Improving the diagnosis of high grade and stage bladder cancer by detecting increased urinary calprotectin expression in tumor tissue and tumor-associated inflammatory response.

Purpose: To investigate whether measurement of urinary calprotectin can serve as a biomarker in the diagnosis of primary bladder cancer and to confirm its diagnostic role in determining high grade and stage disease. Materials and Methods: Urinary calprotectin was measured in spot urine samples from patients with primary bladder cancer and control subjects. To confirm levels in urine, tissue samples were also obtained from bladder tumor and healthy trigone of bladder by transurethral resection in both groups. Finally, calprotectin levels in tissue and urine of the patients and control subjects were compared and their diagnostic potential was investigated in high grade and stage bladder cancers. Results: Of 82 participants, 52 were patients with bladder cancer and 30 were control subjects. The two groups were comparable in terms of age, smoking status, and comorbidities. Tissue and urinary calprotectin levels were significantly higher in the bladder cancer group. In subgroup analyses, urinary calprotectin levels were significantly higher in patients with high-grade, muscle-invasive tumors. After receiver operating characteristic analyses, the sensitivity and specificity of urinary calprotectin was 100% and 96.7%, respectively, in the diagnosis of primary bladder cancer. High grade and stage bladder cancers were detected with sensitivity and specificity of 70% and 74.2%, and 80% and 84.8%, respectively. Conclusions: Urinary calprotectin may be a valuable parameter in the diagnosis of primary bladder cancer with high sensitivity and specificity. Furthermore, it may be useful in the prediction of high grade and stage disease. However, more investigations are needed.

IL-17a and IL-22 Induce Expression of Antimicrobials in Gastrointestinal Epithelial Cells and May Contribute to Epithelial Cell Defense against Helicobacter pylori.

Helicobacter pylori colonization of the human stomach can lead to adverse clinical outcomes including gastritis, peptic ulcers, or gastric cancer. Current data suggest that in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization. Specifically, CD4+ T cell responses impact the pathology elicited in response to H. pylori. Because gastritis is believed to be the initiating host response to more detrimental pathological outcomes, there has been a significant interest in pro-inflammatory T cell cytokines, including the cytokines produced by T helper 17 cells. Th17 cells produce IL-17A, IL-17F, IL-21 and IL-22. While these cytokines have been linked to inflammation, IL-17A and IL-22 are also associated with anti-microbial responses and control of bacterial colonization. The goal of this research was to determine the role of IL-22 in activation of antimicrobial responses in models of H. pylori infection using human gastric epithelial cell lines and the mouse model of H. pylori infection. Our data indicate that IL-17A and IL-22 work synergistically to induce antimicrobials and chemokines such as IL-8, components of calprotectin (CP), lipocalin (LCN) and some ß-defensins in both human and primary mouse gastric epithelial cells (GEC) and gastroids. Moreover, IL-22 and IL-17A-activated GECs were capable of inhibiting growth of H. pylori in vitro. While antimicrobials were activated by IL-17A and IL-22 in vitro, using a mouse model of H. pylori infection, the data herein indicate that IL-22 deficiency alone does not render mice more susceptible to infection, change their antimicrobial gene transcription, or significantly change their inflammatory response.

Low dietary iron intake restrains the intestinal inflammatory response and pathology of enteric infection by food-borne bacterial pathogens.

Orally administrated iron is suspected to increase susceptibility to enteric infections among children in infection endemic regions. Here we investigated the effect of dietary iron on the pathology and local immune responses in intestinal infection models. Mice were held on iron-deficient, normal iron, or high iron diets and after 2 weeks they were orally challenged with the pathogen Citrobacter rodentium. Microbiome analysis by pyrosequencing revealed profound iron- and infection-induced shifts in microbiota composition. Fecal levels of the innate defensive molecules and markers of inflammation lipocalin-2 and calprotectin were not influenced by dietary iron intervention alone, but were markedly lower in mice on the iron-deficient diet after infection. Next, mice on the iron-deficient diet tended to gain more weight and to have a lower grade of colon pathology. Furthermore, survival of the nematode Caenorhabditis elegans infected with Salmonella enterica serovar Typhimurium was prolonged after iron deprivation. Together, these data show that iron limitation restricts disease pathology upon bacterial infection. However, our data also showed decreased intestinal inflammatory responses of mice fed on high iron diets. Thus additionally, our study indicates that the effects of iron on processes at the intestinal host-pathogen interface may highly depend on host iron status, immune status, and gut microbiota composition.

