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This study aimed to develop a local 3 D-printed bioactive graft using poly-caprolacton (PCL) as a drug carrier and 3-O-acetyl-β-boswellic acid (β-ABA) as an anticancer compound. β-ABA-loaded 3 D-printed scaffold was fabricated and physically characterized. The results indicated more desirable mechanical and physical properties of the β-ABA-loaded PCL mat in comparison with the PCL scaffold. Following sustained release of β-ABA, the β-ABA-loaded PCL scaffold revealed selective cytotoxic activity against melanoma cells, while the PCL + ABA with the bolus delivery of β-ABA was toxic against fibroblast cells. Followed by the induction of apoptosis in melanoma cells at the gene level, the result of the western blot showed that the β-ABA-loaded scaffold significantly up-regulated P53 and down-regulated BCL2, with an increment in the ratio of Bax/BCL2. The selective anti-cancer properties of β-ABA-loaded 3 D printed scaffold against melanoma cells indicated that this scaffold could be potentially used as a bioactive graft to improve the melanoma treatment.

Sepsis is a life-threatening disorder that leads to the induction of inflammatory responses and organ failure. Phage therapy is a new approach to controlling infections resistant to common treatments, including sepsis. Several studies have shown the effect of lytic bacteriophages on infection control by reducing the bacterial load. The present study deals with lysogenic bacteriophage M13 on the inflammatory responses caused by cecal ligation and puncture (CLP) induced sepsis in a mouse model.Bacteriophage M13 harvested from ER2738, titrated, and confirmed by TEM analysis. In-vitro toxicity and immunomodulatory effect of bacteriophage M13 were assessed on splenocytes by measurement of cell viability and the production level of cytokines, nitric oxide (NO), and reactive oxygen species (ROS). For in-vivo experiments, eight weeks old male C57BL/6 mice were randomly divided into three groups: CLP + NS (treated with normal saline), CLP + M13 (treated with an intraperitoneal injection of 109 PFU/ml of bacteriophage M13), and sham+ NS (induced surgery but without ligation and puncture, treated with NS). The mice were sacrificed at different time points post-surgery (6,24,48, and 72, n = 10 for each time point of each group). The kidney, liver, and lungs were harvested for histopathological analysis, and blood was obtained for cytokine and liver enzyme assay. The spleen was used to assess the bacterial load using colony forming unit (CFU) assay. The rectal temperature and survival were evaluated during the study.According to the in-vitro results, 109 PFU/ml of bacteriophage M13 was not toxic and did not affect the level of cytokine, NO, and ROS production by splenocytes, but it reduced the inflammatory response of splenocytes in responses to LPS. In-vivo studies indicated that the amount of pro-inflammatory cytokines, liver enzymes, bacterial load, and organ failure were decreased in the CLP + M13 group compared to CLP + NS, whereas the survival rate was increased.These experiments demonstrated that bacteriophage M13 could lessen the consequences related to sepsis in CLP mice and can be considered a therapeutic approach in sepsis.

Among medicinal plants, Acridocarpus orientalis (AO) possesses a remarkable anti-cancer potential, possibly because of its anti-oxidant property. In this study, the leaf and stem extracts from AO were assessed to find the bioactive compound with selective anti-cancer properties. The MTT viability and live and dead assays revealed that around 80% and 98% of 4T1 cells survival were declined after 48 h incubation with leaf and stem extracts, respectively. The leaf extract increased stem cell proliferation by 20% whereas the stem extract inhibited around 22% of stem cells proliferation after 48 h treatment. The live and dead assay of MSCs confirmed that 40% of the MSCs died when treated with AO stem extract. On the other hand, there were no dead cells after two days of treatment with the leaf extract. Followed by the induction of cell cycle arrest in G0/G1-phase, the real-time PCR demonstrated apoptosis properties in 4T1 cells through overexpression of Bax and down-regulation of BCL2 genes. Interestingly, within the pure compounds isolated from AO leaf extract, Morin was responsible for the inhibition of 4T1 cells proliferation as well as MSCs expansion, predicting to play an essential role in the treatment of cancer. The promising in vitro anti-cancer and stem cell-inductive properties of morin isolated from AO extract may provide a great potential to produce selective herbal derived drugs.

