Intestinal Transplantation: Include the Spleen with Intestinal Graft?

The traditional procedure for multivisceral transplant (MVT) is to transplant the stomach, pancreas, intestine, and liver en bloc. During surgery, the native spleen is routinely removed from the recipient, and it usually creates more space in the abdomen to insert the allogeneic graft. Thus, recipients often become asplenic after MVT. Considering all of the risks and benefits, we advocate that temporary transplant of the donor spleen could be the best option for MVT recipients; it could potentially reduce the rate of intestinal allograft rejection without increasing the risk for graft-versus-host disease.

Spleen regeneration after subcutaneous heterotopic autotransplantation in a mouse model.

BACKGROUND: Splenectomy may lead to severe postoperative complications, including sepsis and cancers. A possible solution to this problem is heterotopic autotransplantation of the spleen. Splenic autografts rapidly restore the regular splenic microanatomy in model animals. However, the functional competence of such regenerated autografts in terms of lympho- and hematopoietic capacity remains uncertain. Therefore, this study aimed to monitor the dynamics of B and T lymphocyte populations, the monocyte-macrophage system, and megakaryocytopoiesis in murine splenic autografts. METHODS: The model of subcutaneous splenic engraftment was implemented in C57Bl male mice. Cell sources of functional recovery were studied using heterotopic transplantations from B10-GFP donors to C57Bl recipients. The cellular composition dynamics were studied by immunohistochemistry and flow cytometry. Expression of regulatory genes at mRNA and protein levels was assessed by real-time PCR and Western blot, respectively. RESULTS: Characteristic splenic architecture is restored within 30 days post-transplantation, consistent with other studies. The monocyte-macrophage system, megakaryocytes, and B lymphocytes show the highest rates, whereas the functional recovery of T cells takes longer. Cross-strain splenic engraftments using B10-GFP donors indicate the recipient-derived cell sources of the recovery. Transplantations of scaffolds populated with splenic stromal cells or without them afforded no restoration of the characteristic splenic architecture. CONCLUSIONS: Allogeneic subcutaneous transplantation of splenic fragments in a mouse model leads to their structural recovery within 30 days, with full reconstitution of the monocyte-macrophage, megakaryocyte and B lymphocyte populations. The circulating hematopoietic cells provide the likely source for the cell composition recovery.

Spleen regeneration after subcutaneous heterotopic autotransplantation in a mouse model

BACKGROUND: Splenectomy may lead to severe postoperative complications, including sepsis and cancers. A possible solution to this problem is heterotopic autotransplantation of the spleen. Splenic autografts rapidly restore the regular splenic microanatomy in model animals. However, the functional competence of such regenerated autografts in terms of lympho- and hematopoietic capacity remains uncertain. Therefore, this study aimed to monitor the dynamics of B and T lymphocyte populations, the monocyte-macrophage system, and megakaryocytopoiesis in murine splenic autografts. METHODS: The model of subcutaneous splenic engraftment was implemented in C57Bl male mice. Cell sources of functional recovery were studied using heterotopic transplantations from B10-GFP donors to C57Bl recipients. The cellular composition dynamics were studied by immunohistochemistry and flow cytometry. Expression of regulatory genes at mRNA and protein levels was assessed by real-time PCR and Western blot, respectively. RESULTS: Characteristic splenic architecture is restored within 30 days post-transplantation, consistent with other studies. The monocyte-macrophage system, megakaryocytes, and B lymphocytes show the highest rates, whereas the functional recovery of T cells takes longer. Cross-strain splenic engraftments using B10-GFP donors indicate the recipient-derived cell sources of the recovery. Transplantations of scaffolds populated with splenic stromal cells or without them afforded no restoration of the characteristic splenic architecture. CONCLUSIONS: Allogeneic subcutaneous transplantation of splenic fragments in a mouse model leads to their structural recovery within 30 days, with full reconstitution of the monocyte-macrophage, megakaryocyte and B lymphocyte populations. The circulating hematopoietic cells provide the likely source for the cell composition recovery.

Splenocyte transfer from hypertensive donors eliminates premenopausal female protection from ANG II-induced hypertension.

