Outcomes of Clostridium difficile infection in pediatric solid organ transplant recipients.

BACKGROUND: The incidence of Clostridium difficile infection (CDI) is increasing in the pediatric population. Pediatric recipients of solid organ transplantation (SOT) may be at a higher risk for CDI in part because of chemotherapy and prolonged hospitalization. METHODS: We utilized data from the Healthcare Cost and Utilization Project Kids' Inpatient Database to study the incidence and outcomes related to CDI as a complicating factor in pediatric recipients of SOT. RESULTS: Our results demonstrate that hospitalized children with SOT have increased rates of infection, with the greatest risk for younger children with additional comorbidities and severe illness. The type of transplanted organ affects the risk for CDI, with the lowest incidence observed in renal transplant patients. CONCLUSION: The occurrence of CDI in the pediatric SOT population contributes to a greater length of stay and higher hospital charges. However, CDI is not an independent predictor of increased in- hospital mortality in these patients.

Association of Clostridium difficile infection with outcomes of hospitalized solid organ transplant recipients: results from the 2009 Nationwide Inpatient Sample database.

BACKGROUND: Diarrhea is a frequent and potentially severe complication in solid organ transplant (SOT) recipients. One of the most common infectious etiologies of diarrhea in these patients is Clostridium difficile. Our objective was to investigate the association of C. difficile infection (CDI) with the outcomes of hospitalized SOT patients. METHODS: We extracted all adult cases with discharge diagnoses of SOT or CDI from the United States Nationwide Inpatient Sample, Healthcare Cost and Utilization Project, Agency for Healthcare Research and Quality 2009 database. We collected outcome variables (mortality, length of hospital stay [LOS], hospitalization charges, complications of the transplanted organ, and colectomy), demographic information, and comorbidity data for each of the cases. The data were evaluated using univariate and multiple variable regression analyses. RESULTS: We identified 49,198 cases with SOT of which 2.7% had CDI. Univariate comparisons of cases with SOT + CDI to those with SOT-only revealed significant differences in the evaluated outcomes including in-hospital mortality (7.4% vs. 2.4%, P < 0.001), LOS (median 9 days vs. 4 days, P < 0.001), charges (median $53,808 vs. $31,488, P < 0.001), organ complications (38.1% vs. 33.9%, P < 0.001), and colectomy (1.1% vs. 0.3%, P < 0.001). Using multiple variable regression analyses, in the SOT cohort (SOT-only and SOT + CDI), CDI was independently associated with greater mortality (adjusted odds ratio [aOR] 2.48, 95% confidence interval [CI] = 2.22, 2.76, P < 0.001), longer LOS (difference 9.6 days, 95% CI = 9.3, 9.9, P < 0.001), higher charges (difference $69,647, 95% CI = $66,190, $73,104, P < 0.001), more complications of the transplanted organ (aOR 1.36, 95% CI = 1.28, 1.44, P < 0.001), and increased need for colectomy (aOR 3.10, 95% CI = 2.35, 4.08, P < 0.001). CONCLUSIONS: Our results demonstrate that CDI is associated with overall significantly worse outcomes in hospitalized patients with SOT.

Recombinant AAV serotype 1 transduction efficiency and tropism in the murine brain.

Recombinant adeno-associated virus serotype 2 (rAAV2) vectors have shown promise as therapeutic agents for neurologic disorders. However, intracerebral administration of this vector leads to preferential transduction of neurons and a restricted region of transgene expression. The recently developed rAAV vectors based upon nonserotype 2 viruses have the potential to overcome these limitations. Therefore, we directly compared a rAAV type 1 to a type 2 vector in the murine brain. The vectors were engineered to carry identical genomes (AAV2 terminal repeat elements flanking an enhanced green fluorescent protein expression cassette) and were administered by stereotaxic-guided intracerebral injection. We found that the rAAV1 vector (rAAV1-GFP) had a 13- to 35-fold greater transduction efficiency than that of the rAAV2 vector (rAAV2-GFP). Also, rAAV1-transduced cells were observed at a greater distance from the injection site than rAAV2-transduced cells. Neurons were the predominant cell type transduced by both vector types. However, in contrast to rAAV2-GFP, rAAV1-GFP was capable of transducing glial and ependymal cells. Thus, rAAV1-based vectors have biologic properties within the brain distinct from that of rAAV2. These differences might be capitalized upon to develop novel gene transfer strategies for neurologic disorders.

