The role of hypercoagulability in ischemic colitis.

OBJECTIVE: The aim of this study is to evaluate the role of thrombophilia-hypercoagulability in ischemic colitis (IC). MATERIAL AND METHODS: Thrombophilia and fibrinogen were evaluated in 56 cases of IC and 44 controls with known predisposing factors but no evidence of IC. Thrombophilic factors tested were: protein C (PC), protein S, antithrombin (AT), resistance to activated protein C (APCR), lupus anticoagulant (LA), factor V G1691A mutation (FV Leiden), prothrombin G20210A mutation, methylenetetrahydrofolate reductase (MTHFR) gene C677T and A1298C mutations and plasminogen activator inhibitor-1 (PAI-1) gene 5G/4G and 4G/4G polymorphisms. RESULTS: In IC group were recorded: i) low levels of PC and AT (p = 0.064 and p = 0.022, respectively); ii) low levels of APCR (normal: >2, p = 0.008); iii) high levels of fibrinogen (p = 0.0005); iv) higher number of homozygotes for MTHFR A1298C and C677T mutations (p = 0.061 and p = 0.525 (Pearson chi-square), respectively); v) greater prevalence of 5G/4G and 4G/4G polymorphisms (p = 0.031 (Pearson chi-square)) and vi) higher incidence of LA-positive individuals (p = 0.037, Fischer's exact test). Multivariate analysis was performed to determine the effects of prothrombotic factors in IC. 5G/4G polymorphism of PAI-1 gene (odds ratio (OR) 12.29; 95% confidence interval (CI) 2.26-67.00), APCR (OR 0.089; 95% CI 0.011-0.699) and fibrinogen (OR 1.013; 95% CI 1.003-1.023) were determined as predictors of IC. CONCLUSIONS: This study suggests that hypercoagulability, hereditary or acquired, plays an essential role in the manifestation of IC.

Nocturnal hypertension is associated with an exacerbation of the endothelial damage in preeclampsia.

BACKGROUND: Non-dipping pattern of circadian blood pressure in preeclampsia is associated with an increased risk of cardiovascular disease. The pathogenetic mechanisms of this relationship are still unclear. We investigated whether non-dipping in preeclampsia could relate to endothelial activation or damage. METHODS: Participants, 20 women with normal pregnancy (mean age 29.9 +/- 5.7 years) and 31 women with preeclampsia (mean age 29.1 +/- 5.1 years), underwent 24-hour ambulatory blood pressure monitoring. Plasma levels of von Willebrand factor (vWf), marker of endothelial damage and of soluble adhesion molecules (sVCAM-1, sICAM-1), and markers of endothelial activation were determined using commercially available enzyme-linked immunoassays. RESULTS: Based on whether the nocturnal mean arterial pressure (MAP) relative to the daytime MAP declined by less than 10%, 21 women with preeclampsia were categorized as non-dippers. Compared to healthy pregnant women, patients with preeclampsia showed significantly enhanced levels of vWf (206.9 +/- 40.6 vs. 123 +/- 24 IU/dl;p<0.01) and sVCAM-1 (2,269 +/- 426 vs.1,159.8 +/- 340 ng/ml; p < 0.01). In addition, significantly higher levels of vWf (224.5 +/- 34.9 vs. 170 +/- 23 IU/dl; p < 0.01) and sVCAM-1 (2,405 +/- 421.4 vs. 1,983 +/- 276.7 ng/ml; p = 0.007) were determined, when women with preeclampsia and nocturnal hypertension (non-dippers) were compared to dippers. The results were similar even after adjustment for severity of preeclampsia. In contrast, neither preeclampsia nor dipping status had an effect on sICAM-1 levels. CONCLUSION: Nocturnal hypertension in preeclampsia is associated with elevated levels of molecules related to endothelial damage. Endothelial damage is a recognized pathogenetic factor for atherosclerosis and history of preeclampsia is a risk factor for cardiovascular disease. In this context, possible clinical implications of our findings deserve further investigation.

