A Computer-Assisted Diagnostic Method for Accurate Detection of Early Nondisplaced Fractures of the Femoral Neck.

Nondisplaced femoral neck fractures are sometimes misdiagnosed by radiographs, which may deteriorate into displaced fractures. However, few efficient artificial intelligent methods have been reported. We developed an automatic detection method using deep learning networks to pinpoint femoral neck fractures on radiographs to assist physicians in making an accurate diagnosis in the first place. Our proposed accurate automatic detection method, called the direction-aware fracture-detection network (DAFDNet), consists of two steps, namely region-of-interest (ROI) segmentation and fracture detection. The first step removes the noise region and pinpoints the femoral neck region. The fracture-detection step uses a direction-aware deep learning algorithm to mark the exact femoral neck fracture location in the region detected in the first step. A total of 3840 femoral neck parts in anterior-posterior (AP) pelvis radiographs collected from the China Medical University Hospital database were used to test our method. The simulation results showed that DAFDNet outperformed the U-Net and DenseNet methods in terms of the IOU value, Dice value, and Jaccard value. Our proposed DAFDNet demonstrated over 94.8% accuracy in differentiating non-displaced Garden type I and type II femoral neck fracture cases. Our DAFDNet method outperformed the diagnostic accuracy of general practitioners and orthopedic surgeons in accurately locating Garden type I and type II fracture locations. This study can determine the feasibility of applying artificial intelligence in a clinical setting and how the use of deep learning networks assists physicians in improving correct diagnoses compared to the current traditional orthopedic manual assessments.

A Combination of FPU-Net and Feature Clustering Methods for Accurate Segmentation of Femoral Neck in Radiographic Diagnosis.

In this study, we develop an innovative method that assists computer-aided diagnosis in the determination process of the exact location of the femoral neck junction in plain radiographs. Our algorithm consists of two phases, i.e., coarse prediction and fine matching, which are implemented by supervised deep learning method and unsupervised clustering, respectively. In coarse prediction, standard masks are first produced by a specialist and trained in our proposed feature propagation network (FPU-Net) with supervised learning on the femoral neck dataset. In fine matching, the standard masks are first classified into different categories using our proposed three parameters with unsupervised learning. The predicted mask from FPU-Net is matched with each category of standard masks by calculating the values of intersection of union (IOU), and finally the predicted mask is substituted by the standard mask with the largest IOU value. A total of 4320 femoral neck parts in anterior-posterior (AP) pelvis radiographs collected from China Medical University Hospital database were used to test our method. Simulation results show that, on the one hand, compared with other segmentation methods, the method proposed in this paper has a larger IOU value and better suppression of noise outside the region of interest; on the other hand, the introduction of unsupervised learning for fine matching can help in the accurate localization segmentation of femoral neck images. Accurate femoral neck segmentation can assist surgeons to diagnose and reduce the misdiagnosis rate and burden.

Eradication of multidrug-resistant Acinetobacter baumannii from the respiratory tract with inhaled colistin methanesulfonate: a matched case-control study.

Repeated isolation of multidrug-resistant Acinetobacter baumannii (MDRAB) from respiratory secretions poses a great challenge for infection control. We conducted a retrospective case-control study to evaluate the efficacy and adverse effect of inhaled colistin methanesulfonate (CMS) in the eradication of MDRAB from the respiratory tract. Patients who were admitted to Taipei Veterans General Hospital between February 2009 and June 2010, had at least two sets of monomicrobial culture of MDRAB from respiratory secretions, and remained in hospital for at least 14 days after the first isolation of MDRAB (index day) were included. Patients who received intravenous CMS were excluded. Patients who received CMS inhalation for ≥ 3 days were selected as cases whereas the controls were matched for age and Acute Physiology and Chronic Health Evaluation II score. Thirty-nine cases and controls were identified. The duration of CMS inhalation was 10.9 ± 3.6 days. The use of inhaled CMS was the only independent factor associated with the eradication of MDRAB within 14 days after the index day (OR 266.33; 95% CI 11.26-6302.18, p <0.001), and shortened the duration of MDRAB recovery from the respiratory tract by 13.3 ± 1.45 days. The adverse effects were similar for both groups. The increase of colistin minimal inhibitory concentrations in the last isolate compared with the index isolate from the same patient did not differ between the two groups. In conclusion, our study demonstrated that inhaled CMS enhanced the eradication of MDRAB from the respiratory tract without significant clinical adverse effect or impact on colistin resistance.

