HIGH PREVALENCE OF UNTREATED AND UNDERTREATED VITAMIN D DEFICIENCY AND INSUFFICIENCY IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE.

Inflammatory bowel disease (IBD) patients with vitamin D deficiency show an increased risk of hospital admission, surgery, and loss of response to biologic therapy while high vitamin D levels are identified as a protective factor. Our goal was to investigate the prevalence of untreated and undertreated vitamin D deficiency and factors associated with vitamin D deficiency. In this cross-sectional study, we measured serum vitamin D in a random sample of Caucasian IBD patients. Vitamin D deficiency was defined as <50 nmol/L and insufficiency as 50-75 nmol/L. Supplementation was defined as taking 800-2000 IU vitamin D daily. Untreated patients were defined as not taking supplementation and undertreated group as receiving supplementation but showing vitamin D deficiency or insufficiency despite treatment. Our study included 185 IBD patients, i.e. 126 (68.1%) with Crohn's disease (CD) and 59 (31.9%) with ulcerative colitis (UC). Overall, 108 (58.4%) patients had vitamin D deficiency and 60 (32.4%) patients vitamin D insufficiency. There were 16 (14.8%) and 11 (18.3%) treated patients in vitamin D deficiency and vitamin D insufficiency group, respectively. The rate of untreated patients was 81.7% (n=49) in vitamin D deficiency group and 85.2% (n=92) in vitamin D insufficiency group. Tumor necrosis factor alpha inhibitors were associated with higher serum vitamin D levels in CD and UC, and ileal involvement, ileal and ileocolonic resection with lower levels. In conclusion, not only is vitamin D deficiency common in IBD patients but the proportion of untreated and undertreated patients is considerably high. We suggest regular monitoring of vitamin D levels in IBD patients regardless of receiving vitamin D supplementation therapy.

LEUKOCYTAPHERESIS IN THE MANAGEMENT OF SEVERE STEROID-DEPENDENT ULCERATIVE COLITIS.

Ulcerative colitis (UC) is a multifactorial disease of unknown precise etiology and immunopathogenesis. Peripheral blood granulocytes and monocytes/macrophages are the major sources of cytokines, which regulate inflammation. Leukocytapheresis (LCAP) is a method where blood is processed by apheresis system that removes lymphocytes and plasma before being returned to the body. We report the first case in Croatia where we used LCAP in the treatment of a patient with severe steroid-dependent UC. After 12 LCAP procedures, good clinical response was obtained and there were no significant adverse side effects noticed. The patient remained in clinical remission over two years in which he underwent regular follow ups at outpatient clinic. Over a 10-year follow-up period after LCAP, the patient had only occasional clinical symptoms of disease activity. The clinical course was complicated with the development of metastatic colorectal carcinoma, which points to the importance of regular disease monitoring rather than the increased risk of malignant disease after LCAP. Patients with UC are a demanding group of patients that warrant the search for novel treatment strategies other than conventional pharmacological therapies. Although LCAP is still not a common treatment modality in our daily practice, data from recent studies suggest it to be an effective and safe procedure in the management of active UC patients.

The Role of Vitamin D in Inflammatory Bowel Disease - Assessing Therapeutic and Preventive Potential of Supplementation and Food Fortification.

Inflammatory bowel diseases are a group of chronic inflammatory conditions that affect gastrointestinal tract due to inapt and continuous immune activation in response to a myriad of predisposing factors (most notably genetics, environmental impact and gut microbiota composition). It has been shown that vitamin D status can also play a role in the disease pathogenesis, as its deficiency is commonly observed in two major forms of inflammatory bowel diseases - Crohn's disease and ulcerative colitis. Mounting evidence supports the concept of intricate relationship between gut dysbiosis and vitamin D metabolism, while suboptimal levels of this vitamin have been linked to increased clinical disease relapse rates, inadequate response to drugs, as well as decreased quality of life in patients with Crohn's disease and ulcerative colitis. Consequently, the pertinent question is whether increased vitamin D supplementation and (on a population level) food fortification may bring significant benefit to the affected individuals. In this short review we discuss the synthesis, functions, status and food sources of vitamin D, appraise biotechnological facets of vitamin D status analysis and food fortification, and concentrate on novel developments in the field that describe its influence on intestinal microbiota and inflammatory bowel disease.