Relation between bone marrow angiogenesis and serum levels of angiogenin in patients with myelodysplastic syndromes.

Angiogenesis is implicated in the progression of myelodysplastic syndromes (MDS). Bone marrow microvascular density (MVD), serum angiogenin (ANG) and interleukin 6 (IL-6) were measured in 67 patients with untreated MDS. MVD, ANG and IL-6 were significantly higher in the patient group as a whole when compared to controls (P < 0.01). MVD and ANG were significantly higher in subtypes with a high-risk for leukemic transformation (RAEB, RAEB-t and CMML) than in low-risk subtypes (RA and RARS) (P < 0.01). In the MDS group, a positive correlation was found between ANG and IL-6 (P < 0.001) and also between MVD and IL-6 (P < 0.05). Using multivariate analysis, only IL-6 displayed independent prognostic value and was inversely related to MDS survival.

Pleural effusions in hematologic malignancies.

Nearly all hematologic malignancies can occasionally present with or develop pleural effusions during the clinical course of disease. Among the most common disorders are Hodgkin and non-Hodgkin lymphomas, with a frequency of 20 to 30%, especially if mediastinal involvement is present. Acute and chronic leukemias, myelodysplastic syndromes, are rarely accompanied by pleural involvement. Furthermore, 10 to 30% of patients receiving bone marrow transplantation develop pleural effusions. In cases of hematologic pleural effusions, drug toxicity, underlying infectious, secondary malignant or rarely autoimmune causes should be carefully sought. In most cases, the pleural fluid responds to treatment of the primary disease, whereas resistant or relapsing cases may necessitate pleurodesis.

Expression of the proliferation-associated nuclear protein MIB-1 and its relationship with microvascular density in bone marrow biopsies of patients with myelodysplastic syndromes.

The myelodysplastic syndromes (MDS) are a group of disorders characterized by dysplastic hemopoiesis and an increased risk of leukemic transformation. The process of angiogenesis has been implicated in the pathogenesis and evolution of MDS. In this study the proliferative activity and extent of angiogenesis was examined in bone marrow samples from 54 patients with MDS in relation to clinicopathologic features. Cellular proliferation and microvascular density (MVD) were examined immunohistochemically, using the monoclonal antibody MIB-1 (Ki-67) and an anti-CD34 monoclonal antibody respectively. Serum concentrations of interleukin-6 (IL-6) were measured by ELISA. The results showed that the MIB-1 Labeling Index (MIB-1 LI), MVD and IL-6 increased significantly with advancing severity of disease. Among the MDS-FAB subtypes, MIB-1 LI, MVD and IL-6 were significantly higher in RAEB-t, RAEB and CMML in comparison to RA and RARS (p < 0.0001 in all cases). Similarly, MIB-1 and MVD were increased in patients with score 3 in comparison to scores 0 and 1 in the IPSS system (p < 0.05). All parameters studied were significantly higher in patients versus controls. We conclude that cellular proliferative activity and angiogenesis are associated with disease progression in MDS patients.

Evaluation of bone disease in multiple myeloma: a correlation between biochemical markers of bone metabolism and other clinical parameters in untreated multiple myeloma patients.

INTRODUCTION: Multiple myeloma (MM) is characterised by an uncoupled bone formation/resorption process resulting in osteolysis. Osteocalcin (OC) and bone-specific alkaline phosphatase (BAP) are markers of osteoblastic activity, whereas pyridinoline products and the cross-linked aminoterminal of type I collagen (NTx) reflect bone destruction. In this study, these markers were studied in relation to bone disease severity and other clinical parameters of MM activity. METHODS: Serum calcium, creatinine, CRP, beta 2 microglobulin (b(2)M), M-component, OC, BAP and free urine pyridoline (Pyd) and deoxypyridinoline (Dpd), free urine Dpd and NTx were determined in 38 newly diagnosed MM patients. X-ray examination defined the degree of bone involvement. Patients were classified according to the Durie-Salmon staging system. RESULTS: NTx, free urine Pyd + Dpd, and free urine Dpd increased with increasing degree of bone involvement. NTx was significantly higher in stages II and III compared to stage I (mean values: 100.7, 163.5 and 208.3 nmol BCE/mM creat, respectively, p < 0.002). Free urine Pyd + Dpd correlated positively with b(2)M and CRP. OC was increased in stages I and II compared to III (p < 0.005) and was inversely correlated with NTx, free urine Pyd + Dpd, and free urine Dpd alone. CONCLUSIONS: The measurement of bone turnover markers in MM provides significant information regarding disease progression and should be included in the evaluation of MM patients.

