Secretomic Analysis of Host-Pathogen Interactions Reveals That Elongation Factor-Tu Is a Potential Adherence Factor of Helicobacter pylori during Pathogenesis.

The secreted proteins of bacteria are usually accompanied by virulence factors, which can cause inflammation and damage host cells. Identifying the secretomes arising from the interactions of bacteria and host cells could therefore increase understanding of the mechanisms during initial pathogenesis. The present study used a host-pathogen coculture system of Helicobacter pylori and monocytes (THP-1 cells) to investigate the secreted proteins associated with initial H. pylori pathogenesis. The secreted proteins from the conditioned media from H. pylori, THP-1 cells, and the coculture were collected and analyzed using SDS-PAGE and LC-MS/MS. Results indicated the presence of 15 overexpressed bands in the coculture. Thirty-one proteins were identified-11 were derived from THP-1 cells and 20 were derived from H. pylori. A potential adherence factor from H. pylori, elongation factor-Tu (EF-Tu), was selected for investigation of its biological function. Results from confocal microscopic and flow cytometric analyses indicated the contribution of EF-Tu to the binding ability of H. pylori in THP-1. The data demonstrated that fluorescence of EF-Tu on THP-1 cells increased after the addition of the H. pylori-conditioned medium. This study reports a novel secretory adherence factor in H. pylori, EF-Tu, and further elucidates mechanisms of H. pylori adaptation for host-pathogen interaction during pathogenesis.

Size-dependent attenuation of TLR9 signaling by gold nanoparticles in macrophages.

Gold nanoparticles (GNPs), which are generally thought to be bio-inert and non-cytotoxic, have become one of the most ideal nanomaterials for medical applications. Once engulfed by phagocytes, the immunological effects of GNPs are still of concern and require detailed investigation. Therefore, this study explored the immunological significance of GNPs on TLR-mediated innate immunity in murine macrophages. GNP causes specific inhibition of TLR9 (CpG oligodeoxynucleotides; CpG-ODNs) signal in macrophages. The impaired CpG-ODN-induced TNF-α production is GNP concentration- and size-dependent in murine Raw264.7 cells: a GNP of 4 nm in size is more potent than a GNP of 11, 19, 35, or 45 nm in size. Consistent with cytokine inhibition, the CpG-ODN-induced phosphorylation of NF-κB and JNK as well as NF-κB activation are suppressed by GNPs. GNPs accumulate in lysosomes after phagocytosis and also increase TLR9-associated lysosomal cathepsin expression and activities, but this is irrelevant to TLR9 inhibition by GNPs in our studies. In addition, GNPs affected TLR9 translocation in response to CpG-ODNs and to phagosomes. Further exploring how GNPs inhibited TLR9 function, we found that GNPs could bind to high-mobility group box-1 (which is involved in the regulation of TLR9 signaling) inside the lysosomes. The current studies demonstrate that size-dependent inhibition of TLR9 function by GNP may be attributed to its binding to high-mobility group box-1.

CLEC5A is critical for dengue-virus-induced lethal disease.

Dengue haemorrhagic fever and dengue shock syndrome, the most severe responses to dengue virus (DV) infection, are characterized by plasma leakage (due to increased vascular permeability) and low platelet counts. CLEC5A (C-type lectin domain family 5, member A; also known as myeloid DAP12-associating lectin (MDL-1)) contains a C-type lectin-like fold similar to the natural-killer T-cell C-type lectin domains and associates with a 12-kDa DNAX-activating protein (DAP12) on myeloid cells. Here we show that CLEC5A interacts with the dengue virion directly and thereby brings about DAP12 phosphorylation. The CLEC5A-DV interaction does not result in viral entry but stimulates the release of proinflammatory cytokines. Blockade of CLEC5A-DV interaction suppresses the secretion of proinflammatory cytokines without affecting the release of interferon-alpha, supporting the notion that CLEC5A acts as a signalling receptor for proinflammatory cytokine release. Moreover, anti-CLEC5A monoclonal antibodies inhibit DV-induced plasma leakage, as well as subcutaneous and vital-organ haemorrhaging, and reduce the mortality of DV infection by about 50% in STAT1-deficient mice. Our observation that blockade of CLEC5A-mediated signalling attenuates the production of proinflammatory cytokines by macrophages infected with DV (either alone or complexed with an enhancing antibody) offers a promising strategy for alleviating tissue damage and increasing the survival of patients suffering from dengue haemorrhagic fever and dengue shock syndrome, and possibly even other virus-induced inflammatory diseases.

