(Kur) to human atrial electrophysiology is less prominent than in preclinical models and therefore is likely to be of limited therapeutic value for the prevention of atrial fibrillation.

Plasmablast frequency and trafficking receptor expression are altered in pediatric ulcerative colitis.

BACKGROUND: The incidence of pediatric ulcerative colitis (UC), a chronic autoinflammatory disease of the colon, is on the rise. Although an increased infiltration of B cells from the peripheral blood into the colon occurs in UC, B-cell trafficking is understudied. We hypothesized that the frequency of circulating plasmablasts (PBs) and their trafficking receptor (TR) expression may be indicative of the location and degree of pathology in pediatric UC. METHODS: We conducted multicolor flow cytometry analyses of circulating IgA(+/-) PBs and IgA(+) memory B cells (MBCs) in pediatric UC patients with remission, mild, moderate, and severe state of disease (n = 12), and healthy pediatric (n = 2) and adult donors (n = 11). RESULTS: Compared to healthy donors the average frequency of PBs among total peripheral blood lymphocytes is increased 30-fold during severe UC activity, and positively correlates with Pediatric Ulcerative Colitis Activity Index score, C-reactive protein level, and erythrocyte sedimentation rate. A greater percent of PBs in severe patients express the gut-homing receptors α4ß7 and CCR10, and the inflammatory homing molecule P-selectin ligand (P-sel lig). The percent of IgA(+) MBCs expressing α4ß7, however, is reduced. Furthermore, expression of the small intestine TR CCR9 is decreased on α4ß7(high) PBs, and on α4ß7(high) /CCR10(high) PBs and MBCs in these patients, consistent with preferential cell targeting to the colon. CONCLUSIONS: Peripheral blood PBs with a colon-homing phenotype (α4ß7/CCR10/P-sel lig) are elevated in children with severe UC. Screening this B-cell subset may provide a complementary approach in monitoring disease activity or therapeutic efficacy in pediatric UC.

Bioavailability of alendronate and vitamin D(3) in an alendronate/vitamin D(3) combination tablet.

These studies were designed to demonstrate that the alendronate (ALN) component of an ALN/vitamin D(3) combination tablet was bioequivalent to the 70-mg ALN tablet and that the pharmacokinetic parameters of vitamin D(3) were similar with or without ALN. These were open-label, randomized, 2-part, 2-period, crossover studies. In part I, participants received either a single combination tablet or ALN 70 mg. In part II, participants received either a single combination tablet or vitamin D(3) alone. Results from part I showed that the geometric mean ratio (GMR) for total urinary excretion of ALN for both studies fell within the prespecified bioequivalence bounds. Results from part II showed that the pharmacokinetic profiles of vitamin D(3) with or without ALN were also similar. The combination tablets are bioequivalent to the ALN 70-mg tablet with respect to ALN bioavailability. The bioavailability of vitamin D(3) is similar in the combination tablets and when administered alone. No serious adverse experiences were reported.

Pharmacokinetics of laropiprant and glucuronide metabolite in patients with severe renal insufficiency.

Flushing symptoms limit the use of niacin as an effective treatment for dyslipidemia; laropiprant, a prostaglandin D2 receptor subtype 1 antagonist, reduces niacin-induced flushing and is being developed in combination with niacin. The aims of this study were to both determine the effect of renal insufficiency on plasma pharmacokinetics of laropiprant and to assess safety and tolerability in patients with severe renal insufficiency. This open-label study compared the pharmacokinetics of a single laropiprant 40-mg dose in 8 nondialyzed, severe renal insufficiency patients (RIs) with healthy matched subjects (HSs) (24-hour creatinine clearance <30 mL/min/1.73 m(2) and >80 mL/min/1.73 m(2) for RIs and HSs, respectively). In RIs, laropiprant was well tolerated and the area under the concentration time curve (AUC(0-infinity)) was modestly higher (ratio of geometric least-squares means [GMR] for RIs to HSs was 1.58; 90% confidence interval [CI], 1.06-2.35); neither the maximum laropiprant plasma concentration (C(max)) nor the time to C(max) (T(max)) was significantly affected. The apparent terminal half-life (t(1/2)) was 26.0 and 14.8 hours for RIs and HSs, respectively (P = 0.007). Similarly, for the inactive laropiprant glucuronide metabolite, the GMR for AUC(0-infinity) was 2.17 (90% CI, 1.44-3.27), and the apparent t(1/2) values were 25.3 to 14.5 hours (P = 0.037) in RIs and HSs, respectively. Renal insufficiency had no clinically significant effect on laropiprant pharmacokinetics. Because niacin and its metabolites are excreted through the kidneys, the combination of niacin with laropiprant should be used with caution in patients with renal impairment.

An indispensable role for the chemokine receptor CCR10 in IgA antibody-secreting cell accumulation.

