Node Leukocytes Research Articles

Early Low-Grade Inflammation Induced by High-Salt Diet in Sprague Dawley Rats Involves Th17/Treg Axis Dysregulation, Vascular Wall Remodeling, and a Shift in the Fatty Acid Profile.

Unrestricted increased table salt (NaCl) intake is associated with oxidative stress and inflammation, leading to endothelial dysfunction and atherosclerosis. However, data on salt-induced immunomodulatory effects in the earliest phase of salt loading are scarce. In the present study, an animal model of short-term salt loading was employed, including male Sprague Dawley rats consuming a high-salt diet (HSD; 4% NaCl) or standard laboratory chow (low-salt; LSD; 0.4% NaCl) during a 7-day period. The contribution of angiotensin II (ANGII) suppression was tested by adding a group of rats on a high-salt diet receiving ANGII infusions. Samples of peripheral blood/mesenteric lymph node leukocytes, brain blood vessels, and serum samples were processed for flow cytometry, quantitative real-time PCR, total proteome analysis, and multiplex immunoassay. Data analysis revealed the up-regulation of Il 6 gene in the microcirculation of high-salt-fed rats, accompanied by an increased serum level of TNF-alpha cytokine. The high-salt diet resulted in increased proportion of serum mono-unsaturated fatty acids and saturated fatty acids, reduced levels of linoleic (C18:2 ω-6) and α-linolenic (C18:3 ω-3) acid, and increased levels of palmitoleic acid (C16:1 ω-7). The high-salt diet had distinct, lymphoid compartment-specific effects on leukocyte subpopulations, which could be attributed to the increased expression of salt-sensitive SGK-1 kinase. Complete proteome analysis revealed high-salt-diet-induced vascular tissue remodeling and perturbations in energy metabolism. Interestingly, many of the observed effects were reversed by ANGII supplementation. Low-grade systemic inflammation induced by a HSD could be related to suppressed ANGII levels. The effects of HSD involved changes in Th17 and Treg cell distribution, vascular wall remodeling, and a shift in lipid and arachidonic acid metabolism.

Tumor Vascular Remodeling Affects Molecular Dissemination to Lymph Node and Systemic Leukocytes.

Angiogenic and lymphangiogenic remodeling has long been accepted as a hallmark of cancer development and progression; however, the impacts of this remodeling on immunological responses, which are paramount to the responses to immunotherapeutic treatments, are underexplored. As immunotherapies represent one of the most promising new classes of cancer therapy, in this study, we explore the effects of angiogenic and lymphangiogenic normalization on dissemination of molecules injected into the tumor microenvironment to immune cells in lymph nodes draining the tumor as well as in systemically distributed tissues. A system of fluorescent tracers, size-matched to biomolecules of interest, was implemented to track different mechanisms of tumor transport and access to immune cells. This revealed that the presence of a tumor, and either angiogenic or lymphangiogenic remodeling, altered local retention of model biomolecules, trended toward normalizing dissemination to systemic organs, and modified access to lymph node-resident immune cells in manners dependent on mechanism of transport. More specifically, active cell migration by skin-derived antigen presenting cells was enhanced by both the presence of a tumor and lymphangiogenic normalization, while both angiogenic and lymphangiogenic normalization restored patterns of immune cell access to passively draining species. As a whole, this work uncovers the potential ramifications of tumor-induced angiogenesis and lymphangiogenesis, along with impacts of interrogation into these pathways, on access of tumor-derived species to immune cells. Impact Statement Angiogenic and lymphangiogenic normalization strategies have been utilized clinically to interrogate tumor vasculature with some success. In the age of immunotherapy, the impacts of these therapeutic interventions on immune remodeling are unclear. This work utilizes mouse models of angiogenic and lymphangiogenic normalization, along with a system of fluorescently tagged tracers, to uncover the impacts of angiogenesis and lymphangiogenesis on access of tumor-derived species to immune cell subsets within various organs.

