Signaling lymphocytic activation molecule expression and regulation in human intracellular infection correlate with Th1 cytokine patterns.

Induction of Th1 cytokines, those associated with cell-mediated immunity, is critical for host defense against infection by intracellular pathogens, including mycobacteria. Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein expressed on lymphocytes that promotes T cell proliferation and IFN-gamma production. The expression and role of SLAM in human infectious disease were investigated using leprosy as a model. We found that SLAM mRNA and protein were more strongly expressed in skin lesions of tuberculoid patients, those with measurable CMI to the pathogen, Mycobacterium leprae, compared with lepromatous patients, who have weak CMI against M. leprae. Peripheral blood T cells from tuberculoid patients showed a striking increase in the level of SLAM expression after stimulation with M. leprae, whereas the expression of SLAM on T cells from lepromatous patients show little change by M. leprae stimulation. Engagement of SLAM by an agonistic mAb up-regulated IFN-gamma production from tuberculoid patients and slightly increased the levels of IFN-gamma in lepromatous patients. In addition, IFN-gamma augmented SLAM expression on M. leprae-stimulated peripheral blood T cells from leprosy patients. Signaling through SLAM after IFN-gamma treatment of Ag-stimulated cells enhanced IFN-gamma production in lepromatous patients to the levels of tuberculoid patients. Our data suggest that the local release of IFN-gamma by M. leprae-activated T cells in tuberculoid leprosy lesions leads to up-regulation of SLAM expression. Ligation of SLAM augments IFN-gamma production in the local microenvironment, creating a positive feedback loop. Failure of T cells from lepromatous leprosy patients to produce IFN-gamma in response to M. leprae contributes to reduced expression of SLAM. Therefore, the activation of SLAM may promote the cell-mediated immune response to intracellular bacterial pathogens.

Treatment failure in children in a randomized clinical trial with 10 and 20 days of meglumine antimonate for cutaneous leishmaniasis due to Leishmania viannia species.

Clinical response to supervised treatment of Colombian patients with cutaneous leishmaniasis was evaluated in a randomized controlled trial comparing 10 days versus 20 days of treatment with meglumine antimonate (20 mg Sb/kg/day). Masked examiners evaluated clinical response defined as 100% re-epithelialization of all lesions at 13 weeks and no relapses during 52 weeks of follow-up. The efficacy of meglumine antimonate for 10 days' treatment was 61% (28 of 46) compared to 67% (24 of 36) for 20 days. There was a significantly lower clinical response for children < 5 years in both 10-day (11%) and 20-day (25%) groups compared to patients aged 5-14 years (67% and 75%, respectively) and 15 years or more (81% and 83%, respectively). Overall efficacy of treatment schedules was comparable, but lower than expected, mainly because of low efficacy in children. Pathogenicity of infection and pharmacokinetics may affect the treatment response in children. New therapeutic alternatives should be evaluated in trials that include children and women.

T-cell release of granulysin contributes to host defense in leprosy.

A novel mechanism by which T cells contribute to host defense against microbial pathogens is release of the antimicrobial protein granulysin. We investigated the role of granulysin in human infectious disease using leprosy as a model. Granulysin-expressing T cells were detected in cutaneous leprosy lesions at a six-fold greater frequency in patients with the localized tuberculoid as compared with the disseminated lepromatous form of the disease. In contrast, perforin, a cytolytic molecule that colocalizes with granulysin in cytotoxic granules, was expressed at similar levels across the spectrum of disease. Within leprosy lesions, granulysin colocalized in CD4+ T cells and was expressed in CD4+ T-cell lines derived from skin lesions. These CD4+ T-cell lines lysed targets by the granule exocytosis pathway and reduced the viability of mycobacteria in infected targets. Given the broad antimicrobial spectrum of granulysin, these data provide evidence that T-cell release of granulysin contributes to host defense in human infectious disease.

Induction of direct antimicrobial activity through mammalian toll-like receptors.

The mammalian innate immune system retains from Drosophila a family of homologous Toll-like receptors (TLRs) that mediate responses to microbial ligands. Here, we show that TLR2 activation leads to killing of intracellular Mycobacterium tuberculosis in both mouse and human macrophages, through distinct mechanisms. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide-dependent killing of intracellular tubercle bacilli, but in human monocytes and alveolar macrophages, this pathway was nitric oxide-independent. Thus, mammalian TLRs respond (as Drosophila Toll receptors do) to microbial ligands and also have the ability to activate antimicrobial effector pathways at the site of infection.

Activation of toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10.

