A. Gaafar

T-cell response in human leishmaniasis

In the present communication we provide evidence for the existence of a Th1/Th2 dichotomy in the T-cell response to Leishmania antigens in human leishmaniasis. Our data suggest that the pattern of IL-4 and IFN-gamma response is polarised in these patients. Lymphocytes from individuals recovered from cutaneous leishmaniasis (CL) responded by IFN-gamma production following stimulation with Leishmania antigens whereas cells from patients recovered from visceral leishmaniasis (VL) showed a mixed pattern of IFN-gamma and IL-4 responses. The cells producing these cytokines were predominantly CD4+. Furthermore, IL-10 plays an important role in the development of post kala azar dermal leishmaniasis (PKDL) from VL. The balance between the parasitic-specific T-cell response plays an important regulatory role in determining the outcome of Leishmania infections in humans.

Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis

The T cell response to antigens from Leishmania major promastigotes was investigated in peripheral blood mononuclear cells from Sudanese individuals with a history of cutaneous leishmaniasis (CL), Sudanese individuals with positive DTH reaction in the leishmanin skin test but with no history of skin lesions, and in Danes without known exposure to Leishmania parasites. Proliferation and production of interferon‐gamma (IFN‐γ) and IL‐4 in antigen‐stimulated cultures was measured. Lymphocytes from individuals with a history of CL proliferated vigorously and produced IFN‐γ after stimulation with either a crude preparation of L. major antigens or the major surface protease gp63. These cultures produced no or only little IL‐4. Also cells from leishmanin skin test‐positive donors with no history of CL produced IFN‐γ and no IL‐4 in response to major antigens. Cells from the unexposed Danes were not activated by gp63. The cells from Danish donors produced either IFN‐γ or IL‐4, but not both cytokines after incubation with the crude preparation of L. major antigens. The data show that the T cell response to Leishmania antigens in humans who have had uncomplicated CL or subclinical L. major infection is an IFN‐γ‐producing Th1‐like response.

Evaluation of the polymerase chain reaction in the diagnosis of cutaneous leishmaniasis due to Leishmania major: a comparison with direct microscopy of smears and sections from lesions

We have compared the sensitivity of the polymerase chain reaction (PCR) as a diagnostic tool against conventional microscopical diagnostic techniques in patients with cutaneous leishmaniasis from the Sudan. Twenty-eight patients were diagnosed according to clinical criteria followed by microscopical examination of histological sections and slit or impression smears. The PCR had a sensitivity of 86% when used alone, and 93% when combined with Southern blotting. In contrast, microscopy of histological sections had a sensitivity of 76% and slit and impression smears of only 55% and 48%, respectively. The PCR should be considered as a valuable and sensitive diagnostic tool in the diagnosis of cutaneous leishmaniasis; it has the added advantage of identification of the species of Leishmania causing the lesion.

Dichotomy of the T cell response to Leishmania antigens in patients suffering from cutaneous leishmaniasis; absence or scarcity of Th1 activity is associated with severe infections

The T cell response was studied in 25 patients suffering from cutaneous leishmaniasis caused by Leishmania major with severe (n= 10) and mild (n= 15) disease manifestations. Peripheral blood mononuclear cells (PBMC) from the patients were activated by sonicates of Leishmania promastigotes (LMP) and amastigotes (LDA), and the surface protease gp63. The proliferative responses to Leishmania antigens were lower in patients with severe disease than in patients with mild disease (P= 0·01–0·05), and such a difference was not observed in the response to purified protein derivative of tuberculin (PPD) or tetanus toxoid (TT). LMP‐induced interferon‐gamma (IFN‐γ) production was lower in patients with severe than in patients with mild disease (P < 0·05). When the IL‐4 and IFN‐γ responses of each patient were considered, two response patterns were observed in the cultures activated by the Leishmania sonicates. One response pattern was characterized by high production of IFN‐γ without production of IL‐4 (a Th1‐like pattern), the other was characterized by low IFN‐γ levels which in most cases were associated with IL‐4 production (not a Th1‐like pattern). These patterns could not be distinguished when the cells from the same donors were stimulated by TT and PPD. The percentages of patients with a Th1‐like response pattern after stimulation by LMP in patients with severe and mild disease manifestations were 30% and 80%, respectively. This difference was statistically significant (P= 0·034).

Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP.

The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L. donovani GBP (GBPP) in the diagnosis of L. donovani infections in Sudan. The sensitivity of the rGBP ELISA in diagnosing visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) was 92% and 93%, respectively. In contrast, the sensitivity of the GBPP ELISA was 55% for VL and 63% for PKDL. Plasma antibody reactivity of donors with VL and PKDL remained high for an extended period after the end of treatment. Antibody-reactivity to rGBP and GBPP was detected in 71% and 14% of plasma samples from CL patients, respectively. Plasma from healthy Sudanese donors living in an area endemic for malaria but free of leishmaniasis was negative in both assays.

Antigen-presenting Cells in Human Cutaneous Leishmaniasis due to Leishmania major.

