T cell responses to crude and defined leishmanial antigens in patients from the Lower Amazon region of Brazil infected with different species of Leishmania of the subgenera Leishmania and Viannia

Amazonian localized cutaneous leishmaniasis (LCL) is caused by parasites of the subgenera Leishmania and Viannia . Respectively, these parasites may cause diffuse cutaneous leishmaniasis (DCL) and mucocutaneous leishmaniasis (MCL). This, together with differing skin test responses, suggests some species-specificity in cell mediated immunity. In this study, T cell responses (proliferative and interferon-γ) to crude and defined antigens were examined in paired samples pre and post chemotherapy. Untreated L. (L.) amazonensis LCL patients showed lower responses to crude leishmanial antigens than the L. (V.) spp. group . L. (V.) braziliensis antigen was a more potent stimulator of T cell responses than L. (L.) amazonensis antigen in all patient groups. Few positive responses were seen to the L. (L.) amazonensis glycoprotein GP46. A substantial proportion of LCL patients did respond to the L. (L.) pifanoi amastigote antigens A2, and the surface membrane glycoprotein P8. DCL patients were poor responders to all leishmanial antigens, except GP46. In contrast, MCL patients were good responders to all antigens except GP46 and A2. A significant rise in the response to P8 and A2 antigen was seen post treatment across all LCL and MCL patients, indicating that these antigens might provide suitable vaccine candidates .     

Cross-immunity experiments between different species or strains of Leishmania in rhesus macaques (Macaca mulatta)

This study evaluates cross-immunity in rhesus monkeys (Macaca mulatta) previously infected with one species of Leishmania and have had self-cured disease or were cured by antimony-based therapy upon development of full-blown disease. We found that a self-healing cutaneous leishmaniasis (CL) following experimental infection with Leishmania (Leishmania) major induces significant protection for L. (L.) amazonensis and L. (Viannia) guyanensis, and was dependent on time of re-challenge by L (L.) amazonensis after animals had recovered from primary lesions, but lacked protection against L. (V.) braziliensis. In contrast, monkeys that recovered from L. (V.) braziliensis CL or L. (L.) chagasi visceral leishmaniasis following chemotherapeutic intervention were protected by challenge with L. (V.) braziliensis and L (L.) amazonensis. These findings indicate the relative variability in protection after self-cure or drug-cured experimental leishmaniasis to challenge by heterologous leishmanial parasites. Further studying the immune response may provide information regarding relevant factors influencing cross-protective immunity.     

American tegumentary leishmaniasis: a quantitative analysis of Langerhans cells presents important differences between L. (L.) amazonensis and Viannia subgenus

A quantitative study was conducted on the density of Langerhans cells (LCs) CD1a+ in specimens obtained from patients with American tegumentary leishmaniasis (ATL) lesions without previous treatment, as well as from control healthy individuals. LC density was significantly higher among infected patients when compared to controls and also higher in longer term ones. Regarding parasite quantities, these were proportionally inverse and diminished in chronic patients. Localized cutaneous leishmaniasis (LCL) showed an increase in cell population when compared to diffuse cutaneous leishmaniasis (DCL). A tendency towards density increase was observed in LC Leishmania (Leishmania) amazonensis patients when compared to Leishmania (Viannia) sp. Regarding the delayed hypersensitivity test (DTH, Montenegro skin test), L. (L.) amazonensis demonstrated a peculiar behavior because it is a poor cell immune inducer, presenting--among LCL patients--higher density in negative Montenegro patients than in positive ones. Negative DTH responses are usually poor in LC, although this was not evidenced in this study, possibly due to cell reposition, in order to stimulate immune response. Such results confirm the important role of LC in ATL, while suggesting that L. (L.) amazonensis may be a good model for LC studies as APC in ATL, due to its spectral immunological and clinical behavior.     