Microsphere-adenoviral complexes target and transduce the glomerulus in vivo.

BACKGROUND: Developing new treatments for glomerulonephritis makes the glomerulus a logical target for gene therapy. Microspheres may lodge in the glomerulus, and replication-deficient recombinant adenoviruses are potent mediators of gene transfer. We postulated that adenoviral-microsphere complexes could result in DNA transfer (transduction) into glomerular cells in vivo. METHODS: Two adenoviruses, each one containing a luciferase or beta-galactosidase (beta-gal) transgene expression cassette, were complexed to polystyrene microspheres. To assess in vivo glomerular transduction with this tool, male Sprague-Dawley rats underwent aortic injections with adenovirus linked to 11 or 16 microm diameter microspheres. RESULTS: After 48 hours, adenoviral-microsphere complexes resulted in transduction of up to 19% of glomeruli per kidney section. Endothelial and mesangial cells were transduced with this approach, and transprotein expression persisted for 21 days. Transduction efficiency was greater in the 16 microm group. For all rats, there was a strong correlation between kidney luciferase levels and the number of beta-gal-positive glomeruli (r = 0.87), indicating that transgene expression was primarily glomerular in location. This was supported by reverse transcriptase in situ polymerase chain reaction, which demonstrated glomerular localization of the beta-gal transgene. CONCLUSIONS: The aortic injection of adenoviral-microsphere complexes transduces the glomerulus in vivo and may be a useful tool in developing approaches to gene therapy of glomerular disease.

Expression and activity of human Na+/I- symporter in human glioma cells by adenovirus-mediated gene delivery.

Radioiodide concentrating activity in the thyroid, mediated by human Na+/I- symporter (hNIS), provides a mechanism for effective radioiodide treatment for patients who have invasive, recurrent, and metastatic thyroid cancers after total thyroidectomy. In an attempt to develop hNIS gene transfer for radioiodide therapy for patients with brain tumors, we have constructed recombinant adenoviruses, rAd-CMV-hNIS9 and rAd-CMV-FLhNIS, to express exogenous hNIS in U1240 and U1240Tag human glioma cells. U1240Tag differs from U1240 glioma cells in that it expresses the SV40 large T antigen oncoprotein. In both U1240 and U1240Tag cells, radioiodide uptake (RAIU) activity in the cells infected with rAd-CMV-hNIS9 or rAd-CMV-FLhNIS increases as the adenoviral MOI increases. The protein expression profile of hNIS in infected cells is generally in agreement with their RAIU activity profile. Although the expressed hNIS9 protein appeared to have a shorter half-life than FLhNIS, hNIS9 expression could be maintained by multiple infections in these cells. In addition, we show that hNIS can be expressed and function in a xenografted human glioma by intratumoral injection of rAd-CMV-hNIS9.

Recombinant adeno-associated virus-mediated correction of lysosomal storage within the central nervous system of the adult mucopolysaccharidosis type VII mouse.

The central nervous system (CNS) is a predominant site of involvement in several lysosomal storage diseases (LSDs); and for many patients, these diseases are diagnosed only after the onset of symptoms related to the progressive accumulation of macromolecules within lysosomes. The mucopolysaccharidosis type VII (MPS VII) mice are deficient for the lysosomal enzyme beta-glucuronidase and, by early adulthood, develop a significant degree of glycosaminoglycan storage within neuronal, glial, and leptomeningeal cells. Using this animal model, we investigated whether gene transfer mediated by a recombinant adeno-associated virus (rAAV) vector is capable of reversing the progression of storage lesions within the CNS. Adult MPS VII mice received intracerebral injections of 4 X 10(7) infectious units of a rAAV vector carrying the murine beta-glucuronidase (gus-s(a)) cDNA under the transcriptional direction of the cytomegalovirus immediate-early promoter and enhancer. By 1 month after vector administration, transgene-derived beta-glucuronidase was present surrounding the injection site. Enzyme levels were between 50 and 240% of that found in wild-type mice. This level of beta-glucuronidase activity was sufficient to reduce the degree of lysosomal storage. Moreover, the reduction in storage was maintained for at least 3 months post-rAAV administration. These data demonstrate that rAAV vectors can transduce the diseased CNS of MPS VII mice and mediate levels of transgene expression necessary for a therapeutic response. Thus, rAAV vectors are potential tools in the treatment of the mucopolysaccharidoses and other lysosomal storage diseases.