Clinical significance of circulating endothelial adhesion molecules (sE-selectin and sICAM) in untreated multiple myeloma patients.

BACKGROUND: The expression of adhesion molecules is important for the interaction of myeloma cells with the bone marrow microenvironment. In the current study, serum soluble adhesion molecules (sICAM-1 and sE-selectin) were measured in untreated multiple myeloma (MM) patients in relation with other markers of disease activity. MATERIALS AND METHODS: The study group consisted of 67 patients with MM (classified according to the Durie-Salmon classification) and 15 controls. Interleukin-6 (IL-6), sICAM-1 and sE-selectin concentrations were determined by enzyme-linked immunosorbent assay (ELISA). In addition, the monoclonal protein, erythrocyte sedimentation rate (ESR) and hemoglobin (Hb) concentration were also determined. RESULTS: Serum sICAM-1 level increased significantly at advanced stages of MM and was higher in comparison to controls (p<0.01). sE-selectin increased significantly with advancing stage of the disease, but did not differ from controls. IL-6, ESR and M-component were significantly higher and Hb concentrations lower with advancing stage of disease. There was a positive correlation of IL-6 with sICAM-1 and sE-selectin. CONCLUSIONS: We conclude that serum sICAM-1 differs in multiple myeloma patients from normals and together with sE-selectin increase in parallel to increasing stage of disease, which may reflect a dysregulation and possible involvement of these adhesion molecules in myeloma progression.

Acquired inhibitors to coagulation factors in patients with gastrointestinal diseases.

OBJECTIVES: To study the frequency and specificity of acquired coagulation inhibitors in inflammatory and malignant gastrointestinal diseases. METHODS: In a 10-year period, 511 patients from the island of Crete in Greece were studied, 302 with ulcerative colitis, 112 with Crohn's disease, 82 with gastrointestinal carcinoma and 15 with gastrointestinal lymphoma. Prothrombin time and activated partial thromboplastin time were measured by routine methods. When prothrombin time and/or activated partial thromboplastin time were found to be prolonged, mixture experiments with 25%, 50% and 75% pooled normal human plasma were performed. If clotting times were inadequately corrected, the presence of an acquired inhibitor against a coagulation factor was suggested. Specific coagulation factor assays were then performed with deficient plasmas. RESULTS: Fifteen patients acquired inhibitors to the following coagulation factors within the 10-year observation period: factor IX (four patients); factor X (three patients); factor XII (three patients); factor VIII (two patients); factor XI (two patients); and factor V (one patient). The activity of the above factors varied from < 1% to 10%. Five patients with ulcerative colitis, six with Crohn's disease, two with gastrointestinal lymphoma and two with gastrointestinal carcinoma developed an inhibitor. Only one patient with factor VIII inhibitor presented with severe bleeding and was treated with recombinant human activated factor VII, while the others had no complications. Remission was obtained in all patients after immunosuppressive therapy, chemotherapy or tumour resection. CONCLUSION: An increased incidence of coagulation factor inhibitors was found in patients with gastrointestinal inflammatory and malignant diseases compared to the healthy population. In addition, an increased incidence of these inhibitors was also found in the common population of Crete compared to that found in other areas.

Levels of serum cytokines and acute phase proteins in patients with essential and cancer-related thrombocytosis.

