INTRODUCTION
Primary sclerosing cholangitis (PSC) is a chronic, cholestatic, continuously progressive liver disease of unknown etiology, characterized by localized inflammation, fibrosis, and segmental dilation of the extrahepatic and large intrahepatic bile ducts. The natural course of PSC leads to cholestasis, secondary biliary cirrhosis, portal hypertension, liver failure, and, quite often, cholangiocellular carcinoma (CCC)[1-3].
The causes of PSC remain unknown, and its pathogenesis has not been well studied[4]. It is hypothesized that genetic factors (particularly HLA genes), as well as environmental and immunological factors, play a role in its development[3,5,6]. There is evidence of a hereditary predisposition to this disease. However, according to available data, less than 10% of cases of PSC inheritance can be explained by genes that have been identified as being associated with this disease[3].
Notably, only about half of the genes associated with PSC are also linked to ulcerative colitis (UC) or Crohn’s disease (CD), or both of these conditions[2,3,7]. The most widely accepted current hypothesis regarding the pathogenesis of PSC suggests that it arises as an immune response in individuals with a genetic predisposition following exposure to certain environmental factors or toxic substances, including lipopolysaccharides, which are structural components of bacterial cell walls[8-10].
However, it remains unclear why the pathological process is localized primarily in the extrahepatic and large intrahepatic bile ducts and does not affect the small intralobular and interlobular biliary ducts. The question of the mechanism of formation of multiple, unevenly spaced, concentric strictures, with proximal dilatation of the bile ducts, in these ducts, also remains unanswered. Furthermore, there is no explanation for the strong association between PSC and inflammatory bowel disease (IBD).
To answer these questions, it is logical to assume that there are structures in the bile ducts and colon with similar architecture and function. Simultaneous damage or dysfunction of these structures, caused by certain exogenous or endogenous factors, may trigger the simultaneous development of a pathological process in both the bile ducts and the colon. Identifying these structures would help to understand and explain the association between PSC and IBD, as well as the formation of multifocal biliary concentric strictures in the bile ducts, which develop predominantly in large bile ducts in classic PSC.
We hypothesize that such structures could be peribiliary glands (PBGs) in the bile ducts and crypt-forming glands in the colon. Both types of glands have a similar morphological and functional structure and produce mucin-rich secretions that protect cholangiocytes and colonic epithelial cells. This review compiles materials supporting this hypothesis.
Biliary epithelial cells (BECs) and colonocytes are constantly exposed to various aggressive damaging factors. BECs, which line the bile ducts, are affected by bile components, while colonocytes are exposed to fecal matter and toxic products formed during digestion and the vital activity of microorganisms. This requires specific and effective defense mechanisms to protect against these threats.
FACTORS OF AGGRESSION AND DEFENSE OF COLONOCYTES
Aggressive factors include: (1) Toxic metabolites produced by opportunistic bacteria, which can alter colon wall permeability and colonocyte regeneration processes; (2) The composition and changes in the abundance and ratio of microorganisms in the colon microflora can also affect the permeability and barrier function of the mucous membrane; (3) Endogenous factors, such as bile acids, products of impaired metabolism, pro-inflammatory and vasoactive mediators, can have a damaging effect on the crypt-forming glands and colon epithelium; and (4) Exogenous factors, such as irritating food components and/or medications, can also have a damaging effect on colonocytes.
A pre-epithelial mucus-bicarbonate barrier provides balanced protection of the colon mucosal epithelium against damaging factors. It effectively protects colonocytes from the aggressive effects of antigens, acids, alkalis, enzymes, and other toxic substances, as well as from mechanical damage associated with the passage of fecal matter. The main component of the pre-epithelial mucus protective barrier is mucins. They play a key role in protecting colonocytes from bacteria and other damaging agents. Mucins are glycoproteins synthesized by secretory cells located in the glands forming the crypts.
COLON CRYPTS: CELLULAR COMPOSITION AND GLANDULAR ORGANIZATION
The glands of the large intestine are vital structures responsible for producing mucus. This secret provides reliable protection for colonocytes against various aggressive factors[9-12]. There are between 7.5 million and 12.0 million crypts, which are located between the villi and extend from the surface of the mucous membrane to the underlying muscular layer throughout the entire length of the large intestine, all the way to the anal opening[9]. In adults, the wall of the cecum contains approximately 4.5% of the total number of these glands, the ascending colon contains about 22%, the transverse colon 27%, the descending colon 21%, in the sigmoid colon - 20%, and in the rectum - 5.5% of the glands[9]. The crypts in the right half of the large intestine are deeper, but their density per unit area is slightly lower than in the left sections[9]. The left half of the large intestine, especially the rectum, is characterized by a high density of crypts per unit area. Despite the lower density of crypts, the right half of the large intestine compensates for this with greater depth and total surface area of the crypts.
Normally, crypts are microscopic, straight, blind-ended tubular structures arranged parallel to one another (Figure 1). Their average length is approximately 0.5 mm: Longer in the proximal and shorter in the distal sections of the large intestine[13]. The walls of the glands are lined with a single-layer of epithelium, which is located on the basal membrane. The crypts contain various types of cells[9,14]: (1) Absorptive cells participate in the absorption process; (2) Goblet cells produce mucus containing mucins; (3) Undifferentiated (stem, mesenchymal) cells responsible for regeneration; and (4) Progenitor cells, which develop into mature, differentiated cells from stem cells during their maturation.
Figure 1 Schematic representation of the structural organization and cellular composition of colonic crypts, including the main cell types: Colonocytes, progenitor cells, goblet cells, and stem cells.
The figure demonstrates the tubular organization of the crypt and the distribution of different cell populations.
In the crypts of the cecum and ascending colon, goblet cells account for 44%-49% of all epithelial cells[9]. In the sigmoid colon, their proportion decreases to 33%-44%. The number of undifferentiated cells increases as one approaches the rectum: From 12%-19% in the glands of the cecum to 19%-26% in the glands of the rectum[9]. Stem cells play a key role in regenerating both the glandular and covering epithelium of the mucous membrane. Regenerative processes are particularly important in the distal colon, given that fecal matter becomes denser in this region, increasing the risk of intestinal wall damage.
