Mast Cell Markers Research Articles

P0129 Distinct involvement of plasma cells and monocytes/macrophages in ileal and colonic ulcer formation in patients with Crohn's disease

Abstract Background Despite its interest for the development of personalised medicine, the pathophysiological distinctions between ileal and colonic Crohn’s disease (CD) have been poorly characterised. This study aims to search for immunological differences between ileal and colonic CD. Methods We reanalysed our proteomic dataset1 to extract non-exploited immunological information. Using mass spectrometry-based proteomics, we previously compared the proteome of ulcer edges and adjacent normal mucosa (paired design) in the ileum (n=16 biopsies) and colon (n=16 biopsies) of 16 CD patients (Figure 1). A total of 4,428 and 5,204 proteins were screened in the ileum and colon, respectively. To identify proteins specific to particular immune cell populations, we searched in the Human Proteome Atlas and literature. Results We identified markers specific to plasma cells (IgA1, IgM, IgG1, IgG2, IgG3, IgG4, immunoglobulin J chain [IGJ], and immunoglobulin lambda-like polypeptide 5 [IGLL5]), monocytes/macrophages (cluster of differentiation 14 [CD14]), and mast cells (chymase [CMA1], mast cell carboxypeptidase A [CPA3], and tryptase alpha/beta-1 [TPSAB1]). No specific markers were identified for the other immune cells. The number of increased plasma cell markers (ulcer edges vs adjacent normal mucosa) was higher in the ileum (IgA1, IgM, IgG1, IgG2, IgG3, IGLL5, and IGJ) than in the colon (IgG3) (Figure 2). Of note, immune exclusion markers (IgM, IgA1, IGJ) were increased in the ileum, but not the colon (ulcer edges vs adjacent normal mucosa). The marker of monocytes/macrophages CD14 was increased in the colon, but not in the ileum (ulcer edges vs adjacent normal mucosa) (Figure 2). Mast cells markers (CMA1, CPA3, and TPSAB1) showed no significant change in the ileum or colon (ulcer edges vs adjacent normal mucosa) (Figure 2). Conclusion Plasma cells and monocytes/macrophages may play distinct roles in the formation of ileal and colonic ulcers in patients with CD, suggesting further investigations.

Role of mast cell in locally advanced rectal cancer treated with neoadjuvant chemoradiotherapy

The pro-tumor effects of mast cell (MC) in the tumor microenvironment (TME) are becoming increasingly clear. Recently, MC were shown to contribute to tumor malignancy by supporting the migration of tumor-associated macrophages (TAMs), suggesting a relationship with tumor immunity. In the current study, we aimed to examine the correlation between MC infiltration and neoadjuvant chemoradiotherapy (nCRT) response for locally advanced rectal cancer (LARC). Ninety-five LARC patients who recieved nCRT were enrolled in this study. Protein levels of the MC marker tryptase and TAM marker CD206 were evaluated with immunohistochemistry (IHC). The correlation between MC infiltration and prognostic factors was evaluated. The effects of MCs on the malignant potential were examined using in vitro proliferation and invasion assays with a colorectal cancer (CRC) cell line (HCT-116). Following nCRT, 31.6% of resected LARC patient specimens were positive for MC infiltration by tryptase IHC analysis. MC infiltration was significantly correlated with nCRT response. The 5-year disease-free survival (DFS) rate was significantly lower in the MC-positive group compared with the MC-negative group (52.3% vs. 76.8%). Univariate and multivariate analyses revealed that MC infiltration was the independent prognostic indicator for DFS. MC infiltration was significantly correlated with CD206 expression, and therefore TAMs. In vitro experiments suggested that tumor activated mast cells could promote CRC cell malignant behavior via production of macrophage inhibitory factor. MC infiltration in LARC patients was positively correlated with TAM infiltration and resistance to nCRT, and was also an independent poor prognostic indicator.

Interaction Between Sclerostin and Mast Cells in Fibro-Osseous Lesions of the Jaws.

To assess the sclerostin, β-catenin, and tryptase expression in fibro-osseous lesions (FOL) of the jaws. Immunohistochemistry analysis was performed for these proteins on FOL and non-lesional bone. The sclerostin-positive cells were scored from 0 (no expression) to 3 (high expression). We analyzed 46 FOL biopsies and selected 38 patients. Categorization showed 15 fibrous dysplasia (FD), eight juvenile trabecular ossifying fibroma (JTOF), two psammomatoid ossifying fibroma (PsOF), and 13 FOL. We found more sclerostin-positive cells in fibrous tissue than in bone, showing a phenotype like mast cells with strong dot-cytoplasmic positivity. The analysis of sclerostin-positive cell lesions (scored as 2 and 3) showed also tryptase positivity in 80.9% of 21 biopsies. β-catenin was diffusely expressed on the fibrous component, mostly with cytosol staining. Non-lesional bone showed sclerostin expression in medullary spaces and a few osteocytes. Sclerostin-positive cells are mostly found in the fibrous tissue of FOL, and the tryptase mast cell marker was present in most of the lesions that were positive for sclerostin.

Immunohistochemical analysis and distribution of epithelial mast cells in the rat larynx and trachea.