The incidence of connective tissue diseases such as systemic lupus erythematous (SLE), in adult patients with sickle cell disease (SCD), appears to be increasing. The exact causes underlying this increased risk are still unknown, but a link with B regulatory (Breg) cells is possible as these cells suppress inflammatory responses, and maintain tolerance. Quantitative and qualitative analyses of circulating Breg cells were performed in a cohort of SCD patients with SLE, and their levels were correlated with key soluble mediators promoting autoreactive B cells. We demonstrated that levels of Breg cells were significantly decreased in SCD patients with SLE compared to patients with SCD only or healthy controls. Functional analysis of Breg cells from SCD patients with SLE revealed impairments in IL-10 production that correlated with lower levels of STAT3 phosphorylation, without abnormal expression of IL-10 receptor on Breg cells. On the other hand, BAFF levels were substantially elevated in SCD patients with SLE, but not significantly associated with Breg cell levels. Collectively, these results indicated numerical and functional deficits of Breg cells in SCD patients with SLE and their capacity to maintain tolerance and control inflammation is imbalanced, which leads to the development of autoimmune responses.

This study was designed to evaluate the effect of different concentrations of conjugated equine estrogens (CEE) on the ovarian epithelium of female CD1 mice.Twenty-four female mice at 7 months with irregular estrus cycles were randomly divided into four groups of 6 mice each. Group one was considered as a control group and received a daily dose of 0.5ml of propylene glycol, for three weeks, while those in the treatment groups received a daily dose of 14μg/kg, 28μg/kg and 56μg/kg conjugated equine estrogens, respectively.The results from this study showed a strong correlation between elevated concentrations of CEE and histological changes in ovarian surface epithelium (OSE). They also showed that administration of high-dose estrogen created the conditions for excessive proliferation of OSE which may progress into the development of cysts in the ovaries.This study concluded that high concentrations of CEE may increase the chances of developing epithelial ovarian cancer.

Background and purpose: The formation of postoperative intra-abdominal adhesion band formation may lead to severe complications. This study aimed to evaluate the preventive effect of local administration of frankincense n-hexane extract (FHE) on the formation of postsurgical adhesion bands. Materials and methods FHE was extracted from the resin of a Boswellia sacra tree and its components were identified by gas chromatography-mass spectrometry (GC-MS). In an animal model, the expression levels of TNF-α and TGF-β1 cytokines after application of FHE were assessed to check the inflammatory and fibrotic cues, respectively. Results Following FHE compound analysis, in vivo experiments demonstrated that intraoperative local administration of FHE resulted in the prevention of adhesion band formation. The adhesion grades in the FHE-treated group were significantly lower than those in the negative control (NC) and the positive control (Interceed). The infiltration of inflammatory cells observed by histopathology revealed a significant anti-inflammatory potential of FHE. Furthermore, the gene expression results proved that significant suppression of TNF-α and TGF-β1 was responsible for its antiadhesion properties. Conclusions The study reported the potential of FHE as an ointment for the prevention of adhesion bands.

