Prolonged cold-preservation of nerve allografts

The goal of this study was to determine the effect of varying durations of cold-preservation on the immunogenicity of nerve allografts and their subsequent ability to facilitate neuroregeneration across a short nerve gap. Allografts preserved for 1, 4, and 7 weeks were compared to untreated allografts and isografts. There was a shift from an interferon-gamma-producing cellular response (untreated allografts) to an absence of response (7-week cold-preserved allografts and isografts). There were no detectable alloantibodies by flow cytometry. Histomorphometry distal to the graft showed robust regeneration in the isograft and 7-week cold-preserved groups when compared to the untreated allograft group. Increasing duration of cold-preservation diminished the cellular immune response. This cold-preservation does not preclude subsequent nerve regeneration across a short nerve graft. Prolonged cold-preservation of nerve allograft tissue could serve as a means to produce unlimited graft material for use in peripheral nerve reconstruction.     

Correlation between interleukin-15 and granzyme B expression and acute lung allograft rejection

The levels of interleukin (IL)-15 and granzyme B mRNA expression have been correlated with acute rejection episodes of kidney and heart allografts. Thus, the purpose of this study was to determine whether a correlation exists between the expression of IL-15 and granzyme B and acute lung allograft rejection. Toward this, the levels of IL-15 and granzyme B mRNA expression were determined in bronchoalveolar lavage-derived alveolar macrophages and total cells, respectively, from lung transplant patients with stable lung allograft function and patients undergoing acute rejection episodes. The expression levels of IL-15 mRNA was significantly higher in the patients undergoing acute rejection as compared to patients with stable lung function (P=0.02). The expression levels of granzyme B mRNA was also significantly higher in the patients undergoing acute rejection as compared to patients with stable lung function (P=0.005). The Receiver-Operating-Characteristic curve demonstrated that acute rejection can be predicted with a sensitivity of 94% and specificity of 67% with the use of a cutoff value of 3.1 fg of granzyme B mRNA per microgram of total RNA (or 71% sensitivity and 75% specificity of a cutoff value of 9.1 fg/microg). These data indicate that IL-15 secreted by activated alveolar macrophages and granzyme B secreted by activated CD8+ cytotoxic T lymphocytes play important roles in the process of acute lung allograft rejection.     

Effects of serotoninergic drugs on tremor induced by physostigmine in rats

We investigated the effects of various serotoninergic drugs and serotonin (5-HT) depletion on physostigmine-induced visible tremor in rats. Physostigmine (0.25-1.5 mg/kg) caused dose-dependent tremor, initiated at 3-5 min (latency decreases as dose increases) and lasted for 30-35 min. Serotonin agonists, 8-hydroxy-2-(di-n-propylamino) tetralin (2.5 mg/kg) and buspirone (5 mg/kg) augmented the tremor response caused by physostigmine. The 5-HT(1)/5-HT(2) receptor antagonist, metergoline (1 mg/kg), and 5-HT(2) blocker, cyproheptadine (10 mg/kg) significantly decreased the duration (40%) as well as intensity (45-50%) of physostigmine-tremor. The 5-HT(2a)/5-HT(2c) antagonist ritanserin (5 mg/kg) significantly reduced the duration (60%) without affecting the intensity of the tremor. In 5-HT depleted rats (p-chlorophenylalanine; 300 mg/kg, for 3 days), physostigmine failed to produce tremor. Interestingly, in these animals, administration of a non-specific 5-HT agonist, 5-methoxy-N,N-dimethyl tryptamine, caused high intensity tremor. These results suggest that presence of 5-HT at the pre-synaptic terminals is needed for the tremor response by physostigmine and the response is greatly mediated via post-synaptic 5-HT receptors. The overall data indicated a direct involvement of central 5-HT system in the cholinergic tremor induced by physostigmine.     

Calcium channel agonist, (+/-)-Bay K8644, causes a transient increase in striatal monoamine oxidase activity in Balb/c mice

We investigated in vivo effects of the L-type calcium channel agonist 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl) phenyl] pyridine-3-carboxylic acid ((+/-)-Bay K8644) on mitochondrial monoamine oxidase (MAO) activity and striatal dopamine (DA) content employing fluorimetric and HPLC-electrochemical procedures, respectively. (+/-)-Bay K8644 administration resulted in visible behavioral dysfunctions in mice. A reversible dose-independent inhibition of striatal DA levels and a significant increase in the brain mitochondrial MAO-A and -B activities were observed in animals treated with the calcium agonist. A positive relationship between the rise in the enzyme activity and decrease in DA content in (+/-)-Bay K8644 treated animals indicates a direct, but transient effect of this channel activator on DA metabolism, which may be related to acute behavioral syndromes exhibited following administration of the drug. Moreover, a direct involvement of L-type dihydropyridine sensitive calcium channels is indicated in this action, since nicardipine could effectively attenuate (+/-)-Bay K8644-induced behavioral aberrations, or block the striatal DA depletion and the increase in MAO activity. The present results have far-reaching implications in neuropharmacological research, where co-treatment of calcium channel drugs and MAO inhibitors are warranted.     

