OBJECTIVE

To investigate association of circulating inflammatory factors at the time of colorectal cancer (CRC) surgery with survival.

METHODS

Plasma levels from 174 CRC patients (69 females and 105 men), with median age 70 years (range 29-90), localized in the colon (n = 105) or rectum (n = 69), with stage I (n = 24), stage II (n = 54), stage III (n = 67) and stage IV (n = 29) were measured using commercially available Bio-Plex Pro™ Human Chemokine Panel 40-Plex, including 40 different chemokines, cytokines and interleukins. The prognostic association of each inflammatory factor was analysed as CRC-specific and total mortality.

RESULTS

Out of 174 patients, 66 died during the follow-up, 40 because of CRC specific mortality. High tertile levels of 8 factors were significantly associated with increased CRC-specific mortality, of which CCL1, CCL20, CCL24, CX3CL1, IL-4 and TNF-α remained significant in a multivariate Cox regression analysis. High tertile levels of 14 factors were associated with increased total mortality, of which CCL1, CCL15, CCL20, CX3CL1, CXCL13, IFN-γ, IL-2, IL-4 and IL-10 remained significant after adjustment for clinical covariates. For most of the inflammatory factors the association between higher tertile levels and an increased mortality in general appeared two years after surgery. High tertile levels of TNF-α and CCL24 were exclusively associated with CRC-specific mortality. The distribution of these factors were not associated with TNM stage with exception for CCL20.

CONCLUSIONS

High plasma levels of inflammatory factors are associated with increased risk of mortality among CRC patients and could be potential biomarkers for revealing prognosis.

Understanding the mechanism(s) by which broadly neutralizing antibodies (bNAbs) emerge naturally following infection is crucial for the development of a protective vaccine against human immunodeficiency virus (HIV). Although previous studies have implicated high viremia and associated immune activation as potential drivers for the development of bNAbs, here we sought to unlink the effect of these 2 parameters by evaluating the key inflammatory predictors of bNAb development in HIV-infected individuals who spontaneously control HIV in the absence of antiretroviral therapy ("controllers").

The breadth of antibody-mediated neutralization against 11 tier 2 or 3 viruses was assessed in 163 clade B spontaneous controllers of HIV. Plasma levels of 17 cytokines were screened in the same set of subjects. The relationship of the inflammatory signature was assessed in the context of viral blips or viral RNA levels in peripheral blood or gastrointestinal biopsies from aviremic controllers (<50 copies RNA/mL) and in the context of viral sequence diversity analysis in the plasma of viremic controllers (<50-2000 copies RNA/mL).

A unique inflammatory profile, including high plasma levels of CXCL13, sCD40L, IP10, RANTES, and TNFα, was observed in HIV controllers who developed bNAbs. Interestingly, viral load and tissue viremia, but not intermittent viral blips, were associated with these cytokine profiles. However, viral diversity was not significantly associated with increased breadth in controllers.

These results suggest that low antigenic diversity in the setting of a unique inflammatory profile associated with antigen persistence may be linked to the evolution of neutralizing antibody breadth.

Circulating follicular helper T (cTfh) cells are a novel subset of cluster of differentiation (CD)4+ helper T cells. Interleukin (IL)-21 and C-X-C motif chemokine ligand (CXCL)13 are the principal effectors and chemotactic regulatory factors of Tfh. However, the roles of IL-21 and CXCL13 in gastric cancer have not yet been completely elucidated. The aim of the present study was to investigate the distribution of cTfh cells, and the expression of IL-21 and CXCL13 in patients with gastric cancer was evaluated in order to ascertain the significance and potential mechanisms of these effectors in gastric cancer. A total of 50 patients with gastric cancer were enrolled as the study subjects, with 30 healthy individuals selected as controls. The percentage of cTfh cells (cTfh%) in the peripheral blood was calculated using flow cytometry. They are identified in the present study as CD4+ chemokine C-X-C receptor (CXCR)5+ inducible T cell co-stimulator (ICOS)+ cells. The serum levels of IL-21 and CXCL13 were determined by ELISA. The cTfh% in the peripheral blood and the concentration of IL-21 and CXCL13 in the serum were significantly higher in patients with gastric cancer compared with the control group. cTfh% was significantly higher in patients with lymph node metastasis, Tumor-Node-Metastasis (TNM) stage III-IV and low differentiation. The concentrations of IL-21 and CXCL13 in patients with lymph node metastasis and/or TNM III-IV were significantly higher than in those without lymph node metastasis or with TNM I-II. There was a positive correlation between cTfh%/CXCL13 and IL-21/CXCL13, while there was no correlation between cTfh%/IL-21. cTfh cells and associated factors (IL-21/CXCL13) may be involved in the development and progression of gastric cancer. There may be mutual regulation among cTfh cells, IL-21 and CXCL13.

