Fetal hemoglobin induction during decitabine treatment of elderly patients with high-risk myelodysplastic syndrome or acute myeloid leukemia: a potential dynamic biomarker of outcome.

Hematologic responses to hypomethylating agents are often delayed in patients with myelodysplastic syndrome or acute myeloid leukemia. Fetal hemoglobin is a potential novel bio-marker of response: recently, we demonstrated that a high fetal hemoglobin level prior to decitabine treatment was associated with superior outcome. Here we investigated whether early fetal hemoglobin induction during decitabine treatment also had prognostic value, and studied the potential of decitabine to induce erythroid differentiation and fetal hemoglobin expression in vitro Fetal hemoglobin levels were measured by high-performance liquid chromatography in patients with higher-risk myelodysplastic syndrome (n=16) and acute myeloid leukemia (n=37) before treatment and after each course of decitabine. Levels above 1.0% were considered induced. Patients achieving complete or partial remission as best response had attained a median fetal hemoglobin of 1.9% after two courses of treatment, whereas the median value in patients who did not reach complete or partial remission was 0.8% (P=0.015). Fetal hemoglobin induction after two courses of decitabine treatment was associated with early platelet doubling (P=0.006), and its subsequent decrease with hematologic relapse. In patients with myelodysplastic syndrome, induction of fetal hemoglobin after course 2 of treatment was associated with longer overall survival: median of 22.9 versus 7.3 months in patients with or without induction of fetal hemoglobin, respectively [hazard ratio=0.2 (95% confidence interval: 0.1-0.9); P=0.03]. In vitro decitabine treatment of two bi-potential myeloid leukemia cell lines (K562 and HEL) resulted in induction of an erythroid (not megakaryocytic) differentiation program, and of fetal hemoglobin mRNA and protein, associated with GATA1 gene demethylation and upregulation. In conclusion, fetal hemoglobin may provide a useful dynamic biomarker during hypomethylating agent therapy in patients with myelodysplastic syndrome or acute myeloid leukemia.

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding.

Mutations in hemoglobin can cause a wide range of phenotypic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin α chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) → Leu mutation in α1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (∼16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the α H58L replacement. Mutant α subunits containing Leu-58(E7) autoxidize ∼8 times and lose hemin ∼200 times more rapidly than native α subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the α H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O2, promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant α subunit has an ∼80,000-fold higher affinity for CO than O2, causing it to rapidly take up and retain carbon monoxide, which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.

Elevated fetal haemoglobin is a predictor of better outcome in MDS/AML patients receiving 5-aza-2'-deoxycytidine (Decitabine).

Although azanucleoside DNA-hypomethylating agents (HMAs) are routinely used for the treatment of myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML), very few outcome predictors have been established. Expression of the ß-like globin gene locus is tightly regulated by DNA methylation, is HMA-sensitive in vitro, and fetal haemoglobin (HbF) expression is under study as a potential biomarker for response of MDS patients to azacitidine. We determined HbF expression in 16 MDS and 36 AML patients receiving decitabine (DAC). Pre-treatment HbF was already elevated (>1·0% of total haemoglobin) in 7/16 and 12/36 patients, and HbF was induced by DAC in 81%/54% of MDS/AML patients, respectively. Elevated pre-treatment HbF was associated with longer median overall survival (OS): 26·6 vs. 8·6 months for MDS (hazard ratio [HR] 8·56, 95% confidence interval [CI] 1·74-42·49, P = 0·008, with similarly longer progression-free and AML-free survival), and 10·0 vs. 2·9 months OS for AML (HR 3·01, 95% CI 1·26-7·22, P = 0·014). In a multivariate analysis, the prognostic value of HbF was retained. Time-dependent Cox models revealed that the prognostic value of treatment-induced HbF induction was inferior to that of pre-treatment HbF. In conclusion, we provide first evidence for in vivo HbF induction by DAC in MDS/AML, and demonstrate prognostic value of elevated pre-treatment HbF, warranting prospective, randomized studies.

