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Background. Immunosuppressive therapies have led to improved survival for lung transplant (LT) recipients but these therapies can lead to hypogammaglobulinemia (HGG) and potentially an increased risk of infection. Large prospective studies have not been performed to evaluate the impact of HGG on outcomes for LT recipients. Methods. This is a single-center prospective observational study of LT recipients. Pretransplant and posttransplant IgG levels were measured and related to infection, rejection, antibiotic use, and immunosuppression use. Results. One hundred thirty-three LT recipients were prospectively evaluated. Pretransplant IgG values were higher than IgG values at the time of transplant or any time thereafter (all P < 0.0001). Severe HGG (IgG < 400 mg/dL) was highest at the time of transplant (32.4%) while at 3, 6, 9, and 12 months posttransplant the prevalence of severe HGG was 7.4%, 7.5%, 8.9%, and 6.3%, respectively. Severe HGG was associated with 2 or more pneumonias (P = 0.0006) and increased number of antibiotic courses (P = 0.003) compared with the subjects without severe HGG. Pretransplant IgG level and less than 30% of pretransplant protective pneumococcal antibody levels were identified as pretransplant risk factors for severe HGG. In multivariate analysis, chronic obstructive pulmonary disease as the underlying disease and the use of basiliximab as the induction agent in conjunction with higher prednisone and mycophenolate dosing were most predictive of severe HGG (P = 0.005), whereas the combination of age, severe HGG and number of acute steroid courses were most predictive of total days of pneumonia (P = 0.0001). Conclusions. Our large prospective study identifies risk factors for severe HGG after LT and demonstrates that LT recipients with severe HGG are at increased risk for recurrent pneumonias and more antibiotic courses.

IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3β (GSK3β) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3β. GSK3β was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3β binding site mutant (ST2LS446A) and phosphorylation site mutant (ST2LS442A) are resistant to IL-33–induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33–induced GSK3β activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33–induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3β might serve as a unique strategy to lessen pulmonary inflammation.

The envelope protein (Env) from the CasBrE murine leukemia virus (MLV) can cause acute spongiform neurodegeneration analogous to that induced by prions. Upon central nervous system (CNS) infection, Env is expressed as multiple isoforms owing to differential asparagine (N)-linked glycosylation. Because N-glycosylation can affect protein folding, stability, and quality control, we explored whether unique CasBrE Env glycosylation features could influence neurovirulence. CasBrE Env possesses 6/8 consensus MLV glycosylation sites (gs) but is missing gs3 and gs5 and contains a putative site (gs*). Twenty-nine mutants were generated by modifying these three sites, individually or in combination, to mimic the amino acid sequence in the nonneurovirulent Friend 57 MLV. Three basic viral phenotypes were observed: replication defective (dead; titer < 1 focus-forming unit [FFU]/ml), replication compromised (RC) (titer = 10 2 to 10 5 FFU/ml); and wild-type-like (WTL) (titer > 10 5 FFU/ml). Env protein was undetectable in dead mutants, while RC and WTL mutants showed variations in Env expression, processing, virus incorporation, virus entry, and virus spread. The newly introduced gs3 and gs5 sites were glycosylated, whereas gs* was not. Six WTL mutants tested in mice showed no clear attenuation in disease onset or severity versus controls. Furthermore, three RC viruses tested by neural stem cell (NSC)-mediated brainstem dissemination also induced acute spongiosis. Thus, while unique N-glycosylation affected structural features of Env involved in protein stability, proteolytic processing, and virus assembly and entry, these changes had minimal impact on CasBrE Env neurotoxicity. These findings suggest that the Env protein domains responsible for spongiogenesis represent highly stable elements upon which the more variable viral functional domains have evolved.

Arthritis is among the leading causes of disability in the developed world. There remains no cure for this disease and the current treatments are only modestly effective at slowing the disease's progression and providing symptomatic relief. The clinical effectiveness of current treatment regimens has been limited by short half-lives of the drugs and the requirement for repeated systemic administration. Utilizing gene transfer approaches for the treatment of arthritis may overcome some of the obstacles associated with current treatment strategies. The present review examines recent developments in gene therapy for arthritis. Delivery strategies, gene transfer vectors, candidate genes, and safety are also discussed.

