Small Tissue Fragments Research Articles

Development of patient-derived lymphomoids with preserved tumor architecture for lymphoma therapy screening

The efficacy of anti-cancer therapies depends on the genomic composition of the tumor, its microenvironment, spatial organization, and intra-tumor heterogeneity. B-cell lymphomas are a heterogeneous group of tumors emerging from B-cells at different stages of differentiation and exhibiting tumor-specific interactions with the tumor microenvironment. Thus, the effect of drug treatments can be influenced by the tumor composition and functional interactions among immune cells. Here, we develop a platform to maintain small fragments of human lymphoma tissue in culture for several days, and use them to test response to small molecules. We collect 27 patient samples representative of different lymphoma subtypes, and establish ex vivo tissue fragments that retain histological, cellular, and molecular characteristics of the original tissue, here referred to as lymphomoids. Using lymphomoids, we test sensitivity to several clinically approved drugs in parallel and examine tissue remodeling upon treatment. Moreover, when this information is available, we show that the effect of the inhibitors observed in lymphomoids is consistent with the patients’ response in the clinic. Thus, lymphomoids represent an innovative ex vivo model to assess the effect of anti-cancer therapies while preserving the tissue structure and its components.

6797 Pituitary Apoplexy as initial Presentation of IgG4-Related hypophysitis

Abstract Disclosure: M. Alameri: None. A. Alnuaimi: None. Introduction: IgG4-related hypophysitis is a rare disease due to IgG4 lymphoplasmacytic infiltration of the pituitary gland. It can either be isolated or part of a multisystemic disease. We report a rare case of pituitary apoplexy as first manifestation of IgG4-RD. Case report: A 27-year-old previously healthy male presented to emergency department with severe headache, nausea, vomiting and visual impairment. CT brain showed a large sellar suprasellar mass of 3cm with thick rim enhancement. Blood test showed evidence of central adrenal insufficiency, central hypothyroidism and secondary hypogonadism. Patient was started on hormone replacement therapy for central adrenal insufficiency and central hypothyroidism. Pituitary magnetic resonance imaging (MRI) demonstrated a 3.1 x 2.4 x 2.4 cm well-defined large sellar suprasellar mass with thick rim enhancement with intrinsic heterogeneous T2 signal with hypointense T1 signal on fat-suppressed post contrast imaging consistent with necrotic haemorrhagic suggestive of pituitary apoplexy and background of hypophysitis. Upward displacement of optic chiasm was noted without involvement of cavernous sinus. The patient was initially treated with high-dose steroids aiming for reduction of the pituitary mass. However, eventually, he underwent transsphenoidal surgery due to alternated level of consciousness. Pituitary histopathology showed that vast majority of the tissue is necrotic. A small fragment of viable anterior pituitary tissue is present, which measures up to 5.5 mm in greatest dimension which exhibited marked chronic inflammation, predominantly comprised of small lymphocytes. Plasma cells were present and staining with IgG/IgG4 revealed two high-power fields with clusters of approximately 25 and 50 IgG4 positive plasma cells, respectively. Blood test showed elevated blood IgG4 levels of >2.60 g/L (normal reference range 0.03-2.01 g/L). Case was discussed at Endocrine MDT meeting which agreed that blood tests result along with histopathology findings were in keeping with IgG4-Related hypophysitis as per the Mayo Clinic, Leporati and Japan-Korea proposed criteria. Because blood IgG4 levels were elevated, a systemic evaluation was performed. No evidence of other systemic disease found. High dose steroid therapy was initiated, which reduced remnant inflammatory sellar changes. Discussion and conclusion: IgG4-related pituitary disease should be considered as differential diagnosis in unusual presentations of pituitary apoplexy in the context of sellar suprasellar hypophysitis. Presentation: 6/2/2024

A Minimally Invasive Surgical Procedure to Harvest Palate Periosteum as a Source of Mesenchymal Stromal/Stem Cells for Bone Tissue Engineering.

The aim of this study is to validate a minimally invasive surgical procedure to harvest palate periosteum as a source of tissue for mesenchymal stromal/stem cells. We performed a standardized procedure to harvest the palate periosteum in ten subjects, which consisted of a 3 mm disposable punch and a Molt periosteal elevator to harvest a small full-thickness fragment of soft tissue at the hard palate area, between the upper bicuspids, 3 to 4 mm apical to the cement enamel junction. The one-third inner portion was fragmented, and following standard cell culture procedures, the adherent cells were cultured for three passages, after obtaining 70-90% confluence. Cell morphology analysis, flow cytometry analysis, and viability and osteogenic differentiation assays were performed. In all 10 cases, uneventful healing was observed, with no need for analgesic intake. The evaluation of cell morphology showed elongated spindle-shaped cells distributed in woven patterns. A high viability range was verified as well as an immunophenotype compatible with mesenchymal stem cell lineage. The differentiation assay showed the potential of the cells to differentiate into the osteogenic lineage. These results demonstrate that the minimally invasive proposed surgical technique is capable of supplying enough periosteum source tissue for stem cell culture and bone tissue engineering.

