Fluctuation of BCR-ABL1 qPCRIS level beyond 0.1%IS after stopping tyrosine kinase inhibitor in chronic myeloid leukaemia patients with deep molecular response for at least two years.

Fluctuation of BCR-ABL1 real-time quantitative polymerase chain reaction in International Scale (qPCRIS) level below major molecular response (MMR) (0.1%IS) is a known phenomenon after stopping tyrosine kinase inhibitor (TKI) in chronic myeloid leukaemia (CML) patients who are attempting treatment free remission (TFR). We report here four cases of fluctuation beyond MMR during conduct of a Malaysia Stop TKI Trial (MSIT) to examine the validity of the commonly used relapse criterion - loss of MMR for one reading - aiming to provide evidence in setting relapse criteria for future CML patients who want to attempt TFR.

DNA barcoding of freshwater fishes and the development of a quantitative qPCR assay for the species-specific detection and quantification of fish larvae from plankton samples.

The barcoding of mitochondrial cytochrome c oxidase subunit 1 (coI) gene was amplified and sequenced from 16 species of freshwater fishes found in Lake Wivenhoe (south-eastern Queensland, Australia) to support monitoring of reservoir fish populations, ecosystem function and water health. In this study, 630-650 bp sequences of the coI barcoding gene from 100 specimens representing 15 genera, 13 families and two subclasses of fishes allowed 14 of the 16 species to be identified and differentiated. The mean ± s.e. Kimura 2 parameter divergence within and between species was 0.52 ± 0.10 and 23.8 ± 2.20% respectively, indicating that barcodes can be used to discriminate most of the fish species accurately. The two terapontids, Amniataba percoides and Leiopotherapon unicolor, however, shared coI DNA sequences and could not be differentiated using this gene. A barcoding database was established and a qPCR assay was developed using coI sequences to identify and quantify proportional abundances of fish species in ichthyoplankton samples from Lake Wivenhoe. These methods provide a viable alternative to the time-consuming process of manually enumerating and identifying ichthyoplankton samples.

Identification of seagrasses in the gut of a marine herbivorous fish using DNA barcoding and visual inspection techniques.

Traditional visual diet analysis techniques were compared with DNA barcoding in juvenile herbivorous rabbitfish Siganus fuscescens collected in Moreton Bay, Australia, where at least six species of seagrass occur. The intergenic spacer trnH-psbA, suggested as the optimal gene for barcoding angiosperms, was used for the first time to identify the seagrass in fish guts. Four seagrass species and one alga were identified visually from gut contents; however, there was considerable uncertainty in visual identification with 38 of 40 fish having unidentifiable plant fragments in their gut. PCR and single-strand conformational polymorphism (SSCP) were able to discriminate three seagrass families from visually cryptic gut contents. While effective in identifying cryptic gut content to family level, this novel method is likely to be most efficient when paired with visual identification techniques.

In vivo behavior of peptide-specific T cells during mucosal tolerance induction: antigen introduced through the mucosa of the conjunctiva elicits prolonged antigen-specific T cell priming followed by anergy.

The mucosa of the conjunctiva is an important site of entry for environmental Ags as well as Ags emanating from the eye itself. However, very little is known about T cell recognition of Ag introduced through this important mucosal site. We have characterized the in vivo process of CD4 T cell recognition of Ag delivered via the conjunctival mucosa. Application of soluble OVA to the conjunctiva of BALB/c mice induced potent T cell tolerance. APC-presenting OVA peptide in vivo was only found in the submandibular lymph node and not in other lymph nodes, spleen, or nasal-associated lymphoid tissue. Similarly, in TCR transgenic DO11. 10 adoptive transfer mice, OVA-specific CD4+ T cell clonal expansion was only observed in the submandibular lymph node following conjunctival application of peptide. These experiments thus define a highly specific lymphatic drainage pathway from the conjunctiva. OVA-specific T cell clonal expansion peaked at day 3 following initiation of daily OVA administration and gradually declined during the 10-day treatment period, but remained elevated compared with nontreated adoptive transfer mice. During this period, the T cells expressed activation markers, and proliferated and secreted IL-2 in vitro in response to OVA stimulation. In contrast, these cells were unable to clonally expand in vivo, or proliferate in vitro following a subsequent OVA/CFA immunization. These results suggest that Ag applied to a mucosal site can be efficiently presented in a local draining lymph node, resulting in initial T cell priming and clonal expansion, followed by T cell anergy.

Peptide-specific T cell clonal expansion in vivo following immunization in the eye, an immune-privileged site.

To visualize the primary antigen-specific T cell response to Ag introduced into the eye, we have used an adoptive transfer system in which a limiting number of OVA peptide (323-339)-specific T cells from a TCR-transgenic mouse were transferred into nonirradiated, syngeneic recipients and then tracked in vivo by staining for FACS analysis or immunohistochemistry with the clonotypic mAb KJ1-26. Following posterior chamber injection of Ag, KJ1-26+ cells accumulated primarily in the draining, submandibular lymph node (LN) within 3 days. Although reduced in number, by day 6 these cells were primarily in the paracortical regions and were able to proliferate and secrete IL-2 in response to Ag stimulation. In contrast, following i.v. injection of Ag, the KJ1-26+ cells accumulated in the paracortical regions of the LN to a comparable degree, but did not proliferate or secrete IL-2. The day 3 accumulation of KJ1-26+ cells in the submandibular LN was inhibited if the eye was removed within 5 h after injection of Ag. In the spleen, foci of KJ1-26+ cells were observed in the periarteriolar lymphoid sheaths at day 3; these were not observed to the same degree following other forms of immunization. These results demonstrate that the submandibular LN is the primary site for early clonal expansion of antigen-specific T cells following intraocular Ag administration and that these cells show changes consistent with immunity rather than tolerance.