Green fabrication of agar-conjugated Fe3O4 magnetic nanoparticles.

Magnetic nanoparticles are of great interest both for fundamental research and emerging applications. In the biomedical field, magnetite (Fe(3)O(4)) has shown promise as a hyperthermia-based tumor therapeutic. However, preparing suitable solubilized magnetite nanoparticles is challenging, primarily due to aggregation and poor biocompatibility. Thus methods for coating Fe(3)O(4) NPs with biocompatible stabilizers are required. We report a new method for preparing Fe(3)O(4) nanoparticles by co-precipitation within the pores of agar gel samples. Permeated agar gels were then dried and ground into a powder, yielding agar-conjugated Fe(3)O(4) nanoparticles. Samples were characterized using XRD, FTIR, TGA, TEM and SQUID. This method for preparing agar-coated Fe(3)O(4) nanoparticles is environmentally friendly, inexpensive and scalable.

Ezrin is a negative regulator of death receptor-induced apoptosis.

Ezrin links cortical actin filaments with the cell membrane, and has a critical role in many membrane-initiated events. Fas is directly associated with ezrin, but conflicting results have been reported for the involvement of ezrin in Fas-induced cell death. In this study we show that ezrin was associated with Fas in T cells before stimulation and was released shortly after Fas ligand (FasL) engagement. The knockdown of ezrin moderately increased Fas-triggered or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-triggered cell death in normal T lymphocytes and in H9 cells, but had no effect on death receptor-induced apoptosis in type II cells, such as Jurkat and CEM. Expression of a dominant-negative form of ezrin also led to an increased Fas-induced apoptosis in H9 cells. Ezrin deficiency did not affect the internalization of Fas after Fas ligation. Instead, an enhanced formation of death-inducing signaling complex (DISC) was observed in H9 cells with ezrin knockdown, leading to accelerated caspase-8 activation. Together, our results suggest that ezrin has a negative role in the recruitment of Fas into signaling complexes in type I T cells. Loss of ezrin likely removes the constraint imposed by ezrin and facilitates the assembly of death receptor complex in T cells.

Expression of the triggering receptor expressed on myeloid cells-1 mRNA in a heterogeneous infected population.

This study is to investigate the clinical utility of detection of peripheral blood triggering receptor expressed on myeloid cells (TREM)-1 mRNA as an early indicator of sepsis among critically ill patients and to compare the results of TREM-1 with those of C-reactive protein (CRP). A prospective, non-interventional study of 127 patients with at least two criteria of the systemic inflammatory response (SIRS) was performed. TREM-1 was assessed by real-time quantitative reverse transcription-polymerase chain reaction. The diagnosis of SIRS only was made in 41 patients (32%), and the diagnosis of sepsis was made in other 86 patients (68%). TREM-1 mRNA expression had the comparably discriminative value to differentiate the presence from the absence of infection, with an area under the receiver-operating characteristic curve (AUC) of 0.75 [95% confidence interval (95% CI), 0.67-0.84] than CRP [AUC, 0.72 (95% CI, 0.62-0.81)]. As an indicator of sepsis, a TREM-1 mRNA expression ratio cutoff value of 58.8 had a sensitivity of 72%, a specificity of 71%, a positive likelihood ratio of 2.5 and a negative likelihood ratio of 0.39. Furthermore, TREM-1 mRNA expression was selectively higher in septic patients caused by extracellular bacteria or fungi [112.4 (19.3-680.1)], than in those caused by intracellular bacteria or viruses [18.8 (7.6-53.0), p < 0.001]. There was no difference in plasma CRP levels between both septic groups (p = 0.782). TREM-1 and CRP are similar diagnostic markers of sepsis. The different ability of extracellular and intracellular pathogens to induce TREM-1 expression may provide a potential marker for differential diagnosis.