Levels of Silicon in Maternal, Cord, and Newborn Serum and Their Relation With Those of Zinc and Copper.

BACKGROUND: Evidence of silicon's importance to health has been gradually accumulating. Nevertheless, there are few studies comparing serum silicon levels in newborns with maternal levels. Likewise, little is known concerning the inter-relation between silicon and other trace elements. OBJECTIVE: The present study evaluated maternal and newborn levels of serum silicon and their relation to those of zinc and copper. METHODS: We measured serum silicon, copper, and zinc in 66 pregnant women, in the umbilical cord of their infants, and in 44 newborns, by atomic absorption spectrophotometry. All the samples were from fasted subjects. RESULTS: Serum silicon level in term newborns (20.6 ±â€Š13.2 µmol/L) was significantly higher than in umbilical cord (8.9 ±â€Š3.5 µmol/L; P < 0.0001). Mean serum silicon level in maternal vein (7.7 ±â€Š3.4 µmol/L) was lower than that in umbilical cord, although differences were not significant. We also found higher levels of zinc (P = 0.008) and lower levels of copper (P < 0.0001) in cord blood compared with maternal blood. Umbilical venous/maternal venous level ratios of zinc, copper, and silicon were 1.5 ±â€Š0.5, 0.2 ±â€Š0.1, and 1.3 ±â€Š0.7, respectively. There was a positive correlation between silicon and zinc levels (r = 0.32), and a negative correlation between copper and zinc levels (r = -0.35). CONCLUSIONS: It seems that there is a positive gradient of silicon from the mother to her fetus. Silicon levels were higher in newborn than in cord blood, and correlated significantly with that of zinc but not copper. Additional investigations are needed to further define the role of silicon and its interaction with other trace elements during the perinatal period.

Hb A2-Konz [δ50(D1)Ser → Thr; HBD: c.151T > A]: a new δ chain hemoglobin variant characterized by mass spectrometry and high performance liquid chromatography.

We report a new slow-moving δ chain hemoglobin (Hb) variant, named Hb A2-Konz [δ50(D1)Ser → Thr; HBD: c.151T > A]. It was detected during simultaneous measurement of Hb A1C and Hb A2 by high resolution cation exchange high performance liquid chromatography (HPLC) using a PolyCATA column. Hb A2-Konz comprised 0.8% of total Hb. This new variant was identified by peptide mapping using nanoliquid chromatography electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS) as a serine to threonine substitution at δ50(D1), indicating that the variant was due to a single base change at codon 51 (TCT > ACT) of the δ-globin gene. The new mutant is clinically silent but could lead to a misdiagnosis of ß-thalassemia (ß-thal) based on the level of Hb A2.

Low perforin expression of early differentiated HCV-specific CD8+ T cells limits their hepatotoxic potential.

BACKGROUND & AIMS: Perforin plays a central role in the immunopathogenesis of different viral infections. However, its role in hepatitis C virus (HCV) infection has not been fully understood. Here, we analyzed two closely related questions: first, is CD8+ T cell-mediated killing of HCV-replicating human hepatoma cells mediated by perforin? Second, if so, do HCV-specific CD8+ T cells obtained from chronically HCV infected patients express and upregulate perforin? METHODS: Susceptibility of HCV-replicating human hepatoma cells to the cytotoxic pathway was tested in vitro by addition of perforin substitute streptolysin O and granzyme B and by co-culture experiments with a perforin-expressing HCV-specific CD8+ T cell clone in the presence of perforin or caspase inhibitors. HCV-specific CD8+ T cells were obtained and analyzed for perforin expression and differentiation markers ex vivo from 12 chronically infected patients and 12 patients with resolved HCV infection. RESULTS: HCV-replicating human hepatoma cells were susceptible to cytotoxic killing in vitro and a dominant role of perforin in HCV-specific CD8+ T cell-mediated cytolysis was observed. However, HCV-specific CD8+ T cells obtained ex vivo from chronically HCV infected patients expressed only low levels of perforin and showed an impaired ability to upregulate perforin. This was tightly linked to the distinct differentiation stage of HCV-specific CD8+ T cell differentiation ex vivo since early and intermediate differentiated HCV-specific CD8+ T cells only showed weak perforin expression in contrast to late differentiated CD8+ T cells that displayed strong perforin expression. CONCLUSIONS: Our results suggest that perforin plays a dominant role in CD8+ T cell-mediated lysis of HCV-replicating human hepatoma cells but that lysis may be limited in human chronic viral infection by the low perforin expression of early/intermediate differentiated HCV-specific CD8+ T cells.

