Images were opened in the Aperio Image Scope application by selecting the "Open Image" option. The areas of interest were chosen using the "Pen Tool" option. The specified area was then evaluated by selecting the "Analysis" option. By choosing the "Annotations" option, the number of strongly positive, positive, and weakly positive pixels was calculated.The positivity value has been estimated by dividing the total number of the positive pixels by the total number of the pixels (N Total - N n) / (N Total), where N Total represents the total number of the pixels, (N n) represents the total number of negative pixels, and (N Total - N n) represents the total number of positive pixels. In its analysis, Aperio Image Scope software used the positive pixel method.       

                                                                                                           

Statistical Analysis System- SAS (2012) software has been utilized for the detection of the effects of the different factors in the parameters of the study. T-test has been utilized for significant comparison between the mean values. Chi (-square-square test has been used for multiple comparisons between the percentages (0.05 and 0.01 probability in the present work. The allelic frequency and equilibrium of the studied genes were extracted according to the Hardy-Weinberg equation within the SAS.

    

                      

    

                    

The results of immunohistochemistry staining for (TWEAK/Fn14) , showed a highly significant relationshipin the ileal tissues of patients who have CD with varying degrees according to the intensity of the immune reaction, which represents the intensity of the color, which are distributed between weak, moderate and strong, according to the (Aperio image Scope) program. Where it was 10% weak, 36.67% medium, and 53.33% strong. P-value (patients vs control) were 0.0003 and 0.0001 (P-value 0 ≤ 0.01) Table (3-5) Figs. (22, 23 and 24).

    

                    

    

                      

III - Neutrophil granulocyte infiltration characterizes the active disease. The neutrophils may be found in lamina propria or infect crypt surface epithelium and the lumen of the crypts, resulting in crypt abscesses. The neutrophils aren't commonly found in normal mucosa ³⁶. IV- Epithelial anomalies that are seen in the IBD include depletion of the Mucin, surface epithelial destruction and metaplastic alterations. The reduction of the number of goblet cells or a reduction in intra-cellular mucin amounts has characterized the depletion of the Mucin. The Pyloric gland metaplasia (which is one of the common indicators of chronicity in the ileal Crohn's Disease involvement) and Paneth's cell metaplasia have been defined as metaplastic alteration examples. Surface epithelial damage, like the flattening, localized cell loss, erosions, and ulcers, indicate the activity of the disease. None of those signs have been definitively disease-specific, and they may happen in the UC, Crohn's Disease, or other types of colitis ³⁶.

Due to the disease's patchy inflammatory patterns, endoscopic Crohn's Disease biopsies may exhibit varying inflammation degrees in one biopsy and between biopsies from one segment of the bowel. This is why the presence of focal microscopic alterations like chronic inflammations and focal crypt architectural distortion near normal crypts may be utilized for the diagnosis of Crohn's Disease. The inflammatory infiltrations in Crohn's Disease could be transmural. Deep fissures, fibrosis and ulcers have resulted from the transmural lymphoid aggregates and inflammatory cells infiltrating submucosa and even lamina muscularis. Transmural alterations may only be characterized in the resection due to the fact that the biopsies are more superficial. The existence of the non-caseating epithelioid granulomas results in promoting the diagnosis of Crohn's Disease; however, only in the case where they're characterized with no associations with the ruptured crypts, and identification of the granuloma isn't required strictly for the establishment of the diagnosis ³⁴. The epithelioid cell granuloma can be defined as epithelioid cell granuloma (i.e. the active histiocytes) with or with no multi-nucleated giant cells. Necrosis is not common in the granulomas of Crohn's Disease that are commonly ill-defined. In addition to that, the granulomas are more widespread in children compared to it in adults ³⁸. The uneven villous architecture in the terminal ileum biopsies is one of the Crohn's Disease symptoms; however, when it's continuous with the proximal colitis, it may as well be a backwash ileitis marker in the UC. Pyloric gland metaplasia can occur and is seen in up to 25% of ileal biopsies from CD patients (and quite more often in the ileal samples after the surgical resections); however, only seldom in the patients who have the UC ³³.          

Histopathological evaluation is also useful in detecting early signs of dysplasia and preventing cancer growth. The histopathological examination report must include the results that are suggestive of chronic colitis (such as basal lymphoid aggregates, basal plasmacytosis, crypt architectural/atrophic changes, metaplasia and mucin depletion) in addition to disease activity indication based upon neutrophil infiltration extent (in the lamia propria, crypt abscesses and cryptitis) and the epithelial damages for the symbol (i.e., the erosions). Even though histological remissions are not (yet) utilized as one of the treatment targets in routine clinical practices, it may be helpful for physicians in making decisions in the case of the establishment of a diagnosis and selection of the treatment. For the optimum implementations of histologic index and assessments of histological remissions in clinical settings, consensus agreement involving the clinicians/endoscopists and pathologists is necessary. If possible, a standardized endoscopic biopsy process has to be developed so that the samples for the histological evaluation are consistent across patients and medical facilities.

