严重急性呼吸综合征病毒基因检测和特异性抗体检测的诊断意义分析

目的探讨严重急性呼吸综合征病毒基因检测和特异性抗体检测的诊断意义和价值。方法采用巢式反转录PCR和ELISA法分别检测健康人、普通发热患者、SARS密切接触医务人员及不同时期SARS患者痰标本中SARS病毒核酸和血清标本中特异性抗体IgG和IgM。结果SARS患者住院1～58d期间,采用巢式RT-PCR检测痰SARS病毒基因,52例患者中33例阳性,阳性率63.5%;而采用间接ELISA检测血清抗SARS病毒抗体,52例患者中30例阳性,阳性率为57.7%。2种方法均阳性的有15例,均阴性的有6例。2种病原学诊断方法检测20例正常人、15例发热非SARS患者和20例SARS密切接触医务人员,结果均为阴性。住院1～13d组(92.9%)巢式RT-PCR基因阳性率明显高于ELISA特异性抗体阳性率(7.1%);住院37～58d组巢式RT-PCR基因阳性率(47.4%)则明显低于ELISA特异性抗体阳性率(94.7%)(P<0.01)。结论痰中SARS病毒基因检测可用于SARS的早期诊断,这对于提前确诊和治疗SARS患者,尽早排除疑似病例具有非常关键和现实的意义。血清特异性抗体检测可用于流行病学调查,但对SARS早期诊断和鉴别没有意义。

Development and Characterization of Monoclonal Antibody Specific to Nuclear Protein of Avian Influenza Virus Type A

Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mabs were identified by immunofluorescent assay（IFA） and enzyme linked immunosorbent assay （ELISA）. These five Mabs which were named as AIV-NP-2C3, AIV-NP-6A5, AIV-NP-3 H9, AIV-NP-7B4, AIV-NP-2H4 could react with all viruses of AIV-H9 strains in tests. The result of Western blotting showed that only the 60 ku protein antigen of AIV-H9 could be recognized by the Mabs but never recognized by New castle disease virus, REV and infectious bursa disease virus. The result of preliminary application showed that avian influenza viruses could be deetected bv Mabs in IFA and ELISA. All these Mabs will probably play important roles in preventing and monitoring avian influenza viruses.

Comparison of Multiplex Fluorescent PCR with Serum Type-specific Antibody Detection in Diagnosis of Genital Herpes

Objectives: To compare multiplex fluorescent PCRwith serum type-specific antibody detection in thediagnosis of herpes simplex virus (HSV) infection andto evaluate its significance in the diagnosis of genitalherpes.Methods: We detected HSV infection in 121 speci-mens collected from patients with genital herpesusing both multiplex fluorescent PCR and serum type-specific antibody detection. HSV viral isolation wasused as the standard control.Results: When compared with the viral isolation, thesensitivity and specificity for multiplex fluorescentPCR were 100% and 88.89%, respectively afterdiscrepant analysis. The sensitivity and specificity fortype-specific antibody detection was 77.68 % and77.78 %, respectively. However, the type-specificantibody detected HSV in two asymptomatic patientswhile the multiplex fluorescent PCR couldn’t detectany HSV DNA from those specimens.Conclusions: Multiplex fluorescent PCR is a verysensitive and specific method for detection and typingof HSV in the lesion of genital herpes, it failed todetect HSV DNA from the asymptomatic patients.Serum type-specific antibody detection was a lesssensitive and specific test but could detect the specificantibody from some asymptomatic patients. Thecombination of these two techniques would allow rapid,sensitive and accurate detection and typing of HSVand help clinical diagnosis and epidemiologic survey-ing of genital herpes.