Intratracheal adenovirus-mediated gene transfer is optimal in experimental lung transplantation

OBJECTIVE: Gene transfer to experimental lung grafts has been shown to reduce ischemia-reperfusion injury and acute rejection. The optimal delivery route should produce high lung expression with no inflammation and minimal systemic expression. The goal of this study was to determine the optimal gene transfer route for use in experimental lung transplantation. METHODS: F344 rats were injected with 2.9 x 10(10) plaque-forming units of adenovirus vector encoding beta-galactosidase through intratracheal, intravenous, intraperitoneal, or intramuscular delivery routes and killed 48 hours later. Gene expression was measured by means of enzyme-linked immunosorbent assay. RESULTS: Intratracheal delivery produces significantly greater gene expression in the lung (75,350 +/- 47,288 pg/100 microg of protein, P <.001 vs intravenous, intraperitoneal, and intramuscular routes) and minimal systemic expression (nonsignificant in serum, kidney, liver, spleen, and muscle vs that seen in control animals, P =.016 for heart). Immunohistochemistry staining showed beta-galactosidase expression in the bronchial epithelium of lungs transfected through the intratracheal route with mild inflammation. CONCLUSIONS: Intratracheal gene transfer provides significant expression in the lung with mild to no inflammation and minimal systemic expression. This delivery strategy has tremendous potential in experimental lung transplant models to reduce ischemia-reperfusion injury and acute allograft rejection and should be investigated further.     

Immunology of renal allograft rejection

Allograft rejection remains the critical problem of renal transplantation. The immunologic mechanisms that underlie renal allograft rejection are heterogeneous and involve the humoral and cellular limbs of the immune response. Antibody-mediated hyperacute rejection is now rare owing to improved prospective cross-matching. Chronic rejection, characterized by intrarenal arterial fibrosis, is still poorly understood. Knowledge of the afferent and efferent processes involved in rejection has led to effective therapeutic and experimental strategies that employ monoclonal antibodies and other pharmacologic agents to reverse, or prevent, acute allograft rejection. In addition, allospecific tolerance has been achieved experimentally and clinically in a variety of manners. Preliminary studies on the mechanism of allograft tolerance induced by donor-specific blood transfusions before transplantation suggest a role for an immunoregulatory cell population that specifically down regulates cytotoxic lymphocyte responses to donor antigens in some recipients. A role for noninherited maternal antigens and anti-idiotypic antibodies in down regulating immune responses to allografts have also been reported by several studies. An improved understanding of allograft rejection and tolerance may identify approaches to prolong allograft survival without the morbidity and mortality associated with present-day immunosuppression.     

Concomitant augmentation of type 1 CD4+ and CD8+ T-cell responses during successful interferon-alpha and ribavirin treatment for chronic hepatitis C virus infection

The combined interferon-alpha (IFN-alpha) and ribavirin (IFN-alpha/ribavirin) therapy for chronic hepatitis C virus (HCV) infection results in sustained viral eradication in 31%-64% of the patients. Previous studies have strongly suggested that HCV-specific T-cell responses maybe modulated during this therapy. The objective of this study was to further define the effect of IFN-alpha/ribavirin therapy on type 1 and type 2 HCV-specific CD4(+) and CD8(+) T-cell responses during IFN-alpha/ribavirin therapy. Toward this, serial CD8(+) T-cell responses to HCV-derived epitopes and CD4(+) T-cell responses to the HCV core antigen were analyzed in four patients before (baseline), during (at 24 weeks), and at the end (at 48 weeks) of IFN-alpha/ribavirin therapy. Therapy-induced viral clearance in three patients was associated with a significant augmentation of HCV-specific type 1 CD4(+) and CD8(+) T-cell responses. In contrast, in a patient who did not respond to therapy, a significant HCV-specific CD4(+) Th2 cell reactivity was observed accompanied by a lack of augmentation of the HCV-specific CD8(+) T-cell reactivity. These results indicate that enhancement of HCV-specific CD4(+) and CD8(+) T-cell responses is an important factor in determining the response to the IFN-alpha/ribavirin therapy and the outcome of the HCV infection.     

