Pretransplant Single Antigen Bead-Detected HLA Antibodies in Kidney Transplant Long-term Outcome: A Single-Center Cohort Experience.

Single-antigen bead (SAB) platform permits the identification of antibodies not detectable by complement-dependent lymphocytotoxicity test, but their clinical significance is not completely understood. The aim of this study was to evaluate whether the presence of pretransplant SAB-detected antibodies is associated with the development of allograft failure. This is a single-center cohort study with 10-year follow-up in which 573 kidney recipients with negative pretransplant complement-dependent lymphocytotoxicity crossmatch who received transplants at the Kidney Transplant Center of Policlinico, Milan, from deceased donors between 1996 and 2005 were evaluated. Pretransplant plasma samples were retrospectively analyzed by SAB assay. Survival analyses were performed to assess the risk of allograft failures by SAB-detected antibodies. Pretransplant antibodies were found in 160 (28.0%) recipients, of whom 42 subsequently developed an allograft failure for a survival rate of 70.9% (95% confidence interval [CI), 63.5-78.4). Among those without antibodies, 58 (14.0%) returned to dialysis with a survival rate of 84.7% (95% CI, 81.0-88.4). In Cox regression analyses, patients with SAB-positivity had 2-fold higher risk of allograft failure than those who were SAB-negative (hazard ratio, 2.07; 95% CI, 1.39-2.79). Results did not change after adjustment for putative confounders. In conclusion, in this single-center cohort, 10-year allograft survival rate was significantly influenced by the presence of SAB-detected antibodies.

The distribution of KIR-HLA functional blocks is different from north to south of Italy.

The killer cell immunoglobulin-like receptor (KIR)-human leukocyte antigen (HLA) interaction represents an example of genetic epistasis, where the concomitant presence of specific genes or alleles encoding receptor-ligand units is necessary for the activity of natural killer (NK) cells. Although KIR and HLA genes segregate independently, they co-evolved under environmental pressures to maintain particular KIR-HLA functional blocks for species survival. We investigated, in 270 Italian healthy individuals, the distribution of KIR and HLA polymorphisms in three climatic areas (from cold north to warm south), to verify their possible geographical stratification. We analyzed the presence of 13 KIR genes and genotyped KIR ligands belonging to HLA class I: HLA-C, HLA-B and HLA-A. We did not observe any genetic stratification for KIR genes and HLA-C ligands in Italy. By contrast, in a north-to-south direction, we found a decreasing trend for the HLA-A3 and HLA-A11 ligands (P = 0.012) and an increasing trend for the HLA-B ligands carrying the Bw4 epitope (P = 0.0003) and the Bw4 Ile80 epitope (P = 0.0005). The HLA-A and HLA-B KIR ligands were in negative linkage disequilibrium (correlation coefficient -0.1211), possibly as a consequence of their similar function in inhibiting NK cells. The distribution of the KIR-HLA functional blocks was different along Italy, as we observed a north-to-south ascending trend for KIR3DL1, when coupled with HLA-B Bw4 ligands (P = 0.0067) and with HLA-B Bw4 Ile80 (P = 0.0027), and a descending trend for KIR3DL2 when coupled with HLA-A3 and HLA-A11 ligands (P = 0.0044). Overall, people from South Italy preferentially use the KIR3DL1-HLA-B Bw4 functional unit, while those from the North Italy equally use both the KIR3DL2-HLA-A3/A11 and the KIR3DL1-HLA-B Bw4 functional units to fight infections. Thus, only KIR3DL receptors, which exert the unique role of microbial sensors through the specific D0 domain, and their cognate HLA-A and HLA-B ligands are selectively pressured in Italy according to geographical north-to-south distribution.

Description of two new HLA-C alleles: C*08:63 and C*14:44.

Here, we present two new HLA allelic variants at C locus: HLA-C*08:63 and HLA-C*14:44 detected by sequence-based typing. In both cases, a single-nucleotide mutation in exon 3 is responsible for a change in aminoacid translation. The extremely high polymorphism of human leucocyte antigen (HLA) system in human genome is responsible for the capability to recognize different antigens, including non-self-MHC (Major Histocompatibility Complex) molecules. This very high polymorphism and the improving accuracy of genomic HLA typing methods lead to an exponential increasing of known HLA alleles. Here, we describe the characterization of two new HLA-C alleles identified by sequence-based typing (SBT): HLA-C*08:63 and HLA-C*14:44.