FACTORS OF AGGRESSION AND DEFENSE OF CHOLANGIOCYTES IN THE BILE DUCTS
Bile constitutes an aggressive environment for the cholangiocytes lining the intra- and extrahepatic bile ducts. The bile acids present in bile possess strong detergent properties, which can damage the cell membranes of cholangiocytes. Bile acids have a cytotoxic effect on many types of cells[15]. However, under physiological conditions, despite exposure to very high (millimolar) concentrations of bile acids, the epithelial cells of human bile ducts remain resistant to their cytotoxic effects[16]. This resistance indicates the presence of mechanisms that protect cholangiocytes from the detergent action of bile acids.
The conjugation of bile acids with glycine and taurine, as well as the formation of mixed micelles with cholesterol and phospholipids - these are protective mechanisms that act at the level of hepatocytes, and bile capillaries[16]. A well-known protective factor of the bile duct epithelium is the glycocalyx, which covers cholangiocytes and consists of mucin glycoproteins and bicarbonate (HCO3-)[17]. Under physiological conditions, the primary function of cholangiocytes is the biliary secretion of HCO3-[18]. Cholangiocytes produce HCO3- throughout the entire biliary tree. Mucin glycoproteins are produced by the PBGs, which are found only in the large bile ducts[19].
PBGs
PBGs are small tubule-alveolar structures that are located within and outside the walls of the extrahepatic and large intrahepatic bile ducts (Figure 2). The excretory ducts of these glands open directly into the bile duct lumen (Figure 2). Figure 3 shows how densely the PBGs are located along the biliary tree. The largest number of PBGs (shown in dark green in Figure 3) are located at the point where the cystic duct branches off, in the region of the hepatopancreatic ampulla, as well as in the area of the liver porta[20,21] (Figure 3). Light green indicates areas with low content of PBGs, while uncolored branches of the biliary tree indicate the absence of PBGs. In approximately 40% of cases, intramural PBGs can be detected in the septal bile ducts, which are located between the interlobular and larger (segmental) intrahepatic bile ducts[17,20]. However, PBGs are absent in the gallbladder and in the small intralobular and interlobular bile ducts[20,21] (Figures 2 and 3).
Figure 2
Schematic representation of the location of intramural and extramural peribiliary glands in the large bile ducts.
Figure 3 Density of peribiliary glands distribution in the biliary tree.
Dark green indicates high peribiliary glands (PBGs) density, light green indicates areas with fewer PBGs, and uncolored branches of the biliary tree indicate the absence of PBGs.
Based on their location relative to the wall of the bile duct, PBGs are divided into two types: Intramural and extramural[22,23]. Intramural PBGs are simple tubular mucinous glands. They take the form of shallow invaginations and are randomly distributed within the walls of the extrahepatic and large intrahepatic bile ducts[24] (Figure 2). The mucinous secretion they produce flows directly into the lumen of the bile ducts[22]. Extramural glands consist of branched tubulo-alveolar acini, which may be serous, mucinous, or mixed, and have a lobular structure. These glands are located outside the wall of the bile duct, in the periductal space, and open into the lumen of the extrahepatic and large intrahepatic bile ducts. In the intrahepatic bile ducts, extramural PBGs are found only in the large lobar and segmental ducts[20,25] (Figure 2). Due to their small size, PBGs are usually not detected using standard clinical imaging methods, except in cases of obvious pathological conditions.
PBGs consist of mature and undifferentiated cells that can actively divide and proliferate, both under normal conditions and in various hepatobiliary pathologies[25]. Three main cell types can be identified within these glands[20]: (1) Secretory [mucinous, (PAS+)] cells, which form the seromucinous portion of the PBGs acinus and are responsible for mucin production; (2) Stem (CD34+) cells, which are located on the bottom of PBGs near the fibromuscular layer and involved in the renewal of secretory cells and cholangiocytes of the extrahepatic and large intrahepatic bile ducts[19,24,26]; and (3) Epithelial cells (cholangiocytes), which are located at the place where the PBGs confluence with the lumen of the bile duct. For a long time, it was believed that the sole function of the PBGs was mucin production. However, recent data suggest that these glands contain multipotent stem cells which can differentiate into hepatocytes, cholangiocytes and pancreatic cells[20,21].
Progenitor (intermediate) cells are located in the middle portion of the glandular acini and develop into mature, differentiated cells during maturation from stem cells. The number of proliferating cells decreases as they approach the surface of the gland, as they transform into goblet cells and cholangiocytes[19,27]. The PBGs and their stem cells actively participate in restoring the integrity of extrahepatic and large intrahepatic bile ducts, both under normal conditions and in pathologies of the biliary tract. This active differentiation process occurs during reparative regeneration following duct damage caused by viral infections, cholestasis, or other factors, and is aimed at restoring the structure and function of the biliary tract[22,24,25]. It is hypothesized that PBGs stem cells are involved in the initiation and progression of CCC through the transformation of stem and progenitor cells into cancer cells[28].
MUCINS: THE ROLE OF GOBLET CELLS IN THE PBGs AND COLON CRYPTS
Goblet cells, which are found in the PBGs and the glands of the colon mucosa crypts, are important producers of mucins.
The mucins synthesized by the glands of the colon crypts
The large intestinal mucosa provides reliable protection against the penetration of bacteria and damaging aggressive factors thanks to mucus and the glycocalyx, which are produced by the glands of the colon crypts. The mucus covering the epithelium of the colon consists of two layers: (1) The outer layer is loose, low-adhesive, and abundant in microorganisms; and (2) The inner layer is dense, highly adhesive, and impermeable to microorganisms.
The main structural components of colonic mucus and the glycocalyx are mucins, which constitute a family of highly glycosylated glycoproteins[29,30]. They consist of a protein component (apomucin) and numerous O-linked oligosaccharide chains[29,31]. Two types of mucins are distinguished: Secretory and membrane-associated.
The inner mucus layer consists of a dense network of polymerized type 2 mucin (MUC2) molecules, which are synthesized by goblet cells of the colon crypt glands[29]. This layer adheres tightly to the colon epithelium and cannot be removed by simple aspiration. Under normal conditions, it is impermeable to bacteria and particles larger than 0.5 μm[32].
The outer mucus layer is looser, easily removed, and densely populated by bacteria. It forms as a result of partial degradation and loosening of the mucin network of the inner layer. This process is likely mediated by the intestine’s own proteases, as it can be observed in mice lacking intestinal microflora, and protease inhibitors prevent the formation of the outer mucus layer.
The carbohydrates that make up mucins serve as a nutrient source for commensal bacteria. Many of these bacteria have specialized operons for various types of complex carbohydrates, meaning that the patterns of glycosylated mucins can influence bacterial selection[32,33]. Beneath the mucus layer in the colon lies the glycocalyx - a kind of “sieve” composed of molecules bound to the plasma membrane of the colonocytes. This “sieve” does not allow large molecules and bacteria to pass through.
