• 1

    Modabber F, 1987. The Leishmaniases. Maurice J, Pearce AM, eds. Tropical Diseases Research: A Global Partnership. Eight Programme Report. Geneva: World Health Organization, 99–112.

  • 2

    Bittencourt AL, Barral-Netto M, 1995. Leishmaniasis. Doerr W, Seifert G, eds. Tropical Pathology. Heidelberg: Springer, 597–651.

  • 3

    Berman JD, 1997. Human leishmaniasis: clinical, diagnostic and chemotherapeutic development in the last 10 years. Clin Infect Dis Apr 24 :684–703.

    • Search Google Scholar
    • Export Citation
  • 4

    Barral-Netto M, Machado P, Bittencourt AL, Barral A, 1997. Recent advances in the pathophisiology and treatment of human cutaneous leishmaniasis. Curr Opin Dermatol 4 :51–58.

    • Search Google Scholar
    • Export Citation
  • 5

    Sampaio SA, Castro RM, Dilon NL, Martins JE, 1971. Treatment of mucocutaneous (American) leishmaniasis with amphetericin B: report of 70 cases. Int J Dermatol 10 :179–181.

    • Search Google Scholar
    • Export Citation
  • 6

    Soto-Mancipe J, Grogl M, Berman JD, 1993. Evaluation of pentamidine for the treatment of cutaneous leishmaniasis in Colombia. Clin Infect Dis 16 :417–425.

    • Search Google Scholar
    • Export Citation
  • 7

    Koff AB, Rosen T, 1994. Treatment of cutaneous leishmaniasis. J Am Acad Dermatol 31 :693–708.

  • 8

    Lieschke GL, Burgess AW, 1992. Granulocyte colony-stimulating factor and granulocyte-macrophage-colony stimulating factor. N Engl J Med 327 :99–106.

    • Search Google Scholar
    • Export Citation
  • 9

    Jones TC, 1996. The effect of granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on macrophage function in microbial disease. Med Oncol 13 :141–147.

    • Search Google Scholar
    • Export Citation
  • 10

    Ho JL, Reed SG, Wick EA, Giordano M, 1990. Granulocyte-macrophage and macrophage colony stimulating factors activate intramacrophage killing of Leishmania mexicana amazonensis. J Infect Dis 162 :224–230.

    • Search Google Scholar
    • Export Citation
  • 11

    Al-Zamel F, Al-Shammary FJ, El-Shewemi S, 1996. Enhancement of leishmanicidal activity of human macrophages against Leishmania major and Leishmania donovani infection using recombinant human granulocyte-macrophage colony-stimulating factor. Zentralbl Bakteriol. 285 :92–105.

    • Search Google Scholar
    • Export Citation
  • 12

    Jones TC, 1993. The effects of rhGM-CSF on macrophage function. Eur J Cancer 29 (Suppl 3):10–13.

  • 13

    Marques da Costa R, Aniceto C, Jesus FM, Mendes M, 1994. Quick healing of leg ulcers after molgramostim. Lancet 344 :481–482.

  • 14

    Kurzrock R, Talpaz M, Gutterman JU, 1992. Very low doses of GM-CSF administered alone or with erythropoietin in aplastic anemia. Am J Med 93 :41–48.

    • Search Google Scholar
    • Export Citation
  • 15

    Jaschke E, Zabernigg A, Gattinger C, 1999. Recombinant human granulocyte-macrophage colony-stimulating factor applied locally in low doses enhances healing and prevents recurrence of chronic venous ulcers. Int J Dermatol 38 :380–386.

    • Search Google Scholar
    • Export Citation
  • 16

    Robson MC, Phillips TJ, Falanga V, Odenheimer DJ, Parish LC, Jensen JL, Steed DL, 2001. Randomized trial of topically applied repifermin (recombinant human keratinocyte growth factor-2) to accelerate wound healing in venous ulcers. Wound Repair Regen 9 :347–352.

    • Search Google Scholar
    • Export Citation
  • 17

    Almeida RP, D’Oliveira A Jr, Machado P, Bacellar O, Ko AI, Ribeiro de Jesus A, Mobashery N, Santos JB, Carvalho EM, 1999. Randomized, double-blind study of stibogluconate plus human granulocyte macrophage colony-stimulating factor versus stibogluconate alone in the treatment of cutaneous leishmaniasis. J Infect Dis 180 :1735–1737.

    • Search Google Scholar
    • Export Citation
  • 18

    Santos JB, de Jesus AR, Machado PR, Magalhães A, Salgado S, Carvalho EM, Almeida RP, 2004. Recombinant human GM-CSF applied topically in low doses plus antimony enhances healing of cutaneous leishmaniasis ulcers: a randomized, double-blind study. J Infect Dis 190 :1793–1796.