Prostate cancer stem-like cells (PCSLC) are believed to be responsible for prostate cancer onset and metastasis. Autocrine and microenvironmental signals dictate PCSLC behavior and patient outcome. In prostate cancer patients, IL30/IL27p28 has been linked with tumor progression, but the mechanisms underlying this link remain mostly elusive. Here, we asked whether IL30 may favor prostate cancer progression by conditioning PCSLCs and assessed the value of blocking IL30 to suppress tumor growth. IL30 was produced by PCSLCs in human and murine prostatic intraepithelial neoplasia and displayed significant autocrine and paracrine effects. PCSLC-derived IL30 supported PCSLC viability, self-renewal and tumorigenicity, expression of inflammatory mediators and growth factors, tumor immune evasion, and regulated chemokine and chemokine receptor genes, primarily via STAT1/STAT3 signaling. IL30 overproduction by PCSLCs promoted tumor onset and development associated with increased proliferation, vascularization, and myeloid cell recruitment. Furthermore, it promoted PCSLC dissemination to lymph nodes and bone marrow by upregulating the CXCR5/CXCL13 axis, and drove metastasis to lungs through the CXCR4/CXCL12 axis. These mechanisms were drastically hindered by IL30 knockdown or knockout in PCSLCs. Collectively, these results mark IL30 as a key driver of PCSLC behavior. Targeting IL30 signaling may be a potential therapeutic strategy against prostate cancer progression and recurrence.Significance: IL30 plays an important role in regulating prostate cancer stem-like cell behavior and metastatic potential, therefore targeting this cytokine could hamper prostate cancer progression or recurrence. Cancer Res; 78(10); 2654-68. ©2018 AACR.

B-lymphocytes produce protective antibodies but also contribute to autoimmunity. In particular, marginal zone (MZ) B cells recognize both microbial components and self-antigens. B cell trafficking is critical for B cell activation and is controlled by chemoattactants such as CXCL13 and sphingosine 1-phosphate (S1P). The related tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2) regulate cell migration and adhesion but their roles in B cells are not fully understood. Using a novel Pyk2-selective inhibitor described herein (PF-719), as well as a FAK-selective inhibitor, we show that both Pyk2 and FAK are important for CXCL13- and S1P-induced migration of B-2 cells and MZ B cells. In contrast, LFA-1-mediated adhesion required only Pyk2 whereas activation of the Akt pro-survival kinase required FAK but not Pyk2. Thus Pyk2 and FAK mediate critical processes in B cells and these inhibitors can be used to further elucidate their functions in B cells.

Background: Multiple sclerosis (MS) biomarker identification is important for pathogenesis research and diagnosis in routine clinical practice. Cerebrospinal fluid (CSF) and blood cytokines as potential biomarkers that can inform MS pathogenesis, diagnosis and response to treatment have been assessed in numerous studies. However, there have been no comprehensive meta-analyses to pool cytokine data and to address their diagnostic performance. We systematically reviewed literature with meta-analyses to assess the alteration levels of cytokines and chemokines in MS. Methods: We searched PubMed and Web of Science for articles published between January 1, 1990 and April 30, 2018 for this systematic review and meta-analysis. Data were extracted from 226 included studies encompassing 13,526 MS patients and 8,428 controls. Biomarker performance was rated by a random-effects meta-analysis based on the standard mean difference between cytokine concentration in patients with MS and controls, or patients before and after treatments. Results: Of the 26 CSF cytokines and 37 blood cytokines for potential differentiation between MS patients and controls, the random-effects meta-analysis showed that 13 CSF cytokines and 21 blood cytokines were significantly increased in MS patients in comparison to the controls. Interestingly, TNF-α, CXCL8, IL-15, IL-12p40, and CXCL13 were increased in both blood and CSF of MS patients. For those cytokines analyzed in at least 10 studies, differentiation between case and control was strong for CSF CXCL13, blood IL-2R, and blood IL-23; CSF CXCL8, blood IL-2, and blood IL-17 also performed well in differentiating between MS patients and controls, whereas those of CSF TNF-α and blood TNF-α, CXCL8, IL-12, IFN-γ were moderate. Furthermore, CSF IL-15, CCL19, CCL11, CCL-3, and blood CCL20, IL-12p40, IL-21, IL-17F, IL-22 had large effective sizes when differentiating between MS patients and controls but had a relatively small number of studies (three to seven studies). Conclusion: Our findings clarified the circulating cytokine profile in MS, which provide targets for disease modifying treatments, and suggest that cytokines have the potential to be used as biomarkers for MS.

