Spleen Histology Research Articles

Assessment of protective effect of Vitamin E supplementation on oxidative damage of diabetic rat spleen induced by Streptozotocin

AimsIncreased oxidative stress in diabetes mellitus may lead to splenic damage, contributing to decreased immunity. This study aims to evaluate the potential of vitamin E supplements as a protective agent against spleen injury by examining physiological, hematological, biochemical, and histological changes in diabetic rat model. MethodsDiabetes was induced in male wistar albino rats through an intraperitoneal injection of 50mg/kg Streptozotocin, and the rats were randomly divided into five groups. The rats were then administered varying doses of vitamin E for 14 consecutive days. Assessments included renal function tests, blood glucose levels, complete blood counts, lipid profiles, tissue oxidative stress and spleen histology. An acute toxicity study was also conducted on normal rats. ResultsVitamin E supplementation did not result in any mortality up to 5000mg/kg body weight. The treated diabetic group showed improved metabolic characteristics, such as normal body weight, food and water intake and a reduced spleen index compared to the untreated diabetic group. Significant improvements were observed in hematological parameters like packed cell volume (PCV), hemoglobin concentration, RBC (red blood cells) and WBC (white blood cells) counts. HCA (Hierarchical Cluster Analysis) of these parameters indicated that doses of 100mg/kg and 150mg/kg were most similar to normal condition. Catalase and MDA (Malondialdehyde) assays showed a substantial reduction in oxidative stress in spleen tissue and histopathological examination revealed significant regenerative effects. ConclusionThis study demonstrated that vitamin E supplementation significantly enhanced various metabolic, hematological, biochemical, and histological parameters in the diabetic group compared to those who did not receive the treatment. Among the doses tested, 100mg/kg and 150mg/kg body weight were found to yield the most favorable outcomes.

Immune-enhancing effect of fermented soybean food, Cheonggukjang on cyclophosphamide-treated immunosuppressed rat

Cheonggukjang (CGJ) is a traditional food, made by the fermentation of beans, and it has different recipes for different regions in Korea. However, it has anti-inflammatory, anti-cancer, and anti-obesity effects, and is known to affect changes in the intestinal microbiota. In this study, we investigated the immune-enhancing effects of four type CGJs (one commercial and three transitional CGJs). In the cyclophosphamide (CP)-treated immunosuppressed rat, oral administration of CGJs for 4 weeks was used to investigate weight of body and immune organ, change of microbiota, blood and serum parameters, inflammation pathways (MAPKs and NFκB) and histology of spleen. It showed an immunity-enhancing effect through increase Bacteroidetes in gut, the recovery of complete blood count, levels of cytokines and IgG, activation of inflammatory pathways, and histology of spleen. In conclusion, these results show that the intake of a commercial brand CGJ, and traditional CGJs can maintain or promote the body's immunity.

Unraveling the immune response of the spleen in sepsis using green-synthesized silver nanoparticles from pomegranate peel extracts.

Sepsis is a serious disease characterized by an inappropriate host response to infection, resulting in widespread inflammation and systemic organ failure. The aim of this research is to investigate the possibility of pomegranate peel-derived silver nanoparticles (PGNP) as a potential alternative therapy for sepsis. Characterization using transmission electron microscopy revealed 10-30 nm spherical nanoparticles. In a rat model of sepsis, PGNP treatment improved spleen health, histology, and immune response as compared with septic rats. In rats treated with PGNP during sepsis, significant alterations in oxidative stress markers (p < .01) were observed. These included elevated levels of glutathione (0.63 ± 0.08 mmol/mg protein), reduced concentrations of nitric oxide (8.7 ± 0.8 μ mol/mg protein) and malondialdehyde (2.2 ± 0.3 nmol/mg protein), as well as increased activity of superoxide dismutase (159 ± 33 U/mg protein). Following PGNP administration, gene expression analysis revealed a decrease in spleen IL-1β, IL-6, and TNF-α, highlighting its anti-inflammatory potential. Furthermore, PGNP effectively controlled apoptosis-related genes (Bax, Bcl-2, and Casp3), indicating its role in cellular survival pathways. This study sheds light on the immunological regulation of the spleen during sepsis using PGNP, demonstrating its potential as a new effective treatment approach. The study emphasizes the necessity of continuing to investigate and develop alternative medicines, particularly in light of antibiotic resistance and the global impact of sepsis. RESEARCH HIGHLIGHTS: The study explored the potential medicinal benefits of pomegranate peel-derived silver nanoparticles (PGNP) in the treatment of sepsis. PGNP suppressed pro-inflammatory cytokines and enhanced the immune response. The study recommends PGNP as a viable substitute treatment.

Phytochemical analysis and acute toxicity of Solanum elaeagnifolium extract in Swiss albino mice

