Jornal de Citologia e Histologia

Osteoarthritis (OA) is a degenerative joint disease that affects more than 46 million people in the United States alone. Since mechanisms by which OA ensues are largely unknown, there are no therapeutic targets that effectively prevent and treat the disease. However, growth factors, cytokines and matrix-degrading enzymes are strongly implicated in initiating and aggravating OA lesions. Thus, a molecular understanding of these molecules will provide invaluable information toward the search for novel therapeutic targets for OA. Our genome-wide screen for novel, differentially expressed genes in OA led to the isolation of progranulin (PGRN) as a novel OA-associated growth factor [1]. In subsequent global screen for the binding proteins of PGRN, we found that PGRN bound to TNF Receptors (TNFR). In addition, PGRN blocks the binding of TNFα to TNFR and inhibits TNFα-induced ADAMTS cleavage of cartilage oligomeric matrix protein (COMP) [2]. These previous findings led us to determine whether recombinant PGRN prevents cartilage degradation in the progression of OA in vivo. For this purpose, we took advantage of PGRN knockout mice to generate anterior cruciate ligament (ACL) transection induced OA models which develop severe OA due to the deficiency of PGRN. Importantly, intra-articular injection of rPGRN growth factor significantly prevented the degeneration of cartilage in PGRN-deficient OA model. PGRN treated mice retained cartilage integrity and showed little or no degradation of cartilage matrix in comparison to highly degraded cartilage of non-treated mice (Figure).

Background: The DNA damage checkpoint pathway has been of interest to the field of cancer biology, since checkpoint defects result in the accumulation of altered genetic information, a central feature of carcinogenesis. Little is known about the role that CHK2 (checkpoint kinase 2) gene plays in colorectal cancer tumorigenesis. The purpose of this study was to evaluate CHK2 expression in metastatic colon cancer and correlate it with clinicopathological features and patient survival.
Methods: Tissues of primary tumors were obtained from 58 patients with metastatic colon cancer. The tissue microarray immunohistochemistry was the technique used to evaluate CHK2 expression. Statistical analysis used was SPSS17; p-value was set at <0.050. The relationship between the CHK2 immunohistochemical expression and the patients’ clinical and pathological features as well as survival data was reported.
Results: CHK2 expression was positive in 69% of the cases. CHK2 expression was associated with lymph node status (p=0.012) and survival (p=0.034). Negative CHK2 expression increased the chance of lymph node involvement (Odds ratio: 10.23, p=0.03). The global survival time of CHK2-negative patients was higher (72 versus 59 months); the same trend emerged for progression-free survival time (19 versus 13 months). The survival curves differed depending on CHK2 expression in patients with or without lymph node involvement; survival was lower in CHK2-positive. A larger number of deaths occurred in CHK2-positive. Multivariate regression analysis identified performance status ECOG (p=0.01), synchronous metastasis (p=0.037), tumor cell differentiation (p=0.029) and CHK2 expression (p=0.020) as independent factors for overall survival.
Conclusions: This study demonstrated that positive CHK2 expression in colon cancer indicates aggressiveness and impacts negatively patient survival and outcome. On the other hand, a negative expression indicates dissemination to lymph nodes.

Purpose: Orbital Immunoglobulin 4 (IgG4) related disease (IgG4RD) is a fibro-inflammatory condition that mimics sclerosing orbital inflammatory disease (OID). The recently published IgG4RD consensus criteria included its defining histological features. Using the published criteria, this study aims to describe the frequency of orbital IgG4RD and its histological features in OID biopsies over a 1 year period.
Method: Thirty-seven consecutive orbital biopsies for OID over 1 year were prospectively examined for the features of fibrosis, inflammation, and vasculitis. Immunohistochemistry (IHC) evaluation was performed when significant fibrosis and/or lymphoplasmacytic inflammation ( >25% of the biopsy section) was present.
Results: Ten of 37 (27%) orbital biopsies showed significant fibrosis and/ or lymphoplasmacytic inflammation with the remaining cases showing only non-specific chronic inflammation or reactive lymphoid hyperplasia. Only 3 cases (30%) fulfilled the IgG4RD consensus criteria. The histological patterns included sclerosing dacroadenitis, sclerosing xanthogranulomatous orbital inflammation, and eosinophilic angiocentric fibrosis. Storiform fibrosis was the most common histological feature present (70%), followed by dense lymphoplasmacytic inflammation (60%). When both are present, an almost 2-fold elevation of tissue IgG4 plasma cells and ratio above the diagnostic cut-off was detected. Xanthogranulomatous inflammation and eosinophilia were occasionally present.
Conclusions: Using the consensus criteria, IgG4RD was diagnosed in 30% of our orbital biopsies with significant fibrosis and/ or inflammation and 11% of all OID biopsied in 1 year. Although the storiform fibrosis and lymphplasmacytic inflammation was most commonly seen, associated eosinophilia and xanthogranulomatous inflammation may also be seen in IgG4RD.

