Primary Intrathyroidal Non-Hodgkin Lymphoma: A Case Report.

Primary thyroid lymphoma (PTL) is a rare disease characterized by the appearance of a rapidly growing solid mass in the cervical region. A major risk factor is chronic autoimmune thyroiditis with lymphocytes infiltrating the thyroid gland. The lymphoproliferative disease is seen more frequently in the females. PTL usually develops in the sixth and seventh decades of life. We present a case of a 66-year-old woman with diffuse primary B-cell thyroid lymphoma with no prior evidence of underlying autoimmune thyroid pathology. The initial localization of the lymphoproliferative disease was in the thyroid gland, but the involvement of regional cervical lymph nodes was also found at the time of diagnosis. After histological verification with immunohistochemistry and staging by imaging, chemotherapy was initiated according to the R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin hydrochloride, Oncovin® (vincristine), prednisone) protocol. An excellent therapeutic response was achieved with lymphoma remission after six cycles under the mentioned protocol. Thyroid autoantibodies became positive 18 months after rituximab treatment, possibly reflecting the transient suppressive effects of the immunotherapy. The patient was subsequently kept followed up by a multidisciplinary team in the light of possible lymphoma recurrence and/or development of thyroid dysfunction. This case report demonstrates possible challenges for the diagnosis, treatment, and follow-up of this rare thyroid lesion. At the time of diagnosis, the clinical presentation of the disease, the ultrasound image, and the cytological result may be similar to other low-grade thyroid carcinomas or secondary metastatic involvement of the gland. The initial lack of underlying thyroid autoimmunity makes this distinction even more challenging. Furthermore, despite the rapid resolution, regular long-term monitoring for recurrence is required.

Frantz Tumor: A Case Report of Solid Pseudopapillary Tumor of Pancreas.

The solid pseudopapillary tumor (SPT) is a rare pancreatic lesion that usually affects young and middle-aged patients and has a female predominance and low malignant potential. The exact histogenesis of this tumor is still unclear. We present the case of a 60-year-old female patient with occasional abdominal pain. Positron emission tomography/computed tomography (PET/CT) and magnetic resonance imaging (MRI) revealed a tumor mass in the pancreatic tail. Distal pancreatectomy and splenectomy were performed. The result from the pathology report was solid pseudopapillary neoplasm (SPN). The patient underwent four cycles of adjuvant chemotherapy with gemcitabine, which she tolerated well without complaints. A control computed tomography (CT) scan and PET/CT of the abdomen (five months after the operation) showed a cystic lesion suspicious for local recurrence in the pancreatic tail during the follow-up period. The patient underwent a second surgery operation. Subsequent histological examination showed chronic indurative pancreatitis, areas with steatonecrosis, lipogranulomas, and fibrosis without evidence of relapse. SPT is a rare pancreatic tumor that most commonly affects young women. Although the tumor has locally aggressive characteristics, the prognosis is excellent after surgical excision. Our case emphasizes that this tumor can occur not only in young women but also in older patients. Chronic granulomatous inflammation and indurative pancreatitis can sometimes mimic a relapse on CT and PET/CT image tests.

Improving gene expression analysis efficacy from formalin-fixed paraffin embedded tissues.

INTRODUCTION: Improving RNA isolation and cDNA synthesis techniques has emerged due to advancements in the knowledge of molecular basis of most diseases. This in turn increased the need of higher quantity and quality of the extracted genetic material to be used for a variety of diagnostic tests and experiments. AIM: The aim of the study was to compare three modified methods for RNA extraction from formalin-fixed paraffin embedded (FFPE) biopsied tissue and different cDNA synthesis strategies to facilitate study of gene expression. MATERIALS AND METHODS: Compared RNA extraction methods were: lysis buffer, phenol-based extraction, and combination of both with concomitant use of silica-based spin columns. RNA quantity and purity were estimated spectrophotometrically. Different priming strategies for cDNA synthesis were applied: oligo dT, combination of oligo dT and random hexamer, and gene specific primer. Two-step RT-qPCR of ribosomal protein L37A on preamplified and non-preamplified cDNA templates was performed. RESULTS: The combination of lysis buffer with phenol based extraction gave higher RNA yield. By doing cDNA preamplification, the confidence of detection by qPCR was raised, and efficiency was improved. The preamplified template increased the sensitivity of analysis. CONCLUSIONS: Together, the combination of approaches improved substantially the reproducibility and validity of quantitative gene expression analyses from FFPE tissues.

