Photobiomodulation Increases Viability in Full-Thickness Grafts in Rats Submitted to Nicotine.

BACKGROUND AND OBJECTIVES: Photobiomodulation (PBM) therapy with 830 nm wavelength or 660 wavelength to compare the effects with parameters of 30 mW, 0.028 cm2 , 9.34 seconds, and 3.64 J on the total integration of total skin grafts in rats submitted to nicotine. STUDY DESIGN/MATERIALS AND METHODS: Sixty male Wistar rats were divided in six groups: Sham-skin-grafting surgery; 830 nm-skin-grafting followed by 830 nm irradiation; 660 nm-skin grafting followed by 660 nm irradiation; Nicotine-subjected to subcutaneous nicotine injection (2 mg/kg twice a day for 4 weeks), followed by skin grafting; Group Nicotine/830 nm-similar to Group Nicotine, followed by 830 nm irradiation; Group Nicotine/660 nm-similar to Group Nicotine, followed by 660 nm irradiation. The percentage contraction of the grafting tissue was evaluated through ImageJ®. The thickness of the epidermis, inflammatory infiltrates, and the space between the implanted tissue and receptor bed were determined by histology; and the expression of vascular growth factor and blood vessel density (factor VIII) were evaluated by immunohistochemistry. RESULTS: The PBM at both wavelengths promoted a facilitating effect on the integration of the skin graft under nicotine and had a more significant effect on the thickness of the epidermis and expression of angiogenesis without nicotine at a wavelength of 830 nm. Different wavelengths influence responses related to the viability of cutaneous grafts in rats submitted to nicotine. CONCLUSIONS: The PBM with 830 nm and 660 nm promoted beneficial results in skin grafts submitted to the deleterious action of nicotine. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Selective local irradiation improves islet engraftment and survival in intra-bone marrow islet transplantation.

BACKGROUND: Bone marrow (BM) is as an alternative site for islet transplantation, but it is not an immunoprotected microenvironment and allogeneic islets are rejected. However, the BM, for its structure and anatomic position, offers the possibility to modulate microenvironment by local interventions. We here investigate whether local irradiation is able to improve islet engraftment and prevent rejection in BM in the absence of immunosuppression. METHODS: A model of BM local irradiation was set up. Islets were transplanted in syngeneic and fully major histocompatibility complex-mismatched recipients in control and locally irradiated BM; gain of normoglycemia and time to rejection were evaluated. RESULTS: BM local irradiation proved to be a selective and safe procedure. Syngeneic islet transplantation into locally irradiated BM had better outcome compared with not irradiated recipients in terms of capacity to gain normoglycemia (100% versus 56% in irradiated versus not irradiated mice). In the allogenic setting, glycemia was significantly lower in the first days after transplantation in the group of irradiated mice and local irradiation also delayed time to graft rejection (from 4 ± 1 days for not irradiated to 11 ± 1 days for locally irradiated mice). DISCUSSION: These data indicate that local immunosuppression by irradiation before islet transplantation in BM favors islet engraftment and delays time to rejection.

UVB-induced depletion of donor-derived dendritic cells prevents allograft rejection of immune-privileged hair follicles in humanized mice.

Dendritic cells (DCs) are key targets for immunity and tolerance induction; they present donor antigens to recipient T cells by donor- and recipient-derived pathways. Donor-derived DCs, which are critical during the acute posttransplant period, can be depleted in graft tissue by forced migration via ultraviolet B light (UVB) irradiation. Here, we investigated the tolerogenic potential of donor-derived DC depletion through in vivo and ex vivo UVB preirradiation (UV) combined with the injection of anti-CD154 antibody (Ab) into recipients in an MHC-mismatched hair follicle (HF) allograft model in humanized mice. Surprisingly, human HF allografts achieved long-term survival with newly growing pigmented hair shafts in both Ab-treated groups (Ab-only and UV plus Ab) and in the UV-only group, whereas the control mice rejected all HF allografts with no hair regrowth. Perifollicular human CD3+ T cell and MHC class II+ cell infiltration was significantly diminished in the presence of UV and/or Ab treatment. HF allografts in the UV-only group showed stable maintenance of the immune privilege in the HF epithelium without evidence of antigen-specific T cell tolerance, which is likely promoted by normal HFs in vivo. This immunomodulatory strategy targeting the donor tissue exhibited novel biological relevance for clinical allogeneic transplantation without generalized immunosuppression.

