Critical mass of splenic autotransplant needed for the development of phagocytic activity in rats.

When total splenectomy is inevitable, heterotopic splenic autotransplantation seems to be the only alternative to maintain the functions of the spleen. The present study was carried out to analyse the critical mass of splenic autotransplant (SAT) for the development of phagocytic activity in rats. Wistar rats were submitted to total splenectomy (TS) alone or in combination with slices of SAT ranging from an average rate of 21·9% (one slice) to 100% (five slices) of the total splenic mass implanted into the greater omentum. Sixteen weeks after the beginning of the experiment, the animals were inoculated intravenously with a suspension of Escherichia coli labelled with Tc-99m. After 20 min, the rats were killed and the liver, lung and spleen or SAT, as well as blood samples were removed to determine the percentage of labelled bacteria uptake in these tissues. As the percentage of the total splenic mass contained in the SAT increased, the bacteria remaining in the blood decreased. From the implant of 26% up to the implant of the total splenic mass (100%) there was no difference in the bacteria remaining in the blood between the healthy animals of the control group and those submitted to TS combined with SAT. This finding shows that the critical mass needed for the development of phagocytic activity of macrophages in splenic autotransplants in adult rats is 26% of the total splenic mass.

Observations on the survival and neovascularization of fat grafts interchanged between C57BL/6-gfp and C57BL/6 mice.

BACKGROUND: Autologous fat transplantation has become a prevalent option for soft-tissue augmentation throughout the body. However, there is still much controversy over whether the fat grafts have survived or have been replaced in the recipient sites and over how the vessels grow. METHODS: After C57BL/6-gfp mice and C57BL/6 mice were paired randomly, the inguinal fat was excised and cut into pieces with scissors, and the adipose granules, approximately 0.2 ml (0.195 g), were transplanted subcutaneously with syringes to the dorsa of the paired mice. Samples were obtained at different time intervals: 3 days, 7 days, 2 weeks, 4 weeks, 2 months, 3 months, and 4 months after transplantation. Each fat sample was weighed to evaluate the graft volume. Histology, origin, and densities of neovascularization were examined by immunohistochemical staining. RESULTS: At 4 months, there was no significant difference in either graft survival or histologic evaluation. Histologic evaluation manifested the normal physiologic process of inflammation, neovascularization, remodeling, and maturity at different time intervals. At the endpoint, the immunohistochemical staining of CD34 showed that the difference in capillary density of the fat graft-31.3 ± 3.9 capillaries/mm on the dorsa of the C57BL/6-gfp mice and 29.6 ± 3.2 capillaries/mm on the dorsa of the C57BL/6 mice-was not statistically significant. The α-smooth muscle actin staining indicated that there were neovascularized vessels in both C57BL/6-gfp and C57BL/6 fat grafts. CONCLUSIONS: Fat grafts can survive and neovascularized vessels can grow from the recipient sites. Fat transplantation is feasible and will be applied more widely if fat graft survival is improved.

[Immunological graft rejection after autologous contralateral keratoplasty]. / Immunologisches Transplantatversagen nach autologer kontralateraler Keratoplastik.

Autologous keratoplasty from an amblyopic eye to the fellow oculus ultimus is a rarely used procedure. This is due to the relatively uncommon constellation of pathology. The following article reports the case of a graft rejection after autologous keratoplasty, while the homologous graft on the amblyopic fellow eye remained clear.

Pre-clinical assessment of autologous DC-based therapy in ovarian cancer patients with progressive disease.

