Increased Level of Serum Hepcidin in Female Adolescent Athletes.

OBJECTIVE: To determine the serum hepcidin concentration and standard hematological parameters in a group of female adolescent athletes, compared with a group of nonathlete females. DESIGN: A case-control study. SETTING: A senior high school for athletes in Gothenburg, Sweden. PARTICIPANTS: All female athletes (70), at the school were offered to take part. Fifty-six athletes accepted. From a random sample of age-matched nonathletes, 71 students were recruited to the control group. MAIN OUTCOME MEASURES: Iron deficiency (ID) was determined by levels of serum iron, total iron-binding capacity, transferrin saturation (TS), and ferritin. Serum hepcidin was determined by a mass spectrometry method. All samples were taken at least 12 hours after training. RESULTS: The main result was the finding of a significantly elevated serum hepcidin level in the athlete group, 4.7 nmol/L compared with 3.3 nmol/L (P < 0.001) in the nonathlete group. In the athlete group, the serum iron concentration was significantly lower, 14.0 µmol/L compared with 17.6 µmol/L (P = 0.003) in the nonathlete group. No difference was found regarding TS, total iron binding capacity, and ferritin. There was no difference in the occurrence of ID or iron deficiency anaemia (IDA). CONCLUSIONS: These findings show an increase in serum hepcidin in a large group of female athletes. The elevated hepcidin levels may affect the iron balance of the athletes, adding to the traditional explanation of dietary intake/iron loss balance.

Minimal residual disease assessment in childhood acute lymphoblastic leukaemia: a Swedish multi-centre study comparing real-time polymerase chain reaction and multicolour flow cytometry.

Minimal residual disease (MRD) assessment is a powerful prognostic factor for determining the risk of relapse in childhood acute lymphoblastic leukaemia (ALL). In this Swedish multi-centre study of childhood ALL diagnosed between 2002 and 2006, the MRD levels were analysed in 726 follow-up samples in 228 children using real-time quantitative polymerase chain reaction (RQ-PCR) of rearranged immunoglobulin/T-cell receptor genes and multicolour flow cytometry (FCM). Using an MRD threshold of 0·1%, which was the sensitivity level reached in all analyses, the concordance between RQ-PCR and FCM MRD values at day 29 was 84%. In B-cell precursor ALL, an MRD level of ≥0·1% at day 29 predicted a higher risk of bone marrow relapse (BMR) with both methods, although FCM was a better discriminator. However, considering the higher median MRD values achieved with RQ-PCR, a higher MRD cut-off (≥0·2%) improved the predictive capacity of RQ-PCR. In T-ALL, RQ-PCR was notably superior to FCM in predicting risk of BMR. That notwithstanding, MRD levels of ≥0·1%, detected by either method at day 29, could not predict isolated extramedullary relapse. In conclusion, the concordance between RQ-PCR and FCM was high and hence both methods are valuable clinical tools for identifying childhood ALL cases with increased risk of BMR.

T-cell-mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura.

Chronic idiopathic thrombocytopenic purpura (ITP) is a bleeding disorder that is characterized by increased platelet destruction and is believed to be autoantibody mediated. In this study, CD3+ T cells from ITP patients had increased expression of genes involved in cell-mediated cytotoxicity. In addition, cytotoxic cell-mediated lysis of autologous platelets was shown in active ITP. Our data suggest that T-cell-mediated cytotoxicity is an alternative mechanism for platelet destruction in ITP.

BCR-ABL1 transcript levels increase in peripheral blood but not in granulocytes after physical exercise in patients with chronic myeloid leukemia.