Deferoxamine Preconditioning of Irradiated Tissue Improves Perfusion and Fat Graft Retention.

BACKGROUND: Radiation therapy is a mainstay in the treatment of many malignancies, but collateral damage to surrounding tissue, with resultant hypovascularity, fibrosis, and atrophy, can be difficult to reconstruct. Fat grafting has been shown to improve the quality of irradiated skin, but volume retention of the graft is significantly decreased. Deferoxamine is a U.S. Food and Drug Administration-approved iron-chelating medication for acute iron intoxication and chronic iron overload that has also been shown to increase angiogenesis. The present study evaluates the effects of deferoxamine treatment on irradiated skin and subsequent fat graft volume retention. METHODS: Mice underwent irradiation to the scalp followed by treatment with deferoxamine or saline and perfusion and were analyzed using laser Doppler analysis. Human fat grafts were then placed beneath the scalp and retention was also followed up to 8 weeks radiographically. Finally, histologic evaluation of overlying skin was performed to evaluate the effects of deferoxamine preconditioning. RESULTS: Treatment with deferoxamine resulted in significantly increased perfusion, as demonstrated by laser Doppler analysis and CD31 immunofluorescent staining (p < 0.05). Increased dermal thickness and collagen content secondary to irradiation, however, were not affected by deferoxamine (p > 0.05). Importantly, fat graft volume retention was significantly increased when the irradiated recipient site was preconditioned with deferoxamine (p < 0.05). CONCLUSIONS: The authors' results demonstrated increased perfusion with deferoxamine treatment, which was also associated with improved fat graft volume retention. Preconditioning with deferoxamine may thus enhance fat graft outcomes for soft-tissue reconstruction following radiation therapy.

Small Incision Femtosecond Laser-assisted X-ray-irradiated Corneal Intrastromal Xenotransplantation in Rhesus Monkeys: A Preliminary Study.

BACKGROUND: Gamma-ray irradiation could significantly induce widespread apoptosis in corneas and reduced the allogenicity of donor cornea. And the X-rays may have similar biological effects. The feasibility and effects of X-ray-irradiated corneal lamellae have not been assessed yet. METHODS: Different doses (10 gray unit (Gy), 20 Gy, 50 Gy, 100 Gy) of X-ray irradiated corneal lamellae were collected from SMILE surgery. These corneal lamellae were assessed by physical characterization, hematoxylin and eosin (H-E) staining, Masson's staining, TdT-mediated dUTP nick end labeling (TUNEL), cell viability assay and transmission electron microscopy (TEM). We selected the optimum dose (100Gy) to treat the corneal lamellae to be the grafts. The human grafts and fresh allogeneic monkey corneal lamellae were implanted into rhesus monkeys via the small incision femtosecond laser-assisted surgery, respectively. Clinical examinations and the immunostaining were performed after surgery. RESULTS: There were no significant changes in the transparency of the corneal lamellae, but the absorbency of the corneal lamellae was increased. According to the H-E and Masson's staining results, irradiation had little impact on the corneal collagen. The TUNEL assay and cell viability assay results showed that 100Gy X-ray irradiation resulted in complete apoptosis in the corneal lamellae, which was also confirmed by TEM observations. In the following animal model study, no immune reactions or severe inflammatory responses occurred, and the host corneas maintained transparency for 24 weeks of observation. And the expression of CD4 and CD8 were negative in the all host corneas. CONCLUSION: X-ray irradiated corneal lamellae could serve as a potential material for xenogeneic inlay, and the small incision femtosecond laser-assisted implantation has the potential to become a new corneal transplantation surgical approach.

Low energy X-ray (grenz ray) treatment of purified islets prior to allotransplant markedly decreases passenger leukocyte populations.

