haptoglobins

Summary

Summary: Plasma glycoproteins that form a stable complex with hemoglobin to aid the recycling of heme iron. They are encoded in man by a gene on the short arm of chromosome 16.

Top Publications

  1. Arnold J, Saldova R, Galligan M, Murphy T, Mimura Kimura Y, Telford J, et al. Novel glycan biomarkers for the detection of lung cancer. J Proteome Res. 2011;10:1755-64 pubmed publisher
    ..The combination of all glyco-biomarkers had the highest sensitivity and specificity. This study identifies candidates for further study as potential biomarkers for the disease. ..
  2. Guthrie P, Gaunt T, Abdollahi M, Rodriguez S, Lawlor D, Smith G, et al. Amplification ratio control system for copy number variation genotyping. Nucleic Acids Res. 2011;39:e54 pubmed publisher
    ..The design is generic, simple to set up and economical. Comparisons with real-time PCR and other techniques are made and CNV assays demonstrated for haptoglobin duplicon and 'chemokine (C-C motif) ligand 3-like 1' gene. ..
  3. Banerjee S, Jia Y, Siburt C, Abraham B, Wood F, Bonaventura C, et al. Haptoglobin alters oxygenation and oxidation of hemoglobin and decreases propagation of peroxide-induced oxidative reactions. Free Radic Biol Med. 2012;53:1317-26 pubmed publisher
    ..The negative shift in the redox potential of Hb-Hp and its stabilized ferryl state may be central elements in the protection against Hb-induced oxidative damage afforded by formation of the Hb-Hp complex. ..
  4. Purushothaman K, Purushothaman M, Levy A, Lento P, Evrard S, Kovacic J, et al. Increased expression of oxidation-specific epitopes and apoptosis are associated with haptoglobin genotype: possible implications for plaque progression in human atherosclerosis. J Am Coll Cardiol. 2012;60:112-9 pubmed publisher
    ..56; p < 0.003). These findings provide insights into genetic predisposition to oxidative stress and the relationship between OSEs and macrophage apoptosis that may explain advanced atherosclerosis in human Hp2-2 plaques. ..
  5. Cooper C, Schaer D, Buehler P, Wilson M, Reeder B, Silkstone G, et al. Haptoglobin binding stabilizes hemoglobin ferryl iron and the globin radical on tyrosine ?145. Antioxid Redox Signal. 2013;18:2264-73 pubmed publisher
    ..Hp protects against Hb-induced damage in the vasculature, not by preventing the primary reactivity of heme oxidants, but by rendering the resultant protein products less damaging. ..
  6. Maresca B, Cigliano L, Corsaro M, Pieretti G, Natale M, Bucci E, et al. Quantitative determination of haptoglobin glycoform variants in psoriasis. Biol Chem. 2010;391:1429-39 pubmed publisher
    ..Abundance or structure of specific glycans, which are present in haptoglobin from patients and are different or missing in normal haptoglobin, might be associated with disease activity. ..
  7. Froguel P, Ndiaye N, Bonnefond A, Bouatia Naji N, Dechaume A, Siest G, et al. A genome-wide association study identifies rs2000999 as a strong genetic determinant of circulating haptoglobin levels. PLoS ONE. 2012;7:e32327 pubmed publisher
  8. Ohnishi H, Iihara K, Kaku Y, Yamauchi K, Fukuda K, Nishimura K, et al. Haptoglobin phenotype predicts cerebral vasospasm and clinical deterioration after aneurysmal subarachnoid hemorrhage. J Stroke Cerebrovasc Dis. 2013;22:520-6 pubmed publisher
    ..209; multivariate: P = .905, P = .292). The Hp phenotype seems to be associated with a higher rate of angiographical VS and clinical deterioration by DCI but does not affect the incidence of cerebral infarction and favorable outcome. ..
  9. Vardi M, Blum S, Levy A. Haptoglobin genotype and cardiovascular outcomes in diabetes mellitus - natural history of the disease and the effect of vitamin E treatment. Meta-analysis of the medical literature. Eur J Intern Med. 2012;23:628-32 pubmed publisher
    ..48 to 0.9). This effect was not shown in other Hp types. Hp type 2-2 carries a high risk of cardiovascular events in diabetic patients. A pharmacogenomic approach towards treatment of diabetic patients with vitamin E may be warranted. ..

