Immunogenicity of Whole Mycobacterium intracellulare Proteins and Fingding on the Cross-Reactive Proteins between M.intracellulare and M.tuberculosis

Objectives To evaluate the immunogenicity of Mycobacterium intracellulare proteins and determine the cross-reactive proteins between M.intracellulare and M.tuberculosis.Methods Protein extracts from M.intracellulare were used to immunize BALB/c mice.The antigens were evaluated using cellular and humoral immunoassays.The common genes between M.intracellular and M.tuberculosis were identified using genome-wide comparative analysis,and cross-reactive proteins were screened using immunoproteome microarrays.Results Immunization with M.intracellulare proteins induced significantly higher levels of the cytokines interferon-γ(IFN-γ),interleukin-2(IL-2),interleukin-12(IL-12),interleukin-6(IL-6)and immunoglobulins IgG,IgG1,IgM,and IgG2a in mouse serum.Bone marrow-derived macrophages isolated from mice immunized with M.intracellulare antigens displayed significantly lower bacillary loads than those isolated from mice immunized with adjuvants.Whole-genome sequence analysis revealed 396 common genes between M.intracellulare and M.tuberculosis.Microchip hybridization with M.tuberculosis proteins revealed the presence of 478 proteins in the serum of mice immunized with M.intracellulare protein extracts.Sixty common antigens were found using both microchip and genomic comparative analyses.Conclusion This is the advanced study to investigate the immunogenicity of M.intracellulare proteins and the cross-reactive proteins between M.intracellulare and M.tuberculosis.The results revealed the presence of a number of cross-reactive proteins between M.intracellulare and M.tuberculosis.Therefore,this study provides a new way of identifying immunogenic proteins for use in tuberculosis vaccines against both M.intracellulare and M.tuberculosis in future.

Evaluation of cross-reactive antibody response to HVR1 in chronic hepatitis C

AIM:To evaluate the presence and cross-reactive anti-bodies against hypervariable region 1(HVR1) in hepatitis C virus(HCV) infected patients and its relationship with the progression of the disease.METHODS:Sixteen representative HVR1 proteins selected from a unique set of 1600 natural sequences were used to semiquantitate the cross-reactivity of HVR1 antibodies in the sera of HCV patients.Fifty-five chronic HCV patients including 23 with asymptomatic mild hepatitis,18 with chronic hepatitis and 16 with liver cirrhosis patients were studied.RESULTS:The degree of the cross-reactivity of anti-HVR1 antibodies in 23 patients with mild asymptomatic hepatitis was 3.09 ± 2.68,which was signif icantly lower than in those with chronic hepatitis(5.44 ± 3.93,P < 0.05) and liver cirrhosis(7.44 ± 3.90,P < 0.01).No correlation was observed between the broadness of the cross-reactivity anti-HVR1 antibodies and patient's age,infection time,serum alanine aminotransferase activity,or serum HCV-RNA concentration.It was the breath of cross-reactivity rather than the presence of anti-HVR1 antibody in HCV sera that was associated with the progression of liver disease.CONCLUSION:The broadly cross-reactive HVR1 antibodies generated in natural HCV patients can not neutralize the virus,which results in persistent infection in patients with chronic hepatitis.

Diagnosis of West Nile virus infections:Evaluation of different laboratory methods

