Efficiency of neutral honey as a tissue fixative in histopathology

In the routine laboratory, 10% neutral buffered formalin (NBF) is the fixative of choice. However, formalin is a human carcinogen. To the best of our knowledge, neutral honey, not natural or artificial honey, has not been tested to fix histological tissues. This study aimed to examine the efficiency of neutral buffered honey and other types of honey fixatives to fix histological tissues. The most two natural common Omani honey were used as fixatives, namely Sumar and date. We tested samples of rat liver, kidney, and stomach. Nine types of fixatives were used. All tissues were treated equally. The evaluation was performed blindly by three senior biomedical scientists who work in a histopathology laboratory. Hematoxylin and eosin showed adequate staining in all groups when compared to 10% NBF. The intensity and specificity of Jones Methenamine silver stain in 10% Sumer and Date honey and 10% alcoholic Sumer honey showed similar findings of 10% NBF. The specificity and intensity of all groups for Periodic acid–Schiff were comparable with 10% neutral buffered formalin accepts for 10% Sumer honey and 10% Alcoholic Date honey. However, all honey groups showed weak staining for the reticulin fibers using Gordon and Sweets method. Vimentin showed comparable findings with 10% NBF as there were no significant differences. The findings of this study are promising. Further in depth research on honey as a possible safe substitute fixative for formalin should be conducted.

Morphological aspects of orbital defect reconstruction in rats with elastin-based biomaterial

The objective of the study was to identify morphological aspects of replacement of xenogeneic decellularized elastin matrix (ХDEM) transplanted into a bone defect of the upper orbital wall in rats. Materials and methods. The experiment was performed on 60 Wistar rats with artificially created 7×4 mm defect in the upper edge of their orbit. In the experimental group (n=30), DХEM was placed in the defect area. Its size matched the size of the defect, and it was attached with a suture material (50 μm silk). Soft tissues were sutured layer by layer in the control group (n=30). Tissue excision was performed after 1, 3 and 12 months. Histological, immunohistochemical and electron microscopic methods were employed. Results. We were gradually replacing DХEM with bone tissue against the background of a pronounced reaction of CD68+/MMP-9+ macrophages, which implied its resorption and lysis. Osteogenesis occurred via intramembranous ossification and endochondral ossification, which was preceded by centripetal migration of endothelial kidneys with subsequent differentiation into capillaries and overgrowth of loose fibrous connective tissue containing progenitor cells. The microenvironment, represented by reticulin fibers, TGF-β1, and sulfated glycosaminoglycans, could contribute to the differentiation of progenitor cells in the osteogenic direction and to osteogenesis per se. In the control group, the defect remained open throughout the experiment. Conclusion. Decellularized biomaterial, based on elastin matrix, has osteoconductive and osteoinductive properties and can serve an adequate biomimetic for reconstruction of the bone defects.

Calreticulin del52 and ins5 knock-in mice recapitulate different myeloproliferative phenotypes observed in patients with MPN

Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations, including the two most frequent 52 bp deletion (del52) and 5 bp insertion (ins5), induce a frameshift to the same alternative reading frame generating new C-terminal tails. In patients, del52 and ins5 induce two phenotypically distinct myeloproliferative neoplasms (MPNs). They are equally found in ET, but del52 is more frequent in PMF. We generated heterozygous and homozygous conditional inducible knock-in (KI) mice expressing a chimeric murine CALR del52 or ins5 with the human mutated C-terminal tail to investigate their pathogenic effects on hematopoiesis. Del52 induces greater phenotypic changes than ins5 including thrombocytosis, leukocytosis, splenomegaly, bone marrow hypocellularity, megakaryocytic lineage amplification, expansion and competitive advantage of the hematopoietic stem cell compartment. Homozygosity amplifies these features, suggesting a distinct contribution of homozygous clones to human MPNs. Moreover, homozygous del52 KI mice display features of a penetrant myelofibrosis-like disorder with extramedullary hematopoiesis linked to splenomegaly, megakaryocyte hyperplasia and the presence of reticulin fibers. Overall, modeling del52 and ins5 mutations in mice successfully recapitulates the differences in phenotypes observed in patients.

