Comparative study on English immunology textbooks / 中华医学教育探索杂志

Immunology is a kind of important basic subject for medical education, involving theories and technologies covering many aspects of disease occurrence, diagnosis, prevention and treatment, and is also the source of new means of medical treatment in the future. Therefore, successful teaching of medical immunology is associated with high-level medical undergraduate education. An appropriate textbook is critical for curriculum construction of immunology. However, little analysis of immunology textbooks has been published. We selected several famous and popular English immunology textbooks and compared their teaching objectives, methods, and contents, concluding the features and targeting readers of each textbooks. Our research results could provide some advices for students to learn immunology theory and researchers to utilize immunology methods, and also give a glimpse of the development trend and direction of immunology.

Human monoclonal antibodies block the binding of SARS-CoV-2 spike protein to angiotensin converting enzyme 2 receptor

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel corona virus disease (COVID-19). To date, no prophylactic vaccines or approved therapeutic agents are available for preventing and treating this highly transmittable disease. Here we report two monoclonal antibodies (mAbs) cloned from memory B cells of patients recently recovered from COVID-19, and both mAbs specifically bind to the spike (S) protein of SARS-CoV-2, block the binding of receptor binding domain (RBD) of SARS-CoV-2 to human angiotensin converting enzyme 2 (hACE2), and effectively neutralize S protein-pseudotyped virus infection. These human mAbs hold the promise for the prevention and treatment of the ongoing pandemic of COVID-19.

The Novel Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2) Directly Decimates Human Spleens and Lymph Nodes

While lymphocytopenia is a common characteristic of patients infected by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the mechanisms responsible for this depletion are unclear. Through careful inspection of the spleens and lymph nodes (LNs) from six cases with postmortem examinations, we observed that SARS-CoV-2 could directly infect secondary lymphoid organs to induce cell death. Immunohistochemistry demonstrated ACE2 (angiotensin-converting enzyme 2), the potential receptor of SARS-CoV-2, expresses on tissue-resident CD169+ macrophages in spleens and LNs. Immunofluorescent staining confirmed that viral nucleocaspid protein (NP) can be found in ACE2+ cells, CD169+ macrophages, but not in CD3+ T cells or B220+ B cells in spleens and LNs. SARS-CoV-2 infection induces severe tissue damage including lymph follicle depletion, splenic nodule atrophy, histiocyte hyperplasia and lymphocyte reductions. Moreover, in situ TUNEL staining illustrated that viral infection leads to severe lymphocyte apoptosis, which might be mediated by viral antigens inducing Fas upregulation. Furthermore, SARS-CoV-2 also triggers macrophages to produce IL-6, a proinflammatory cytokine that directly promotes lymphocyte necrosis. Collectively, these results demonstrate that SARS-CoV-2 directly neutralizes human spleens and LNs through infecting tissue-resident CD169+ macrophages.

Diagnosis of Acute Respiratory Syndrome Coronavirus 2 Infection by Detection of Nucleocapsid Protein

BACKGROUNDNucleic acid test and antibody assay have been employed in the diagnosis for SARS-CoV-2 infection, but the use of viral antigen for diagnosis has not been successfully developed. Theoretically, viral antigen is the specific marker of the virus and precedes antibody appearance within the infected population. There is a clear need of detection of viral antigen for rapid and early diagnosis. METHODSWe included a cohort of 239 participants with suspected SARS-CoV-2 infection from 7 centers for the study. We measured nucleocapsid protein in nasopharyngeal swab samples in parallel with the nucleic acid test. Nucleic acid test was taken as the reference standard, and statistical evaluation was taken in blind. We detected nucleocapsid protein in 20 urine samples in another center, employing nasopharyngeal swab nucleic acid test as reference standard. RESULTSWe developed a fluorescence immunochromatographic assay for detecting nucleocapsid protein of SARS-CoV-2 in nasopharyngeal swab sample and urine within 10 minutes. 100% of nucleocapsid protein positive and negative participants accord with nucleic acid test for same samples. Further, earliest participant after 3 days of fever can be identified by the method. In an additional preliminary study, we detected nucleocapsid protein in urine in 73.6% of diagnosed COVID-19 patients. CONCLUSIONSThose findings indicate that nucleocapsid protein assay is an accurate, rapid, early and simple method for diagnosis of COVID-19. Appearance of nucleocapsid protein in urine coincides our finding of the SARS-CoV-2 invading kidney and might be of diagnostic value.

