Anti-PD1 antibody and not anti-LAG-3 antibody improves the antitumor effect of photodynamic therapy for treating metastatic breast cancer

Photodynamic therapy(PDT)has limited effects in treating metastatic breast cancer.Immune checkpoints can deplete the function of immune cells;however,the expression of immune checkpoints after PDT is unclear.This study investigates whether the limited e±cacy of PDT is due to upregulated immune checkpoints and tries to combine the PDT and immune checkpoint inhibitor to observe the e±cacy.A metastatic breast cancer model was treated by PDT mediated by hematoporphyrin derivatives(HpD-PDT).The anti-tumor effect of HpD-PDT was observed,as well as CD4þT,CD8þT and calreticulin(CRT)by immunohistochemistry and immunofluorescence.Immune checkpoints on T cells were analyzed byflow cytometry after HpD-PDT.When combining PDT with immune checkpoint inhibitors,the antitumor effect and immune effect were assessed.For HpD-PDT at 100 mW/cm2 and 40,60 and 80 J/cm2,primary tumors were suppressed and CD4þT,CD8þT and CRT were elevated;however,distant tumors couldn't be inhibited and survival could not be prolonged.Immune checkpoints on T cells,especially PD1 and LAG-3 after HpD-PDT,were upregulated,which may explain the reason for the limited HpD-PDT effect.After PDT combined with anti-PD1 antibody,but not with anti-LAG-3 antibody,both the primary and distant tumors were signi-cantly inhibited and the survival time was prolonged,additionally,CD4þT,CD8þT,IFN-þCD4þT and TNF-þCD4þT cells were signi-cantly increased compared with HpD-PDT.HpD-PDT could not combat metastatic breast cancer.PD1 and LAG-3 were upregulated after HpD-PDT.Anti-PD1 antibody,but not anti-LAG-3 antibody,could augment the antitumor effect of HpD-PDT for treating metastatic breast cancer.

Autoantibody profiles in autoimmune hepatitis and chronic hepatitis C identifies similarities in patients with severe disease

To determine how the auto-antibodies (Abs) profiles overlap in chronic hepatitis C infection (CHC) and autoimmune hepatitis (AIH) and correlate to liver disease.METHODSLevels of antinuclear Ab, smooth muscle antibody (SMA) and liver/kidney microsomal-1 (LKM-1) Ab and markers of liver damage were determined in the sera of 50 patients with CHC infection, 20 AIH patients and 20 healthy controls using enzyme linked immunosorbent assay and other immune assays.RESULTSWe found that AIH patients had more severe liver disease as determined by elevation of total IgG, alkaline phosphatase, total serum bilirubin and serum transaminases and significantly higher prevalence of the three non-organ-specific autoantibodies (auto-Abs) than CHC patients. Antinuclear Ab, SMA and LKM-1 Ab were also present in 36% of CHC patients and related to disease severity. CHC cases positive for auto-Abs were directly comparable to AIH in respect of most markers of liver damage and total IgG. These cases had longer disease duration compared with auto-Ab negative cases, but there was no difference in gender, age or viral load. KLM-1+ Ab CHC cases showed best overlap with AIH.CONCLUSIONAuto-Ab levels in CHC may be important markers of disease severity and positive cases have a disease similar to AIH. Auto-Abs might have a pathogenic role as indicated by elevated markers of liver damage. Future studies will unravel any novel associations between these two diseases, whether genetic or other.

Preparation of anti-canine interleukin-31 receptor alpha polyclonal antibody and evaluation of its therapeutic efect in canine atopic dermatitis

Canine atopic dermatitis(CAD)is a prevalent genetically susceptible infammatory and pruritic allergic skin condition afecting not only the health of dogs but also the quality of life of their owners.Interleukin-31(IL-31)and interleukin-31 receptor alpha(IL-31RA)are essential for the development of pruritus in primates and mice.Hence,it is expected that inhibiting IL-31RA will be an efective approach to alleviate pruritus.The purpose of the study was to produce anti-canine IL-31RA polyclonal antibodies(anti-IL-31RA pAbs)and evaluate their efcacy in inhibiting house dust mite(HDM)-evoked pruritic responses.Dogs were immunized with antigens formed by IL-31RA recombinant short peptides coupled to BSA to produce anti-IL-31RA pAbs.The CAD model was developed by using HDM allergen stimulation,and the efects of IL-31RA pAbs on the reduction of pruritus in CAD model dogs were examined.The Canine Atopic Dermatitis Extent and Severity Index(CADESI)-4 and pruritus Visual Analog Scale(pVAS)were utilized to evaluate pruritic responses,and skin tissue samples were collected from the inguinal area for pathological assessment of skin infammatory cell infltration.The results showed that anti-IL-31RA pAbs with high titers(1:128,000)and specifcity were efectively produced.In the CAD model group,the severity of skin damage,pruritus score,infammatory cell infltration and level of infammatory factors were considerably elevated.Anti-IL-31RA pAbs relieved pruritic behavior and dermatitis in dogs compared to placebo-treated dogs.In conclusion,anti-IL-31RA pAbs efectively suppressed CAD in vivo and are anticipated to be an efective novel treatment for pruritic skin disorders such as CAD.