Comportamientos que favorecen la dinámica de reinfestación de Triatomainfestans del Chaco paraguayo / Behaviors that favor reinfestation by Triatoma infestans in the Paraguayan Chaco

El Chaco central paraguayo es unaregión de alto nivel de reinfestación porTriatoma infestans. La población indígena que la habita tiene alta vulnerabilidad por factores culturales y medio ambientales que dificultan acceso y trabajo en la zona. Se propusoconocer factores psicosociales asociadosal proceso de reinfestación para desarrollar tareas de vigilanciacomunitaria. Estudio transversal, con enfoques cualitativo y cuantitativo.Treinta y seis punto siete por ciento (96)de la población de estudio realizómejoras en viviendas; 41,6 % (40)mejoró revoque en paredes. Poblaciónubica al vector en el monte, entre leñas,hojas secas, agujeros de árboles, pozosde topos o tatú; alrededor de animales domésticos, techos de viviendas, gallineros, chiquero de cabras y cerdos.Comprometiendo el traslado pasivo de vinchucas se encontró la recolección deleña 98,5 % (266), del monte, 97,7 %(261) el cambio de lugar de ropas, cajas y comida en las viviendas, 54,7 % (146). Se asoció (p< 0,0005) vivienda mejorada con revoque en paredes yno infestación; viviendas con animales(p< 0,03) e infestación; actitud positivapara eliminar el vector (p<0,04) y no infestación. Comportamientos que comprometen traslado y permanencia de vinchucas fueron acarreo de leña, almacenamiento de comidas yacumulación de ropa y cajas. Paredes revocadas y presencia de animales domésticos se correlacionaron a infestación y actitudes positivas paraeliminación de la vinchuca con viviendas sin reinfestación. Todos ellos sonfactores estratégicos para tareas deprevención y vigilancia con participación comunitaria.

Identification of two new HLA-DRB1 alleles, DRB1*040405 and DRB1*1190.

We describe here two novel DRB1 alleles, officially named *040405 and *1190. DRB1*040405 differs from DRB1*0404 for one point mutation at codon 72 with no coding changes. DRB1*1190 is identical to DRB1*110101 except for a nucleotide substitution at codon 24 which causes an aminoacidic mutation from valine to methionine. Over time we have been witnessing the identification of a great number of new HLA alleles. DRB1 allelic variability is mostly present in the second exon and more that 760 alleles have been so far identified. Here, we report the description of two novel DRB1 alleles, named *040405 and *1190, and identified in two Caucasoid subjects.

Identification of Trypanosoma cruzi sublineages by the simple method of single-stranded conformation DNA polymorphism (SSCP).

Fifty-eight stocks of Trypanosoma cruzi from Latin America were genetically characterized using the methods of the polymerase chain reaction (PCR) and the single-stranded conformation DNA polymorphism (SSCP) with four genes, mini-exon, 24Salpha rRNA, 18Sr RNA, cruzipain, and a RAPD fragment DNA region, P7-P8. All the isolates examined were assigned to T. cruzi I or subgroups of T. cruzi II by these methods. From these results, the SSCP analysis, which was simple to perform and highly sensitive to sequence variation, seemed to be a good modality for characterizing T. cruzi, particularly for subgroups of T. cruzi II. However, in several isolates of T. cruzi II, the subgroups determined with the SSCP of 24Salpha rRNA were not consistent with those determined with other genes, the SSCP of 18S rRNA and cruzipain, and the PCR of P7-P8, possibly because of the occurrence of rare genetic exchanges or mutations or both in natural populations of this parasite. The SSCP patterns of 24Salpha rRNA and 18S rRNA were highly variable in the T. cruzi I isolates; therefore, analyses using both genes are considered to be one possible method for the characterization of isolates within T. cruzi I.

Identification of a new HLA-B variant, HLA-B*5615.

A new HLA-B allele, B*5615, has been identified in a Caucasian individual by sequence-based typing. This allele shows a sequence identical to that of HLA-B*5601 except for two nucleotide substitutions that cause a change from TTA to TAC at codon 116 and an amino acidic change from Leucine to Tyrosine in the mature protein.

Screening of New Caledonian and Vanuatu medicinal plants for antiprotozoal activity.

