Thomas Schmitt

Adeno-associated virus (AAV) is a small non-enveloped virus, belonging to the family Parvoviridae. It was first discovered in 1965 from contaminants of adenovirus isolates. It has an icosahedral structure on the outside and a diameter of about 26 nm. Its capsid protein is composed of It is composed of three proteins: VP1, VP2 and VP3. The genome of AAV is a single-stranded linear DNA, approximately 4700 bp, including two upstream and downstream open reading frames (ORFs): Rep and Cap. They are located between 2 T-shaped inverted terminal repeats (ITRs) each consisting of 145 nucleotides. The role of ITR is to serve as the origin of viral replication and packaging signal. The Rep gene is involvedin viral replication and integration, encoding viral replication proteins, and the Cap gene is responsible for encoding the three viral capsid proteins. The natural wild-type adeno-associated virus that exists in nature has Rep and Cap genes on its genome, while the experimental AAV vector is an artificially modified plasmid based on wild adeno-associated virus and does not have Rep and Cap genes on its genome. Therefore, also called recombinant adeno-associated virus (rAAV). Unless otherwise specified, the abbreviation AAV generally refers to the modified AAV vector. There are many serotypes of AVV. Currently, 12 AAV serotypes have been isolated from humans and monkeys. Different AAV serotypes have different capsid protein spatial structures, sequences and tissue specificities. Therefore, the cell surface receptors they recognize and bind to are also very different. , which also results in different tissue types, cell types and infection efficiencies transfected by different serotypes. In the process of applying AAV virus, it is necessary to select the AAV virus of the corresponding serotype according to different tissues and organs. Because adeno-associated virus has the advantages of wide host range, high safety, low immunogenicity, stable expression and stable physical properties, it has been widely used in basic research and clinical trials, and adeno-associated virus vectors have become one of the most commonly used gene therapy vectors in the world. 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.

Procalcitonin (PCT) is the propeptide of calcitonin. The production of procalcitonin during infection and sepsis does not depend on calcitonin levels, but is closely related to the release of endotoxin and inflammatory mediators in bacterial infection. Traditional biological markers, clinical symptoms, and signs lack sufficient sensitivity and specificity to guide treatment decisions for infectious diseases. As a recently applied biomarker, procalcitonin is increasingly supported by increasing evidence that detecting serum procalcitonin concentration can be used to early diagnose the type of infectious pathogen, evaluate the severity of infection, guide medication, and determine prognosis. Procalcitonin has become the most useful biomarker in the management of infection and sepsis in most parts of the world. Figure 1. Specific mediators, metabolic pathways, and organs responsible for procalcitonin production in response to bacterial infections or inflammation compared to normal physiological conditions. The diagnosis of infection has always been a major problem faced by clinicians. Although there are many monitoring methods, there is still a lack of sensitive and specific dynamic monitoring indicators. Since it was first discovered in 1993 that the concentration of procalcitonin in the blood of patients with sepsis was significantly elevated, procalcitonin has become an important marker for the diagnosis of bacterial infection. Procalcitonin has higher accuracy and specificity than traditional biological markers. At the same time, PCT concentration is related to the severity of the disease and can be used to guide antibacterial drug treatment. Production and Release of Procalcitonin Procalcitonin is the propeptide of calcitonin and consists of 116 amino acids. Normally procalcitonin is encoded by the Calc-1 gene located on the short arm of chromosome 11 in thyroid C cells. In thyroid C cells, the Calc-1 gene is translated into a PCT precursor peptide containing 141 amino acids, and then enters the endoplasmic reticulum to form PCT under the action of glycosylase and specific enzymes. Subsequently, PCT is processed by specific endopeptidase, cleaved into N-PCT, calcitonin and calstatin. Under normal circumstances, PCT is almost not secreted from cells, and the blood content is <0.1ng/ml. Once bacteria invade the body, the PCT concentration in the blood can quickly increase to 5,000 times. The reason for this is that some studies believe that bacterial infection stimulates specific transcription factors in tissues to activate the Cala-1 regulatory gene, thereby activating PCT transcription. Others believe that under normal circumstances, PCT transcription is inhibited by specific transcription factors. After bacterial infection, this inhibitory factor is cleaved, resulting in PCT transcription. Unlike C-reactive protein, almost all peripheral tissues are involved in the production of PCT during bacterial infection. Studies on thyroidectomy patients have found that blood PCT concentrations remain high during bacterial infection. Researchers injected endotoxin into baboons and found that PCT-mRNA was expressed in almost all tissues. The liver and kidney were the main tissues producing PCT 6 hours after the injection of endotoxin. About 24 hours after the injection, the lungs, stomach, the heart produces PCT at its peak. The half-life of PCT in the circulation is approximately 22 to 26 hours and does not depend on renal excretion. It is controversial whether peripheral blood cells, in addition to parenchymal cells, participate in the production of PCT during bacterial infection. Scientists studied the peripheral blood of 17 patients with moderate to severe infections and found that PCT could be detected in monocytes and lymphocytes. By intracellular antibody staining, the researchers confirmed the expression of PCT in monocytes and neutrophils. However, the researcher believes that peripheral blood mononuclear cells cannot produce PCT even when stimulated by endotoxins and cytokines. Other investigators found that PCT-mRNA was not detected in endotoxin-stimulated macrophages or viable isolated peripheral blood mononuclear cells. Therefore, whether peripheral blood cells are involved in the release of PCT during bacterial infection still requires further study. Current research suggests that there are two pathways for bacteria to induce the release of PCT, namely the direct and indirect pathways. In the direct pathway, bacterial tissue structures (DNA, fimbriae, peptidoglycan, etc.) directly induce intracellular signaling to release PCT. In the indirect pathway, pathogens stimulate the body to produce intermediaries (such as pro-inflammatory cytokines) that then act on target cells to produce PCT. Some researchers believe that during bacterial infection, the pathogen directly acts on the specific microorganism-related receptor at the No. 5 start site of the Calc-1 gene, causing a large amount of PCT to be released. On the contrary, another researcher found that during bacterial infection, bacterial endotoxin stimulates the body to produce a series of pro-inflammatory cytokines (such as TNFa, IL-1β, IL-8, IL-6) that stimulate tissues other than the thyroid gland (intestine, lung , immune cells) release PCT into the blood. Both clinical and animal studies have confirmed that direct injection of endotoxin results in the release of PCT in the blood. Some researchers add endotoxin to cells cultured in vitro can cause intracellular production of PCT. Intravenous injection of TNFa and IL-2 into cancer patients causes rapid and large release of PCT. Interestingly, infusion of anti-TNFa monoclonal antibodies into human leukocytes inhibited bacterial-induced stimulation of PCT release. Another study found that IFN-r inhibits the release of PCT. Application of PCT in Infectious Diseases Differentiate bacterial infections from non-bacterial infectious diseases The Infectious Diseases Society of America and the American Society of Critical Care Medicine jointly recommend PCT as an auxiliary diagnostic marker to distinguish sepsis from non-infectious systemic inflammatory responses. When bacterial infection causes a systemic inflammatory reaction, PCT concentration will increase significantly. However, when viral infection, cancer fever, transplant-host rejection and other inflammatory reactions occur, the blood PCT concentration will not increase or only increase slightly. Evaluate the degree of infection and prognosis Blood PCT concentration is not only a specific indicator of infectious diseases, but also can monitor the severity of host infection and judge prognosis by continuously monitoring PCT levels. The concentration of PCT is related to the severity of bacterial infection and bacterial load. Application of PCT in Infections in Special Populations Neonatal bacterial infection is one of the most important health problems, and early diagnosis and treatment are very important due to the non-specific symptoms and signs and high mortality rate after neonatal bacterial infection. Studies found that neonates with sepsis, urinary tract infection, and meningitis had significantly higher blood PCT concentrations. Current research has confirmed the value of PCT in neonatal infections. Based on these research results, the sensitivity and specificity of PCT They were 76.9% and 100% respectively, and the positive prediction rate and negative prediction rate were 100% and 78% respectively.

