Variable severity of β-thalassemia patients of Eastern India: effect of α-thalassemia and XmnI polymorphism

Sixty-four thalassemia and E-β thalassemia patients were studied for factors that modulate the severity of the disease; i. e., mutation of β-globin gene, presence of α-deletion, and presence of an XnmI site at the −158 position of the Gγ gene. Presence of α-deletion and/or homozygosity for the XmnI site was in general associated with less-severe disease. About 12% of the patients harbored single α-gene deletion, and the gene frequency of the XnmI polymorphism in these patients is 0.48. Received: 16 January 2001 / Accepted: 18 September 2001     

Production and characterization of monoclonal antibodies against α-globin chain-containing human hemoglobins for detecting α-thalassemia disease

Monoclonal antibodies against α-globin containing human Hbs, named AMS-Alpha1 and AMS-Alpha 2, were produced by the hybridoma technique using spleen cells enriched by the newly developed B lymphocyte enrichment protocol. These two monoclonal antibodies were of IgM class, reacting to only intact form of human Hbs A, A₂, E, and F, which contain α-globin chain. By the indirect ELISA, the AMS-Alpha1 and AMS-Alpha 2 quantified less amount of α-globin chain containing hemoglobins in HbH disease than the SEA-α thalassemia 1 carriers and normal individuals. It was thus anticipated that these monoclonal antibodies can be used for detecting Hb Bart’s hydrops fetalis in which no α-globin chain is produced.     

Characteristics of Radioimmunoassays for the α- and β-Subunits of Human Luteinizing Hormone

Abstract

The binding characteristics and specificities of the National Hormone and Pituitary Program (NHPP) kits for the radioimmunoassay of the alpha- and beta-subunits of human luteinizing hormone (hLH-α and hLH-β) were studied, as well as the specificities of the anti-hLH and anti-human follicle stimulating hormone (anti-hFSH) antisera distributed by the same organization. The affinity constants of the anti-hLH-α and anti-hLH-β antisera were calculated at 157 ± 8.4 nM^?1 and 109 ± 7.4 nM^?1, respectively. Both antisera were highly specific with regard to the other subunit. However, in the homologous hLH-α RIA, native hLH cross-reacted at 21.9%, hFSH at 17.5% and hTSH at 7.9%. The alpha-subunit of the human chorionic gonadotropin, hCG-α, was equipotent with the hLH-α standard in this assay. In the homologous hLH-β RIA, hLH showed a cross-reactivity of 14.7% while the cross-reactivities of hCG-β, hFSH and hTSH were 3.5%, 1.2% and 0.6%, respectively. The anti-hFSH antiserum was highly specific, while the anti-hLH antiserum showed non parallel competition curves. With this knowledge of the specificity of each antiserum, corrections can be properly made for the assays of hLH, hLH-α and hLH-β while the hFSH RIA can be used without correction for the presence of the three other components.     

BACH1 regulates erythrophagocytosis and iron-recycling in β-thalassemia

Macrophages play essential roles in erythrophagocytosis and iron recycling. β-thalassemia is characterized by a genetic defect in hemoglobin synthesis, which increases the rate of iron recycling. We previously showed that reduced expression of the BTB and CNC homolog 1 (BACH1) gene leads to increased phagocytosis of abnormal RBCs by activated monocytes. However, the mechanisms underlying this abnormal RBC clearance remained unclear. Herein, the spleen and bone marrow cells of β-thalassemic mice were examined for erythrophagocytosis CD markers and iron-recycling genes. Higher expression levels of CD47 and CD163 on RBCs and macrophages, respectively, were observed in β-thalassemic mice than in wild-type cells. The decreased expression of BACH1 caused an increase in Nrf2, Spic, Slc40a1, and HMOX1 expression in splenic red pulp macrophages of thalassemic mice. To investigate BACH1 regulation, a macrophage cell line was transfected with BACH1-siRNA. Decreased BACH1 expression caused an increase in CD163 expression; however, the expression levels were lower when the cells were cultured in media supplemented with β-thalassemia/HbE patient plasma. Additionally, the iron recycling-related genes SPIC, SLC40A1, and HMOX1 were significantly upregulated in BACH1-suppressed macrophages. Our findings provide insights into BACH1 regulation, which plays an important role in erythrophagocytosis and iron recycling in thalassemic macrophages.     

