Angioimmunoblastic T-cell lymphoma (AITL) is frequently associated with skin lesions, but epidermotropic cutaneous involvement has never been described. A 37-year-old man presented with erythematous and pruriginous plaques, clinically suggestive of mycosis fungoides, distributed all over the body, 3 weeks after the last line of a polychemotherapy, given for an AITL diagnosed 1 year earlier on a lymph node biopsy. Skin biopsy showed an epidermotropic CD4(+) T-cell lymphoma, so that a diagnosis of mycosis fungoides was first proposed. Further investigations showed that atypical lymphocytes strongly expressed CD10 and markers of follicular helper T cells (T(FH) ) including PD1, BCL-6 and CXCL13. The diagnosis of an unusual epidermotropic cutaneous localization of the AITL was finally made, supported by the presence of the same T-cell clone in the initial lymph node biopsy and the skin. We therefore recommend performing markers of T(FH) cells in patients with unusual epidermotropic cutaneous T-cell lymphomas, particularly if they have any clinical features suggestive of AITL.

BACKGROUND

Examine lymphatic malformation lymphoid aggregates for the expression of tertiary lymphoid organ markers. Determine how lymphoid aggregate density relates to lymphatic malformation clinical features.

RESULTS

Retrospective cohort study. Tissue and clinical data were reviewed from 29 patients in the Vascular Anomaly Database who represented the spectrum of head and neck lymphatic malformations and had >5 years of follow-up. Archived formalin-fixed, paraffin-embedded lymphatic malformation tissue was immunohistochemically stained with antibodies for tertiary lymphoid organ markers, which included follicular and mature myeloid dendritic cells, high endothelial venules, segregated B and T-cells, lymphatic endothelial cells, and lymphoid homing chemokines (CXCL13, CCL21). Lymphoid aggregate density (count/mm(2)) was quantified by 2 independent, blinded reviewers. Lymphoid aggregate density and lymphatic malformation clinical features were characterized using analysis of variance. Larger lymphatic malformation tissue lymphoid aggregates stained consistently for tertiary lymphoid organ markers. In oral cavity and neck specimens from the same patients (n = 9), there were more tertiary lymphoid organ in oral cavity than in neck specimens (p = 0.0235). In lymphatic malformation neck tissue, de Serres stage 4 lymphatic malformations displayed the highest tertiary lymphoid organ density. No significant association was seen between tertiary lymphoid organ density and other clinical features.

CONCLUSIONS

This study demonstrates that some lymphoid aggregates within lymphatic malformations represent tertiary lymphoid organs. There was an association between tertiary lymphoid organ density and lymphatic malformation location. Further study is required to define the role of lymphoid neogenesis and tertiary lymphoid organ formation in lymphatic malformation pathogenesis.

OBJECTIVE

To evaluate the stiffness of parotid and submandibular glands using elastography ultrasound and to correlate it with B-mode ultrasonographical, clinical and serological features, salivary profibrotic and inflammatory chemokines, and salivary gland fibrosis.

METHODS

We performed B-mode and elastography ultrasound of major salivary glands of 26 patients with primary Sjögren's syndrome. We registered the shear wave velocity (SWV) and correlated it with the morphologic ultrasonographic changes assessed by the Hocevar scale. We assessed the European League Against Rheumatism (EULAR) Sjögren's Syndrome Disease Activity Index (ESSDAI), EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI), non-stimulated whole salivary flow rate (NSWSF), C3 and C4 levels, anti-Ro/La antibodies, salivary inflammatory (C-X-C motif ligand 13 [CXCL13], CXCL10, CXCL8, C-C motif ligand 2 [CCL2], interleukin 10 [IL-10] and IL-6) and pro-fibrotic (CXCL14, CCL28, tumor necrosis factor-related apoptosis-inducing ligand and transforming growth factor β) chemokines and cytokines and evaluated the presence of fibrosis in the minor salivary gland.

RESULTS

Ninety-two percent of patients were women; mean age was 51.1 ± 11 years; median disease duration was 6.1 years; 92.3% had oral symptoms and 26.9% fibrosis. The median B-mode score was 22.2 points and the median SWV 2.5 m/s (τ = 0.53, P = 0.001). The SWV correlated with the NSWSF (τ = -0.53, P = 0.001), ESSDAI (τ = 0.31, P = 0.03), glandular ESDDAI domain (τ = 0.36, P = 0.02), C4 levels (τ = -0.32, P = 0.04), salivary CXCL13 (τ = 0.29, P = 0.03) and CXCL10 (τ = 0.30, P = 0.003), but not with age and fibrosis.

