Mononuclear Infiltration Research Articles

Immune response profiles from humans experimentally exposed to Phlebotomus duboscqi bites.

Cutaneous leishmaniasis is a neglected vector-borne parasitic disease prevalent in 92 countries with approximately one million new infections annually. Interactions between vector saliva and the human host alter the response to infection and outcome of disease. To characterize the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector, we repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining. A variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens. Our results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine.

Infiltration by monocytes of the central nervous system and its role in multiple sclerosis: reflections on therapeutic strategies.

Mononuclear macrophage infiltration in the central nervous system is a prominent feature of neuroinflammation. Recent studies on the pathogenesis and progression of multiple sclerosis have highlighted the multiple roles of mononuclear macrophages in the neuroinflammatory process. Monocytes play a significant role in neuroinflammation, and managing neuroinflammation by manipulating peripheral monocytes stands out as an effective strategy for the treatment of multiple sclerosis, leading to improved patient outcomes. This review outlines the steps involved in the entry of myeloid monocytes into the central nervous system that are targets for effective intervention: the activation of bone marrow hematopoiesis, migration of monocytes in the blood, and penetration of the blood-brain barrier by monocytes. Finally, we summarize the different monocyte subpopulations and their effects on the central nervous system based on phenotypic differences. As activated microglia resemble monocyte-derived macrophages, it is important to accurately identify the role of monocyte-derived macrophages in disease. Depending on the roles played by monocyte-derived macrophages at different stages of the disease, several of these processes can be interrupted to limit neuroinflammation and improve patient prognosis. Here, we discuss possible strategies to target monocytes in neurological diseases, focusing on three key aspects of monocyte infiltration into the central nervous system, to provide new ideas for the treatment of neurodegenerative diseases.

Abstract 2591: Nonclinical ocular toxicity of a maytansinoid payload-antibody drug conjugate: Ocular tissue distribution, lesion pathogenesis, and mitigation

Abstract Background: Antibody-drug conjugates (ADCs) have emerged as a compelling therapeutic modality in oncology with several recent regulatory approvals. However, on and off-target toxicities require further evaluation. Corneal microcyst-like epithelial changes (MECs) are a well-known off-target class effect of ADCs with microtubulin-acting payloads, with varying degrees of severity. Manifestation of MECs is generally unrelated to antibody target and occurs in the absence of corneal target expression. Methods: An ocular toxicology model in Dutch belted rabbits was established using a humanized monoclonal antibody not cross-reactive in rabbits conjugated through an SPDB linker to the maytansinoid drug, DM4. Radiolabeled ADC was administered once and tissues were collected for radioanalysis at multiple time points to evaluate the contribution of ADC exposure and ocular tissue distribution to corneal toxicity. Non-radiolabeled ADC was administered once or twice to evaluate onset and characterization of MECs using slit lamp microscopy and histopathology. Furthermore, potential prophylactic methods for reducing MECs were evaluated. Results: The drug radioactivity was measured over 14 days and was widely distributed with elimination from most ocular tissues (except cornea) similar to blood/plasma elimination (T1/2 of 4.5-7.5 days). In contrast, the corneal layers had an apparent absorption phase followed by a relatively stable concentration plateau through 14 days. Furthermore, preferential distribution to the corneal epithelium was evident by high tissue-to-plasma ratio (1.6 vs <0.5 for other ocular tissues, including other corneal layers). Generally, MECs increased in frequency (number of eyes affected) and decreased in time to onset with increasing dose. Ophthalmic observations of MECs began in the perilimbal cornea with axial migration over time and were reflected by the histopathologic findings in the corneal epithelium of single cell necrosis, increased mitosis, mononuclear cell infiltration, and/or atrophy. A topical ophthalmic vasoconstrictor (brimonidine tartrate 0.2%) delayed the onset of MECs compared to controls (8% vs. 67% of eyes at 7 days; 33% vs. 100% at 10 days) and mitigated severity of some histopathological findings (reduced or absent corneal epithelial atrophy, punctate erosion, pigment, and/or mononuclear cell infiltration). In contrast, corticosteroid administration (prednisolone acetate 1%) did not reduce MECs clinically but did reduce the histologic incidence and severity of corneal pigment and mononuclear cellular infiltration. Conclusion: Collectively, this rabbit model reproduces the MECs associated with maytansinoid ADCs, demonstrates selective long-term corneal epithelium exposure, and provides data to support clinical testing of vasoconstrictor drops as ocular prophylaxis. Citation Format: Ali Warbington, Olga Ab, Sribalaji Lakshmikanthan, Shana Dalton, Paul Miller, Edward Stevens, Patrick Zweidler-McKay, Grace Lytle. Nonclinical ocular toxicity of a maytansinoid payload-antibody drug conjugate: Ocular tissue distribution, lesion pathogenesis, and mitigation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2591.