Dendritic cell-based vaccines offer promise for therapy of ovarian cancer. Previous studies have demonstrated that oxidation of several antigens, including ovarian cancer cells, using hypochlorous acid strongly enhances their immunogenicity and their uptake and presentation by dendritic cells. The response of T cells and dendritic cells to autologous tumour from patients with active disease has not previously been investigated. Monocyte-derived dendritic cells were generated from patients with active disease and activated by co-culture with oxidised tumour cells and the TLR agonist poly I:C. The dendritic cells showed an activated phenotype, but secreted high levels of TGFß. Co-culture of the antigen-loaded dendritic cells with autologous T cells generated a population of effector T cells that showed a low level of specific lytic activity against autologous tumour, as compared to autologous mesothelium. The addition of neutralising antibody to TGFß in DC/T cell co-cultures increased the levels of subsequent tumour killing in three samples tested. Co-culture of monocytes from healthy volunteers with the ovarian cell line SKOV-3 prior to differentiation into dendritic cells reduced the ability of dendritic cells to stimulate cytotoxic effector cells. The study suggests that co-culture of dendritic cells with oxidised tumour cells can generate effector cells able to kill autologous tumour, but that the high tumour burden in patients with active disease may compromise dendritic cell and/or T cell function.

Optimization of autologous stem cell transplantation for systemic sclerosis -- a single-center longterm experience in 26 patients with severe organ manifestations.

OBJECTIVE: Autologous stem cell transplantation (aSCT) for systemic sclerosis (SSc) has been shown to be effective in recent reports. This aggressive approach and the disease itself are associated with a high mortality. We report our experiences in 26 consecutive patients. METHODS: Between 1997 and 2009, 26 patients were scheduled for aSCT. Our standard transplant regimen consists of cyclophosphamide (CYC) and granulocyte colony-stimulating factor (GCSF) for mobilization and CYC plus antithymocyte globulin for conditioning before the retransfusion of CD34 selected stem cells. The major outcome variable was the response to treatment [reduction of modified Rodnan skin score (mRSS) by 25%] at Month 6. Secondary endpoints were the transplant-related mortality and the progression-free survival. RESULTS: Significant skin and lung function improvement of the mRSS was achieved in 78.3% of patients at Month 6. The overall response rate was 91%, as some patients improved even after Month 6. Three patients died between mobilization and conditioning treatment, 2 due to severe disease progression and 1 whose death was considered treatment-related (i.e., GCSF or CYC toxicity). Depending on definitions, transplant-related mortality was 4% and treatment-related mortality 11%. Seven patients experienced a relapse during the 4.4 years of followup. The progression-free survival was 74%. Four patients died during followup and the most frequent causes of death were pulmonary and cardiac complications of SSc. CONCLUSION: aSCT led to significant improvement in most patients with SSc. The procedure requires further optimization; hence we are modifying our screening and treatment strategy. To minimize infectious complications, CYC for mobilization and GCSF were reduced. We intensified our screening for cardiac involvement and modified our conditioning regimen in case of cardiac involvement.

Immune mechanisms underlying the beneficial effects of autologous hematopoietic stem cell transplantation in multiple sclerosis.

A recent phase I/II clinical trial drew serious attention to the therapeutic potential of autologous hematopoietic stem cell transplantation (AHSCT) in multiple sclerosis. However, questions were raised as to whether these beneficial effects should be attributed to the newly reconstituted immune system per se, or to the lymphoablative conditioning regimen-induced immunosuppression, given that T-cell depleting combinational drug therapies were used in the study. We discuss here the possibility that both AHSCT and T-cell depleting therapies may re-program alternatively the immune system, and why transplantation of CD34+ hematopoietic stem cells may offer AHSCT a possible advantage regarding long-term remission.

Autologous versus allogeneic cell-based vaccines?