Mast cells (MCs) in rat airways have been classified into two subtypes: epithelial MCs and connective tissue MCs (CTMCs). However, the immunohistochemical characteristics, cellular morphology, and distribution of epithelial MCs in the upper airways remain unclear. The present study investigated the morphological characteristics and distribution of epithelial MCs using 5-hydroxytryptamine (5-HT) and other immunohistochemical markers in sectioned or whole-mount preparations of the rat larynx and trachea. A double immunofluorescence analysis revealed the colocalization of 5-HT immunoreactivity with c-kit, a stem cell factor receptor commonly used as a MC marker, in both epithelial MCs and CTMCs. Dopa decarboxylase, an enzyme involved in 5-HT synthesis, was detected in both subtypes, suggesting their ability to synthesize and release 5-HT. Tryptase and histidine decarboxylase (a biosynthetic enzyme of histamine), which are well-known mediators of MCs, were exclusive to CTMCs. Epithelial MCs were pleomorphic with long cytoplasmic processes, whereas CTMCs were round and lacked cytoplasmic processes. The density of epithelial MCs was significantly higher in the glottis and cranial part of the trachea than in the epiglottis and other parts of the trachea. The present results showed that the morphology and immunohistochemical characteristics of epithelial MCs were different from those of CTMCs in the rat larynx and trachea, and variform epithelial MCs were predominantly located at the entrance of the upper airways. Epithelial MCs may release 5-HT to regulate innate immune responses by modulating epithelial cell functions at the entrance gate of the upper airways.

Ligand-independent function of β2-adrenergic receptor affects IgE-mediated Ca2+ influx in mast cells

BackgroundMast cells are key effector cells that elicit immunoglobulin E (IgE)-mediated allergic inflammations. Allergen cross-linking of IgE bound to the high-affinity IgE receptor, FcεRI, on mast cells triggers signaling cascades that activate signal proteins and evoke extracellular Ca2+ influx, which are crucial for cytokine production. The β2-adrenergic receptor (Adrb2) on mast cells negatively regulates FcεRI signaling, as demonstrated by the inhibition of IgE/antigen (Ag)-induced activation by Adrb2 agonists. ObjectiveAlthough β2-adrenergic-related reagents are known to influence mast cell functions, the specific intrinsic role of Adrb2 in these cells is not fully understood, potentially because of off-target effects. In this study, the additional roles of Adrb2 in mast cells were investigated, specifically the involvement of Adrb2 in FcεRI signaling, using Adrb2−/− mice. MethodsAdrb2−/− mice were used to investigate the roles of Adrb2 in mast cells by examining bone marrow-derived mast cells (BMMCs) for surface expression of mast cell markers, granule numbers, and gene expression of mast cell proteases. Cytokine production, Ca2+ influx, and nuclear factor of activated T cells (NFAT) nuclear translocation were measured in Adrb2−/− and Adrb2+/+ BMMCs upon IgE/Ag stimulation. ResultsAdrb2−/− did not affect the generation of BMMCs, their surface expression of mast cell markers, granule numbers, or gene expression of mast cell proteases, indicating that the absence of Adrb2 had no adverse effect on mast cell development. However, Adrb2−/− BMMCs exhibited reduced tumor necrosis factor α (TNFα) production and diminished Ca2⁺ influx upon IgE/Ag stimulation, which correlated with decreased NFAT translocation. Restoration of Adrb2 in Adrb2−/− BMMCs rescued cytokine production. Notably, FcεRI-mediated phosphorylation of the phospholipase PLCγ1 and mitogen-activated protein kinases (MAPKs) remained unchanged in the absence of Adrb2. ConclusionThese results suggest that Adrb2 has a novel ligand-independent function, increasing Ca2+ entry in mast cells when stimulated with IgE/Ag.

(026) CORRELATIONS BETWEEN IMMUNOHISTOCHEMICAL AND PAIN-RELATED ASSESSMENTS IN NEUROPROLIFERATIVE VESTIBULODYNIA: AN UPDATE IN 102 PATIENTS

Abstract Introduction Neuroproliferative vestibulodynia (NPV) is confirmed by immunohistochemical (IHC) staining of excised vestibular specimens. Compared to controls, specimens from NPV patients contain >8 mast cells per high-power field (HPF) and increased density of nerve endings. A 2024 publication of 65 patients with IHC confirmed diagnosis of NPV assessed correlations between patient-reported pain-related measures and computer-assisted histometry of excised vestibulectomy specimen IHC stained with CD117 (marker for mast cells) and PGP9.5 (marker for nerves). There were no statistically significant correlations between mean fractional area of immunopositive staining and multiple pain-related assessment scores; we hypothesized that there was a threshold of mast cell and nerve accumulation, above which there was no increase in pain scores. Objective To determine whether reviewing additional charts of NPV patients would result in statistically significant correlations between immunopositive staining and pain-related assessments. Methods Charts of patients who underwent complete vestibulectomy between January 2023 and February 2024 were reviewed and their data added to the original set from the 2024 publication. Tissue sections from the 1:00-11:00 and/or 12:00 regions of the vestibule were examined. High-resolution images were captured using a microscope at 100x and 200x magnification. At least 2 photomicrographs/magnification were taken for each tissue section, including epithelial basement membrane and adjacent subepithelium, representative of the majority of immunostained tissue (Fig. 1). Fractional area of positive immunostaining of all photomicrographs was determined using computer-assisted histometry by ImageJ. Mean fractional area was determined from 3 measurements of each photomicrograph. Correlations were made between density of IHC staining and the following assessments: Female Sexual Function Index (FSFI) pain domain, Short Form McGill Pain Questionnaire (SF-MPQ), cotton-tipped swab testing (1:00–11:00 and 12:00 vestibule), and patient global impression of improvement (PGI-I). Data were analyzed using Spearman correlations and the Mann–Whitney test. Results 37 charts of NPV patients were added to the original 65, resulting in 102 patients reviewed who underwent vestibulectomy between November 2019 and February 2024, for a total of 1091 photomicrographs analyzed. In the previous study, there were no statistically significant correlations between CD117- or PGP9.5-immunopositive mean fractional area and the FSFI pain domain, SF-MPQ scores, cotton-tipped swab test pain score, or PGI-I. Adding IHC data from the additional 37 patients confirmed no statistically significant correlations between CD117 or PGP9.5 immunopositive mean fractional area and the multiple pain assessments (Fig. 2). Conclusions The lack of correlations between immunopositive staining and multiple clinical assessment scores in patients with severe vestibular pain suggests a non-linear threshold for mast cell accumulation and nerve proliferation that results in pain symptoms for NPV patients. Adding data from an additional 37 patients for a total of 102 patients did not change this observation. We hypothesize that once a theoretical threshold for accumulation of mast cells and nerves is reached, additional accumulation does not result in worsened clinical symptoms. This can account for the variability in pain scores among NPV patients and explain why a complete vestibulectomy may be the appropriate treatment no matter the results of the multiple pain assessments once pain is experienced from clinically suspected NPV. Disclosure No.