CONCLUSIONS

WV correlated with the B-mode ultrasound score, systemic and glandular activity and in a large degree with CXCL10, an inflammatory chemokine, but not with fibrosis. An increased SWV might represent chronic glandular inflammation rather than fibrotic changes in these patients.

BACKGROUND

Chronic lung infection with Pseudomonas aeruginosa remains a major cause of mortality and morbidity among individuals with CF. Expression of mediators promoting recruitment and differentiation of B cells, or supporting antibody production is poorly understood yet could be key to controlling infection.

METHODS

BAFF was measured in BAL from children with CF, both with and without P. aeruginosa, and controls. Mice were intra-nasally infected with P. aeruginosa strain LESB65 for up to 7 days. Cellular infiltration and expression of B cell chemoattractants and B cell differentiation factor, BAFF were measured in lung tissue.

RESULTS

BAFF expression was elevated in both P. aeruginosa negative and positive CF patients and in P. aeruginosa infected mice post infection. Expression of the B cell chemoattractants CXCL13, CCL19 and CCL21 increased progressively post infection.

CONCLUSIONS

In a mouse model, infection with P. aeruginosa was associated with elevated expression of BAFF and other B cell chemoattractants suggesting a role for airway B cell recruitment and differentiation in the local adaptive immune response to P. aeruginosa. The paediatric CF airway, irrespective of pseudomonal infection, was found to be associated with an elevated level of BAFF implying that BAFF expression is not specific to pseudomonas infection and may be a feature of the CF airway. Despite the observed presence of a potent B cell activator, chronic colonisation is common suggesting that this response is ineffective.

To ensure safety tolerance induction protocols are accompanied by conventional immunosuppressive drugs (IS). But IS such as calcineurin inhibitors (CNI), for example, cyclosporin A (CsA), can interfere with tolerance induction. We investigated the effect of an additional transient CsA treatment on anti-CD4mAb-induced tolerance induction upon rat kidney transplantation. Additional CsA treatment induced deteriorated graft function, resulting in chronic rejection characterized by glomerulosclerosis, interstitial fibrosis, tubular atrophy and vascular changes. Microarray analysis revealed enhanced intragraft expression of the B cell attracting chemokine CXCL13 early during CsA treatment. Increase in CXCL13 expression is accompanied by enhanced B cell infiltration with local and systemic IgG production and C3d deposition as early as 5 days upon CsA withdrawal. Adding different CNIs to cultures of primary mesangial cells isolated from glomeruli resulted in a concentration-dependent increase in CXCL13 transcription. CsA in synergy with TNF-α can enhance the B cell attracting and activating potential of mesangial cells. Transient B cell depletion or transfer of splenocytes from tolerant recipients 3 weeks after transplantation could rescue tolerance induction and did inhibit intragraft B cell accumulation, alloantibody production and ameliorate chronic rejection.

Chronic systemic inflammation contributes to the development of adverse health conditions, yet the influence of fixed and modifiable risk factors on many serologic biomarkers of inflammation remains largely unknown. Serum concentrations of twenty-three biomarkers, including C-reactive protein (CRP), cytokines (CXCL11, CXCL8, CXCL10, CCL2, CCL13, CCL4, CCL17, CXCL13, IL-10, IL-12p70, IL-6, TNF-α, IL-2, IFN-γ, IL-1β, GM-CSF, BAFF), and soluble immune receptors (sCD14, sIL-2Rα, sCD27, sgp130, sTNF-R2) were measured longitudinally using multiplexed immunometric assays in 250 HIV-uninfected men followed in the Multicenter AIDS Cohort Study (1984-2009). Generalized gamma regression was used to determine the statistical significance of factors associated with each biomarker. After accounting for age, race, and education, and for analysis of multiple biomarkers, higher concentrations of specific individual biomarkers were significantly (P<0.002) associated with hypertension, obesity, hepatitis C infection, stimulant use, and diabetes and lower concentrations with hypercholesterolemia. These associations should be taken into account in epidemiological studies of these biomarkers, and may provide potential targets for disease prevention and treatment.

UNASSIGNED

Previous studies documented that humoral immune responses participated in neurological damage in neurosyphilis patients. However, the mechanisms that trigger and maintain humoral immunity involved in neurosyphilis remain unknown.