Paraganglioma in the spinal cord histologically mimicking ependymoma - a case report.

Paraganglioma is a tumour lesion of neuroectodermal origin that occurs at various places in the human body, but is rarely observed in the spinal cord. Usually, it presents in the lumbar region (cauda equine and filum terminal) as a slow-growing painless tumour mass that causes local compression.

Rare Finding of Inflammatory Fibroid Polyp of the Duodenum: A Complete Diagnostic and Pathological Workup.

Inflammatory fibroid polyps (IFP) are solitary benign tumors rarely found in the gastrointestinal (GI) tract. Additionally, duodenal polyps are diagnosed incidentally. We present a case of a 51-year-old female admitted to the department with an initial diagnosis of duodenal polyp on gastroscopy, CT, and positron-emission tomography (PET). COVID-19 pandemics was the reason for delayed treatment which allowed the lesion to progress and almost double its size in an eight-month period. We performed conventional duodenotomy and excision of the polyp. Diseases like gastrointestinal stromal tumor (GIST), inflammatory myofibroblastic tumor, and inflammatory polyp of Crohn's disease must be considered in the differential diagnosis of IFP because they could be observed in the same location.

Temporal Patterns of COVID-19-Associated Pulmonary Pathology: An Autopsy Study.

Introduction The novel coronavirus variant - severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) and the disease it causes clinically (novel coronavirus disease 2019 or COVID-19) have placed medical science into a frenzy due to the significant morbidity and mortality, as well as the myriad of clinical complications developing as a direct result of infection. The most notable and one of the most severe changes in COVID-19 develops in the lungs. Materials and methods All cases of real-time polymerase chain reaction (rtPCR)-proved COVID-19 subjected to autopsy were withdrawn from the central histopathology archive of a single tertiary medical institution - St. Marina University Hospital - Varna, Varna, Bulgaria. Pulmonary gross and histopathology changes observed on light microscopy with hematoxylin and eosin as well with other histochemical and immunohistochemical stains were compared with the time from patient-reported symptom onset to expiration, to compare the extent and type of changes based on disease duration. Results A total of 27 autopsy cases fit the established criteria. All cases clinically manifested with severe COVID-19. From the selected 27 cases, n=14 were male and n=13 were female. The mean age in the cohort was 67.44 years (range 18-91 years), with the mean age for males being 68.29 (range 38-80 years) and the mean age for females being 66.54 (range 18-91 years). Gross changes in patients who expired in the first 10 days after disease onset showed a significantly increased mean weight - 1050g, compared to a relatively lower weight in patients expiring more than 10 days after symptom onset - 940g. Histopathology changes were identified as intermittent (developing independent from symptom onset and persisting) - diffuse alveolar damage with hyaline membranes - acute respiratory distress syndrome, endothelitis with vascular degeneration and fibrin thrombi; early (developing within the first week, but persisting) - type II pneumocyte hyperplasia, alveolar cell multinucleation and scant interstitial mononuclear inflammation; intermediate (developing within the late first and second weeks) - Clara cell hyperplasia and late (developing after the second week of symptom onset) - respiratory tract and alveolar squamous cell metaplasia and fibrosis. Conclusion COVID-19-associated pulmonary pathology, both gross and histopathology, show a time-related dynamic with persistent early and a myriad of later developing dynamic changes in patients with severe disease. These changes underline both the severity of the condition, as well as the mechanisms and the probability of long-lasting severe complications in patients with post-COVID syndrome.

Characterization of stitch adhesions: Fibronectin-containing cell-cell contacts formed by fibroblasts.

Fibronectin is a multifunctional, extracellular matrix glycoprotein that exists either as an insoluble multimeric fibrillar component of the extracellular matrix or as a soluble monomer. Cells attach to fibronectin through transmembrane integrin receptors and form a variety of cell-matrix contacts. Here we show that primary fibroblasts can use fibronectin to organize a specific cell-cell contact - "stitch adhesions." This contact is formed by short parallel fibronectin fibrils connecting adjacent cells above the level of the focal adhesions that attach the cells to the substrate. Stitch adhesions contain integrin α5ß1 but not αVß3, align with actin filament bundles, and contain talin, tensin, α-actinin, vinculin, paxillin and a phosphorylated form of focal adhesion kinase. This combination of components differs from the described constituents of the known cell adhesions. Stitch adhesions are organized when protein synthesis and secretion are inhibited by cycloheximide and exogenous fibronectin is provided to the cells. The adhesion stitches described here provide an attractive model system for studying fibronectin fibrillogenesis and the mechanisms governing the formation of cellular adhesions.