IntroductionThe curative effects of Solanum elaeagnifolium have aroused the interest of botanical medicine researchers. However, no research has been carried out into its potential toxicity. The aim of this study was firstly to screen the secondary metabolites contained in the leaves and fruits of S. elaeagnifolium and their mineralogical composition, and secondly to assess the acute oral toxicity of S. elaeagnifolium in Swiss albino mice. MethodsThe amount of minerals in the plant powder was assessed by inductively coupled plasma atomic emission spectrometry, and phytochemical screening was performed. The hydroethanolic extracts of the leaves and fruits of this plant were prepared using the maceration method. Acute oral toxicity was assessed according to the OECD guideline 423. Mice were given leaf extracts (SEFE) and fruit extracts (SEFR) once at doses of 500 mg/kg, 1000 mg/kg and 2000 mg/kg in an acute toxicity study. The animals were then monitored for 14 days. The animals were then monitored for 14 days. To rule out any potential toxicity, the general behavior of the animals, the clinical symptoms of poisoning, body weight, biochemical and hematological markers, and liver, kidney, and spleen histology were examined. ResultsThe two different plant extracts comprised very important mineralogical elements such as Mg, Zn, and k, as well as secondary metabolites such as polyphenols and flavonoids, these chemical elements were shown to have very important biological activities. An oral administration of a hydroethanolic extract of S. elaeagnifolium leaves and fruits at a dose of 2000 mg/kg resulted in toxicity and death in mice. We found no significant difference in the weight of the mice. For doses less than 2000 mg/kg, biochemical markers in the liver, kidneys, and hematology did not differ significantly from those in the control group. Furthermore, there were no changes in hematological markers. Furthermore, the architecture of the liver, spleen, and kidney was normal and histopathological analysis did not reveal significant adverse effects. ConclusionsThese findings indicate that the plant harbors a significant array of secondary metabolites and minerals. S. elaeagnifolium exhibited signs of toxicity and induced histological, hematological, and biochemical alterations at a dose of 2000 mg/kg body weight. These comprehensive studies will help to improve our understanding of S. elaeagnifolium safety and potential utility, laying the groundwork for its safe use in a variety of settings.

ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome.

Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic signs, including short stature, thrombocytopenia, and hyposplenism. TAM/STRMK is caused by gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1, both of which regulate Ca2+ homeostasis through the ubiquitous store-operated Ca2+ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca2+. There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca2+ influx. Compared with Stim1R304W/+ littermates, Stim1R304W/+Orai1R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics. Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1R304W/+ muscle and revealed a major restoration of gene expression in Stim1R304W/+Orai1R93W/+ mice. Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.

CYP26 Activity Maintains the Bone Marrow Niche and Stem Cell Homeostasis

Vitamin A and its active compound, retinoic acid (RA), manifest pleomorphic activities in hematopoiesis. Vitamin A homeostasis is maintained systemically through hepatic CYP26. Emerging data suggests that CYP26 mediated RA metabolism also plays a role in controlling hematopoietic stem and progenitor cells (HSPCs) behavior (Ghiaur G et al. PNAS 2013, Schonberger K et al. Cell Stem Cell 2022). We generated Cyp26a1/ Cyp26b1 inducible knockout (CYP26KO) using ROSA26CreERT Cyp26a1/ Cyp26b1 conditional mice to study the impact on hematopoiesis and the bone marrow microenvironment (BME). Tamoxifen induces complete CYP26KO in these mice and subsequently higher retinoid levels and systemic inflammation (Snyder JM et al. FASEB J 2020). Here, we characterize the hematopoietic, bone and bone marrow (BM) phenotype of these mice. Compared to the CYP26 wild type controls (CTR), we observed leukocytosis and mild anemia as well as signs of extramedullary hematopoiesis in CYP26KO mice (Table). Spleen histology and flow cytometry showed an increased number of all mature elements, including megakaryocytes as well as increased absolute numbers of all hematopoietic progenitor populations (HPCs): LSK, CMP, GMP and MEP (Table). BM analysis showed decreased cellularity and myeloid biased in CYP26KO mice (Table). There was an increased frequency of colony forming unit cell (CFU-C) in the BM of CYP26KO mice but overall preserved absolute numbers when accounted for cellularity (Table). We observed increased frequency of LSK (Table) but decreased numbers of CMP, GMP and MEP in the BM of CYP26KO mice. Both CFU assay and flow cytometry showed myeloid biased HPCs found in the BM of CYP26KO mice (Table). When analyzing the stem cell compartment, short term stem cells and multipotent progenitor cells number were decreased in CYP26KO BM (p&amp;lt;0.01), but the long-term HSCs (LT-HSC) were relatively preserved (0.04 ± 0.01 vs. 0.17 ± 0.04 x10 6/femur, CTR vs CYP26KO, p&amp;lt;0.05). Both flow cytometry and CFU assay confirmed the presence of circulating HPCs in CYP26KO mice (Table). Thus, hematopoiesis of CYP26KO mice migrated away from the BM and into extramedullary tissues. The hematopoietic phenotype was most likely due to the cell extrinsic role of CYP26 since bone marrow transplantation experiments confirmed that a CYP26KO recipient (regardless of the genotype of the hematopoietic cells) and not CYP26 wild type recipient recapitulates the observed hematopoietic findings. This observation, together with necropsy findings of thin, frail bones in CYP26KO mice, prompted a closer look at the BM matrix/microenvironment of these mice. CYP26KO mice had smaller BM cavities, decreased trabecular bone and an increased fat/bone marrow volume ratio (Table). Consistent with these findings, CYP26KO mesenchymal stroma cells (MSC) had no osteoblastic differentiation potential in vitro but showed preserved adipogenic potential. The number of CFU-fibroblasts was decreased in the BM of CYP26KO mice but were found circulating in the PB and were dramatically increased in the spleen of these mice (Table). Single cell RNAseq showed quantitative and qualitative alterations in the BME populations with relative reduction of Cxcl12 abundant MSC2, which explained the qRT-PCR observation of lower levels of Cxcl12 mRNA in MSC isolated from CYP26KO mice. More so, Spp1 mRNA was downregulated in various MSC populations of CYP26KO bone marrow, consistent with their osteopenia and with potential implications for the observed HSPC phenotype. Receptor ligand analysis revealed that various components of the BME have impaired interactions with the extracellular matrix. Thus, the leading hypothesis is that absence of CYP26 leads to a dysregulated BME and to migration of a HSCs-niche unit out of the BM and into secondary hematopoietic organs. Interestingly, scRNAseq experiments uncovered upregulation of S100a8/9 in the CYP26KO neutrophil progenitors, which may be in response to systemic stress. The observed bone alterations (osteopenia and increased BM fat) coupled with noted changes in hematopoiesis (i.e. myeloid bias and inflammatory stress) may represent early signs of aging of the bone and bone marrow and suggest a central role of retinoid homeostasis in this process.

