Figure 1. AAV gene transfer therapy mechanism of action Following gene transfer therapy administration.

After injecting AAV into animals, the desired effect is not achieved, or the overexpression or knockdown of the target gene cannot even be detected. What are the reasons?
This issue involves many possibilities, which need to be eliminated one by one and comprehensive consideration of various influencing factors:
Virus issues: Whether the size of the foreign gene sequence is within the packaging capacity of AAV. The packaging capacity of AAV usually does not exceed 4.7kb. After removing components such as promoters, fluorescent tags, and protein tags, the actual capacity of foreign genes is generally within Under 2kb. Furthermore, whether the virus titer meets the requirements, it is recommended that viruses stored for more than half a year should be re-measured before use. Also, the storage method must be appropriate and avoid repeated freezing and thawing. If you plan to use it within a week, you can store it at 4°C. For long-term storage, you need to aliquot and store it at -80°C.
Tissue/cell specificity issues: AAV has serotypes with different tissue tropisms and can carry specific promoters. Different tissue sites may have different injection methods and injection volumes. These are all factors in AAV infection efficiency. and important influencing factors of expression characteristics.
Detection time issue: Whether the detection time point is during the peak period of AAV expression, testing too early or too late may not achieve the desired results.
Background expression level of the target gene: Before constructing AAV to regulate gene expression, has the background expression level of the target gene in the target tissue or cells been detected? Higher background expression will cause overexpression, and lower background expression will cause interference. The regulatory effect may not be observed.
Problems with the method of sampling: whether the time, method, and storage conditions for collecting animal tissues or cells are appropriate, such as whether to choose paraffin sections or frozen sections; if you want to specifically detect a certain type of cells, you can use flow sorting and other methods to separate the target cells from Separate it from the tissue and then test it.
 Detection method issues: Testing the expression level of the target gene usually involves qPCR (mRNA level) and WB (protein level). One issue to consider is whether AAV is expected to infect the entire tissue or a certain cell in the tissue. The latter It is necessary to isolate the target cells and then detect the expression level. In addition, the quality of the reagents, primers, and antibodies used in the detection should also be considered. If AAV has a fluorescent label, fluorescence can also be directly observed through in vivo imaging and tissue frozen sections to reflect the infection efficiency. It is recommended to use different methods and levels to detect AAV infection efficiency and target gene regulation effects.
Influence of regulatory factors of tissue cells themselves: In fact, in most cases, there is not a linear relationship between mRNA and protein expression levels. This process involves factors such as translation regulation, protein modification, and protein half-life. In addition, there may be a tendency for compensatory expression to increase or decrease, leading to undetectable significant changes in the protein.
Other issues: Whether the selection and health status of experimental animals meet the needs, whether the experimental operation technology is proficient, and the correct use of instruments are also keys to the success or failure of the experiment.

The post Why the Desired Effect is not Achieved After Using AAV? first appeared on Creative Diagnostics.

Figure 1. Engineering the AAV cassette.(Li C, et al.; 2020)

Q: Are there any biosafety issues when using AAV viruses?

A: To date, AAV has not been found to be pathogenic in humans. It is reported that 8 out of 10 people will be infected with AAV during their lifetime without causing related diseases. We generally use recombinant AAV (rAAV), which removes 96% of the wild-type AAV genome, further ensuring safety. rAAV generally does not have the ability to replicate and has a low host genome integration rate, small molecular weight, and extremely low immunogenicity, so it is highly safe. It is not only used in animal research, but also is a very safe viral vector even when used clinically. The clinical application of rAAV in gene therapy is a good example. In addition, NIH also pointed out that AAV belongs to the RG1 category of biological agents, which are low-risk biological agents that are not related to human and animal diseases. Therefore, just pay attention to routine safety protection and disinfection during AAV experimental operations.

Q: What do AAV serotypes mean?

A: AAV serotype refers to the protein shell that wraps genetic material, that is, the capsid protein, encoded by the Cap gene, which is the key for the virus to recognize and bind to cell surface receptors. The difference between different serotypes lies in the differences in the amino acid sequence and spatial structure of the capsid protein, which results in different serotypes having different tropisms for specific tissues or cells. AAV serotypes can be directly expressed as AAV2, AAV5, AAV9, etc. Another point that needs to be made clear is that the core skeleton of almost all recombinant AAV viral vectors is derived from the AAV2 subtype, so the complete AAV serotype can be expressed as, for example, AAV2 /2, AAV2/5, AAV2/9, etc.

Q: What are the roles of specific AAV serotypes and specific promoters?

A: AAV serotypes are based on the characteristics of virus infection, and the role of specific promoters is at the level of gene expression after virus infection. Generally speaking, specific promoters refer to tissue-specific promoters or organ-specific promoters. They are promoters derived from genes that are only expressed in certain specific tissues or organs, and the promoter activity is relatively good. With the blessing of its specific expression function, AAV with specific serotypes has stronger targeting ability. However, one thing that needs to be clear is that the regulation of cell expression in organisms is very complex. In actual application, not all specific promoters will strictly follow expression specificity. To a certain extent, it can be defined as relative specificity, so sometimes in animal experiments, it is possible to find that relevant genes are expressed in some non-target tissues, which is a promoter expression leakage incident. In this case, attempts can be made to further increase targeting specificity through in situ tissue injection.

Q: How long does it take to observe the effects after AAV injection into animals?

A: AAV is a single-stranded DNA virus. After infecting cells, it needs to form a double-stranded DNA form in the cell for gene expression. This process is generally long, and expression begins 1-2 weeks after the virus is injected, but the expression level at this time is relatively low. It is not suitable for sampling and testing, and it is usually recommended to conduct subsequent testing after 3-4 weeks. For AAV that wants to express quickly, you can choose scAAV, which is a self-complementary AAV whose DNA is double-stranded. Compared with standard single-stranded ssAAV, scAAV vectors do not require the step of replicating single-stranded DNA into double-stranded DNA to perform transcription, so gene expression It is more rapid and can reach the peak expression 3-5 days after injection, and the expression level is higher. However, their packaging capacity (<2.5Kb) is half that of ssAAV (<4.8Kb), limiting the genes that can be successfully packaged and the number of regulatory elements that the vector can have.

