High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development.

Large oncosomes in human prostate cancer tissues and in the circulation of mice with metastatic disease.

Oncosomes are tumor-derived microvesicles that transmit signaling complexes between cell and tissue compartments. Herein, we show that amoeboid tumor cells export large (1- to 10-μm diameter) vesicles, derived from bulky cellular protrusions, that contain metalloproteinases, RNA, caveolin-1, and the GTPase ADP-ribosylation factor 6, and are biologically active toward tumor cells, endothelial cells, and fibroblasts. We describe methods by which large oncosomes can be selectively sorted by flow cytometry and analyzed independently of vesicles <1 μm.

Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection.

Phenotypes that might otherwise reveal a gene's function can be obscured by genes with overlapping function. This phenomenon is best known within gene families, in which an important shared function may only be revealed by mutating all family members. Here we describe the 'green monster' technology that enables precise deletion of many genes.

APRIL-deficient mice have normal immune system development.

APRIL (a proliferation-inducing ligand) is a member of the tumor necrosis factor (TNF) superfamily. APRIL mRNA shows high levels of expression in tumors of different origin and a low level of expression in normal cells. APRIL shares two TNF receptor family members, TACI and BCMA, with another TNF homolog, BLyS/BAFF.

Development of a frozen cell array as a high-throughput approach for cell-based analysis.

Recent advances in molecular biology, human genetics, and functional genomics tremendously increase the number of molecular targets available for potential therapeutic and diagnostic use. To complement DNA array data, cost-efficient high-throughput technologies providing reliable information at the protein level need to be developed. Here we describe the generation of a frozen cell array that required the use of single cell suspensions and could serve various applications such as the analysis of specific antibody or ligand binding to a large panel of different cell types.

Prostate stem cell antigen as therapy target: tissue expression and in vivo efficacy of an immunoconjugate.

We conducted an expression analysis of prostate stem cell antigen (PSCA)in normal urogenital tissues, benign prostatic hyperplasia (n = 21), prostatic intraepithelial neoplasia (n = 33), and primary (n = 137) and metastatic (n = 42) prostate adenocarcinoma, using isotopic in situ hybridization on tissue microarrays. In normal prostate, we observe PSCA expression in the terminally differentiated, secretory epithelium; strong expression was also seen in normal urothelium. Forty-eight percent of primary and 64% of metastatic prostatic adenocarcinomas expressed PSCA RNA.

Tumor-cell resistance to death receptor--induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax.

The importance of Bax for induction of tumor apoptosis through death receptors remains unclear. Here we show that Bax can be essential for death receptor--mediated apoptosis in cancer cells. Bax-deficient human colon carcinoma cells were resistant to death-receptor ligands, whereas Bax-expressing sister clones were sensitive.

Vascular endothelial-junctional adhesion molecule (VE-JAM)/JAM 2 interacts with T, NK, and dendritic cells through JAM 3.

Screening expressed sequence tag databases for endothelial-specific homologs to human junctional adhesion molecule (JAM) and A33-Ag, we identified a protein of 298 aa that represents the recently described vascular endothelial-JAM (VE-JAM)/JAM 2. We confirmed VE-JAM/JAM 2 expression to be restricted to the high endothelial venule of tonsil and lymph nodes, and we further expanded the localization to the endothelium of arterioles in and around inflammatory and tumor foci. In our functional characterizations of VE-JAM/JAM 2, we discovered that it can function as an adhesive ligand for the T cell line J45 and can interact with GM-CSF/IL-4-derived peripheral blood dendritic cells, circulating CD56(+) NK cells, circulating CD56(+)CD3(+) NK/T cells, and circulating CD56(+)CD3(+)CD8(+) cytolytic T cells.

Forced expression of murine IL-17E induces growth retardation, jaundice, a Th2-biased response, and multiorgan inflammation in mice.

IL-17 is a proinflammatory cytokine, and its in vivo expression induces neutrophilia in mice. IL-17E is a recently described member of an emerging family of IL-17-related cytokines. IL-17E has been shown to bind IL-17Rh1, a protein distantly related to the IL-17R, suggesting that IL-17E probably possesses unique biological functions.

Mental stimulation increases circulating CD4-positive T lymphocytes: a preliminary study.

To stimulate the dorsolateral frontal cortex, 12 healthy, adult, human females played contract bridge for 1.5 h between initial and final blood sample collections. Flow cytometric analyses of samples, performed in triplicate, showed a significant increase in CD4-positive T lymphocytes.

Death receptor recruitment of endogenous caspase-10 and apoptosis initiation in the absence of caspase-8.

Caspase-8 is believed to play an obligatory role in apoptosis initiation by death receptors, but the role of its structural relative, caspase-10, remains controversial. Although earlier evidence implicated caspase-10 in apoptosis signaling by CD95L and Apo2L/TRAIL, recent studies indicated that these death receptor ligands recruit caspase-8 but not caspase-10 to their death-inducing signaling complex (DISC) even in presence of abundant caspase-10. We characterized a series of caspase-10-specific antibodies and found that certain commercially available antibodies cross-react with HSP60, shedding new light on previous results.