Computed tomography angiography of liver alveococcosis: a case report

BackgroundAlveococcosis, helminthiasis caused by the larvae of Alveococcus multilocularis, is characterized by the formation of parasitic nodes in the liver. This clinical case is a rare occurrence of liver alveococcosis in Uzbekistan.Case presentationWe present a case of a 33-year-old Asian woman from Uzbekistan who complained of discomfort in the epigastric region and right hypochondrium, along with general weakness. She had been experiencing symptoms for 5 months when a routine ultrasound examination revealed a structural formation in the liver. Patient was investigated by using contrast enhanced computed tomography (CT) and diagnosed with liver alveococcosis with multiple lesions. Patient underwent diagnostic laparotomy with obtaining gross specimen, biomaterial was examined microscopically and found that there were small fragments of fibrous tissue determined together with small groups of cystic formations and walls consisted of chitin. Moreover, contrast enhanced CT allowed us to differentiate liver alveococcosis from cavernous hemangioma, hepatocellular carcinoma, and liver metastases from unknown source.ConclusionContrast enhanced CT plays a major role in differentially diagnosing liver alveococcosis and makes it the first line method of choice for the consideration of the future treatment and surgical interventions.

Применение секретома ММСК для стимуляции репаративного остеогенеза: экспериментальное исследование

The article is devoted to the study of the effect obtained by using a regenerative medicine drug (secretome of multipotent mesenchymal stromal cells) to stimulate reparative regeneration of a long tubular bone after a fracture. The presented materials are a fragment of comprehensive studies on the mechanisms of reparative regeneration of bone tissue under the conditions of the use of cellular products. The work was carried out on outbred white rats, in which the state of the bone regenerate was studied on the 30th and 120th days after injury. After the fracture, the animals were kept under normal conditions, without immobilization or restriction of motor activity. The work used methods of anatomical preparation and light microscopy. Studies have shown that the use of a secretome was accompanied by a more reliable consolidation of fragments behind the ossification of the callus, in the structure of which by the final period of the experiment lamellar bone tissue predominated, against which foci were observed reticulofibrous bone tissue and small fragments of cartilaginous tissue with signs of osteolysis and replacement with bone tissue. In the control group, the reparative process occurred more slowly, foci of cartilaginous tissue remained in the structure of the regenerate for a long time, and the bone tissue was represented mainly by reticulofibrous tissue. A significant amount of connective tissue was revealed between the fragments. All this indicates instability and unreliable consolidation of fragments. Apparently, the secretome of multipotent mesenchymal stromal cells stimulates the repair of bone tissue in fractures by improving the trophic supply in the healing area, against the background of which the formation and remodeling of callus is activated.

Histopathological assessment of depth of coagulation necrosis with Holmium, Moses, and Thulium fiber lasers in human prostate tissue.

To evaluate coagulation necrosis depth (CND) of Holmium (HL), Moses (ML), and Thulium fiber laser (TFL) in ex vivo human prostate tissue at various energy settings. After endoscopic HL enucleation, small prostate tissue fragments were removed from the bladder with graspers and used for study. Immediately after surgery, a single incision was made on the surface of the tissue kept under normal saline at room temperature using a hand-held 550-µm laser fiber. Variable energy settings were tested for all three lasers. Two pathologists measured the CND with light microscopy using ocular micrometer. Impact of various laser settings on CND was analyzed. The differences in CND of all three lasers at similar laser power were compared. Mean CND was 0.56 ± 0.53mm for long-pulse HL, 0.54 ± 0.53mm for ML, 0.67 ± 0.67mm for low-pulse TFL, and 0.81 ± 0.78mm for high-pulse TFL. There was no significant difference between mean CND of HL and ML at various laser settings ranging from 10 to 120 W and CND with long- and short-pulse settings of TFL at settings from 10 to 60 W. There was a trend of increasing CND in HL and ML with increasing laser power; however, it was not statistically significant. TFL had similar tissue effects as HL and ML. There is no significant difference in CND of HL, ML, and TFL in ex vivo human prostate tissue. Other factors besides laser type and settings need to be studied to explain clinical differences among various lasers used for prostate enucleation.

First Report of Bacterial Canker of Pecan Caused by Pseudomonas syringae pv. syringae in China.

