Potential Utility of Fecal Calprotectin in Discriminating Colorectal Polyps From Other Major Etiologies in Children Presenting With Isolated Hematochezia.

BACKGROUND: Colorectal polyps are the most common cause of isolated hematochezia in children, which requires a colonoscopy for diagnosis. We aimed to investigate the potential utility of fecal calprotectin (FC) in assessing colorectal polyps detected by colonoscopy among children presenting with isolated hematochezia. METHODS: Pediatric patients of the age of < 18 years who had undergone both colonoscopy and FC tests for isolated hematochezia from June 2016 to May 2020 were included in the present multicenter, retrospective, cross-sectional study. Comparative analysis was conducted between major causes of isolated hematochezia and FC cut-offs for discriminating colorectal polyps were explored. RESULTS: A total 127 patients were included. Thirty-five patients (27.6%) had colorectal polyps, followed by anal fissure (14.2%), ulcerative colitis (UC; 12.6%), and others. A significant difference in FC levels was observed between patients with colorectal polyps (median, 278.7 mg/kg), anal fissures (median, 42.2 mg/kg), and UC (median, 981 mg/kg) (P < 0.001). According to receiver operating characteristic curve analysis, among patients diagnosed with colorectal polyp or anal fissure, the most accurate FC cut-off for discriminating colorectal polyps from anal fissures on colonoscopy was 225 mg/kg (sensitivity, 59.4%; specificity, 94.4%; positive predictive value [PPV], 95.0%; negative predictive value [NPV], 56.7%; area under the curve [AUC], 0.8; 95% confidence interval [CI], 0.678-0.923; P < 0.001), while among patients diagnosed with colorectal polyp or UC, the most accurate FC cut-off for discriminating colorectal polyps from UC on colonoscopy was 879 mg/kg (sensitivity, 81.2%; specificity, 56.2%; PPV, 78.8%; NPV, 60.0%; AUC, 0.687; 95% CI, 0.521-0.852; P < 0.001). CONCLUSION: FC may assist in assessing the cause of lower gastrointestinal tract bleeding in children who present with isolated hematochezia.

In Vitro Inhibitory Effect of Recombinant Human Calprotectin on Nalm6 Leukemia Cell Line.

BACKGROUND & PURPOSE: In evaluating new drugs for the treatment of various types of cancer, investigations have been made to discover a variety of anti-tumor compounds with less side effects on normal cells. Investigations have shown that the heterodimers S100A8 and S100A9 inhibit the enzyme casein kinase 2 and then prevent the activation of the E7 oncoprotein. Therefore, the aim of this study was to evaluate the effect of calprotectin as an antitumor compound on the Nalm6 (B cell precursor leukemia cell line). MATERIALS & METHODS: Transformation of genes encoding S100A8 and S100A9 human, designed in the pQE32 plasmid, was performed by the thermal shock method into E. coli M15 bacteria. After bacterial growth in LB medium, the expression of two S100A8 and S100A9 subunits, the solubility of the protein by SDS-PAGE method was determined. Finally, the S100A8 / A9 complex was equally placed in the microtube. In the next step, the cytotoxic effects of calprotectin produced on the Nalm6 cell line were evaluated using the wst1 test. Then, the apoptosis in these cells was measured using flow cytometry methods with Annexin-V coloration. RESULTS: In the current study, the results showed that the cytotoxic effects of Calprotectin are time and concentration- dependent. Therefore, it can reduce the tumor expression and had a beneficial effect by induced apoptosis in Nalm6 cell line. CONCLUSION: Calprotectin has an anti-tumor effect on the Nalm6 cell line by increasing apoptosis.

Patient-performed extraction of faecal calprotectin.

BACKGROUND: Faecal (f-) calprotectin is a widely used marker for intestinal inflammation. However, extraction procedure is time consuming and cumbersome. The main aim of this study was to evaluate patient-performed extraction of f-calprotectin compared to extraction performed in the laboratory. METHODS: A total of 81 adult patients with an established diagnosis of inflammatory bowel disease provided two samples from the same bowel movement, one conventional faeces sample and one sample with a patient administered extraction device. A laboratory technician extracted the conventional faeces sample with the same extraction device. RESULTS: F-calprotectin results from the laboratory-performed extraction and the patient-performed extraction correlated significantly, with a Spearman rank correlation coefficient of 0.92. Method comparison showed a slope of 1.20 (95% confidence interval 1.08-1.36) with intercept of -0.30 (95% confidence interval -9.00 to 4.62). This demonstrates a small proportional difference between the results from the home extracted samples and the results from the laboratory extracted samples, where the home extracted samples are slightly higher. However, six of the 81 patients had made obvious mistakes in the extraction process and their samples were excluded from the study. CONCLUSIONS: Patient administered extraction of f-calprotectin can be a realistic alternative for selected patients. However, instructions must be very precise to avoid mistakes.

