Reciprocal Donor-Recipient Strain Combinations Present Different Vascularized Composite Allotransplantation Outcomes in Rodent Models.

BACKGROUND: Although vascularized composite allotransplantation (VCA) has been the focus of many animal studies, further research is needed to determine the potential for a generalized model and immunosuppression regimen that applies across different donor-recipient combinations. In this study, the authors evaluated the outcome of VCAs performed on reciprocal rodent donor-recipient combinations. METHODS: VCA was performed in rats using Lewis and Brown Norway (BN) donor-recipient pairs, under the previously reported antilymphocyte serum/cyclosporine/adipose-derived stem cell regimen. Similarly, a published co-stimulatory blockade/rapamycin regimen was performed on the mouse VCA model between Balb/C and C57BL/6 strains. RESULTS: To accommodate the active behaviors of BN recipients, the allograft had to be modified and inset to the neck instead of to the groin. The tolerogenic regimen did not provide the same benefits for BN rats as it did for Lewis recipients. Increasing antilymphocyte serum dose and extending the duration of cyclosporine administration from 10 to 21 days significantly prolonged allograft survival and induced donor-specific tolerance. In mice, the co-stimulatory blockade/rapamycin regimen produced inferior VCA outcomes in BALB/c recipients than in C57BL/6 recipients. In both rats and mice, the authors identified an association between the tolerance outcome and the peripheral chimerism measured on postoperative day 30. CONCLUSIONS: Reciprocal donor-recipient combinations led to different responses toward the immunosuppression regimen and varied VCA outcomes. Sustained donor chimerism that remained in circulation for 1 month after surgery supported long-term VCA survival. Modification of the model and immunosuppression regimen accordingly is recommended. CLINICAL RELEVANCE STATEMENT: Various donor-recipient combinations respond differently to the immunosuppression regimens. Maintaining donor chimerism for 30 days after surgery improves VCA survival. It is recommended to tailor the immunosuppression regimen based on the recipient's background to optimize outcomes.

Predicting Outcomes of Rat Vascularized Composite Allotransplants through Quantitative Measurement of Chimerism with PCR-Amplified Short Tandem Repeat.

Chimerism has been associated with the induction and maintenance of tolerance to vascularized composite allotransplants (VCA). Although most VCA studies have examined chimerism using flow cytometry, we proposed that precision in the measurement of chimerism may be better approximated when complimentary polymerase chain reaction (PCR) is applied to a specific short tandem repeat (STR). We identified a STR, D10Rat25, which exhibited a ~20 bp difference in length between two rat strains (BN and LEW) often utilized as the donor and recipient in many allotransplantation studies. D10Rat25 was PCR-amplified and quantified with capillary electrophoresis. With pure LEW and BN DNA, a standard curve was constructed to measure chimerism with good linearity. When applied to rat VCA, the relationship between systematic (in peripheral blood) or local (at specific organ/tissues) chimerism to allograft outcomes was noted. We found that peripheral chimerism was elevated by up to ~9% postoperative month 1 (POM 1) but then reduced regardless of the final VCA outcome. However, differences in VCA skin chimerism between early rejection and POM 1 (shown as ΔChimerismPOM1-ER) were notable with respect to VCA outcomes. ROC analysis identified the optimum cutoff value as 17.7%. In summary, we have developed a reliable method to quantify the percentage of BN cells/DNA in BN-LEW chimeras. The detection limit was characterized, and the acquired data were comparable with flow cytometry. This method can be applied to solid organ and composite tissue allotransplantation studies.

Bioimaging of alloantigen-stimulated regulatory T cells in rat vascularized composite allotransplantation.

