Immunohistochemical Methods Research Articles

Pathological Investigation of Double–Stranded DNA Breaks and DNA Oxidation in Natural Infection with Mycobacterium avium subspecies paratuberculosis in Goats

Paratuberculosis, created by Mycobacterium avium subspecies paratuberculosis (MAP), manifests as a chronic affliction marked by persistent diarrhoea and granulomatous enteritis, pervasive in both domestic and global wild ruminants. In this investigation, DNA disruption in lesioned tissues of goat as natural infecte with MAP was pathologically assessed. Accordingly, goats manifesting symptoms suggestive to paratuberculosis, including pronounced emaciation and continual episodic diarrhoea, were subjected to an ELISA diagnostic procedure to ascertain the presence of MAP. This diagnostic approach confirmed the presence of the infectious agent in 20 patients. These patients were subsequently euthanized, and tissue samples from intestinal and regional lenf nods. It were subjected to Hematoxylin and Eosin (HE) staining for histopathological investigatıon, Ziehl Neelsen (ZN) staining to identify acid–fast mycobacteria, γ–H2AX to discern disruptions in double stranded DNA, and 8–Ohdg to detect DNA oxidation by immunohistochemical (IHC) method. Gross anatomical observation serous adipose atrophy, augmented dimensions of mesenterial lymphatic nodes, mucosal hypertrophy and non–retractable mucosal undulations. Histological assessment highlighted epithelial cellular degeneration, an abundance of epithelioid macrophages, lymphocytes, plasmocytes, infiltrating in mucosa. Acid–fast entities, discernible through ZN staining, appeared as luminescent red conglomerates in intestinal and mesenterial tissue. The immunohistochemical analyses evinced positive results for both γ–H2AX and 8–Ohdg across all sampled tissues. Intriguingly, this investigation presented the inaugural global evidence of γ–H2AX and 8–Ohdg expression in a natural MAP infection, demonstrating that this pathological agent precipitates DNA degradation and oxidation, thereby augmenting comprehension of the disease’s pathogenesis.

Knockdown of S100A2 inhibits the aggressiveness of endometrial cancer by activating STING pathway

Background Endometrial cancer is a kind of gynaecological cancer. S100A2 is a newfound biomarker to diagnose endometrial cancer. This study was to investigate the role of S100A2 on regulating migration and invasion of endometrial cancer. Methods The mRNA and protein levels of S100A2 were obtained by quantitative real-time polymerase chain reaction, immunohistochemistry and western blot methods. Cell viability was measured by the Cell Counting Kit-8 assay. Cell migration and invasion were quantified using transwell assays. Western blot assay was conducted to quantify protein expressions of epithelial to mesenchymal transition-related proteins (N-cadherin and E-cadherin). Furthermore, in vivo tumour formation experiments were performed to evaluate the role of S100A2 on tumour xenografts. Results S100A2 was significantly up-regulated in endometrial cancer tissues. Knockdown of S100A2 inhibited cell viability, migration and invasion of endometrial cancer cells. Meanwhile, STING pathway was activated by the inhibited S100A2. STING inhibitor C-176 significantly reversed the effects of S100A2 knockdown on aggressive behaviours of endometrial cancer cells. Inhibition of S100A2 dramatically suppresses the tumour growth in vivo. Conclusions S100A2 functions as an oncogene in endometrial cancer. Targeting S100A2 may be a promising therapeutic method to treat endometrial carcinoma.

Piracetam mitigates nephrotoxicity induced by cisplatin via the AMPK-mediated PI3K/Akt and MAPK/JNK/ERK signaling pathways

AimsCisplatin (CDDP) is commonly employed as an antineoplastic agent, but its use is significantly limited by the occurrence of dose-dependent nephrotoxicity, the detailed mechanisms of which remain unclear. This research is aimed to explore the molecular mechanisms of Piracetam (PIR)’s protective effects on nephrotoxicity resulting from CDDP exposure and to elucidate the mechanisms responsible for these effects. Main methodsPIR was given in dosages of 100 and 300 mg/kg body weight for a duration of 15 days; concurrently, on the last day, a single 10 mg/kg dose of CDDP was delivered via intraperitoneal injection. Forty-eight hours post-CDDP injection, the animals were sacrificed to assess nephrotoxicity. Blood samples and renal tissues were taken for biochemical and histopathological investigations. Serum creatinine and blood urea nitrogen (BUN) were measured. AMP-activated protein kinase (AMPK), caspase-9 and nuclear factor kappa b p65 (NF-κB p65) were assessed by immunohistochemistry method. Enzyme-linked immunosorbent assay (ELISA) analysis was employed to determine cytochrome c (Cyt. c), Bcl-2-associated X-protein (BAX), caspase-3, nuclear factor erythroid 2–related factor 2 (Nrf2), Heme oxygenase-1 (HO-1), superoxide dismutase (SOD), tumor necrosis factor alpha (TNF-α), myeloperoxidase (MPO), and interleukin-1β (IL-1β) levels in renal tissue homogenates. The mRNA levels of tumor protein P53 (TP53), phosphatidylinositol-3 kinase (PI3K), protein kinase B (Akt), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK) were tested by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, histopathological evaluations of the renal tissues and the binding affinity of PIR to AMPK by molecular docking were also performed. Key findingsPre-treatment with PIR enhanced renal function markers such as urea and creatinine, mitigated histological damage, and diminished inflammatory cell presence in renal tubules. PIR demonstrated antioxidant effects by reestablishing the equilibrium between pro-oxidants and antioxidants such as MPO, HO-1, Nrf2, as well as SOD. Furthermore, PIR inhibited the inflammatory pathways through the MAPK/NF-κB pathway. Additionally, PIR counteracted the CDDP-induced decline in PI3K/Akt activity and hindered caspase-dependent apoptotic processes. SignificanceIn summary, PIR appears to be an effective therapeutic strategy for reducing CDDP-induced nephrotoxicity, attributed to its antioxidant, anti-inflammatory, and antiapoptotic mechanisms. Consequently, PIR may serve as a complementary treatment alongside CDDP to alleviate nephrotoxicity associated with CDDP.