Elevated serum decoy receptor 3 with enhanced T cell activation in systemic lupus erythematosus.

Decoy receptor 3 (DcR3/TR6) is a decoy receptor for the Fas ligand (FasL) and can inhibit FasL-induced apoptosis. It has been reported recently that DcR3 can induce T cell activation via co-stimulation of T cells, suggesting that DcR3 may be involved in the pathophysiology of autoimmune diseases. This study aims to analyse the serum DcR3 in patients with systemic lupus erythematosus (SLE) and to investigate the role of DcR3 in the pathogenesis of SLE. Significantly elevated serum DcR3 was observed in SLE patients, and the mean serum DcR3 level was significantly higher for those with active disease [SLE disease activity index (SLEDAI) >/= 10] compared with that in patients with inactive disease (SLEDAI < 10). In addition to reducing activation-induced cell death in activated T cells via neutralization of the FasL, soluble DcR3-Fc enhanced T cell proliferation and increased interleukin-2 and interferon-gamma production via co-stimulation of T cells. Moreover, enhanced T cell reactivity to DcR3-induced co-stimulation was demonstrated in lymphocytes from patients with SLE, suggesting the elevated serum DcR3 may associate with enhanced T cell activation in vivo. These findings are the first to demonstrate that serum DcR3 concentrations are increased in SLE patients, and this may imply a possible role of DcR3 in the pathogenesis of SLE via enhanced T cell hyperreactivity and reduced apoptosis in activated T cells.

Molecular cloning and gene expression of the gonadotropin-releasing hormone receptor in the orange-spotted grouper, Epinephelus coioides.

The objective of this study was to investigate the molecular mechanisms of gonadotropin-releasing hormone receptor (GnRH-R) involved in the endocrine regulation of reproduction in the orange-spotted grouper, Epinephelus coioides. The full-length cDNA encoding GnRH-R type I was successfully cloned from the pituitary by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods in the grouper. The complete GnRH-R type I cDNA is 1607 bp, which includes an open reading frame of 1092 bp encoding a protein of 364 amino acids, a seven-alpha helix transmembrane domain, a N-terminal extracellular domain, and a C-terminal cytoplasmic domain. The expression of GnRH-R type I was found to be highest in the pituitary. An intramuscular injection of various GnRH types in vivo was attempted. The expression of GnRH-R type I was stimulated by a single injection of salmon GnRH, while in the case of chicken GnRH II treatment, the expression of GnRH-R type I was inhibited. This suggests that the action of chick GnRH II is probably enhanced through the GnRH receptor of different forms. Furthermore, none of them were expressed by an injection of seabream GnRH, and this is likely attributed to the injection dose being below the threshold level, and this remains to be further examined. In conclusion, GnRHs of various types are effective in stimulating the expression of gonadotropins through various forms of the GnRH-R, and multiple forms of the receptor gene likely exist in teleosts.

Involvement of norepinephrine in the hyperglycemic responses of the freshwater giant prawn, Macrobrachium rosenbergii, under cold shock.

Hyperglycemic response of freshwater giant prawn, Macrobrachium rosenbergii, under acute cold shock was investigated, and the involvement and stimulatory pathways of norepinephrine (NE) on induced-glycemia were further examined. Remarkable elevations in hemolymph glucose at comparable magnitude were observed in both intact and eyestalkless prawn under cold treatments, suggesting that hyperglycemic response of this species is not solely mediated through the actions of crustacean hyperglycemic hormone released from X-organ sinus gland complex on the target tissues, but NE is involved. Positive and significant correlations were noted between the hemolymph glucose titers and NE contents in both thoracic ganglia and the hemolymph, suggesting that NE plays a significant role in the hyperglycemic responses of this species under cold. Depressive effects of various adrenoceptor antagonists monitored in vivo and in vitro further suggest that the action of NE is primarily mediated through both alpha1- and beta1-adrenoceptors.