Sage (Salvia officinalis) alleviates trazadone induced rat cardiotoxicity mediated via modulation of autophagy and oxidative stress.

The antidepressant drug trazodone (TRZ) is commonly used for treating depression, anxiety, and insomnia, however, it causes cardiotoxicity, which is one of its limitations. The objective of this work was to investigate the impact of sage (Salvia officinalis) in rats against cardiotoxicity induced by TRZ and to investigate the mechanisms involved in its cardio-protective properties through autophagy and oxidative stress. Fifty male albino rats were split randomly into five experimental groups: control group, sage oil group (100 mg/kg), TRZ group (20 mg/kg), protective group, and curative group. Cardiac function biomarkers (aspartate aminotransferase [AST], creatine kinase-MB [CK-MB], and cardiac troponin T [cTnI]) were assessed in serum. Oxidative stress and inflammatory biomarkers in cardiac tissue (total antioxidant capacity, malondialdehyde, and tumor necrosis factor-α) were evaluated. Heart tissues were subjected to histological, immunohistochemical, and ultrastructural evaluations. DNA damage also evaluated. Significant rise in the levels of AST, CK-MB, and cTnI were observed with enhanced autophagy along with marked histopathological changes in the form of interrupted muscle fibers with wide interstitial spaces with areas of hemorrhage and extravasated blood and interstitial mononuclear cellular infiltration in TRZ group. DNA damage was also significantly increased in TRZ group. However, administration of sage in both protective and curative groups show marked improvement of the cardiac alterations. In conclusion, sage ameliorated the alterations in the heart induced by trazadone through modulation of autophagy and oxidative stress.

CCR5/CXCR3 antagonist TAK-779 prevents diffuse alveolar damage of the lung in the murine model of the acute respiratory distress syndrome.

Introduction: The acute respiratory distress syndrome (ARDS), secondary to viral pneumonitis, is one of the main causes of high mortality in patients with COVID-19 (novel coronavirus disease 2019)-ongoing SARS-CoV-2 infection- reached more than 0.7 billion registered cases. Methods: Recently, we elaborated a non-surgical and reproducible method of the unilateral total diffuse alveolar damage (DAD) of the left lung in ICR mice-a publicly available imitation of the ARDS caused by SARS-CoV-2. Our data read that two C-C chemokine receptor 5 (CCR5) ligands, macrophage inflammatory proteins (MIPs) MIP-1α/CCL3 and MIP-1β/CCL4, are upregulated in this DAD model up to three orders of magnitude compared to the background level. Results: Here, we showed that a nonpeptide compound TAK-779, an antagonist of CCR5/CXCR3, readily prevents DAD in the lung with a single injection of 2.5mg/kg. Histological analysis revealed reduced peribronchial and perivascular mononuclear infiltration in the lung and mononuclear infiltration of the wall and lumen of the alveoli in the TAK-779-treated animals. Administration of TAK-779 decreased the 3-5-fold level of serum cytokines and chemokines in animals with DAD, including CCR5 ligands MIP-1α/β, MCP-1, and CCL5. Computed tomography revealed rapid recovery of the density and volume of the affected lung in TAK-779-treated animals. Discussion: Our pre-clinical data suggest that TAK-779 is more effective than the administration of dexamethasone or the anti-IL6R therapeutic antibody tocilizumab, which brings novel therapeutic modality to TAK-779 and other CCR5 inhibitors for the treatment of virus-induced hyperinflammation syndromes, including COVID-19.