Q: How to improve infection efficiency when AAV is used to infect animals?

A: Excluding the influence of the status of the animal itself, from the perspective of AAV use alone, we need to select the appropriate AAV serotype, injection method, injection site, and injection volume based on the target of infection, and then by consulting references and virus usage instructions. Generally, AAV with a higher titer will be more effective than the same amount of AAV with a lower titer.

Q: Why is no fluorescence or weak fluorescence observed after AAV infects animals?

A: Here we need to consider the characteristics of the fluorescent protein, such as the brightness and stability of the fluorescent protein. The organic solvents used in the process of making tissue sections may change the structure of the fluorescent protein, causing fluorescence quenching; GFP is easily quenched in an acidic environment.

Q: Can fluorescence be directly observed on paraffin tissue sections to determine infection efficiency?

A: Can’t. Although paraffin sections can better preserve the complete tissue structure, the production process requires the use of organic solvents such as alcohol and xylene, which will cause the conformation of the fluorescent protein to change and prevent it from being excited to produce fluorescence. This situation can be observed by immunofluorescence staining of fluorescent proteins.

Q: When it is necessary to observe in vivo imaging of animals, is it better to carry a fluorescent protein gene or a luciferase gene in AAV?

A: In terms of comparison between fluorescent proteins and luciferase, luciferase is more suitable for in vivo imaging observation. It has stronger fluorescence penetration and is catalyzed by its substrate to emit light (i.e. bioluminescence), so the observed background is low and The effect is good, and the substrate has almost no impact on the physiological state of animals; fluorescent proteins require excitation light of a specific wavelength to excite fluorescence. Interestingly, animal hair and other tissues will also exhibit fluorescence, resulting in background that affects observation; By removing hair from the observation area, the background can be reduced.

Q: Can adeno-associated viruses (AAV) be used for genome editing?

A: The answer is yes. Using Crispr/Cas9 technology for in vivo knockout through AAV, a problem that needs to be solved is how to effectively load gene editing tools. At present, many Cas proteins with smaller genes have been discovered, such as SaCas9 (Staphylococcus aureus Cas9), Nme2Cas9 (Neisseria meningitides Cas9), etc. These Cas proteins can be effectively loaded into AAV vectors to achieve targeted gene editing in vivo. Another method is to use the cre-loxp system for knockout. The premise is that flox animals must be available, and then used with AAV-cre to achieve target gene knockout.

Q: Animals need to be modeled. When is the appropriate time to inject AAV?

A: The peak expression of AAV is 3-4 weeks after injection. The injection timing is then calculated based on the time of animal modeling and the purpose of injecting AAV. For example, if AAV regulates gene expression and thus plays a therapeutic role, the peak expression period of AAV must be controlled in synchronization with the modeling process; if it is for prevention, then it may be necessary to keep AAV at the peak expression period before or early in the modeling process. Of course, specific issues still need to be analyzed in detail and judged based on the characteristics of the model. It should be noted that there is no specific time frame for how long the peak expression of AAV can be maintained; if the modeling time is long, a follow-up injection can be performed 1-2 months after the first injection to maintain a long-term high expression state.

Q: Can AAV be used to infect cells in vitro?

A: In general, it is not recommended to use AAV to directly infect cells in vitro. Although AAV can maintain long-term gene transcription expression, in vitro cell lines usually proliferate quickly, causing the AAV infected into the cells to be continuously diluted as the cells divide, further reducing its expression level and thus limiting the expression ability of AAV. If it must be used for in vitro cell infection, it is best to use specific serotypes (such as AAV-DJ type), and the infection process requires a very high MOI (104~106). The specifics need to be explored in preliminary experiments.

The post Adeno-Associated Virus Vector (AAV) Application Q&A first appeared on Creative Diagnostics.

Figure1. The Production of biological products.

Protein A, G and L residues

Affinity chromatography for Protein A, G and L is a common method for purifying therapeutic antibodies. They can separate the proteins on the surface of engineered bacteria very well. However, they will shed to a certain extent during repeated use, and the remaining Protein A, G, and L in the product will cause adverse immune responses. The complex of it and antibody can activate the immune response of Fc-carrying leukocytes and complement system in vivo, resulting in oxidation and allergic reactions. Therefore, it is necessary to detect whether Protein A, G and L remain in the product during the production process. Among them, the commonly used method is ELISA detection.

dsRNA Residue

In the process of preparing mRNA vaccines based on enzyme reactions, abnormal in vitro transcription reactions (IVT) may also introduce dsRNA contaminants. Detection and removal of dsRNA contamination generated in in vitro reactions is more important for RNA vaccine production than viral-derived dsRNA. IVT mRNA mixed with dsRNA contamination will cause a strong type I interferon-mediated antiviral response, upregulate the expression of key enzymes such as PKR and OAS, resulting in the inhibition of cellular translation and the degradation of intracellular mRNA and ribosomal RNA. Correspondingly, the introduction of an FPLC purification process that can efficiently remove dsRNA contamination can increase IVT mRNA-mediated protein expression by 1000-fold in primary human DC cells. Therefore, proper and efficient IVT mRNA purification during mRNA vaccine production is critical for maximizing production of DC antigenic proteins and avoiding unnecessary activation of autoimmune responses.

Host Cell DNA Residue

Recombinant protein drugs, antibody drugs, vaccines and other products in biological products are expressed and produced by continuously passaged animal cell lines. Although they have undergone strict purification processes, there may still be residual host cell DNA in the products. The risks associated with host cell residual DNA in viral vaccines are mainly infectivity and carcinogenicity, if retroviral proviruses are present, their genes integrated into the recipient genome may cause infection; if there are viruses or oncogenes in the passaged cell matrix, may have the potential risk of causing tumors. Therefore, it is very important to establish a suitable detection method for residual DNA in host cells to monitor the safety and quality controllability of biological products.

