Elevated calprotectin levels reveal bowel inflammation in spondyloarthritis.

INTRODUCTION: Microscopic bowel inflammation is present in up to 50% of patients with spondyloarthritis (SpA) and is associated with more severe disease. Currently no reliable biomarkers exist to identify patients at risk. Calprotectin is a sensitive marker of neutrophilic inflammation, measurable in serum and stool. OBJECTIVES: To assess whether serum and faecal calprotectin in addition to C-reactive protein (CRP) can be used to identify patients with SpA at risk of microscopic bowel inflammation. METHODS: Serum calprotectin and CRP were measured in 125 patients with SpA. In 44 of these patients, faecal samples were available for calprotectin measurement. All 125 patients underwent an ileocolonoscopy to assess the presence of microscopic bowel inflammation. RESULTS: Microscopic bowel inflammation was present in 53 (42.4%) patients with SpA. Elevated serum calprotectin and CRP were independently associated with microscopic bowel inflammation. Faecal calprotectin was also significantly higher in patients with microscopic bowel inflammation. Patients with CRP and serum calprotectin elevated had a frequency of bowel inflammation of 64% vs 25% in patients with low levels of both. When either CRP or serum calprotectin was elevated, the risk was intermediate (40%) and measuring faecal calprotectin provided further differentiation. Hence we suggest a screening approach where initially serum calprotectin and CRP are assessed and, if necessary, faecal calprotectin. The model using this scenario provided an area under the ROC curve of 74.4% for detection of bowel inflammation. CONCLUSIONS: Calprotectin measurements in stool and serum, in addition to CRP, may provide a promising strategy to identify patients with SpA at risk of bowel inflammation and could play a role in overall patient stratification.

Need for objective and reproducible criteria in histopathological assessment of total mesorectal excision specimens: lessons from a national improvement project.

AIM: Data on quality control of the pathologic evaluation of total mesorectal excision (TME) specimens are scarce. We aimed to assess differences between evaluation by local pathologists participating in PROject on CAncer of the REctum (PROCARE; a Belgian improvement project on rectal cancer) and by a review panel of experts. METHOD: Based on photographic material and histopathology slides, a Review Committee of gastrointestinal expert pathologists re-evaluated the mesorectal plane, the tumour differentiation grade, the (y)pT stage and the tumour regression grade in 444 patients previously routinely assessed by local pathologists. RESULTS: The surgical plane was reported in 89% of patients and the circumferential resection margin in 88% of patients by the local pathologist. The median number of lymph nodes harvested in patients undergoing neoadjuvant radiochemotherapy was 11 and 14 in the other patients. The Review Committee downgraded the surgical plane from (intra)mesorectal to intramuscular in 17% of patients, and upgraded it from intramuscular to (intra)mesorectal in 27%. Tumour differentiation grade, T stage and tumour regression grade differed between local pathologists and the Review Committee in 15%, 10% and 38%, respectively, of patients. T stage was upgraded, mainly from T2 to T3, in 8% of patients. Tumour regression was judged by the Review Committee to be less advanced in 15% of patients. CONCLUSION: Acknowledging some shortcomings, this study gives a realistic view of clinical practice. There are differences in interpretation with regard to both macroscopic and microscopic analysis of TME specimens. These findings indicate a need for more objective and reproducible criteria in histopathology. Being aware of this is a first step for improvement.

Clinicopathological significance of indoleamine 2,3-dioxygenase 1 expression in colorectal cancer.

BACKGROUND: Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolising enzyme that induces immune tolerance by modulating T-cell responses. Carcinomas may create an immunosuppressive state via IDO1 expression. Here we examined a possible contribution of IDO1 on this phenomenon and investigated whether IDO1 has prognostic value in colorectal cancer (CRC). METHODS: IDO1 expression was investigated by quantitative PCR and western blotting in three colon cancer cell lines, in basal state and after interferon (IFN)-γ stimulation. Semi-quantitative immunohistochemistry was used to evaluate IDO1 expression in 265 pT1-4N0-2Mx-staged CRCs. Results were related to clinical variables and correlated with amounts of CD3(+) and CD8(+) T lymphocytes, which were quantitatively evaluated using image analysis. RESULTS: In vitro expression of IDO1 depended on IFN-γ stimulation. Higher IDO1 expression at the tumour invasion front was an independent adverse prognostic factor in pT1-4N1Mx-staged CRC. It was associated with overall survival (P=0.001) and with metachronous metastases (P=0.018). IDO1 expression was not associated with the presence of CD3(+) or CD8(+) T lymphocytes. CONCLUSION: Higher IDO1 expression at the tumour invasion front is involved in CRC progression and correlates with impaired clinical outcome, suggesting that IDO1 is an independent prognostic indicator for CRC.