Current Perspectives on Uterine Fibroids

In this paper, we aimed to: 1) discuss the approaches for increasing the effectiveness of organ-preserving surgical treatment of uterine fibroids; 2) analyse pre-operative preparation options for the formation of a restitutive scar during myomectomy. The article addresses the epidemiology of uterine fibroids in women, particularly in the reproductive age, and discussed the role of inflammation, disrupted nutrition, and proteolysis in the development of myomatous nodules. We also consider the terminology, classification, primary clinical symptoms of uterine fibroids, and discuss the causes of uterine scar incompetence. Various growth factors and collagen types have a differential impact on myometrial and on the formation of a restitutive scar. Finally, we talk about the histological and immunohistochemical methods in the diagnosis of uterine scar incompetence.

Pathomorphological parameters of sepsis-associated encephalopathy in deceased septic patients without purulent lesions to the brain

Sepsis-associated encephalopathy (SAE) clinically manifests by delirium and decreased consciousness less than 15 points on Glasgow Coma Scale. SAE pathophysiology includes neuroinflammation, ischemic-hypoxic and dysmetabolic mechanisms. Despite the high frequency and the important role in thanatogenesis, pathomorphological criteria of SAE remain to be defined. The aim of the study was to specify the key pathomorphological parameters of sepsis-associated encephalopathy in deceased septic patients without purulent lesions to the brain by defining the changes of neurogliovascular unit and the level of tissue ammonia. Material and methods. Using pathohistological, histochemical, and immunohistochemical methods we studied cerebral cortex and white matter, hippocampus, thalamus, and cerebellum of 35 deceased septic patients with SAE in comparison with the control group, which included 30 patients who died from acute cardiovascular failure without CNS pathology. Results. In SAE, small foci of encephalolysis due to thrombosis of microvessels, ischemic-hypoxic and apoptotic changes in neurons are associated with the following parameters that are reliably (p < 0.05) different from the control group: higher (up to 199.48 %) level of tissue ammonia and increased number (up to 316.07 %) of caspase-3+ apoptotic neurons in the cortex, hippocampus, thalamus, and cerebellum; in all studied brain regions, an increased expression level of astrocytic glial fibrillary acidic protein (up to 192.69 %), glutamine synthetase (up to 134.41 %) and aquaporin-4 (up to 400.8 %); significant (up to 947.01 %) expansion of perivascular and pericellular “edematous” spaces, increased (up to 479.58 %) immunopositive area of extravascular CD68+ microgliocytes and increased (up to 374.43 %) proportion of CD68+ ameboid microgliocytes, increased (up to 3.66 times) number of Alzheimer type 2 astrocytes in cerebral cortex, thalamus, and cerebellum; increased (up to 2 times) number of amyloid bodies in the thalamus and cerebellum. Conclusions. The obtained data indicate that the delirious state, loss of consciousness and other manifestations of SAE are associated with ischemic-hypoxic and ammonia-induced ischemic and apoptotic changes of the brain neurones; small foci of encephalolysis; adaptive remodeling and dystrophic changes of astrocytes; microglial reactiveness with increased proportion of phagocytic microgliocytes; brain edema and dysfunctional glymphatics.