BACKGROUND: Tipping the balance toward regulatory T cells (Tregs) through adoptive cell therapy has shown promise to induce transplantation tolerance. Although such strategy has been explored in many mice organ transplantation studies, less knowledge was available in rat systems. Furthermore, the behaviors of the transferred cells have not been well studied in real-time fashion. METHODS: Tregs from naïve LEW rats were purified in two steps with the autoMACS system. Immunosuppression potential of these cells was examined with mixed lymphocyte reaction. Following stimulation by the alloantigen in vitro, the purified Tregs were infused into the recipients of vascularized composite allotransplantation (VCA). Secondary allogeneic skin grafting challenge was performed on the recipients with long-term survived VCA. Live optical imaging was performed to track luciferase-expressing Tregs following infusion to the VCA recipients. Expression of relevant molecules was studied by flow cytometry or quantitative RT-PCR. RESULTS: Rat Tregs were enriched following two-step cell sorting and showed immunosuppressive capacity. Upon infusion into the VCA recipients that have been treated with antilymphocyte serum and short-term Cyclosporin A, the antigen-stimulated Tregs significantly prolonged VCA survival and induced donor-specific tolerance. Tracking of the infused bioluminescent Tregs showed their specific homing to lymph nodes, and then to the VCAs. Following secondary skin grafting, Tregs specifically gathered at the donor-derived skin that was not rejected by the recipient. The in vivo migratory pattern coincided with the altered expression of cell surface molecules of CD62L, CD103, CD134, and CD278, following donor-antigen stimulation. Elevated expression of CCR4 and CCL22 in allograft may also participate in recruiting Tregs for maintenance of VCA survival and promoting donor-specific tolerance. CONCLUSION: Sorted Tregs induced donor-specific tolerance to VCA in rats. Live cell tracking demonstrated that activated CD4+CD25+FoxP3+ Tregs targeted primarily to the lymph nodes and VCA. The Tregs migrated to the secondary grafted donor skin and contributed to the maintenance of donor-specific tolerance. These behaviors were associated with phenotypic changes induced by donor antigen stimulation. Increased expression of CCR4 and CCL22 in VCA skin may also be relevant.

Large-Scale Analysis of Antimicrobial Activities in Relation to Amphipathicity and Charge Reveals Novel Characterization of Antimicrobial Peptides.

It has been unclear to which antimicrobial activities (e.g., anti-gram-positive bacterial, anti-gram-negative bacterial, antifungal, antiparasitic, and antiviral activities) of antimicrobial peptides (AMPs) a given physiochemical property matters most. This is the first computational study using large-scale AMPs to examine the relationships between antimicrobial activities and two major physiochemical properties of AMPs-amphipathicity and net charge. The results showed that among all kinds of antimicrobial activities, amphipathicity and net charge best differentiated between AMPs with and without anti-gram-negative bacterial activities. In terms of amphipathicity and charge, all the AMPs whose activities were significantly associated with amphipathicity and net charge were alike except those with anti-gram-positive bacterial activities. Furthermore, the higher the amphipathic value, the greater the proportion of AMPs possessing both antibacterial and antifungal activities. This dose-response-like pattern suggests a possible causal relationship-dual antibacterial and antifungal activities of AMPs may be attributable to amphipathicity. These novel findings could be useful for identifying potent AMPs computationally.

Nonvolatile reconfigurable sequential logic in a HfO2 resistive random access memory array.

Resistive random access memory (RRAM) based reconfigurable logic provides a temporal programmable dimension to realize Boolean logic functions and is regarded as a promising route to build non-von Neumann computing architecture. In this work, a reconfigurable operation method is proposed to perform nonvolatile sequential logic in a HfO2-based RRAM array. Eight kinds of Boolean logic functions can be implemented within the same hardware fabrics. During the logic computing processes, the RRAM devices in an array are flexibly configured in a bipolar or complementary structure. The validity was demonstrated by experimentally implemented NAND and XOR logic functions and a theoretically designed 1-bit full adder. With the trade-off between temporal and spatial computing complexity, our method makes better use of limited computing resources, thus provides an attractive scheme for the construction of logic-in-memory systems.

Analysis and prediction of the critical regions of antimicrobial peptides based on conditional random fields.