SUMMARY: In this study the consequences of prenatal exposure to tobacco smokes on the histo-morphological changes of cerebellum was assessed by comparing the smoker mice to the nonsmoker mice. A total of 30 pregnant cd-1 mice were divided into three groups of 10 mice each and with two replicates per group (5 mice each). Following acclimation for five days, the mice were placed in a special modified smoking machine for 2 hours per day over a two- and three-week period for group two and group three, respectively. Group one was considered as a control group. Mice in the control group were exposed simultaneously to fresh air from the room, while those in the treatment groups were exposed to tobacco smoke from six commercial filter cigarettes, containing 0.8 mg of nicotine, 10 mg of tar, and 10 mg of carbon monoxide, for three 1-hour exposure periods every day for three weeks. The mice in the control group were exposed to room air for three 1-hour periods every day for the same period of three weeks. The results from this study showed a correlation between maternal smoking and histological changes in Neuron purkinjense (Purkinje cells) of the cerebellum. They also showed that prenatal smoking period may have caused more damage in the histology and structure of Neuron purkinjense in some juvenile mice. An increased incidence of morphology damage of the cerebellum’s Neuron purkinjense’ structures was also observed in fetuses with prolonged exposure to tobacco smoking. Exposure of in utero maternal smoking may interfere with brain biological development parameters, giving rise to structural abnormalities of the cerebellum. This study concluded that tobacco smoke exposure to pregnant mice may affect neurodevelopment which may induce behavioural changes as a result of reduced cerebellar size and function.

This study was designed to develop a rapid, sensitive, accurate, and inexpensive Fourier Transform Infrared Reflectance (FT-IR) Spectroscopy coupled with Principle Component Analysis (PCA) as a detection technique to evaluate the solubility of 11-Keto-β-Boswellic acid (KBA), from the gum resin extracted from the Omani frankincense, (Boswellia sacra) in the liver of STZ induced diabetic mice. This study also investigated the effect of KBA on the histological changes of hepatocytes of diabetic mice. Liver tissue samples from three groups of mice included normal control group, diabetic control group and diabetic group treated IP with KBA were scanned with FT-IR spectrophotometer in the reflection mode. FT-IR Spectra were collected in the wavenumber range from 400 to 4000cm-1 using ATR accessorry. The results of FT-IR Spectra were analyzed by using multivariate method Principle Component Analysis. The PCA score plot is an exploratory multivariate method indicated that there was a complete segregation among the three groups of liver samples based on change in variation of position of wavenumber in FT-IR spectra, which revealed that there is a clear effect of KBA solubility on treatments. The histological features showed an improvement in the liver tissues with normal structures of hepatocytes with exhibiting mild vacuolations in their cytoplasm. In conclusion, reflectance FT-IR spectroscopy coupled with PCA could be deployed as a new detection method for rapid, low cost and non-destructive method for evaluating of treatment effects in diseased liver tissue based on the solubility of KBA. Histological findings demonstrated the protective effective of KBA on improving the morphology of liver tissue in diabetic mice which resulted in complete recovery to the damage observed in diabetic control group.Summary StatementReflectance FT-IR spectroscopy coupled with PCA has been deployed as a new rapid, inexpensive and non-destructive detection method to examine the solubility of 11-keto-β-Boswellic acid (KBA) in streptozotocin (STZ) induced-diabetes mice liver tissue following intraperitoneal treatment. Moreover, microscopic study of liver tissue histopathology revealed that KBA has a protecting effect against STZ damage.

The aim of this study was to evaluate a bioactive multilayer wound dressing, based on chitosan and alginate. To enhance healing potential, Dracaena Cinnabari and Aloe Vera were loaded as separate layers into the scaffold. The bare and bioactive multilayered scaffolds were fabricated by an iterative layering freeze-drying technique. Following of topographical, chemical, and physical assessment, the performance of the scaffolds was evaluated in vitro and in vivo. The results revealed adequate attachment, and proliferation of human foreskin fibroblasts, indicating excellent biocompatibility of the bioactive scaffold. In vivo, the performance of the multi-layered scaffold loaded with the bioactive materials was comparable with Comfeel plus®. The wounds treated with the bioactive scaffold exhibited superior hypergranulation, fibroblast maturation, epithelization, and collagen deposition, with minimal inflammation, and crust formation. It is concluded that the synergism of extracellular matrix-mimicking multi-layered scaffolding with Aloe Vera and Dracaena Cinnabari could be considered as a supportive wound dressing.