Disseminated cutaneous leishmaniasis due to Leishmania(Leishmania) amazonensis in human immunodeficiency virus(HIV)-infected patients: A report of two cases

Rationale: Co-infection of human immunodeficiency virus(HIV) and Leishmania spp. has impact on clinical and therapeutic outcomes of leishmaniases. Most studies do not present the identification of Leishmania species causing American tegumentary leishmaniasis in co-infections. In the Americas, Leishmania(L.) Viannia(V.) braziliensis and L.(V.) guyanensis have been identified. Patient concerns: In this study, two cases of American tegumentary leishmaniasis in patients infected with HIV are described. Patients presented several lesions with rapid dissemination and mucosal involvement. Diagnosis: Disseminated cutaneous leishmaniasis caused by L. amazonensis was identified by molecular test. Interventions: The patients were treated with conventional therapies for HIV infection and American tegumentary leishmaniasis. Outcomes: In co-infection, the clinical manifestations are atypical and the treatment response can be impaired. Lessons: These cases show that HIV infection impacts L. amazonensis infection and point to the relevance of identifying Leishmania species, which can lead to a better patient management.     

Species diversity of Leishmania (Viannia) parasites circulating in an endemic area for cutaneous leishmaniasis located in the Atlantic rainforest region of northeastern Brazil

Objectives  To identify the aetiological agents of cutaneous leishmaniasis and to investigate the genetic polymorphism of Leishmania (Viannia) parasites circulating in an area with endemic cutaneous leishmaniasis (CL) in the Atlantic rainforest region of northeastern Brazil.
Methods  Leishmania spp. isolates came from three sources: (i) patients diagnosed clinically and parasitologically with CL based on primary lesions, secondary lesions, clinical recidiva, mucocutaneous leishmaniasis and scars; (ii) sentinel hamsters, sylvatic or synanthropic small rodents; and (iii) the sand fly species Lutzomyia whitmani . Isolates were characterised using monoclonal antibodies, multilocus enzyme electrophoresis (MLEE) and polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region rDNA locus.
Results  Seventy-seven isolates were obtained and characterised. All isolates were identified as Leishmania (Viannia) braziliensis serodeme 1 based on reactivity to monoclonal antibodies. MLEE identified 10 zymodemes circulating in the study region. Most isolates were classified as zymodemes closely related to L. (V.) braziliensis, but five isolates were classified as Leishmania (Viannia) shawi . All but three of the identified zymodemes have so far been observed only in the study region. Enzootic transmission and multiclonal infection were observed.
Conclusions  Our results confirm that transmission cycle complexity and the co-existence of two or more species in the same area can affect the level of genetic polymorphism in a natural Leishmania population. Although it is not possible to make inferences as to the modes of genetic exchange, one can speculate that some of the zymodemes specific to the region are hybrids of L. (V.) braziliensis and L. (V.) shawi .     

Antibody response in patients with cutaneous leishmaniasis infected by Leishmania (Viannia) braziliensis or Leishmania (Viannia) guyanensis in Brazil

The antibody response against Leishmania (Leishmania) amazonensis crude antigen was measured through the indirect immunofluorescent assay (IFA) and the immunoenzymatic assay (ELISA) in 114 patients with cutaneous leishmaniasis (CL) in Brazil. Fifty-four patients were infected by Leishmania (Viannia) braziliensis, and 60 patients had L. (V.) guyanensis infection. Patients were comparable by age, sex, disease duration and the Montenegro skin test diameter. L. (V.) braziliensis-infected patients showed significant lower number of ulcerated lesions, greater ulcerated area and higher proportion of lymph node enlargement. Sensitivity of IFA was 79.6% (95% CI 66.1-88.9) and 71.7% (95% CI 58.4-82.2) for L. (V.) braziliensis and L. (V.) guyanensis-infected patients, respectively (P=0.324). Sensitivity of ELISA was 98.2% (95% CI 88.8-99.9) and 85.0% (95% CI 72.9-92.5) for L. (V.) braziliensis and L. (V.) guyanensis-infected patients, respectively (P=0.018). Significant differences were observed in the magnitude of the antibody response before treatment with higher levels detected in L. (V.) braziliensis-infected patients by both serologic techniques. Eighty-four patients had serologic evaluations before and 12 weeks after treatment with meglumine antimoniate, 20 mg/kg/day for 20 days. Significant lower optic density values were observed after treatment with both species independent of cure or failure. Our data showed that L. (V.) braziliensis induces a higher antibody response against L. (L.) amazonensis antigens than L. (V.) guyanensis and that down-modulation of the antibody response occurs shortly during disease evolution after treatment. Moreover the data support the use of ELISA as a better tool for detection of antibodies in CL.     