Neutrophil-mediated type IV collagen degradation is elevated in patients with mild endoscopic ulcerative colitis reflecting early mucosal destruction.

Neutrophils play a significant role in sustaining chronic inflammation in Inflammatory Bowel Disease. The intestinal basement membrane acts as a barrier for immunological homeostasis, where the α3 and α4 chains of type IV collagen are expressed on the mucosal surface. We wanted to develop a biomarker reflecting early tissue injury, providing an opportunity for intervention. Two competitive enzyme-linked immunosorbent assays (ELISAs) quantifying human neutrophil elastase (HNE) degraded neo-epitopes of COL4A3 and COL4A4 were developed and investigated in two observational cohorts (n = 161, n = 100). A biomarker of MMP-mediated degradation of COL4A1 (C4M) was used for comparison. In Cohort 1, patients with mild endoscopic ulcerative colitis showed elevated levels of C4A3-HNE compared to those with severe disease. C4M had a strong positive correlation with disease activity. C4A3-HNE/C4M provided superior discrimination between mild and severe endoscopic disease and negatively correlated to disease activity. In Cohort 2, C4A4-HNE and C4A4-HNE/C4M showed similar trends. C4A3-HNE and C4A4-HNE possibly reflect early intestinal tissue injury. Combining the markers with a biomarker of another α-chain of the same collagen provides information on two distinct stages of mucosal damage. These biomarkers may be used to monitor disease flare-up in patients in remission, reducing the need for frequent endoscopic procedures.

Blood-Based Biomarkers Reflecting Protease 3 and MMP-12 Catalyzed Elastin Degradation as Potential Noninvasive Surrogate Markers of Endoscopic and Clinical Disease in Inflammatory Bowel Disease.

Chronic inflammation in inflammatory bowel disease (IBD) triggers significant extracellular matrix remodeling, including elastin remodeling, leading to severe clinical complications. Novel methods to assess intestinal tissue destruction may act as surrogate markers of endoscopic disease activity, relieving patients of invasive endoscopy. We explored the noninvasive blood-based biomarkers ELP-3 and ELM-12, measuring elastin degradation in IBD. In a study involving 104 Crohn's disease (CD), 39 ulcerative colitis (UC), and 29 healthy donors, we assessed these biomarkers' association with endoscopic and clinical disease activity using ELISA. Patients were evaluated based on the SES-CD and CDAI for CD patients and modified MES and partial Mayo for UC patients. ELP-3 and ELM-12 were elevated in patients with IBD. Discerning CD patients in endoscopic remission and mild from moderate to severe, ELP-3 provided an AUC of 0.69 and ELM-12 an AUC of 0.73. The ELP-3 biomarker was associated with UC patients and provided the highest diagnostic power of 0.87 for remission vs. active clinical disease. The data suggest an association of ELP-3 with active CD and ELM-12 with endoscopic remission in CD patients. Additionally, ELP-3 could identify UC patients with active clinical disease from patients in remission. The noninvasive biomarkers ELP-3 and ELM-12 could be potential surrogate biomarkers of elastin degradation and endoscopic and clinical disease markers.

Case report: A patient with mitochondrial neurogastrointestinal encephalomyopathy and chronic intestinal failure.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare disorder commonly diagnosed in later disease stages when it prominently manifests as malnutrition. We report on a female patient diagnosed with MNGIE at the age of 36. She was severely malnourished due to loss of resorptive surface after several surgical procedures, gastrointestinal dysmotility, and small intestinal bacterial overgrowth. Therefore, early and aggressive total parenteral nutrition was introduced. Although no reports have shown that nutritional support can modify the clinical outcome, this case suggests that adequate nutritional support, particularly parenteral nutrition, supervised by an experienced nutritional team, may prolong the lifespan of patients with MNGIE.

Inflammatory Biomarkers of Extracellular Matrix Remodeling and Disease Activity in Crohn's Disease and Ulcerative Colitis.

