5 research outputs found
PTEN transcript variants caused by illegitimate splicing in âagedâ blood samples and EBV-transformed cell lines
PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. Mutations occur in either heritable or sporadic fashion. Sequencing of cDNA from patients and normal individuals often reveals splicing variants (SVs) of PTEN, some of which are non-mutation related. To investigate whether these SVs were the result of illegitimate splicing (a general decrease of fidelity in splicing site selection in âagedâ samples), we tested âagedâ blood from individuals who had normal PTEN transcripts in their âfreshâ mononuclear cells. Blood from 20 normal individuals was collected and split into two aliquots. Total RNA and DNA were extracted immediately (âfreshâ) and 48Â h later (âagedâ), respectively. Using RT-PCR, subcloning and sequencing, we found seven types of SVs. No mutation was detected in the related intronâexon flanking region in genomic DNA in either âfreshâ or âagedâ samples. Some of the SVs were also consistently present in both the âfreshâ and âagedâ EBV-transformed lymphoblastoid cells from six normal individuals. Western blot data indicated that the PTEN protein level (in full length) was not altered in the âfreshâ EBV-transformed lymphoblastoid cells with SVs. In conclusion, our data demonstrate that PTEN illegitimate splicing often occurs in âagedâ blood and EBV-transformed lymphoblastoid cells. Therefore, it is critical to note the time point of RNA extraction when investigating for PTEN aberrant transcripts. We hope that our data will increase awareness about the sample status, because gene expression data may be potentially flawed from âagedâ samples, particularly when dealing with clinical samples
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Antibody, but not Bâcellâdependent antigen presentation, plays an essential role in preventing Chlamydia systemic dissemination in mice
The obligate intracellular bacterium Chlamydia trachomatis causes the most prevalent bacterial sexually transmitted infection worldwide. CD4 T cells play a central role in the protective immunity against Chlamydia female reproductive tract (FRT) infection, while B cells are thought to be dispensable for resolution of primary Chlamydia infection in mouse models. We recently reported an unexpected requirement of B cells in local Chlamydia-specific CD4 T-cell priming and bacterial containment within the FRT. Here, we sought to tackle the precise effector function of B cells during Chlamydia primary infection. Using mixed bone marrow chimeras that lack B-cell-dependent Ag presentation (MHCIIB-/- ) or devoid of circulating antibodies (AID-/- Ă ÎŒS-/- ), we show that Chlamydia-specific CD4 T-cell expansion does not rely on Ag presentation by B cells. Importantly, we demonstrate that antibody, but not B-cell-dependent Ag presentation, is required for preventing systemic bacterial dissemination following Chlamydia FRT infection
Bortezomib down-regulates the cell-surface expression of HLA class I and enhances natural killer cellâmediated lysis of myeloma
Human leukocyte antigen class I molecules expressed by tumor cells play a central role in the regulation of natural killer (NK) cellâmediated immune responses. The proteasome inhibitor bortezomib has demonstrated significant activity in multiple myeloma (MM). We hypothesized that treatment of MM with bortezomib results in the reduction of cell-surface expression of class I and thereby sensitizes MM to NK cellâmediated lysis. Here we report that bortezomib down-regulates class I in a time- and dose-dependent fashion on all MM cell lines and patient MM cells tested. Downregulation of class I can also be induced in vivo after a single dose of 1.0 mg/m2 bortezomib. Bortezomib significantly enhances the sensitivity of patient myeloma to allogeneic and autologous NK cellâmediated lysis. Further, the level of decrease in class I expression correlates with increased susceptibility to lysis by NK cells. Clinically relevant bortezomib concentrations do not affect NK-cell function. Our findings have clear therapeutic implications for MM and other NK cellâsensitive malignancies in the context of both allogeneic and autologous adoptively transferred NK cells