Premenopausal females are protected from angiotensin II (ANG II)-induced hypertension following the adoptive transfer of T cells from normotensive donors. For the present study, we hypothesized that the transfer of hypertensive T cells (HT) or splenocytes (HS) from hypertensive donors would eliminate premenopausal protection from hypertension. Premenopausal recombination-activating gene-1 (Rag-1)-/- females received either normotensive (NT) or hypertensive cells 3 wk before ANG II infusion (14 days, 490 ng/kg/min). Contrary to our hypothesis, no increase in ANG II-induced blood pressure was observed in the NT/ANG or HT/ANG groups. Flow cytometry demonstrated that renal FoxP3+ T regulatory cells were significantly decreased, and immunohistochemistry showed an increase in renal F4/80+ macrophages in the HT/ANG group, suggesting a shift in the renal inflammatory environment despite no change in blood pressure. Renal mRNA expression of macrophage chemoattractant protein-1 (MCP-1), endothelin-1 (ET-1), and G protein-coupled estrogen receptor-1 (GPER-1) was significantly decreased in the HT/ANG group. The adoptive transfer of hypertensive splenocytes before ANG II infusion (HS/ANG) eliminated premenopausal protection from hypertension and significantly decreased splenic FoxP3+ T regulatory cells compared with females that received normotensive splenocytes (NS/ANG). Expression of macrophage inflammatory protein 1α/chemokine (C-C motif) ligand 3 (MCP-1/CCL3), a potent macrophage chemokine, was elevated in the HS/ANG group; however, no increase in renal macrophage infiltration occurred. Together, these data show that in premenopausal females, T cells from hypertensive donors are not sufficient to induce robust ANG II-mediated hypertension; in contrast, transfer of hypertensive splenocytes (consisting of T/B lymphocytes, dendritic cells, and macrophages) is sufficient. Further work is needed to understand how innate and adaptive immune cells and estrogen signaling coordinate to cause differential hypertensive outcomes in premenopausal females.NEW & NOTEWORTHY Our study is the first to explore the role of hypertensive T cells versus hypertensive splenocytes in premenopausal protection from ANG II-induced hypertension. We show that the hypertensive status of T cell donors does not impact blood pressure in the recipient female. However, splenocytes, when transferred from hypertensive donors, significantly increased premenopausal recipient blood pressure following ANG II infusion, highlighting the importance of further investigation into estrogen signaling and immune cell activation in females.

An aged immune system drives senescence and ageing of solid organs.

Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly1,2. To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1, which encodes a crucial DNA repair protein3,4, in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence5-7 in the immune system only. We show that Vav-iCre+/-;Ercc1-/fl mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice8-10. Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre+/-;Ercc1-/fl or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre+/-;Ercc1-/fl mice with rapamycin reduced markers of senescence in immune cells and improved immune function11,12. These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.

Malignant subclone drives metastasis of genetically and phenotypically heterogenous cell clusters through fibrotic niche generation.

A concept of polyclonal metastasis has recently been proposed, wherein tumor cell clusters break off from the primary site and are disseminated. However, the involvement of driver mutations in such polyclonal mechanism is not fully understood. Here, we show that non-metastatic AP cells metastasize to the liver with metastatic AKTP cells after co-transplantation to the spleen. Furthermore, AKTP cell depletion after the development of metastases results in the continuous proliferation of the remaining AP cells, indicating a role of AKTP cells in the early step of polyclonal metastasis. Importantly, AKTP cells, but not AP cells, induce fibrotic niche generation when arrested in the sinusoid, and such fibrotic microenvironment promotes the colonization of AP cells. These results indicate that non-metastatic cells can metastasize via the polyclonal metastasis mechanism using the fibrotic niche induced by malignant cells. Thus, targeting the fibrotic niche is an effective strategy for halting polyclonal metastasis.

Histopathology of Human Donor Spleen Utilized as a Desensitization Tool Before Intestinal Transplantation.