Analysis of CD8+ T-cell-mediated inhibition of hepatitis C virus replication using a novel immunological model.

BACKGROUND & AIMS: Virus-specific CD8+ T cells are required for the control of hepatitis C virus (HCV) infection. We investigated the extent to which different effector functions of CD8+ T cells contribute to the inhibition of viral replication. METHODS: We developed a novel immunologic model by stably transducing the HLA-A2 gene into the replicon system, matching the epitope sequence of the replicon to the sequence targeted by an HCV-specific CD8+ T-cell clone. Luciferase activity was then measured to quantitate HCV RNA replication. RESULTS: HCV-specific CD8+ T cells strongly inhibited viral replication, through cytolytic and noncytolytic mechanisms, in a dose-dependent manner. HCV replication was almost completely inhibited at an effector-to-target ratio of 1:1 with significant cytotoxicity; however, >95% viral inhibition occurred at ratios as low as 1:100. Importantly, no cytotoxicity was observed at low effector-to-target ratios, indicating a dominant effect of noncytolytic effector functions that was confirmed by Transwell experiments. Neutralization experiments revealed that interferon gamma mediates the noncytolytic inhibition. CONCLUSIONS: Only a very few HCV-specific CD8+ T cells are required to inhibit HCV replication; inhibition occurs primarily by noncytolytic effector functions.

The role of X-inactivation in the gender bias of patients with acquired alpha-thalassaemia and myelodysplastic syndrome (ATMDS).

Alpha thalassaemia myelodysplastic syndrome (ATMDS) is an unusual complication of chronic myeloid malignancy that is associated with a striking red cell phenotype. It represents an acquired form of alpha-thalassaemia that most commonly arises in the context of myelodysplasia. It has recently been shown that this condition occurs in association with somatic mutations of a known X-encoded trans-acting regulator of alpha globin gene (HBA) expression, ATRX. There is an unexplained, strong male preponderance of individuals with the ATMDS phenotype with a >5:1 male-female ratio and furthermore, all the somatic ATRX mutations described to date have been in males. Here we report the identification, in a single centre, of two females with ATMDS and mutations in the ATRX gene, proving that ATMDS associated with such mutations may occur, albeit rarely, in females. It seemed possible that females might be less likely to develop ATMDS if the inactivated copy of the ATRX gene (ATRX) became progressively re-activated throughout life. This study ruled out this hypothesis by investigating the pattern of ATRX inactivation in a cross-sectional analysis of normal females at ages ranging from newborn to 90 years.

Haemoglobin Noah Mehmet Oeztuerk (alpha(2) delta(2)143 (H21)His-->Tyr: A novel delta-chain variant in the 2,3-DPG binding site.

A new delta-chain variant, delta143 (H21) His-->Tyr or Hb Noah Mehmet Oeztuerk, was discovered during the investigation of the cause of hemolytic anaemia in a 6-month-old infant of Turkish descent. It was detected by Cation exchange high-performance liquid chromatography (CE-HPLC) using PolyCAT A column. P(50) was 20.6+/-0.60 mmHg and 29.3+/-0.40 mmHg for the carrier and the wild-type, respectively. This suggests an increase in oxygen affinity. On routine CE-HPLC Hb A(2) was low (1.2%) and the variant was not detected. An extended family study revealed that the variant was not associated with the anaemia or with any other clinical abnormality.

