Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients

BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.     

Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite cGMP-dependent protein kinase as novel anticoccidial agents

Several analogs of 2,3-diaryl pyrroles were synthesized and evaluated as inhibitors of Eimeria tenella cGMP-dependent protein kinase and in in vivo anticoccidial assays. A 4-fluorophenyl group enhances both in vitro and in vivo activities. The most potent analogs are the 5-(N-methyl, N-ethyl, and N-methylazetidine methyl) piperidyl derivatives 12, 23, and 34. These compounds have a broad spectrum of activity. Based on the in vivo efficacy and cost of synthesis, the N-ethyl analog 23 was chosen as a novel anticoccidial agent for a field trial.     

MAP1A light chain 2 interacts with exchange protein activated by cyclic AMP 1 (EPAC1) to enhance Rap1 GTPase activity and cell adhesion

We have recently demonstrated that light chain 2 (LC2) of the microtubule-associated protein MAP1A interacts with the cyclic AMP (cAMP)-binding domain of exchange protein directly activated by cyclic AMP 1 (EPAC1). In the present study we used a simultaneous expression system and found that LC2 enhances both basal and 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3':5'-cyclic monophosphate (8-CPT-2Me-cAMP)-stimulated Rap1 activation by EPAC1. LC2 is known to stabilize microtubules; therefore we examined whether microtubules enhanced Rap1 activation by LC2. Nocodazole inhibited Rap1 activity in cells transfected with EPAC1 alone but had little effect on Rap1 activity in cells transfected with both EPAC1 and LC2. This indicates that part of the actions of LC2 in enhancing EPAC1 activity may be through stabilization of microtubules. We also found that in cells transfected with LC2, Rap1 was more sensitive to activation by 8-CPT-2Me-cAMP. Moreover, LC2 enhanced the ability of transfected and endogenous EPAC1 to interact with cyclic AMP-agarose, indicating that LC2 elicits conformational changes in the cAMP domain of EPAC1, enhancing its ability to be activated by cyclic AMP. We also found that disruption of the interaction of endogenous EPAC1 and LC2 with antibodies to the cAMP domain of EPAC1 abolished Rap1 activity in PC12 cell lysates, demonstrating the importance of LC2 for EPAC1 activation in these cells. Consistent with a role of EPAC1 in controlling integrin activity, we found that cell adhesion to laminin was enhanced in LC2- and EPAC1-transfected cells stimulated with 8-CPT-2Me-cAMP. LC2 is therefore a biological enhancer of EPAC1 activity toward Rap1 and associated downstream signaling mechanisms.     

Genetic analysis of completely sequenced disease-associated MHC haplotypes identifies shuffling of segments in recent human history

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II-related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR-DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations.     

Improving performance of HVAC systems to reduce exposure to aerosolized infectious agents in buildings; recommendations to reduce risks posed by biological attacks

The prospect of biological attacks is a growing strategic threat. Covert aerosol attacks inside a building are of particular concern. In the summer of 2005, the Center for Biosecurity of the University of Pittsburgh Medical Center convened a Working Group to determine what steps could be taken to reduce the risk of exposure of building occupants after an aerosol release of a biological weapon. The Working Group was composed of subject matter experts in air filtration, building ventilation and pressurization, air conditioning and air distribution, biosecurity, building design and operation, building decontamination and restoration, economics, medicine, public health, and public policy. The group focused on functions of the heating, ventilation, and air conditioning systems in commercial or public buildings that could reduce the risk of exposure to deleterious aerosols following biological attacks. The Working Group's recommendations for building owners are based on the use of currently available, off-the-shelf technologies. These recommendations are modest in expense and could be implemented immediately. It is also the Working Group's judgment that the commitment and stewardship of a lead government agency is essential to secure the necessary financial and human resources and to plan and build a comprehensive, effective program to reduce exposure to aerosolized infectious agents in buildings.     

Conformational Dynamics of the Escherichia coli DNA Polymerase Manager Proteins UmuD and UmuD′

The expression of Escherichia coli umuD gene products is upregulated as part of the SOS response to DNA damage. UmuD is initially produced as a 139-amino-acid protein, which subsequently cleaves off its N-terminal 24 amino acids in a reaction dependent on RecA/single-stranded DNA, giving UmuD′. The two forms of the umuD gene products play different roles in the cell. UmuD is implicated in a primitive DNA damage checkpoint and prevents DNA polymerase IV-dependent − 1 frameshift mutagenesis, while the cleaved form facilitates UmuC-dependent mutagenesis via formation of DNA polymerase V (UmuD′₂C). Thus, the cleavage of UmuD is a crucial switch that regulates replication and mutagenesis via numerous protein-protein interactions. A UmuD variant, UmuD3A, which is noncleavable but is a partial biological mimic of the cleaved form UmuD′, has been identified. We used hydrogen-deuterium exchange mass spectrometry (HXMS) to probe the conformations of UmuD, UmuD′, and UmuD3A. In HXMS experiments, backbone amide hydrogens that are solvent accessible or not involved in hydrogen bonding become labeled with deuterium over time. Our HXMS results reveal that the N-terminal arm of UmuD, which is truncated in the cleaved form UmuD′, is dynamic. Residues that are likely to contact the N-terminal arm show more deuterium exchange in UmuD′ and UmuD3A than in UmuD. These observations suggest that noncleavable UmuD3A mimics the cleaved form UmuD′ because, in both cases, the arms are relatively unbound from the globular domain. Gas-phase hydrogen exchange experiments, which specifically probe the exchange of side-chain hydrogens and are carried out on shorter timescales than solution experiments, show that UmuD′ incorporates more deuterium than either UmuD or UmuD3A. This work indicates that these three forms of the UmuD gene products are highly flexible, which is of critical importance for their many protein interactions.     