Extracellular matrix (ECM) homeostasis is highly affected in active inflammatory bowel disease (IBD). The aim of the study was to investigate serological biomarkers of type III, IV, and V collagen degradation and formation, and their association with disease activity in IBD. ECM remodeling serum biomarkers were measured in 162 IBD patients, 110 with Crohn's disease (CD) and 52 with ulcerative colitis (UC), and in 29 healthy donors. Biomarkers of type III collagen degradation (C3M) and formation (PRO-C3), type IV collagen degradation (C4M) and formation (PRO-C4), and type V collagen formation (PRO-C5) were measured using ELISA. Inflammatory activity was assessed using endoscopic, clinical, and biochemical activity indices. The highest diagnostic value was identified in discriminating endoscopically moderate to severe disease in CD (PRO-C3, C3M/PRO-C3, and C4M with AUC of 0.70, 0.73, and 0.69, respectively) and UC (C3M, C3M/PRO-C3, and C4M with AUC of 0.86, 0.80, and 0.76, respectively). C4M and C3M/PRO-C3 in combination yielded AUC of 0.93 (0.66-0.90) in CD and 0.94 (0.65-0.99) in UC. This study confirmed that ECM remodeling reflected disease activity in CD and UC. A combination of C4M, C3M, and PRO-C3 biomarkers may potentially be considered as a biomarker differentiating moderate to severe endoscopic disease.

Gut microbiota in mucosa and feces of newly diagnosed, treatment-naïve adult inflammatory bowel disease and irritable bowel syndrome patients.

The knowledge on how gut microbes contribute to the inflammatory bowel disease (IBD) at the onset of disease is still scarce. We compared gut microbiota in newly diagnosed, treatment-naïve adult IBD (Crohn's disease (CD) and ulcerative colitis (UC)) to irritable bowel syndrome (IBS) patients and healthy group. Mucosal and fecal microbiota of 49 patients (13 UC, 10 CD, and 26 IBS) before treatment initiation, and fecal microbiota of 12 healthy subjects was characterized by 16S rRNA gene sequencing. Mucosa was sampled at six positions, from terminal ileum to rectum. We demonstrate that mucosal microbiota is spatially homogeneous, cannot be differentiated based on the local inflammation status and yet provides bacterial footprints superior to fecal in discriminating disease phenotypes. IBD groups showed decreased bacterial diversity in mucosa at all taxonomic levels compared to IBS. In CD and UC, Dialister was significantly increased, and expansion of Haemophilus and Propionibacterium characterized UC. Compared to healthy individuals, fecal microbiota of IBD and IBS patients had increased abundance of Proteobacteria, Enterobacteriaceae, in particular. Shift toward reduction of Adlercreutzia and butyrate-producing taxa was found in feces of IBD patients. Microbiota alterations detected in newly diagnosed treatment-naïve adult patients indicate that the microbiota changes are set and detectable at the disease onset and likely have a discerning role in IBD pathophysiology. Our results justify further investigation of the taxa discriminating between disease groups, such as H. parainfluenzae, R. gnavus, Turicibacteriaceae, Dialister, and Adlercreutzia as potential biomarkers of the disease.

Cervix cancer screening in Croatia within the European Cervical Cancer Prevention Week.

Croatia still has opportunistic screening and the organized national screening has been planned. The European Cervical Cancer Prevention Week was held twice in Croatia, in January 2008 and 2009. Within the first one in 2008, information campaign "For All Women" via mass media was held, and women were invited to the organized free gynecological examination and Papanicolaou test (Pap test) in the University Department of Gynecology and Obstetrics, Zagreb University Hospital Center. Following invitation 481 women attended the testing; the median age was 55 years. There were more women aged > or = 50 (n = 353), with the highest participation in the age group 55-59 years (n = 94). Some women came because of subjective symptoms (n = 10), but the majority of them came only for testing (n = 471). According to history of previous cytological testing, 400 women have had > or = 1 negative findings, 71 women have had > or = 1 positive findings, 9 women attended Pap test for the first time, and 1 woman does not know about previous testing. Cervical cytology was abnormal in 35 women (7.28%), the median age was 42 years with the highest proportion in the age group 30-34 years (n = 7); among all of them 21 women (60%) had no abnormal Pap test previously. The findings were: Atypical squamous cells of undetermined significance--ASC-US (n = 9), ASC cannot exclude high-grade squamous intraepithelial lesion--ASC-H (n = 1), cervical intraepithelial neoplasia--CIN 1 (n = 13), CIN 2 (n = 1), CIN 3 (n = 6), carcinoma planocellulare (n = 2), atypical glandular cells--AGC-favor reactive endocervical cells (n = 3). Among women aged < or = 49 there were 20.47% abnormal findings and among those aged > or = 50, 2.55%. According to 21 positive Pap tests previously, among women aged < or = 49 there were 30.71% while among those aged > or = 50 there were 9.07%. Within the European Cervical Cancer Prevention Week in 2009, employed women from one national company were invited by internal information to the same procedure. A smaller group of younger asymptomatic women came for testing (n = 53), median age 39 years. According to history of previous cytological testing, 50 women have had > or = 1 negative findings, 3 women have had > or = 1 positive findings. In this study, Pap test was positive in 3.77% (n = 2). National screening programme should be focused on the participation of all personally invited women, especially younger age groups and under-screened women. Well designed information campaign should be implemented in national screening programme.