Clinical validation of a new blood collection tube for the accuracy of total homocysteine measurement by different methods.

OBJECTIVE: To evaluate the clinical use of Homocysteine-Primavette, a new blood collection medium for total homocysteine (tHcy) assay. METHODS: The agreement between baseline tHcy and tHcy in stabilized samples (40 h) was assessed for FPIA, HPLC, GC-MS, LC-MS, and ICL. RESULTS: tHcy concentrations in whole blood were stable for 40 h in Hcy-Primavette tubes. CONCLUSION: Primavette tubes are a good alternative for the accurate tHcy measurement and no readjustment of reference intervals is needed.

Biomarkers of exercise-induced myocardial stress in relation to inflammatory and oxidative stress.

Increased concentrations of biomarkers reflecting myocardial stress such as cardiac troponin I and T and brain natriuretic peptide (BNP) have been observed following strenuous, long-lasting endurance exercise. The pathophysiological mechanisms are still not fully elucidated and the interpretations of increased post-exercise concentrations range from (i) evidence for exercise-induced myocardial damage to (ii) non-relevant spurious troponin elevations, presumably caused by assay imprecision or heterophilic antibodies. Several lines of evidence suggest that inflammatory processes or oxidative stress could be involved in the rise of NT-proBNP and Troponin observed in critically ill patients with sepsis or burn injury. We tested the hypothesis that inflammatory or oxidative stress is also responsible for exercise-induced cardiomyocyte strain in a large cohort of triathletes following an Ironman triathlon. However, the post-race increase in cardiac troponin T and NT-proBNP was not associated with several markers of exercise-induced inflammation, oxidative stress or antioxidant vitamins. Therefore, we clearly need more studies with other inflammatory markers and different designs to elucidate the scientific background for increases in myocardial stress markers following strenuous endurance events.

Developing a reference system for the IFCC standardization of HbA2.

The importance of hemoglobin A2 (HbA2) as an indicator of the presence of ß-thalassemia was established many years ago. However, clinical application of recommended HbA2 cut off values is often hampered due to poor equivalence of HbA2 results among methods and laboratories. Thus, the IFCC standardization program for HbA2 was initiated in 2004 with the goal of achieving a complete reference system for this measurand. HbA2 standardization efforts are still in progress, including the development of a higher-order HbA2 reference measurement procedure and the preparation of a certified reference material in collaboration with the IRMM. Here, we review the past, present and future of HbA2 standardization and describe the current status of HbA2 testing.

Characterization of hemoglobin Würzburg (alpha2beta2 4(A1)Thr-->Asn), a new electrophoretically silent variant, by mass spectrometry and molecular modeling studies.

The first hemoglobin (Hb) variant carrying a mutation at beta4 was identified as beta4(A1)Thr-->Asn or Hb Würzburg and constituted 38% of the total hemoglobin. It showed a slightly elevated oxygen affinity and a slightly decreased cooperativity index (n50 = 2.3 versus n50 = 2.8). The analysis of the electrostatic potential showed an increased negative charge at the site of the mutation with a displacement of beta6(A3)Glu by 1.3A. The replacement of threonine by asparagine seems to stabilize the R conformation. This may explain partially both the high affinity and the reduction in cooperativity.

Factors influencing [F-18] 2-fluoro-2-deoxy-D-glucose (F-18 FDG) uptake in melanoma cells: the role of proliferation rate, viability, glucose transporter expression and hexokinase activity.

