Trabecular and cortical bone involvement in rheumatoid arthritis by DXA and DXA-based 3D modelling.

Rheumatoid arthritis (RA) patients had a higher risk of developing low bone mineral density (BMD) or osteoporosis. RA patients on classic disease-modifying antirheumatic drug (c-DMARD) therapy showed significantly lower BMD than controls, while no significant differences in most parameters were found between RA patients receiving biological disease-modifying antirheumatic drugs (b-DMARDs) and controls. The 3D analysis allowed us to find changes in the trabecular and cortical compartments. INTRODUCTION: To evaluate cortical and trabecular bone involvement of the hip in RA patients by dual-energy X-ray absorptiometry (DXA) and 3D analysis. The secondary end-point was to evaluate bone involvement in patients treated with classic (c-DMARD) or biological (b-DMARD) disease-modifying antirheumatic drug therapies and the effect of the duration of the disease and corticosteroid therapy on 3D parameters. METHODS: A cross-sectional study of 105 RA patients and 100 subjects as a control group (CG) matched by age, sex, and BMI was carried out. BMD was measured by DXA of the bilateral femoral neck (FN) and total hip (TH). The 3D analyses including trabecular and cortical BMD were performed on hip scans with the 3D-Shaper software. RESULTS: FN and TH BMD and trabecular and cortical vBMD were significantly lower in RA patients. The c-DMARD (n = 75) group showed significantly lower trabecular and cortical vBMD than the CG. Despite the lower values, the b-DMARD group (n = 30) showed no significant differences in most parameters compared with the CG. The trabecular and cortical 3D parameters were significantly lower in the group with an RA disease duration of 1 to 5 years than in the CG, and the trabecular vBMD was significantly lower in the group with a duration of corticosteroid therapy of 1 to 5 years than in the CG, while no significant differences were found by standard DXA in the same period. CONCLUSIONS: RA patients had a higher risk of developing low BMD or osteoporosis than controls. RA patients receiving c-DMARD therapy showed significantly lower BMD than controls, while no significant differences in most parameters were found between RA patients receiving b-DMARDs and controls. 3D-DXA allowed us to find changes in trabecular and cortical bone compartments in RA patients.

Prevalence, serologic and genetic studies of high expressers of the blood group A antigen on platelets*.

OBJECTIVE/AIM: The aim of this study is to describe the distribution of the platelet blood group A antigenicity in Euro-Brazilians (EUBs) and Afro-Brazilians (AFBs). BACKGROUND: A small but significant proportion of individuals express high levels of A or B antigen on their platelets corresponding to the erythrocyte ABO group. The mechanism of increased antigen expression has not been elucidated. MATERIAL/METHODS: A cohort of 241 blood group A donors was analysed by flow cytometry. Although mean fluorescence intensity (MFI) is a typical continuous variable, platelets were screened and divided into two categories: low expressers (LEs) and high expressers (HEs). A three-generation family was investigated looking for an inheritance mechanism. RESULTS: The prevalence of the HE platelet phenotype among group A(1) donors was 2%. The mean of MFI on platelets of A(1) subgroup of EUBs differs from that of AFBs (P = 0·0115), whereas the frequency of the HE phenotype was similar between them (P = 0·5251). A significant difference was found between sexes (P = 0·0039). Whereas the serum glycosyltransferase from HE family members converted significantly more H antigen on group O erythrocytes into A antigens compared with that in LE serum, their ABO, FUT1 and FUT2 genes were consensus. The theoretically favourable, transcriptionally four-repeat ABO enhancer was not observed. CONCLUSION: The occurrence of HE in several members suggests familial aggregation. Indeed, in repeated measures, stability of the MFI values is suggesting an inherited condition. Factors outside the ABO locus might be responsible for the HE phenotype. Whether the real mechanism of inheritance is either of a polygenic or of a discrete Mendelian nature remains to be elucidated.

Immunotherapy with the saponin enriched-Leishmune vaccine versus immunochemotherapy in dogs with natural canine visceral leishmaniasis.