BSA Residue

The production of biologics often requires cell culture, and bovine serum albumin (BSA) is used as a nutrient in cell and microbial culture. A large amount of bovine serum albumin has removed most of the BSA through steps such as washing and purification during the production process, but trace amounts of BSA still exist in the finished biological preparations. As a foreign protein, BSA may cause severe allergic reactions after entering the human body and affect the safety of biological products. Therefore, strictly controlling the content of BSA in products is one of the key tasks in the production of biological products. In addition to vaccines, biological agents such as cell therapy, stem cells, and tissue engineering products will also be tested for BSA residues to ensure the safety of related biological products.

CHO Host Cell Protein (HCP) Residue

Host cell proteins (HCPs) are process-related impurities expressed by host cells used to produce biopharmaceutical proteins. During purification, most of the HCPs are removed (>99%), but residual HCP amounts remain in distributed products such as monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), therapeutic proteins, vaccines, and other protein-based biopharmaceuticals. National regulatory mandates such as FDA require that biopharmaceuticals must be analyzed and purified to reduce the host cell protein HCP to acceptable levels.

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Molecular Biology Methods

There are multiple virulence genes in Legionella pneumophila, including mip, ira, lvr, ot, icm, lvh, cpx, and rtxA. Mip-encoded protein can resist intracellular killing against mammalian and protozoan phagocytes; cpxRA regulates the essential factor for the secretion of effector proteins by the type IVB secretion system; the methyltransferase encoded by the iraA locus is required for bacterial intracellular infection The hypothetical iron-peptide transporter encoded by the iraB locus can be involved in the uptake of high-valent iron by means of iron-loaded peptides; the lvr gene is responsible for the regulation of lvh family secreted proteins; rtxA encodes proteins related to adhesion, cytotoxicity and pore formation. Lvh encodes a protein derived from the type IVA secretion system, which helps to bind virulence; dot/icm encodes a protein derived from the type IVB secretion system, which is responsible for the replication of Legionella in host cells. Virulence genes are distinct regions of DNA that are present in the genome of pathogenic bacteria but not present in the same or related non-pathogenic strains. The pathogenicity of Legionella is closely related to its virulence, and its pathogenicity is determined according to the amplification results of pathogenic virulence genes. The polymerase chain reaction (PCR) is a molecular biology technique used to amplify the target nucleotide sequence, and the target gene to be amplified can be amplified several million times within 2 to 3 hours. After the reaction, the amplified product was separated by agarose gel electrophoresis, and the result was positive, that is, the existence of the gene was confirmed. Although the quality of template nucleic acid extraction is not high and false negative results may occur, this method has strong specificity and high sensitivity, and is the basis for other molecular biology experiments.

In addition, PCR-based pathogenicity detection methods for Legionella include multi-locus variable number tandem repeat analysis to detect virulence genotype, micro-DNA microarray to detect Legionella virulence genes, and use of genetic markers to identify Legionella clinical and strains environmental conditions.

Immunology-based Assays

ELISA

Enzyme-linked immunosorbent assay (ELISA) is a simple and rapid technique for the detection and quantification of antibodies or antigens attached to solid surfaces. The substrate to which the enzyme is added produces a color change or light signal that correlates to the amount of antigen present in the original sample. Therefore, the total amount of antigen in the sample can calculate based on the direct relationship between the intensity of the color or the chemical signal and the amount of antigen in the sample amount or concentration. This method is simple to operate and has high sensitivity. However, the method has high requirements for technicians to operate. In addition, recombinant antigens are used in the method, and the specificity is not high and false positive results are likely to occur. A key feature of the pathogenesis of L. pneumophila is the rapid influx of neutrophils into the lung, and IL-1α is a key initiator of neutrophil recruitment to the lungs of L. pneumophila-infected mice. The researchers used this technique to detect the amount of IL-1α released from mice infected with Legionella pneumophila and found that neutrophil recruitment was in response to the virulent Legionella pneumophila, and determined the relationship between the amount of IL-1α released and the strain.

Western blot

Western blotting is a method based on the specificity of antigen-antibody binding proteins. The mixed antigen samples are separated by one-dimensional or two-dimensional electrophoresis on the gel plate, and the antigen components in the gel are transferred to the blotting paper under the natural adsorption force of the blotting paper or other external forces, and then the immobilized matrix membrane is combined with the blotting paper. And then, antigen-immobilized matrix membranes were detected and analyzed by substrate development and autoradiography. Western blot analysis is simple, time-saving and labor-saving, and can identify multiple proteins at the same time. Previous studies have found that flagella and their secreted proteins contribute to the movement, host finding, biofilm colonization and virulence of L. pneumophila. Bacterial virulence can thus be determined by detecting the L. pneumophila flagella and their secreted proteins.

LDH Cytotoxicity Assay

Lactate dehydrogenase (LDH) is a stable protein that exists in the cytoplasm of normal cells. When the cell membrane is damaged, LDH is released outside the cell. There is a good correlation between the cytotoxicity of LDH and the blood concentration of animal mortality and human death. The LDH cytotoxicity detection kit can be used to detect the activity of LDH in the cell supernatant on a 500nm microplate reader to determine the degree of cell damage, thereby realizing the detection of the toxicity of Legionella. This assay is sensitive, simple to operate, and accurate; however, the assessment of cytotoxicity is based on quantification of cell damage, so it is necessary to determine the optimal amount of cells.

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Pharmacological Action

Anticoagulant and Antithrombotic Effect

The study found that recombinant hirudin has obvious antithrombotic effect, and both injection and oral administration can exert the drug effect. In animal experiments, for venous thrombosis and arterial thrombosis in rats and disseminated intravascular coagulation in rabbits, recombinant hirudin can significantly prolong the activated partial thromboplastin time, and inhibit the level of fibrinogen and antithrombin activity, so as to achieve anti-inflammatory effects, anti-coagulation and anti-antithrombotic effects. Its anti-venous thrombosis intensity is about 4 times that of heparin, and the dose of recombinant hirudin is negatively correlated with the dry weight of thrombus. Its mechanism of action may be related to affecting the extrinsic coagulation system and promoting fibrinolysis: 1) Recombinant hirudin can prolong plasma prothrombin time (PT) and increase the release of tissue plasminogen activator (t-PA) , while inhibiting the release of PAI-1, and has endothelial protection; 2) Recombinant hirudin can effectively inhibit thrombin, especially interfere with or hinder the formation of thrombin-thrombomodulin complex (Th-TM). Inhibit the activation of TAFI by Th or Th-TM, reduce the production of TAFIa, and then achieve the purpose of antithrombotic.