AB1049 CLINICAL SPECTRUM AND THERAPEUTIC MANAGEMENT OF AUTO-IMMUNE MYELOFIBROSIS: A NATION-WIDE STUDY OF 30 CASES

Background:Little is known about autoimmune myelofibrosis (AIMF), a rare entity that can occur alone or in association with another autoimmune disease (AID) and is responsible for bone marrow (BM) failure and life-threatening complications.Objectives:We conducted a nationwide retrospective observational study of AIMF cases to better characterize the epidemiology, clinical presentation and evolution of this rare entity.Methods:The aim of the study was to analyze the characteristics of AIMF and the nature and indication of treatments currently used. Response to treatment was evaluated by the revised Tefferi et al. response criteria.Results:Among 30 cases of AIMF, the sex ratio (F/M) was 4:1 and the median age at diagnosis was 37 years (interquartile range 30–49). AIMF was diagnosed after the onset of an associated AID in 12 cases and concomitant to an AID in the remaining 18 cases. The most frequently associated AID was systemic lupus erythematous, followed by Sjögren syndrome. All cases consisted of reticulin fibers, and no collagen fibrosis was described. More than 50% of cases showed complete response after first-line therapy, with glucocorticoids (GC) in 28 cases. Half of the cases had treatment complications mainly related to GC therapy.Conclusion:Diagnosis of AIMF remains challenging in the absence of a validated set of diagnosis criteria, and must always be searched in the presence of hematological abnormalities at onset or during follow-up of AID. Clinical context, search for mutations and pathology findings can help differentiating this rare disease from a clonal pathology. GC is currently an effective first-choice therapy for AIMF, but a high rate of GC dependency and long-term complications indicate the need to find new sparing drugs.Disclosure of Interests:PHILIPPE MERTZ: None declared, Emilie Chalayer: None declared, Jean Sibilia: None declared, Jacques-Eric Gottenberg Grant/research support from: BMS, Pfizer, Consultant of: BMS, Sanofi-Genzyme, UCB, Speakers bureau: Abbvie, Eli Lilly and Co., Roche, Sanofi-Genzyme, UCB, Anne-Sophie Korganow: None declared, Laurent Arnaud: None declared, Thierry Martin: None declared

Evaluation of angiogenesis as a prognostic marker in prostatic neoplasm especially carcinoma of prostate

Background: Prostatic carcinoma shows an unusually wide range of biological potential with well-known disparity between incidence and mortality for disease. Clinico-pathological studies suggests that angiogenesis and tumor neovascularity contributes to pathogenesis of prostate cancer. The aim of this study to present study was done to assess the validity of angiogenesis as a suitable prognostic marker in various prostatic disease specially the neoplasm’s including the malignant ones. Settings and design are Retrospective study.Methods: The present study of evaluation of angiogenesis as a prognostic marker in prostatic neoplasm especially carcinoma of prostate was done with 40 biopsy sample. The biopsy sample were obtained by suprapubic prostatectomy specimen and trans rectal needle biopsy specimen. Tissue sections were subjected to routine H and E staining. For demonstration of angiogenesis staining for reticulin fibers was applied.Results: The microvessels density increases as the severity of lesion increases from benign to pre-neoplastic to frankly malignant. The micro vessel density in malignant lesions is approximately thrice that in benign lesions. Conclusions: Very few studies have been done in prostatic lesions Hence an attempt is made to demonstrate and correlate angiogenesis as a tumour marker.

Evaluation of antifibrotic and antifungal combined therapies in experimental pulmonary paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the Paracoccidioides genus. Most of the patients with chronic form present sequelae, like pulmonary fibrosis, with no effective treatment, leading to impaired lung functions. In the present study, we aimed to investigate the antifibrotic activity of three compounds: pentoxifylline (PTX), azithromycin (AZT), and thalidomide (Thal) in a murine model of pulmonary PCM treated with itraconazole (ITC) or cotrimoxazole (CMX). BALB/c mice were inoculated with P. brasiliensis (Pb) by the intratracheal route and after 8 weeks, they were submitted to one of the following six treatments: PTX/ITC, PTX/CMX, AZT/ITC, AZT/CMX, Thal/ITC, and Thal/CMX. After 8 weeks of treatment, the lungs were collected for determination of fungal burden, production of OH-proline, deposition of reticulin fibers, and pulmonary concentrations of cytokines and growth factors. Pb-infected mice treated with PTX/ITC presented a reduction in the pulmonary concentrations of OH-proline, associated with lower concentrations of interleukin (IL)-6, IL-17, and transforming growth factor (TGF)-β1 and higher concentrations of IL-10 compared to the controls. The Pb-infected mice treated with AZT/CMX exhibited decreased pulmonary concentrations of OH-proline associated with lower levels of TGF-β1, and higher levels of IL-10 compared controls. The mice treated with ITC/Thal and CMX/Thal showed intense weight loss, increased deposition of reticulin fibers, high pulmonary concentrations of CCL3, IFN-γ and VEGF, and decreased concentrations of IL-6, IL-1β, IL-17, and TGF-β1. In conclusion, our findings reinforce the antifibrotic role of PTX only when associated with ITC, and AZT only when associated with CMX, but Thal did not show any action upon addition.