OBJECTIVES: To describe the histopathologic and immunophenotypic features of a temporary splenic allograft exposed to massive donor-specific antibody (DSA) insult. METHODS: A human cadaveric donor splenic allograft was temporarily transplanted in a highly sensitized patient with the intention of removing DSA before intestinal transplantation from the same donor. Before splenic transplant, the patient had several preformed cytotoxic DSAs that resulted in positive flow cytometric and complement-dependent cytotoxicity crossmatch. The splenic allograft was removed before intestinal transplantation and evaluated by H&E and immunohistochemical stains. RESULTS: Explanted donor splenic allograft showed several histopathologic changes: expanded red pulp secondary to congestion and marked neutrophilic and macrophage infiltration in the sinusoids, numerous neutrophilic microabscesses, and focal capillaritis. The C4d and IgG immunohistochemical stains were diffusely positive in the endothelial lining of the capillaries and sinusoidal lining, indicating diffuse IgG deposition and complement activation. CONCLUSIONS: We propose that the noted changes are features of splenic acute antibody-mediated rejection (AMR). Additional cases are required to determine all the features of splenic AMR. To our knowledge, this is the first report of histopathologic changes in donor spleen after exposure to DSA for a short duration.

Single segment of spleen autotransplantation, after splenectomy for trauma, can restore splenic functions.

BACKGROUND: Splenectomy is sometimes necessary after abdominal trauma, but splenectomized patients are at risk of sepsis due to impaired immunological functions. To overcome this risk, autotransplantation of the spleen by using a new technique has been proposed, but so far, a demonstration of functionality of the transplanted tissue is lacking. METHODS: We therefore evaluated 5 patients who underwent a splenic autotransplant in comparison with 5 splenectomized patients without splenic autotransplant and 7 normal subjects. RESULTS: We confirmed that the patients not undergoing autotransplantation, when compared to normal subjects, had a higher platelet count, higher percentage of micronucleated reticulocytes (p = 0.002), increased levels of naive B lymphocytes (p = 0.01), a defect of class-switched memory (p = 0.001) and class-unswitched memory B cells (p = 0.002), and increased levels of PD1 on T lymphocytes CD8+ (p = 0.08). In contrast, no significant differences for any of the abovementioned parameters were recorded between patients who underwent spleen autotransplantation and normal subjects. CONCLUSION: These findings suggest that splenic autotransplantation is able to restore an adequate hemocatheretic activity as well as recover the immunological deficit after splenectomy.

Splenocyte transfer exacerbates salt-sensitive hypertension in rats.

NEW FINDINGS: What is the central question of this study? Recruitment of immune cells to the kidney potentiates hypertensive pathology, but more refined methods are needed to assess these cells functionally. Adoptive transfer studies of immune cells have been limited in rat models and especially in the study of salt-sensitive hypertension. We tested the hypothesis that splenocyte transfer into T-cell-deficient rats is sufficient to exacerbate salt-sensitive hypertension. What is the main finding and its importance? We demonstrate that transfer of splenocytes into T-cell-deficient animals exacerbates salt-sensitive hypertension, and an enrichment in the CD4+ compartment specifically induces this phenomenon. ABSTRACT: Increasing evidence of immune system activation during the progression of hypertension and renal injury has led to a need for new methods to study individual cell types. Transfer of immune cells serves as a powerful tool to isolate effects of specific subsets. Transfer studies in Rag1-/- mice have demonstrated an important role of T-cell activation in hypertension, but this approach has yielded limited success in rat models. Using the T-cell-deficient Dahl salt-sensitive (SS) rat, SSCD247-/- , we hypothesized that splenocyte transfer from SS wild-type animals into SSCD247-/- animals would populate the T-cell compartment. The Dahl SS background provides a model for studying salt-sensitive hypertension; therefore, we also tested whether the dietary salt content of the donor would confer differential salt sensitivity in the recipient. To test this, donors were maintained on either a low-salt or a high-salt diet, and at postnatal day 5 the recipients received splenocyte transfer from one of these groups before a high-salt diet challenge. We showed that splenocyte transfer elevated blood pressures while rats were fed low salt and exacerbated the salt-sensitive increase in pressure when they were fed fed high salt. Furthermore, transfer of splenocytes conferred exacerbated renal damage. Lastly, we confirmed the presence of T cells in the circulation and in the spleen, and that infiltration of immune cells, including T cells, macrophages and B cells, into the kidney was elevated in those receiving the transfer. Interestingly, the source of the splenocytes, from donors fed either a low-salt or a high-salt diet, did not significantly affect these salt-sensitive phenotypes.