The differential expression of chemokines and chemokine receptors, by tissues and leukocytes, respectively, contributes to the specific accumulation of leukocyte subsets to different tissues. CCR10/CCL28 interactions are thought to contribute to the accumulation of IgA Ab-secreting cells (ASC) to mucosal surfaces, such as the gastrointestinal tract and the lactating mammary gland. Although the role of CCL28 in lymphocyte homing is well established, direct in vivo evidence for CCR10 involvement in this process has not been previously shown. In this study, we describe the generation of a CCR10-deficient mouse model. Using this model, we demonstrate that CCR10 is critical for efficient localization and accumulation of IgA ASC to the lactating mammary gland. Surprisingly, IgA ASC accumulation to the gastrointestinal tract is minimally impacted in CCR10-deficient mice. These results provide the first direct evidence of CCR10 involvement in lymphocyte homing and accumulation in vivo, and demonstrate that reliance on CCR10-mediated recruitment of IgA ASC varies dramatically within mucosal tissues.

Case study of the morphologic variation of circulating tumor cells.

We report a detailed cytomorphologic evaluation of the circulating component of widely metastatic breast carcinoma. A previously healthy 38-year-old woman was diagnosed with breast cancer. Wide local excision revealed a 1.7-cm infiltrating ductal adenocarcinoma, BSR score 7/9 with angiolymphatic invasion, and 4/20 lymph nodes positive for carcinoma. Five years later, a bone marrow biopsy revealed involvement of bone marrow by metastatic breast carcinoma, and shortly thereafter, metastases were identified in the liver and lung hilum. She enrolled in a clinical investigation for the detection of circulating tumor cells (CTCs) in breast carcinoma. A total of 659 CTCs were identified in a 10-mL blood sample using an immunofluorescent protocol targeting cytokeratins and detected using fiber-optic array scanning technology. The detected CTCs were subsequently stained with a Wright-Giemsa stain, and representative cells were evaluated in detail by light microscopy for morphologic evaluation. We find that the patient's CTCs exhibit a high degree of pleomorphism including CTCs with high and low nuclear-to-cytoplasmic ratios along with CTCs exhibiting early and late apoptotic changes. In addition, in comparison with her tumor cells in other sites, the full morphologic spectrum of cancer cells present in primary and metastatic tumor is also present in peripheral blood circulation.

Redundant role of chemokines CCL25/TECK and CCL28/MEC in IgA+ plasmablast recruitment to the intestinal lamina propria after rotavirus infection.

Rotaviruses (RV) are the most important cause of severe childhood diarrheal disease. In suckling mice, infection with RV results in an increase in total and virus-specific IgA(+) plasmablasts in the small intestinal lamina propria (LP) soon after infection, providing a unique opportunity to study the mechanism of IgA(+) cell recruitment into the small intestine. In this study, we show that the increase in total and RV-specific IgA(+) plasmablasts in the LP after RV infection can be blocked by the combined administration of Abs against chemokines CCL25 and CCL28, but not by the administration of either Ab alone. RV infection in CCR9 knockout mice still induced a significant accumulation of IgA(+) plasmablasts in the LP, which was blocked by the addition of anti-CCL28 Ab, confirming the synergistic role of CCL25 and CCL28. The absence of IgA(+) plasmablast accumulation in LP following combined anti-chemokine treatment was not due to changes in proliferation or apoptosis in these cells. We also found that coadministration of anti-CCL25 and anti-CCL28 Abs with the addition of anti-alpha(4) Ab did not further inhibit IgA(+) cell accumulation in the LP and that the CCL25 receptor, CCR9, was coexpressed with the intestinal homing receptor alpha(4)beta(7) on IgA(+) plasmablasts. Finally, we showed that RV infection was associated with an increase in both CCL25 and CCL28 in the small intestine. Hence, our findings indicate that alpha(4)beta(7) along with either CCR9 or CCR10 are sufficient for mediating the intestinal migration of IgA(+) plasmablasts during RV infection.

High speed detection of circulating tumor cells.

Epithelial tumor cells circulate in peripheral blood at ultra-low concentrations in cancer patients. We have developed an instrument capable of rapid and accurate detection of rare cells in circulation utilizing fiber-optic array scanning technology (FAST). The FAST cytometer can locate immunofluorescently labeled rare cells on glass substrates at scan rates 500 times faster than conventional automated digital microscopy. These high scan rates are achieved by collecting fluorescent emissions using a fiber bundle with a large (50 mm) field of view. Very high scan rates make possible the ability to detect rare events without the requirement for an enrichment step. The FAST cytometer was used to detect, image and re-image circulating tumor cells in peripheral blood of breast cancer patients. This technology has the potential to serve as a clinically useful point-of-care diagnostic and a prognostic tool for cancer clinicians. The use of a fixed substrate permits the re-identification and re-staining of cells allowing for additional morphologic and biologic information to be obtained from previously collected and identified cells.

Maturation and trafficking markers on rotavirus-specific B cells during acute infection and convalescence in children.