A series of new 11-keto-β-boswellic acid were partially-synthesized by modifying the hydroxyl and carboxylic acid functional groups of ring A. The structures of the new analogs were confirmed by detailed spectral data analysis. Compounds

Background The development of anti-cancer drugs with the ability to inhibit brain metastasis through the blood-brain barrier (BBB) is substantially limited due to the lack of reliable in vitro models. Main methods In this study, the Geltrex™-based Transwell® and microfluidic BBB models were applied to screen the effect of β-boswellic acid (β-BA) on the metastasis of MDA-MB-231 cells through the BBB in static and dynamic conditions, respectively. Major results The toxicity assay revealed that β-BA deteriorates MDA-MB-231 cells, while β-BA had no detectable toxic effects on human umbilical vein endothelial cells (HUVECs) and astrocytes. Trans-endothelial electrical resistance evaluation showed sustainable barrier integrity upon treatment with β-BA. Vimentin expression in HUVECs, evaluated using western blot, confirmed superior barrier integrity in the presence of β-BA. The obtained results were confirmed using an invasion study with a cell tracker and a scanning electron microscope. β-BA significantly inhibited metastasis by 85%, while cisplatin (Cis), a positive control, inhibited cancer cell migration by 12% under static conditions. Upon applying a dynamic BBB model, it was revealed that β-BA-mediated metastasis inhibition was significantly higher than that mediated by Cis. Conclusions and implications In summary, the current study proved the anti-metastatic potential of β-BA in both static and dynamic BBB models. This article is protected by copyright. All rights reserved.

The Arabian leopard (

Keratin-based biomaterials can be considered as the beneficial platform for designing suitable wound dressers. One of the most common natural materials that are composed of keratin and lipid is snakes shed skin. In this new study, shed skin of two different Omani snakes; Bitis arietans snake (Puff adder, “P”) and Telescopus dhara snake (Arabian Cat snake, “C”); were examined as wound dresser. The presence of lipid and keratin were assessed using Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR). 3- Layered filamentous morphology of the P shed skin and the 3-layered compact structure of C shed skin were analyzed by Scanning Electron Microscopy (SEM). X-Ray powder Diffraction (XRD) analysis proved two main peaks corresponding to the α-helix and β-sheet of the protein. Crystallinity index (CI) of P and C shed skins were obtained from XRD peaks 42.85 and 28.57%. The in vivo and histopathological results indicated that skin reconstruction was effectively improved under P shed skin treatment as compared with negative and positive control (PC) and C groups. Superior histopathological scores were the beneficial properties of P group over the positive control. The synergistic effect of natural extracellular matrix-mimicking structure and keratin beneficial proteins for wound healing could develop a natural substrate for wound healing in the clinical setting.

GVHD is a major complication after allo-SCT. In GVHD, some tissues like liver, intestine and skin are infiltrated by donor T cells while others like muscle are not. The mechanism underlying targeted tropism of donor T cells is not fully understood. In the present study, we aim to explore differences in gene expression profile among target versus non-target tissues in a mouse model of GVHD based on chemotherapy conditioning. Expression levels of JAK-signal transducers and activators of transcription (STAT), CXCL1, ICAM1 and STAT3 were increased in the liver and remained unchanged (or decreased) in the muscle and kidney after conditioning. At the start of GVHD the expression levels of CXCL9, ITGb2, SAA3, MARCO, TLR and VCAM1 were significantly higher in the liver or kidney compared with the muscle of GVHD animals. Moreover, biological processes of inflammatory reactions, leukocyte migration, response to bacterium and chemotaxis followed the same pattern. Our data show that both chemotherapy and allogenicity exclusively induce expression of inflammatory genes in target tissues. Moreover, gene expression profile and histopathological findings in the kidney are similar to those observed in the liver of GVHD mice.