Scientific Experts

More Information

Publications62

  1. Guthrie P, Rodriguez S, Gaunt T, Lawlor D, Smith G, Day I. Complexity of a complex trait locus: HP, HPR, haemoglobin and cholesterol. Gene. 2012;499:8-13 pubmed publisher
  2. Abdullah M, Kahler D, Vock C, Reiling N, Kugler C, Drömann D, et al. Pulmonary haptoglobin and CD163 are functional immunoregulatory elements in the human lung. Respiration. 2012;83:61-73 pubmed publisher
  3. Kumar D, Thota B, Shinde S, Prasanna K, Hegde A, Arivazhagan A, et al. Proteomic identification of haptoglobin ?2 as a glioblastoma serum biomarker: implications in cancer cell migration and tumor growth. J Proteome Res. 2010;9:5557-67 pubmed publisher
    ..Thus, we have identified haptoglobin as a GBM-specific serum marker with a role on glioma tumor growth and migration. ..
  4. Lynch E, Earley B, McGee M, Doyle S. Effect of abrupt weaning at housing on leukocyte distribution, functional activity of neutrophils, and acute phase protein response of beef calves. BMC Vet Res. 2010;6:39 pubmed publisher
    ..Together with the changes in lymphocyte subsets, the results suggest that there was a greater transitory reduction in immune function at housing in abruptly weaned than non-weaned beef calves. ..
  5. Pan J, Cheng T, Shih C, Chiang S, Chou S, Mao S, et al. Haptoglobin phenotypes and plasma haptoglobin levels in patients with abdominal aortic aneurysm. J Vasc Surg. 2011;53:1189-94 pubmed publisher
    ..024). Our findings suggest that plasma Hp concentrations are elevated in patients with AAA, particularly those with the Hp 2-2 phenotype. ..
  6. Chavez Bueno S, Beasley J, Goldbeck J, Bright B, Morton D, Whitby P, et al. 'Haptoglobin concentrations in preterm and term newborns'. J Perinatol. 2011;31:500-3 pubmed publisher
    ..033). HPT is detectable from birth in PT and T newborns. HPT concentrations increase in bacteremic newborns. HPT levels may have clinical utility in the evaluation of neonatal sepsis. ..
  7. Alayash A. Haptoglobin: old protein with new functions. Clin Chim Acta. 2011;412:493-8 pubmed publisher
    ..It may prove necessary to explore these protective clearing mechanisms to counter the toxicity associated with free Hb when used as oxygen therapeutics in hemolytic anemias and in RBC storage lesions. ..
  8. Møller H. Soluble CD163. Scand J Clin Lab Invest. 2012;72:1-13 pubmed publisher
    ..This review summarizes the current knowledge on the regulation of sCD163 in normal and pathological states and also deals with analytical aspects of sCD163 measurements in biological samples. ..
  9. Pimenova T, Pereira C, Gehrig P, Buehler P, Schaer D, Zenobi R. Quantitative mass spectrometry defines an oxidative hotspot in hemoglobin that is specifically protected by haptoglobin. J Proteome Res. 2010;9:4061-70 pubmed publisher
    ..Taken together, these data suggest differences in oxidative modifications resulting from peroxide induced heme emanated free radical distribution in tetrameric compared to Hp1-1/Hp2-2 stabilized dimeric Hb. ..
  10. Froehler M, Kooshkabadi A, Miller Lotan R, Blum S, Sher S, Levy A, et al. Vasospasm after subarachnoid hemorrhage in haptoglobin 2-2 mice can be prevented with a glutathione peroxidase mimetic. J Clin Neurosci. 2010;17:1169-72 pubmed publisher
    ..7%). We conclude that vasospasm after SAH in Hp-2 mice can be prevented with SYI-2074, suggesting that oxidative stress contributes significantly to vasospasm. ..