Abstract P3-08-67: Prognostic value of the neutrophil-lymphocyte, platelet-lymphocyte and monocyte-lymphocyte ratio breast cancer patients according to HER2 overexpression

Abstract Introduction: HER2 gene amplificationis associated with shorter disease-free and overall survival in breast cancer.The prognostic value of the NLR, PLR, LMR or MLR has been also reported for many types of cancer. The aim of the present study was to assess the blood the neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR) and monocyte-lymphocyte ratio (MLR) as a prognostic factors in breast cancer patients (BC) according to HER2 overexpression. Material and Methods: A the retrospective analysis of 529 BC patients who were treated at COI (Gliwice, Poland) between January 2005 and June 2018 was performed. The prognostic value (OS) of the pre-treatment PLR, NLR and MLR was assessed by univariate and multivariate analysis. The cut-off values were determined using receiver operating characteristic curves. Based on the cut-off values determined, the NLR was considered as ‘elevated’ at >1.68, the MLR value was ‘elevated’ at >0.23 and the PLR was considered ‘elevated’ at>119.0. Result: The 5-year OS rates in HER2 positive and HER2 negative BC were 86.5 and 90.4%, respectively.The 5-year OS in the NLR >1.68 subgroup with HER2 (83.0 vs. 93.0%; P=0.579) and without HER2 overexpression (88.7 vs. 92.9%; P=0.159) were lower compared with a patients with NLR <1.68. Similarly, 5-year OS was lower in patients with a PLR of >119.0 with HER2 (85.9vs. 87.8%; P=0.782) and without HER2 overexpression (88.1 vs. 94.0%; P=0.060) compared with that in patients with a PLR of ≤119.0. The 5-year OS was similar in patients with a MLR of >0.23 with HER2 overexpression compared with that in patients with a MLR of ≤119.0 (86.1 vs. 86.7%; P=0.745). In group with a MLR of >0.23 without HER2 overexpression OS was lower compared with that in patients with a MLR of ≤0.23 (89.5 vs. 91.7%; P=0.185). Univariate Cox regression analyses of OS showed that in subgroup with HER2 overexpression factors such as tumor size (T3-4 vs. T1-2, HR=2.47; P=0.008), the presence of lymph node metastases (N+ vs.N0, HR=3.15; P=0.006) and estrogen receptor status (ER(+) vs. ER(-), HR=0.50; P=0.039) were statistically significant. Factors such as NLR, PLR and MLR were not statistically significant.Multivariate analysis revealed that in group breast cancer patients with HER2 overexpression negative prognostic factors were: tumor size and lymph node metastases. In contrary, positive prognostic factor was positive steroid receptor status (ER+).Univariate Cox regression analyses of OS showed that in subgroup without HER2 overexpression factors such as tumor size (T3-4 vs. T1-2, HR=3.89; P=0.0001), the presence of lymph node metastases (N+ vs.N0, HR=3.16; P=0.001), tumor grade (G3 vs. G1-2, HR=2.59; P=0.005), estrogen receptor status (ER(+) vs. ER(-), HR=0.43; P=0.012), progesterone receptor status (PR(+) vs. PR(-), HR=0.31; P=0.001), leukocytes >6.22 (HR=2.11; P=0.041), monocytes >0.52 (HR=2.24; P=0.018) and PLT>291 (HR=2.24; P=0.024) were statistically significant. Factors such as NLR (HR=1.66; P=0.166), PLR (HR=2.03; P=0.067) and MLR (HR=1.61; P=0.192) were not statistically significant. Multivariate analysis revealed that significant negative prognostic factors in without HER2 overexpression breast cancer patients were: tumor size, lymph node metastases and leukocyte number. In contrary, positive prognostic factor were positive steroid receptor status (PR+) and lymphocytes number. Conclusion: Elevated PLR, NLR and MLR worsens insignificantly prognosis in group of patients with tumors without HER2 overexpression. The presence of elevated MLR, PLR, NLR did not influence overall survival (prognosis) in group with HER2 overexpression. In both subgroups (HER2 positive and HER2 negative) common negative prognostic factors were tumor size and lymph node metastases. Citation Format: Joanna Huszno, Zofia Kolosza, Jolanta Mrochem Kwarciak, Aleksander Zajusz. Prognostic value of the neutrophil-lymphocyte, platelet-lymphocyte and monocyte-lymphocyte ratio breast cancer patients according to HER2 overexpression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-67.