Potential implications of Helicobacter pylori-related neutrophil-activating protein

Helicobacter pylori （H. pylori） virulence factors pro- mote the release of various chemoattractants/inflam- matory mediators, including mainly the neutrophil- attractant chemokine interleukin-8 and neutrophil- activating protein （NAP）, involved in H. pylor/-induced gastric pathologies. Co-administration of Chios mastic gum （CMG）, which inhibits H. pylor/NAP, with an H. pylori eradication regimen might add clinical benefits against H. pylori-related gastric pathologies, but pos- sibly not CMG as main therapy. Although H. pylori NAP and other H. pylori-related cytotoxins [i.e., vaculating cytotoxin （VacA）] appear to play a major role in gener- ating and maintaining the H. pylori-associated gastric inflammatory response and H. pylor/NAP is a promising vaccine candidate against H. pylori infection （H. pylori-1）, concerns regarding its potential drawbacks, particularly neurogenic ones, due to possible cross- mimicry, should be considered. Possible cross-mimicry between H. p, vlor/ NAP and/or bacterial aquaporin （AQP） and neural tissues may be associated with the anti-AQP-4 antibody-related neural damage in multiple sclerosis （MS）/neuromyelitis optica patients. Moreover, the sequence homology found between H. pylori VacA and human Na＋/K＋-ATPase A subunit suggests that antibodies to VacA involve ion channels in abaxonal Schwann cell plasmalemma resulting in demyelination in some patients. A series of factors have been im- plicated in inducing blood-brain barrier （BBB） disrup- tion, including inflammatory mediators （e.g., cytokines and chemokines induced by H. pylor/-I） and oxidative stress. BBB disruption permits access of AQP4-specific antibodies and T lymphocytes to the central nervous system, thereby playing a major role in multiple sclero- sis pathogenesis. Relative studies show a strong asso- ciation between H. pylori-I and MS. H. pylor/-I induces humoral and cellular immune responses that, owing to the sharing of homologous epitopes （molecular mim- icry）, cross-react with components of nerves, thereby contributing and perpetuating neural tissue damage. Finally, H. pylori NAP also plays a possible pathoge- netic role in both gastric and colon oncogenesis.

Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus

Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstmctural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV（Mammalian orthoreovimses）, which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region （335-742） was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28℃. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80（335-742） protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody （mouse） and NS80〈335.742） specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.

Detection and evaluation of antibodies against neutrophil-activating protein of Helicobacter pylori in patients with gastric cancer

AIM： To detect and evaluate the antibodies against Helicobacter pylori （H pylori） neutrophil-activating protein （HP-NAP） in patients with gastric cancer and other gastroduodenal diseases.METHODS： Recombinant HP-NAP was prepared from a prokaryotic expression system in Escherichia coll. Serum positivity and level of HP-NAP-specific antibodies in sera from 43 patients with gastric cancer, 28 with chronic gastritis, 28 with peptic ulcer, and 89 healthy controls were measured by rHP-NAP-based ELISA. rHP-NAP-stimulated production of interleukin-8 （IL-8） and growth-related oncogene （GROα） cytokines in the culture supernatant of SGC7901 gastric epithelial cells was also detected.RESULTS： The serum positivity and mean absorbancevalue of HP-NAP-specific antibodies in the gastriccancer group （97.7% and 1.01 ± 0.24） were significantly higher than those in the chronic gastritisgroup （85.7% and 0.89 ± 0.14, P 〈 0.005） and healthy control group （27.7% and 0.65 ± 0.18, P 〈 0.001）. The sensitivity and specificity of ELISA for the detection of HP-NAP-specific antibodies were 95.5% and 91.5%, respectively. HP-NAP could slightly upregulate IL-8 production in gastric epithelial cell lines but had no effect on GROα production.CONCLUSION： Infection with virulent H py/ori strains secreting HP-NAP is associated with severe gastroduodenal diseases, and HP-NAP may play a role in the development of gastric carcinoma, rHP-NAP- based ELISA can be used as a new method to detect H pylori infection. The direct effect of HP-NAP on gastric epithelial cells may be limited, but HP-NAP may contribute to inflammatory response or carcinogenesis by activating neutrophils.