  11. Vardi M, Levy A. Is it time to screen for the haptoglobin genotype to assess the cardiovascular risk profile and vitamin E therapy responsiveness in patients with diabetes?. Curr Diab Rep. 2012;12:274-9 pubmed publisher
  12. Etzerodt A, Kjolby M, Nielsen M, Maniecki M, Svendsen P, Moestrup S. Plasma clearance of hemoglobin and haptoglobin in mice and effect of CD163 gene targeting disruption. Antioxid Redox Signal. 2013;18:2254-63 pubmed publisher
    ..The new data further underscore the fact that the Hp system in man seems to have a broader and more sophisticated role. This has major implications in the translation of data on Hb metabolism from mouse to man. ..
  13. Carlsson M, Cederfur C, Schaar V, Balog C, Lepur A, Touret F, et al. Galectin-1-binding glycoforms of haptoglobin with altered intracellular trafficking, and increase in metastatic breast cancer patients. PLoS ONE. 2011;6:e26560 pubmed publisher
    ..In conclusion, galectin-1 detects a new type of functional biomarker for cancer: a specific type of glycoform of haptoglobin, and possibly other serum glycoproteins, with a different function after uptake into tissue cells. ..
  14. Bastos B, Meyer R, Guimarães J, Ayres M, Guedes M, Moura Costa L, et al. Haptoglobin and fibrinogen concentrations and leukocyte counts in the clinical investigation of caseous lymphadenitis in sheep. Vet Clin Pathol. 2011;40:496-503 pubmed publisher
    ..Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a disease that affects small ruminants and is responsible for economic losses, including condemnation of carcasses and damaged hides...
  15. Aronowski J, Zhao X. Molecular pathophysiology of cerebral hemorrhage: secondary brain injury. Stroke. 2011;42:1781-6 pubmed publisher
  16. Hämäläinen P, Saltevo J, Kautiainen H, Mäntyselkä P, Vanhala M. Erythropoietin, ferritin, haptoglobin, hemoglobin and transferrin receptor in metabolic syndrome: a case control study. Cardiovasc Diabetol. 2012;11:116 pubmed publisher
    ..However, erythropoietin levels are related only with abdominal obesity. Higher serum erythropoietin concentrations may suggest underlying adipose tissue hypoxemia in MetS. ..
  17. Schaer D, Buehler P, Alayash A, Belcher J, Vercellotti G. Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins. Blood. 2013;121:1276-84 pubmed publisher
  18. Delanghe J, Langlois M, De Buyzere M. Haptoglobin polymorphism: a key factor in the proatherogenic role of B cells?. Atherosclerosis. 2011;217:80-2 pubmed publisher
    ..Findings in animal models (where no haptoglobin polymorphism is present) cannot always be extrapolated to humans. ..
  19. Farbstein D, Levy A. The genetics of vascular complications in diabetes mellitus. Cardiol Clin. 2010;28:477-96 pubmed publisher
    ..Use of the knowledge of these genetic differences among individuals in targeting drug therapy (pharmacogenomics) is also discussed. ..
  20. Bannikov G, Hinds C, Rajala Schultz P, Premanandan C, Rings D, Lakritz J. Serum haptoglobin-matrix metalloproteinase 9 (Hp-MMP 9) complex as a biomarker of systemic inflammation in cattle. Vet Immunol Immunopathol. 2011;139:41-9 pubmed publisher
    ..The Hp-MMP 9 ELISA may be the serological test of choice in the determination of systemic inflammation associated with bacterial sepsis. ..
  21. Kim H, Choi J, Park K, Kim H, Min Y, Kim M, et al. Anaphylactic transfusion reaction in a patient with anhaptoglobinemia: the first case in Korea. Ann Lab Med. 2012;32:304-6 pubmed publisher
    ..Consequently, anaphylactic transfusion reactions did not develop. Here, we report the first case of anhaptoglobinemia causing anaphylactic transfusion reaction in Korea. ..
  22. Rodriguez S, Williams D, Guthrie P, McArdle W, Smith G, Evans D, et al. Molecular and population analysis of natural selection on the human haptoglobin duplication. Ann Hum Genet. 2012;76:352-62 pubmed publisher
    ..1. Taken together, our results represent the first molecular analysis of natural selection in the Hp CNV genetic region. ..
  23. Soejima M, Egashira K, Kawano H, Kawaguchi A, Sagawa K, Koda Y. Rapid detection of haptoglobin gene deletion in alkaline-denatured blood by loop-mediated isothermal amplification reaction. J Mol Diagn. 2011;13:334-9 pubmed publisher
    ..In addition, the simple preparation of blood samples using NaOH saves time and effort for various genetic tests. ..