Leishmune, the first licensed vaccine for prophylaxis against canine visceral leishmaniasis (CVL) and is also immunotherapeutic when used with double saponin adjuvant concentration. The Leishmune therapeutic vaccine was assessed for immunotherapy (IT) in 31 infected dogs and for immunochemotherapy (ICT) in combination with allopurinol or amphotericinB/allopurinol, in 35 dogs. Compared to infected untreated control dogs, at month 3, both treatments increased the proportion of dogs showing intradermal response to Leishmania antigen to a similar extent (from 8 to 67%, in the IT and to 76%, in the ICT groups), and conversely reduced from 100 to 38% (IT) and to 18% (ICT) the proportion of symptomatic cases, from 54 to 12% (IT) and to 15% (ICT) the proportion of parasite evidence in lymph nodes and from 48 to 19% (IT) and 12% (ICT) the proportion of deaths, indicating that the immunotherapy with enriched-Leishmune vaccine promotes the control of the clinical and parasitological signs of CVL rendering most dogs asymptomatic although PCR positive. By month 8, negative lymph node PCR results were obtained in 80% of the ICT-treated dogs, but only in 33% of the IT group (p=0.0253), suggesting that the combination of additional chemotherapy with Leishmune-enriched saponin vaccination abolished, not only the symptoms but also the latent infection condition, curing the dogs. The animals were followed up until 4.5 years after the beginning of the experiment and, compared to the untreated control group at month 3 (12/25 dogs; 48%), a decrease in the rate of CVL deaths was only seen after ICT treatment (7/35 dogs; 20%; 0.0273) but not after IT treatment (10/31 dogs; 32%; p=0.278), pointing out an additional advantage of the ICT treatment with the enriched-Leishmune in the control and cure of CVL.

Immunotherapy against experimental canine visceral leishmaniasis with the saponin enriched-Leishmune vaccine.

In order to assess the immunotherapeutic potential on canine visceral leishmaniasis of the Leishmune vaccine, formulated with an increased adjuvant concentration (1mg of saponin rather than 0.5mg), 24 mongrel dogs were infected with Leishmania (L.) chagasi. The enriched-Leishmune vaccine was injected on month 6, 7 and 8 after infection, when animals were seropositive and symptomatic. The control group were injected with a saline solution. Leishmune-treated dogs showed significantly higher levels of anti-FML IgG antibodies (ANOVA; p<0.0001), a higher and stable IgG2 and a decreasing IgG1 response, pointing to a TH1 T cell mediated response. The vaccine had the following effects: it led to more positive delayed type hypersensitivity reactions against Leishmania lysate in vaccinated dogs (75%) than in controls (50%), to a decreased average of CD4+ Leishmania-specific lymphocytes in saline controls (32.13%) that fell outside the 95% confidence interval of the vaccinees (41.62%, CI95% 43.93-49.80) and an increased average of the clinical scores from the saline controls (17.83) that falls outside the 95% confidence interval for the Leishmune immunotherapy-treated dogs (15.75, CI95% 13.97-17.53). All dogs that received the vaccine were clustered, and showed lower clinical scores and normal CD4+ counts, whereas 42% of the untreated dogs showed very diminished CD4+ and higher clinical score. The increase in clinical signs of the saline treated group was correlated with an increase in anti-FML antibodies (p<0.0001), the parasitological evidence (p=0.038) and a decrease in Leishmania-specific CD4+ lymphocyte proportions (p=0.035). These results confirm the immunotherapeutic potential of the enriched-Leishmune vaccine. The vaccine reduced the clinical symptoms and evidence of parasite, modulating the outcome of the infection and the dog's potential infectiosity to phlebotomines. The enriched-Leishmune vaccine was subjected to a safety analysis and found to be well tolerated and safe.

Assessment of the monoterpene, glycidic and triterpene-moieties' contributions to the adjuvant function of the CP05 saponin of Calliandra pulcherrima Benth during vaccination against experimental visceral leishmaniasis.

