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The quantitative simultaneous description of both variable region gene usage and antigen specificity of immunoglobulin repertoires is a major goal in immunology. Current quantitative assays are labor intensive and depend on extensive gene expression cloning prior to screening for antigen specificity. Here we describe an alternative method based on high efficiency single B cell cultures coupled with RT-PCR that can be used for rapid characterization of immunoglobulin gene segment usage, clonal size and antigen specificity. This simplified approach should facilitate the study of antibody repertoires expressed by defined B cell subpopulations, the analysis of immune responses to self and nonself-antigens, the development and screening of synthetic antibodies and the accelerated study and screening of neutralizing antibodies to pathogenic threats., (Published by Elsevier B.V.)

Curcumin is a phenolic natural product isolated from the rhizome of Curcuma longa (tumeric). It was previously described that curcumin had a potent anti-inflammatory effect and inhibited the proliferation of a variety of tumor cells. In the present study, we investigated the inhibitory effects of curcumin on the response of normal murine splenic B cells. Curcumin inhibited the proliferative response of purified splenic B cells from BALB/c mice stimulated with the Toll-like receptor ligands LPS and CpG oligodeoxynucleotides. LPS-induced IgM secretion was also inhibited by curcumin. The proliferative response induced by either the T-independent type 2 stimuli anti-delta-dextran or anti-IgM antibodies was relatively resistant to the effect of curcumin. We investigated the intracellular signaling events involved in the inhibitory effects of curcumin on murine B cells. Curcumin did not inhibit the increase in calcium levels induced by anti-IgM antibody. Western blotting analysis showed that curcumin inhibited TLR ligands and anti-IgM-induced phosphorylation of ERK, IkappaB and p38. Curcumin also decreased the nuclear levels of NFkappaB. Our results suggested that curcumin is an important inhibitor of signaling pathways activated upon B cell stimulation by TLR ligands. These data indicate that curcumin could be a potent pharmacological inhibitor of B cell activation.

Rho GTPases are members of the Ras superfamily and are involved in signal transduction pathways, including maintenance of cell morphology and motility, cell cycle progression, and transcription activation. We report the molecular identification in trypanosomatids (Trypanosoma cruzi) of the first member of the Rho family. The cloned Rho protein, TcRho1, shares approximately 40% homology with other members of the Rho family. Southern blot analysis revealed that TcRHO1 is a single copy gene per haploid genome, and Northern blot assays showed a transcript of 1200 nucleotides in length. Mapping the 5'-untranslated region of TcRHO1 transcripts revealed at least five different transcripts derived from differential trans-splicing. Three of the five transcripts contain the trans-splicing site within the coding region of the TcRHO1 gene. TcRho1 also contains the C-terminal sequence CQLF (CAAX motif), which is predicted to direct post-translation prenylation of the cysteine residue. A synthetic peptide containing this C-terminal motif, when tested against Q-Sepharose chromatography fractions from T. cruzi cytosol, was shown to be efficiently farnesylated, but not geranylgeranylated, despite the fact that the CAAX motif with X = Phe specifies geranylgeranylation by mammalian protein geranylgeranyltransferase I. Furthermore, immunoblot analyses of epimastigote protein with anti-S-farnesylcysteine methyl ester and anti-TcRho1 antisera strongly suggested that TcRho1 is farnesylated in vivo. The farnesylation of proteins such as Rho GTPases could be the basis for the selective cytotoxic action of protein farnesyltransferase inhibitors on trypanosomatids versus mammalian cells.

The intestinal microbiota, a barrier to the establishment of pathogenic bacteria, is also an important reservoir of opportunistic pathogens. It plays a key role in the process of resistance-genes dissemination, commonly carried by specialized genetic elements, like plasmids, phages, and conjugative transposons. We obtained from strains of enterobacteria, isolated from faeces of newborns in a university hospital nursery, indication of phenotypical gentamicin resistance amplification (frequencies of 10(-3) to 10(-5), compatible with transposition frequencies). Southern blotting assays showed strong hybridization signals for both plasmidial and chromosomal regions in DNA extracted from variants selected at high gentamicin concentrations, using as a probe a labeled cloned insert containing aminoglycoside modifying enzyme (AME) gene sequence originated from a plasmid of a Klebsiella pneumoniae strain previously isolated in the same hospital. Further, we found indications of inactivation to other resistance genes in variants selected under similar conditions, as well as, indications of co-amplification of other AME markers (amikacin). Since the intestinal environment is a scenario of selective processes due to the therapeutic and prophylactic use of antimicrobial agents, the processes of amplification of low level antimicrobial resistance (not usually detected or sought by common methods used for antibiotic resistance surveillance) might compromise the effectiveness of antibiotic chemotherapy.