Cytologic characteristics of adenoid cystic carcinoma of the cervix uteri--case report.

Adenoid cystic carcinoma is a rare malignancy, usually found in the salivary glands, although this unusual tumor can be found at other sites of the body. In particular, regarding adenoid cystic carcinoma of the cervix (ACCC) most frequently reports are given for postmenopausal women. In this respect, our work is one among very few in the literature that considers a cytologic picture of this uncommon cervix carcinoma. The case of 74 year old patient with postmenopausal bleeding is described. Both Pap smear and air dried smear of the uterine cervix were analyzed. The cytologic findings revealed very few small clusters of abnormal glandular cells, as well as some amorphous eosinophilic globule-like material, with granulomatous and necrotic background. The latter includes a lot of histiocytes, multinucleated giant cells, large aggregates of epitheloid cells and lymphocytes. Histology revealed the diagnosis of ACCC. We emphasize the importance of careful screening of Pap smear that might be crucial in the case of suspicious clinical findings in postmenopausal women, when the possibility of ACCC has to be considered.

Cytology of cervical intraepithelial glandular lesions.

Cytological criteria for the identification of glandular intraepithelial lesions (GIL) have not yet been fully described, especially for the precursors of adenocarcinoma in situ (AIS), thus these lesions may frequently remain unrecognized. As most patients diagnosed with AIS or mild to moderate GIL (grades I, II) are free from clinical symptoms, cytology has a very responsible role in the detection of these lesions. The aim of the study was to achieve the most appropriate cytologic diagnosis of intraepithelial lesions of endocervical columnar epithelium, analyzing the cytology findings in patients with histologically verified AIS and GIL (I, II). The value of cytology in the detection and differential diagnosis was assessed in 123 patients with definitive histologic diagnosis of glandular lesions (AIS, n = 13; GIL I, n = 11; and GIL II, n = 7), and glandular lesions associated with squamous component (AIS associated with cervical intraepithelial neoplasia (CIN) or invasive squamous cell carcinoma (SCC), n = 58; GIL I or GIL II associated with CIN, n = 28; and GIL associated with microinvasive squamous carcinoma (MIC), n = 6). In 95.1% of patients, lesions were detected by cytologic analysis that indicated additional diagnostic procedure. In terms of differential diagnosis, cytology showed higher accuracy in predicting lesion severity vs. type of epithelial alteration (75.6% vs. 55.3%) and abnormalities of columnar epithelium (95.7%; vs. 74.2%). The accuracy of cytology was higher in pure (AIS, 61.5% and GIL I, II, 22.2%) than in mixed lesions (25.9% and 20.6%). Continuous improvement in cervical specimens and cytodiagnostic skills, better understanding of intraepithelial adenocarcinoma and precursors, and their inclusion in the classification of cytologic and histologic findings are expected to upgrade the detection of these lesions, and to reduce the invasive cervical adenocarcinoma morbidity and mortality.

Digital morphometry of cytologic aspirate endometrial samples.