Alteration in α- and β- adrenoceptor profile of rabbit-knee-joint blood vessels due to chronic inflammation

Experiments were performed to investigate the nature of α- and β-adrenoceptors in blood vessels supplying the posterior capsule of the chronically inflamed rabbit knee joint, and results were compared to the finding from previous experiments on the normal and acutely inflamed joint to assess any alteration which may occur in the adrenoceptor profile due to the chronic inflammation process. Electrical stimulation of the posterior articular nerve resulted in vasoconstriction that was completely blocked by phentolamine. This constrictor response was almost equally inhibited by prazosin and yohimbine. The dose–response curves to close intraarterial injection of α-adrenoceptor agonists showed a rank-order potency of adrenaline = clonidine = phenylephrine. The adrenaline dose–response curve was shifted to the right by administration of α-antagonists with a rank-order potency of phentolamine = prazosin = yohimbine. At this stage of experiments, there was an equal response of α₁- and α₂- adrenoceptors in blood vessels of the chronically inflamed rabbit knee joint. In another group of animals, the neurally mediated vasodilatation, which appeared after administration of phentolamine, was completely blocked by propranolol and was reduced significantly by ICI118551, but the effect of atenolol was not significant. The dose–response curve to close intraarterial injection of β-adrenoceptor agonists showed a rank-order potency of: isoprenaline > salbutamol > dobutamine. The isoprenaline dose–response curve was shifted to the right by administration of β-antagonists with rank-order potency of propranolol > ICI118551 > atenolol. These experiments showed a greater β₂-adrenoceptor response than β₁-adrenoceptor response in chronically inflamed rabbit-knee-joint blood vessels. Overall, compared to previous experiments on normal joint in which α₂- and β₁-adrenoceptor responses predominated, and in acutely inflamed joint in which an equal α₁/α₂ and β₁/β₂ response was shown, in chronically inflamed joint the sympathetic constriction response was returned toward normal. No more α-adrenoceptor shift had happened, and the shift of β₁ to β₂ response continued.     

Interferon β-1a and β-1b for patients with multiple sclerosis: updates to current knowledge

Introduction: Although multiple sclerosis (MS) remains incurable, interferon beta (IFNβ) has been at the forefront of treatment for many years. Different formulations of IFNβ allow for different levels of exposure: low-dose/frequency with some agents, and high-dose/frequency with others.

Areas covered: This review article discusses existing and emerging efficacy and safety data for IFNβ in MS. Clinical evidence of IFNβ efficacy has been generated and accumulated over many decades. During this time, key clinical trials have demonstrated the benefits of high-dose and/or high-frequency dosing of IFNβ-1a or β-1b, compared with lower levels of exposure, on outcome measures such as relapse rates, disability progression, disease progression and magnetic resonance imaging lesion outcomes.

IFNβ therapy is well tolerated and has one of the best characterized safety profiles of all first line therapies. The overall severity of adverse events (AEs) does not appear to be affected by different IFNβ exposures. Typical AEs that patients may experience with IFNβ are mild, reversible and manageable.

Expert commentary: IFNβ is one of the best characterized treatments for MS, with a large body of clinical and real-world evidence supporting the risk-benefit profile. High-dose/frequency regimens may provide better long-term outcomes.     

ThalInd, a β-thalassemia and hemoglobinopathies database for India: defining a model country-specific and disease-centric bioinformatics resource

Web-based informatics resources for genetic disorders have evolved from genome-wide databases like OMIM and HGMD to Locus Specific databases (LSDBs) and National and Ethnic Mutation Databases (NEMDBs). However, with the increasing amenability of genetic disorders to diagnosis and better management, many previously underreported conditions are emerging as disorders of public health significance. In turn, the greater emphasis on noncommunicable disorders has generated a demand for comprehensive and relevant disease-based information from end-users, including clinicians, patients, genetic epidemiologists, health administrators and policymakers. To accommodate these demands, country-specific and disease-centric resources are required to complement the existing LSDBs and NEMDBs. Currently available preconfigured Web-based software applications can be customized for this purpose. The present article describes the formulation and construction of a Web-based informatics resource for β-thalassemia and other hemoglobinopathies, initially for use in India, a multiethnic, multireligious country with a population approaching 1,200 million. The resource ThalInd (http://ccg.murdoch.edu.au/thalind) has been created using the LOVD system, an open source platform-independent database system. The system has been customized to incorporate and accommodate data pertinent to molecular genetics, population genetics, genotype-phenotype correlations, disease burden, and infrastructural assessment. Importantly, the resource also has been aligned with the administrative health system and demographic resources of the country.     