The glycocalyx also contains mucins that have a transmembrane domain and protrude 200-1500 nm above the surface of the epithelial cell[30]. The stability of the mucus layer is maintained by a balance between its secretion by goblet cells of colon crypts and its degradation by proteases and glycosidases, as well as by mechanical flushing with intestinal contents[29,30]. The glycocalyx performs several important functions: It prevents bacterial adhesion and invasion, protects the epithelium from mechanical damage, and participates in cellular signal transmission.
In the large intestine of a healthy person, the primary secretory mucin produced by goblet cells throughout the crypts is MUC2. Another secretory mucin that plays an important role in the intestine is MUC5B, which possesses unique antimicrobial properties. However, its expression in the colon is weak and remains at a low basal level. Along with the dominant MUC2, a group of membrane-bound mucins - MUC1, MUC3, MUC4, MUC12, MUC13 and MUC17 - is actively synthesized in the glands of the crypts in the large intestine under normal conditions. These mucins form an important protective glycocalyx layer.
The mucins synthesized by the PBGs
One important function of the PBGs is to produce mucin glycoproteins. These glycoproteins, together with bicarbonate (HCO3-), protect the cholangiocytes that line the large bile ducts from the aggressive, detergent-like effects of bile acids[18,20]. Experimental data indicate that the glycocalyx covering the apical surface of large cholangiocyte membranes contains glycosylated mucins and other glycan-containing membrane glycoproteins. The glycocalyx stabilizes the “biliary bicarbonate umbrella”, thereby helping to protect cholangiocytes from the detergent action of bile acids[34].
The key mucins comprising the glycocalyx of large bile ducts are MUC3 and, possibly, MUC5B, synthesized by the PBGs. The mucins produced by the PBGs, together with HCO3-, provide protection for cholangiocytes only in extrahepatic and large intrahepatic bile ducts[17]. Intralobular, interlobular, and many septal bile ducts lack PGBs, resulting in the absence of a protective mucin-containing secretion in these ducts[19] (Figure 2). As a result, at the level of small bile ducts, cholangiocytes are protected only by HCO3-[17-19,34-36].
Under normal conditions, there is a balance between aggressive factors, such as bile acids, and protective mechanisms, such as the secretion of bicarbonate and mucin glycoproteins. Therefore, the mucins produced by the PBGs and the glands of the colon crypts are highly specialized secretions, perfectly adapted to their location. Mucins from the PBGs, together with bicarbonate, provide protection for the cholangiocytes, while mucins from the colon crypts serve not only an important protective function for colonocytes but also act as a “lubricant” and nutrient medium for commensal microbes.
It is worth noting that two common types of mucins can be distinguished in the glycocalyx covering the epithelial cells of the bile ducts, as well as in the glycocalyx covering colonocytes: Membrane-bound MUC3 and secreted MUC5B. Impaired secretion or a change in the structure and conformation of these mucins can play a role in damage to both the PBGs of the bile ducts and the colon crypt glands. This, in turn, can lead to the simultaneous development of a pathological process in the liver and the large intestine.
Current data indicate an association of PSC with a mutation of the FUT2 gene, encoding the enzyme fucosyltransferase 2, which is involved in the synthesis of mucin - a component that ensures the stability of glycocalyx[31,37]. Dysregulation of expression of the FUT2 gene, leading to the synthesis of abnormal mucin and destabilization of the glycocalyx, can initiate in the PBGs a cascade of pathological changes, including uncontrolled cell proliferation, impaired apoptosis, and pathological fibrogenesis. In genetically predisposed individuals, these mechanisms may represent key pathogenic factors in the development of both isolated PSC and PSC-IBD. Furthermore, it is likely that the FUT2 mutation may serve as a potential biomarker for assessing the risk of occurrence and progression of both isolated PSC and PSC-IBD, requiring further validation in prospective studies.
Fucosyltransferase 2 and mucins in PSC
The FUT2 enzyme plays a key role in the incorporation of fucose into the sugars of mucin glycoproteins and glycolipids, which are components of secreted and membrane-bound mucins synthesized by PBGs and glands of the colon crypts[31]. Fucosylation of glycoproteins promotes cell-cell interactions, including those between host cells and bacteria[38-40].
Mucins that are fucosylated by FUT2 serve as a primary nutrient source and, consequently, as a selective substrate for commensal bacteria. These bacteria break down complex carbohydrates and contribute to the formation of a healthy gut microbiota[40]. Fucose also influences the properties of mucins, such as their hydrophilicity, charge, and intermolecular interactions.
Since the same enzyme, FUT2, is involved in mucin synthesis in both the PBGs and the glands of the colon crypts, disruption of fucosylation processes may weaken the protective properties of the glycocalyx mucin layer in both the bile ducts and the colon mucosa. Therefore, the described mutation in the FUT2 gene in PSC poses a common threat to two critically important mucosal barriers: The colonic barrier and the biliary barrier in the large bile ducts. This dual vulnerability may explain the high frequency of PSC co-occurrence with IBD.
The FUT2 gene encodes the enzyme fucosyltransferase 2, which is likely to play an important role in the pathogenesis of PSC in genetically predisposed individuals[37,41,42]. Nonsynonymous mutations, known as missense mutations, have been identified in this gene in patients with PSC[31,37]. These mutations lead to changes in the protein’s structure, which, in turn, can result in the loss or alteration of its function.
The most common mutation in the gene is rs601338 (G428A), which renders the enzyme inactive[37]. Mutated alleles (A) of this gene (AA or AG genotypes) were identified in 67.9% of patients with PSC, which significantly exceeds the frequency in the general population (approximately 20%). This underscores the importance of this genetic risk factor in the development of the disease[31,37]. In carriers of inactive FUT2 alleles, the risk of developing PSC increases by approximately 1.5-fold[31,37]. This is one of the most reproducible genetic associations in PSC[31,37].
It can be hypothesized that the relationship between a mutation in the FUT2 gene and the pathogenesis of PSC may occur due to impaired mucins synthesis by the PBGs and the colonic crypt glands. A mutation in the FUT2 gene presumably can lead to disruption of terminal fucosylation, which, in turn, may cause the synthesis and incorporation of structurally abnormal mucins into the glycocalyx of the colon and bile ducts in genetically predisposed individuals.