Aberrant spindle structures responsible for recurrent human metaphase I oocyte arrest with attempts to induce meiosis artificially.

BACKGROUND In some couples, not all retrieved oocytes mature, even after prolonged in vitro culture. The underlying mechanisms are not known, although ionophore treatment may alleviate metaphase I (MI) arrest in some mouse strains. We attempted to induce first polar body (PB) extrusion and fertilization using assisted oocyte activation (AOA) after ICSI in maturation-resistant human MI oocytes. METHODS Four ICSI patients are described in this retrospective study. A pilot study tested the calcium ionophore ionomycin (10 µM) on donated MI oocytes from patients with a normal number of metaphase II (MII) oocytes. Subsequently, ionomycin was used to induce first PB extrusion in two patients showing maturation-resistant MI oocytes. AOA, by calcium injection and ionomycin exposure, was applied when mature oocytes were available. Oocytes were analysed by polarized microscopy and immunostaining. RESULTS Ionomycin induced the first PB extrusion in MI oocytes from patients with a normal number of retrieved MII oocytes, while extended in vitro culture failed to achieve the MII stage. Similarly, ionomycin induced first PB extrusion in one of two patients with recurrent maturation-resistant MI oocytes. Use of ICSI combined with AOA on MII oocytes matured in vitro or in vivo resulted in failed or abnormal fertilization with no further embryo cleavage potential. Highly abnormal spindle and chromosome configurations were observed in MI maturation-resistant oocytes, in contrast to control MI oocytes. CONCLUSIONS Ionophore induced first PB extrusion in MI oocytes from patients without maturation arrest but to a lower extent in maturation-resistant MI oocytes. Immunofluorescence staining and confocal analysis revealed, for the first time, highly abnormal spindle/chromosomal structures that may be responsible for this maturation arrest.

Xenotransplantation of cryopreserved human ovarian tissue into murine back muscle.

BACKGROUND: Ovarian tissue (OT) cryopreservation and transplantation are options for fertility preservation in young female cancer patients. METHODS: We investigated xenotransplantation of human OT into back muscle (B) of severe combined immunodeficiency mice. OT follicle content was evaluated by stereomicroscopy and pre-transplantation. Xenograft survival, follicular development (with/without FSH administration), apoptosis and vascularization were compared in B- versus K-site (under the kidney capsule) several times after grafting using histology, immunohistochemistry and magnetic resonance imaging. In vitro maturation (IVM) was also performed. RESULTS: Anastomoses which developed from existing human and invading murine vessels were seen in OT at both sites, but angiogenesis was more prominent at the B- than K-site (P < 0.001). Vascularization and follicle size were correlated in the B-group (Spearman's coefficient 0.73; P < 0.001). FSH increased early (8 days) micro-vessel formation in B but not in K grafts (P < 0.0001, versus no FSH). B-site grafts showed a better histological morphology and survival (P = 0.0084), formation of larger antral follicles (P = 0.005), more metaphase-II (MII) oocytes, growing follicles (P = 0.028) and slightly fewer apoptotic follicles than K grafts. One MI oocyte from B underwent IVM and reached MII stage next day. CONCLUSIONS: To our knowledge, this is the first report of MII and IVM-MII oocytes obtained from B xenografts. We report the largest oval-shaped antral follicles containing an MII oocyte obtained after OT xenotransplantation to date. Xenografting in the mouse B should be further explored as a method for human OT transplantation.

Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men.

BACKGROUND: In mammals, oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta (PLCzeta). However, it still remains to be conclusively shown that PLCzeta is the endogenous agent of oocyte activation. Some types of human infertility appear to be caused by failure of the sperm to activate and this may be due to specific defects in PLCzeta. METHODS AND RESULTS: Immunofluorescence studies showed PLCzeta to be localized in the equatorial region of sperm from fertile men, but sperm deficient in oocyte activation exhibited no specific signal in this same region. Immunoblot analysis revealed reduced amounts of PLCzeta in sperm from infertile men, and in some cases, the presence of an abnormally low molecular weight form of PLCzeta. In one non-globozoospermic case, DNA analysis identified a point mutation in the PLCzeta gene that leads to a significant amino acid change in the catalytic region of the protein. Structural modelling suggested that this defect may have important effects upon the structure and function of the PLCzeta protein. cRNA corresponding to mutant PLCzeta failed to induce calcium oscillations when microinjected into mouse oocytes. Injection of infertile human sperm into mouse oocytes failed to activate the oocyte or trigger calcium oscillations. Injection of such infertile sperm followed by two calcium pulses, induced by assisted oocyte activation, activated the oocytes without inducing the typical pattern of calcium oscillations. CONCLUSIONS: Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

Prediction of recurrent disease by cytology and HPV testing after treatment of cervical intraepithelial neoplasia.