Pecan (Carya illinoinensis) is a world-famous nut tree that is widely cultivated in China, especially in Jiangsu Province (Zhang et al. 2015). In April 2022, cankers on trunks were recorded in pecan (cv. Pawnee) fields located in Taizhou (32°27'58″ N, 120°0'49″ E), Jiangsu. Cankers on the trunks resulted in wilt of the plants. Usually, the color of infected bark on the trunk became darker than the healty bark. When the outer bark was peeled away, the inner tissues were water-soaked, often with reddish streaks. In the surveyed orchards, disease incidence ranged from 10 to 20% among young saplings (about 200 three-year-old trees). While no fungal mycelium or spores were found in the diseased areas by microscope, bacterial colonies were isolated by surface-sterilizing small fragments (25 mm2) of symptomatic tissue in 0.5% NaOCl, rinsing the sections twice in sterilized water, and then streaking them on Luria-Bertani (LB) plates. More than 20 bacterial isolates were obtained and all isolates induced a hypersensitive response on Nicotiana tabacum. All isolates were fluorescent on King's medium B, and were gram-negative based on lysis by KOH. Isolates were positive for levan formation, negative for oxidase and arginine dihydrolase, and did not cause soft rot on potato slices. Based on above information, the isolates thus belonged to Lelliot's LOPAT group 1, P. syringae (Lelliott and Stead 1988). The 16S rRNA sequences of five representative isolates (accession numbers OP175939-OP175943) were amplified by PCR, sequenced, and compared with the NCBI GenBank database (Weisburg et al. 1991; Sarkar and Guttman 2004), finding a 99.92% genetic similarity with a previously reported 16S rRNA sequence of a Pseudomonas syringae pv. syringae (Pss) isolate (accession numbers NW389777). Additional housekeeping genes gap1(accession numbers OP186937-OP186941), rpoD (accession numbers OP186952-OP186956), gyrB (accession numbers OP186947-OP186951), and gltA (accession numbers OP186942-OP186946) were PCR-amplified and sequenced as reported by Hwang et al. (2005), followed by multilocus sequence typing analysis (MLSA). Molecular phylogenetic trees (MEGA vesion 6.0, maximum likelihood with Jukes-Cantor model, 1,000 bootstraps) were generated based on each of these five DNA regions and revealed that all five isolates were clustered together with the strains in P. syringae genomospecies 2, and grouped these isolates with Pss in the PAMDB database (Hwang et al. 2005). As a result, these isolates were identified as Pss. Pathogenicity on pecan (cv. Pawnee) was confirmed by cutting the trunks of two-year-old pecan trees with sterilized blades dipped in cell suspensions containing 107 CFU/ml of each isolate. Plants inoculated in a similar manner with sterile water served as negative controls. The inoculated plants were incubated in a greenhouse maintained at 25°C and 80% relative humidity. After 7 to 8 days, all inoculated plants showed the symptoms of necrosis previously described for the original field plants, while the control plants did not show symptoms. The bacteria reisolated from the inoculated plants were identified as Pss using the LOPAT tests. These results and the sequence analysis of the 16S rRNA and four housekeeping genes described above, fulfilled Koch's postulates. No target bacteria were isolated from the control plants. To our knowledge, this is the first report of Pseudomonas syringae pv. syringaecausing bacterial canker of pecan worldwide. The identification of this pathogen will allow the study of strategies for managing the disease.

"Diagnostic Value of Processing Small Fragments of Bone Marrow Tissue Obtained During an Unsuccessful Attempt to Obtain an Adequate Trephine (Core) Biopsy Specimen"

"Diagnostic Value of Processing Small Fragments of Bone Marrow Tissue Obtained During an Unsuccessful Attempt to Obtain an Adequate Trephine (Core) Biopsy Specimen"

Slide-to-Slide Tissue Transfer and Array Assembly From Limited Samples for Comprehensive Molecular Profiling

Tissue microarrays (TMA) have become an important tool in high-throughput molecular profiling of tissue samples in the translational research setting. Unfortunately, high-throughput profiling in small biopsy specimens or rare tumor samples (eg, orphan diseases or unusual tumors) is often precluded owing to limited amounts of tissue. To overcome these challenges, we devised a method that allows tissue transfer and construction of TMAs from individual 2- to 5-μm sections for subsequent molecular profiling. We named the technique slide-to-slide (STS) transfer, and it requires a series of chemical exposures (so-called xylene-methacrylate exchange) in combination with rehydrated lifting, microdissection of donor tissues into multiple small tissue fragments (methacrylate-tissue tiles), and subsequent remounting on separate recipient slides (STS array slide). We developed the STS technique by assessing the efficacy and analytical performance using the following key metrics: (a) dropout rate, (b) transfer efficacy, (c) success rates using different antigen-retrieval methods, (d) success rates of immunohistochemical stains, (e) fluorescent in situ hybridization success rates, and (f) DNA and (g) RNA extraction yields from single slides, which all functioned appropriately. The dropout rate ranged from 0.7% to 6.2%; however, we applied the same STS technique successfully to fill these dropouts (“rescue” transfer). Hematoxylin and eosin assessment of donor slides confirmed a transfer efficacy of >93%, depending on the size of the tissue (range, 76%-100%). Fluorescent in situ hybridization success rates and nucleic acid yields were comparable with those of traditional workflows. In this study, we present a quick, reliable, and cost-effective method that offers the key advantages of TMAs and other molecular techniques—even when tissue is sparse. The perspectives of this technology in biomedical sciences and clinical practice are promising, given that it allows laboratories to create more data with less tissue.