Anti-OX40 Antibody Combined with HBc VLPs Delays Tumor Growth in a Mouse Colon Cancer Model

Objective Combination immunotherapy strategies targeting OX40,a co-stimulatory molecule that can enhance antitumor immunity by modulating the proliferation,differentiation,and effector function of tumor-infiltrating T cells,have attracted much attention for their excellent therapeutic effects.In this study,we aimed to evaluate the antitumor efficacy of combined anti-OX40 and hepatitis B core viruslike particles(HBc VLPs)therapy using a mouse colon cancer model.Methods Humanized B-h OX40 mice were injected subcutaneously with MC38 colon tumor cells and treated with HBc VLPs+anti-h OX40 antibody.Tumor growth was monitored.Flow cytometric analysis was performed to evaluate the populations of T cell subsets in the tumors.Results The combination of anti-OX40 with HBc VLPs resulted in a significant delay in tumor growth,suggesting that a potent antitumor immunity was induced by the combination therapy.Further studies revealed that HBc VLPs+anti-OX40 treatment induced a significant increase in effector T cells(Teffs)and a significant decrease in regulatory T cells(Tregs)in the tumor microenvironment(TME),which accounted for the synergistic antitumor effect of anti-OX40 in combination with HBc VLPs.Conclusion Combination therapy of anti-h OX40 and HBc VLPs provides synergistic antitumor activity in colon cancer-bearing mice,which may represent a potential design strategy for cancer immunotherapy.

Antibody Levels and Infection Status of Pertussis in the Population under Pertussis Resurgence in Guangxi in 2018:A Cross-Sectional Survey

Objective Pertussis cases have increased markedly since 2018 in Guangxi.The aim of this study was to evaluate antibody levels and the infection status of pertussis in the resident population.Method A total of 10,215 serum samples from residents were collected from August-November 2018 and tested for anti-pertussis IgG and toxin IgG using the enzyme-linked immunosorbent assay(ELISA).Results Of the collected samples,1,833(17.94%)tested positive for anti-pertussis IgG,with the median concentration of 16.06 IU/mL.Antibody level<10 IU/mL accounted for more than 60%in children under 4 years of age,but declined with age,whereas the percentages of the other three levels(10-40,40-50,and≥50 IU/mL)increased almost with age(P<0.001).Moreover,7,924 samples were selected for anti-pertussis toxin IgG,of which 653(8.24%)tested positive(≥40 IU/mL)with the median concentration of 5.89 IU/mL,and 204 participants(2.56%)had recent pertussis infection(≥100 IU/mL).Among the different age groups,the highest rates of positivity and recent infection were observed at 11-20 years of age,the lowest positivity rate at 5 years of age,and the lowest recent infection rate at 4 years of age(P<0.001,P=0.005,respectively).Conclusion The survey results showed that all age groups in Guangxi lacked immunity against pertussis,which was one of the main factors contributing to the resurgence of pertussis in 2018.In addition,the prevalence of pertussis is relatively high in Guangxi,and its incidence is seriously underestimated,especially in adolescents and adults.

Antibody-platinum(IV)prodrugs conjugates for targeted treatment of cutaneous squamous cell carcinoma

Antibody-drug conjugates(ADCs)are a new type of targeting antibodies that conjugate with highly toxic anticancer drugs via chemical linkers to exert high specificity and efficient killing of tumor cells,thereby attracting considerable attention in precise oncology therapy.Cetuximab(Cet)is a typical antibody that offers the benefits of good targeting and safety for individuals with advanced and inoperable cutaneous squamous cell carcinoma(cSCC);however,its anti-tumor activity is limited to a single use.Cisplatin(CisPt)shows good curative effects;however,its adverse effects and non-tumor-targeting ability are major drawbacks.In this study,we designed and developed a new ADC based on a new cytotoxic platinum(IV)prodrug(C8Pt(IV))and Cet.The so-called antibody-platinum(IV)prodrugs conjugates,named Cet-C8Pt(IV),showed excellent tumor targeting in cSCC.Specifically,it accurately delivered C8Pt(IV)into tumor cells to exert the combined anti-tumor effect of Cet and CisPt.Herein,metabolomic analysis showed that Cet-C8Pt(IV)promoted cellular apoptosis and increased DNA damage in cSCC cells by affecting the vitamin B6 metabolic pathway in tumor cells,thereby further enhancing the tumor-killing ability and providing a new strategy for clinical cancer treatment using antibody-platinum(IV)prodrugs conjugates.

