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Honeybees, Apis mellifera, play an important role in the web of life. We rely on bees for pollinating approximately one-third of our crops, including multiple fruits, vegetables, nuts, and seeds.1,2 Bees are also instrumental in the propagation of other plants, flower nectar, and flower pollen. A. mellifera, the European honeybee, is the main pollinator in Europe and North America, but other species, including A. cerana, A. dorsata, A. floria, A. andreniformis, A. koschevnikov, and A. laboriosa, yield honey.3 Honey, propolis, and royal jelly, along with beeswax and bee pollen, are among some of the celebrated bee products that have been found to confer health benefits to human beings.4,5 Bee venom, a toxin bees use for protection, is a convoluted combination of peptides and toxic proteins such as phospholipase A2 (PLA2) and melittin that has garnered significant scientific attention of late and is used to treat various inflammatory conditions.6-8 This column will focus on the investigation of the use of bee venom to treat atopic dermatitis (AD) and acne.
Atopic dermatitis
In 2013, Kim et al. assessed the impact of bee venom on AD-related symptoms in mice, finding that it attenuated the effects of AD-simulating compounds in 48 of 80 patients injected subcutaneously. They concluded that bee venom acted by suppressing mast cell degranulation and proinflammatory cytokine expression.9 Three years later, You et al. conducted a double-blind, randomized, base-controlled multicenter study of 136 patients with AD to ascertain the effects of a bee venom emollient. For 4 weeks, patients applied an emollient with bee venom and silk protein or a vehicle lacking bee venom twice daily. Eczema area and severity index (EASI) scores were significantly lower in the bee venom group, as were the visual analogue scale (VAS) scores. The investigators concluded that bee venom is an effective and safe therapeutic choice for treating patients with AD.10 Further, in 2018, Shin et al. demonstrated that PLA2 derived from bee venom mitigates atopic skin inflammation via the CD206 mannose receptor. They had previously shown in a mouse model that PLA2 from bee venom exerts such activity against AD-like lesions induced by 2,4-dinitrochlorobenzene (DNCB) and house dust mite (Dermatophagoides farinae) extract.11 Gu et al. observed later that year that intraperitoneal administration of bee venom eased the symptoms of ovalbumin-induced AD-like skin lesions in an experimental mouse model. Bee venom also lowered serum immunoglobulin E levels and suppressed infiltration of eosinophils and mast cells. They concluded that bee venom is a viable alternative for attenuating the allergic skin inflammation characteristic of AD.12 At the end of 2018, An et al. reported on the use of an in vivo female Balb/c mouse AD model in which 1-chloro-DNCB acted as inducer in cultures of human keratinocytes, stimulated by TNF-alpha/IFN-gamma. The investigators found that bee venom and melittin displayed robust antiatopic effects as evidenced by reduced lesions. The bee products were also found to have hindered elevated expression of various chemokines and proinflammatory cytokines. The authors suggested that bee venom and melittin appear to warrant consideration as a topical treatment for AD.13 In 2019, Kim et al. demonstrated in mice that bee venom eases the symptoms of AD by inactivating the complement system, particularly through CD55 induction, which might account for its effectiveness in AD treatment in humans, they suggested.6 Early in 2020, Lee et al. demonstrated in a Balb/c mouse model that bee venom appears to be a possible therapeutic macromolecule for treating phthalic anhydride-induced AD.7
