Lorita Dudus

CD4(+) T-lymphocytes mediate ischemia/reperfusion-induced inflammatory responses in mouse liver.

The success of orthotopic liver transplantation is dependent on multiple factors including MHC tissue compatibility and ischemic/reperfusion injury. Ischemic/reperfusion (I/R) injury in the liver occurs in a biphasic pattern consisting of both acute phase (oxygen free radical mediated) and subacute phase (neutrophil-mediated) damage. Although numerous studies have given insights into the process of neutrophil recruitment after I/R injury to the liver, the exact mechanism that initiates this subacute response remains undefined. Using a T cell-deficient mouse model, we present data that suggests that T-lymphocytes are key mediators of subacute neutrophil inflammatory responses in the liver after ischemia and reperfusion. To this end, using a partial lobar liver ischemia model, we compared the extent of reperfusion injury between immune competent BALB/c and athymic nu/nu mice. Studies evaluating the extent of liver damage as measured by serum transaminases (GPT) demonstrate similar acute (3-6 h) post-I/R responses in these two mouse models. In contrast, the subacute phase (16-20 h) of liver injury, as measured by both serum GPT levels and percent hepatocellular necrosis, was dramatically reduced in T cell-deficient mice as compared with those with an intact immune system. This reduction in liver injury seen in nu/nu mice was associated with a 10-fold reduction in hepatic neutrophil infiltration. Adoptive transfer of T cell-enriched splenocytes from immune competent mice was capable of reconstituting the neutrophil-mediated subacute inflammatory response within T cell-deficient nu/nu mice. Furthermore, in vivo antibody depletion of CD4(+) T-lymphocytes in immune competent mice resulted in a reduction of subacute phase injury and inflammation as measured by serum GPT levels and neutrophil infiltration. In contrast, depletion of CD8(+) T-lymphocytes had no effect on these indexes of subacute inflammation. Kinetic analysis of T cell infiltration in the livers of BALB/c mice demonstrated a fivefold increase in the number of hepatic CD4(+) T-lymphocytes within the first hour of reperfusion with no significant change in the number of CD8(+) T-lymphocytes. In summary, these results implicate CD4(+) T-lymphocytes as key regulators in initiating I/R-induced inflammatory responses in the liver. Such findings have implications for therapy directed at the early events in this inflammatory cascade that may prove useful in liver transplantation.

Redox gene therapy for ischemia/reperfusion injury of the liver reduces AP1 and NF-κB activation

Liver transplantation is the only therapeutic strategy for many inherited and acquired diseases. The formation of reactive oxygen species following ischemia/reperfusion is a cause of hepatocellular injury during transplantation. This report describes the therapeutic application of mitochondrial superoxide dismutase gene transfer to the liver for acute ischemia/reperfusion injury. Recombinant adenoviral expression of mitochondrial superoxide dismutase in mouse liver prior to lobar ischemia/reperfusion significantly reduced acute liver damage and associated redox activation of both NF-κB and AP1. These immediate early transcription factors represent common pathways by which cells respond to environmental stress. This work provides the foundation for redox-mediated gene therapies directed at ameliorating ischemia/reperfusion injury and associated acute rejection in orthotopic liver transplantation.

Persistent transgene product in retina, optic nerve and brain after intraocular injection of rAAV

Recombinant adeno-associated virus (rAAV) is a promising vector for retinal application as it transduces photoreceptors and retinal pigment epithelium cells efficiently and in a stable fashion. Because rAAV also transduces retinal ganglion cells, we reasoned that ocular application of rAAV might result in delivery of transgenic protein to the CNS. Here we describe high levels of green fluorescent protein (GFP) persisting at least 6 months in optic nerves and brains of mice and dogs after intravitreal delivery of rAAV-GFP. There was no clinical or histological evidence of inflammatory response although a mild humoral Th-2 response to viral capsid proteins was detected. These findings have important implications with respect to therapeutic applications of rAAV.

Isolation of Differentially Expressed cDNAs During Ferret Tracheal Development: Application of Differential Display PCR

The technique of differential display polymerase chain reaction (DD-PCR) was used to identify cDNA sequences, which are temporally expressed during ferret tracheal airway development. Such differentially expressed cDNAs may ultimately prove to be useful markers in elucidating mechanisms of epithelial differentiation and submucosal gland development in the airway. Using two sets of oligonucleotide primers 15 differentially amplified cDNAs were isolated by comparative reverse transcriptase (RT) PCR of 6-h and 3-day postnatal tracheal poly-A mRNA. In situ hybridization was used to assess the reliability of this method and confirm the differential mRNA expression patterns of cloned cDNAs. Results of in situ hybridization analysis demonstrated that 10 of the 15 cDNA sequences gave a temporally regulated pattern of expression, which was concordant with that of the differential display. Furthermore, sequence analysis of the 15 isolated cDNAs revealed that the majority of clones were amplified from two inverted decamer primers. These findings demonstrate the lack of poly-T priming in the differential display reaction, which suggests that this method may yield substantially more information regarding the coding sequence of cloned genes. In support of this observation, 6 of the 15 cDNA sequences contained one complete open reading frame. Although the majority of cDNAs demonstrated no homology to sequence data bases at the DNA or amino acid level, clone FT-4, which demonstrated a differential expression pattern limited to 3-day tracheal time points, was composed of a 10-amino acid repeat domain that was structurally similar to neuropeptide anthoRFamide and barley D hordein seed protein. A second interesting clone, FT-3, demonstrated an infrequent pattern of expression within a subset of epithelial cells limited to early developmental time points (6 h) and was dramatically reduced by 3 days postnatally. Several additional clones with no homologies to previously cloned genes demonstrated expression patterns that were also temporally regulated throughout tracheal development. Although the function of these temporally regulated genes has not been determined, these genes may ultimately prove to be useful markers of cellular differentiation during tracheal development.