Connexin26 Modulates Radiation-Induced Skin Damage by Regulating Chemokine CCL27 through MAPK Signaling.

Connexin26 (Cx26) plays an important role in ionizing radiation-induced damage, and CC chemokine ligand 27 (CCL27) regulates the skin immune response. However, the relationship between Cx26 and CCL27 in radiation-induced skin damage is unclear. After X-ray irradiation, clonogenic survival and micronucleus formation were assessed in immortalized human keratinocytes (HaCaT). Proteins in the mitogen activated protein kinase (MAPK) signaling pathway and CCL27-related proteins were detected by immunoblotting. HaCaTCx26-/- cells were constructed to verify the effects of Cx26 on CCL27 secretion. A mouse model was established to examine the expression of CCL27 and skin inflammation in vivo. The degree of skin injury induced by 6 MV of X rays was closely related to CCL27. The phosphorylation of ERK, p38 and NF-κB was significantly increased in irradiated cells. The secretion of CCL27 was significantly decreased in HaCaT wild-type cells relative to HaCaTCx26-/- cells. Whereas cell survival fractions decreased, and the micronuclei formation rate increased as a function of increasing X-ray dose in HaCaT cells, the opposite trend occurred in HaCaTCx26-/- cells. Our findings show that Cx26 likely plays a role in the activation of the MAPK and NF-κB/COX-2 signaling pathways and regulates the secretion of CCL27 in keratinocytes after X-ray radiation-induced skin damage.

The CCL27-CCR10 axis contributes to promoting proliferation, migration, and invasion of lung squamous cell carcinoma.

Lung cancer is characterized by its high mortality and morbidity. A deep understanding of the molecular mechanisms of lung cancer tumorigenesis helps to develop novel lung cancer diagnostic and therapeutic strategies. However, the picture of the associated molecular landscape is not yet complete. As understood, chemokine-receptor interactions contribute much to lung cancer tumorigenesis, in which CCR10 also plays an important role. This study aimed to expand the knowledge of CCR10 in lung squamous cell carcinoma (LUSC) in the manner of molecular mechanism and biological functions. Using GEPIA database, the survival analysis between LUSC patients with high and low CCR10 expressions was performed, showing that CCR10 could be regarded as a risk factor for LUSC patients. Subsequently, CCR10 protein and mRNA expressions in LUSC were examined by qRT-PCR and western blot respectively. The results indicated that CCR10 was highly expressed in LUSC cells. The results of CCK-8, colony formation, and Transwell assays presented that CCL27, the ligand of CCR10, promoted proliferative, migratory, and invasive abilities of LUSC cells by activating CCR10. Also, the PI3K/AKT signaling pathway was verified as the involved pathway by western blot. Overall, it could be concluded that the CCL27-CCR10 regulatory axis can activate the PI3K/AKT pathway fostering the malignant features of LUSC cells.

Progranulin promotes osteogenic differentiation of human periodontal ligament stem cells via tumor necrosis factor receptors to inhibit TNF-α sensitized NF-kB and activate ERK/JNK signaling.

OBJECTIVE: To investigate the molecular mechanism of Progranulin (PGRN) in promoting osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in inflammatory environment. BACKGROUND: Progranulin is an antagonist of tumor necrosis factor (TNF) receptors (TNFRs) and is known to promote inflammatory periodontal bone defect regeneration. METHODS: TNFR1- and TNFR2-silenced hPDLSCs designed as hPDLSCs-sh-TNFR1 and hPDLSCs-sh-TNFR2 were cultured with osteoinductive medium containing TNF-α and (or) PGRN. Immunofluorescence, quantitative real-time PCR, and western blot were used to, respectively, detect expressions of TNFR1\TNFR2 and osteogenic differentiation markers as well as phosphorylation level in NF-κB\MAPK-related pathways. RESULTS: Immunofluorescence and real-time PCR showed that TNFR1 and TNFR2 positively expressed in hPDLSCs. TNF-α stimulation could significantly decrease the expressions of ALP and RUNX2 in hPDLSCs, whereas PGRN treatment could significantly enhance their expressions, and reverse TNF-α-mediated expression suppression of ALP and RUNX2 in hPDLSCs. In hPDLSCs-sh-TNFR1, TNF-α mediated osteogenic inhibition decreased, but both TNF-α + PGRN and alone PGRN significantly promoted expression of ALP and RUNX2. PGRN significantly enhanced expression of P-ERK1/2 and P-JNK, while corresponding inhibitors eliminated PGRN-stimulated osteogenic differentiation. In hPDLSCs-sh-TNFR2, no significant difference existed in osteogenic markers and P-JNK expression between the PGRN group and the control group. However, PGRN still activated P-ERK1/2 expression. Besides, PGRN antagonized TNF-α-enhanced NF-κB P65 expression. CONCLUSION: Progranulin promotes osteogenic differentiation of hPDLSCs via TNFR1 to inhibit TNF-α-sensitized NF-κB and via TNFR2 to activate JNK signaling. The mechanism by which PGRN activates ERK signaling remains to be explored.