Chromophobe renal cell carcinoma—chromosomal aberration variability and its relation to Paner grading system: an array CGH and FISH analysis of 37 cases

Genetically, chromophobe renal cell carcinoma (ChRCC) is characterized by multiple chromosomal changes, especially losses. The most common losses include chromosomes 1, 2, 6, 10, 13, 17, and 21. The Fuhrman grading system lacks prognostic relevance for ChRCC, and recently, a new grading system for ChRCC was proposed by Paner. The objective of this study was to map the spectrum of chromosomal aberrations (extent and location) in a large cohort of ChRCCs and relate these findings to the Paner grading system (PGS). A large cohort of ChRCC was reviewed and graded according to the PGS. All the cases were reevaluated and separated into groups according to their PGS. The final study set was 37 patients. ChRCCs were divided into PG 1–3, sarcomatoid, and aggressive groups. “Aggressive ChRCCs” were designated cases with known metastatic activity, local recurrence, aggressive growth to the adjacent organs, or invasive growth into the renal sinus (with/without angioinvasion). Sarcomatoid tumors were divided into their epithelial and sarcomatoid component (further molecular genetic analyses were performed separately). Array comparative genome hybridization and/or fluorescence in situ hybridization analysis was applied to 42 samples from the 37 cases. Multiple losses, as well as gains, were detected in different chromosomes. Regardless of the PGS groups, the most frequently detected losses involved chromosomes 1 (27/37), 2 (26/37), 6 (23/37), 10 (26/37), 13 (19/37), and 17 (24/37). Loss of chromosome 21 was found in 12/37 cases. The most frequently detected gains were found on chromosomes 4 (22/37), 7 (24/37), 15 (20/37), 19 (22/37), and 20 (21/37). Cluster analysis showed that there is no relation between PGS and particular pattern of chromosomal changes (losses or gains) in ChRCCs. Conclusions are as follows: (1) ChRCCs showed a significantly broader spectrum of chromosomal aberrations than previously recognized. While previously published chromosomal losses were found in our cohort, gains of multiple chromosomes were also identified in a high percentage. The most frequently detected gains involved chromosomes 4, 7, 15, 19, and 20. (2) There is no relation between chromosomal numerical changes and Paner grading system.     

Effect of ascorbate and α-tocopherol on resistance of β-carotene to oxidation

The efficiency of ascorbate and α-tocopherol as stabilizers of β-carotene, which is widely used in complex therapy and prevention of some diseases accompanied by oxidative stress, was studied. The latency of induced β-carotene oxidation linearly depends on ascorbate concentration, while steady-state rate nonlinearly depends on the concentration of α-tocopherol, which attests to involvement of antioxidants in various stages of chain oxidation of β-carotene. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 7, pp. 59–62, July, 2000     

Noradrenaline release in the locus coeruleus modulates memory formation and consolidation; roles for α- and β-adrenergic receptors

Noradrenaline, essential for the modulation of memory, is released in various parts of the brain from nerve terminals controlled by the locus coeruleus (LoC). Noradrenaline release consequent upon input from higher brain areas also occurs within the LoC itself. We examined the effect of noradrenaline on adrenergic receptors in the LoC on memory processing, using colored bead discrimination learning in the young domestic chick. We have shown previously that the release of noradrenaline in the hippocampus and cortex (mesopallium) is essential for acquisition and consolidation of short-term to intermediate and to long-term memory. Noradrenaline release within the LoC is triggered by the glutamatergic input from the forebrain. Inhibition by LoC injection of NMDA or AMPA receptor antagonists is rescued by injection of β2-and β3-adrenoceptor (AR) agonists in the hippocampus. We show that inhibition of α2A-ARs by BRL44408 in the LoC up to 30 min post-training consolidates weakly-reinforced learning. Conversely activation of α2A-ARs in the LoC at the times of consolidation between short-term and intermediate and long-term memory caused memory loss, which is likely to be due to a decreased release of noradrenaline within these two time windows. The α2A-AR antagonist will block presynaptic inhibitory receptors leading to an increase in extracellular noradrenaline. This interpretation is supported by the actions of noradrenaline uptake blockers that produce the same memory outcome. BRL44408 in the mesopallium also caused memory enhancement. β2-ARs are important in the first time window, whereas α1-, α2C-and β3-ARs are important in the second time window. The results reveal that for successful memory formation noradrenaline release is necessary within the LoC as well as in other brain regions, at the time of consolidation of memory from short-term to intermediate and from intermediate to long-term memory.     