Prognostic Significance of Peripheral T‐Cell Subsets in Laryngeal Squamous Cell Carcinoma

ObjectivesThe role of the immune system in head and neck squamous cell carcinoma is controversial. The aim of our study was to analyze full blood counts and distribution of T cell subsets in patients affected by laryngeal squamous cell cancer (LSCC) and their association with clinical variables and survival.Study designRetrospective study.MethodsWe analyzed the levels of platelets, lymphocytes, and neutrophils, as well as the CD4+, CD8+, and CD3+ T‐cell subpopulations by cytofluorometry in LSCC patients. A cohort of healthy patients was used as control group. The disease‐specific survival (DSS) was considered as survival outcome.ResultsSixty‐five LSCC patients and 48 controls were enrolled. In LSCC patients, neutrophils were higher than in the healthy group (P < .0001). The neutrophil‐to‐lymphocyte ratio (NLR) and the platelet‐to‐lymphocyte ratio (PLR) were both higher in LSCC patients (P < .0001). In patients treated for recurrent disease, the CD8+/CD3+ ratio was increased (P = .02), while the CD4+/CD8+ (P = .03) and CD4+/CD3+ (P = .04) ratios were lower. In patients with lymph node metastases, leukocytes (P = .03), CD3+ (P = .04), and CD4+ (P = .0098) were all higher. Among Stages III‐IV patients, low lymphocyte and low leukocyte count were associated with worse DSS.ConclusionOur data demonstrate that NLR and PLR are significantly increased in LSCC. Lower CD4+/CD8+ and CD3+/CD8+ ratios are related to recurrent disease and a higher level of CD3+ and CD4+ is associated with nodal metastasis.Level of Evidence4

RELT family members activate p38 and induce apoptosis by a mechanism distinct from TNFR1

Receptor Expressed in Lymphoid Tissues (RELT) is a human Tumor Necrosis Factor Receptor (TNFR) family member that has two identified homologous binding partners, RELL1 and RELL2. This study sought to further understand the pattern of RELT expression, the functional role of RELT family members, and the mechanism of RELT-induced apoptosis. RELT protein expression was detected in the spleen, lymph node, brain, breast and peripheral blood leukocytes (PBLs). A smaller than expected size of RELT was observed in PBLs, suggesting a proteolytically cleaved form of RELT. RELL1 and RELL2 overexpression activated the p38 MAPK pathway more substantially than RELT in HEK-293 cells, and this activation of p38 by RELT family members was blocked by dominant-negative mutant forms of OSR1 or TRAF2, implicating these molecules in RELT family member signaling. RELT was previously shown to induce apoptosis in human epithelial cells despite lacking the characteristic death domain (DD) found in other TNFRs. Seven deletion mutants of RELT that lacked differing portions of the intracellular domain were created to assess whether RELT possesses a novel DD. None of the deletion mutants induced apoptosis as efficiently as full-length RELT, a result that is consistent with a novel DD being located at the carboxyl-terminus. Interestingly, induction of apoptotic morphology by RELT overexpression was not prevented when signaling by FADD or Caspase-8 was blocked, indicating RELT induces apoptosis by a pathway distinct from other death-inducing TNFRs such as TNFR1. Collectively, this study provides more insights into RELT expression, RELT family member function, and the mechanism of RELT-induced death.

M1 polarization and the effect of PGE2 on TNF-α production by lymph node cells from dogs with visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is caused by the intracellular parasite Leishmania infantum. Increased levels of arginase, nitric oxide (NO2 ) and prostaglandin E2 (PGE2 ) can play a regulatory role regarding the immune response in CVL cases. This study aimed to evaluate the arginase activity in adherent macrophages cultured from the lymph nodes of healthy and naturally infected dogs and to examine the NO2 and PGE2 levels in the supernatant of these cultures. In addition, the regulatory effect of PGE2 on the production of tumour necrosis factor (TNF-α) and interleukin-10 (IL-10) in supernatants from the total lymph node was observed in leucocyte cultures. The arginase activity was lower in the adherent macrophages cultured from the lymph nodes of naturally infected dogs and there were higher concentrations of NO2 and PGE2 in the supernatants of these cultures. Higher TNF-α and IL-10 concentrations were observed in supernatants from total lymph node leucocytes cultures, from infected dogs, and the presence of indomethacin only decreased TNF-α in the supernatant of these cultures. We conclude that the low arginase activity in macrophages suggested that M1 polarization and PGE2 were participating in the immune response and were increasing TNF-α in CVL.

