Immunosenescence does not abrogate engraftment of murine allogeneic bone marrow.

BACKGROUND: Allogeneic bone marrow transplantation is under investigation for a range of nonmalignant indications, including tolerance induction through mixed chimerism. This strategy has so far been tested experimentally only in young recipients. Due to immunosenescence, older patients have an increase in memory T cells (TMEM) as well as other alterations to their immune system, which may influence the potential to induce tolerance. We therefore investigated the impact of immunosenescence on chimerism-based tolerance induction. METHODS: Groups of young (2 months) and old (12 months) C57BL/6 recipients received BALB/c bone marrow under nonmyeloablative (3 Gy) and minimal (1 Gy) total body irradiation and treatment with costimulation blockade, T-cell depletion, or rapamycin. Multilineage chimerism, clonal deletion, and lymphocyte subsets were analyzed by flow cytometry. Tolerance was assessed by skin and heart grafts and enzyme-linked immunospot, intracellular cytokine, and mixed lymphocyte reaction assays. RESULTS: Unexpectedly, chimerism and tolerance were established in old recipients with comparable-and in some cases increased-efficacy as in young recipients employing costimulation blockade-based or T-cell depletion-based conditioning with 1 or 3 Gy total body irradiation. TMEM reactivity in (naïve) old mice was augmented in response to polyclonal but not to allogeneic stimulation, providing a mechanistic underpinning for the susceptibility to chimerism induction despite increased TMEM frequencies. Tolerance in old recipients was associated with peripheral and central clonal deletion and a higher frequency of regulatory T cells. CONCLUSION: Advanced age does not impair bone marrow engraftment, thereby widening the clinical potential of experimental protocols inducing transplantation tolerance through mixed chimerism.

Graft-specific immune cells communicate inflammatory immune responses after brain death.

BACKGROUND: Donor brain death (BD) triggers inflammatory graft activation that leads to impaired graft quality and outcome. We used a mouse BD model to investigate graft inflammation in cardiac transplants from immune-competent and immune-deficient donor animals. Effects of donor T-cell depletion were tested in an additional group of cardiac transplant recipients. METHODS: We analyzed systemic and graft-specific inflammatory activation after BD in donors and in syngeneic recipients of hearts retrieved from BD donors. To dissect the role of donor-specific immune cells in communicating BD-triggered inflammation, immune-deficient T-cell-, B-cell-, and natural killer cell-deficient Rag2/double knockout mice and naïve C57BL6 treated with anti-thymocyte globulin (Thymoglobulin; Genzyme Transplant, Cambridge, MA) were observed. RESULTS: Donor BD boosted lymphocyte activation in donors and recipients of syngeneic BD grafts. Lymphocyte activation was mitigated in recipients of immune-deficient and Thymoglobulin-treated BD donor grafts. Likewise, systemic and intra-graft levels of inflammatory cytokines interleukin -1, interleukin-6, interferon-γ, and tumor necrosis factor-α were significantly reduced in immune-deficient and anti-thymocyte globulin-treated recipients. Dense lymphocyte infiltrates were detected in the hearts from untreated BD donors; in contrast, the hearts from donors treated with Thymoglobulin demonstrated a preserved structure with minimal infiltrates comparable with naïve controls. CONCLUSION: BD triggers inflammatory graft activation communicated through intra-graft immune cells. Donor treatment with Thymoglobulin prevented inflammatory immune activation and improved graft quality to levels comparable to living donor organs.

Inflammatory immune responses in a reproducible mouse brain death model.

BACKGROUND: Brain death impairs donor organ quality and accelerates immune responses after transplantation. Detailed aspects of immune activation following brain death remain unclear. We have established a mouse model and investigated the immediate consequences of brain death and anesthesia on immune responses. METHODS: C57JBl/6 mice (n=6/group) were anesthetized with isoflurane (ISF) or ketamine/xylazine (KX); subsequently, animals underwent brain death induction and were followed for 3h under continuous ventilation. Blood pressure was monitored continuously and animals were resuscitated with normal saline to achieve normotension. Immune activation in brain dead animals was analyzed by IFNγ-ELispot, MLR, and flow-cytometry. Sham-operated and naïve animals served as controls. RESULTS: Blood pressure remained stable in both BD/KX and BD/ISF animals during the 3h observation time. Brain death was linked to systemic immune activation: IFNγ-expression of splenocytes and lymphocyte proliferation rates was significantly elevated subsequent to brain death (p<0.02, <0.01); T-cell activation markers CD28 and CD69 had increased in brain dead animals (p<0.03, <0.02). Isoflurane treatment in sham controls throughout the observation period (3.5h) revealed anesthesia associated IFNγ-expression and lymphocyte activation which were not observed when animals were treated with ketamine/xylazine (p<0.04, <0.009). CONCLUSIONS: This study reports on a reproducible and hemodynamically stable brain death mouse model. Hemodynamic stability was not impacted through either isoflurane or ketamine/xylazine induction. Of clinical relevance, prolonged anesthesia with isoflurane had been linked to pro-inflammatory cytokine activation. Brain death caused systemic immune activation in organ donors.