Immunogenicity and protective efficacy of Vibrio harveyi pcFlaA DNA vaccine in Epinephelus awoara

The FlaA gene from Vibrio harveyi marker, was cloned into the eukaryotic expression with a short nucleotide sequence encoding the Flag vector pcDNA3.1（＋） （designated as pcFlaA）. Ninety grouper （Epinephelus awoara） were separated into three equal size groups. An experimental group was immunized with pcFlaA, Control I group was immunized with the vector pcDNA3.1（＋）, and Control 1I group was immunized with PBS. The expression of pcFlaA mRNA and protein was examined using reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry. We also evaluated the immunogenicity and protective efficacy of pcFlaA against V. harveyi by measuring the lymphocyte proliferation response and serum levels of specific antibody and conducting a bacterial challenge test. We successfully transfected the fish muscle with pcFlaA. The pcFlaA mRNA and protein was expressed in the muscle cells for up to one month following injection. The proliferation response of lymphocytes in fish immunized with pcFlaA was significantly higher than in control group II. Furthermore, the immunized fish generated specific antibody. The vaccination also resulted in significantly higher survival during the bacterial challenge test.

Preparation of monoclonal antibody to P53 and its clinical application

Objective：The aim of this study was to prepare monoclonal antibody against P53, a kind of tumor suppressor protein,and use the antibody initial y in clinical immunoassay. Methods：Monoclonal antibody was prepared and identified via the classic protocol of monoclonal antibody preparation. Identified monoclonal antibodies were purified by af inity chro-matography. Antibody titer was determined by enzyme linked immunosorbent assay （ELISA）. The specific binding activity of antibody was detected by Western blotting and immunohistochemistry. Results：Three strains of monoclonal antibodies named 1P15, 2P37 and 3P40 were obtained and purified by af inity chromatography. The purity of antibodies was higher than 90%. The titers of antibodies were more than 1：6000. Western blot and immunohistochemistry assay showed that the specific antibody can combine with endogenous P53 protein in the tumor celllines and determine the expression of P53 in tumor tis-sue. Conclusion：Three strains of monoclonal antibodies with high af inity to P53 were successful y established, which can be used for detecting the expression of P53 in tumor cells or tissue.

A Woman with Psychogenic Non-epileptic Seizures and Pelvic Mass

MOST cases of encephalitis are caused by viruses but a few have an immunological basis, such as paraneoplastic encephalitis, with specific antibodies identified. One recently characterized encephalitis caused by antibodies is anti-N- methyl-D-aspartate （NMDA） receptor encephalitis. It is a form of paraneoplastic limbic encephalitis associated with ovarian teratoma and has recently been described.The NMDA receptor mediates excitatory neurotransmission. It is important for synaptic plasticity, and thus for higher function such as learning and memory. This disorder results in prominent psychiatric symptoms followed by a rapid decline of the level of consciousness, central hypoventilation, seizures, involuntary movements and dysautonomia.

Development of Co-agglutination Method for Viral Nervous Necrosis from Grouper （Epinephelus sp,） in Batam

Viral nervous necrosis （VNN） has been reported to infect larvae and juvenile of humpback grouper, and until now, this virus is one of main problem for humpback grouper. Co-agglutination test proved to be a simple, rapid and reliable diagnostic test suitable for use in the field or laboratory without any special apparatus. The study aimed to study application of a co-agglutination test using Staphylococci sensitized with specific antibody for the diagnostic of VNN in grouper. First, brain and eye organ samples from diseased fish are homogenized with phosphate buffer saline （PBS） of pH 7.2. Then, the supernatant is collected after centrifugation at 8,000 rpm for 20 min, and finally one drop of the supematant and one drop of anti VNN antibody sensitized Staphylococci suspension are mixed on a glass slid and observation of the agglutination is performed after 5-10 min. The result shows that co-agglutination technique detects positive VNN in the brain and eye samples. The co-agglutination technique may provide valid result in a very short time as compared with complex method, such as enzyme-linked immunosorbant essay （ELISA） and fluorescient antibody technique （FAT） that require a high cost. Thus, this test can detect VNN faster, simple and more economic.

