c-Rel gain in B cells drives germinal center reactions and autoantibody production.

Single-nucleotide polymorphisms and locus amplification link the NF-κB transcription factor c-Rel to human autoimmune diseases and B cell lymphomas, respectively. However, the functional consequences of enhanced c-Rel levels remain enigmatic. Here, we overexpressed c-Rel specifically in mouse B cells from BAC-transgenic gene loci and demonstrate that c-Rel protein levels linearly dictated expansion of germinal center B (GCB) cells and isotype-switched plasma cells. c-Rel expression in B cells of otherwise c-Rel-deficient mice fully rescued terminal B cell differentiation, underscoring its critical B cell-intrinsic roles. Unexpectedly, in GCB cells transcription-independent regulation produced the highest c-Rel protein levels among B cell subsets. In c-Rel-overexpressing GCB cells this caused enhanced nuclear translocation, a profoundly altered transcriptional program, and increased proliferation. Finally, we provide a link between c-Rel gain and autoimmunity by showing that c-Rel overexpression in B cells caused autoantibody production and renal immune complex deposition.

GP130 activation induces myeloma and collaborates with MYC.

Multiple myeloma (MM) is a plasma cell neoplasm that results from clonal expansion of an Ig-secreting terminally differentiated B cell. Advanced MM is characterized by tissue damage that involves bone, kidney, and other organs and is typically associated with recurrent genetic abnormalities. IL-6 signaling via the IL-6 signal transducer GP130 has been implicated as an important driver of MM pathogenesis. Here, we demonstrated that ectopic expression of constitutively active GP130 (L-GP130) in a murine retroviral transduction-transplantation model induces rapid MM development of high penetrance. L-GP130-expressing mice recapitulated all of the characteristics of human disease, including monoclonal gammopathy, BM infiltration with lytic bone lesions, and protein deposition in the kidney. Moreover, the disease was easily transplantable and allowed different therapeutic options to be evaluated in vitro and in vivo. Using this model, we determined that GP130 signaling collaborated with MYC to induce MM and was responsible and sufficient for directing the plasma cell phenotype. Accordingly, we identified Myc aberrations in the L-GP130 MM model. Evaluation of human MM samples revealed recurrent activation of STAT3, a downstream target of GP130 signaling. Together, our results indicate that deregulated GP130 activity contributes to MM pathogenesis and that pathways downstream of GP130 activity have potential as therapeutic targets in MM.

Genetic variation and recurrent parasitaemia in Peruvian Plasmodium vivax populations.

BACKGROUND: Plasmodium vivax is a predominant species of malaria in parts of South America and there is increasing resistance to drugs to treat infections by P. vivax. The existence of latent hypnozoites further complicates the ability to classify recurrent infections as treatment failures due to relapse, recrudescence of hyponozoites or re-infections. Antigen loci are putatively under natural selection and may not be an optimal molecular marker to define parasite haplotypes in paired samples. Putatively neutral microsatellite loci, however, offer an assessment of neutral haplotypes. The objective here was to assess the utility of neutral microsatellite loci to reconcile cases of recurrent parasitaemia in Amazonian P. vivax populations in Peru. METHODS: Patient blood samples were collected from three locations in or around Iquitos in the Peruvian Amazon. Five putatively neutral microsatellite loci were characterized from 445 samples to ascertain the within and amongst population variation. A total of 30 day 0 and day of recurrent parasitaemia samples were characterized at microsatellite loci and five polymorphic antigen loci for haplotype classification. RESULTS: The genetic diversity at microsatellite loci was consistent with neutral levels of variation measured in other South American P. vivax populations. Results between antigen and microsatellite loci for the 30 day 0 and day of recurrent parasitaemia samples were the same for 80% of the pairs. The majority of non-concordant results were the result of differing alleles at microsatellite loci. This analysis estimates that 90% of the paired samples with the same microsatellite haplotype are unlikely to be due to a new infection. CONCLUSIONS: A population-level approach was used to yield a better estimate of the probability of a new infection versus relapse or recrudescence of homologous hypnozoites; hypnozoite activation was common for this cohort. Population studies are critical with the evaluation of genetic markers to assess P. vivax biology and epidemiology. The additional demonstration of microsatellite loci as neutral markers capable of distinguishing the origin of the recurrent parasites (new infection or originating from the patient) lends support to their use in assessment of treatment outcomes.

A FRET-based real-time PCR assay to identify the main causal agents of New World tegumentary leishmaniasis.

In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL). The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR) assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) peruviana and L. (V.) lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST). In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST) data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America.

A20 and CYLD do not share significant overlapping functions during B cell development and activation.