Anti-Atherosclerotic Effect

Recombinant hirudin can improve apolipoprotein E knockout (ApoE-/-) mice atherosclerosis, regulate serum lipid levels, inhibit vascular endothelial proliferation, and reduce collagen fiber proliferation, thereby improving ApoE-/- mice Studies have found that recombinant hirudin can also reduce the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP), and promote the expression of CD36 and Toll-like receptor 4 (TLR4) /NF-κB, thereby improving the chronic inflammatory response of aortic atherosclerosis. Recombinant hirudin may also delay the occurrence and development of atherosclerosis by down-regulating the expression of matrix sympathetic molecule 1, Orai1 protein, and transient receptor potential channel 1.

Antitumor Effect

The study found that recombinant hirudin can significantly inhibit the proliferation of human hepatoma SMMC-7721 cells and promote their apoptosis, and can effectively reverse the growth promotion and apoptosis resistance caused by thrombin. The molecular mechanism may be that recombinant hirudin significantly reduces the apoptosis. The anti-apoptotic gene Bcl-2 mRNA and protein expression in tumor cells was increased, and the pro-apoptotic gene Bax mRNA and protein expression was increased. In addition, some studies have found that recombinant hirudin can reduce the expression of protease-activated receptor (PAR-1) in laryngeal cancer cells, thereby inhibiting the expression of VEGF and matrix metalloproteinase-2 (MMP-2), thereby inhibiting the expression of laryngeal cancer cells and metastases,  and ultimately exert an anti-tumor effect.

Reduce Brain Damage

Studies have found that recombinant hirudin can significantly reduce cerebral edema in rats with cerebral hemorrhage by specifically inhibiting thrombin and intervening in the inflammatory response of surrounding tissue of cerebral edema, and can improve the neurological deficit of rats for a long time, and significantly reduce the residual rate of cerebral hemorrhage caused by cerebral hemorrhage. Recombinant hirudin may reduce brain injury by intervening in the inflammatory tissue surrounding hematoma in rat cerebral hemorrhage by reducing the expression of TNF-α and ICAM-1.

Improves the Survival Rate of Random Skin Flaps

In the rabbit ear vein congestion model, the local application of recombinant hirudin can induce the expression of bFGF and promote the proliferation of microvessels, thereby improving the flap congestion state and improving the survival rate of the flap with venous congestion. The study found that recombinant hirudin can reduce the local malondialdehyde (MDA) level in the random flap congestion model rat, reduce the local inflammatory response of the flap, reduce the local endothelin level of the congestion flap, and improve the survival rate of the random flap. Another study found that for skin avulsion injury in rats, local application of recombinant hirudin can effectively reduce the effect of adhesion factor CD11b/CD18 in blood and ICAM-1 in tissue, reduce tissue inflammatory response, and improve the effect of vascular endothelial cell injury in ischemia-reperfusion process, thereby significantly improving the healing rate of skin flaps after skin avulsion injury.

Relieve Renal Interstitial Fibrosis

The researchers used unilateral tubal ligation to prepare a mouse model of renal fibrosis. After treatment with recombinant hirudin, the researchers found that the renal function of the mice was improved, the renal cortex was thinned, the degree of renal lesions was reduced, and the positive staining area of collagen fibers in the renal interstitium was found. The expression levels of α-smooth muscle actin (α-SMA) and fucosyltransferase-8 (FUT-8) in renal tissue were significantly reduced, indicating that recombinant hirudin could alleviate renal interstitial fibrosis.

Anti-Cataract Effect

Scientists used D-galactose and sodium selenite to induce cataract models in lactating rats respectively, used recombinant hirudin for intervention treatment, and used rabbits to conduct eye irritation experiments to test the safety of recombinant hirudin. The results showed that recombinant hirudin could reduce the turbidity and MDA level of the rat lens, and increase the level of superoxide dismutase (SOD), and had good preventive and therapeutic effects on both sugar-induced cataracts and selenium-induced cataracts, and it was safe and non-irritating. Another study found that in the human lens epithelial cell cataract model induced by high concentration of galactose, recombinant hirudin can reduce the expression of heat shock protein 70 (Hsp70), Hsp27 and αB crystal protein, and significantly alleviate the tolerance of Hsp induced by high concentration of galactose. That is, reducing the damage of high concentrations of galactose to lens epithelial cells, thereby protecting lens epithelial cells.

Other Functions

The study found that recombinant hirudin can alleviate experimental autoimmune encephalomyelitis (EAE) in mice, and the mechanism of action may be related to the inhibition of MMP-9 and inducible nitric oxide synthase (iNOS) mRNA expression in mouse brain tissue, thereby reducing MMP-9 and NO levels. Cigarette smoking combined with intratracheal injection of lipopolysaccharide (LPS) to establish a rat model of chronic obstructive pulmonary disease (COPD). Recombinant hirudin can reduce airway remodeling in COPD rats after intervention. TNF-α levels in bronchopulmonary tissue, reduced phosphorylation of extracellular protein-regulated kinase (p-EPK) expression.

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Structure, Function and Features

Natural hirudin is an acidic single-chain polypeptide with a relative molecular mass of about 7000. It consists of 64-66 amino acids and contains 3 disulfide bonds without the presence of polysaccharides. At present, 7 isomers have been isolated and identified, which are active High, stable structure, not easy to inactivate and so on. Natural hirudin contains six cysteine ​​residues with similar three-dimensional structures and distribution positions, the thrombin active binding site is located at the N-terminal of its structure, and the thrombin fibrinogen binding site is located at the C-terminal of its structure. Amino acid residues in the middle region play a regulatory role. Due to the special structure of its peptide chain, natural hirudin has a strong anticoagulant effect. Its penetration ability and stability are strong, it will not be destroyed by trypsin and chymotrypsin, and it will not decompose even if the pH and temperature change slightly, it can be taken orally, and the decomposed fragments also have anticoagulant effect, But under some special conditions, such as the coexistence of strong alkali and high temperature, it will lead to irreversible inactivation.