Modified Multivisceral Transplantation with Native Spleen Removal in Rats.

BACKGROUND: Modified multivisceral transplantation (MMVTx) refers to the use of a graft that includes all abdominal organs except the liver. The use of this type of transplant in children and adults expanded over the last years with good results. However, long-term survival in experimental models has not been reported. Our aim is to describe in detail some technical modifications of MMVTx to obtain long-term survival. MATERIALS AND METHODS: Syngeneic (Lewis-Lewis) heterotopic MMVTx was performed in 16 male rats (180-250 g). All procedures were performed under isoflurane anesthesia. The graft consisted of stomach, duodenopancreatic axis, spleen, and small bowel. The vascular pedicle consisted of a conduit of aorta, including the celiac trunk and the superior mesenteric artery (SMA), and the portal vein (PV). The engraftment was performed by end-to-side anastomosis to the infra-renal cava vein and aorta. After reperfusion, the graft was accommodated in the right side of the abdomen, and a terminal ileostomy performed. The native spleen was removed. RESULTS: Donor and recipient time was 39 ± 4.4 minutes and 69 ± 7 minutes, respectively; venous and arterial anastomosis time was 14 ± 1 minutes and 12.3 ± 1 minutes, respectively. Total ischemia time was 77.2 ± 7.9 minutes. Survival was 75% (12/16), six were sacrificed after 2 hours, and six were kept alive for long-term evaluation (more than 1 week). CONCLUSION: Long-term survival is reported after heterotopic MMVTx in rats. The heterotopic MMVTx with native spleen removal would potentially improve the existent models for transplant research. The usefulness of this model warrants further confirmation in allogeneic experiments.

Hepatic stellate cells regulate hepatic progenitor cells differentiation via the TGF-ß1/Jagged1 signaling axis.

Hepatic stellate cells (HSCs) play an important microenvironmental role in hepatic progenitor cells (HPCs) differentiation fate. To reveal the specific mechanism of HSCs induced by transforming growth factor ß1 (TGF-ß1) signaling in HPCs differentiation process, we used Knockin and knockdown technologies induced by lentivirus to upregulate or downregulate TGF-ß1 level in mouse HSCs (mHSCs) (mHSCs-TGF-ß1 or mHSCs-TGF-ßR1sih3). Primary mouse HPCs (mHPCs) were isolated and were cocultured with mHSCs-TGF-ß1 and mHSCs-TGF-ßR1sih3 for 7 days. Differentiation of mHPCs was detected by quantitative reverse transcriptase polymerase chain reaction analysis and immunofluorence in vitro. mHPCs-E14.5 cell lines and differently treated mHSCs were cotransplanted into mice spleens immediately after establishment of acute liver injury model for animal studies. Engraftment and differentiation of transplanted cells as well as liver function recovery were measured at the seventh day via different methods. mHSCs-TGF-ß1 were transformed into myofibroblasts and highly expressed Jagged1, but that expression was reduced after blockage of TGF-ß1 signaling. mHPCs highly expressed downstream markers of Jagged1/Notch signaling and cholangiocyte markers (CK19, SOX9, and Hes1) after coculture with mHSCs-TGF-ß1 in vitro. In contrast, mature hepatocyte marker (ALB) was upregulated in mHPCs in coculture conditions with mHSCs-TGF-ßR1sih3. At the seventh day of cell transplantation assay, mHPCs-E 14.5 engrafted and differentiated into cholangiocytes after cotransplanting with TGF-ß1-knockin mHSCs, but the cells had a tendency to differentiate into hepatocytes when transplanted with TGF-ßR1-knockdown mHSCs, which corresponded to in vitro studies. HSCs play an important role in regulating HPCs differentiation into cholangiocytes via the TGF-ß1/Jagged1 signaling axis. However, HPCs have a tendency to differentiate into hepatocytes after blockage of TGF-ß1 signaling in HSCs.