GLP-2 (glucagon-likepeptide-2) was discovered and named by the Lilly Laboratory in the United States when cloning the proglucagon gene (PG). In 1996, Drucker et al. discovered that the active ingredient in proglucagon-derived peptide (PGDP) that promotes intestinal mucosal growth is GLP-2. Its effect is stronger than other growth factors that have been discovered, and its growth-promoting effect is organ-specific (limited to the stomach and intestine), GLP-2 began to be valued and studied. Subsequent animal experiments and human studies have shown that the main function of GLP-2 is to stimulate the proliferation of intestinal mucosal crypt cells and inhibit their apoptosis, thereby promoting the growth of intestinal mucosa and regeneration and repair after injury.GLP-2 can also inhibit the secretion of gastric acid and motility, increase the blood supply of intestine, improve the barrier function of the intestine, and promote the absorption of nutrients in the intestine. GLP-2 and Glucagon-Derived Peptides For mammals, GLP-2 uses the proglucagon gene (PG) as a template and is transcribed in pancreatic A cells, intestinal L cells, hypothalamus, brainstem and other central neurons, and is expressed and translated. It is processed into a polypeptide containing 33 amino acids with a molecular weight of 3900. The proglucagon gene includes 6 exons and 5 introns, and its mRNA is translated into a single-chain precursor protein containing 160 amino acids. It is tissue-specific and is activated by prohormone convertase ( PCs) undergo post-translational processing to generate a series of proglucagon-derived peptides (PGDP) with different biological activities. PGDP includes glucagon (glucagon), proglucagon fragment (MPGF), and glucagon-related pancreatic polypeptide (GRPP) secreted by pancreatic A cells, as well as enteroglucagon and oxyntomodulin in the intestine and brain, glucagon-like peptide-1 (GLP-1), glucagon-like peptide 2 (GLP-2), intermediate peptide 1 (IP-1) and intermediate peptide 2 (IP-2), etc. The main form of GLP-2 in the blood circulation is complete GLP-2, and there is also partial GLP-2 formed after the first two amino acid residues at the amino terminus are hydrolyzed by diacyl peptidase IV (DPPIV).This form of GLP-2 is inactive. Figure 1.Main biological actions of GLP-1 and GLP-2. Physiological Functions and Pharmacological Effects of GLP-2 Different from other polypeptide growth factors, GLP2 is an intestinal epithelial-specific growth factor with intestinal specificity and stronger growth-promoting effect. Researchers used subcutaneous injection to compare the growth-promoting effects of GLP-2 EGF (epidemal growth factor), IGF-1 (in-sulin-growth factor-1) and GH (growth homone) on normal intestinal mucosa in mice., found that GLP2 has the most obvious effect. In the study on the effect of intestinal adaptation in short-intestine rats, it was found that the GLP-2 group had a significantly higher effect on increasing the weight of the jejunal intestine and the height of villi than the GH group and the KGF (keratinocyte growth factor) group.In addition, research data shows that GLP-2 mainly promotes the proliferation of intestinal epithelial cells and has no effect on the proliferation of other important organ tissues such as heart, liver, and kidney cells. No pathological changes have been seen in these tissues after long-term application of GLP-2. GLP-2 Promotes Normal Small Intestinal Growth GLP-2 can promote the increase in the weight and length of the stomach and small intestine of newborn rats during the lactation period. GLP2RmRNA is expressed at high levels in the stomach and small intestine of rat embryos and neonatal stages. The concentration of GLP2 in plasma increases before the birth of fetal pigs. It is significantly increased and maintained at a high level throughout the entire period of breast milk nutrition, which suggests that GLP2 may play an important role in the development and maturation of the gastrointestinal tract. GLP2 Promotes Recovery of Damaged Intestinal Mucosa A phase I clinical trial using teduglutide (a GLP-2 analogue resistant to DPPIV degradation) to treat patients with ulcerative colitis showed that the remission rate of ulcerative colitis in the treatment group was higher than that of the placebo group, and the therapeutic effect was dose-dependent. GLP-2 Affects Gastric Acid Secretion and Gastrointestinal Motility GLP2 inhibits human gastric acid secretion caused by sham feeding. Sham feeding causes gastric acid secretion to increase approximately 5 times. This increase is reduced by 65% in the GLP-2 perfusion group compared with the normal saline perfusion group, indicating that GLP-2 is a powerful inhibitory factor for human gastric acid secretion. As an inhibitory factor, animal experiments have also proven that GLP-2 can inhibit gastric emptying in pigs. GLP-2 Increases Intestinal Blood Supply In newborn piglets fed total parenteral nutrition, administration of GLP-2 can increase portal blood flow by 25%, and this reaction is dependent on nitric oxide. Interesting, GLP-2 increases portal blood flow only in the small intestine, because it was found that the increase in hemoglobin content and the expression of GLP-2 receptors were mainly concentrated in the small intestine. GLP2 and Feeding Studies have found that the plasma level of GLP-2 in healthy people increases significantly after eating, but physiological levels of GLP2 have no significant effect on appetite and energy intake. GLP-2 Inhibits Bone Resorption Porosis may be the result of bone loss due to an imbalance in bone remodeling, which causes bone resorption to exceed bone formation. Common treatments inhibit bone resorption by reducing osteoclast number, activity, and longevity, but subsequently bone formation is also inhibited. The study found that exogenous GLP-2 caused a sharp and sustained reduction in bone resorption but had no effect on immediate bone formation. It can be seen that GLP-2 can affect the balance of bone remodeling, and the tilt of this balance toward osteogenesis may eventually lead to an increase in bone mass and bone strength, thus providing a new model for the treatment of osteoporosis. GLP-2 treatment reduces bone resorption but does not affect bone formation, and may have a more positive impact on bone health than therapies that reduce bone resorption and bone formation at the same time. Post-receptor Signal Transduction Mechanism of GLP-2 A large number of studies have shown that GLP2 regulates the proliferation of intestinal epithelial cells and inhibits their apoptosis by acting on the GLP2 receptor (GLP-2R), thereby protecting intestinal cells. The human GLP2R gene is located on chromosome 17p13.3. Cloning of human and rat cDNA-encoded GLP2R shows that it is a member of the G protein-coupled receptor superfamily, with 7 transmembrane domains, similar to GLP-1 glucagon and GIP (Glucose-dependent insulinotropic polypeptide) receptors have a high degree of homology. A series of studies using immunohistochemistry, in situ hybridization, confocal laser microscopy, RT-PCR and Westem blot analysis separately and/or in combination showed that GLP-2R is expressed in intestinal epithelial cells, gastric epithelial cells, intestinal intrinsic neurons. It is expressed in enteroendocrine cells, intestinal, submucosal myofibroblasts, vagal afferent fibers, cerebral cortex, cerebellum, hypothalamus, amygdala, hippocampus, dentate gyrus and lungs. The distribution density of GLP2R in the gastrointestinal tract is in the jejunum, duodenum, ileum, colon, and stomach. However, the signal transduction mechanism of GLP2R is still unclear, mainly due to the lack of cell models for studying GLP-2R.