Antibody Titer of Newcastle Disease in Vaccinated and Non-vaccinated Local Chicken of Cambodia

The experiment was conducted at Veterinary Research Station of Faculty of Veterinary Medicine,Royal University of Agriculture.The experimental period lasted 60 days,starting from October 1st to November 30th 2022.CRD(Completely Randomized Design)was used with 2 treatments/groups,vaccination group and non-vaccination group“control”,and 6 replications.The vaccination groups received two times of vaccination by dropping into the ocular at 7 days and 21 days.Meanwhile,blood samples were collected 3 times to detect the antibody level of ND(Newcastle Disease)and contained 21 days old,35 days old and 49 days old chicks.The ELISA(Enzyme-Linked Immunosorbent Assay)was performed to detect the antibody of those 2 groups.The result of finding showed that the S/P(Sample to Positive)ratio of control at 21 days,was very low,even in 3rd quartile,which was below the threshold.However,the vaccination group was relatively high,even in 1st quartile,which was higher than the threshold.At 35 days,S/P ratio of control group was still very low,but a bit higher than at 21 days.Meanwhile,the vaccination group was still high,even in 1st quartile,and two-time higher than at 21 days,but an increasing number of samples developed less antibody than threshold,accounting for 12.22%.At 49 days,the control group was still very low,even in 3rd quartile,but a bit higher than at 21 days and 35 days,and was close to the threshold.The vaccination group was still relatively high,even in 1st quartile but lower than three times comparing to 35 days.However,in this age,the number of chickens that developed antibody seemed to be increased in the control group,vice versa for vaccination group.The average S/P ratio was different significant(p<0.001),where vaccination had higher S/P ratio than control.It was similar finding for log-titer,the vaccination had higher figure(p<0.001).The risk of infection of ND was higher in control group,but it will reduce by increasing the age of chicken,while vaccination group was decreased by increasing age,especially at 49 days and we need to consider another vaccination to get full protection.

Quantitative hepatitis B core antibody and quantitative hepatitis B surface antigen:Novel viral biomarkers for chronic hepatitis B management

The management of hepatitis B virus(HBV)infection now involves regular and appropriate monitoring of viral activity,disease progression,and treatment response.Traditional HBV infection biomarkers are limited in their ability to predict clinical outcomes or therapeutic effectiveness.Quantitation of HBV core antibodies(qAnti-HBc)is a novel non-invasive biomarker that may help with a variety of diagnostic issues.It was shown to correlate strongly with infection stages,hepatic inflammation and fibrosis,chronic infection exacerbations,and the presence of occult infection.Furthermore,qAnti-HBc levels were shown to be predictive of spontaneous or treatment-induced HBeAg and HBsAg seroclearance,relapse after medication termination,re-infection following liver transplantation,and viral reactivation in the presence of immunosuppression.qAnti-HBc,on the other hand,cannot be relied on as a single diagnostic test to address all problems,and its diagnostic and prognostic potential may be greatly increased when paired with qHBsAg.Commercial qAnti-HBc diagnostic kits are currently not widely available.Because many methodologies are only semi-quantitative,comparing data from various studies and defining universal cut-off values remains difficult.This review focuses on the clinical utility of qAnti-HBc and qHBsAg in chronic hepatitis B management.

A Comparative Study on Detection of the Expression of Alternative Oxidase in Tobacco Callus with the Monoclonal Antibody Against Alternative Oxidase and Antibody Against-Synthetic Polypeptide Antibody

从经过不同温度处理的烟草 (NicotianarusticaL .)愈伤组织中提取并纯化线粒体蛋白 ,分别与交替氧化酶的单克隆抗体和抗合成多肽抗体进行免疫杂交。结果表明 :交替氧化酶的含量随温度的下降而显著上升 ;单克隆抗体的特异性较高于抗合成多肽抗体 ,但后者与交替氧化酶同样有良好的亲和性。因此 ,用合成多肽方法制备的抗体可以用于交替氧化酶的研究中。

Strategies to enhance monoclonal antibody uptake and distribution in solid tumors