BACKGROUND The diagnosis of West Nile virus(WNV)is challenging due to short-term and low-level viremia,flavivirus cross-reactivity,and long immunoglobulin M(IgM)persistence.AIM To evaluate different methods for WNV detection[reverse transcription-polymerase chain reaction(RT-PCR),IgM/IgG antibodies,IgG avidity]in serum,cerebrospinal fluid(CSF),and urine samples of patients with confirmed WNV infection.METHODS The study included patients with confirmed WNV neuroinvasive infection(n=62),asymptomatic WNV seropositive individuals(n=22),and individuals with false-positive WNV IgM antibodies(n=30).WNV RNA was detected using RT-PCR.A commercial ELISA was used to detect WNV IgM/IgG antibodies with confirmation of cross-reactive samples using a virus neutralization test(VNT).IgG-positive samples were tested for IgG avidity.RESULTS The WNV-RNA detection rates were significantly higher in the urine(54.5%)/serum(46.4%)than in CSF(32.2%).According to the sampling time,the WNV-RNA detection rates in urine collected within 7 days/8-14/≥15 days were 29.4/66.6/62.5%(P=0.042).However,these differences were not observed in the CSF.The median RT-PCR cycle threshold values were significantly lower in urine(32.5,IQR=28-34)than in CSF(34.5,IQR=33-36).The frequency of positive WNV IgM and IgG significantly differed according to the sampling time in serum but not in CSF.Positive IgM/IgG antibodies were detected in 84.3/9.3%of serum samples collected within 7 days,100/71.1%of samples collected 8-14,and 100%samples collected after≥15 days.Recent WNV infection was confirmed by low/borderline avidity index(AI)in 13.6%of asymptomatic individuals.A correlation between ELISA and AI was strong negative for IgM and strong positive for IgG.No significant correlation between ELISA IgG and VNT was found.CONCLUSION The frequency of WNV RNA and antibody detection depends on the sampling time and type of clinical samples.IgG avidity could differentiate recent WNV infections from long-persisting IgM antibodies.

Cross-reactivity between aeroallergens and food allergens

In patients with respiratory allergy,cross-reactivity between aeroallergens and foods may induce food allergy,symptoms ranging from oral allergy syndrome to severe anaphylaxis.Clinical entities due to Ig E sensitization to cross-reactive aeroallergen and food allergen components are described for many sources of plant origin(pollen-food syndromes and associations,such as birch-apple,cypress-peach and celery-mugwortspice syndromes,and mugwort-peach,mugwortchamomile,mugwort-mustard,ragweed-melon-banana,goosefoot-melon associations),fungal origin(Alternariaspinach syndrome),and invertebrate,mammalian or avian origin(mite-shrimp,cat-pork,and bird-egg syndromes).Clinical cases of allergic reactions to ingestion of food products containing pollen grains of specific plants,in patients with respiratory allergy to Asteraceae pollen,especially mugwort and ragweed,are also mentioned,for honey,royal jelly and bee polen dietary supplements,along with allergic reactions to foods contaminated with mites or fungi in patients with respiratory allergy to these aeroallergens.Medical history and diagnosis approach may be guided by the knowledge about the diverse cross-reacting allergens involved,and by the understanding of these clinical entities which may vary significantly or may be overlapping.The association between primary Ig E sensitization with respiratory symptoms to inhaled allergens and food allergy due to cross-reactive allergen components is important to assess in allergy practice.The use of molecular-based diagnosis improves the understanding of clinically relevant Ig E sensitization to cross-reactive allergen components from aeroallergen sources and foods.

Studies on Preparation and Characteristics of Monoclonal Antibodies Against Enrofloxacin and Cross-Reactivity of Related Fluoroquinolones

An ester activation method was employed to couple enrofloxacin(ENFX) to the carrier proteins BSA and OVA. The conjugates ENFX-BSA and ENFX-OVA were identified with an UV spectrophotometer and amino acid automation analysis instrument, and resulted in conjugates with 48 ENFX molecules per carrier molecule(BSA). Splenocytes from mice immunized with ENFX-BSA were fused with SP2/0 myeloma cells and hybridomas secreting antibodies against enrofloxacin were selected and cloned. Two stable monoclonal antibodies, 2C5, 5D5 of the subclass IgG2a, were isolated. Using antibody 5D5, an indirect competitive inhibition enzyme-linked immunosorbent assay (Ci-ELISA) was developed for the quantitative detection of enrofloxacin and its metabolites. The IC50 of the standard curve was 21.67 ng mL-1 and the limit of detection for enrofloxacin was 0.13 ng mL-1. This method was sensitive and had a linear range from 0.13 to 10 000 ng mL-1 (r= -0.9782). Monoclonal antibody 5D5 exhibited high relative affinity to enrofloxacin, and the cross-reactivities with ciprofloxacin, marbofloxacin, sarafloxacin and danorfloxacin were 110.8, 27.40, 71.05 and 37.41%, respectively. Three non-fluoroquinolones of cefadroxil, chloramphenicol, sulfadimethoxine were tested and there was no cross-reaction between them.

