Thromboses represent a major cause of morbidity and mortality in Polycythemia Vera (PV) but the contributing mechanisms are not fully described.
Patients and methods
To evaluate whether environmental conditions such as altitude/hypoxia could impact thromboses history, we retrospectively analyzed thrombosis history in 71 PV patients living at an elevation of 5,000 feet or more in the SLC area (SLC) and 166 PV patients living near sea level in the Baltimore area (BLM). The SLC cohort was older with a longer disease duration. No significant differences in type of anticoagulation therapy or prothrombotic factors were present between the two cohorts. After adjusting for age, sex and disease duration, SLC patients experienced an estimated 3.9-fold increase in the odds of a history of thromboses compared to BLM patients (95% confidence interval 1.8-7.6; p = 0.0004). A history of cardiovascular event was present in 58% of the SLC patients compared to 27% of the BLM patients (p<0.0001). Before diagnosis thromboses occurred in 18% and 4% of the SLC and BLM groups respectively (p =0.003). No correlation between JAK2V617F allele burden and thrombosis was observed in this study.
This retrospective study suggests that even moderate hypoxia associated with 5,000 feet elevation should be considered as independent prothrombotic risk factor. This observation needs to be confirmed by prospective studies.
In the current study, we developed a HPLC method to quantitatively measure the permeability of the BpT-based chelators, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT), across human colorectal adenocarcinoma (Caco-2) monolayers as a model of gut absorption. In aqueous solution, Bp4eT and Bp4aT formed inter-convertible Z and E isomers that were resolved by HPLC. Peak area was linear with respect to chelator concentration. Acceptable within-day and between-day precision (<22%) and accuracy (85–115% of true values) were obtained over a range of 1.0 – 100 µM for Bp4eT and 1.5 – 300 µM for Bp4aT. Limits of detection were 0.3 µM and 1 µM for Bp4eT and Bp4aT, respectively, while corresponding limits of quantification were 1 µM and 5 µM. Both chelators showed significant ability to chelate iron in THP-1 cells using a calcein-based assay and no apparent cytotoxicity was observed within 24 h. Ratios of the apical to basolateral and basolateral to apical transport for Bp4eT were 1.10 and 0.89 at 100 µM and 300 µM respectively, indicating equal bi-directional movement of the compounds. Similarly, ratios were 0.77 and 0.92 for Bp4aT, respectively. This study demonstrates that Bp4eT and Bp4aT can be efficiently transported through Caco-2 cells and can potentially be formulated for oral delivery.
Bivariate mixture modeling was used to analyze joint population distributions of transferrin saturation (TS) and serum ferritin concentration (SF) measured in the Hemochromatosis and Iron Overload Screening (HEIRS) Study. Four components (C1, C2, C3, and C4) with successively age-adjusted increasing means for TS and SF were identified in data from 26,832 African Americans, 12,620 Asians, 12,264 Hispanics, and 43,254 whites. The largest component, C2, had normal mean TS (21% to 26% for women, 29% to 30% for men) and SF (43–82 μg/L for women, 165–242 μg/L for men), which consisted of component proportions greater than 0.59 for women and greater than 0.68 for men. C3 and C4 had progressively greater mean values for TS and SF with progressively lesser component proportions. C1 had mean TS values less than 16% for women (<20% for men) and SF values less than 28 μg/L for women (<47 μg/L for men). Compared with C2, adjusted odds of iron deficiency were significantly greater in C1 (14.9–47.5 for women, 60.6–3530 for men), adjusted odds of liver disease were significantly greater in C3 and C4 for African-American women and all men, and adjusted odds of any HFE mutation were increased in C3 (1.4–1.8 for women, 1.2–1.9 for men) and in C4 for Hispanic and white women (1.5 and 5.2, respectively) and men (2.8 and 4.7, respectively). Joint mixture modeling identifies a component with lesser SF and TS at risk for iron deficiency and 2 components with greater SF and TS at risk for liver disease or HFE mutations. This approach can identify populations in which hereditary or acquired factors influence metabolism measurement.