Evaluating the therapeutic effect of vitamin D and nerolidol on lung injury due to experimental myocardial infarction: The potential role of asprosin and spexin

Injury to internal organs caused by myocardial infarction (MI), although often neglected, is a very serious condition which damages internal organs especially the lungs. Changes in microcirculation can begin with acute lung injury and result in severe respiratory failure. The aim of this study was to create new approaches that will explain the pathophysiology and treatment of the disease by examining the therapeutic effects of vitamin D (VITD) and Nerolidol (NRD) on the injuries of the lungs caused by MI, and their relationship with asprosin / spexin proteins. MethodsSix groups of seven experimental animals each were constituted. Control, VITD (only 50 IU/day during the experiment), NRD (only 100 mg/kg/day during the experiment), MI (200 mg/kg isoproterenol was administered to rats as a single dose subcutaneously), MI+VITD (200 mg/kg isoproterenol +50 IU/day) and MI+NRD (200 mg/kg isoproterenol +100 mg/kg/day) were the six (6) groups constituted. Tissues were analyzed using histopathological and immunohistochemical methods, whereas serum samples were analyzed using ELISA method. ResultsThe result of the histopathological study for the MI group showed an observed increase in inflammatory cells, congestion, interalveolar septal thickening, erythrocyteloaded macrophages and fibrosis in the lung tissues. The treatment groups however recorded significant differences with regards to these parameters. In the immunohistochemical analysis, expressions of asprosin and spexin were observed in the smooth muscle structures and interalveolar areas of the vessels and bronchioles of the lung, as well as the bronchiole epithelium. There was no significant difference between the groups in terms of asprosin and spexin expression in the bronchiol epithelium.When immunohistochemical and serum ELISA results were examined, it was observed that asprosin levels increased significantly in the lung tissues of the MI group compared to the control group, decreased significantly in the treatment groups treated with Vitamin D and NRD after MI. While spexin decreased significantly in the MI group compared to the control group, it increased significantly in the MI+VİTD group, but did not change in the MI+NRD group. ConclusionIt was observed that serious injuries occurred in the lungs due to myocardial infarction and that, VITD and NRD treatments had a curative effect on those injuries. It was also observed that Asprosin and Speksin proteins can have effect on mechanisms of both injury and therapy of the lung. Furthermore, the curative effects of VITD are dependent on the expression of asprosin and spexin; whereas the observation indicated that nerolidol could be effective through asprosin-dependent mechanisms and specisin by independent mechanisms.

Complex Assessment of the Functional State of the Urinary System in Preclinical Studies. Part 1. Instrumental and Laboratory Assessment Methods (Review)

INTRODUCTION. Functional examination of the urinary system, and particularly the kidneys, is an important challenge in preclinical studies. Currently, there is no generally recognised and detailed approach to drug-induced nephrotoxicity detection in vivo, nor are there clear criteria for its assessment.AIM. This study aimed to analyse and systematise instrumental and laboratory methods for the assessment of urinary system function in laboratory animals and to identify the basic principles for studying drug-induced nephrotoxic effects.DISCUSSION. The study analysed the advantages and limitations of the methods used to study the nephrotoxicity of medicinal products, with considerations for the use of these methods in small and large laboratory animals. The effects of a test substance on the urinary system should first be evaluated using minimally invasive methods. One of these methods is urinalysis. For urinalysis, important considerations include the sampling technique, the volume of the biomaterial, and the turnaround time between urine collection and analysis. Ultrasonography is the most accessible instrumental method in preclinical studies. Ultrasonography can assess organ position, size, structure, and echogenicity and detect abnormalities and changes in real time. Dif ferent method settings are preferred for each species of laboratory animal. Further analysis can include macroscopic examination of organs, measurement of their masses, and microscopic analysis of tissues. Visual assessment should cover the size, colour, and consistency of the ureters, bladder, and kidneys. Nephrotoxicity may manifest as increased apoptosis, vacuolation of renal tubular epithelial cells, epithelial degeneration or dystrophy, oedema, diapedesis-associated haemorrhages, acute tubular and papillary necrosis, necrosis of the Bowman–Schumlansky capsule, casts and crystals in the tubular lumen, glomerulopathy with the corresponding changes, and inflammatory and vascular reactions.CONCLUSIONS. The study analysed and systematised instrumental and laboratory methods for assessing the functional state of the urinary system in preclinical studies. The authors outlined the basic principles for a structured and comprehensive study of the potential nephrotoxicity of novel medicines. The assessment of nephrotoxicity should start with simple and minimally invasive laboratory and instrumental methods, which include general urinalysis and microscopic examination of urine sediment. These methods can detect organ dysfunction that has not yet presented with an associated anatomical lesion. A more in-depth analysis should involve histological and immunohistochemical methods to examine the urinary tissues of laboratory animals.