  24. Roche C, Dantsker D, Alayash A, Friedman J. Enhanced nitrite reductase activity associated with the haptoglobin complexed hemoglobin dimer: functional and antioxidative implications. Nitric Oxide. 2012;27:32-9 pubmed publisher
    ..These results in conjunction with the earlier redox properties of the Hb-Hp are discussed in terms of limiting the pathophysiological consequences of acellular Hb in the circulation. ..
  25. Sammour R, Nakhoul F, Levy A, Miller Lotan R, Nakhoul N, Awad H, et al. Haptoglobin phenotype in women with preeclampsia. Endocrine. 2010;38:303-8 pubmed publisher
    ..Haptoglobin 1-1 might have a protective role in preeclampsia. Further work is needed with more Hp 1-1 subjects before we can conclude on the possible use of Haptoglobin phenotype to assess the risk of preeclampsia. ..
  26. Marquez L, Shen C, Cleynen I, De Hertogh G, Van Steen K, Machiels K, et al. Effects of haptoglobin polymorphisms and deficiency on susceptibility to inflammatory bowel disease and on severity of murine colitis. Gut. 2012;61:528-34 pubmed publisher
    ..Experimental models of colitis showed that Hp has a protective role in inflammatory colitis, most likely by inhibiting the production of Th1 and Th17 cytokines. ..
  27. Simpson M, Snell Bergeon J, Kinney G, Lache O, Miller Lotan R, Anbinder Y, et al. Haptoglobin genotype predicts development of coronary artery calcification in a prospective cohort of patients with type 1 diabetes. Cardiovasc Diabetol. 2011;10:99 pubmed publisher
    ..Hp genotype did not predict CAC progression in individuals without diabetes. Hp genotype may aid prediction of accelerated coronary atherosclerosis in subjects with type 1 diabetes. ..
  28. Moreno P, Purushothaman M, Purushothaman K. Plaque neovascularization: defense mechanisms, betrayal, or a war in progress. Ann N Y Acad Sci. 2012;1254:7-17 pubmed publisher
  29. Spagnuolo M, Cigliano L, Maresca B, Pugliese C, Abrescia P. Identification of plasma haptoglobin forms which loosely bind hemoglobin. Biol Chem. 2011;392:371-6 pubmed publisher
    ..LB-Hpt and TB-Hpt displayed comparable levels of nitrotyrosine residues, but differences in glycan chains. Changes in LB-Hpt level might be associated with changes in Hpt functions. ..
  30. Kasvosve I, Speeckaert M, Speeckaert R, Masukume G, Delanghe J. Haptoglobin polymorphism and infection. Adv Clin Chem. 2010;50:23-46 pubmed
    ..g., antibody-like properties, immunomodulation, iron metabolism). More studies on the interplay of haptoglobin polymorphism, vaccination, and susceptibility or resistance to common infections seem warranted. ..
  31. Wobeto V, Pinho P, Souza J, Zaccariotto T, Zonati M. Haptoglobin genotypes and refractory hypertension in type 2 diabetes mellitus patients. Arq Bras Cardiol. 2011;97:338-45 pubmed
    ..Our results suggest that type-2 diabetes mellitus patients with the Hp2-1 genotype may have higher chances of developing refractory hypertension. Further studies in other diabetic populations are required to confirm these findings. ..
  32. Cahill L, El Sohemy A. Haptoglobin genotype modifies the association between dietary vitamin C and serum ascorbic acid deficiency. Am J Clin Nutr. 2010;92:1494-500 pubmed publisher
    ..The findings suggest that the greater antioxidant capacity of Hp1 might spare serum ascorbic acid. ..
  33. Tsai H, Boonyapranai K, Sriyam S, Yu C, Wu S, Khoo K, et al. Glycoproteomics analysis to identify a glycoform on haptoglobin associated with lung cancer. Proteomics. 2011;11:2162-70 pubmed publisher
    ..These preliminary findings suggest that the specific glycoform of Hp may be useful as a marker to monitor lung cancer progression. ..