The CP05 saponin from Calliandra pulcherrima Benth, shows remarkable similarities to the QS21 saponin of Quillaja saponaria Molina. Both shared a monoterpene hydrophobic moiety, a glycidic chain attached to the triterpene C28, and three sugars attached to C3. Different from QS21, the CP05 does not show the aldehyde group in triterpene C4 involved in TH1 response. Balb/c mice were immunized either intact saponin (CP05), the monoterpene-deprived (BS), the C28 carbohydrate-deprived (HS) or the sapogenin fraction, in formulation with the FML antigen of Leishmania donovani and challenged with 2 x 10(8) amastigotes of L. chagasi. While the CP05 induced 90% survival and 92.1% parasite reduction, a 100% survival and 94.1% protection were detected after the BS-vaccine treatment, indicating that the monoterpene acylated moiety, absent in the BS vaccine, is not necessary for the induction of a protective global TH1 response. Only the DTH response of BS vaccines was mildly lower than that of CP05 vaccinees. Maximal anti-FML antibody, CD4(+) and CD8(+) Leishmania specific lymphocytes, IFN-gamma splenocyte secretion, reduction in parasite load and survival was also detected for the BS vaccine. The HSFML vaccine showed diminished responses in all tested variables, except for IFN-gamma secretion, indicating that the integrity of the carbohydrate moiety attached to C28 is mandatory for the these functions. No protection was induced by the sapogenin-FML indicating that the CP05 triterpene which lacks the C4 aldehyde group, is not a immunostimulating compound. No contribution to protection was detected in the CP05 saponin treated control group supporting the specificity of the FML antigenic preparation.

Acylated and deacylated saponins of Quillaja saponaria mixture as adjuvants for the FML-vaccine against visceral leishmaniasis.

The adjuvant of the FML-vaccine against murine and canine visceral leishmaniasis, the Riedel de Haen saponin mixture, was fractionated by ion exchange chromatography on DEAE-cellulose to afford one TLC homogeneous Quillaja saponaria Molina QS21 saponin fraction (18.0%), a mixture of two deacylsaponins (19.4%), sucrose (39.9%), sucrose and glucose (19.7%), rutin (0.8%) and quercetin (2.2%), that were identified by comparison of 1H and 13C NMR spectroscopy. The QS21 shows the typical aldehyde group in C-23 (65% equatorial) and a normonoterpene moiety acylated in C-28. The deacylsaponins show the aldehyde group but do not have the normonoterpene moiety. Balb/c mice were vaccinated with 150 microg of FML antigen of Leishmania donovani and 100 microg of each obtained fraction and further challenged by infection with 10(8) amastigotes of Leishmania chagasi. The safety analysis and the effect on humoral and cellular immune responses and in clinical signs showed that the QS21 saponin and the deacylsaponins are the most active adjuvant compounds of the Riedel the Haen saponin mixture. Both induced the highest and non-significantly different increases in DTH, CD4+ T lymphocytes in spleen, IFN-gamma in vitro, body weight gain and the most pronounced reduction of parasite burden in liver (95% for QS21 and 86% for deacylsaponins; p>0.05). While the QS21 showed mild toxicity, significant adjuvant effect on the anti-FML humoral response before and after infection, and decrease in liver relative weight, the deacylsaponins showed no toxicity, less haemolysis and antibody and DTH responses increased mainly after infection, still inducing a stronger Leishmania-specific in vitro splenocyte proliferation. Our results confirm in the Riedel de Haen saponin extract the presence of deacylsaponins normonoterpene-deprivated which are non-toxic and capable of inducing a specific and strong immunoprotective response in vaccination against murine visceral leishmaniasis.

The mutation G298A®Ala100Thr on th coding sequence of the Duffy antigen/chemokine receptor gene in non-caucasian Brazilians

Ala100Thr has been suggested to be a Caucasian genetic marker on the FY*B allele. As the Brazilian population has arisen from miscegenation among Portuguese, Africans, and Indians, this mutation could possibly be found in Euro- and Afro-Brazilians, or in Brazilian Indians. Fifty-three related individuals and a random sample of 100 subjects from the Brazilian population were investigated using the polymerase chain reaction and four restriction fragment length polymorphisms. Confirming the working hypothesis, among the related individuals three Afro-Brazilians (two of them a mother and daughter) and a woman of Amerindian descent had the Ala100Thr mutation on the FY*B allele. Five non-related Euro-Brazilians also carried the mutation. All nine individuals presented the Fy(a-b+) phenotype. We conclude that the Ala100Thr mutation can occur in populations other than Caucasians and that this mutation does not affect Duffy expression on red blood cells. Gene frequencies for this allele in the non-related individuals were in agreement with those of other populations. The Duffy frequencies of two Amerindian tribes were also investigated.