Immunostimulant, hepatoprotective, and nephroprotective potential of Bacillus subtilis (NMCC-path-14) in comparison to dexamethasone in alleviating CFA-induced arthritis.

To investigate and compare efficacy as well as safety of Bacillus subtilis and dexamethasone (Dexa) in complete Freund's adjuvant (CFA)-induced arthritis, we used glucocorticoid monotherapy (Dexa 5mg/kg/day) and B. subtilis (1 × 108CFU/animal/day p.o) as pre-treatment and concurrent treatment for a duration of 35days. Specific emphasis was on chronic aspect of this study since long-term use of Dexa is known to produce undesirable side effects. Treatment with Dexa significantly attenuated the arthritic symptoms but produced severe side effects like weight loss, increased mortality, immunosuppression, and altered histology of liver, kidney, and spleen. Oxidative stress was also elevated by Dexa in these organs which contributed to the damage. Treatment with B. subtilis improved symptoms of arthritis without producing any deleterious side effects as seen with Dexa therapy. Immunohistochemistry (IHC) profile revealed decreased expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interleukin (IL)-1β, tumor necrosis factor alpha (TNF-α), and increased nuclear factor erythroid 2-related factor 2 (Nrf-2) expression by B. subtilis and Dexa treatment in ankle joint of arthritic mice. Radiological scores were also improved by both treatments. This study concludes that B. subtilis could be an effective alternative for treating arthritis than Dexa since it does not produce life-threatening side effects on prolong treatment.

Effect of &lt;i&gt;Aloe barbadensis&lt;/i&gt; root extract on the histology of some vital organs of male rats assessed for aphrodisiac activity

Histopathology is an essential tool in assessing the untoward effect of toxicants, synthetic drugs, and herbal medicines in toxicological animal studies. Therefore, the effect of the oral administration of graded doses of the ethanol extract of A. barbadensis root on the histology and organ-body weight ratios of some vital rat organs was evaluated. Seventy-five (75) healthy and matured male rats of 140-270 g body weights were used for this study. The rats were randomized entirely into five groups of 15 and orally given treatment using the orogastric tube. The animals were placed into Groups A, B, C, D, and E. Group A was administered the diluent (2 mL of distilled water) while groups B, C and D were given 100, 200 and 400 mg/kg of the extract for 14 days. Group E was given the standard drug-Sildenafil citrate (5 mg/kg). The vital organs (liver, kidney, heart, and spleen) were harvested, prepared, and stained after sacrificing the animals. The tissue sections were subjected to histopathological studies, and the photomicrographs of the tissues were captured appropriately. The organ-body weights were determined using an electronic weighing balance. The histopathological examination revealed no apparent abnormalities in the vital organs; instead, a vasoactive effect of active congestion was observed. The liver and spleen's histology showed a mild activation of the local immune system. The extract had no significant effect (P&gt;0.05) on the organ-body weight ratios. The study showed that the extract had no untoward effect on the selected essential organs within the dose range explored.

Serum biochemistry, meat quality and oxidative stability in broiler chicken supplemented with a novel phytogenic feed additive formulated from P. betle, P. nigrum, A. lanata and C. dactylon

The present study investigated the effect of a phytogenic feed additive (PFA) formulated with Aerva lanata, Piper betle, Cynodon dactylon and Piper nigrum on the haematological and serum biochemical profiles in broiler chicken, and its efficacy to improve quality and oxidative stability of chicken meat. In a six-week experiment, a total of 192 day-old broiler chicks were subjected to four dietary treatments that included, basal diet+ chlortetracycline; only basal diet without chlortetracycline and PFA; and basal diet + 1 or 2% PFA. Each treatment group included six replicates of eight birds per replicate. The results showed improved albumin and cholesterol in serum of birds fed 1% PFA. The meat of birds supplemented 1% PFA showed a significant reduction in drip loss after 1 and 4 days of storage. The lipid peroxidation of breast meat measured as malondialydehyde concentration was not altered by dietary treatments at different periods of storage. Feeding 1% PFA did not induce any toxic effects on liver, kidney and spleen histology. It was concluded that the PFA when used as a feed additive improved serum biochemistry and meat quality in broiler chickens.