Background: Triclosan (TCS) is widely used broad spectrum bactericide. Ellagic acid (EA) has radical scavenging properties.
Aim of the work: to assess the structure of the renal cortex after TCS administration and to determine the possible protective role of EA.
Materials and Methods: Thirty adult male albino rats were divided into three groups; control group, TCS treated group: TCS was administered 200 mg/kg, once daily for six week, by oral gavages. TCS-EA treated group: received in the same doses of TCS and EA (30 mg/kg) for six weeks. Blood urea nitrogen (BUN), serum creatinine (Sc) and uric acids (UA) were measured. Renal cortex samples were processed for light and electron microscope examination.
Results: Examination of TCS treated group revealed increased glomerular cellularity in some corpuscles. Podocytes showed effacement of the foot processes and focal thickening of the glomerular basement membrane. Some tubules showed marked cellular disorganization. Disoriented basal mitochondria, areas of rarified cytoplasm and electron-dense bodies were noticed. Intense positive caspase-3 reaction was expressed in the tubular cells. TCS-EA showed improvement of morphological organization of renal cortex glomeruli, proximal and distal tubules with moderately expressed caspase-3 in renal tubules Statistical analysis showed significant increase of BUN, SC and BUN in TCS treated group in comparison with the control group.Their levels in TCS-EA treated group showed a significant decrease in comparison with TCS treated group
Conclusion: In conclusion, TCS leads to alterations in the histological structure and functions of renal cortex of albino rats and EA supplementation could protect from these changes.

Monosodium glutamate (MSG), a flavor enhancer, is used in modern nutrition to improve food palatability. The objectives of the current study were to investigate the effect of MSG on thymus as well as spleen structures and functions. Also, to evaluate the possibility of recovery after cessation of administration. Adult male rats were divided into three groups: control, MSG (3 g MSG/kg body weight daily for 8 weeks by oral gavages), and Recovery (MSG for same period and then left untreated for additional 4 weeks). The results showed that MSG treatments significantly increased serum interleukin (IL)-1β as well as thymic and splenic malondialdehyde and decreased serum levels of IL-10 and also reduced glutathione (GSH) levels and both catalase and superoxide dismutase activities in the thymus and spleen. Histological examination showed that MSG induced a remarkable disruption in the lobular architecture of the thymus with marked decrease of the T lymphocytes with darkly stained nuclei and dilated blood sinusoid in the cortical region. Medullary region were enlarged and repopulated with small lymphocytes and dilated blood sinusoids. The cortical-medullary differentiation was difficult to be determined. Small sized splenic lymphatic follicles with absence of germinal centers and large congested blood vessels were also noticed. The differentiation between the red and the white pulps was indistinct. Recovery groups showed preserved thymic lobular architecture with repopulation of the cortical thymocytes enclosing the paler staining medulla .Splenic lymphatic follicles of different sizes with absence of germinal centers were noticed. Marginal zone is differentiated from the red pulp. Immunohistochemical staining of MSG group demonstrated a marked decrease in CD3-positive T-lymphocytes in both thymus and spleen that significantly increased in recovery group. Taken together, the data showed that MSG consumption may have immunotoxic effects on the thymus and spleen of adult rats which is reversible but the normal structure of the spleen would need time to be regained. It is recommended that further studies aimed at corroborating these findings be carried out.