Sixty-seven extracts of 30 medicinal plants traditionally used in New Caledonia or Vanuatu by healers to treat inflammation, fever and in cicatrizing remedies were evaluated in vitro for their antiprotozoal activity against Leishmania donovani, Leishmania amazonensis and Trypanosoma cruzi. Among the selected plants, Pagiantha cerifera was the most active against both Leishmania species; four extracts were active against promastigotes of Leishmania donovani at EC(50) values inferior to 5 microg/ml. Garcinia pedicillata extract had an EC(50) value of 12.5 microg/ml against intracellular amastigotes of Leishmania amazonensis. Alone Amborella trichopoda reduced by more of 80% the trypomastigotes of Trypanosoma cruzi in the blood.

Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects.

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb-e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb-e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb-e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.

Identification of two new HLA-B alleles, HLA-B*0732 and HLA-B*5809, by sequence-based typing.

Here, we have described the characterization of two novel human leukocyte antigen-B (HLA-B) alleles. The new alleles, HLA-B*0732 and HLA-B*5809, were identified in Italian Caucasian individuals. B*0732 differs from HLA-B*0708 by one nucleotidic change at position 412 (from G to A) in exon 3, leading to an amino acidic substitution from Asp (GAC) to Asn (AAC) at codon 114. The sequence of B*5809 is identical to that of HLA-B*5801, except for a point mutation at position 583 in exon 3, where a T is substituted by a C. This change leads to an amino acidic substitution from Tyr (TAC) to His (CAC) at codon 171.

Characterization of two new HLA-B alleles by sequence-based typing: HLA-B*0817 and HLA-B*1311.

In this brief communication we report the characterization of two new HLA-B variants officially named HLA-B*0817 and HLA-B*1311. The HLA-B*0817 allele was identified in a Caucasoid male candidate for renal transplantation in the North Italy Transplant program. The nucleotidic sequence of exons 2, 3 and 4 of this novel allele is identical to that of HLA-B*0804 except for three point mutations in exon 2: from A to G at position 259, from C to G at position 261 and from G to A at position 302. These mutations are responsible for two aminoacidic substitutions [Asn (r) Glu, codon 63, and Ser (r) Asn, codon 77]. HLA-B*1311 was found in a volunteer donor belonging to National Marrow Donor Program(R). This new variant is identical to that of HLA-B*1301 except for three nucleotide substitutions at positions 353, 355 and 369 leading to two aminoacidic variations from Ile to Thr at codon 94 and from Ile to Leu at codon 95 and a silent mutation at codon 99.

Characterization of a new HLA-DRB3 allele, DRB3*0217, by direct sequencing.

We report the identification of a novel DRB3*02 using sequence-based typing (SBT). This new allele, officially named DRB3*0217, was detected while performing HLA high resolution typing of a bone marrow recipient and his siblings. DNA sequencing demonstrated the presence of a nucleotide substitution in exon 2 at position 199 where a C was substituted by a T. This point mutation at codon 67 (CTC-->TTC) has resulted in an amino acid substitution from Leucine to Phenylalanine.

Leishmanicidal activity of two canthin-6-one alkaloids, two major constituents of Zanthoxylum chiloperone var. angustifolium.

The crude alkaloidal extract of Zanthoxylum chiloperone stem bark exhibited in vitro activity against various strains of Leishmania ssp. at 100 microg/ml. Two active major constituents were isolated and identified as canthin-6-one and 5-methoxycanthin-6-one. The effect of these compounds was also tested in an in vivo assay using BALB/c mice infected with Leishmania amazonensis. The mice were treated for 5 weeks postinfection with these alkaloids by oral (14 days) or intralesional route (4 days) at 10 mg/kg daily. The reference drug, N-methylglucamine antimonate was administered by subcutaneous injections at 100 mg/kg for 10 days. Intralesional administration of canthin-6-one reduced the parasite burden but not significantly when it was compared with the untreated group, while the reference drug reduced by 91% the parasite loads in the lesion.

Experimental treatment of chronic Trypanosoma cruzi infection in mice with 2-n-propylquinoline.