Real-Time Fluorescence Imaging to Identify Cholangiocarcinoma in the Extrahepatic Biliary Tree Using an Enzyme-Activatable Probe

Introduction: Complete resection is the only possible treatment for cholangiocarcinoma in the extrahepatic biliary tree (eCCA), although current imaging modalities are limited in their ability to accurately diagnose longitudinal spread. We aimed to develop fluorescence imaging techniques for real-time identification of eCCA using an enzyme-activatable probe, which emits fluorescence immediately after activation by a cancer-specific enzyme. Methods: Using lysates and small tissue fragments collected from surgically resected specimens, we selected the most specific probe for eCCA from among 800 enzyme-activatable probes. The selected probe was directly sprayed onto resected specimens and fluorescence images were acquired; these images were evaluated for diagnostic accuracy. We also comprehensively searched for enzymes that could activate the probe, then compared their expression levels in cancer and non-cancer tissues. Results: Analyses of 19 samples (four cancer lysates, seven non-cancer lysates, and eight bile samples) and 54 tissue fragments (13 cancer tissues and 41 non-cancer tissues) revealed that PM-2MeSiR was the most specific fluorophore for eCCA. Fluorescence images of 7 patients were obtained; these images enabled rapid identification of cancerous regions, which closely matched histopathology findings in 4 patients. Puromycin-sensitive aminopeptidase was identified as the enzyme that might activate the probe, and its expression was upregulated in eCCA. Conclusion: Fluorescence imaging with PM-2MeSiR, which may be activated by puromycin-sensitive aminopeptidase, yielded generally high accuracy. This technique may be useful for real-time identification of the spread of eCCA during surgery and endoscopic examinations.

Analogy between the oral microbial flora found at the level of carious lesions, endodontic spaces and prosthetic components

Nowadays, eating habits and lifestyles are moving towards a diet high in sugars and carbohydrates, which is extremely harmful to both oral integrity and systemic health. The aim of the study was to identify the variations between the bacterial plaque formed in carious lesions, infected root canals and prosthetic components, in comparison with commensal oral microflora. The study was carried out on a group of 21 patients aged between 7 and 84 years, 13 women and 8 men, with various oral diseases, which were diagnosed during the specialized examination. The preparation of the medical record, the signing of the informed consent, the anamnesis and the consultation were carried out in the private dental office where the patients presented for a personalized treatment plan, and the examination of the pathological products and the interpretation of the results obtained in the Microbiology Discipline, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania. The study carried out on the preparations taken from patients with carious lesions reveals the presence of numerous G+ cocci arranged in short chains (23%), frequent G+ cocci in diplo, thin G+ bacilli with rounded ends and G+ cocci in diplo, arranged in small piles (15 %), and values lower than 8% are represented by G+ in diplo cocci, lanceolate, rare G+ in diplo cocci, very rare fusiform bacilli and dentinal fragments. The study carried out on the preparations taken from the endodontic space showed a remarkable percentage of epithelial cells and leukocytes (28%), rare fusiform bacilli, very small fragments of epithelial tissue, thin G+ bacilli with rounded ends, G+ cocci in diplo and in small piles and buns arranged in short chains (5-6%). The study carried out on the preparations taken from the level of prosthetic components, recorded a relatively high number of epithelial cells (19%), G+ cocci arranged in diplo, lanceolate and in small piles (13%), thin G+ bacilli, with rounded ends (10 %), cocci in short chains (10%) and rare polymorphonuclear leukocytes (10%), while at the opposite pole, squamous epithelial cells, fusobacteria and pleomorphic forms (3%) are found.

Transplantation of small ovarian tissue fragments using pipelle device is effective: method evaluation and reproductive outcomes.