Acne
In 2013, in vitro experiments by Han et al. showed that purified bee venom exhibited antimicrobial activity, in a concentration-dependent manner, against Cutibacterium acnes (or Propionibacterium acnes). They followed up with a small randomized, double-blind, controlled trial with 12 subjects who were treated with cosmetics with pure bee venom or cosmetics without it for two weeks. The group receiving bee venom experienced significantly fewer inflammatory and noninflammatory lesions, and a significant decline in adenosine triphosphate levels (a 57.5% reduction) was noted in subjects in the bee venom group, with a nonsignificant decrease of 4.7% observed in the control group. The investigators concluded the purified bee venom may be suitable as an antiacne agent.14 Using a mouse model, An et al. studied the therapeutic effects of bee venom against C. acnes–induced skin inflammation. They found that bee venom significantly diminished the volume of infiltrated inflammatory cells in the treated mice, compared with untreated mice. Bee venom also decreased expression levels of tumor necrosis factor (TNF)-α, and interleukin (IL)-1beta and suppressed Toll-like receptor (TLR)2 and CD14 expression in C. acnes–injected tissue. The investigators concluded that bee venom imparts notable anti-inflammatory activity and has potential for use in treating acne and as an anti-inflammatory agent in skin care.15
In 2015, Kim et al. studied the influence of bee venom against C. acnes–induced inflammation in human keratinocytes (HaCaT) and monocytes (THP-1). They found that bee venom successfully suppressed the secretion of interferon-gamma, IL-1beta, IL-8, and TNF-alpha. It also galvanized the expression of IL-8 and TLR2 in HaCaT cells but hampered their expression in heat-killed C. acnes. The researchers concluded that bee venom displays considerable anti-inflammatory activity against C. acnes and warrants consideration as an alternative to antibiotic acne treatment.16 It is worth noting that early that year, in a comprehensive database review to evaluate the effects and safety of a wide range of complementary treatments for acne, Cao et al. found, among 35 studies including parallel-group randomized controlled trials, that one trial indicated bee venom was superior to control in lowering the number of acne lesions.17
Conclusion
More research, in the form of randomized, controlled trials, is required before bee venom can be incorporated into the dermatologic armamentarium as a first-line therapy for common and vexing cutaneous conditions. Nevertheless, .
Dr. Baumann is a private practice dermatologist, researcher, author, and entrepreneur who practices in Miami. She founded the Cosmetic Dermatology Center at the University of Miami in 1997. Dr. Baumann has written two textbooks and a New York Times Best Sellers book for consumers. Dr. Baumann has received funding for advisory boards and/or clinical research trials from Allergan, Galderma, Revance, Evolus, and Burt’s Bees. She is the CEO of Skin Type Solutions Inc., a company that independently tests skin care products and makes recommendations to physicians on which skin care technologies are best. Write to her at dermnews@mdedge.com.
References
1. Walsh B. The plight of the honeybee: Mass deaths in bee colonies may mean disaster for farmers – and your favorite Foods. Time Magazine, 2013 Aug 19.
2. Klein AM et al. Proc Biol Sci. 2007 Feb 7;274(1608):303-13. doi: 10.1098/rspb.2006.3721.
3. Ediriweera ER and Premarathna NY. AYU. 2012 Apr;33(2):178-82. doi: 10.4103/0974-8520.105233.
4. Baumann, L. Honey/Propolis/Royal Jelly. In Cosmeceuticals and Cosmetic Ingredients. New York:McGraw-Hill; 2014:203-212.