Protective vaccination against murine visceral leishmaniasis using aldehyde-containing Quillaja saponaria sapogenins.

The presence of aldehyde groups at C-23 and C-24 of the triterpen aglycon moiety was disclosed in 1H NMR spectra of both the Riedel de Haen saponin (R) (delta 9.336) and Quillaja saponaria QuilA saponin (delta 9.348). The sign of the C-28 acylated linked moiety (delta 176) was present in both saponins, while the delta 171 at C-28 (carboxy group) corresponding to the deacylated saponin, was only detected in the QuilA preparation, indicating 50% of hydrolysis of the ester moiety, probably due to the storage in aqueous solution. The normoterpen moiety was present in both saponins (signals at delta 14-18). The chemical removal of saponin glicidic moieties gave rise to their sapogenin fractions. Their 1H NMR spectra showed the presence of two signals (delta 9.226 and 9.236) for sapogenin R and two signals (delta 9.338 and 9.352) for the QuilA sapogenin. The intensity of the signals suggested two conformational isomers of sapogenin R in the ratio 53% of equatorial aldehyde group to 47% of axial aldehyde group, and two conformational isomers of QuilA sapogenin in the ratio 76% of equatorial aldehyde group to 24% of axial aldehyde group. The chemical treatment abolished the saponin slight in vivo toxicity, reduced their hemolytic potential, did not affect their aldehyde contents, but gave rise to an enriched axial aldehyde-containing sapogenin R with enhanced potential on antibody humoral response (anti-IgM, IgG, IgG1, IgG2a, IgG2b and IgG3) and to an enriched equatorial aldehyde-containing QuilA-sapogenin that induced a mainly cellular specific immune response (increased intradermal response to leishmanial antigen and IFNgamma sera levels) and effective protection against murine infection by L. donovani (77% reduction in liver parasitic load). Our results suggest that the Riedel de Haen saponin is probably a Quillaja saponaria saponin.

A novel blood group B subgroup: serological and genetic studies.

A discrepancy in the ABO blood groups between a newborn child and her parents was identified. Serological and DNA investigative techniques were performed. A weak variant of B (B(w)) was detected on the erythrocytes of the child, her grandmother and great-uncle. Adsorption-elution studies showed that their erythrocytes adsorb and yield anti-B on elution. The B(w) antigenic strength of the A(1)B(w) cells of her mother and maternal aunt was reduced when compared to that of the A(2)B(w) from another family member. Only one of 15 different anti-B sera agglutinated the A(1)B(w) erythrocytes. Agglutinin anti-B that reacted strongly with normal B erythrocytes and did not agglutinate the B(w) cells, was found in the sera of the A(1)B(w) individuals. The B(w) serum glycosyltransferase could not convert O cells into B cells and no B substance was found in saliva. All family members with the B(w)/AB(w) phenotypes were heterozygous for a B allele and DNA sequencing revealed a novel missense mutation in exon 7 of the B allele (556A > G), resulting in M186V. This substitution changes a highly conserved region of the enzyme, proposed to be a disordered loop near the enzyme cleft, and is expected to diminish the enzyme's activity, leading to this B(w) phenotype.

Long lasting protection against canine kala-azar using the FML-QuilA saponin vaccine in an endemic area of Brazil (São Gonçalo do Amarante, RN).