The Arabian leopard (Panthera pardus nimr) is critically endangered by the International Union for Conservation of Nature, with an effective population of 150-250 across its entire range in the Arabian Peninsula. Isolation and preservation of multipotent mesenchymal stem cells is beneficial both for medical and research purposes. The optimal protocol for collection, handling, culturing and preserving the Arabian leopard mesenchymal stem cells acquired from bone marrow was established. Anesthesia with combination of medetomidine and tiletamine-zozalepam is the safest option even for old animals with concurrent disease including chronic kidney disease.Keywords: Anesthesia, Arabian leopard, Bone marrow, Mesenchymal stem cells, Panthera pardus nimr

Programmed cell death ligand-1 (PD-L1/CD274) is an immunomodulatory molecule involved in cancer and complications of bone marrow transplantation, such as graft rejection and graft-versus-host disease. The present study was designed to assess the dynamic expression of this molecule after hematopoietic stem cell transplantation in relation to acute graft-versus-host disease. Female BALB/c mice were conditioned with busulfan and cyclophosphamide and transplanted with either syngeneic or allogeneic (male C57BL/6 mice) bone marrow and splenic cells. The expression of PD-L1 was evaluated at different time points employing qPCR, western blot and immunohistochemistry. Allogeneic- but not syngeneic-transplanted animals exhibited a marked up-regulation of PD-L1 expression in the muscle and kidney, but not the liver, at days 5 and 7 post transplantation. In mice transplanted with allogeneic bone marrow cells, the enhanced expression of PD-L1 was associated with high serum levels of IFNγ and TNFα at corresponding intervals. Our findings demonstrate that PD-L1 is differently induced and expressed after allogeneic transplantation than it is after syngeneic transplantation, and that it is in favor of target rather than non-target organs at the early stages of acute graft-versus-host disease. This is the first study to correlate the dynamics of PD-L1 at the gene-, protein- and activity levels with the early development of acute graft-versus-host disease. Our results suggest that the higher expression of PD-L1 in the muscle and kidney (non-target tissues) plays a protective role in skeletal muscle during acute graft-versus-host disease.

Recent observational cohort studies indicated that the incidence of connective tissue diseases such as systemic lupus erythematous (SLE), in adult patients with sickle cell disease (SCD), appears to be increasing. The exact causes underlying this increased risk are still unknown, but patients with SCD are at risk of infections due to immune dysfunction, and a link with regulatory B (Breg) cells is possible as these cells suppress inflammatory responses, maintain tolerance and prevent the development of SLE in animal models via the production of interleukin-10 (IL-10). Herein, we numerically and functionally evaluated Breg cells in a well-defined group of SCD patients. The classification for SCD was based on patients' history, clinical examination, hematological and radiological findings. The American College of Rheumatology (ACR) criteria were used to classify and diagnose SLE patients with or without SCD. Patients were stratified into three groups including SCD patients with SLE (n=21), patients with SCD only (n=24) and patients with SLE only (n=24). Normal healthy individuals (n=24) were used as controls. Circulating levels of Breg cells were prospectively assessed by immuno-phenotyping using specific surface markers, CD19, CD24 and CD38 by flow cytometry. The functional properties were evaluated by IL-10 production and STAT3 phosphorylation after stimulation and cell culture. Unstained cells and n-1 monoclonal antibodies along with live and dead stain were used as controls in all experiments. Comparisons among study groups were performed using ANOVA and unpaired t test, while the Spearman's correlation was used to assess associations. The demographic data were similar among the study groups. All participants were free of infections such as human immunodeficiency virus, hepatitis B and C virus and syphilis. SCD patients with SLE and patients with SLE only were positive for autoimmune markers and showed an average ACR score of five and six respectively. The frequency of Breg cells, as phenotypically defined as CD19+CD24hiCD38hi, ranged between 1.5-8% in healthy controls. The circulating levels of Breg cells were significantly reduced in SCD patients with or without SLE and patients with SLE only when compared to the healthy controls (p Disclosures No relevant conflicts of interest to declare.