Devitalized tumor cells either autologous or allogeneic have been used as anti-cancer vaccines with the purpose of facilitating the induction of an immune response able to destroy growing tumor cells since the identification of tumor antigens was deemed not to be necessary, particularly in the autologous system. Such vaccines were tested first in animal models and then in the clinics as unmodified tumor cells or after insertion of genes coding for factors known to increase the immune response against tumors. These vaccines were usually given by subcutaneous injections along with different immunological adjuvants. Such immunization approaches were found to be effective in mice when carried out in a tumor preventive setting but significantly less in the therapeutic context, that is, in the presence of an established tumor. By analyzing several clinical trials of vaccination using either autologous or allogeneic unmodified and gene-modified tumor cells published in the last 10 to 15 years, we conclude for a lack of sufficient evidence for efficacy of this strategy in inducing both a strong immune response and a therapeutic response. A potential variant of this strategy is the direct intratumoral injection of immunostimulatory genes delivered by vectors in vivo. But even this approach failed to provide a statistically significant clinical benefit for the cancer patients.We also point out the inherent drawbacks of the tumor cell-based vaccine strategy that include (a) a limited frequency by which human tumor lines can be obtained from clinical samples, (b) the low number of available cells for vaccination, (c) the release of immune-suppressive factors by tumor cells, and (d) the cost and time necessary for standardization and collecting/expanding a number of cells according to the approved regulatory requirements. Thus, taking into consideration the new developments in cancer vaccines, we believe that tumor cell-based vaccines should be dismissed as anti-cancer vaccines unless a clear benefit could be demonstrated by the few ongoing trials of combination with new immunomodulating reagents (eg, anti-CTLA4, PD-1, chemotherapy).

Successful cross-presentation of allogeneic myeloma cells by autologous alpha-type 1-polarized dendritic cells as an effective tumor antigen in myeloma patients with matched monoclonal immunoglobulins.

For wide application of a dendritic cell (DC) vaccination in myeloma patients, easily available tumor antigens should be developed. We investigated the feasibility of cellular immunotherapy using autologous alpha-type 1-polarized dendritic cells (αDC1s) loaded with apoptotic allogeneic myeloma cells, which could generate myeloma-specific cytotoxic T lymphocytes (CTLs) against autologous myeloma cells in myeloma patients. Monocyte-derived DCs were matured by adding the αDC1-polarizing cocktail (TNFα/IL-1ß/IFN-α/IFN-γ/poly-I:C) and loaded with apoptotic allogeneic CD138(+) myeloma cells from other patients with matched monoclonal immunoglobulins as a tumor antigen. There were no differences in the phenotypic expression between αDC1s loaded with apoptotic autologous and allogeneic myeloma cells. Autologous αDC1s effectively took up apoptotic allogeneic myeloma cells from other patients with matched subtype. Myeloma-specific CTLs against autologous target cells were successfully induced by αDC1s loaded with allogeneic tumor antigen. The cross-presentation of apoptotic allogeneic myeloma cells to αDC1s could generate CTL responses between myeloma patients with individual matched monoclonal immunoglobulins. There was no difference in CTL responses between αDC1s loaded with autologous tumor antigen and allogeneic tumor antigen against targeting patient's myeloma cells. Our data indicate that autologous DCs loaded with allogeneic myeloma cells with matched immunoglobulin can generate potent myeloma-specific CTL responses against autologous myeloma cells and can be a highly feasible and effective method for cellular immunotherapy in myeloma patients.

Anti-HMGB1 neutralization antibody improves pain-related behavior induced by application of autologous nucleus pulposus onto nerve roots in rats.

STUDY DESIGN: Controlled, interventional, animal study. OBJECTIVE: To examine the involvement of high-mobility group box 1 (HMGB1) in the neuropathic pain state induced by the application of nucleus pulposus onto the dorsal root ganglion (DRG) and to investigate the effect of HMGB1 neutralization antibody in the pathogenesis. SUMMARY OF BACKGROUND DATA: HMGB1 is a potent proinflammatory mediator when present extracellularly, and anti-HMGB1 neutralization antibody inhibits inflammation, cytokine expression, and macrophage activation. METHODS: Thirty-nine adult female Sprague-Dawley rats (200-300 g) were used. The left L5/6 facet joint was removed, and the L5 DRG was exposed. Nucleus pulposus harvested from the tail was applied to the left L5 DRG. Then, 400 µg of anti-HMGB1 neutralization antibody was administered intraperitoneally after surgery. Behavioral testing using von Frey hairs was performed to investigate the mechanical withdrawal threshold. Neuronal damage was investigated by counting the number of activating transcription factor 3 (ATF3) neurons. The expressions of tumor necrosis factor-α (TNF-α) and HMGB1 were measured by double-labeled immunohistochemistry and immunoblotting. RESULTS: Immunoblotting of harvested nucleus pulposus revealed HMGB1 in the nucleus pulposus. Double-labeled immunohistochemistry revealed that macrophages in the applied nucleus pulposus expressed HMGB1 and TNF-α. Administration of anti-HMGB1 neutralization antibody significantly reduced the TNF-α expression in the DRG and improved the pain-related behavior from day 2 to day 14. CONCLUSION: HMGB1 appears to play an important role in the development of pain-related behavior induced by the application of nucleus pulposus onto the DRG. HMGB1 from applied nucleus pulposus and actively secreted from macrophages would act as a proinflammatory mediator together with proinflammatory cytokines. HMGB1 blocking therapy might become a new treatment method for neuropathic pain.