Naturally exposed dogs of an endemic area were vaccinated with the fucose mannose ligand (FML) antigen of Leishmania donovani in formulation with QuilA saponin. The 100% of vaccinees were seropositive to FML and showed intradermal reaction to L. donovani lysate, 2 months after vaccination. The absorbency values and size of intradermal reaction were both significantly higher in vaccinees than in controls along a 3.5 years period (ANOVA, P<0.0001). The 25% of the control animals (two dogs on the first year and six dogs on the fourth year, respectively) and 5% of the vaccinees (one dog during the fourth year) developed clinical and fatal disease until the end of experiment. This difference was significant (chi(2)=3.93, P<0.05). This means that 95% protection against kala-azar was achieved in vaccinees, after FML-QuilA vaccination (80% of vaccine efficacy (VE)). Leishmania infection was also confirmed, 3.5 years after vaccination, in saline controls that showed positive polymerase chain reaction (PCR) for Leishmania DNA and FML-serology with no intradermal reaction. Higher seropositivities and intradermal reactions with no Leishmanial DNA were detected in vaccinees. The FML-QuilA vaccine induced a significant, long lasting and strong protective effect against canine kala-azar in the field.

Impact of canine control on the epidemiology of canine and human visceral leishmaniasis in Brazil.

Brazil is the only country endemic for zoonotic visceral leishmaniasis (ZVL) that regularly conducts epidemiologic and prophylactic control programs that involve the treatment of human cases, insect vector control, and the removal of seropositive infected dogs. This report reviews 60 studies reporting data on the efficacy of these recommended control tools and concludes that in Brazil 1) eradication of the disease in Minas Gerais was achieved by the concomitant use of the three control methods, 2) although seropositivity by an immunofluorescent assay is not completely related to infectiousness, the removal of seropositive dogs leads to a significant reduction of canine and human incidence, 3) improvement of the sensitivity of the diagnostic tool used for canine control should optimize the efficacy of control, and 4) although difficult and expensive, the public health dog control campaigns performed in Brazil reduced the incidence of ZVL and should be maintained since treatment of dogs is an unrealistic intervention, both because of its prohibitive cost and relatively poor effectiveness.

New genetic data on Amerindians from the Paraguayan Chaco.

New data on 17 blood group and protein genetic systems obtained among the Ayoreo and Lengua Indians of Paraguay are presented. They include the first report on the red cell band-3 protein investigated among South American Indians. This information was integrated with previous results available for these two and four other groups. Five of the six populations reside in the Chaco area, while the sixth was included as an outgroup living elsewhere in Paraguay. Four of the five Chaco tribes exhibit good genetic homogeneity, but the Ayoreo are somewhat different. The results confirm the Chaco as a distinct biological (as well as cultural and economic) region, which should be considered in evaluations of genetic variability among South American Indians.

Occurrence of Leishmania donovani parasitemia in plasma of infected hamsters.

Intracardiac transfusion of plasma, mononuclear cell fraction and blood of infected hamster donors induced visceral leishmaniasis in normal hamster receptors. At the moment of transfusion, the donors already showed all the typical signs of the disease: ascites, cachexia, as well as splenomegaly and a high parasite load in the spleen and liver. All transfused hamsters developed typical visceral leishmaniasis between 90 and 120 days, indicating that all blood products were infectious. Transfusion of the mononuclear cell fraction induced the highest values of parasitic load (spleen, 766 Leishman Donovan Units (LDU); liver, 2650 LDU), splenomegaly and hepatomegaly (spleen-liver/body relative weight: 1.130 and 6.870, respectively). Animals that received the plasma fraction also developed visceral leishmaniasis, showing similar parasitic load (spleen, 107 LDU; liver, 220 LDU) and spleen-liver/body relative weight (1.005 and 6.35, respectively) than those transfused with whole blood. The finding of typical Leishmania donovani infection in animals transfused with plasma demonstrates the possibility of the extracellular location of parasites, free in this blood fraction deprived of red and white blood cells. Fluorescence-assisted cell sorter analysis (FACS) of plasma showed the presence of particles corresponding in size to amastigotes, which fluoresced strongly with the serum of a patient with Kala-azar (73%), but not with normal serum.

Genetic relationships between Amerindian populations of Argentina.