Recent data suggests that invariant natural killer T (iNKT) cells, a unique subset of T lymphocytes that express markers characteristic of both T cells and natural killer cells, play a pivotal role in the pathogenesis of sickle cell disease (SCD). Indeed, these cells are being considered as a potential target to prevent or treat acute crises of SCD in adult patients. However, the phenotypic and functional characteristics of circulating iNKT cell subsets in children with SCD remain unknown. Herein, we functionally evaluated iNKT cell subsets in relation to myeloid dendritic (mDC) and plasmacytoid dendritic (pDC) cells and markers of SCD severity in well-defined groups of children with SCD. The classification criteria for SCD was based on the history of patients, clinical examination, hematological and radiological findings. Blood iNKT cell subsets were prospectively studied in 68 children with SCD and normal controls using peripheral blood mononuclear cells, 10-color flow cytometry and culture assays. The iNKT cell subsets were identified by the positive-staining of Vα24Jα18 T cell receptor alpha chain, along with CD3, CD4 and CD8 surface markers and the intra-cellular cytokine production of interferon-gamma (Th1-like), interleukin-4 (Th2-like) and interleukin-17 (Th17-like) cells. The mDC and pDC cells were phenotypically characterized by the expression of HLA-DR, CD123, CD11c, Lin and CD1d, a non-classical molecule that induces the activation of iNKT cells. SCD patients were stratified into three groups including surgically splenectomized (n=23), large spleen (n=21) and steady state (n=24). Comparisons among study groups were performed using ANOVA and unpaired t test, while the Spearman's correlation was used to assess associations. Compared to normal individuals, splenectomized and steady state subjects, large spleen patients exhibited significantly higher levels of CD3iNKT, CD4iNKT and CD8iNKT cells (P=0.04). There were no differences in the levels of iNKT cell subsets between splenectomized and steady state subjects (P=0.56). The levels of mDC and pDC cells were also similar among the study groups (P=0.70). Interestingly, the large spleen patients tend to have higher CD4, but not CD8, Th1-like, Th2-like and Th17-like iNKT cells compared to splenectomized and steady state subjects (p=0.08). The expression levels of CD1d on both mDC and pDC were equivalent among study groups. The levels of CD3iNKT cells were negatively associated with baseline haemoglobin F levels (r=0.45, P=0.04) but not with age, time since splenectomy, spleen size or total hemoglobin levels at phlebotomy. Collectively, these results further advance the functional characterization of circulating iNKT cell subsets in children with SCD and reveal that surgically splenectomized patients have preserved function of iNKT cell subsets that are to be considered for clinical purposes. Disclosures No relevant conflicts of interest to declare.