Nesfatin/nucleobindin-2 (NUCB2), a precursor protein of anorexigenic protein, nesfatin-1, is ubiquitously expressed in the body. The finding that peripheral administration of nesfatin-1 increases blood pressure indicates a possible involvement of nesfatin-1 in the regulation of blood pressure. The present studies were undertaken to investigate a possible involvement of nesfatin/NUCB2 in the regulation of blood pressure. The immunoreactivity against nesfatin/NUCB2 was selectively found in vascular endothelial cells of aorta, pulmonary artery and renal artery, cardiac muscle and skeletal striated muscle cells, but not in vascular smooth muscle cells at all. Furthermore, the immunoreactivity against nesfatin/NUCB2 was selectively found in renal collecting duct cells, which contain aquaporin (AQP)-2 and/or epithelial sodium channel (ENaC). In medullary collecting ducts, cells expressing nesfatin/ NUCB2 co-expressed AQP-2, but did not co-expressed AQP-2 in renal cortical collecting ducts. On the other hand, collecting ductal cells expressing ENaC were totally compatible with those expressing nesfatin/NUCB2 in both renal medullary and cortical collecting ducts. Thus, there is a possibility that nesfatin/NUCB2 is involved in the regulation of blood pressure through increased water reabsorption in the kidney.

Complex diseases such as heart disease, stroke, cancer, and aging are the primary causes of death in the US. These diseases cause heterogeneous conditions among cells, conditions that cannot be measured in tissue homogenates and require single cell approaches. Understanding protein levels within tissues is currently assayed using various molecular biology techniques (e.g., Western blots) that rely on milligram to gram quantities of tissue homogenates or immunofluorescent (IF) techniques that are limited by spectral overlap. Tissue homogenate studies lack references to tissue structure and mask signals from individual or rare cellular events. Novel techniques are required to bring protein measurement sensitivity to the single cell level and offer spatiotemporal resolution and scalability. We are developing a novel approach to protein quantification by exploiting the inherently low concentration of rare earth elements (REE) in biological systems. By coupling REE-antibody immunolabeling of cells with laser capture microdissection (LCM) and ICP-QQQ, we are achieving multiplexed protein measurement in histological sections of single cells. This approach will add to evolving single cell techniques and our ability to understand cellular heterogeneity in complex biological systems and diseases.

The Tunica albuginea surrounding the testis of the great grey kangaroo was thick and there were large number of Leydig cells present in between the seminiferous tubules. These findings were the striking features among other histological findings of an 18 year old kangaroo which was kept in captivity in Los Angeles zoo all his life. Eighteen year old great grey kangaroo testes, epididymis and ductus deferens were collected and processed using standard histologic techniques. The animal was kept all his life in captivity in LA zoo. Two different stains were used to differentiate tissues and cells. Histological characteristics of all organs under study were found to be similar to other animal species of younger age with few striking exceptions. The Tunica albuginea was very thick and large number of Leydig cells was present between the seminiferous tubules. Animals in captivity with excellent care and veterinary services will continue to be fertile and actively producing sperms when compared to animals of the same age live in the wild.

A significant proportion of follicular lymphoma transform to a higher-grade lymphoma, most commonly, diffuse large B-cell lymphoma. This histologic transformation usually shows diffuse sheets of large cells; however, variant morphologic patterns are also known to occur. We report a case of a 70-year old woman who presented with an unusual large cell transformation of follicular lymphoma with a T-cell and histiocyte-rich background and limited foci of confluent large B-cells. The atypical growth pattern in the excised lymph node led to a broad differential diagnosis that not only included diffuse large B-cell lymphoma but also T-cell lymphoma and nodular lymphocyte predominant Hodgkin lymphoma. Ancillary studies confirmed a neoplastic and clonal B-cell process and immunochemotherapy was initiated. This case illustrates the need for recognition of variant morphologic patterns in large cell transformation of follicular lymphoma for appropriate classification and clinical management.

Background: More than half of women present nipple discharge during reproductive age.
Case: This case is about a 36 years old woman with unilateral spontaneous hyaline nipple discharge associated with breast pain. The study of scintimammography was compatible with multifocal proliferative lesion in situ. It was performed cytological smear of nipple discharge. It was paucicellular smear represented by cluster of ductal cells in three-dimensional design with hyperchromatic nuclei in the presence of myoepithelial cells. Red cells and signs of necrosis were not observed. The diagnosis of in situ ductal carcinoma was confirmed in biopsy and mastectomy specimen through the expression of calponin in myoepithelial cells at immunohistochemistry. It is known that the cytological examination of nipple discharge has low sensitivity and specificity. However, it is an easy and inexpensive procedure. Suspicious or positive results, may be important for guidance workup of patients in order to perform earlier diagnosis of malignancy.
Conclusion: this case demonstrates that in situ ductal carcinoma can be characterized by positive nipple discharge, and cytology sample is an important tool for the diagnosis of suspicion of malignancy and further diagnostic investigation.

