OBJECTIVE: To investigate the role of multiple serological methods in the identification of complex antibodies. METHODS: The blood group antigens were detected by saline and microcolumn agglutination methods. The saline method was used to screen and identify IgM-type antibodies in the patient's serum, while the polybrene, anti-globulin, microcolumn agglutination, enzymic and absorption-elution methods were used to screen and identify IgG-type antibodies. RESULTS: The patient was B/CCDee/Jk(a-b+)/Fy(a-b+) blood type. The serum reacted with panel cells, and the reaction presented anti-E pattern in the saline medium. It was fully positive in the microcolumn agglutination card, except 2 negative ones after using papain to treat the panel cells. Referring to the pattern table, it was concluded that there existed anti-c, anti-E, and anti-Jk^a antibodies, and one antibody corresponding to an antigen that was easily destroyed by papain. The red blood cells with specific phenotype were selected for absorption-elution to identify IgG-type anti-c, anti-E, anti-Jk^a and anti-Fy^a antibodies. CONCLUSION It is confirmed that IgM-type anti-E, and IgG-type anti-c, anti-E, anti-Jk^a and anti-Fy^a antibodies exist in the patient's serum by multiple serological methods.
Humans ; Papain ; Blood Group Antigens ; Erythrocytes ; Immunoglobulin G ; Immunoglobulin M

OBJECTIVE: Jingfang Granules have been recommended for the prevention and treatment of corona virus disease 2019 (COVID-19). Through chemical analysis and bioactivity evaluation, this study aims to elucidate the potential effective components of Jingfang Granules. METHODS: The inhibitory acti-vities of Jingfang Granules extract against 3-chymotrypsin-like protease (3CL^pro), papain like protease (PL^pro), spike protein receptor-binding domain (S-RBD) and human cyclooxygenase-2 (COX-2) were evaluated using enzyme assay. The antitussive effects were evaluated using the classical ammonia-induced cough model. The chemical constituents of Jingfang Granules were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometry (LC/MS). The 3CL^pro and PL^pro inhibitory activities of the major compounds were determined by enzyme assay, molecular docking, and site-directed mutagenesis. RESULTS: Jingfang Granules exhibited 3CL^pro and PL^pro inhibitory activities, as well as COX-2 inhibitory and antitussive activities. By investigating the MS/MS behaviors of reference standards, a total of fifty-six compounds were characterized in Jingfang Granules. Sixteen of them were unambiguously identified by comparing with reference standards. The contents of the 16 major compounds were also determined, and their total contents were 2 498.8 μg/g. Naringin, nodakenin and neohesperidin were three dominating compounds in Jingfang Granules, and their contents were 688.8, 596.4 and 578.7 μg/g, respectively. In addition, neohesperidin and naringin exhibited PL^pro inhibitory activities, and the inhibition rates at 8 μmol/L were 53.5% and 46.1%, respectively. Prim-O-glucosylcimifugin showed significant inhibitory activities against 3CL^pro and PL^pro, and the inhibitory rates at 8 μmol/L were 76.8% and 78.2%, respectively. Molecular docking indicated that hydrogen bonds could be formed between prim-O-glucosylcimifugin and amino acid residues H163, E166, Q192, T190 of 3CL^pro (binding energy, -7.7 kcal/mol) and K157, D164, R166, E167, T301 of PL^pro(-7.3 kcal/mol), respectively. Site-directed mutagenesis indicated amino acid residue K157 was a key active site for the interaction between prim-O-glucosylcimifugin and PL^pro. CONCLUSION Prim-O-glucosylcimifugin, neohesperidin, and naringin as the major compounds from Jingfang Granules could inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus proteases 3CL^pro and PL^pro. The results are valuable for rational clinical use of Jingfang Granules.
Amino Acids ; Ammonia ; Antitussive Agents ; COVID-19 ; Chymases ; Coronavirus 3C Proteases ; Cyclooxygenase 2 ; Cyclooxygenase 2 Inhibitors ; Cysteine Endopeptidases/metabolism* ; Humans ; Molecular Docking Simulation ; Papain ; Peptide Hydrolases ; SARS-CoV-2 ; Spike Glycoprotein, Coronavirus ; Tandem Mass Spectrometry