An integrated healthcare enterprise information portal and healthcare information system framework.

The paper presents an integrated, distributed Healthcare Enterprise Information Portal (HEIP) and Hospital Information Systems (HIS) framework over wireless/wired infrastructure at National Taiwan University Hospital (NTUH). A single sign-on solution for the hospital customer relationship management (CRM) in HEIP has been established. The outcomes of the newly developed Outpatient Information Systems (OIS) in HIS are discussed. The future HEIP blueprints with CRM oriented features: e-Learning, Remote Consultation and Diagnosis (RCD), as well as on-Line Vaccination Services are addressed. Finally, the integrated HEIP and HIS architectures based on the middleware technologies are proposed along with the feasible approaches. The preliminary performance of multi-media, time-based data exchanges over the wireless HEIP side is collected to evaluate the efficiency of the architecture.

Stearoyl-CoA desaturase expression and fatty acid composition in milkfish (Chanos chanos) and grass carp (Ctenopharyngodon idella) during cold acclimation.

Desaturation of fatty acids is an important adaptation mechanism for fish to maintain membrane fluidity under thermal stress. To comprehend the temperature adaptation mechanism in fish, we investigated the difference in the changes of stearoyl-CoA desaturase expression and fatty acid composition between milkfish and grass carp under cold acclimation. We find that in both fish the proportions of unsaturated fatty acids at 15 degrees C are all higher than those at 25 degrees C. In milkfish Delta(9)-desaturation index (ratios of 16:1/16:0 and 18:1/18:0) increases significantly in the beginning of cold acclimation at 15 degrees C and decreases afterward, but in grass carp it increases slightly in the beginning of cold acclimation followed by a sustained dramatic increase. Similarly, activity of stearoyl-CoA desaturase in milkfish increases significantly in the beginning, peaks at day 4, and then decreases constantly, but in grass carp it increases gradually in the first week, rises dramatically afterward, and then maintains a very high level. The change of stearoyl-CoA desaturase activity is parallel to the change of Delta(9)-desaturation index in both milkfish and grass carp, but it is one day earlier than Delta(9)-desaturation index in milkfish. The difference of adaptation capability between milkfish and grass carp under cold stress is further evidenced by RT-PCR and Northern blot analysis of stearoyl-CoA desaturase gene expression.

Decoy receptor 3 (DcR3) induces osteoclast formation from monocyte/macrophage lineage precursor cells.

Recent evidence indicates that the decoy receptor 3 (DcR3) of the TNF receptor superfamily, which initially though prevents cytokine responses of FasL, LIGHT and TL1A by binding and neutralization, can modulate monocyte function through reverse signaling. We show in this work that DcR3 can induce osteoclast formation from human monocytes, murine RAW264.7 macrophages, and bone marrow cells. DcR3-differentiated cells exhibit characteristics unique for osteoclasts, including polynuclear giant morphology, bone resorption, TRAP, CD51/61, and MMP-9 expression. Consistent with the abrogation of osteoclastogenic effect of DcR3 by TNFR-Fc, DcR3 treatment can induce osteoclastogenic cytokine TNF-alpha release through ERK and p38 MAPK signaling pathways. We conclude that DcR3 via coupling reverse signaling of ERK and p38 MAPK and stimulating TNF-alpha synthesis is a critical regulator of osteoclast formation. This action of DcR3 might play an important role in significant osteoclastic activity in osteolytic bone metastases.

cDNA nucleotide sequence coding for stearoyl-CoA desaturase and its expression in the zebrafish (Danio rerio) embryo.