MiRNA-27a-3p promotes osteogenic differentiation of human mesenchymal stem cells through targeting ATF3.

OBJECTIVE: To elucidate whether miRNA-27a-3p can promote osteogenic differentiation of hMSCs by targeting ATF3, thus alleviating osteoporosis symptoms. PATIENTS AND METHODS: The serum levels of miRNA-27a-3p in osteoporosis patients (n=20) and normal controls (n=20) were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Human bone marrow mesenchymal stem cells (hMSCs) were subjected to osteogenic differentiation for 1, 3 and 7 days. Subsequently, mRNA levels of miRNA-27a-3p, ALP, and Bglap in hMSCs were determined by qRT-PCR. The regulatory effects of miRNA-27a-3p levels and the mRNA levels of ALP, Bglap, and Runx2 were detected. After the overexpression or knockdown of miRNA-27a-3p, we evaluated the changes in the osteogenic differentiation by alizarin red staining and ALP staining. Through Dual-Luciferase Reporter Gene Assay, we verified the binding relationship between miRNA-27a-3p and ATF3. Rescue experiments were finally conducted to prove whether miRNA-27a-3p regulated the osteogenic differentiation by targeting ATF3. RESULTS: The serum level of miRNA-27a-3p remained lower in osteoporosis patients relative to controls. With the prolongation of osteogenic differentiation, the mRNA levels of miRNA-27a-3p, ALP, and Bglap gradually increased. The overexpression of miRNA-27a-3p upregulated mRNA and the protein levels of osteogenesis-related genes, increased ALP activity, and enhanced mineralization capacity. The knockdown of miRNA-27a-3p obtained the opposite trends. MiRNA-27a-3p could target ATF3, and the overexpression of ATF3 reversed the promotive effects of miRNA-27a-3p on osteogenic differentiation. CONCLUSIONS: MiRNA-27a-3p promotes the differentiation of hMSCs into osteoblasts by targeting ATF3, thus alleviating osteoporosis symptoms.

Impaired cutaneous T-cell attracting chemokine elevation and adipose-derived stromal cell migration in a high-glucose environment cause poor diabetic wound healing.

Diabetic wound care is a major health care concern. The major cause of non-healing of wounds in patients with diabetes mellitus (DM) patients mainly involves poor glycemic control, which hinders the migration of progenitor cells including mesenchymal stem cells to the wound site. In this study, we introduced adipose-derived stromal cells (ADSCs) into wound sites and demonstrated that the local transplantation of ADSCs accelerated DM-related wound healing. Furthermore, the migration ability of ADSCs, which diminishes in a high-glucose environment, was partially restored by the exogenous replenishment of the cutaneous T-cell attracting chemokine (CTACK/CCL27). Our findings suggest that CTACK is a potential novel therapeutic target in DM-related wound healing.

Reconstructed human keloid models show heterogeneity within keloid scars.