Cross-Reaction between Gliadin and Different Food and Tissue Antigens

A subgroup of coeliac disease patients continues to experience symptoms even on a gluten-free diet (GFD). We attempted to determine whether these symptoms could be due to either cross-contamination with gluten-containing foods or cross-reactivity between α-gliadin and non-gluten foods consumed on a GFD. We measured the reactivity of affinity-purified polyclonal and monoclonal α-gliadin 33-mer peptide antibodies against gliadin and additional food antigens commonly consumed by patients on a GFD using ELISA and dot-blot. We also examined the immune reactivity of these antibodies with various tissue antigens. We observed significant immune reactivity when these antibodies were applied to cow’s milk, milk chocolate, milk butyrophilin, whey protein, casein, yeast, oats, corn, millet, instant coffee and rice. To investigate whether there was cross-reactivity between α-gliadin antibody and different tissue antigens, we measured the degree to which this antibody bound to these antigens. The most significant binding occurred with asialoganglioside, hepatocyte, glutamic acid decarboxylase 65, adrenal 21-hydroxylase, and various neural antigens. The specificity of anti-α-gliadin binding to different food and tissue antigens was demonstrated by absorption and inhibition studies. We also observed significant cross-reactivity between α-gliadin 33-mer and various food antigens, but some of these reactions were associated with the contamination of non-gluten foods with traces of gluten. The consumption of cross-reactive foods as well as gluten-contaminated foods may be responsible for the continuing symptoms presented by a subgroup of patients with coeliac disease. The lack of response of some CD patients may also be due to antibody cross-reactivity with non-gliadin foods. These should then be treated as gluten-like peptides and should also be excluded from the diet when the GFD seems to fail.

Visual Detection of Vibrio parahaemolyticus using Combined CRISPR/Cas12a and Recombinase Polymerase Amplification

Objective To establish an ultra-sensitive,ultra-fast,visible detection method for Vibrio parahaemolyticus(VP).Methods We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a(CRISPR/Cas12a)combined with recombinase polymerase amplification and visual detection(CRISPR/Cas12a-VD).Results CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10^(-18)M(single molecule detection)within 30 min without cross-reactivity against other bacteria.When detecting pure cultures of VP,the consistency of results reached 100%compared with real-time PCR.The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10^(2)CFU/g.Conclusion The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods,such as real-time PCR,and has great potential for preventing the spread of pathogens.

Reaction of antibodies to Campylobacter jejuni and cytolethal distending toxin B with tissues and food antigens

BACKGROUND The bacteria Campylobacter jejuni(C. jejuni) is commonly associated with GuillaneBarré syndrome(GBS) and irritable bowel syndrome(IBS), but studies have also linked it with Miller Fisher syndrome, reactive arthritis and other disorders, some of which are autoimmune. It is possible that C. jejuni and its toxins may be crossreactive with some human tissues and food antigens, potentially leading to autoimmune responses.AIM To measure the immune reactivity of C. jejuni and C. jejuni cytolethal distending toxin(Cdt) antibodies with tissue and food antigens to examine their role in autoimmunities.METHODS Using enzyme-linked immunosorbent assay(ELISA) methodology, specific antibodies made against C. jejuni and C. jejuni Cdt were applied to a variety of microwell plates coated with 45 tissues and 180 food antigens. The resulting immunoreactivities were compared to reactions with control wells coated with human serum albumin(HSA) which were used as negative controls and with wells coated with C. jejuni lysate or C. jejuni Cdt which served as positive controls.RESULTS At 3 SD above the mean of control wells coated with HSA or 0.41 OD, the mouse monoclonal antibody made against C. jejuni showed moderate to high reactions with zonulin, somatotropin, acetylcholine receptor, β-amyloid and presenilin.This immune reaction was low with an additional 25 tissue antigens including asialoganglioside, and the same antibody did not react at all with another 15 tissue antigens. Examining the reaction between C. jejuni antibody and 180 food antigens, we found insignificant reactions with 163 foods but low to high immune reactions with 17 food antigens. Similarly, we examined the reaction of C. jejuni Cdt with the same tissues and food antigens. The strongest reactions were observed with zonulin, intrinsic factor and somatotropin. The reaction was moderate with 9 different tissue antigens including thyroid peroxidase, and reaction was low with another 10 different antigens, including neuronal antigens.The reaction of C. jejuni Cdt antibody with an additional 23 tissue antigens was insignificant. Regarding the reaction of C. jejuni Cdt antibody with different food antigens, 160 out of 180 foods showed insignificant reactions, while 20 foods showed reactions ranging from low to high.CONCLUSION Our findings indicate that C. jejuni and its Cdt may play a role in inflammation and autoimmunities beyond the gut.