Molecular Mechanisms of Melatonin Protection from Gastric Mucosal Apoptotic Injury in Experimental Burns.

Melatonin, a basic secretory pineal gland product, is a nontoxic, multifunctional molecule. It has antioxidant and anti-apoptotic activities and protects tissues from injury. The objective of the present study was to determine the molecular mechanism of melatonin anti-apoptotic effect on gastric injury in a rat burn model. We hypothesized that melatonin gastric protection may be related to the activation of transcription erythroid 2-related factor 2 (Nrf2). Using a 30% total body surface area (TBSA) rat burn model, melatonin (10 mg/kg, i.p.) was injected immediately and 12 h after thermal skin injury. Via light immunohistochemistry, we determined the tissue level of 4-hydroxy-2-nonenal (4-HNE) as a marker of lipid peroxidation, Bcl-2 and Bax as apoptosis-related proteins, and Nrf2. Results are presented as medians (interquartile range (IQR)). Thermal trauma in burned animals, compared with the controls, increased the expression of pro-apoptotic Bax protein (1.37 (0.94-1.47)), decreased anti-apoptotic Bcl-2 protein (1.16 (1.06-1.23), p < 0.001) in epithelial cells, and elevated Bax/Bcl-2 ratios (p < 0.05). Tissue 4-HNE and Nrf2 levels were increased following severe burns (1.55 (0.98-1.61) and 1.16 (1.01-1.25), p < 0.05, respectively). Melatonin significantly decreased 4-HNE (0.87 (0.74-0.96), p < 0.01) and upregulated Nrf2 (1.55 (1.52-1.65), p < 0.001) levels. It also augmented Bax (1.68 (1.5-1.8), p < 0.001) and Bcl-2 expressions (1.96 (1.89-2.01), p < 0.0001), but reduced Bax/Bcl-2 ratios (p < 0.05). Our results suggest that experimental thermal trauma induces oxidative gastric mucosal injury. Melatonin manifests a gastroprotective effect through Nrf2 activation, lipid peroxidation attenuation, and Bax/Bcl-2 ratio modification as well.

Anorectal Melanoma - a Histopathological Case Report and a Review of the Literature.

Primary melanomas of the anus and rectum are rare neoplasms with aggressive behavior, accounting for 0.1%-4.6% of anal canal tumors. Mucosal melanomas account for approximately 1.2% of all melanomas, of which fewer than 25% are anorectal. Histological evaluation with immunohistochemical stains like HMB-45, S-100, vimentin and Melan A is required for definitive diagnosis. The 5-year survival rate for anorectal melanomas (AM) was reported to be as low as < 20%, in contrast to the value of approximately 80% for cutaneous melanomas. Furthermore, up to 67% of patients are found to have distant metastases at the time of their initial diagnosis with AM. Since the chemotherapy treatment possibilities are limited, patients usually undergo mutation detection tests giving the opportunity of targeted therapy. Herein we report a case of a patient with anorectal melanoma, diagnosed in stage II and the pathomorphological and mutation status finding, together with their correlation to tumor behavior and patient prognosis.

Role of the pretreatment 18F-fluorodeoxyglucose positron emission tomography maximal standardized uptake value in predicting outcomes of colon liver metastases and that value's association with Beclin-1 expression.

The current study sought to evaluate the predictive and prognostic performance of the maximum standardized uptake value (SUVmax) prior to treatment in 43 patients with colon cancer and unresectable liver metastases. Patients with colon cancer who underwent 18F-FDG-PET/computed tomography (CT) scans for staging before the start of first-line 5-fluorouracil-based chemotherapy were retrospectively analyzed. Expression of Beclin-1 in cancer cells was evaluated in primary tumors using immunohistochemical staining. The pretreatment SUVmax for liver metastases was not able to predict progression-free survival but was significantly associated with poorer overall survival, with a hazard ratio of 2.05 (95 % CI, 1.016-4.155). Moreover, a negative correlation was noted between SUVmax and expression of a marker of autophagy - Beclin-1 (rho = -0.42, p = 0.006). This suggests that the pretreatment SUVmax in 18F-FDG PET/CT is a useful tool to help predict survival outcome in patients with colon cancer and unresectable liver metastases and may significantly distinguish between patients with low and high levels of Beclin-1 expression (AUC = 0.809, 95% CI: 0.670-0.948, p = 0.001).