To assess the feasibility, effectiveness, and reproductive outcomes of transplantation of tiny cryopreserved ovarian pieces through a pipelle cannula during laparoscopic surgery. A retrospective study of patients who underwent ovarian tissue transplantation for fertility restoration between 2004 and 2022. The "pipelle group" had their ovarian cortex cut into tiny pieces of ~ 1-2 mm3 before cryopreservation. The pieces were too small to be handled and transplanted via standard laparoscopic tools. Transplantation was performed using a pipelle cannula during laparoscopic surgery. The "control group" underwent transplants of ovarian cortex pieces 1-2mm thick, measuring approximately 25-50 mm2 pieces, using standard procedures. The pipelle group consisted of 4 patients aged 19, 21, 27, and 28years old at ovarian tissue cryopreservation (OTC). The control group consisted of 14 patients aged 21-30years old. All pipelle patients restored their endocrine activity, and all of them conceived. FSH levels dropped during the first 3months following the pipelle transplant. IVF cycle outcomes were similar for both groups. All patients from the pipelle group conceived, resulting in 5 pregnancies and 4 live births (one patient had 2 deliveries, and one additional pregnancy is ongoing), compared to the control group, where 8 patients achieved a total of 20 pregnancies and 18 live births. Pipelle transplantation for tiny cryopreserved ovarian pieces is feasible and effective. This study opens a door for patients who had their ovaries cut into small pieces and may even simplify the procedure in some instances, making ovarian transplant more accessible. (#6531-19-SMC) [18/09/2019].

Severe dieback and mortality of wild olive trees associated with Phytophthora species in Italy

Wild olive (Olea europaea var. sylvestris) is a sclerophyllous drought-tolerant tree native to the Mediterranean basin, which is used as a hardy and disease-resistant rootstock on which scions of cultivated olive varieties are grafted (Breton et al., 2006). In Sardinia, Italy, wild olive is a significant component of the agroforestry landscape, and its ancient origin is proven by the presence of several millennial trees. In March 2022, extensive dieback and mortality of wild olive trees were observed in an area of c. 200 ha, located 5 km south from Paulilatino in central Sardinia (Fig. 1). Three separate plots were examined and in each the trees showed severe leaf chlorosis, wilting or defoliation of the whole crown (Fig. 2). Often these symptoms were associated with root rot and necrotic lesions on the feeder roots (Fig. 3). In the investigated sites, estimated tree mortality and disease incidence were 60 and 80%, respectively. Rhizosphere soil was sampled from a total of 16 unhealthy trees and subjected to a baiting technique, using fresh Ceratonia siliqua leaves as baits (Perez-Sierra et al., 2022). Isolations were also made by plating small fragments of root phloem tissue from lesion margins onto Phytophthora selective medium (SMA) (Brasier et al., 2005). A homothallic Phytophthora species was consistently isolated from 14 of the sampled trees. An additional Phytophthora species was detected from the rhizosphere of four diseased trees. Developing colonies were transferred onto carrot agar, and DNA extracted for amplification and sequencing of ITS and cox1 genes. A BLAST search in GenBank showed 99–100% identity with sequences of the ex-type cultures of P. bilorbang (CBS131653) and P. pseudocryptogea (CBS139749). Sequences of a representative isolate for each species were submitted to GenBank (Accession Nos. OP801844, OP801845, OP856579 and OP856580). The pathogenicity of both Phytophthora species was verified using six-month-old wild olive seedlings and the soil infestation method (Perez-Sierra et al., 2022). Three weeks after inoculation with P. bilorbang and P. pseudocryptogea, all seedlings showed wilting and dieback, and the fine root weight of inoculated plants was reduced by 68 and 42%, respectively, in relation to non-inoculated controls (Fig. 4). Both species were re-isolated from necrotic roots, thus fulfilling Koch's postulates. This is the first report of P. bilorbang on wild olive worldwide. However, this species was recently detected in Calabria, Italy, causing root rot on 15-year-old cultivated olive trees originating from Sardinia (Santilli et al., 2020). In contrast, P. pseudocryptogea was previously reported on wild olive in Spain together with other Phytophthora species (González et al., 2019). Further investigations are required to better understand the epidemiology of the disease and other possible factors involved in the decline. A consequence of this damaging new disease is that further use of wild olive as a source of rootstock for grafting olive varieties could be limited. We thank the local municipality of Paulilatino for their logistic support in the field surveys.