Leishmania (Viannia) braziliensis is the predominant species infecting patients with American cutaneous leishmaniasis in the State of Minas Gerais, Southeast Brazil

Skin biopsies from 53 patients with American cutaneous leishmaniasis (ACL) from the State of Minas Gerais, Brazil, were used for a characterization of the Leishmania parasites. A pair of primers flanking the conserved region of the Leishmania minicircle kDNA was used to obtain amplified DNA via the polymerase chain reaction. The amplified products were subsequently hybridized with Leishmania subgenus-specific radiolabeled probes. Parasites from 49 out of 53 samples (92.5%) were characterized as belonging to the subgenus Viannia and four (7.5%) as belonging to the subgenus Leishmania. Clinical, epidemiological and molecular evidence allow us to conclude that Leishmania (V.) braziliensis and Leishmania (L.) amazonensis are the species present in the patients studied and that L. (V.) braziliensis is the predominant species in the State of Minas Gerais, Brazil.     

Disseminated cutaneous leishmaniasis in a field clinic in Bahia, Brazil: a report of eight cases

Eight Bahian patients with cutaneous leishmaniasis who had 20 or more ulcerative lesions of short duration are described. Of five identifications of isolated parasites, four were Leishmania braziliensis braziliensis and one was L. mexicana amazonensis. All but one had positive Montenegro tests initially, and all did after treatment. All had circulating anti-leishmanial antibodies and five responded well to glucantime therapy suggesting a functioning immune response. This is quite different to the anergic hansenoid leishmaniasis seen with L. mexicana amazonensis infections in Brazil. Possible reasons for the occurrence of this type of leishmaniasis are briefly discussed.     

Antileishmanial activity of azithromycin against Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi

Azithromycin, an azalide antibiotic, is highly concentrated within different phagocytic cells, especially macrophages. The potential antileishmanial activity of azithromycin against three species of Leishmania from the New World was assessed using in vitro models. Azithromycin decreased viability of promastigote cultures of Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi as determined by the colorimetric Alamar blue assay. In amastigote intracellular cultures, a significant decrease in infected macrophages counts was observed for all three species with IC(50) of 20.83 (27 micromol/L), 2.18 (2.7 micromol/L), and 6.12 (7.8 micromol/L) microg/mL, respectively. Azithromycin showed in vitro activity against L. (L.) amazonensis, L. (V.) braziliensis, and L. (L.) chagasi and may offer an alternative to current leishmaniasis treatment.     

Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease

One hundred fourteen Leishmania isolates from patients with different clinical forms of leishmaniasis in the State of Bahia, Brazil, were characterized by indirect radioimmune binding assay using specific monoclonal antibodies (serodeme analysis). Seventy-five of these isolates were also analyzed by enzyme electrophoresis, based on 11 enzyme loci; parasite species were compared, according to their characteristic zymodemes, to those of WHO Leishmania reference strains. All isolates could be classified into one of three species: Leishmania amazonensis (n = 40), L. braziliensis (n = 39) or L. chagasi (n = 35). The most interesting information obtained from this study is the realization that L. amazonensis is capable of producing a wide spectrum of disease in humans. Infection with this parasite was associated with many different clinical presentations, including cutaneous leishmaniasis [CL] (20/49 cases), mucocutaneous leishmaniasis [MCL] (5/13 cases) and, of special note, visceral leishmaniasis [VL] (11/46 cases), as well as four cases of post kalaazar dermal leishmaniasis [PKDL]. In situ tissue parasite characterization, by immunoperoxidase assay and employing anti-L. amazonensis amastigote monoclonal antibodies, confirmed the infection with this species in two cases of CL, one case of DCL, one case of MCL and one case of PKDL. Our results also demonstrate the difficulty of parasite differentiation based on clinical grounds, since at least L. amazonensis infection can be associated with all types of leishmanial diseases, and different Leishmania species may be associated with indistinguishable clinical presentations. Since leishmanial parasites may vary in their biological behavior or in their response to treatment, it is important that their identification be made by reliable methods.     