NFS-26.RB1-ALTERED SUBTYPE (GROUP A) PINEOBLASTOMA AS A PART OF «TRILATERAL RETINOBLASTOMA» PHENOMENON: NEURO-ONCOLOGICAL AND OPHTHALMIC-ONCOLOGICAL VIEW

Abstract BACKGROUND RB1-altered subtype (Group A) pineoblastoma as a part of «Trilateral retinoblastoma» is an extremely rare phenomenon. But over the past 10 years, the incidence of this disease has been increasing. Optimal therapeutic strategy is unknown. The aim of this study was to estimate the incidence of this phenomenon among patients with retinoblastoma, age at onset of retinoblastoma/RB1-altered pineoblastoma and genetic, gender, age predisposition. DESIGN/METHODS From 2019 to 2023 years 256 pediatric patients with de novo retinoblastoma were treated in the FSBI «N.N.Blokhin National Medical Research Center of Oncology». RB1-altered pineoblastoma was revealed in 9 cases (3,5% of all patients, 9,5% among patients with bilateral retinoblastoma, 6,6% for patient with the onset of retinoblastoma under the age of 12 months). Diagnosis was determined according to familial history, retinal camera and MRI data, histology, immunohistochemistry and Illumina Infinium methylation EPIC Bead Chip method (FFPE tumor samples), NGS and MLPA methods for detection of RB1 germline mutation status (blood samples). Therapeutic options for patients with RB1-altered subtype pineoblastoma were presented as like–COGARET0321, like–HIT–MED2017/2020, selective intra-arterial chemotherapy, intravitreal chemotherapy, transpupillary thermotherapy, brachytherapy. RESULTS Gender ratio was 1:2 (3 male and 6 female). In all cases, intraocular retinoblastoma was bilateral. Family history was revealed in two cases. Age at onset of retinoblastoma: less than 6 months in 8 cases, all cases less 2 years, median 3±1,9 months [1,5-20,5]. Median age at onset of RB1-altered subtype pineoblastoma was 20,5±7,3 months [1,5-57,5]. A synchronous variant of the disease was identified in 5 cases, metachronous in 4. Pineoblastoma of pineal region was detected in 7 cases, suprasellar region in 2. Six out of 9 patients are alive, median follow-up 16,5±5,1 months [1,5-43,0]. CONCLUSION It is necessary to expand both regional and international interdisciplinary cooperation to develop an optimal algorithm for the diagnosis and treatment of this disease.

Chemical Composition and Neuroprotective Properties of Indonesian Stingless Bee (Geniotrigona thoracica) Propolis Extract in an In-Vivo Model of Intracerebral Hemorrhage (ICH).

Stroke is the world's second-leading cause of death. Current treatments for cerebral edema following intracerebral hemorrhage (ICH) mainly involve hyperosmolar fluids, but this approach is often inadequate. Propolis, known for its various beneficial properties, especially antioxidant and anti-inflammatory properties, could potentially act as an adjunctive therapy and help alleviate stroke-associated injuries. The chemical composition of Geniotrigona thoracica propolis extract was analyzed by GC-MS after derivatization for its total phenolic and total flavonoid content. The total phenolic content and total flavonoid content of the propolis extract were 1037.31 ± 24.10 μg GAE/mL and 374.02 ± 3.36 μg QE/mL, respectively. By GC-MS analysis, its major constituents were found to be triterpenoids (22.4% of TIC). Minor compounds, such as phenolic lipids (6.7% of TIC, GC-MS) and diterpenic acids (2.3% of TIC, GC-MS), were also found. Ninety-six Sprague Dawley rats were divided into six groups; namely, the control group, the ICH group, and four ICH groups that received the following therapies: mannitol, propolis extract (daily oral propolis administration after the ICH induction), propolis-M (propolis and mannitol), and propolis-B+A (daily oral propolis administration 7 days prior to and 72 h after the ICH induction). Neurocognitive functions of the rats were analyzed using the rotarod challenge and Morris water maze. In addition, the expression of NF-κB, SUR1-TRPM4, MMP-9, and Aquaporin-4 was analyzed using immunohistochemical methods. A TUNEL assay was used to assess the percentage of apoptotic cells. Mannitol significantly improved cognitive-motor functions in the ICH group, evidenced by improved rotarod and Morris water maze completion times, and lowered SUR-1 and Aquaporin-4 levels. It also significantly decreased cerebral edema by day 3. Similarly, propolis treatments (propolis-A and propolis-B+A) showed comparable improvements in these tests and reduced edema. Moreover, combining propolis with mannitol (propolis-M) further enhanced these effects, particularly in reducing edema and the Virchow-Robin space. These findings highlight the potential of propolis from the Indonesian stingless bee, Geniotrigona thoracica, from the Central Tapanuli region as a neuroprotective, adjunctive therapy.

Immunohistochemical Analysis of Dentigerous Cysts and Odontogenic Keratocysts Associated with Impacted Third Molars-A Systematic Review.