The elongation factor EF-1alpha is one of the most studied components of the translation machinery owing to its abundance and possible role in other cellular functions. EF-1alpha mediates the correct coupling of the aminoacyl-tRNA on the A site of the ribosome in a GTP-dependent reaction. We have previously described an EF-1alpha DNA sequence in Leishmania amazonensis, pLEF11 (accession No. M92653), using PCR. In this paper we describe the DNA sequence and genomic organization of L. braziliensis EF-1alpha gene. Southern blot analysis revealed that EF-1alpha is organized as a 2 kb tandem repeat. The pLEF11 probe recognized a 1.8 kb mRNA from promastigotes in Northern blots. A clone containing the first copy and a half of the EF-1alpha tandem repeat was isolated by screening a L. braziliensis genomic library. Southern blot analysis showed that the isolated clone (lambda2.2) presented the same hybridization profile as that of a genomic blot. The partial sequencing of clone lambda2.2 spans 2959 nucleotides in length, which has two open reading frames separated by a putative non-coding region. The nucleotide and the predicted peptide sequence of the first coding region presented approximately 80% identity with other eukaryotic EF-1alpha genes. The sequence also displayed the four consensus motifs corresponding to the GTP-binding site (G1, G2, G3 and G4). Computer analysis of the sequence of both coding regions revealed three divergent nucleotides, which generated two changes at the amino acid level. One was found to be located in the G2 domain. The non-coding region of the EF-1alpha gene sequence showed potential regulatory elements such as polypyrimidine tracks, chi-homologous sequences and stem-loop forming sequences.

Sequence analysis of Leishmania (Viannia) kDNA minicircles and analysis of multiple sequence alignments of the conserved region (minirepeats) of five distinct minicircles from L. (V.) braziliensis species with corresponding sequences derived from other dermotropic leishmanias indicated the presence of a sub-genus specific sequence. An oligonucleotide bearing this sequence was designed and used as a molecular probe, being able to recognize solely the sub-genus Viannia species in hybridization experiments. A dendrogram reflecting the homologies among the minirepeat sequences was constructed. Sequence clustering was obtained corresponding to the traditional classification based on similarity of biochemical, biological and parasitological characteristics of these Leishmania species, distinguishing the Old World dermotropic leishmanias, the New World dermotropic leishmanias of the sub-genus Leishmania and of the sub-genus Viannia.

A vinblastine-resistant Leishmania amazonensis cell line (RV100) which exhibits cross-resistance to the unrelated drug adriamycin, and thus is considered to be multidrug resistant (MDR), was isolated after stepwise selection with increasing concentrations of vinblastine. This phenotype was partially reverted by the calcium channel antagonist verapamil. Drug transport studies using the hydrophobic fluorescent dye rhodamine 123 demonstrated that the MDR cell line has a reduced dye accumulation due to an increased efflux. Furthermore, DNA and RNA hybridization studies demonstrated that a gene (lamdr1), homologous to ldmdr1 and lemdr1, was overexpressed and amplified within 27 kb extrachromosomal DNA circles (V-circles) in these cells. An independent cell line, RA5000, which was selected for resistance to adriamycin and was not cross-resistant to vinblastine, accumulated normal levels of rhodamine 123 and did not contain amplified DNA or overexpressed RNA of mdr-related sequences.

During a field investigation carried out in Baturite, Brazil from 1989 to 1991, sand flies, sympatric rodents, domestic dogs and humans were surveyed for leishmaniasis. Twenty strains of Leishmania were isolated by in vitro culture from Lutzomyia whitmani, three strains were obtained from Rattus rattus, two strains from dogs, and five strains from humans. The isolates were characterized by isoenzyme electrophoresis by hybridization with kinetoplast DNA-specific probes. All the samples were identified as L. (Viannia) braziliensis. The importance of these results in the dynamics of the Leishmania infection in this focus is discussed.