TBK1-associated protein in endolysosomes (TAPE)/CC2D1A is a key regulator linking RIG-I-like receptors to antiviral immunity.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are key RNA viral sensors for triggering antiviral immunity. The underlying mechanisms for RLRs to trigger antiviral immunity have yet to be explored. Here we report the identification of TAPE (TBK1-associated protein in endolysosomes) as a novel regulator of the RLR pathways. TAPE functionally and physically interacts with RIG-I, MDA5, and IPS-1 to activate the IFN-ß promoter. TAPE knockdown impairs IFN-ß activation induced by RLRs but not IPS-1. TAPE-deficient cells are defective in cytokine production upon RLR ligand stimulation. During RNA virus infection, TAPE knockdown or deficiency diminishes cytokine production and antiviral responses. Our data demonstrate a critical role for TAPE in linking RLRs to antiviral immunity.

Dengue virus infection induces autophagy: an in vivo study.

BACKGROUND: We and others have reported that autophagy is induced by dengue viruses (DVs) in various cell lines, and that it plays a supportive role in DV replication. This study intended to clarify whether DV infection could induce autophagy in vivo. Furthermore, the effect of DV induced autophagy on viral replication and DV-related pathogenesis was investigated. RESULTS AND CONCLUSIONS: The physiopathological parameters were evaluated after DV2 was intracranially injected into 6-day-old ICR suckling mice. Autophagy-related markers were monitored by immunohistochemical/immunofluorescent staining and Western blotting. Double-membrane autophagic vesicles were investigated by transmission-electron-microscopy. DV non-structural-protein-1 (NS1) expression (indicating DV infection) was detected in the cerebrum, medulla and midbrain of the infected mice. In these infected tissues, increased LC3 puncta formation, LC3-II expression, double-membrane autophagosome-like vesicles (autophagosome), amphisome, and decreased p62 accumulation were observed, indicating that DV2 induces the autophagic progression in vivo. Amphisome formation was demonstrated by colocalization of DV2-NS1 protein or LC3 puncta and mannose-6-phosphate receptor (MPR, endosome marker) in DV2-infected brain tissues. We further manipulated DV-induced autophagy by the inducer rapamycin and the inhibitor 3-methyladenine (3MA), which accordingly promoted or suppressed the disease symptoms and virus load in the brain of the infected mice.We demonstrated that DV2 infection of the suckling mice induces autophagy, which plays a promoting role in DV replication and pathogenesis.

A microfluidic immunomagnetic bead-based system for the rapid detection of influenza infections: from purified virus particles to clinical specimens.

Seasonal and novel influenza infections have the potential to cause worldwide pandemics. In order to properly treat infected patients and to limit its spread, a rapid, accurate and automatic influenza diagnostic tool needs to be developed. This study therefore presents a new integrated microfluidic system for the rapid detection of influenza infections. It integrated a suction-type, pneumatic-driven microfluidic control module, a magnetic bead-based fluorescent immunoassay (FIA) and an end-point optical detection module. This new system can successfully distinguish between influenza A and B using a single chip test within 15 min automatically, which is faster than existing devices. By utilizing the micromixers to thoroughly wash out the sputum-like mucus, this microfluidic system could be used for the diagnosis of clinical specimens and reduced the required sample volume to 40 µL. Furthermore, the results of diagnostic assays from 86 patient specimens have demonstrated that this system has 84.8 % sensitivity and 75.0 % specificity. This developed system may provide a powerful platform for the fast screening of influenza infections.

Dengue virus-induced autoantibodies bind to plasminogen and enhance its activation.