To restore the protective properties of the glycocalyx layer, the PBGs and the colon crypts glands begin to compensatorily intensively produce additional mucins. Increased mucin synthesis activates stem cells to increase the number of goblet cells that produce mucin glycoproteins. Since the FUT2 gene mutation is a constantly active factor causing the formation of defective mucins, this leads to continuous activation of stem cells and an increase in their differentiation into goblet cells. This process results in the proliferation of the PBGs and the colon crypt glands.
Thus, a mutation in the FUT2 gene, which is present in both the PBGs and the colon crypt glands, can lead to a defect in mucin synthesis in the liver and the large intestine, creating two independent sites of vulnerability. Unfortunately, data on the FUT2 genetic mutation in PSC are scarce. For example, we were unable to find data in the scientific literature on the prevalence of the FUT2 genetic mutation in isolated PSC, in PSC co-occurring with UC, and in isolated UC without PSC.
If this hypothesis is correct, then the combination of PSC and IBD caused by a mutation in the FUT2 gene represents a single systemic pathological process. Perhaps this assumption explains the unique IBD phenotype in patients with PSC, which is actively discussed by the scientific community[7,43,44].
In addition to the key FUT2 gene, associations have been identified in PSC with mutations in other genes that also may significantly impact the synthesis, maturation, and function of mucins: (1) Galactose-3-O-sulfotransferase 2 - participates in the sulfation of mucin oligosaccharide chains; and (2) Glucosaminyl (N-acetyl) transferase 4 - responsible for the synthesis of branched O-glycans (complex sugar chains) in mucins.
Mutations in these genes are not as common in PSC and do not have as significant an impact on its development as mutations in the FUT2 gene. Nevertheless, they indicate a genetic predisposition to defects in the synthesis of fully functional protective mucin glycoproteins in both the colon and the bile ducts. When the proliferation process becomes chronic and progressive, it can lead to the development of fibrotic and neoplastic processes, which are frequently observed in patients with PSC and PSC co-occurring with IBD.
CONCENTRIC STRICTURES IN PSC: PATHOGENETIC SIGNIFICANCE OF PBGs
The clinical presentation of PSC is highly variable. Most often, the onset of clinical signs is driven by disease complications, such as strictures with proximal dilatation of the bile ducts, and the resulting cholestasis, bacterial cholangitis, and liver cirrhosis[2,45-47]. The formation of bile duct strictures is a common complication of the classic form of PSC, which affects the large bile ducts. In 45%-60% of patients with PSC, the formation of a dominant stricture is observed[37]. Notably, the fibrotic process in the bile ducts correlates spatially with the locations of the PBGs, suggesting their involvement in PSC pathogenesis.
In the classic form of the disease, strictures typically form in the extrahepatic and larger intrahepatic bile ducts, where both intramural and extramural PBGs are located. PSC with involvement of small (septal) intrahepatic bile ducts, where intramural PBGs are predominantly present, is less common. Small intralobular and interlobular bile ducts, which lack PBGs completely, are never affected by PSC.
Multifocal strictures indicate focal rather than diffuse damage to bile ducts. Local concentric stenoses caused by strictures result from periductal fibrosis, which, according to the proposed hypothesis, originates in the PBGs. The exact mechanism of periductal fibrosis in PSC remains incompletely understood.
Scientific evidence indicates that PBGs are key to the development of the fibrosis and underlies multifocal concentric strictures. Under physiological conditions, PBGs maintain a homeostatic balance between stem cell proliferation and goblet cell apoptosis. In PSC, persistent and prolonged proliferation in the PBGs, caused by impaired synthesis of protective mucins, may lead to an imbalance of these processes. This imbalance can lead to fibrogenesis, directly linking PBGs dysfunction to the development of fibrosis[48].
Periductal fibrosis in PSC: Key mechanisms
According to the theory of fibrogenesis, the mechanism of fibrosis development in PSC can be explained by the progressive proliferation and uncontrolled hyperplasia of the PBGs in large bile ducts[24,48]. Carpino et al[24] convincingly demonstrated that, in PSC, stem cells of the PBGs are activated and acquire the myofibroblast phenotype. Their data indicate a significant increase in the area occupied by α-smooth muscle actin (α-SMA)-positive myofibroblasts in large bile ducts affected by fibrosis (15.8 ± 2.3 vs 0.8 ± 1.1 in normal ducts; P < 0.05). The expansion of the PBGs area and the area occupied by α-SMA-positive myofibroblasts in fibrotic large bile ducts indirectly indicates active local proliferation processes.
Myofibroblasts exhibit high fibrogenic activity: They proliferate rapidly and, due to α-SMA expression, become contractile, enabling them to migrate and colonize the interstitial space[49]. The locally formed numerous myofibroblast populations activate type I collagen synthesis, which forms a fibrous structure, thickening the wall around the bile duct, forming a stricture that leads to bile duct stenosis. Notably, the wall thickness of bile ducts affected by fibrosis (861.1 ± 299.8 μm; P < 0.05) in PSC is statistically significantly greater than the wall thickness in normal bile ducts (100.3 ± 15.0 μm) and in adjacent bile ducts without fibrosis (103.7 ± 20.5 μm)[24]. Local increased proliferation and hyperplasia of PBGs in PSC are confirmed by a statistically significant elevation of the area occupied by these glands in large fibrotic bile ducts (5.9 ± 2.1 vs 1.64 ± 0.51 in the control and 1.8 ± 0.7 in large ducts without fibrosis; P < 0.02)[24]. Moreover, the wall thickness of the bile duct with fibrosis positively correlates with the area occupied by α-SMA-positive myofibroblasts (r = 0.855; P = 0.014) and the PBGs area (r = 0.811; P = 0.015)[24].
Excessive myofibroblast activity leads to remodeling of the periductal space. Local periductal fibrosis affects specific bile duct segments, resulting in stricture formation and ductal stenosis. The formation of bile duct strictures in PSC appears to be the final stage of chronic proliferation and fibrosis of the extramural PBGs.
Studies by Carpino et al[24] indicate that the primary pathological process in PSC develops in the PBGs, with subsequent involvement of cholangiocytes. As the disease progresses, the area of proliferating PBGs expands. Cholangiography reveals multifocal, ring-shaped strictures alternating with segments of normal or slightly dilated bile ducts containing sac-like protrusions resembling diverticula[45,50].