Therapeutic efficacy of pH-dependent release formulation of mesalazine on active ulcerative colitis resistant to time-dependent release formulation: analysis of fecal calprotectin concentration.

PURPOSE: Few reports have compared the clinical efficacy of a pH-dependent release formulation of mesalazine (pH-5-ASA) with a time-dependent release formulation (time-5-ASA). We examined whether pH-5-ASA is effective for active ulcerative colitis (UC) in patients resistant to time-5-ASA. METHODS: We retrospectively and prospectively analyzed the efficacy of pH-5-ASA in mildly to moderately active UC patients in whom time-5-ASA did not successfully induce or maintain remission. The clinical efficacy of pH-5-ASA was assessed by clinical activity index (CAI) before and after switching from time-5-ASA. In addition, the efficacy of pH-5-ASA on mucosal healing (MH) was evaluated in a prospective manner by measuring fecal calprotectin concentration. RESULTS: Thirty patients were analyzed in a retrospective manner. CAI was significantly reduced at both 4 and 8 weeks after switching to pH-5-ASA. In the prospective study (n=14), administration of pH-5-ASA also significantly reduced CAI scores at 4 and 8 weeks in these patients who were resistant to time-5-ASA. In addition, fecal calprotectin concentration was significantly decreased along with improvement in CAI after switching to pH-5-ASA. CONCLUSIONS: Our results suggest that pH-5-ASA has clinical efficacy for mildly to moderately active patients with UC in whom time-5-ASA did not successfully induce or maintain remission.

Calprotectin induces cell death in human prostate cancer cell (LNCaP) through survivin protein alteration.

Calprotectin (CP), an abundant heterodimeric cytosolic protein of neutrophils, conveys a variety of functions such as tumor cell growth arrest and antimicrobial activity. We investigated CP activity and its possible apoptosis-inducing mechanism of action against an antiandrogen therapy-resistance prostate cancer cell line LNCaP. Cell viability and Annexin V FITC assays were performed in order to investigate its cell death activity and apoptosis, respectively. In order to address cell death inducing mechanism(s), immunocytochemistry and immunobloting analysis, reactive oxygen species (ROS) and nitric oxide (NO) measurements were performed. The effective concentration of CP against LNCaP promoting LNCaP cell death was 200 µg/mL. ROS and NO levels of cells remarkably were enhanced following treatment with 50 and 100 µg/mL of CP, respectively. Protein expression of anti-apoptotic protein survivin was significantly decreased after administration of tumor cells with CP. Our data indicate that CP regulates the LNCaP cells viability via survivin-mediated pathway and ROS and NO enhancement. Thus, inhibition of survivin expression, enhancement of ROS and NO level by CP or other similar pharmaceutical agents might be effective in lowering the malignant proliferation of human prostate cancer cells.

Comparison of two immunoassays for measurement of faecal calprotectin in detection of inflammatory bowel disease: (pre)-analytical and diagnostic performance characteristics.