Grenz rays, or minimally penetrating X-rays, are known to be an effective treatment of certain recalcitrant immune-mediated skin diseases, but their use in modulating allograft rejection has not been tested. We examined the capacity of grenz ray treatment to minimize islet immunogenicity and extend allograft survival in a mouse model. In a preliminary experiment, 1 of 3 immunologically intact animals demonstrated long-term acceptance of their grenz ray treated islet allograft. Further experiments revealed that 28.6% (2 of 7) grenz ray treated islet allografts survived >60 d. A low dose of 20Gy, was important; a 4-fold increase in radiation resulted in rapid graft failure, and transplanting a higher islet mass did not alter this outcome. To determine whether increased islet allograft survival after grenz treatment would be masked by immunosuppression, we treated the recipients with CTLA-4 Ig, and found an additive effect, whereby 17.5% more animals accepted the graft long-term versus those with CTLA-4 Ig alone. Cell viability assays verified that islet integrity was maintained after treatment with 20Gy. As well, through splenocyte infiltration analysis, donor CD4+ T cell populations 24-hours after transplant were decreased by more than16-fold in recipients receiving irradiated islets compared with control. Donor CD8+ T cell populations, although less prevalent, decreased in all treatment groups compared with control. Our results suggest that brief treatment of isolated islets with low energy grenz rays before allotransplantation can significantly reduce passenger leukocytes and promote graft survival, possibly by inducing donor dendritic cells to differentiate toward a tolerogenic phenotype.

Collagen cross-linking treatment increases adhesion in mock corneal grafts.

PURPOSE: We tested the hypothesis that collagen cross-linking (CXL) could be used to promote adhesion in mock corneal grafts. METHODS: Donated human corneal tissue underwent epithelial debridement and was cut into sections measuring 4mm×3mm. Paired sections were sutured together with 10-0 vicryl, forming mock corneal grafts. Looped 6-0 sutures were placed at each end to facilitate tension measurement. Mock grafts underwent CXL before being cultured for 2days in Eagle's MEM culture medium. Control mock grafts did not undergo CXL treatment before culture. Tissue was obtained from 4 donors and a maximum of 2 controls and 2 treated grafts was obtained from each donor. Following the culture period, the 10-0 sutures were cut. The mock grafts were mounted on force transducers and were put under increasing tension until eventually the sections were pulled apart. RESULTS: The mean applied stress required to generate graft failure was calculated for all mock grafts±standard error of the mean. In the control group 0.236±0.09mPa of applied stress was required to cause graft failure, in comparison to 0.691±0.12mPa in the treated group. A paired t-test showed this result to be significant, (p=0.0087). CONCLUSION: The results of our study are consistent with our hypothesis that CXL treatment could be used to promote early adhesion between separate sections of corneal tissue.

The Effects of Preoperative Radiotherapy on Head and Neck Free Flap Anastomosis Success.

PURPOSE: The long-term survival benefit of neoadjuvant radiotherapy for oropharyngeal tumors is controversially discussed in the literature. To bring more light into this important debate, we evaluated our cases. PATIENTS AND METHODS: In this retrospective study the main focus was the relationship between neoadjuvant irradiation and anastomosis failure. Other influencing factors including previous operative treatment and tumor stage and type also were studied. RESULTS: In total, 7 anastomoses failed (total failure rate, 8.4%; n = 83); 1 of the 7 underwent neoadjuvant irradiation (failure rate, 3.1%; n = 32). All 7 anastomosis failures were in squamous cell carcinoma cases, with 5 of them staged as T4. CONCLUSION: Our data suggest that neoadjuvant radiotherapy for oropharyngeal carcinoma does not increase the risk of postoperative anastomosis failure compared with surgical reconstruction alone. Furthermore, the data suggest a correlation between the stage and type of tumor.

Splenic Irradiation for the Treatment of Severe Antibody-Mediated Rejection.

Patients requiring desensitization prior to renal transplantation are at risk for developing severe antibody-mediated rejection (AMR) refractory to treatment with plasmapheresis and intravenous immunoglobulin (PP/IVIg). We have previously reported success at graft salvage, long-term graft survival and protection against transplant glomerulopathy with the use of eculizumab and splenectomy in addition to PP/IVIg. Splenectomy may be an important component of this combination therapy and is itself associated with a marked reduction in donor-specific antibody (DSA) production. However, splenectomy represents a major operation, and some patients with severe AMR have comorbid conditions that substantially increase their risk of complications during and after surgery. In an effort to spare recipients the morbidity of a second operation, we used splenic irradiation in lieu of splenectomy in two incompatible live donor kidney transplant recipients with severe AMR in addition to PP/IVIg, rituximab and eculizumab. This novel approach to the treatment of severe AMR was associated with allograft salvage, excellent graft function and no short- or medium-term adverse effects of the radiation therapy. One-year surveillance biopsies did not show transplant glomerulopathy (tg) on light microscopy, but microcirculation inflammation and tg were present on electron microscopy.