In vivo biotinylation of the major histocompatibility complex (MHC) class II/peptide complex by coexpression of BirA enzyme for the generation of MHC class II/tetramers

Success in generation of major histocompatibility complex (MHC) tetramer relies on application of a key technique, biotinylation of MHC molecule specifically on a single lysine residue using the BirA enzyme. However, in vitro biotinylation of MHC-BSP (BirA enzyme substrate peptide) fusion protein using BirA enzyme is laborious and is prone to losses of target proteins to unacceptable levels. To circumvent this problem, an in vivo biotinylation strategy was developed where the BirA enzyme was coexpressed with target protein, HLA-DR2BSP/MBP, in an insect cell expression system. Bacterial BirA enzyme expressed in Drosophila melanogaster 2 (D. Mel-2) cell lines was biologically functional and was able to biotinylate secretary target protein (on specific lysine residue present on the BSP tag). Biotinylation efficiency was maximized by providing exogenous d-biotin in the culture medium and optimization of the expression vector ratios for cotransfection. By limiting dilution cloning, a clone was identified where the expressed DR2BSP/MBP protein was completely biotinylated. DR2BSP/MBP protein expressed and purified from such a clone was ready to be tetramerized with streptavidin to be used for staining antigen-specific T cells.     

Analysis of graft-versus-host disease (GVHD) and graft rejection using MHC class I-deficient mice

GVHD is a major complication in allogeneic bone marrow transplantation (BMT). MHC class I mismatching increases GVHD, but in MHC-matched BMT minor histocompatibility antigens (mH) presented by MHC class I result in significant GVHD. To examine the modification of GVHD in the absence of cell surface MHC class I molecules, β₂-microglobulin-deficient mice (β₂m-/-) were used as allogeneic BMT recipients in MHC- and mH-mismatched transplants. β₂m-/- mice accepted MHC class I-expressing BM grafts and developed significant GVHD. MHC (H-2)-mismatched recipients developed acute lethal GVHD. In contrast, animals transplanted across mH barriers developed indolent chronic disease that was eventually fatal. Engrafted splenic T cells in all β₂m-/- recipients were predominantly CD3⁺ αβ TCR⁺ CD4⁺ cells (15–20% of all splenocytes). In contrast, CD8⁺ cells engrafted in very small numbers (1–5%) irrespective of the degree of MHC mismatching. T cells proliferated against recipient strain antigens and recognized recipient strain targets in cytolytic assays. Cytolysis was blocked by anti-MHC class II but not anti-CD8 or anti-MHC class I monoclonal antibodies (MoAbs). Cytolytic CD4⁺ T cells induced and maintained GVHD in mH-mismatched β₂m-/- mice, supporting endogenous mH presentation solely by MHC class II. Conversely, haematopoietic β₂m-/- cells were unable to engraft in normal MHC-matched recipients, presumably due to natural killer (NK)-mediated rejection of class I-negative cells. Donor-derived lymphokine-activated killer cells (LAK) were unable to overcome graft rejection (GR) and support engraftment.     

Anti-HLA class I antibody binding to airway epithelial cells induces production of fibrogenic growth factors and apoptotic cell death: a possible mechanism for bronchiolitis obliterans syndrome

Development of anti-HLA class I antibodies is associated with bronchiolitis obliterans syndrome (BOS) after lung transplantation. BOS is characterized histologically by significant fibrosis and airway epithelial cell (AEC) apoptosis. Thus, this study was designed to determine whether anti-HLA class I antibodies can activate AECs to produce growth factors and to undergo apoptosis. KCC-266 AECs were activated with the W6/32 anti-HLA class I monoclonal antibody. Proliferation and apoptosis levels were determined after 24, 48, and 72 hours. The induction of fibroblast and bronchial smooth muscle cell proliferation by anti-HLA class I activated AECs was assessed in the presence of neutralizing antibodies against various growth factors. The anti-HLA class I induced AEC proliferation after 24 hours followed by significant induction of apoptosis after 48 hours. Anti-HLA class I activated AECs produced soluble growth factors that stimulated fibroblasts but not bronchial smooth muscle cells. The stimulation of fibroblast proliferation was inhibited by antibodies against platelet-derived growth factor, heparin-binding epidermal growth factor, insulin-like growth factor 1, and basic fibroblast growth factor. The results from this study suggest that anti-HLA class I alloantibodies may play an important role in the pathogenesis of BOS by inducing proliferation, growth factor production, and apoptotic cell death in AECs.