The Effect of Administration of Ethanol Extract of Mimosa pudica L. Herbs on The Organ Index and Histology of Spleen of Male Wistar Rats

Mimosa Pudica L. is one of the plants that can be used as a traditional medicine which can be used also as analgesic and anti-inflammatory. In the studies of acute toxicity test, Mimosa Pudica L did not cause toxic. This study aimed to investigate the sub-chronic toxicity of Mimosa Pudica L to the spleen index and histology of the spleen of mice which included the observation of follicular diameter and the center of the germ. This test method referred to the OECD 407 (1995) conducted for 28 days with 7 treatment groups and consist of 1 control group , 3 treatment groups and three groups of satellites with a dose of 400 mg / kg , 600 mg / kg and 900 mg / kg. In the satellite group, there was an advance observation for 14 days without any treatment to see whether there was an effect of improvement or not. The results showed that the average value of the index spleen was getting smaller. The data were analyzed with test of homogenety of variances (levene test) showed a similarity of data variance between groups (P &gt; 0.050). The mean value of the index spleen showed there was no significant difference between groups (P &gt; 0.05). The highest mean value of the follicle diameter was in the negative control. Data was analyzed by non- parametric tests Kruskal - Wallis because it did not show the similarity of variance (P &lt; 0.05). The results showed that there was no significant difference between groups (P &gt; 0.05). The satellite group with the dose of 600 mg / kg gave the effect of improvement on the center germinal diameter. The results of the study conveniently indicated that there was no significant difference between groups (P &gt; 0.05). The conclusion of this study was the ethanol extract of the herb Mimosa Pudica L. orally did not leave a distinct meaningful difference or did not affect the spleen index and picture spleen histology.

Safety of Allogeneic Mesenchymal Stem Cell Seeding of NeuraGen Nerve Guides in a Rabbit Model.

Mesenchymal stem cells (MSCs) stimulate nerve and tissue regeneration and are primed for clinical translation. Application of autologous MSCs is limited by requirements for expedient harvesting procedures, proliferative expansion to increase number of cells, and reduced regenerative potential due to aging or pathological conditions. Because MSCs are immune privileged, allogeneic MSCs may serve as "off-the-shelf" cell-based reconstructive treatments to support nerve repair. Therefore, we examined the safety and immune response parameters of allogeneic MSCs seeded on NeuraGen® Nerve Guides (NNGs) in a rabbit model. NNGs with or without allogeneic rabbit MSCs were applied to rabbit sciatic nerves. Randomly assigned treatment included group I (no surgery control, n = 3) or groups II and III (sciatic nerve wrapped with unseeded or allogeneic MSC-seeded NNGs; n = 5/group). Rabbits were euthanized after 2 weeks to monitor functional recovery by histological evaluation of sciatic nerves and tibialis anterior (TA) muscle. Host reactions to allogeneic MSCs were analyzed by assessment of body and tissue weight, temperature, as well as hematological parameters, including white blood cell count (WBC), spleen histology, and CD4+ and CD8+ T lymphocytes. Histological analyses of nerves and spleen were all unremarkable, consistent with absence of overt systemic and local immune responses upon allogeneic MSC administration. No significant differences were observed in WBC or CD4+ and CD8+ T lymphocytes across unseeded and seeded treatment groups. Thus, allogenic MSCs are safe for use and may be considered in lieu of autologous MSCs in translational animal studies as the basis for future clinical nerve repair strategies. Impact statement Autologous mesenchymal stem cells (MSC) have been reported to enhance nerve regeneration when used in conjunction with nerve graft substitutes. However, autologous stem cell sources delay treatment and may be susceptible to age- or disease-related dysfunctions. In this study, we investigated the safety of allogeneic MSCs and the optimal number of cells for nerve conduit delivery in a rabbit model. When compared with unseeded nerve conduits, allogeneic MSC-seeded conduits did not induce a systemic or local immune response. The findings of this study will ultimately facilitate the clinical translation of a universal donor cell-based treatment option for nerve defects.

Effect of sleep deprivation and daylight restriction on the immune response to Escherichia coli-induced septicemia in Wistar rats

ABSTRACT Circadian rhythms influence the immune response. Its disruption can therefore alter the immune response of the host to pathogens. This study was designed to investigate the effects of two circadian disruptors, sleep-deprivation (SD) and daylight-restriction (DLR), on the immune response to septicemia which was induced using low-dose Escherichia coli (0.1 mL i.p.) (ECIS). Forty-eight Wistar rats (150–180 g) were grouped into six (n = 8): I(control), II(control+ECIS), III(SD12hrs+ECIS), IV(SD96hrs+ECIS), V(DLR12hrs+ECIS), and VI(DLR96hrs+ECIS) respectively. Following either SD or DLR in experimental groups, ECIS was induced and systemic immune response was evaluated after 7 days. Innate (leucocytes, eosinophils, IFN-γ and TNF-α) and adaptive (immunoglobulin -G and -M) immune responses was altered in short and long-term sleep deprived ECIS groups while only innate immune responses were impaired in the short-term daylight-restricted group. Spleen histology exhibited pathologies that worsened with increasing durations of SD and DLR. In conclusion, the immune response in Wistar rats to systemic infection is weakened to a greater extent by sleep-deprivation than by daylight-restriction.

Dual Inhibition of NF-Kb and JAK/STAT Pathways Reverts Myelofibrosis-like Phenotype in an In Vivo Murine Model