A PCR based assay was designed in order to amplify putative ras gene sequences of the GTPase superfamily eventually present in Leishmania amazonensis and Trypanosoma cruzi. A set of primers corresponding to the conserved motifs G1 and G3 of the GTP binding proteins was synthesized. Sequencing of six PCR products (three from Leishmania and three from Trypanosoma) identified, however, two other different GTPases. The 270 bp L. amazonensis clone, pLef-11, shared an amino acid identity of around 80% with an eukaryotic elongation factor 1a of protein synthesis. On the other hand, the 168 bp T. cruzi clone, pTCr1, demonstrated over 60% amino acid identity to ras-related proteins of the rab-YPT-SEC4 family involved in control of vesicular traffic. To our knowledge, this is the first report of GTP binding protein genes in trypanosomatids.

Domestic dogs are considered to be a major reservoir of Leishmania donovani chagasi in northeast Brazil, and the elimination of infected dogs is an important part of the control program. We assessed 2 serological methods, IFA and ELISA. Of 405 dogs, 8% were positive by IFA obtained from blood collected by drying onto filter paper followed by elution, 17% were positive by IFA performed using sera, and 38% were positive by ELISA on the same sera. Thirty-five dogs, seropositive by 1 or more of the above tests, were killed and touch preparations were made of liver, spleen, and mesenteric lymph nodes. Samples were cultured in enriched NNN media. The ELISA recognized all dogs with proven infection; IFA detected 10 of 12. Eleven dogs were positive by touch preparations and 7 by culture. In addition, kDNA hybridization was undertaken with probes to L. donovani chagasi, L. braziliensis ssp., and L. mexicana amazonensis. Positive results were obtained from tissue in 19 instances, but 10 culture positive specimens were not recognized.

Several efforts have been made in order to develop more precise and sensitive methods in the identification of Leishmania parasites. We report here the identification of cloned subfragments of minicircle kinetoplast DNA (kDNA) isolated from L. donovani, WR352, which show different taxonomic specificities. Analysis of these fragments demonstrates a significant sequence diversity within the kDNA minicircle. For example, one cloned fragment was found to be present in all visceral Leishmania species tested, but was not present in any of the cutaneous Leishmania species. Another cloned fragment was only found in the strain from which it had been derived, and was not present in any of the other strains tested. In similar experiments with the New World visceral leishmanias (L. chagasi, WR518) several different cloned kDNA fragments were found to react with all of isolates of the L. chagasi tested, but not with any cutaneous Leishmania species, either from the Old World or the New World. It is of interest to note that these cloned L. chagasi kDNA fragments reacted with isolates of African visceral Leishmania species but not with isolates from India.

Leishmania parasites were isolated from humans and canines in foci of cutaneous and visceral leishmaniasis. After in vitro cultivation the parasites were examined by the following biochemical techniques: (i) restriction analysis of kinetoplast DNA (kDNA) also known as schizodeme analysis (Morel et al., 1980); (ii) zymodeme analysis (Barret et al., 1980); by agarose gel electrophoresis and (iii) isoelectricfocusing in polyacrylamide gels. The strains of cutaneous and visceralizing leishmanias studied could be differentiated by schizodeme analysis, using the endonuclease MspI, into three complexes agreeing with those accepted for human New World leishmaniasis. In the municipality of Rio de Janeiro, isolates from a focus of cutaneous leishmaniasis were identified as L. braziliensis braziliensis and from a focus of visceral leishmaniasis were identified as L. donovani by zymodeme characterization. Identical restriction enzyme profiles of kDNA from human and canine isolates indicated that in the cutaneous focus at Jacarepaguá, Rio de Janeiro, the same strain was probably circulating in both the canine and human populations. This suggests a possible role for dogs as a reservoir host for L. braziliensis braziliensis. In addition, our results confirm the importance of dogs as reservoirs in visceral leishmaniasis. The stability of the electrophoretic patterns of restriction digest ("fingerprints") of Leishmania kDNA as well as differences in the sensitivity of the techniques used were demonstrated. Strains from widely different geographical areas as well as strains maintained in vivo and in vitro showed identical kDNA restriction patterns, while strains showing similar banding patterns by enzyme electrophoresis could be differentiated by schizodeme analysis. These results demonstrate the usefulness of an integrated biochemical approach in the identification of Leishmania.