| Abstract |
1. Inflammatory Bowel Diseases (IBD)
IBD is a group of diseases characterized by chronic, relapsing destructive inflammation of the gastrointestinal tract. The clinical presentation includes a variety of symptoms such as chronic or recurrent diarrhea, often with mixture of blood, abdominal pain, fever and anemia. Moreover, not infrequently symptoms are accompanied by extraintestinal manifestations of the skin (pyoderma gangrenosum, erythema nodosum), joints (arthralgia, peripheral arthritis, spondylitis) or eye (iridocyclitis, uveitis). IBD can be divided into two main forms: Crohn’s Disease (CD) and Ulcerative Colitis (UC). In the West, the incidence and prevalence of IBD has increased in the past 50 years, up to 8–14/100,000 and 120–200/100,000 persons, respectively, for UC and 6–15/100,000 and 50–200/100,000 persons, respectively, for CD1. Family history is an important risk factor for IBD onset, with peak incidence in early adult period of life, although individuals of all ages can be affected.
IBD are considered to be the result of a continuous, excessive inflammatory response to symbiotic bacteria, in a genetically predisposed host2,3. The first genome-wide association studies in IBD were published 10 years ago and since then many areas of genetic predisposition have been found on chromosomes 1, 3, 4, 5, 6, 7, 10, 12, 14, 16, 19 and X. Hugot et al.4 first announced in 1996 a correlation with chromosome 16q and five years later, the underlying gene CARD15 (or NOD2) was identified. More recently, over one year, seven genome-wide association studies have been published, and many additional genes for CD and UC have been identified (ATG16L1, IL23R, PTGER4, IRGM, NELL1). Data from immunological, microbiological and genetic studies favor an impaired interaction between host and microbes in the pathogenesis of IBD5. Microbial aberrations observed in patients with IBD, with consequent disruption of the intestinal flora, may be due to colonization by enteric pathogens, to host inflammatory response or to combination6. Furthermore, recent data, concerning pathogenesis of IBD, support the hypothesis of a significant interaction among 3 fundamental biological pathways of the cell: autophagy, microbial sensing and endoplasmic reticulum stress7. Other factors that play an important role in the pathogenesis of IBD are hypoxia, metabolic factors8, inflammatory cytokines9, and disorders of homeostasis regulation10.
2. Anemia of IBD
Anemia is one of the most common extraintestinal manifestations of IBD, with a strong impact on quality of life. According to the World Health Organization (WHO), anemia is defined as hemoglobin level below 120 g/L in women and 130 g/L in men, at sea level. The main cause of anemia in IBD patients is iron deficiency due to chronic blood loss and decreased absorption of iron from the intestinal mucosa, due to inflammation. However, anemia in IBD appears to have a multifactorial etiology, and frequently appears as the result of a combination of iron deficiency (most common cause) and anemia of inflammation or anemia of chronic disease (second most common cause). Iron deficiency takes place when loss of iron exceeds the amount of iron absorbed from the intestine. This situation of negative iron balance occurs when not enough iron is consumed, when iron absorption is suspended, when body requirements in iron are increased (e.g. during pregnancy or puberty) or due to iron loss, because of bleeding/blood loss. The main cause of iron deficiency is blood loss from the urogenital system in women or the gastrointestinal tract in both sexes. The anemia of IBD is mainly due to iron deficiency. Many of the above pathogenetic mechanisms play a role in the development of iron deficit in IBD. Another factor that influences the absorption of food iron is increased levels of hepcidin (as a result of chronic inflammation), leading to reduced outflow of iron from enterocytes. However, chronic blood loss from ulcerated intestinal mucosa remains the main cause of negative iron balance in IBD11.
