The use of UV-C radiation for terminal disinfection of pathogenic Gram-negative rods: a pilot study.

In this controlled study, we found that exposure to ultraviolet-C (UV-C) radiation was able to arrest the growth of selected pathogenic enteric and nonfermenting Gram-negative rods. Further studies are needed to confirm the clinical efficacy and determine optimal implementation strategies for utilizing UV-C terminal disinfection.

Capture and stimulated release of circulating tumor cells on polymer-grafted silicon nanostructures.

A platform for capture and release of circulating tumor cells is demonstrated by utilizing polymer grafted silicon nanowires. In this platform, integration of ligand-receptor recognition, nanostructure amplification, and thermal responsive polymers enables a highly efficient and selective capture of cancer cells. Subsequently, these captured cells are released upon a physical stimulation with outstanding cell viability.

C5L2 is critical for the biological activities of the anaphylatoxins C5a and C3a.

Complement-derived anaphylatoxins regulate immune and inflammatory responses through G-protein-coupled receptor (GPCR)-mediated signalling. C5L2 (also known as GPR77) is a relatively new GPCR thought to be a non-signalling receptor binding to C5a, on the basis of sequence information and experimental evidence. Here we show, using gene targeting, that C5L2 is required to facilitate C5a signalling in neutrophils, macrophages and fibroblasts in vitro. Deficiency of C5L2 results in reduced inflammatory cell infiltration, suggesting that C5L2 is critical for optimal C5a-mediated cell infiltration in certain in vivo settings. C5L2 is also involved in optimizing C3a-induced signals. Furthermore, like mice incapable of C3a/complement 3a receptor (C3aR) signalling, C5L2-deficient mice are hypersensitive to lipopolysaccharide (LPS)-induced septic shock, show reduced ovalbumin (OVA)-induced airway hyper-responsiveness and inflammation, and are mildly delayed in haematopoietic cell regeneration after gamma-irradiation. Our data indicate that C5L2 can function as a positive modulator for both C5a- and C3a-anaphylatoxin-induced responses.

Mapping of determinants required for the function of the HIV-1 env nuclear retention sequence.

Control of HIV-1 RNA processing and transport are critical to the successful replication of the virus. In previous work, we identified a region within the HIV-1 env that is involved in mediating nuclear retention of unspliced viral RNA. To define this sequence further and identify elements required for function, deletion mutagenesis was carried out. Progressive 5' and 3' deletions map the nuclear retention sequence (NRS) within the intron between nts 8281 and 8381. While deletion of sequences comprising the 3'ss had no effect, removal of the 5'ss resulted in cytoplasmic accumulation of unspliced RNA. Sequence analysis determined that the region corresponding to the NRS is highly conserved among HIV-1 strains. To evaluate whether this NRS interacts with cellular factors, RNA electrophoretic mobility shift assays (REMSA) were performed. We show that the NRS specifically interacts with cellular factors present in HeLa nuclear extracts, and, by UV crosslinking, correlates with the binding of a 49-kDa protein. Immunoprecipitation of the UV crosslinked products determined that this 49-kDa protein corresponds to hnRNP C.

Overcoming the immune response to permit ex vivo gene therapy for spine fusion with human type 5 adenoviral delivery of the LIM mineralization protein-1 cDNA.