It was reported previously that 2-n-propylquinoline was active against the epimastigote form of Trypanosoma cruzi. The effects of oral treatments with benznidazole and 2-n-propylquinoline were evaluated in Balb/c mice infected with T. cruzi chronically. The reference drug and 2-n-propylquinoline were administered 60 days post-infection for 30 days at 25 mg/mL. At 35 days post-treatment, the serological tests (ELISA) of the 2-n- propylquinoline-treated mice were significantly different from the controls (p = 0.01) and the benznidazole-treated mice (p = 0.03), while this was not the case at 85 days post-treatment. These results are encouraging for continuing the investigation of other analogues of 2-n-propylquinoline in experimental chronic Chagas' disease.

Chagas disease prevention through improved housing using an ecosystem approach to health.

This Chagas disease prevention project via housing improvement aims to determine the efficiency of different interventions in vector control. The following study describes the target communities, disease magnitude, and housing improvements. Transmission levels are analysed from an ecological and socioeconomic perspective. Special interest was focused on the peridomicile as the origin of domiciliary reinfestation. In the original project, three intervention programs were proposed, one for each of the three communities: (a) an insecticide spraying program; (b) a housing improvement program; and (c) a combined program of spraying and housing improvement. The three communities currently have different risks of exposure to triatominae reinfestation as a consequence of the type of intervention carried out. A new multidisciplinary approach which integrates participatory, community-based research and socioeconomic dimensions will allow to determine the efficiency of models for territorial ordering, community education, and environmental interventions in Chagas disease control.

Isoenzyme profiles of Trypanosoma cruzi stocks from different areas of Paraguay.

Twenty one Trypanosoma cruzi stocks from humans, domiciliary triatomines and one sylvatic animal of different areas of Paraguay were subjected to isoenzyme analysis. Thirteen enzyme systems (15 loci in total) were studied. MN cl2 (clonets 39) and SO34 cl4 (clonets 20) were used as references. Relationships between stocks were depicted by an UPGMA dendrogram constructed using the Jaccard's distances matrix. Among the Paraguayan stocks 14 zymodemes were identified (Par1 to Par14), Par 5 being the most frequent. Polymorphism rate and clonal diversity were 0.73 and 0.93, respectively. Average number of alleles per polymorphic locus was 2.5 (range 2-4). These measurements show a high diversity, which is confirmed by the dendrogram topology. All stocks belong to the same lineage, as MN cl2 reference strain (T. cruzi II). Moreover three distinct subgroups were identified and two of them correspond to Brazilian and Bolivian zymodemes, respectively. The third subgroup, the most common in Paraguay, is related to Tulahuen stock. The large geographical distribution of some zymodemes agrees with the hypothesis of clonality for T. cruzi populations. However sample size was not adequate to detect genetic recombination in any single locality.

Cryptofolione derivatives from Cryptocarya alba fruits.

Cryptofolione (1) and the new cryptofolione derivative 6-(4,6-dimethoxy-8-phenyl-octa-1,7-dienyl)-4-hydroxy-tetrahydro-pyran-2-one (2) were isolated from the fruits of Cryptocarya alba. The structures were elucidated by spectroscopic methods. Cryptofolione showed activity towards Trypanosoma cruzi trypomastigotes, reducing their number by 77% at 250 microg mL(-1). Cryptofolione showed moderate cytotoxicity in both macrophages and T. cruzi amastigotes. It also displayed a mild inhibitory effect on the promastigote form of Leishmania spp. As both cytotoxic and trypanocidal effects are similar, the compound presented little selectivity in our assay models.

Efficacy of the bisbenzylisoquinoline alkaloids in acute and chronic Trypanosoma cruzi murine model.

We have shown previously that daphnoline and cepharanthine are active against Trypanosoma cruzi and inhibited trypanothione reductase. The effects of oral treatments with daphnoline, cepharanthine and benznidazole were examined in Balb/c mice infected with T. cruzi acutely and chronically. In acute infections, parasitaemia was significantly reduced in the daphnoline-treated mice compared with controls and benznidazole-treated mice. The parasitological cure rate was increased in mice treated with daphnoline. Fifty days after infection, the negative serological response in both models was significantly different for the three tested drugs. Daphnoline showed the highest negative serological rate (48%). In chronically infected mice treated with daphnoline, we were unable to detect parasites in 70% of mice. The results obtained of oral treatment of daphnoline suggest that this bisbenzylisoquinoline may be useful in the treatment of acute and chronic Chagas' disease. This was not seen with cepharanthine, an excellent trypanothione reductase inhibitor.