Anemia of chronic disease (ACD) seems to be the result of stimulation of the immune system from the underlying process and specific inflammatory cytokines, including tumor necrosis factor-alpha (TNFα), interferon-γ (IFNγ) and interleukin (IL)-1, 6, 8 and 10. So far, 3 main pathophysiological factors, underlying ACD, have been identified. All are induced by stimulation of the intrinsic immune mechanism: First, inflammation leads to iron retention within cells of the reticuloendothelial system, resulting in limited availability of iron for erythropoietic progenitor cells. Secondly, proliferation and differentiation of erythropoietic progenitor cells are down-regulated by cytokines and acute phase proteins. Thirdly, reduced production and biological activity of endogenous EPO induces development of ACD. Briefly, in ACD, several different pathophysiological mechanisms (corresponding cytokines) seem to be involved12,13:
1. Reduced life span of erythrocytes due to dyserythropoiesis, increased destruction rate and increased phagocytosis (TNFα).
2. Poor response of EPO production, for the corresponding degree of anemia, in many but not all patients (IL-1 & TNFα).
3. Decreased sensitivity of erythroid cells against EPO stimulation (IFNγ, IL-1, TNFα, hepcidin).
4. Inhibition of maturation and differentiation of erythroid cells (IFNγ, IL-1, TNFα, alpha1-antitrypsin).
5. Abnormal iron homeostasis due to increased expression of DMT1 (IFNγ) and TfR (IL-10) in macrophages, decreased expression of ferroportin 1 (elevated hepcidin concentration due to IFNγ and IL-6) in enterocytes (inhibition of iron absorption) and macrophages (suspension of iron recirculation), and increased rate of ferritin synthesis (TNFα, IL-1, IL-6, IL-10) (increased iron storage).
Diagnostic approach to anemia in IBD, particularly concerning distinguish of iron deficiency anemia (IDA) from ACD, includes many well established and new generation laboratory markers, which, in combination, may diagnose the degree of involvement of each type of anemia, with great accuracy. Ferritin, with transferrin saturation and CRP, is the minimum group of tests required for IBD patients with anemia. The combination of serum ferritin and soluble transferrin receptor (sTfR) levels, can be used to identify iron deficiency (high sTfR, low ferritin), inflammation (normal sTfR and ferritin) or mixed states (increased sTfR, normal ferritin). Moreover, ferritin levels <30 micrograms/L (in combination with transferrin saturation <16%) is an established marker of iron deficiency, in patients with inflammation. In the presence of inflammation, the lower limit of serum ferritin, which corresponds to normal iron stores, is 100 μg/L, so that diagnostic criteria for ACD is serum ferritin ≥100 μg/L and transferrin saturation <16%. When ferritin levels are between 30 and 100 μg/L, a combination of true IDA and ACD is probable. Finally, serum ferritin can be used in the form of sTfR/log ferritin ratio, as a useful tool for IDA exclusion, when it is 7lt;114. Iron metabolism regulators, hepcidin and prohepcidin, are under evaluation and it seems to play a central role in the development of anemia in IBD, but are rather inadequate to separate IDA from ACD. In addition, a variety of red cell and reticulocyte indices play a supporting role in the
diagnosis of anemia in IBD, are still under evaluation, whereas some of them seem to reflect different pathophysiological mechanisms of anemia, very precisely15.
Therapeutic approach to anemia in IBD involves iron replacement therapy (oral or intravenous), administration of erythropoietic agents, while recent trends include treatment of ACD. Hepcidin antagonists are expected to help patients with disorders of over-expression of this hormone, presented as IDA, on a basis of ACD16.
3. Clinical studies
The aims of clinical studies in this thesis were:
1. The survey of epidemiological, clinical and laboratory data on anemia in patients with IBD, who are monitored in the Gastroenterology Clinic at University Hospital of Heraklion.
2. The investigation of iron homeostasis disorders and their correlation with the mechanisms of inflammation in patients with IBD, with particular emphasis on the role of the protein hepcidin.
3. The investigation of erythropoiesis disorders in patients with IBD, with emphasis on the role of conventional and new generation blood markers in the diagnosis of erythropoiesis and iron homeostasis disorders.