Partial overlap of anti-mycobacterial,and anti-Saccharomyces cerevisiae mannan antibodies in Crohn's disease

AIM： To test whether humoral immune reaction against mycobacteria may play a role in anti- Saccharomyces cerevisiae antibodies （ASCA） generation in Crohn＇s disease （CD） and/or whether it correlates with clinical subtypes. METHODS： The dominant ASCA epitope was detected by Galanthus nivalis lectin （GNL）-binding assay. ASCA and IgG against mycobacterial lysates （M avium, M smegmatis, M chelonae, M bovis BCG M avium ssp. paratuberculosis （MAP）] or purified lipoarabinomannans （LAM） were detected by ELISA. ASCA and anti-mycobacterial antibodies were affinity purified to assess cross-reactivities. Anti-mycobacterial IgG were induced by BCG-infection of mice. RESULTS： GNL bound to different extents to mycobacterial lysates, abundantly to purified mannosecapped （Man） LAM from M tuberculosis, but not to uncapped LAM from M srnegrnatis. Fifteen to 45% of CD patients but only 0%-6% of controls were seropositive against different mycobacterial antigens. Anti-mycobacterial IgG correlated with ASCA （r = 0.37-0.64; P = 0.003-P 〈 0.001）. ASCA-positivity and deficiency for mannan-binding lectin synergistically associated with anti-mycobacterial IgG. In some patients, anti-mycobacterial antibodies represent crossreactive ASCA. Vice-versa, the predominant fraction of ASCA did not cross-react with mycobacteria. Finally, fistulizing disease associated with antibodies against M avium, M smegmatis and MAP （P = 0.024, 0.004 and 0.045, respectively）. CONCLUSION： Similar to ASCA, seroreactivity against mycobacteria may define CD patients with complicated disease and a predisposition for immune responses against ubiquitous antigens. While in some patients anti-mycobacterial antibodies strongly cross-react with yeast mannan; these cross-reactive antibodies only represent a minor fraction of total ASCA. Thus, mycobacterial infection unlikely plays a role in ASCA induction.

Viral infections in orthopedics: A systematic review and classification proposal

BACKGROUND Although the impact of microbial infections on orthopedic clinical outcomes is well recognized,the influence of viral infections on the musculoskeletal system might have been underestimated.AIM To systematically review the available evidence on risk factors and musculoskeletal manifestations following viral infections and to propose a pertinent classification scheme.METHODS We searched MEDLINE,Cochrane Central Register of Controlled Trials(CENTRAL),the Reference Citation Analysis(RCA),and Scopus for completed studies published before January 30,2021,to evaluate risk factors and bone and joint manifestations of viral infection in animal models and patient registries.Quality assessment was performed using SYRCLE's risk of bias tool for animal studies,Moga score for case series,Wylde score for registry studies,and Newcastle-Ottawa Scale for case-control studies.RESULTS Six human and four animal studies were eligible for inclusion in the qualitative synthesis.Hepatitis C virus was implicated in several peri-and post-operative complications in patients without cirrhosis after major orthopedic surgery.Herpes virus may affect the integrity of lumbar discs,whereas Ross River and Chikungunya viruses provoke viral arthritis and bone loss.CONCLUSION Evidence of moderate strength suggested that viruses can cause moderate to severe arthritis and osteitis.Risk factors such as pre-existing rheumatologic disease contributed to higher disease severity and duration of symptoms.Therefore,based on our literature search,the proposed clinical and pathogenetic classification scheme is as follows:(1)Viral infections of bone or joint;(2)Active bone and joint inflammatory diseases secondary to viral infections in other organs or tissues;and(3)Viral infection as a risk factor for post-surgical bacterial infection.