First report of co-infections of Marek's disease virus and chicken infectious anaemia virus in poultry flocks in Nigeria

Marek's disease (MD) and chicken infectious anaemia (CIA) are viral immunosuppressive diseases of poultry caused by the MD virus (MDV) and CIA virus (CIAV) respectively. Despite vaccination against MD, the incidence of the disease in vaccinated poultry flocks in Nigeria persists. However, underlying factors like co-infection with CIAV have not been investigated in the country. This study was designed to investigate possible co-infections of MDV and CIAV in poultry flocks in Nigeria. In 2016, tumorous tissue samples were collected from suspected cases of MD at necropsy in Jos, Plateau State, Nigeria. The samples collected were fixed in formalin for histopathological examination, genomic DNA was extracted from a second part and analysed by polymerase chain reaction (PCR), targeting the meq and VP1 genes of the MDV and CIAV, respectively. The histology results revealed that the cutaneous and proventricular lymphomas were characterized by large numbers of mononuclear cellular infiltrates admixed with heterophils. The PCR results revealed that MDV was detected in 66.7% (16/24), CIAV in 45.8% (11/24), and co-infections of MDV and CIAV were detected in 45.8% (11/24) of the samples analysed. In addition, co-infections of MD and CIA were recorded in 100% (6/6) and 27.7% (5/18) of broilers and layer/pullet’ samples respectively. Phylogenetic analysis of the meq gene sequences revealed that the Nigerian MDV clusters with very virulent MDV from Egypt and Italy. While, CIAV sequences were genotype II and genotype III and clustered with CIAVs from Cameroon and China. This is the first report of co-infections of MD and CIA in Nigeria.

Pharmacokinetics, pharmacodynamics, and toxicity of a PD-1-targeted IL-15 in cynomolgus monkeys.

PF-07209960 is a novel bispecific fusion protein composed of an anti-PD-1 antibody and engineered IL-15 cytokine mutein with reduced binding affinity to its receptors. The pharmacokinetics (PK), pharmacodynamics (PD), and toxicity of PF-07209960 were evaluated following once every other week subcutaneous (SC) or intravenous (IV) administration to cynomolgus monkeys in a repeat-dose PKPD (0.01-0.3 mg/kg/dose) and GLP toxicity study (0.1-3 mg/kg/dose). PF-07209960 showed dose dependent pharmacokinetics with a terminal T1/2 of 8 and 13 hours following IV administration at 0.03 and 0.1 mg/kg, respectively. The clearance is faster than a typical IgG1 antibody. Slightly faster clearance was also observed following the second dose, likely due to increased target pool and formation of anti-drug antibodies (ADA). Despite a high incidence rate of ADA (92%) observed in GLP toxicity study, PD-1 receptor occupancy, IL-15 signaling (STAT5 phosphorylation) and T cell expansion were comparable following the first and second doses. Activation and proliferation of T cells were observed with largest increase in cell numbers found in gamma delta T cells, followed by CD4+ and CD8+ T cells, and then NK cells. Release of cytokines IL-6, IFNγ, and IL-10 were detected, which peaked at 72 hours postdose. There was PF-07209960-related mortality at ≥1 mg/kg. At scheduled necropsy, microscopic findings were generalized mononuclear infiltration in various tissues. Both the no observed adverse effect level (NOAEL) and the highest non severely toxic dose (HNSTD) were determined to be 0.3 mg/kg/dose, which corresponded to mean Cmax and AUC48 values of 1.15 μg/mL and 37.9 μg*h/mL, respectively.

First report of Myxobolus neurofontinalis (Bivalvulida: Myxobolidae) infecting anadromous Brook Trout from Prince Edward Island, Canada.

During routine histological examination of tissues from mortality events of anadromous Brook Trout Salvelinus fontinalis from Prince Edward Island (PEI), Canada, myxospores consistent with Myxobolus were observed infecting the central nervous system. The objective of this study was to identify the species of Myxobolus infecting the nervous system of anadromous Brook Trout from PEI, Canada. Myxospore morphology, small subunit (SSU) ribosomal DNA (rDNA) sequence data, and histology were used to identify myxospores isolated from infected Brook Trout. Myxospore measurements from the PEI samples matched those reported in the description of Myxobolus neurofontinalis from North Carolina. A 1057-bp fragment of the SSU rDNA from myxospores collected from Brook Trout in PEI was identical to an isolate of M. neurofontinalis (MN191598) collected previously from the type locality, New River basin, North Carolina. Histological sections confirmed infections were intercellular in the central nervous system. Minimal host response was observed, with only sparse mononuclear inflammatory infiltrates present at the periphery of and within dispersed myxospores, suggesting that infections are not pathogenic to Brook Trout. Myxospores were identified as M. neurofontinalis, which was previously described from the central nervous system of Brook Trout from the New River basin, North Carolina, USA. This constitutes the first time M. neurofontinalis has been documented outside of the New River basin in North Carolina.

Biofilm formation is a risk factor for late and delayed complications of filler injection.