(024) FIRST TIME ASSESSMENT OF HYMENAL TISSUE IMMUNOHISTOCHEMICAL STAINING FROM PATIENTS WITH NEUROPROLIFERATIVE VESTIBULODYNIA

Abstract Introduction Embryologically, the hymen forms at the junction of the endodermal urogenital sinus and the mesodermal primitive vagina. During fetal development the hymen becomes surrounded by the endodermal vestibule distally and the endodermal vagina proximally (Fig. 1) and therefore, is likely to also be of endodermal origin. Neuroproliferative vestibulodynia (NPV) is a provoked vestibular pain disorder associated with excess immunohistochemical (IHC) staining with CD117, a mast cell marker, throughout the entire 1:00-11:00 o’clock as well as 12:00 o’clock vestibular regions. Although the hymen is surrounded by endodermal tissue, to the best of our knowledge, it has not been established whether hymenal tissue of patients with NPV also would have excess IHC staining consistent with mast cells. This is clinically relevant, as some patients have persistent post-operative pain in the region of the unexcised hymen when vestibulectomy is not complete. Objective We wished to assess, for the first time, CD 117 immunostaining in the hymenal tissue of NPV patients and compare the IHC data with that of excised vestibular tissue. Methods Medical records from the patient cohort who underwent complete vestibulectomy between April 1, 2024 and May 31, 2024 were reviewed. For this patient cohort, three separate specimens were sent to pathology for analysis: i) 1:00-11:00 vestibule, ii) 12:00 vestibule and iii) hymenal tissue that had been sharply separated from the 1:00-11:00 excised vestibular specimen (Fig. 2). Slides of IHC-stained vestibular tissue specimens from the 1:00-11:00 and 12:00 vestibule as well as hymen were examined, and images captured using a 3.5 megapixel digital camera attached to a light microscope at 100× magnification. A minimum of 2 photomicrographs were taken for each stained slide, identifying representative regions of the epithelial basement membrane and adjacent subepithelium. A total of 48 photomicrographs of slides stained for CD117 were assessed. Each photomicrograph was assigned a unique identification number. Manual counting of CD117-immunopositive stained cells was performed on these photomicrographs for comparison to Bornstein’s data that >8 mast cells per high power field confirmed the diagnosis of NPV. Results A total of 8 NPV patients (mean age 28 years, range 21–42) were included in the chart review. All had symptoms consistent with NPV, and all had other conditions associated with aberrant mast cell activity. Both 1:00– 11:00 and 12:00 vestibular tissue specimens had >8 mast cells per high power field. The hymenal tissue in all 8 patients also contained >8 mast cells per field at the same magnification (Fig. 3). Median manual mast cell count values were similar in 1:00-11:00 o’clock and 12:00 vestibular regions and the hymen: 45 (range 21-67), 39 (range 19-63) and 43 (range 18-68) mast cells per 100× field, respectively. Conclusions NPV is a field disease of the endoderm, which based on these data, includes all hymenal tissue. To maximize success from complete vestibulectomy for NPV, excision of all hymenal tissue should be considered. Disclosure No.

Granzyme B Expression in Conjunctiva of Patients with Pterygium.

Pterygium is often associated with chronic ultraviolet (UV) radiation exposure and characterized by the overgrowth of conjunctiva and extracellular matrix (ECM) remodeling. Notably, several studies in the skin have demonstrated that chronic UV radiation can upregulate Granzyme B (GrB) expression and increase ECM degradation. The aim of this study was to compare GrB expression between pterygium and healthy controls and to further link this GrB expression to mast cells. Post-mortem pterygium tissues and conjunctival tissues from age-matched controls were used to assess GrB expression via immunofluorescence and microscopy. We found a significantly higher density of GrB+ cells from pterygium specimens compared to healthy controls. Furthermore, many of the GrB+ cells in pterygium specimens co-expressed tryptase, a mast cell marker. These findings suggest a role for conjunctival mast cell-secreted GrB in the pathogenesis of pterygium and highlight GrB as a possible therapeutic target in delaying or halting pterygium progression.