UNASSIGNED

Using flow cytometry, expression of B cells was measured in neurosyphilis and non-neurosyphilis. Expression of immunoglobulin indices and chemokine ligand CXCL13 was detected by enzyme-linked immunosorbent assay. The migration and inhibition assays were evaluated by modified chamber assays. The presence of CXCL13+ cells, cluster of differentiation (CD)20+ B cells, CD3+ T cells, CD138+ plasma cells and CD35+ follicular dendritic cells was studied by immunohistochemistry.

UNASSIGNED

Enrichment of B cells was observed and activated in the cerebrospinal fluid (CSF) of neurosyphilis patients. Immunoglobulin indices were increased and associated with the progress to neurosyphilis. High expression of CSF CXCL13 mediated B cell migration both in vitro and in vivo. There was a positive correlation among the CSF B cells, immunoglobulin indices, and CSF CXCL13 levels. Ectopic germinal centers (EGCs), important structures for humoral immunity, were observed in the intracranial syphilitic gumma.

UNASSIGNED

CXCL13/CXCR5 mediated the aggregation of B cells, that directed the aberrant humoral immune responses via the formation of EGCs, which suggests a molecular mechanism of neurological damage in neurosyphilis.

B cells are involved in immune thrombocytopenia (ITP) pathophysiology by producing antiplatelet auto-antibodies. However more than a half of ITP patients do not respond to B cell depletion induced by rituximab (RTX). The persistence of splenic T follicular helper cells (TFH) that we demonstrated to be expanded during ITP and to support B cell differentiation and antiplatelet antibody-production may participate to RTX inefficiency. Whereas it is well established that the survival of TFH depends on B cells in animal models, nothing is known in humans yet. To determine the effect of B cell depletion on human TFH, we quantified B cells and TFH in the spleen and in the blood from ITP patients treated or not with RTX. We showed that B cell depletion led to a dramatic decrease in splenic TFH and in CXCL13 and IL-21, two cytokines predominantly produced by TFH. The absolute count of circulating TFH and serum CXCL13 also decreased after RTX treatment, whatever the therapeutic response. Therefore, we showed that the maintenance of TFH required B cells and that TFH are not involved in the inefficiency of RTX in ITP.

Lymphoid tissue organizer (LTo) cells, identified in mouse and human embryos, are thought to be precursors of stromal cells in secondary lymphoid organs. Whether LTo cells are present in human adults, however remains unknown. We obtained 15 stromal cell lines from tonsils from children who underwent tonsillectomy, and studied the antigen phenotype of these tonsil stromal cell (TSC) lines by flow cytometry and RT-PCR. Cell lines met the minimal criteria proposed by the International Society for Cellular Therapy to define human mesenchymal stem/stromal cells (MSCs): plastic-adherent capacity; expression of CD73, CD90 and CD105, lack of CD45, CD19 and HLA-DR; and capacity to differentiate into adipocytes, osteoblasts and chondrocytes. Furthermore, our TSC lines exhibited an antigen phenotype and functional characteristics very similar to those seen in murine embryo LTo cells: they expressed chemokines CCL19, CCL21 and CXCL13, cytokines TRANCE and IL-7, and adhesion molecules ICAM-1, mucosal addressin cell adhesion molecule (MadCAM)-1 and VCAM-1. The expression of LTo cell-associated markers and functions were upregulated by lymphotoxin (LT)α1β2 and TNF, two cytokines involved in the development and maturation of secondary lymphoid tissues. Our results show that TSCs are tonsil MSCs that differentiate into LTo-like cells in response to the effects of these cytokines.

CXCL13 is a constitutively expressed chemokine that controls migration of immune cells to lymphoid follicles. Previously, we found CXCL13 mRNA levels increased in rhesus macaque spleen tissues during AIDS. This led us to examine the levels and locations of CXCL13 by detailed in situ methods in cynomolgus macaque lymphoid and intestinal tissues. Our results revealed that there were distinct localization patterns of CXCL13 mRNA compared to protein in germinal centers. These patterns shifted during the course of simian immunodeficiency virus (SIV) infection, with increased mRNA expression within and around follicles during AIDS compared to uninfected or acutely infected animals. Unexpectedly, CXCL13 expression was also found in abundance in Paneth cells in crypts throughout the small intestine. Therefore, we expanded our analyses to include chemokines and antimicrobial peptides (AMPs) not previously demonstrated to be expressed by Paneth cells in intestinal tissues. We examined the expression patterns of multiple chemokines, including CCL25, as well as α-defensin 6 (DEFA6), β-defensin 2 (BDEF2), rhesus θ-defensin 1 (RTD-1), and Reg3γ in situ in intestinal tissues. Of the 10 chemokines examined, CXCL13 was unique in its expression by Paneth cells. BDEF2, RTD-1, and Reg3γ were also expressed by Paneth cells. BDEF2 and RTD-1 previously have not been shown to be expressed by Paneth cells. These findings expand our understanding of mucosal immunology, innate antimicrobial defenses, homeostatic chemokine function, and host protective mechanisms against microbial translocation.