A rapid detection for α-thalassemia by PCR combined with dissociation curve analysis

Deletion mutations of 3.7 kb and 4.2 kb of α-globin gene are the most common causes of α-thalassemia (-α^3.7/, -α^4.2/). A simple, rapid assay by using a single-tube PCR to detect the two deletions has been needed. In this study, a pair of shared primers was designed for α2 and α1 gene but with length-different amplicons (159 bp and 409 bp). On the dissociation curve analysis profile after PCR, there shows two obvious peaks which represent the two different amplicons. Relative copy number of α2 and α1 gene can be deduced from the ratio of the two peaks. A comprehensive diagnosis for α-thalassemia 10 genotypes of deletions can be achieved when combined with a single-tube duplex PCR for detecting −−SEA and non-deletional alleles of αα or α^Tα. Besides, a single-tube multiplex PCR, which is a cost-effective version of dual-priming-oligonucleotide based system, was designed for two common mutations of α-thalassemia in China (Hb Constant Spring and Hb Quong Sze), and these two mutations can be identified in samples by use of dissociation curve analysis. In all, using above three PCRs followed by dissociation curve analysis, three deletions and two mutations of α-thalassemia in the populations of southern China and Southeast Asia can be detected for molecular diagnosis or prenatal diagnosis. A blinded study of 163 samples was performed using this new assay and it was concordant with the original methods. This comprehensive molecular assay is simple, rapid, automatic and cost-effective, and can be used to diagnose α-thalassemia in this geographical area.     

Homozygous deletions of a copy number change detected by array CGH: a new cause for mental retardation?

We describe two unrelated patients with mental retardation and normal karyotypes found to have relatively large homozygous deletions (>150 kb) of different regions detected by array comparative genomic hybridization (aCGH). Patient 1 showed a 157-214 kb deletion at 8q24.2, containing BAC clone RP11-17M8. This patient was born to phenotypically normal parents and has microcephaly, distinctive craniofacial features, brachymetacarpia, brachymetatarsia and severe mental retardation. This BAC clone is listed as a copy number variant on the Database of Genomic Variants (http://projects.tcag.ca/variation/). Heterozygosity for the deletion was found in the mother (father is deceased) and uniparental disomy of chromosome 8 was excluded. Patient 2 showed a 812-902 kb deletion at 12q21.1, containing BAC clone RP11-89P15. This region was not listed in any public database as a known variant. This patient has mild craniofacial dysmorphic features, bifid uvula, peripheral pulmonic stenosis and developmental delay. Heterozygosity for this deletion was confirmed in the phenotypically normal parents and two normal siblings, but surprisingly, homozygosity for the deletion in an apparently normal younger sibling brings into question whether this large homozygous copy number change (CNC) is causal. Homozygous deletions of CNCs have not previously been reported in association with a phenotype or mental retardation. These cases represent homozygosity for presumably benign CNCs, and while causality for the phenotypes cannot be confirmed, similar deletions are bound to be identified more frequently as aCGH is used with increasing regularity. Such homozygous deletions should be viewed as potentially clinically relevant.     

Haemolysis caused by alterations of α- and β-spectrin after 10 to 35 min of severe exercise

The pathophysiology of exercise related haemolysis is not thoroughly understood. We investigated whether exercise related haemolysis (1) is associated with alterations of red blood cell (RBC) membrane proteins similar to those found in inherited anaemic diseases, (2) can be induced with a non-running exercise mode, (3) is related to exercise intensity, and (4) coincides with indicators of oxidative stress. In ten triathletes [median (P25/P75-percentiles) age: 28.0 (26.3/28.5) years, height: 1.84 (1.78/1.87) m, body mass: 78.5 (74.8/80.8) kg, maximal oxygen uptake: 60.0 (57.3/64.8) ml kg⁻¹ min⁻¹], haptoglobin, α- and β-spectrin bands, malondialdehyde (MDA) and H₂O₂-induced chemiluminescence (H₂O₂-Chem) were determined immediately pre- and post-both, a 35 min low intensity and a high intensity cycling exercise [240 (218/253) vs 290 (270/300) W, P<0.05) requiring similar amounts of metabolic energy [28.3 (25.9/29.9) vs 24.9 (18.4/30.5) kJ kg⁻¹, P>0.05]. At high exercise intensity haptoglobin [1.10 (0.81/2.53) vs 1.01 (0.75/2.00) g l⁻¹] decreased (P<0.05) whilst MDA [2.80 (2.65/3.20) vs 3.13 (2.78/3.31) nmol ml⁻¹] and H₂O₂-Chem [29.70 (22.55/37.10) vs 37.25 (35.20/52.63) rel. U min] increased (P<0.05), coinciding with the disappearance of the spectrin bands in six out of ten gels. No corresponding changes were found at low intensity exercise. Ten to 35 min of non-running exercise in a regularly used intensity domain causes intra-vascular haemolysis associated with alterations in the RBC membrane proteins similar to those found after in vitro oxidative stress and in inherited anaemic diseases like Sphaerocytosis and Fanconi’s anaemia.     