Type 2 Diabetes (T2D) is a global epidemic that affects hundreds of millions of individuals. The pancreatic β-cell has long been the focus of studies pertaining to T2D, but the therapies that exist to date are inadequate. Devising new therapies will require a more detailed understanding of the pathogenesis of T2D and of the normal function of the β-cell. In particular, there is a great need to understand the signaling pathways that govern normal β-cell function and that become dysfunctional during the progression to diabetes. The Transforming Growth Factor β (TGF-β) signaling pathway is implicated in nearly all tissue types in the body, and has been shown to play a role in pancreas development and homeostasis, including β-cell regeneration after pancreatic insult. TGF-β exerts its cellular effects through transcriptional activity of downstream SMAD molecules, as well as through cross-talk with other signaling pathways. Accumulating evidence suggests that β-cell failure in T2D is a multifaceted process that may include islet inflammation, increased β-cell apoptosis, reduced β-cell proliferation, and/or β-cell dedifferentiation to a progenitorlike state. This review details the known roles of TGF-β signaling in dedifferentiation-induced and inflammationinduced β-cell failure, and draws on the apparent coordinated regulation of β-cell proliferation and the β-cell differentiation state to offer new hypotheses about β-cell failure in T2D.

A history of a gradual increase in swelling mass was presented in the sublingual gland of a 72-year-old female. The inadequate cytologic specimens retrospectively contained some clusters of three-dimensional monomorphic and round basaloid cells having hyperchromatic small nuclei and scant cytoplasm, along with a small amount of spherical globules of amorphous material and myxoid stroma, adjacent to few squamous metaplastic tumor nests. We first interpreted it as a pleomorphic adenoma, basal cell adenoma (BCA), or adenoid cystic carcinoma (ACC). A tumor extirpation was performed, and gross examination revealed a non-capsulated and ill-defined tumor lesion, looking grayish to yellowish-white, focally associated with fat invasion. On microscopic examination, the tumor was predominantly composed of a proliferation of small to medium-sized mildly atypical epithelial cells having very rare mitoses, often arranged in a cribriform or alveolar growth pattern with peripheral nuclear palisading and scattered squamous differentiation, embedded in a prominent eosinophilic hyaline material. Therefore, we finally made a diagnosis of invasive basal cell adenocarcinoma (BCAC), defined as the malignant counterpart of benign BCA. We should be aware that owing to its characteristic features, cytopathologists might be able to determine correct diagnosis, based on multiple and adequate samplings.

Metastasis to the breast from extra mammary sites is very rare, constituting only 1 to 2 percent of metastasis. Lymphoma, malignant melanoma and bronchial carcinoma are the malignancies which metastasize to the breast. Metastasis to the breast in a case of primary anorectal melanoma is rare with only few cases reported in literature. We present a case of a 40 year old female who presented with a lump in the left breast. She had history of malignant melanoma of anorectal region for which she underwent anterior resection 6 months back. We did FNAC of left breast lump and found metastasis. Unfortunately patient died within few days.

Heterotopic ossification can be found in neovascularized aortic valves with end-stage valvular disease, but its histopathological characteristics are not fully understood. The present study found ossification and neovessels in 21 (28%) and 54 (72%) of 75 surgically removed dysfunctional aortic valves, respectively; there was a significant association between them (P<0.001). Fatty bone marrow was found in 5 aortic valves (6.7%) and always contained neovessels. Neovessels could be divided into thick-walled, arteriole-like vessels (ThKA-Vs) and thin-walled, capillarylike vessels (ThNC-Vs). ThKA-Vs would be a hallmark of rheumatic disease, were always accompanied by ThNCVs, and were closely related to ossification (P=0.046). ThNC-Vs may be in part branches of ThKA-Vs, but were also identified in 23 aortic valves without ThKA-Vs. In 44 non-rheumatic dysfunctional aortic valves, such ThNC-Vs only were also associated with ossification (P=0.004). In addition, the present study revealed vessel-independent focal/ minute ossification without nearby neovessels in 6 dysfunctional aortic valves, and also in 4 of 75 age- and sexmatched postmortem non-dysfunctional aortic valves with no neovessels or prominent calcification/thickening; there were no significant differences in the incidence between them. Neovascularized vessels would consist of rheumatic neovessels (ThKA-Vs and their branches) and non-rheumatic neovessels (ThNC-Vs only), and the presence of neovessels is one of the risk factors for aortic valvular ossification. However, aortic valvular ossification can occur in various pathogeneses, such as rheumatic neovessel-dependent ossification, non-rheumatic neovessel-dependent ossification, and rarely vessel-independent focal/minute ossification.