The rarity of infection with Leishmania (Viannia) braziliensis among patients from the Manaus region of Amazonas state,Brazil, who have cutaneous leishmaniasis

The frequency of Leishmania ( Viannia) braziliensis infection was assessed in 79 of the 138 patients with cutaneous leishmaniasis who attended a reference outpatient unit in Manaus, Amazonas state, between the August and December of 1997. The disease was characterized by one or more cutaneous ulcers, the skin lesions being frequently associated with satellite lymph-node enlargement. All parasite isolates were identified using monoclonal antibodies and enzyme electrophoresis. Only two (2.8%) of the 71 patients from whom parasites were successfully isolated were found to be infected with L. ( V.) braziliensis, the other 69 isolates being identified, from their isoenzyme profiles, as L. ( V.) guyanensis. In the Manaus region, therefore, almost all human cutaneous leishmaniasis is the result of infection with L. (V.) guyanensis, and L. ( V.) braziliensis is a relatively rare cause of the disease.     

The relationship of Leishmania braziliensis subspecies and immune response to disease expression in New World leishmaniasis

Multivariate analyses of clinical presentation, subspecies identity of the causal organism, and the Leishmania-specific immune response parameters (indirect fluorescent antibody test [IFAT], cutaneous delayed type hypersensitivity [DTH], and in vitro lymphocyte transformation [LT]) of 441 patients with tegumentary leishmaniasis were used to examine the human host-parasite interaction in L. braziliensis infection. Mucocutaneous disease (P less than .002) and L. braziliensis braziliensis infection (P less than .001) were independently associated with significantly higher IFAT titers and cutaneous DTH than were cutaneous disease or L. braziliensis panamensis infection. Lesion size was also correlated with IFAT titer (P. less than .001). Although time of lesion evolution was highly correlated with all parameters, differences associated with subspecies and disease form were independent of lesion duration (three-way analysis of variance). In contrast with the cutaneous DTH response, the in vitro lymphocyte proliferative response to Leishmania antigen did not correlate with disease form and only weakly with infecting subspecies when time of evolution and subspecies were controlled. The association of mucosal disease presentation with a particular subspecies and the independent correlation of both variables with heightened IFAT titers and cutaneous DTH to Leishmania antigen supports the possibility of immune mechanisms of pathogenesis in human tegumentary leishmaniasis.     

Usefulness of sampling with cotton swab for PCR-diagnosis of cutaneous leishmaniasis in the New World

In this study, we tested the polymerase chain reaction (PCR)-method to diagnose cutaneous leishmaniasis (CL) by taking exudate materials from lesions with cotton swabs, using our previously tested (PCR) panel comprised of Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) mexicana and L. (L.) amazonensis. The objectives of the present study were to improve the sampling method convenient for the patients and to test the usefulness of samples taken with cotton swabs. Sixteen patients were clinically diagnosed to have CL including one case of diffuse cutaneous leishmaniasis (DCL) in Ecuador and the causative Leishmania parasites were identified by PCR. All the 12 samples from CL patients of La Mana, positive for Leishmania DNA, were identified as L. (V.) panamensis, while two from CL of Huigra and one from DCL of San Ignacio were L. (L.) mexicana. In the field condition, taking biopsy material is not only painful but sometimes causes iatrogenic bacterial infections. Considering the sensitivity of the test, and convenient sampling procedure, it may be suggested that collection of exudates using cotton swabs may be a better alternative to biopsy sample for PCR-diagnosis of CL.     

Immunological selection for Leishmania (Viannia) braziliensis antigens

Comparative ELISA and selective immunoblotting procedures were used in attempts to identify differential serological indicators of infection with the Leishmania (Viannia) braziliensis complex, infection with the L. braziliensis species, and therapeutic cure of localized or mucocutaneous leishmaniasis (LCL or MCL). Although mean ELISA absorbance values were significantly higher for MCL sera than for LCL sera, absorbance could not be used as a reliable indicator of the clinical form of disease. Immunoblotting profiles were similar with sera from MCL and LCL. Pre-adsorption with heterologous trypanosomatid antigens indicated that recognition of antigens of about 56, 60, 66, 72, 88 and 110 kDa might be specific to the subgenus Viannia. In two-colour, sequential, dual ELISA-based immunoblotting, no antigens recognized only by sera from MCL patients were detected. After glucantime therapy, immunoblotting profiles with LCL sera were reduced both in intensity and in the range of antigens detected; a 104-kDa antigen was newly detected with post-treatment LCL sera. Overall, the results show the value of differential immunological detection strategies and support the close relationship between species of the subgenus Viannia but fail to indicate a prognostic antigen for MCL.     