    • Search Google Scholar
    • Export Citation
  • 19

    Robson M, Kucukcelebi A, Carp SS, 1994. Effects of granulocyte-macrophage colony-stimulating factor on wound contraction. Eur J Clin Microbiol Infect Dis 13 :41–46.

    • Search Google Scholar
    • Export Citation
  • 20

    Kaplan G, Walsch G, Guido LS, Meyn P, Burkhardt RA, Abalos RM, Barker J, Frindt PA, Fajardo TT, Celona R, 1992. Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony–stimulating factor. Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing. J Exp Med 175 :1717–1728.

    • Search Google Scholar
    • Export Citation
  • 21

    Kucukcelebi A, Carp SS, Haward PG, 1992. Granulocyte-macrophage colony-stimulating factor reverses the inhibition of wound contraction caused by bacterial contamination. Wounds 4 :241–247.

    • Search Google Scholar
    • Export Citation
  • 22

    Doherty TM, Coffman RL, 1993. Leishmania antigens presented by GM-CSF-derived macrophages protect susceptible mice against challenge with Leishmania major. J Immunol 150 :5476–5483.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 
 
 

 

 

 

 

 

 

SUCCESSFUL TREATMENT OF REFRACTORY CUTANEOUS LEISHMANIASIS WITH GM-CSF AND ANTIMONIALS

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  • 1 Serviço de Imunologia, Hospital Universitário Professor Edgard Santos, Universidade Federal da Bahia, Salvador, Bahia, Brazil; Instituto de Investigação em Imunologia (iii)/CNPq, São Paulo, Brazil

Therapeutic failure in the treatment of cutaneous leishmaniasis (CL) occurs in 5% of patients infected by Leishmania braziliensis. This study evaluates the use of topically applied granulocyte macrophage colony-stimulating factor (GM-CSF) combined with the standard dose of antimony to treat refractory cases of CL. Five patients who had received three courses or more of antimony were enrolled in an open-label clinical trial. One to 2 mL of the GM-CSF solution (10 μg/mL in 0.9% saline) was reapplied topically, and dressings were changed three times per week for 3 weeks, associated with standard parenteral antimony (20 mg kg−1 day−1 for 20 days). All the patients healed their CL ulcers; 3 healed within 50 days (21, 27, and 44 days) and 2 in 118 and 120 days after beginning therapy. There were no side effects. This study shows that combined topically applied GM-CSF and antimony can be effective and well tolerated in the treatment of relapsed CL.

INTRODUCTION

Cutaneous leishmaniasis (CL), caused by Leishmania spp., is a sandfly-transmitted protozoal disease endemic in many tropical countries of the Americas, Africa, and Asia. It is estimated that 400,000 new cases occur each year worldwide.1 In areas of Leishmania braziliensis transmission, the typical initial clinical manifestation is a single skin ulceration, localized predominantly in the lower limbs, areas commonly exposed to phlebotomine bites.2 Weeks to years after the onset of the cutaneous disease, mucosal lesion(s) involving the nasal mucosa, palate, pharynx, larynx, and/or vocal cords may develop.3

The pentavalent antimonials have been the treatment of choice for leishmaniasis for more than 50 years despite the need for daily parenteral injections for 20 to 30 days. There is a prolonged healing time of 3 to 4 months, and serious side effects include pancreatitis, liver enzyme abnormalities, and cardiac arrhythmia.3,4 Alternative drugs, such as amphotericin B and pentamidine, are of greater toxicity; however, amphotericin B is much less toxic and very effective if used in liposomes, although it is more expensive.5,6 Therapeutic failure is observed in about 5% of the patients with cutaneous ulcers due to L. braziliensis.7 The granulocyte macrophage colony-stimulating factor (GM-CSF) is a multipotential growth factor for marrow stem cells.8 Both Th1 and Th2 lymphocyte subsets respond to GM-CSF.9 In vitro, GM-CSF has been shown to activate macrophages to kill leishmania.10,11 Besides its microbicidal activity, GM-CSF is known also to stimulate fibrosis and tissue wound healing.12,13 It has been shown that GM-CSF used in low doses has an effect in platelet function in the bone marrow.14 Moreover, GM-CSF locally applied in low doses improves the healing of chronic venous ulcer15 and also another cytokine, keratinocyte growth factor-2, applied locally accelerated wound healing in chronic venous ulcer.16 In double-blind randomized trials, we have shown that GM-CSF injected intralesional17 or applied topically18 as adjuvant therapy to antimony accelerates cutaneous leishmaniasis ulcer healing.16 The currentt study was designed to evaluate the effect of topically applied low doses of GM-CSF plus standard antimony in the healing of cutaneous leishmaniasis ulcers refractory to antimony.