Fibrosis of the PBGs in PSC of small (septal) bile ducts
In clinical practice, a special form of PSC of small bile ducts is distinguished. This form is characterized by signs of cholestasis and a typical histological pattern. However, it shows no strictures in the large bile ducts on cholangiography[46,51,52]. This type of PSC is less common, occurring in approximately 10%-20% of cases, and has a more favorable prognosis[52]. We hypothesize that in this form of PSC, the intramural PBGs located within the wall of small septal bile ducts are damaged. However, this assumption remains hypothetical and requires rigorous clinical and morphological validation through further research. Since intramural PBGs are present in only 40% of septal bile ducts, the prevalence of this disease form is significantly lower.
The mechanism of fibrosis development in PSC of the small bile ducts is similar to that developed in the extramural PBGs during classic form of PSC: (1) Activation of stem cells and uncontrolled hyperplasia in intramural PBGs of small septal bile ducts; (2) Acquisition of the myofibroblast phenotype by stem cells; (3) Synthesis of type I collagen; and (4) Formation of local concentric fibrosis, which is described histologically as “onion-skin”[1,52].
In the PSC of small bile ducts, damage to the intramural PBGs is accompanied by the formation of a concentric type “onion-skin” fibrosis surrounding the bile duct. This leads to obstruction of its lumen and development of ductulopenia. In a small number of patients (12%) with PSC of small bile ducts, the disease progresses to the classic form involving large bile ducts[53-55]. Based on the proposed hypothesis, in this case, both intramural and extramural PBGs of extrahepatic and large intrahepatic bile ducts become involved in the pathological process. The appearance of a morphological picture of fibrosis of the “onion-skin” type in large bile ducts probably indicates damage to the intramural PBGs in large intrahepatic bile ducts. Additionally, extramural PBGs are also involved in the pathological process, leading to the formation of local strictures.
Development of cholestasis in PSC
In PSC, bile duct narrowing occurs due to both local fibrous strictures and the development of ductulopenia. This impedes the normal bile flow, causing cholestasis and dilation of the bile ducts proximal of the stricture and/or ductulopenia. Studies show that areas of bile duct dilation in PSC are not associated with damage to extra- or intramural PBGs or cholangiocytes[24]. For this reason, in bile ducts without fibrosis, the area of PBGs, the number of α-SMA-positive myofibroblasts and wall thickness of bile duct remain within normal limits[24]. This suggests that non-fibrotic bile ducts are only minimally involved in the pathological process, particularly in the early stages of PSC[46,56]. This is precisely why a blind liver biopsy in patients with PSC can yield ambiguous results, which can vary significantly depending on the tissue sampling site[46,56]. For this reason, biopsy often proves insufficiently informative[46,56]. It is important to note that dysplastic cells are not detected in the intralobular and interlobular bile ducts, where PBGs is absent[24].
The presented data convincingly demonstrate that in PSC, the pathological process may begin in the PBGs. Local bile duct damage in the form of multifocal biliary concentric strictures and ductulopenia is secondary. Most likely, strictures form at the sites where the PBGs open into the lumen of the bile duct. Cholestasis, which develops proximal to the site of stricture, eventually leads to damage to cholangiocytes and hepatocytes. This is accompanied by cellular senescence, apoptosis and autophagy, as confirmed by the presence of the relevant markers in liver samples and blood plasma from patients with PSC[24,49]. Over time, persistent cholestasis drives disease progression to cirrhosis, with consequent hepatocellular failure and portal hypertension. Furthermore, cholestasis, associated with strictures and ductulopenia, frequently raises the risk and increases the likelihood of the development of cholangitis.
Cholangitis and FUT2 mutations in PSC
The synthesis of physiologically defective fucosylated glycans in PSC predisposes to an increased risk of microbial invasion of bile. Mutations in the FUT2 gene, identified in patients with PSC, are a significant genetic factor that links impaired fucosylation with the development and spread of various microorganisms in bile. The rs601338-FUT2 genotype is closely associated with episodes of cholangitis, fungal infection, and the frequency of dominant strictures, which are three clinical features of PSC[37,57,58]. The FUT2 mutation in these patients directly correlates with an elevated risk of both dominant strictures and cholangitis caused by bacterial and Candida infections of the bile. An inactivating mutation in the FUT2 gene leads to the synthesis of functionally abnormal fucosylated glycans for the glycocalyx, which disrupts its protective properties. As a result, patients with mutant FUT2 alleles (rs601338) exhibit a statistically significant increase in the frequency of cholangitis episodes (P = 0.0025)[37,57]. Carriers of mutant alleles also show a marked increase in the frequency of Candida fungi detection in bile (P = 0.025), which is an additional factor complicating the course of the disease[37,57]. Thus, the FUT2 gene mutation not only contributes to the formation of a defective protective barrier for the bile ducts, but actively shapes the biliary microbiome, promoting an aggressive fungal-bacterial community that exacerbates disease severity[10,37,57].
THE UNIQUE PHENOTYPE OF IBD IN PSC
Given the frequent co-occurrence of PSC with IBD, intestinal symptoms often precede or coincide the onset of liver disease[4,7,51]. The prevalence of IBD in patients with PSC reaches 90%[7,10]. Patients with PSC exhibit a specific IBD phenotype characterized by pancolitis with relative sparing of the rectum, as well as reverse ileitis[7,43]. Typical symptoms observed in PSC-associated IBD include: Loose stools, sometimes with blood, abdominal discomfort, a rise in temperature, and weight loss. However, these symptoms are less pronounced than in isolated UC and CD. In some patients, IBD onset in combination with PSC may be asymptomatic or present with minimal clinical manifestations until the late stages of the disease[59-61]. More than half of patients with IBD and PSC have a mild course of colitis[62,63]. Furthermore, the endoscopic activity of IBD in PSC is usually lower than in isolated forms of UC and CD[62-64]. Another endoscopic feature of IBD in PSC is that rectal involvement is less frequent and less severe than in isolated UC[3,7].
However, histological examination of biopsy material from patients with IBD and PSC reveals high inflammatory activity in 97% of cases, accompanied by marked infiltration, crypt damage, and an increase in the number of goblet cells in the right parts of the colon[3,65]. Thus, a characteristic feature of colitis in patients with combined PSC and IBD is a proximal inflammatory gradient in the colon, with less pronounced endoscopic activity and more pronounced morphological activity in the caecum and ascending colon[63,66,67]. There is evidence of high expression of angiotensin-converting enzyme (ACE) and ACE2, the main components of the renin-angiotensin system (RAS), in the ileum and colon[68-71]. Dysregulation of these RAS components may have potential implications for inflammation and fibrosis in patients with IBD[68-71].