We have previously studied B cells, from people and mice, that express rotavirus-specific surface immunoglobulin (RV-sIg) by flow cytometry with recombinant virus-like particles that contain green fluorescent protein. In the present study we characterized circulating B cells with RV-sIg in children with acute and convalescent infection. During acute infection, circulating RV-sIgD(-) B cells are predominantly large, CD38(high), CD27(high), CD138(+/-), CCR6(-), alpha4beta7(+), CCR9(+), CCR10(+), cutaneous lymphocyte antigen-negative (CLA(-)), L-selectin(int/-), and sIgM(+), sIgG(-), sIgA(+/-) lymphocytes. This phenotype likely corresponds to gut-targeted plasma cells and plasmablasts. During convalescence the phenotype switches to small and large lymphocytes, CD38(int/-), CD27(int/-), CCR6(+), alpha4beta7(+/-), CCR9(+/-) and CCR10(-), most likely representing RV-specific memory B cells with both gut and systemic trafficking profiles. Of note, during acute RV infection both total and RV-specific murine IgM and IgA antibody-secreting cells migrate efficiently to CCL28 (the CCR10 ligand) and to a lesser extent to CCL25 (the CCR9 ligand). Our results show that CCR10 and CCR9 can be expressed on IgM as well as IgA antibody-secreting cells in response to acute intestinal infection, likely helping target these cells to the gut. However, these intestinal infection-induced plasmablasts lack the CLA homing receptor for skin, consistent with mechanisms of differential CCR10 participation in skin T versus intestinal plasma cell homing. Interestingly, RV memory cells generally lack CCR9 and CCR10 and instead express CCR6, which may enable recruitment to diverse epithelial sites of inflammation.

CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells.

The dissemination of IgA-dependent immunity between mucosal sites has important implications for mucosal immunoprotection and vaccine development. Epithelial cells in diverse gastrointestinal and nonintestinal mucosal tissues express the chemokine MEC/CCL28. Here we demonstrate that CCR10, a receptor for MEC, is selectively expressed by IgA Ab-secreting cells (large s/cIgA(+)CD38(hi)CD19(int/-)CD20(-)), including circulating IgA(+) plasmablasts and almost all IgA(+) plasma cells in the salivary gland, small intestine, large intestine, appendix, and tonsils. Few T cells in any mucosal tissue examined express CCR10. Moreover, tonsil IgA plasmablasts migrate to MEC, consistent with the selectivity of CCR10 expression. In contrast, CCR9, whose ligand TECK/CCL25 is predominantly restricted to the small intestine and thymus, is expressed by a fraction of IgA Ab-secreting cells and almost all T cells in the small intestine, but by only a small percentage of plasma cells and plasmablasts in other sites. These results point to a unifying role for CCR10 and its mucosal epithelial ligand MEC in the migration of circulating IgA plasmablasts and, together with other tissue-specific homing mechanisms, provides a mechanistic basis for the specific dissemination of IgA Ab-secreting cells after local immunization.

A common mucosal chemokine (mucosae-associated epithelial chemokine/CCL28) selectively attracts IgA plasmablasts.

IgA immunoblasts can seed both intestinal and nonintestinal mucosal sites following localized mucosal immunization, an observation that has led to the concept of a common mucosal immune system. In this study, we demonstrate that the mucosae-associated epithelial chemokine, MEC (CCL28), which is expressed by epithelia in diverse mucosal tissues, is selectively chemotactic for IgA Ab-secreting cells (ASC): MEC attracts IgA- but not IgG- or IgM-producing ASC from both intestinal and nonintestinal lymphoid and effector tissues, including the intestines, lungs, and lymph nodes draining the bronchopulmonary tree and oral cavity. In contrast, the small intestinal chemokine, TECK (CCL25), attracts an overlapping subpopulation of IgA ASC concentrated in the small intestines and its draining lymphoid tissues. Surprisingly, T cells from mucosal sites fail to respond to MEC. These findings suggest a broad and unifying role for MEC in the physiology of the mucosal IgA immune system.

The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells.

Immunoglobulin A (IgA) provides protection against pathogens at mucosal surfaces. Chemotactic responses have been hypothesized to target IgA plasma cells involved in mucosal immune responses. We show here that thymus-expressed chemokine (TECK, CCL25) is a potent and selective chemoattractant for IgA antibody-secreting cells (ASC), efficiently recruiting IgA-producing cells from spleen, Peyer's patches, and mesenteric lymph node. Cells secreting IgA antibody in response to rotavirus, an intestinal pathogen, also respond well. In contrast, IgG- and IgM-ASC respond poorly. Epithelial cells in the small intestines, a principal site of IgA-ASC localization and IgA production in the body, highly and selectively express TECK. The migration of IgA-ASC to the intestinal epithelial cell chemokine TECK may help target IgA-producing cells to the gut wall, thus helping define and segregate the intestinal immune response.