There are few descriptions of young adults with self-reported hemochromatosis or iron overload (H/IO). We analyzed initial screening data in 7,343 HEmochromatosis and IRon Overload Screening (HEIRS) Study participants ages 25–29 years, including race/ethnicity and health information; transferrin saturation (TS) and ferritin (SF) measurements; and HFE C282Y and H63D genotypes. We used denaturing high-pressure liquid chromatography and sequencing to detect mutations in HJV, TFR2, HAMP, SLC40A1, and FTL. Fifty-one participants reported previous H/IO; 23 (45%) reported medical conditions associated with H/IO. Prevalences of reports of arthritis, diabetes, liver disease or liver cancer, heart failure, fertility problems or impotence, and blood relatives with H/IO were significantly greater in participants with previous H/IO reports than in those without. Only 7.8% of the 51 participants with previous H/IO reports had elevated TS; 13.7% had elevated SF. Only one participant had C282Y homozygosity. Three participants aged 25–29 years were heterozygous for potentially deleterious mutations in HFE2, TFR2, and HAMP promoter, respectively. Prevalences of self-reported conditions, screening iron phenotypes, and C282Y homozygosity were similar in 1,165 participants aged 30 years or greater who reported previous H/IO. We conclude that persons who report previous H/IO diagnoses in screening programs are unlikely to have H/IO phenotypes or genotypes. Previous H/IO reports in some participants could be explained by treatment that induced iron depletion before initial screening, misdiagnosis, or participant misunderstanding of their physician or the initial screening questionnaire.
How often elevated serum ferritin in primary-care patients reflects increased iron stores (normally 0.8 g in men, 0.4 g in women) is not known. The Hereditary Hemochromatosis and Iron Overload Screening (HEIRS) study screened 101,168 primary-care participants (44% Caucasians, 27% African-Americans, 14% Asians/Pacific Islanders, 13% Hispanics, 2% others). Follow-up clinical evaluation was performed in 302 of 333 HFE C282Y homozygotes regardless of iron measures and 1,375 of 1,920 nonhomozygotes with serum ferritin >300 μg/L (men), >200 μg/L (women) and transferrin saturation >50% (men), >45% (women). Quantitative phlebotomy was conducted in 122 of 175 C282Y homozygotes and 122 of 1,102 nonhomozygotes with non-transfusional serum ferritin elevation at evaluation. The estimated prevalence in the Caucasian population of C282Y homozygotes with serum ferritin >900 μg/L at evaluation was 20 per 10,000 men and 4 per 10,000 women; this constellation was predictive of iron stores >4 g in men and >2 g in women. The estimated prevalence per 10,000 of non-C282Y homozygotes with serum ferritin >900 μg/L at evaluation was 7 among Caucasians, 13 among Hispanics, 20 among African Americans, and 38 among Asians and Pacific Islanders, and this constellation was predictive of iron stores >2 g but <4 g. In conclusion, serum ferritin >900 μg/L after initial elevations of both serum ferritin and transferrin saturation is predictive of mildly increased iron stores in multiple ethnic populations regardless of HFE genotype. Serum ferritin >900 μg/L in male C282Y homozygotes is predictive of moderately increased iron stores.
The ferroportin (FPN1) Q248H polymorphism has been associated with increased serum ferritin (SF) levels in sub-Saharan Africans and in African Americans (AA). AA participants of the HEIRS Study who did not have HFE C282Y or H63D who had elevated initial screening SF (≥300 μg/L in men and ≥200 μg/L in women) (defined as cases) were frequency-matched to AA participants with normal SF (defined as controls) to investigate the association of the Q248H with elevated SF. 10.4% of cases and 6.7% of controls were Q248H heterozygotes (P = 0.257). Q248H homozygosity was observed in 0.5% of the cases and none of the controls. The frequency of Q248H was higher among men with elevated SF than among control men (P = 0.047); corresponding differences were not observed among women. This appeared to be unrelated to self-reports of a previous diagnosis of liver disease. Men with elevated SF were three times more likely than women with elevated SF to have Q248H (P = 0.012). There were no significant differences in Q248H frequencies in men and women control participants. We conclude that the frequency of the FPN1 Q248H polymorphism is greater in AA men with elevated SF than in those with normal SF.
genetics; mutation; transferrin saturation
HFEC282Y homozygotes have an increased risk for developing increased iron stores and related disorders. It is controversial whether dietary iron restrictions should be recommended to such individuals.
To determine whether dietary iron content influences iron stores in HFEC282Y homozygotes as assessed by serum ferritin concentration.