Genetically engineered drugs are introduced into host cells such as Escherichia coli, yeast or mammalian cells through recombinant technology to construct engineering strains or cell strains, and then undergo specific expression, assembly, folding and post-translational modification processes to produce drugs with corresponding Bioactive macromolecules. Endogenous proteins derived from host cells are called host cell proteins (HCPs). Their composition is complex. Depending on the selected host cells and production processes, the isoelectric point (3-11), hydrophobicity, relative the molecular mass (generally 5000~250000) distribution varies significantly. Figure 1. Autologous and allogeneic T cell immunotherapy. Cellular HCPs in genetically engineered drugs may cause different human reactions when administered, because specific HCPs have potential “adjuvant effects”, generate immune responses against impurities in HCPs, or cause allergic reactions or even anaphylactic shock. In addition to safety issues, the presence of HCPs can also affect product quality. For example, HCPs can trigger aggregation or fragmentation of therapeutic proteins. In addition, it was found that certain HCPs promote the degradation of polysorbates, a type of non-ionic surfactant widely used in the preparation of buffers to stabilize proteins. The degradation of polysorbates will affect the stability of protein drugs. The protease activity in HCPs can also affect the protein composition in the culture supernatant, thereby affecting the subsequent protein purification process or the stability of long-term storage of protein drugs. In addition, HCPs may undergo post-translational modifications, making quantification and characterization more difficult. In view of the fact that cellular HCPs in genetically engineered drugs may cause safety and effectiveness issues, the detection of their removal efficiency and cell limit is an important parameter for drug release and clinical research, and is one of the critical product quality attributes (CQA) of drug quality control. Therefore, although only a few clinical adverse events have so far been attributed to HCPs impurities, in order to ensure product safety, HCPs must be accurately characterized and quantitatively analyzed through highly sensitive analytical methods. Factors Influencing HCPs The quantity and composition of HCPs are affected by the entire production process. Among them, the expression pathway is very important. Depending on the host cells and culture conditions, the types of HCPs vary greatly, ranging from hundreds to thousands. Many host cells such as E. coli, mammalian cells, NSO, SP2/0 and the human embryonic kidney cell line HEK293 have been used for biopharmaceutical production. During mammalian cell culture, recombinant proteins are often secreted from cells into cell culture fluid (CCF), which contains HCPs. In addition, due to the death of some cells, soluble intracellular proteins are released into CCF, and some artificial operations (such as centrifugation, filtration, etc.) can also cause cell lysis. Therefore, harvested CCF usually contains secreted HCPs and intracellular HCPs. When this protein mixture is incubated in a fermenter, other changes in HCPs occur due to enzymatic activity (such as proteases or sialidases). The main factors affecting HCPs are shown in the table below: Production Process Influencing Factors of HCPs Upstream Processes Amino Acid Sequence of Drug Protein Host Cell Culture Process?Media Composition?Feeding Method? Temperature? Cell Viability at Harvest Time Of Culture Cells Amplification Process Downstream Processes Cell Stability/Shear Sensitivity Purification Process Conditional Changes During Analysis HCPs analysis provides information about the composition of substances entering the downstream process and the removal rate of HCPs at each purification step. In some cases, HCPs can even be combined with or co-purified with certain protein drugs. Process characterization and validation studies are needed to illustrate which process steps can remove HCPs and also to demonstrate the stability of these steps in removing HCPs. Therefore, detection of HCPs is an important part of purification process development and helps ensure production consistency. Finally, stable and highly sensitive detection methods are needed to detect HCPs of drug proteins. Taking the CHO expression system as an example to introduce the analysis method of HCPs: Quality Control Content and Requirements Currently, there is no unified international standard for cell limits for HCPs, and biopharmaceutical companies use risk control to control the cell volume of HCPs. After the production and purification of any genetically engineered drug, trace amounts of HCPs are usually found in the final drug protein. The US FDA stipulates that when using a highly sensitive method to detect HCPs in pharmaceuticals, the content should be lower than the relative value of the lower detection limit of 100ppm (100ng·mg-1 total protein), which is usually considered an important reference point for process development. HCPs levels should be monitored in: 1) preclinical batches used for toxicological evaluation; 2) all batches during clinical development; and 3) process validation samples during final production. Research Methods During process development and production, appropriate methods must be selected for the detection of impurities such as HCPs, and their quantitative methods have always received attention. Currently, qualitative or semi-quantitative methods may be sufficient for process sample and batch testing. Typically, methods used for HCPs analysis include immunoassay methods (such as Western Blot and ELISA) or other methods (such as electrophoresis and mass spectrometry), and multiple techniques can be studied in parallel or in combination. In addition, new detection methods have emerged but are not yet widely used due to their immaturity. ELISA Method Among methods for HCPs analysis, anti-HCPs-ELISA is considered the gold standard. This method (1-100ppm) is suitable for product development and process control. However, unlike conventional ELISA, the target of anti-HCPs ELISA is not single, and the total HCPs “antigen” is a complex protein group. Therefore, when producing HCPs antibodies/standard reagents, it is necessary to ensure: 1) a specific production cell line and production process; 2) its protein concentration must be accurately quantified. The general method for preparing HCPs antibodies is to use host cells containing empty expression vectors to prepare antigen-immunized animals, obtain antiserum, and isolate and purify polyclonal antibodies for ELISA detection. Since the immunogenicity of HCPs as antigens depends on factors such as the structure, properties, abundance, and relative molecular mass of the protein itself, there will be different detection results during the analysis process, which to a certain extent will lead to inaccurate quantitative results. In addition, commercial kits cannot cover all process-specific HCPs and cannot identify changes in HCPs caused by process changes, etc. If other binding and detection antibodies are used, it is recommended to test the detection antibody coverage. HCPs coverage assessment helps evaluate the antibody’s ability to identify HCPs in the standard as well as those present in the manufacturing process and protein drug product. The design and validation of immunoassays for HCPs is a challenge due to: 1) the method has a narrow dynamic range (<100), long development cycle, high cost, and high risk of failure; 2) a wide variety of possible kinds of HCPs in genetically engineered drugs; 3 ) Generally, polyclonal antibody reagents are used for detection; 4) Different proteins have similar epitopes, leading to false positives; 5) Hook effect, high-abundance proteins lead to detection saturation; 6) The antigen composition of HCPs should be comprehensive enough to withstand changes in the normal production process of the product; 7) There is a lack of completely matching quantitative standards. Since the HCPs test is used to detect samples containing HCPs, any cross-reactivity between anti-HCPs antibodies and drug proteins will affect the test and lead to biased results. Therefore, contamination by HCPs antigens must be avoided. In the future, it can be expected that ELISA will remain the gold standard for detecting HCPs, but in order to make this detection method more accurate, it is crucial to select appropriate antibodies. Gel Electrophoresis/Western-Blot Method Gel electrophoresis is commonly used in biopharmaceutical development laboratories to perform semi-quantitative analysis of different proteins present in a sample. For HCPs analysis, one-dimensional SDS-PAGE gels do not have high resolution but are used in combination with Western-Blot. Two-dimensional electrophoresis (2-DE) is commonly used for upstream or downstream process development and characterization, with its enhanced ability to resolve different HCPs on a single gel. The principle of 2-DE is to separate based on isoelectric point (first dimension) first, and then separate based on size (second dimension). Spot analysis in gel does not require immunoblotting, avoids the problem of membrane transfer, and can achieve the separation of trace HCPs impurities and products. Provides approximate relative molecular mass and isoelectric point information of protein spots, but excess protein may obscure HCPs spots. Mass spectrometry is often used to further identify in-gel proteins. Often, gel electrophoresis is used as a complementary method for the detection of non-immunogenic HCPs. Western-Blot is used for consistent screening of large numbers of samples and detection of unknown proteins that react with anti-HCPs antibodies. It is not only suitable for detecting HCPs, but also provides approximate information on the relative molecular weight of HCPs and can be used as part of a GMP control system. However, Western-Blot also has big problems: 1) It requires high-quality HCPs polyclonal antibodies; 2) A large amount of overloaded target proteins in the gel will cause non-specific staining of the bands, thereby masking the possible bands of HCPs; 3) SDS-induced protein denaturation may lead to the loss of conformational epitopes; 4) Western-Blot cannot accurately quantify HCPs, and the sensitivity of the experiment depends on the quality of the polyclonal antibody. Proximity Ligation Analysis (PLA) In the PLA experiment, oligonucleotide chains (i.e., PLA probes) are labeled on 2 antibodies targeting different epitopes. These probes are connected by ligase when they are close to each other, and then amplified by qPCR to quantify the target proteins (i.e. HCPs). Biosensor Method Researchers report the use of biosensors for the detection of typical impurities in biopharmaceutical production. Biosensors can measure target molecules online making them promising analytical tools in process analytical technology (PAT). However, unlike traditional immunoassays, biosensors require regeneration, so the use of weak-affinity antibodies is recommended, but at the expense of sensitivity. Mass Spectrometry In recent years, mass spectrometry (MS) has been increasingly used in the detection of HCPs to help identify possible risk factors and evaluate purification processes. Although ELISA is most commonly used to detect the total amount of HCPs, it cannot identify HCPs outside the detection range of the ELISA kit, and compared with traditional ELISA methods, the liquid chromatography mass spectrometry (LC-MS) method has a short development time, It can specifically confirm and quantify all HCPs, detect all impurity proteins in an unbiased manner, and has the advantages of high method dynamic range (up to 6 orders of magnitude) and fast method adjustment.