This systematic review investigates the diagnostic, prognostic, and therapeutic implications of immunohistochemical markers in dentigerous cysts (DCs) and odontogenic keratocysts (OKCs) associated with impacted third molars. A comprehensive search strategy was employed across major databases including MEDLINE/PubMed, EMBASE, and Web of Science, from the inception of the databases to March 2024. Keywords and Medical Subject Heading (MeSH) terms such as "dentigerous cysts", "odontogenic keratocysts", "immunohistochemistry", "Ki-67", and "p53" were used. The PRISMA 2020 guidelines were followed to ensure methodological rigor. Inclusion criteria encompassed studies on humans and animals providing definitive diagnoses or specific signs and symptoms related to DCs and OKCs, with results on protein expression derived from immunohistochemistry, immune antibody, proteomics, or protein expression methods. Of the 159 studies initially identified, 138 met the inclusion criteria. Our analysis highlighted significantly higher expressions of Ki-67 (22.1% ± 4.7 vs. 10.5% ± 3.2, p < 0.001), p53 (15.3% ± 3.6 vs. 5.2% ± 1.9, p < 0.001), and Bcl-2 (18.4% ± 3.2 vs. 8.7% ± 2.4, p < 0.001) in OKCs compared to DCs, indicating a higher proliferative index, increased cellular stress, and enhanced anti-apoptotic mechanisms in OKCs. Additionally, PCNA levels were higher in OKCs (25.6% ± 4.5 vs. 12.3% ± 3.1, p < 0.001). Genetic mutations, particularly in the PTCH1 gene, were frequently observed in OKCs, underscoring their aggressive behavior and potential malignancy. The findings emphasize the significant role of immunohistochemical markers in distinguishing between DCs and OKCs, with elevated levels of Ki-67, p53, Bcl-2, and PCNA in OKCs suggesting a higher potential for growth and recurrence. Genetic insights, including PTCH1 mutations, further support the need for personalized treatment approaches. These markers enhance diagnostic accuracy and inform targeted therapeutic strategies, potentially transforming patient management in oral and maxillofacial surgery.

Molecular mechanisms of thrombotic complications in glial tumors

Cancer patients are at increased risk of venous thromboembolic complications (VTE), which are the second leading cause of death in these patients. Moreover, the maximum incidence of VTE occurs in tumors of the central nervous system [1]. Research is being conducted to identify laboratory biomarkers of increased risk of VTE, but to date data on their role are ambiguous. Patients with a mutation in the isocitrate dehydrogenase (IDH) gene are at lower risk of VTE, and the IDH mutation is associated with suppression of the production of tissue factor (TF) and podoplanin, the most studied molecules responsible for the occurrence of thromboembolic complications [2]. The main specific markers of VTE are based on immunohistochemical methods, which are possible only with histological examination of the tumor material. Therefore, it is important to find noninvasive biomarkers that could be used to assess the risk of venous thromboembolic complications. In this review, we will focus on highlighting the accumulated knowledge on this theme.

Hesperetin alleviates pulmonary injury in a blunt chest trauma-induced pulmonary contusion model in rats

Pulmonary contusion (PC), a condition that occurs frequently in severe thoracic injuries, is a significant contributor to mortality in those under the age of 40. Hesperetin, a natural flavonoid derivative of hesperidin, is a substance found in various citrus fruits such as oranges and grapefruits and possesses a variety of biological activities, including antiapoptotic, antioxidant, and anticancer effects. In the current study, we investigated the effect of hesperetin on pulmonary tissue structure, expression of some pro-inflammatory cytokines, and mediators in a PC model induced by blunt chest trauma. In this study, 18 adult male Wistar albino rats (8 &amp;ndash; 10 weeks, 250 &amp;ndash; 300 g) were used. The rats were divided into three groups: control, PC, and PC + hesperetin. Hesperetin administration (100 mg/kg/day oral) was completed for 7 days following induction of the model. The wet/dry weight ratio of pulmonary tissue was determined. Tumor necrosis factor alpha (TNF&amp;alpha;) and malondialdehyde (MDA) in lung tissues, serum interleukin (IL)-6, and IL-1&amp;beta; levels were determined using the enzyme-linked immunosorbent assays. Pulmonary tissue specimens were examined histologically using hematoxylin-eosin and Masson trichrome staining. Inducible nitric oxide synthase (iNOS) activity was determined using immunohistochemical methods. Hesperetin administration reduced TNF&amp;alpha; and iNOS activity, serum IL-1&amp;beta;, IL-6, MDA, and wet/dry weight ratio in pulmonary tissue to improve pulmonary function. Our results showed that administration of hesperetin prevents activation of local inflammatory mediators, thereby obstructing the proinflammatory cytokine cascade and tissue injury.

Multi-Etiological Abortion due to Campylobacter spp. and Chlamydia abortus in a Sheep

In this case, multi-etiologic abortion due to Campylobacter spp. and Chlamydia abortus was determined in an aborted sheep fetus sent to Konya Veterinary Control Institute (KVKE) from a sheep farm operating in Aksaray province in 2020. The presence of Campylobacter spp. was determined by the bacterial isolation method. C.abortus was identified by qPCR and immunohistochemistry (IHC) methods. This study should be published in order to emphasize that in the fight against sheep abortions, multi-etiologic abortions involving multiple agents should be taken into consideration in addition to a single etiologic agent, and a multidisciplinary approach should be used in the diagnosis to identify the abortion agents.