Essential thrombocytosis (ET) is a myeloproliferative disorder resulting in an increased production of abnormal platelets. Reactive thrombocytosis (RT) is occasionally observed in clinical situations including chronic inflammation and malignancy. The aim of the present study was to evaluate the discriminatory efficiency of various laboratory tests in patients with ET and cancer-related RT. Forty-five patients with ET, 52 patients with RT, and 25 age-matched normal individuals comprised the study population. Plasma interleukin-1 alpha (IL-1a), IL-2, IL-6, tumor necrosis factor alpha (TNF-a), platelets, hematocrit, hemoglobin, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), lactate dehydrogenase (LDH) and ferritin were determined. We found increased levels of ferritin, LDH, CRP, ESR, IL-1a, and IL-6 in RT compared with ET (p < 0.01 to p < 0.0005). Hemoglobin, hematocrit, and platelets were significantly lower in RT than in ET (p < 0.0005). Furthermore, ferritin and ESR were negatively correlated with Hct, hemoglobin, and TNF-a, whereas ferritin was positively correlated with ESR, IL-1a, IL-6, and CRP, and IL-1a was positively correlated with IL-6. We consider that the aforementioned parameters should be included in the investigation of unexplained thrombocytosis for the differentiation of essential from cancer related thrombocytosis.

Septic shock due to visceral leishmaniasis, probably transmitted from blood transfusion.

A case of visceral leishmaniasis (VL) in a 77-year-old woman, with renal failure on haemodialysis, admitted in the intensive care unit (ICU) with vascular instability requiring vassopressor treatment, is presented. Initially, no co-infection could be detected. The patient initially responded well when liposomal amphotericin B was administered, after bone marrow demonstrated multiple intra-cellular Leishmania amastigotes and extra-cellular promastigotes. However, the patient died from uncontrolled septic shock from a secondary bacterial infection, the tenth day of admission. To our knowledge, vascular instability has not been reported in VL. Moreover, non-vector transmission was also suspected in this case. The patient had undergone cholecystectomy three months earlier, during which two blood units had been transfused; IgG anti-Leishmania antibodies at a high titer were detected in one of the two healthy blood donors, later.

Markers of cell activation and apoptosis in bone marrow mononuclear cells of patients with autoimmune hepatitis type 1 and primary biliary cirrhosis.

BACKGROUND/AIMS: We have reported quantitative and qualitative differences in bone marrow (BM) progenitor cells in autoimmune hepatitis type-1 (AIH-1) and primary biliary cirrhosis (PBC). This study investigated the apoptotic features and cytokine suppressors of haematopoiesis in long-term cultures of BM mononuclear cells (BMMCs) from AIH-1 and PBC patients. METHODS: Apoptotic markers and CD14 expression were evaluated in 13 AIH-1 patients, 13 PBC patients, 12 cirrhotic controls and 10 healthy subjects. TNF-alpha, TGF-beta and IFN-gamma were determined using ELISAs. RESULTS: All apoptotic markers and CD14 were increased in AIH-1 and PBC compared to controls (P<0.0001). Fas+ cells were positively correlated (P=0.0001) with apoptotic cells in AIH-1 and PBC. TNF-alpha and IFN-gamma were higher in AIH-1 (P=0.003 and P=0.001) and PBC (P=0.0001) compared to controls. No differences were found between the control groups. CONCLUSIONS: We demonstrate for the first time that the apoptotic process, macrophage activation and the production of cytokine suppressors of haematopoiesis in BMMCs from AIH-1 and PBC patients are higher compared to controls. The Fas-FasL pathway is likely to be involved in the apoptotic process; the increased levels of selected cytokines may contribute to Fas-FasL stimulation. Cirrhosis appears unlikely to be the cause of the above findings.

Autoimmune hepatitis type 1 and primary biliary cirrhosis have distinct bone marrow cytokine production.