Machine learning-driven mast cell gene signatures for prognostic and therapeutic prediction in prostate cancer

BackgroundThe role of Mast cells has not been thoroughly explored in the context of prostate cancer's (PCA) unpredictable prognosis and mixed immunotherapy outcomes. Our research aims to employs a comprehensive computational methodology to evaluate Mast cell marker gene signatures (MCMGS) derived from a global cohort of 1091 PCA patients. This approach is designed to identify a robust biomarker to assist in prognosis and predicting responses to immunotherapy. MethodsThis study initially identified mast cell-associated biomarkers from prostate adenocarcinoma (PRAD) patients across six international cohorts. We employed a variety of machine learning techniques, including Random Forest, Support Vector Machine (SVM), Lasso regression, and the Cox Proportional Hazards Model, to develop an effective MCMGS from candidate genes. Subsequently, an immunological assessment of MCMGS was conducted to provide new insights into the evaluation of immunotherapy responses and prognostic assessments. Additionally, we utilized Gene Set Enrichment Analysis (GSEA) and pathway analysis to explore the biological pathways and mechanisms associated with MCMGS. ResultsMCMGS incorporated 13 marker genes and was successful in segregating patients into distinct high- and low-risk categories. Prognostic efficacy was confirmed by survival analysis incorporating MCMGS scores, alongside clinical parameters such as age, T stage, and Gleason scores. High MCMGS scores were correlated with upregulated pathways in fatty acid metabolism and β-alanine metabolism, while low scores correlated with DNA repair mechanisms, homologous recombination, and cell cycle progression. Patients classified as low-risk displayed increased sensitivity to drugs, indicating the utility of MCMGS in forecasting responses to immune checkpoint inhibitors. ConclusionThe combination of MCMGS with a robust machine learning methodology demonstrates considerable promise in guiding personalized risk stratification and informing therapeutic decisions for patients with PCA.

(089) CORRELATIONS BETWEEN IMMUNOHISTOCHEMICAL STAINING AND PAIN-RELATED ASSESSMENTS IN PATIENTS WITH NEUROPROLIFERATIVE VESTIBULODYNIA

Abstract Introduction Mast cell accumulation and nerve proliferation has been hypothesized to lead to pain symptoms for patients with neuroproliferative vestibulodynia (NPV). A clinically suspected diagnosis of NPV can be confirmed by examining immunohistochemical (IHC) staining of excised vestibular specimens. Compared to controls, specimens from NPV patients have been found to contain >8 mast cells per high-power field (HPF) and increased density of nerves. Objective To assess for correlations between patient reported pain-related measures and computer-assisted histometry of excised vestibulectomy specimen IHC staining with CD117 (marker for mast cells) and PGP9.5 (marker for nerves) in a cohort of histopathologically confirmed NPV patients. Methods Patients (n=65) with lifelong or acquired NPV undergoing vestibulectomy had IHC-staining of excised vestibular tissue. Tissue sections from the 1:00-11:00 and/or 12:00 regions of the vestibule were examined and high-resolution images were captured using a microscope at 100x and 200x magnification. A minimum of 2 photomicrographs per magnification were taken for each tissue section that included epithelial basement membrane and adjacent subepithelium representative of the majority of immunostained tissue. Fractional area of positive immunostaining of all photomicrographs was determined using computer-assisted histometry by ImageJ. Mean fractional area was determined from 3 separate measurements of each photomicrograph. Correlations were made between density of IHC staining and the following assessments: pain domain of the Female Sexual Function Index (FSFI), the Short Form McGill Pain Questionnaire (SF-MPQ), and cotton-tipped swab testing. We also examined correlations with the Patient Global Impression of Improvement (PGI-I), a treatment response index and indirect assessment of pain. Data were analyzed using Spearman correlations and the Mann-Whitney test. Results The study cohort had a median age of 26 (IQR = 23-31) years and experienced symptoms for a median duration of 11 (IQR = 6-15) years. While all patients reported high levels of pain (median FSFI pain domain score = 0.0), there were no correlations between CD117- or PGP9.5-immunopositive mean fractional area and FSFI pain domain or SF-MPQ scores. There was no difference in immunopositive mean fractional area between lifelong and acquired NPV. A positive correlation between fractional area of CD117 immunostaining in the 1:00-11:00 region of the vestibule and cotton-tipped swab test pain score approached but did not reach statistical significance (r = 0.24, p = 0.063). No correlations were observed between immunostaining and PGI-I or cotton-tipped swab testing scores. Likewise, SF-MPQ domain scores had no correlation to PGI-I or cotton-tipped swab testing. Median cotton-tipped swab testing pain scores were significantly higher (p < 0.001) in patients with lifelong NPV (7.6) compared to those with acquired NPV (5.7). Conclusions Our findings suggest that the high levels of pain experienced by NPV patients over a prolonged period of time may be dissociated from the density of mast cells and nerves in vestibular tissue. We postulate that any potential threshold of mast cell accumulation and increased nerve density that results in symptoms of vestibulodynia has been exceeded in our patient cohort. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Dare Bioscience; Pfizer; Prometheus Laboratories; Sprout Pharmaceuticals.