Pharmacological Effects of Natural Hirudin

Anticoagulant and Antithrombotic Effect

In animal experiments, it was found that natural hirudin can accelerate the blood flow rate at the blood stasis site, improve the abnormal blood rheology, inhibit platelet aggregation, improve the vascular patency rate, and have a significant antithrombotic effect.

Anti-Fibrotic Effect

Some studies have found that natural hirudin can inhibit the growth of gingival fibroblasts and tendon fibroblasts, and is effective in renal interstitial fibrosis, renal tubular epithelial cell fibrosis, pulmonary fibrosis, and idiopathic interpulmonary fibrosis (IPF). Has inhibitory effect. The possible mechanisms of natural hirudin against fibrosis include three aspects: 1) Regulating the secretion of basic fibroblast growth factor (bFGF) and transforming growth factor-β1 (TGF-β1), inhibiting the growth, proliferation and further development of fibroblasts Inhibit scar formation, inhibit the production and activity of plasminogen activator inhibitor-1 (PAI-1), reduce fibrin deposition, and promote collagen dissolution; 2) Reduce monocyte Chemoattractant Protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), p38 mitogen-activated protein kinase (p38MAPK) expression; 3) Decreased protein kinase B (Akt), phosphorylated Akt ( p-Akt) protein expression, slowing down the progression of IPF.

Regulates Microvascular Regeneration

Natural hirudin can promote the expression of vascular endothelial cell growth factor in random skin flaps and increase its survival rate to promote the formation of new microangiogenesis. Vascular endothelial growth factor (VEGF)/Notch signaling pathway, as a classical pro-angiogenic molecular pathway, can promote the differentiation of human pluripotent stem cells into adult microvascular endothelial cells (HMVECs). Some studies have found that natural hirudin can regulate angiogenesis.     At a low concentration of 4 ATU/mL (ATU is the amount of inactivating 1 NIH unit of thrombin at 37 °C), it may promote the proliferation of HMVECs and promote the proliferation of HMVECs through the VEGF-Notch signaling pathway, and angiogenesis; at a high concentration of 7 ATU/mL, it mainly exerts antithrombin and anti-inflammatory effects, and inhibits angiogenesis by improving microcirculation. The study found that natural hirudin can also improve the survival rate of rat skin flaps through the protease-activated receptors (PARs)/p38/nuclear factor-κB (NF-κB) pathway.

Antitumor Effect

Studies have found that natural hirudin can effectively promote the apoptosis of mouse hemangioma EOMA cells and inhibit their proliferation in vitro, and there is an obvious dose-response relationship. The study found that natural hirudin may inhibit the proliferation, migration and invasion of liver cancer HepG2 cells by down-regulating the expression of VEGF. In addition, other results showed that natural hirudin may inhibit the proliferation of nasopharyngeal carcinoma CNE2 cells by inducing cancer cell apoptosis and arresting the cell cycle at G2/M, which may be related to the up-regulation of Bax and p21 mRNA expression by natural hirudin.

Improve Inflammatory Obstruction of Fallopian Tubes

The pelvic injection of natural hirudin was given to the rats with salpingitis obstructed, and metronidazole was used as the positive control group. After hematoxylin-eosin (HE) staining, the morphology and structure of the fallopian tubes of the rats before and after the experiment were observed, and the expression of VEGF protein in fallopian tubes were detected. The results showed that after pelvic injection of natural hirudin, the structure of the fallopian tubes of rats was significantly changed and improved, indicating that natural hirudin has a significant therapeutic effect on salpingitis obstruction. Treatment offers new ideas.

Anti-gout Effect

The mouse hyperuricemia model, the mouse chronic hyperuricemia model and the rat acute gout inflammation model were used, and allopurinol tablets were used as the positive control to observe the effect of natural hirudin on hyperuricemia and anti-gout effect. The results show that natural hirudin has significant anti-hyperuricemia and anti-gout effects, can reduce serum urea nitrogen levels, significantly inhibit the expression of human glucose-facilitated transporter 9 (GLUT9), and alleviate renal pathological damage.

Improve Kidney Function

Natural hirudin can reduce renal injury and improve renal function in diabetic nephropathy model mice. There are three possible mechanisms: 1) Inhibit the expression of transient receptor potential cation channel 6 (TRPC6) to reduce calcium influx, Protect renal podocytes, thereby improving renal function; 2) Reduce RhoA protein, protect podocyte cytoskeleton, and maintain podocyte cytoskeleton morphology and density; 3) Inhibit the expression of VEGF and TGF-β1 to improve renal function.

Other Function

Through the vascular ring experiment and the kinetic model of large-conductance calcium-activated potassium channel (BKCa), it was found that natural hirudin has a vasodilatory effect, which may be achieved by activating BKCa channel. In additon, studies have found that natural hirudin can reduce cerebral edema in rats with cerebral hemorrhage, reduce the rate of brain cell apoptosis, and relieve neurological impairment. Its anti-apoptotic mechanism of brain cells may be related to the inhibition of JAK2/STAT3 signaling pathway.

The post Application Research of Natural Hirudin first appeared on Creative Diagnostics.

Urea Breath Test UBT

UBT uses the urease activity of Hp to decompose urea into ammonia and carbon dioxide. The subjects took the labeled ¹³C or ¹⁴C urea capsules, and the exhaled ¹³CO₂ or ¹⁴CO₂ was measured by a high-sensitivity specific instrument to determine whether there was Hp infection. Because the 13C-UBT test is not radioactive, it is more suitable for children and pregnant women, but in fact the ¹⁴C-UBT radiation dose is very small. In most studies, the sensitivity and specificity of ¹⁴C-UBT are more than 90%. UBT is easy to operate, has low technical requirements for inspectors, and has good compliance by inspected personnel, so it is popularized and used in clinical work. UBT has been in use for approximately 30 years and is still the most popular, accurate and universal non-invasive test for diagnosing Helicobacter pylori infection.