The ubiquitin-editing enzyme A20 (TNFAIP3) and the deubiquitinase CYLD are central negative regulators of NF-κB signaling. Both can act by removing nonproteolytic K63-linked polyubiquitin chains from an overlapping set of signaling molecules. In B cells, A20 deficiency results in hyperactivity, loss of immune homeostasis, inflammation, and autoimmunity. The reported consequences of CYLD deficiency are controversial, ranging from an absence of effects to dramatic B cell hyperplasia. These differences could be due to varying compensation for the loss of CYLD function by A20. Therefore, to explore potential overlapping physiological functions between A20 and CYLD, we generated and characterized A20/CYLD double-deficient B cells. Interestingly, the lack of both A20 and CYLD did not exacerbate the developmental defects and hyperresponsive activity of A20-deficient B cells. In addition, the extent of B cell activation after in vitro stimulation with anti-CD40, LPS, and CpG was comparable in B cells lacking A20/CYLD and A20 alone. However, in response to BCR cross-linking, we observed small but reproducible additive effects of the lack of A20 and CYLD. Taken together, our results demonstrate that A20 and CYLD do not share significant functions during B cell development and activation.

B cells lacking the tumor suppressor TNFAIP3/A20 display impaired differentiation and hyperactivation and cause inflammation and autoimmunity in aged mice.

The ubiquitin-editing enzyme A20/TNFAIP3 is essential for controlling signals inducing the activation of nuclear factor-κB transcription factors. Polymorphisms and mutations in the TNFAIP3 gene are linked to various human autoimmune conditions, and inactivation of A20 is a frequent event in human B-cell lymphomas characterized by constitutive nuclear factor-κB activity. Through B cell-specific ablation in the mouse, we show here that A20 is required for the normal differentiation of the marginal zone B and B1 cell subsets. However, loss of A20 in B cells lowers their activation threshold and enhances proliferation and survival in a gene-dose-dependent fashion. Through the expression of proinflammatory cytokines, most notably interleukin-6, A20-deficient B cells trigger a progressive inflammatory reaction in naive mice characterized by the expansion of myeloid cells, effector-type T cells, and regulatory T cells. This culminates in old mice in an autoimmune syndrome characterized by splenomegaly, plasma cell hyperplasia, and the presence of class-switched, tissue-specific autoantibodies.

Genome scanning of Amazonian Plasmodium falciparum shows subtelomeric instability and clindamycin-resistant parasites.

Here, we fully characterize the genomes of 14 Plasmodium falciparum patient isolates taken recently from the Iquitos region using genome scanning, a microarray-based technique that delineates the majority of single-base changes, indels, and copy number variants distinguishing the coding regions of two clones. We show that the parasite population in the Peruvian Amazon bears a limited number of genotypes and low recombination frequencies. Despite the essentially clonal nature of some isolates, we see high frequencies of mutations in subtelomeric highly variable genes and internal var genes, indicating mutations arising during self-mating or mitotic replication. The data also reveal that one or two meioses separate different isolates, showing that P. falciparum clones isolated from different individuals in defined geographical regions could be useful in linkage analyses or quantitative trait locus studies. Through pairwise comparisons of different isolates we discovered point mutations in the apicoplast genome that are close to known mutations that confer clindamycin resistance in other species, but which were hitherto unknown in malaria parasites. Subsequent drug sensitivity testing revealed over 100-fold increase of clindamycin EC(50) in strains harboring one of these mutations. This evidence of clindamycin-resistant parasites in the Amazon suggests that a shift should be made in health policy away from quinine + clindamycin therapy for malaria in pregnant women and infants, and that the development of new lincosamide antibiotics for malaria should be reconsidered.

Dynamics of malaria drug resistance patterns in the Amazon basin region following changes in Peruvian national treatment policy for uncomplicated malaria.

Monitoring changes in the frequencies of drug-resistant and -sensitive genotypes can facilitate in vivo clinical trials to assess the efficacy of drugs before complete failure occurs. Peru changed its national treatment policy for uncomplicated malaria to artesunate (ART)-plus-mefloquine (MQ) combination therapy in the Amazon basin in 2001. We genotyped isolates collected in 1999 and isolates collected in 2006 to 2007 for mutations in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes, multidrug resistance gene 1 (Pfmdr-1), the chloroquine (CQ) resistance transporter gene (Pfcrt), and the Ca(2+) ATPase gene (PfATP6); these have been shown to be involved in resistance to sulfadoxine-pyrimethamine (SP), MQ, CQ, and possibly ART, respectively. Microsatellite haplotypes around the Pfdhfr, Pfdhps, Pfcrt, and Pfmdr-1 loci were also determined. There was a significant decline in the highly SP resistant Pfdhfr and Pfdhps genotypes from 1999 to 2006. In contrast, a CQ-resistant Pfcrt genotype increased in frequency during the same period. Among five different Pfmdr-1 allelic forms noted in 1999, two genotypes increased in frequency while one genotype decreased by 2006. We also noted previously undescribed polymorphisms in the PfATP6 gene as well as an increase in the frequency of a deletion mutant during this period. In addition, microsatellite analysis revealed that the resistant Pfdhfr, Pfdhps, and Pfcrt genotypes have each evolved from a single founder haplotype, while Pfmdr-1 genotypes have evolved from at least two independent haplotypes. Importantly, this study demonstrates that the Peruvian triple mutant Pfdhps genotypes are very similar to those found in other parts of South America.