- Email: [email protected]
Randomised placebo-controlled trial of recombinant interleukin-2 in chronic uraemic patients who are non-responders to hepatitis B vaccine
pulmonary capillary bed is a particularly important site for neutrophil margination,’ we are, to confirm this possibility, examining the leucocyte flux across the lung in response to
4
transfusion.
6
5
Mayne EE, Fitzpatrick J, Nelson SD. Leucocytosis following administration of cryoprecipitate. Acta Haematol 1970; 44: 155-60. Lee RG. Wintrobe’s clinical hematology. 9th ed. Philadelphia: Lea and Febiger, 1993. Muir AL, Cruz M, Martin BA, et al. Leukocyte kinetics in the human lung: Role of exercise and catecholamines. J Appl Physiol 1984; 57: 711-19.
References 1
2
3
American College of Chest Physicians-Society of Critical Care Medicine Consensus Conference. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest 1992; 101: 1644-55. Knaus WA, Draper EA, Wagner DP, Zimmerman JE. APACHE II: severity of disease classification system. Crit Care Med 1985; 13: 818-29. Crowder MJ, Hand DJ. Analysis of repeated measures. New York: Chapman and Hall, 1990.
a
Randomised placebo-controlled trial of recombinant interleukin-2 in chronic uraemic patients who are non-responders to hepatitis B vaccine
Natural interleukin-2 at low dose has been reported to overcome the non-responsiveness of patients with chronic uraemia to hepatitis B vaccine. Therefore, we revaccinated 52 previous such non-responders (24 on maintenance dialysis) with 20 )Jbg of recombinant preS2-containing hepatitis B vaccine with human recombinant interleukin-2 (1 MU) or placebo (randomly allocated). Seroconversion rates (74 vs 80%, respectively) and proportion of patients who elicited anti-HBs titres of 10 mIU/mL or more (56 vs 68%) were similar in both groups. Our results do not confirm local injection of interleukin-2 as an effective immunoadjuvant to hepatitis B vaccine in uraemic
patients. Lancet 1994; 344: 856-57
Patients with chronic uraemia, whether dialysed or not, have an impaired immune response to hepatitis B vaccines, with lower seroconversion rates and anti-HBs titres than healthy subjects.1,2 Despite reinforced vaccination protocols, 20-40% of such patients fail to produce protective titres and, even in responders, the duration of protection is shorter than in healthy subjects.3,4 Indeed, the uraemia-associated state of immunodeficiency is present from an early stage of renal failure and is not improved by dialysis.5 Deficient interleukin-2 production, due to impaired monocyte functionby lymphocytes from uraemic patients may have a central role, and the addition of interleukin-2 restores the proliferative response of lymphocytes in vitro.7 In a pilot study, Meuer et al8 reported a beneficial effect of low-dose (254X 105 U) natural, human interleukin-2 associated with revaccination in 10 dialysis patients who had not previously responded to a fullcourse vaccination protocol. This result prompted us to evaluate the immunoadjuvant effect of a recombinant, human interleukin-2 combined with a booster dose of hepatitis B vaccine in previously non-responsive uraemic
patients. 856
Program of Critical Care Medicine (J C Fenwick MD, M Cameron RN, J J Ronco MD, B R Wiggs MSc, M G Tweeddale MB), and Division of Hematopathology (S C Naiman MD, L P Haley MD), Vancouver General and St Paul’s Hospitals, University of British Columbia, Vancouver, British Columbia, Canada Correspondence to: Dr John C Fenwick, Intensive Care Unit, Vancouver General Hospital, Room 2438, LSP II, 855 West 12th Avenue, Vancouver BC, Canada, V5Z 1M9
We included, in a multicentre, placebo-controlled randomised trial, 52 patients with chronic uraemia (age 25-87 years; 35 males) including 28 (20 males) predialysis patients followed up at Hopital Necker and 24 (15 males) dialysis patients treated in six nephrology units in the Paris area. They had produced no detectable antiHBs, or at a titre constantly lower than 10 mIU/mL after at least four injections of either Hevac B or GenHevac B (Pasteur-Merieux). After informed consent, 27 were randomised to receive one injection of recombinant preS2-containing vaccine plus human recombinant interleukin (Roussel Uclaf), whereas 25 received the vaccine plus a placebo comprising the same vehicle components. GenHevac B (20 jjbg per dose in 0-5 mL) was injected
subcutaneously in the deltoid area, and interleukin-2 (1 MU in 0-2 mL) or 0-2 mL placebo was injected 5 minutes later by the same route as close as possible to the vaccine needletrack. All patients were kept under observation for the next 2 hours and were instructed to report any local or systemic side-effect during the next 7 days. Blood samples were taken just before and 28 days after vaccination for anti-HBs titration. An anti-HBs titre of 2 mIU/mL or more defined seroconversion, and patients were defined as responders when a protective level (10 mIU/mL) was achieved.