Myosin5a tail associates directly with Rab3A-containing compartments in neurons.

Myosin-Va (Myo5a) is a motor protein associated with synaptic vesicles (SVs) but the mechanism by which it interacts has not yet been identified. A potential class of binding partners are Rab GTPases and Rab3A is known to associate with SVs and is involved in SV trafficking. We performed experiments to determine whether Rab3A interacts with Myo5a and whether it is required for transport of neuronal vesicles. In vitro motility assays performed with axoplasm from the squid giant axon showed a requirement for a Rab GTPase in Myo5a-dependent vesicle transport. Furthermore, mouse recombinant Myo5a tail revealed that it associated with Rab3A in rat brain synaptosomal preparations in vitro and the association was confirmed by immunofluorescence imaging of primary neurons isolated from the frontal cortex of mouse brains. Synaptosomal Rab3A was retained on recombinant GST-tagged Myo5a tail affinity columns in a GTP-dependent manner. Finally, the direct interaction of Myo5a and Rab3A was determined by sedimentation velocity analytical ultracentrifugation using recombinant mouse Myo5a tail and human Rab3A. When both proteins were incubated in the presence of 1 mm GTPγS, Myo5a tail and Rab3A formed a complex and a direct interaction was observed. Further analysis revealed that GTP-bound Rab3A interacts with both the monomeric and dimeric species of the Myo5a tail. However, the interaction between Myo5a tail and nucleotide-free Rab3A did not occur. Thus, our results show that Myo5a and Rab3A are direct binding partners and interact on SVs and that the Myo5a/Rab3A complex is involved in transport of neuronal vesicles.

Scaled cortical impact in immature swine: effect of age and gender on lesion volume.

The piglet scaled cortical impact model creates a focal contusion using a skull-mounted, spring-loaded blunt indentation device scaled to achieve identical tissue strains in subjects with different brain sizes. Preliminary data showed that contusion size increased proportional to subject age. This study details the results from a new, larger series of subjects of three ages, and compares the effect of age and additional host and physiologic variables on injury response. Sixty-seven subjects, including infant (5- to 7-day-old), "toddler" (1-month-old), and early adolescent (4-month-old) swine underwent scaled cortical impact under strict anesthetic protocols. Serum glucose, testosterone, and 17beta-estradiol levels were measured. Lesion size was measured at 1 week post injury, as the ratio of the lesion area over the area of the contralateral hemisphere. Adolescent subjects had lesions over eight times larger than infants (p < 0.0001). Lesion volumes were larger in toddlers than in infants, most significantly for males (p < 0.05). Adolescent subjects were warmer on average, but there was no correlation between temperature and lesion volume within any age group. Serum glucose did not differ among ages. Infant males had the highest levels of circulating sex steroids. In this model, age was the most robust predictor of lesion size. Temperature had an effect, but did not explain all the variability seen among age groups. There was an interaction among gender, hormone levels, and lesion size in younger subjects. Characterization of these variables allows use of this model for treatment trials for subjects at different stages of maturation.

Tensile and tribological properties of high-crystallinity radiation crosslinked UHMWPE.

Osteolysis due to particulate wear debris associated with ultrahigh molecular weight polyethylene (UHMWPE) components of total joint replacement prostheses has been a major factor determining their in vivo lifetime. In recent years, radiation crosslinking has been employed to decrease wear rates in PE components, especially in acetabular cups of total hip replacement prostheses. A drawback of radiation crosslinking is that it leads to a crosslinked PE (or XPE) with lower mechanical properties compared with uncrosslinked PE. In contrast, high-crystallinity PEs are known to have several mechanical properties higher than conventional PE. In this study, we hypothesized that increasing the crystallinity of radiation crosslinked and remelted XPE would result in an increase in tensile properties without compromising wear resistance. High-pressure crystallization was performed on PE and XPE and analyzed for the resulting morphological alterations using differential scanning calorimeter, low voltage scanning electron microscopy, and ultrasmall angle X-ray scattering. Uniaxial tensile tests showed that high-pressure crystallization increased the tensile modulus and yield stress in both PE and XPE, decreased the ultimate strain and ultimate stress in PE but had no significant effect on ultimate strain or ultimate stress in XPE. Multidirectional wear tests demonstrated that high-pressure crystallization decreased the wear resistance of PE but had no effect on the wear resistance of XPE. In conclusion, this study shows that high-pressure crystallization can be effectively used to increase the crystallinity and modulus of XPE without compromising its superior wear resistance compared with PE.