The pro-apoptotic and pro-inflammatory effects of calprotectin on human periodontal ligament cells.

Calprotectin, a heterodimer of S100A8 and S100A9 subunits, is associated with inflammatory disorders such as rheumatoid arthritis and cystic fibrosis. Although calprotectin levels are increased significantly in the gingival crevicular fluid (GCF) of periodontitis patients, its effects on periodontal ligament cells (PDLCs) remain largely unknown. The aim of this study was to evaluate calprotectin levels in the GCF of generalized aggressive periodontitis (AgP) patients and to investigate the effects of recombinant human calprotectin (rhS100A8/A9) and its subunits (rhS100A8 and rhS100A9) in PDLCs. Both the concentration and amount of crevicular calprotectin were significantly higher in the AgP group compared with healthy controls. In addition, the GCF calprotectin levels were correlated positively with clinical periodontal parameters including bleeding index, probing depth, and clinical attachment loss. rhS100A8/A9 promoted cell apoptosis, whereas rhS100A8 and rhS100A9 individually exerted little effect on apoptosis in PDLCs. rhS100A9 and rhS100A8/A9 increased the activation of nuclear factor-κB (NF-κB) by promoting the nuclear translocation of p65 in PDLCs, subsequently inducing expression of the pro-inflammatory cytokines IL-6, IL-8, TNFα, and COX2. Treatment with an NF-κB inhibitor partially reversed the rhS100A9- and rhS100A8/A9-induced upregulation of the pro-inflammatory cytokines. rhS100A9, and not rhS100A8, was mainly responsible for the pro-inflammatory role of calprotectin. Collectively, our results suggest that calprotectin promotes apoptosis and the inflammatory response in PDLCs via rhS100A9. These findings might help identify novel treatments for periodontitis.

Gingival crevicular fluid calprotectin, osteocalcin and cross-linked N-terminal telopeptid levels in health and different periodontal diseases.

AIM: The aim of the present study was to investigate gingival crevicular fluid (GCF) calprotectin, osteocalcin and cross-linked N-terminal telopeptide (NTx) levels in health along with different periodontal diseases. MATERIAL AND METHODS: Twenty chronic periodontitis (CP), 20 generalized aggressive periodontitis (G-AgP), 20 gingivitis and 20 healthy subjects were included. Probing depth, clinical attachment level, plaque index and papillary bleeding index was recorded. GCF calprotectin, osteocalcin and NTx levels were analyzed by enzyme-linked immunosorbent assay (ELISA). RESULTS: CP, G-AgP and gingivitis groups had higher GCF calprotectin total amount compared to healthy subjects (p< 0.008). CP and G-AgP groups had similar, but higher levels compared to gingivitis groups (p< 0.008). CP and G-AgP groups had lower GCF osteocalcin total amount compared to gingivitis and healthy groups (p< 0.008). CP group had higher GCF NTx but lower osteocalcin total amount and osteocalcin/NTx ratio than the G-AgP group (p< 0.008). CONCLUSIONS: Our results suggest that elevated GCF calprotectin levels play a role as a reliable inflammatory marker in the pathogenesis of periodontal disease. Fluctuating GCF levels of osteocalcin and NTx might point out to the abnormal bone turnover in periodontitis. Our data document for the first time the role of NTx in the pathogenesis of different periodontal diseases.

Local calprotectin production in colorectal cancer and polyps--active neutrophil recruitment in carcinogenesis.