Patients in the interleukin-2 and placebo groups did not differ at inclusion in age (mean 60-6 [SD 11 8] vs 57.3 [15.7] years), sex distribution, bodyweight, time on haemodialysis, or in the total number of vaccine injections they had previously received (6-9 [6’7] vs 5-9 [2-8]). 26 patients had no detectable antibody at inclusion; the other 26 had a low titre (2-9 mIU/mL). 5 (18%) of the 27 patients in the interleukin-2 group complained within the first 24 hours of pain and erythema at the site of injections, and 2 also reported headache, nausea, and malaise, which resolved spontaneouslywithin 24-48 hours. In the placebo group, 2 (8%) reported only local reaction. No patient reported body temperature above 38°C. At day 28 post-vaccination, 74% of patients who received interleukin-2 and 80% of those who received placebo had seroconverted (table). 56% of patients in the interleukin-2 group compared with 68% in the placebo group had an anti-HBs of 10 mIU/mL or more. Geometric mean titres (GMTs) in responders did not differ between groups, nor did the mean anti-HBs titre (table). As expected, the overall proportion of patients who developed a protective antibody titre was higher in patients who had detectable, low-titre anti-HBs at inclusion than in patients without detectable antibody, but there was no difference between interleukin-2 and
References 1
2
day 28 In patients who received with hepatitis B vaccine placebo
Table : Antl-HBs response at
interleukin-2
or
3
4
placebo recipients. The proportion of responders was similar in predialysis and in dialysis patients (57% in both groups in predialysis patients; 54 vs 82% in dialysis patients). There was no difference in response in patients whose original vaccination was with the S vaccine (Hevac B) compared with those who initially received the pre-S2
5
6
vaccine.
Thus,
at
variance with Meuer
et
al,8
our
multicentre
placebo-controlled randomised trial did not find a significant effect for interleukin-2 in the enhancement of the response to hepatitis B revaccination in chronic uraemic patients who did not respond to a previous fullcourse vaccination. The proportion of our patients who acquired seropositive anti-HBs was similarly high, whether they had received interleukin-2 or placebo. In both groups, the proportion of responders was similar to the rate of 60% observed by Meuer et al in their patients who received interleukin-2, whereas only 12% of their patients who received vaccine alone (but without placebo) responded. Therefore, we cannot exclude that the adjuvant effect of the natural, human interleukin-2 preparationS was, at least in part, due to the mechanical injury of injection and subsequent inflammatory reaction rather than to an effect of the cytokine itself. The low response rate obtained in patients who received only the vaccine booster could be due to the weak immunogenic potency of the vaccine they used. The recombinant preS2-containing vaccine we used is highly immunogenic in uraemic patients.4 We preferred the subcutaneous route for interleukin-2 injection because the safety of recombinant interleukin-2 has been well-documented for this route. Obviously, the subcutaneous route for the vaccine was immunogenic, in our view of the consistently high rate of seroprotection achieved in our patients whether or not they received interleukin-2 with GenHevac B. Another controlled study has also failed to confirm an immunoadjuvant effect of a human recombinant interleukin-2 preparation with an S-vaccine (Engerix) in the response of dialysis patients who were previously non-responders to hepatitis B vaccination.9 Therefore, our conclusions can be generalised to all vaccines, of either the S or pre-S type. In another randomised trial, recombinant interleukin-2 did not augment the primary response to hepatitis B vaccine in healthy adults.’" Thus, we could not confirm an immunoadjuvant effect of human recombinant interleukin-2 on the response to a booster injection of hepatitis B vaccine in previously nonresponder uraemic patients. Our findings stress the need for placebo-controlled studies to evaluate accurately the effects of immunoadjuvants on the response to hepatitis B vaccines. We thank our colleagues who contributed to the study: Dr M C Geffnaud, Dr B Lebkiri and Dr P Chauveau, Hopital Necker, Paris; Dr A Deschamps and Dr B Zins, Clinique du Parc Monceau, Paris; DrJP Juquel and Dr N Simon, AURA, Paris; Dr H Fessi, Hôpital Bichat, Paris; Dr A Meyrier, Hopital Avicenne, Bobigny; and Dr D Dahmane, Hôpital Rene Dubos, Pontoise.