Importance of blood graft characteristics in auto-SCT: implications for optimizing mobilization regimens.

Although blood stem cells have been widely used to support high-dose therapy in the autologous setting, limited data are available on the effects of graft characteristics in patient outcomes other than haematopoietic engraftment. Retrospective studies suggest that patients who mobilize more CD34(+) cells have better outcomes than do patients who mobilize less well. Furthermore, immunological reconstitution may be important in terms of post-transplant outcome and is apparently affected by graft composition. There is accumulating evidence that the mobilization regimen used may be an important determinant of graft content. Plerixafor has been recently introduced combined with G-CSF in patients who mobilize poorly. In addition to enhancing mobilization of CD34(+) cells, there are indications that plerixafor may also affect other graft components. A combination of chemotherapy plus G-CSF with plerixafor has been shown to be very effective in stem-cell mobilization, but more data are needed in regard to other graft characteristics in this setting. Prospective studies are needed to evaluate whether higher CD34(+) doses or other modifications to graft composition translate into better long-term outcomes in the autologous setting. These studies are not only important in regard to defining the optimal stem-cell graft in the autologous setting, but also in identifying the optimal mobilization regimen.

Restoration of the immune balance by autologous bone marrow transplantation in juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) is one of the most frequent autoimmune diseases in childhood and is characterized by chronic inflammation of the synovial fluid in joints. Several drugs are available for the treatment of JIA, including various biological agents that interfere with critical cytokine pathways. Though very effective in suppressing disease activity, none of these drugs can cure the disease and induce a lasting medication free remission. A small proportion of JIA patients will become or are unresponsive to any form of medical treatment. For these severely ill patients autologous bone marrow transplantation (aBMT) is a last resort treatment. aBMT is remarkably effective in suppressing disease activity, with beneficial outcome reported in around 70% of these previously refractory patients. Moreover aBMT is the only treatment that can induce a lasting medication-free-disease remission in these patients. In the very long term (after 7 years of remission) however, some disease relapses are observed, with the disease returning in a less severe form compared to prior aBMT. The exact mechanism of how aBMT is inducing this lasting disease remission is still largely unknown, but data from both animal models and humans suggest a prominent role for regulatory T cells. In this review we reviewed the current views of the cellular mechanisms that lay beneath disease induction of JIA and the disease remission caused by aBMT therapy.

[Differential atticoanthrotomy with type 1 tympanoplasty as the method of choice for the treatment of chronic perforating otitis media with expressed mucositis].