AIM: The aim of this study is to examine the expression level and localization of calprotectin in cancer tissue, tumor-adjacent mucosa, and polyps in colonic biopsies. Calprotection expression was correlated with neutrophil infiltration, markers of bacteremia, and systemic inflammation. MATERIALS AND METHODS: Patients with colorectal cancer (n = 28) and adenoma (n = 38) were compared with healthy controls (n = 33). Calprotectin expression levels were measured by ELISA, and its localization was visualized by immunohistochemistry and correlated with the degree of neutrophil infiltration (visualized by Esterase staining). The expression of tumor necrosis factor (TNF)-alpha, procalcitonin, endotoxemia, carcinoembryonic antigen (CEA), and C-reactive protein was also investigated. RESULTS: Mucosal calprotectin was expressed in significantly higher concentrations in carcinoma (94.2 ± 31.2 ng/mg total protein) and adenoma (122.8 ± 60.3 ng/mg total protein) in comparison with mucosal biopsies from healthy controls (20.4 ± 5.4 ng/mg total protein), tumor-adjacent mucosa from patients with colorectal carcinoma (21.6 ± 5.1 ng/mg total protein), and adenoma (45 ± 14.6 ng/mg total protein, all p < 0.05). Immunohistochemistry showed calprotectin reactivity mainly in granulocytes and macrophages with only singular reactive epithelial cells. Positive staining (quantified by the number of positive cells per square millimeter) was markedly increased in carcinoma tissue (85 ± 21.5) and in adenoma (67.5 ± 20) as compared with tumor-adjacent epithelia (18.8 ± 4.3, p = 0.0007, p = 0.003, respectively), and there was a highly significant correlation, r = 0.89, p = 0.001) between calprotectin staining and neutrophil infiltration. No significant differences were found in the systemic levels of TNF-alpha, procalcitonin, and endotoxemia, whereas CEA and C-reactive protein levels were significantly higher in the cancer group (p < 0.05). CONCLUSION: Our results support the evidence that increased calprotectin expression is an early step in the neoplastic transformation during colorectal carcinogenesis. Moreover, its expression is closely related to an inflammatory response and points out a possible biological link between inflammation and neoplastic transformation in colorectal cancer.

Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.

BACKGROUND: Decreased epithelial expression of mRNA for S100A7 (psoriasin) and S100A8/A9 (calprotectin) has been reported in patients with chronic rhinosinusitis (CRS). OBJECTIVES: We sought to assess whether the expression of S100 proteins is also altered in the sinonasal cavity of patients with CRS. METHODS: We determined levels of S100 proteins in nasal lavage fluid and sinonasal tissue extracts from patients with CRS using ELISA and immunohistochemical analysis of nasal polyp tissue from patients with CRS with nasal polyps and uncinate tissue from healthy control subjects, patients with CRSsNP, and patients with CRSwNP. RESULTS: Expression levels of S100 proteins were decreased compared with those seen in control subjects in nasal lavage fluid from both CRS groups (P < .05). Similarly, tissue expression of these proteins assessed by means of immunohistochemistry demonstrated clear reductions, primarily in the epithelial lining. Interestingly, levels of calprotectin were increased in nasal polyp tissue despite lower levels in lavage fluid. Levels of calprotectin in nasal tissues were correlated with levels of neutrophils, as assessed by means of quantification of neutrophil elastase. CONCLUSIONS: Several S100 proteins are in the epidermal differentiation complex of genes and have been demonstrated to play a role in maintenance of barrier function and formation of an antimicrobial shield. We demonstrate significantly decreased levels of expression of S100 proteins in the epithelium of patients with CRS, which might lead to diminished innate immune responses and barrier function. Increased levels of calprotectin in nasal polyp tissue might reflect neutrophil recruitment and a compensatory mechanism. Future studies will be important to determine whether reduced levels of S100 proteins lead to decreased antimicrobial responses in the upper airways and sinuses and whether this reduction plays a causative role in CRS pathogenesis and susceptibility to infectious disease.