The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics. Drugging the multi-functional papain-like protease (PLpro) domain of the viral nsp3 holds promise. However, none of the known coronavirus PLpro inhibitors has been shown to be in vivo active. Herein, we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity, including against the Sarbecoviruses (SARS-CoV-1 and SARS-CoV-2), Merbecovirus (MERS-CoV), as well as the Alphacoronavirus (hCoV-229E and hCoV-OC43). Importantly, F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice. F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage, as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity. Despite the significant difference of substrate recognition, mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue, whereas an allosteric inhibitor of MERS-PLpro interacting with its 271E position. Our proof-of-concept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anti-coronavirus agents. The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks.
Animals ; Coronavirus Papain-Like Proteases/antagonists & inhibitors* ; Cricetinae ; Humans ; Mice ; Pandemics ; SARS-CoV-2/enzymology* ; COVID-19 Drug Treatment

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M
Antiviral Agents/therapeutic use* ; Binding Sites ; COVID-19/virology* ; Coronavirus Papain-Like Proteases/metabolism* ; Crystallography, X-Ray ; Drug Evaluation, Preclinical ; Drug Repositioning ; High-Throughput Screening Assays/methods* ; Humans ; Imidazoles/therapeutic use* ; Inhibitory Concentration 50 ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; Naphthoquinones/therapeutic use* ; Protease Inhibitors/therapeutic use* ; Protein Structure, Tertiary ; Recombinant Proteins/isolation & purification* ; SARS-CoV-2/isolation & purification*

A 67-year-old man previously diagnosed with ulcerative colitis complained of difficulty in defecation and underwent balloon dilatation of rectum, but the procedure failed. The patient was transferred to a surgical department for further treatment. Before surgery, his red cells were typed A, Rh(D) positive. The antibody screening test was positive and the results of the identification tests were atypical. The reactivity was similar to anti-Le(b) antibody; however, the antibody showed panreactivity against papainized red cells. It showed stronger reactivity against O red cells than A Le(a−b+) red cells, and we concluded that the antibody was anti-Le(bH). After reexamination, his Lewis phenotype was found to be Le(a−b−). His FUT2 and FUT3 were analyzed to confirm his Lewis blood type, and c.59T>G and c.1067T>A variants were found on the FUT3. Therefore, the patient's Lewis blood type was concluded as Le(a−b−).
Aged ; Colitis, Ulcerative ; Defecation ; Dilatation ; Humans ; Mass Screening ; Papain ; Phenotype ; Rectum ; Ulcer

Papain is a proteolytic enzyme which is widely used in food industry, pharmaceuticals, and cosmetics. Occupational and non-occupational papain allergies have previously been documented; however, there are limited publications about papain allergy with its relative fruit allergy. Here, we present a case of occupational, IgE-mediated papain allergy with kiwi fruit and fig fruit allergy. A 53-year-old man suffered from rhinitis for several years, with the onset of his symptoms coinciding with the time he started to work at a sausage processing plant where papain is often used as a meat tenderizer. He began to experience symptoms of chest tightness, shortness of breath and wheezing shortly after starting work 5 years ago. Furthermore, he experienced several episodes of oral itching, and tongue and oropharyngeal angioedema after injestion of kiwi fruit and fig fruit. The patient had a lifelong history of allergic conjunctivitis, allergic rhinitis, and childhood asthma. Specific IgE was positive to kiwi fruit, papain and chymopapain (2.95 kUA/L, >100 kUA/L, and 95.0 kUA/L, respectively). Similar bands at 10-15 kDa in blotting with papain and kiwi fruit extracts were found. This patient showed a potential association between papain allergy and sensitization to kiwi fruit. We also reviewed 13 patients with papain allergy published in the literature, with 85% (11/13) of the patients sensitized through the respiratory tract, and 40% (4/11) having atopy. Further studies should focus on the determination of cross-reactive allergens between papain and its fruit relatives, and the prevalence of food allergy in patients with papain allergy should be investigated in a relatively large cohort.
Allergens ; Angioedema ; Asthma ; Asthma, Occupational* ; Chymopapain ; Cohort Studies ; Conjunctivitis, Allergic ; Dyspnea ; Food Hypersensitivity ; Food Industry ; Fruit ; Humans ; Hypersensitivity* ; Immunoglobulin E ; Meat ; Middle Aged ; Papain* ; Plants ; Prevalence ; Pruritus ; Respiratory Sounds ; Respiratory System ; Rhinitis ; Thorax ; Tongue

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.
Dimerization ; HEK293 Cells ; Humans ; I-kappa B Kinase ; metabolism ; Interferon Regulatory Factor-3 ; metabolism ; Interferon Type I ; antagonists & inhibitors ; metabolism ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Papain ; metabolism ; Peptide Hydrolases ; chemistry ; metabolism ; Phosphorylation ; Protein Binding ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases ; metabolism ; SARS Virus ; enzymology ; Signal Transduction ; TNF Receptor-Associated Factor 3 ; metabolism ; Ubiquitination

Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to coronavirus regulation of host innate responses. Here, we show that the membrane-associated papain-like protease PLP2 (PLP2-TM) of coronaviruses acts as a novel autophagy-inducing protein. Intriguingly, PLP2-TM induces incomplete autophagy process by increasing the accumulation of autophagosomes but blocking the fusion of autophagosomes with lysosomes. Furthermore, PLP2-TM interacts with the key autophagy regulators, LC3 and Beclin1, and promotes Beclin1 interaction with STING, the key regulator for antiviral IFN signaling. Finally, knockdown of Beclin1 partially reverses PLP2-TM's inhibitory effect on innate immunity which resulting in decreased coronavirus replication. These results suggested that coronavirus papain-like protease induces incomplete autophagy by interacting with Beclin1, which in turn modulates coronavirus replication and antiviral innate immunity.
Apoptosis Regulatory Proteins ; antagonists & inhibitors ; genetics ; immunology ; Autophagy ; Beclin-1 ; Coronavirus NL63, Human ; genetics ; immunology ; Gene Expression Regulation ; HEK293 Cells ; HeLa Cells ; Host-Pathogen Interactions ; immunology ; Humans ; Immune Evasion ; Immunity, Innate ; Interferon-gamma ; genetics ; immunology ; Lysosomes ; metabolism ; virology ; MCF-7 Cells ; Membrane Fusion ; Membrane Proteins ; antagonists & inhibitors ; genetics ; immunology ; Microtubule-Associated Proteins ; genetics ; immunology ; Papain ; genetics ; immunology ; Phagosomes ; metabolism ; virology ; RNA, Small Interfering ; genetics ; immunology ; Signal Transduction ; Virus Replication

OBJECTIVES: This study aimed to evaluate the capacity of 2% chlorhexidine gel associated with 8% papain gel in comparison with 5.25% sodium hypochlorite in bovine pulp tissue dissolution. MATERIALS AND METHODS: Ninety bovine pulps of standardized sizes were used and fragmented into 5-mm sizes. The fragments were removed from the root middle third region. They were divided into 6 experimental groups (n = 15), 1) 8% papain; 2) 2% chlorhexidine; 3) 2% chlorhexidine associated with 8% papain; 4) 0.9% saline solution; 5) 2.5% sodium hypochlorite; and 6) 5.25% sodium hypochlorite. The pulp fragments were weighed and put into immobile test tubes for dissolution for time intervals of 30, 60, 90, and 120 min. RESULTS: The 5.25% sodium hypochlorite had greater dissolution potential than the pure papain, and when associated with chlorhexidine, both promoted greater dissolution than did the saline solution and 2% chlorhexidine groups (p < 0.05). The 2.5% sodium hypochlorite promoted dissolution to a lesser extent than the groups with papain within a period of 30 min (p < 0.05), but, was comparable to the saline solution and chlorhexidine. After 120 min, the 2.5% and 5.25% sodium hypochlorite promoted dissolution of 100% of the pulp fragments, and papain, 61%, while chlorhexidine associated with papain and chlorhexidine alone dissolved only 55% and 3%, respectively. CONCLUSIONS: The 8% papain in gel, both alone and in association with chlorhexidine, was able to dissolve bovine pulp tissue, but to a lesser extent than did 5.25% sodium hypochlorite.
Chlorhexidine* ; Endodontics ; Papain* ; Sodium Chloride ; Sodium Hypochlorite

How Th2 immunity develops in vivo remains obscure. Basophils have been considered key innate cells producing IL-4, a cytokine essential for Th2 immunity. Increasing evidence suggests that basophils are dispensable for the initiation of Th2 immunity. In this study, we revisited the role of basophils in Th2 immune responses induced by various types of adjuvants. Mice deficient in IL-3 or IL-3 receptor, in which basophil lymph node recruitment is completely abolished, fully developed wild type level Th2 CD4 T cell responses in response to parasite antigen or papain immunization. Similar finding was also observed in mice where basophils are inducibly ablated. Interestingly, IL-4-derived from non-T cells appeared to be critical for the generation of IL-4-producing CD4 T cells. Other Th2 promoting factors including IL-25 and thymic stromal lymphopoietin (TSLP) were dispensable. Therefore, our results suggest that IL-3- and basophil-independent in vivo Th2 immunity develops with the help of non-T cell-derived IL-4, offering an additional mechanism by which Th2 type immune responses arise in vivo.
Animals ; Basophils ; Immunization ; Interleukin-3 ; Interleukin-4* ; Lymph Nodes ; Mice ; Papain ; Parasites ; Receptors, Interleukin-3 ; T-Lymphocytes