¹⁵N-urinary Ammonia Excretion Test

The principle is the same as that of UBT. After subjects take orally 15N-labeled urea, it is decomposed by urease, and then the labeled 15NH3 is absorbed by the small intestine and then excreted in the urine. Urine was collected 2 hours after taking the medicine, and the excretion rate of 15N-ammonia in the urine was detected by mass spectrometry to determine the degree of Hp infection in the stomach. This method also has the advantages of convenience, non-invasiveness and less pain for subjects, and compared with 14C-UBT, 15N excretion test is non-radioactive. However, specimen collection, storage and transportation are important reasons that affect its accuracy. In addition, because 15N-ammonia needs to be metabolized in the liver and excreted by the kidneys, there are certain contraindications for patients with liver and kidney insufficiency and false negative results are prone to occur. Some studies believe that probiotics have a certain ammonia-lowering effect, so false negative results may occur. Researchers have shown that the 15N excretion test has a specificity of 81% and a sensitivity of 89%. Because its accuracy is lower than that of UBT, it is rarely used clinically.

Stool Antigen Test Hp-SAT

Hp colonizes on the epithelial surface of the gastric antrum mucosa. As the epithelium of the gastric mucosa is renewed every 3 days, Hp metabolites and dead bacteria are excreted in the feces as specific antigens. Stool antigen detection mainly uses enzyme immunoassay (EIA) and immunochromatography (ICA) to detect related antigens. A retrospective study reported the specificity (93%), sensitivity (91%), positive predictive value (92%) and negative predictive value (93%) of SAT, and it was considered that the coincidence rate of HpSAT with serological examination or UBT exceeded 90%. Some scholars believe that HpSAT based on enzyme immunoassay is more reliable and accurate than the test results based on immunochromatography. Compared with other Hp detection methods, SAT does not require subjects to take any reagents orally and collect test specimens for a long time. The operation is simple and suitable for any age. For subjects with poor compliance or unable to cooperate with the test, SAT is the most beneficial test method.

Serological Testing

Studies have tested Hp’s IgA, IgG and IgM antibodies, but only the IgG antibody test is reliable. Scientists have pointed out that the sensitivity, specificity, and accuracy of the rapid colloidal gold method for detecting Hp are 94.1%, 88.9%, and 93.3%, respectively, and there is no statistically significant difference compared with the ¹³C breath test. There are also certain differences in the application of different serological testing methods. Through research, scientists believe that the rapid colloidal gold detection method has a higher correlation with the current Hp infection than the western blot method, and is suitable for the preliminary screening of Hp infection; while the western blot method can be used for virulence typing, which is more suitable For the choice of clinical drug treatment. Studies have shown that specific antibodies will only appear in the blood several weeks after the human body is infected with Hp, and the antibodies in the serum can be maintained for more than 6 months after the eradication of Hp. Therefore, a positive serological antibody cannot be used as a basis for current infection, and a negative antibody cannot be used as a non-infection. Because of the possibility of infection, the false negative result of early antibody did not reach the detection threshold. Therefore, when choosing a serological antibody test as a clinical diagnosis result, it is recommended to make a diagnosis in conjunction with UBT. Because of this, researchers proposed that SAT is the only Hp test that is not affected by PPIs, bismuth, antibiotics, and gastric bleeding. If the above-mentioned drug treatment cannot be stopped, SAT is considered to be one of the useful detection methods.

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Endoscopy

In recent years, endoscopy technology has gradually matured and continuously improved, which makes it possible to use endoscopy to diagnose Hp infection. Some scholars believe that endoscopic manifestations such as gastric mucosal swelling, punctate erythema, variegated changes, fold enlargement, and muddy mucus can help diagnose Hp infection. In recent years, the observation of abnormal gastric mucosa through narrow-band imaging, confocal laser microendoscope and linkage imaging technology provides a basis for the diagnosis of Hp. Japanese endoscopy experts proposed that after Hp infection, under magnifying gastroscopy, it can be observed that the density of the veins in the gastric body collection decreases, and the texture becomes blurred, disordered, or even disappeared. Related studies have shown that observing the collecting veins of the gastric body has better specificity than observing the collecting veins of the gastric antrum. The narrow band imaging technique (NBI) diagnosis of Hp infection mainly observes gastric pits, which are tortuous, thick, and elongated, with obvious increase in branch curvatures, and interconnections in a dendritic shape. Confocal laser microscopy (CLE) provides gastric submucosal analysis and histological examination. CLE uses white and bright round spots, neutrophil infiltration, and crypt abscess as the diagnostic criteria for Hp infection, and the accuracy and specificity are more than 90%. Studies have shown that there is no Hp colonization in the metaplastic epithelium. CLE can identify goblet cells and small intestinal villi cells to determine whether there is local metaplasia and avoid biopsy at the metaplasia site, which affects the detection rate of Hp.

Rapid Urease Test-RUT

RUT is a detection method that can be performed in liquid or solid media, and there is no statistically significant difference in specificity between the two. RUT has the advantages of speed, simplicity and relatively high accuracy. However, RUT sensitivity is affected by the density and morphology of bacteria in the biopsy. Researchers believe that each biopsy specimen requires at least 104 microorganisms to reach the detection threshold of RUT, but a considerable proportion of patients may have a cell density below this limit and false negatives. In addition, treatment with PPIs, antibiotics, and bismuth agents may lead to false negative results. When the patient’s saliva is excessively secreted or the bile is refluxed to the stomach, it may contaminate the gastric biopsy specimen and cause a false positive result of RUT. The presence of other urease-producing bacteria in the stomach, such as Staphylococcus cephalosporium, may also cause false positive results.

Polymerase Chain Reaction (PCR) Detection

PCR is not only used for bacterial detection, but also for broad-spectrum infection detection, new infection assessment, bacterial genotype identification, antibiotic resistance and epidemiological research. PCR detection of Helicobacter pylori has the advantages of simplicity, accuracy, rapidness, automation and efficiency. Accurate primer design and correct gene selection are the key to successful PCR reactions. Commonly used primers for Hp detection include 16srRNA, 23srRNA, CagA, VacA, ureA, ureB, ureC and Hsp60 genes. The test samples for Hp include fresh gastric mucosal tissue, tissue specimens that have undergone RUT, tissue specimens embedded in wax blocks, gastric juice, saliva, feces, and environmental samples. The researchers used fresh gastric mucosal tissue to perform PCR with 100% specificity and sensitivity, but the specificity of stool specimens was only 25%. The PCR and aniline blue staining of the tissues embedded in the wax block were compared, and the positive rate of Hp in the tissue wax block was 64.2%, which was higher than that of the aniline blue staining method. Because fixatives are needed in the process of tissue embedding, it may cause RNA fragmentation and is not suitable for RNA isolation, so there are some false negative results. With the completion of the whole genome sequencing of Hp, PCR technology is widely used in the diagnosis, genotyping, epidemiological investigation and prognostic judgment of Hp infection, and it has high clinical application value.