Staphylococcus aureus (S. aureus) is an important class of food-borne pathogens that cause food contamination and food poisoning, and is also a symbiont of human skin and mucous membranes. Studies have found that about 20%~30 % of healthy individuals carry Staphylococcus aureus. Animal foods are susceptible to Staphylococcus aureus infection, such as milk, butter, cured ham, etc. Food poisoning incidents caused by Staphylococcus aureus have been frequently reported, and more than 95% of them are caused by enterotoxin. According to reports, the United States and Canada accounted for 33% and 45% of food poisoning incidents caused by staphylococcal enterotoxins (SEs), respectively. Epidemiological studies have shown that Staphylococcus aureus enterotoxin A can be produced under more suitable environmental conditions (temperature 7~47, pH 4?~10) and growth phase (logarithmic growth phase or transition to stable phase) Staphylococcal enterotoxin A(SEA), Staphylococcal enterotoxin B (SEB), Staphylococcal enterotoxin C1 (SEC1), Staphylococcal enterotoxin D (SED) and other about 23 serotypes, they are similar in structure and function with molecular weight of 27.5~30 kDa. Among the above-mentioned SEs, SEB is one of the most common biotoxins reported so far, and it is also a typical biotoxin that causes food poisoning. In order to detect SEB, researchers have developed different detection methods, among which the main detection methods are biological methods, immunological methods, gene probe methods, instrumental analysis methods and biosensing methods. Figure 1. New insights into Staphylococcus aureus immune evasion. Structural Properties and Hazards of SEB SEB is mostly found in spoiled milk and meat products in food. According to relevant research reports, the content of SEB in the culture supernatant of Staphylococcus aureus (strain S-6) is as high as 0.2 mg/mL, and the semi-lethal dose LD50 of mice is 0.02 µg/kg. The molecular weight of SEB is 28.4 KDa, which contains 239 amino acid fragments. The structure of SEB is mainly composed of 2 regions, of which 129 amino acid residues in region 1 contain 2 β-sheets and 3 α-helices (α1, α2 and α3). The 2 β-sheets form a cylindrical structure, the inner wall is lined with hydrophilic residues, and they form “crosses” with each other. Region 2 is divided into 2 different parts, one part contains 2 α-helices (α4 and α5) and 2 very short β-strands, and the other part has α-helices located between the curved sheet and β cylinder. One side of α4 and α5 is exposed to the solvent, and the two domains are connected by a ring structure. Compared with other enterotoxins, SEB has stronger heat resistance, its structure and function do not change significantly after being heated at 100 ? for 30 min, and it can resist the hydrolysis of intestinal proteases. Therefore, enterotoxin-contaminated food can still cause potential toxic effects even after being cooked and digested by the human body. As a superantigen substance, enterotoxin B simultaneously forms a bridge by cross-linking with major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs), Stimulates T cells in a manner that does not involve antigen recognition by generating receptor complexes with MHC II sites on APCs and T cells. It can effectively bypass the treatment of conventional APCs, induce T cells to produce cytokines, and cause the activation and proliferation of T cell subsets. Related studies have further confirmed that SEB increases the expression of components of the arachidonic acid pathway, leading to inflammation, edema and shock. Current studies have found that ingesting a small amount of SEB in the human body can cause immune disorders, organ damage and other symptoms, and the clinical manifestations are fever, respiratory diseases (cough, dyspnea and chest pain) and digestive tract diseases. Therefore, it is of great significance to study the highly sensitive and rapid detection method of SEB for the clinical diagnosis, prevention and control of food poisoning. Method for the Detection of Staphylococcus aureus Enterotoxin B Biological Detection Method The detection method of Staphylococcus aureus enterotoxin B was initially based on related detection methods such as zoological experiments. Through intraperitoneal injection and feeding of samples added with enterotoxin B to the tested animals, the abnormal physiological reactions and activities of the animals were observed and recorded. The existence and toxicity of enterotoxin B are qualitatively analyzed. This type of experiment is intuitive in the interpretation of the results, but the disadvantages such as poor specificity limit the application of this method. Immunoassay Immunological detection is mainly based on the principle of specific combination of antigen and antibody, including immunoagglutination test, agarose diffusion method and enzyme-linked immunosorbent assay. However, the detection sensitivity of these methods is only 0.1 ng/mL, and the sample preparation and detection take a long time. In addition, this method needs to ensure the homogeneity of monoclonal antibody structure and avoid cross-reaction between each other. Immunoagglutination Test The immunoagglutination test is the agglutination produced by the combination of antigen and antibody in the presence of electrolytes. These include the reverse indirect hemagglutination test and the reverse passive latex agglutination test. The former uses the antiserum of enterotoxin B to adsorb on the surface of red blood cells of animals. When the test specimen contains SEB, hemagglutination occurs. This method can interpret the results in about 2 hours, and the detection sensitivity is greatly improved compared with the previous method; the latter adsorbs the specific anti-SEB antibody on the latex particles, and when the electrolyte and SEB exist at the same time, the latex particles agglutinate phenomenon, the detection time is about 18 h. 2. Agarose Diffusion Method Immuno-agar diffusion method refers to the diffusion of soluble antigens and antibodies in semi-solid agar. It is not suitable for the detection of low concentration SEB (below ng level). If the antigen and antibody react according to a certain concentration relationship, a white precipitate line appears on the surface of the plate, which is a positive result, and the standard curve is made by the logarithm of the distance of the SEB diffusion agarose layer and the antigen concentration, which is used for the indirect quantification of SEB . The method is simple to operate and does not require any equipment, but the disadvantages of long detection time and low sensitivity limit the application of this method. 3. Immunolabeling Technique Immunolabeling techniques can be divided into detection techniques such as enzyme-linked immunosorbent immunoassay, radioimmunoassay, and immunofluorescence. It is based on the traditional antigen-antibody reaction, by modifying fluorescent groups on antibodies such as hydroxyfluorescein (financial analyst meeting, FAM), Fluoresceine isothiocyanate (FITC), water-soluble indocyanine-type fluorescein (cyanine-5, Cy5), etc., radioactive isotopes, horseradish peroxidase (horseradish peroxidase, HRP) or alkaline phosphatase (alkaline phosphatase, AP) and other trace substances, using relevant equipment to output signal intensity to achieve the purpose of detection. Enzyme-linked Immunoassay Technology Enzyme linked immunosorbent assay (ELISA) is based on the antigen-antibody reaction of a solid-phase carrier (such as polystyrene, etc.), and the color development of the substrate is catalyzed by HRP or AP, thereby amplifying the detection signal. Double-antibody sandwich and indirect competition methods are usually used to detect SEB, and the former can obtain lower detection sensitivity and wider linear range. At present, enzyme-linked immunoassay technology has been widely used in actual detection, and a targeted detection kit has been developed. Radioimmunoassay Technology Radioimmunoassay is based on the competitive reaction of labeled antigen (labeled with radioactive isotope) and non-labeled antigen to antibody, and is used to detect the antigen concentration of the sample to be tested. Immunofluorescence Detection Technique Immunofluorescence detection technology uses fluorescent substances (FITC, Cy5, quantum dots, upconversion particles, etc.) as signal molecules labeled on antibodies, and the concentration of SEB is indirectly measured by the fluorescence intensity of the labeled antibody bound to SEB, usually using quantitative determination by photometer. Colloidal Gold Test Strip Detection Technology Colloidal gold-labeled immune test strip mainly utilizes the excellent physical and chemical properties of colloidal gold. Since the surface of colloidal gold has a negative charge, under alkaline conditions, the antibody can be immobilized on the surface by physical adsorption without affecting the biological activity of the antibody itself. This method can quickly interpret the results under naked eye conditions, and is generally used for preliminary screening on the spot. Gene Probe Method Enterotoxin B is transcribed and translated from the toxin expression gene sequence seb of Staphylococcus aureus. By detecting the enterotoxin B gene in Staphylococcus aureus in food, it can be used for rapid and high-throughput detection of positive strains of Staphylococcus aureus. Specifically, the method mainly uses nucleic acid amplification techniques, including polymerase chain amplification and nucleic acid isothermal amplification techniques. Instrumental Analysis Method With the continuous improvement of the current detection instruments and equipment, many scholars are no longer limited to the traditional immunology and other related methods, but instead use modern large-scale precision instruments for qualitative and quantitative detection of enterotoxin B. Some instrumental analysis methods can directly reveal the molecular structure of toxins. Biosensing Detection Method Biosensor (biosensor) connects biochemical reactions with transducers through physical and chemical instruments, and expresses them in the form of electricity. It mainly recognizes biological components of specific substances (such as enzymes, antibodies, nucleic acids, lectins, Cells or tissues) and biological reaction signal amplification are organically combined, which has the advantages of less sample usage and fast analysis speed, and can be used for simultaneous detection of multiple substances. At present, the biosensors used for detection mainly include electrochemical sensors, surface plasmon resonance and piezoelectric crystal sensors.