Introduction: In JAK-STAT-mediated inflammatory cytokine production in myeloproliferative neoplasms (MPN), both malignant and non-malignant clones contribute. The NF-κB pathway is a mediator of inflammation-induced carcinogenesis. We have previously demonstrated the role of miR-146a, a NF-κB repressor through IRAK1/TRAF6, in the progression of MPN. Indeed, the rs2431697 TT genotype, associated with lower miR-146a levels, contributes to early progression to secondary myelofibrosis (MF); and miR-146a-/- (KO) mice, with MF-like phenotype, show an increase in p-Stat3 with aging (Ferrer-Marin F. et al. Leukemia 2020). Here, we postulate that in this inflammation-driven MF murine model, inhibition of both NF-κB and JAK/STAT pathways can synergistically dampen the inflammatory state and reverse the MF phenotype. Methods: 11-month-old miR-146a-/- micewere 1-month-oral ad libitum treated with either ruxolitinib (JAK1/2 inhibitor) at 75 mg/kg/day, BMS-345541 (BMS) (NF-κB pathway inhibitor at IKKα/β level) at 100 mg/kg/day, both inhibitors at the same concentrations, or pacritinib (JAK2 and NF-κB pathway inhibitor at IRAK1 level) at 150 mg/kg/day. Untreated KO and wild-type (WT) mice were used as controls (n=7-15 mice/group). We evaluated: cell counts in peripheral blood; proinflammatory plasma cytokines by Luminex; size, weight and myeloid (CD11b) and lymphoid (CD19) populations by flow cytometry in spleen; histology of spleen and bone marrow (BM) by hematoxylin-eosin and reticulin staining. SET2 cells were used as an in vitro human model of JAK2V617F clonal hematopoiesis for immunoblotting quantification of pTyrSTAT3 (JAK/STAT pathway) and IRAK1 and nuclear p-p65 (NF-κB pathway). Results: As expected, compared to WT mice, KO mice exhibited monocytopenia, anemia and thrombocytopenia (Table 1), splenomegaly and severe reticulin fibrosis with osteosclerosis in BM (Fig.1). Moreover, in line with their inflammatory phenotype, plasma IL6, IL1β and TNFα levels were elevated in KO (vs WT) mice (Table 1). Besides splenomegaly, KO mice had spleen architecture loss, decreased white pulp and increased megakaryocytes (Fig.1), as a sign of extramedullary hematopoiesis. Consistent with histology, at a cellular level, KO mice showed significantly increased myeloid (CD11b+) and decreased lymphoid (CD19+) populations in the spleen. NF-κB pathway inhibitors (BMS and pacritinib) improved the thrombocytopenia, but only pacritinib improved monocyte counts without worsening anemia; by contrast, ruxolitinib did not improve monocytes or platelet counts, and the combination of ruxolitinib/BMS worsened anemia (Table 1). Regarding the proinflammatory cytokines, IL6 levels were significantly decreased with ruxolitinib; IL1β levels decreased whenever NF-κB pathway was inhibited, although it only showed significance with BMS; and TNFα levels significantly decreased with all treatments, with only a tendency for ruxolitinib/BMS (Table 1). Although all treatments decreased splenomegaly and partially recovered spleen architecture, only pacritinib and BMS (NF-κB inhibitors) diminished splenic megakaryocytic counts (Fig.1). NF-κB inhibitors [pacritinib and BMS (+/- ruxolitinib)] decreased the number of mice with severe reticulin fibrosis or moderate/severe osteosclerosis in the BM, whereas ruxolitinib alone did not have this effect. Unlike pacritinib, however, the combination of ruxolitinib/BMS induced BM aplasia (Fig.1). Finally, in SET2 cells, monotherapy with ruxolitinib or BMS inhibited JAK-STAT or NF-κB signaling pathways, respectively, but only pacritinib and the combination were able to inhibit both pathways. Conclusions: miR-146a-/- mice constitute a useful model of MPN without driver mutations to evaluate the role of inflammation in these neoplasms. The dual inhibition of NF-κB and JAK/STAT with pacritinib reduced inflammatory cytokines, reverted monocytopenia and thrombocytopenia, and improved BM fibrosis without worsening anemia. While some of these benefits could be recapitulated in vivo by combining ruxolitinib with an NF-κB inhibitor, this combination resulted in worsening anemia and BM aplasia. These results highlight the therapeutic possibilities of dual JAK/NF-κB inhibitors, such as pacritinib, in patients with MF. Funding: PI18/00316; PI20/00136; PI21/00347; 20644/JLI/18; FPU18/03189. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Haploinsufficiency of Calreticulin Causes Dysregulation of Hematopoiesis and Results in a Myeloproliferative Phenotype in an in-Vivo Mouse Model