In chronic myeloid leukemia (CML) treatment response is determined by measurements of BCR-AB1L transcripts in peripheral blood by quantitative real-time PCR (qRT-PCR) and a 2-5 fold increase is considered a warning sign. The BCR-ABL1 gene is mainly expressed in myeloid cells whereas quantification of BCR-ABL1 is performed on the nucleated cell fraction of peripheral blood. Hence, leukocyte composition of the nucleated cell fraction may affect the result of BCR-ABL1 quantification. The aim of this study was to investigate if changes in leukocyte composition of peripheral blood had any effect on BCR-ABL1 transcript levels in CML patients. Six CML patients in complete cytogenetic remission (CCgR) performed a maximal physical exercise test. Blood samples were collected before exercise, at maximal exhaustion and after exercise. A biphasic increase in leukocyte count was observed and the relative proportion of granulocytes in peripheral blood changed significantly after exercise compared with baseline (p < 0.001). The BCR-ABL1 transcript level increased significantly following exercise, in nucleated cell fraction of peripheral blood (p < 0.05) but not in isolated granulocytes. In the nucleated cell fraction, the mean BCR-ABL1 transcript level was 3.3-fold (range 0.7-6.8) higher 180 min after exercise compared with baseline (p < 0.01). In conclusion, physical exercise induced significant increases in BCR-ABL1 transcript levels concomitant with changes in leukocyte content of peripheral blood. We therefore suggest that variations in leukocyte composition of peripheral blood, causing pre-analytic variations that affect BCR-ABL1 quantification, have to be accounted for. Consequently, small variations in BCR-ABL1 transcript levels should be interpreted cautiously in CML patients in CCgR.

First case of human "Candidatus Neoehrlichia mikurensis" infection in a febrile patient with chronic lymphocytic leukemia.

An immunocompromised patient presented with febrile episodes, an erysipelas-like rash, and thromboembolic complications. Amplification of 16S rRNA gene sequences from blood and sequence analysis revealed "Candidatus Neoehrlichia mikurensis." We report the first case of human disease caused by "Ca. Neoehrlichia mikurensis."

Recruitment of T cells into bone marrow of ITP patients possibly due to elevated expression of VLA-4 and CX3CR1.

In idiopathic thrombocytopenic purpura (ITP), platelets are destroyed in the spleen, liver, and bone marrow (BM) by autoantibodies and cytotoxic T cells. In a DNA microarray screen of peripheral blood T cells, we found that VLA-4, CX3CR1, and CXCR4, involved in T-cell homing, had increased expression in ITP patients compared with controls. However, we only found increased protein expression of VLA-4 on T cells from peripheral blood by flow cytometry. To address a possible recruitment of T cells into the organs involved in platelet destruction, we analyzed T cells in BM. In BM, T-cell surface expression of VLA-4 and CX3CR1 was increased in ITP patients compared with controls. Furthermore, the number of CD3(+) T cells in BM, but not in blood, was increased in ITP patients compared with controls. This finding was confirmed by immunohistochemistry of BM biopsies. The number of regulatory T cells (CD4(+)/CD25(bright)) was decreased in the BM of ITP patients, whereas Fas expression was increased. In conclusion, ITP is associated with accumulation and activation of T cells in the BM. Recruitment of T cells into the target organ (eg, BM) is plausible and may be facilitated through increased VLA-4 and CX3CR1 expression. These molecules might serve as new treatment targets in ITP.

Applicability of IG/TCR gene rearrangements as targets for minimal residual disease assessment in a population-based cohort of Swedish childhood acute lymphoblastic leukaemia diagnosed 2002-2006.

Minimal residual disease (MRD) detection during the early treatment phase has become an important stratification parameter in many childhood acute lymphoblastic leukaemia (ALL) treatment protocols. Here, we aimed to address the applicability of rearranged antigen-receptor genes as potential MRD markers using real-time quantitative polymerase chain reaction (RQ-PCR) in a Swedish population-based cohort. From 334 childhood ALL cases diagnosed during 2002-2006, we analysed 279 diagnostic samples (84%) by screening for rearranged immunoglobulin (IG) and T-cell receptor (TCR) genes. Allele-specific oligonucleotides were designed, and the sensitivity and quantitative level was determined for each target. Overall, clonal IG/TCR rearrangements were detected in 97% (236/244) of B-cell precursor ALL (BCP ALL) and 94% (33/35) of T-ALL. A sensitive RQ-PCR analysis (< or = 10(-4)) was obtained in 89% (216/244) of BCP ALL and in 74% (26/35) of T-ALL, whereas two sensitive targets were only available in 47% (115/244) of BCP ALL and 29% (10/35) of T-ALL cases. With the stratification threshold of > or = 10(-3), which is applied in the current Nordic treatment protocol (NOPHO-ALL 2008) for the identification of high-risk patients, 93% of BCP ALL and 86% of T-ALL reached this quantitative range by at least one target gene. Taken together, this national retrospective study demonstrates that an IG/TCR target for MRD monitoring can be identified in the majority of childhood ALL cases, whereas identification of a second sensitive target gene needs to be improved.