4. The investigation of the effects of therapeutic interventions in the above mechanisms and parameters.
The aim of the first study17 was measurement of serum hepcidin and prohepcidin levels in patients with IBD, comparison with healthy controls (HC) and correlation with established parameters of anemia evaluation and inflammation profile of each patient. 100 patients with IBD were included in the study: 49 with ulcerative colitis [UC], 51 with Crohn’s Disease [CD], mean age 49 years, male / female: 58/42, mean disease duration 8 years. These patients were compared with 102 HC of adjusted age. Serum concentrations of hepcidin-25 and prohepcidin were measured using a commercially available ELISA kit (enzyme-linked immunosorbent assay) (DRG International, Inc., New Jersey, USA). The prevalence of anemia in this group of patients with IBD was 42% (41.2% for UC and 42.9% for CD). Mean hepcidin levels were significantly higher in patients with UC and CD, compared to HC (P 7lt;0.0001). Mean prohepcidin levels were significantly lower in patients with IBD,
compared to HC (P = 0.03). In univariate analysis, there was a significant negative correlation (r =- 0.36, P = 0.0003) of serum hepcidin and positive (r = 0.65, P<0.0001) of serum prohepcidin levels with hemoglobin levels. In patients with IBD, a significant correlation of hepcidin (r = 0.34, P = 0.0007) and prohepcidin (r =- 0.21, P = 0.04) with ferritin levels was also found. Serum hepcidin correlated with disease activity (for UC, r = 0.36, P = 0.009) and C-reactive protein (CRP) (r = 0.29, P = 0.004). After multivariate analysis, serum hepcidin levels showed significant correlation with ferritin (P = 0.0008) and disease activity (for UC, P = 0.004). This is the first study that investigates both serum hepcidin and prohepcidin levels in patients with IBD. Higher levels of hepcidin and lower of prohepcidin in patients with IBD compared to HC, underline the crucial role played by these two hormones in IBD. The strong correlation (positive for hepcidin and negative for prohepcidin) with hemoglobin in univariate analysis indicates a significant contribution to anemia mechanisms, whereas multivariate analysis reveals that these levels per se seem inadequate in separating IDA from ACD. This is exacerbated by the lack of correlation between hepcidin / prohepcidin and Tsat & sTfR, which are well established indices for classification of anemia. In contrast, we found a positive correlation between hepcidin and ferritin, something that is rather expected, since both are acute phase proteins and their levels are increased during active inflammatory process.
In the second study18, in the same patient and HC population, the utility of a conventional index (RDW) and four new generation indices (IRF, RSF, RDWR-CV, RDWR-SD) in the evaluation of anemia of IBD was investigated. Red cell and reticulocyte indices were measured using the Coulter LH 780 hematology analyzer (Beckman Coulter, Inc., California, USA). 30 patients (30%) had IDA (ferritin <30 micrograms / L & Tsat <16%), 4 patients (4%) had ACD (ferritin≥ 100 micrograms / L & Tsat<16%) and 8 patients (8%) had mixed IDA / ACD (ferritin = 30-100 μg / L & Tsat <16%). We found a significant correlation between red cell distribution width (RDW), red blood cell size factor (RSF) and reticulocyte distribution width-coefficient of variation (RDWR-CV) with Tsat and sTfR, but not with ferritin levels. Patients with IDA had significantly higher levels of RDW and RDWR-CV and significantly lower levels of RSF, compared with patients without IDA. High values of RDW (sensitivity 93%, specificity 81%) and low values of RSF (sensitivity 83%, specificity 82%) were the best markers for diagnosis of IDA. There was a significant
correlation of RDWR-CV and RDWR-SD (Standard Deviation) with disease activity and CRP levels. Concerning new indices, this is the first evaluation study in this group of patients. It seems that RDW may be proposed as a suitable index for the differential diagnosis between IDA and ACD, in patients with active IBD. However, this study failed to confirm this hypothesis because of the small number of patients with ACD, making any clear conclusions unsafe. IRF is the ratio of immature reticulocytes to the total number of them. In our study, IRF was significantly increased in patients compared to HC, but both groups were within normal limits, according to manufacturer's range (0.20-0.40). This finding seems reasonable, taking into account the continuous blood loss in patients with IBD, often subclinical, as a result of chronic inflammation in the intestine, thus leading to increased driver signal for erythropoiesis in the bone marrow, even under conditions of low iron availability, due to reduced absorption. It seems that IRF is an indicator