STUDY DESIGN: An animal study in immune competent rabbits and athymic rats was conducted. OBJECTIVES: To develop an animal model for simulation of previous human Type 5 adenovirus (Ad5) exposure, to determine the impact of adenoviral pre-exposure on spine fusion induced with ex vivo Ad5-LMP-1, and to test strategies for overcoming any potential immune response. SUMMARY OF BACKGROUND DATA: Cells transduced with adenovirus containing the osteoinductive LMP-1 cDNA (Ad5-LMP-1) can induce spine fusion in rabbits. Because up to 80% of the human population has been exposed to adenovirus, immune responses to the vector may limit this strategy in humans. Few studies have modeled previous adenoviral exposure and tested strategies to circumvent it. METHODS: Adult New Zealand white rabbits were injected with 10 or 10 viral particles of Ad5-LacZ. At 4 or 16 weeks after Ad5 injection, autologous buffy coats were prepared from peripheral blood, and 4 million cells per side were infected ex vivo for 10 minutes with Ad5-LMP-1 (multiplicity of infection = 4). Cells were implanted on a collagen matrix instead of an autograft for posterolateral lumbar arthrodesis. Unimmunized rabbits served as control subjects. Additional immunized rabbits underwent arthrodesis at 4 weeks with increased cell number (10 million) and viral dose (multiplicity of infection = 10), or with both parameters increased. The rabbits were killed at 4 weeks, and the spines were assessed by palpation and radiograph. A parallel study was performed in athymic rats using immunized rabbits for the donor cells. RESULTS: All the unimmunized rabbits had solid spine fusions. None of the rabbits arthrodesed 4 weeks after Ad5 pre-exposure achieved fusion. At 4 weeks after Ad5 exposure, increasing the multiplicity of infection to 10 did not overcome the immune response (0/3 fused), but increasing the cell number to 10 million (2/3 fused) or increasing both cell number and multiplicity of infection (3/3 fused) did overcome the immune effects. Delaying arthrodesis until 16 weeks after Ad5 pre-exposure also overcame the immune response (3/3 fused). Similar results were seen in the athymic rat ectopic implant model, suggesting that the immune effect was mediated by humoral antibodies rather than a T-cell response. CONCLUSIONS: Two model systems were developed that simulate previous exposure to human Ad5 and could separate the cellular and humoral components of the response. There was a dose-dependent inhibition of ex vivo Ad5-LMP-1 gene transfer to cells from animals previously exposed to human Ad5. Data suggested that the inhibition of Ad5 infection was caused by humoral antibodies rather than a T-cell-based response. Minor modifications in the gene transfer protocol, such as doubling the viral dose or number of cells infected, or increasing the infection time, could overcome the immune response for an ex vivo approach.

SLC7A9 mutations in all three cystinuria subtypes.

BACKGROUND: Cystinuria is an inherited disorder of cystine and dibasic amino acid transport in kidney. Subtypes are defined by the urinary cystine excretion patterns of the obligate heterozygous parents: Type I/N (fully recessive or silent); Type II/N (high excretor); Type III/N (moderate excretor). The first gene implicated in cystinuria (SLC3A1) is associated with the Type I urinary phenotype. A second cystinuria gene (SLC7A9) was recently isolated, and mutations of this gene were associated with dominant (non-Type I) cystinuria alleles. Here we report genotype-phenotype studies of SLC7A9 mutations in a cohort of well-characterized cystinuria probands and their family members. METHODS: Individual exons of the SLC7A9 gene were screened by single strand conformation polymorphism (SSCP) analysis and sequencing of abnormally migrating fragments. RESULTS: Seven mutations were identified. A single bp insertion (799insA) was present in four patients: on Type III alleles in two patients and on Type II alleles in two patients. These results suggest that Type II and Type III may be caused by the same mutation and, therefore, other factors must influence urinary cystine excretion. A 4bp deletion in intron 12 (IVS12+4delAGTA) and a missense mutation (1245G-->A, A354T) were identified on Type III alleles. A nonsense codon (1491G-->T, E436X) and a possible splicing mutation (IVS9-17G-->A) were seen in a Type I/III patient, but the mutations could not be assigned to particular alleles. Of additional interest were two missense mutations (316T-->C, I44T and 967C-->T, P261L) linked to Type I alleles. CONCLUSION: Our results provide evidence that some SLC7A9 mutations may be associated with fully recessive (Type I) forms of cystinuria. We also demonstrate SLC7A9 mutations in dominant Types II and III cystinuria. The finding of SLC7A9 mutations in all three subtypes underscores the complex interactions between specific cystinuria genes and other factors influencing cystine excretion. A simpler phenotypic classification scheme (recessive and dominant) for cystinuria is warranted.