Keloid scars are often described as having an actively growing peripheral margin with a regressing centre. The aim of this study was to examine the possible heterogeneity within keloids and the involvement of different regions within and around keloid scars in the pathogenesis, using an in vitro keloid scar model. In vitro skin models were constructed from keratinocytes and fibroblasts from normal skin and different regions within and around keloid scars: periphery, centre, and (adjacent) surrounding-normal-skin regions. Additionally, fibroblasts were isolated from the superficial-central and deep-central regions of the keloid and combined with central keratinocytes. All keloid regions showed increased contraction compared to normal skin models, particularly in central regions. Myofibroblasts were present in all keloid regions but were more abundant in models containing central-deep keloid fibroblasts. Secretion of anti-fibrotic HGF and extracellular matrix collagen IV gene expression was reduced in the central deep keloid compared to normal skin. No significant differences between peripheral and central regions within keloids were observed for inflammatory cytokine CCL20, CCL27, CXCL8, IL-6 and IL-18 secretion. Parameters for surrounding-normal-skin showed similarities to both non-lesional normal skin and keloids. In conclusion, a simple but elegant method of culturing keloid-derived keratinocytes and fibroblasts in an organotypic 3D scar model was developed, for the dual purpose of studying the underlying pathology and ultimately testing new therapeutics. In this study, these tissue engineered scar models show that the central keloid region shows a more aggressive keloid scar phenotype than the periphery and that the surrounding-normal-skin also shares certain abnormalities characteristic for keloids.

Hybrid training system-induced myokine secretion in healthy men.

The hybrid training system (HTS) is a special and compact system for effective skeletal muscle training by a combined application of volitional and electrical muscle contraction. Lower limbs' muscle training using HTS has been reported to increase not only muscle strength but also plasma interleukin-6 levels; however, little is known in other cytokines. In this study, we measured 52 cytokines and creatine phosphokinase-MM in the serum of 16 healthy men before and after lower limbs' muscle training by the knee flexion and extension using HTS. Skeletal muscle volume-corrected serum concentrations of cutaneous T-cell-attracting chemokine, erythropoietin, and tumor necrosis factor-related apoptosis-inducing ligand increased immediately after the training. These increased cytokines have been reported to play important roles in wound healing, neuroprotection, and cardiovascular protection.

Acteoside relieves mesangial cell injury by regulating Th22 cell chemotaxis and proliferation in IgA nephropathy.

The existing therapies of IgA nephropathy are unsatisfying. Acteoside, the main component of Rehmannia glutinosa with anti-inflammatory and anti-immune effects, can improve urinary protein excretion and immune disorder. Th22 cell is involved in IgA nephropathy progression. This study was determined to explore the effect of acteoside on mesangial injury underlying Th22 cell disorder in IgA nephropathy. Serum Th22 cells and urine total protein of patients with IgA nephropathy were measured before and after six months treatment of Rehmannia glutinosa acteoside or valsartan. Chemotactic assay and co-culture assay were performed to investigate the effect of acteoside on Th22 cell chemotaxis and differentiation. The expression of CCL20, CCL22 and CCL27 were analyzed. To explore the effect of acteoside on mesangial cell injury induced by inflammation, IL-1, IL-6, TNF-α and TGF-ß1 were tested. Results showed that the proteinuria and Th22 lymphocytosis of patients with IgA nephropathy significantly improved after combination treatment of Rehmannia glutinosa acteoside and valsartan, compared with valsartan monotherapy. In vitro study further demonstrated that acteoside inhibit Th22 cell chemotaxis by suppressing the production of Th22 cell attractive chemokines, i.e., CCL20, CCL22 and CCL27. In addition, acteoside inhibited the Th22 cell proliferation. Co-culture assay proved that acteoside could relieve the overexpression of pro-inflammatory cytokines, and prevent the synthesis of TGF-ß1. TGF-ß1 level in mesangial cells was positively correlated with the Th22 cell. This research demonstrated that acteoside can alleviate mesangial cell inflammatory injury by modulating Th22 lymphocytes chemotaxis and proliferation.

CCR10 activation stimulates the invasion and migration of breast cancer cells through the ERK1/2/MMP-7 signaling pathway.