5. Cornara L et al. Front Pharmacol. 2017 Jun 28;8:412. doi: 10.3389/fphar.2017.00412.
6. Kim Y et al. Toxins (Basel). 2019 Apr 26;11(5):239. doi: 10.3390/toxins11050239.
7. Lee YJ et al. Inflammopharmacology. 2020 Feb;28(1):253-63. doi: 10.1007/s10787-019-00646-w.
8. Lee G and Bae H. Molecules. 2016 May 11;21(5):616. doi: 10.3390/molecules21050616.
9. Kim KH et al. Int J Clin Exp Pathol. 2013 Nov 15;6(12):2896-903.
10. You CE et al. Ann Dermatol. 2016 Oct;28(5):593-9. doi: 10.5021/ad.2016.28.5.593.
11. Shin D et al. Toxins (Basel). 2018 Apr 2;10(4):146. doi: 10.3390/toxins10040146.
12. Gu H et al. Mol Med Rep. 2018 Oct;18(4):3711-8. doi: 10.3892/mmr.2018.9398.
13. An HJ et al. Br J Pharmacol. 2018 Dec;175(23):4310-24. doi: 10.1111/bph.14487.
14. Han SM et al. J Integr Med. 2013 Sep;11(5):320-6. doi: 10.3736/jintegrmed2013043.
15. An HJ et al. Int J Mol Med. 2014 Nov;34(5):1341-8. doi: 10.3892/ijmm.2014.1933.
16. Kim JY et al. Int J Mol Med. 2015 Jun;35(6):1651-6. doi: 10.3892/ijmm.2015.2180.
17. Cao H et al. Cochrane Database Syst Rev. 2015 Jan 19;1:CD009436. doi: 10.1002/14651858.CD009436.pub2.
Honeybees, Apis mellifera, play an important role in the web of life. We rely on bees for pollinating approximately one-third of our crops, including multiple fruits, vegetables, nuts, and seeds.1,2 Bees are also instrumental in the propagation of other plants, flower nectar, and flower pollen. A. mellifera, the European honeybee, is the main pollinator in Europe and North America, but other species, including A. cerana, A. dorsata, A. floria, A. andreniformis, A. koschevnikov, and A. laboriosa, yield honey.3 Honey, propolis, and royal jelly, along with beeswax and bee pollen, are among some of the celebrated bee products that have been found to confer health benefits to human beings.4,5 Bee venom, a toxin bees use for protection, is a convoluted combination of peptides and toxic proteins such as phospholipase A2 (PLA2) and melittin that has garnered significant scientific attention of late and is used to treat various inflammatory conditions.6-8 This column will focus on the investigation of the use of bee venom to treat atopic dermatitis (AD) and acne.
Atopic dermatitis
In 2013, Kim et al. assessed the impact of bee venom on AD-related symptoms in mice, finding that it attenuated the effects of AD-simulating compounds in 48 of 80 patients injected subcutaneously. They concluded that bee venom acted by suppressing mast cell degranulation and proinflammatory cytokine expression.9 Three years later, You et al. conducted a double-blind, randomized, base-controlled multicenter study of 136 patients with AD to ascertain the effects of a bee venom emollient. For 4 weeks, patients applied an emollient with bee venom and silk protein or a vehicle lacking bee venom twice daily. Eczema area and severity index (EASI) scores were significantly lower in the bee venom group, as were the visual analogue scale (VAS) scores. The investigators concluded that bee venom is an effective and safe therapeutic choice for treating patients with AD.10 Further, in 2018, Shin et al. demonstrated that PLA2 derived from bee venom mitigates atopic skin inflammation via the CD206 mannose receptor. They had previously shown in a mouse model that PLA2 from bee venom exerts such activity against AD-like lesions induced by 2,4-dinitrochlorobenzene (DNCB) and house dust mite (Dermatophagoides farinae) extract.11 Gu et al. observed later that year that intraperitoneal administration of bee venom eased the symptoms of ovalbumin-induced AD-like skin lesions in an experimental mouse model. Bee venom also lowered serum immunoglobulin E levels and suppressed infiltration of eosinophils and mast cells. They concluded that bee venom is a viable alternative for attenuating the allergic skin inflammation characteristic of AD.12 At the end of 2018, An et al. reported on the use of an in vivo female Balb/c mouse AD model in which 1-chloro-DNCB acted as inducer in cultures of human keratinocytes, stimulated by TNF-alpha/IFN-gamma. The investigators found that bee venom and melittin displayed robust antiatopic effects as evidenced by reduced lesions. The bee products were also found to have hindered elevated expression of various chemokines and proinflammatory cytokines. The authors suggested that bee venom and melittin appear to warrant consideration as a topical treatment for AD.13 In 2019, Kim et al. demonstrated in mice that bee venom eases the symptoms of AD by inactivating the complement system, particularly through CD55 induction, which might account for its effectiveness in AD treatment in humans, they suggested.6 Early in 2020, Lee et al. demonstrated in a Balb/c mouse model that bee venom appears to be a possible therapeutic macromolecule for treating phthalic anhydride-induced AD.7