Neural crest cell-specific deletion of Rac1 results in defective cell-matrix interactions and severe craniofacial and cardiovascular malformations

The small GTP-binding protein Rac1, a member of the Rho family of small GTPases, has been implicated in regulation of many cellular processes including adhesion, migration and cytokinesis. These functions have largely been attributed to its ability to reorganize cytoskeleton. While the function of Rac1 is relatively well known in vitro, its role in vivo has been poorly understood. It has previously been shown that in neural crest cells (NCCs) Rac1 is required in a stage-specific manner to acquire responsiveness to mitogenic EGF signals. Here we demonstrate that mouse embryos lacking Rac1 in neural crest cells (Rac1/Wnt1-Cre) showed abnormal craniofacial development including regional ectodermal detachment associated with mesenchymal acellularity culminating in cleft face at E12. Rac1/Wnt1-Cre mutants also displayed inappropriate remodelling of pharyngeal arch arteries and defective outflow tract septation resulting in the formation of a common arterial trunk (‘persistent truncus arteriosus’ or PTA). The mesenchyme around the aortic sac also developed acellular regions, and the distal aortic sac became grossly dysmorphic, forming a pair of bilateral, highly dilated arterial structures connecting to the dorsal aortas. Smooth muscle cells lacking Rac1 failed to differentiate appropriately, and subpopulations of post-migratory NCCs demonstrated aberrant cell death and attenuated proliferation. These novel data demonstrate that while Rac1 is not required for normal NCC migration in vivo, it plays a critical cell-autonomous role in post-migratory NCCs during craniofacial and cardiac development by regulating the integrity of the craniofacial and pharyngeal mesenchyme.     

Low mannose-binding lectin function is associated with sepsis in adult patients

Mannose-binding lectin (MBL) is an innate immune system pattern recognition molecule that kills a wide range of pathogens via the lectin complement pathway. MBL deficiency is associated with severe infection but the best measure of this deficiency is undecided. We investigated the influence of MBL functional deficiency on the development of sepsis in 195 adult patients, 166 of whom had bloodstream infection and 35 had pneumonia. Results were compared with 236 blood donor controls. MBL function (C4b deposition) and levels were measured by enzyme-linked immunosorbent assay. Using receiver-operator characteristics of MBL function in healthy controls, we identified a level of <0.2 U microL(-1) as a highly discriminative marker of low MBL2 genotypes. Median MBL function was lower in sepsis patients (0.18 U microL(-1)) than in controls (0.48 U microL(-1), P<0.001). MBL functional deficiency was more common in sepsis patients than controls (P<0.001). MBL functional deficient patients had significantly higher sequential organ failure assessment (SOFA) scores and higher MBL function and levels were found in patients with SOFA scores predictive of good outcome. Deficiency of MBL function appears to be associated with bloodstream infection and the development of septic shock. High MBL levels may be protective against severe sepsis.     

Two processivity clamp interactions differentially alter the dual activities of UmuC

DNA polymerases of the Y family promote survival by their ability to synthesize past lesions in the DNA template. One Escherichia coli member of this family, DNA pol V (UmuC), which is primarily responsible for UV-induced and chemically induced mutagenesis, possesses a canonical beta processivity clamp-binding motif. A detailed analysis of this motif in DNA pol V (UmuC) showed that mutation of only two residues in UmuC is sufficient to result in a loss of UV-induced mutagenesis. Increased levels of wild-type beta can partially rescue this loss of mutagenesis. Alterations in this motif of UmuC also cause loss of the cold-sensitive and beta-dependent synthetic lethal phenotypes associated with increased levels of UmuD and UmuC that are thought to represent an exaggeration of a DNA damage checkpoint. By designing compensatory mutations in the cleft between domains II and III in beta, we restored UV-induced mutagenesis by a UmuC beta-binding motif variant. A recent co-crystal structure of the 'little finger' domain of E. coli pol IV (DinB) with beta suggests that, in addition to the canonical beta-binding motif, a second site of pol IV ((303)VWP(305)) interacts with beta at the outer rim of the dimer interface. Mutational analysis of the corresponding motif in UmuC showed that it is dispensable for induced mutagenesis, but that alterations in this motif result in loss of the cold-sensitive phenotype. These two beta interaction sites of UmuC affect the dual functions of UmuC differentially and indicate subtle and sophisticated polymerase management by the beta clamp.