Stained Microscopic Examination of Tissue Sections

Histological examination is considered to be the gold standard for direct diagnosis of Hp in gastric mucosa. Scientists believe that the sensitivity and specificity of histological diagnosis of Helicobacter pylori are higher than UBT and RUT. In addition to directly observing rod-shaped and spherical bacteria, this detection method can also provide basic data on different types of gastritis, atrophy, dysplasia, metaplasia, and malignant tumors. At present, the more commonly used methods for detecting Hp include: Giemsa staining, H&E staining, W-S staining and immunohistochemical staining. In addition to directly observing the bacteria outside the body, H&E staining also helps to assess the severity of inflammation. However, the bacteria and the background in H&E staining are blue-purple, which is difficult to distinguish with the naked eye, and false negative results may be caused by the lack of experience of the observer. W-S staining is brown-black with silver stained particles attached to the bacteria, and the background is brown, which is easier to distinguish and has high sensitivity. However, too many silver-stained particles are often mixed with the bacteria, resulting in false positive results. The immunohistochemical staining of the bacterial body is brown, the background is blue, and the contrast is clear and easy to observe. But the disadvantage is that the reagents are expensive and the staining time is long. A sensitive detection study for the three detection methods found that the sensitivity and specificity of the three methods were H&E (94%, 97%), WS staining (97%, 98%), and immunohistochemistry (100%, 100%). Immunohistochemical staining has the highest sensitivity, specificity, and accuracy, and has low requirements for doctors to read the film. It is worthy of promotion in primary hospitals. The limitation of histological examination lies in the dependence on the sampler and the observer, and there are certain experience and ability requirements for staining techniques and identification of pathogens. In addition, the sensitivity of histology is often affected by the location, quantity and size of the biopsy tissue. The deformation of Hp caused by various factors reduces the recognition of conventional staining methods and causes false negative results.

Bacterial Culture Test

It is a challenging task to successfully isolate, extract and culture Hp from gastric biopsy specimens. Although Hp culture has extremely high specificity. However, due to strict culture conditions, Hp culture is less sensitive and the culture time is longer. Hp culture is affected by many factors, such as the quality of clinical specimens, the time interval between sampling and culture, and unsuitable transportation conditions (temperature, air exposure time, etc.). The sensitivity of cultured Hp from gastric biopsy specimens is above 90% and the specificity is 100% under optimal conditions. However, some researchers believe that even experienced experimenters can only cultivate the bacteria from 50% to 70% of the infected tissue. A large number of studies have shown that it is very difficult to cultivate Hp from feces, saliva and vomit because of the presence of symbiotic bacteria composed of other microorganisms that hinder the growth of Hp. Therefore, these methods are not recommended for clinical diagnosis.

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Food allergy has become a global public health problem. According to reports, the incidence of allergic reactions in adults is about 3% to 4%, and the incidence in children is as high as 6%. At present, there are more than 180 types of allergic foods identified. According to the UN Food and Agriculture Organization’s report, more than 90% of food allergens are found in milk, eggs, fish, crustacean aquatic products, peanuts, soybeans, nuts and wheat. Food allergic reactions are very harmful to the body, which can affect the respiratory system, gastrointestinal system, central nervous system, skin, muscles and bones, etc., and may even cause anaphylactic shock and endanger life. At present, allergy research focuses on the sensitization detection of food allergens. In view of the different sensitivity of each individual’s immune response to allergens, the sensitization evaluation of food allergens is of great significance.

The material basis of food allergies includes food allergens and specific IgE. According to different evaluation objects, the sensitization evaluation methods of food allergens are divided into two categories: serological methods and cytological methods. The serological method is mainly to evaluate the sensitization of the protein according to the binding capacity of IgE in the positive serum. The cytological method evaluates the sensitization of immune cells based on the protein stimulated to produce inflammatory mediators. According to the different places where the evaluation method is performed, it can be divided into in vivo method, in vitro method and bioinformatics comparison method. This article focuses on methods for evaluating the sensitization of food allergens in the body.

The in vivo method uses a very small amount of food allergens to stimulate the body, and then evaluates the sensitization of food allergens based on whether the body has symptoms of allergic reactions or detecting the level of IgE in the body. It mainly includes double-blind placebo-controlled food challenge experiment (DBPCFC), skin experiment (ST) and animal models.

Double-blind Placebo-Controlled Food Challenge Experiment 

The double-blind placebo-controlled food challenge experiment (DBPCFC) uses suspicious food to stimulate the patient, and then determines whether the food is allergenic according to the patient’s symptoms. This method is considered to be the gold standard for diagnosing food allergies, and it can also be used to determine the threshold of allergenic foods. In the absence of an allergic reaction, it is not sufficient to determine an exclusion diet based on the results of IgE detection by serological methods, and the use of DBPCFC to determine food allergens can effectively stop most children’s unnecessary diet exclusion. DBPCFC generally needs to be carried out in a hospital or outpatient department. It requires medical staff and equipment capable of dealing with severe allergic reactions, and the operation and testing by experienced nursing staff. The most important thing in the experiment is to control double-blind, experimental dose and placebo. Double-blind control requires that the difference between the tested food and the placebo be eliminated as much as possible, that is, the intermedium can mask the taste, texture and odor of the tested food, which limits the experimental dose, but can minimize the subject’s subjective factors to the experiment The impact of results. For most foods, patients, and clinical conditions, it is appropriate to use 3, 10, 30, 100, 300, 1,000, and 3,000 mg of challenge agents every 20 minutes. For liquid and pasty foods, the dosage cannot exceed 250 mL; for solid foods, the dosage cannot exceed 125 g.