Dengue virus infection can lead to life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) in patients. Abnormal activation of the coagulation and fibrinolysis system is one of the hallmarks associated with DHF/DSS patients. However, the mechanisms that cause pathology in DHF/DSS patients are still unclear. Because conversion of plasminogen (Plg) to plasmin (Plm) is the first step in the activation of fibrinolysis, Abs against Plg found in DHF/DSS patients may be important. Therefore, to investigate the specificity, function, and possible origin of these Abs, we generated several Plg cross-reactive mAbs from DENV-immunized mice. An IgG mAb, 6H11, which recognizes an epitope associated with a dengue envelope protein, demonstrated a high level of cross-reactivity with Plg. The 6H11 Ab was further characterized with regard to its effect on Plg activation. Using Plm-specific chromogenic substrate S-2251, we found that mAb 6H11 demonstrated serine protease activity and could convert Plg directly to Plm. The serine protease activity of mAb 6H11 was further confirmed using serine protease chromogenic substrate S-2288. In addition, we found several Plg cross-reactive mAbs that could enhance urokinase-induced Plg activation. Lastly, mAb 6H11 could induce Plm activity and increase the level of D-dimer (a fibrin degradation product) in both human and mouse platelet-poor plasma. Taken together, these data suggest DENV-induced Plg cross-reactive Abs may enhance Plg conversion to Plm, which would be expected to contribute to hyperfibrinolysis in DHF/DSS patients.

Factors contributing to the disturbance of coagulation and fibrinolysis in dengue virus infection.

Hemorrhage is one of the hallmarks of dengue hemorrhagic fever. However, the mechanisms that cause hemorrhage are unclear. In this review we focus on the possible factors that may be involved in the disturbance of coagulation and fibrinolysis during dengue virus (DENV) infection. Factors such as autoantibodies and cytokines induced by DENV infection as well as hemostatic molecules expressed on DENV-infected cells, and DENV viral proteins may all contribute to the defect of hemostasis during DENV infection. It is the combination of these viral and host factors that may tilt the balance of coagulation and fibrinolysis toward bleeding in dengue patients.

Autoimmunity in dengue pathogenesis.

Dengue is one of the most important vector-borne viral diseases. With climate change and the convenience of travel, dengue is spreading beyond its usual tropical and subtropical boundaries. Infection with dengue virus (DENV) causes diseases ranging widely in severity, from self-limited dengue fever to life-threatening dengue hemorrhagic fever and dengue shock syndrome. Vascular leakage, thrombocytopenia, and hemorrhage are the major clinical manifestations associated with severe DENV infection, yet the mechanisms remain unclear. Besides the direct effects of the virus, immunopathogenesis is also involved in the development of dengue disease. Antibody-dependent enhancement increases the efficiency of virus infection and may suppress type I interferon-mediated antiviral responses. Aberrant activation of T cells and overproduction of soluble factors cause an increase in vascular permeability. DENV-induced autoantibodies against endothelial cells, platelets, and coagulatory molecules lead to their abnormal activation or dysfunction. Molecular mimicry between DENV proteins and host proteins may explain the cross-reactivity of DENV-induced autoantibodies. Although no licensed dengue vaccine is yet available, several vaccine candidates are under development. For the development of a safe and effective dengue vaccine, the immunopathogenic complications of dengue disease need to be considered.

TBK1-associated protein in endolysosomes (TAPE) is an innate immune regulator modulating the TLR3 and TLR4 signaling pathways.

The innate immune system elicits the first wave of immune responses against pathogen infection. Its operational modes are complex and have yet to be defined. Here, we report the identification of an innate immune regulator termed TAPE (TBK1-associated protein in endolysosomes), previously known as CC2D1A/Freud-1/Aki-1, which modulates the TLR3 and TLR4 pathways. We found that TAPE activated the TBK1, NF-κB, and ERK pathways leading to IFN-ß and inflammatory cytokine induction. TAPE was shown to colocalize with endosomal marker Rab5 and lysosomal marker LAMP1 in mammalian cells, suggesting that TAPE resided in endolysosomes. Knockdown of TAPE selectively impaired the TLR3 and endocytic TLR4 pathways to IFN-ß induction. Furthermore, TAPE interacted and synergized with Trif to activate IFN-ß. TAPE knockdown failed to block Trif-mediated IFN-ß induction, whereas Trif knockdown impaired the TLR3 and TAPE cooperation on IFN-ß induction, suggesting that TAPE acts upstream of Trif. Together, our data demonstrate a central role for TAPE in linking TLR3 and TLR4 to innate immune defenses at an early step.

Loss of outer membrane protein C in Escherichia coli contributes to both antibiotic resistance and escaping antibody-dependent bactericidal activity.

