The composition of T cell subtypes in duodenal biopsies are altered in coeliac disease patients.

One of the hallmarks of Celiac disease (CD) is intraepithelial lymphocytosis in the small intestine. Until now, investigations to characterize the T cell subpopulations within the epithelial layer have not discriminated between the heterodimeric co-receptor molecule, CD8αß, and the possibly immunoregulatory CD8αα homodimer molecule. Besides TCRαß+ CD4+ cells, no other phenotypes have been shown to be gluten-reactive. Using flow cytometry on lymphocytes from duodenal biopsies, we determined that the number of B cells (CD3- CD19+) and the number of CD3+ CD4- CD8- double-negative (DN) T cells were elevated 6-7 fold in children with CD. We next isolated and quantified intraepithelial lymphocytes (IELs) from biopsies obtained from patients (both children and adults) with CD, potential CD and non-CD controls. Flow cytometric analysis of the duodenal T cell subpopulations was performed including the markers TCRαß, TCRγδ, CD4, CD8α and CD8ß. Proportions of γδ T cells and CD8αß+ cells among IELs were increased in CD patients, whereas proportions of CD4+ CD8αα+ and CD4+ single-positive T cells were decreased. Additionally, two gluten-reactive T cell lines (TCLs) derived from CD biopsies were analyzed for changes in proportions of T cell subsets before and after gluten stimulation. In a proliferation assay, dividing cells were tracked with carboxyfluorescein succinimidyl ester (CFSE), and both αß and γδ T cells proliferated in response to gluten. Changes in duodenal T cell subpopulations in potential CD patients followed the same pattern as for CD patients, but with less pronounced effect.

Treatment of both native and deamidated gluten peptides with an endo-peptidase from Aspergillus niger prevents stimulation of gut-derived gluten-reactive T cells from either children or adults with celiac disease.

Celiac disease (CD) is characterized by an inappropriate immunological reaction against gluten driven by gluten-specific CD4+ T cells. We screened 25 proteases and tested 10 for their potential to degrade gluten in vitro. Five proteases were further tested for their ability to prevent the proliferative response by a gluten-specific CD4+ T cell clone and seven gluten-reactive T cell lines to protease-digested gluten peptides. A proline-specific endo-peptidase from Aspergillus niger (AnP2) was particularly efficient at diminishing proliferation after stimulation with cleaved antigen, and could completely block the response against both native and deamidated gluten peptides. We found that AnP2 was efficient down to a 1:64 protease:substrate ratio (w:w). When AnP2 was tested in assays using seven gluten-reactive T cell lines from individual CD patients (three adults and four children), the response to gluten was diminished in all cases. Our study indicates a therapeutic benefit of AnP2 to CD patients.

[Biclonal or biphenotypic chronic lymphotic leukemia? An answer brought by the treatment]. / Leucémie lymphoïde chronique biclonale ou biphénotypique ? Une réponse par le traitement.

A 51-years-old patient with polyadenopathies presents a non monotypic chronic lymphocytic leukemia (CLL): lymphocytes B CD5+, CD23+, CD22-,CD79b dim, FMC7- express κ (71%) and λ (21%) light chains without coexpression. The caryotype shows two clones with one in evolution. Lymphoid clonality analysis by PCR (polymerase chain reaction) of the heavy and light chains genes of immunoglobulins shows two monoclonal rearrangements for each kind of chains. Lack of lymphoid cells sorting in function of the light chain doesn't allow us to conclude between a biclonal CLL and a biallelic monoclonal CLL. New adenopathies appearance in this patient, one year and a half after the end of his chemotherapy, leads to realisation of a new immunophenotyping: CLL is now monotypic with κ light chains expressed at 95%. In the new caryotype, only one clone is still present: it is the clone in evolution in the first caryotype and which presents additional abnormalities. This evolution allows us to assert the biclonality of the primitive CLL, the two clones evolving differently under treatment. In conclusion, the patient presented a biclonal CLL with one clone responding to treatment and the other one resistant.