Human immunodeficiency virus infection induces lymphoid fibrosis in the BM-liver-thymus-spleen humanized mouse model.

A major pathogenic feature associated with HIV infection is lymphoid fibrosis, which persists during antiretroviral therapy (ART). Lymphoid tissues play critical roles in the generation of antigen-specific immune response, and fibrosis disrupts the stromal network of lymphoid tissues, resulting in impaired immune cell trafficking and function, as well as immunodeficiency. Developing an animal model for investigating the impact of HIV infection-induced lymphoid tissue fibrosis on immunodeficiency and immune cell impairment is critical for therapeutics development and clinical translation. Said model will enable in vivo mechanistic studies, thus complementing the well-established surrogate model of SIV infection-induced lymphoid tissue fibrosis in macaques. We developed a potentially novel human immune system-humanized mouse model by coengrafting autologous fetal thymus, spleen, and liver organoids under the kidney capsule, along with i.v. injection of autologous fetal liver-derived hematopoietic stem cells, thus termed the BM-liver-thymus-spleen (BLTS) humanized mouse model. BLTS humanized mouse model supports development of human immune cells and human lymphoid organoids (human thymus and spleen organoids). HIV infection in BLTS humanized mice results in progressive fibrosis in human lymphoid tissues, which was associated with immunodeficiency in the lymphoid tissues, and lymphoid tissue fibrosis persists during ART, thus recapitulating clinical outcomes.

INFLUENCE OF SPLENIC IMPLANTS IN THE SUBCUTANEOUS TISSUE ON ASPLENIC ANIMALS SURVIVAL.

BACKGROUND: The best site for splenic implant was not defined, mainly evaluating the functionality of the implant. AIM: To evaluate the effects of autogenous splenic implantation on the subcutaneous tissue in the survival of splenectomized rats. METHOD: Twenty-one randomly assigned rats were studied in three groups (n=7): group 1 - manipulation of the abdominal cavity and preservation of the spleen; group 2 - total splenectomy; group 3 - splenectomy and implant of the tissue removed in the subcutaneous. The animals were followed for 90 days postoperatively. RESULTS: There was a higher mortality in groups 2 (p=0.0072) and 3 (p=0.0172) in relation to group 1. There was no difference between groups 2 and 3 (p=0.9817). CONCLUSION: The splenic implant in the subcutaneous is ineffective in the survival of rats submitted to splenectomy.

Should splenic autotransplantation be considered after total splenectomy due to trauma?

Trauma is a public health problem and the most common cause of death in people under the age of 45. In blunt abdominal trauma, the spleen is the most commonly injured organ. Splenectomy remains the most common treatment, especially in high-grade lesions, despite increased nonoperative treatment. Removal of the spleen leads to increased susceptibility to infections due to its role in the immune function. Postsplenectomy sepsis is an important complication and presents a high mortality rate. Patients undergoing splenectomy should be immunized for encapsulated germs, as these are the agents most commonly associated with such infections. Splenic autotransplantation is a simple procedure, which can be an alternative to reduce infection rates consequent to total splenectomy, and reduce costs related to hospitalizations. This review aims to provide evidence-based information on splenic autotransplantation and its impact on the prognosis of patients undergoing total splenectomy. We searched the Cochrane Library, Medline/PubMed, SciELO and Embase, from January 2017 to January 2018 and selected articles in English and Portuguese, dated from 1919 to 2017. We found that the adjusted risk of death in splenectomized patients is greater than that of the general population, and when total splenectomy is performed, splenic autotransplantation is the only method capable of preserving splenic function, avoiding infections, especially postsplenectomy sepsis. Health professionals should be familiar with the consequences of the method chosen to manage the patient suffering from splenic trauma.

Ethylenecarbodiimide-fixed splenocytes carrying whole islet antigens decrease the incidence of diabetes in NOD mice via down-regulation of effector memory T cells and autoantibodies.

