Experimental study of environmental tobacco smoke particles under actual indoor environment.

Environmental tobacco smoke (ETS) is a major source of human exposure to airborne particles. In order to provide more information necessary for human exposure investigations, the aim of the work presented here is to investigate experimentally the variation of the ETS particle concentration and size distribution under an actual indoor environment, in a room of 30 m3, using human smokers. The effect of number of cigarettes and brands of cigarettes, the effect of sampling location and the effect of ventilation rates were investigated. The results indicated little difference in the geometric mean diameter (GMD) of the ETS particles from those in background air. Under a ventilation rate of 0.03 m3/s, the concentration of the ETS particles reached a peak value at the sampling point shortly after completing the smoking process. The GMD first increased due to coagulation and diffusion deposition, and finalize decreased due to the effect of ventilation. Smoking two cigarettes at the same time would increase the initial concentration and led to an increase in GMD of the ETS particles. Two different brands of cigarette with different tar contents released ETS particles of different GMDs but similar particle concentrations. Spatial variation in particle concentration was obvious only in the first 600 s of the tests and tended to fade out subsequently. Stronger ventilation would reduce the concentration and GMD of the particles.

Activation of innate immunity in nonhuman primates following intraportal administration of adenoviral vectors.

The innate immune response to intraportally infused adenoviral vector was evaluated in rhesus monkeys. A first-generation adenovirus-expressing lacZ (Ad-lacZ) was administered at a dose just below that which causes severe morbidity. The response to vector was evaluated for the initial 24 h following infusion. Clinical findings during this time were primarily limited to petechiae, consistent with the development of thrombocytopenia and biochemical evidence of disseminated intravascular coagulation. Serum transaminases were elevated and a lymphopenia developed. Tracking of fluorescent-labeled vector demonstrated distribution to macrophages and dendritic cells of the spleen and Kupffer cells of the liver. A systemic release of the cytokine IL-6 occurred soon after vector infusion. Analysis of splenic cells revealed acute activation of macrophages and dendritic cells followed by massive apoptosis. Bone marrow cultures demonstrated normal erythroid and primitive progenitors with a significant decrease in myeloid progenitors. Similar findings, except the abnormality in bone marrow cultures, were observed in monkeys who received an identical dose of Ad-lacZ in which vector genes were inactivated with psoralen and UV irradiation. These data suggest that inadvertent targeting of antigen-presenting cells following intraportal infusion of vector leads to a systemic cytokine syndrome which may be triggered by the viral capsid proteins.

Biology of E1-deleted adenovirus vectors in nonhuman primate muscle.

Adenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors.

Route of administration determines induction of T-cell-independent humoral responses to adeno-associated virus vectors.

Vectors based on adeno-associated viruses (AAV) type 2 show promise for treating chronic diseases because transgene expression appears to be stable. This study evaluated the impact of humoral immunity to the capsid proteins on vector uptake by hepatocytes following an intravascular approach. Route of vector administration in mice had a qualitative effect on antivector B cell responses. Administration of vector into the tail vein resulted in T-cell-dependent (TD) B cell responses that were completely inhibited with depleting CD4 antibody. Delivery of vector into the portal circulation via the spleen yielded B cell response that were partially T cell independent (TI) rendering strategies based on T cell inhibition ineffective in allowing vector readministration. The TI B cell response was short lived in comparison to the TD response. Rhesus monkeys produced a B cell memory response to intraportal vector which appeared to be T cell dependent based on Ig isotypes. Gene therapy strategies that require AAV vector readministration should consider vector biodistribution and its impact on B cell activation.

Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle.

Adeno-associated virus (AAV) is being developed as a vector capable of conferring long-term gene expression, which is useful in the treatment of chronic diseases. In most therapeutic applications, it is necessary to readminister the vector. This study characterizes the humoral immune response to AAV capsid proteins following intramuscular injection and its impact on vector readministration. Studies of mice and rhesus monkeys demonstrated the formation of neutralizing antibodies to AAV capsid proteins that persisted for over 1 year and then diminished, but this did not prevent the efficacy of vector readministration. More-detailed studies strongly suggested that the B-cell response was T cell dependent. This was further evaluated with a blocking antibody to human CD4, primatized for clinical trials, in a biologically compatible mouse in which the endogenous murine CD4 gene was functionally replaced with the human counterpart. Transient pharmacologic inhibition of CD4 T cells with CD4 antibody prevented an antivector response long after the effects of the CD4 antibody diminished; readministration of vector without diminution of gene expression was possible. Our studies suggest that truly durable transgene expression (i.e., prolonged genetic engraftment together with vector readministration) is possible with AAV in skeletal muscle, although it will be necessary to transiently inhibit CD4 T-cell function to avoid the activation of memory B cells.

Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer.

Stable delivery of a therapeutic protein under pharmacologic control was achieved through in vivo somatic gene transfer. This system was based on the expression of two chimeric, human-derived proteins that were reconstituted by rapamycin into a transcription factor complex. A mixture of two adeno-associated virus vectors, one expressing the transcription factor chimeras and one containing erythropoietin (Epo) under the control of a promoter responsive to the transcription factor, was injected into skeletal muscle of immune-competent mice. Administration of rapamycin resulted in 200-fold induction of plasma Epo. Stable engraftment of this humanized system in immune-competent mice was achieved for 6 months with similar results for at least 3 months in a rhesus monkey.

Provision of HPA-1a (PlA1)-negative platelets for neonatal alloimmune thrombocytopenia: screening, testing, and transfusion protocol.

HPA-1a-negative platelet products are not routinely available for newborns with alloimmune thrombocytopenia. In this article we describe a program established to identify normal pheresis donors who are HPA-1a-negative and to organize their future donations so that our regional blood center would always have an HPA-1a-negative platelet product available. The solid phase red cell adherence assay was used for initial screening of platelet pheresis products. HPA-1a-negative donors were confirmed with the platelet suspension immunofluorescence test using three anti-HPA-1a sera. Screening of 2600 plateletpheresis donor samples identified 40 HPA-1a-negative donors. Of these, 36 are active and are coded for recognition on the daily pheresis inventory sheet. Theoretically, assuming four donations per year and donors' cooperation with scheduling, these 36 donors would enable us to have at least one HPA-1a-negative product available every day. In addition, a decision tree for patient management using platelet serology and availability of HPA-1a-negative products was developed. The GTI-PAK trade mark 12 is the major technique used for serologic screening of mothers of patients thought to have neonatal alloimmune thrombocytopenia. By screening pheresis donors and developing a clinical decision tree, HPA-1a-negative products, a rare resource, can be fully utilized.