Optimal melanin removal methods for HE staining, immunohistochemistry and molecular detection

Objective: To seek the optimal melanin-removal method for hematoxylin and eosin (HE) staining, immunohistochemistry and molecular detection. Methods: Thirty-eight paraffin tissue samples of malignant melanoma diagnosed at the Fujian Cancer Hospital, Fuzhou, China between January 2018 and March 2022 were collected and used to make a tissue microarray. Melanin in these cases was removed using warm hydrogen peroxide, double oxidation depigmentation, modified potassium permanganate-oxalic acid or trichloroisocyanuric acid, followed by HE staining. The cases were divided into two cohorts: one was subject to the one of the above four methods to remove melanin first, followed by immunohistochemistry (SOX-10, Ki-67, HMB45 and Melan A), while the other was subject to immunohistochemical staining first and then a melanin removal. Following that, seventeen melanin-rich paraffin tissue samples were collected and depigmented using the methods described above. DNA extraction was then done, followed by assessments of DNA content and quality. Moreover, the completeness of melanin removal, the effect on HE and immunohistochemical staining, and the quality of DNA were compared between the depigmented methods. Results: Regarding the effectiveness of melanin removal, the modified potassium permanganate-oxalic acid and the warm hydrogen peroxide methods were the most effective, and both showed residual melanin in only 5.26% (2/38) of the cases. The trichloroisocyanuric acid method showed residual melanin in 10.53% (4/38) of the cases. The worst was the double oxidation depigmentation method, which showed pigment residue in 15.79% (6/38) of the cases. For HE staining, the percentage of good staining with the warm hydrogen peroxide method was 92.11%, higher than the other three methods. For immunohistochemical staining, the mean staining scores of immunohistochemistry first followed by melanin removal with modified potassium permanganate-oxalic acid, double oxidation and trichloroisocyanuric acid were 20.84, 26.63 and 35.02, respectively. These immunohistochemical staining scores were higher than those of melanin removal first followed by immunohistochemistry (8.70, 15.41 and 21.22, respectively). The mean staining score of melanin removal by warm hydrogen peroxide method followed by immunohistochemistry was 33.57, superior to that of immunohistochemistry followed by the melanin removal (19.96). Moreover, the staining scores of HMB45, MelanA and Ki-67 with immunohistochemical staining followed by trichloroisocyanuric acid method were 36.45, 33.79, and 36.24, respectively, while the staining score of SOX10 with melanin removal by warm hydrogen peroxide followed by immunohistochemistry was 34.39. The DNA was significantly degraded by modified potassium permanganate-oxalic acid, double oxidation depigmentation and trichloroisocyanuric acid, whereas the mean concentration of DNA extracted after melanin removal by hydrogen peroxide method was 59.59 μg/L, substantially higher than that of DNA extracted without melanin removal (30.3 μg/L, P=0.001). The A260/A280 of DNA extracted after melanin removal by hydrogen peroxide was between 1.8 and 2.0 in all cases, and the A260/A230 was above 2.0 in sixteen cases, suggesting high purity of DNA. However, the DNA extracted without removing the melanin showed poor purity, with A260/A280 below 1.8 in eight cases and A260/A230 below 2.0 in sixteen cases. Conclusions: Warm hydrogen peroxide showed the least melanin residue, superior HE staining and a minimal effect on DNA purity/quality compared to the other three methods. It thus appears most suitable for PCR, NGS and other molecular detection. Melanin removal with trichloroisocyanuric acid after immunohistochemical staining has the least melanin residual, and thus could be the most convenient and efficient. However, it is noted that the efficacy of the same depigmentation method varies with different antibodies. Therefore, the optimal depigmentation method should be selected based on the specific markers of interest.

Histochemical and immunohistochemical investigation of the number and localization of mast cells in the feline tongue.

This is the first study to describe the subtypes, number and distribution of mast cells (MC) in cat tongue by histochemical and immunohistochemical methods. Six male adult felines' tongue tissue samples consist of the study's material. Samples were fixed in 10% formaldehyde. MC number and distribution in the feline tongue were assessed using toluidine blue. Also, sections taken from blocks were stained in alcian blue/safranin O (AB/SO) combined dyes to determine the MC subtypes. The Streptavidin biotin complex method using anti-chymase and anti-tryptase primary antibodies was used for immunohistochemistry. Metachromatic MCs were mainly observed in the lamina propria close to the multilayered keratinized stratified squamous epithelium. The high number of MCs in this region may be because the dorsal surface of the tongue plays an essential role in the defence system of tongue tissue and, thus, of the body as a whole. Additionally, the number of MCs stained with AB (+) (1.7 ± 0.08) in the feline tongue was statistically higher than those with SO (+) (0.18 ± 0.02). This might be interpreted as an indication that MC heterogeneity may be due not only to their staining properties but also to their localization. It is also conceivable that the high histamine content may be a factor in this. Tryptase-positive MCs were found in the loose connective tissue around blood vessels, between the glands, as solitary cells, or in groups of several cells. Chymase-positive MCs were observed more individually rather than in groups. Moreover, chymase-positive MCs were detected to be located in the filiform papillae subepithelial and in the blood vessels' immediate vicinity. Animals often lick themselves to clean themselves and promote healing. For this reason, it is very important to protect the tongue, which is in direct contact with the external environment, against foreign agents. Considering both the functional and protective properties of the tongue, we concluded that MCs may play a role in oral cavity immunity and protective effect.

