Oligodendrocyte mitochondria from 21-dayold Sprague-Dawley male rats were incubated with 100 nM[3lH]cholesterol. It yielded [3lH]pregnenolone at a rate of 2.5 ± 0.7 and 5-[3Hjpregnene-3fi,20a-diol at a rate of 2.5 ± 1.1 pmol per mg of protein per hr. Cultures of glial cells from 19-to 21-day-old fetuses (a mixed population of astrocytes and oligodendrocytes) were incubated for 24 hr with [3H]mevalonolactone.[3H]Cholesterol, [3H]pregnenolone, and 5-[3HJpreg-nene-3fi,20a-diol were characterized in cellular extracts. The formation of the 3H-labeled steroids was increased by dibutyryl cAMP (0.2 mM) added to the culture medium. The active cholesterol side-chain cleavage mechanism, recently suggested immunohistochemically and already observed in cultures of C6 glioma cells, reinforces the concept of "neurosteroids" applied to A5-3fi-hydroxysteroids previously isolated from brain.We have characterized two A5-3f3-hydroxysteroid metabolites of cholesterol, pregnenolone (3,8-hydroxy-5-pregnene-20-one) and dehydroepiandrosterone (3f3-hydroxy-5-androstene-17-one), in the brain of several mammalian species (rat, mouse, monkey, and occasionally pig and human). Definitive identification of the steroid moiety was made in rat brain extracts by gas/liquid chromatography-mass spectrometry (1-3). Pregnenolone has also been identified in acetone powder of rabbit brain by its mass spectrum (4). The A5-3p8-hydroxysteroids persisted in brain after removal of steroidogenic organs, and we therefore proposed that their formation or accumulation in the rat brain depends on in situ mechanisms unrelated to the peripheral endocrine gland system. However, both S. Lieberman's and our group were unable up to now to conclusively demonstrate the biosynthesis of pregnenolone in brain (3, 4). Pregnenolone is the key steroid synthesized from cholesterol in steroidogenic glands (5, 6). The oxidative side-chain cleavage of cholesterol is operated by a specific enzyme complex, which includes cytochrome P-450,CC (7). We have used specific antibodies to the bovine adrenal P-450 for the immunohistochemical localization of the enzyme in the adult rat brain, and we have found that the white matter was selectively immunostained throughout the brain (8 . The latter is a competitive inhibitor of 3,3-hydroxysteroid dehydrogenase activity, which might decrease pregnenolone levels in our experiments. The final protein concentration was 0.8 mg/ml for brain (corresponding to -30 x 106 cells) and 1-2 mg/ml for adrenal mitochondria.[3H]Cholesterol was added (5 x 106 cpm, 0.1 nmol) after suspension in 0.1 ml of Tris buffer (pH 7.4) containing 20 ,ug of Tween 80 (13). The reaction was initiated by the addition of 10 mM isocitrate to generate NADPH. Alternatively, a NADPHgenerating system was used: 0.75 mg of NADP+, 2.5 mg of glucose 6-phosphate, 0.5 unit of glucose-6-phosphate dehydrogenase (from Bakers' yeast; 260 units per mg of protein; Sigma), 95 ,g of MgCl2 in a total vol of 0.1 ml of phosphate buffer (0.1 M) (pH 7.4). The reaction was initiated by the *O...

Treatment failure and symptomatic relapse are major concerns in American tegumentary leishmaniasis (TL). Such complications are seen frequently in Leishmania guyanensis infections, in which patients respond variously to first-line antileishmanials and are more prone to develop chronic cutaneous leishmaniasis. The factors underlying this pathology, however, are unknown. Recently, we reported that a double-stranded RNA virus, Leishmania RNA virus 1 (LRV1), nested within L. guyanensis parasites is able to exacerbate experimental murine leishmaniasis by inducing a hyperinflammatory response. This report investigates the prevalence of LRV1 in human L. guyanensis infection and its effect on treatment efficacy, as well as its correlation to symptomatic relapses after the completion of first-line treatment. In our cohort of 75 patients with a diagnosis of primary localized American TL, the prevalence of LRV1-positive L. guyanensis infection was elevated to 58%. All patients infected with LRV1-negative L. guyanensis were cured after 1 dose (22 of 31 [71%]) or 2 doses (31 of 31 [100%]) of pentamidine. In contrast, 12 of 44 LRV1-positive patients (27%) presented with persistent infection and symptomatic relapse that required extended therapy and the use of second-line drugs. Finally, LRV1 presence was associated with a significant increase in levels of intra-lesional inflammatory markers. In conclusion, LRV1 status in L. guyanensis infection is significantly predictive (P = .0009) of first-line treatment failure and symptomatic relapse and has the potential to guide therapeutic choices in American TL.

Buruli disease, caused by Mycobacterium ulcerans, is the third most important mycobacterial disease in humans besides tuberculosis and leprosy. We have compared systemic and intralesional cytokine production in patients presenting with a nodular form and a necrotizing, ulcerative form of the disease. Gamma interferon (IFN-␥) levels in response to whole M. ulcerans and Mycobacterium bovis BCG bacilli and in response to purified Ag85 protein from BCG were lower in peripheral blood mononuclear cells (PBMC) cultures from Buruli disease patients than in PBMC from healthy purified protein derivative-positive contacts. Interleukin-4 (IL-4) and IL-13 content was below the detection threshold in these PBMC cultures. IFN-␥ production after stimulation with M. ulcerans was significantly lower (P < 0.05) in PBMC cultures from patients with ulcers than in those from patients with nodules. On the other hand, PBMC from Buruli disease patients produced significant levels of IL-10 in response to M. ulcerans (but not to M. bovis BCG) and production was highest in patients with the ulcerative form. Third, semiquantitative reverse transcription-PCR analysis demonstrated a similar difference in the local, intralesional cytokine profile for the two forms of the disease: high IFN-␥ but low IL-10 mRNA levels in nodular lesions and high IL-10 but low IFN-␥ mRNA levels in ulcerative lesions. Intralesional IL-4 and IL-13 mRNA levels were low and only detected in patients with the ulcerative form. Our results indicate, although they do not formally prove, that production of IL-10 rather than production of IL-4 or IL-13 by Th2-type T cells may be involved in the low M. ulcerans-specific IFN-␥ response in Buruli disease patients.