Serum ferritin concentration was measured and a dietary iron questionnaire was completed as part of the evaluation of 213 HFEC282Y homozygotes who were identified through screening of >100,000 primary care patients at five HEmochromatosis and IRon Overload Screening (HEIRS) Study Field Centers in the United States and Canada.
No significant relationships between serum ferritin concentration and dietary heme iron content, dietary nonheme iron content or reports of supplemental iron use were found.
These results do not support recommending dietary heme or nonheme iron restrictions for HFEC282Y homozygotes diagnosed through screening in North America.
Haemochromatosis; Hemochromatosis; Iron overload; Iron supplementation
Platelets are activated in sickle cell disease (SCD), and particularly during vaso-occlusive episodes (VOE). Thrombospondin-1 (TSP1), a major secretory product of activated platelets, is increased in the circulation in VOE and binds to sickle red blood cells (RBC) promoting vascular adhesion. Thus, we hypothesized that TSP1 may represent a plasma biomarker of disease severity in SCD. We tested the plasma collected from patients in steady state (n = 27) and VOE (n = 14), as well as healthy controls (n = 17) at the University of Pittsburgh Medical Center (UPMC), and from patients in steady state enrolled in the walk-PHaSST clinical trial (n = 483). We found that TSP1 levels were increased in VOE in the UPMC cohort. Among steady-state patients at UPMC, TSP1 values correlated positively with lifetime history of acute chest syndrome (r = 0.72, P < 0.0001) and hemoglobin concentration (r = 0.49, P = 0.01), and negatively with markers of hemolysis, such as LDH (r = −0.50, P = 0.009). Analysis of the walk-PHaSST cohort also showed a positive association between TSP1 levels and hydroxyurea use (r = 0.14, P = 0.003), and confirmed the negative associations with the severity of hemolysis. Our results suggest that TSP1 levels are associated with more VOE, hydroxyurea use and lower rates of hemolysis. High TSP1 concentrations may indicate higher risk of the viscosity/vaso-occlusion phenotype of SCD.
We analyzed entry data from 163 adult hemoglobin SS and Sβ0 thalassemia patients enrolled in the prospective Sickle Cell Pulmonary Hypertension Screening Study and stratified their ECHO-determined tricuspid regurgitant jet velocity (TRV) and serum creatinine concentration according to three blood pressure categories. TRV was ≥2.5 m/sec in 27% of the patients with systolic blood pressure (SBP) <120 mm Hg and diastolic blood pressure (DBP) <70 mm Hg, in 37% with SBP 120–139 mm Hg or DBP 70–89 mm Hg, and in 93% with SBP 140 mm Hg or DBP 90 mm Hg or higher (P <0.0005 for trend). Serum creatinine concentration was 1.0 mg/dL or higher in 7% of patients with SBP <120 mm Hg and DBP <70 mm Hg, in 17% with SBP 120–139 mm Hg or DBP 70–89 mm Hg and 50% with SBP 140 mm Hg or DBP 90 mm Hg or higher (P <0.0005 for trend). Over two years of follow-up, there were trends for more frequent progression to elevated TRV (P = 0.073) or creatinine (P = 0.038) values according to the higher systemic blood pressure categories. Our findings suggest that systolic SBP 120–139 mm Hg or DBP 70–89 mm Hg defines a category of relative systemic hypertension in patients with sickle cell disease that is associated with increased risk for pulmonary hypertension and renal dysfunction. Whether antihypertensive and/or nitric oxide donor therapy in sickle cell disease patients with relative hypertension prevents these and other complications should be determined by clinical trials.
Alcohol consumption is associated with increased iron stores. In sub-Saharan Africa, high dietary ionic iron and the ferroportin Q248H allele have also been implicated in iron accumulation. We examined the associations of ferroportin Q248H, alcohol and dietary iron with serum ferritin, aspartate aminotransaminase (AST) and alanine aminotransaminase (ALT) concentrations in African Americans.
Inner-city African Americans (103 men, 40 women) were recruited from the community according to reported ingestion of >4 alcoholic drinks per day or <2 per week. Typical daily heme iron, non-heme iron and alcohol were estimated using University of Hawaii’s multiethnic dietary questionnaire. Based on dietary questionnaire estimates we established categories of < versus ≥56 g alcohol per day, equivalent to 4 alcoholic drinks per day assuming 14 g alcohol per drink.