IT-01 * PD1/PDL1 INTERACTIONS MEDIATE SUPPRESSION OF ANTI-TUMOR IMMUNE ACTIVITY IN GLIOMA

Recent work has demonstrated that the interaction of the Programed Death-1 (PD1) negative costimulatory molecule with its ligand, PDL1 in the tumor microenvironment, plays an immunoregulatory role that may prevent the development of a successful anti-tumor response. We hypothesized that dendritic cell vaccination (DCVax) would induce a compensatory upregulation of PDL1 within the tumor microenvironment as a result of increased tumor infiltrating lymphocytes (TILs). We also hypothesized that simultaneous blockade of the PD1/ PDL1 interaction would promote a successful antitumor response. To test this, immunocompetent C57BL/6 mice were implanted with GL261 glioma cells in the brain. Mice were then stratified into 4 treatment groups (Tx on days 3 and 13 post-implant): (1) Control; (2) IP murine anti-PD1 mAb (10mg/kg) only; (3) subcutaneous GL261 lysate-pulsed DCVax only; and (4) combination (anti-PD1 mAb plus DCVax). Overall survival was quantified. On post-implant days 15 and 20, mice were imaged using a Bruker 7T MR scanner to obtain anatomical, perfusion, and pH-weighted data. On post-implant days 16 and 21, mice were imaged with [18F]-FAC PET to evaluate the lymphocyte response. Tumor-bearing hemisphere, spleen, and lymph node (LN) leukocytes were then harvested, processed, and stained for FACS analysis. Median survival of the combination group was significantly greater than the DCVax or PD-1 treatment groups (p < 0.005). MRI showed decreased tumor volume and inflammation in the combination group. FACS analysis and [18F]-PET demonstrated significantly enhanced numbers of activated lymphocytes accumulating within the cervical LN and tumor in PD-1 mAb, DCVax, and combination treatment groups. Elevated PDL1 expression on tumor-associated APCs was also noted in the DCVax group, but specifically lower in the combination group. We propose that PDL1 is upregulated within the tumor in response to immunotherapy, but can be blocked to restore effective anti-tumor immune responses.

Artificial neural networks predict survival from pancreatic cancer after radical surgery

BackgroundArtificial neural networks (ANNs) are nonlinear pattern recognition techniques that can be used as a tool in medical decision making. The objective of this study was to develop an ANN model for predicting survival in patients with pancreatic ductal adenocarcinoma (PDAC). MethodsA flexible nonlinear survival model based on ANNs was designed by using clinical and histopathological data from 84 patients who underwent resection for PDAC. ResultsSeven of 33 potential risk variables were selected to construct the ANN, including lymph node metastasis, differentiation, body mass index, age, resection margin status, peritumoral inflammation, and American Society of Anesthesiologists grade. Three variables (ie, lymph node metastasis, leukocyte count, and tumor location) were significant according to Cox regression analysis. Harrell's concordance index for the ANN model was .79, and for Cox regression it was .67. ConclusionsFor the first time, ANNs have been used to successfully predict individual long-term survival for patients after radical surgery for PDAC.

Protocols for vaginal inoculation and sample collection in the experimental mouse model of Candida vaginitis.

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors. An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida. This model has also been employed to test potential antifungal therapies in vivo. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice. Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.