Effect of hydrophilic polymers on the wettability, static and dynamic, of solid substrate covered by confluent monolayer of air-damaged SIRC cells.

The aim of this study was to evaluate the possible implementation of hydrophilic polymers as recovery agents in air-damaged corneal cells. The sessile bubble technique was implemented to measure the wetting properties of four selected polymers: hydroxyethyl cellulose (HEC), sodium chondroitin sulphate (SCS), hydroxypropyl-methylcellulose (HPMC) and poloxamer F127 (PO12), at equilibrium conditions and in the case of advancing and receding contact angle. For testing the wetting properties of the polymers, glass slides covered with a confluent monolayer of Statens Seruminstitut rabbit cornea (SIRC) cells were used. HEC showed best properties for a broad concentration range, as the polymer showed capability to maintain low values of the static (equilibrium) contact angle (average static contact angle - 36.07˚, compared to average static compact angles of HPMC - 38.44˚, PO12 - 38.92˚ and SCS - 37.85˚), i.e. better wettability. Sessile bubble technique provides quick, relatively simple and reliable approach for testing surface properties of the listed polymers. The nature of the surface damage produced by the exposition of SIRC cells was used as a plausible model of evaporative dry eye syndrome, and thus the results may have clinical implementation.

Comparison of the activity levels and localization of dipeptidyl peptidase IV in normal and tumor human lung cells.

Dipeptidyl peptidase IV (DPPIV) was studied in three human lung cells - P (fetal lung-derived cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma) using a fluorescent cytochemical procedure developed on the basis of the substrate 4-(glycyl-L-prolyl hydrazido)-N-hexyl-1,8-naphthalimide. The observed differences in the enzyme expression were confirmed by measuring the enzyme hydrolysis of glycyl-L-prolyl-para-nitroanilide. The surface and total dipeptidyl peptidase activities of P cells were correspondingly 7-8 and 3-10 times higher than those of SK-MES-1 and A549 cells. The ratio surface per total activity showed that in P (95%) and A549 (93%) cells the enzyme is associated with the plasmalemma while in SK-MES-1 cells (35%) it is bound to intracellular membranes. In order to compare the results from cell cultures with those in human tumor, the enzyme activity was investigated in cryo-sections of three cases of diagnosed squamous lung carcinoma. DPPIV activity was restricted to the connective tissue stroma surrounding the DPPIV-negative tumor foci.

Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix.

The three-dimensional (3D) cell culture approach offers a means to study cells under conditions that mimic an in vivo environment, thus avoiding the limitations imposed by the conventional two-dimensional (2D) monolayer cell cultures. By using this approach we demonstrated significant differences in the plasma membrane phospholipid composition and susceptibility to oxidation in cells cultured in three-dimensional environment compared to conventional monolayer cultures. The plasma membrane sphingomyelin (SM), which is a functionally active membrane phospholipid, was markedly increased in plasma membranes of 3D cells. To analyze the mechanisms underlying SM accumulation, we determined the activities of sphingolipid-metabolizing enzymes like neutral sphingomyelinase and ceramidase, which are also related to cellular redox homeostasis and to oxidative stress. Fibroblasts cultured in three-dimensional environment showed different redox potential and lower lipid susceptibility to oxidative damage compared to monolayer cells. The relative content of unsaturated fatty acids, which serve as targets of oxidative attack, was observed to be higher in major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, in plasma membranes of 3D cells. The possibility that the higher level of SM, might be responsible for the lower degree of oxidation of 3D phospholipids was tested by selective reduction of SM through treatment with exogenous sphingomyelinase. The results showed that the decrease of plasma membrane SM was accompanied by an increase of the lipid peroxides in both 2D and 3D cells. We presume that culturing as a monolayer is stressful for the cells and leads to activation of certain stress-related enzymes, resulting in reduction of the SM level. Our results show that the lower content of plasma membrane SM in cells cultured as a monolayer renders the phospholipid molecules more susceptible to oxidative stress.

Cell culturing in a three-dimensional matrix affects the localization and properties of plasma membrane cholesterol.