Human Kidney is a Target for Novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection

BACKGROUNDThe outbreak of a novel coronavirus (SARS-CoV-2, previously provisionally named 2019 novel coronavirus or 2019-nCoV) since December 2019 in Wuhan, China, has become an emergency of major international concern. Apart from the respiratory system, it is unclear whether SARS-CoV-2 can also directly infect other tissues such as the kidney or induce acute renal failure. METHODSWe conducted a retrospective analysis of estimated glomerular filtration rate (eGFR) along with other clinical parameters from 85 patients with laboratory-confirmed COVID-19 admitted to a hospital in Wuhan from January 17, 2020 to March 3, 2020. Kidney tissues from six patients with postmortem examinations were analyzed by Hematoxylin and Eosin (H&E) and in situ expression of viral nucleocaspid protein (NP) antigen, immune cell markers (CD8, CD68 and CD56) and the complement C5b-9 was detected by immunohistochemistry. Moreover, the viral particles in kidneys were also investigated by transmission electronic microscope (EM). RESULTS27.06% (23/85) patients exhibited acute renal failure (ARF). The eldery patients and cases with comorbidities such as hypertension and heart failure more easily developed ARF (65.22% vs 24.19%, p< 0.001; 69.57% vs 11.29%, p< 0.001, respectively). H&E staining demonstrated kidney tissues from postmortems have severe acute tubular necrosis and lymphocyte infiltration. Immunohistochemistry showed that SARS-CoV-2 NP antigen was accumulated in kidney tubules. EM observation also demonstrated that viruses-like particles are visible in the kidneys. Viral infection not only induces CD68+ macrophages infiltrated into tubulointerstitium, but also enhances complement C5b-9 deposition on tubules. CONCLUSIONSSARS-CoV-2 induces ARF in COVID-19 patients. Viruses directly infect human kidney tubules to induce acute tubular damage. The viruses not only have direct cytotoxicity, but also initiate CD68+ macrophage together with complement C5b-9 deposition to mediate tubular pathogenesis.

Reduction and Functional Exhaustion of T Cells in Patients with Coronavirus Disease 2019 (COVID-19)

BACKGROUNDThe outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed great threat to human health, which has been declared a public health emergency of international concern (PHEIC) by the WHO. T cells play a critical role in antiviral immunity but their numbers and functional state in COVID-19 patients remain largely unclear. METHODSWe retrospectively reviewed the counts of total T cells, CD4+, CD8+ T cell subsets, and serum cytokine concentration from inpatient data of 522 patients with laboratory-confirmed COVID-19, admitted into two hospitals in Wuhan from December 2019 to January 2020, and 40 healthy controls, who came to the hospitals for routine physical examination. In addition, the expression of T cell exhaustion markers PD-1 and Tim-3 were measured by flow cytometry in the peripheral blood of 14 COVID-19 cases. RESULTSThe number of total T cells, CD4+ and CD8+ T cells were dramatically reduced in COVID-19 patients, especially among elderly patients ([>=]60 years of age) and in patients requiring Intensive Care Unit (ICU) care. Counts of total T cells, CD8+T cells or CD4+T cells lower than 800/L, 300/L, or 400/L, respectively, are negatively correlated with patient survival. Statistical analysis demonstrated that T cell numbers are negatively correlated to serum IL-6, IL-10 and TNF- concentration, with patients in decline period showing reduced IL-6, IL-10 and TNF- concentrations and restored T cell counts. Finally, T cells from COVID-19 patients have significantly higher levels of the exhausted marker PD-1 as compared to health controls. Moreover, increasing PD-1 and Tim-3 expression on T cells could be seen as patients progressed from prodromal to overtly symptomatic stages, further indicative of T cell exhaustion. CONCLUSIONST cell counts are reduced significantly in COVID-19 patients, and the surviving T cells appear functionally exhausted. Non-ICU patients, with total T cells, CD8+T cells CD4+T cells counts lower than 800/L, 300/L, and 400/L, respectively, may still require aggressive intervention even in the immediate absence of more severe symptoms due to a high risk for further deterioration in condition.