Acute monocytic leukemia with coexpression of minor BCR-ABL1 and PICALM-MLLT10 fusion genes along with overexpression of HOXA9.

The t(9;22)(q34;q11) translocation occurs in chronic myeloid leukemia (CML) and adult B-cell acute lymphoblastic leukemia (ALL), leading to fusion of BCR to ABL1 and constitutive activation of ABL1 tyrosine kinase activity. The main BCR-ABL1 breakpoints result in P190 BCR-ABL1 or P210 BCR-ABL1 fusion proteins. The latter is found in almost all cases of CML and in one third of the cases of t(9;22)-positive adult B-ALL. P190 BCR-ABL1 is found in the remaining two thirds of t(9;22)-positive adult B-ALL cases but only exceptionally in CML. We describe here the first case of t(9;22)(q34;q11) associated with t(10;11)(p13;q14) in acute monocytic leukemia. The recurrent t(10;11)(p13;q14) translocation, usually found in acute myeloid leukemia (AML) and T-ALL, merges PICALM to MLLT10. RT-PCR enabled identification of PICALM-MLLT10 and BCR-ABL1 e1-a2 fusion transcripts; in the context of chronic and acute myeloid leukemia, the latter usually has a monocytic presentation. We also identified overexpression of HOXA9, a gene essential to myeloid differentiation that is expressed in PICALM-MLLT10 and MLL-rearranged acute leukemias. This case fits with and extends a recently proposed multistage AML model in which constitutive activation of tyrosine kinases by mutations (BCR-ABL1) are associated with deregulation of transcription factors central to myeloid differentiation (HOXA9 secondary to PICALM-MLLT10).

Real-time PCR for chloroquine sensitivity assay and for pfmdr1-pfcrt single nucleotide polymorphisms in Plasmodium falciparum.

Plasmodium falciparum drug resistance is a major problem in malaria endemic areas. Molecular markers and in vitro tests have been developed to study and monitor drug resistance. However, none, used alone, can provide sufficient data concerning the level of drug resistance and to issue precise guidelines for drug use policies in endemic areas. We propose real-time PCR for the simultaneous detection of pfcrt and pfmdr1 genes mutations and to determine the half-maximal inhibitory response (IC(50)) of antimalarial drug. Using hybridization probes and SybrGreen technology on LightCycler instrument, point mutations of pfcrt and pfmdr1 genes have been successfully detected in 161 human blood samples and determination of IC values was applied to chloroquine-sensitive and chloroquine-resistant strains. Moreover, mixed infections caused by P. falciparum clones with wild-type or mutant alleles could be efficiency separated. The aim of this study was not to provide definitive data concerning the rate of mutations in an endemic area, but to describe a powerful method allowing the quantification of DNA for IC(50) determination and the detection of major pfmdr1 and pfcrt mutations.

Simultaneous identification of the four human Plasmodium species and quantification of Plasmodium DNA load in human blood by real-time polymerase chain reaction.

The incidence of imported malaria cases in travellers returning from endemic areas has considerably increased over the last few years. The microscopical examination of stained blood films is the gold standard method to confirm clinical suspicion of malaria but diagnosis is difficult in the case of mixed infections, low-grade parasitaemia, or forms altered by uncompleted treatment. We have developed a real-time polymerase chain reaction (PCR) for the simultaneous identification of the 4 human Plasmodium spp. and quantification of Plasmodium DNA in human blood. The rapid turnaround and reduction in the risk of PCR product carryover are major advantages compared with conventional PCR. In combination with conventional tests, this method could be a powerful tool for the diagnosis of malaria infections among travellers from endemic areas and during the follow-up of patients in reference centres involved in travel and tropical medicine. Quantitative real-time PCR could also be used for the follow-up of patients during drug resistance studies managed by national malaria programmes, the testing of new drugs, and vaccine trials.