Acne
In 2013, in vitro experiments by Han et al. showed that purified bee venom exhibited antimicrobial activity, in a concentration-dependent manner, against Cutibacterium acnes (or Propionibacterium acnes). They followed up with a small randomized, double-blind, controlled trial with 12 subjects who were treated with cosmetics with pure bee venom or cosmetics without it for two weeks. The group receiving bee venom experienced significantly fewer inflammatory and noninflammatory lesions, and a significant decline in adenosine triphosphate levels (a 57.5% reduction) was noted in subjects in the bee venom group, with a nonsignificant decrease of 4.7% observed in the control group. The investigators concluded the purified bee venom may be suitable as an antiacne agent.14 Using a mouse model, An et al. studied the therapeutic effects of bee venom against C. acnes–induced skin inflammation. They found that bee venom significantly diminished the volume of infiltrated inflammatory cells in the treated mice, compared with untreated mice. Bee venom also decreased expression levels of tumor necrosis factor (TNF)-α, and interleukin (IL)-1beta and suppressed Toll-like receptor (TLR)2 and CD14 expression in C. acnes–injected tissue. The investigators concluded that bee venom imparts notable anti-inflammatory activity and has potential for use in treating acne and as an anti-inflammatory agent in skin care.15
In 2015, Kim et al. studied the influence of bee venom against C. acnes–induced inflammation in human keratinocytes (HaCaT) and monocytes (THP-1). They found that bee venom successfully suppressed the secretion of interferon-gamma, IL-1beta, IL-8, and TNF-alpha. It also galvanized the expression of IL-8 and TLR2 in HaCaT cells but hampered their expression in heat-killed C. acnes. The researchers concluded that bee venom displays considerable anti-inflammatory activity against C. acnes and warrants consideration as an alternative to antibiotic acne treatment.16 It is worth noting that early that year, in a comprehensive database review to evaluate the effects and safety of a wide range of complementary treatments for acne, Cao et al. found, among 35 studies including parallel-group randomized controlled trials, that one trial indicated bee venom was superior to control in lowering the number of acne lesions.17
Conclusion
More research, in the form of randomized, controlled trials, is required before bee venom can be incorporated into the dermatologic armamentarium as a first-line therapy for common and vexing cutaneous conditions. Nevertheless, .
Dr. Baumann is a private practice dermatologist, researcher, author, and entrepreneur who practices in Miami. She founded the Cosmetic Dermatology Center at the University of Miami in 1997. Dr. Baumann has written two textbooks and a New York Times Best Sellers book for consumers. Dr. Baumann has received funding for advisory boards and/or clinical research trials from Allergan, Galderma, Revance, Evolus, and Burt’s Bees. She is the CEO of Skin Type Solutions Inc., a company that independently tests skin care products and makes recommendations to physicians on which skin care technologies are best. Write to her at dermnews@mdedge.com.
References
1. Walsh B. The plight of the honeybee: Mass deaths in bee colonies may mean disaster for farmers – and your favorite Foods. Time Magazine, 2013 Aug 19.
2. Klein AM et al. Proc Biol Sci. 2007 Feb 7;274(1608):303-13. doi: 10.1098/rspb.2006.3721.
3. Ediriweera ER and Premarathna NY. AYU. 2012 Apr;33(2):178-82. doi: 10.4103/0974-8520.105233.
4. Baumann, L. Honey/Propolis/Royal Jelly. In Cosmeceuticals and Cosmetic Ingredients. New York:McGraw-Hill; 2014:203-212.