7
8
9
Crosnier J,
Jungers P, Couroucé AM, et al. Randomised placebo-controlled trial of hepatitis B surface antigen vaccine in French haemodialysis units: II, haemodialysis patients. Lancet 1981; i: 797-800. Köhler H, Arnold W, Renschin G, Dormeyer HH, Meyer zum Büschenfelde KH. Active hepatitis B vaccination of dialysis patients and medical staff. Kidney Int 1984; 25: 124-38. Benhamou E, Couroucé AM, Jungers P, et al. Hepatitis B vaccine: randomized trial in immunogenicity in hemodialysis patients. Clin Nephrol 1984; 21: 143-47 Jungers P, Chauveau P, Couroucé AM, et al. Immunogenicity of the recombinant Genhevac B Pasteur Vaccine against hepatitis B in chronic uremic patients. J Infect Dis 1994; 169: 399-402. Kurz P, Köhler H, Meuer S, Hütteroth T, Meyer zum Büschenfelde KH. Impaired cellular immune responses in chronic renal failure: evidence for a T cell defect. Kidney Int 1986; 29: 1209-13. Meuer SC, Hauer M, Kurz P, Meyer zum Büschenfelde KH, Köhler H. Selective blockade of the antigen-receptor mediated pathway of T cell activation in patients with impaired primary immune responses. J Clin Invest 1986; 80: 743-49. Dumann H, Meuer S, Meyer zum Büschenfelde KH, et al. Hepatitis B vaccination and interleukin-2 receptor expression in chronic renal failure. Kidney Int 1990; 38: 1164-68. Meuer SC, Dumann H, Meyer zum Büschenfelde KH, Köhler H. Low-dose interleukin-2 induces systemic immune responses against HBsAg in immunodeficient non-responders to hepatitis B vaccination. Lancet 1989; i: 15-18 Blankenstijn PJ, Van Hattum J, Boland GJ, et al. Hepatitis B vaccination of non-responder dialysis patients with recombinant hepatitis B vaccine in combination with interleukin-2. J Am Soc Nephrol
1993; 4: 334A. 10 Rose RM, Rey-Martinez J, Croteau C, et al. Failure of recombinant Interleukin-2 to augment the primary humoral response to a recombinant hepatitis B vaccine in healthy adults. J Infect Dis 1992; 165: 775-77.
Department of Nephrology, Hôpital Necker, 75743 Paris, Cedex 15, France (Prof P Jungers MD); Immunology Department, Roussel-Uclaf, Romainville (P Devillier MD); Medical Department, Pasteur-Mérieux sérums et vaccins, Marnes la Coquette (H Salomon PhD, J E Cerisier PhD); and Institut National de Transfusion Sanguine, Paris (A M Courouce PhD)
Correspondence to: Prof Paul Jungers
syndrome in Leber’s hereditary optic neuropathy Pre-excitation
Pre-excitation
syndrome is common in families with Leber’s hereditary optic neuropathy (LHON). 24 Finnish families with LHON were screened for the 11778 and the 3460 mitochondrial DNA mutations. 5 of 30 individuals with LHON and the 11778 mutation had the Wolff-ParkinsonWhite pre-excitation syndrome. None of 10 with the 3460 mutation or of 11 with "other" mutations had this syndrome. Overall, 5 of 51 LHON patients and 9 of 112 symptom-free maternal relatives had Wolff-Parkinson-White syndrome (9%). In paternal relatives, the frequency was 1.6%. Mitochondrial DNA causal for LHON may contribute
pre-excitation syndrome. Lancet 1994; 344: 857-58
to
hereditary optic neuropathy (LHON) is a mitochondrially inherited disorder that causes subacute blindness, especially in young men. Half the families with LHON harbour a mitochondrial DNA (mtDNA) point Leber’s
mutation
at
nucelotide 11778 in the ND4 subunit of
857
4
transfusion.