Background: CXCL13 is preferentially secreted by Follicular Helper T cells (T_FH) to attract B cells to germinal centers. Plasma levels of CXCL13 have been reported to be elevated during chronic HIV-infection, however there is limited data on such elevation during early phases of infection and on the effect of ART. Moreover, the contribution of CXCL13 to disease progression and systemic immune activation have been partially defined. Herein, we assessed the relationship between plasma levels of CXCL13 and systemic immune activation. Methods: Study samples were collected in 114 people living with HIV (PLWH) who were in early (EHI) or chronic (CHI) HIV infection and 35 elite controllers (EC) compared to 17 uninfected controls (UC). A subgroup of 11 EHI who initiated ART and 14 who did not were followed prospectively. Plasma levels of CXCL13 were correlated with CD4 T cell count, CD4/CD8 ratio, plasma viral load (VL), markers of microbial translocation [LPS, sCD14, and (1→3)-β-D-Glucan], markers of B cell activation (total IgG, IgM, IgA, and IgG1-4), and inflammatory/activation markers like IL-6, IL-8, IL-1β, TNF-α, IDO-1 activity, and frequency of CD38⁺HLA-DR⁺ T cells on CD4⁺ and CD8⁺ T cells. Results: Plasma levels of CXCL13 were elevated in EHI (127.9 ± 64.9 pg/mL) and CHI (229.4 ± 28.5 pg/mL) compared to EC (71.3 ± 20.11 pg/mL), and UC (33.4 ± 14.9 pg/mL). Longitudinal analysis demonstrated that CXCL13 remains significantly elevated after 14 months without ART (p < 0.001) and was reduced without normalization after 24 months on ART (p = 0.002). Correlations were observed with VL, CD4 T cell count, CD4/CD8 ratio, LPS, sCD14, (1→3)-β-D-Glucan, total IgG, TNF-α, Kynurenine/Tryptophan ratio, and frequency of CD38+HLA-DR+ CD4 and CD8 T cells. In addition, CMV+ PLWH presented with higher levels of plasma CXCL13 than CMV- PLWH (p = 0.005). Conclusion: Plasma CXCL13 levels increased with HIV disease progression. Early initiation of ART reduces plasma CXCL13 and B cell activation without normalization. CXCL13 represents a novel marker of systemic immune activation during early and chronic HIV infection and may be used to predict the development of non-AIDS events.

The colorectal tumor microenvironment contains a diverse population of myeloid cells that are recruited and converted to immunosuppressive cells, thus facilitating tumor escape from immunoediting. We have identified a genetically and functionally distinct subset of dynamic bone marrow myeloid cells that are characterized by histidine decarboxylase (HDC) expression. Lineage tracing in Hdc-CreERT2;R26-LSL-tdTomato mice revealed that in homeostasis, there is a strong bias by HDC+ myeloid cells toward the CD11b+Ly6Ghi granulocytic lineage, which was accelerated during azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colonic carcinogenesis. More importantly, HDC+ myeloid cells strongly promoted colonic tumorigenesis, and colon tumor progression was profoundly suppressed by diphtheria toxin A (DTA)-mediated depletion of HDC+ granulocytic myeloid cells. In addition, tumor infiltration by Foxp3+ regulatory T cells (Tregs) was markedly impaired following HDC+ myeloid cell depletion. We identified an HDC+ myeloid-derived Cxcl13/Cxcr5 axis that mediated Foxp3 expression and Treg proliferation. Ablation of HDC+ myeloid cells or disruption of the Cxcl13/Cxcr5 axis by gene knockdown impaired the production and recruitment of Tregs. Cxcl13 induction of Foxp3 expression in Tregs during tumorigenesis was associated with Stat3 phosphorylation. Overall, HDC+ granulocytic myeloid cells affect CD8+ T cells directly and indirectly through the modulation of Tregs and thus appear to play key roles in suppressing tumoricidal immunity.