MATERIALS AND METHODS

All patients lived in Corte de Pedra or surrounding communities, an endemic area of tegumentary leishmaniasis in the state of Bahia, Brazil. Each of the five patients had a confirmation of leishmaniasis by typical leishmania skin lesion and a positive skin test with Leishmania antigen or by parasite isolation in culture of lesion aspirates. Previous studies from our group have demonstrated L. braziliensis to be the etiologic agent in this region, and in the current study Leishmania was typed by using monoclonal antibody and zymodema analysis. All the patients had received at least three courses of intravenous pentavalent antimony in a dose of 20 mg kg−1 day−1 for 20 days and had at least three episodes of relapse of their cutaneous lesions. Additionally, the last treatment was done under observation in our clinic. After documentation of persistence of active lesions 90 days after the last therapy, the patients were considered refractory to antimony therapy and were enrolled in the study. This study was approved by the human ethical committee for research of the Hospital Universitário Prof. Edgard Santos, and informed consent was obtained from patients or guardians. The patients were treated topically with a commercially available GM-CSF (Leucomax; Novatis, São Paulo, Brazil) diluted to a final concentration of 10 μg/mL in saline.15,18 Ulcers were cleansed with 0.9% sodium chloride solution, and then 1 to 2 mL of the GM-CSF solution (10 μg/mL in 0.9% sodium chloride solution) was applied topically. A 1-mL portion was enough to cover a 10-cm2 area. A nonadhesive wound compress (Adaptic; São Paulo, Brazil) was secured over the area with a cotton bandage and a short elastic compression bandage (Coban; 3M, São Paulo, Brazil) was applied. The GM-CSF solution was reapplied, and dressings were changed three times per week for 3 weeks.15,18 All patients received intravenous pentavalent antimonial (meglumin antimoniate) 20 mg per kg of body weight daily for 20 days. The patients were evaluated on Days 30, 60, 90, 120, 180, and 360 after treatment onset. On all occasions, two separate medical doctors examined the patients. A cure was defined to be a patient whose previous ulcer (crater and border) had undergone complete re-epithelialization.

RESULTS

Complete healing was observed in all five patients included in the study. Table 1 shows the demographic data and time of healing of the 5 patients, 3 males and 2 females, with ages ranging from 14 to 25 years. The largest diameter size of the ulcer ranged from 14 to 120 mm. The majority of the lesions were below the waist and on the lower limbs (four patients). The other patient presented a lesion on the right arm. Four patients had positive Leishmania skin test, and one had negative test, but the parasite was cultivated from the lesion and was typed as L. braziliensis. The last course of antimony treatment had been given more than 90 days prior the inclusion in the study. Each of the five patients demonstrated complete healing by use of topical GM-CSF and antimony.

All patients cured with one new course of antimony combined to GM-CSF. Three patients had their ulcers healed before 50 days with a mean healing time of 66 ± 49 days (Days 21, 27, 44). The other two patients required 118 and 120 days for complete healing. Continuous follow-up of all cases reported in this study was performed over a period of at least 12 months after healing, and no relapse was observed. No systemic side-effects or contact allergic reactions from the therapy were reported by the patients or observed by the investigators.

DISCUSSION

This open clinical trial indicates that successful healing can be accomplished by the use of topically applied GM-CSF as adjuvant therapy to pentavalent antimony to treat cutaneous leishmaniasis patients refractory to antimony therapy. Our previous double-blind randomized data also showed that GM-CSF injected intralesionally or used topically as adjuvant therapy to antimony17,18 can be useful to reduce the healing time of cutaneous leishmaniasis ulcers. Previous studies have shown the ability of GM-CSF to improve wound healing. Robson and others19 demonstrated that topically applied GM-CSF, in a dose of 1 μg/cm2 of wound areas, is as effective as a dose of 10 μg/cm2 to reverse the inhibition of wound contraction caused by bacterial contamination in rats. Locally applied GM-CSF has also been useful in the treatment of chronic venous ulcers.15

