Fatal anaphylactic shock is common in forensic practice. However, it is difficult to diagnose for lacking specific pathological and morphologic changes in forensic autopsy. The application of some biochemical indicators is of great significance. This paper reviews the biological characteristics of some biochemical indicators and detection methods. The forensic application, problems and prospects of these indicators are also introduced in details. The stable biochemical indicators, IgE, tryptase and chymase, show great potential and advantages in the identification of fatal anaphylactic shock in forensic medicine.
Anaphylaxis/metabolism* ; Autopsy ; Biomarkers ; Chymases ; Forensic Medicine ; Humans ; Tryptases

To screen potential hamster chymase 2 inhibitors, a high-throughput screening (HTS) model was established. Recombinant hamster chymase 2 with active form was cloned and expressed in E. coli. The HTS model with total volume of 50 microL in 384-well microplate was based on fluorescence analysis and was proved sensitive as well as specific (Z' = 0.84). A total of 40 080 samples (including 28 060 compounds and 12 020 natural products) were screened, and 613 samples with inhibition greater than 90% were selected for further rescreening. Finally, compounds J16647 and J16648 were identified with high inhibitory activity on chymase 2, and whose IC50 values were 0.823 and 0.690 micromol x L(-1), respectively.
Animals ; Chymases ; analysis ; antagonists & inhibitors ; Cricetinae ; Enzyme Inhibitors ; analysis ; pharmacology ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; methods ; Inhibitory Concentration 50 ; Rats ; Structure-Activity Relationship

BACKGROUNDLittle is known about the role of dual angiotensin II forming pathways during heart failure. In the present study, the changes of chymase and angiotensin converting enzyme (ACE) expressions in the failing hearts of hamsters were analysed.

METHODSHeart failure was induced by ligation of left anterior descending branch of the coronary artery. Chymase, ACE and angiotensin II type 1 receptor (AT1R) mRNA levels were analysed by reverse transcription polymerase chain reaction (RT-PCR). The activities of chymase and ACE were determined by radioimmunoassay (RIA). Myocardial collagen fibre analysis was performed under optical microscope.

RESULTSLeft ventricular systolic pressure (LVSP) and maximum left ventricular developed pressure increase rate (dp/dtmax, mmHg/s) gradually moved lower at 2, 3, 4 and 8 weeks after operation. On the other hand, left ventricular end-diastolic pressure (LVEDP) increased gradually after operation. Compared with the control group (3.55 +/- 0.06, 4.79 +/- 0.70), the heart weight/body weight ratio in operation group had increased significantly at 4 weeks and 8 weeks (4.28 +/- 0.43, 6.17 +/- 0.73) (P < 0.01). Collagen staining showed that the quantity of myocardial collagen fibre increased significantly in the operation group. RT-PCR showed that the chymase mRNA level in the operation group was consistently greater than that in the control group. AT1R mRNA level was also increased significantly at 3 weeks and 4 weeks, both being 1.3 times that of the control group (P < 0.01), whereas ACE mRNA level was not changed. Higher activity of chymase was detected in operation group, being 4, 8, 13 and 19 times that of the control group at 2, 3, 4 and 8 weeks (P < 0.01), respectively. ACE activity was also significantly higher at the same time, being 7, 10, 10 and 3.5 times that of the control (P < 0.01). Angiotensin II (Ang II) level in operation group increased significantly, being 2.5, 2.7, 3.5 and 2 times that of the control group at 2, 3, 4 and 8 weeks, respectively (P < 0.01).

CONCLUSIONSA dual Ang II forming pathway from both ACE and chymase in the hamster hearts plays an important role during the development of heart failure. At the decompensatory stage, the reduction of AngII level may be associated with the decrease of ACE activity.

Angiotensin II ; analysis ; Animals ; Body Weight ; Chymases ; Cricetinae ; Heart Failure ; metabolism ; Male ; Myocardium ; metabolism ; Peptidyl-Dipeptidase A ; genetics ; physiology ; RNA, Messenger ; analysis ; Receptor, Angiotensin, Type 1 ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Serine Endopeptidases ; genetics ; physiology ; Ventricular Function, Left