Among 143 participants, 77% drank <56 g alcohol/day and 23% ≥56 g/d as estimated by the questionnaire. The prevalence of ferroportin Q248H was 23.3% with alcohol >56 g/d versus 7.5% with lower amounts (P=0.012). Among subjects with no history of HIV disease, serum ferritin concentration had positive relationships with male gender (P=0.041), alcohol consumption (P=0.021) and ALT concentration (P=0.0001) but not with dietary iron intake or ferroportin Q248H. Serum AST and ALT concentrations had significant positive associations with male gender and hepatitis C seropositivity but not with alcohol or dietary iron intake or ferroportin Q248H.
Our findings suggest a higher prevalence of ferroportin Q248H with greater alcohol consumption, and this higher prevalence raises the possibility that the allele might ameliorate the toxicity of alcohol. Our results suggest that alcohol but not dietary iron contributes to higher body iron stores in African Americans. Studies with larger numbers of participants are needed to further clarify the relationship of ferroportin Q248H with the toxicity of alcohol consumption.
Background. The mechanisms of severe malarial anemia and cerebral malaria, which are extreme manifestations of Plasmodium falciparum malaria, are not fully understood.
Methods. Children aged <6 years from southern Zambia presenting to the hospital with severe malarial anemia (n = 72), cerebral malaria (n = 28), or uncomplicated malaria (n = 66) were studied prospectively. Children with overlapping severe anemia and cerebral malaria were excluded.
Results. Low interleukin 10 concentrations had the strongest association with severe anemia (standard β = .61; P < .001) followed by high tumor necrosis factor α and sFas concentrations, low weight-for-age z scores, presence of stool parasites, and splenomegaly (standard β = .15–.25; P ≤ .031); most of these factors were also associated with lower reticulocytes. Greater parasitemia was associated with higher interleukin 10 and tumor necrosis factor α concentrations, whereas sulfadoxizole/pyrimethamine therapy and lower weight-for-age z scores were associated with lower interleukin 10 levels. Thrombocytopenia and elevated tissue plasminogen activator inhibitor 1 levels had the strongest associations with cerebral malaria (standard β = .37 or .36; P < .0001), followed by exposure to traditional herbal medicine and hemoglobinuria (standard β = .21–.31; P ≤ .006).
Conclusions. Predictors of severe malarial anemia (altered immune responses, poor nutrition, intestinal parasites, and impaired erythropoiesis) differed from those of cerebral malaria (thrombocytopenia, herbal medicine, and intravascular hemolysis). Improved preventive and therapeutic measures may need to consider these differences.
Iron deficiency and the Q248H mutation in the gene, SLC40A1, that encodes for the cellular iron exporter, ferroportin, are both common in African children. The iron status of macrophages influences the pro-inflammatory response of these cells. We hypothesized that Q248H mutation may modify the inflammatory response by influencing iron levels within macrophages.
The Q248H mutation and circulating concentrations of ferritin, C-reactive protein and selected pro-inflammatory cytokines (interleukin-12, interferon-γ, TNF-α, and macrophage migration inhibitory factor) and anti-inflammatory cytokines (interleukin-4 and interleukin-10) were measured in 69 pre-school children recruited from well-child clinics in Harare, Zimbabwe.
In multivariate analysis, both ferroportin Q248H and ferritin <10 ug/L were associated with significantly lower circulating concentrations of tumor necrosis factor-α. Ferroportin Q248H but not low iron stores was associated with lower circulating macrophage migration inhibitory factor as well. Anti-inflammatory cytokine levels were not significantly associated with either ferroportin Q248H or iron status.
Ferroportin Q248H and low iron stores are both associated with lower circulating tumor necrosis factor-alpha, while only ferroportin Q248H is associated with lower circulating macrophage migration inhibitory factor. Whether the reduced production of tumor necrosis factor-α observed in ferroportin Q248H heterozygotes may be of significance in anemia of chronic disease is yet to be determined.
The genetic bases of the highly variable degrees of anaemia and haemolysis in persons with Hb SS are not fully known, but several studies have indicated that G6PD deficiency is not a factor. The G6PD202A and G6PD376G alleles and α-thalassaemia were determined by molecular genetic testing in 261 children and adolescents with Hb SS in a multicentre study. G6PD202A,376G (G6PD A-) was defined as hemizygosity for both alleles in males and homozygosity in females. Among the participants 41% were receiving hydroxycarbamide.