We have recently reported differences in the hematopoiesis between autoimmune hepatitis type 1 (AIH-1) and primary biliary cirrhosis (PBC). In view of the notion that cytokines are regulators of hematopoiesis, we investigated in our tertiary center the cytokine production in the bone marrow (BM) of the same consecutive cohort of patients (13 AIH-1, 13 PBC, 10 healthy and 7 patients with cirrhosis due to chronic hepatitis B). Interferon-gamma (IFN-gamma), interleukin-4 (IL-4), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) were determined in the supernatants of long-term BM cultures by ELISAs. IL-4, TNF-alpha and TGF-beta were found significantly increased in the BM of PBC patients compared to AIH-1 and both control groups. AIH-1 patients had significantly higher BM IL-10 compared to PBC patients and higher IL-10, IL-4 and TNF-alpha compared to controls. BM IFN-gamma was significantly higher in PBC and AIH-1 patients compared to controls. In AIH-1 patients, IL-10 was positively correlated with CD34+, CD34+/CD38- and CD34+/CD38+ cell proportions. In conclusion, the BM cytokine microenvironment of PBC and AIH-1 patients differs significantly compared to that of healthy individuals and cirrhotic patients of non-autoimmune etiology. Differences were also found between patients with PBC and AH-1. The implication of BM in the pathogenesis of autoimmune liver diseases is possible and needs further investigation.

The isoflavone genistein inhibits proliferation and increases histamine content in human leukemic mast cells.

Mast cells are involved in allergic inflammation and some rare disorders such as systemic mastocytosis and mast cell leukemia. Certain naturally occurring flavonoids have been shown to inhibit mast cell activation and promote maturation of secretory granules. Here, we report that the isoflavone genistein inhibited the growth of human leukemic mast cells (HMC-1) by 68.8, 51.6, and 30.2% at 10(-4), 10(-5), and 10(-6) M, respectively, at day 3 (p < 0.001). Genistein at 10(-4) M increased the histamine content per 2 x 10(5) cells at day 3 from 5.9 +/- 1.2 micrograms/mL to 11.1 +/- 1.3 micrograms/mL (n = 6; p < 0.0001). These results indicate that genistein can inhibit proliferation and induce maturation of HMC-1 cells.

Ki-67 proliferation index: correlation with prognostic parameters and outcome in multiple myeloma.

The nuclear protein Ki-67 is a proliferation index, as it is expressed only by dividing cells. In this study, we investigated the clinical significance of Ki-67 determination on bone marrow biopsies of 35 patients with newly diagnosed multiple myeloma (MM). We examined the correlation of Ki-67 with other MM proliferation-related factors: interleukin-6 (IL-6), IL-10, bone marrow infiltration by plasma cells, serum lactate dehydrogenase (LDH), and beta 2 microglobulin (b2M). Ki-67 expression was also correlated with the survival rate of the patients. The results showed that Ki-67 expression increases with increasing stage of disease according to Durie-Salmon (classification stage III vs. I and II, p < 0.001). Furthermore, infiltration, IL-6, LDH, and b2M increase significantly with advancing stage of disease (p < 0.004). All parameters studied were significantly higher in patients versus controls. Ki-67 correlated with IL-6 (r: 0.422, p < 0.01), LDH (r: 0.437, p < 0.01), and b2M (r: 0.478, p < 0.004). There was a marked difference in survival between patients with MM with Ki-67 greater than 8% and patients with Ki-67 less than 8%, in favor of the latter (p < 0.07). We conclude that Ki-67 determination during routine pathological analysis of bone marrow in newly diagnosed MM could provide useful information about the proliferative activity and prognosis of the disease.

Hemopoietic progenitor cells and bone marrow stromal cells in patients with autoimmune hepatitis type 1 and primary biliary cirrhosis.