Abstract 4671 Background: Omega-3 has been reported to enhance the effects of some cancer chemotherapeutic agents and to play a role in the immune system as immunoregulator and immunosuppressant. The effect of a diet rich in omega-3 during immunosuppressed states of anticancer treatment and on bone marrow transplantation (BMT) outcome is not well known. Aims: To study the effect of omega-3 on busulfan-cyclophosphamide (Bu-Cy) conditioning regimen. Moreover, to evaluate the effect of omega-3 on the outcome of BMT after Bu-Cy conditioning. Methods: We evaluated the effects of menhadenfish oil (omega-3 rich) on the myeloablative treatment with Bu-Cy as well as on the outcome of BMT in mice compared to the effects of a diet containing corn oil (omega-6 rich) or to standard chow. Animals were divided into three groups, Group 1, Group 2 and Group 3. In all the groups, female BALB/c mice, 8–12 weeks old, were randomly selected to receive a diet enriched with fish oil, a diet enriched with corn oil or standard chow. The mice were fed for two weeks prior to conditioning. Thereafter, all the mice were injected IP with 80 mg/kg busulfan (Bu) for four days (day –7 to day –4) followed by 200 mg/kg cyclophosphamide (Cy) for two days (days –3 and −2). The mice in groups 1 and 2 were killed on day 0 (the proposed day for transplantation) and spleen and femur bone marrow cells were harvested. Organs were analysed with flow cytometry to evaluate the lymphoid and myeloid cells phenotype. Also, regulatory T cell number and function were studied. Mice in Group 3 were transplanted on day 0 with allogeneic bone marrow cells from 8–12 weeks old male C57BL/6 mice (2.0 × 107 bone marrow cells and 3.0 × 107spleen cells). Results: Fish oil-enriched chow significantly suppressed the function (Figure 1) of CD4+CD25+FoxP3+ regulatory T (Treg) cells compared to standard chow and corn oil fed mice at day 0. However, the percentage of FoxP3+ cells was significantly elevated in mice fed corn oil compared to standard chow. In concert with myeloablative chemotherapy, fish oil enhanced the suppressive effect of Bu-Cy on CD11b+ myeloid and CD11c+CD86+ dendritic cell populations in the bone marrow and spleen compared to the other dietary groups. Recipient mice fed fish oil had lower survival after BMT (Figure 2). A higher survival was observed for corn oil-fed recipients, but this survival rate was still inferior to that of recipients fed standard chow. Feeding recipients omega-3 enriched diet was associated with a more pronounced acute graft versus host disease (aGVHD). BM and spleen from mice fed corn oil demonstrated lower chimerism. Conclusion: Fish oil enhances the immunosuppressive effect of the conditioning regimen (Bu-Cy) in mice. BMT recipients fed a diet rich in polyunsaturated fatty acids either have lower engraftment (corn oil) or react with a lethal aGVHD (fish oil) associated with a suppression of Treg cells. These findings may have clinical implications. PUFA supplementation should be reconsidered and caution is advised in patients undergoing stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Introduction: Several studies reported the importance of nutrition on SCT outcome. Many food components have been shown to affect the immune system and to interact with a number of drugs used in conditioning and in prophylactic treatment during SCT. Omega-3 fatty acids have immuno-modulatory effects via improving endothelial function, protection against oxidative DNA damage and by suppressing CD4+ T-cell function and proliferation. In the present study we examined the effects of omega-3 on the immune system in mice undergoing SCT. Method: Female BALB/c mice, 8 – 12 weeks, were randomised to receive either diet enriched with menhaden fish oil (omega-3) or, corn oil (rich in omega-6). Both groups were fed for two weeks prior to conditioning (Day 0 was set to be the day of transplantation). Both groups injected intra-peritoneally with 80mg/kg busulfan (Bu) for four days (day –7 to day –4) followed by 200mg/kg cyclophosphamide (Cy) for two days (days –3 & -2). At day 0, half of the mice of both groups were killed and bone marrow, spleen and thymus were harvested and analysed with FACS to evaluate the lymphoid and myeloid cell phenotype. Serum was collected for analysis of cytokines. The remaining mice in both groups were transplanted with allogeneic bone marrow from 8 – 12 weeks male C57BL/6 mice (2.0 × 107 bone marrow cells and 3.0 × 107 spleen cells). Results: Mice fed an omega 3-rich diet showed significant decrease in bone marrow and spleen cellularity compared to that observed in the corn oil group. In particular, the splenic B-cell population was severely affected, showing a 50% decrease in the omega-3 group compared to that seen in corn oil group. The T-cell populations of omega-3 fed mice were also affected, with a significant decrease by 10 to 20 % in the number of CD4+ cells. In contrast, no difference was observed for CD8+ cells, compared to corn oil-fed mice. In the thymus, the number of activated T-cells was decreased by 50% in the omega-3 compared to that observed in corn oil group. NK cells were decreased by 50% in the spleens of omega-3 fed mice. In bone marrow, the myeloid cells (CD11b+) were significantly lower in omega-3 fed mice compared to corn oil-fed controls. All transplanted mice that had received an omega-3 enriched diet died within the first week. These data can be compared to our previous results where more than 60% of mice receiving standard diet survive during the first week and of which at least 20% reach day+21. Conclusion: Omega-3 administration as a dietary supplement for two weeks prior to conditioning regimen showed significant effects on the immune system in terms of a pronounced alteration in the balance of different cells lineages in both bone marrow and spleen. Rapid death of omega-3 fed animals after allogeneic transplantation was observed. Further studies are warranted to investigate the role of omega-3 on the outcome of stem cell transplantation.