OBJECTIVE: To explore the expression of tryptase and chymase in human lung tissue of anaphylactic shock and its value for forensic medicine. METHODS: With ten carbon monoxide poisoning cases as control group, the levels of tryptase and chymase were observed by immunofluorescence and analyzed using the Image Analyze and the Image-pro plus 5.0.2. The positive mast cells were counted and the levels of the tryptase and chymase were calculated respectively. RESULTS: There was a statistically significant difference (P < 0.05) for the tryptase and chymase concentrations in the lung tissue between the anaphylactic shock group and the control group. CONCLUSION The levels of the tryptase and the chymase expression are greatly increased in human lung tissue of anaphylactic shock, which may provide the morphological evidence and reference for the diagnosis of anaphylactic shock in forensic practice.
Adolescent ; Adult ; Anaphylaxis/pathology* ; Cadaver ; Carbon Monoxide Poisoning/pathology* ; Child ; Child, Preschool ; Chymases/metabolism* ; Female ; Fluoroimmunoassay/methods* ; Forensic Pathology ; Humans ; Infant ; Lung/pathology* ; Male ; Mast Cells/enzymology* ; Middle Aged ; Staining and Labeling ; Tryptases/metabolism* ; Young Adult

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

Mucosal mast cell-derived chondroitin sulphates (sulphated proteoglycans) were assayed in gut washings and homogenate of FcRgamma-knockout (KO) and wild-type (WT) C57BL/6 mice challenged with Strongyloides venezuelensis in order to assess their possible role in secondary immunity against enteric nematodes. Groups of immune KO and WT mice were challenged by oral gavage with 300 infective larvae (L3). Establishment of infection was assessed by daily faecal analysis to determine the number of eggs per gram of faeces (EPG) and by adult worm recovery on days 5 and 13 post challenge. Mucosal mast cell (MMC) counts were done on days 5 and 13 post challenge while MMC-derived chondroitin sulphates in gut washings (days 1 and 5) and homogenate (day 8) were assayed by high performance liquid chromatography (HPLC). Results showed that patent infection occurred in challenged KO but not WT mice despite significantly higher mastocytosis in jejunal sections of KO than WT mice (p<0.001). Similarly but against prediction, significantly higher concentration of MMC-derived chondroitin sulphates was observed in gut homogenate of KO than WT mice (p<0.05). In contrast, significantly higher concentration of chondroitin sulphates was observed in gut washings of WT than KO mice (p<0.05). These results suggest that MMC in KO mice failed to release sufficient amount of sulphated proteoglycans into the gut lumen as did the WT mice, which may have been part of the hostile environment that prevented the establishment in and eventual expulsion of adult S. venezuelensis from the gut of WT mice following challenge.
Animals ; Cell Count/veterinary ; Chondroitin Sulfates/*immunology/metabolism ; Chymases ; Feces/parasitology ; Intestinal Diseases, Parasitic/immunology/*veterinary ; Intestinal Mucosa/cytology/immunology/parasitology ; Jejunum/cytology/immunology/parasitology ; Male ; Mast Cells/immunology/metabolism/*parasitology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Parasite Egg Count/veterinary ; Receptors, IgG/*immunology ; Serine Endopeptidases/blood/immunology ; Specific Pathogen-Free Organisms ; Strongyloides/*immunology ; Strongyloidiasis/immunology/parasitology/*veterinary

OBJECTIVE: To explore the application value of serum total IgE, tryptase and chymase in the identification of death caused by drug anaphylactic shock. METHODS: The general information from 235 cases of non-drug anaphylactic shock and 32 cases of drug anaphylactic shock were analyzed. The serum IgE level had been detected in the cases. Ten cases caused by coronary disease and 10 cases caused by sudden manhood death syndrome were selected from non-drug anaphylactic shock cases for the control group. Expressions of tryptase and chymase in the lung and heart were detected using immunohistochemistry method. The number and IOD of positive mast cells were counted. RESULTS: In the drug anaphylactic shock group, the IgE value of 18 samples (56.25%) was significantly higher than the normal upper limit of 120 IU/mL. In the non-drug anaphylactic shock group, the IgE value of 67 samples (28.51%) was higher than 120 IU/mL. The expressions of tryptase and chymase were significantly increased in lung and myocardial tissue in drug anaphylactic shock group (P < 0.05). CONCLUSION Tryptase and chymase are more superior than that of the serum total IgE in the diagnosis of death caused by drug anaphylactic shock, and are more suitable in forensic practice.
Adolescent ; Adult ; Aged ; Anaphylaxis/pathology* ; Autopsy ; Case-Control Studies ; Cause of Death ; Child ; Child, Preschool ; Chymases/metabolism* ; Death, Sudden, Cardiac/pathology* ; Drug Hypersensitivity ; Female ; Forensic Pathology ; Humans ; Immunoglobulin E/blood* ; Immunohistochemistry ; Infant ; Lung/pathology* ; Male ; Middle Aged ; Myocardium/pathology* ; Tryptases/metabolism* ; Young Adult