(042) IMMUNOSTAINING FOR MARKERS OF MAST CELLS AND NERVES IN BOTH EXCISED VESTIBULAR AND ENDOMETRIOSIS IMPLANT TISSUES IN A SINGLE PATIENT

Abstract Introduction Neuroproliferative vestibulodynia (NPV) is a chronic genital pain condition characterized by severe vestibular allodynia and hyperalgesia. The clinically suspected diagnosis of NPV is confirmed in excised vestibular tissue by immunohistochemical staining of > 8 cells per 100x magnification positively immunostained for CD 117, a mast cell marker, and 10 times the area compared to controls positively immunostained for PGP 9.5, a neuronal marker, Endometriosis, on the other hand, is a chronic pelvic pain condition associated with growth of ectopic endometrial-like tissue outside the uterus. The clinically suspected diagnosis is confirmed in excised endometriosis tissue revealing, in some patients, endometrial type glands and/or stroma. In patients with NPV and endometriosis, it is hypothesized that excess mast cells release cytokines and growth factors that promote neuroproliferation in their respective tissues, resulting in chronic genital and pelvic pain symptoms, respectively. In a study involving 65 patients with pathologically confirmed NPV, 63% reported having additional conditions associated with aberrant mast cell activity, including endometriosis. The conditions of NPV and endometriosis may both be focal to the vestibule and pelvis, respectively, but both may also share pathologies of aberrant mast cell activity with other systemic disorders. Objective To perform immunostaining for markers of mast cells and nerves in excised vestibular and endometriosis implant tissues from a single patient. Methods Fixed, paraffin-embedded vestibular tissue specimens from the 1:00-11:00 and 12:00 vestibule, and from excised endometriosis fibroadipose tissue on the rectosigmoid colon, were sectioned and immunostained for CD 117 (C-kit) and PGP 9.5. The immunostained slides were examined and digital photomicrographs taken. A minimum of 2 photomicrographs were examined at 100x magnification for each stained slide, identifying representative regions of the excised tissue. Manual counting of CD117 and PGP 9.5 immunopositive profiles, cells/nerves per 100x magnification, was performed for each photomicrograph. Results A 23-year-old woman presented with a history marked by painful and prolonged menstrual periods, inability to insert tampons, and discomfort with both entry and deep thrusting during intercourse. After excluding other forms of vestibulodynia, she underwent complete 1:00-11:00 and 12:00 subepithelial vestibulectomy with vaginal advancement flap reconstruction. Post-operatively, she reported “very much better” symptoms, with a reduction of entrance dyspareunia, but continued to experience deep thrusting pain with penetrative sexual activity. She subsequently underwent endometriosis excision surgery with positive lesions identified in 12 locations in the pelvis and retroperitoneum. Tissue stained for CD117 and PGP9.5 analyzed by manual counting exhibited > 20 immunopositive cells and nerves per 100x microscopic field in the epithelial basement membrane and adjacent subepithelial regions of the 1:00-11:00 and 12:00 vestibule, as well as in the endometriosis fibroadipose tissue on the rectosigmoid colon. Conclusions We report for the first time, in a single patient diagnosed with both NPV and endometriosis, immunostaining findings of high densities of mast cells and nerves in their respective tissues. These findings reinforce the hypothesis that NPV and endometriosis are linked to a broader condition associated with aberrant mast cell activity. Disclosure No.

Diagnostic and prognostic significance of mast cell markers in HIV/AIDS: Current insights and future directions.

Human immunodeficiency virus (HIV) infection continues to pose significant global health challenges, necessitating advancements in diagnostic and prognostic approaches to optimize disease management. While primarily recognized for their roles in allergic responses, mast cells have emerged as potential markers with diagnostic and prognostic significance in the context of HIV/AIDS. This paper aims to synthesize current insights and delineate future directions regarding the utility of mast cell markers in diagnosing HIV infection, predicting disease progression, and guiding therapeutic strategies. Mast cells, equipped with distinct markers such as tryptase, chymase, carboxypeptidase A3, and c-kit/CD117 receptors, exhibit tissue-specific expression patterns that offer potential as diagnostic indicators for HIV infection. Understanding the dynamics of these markers in different tissues and body fluids holds promise for accurate HIV diagnosis, disease staging, and monitoring treatment responses. Moreover, the prognostic significance of mast cell markers in HIV/AIDS lies in their potential to predict disease progression, immune dysregulation, and clinical outcomes. The integration of mast cell markers into clinical applications offers promising avenues for refining diagnostic assays, patient monitoring protocols, and therapeutic strategies in HIV/AIDS. Future research directions involve the development of novel diagnostic tools and targeted therapies based on mast cell-specific markers, potentially revolutionizing clinical practice and enhancing patient care in the management of HIV/AIDS. Continued investigations into mast cell markers' diagnostic and prognostic implications hold immense potential to advance our understanding and improve outcomes in HIV/AIDS management.

(048) PREVALENCE OF CONDITIONS INVOLVING ABERRANT MAST CELL ACTIVITY MAY HELP IDENTIFY PATIENTS WITH NEUROPROLIFERATIVE VESTIBULODYNIA