Effects of Bacterial Endotoxin on α-Synuclein Expression in the Lymph Node Leukocytes of Rats

The effects of bacterial LPS on the expression of early apoptosis marker (annexin V) and immunoreactive α-synuclein in mononuclear leukocytes of the mesenteric lymph nodes of rats were studied in vivo and in vitro. Injection of LPS increased the number of lymphocytes with high expression of immunoreactive α-synuclein in cell nucleus and cytoplasm (by 68.6±8.1%) and stimulated apoptosis in this population (by 96.0±3.6% of the control). The expression of α-synuclein in macrophages increased only in the cytoplasm and this increase was not paralleled by stimulation of apoptosis in these cells. The data indicate that LPS-induced elevation of α-synuclein in lymphocytes is a component of the mechanism of programmed death in these cells. The increase of α-synuclein expression in macrophages can be related to processes associated with their stimulation.

An intestinal epithelial defect conferring ER stress results in inflammation involving both innate and adaptive immunity.

We recently characterized Winnie mice carrying a missense mutation in Muc2, leading to severe endoplasmic reticulum stress in intestinal goblet cells and spontaneous colitis. In this study, we characterized the immune responses due to this intestinal epithelial dysfunction. In Winnie, there was a fourfold increase in activated dendritic cells (DCs; CD11c+ major histocompatibility complex (MHC) class IIhi) in the colonic lamina propria accompanied by decreased colonic secretion of an inhibitor of DC activation, thymic stromal lymphopoietin (TSLP). Winnie also displayed a significant increase in mRNA expression of the mucosal TH17 signature genes Il17a, IL17f, Tgfb, and Ccr6, particularly in the distal colon. Winnie mesenteric lymph node leukocytes secreted multiple TH1, TH2, and TH17 cytokines on activation, with a large increase in interleukin-17A (IL-17A) progressively with age. A major source of mucosal IL-17A in Winnie was CD4+ T lymphocytes. Loss of T and B lymphocytes in Rag1-/- × Winnie (RaW) crosses did not prevent spontaneous inflammation but did prevent progression with age in the colon but not the cecum. Adoptive transfer of naive T cells into RaW mice caused more rapid and severe colitis than in Rag1-/-, indicating that the epithelial defect results in an intestinal microenvironment conducive to T-cell activation. Thus, the Winnie primary epithelial defect results in complex multicytokine-mediated colitis involving both innate and adaptive immune components with a prominent IL-23/TH17 response, similar to that of human ulcerative colitis.

W1674 Diagnostic Laparoscopy for Pancreatic Cancer in an MRI Driven Practice: What's it Worth?

tion) OR time, estimated blood loss and transfusions, tumor size and histology, lymph node status, postoperative hematocrit and leukocyte count and postoperative NSAID use. We defined DGE as failure to tolerate a regular diet by postoperative day seven. Data was analyzed with alpha=0.05 using Fisher exact test for categorical variables and Mann-Whitney U test for continuous variables. RESULTS: A total of 114 patients with DGE were compared with 134 patients without DGE. Patients who suffered DGE were significantly more likely to have smaller tumors (p=0.015) and fewer positive lymph nodes (p=0.025), while patients without DGE were more likely to have received preoperative chemotherapy (p= 0.005) or preoperative radiation (p= 0.010). A possible explanation for these results is that patients with malignant disease have less normal functioning pancreatic parenchyma and are consequently less at risk for digestive symptoms resulting from pancreatic injury and inflammation. In addition, DGE was associated with an infection during hospitalization (p=0.008.) Hematocrit on the third postoperative day was significantly lower (p=0.019) for patients who experienced DGE , excluding patients who experienced post-operative bleeding. We hypothesize that DGE patients experienced more postoperative third space sequestration and bowel edema, resulting in greater hemodilution when sequestered fluid redistributed back into the vasculature. CONCLUSIONS: Contrary to our expectations, patients with smaller tumors and benign disease were more likely to experience DGE than patients with more advanced, malignant disease. As our ability to diagnose premalignant lesions improves, there will likely be more PD performed for benign disease, with a subsequent increase in the frequency of patients with DGE.