Type 1 diabetes mellitus (T1DM) is a syndrome of loss of glucose homeostasis caused by the loss of ß cell chronic autoimmunity against islet cells. Islet-specific epitopes coupled antigen presenting cells by Ethylenecarbodiimide (ECDI) is a promising strategy to induce antigen-specific tolerance. However, single epitope induced tolerance is insufficient to prevent the onset of T1DM. The aim of this study is to evaluate the efficacy of whole islet antigens in preventing the onset and progression of T1DM and identify the underlying immune mechanism in NOD mice. In this study, the whole islet antigens, derived from islet lysate isolated from BALB/c mice, were coupled to splenocytes of BALB/c mice by ECDI fixation (SP-Islet lysate), and then intravenously administrated to NOD mice. The results showed that, compared with control group, SP-Islet lysate group significantly decreased T1DM incidence and improved the survival of NOD mice. SP-Islet lysate treated mice had reduced insulitis score and autoantibody levels, and improved glucose tolerance and insulin/glucagon production. Furthermore, the effector memory T cells (TEMs) were downregulated and regulatory T cells (Tregs) were upregulated by the SP-Islet lysate treatment, with reduced populations of Th1&Th17 cells. In conclusion, ECDI-fixed splenocytes carrying whole islet antigens effectively prevented the onset of T1DM in NOD mice, via suppressing the production of autoantibodies and inducing anergy of autoreactive T cells.

Administration of donor splenocytes via the respiratory tract generates CD8α+ regulatory dendritic cells and induces hyporesponsiveness to fully allogeneic cardiac grafts.

BACKGROUND: We previously showed that pretreatment with intratracheal delivery (ITD) of alloantigen induced prolonged cardiac allograft survival and generated regulatory T cells (Tregs) in mice. In this study, we examined the role of splenic dendritic cells (DCs) in the ITD model. METHODS: CBA mice were treated with ITD from C57BL/10 splenocytes and 7 days later received transplantation of C57BL/10 hearts. In adoptive transfer studies, splenic DCs from ITD-treated mice were transferred into naïve CBA recipients that received C57BL/10 hearts immediately after the transfer. In addition, to determine the role of splenic DCs isolated from ITD-treated mice, the cells were incubated under stimulation with lipopolysaccharide (LPS). RESULTS: ITD-treated CBA recipients had markedly prolonged allograft survival (median survival time [MST], 67 days) while naïve recipients rejected allografts acutely (MST, 8 days). In adoptive transfer studies, CBA recipients of the transfer of splenic DCs from ITD-treated mice had prolonged allograft survival (MST, 85 days), while CBA recipients of the transfer of splenic DCs from naïve mice did not have prolonged allograft survival (MST, 8 days). In another transfer study, CBA recipients of the transfer of splenic CD8α+ DCs from ITD-treated mice had prolonged allograft survival (MST, 79 days), while those receiving splenic CD8α- DCs from ITD-treated mice did not have prolonged allograft survival (MST, 8 days). In vitro studies showed that ITD-treated splenic DCs produced more IL-10 and less IL-12 than naïve splenic DCs under stimulation with LPS. CONCLUSIONS: ITD pretreatment induces regulatory DCs, which produce high amounts of IL-10 resulting in the prolongation of graft survival in our model.

Intraperitoneal oxaliplatin administration inhibits the tumor immunosuppressive microenvironment in an abdominal implantation model of colon cancer.

Recent studies have demonstrated that some chemotherapeutic drugs can enhance antitumor immunity by eliminating and inactivating immunosuppressive cells. Oxaliplatin (OXP) induces immunogenic cell death by increasing the immunogenicity of cancer cells. However, the effects of OXP on the tumor immunosuppressive microenvironment remain unclear. The aim of the present study was to evaluate the antitumor activity of OXP by intraperitoneal (i.p.) administration in an abdominal implantation model of colon cancer and tested the tumor immune microenvironment to observe whether OXP affects the local immune inhibitory cell populations. Abdominal metastasis models were established by inoculation of CT26 cells. The antitumor efficacy of OXP and the tumor immune microenvironment were evaluated. The tumors and spleens of mice were harvested for flow cytometric analysis. Cluster of differentiation (CD)­8+CD69+ T cells, regulatory T cells (Tregs), CD11b+F4/80high macrophages and myeloid­derived suppressor cells (MDSCs) were evaluated by flow cytometric analysis. In vivo i.p. administration of OXP inhibited tumor growth in the abdominal metastasis model. Furthermore, OXP was observed to increase tumor­infiltrating activated CD8+ T cells in tumors, decrease CD11b+F4/80high macrophages in tumors and decrease MDSCs in the spleen. These results suggested that i.p. administration of OXP alone may inhibit tumor cell growth and induce the antitumor immunostimulatory microenvironment by eliminating immunosuppressive cells.