We compared the chemokine/receptor expression in skin biopsies of discoid (SLE/DLE) and subacute lupus (SLE/SCLE) and correlated it with tissue and circulating effector CD4 T cells/regulatory cells. Skin biopsies and peripheral blood from 9 active SLE/DLE patients, 9 SLE/SCLE patients, 5 control SLE patients without cutaneous lesions and 10 control healthy donors were included. Clinical skin activity was measured by Cutaneous Lupus Erythematosus Disease Area and Severity Index scoring, and systemic activity was measured by a modified SLEDAI-2K excluding the cutaneous items. Pain and pruritus were evaluated by a 10-point visual analogue scale. To determine the frequencies of CXCL-10/CXCR3-, CCL2/CCR2-, CCL17/CCR4-, CCL20/CCR6-, CCL27/CCR10-, CXCL8/CXCR1-, CXCL13/CXCR5-, IL-22-, CD4/IL-17A-, CD4/IL-4-, CD4/IFN-γ-, CD123/IDO-, CD25/Foxp3-, and CD20/IL-10-expressing cells, double immunostaining procedures were performed. Circulating CD4+/CD161-/IL-22+, CD4+/CD161+/IL-17+, CD4+/CD25-/IL-4+, CD4+/CD25-/IFN-γ+, CD4+/CD25hi/Foxp3+, CD3+/CD19+/CD38hi/IL-10+, and CD123+/CD196+/IDO + cells were analyzed by flow cytometry. RESULTS: In the tissue, CXCL10, CXCR5, and CCL20 expression and IL-22+, CD4+/IL-17+, CD4+/IFN-γ + and CD123+/IDO + cell percentages were increased in SLE/DLE versus SLE/SCLE. Circulating CD4+/CD161-/IL-22+, CD4+/CD161+/IL-17+, CD4+/CD25-/IFN-γ+, CD19+/CD38hi/IL-10 + and CD123+/CD196+/IDO + cell percentages were higher in SLE/DLE versus SLE/SCLE. In the tissue, we found positive correlations between CXCR3 and CD4+/IL-17 + cells; CCR2 and CD4+/IFN-γ + cells; and CCR10 and CD123+/IDO + cells in the SLE/DLE patients and between CXCL13 and CD20+/IL-10 + cells in the SLE/SCLE patients. In the peripheral blood, we determined positive correlations between CXCR5 and CD4+/CD25-/IFN-γ + cells; CCL17 and CD4+/CD161-/IL-22 + cells; and CCL17 and CD4+/CD161+/IL-17 + cells in the SLE/DLE patients and between CXCR5 and CD3+/CD19+/CD38hi/IL-10 + cells; CCR2 and CD4+/CD25hi/Foxp3 + cells; and CXCR1 and CD4+/CD25hi/Foxp3 + cells in the SLE/SCLE patients. Positive correlations between the pain score and the expression of CCL2 and CCR6 expression were found in the SLE/SCLE patients. In conclusion, the correlations between the expression of chemokines/receptors and subpopulations of effector/regulatory cells showed differential responses among the cutaneous pathologies.

Innate immune responses provoke the accumulation of leukocytes at sites of inflammation. In addition to monocytes and granulocytes, B cells also participate in antimicrobial innate immune responses; however, the mechanisms for accumulation of B cells to sites of inflammation are not well understood. To study B cell accumulation following systemic inflammation, we used a model synthetic ligand that stimulates a specific pattern recognition molecule, nucleotide-binding oligomerization domain-containing protein 1 (Nod1). Upon exposure to Nod1 agonists, both B cells and neutrophils rapidly accumulate within the spleen, and dendritic cells migrate into the periarterial lymphoid sheath. Nod1 stimulation led to a marked increase in several chemokines within the spleen, including CXCL13, CCL2, and CCL20. Whereas the lymphotoxin pathway was critical for the induction of the B cell chemoattractant CXCL13 in response to Nod1 agonists, B cell accumulation within the spleen following Nod1-induced systemic inflammation was independent of the lymphotoxin pathway. In contrast, a CCR6/CCL20 chemokine loop instructed rapid increase of B cells in the spleen in response to systemic administration of Nod1 agonists in a TNF-α-dependent manner. Moreover, CCR6 was required to regulate Nod1-mediated B cell responses. These results reveal a novel mechanism of B cells during inflammation and shed light on how B cells participate in innate immune responses to microbial stimulation.