Background.JAK2V617F, MPLW515K/L and CALR exon9 mutations drive the development of Philadelphia negative Myeloproliferative Neoplasms (MPN). While JAK2 and MPL knock out (KO) leads to severe defects of hematopoiesis, the role of Calreticulin (CALR) deregulation is not fully understood, however recent reports demonstrated that CALR deletion leads to neoplastic transformation (Shide K et al Blood, 2020; Balliu M et al Blood Adv., 2021). Aim. To investigate the role of CALR in hematopoiesis and MPN development, we generated and characterized a Calr KO mouse model. Methods. We developed a Calr+/- mouse model by crossing mice carrying a floxed allele targeting Calr intron 3 and 7 (Calr+/flx) with Vav-Cre mice. The phenotypic characterization was performed by blood counts analysis, bone marrow (BM) and spleen histology, flow-cytometry and clonogenic assay by plating 2x104 cells for CFU-GEMM, CFU-GM and BFU-E and 2.5x105 for CFU-Mk. Proteome profiler membrane-based array and RNA-sequencing were employed to profiling BM protein and gene expression. Results. Sanger sequencing of BM cells of Calr+/- mice confirmed the deletion of Calr exons 4-7, resulting in the downregulation of Calr mRNA (-0.56-fold ±0.1) and decreased protein content (-0.47-fold ±0.21) compared to Calr+/flx mice (hence, controls). The productivity, body weight and lifespan were comparable between Calr+/- and controls. Calr+/- mice showed increased platelet count (median: 1360, range: 1156-1986 x109/L), 1.2 to 1.5-fold compared to controls, starting from month 4 (p=0.007) up to the latest time point available at 12 months (p=0.03). Erythrocytes count of Calr+/- increased after month 6 up to month 12 (median:10.3, range:9.9-10.6 x1012/L)(P=0.004) paralleled by an upward trend of hematocrit (median:47%, range:42-51%) and hemoglobin (median:18.3, range:14.2-20.1 g/L) levels. The spleen index (spleen:body weight ratio x100) resulted increased in Calr+/- (mean: 0.38±0.09) compared to controls (mean: 0.3±0.08) (P=0.039). Histology revealed an augmented number of poly-lobulated megakaryocytes, most of which found in clusters, in BM of Calr+/- mice. Most Calr+/- mice showed a progressive loss of spleen architecture with resulting loss of clear demarcation between red and white pulp combined with focal fibrosis, especially in aged mice. Calr haploinsufficiency conferred an augmented clonogenic capacity to BM and, to a lesser extent, splenic progenitors, in semisolid culture assay. BM Calr+/- CFU-GEMM, CFU-GM and CFU-Mk were in median 8.9 (5-15), 102.7 (49-157) and 16 (9.7-13.6) and 5.7 (3 -10) (P=0.007), 80.2 (33-159) (P=0.0031) and 11.9 (12.7-22) (P=0.048) in Calr+/flx, respectively. Of note, BM BFU-E count was 1.3-fold higher in Calr +/- than controls (P=0.009). Calr+/- splenic progenitors showed an enhanced clonogenic potential, strikingly for CFU-GEMM (P=0.0064). We observed that Calr+/- myeloid progenitors were significantly expanded when compared to controls: BM Mk precursors (FcγRlow/cKIT+/CD41+) and both BM and spleen LSK (Lin-/Sca1+/cKIT+) were, respectively, +1.6-fold (P=0.048), +1.4-fold (P=0.042) and +2-fold (P=0.008) than controls. Proteomic array performed on BM cells identified a set of deregulated proteins, mainly represented by cytokines (IL1α, IL4, IL6), chemokines (CXCL5, CXCL12, C1qR, VEGF, Endoglin) and cell adhesion molecules (VCAM, ICAM). These observations were strengthened by the result of transcriptomics analysis on bulk RNA-sequencing from Calr+/- and Calr+/flx mice BM cells highlighting a number of differentially expressed RNAs (increased n=385, decreased n=350) that resulted enriched in signaling pathways regulating chemokines and cell adhesion molecules, including "cell chemotaxis” (GO:0060326), "leukocyte migration” (GO:0050900), "regulation of cell-cell adhesion” (GO:0022407); cytokines, as determined by "Cytokine-cytokine receptor interaction” (KEGG:mmu04060); and Ca2+ homeostasis, including "cellular calcium ion homeostasis” (GO:0006874), "regulation of cytosolic calcium ion concentration” (GO:0051480 ). Conclusion. Finding from this in-vivo model suggest that haploinsufficiency of Calr induces dysregulation of hematopoiesis, ultimately resulting in a mild-ET phenotype. These findings will be validated in larger series and integrated with ongoing experiments to outline the molecular mechanisms underlying the CALR-driven MPN phenotype.

Mutational Permutations of a Single Amino Acid Exert Divergent Phenotypic Effects in Human, Mice, and Cellular Models of SAMD9L Bone Marrow Failure Disorder