In vitro cellular drug sensitivity at diagnosis is correlated to minimal residual disease at end of induction therapy in childhood acute lymphoblastic leukemia.

Leukemic cells from 85 children with newly diagnosed precursor B-lineage ALL were tested for in vitro drug sensitivity to a panel of anti-cancer drugs. Minimal residual disease (MRD) was measured by RQ-PCR. There was a significant correlation between MRD day 29 and in vitro sensitivity to prednisolone (p<0.001) and doxorubicin (p=0.017), drugs administered during induction therapy. In patients with t(12;21) (n=20), in vitro sensitivity to doxorubicin was an independent factor for MRD <0.1% (p=0.031; R(2)=0.66). Thus, data show that in vitro drug sensitivity at diagnosis is correlated to cell kill during induction therapy as measured by MRD day 29.

Quality control of flow cytometry data analysis for evaluation of minimal residual disease in bone marrow from acute leukemia patients during treatment.

Low levels of leukemia cells in the bone marrow, minimal residual disease (MRD), are considered to be a powerful indicator of treatment response in acute lymphatic leukemia (ALL). A Nordic quality assurance program, aimed on standardization of the flow cytometry MRD analysis, has been established before implementation of MRD at cutoff level 10 as one of stratifying parameters in next Nordic Society of Pediatric Hematology and Oncology (NOPHO) treatment program for ALL. In 4 quality control (QC) rounds 15 laboratories determined the MRD levels in 48 follow-up samples from 12 ALL patients treated according to NOPHO 2000. Analysis procedures were standardized. For each QC round a compact disc containing data in list-mode files was sent out and results were submitted to a central laboratory. At cutoff level 10, which will be applied for clinical decisions, laboratories obtained a high concordance (91.6%). If cutoff level 10 was applied, the concordance would be lower (85.3%). The continuing standardization resulted in better concordance in QC3 and QC4 compared with QC1 and QC2. The concordance was higher in precursor B as compared with T-cell ALL. We conclude that after standardization, flow cytometry MRD detection can be reliably applied in international, multicenter treatment protocols.

Serum biomarkers for atrophic gastritis and antibodies against Helicobacter pylori in the elderly: Implications for vitamin B12, folic acid and iron status and response to oral vitamin therapy.

OBJECTIVES: To investigate the prevalence of serological markers for chronic atrophic gastritis (AG) and Helicobacter pylori antibodies (HPAb) in an elderly population, and to examine the interrelationship and significance for cobalamin, folic acid and iron status and response to oral vitamin therapy. MATERIAL AND METHODS: The study included community-dwelling subjects (n=209), mean age 76 years, randomized to 4 month of oral daily treatment with 0.5 mg cyanocobalamin, 0.8 mg folic acid and 3 mg vitamin B(6) or placebo (double-blind). Biochemical tests were carried out before and after treatment. RESULTS: AG, as indicated by a pepsinogen I/II ratio <2.9, occurred in 14% (26/190) and HPAb in 54% (102/190) of the subjects. AG subjects had higher levels of serum methylmalonic acid (MMA) (p<0.001), plasma homocysteine (tHcy) (p<0.05), lower haemoglobin (Hb) (p<0.01) and a higher prevalence of vitamin B(12) deficiency (p<0.01). HPAb was associated with AG, whereas AG subjects without HPAb had higher tHcy and MMA levels. There was no correlation between AG and iron status. Oral vitamin treatment led to greater (albeit non-significant) improvements in MMA, tHcy and total cobalamins in AG subjects compared to non-AG subjects. CONCLUSIONS: AG is a common condition and is a significant determinant of vitamin B(12) status. AG is correlated to HPAB and lower Hb. Elderly AG subjects respond at least as well as non-AG subjects to oral treatment with B-vitamins in the doses employed.