OBJECTIVE

To evaluate the role of the follicular helper T (T(FH)) cell markers, CD10, BCL6, programmed death-1 (PD-1) and CXCL13, in the differential diagnosis of nodal peripheral T cell lymphomas (PTCLs) and to determine whether PTCL subtypes other than angioimmunoblastic T cell lymphoma (AITL) express T(FH) cell markers.

METHODS

162 nodal PTCL specimens and 53 other lymphoid pathology specimens were collected. Immunohistochemistry for CD10, BCL6, PD-1 and CXCL13 was performed on tissue microarray sections. Morphological feature analysis and double labelling assay were also performed.

RESULTS

For AITL cases, the rate of CD10, BCL6, PD-1 and CXCL13 expression was 75.0% (36/48), 66.7% (32/48), 93.8% (45/48) and 97.9% (47/48), respectively. Expression of CD10, PD-1 and CXCL13 in the AITL group was significantly higher than in other nodal PTCLs and the control group (p<0.05). The rate of coexpression of three or four (≥3) markers was 83.3% for AITL cases, which was significantly higher than that for any of the non-AITL cases (0-4.9%; p<0.05). The rate of coexpression of PD-1 and CXCL13 (91.7%, 44/48) was significantly higher than that of CD10 and BCL6 (56.3%, 27/48) (p=0.000) in the AITL group. Seventeen cases of PTCL not otherwise specified (PTCL, NOS) expressed CXCL13, including both cases of the follicular variant of PTCL, NOS (FVPTCL, NOS), three of the four cases of the lymphoepithelioid variant of PTCL, NOS (LVPTCL, NOS), and the remaining 12 cases which displayed one or more features of AITL.

CONCLUSIONS

The combined detection of CD10, BCL6, PD-1 and CXCL13 has high specificity and sensitivity for the differential diagnosis of AITL. PD-1 and CXCL13 are more sensitive, superior diagnostic markers for AITL than CD10 and BCL6. Currently, T(FH) cell markers are the only available markers that show high specificity for AITL. LVPTCL, NOS and/or FVPTCL, NOS may also arise from T(FH) cells and fall within the spectrum of AITL.

C-X-C ligand 13 (CXCL13), a regulator of mucosal immunity, is secreted by human endometrial epithelium and may be involved in embryo implantation. However, cyclic expression of human endometrial CXCL13 in health and disease is not well studied. This study examines cycle stage-specific endometrial CXCL13 expression in normal humans when compared to those with biopsy-confirmed, stage 1 to 4 endometriosis using real-time reverse transcriptase, real-time polymerase chain reaction and immunohistochemistry. Eutopic endometrial CXCL13 expression was also compared between normal, control Rhesus macaques, and macaques with advanced endometriosis. In healthy women, CXLC13 messenger RNA expression was minimal in the proliferative phase and maximal in the secretory phase. However, in the presence of endometriosis, proliferative-phase endometrial expression markedly increased in both humans and rhesus subjects (P < .05). The cross-species and cross-stage concordance suggests a pathophysiologic role for CXCL13 in endometriosis and its use as a biomarker for disease.