Antimicrobial peptides (AMPs) are potent drug candidates against microbes such as bacteria, fungi, parasites, and viruses. The size of AMPs ranges from less than ten to hundreds of amino acids. Often only a few amino acids or the critical regions of antimicrobial proteins matter the functionality. Accurately predicting the AMP critical regions could benefit the experimental designs. However, no extensive analyses have been done specifically on the AMP critical regions and computational modeling on them is either non-existent or settled to other problems. With a focus on the AMP critical regions, we thus develop a computational model AMPcore by introducing a state-of-the-art machine learning method, conditional random fields. We generate a comprehensive dataset of 798 AMPs cores and a low similarity dataset of 510 representative AMP cores. AMPcore could reach a maximal accuracy of 90% and 0.79 Matthew's correlation coefficient on the comprehensive dataset and a maximal accuracy of 83% and 0.66 MCC on the low similarity dataset. Our analyses of AMP cores follow what we know about AMPs: High in glycine and lysine, but low in aspartic acid, glutamic acid, and methionine; the abundance of α-helical structures; the dominance of positive net charges; the peculiarity of amphipathicity. Two amphipathic sequence motifs within the AMP cores, an amphipathic α-helix and an amphipathic π-helix, are revealed. In addition, a short sequence motif at the N-terminal boundary of AMP cores is reported for the first time: arginine at the P(-1) coupling with glycine at the P1 of AMP cores occurs the most, which might link to microbial cell adhesion.

A novel rat full-thickness hemi-abdominal wall/hindlimb osteomyocutaneous combined flap: influence of allograft mass and vascularized bone marrow content on vascularized composite allograft survival.

Vascularized bone marrow transplantation (VBMT) appears to promote tolerance for vascularized composite allotransplantation (VCA). However, it is unclear whether VBMT is critical for tolerance induction and, if so, whether there is a finite amount of VCA that VBMT can support. We investigated this with a novel VCA combined flap model incorporating full-thickness hemiabdominal wall and hindlimb osteomyocutaneous (HAW/HLOMC) flaps. Effects of allograft mass (AM) and VBMT on VCA outcome were studied by comparing HAW/HLOMC VCAs with fully MHC-mismatched BN donors and Lewis recipients. Control groups did not receive treatments following transplantation. Treatment groups received a short course of cyclosporine A (CsA), antilymphocyte serum, and three doses of adipocyte-derived stem cells (POD 1, 8, and 15). The results showed that all flaps in control allogeneic groups rejected soon after VCAs. Treatment significantly prolonged allograft survival. Three of eight recipients in HLOMC treatment group had allografts survive long-term and developed donor-specific tolerance. Significantly higher peripheral chimerism was observed in HLOMC than other groups. It is concluded that the relative amount of AM to VBMT is a critical factor influencing long-term allograft survival. Accordingly, VBMT content compared with VCA mass may be an important consideration for VCA in humans.

Syngeneic adipose-derived stem cells with short-term immunosuppression induce vascularized composite allotransplantation tolerance in rats.

BACKGROUND AIMS: A clinically applicable tolerance induction regimen that removes the requirement for lifelong immunosuppression would benefit recipients of vascularized composite allotransplantation (VCA). We characterized the immunomodulatory properties of syngeneic (derived from the recipient strain) adipocyte-derived stem cells (ADSCs) and investigated their potential to induce VCA tolerance in rats. METHODS: ADSCs were isolated from Lewis (LEW, RT1A(l)) rats; their immunomodulatory properties were evaluated by means of mixed lymphocyte reactions in vitro and VCAs in vivo across a full major histocompatibility complex mismatch with the use of Brown-Norway (BN, RT1A(n)) donor rats. Two control and four experimental groups were designed to evaluate treatment effects of ADSCs and transient immunosuppressants (anti-lymphocyte globulin, cyclosporine) with or without low-dose (200 cGy) total body irradiation. Flow cytometry was performed to quantify levels of circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). RESULTS: Cultured syngeneic ADSCs exhibited CD90.1(+)CD29(+)CD73(+)CD45(-)CD79a(-)CD11b/c(-) phenotype and the plasticity to differentiate to adipocytes and osteocytes. ADSCs dramatically suppressed proliferation of LEW splenocytes against BN antigen and mitogen, respectively, in a dose-dependent fashion, culminating in abrogation of allo- and mitogen-stimulated proliferation at the highest concentration tested. Accordingly, one infusion of syngeneic ADSCs markedly prolonged VCA survival in LEW recipients treated with transient immunosuppression; of these, 66% developed tolerance. Total body irradiation provided no additional VCA survival benefit. An important role for Tregs in tolerance induction/maintenance was suggested in vivo and in vitro. CONCLUSIONS: Treatment comprising syngeneic ADSCs and transient immunosuppression (i) increased levels of circulating Tregs and (ii) induced tolerance in 66% of recipients of major histocompatibility complex-mismatched VCAs.