The prevalence of G6PD202A,376G was 13.6% in males and 3.3% in females with an overall prevalence of 8.7%. G6PD202A,376G was associated with a 10 g/l decrease in haemoglobin concentration (P=0.008) but not with increased haemolysis as measured by lactate dehydrogenase, bilirubin, aspartate-aminotransferase, reticulocyte count or a haemolytic component derived from these markers (P>0.09). Similar results were found within a sub-group of children who were not receiving hydroxycarbamide. By comparison, single and double α-globin deletions were associated with progressively higher haemoglobin concentrations (P=0.005 for trend), progressively lower values for haemolytic component (P=0.007), and increased severe pain episodes (P<0.001).
In conclusion, G6PD202A,376G may be associated with lower haemoglobin concentration in sickle cell anaemia by a mechanism other than increased haemolysis.
sickle cell anaemia; G6PD; haemolysis; alpha-thalassaemia; haemoglobin concentration
N-terminal (NT) pro-brain natriuretic peptide (proBNP) ≥160 ng/l has a 78% positive predictive value for pulmonary hypertension and is associated with increased mortality in US sickle cell disease patients, but the importance in sickle cell disease patients in Africa is not known. In a cross-sectional study at Ahmadu Bello University Teaching Hospital, Shika-Zaria, Nigeria, we studied 133 hydroxycarbamide-naïve Nigerian sickle cell anaemia patients aged 18-52 years at steady-state and 65 healthy controls. Twenty-six percent of patients versus 5% of controls had NT-proBNP ≥160 ng/l (P=0.0006). By logistic regression among the patients, human immunodeficiency virus seropositivity, higher serum ferritin and lower haemoglobin or higher lactate dehydrogenase independently predicted elevated NT-proBNP. After adjustment for haemoglobin concentration, elevated NT-proBNP concentration was associated with an estimated 7.8-fold increase in the odds of severe functional impairment, defined as an inability to walk more than 300 m in six min (95% confidence interval 1.5-32.6; P=0.005). Similarly, elevated tricuspid regurgitation velocity was associated with an estimated 5.6-fold increase in the odds of functional impairment (95% confidence interval 1.5-21.0; P=0.011). In conclusion, NT-proBNP elevation is common and is associated with markers of anaemia, inflammation and iron status and with severe functional impairment among sickle cell anaemia patients in Nigeria.
sickle cell disease; N-terminal pro-brain natriuretic peptide; six-minute walk; haemolysis; Africa
Hepatitis C virus (HCV) infection may be associated with thrombocytopenia and increased iron stores in patients receiving medical care. We aimed to determine how often changes in hematologic, iron metabolic and inflammatory markers occur in individuals with undiagnosed HCV in the community.
Inner-city African Americans (n=143) were recruited from the community according to reported ingestion of alcohol. They were divided broadly into those who drank more or less than 56 g alcohol/day as assessed by dietary questionnaire. HCV serology was determined and laboratory values were compared according to HCV seropositivity in analyses that adjusted for alcohol consumption.
The prevalence of HCV seropositivity was 23% among men and 29% among women. Levels of hepatocellular enzymes were higher with HCV seropositivity (P <0.0001) but hemoglobin concentrations, white blood cell and platelet counts and serum ferritin concentrations did not differ. The globulin fraction of the serum protein concentration (P=0.002) was increased with HCV seropositivity as expected with chronic inflammation. However, erythrocyte sedimentation rate and serum iron and haptoglobin levels did not differ significantly according to HCV status. Furthermore, multivariate analysis revealed that C-reactive protein was decreased and transferrin concentration was increased with both HCV and alcohol consumption (P<0.014).
Previously undiagnosed HCV seropositivity has little effect on the complete blood count and body iron stores but appears to perturb the response to an inflammatory stimulus, causing reduced rather than increased circulating CRP concentrations and increased rather than decreased transferrin concentrations.
HCV infection; African American; CRP; Iron metabolism
The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). CDK9 activity is regulated by protein phosphatase-1 (PP1) which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD in vitro and in vivo. Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by (32P) in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D – inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription.