5. Cornara L et al. Front Pharmacol. 2017 Jun 28;8:412. doi: 10.3389/fphar.2017.00412.
6. Kim Y et al. Toxins (Basel). 2019 Apr 26;11(5):239. doi: 10.3390/toxins11050239.
7. Lee YJ et al. Inflammopharmacology. 2020 Feb;28(1):253-63. doi: 10.1007/s10787-019-00646-w.
8. Lee G and Bae H. Molecules. 2016 May 11;21(5):616. doi: 10.3390/molecules21050616.
9. Kim KH et al. Int J Clin Exp Pathol. 2013 Nov 15;6(12):2896-903.
10. You CE et al. Ann Dermatol. 2016 Oct;28(5):593-9. doi: 10.5021/ad.2016.28.5.593.
11. Shin D et al. Toxins (Basel). 2018 Apr 2;10(4):146. doi: 10.3390/toxins10040146.
12. Gu H et al. Mol Med Rep. 2018 Oct;18(4):3711-8. doi: 10.3892/mmr.2018.9398.
13. An HJ et al. Br J Pharmacol. 2018 Dec;175(23):4310-24. doi: 10.1111/bph.14487.
14. Han SM et al. J Integr Med. 2013 Sep;11(5):320-6. doi: 10.3736/jintegrmed2013043.
15. An HJ et al. Int J Mol Med. 2014 Nov;34(5):1341-8. doi: 10.3892/ijmm.2014.1933.
16. Kim JY et al. Int J Mol Med. 2015 Jun;35(6):1651-6. doi: 10.3892/ijmm.2015.2180.
17. Cao H et al. Cochrane Database Syst Rev. 2015 Jan 19;1:CD009436. doi: 10.1002/14651858.CD009436.pub2.
Honeybees, Apis mellifera, play an important role in the web of life. We rely on bees for pollinating approximately one-third of our crops, including multiple fruits, vegetables, nuts, and seeds.1,2 Bees are also instrumental in the propagation of other plants, flower nectar, and flower pollen. A. mellifera, the European honeybee, is the main pollinator in Europe and North America, but other species, including A. cerana, A. dorsata, A. floria, A. andreniformis, A. koschevnikov, and A. laboriosa, yield honey.3 Honey, propolis, and royal jelly, along with beeswax and bee pollen, are among some of the celebrated bee products that have been found to confer health benefits to human beings.4,5 Bee venom, a toxin bees use for protection, is a convoluted combination of peptides and toxic proteins such as phospholipase A2 (PLA2) and melittin that has garnered significant scientific attention of late and is used to treat various inflammatory conditions.6-8 This column will focus on the investigation of the use of bee venom to treat atopic dermatitis (AD) and acne.
Atopic dermatitis
In 2013, Kim et al. assessed the impact of bee venom on AD-related symptoms in mice, finding that it attenuated the effects of AD-simulating compounds in 48 of 80 patients injected subcutaneously. They concluded that bee venom acted by suppressing mast cell degranulation and proinflammatory cytokine expression.9 Three years later, You et al. conducted a double-blind, randomized, base-controlled multicenter study of 136 patients with AD to ascertain the effects of a bee venom emollient. For 4 weeks, patients applied an emollient with bee venom and silk protein or a vehicle lacking bee venom twice daily. Eczema area and severity index (EASI) scores were significantly lower in the bee venom group, as were the visual analogue scale (VAS) scores. The investigators concluded that bee venom is an effective and safe therapeutic choice for treating patients with AD.10 Further, in 2018, Shin et al. demonstrated that PLA2 derived from bee venom mitigates atopic skin inflammation via the CD206 mannose receptor. They had previously shown in a mouse model that PLA2 from bee venom exerts such activity against AD-like lesions induced by 2,4-dinitrochlorobenzene (DNCB) and house dust mite (Dermatophagoides farinae) extract.11 Gu et al. observed later that year that intraperitoneal administration of bee venom eased the symptoms of ovalbumin-induced AD-like skin lesions in an experimental mouse model. Bee venom also lowered serum immunoglobulin E levels and suppressed infiltration of eosinophils and mast cells. They concluded that bee venom is a viable alternative for attenuating the allergic skin inflammation characteristic of AD.12 At the end of 2018, An et al. reported on the use of an in vivo female Balb/c mouse AD model in which 1-chloro-DNCB acted as inducer in cultures of human keratinocytes, stimulated by TNF-alpha/IFN-gamma. The investigators found that bee venom and melittin displayed robust antiatopic effects as evidenced by reduced lesions. The bee products were also found to have hindered elevated expression of various chemokines and proinflammatory cytokines. The authors suggested that bee venom and melittin appear to warrant consideration as a topical treatment for AD.13 In 2019, Kim et al. demonstrated in mice that bee venom eases the symptoms of AD by inactivating the complement system, particularly through CD55 induction, which might account for its effectiveness in AD treatment in humans, they suggested.6 Early in 2020, Lee et al. demonstrated in a Balb/c mouse model that bee venom appears to be a possible therapeutic macromolecule for treating phthalic anhydride-induced AD.7
