Inducible nitric oxide synthase and cytokine pattern in lesions of patients with American cutaneous leishmaniasis.

American tegumentary leishmaniasis has three forms: localized (LCL), found in resistant individuals; diffuse (DCL), found in susceptible individuals; and intermediate cutaneous leishmaniasis (ICL), found in individuals with exacerbated immunity. We evaluated cytokines and inducible nitric oxide synthase (iNOS) in lesions of LCL, ICL and DCL using immunohistochemistry. LCL granulomas showed a preponderance of interferon (IFN)-gamma and interleukin (IL)-12 expression, whereas ICL granulomas had more IL-4-, IL-10- and mainly transforming growth factor (TGF)-beta1-expressing cells. Higher densities of iNOS+ cells were observed in ICL and LCL than in DCL. iNOS was also expressed in keratinocytes of LCL and ICL lesions, and in epidermal dendritic cells of ICL lesions. In LCL and ICL, most keratinocytes expressed IL-12 and a portion expressed IFN-gamma. IL-12+ and IFN-gamma+ dendritic cells were absent or sparse in LCL and ICL epidermis. Our results show the importance of iNOS, IL-12 and INF-gamma in LCL and ICL lesions, emphasizing the existence of a mixed cytokine pattern in ICL different from the Th1 and Th2 responses established in LCL and DCL lesions.

Acute immobilization stress induces clinical and neuroimmunological alterations in experimental murine cutaneous leishmaniasis.

BACKGROUND: The skin is an important component of the neuroendocrine-immune axis. Several studies have shown that stress exacerbates skin disorders, affecting the function of sebaceous glands, keratinocytes, epidermal Langerhans cells and other cells, having an impact on the pathogenesis of many immunologically associated skin diseases. In American cutaneous leishmaniasis, we have shown the importance of the epidermis as a regulatory site, with the key participation of Langerhans cells. OBJECTIVES: To analyse the effect of acute immobilization stress on Langerhans cells, substance P (SP), calcitonin gene-related peptide (CGRP) and the natural course of infection in a murine model of cutaneous leishmaniasis. METHODS: BALB/c mice, susceptible to Leishmania infection, were placed under acute stress by immobilization (confinement) for 2 or 8 h before inoculation with L. mexicana (MHOM/BZ/82/BEL21). An avidin-biotin immunoperoxidase technique was used for cell and neuropeptide identification. RESULTS: The stressed animals became more susceptible to the parasite infection, which was manifested by acceleration and exacerbation of the lesions. In addition, the stressed animals showed morphological alterations (spherical bodies and shortened dendrites) and decreased numbers of epidermal Langerhans cells, when compared with control L. mexicana-infected mice. Mice stressed for 8 h showed greater and antidromic immunoreactivity to CGRP and SP at the time of infection. Moreover, the single inoculation of parasites caused a decrease of CGRP innervation. CONCLUSIONS: Acute immobilization stress induces an immunosuppressive state that further favours Leishmania invasion in susceptible animals.

Nitric oxide and cellular immunity in experimental cutaneous leishmaniasis.

We examined the local and systemic production of nitric oxide (NO) and the pattern of cytokine during the course of Leishmania mexicana infection in susceptible BALB/c and resistant C57BL/6 mice. NO derivatives were measured in serum, and the expression of inducible nitric oxide synthase (iNOS), interferon (IFN-gamma), interleukin (IL-4) and epidermal Langerhans cells (LC) was measured in the lesions by immunohistology. Circulating NO concentrations, iNOS+ cell density, IFN-gamma+ Th1 cells and CD205+ Langerhans cells were higher in early lesions of resistant C57BL/6 mice. In contrast, susceptible BALB/c mice developed chronic and progressive lesions with a predominance of IL-4+ Th2 cells. In both susceptible and resistant mice, lesion size and lymph node volume followed a similar course. The early local and systemic production of NO in resistant mice may be related with the premature production of IFN-gamma observed, contributing to the resolution of the lesion.

Microwave irradiation for rapid epidermis-dermis separation and improved epidermal cell immunodetection.

We explored the effects of microwave irradiation on epidermal-dermal separation and subsequent immunostaining of epidermal cells. Epidermal sheets were obtained after incubation in 0.02 M EDTA in PBS and microwave irradiation with 4 pulses of 420 watts for 5 sec, with a total incubation period of 4 min. The control epidermal sheets were immunostained for Langerhans cells and dendritic epidermal T cells using a conventional immunoperoxidase method. The experimental immunodetection of these cells was assisted by incubating the primary antibodies for 10 min at 70 watts. We showed a simple and rapid method for separation of the epidermal-dermal junction and immunostaining of epidermal cells with optimal morphological preservation.