Physiological regulation of cellular iron involves iron export by the membrane protein, ferroportin, the expression of which is induced by iron and negatively modulated by hepcidin. We previously showed that iron chelation is associated with decreased HIV-1 transcription. We hypothesized that increased iron export by ferroportin might be associated with decreased HIV-1 transcription, and degradation of ferroportin by hepcidin might in turn induce HIV-1 transcription and replication. Here, we analyzed the effect of ferroportin and hepcidin on HIV-1 transcription.
Expression of ferroportin was associated with reduced HIV-1 transcription in 293T cells and addition of hepcidin to ferroportin-expressing cells counteracted this effect. Furthermore, exposure of promonocytic THP-1 cells to hepcidin was associated with decreased ferroportin expression, increased intracellular iron and induction of reporter luciferase gene expression. Finally, exposure of human primary macrophages and CD4+ T cells to hepcidin and iron was also associated with induction of viral production.
Our results suggest that the interplay between ferroportin-mediated iron export and hepcidin-mediated degradation of ferroportin might play a role in the regulation of HIV-1 transcription and may be important for understanding of HIV-1 pathogenesis.
Chuvash polycythemia results from a homozygous 598C>T mutation in exon 3 of the von Hippel-Lindau (VHL) gene. This disrupts the normoxia pathway for degrading hypoxia inducible factor (HIF)-1α and HIF-2α causing altered expression of HIF-1 and HIF-2 inducible genes. As hypoxia induces expression of proinflammatory cytokines, we hypothesized that there might be an elevation of Th1 cytokines in the setting of Chuvash polycythemia. We analyzed plasma concentrations of Th1 (interleukins-2 and 12, interferon-γ, granulocyte-monocyte colony-stimulating factor, tumor necrosis factor-α) and Th2 cytokines (interleukins-4, 5, 10, and 13) using the Bio-Plex multiplex suspension array system in 34 VHL598C>T homozygotes and 32 VHL wild-type participants from Chuvashia. Concentrations of all the Th1 and Th2 cytokines measured were elevated in the VHL598C>T homozygotes compared with the control wild-type participants, but the ratios of Th1 to Th2 cytokines did not differ by genotype. In parallel, peripheral blood concentrations of CD4 positive T-helper cells and CD4/CD8 ratio were lower in the VHL598C>T homozygotes. In conclusion, the up-regulated hypoxic response in Chuvash polycythemia is associated with increased plasma products of both the Th1 and Th2 pathways, but the balance between the two pathways seems to be preserved.
In Chuvash polycythemia, homozygous von Hippel-Lindau (VHL) 598C>T leads to increased hypoxia inducible factor-1α and 2α, thromboses and lower systemic blood pressures. Circulating homocysteine, glutathione, γ-glutamyltransferase and cysteinylglycine concentrations were higher in 34 VHL598C>T homozygotes than in 37 normal controls and cysteine was lower. Multivariate analysis showed elevated homocysteine independently associated with higher mean systemic blood pressures and elevated glutathione was associated with lower pressures to a similar degree. Among VHL598C>T homozygotes, homocysteine was elevated with low and normal folate concentrations, consistent with a possible defect in the remethylation pathway. The elevated glutathione and γ-glutamyltranserase levels correlated positively with cysteinylglycine, consistent with possible upregulation of a glutathione synthetic enzyme and γ-glutamyltransferase. Cysteinylglycine correlated inversely with cysteine, consistent with possible reduced cysteinyldipeptidase activity. We conclude that up-regulated hypoxia-sensing may influence multiple steps in thiol metabolism. The effects of the resultant elevated levels of homocysteine and glutathione on systemic blood pressure may largely balance each other out.
VHL; polycythemia; homocysteine; folate; glutathione
Heme carrier protein 1 (HCP1) has been identified as a possible heme carrier by in vitro analysis. To determine the association of mutations within the HCP1 gene with iron phenotypes, we examined the entire coding region of the HCP1 gene in 788 US and Canadian participants selected from the Hemochromatosis and Iron Overload Screening (HEIRS) Study using denaturing high-performance liquid chromatography. We sequenced the exon and flanking intronic regions if variants were detected. We tested 298 non-C282Y homozygotes from four racial/ethnic backgrounds (White, Black, Asian, and Hispanic) selected because they had high serum ferritin (SF) and transferrin saturations (TS). As controls, we chose 300 other random participants of the same racial/ethnic backgrounds from the same geographic locations. From the 333 HEIRS Study C282Y homozygotes, we selected 75 based on high SF and TS, 75 based on low SF and TS; 75 were selected randomly as controls. Thirty-five of the randomly selected C282Y homozygotes were also included in the high and the low SF and TS groups due to numerical limitations. We identified eight different HCP1 genetic variants; each occurred in a heterozygous state. Except one, each was found in a single HEIRS Study participant. Thus, HCP1 variants are infrequent in the populations that we tested. Five HEIRS Study participants had non-synonymous, coding region HCP1 variants. Each of these five had TS above the 84th gender- and ethnic/racial group-specific percentile (TS percentiles: 84.7, 91.3, 97.9, 99.5, and 99.9).