  34. Kaempfer T, Duerst E, Gehrig P, Roschitzki B, Rutishauser D, Grossmann J, et al. Extracellular hemoglobin polarizes the macrophage proteome toward Hb-clearance, enhanced antioxidant capacity and suppressed HLA class 2 expression. J Proteome Res. 2011;10:2397-408 pubmed publisher
    ..Enhanced Hb clearance and antioxidant capacity together with reduced antigen presentation might therefore be essential qualities of Hb polarized macrophages in wounded tissues and hemorrhage or atherosclerotic plaques. ..
  35. Lioupis C, Barbatis C, Drougou A, Koliaraki V, Mamalaki A, Klonaris C, et al. Association of haptoglobin genotype and common cardiovascular risk factors with the amount of iron in atherosclerotic carotid plaques. Atherosclerosis. 2011;216:131-8 pubmed publisher
    ..Current evidence and pathophysiological considerations suggest that the increased intraplaque iron deposition may be associated with increased oxidative stress, affecting the stability of the carotid plaque. ..
  36. Park S, Lee S, Kawasaki N, Itoh S, Kang K, Hee Ryu S, et al. ?1-3/4 fucosylation at Asn 241 of ?-haptoglobin is a novel marker for colon cancer: a combinatorial approach for development of glycan biomarkers. Int J Cancer. 2012;130:2366-76 pubmed publisher
    ..In conclusion, the level of ?1-3/4 fucosyl epitope at Asn 241 of ?-Hp is potentially useful as a novel marker for colon cancer. ..
  37. Mendonça V, Luz N, Santos N, Borges V, Goncalves M, Andrade B, et al. Association between the haptoglobin and heme oxygenase 1 genetic profiles and soluble CD163 in susceptibility to and severity of human malaria. Infect Immun. 2012;80:1445-54 pubmed publisher
    ..The validation of these results could support the development of targeted therapies and aid in reducing the severity of malaria. ..
  38. Fischer C, Drillich M, Odau S, Heuwieser W, Einspanier R, Gabler C. Selected pro-inflammatory factor transcripts in bovine endometrial epithelial cells are regulated during the oestrous cycle and elevated in case of subclinical or clinical endometritis. Reprod Fertil Dev. 2010;22:818-29 pubmed publisher
    ..These results suggest that CXCL5, IL1B, IL8 and TNF may represent potential marker genes for the detection of cows with subclinical endometritis and for monitoring new therapeutic approaches. ..
  39. Baek J, D Agnillo F, Vallelian F, Pereira C, Williams M, Jia Y, et al. Hemoglobin-driven pathophysiology is an in vivo consequence of the red blood cell storage lesion that can be attenuated in guinea pigs by haptoglobin therapy. J Clin Invest. 2012;122:1444-58 pubmed publisher
    ..Hb sequestration by Hp might therefore be a therapeutic modality for enhancing transfusion safety in severely ill or massively transfused patients...
  40. Levy N, Vardi M, Blum S, Miller Lotan R, Afinbinder Y, Cleary P, et al. An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype. Clin Chem Lab Med. 2013;51:1615-22 pubmed publisher
    ..The Hp ELISA represents a user-friendly, rapid and highly accurate diagnostic tool for determining Hp phenotypes. This test will greatly facilitate the typing of thousands of samples in ongoing clinical studies. ..
  41. Andersen C, Torvund Jensen M, Nielsen M, de Oliveira C, Hersleth H, Andersen N, et al. Structure of the haptoglobin-haemoglobin complex. Nature. 2012;489:456-9 pubmed publisher
    ..Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin-haemoglobin for CD163 (ref. 4)...
  42. Etzerodt A, Moestrup S. CD163 and inflammation: biological, diagnostic, and therapeutic aspects. Antioxid Redox Signal. 2013;18:2352-63 pubmed publisher
    ..The diagnostic and therapeutic properties of CD163 await further clinical studies and regulatory approval before implementation in the clinic. ..
  43. Widener J, Nielsen M, Shiflett A, Moestrup S, HAJDUK S. Hemoglobin is a co-factor of human trypanosome lytic factor. PLoS Pathog. 2007;3:1250-61 pubmed
    ..We propose that infection of humans by T. b. brucei causes hemolysis that triggers the activation of TLF by the formation of Hpr-Hb complexes, leading to enhanced binding, trypanolytic activity, and clearance of parasites. ..