CCR10, a member of the chemokine receptor subfamily, is overexpressed in several tumors and play a crucial role in cancer development and progression. However, the functions of CCR10 in breast cancer are unknown. Here, we detected the protein levels of CCR10 in breast cancer cells by western blotting, and examined CCR10 expression in breast cancer tissues via immunohistochemical assay. The results showed that CCR10 expression was elevated in breast cancer MCF7, BT-474 and MDA-MB-231 cells. Further, 63 of 89 cases (70.8%) had positive CCR10 staining, and the CCR10 level was closely related to capsular invasion, lymph node metastasis and tumor stage. Moreover, CCL27, the ligand of CCR10, dose-dependently stimulated the invasion and migration of breast cancer cells, and promoted MMP-7 expression and ERK1/2 activation. CCR10 knockdown in breast cancer cells through siRNA transfection attenuated CCL27-induced cell invasiveness, and suppressed MMP-7 expression and ERK1/2 activation. Additionally, blocking the ERK1/2 pathway inhibited the CCL27/CCR10-promoted cell invasion of breast cancer cells. Together, these data suggest that CCL27/CCR10 interaction induces the ERK1/2 pathway, which then increases MMP-7 expresion and subsequently promotes breast cancer cell invasion and migration. Thus, CCR10 may be a key regulator in breast cancer cell invasion and migration.

High CCL27 immunoreactivity in 'supratumoral' epidermis correlates with better prognosis in patients with cutaneous malignant melanoma.

AIMS: It has been proposed that the expression of chemokines and chemokine receptors by melanoma cells may have a role in tumour immune escape. Chemokine CCL27 is reported to be expressed specifically on the epidermal keratinocytes. The implication of CCL27 in cutaneous melanomas is currently unresolved. It has been suggested that CCL27 expression in melanomas can induce antitumoral immunity, and that CCL27 may suppress tumour growth probably due to the local lymphocyte recruitment. METHODS: We studied CCL27 chemokine expression in three different concentric epidermal areas covering the primary cutaneous melanoma in patients with a long clinical follow-up. Our study included 91 cases of primary melanomas of the skin diagnosed during the 10-year period 1992-2002, and a minimum clinical follow-up of 10 years. RESULTS: We evaluated three different concentric and easily reproducible areas in the epidermis: the area covering melanoma (which we called 'supratumoral'), the area adjacent to the tumour ('peritumoral') and the most peripheral epidermal area ('peripheral'). Only CCL27 expression in supratumoral epidermis correlated with clinical outcome. CONCLUSIONS: Our study showed that a higher immunostaining of CCL27 in supratumoral epidermis is associated with longer progression-free interval and melanoma-specific survival.

Ionizing radiation promotes CCL27 secretion from keratinocytes through the cross talk between TNF-α and ROS.

The skin-associated chemokine CCL27 and its receptor CCR10 mediate the immune response of skin-homing T cells. The CCL27 secreted from keratinocytes was reportedly involved in inflammatory skin diseases such as atopic dermatitis, contact dermatitis, and psoriasis. However, whether ionizing radiation increases the levels of CCL27 secretion still remains unclear. In HaCaT cells, a human keratinocyte cell line, CCL27 secretion was markedly increased after X-ray irradiation. We further found that irradiation boosted the generation of reactive oxygen species (ROS), which was concomitant with the release of tumor necrosis factor-alpha (TNF-α). Moreover, alteration of ROS in irradiated HaCaT cells correlated with TNF-α secretion, indicating a positive loop of TNF-α secretion and ROS generation. This positive loop regulated the secretion of CCL27 from irradiated cells. We therefore concluded that the cross talk between TNF-α and ROS after keratinocytes was exposed to radiation, triggered CCL27 secretion for subsequent inflammation response.

CCR10/CCL27 crosstalk contributes to failure of proteasome-inhibitors in multiple myeloma.