Abstract 3274: Loss of interleukin 10 (IL-10) decreases mouse mammary gland development and lactogenic differentiation

Abstract Increased expression of IL-10 has been associated with a decreased breast cancer risk but the mechanism is not clear. The purpose of this study was to examine the effects of IL-10 loss on mouse mammary gland development. Female wild-type and IL-10 knockout mouse littermates born to and raised by IL-10 +/− mothers were maintained under specific pathogen-free (SPF) condition with NIH31 diet. They were sacrificed at days 21, 50-55, 80, and 150 of postnatal age, and at day 2 of lactation. IL-10 loss had no effect on body weight at any age. Whole mounts and paraffin sections of mammary glands from wild type and IL-10 knockout mice were compared for changes in development, differentiation, and the presence of any preneoplastic histologic changes. Interactions between genotype and estrus stage were also examined. Despite its role as an anti-inflammatory immune regulator, IL-10 loss did not result in the enlargement of the intramammary lymph node or frank leukocyte infiltration under SPF condition. No significant differences in mammary gland development were observed in prepubertal glands (day 21). In the pubertal gland at day 50-55 of age, loss of IL-10 was associated with significantly decreased epithelial area, decreased terminal end buds, and decreased adipose tissue. This decrease in peripheral structures and terminal end buds was maintained at day 80 and day 150 of age. Pregnancy and lactogenesis was unable to rescue this phenotype: at day 2 of lactation, the IL-10 knockout mouse maintained a decreased lobular complexity and glandular area, accompanied by abnormal retention of large adipocytes, compared to wild-type mice, consistent with published reports of decreased pup growth in pups born to and raised by IL-10 knock-out mother. Lactating IL-10 knockout mice had a significant increase in abundance of cysts, but no other signs of hyperplasia or neoplasia were present. In conclusion, IL-10 loss decreases epithelial branching in the resting gland, resulting in a compromised differentiation during lactogenesis with decreased glandular area. Although the loss of IL-10 alone was not sufficient to induce frank neoplastic changes in the resting mouse mammary gland, our observation suggests a epithelial differentiation-promoting role of IL-10 which is consistent with its association with a decreased breast cancer risk. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3274.

CXCL10 blockade protects mice from cyclophosphamide-induced cystitis

BackgroundAlterations in serum CXCR3 ligand levels were examined in interstitial cystitis (IC) patients; similar expression patterns in serum as well as CXCR3, CXCR3 ligands, and cytokines expressed by peripheral and local leukocyte subpopulations were characterized during cyclophosphamide (CYP)-induced acute cystitis in mice.ResultsSerum levels of monokine-induced by interferon-γ (IFN-γ) (MIG/CXCL9), IFN-γ-inducible protein-10 (IP-10/CXCL10), and IFN-γ-inducible T cell α chemoattractant (I-TAC/CXCL11) were elevated in patients with IC. These clinical features closely correlated with CYP-induced cystitis in mice. Serum levels of these CXCR3 ligands and local T helper type 1 (Th1) cytokines were also increased. We demonstrate that CXCR3 as well as CXCL9, CXCL10 and CXCL11 mRNA were significantly expressed by urinary bladder lymphocytes, while CXCR3 and CXCL9 transcripts were significantly expressed by iliac lymph node leukocytes following CYP treatment. We also show that the number of CD4+ T cells, mast cells, natural killer (NK) cells, and NKT cells were increased at systemic (spleen) and mucosal (urinary bladder and iliac lymph nodes) sites, following CYP-induced cystitis in mice. Importantly, CXCL10 blockade attenuated these increases caused by CYP.ConclusionAntibody (Ab)-mediated inhibition of the most abundant serum CXCR3 ligand, CXCL10, in mice decreased the local production of CXCR3 ligands as well as Th1 cytokines expressed by local leukocytes, and lowered corresponding serum levels to reduce the severity of CYP-induced cystitis. The present study is among the first to demonstrate some of the cellular and molecular mechanisms of chemokines in cystitis and may represent new drug target for this disease.

Molecular cloning and characterization of equine Toll-like receptor 9

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4 + and CD8 + T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.

High Expression of Human Leukocyte Formin Protein in T Non-Hodgkin's Lymphomas and in CD19− Cell Population of Normal Tonsils.

