INTRODUCTIONSince undetectable BCR-ABL mRNA transcription does not always indicate eradication of the Ph+ CML clone and since transcriptionally silent Ph+ CML cells exist, quantitation by genomic PCR of bcr-abl genes can be clinically useful. Furthermore, hotspot mutations in the Abelson tyrosine kinase (ABLK) domain of the bcr-abl gene translocation in Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) cells confer resistance on the specific kinase blocking agent, STI571.

MATERIALS AND METHODSGenomic DNA from K562, CESS and patient CML cells were amplified using rapid cycle quantitative real-time polymerase chain reaction for the gene regions spanning the mutation hotspots. In assays for ABLK exons 4 or 6, exonic or intronic PCR primers were used.

RESULTSWe show that separation of cycle threshold (CT) values for log-fold amplicon quantification was 2.9 cycles for ABLK exon 4, and 3.8 cycles for exon 6 with rapid amplification times. K562 CML cells were found to have a approximately 2 log-fold ABLK gene amplification. In contrast, patient CML cells had CT differences of 2.2 for both exon, suggesting that there was no significant ABLK gene amplification. DNA sequencing confirmed that neither K562 nor patient CML cells contained ABLK hotspot mutations. Messenger RNA transcription analysis permitted the assessment of BCR-ABL transcription, which was qualitatively correlated to genomic amplification.

CONCLUSIONSThis novel Q-PCR assay was found to have high fidelity and legitimacy, and potentially useful for monitoring minimal residual disease, transcriptionally silent Ph+ CML cells, and bcr-abl gene amplification.

Chronic Disease ; Drug Resistance ; genetics ; Fusion Proteins, bcr-abl ; genetics ; Gene Amplification ; Genes, abl ; genetics ; Hematologic Neoplasms ; genetics ; Humans ; Leukemia, Myeloid ; genetics ; Mutation ; Protein-Tyrosine Kinases ; genetics ; RNA, Messenger ; Reverse Transcriptase Polymerase Chain Reaction

Interleukin-27 (IL-27) has a pleiotropic role either as a pro-inflammatory or anti-inflammatory cytokine in inflammatory related diseases. The role and involvement of IL-27 during malaria was investigated and the effects of modulating its release on the production of major inflammatory cytokines and the histopathological consequences in major affected organs during the infection were evaluated. Results showed that IL-27 concentration was significantly elevated throughout the infection but no positive correlation with the parasitaemia development observed. Augmentation of IL-27 significantly elevated the release of anti-inflammatory cytokine, IL-10 whereas antagonising and neutralising IL-27 produced the opposite. A significant elevation of pro-inflammatory cytokines (IFN-γ and IL-6) was also observed, both during augmentation and inhibition of IL-27. Thus, it is suggested that IL-27 exerts an anti-inflammatory activity in the Th1 type response by signalling the production of IL-10 during malaria. Histopathological examination showed sequestration of PRBC in the microvasculature of major organs in malarial mice. Other significant histopathological changes include hyperplasia and hypertrophy of the Kupffer cells in the liver, hyaline membrane formation in lung tissue, enlargement of the white and red pulp followed by the disappearance of germinal centre of the spleen, and tubular vacuolation of the kidney tissues. In conclusion, it is suggested that IL-27 may possibly acts as an anti-inflammatory cytokine during the infection. Modulation of its release produced a positive impact on inflammatory cytokine production during the infection, suggesting its potential in malaria immunotherapy, in which the host may benefit from its inhibition.