Angioimmunoblastic T-cell lymphoma is known to frequently involve bone marrow. However, the histologic and immunophenotypic features of angioimmunoblastic T-cell lymphoma at this site are poorly defined. We assessed 27 bone marrow specimens involved by angioimmunoblastic T-cell lymphoma from 20 patients. Histologically, bone marrow involvement was predominantly multifocal (74%) and exhibited a nodular pattern (78%), often associated with other patterns. Using immunohistochemistry, programed death-1 and CD10 were expressed by atypical lymphocytes in 17 (85%) of 20 and 5 (18.5%) of 27 specimens, respectively. CXCL13 was not expressed by atypical lymphocytes in all cases but did stain stromal cells consistent with follicular dendritic cells in 1 case. BCL-6 as a single antibody was difficult to interpret because many normal bone marrow cells are dimly positive, but BCL-6/CD3 dual staining highlighted BCL-6+ T-cells in all cases assessed. Antibodies specific for CD21 and CD35 did not highlight follicular dendritic cells in any biopsy specimens. Flow cytometry immunophenotyping revealed a CD3+CD10+ T-cell population in 2 (25%) of 8 cases assessed. We conclude that the recognition and classification of angioimmunoblastic T-cell lymphoma in bone marrow are made difficult by the uncommon expression of CD10 (25%), rarity of follicular dendritic cells, and lack of CXCL13 expression at this site. This is most likely attributable to the very different microenvironment of the bone marrow relative to lymph nodes and, in particular, the absence of follicles in bone marrow. By contrast, programed death-1 immunohistochemical staining and double labeling using antibodies specific for BCL-6 and CD3 were helpful in appreciating the follicular T-helper cell immunophenotype of angioimmunoblastic T-cell lymphoma.

The identification of chemokines has profoundly changed the way we interpret the immune response, elucidating the mechanism by which inflammatory cells are recruited to the site of infection by local secretion of chemoattractants such as CXC chemokine ligand 8 (CXCL8)/interleukin-8, chemokine ligand 2 (CCL2)/monocyte chemoattractant protein 1. This novel view of the immune response has been remodeled further following observations that lymphoid tissue development derives from the coordinated secretion of homeostatic chemokines such as CCL19, CCL21, and CXCL13, which mediate recruitment and clustering of the cells involved in lymphoid organogenesis. The study of primary immunodeficiencies has demonstrated that the number of circulating leukocytes is dependent on migration amongst bone marrow, blood circulation, and inflamed tissues. Defects of leukocyte adhesion and chemotaxis as a result of mutations of beta2-integrins lead to abnormal leukocytosis and susceptibility to skin infections, as observed in leukocyte adhesion deficiency. Conversely, neutropenia in children with myelokathexis is a result of leukocyte retention in the bone marrow because of the mutations of CXC chemokine receptor 4, which affect the capacity of cells to recirculate between blood and bone marrow. Moreover, the identification of the genetic basis of primary immunodeficiencies has shown that many primary immunodeficiencies such as Wiskott-Aldrich syndrome and common variable immunodeficiencies are characterized by altered migration of leukocytes and/or disregulation of cellular response to chemokines. This paper will be focused on the interpretation of primary immunodeficiencies as defects in leukocyte circulation between blood and primary and secondary organs.

OBJECTIVE

To investigate chemokines and their receptors gene expression in the intra-abdominal adipose tissue of diabetic/obese mice.

METHODS

KKAy mice were fed either by a high-fat diet (HFD) or a low-fat diet (LFD) and obese characteristics were analyzed. Various adipose tissues were isolated from HFD-fed obese KKAy mice and from obese controls. We carried out RT-PCR, GeneChip microarray, and real-time PCR analyses on samples derived from the adipose tissues.

RESULTS

The HFD-feded obese KKAy mice had the physiological characteristics of obese animal and had increased levels of the transcripts of several chemokine and chemokine receptor genes, such as CCL5, CCL19, CCL25, CXCL10, CXCL13, CCR6, and CCR7, in their intra-abdominal adipose tissue. The strong expression of CCR6 and CCR7 was verified by microarray and quantitative real-time PCR analysis. The HFD increased CCR6 and CCR7 expression only in mesenteric (ME) adipose tissue, not in subcutaneous (SC) adipose tissue.

CONCLUSIONS

Since the enhanced expression of such molecules is likely to contribute to the inflammation in chronic inflammatory disease, our data suggest that the increased levels of CCR6 and CCR7 are involved in the inflammation response in the intra-abdominal adipose tissue of the obese/diabetic mice.