Exploring GZMK as a prognostic marker and predictor of immunotherapy response in breast cancer: unveiling novel insights into treatment outcomes

BackgroundGranzyme K (GZMK) is a crucial mediator released by immune cells to eliminate tumor cells, playing significant roles in inflammation and tumorigenesis. Despite its importance, the specific role of GZMK in breast cancer and its mechanisms are not well understood.MethodsWe utilized data from the TCGA and GEO databases and employed a range of analytical methods including GO, KEGG, GSEA, ssGSEA, and PPI to investigate the impact of GZMK on breast cancer. In vitro studies, including RT-qPCR, CCK-8 assay, cell cycle experiments, apoptosis assays, Celigo scratch assays, Transwell assays, and immunohistochemical methods, were conducted to validate the effects of GZMK on breast cancer cells. Additionally, Cox regression analysis integrating TCGA and our clinical data was used to develop an overall survival (OS) prediction model.ResultsAnalysis of clinical pathological features revealed significant correlations between GZMK expression and lymph node staging, differentiation grade, and molecular breast cancer subtypes. High GZMK expression was associated with improved OS, progression-free survival (PFS), and recurrence-free survival (RFS), as confirmed by multifactorial Cox regression analysis. Functional and pathway enrichment analyses of genes positively correlated with GZMK highlighted involvement in lymphocyte differentiation, T cell differentiation, and T cell receptor signaling pathways. A robust association between GZMK expression and T cell presence was noted in the breast cancer tumor microenvironment (TME), with strong correlations with ESTIMATEScore (Cor = 0.743, P < 0.001), ImmuneScore (Cor = 0.802, P < 0.001), and StromalScore (Cor = 0.516, P < 0.001). GZMK also showed significant correlations with immune checkpoint molecules, including CTLA4 (Cor = 0.856, P < 0.001), PD-1 (Cor = 0.82, P < 0.001), PD-L1 (Cor = 0.56, P < 0.001), CD48 (Cor = 0.75, P < 0.001), and CCR7 (Cor = 0.856, P < 0.001). Studies indicated that high GZMK expression enhances patient responsiveness to immunotherapy, with higher levels observed in responsive patients compared to non-responsive ones. In vitro experiments confirmed that GZMK promotes cell proliferation, cell division, apoptosis, cell migration, and invasiveness (P < 0.05).ConclusionOur study provides insights into the differential expression of GZMK in breast cancer and its potential mechanisms in breast cancer pathogenesis. Elevated GZMK expression is associated with improved OS and RFS, suggesting its potential as a prognostic marker for breast cancer survival and as a predictor of the efficacy of immunotherapy.

YY1 mediated DCUN1D5 transcriptional activation promotes triple-negative breast cancer progression by targeting FN1/PI3K/AKT pathway

Triple-negative breast cancer (TNBC) is more aggressive and has a higher metastasis rate compared with other subtypes of breast cancer. Due to the lack of drug-targetable receptors, chemotherapy is now the only available systemic treatment for TNBC. However, some patients might still develop drug resistance and have poor prognosis. Therefore, novel molecular biomarkers and new treatment targets are urgently needed for patients with TNBC. To provide molecular insights into TNBC progression, we investigated the function and the underlying mechanism of Defective in cullin neddylation 1 domain containing 5 (DCUN1D5) in the regulation of TNBC. By TCGA dataset and surgical specimens with immunohistochemical (IHC) staining method, DCUN1D5 was identified to be significantly upregulated in TNBC tumor tissues and negatively associated with prognosis. A series of in vitro and in vivo experiments were performed to confirm the oncogenic role of DCUN1D5 in TNBC. Overexpression of FN1 or PI3K/AKT activator IGF-1 could restore the proliferative and invasive ability induced by DCUN1D5 knockdown and DCUN1D5 could act as a novel transcriptional target of transcription factor Yin Yang 1 (YY1). In conclusion, YY1-enhanced DCUN1D5 expression could promote TNBC progression by FN1/PI3K/AKT pathway and DCUN1D5 might be a potential prognostic biomarker and therapeutic target for TNBC treatment.

Differential expression of WNT5A long and short isoforms in non-muscle-invasive bladder urothelial carcinoma.