Abstract Introduction Neuroproliferative vestibulodynia (NPV) is a provoked genital pain characterized by severe allodynia, hyperalgesia and entry dyspareunia. Bornstein et al. determined the parameters for diagnostic confirmation of NPV to be >8 cells positively immunostained for CD117 (mast cell marker) in a field viewed at 100× magnification. Since the cellular pathology of NPV is not visible during physical examination and lies underneath the vestibular epithelium, many patients with NPV have been examined by multiple clinicians and have undergone numerous unsuccessful diagnostic tests and treatments. We noted that patients with documented NPV by histomorphometric quantification of mast cells reported other concomitant medical conditions associated with aberrant mast cell activity. Thus, taking a detailed history of patients with suspected NPV for the presence of other mast cell related conditions may assist in the identification of NPV. Objective In patients with a histopathologically confirmed diagnosis of NPV, we wished to assess the prevalence of concomitant aberrant mast cell activity conditions. Methods An IRB exempt chart review of people with vulvas who underwent vestibulectomy between June 1, 2019 and October 31, 2023 was performed at our facility. Histories from these patients, all of whom had histopathologically confirmed NPV after observing >8 positive CD117 stained cells/high power field in the sub-epithelial stroma, were reviewed. Conditions of aberrant mast cell activity reported by patients as part of their history was noted. Focus was on the following eight conditions: asthma, chronic sinusitis, endometriosis, food allergy/intolerance, interstitial cystitis, irritable bowel syndrome, migraine, and postural orthostatic tachycardia syndrome. Results A total of 88 NPV patients were included in this chart review. While 34 (39%) reported no other aberrant mast cell activity, 54 (51%) had at least one concomitant condition. A total of 33 (38%), 13 (15%), 3 (6%), 1 (1%) and 2 (2%) had one, two, three, four and five other conditions involving aberrant mast cell activity, respectively. The most common conditions were migraine (n = 21; 24%), interstitial cystitis (n = 17; 19%) and endometriosis (n = 15; 17%), followed by food allergy/intolerance (n = 12; 14%), irritable bowel syndrome (n = 9; 10%), asthma (n = 9; 10%), chronic sinusitis (n = 5; 6%) and postural orthostatic tachycardia syndrome (n = 2; 2%). As these data are based on self-report during patient intake, and not on a complete assessment of all aberrant mast cell activity conditions, actual prevalence may be higher. There was no correlation between the number of concomitant conditions with aberrant mast cell activity reported and the density of CD117-immunopositive staining in the overall NPV cohort or within the subgroups with either lifelong or acquired NPV. Conclusions Patients with histopathologically confirmed NPV have a high prevalence of concomitant conditions associated with aberrant mast cell activity, possibly due to a genetic predisposition, making them more susceptible to mast cell conditions. While more studies are needed, NPV is likely part of a broader systemic collection of conditions associated with aberrant mast cell activity and may serve to help diagnose and direct treatment of NPV in the future. Disclosure No.

Immunohistochemical staining with CD117 and PGP9.5 of excised vestibular tissue from patients with neuroproliferative vestibulodynia.

Neuroproliferative vestibulodynia (NPV), a provoked genital pain characterized by severe allodynia and hyperalgesia, is confirmed in excised vestibular tissue by immunohistochemical staining (>8 CD117-positive immunostained cells/100× microscopic field) rather than by hematoxylin and eosin staining. In this study we sought to assess immunostaining of tissue samples obtained during vestibulectomy surgery and to correlate results with patient outcomes. Patients (n = 65) meeting criteria for NPV who underwent vestibulectomy during the period from June 2019 through December 2022 formed the study cohort. We performed assessment of pathology of vestibular tissues by use of immunohistochemical staining, including quantitation of mast cells by CD117 (mast cell marker) and nerve fibers by protein gene product (PGP) 9.5 (neuronal marker). We analyzed 725 photomicrographs of immunostained tissue sections (100× and 200×) by manual counting and computer-assisted histometry and correlated these data to clinical assessments. Outcomes included density of CD117 and PGP9.5 immunostaining in the 1:00-11:00 o'clock and 12:00 o'clock vestibular regions, and patient-reported outcomes assessing sexual function, pain, distress, and symptom improvement. All 65 NPV patients (median age 26 years), 45 with lifelong and 20 with acquired NPV, had severe pain documented by PROs and vulvoscopy and had >8 CD117-immunopositive cells/100× microscopic field. Median cell count values were similar in the 1:00-11:00 o'clock and 12:00 vestibular regions (28.5 and 29.5/100× field, respectively). Likewise, the marker) and nerve fibers by protein gene product (PGP) 9.5 (neuronal marker). We analyzed 725 photomicrographs of immunostained tissue sections (100× and 200×) by manual counting and computer-assisted histometry and correlated these data to clinical assessments. Outcomes included density of CD117 and PGP9.5 immunostaining in the 1:00-11:00 o'clock and 12:00 o'clock vestibular regions, and patient-reported outcomes assessing sexual function, pain, distress, and symptom improvement. All 65 NPV patients (median age 26 years), 45 with lifelong and 20 with acquired NPV, had severe pain documented by PROs and vulvoscopy and had >8 CD117-immunopositive cells/100× microscopic field. Median cell count values were similar in the 1:00-11:00 o'clock and 12:00 vestibular regions (28.5 and 29.5/100× field, respectively). Likewise, the median area of CD117 immunostaining was similar in both regions (0.69% and 0.73%). The median area of PGP9.5 immunostaining was 0.47% and 0.31% in these same regions. Pain scores determined with cotton-tipped swab testing were nominally higher in lifelong vs acquired NPV patients, reaching statistical significance in the 1:00-11:00 o'clock region (P < .001). The median score for the McGill Pain Questionnaire affective subscale dimension was also significantly higher in lifelong vs acquired NPV patients (P = .011). No correlations were observed between hematoxylin and eosin results and density of mast cells or neuronal markers. Of note, 63% of the patient cohort reported having additional conditions associated with aberrant mast cell activity. The pathology of NPV is primarily localized to the vestibular epithelial basement membrane and subepithelial stroma with no visible vulvoscopic findings, making clinical diagnosis challenging. Strengths of this study include the large number of tissues examined with what is to our knowledge the first-ever assessment of the 12:00 vestibule. Major limitations are specimens from a single timepoint within the disease state and lack of control tissues. Performing immunohistochemical staining of excised vestibular tissue with CD117 and PGP9.5 led to histometric confirmation of NPV, indications that NPV is a field disease involving all vestibular regions, validation for patients whose pain had been ignored and who had experienced negative psychosocial impact, and appreciation that such staining can advance knowledge.