Outer membrane proteins (OMPs) serve as the permeability channels for nutrients, toxins, and antibiotics. In Escherichia coli, OmpA has been shown to be involved in bacterial virulence, and OmpC is related to multidrug resistance. However, it is unclear whether OmpC also has a role in the virulence of E. coli. The aims of this study were to characterize the role of OmpC in antimicrobial resistance and bacterial virulence in E. coli. The ompC deletion mutant showed significantly decreased susceptibility to carbapenems and cefepime. To investigate the survival of E. coli exposed to the innate immune system, a human blood bactericidal assay showed that the ompC mutant increased survival in blood and serum but not in complement-inactivated serum. These effects were also demonstrated in the natural selection of OmpC mutants. Also, C1q interacted with E. coli through a complex of antibodies bound to OmpC as a major target. Bacterial survival was increased in the wild-type strain in a dose-dependent manner by adding free recombinant OmpC protein or anti-C1q antibody to human serum. These results demonstrated that the interaction of OmpC-specific antibody and C1q was the key step in initiating the antibody-dependent classical pathway for the clearance of OmpC-expressing E. coli. Anti-OmpC antibody was detected in human sera, indicating that OmpC is an immunogen. These data indicate that the loss of OmpC in E. coli is resistant to not only antibiotics, but also the serum bactericidal effect, which is mediated from the C1q and anti-OmpC antibody-dependent classical pathway.

Endothelial cell surface expression of protein disulfide isomerase activates ß1 and ß3 integrins and facilitates dengue virus infection.

Infection with dengue virus (DENV) causes diseases ranging from mild dengue fever to severe hemorrhage or shock syndrome. DENV infection of endothelial cells may cause cell apoptosis or vascular leakage and result in clinical illness of hemorrhage. However, the endothelial cell molecules involved in DENV infection and the mechanisms governing virus-cell interactions are still uncertain. Since protein disulfide isomerase (PDI) reducing function at the cell surface was shown to be required for entry of certain viruses and bacteria, we explored the role of PDI expressed on endothelial cell surface in DENV infection. Using siRNA to knock down PDI, DENV infection was reduced which could be reversed by treating cells with a reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). DENV-induced PDI surface expression was mediated through the Lys-Asp-Glu-Leu (KDEL) receptor-Src family kinase signal pathway. Furthermore, cell surface PDI colocalized with ß1 and ß3 integrins after DENV infection, and the activation of integrins was blocked by PDI inhibition. Finally, blockade of PDI inhibited DENV entry into endothelial cells. Our findings suggest a novel mechanism whereby surface PDI which causes integrin activation is involved in DENV entry, and DENV infection further increases PDI surface expression at later time points. These findings may have implications for anti-DENV drug design.

Immature dengue virus: a veiled pathogen?

Cells infected with dengue virus release a high proportion of immature prM-containing virions. In accordance, substantial levels of prM antibodies are found in sera of infected humans. Furthermore, it has been recently described that the rates of prM antibody responses are significantly higher in patients with secondary infection compared to those with primary infection. This suggests that immature dengue virus may play a role in disease pathogenesis. Interestingly, however, numerous functional studies have revealed that immature particles lack the ability to infect cells. In this report, we show that fully immature dengue particles become highly infectious upon interaction with prM antibodies. We demonstrate that prM antibodies facilitate efficient binding and cell entry of immature particles into Fc-receptor-expressing cells. In addition, enzymatic activity of furin is critical to render the internalized immature virus infectious. Together, these data suggest that during a secondary infection or primary infection of infants born to dengue-immune mothers, immature particles have the potential to be highly infectious and hence may contribute to the development of severe disease.

Cytokine immunopathogenesis of enterovirus 71 brain stem encephalitis.