The bone marrow microenvironment plays a decisive role in multiple myeloma progression and drug resistance. Chemokines are soluble mediators of cell migration, proliferation and survival and essentially modulate tumor progression and drug resistance. Here we investigated bone marrow-derived chemokines of naive and therapy-refractory myeloma patients and discovered that high levels of the chemokine CCL27, known so far for its role in skin inflammatory processes, correlated with worse overall survival of the patients. In addition, chemokine levels were significantly higher in samples from patients who became refractory to bortezomib at first line treatment compared to resistance at later treatment lines.In vitro as well as in an in vivo model we could show that CCL27 triggers bortezomib-resistance of myeloma cells. This effect was strictly dependent on the expression of the respective receptor, CCR10, on stroma cells and involved the modulation of IL-10 expression, activation of myeloma survival pathways, and modulation of proteasomal activity. Drug resistance could be totally reversed by blocking CCR10 by siRNA as well as blocking IL-10 and its receptor.From our data we suggest that blocking the CCR10/CCL27/IL-10 myeloma-stroma crosstalk is a novel therapeutic target that could be especially relevant in early refractory myeloma patients.

Gingiva Equivalents Secrete Negligible Amounts of Key Chemokines Involved in Langerhans Cell Migration Compared to Skin Equivalents.

Both oral mucosa and skin have the capacity to maintain immune homeostasis or regulate immune responses upon environmental assault. Whereas much is known about key innate immune events in skin, little is known about oral mucosa. Comparative studies are limited due to the scarce supply of oral mucosa for ex vivo studies. Therefore, we used organotypic tissue equivalents (reconstructed epithelium on fibroblast-populated collagen hydrogel) to study cross talk between cells. Oral mucosa and skin equivalents were compared regarding secretion of cytokines and chemokines involved in LC migration and general inflammation. Basal secretion, representative of homeostasis, and also secretion after stimulation with TNFα, an allergen (cinnamaldehyde), or an irritant (SDS) were assessed. We found that proinflammatory IL-18 and chemokines CCL2, CCL20, and CXCL12, all involved in LC migration, were predominantly secreted by skin as compared to gingiva. Furthermore, CCL27 was predominantly secreted by skin whereas CCL28 was predominantly secreted by gingiva. In contrast, general inflammatory cytokines IL-6 and CXCL8 were secreted similarly by skin and gingiva. These results indicate that the cytokines and chemokines triggering innate immunity and LC migration are different in skin and gingiva. This differential regulation should be figured into novel therapy or vaccination strategies in the context of skin versus mucosa.

Proposing the use of dental pulp stem cells as a suitable biological model of neurofibromatosis type 1.

PURPOSE: This study aims to propose the dental pulp stem cells (DPSCs) as a model for studying two features related to neurofibromatosis type 1 (NF1), i.e. augmented proliferative capacity and altered osteogenic differentiation. METHODS: We isolated a DPSC from the pulp of deciduous teeth of a 6-year-old NF1 patient and two other healthy children of similar age. Cell proliferation was assayed by counting with a haemocytometer after successive cell re-plating. In order to compare osteogenic differentiation, we used osteoblast-differentiating medium and quantified alizarin stain, which relates to degree of calcification, and evaluated the expression of osteoblastic markers by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The DPSCs isolated from the NF1 patient displayed a greater rate of proliferation when compared to the control cells. Osteogenic differentiation occurred as expected for both NF1 and control, which concerned cell morphology and expression of osteoblast marker genes ALP, BMP2, BMP4, OCN and SPP1. However, alizarin staining denoted a markedly lower calcification level in the cells from the NF1-diagnosed child, considering that less calcium deposits were visualized under light microscopy and a smaller amount of alizarin could be quantified by spectrophotometry after extraction from the stained cells. CONCLUSION: DPSCs seem to be useful as a model for studying NF1 and predicting prognosis of patients, since their in vitro behaviour seems to mimic at least two features of this disorder: higher tendency to develop bone abnormalities and neoplastic cell proliferation.

Microarray analysis of gene expression by microdissected epidermis and dermis in mycosis fungoides and adult T-cell leukemia/lymphoma.