Unlike cervical cytology, morphological cytology criteria in the differential diagnosis of endometrium have not yet been clearly defined, and methods to allow for more precise evaluation of endometrium status have been searched for. The aim of the present study was to assess the value of morphometric nucleus analysis of cytologic aspirate endometrial samples in proliferative, hyperplastic and malignant endometrium by use of digital image analysis. Morphometric analysis was performed on archival cytologic aspirate endometrial samples (at least 10 per group) stained according to Papanicolaou (n = 77) and May-Grünwald-Giemsa (MGG; n = 80) with the following histopathologic diagnoses: proliferative endometrium, hyperplasia simplex, hyperplasia complex, hyperplasia complex atypica, and adenocarcinoma endometriodes endometrii (grade I, II and III). Interactive image analysis (nuclear area, convex area, perimeter, maximum and minimum radius, length and breadth, as well as nucleus form factor and elongation factor) was performed by use of the SFORM software (VAMSTEC, Zagreb) on at least 50 (Papanicolaou stain) and 100 (MGG stain) well preserved endometrial epithelial cell nuclei without overlapping, at magnification of x1000. Statistical data analysis was done by use of the Statistica Ver. 6 statistical package. Multivariate analysis (ANOVA) distinguished malignant, hyperplastic and proliferative endometrium according to all morphometric variables with both staining methods (p < 0.05). However, on interactive testing of the groups (Kruskal-Wallis test), hyperplasias without atypia yielded no significant differences (p > 0.05) from atypical hyperplasia, adenocarcinoma and proliferative endometrium only according to the nucleus form factor and elongation factor (Papanicolaou stain), whereas malignant and atypical hyperplastic endometrium (MGG stain) differed statistically significantly (p < 0.05) from proliferative and hyperplastic endometrium without atypia according to all study parameters except for the nucleus form factor (p > 0.05). According to the cytologic staining method, morphometric parameters were considerably higher in MGG stained endometrial samples, reaching the level of statistical significance (p < 0.05) except for the nucleus form factor and elongation factor (p > 0.05) in the groups of hyperplasia simplex and complex, well differentiated adenocarcinoma (form factor) and atypical hyperplasia (elongation factor). A combination of cytomorphology and the morphometric variables assessed in this study can yield useful information on the cytologic state of endometrium, with special reference to the possible differentiation of the group of hyperplasia without atypia from the group of adenocarcinoma and atypical hyperplasia.

Liquid-based cytology--new possibilities in the diagnosis of cervical lesions.

Liquid-based cytology (LBC) enables the use of supplementary methods in the diagnosis and prognosis of cervical lesions. The aim of this study was to analyze the correlation between p16-INK4a immunoexpression in ThinPrep cervical cytologic samples and human papillomavirus (HPV) detection by polymerase chain reaction (PCR) from the same sample. LBC-ThinPrep (Cytyc, USA) cervical cytology samples, prepared and stained by Papanicolaou method, were analyzed using modified Bethesda cytologic classification named "Zagreb 2002". A second ThinPrep slide, prepared from the same sample, was immunostained for p16INK4a using CINtec p16INK4a Cytology Kit (DakoCytomation, Denmark). Increased expression of the high-risk (HR) HPV E6 and E7 oncogenes results in a highly specific increase in p16 protein expression and overexpression of p16INK4a acts as a potential biomarker for cervical cancer progression from premalignant lesions. Brown nuclear and/or cytoplasmic staining of abnormal cells was considered a positive result. Residual material was used for 13 HR HPV-DNA detection by the PCR based AMPLICOR HPV test (Roche Molecular Systems). A total of 120 ThinPrep Pap tests with the following cytologic diagnoses: 17 within normal limits, 17 atypical squamous cell (ASC) (7 ASC of undetermined significance /ASCUS/ and 10 ASC of high-grade squamous intraepithelial lesions cannot be excluded /ASC-H/), 26 low-grade squamous intraepithelial lesions (LSIL) corresponding cervical intraepithelial neoplasia (CIN) 1, 57 high-grade SIL (HSIL) i.e. 24 CIN II and 33 CIN III and 3 squamous cell carcinoma (SCC) were included in the study. All CIN III (n = 33) and SCC (n = 3) specimens expressed p16INK4a immunoreactivity, whereas the HR HPV test was positive in 97% (32/33) of CIN III and 100% (3/3) of SCC specimens. The p16INK4a biomarker was positive in 87.5% (21/24) of CIN II and 69% (18/26) of CIN I, while the HR HPV was positive in 75% (18/24) of CIN II and 50% (13/26) of CIN I. In ASCUS cytology, p16INK4a and HR HPV showed the same rate of positivity (28.5%; 2/7). Expression of p16INK4a was detected in all cytologic (10/10) ASC-H lesions, in contrast to HR HPV detected in only 20% (2/10) of ASC-H cases. These data suggest the p16INK4a evaluation in ThinPrep cervical samples to be significantly associated with HR HPV testing by PCR in the same sample for the diagnosis of HSIL lesions and cervical carcinomas. A prospective study with longer follow up may clarify the predictive values in the management of LSIL and ASC diagnosis.