Wnt ligands belong to a family of secreted glycoproteins in which binding to a range of receptors/co-receptors activates several intracellular pathways. WNT5A, a member of the Wnt family, is classified as a non-canonical Wnt whose activation triggers planar cell polarity (PCP) and Ca+2 downstream pathways. Aberrant expression of WNT5A has been shown to play both protective and harmful roles in an array of conditions, such as inflammatory disease and cancer. In the present study, using histological, immunohistochemical, and molecular methods, we investigated the expression of two isoforms of WNT5A, WNT5A-Short (WNT5A-S) and WNT5A-Long (WNT5A-L) in bladder urothelial carcinoma (UC). Three UC cell lines (RT4, J82, and T24), as well as a normal urothelial cell line, and formalin-fixed, paraffin-embedded (FFPE) transurethral resection (TUR) tissue samples from 17 patients diagnosed with UC were included in the study. WNT5A-L was the predominantly expressed isoform in urothelial cells, although WNT5A-S was also detectable. Further, although no statistically significant difference was found between the percentage of WNT5A-S transcripts in low-grade versus high-grade tumors, we did find a difference between the percentage of WNT5A-S transcripts found in non-invasion versus invasion of the lamina propria, subgroups of non-muscle-invasive tumors. In conclusion, both WNT5A-S and WNT5A-L isoforms are expressed in UC, and the percentage of their expression levels suggests that a higher proportion of WNT5A-S transcription may be associated with lamina propria invasion, a process preceding muscle invasion.

Clinical Impact of Connexin 43 Deregulation on Myocardial Infraction.

Coronary artery disease (CAD) is a major and multifaceted health problem but also the first cause of death in modern Western societies. Furthermore, myocardial infarction (MI) constitutes a challenge for analysis in the field of molecular mechanisms, early diagnosis and therapeutic approaches, as its incidence increases every year worldwide. Concerning the histopathological diagnosis in the corresponding cases, a variety of immunohistochemistry (IHC) markers and methods are available to support conventional histology diagnosis. Immunohistochemistry techniques are effective for use in forensic pathology, expanding the limits of differential diagnoses in borderline cases, as they can be applied to tissue samples fixed in formalin and embedded in paraffin. The purpose of the current review was to explore the role of connexin 43 (gene locus: 6q22.31) as a reliable biomarker of myocardial disease/infarction and its impact on MI pathology. A systematic review of the literature was carried out based on the international database PubMed. The majority of medical data referred to articles published after the year 2020, whereas specific references of great importance and value were also included. The following keywords were used: coronary, artery, myocardial, infarction, connexin and immunohistochemistry. A pool of 38 significant articles focused on the mechanisms and novel experimental biomarkers was selected for the present study at the basis of combining molecular knowledge with new clinical features in CAD, and MI histodiagnosis. The role of connexin 43 - as a significant gap junction intermediate protein - in MI pathology, clinical symptoms and prognosis is critical because its dysfunction is involved in myocardial conduction and the onset of ventricular arrhythmias due to a crucial interruption of the intra-cardiomyocyte's conjunction.

Employing semi-quantitative methods to classify breast cancer markers (ER, PR, HER2, Ki67) expression, using the RT-qPCR based APIS Breast Cancer Subtyping Kit.

e13091 Background: Accurate determination of breast cancer (BC) biomarker status is crucial for guiding patient management decisions. Histopathologists commonly use semi-quantitative scoring systems to convert subjective observations of immunohistochemistry (IHC) marker expression into quantitative data, allowing a more detailed categorisation of marker status. The APIS Breast Cancer Subtyping Kit (BCSK) is an in vitro diagnostic test, detecting the relative expression of seven mRNA target genes ( ESR1, PGR, ERBB2, MKI67, CCNA2, PCNA, and KIF23) from invasive BC tissue. The APIS BCSK reports a positive/negative result for each biomarker alongside a molecular classification. Here, we demonstrate that by implementing additional RNA expression cut-off values, it is possible to generate a semi-quantitative result by further stratifying target expression into negative, low, medium, and high status using the APIS BCSK. Methods: 368 formalin-fixed paraffin-embedded (FFPE) samples of BC cases were used, obtained by core needle biopsy or resection. IHC scores were correlated with copy numbers as determined by an independently validated digital PCR (dPCR) assay and were used to set copy number cut-off values corresponding to IHC classification. The generated dPCR cut-off values were used to evaluate the corresponding ∆Ct values (RNA expression results derived from the APIS BCSK). For each target, three ΔCt cut-offs were established, facilitating classification into negative, low, medium, and high expression. Results: The semi-quantitative approach successfully classified target expression levels ( ESR1, PGR, ERBB2, MKI67). Overlap in ΔCt values was observed in central IHC categories for all targets, likely due to tumor heterogeneity and differing measurement methods for IHC and RT-qPCR. Despite these discrepancies, the derived cut-off values define a semi-quantitative scale for each target (Table). Conclusions: This study has established a methodology that utilises target copy number values and IHC protein quantification to establish ΔCt cut-off points. For each target we have defined four distinct groups as negative, low, medium, and high expression. This approach provides a semi-quantitative scale for evaluating each target with the APIS BCSK and serves as a valuable tool for classifying and understanding target expression levels. [Table: see text]