Tuberculosis can affect any organ or tissue in the abdomen such as gastrointestinal tract, peritoneum, lymphatic system and solid organs. Lymphadenopathy is the most common manifestation of abdominal tuberculosis which can be the only sign of the disease, especially in the periportal region or combined with peritoneal or solid organ involvement. There are a number of causes for prehepatic noncirrhotic portal hypertension, but there were a few reports of prehepatic portal hypertension associated with tuberculosis. Here we report a rare case of intra-abdominal tuberculosis as a cause for portal hypertension and recurrent gastrointestinal bleeding. Enlarged lymph nodes at hepatic hilum compressed the portal vein which caused portal hypertension and portal hypertensive gastropathy and enteropathy. The patient was managed by anti-tuberculosis therapy followed by splenectomy, surgical portaazygous devascularization and splenorenal shunt. In the 2-year followup after operation, the patient was free of symptoms and had cavernous portal vein transformation on CT, and disappearance of varices and portal hypertensive gastroenteropathy on EGDs.

Background: To make an update on the state of knowledge on perianal extramammary Paget’s disease.
Case presentation: We report one case as an illustration of this rare, slow-growing and occasionally metastasizing entity. The standard treatment is a local surgical excision. Radiation therapy is recommended as primary treatment in inoperable patients, and as alternative to surgery for recurrence disease previously excised or in case of mutilating surgery.
Conclusions: Definitive conclusion cannot be drawn. Clinical trials are required to test whether radiation therapy is advantageous as primary treatment approach, compared to surgery.

Background: Previous reports have been published describing the role of protein markers in the development and progression of colorectal cancers (CRC). In those investigations, Nuclear Factor kappaB (NFκB) and the Hypoxia Inducible Factor-1α (HIF-1α) have emerged as markers of clinical interest. The aim of this study was to evaluate their expression in patients who underwent surgery for CRC in the Guadeloupe Teaching Hospital (GlpeTH).
Methods: Tumour tissues and the normal mucosa from 67 patients of GlpeTH were immunohistochemically evaluated using antibodies against HIF-1α and NFκBp65. Additionally; we examined the expression of Vascular Endothelial Growth factor (VEGF), Vascular Cell Adhesion Molecule-1 (VCAM-1) and P-selectin, which was regulated by HIF-1α and NFκBp65, respectively.
Results: Expression of NFκBp65 and HIF-1α, mainly located in the cytoplasm of the cancer cells and P-selectin, located on endothelial cells of blood vessels, was positively observed in 74.6%, 73.1% and 23.9% among the patients, respectively. Unexpectedly, expression of VEGF and VCAM-1 was weak, 6% and 3% respectively. HIF-1α did not associate either with clinicopathological parameters or NFκBp65 expression, whereas the latter significantly did with invasion depth (p=0.017). Regarding P-selectin, its expression was not correlated with that of NFκBp65, but significantly was with TNM stage (p=0.022).
Conclusion: Expression of VEGF and VCAM-1 may depend of other tumour environmental factors. NFκB, more frequently expressed in T3-T4 stages and P-selectin less in aggressive CRCs (TNM III and IV) are more valuable protein markers than HIF-1α, to characterize CRC in GlpeTH patients.

The staining ability of Allium cepa (red onion) skin extract on tissue sections was determined. The dried red onion skin was ground into small pieces and 300 g (150 g for aqueous and 150 g for ethanolic extraction) of the milled dried red onion skin was used for extraction. Dark red-pigmented granules were obtained. The aqueous and ethanolic extract of mordanted (Potassium aluminum alum and ferric chloride), non-mordanted and acidified solution prepared from the extract were used to stain tissue sections. Cytoplasm of cells and connective tissues were remarkably stained in shades of redish to yellowish brown colours. Preliminary phytochemical screening of these extracts reveals that it contained alkaloid steroid ring, saponin, Tannin, Flavonoid, Cardiac glycoside and anthraquinone. The presence of pigments, saponin, tannin, contributed to the staining ability of the extract.