6
5
Mayne EE, Fitzpatrick J, Nelson SD. Leucocytosis following administration of cryoprecipitate. Acta Haematol 1970; 44: 155-60. Lee RG. Wintrobe’s clinical hematology. 9th ed. Philadelphia: Lea and Febiger, 1993. Muir AL, Cruz M, Martin BA, et al. Leukocyte kinetics in the human lung: Role of exercise and catecholamines. J Appl Physiol 1984; 57: 711-19.
References 1
2
3
American College of Chest Physicians-Society of Critical Care Medicine Consensus Conference. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest 1992; 101: 1644-55. Knaus WA, Draper EA, Wagner DP, Zimmerman JE. APACHE II: severity of disease classification system. Crit Care Med 1985; 13: 818-29. Crowder MJ, Hand DJ. Analysis of repeated measures. New York: Chapman and Hall, 1990.
a
Randomised placebo-controlled trial of recombinant interleukin-2 in chronic uraemic patients who are non-responders to hepatitis B vaccine
Natural interleukin-2 at low dose has been reported to overcome the non-responsiveness of patients with chronic uraemia to hepatitis B vaccine. Therefore, we revaccinated 52 previous such non-responders (24 on maintenance dialysis) with 20 )Jbg of recombinant preS2-containing hepatitis B vaccine with human recombinant interleukin-2 (1 MU) or placebo (randomly allocated). Seroconversion rates (74 vs 80%, respectively) and proportion of patients who elicited anti-HBs titres of 10 mIU/mL or more (56 vs 68%) were similar in both groups. Our results do not confirm local injection of interleukin-2 as an effective immunoadjuvant to hepatitis B vaccine in uraemic
patients. Lancet 1994; 344: 856-57
Patients with chronic uraemia, whether dialysed or not, have an impaired immune response to hepatitis B vaccines, with lower seroconversion rates and anti-HBs titres than healthy subjects.1,2 Despite reinforced vaccination protocols, 20-40% of such patients fail to produce protective titres and, even in responders, the duration of protection is shorter than in healthy subjects.3,4 Indeed, the uraemia-associated state of immunodeficiency is present from an early stage of renal failure and is not improved by dialysis.5 Deficient interleukin-2 production, due to impaired monocyte functionby lymphocytes from uraemic patients may have a central role, and the addition of interleukin-2 restores the proliferative response of lymphocytes in vitro.7 In a pilot study, Meuer et al8 reported a beneficial effect of low-dose (254X 105 U) natural, human interleukin-2 associated with revaccination in 10 dialysis patients who had not previously responded to a fullcourse vaccination protocol. This result prompted us to evaluate the immunoadjuvant effect of a recombinant, human interleukin-2 combined with a booster dose of hepatitis B vaccine in previously non-responsive uraemic
patients. 856
Program of Critical Care Medicine (J C Fenwick MD, M Cameron RN, J J Ronco MD, B R Wiggs MSc, M G Tweeddale MB), and Division of Hematopathology (S C Naiman MD, L P Haley MD), Vancouver General and St Paul’s Hospitals, University of British Columbia, Vancouver, British Columbia, Canada Correspondence to: Dr John C Fenwick, Intensive Care Unit, Vancouver General Hospital, Room 2438, LSP II, 855 West 12th Avenue, Vancouver BC, Canada, V5Z 1M9
We included, in a multicentre, placebo-controlled randomised trial, 52 patients with chronic uraemia (age 25-87 years; 35 males) including 28 (20 males) predialysis patients followed up at Hopital Necker and 24 (15 males) dialysis patients treated in six nephrology units in the Paris area. They had produced no detectable antiHBs, or at a titre constantly lower than 10 mIU/mL after at least four injections of either Hevac B or GenHevac B (Pasteur-Merieux). After informed consent, 27 were randomised to receive one injection of recombinant preS2-containing vaccine plus human recombinant interleukin (Roussel Uclaf), whereas 25 received the vaccine plus a placebo comprising the same vehicle components. GenHevac B (20 jjbg per dose in 0-5 mL) was injected
subcutaneously in the deltoid area, and interleukin-2 (1 MU in 0-2 mL) or 0-2 mL placebo was injected 5 minutes later by the same route as close as possible to the vaccine needletrack. All patients were kept under observation for the next 2 hours and were instructed to report any local or systemic side-effect during the next 7 days. Blood samples were taken just before and 28 days after vaccination for anti-HBs titration. An anti-HBs titre of 2 mIU/mL or more defined seroconversion, and patients were defined as responders when a protective level (10 mIU/mL) was achieved.