GM-CSF may decrease the healing time of cutaneous leishmaniasis ulcers by three potential mechanisms: increasing parasite killing by directly activating macrophages,1012 enhancing scar formation,19 and modulating immunologic balance.20 Previous studies have shown that GM-CSF activates macrophages to kill Leishmania in vitro.10,11 GM-CSF has been described to improve healing and scarring of cutaneous lesions caused by agents other than Leishmania.21 Moreover, Doherty and others have shown that transfer of macrophages primed with GM-CSF and Leishmania antigens protected naive BALB/c against challenge with Leishmania major.22 The proposed mechanism was the induction of a cell-mediated immune response to Leishmania antigen due to differentiation of CD4+ T cells to Th1. The healing of relapsing cutaneous leishmaniasis ulcers brought exceptional benefits to patients who had been through repeated courses of antimony. These patients are usually treated with other drugs with higher toxicity, such as amphotericin B, which needs to be administered during hospitalization; however, the drug is much less toxic and very effective if used in liposomes. Locally applied GM-CSF in conjunction with antimony should be evaluated in further studies in severe cases of leishmaniasis and in patients who do not respond to repeated courses of antimony.

Table 1

Clinical data of the cutaneous leishmaniasis patients resistant to antimony therapy and response to topically applied GM-CSF

PatientAgeSexDisease duration (months)Previous courses of antimony treatmentUlcer size (mm)Leishmania skin test (mm)Healing time with topical GM-CSF (days)
* The parasite was cultivated from the lesion.
1326718F1603141521
1324916F24063517118
1389414M903120Negative*44
1135319M4804301127
1292425M30063012120
Mean ± SD18 ± 425 ± 154.4 ± 1.546 ± 4214 ± 366 ± 49

*

Address correspondence to Edgar M. Carvalho, Serviço de Imunologia, Hospital Universitário Prof. Edgard Santos, UFBA, Rua João das Botas s/n, Canela, 40110-160, BA, Brazil. E-mail: edgar@ufba.br; imuno@ufba.br

Authors’ addresses: Roque P. Almeida (E-mail: roque@ufba.br), Jussamara Brito (E-mail: imuno@ufba.br), Paulo L. Machado (E-mail: imuno@ufba.br), Amélia R. de Jesus (E-mail: amelia@ufba.br), Albert Schriefer (E-mail: imuno@ufba.br), Luiz Henrique Guimarães (E-mail: umuno@ufba.br), Edgar M. Carvalho (E-mail: edgar@ufba.br), Serviço de Imunologia, Hospital Universitário Prof. Edgar Santos, UFBA, Rua João das Botas s/n, Canela, 40110-160, BA, Brazil.

Acknowledgments: The authors thank the patients included in this study. The authors thank Maria Neuza S. Souza, health post assistant, who applied the medication. We also thank Ednaldo Lago and the personnel from Corte de Pedra Health Post for their assistance and Mrs. Elbe M. S. Silva for preparing the manuscript.

Financial support This study was supported by the Brazilian Research Council (CNPq) and Fundação de Apoio à Pesquisa do Estado da Bahia (FAPESB). Dr. Edgar M. Carvalho is a senior investigator of CNPq.

REFERENCES

  • 1

    Modabber F, 1987. The Leishmaniases. Maurice J, Pearce AM, eds. Tropical Diseases Research: A Global Partnership. Eight Programme Report. Geneva: World Health Organization, 99–112.

  • 2

    Bittencourt AL, Barral-Netto M, 1995. Leishmaniasis. Doerr W, Seifert G, eds. Tropical Pathology. Heidelberg: Springer, 597–651.

  • 3

    Berman JD, 1997. Human leishmaniasis: clinical, diagnostic and chemotherapeutic development in the last 10 years. Clin Infect Dis Apr 24 :684–703.

    • Search Google Scholar
    • Export Citation
  • 4

    Barral-Netto M, Machado P, Bittencourt AL, Barral A, 1997. Recent advances in the pathophisiology and treatment of human cutaneous leishmaniasis. Curr Opin Dermatol 4 :51–58.

    • Search Google Scholar
    • Export Citation
  • 5

    Sampaio SA, Castro RM, Dilon NL, Martins JE, 1971. Treatment of mucocutaneous (American) leishmaniasis with amphetericin B: report of 70 cases. Int J Dermatol 10 :179–181.

    • Search Google Scholar
    • Export Citation
  • 6

    Soto-Mancipe J, Grogl M, Berman JD, 1993. Evaluation of pentamidine for the treatment of cutaneous leishmaniasis in Colombia. Clin Infect Dis 16 :417–425.

    • Search Google Scholar
    • Export Citation
  • 7

    Koff AB, Rosen T, 1994. Treatment of cutaneous leishmaniasis. J Am Acad Dermatol 31 :693–708.