Interestingly, FUT2 gene expression is also higher in the proximal colon[72]. This is believed to be associated with the need to produce more glycans, which serve as a nutrient substrate for the resident microbiota in this region. In PSC-associated IBD, a decrease in FUT2 gene expression is observed. It is logical to assume that this causes structural and functional changes that are more pronounced in the proximal and less pronounced in the distal colon. Therefore, more pronounced morphological changes are observed in these regions, thereby determining disease activity predominantly in the right colon. The degree of involvement of the intestinal crypt glands in the pathological process may vary. Therefore, damage to the colonic mucosa stroma may not be diffuse but focal (zonal). A distinctive feature of colon involvement in PSC is the minimal clinical and endoscopic manifestations, which are discordant with the high morphological activity of colitis. Often, this colitis is an incidental finding during ileocolonoscopy[73].
FIBROSIS OF THE COLONIC MUCOSA STROMA IN IBD CO-OCCURRENCE WITH PSC
Fibrosis of the colonic mucosal stroma is a common feature of PSC-associated IBD. Notably, colonic mucosal fibrosis is observed in many patients with classical PSC, even in the absence of overt IBD signs[73]. This fibrosis can be detected via histological examination of colon biopsy specimens and is often associated with eosinophilia in peripheral blood[3,66,74]. The association between IBD and PSC is so strong that the term “PSC-IBD phenotype” has appeared in the scientific literature[7,75,76]. However, in isolated UC, colonic mucosal fibrosis occurs in only 5% of cases[76]. Despite the frequent development of colonic mucosal fibrosis in PSC, the clinical course of IBD in these patients is relatively mild[66,77]. This may be due to the fact that, in the development of both classic PSC and PSC combined with IBD, the glands of the colon crypts are involved in the pathological process not diffusely, but zonally. The mechanism of fibrosis development can be schematically represented as follows: FUT2 mutation → apoptosis/regeneration imbalance → stem cell activation → proliferation → transdifferentiation into myofibroblasts → collagen synthesis → fibrosis.
Since myofibroblasts are resistant to apoptotic stimuli, and the fibrous collagenous tissue they produce is resistant to degradative enzymes, their excessive activity is a major driver of fibrosis development and of the remodeling of the colonic mucosa stroma[78]. Nielsen et al[78] analyzed four highly specific serological markers reflecting extracellular matrix remodeling processes in PSC. These markers reflect the dynamic balance between two opposing processes: (1) Fibrogenesis: PRO-C3 (interstitial matrix collagen type III) and PRO-C5 (interstitial matrix collagen type V); and (2) Collagen degradation: C3M (marker of collagen type III degradation) and C4M (marker of collagen type IV degradation).
The authors demonstrated that in PSC, markers of fibrosis formation are important prognostic factors and are stronger predictors of fibrosis development than degradation markers. Furthermore, they noted that initial bile duct injury in PSC is accompanied by an elevation in C4M levels, which is a marker of collagen degradation. This may reflect the predominance of collagen degradation processes over fibrogenesis in the early stages of the disease, representing a compensatory response aimed at preventing the development and progression of fibrosis in the PBGs of bile ducts and colonic crypt glands. Conversely, levels of interstitial matrix formation markers (PRO-C3 and PRO-C5) increase as the disease progresses, reflecting the advancement of fibrosis in later stages[78].
MESENCHYMAL STEM CELLS OF PBGs AND COLON CRYPT GLANDS IN THE DEVELOPMENT OF CCC AND COLORECTAL CANCER
The development of CCC and colorectal cancer (CRC) in PSC is associated with the activation of mesenchymal stem cells. Activation of stem cells in PBGs and colon crypt glands can stimulate the Hedgehog (Hh) signaling pathway, which plays a crucial role in embryonic development. In adult tissues, this pathway is largely inactive, except in cases where it is required to actively maintain regenerative processes[79]. In PSC, persistent regenerative and proliferative processes in PBGs and colon crypt glands lead to pathological autocrine activation of the Hh signaling pathway. As a result, this pathway is involved not only in fibrogenesis but also in carcinogenesis[80,81]. Within stem cells, an uncontrolled intracellular signaling cascade is triggered, leading to alterations in processes associated with continuous proliferation. This results in hyperplasia and dysplasia of cells[82]. Continuous, uncontrolled autocrine stimulation of the Hh pathway causes persistent, unrestrained self-renewal of stem cells in PBGs and colon crypt glands, which may block differentiation. As a result, this can lead to malignant transformation of stem cells, accompanied by the development of CCC or CRC[80-82].
Development of CCC in PSC
A growing body of scientific evidence suggests that PBGs in PSC play a crucial role in active cell proliferation, which can lead to the malignant transformation of stem cells[24,83,84]. Liver biopsy specimens from patients with PSC reveal progressive hyperplasia and mucinous metaplasia of the PBGs[24]. These processes are observed exclusively in large bile ducts with fibrosis and are absent in ducts without fibrosis[24]. In PSC, activation of stem cells in the depth of the acini of PBGs promotes metaplasia development, which may initiate CCC[24,85]. It should be noted that PSC is associated with a very high incidence of CCC, whereas hepatocellular carcinoma and gallbladder cancer are extremely rare in this disease[86]. This can be explained by the absence of PBGs in the gallbladder; therefore, the gallbladder is not involved in the process of malignant transformation in PSC. These data indirectly suggest that the cause of CCC in PSC is most likely the malignant transformation of stem cells in the PBGs.
CCC has not been reported in patients with small-duct PSC[54,87]. It is likely that the proliferative activity and the number of stem cells in the intramural PBGs of the small septal bile ducts are not so large as to cause a malignant transformation. They may be sufficient for the development of periductular fibrosis and ductulopenia. Schematically, the development of CCC can be described as: Activation of stem cells in the acini of the PBGs → hyperplasia/metaplasia of the PBGs → initiation of cholangiocarcinogenesis.