OBJECTIVE: To assess the role of human papillomavirus (HPV) testing and cytology as predictors of residual/recurrent disease after treatment of high-grade cervical intraepithelial lesions. METHODS: One hundred and thirty-eight women with cervical intraepithelial neoplasia (CIN) grade 2/3 lesion on biopsy were included in a prospective follow-up study in Belgium and Nicaragua. All women were treated with loop electrosurgical excision procedure (LEEP) and follow-up visits took place at 6 weeks, 6 months, 1 year and 2 years. During these visits, a Papanicolaou (Pap) smear test was taken, colposcopy was performed and specimens were collected for HPV testing. Cytology, high-risk (HR) HPV presence, persistent HR HPV infection and combinations of these tests at different time points during follow-up were correlated with histologically confirmed residual/recurrent disease. RESULTS: Thirteen patients (9%) developed residual/recurrent disease during follow-up. Abnormal cytology at 6 weeks after treatment was significantly correlated with residual/recurrent disease. Nine of thirty-seven patients with abnormal cytology at 6 weeks had recurrent disease versus three of seventy with a normal cytology [odds ratio (OR): 7.2; 95% confidence interval (CI): 1.8-28.5; P = 0.003). Sensitivity of this test was 75.0%, specificity 70.5%. Combining abnormal cytology and the presence of HR HPV within the first 6 months after treatment gave the best correlation with residual/recurrent disease: of the 54 women with abnormal cytology and/or HR HPV presence within the first 6 months, 11 developed residual/recurrent disease (OR 10.2; 95% CI: 2.2-48.3). Sensitivity of this combination was 84.6% and specificity 65.0%. CONCLUSION: Cytology remains the cornerstone in the early follow-up after LEEP for CIN lesions of the cervix. HPV testing can add value as it increases the sensitivity of cytology in concomitant testing within the first 6 months.

Natural history and clearance of HPV after treatment of precancerous cervical lesions.

AIM: To assess the clearance rate of human papillomavirus (HPV) after out-patient treatment of cervical intraepithelial neoplasia (CIN). METHODS AND RESULTS: A total of 122 Nicaraguan women with HPV DNA-positive and histologically confirmed CIN lesions were included in the study. Fifty-five patients with CIN1 and 67 with CIN2-3 were treated by cryotherapy and loop electrosurgical excision procedure (LEEP), respectively. Follow-up visits were scheduled at 6 weeks, 6 months, 1 year and 2 years. Investigations included cytology, HPV DNA testing and colposcopy/biopsy if needed. The clearance rate of HPV was calculated by multivariate logistic regression. Immediately after treatment, a pronounced decrease in presence of HPV was observed in both groups, with a significantly higher clearance in the LEEP group than in the cryotherapy group (P = 0.019). Subsequently, clearance continued over time and was similar between the cryotherapy group and the LEEP group (P = 0.73). Approximately the same detection rates were obtained for persistence of all HPV types and for high-risk types separately: 43.9, 37.6, 29.9 and 17.7% in the cryotherapy group and 24.9, 20.3, 15.3 and 8.4% in the LEEP group at 6 weeks, 6 months, 1 year and 2 years, respectively. CONCLUSIONS: Out-patient treatment of precancerous lesions of the cervix usually results in clearance of HPV. Both LEEP and cryotherapy are highly effective methods of eradicating HPV. HPV DNA testing may have added value in the follow-up of patients.