BACKGROUND/AIMS: Autologous hematopoietic stem cell transplantation has been used in severe cases of autoimmunity. We investigated whether hemopoietic progenitor cells and/or bone marrow (BM) microenvironment are affected in autoimmune hepatitis type-1 (AIH-1) and primary biliary cirrhosis (PBC). METHODS: We studied 13 AIH-1 patients, 13 PBC patients, 12 cirrhotic controls (CC) and ten healthy controls (HC). Flow cytometry, expansion cultures, long-term BM cultures and clonogenic progenitor cell assays were used. Stromal cell function was assessed in long-term BM cultures recharged with normal CD34+ cells. RESULTS: AIH-1 had increased CD34+, CD34+/CD38+ and CD34+/CD38- cells compared to all groups (P<0.001). PBC had lower progenitor cells compared to controls (P<0.005). No differences were found between CC and HC. Committed progenitor cells in non-adherent cell fraction were increased in AIH-1 (P<0.05) but decreased in PBC compared to controls (P<0.05). Granulocyte-macrophage colony forming units (CFU) and erythroid-burst CFU were increased in AIH-1 compared to all groups (P<0.001). PBC had these CFUs decreased compared to controls (P<0.005). Stromal cells failed to support normal hemopoiesis in PBC. CONCLUSIONS: We demonstrated for the first time that AIH-1 had increased hemopoietic progenitor cells and normal stromal function. In PBC, progenitor cells and BM microenvironment were defective. Further studies will determine the significance of these novel findings.

Bone marrow microvascular density and angiogenic growth factors in multiple myeloma.

There is evidence that angiogenesis plays an important role in the progression of multiple myeloma (MM). Hepatocyte growth factor (HGF) and tumor necrosis factor-alpha (TNF-alpha) are cytokines that potently stimulate angiogenesis. We evaluated the microvascular density (MVD) of bone marrow biopsies (after immunostaining with anti-CD34 antibodies) and serum levels of HGF and TNF-alpha in 43 patients with newly diagnosed MM. Twenty-four of these patients reached a plateau phase after treatment and were reevaluated for MVD, HGF and TNF-alpha. MVD values and serum levels of HGF and TNF-alpha were elevated in newly diagnosed MM patients in comparison with healthy controls. Pre-treatment MVD, HGF and TNF-alpha increased with advancing stage of MM disease. In patients reaching the plateau phase, a significant reduction in MVD, HGF and TNF-alpha levels occurred. A positive correlation was noted between pre-treatment MVD and serum levels of TNF-alpha and lactic dehydrogenase but not with HGF. However, HGF strongly correlated with beta2-microglobulin (beta2M), TNF-alpha and lactate dehydrogenase (LDH). We conclude that angiogenesis in MM, as expressed by the bone marrow MVD and the serum levels of angiogenic molecules such as HGF and TNF-alpha, increases with advancing clinical stage and decreases after effective chemotherapy.

Serum level of interleukin-16 in multiple myeloma patients and its relationship to disease activity.

Interleukin-16 (IL-16) is a chemoattractant of CD4+ lymphocytes, and it has been implicated in the pathogenesis of various inflammatory diseases. There is evidence that it may have a role in multiple myeloma (MM). In the present study, we determined the serum level of IL-16 both before and after treatment of MM and related it to inflammatory markers and survival. Forty-eight newly diagnosed MM patients were included in the study. Disease stage was defined using the Durie-Salmon classification system (10 patients were in stage I, 19 in stage II, and 19 in stage III). After standard treatment, 22 patients reached the plateau phase and were re-evaluated. The following serum parameters were measured: IL-16, IL-6, alpha-1 antitrypsin (alpha1AT), and C-reactive protein (CRP). Survival was determined as the number of months elapsed since original diagnosis. The mean +/- SD of serum IL-16 was 343 +/- 195 pg/ml in the pre-treatment MM group and 101 +/- 30 pg/ml in the control group. All measured parameters were higher in the patient group compared to healthy controls. Furthermore, IL-16, IL-6, alpha1AT, and CRP were significantly increased with increasing stage of disease, from stage I to stage III (P<0.01). All parameters decreased significantly following effective chemotherapy (P<0.002). Patients with a high level of IL-16 (>430 pg/ml) displayed an inferior survival time in comparison to those with lower levels of IL-16. In the pre-treatment group, IL-16 correlated with alpha1AT and IL-6 (r=0.374, P<0.01 and r=0.454, P<0.002, respectively). IL-16 may play a role in multiple myeloma; however, further functional studies are required.