Biofilm formation is a major cause of delayed-graft complications. Similarly to implants, dermal fillers carry the risk of biofilm formation, which can lead to the development of nodules, chronic inflammatory reactions, abscesses and other complications. In this study, we investigated the late or delayed complications associated with biofilm formation on dermal fillers. In this retrospective analysis, we analyzed all cases of complications caused by filler injections at a single center between January 2017 and December 2022, the majority of which comprised nodule formation and chronic persistent inflammatory reactions. The risk of biofilm formation with fillers was summarized and analyzed based on the results of bacterial culture and pathological examination. Sixty-one patients were enrolled, including 42 cases of nodule formation, 15 of chronic inflammatory reactions, and 4 of active infection. Bacterial culture of the tissue samples obtained from seven patients after surgical treatment were positive, and comprised four cases of Staphylococcus aureus, one case of Staphylococcus epidermidis, one case of Staphylococcus saprophyticus and one case of Mycobacterium abscessus. The corresponding histopathological results indicated extensive mononuclear lymphocyte infiltration, with a giant cell reaction in the fibrous connective tissue. The results of this study suggest that biofilm formation is a significant risk factor for late and delayed complications following filler injection, and is caused by the contamination of resident bacteria and recessive infection at the injection site.

Gross and Histopathological Changes in Layers Experimentally Infected with Escherichia coli (APEC serotype O1KI)

Background: Avian pathogenic Escherichia coli (APEC) induces colibacillosis, an acute and systemic disease, resulting in substantial economic losses in the poultry sector. Aim: This study determined the gross and microscopic changes associated with Escherichia coli (E. coli) (APEC serotype O1KI) infection in layer chickens. Methods: A total of 10 layer chickens (20 weeks old) were acquired and assigned, at random, into two groups (infected and control) of 5 layers each. Each bird in the infected group was challenged with 0.5 ml of bacterial aliquot containing 109 colony-forming units (CFU) of the bacteria, administered intra-tracheally. The birds from both groups were humanely sacrificed on tenth day post-infection and subjected to post-mortem examination for gross and microscopic changes. Results: Gross lesions observed in the infected chickens included congested lungs, enlarged liver and kidney, hyperaemic intestine, congested ovarian follicles and yellowish fibrinous material in the infected group. While microscopic lesions observed were congested ovarian blood vessels, mononuclear cellular infiltration and generalised necrosis of the liver, kidney, and heart. Conclusion: This study revealed various lesions of congested lungs, enlarged liver, hyperaemic intestine, enlarged kidney, and congested ovarian follicles, yellowish fibrinous material, congested blood vessels of the oviduct that were typical of the natural infection and may be attributable to colisepticaemia.

Biological evaluation of critical bone defect regeneration using hydroxyapatite/ alginate composite granules.

to evaluate biocompatibility and osteogenic potential of hydroxyapatite/alginate composite after its implantation on rat calvarian critical bone defect. thirty adults male Wistar rats were randomly distributed into two groups: GHA - critical bone defect filled with hydroxyapatite/alginate composite granules (HA/Alg) and CG - critical bone defect without biomaterial; evaluated at biological points of 15, 45 and 120 days. the histomorphometrically analyses for GHA showed osteoid matrix deposition (OM) among the granules and towards the center of the defect in centripetal direction throughout the study, with evident new bone formation at 120 days, resulting in filling 4/5 of the initial bone defect. For CG, this finding was restricted to the edges of the bone margins and formation of connective tissue on the residual area was found in all biological points. Inflammatory response on GHA was chronic granulomatous type, discrete and regressive for all biological points. Throughout the study, the CG presented mononuclear inflammatory infiltrate diffuse and regressive. Histomorphometry analyses showed that OM percentage was evident for GHA group when compared to CG group in all analyzed periods (p > 0.05). the biomaterial evaluated at this study showed to be biocompatible, bioactive, osteoconductive and biodegradable synchronously with bone formation.

Histopathological Approach to Camelid Hepatitis in One-Humped Camel Slaughter in Aswan Governorate, Egypt

Abstract Many liver illnesses in camels are not recognized ante-mortem and are frequently missed due to vague or nonspecific symptoms. So, determining the histological appearance is a very important first step in the study of camel hepatitis. This study documents the histological changes associated with acute hepatitis in liver samples from camels (Camelus dromedaries) in Egypt's Aswan district. Liver samples were kept in 10 mL of formalin. The paraffin liver slices were stained on slides with hematoxylin and eosin. This is a common histological method for defining various tissue types and morphological changes. Acute hepatitis was detected in three out of the 43 camels slaughtered at the Aswan Government's Draw slaughterhouse. Acute hepatitis is characterized by a combination of inflammation, hepatocellular apoptosis, necrosis, and a varied mononuclear infilammatory infiltration, mainly lymphocytic, accompanied by a low number of plasma cells. Kupffer-cell activation, bile-duct damage, and the ductular reaction are additional characteristics.