  • 8

    Lieschke GL, Burgess AW, 1992. Granulocyte colony-stimulating factor and granulocyte-macrophage-colony stimulating factor. N Engl J Med 327 :99–106.

    • Search Google Scholar
    • Export Citation
  • 9

    Jones TC, 1996. The effect of granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on macrophage function in microbial disease. Med Oncol 13 :141–147.

    • Search Google Scholar
    • Export Citation
  • 10

    Ho JL, Reed SG, Wick EA, Giordano M, 1990. Granulocyte-macrophage and macrophage colony stimulating factors activate intramacrophage killing of Leishmania mexicana amazonensis. J Infect Dis 162 :224–230.

    • Search Google Scholar
    • Export Citation
  • 11

    Al-Zamel F, Al-Shammary FJ, El-Shewemi S, 1996. Enhancement of leishmanicidal activity of human macrophages against Leishmania major and Leishmania donovani infection using recombinant human granulocyte-macrophage colony-stimulating factor. Zentralbl Bakteriol. 285 :92–105.

    • Search Google Scholar
    • Export Citation
  • 12

    Jones TC, 1993. The effects of rhGM-CSF on macrophage function. Eur J Cancer 29 (Suppl 3):10–13.

  • 13

    Marques da Costa R, Aniceto C, Jesus FM, Mendes M, 1994. Quick healing of leg ulcers after molgramostim. Lancet 344 :481–482.

  • 14

    Kurzrock R, Talpaz M, Gutterman JU, 1992. Very low doses of GM-CSF administered alone or with erythropoietin in aplastic anemia. Am J Med 93 :41–48.

    • Search Google Scholar
    • Export Citation
  • 15

    Jaschke E, Zabernigg A, Gattinger C, 1999. Recombinant human granulocyte-macrophage colony-stimulating factor applied locally in low doses enhances healing and prevents recurrence of chronic venous ulcers. Int J Dermatol 38 :380–386.

    • Search Google Scholar
    • Export Citation
  • 16

    Robson MC, Phillips TJ, Falanga V, Odenheimer DJ, Parish LC, Jensen JL, Steed DL, 2001. Randomized trial of topically applied repifermin (recombinant human keratinocyte growth factor-2) to accelerate wound healing in venous ulcers. Wound Repair Regen 9 :347–352.

    • Search Google Scholar
    • Export Citation
  • 17

    Almeida RP, D’Oliveira A Jr, Machado P, Bacellar O, Ko AI, Ribeiro de Jesus A, Mobashery N, Santos JB, Carvalho EM, 1999. Randomized, double-blind study of stibogluconate plus human granulocyte macrophage colony-stimulating factor versus stibogluconate alone in the treatment of cutaneous leishmaniasis. J Infect Dis 180 :1735–1737.

    • Search Google Scholar
    • Export Citation
  • 18

    Santos JB, de Jesus AR, Machado PR, Magalhães A, Salgado S, Carvalho EM, Almeida RP, 2004. Recombinant human GM-CSF applied topically in low doses plus antimony enhances healing of cutaneous leishmaniasis ulcers: a randomized, double-blind study. J Infect Dis 190 :1793–1796.

    • Search Google Scholar
    • Export Citation
  • 19

    Robson M, Kucukcelebi A, Carp SS, 1994. Effects of granulocyte-macrophage colony-stimulating factor on wound contraction. Eur J Clin Microbiol Infect Dis 13 :41–46.

    • Search Google Scholar
    • Export Citation
  • 20

    Kaplan G, Walsch G, Guido LS, Meyn P, Burkhardt RA, Abalos RM, Barker J, Frindt PA, Fajardo TT, Celona R, 1992. Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony–stimulating factor. Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing. J Exp Med 175 :1717–1728.

    • Search Google Scholar
    • Export Citation
  • 21

    Kucukcelebi A, Carp SS, Haward PG, 1992. Granulocyte-macrophage colony-stimulating factor reverses the inhibition of wound contraction caused by bacterial contamination. Wounds 4 :241–247.

    • Search Google Scholar
    • Export Citation
  • 22

    Doherty TM, Coffman RL, 1993. Leishmania antigens presented by GM-CSF-derived macrophages protect susceptible mice against challenge with Leishmania major. J Immunol 150 :5476–5483.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Edgar M. Carvalho, Serviço de Imunologia, Hospital Universitário Prof. Edgard Santos, UFBA, Rua João das Botas s/n, Canela, 40110-160, BA, Brazil, Telephone: (55.71) 237-7353, Fax: (55.71) 245-7110, E-mail: edgar@ufba.br; imuno@ufba.br.
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