of rather limited utility in the evaluation of anemia in IBD, which was also confirmed by the results of our study, where there was no correlation with ferritin, Tsat and sTfR, and no difference between patients with and without IDA. RSF connects MCV with MRV. The results of our study support the hypothesis that RSF seems to be a sensitive real-time parameter for early detection of erythropoiesis disorders in functional iron deficiency, in patients with IBD. In our study RDWR-SD was significantly elevated in patients compared with HC (p <0.0001), and RDWR-CV was increased both in patients (compared to HC) and patients with IDA (compared to those with non-IDA). Given the strong negative correlation of the latter with Tsat and positive correlation with sTfR, it seems that RDWR-CV is a sensitive indicator of the reticulocytes population dispersion, reflecting a real-time monitoring of reticulocytes production, beyond the established reticulocyte number index, which rather seems to correspond to a single moment in the reticulocytes life-cycle. Furthermore, both RDWR-CV and RDWR-SD correlated well with disease activity and CRP, a promising finding on the basis that these indices could be combined with others (corresponding only to iron deficiency), so to separate the subgroup of patients with pure IDA (not mixed with ACD). In conclusion, this is the first study to demonstrate the usefulness of the reticulocytes indices in the evaluation of anemia in patients with IBD. RSF and RDWR, alone or in combination with RDW and other well-established indices of anemia and inflammation (Tsat, sTfR, CRP), show great promise as new sensitive tools, providing the clinician with a lot of data from a fairly simple and inexpensive full blood count.
The third study19,20, also in the same group of patients and HC, investigated the use of soluble transferrin receptor (sTfR) and sTfR-ferritin ratio (sTfR-F, sTfR/log10 ferritin) in the evaluation of anemia in patients with IBD. In this study it was found that patients with IDA had significantly higher levels of sTfR, compared to those without IDA (i.e. with other types of anemia or without anemia). Similarly, patients with IDA had significantly higher sTfR-F levels, compared to those without IDA. Furthermore, patients with IDA had significantly higher sTfR & sTfR-F levels, compared to patients with ACD (including mixed IDA / ACD) (P <0.0001). The sensitivity of high sTfR for diagnosing IDA, with cutoff level 1.8 mg / L was 81%, specificity 80%, PPV 63% and NPV 91%. Similarly, the sensitivity of high sTfR-F for the diagnosis of IDA with 1.4 as cutoff limit was 91%, specificity 92%, while the PPV was 83% and NPV 96%. No significant correlation between sTfR or sTfR-F and CRP levels was found. This is the first study evaluating sTfR-F index in patients with IBD. This study showed that sTfR-F appears quite effective in the detection and diagnosis of IDA in patients with IBD. The detection rate is higher than that of sTfR and certainly higher than other existing indices. It is important to add that the clinical use of this index rather adds (and not replaces) to the value of other well established markers such as ferritin, transferrin and Tsat, in the diagnosis of IDA. Therefore, sTfR-F can be recommended as an additional parameter, which can improve the diagnosis of IDA in patients with IBD.
The fourth study21 was focused on the safety and efficacy of total dose low-MW iron dextran infusion in patients with anemia and IBD. The study included 50 patients with IBD (27 women, 35 with CD, 15 with UC). The [mean ± standard deviation] of hemoglobin and ferritin level before infusion was 9.88 ± 1.42 g / dL and 13.9 ± 10.9 ng / ml, respectively. Following administration of a 25 mg test dose, low molecular weight iron dextran was then infused intravenously in one single session, with a dose calculated according to the iron deficit. In all patients clinical and laboratory parameters were recorded, before and 4 weeks after treatment. 4 patients (8%) experienced adverse events during the test dose and were not given full dose. Only one patient had an allergic reaction during infusion of total dose. In the remaining 45 patients, the mean dose of iron administered was 1075 ± 269 mg. The average rise in hematocrit and hemoglobin at week 4 was 4.9 ± 1.9% and 1.7 ± 0.8 g / dL, respectively. Hematopoietic response was observed in 23 of 45 patients (51.1%). The results of this study suggest that IV administration of low-MW iron dextran may
be useful in the treatment of IDA in IBD. This approach is effective, simple, well accepted and friendly to patients. A minority of patients (approximately 10%) may develop allergic reactions, and should be excluded from total dose infusion.
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