Selective inhibition of interleukin 6 receptor decreased inflammatory cytokines and increased proteases in an experimental model of critical calvarial defect.

Considering the lack of consensus related to the impact of selective IL-6 receptor inhibition on bone remodeling and the scarcity of reports, especially on large bone defects, this study proposed to evaluate the biological impact of the selective inhibitor of interleukin-6 receptor (tocilizumab) in an experimental model of critical calvarial defect in rats. In this preclinical and in vivo study, 24 male Wistar rats were randomly divided into two groups (n=12/group): defect treated with collagen sponge (CG) and defect treated with collagen sponge associated with 2 mg/kg tocilizumab (TCZ). The defect in the parietal bone was created using an 8-mm diameter trephine drill. After 90 days, the animals were euthanized, and tissue samples (skull caps) were evaluated through micro-CT, histological, immunohistochemistry, cytokines, and RT-qPCR analyses. Tocilizumab reduced mononuclear inflammatory infiltration (P<0.05) and tumor necrosis factor (TNF)-α levels (P<0.01) and down-regulated tissue gene expression of BMP-2 (P<0.001), RUNX-2 (P<0.05), and interleukin (IL)-6 (P<0.05). Moreover, it promoted a stronger immunostaining of cathepsin and RANKL (P<0.05). Micro-CT and histological analyses revealed no impact on general bone formation (P>0.05). The bone cells (osteoblasts, osteoclasts, and osteocytes) in the defect area were similar in both groups (P>0.05). Tocilizumab reduced inflammatory cytokines, decreased osteogenic protein, and increased proteases in a critical bone defect in rats. Ninety days after the local application of tocilizumab in the cranial defect, we did not find a significant formation of bone tissue compared with a collagen sponge.

Efficacy of dietary supplements of Glycyrrhiza glabra (Licorice) and maduramicin alone or in combination with Eimeria tenella infected chicks: A clinical study and molecular docking.

Coccidiosis is one of the most economically significant poultry diseases worldwide, caused by the pathogenic Eimeria species, and is characterized by decreased weight gain (WG) and failure to grow due to malabsorption, low feed conversion rate, bloody diarrhea, and dehydration. This study investigated the effectiveness of licorice root extract (LRE) in controlling cecal coccidiosis to determine whether its combination with maduramicin could help alleviate the pathological, biochemical, and histopathological effects of cecal coccidiosis in Sasso broiler chicks. A total of 125 one-day-old Sasso broiler chicks were categorized into five equal groups (n = 25), each consisting of five replicates (n = 5 per replicate). G1-LE received a basal diet supplemented with LRE (3 g/kg); G2-ME received a basal diet containing maduramycin (0.5 g/kg); and G3-LME received a basal diet containing LRE and maduramicin together with the same rates. G4-E (positive control) and G5-N (negative control) received no additives in their feed. Birds in groups (G1-4) were challenged on day 14 of the experiment by orally intercropping a 1 ml suspension of Eimeria tenella sporulated oocysts. Groups of birds fed on LRE and maduramicin separately or together appeared to be in good condition where no deaths or clinical abnormalities were observed, based on the analysis of clinicopathological examination. Compared with the G4-E positive control, the dropping scoring and oocyst shedding of groups G1-LE, G2-ME, and G3-LME along the 10th-day post-challenge (dpc), as well as macroscopic and microscopic lesions scoring at the 7th dpc, was considerably lower. The dual supplementation use of LRE and maduramicin in G3-LME's reduced the harmful effects of coccidian, which appeared only as a mononuclear cellular infiltration and a small number of oocysts invading the intestinal glands. Molecular docking revealed that LRE and maduramicin interacted with E. tenella DNA polymerase, E. tenella apical membrane antigen 1, and microneme protein binding sites resulting in reduced E. tenella replication and invasion. The inclusion of LRE and maduramicin, individually or in combination, in the diet might effectively mitigate the detrimental effects of coccidiosis.