A total of 495 individuals from five different Argentinian tribes was examined for variation in 23 blood group and protein genetic systems, and the results were integrated with previous data on some of these systems. These tribes generally present RH * R1, PGM1 * 1, and ACP * A frequencies lower and RH * R2, ESD * 1, and GLO * 1 prevalences higher than those observed in other South American Indian groups. Earlier studies with mitochondrial DNA showed that haplogroup A was present in low frequencies in these tribes, but haplogroup B showed a high prevalence among the Mataco. Average heterozygosities are very similar in the five tribes, while estimates of non-Indian ancestry are generally low. Both the blood group and protein, as well as the mtDNA data sets, divide the five tribes into two groups, and the relationships obtained with the blood group and protein systems are exactly those expected on the basis of geography and language. However, the topology obtained with the mtDNA results was different, possibly due to sampling effects or diverse patterns of exchange between the groups related to sex.

Vaccination of Balb/c mice against experimental visceral leishmaniasis with the GP36 glycoprotein antigen of Leishmania donovani.

Leishmania donovani GP36 glycoprotein is the main antigen of the FML Fucose Mannose Ligand (FML) complex specifically recognized by sera of kala-azar human patients. The GP36 was isolated by chemical elution + sonication and used for Balb/c mouse vaccination in combination with saponin, by the s.c. route, inducing a strong and specific protective effect against experimental visceral leishmaniasis shown by the increase of: specific IgG antibodies (82.6%), mainly IgG2a, the delayed type of hypersensitivity to promastigote lysate (37.8%, P < 0.001), the in vitro cellular proliferative response to GP36 of ganglia lymphocytes (53.5%, P < 0.005) and the decrease of liver parasite burden (68.1%, P < 0.025). Saponin treated controls reacted significantly differently from GP36 vaccinated animals at all the assayed variables (P < 0.05). GP36 induced significant protection against murine visceral leishmaniasis at concentrations commonly used for vaccination with recombinant antigens.

Short report: occurrence of Leishmania donovani DNA in donated blood from seroreactive Brazilian blood donors.

Human visceral leishmaniasis (kala-azar) transmitted by blood transfusion has been described in previous reports. Seroprevalence of antibodies to Leishmania donovani was shown to be related to prior blood transfusions in multiply transfused hemodialysis patients in Natal, Rio Grande do Norte, Brazil. In this study, a possible correlation between seroreactivity and the presence of L. donovani DNA was investigated in asymptomatic healthy blood donors. Sera were tested using the fucose mannose ligand (FML) ELISA, which was shown to have a sensitivity of 100%, a specificity of 96-100%, reliability, and diagnostic and prognostic potential for the detection of human and canine kala-azar, respectively. Leishmanial DNA was assessed by the polymerase chain reaction (PCR) and dot-blot hybridization techniques in blood and bone marrow samples. Among 21 FML-seroreactive asymptomatic blood donors, 5 (24%) were positive by the PCR and 9 (43%) were positive in a dot-blot assay of blood samples, showing a significant correlation (chi2 = 14.24, P < 0.01). No Leishmania DNA was detected in 20 FML non-reactive blood donors. Our results point to the need for control of transmission of kala-azar by blood transfusion in areas endemic for this disease.

A phase III trial of efficacy of the FML-vaccine against canine kala-azar in an endemic area of Brazil (São Gonçalo do Amaranto, RN).

Protection against canine kala-azar was investigated in naturally exposed dogs of an endemic area, vaccinated with the fucose mannose ligand (FML)-vaccine of Leishmania donovani. A total of 97% of vaccinees were seropositive to FML and 100% showed intradermal reaction to L. donovani lysate, 7 months after vaccination. The absorbency values and size of intradermal reaction were both significantly higher in vaccinees than in controls (ANOVA, P<0.0001). After 2 years, 92% (chi(2)=6.996; P<0.0025) protection was achieved: only 8% of vaccinees showed mild signs of kala-azar with no deaths while 33% of controls developed clinical or fatal disease. The FML-vaccine induced a significant, long-lasting and strong protective effect against canine kala-azar in the field.

Vaccination of Swiss Albino mice against experimental visceral leishmaniasis with the FML antigen of Leishmania donovani.