OBJECTIVE

Little is known about the pathogenic mechanisms of autoimmune pancreatitis (AIP), an increasingly recognized, immune-mediated form of chronic pancreatitis. Current treatment options are limited and disease relapse is frequent. We investigated factors that contribute to the development of AIP and new therapeutic strategies.

METHODS

We used quantitative polymerase chain reaction, immunohistochemical, and enzyme-linked immunosorbent analyses to measure the expression of cytokines and chemokines in tissue and serum samples from patients with and without AIP. We created a mouse model of human AIP by overexpressing lymphotoxin (LT)α and β specifically in acinar cells (Ela1-LTab mice).

RESULTS

Messenger RNA levels of LTα and β were increased in pancreatic tissues from patients with AIP, compared with controls, and expression of chemokines (CXCL13, CCL19, CCL21, CCL1, and B-cell-activating factor) was increased in pancreatic and serum samples from patients. Up-regulation of these factors was not affected by corticosteroid treatment. Acinar-specific overexpression of LTαβ (Ela1-LTαβ) in mice led to an autoimmune disorder with various features of AIP. Chronic inflammation developed only in the pancreas but was sufficient to cause systemic autoimmunity. Acinar-specific overexpression of LTαβ did not cause autoimmunity in mice without lymphocytes (Ela1-LTab/Rag1(-/-)); moreover, lack of proinflammatory monocytes (Ela1-LTab/Ccr2(-/-)) failed to prevent AIP but prevented early pancreatic tissue damage. Administration of corticosteroids reduced pancreatitis but did not affect production of autoantibodies, such as antipancreatic secretory trypsin inhibitor in Ela1-LTab mice. In contrast, inhibition of LTβR signaling reduced chemokine expression, renal immune-complex deposition, and features of AIP in Ela1-LTab mice.

CONCLUSIONS

Overexpression of LTαβ specifically in acinar cells of mice causes features of AIP. Reagents that neutralize LTβR ligands might be used to treat patients with AIP.

The aims of the study were to determine and compare the concentration of CXCL13 in cerebrospinal fluid (CSF) of patients with Lyme neuroborreliosis (LNB) and various other neurological disorders applying a Luminex based assay and ELISA, and to find factors associated with CXCL13 concentration. CSF samples obtained from four clinically well-defined groups of patients (proven LNB, suspected LNB, tick-borne encephalitis (TBE), and aseptic meningitis/meningoencephalitis other than TBE) - 25 samples per group - were analyzed. The performance of the Luminex recomBead CXCL13 assay (Microgen, Neuried, Germany) and ELISA (Euroimmun, Lübeck, Germany) was assessed by receiver operating characteristics. CXCL13 cut-off values were presented as functions of CSF lymphocyte/monocyte counts. Demographic variables, CSF findings, and history of erythema migrans were assessed as possible predictors for CXCL13 CSF concentrations by a general linear model. The calculated cut-off values determined by the maximum of the Youden index were >131 pg/mL for recomBead and >259 pg/mL for the ELISA. RecomBead showed a sensitivity of 88% (68.8-97.5%) and a specificity of 94% (83.5-98.7%). For the ELISA the corresponding values were 84% (63.9-95.5%) and 98% (89.4-99.9%). The CXCL13 concentration positively correlated with CSF lymphocyte/monocyte count and Borrelia-specific intrathecal antibody index (p < 0.05). High CXCL13 concentrations were only found in the group with proven LNB. CXCL13 levels above cut-off value were established in some patients with viral meningitis/meningoencephalitis but were not detected in patients with suspected LNB without pleocytosis. Applying a linearized cut-off of the CXCL13 concentration in the CSF which is dependent on the CSF cell count is a novel approach in the laboratory diagnosis of LNB.