Acne
In 2013, in vitro experiments by Han et al. showed that purified bee venom exhibited antimicrobial activity, in a concentration-dependent manner, against Cutibacterium acnes (or Propionibacterium acnes). They followed up with a small randomized, double-blind, controlled trial with 12 subjects who were treated with cosmetics with pure bee venom or cosmetics without it for two weeks. The group receiving bee venom experienced significantly fewer inflammatory and noninflammatory lesions, and a significant decline in adenosine triphosphate levels (a 57.5% reduction) was noted in subjects in the bee venom group, with a nonsignificant decrease of 4.7% observed in the control group. The investigators concluded the purified bee venom may be suitable as an antiacne agent.14 Using a mouse model, An et al. studied the therapeutic effects of bee venom against C. acnes–induced skin inflammation. They found that bee venom significantly diminished the volume of infiltrated inflammatory cells in the treated mice, compared with untreated mice. Bee venom also decreased expression levels of tumor necrosis factor (TNF)-α, and interleukin (IL)-1beta and suppressed Toll-like receptor (TLR)2 and CD14 expression in C. acnes–injected tissue. The investigators concluded that bee venom imparts notable anti-inflammatory activity and has potential for use in treating acne and as an anti-inflammatory agent in skin care.15
In 2015, Kim et al. studied the influence of bee venom against C. acnes–induced inflammation in human keratinocytes (HaCaT) and monocytes (THP-1). They found that bee venom successfully suppressed the secretion of interferon-gamma, IL-1beta, IL-8, and TNF-alpha. It also galvanized the expression of IL-8 and TLR2 in HaCaT cells but hampered their expression in heat-killed C. acnes. The researchers concluded that bee venom displays considerable anti-inflammatory activity against C. acnes and warrants consideration as an alternative to antibiotic acne treatment.16 It is worth noting that early that year, in a comprehensive database review to evaluate the effects and safety of a wide range of complementary treatments for acne, Cao et al. found, among 35 studies including parallel-group randomized controlled trials, that one trial indicated bee venom was superior to control in lowering the number of acne lesions.17
Conclusion
More research, in the form of randomized, controlled trials, is required before bee venom can be incorporated into the dermatologic armamentarium as a first-line therapy for common and vexing cutaneous conditions. Nevertheless, .
Dr. Baumann is a private practice dermatologist, researcher, author, and entrepreneur who practices in Miami. She founded the Cosmetic Dermatology Center at the University of Miami in 1997. Dr. Baumann has written two textbooks and a New York Times Best Sellers book for consumers. Dr. Baumann has received funding for advisory boards and/or clinical research trials from Allergan, Galderma, Revance, Evolus, and Burt’s Bees. She is the CEO of Skin Type Solutions Inc., a company that independently tests skin care products and makes recommendations to physicians on which skin care technologies are best. Write to her at dermnews@mdedge.com.
References
1. Walsh B. The plight of the honeybee: Mass deaths in bee colonies may mean disaster for farmers – and your favorite Foods. Time Magazine, 2013 Aug 19.
2. Klein AM et al. Proc Biol Sci. 2007 Feb 7;274(1608):303-13. doi: 10.1098/rspb.2006.3721.
3. Ediriweera ER and Premarathna NY. AYU. 2012 Apr;33(2):178-82. doi: 10.4103/0974-8520.105233.
4. Baumann, L. Honey/Propolis/Royal Jelly. In Cosmeceuticals and Cosmetic Ingredients. New York:McGraw-Hill; 2014:203-212.
5. Cornara L et al. Front Pharmacol. 2017 Jun 28;8:412. doi: 10.3389/fphar.2017.00412.
6. Kim Y et al. Toxins (Basel). 2019 Apr 26;11(5):239. doi: 10.3390/toxins11050239.
7. Lee YJ et al. Inflammopharmacology. 2020 Feb;28(1):253-63. doi: 10.1007/s10787-019-00646-w.
8. Lee G and Bae H. Molecules. 2016 May 11;21(5):616. doi: 10.3390/molecules21050616.
9. Kim KH et al. Int J Clin Exp Pathol. 2013 Nov 15;6(12):2896-903.
10. You CE et al. Ann Dermatol. 2016 Oct;28(5):593-9. doi: 10.5021/ad.2016.28.5.593.
11. Shin D et al. Toxins (Basel). 2018 Apr 2;10(4):146. doi: 10.3390/toxins10040146.
12. Gu H et al. Mol Med Rep. 2018 Oct;18(4):3711-8. doi: 10.3892/mmr.2018.9398.
13. An HJ et al. Br J Pharmacol. 2018 Dec;175(23):4310-24. doi: 10.1111/bph.14487.
14. Han SM et al. J Integr Med. 2013 Sep;11(5):320-6. doi: 10.3736/jintegrmed2013043.
15. An HJ et al. Int J Mol Med. 2014 Nov;34(5):1341-8. doi: 10.3892/ijmm.2014.1933.
16. Kim JY et al. Int J Mol Med. 2015 Jun;35(6):1651-6. doi: 10.3892/ijmm.2015.2180.
17. Cao H et al. Cochrane Database Syst Rev. 2015 Jan 19;1:CD009436. doi: 10.1002/14651858.CD009436.pub2.