Ten years of Croatian national guidelines for use of eicosapentaenoic acid and megestrol acetate in cancer cachexia syndrome - Evaluation of awareness and implementation among Croatian oncologists.

BACKGROUND & AIMS: Cancer cachexia (CC) syndrome and anorexia-cachexia syndrome are common terms used to describe changes in metabolism with increased inflammatory activity and can progressively develop through various stages such as pre-cachexia; cachexia; and refractory cachexia. Therefore in year 2007 Croatian guidelines for use of eicosapentaenoic acid and megestrol acetate in cancer cachexia syndrome were published. Aim of this study was to assess the awareness and implementation of Croatian guidelines for use of eicosapentaenoic acid (EPA) and megestrol acetate (MA) into clinical practice among Croatian oncologists approximately 10 years after the publication, but also to point out the importance of adequate recognition and treatment of CC. METHODS: Survey with questions was designed to assess the awareness and implementation of Croatian guidelines for use of EPA and MA into clinical practice and was distributed among all Croatian oncologists in secondary and tertiary hospital centers. Survey was conducted in January 2011 (40 months following release of the guidelines), February 2013 and June 2018, and were formed in a way of yes/no answers. Additional multiple choice questions that focus on the implementation of guidelines were added in June 2018. RESULTS: A total of 128 oncologists completed a questionnaire. There was no statistically significant difference in follow up period (2011-2018) of percentage of oncologists that are familiar with Croatian guidelines for use of EPA and MA in CC, percentage of oncologists in which Croatian national guidelines changed their approach in treating patients with CC syndrome and proportion of oncologists that are using MA, enteral nutrition formulas with EPA or their combination. Most of the oncologists 38% (N = 44) are using >2.2 g of EPA per day. Nutritional support is prescribed in 25-50% of patients by 42% (N = 48) of oncologists and most of the oncologists (35%, N = 41) start with nutritional support when a body mass loss is >5%. Oncologists mostly recommend patients to use nutritional support during 1 year or more (43%, N = 49) or two months to 1 year (42%, N = 48). Compliance of patients with malignant diseases for using nutritional support was mostly evaluated as medium (69%, N = 60). CONCLUSIONS: Results have shown that majority of oncologists who filled the questionnaire believe that the Croatian national guidelines for use of EPA and MA in CC syndrome changed their approach in treating patients with CC, but also that there are several targeted issues that can be significantly improved. The awareness of and adherence to national guidelines was maintained at high level even 11 years after the guidelines were published.

ATP7B Gene Mutations in Croatian Patients with Wilson Disease.

AIMS: Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, characterized by its accumulation in tissues which results in hepatic, neurological, and/or psychiatric symptoms. The aim of this study was to investigate the genetics of WD in Croatian patients. METHODS: Correlation of the clinical presentation subtype and the age at onset of the diagnosis of WD with the ATP7B genotype was investigated in a group of Croatian WD patients. DNA from peripheral blood samples was tested for the p.His1069Gln by direct mutational analysis and other polymorphisms were identified by sequence analysis of coding and flanking intronic regions of ATP7B gene. RESULTS: In the group of 75 WD patients of Croatian origin, 18 different mutations in ATP7B gene were detected, three of which were novel. The p.His1069Gln mutation was most frequent, being detected in 44 Croatian WD patients (58.7%). Most ATP7B mutations (90.4%) were located in exons 5, 8, 13, 14, and 15. CONCLUSIONS: Clinical diagnosis of WD was confirmed in 59 patients by detecting mutations on both ATP7B alleles. The age at onset of WD and the type of WD clinical presentation showed no significant correlation with the ATP7B genotype.