BACKGROUND: Symptoms of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) can overlap. Faecal calprotectin has recently been established to be a non-invasive marker for neutrophilic intestinal inflammation. We compared two devices for extraction of faecal calprotectin. Based on these results, two immunoassays for measurement of faecal calprotectin were evaluated. METHODS: Samples were extracted using the Thermo Fisher extraction device (Thermo Fisher Scientific) and Smart Pep extraction device (Roche Diagnostics) and measured with the EliA Calprotectin immunoassay (Thermo Fisher Scientific) on ImmunoCAP 250. The performance of both assays was investigated by enrolling 183 consecutive patients (79 males, 104 females; median age 32 years) with clinical suspicion of IBD. Faecal calprotectin was measured using a recently launched immunoassay, EliA Calprotectin in comparison with an established immunochomatographic point-of-care-test (POCT, Quantum Blue Calprotectin; Bühlmann). Results were compared with endoscopic and histological findings. RESULTS: The use of the Thermo Fisher extraction device resulted in an underestimation of faecal calprotectin concentrations, especially in liquid stool samples. IBD was diagnosed in 51/183 patients (27.9%) [Crohn's disease (CD, n=37), ulcerative colitis (UC, n=14)]. After adjusting the optimal cut-off for detection of IBD using receiver operating curve analysis, a sensitivity of 94.1% and 90.2% and specificity of 87.9% and 90.9% for the EliA and POCT assay, respectively, were obtained. CONCLUSIONS: The Thermo Fisher device is not reliable for extraction of faecal calprotectin. The performance characteristics of the EliA Calprotectin assay are statistically equivalent to the Bühlmann POCT.

Imaging mass spectrometry for assessing temporal proteomics: analysis of calprotectin in Acinetobacter baumannii pulmonary infection.

Imaging MS is routinely used to show spatial localization of proteins within a tissue sample and can also be employed to study temporal protein dynamics. The antimicrobial S100 protein calprotectin, a heterodimer of subunits S100A8 and S100A9, is an abundant cytosolic component of neutrophils. Using imaging MS, calprotectin can be detected as a marker of the inflammatory response to bacterial challenge. In a murine model of Acinetobacter baumannii pneumonia, protein images of S100A8 and S100A9 collected at different time points throughout infection aid in visualization of the innate immune response to this pathogen. Calprotectin is detectable within 6 h of infection as immune cells respond to the invading pathogen. As the bacterial burden decreases, signals from the inflammatory proteins decrease. Calprotectin is no longer detectable 96-144 h post infection, correlating to a lack of detectable bacterial burden in lungs. These experiments provide a label-free, multiplexed approach to study host response to a bacterial threat and eventual clearance of the pathogen over time.

Calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin.

Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineate the zinc-binding properties of CP. Unique optical absorption and EPR spectroscopic signatures for the interfacial His(3)Asp and His(4) sites of human calprotectin are identified by using Co(II) as a spectroscopic probe. Zinc competition titrations employing chromophoric Zn(II) indicators provide a 2:1 Zn(II):CP stoichiometry, confirm that the His(3)Asp and His(4) sites of CP coordinate Zn(II), and reveal that the Zn(II) affinity of both sites is calcium-dependent. The calcium-insensitive Zn(II) competitor ZP4 affords dissociation constants of K(d1) = 133 ± 58 pM and K(d2) = 185 ± 219 nM for CP in the absence of Ca(II). These values decrease to K(d1) ≤ 10 pM and K(d2) ≤ 240 pM in the presence of excess Ca(II). The K(d1) and K(d2) values are assigned to the His(3)Asp and His(4) sites, respectively. In vitro antibacterial activity assays indicate that the metal-binding sites and Ca(II)-replete conditions are required for CP to inhibit the growth of both Gram-negative and -positive bacteria. Taken together, these data provide a working model whereby calprotectin responds to physiological Ca(II) gradients to become a potent Zn(II) chelator in the extracellular space.

Detection of calprotectin and its correlation to the accumulation of neutrophils within equine large colon during ischaemia and reperfusion.

REASON FOR PERFORMING STUDY: The cytosolic protein complex, calprotectin, is abundant in neutrophils and could be used to improve the ability to localise and assess neutrophil infiltration in the equine intestine during ischaemia and reperfusion (I/R), but further study is required. OBJECTIVES: To assess the number of calprotectin-containing cells by immunohistochemistry in correlation with direct counting and scoring of neutrophils in the equine colon during I/R. METHODS: One and 2 h ischaemia of the left dorsal colon were induced, followed by 30 min reperfusion under general anaesthesia or by 18 h reperfusion after anaesthetic recovery. Biopsies were processed for light microscopy and stained with H/E for detection of neutrophils. To identify calprotectin-containing cells, immunohistochemistry was performed on formalin-fixed tissues with the murine MAC 387 antibody and a biotin-free peroxidase staining procedure. The number of neutrophils within submucosal venules and the colonic mucosa were calculated and compared with the number of calprotectin-positive cells. RESULTS: The number of calprotectin-positive cells within submucosal venules and within the colonic mucosa correlated significantly with the accumulation of neutrophils within the corresponding tissue segments. Within the submucosal venules, both calprotectin-positive cells and H/E-stained neutrophils increased with duration of ischaemia and peaked after 30 min of reperfusion. After 18 h reperfusion the number of these cells declined within the vessels. After 2 h ischaemia, neutrophils started to migrate into the mucosa towards the epithelium, with a significant increase over time during reperfusion, and peak infiltration after 18 h reperfusion. CONCLUSIONS: Neutrophil infiltration into the colon after I/R is a time-dependent process, involving migration through the submucosa towards the epithelium.