Patients in the interleukin-2 and placebo groups did not differ at inclusion in age (mean 60-6 [SD 11 8] vs 57.3 [15.7] years), sex distribution, bodyweight, time on haemodialysis, or in the total number of vaccine injections they had previously received (6-9 [6’7] vs 5-9 [2-8]). 26 patients had no detectable antibody at inclusion; the other 26 had a low titre (2-9 mIU/mL). 5 (18%) of the 27 patients in the interleukin-2 group complained within the first 24 hours of pain and erythema at the site of injections, and 2 also reported headache, nausea, and malaise, which resolved spontaneouslywithin 24-48 hours. In the placebo group, 2 (8%) reported only local reaction. No patient reported body temperature above 38°C. At day 28 post-vaccination, 74% of patients who received interleukin-2 and 80% of those who received placebo had seroconverted (table). 56% of patients in the interleukin-2 group compared with 68% in the placebo group had an anti-HBs of 10 mIU/mL or more. Geometric mean titres (GMTs) in responders did not differ between groups, nor did the mean anti-HBs titre (table). As expected, the overall proportion of patients who developed a protective antibody titre was higher in patients who had detectable, low-titre anti-HBs at inclusion than in patients without detectable antibody, but there was no difference between interleukin-2 and
References 1
2
day 28 In patients who received with hepatitis B vaccine placebo
Table : Antl-HBs response at
interleukin-2
or
3
4
placebo recipients. The proportion of responders was similar in predialysis and in dialysis patients (57% in both groups in predialysis patients; 54 vs 82% in dialysis patients). There was no difference in response in patients whose original vaccination was with the S vaccine (Hevac B) compared with those who initially received the pre-S2
5
6
vaccine.
Thus,
at
variance with Meuer
et
al,8
our
multicentre
placebo-controlled randomised trial did not find a significant effect for interleukin-2 in the enhancement of the response to hepatitis B revaccination in chronic uraemic patients who did not respond to a previous fullcourse vaccination. The proportion of our patients who acquired seropositive anti-HBs was similarly high, whether they had received interleukin-2 or placebo. In both groups, the proportion of responders was similar to the rate of 60% observed by Meuer et al in their patients who received interleukin-2, whereas only 12% of their patients who received vaccine alone (but without placebo) responded. Therefore, we cannot exclude that the adjuvant effect of the natural, human interleukin-2 preparationS was, at least in part, due to the mechanical injury of injection and subsequent inflammatory reaction rather than to an effect of the cytokine itself. The low response rate obtained in patients who received only the vaccine booster could be due to the weak immunogenic potency of the vaccine they used. The recombinant preS2-containing vaccine we used is highly immunogenic in uraemic patients.4 We preferred the subcutaneous route for interleukin-2 injection because the safety of recombinant interleukin-2 has been well-documented for this route. Obviously, the subcutaneous route for the vaccine was immunogenic, in our view of the consistently high rate of seroprotection achieved in our patients whether or not they received interleukin-2 with GenHevac B. Another controlled study has also failed to confirm an immunoadjuvant effect of a human recombinant interleukin-2 preparation with an S-vaccine (Engerix) in the response of dialysis patients who were previously non-responders to hepatitis B vaccination.9 Therefore, our conclusions can be generalised to all vaccines, of either the S or pre-S type. In another randomised trial, recombinant interleukin-2 did not augment the primary response to hepatitis B vaccine in healthy adults.’" Thus, we could not confirm an immunoadjuvant effect of human recombinant interleukin-2 on the response to a booster injection of hepatitis B vaccine in previously nonresponder uraemic patients. Our findings stress the need for placebo-controlled studies to evaluate accurately the effects of immunoadjuvants on the response to hepatitis B vaccines. We thank our colleagues who contributed to the study: Dr M C Geffnaud, Dr B Lebkiri and Dr P Chauveau, Hopital Necker, Paris; Dr A Deschamps and Dr B Zins, Clinique du Parc Monceau, Paris; DrJP Juquel and Dr N Simon, AURA, Paris; Dr H Fessi, Hôpital Bichat, Paris; Dr A Meyrier, Hopital Avicenne, Bobigny; and Dr D Dahmane, Hôpital Rene Dubos, Pontoise.