Pulmonary hypertension and left ventricular diastolic dysfunction are complications of sickle cell disease. Pulmonary hypertension is associated with hemolysis and hypoxia, but other unidentified factors are likely involved in pathogenesis as well.
Design and Methods
Plasma concentrations of three angiogenic markers (fibroblast growth factor, platelet derived growth factor–BB [PDGF-BB], vascular endothelial growth factor [VEGF]) and seven inflammatory markers implicated in pulmonary hypertension in other settings were determined by Bio-Plex suspension array in 237 children and adolescents with sickle cell disease at steady state and 43 controls. Tricuspid regurgitation velocity (which reflects systolic pulmonary artery pressure), mitral valve E/Edti ratio (which reflects left ventricular diastolic dysfunction), and a hemolytic component derived from four markers of hemolysis and hemoglobin oxygen saturation were also determined.
Plasma concentrations of interleukin-8, interleukin-10 and VEGF were elevated in the patients with sickle cell disease compared to controls (P≤0.003). By logistic regression, greater values for PDGF-BB (P = 0.009), interleukin-6 (P = 0.019) and the hemolytic component (P = 0.026) were independently associated with increased odds of elevated tricuspid regurgitation velocity while higher VEGF concentrations were associated with decreased odds (P = 0.005) among the patients with sickle cell disease. These findings, which are consistent with reports that PDGF-BB stimulates and VEGF inhibits vascular smooth muscle cell proliferation, did not apply to E/Etdi.
Circulating concentrations of angiogenic and pro-Inflammatory markers are altered in sickle cell disease children and adolescents with elevated tricuspid regurgitation velocity, a subgroup that may be at risk for developing worsening pulmonary hypertension. Further studies to understand the molecular changes in these children are indicated.
HIV transcription is induced by the HIV-1 Tat protein, in concert with cellular co-factors including CDK9, CDK2, NF-κB, and others. The cells of most of the body’s organs are exposed to ~3–6% oxygen, but most in vitro studies of HIV replication are conducted at 21% oxygen. We hypothesized that activities of host cell factors involved in HIV-1 replication may differ at 3% versus 21% O2, and that such differences may affect HIV-1 replication. Here we show that Tat-induced HIV-1 transcription was reduced at 3% O2 compared to 21% O2. HIV-1 replication was also reduced in acutely or chronically infected cells cultured at 3% O2 compared to 21% O2. This reduction was not due the decreased cell growth or increased cellular toxicity and also not due to the induction of hypoxic response. At 3% O2, the activity of CDK9/cyclin T1 was inhibited and Sp1 activity was reduced, whereas the activity of other host cell factors such as CDK2 or NF-κB was not affected. CDK9-specific inhibitor ARC was much less efficient at 3% compared to 21% O2 and also expression of CDK9/cyclin T1-dependent IκB inhibitor α was repressed. Our results suggest that lower HIV-1 transcription at 3% O2 compared to 21% O2 may be mediated by lower activity of CDK9/cyclin T1 and Sp1 at 3% O2 and that additional host cell factors such as CDK2 and NF-κB might be major regulators of HIV-1 transcription at low O2 concentrations.
Patients with hemochromatosis may suffer organ damage from iron overload, often with serious clinical consequences.
To assess prevalences of self-reported symptoms and clinical signs and conditions in persons homozygous for the hemochromatosis gene (HFE) mutation (C282Y) identified by screening.
Participants were adults 25 years of age or older enrolled in the Hemochromatosis and Iron Overload Screening (HEIRS) Study. C282Y homozygotes (n=282) were compared with control participants without the HFE C282Y or H63D alleles (ie, wild type/wild type; n=364).