Inflamed intestinal mucosa features a specific epithelial expression pattern of indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the first step in the degradation of tryptophan, an essential amino acid. During inflammation IDO can be induced in different cell types resulting in local tryptophan depletion. This inhibits T cell proliferation and may induce apoptosis. High expression of IDO was previously found in inflammatory bowel disease and is thought to represent a mechanism for downregulation of the local immune response. Our aim is to investigate the expression pattern of IDO in normal and inflamed murine and human intestinal mucosa. Immunohistochemical staining for IDO was performed on paraffin sections of colon of two mouse models for colitis and their controls and on paraffin sections of human ileum and colon in normal and two different inflammatory conditions, namely inflammatory bowel disease and diverticulitis. IDO immunohistochemistry showed similar results in murine and human tissue. In normal, as well as in inflamed mucosa, some mononuclear cells, fibroblasts and endothelial cells were positive for IDO. In inflamed mucosa a specific expression pattern of epithelial IDO was found where epithelial cells flanking ulcers or bordering crypt abscesses showed high IDO expression. Moreover, in human intestinal inflammation, IDO was expressed in ulcer associated cell lineage. Since bacterial invasion is more pronounced in erosions and in crypt abscesses and since IDO activity and the resulting local tryptophan depletion can cause growth arrest of several tryptophan-dependent microorganisms, IDO expression in the vicinity of interruptions of the epithelial barrier may point to a role for IDO as a local anti-infectious agent. Furthermore, expression of IDO at the margin of ulcerations and in the reparative ulcer-associated cell lineage suggests involvement of IDO in repair processes.

Focal up-regulation of E-cadherin-catenin complex in inflamed bowel mucosa but reduced expression in ulcer-associated cell lineage.

The E-cadherin-catenin complex is important for the maintenance of epithelial architecture. We studied its expression in Crohn disease, ulcerative colitis, acute ileitis, and controls. Immunohistochemical stainings for E-cadherin, alpha-catenin, beta-catenin and gamma-catenin were performed. E-cadherin messenger RNA (mRNA) was detected using riboprobes. In active inflammation, there was up-regulation of the complex. In particular, epithelium adjacent to ulcers showed increased expression of protein and mRNA, but in ulcer-associated cell lineage, the intensity of staining was weak to negative. In focal inflammation, up-regulation was found in affected areas. Reparative epithelium growing over denuded areas showed weaker expression. Since structural or functional perturbation in any of the molecules of the E-cadherin-catenin complex results in loss of intercellular adhesion, the preexistent epithelium may benefit from up-regulation to try to maintain its normal architecture under inflammatory conditions. Reduced expression in reparative epithelium and ulcer-associated cell lineage could facilitate the motility of these cells.

Immunohistochemical analysis of E-cadherin expression in human colorectal tumours.

The homotypic homophilic cell-cell adhesion molecule E-cadherin is crucial in the organization and maintenance of most epithelia. The expression of E-cadherin was studied immunohistochemically in various human colorectal tumours. Therefore we stained 1 tubular adenoma with low grade dysplasia, 18 adenocarcinomas with different histologic degrees of differentiation and invasion, and 1 metastasis using a modified peroxidase-anti-peroxidase technique. In the adenoma as well as in all well differentiated adenocarcinomas we found E-cadherin immunopositivity at the cell membrane of almost all cancer cells. The immunopositivity of E-cadherin was clearly weaker and sometimes even absent in isolated neoplastic cells and glands of less differentiated adenocarcinomas. The moderately differentiated adenocarcinomas showed an intermediate staining pattern. These findings are in line with experimental evidence that downregulation of E-cadherin favours invasion, eventually leading to metastasis.

Combined leiomyomatosis of the small intestine and colon.

We report a case of leiomyomatosis that involved the small intestine and colon. On gross examination, the bowel wall was irregularly thickened at various levels. Histologically, the lesions were characterized by multifocal, diffuse tumor masses that were present in the muscular layers. These masses consisted of proliferating smooth-muscle cells that often surrounded prominent blood vessels. Cytologic atypia was not observed, and the mitotic count was low, illustrating the benign nature of the tumors. Findings from immunohistochemical analysis of the lesions were essentially negative; vimentin, cytokeratin, actin, desmin, and S100 stains showed no reactivity in the tumor cells, despite positive internal controls. By reviewing the literature, we found several reports on related lesions that occurred in the gastrointestinal tract; diffuse leiomyomatosis of the esophagus and the colon have been reported. We did not find any report on the small intestinal variant of the disease; therefore, it seemed useful to term this new localization as leiomyomatosis of the small intestine and colon.

Host reaction against empty alginate-polylysine microcapsules. Influence of preparation procedure.