The characteristic histopathological feature of mycosis fungoides (MF) and adult T-cell leukemia/lymphoma (ATLL) is epidermotropism. To identify the mechanism for epidermotropism of lymphoma cells, total RNAs were obtained from skin biopsies of epidermis and dermis of MF and ATLL patients by means of laser capture microdissection, and used for subsequent complementary DNA (cDNA) microarray experiments. This procedure has made it possible for us to observe and evaluate the regional environment of MF and ATLL. Hierarchical cluster analysis revealed that the cDNAs could be clearly differentiated into MF and ATLL. CCL27 was expressed in the dermis generated from keratinocytes, CCR4/CCR6/CCR7/CCR10/cutaneous lymphocyte-associated antigen (CLA) lymphoma cells in the dermis, and CCL21 in the extracellular matrix (stroma). Lymphotoxin (LT) ß and CCL21 expression was significantly higher and that of CCR10 relatively for MF, while CCR4 and CLA expression was relatively higher for ATLL. In the epithelium, keratinocytes expressed CCL20/CCL27, and lymphoma cells CCR4/CCR6/CCR10, while CCR4, CCR6, CCL20 and CCL27 expression was relatively higher for ATLL than MF. The dermis of MF, but not that of ATLL, showed correlation between CCR7 and CCL21. These findings support the suggestion that chemokines and chemokine receptors are involved in the pathogenesis of MF and ATLL, indicate that cutaneous homing seems to be different for MF and ATLL, and point to the possibility that cutaneous T-cell lymphomas originate in regulatory T cells, especially in the case of ATLL.

Differential response of human adipose tissue-derived mesenchymal stem cells, dermal fibroblasts, and keratinocytes to burn wound exudates: potential role of skin-specific chemokine CCL27.

Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell-cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006-9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation factors. Our findings have implications for the choice of cell type (ASC or dermal fibroblast) to be used in regenerative medicine strategies and indicate the importance of taking into account interactions with the wound bed when developing advanced therapies for difficult-to-close cutaneous wounds.

Analysis of chemotactic molecules in bone marrow-derived mesenchymal stem cells and the skin: Ccl27-Ccr10 axis as a basis for targeting to cutaneous tissues.

BACKGROUND AIMS: Adult stem cells produce a plethora of extracellular matrix molecules and have a high potential as cell-based therapeutics for connective tissue disorders of the skin. However, the primary challenge of the stem cell-based approach is associated with the inefficient homing of systemically infused stem cells to the skin. METHODS: We examined chemotactic mechanisms that govern directional migration of mesenchymal stem cells (MSCs) into the skin by conducting a comprehensive expression analysis of chemotactic molecules in MSCs and defined cutaneous tissues from normal and hereditary epidermolysis bullosa (EB)-affected skin. RESULTS: Analysis of chemokine receptors in short-term and long-term MSC cultures showed tissue culture-dependent expression of several receptors. Assessment of epidermis-derived and dermis-derived chemokines showed that most chemotactic signals that originate from the skin preferentially recruit different sets of leukocytes rather than MSCs. Analysis of the chemotactic molecules derived from EB-affected non-blistered skin showed only minor changes in expression of selected chemokines and receptors. Nevertheless, the data allowed us to define the Ccl27-Ccr10 chemotactic axis as the most potent for the recruitment of MSCs to the skin. Our in vivo analysis demonstrated that uniform expression of Ccr10 on MSCs and alteration of Ccl27 level in the skin enhance extravasation of stem cells from circulation and facilitate their migration within cutaneous tissue. CONCLUSIONS: Collectively, our study provides a comprehensive analysis of chemotactic signals in normal and EB-affected skin and proof-of-concept data demonstrating that alteration of the chemotactic pathways can enhance skin homing of the therapeutic stem cells.

Presence of CTAK/CCL27, MCP-3/CCL7 and LIF in human colostrum and breast milk.

Human colostrum and breast milk are known to contain high levels of cytokines and chemokines, which are thought to contribute to the development of the newborn. The aim of this study was to investigate the difference in the presence and levels of 21 soluble cytokines and chemokines in paired samples of human colostrum (day 2 after delivery) and breast milk (day 4-5 after delivery) by using the multiplex technology. Of the 21 cytokine investigated in 10 pairs of samples, only ß-NGF was absent in both colostrum and milk, while INF-α2, SCF and TNF-ß were present in colostrum but not in human milk. As a general rule, colostrum contained higher concentrations of cytokines and chemokines with respect to breast milk. The majority of cytokines, detected in colostrum alone or in colostrum and human milk (IL-1α, IL-2Rα, IL-3, IL-16, IL-18, GRO-α, HGF, IFN-α2, M-CSF, MIF, MIG, TNF-ß, SDF-1α, TRAIL) have been described in previous studies, while for the first time we describe the presence of additional cytokines either in colostrum alone (SCF) or in both colostrum and breast milk (CTAK/CCL27, MCP-3/CCL7, LIF). Our data confirm and expand previous studies showing that some cytokines/chemokines, which might contribute to the development of the gastro-intestinal and nervous systems, are overexpressed in human colostrum and breast milk, and might contribute to the development of these systems.