A cDNA sequence of stearoyl-CoA desaturase (SCD) was determined from zebrafish (Danio rerio) and compared to the corresponding genes in several teleosts. Zebrafish SCD cDNA has a size of 1,061 bp, encodes a polypeptide of 325 amino acids, and shares 88, 85, 84, and 83% similarities with tilapia (Oreochromis mossambicus), grass carp (Ctenopharyngodon idella), common carp (Cyprinus carpio), and milkfish (Chanos chanos), respectively. This 1,061 bp sequence specifies a protein that, in common with other fatty acid desaturases, contains three histidine boxes, believed to be involved in catalysis. These observations suggested that SCD genes are highly conserved. In addition, an oligonucleotide probe complementary to zebrafish SCD mRNA was hybridized to mRNA of approximately 396 bases with Northern blot analysis. The Northern blot and RT-PCR analyses showed that the SCD mRNA was expressed predominantly in the liver, intestine, gill, and muscle, while a lower level was found in the brain. Furthermore, we utilized whole-mount in situ hybridization and real-time quantitative RT-PCR to identify expression of the zebrafish SCD gene at five different stages of development. This revealed that very high levels of transcripts were found in zebrafish at all stages during embryogenesis and early development.

Molecular cloning and sequence analysis of stearoyl-CoA desaturase in milkfish, Chanos chanos.

Stearoyl-CoA desaturase (EC 1.14.99.5) is a key enzyme in the biosynthesis of polyunsaturated fatty acids and the maintenance of the homeoviscous fluidity of biological membranes. The stearoyl-CoA desaturase cDNA in milkfish (Chanos chanos) was cloned by RT-PCR and RACE, and it was compared with the stearoyl-CoA desaturase in cold-tolerant teleosts, common carp and grass carp. Nucleotide sequence analysis revealed that the cDNA clone has a 972-bp open reading frame encoding 323 amino acid residues. Alignments of the deduced amino acid sequence showed that the milkfish stearoyl-CoA desaturase shares 79% and 75% identity with common carp and grass carp, and 63%-64% with other vertebrates such as sheep, hamsters, rats, mice, and humans. Like common carp and grass carp, the deduced amino acid sequence in milkfish well conserves three histidine cluster motifs (one HXXXXH and two HXXHH) that are essential for catalysis of stearoyl-CoA desaturase activity. However, RT-PCR analysis showed that stearoyl-CoA desaturase expression in milkfish is detected in the tissues of liver, muscle, kidney, brain, and gill, and more expression sites were found in milkfish than in common carp and grass carp. Phylogenic relationships among the deduced stearoyl-CoA desaturase amino acid sequence in milkfish and those in other vertebrates showed that the milkfish stearoyl-CoA desaturase amino acid sequence is phylogenetically closer to those of common carp and grass carp than to other higher vertebrates.

Enhanced proliferation and increased IFN-gamma production in T cells by signal transduced through TNF-related apoptosis-inducing ligand.

TNF-related apoptosis-inducing ligand (TRAIL, also called Apo2L), a novel member of TNF superfamily, induces apoptosis in transformed cell lines of diverse origin. TRAIL is expressed in most of the cells, and the expression is up-regulated in activated T cells. Four receptors for TRAIL have been identified, and there is complex interplay between TRAIL and TRAIL receptors in vivo. The actual biological function of TRAIL/TRAIL receptor is still not clear. Growing evidence has demonstrated that members of TNF superfamily transduce signals after engagement with their receptors. Cross-linking of TRAIL by plate-bound rTRAIL receptor, death receptor 4-Fc fusion protein enhanced T cell proliferation and increased IFN-gamma production in conjunction with immobilized suboptimal anti-CD3 stimulation in mouse splenocytes. The increase of T cell proliferation by death receptor 4-Fc was dose dependent, and this effect could be blocked by soluble rTRAIL proteins, indicating the occurrence of reverse signaling through TRAIL on T cell. The enhanced secretion of IFN-gamma mediated via TRAIL could be blocked by SB203580, a p38 mitogen-activated protein kinase-specific inhibitor. Thus, in addition to its role in inducing apoptosis by binding to the death receptors, TRAIL itself can enhance T cell proliferation after TCR engagement and signal the augmentation of IFN-gamma secretion via a p38-dependent pathway. This provides another example of reverse signaling by a member of TNF superfamily. In conclusion, our data suggest that TRAIL can itself transduce a reverse signal, and this may shed light on the biological function of TRAIL.