Detecting Changes in Mast Cell Numbers Versus Activation in Human Disease: A Roadblock for Current Biomarkers?

The pathophysiology of mast cell (MC)-driven disorders is diverse, ranging from localized reactions to systemic disorders caused by abnormal accumulation and activation in multi-organ systems. Prompt and accurate diagnosis is critically important, both for informing treatment and objective assessment of treatment outcomes. As new therapeutics are being developed to deplete MCs or silence them (e.g., by engaging inhibitory receptors that block activation), new biomarkers are needed that can distinguish between MC activation vs. burden. Serum tryptase is the gold standard for assessing both mast cell burden and activation; however, commercial tryptase assays have limitations related to timing of release, lack of discernment between inactive (α) and active (β) forms of tryptase, and interpatient variability of baseline levels. Alternative approaches to measuring MC activation include urinary mast cell mediators, flow cytometry-based assays or gene expression profiling. Additional markers of MC activation are needed for use in clinical diagnostics, to help selection of treatment of MC diseases, and for assessing outcomes of therapy. We review the spectrum of disorders with known or suspected MC contribution, describe the utility and limitations of current MC markers and assays, and discuss the need for new markers that can differentiate between MC activation and burden.

Expression of CD25, mast cell markers and T-cell markers in eosinophilic esophagitis

While eosinophilic esophagitis (EOE) is defined by histologic presence of eosinophils, a few studies have established the presence of mast cells in EOE and even shown their correlation with symptom persistence despite resolution of eosinophils. Expression of aberrant mast cell markers CD25 and CD2 have not been studied in EOE. This study quantifies the number of hotspot cells per high power field expressing CKIT/CD117, tryptase, CD25, CD2 and CD3 by immunohistochemical stains in endoscopic esophageal biopsies of the following three cohorts: (1) established and histologically confirmed EOE, (2) suspected EOE with biopsies negative for eosinophils, and (3) no history of or suspicion for EOE with histologically unremarkable biopsies. In this study, mast cells were highlighted by CKIT and tryptase in EOE, and not seen in other clinically mimicking cases. There were also significantly higher densities of CD25 and pan-T-cell marker staining in EOE cases. These findings suggest an inflammatory cellular milieu in EOE, beyond just eosinophils, that can be demonstrated by immunohistochemistry, and that invite further study into the role that these cells may play in EOE.

Steroid responsiveness in alcohol-associated hepatitis is linked to glucocorticoid metabolism, mitochondrial repair, and heat shock proteins.

Alcohol-associated hepatitis (AH) is one of the clinical presentations of alcohol-associated liver disease. AH has poor prognosis, and corticosteroids remain the mainstay of drug therapy. However, ~40% of patients do not respond to this treatment, and the mechanisms underlying the altered response to corticosteroids are not understood. The current study aimed to identify changes in hepatic protein expression associated with responsiveness to corticosteroids and prognosis in patients with AH. Patients with AH were enrolled based on the National Institute on Alcohol Abuse and Alcoholism inclusion criteria for acute AH and further confirmed by a diagnostic liver biopsy. Proteomic analysis was conducted on liver samples acquired from patients with AH grouped as nonresponders (AH-NR, n = 7) and responders (AH-R, n = 14) to corticosteroids, and nonalcohol-associated liver disease controls (n = 10). The definition of responders was based on the clinical prognostic model, the Lille Score, where a score < 0.45 classified patients as AH-R and a score > 0.45 as AH-NR. Primary outcomes used to assess steroid response were Lille Score (eg, improved liver function) and survival at 24 weeks. Reduced levels of the glucocorticoid receptor and its transcriptional co-activator, glucocorticoid modulatory element-binding protein 2, were observed in the hepatic proteome of AH-NR versus AH-R. The corticosteroid metabolizing enzyme, 11-beta-hydroxysteroid dehydrogenase 1, was increased in AH-NR versus AH-R along with elevated mitochondrial DNA repair enzymes, while several proteins of the heat shock pathway were reduced. Analysis of differentially expressed proteins in AH-NR who survived 24 weeks relative to AH-NR nonsurvivors revealed several protein expression changes, including increased levels of acute phase proteins, elevated coagulation factors, and reduced mast cell markers. This study identified hepatic proteomic changes that may predict responsiveness to corticosteroids and mortality in patients with AH.

(004) Correlations Between Immunohistochemical Staining and Pain-Related Assessments in Patients with Neuroproliferative Vestibulodynia