OBJECTIVE

Sjogren's syndrome (SS) is an autoimmune disease with features of both over-production of specific autoantibodies and organ-specific disorders, mainly sialadenitis and dacryoadenitis. However, little is known about the factors that contribute to lymphocytic infiltration of SS.

METHODS

Minor salivary gland (MSG) tissue was obtained from 83 patients with primary SS (pSS) and 95 patients with secondary SS and examined pathologically, and correlation between infiltrated immune cells and histological features was evaluated.

RESULTS

Plasmacytoid dendritic cells (pDCs) were increased in MSG of SS compared to Sicca syndrome. The density of pDCs was characteristically correlated with the accumulation of CXCL13(+)CD68(+) macrophages and CXCR5(+)CD19(+) B in the MSG of pSS. In vitro analysis indicated that Type I interferon (IFN) enhanced CXCL13 production by macrophages. Type I IFN was mainly expressed in pDCs and its expression was correlated with the accumulation of CXCL13(+) macrophages in the MSG of pSS.

CONCLUSIONS

Our histological findings suggest the possible mechanism of type I IFN-CXCL13 axis during the pathological processes of acute/chronic salivary inflammation in SS; local production of type I IFN by pDCs, induction of CXCL13 production in macrophages by type I IFN, induction of accumulation of CXCR5(+)CD19(+) B cells by CXCL13 in the MSG.

BACKGROUND

Natalizumab is approved for treatment of active forms of relapsing-remitting multiple sclerosis (MS) based on a pivotal phase III study comprising patients aged 18-50 years. The effect of natalizumab has not been specifically studied in older patients.

OBJECTIVE

We analyzed age-dependent effects on treatment-related outcome measures in 1872 patients, 189 of whom were aged 50 or more, included in the Swedish post-marketing natalizumab surveillance program.

METHODS

In three MS centers registry data for patients aged >50 years were validated.

RESULTS

At baseline older patients had longer disease duration, higher Expanded Disability Status Scale (EDSS) and lower Symbol Digit Modality Test (SDMT) scores than younger patients. The influence from natalizumab on outcome measures was significantly reduced and 18.7% of patients >50 years stopped treatment for lack of effect compared to 7.7% in the younger age group. At baseline, the cerebrospinal fluid levels of the chemokine CXCL13 and the leukocyte cell count were negatively correlated with age in a smaller subgroup of patients.

CONCLUSIONS

These results were in agreement with previous findings suggesting that inflammation is more pronounced in younger patients and therefore the beneficial effects of potent anti-inflammatory treatments are subsiding with older ages.

Painful diabetic neuropathy (PDN) is a severely debilitating chronic pain syndrome. Spinal chemokine CXCL13 and its receptor CXCR5 were recently demonstrated to play a pivotal role in the pathogenesis of chronic pain induced by peripheral tissue inflammation or nerve injury. In this study we investigated whether CXCL13/CXCR5 mediates PDN and the underlying spinal mechanisms. We used the db/db type 2 diabetes mice, which showed obvious hyperglycemia and obese, long-term mechanical allodynia, and increased expression of CXCL13, CXCR5 as well as pro-inflammatory cytokines TNF-α and IL-6 in the spinal cord. Furthermore, in the spinal cord of db/db mice there is significantly increased gliosis and upregulated phosphorylation of cell signaling kinases, including pERK, pAKT and pSTAT3. Mechanical allodynia and upregulated pERK, pAKT and pSTAT3 as well as production of TNF-α and IL-6 were all attenuated by the noncompetitive NMDA receptor antagonist MK-801. If spinal giving U0126 (a selective MEK inhibitor) or AG490 (a Janus kinase (JAK)-STAT inhibitor) to db/db mice, both of them can decrease the mechanical allodynia, but only inhibit pERK (by U0126) or pSTAT3 (by AG490) respectively. Acute administration of CXCL13 in C57BL/6J mice resulted in exacerbated thermal hyperalgesia and mechanical allodynia, activation of the pERK, pAKT and pSTAT3 pathways and increased production of pro-inflammatory cytokines (IL-1β, TNF-α and IL-6), which were all attenuated by knocking out of Cxcr5. In all, our work showed that chemokine CXCL13 and its receptor CXCR5 in spinal cord contribute to the pathogenesis of PDN and may help develop potential novel therapeutic approaches for patients afflicted with PDN.