Mammalian Toll-like receptors (TLRs) are required for cell activation by bacterial lipoproteins (bLP) and LPS. Stimulation of monocytes with bLP and LPS results in a TLR-dependent induction of immunomodulatory genes leading to the production of pro-inflammatory cytokines. In this paper, we compared the expression and response of TLRs on monocytes and dendritic cells (DC). TLR2, but not TLR4, was detected on peripheral blood monocytes and DC, in lymphoid tissue CD1alpha+ DC as well as on in vitro monocyte-derived DC. Upon stimulation with bLP or LPS, monocytes produced IL-12 and IL-10 at similar levels, whereas monocyte-derived DC produced comparable levels of IL-12, but little IL-10. Greater than 90% of the bLP-induced production of IL-12 was blocked by anti-TLR2 mAb. Thus, DC express TLR2 and activation of this receptor by bLP provides an innate mechanism by which microbial pathogens preferentially activate cell-mediated immunity.

Evidence for human CD4+ T cells in the CD1-restricted repertoire: derivation of mycobacteria-reactive T cells from leprosy lesions.

Both the CD4-CD8- (double negative) and CD4-CD8+ T cell lineages have been shown to contain T cells which recognize microbial lipid and glycolipid Ags in the context of human CD1 molecules. To determine whether T cells expressing the CD4 coreceptor could recognize Ag in the context of CD1, we derived CD4+ T cell lines from the lesions of leprosy patients. We identified three CD4+ Mycobacterium leprae-reactive, CD1-restricted T cell lines: two CD1b restricted and one CD1c restricted. These T cell lines recognize mycobacterial Ags, one of which has not been previously described for CD1-restricted T cells. The response of CD4+ CD1-restricted T cells, unlike MHC class II-restricted T cells, was not inhibited by anti-CD4 mAb, suggesting that the CD4 coreceptor does not impact positive or negative selection of CD1-restricted T cells. The CD4+ CD1-restricted T cell lines produced IFN-gamma and GM-CSF, the Th1 pattern of cytokines required for cell-mediated immunity against intracellular pathogens, but no detectable IL-4. The existence of CD4+ CD1-restricted T cells that produce a Th1 cytokine pattern suggests a contributory role in immunity to mycobacterial infection.

Insulin-like growth factor-I, but not growth hormone, is dependent on a high protein intake to increase nitrogen balance in the rat.

The aim of the present study was to investigate the influence of dietary protein level on the protein anabolic effects of growth hormone (GH) and insulin-like growth factor-I (IGF-I). Female growing rats were fed on either a high- or a low-protein diet with crude protein contents of 222 and 83 g/kg respectively. The diets contained the same amount of metabolizable energy (15.1 MJ/kg) and were given during a 14 d period. During the same time, three groups of rats (n 8) on each diet received subcutaneous infusions of either saline, recombinant human GH (rhGH) or recombinant human IGF-I (rhIGF-I). rhGH and rhIGF-I were given in doses of 360 and 500 micrograms/d respectively. The low-protein diet alone reduced significantly (P < 0.05) IGF-I concentrations in serum and in tissue taken from the gastrocnemius muscle as well as IGF-I mRNA from the same muscle. The responses to rhGH and rhIGF-I in terms of muscle IGF-I and its mRNA were variable. However, when rhIGF-I was infused into rats on the high-protein diet, significantly elevated levels of IGF-I in muscle tissues could be observed. This was associated with a significantly (P < 0.05) increased N balance, whereas rhGH significantly (P < 0.05) enhanced the N balance in rats on the low-protein diet. Thus, it can be concluded that the level of dietary protein ingested regulates not only the effect of IGF-I on whole-body N economy but also the regulation of IGF-I gene expression in muscles. The exact mechanism by which GH exerts its protein anabolic effect, however, remains to be elucidated.

Positive IgG Western blot for Borrelia burgdorferi in Colombia.

In order to evaluate the presence of specific IgG antibodies to Borrelia burgdorferi in patients with clinical manifestations associated with Lyme borreliosis in Cali, Colombia, 20 serum samples from patients with dermatologic signs, one cerebrospinal fluid (CSF) sample from a patient with chronic neurologic and arthritic manifestations, and twelve serum samples from individuals without clinical signs associated with Lyme borreliosis were analyzed by IgG Western blot. The results were interpreted following the recommendations of the Centers for Diseases Control and Prevention (CDC) for IgG Western blots. Four samples fulfilled the CDC criteria: two serum specimens from patients with morphea (localized scleroderma), the CSF from the patient with neurologic and arthritic manifestations, and one of the controls. Interpretation of positive serology for Lyme disease in non-endemic countries must be cautious. However these results suggest that the putative "Lyme-like" disease may correlate with positivity on Western blots, thus raising the possibility that a spirochete genospecies distinct from B. burgdorferi sensu stricto, or a Borrelia species other than B. burgdorferi sensu lato is the causative agent. Future work will focus on a survey of the local tick and rodent population for evidence of spirochete species that could be incriminated as the etiologic agent.