Skin Test 

The skin test is an immunological test by means of the reaction of antigens and antibodies in or on the skin. There are two types of skin tests, one is the skin prick test (SPT) and the other is the intradermal test (IDST). The sensitivity of SPT is highly consistent with the clinical results, and the specificity is good, but it is not as sensitive as the intradermal experiment. SPT generally selects healthy and complete skin on the inside of the forearm or on the back. Studies have shown that the back is more suitable for SPT than the inside of the forearm. Not all patients are suitable for SPT, such as those with severe urticaria.

Animal model 

The goal of this method is to provide a model that can provide useful and reliable information when combined with other relevant data to help make a reasonable evaluation. However, there is no animal that can meet all the requirements of an ideal animal model. Commonly used animal models are rats, dogs, and pigs. When selecting, it is necessary to clarify the operating characteristics, limitations, and reliability in different environments. Commonly used mouse species are BALB/c inbred mice and brown Norwegian mice. The advantages of the mouse model are its small size, short reproduction cycle, similar to human immune mechanism, and rich related immunological information; its disadvantage is that allergic reactions are complex, and it is difficult to find reliable reaction markers to reflect the potential sensitization of the protein. Therefore, it is necessary to detect related serological indicators.

The post Food Allergen Sensitization Assessment Method-In Vivo Measurement first appeared on Creative Diagnostics.

Serological Methods 

The serum of allergic patients contains IgE antibodies that can specifically bind to the allergen. The potential sensitization of the protein can be evaluated based on the binding ability of the tested protein to IgE in human serum. In order to overcome the dependence on human serum, some immunoassay methods that rely on the antiserum of animals such as mice and rabbits have gradually developed. Currently commonly used serological methods mainly include radioallergen adsorption inhibition test (RAST inhibition test), enzyme-labeled allergen adsorption inhibition test (EAST inhibition test), western blotting and enzyme-linked immunosorbent assay (ELISA).

RAST and EAST

Inhibition experiments RAST and EAST are currently widely used methods for studying allergic reactions in the world, with high sensitivity and accuracy, and at the same time solve the problem of cross-reactions between different food allergens. Therefore, they are the key technology to evaluate the total sensitization of allergens. However, because the RAST and EAST inhibition experiments rely heavily on human serum, and the serum is difficult to ensure consistency, it is difficult to standardize RAST and EAST; and the radioactive hazards during the RAST experiment are serious and damage the health of the staff. Therefore, in the method for detecting food allergens recommended by FAO/WHO revised in 2001, the above two methods are not recommended. At present, food allergen detection basically uses the UniCAP system for RAST inhibition experiments, which can also perform fluorescence enzyme-linked immunoassay.

Western Blot

Western blotting is a hybridization technique that combines high-resolution gel electrophoresis and immunochemical analysis techniques. This method has the advantages of large analysis capacity, high sensitivity, and strong specificity. However, the operation is complicated and time-consuming, and is often combined with ELISA. Used to detect the allergenicity of allergic foods.

Enzyme-linked Immunosorbent Assay (ELISA)

ELISA is a new technology developed in the late 1990s. The method has high sensitivity, good specificity and easy operation. However, ELISA can only determine the specific binding of antigen and antibody, and cannot determine the sensitization of food allergens, and further testing with other methods is needed to determine the sensitization of food allergens. According to different operations, ELISA is mainly divided into double antibody sandwich method, indirect method, capture method and competition method. Among them, double antibody sandwich ELISA and competitive ELISA are the most widely used. ELISA can not only perform qualitative analysis, but also quantitative analysis. In recent years, ELISA technology has developed rapidly, and its sensitivity and specificity have been improved. Competitive ELISA can also be used to quantitatively detect food allergens. In the EL I S A reaction, the smaller the I C 50 value, the stronger the sensitization of food allergens, and vice versa. When detecting multiple food allergens at the same time, cross-reactions may occur and affect the accuracy of the results. However, sandwich ELISA can overcome cross-reactions among food allergens.

Cytological Methods

In a food allergic reaction, immune cells will undergo a series of changes, including the release of cytokines and T cell proliferation. The sensitization of food allergens can be evaluated by detecting a series of changes in immune cells. Generally speaking, the more cytokines are released, the proliferation of T cells is violent, and the sensitization of food allergens is stronger, and vice versa.

Histamine Release Test (HRT)

IgE-mediated allergic reactions can cause a large amount of histamine to be released in the body, and the amount of histamine released can be measured by fluorescence RAST. However, because the histamine determination must be completed within one day after the blood draw, and the serum source of allergic patients is insufficient, and the experiment is easily affected by the environment and operating conditions, the practical application of this method is greatly restricted.

Basophil Activity Test and Degranulation Experiment

The activity of basophils and the amount of degranulation are closely related to the sensitization of allergens. The stronger the sensitization of the allergen, the stronger the activity of basophils and the greater the amount of degranulation. CD63 and CD203c are often used as markers for cross-linking of allergens and IgE to trigger basophil activation. Studies have shown that the detection of CD63 and CD203c through the kit can effectively diagnose children’s egg allergy and milk allergy. The use of basophil activity test technology to detect food allergens has good specificity and high sensitivity, and the test results for patients receiving antihistamine therapy are still reliable. It is a supplement to traditional quantitative determination of IgE levels and skin experiments. The current challenge is how to standardize this method to make it the mainstream method for detecting the sensitization of food allergens and apply it to the evaluation of the sensitization of food allergens.

T Cell Response Analysis 
An important stage of IgE-mediated food allergy is T cell proliferation and the production of cytokines (IL-10, IL-13, IFN-γ and TNF-α). The peripheral blood mononuclear cells of allergy patients contain a small amount of specific T cells, which can be used as a source of T cell clones.  By measuring the proliferation of T cells and the release of cytokines, the potential sensitization of proteins and the epitopes of T cells can be studied.  Studies have found that when T cells are stimulated by peanut extract, they will produce Th1 and Th2 cytokines, but when they are stimulated again by peanut allergen, allergic individuals will only strengthen Th2 response. Therefore, the response of T cells can be used to evaluate the sensitization of food allergens. This method can be used in conjunction with animal experiments to explain the structure that determines protein sensitization, but T cell response analysis is difficult and time-consuming, and it is rarely used in research.

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