BACKGROUND

Although rectal bleeding in infancy (RBI) is not a rare phenomenon, the clinical course of RBI is not fully understood.

METHODS

To investigate the outcome and pathogenesis of RBI, especially when concomitant with food-protein-induced proctocolitis (FPIP) and neonatal transient eosinophilic colitis (NTEC), 22 neonates with rectal bleeding with FPIP and NTEC from January 2008 to June 2012 were enrolled and their clinical course and mechanisms of inflammation were examined.

RESULTS

Thirteen infants showed rectal bleeding after feeding and were diagnosed with FPIP, and nine infants showed rectal bleeding before feeding and were diagnosed with NTEC. Elevated peripheral white blood cell (12,685 ± 3754/μl and 30,978 ± 16,166/μl) and eosinophil (1084 ± 816/μl and 4456 ± 3341/μl) were confirmed in FPIP and NTEC, respectively. Colonoscopy revealed nodular lymphoid hyperplasia, a pale mucosal surface and oozing with diffuse infiltration of neutrophils, lymphocytes, and eosinophils in both groups. Reverse transcription polymerase chain reaction analysis revealed enhanced expression of the interleukin-6, CCL11, and CXCL13 genes, where CXCL13 expression was more prominent in FPIP. Mucosal infiltration by CD3- and immunoglobulin-A- but not immunoglobulin-E-positive cells was confirmed. Among them, only one infant with FPIP developed milk allergy, whereas none with NTEC had developed milk allergy at the age of 1 year.

CONCLUSIONS

FPIP in infancy and NTEC are similar diseases and interleukin-6, CCL11, and CXCL13 may play a major role in the pathogenesis of rectal bleeding. Although the involvement of allergic reaction is possible, milk allergy was not a common outcome after 1 year of follow up.

Chemokines play an essential role in immune and inflammatory reactions via the recruitment of leukocytes. Studying the role of chemokines in vivo is complicated by the redundancy of their action and by their promiscuous receptor usage. The simultaneous analysis of several chemokines is, therefore, advantageous in order to obtain a comprehensive view of chemokine participation in inflammatory and infectious processes. At present, no multi-probe detection systems are available for the analysis of recently described chemokines. In this study, new multi-probe RNase protection assay (RPA) template sets were developed for the analysis of murine chemokines. Chemokine cDNA fragments were generated by RT-PCR and individually subcloned into the plasmid pGEM-T providing a T7 promotor. In this way, two multi-probe template sets were constructed each containing six chemokine sequences (CXCL12/SDF-1, XCL1/lymphotactin, CCL20/exodus-1, CCL25/TECK, CX3CL1/fractalkine, CXCL1/KC, and CCL20/MDC, CXCL9/MIG, CCL9/10/MIP-1gamma, CXCL13/BLC, CCL12/MCP-5, CCL19/ELC, respectively) and templates for the two house-keeping genes L32 and GAPDH. The evaluation of these RPA template sets in various murine models demonstrated their suitability for the analysis of the above chemokines both under constitutive and infection-induced conditions. To reduce the personal radiation hazard, we found that 32P could be replaced by 33P without any loss of assay-sensitivity. These new RPA multi-probe sets provide valuable tools for the simultaneous quantitative determination of gene expression of multiple murine chemokines of both constitutive and inducible type.

S100A2 is a homodimeric protein that undergoes oxidative cross-linking and translocation from the nucleus to the cytosol in the context of oxidative stress. Suggestive of a role for S100A2 in the cutaneous response to ultraviolet light, we found altered S100A2 immunostaining in photodamaged human skin, and crosslinking of S100A2 after ultraviolet A (UVA) irradiation of normal human keratinocytes (NHK). Skin from mice, rats, and rabbits did not contain S100A2 protein, whereas skin samples from pigs, frogs and humans were strongly positive. Survival after UVA irradiation was significantly greater in NHK compared to mouse keratinocytes, suggesting a protective role for S100A2. To test this hypothesis in vivo, we expressed S100A2 in SKH2/J hairless mice under the control of a bovine keratin 5 promoter, and compared responses of TG and WT mice from 1 to 7 days after a single dose (0.5-1 MED) of solar-simulated radiation (SSR) from UVA-340 bulbs. WT and TG mice manifested a similarly robust response to SSR, characterized by epidermal hyperplasia, marked induction of p21(WAF), and a twofold increase in p53. Thymine dimers (TD) were markedly increased in the epidermis and the dermis, but while over 95% of the epidermal TD were removed by 5-6 days, elevated dermal TD persisted nearly unchanged for 7 days. Global transcriptional profiling of WT and TG mice revealed strong induction of multiple transcripts, including keratins K6 and K16, defensin beta 3, S100A8, S100A9, Sprr2i and Sprr2f. However, the only S100A2-dependent difference we observed was an induction of Cxcl13 transcripts in TG, but not WT mice (4.4-fold vs. 0.7-fold, n = 3, P = 0.022). This finding was confirmed in an independent set of mice analyzed by quantitative RT-PCR (8.8-fold vs. 1.2-fold, n = 4, P = 0.001). The finding of persistent dermal DNA damage after suberythemal doses of SSR merits further study.