First report of black rot caused by Xanthomonas nasturtii on watercress in Spain and Portugal

Xanthomonas nasturtii was first identified as the cause of black rot of watercress (Nasturtium officinale) on plants grown in Florida, USA (Vicente et al., 2017). Similar symptoms had been reported earlier in Hawaii (McHugh & Constantinides, 2004) and an unidentified Xanthomonas sp. was isolated in Portugal in 2003 from wild watercress (Cruz et al., 2017). Since 2017, watercress crops in southern Spain have occasionally exhibited small yellow leaf lesions around the hydathodes, leaf spots, V-shaped leaf lesions, wilt, distortion and senescence (Fig. 1). Symptoms were more frequent during mild and humid periods from November to February, reducing yields by up to 60% and leading to rejection of some crops (Lascelles, 2019). In 2021, symptoms were seen on watercress produced in Portugal and sold in the UK. Isolations were made from watercress leaves showing typical symptoms between 2017 and 2021 by excising and macerating small tissue fragments in sterile water and plating on King's medium B (KB). Seed produced in Spain was tested using a method based on the International Seed Testing Association method for detection of Xanthomonas campestris pv. campestris. Mixtures of bacterial colonies were observed on the plates after incubation for 48–72 hours at 28°C and colonies resembling Xanthomonas (yellow, mucoid) were sub-cultured. Isolates were purified and stored at -76°C in nutrient broth with 150 g/l glycerol (Vicente et al., 2017) and/or at -80°C in Protect® (Protect System, UK). In contrast to the type strain (NCPPB 4600) from Florida, isolates from Spain and Portugal did not cause browning of the KB medium (Fig. 2). Pathogenicity was tested on plants of three watercress accessions and Savoy cabbage cv. Wirosa F1 (Bejo Zaden B.V., The Netherlands) by stabbing with an insect pin charged with bacteria (Vicente et al., 2017). The Xanthomonas-like isolates were pathogenic to all three watercress accessions (Fig. 3) whereas no symptoms were observed on cabbage. To complete Koch's postulates, re-isolations were made from symptomatic watercress leaves inoculated with isolates WHRI 10015A (from Spain) and 10049A (from Portugal). Isolates with the same morphological characteristics were obtained. Fatty acid profiling (Weller et al., 2000) was performed on isolates grown on trypticase soy agar plates at 28°C for 48 hours; comparisons with libraries that did not include X. nasturtii, indicated the highest matches of 0.38 to 0.68 with X. axonopodis and X. campestris. DNA was extracted as outlined in Vicente et al. (2017). The partial 16S rRNA gene was amplified and sequenced for isolates from Spain and confirmed their identity as a Xanthomonas species. The partial gyrB gene was amplified and sequenced for selected isolates according to Parkinson et al. (2007) (Table 1). The phylogeny (Fig. 4) confirmed that isolates from Spain and Portugal belong to X. nasturtii and shows that there is some genetic diversity amongst them. This is the first report of X. nasturtii on watercress crops in Europe. We suspect that black rot of watercress may be widespread in production areas of the Iberian Peninsula and, as the crop is grown in water beds, the pathogen is likely to spread relatively easily. Based on detection in a watercress seed lot produced in Spain, the pathogen may be seed borne and therefore it is important to test watercress seed to avoid introducing the disease into clean beds and new areas/countries. This work was funded by Royalcress S.A., The Watercress Company, UKRI, Defra and the Scottish Government.

KERATOCYST OF THE BUCCAL MUCOSA: A CASE REPORT

Keratocyst of the buccal mucosa is rare, with approximately 20 cases reported to date. A 58-year-old male patient was referred for evaluation of an exuberant swelling of the midface of 2 years' duration. Intraoral examination revealed an asymptomatic tender swelling in the right buccal mucosa, with a smooth surface and normal coloration, measuring 5 cm. Extraosseous location was confirmed by panoramic radiographic examination. Aspiration revealed a yellowish creamy material. Incisional biopsy was performed, and multiple small soft tissue fragments were microscopically evaluated. There was a cyst lined with 6 cell layers composed of columnar palisading basal cells and a corrugated parakeratinizing surface. The capsule was composed of fibrous connective tissue with blood vessels, areas of hemorrhage, nerve bundles, and absence of pilosebaceous units. The final diagnosis was of keratocyst of the buccal mucosa. The odontogenic origin of keratocyst from buccal mucosa is still debatable.

First Report of Sea Buckthorn Stem Wilt Caused by Fusarium proliferatum in Liaoning, China.