The FML antigen of Leishmania donovani in combination with saponin, aluminum hydroxide (Al(OH)3) and Freund's incomplete adjuvant (FIA) was used in vaccines tested in an outbred murine model of visceral leishmaniasis, either through intraperitoneal or subcutaneous routes. The humoral response was significantly higher in the groups treated with FML + saponin or FML + Al(OH)3 than in controls, both before and after the infection. Animals immunized by the i.p. route developed higher antibody titres. A significant and specific reduction of parasitic load in relation to saline (85%, p < 0.01) and saponin (p < 0.025) controls, was seen in animals treated with FML + saponin by the i.p. Coincidentally with this reduction, an increase in antibodies of the IgG2a subtype was detected only in animals treated with FML + saponin i.p. A reduction of 88% in parasitic load was achieved by the combination of FML + Al(OH)3 (s.c.), but the Al(OH)3 treatment itself accounted for 68% of this protection. In our conditions, vaccination with FML + saponin i.p. was superior to other treatments and had no toxic effect due to saponin.

Prevalence of anti-Leishmania donovani antibody among Brazilian blood donors and multiply transfused hemodialysis patients.

The prevalence of anti-Leishmania donovani antibodies was investigated in 1,500 Brazilian blood donors and multiply transfused hemodialysis patients. Sera were tested using the fucose-mannose ligand (FML) ELISA, which was shown to have 100% sensitivity and 96% specificity for kala-azar. Among 1,194 volunteer blood donors, seroreactivity was 9%, increasing to 25% in a periurban kala-azar focus. However, higher positivity (37%) was found in multiply transfused hemodialysis patients from Natal, where kala-azar is constantly present in low numbers (endemic), with sporadic outbreaks in localized regions (endemic and epidemic). Risk factors included blood transfusion, which was significantly associated with the presence of anti-Leishmania antibodies (chi2 = 8.567, P < 0.005), but did not include potential exposure to sandfly bites (chi2 = 0.033, P > 0.1). The prevalence significantly decreased to 7% in hemodialysis patients from Rio de Janeiro, where kala-azar is only occasionally seen, and was 0% in patients undergoing continuous ambulatorial peritoneal dialysis. The prospective analysis of 27 FML-seroreactive donors from Natal revealed amastigotes of Leishmania in the bone marrow of one subject while four had clinical complaints, including splenomegaly and hepatosplenomegaly. Our results point to the need for control of blood transfusion as a possible route for transmission of kala-azar in endemic areas.

Haemolytic activities of plant saponins and adjuvants. Effect of Periandra mediterranea saponin on the humoral response to the FML antigen of Leishmania donovani.

An 87.7% (P < 0.01) and 84% (P < 0.001) of protection against visceral leishmaniasis was achieved in CB hamsters and Balb/c mice, respectively, with saponin combined to the fucose-mannose ligand of Leishmania donovani (FML). However, an undesirable haemolytic effect was described for several saponins. Aiming to improve the formulation with FML/saponin, we comparatively analysed the haemolytic potential of recently characterized plant saponins and currently used adjuvants. The haemolytic activity of steroidic saponins from Agave sisalana; Smilax officinalis as well as commercial saponin (Riedel De Haën's), was higher than that of triterpenoid ones (Bredemeyera floribunda; Periandra mediterranea) and the Freund's complete adjuvant. The concentration resulting in 50% haemolysis was 500 micrograms ml-1 for aluminum hydroxide. The low haemolytic effect of P. mediterranea saponin was abolished by removal of its glycidic moiety and its sapogenin fraction as well as the Freund's Incomplete Adjuvant were non-haemolytic within this range. Furthermore, the adjuvant effect of three doses of P. mediterranea saponin injected with the FML antigen of L. donovani, was assayed in mice, either by the intraperitoneal (i.p.) or the subcutaneous (s.c.) route. The anti-FML IgG antibody levels increased and detectable levels were observed up to 3 months in the s.c. group. The response was expanded in both groups after an injection with a fourth vaccine dose. The IgG response showed increased levels of IgG2a only in the i.p. group, while IgG2b and IgG1 but not IgG3 antibodies were higher than controls in both groups. In conclusion, the results suggest that the recently described triterpenoid fractions of P. mediterranea can be safely used as adjuvant with low or non-haemolytic effect.