Furthermore, agreeing on a specific scoring criterion would make comparing facilities easier. Recent findings in CD ileocecal resection specimens suggested that it can be advantageous to routinely report on active inflammations, plexitis, and granulomas in the margins of resection for advising the clinical follow-up post-surgery ³⁹. The latest advancements in using the histologic indexes in clinical trials and their ability to assist as a predictive factor for guiding disease management even after diagnosis was established visibly prove that histopathology is still one of the significant factors in controlling the IBD ⁴⁰.

            

The present statistical results of immunohistochemistry for the marker of Mycobacterium avium paratuberculosis (MAP), showed that there was a highly significant association with Crohn's patients according to the p-value that was 0.0052 and 0.0001(patients vs. control) (P-value 0 ≤ 0.01) table (1). And the results were distributed between weak, medium and strong, according to the program (Aperio image Scope). Where 10% were weak, 43.33% moderate, and 46.67% strong because of the severity of the infection, disease stage, taking antibiotics, immune-enhancing drugs and because of the severity of the infection as well as external environmental factors such as food, heat and sterilization conditions. This is consistent with the MAP Marker's relationship with CD ⁴¹. As the twenty-first century arrived, a growing body of parallel studies had characterized 2 main bacteria groups as candidates for gross inflammatory disease pathogenesis of intestines in Crohn's Disease. This is a typical intestinal flora group ⁴². An abnormal gut flora, like the adherent invasive E. coli (AIEC) ⁴³. Furthermore, given the global spread of Crohn's Disease and its serious implications for public safety in addition to the cumulative individual suffering, scientists and clinicians in the research area must recognize that accurate evidence that has been obtained from every one of the 3 evidence forms is convergent and that there aren't any conflicts between them. Based on experimental and clinical evidence, there isn't any reason to suspect that bacteria from normal gut microbes community can infect and inflame the gut wall, and this occurs in Crohn's Disease.

Nonetheless, without another distinct activating factor, the unexpected emergence and increase of Crohn's Disease in human societies worldwide due to an epidemic of normal intestinal flora appear highly suspicious. The gut bacteria serves as a nursery for horizontal gene transfer ⁴⁴. We've seen the pathological effects of this modification in common gut bacteria like E. coli that may be enterohaemorrhagic, enteropathogenic, enteroaggregative, enterotoxigenic, and, more lately, the entire adherent and infective AIEC ⁴⁵. Those changes are typically the result of some sort of external selection pressure. Recent studies reveal that typical gut flora, like E. coli, may be significant predictors ⁴⁶. MAP's status as a known multi-host inflammatory bowel pathogen identifies it from all other harmful microorganisms as the primary cause of Crohn's Disease. MAP has been shown in numerous animals, including primates, to have the unique capability for initiating and sustaining chronic inflammation of small bowel of various histological types. MAP infection causes both local and systemic immunological dysfunction in animals. In addition to that, it is neuropathogenic as well, particularly to the non-myelinated neurons, and small intestine disease has been linked to chronic enteric neuropathy ⁴⁷. Despite its widespread pathogenicity, MAP infection in animals can last for years without inevitably progressing into clinical illness.

When tried-and-true methods were used, the majority of the individuals who have Crohn's Disease have been found infected with MAP ⁴⁸. In layman's terms, most individuals with chronic inflammatory bowel disease (CD) have been infected with the Mycobacterium, one of the known IBD causes.                                                           

MAP infects the gut extensively in Crohn's Disease and can be found in the more normal-looking digestive tract as well as highly inflamed and infected parts of the intestine ⁴⁹. Immunological responses of CD-4 T cell lines derived from patients' intestines appear to be dominated by MAP antigens ⁵⁰. Bacterial intracellular death is inhibited by mannans produced by MAP ⁵¹.