The authors report the results of surgical treatment of 37 patients (42 ears) presenting with chronic perforating otitis media and grade I-III mucositis. All the patients were treated by differential atticoanthrotomy with type 1 tympanoplasty. Most of them suffered seriously disturbed ventilation and drainage functions of the Eustachian tube. Mean bone conduction and air conduction audibility thresholds for the voice frequency (0.5-2 kHz) prior to surgery were 12.5 +/- 6.7 and 12.0 +/- 4.2, 14.2 +/- 4.6 and 45.1 +/- 11.7 dB, 46.2 +/- 8.4 and 58.4 +/- 8.6 dB, respectively. Large defects of the tympanic membrane necessitated the use of a composite flap (autofascia and autocartilage) as the tympanic membrane autograft. Excellent survival of the tympanic autograft was documented in 33 (78.6%) patients. The remaining 9 patients had perforations in the neotympanic membrane of different size. Mean bone conduction and air conduction audibility thresholds for the voice frequency upon discharge from the clinic were 14.3 +/- 4.2, 14.0 +/- 4.4, 16.2 +/- 4.2 and 35.9 +/- 12.4 dB, 36.2 +/- 10.4, 46.6 +/- 8.2 dB, respectively. The functional outcome of the surgical treatment further improved in half a year due to the fact that the sound-conducting system acquired a higher rigidity (mean audibility thresholds for air conduction became 32.6 +/- 8.4, 34.0 +/- 6.8, and 42.2 +/- 8.4 dB. Audibility thresholds for bone conduction remained practically unaltered compared with their vales upon discharge from the clinic.

Pretransplant kidney-specific treatment to eliminate the need for systemic immunosuppression.

BACKGROUND: Despite significant side effects, chronic systemic immunosuppression remains the backbone of clinical transplantation. We investigated the feasibility of preventing early allorecognition in canine renal allografts using a nonsystemic pretreatment. METHODS: The renal vasculature was treated with a bioengineered interface consisting of a nano-barrier membrane during 3 hr of ex vivo warm perfusion. RESULTS: Preliminary feasibility of the immunocloaking technology was established by the following criteria: it is possible to achieve approximately 90% coverage of the vasculature with nano-barrier membrane after 3 hr of ex vivo warm perfusion; covering the luminal surfaces prevents allorecognition as determined by mixed lymphocyte-vascular endothelial reaction; covering the luminal surfaces does not negatively affect renal function as determined by autotransplant outcomes; and graft rejection is significantly postponed in canine kidneys treated with the immunocloaking technology. In the absence of systemic immunosuppression, untreated control dogs experienced a mean onset of rejection on day 6, whereas in the treated dogs with modified renal vascular luminal surfaces, the mean onset of rejection was significantly delayed until day 30. CONCLUSIONS: The ability to postpone, or eventually eliminate, the allorecognition that occurs immediately on reperfusion could provide a new window of opportunity to introduce adjunct therapies to support tolerance induction. To our knowledge, this is the first time significantly prolonged canine renal allograft survival has been achieved in the absence of systemic immunosuppression or immunologic manipulation of the recipient.

[Safety and efficacy of vaccination in children after stem cell transplantation. Part 1]. / Bezpieczenstwo i skutecznosc szczepien ochronnych prowadzonych u dzieci po przeszczepieniu komórek krwiotwórczych. Doniesienie 1.

UNLABELLED: The aim of the study was the evaluation of safety and efficacy of vaccination in children after stem cell transplantation. PATIENTS AND METHODS: 21 patients, 1.4-22 (average 7.8) years old, 13 boys and 8 girls after autologous (11-52%) and allogeneic (10-48%) transplantation were included in the vaccination protocol. Indications for transplantation were: neoplastic disease--16, immunodeficiencies--3 and aplastic anaemia 2 cases. Time between transplantation and beginning of vaccination protocol was 0.8-4 (average 1.5) years. Vaccination protocol was constructed on the basis of the European Group for Blood and Marrow Transplantation indications. We have evaluated: (1) quality of recipient immune reconstitution and protection against common pathogens (2) immunogenicity of revaccination schedule; (3) safety of the vaccination programme. RESULTS: With the exception of one patient presenting with repeated fever, lymph node enlargement, muscle and joint pain, no important side effects were observed. Meningococcial meningitis developed in one patient who refused vaccination. The mean concentrations of antibodies in the plasma before and after vaccination were as follows: anti-diphteria (54; 2285), anti-tetanus (136; 3149) and anti-hepatitis B virus (anti-HBs: 24; 474) IU/ml. CONCLUSIONS: (1) Vaccination in patients after transplantation is efficient and well tolerated. (2) Significant increase of antibody level was detected. (3) Any delay in beginning the vaccination can result in life threatening complications.