Affinity peptide developed by phage display selection for targeting gastric cancer

AIM:To develop an affinity peptide that binds to gastric cancer used for the detection of early gastric cancer.METHODS:A peptide screen was performed by biopanning the PhD-12 phage display library,clearing non-specific binders against tumor-adjacent normal appearing gastric mucosa and obtaining selective binding against freshly harvested gastric cancer tissues.Tumortargeted binding of selected peptides was confirmed by bound phage counts,enzyme-linked immunosorbent assay,competitive inhibition,fluorescence microscopy and semi-quantitative analysis on immunohistochemistry using different types of cancer tissues.RESULTS:Approximately 92.8% of the non-specific phage clones were subtracted from the original phage library after two rounds of biopanning against normal-appearing gastric mucosa.After the third round of positive screening,the peptide sequence AADNAKTKSFPV(AAD) appeared in 25%(12/48) of the analyzed phages.For the control peptide,these values were 6.8 ± 2.3,5.1 ± 1.7,3.5 ± 2.1,4.6 ± 1.9 and 1.1 ± 0.5,respectively.The values for AAD peptide were statistically signif icant(P < 0.01) for gastric cancer as compared with other histological classif ications and control peptide.CONCLUSION:A novel peptide is discovered to have a specific binding activity to gastric cancer,and can be used to distinguish neoplastic from normal gastric mucosa,demonstrating the potential for early cancer detection on endoscopy.

Microfluidic Immunoprecipitation for Post-Translational Modified Protein Purification

In this paper, we report an antibody functionalized microimmunopreci- pitation （IX IP） method used for enrich lowabundant post-translational modified （PT~ proteins. The device is fabricated by inert, nontoxic and disposable polydimethylsiloxane （PDMS） using a silane-based chemical modification protocol, which yield antibody- terminated PDMS surfaces. In this study, the IX IP device is specifically designed for the purification of carbonylated protein, a representative example here to illustrate the potential applications for any other PTMs, which could be immuno-tagged by specific antibodies. The test model in vitro oxidized bovine serum albumin （BSA） was first derivitized by dinitrophenylhydrazide （DNPH） and then captured by the anti-DNP immobilized on this Ix lP device. The surface functional group mapping was systematically analyzed and validated by fluorescence microscopy. Quantitative study of DNP-derivatized carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this proteome enrichment Ix IP device can be assembled with other lab-on-a-chip components, such as microelectrophoresis or micro-chromatographic devices for follow-up protein analysis. This selective enrichment of modified proteins greatly facilitates the study of low abundant protein biomarkers discovery.

Antigenicity of Synthetic Peptides Derived from Plasmodium Apoptosis-Linked Pathogenicity Factors

Background： Plasmodium falciparum malaria remains a major life-threatening disease. Recently, the Plasmodium apoptosis-linked pathogenicity factors （PALPF） have been identified. These antigens PALPF are expressed only by P falciparum-infected erythrocytes triggering endothelial cell apoptosis （apoptogenic）. Methods： We designed ten synthetic peptides （PI to P10） from PALPF： PF07 0032, PF10_0226, PFI0130c, PFD0875c and MAL13P1.206, and analyzed their antigenicity with an ELISA method using plasma samples from subjects living in Dienga, Gabon. Results： Four peptides showed good reactivity with human antibodies. The prevalence rate of specific IgG was 61%, 51%, 44% and 34% for P5, P6, P4 and P2, respectively. The median optical density of total IgG anti-P2 was higher than that directed against P4 and P6 （P = 0.009; P = 0.012 respectively）. The prevalence rate oflgG subclasses determined with plasma samples recognizing peptide 5 for IgGl, 2, 3 and 4 isotypes was 69%, 45%, 76% and 62%, respectively. All the subjects had at least one immunoglobulin subclass, while 13 （44%） had both IgG1 and IgG3 antibodies. There was no significant difference in the prevalence rate of anti-P5 IgG1, IgG3 and IgG4. Conclusion： These results warrant further immunogenicity studies of peptides 2, 4, 5 and 6 with a view of a tentative to antimalarial vaccine development.