Development of CRC in PSC with concomitant IBD
CRC is the most dangerous and common complication of IBD combined with PSC[88-92]. Although these patients exhibit a relatively mild clinical course of IBD, including a lower rate of colectomy, they have a higher incidence of CRC[43]. One of the early predictors of potential CRC development is the detection of non-conventional forms of dysplasia (hypermucinous, crypt-cell, and goblet-cell-deficient) in histological examination of colonic mucosa biopsy specimens[93]. A distinctive feature of CRC in IBD-PSC patients is its location in the proximal colon, where the density of goblet cells in the crypt glands is highest[94,95]. In both isolated PSC and PSC combined with IBD, CRC development is most likely associated with dysplasia, which results from stem cell transformation in the glands of the colon crypts[96]. Crypts are part of the transformation zone, and metaplastic processes within them in the IBD with PSC carry a risk of CRC development[97]. As colitis progresses, structural changes occur in the crypts: They become shorter, and the distance between them becomes irregular[13,96,98,99]. Progressive proliferative processes in the colon crypt glands lead to the development of non-conventional forms of dysplasia[93].
According to the World Health Organization definition, dysplasia is a morphological manifestation of neoplastic transformation without signs of invasive growth and is the most important risk marker for cancer development in patients with IBD and PSC[96,100]. Changes in the crypts in the form of non-conventional dysplasia become increasingly noticeable as the disease progresses. These non-conventional dysplasia forms, on the one hand, indicate involvement of the crypts in the pathological process, and, on the other hand, signal a high risk of progressive neoplasia[96,100]. The vast majority (up to 96%) of non-conventional dysplasia in patients with IBD and PSC, as well as in isolated PSC, manifests as flat, invisible lesions, which may subsequently progress to CRC[94]. Patients with isolated PSC without IBD have a lower risk of CRC development compared to those with combined pathology[93]. The presented data indirectly suggest that stem cells of the colon crypt glands are involved in the process of malignant transformation and CRC development.
SEROLOGICAL MARKERS OF PSC
The most extensively studied autoantibodies in PSC are antineutrophil cytoplasmic antibodies (ANCA). They are detected in 65%-95% of patients with this disease[43,101]. ANCA are classified into two types, perinuclear (pANCA) and cytoplasmic (cANCA), depending on their staining pattern in immunohistochemical assays. In PSC, pANCA often react not with classical antigens but with antigens of the neutrophil nuclear envelope. Because of this, they are termed atypical pANCA. Serum levels of these antibodies are detected in: (1) 26%-94% of PSC patients; (2) 33%-83% of UC patients; and (3) 0%-27% of CD patients[102,103].
ANCA-associated vasculitis in PSC
Atypical pANCA, detected in approximately two-thirds of PSC patients[104,105], serve as markers of ANCA-associated vasculitis (small-vessel vasculitis)[106,107]. Detection of atypical pANCA in PSC patients indicates involvement of the neuro-vascular plexus in progressive fibrotic changes which happen in both the periductal regions of bile ducts and the mucosal stroma of the colon. The resulting blood flow impairment leads to local hypoxia, which damages PBGs cells and colonic crypt glands, and thereby exacerbates local fibrotic processes.
PBGs, which actively contribute to fibrogenesis and biliary stricture development in classical PSC, have a close anatomical and pathophysiological association with the periductal neurovascular plexus. This plexus - including branches of the hepatic artery and vein, lymphatic vessels, and nerve fibres - is concurrently involved in the underlying fibrotic pathological process. The involvement of the peribiliary neurovascular plexus in the pathological fibrotic process leads to the development of ANCA-associated small-vessel vasculitis, accompanied by the appearance of atypical pANCA. A correlation exists between the level of atypical pANCA and the degree of bile duct narrowing caused by the strictures[103]. According to various authors, the median prevalence of atypical pANCA in PSC is 63%, with values ranging from 26% to 94%[43,101,103]. This wide variation can be explained by the differences in patient groups across studies. These groups included individuals with different levels of periductal fibrosis progression and different numbers of strictures. This led to varying degrees of involvement of the periductal neurovascular plexus in the pathological process.
Atypical pANCA are also detected in small-duct PSC, where “onion-skin” type periductular fibrosis develops, which also involves the periductal neurovascular plexus[108]. Therefore, the presence of pANCA should be considered not only as an indicator of the development of periductular vasculitis of small vessels[106,107], but also as a marker of the progression of periductular fibrosis in PBGs with the development of strictures and/or ductulopenia in PSC. Since all ANCA immunoglobulin subtypes in the serum and bile of PSC patients reflect the periductal fibrotic process, their levels do not correlate with any laboratory parameters associated with cholestasis and hepatocyte damage[105]. However, a positive immunoglobulin G (IgG) ANCA level in bile correlates with disease activity and the presence of dominant strictures, and thus may serve as a prognostic marker of disease activity[105]. Fibrosis formation in the stroma of the colonic mucosa is also associated with neurovascular plexus involvement in the pathological process. This is supported by positive atypical pANCA test results in patients with IBD concurrent with PSC[109].
Involvement of the neurovascular plexus in the pathological process in PSC plays an important role in the spread of metastases during the development of CCC and CRC[22,110]. The involvement of nerve fibers in the pathological process may be accompanied by unpleasant sensations in the right upper quadrant in PSC and abdominal discomfort in PSC associated with IBD.
IgA antibodies to glycoprotein 2 in PSC
According to various authors, IgA antibodies to glycoprotein 2 (GP2) are detected in the blood plasma of 31%-72% of patients with PSC. These antibodies are either absent or present in less than 3% of healthy individuals[102,111,112]. A study aimed at detecting IgG antibodies to various GP2 isoforms showed that they have lower sensitivity and specificity compared to IgA antibodies to the same GP2 isoforms[113].
GP2 was previously thought to be a protein secreted by pancreatic acinar cells and stored in granules on their membrane. Studies have also shown that it can be found in M cells of the intestinal lymphoid tissue[8]. Lopens et al[114] demonstrated that GP2 is also expressed by PBGs in PSC and acts as a specific antigen that binds to IgA autoantibodies. In their study, the authors used three methods: Enzyme-linked immunosorbent assays, immunoblotting, and mass spectrometry to confirm the presence of GP2 in the bile of patients with PSC. Biliary GP2 is detected in the bile of all patients with PSC, whereas it is absent in the bile of healthy individuals[114]. Another important finding of this study was the identification of a correlation between the morphological changes in the PBGs in patients with PSC and the presence of GP2 in the bile[114].
Glycoproteins constitute a large group of proteins with diverse functions: They serve as structural molecules, act as protective agents, participate in substance transport, facilitate cell-cell recognition, and more. One of these functions, the structural one, may determine the role of GP2 in the development of PSC. The presence of GP2 in the bile of PSC patients may indicate its potential role in fibrogenesis, which is actively occurring in PBGs in this disease.