MMP-9 activity is induced by Leishmania braziliensis infection and correlates with mucosal leishmaniasis

Infection of humans with Leishmania braziliensis typically results in localized cutaneous leishmaniasis (LCL). Rarely, after months or years of apparent clinical cure, some patients develop the destructive mucosal leishmaniasis (ML). ML results from L. braziliensis dissemination, probably via phagocytic cells. As the preferred cells for Leishmania spp. colonization, macrophages are critical to infection control, and may contribute to parasite dissemination. However, the host factors that determine this outcome are unknown. Matrix metalloproteinase 9 (MMP-9) is known to be important for immune cell migration, macrophage recruitment, and effective granuloma formation. Moreover, MMP-9 has been involved in Mycobacterium tuberculosis dissemination. Here, we demonstrate that in vitro infection of human macrophages with L. braziliensis increased the secretion and activation of MMP-9. We also demonstrate that macrophages from healthy cured individuals with previous history of ML had increased MMP-9 activity compared to LCL cured individuals. These findings may represent a fundamental difference in host innate immunity that could contribute to the clinical leishmaniasis presentation.     

Endemically exposed asymptomatic individuals show no increase in the specific Leishmania (Viannia) panamensis-Th1 immune response in comparison to patients with localized cutaneous leishmaniasis

In Colombia, most cases of human cutaneous leishmaniasis are caused by Leishmania (Viannia) panamensis. Interestingly, up to 30% of the exposed population do not suffer from clinical leishmaniasis although it is likely that they are continuously infected with Leishmania parasites. Since it is believed that the induction of efficient Th1 immune responses protects against Leishmania infections both in humans and in animal models, we determined if endemically exposed asymptomatics showed stronger Leishmania-specific Th1 immune responses than patients with active localized cutaneous leishmaniasis (LCL). We found that Montenegro skin test responses were slightly higher among asymptomatic individuals compared to patients suffering from LCL. However, PBMC from patients with LCL showed similar Leishmania-specific proliferative responses compared to PBMC from asymptomatic individuals. Furthermore, PBMC from both groups also secreted similar amounts of IFN-gamma, IL-12p40 and IL-10 after in vitro exposure to L. panamensis. No IL-4 was detected in the supernatants. Taken together our results suggest that lack of LCL development in endemically exposed asymptomatics cannot be explained by stronger systemic anti-Leishmania Th1 immune responses or decreased Th2 responses in these individuals in comparison to individuals who develop LCL. It may be possible that other mechanisms are responsible for resistance to cutaneous leishmaniasis in Colombia in endemically exposed asymptomatics.     

Species assignation of Leishmania from human and canine American tegumentary leishmaniasis cases by multilocus enzyme electrophoresis in North Argentina

Sixteen Leishmania stocks, 15 isolated from patients with cutaneous (CL), mucocutaneous (MCL), or recurrent cutaneous leishmaniasis, plus one from a dog with CL in Salta and Corrientes Provinces, Argentina, were studied by multilocus enzyme electrophoresis. Thirteen of the stocks from humans were grouped in two zymodemes; nine termed as KMS 1, four as KMS 2, and assigned to Leishmania (Viannia) braziliensis. Two additional stocks from CL cases expressed a KMS 4 enzyme profile, corresponding to L. (V.) guyanensis. Although the parasites from the dog were also assigned to L. (V.) braziliensis, its zymodeme, KMS 3, was not expressed in any of the current human isolates. The characterization of Leishmania from a dog was done for the first time in Argentina. The importance of the intraspecific polymorphism in the induction of clinical forms and in the host-reservoir concept is briefly discussed, based on the zymodeme data of isolates from humans and dogs. The presence of L. (V.) guyanensis was confirmed in the country.     