Resolution of bleomycin-induced murine pulmonary fibrosis via a splenic lymphocyte subpopulation.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive disease with high mortality, and the pathogenesis of the disease is still incompletely understood. Although lymphocytes, especially CD4+CD25+FoxP3+ regulatory T cells (Tregs), have been implicated in the development of IPF, contradictory results have been reported regarding the contribution of Tregs to fibrosis both in animals and humans. The aim of this study was to investigate whether a specific T cell subset has therapeutic potential in inhibiting bleomycin (BLM)-induced murine pulmonary fibrosis. METHODS: C57BL/6 mice received BLM (100 mg/kg body weight) with osmotic pumps (day 0), and pulmonary fibrosis was induced. Then, splenocytes or Tregs were adoptively transferred via the tail vein. The lungs were removed and subjected to histological and biochemical examinations to study the effects of these cells on pulmonary fibrosis, and blood samples were collected by cardiac punctures to measure relevant cytokines by enzyme-linked immunosorbent assay. Tregs isolated from an interleukin (IL)-10 knock-out mice were used to assess the effect of this mediator. To determine the roles of the spleen in this model, spleen vessels were carefully cauterized and the spleen was removed either on day 0 or 14 after BLM challenge. RESULTS: Splenocytes significantly ameliorated BLM-induced pulmonary fibrosis when they were administered on day 14. This effect was abrogated by depleting Tregs with an anti-CD25 monoclonal antibody. Adoptive transfer of Tregs on day 14 after a BLM challenge significantly attenuated pulmonary fibrosis, and this was accompanied by decreased production of fibroblast growth factor (FGF) 9-positive cells bearing the morphology of alveolar epithelial cells. In addition, BLM-induced plasma IL-10 expression reverted to basal levels after adoptive transfer of Tregs. Moreover, BLM-induced fibrocyte chemoattractant chemokine (CC motif) ligand-2 production was significantly ameliorated by Treg adoptive transfer in lung homogenates, accompanied by reduced accumulation of bone-marrow derived fibrocytes. Genetic ablation of IL-10 abrogated the ameliorating effect of Tregs on pulmonary fibrosis. Finally, splenectomy on day 0 after a BLM challenge significantly ameliorated lung fibrosis, whereas splenectomy on day 14 had no effect. CONCLUSIONS: These findings warrant further investigations to develop a cell-based therapy using Tregs for treating IPF.

Splenic implant preservation after conservation in lactated Ringer´s solution.

OBJECTIVE: to evaluate the morphology and function of autogenous splenic tissue implanted in the greater omentum, 24 hours after storage in Ringer-lactate solution. METHODS: we divided 35 male rats into seven groups (n=5): Group 1: no splenectomy; Group 2: total splenectomy without implant; Group 3: total splenectomy and immediate autogenous implant; Group 4: total splenectomy, preservation of the spleen in Ringer-lactate at room temperature, then sliced ​​and implanted; Group 5: total splenectomy, ​​spleen sliced and preserved in Ringer-lactate at room temperature before implantation; Group 6: total splenectomy with preservation of the spleen in Ringer-lactate at 4°C and then sliced ​​and implanted; Group 7: total splenectomy and the spleen sliced for preservation in Ringer-lactate at 4°C before implantation. After 90 days, we performed scintigraphic studies with Tc99m-colloidal tin (liver, lung, spleen or implant and clot), haematological exams (erythrogram, leucometry, platelets), biochemical dosages (protein electrophoresis) and anatomopathological studies. RESULTS: regeneration of autogenous splenic implants occurred in the animals of the groups with preservation of the spleen at 4ºC. The uptake of colloidal tin was higher in groups 1, 3, 6 and 7 compared with the others. There was no difference in hematimetric values ​​in the seven groups. Protein electrophoresis showed a decrease in the gamma fraction in the group of splenectomized animals in relation to the operated groups. CONCLUSION: the splenic tissue preserved in Ringer-lactate solution at 4ºC maintains its morphological structure and allows functional recovery after being implanted on the greater omentum.