Most in vitro studies use 2-dimensional (2D) monolayer cultures, where cells are forced to adjust to unnatural substrates that differ significantly from the natural 3-dimensional (3D) extracellular matrix that surrounds cells in living organisms. Our analysis demonstrates significant differences in the cholesterol and sphingomyelin content, structural organization and cholesterol susceptibility to oxidation of plasma membranes isolated from cells cultured in 3D cultures compared with conventional 2D cultures. Differences occurred in the asymmetry of cholesterol molecules and the physico-chemical properties of the 2 separate leaflets of plasma membranes in 2D and 3D cultured fibroblasts. Transmembrane distribution of other membrane phospholipids was not different, implying that the cholesterol asymmetry could not be attributed to alterations in the scramblase transport system. Differences were also established in the chemical activity of cholesterol, assessed by its susceptibility to cholesterol oxidase in conventional and "matrix" cell cultures. The influence of plasma membrane sphingomyelin and phospholipid content on cholesterol susceptibility to oxidation in 2D and 3D cells was investigated with exogenous sphingomyelinase (SMase) and phospholipase C (PLC) treatment. Sphingomyelin was more effective than membrane phospholipids in protecting cholesterol from oxidation. We presume that the higher cholesterol/sphingomyelin molar ratio is the reason for the higher rate of cholesterol oxidation in plasma membranes of 3D cells.

Surface properties and behavior of lipid extracts from plasma membranes of cells cultured as monolayer and in tissue-like conditions.

The differences in the surface active properties of native lipids extracted from plasma membranes of cells cultured as a monolayer and in three-dimensional (3D) matrix were investigated. This experimental model was chosen because most of the current knowledge on cellular physiological processes is based on studies performed with conventional monolayer two-dimensional (2D) cell cultures, where cells are forced to adjust to unnaturally rigid surfaces that differ significantly from the natural matrix surrounding cells in living organisms. Differences between monolayer and 3D cells were observed in the lipid composition of plasma membranes and especially in the level of the two major microdomain-forming lipids--sphingomyelin (SM) and cholesterol, which were significantly elevated in 3D cells. The obtained results showed that culturing of cells in in vivo-like environment affected the surface active properties of plasma membrane lipids at interfaces which might influence certain membrane-associated interface processes. The detected differences in the lipid levels in 2D and 3D cell extracts affected significantly the behavior of the model lipid monolayers at the air-water interface (Langmuir monolayers) which resulted in different values of the monolayer equilibrium (gamma(eq)) and dynamic (gamma(max), gamma(min)) surface tension and surface potential. Compensation of the SM content in extracts of 2D cell cultures up to a level close to the one measured in 3D cells approximated the monolayer properties to the values observed for 3D cells. These results implied that the interactions between the cells and the surrounding medium affected the level of plasma membrane SM and other lipids, which had a strong impact on the surface properties of lipid monolayers, such as gamma(eq), gamma(max), and gamma(min), the compression/decompression curve shape, the hysteresis area during cycling of the monolayers, etc. We suggest that the elevated content of SM observed in plasma membranes of 3D fibroblasts could be responsible for an increased rigidity and possibly reduced permeability of cells cultured in 3D environment. The current results provide useful information that should be taken into account in the interpretation of the membrane physico-chemical properties of cells cultured under different conditions.

Three-dimensional matrix induces sustained activation of ERK1/2 via Src/Ras/Raf signaling pathway.

Research in cell signaling often depends on tissue culture, but the artificial substrates used to grow cells in vitro are likely to distort the conclusions, particularly when adhesion-mediated signaling events are investigated. Studies of signal transduction pathways operating in cells grown in three-dimensional (3D) matrices provide a better system, giving a closer insight of the cell signaling in vivo. We compared the steady-state levels of ERK1/2 activity in primary human fibroblasts, induced by cell-derived 3D fibronectin matrix or fibronectin, coated on flat surfaces. 3D environment caused ERK1/2 stimulation concomitant with a 2.5-fold increase in Ras GTP loading and Src activation. Under these conditions FAK autophosphorylation was suppressed. Treatment with Src inhibitor PP2 abolished these effects indicating that 3D fibronectin matrix activated ERK1/2 through Src/Ras/Raf pathway, bypassing FAK. These observations suggest that within in vivo-like conditions Src may have a leading role in the induction of sustained ERK1/2 activation.