Application experience of bioresorbable implants in correcting the volume of the pleural cavity and their biocompatibility assessment

Objective: To substantiate experimentally the possibility of using bioresorbable biopolymer pleural implants made of polylactide and polycaprolactone (PLA/PCL) to correct the volume of the chest. Materials and methods. Rabbits were used as experimental animals — mature females of the Chinchilla breed, age 1–1.5 years, weight 3500–4300 g. The number of individuals were 5. For studying changes in implants and local tissue reactions in vivo, as close as possible to clinical conditions, the method of intrapleural implantation was used. Bio-resorbable highly porous samples based on polylactide and polycaprolactone (PLA/PCL) with different polymer ratios and porosity degrees were used as implants. The implant and local tissue reactions were evaluated over five implantation periods: 7, 21, 54, 66 and 78 weeks. Results. The study of morphology in implants during a long experiment showed good biocompatibility, which was confirmed by the absence of necrosis in implants and surrounding tissues. In the first and second implantation periods minimal reactive changes were detected in the adjacent soft tissues (lung, pleura, soft tissues of the chest wall), existing as a focal fibrosis of muscle fibers, focal mononuclear infiltration of fibrous tissue. Conclusion: As a result of studying morphological changes in the implant and adjacent tissues of the chest wall, the absence of necrosis and purulent-inflammatory changes both in the implant itself and in the surrounding tissues of the chest wall and lung was confirmed. It has been proven that a highly porous pleural implant based on polylactide and polycaprolactone is a safe and promising material for correcting the volume of the chest, which can be in demand both in general thoracic surgery and for the surgical treatment of patients with advanced pulmonary tuberculosis.

Effects of ozone therapy applied topically, by bagging, or both on the healing of clean wounds induced in rat's skin.

This study aimed to evaluate the effects of ozone therapy applied topically and/or by bagging on the healing of clean wounds induced in rat's skin. One hundred and twenty male rats of about 16 weeks old was divided into five groups: G1) saline solution (0.9%); G2) sunflower oil; G3) ozonated sunflower oil; G4) ozone bagging; G5) association of ozonated sunflower oil and ozone bagging. The wounds were evaluated through macroscopic, morphometric, histopathologic, and tensile strength analyses. Analysis among groups showed a lower percentage of wound contraction in G1 compared to G4 only in M7D. The tensile strength of the wounds showed differences among groups in the seventh (M7D) and the 14th (M14D) postoperative day, and among time points in G1 (M14D > M7D). The elongation of the wounds showed differences in G3 (M7D > M14D). Histological evaluation of the wounds showed significant change in bleeding, mixed to mononuclear infiltrate, congestion, and tissue disorganization for tissue organization between groups and time points. Ozone therapy applied topically and/or by bagging was not deleterious to the healing of clean wounds induced in rat's skin, but ozone bagging showed the best contribution to the healing process.

PATHOMORPHOLOGICAL CHANGES IN THE ORGANS OF CATS CAUSED BY ALIMENTARY LYMPHOMA. CLINICAL CASES

The article presents the results of clinical, ultrasonographic, pathomorphological and immunohistochemical studies of two random cases of alimentary lymphoma in 9- and 12-year-old domestic cats. Clinical signs of the disease were similar and characterized by anorexia, cachexia, vomiting and diarrhea that appeared in different periods of the disease. The mucous membranes of the oral cavity and eyes were icteric, the abdomen was visually enlarged, palpation revealed nodular thickenings in the abdominal cavity. During the ultrasound diagnostics, diffuse multiple non-encapsulated anechoic formations with uneven edges and growth of tumorous tissue into the intestinal wall were visualized. In another cat, there was an encapsulated formation of a hypoechoic fine-grained structure with well-defined vascularization.&#x0D; From the moment of the preliminary diagnosis until the very death, the cats were kept in the clinic under supervision. After the death, an autopsy was performed with the selection of material for histopathological examination. Organs’ fragments were fixed in 10% aqueous solution of neutral formalin and Carnois fluid. Histological preparations were made according to the generally accepted methods. Immunophenotyping of lymphoma type was performed using CD3 (T-cell) and CD79a (B-cell) markers. During the pathological autopsy, an increase in mesenteric lymph nodes, their compaction, and focal thickening of the small intestine wall were revealed. The histological examination showed that jejunum was the most infiltrated intestinal segment, in which massive, diffusely placed monomorphic lymphoblastic cells were localized in the mucous membrane lamina, submucosa and muscle layers. The submucosal layer vessels were sharply dilated, filled with erythrocytes; the muscles of the inner circular layer were swollen. The outer (longitudinal) muscle layer of the jejunum thickened significantly and was intensively infiltrated by lymphoblastic cells.&#x0D; Typical diffuse lymphoblastic proliferations were also detected in the cortical substance of lymph nodes and lymphoid nodules. Stasis and significant expansion of the marginal sinus vessels were visualized too. In addition, moderate mononuclear lymphoblastic infiltrates were detected in the pancreas parenchyma with their perivascular location. Immunohistochemically, a significant positive expression of CD3 cells was detected both in the jejunum mucous membrane and the marginal sinus area, around the germinal centers of the lymph nodes, which indicated a T-cell type of lymphoma.