To examine the cytokine/chemokine profile of cerebrospinal fluid (CSF) during acute herpes simplex virus-induced N-methyl-d-aspartate receptor (NMDAR) autoimmunity and in chronic/relapsing post-herpes simplex virus encephalitis (HSE) neurological syndromes.

We measured longitudinal serial CSF cyto-/chemokines (n=34) and a glial marker (calcium-binding astroglial protein, S100B) in one patient during acute HSE and subsequent anti-NMDAR encephalitis, and compared the results with those from two patients with anti-NMDAR encephalitis without preceding HSE. We also compared cyto-/chemokines in cross-sectional CSF samples from three children with previous HSE who had ongoing chronic or relapsing neurological symptoms (2yr 9 mo-16y after HSE) with those in a group of children having non-inflammatory neurological conditions (n=20).

Acute HSE showed elevation of a broad range of all T-helper-subset-related cyto-/chemokines and S100B whereas the post-HSE anti-NMDAR encephalitis phase showed persistent elevation of two of five T-helper-1 (chemokine [C-X-C motif] ligand 9 [CXCL9], CXCL10), three of five predominantly B-cell (CXCL13, CCL19, a proliferation-inducing ligand [APRIL])-mediated cyto-/chemokines, and interferon-α. The post-HSE anti-NMDAR encephalitis inflammatory response was more pronounced than anti-NMDAR encephalitis. All three chronic post-HSE cases showed persistent elevation of CXCL9, CXCL10, and interferon-α, and there was histopathological evidence of chronic lymphocytic inflammation in one biopsied case 7 years after HSE. Two of three chronic cases showed a modest response to immune therapy.

HSE-induced anti-NMDAR encephalitis is a complex and pronounced inflammatory syndrome. There is persistent CSF upregulation of cyto-/chemokines in chronic or relapsing post-HSE neurological symptoms, which may be modifiable with immune therapy. The elevated cyto-/chemokines may be targets of monoclonal therapies.

A DNA microarray-based gene expression analysis study was performed with bovine spongiform encephalopathy (BSE) transgenic mice. Several genes were found to be overexpressed including the lysosomal enzyme cathepsin C, the chemokine CXCL13 and a number of genes related to cellular proliferation. The brains from terminal stage, BSE inoculated, 'bovinized', transgenic mice were subjected to immunohistochemistry with antibodies against these two proteins and Ki-67, a cell proliferation marker, to assess the biological relevance of the gene expression changes. Differential expression of cathepsin C and CXCL13 proteins and increased expression of Ki-67 was observed. These changes were localized to areas of deposition of PrP(res) and spongiform change and to areas showing an astroglial and microglial response. These findings suggest that these proteins are involved in the mechanisms leading to the establishment of transmissible spongiform encephalopathy.

Peripheral T-cell lymphoma (PTCL) with a nodular pattern of growth is uncommon and may be misdiagnosed initially as a B-cell lymphoma or reactive process. We report a case of a rapidly growing PTCL with a distinctly nodular pattern in an axillary lymph node from an 89-year-old man. Immunohistochemical stains for CD21, CD23, and CD35 highlighted an extensive dendritic cell network that imparted the nodular appearance and, in addition, was associated intimately with the neoplastic cells. The neoplastic cells otherwise had an immunophenotype similar to previously reported cases of PTCL with a nodular pattern and germinal center origin (CD3+, CD4+, CD5+, bcl-6+, CD31+, subset CD10+, subset CXCL13+, and subset CD79a+). Molecular studies confirm a clonal T-cell receptor g gene rearrangement. This case emphasizes unusual morphologic features in a PTCL that may be mistaken for follicular lymphoma or a tumor of follicular dendritic cell origin.