Overexpression of the human 2-oxoglutarate carrier lowers mitochondrial membrane potential in HEK-293 cells: contrast with the unique cold-induced mitochondrial carrier CGI-69.

Using differential mRNA expression analysis, a previously uncharacterized gene was found to be up-regulated 2-fold in brown adipose tissue (BAT) of mice exposed to cold (4 degrees C) for 48 h. Contig and homology analysis revealed that the gene represents the murine orthologue to a sequence from a public database encoding a putative human protein (CGI-69). The presence of mitochondrial carrier domains in the human protein, its transmembrane topology and cold-induction of the mouse CGI-69 gene in BAT prompted an analysis of the idea that CGI-69 may represent a new uncoupling protein (UCP) functional homologue.

Characterization of novel UCP5/BMCP1 isoforms and differential regulation of UCP4 and UCP5 expression through dietary or temperature manipulation.

Mitochondrial uncoupling proteins have been implicated in the maintenance of metabolic rate and adaptational thermoregulation. We recently reported the identification of a brain-specific mitochondrial uncoupling protein homologue, UCP4. Here we characterized another newly described member of the uncoupling protein family, termed UCP5 (also called BMCP1).

Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5.

Fas (APO-1/CD95) and tumor necrosis factor receptor 1 (TNFR1) trigger apoptosis by recruiting the apoptosis initiator caspase-8 through the adaptor FADD. Fas binds FADD directly, whereas TNFR1 binds FADD indirectly, through TRADD. TRADD alternatively recruits the NF-kappaB-inducing adaptor RIP.

Selective modulation of human natural killer cells in vivo after prolonged infusion of low dose recombinant interleukin 2.

The immunologic consequences of prolonged infusions of rIL-2 in doses that produce physiologic serum concentrations of this cytokine were investigated. rIL-2 in doses of 0.5-6.

Follicular dendritic cells inhibit human B-lymphocyte proliferation.

In germinal centers, B lymphocytes are intimately associated with follicular dendritic cells (FDCs). It has been hypothesized that FDCs are involved in the regulation of B-cell growth and differentiation through cell-cell interactions. In this study, highly enriched preparations of FDCs were isolated by cell sorting using the FDC restricted monoclonal antibody DRC-1.

Clinical and immunologic effects of prolonged infusion of low-dose recombinant interleukin-2 after autologous and T-cell-depleted allogeneic bone marrow transplantation.

Bone marrow transplantation (BMT) can produce prolonged clinical remission in some patients with hematologic malignancies. Unfortunately, disease relapse may occur despite BMT. Studies in animal models and clinical experience have provided evidence that immunologic factors play an important role in preventing relapse post-BMT.

Differential responses to interleukin 2 define functionally distinct subsets of human natural killer cells.

Human natural killer (NK) cells can be subdivided into two populations based on the density of cell surface CD56 antigen. The great majority (approximately 90%) of NK cells express CD56 at low levels (the CD56dim phenotype), whereas a small NK cell subset (approximately 10%) exhibits approximately fivefold greater density of surface CD56. Exposure to exogenous interleukin 2 (IL 2) induces tenfold greater proliferation of CD56bright cells compared to CD56dim lymphocytes, even though both subsets constitutively express similar levels of intermediate affinity IL 2 receptor (IL 2R) p75 chains.

Cell surface modulation of CD26 by anti-1F7 monoclonal antibody. Analysis of surface expression and human T cell activation.

In this paper, we examined in detail the ability of anti-1F7 to modulate 1F7 (CD26) surface expression as well as analyzed the functional relationship between the surface expression of CD3, CD2, and CD26 and human T cell activation. We showed that anti-1F7-induced modulation is an energy-dependent process that occurs via capping and internalization of the Ag-antibody complex. Although the recovery rate for Ag reexpression of 1F7 following optimal modulation is relatively delayed, reexpression of 1F7 is greatly accelerated following phorbol ester treatment.

Fatal liver disease associated with alpha 1-antitrypsin deficiency PiM1/PiMduarte.

A 53-yr-old man with a rare form of partial alpha 1-antitrypsin deficiency, PiM1/PiMduarte, died of endstage cirrhosis. Typical cytoplasmic alpha 1-antitrypsin globules were present in the hepatocyte cytoplasm. Initial protease inhibitor phenotyping on the patient was reported as normal PiM1 in more than one laboratory.

Splenic artery aneurysm-pancreatic duct fistula.

Massive hemorrhage as a sequela of chronic pancreatitis may originate from many sources. This report documents a rare case of communication between the pancreatic ductal system and a pseudoaneurysm of the splenic artery. The multidisciplinary management of this unusual case required the combined expertise of the endoscopist, radiologist and surgeon.