The incidence and prevalence of diabetes are increasing year by year globally, and patients with diabetes are at increased risk of cardiovascular disease and heart failure. Diabetic cardiomyopathy (DCM) refers to the presence of abnormal myocardial structure and performance in the absence of other heart-related risk factors (such as coronary artery-related disease, hypertension, and severe valvular disease). DCM can lead to cardiac systolic or diastolic dysfunction, further causing heart failure with reduced ejection fraction and heart failure with normal ejection fraction, seriously affecting the patient’s quality of life. The pathogenesis of DCM is particularly complex. Current research has discovered multiple mechanisms of action, including metabolic disorders, insulin resistance, formation of advanced glycation end products, mitochondrial damage, oxidative stress, endoplasmic reticulum stress (ERS), Inflammatory response and cell apoptosis, etc. Glucagon-like peptide-1 (GLP-1) receptor agonist is an incretin secreted by intestinal L cells. It reduces the blood sugarof diabetes in patients with diabetes by increasing glucose-dependent insulin secretion and inhibiting glucagon secretion, and also regulates a variety of cardiovascular risk factors, thereby inhibiting the process of atherosclerosis and reducing the occurrence of coronary events and cardiovascular death. GLP-1 receptor agonists have a longer half-life, are more effective than many oral hypoglycemic drugs in clinical trials, and have a low risk of hypoglycemia. This article reviews the effects of GLP-1 receptor agonists on DCM to provide reference value for further research on GLP-1 receptor agonists. Figure 1. Major cardiometabolic actions of GLP1. GLP-1 Receptor Agonists Affect DCM by Regulating Metabolic Disorders Hyperglycemia and insulin resistance are the most significant features of DCM. In addition, they are also accompanied by changes in myocardial structure and function caused by calcium imbalance, abnormal lipid metabolism, lipid deposition, and the combined effects of multiple hormone changes. The metabolic disorder of myocardial cells is mainly caused by the increase in blood sugar levels caused by insulin resistance. When diabetes occurs, there are conversion obstacles in both the anaerobic glycolysis and aerobic oxidation of glucose in the body. The body’s utilization of glucose is reduced, lipolysis is increased, and excessive free Fatty acids are absorbed by cardiomyocytes, reducing the heart’s ability to use glucose. At this time, the heart mainly relies on fatty acid oxidation to provide energy, but high oxygen consumption is accompanied by low energy metabolism efficiency. The accumulation of free fatty acids in the form of triglycerides promotes fatty degeneration of the myocardium. Therefore, long-term hyperglycemic environment, abnormal lipid metabolism, and lipid deposition lead to the occurrence and development of DCM in diabetic patients. Multiple studies have shown that GLP-1 receptor agonists can directly or indirectly improve the function of cardiomyocytes by controlling the above multiple metabolic factors and related signaling pathways. ?GLP-1 Receptor Agonists Affect DCM by Improving ERS In diabetic patients, the endoplasmic reticulum dysfunction caused by cardiac oxidative stress, abnormal lipid metabolism and inflammatory response is called ERS when the body is in a high glucose state. Pathophysiological factors such as ischemia, hypoxia, oxidative stress, or calcium homeostasis disorders can disrupt endoplasmic reticulum homeostasis, cause endoplasmic reticulum dysfunction, and lead to the accumulation of unfolded or misfolded proteins, thus inducing ERS and triggering unfolded protein response, thereby activating the corresponding endoplasmic reticulum and calcium regulatory mechanisms and other signal transduction pathways to restore endoplasmic reticulum homeostasis. When the ERS intensity is too high or lasts for a long time, the endoplasmic reticulum cannot restore homeostasis. At this time, multiple pathways induce cell apoptosis, which together with multiple factors lead to cardiomyopathy and heart failure. Increasing evidence shows that ERS is associated with diabetes, endothelial dysfunction, and atherosclerosis. GLP-1 receptor agonists can inhibit ERS and reduce intracellular calcium concentration through indirect effects, thereby reducing myocardial damage. Their effects may be related to the inactivation of the ERS signaling pathway. GLP-1 Receptor Agonists Affect DCM by Interfering with Inflammatory Response Oxidative stress is an important factor driving the inflammatory response. High glucose status and insulin resistance in diabetic patients promote cell metabolism disorders and release a large amount of reactive oxygen species, which in turn activates nuclear factor κB, induces the expression of inflammatory factors, damages vascular endothelial cells, and causes abnormal proliferation of smooth muscle cells. It will affect the structure and function of the heart in the long term, leading to the occurrence and development of DCM. GLP-1 receptor agonists can inhibit the inflammatory pathway through direct or indirect effects, activate the AMP-activated protein kinase (AMPK) pathway, reduce the activity of nuclear factor κB, reduce the production and release of inflammatory mediators, inhibit the differentiation of inflammatory monocytes and macrophages, and Reduce heart and blood vessel inflammation, etc. GLP-1 Receptor Agonists affect DCM by protecting Mitochondria and Improving Oxidative Stress The tissue damage of DCM mainly has the following five mechanisms: (1) Increased metabolism of glucose and other sugars through the polyol pathway; (2) Formation of intracellular advanced glycation end products; (3) Advanced glycation end products Increased expression of receptors and ligands; (4) activation of protein kinase C subtype; (5) overexpression of hexosamine pathway activity. Mitochondria are one of the main sources of reactive oxygen species in cardiomyocytes, and reactive oxygen species are the upstream products of the above five pathways. In the body’s high-glucose state, due to changes in metabolic substrates, the oxidation of free fatty acids increases the transfer of reducing equivalents, which can damage the ability of the electron transport chain, increase the generation of reactive oxygen species, excessive reactive oxygen species deposition, or weaken the antioxidant mechanism. The redox imbalance leads to oxidative stress; this process and the high glucose state can simultaneously promote oxidative stress and the body’s inflammatory response, damage myocardial cells, and then lead to myocardial fibrosis, ventricular hypertrophy, coronary microvascular damage, cardiac systolic and diastolic function obstacles, eventually developing into DCM. In the entire pathogenesis of DCM, the effects of GLP-1 receptor agonists on metabolic disorders, accumulation of reactive oxygen species, ERS, and inflammatory responses all require the oxidative stress link. The oxidative stress link can be called the key to the pathogenesis of DCM. Therefore, intervening in the oxidative stress process is an important step in improving the prognosis of DCM. Studies have shown that exenatide reduces oxidative stress in cardiomyocytes and improves mitochondrial function by activating autophagy; it can also remove dysfunctional mitochondria to inhibit cardiomyocyte apoptosis, achieve anti-apoptosis and antioxidant effects, and protect cardiac function. Exenatide can also inhibit the activation of caspase-1, indirectly attenuating mitochondrial damage and reducing reactive oxygen species deposition, thereby attenuating oxidative stress.