The dynamic movement of B cells increases the probability of encountering specific antigen and facilitates cell-cell interactions required for mounting a rapid antibody response. B1a and B1b cells are enriched in the coelomic cavity, contribute to T-cell-independent (TI) antibody responses, and increase in number upon antigen exposure. B1 cell movement is largely governed by Cxc ligand 13 (Cxcl13), and mice deficient in this chemokine have a severe reduction in peritoneal B1 cells. In this study, we examined the role of Cxcl13-dependent B cell migration using Borrelia hermsii infection or intraperitoneal immunization with pneumococcal polysaccharide or 4-hydroxy-3-nitrophenyl-acetyl (NP)-Ficoll, all of which induce robust antibody responses from B1b cells. Surprisingly, we found that antibody responses to B. hermsii or to FhbA, an antigenic target of B1b cells, and the resolution of bacteremia were indistinguishable between wild-type and Cxcl13-/- mice. Importantly, we did not observe an expansion of peritoneal B1b cell numbers in Cxcl13-/- mice. Nonetheless, mice that had resolved infection were resistant to reinfection, indicating that the peritoneal B1b cell reservoir is not required for controlling B. hermsii. Furthermore, despite a reduced peritoneal B1b compartment, immunization with pneumococcal polysaccharide vaccine yielded comparable antigen-specific antibody responses in wild-type and Cxcl13-/- mice and conferred protection against Streptococcus pneumoniae. Likewise, immunization with NP-Ficoll elicited similar antibody responses in wild-type and Cxcl13-/- mice. These data demonstrate that homing of B1 cells into the coelomic cavity is not a requirement for generating protective TI antibody responses, even when antigen is initially localized to this anatomical compartment.

BACKGROUND

Post-therapeutic follow-up is essential to confirm cure and to detect early treatment failures in patients affected by sleeping sickness (HAT). Current methods, based on finding of parasites in blood and cerebrospinal fluid (CSF) and counting of white blood cells (WBC) in CSF, are imperfect. New markers for treatment outcome evaluation are needed. We hypothesized that alternative CSF markers, able to diagnose the meningo-encephalitic stage of the disease, could also be useful for the evaluation of treatment outcome.

RESULTS

Cerebrospinal fluid from patients affected by Trypanosoma brucei gambiense HAT and followed for two years after treatment was investigated. The population comprised stage 2 (S2) patients either cured or experiencing treatment failure during the follow-up. IgM, neopterin, B2MG, MMP-9, ICAM-1, VCAM-1, CXCL10 and CXCL13 were first screened on a small number of HAT patients (n = 97). Neopterin and CXCL13 showed the highest accuracy in discriminating between S2 cured and S2 relapsed patients (AUC 99% and 94%, respectively). When verified on a larger cohort (n = 242), neopterin resulted to be the most efficient predictor of outcome. High levels of this molecule before treatment were already associated with an increased risk of treatment failure. At six months after treatment, neopterin discriminated between cured and relapsed S2 patients with 87% specificity and 92% sensitivity, showing a higher accuracy than white blood cell numbers.

CONCLUSIONS

In the present study, neopterin was highlighted as a useful marker for the evaluation of the post-therapeutic outcome in patients suffering from sleeping sickness. Detectable levels of this marker in the CSF have the potential to shorten the follow-up for HAT patients to six months after the end of the treatment.

Leptin is a pleiotropic hormone proposed to link nutritional status to the development of strong Th1 immunity. Because Mycobacterium tuberculosis control is affected by starvation and diabetes, we studied the role of the leptin receptor in regulating distinct immune cells during chronic infection. Infected db/db mice, bearing a natural mutation in the leptin receptor, have a markedly increased bacterial load in their lungs when compared with that of their wild-type counterparts. In response to M. tuberculosis infection, db/db mice exhibited disorganized granulomas, neutrophilia, and reduced B cell migration to the lungs, correlating with dysfunctional lung chemokine responses that include XCL1, CCL2, CXCL1, CXCL2, and CXCL13. In a db/db lung, myeloid cells were delayed in their production of inducible NO synthase and had reduced expression of MHC I and II. Although the Th1 cell response developed normally in the absence of leptin signaling, production of pulmonary IFN-γ was delayed and ineffective. Surprisingly, a proper immune response took place in bone marrow (BM) chimeras lacking leptin receptor exclusively in BM-derived cells, indicating that leptin acts indirectly on immune cells to modulate the antituberculosis response and bacterial control. Together, these findings suggest that the pulmonary response to M. tuberculosis is affected by the host's nutritional status via the regulation of non-BM-derived cells, not through direct action of leptin on Th1 immunity.

