Development of a novel monoclonal antibody that binds to most HLA-A allomorphs in a conformation-dependent yet peptide-promiscuous fashion.

Specificity analyses of peptide binding to human leukocyte antigen (HLA)-A molecules have been hampered due to a lack of proper monoclonal antibodies (mAbs) for certain allomorphs, such as the prevalent HLA-A1 for Caucasians and HLA-A11 for Asians. We developed a mAb that recognizes a conformational epitope common to most HLA-A allomorphs. The mAb, named A-1, does not discriminate peptides by amino acid sequences, making it suitable for measuring peptide binding. A stabilization assay using TAP-deficient cell lines and A-1 was developed to investigate the specificity of peptide binding to HLA-A molecules. Regarding the evolution of HLA-A genes, the A-1 epitope has been conserved among most HLA-A allomorphs but was lost when the HLA-A gene diversified into the HLA-A*32, HLA-A*31, and HLA-A*33 lineages together with HLA-A*29 after bifurcating from the HLA-A*25 and HLA-A*26 branchs. The establishment of A-1 is expected to help researchers investigate the peptide repertoire and develop computational tools to identify cognate peptides. Since no HLA-A locus-specific mAb has been available, A-1 will also be useful for analyzing the locus-specific regulation of the HLA gene expression.

Rapid production of antigen-specific monoclonal antibodies from a variety of animals.

BACKGROUND: Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. RESULTS: We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. CONCLUSIONS: Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

O-acetylesterase activity of Bifidobacterium bifidum sialidase facilities the liberation of sialic acid and encourages the proliferation of sialic acid scavenging Bifidobacterium breve.

Bifidobacterium bifidum possesses two extracellular sialidases (SiaBb1 and SiaBb2) that release free sialic acid from mucin sialoglycans, which can be utilized via cross-feeding by Bifidobacterium breve that, otherwise, is prevented from utilizing this nutrient source. Modification of sialic acids with O-acetyl esters is known to protect mucin glycans from degradation by bacterial sialidases. Compared to SiaBb2, SiaBb1 has an additional O-acetylesterase (Est) domain. We aimed to elucidate the role of the SiaBb1 Est domain from B. bifidum in sialic acid cross-feeding within Bifidobacterium. Pre-treatment of mucin secreted from bovine submaxillary glands (BSM) using His6 -tagged-Est and -SiaBb2 released a higher amount of sialic acid compared to the pre-treatment by His6 -SiaBb2. Growth of B. breve increased with an increase in nanE expression when supplemented with both His6 -Est- and His6 -SiaBb2-treated BSM. These results indicate that the esterase activity of the SiaBb1 Est domain enhances the efficiency of SiaBb2 to cleave sialic acid from mucin. This free sialic acid can be utilized by coexisting sialic acid scavenging B. breve via cross-feeding. Here, we provide the molecular mechanism underlying the unique sialoglycan degradation property of B. bifidum which is mediated by the complementary activities of SiaBb1 and SiaBb2 in the context of sialic acid cross-feeding.

Minimum length of homology arms required for effective Red/ET recombination.

The original protocol of Red/ET recombination requires 50-bp sequence homology with target vector on both sides of the DNA fragment. To make it more cost effective, we investigated to determine the minimal length of homology required for the system to work. We found that a homology of 9-bp was sufficient to achieve homologous recombination with more than 50% efficiency.

Cells of origin of squamous epithelium, dysplasia and cancer in the head and neck region after bone marrow transplantation.

Secondary solid tumors that occur after hematopoietic stem cell transplantation (HSCT) are late complications of HSCT. Previously, secondary solid tumors were considered to be recipient-derived cells because transplanted cells do not contain epithelial cells. Recently, however, not only donor­derived epithelial cells but also donor-derived secondary solid tumors have also been reported in mice and humans. It means that circulating bone marrow-derived stem cells (BMDCs) including hematopoietic stem cells include the stem cells of many tissue types and the precancerous cells of many solid tumors. In most reports of donor-derived secondary solid tumors, however, tumors contained a low proportion of BMDC-derived epithelial cells in mixed solid tumor tissues. To our knowledge, there are only five known cases of completely donor-derived tumor tissues, i.e., four oral SCCs and a pharyngeal SCC. In this study, we analyzed five human clinical samples of solid tumors, i.e., two esophageal squamous cell carcinomas (SCCs), two oral SCCs and a tongue carcinoma. In the oral and tongue, completely donor-derived tissues were not observed, but in esophagus a completely donor-derived esophageal epidermis and SCC were observed for the first time. In addition, in another esophageal SCC patient, a completely donor-derived dysplasia region of esophageal epidermis was observed near recipient-derived SCC. This study suggests that BMDC-derived cells include the stem cells of esophageal epidermis and the precancerous cells of esophageal SCC and can differentiate into esophageal epithelium and esophageal SCC.

Reduced level of the BCL11B protein is associated with adult T-cell leukemia/lymphoma.

BACKGROUND: Adult T-cell leukemia/lymphoma (ATLL) develops in a small proportion of human T-cell leukemia virus type I (HTLV-I)-infected individuals. However, the mechanism by which HTLV-I causes ATLL has not been fully elucidated. To provide fundamental insights into the multistep process of leukemogenesis, we have mapped the chromosomal abnormalities in 50 ATLL cases to identify potential key regulators of ATLL. RESULTS: The analysis of breakpoints in one ATLL case with the translocations t(14;17)(q32;q22-23) resulted in the identification of a Kruppel zinc finger gene, BCL11B, which plays a crucial role in T-cell development. Among the 7 ATLL cases that we examined by immunofluorescence analysis, 4 displayed low and one displayed moderate BCL11B signal intensities. A dramatically reduced level of the BCL11B protein was also found in HTLV-I-positive T-cell lines. The ectopic expression of BCL11B resulted in significant growth suppression in ATLL-derived cell lines but not in Jurkat cells. CONCLUSIONS: Our genetic and functional data provide the first evidence that a reduction in the level of the BCL11B protein is a key event in the multistep progression of ATLL leukemogenesis.

HELIOS-BCL11B fusion gene involvement in a t(2;14)(q34;q32) in an adult T-cell leukemia patient.

To provide fundamental insights into the leukemogenesis of adult T-cell leukemia/lymphoma (ATLL), we performed a molecular analysis of the chromosomal abnormalities in one ATLL case with a novel reciprocal translocation: t(2;14)(q34;q32). Using fluorescence in situ hybridization with cosmid probes derived from the 14q32 region, we characterized the rearranged 14q32 allele. Molecular cloning of the breakpoint revealed that the reciprocal translocation fused the 5' proximal region of the B-cell lymphoma 11B (BCL11B) gene segment (on 14q32) to the third intron of the HELIOS gene (on 2q34). Reverse transcription-polymerase chain reaction analysis of the leukemia cells revealed that a substantial level of the HELIOS-BCL11B fusion mRNA was expressed relative to the level of wild-type (WT)-BCL11B derived from the intact allele. In contrast, an aberrant HELIOS isoform was detected at a low level of expression compared to the expression of normal HELIOS isoforms. Functional analysis of the HELIOS-BCL11B fusion protein revealed reduced transcriptional suppression activity compared to that of the WT-BCL11B due to the loss of the N-terminal friend of GATA-repression motif, which functions as a metastasis-associated protein 2 binding site. We also found abnormal subnuclear localization of the ectopically expressed fusion protein compared to the localization of WT-BCL11B to subnuclear speckles in HEK293T cells. Our results suggest that dysfunction of the BCL11B gene plays an important role in the development of ATLL.