On-column Refolding of Diphtheria Toxin Variant CRM197 by Different Metal-Chelating Affinity Chromatography Matrices

We have previously shown that the diphtheria toxin variant CRM 197 （cross-reacting material 197） can be overexpressed in Escherichia coli at high levels, yielding insoluble aggregates, which were solubilized using urea. This study reports a comparison of three matrices suitable for the purification and refolding of recombinant CRM197 by metal-chelating affinity chromatography, Moreover, we show that refolded CRM197 features enzymatic activity.

Heterologous Soluble Expression of Recombinant OmpR of <i>Aeromonas hydrophila</i>and Its Immunogenic Potential

Aeromonas hydrophila, a gram negative bacterium is a major fish pathogen and causes major economic losses to aquaculture industry. Outer membrane proteins play a significant role in its survival during different environmental conditions and bacterial pathogenesis. The outer membrane protein R (OmpR) is a member of the two-component regulatory system of Aeromonas hydrophila which differentially regulates the expression of OmpF or OmpC depending on the osmolarity conditions. Role of OmpR has been demonstrated in its virulence in other infectious bacteria and it is found to be a potential drug target/vaccine candidate. However, the OmpR of A. hydrophila has not been characterized. In the present study, we report recombinant expression, purification of the OmpR of A. hydrophila strain Ah17 in salt inducible E. coli GJ1158 cells. Leaky expression of rOmpR was confirmed by Western blot analysis using anti-6 × His antibody. The histidine tagged recombinant OmpR (rOmpR) (~29 kDa) was purified using Ni-NTA affinity chromatography from the soluble fraction of induced E. coli cells. The rOmpR was found to be highly immunogenic with end point titres of greater than 1:80,000. The anti-rOmpR antisera were capable of agglutinating live A. hydrophila cells, thus showing vaccine potential of the rOmpR.

Comparison of indirect immunofluorescence and western blot method in the diagnosis of hantavirus infections

BACKGROUND Serologic cross-reactivity between hantaviruses often complicates the interpretation of the results.AIM To analyze the diagnostic value of indirect immunofluorescence assay(IFA)and western blot(WB)in the diagnosis of hantavirus infections.METHODS One hundred eighty-eight serum samples from Puumala(PUUV)and Dobrava(DOBV)orthohantavirus infected patients were analyzed.Serology was performed using commercial tests(Euroimmun,Lübeck,Germany).RESULTS Using IFA,49.5%of acute-phase samples showed a monotypic response to PUUV,while 50.5% cross-reacted with other hantaviruses.The overall cross-reactivity was higher for immunoglobulin G(IgG)(50.0%)than for immunoglobulin M(IgM)(25.5%).PUUV IgM/IgG antibodies showed low/moderate reactivity with orthohantaviruses Hantaan(12.3%/31.5%),Seoul(7.5%/17.8%),DOBV(5.4%/28.1%),and Saaremaa(4.8%/15.7%).Both DOBV IgM and IgG antibodies were broadly reactive with Hantaan(76.2%/95.2%),Saaremaa(80.9%/83.3%),and Seoul(78.6%/85.7%)and moderate with PUUV(28.5%/38.1%).Using a WB,serotyping was successful in most cross-reactive samples(89.5%).CONCLUSION The presented results indicate that WB is more specific than IFA in the diagnosis of hantavirus infections,confirming serotype in most IFA cross-reactive samples.

Occurrence of False-Positive Tests and Cross-reactions Between COVID-19 and Dengue With Implications During Diagnosis:A Mixed Evidence Synthesis

This review aimed to assess the occurrence of false-positive serological reaction between dengue and coronavirus disease 2019(COVID-19)and its implications for diagnosis.Evidence syntheses were conducted by systematically reviewing available literature using multiple databases,including Web of Science,PubMed,Google Scholar and medRxiv.Among a total of 16 presented cases from clinical settings,cross-reaction to COVID-19 serological tests was observed in two(12.5%)dengue-positive patients,while 14 patients(87.5%)confirmed positive for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)showed a cross-reaction with dengue serological tests,leading to misdiagnosis and mismanagement by attending clinicians.Of 1789 SARS-CoV-2-positive sera,cross-reaction to dengue serological tests was observed in 180 sera(10%),which is higher than the cross-reaction observed for SARS-CoV-2 in archived pre-COVID-19 sera positive for a dengue infection(75 of 811,9.2%,P=0.674).Clinicians in tropical regions are therefore advised to interpret serological tests with caution and use a more pragmatic approach to triage these infections.