Signal transduction pathways regulating NF-kappaB activation essential for microenvironment formation in secondary lymphoid organs remain to be determined. We investigated the effect of a deficiency of TNFR-associated factor 6 (TRAF6), which activates the classical NF-kappaB pathway, in splenic microenvironment formation. Two-week-old TRAF6-deficient mice showed severe defects in B cell follicle and marginal zone formation, similar to mutant mice defective in lymphotoxin (Lt) beta receptor (LtbetaR) signal induction of nonclassical NF-kappaB activation. However, analysis revealed a TRAF6 role in architecture formation distinct from its role in the early neonatal Lt signaling pathway. LtbetaR signal was essential for primary B cell cluster formation with initial differentiation of follicular dendritic cells (FDCs) in neonatal mice. In contrast, TRAF6 was dispensable for progression to this stage but was required for converting B cell clusters to B cell follicles and maintaining FDCs through to later stages. Fetal liver transfer experiments suggested that TRAF6 in radiation-resistant cells is responsible for follicle formation. Despite FDC-specific surface marker expression, FDCs in neonatal TRAF6-deficient mice had lost the capability to express CXCL13. These data suggest that developmentally regulated activation of TRAF6 in FDCs is required for inducing CXCL13 expression to maintain B cell follicles.

We have previously shown that overexpression of BLyS/BAFF was associated with increased relative frequencies of innate "precursor" marginal zone (MZ)-like B-cells in the blood of HIV-1-infected rapid and classic progressors. However, along with relatively normal BLyS/BAFF expression levels, these cells remain unaltered in elite-controllers (EC), rather, percentages of more mature MZ-like B-cells are decreased in the blood of these individuals. Fluctuations in frequencies of blood MZ-like B-cell populations may reflect migratory patterns associated with disease progression status, suggesting an important role for these cells in HIV-1 pathogenesis. We have therefore longitudinally measured plasma levels of B-tropic chemokines by ELISA-based technology as well as their ligands by flow-cytometry on blood B-cell populations of HIV-1-infected individuals with different rates of disease progression and uninfected controls. Migration potential of B-cell populations from these individuals were determined by chemotaxis assays. We found important modulations of CXCL13-CXCR5, CXCL12-CXCR4/CXCR7, CCL20-CCR6 and CCL25-CCR9 chemokine-axes and increased cell migration patterns in HIV progressors. Interestingly, frequencies of CCR6 expressing cells were significantly elevated within the precursor MZ-like population, consistent with increased migration in response to CCL20. Although we found little modulation of chemokine-axes in EC, cell migration was greater than that observed for uninfected controls, especially for MZ-like B-cells. Overall the immune response against HIV-1 may involve recruitment of MZ-like B-cells to peripheral sites. Moreover, our findings suggest that "regulated" attraction of these cells in a preserved BLyS/BAFF non-inflammatory environment, such as encountered in EC could be beneficial to the battle and even control of HIV.

Systemic lupus erythematosus is clinically characterized by episodes of flare and remission. In patients, cutaneous exposure to ultraviolet light has been proposed as a flare trigger. However, induction of flare secondary to cutaneous exposure has been difficult to emulate in many murine lupus models. Here, we describe a system in which epidermal injury is able to trigger the development of a lupus nephritis flare in New Zealand Mixed (NZM) 2328 mice. 20-week old NZM2328 female mice underwent removal of the stratum corneum via duct tape, which resulted in rapid onset of proteinuria and death when compared to sham-stripped littermate control NZM2328 mice. This was coupled with a drop in serum C3 concentrations and dsDNA antibody levels and enhanced immune complex deposition in the glomeruli. Recruitment of CD11b(+)CD11c(+)F4/80(high) macrophages and CD11b(+)CD11c(+)F4/80(low) dendritic cells was noted prior to the onset of proteinuria in injured mice. Transcriptional changes within the kidney suggest a burst of type I IFN-mediated and inflammatory signaling which is followed by upregulation of CXCL13 following epidermal injury. Thus, we propose that tape stripping of lupus-prone NZM2328 mice is a novel model of lupus flare induction that will allow for the study of the role of cutaneous inflammation in lupus development and how crosstalk between dermal and systemic immune systems can lead to lupus flare.