Despite the significant resources dedicated to the development of monoclonal antibody(m Ab)therapies for solid tumors,the clinical success,thus far,has been modest.Limited efficacy of m Ab in solid tumors likely relates to unique aspects of tumor physiology.Solid tumors have an aberrant vasculature and a dense extracellular matrix that slow both the convective and diffusive transport of m Abs into and within tumors.For m Abs that are directed against cellular antigens,high antigen expression and rapid antigen turnover can result in perivascular cells binding to and eliminating a significant amount of extravasated m Ab,limiting m Ab distribution to portions of the tumor that are distant from functional vessels.Many preclinical investigations have reported strategies to improve m Ab uptake and distribution;however,to our knowledge,none have translated into the clinic.Here,we provide an overview of several barriers in solid tumors that limit m Ab uptake and distribution and discuss approaches that have been utilized to overcome these barriers in preclinical studies.

Construction of human Fab library and screening of a single-domain antibody of amyloid-beta 42 oligomers

Screening humanized antibodies from a human Fab phage display library is an effective and quick method to obtain beta-amyloid oligomers. Thus, the present study prepared amyloid-beta 42 oli- gomers and constructed a have human Fab phage display library based on blood samples from six healthy people. After three rounds of biopanning in vitro, a human single-domain antibody that spe- cifically recognized amyloid-beta 42 oligomers was identified. Western blot and enzyme-linked im- munosorbent assay demonstrated this antibody bound specifically to human amyloid-beta 42 tetramer and nonamer, but not the monomer or high molecular weight oligomers. This study suc- cessfully constructed a human phage display library and screened a single-domain antibody that specifically recognized amyloid-beta 42 oligomers.

Prokaryotic Expression of Rubber Elongation Factor Gene and Preparation of Its Polyclonal Antibody

[Objective] The aim of this study was to prepare the recombination protein of rubber elongation factor and its polyclonal antibodies.[Method] The encoding gene of rubber elongation factor(REF)was amplified by RT-PCR,and cloned into the prokaryotic expression vector pDEST17 to transform into Escherichia coil BI2I-AI.The recombinant protein induced by L-Arabinose was purified by the affinity chromatography.As the immunogen,the recombination protein was used to immunize mice for preparing polyclonal antibodies,while ELISA and Western blot hybridization were used to detect the titers and specificity.[Result] The purified recombination protein of REF with high expression was used to immunize house mice for preparing polyclonal antibodies with high titer and specificity.The western blot hybridization showed that the antibody could recognize the natural REF from latex.[Conclusion] The recombination protein of REF was successfully obtained and the mouse anti REF antibody with high titer and specificity was prepared,which lays a basis for further studies on biological functions of rubber elongation factor and other membrane proteins in rubber particles.

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.

Establishment of Monoclonal Antibody Competitive ELISA Using Monoclonal Antibody Against VP1 Protein of Asia 1 Type Foot-and-Mouth Disease Virus

Using the purified VP1 protein of Asia 1 type foot-and-mouth disease virus as the antigen, the purified monoclonal antibody was labeled by the sodium periodate method and the monoclonal antibody competitive ELISA was established in this study. Ten positive porcine foot-and-mouth disease serums and more than two hundreds negative serum were tested, and the results were the same as the background of samples. The sensitivity test and replicate test indicated that this method was stable and sensitive, which was suitable for monitoring Asia 1 type porcine foot-and-mouth disease virus antibody.

Preparation and Identification of Specific Monoclonal Antibody against Porcine Circovirus Type 2

BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay （IFA） indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.

Antibody Detection on Mycoplasma capricolum subsp. Capripneumoniae in Qinghai Province

[Objective] The aim was to investigate the prevalence of Mycoplasma capricolum subsp. Capripneumoniae in Qinghai Province. [Method] By using indirect hemagglutination test kit for detecting Mycoplasma capricolum subsp. Capripneumoniae,208 goat serums were detected. [Result] The positive rate of goat sera was 16.3%,and the positive rate of sera from different regions ranged from 6.7% to 24.3%. [Conclusion] The infection rate of Mycoplasma capricolum subsp. Capripneumoniae was high in Qinghai Province,so it is necessary to strengthen the prevention and control of this disease.