Abstract Introduction A clinically suspected diagnosis of neuroproliferative vestibulodynia (NPV) can be confirmed by examining immunohistochemical (IHC) staining of excised vestibular specimens. Compared to controls, specimens from NPV patients have been found to contain &amp;gt;8 mast cells per high-power field (HPF) and increased density of nerves. Objective To assess for correlations between patient reported pain-related measures and computer-assisted histometry of excised vestibulectomy specimen IHC staining with CD117 (marker for mast cells) and PGP9.5 (marker for nerves) in a cohort of histopathologically confirmed NPV patients. Methods Patients (n=65) with lifelong or acquired NPV undergoing vestibulectomy had IHC-staining of the vestibular tissue. Tissue sections from the 1:00-11:00 and/or 12:00 regions of the vestibule were examined and high-resolution images were captured using a microscope at 100x and 200x magnification. A minimum of 2 photomicrographs per magnification were taken for each tissue section that included epithelial basement membrane and adjacent subepithelium representative of the majority of immunostained tissue. Fractional area of positive immunostaining of all photomicrographs was determined using computer-assisted histometry by ImageJ. Mean fractional area was determined from 3 separate measurements of each photomicrograph. Correlations were made between density of IHC staining and the following assessments: pain domain of the Female Sexual Function Index (FSFI), the Short Form McGill Pain Questionnaire (SF-MPQ), and cotton-tipped swab testing. We also examined correlations with the Patient Global Impression of Improvement (PGI-I), a treatment response index and indirect assessment of pain. Data were analyzed using Spearman correlations and the Mann-Whitney test. Results While all patients reported high levels of pain, there were no correlations between CD117- or PGP9.5-immunopositive mean fractional area and FSFI pain domain or SF-MPQ scores. There was no difference in immunopositive mean fractional area between lifelong and acquired NPV. A positive correlation between fractional area of CD117 immunostaining in the 1:00-11:00 region of the vestibule and cotton-tipped swab test pain score approached but did not reach statistical significance (r = 0.24, p = 0.063). Fractional area of PGP9.5 immunostaining was negatively correlated with PGI-I scores (r = -0.50, p = 0.006) in the 1:00-11:00 region of the vestibule. Lower PGI-I scores are indicative of greater improvement after surgical intervention. No other correlations were observed between immunostaining and PGI-I or cotton-tipped swab testing scores. Likewise, SF-MPQ domain scores had no correlation to PGI-I or cotton-tipped swab testing. Median cotton-tipped swab testing pain scores were significantly higher (p &amp;lt; 0.001) in patients with lifelong NPV (7.6) compared to those with acquired NPV (5.7). Conclusions The lack of correlations between immunopositive staining and multiple pain-related assessment scores suggests a non-linear threshold for mast cell accumulation and nerve proliferation that produces pain symptoms for all NPV patients. Cotton-tipped swab pain scores were more closely associated with density of CD117 immunostaining, while clinical improvement in pain symptoms was significantly correlated with density of PGP9.5 immunostaining. The fact that patients with lifelong NPV had significantly more pain on cotton-tipped swab testing than the acquired NPV subgroup may be related to duration of the condition. Disclosure No.

Influence of maternal rats' hypothyroidism on morpho-functional peculiarities and glycome of progeny skin.

We studied the influence of maternal hypothyroidism on progeny skin morphogenesis by means of histological, histochemical and lectin-histochemical methods. Hypothyroid conditions in rats were achieved by daily food supplementation with antithyroid drug Mercazolil. The experiment was conducted on 10 control and 10 hypothyroid rats, which delivered 70 and 46 offsprings, respectively. We discovered that maternal hypothyroidism induces the accumulation of mast cells (MCs) in the skin of progeny on the 1st, 10th and 20th postnatal days, with decrease of these cell's count returning to control level on 40th postnatal day. These results indicate that offsprings developing under conditions of maternal hypothyroidism are a risk group for changes in immune status and the occurrence of allergic reactions. The stratum corneum of epidermis, its lipid barrier as well as pilosebaceous units, in both control and experimental group animals, at the early stages of postnatal ontogenesis are enriched with carbohydrate determinants of αDMan, βDGal, βDGal(1-3)DGalNAc, αLFuc, αDGalNAc, αDGlcNAc, Neu5Ac. Galanthus nivalis agglutinin (GNA) is a selective histochemical marker of MCs, while Lactarius torminosus fungus agglutinin (LTFA) is a selective label of Langerhans cells. Maternal hypothyroidism resulted in reduction of lectin binding with the structural components of progeny skin and its derivatives. We speculate that alterations in glycoconjugate processing and degradation sequences have an impact on the cell signaling, formation of adhesive contacts, cellular proliferation and differentiation. The lectin set we used clearly demonstrated specific labeling of cellular subpopulations, monitoring glycoconjugates processing and degradation under physiological and pathological conditions in all skin components.

Identification of Structures Labeled by Indocyanine Green in the Rat Choroid and Retina Can Guide Interpretation of Indocyanine Green Angiography.

Indocyanine green (ICG) is an albumin and lipoprotein binding dye absorbing in the far red used in angiography to visualize choroidal vessels (ICG angiography [ICGA]). To guide interpretation, ICG transport in the choroid, RPE, and retina of rats was studied. Two conditions were used: RPE/choroid organoculture, incubated for 45 minutes in DMEM medium, 1% fetal bovine serum containing 0.25 mg/mL ICG and RPE/choroid and neural retina flat-mounts at 1 and 6 hours after intravenous ICG injection. Early and late sequences of ICGA were recorded until 6 hours. Ultra-deep red confocal microscope was used to localize ICG in flat-mounts and immunohistochemistry was performed for caveolin-1, tryptase (mast cell marker), and tubulin β3 (a nerve marker). In the organoculture, ICG penetrated homogeneously in the cytoplasm and stained the membranes of the RPE. At 1 hour after intravenous injection, ICG appeared in fine granules in RPE, partly labeled with caveolin-1 and decreasing at 6 hours. At 1 hour and 6 hours, ICG was found in the retinal vessels, faintly in the inner retina, and in the photoreceptor outer segments at 6 hours. In the choroid, ICG colocalized with mast cells, immunostained with tryptase, and accumulated along the large tubulin β3-labeled nerve bundles. The hypothesis was raised on the interpretation of late ICGA infrared photography in case of transthyretin amyloidosis with neuropathy. Beside being a vascular dye, ICG is transported from the vessels to the RPE toward the outer retina. It stains mast cells and large choroidal nerves. These observations could help the analysis of ICGA images.