Myofibroblasts, which form from stem cells of PBGs and colon crypt glands, actively synthesize collagen. There is a high probability that during fibrogenesis and collagen synthesis, there is an unusual (immunoreactive) GP2. Notably, immunoblotting of bile samples from PSC patients predominantly reveals a high-molecular-weight isoform of GP2[114]. These data suggest a potential role for this specific GP2 variant in the pathophysiology of PSC. Perhaps this protein contains autoantigenic epitopes that have immunoreactive antigenic properties capable of eliciting an immune response[113]. A “healthy” immune system may recognize this immunoreactive antigen as a foreign protein and start to produce autoantibodies in response.
If this is the case, the presence of IgA antibodies to GP2 in PSC may serve as a serological marker for fibrotic changes in both the PBGs of the large bile ducts and the glands of the crypts in the colon mucosa. This hypothesis is supported by the following observations: (1) IgA antibodies to GP2 are detected in both isolated PSC and in cases of PSC combined with IBD[43]; (2) The wide variation in antibody levels reported by different authors indirectly supports this hypothesis: Different degrees of fibrosis correspond to different antibody levels[43,113,114]; and (3) IgA antibodies to GP2 are significantly less common in patients with PSC combined with CD[113].
The presence of IgA antibodies to GP2 in PSC is associated with an increased risk of CCC and higher mortality[115,116]. Studies by Tornai et al[8] demonstrate that patients with IgA antibodies to GP2 have a more severe disease phenotype and lower survival rates, which is thought to be due to increased fibrogenesis or the development of CCC. Similar results regarding the association between anti-GP2 IgA and CCC were obtained by Jendrek et al[116] and Wunsch et al[117].
The presence of atypical pANCA and IgA antibodies to GP2 in the blood of patients with PSC is a significant indicator of the risk of fibrosis development and its progression in the PBGs of the bile ducts and colon crypt glands, as well as the possibility of developing CCC[102]. The hypothesis suggesting that a mutation in the FUT2 gene may potentially play a pathogenic role in both isolated PSC and PSC concurrent with IBD is consistent with the proposition that PSC is not a true classical autoimmune disease[118].
CONCLUSION
PSC is described as an idiopathic chronic cholangiopathy characterized by inflammation and fibrosis of the bile ducts. It remains unclear why the pathological process only affects extrahepatic and large intrahepatic bile ducts, while sparing small intralobular and interlobular ducts. Moreover, in the classic form of PSC, the large bile ducts show not diffuse but only local damage, leading to the formation of discretely located concentric fibrotic strictures. Proximal to these fibrotic strictures, dilation of the bile ducts is observed. At the same time, fibrous processes are not observed in these areas. The strong association between PSC and IBD also remains unexplained. The authors of this review, after analyzing the scientific data available in the literature, have formulated a hypothesis that explains the mechanisms underlying the development of major clinical and laboratory signs observed in PSC. According to this hypothesis, there is a common morphofunctional structure in the liver and colon, the simultaneous dysfunction of which may potentially trigger pathological changes in both the liver and the colon among genetically predisposed individuals. In the liver, this structure is the PBGs, and in the large intestine, the crypt glands of the colonic mucosa.
Figure 4 shows a schematic presentation of the mechanism responsible for the development of the main clinical and laboratory signs of PSC, based on the hypothesis that in this disease the pathological process originates in the PBGs of the bile ducts and in the colon crypt glands. The triggering factor of structural and functional changes occurring in the PBGs and crypt glands of the colon is likely to be mutations in genes responsible for the synthesis of enzymes involved in the production of protective mucins.
Figure 4 Schematic illustration of the pathogenesis underlying the development of the main clinical and laboratory manifestations in primary sclerosing cholangitis.
PSC: Primary sclerosing cholangitis; PBGs: Peribiliary glands; Hh: Hedgehog; GP2: Glycoprotein 2; IgA: Immunoglobulin A; pANCA: Perinuclear antineutrophil cytoplasmic antibodies; ANCA: Antineutrophil cytoplasmic antibodies.
The hypothesis that in PSC the pathological process may simultaneously emerge in the PBGs of the liver and the colon crypt glands helps explain the close association between PSC and IBD. PBGs and colon crypt glands play an important role not only in mucin production but also in shaping the qualitative and quantitative composition of the microbiota. A mutation in the FUT2 gene in PSC contributes to the formation of an aggressive biliary microbiota consisting of fungi and bacteria. In the colon, the identified mutation influences the development of gut dysbiosis. This hypothesis of the concurrent involvement of PBGs in the liver and crypt glands of the colon also explains why PSC can develop de novo several years after a total colectomy and why IBD may first manifest several years later in patients who have undergone liver transplantation for PSC.
Although the proposed hypothesis sheds light on many aspects of clinical manifestations of PSC and associated complications, further confirmatory studies are undoubtedly needed. Nevertheless, the new perspective on PSC pathogenesis presented in this review opens avenues for the development of early diagnostic methods and the identification of novel therapeutic approaches for this disease.
Limitations of the hypothesis
The proposed hypothesis has several significant limitations that require further investigation and validation: (1) Although the hypothesis effectively explains the relationship between PSC and IBD through shared morphofunctional structures, it does not elucidate the primary etiological trigger that initiates the pathological process in genetically predisposed individuals; (2) The role of FUT2 gene mutations in the development of PSC requires further in-depth study and confirmation. The scientific literature provides insufficient data on the prevalence of this genetic mutation in isolated PSC, PSC concurrent with IBD, and isolated IBD without PSC; and (3) While the proposed pathogenetic scheme explains the mechanism of development of certain clinical and laboratory manifestations of PSC and associated IBD, the causal relationships outlined in the hypothesis require additional experimental and clinical validation. Despite these limitations, the hypothesis provides a novel perspective on the pathogenesis of PSC and opens new avenues for research in the following areas: (1) Development of early diagnostic methods; (2) Identification of innovative therapeutic approaches; and (3) Conducting further research studies.
Peer review: Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Gastroenterology and hepatology
Country of origin: Russia
Peer-review report’s classification
Scientific quality: Grade B, Grade B, Grade B
Novelty: Grade B, Grade B, Grade B
Creativity or innovation: Grade B, Grade B, Grade B
Scientific significance: Grade B, Grade B, Grade B
P-Reviewer: He XS, Academic Fellow, PhD, China; Lindner C, MD, Researcher, Chile S-Editor: Wang JJ L-Editor: A P-Editor: Wang CH