Research capacity training for graduate students in basic medical sciences with the guideline of translational medicine / 中华医学科研管理杂志

In China,few research findings in basic medicine could be translated to clinical practice,which is known as the disconnection between basic research and clinical application.Such phenomenon is mainly due to lack of ability in translational medicine,particularly,lack of training in clinical research and translational medicine.Now the emergence of translational medicine has put forward new requirements for graduate education in medical school,and demanding that the patient-centered concept be strengthened.As the major force of scientific and technological innovation,graduate students should receive long-term systematic trainings and conduct original researches.In addition,advanced courses in clinical research and translational medicine should be offered to graduate students ;furthermore,translation research platforms should be built up for them so as to improve the capacity of scientific research and innovation.

Construction,prokaryotic expression and purification of FimH1-156 fusion protein / 重庆医学

Objective To construct and express a prokaryotic expression vector carrying the gene of FimH 1-156 that comprises human lysosome membrane protein 2 P41-49 gene ,and to express and purify the fusion protein .Methods FimH1-156 gene was cloned from plasmid pPKL241 by PCR ,and inserted into vector pET-28a(+ ) to obtain prokaryotic expression plasmid pET-28a-FimH . After transforming Escherichia coli BL21(DE3) with pET-28a-FimH ,fusion protein FimH1-156 was expressed under induction .The target fusion protein was purified ,and its antigenicity was detected through Western blot .Results The expressed recombinant pro-tein was purified ,the expression of protein was the highest when IPTG was 1 mmol/L and 4h after induction ,it was expressed as include body form ,and the expressed protein was identified to react with monoclonal antibodies 6 × His by Western blotting .Conclu-sion We cloned FimH1-156 fusion protein expressed genes successfully ,constructed prokaryotic expression vector ,and won the in-clusion body purification of FimH1-156 fusion protein .

Medical Ph.D candidate cultivating models in two universities of America and Canada / 中华医学教育探索杂志

Ph.D candidate education is the highest level of higher education. Training model of Ph.D candidate in Medical College of Georgia and University of Manitoba)has vivid characters compared with that in China,which is reflected by the training objective,qualification of students and tutors,culti-vating procedures and admission requirements for graduation. This kind of cultivating model performs stringent selection and can gradually pick out persons who are really fit for the scientific research. Ph.D candidate quality in the two universities is guaranteed by systemic and deep courses learning,immediate update of knowledge and strict evaluation system. The goal of this article is to provide experience and ref-erence for improving the education quality of medical Ph.D candidates in China.

Transposition thinking in medical immunology teaching / 中华医学教育探索杂志

Medical Immunology is a multidisciplinary basic discipline,but its' abstract and complex leading most beginners daunting.Teachers should emphasize on ‘ transposition thinking' and stick the teaching concept of ‘ students-centered and teacher-guided learning' to the whole teaching process.Teacher should always make transposition thinking on how to stimulate students learning interest,how to guide students to learn autonomously,and how to make problem-based learning.This is important to build self-learning platform for the students and to achieve‘teaching' and ‘learning'win-win.

Practice and optimization of Proteomics curriculum for postgraduates in medical college and university / 中华医学教育探索杂志

The curriculum system of Proteomics was analyzed based on the teaching practice,the characteristics of ability training and gradation teaching were summarized and the prospect of curriculum optimization was proposed.These measures were conceived to enrich the course content and teaching methods for Proteomics course.

Immunogenicity of the immunodominant cytotoxic T lymphocyte epitope E749-57 in HPV16 oncoprotein E7 chaperoned by HSP110 / 中华皮肤科杂志

Objective To investigate the immunogenicity of immunodominant cytotoxic T lymphocyte epitope E749-57 of human papilloma virus (HPV) 16 oncoprotein E7 chaperoned by heat shock protein (HSP)110. Methods Mouse HSP110 gene was cloned into prokaryotic expression vector pQE-80L for the expression of HSP110 protein, which was purified using Ni-NTA column. SDS-PAGE and Western-blot were conducted to confirm the purified mHSP110 protein, which was subsequently incubated with E749-57 peptide under heat shock condition, and high-performance liquid chromatography (HPLC) was used to evaluate the binding efficiency of the recombinant protein and E749-57 peptide. Twenty mice were divided into 4 groups to be immunized with mHSP110 protein, E749-57 peptide, mHSP110-E749-57 complex and phosphate buffered saline (PBS),respectively. Two weeks after the last immunization, spleen cells were collected from the immunized mice and divided into 2 parts: one were stimulated by E749-57 peptide followed by the detection of CD8+ INF-γ+ T cells with flow cytometry; the other one were subjected to MTT analysis for the estimation of cell proliferation. The mHSP110-E749-57 complex was also used to immunize TC-1 tumor bearing mice to observe its anti-tumor effect.Results The full-length 2577 bp-sized mHSP110 gene was amplified from mouse liver cDNA and cloned into pQE-80L vector. Direct sequencing confirmed the correctness of the cloning. SDS-PAGE and Western-blot demonstrated the successful purification of mHSP110. HPLC assay showed that the purified mHSP110 protein could bind with E749-57 to form a relatively stable protein complex. The percentage of IFN-γ+ CD8+ T cells in and proliferation index of spleen cells from the complex-immunized mice were statistically higher than those from the other 3 groups of mice. Moreover, the complex could obviously inhibit the growth of TC-1 tumor in mice. Conclusion The mHSP110-E749-57 complex could enhance the generation of specific cytotoxic T lymphocytes and exert anti-tumor effects in mice.