Enterovirus 71 (EV71) is one of the most important causes of herpangina and hand, foot, and mouth disease. It can also cause severe complications of the central nervous system (CNS). Brain stem encephalitis with pulmonary edema is the severe complication that can lead to death. EV71 replicates in leukocytes, endothelial cells, and dendritic cells resulting in the production of immune and inflammatory mediators that shape innate and acquired immune responses and the complications of disease. Cytokines, as a part of innate immunity, favor the development of antiviral and Th1 immune responses. Cytokines and chemokines play an important role in the pathogenesis EV71 brain stem encephalitis. Both the CNS and the systemic inflammatory responses to infection play important, but distinctly different, roles in the pathogenesis of EV71 pulmonary edema. Administration of intravenous immunoglobulin and milrinone, a phosphodiesterase inhibitor, has been shown to modulate inflammation, to reduce sympathetic overactivity, and to improve survival in patients with EV71 autonomic nervous system dysregulation and pulmonary edema.

The serotype-independent but concentration-dependent enhancing antibodies among Thai dengue patients.

Antibody-dependent enhancement of infection (ADE) is central to explaining the development of severe disease at the end of post-dengue virus infection. Non-neutralizing anti-dengue antibodies bound to the dengue virion enhances the virus entrance into the target cells via the Fc receptor. The titer of enhancing antibodies in dengue patients is not determined during dengue virus infection. Sensitive flow cytometry detecting dengue virus-infected K562 cells was used to quantitate enhancing activity among Thai DF and DHF patients against four serotypes and the patient's dengue isolate. The titer was defined as the reciprocal of the final dilution that loses enhancing activity. The serum of Thai patients confirmed to have dengue infection were found to have high titers of enhancing antibodies and increased gradually through the convalescent phase of infection. The enhancing antibody titers were not different among the four serotypes or from the infecting isolate. The anti-dengue antibodies from dengue patients can enhance dengue virus infections in a concentration-dependent, serotype-independent manner.

ACSL3 and GSK-3ß are essential for lipid upregulation induced by endoplasmic reticulum stress in liver cells.

The endoplasmic reticulum (ER) is essential for lipid biosynthesis, and stress signals in this organelle are thought to alter lipid metabolism. Elucidating the mechanisms that underlie the dysregulation of lipid metabolism in hepatocytes may lead to novel therapeutic approaches for the treatment of lipid accumulation. We first tested the effects of several inhibitors on lipid dysregulation induced by tunicamycin, an ER stress inducer. Triacsin C, an inhibitor of long-chain acyl-CoA synthetase (ACSL) 1, 3, and 4, was the most potent among these inhibitors. We then analyzed the expression of the ACSL family during ER stress. The expression of ACSL3 was induced by ER stress in HuH-7 cells and in mice livers. ACSL3 shRNA, but not ACSL1 shRNA, inhibited the induction of lipid accumulation. GSK-3ß inhibitors attenuated ACSL3 expression and the lipid accumulation induced by ER stress in HuH-7 cells. shRNA that target GSK-3ß also inhibited the upregulation of ACSL3 and lipid accumulation in HuH-7 and HepG2 cells. The hepatitis B virus mutant large surface protein, which is known to induce ER stress, increased the lipid content of cells. Similarly, Triacsin C, and GSK-3ß inhibitors abrogated the lipid dysregulation caused by the hepatitis B virus mutant large surface protein. Altogether, ACSL3 and GSK-3ß represent novel therapeutic targets for lipid dysregulation by ER stress.

Macrophage migration inhibitory factor induced by dengue virus infection increases vascular permeability.

Dengue virus (DENV) infection can cause mild dengue fever or severe dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Serum levels of the macrophage migration inhibitory factor (MIF) have been shown to be correlated with severity and mortality in DENV patients, but the pathogenic roles of MIF in DHF/DSS are still unclear. Increase in vascular permeability is an important hallmark of DHF/DSS. In this study, we found that DENV infection of the human hepatoma cell line (Huh 7) induced MIF production. Conditioned medium collected from DENV-infected Huh 7 cells enhanced the permeability of the human endothelial cell line (HMEC-1) which was reduced in the presence of a MIF inhibitor, ISO-1 or medium from DENV-infected MIF knockdown Huh 7 cells. To further identify whether MIF can alter vascular permeability, we cloned and expressed both human and murine recombinant MIF (rMIF) and tested their effects on vascular permeability both in vitro and in vivo. Indirect immunofluorescent staining showed that the tight junction protein ZO-1 of HMEC-1 was disarrayed in the presence of rMIF and partially recovered when cells were treated with ISO-1 or PI3K/MEK-ERK/JNK signaling pathway inhibitors such as Ly294002, U0126, and SP600215. In addition, ZO-1 disarray induced by MIF was also recovered when CD74 or CXCR2/4 expression of HMEC-1 were inhibited. Last but not least, the vascular permeabilities of the peritoneal cavity and dorsal cutaneous capillary were also increased in mice treated with rMIF. Taken together; these results suggest that MIF induced by DENV infection may contribute to the increase of vascular permeability during DHF/DSS. Therapeutic intervention of MIF by its inhibitor or neutralizing antibodies may prevent DENV-induced lethality.