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease with an insidious onset. Clinically, it is characterized by comprehensive dementia manifestations such as memory impairment, aphasia, apraxia, agnosia, impairment of visuospatial skills, executive dysfunction, and personality and behavioral changes. Alzheimer’s disease is progressive and irreversible and can only be delayed with medication. Once it develops into moderate or severe disease, it will cause significant harm to the lives of patients and their families. Figure 1. Schematic overview of invasive biomarkers. Pathological Mechanism of AD The pathological mechanism of AD is currently unclear, and is currently summarized as neurodegenerative changes and inflammatory response. Neurodegenerative changes involve neuronal degeneration, nerve cell apoptosis, and the generation of new neurons. Inflammatory response refers to the inflammatory response in the brain of AD patients, including the release of activated cytokines and inflammatory cytokines. In addition, many studies have shown that the pathological mechanism of AD is also affected by protein oxidative stress. Oxidative stress refers to the production of free radicals and the increase in reactive oxygen species under conditions of intracellular hypoxia or anoxia, leading to the destruction of cell structure and function. Studies have found that oxidative stress can affect the apoptosis and degeneration of nerve cells in the brains of AD patients, thus increasing the severity of the disease. There are reports that AD is also affected by neurotransmitters. The level of neurotransmitters in the brains of AD patients is lower than that of normal people. This may be one of the reasons for the cognitive impairment of AD patients. In addition, AD patients have obvious amyloidosis in their brains. Amyloidosis refers to a variety of pathological changes that occur in the brains of AD patients, including amyloid crystals, neuronal degeneration, and nerve cell apoptosis. Such lesions may be a key cause of AD and serve as diagnostic markers for AD. AD Diagnostic Markers Apolipoprotein Eε4 Apolipoprotein Eε4 (APOEε4) is a very clear risk gene for late-onset AD, which may advance the age of onset and accelerate the development of cognitive decline. It has been recognized as a genetic marker of AD. ApoE is a key component of plasma lipoproteins, which can produce β-amyloid peptide and maintain neurological function. β?amyloid Under the action of hydrolyzing amyloid precursor protein (APP), β? and γ?secretase can produce β?amyloid protein (Aβ). Such polypeptides are distributed in many cells and can also be found in blood, cerebrospinal fluid (CSF) and brain interstitial fluid. The most widespread subtypes of Aβ are Aβ40 and 42. The latter are neurotoxic and are related to the formation of AD. Most of them are generated from microglia and astrocytes, have hydrophobic characteristics, and play a major role in plaque formation. Aβ42 in CSF is generally considered to be a biomarker of AD and is helpful in diagnosing preclinical AD. Lower levels of Aβ42 in the cerebrospinal fluid are associated with higher Aβ plaques in the brain, and the concentration levels of Aβ42 in the cerebrospinal fluid of AD patients are significantly reduced compared with normal controls. Currently, the detection of Aβ42 in cerebrospinal fluid can be carried out using immunological technology or mass spectrometry. However, the specificity of Aβ42 in detecting AD needs to be improved. Some studies have shown that the ratio of Aβ42/Aβ40 can be used to judge amyloid deposition in the brain in the early stages of the disease. The specificity of this method is significantly better than that of measuring Aβ42 alone. However, the detection of cerebrospinal fluid is invasive and is not conducive to early screening. Currently, more and more scholars are beginning to explore blood biomarkers. Aβ protein can be measured in plasma, but the Aβ concentration in plasma will be affected by factors such as platelets, and its correlation with AD is not as good as the Aβ concentration in cerebrospinal fluid. However, gratifyingly, studies have shown that using mass spectrometry to detect plasma Aβ concentration can show a significant correlation with cerebral amyloidosis. Therefore, exploring more effective measurement technologies is the core of breakthroughs in blood screening. Tau The microtubule system is a component of the neuronal cytoskeleton, and its composition contains tubulin and microtubule-related proteins, with the latter having a higher content of Tau protein. The main function of Tau protein is to bind tubulin and promote the production of microtubules. If Tau is over-phosphorylated, it can accumulate in cells, damage the function of microtubules, and damage the cytoskeleton or axonal transport of nerve cells, thus triggering neurodegeneration. It is generally believed that total?t tau (T?t tau) and phosphorylated?Tau (P?tau) concentrations are related to AD. The increase in T-tau reflects the degree of degeneration and damage of neurons and axons. High levels of P-tau suggest the formation of neurofibrillary tangles. CSF P-tau is regarded as the most specific marker of AD, and the concentration of CSF P-tau in AD patients will be significantly increased. Studies have shown that P-tau protein can be used to judge the conversion of MCI to AD, and therefore can be used as a key marker for early clinical diagnosis. Nowadays, P-tau levels in CSF can be measured through immunological methods such as ELISA. Neurofilament lightweight peptide (NFL) NFL is a peptide encoded by the NEFL gene. It is an intrinsic cytoskeletal protein that can be used as a biomarker of axonal injury and can be obtained in both cerebrospinal fluid and plasma. Neurons in the brain interstitium and cerebrospinal fluid release NFL after death, and then enter the blood. The serum or plasma level of NFL is closely related to CSF ??NFL, and is increased in both familial and sporadic AD. In familial AD, patients show increased concentrations of NFL 10 years before the onset of significant clinical symptoms. . Therefore, plasma or serum NFL may be a reliable blood biomarker for neurodegenerative diseases in AD and other neurodegenerative diseases. Plasma glial fibrillary acidic protein (GFAP) GFAP is a key indicator used to monitor astrocyte viability. Previous studies have shown that GFAP is significantly increased in the brain and CSF of AD patients, suggesting that GFAP may become a potential biomarker of AD. Studies have shown that serum GFAP levels in AD patients are significantly higher than those in normal controls. In addition, reports also suggest that the plasma concentration of early-onset AD patients is significantly higher than that of normal controls. While the increase in GFAP concentration in the plasma of late-onset AD patients is not as significant as that of early-onset AD, it is also significantly higher than that of normal controls group. Therefore, GFAP is expected to become a member of the blood AD markers and provide new clues for the clinical diagnosis of AD. Neurogranin (Ng) Ng is also a potential biomarker for early diagnosis of AD and is usually expressed in granular structures in pyramidal cells in the hippocampus and cortex. Ng has been shown to be related to synaptic plasticity, synaptic regeneration and other functions mediated by the calcium/calmodulin signaling pathway. Some scholars have compared the levels of neurogranin in the cerebrospinal fluid of patients with AD, frontotemporal dementia, Lewy body dementia, Parkinson’s disease, etc. The results show that the concentration of neurogranin in the cerebrospinal fluid of AD patients is significantly higher than that of other diseases, further illustrating that Higher Ng protein concentration in cerebrospinal fluid has a specific screening effect on AD.