While characterizing the new SAMD9 and SAMD9L - related bone marrow failure syndromes, we found that different germline pathogenic mutations can occur at the same amino acid position (Sahoo, Nat, Medicine 2021). Here, we explore this phenomenon by studying a family with triple allelic mosaicism in SAMD9L and two mutational permutations of the same amino acid in different children. We applied genomic methods (exome sequencing, Sanger, error-corrected sequencing, FISH, single-cell DNA sequencing (scDNAseq), ddPCR, and PacBio haplotype sequencing), developed stable mutant HEK293 cell lines, created V1512M and V1512Lmutant inducible pluripotent stem cells (iPSC) and ortholog mutant mouse models with Samd9l heterozygous (V1507Mhet, V1507Lhet) and homozygous (V1507Lhom) mutations using CRISPR/Cas9 engineering. In a family with SAMD9L-related bone marrow failure, the affected girl carried heterozygous c.4534G>A (V1512M) mutation, while her brother was heterozygous for c.4534G>T (V1512L) mutation. Both mutations were germline and confirmed to arise from the same haplotype. The child with V1512M also carried somatic rescue events (del7q, acquired SAMD9L mutation) and had a more severe clinical phenotype. The asymptomatic mother who had normal diploid karyotype was a triple-allelic mosaic. The SAMD9L wildtype (WT) allele was present at ~50% frequency in all tissues, while both mutant alleles present in the remaining 50% of reads "competed" in their allelic distribution depending on the tissue origin (hematopoietic cells: V1512M ~9% and V1512L ~41%, hair follicles: both at ~25%). ScDNAseq revealed 3 separate clonal populations in blood, all with biallelic configuration: SAMD9L V1512M in 14%, V1512L in 68%, and WT (with acquired uniparental isodisomy 7q) in 18% of cells. Because her parents were SAMD9L WT, one of the mutations likely arose de novo and underwent failed rescue attempt during embryogenesis leading to a second mutation. Overexpression studies in HEK293 cells demonstrated significant growth inhibition of mutants (V1512M more severe than V1512L) compared to WT. Hematopoietic progenitor cells (HPC) derived from mutated iPSC had severely compromised proliferative capacity, and differentiation of V1512M and V1512L HPC yielded significantly fewer CD235+CD71+ erythroid cells and severely decreased CD45+CD33+ myeloid cells; in both lineages, V1512M mutation had the most severe phenotype. We also observed divergent mutational phenotypes in mice: two-thirds (13/19) of the Samd9l V1507Mhet pups died before weaning and the vast majority died within the first 2 weeks of life. On the contrary, V1507Lhet mice had overall survival equal to WT mice, while V1507Lhom mice had a trend of decreased survival with 40% of mice dying by 19 weeks. At baseline, Samd9l V1507Mhet mice showed severe growth retardation with multisystem involvement (kidney, ovary, and alimentary tract), while V1507Lhet and V1507Lhom mice had no such abnormalities. The histology of BM and spleen in >7 weeks old V1507Mhet mice showed decreased erythroid and increased myeloid precursors in BM with extramedullary hematopoiesis. Significant multi-lineage peripheral blood (PB) cytopenia were observed in V1507Mhet mice compared to WT littermates. V1507Lhet mice had normal PB counts and BM phenotype comparable to WT, while V1507Lhom mice had isolated cytopenia and reduced numbers of BM erythroid and B-cells, and an increase in myeloid cells. Next, to induce replicative stress in V1507Lhom model, we performed non-competitive BM transplantation (BMT) into lethally irradiated B6.SJL/J WT recipients. At 12 weeks post BMT, there was a significant reduction of total V1507Lhom cell numbers with a decrease in B-cells, increase in myeloid cells, and no difference in T-cells. For V1507Mhet mice, we performed competitive BMT (because of the severe baseline phenotype), revealing significant changes post BMT, like V1507Lhom transplants, yet more severe and arising earlier. Altogether, our data suggest that Samd9l V1507Mhet and V1507Lhom cells have lower erythroid and B-cell output and a shift towards myelopoiesis. To our knowledge, this is the first report of germline triple-allelic mosaicism with bi-allelic segregation in humans. Our study establishes how different mutational permutations of the same amino acid exert divergent phenotypic effects in humans, mice, and cellular models of SAMD9L bone marrow failure disorder.

Hematological Profile and Spleen Histology Improvement in Diabetic Rats Treated with PLGA Nanoparticles-Ethanol Extract of Jengkol (Archidendron pauciflorum) Fruit Peel

Jengkol (Archidendron pauciflorum) fruit peel-ethanol extract (JFP) contains several chemical compounds that act as antioxidant agents and are considered to have the potential to treat chronic disorders such as diabetes mellitus. Plant extract is generally applied in polylactic-co-glycolic acid (PLGA) nanoparticles due to its efficiency in entering the target organs. This study aims to investigate the effect of PLGA nanoparticles-ethanol extract of jengkol fruit peel (PLGA nanoparticle-JFP) on improving the hematological profile and the spleen histology of streptozotocin-induced diabetic rats. The treatment group was divided into six groups: (1) control group, (2) streptozotocin control (STZ); (3) 10 mg/kg BW of glibenclamide (GLB); (4) 770 mg/kg BW of jengkol fruit peel-ethanol extract (JFP1); (5) 110 mg/kg BW of JFP (JFP2); and (6) 770 mg/kg BW PLGA nanoparticle-JFP (PLGA-JFP). PLGA-JFP group showed a significant increase in erythrocytes and leukocytes counts (7.73x106 ± 0.02 cell/mm3 and 9.68x103 ± 3.0 cell/mm3 respectively), a decrease in lymphocytes and neutrophils percentage (66.5%±0.5% and 28±1.4% respectively) compared to the STZ group, and no significant difference in monocyte, eosinophils, and basophils percentage within groups. On the spleen histology, the white pulp diameter and the red pulp area showed significantly smaller (168.31±10.69μm and 8,01.28± 480.33 μm2 respectively) compared to the STZ group. Almost all parameters showed no significant difference compared to the GLB group but are significantly different from the control group. The administration of PLGA nanoparticle-JFP was proven to reverse hematological parameters and improve the spleen histology but has not yet reversed the diabetic rats’ condition back to normal.

Administration of Water Extract of Stachytarpheta Jamaicensis L. Vahl Affects Haematological Profile on Animal Induced Imiquimod

Psoriasis is an autoimmune disease that occurs in the skin. An example of a drug that can induce psoriasis is imiquimod. This study aims to determine the effect of water extract of Stachytarpheta jamaicensis L. Vahl. leaves on haematological profile, spleen organ index and spleen histology in male Balb/c mice induced imiquimod. The extract was made by boiling water. The animals divided into 6 groups: normal, control, methotrexate 0.2 mg/kg BW, the extract at doses of 25, 50 and 100 mg/kg BW. Measurement of the haematological profile was carried out in the end of the srudy and the parameters were white blood cells, lymphocytes, mid-sized cells (MID), granulocytes, red blood cells, haemoglobin, haematocrit and platelets. The results showed that the extract at all doses can increase white blood cells including lymphocytes, MID, granulocytes, red blood cells, haemoglobin, haematocrit and platelets compared to control group (p<0.05). The result also showed that the extract at all dose can prevent enlargement of the spleen and reduce the level of lymphocyte infiltration in the spleen. It can be concluded that the water extract of S.jamaicensis leaves affects the haematological profile and spleen organ on animal induced imiquimod.