Introduction: Cytological features obtained from fine needle aspiration cytology(FNAC) are very essential for the preoperative diagnosis in breast carcinoma. The quantitative evaluation of nuclear size and shape using FNAC material can be utilized as a diagnostic tool in breast tumor. The main objectives of our study were (a) to evaluate major axis (MAJX), minor axis (MINX), nuclear area (NA), nuclear perimeter (NP) and nuclear aspect ratio (NAR) using morphometric techniques, (b) to compare these nuclear parameters with their variability in benign and malignant cases and evaluate suitable cut off values and (c) to study the correlation of these parameters with cytological grades.
Materials and Methods: Nuclear parameters were assessed in 50 cases of invasive ductal carcinoma and 50 cases of benign breast tumors by image morphometric technique on FNAC slides and statistical analysis was performed.
Results: Mean MAJX, MINX, NA, NP and their variability were significantly greater (p<0.001) in malignant cases than the benign unlike NAR. All nuclear parameters showed positive correlation with their variability. Cytological grade exhibited mild positive correlation with MAJX, MINX, NA and NP except NAR. The cut off values with sensitivity=1.00 for the differentiation of malignant from benign were: (a) MAJX>10.70 micron (specificity=0.98), (b) MINX>7.53 micron (specificity=0.94), (c) NA>60.61 micron2 (specificity=0.98) and (d) NP>27.81 micron (specificity=0.96).
Conclusion: Morphometric parameters related to nuclear size and variability evaluated from FNAC material were significantly larger in malignant cases than the benign and they can be gainfully exploited in the diagnosis of breast carcinoma. Again these parameters showed mild positive correlation with the cytological grades.

Objective: To evaluate the efficacy and clinical outcome of hypofractionated stereotactic radiotherapy (HSRT) in non-small-cell lung cancer (NSCLC) cancer patients with orbital metastasis.
Case report: A 61-year-old man was admitted to the hospital referring right ptosis and dry hacking cough. HSRT was performed for the orbital metastasis followed by chemotherapy with cisplatin (60 mg/m2) and pemetrexed (500 mg/m2) on day 1 every 21 days for 4 cycles and maintenance pemetrexed (500 mg/m2) on day 1 every 21 days. Control TC scan revealed a complete response of the orbital mass.
Conclusion: HSRT should be considered a valid therapeutic alternative in the management of selected patient with orbital metastases.

Background and Objectives: Mucositis can be a dose-limiting toxicity of cancer chemotherapy with direct effects on patient survival; therefore an effective intervention is considered a high priority in cancer patient. The aim of the present study was to evaluate the effectiveness of honey as a preventive treatment for the methotrexate (MTX) induced oral mucositis.
Materials and Methods: In current study forty females Albino rats, weighing 250-300 g were used in the study. For the induction of oral mucositis, 60 mg/kg of MTX was administered intraperitoneally to each animal in the study group at day 4. The control animals were intraperitoneally injected by normal saline in the same manner and dose like MTX. At the beginning of the experiment, the rats in each group were randomly divided into two groups: Distilled water treated group and honey treated group (10 animals each). A volume of distilled water equal to honey was given by intragastric gavage tube, while the other group was gavaged with honey at a dose of 2.5 g /kg two times daily (with a total of 5 g/kg/day). The animals were sacrificed at day 8. In each experiment, the middle third of tongue was removed for histopathological and immunohistochemical analysis using Ki-67 and Bcl-2 immunolabeling.
Results: The result showed that, the MTX/honey group showed a significant increase in the thickness of the epithelium (p<0.01), significant decrease in the number of congested blood vessels in the connective tissue of rat tongue mucosa (p<0.01), non significant increase in the Ki-67 immune expression (p>0.01), and significant increase in Bcl- 2 immune expression (p<0.01) in comparison with the methotrexate/water group.
Conclusion: Natural honey at a concentration of (5 g/ kg/day) produced protection against methotrexate induced tongue mucositis and therefore can be used as a protective natural product to oral mucosa against methotrexate induced cytotoxicity.