Herpes simplex virus (HSV) belongs to the herpesvirus alpha subfamily and has a unique 4-layer structure. The core of HSV is a linear double-stranded DNA of about 152 000 bp, surrounded by an icosahedral capsid. The outer capsid is a membrane, which contains more than 20 important proteins that can regulate the virus replication cycle. It is a characteristic structure of herpes viruses, can connect the capsid and envelope to form a complete virus particle. The outermost layer of HSV is the lipid bilayer envelope, which contains at least 12 viral membrane proteins (gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM, gN). Entry into host cells and viral immune evasion are both critical. Figure 1. Schematic Representation of Acute and Latent Herpes Simplex Virus-1 (HSV-1) Infection in Humans. HSV Type There are 2 serotypes of HSV: herpes simplex virus ?1 (HSV?1) and HSV 2, which share 83% homology in their amino acid sequences. HSV?1 infection can cause inflammation of the lips in the human body. When the infection is severe, it can induce conjunctivitis and lead to blindness. It may also invade nerve cells and damage the nervous system, leading to encephalitis. HSV ?2 is the main causative agent of genital herpes. Once infected, patients will carry the virus for life and develop periodic genital herpetic lesions. Recent epidemiological studies have shown that genital damage caused by HSV?2 infection increases the risk of HSV?2 carriers contracting and transmitting human immunodeficiency virus (HIV). Even if viral shedding occurs without causing genital damage, will significantly increase the probability of HIV transmission. Different herpesviruses have similar viral structures and can enter host cells through similar membrane fusion mechanisms. Membrane fusion requires the interaction of four basic glycoproteins (gB, gD, gH and gL) on the virus surface. The complex composed of these four glycoproteins is called the core fusion mechanism. They are usually located on the surface of the viral envelope and can pass through the cascade The action transmits the activation signal from gD to gB, causing conformational changes in gB, forming a fusion pore on the host cell membrane to achieve membrane fusion, allowing the virus to successfully release the nucleocapsid and DNA into the host cell for a new round of replication. The Mechanism of HSV Entry into Host Cells HSV can infect a variety of cells including neurons, epithelial cells, and fibroblasts with the help of fusion proteins. In the current research model, specific binding of HSV to receptors on host cells can cause conformational changes in viral surface glycoproteins, thereby mediating HSV entry into cells. The mechanism of action is achieved through two similar pathways: 1. Entering cells through the direct fusion of the viral envelope and the host cell plasma membrane, and the virus uses this pathway to enter fibroblasts and lymphocytes; 2. The virus triggers the invagination of the host cell plasma membrane to form endosomes, Endocytosis into the cytoplasm is the pathway used by the virus to enter neuronal cells. However, the specific entry route may depend on the type of host cell surface receptors, such as the paired immunoglobulin-like receptor α (PILRα) of gB, and the integrin receptors αvβ3, αvβ6, and αvβ8 of gH?gL, which may be related to It is related to the role of virus non-essential envelope proteins (gC, gE, gG, gI, gJ, gK, gM, gN), and the number of receptors on the host cell membrane may also affect the entry method of the virus.

The tumor microenvironment is composed of tumor cells and non-tumor cells (including fibroblasts, vascular and lymphatic endothelial cells, various immune cells) and extracellular components (such as extracellular matrix, cytokines, inflammatory factors, chemokines, etc.) in tumor tissues. Factors, etc.) interact to form a dynamic and complex network structure. Among them, chemokine receptors can participate in a variety of physiological and pathological processes after binding to their ligands, such as cell growth, development, differentiation, apoptosis, tissue damage and repair, etc. Chemokine receptors and their ligands can not only play an anti-tumor role in tumors by chemotaxis, activating immune cells or inhibiting vascular proliferation, but can also stimulate tumor growth, chemoattract tumor cells, and promote tumor blood vessel growth and promote the degradation of extracellular matrix to achieve the effect of promoting tumor growth, invasion and metastasis. Figure 1. CXCL13/CXCR5-associated immune activities across tissues. Biological Characteristics of CXCR5/CXCL13 C-X-C motif chemokine ligand 13 (CXCL13) is an important member of CXC-like chemokines, mainly expression and secretion of T lymphoid follicular helper cells, dendritic cells and part of the mesenchyme cells in secondary lymphoid tissue. CXCL13 is the only ligand of the G-protein coupled receptor family CXCR5. The CX-CR5/CXCL13 axis plays a pivotal role in the progression of many cancers. CXCL13 can promote tumor growth and invasion through the PI3K/AKT signaling pathway, or enhance anti-tumor immune responses by increasing tumor immune localization. Common CXCR5/CXCL13 axis-related immune cells include B cells, macrophage M1, NK cells, CD8+T cells, follicular helper T cells (THF), γδT cells, and those expressing CX-CR5 CD8+T cell subset, called follicles Cytotoxic T cells (Tfc). The Role of CXCR5/CXCL13 in Different Tumors The role of CXCR5/CXCL13 axis in solid tumors Increased expression of CX-CR5/CXCL13 is associated with tumor occurrence and development in breast cancer, lung cancer, gastric cancer, clear cell renal cell carcinoma, and osteosarcoma. Studies have shown that in HER2+ breast cancer patients, increased CXCL13 expression has a better survival rate. Studies on female mice suggest that CX-CL13 has an inhibitory effect on tumor growth, which may be related to the CXCR5/ERK signaling pathway. This has important implications for immunotherapy in triple-negative breast cancer patients. CXCR5 and CXCL13 may be involved in the occurrence, metastasis and recurrence of advanced colon cancer through the PI3K/AKT signaling pathway. In mouse gastric cancer, CD40 can upregulate the chemokine receptor CXCR5, thereby inhibiting MDSC recruitment and T cell proliferation and promoting tumor immune escape. Bioinformatics analysis showed that low expression of CXCL13 indicates a good prognosis in ovarian cancer. DNA methylation-dependent down-regulation of CXCL13 may promote the occurrence and development of cervical cancer. In addition, CXCL13 promotes the invasion and metastasis of pancreatic ductal carcinoma by activating the ERK1/2 pathway and increasing the expression of ETV4. The role of CXCR5/CXCL13 axis in hematological malignancies Chemokine receptors affect the migration and homing of malignant lymphocytes in the blood tumor microenvironment, and can promote the growth and drug resistance of tumor cells. Among hematological malignancies, especially chronic lymphocytic leukemia (B-CLL) and acute lymphoblastic leukemia (B-ALL), there is important evidence that the expression of CXCR5/CXCL13 in PCNSL tumor tissue is 100%. In addition, elevated serum CXCL13 levels are associated with an increased risk of B-cell non-Hodgkin lymphoma (NHL) in HIV-infected patients. The p53 deletion may participate in the organ invasion and chemotherapy resistance of multiple myeloma through the miR-19a/CXCR5 pathway. Bone marrow mesenchymal stem cells express high levels of the chemokine CXCL13 in the bone marrow microenvironment, which affects the invasion, drug resistance and proliferation of bone marrow mesenchymal stem cell lines U266 and LP-1 through the CXCL13-mediated signaling pathway. In addition, the CXCL13 pathway also up-regulated the mRNA and protein expression levels of BTK, NF-κB, BCL-2 and MDR-1 in U266 and LP-1 cells, suggesting CXCR The 5/CXCL13 axis may provide new insights into the immunotherapy of multiple myeloma. Applications Related to CXCR5/CXCL13 Axis and Tumor Immunotherapy CXCR5 /CXCL13-B Cells and Tumor Immune B cells through the use of B cell receptors (BCR) to identify the function of APC, which is very important for the stable state of exogenous antigen and its own antigen to maintain the body’s immune system. Intratumoral CXCR5+ tumor-infiltrating lymphocytes (TILs) were found in chronic inflammatory breast cancer, mainly B cells, and their abundance was significantly correlated with CXCL13 gene expression, suggesting that CXCL13 may promote and guide the infiltration and migration of B-TILs in the tumor. CXCR5/CXCL13 not only participates in the occurrence and development of tumors, but also forms a network structure with various cells in the tumor microenvironment to exert anti-tumor immune effects. B cells play an important role in humoral immunity. For example, in patients with colorectal cancer, increased intratumoral CXCL13 is associated with abundant T cell and B cell tumor infiltration and prolonged patient survival. Injecting CXCL13 into the colon submucosa of mice containing colorectal cancer can effectively prevent tumor growth. TABs (TABs) recruited by Th-CXCL13 cells in patients with nasopharyngeal carcinoma induce plasma cell differentiation and immunoglobulin production through the interaction of IL-21 and CD84, which may predict better survival rate. In addition, CD4+T cells can affect the infiltration of B lymphocytes in HPV+ head and neck squamous cell carcinoma through the CXCL13/CXCR5 axis. CXCR5/CXCL13-T cells and Tumor Immunity CD8+T cells are considered to be the main cytotoxic lymphocytes with anti-tumor effects. The occurrence of tumors is related to the functional exhaustion of CD8+T cells. In chronic viral infection and follicular lymphoma, CXCR5+CD8+T cells have stronger pro-inflammatory functions than CXCR5-CD8+T cells. Studies have found that CXCR5+CD8+T cells are an important CD8+T cell subset in colorectal tumors and have the potential to promote anti-tumor immunity. The recruitment of anti-tumor immune cells depends largely on the expression of chemokine receptors. In colorectal cancer, the frequency of CCR2-expressing and CXCR5-expressing cells was significantly down-regulated in CD4+ and CD8+ T lymphocyte populations, respectively, suggesting T cells anti-tumor function is reduced. Studies have shown that CXCR5+CD8+T cells are abundantly infiltrated in gastric cancer, muscle-invasive bladder cancer, serous ovarian cancer, head and neck squamous cell carcinoma, and pancreatic cancer, suggesting a good prognosis.