The Hypomorphic Gata1low Mutation Induces Fibrosis in Multiple Organs

Mice carrying the hypomorphic mutation which reduces the transcription factor GATA1 in megakaryocytes (Gata1low mice), develop by 8-months myelofibrosis a phenotype resembling primary myelofibrosis, the most severe of myeloproliferative neoplasms (Vannucchi et al Blood 2002;100:1123). The high levels of TGF-β expressed by the abnormal Gata1low megakaryocytes that drive myelofibrosis in these mice alter the bone marrow (BM) expression profiling up-regulating expression of the transcription factor c-Jun (Ciaffoni et al BCD 2015; 54:234). Recently, Dr. Weissman reported that c-Jun over-expression in response to TGF-β activation induces in mice fibrosis in multiple organs (Werning et al PNAS 2017, 114, 4757), suggesting that also Gata1low mice may develop multi-organ fibrosis. To test this hypothesis, we compared morphology (by haematoxylin-eosin staining) and fibrosis (reticulin fibres by trichrome Mallory and collagen fibres by Gomory or Sirius Red Picrate staining) of organs from Gata 1low and wild-type (WT) mice at 1-, 8- and 15-months of age (3 mice/group). With age, the organs from WT mice presented histological abnormalities consistent with the mild one expected for being associated with aging and were seldom positive for fibrosis. By contrast, all the organs from Gata1low mice analysed had profound abnormal morphologies with reticulin or collagen fibers detectable, in addition to BM and spleen, in skin, lung, and kidney in an age-specific fashion (Fig 1). Gata1lowskin was thicker than normal and the connective layer presented numerous reticulin strikes already by 1-month and heavily dense connective regions strongly positive for collagen fibres by 8-15 months, resembling the skin from scleroderma patients. In lung, the alveoli had thickened walls with reticulin fibers detectable by 8-months and collagen bundles by 15-months near the bronchus walls. The abnormal morphology of kidney included reduced numbers of glomeruli and poorly organized cortical parenchyma. Reticulin and collagen fibres were observed in the medullary and nephron region by 8- and 15-months, respectively. In heart, cardiomyocytes presented a strong reduction of intercalary disks by 8-months and reticulin fibers were detectable at 15-months, suggesting that this mild fibrosis is driven by lung insufficiency. The liver presented abnormal localization and morphology of hepatocytes and presence of extramedullary hematopoiesis in perisinusoid areas. Sirius Red Picrate staining revealed few reticulin fibres mostly within erythroid islands. To clarify the mechanisms leading to multi-organ fibrosis in Gata1low mice, the transcription signature of 8-months Gata1low BM was compared with that published for murine liver, lung and kidney fibrosis (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? token = ijgpwgsyjhwpdoj&acc = GSE89630). Gata1low BM presented abnormal expression of 1724 genes (821 up- and 903 down). Pathway analyses indicated that most of the down genes were in the Gata1 pathway while the most prominent up genes were in the c-Jun, EZH2, SCL and p53 pathways. Some of the gene abnormal in Gata1low BM were also abnormal in liver (64), lung (29) and kidney (432) fibrosis. Only 28 genes were abnormal in more than one organ (25 liver and kidney, 7 lung). These common genes were obvious markers for fibrosis (5 collagen genes, fibronectin, TGF-β) and did not include c-Jun. Among genes up in Gata1low BM, kidney and liver there was lipocalin 2 (LCN2), a growth factor overexpressed in primary myelofibrosis patients (Lu et al Blood 2015;126:972) that exerts positive and negative effects, respectively, on fibrosis in BM and liver. By comparing the plasma levels of LCN2 in Gata1low and WT mice at 8-9 -and 15-17-months (8-29 mice/group), we determined that levels of LNC2 do not change with age in WT mice but increase by 3-fold in old Gata1low mice (p=0.028), explaining why liver was not one of the organs in which the mutants develop fibrosis. In conclusion, in addition to myelofibrosis, Gata1low mice develop fibrosis in skin, lung and kidney but not in heart and liver and represent genetic models for studies on the pathogenesis of fibrosis in multiple organs. Moreover, these results suggest that although the initiation factor(s) for fibrosis in the various organs are likely different, most of them are expressed by Gata1low megakaryocytes highlighting the importance for studies on the secretome profile of these cells. Disclosures No relevant conflicts of interest to declare.