Development of Highly Specific Enzyme Immunoassay for Monitoring Serum Digitoxin Level in Patients

We previously developed radioimmunoassays (RIAs) for digitoxin and digoxin using antisera raised against digitoxin 3’-hemisuccinate-bovine serum albumin and digoxin 3’-hemisuccinate-bovine serum albumin conjugates, respectively. Very recently, we converted the RIA for digoxin to an enzyme immunoassay (EIA) system. Here, we aimed to convert the RIA for digitoxin to an EIA suitable for measuring serum digitoxin level in patients, using digitoxin 3’-hemisuccinate-β-D-galactosidase as an enzyme-labeled antigen. The developed EIA showed a quantification range of 1 to 70 ng/mL and exhibited high specificity for digitoxin, with low cross-reactivity to digitoxin metabolites. Compared with a commercial anti-digitoxin antiserum clinically used to monitor serum digitoxin level in patients, our antiserum showed much higher specificity for intact digitoxin. Intra- and inter-assay variations were less than 10.0% and 8.5%, respectively. Recovery was within the range of 93.7% - 107.5%. Mean digitoxin concentrations measured in serum samples (n = 26) from digitoxin-treated patients by EIA using our new antiserum and the commercial anti-digitoxin antiserum were 11.0 and 13.8 ng/mL, respectively. The present EIA, which is superior to RIA in terms of convenience and disposal of waste materials, is expected to be practically useful for clinical monitoring of intact digitoxin in serum.

In search of a pan-coronavirus vaccine:next-generation vaccine design and immune mechanisms

Members of the coronaviridae family are endemic to human populations and have caused several epidemics and pandemics in recent history.In this review,we will discuss the feasibility of and progress toward the ultimate goal of creating a pan-coronavirus vaccine that can protect against infection and disease by all members of the coronavirus family.We will detail the unmet clinical need associated with the continued transmission of SARS-CoV-2,MERS-CoV and the four seasonal coronaviruses(HCoV-OC43,NL63,HKU1 and 229E)in humans and the potential for future zoonotic coronaviruses.We will highlight how first-generation SARS-CoV-2 vaccines and natural history studies have greatly increased our understanding of effective antiviral immunity to coronaviruses and have informed next-generation vaccine design.We will then consider the ideal properties of a pan-coronavirus vaccine and propose a blueprint for the type of immunity that may offer cross-protection.Finally,we will describe a subset of the diverse technologies and novel approaches being pursued with the goal of developing broadly or universally protective vaccines for coronaviruses.

Cross-Reaction of SARS-CoV Antigen with Autoantibodies in Autoimmune Diseases

To investigate the significance of the SARS-associated coronavirus (SARS-CoV) antibody,detected by ELISA and indirect immunofluorescence assays (IFA) for the SARS-CoV Vero E6 cell lysates,in non-SARS subjects, 114 serum samples from healthy controls and 104 serum specimens from autoimmune disease patients were collected.The results of ELISA showed that among 114 sera from healthy controls,4 (3.5%) were positive of SARS-CoV-IgG antibody and 114 (100%) were all negative of SARS-CoV-IgM antibody;the specificity of SARS-CoV-IgG antibody for SARS patients was 96.5%,but the specificity of both SARS-CoV-IgG and -IgM antibodies for SARS patients was 100%.In 58 cases with SLE,positive rates of SARS-CoV-IgG and -IgM antibodies were 32.8% (19/58) and 8.6% (5/58),respectively,in which 11 cases (19%) were positive of both SARS-CoV-IgG and -IgM antibodies;in 10 cases with SS,positive rate of both SARS-CoV-IgG and -IgM antibodies was 10% (1/10);in 16 cases with MCTD,positive rate of SARS-CoV-IgG was 37.5% (6/16),positive rate of both SARS-CoV-IgG and -IgM antibodies was 6.3% (1/16);in 20 cases with RA,one case was positive (5%) of SARS-CoV-IgG.However,of all samples with positive SARS-CoV-IgG and -IgM antibodies for autoimmune diseases and healthy controls,SARS-CoV RNA and antibodies were all negative by RT-PCR and IFA.All sera for negative or positive ELISA results were also negative or positive results using ELISA with Vero E6 cells lysates.These studies showed that SARS-CoV Vero E6 cell lysates for the ELISA to detect SARS-CoV antibodies could lead to the false-positive reactions or cross-reactions of SARS-CoV antibodies in non-SARS diseases and healthy controls,and the false-positive reactions or cross-reactions were related to Vero E6 cell lysates and autoantibodies in non-SARS population.Cellular & Molecular Immunology.2004;1(4): 304-307.