Microcapsules, prepared with alginate and polylysine, were injected intraperitoneally into mice and the number of peritoneal leucocytes as well as the cells sticking to the capsule wall were counted after 4-28 days. A significant increase in host reaction was observed when the microcapsules contained an outer layer of polylysine as compared with calcium alginate beads without polylysine or microcapsules coated with an outer layer of alginate. The alginate sources influenced the host reaction significantly. After an intraperitoneal residence of 4 days, the microcapsules were mainly surrounded by macrophages. After 28 days, several cell layers surrounded the microcapsules; macrophages, multinucleate giant cells, fibroblasts and mesothelial cells.

Host reaction against alginate-polylysine microcapsules containing living cells.

Syngeneic and nude mice were injected intraperitoneally with saline, empty microcapsules, aggregates of tumourogenic MO4 cells, encapsulated non-tumourogenic MO cells and encapsulated MO4 cells. The host reaction 4 days after injection, was evaluated by counting the leucocytes in the peritoneal cavity and the cells sticking to recovered microcapsules. A significantly lower number of peritoneal leucocytes was found in mice injected with empty microcapsules or encapsulated MO cells as compared with encapsulated MO4 cells. Histological evaluation showed a significantly higher number of cells sticking to microcapsules containing cells as compared with empty microcapsules. These observations showed that the cellular host reaction against intraperitoneal implants can be evaluated by counting the leucocytes in the peritoneal lavage and histology of recovered capsules.

Oncocytoma of the nasopharynx.

The case of a patient with a nasopharyngeal oncocytoma is described. The diagnostic and therapeutic aspects of oncocytoma are reviewed in light of the present literature.

M cells are damaged and increased in number in inflamed human ileal mucosa.

Ileocolonoscopy and biopsies of patients with spondylarthropathy revealed gut inflammation in 62% of the cases. In order to better understand the pathogenetic mechanisms of spondylarthropathy related gut inflammation the follicle associated epithelium was examined. Biopsies from 9 controls and 18 patients with spondylarthropathy were studied by electron microscopy. Membranous (M) cells were investigated in normal and inflamed ileum. In normal mucosa M cells were scarce whereas in inflamed mucosa their number was increased (up to 24% of follicle associated epithelial cells). They showed a thin rim of cytoplasm covering groups of lymphocytes. In chronic ileitis necrotic M cells, ruptures of M cell cytoplasm and lymphocytes entering the gut lumen were observed. The bursts of M cells at the top of the lymphoid follicles lead to interruption of the gut epithelial lining and give luminal content access to the lymphoid tissue. This pathogenetic mechanism may cause aphthoid ulcers.

Belgian consensus guidelines for follow-up of women with cervical cytological abnormalities.

In medical care cervical cancer screening is important because it enables the detection of precancer and cancer at an early stage. By adequate treatment after a screening-detected lesion it helps to reduce the mortality related to cervical cancer. Worldwide, many millions of women have smears taken at a more or less regular base and of these, approximately 7% are abnormal, and follow-up is thus required.As this represents an important cost in medical health care and has serious consequences for the affected women, it is important to have uniform and clear guidelines to allow an optimal follow-up and clinical management. A system for the uniform reporting of cervical cytology has been designed by the National Cancer Institute (U.S.A.) and resulted in the Bethesda System 1991. The present paper and the terminology used are based on the Bethesda System revised in 2001. It explains the guidelines, based on the 2001 Bethesda System and the 2004 consensus guidelines for the management of women with cervical cytological abnormalities, as developed by the members of the Board of the Belgian Society of Clinical Cytology, and adapted to the Belgian situation.

Murine M cells express annexin V specifically.

The specialized epithelium covering the lymphoid follicles of Peyer's patches in the gut mediates transcytosis of antigens to the underlying immune cells, mainly through the membranous, or M, cells. At present, the molecular processes involved in the mucosal immune response, and in antigen transport across the follicle-associated epithelium (FAE) and M cells, are poorly understood. To characterize FAE and M cells, we compared the gene expression profiles of small intestine FAE and villus epithelium (VE) in BALB/c mice by microarray analysis; 91 genes were found to be up-regulated and four down-regulated at least two-fold (p<0.01) in the FAE. The differential expression of a subset of these genes was shown to be confirmed by quantitative RT-PCR. Using immunohistochemistry on BALB/c Peyer's patches, cathepsin H and clusterin expression was increased in the FAE compared to the VE. Moreover, we demonstrated M cell-specific expression of annexin V, which has recently been reported to be important in endocytic transport and membrane scaffolding, suggesting that annexin V has a function in M cell-mediated transcytosis.