Diet supplemented with African nutmeg (Monodora myristica) has antidepressant action in a rodent model of chronic unpredictable mild stress by regulating the vitamins, lymphocytes, platelets and immune organs

Some of the problems associated with the current treatment protocol for depression include the negative perception associated with existing therapies for depression in some religions and beliefs, as well as negative impacts, a slow onset of therapeutic action, and the misuse of prescription, which may lead to suicidal attempts. The use of dietary therapy may offer a novel alternative approach to addressing these concerns. The study was carried out to determine whether including M. myristica (African nutmeg) seed in the diet lowers the risk of depression as well as leads to a change in some biochemical, haematological, and immunological parameter that arises in stressed animal models. Twenty male and female wistar rats were randomly assigned to four groups (n = 5); group I were stressed and fed alternative diet, group II were fed alternative diet only, group III were stressed and received African nutmeg diet while group IV were fed African nutmeg diet only. The fat, fibre and protein in African nutmeg were matched with corn oil, protein casein and a pure fibre source. Animal feed and stressors were administered once daily for five consecutive weeks from the 2nd week. Sucrose preference, open field, tail suspension and forced swim test were used to evaluate behavioural activity. In addition, biochemical parameters (serum protein, albumin, urea, creatinine, total and conjugated bilirubin, triglycerides, total cholesterol, HDL cholesterol, LDL cholesterol, vitamin B9 and vitamin B12), haematological parameters and immunological parameters, (Immune organ index and histology of thymus and spleen) were assessed. African nutmeg diet exhibited antidepressant-like action in the forced swim, open field, tail suspension and sucrose preference tests. Administration of the African nutmeg diet effectively increased the levels of lymphocytes, platelets, vitamin B9, vitamin B12 and immune function in chronically stressed rats. The findings serve as evidence that including African nutmeg (M. myristica) seed in the diet of rodents subjected to Chronic Unpredictable Mild Stress may reduce depression. Thus African nutmeg inclusion in the diet may serve as a dietary alternative in the management of depression at least in experimental animals.

Traditional Chinese medicine prescriptions (XJZ, JSS) ameliorate spleen inflammatory response and antioxidant capacity by synergistically regulating NF-κB and Nrf2 signaling pathways in piglets.

Weaning transition generally impairs the immune system, inducing immune disturbance, which may be associated with post-weaning diarrhea and high mortality in piglets. The spleen is a pivotal lymphatic organ that plays a key role in the establishment of the immune system. Traditional Chinese medicine (TCM) prescriptions, XiaoJianZhong (XJZ) and Jiansananli-sepsis (JSS), are widely used prescriptions for treating spleen damage and diarrhea. Here, we hypothesized that XJZ and JSS maintain the spleen physiological function by ameliorating antioxidant capacity and inflammatory response in weaned piglets. In this study, 18 weaned piglets were assigned to the Control, XJZ and JSS groups. By hematoxylin and eosin staining, hematological analysis, flow cytometric analysis, qRT-PCR and western blot, the effects of both TCM prescriptions on the spleen antioxidant defense system and inflammatory pathway were explored. Results showed that both TCM treatment significantly ameliorated the weaning-induced morphological damage in piglets, as evidenced by clearer and more perfect spleen histology, as well as higher relative area of white pulp. Meanwhile, both XJZ and JSS exerted better blood parameters, as supported by the changes of monocyte level and lymphocyte subpopulations CD4+/CD8+ ratio. Furthermore, the levels of inflammatory markers, IL1β, IL6, IL8, and TNF-α in the spleen were markedly decreased after supplemented with both TCM prescriptions. Importantly, the inhibition of nuclear factor-kappaB (NF-κB) and its downstream effector genes (IL6, IL8, and TNF-α) in both XJZ and JSS treatment groups further confirmed alleviation of inflammatory responses in the spleen. In addition, both XJZ and JSS enhanced the antioxidant capacity of the spleen by activating the nuclear factor erythroid 2-related factor 2 (Nrf2)-activated antioxidant defense system. Notably, the results of PCA and network correlation analysis indicated that XJZ and JSS treatment altered the expression profiles of inflammatory and antioxidant-related factors in the spleen of weaned-piglets, which may involve the synergy of NF-κB and Nrf2 signaling pathways. In summary, our study showed that TCM prescriptions, XJZ and JSS could ameliorate inflammatory response and antioxidant capacity in the spleen by synergistically regulating NF-κB and Nrf2 signaling pathways in piglets.

Condensed Fuzheng extract increases immune function in mice with cyclophosphamide-induced immunosuppression.

Our general purpose was to examine the effect of condensed Fuzheng extract (CFE) on the alleviation of immunosuppression. A mouse model of immunosuppression was established by intraperitoneal injection of CTX. A healthy control group received no CTX and no CFE; different intragastric doses of CFE were administered to three groups of mice for 28 days (4500, 2250, or 1125 mg/kg/day); a negative control received CTX alone, and a positive control received CTX and levamisole hydrochloride. We evaluated the effects of CFE on the immune system organs, cells, and molecules by comparing the different groups. CFE significantly improved immune system organs (spleen and thymus indices and histology), stimulated immune cell activities (number of white blood cells and lymphocytes, phagocytosis of mononuclear phagocytes, proliferation of splenic lymphocytes, antibody formation, and NK cell activity), and increased the levels of immunoglobulins (IgA, IgG, and IgM) and cytokines (IL-2 and IFN-γ). Thus CFE effectively alleviated CTX-mediated immunosuppression and oxidative stress and enhanced the immunological functions of mice.