Cooperative Somatic Alterations in XPO1 and TNFAIP3 (A20) Promote Immune Modulation and Tissue Infiltration in Primary Mediastinal B Cell Lymphoma

Background: Primary Mediastinal B cell lymphoma (PMBCL) is an aggressive large B cell non-Hodgkin lymphoma typically presenting as a mass in the anterior mediastinum in early adulthood, disproportionately affecting females (2:1). Previous studies have identified constitutive activation of the NF-κB pathway and immune evasion as hallmark features of PMBCL. The master negative regulator of canonical NF-κB, TNFAIP3 (A20), is inactivated in ~25-60% of PMBCL cases via disruptive mutations or deletions. Gains of the 2p16.1-15 locus ( REL, XPO1, BCL11A) are seen in ~40-75% of PMBCL cases. Interestingly, PMBCL cases with 2p16.1-15 gain/amplification display concomitant increases in XPO1 expression but lack correlative increases in REL or BCL11A. In addition, XPO1 mutations are seen in ~15-33% of PMBCL cases, with the most prevalent mutation (E571K) implicated in NF-KB activation. Despite the established occurrence of genetic events impacting XPO1 and A20 in PMBCL, the cooperative potential of these alterations remains unexplored. Consequently, we conducted an extensive review of literature describing the PMBCL genomic landscape and utilized in vivo models to examine the combined potential of XPO1 and A20 alterations to promote lymphomagenesis. Methods: Genomic data was obtained from NGS analyses from a total of 233 PMBCL patients across 6 independent published research studies and analyzed for cooccurrence of A20 &amp; XPO1 alterations (Camus et al. 2022; Chapuy et al. 2019; Lacy et al. 2020; Mareschal et al. 2015; Mottock et al. 2019; Tuveri et al 2022). For in vivo analysis, we developed a murine model of B cell specific overexpression of wildtype (WT) or E571K- XPO1 with CD19.cre driven depletion of A20 (Eµ-XPO1xA20 KO). Whole body histopathologic evaluation of FFPE tissue with H&amp;E staining was performed on 6-month-old, age and sex matched littermate mice. Results: We found co-occurrence of inactivating events in A20 and XPO1 missense mutations impacting 15% of PMBCL evaluated cases, with consistent results seen across all studies (range: 13.8-20%). As expected, A20 mutations/del were more prevalent than XPO1 mutations across all studies (OR 2.46, 95% CI 0.84-7.18, p = 0.02). XPO1 mutated cases trended towards a greater likelihood of having co-occurring A20 mutations (OR 8.51, 95% CI 0.84-86.47, p = 0.08). XPO1 mutations and gain (2p16) were found to be mutually exclusive events with gains co-occurring with A20 mutations/del in ~17% of PMBCL cases evaluated. Analysis of survival of PMBCL patients with cooccurring A20 inactivating alterations and XPO1 mutations is ongoing although preliminarily trend towards reduced mean 2-year survival (p=0.056) compared to all others are observed. Taken together, co-occurring genetic alterations effecting A20 and XPO1 are prevalent in PMBCL the alternative biology stemming from potential cooperative mechanisms may represent an aggressive subgroup of PMBCL. To evaluate concurrent B cell-specific alterations of A20 and XPO1 in vivo we utilized Eµ-XPO1xA20 KO mouse models. Performing histopathology in 6-month-old mice we observed enlarged lymph nodes due to marked increases in lymphocytes within the cortex, paracortex, medulla, and sinuses in mice with loss of functional A20, particularly in Eµ-XPO1xA20 KO mice with overexpression of WT XPO1. Increased lymphocyte populations drove overall enlargement of the node (mandibular, mesenteric, pancreatic, and mediastinal) and distorted normal architecture, featuring small lymphocytes populating sinus regions and larger lymphocytes populating areas resembling follicles. Eµ-XPO1(WT &amp; E571K)xA20 KO mice additionally featured mild mononuclear infiltrates in the lung and salivary glands; all of which were observed with increased severity compared to A20KO, Eµ-XPO1, or non-transgenic littermates. Significance: We identified co-occurring genetic lesions impacting A20 and XPO1 in PMBCL tumors and show that these alterations synergize in vivo leading to nodal/extranodal lymphocyte infiltration. The immunomodulatory effects appear to be driven by the cooperation of XPO1 overexpression, more so than mutation, and A20 inactivation in young mice. Further investigation of these models will allow elucidation of underlying cooperative mechanisms and reveal targetable pathways in PMBCL.