Purification and partial characterization of canine calprotectin.

Calprotectin (CP) is an abundant protein in human neutrophilic granulocytes and macrophages. In humans, serum, urine, and fecal concentrations of neutrophil-derived proteins, such as CP are used as markers of disease activity for conditions associated with increased neutrophil activity, such as inflammatory bowel disease. The aims of the present study were to purify and partially characterize CP in the dog (Canis familiaris) as a prelude to the development of an immunoassay for the quantification of canine serum, urine, and fecal CP in dogs with inflammatory conditions. Leukocytes were isolated from whole blood by dextran sedimentation, and canine CP (cCP) was extracted from the cytosol fraction by repeated freezing--thawing--sonication, followed by further purification using anion- and cation-exchange column chromatography. The overall yield of the purification protocol was 3.7mg cCP per 600ml whole blood. The relative molecular masses of the two proteins representing cCP (cS100A8 and cS100A9) were estimated at 10,340 and 14,628, respectively. Isoelectric focusing revealed two bands with isoelectric points of 6.4 and 6.2 for the heterodimeric protein. The approximate specific absorbance of cCP at 280nm was 0.872 for a 1mg/ml solution. The amino acid sequence of the first 13 N-terminal residues of cS100A8 was Met-Leu-Thr-Glu-Leu-Glu-Ser-Ala-Ile-Asn-Ser-Leu-Ile, whereas the N-terminus of cS100A9 was blocked. Identity of both cS100A8 and cS100A9 was confirmed by tryptic peptide mass fingerprinting followed by peptide sequencing. Antibacterial activity of cCP against Escherichia coli was shown to be concentration-dependent and was reversible upon addition of micromolar amounts of zinc. We conclude that cCP can be successfully purified from canine whole blood using this reproducible, rapid and efficient method.

Human neutrophil calprotectin reduces the susceptibility of Borrelia burgdorferi to penicillin.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is susceptible to killing by a variety of polymorphonuclear leukocyte (PMN) components. Some are most effective against metabolically active B. burgdorferi. The abundant PMN cytoplasmic protein calprotectin, elevated 10- to 100-fold in inflammation, inhibits the growth of spirochetes through chelation of the essential cation, Zn. Since the action of some therapeutic antibiotics depends on bacterial division, we investigated the antibiotic sensitivities of spirochetes in calprotectin. In physiologic calprotectin, B. burgdorferi is not eliminated by therapeutic doses of penicillin G; in contrast, doxycycline is effective. Calprotectin may modify the clearance of spirochetes at sites of inflammation.

Calprotectin, an abundant cytosolic protein from human polymorphonuclear leukocytes, inhibits the growth of Borrelia burgdorferi.

We previously showed that numerous polymorphonuclear leukocyte (PMN) granule components efficiently kill Borrelia burgdorferi, the agent of Lyme disease. In addition, motile, granule-poor cytoplasts (U-Cyt) from human blood PMN can exert anti-Borrelia activity against opsonized B. burgdorferi independently of oxidative mechanisms. Here we show that lysates of U-Cyt also possess anti-Borrelia activity, a portion of which comes from the abundant cytosolic protein calprotectin. The anti-Borrelia activity of U-Cyt lysates and recombinant calprotectin was partially or completely reversed by specific antibody to calprotectin and by Zn(2+), a cation essential for the growth of B. burgdorferi and known to inhibit the antimicrobial activity of calprotectin. Quantitative microscopic and regrowth assays revealed that calprotectin acted in a bacteriostatic fashion against B. burgdorferi. We conclude that calprotectin, a potent bacteriostatic agent from a cell primarily recognized for its oxidative and granular antibacterial mechanisms, may play a modulatory role in infection by the Lyme spirochete, particularly at sites of acute inflammation.