Intermediate or chronic cutaneous leishmaniasis: leukocyte immunophenotypes and cytokine characterisation of the lesion.

The American cutaneous forms of leishmaniasis include immune-responder individuals with localised cutaneous leishmaniasis (LCL) and non-responder individuals with diffuse cutaneous leishmaniasis (DCL). Patients with intermediate or chronic cutaneous leishmaniasis (ICL) have increased morbidity due to the length of their illness, atypical forms and areas of compromise. In the present study, we evaluated the expression of the leukocyte antigens (CD4, CD8, CLA: cutaneous lymphocyte antigen, CD69, CD83 and CD1a) and cytokines (IFN-gamma, IL-4, IL-10 and TGF-beta 1) in the lesions of patients with ICL (n = 18) using an immunocytochemical procedure. ICL results were compared with the information for LCL (n = 19) and DCL (n = 4). The numbers of CD4+ and CD8+ T cells in ICL were similar to those of LCL lesions, but significantly different (P < or = 0.05) from DCL lesions. LCL lesions have about half the numbers of early activated CD69+ cells as ICL, but most are CLA+ skin homing memory T cells, whereas ICL lesions have the highest number of CD69+ T cells, but about one-third of these cells expressed CLA. This suggests that the granuloma of ICL patients contains many activated T cells that are unprimed to cutaneous-launched antigens, thus contributing to an aberrant immune response. In contrast, DCL granulomas presented the lowest numbers of activated CD69+ and CLA+ cells, associated with the characteristic tolerogenic state of these patients. The immunolocalisation of cytokines showed a mixed cytokine pattern in ICL lesions with many positive cells for IL-10, TGF-beta 1, IL-4 and IFN-gamma, with a preponderance of the first two, and different from the prevalent Th1 and Th2 responses associated with LCL and DCL lesions, respectively. CD1a+ Langerhans cells were decreased (P < or = 0.05) in both ICL (271 +/- 15 cells/mm2) and DCL (245 +/- 19 cells/mm2) as compared to LCL (527 +/- 54 cells/mm2) epidermis. The percentage of IL-10+ epidermal Langerhans cells in ICL (33.69), from the total CD1a+ population, was higher than in LCL (17.45). In addition, fewer CD83+ primed Langerhans cells were present in ICL epidermis. The diminished participation of epidermal Langerhans cells, causing a defective signalling by the epidermis, in ICL lesions may account for the tissue-damaging state observed in these patients.

Ultrastructure of hepatocyte abnormalities in perimetastatic areas.

The ultrastructural pathology of non-invaded hepatocytes located in close proximity to metastases from diverse gastrointestinal carcinomas was studied. Observed abnormalities included swelling of rough and smooth endoplasmic reticulum, proliferation of lysosomes, and mitochondrial alterations as presence of granules and paracrystalline inclusions, marked pleomorphism, and lack of cristae. These results show that, contrary to the classical conception, the non-invaded cells surrounding primary tumours or their metastases could be abnormal.

Histochemical and ultrastructural study of skeletal muscle in patients with sepsis and multiple organ failure syndrome (MOFS).

Muscle biopsies for histochemical and ultrastructural analysis were obtained from seven critically ill patients admitted to the Intensive Care Unit of the "Domingo Luciani" Hospital, Caracas, Venezuela. The sample included two patients with sepsis of abdominal origin, and five that presented sepsis/MOFS, with renal, hepatic, and respiratory disturbances and muscular weakness. Sections were examined for myosin adenosine triphosphatase (ATPase) after pre-incubation with both acid buffer (pH 4.37 and 4.6) and alkaline buffer (pH 10.3), for reduced nicotinamide dinucleotide diaphorase (NADHd), and for alpha-glycerophosphate dehydrogenase (alpha-GPDH). Sections were stained with hematoxilin and eosin to look for pathological changes and examined with a transmission electron microscope. Skeletal muscle of patients in early stage of sepsis showed a normal aspect with light microscopy, but at the ultrastructural level some of the fibres showed atrophy and some capillaries looked altered. Patients with sepsis/MOFS exhibited an evident muscle disorder with oedema, infiltrate, atrophy and segmental necrosis. All fibre types showed decrease in diameter; specially fibre types IIA and IIB. Intramuscular capillaries were thickened and occluded, indexes of capillarity were slightly reduced, and fibre oxidative activity was decreased. At ultrastructural level fibres showed severe atrophy, contractile system disorganization and segmental necrosis. Capillaries were also altered and the mononuclear cell infiltrate was abundant and represented by macrophages, lymphocytes and mastocytes.