Preparation and Preliminary Identification of Fluorescein Labeled Monoclonal Antibody against Canine Distemper Virus

[Objective] The aim of the present study was to develop a direct immunofluorescence method for the diagnosis of canine distemper （CD） with FITC-conjugated monoclonal antibodies （FITC-McAb）.[ Metbod] The McAb against CDV, designated as CE3, was purified with protein G and labeled with FITC through agitation method. After purification and identification, the optimal working concentration of FITC-labeled CE3 was determined. Then 61 clinical samples of suspected canine distemper were detected by direct immunofluorescence assay. [ Result] The absorption test, blocking test and specificity test showed that the labeled antibody had high specificity and sensitivity, but didn＇t have cross reaction with canine parvovirus （CPV）, canine parainfluenza virus （CPIV）, canine adenovirus （CAV） and rabies virus （RV）. The optimal working concentration was 1：80. The positive rate of clinical suspected samples was 48%. [ Conclusion] The direct immunofluorescence assay developed in this study was rapid, specific and convenient, and had great significance for the early diagnosis of canine distemper.

A MONOCLONAL ANTIBODY RECOGNIZING NON DERIVATIVE 13 HYDROXY GIBBERELLINS AND THEIR GLUCOSIDES *

The production and characterization of a monoclonal antibody (MAb AB10) against GA 3 glucoside as well as GA 3 is described. MAb AB10 was derived from an immunogen in which human serum albumin (HSA) was linked to GA 3 at carbon 3. This antibody showed high affinity for GA 3 glucoside as well as for 13 hydroxy gibberellins (GA 1, GA 3, GA 5, etc). The affinity of MAb AB10 for 13 hydroxy GAs was significantly reduced by methylation of the 7 oic acid but not by glycosylation of 3 hydroxyl group. Based on this antibody, both of competitive enzyme linked immunosorbent assays (ELISAs) for GA 3 glucoside and for GA 3 were developed. These two ELISAs displayed linear detection ranges from 0 2 pmol to 20 pmol. Using these assays, the fluctuation of GA 3 like and GA 3 glucoside like substances in the leaves of Rumex japonicus was investigated. The results indicated that the glycosylation of free GAs was connected with leaf senescence and that the function of 6 benzyl amino purine in retarding the leaf senescence was probably related to delaying the process of glycosylation of free GAs.

Clinical evaluation of serum antimitochondrial antibody-negative primary biliary cirrhosis

BACKGROUND: Primary biliary cirrhosis (PBC) is cha- racterized by frequent presence of antimitochondrial anti- bodies (AMAs). The sensitivity and specificity of AMA for PBC are both greater than 90%-95%, so the presence of AMA in serum is the major hallmark in PBC. However, it has long been recognized that in 5%-10% of patients the clinical, biochemical and histological features are diagnos- tic for PBC, but their sera are consistently tested negative for AMA/AMA-M2. This study aimed to evaluate whether the presence of AMA alters the clinical, serological and his- tological features of the disease. METHODS: Clinical data of 70 patients clinically and/or histologically diagnosed with PBC were reviewed. AMA- negative and AMA-positive patients were compared in terms of clinical, biochemical, immunological and histo- logical features. RESULTS: At presentation, 11 patients were serum AMA/ AMA-M2 negative. At the initial visit, AMA-negative and AMA-positive patients were similar in terms of age, sex, clinical manifestations, liver biochemistries and histological findings. The mean level of serum immunoglobulin M (IgM) was significantly lower in AMA-negative PBC pa- tients than in AMA-positive PBC patients (2851±1418 mg/L vs 6361 ±4928 mg/L, P =0.033). Serum antinuclear anti- bodies ( ANA) and/or smooth muscle antibodies ( SMA) were more frequently positive in the AMA-negative PBC patients than in the AMA-positive patients (81.8% vs 40.7%, P =0.031). CONCLUSION: AMA-negative PBC patients are characte- rized by relatively lower levels of serum IgM and a higher prevalence of serum ANA/SMA and are not associated with substantial differences in the clinical, biochemical and histo- logical spectrum of the disease.

Generation of high affinity human single-hain antibody against PreSl of hepatitis B virus from immune phage-display antibody library

BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filamentous phage, from which high affinity completely humanized ScFv against PreS1 of hepatitis B virus could be screened and characterized. METHODS: A combinatorial library of phage-display hu- man ScFv genes, which were derived from peripheral blood lymphocytes immunized by peptide PreS1 in vitro, was constructed. The library contained 7 × 108 clones. RESULTS: After 3 rounds panning, a high affinity (K = 10-7-10-8 mol/L) ScFv specific to PreS1 was obtained. Sequence analysis showed that the VH belonged to the VH4 family and Vλ to Vλ4. CONCLUSIONS: The described ScFv may provide a more satisfactory therapy. This application further illustrates that the method of in vitro antigen stimulation is expeditious for the source of human immune antibody library.