Occupational Asthma Caused by Inhalable Royal Jelly ancl Its Cross-reactivity with Honeybee Venom

Royal jelly is a honeybee nutriment secreted from the glands in the hypopharynx of worker bees essential in the development of queen bees. Ingestion of royal jelly has been reporied to trigger rhinitis, asthma, and anaphylaxis, but occupational asthrna occurring after inhalation of volatile royal jelly is rare.

Cross-reactivity of anti-programmed death ligand 2 polyclonal antibody in mouse tissues

The inhibitory co-receptor programmed death 1 （PD-1, encoded by pdcdl） and its two ligands PD-L1 and PD-L2 comprise an important immune inhibitory signaling pathway for defense against microbes and for self-tolerance. Unlike other members of the B7-CD28 family, expression of PD-L1 and PD-L2 is not limited to the immune system. In this study, we determined that a polyclonal antibody （pAb） （R＆D Systems） against extracellular domains of mouse PD-L2 （mPD-L2） could recognize anti- gen（s） in diverse mouse tissues, including the anterior and intermediate pituitary gland, olfactory bulbs and olfactory epitheli- um, tongue epithelium, keratinized epithelial ceils and skin and whisker hair follicles. These findings differed from previous reports of mPD-L2 localization. Reverse transcription PCR and Western blot analyses, however, were unable to detect any mPD-L2 transcripts or proteins of the 25-kD predicted molecular weight in RNA and protein extracts, respectively, from the above tissues, suggesting that the anti-mPD-L2 pAb cross-reacts with certain novel antigen（s）. Developmental studies revealed that the earliest expression of mPD-L2-1ike antigen was in the olfactory epithelium at embryonic day 12.5 （E12.5）. At E14.5, mPD-L2-1ike antigen was present in the skin, tongue and follicles of the skin and whiskers. The distribution patterns of mPD-L2-1ike antigen remained similar from E18.5 to adulthood. The results of bioinformatic analysis and other experiments suggested neural cell adhesion molecule and hemicentin-1 as candidate proteins with cross-reactivity to the anti-mPD-L2 pAb. These results demonstrate that care is required in interpreting staining patterns generated when anti-PD-L2 pAb is used to lo-cate PD-L2-expressing cells in the central nervous system and epithelial tissues, such as the olfactory epithelium. In addition, this anti-PD-L2 pAb may be used as an alternative antibody for labeling the olfactory epithelium during embryonic develop-ment in mice.

HIV/AIDS vaccine development： are we walking out from the dark？

The need for AIDS vaccine has been emphasized by the increase of HIV-1 prevalence in sexual transmission which bridges the spreading of HIV-1 from high-risk population to other populations. After more than two-decade intensive effort on the AIDS vaccine development, it remains elusive whether and how an effective vaccine will be achieved. Recent data released from a phase Ⅲ trial in Thailand showed a partial protection might be accomplishable by（R）the ＂prime-boost＂combination of two vaccines： ALVAC（R） HIV vaccine （the prime）, and AIDSVAX（R） B/E vaccine （the boost）.1 This unprecedented large clinical trial observed that the prime-boost combination lowered the rate of HIV infection by 31.2% compared to placebo based on the modified intent-to-treat population （n=51 vs. n=74,respectively; P=0.04）. However, debating on the efficacy interpretation of this trial arose among field scientists.Furthermore, how to improve the efficacy will become the most important question to be tackled. Here we reviewed the recent publications and summarized the major progress achieved.