Influence of Leishmania (Viannia) species on the response to antimonial treatment in patients with American tegumentary leishmaniasis

BACKGROUND: Pentavalent antimonials (SbV) are the first-line chemotherapy for American tegumentary leishmaniasis (ATL). There are, however, reports of the occurrence of treatment failure with these drugs. Few studies in Latin America have compared the response to SbV treatment in ATL caused by different Leishmania species. METHODS: Clinical parameters and response to SbV chemotherapy were studied in 103 patients with cutaneous leishmaniasis (CL) in Peru. Leishmania isolates were collected before treatment and typed by multilocus polymerase-chain-reaction restriction fragment-length polymorphism analysis. RESULTS: The 103 isolates were identified as L. (Viannia) peruviana (47.6%), L. (V.) guyanensis (23.3%), L. (V.) braziliensis (22.3%), L. (V.) lainsoni (4.9%), L. (Leishmania) mexicana (1%), and a putative hybrid, L. (V.) braziliensis/L. (V.) peruviana (1%). L. (V.) guyanensis was most abundant in central Peru. Of patients infected with the 3 former species, 21 (21.9%) did not respond to SbV chemotherapy. The proportions of treatment failure (after 12 months of follow-up) were 30.4%, 24.5%, and 8.3% in patients infected with L. (V.) braziliensis, L. (V.) peruviana, and L. (V.) guyanensis, respectively. Infection with L. (V.) guyanensis was associated with significantly less treatment failure than L. (V.) braziliensis, as determined by multiple logistic regression analysis (odds ratio, 0.07 [95% confidence interval, 0.007-0.8]; P=.03). CONCLUSIONS: Leishmania species can influence SbV treatment outcome in patients with CL. Therefore, parasite identification is of utmost clinical importance, because it should lead to a species-oriented treatment.     

Experimental cutaneous leishmaniasis. III. Histopathological aspects of the developmental behavior of the cutaneous lesion induced in Cebus apella (Primates: Cebidae) by Leishmania (Viannia) lainsoni, L. (V.) braziliensis and L. (Leishmania) amazonensis

We have studied the histopathological aspects related to the evolution of cutaneous lesions experimentally produced in the monkey Cebus apella (Primates: Cebidae) by Leishmania (V.) lainsoni, L. (V.) braziliensis and L. (L.) amazonensis. Microscopical examination of a series of biopsies obtained from these animals showed the kinetics of the cutaneous lesions regarding three species of Leishmania inoculated, as follows: 1) an initial non-specific chronic inflammatory infiltrate; 2) macrophagic nodules; 3) necrosis of parasitized phagocytic cells; 4) epitheliode granuloma; 5) absorption of the necrotic area (sometimes forming "foreign-body granuloma"); 6) a non-specific residual inflammatory infiltration; and 7) cicatrization. These pathological processes are, of course, responsible for both development and resolution of the leishmaniotic lesion. We also discuss some immunopathological mechanisms probably related with the sequential events, and that could be also responsible for the different clinical aspects found in man.     

Monocyte cytokine and costimulatory molecule expression in patients infected with Leishmania mexicana

Leishmania mexicana causes localized and diffuse cutaneous leishmaniasis. Patients with localized cutaneous leishmaniasis (LCL) develop a benign disease, whereas patients with diffuse cutaneous leishmaniasis (DCL) suffer from a progressive disease associated with anergy of the cellular response towards Leishmania antigens. We evaluated the production of the interleukins (IL) IL-12, IL-15, IL-18 and tumour necrosis factor-alpha (TNF-alpha) and the expression of the costimulatory molecules CD40, B7-1 and B7-2 in monocytes from LCL and DCL patients, stimulated in vitro with Leishmania mexicana lipophosphoglycan (LPG) for 18 h. LCL monocytes significantly increased TNF-alpha, IL-15 and IL-18 production, and this increase was associated with reduced amounts of IL-12. DCL monocytes produced no IL-15 or IL-18 and showed a decreasing tendency of TNF-alpha and IL-12 production as the severity of the disease increased. No difference was observed in the expression of CD40 and B7-1 between both groups of patients, yet B7-2 expression was significantly augmented in DCL patients. It remains to be established if this elevated B7-2 expression in DCL patients is cause or consequence of the Th2-type immune response that characterizes these patients. These data suggest that the diminished ability of the monocytes from DCL patients to produce cell-activating innate proinflammatory cytokines when stimulated with LPG is a possible cause for disease progression.