MAP infection results in causing primary microscopic inflammation, specific immunological dysregulation and enteric neuropathy ⁴⁹. Mucosal integrity, as well as other critical intestinal functions, are jeopardized. The visible components of gross chronic inflammation disease are caused by a dysregulated neuro-immune response to secondary gut flora penetration into the gut wall, including the normal intestinal bacteria and the ones that have undergone changes that lead to more invasive phenotypes, such as AIEC. Even though MAP has been discovered in human gut granulomas ⁴⁷. The occurrence or lack of those and other characteristics of the CD's variable histopathological image is determined primarily by big-scale responses to secondary co-pathogens like MAP, which is commonly recovered in the culture from the Crohn's Disease tissues ⁵². As a result, the three current lines of research intersect in a two-tiered co-operative disease pathogenesis. The evidence for the link between MAP pathogens and CD is solid and effective and agrees with the previous investigation ^41,53. The present study utilized immunohistochemistry, which reflects true and specific immune reactions between anti-MAP and MAP using the Aprio imaging Scope program. In conjunction with other approved methods, this type of diagnosis can be one of the most dependable methodologies for specific diagnostic CD.

This immunohistochemistry investigation results show that the expression of TWEAK and its receptor Fn14 has been elevated in enteritis or (inflammatory bowel disease) tissue, particularly Crohn's disease. TWEAK protein expression was significantly higher in the Crohn's disease group than in the seemingly healthy group, as demonstrated by rigorous statistical analysis based on Aperio scope images, Where it was 10% weak, 36.67% medium, and 53.33% strong. P-value (patients vs control) were 0.0003 and 0.0001 (P-value 0 ≤ 0.01), and these differences in the proportions between patients may be due to the different stage of the disease and its severity, or Marker-inhibiting drugs, the person's immune status, and external factors. According to TWEAK and Fn-14, expression typically increases in response to stresses, tissue damage, or remodeling ⁵⁴. TWEAK has been expressed widely by monocytes, natural killer (NK) cells, and dendritic cells, with macrophages/monocytes being the main soluble TWEAK (sTWEAK) source in inflammatory body tissue ⁵⁵. In the current study, Fn14 expression has been found to be much higher in Enteritis patients than in seemingly healthy and healthy tissues; in comparison, Fn14 expression was relatively weak in the control. However, it can be strongly induced in injured and sick tissues by a range of different growth factors ²⁸. Fn14 can be found in many tissues, which include the colon, skin, small intestine, heart, brain, kidney, striated muscle, and pancreas ^{26, 27}. TWEAK and Fn14 expression in healthy tissue is quite minimal. This is proven by the fact that the most frequently used CD treatments rely on steroids as well as the monoclonal antibodies against the TNF ²⁴. Overexpression of TWEAK During chronic inflammation, the digestive system is one of the tissue types most affected by the TWEAK/Fn-14 ²⁹. Following mucosal barrier collapse continued Fn-14 stimulation on the intestinal epithelial cells may result in increased gut immune responses against the commensal flora. As a result, healing is slowing down, tissue repair is compromised, and fibrosis develops ⁵⁶. Although TWEAK/Fn-14 has been shown to have similar effects in models of enteritis caused by recurrent acute damage, the role of TWEAK/Fn14 in a more immunologically driven inflammation is unknown. The current study supports previous findings that showed increased expressions of TWEAK and Fn-14 in the Crohn's Disease tissue lesions compared to healthy controls ⁵⁷. Finally, to validate the current findings, we looked at the expressions of Fn-14 TWEAK in the ileal tissues of the IBD and non-IBD patients. TWEAK and Fn14 were significantly overexpressed during active inflammation in ileal biopsy specimens from CD patients compared to healthy humans. These findings support the theory that the TWEAK/Fn-14 signaling pathways have been activated as a defense mechanism during acute and chronic inflammation. However, over-activation in IBD could result in increased gut immune responses against intestinal flora. As a result, healing is slowed, tissue repair is compromised, and fibrosis develops ^{58, 59}. Our findings differ from those of Kawashima and his team ⁵⁵, TWEAK/Fn-14 levels have been higher in patients with active UC but not in CD patients or healthy controls for two reasons. First, only four CD patients have been included in the studies, compared to 30 in the current report. Second, the current studies primarily used small intestine tissue specimens, whereas previous studies only looked at colonic tissues. TWEAK/Fn-14 over-expression was discovered in tissue lesions obtained from patients with active CD, according to the current study. ⁵⁶. To summarize, the current findings show significant TWEAK/Fn-14 overexpression in CD patients' small intestine tissue lesions, as well as a mechanistic role for Fn-14 signaling in CD pathogenesis. With the introduction of new TWEAK/Fn14-targeting medications ²⁶.

Funding: Self-Funding.

Acknowledgments:

Conflicts of Interest: The authors declare no conflict of interest.

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Received: 26 December 2022 / Accepted: 15 March 2023 / Published:15 June 2023

Citation: Sameer A F, Barraj A H, Mahmood H J. Investigation of Crohn's Disease by Immunohistochemistry Technique in Iraqi Patients. Revis Bionatura 2023;8 (2) 2. http://dx.doi.org/10.21931/RB/2023.08.02.2