Sea buckthorn（Hippophae rhamnoides L.） is a flowering shrub native to cold-temperate regions of Eurasia, which is also valuable for its berries and leaves containing various vitamins and flavonoids (Pundir et al. 2021). In late June 2020, high mortality (more than 70%) was observed in sea buckthorn in a 1.6-ha seedling nursery in Chaoyang City, Liaoning province, China, where 16 Chinese and Russian cultivars (cv.) had been planted since 2014 (cv. Shenqiuhong, eshi01 through eshi15). The mortality of two introduced sea buckthorn varieties (eshi02, eshi04) was 100% (125 trees died in total). The symptoms include massive drooping leaves and dried-up stems on 6-year-old infected trees. Pieces of tree roots and stems with brown discoloration in the xylem vessels were selected. Small tissue fragments (0.2-0.5 cm) were surface disinfested (3 min in 75% ethanol, rinsed with sterile distilled water), air-dried, and placed on potato dextrose agar (PDA) medium for 5 days at 25°C in the dark. A fungus was consistently isolated from both diseased roots and stems tissues, and a representative isolate (LC-1) was harvested. Genomic DNA was extracted for amplification and sequencing of the partial translation elongation factor-1α (EF1 and EF2 primers, accession Nos. MZ669853) (O'Donnell et al. 1998) and RNA polymerase II second largest subunit (RPB2) (7cf/11aR primers, accession Nos. MZ669854) (O'Donnell et al. 2007). The sequences were further analyzed at the Fusarium MLST (https://fusarium.mycobank.org/) for identity confirmation, and showed 99.8% (over 95.2% query coverage) and 96.4% (over 88.4% query coverage) similarity to Fusarium proliferatum (NRRL 13584, 13591). Isolates on Spezieller Nahrstoffarmer agar (SNA) produced abundant aerial white mycelia and yellow pigmentation. The 30 macroconidia measured ranged from 28.5 - 62.5 × 3.2 - 5.4 μm, were thin, slender, with 3-5 septa. The aseptate microconidia ranged from 4.7 - 13.6 × 2.2 - 4.3 μm (n = 30). Pathogenicity tests were performed on healthy, potted 1-year-old sea buckthorn seedlings (cv. eshi05) using two isolates in a greenhouse at 25 °C, 80% relative humidity, and 12-hour light/dark photoperiod. Ten potted seedlings were inoculated on the stems by placing a 5-mm-diameter mycelial plug (5-day-old PDA cultures for each isolate) into the surface of a wound created with a needle, and the inoculation sites were covered with Parafilm to maintain moisture. Ten seedlings were inoculated with PDA plugs as controls. Six to ten days after inoculation, color of the leaves in the middle of the stems was variegated, and then dark necrotic lesions on leaf margins were observed. Three weeks after inoculation, 80% of inoculated stems were wilted, while control plants remained asymptomatic. The pathogen was consistently re-isolated and the recovered isolates were identified as F. proliferatum by amplifying the EF-1α gene. The typical symptoms on inoculated plants were dark to brown necrotic lesions on chlorotic leaves initially, and black withered stems in the terminal stage, similar to those observed on sea buckthorn trees infected with Fusarium sporotrichioides in Gansu and Heilongjiang provinces (Song et al. 2010; Xia et al. 2021). To our knowledge, this is the first report of sea buckthorn stem wilt caused by F. proliferatum in Liaoning province, China, which will be beneficial for expanding knowledge of Fusarium disease in sea buckthorn and provide more information for sustainable disease management in sea buckthorn.

Tumor-Infiltrating T Cells Can Be ExpandedSuccessfully from Primary Uveal Melanoma after Separation from Their TumorEnvironment.

PurposeTo evaluate whether expanded tumor-infiltrating lymphocytes (TILs) can be obtained from primary uveal melanoma (UM) for potential use as adjuvant treatment in patients at risk of developing metastatic disease.DesignExperimental research study.ParticipantsFreshly obtained primary UM from 30 patients.MethodsThree different methods were used to expand TILs: (1) direct culture from small fragments of fresh tumor tissue, (2) single-cell tissue preparation by enzymatic digestion and subsequent enrichment of mononuclear cells, and (3) selection of CD3+ T cells using magnetic beads. Surface expression of costimulatory and inhibitory T-cell markers and T-cell reactivity against autologous tumor cells was assessed. Clinical, histopathologic, genetic, and immunologic characteristics of the tumors were compared with the capacity to expand TILs and with their reactivity against autologous tumor cells.Main Outcome MeasuresThe feasibility of expanding TILs from primary UM, testing their reactivity to autologous UM cells, and evaluating the impact of an immunomodulatory environment.ResultsDirect culture of tumor parts led to successful TIL culture in 4 of 22 tumors (18%), enrichment of mononuclear cells gave rise to TILs in 5 of 12 tumors (42%), while preselection of CD3+ T cells with magnetic beads resulted in TIL expansion in 17 of 25 tumors (68%). In 8 of 17 tumors (47%), the TIL cultures comprised UM-reactive T cells. The presence of UM-reactive T cells among TILs was not related to clinical, histologic, genetic, or immunological tumor characteristics. Interestingly, RNA-Seq analysis showed that approximately half of the UM tumors displayed an increased expression of immunomodulatory molecules related to T-cell suppression, such as galectin 3, programmed death-ligand 1, cytotoxic T-lymphocyte-associated protein 4, indoleamine 2,3-dioxygenase 1, and lymphocyte activating 3, potentially explaining why T cells require optimal removal of tumor components for expansion.ConclusionsThe need to separate TILs from their tumor microenvironment for their successful expansion and the presence of UM-reactive T cells among TILs suggests that these UM-reactive T cells are strongly suppressed in vivo and that UM is immunogenic. These findings indicate that adoptive TIL therapy could be an option as an adjuvant treatment in primary UM patients at high risk of developing metastatic disease.