Absence of L1 in pancreatic masses distinguishes adenocarcinomas from poorly differentiated neuroendocrine carcinomas.

BACKGROUND: Pancreatic adenocarcinoma is a tumor with fatal outcome. Cell adhesion molecules, such as L1 (CD171), have an essential function in tumor progression. L1 has been shown to be specifically expressed in poorly differentiated neuroendocrine carcinomas of the pancreas. The aim of this study was to determine the expression of L1 in pancreatic adenocarcinomas to evaluate whether L1 might differentiate between pancreatic carcinomas of neuroendocrine and ductal origin. MATERIALS AND METHODS: L1 expression was retrospectively analyzed in 111 cases of pancreatic adenocarcinomas by immunohistochemistry on paraffin sections of primary tumors. Staining was performed by the peroxidase technique with monoclonal antibody against human L1. All tumors were classified according to the most recent TNM classification. RESULTS: The focal expression of L1 was detected in 2 (2%) out of 111 pancreatic carcinomas only, the remaining 109 (98%) being L1-negative. No expression was found in acinar or ductal cells of normal pancreatic tissue. CONCLUSION: Our data suggest that L1 is expressed in few cases of pancreatic ductal adenocarcinoma. Since L1 was previously found to be expressed specifically in neuroendocrine pancreatic carcinomas, its absence in unclear pancreatic masses might hint at a ductal origin for a malignant pancreatic tumor.

Myeloid-related protein (MRP)8/14 (calprotectin) and its subunits MRP8 and MRP14 in plaque-induced early gingival inflammation.

BACKGROUND: The inflammatory myeloid-related protein, MRP8/14, also called calprotectin, and its subunits MRP8 and MRP14 have been detected and identified recently in gingival crevicular fluid (GCF). It has been suggested that the type and phase of inflammation can be discriminated on the basis of differences in the expression of calprotectin and its subunits, released during activation and/or death of granulocytes and monocytes. The purpose of this study was to quantify calprotectin and its subunits (MRPs) simultaneously in the GCF during the initial phase of experimentally induced gingivitis, and to examine their inter- and intra-individual variations. MATERIAL AND METHODS: Fifteen healthy non-smoking subjects, aged 18-30, were involved in this study. An initial hygiene phase (days -11 to 0) was followed by 10 days of undisturbed plaque accumulation. At days -11, -3, 0, 10, 11, clinical parameters were recorded and GCF samples collected with Durapore strips from 12 sites in each subject. Quantitative analyses of total proteins, MRP8/14, MRP14 and MRP8 were performed by ELISA procedures. RESULTS: During the experimental phase with no oral hygiene (days 0-10), the clinical parameters Plaque Index, Gingival Index (GI) and bleeding on probing increased as expected, confirming that plaque accumulation leads to gingival inflammation. Levels of the MRPs were individually variable. They increased with plaque accumulation in one-half of the subjects, and decreased in the other subjects. The levels of MRP8/14 and MRP14 at subject recruitment (day -11) could predict a significant part of the GI at day 10. Only minute amounts of the subunits MRP8 and MRP14 were detected in comparison with the complex MRP8/14 throughout the experiment. Considerable variations were noted among sites within subjects. CONCLUSION: The expression of calprotectin in the early phase of experimental gingivitis is variable between subjects, and two groups of subjects can be differentiated according to their response patterns. Clinical parameters at the very first visit (day -11) seemed to be different in the two response groups. The results of the present investigation indicate that the inflammatory response to plaque accumulation depends on the initial status of the subjects, which may not be leveled out by the introduction of perfect oral hygiene. Whether these patterns reflect a different susceptibility to periodontal diseases remains to be determined.

Immunohistochemical localization of macrophage CD68+, HLA-DR+, L1+ and CD44+ subsets in uterine endometrium during different phases of menstrual cycle.