Expression of haematogenous and lymphogenous chemokine receptors and their ligands on uveal melanoma in association with liver metastasis.

PURPOSE: Chemokine receptors and their ligands are involved in a number of cell processes, including normal cell trafficking as well as metastasis in cancer. During metastasis, they are thought to play a role in determining cancer cell distribution and target organs. The aim of this study was to examine the expression of the chemokine receptors CXCR4, CCR7 and CCR10 as well as their respective chemokine ligands (CXCL12, CCL19, CCL27) in human uveal melanomas. METHODS: Seventy formalin-fixed paraffin-embedded uveal melanoma specimens from patients treated in 1996-1997 were examined using immunohistochemistry and evaluated using an immune reactive score (IRS). RESULTS: The chemokine receptors CXCR4, CCR7 and CCR10 were primarily expressed in the cytoplasm of uveal melanoma cells, with CXCR4 (average IRS 8.2) and CCR7 (average IRS 5.7) showing the strongest expression, respectively. The chemokine ligand CCL19 demonstrated a moderate expression (average IRS 5.3), whereas the expression of receptor CCR10 (average IRS of 3.4), ligand CCL27 (average IRS 2.5) and ligand CXCL12 (average IRS 0.6) by uveal melanoma cells was low. A significant association between liver metastases and chemokine expression was found for CCR7 expression (p = 0.037) only. Comparison of liver metastasis and choroid uveal melanoma (35.3%, n = 12 of 34) versus ciliary body involvement (72.7%, n = 8 of 11) was significant (p = 0.030). CONCLUSION: Chemokine receptors are more strongly expressed on uveal melanoma cells than their ligands. Our results show new aspects of the metastatic process in uveal melanoma.

Serum and tissue CTACK/CCL27 chemokine levels in early mycosis fungoides may be correlated with disease-free survival following treatment with interferon alfa and psoralen plus ultraviolet A therapy.

BACKGROUND: Neoplastic T-cell recruitment into the skin is a critical step in the pathogenesis of mycosis fungoides (MF), and the cutaneous T-cell attracting chemokine, CTACK/CCL27, might be involved. OBJECTIVES: To investigate the clinical and prognostic significance of CTACK/CCL27 levels in patients with early-stage MF. METHODS: Serum samples and skin biopsy specimens were collected from 15 patients at the time of diagnosis and after the end of treatment with psoralen plus ultraviolet A/interferon alfa-2b combination therapy. Serum samples were also collected from 20 healthy donors as controls. CTACK/CCL27 serum levels were analysed by enzyme-linked immunosorbent assays. CTACK/CCL27 tissue expression was determined by immunohistochemistry on skin biopsy specimens taken at diagnosis and after therapy. Event-free survival was taken as the primary clinical outcome. RESULTS: In patients with MF at diagnosis, CTACK/CCL27 serum levels were not significantly different from healthy controls, whereas CTACK/CCL27 expression in the skin was increased in 87% of cases compared with normal controls. After therapy, all patients obtained a clinical complete remission, serum levels did not change significantly and tissue expression remained abnormal in 80% of patients, even if complete histological remission was recorded. Serum levels were not significantly different in cases with different intensity of cutaneous immunostaining. Eight patients experienced a relapse: the combination of high CTACK/CCL27 levels both in sera and skin increased the probability of experiencing an event at 51 months from 36% to 83%. CONCLUSIONS: Our data seem to indicate that CTACK/CCL27 levels in skin and sera after therapy might be correlated with risk of recurrence.