Anti-CD45 radioimmunotherapy without TBI before transplantation facilitates persistent haploidentical donor engraftment.

Many patients with hematologic malignancies cannot tolerate hematopoietic cell transplantation (HCT), whereas others may not have a compatible human leukocyte antigen-matched donor. To overcome these limitations, we optimized a conditioning regimen employing anti-CD45 radioimmunotherapy (RIT) replacing total body irradiation (TBI) before haploidentical HCT in a murine model. Mice received 200 to 400 µCi (90)Y-anti-CD45 antibody (30F11), with or without fludarabine (5 days starting day -8), with cyclophosphamide (CY; days -2 and +2) for graft-versus-host disease prophylaxis, and 1.5 × 10(7) haploidentical donor bone marrow cells (day 0). Haploidentical bone marrow transplantation (BMT) with 300 µCi (90)Y-anti-CD45 RIT and CY, without TBI or fludarabine, led to mixed chimeras with 81.3 ± 10.6% mean donor origin CD8(+) cells detected 1 month after BMT, and remained stable (85.5 ± 11% mean donor origin CD8(+) cells) 6 months after haploidentical BMT. High chimerism levels were induced across multiple hematopoietic lineages 28 days after haploidentical BMT with 69.3 ± 14.1%, 75.6 ± 20.2%, and 88.5 ± 11.8% CD3(+) T cells, B220(+) B cells, and CD11b(+) myeloid cells, respectively. Fifty percent of SJL leukemia-bearing mice treated with 400 µCi (90)Y-DOTA-30F11, CY, and haploidentical BMT were cured and lived >200 days. Mice treated with 200 µCi (90)Y-DOTA-30F11 had a median overall survival of 73 days, while untreated leukemic mice had a median overall survival of 34 days (P < .001, Mantel-Cox test). RIT-mediated haploidentical BMT without TBI may increase treatment options for aggressive hematologic malignancies.

Local allograft irradiation as an adjunct for treating severe resistant rejection after liver transplantation in adults.

Acute rejection after liver transplantation occurs in one-third of all recipients and can be managed with conventional rejection therapy in the majority of cases. In rare instances, patients with severe acute rejection may be refractory to or have contraindications for conventional therapies. This case series evaluates the role of local allograft irradiation (LAI) as an adjunct for patients with rejection that is refractory to or contraindicated for conventional therapies. Additionally, the literature on the use of radiation therapy for reversing rejection in solid organ transplantation is reviewed. Five patients underwent 9 LAI treatments: 2 had refractory rejection, and 1 each had a malignancy, a concurrent life-threatening infection, and serum sickness with antibody therapy. Conventional rejection therapies included steroids, calcineurin inhibitors, and antithymocyte globulin. LAI consisted of 3 cycles of 1.5 Gy directed toward the liver allograft. Two of the 5 patients remained alive with excellent graft function. Six of the 9 treatments were successful in rescuing the liver allograft (reversing the rejection episode). Treatment success was associated with lower pretreatment serum bilirubin levels and higher pretreatment alanine aminotransferase levels. Compared with patients with immunosuppression-responsive severe acute rejection, those requiring LAI trended toward a later onset of first rejection. In conclusion, local irradiation of liver allografts can be a useful adjunct in patients for whom conventional options have been exhausted or cannot be used. The ability of LAI to reverse allograft dysfunction and promote patient survival appears to be greatest before the onset of severe cholestatic injury.

Infusion of dendritic cells carrying donor lymphocytes treated with 8-methoxypsoralen and ultraviolet A light induces CD19+ IL-10+ regulatory B cells and promotes skin allograft survival.