As a tumor suppressor, p53 is one of the most commonly mutated genes in human cancers. Due to its important role in tumor suppression, p53 has attracted great interest from researchers in drug development. As a transcription factor, p53 is directly or indirectly involved in regulating many genes, including cell cycle arrest, apoptosis, senescence, and DNA repair. In recent years, p53 has become one of the most important and attractive drug targets in cancer treatment. This article will review research on p53-based gene therapy, targeted therapy and immunotherapy. Figure 1. The p53 pathway. Overview of the Structure and Function of p53 Gene The human p53 gene is located on chromosome 17p13.1, contains 11 exons, encodes 393 amino acids, and has a relative molecular mass of 43,700. p53 is a sequence-specific DNA-binding protein with two transactivation domains at its N-terminus, followed by a conserved proline-rich domain, a central DNA-binding domain, and finally a The C terminus encodes its nuclear localization signal and an oligomerization domain required for transcriptional activity. p53 mutations or deletions occur in about half of cancer patients, and it is one of the most commonly mutated genes in cancer. p53 mutations mainly occur in the DNA-binding domain, and several amino acid residues have a much higher mutation frequency than other amino acids. These residues are often called hotspot TP53 mutations, among which R175, G245, R248, R249, R273, and R282 are the major hot spot. As a tumor suppressor, p53 can regulate cell division, prevent cells with mutated or damaged DNA from dividing, and transmit apoptosis signals to these cells, thereby preventing tumor formation. p53 can respond to cellular stress or DNA damage, activate a variety of transcription targets, and coordinate a variety of responses, including cell cycle arrest, DNA repair, antioxidant effects, anti-angiogenesis effects, metabolism, autophagy, senescence, and apoptosis. Under non-stress conditions, p53 is ubiquitinated by E3 proteins such as mouse two-microbody protein 2 (MDM2), constitutive photomorphogen 1 (COP1), RING domain family member 2 (PIRH2), and triple motif protein 24 (TRIM24), and subsequent degradation by the proteasome maintains p53 protein at a low level. Another important negative regulator of p53 is mouse double microbody protein X(MDM4) can inhibit cell cycle progression in a variety of ways, one of which is to upregulate p21 protein expression. Subsequently, p21 protein can bind to cell cycle proteins, causing cell cycle G1 phase arrest. In addition, p53 can also bind to other target genes, such as 14-3-3σ and cell division cycle protein 25 (CDC25), to arrest the cell cycle in the G2/M phase. In addition, p53 also plays an active role in many different types of DNA repair, including nucleotide excision repair, base excision repair, mismatch repair and non-homologous end joining. Beyond this, p53 mutations provide a selective advantage to tumor cells, allowing them to circumvent cell cycle checkpoints, avoid apoptosis and senescence, and proliferate under conditions where normal cells cannot proliferate. Targeting MDM2 and MDMX MDM2 inhibitors MDM2 is a major negative regulator of p53. In cancer cells carrying WT p53, MDM2 is often overexpressed through gene amplification or transcriptional upregulation. Therefore, MDM2 is a good target for cancer treatment. Common inhibitors of MDM2 are Nutlin3a and Nutlin-3a-aa, which are cis-imidazole compounds that can induce p53 activation in WT p53 cancer cells but have no effect in mutant p53 (mutp53) cells. Nutlin- 3a-aa is more active than Nutlin-3a against purified wild-type MDM2 and is more effective in increasing p53 levels and releasing the transcription of p53 target genes from MDM2-induced repression. MDMX inhibitors Like MDM2, as a negative regulator of p53, MDMX has also attracted great attention from inhibitor developers, but few inhibitors have been discovered so far, and most of them are short peptides that inhibit MDMX activity. To inhibit the interaction between MDMX and p53, the researchers designed a highly specific short peptide (SAH-p53-8) that can activate p53 in MDMX-dependent cancer cells and induce apoptosis in vivo. However, later studies showed that SAH-p53-8 binds very tightly to serum, which limits its entry into tumor cells and further clinical development. Targeting p53 mutants Most tumor cells contain p53 gene alterations, including mutations, deletions, and translocations. However, the incidence of p53 gene mutations is higher than deletions and translocations. p53 mutations not only lead to changes in p53 function, but also confer new functions to these mutants, called gain of function (GOFs). To target cancer cells with p53 mutations, several therapies have been developed. Activate WT p53 Activity As a tumor suppressor gene, reactivating the activity of WT p53 is a better treatment method. The p53-Y220C mutant is an excellent example of developing mutant p53 therapeutics through protein stabilization. Some studies have found that small molecule compounds PK083 and PK7080, which target the unique surface cracks produced by the Y220C mutation, can bind to the Y220C mutant, restore the wild conformation, and induce Y220C-dependent cell cycle arrest and apoptosis. Studies have shown that the correct folding of p53 requires the presence of zinc, and the lack of zinc in the DNA-binding domain leads to misfolding of the protein. By providing zinc to certain p53 mutants such as R175H, these mutants can restore the conformation and function of WT p53, once again functioning as a tumor suppressor gene. GOFs that Inhibit Mutant p53 Most p53-based drug development targets WT p53 activity in cancer cells, but there are also attempts to eliminate mutp53 GOFs activity by targeting mutp53 for rapid degradation. Since the introduction of mutp53 GOFs, many mechanisms of mutp53 GOFs have been proposed, such as: BAG5 interacts with mutp53 protein and promotes the accumulation of mutp53 protein, resulting in GOFs promoting cell proliferation, tumor growth, cell migration and chemical resistance. p53 mutants down-regulate solute carrier family member SLC7A11 (solute carrier family 7 member 11) through the epidermal growth factor receptor (EGFR)/extracellular regulated protein kinase (ERK) cycle or through transcriptional regulatory protein 1 (BACH1)-mediated down-regulation, enhances the invasion and metastasis of cancer cells. A general strategy for GOFs targeting mutant p53 is to utilize existing therapeutics that target pathways or genes affected by mutant p53, such as EGFR inhibitors, statins of the metovalerate pathway, and inhibitors of mixed lineage leukemias (MLLs). Furthermore, there is emerging evidence that the interaction between mutp53 and the tumor microenvironment may shape the GOFs of mutant p53. The GOFs of p53 play a key role in the occurrence and development of cancer. A comprehensive understanding of the GOFs of p53 will help provide a better theoretical basis for personalized drug therapy in cancer treatment. Targeting Mutant p53 Stability One strategy to target p53 mutants is to reduce their stability. Some p53 mutants are oncogenic, and reduced levels can lead to cancer cell death. Studies have found that the stability of mutant p53 can be enhanced by the heat shock protein (HSPs) family, and the binding of the HSPs complex prevents the degradation of mutant p53 by E3 ubiquitin ligases such as MDM2 and HSP70-interacting protein (CHIP).