Expression of human Fas ligand on mouse beta islet cells does not induce insulitis but is insufficient to confer immune privilege for islet grafts.

Fas (CD95) and Fas ligand (FasL/CD95L) are involved in programmed cell death and the regulation of host immune responses. FasL has been shown to provide immune privilege, thus prolonging the survival of unmatched grafts in a variety of tissues, such as eyes and testis. In murine FasL (mFasL) transgenic mice, FasL provoked granulocyte infiltration and insulitis in the pancreas. We intended to study whether the expression of human FasL, instead of mFasL, on mouse beta islet cells could avoid granulocyte infiltration, and whether islet cells transgenic for FasL could be used in islet transplantation. We produced transgenic mice in which the human FasL transgene was driven by rat insulin promoter and was expressed exclusively in the pancreas islet cells in ICR mice. In contrast to mFasL transgenic mice, histochemical staining showed that the pancreas was intact in human FasL transgenic ICR mice. However, when human FasL transgenic islet cells were transplanted into allogeneic mice with streptozotocin-induced diabetes, human FasL appeared not to prolong graft survival. Intensive granulocyte infiltration into the islet grafts was observed in recipients (Balb/c mice) which received islet grafts from human FasL transgenic mice, but not from nontransgenic, allogeneic ICR mice on day 31. Our observations suggest that FasL alone is insufficient to confer immune protection, and that other environmental factors might contribute to the formation of immune privilege sites in vivo

Involvement of protein kinases in the potentiation of lipopolysaccharide-induced inflammatory mediator formation by thapsigargin in peritoneal macrophages.

We have explored the regulatory roles played by Ca2+-dependent signaling on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6) release in mouse peritoneal macrophages. To elevate intracellular Ca2+, we used thapsigargin (TG) and UTP. Although LPS alone cannot stimulate NO synthesis, co-addition with TG, which sustainably increased [Ca2+]i, resulted in NO release. UTP, via acting on P2Y6 receptors, can stimulate phosphoinositide (PI) turnover and transient [Ca2+]i increase, however, it did not possess the NO priming effect. LPS alone triggered the release of PGE2, TNF-alpha, and IL-6; all of which were potentiated by the presence of TG, but not of UTP. The stimulatory effect of LPS plus TG on NO release was inhibited by the presence of Ro 31-8220, Go6976, KN-93, PD 098059, or SB 203580, and abolished by BAPTA/AM and nuclear factor kappaB (NF-kappaB) inhibitor, PDTC. PGE2, TNF-alpha, and IL-6 release by LPS alone were attenuated by Ro 31-8220, Go6976, PD 098059, SB 203580, and PDTC. Using L-NAME, soluble TNF-alpha receptor, IL-6 antibody, NS-398, and indomethacin, we performed experiments to understand the cross-regulation by the four mediators. The results revealed that TNF-alpha up-regulated NO, PGE2, and IL-6 synthesis; PGE2 up-regulated NO, but down-regulated TNF-alpha synthesis; and PGE2 and IL-6 mutually up-regulated reciprocally. Taken together, murine peritoneal macrophages required a sustained [Ca2+]i increase, which proceeds after TG, but not UTP, stimulation, to enhance LPS-mediated release of inflammatory mediators, particularly for NO induction. Activation of PKC-, ERK-, and p38 MAPK-dependent signaling also are essential for LPS action. The positive regulatory interactions among these mediators might amplify the inflammatory response caused by endotoxin.