Introduction: Polyclonal anti-thymocyte globulin (ATG) is mainly used in hematopoietic stem cell transplantation, as a prevention for graft rejection as well as for GVHD. Immunosuppressive properties of ATG have been considered primarily from the depletion of peripheral lymphocytes. However direct or indirect effects of ATG on other immune components still is controversial. In the present study we evaluated the immunopathologic effects of ATG on immune system in a mouse model. Methods Ten to fourteen weeks old BALB/c and C57BL/6 mice, were injected intra- peritoneally or intravenously with different doses of ATG (0.2mg/kg -25mg/kg) at three dosage schedules. Considering bone marrow transplantation as day 0, ATG were given at days -10, -7 and -5 or -7, -5 and -1 or -5, -3 and -1. At day 0, mice were killed; spleen (SP), bone marrow (BM), lymph nodes (LN) and thymus were removed and analyzed for T-, B-, DC, NK-, T-reg and myeloid cells. To evaluate the efficacy of immunosuppressive effect of ATG, a group of BALB/c mice were conditioned using busulfan (Bu) and ATG and compared to a control group of BALB/c conditioned with Bu (80mg/kg) followed by cyclophosphamide (200mg/kg). Both groups transplanted with BM and spleen cells from C57BL/6 and followed for engraftment and/or GVHD. Results The administration of ATG (0.2- 4.5mg/kg) has resulted in an increase in spleen cellularity while in lymph node and thymus a decreased cellularity was observed. We have found that injecting 4.5mg/kg of ATG at day -7, -5 and -3 significantly decreased T-cell population in spleen and LN compare to Bu-Cy conditioning. The ratio CD4/CD8 increased after ATG treatment showing that CD8 cells are six-fold more sensitive to ATG treatment compared to CD4 lymphocyte. Interestingly T-reg cell population increase after ATG at day 0, however, the increment was negatively correlated with the administration time and positively correlated with the dose. ATG treatment has resulted in an increase in B-cell population by two- and three- fold in spleen and lymph nodes, respectively. Moreover, 1.2- to 3.5-fold increase in DCs, NK and myeloid cells was observed in SP and LN. Thymus cellularity and cell phenotype was less affected while BM cellularity was not affected by ATG treatment. No differences in the ATG effect on cellularity or cell phenotype between IP and IV route was observed. Despite the high immunosuppressive effect observed in T-cell population compared to that seen in Bu-Cy conditioning, no chimerism was observed when Cy was substituted with ATG (4.5 – 10 mg/kg). Conclusion No donor chimerism could be obtained using ATG as a single agent up to 25mg/kg. ATG dose and the administration time are important factors affecting the repopulation of residual T-cells in spleen and lymph node that have to be considered in bone marrow transplantation setting.

Graft versus host disease (GVHD) is the major limiting factor after Hematopoietic stem cell transplantation (HSCT). In this study, we report a HSCT-patient who developed arthritis 10-month after allogeneic sibling transplantation, treated with glucosamine-hydrochloride and developed severe acute GVHD within three weeks after the administration of glucosamine. Another HSCT-recipient with an increase in CD33+cells received one dose of donor lymphocyte infusion (DLI). Due to the lack of DLI and based on our experience from the first case, the patient was treated with glucosamine. No sign of relapse was observed in the second patient despite increased number of CD33+ for more than three years. The glucosamine effect was evaluated in seven individuals given the drug for four weeks. The effect of glucosamine treatment was examined by mixed-lymphocyte-reaction (MLR) and the levels of soluble IL-2 receptor (sIL- 2R), TNF-α and IFN-γ were determined. Glucosamine administration exhibited an enhancement in the allogeneic MLR and an increase in the serum levels of sIL-2R, but a decrease in the serum levels of inflammatory cytokines TNF-α and IFN-γ. We conclude that glucosamine may act as an activator of the immune system. In HSCT-patients, glucosamine-mediated immune activation might result in either beneficial or deleterious outcomes. Thus, precautions should be taken when glucosamine is prescribed to HSCT-patients.