We recently described a new gene, denominated human leukocyte formin (hlf), which was overexpressed in lymphoid malignancies and cancer cell lines. In contrast, a low expression of the hlf protein was observed in lymph node and peripheral blood leukocytes in normal tissues. Interestingly, the hlf protein associates with Akt, a protein kinase of an important pathway for cell survival. In order to better characterize the expression of the hlf protein we performed Western blotting in the lymphocytes isolated from 4 tonsils from adult patients obtained during routine tonsillectomy, at the Department of Hematology, Clinic Hospital, Barcelona. Results demonstrated that the CD19− cell population of the tonsil displayed a higher expression of this protein when compared with CD19+ cells. In addition, CD19+ cells were separated into two subpopulations: CD27+ (memory cells) and CD27− (naïve cells), and the CD19+/CD27+ cell presented a higher expression when compared with naïve B cells. Furthermore, we performed Western blotting analysis in frozen biopsies of non-Hodgkin's lymphoma (NHL) patients obtained from the Department of Pathology, Purpan Hospital (Toulouse, France). The lymph node biopsies were performed at the time of clinical diagnosis and the initial diagnosis was confirmed by immunohistochemical analysis and classified according to REAL classification. Fifty-four patients were studied with ages ranging between 28 and 93 years (median 57 years). The histologic types were: 22 follicular NHL, 15 diffuse large B-cell NHL, 17 T cell NHL (non-otherwise specified). Five reactive lymph nodes were also studied. The expression of the hlf protein was detected in all lymphoma samples studied and also in the 5 reactive lymph nodes. The hlf expression, however, was higher in T cell NHL when compared with the others NHL and reactive lymph nodes (T cell NHL vs reactive lymph node, p=0.002; T cell NHL vs follicular NHL, p=0.0001; T cell NHL vs diffuse large B-cell, p=0.012; Mann Whitney test). The hlf protein may be involved in the anti-apoptosis mechanisms, as it is expressed in all types of lymphoproliferative samples and it is associated with Akt, a pathway that is constitutively activated in some hematologic malignancies. Indeed, the ortholog protein described in mice, presents a role in the protection of the cells from apoptosis, but the pathway is unknown. This report provides a more detailed description of the expression of hlf protein in normal lymphocytes and supports the hypothesis that the hlf protein has a role in the cancer molecular pathology of hematologic malignancies.

Expression of WNT7A in human normal tissues and cancer, and regulation of WNT7A and WNT7B in human cancer

WNT signals are transduced through seven-transmembrane-type WNT receptors encoded by Frizzled (FZD) genes to the beta-catenin - TCF pathway, the JNK pathway or the Ca2+-releasing pathway. WNT signaling molecules are potent targets for diagnosis of cancer (susceptibility, metastasis, and prognosis), for prevention and treatment of cancer, and for regenerative medicine or tissue engineering. We have so far cloned and characterized human WNT signaling molecules WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14, WNT14B/WNT15, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD10, FRAT1, FRAT2, NKD1, NKD2, VANGL1/STB2, ARHU/WRCH1, ARHV/WRCH2, GIPC2, GIPC3, betaTRCP2/FBXW1B, SOX17, and TCF-3 using bioinformatics, cDNA-library screening, and cDNA-PCR. Here, expression of WNT7A in human normal tissues and cancer, and regulation of WNT7A and WNT7B in human cancer were investigated. WNT7A was highly expressed in fetal lung, adult testis, lymph node, and peripheral blood leukocytes. WNT7A was relatively highly expressed in temporal lobe, occipital lobe, parietal lobe, paracentral gyrus of cerebral cortex, caudate nucleus, hippocampus, medulla oblongata and putamen within adult brain. WNT7A was highly expressed in SW480 (colorectal cancer), BxPC-3 and Hs766T (pancreatic cancer), and was also expressed in MKN7 and MKN45 (gastric cancer). WNT7B rather than WNT7A was expressed in MCF-7 (breast cancer) and NT2 (embryonal tumor). beta-estradiol did not affect expression levels of WNT7A and WNT7B in MCF-7 cells. WNT7B, but not WNT7A, was slightly up-regulated by all-trans retinoic acid in NT2 cells.