Molecular cloning of full-length cDNA encoding delta-9 desaturase through PCR strategies and its genomic organization and expression in grass carp (Ctenopharyngodon idella).

Desaturases are enzymes that catalyze double bond formation in fatty acids, which is a critical step in the synthesis of unsaturated fatty acids in organisms. Desaturase cDNA has been cloned from various species. Here we report the cloning of a full-length cDNA of Delta(9)-desaturase from grass carp (Ctenopharyngodon idella), using a combination of PCR techniques: reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The resolved cDNA encompasses 2420 bp, containing an open reading frame corresponding to 324 amino acids. The deduced amino acid sequence shares high homology with those of mammalian desaturases. Northern blot and RT-PCR analyses demonstrated a high abundance of the transcript in liver tissue but low abundance in brain tissue. Furthermore, the structure of the gene has been resolved by screening its cognate genomic DNA library. The analysis shows that this gene is composed of six exons and five introns, encompassing a region of 8.5 kb. In particular, the last exon contains a length of the 3' untranslated region as long as 1382 bp. Although the primary sequence and the genomic organization are phylogenetically conserved between fish and mammals, the regulation of the gene expression appears to be divergent among species.

Enhanced secretion of IFN-gamma by activated Th1 cells occurs via reverse signaling through TNF-related activation-induced cytokine.

Growing evidence has demonstrated that members of TNF superfamily transduce signals after engagement with their receptors. TNF-related activation-induced cytokine (TRANCE), a member of TNF superfamily, is preferentially expressed on the surface of activated CD4(+) Th1 cells. The soluble receptor activator of NF-kappaB (RANK).Fc fusion protein suppresses IFN-gamma secretion by activated Th1 cells, but does not affect IL-4 secretion by Th2 cells. The suppressive effect on IFN-gamma secretion is observed when Th1 cells are activated by APCs, but not by immobilized anti-TCR beta mAb. In contrast, immobilized RANK.Fc fusion protein augments IFN-gamma secretion by Th1 cells, indicating the occurrence of reverse signaling through TRANCE during T cell/APC interaction. The enhanced secretion of IFN-gamma mediated via TRANCE correlates with the activation of p38 mitogen-activated protein kinase and is blocked by SB203580, a p38 mitogen-activated protein kinase-specific inhibitor. Thus, in addition to its role in activating dendritic cells by binding to the receptor RANK, TRANCE itself can signal the augmentation of IFN-gamma secretion via a p38-dependent pathway, and this provides yet another example of reverse signaling by a member of TNF superfamily.

Enhanced antitumor immunity by fusion of CTLA-4 to a self tumor antigen.

The idiotypic determinant (Id) of the immunoglobulin expressed by a B-cell malignancy can serve as an effective tumor-specific antigen but is only weakly immunogenic. This study demonstrates that the immunogenicity of the tumor Id protein can be dramatically increased by directing it to antigen-presenting cells (APCs). Cytotoxic T-lymphocyte antigen 4 (CTLA-4) present on activated T cells has a strong binding affinity to both B7-1 and B7-2 molecules, which are primarily expressed on APCs. After construction of a fusion protein consisting of Id and CTLA-4 (Id-CTLA4), mice immunized with the fusion protein induced high titers of Id-specific antibody and T-cell proliferative responses without adjuvants and were protected from lethal tumor challenge. The Id-CTLA4 fusion protein was so potent that even low doses (down to 0.1 microg) of the immunogen were able to elicit strong antibody responses. By using an Id-CTLA4 mutant protein, the ability to bind B7 molecules on APCs was shown to be required for the enhanced immunogenicity of Id-CTLA4. These findings demonstrate that fusing CTLA-4 to a potential tumor antigen represents an effective approach to prime antitumor immunities in vivo and may be applicable to the design of vaccines for a variety of other diseases. (Blood. 2000;96:3663-3670)