Splenic implant preservation after conservation in lactated Ringer´s solution / Preservação de implante esplênico autógeno após conservação em solução de Ringer-lactato

ABSTRACT Objective: to evaluate the morphology and function of autogenous splenic tissue implanted in the greater omentum, 24 hours after storage in Ringer-lactate solution. Methods: we divided 35 male rats into seven groups (n=5): Group 1: no splenectomy; Group 2: total splenectomy without implant; Group 3: total splenectomy and immediate autogenous implant; Group 4: total splenectomy, preservation of the spleen in Ringer-lactate at room temperature, then sliced ​​and implanted; Group 5: total splenectomy, ​​spleen sliced and preserved in Ringer-lactate at room temperature before implantation; Group 6: total splenectomy with preservation of the spleen in Ringer-lactate at 4°C and then sliced ​​and implanted; Group 7: total splenectomy and the spleen sliced for preservation in Ringer-lactate at 4°C before implantation. After 90 days, we performed scintigraphic studies with Tc99m-colloidal tin (liver, lung, spleen or implant and clot), haematological exams (erythrogram, leucometry, platelets), biochemical dosages (protein electrophoresis) and anatomopathological studies. Results: regeneration of autogenous splenic implants occurred in the animals of the groups with preservation of the spleen at 4ºC. The uptake of colloidal tin was higher in groups 1, 3, 6 and 7 compared with the others. There was no difference in hematimetric values ​​in the seven groups. Protein electrophoresis showed a decrease in the gamma fraction in the group of splenectomized animals in relation to the operated groups. Conclusion: the splenic tissue preserved in Ringer-lactate solution at 4ºC maintains its morphological structure and allows functional recovery after being implanted on the greater omentum.

RESUMO Objetivo: avaliar morfologia e função de tecido esplênico autógeno, implantado no omento maior, 24 horas após conservação em solução de Ringer-lactato. Métodos: foram estudados 35 ratos machos, distribuídos em sete grupos (n=5): Grupo 1: sem esplenectomia; Grupo 2: esplenectomia total sem implante; Grupo 3: esplenectomia total e implante autógeno imediato; Grupo 4: esplenectomia total, preservação do baço em Ringer-lactato à temperatura ambiente, em seguida, fatiado e implantado; Grupo 5: esplenectomia total, baço fatiado e preservado em Ringer-lactato à temperatura ambiente antes de ser implantado; Grupo 6: esplenectomia total com preservação do baço em Ringer-lactato a 4°C e, em seguida, fatiado e implantado; Grupo 7: esplenectomia total e baço fatiado, para preservação em Ringer-lactato a 4°C antes de ser implantado. Após 90 dias, realizaram-se estudos cintilográficos com estanho coloidal-Tc99m (fígado, pulmão, baço ou implante e coágulo), hematológicos (eritrograma, leucometria, plaquetas), bioquímicos (eletroforese de proteínas) e anatomopatológicos. Resultados: ocorreu regeneração dos implantes esplênicos autógenos nos animais dos grupos com preservação do baço a 4ºC. A captação de estanho coloidal foi superior nos grupos 1, 3, 6 e 7 em relação aos demais. Não houve diferença nos valores hematimétricos nos sete grupos. A eletroforese de proteínas mostrou diminuição da fração gama no grupo de animais esplenectomizados em relação aos grupos operados. Conclusão: o tecido esplênico conservado em solução de Ringer-lactato à temperatura de 4ºC mantém sua estrutura morfológica e permite a recuperação funcional após ser implantado sobre o omento maior.