Molecular characterization and expression of a novel human leukocyte cell-surface marker homologous to mouse Ly-9

Ly-9 is a mouse cell-surface glycoprotein that is selectively expressed on thymocytes and on mature T and B lymphocytes. Ly-9 belongs to the CD2 subset of the immunoglobulin superfamily, an emerging family of cell signaling receptors. Recently, a partial human Ly-9 complementary DNA (cDNA) sequence has been described. Full-length cDNA clones were isolated that included the initiation codon, the sequence encoding the full signal peptide, and 14 amino acids more in the cytoplasmic domain than in the previously reported clone. The predicted extracellular domain of human Ly-9 contains 4 immunoglobulinlike domains, similar to those in mouse Ly-9. Northern blot analysis revealed that the human Ly-9 messenger RNA (2.6 kb) is expressed predominantly in lymph node, spleen, thymus, and peripheral blood leukocytes. Four monoclonal antibodies (mAbs) were raised against human Ly-9 by immunizing mice with the pre-B-cell line 300.19 stably transfected with human Ly-9 full-length cDNA. These mAbs strongly stained the surfaces of cells transfected with human Ly-9 cDNA but not of untransfected cells. Human Ly-9 expression was restricted to T and B lymphocytes and thymocytes, with the highest levels of expression on CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes. Monocytes, granulocytes, platelets, and red blood cells were uniformly negative for Ly-9. These mAbs immunoprecipitated major polypeptides of 120 kd from the transfected cells and 120 kd and 100 kd from B-cell line Daudi, probably because of the cell-surface-expressed isoforms. These data demonstrate that human Ly-9 is a new marker for the study of normal and malignant leukocytes. (Blood. 2001;97:3513-3520)

RELT, a new member of the tumor necrosis factor receptor superfamily, is selectively expressed in hematopoietic tissues and activates transcription factor NF-κB

AbstractThe members of tumor necrosis factor receptor (TNFR) superfamily have been designated as the “guardians of the immune system” due to their roles in immune cell proliferation, differentiation, activation, and death (apoptosis). This study reports the cloning of a new member of the TNFR superfamily, RELT (ReceptorExpressed in Lymphoid Tissues). RELT is a type I transmembrane glycoprotein with a cysteine-rich extracellular domain, possessing significant homology to other members of the TNFR superfamily, especially TNFRSF19, DR3, OX40, and LTβ receptor. The messenger RNA of RELT is especially abundant in hematologic tissues such as spleen, lymph node, and peripheral blood leukocytes as well as in leukemias and lymphomas. RELT is able to activate the NF-κB pathway and selectively binds tumor necrosis factor receptor-associated factor 1. Although the soluble form of RELT fusion protein does not inhibit the one-way mixed lymphocyte reaction, immobilized RELT is capable of costimulating T-cell proliferation in the presence of CD3 signaling. These results define a new member of the TNFR superfamily that may be a potential regulator of immune responses.

Cloning, Characterization, and Tissue Distribution of Mouse Phosphodiesterase 7A1

We have cloned a cDNA representing mouse phosphodiesterases (PDE) 7A1. The open reading frame encodes a protein of 482 amino acids with a predicted molecular mass of 55417. Like human PDE7A variants, mouse PDE7A1 and A2 are 5′ splice variants from a common gene. The distinct N-terminal sequence of mouse PDE7A1 is highly homologous to the corresponding sequence of human PDE7A1 with a similarity of 98% but not to that of mouse PDE7A2 (with a similarity of 12%), and is more hydrophilic than that of mouse PDE7A2. Mouse PDE7A1 expressed in SF9 cells has been compared with human PDE7A1 under identical conditions. Mouse PDE7A1 has a Km for cAMP of 0.2 μM, an optimal pH of 7.5, an IC50 value of 14 μM for 3-isobutyl-1-methylxanthine (IBMX), and is dependent on Mg2+ for activity. All these characteristics are very similar to those of human PDE7A1. In mice, PDE7A1 is expressed in tissues of the immune system (lymph node, thymus, spleen, and blood leukocyte), testis, brain, kidney and lung but not in skeletal muscle, heart, embryo, or liver, while PDE7A2 is expressed in skeletal muscle, heart, embryo, and kidney, but not in the other tissues. This tissue distribution profile is very similar to that in humans, and hence suggests that PDE7A1 and 7A2 might play a similar role in different species.