Non-specific Arm Pain

Non-specific arm pain is a special clinical condition that can occur in work-related activities that involve maintaining a static position for prolonged periods or repetitive and frequent movements of the hand or entire arm. Such activities include typing on a keyboard, maneuvering a computer mouse, playing musical instruments （such as piano and guitar） and many forms of manual labor. The pain is dull and diffuse; It is localized in the forearm or in the hand but quickly can expand to the entire extremity. Non-specific arm pain is the most frequent type of work-related pain after lower-back pain. It thus has important socio-economic significance as a major cause of absence from work. The designation of ＂non-specific＂ originates from the fact that it has no obvious signs of tissue damage, unlike the ＂specific＂ pain accompanying carpal tunnel syndrome, tenosinovitis de Quervain, or lateral epicondylitis. Suggested causes of the pain include microtrauma of soft tissue followed by an inflammatory reaction, ischemia, fatigue, hyper-sensitization of nociceptors, focal dystonia of the hand and/or psychological stress. Treatment consists of application of NSAIDs, physical modalities, stretching and aerobic exercises. Prevention focuses on ergonomic modification during manual labor or work on a computer.

Clinical application of specific antibody against glypican-3 for hepatocellular carcinoma diagnosis

Glypican-3(GPC3) is a promising tumor marker for hepatocellular carcinoma(HCC) diagnosis with high sensitivity and specificity.The aim of this study was to establish an immunohistochemical detection method for GPC3 using the 7D11 monoclonal antibody(7D11 mAb) and evaluate its application for HCC diagnosis.The feasibility of the 7D11 mAb was evaluated by immunohistochemistry performed on adjacent normal liver and intrahepatic cholangiocarcinoma(ICC) samples,Furthermore,the serum GPC3 levels were evaluated in 40 HCC patients,7 ICC patients and 50 healthy donors.The results showed that GPC3 was expressed in 85% of HCC tissues(34/40),but was undetectable in ICC tissues and adjacent normal tissues.GPC3 was significantly increased in the serum of HCC patients(17/40,42.5%) but was undetectable in the serum of ICC patients(0/7,0%) and healthy donors(0/50,0%).This prospective study evaluated the clinical usefulness of 7D11 mAb for GPC3 detection in HCC patients.In conclusion,the use of 7D11 mAb might be good for GPC3 large-scale applications for clinical diagnosis of HCC.

Synthesis and characteristics of a novel artificial hapten using the copper mercaptide of penicillenic acid from penicillin G for immunoassay of heavy metal ions

In this paper, we describe the synthesis of a novel copper ion hapten using the copper mercaptide of penicillenic acid （CMPA） derived from penicillin. Results from tests with immune rabbits indicate that： （i） A new antigen synthesized with CMPA has good stability and is safe for immunizing animals with no toxic phenomena being found in animal experiments; （ii） the immunogenic antigen （CMPA-BSA） can stimulate the immune system to produce specific antibodies with high titrations, up to 150000; and （iii） antibodies in antisera showed higher affinity to OVA-GSH-CuC1 than OVA-GSH, which indicates that the antibodies have specific affinity towards copper ions. These results confirm that the novel hapten and relevant antigen for copper ion have been successfully synthesized, giving progress towards an immunoassay for copper ions in environmental and food samples.