Using human (SK-MEL 23, SK-MEL 24 and G361) and murine (B16) melanoma cell lines, the coregulatory potential of the uptake of the positron emission tomography (PET) tracer, [Fluorine-18] 2-fluoro-2-deoxy-D-glucose (F-18 FDG) has been investigated in relationship to tumor characteristics. Comparative studies among the four melanoma cell lines demonstrated that the lowest FDG uptake in SK-MEL 24 corresponded strongly to the data for DT (population doubling time) and MTT (tetrazolium salt) cell viability as well as hexokinase (HK) activity, but was not related to the glucose transporter 1 (GLUT 1) expression level. Furthermore, the FDG uptake in each melanoma cell line measured by cell cycle kinetics was significantly positively correlated to both the proliferation index (PI=S/G2M phase fractions) and the cell viability, though with one exception relating to the PI of the lowest FDG uptake cell line, SK-MEL 24. No positive correlation was found between the expression of GLUT 1 and FDG uptake in any individual cell line. However, the HK activities in SK-MEL 23 and 24 showed considerable positive relationships with FDG uptake. Our present study suggests that both the proliferation rate and the cell viability of melanoma cells may be key factors for FDG uptake and that HK activity, rather than GLUT 1 expression, seems to be a major factor.

Zidovudine induces visceral mitochondrial toxicity and intra-abdominal fat gain in a rodent model of lipodystrophy.

BACKGROUND: The use of zidovudine is associated with a loss of subcutaneous adipose tissue (SAT). We assessed if zidovudine treatment also affects visceral adipose tissue (VAT) and if uridine supplementation abrogates the adverse effects of zidovudine on VAT. METHODS: Rats were fed zidovudine for 21 weeks with or without simultaneous uridine supplementation. Control animals did not receive zidovudine, or were treated with uridine alone. Changes in SAT and VAT were monitored by magnetic resonance imaging. Adipose tissue was examined for structural and molecular signs of mitochondrial toxicity. RESULTS: Zidovudine induced lipoatrophy in SAT and fat hypertrophy in VAT. Compared with controls zidovudine-exposed VAT adipocytes had increased diameters, microvesicular steatosis and enlarged mitochondria with disrupted crystal architecture on electron microscopy. VAT adipocyte mitochondrial DNA (mtDNA) copy numbers were diminished, as were mtDNA-encoded respiratory chain proteins. The 'common' mtDNA deletion was detected in high frequencies in zidovudine treated animals, but not in the controls. Although mtDNA depletion was more profound in SAT compared with VAT, the 'common' deletion tended to be more frequent in the VAT than in the SAT. Uridine coadministration abrogated all effects of zidovudine on VAT and SAT pathology. CONCLUSIONS: Zidovudine induces a gain of intra-abdominal fat in association with quantitative and qualitative alterations of the mitochondrial genome and impaired expression of mtDNA-encoded respiratory chain components, indicating that zidovudine may contribute to abdominal fat hypertrophy in HIV-infected patients. In this rodent model, uridine supplementation abrogates both SAT and VAT pathology induced by zidovudine.

Muscle-fiber transdifferentiation in an experimental model of respiratory chain myopathy.

INTRODUCTION: Skeletal muscle fiber composition and muscle energetics are not static and change in muscle disease. This study was performed to determine whether a mitochondrial myopathy is associated with adjustments in skeletal muscle fiber-type composition. METHODS: Ten rats were treated with zidovudine, an antiretroviral nucleoside reverse transcriptase inhibitor that induces a myopathy by interfering with mitochondrial functions. Soleus muscles were examined after 21 weeks of treatment. Ten untreated rats served as controls. RESULTS: Zidovudine induced a myopathy with mitochondrial DNA depletion, abnormalities in mitochondrial ultrastructure, and reduced cytochrome c oxidase activity. Mitochondrial DNA was disproportionally more diminished in type I compared with type II fibers, whereas atrophy predominated in type II fibers. Compared with those of controls, zidovudine-exposed soleus muscles contained an increased proportion (256%) of type II fibers, whereas neonatal myosin heavy chains remained repressed, indicating fiber-type transformation in the absence of regeneration. Microarray gene-expression analysis confirmed enhanced fast-fiber isoforms, repressed slow-fiber transcripts, and reduced neonatal fiber transcripts in the mitochondrial myopathy. Respiratory chain transcripts were diminished, whereas the enzymes of glycolysis and glycogenolysis were enhanced, indicating a metabolic adjustment from oxidative to glycolytic capacities. A coordinated regulation was found of transcription factors known to orchestrate type II fiber formation (upregulation of MyoD, Six1, Six2, Eya1, and Sox6, and downregulation of myogenin and ERRγ). CONCLUSIONS: The type I to type II fiber transformation in mitochondrial myopathy implicates mitochondrial function as a new regulator of skeletal muscle fiber type.