In this study biopsies from skin lesions and draining lymph nodes of patients suffering from cutaneous leishmaniasis caused by Leishmania major were examined by immunohistochemistry, and by light and electron microscopy to identify the types of antigen‐presenting cells (APC) and their location. APC, identified morphologically and by their expression of specific cell markers, included Langerhans cells, macrophages, follicular dendritic cells, and interdigitating reticulum cells of the paracortex of lymph nodes. These cells expressed MHC class II antigens and contained Leishmania antigen. Since some keratinocytes and endothelial cells also showed these characteristics, they may also act as APC. By examining tissue samples from skin lesions and draining lymph nodes it was possible to follow the probable route of trafficking of various inflammatory cells between the skin lesion and lymph nodes. Leishmania antigen containing Langerhans cells were found in the epidermis, dermis and the regional lymph nodes. We believe these cells translocate from the epidermis to the dermis, where they take up antigen and migrate to the paracortex of the regional lymph nodes. There they are intimately associated with cells of the paracortex, and could be involved in the generation of Leishmania‐specific T memory cells. LFA‐1 ‐positive T cells of the CD45RO phenotype were found in the skin lesion. Venular endothelium in the skin lesions expressed intercellular adhesion molecule‐1 (ICAM‐1), which is the ligand for LFA‐1. The migration of lymphocytes from the vascular lumen to the site of inflammation is possibly a result of the interaction of these two adhesion molecules.

Sporotrichoid cutaneous leishmaniasis due to Leishmania major of different zymodemes in the Sudan and Saudi Arabia: a comparative study

Sporotrichoid cutaneous leishmaniasis is due to dissemination of amastigotes via the lymphatics to the subcutaneous tissues. A comparison was made between the potential to disseminate by this route of 2 parasites of different zymodemes in Sudan and Saudi Arabia. In Sudan cutaneous leishmaniasis is caused by Leishmania major zymodeme LON-1, and in Saudi Arabia by L. major LON-4. Sporotrichoid leishmaniasis was significantly more common in Sudan, occurring in 23% of patients compared with 10% in Saudi Arabia. Lymph node involvement was slightly more prevalent in the Sudan. Clinical and pathological differences between subcutaneous nodules, particularly when they ulcerate, and multiple primary cutaneous lesions are described and treatment of localized and sporotrichoid leishmaniasis is discussed. The pathological features of the primary lesions in the Sudan and Saudi Arabia were similar.

Necrotizing and suppurative lymphadenitis in Leishmania major infections

The pathology of lymph nodes and subcutaneous nodules in 6 patients with cutaneous leishmaniasis (Oriental sore) due to Leishmania major is described in this paper. In 3 patients enlarged epitrochlear lymph nodes were found to be associated with primary skin lesions in the forearm. The lymph node in one patient showed a necrotizing granulomatous reaction that simulated tuberculous lymphadenitis. Leishmania parasites were, however, found in sections of the node, and staining for mycobacteria was negative. The second patient presented with an abscess and a discharging sinus in the epitrochlear region. Parasites were found in smears of the pus and cultures for bacteria were negative. The lesion healed with antimonial therapy. In the third patient the lesion resembled cat‐scratch disease and showed stellate abscesses and granulomas. Leishmania parasites were also identified in the sections. Sections of a subcutaneous nodule from the fourth patient showed a necrotizing granuloma. The lesion healed spontaneously and the patient became leishmanin‐positive. In two other patients fine needle aspiration of the subcutaneous nodules showed parasites, granuloma and necrosis.

Humoral and cellular immune responses to glucose regulated protein 78 – a novel Leishmania donovani antigen

The recently cloned glucose regulated protein 78 (GRP78) of Leishmania donovani has been suggested as a new and promising Leishmania vaccine candidate. We assessed antibody and T‐cell reactivity to GRP78 in an enzyme‐linked immunosorbent assay (ELISA) and in lymphoproliferative assays. Serological evaluation of plasma samples obtained in Sudan revealed that 89% of patients with visceral leishmaniasis (VL), 78% with post kala‐azar dermal leishmaniasis (PKDL), and 85% with cutaneous leishmaniasis (CL) had antibody reactivity to this Leishmania antigen. Plasma from healthy Sudanese individuals living in an area endemic for malaria but free of leishmaniasis and plasma from healthy Danes was negative in the assay. GRP78 antibody was detected in 10% and 5% of plasma samples from Sudanese and Ghanaian malaria patients, respectively, whereas 35% of plasma samples from otherwise healthy Sudanese individuals with a positive leishmanin skin test showed antibody reactivity to recombinant GRP78 (rGRP78). In lymphoproliferative assays, 9 of 13 isolates of peripheral blood mononuclear cells (PBMC) from individuals previously infected with L. donovani and one of three individuals previously infected with L. major showed a response to rGRP78, whereas PBMC isolates from Danish control individuals did not respond. These findings, in addition to our previous observations in experimental CL ( Jensen et al. 2001 ), confirm GRP78 as a possible vaccine antigen.