Prediction of the secondary structure and B cell epitopes for the Izumo protein of Homo Sapiens / 中国免疫学杂志

Objective:To predict and analyze the secondary structure and B cell epitopes of Izumo protein.Methods: The secondary structure and flexible regions of Izumo protein were predicted by the methods of Chou-Fasman,Gamier-Robson and Karplus-Schulz.Moreover,hydrophilicity plot,surface probability plot and antigenic index of Izumo protein were predicted by the methods of Kyte-Doolitde,Emini and Jameson-Wolf,respectively.Results: Izumo protein contained moreαhelix regions.There were several centers ofαhelix in the regions of 6-17,30-40,88-99,103-120,153-160,173-188,249-260,283-297,334-338 and 339-346 of Izumo protein,and several centers of βsheet in the regions of 21-25,198-200,245-248 and 320-323.Moreover,many distinct B cell epitopes in Izumo protein possibly localized in the regions of 3642,62-66,94-99,118-122,129-132,151-154,161-164,173-177,205-208,212-216,256-265,271-276,283-288,314-318 and 336-350.Conclusion:These results are helpful for identification of the dominant B cell epitopes and the functional domains of Izumo protein.

Toward the quantitative prediction of T-cell epitopes: QSAR studies on peptides having affinity with the class I MHC molecular HLA-A*0201.

It would be useful for vaccine development to develop a method of rapidly identifying peptide epitopes. In this paper, the empirical three-dimensional quantitative structure-affinity relationship (3D-QSAR) methods were used to study the relationship between the three dimensional structural parameters (the isotropic surface area, ISA, and the electronic charge index, ECI) of the HLA-A*0201 binding peptide and the HLA-A*0201/peptide binding affinities. A set of 102 peptides having affinity with the class I MHC HLA-A*0201 molecule was used as training set. A test set of 40 peptides was used to determine the predictive value of the models. The 3D-QSAR models yielded a q2 = 0.5724 and a high rpred2 = 0.6955. The standard regression coefficients indicated that the hydrophobic interactions played an important role in peptide-MHC molecule binding and predicted the specific amino acid residue essential at a certain position of the peptide. The approach tested in the current paper is highly complementary to many of the methods described in references and possesses good predictability. It is a rapid and convenient method to detect high affinity peptide epitopes.

Differential microRNA expression profile of HepG2 cells induced by HBV integration / 第三军医大学学报

Objective To investigate the differential expressions of microRNA after HBV integration. Methods A human microRNA microarray was used to analyze the microRNA expression profile in HepG2.2.15 cell line, a novel model of HBV infection. HepG2 cells served as a mock control. Then the microRNA with differential expression were proved using real-time PCR. Results HBV infection induced 6 microRNA down-regulated (hsa-miR-30a-5p, hsa-miR-24, hsa-let-7a, hsa-let-7c, hsa-let-7f and hsa-miR-23b) and 3 microRNA up-regulated (hsa-miR-194, hsa-miR-200a and hsa-miR-345). Conclusion HBV integration can induce the changes of microRNA expression profile in hepatocytes.