PUVA-SP DCs are immature dendritic cells (imDCs) that have taken up donor splenic lymphocytes treated with 8-methoxypsoralen and ultraviolet A light (PUVA-SPs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient imDCs, and infusion of PUVA-SP DCs can induce CD4(+)CD25(high)Foxp3(+) regulatory T cells (Treg). However, the regulatory effect of PUVA-SP DCs on B cells is poorly understood. In this study, we compared PUVA-SP DCs with imDCs in terms of lipopolysaccharide (LPS) responsiveness and flow cytometric analysis of interleukin (IL) 10 expression in splenic CD19(+)B cells. Our results demonstrate that PUVA-SP DCs can suppress subsequent LPS-induced DC maturation and that infusion of PUVA-SP DCs, in the absence of an immunosuppressant, significantly promotes skin allograft survival. This effect was associated with up-regulation of circulating regulatory B cells exhibiting preferential IL-10 secretion. Our results suggest that effective treatments involving infusion of PUVA-SP DCs is likely related to the modulation not only of T-cell and Treg functions but also of B-cell and regulatory B-cell (Breg) functions.

[Effect of different irradiation doses on the establishment of murine cGVHD model after MHC matched spleen stem cell transplantation].

This study was aimed to investigate the effect of different irradiation doses on the establishment of murine cGVHD model after MHC matched spleen stem cell transplantation. The male mouse BALB/c(H)-2d was totally irradiated with different radiation dose of (60)Co (TBI), then was infused with the same number of splenocytes from MHC matched DBA/2 male mice. After transplantation, the bodyweight, general appearance, hair changes, survival time and pathological damage were observed. The results indicated that compared to the control group (0 Gy) and the 7.0 Gy group, the mice irradiated with 7.5 Gy and 8.0 Gy showed cGVHD symptoms and obvious pathological damage. At the end of experiments (60 d after transplantation), all mice irradiated by 7.5 Gy survived while only 60% animals survived in the 8.0 Gy group. It is concluded that under infusion of 10(8) MHC matched splenocytes per mouse, 7.5 Gy irradiation is appropriate to efficiently establish cGVHD model. This study laid an important foundation for further studying the pathogenesis, biological characteristics, and intervention factors of cGVHD.

Double umbilical cord blood transplantation after novel myeloablative conditioning using a regimen of fludarabine, busulfan, and total lymphoid irradiation.

We conducted a pilot study evaluating double umbilical cord blood transplantation (dCBT) after myeloablative conditioning with fludarabine and busulfan 3.2 mg/kg i.v. × 4, followed by total lymphoid irradiation at 400 cGy (FluBu4/TLI) for any indicated hematological disorder for patients without a suitable donor. Twenty patients with predominantly high-risk disease underwent dCBT according to protocol. The regimen was well tolerated, with mucositis as the primary observed toxicity (n = 19). The cumulative incidence of neutrophil engraftment was 89% (95% confidence interval [CI], 64% to 97%), with a median time to recovery of 16 days (range, 12 to 31 days). All evaluable patients with neutrophil engraftment achieved complete donor chimerism by day 40. The cumulative incidence of grades III and IV acute graft-versus-host disease (GVHD) at day 100 was 10% (95% CI, 2% to 27%), and the cumulative incidence of chronic GVHD was 35% (95% CI, 16% to 55%) by the end of the study. At 1 year, the cumulative incidence of treatment-related mortality (TRM) was 35% (95% CI, 16% to 55%). The leading cause of nonrelapse mortality was acute GVHD (n = 4), followed by graft failure (n = 2) and chronic GVHD (n = 1). TRM was significantly associated with a pretransplantation hematopoietic cell transplantation-specific comorbidity index score ≥ 3 (P = .005). At 1 year, disease relapse occurred in 6 patients and overall survival was 40% (95% CI, 19% to 60%). We conclude that FluBu4/TLI is an adequate preparative regiment before dCBT, providing high engraftment rates and relatively early neutrophil recovery. The best survival outcomes were seen in patients without significant comorbidities before transplantation, and outcomes are comparable to previously published dCBT studies.

Histological assessment of non-ablative laser stimulation of tissue repair in acellular dermal grafts.