Diagnosing infections—current and anticipated technologies for point-of-care diagnostics and home-based testing

In recent years, we have witnessed many transitions in healthcare systems around the globe. For example, population expansion and ageing, and the human immunodeficiency virus (HIV)-AIDS epidemics, have exerted pressure to decentralize the practice of healthcare outside of traditional settings to bring care to those in need. Upstream of patient management, diagnosis is aimed at adequately orienting medical decisions, and considerable efforts have been made to make this process faster and more efficient. However, there are several diseases and medical conditions that may/will benefit from technologies and tests that can be performed closer to the patient, at the point of care or even in the home. In this review, and in light of the paradox that technology and assay developers and healthcare officials must take into consideration for advancing human health in developed and developing countries, we present an overview of rapid diagnosis of infectious diseases at the point of care and of technologies that may contribute to enhancement of the worldwide point-of-care testing market.     

Globin gene structure in a reptile supports the transpositional model for amniote α- and β-globin gene evolution

The haemoglobin protein, required for oxygen transportation in the body, is encoded by α- and β-globin genes that are arranged in clusters. The transpositional model for the evolution of distinct α-globin and β-globin clusters in amniotes is much simpler than the previously proposed whole genome duplication model. According to this model, all jawed vertebrates share one ancient region containing α- and β-globin genes and several flanking genes in the order MPG-C16orf35-(α-β)-GBY-LUC7L that has been conserved for more than 410 million years, whereas amniotes evolved a distinct β-globin cluster by insertion of a transposed β-globin gene from this ancient region into a cluster of olfactory receptors flanked by CCKBR and RRM1. It could not be determined whether this organisation is conserved in all amniotes because of the paucity of information from non-avian reptiles. To fill in this gap, we examined globin gene organisation in a squamate reptile, the Australian bearded dragon lizard, Pogona vitticeps (Agamidae). We report here that the α-globin cluster (HBK, HBA) is flanked by C16orf35 and GBY and is located on a pair of microchromosomes, whereas the β-globin cluster is flanked by RRM1 on the 3′ end and is located on the long arm of chromosome 3. However, the CCKBR gene that flanks the β-globin cluster on the 5′ end in other amniotes is located on the short arm of chromosome 5 in P. vitticeps, indicating that a chromosomal break between the β-globin cluster and CCKBR occurred at least in the agamid lineage. Our data from a reptile species provide further evidence to support the transpositional model for the evolution of β-globin gene cluster in amniotes.     

Optimization of the method for α-l-fucosidase, β-d-galactosidase and β-d-glucuronidase determination in serum from hemolyzed blood

Purpose

Adaptation of the colorimetric method for the determination of β-d-galactosidase, β-d-glucuronidase and α-l-fucosidase activities in serums from hemolyzed blood, the material currently being discarded.

Nanoprobes for in vitro diagnostics of cancer and infectious diseases

The successful and explosive development of nanotechnology is significantly impacting the fields of biology and medicine. Among the spectacular developments of nanobiotechnology, interest has grown in the use of nanomaterials as nanoprobes for bioanalysis and diagnosis. Herein, we review state-of-the-art nanomaterial-based probes and discuss their applications in in vitro diagnostics (IVD) and challenges in bringing these fields together. Major classes of nanoprobes include quantum dots (QDs), plasmonic nanoparticles, magnetic nanoparticles, nanotubes, nanowires, and multifunctional nanomaterials. With the advantages of high volume/surface ratio, surface tailorability, multifunctionality, and intrinsic properties, nanoprobes have tremendous applications in the areas of biomarker discovery, diagnostics of infectious diseases, and cancer detection. The distinguishing features of nanoprobes for in vitro use, such as harmlessness, ultrasensitivity, multiplicity, and point-of-care use, will bring a bright future of nanodiagnosis.