  44. Levy A, Asleh R, Blum S, Levy N, Miller Lotan R, Kalet Litman S, et al. Haptoglobin: basic and clinical aspects. Antioxid Redox Signal. 2010;12:293-304 pubmed publisher
    ..A pharmacogenomic strategy of administering high dose antioxidants specifically to Hp 2-2 DM individuals may be clinically effective. ..
  45. Eckersall P, Lawson F, Kyle C, Waterston M, Bence L, Stear M, et al. Maternal undernutrition and the ovine acute phase response to vaccination. BMC Vet Res. 2008;4:1 pubmed publisher
    ..The differences in response of Hp and SAA to primary and secondary vaccinations indicate that the cytokine driven APP response mechanisms vary with individual APP. ..
  46. Nielsen M, Moestrup S. Receptor targeting of hemoglobin mediated by the haptoglobins: roles beyond heme scavenging. Blood. 2009;114:764-71 pubmed publisher
    ..In conclusion, variant human homologous hemoglobin-binding proteins that collectively may be designated the haptoglobins have diverted from the haptoglobin gene...
  47. Lai I, Lin K, Larsson M, Yang M, Shiau C, Liao M, et al. A unique tetrameric structure of deer plasma haptoglobin--an evolutionary advantage in the Hp 2-2 phenotype with homogeneous structure. FEBS J. 2008;275:981-93 pubmed publisher
    ..A mechanism involved in forming Hp tetramers is proposed and discussed, and the possibility is raised that the evolved tetrameric structure of deer Hp might confer a physiological advantage. ..
  48. Vallelian F, Pimenova T, Pereira C, Abraham B, Mikolajczyk M, Schoedon G, et al. The reaction of hydrogen peroxide with hemoglobin induces extensive alpha-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways. Free Radic Biol Med. 2008;45:1150-8 pubmed publisher
    ..Failed Hb clearance, inadequate HO-1 expression, and the subsequent accumulation of oxidatively damaged Hb species might thus contribute to pathologies related to oxidative stress. ..
  49. Hoagland L, Campa M, Gottlin E, Herndon J, Patz E. Haptoglobin and posttranslational glycan-modified derivatives as serum biomarkers for the diagnosis of nonsmall cell lung cancer. Cancer. 2007;110:2260-8 pubmed
    ..The markers increase with stage, suggesting they may also be useful in stratifying patients at presentation and in following patients after treatment. ..
  50. Salvatore A, Cigliano L, Carlucci A, Bucci E, Abrescia P. Haptoglobin binds apolipoprotein E and influences cholesterol esterification in the cerebrospinal fluid. J Neurochem. 2009;110:255-63 pubmed publisher
    ..The results suggest that Hpt might save the function of ApoA-I and ApoE for cholesterol esterification, a process contributing to cholesterol elimination from the brain. ..
  51. Schaer C, Vallelian F, Imhof A, Schoedon G, Schaer D. CD163-expressing monocytes constitute an endotoxin-sensitive Hb clearance compartment within the vascular system. J Leukoc Biol. 2007;82:106-10 pubmed
    ..Disruption of monocyte Hb-Hp clearance may increase Hb-Hp toxicity and contribute to the pathogenesis of systemic inflammatory diseases associated with reduced monocyte CD163 expression. ..
  52. Teye K, Soejima M, Quaye I, Pang H, Tsuneoka M, Koda Y, et al. Haptoglobin gene promoter polymorphism and haplotypes are unique in different populations. Hum Biol. 2006;78:121-6 pubmed
    ..Six base substitutions at the promoter region were population specific. Only 3 out of 18 haplotypes were shared among the populations. A probable application of HP in human population genetics appears legitimate. ..
  53. Ruutu T, Barosi G, Benjamin R, Clark R, George J, Gratwohl A, et al. Diagnostic criteria for hematopoietic stem cell transplant-associated microangiopathy: results of a consensus process by an International Working Group. Haematologica. 2007;92:95-100 pubmed
    ..The sensitivity and specificity of this definition exceed 80%. The Working Group recommends that the presented criteria of TAM be adopted in clinical use, especially in scientific trials. ..