Expression of human hDAF in CHO cells and its decay-accelerating activity / 第三军医大学学报

Objective To obtain Chinese hamsterovary (CHO) cell line expressing human decay accelerating activity (hDAF) stably and to observe the protective effect of hDAF on heterologous cells under the circumstance of complement activation. Methods The eukaryotic expression vector DAF pcDNA3.1 was constructed and then transfected into CHO cells by lipofection. Monoclones of cells expressing hDAF stably were screened by the method of limiting dilution. hDAF expression was detected by flow cytometry. The decay accelerating activity of hDAF was determined by assay of C3 deposition and 51Cr release. Results The expression vector DAF pcDNA3.1 was successfully constructed, and monoclones of cells expressing hDAF were obtained. CHO cells expressing hDAF could decrease C3 deposition and attenuate the killing effect of activation of the complement system. Conclusion We have obtained CHO cell clones expressing hDAF stably, which is helpful for the further studies of the relationship of the structure with the functions of hDAF.

Establishment and evaluation of animal tumor model expressing HER2/neu / 第三军医大学学报

Objective To establish animal tumor model expressing human oncogene HER2/neu and to evaluate the model by immunological method. Methods The recombinant eukaryotic plasmid expressing HER2/neu gene was transfected into P815 tumor cells. The tumor cell line P815 expressing HER2/neu gene stably was screened by limiting dilution method. The expression of target gene in host cells was determined by Western blotting. Following the establishment of animal tumor model expressing HER2 antigen in DBA/2 mice, tumor specific response was induced in the spleen cells of DBA/2 mice which were inoculated with recombinant plasmids. Results P815 cell line expressing HER2/neu oncogene stably was obtained. HER2/neu specific CTLs could be induced from immunized DBA/2 mice. Conclusion HER2/neu expressing tumor animal model is established successfully and the in vivo immune response can be induced after immunization with HER2/neu genetic vaccine.

Preparation of recombinant alpha-virus / 第三军医大学学报

Objective To explore a new simpler method for the preparation of recombinant alpha virus as a novel vaccine at the DNA level. Methods Plasmids expressing ? gal protein and helper plasmids were transfected into BHK cells. Virus in culture supernatant of the transfected BHK cells were collected and purified and used to infect BHK cells in vitro to identify the expression of target gene and the titre of the recombinant virus. Results Recombinant virus with high titre, prepared by this method, could be expressed well in mammalian cells in vitro . Conclusion High titre recombinant alpha virus can be produced at the DNA level and this method can be applied for vaccine preparation and gene therapy.

Mutation Site of Cu/Zn superoxide dismutase in an amyotrophic lateral sclerosis family / 医学研究生学报

Objective: To identify the mutation points of Cu/Zn superoxide dismutase(SOD1) gene in an amyotrophic lateral sclerosis(ALS) family with a unique phenotype,and to compare the value of single strand conformation polymorphism(SSCP) and denaturing high performance liquid chromatography(DHPLC). Methods: Five exons of SOD1 gene were amplified by PCR. The difference of these products were analyzed by PCR-SSCP and DHPLC.DNA sequencing was used to examine the mutation. Results: ①Mutations were found in exons 2 and 5 in several family members.DNA sequencing revealed that a base pair insertion occurred in the codon area of exon 2 and in the non-codon area of exon 5.②The results of DHPLC tests proved double peaks in one member with ALS symptoms(Ⅲ1),which indicated the possibility of mutation in SOD1 exon 4.DNA sequencing revealed that there was a heterozygote,with a mutation of GAA to GGA in exon 4 in the member with double peak. Conclusion: ①The mutations in exons 2,4,5 were proved.Insertion of exon 2 may be responsible for the disease of the ALS family in Chongqing.②Compared with PCR-SSCP,DHPLC technique has been proven to be a rapid and reliable method for screening mutation site in large samples.

Immunity suppressive effect and possible mechanism of SEB as a superantigen / 第三军医大学学报

Objective To study the immunity suppressive effect of the staphylococcal enterotoxin as a super-antigen and investigate its mechanism.Methods BALB/c mice aged 8-12 weeks were randomly assigned to receive 0.2 ml injection of 50 ?g/ml staphylococcal enterotoxin B(SEB)(n=20) or 0.2 ml physiological saline(n=20).One day later,all mice were sacrifice to collect the splenocytes which were employed to detect the expression of TGF-?1 and to countthe cells expressing CD4 and CD25 by flow cytometry(FCM).Results FMC showed that a remarkable increase of cells that expressed CD4 and CD25 in the SEB-primed splenocytes as compared with the saline primed splenocytes.Conclusion SEB,which is used as a superantigen in vivo,can induce the regulatory cells bearing suppressive activity.This may be partial mechanism of SEB-induced hyporesponsiveness.