Localization and characterization of the hypothetical protein CT440 in Chlamydia trachomatis-infected cells

The inclusion membrane proteins play potentially important roles in chlamydial biology and pathogenesis.Here we localized and characterized the hypothetical protein CT440 in Chlamydia trachomatis-infected cells.The open reading frame(ORF) encoding the CT440 protein from the C.trachomatis serovar D genome was cloned into the prokaryotic expression vector pGEX-6p and expressed as a glutathione-S-transferase(GST) fusion protein in E.coli XL1-Blue.The CT440 fusion protein was used to immunize mice to raise antigen-specific antibody.After verification by Western blot and immunofluorescence assay(IFA),the specific antibody was used to localize the endogenous CT440 protein and to detect its expression pattern in Chlamydia-infected cells.Cytosolic expression of CT440 in HeLa cells was also carried out to evaluate the effect of the CT440 protein on the subsequent chlamydial infection.The results showed that the hypothetical protein CT440 was localized in the C.trachomatis inclusion membrane,and was detectable 12 h after chlamydial infection.Expression of CT440 in the cytoplasm did not inhibit the subsequent chlamydial infection.In summary,we have identified a new inclusion membrane protein that may be an important candidate for understanding C.trachomatis pathogenesis.

Efficacy of seasonal pandemic influenza hemagglutinin DNA vaccines delivered by electroporation against aseasonal H1N1 virus challenge in mice

Prophylactic DNA vaccines against the influenza virus are promising alternatives to conventional vaccines. In this study, we generated two candidate gene-based influenza vaccines encoding either the seasonal or pandemic hemagglutinin antigen （HA） from the strains A/New Caledonia/20/99 （HIN1） （pV1AS） and A/Califorrtia/04/2009 （H1N1） （pVEH1）, respectively. After verifying antigen expression, the immunogenicity of the vaccines delivered intramuscularly with electroporation was tested in a mouse model. Sera of immunized animals were tested in hemagglutination inhibition assays and by ELISA for the presence of HA-specific antibodies. HA-specific T-cells were also measured in IFN-γ ELISpot assays. The protective efficacy of the candidate influenza vaccines was evaluated by measuring mortality rates and body weight after a challenge with 100 LD50 of mouse-adapted A/New Caledonia/20/99 （H1N1）. Mice immunized with either one of the two vaccines showed significantly higher T cell and humoral immune responses （P〈0.05） than the pVAX1 control group. Additionally, the pV1A5 vaccine effec- tively protected the mice against a lethal homologous mouse-adapted virus challenge with a survival rate of 100% compared with a 40% survival rate in the pVEH1 vaccinated group （P〈0.05）. Our study indicates that the seasonal influenza DNA vac- cine completely protects against the homologous A/New Caledonia/20/99 virus （H1N1）, while the pandemic influenza DNA vaccine only partially protects against this virus.

Screening and identification of human Zn T8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library

Zinc transporter 8 （ZnT8） is a major autoantigen and a predictive marker in type 1 diabetes （T1D）. To investigate ZnT8-specific antibodies, a phage display library from TID was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment （scFv） phage display library consists of approximately 1 ~ l0s clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Topl0F＇ and then purified by affin- ity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D.

NK cell-based cancer immunotherapy: from basic biology to clinical application

Natural killer(NK) cells, which recognize and kill target cells independent of antigen specificity and major histocompatibility complex(MHC) matching, play pivotal roles in immune defence against tumors. However, tumor cells often acquire the ability to escape NK cell-mediated immune surveillance. Thus, understanding mechanisms underlying regulation of NK cell phenotype and function within the tumor environment is instrumental for designing new approaches to improve the current cell-based immunotherapy. In this review, we elaborate the main biological features and molecular mechanisms of NK cells that pertain to regulation of NK cell-mediated anti-tumor activity. We further overview current clinical approaches regarding NK cell-based cancer therapy, including cytokine infusion, adoptive transfer of autologous or allogeneic NK cells, applications of chimeric antigen receptor(CAR)-expressing NK cells and adoptive transfer of memory-like NK cells. With these promising clinical outcomes and fuller understanding the basic questions raised in this review, we foresee that NK cell-based approaches may hold great potential for future cancer immunotherapy.