The expression of NK cell inhibitory receptors on cytotoxic T cells in B-cell chronic lymphocytic leukaemia (B-CLL).

Immune surveillance of tumours is mediated by cytotoxic T cells (CTL) that recognise tumour antigen. Reduced reactivity of CTL towards tumour cells could thus lead to disease progression and loss of tumour control. In B-cell chronic lymphocytic leukaemia (B-CLL), the function of tumour-reactive CTL seems to correlate inversely to disease stage. Inhibitory NK cell receptors are known to suppress the CTL response upon interaction with major histocompatibility complex (MHC) class I and increased expression of such receptors on CTL may inhibit the anti-tumour response. So, the aim of this study was to investigate the expression of NK cell inhibitory receptors on CTL in B-CLL patients and if such expression correlated to disease stage. CD8+ T cells from B-CLL patients in Binet stage A (n = 26) and stage C (n = 14) and healthy controls (n = 14) were analysed for the expression of killer immunoglobulin-like receptors (KIR) CD158a (KIR2DL1), CD158b (KIR2DL2), CD158e (KIR3DL1) and the C-type lectin receptor CD94, by flow cytometry analysis. Patients with advanced disease (Binet stage C) had a significantly greater percentage of CTL expressing CD158b, CD158e and CD94 than patients with non-progressive disease (Binet stage A) and healthy controls. Stage C patients also had a significantly higher percentage of CTL expressing CD158a than stage A patients. No statistically significant differences were found between Binet A patients and healthy controls. Our results suggest that increased expression of KIR and CD94 on CTL in advanced stage B-CLL may potentially contribute to the impaired anti-tumour immune response in these patients.

Early hematopoiesis in multiple sclerosis patients.

Contemporary evidence supports that MS immunopathology starts in the peripheral lymphatic system. However, the site and character of crucial initiating events are unknown. We examined subsets of the first stages of blood cells in the bone marrow of 9 MS patients and 11 neurologically healthy controls using FACS analysis. The proportion of natural killer T cells was lower (P=0.045) in the bone marrow of MS patients, but proportions of hematogenous stem cells, myeloblasts, and B cell precursor subsets in the bone marrow did not differ between MS patients and controls. In this pilot study with a limited number of samples we found no deviation of the early B cell lineage in bone marrow from MS patients.

Disturbed apoptosis of T-cells in patients with active idiopathic thrombocytopenic purpura.

Idiopathic thrombocytopenic purpura (ITP) is an organ specific autoimmune disorder in which T-lymphocyte abnormalities have pathogenetic importance. In a DNA microarray screen of CD3+ T-lymphocytes from ITP patients and healthy controls we found an altered expression of genes associated with apoptosis, e.g. A20, caspase-8 and Bax. This together with our previous findings of increased gene expression of Fas, interferon-g and IL-2 receptor beta (IL2RB) indicated an altered activation induced cell death (AICD) of T-cells in ITP. Using a proliferation assay we found that CD3+ lymphocytes from ITP patients were significantly more resistant to dexamethasone induced suppression compared to normal lymphocytes. We also found that cultured CD3+ lymphocytes from ITP patients in remission were more susceptible to apoptosis both in the presence and absence of dexamethasone compared to cells from patient with active ITP and healthy controls, as indicated by increased staining of AnnexinV binding. Our findings suggest that apoptotic resistance of activated T-lymphocytes in patients with active ITP may lead to defective clearance of autoreactive T-lymphocytes through AICD, which might cause a continued immune destruction of platelets. Conversely, a loss of resistance to AICD in ITP patients in remission might be an important mechanism for the achievement of remission.

Serum estradiol associates with blood hemoglobin in elderly men: the MrOS Sweden study.