Comparative study between the effect of mesenchymal stem cells microvesicles versus ozone on induced liver injury in adult male albino rats (Histological & Immunohistochemical study)

Liver disease accounts for approximately 2 million deaths er year worldwide. Liver fibrisis results from chronic injury to the liver. If not effectively treated in time, liver fibrosis may transform into liver cirrhosis. MVs are recognized as potential biomarkers and important theraputic tools for a wide sectrum of diseases. Medical ozone has the ability to protect the body against pathological conditions caused by oxidative stress. The influence of ozone and MVs on CCL4 induced liver fibrosis was investigated in this study. Forty-eight adult male albino rats were divided into four equal groups. I control, II CCL4 group, III ozone and IV microvesicles groups. Liver fibrosis was induced in group II, III & IV using 12 SC injections (0.5 ml/kg body weight) of CCL4 dissolved in olive oil twice ber week for weeks. Blood samples were obtained to estimate serum ALT & AST. Liver tissues were processed for measurment of GSH & SOD, light and electron microscopic examination. H&E staine sections og group II showed dilated congested sinusoids and centralveins, mononuclear infiltrations, vacuolations and dark nuclei. Ultrastructurally, group II revealed irregular heterochromatic nuclei of hepatocytes, small scanty mitochondria & vacuolations. Morphometric & statistical analyses were performed. Group III showed some improvement, however, group IV showed more imrovement. The results indicates that MVs caused marked improvement than ozone against CCL4 induced liver damage via antioxidant & antiinflammatory properties.

Abstract 13494: Histopathological Analysis Indicates a Significant Role of Myocardial Perfusion Disturbance in Tissue Lesion in Chronic Chagas’ Cardiomyopathy Experimental Model

Introduction: Coronary microvascular perfusion disturbance (MPD) is frequently found in Chronic Chagas' cardiomyopathy (CCC), but its role in tissue lesion and left ventricular dysfunction progression is still unclear. Hypothesis: As MPD is associated with more severe subendocardial myocardial injury, we tested the hypothesis that histopathological changes in subendocardial layer could be quantitatively correlated to MPD in experimental model of CCC in hamsters. Methods: Female Syrian hamsters (n=23) infected with 3.5x10 4 blood trypomastigote forms of T. cruzi ( Y strain ) and their respective controls (n=6) were studied. At 8 months after infection, the animals underwent in vivo high-resolution myocardial rest perfusion scintigraphy with 99m Tc-Sestamibi and transthoracic echocardiography. After euthanasia, the myocardial tissue was processed for quantitative histopathological analysis of mononuclear infiltrate index (MII) using hematoxylin-eosin staining and myocardial fibrosis using picrosirius staining in LV wall layers: subendocardium, mesocardium and subepicardium. Results: Fourteen animals (61%) presented rest MPD. The area of MPD was negatively correlated with LVEF (R=-0.5; p=0.02), and positively correlated with LV diastolic diameter (R=0.83; p&lt;0.01). Animals with MPD exhibited increased extent of myocardial fibrosis in the subendocardial layer (0.67 + 0.18%) as compared to control animals (0.47 + 0.15%), p=0.03. However, the extent of total transmural fibrosis or fibrosis in other myocardial layers were similar among groups. The group with MPD presented higher values of MII in every myocardial layer as compared to controls and animals without MPD. However, only the MII in the subendocardial layer presented positive correlation with the extent of rest MPD (R=0.55, p=0.001). Conclusions: Our results indicate that MPD is significantly correlated with histopathological changes located in the subendocardial layer of LV walls. These results suggest that MPD may act as an amplifying and localizing factor for inflammation and fibrosis, suggesting a relevant role for myocardial hypoperfusion in the mechanisms of myocardial injury in CCC.