IL-18 promotes type 1 cytokine production from NK cells and T cells in human intracellular infection.

We investigated the role of IL-18 in leprosy, a disease characterized by polar cytokine responses that correlate with clinical disease. In vivo, IL-18 mRNA expression was higher in lesions from resistant tuberculoid as compared with susceptible lepromatous patients, and, in vitro, monocytes produced IL-18 in response to Mycobacterium leprae. rIL-18 augmented M. leprae-induced IFN-gamma in tuberculoid patients, but not lepromatous patients, while IL-4 production was not induced by IL-18. Anti-IL-12 partially inhibited M. leprae-induced release of IFN-gamma in the presence of IL-18, suggesting a combined effect of IL-12 and IL-18 in promoting M. leprae-specific type 1 responses. IL-18 enhanced M. leprae-induced IFN-gamma production rapidly (24 h) by NK cells and in a more sustained manner (5 days) by T cells. Finally, IL-18 directly induced IFN-gamma production from mycobacteria-reactive T cell clones. These results suggest that IL-18 induces type 1 cytokine responses in the host defense against intracellular infection.

Mucosal leishmaniasis due to Leishmania (Viannia) panamensis in Colombia: clinical characteristics.

The Leishmaniases are a group of diseases whose clinical presentation is in part determined by the infecting species. Until recently, mucosal leishmaniasis was attributed exclusively to Leishmania (Viannia) braziliensis; however, the capacity of other species of the subgenus Viannia to invade mucosal tissue has been documented. This report examines the clinical characteristics of 23 parasitologically diagnosed patients with mucosal leishmaniasis due to L. (V.) panamensis from the Pacific Coast of Colombia seen at CIDEIM between 1985 and 1996. Most of the mucosal lesions 74% (17 of 23) were mild, with a short time of evolution (median = 2.5 months) and were present concomitantly with an active cutaneous lesion in 61% (14 of 23) of the cases. The simultaneous presentation of mucosal and active cutaneous lesions contrast with classical descriptions of mucosal leishmaniasis caused by L. (V.) braziliensis, and highlights the importance of early diagnosis of mucosal disease by the examination of mucosa in all cases of cutaneous leishmaniasis.

Changes in expression of signal transduction proteins in T lymphocytes of patients with leprosy.

Advanced stages of mycobacterial diseases such as leprosy and tuberculosis are characterized by a loss of T-cell function. The basis of this T-cell dysfunction is not well understood. The present report demonstrates major alterations in the expression of signal transduction molecules in T cells of leprosy patients. These alterations were most frequently observed in lepromatous leprosy (LL) patients. Of 29 LL patients, 69% had decreased T-cell receptor zeta-chain expression, 48% had decreased p56(lck) tyrosine kinase, and 63% had a loss of nuclear transcription factor NF-kappaB p65. An electrophoretic mobility shift assay with the gamma interferon core promoter region revealed a loss of the Th1 DNA-binding pattern in LL patients. In contrast, tuberculoid leprosy patients had only minor signal transduction alterations. These novel findings might improve our understanding of the T-cell dysfunction observed in leprosy and other infectious diseases and consequently might lead to better immunologic evaluation of patients.

Reliability of the western ligand blot method for the simultaneous relative estimations of serum insulin-like growth factor binding proteins (IGFBPs).