Previous research has shown that CXCR5^-/- mice develop retinal degeneration (RD) with age, a characteristic related to age macular degeneration (AMD). RD in these mice is not well-understood, and in this study, we sought to characterize further the RD phenotype and to gain mechanistic insights into the function of CXCR5 in the retina. CXCR5^-/- and WT control mice were used. Fundus images demonstrated a significant (p < 0.001) increase of hypo-pigmented spots in the retina of aged CXCR5^-/- mice compared with WT control mice. PAS staining indicated localization of deposits in the sub-retinal pigment epithelia (RPE) layer. AMD-associated proteins Cryab, amyloid beta, and C3d were detected within the RPE/sub-RPE tissues by immunofluorescence (IF). In addition, western blot analysis of COX-2, Arg1, and VEGF-a revealed an increase in the signaling of these molecules within the RPE/choroid complex. Transmission electron microscopy (TEM) indicated a drusen-like structure of sub-RPE deposits with an accumulation of vacuolated cellular debris. Loss of photoreceptors was detected by peanut lectin staining and was corroborated by a reduction in MAP2 signaling. Loss of blood-retinal barrier integrity was demonstrated by a reduction of ZO-1 expression. Inflammatory cells were detected in the sub-RPE space, with an increase in IBA-1 positive microglia cells on the surface of the RPE. Mass spectrometry analysis of CXCR5^-/- mouse RPE/choroid proteins extracts, separated by SDS-page and incubated with autologous serum, identified autoantibodies against AMD-associated proteins: Cryaa, Cryab, and Anxa2. In vitro evaluations in BV-2 cell culture indicated a significant increase in production of Arg-1 (p < 0.001) and COX-2 (p < 0.01) in the presence of anti-CXCR5 antibody when compared with Igg-treated control BV-2 cells stimulated with IL-4 and TNFα/IFNγ, respectively. Anti-CXCR5 antibody treatment without stimulating agents did not affect Arg-1 and COX-2 expression; this suggests that CXCR5 may have a regulatory role in microglia cells activation. These results indicate that with age, CXCR5^-/- mice develop RD characterized by microglia dysfunction, increased production of CXCL13 in the RPE progressive photoreceptor, neuronal loss, and sub-RPE deposition of cellular debris, resulting in the production of immunogenic proteins and autoimmune-mediated RD.

BACKGROUND

Immune thrombocytopenia (ITP) is an acquired and autoimmune disease of adults and children characterized by decreased platelet production. CXC chemokine ligand-13 (CXCL13) participates in multiple immunological responses. However, it is still unknown the relationship between CXCL13 and ITP.

METHODS

Plasma CXCL13 was detected in ITP (n = 30) children. CD4+ T cells was isolated from peripheral blood mononuclear cells (PBMCs) from healthy volunteers. Treated CD4+ T cells with dexamethasone and/or miR-125-5p mimic/inhibitor, to observe the regulation of CXCL13.

RESULTS

Compared with controls, ITP children had elevated plasma CXCL13, the concentration of which was reduced after treatment. In vitro, dexamethasone decreased CXCL13 level in in dose- dependent and in time-dependent manner. MiR-125-5p mimic decreased CXCL13 level and miR-125-5p inhibitor increased CXCL13 level in CD4+ T cells. CXCL13 was implied to be target gene of miR-125-5p. MiR-125-5p inhibitor also canceled dexamethasone induced decrease of CXCL13.

CONCLUSIONS

CXCL13 is the target gene of miR-125-5p, which is possibly involved in the pathological process of ITP.

Chemokines are small, secreted cytokine peptides that have the ability to recruit a wide range of immune cells to sites of infection and disease. A novel CXC chemokine was obtained from Japanese flounder Paralichthys olivaceus. This chemokine cDNA contains an open reading frame of 333 nucleotides encoding 111 amino acid residues containing four conserved cysteine residues. The gene is composed of four exons and three introns as are those of mammalian and fish CXC chemokines. Results of homology and phylogenetic analysis revealed that the Japanese flounder CXC chemokine is closest to CXCL13 subgroup. The gene was expressed in immune-related organs, including head kidney, trunk kidney, spleen and peripheral blood leukocytes (PBLs). Japanese flounder CXC chemokine gene expression was observed at 3 and 6h after induction by LPS, but not at 3 and 6h after induction by poly I:C. These results suggest that the Japanese flounder CXC chemokine is probably associated with inflammatory as well as homeostatic functions.