Previously diagnosed C282Y homozygotes and newly diagnosed homozygotes with elevated serum ferritin levels had higher prevalences of certain symptoms such as chronic fatigue (OR 2.8; 95% CI 1.34 to 5.95, and OR 2.0; 95% CI 1.07 to 3.75, respectively), and had more hyperpigmentation on physical examination (OR 4.7; 95% CI 1.50 to 15.06, and OR 3.7; 95% CI 1.10 to 12.16, respectively) and swelling or tenderness of the second and third metacarpophalangeal joints (OR 4.2; 95% CI 1.37 to 13.03, and OR 3.3; 95% CI 1.17 to 9.49, respectively) than control subjects. Joint stiffness was also more common among newly diagnosed C282Y homozygotes with elevated serum ferritin than among control subjects (OR 2.7; 95% CI 1.38 to 5.30). However, the sex- and age-adjusted prevalences of self-reported symptoms and signs of liver disease, heart disease, diabetes and most other major clinical manifestations of hemochromatosis were similar in C282Y homozygotes and control subjects.
Some symptoms and conditions associated with hemochromatosis were more prevalent among C282Y homozygotes identified by screening than among control subjects, but prevalences of most outcomes were similar in C282Y homozygotes and controls in this primary care-based study.
Complications; Cross-sectional study; Hemochromatosis; HFE; Iron overload; Prevalence
Low steady state haemoglobin oxygen saturation in patients with sickle cell anaemia has been associated with the degree of anaemia and haemolysis. How much pulmonary dysfunction contributes to low saturation is not clear. In a prospective study of children and adolescents with sickle cell disease aged 3–20 years at steady state and matched controls, 52% of 391 patients versus 24% of 63 controls had steady state oxygen saturation <99% (P < 0·0001), 9% of patients versus no controls had saturation <95% (P = 0·008) and 8% of patients versus no controls had exercise-induced reduction in saturation ≥3%. Decreasing haemoglobin concentration (P ≤ 0·001) and increasing haemolysis (P ≤ 0·003) but not pulmonary function tests were independent predictors of both lower steady-state saturation and exercise-induced reduction in saturation. Neither history of stroke nor history of acute chest syndrome was significantly associated with lower steady-state oxygen saturation or exercise-induced reduction in saturation. Tricuspid regurgitation velocity was higher in patients with lower steady state haemoglobin oxygen saturation (P = 0·003) and with greater decline in oxygen saturation during the six-minute walk (P = 0·022). In conclusion, lower haemoglobin oxygen saturation is independently associated with increasing degrees of anaemia and haemolysis but not pulmonary function abnormalities among children and adolescents with sickle cell disease.
sickle cell disease; paediatric; oxygen saturation; six-minute walk; pulmonary hypertension
HIV-1 replication is induced by the excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication by reducing proliferation of infected cells. Treatment of the cells with DFO and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) inhibit expression of proteins that regulate cell-cycle progression, including cycle-dependent kinase 2 (CDK2). Our recent studies showed that CDK2 participates in HIV-1 transcription and viral replication suggesting that inhibition of CDK2 by iron chelators might also affect HIV-1 transcription. Here we evaluated the effect of a clinically approved orally effective iron chelator, 4-[3,5-bis-(hydroxyphenyl) -1,2,4-triazol-1-yl]-benzoic acid (ICL670) and 311 on HIV-1 transcription. Both ICL670 and 311 inhibited Tat–induced HIV-1 transcription in CEM-T cells, 293T and HeLa cells. Neither ICL670 nor 311 induced cytotoxicity at concentrations that inhibited HIV-1 transcription. The chelators decreased cellular activity of CDK2 and reduced HIV-1 Tat phosphorylation by CDK2. The ICL670 and 311 did not decrease CDK9 protein level but significantly reduced association of CDK9 with cyclin T1 and reduced phosphorylation of Ser-2 residues of RNA polymerase II C-terminal domain. In conclusion, our findings add to the evidence that iron chelators can inhibit HIV-1 transcription by deregulating inhibiting CDK2 and CDK9. Further consideration should be given to the development of iron chelators for future anti-retroviral therapeutics.
HIV-1; desferrioxamine (DFO); 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311); 4-[3,5-bis-(hydroxyphenyl) -1,2, 4-triazol-1-yl]-benzoic acid (ICL670); transcription; CDK2; CDK9