It is well established that nutrition is an important regulator of both serum insulin-like growth factor-I (IGF-1) and its binding proteins (IGFBPs). The Western ligand blot method (WLB) for simultaneous determinations of IGFBPs in serum or plasma samples was evaluated and validated with emphasis on its reproducible capabilities. After electrophoretic separation and transfer, the membranes were incubated with a mixture of recombinant labeled human (GF-I/IGF-II(rhIGF-I/rhlGF-II) and band intensities measured by autoradiography. The typical electrophoretic profile for pig serum, as determined with molecular weight markers, showed four mainbands of approximately 42-39, 32, 30-28 and 24 kDa which seemed to correspond to IGFBP-3, IGFBP-2, IGFBP-1 and IGFBP-4 respectively. Likewise, a triplet of approximately 42-39 kDa (IGFBP-3), a broad area called IGFBP-30 region (most probably IGFBP-1, -2 and -3 variants) and a third band of approximately 24 kDa (IGFBP-4) were seen in rat samples. Determination of IGFBP-2 and -1 in rat serum samples, as two separate bands on 12% gels was difficult due to their close electrophoretic migration and possibly to the reported lower levels of IGFBP-2 in adult rat serum. Dilutions tested on 0.2 micron nitrocellulose membranes with samples volumes between 0.25 to 1.5 microliters (1:10-1:60 dilutions), showed IGFBPs curves with good linearity which suggest first, that there exist a quantitative relation between the amount of each protein and the densitometric response and second, that the transfer of the proteins was linear across the range of 0.25 to 1.5 microliters (1:10-1:60 dilutions). Moreover, the results also suggest that losses were equally spread and that the proteins retained their binding properties after the transfer process. Reproducibility showed intraassay coefficients of variation (CVs) of 15% or lower using either a transfer device without cooling system or a combination of a transfer device with cooling system and manually defined band boundaries. In summary, it was shown that the optimized experimental conditions here described for the WLB method, allow reliable simultaneous measurements of the main pig and rat serum IGFBPs and therefore, could be utilised to detect changes in the serum profile after dietary manipulations.

Lepromatous and tuberculoid leprosy: clinical presentation and cytokine responses.

OBJECTIVE: This study analyzes the major clinical characteristics of patients with active leprosy in relation to the in vitro immune response to the T-lymphocyte activator anti-CD3. METHODS: Thirty-eight patients with an established diagnosis of leprosy were classified according to the Ridley and Jopling table. Peripheral blood mononuclear cells from both lepromatous leprosy (LL) and tuberculoid leprosy (TL) patients and healthy controls were used to evaluate lymphocyte proliferation; immunoenzymatic assays were used to evaluate cytokine production (IL-1, IL-2, IL-4, IL-6, IL-10, IFN-gamma). RESULTS: Peripheral blood mononuclear cells from both LL and TL patients displayed blastogenic responses to anti-CD3. The cytokines IL-1 beta, IL-6, IL-10, and IFN-gamma were detected in culture supernatants. Endogenous production of IL-1 beta was significantly higher in cell cultures from patients with the lepromatous form of the disease compared to those with tuberculoid leprosy. Production of IL-6 in response to anti-CD3 was observed in a significantly higher proportion of LL than TL patients (P = 0.0025). Gamma-interferon production did not differ between TL and LL, but a direct correlation was observed between time of multidrug treatment and IFN production in vitro (P = 0.016). Interleukin-10 was detected in culture supernatants of lymphocytes activated by anti-CD3 from both patient groups, but not from healthy controls. CONCLUSIONS: The findings of this study suggest that patients with the two distinct forms of leprosy are capable of responding to a polyclonal T-lymphocyte stimulus such as anti-CD3 and provide evidence suggestive of alterations in the immune responses mediated by cytokines that may contribute to the spectrum of disease and response to treatment.

Rabies reference vaccine for use as a regional standard for Latin America and the Caribbean countries.

A candidate rabies reference vaccine of suckling mouse brain (SMB) origin was prepared and standardized at the Pan American Zoonoses Center (PAHO/WHO) and evaluated in a collaborative study involving seven laboratories. On the basis of three different tests, its potency, immunogenicity, and stability were demonstrated to be satisfactory. The vaccine was proposed for consideration of the Latin American and Caribbean countries as a regional standard to determine the potency of SMB vaccines, the most widely used in the Region.

Rabies reference vaccine for use as a regional standard for Latin America and the Caribbean countries

A candidate rabies reference vaccine of suckling mouse brain (SMB) origin was prepared and standardized at the Pan American Zoonoses Center (PAHO/WHO) and evaluated in a collaborative study involving seven laboratories. On the basis of three different tests, its potency, immunogenicity, and stability were demonstrated to be satisfactory. The vaccine was proposed for consideration of the Latin American and Caribbean countries as a regional standard to determine the potency of SMB vaccines, the most widely used in the region. (AU)

A yeast-derived antigen from Paracoccidioides brasiliensis useful for serologic testing.

Antigens prepared from P. brasiliensis yeast cells subjected to ultrasonic treatment proved reliable in the serological diagnosis of paracoccidioidomycosis. Detection of antibodies was possible in over 90% from paracoccidioidomycosis patients in tests with agar gel immunodiffusion and counterimmunoelectrophoresis. Specificity was high and only histoplasmosis sera produced cross reactions, albeit at a lower frequency (10%). The new antigens compared favorably to the standard yeast culture filtrate antigen used in the past and they have the advantage of being reproducible. Proper control of proteolysis is required if activity is to be preserved.