Exogenous interleukin-6, interleukin-13, and interferon-γ provoke pulmonary abnormality with mild edema in enterovirus 71-infected mice.

BACKGROUND: Neonatal mice developed neurological disease and pulmonary dysfunction after an infection with a mouse-adapted human Enterovirus 71 (EV71) strain MP4. However, the hallmark of severe human EV71 infection, pulmonary edema (PE), was not evident. METHODS: To test whether EV71-induced PE required a proinflammatory cytokine response, exogenous pro-inflammatory cytokines were administered to EV71-infected mice during the late stage of infection. RESULTS: After intracranial infection of EV71/MP4, 7-day-old mice developed hind-limb paralysis, pulmonary dysfunction, and emphysema. A transient increase was observed in serum IL-6, IL-10, IL-13, and IFN-γ, but not noradrenaline. At day 3 post infection, treatment with IL-6, IL-13, and IFN-γ provoked mild PE and severe emphysema that were accompanied by pulmonary dysfunction in EV71-infected, but not herpes simplex virus-1 (HSV-1)-infected control mice. Adult mice did not develop PE after an intracerebral microinjection of EV71 into the nucleus tractus solitarii (NTS). While viral antigen accumulated in the ventral medulla and the NTS of intracerebrally injected mice, neuronal loss was observed in the ventral medulla only. CONCLUSIONS: Exogenous IL-6, IL-13, and IFN-γ treatment could induce mild PE and exacerbate pulmonary abnormality of EV71-infected mice. However, other factors such as over-activation of the sympathetic nervous system may also be required for the development of classic PE symptoms.

Endothelial cells are damaged by autophagic induction before hepatocytes in Con A-induced acute hepatitis.

We have reported both T-cell-dependent and -independent hepatitis in immunocompetent and immunodeficiency mice, respectively, after intravenous injection of Con A in mice. The mode of hepatocyte cell death is different: autophagy for T-cell-independent hepatitis in contrast to apoptosis for T-cell-dependent one. In this study, we further demonstrate that liver blood vessels are the first target in both modes. The infused Con A bond to the hepatic vascular endothelial cells and cause its damage with autophagy. Before the elevation of the serum alanine aminotransferase at 6 h post-injection, the plasma leakage and hemorrhage occur at 1-3 h without inflammation. Con A induces autophagy of endothelial cells and hemorrhage that is enhanced by IFN-gamma. Using the endothelial cell line HMEC-1, a dose- and time-dependent cell death with autophagic LC3-II (microtubule-associated protein light chain 3) conversion was induced by Con A and was enhanced by IFN-gamma. In conclusion, Con A induced autophagy on hepatic endothelial cells; the damage of liver blood vessel occurs before the induction of T-cell-dependent hepatitis via apoptosis or T-cell-independent hepatitis via autophagy.

A suction-type microfluidic immunosensing chip for rapid detection of the dengue virus.

The enzyme-linked immunosorbent assay (ELISA) is widely used in medical diagnostics. In order to reduce the diagnosis time and to lower the consumption of sample/reagents in an ELISA assay, a suction-type, automatic, pneumatically-driven microfluidic chip has been designed and fabricated in this study. The microfluidic chip integrates a multi-functional micro-transport/mixing unit, for transporting metering and mixing of samples and reagents in order to automatically perform the entire ELISA protocol. A new surface modification has been adopted which allows for a high processing capacity. The detection sensitivity for the dengue virus is found to be 10(1) PFU/ml, which is much better than a conventional ELISA assay (10(3) PFU/ml). The entire assay time is only 30 min, which is much faster than with 96-well microtiter plates (4 h). The consumed sample and reagent volume is only 12 µl, which is less than a conventional assay (100 µl). The development of this microfluidic chip may be promising for other immunosensing applications.