CONTEXT: Blood hemoglobin (Hb) declines with age in healthy elderly men, in whom decreasing T has been regarded as part of normal aging. However, the association between Hb and serum estradiol is incompletely known. OBJECTIVE: To determine whether estradiol is associated with anemia/Hb and established determinants of Hb in elderly men without prostate cancer. DESIGN, SETTING, AND PARTICIPANTS: The MrOS (Osteoporotic Fractures in Men) is a population-based study (n = 918; median age, 75.3 y; range, 70-81 y). MAIN OUTCOME MEASURES: We evaluated total estradiol in relation to Hb and adjusted for potential confounders (ie, age, body mass index [BMI], erythropoietin [EPO], total T, cystatin C, and iron and B-vitamin status). RESULTS: Estradiol correlated negatively with age (r = -0.14; P < .001). Hb correlated (age adjusted) positively with estradiol (r = 0.21; P < .001) and T (r = 0.10; P < .01). Independent predictors for Hb in multivariate analyses were estradiol, EPO, BMI, transferrin saturation, cystatin C, and free T4, but not T. After exclusion of subjects with Hb <130 g/L and/or T < 8 nmol/L (n = 99), the correlation between Hb and T was no longer significant, whereas the associations between Hb and estradiol remained. After adjusting for age, BMI, and EPO, men with lower estradiol levels were more likely to have Hb in the lowest quartile of values (odds ratio per SD decrease in estradiol = 1.61 [95% confidence interval, 1.34-1.93]). Anemic subjects (Hb < 130 g/L) had lower mean estradiol than nonanemic subjects (67.4 vs 79.4 pmol/L; P < .001). CONCLUSIONS: Estradiol correlated positively and independently with Hb. Decreased estradiol might partly explain the age-related Hb decline observed in healthy elderly men.

Holotranscobalamin is not influenced by decreased renal function in elderly men: the MrOS Sweden study.

BACKGROUND: Subclinical cobalamin deficiency is common in the elderly, but the sensitivity and specificity of serum total cobalamin for this diagnosis is poor. Serum holotranscobalamin (holoTC), a measure of biologically available cobalamin, is considered a better marker for early cobalamin depletion than total cobalamin. However, in elderly populations, health-related reference intervals for holoTC and correlations to renal function are not entirely clear. METHODS: HoloTC was determined with an automated microparticle enzyme immunoassay (AxSYM®) in 790 elderly non-vitamin-supplemented Swedish men, median age 75.3 years. Renal function was assessed with creatinine, cystatin C and estimated glomerular filtration rate (eGFR calculated from creatinine). RESULTS: Median holoTC was 51.8 pmol/L, the health-related reference interval 19.6-132.3 pmol/L. There was no significant difference in mean holoTC in probands with normal compared to high creatinine (P = 0.80) and cystatin C (P = 0.82). No significant differences between the quartiles of creatinine or cystatin C in mean of log holoTC were seen. HoloTC correlated strongly with total cobalamin (r = 0.69, P < 0.001), weaker with eGFRcreatinine (r = -0.09, P < 0.05) and creatinine (r = 0.09, P < 0.05), the latter correlation was only seen in subjects with creatinine <100 µmol/L. HoloTC correlated negatively with plasma total homocysteine (r = -0.24, P < 0.001), but not with cystatin C and age. CONCLUSIONS: Serum holoTC in healthy elderly men shows the same distribution as earlier described for a younger reference population. In this group of elderly subjects, holoTC did not correlate to reduced renal function. Thus, holoTC appears to be a promising tool for evaluating cobalamin status also in elderly populations.

The autocrine motility factor receptor is overexpressed on the surface of B cells in Binet C chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a clinical spectrum reaching from discrete lymphocytosis to extensive enlargement of lymph nodes, spleen and liver, and bone marrow failure. The aim of this study was to identify genes that differentiate between patients with disease stage A vs. C according to Binet in order to better understand the disease. To achieve this, we performed DNA microarray analysis on B cells from CLL patients with stage A and C according to Binet and matched controls. Between CLL patients and controls, there were 1,528 differentially expressed genes and 360 genes were differentially expressed between Binet A and C patients. Due to the sheer number of regulated genes, we focused on the autocrine motility factor receptor (AMFR). AMFR has not previously been investigated in hematological disorders, but high expression of AMFR correlates with a more advanced stage and invasive potential in several human tumors. AMFR mRNA expression was higher in Binet A compared with Binet C patients (P=0.0053) and healthy controls (P=0.0051). Total AMFR protein was higher in Binet A patients compared to Binet C as analyzed by intracellular flow cytometry. However, AMFR exist both in the ER involved in protein degradation and on the cell surface involved in metastasis and cell motility. Cell surface AMFR was increased in Binet C compared with Binet A+B (P=0.016). In conclusion, the mRNA levels reflect the total amount of AMFR, whereas cell surface expression is associated with progression in CLL.