7
8
9
Crosnier J,
Jungers P, Couroucé AM, et al. Randomised placebo-controlled trial of hepatitis B surface antigen vaccine in French haemodialysis units: II, haemodialysis patients. Lancet 1981; i: 797-800. Köhler H, Arnold W, Renschin G, Dormeyer HH, Meyer zum Büschenfelde KH. Active hepatitis B vaccination of dialysis patients and medical staff. Kidney Int 1984; 25: 124-38. Benhamou E, Couroucé AM, Jungers P, et al. Hepatitis B vaccine: randomized trial in immunogenicity in hemodialysis patients. Clin Nephrol 1984; 21: 143-47 Jungers P, Chauveau P, Couroucé AM, et al. Immunogenicity of the recombinant Genhevac B Pasteur Vaccine against hepatitis B in chronic uremic patients. J Infect Dis 1994; 169: 399-402. Kurz P, Köhler H, Meuer S, Hütteroth T, Meyer zum Büschenfelde KH. Impaired cellular immune responses in chronic renal failure: evidence for a T cell defect. Kidney Int 1986; 29: 1209-13. Meuer SC, Hauer M, Kurz P, Meyer zum Büschenfelde KH, Köhler H. Selective blockade of the antigen-receptor mediated pathway of T cell activation in patients with impaired primary immune responses. J Clin Invest 1986; 80: 743-49. Dumann H, Meuer S, Meyer zum Büschenfelde KH, et al. Hepatitis B vaccination and interleukin-2 receptor expression in chronic renal failure. Kidney Int 1990; 38: 1164-68. Meuer SC, Dumann H, Meyer zum Büschenfelde KH, Köhler H. Low-dose interleukin-2 induces systemic immune responses against HBsAg in immunodeficient non-responders to hepatitis B vaccination. Lancet 1989; i: 15-18 Blankenstijn PJ, Van Hattum J, Boland GJ, et al. Hepatitis B vaccination of non-responder dialysis patients with recombinant hepatitis B vaccine in combination with interleukin-2. J Am Soc Nephrol
1993; 4: 334A. 10 Rose RM, Rey-Martinez J, Croteau C, et al. Failure of recombinant Interleukin-2 to augment the primary humoral response to a recombinant hepatitis B vaccine in healthy adults. J Infect Dis 1992; 165: 775-77.
Department of Nephrology, Hôpital Necker, 75743 Paris, Cedex 15, France (Prof P Jungers MD); Immunology Department, Roussel-Uclaf, Romainville (P Devillier MD); Medical Department, Pasteur-Mérieux sérums et vaccins, Marnes la Coquette (H Salomon PhD, J E Cerisier PhD); and Institut National de Transfusion Sanguine, Paris (A M Courouce PhD)
Correspondence to: Prof Paul Jungers
syndrome in Leber’s hereditary optic neuropathy Pre-excitation
Pre-excitation
syndrome is common in families with Leber’s hereditary optic neuropathy (LHON). 24 Finnish families with LHON were screened for the 11778 and the 3460 mitochondrial DNA mutations. 5 of 30 individuals with LHON and the 11778 mutation had the Wolff-ParkinsonWhite pre-excitation syndrome. None of 10 with the 3460 mutation or of 11 with "other" mutations had this syndrome. Overall, 5 of 51 LHON patients and 9 of 112 symptom-free maternal relatives had Wolff-Parkinson-White syndrome (9%). In paternal relatives, the frequency was 1.6%. Mitochondrial DNA causal for LHON may contribute
pre-excitation syndrome. Lancet 1994; 344: 857-58
to
hereditary optic neuropathy (LHON) is a mitochondrially inherited disorder that causes subacute blindness, especially in young men. Half the families with LHON harbour a mitochondrial DNA (mtDNA) point Leber’s
mutation
at
nucelotide 11778 in the ND4 subunit of
857