The 4070A amphotropic murine leukemia virus (A-MuLV) has been variably reported to harbor neurovirulence determinants within its env gene. In this report we reexamined this issue by applying two approaches previously demonstrated to amplify murine leukemia virus neurovirulence. The first approach involved introducing the 4070A env gene into the background of Friend virus clone FB29 to enhance peripheral virus replication kinetics and central nervous system entry. The resulting chimeric virus, FrAmE, exhibited widespread vascular infection throughout the central nervous system (CNS); however, parenchymal infection was quite limited. Neither clinical neurological signs nor spongiform neurological changes accompanied FrAmE CNS infection. To overcome this CNS entry limitation, 4070A and FrAmE were delivered directly into the CNS via transplantation of infected C17.2 neural stem cells (NSCs). Significantly, NSC dissemination of either 4070A or FrAmE resulted in widespread, high-level amphotropic virus expression within the CNS parenchyma, including the infection of microglia, the critical target required for inducing neurodegeneration. Despite the extensive CNS infection, no associated clinical neurological signs or acute neuropathological changes were observed. Interestingly, we observed the frequent appearance of circulating polytropic (MCF) virus in the serum of amphotropic virus-infected animals. However, neither peripheral inoculation of an amphotropic/MCF virus mixture nor transplantation of NSCs expressing both amphotropic and MCF viruses induced acute clinical neurological signs or spongiform neuropathology. Thus, the results generated in this study suggest that the 4070A env gene is not inherently neurovirulent. However, the frequent appearance of endogenous MCF viruses suggests the possibility that the interactions of amphotropic viruses with endogenous retroviral elements could contribute to the development of retrovirus-induced neurodegenerative disease.

Certain murine leukemia viruses (MLVs) can induce progressive noninflammatory spongiform neurodegeneration similar to that caused by prions. The primary MLV determinants responsible have been mapped to within the env gene; however, it has remained unclear how env mediates disease, whether non-Env viral components are required, and what central nervous system (CNS) cells constitute the critical CNS targets. To address these questions, we examined the effect of transplanting engraftable C17.2 neural stem cells engineered to pseudotype, disseminate, and trans -complement neurovirulent (CasBrE, CasE, and CasES) or non-neurovirulent (Friend and SFF-FE) env sequences (SU or SU/TM) within the CNS using either the “non-neurovirulent” amphotropic helper virus, 4070A, or p gag-pol gpt (a nonpackaged vector encoding Gag-Pol). These studies revealed that acute MLV-induced spongiosis results from two separable activities of Env. First, Env causes neuropathology through unique viral targeting within the CNS, which was efficiently mediated by ecotropic Envs (CasBrE and Friend), but not 4070A amphotropic Env. Second, Env induces spongiosis through a toxin activity that is MLV-receptor independent and does not require the coexpression of other viral structural proteins. CasBrE and 4070A Envs possess the toxin activity, whereas Friend Env does not. Although the identity of the critical viral target cell(s) remains unresolved, our results appear to exclude microglia and oligodendrocyte lineage cells, while implicating viral entry into susceptible neurons. Thus, MLV-induced disease parallels prionopathies in that a single protein, Env, mediates both the CNS targeting and the toxicity of the infectious agent that manifests itself as progressive vacuolar neurodegeneration.

Background Severe asthma remains poorly characterized, although it likely consists of at least 1 phenotype with features of T H 2-like inflammation. IL1RL1 , encoding both the IL-33 receptor, ST2L, and decoy receptor, sST2, has been genetically associated with asthma, though the mechanism for susceptibility remains unknown. Objective Given previous data supporting a role for IL1RL1 in T H 2 inflammation, we hypothesized that ST2L expression might be increased in T H 2-like asthma and that expression levels would be associated with single nucleotide polymorphisms in IL1RL1 , possibly explaining its genetic relationship with asthma. We also sought to evaluate the regulation of ST2L and sST2 in vitro . Methods Endobronchial brushings and biopsies were obtained and expression of ST2L compared by severity levels, as well as by T H 2-like biomarkers. Subjects were genotyped and the relationship of dichotomous expression of ST2L and sST2 to single nucleotide polymorphisms in IL1RL1 were determined. Epithelial cells were grown in air-liquid interface culture, and ST2L and sST2 responses to IFN-γ and IL-13 were evaluated. Results ST2L expression was increased in severe asthma ( P = .02) and associated with multiple indicators of T H 2-like inflammation, including blood eosinophils ( P = .001), exhaled nitric oxide ( P = .003), and epithelial CLCA1 ( P P = .001) mRNA expression. Multiple single nucleotide polymorphisms in IL1RL1 were found in relation to dichotomous expression of both ST2L and sST2. sST2 expression was associated with IFN-γ expression in bronchoalveolar lavage, while inducing its expression in vitro in primary human epithelial cells. Conclusion Both pathologic and genetic approaches support a role for IL1RL1 in severe asthma, as well as T H 2-lke asthma, suggesting that targeting this pathway may have therapeutic benefits.