Different tissue macrophage subsets were immunohistochemically examined in normal endometrial samples collected from proliferative (n=4), peri-ovulatory (n=6) and secretory (n=8) phases of menstrual cycles in women. The different macrophage subsets, namely CD68 (pan macrophage marker), CD44 (transmembrane adhesion molecule), HLA-DR (transmembrane heterodimeric protein involved in antigen presentation) and L1 (calprotectin)-positive cells, as well as, CD45 (common leucocytic antigen)-positive cells were examined on the basis of immunohistochemical staining, and areas of immunoprecipitation were analyzed morphometrically using computer-assisted video imaging system. The stage-specific distribution of receptors for estrogen (ER) and progesterone (PR) in endometrial cells were examined and morphometrically analyzed. There was an increase in the number of CD45+ cells (P < 0.01) and CD68+ cells (P < 0.05) in secretory phase endometrium compared with proliferative and peri-ovulatory phases. There was no remarkable cycle dependent pattern in HLA-DR+ and L1+ cells. However, there was an increase in CD44 immunopositive area in peri-ovulatory (P < 0.05) and in secretory (P < 0.01) phases of endometrium compared with proliferative phase endometrium. A higher (P < 0.01) degree of immunopositivity for ER was observed during peri-ovulatory phase, and for PR, during peri-ovulatory (P < 0.05) and secretory (P < 0.01) phases compared with proliferative phase of cycle. Positive correlations between areas occupied by (i) CD68+ cells and PR (P < 0.01), (ii) HLA-DR+ and L1+ cells (P < 0.05), (iii) CD45+ and CD68+ cells (P < 0.01), (iv) CD45+ and L1+ cells (P < 0.05), and (v) PR and L1+ cells (P < 0.05) were obtained. It appears that the recruitment of different macrophage subsets in human endometrium involves a complex set of endocrine and paracrine factors.

Changes in L1 and NCAM expression in the rat suprachiasmatic nucleus during growth and after orbital enucleation.

Mammals possess a master circadian clock in the hypothalamic suprachiasmatic nucleus (SCN). In order to clarify the roles of the L1 adhesion molecule (L1) and neural cell adhesion molecule (NCAM), both members of the immunoglobulin superfamily, in the organization of the clock core, changes in the expression of these molecules in the SCN during the growth of rats were examined by immunohistochemistry. On postnatal day 7, L1 and NCAM were chiefly expressed in the region surrounding the SCN, but not in the SCN itself. In subsequent weeks, however, expression of both molecules shifted predominately to the SCN. This change seemed to parallel immunoreactivity increases in the SCN of synaptotagmin, a synapse marker, and of phosphotyrosine, a possible factor in the photic entrainment of the SCN clock. To further elucidate the roles of the L1 and NCAM adhesion molecules in the formation and maintenance of retinal neural projection into the SCN, the effects of orbital enucleation on their expression in the SCN were examined. L1 expression decreased on days 1 and 2 after the operation, in parallel with reductions in the tyrosine phosphorylation of several proteins, but recovered to the control level by the second week. In contrast, the expression of NCAM showed little change following orbital enucleation. These results suggest that L1 and NCAM are involved in the morphological organization of the SCN during the developmental stage, and that expression of L1 also contributes to the formation of the SCN network in a manner that is dependent on the retinal neural input to it.

Calprotectin (S100A8/S100A9), an inflammatory protein complex from neutrophils with a broad apoptosis-inducing activity.