AIM: The objective of this study was to compare integration of AlloDerm® acellular dermal grafts in animals subjected to non-ablative laser irradiation and animals not exposed to this therapy. METHODS: Standardized AlloDerm® fragments measuring 5 mm² were grafted into the subcutaneous tissue overlying the calvaria in 32 Wistar rats. Laser therapy (685 ηm), at a dose of 4 J/cm2 per session, was applied immediately after surgical intervention and every 48 hours thereafter for a total of four applications. RESULTS: Analysis of histology slides revealed significantly greater edema in the control group. There was no neutrophil infiltration in the laser-irradiated group at any point during the study period, whereas such infiltration was present in control animals at three of the four points of observation. In the laser therapy group, lymphocyte infiltration was observed from day 1, whereas in the control group, it was only apparent from day 3. Vascularization was substantially greater in the control group. In the experimental group, the AlloDerm® graft was completely replaced by fibrous tissue. CONCLUSION: These findings suggest that add-on non-ablative laser therapy is an effective stimulator of healing and graft integration after placement of AlloDerm® acellular dermal grafts.

Evaluation of engraftment and immunological tolerance after reduced intensity conditioning in a rhesus hematopoietic stem cell gene therapy model.

Reduced intensity conditioning (RIC) is desirable for hematopoietic stem cell (HSC) targeted gene therapy; however, RIC may be insufficient for efficient engraftment and inducing immunological tolerance to transgenes. We previously established long-term gene marking in our rhesus macaque autologous HSC transplantation model following 10 Gy total body irradiation (TBI). In this study, we evaluated RIC transplantation with 4 Gy TBI in two rhesus macaques that received equal parts of CD34(+) cells transduced with green fluorescent protein (GFP)-expressing lentiviral vector and empty vector not expressing transgenes. In both animals, equivalently low gene marking between GFP and empty vectors was observed 6 months post-transplantation, even with efficient transduction of CD34(+) cells in vitro. Autologous lymphocyte infusion with GFP marking resulted in an increase of gene marking in lymphocytes in a control animal with GFP tolerance, but not in the two RIC-transplanted animals. In vitro assays revealed strong cellular and humoral immune responses to GFP protein in the two RIC-transplanted animals, but this was not observed in controls. In summary, 4 Gy TBI is insufficient to permit engraftment of genetically modified HSCs and induce immunological tolerance to transgenes. Our findings should help in the design of conditioning regimens in gene therapy trials.

Transplanted germ cells persist long-term in irradiated ram testes.

Testicular germ cell transplantation provides a tool to study transgenesis, spermatogenesis and to increase production efficiency in livestock industries. Isolated testicular germ cells can be transplanted into testes of livestock breeds to generate sperm of donor origin. In sheep, methods have been developed previously to isolate cell populations from ram testes and transplant these into irradiated testes of recipient rams. This has resulted in rams producing sperm derived from the donor cells and a number of the recipient animals have produced donor-derived offspring from the introduced spermatogonial cells. Microsatellite genotyping data presented here demonstrates that these rams continue to produce sperm of donor origin for at least 5 years post-transplantation. This research provides new evidence of the stability of transplanted germ cells in a commercially important species, and with further refinements to cell isolation, transplantation and recipient preparation, this technology should find use in breeding systems to increase livestock production efficiency.

Comparing high and low total body irradiation dose rates for minimum-intensity conditioning of dogs for dog leukocyte antigen-identical bone marrow grafts.

We tested the hypothesis that total body irradiation (TBI) given at a high dose rate would be more immunosuppressive and lead to a higher incidence of stable hematopoietic cell engraftment after suboptimal levels of conditioning irradiation compared with TBI at a low dose rate. We assessed the engraftment success of dog leukocyte antigen-identical bone marrow transplantation in recipients of 100, 150, and 200 cGy TBI administered at a rate of 7 or 70 cGy/min. Dogs received donor marrow on the same day as TBI and were subsequently treated with postgraft immunosuppression consisting of mycophenolate mofetil (for 28 days) and cyclosporine (for 37 days). Donor chimerism was monitored until the end of study and was characterized by either graft rejection or stable engraftment. Increasing the radiation dose rate from the traditional 7 cGy/min to 70 cGy/min did not lead to increased engraftment success at any of the irradiation doses tested. The dose rate of 70 cGy/minute was no more hematotoxic than the rate of 7 cGy/minute. TBI delivered at a high dose rate was well tolerated but was not associated with a better rate of allogeneic hematopoietic cell engraftment compared with TBI delivered at a lower dose rate.