Studying Mechanical Oscillations During Whole-Body Regeneration in Hydra.

Cells of the freshwater cnidarian Hydra possess an exceptional regeneration ability. In small groups of these cells, organizer centers emerge spontaneously and instruct the patterning of the surrounding population into a new animal. This property makes them an excellent model system to study the general rules of self-organization. A small tissue fragment or a clump of randomly aggregated cells can form a hollow spheroid that is able to establish a body axis de novo. Interestingly, mechanical oscillations (inflation/deflation cycles of the spheroid) driven by osmosis accompany the successful establishment of axial polarity. Here we describe different approaches for generating Hydra tissue spheroids, along with imaging and image analysis techniques to investigate their mechanical behavior.

Multi-System Histiocytosis of Langerhans Cells in Boys Aged 2 Years (Histopathology Review)

Langerhans cell histiocytosis (HSL) or histiocytosis X, eosinophilic granuloma, letterer siwe disease, hand schuller christian disease and hashimoto pritzker disease is a disorder caused by the accumulation and proliferation of langerhans cells in various organs of the body that causes tissue damage. The highest prevalence of HSL occurs in children less than 3 years old, with a ratio of 2:1 for boys and girls. The classic appearance of skin manifestations is an eruption resembling seborrheic dermatitis in the folds, axillae, scalp, retroauricular and trunk areas. The appearance of the lesion is yellow to brownish red papules, purpuric and crusted papules that can occur together with erosion. Histopathological examination with staining with S100 or cluster of differentiation 1a (CD1a) revealed Langerhans cell infiltration. The results of immunohistochemistry with IHC S-100 staining, CD1a showed a picture of Langerhans cells and histiocytes. The purpose of this paper is to study the histopathological features of HSL to better than to diagnosis and treatment of HSL. A 2-year-old boy was consulted with the chief complaint of itchy red nodules on the head, chest and back area. Complaints accompanied by diarrhea, often feel thirsty and hungry and lose weight. Dermatological status in the facial region, the anterior and posterior trunks showed lenticular papules with umbilication (pin points) with erosions covered with necrotic tissue above, on the scalp, facial, retroauricular and trunk regions, yellow to brownish papules were crusted with erosions. Histopathology with hematoxylin and eosin (HE) staining showed the presence of small fragments of skin tissue, the epidermis was partially composed intact, the dermis was filled with histiocytes, plasma cells, eosinophils, mast cell impressions and polymorphonuclear leukocytes. Immunohistochemical examination with staining IHC S-100 and CD1a showed positive. Histiocytosis is a group of disorders characterized by the accumulation of cells derived from dendritic cells accompanied by inflammation and causing tissue damage. Histopathological and immunohistochemical examinations serve to establish the diagnosis. The results of immunohistochemical examination with IHC S-100 staining, CD1a will show a picture of Langerhans cells and histiocytes

Three-dimensional cell culture for the study of nasal polyps

ObjectivesThree-dimensional (3D) cell cultures have many applications such as stem cell biology research, new drug discovery, cancer, and Chronic Rhinosinusitis with Nasal Polyps (CRSwNP). This disease is characterized by a significant impact on quality of life and productivity. The diversity of factors that act in the progression of CRSwNP point to the creation of a cell culture model that allows the integration of different cell types with extracellular matrix. This work aimed to create a cell culture model in 3 dimensions (spheroids) for the study of Nasal Polyposis. MethodsNasal polyp tissue from patients diagnosed with CRSwNP was mechanically dissociated using tweezers and a scalpel and the solution containing cells and small aggregates of nasal polyps was transferred to a Petri dish containing 5 mL of culture medium at the concentration of 106 cells/mL. ResultsThe spheroids were cultivated for 20 days, fixed and analyzed using confocal microscopy. In a 3D culture environment, the spheroids were formed both by clustering cells and from small tissue fragments. In the cultures analyzed, the ciliary beat was present from the dissociation of the cells up to 20 days in culture. ConclusionOur findings also point to these characteristics showing the environment generated in our study, the cells remained differentiated for a longer time and with ciliary beating. Thus, this work shows that nasal polyp-derived cells can be maintained in a 3D environment, enabling better strategies for understanding CRSwNP in situations similar to those found in vivo. Level of evidenceLaboratory studies.