Objective An ideal gene transfer vector for chronic inflammatory diseases such as rheumatoid arthritis (RA) would provide local transgene expression only when the disease is active. To determine whether adeno-associated virus (AAV) possesses this ability, the effects of inflammatory cytokines on transgene expression were evaluated in human RA fibroblast-like synoviocytes (FLS). Methods Human FLS were infected with AAV in the presence or absence of inflammatory cytokines or synovial fluid obtained from patients with RA. Transgene expression was monitored by either enzyme-linked immunosorbent assay or flow cytometry. Transgene messenger RNA (mRNA) was measured by real-time quantitative reverse transcription–polymerase chain reaction. Results Inflammatory cytokines increased transgene expression in FLS by up to 60-fold. Synovial fluid from patients with RA, but not from patients without arthritis, was also able to increase expression in synoviocytes. Protein expression correlated with transgene mRNA levels. The enhanced expression required the continued presence of cytokines because, upon removal, transgene expression returned to baseline levels. Expression could be repeatedly reinduced by reexposure to cytokines. The effect was not promoter specific and was demonstrated to be phosphatidylinositol 3-kinase–dependent. Conclusion These results suggest that expression of a therapeutic transgene can be controlled by the presence of inflammation following AAV gene transfer, making it an attractive vector for chronic inflammatory diseases such as RA.

To explore the potential applicability of recombinant adeno-associated virus (rAAV) vectors in the treatment of rheumatoid arthritis (RA), primary human fibroblast-like synoviocytes (FLS) derived from patients with RA were infected with rAAV encoding mouse IL-10 under the control of the CMV promoter. Addition of the proteasome inhibitor carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (zLLL) to the cultures dramatically enhanced expression of the IL-10 transgene, in a dose-dependent manner. The increased expression was transient, peaking at 3 days and returning to near baseline by 7 days. The enhancement was observed even when zLLL was added 13 days after infection with rAAV. The effect of zLLL was not specific to either the mIL-10 transgene or the CMV promoter, as similar findings were observed using an rAAV construct encoding alpha1-anti-trypsin under the control of the chick beta-actin promoter or GFP, driven by the CMV promoter. Transgene expression could be repeatedly induced by reexposure to zLLL. Transgene mRNA levels increased in parallel with protein levels. Transgene expression could also be repeatedly induced in vivo by administering zLLL to SCID mice previously injected with rAAV-infected FLS. These data demonstrate that proteasome inhibition can dramatically enhance transgene expression in human RA FLS following infection with rAAV and suggest a possible approach to regulating synovial transgene expression in vivo.

Background Combined immunodeficiency with multiple intestinal atresias (CID-MIA) is a rare hereditary disease characterized by intestinal obstructions and profound immune defects. Objective We sought to determine the underlying genetic causes of CID-MIA by analyzing the exomic sequences of 5 patients and their healthy direct relatives from 5 unrelated families. Methods We performed whole-exome sequencing on 5 patients with CID-MIA and 10 healthy direct family members belonging to 5 unrelated families with CID-MIA. We also performed targeted Sanger sequencing for the candidate gene tetratricopeptide repeat domain 7A ( TTC7A ) on 3 additional patients with CID-MIA. Results Through analysis and comparison of the exomic sequence of the subjects from these 5 families, we identified biallelic damaging mutations in the TTC7A gene, for a total of 7 distinct mutations. Targeted TTC7A gene sequencing in 3 additional unrelated patients with CID-MIA revealed biallelic deleterious mutations in 2 of them, as well as an aberrant splice product in the third patient. Staining of normal thymus showed that the TTC7A protein is expressed in thymic epithelial cells, as well as in thymocytes. Moreover, severe lymphoid depletion was observed in the thymus and peripheral lymphoid tissues from 2 patients with CID-MIA. Conclusions We identified deleterious mutations of the TTC7A gene in 8 unrelated patients with CID-MIA and demonstrated that the TTC7A protein is expressed in the thymus. Our results strongly suggest that TTC7A gene defects cause CID-MIA.

A major drawback to current gene transfer strategies is the inability to regulate transgene expression. An ideal vector would provide local transgene expression at the site of the disease only when the disease is active. Recombinant adeno-associated virus (rAAV) appears to possess these important qualities. We monitored the effects of inflammatory cytokines on transgene expression in human rheumatoid arthritis fibroblast-like synoviocytes (FLS) infected with rAAV. Synovial cell cultures were infected with rAAV under control of the cytomegalovirus (CMV) promoter in the presence or absence of TNF|[alpha]|, IL-1|[beta]|, and/or IL-6. These cytokines were found to act synergistically to increase transgene expression in rAAV infected FLS up to 1000 fold. The enhanced expression required the continued presence of cytokines, although cytokine removal and re-addition demonstrated a remarkable ability of the cytokines to regulate transgene expression. The effect was most pronounced on cells infected with a double stranded version of AAV in which a rate-limiting step of viral transduction (second strand synthesis) is bypassed, and the effect was not promoter specific, as similar results were obtained using rAAV under control of the chicken beta actin promoter. mRNA levels of the transgene were demonstrated to increase up to 25-fold upon cytokine addition. The observed phenomenon was also phosphatidylinositol 3-kinase (PI3K) dependent, as addition of wortmannin reversibly inhibited cytokine-mediated increases in transgene expression. These results suggest that expression of a therapeutic transgene can be controlled by the presence of inflammation following AAV-mediated gene transfer, making AAV an attractive gene transfer vector for chronic inflammatory diseases such as rheumatoid arthritis.