Calprotectin, a complex of two calcium-binding proteins that belong to the S100 protein family, is abundant in the cytosolic fraction of neutrophils. A high level of calprotectin reportedly exists in extracellular fluid during various inflammatory conditions, such as rheumatoid arthritis, cystic fibrosis and abscesses. However, the exact biological role(s) of the factor is now under investigation. We recently observed that neutrophils contain a factor that shows growth-inhibitory and apoptosis-inducing activities against various cell types including tumor cells and normal fibroblasts, and we identified that factor as calprotectin. The findings suggest that calprotectin exerts a regulatory activity in inflammatory processes through its effect on the survival or growth states of cells participating in the inflammatory reaction. It is also possible that calprotectin, at a high concentration, might have a deleterious effect on fibroblasts and influence the recovery of inflammatory tissue. Therefore, the protein factor may be a new drug target to control inflammatory reactions. We found that a few of the Amaryllidaceae alkaloids effectively inhibited the growth-inhibitory and apoptosis-inducing activities of calprotectin. In this article, we focus on the biological functions of calprotectin in extracellular fluids, focusing on its apoptosis-inducing activity.

Effect of Porphyromonas gingivalis lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1beta on calprotectin release in human monocytes.

BACKGROUND: Calprotectin is a major cytosolic protein of monocytes, granulocytes, and epithelial cells. It is known that calprotectin is released in inflammatory tissues and detected in gingival crevicular fluid (GCF) of periodontitis patients at high levels. The origin of calprotectin in GCF and its regulation in periodontal disease are unknown. In this study, we investigated the distribution of calprotectin in gingival tissue with inflammation and the induction of calprotectin release from human monocytes by lipopolysaccharide of Porphyromonas gingivalis (P-LPS), tumor necrosis factor-alpha (TNF-alpha), or interleukin-1beta (IL-1beta). METHODS: Gingival tissues were obtained from a healthy donor and a periodontitis patient and calprotectin in gingival tissues was examined by immunohistochemical staining. Monocytes were isolated from the peripheral blood of healthy donors and cultured with P-LPS, TNF-alpha or IL-1beta for 30 minutes to 4 hours. The content of calprotectin in the cell and medium fractions was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Calprotectin was markedly detected at the epithelial and adjacent connective tissue with many inflammatory cells in the gingival tissue from the periodontitis patient. P-LPS increased calprotectin release from monocytes to the maximum level after 30 minutes of treatment and its level was elevated to about 2- to 3-fold of the control level in a dose-dependent manner (1 to 1,000 ng/ml). When the effect of TNF-alpha and IL-1beta on calprotectin release was investigated, calprotectin release significantly increased to about 2.2- and 1.5-fold that of the control level, respectively. CONCLUSION: These results demonstrate that calprotectin release from monocytes is induced by P-LPS, TNF-alpha, and IL-1beta, which in turn, cause and aggravate periodontal disease.

Keratinocyte injury in drug-induced toxic epidermal necrolysis: Simultaneous but distinct topographic expression of CD95R and calprotectin.

Keratinocyte injury in drug-induced toxic epidermal necrolysis (TEN) is attributed to a dysregulation in the complex apoptotic machinery. This study was performed to investigate some aspects of the apoptotic pathomechanism involving calprotectin and the CD95 receptor (CD95R, FasR) in TEN. The expression of these two molecules corresponds to calcium-dependent and calcium-independent processes, respectively. Biopsies were collected from blistering skin in 21 TEN patients and from clinically uninvolved skin in 16 of these patients. Immunohistochemistry was performed using specific antibodies directed to CD95R and calprotectin. Half (8/16) of the biopsy specimens taken from clinically uninvolved sites showed the expression of calprotectin throughout the epidermis or restricted to the suprabasal layers. In these samples, a strong CD95R immunoreactivity was often restricted to the basal layer (8/16). Calprotectin was present in all biopsies of bullous skin, especially in suprabasal layers (15/21) or throughout the epidermis (6/21). By contrast, the prominent expression of CD95R was confined almost exclusively to the basal layer (15/21), and more rarely throughout the epidermis (2/21), and it remained sometimes unexpressed (4/21). A clear-cut boundary was often present between the areas labeled by the two antibodies with exceptional overlap between them. The simultaneous expression of calprotectin and CD95R in TEN at distinct levels of the epidermis indicates that the pathomechanism leading to keratinocyte death does not belong to a single process in TEN. Their expression in clinically uninvolved skin suggests that these processes are early and widespread events in TEN.