The FMCA-GM assays, high throughput non-clonogenic alternatives to CFU-GM in preclinical hematotoxicity testing.

One of the most common dose limiting adverse effects in cancer treatment is myelotoxicity. The aim of this study was to develop an in vitro method for measuring potential myelotoxic properties of a drug candidate in a high throughput setting. Human CD34(+) progenitor cells from umbilical cord blood were plated in 384-well microplates with drugs in liquid culture, supplemented with specific cytokines for the granulocytopoietic-macrophage lineage. After 7 or 14 days of proliferation and differentiation the cells were analyzed using the automated non-clonogenic fluorometric microculture cytotoxicity assay (FMCA). Two types of assays setups were evaluated, the FMCA-GM7 where cells were exposed to drugs directly after thawing and cytotoxicity measured on day 7 in contrast to the FMCA-GM14 where the cells were cultured 7 days prior to plating and drug exposure, with viability analysis on day 14 of differentiation. Drug sensitivity was similar in both assays and method validation was performed using 24 drugs with known myelotoxic profile (acyclovir, bortezomib, busulfan, carboplatin, chloramphenicol, chlorpromazine, cisplatin, cytarabine, clozapine, doxorubicin, erlotinib, etoposide, 5-fluorouracil, fludarabine, gefitinib, gemcitabine, hydroxyurea, imatinib, lomustine, melphalan, sorafenib, sunitinib, taxol and 6-thioguanine). The 50% inhibitory concentrations (IC(50)) from the FMCA-GM7 and the FMCA-GM14 correlated highly (r = 0.83) and (r = 0.82), respectively, with IC(50) from the established clonogenic assay (CFU-GM), obtained from the literature. The current data suggests that the FMCA-GM could offer a simple and robust alternative to the CFU-GM assay in preclinical hematotoxicity studies.

Newly diagnosed bladder cancer: the relationship of initial symptoms, degree of microhematuria and tumor marker status.

PURPOSE: We recorded initial symptoms and evaluated the frequency and intensity of hematuria in patients with newly diagnosed bladder cancer. We also evaluated and compared the sensitivity of bladder wash cytology, NMP22 (Matritech, Newton, Massachusetts), BTA Stat (Bion Diagnostic Sciences, Redmond, Washington) and UBC antigen (IDL Biotech, Sollentona, Sweden) with hematuria dipsticks and flow cytometry for determining the size of erythrocytes in urine. MATERIALS AND METHODS: Urine samples were collected from 92 patients with newly diagnosed bladder cancer, 64 with idiopathic microhematuria and 42 with nephritis. Urine was analyzed for NMP22, BTA Stat, UBC and erythrocytes size using flow cytometry. Bladder wash cytology was done at cystoscopy. Urine was analyzed for microhematuria with hematuria dipsticks at home for 7 consecutive days immediately before the operation and in the hospital on the day of surgery. RESULTS: Sensitivity was 75% for NMP22, 78% for BTA Stat, 64% for UBC and 61% for flow cytometry at 73% specificity. Cytology had 42% sensitivity at 97% specificity. Tumor size, grade and stage had a statistically significant influence on NMP22, BTA Stat, UBC and cytology. Of the patients 75% had microhematuria on the day of the operation and 75% had hematuria at least 1 of 7 days when tested at home the last week before transurethral bladder resection. The 70% of all patients with macroscopic hematuria as the initial symptom did not seem to differ from those without the condition in tumor size, grade, stage or tumor marker levels. CONCLUSIONS: Flow cytometry was not well enough able to distinguish patients with bladder cancer from controls. The sensitivity of all tested markers, including hematuria dipsticks, was high for large and high grade, high stage tumors. Further studies are needed to evaluate whether a marker could be used to determine priority among patients referred due to microhematuria.