Hb Riesa or ß93 (F9) Cys→Ser, a new electrophoretically silent haemoglobin variant interfering with haemoglobin A1c measurement.

A new ß variant was found in a German diabetic patient whose blood samples appeared to contain 45% Hb A(1c) using Bio-Rad Variant V-II A1c-analyzer but 7.6% on boronate affinity chromatography. Structural studies using, HPLC, mass spectrometry, and the genomic DNA analysis revealed a new substitution in which the cysteine residue at position ß93 was replaced by serine. The variant was named Hb Riesa or ß93 (F9) Cys→Ser and accounted for 54.3% of the total haemoglobin. This suggests that the protein-synthesis processes for the mutant could be slightly more promoted than those of the wild-type. Hb Riesa is clinically and electrophoretically silent.

A polymer-based DNA biochip platform for human papilloma virus genotyping.

Genotyping of the human papilloma virus (HPV) is from a clinical point of view an important diagnostic task as some genotypes play a major role in the development of cervical carcinoma. So far PCR combined with blotting or in situ labelling is known to be the most accurate and sensitive method for detection and genotyping of HPV infection in clinical samples. However, specificity, cost-efficiency and sensitivity are not always satisfactory. A novel DNA biochip is described based on a plastic substrate, onto which small polymer droplets and single-stranded DNA are printed in the form of microarrays. Immobilisation of all compounds on the chip surface is achieved by a short UV-irradiation process, inducing photochemical reactions in the polymer. The chip designed for this study contains 36 probes for determining 12 common, different HPV genotypes. After isolation of the DNA, PCR and biochip read-out, the chip allows for genotyping of the most common virus strains, which, according to current prevalence studies, cover 85-95% of all infections. Following this approach as little as 10 virus copies can be detected within a short exposure time. Even using paraffin-embedded material and 10(4) copies per PCR are sufficient to allow rapid and reliable HPV genotyping.

Haemoglobin Hokusetsu [beta52 (D3)] Asp-->Gly in German families associated with inclusion body.

Inclusion bodies associated with Hb Hokusetsu have never been published. We investigated the autoxidation of this variant as a cause for the inclusion bodies in three unrelated families. Moreover, haplotype analysis was carried out to unravel the origin of this variant also found in the Japanese population. The presence of inclusion bodies was revealed by incubating the fresh peripheral blood with brilliant cresyl blue. We further characterised this variant using mass spectrometry and DNA analysis. The generation of superoxide radical (ROS) during the autoxidation was assayed by electron spin resonance spectrometry. Inclusion bodies were seen in about 25% of red cells. Hb Hokusetsu turned out to be less thermostable than the control. It showed a tenfold-enhanced ROS formation versus control. The analysis of the beta-globin haplotypes for the three unrelated families showed that Hb Hokosetsu was linked with haplotype I (5' + - - - - + + 3'). This is the first case published in the German population. The inclusion bodies could be due to the instability of the variant. This is supported by the increased autoxidation. The absence of anaemia evokes an elimination of the inclusion bodies by the proteolytic mechanism of the red cells. The association of the variant in three unrelated families with the five polymorphisms of haplotype I indicates a single common mutation event. In the presence of Hb Hokusetsu, HbA 1C standard methods used to assess glycaemic control are mistaken.