A phase I study of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fibrosis.

A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation, which diminished to undetectable levels by day 43. Adenovirus-specific cell-mediated T cells were induced in most subjects, although only mild increases in systemic humoral immune response were observed. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors but that efficiency is low and of short duration in the native CF airway.

Adeno-associated virus-mediated gene transfer to the brain: duration and modulation of expression.

Adeno-associated virus (AAV) is a promising vector for central nervous system (CNS) gene transfer, but a number of issues must be addressed if AAV is to be used for widespread delivery throughout the CNS. Our aim was to test the effect of dose, route of delivery, and hydroxyurea treatment on brain expression of beta-galactosidase activity after cerebral inoculation with an rAAV-lacZ vector (rAAV-beta-gal). We also wished to test whether an immune response appeared against the vector and the transgene product. We found in BALB/c mice that beta-Gal expression increased during the first 2 months after inoculation, then decreased slightly by 4 months, and continued out to 6, 12, and 15 months in single animals. Cerebral injection produced localized beta-Gal expression that did not diffuse to other regions despite a fivefold increase in injection volume. Intraventricular injection resulted in negligible transduction. Antibodies to AAV capsid protein and beta-Gal appeared at low levels at 2 and 4 months, but correlated poorly with beta-Gal expression and did not prevent readministration of rAAV-beta-gal. Hydroxyurea treatment did not result in increased transduction in vivo. We conclude that our study confirms rAAV vectors as having considerable potential for CNS gene transfer; however, several important problems must be addressed if this vector system is to be used for long-term transduction of the entire brain. Sustained, regulatable expression will be needed if rAAV is to be used in the treatment of chronic CNS disease. The difficulty in delivering AAV to diverse regions of the brain is an important problem that must be overcome if these vectors are to be used for anything beyond localized transduction.

Gene transfer into the CNS using recombinant adeno-associated virus: analysis of vector DNA forms resulting in sustained expression.

Recombinant adeno-associated virus (rAAV) vectors have shown significant promise as vehicles for in vivo gene transfer, particularly for transduction of organs composed primarily of non-dividing cells (i.e., muscle, CNS, and liver). However, the mechanistic basis for this desirable property remains unclear. To investigate the fate of rAAV genomes in mouse brain, we stereotactically injected an rAAV vector carrying the E. coli lacZ gene into the caudate of BALB/c mice and demonstrate efficient transduction of mouse brain cells that possess cellular morphology consistent with post-mitotic neurons. We observed a significant increase in beta-galactosidase expression from 5 to 56 days after injection that paralleled the disappearance of single-stranded DNA input genomes. Analysis of in vivo viral DNA forms over time out to 5 months after inoculation revealed that rAAV genomes associated with high molecular weight mouse chromosomal DNA by 14 days after injection and persisted for the length of this study. The pattern of Southern hybridization was consistent with random viral integration in predominantly head-to-tail concatameric arrays. Importantly, we also documented an additional DNA species that appears to be a monomeric episomal circular form based on nuclease sensitivity assays. These data are the first to document the existence of multiple vector DNA forms present within the adult murine brain following direct rAAV inoculation and therefore, provide insight into the molecular events that ultimately result in long-term rAAV mediated transgene expression.

Successful DNA transfer in cultured human mesangial cells using replication deficient recombinant adenovirus.

BACKGROUND: Efficient transfer of DNA into human mesangial cells is an essential first step in the development of gene therapies for mesangial cell-mediated glomerulopathies. In the present studies, we assessed the ability of replication deficient recombinant adenovirus to transfer DNA (transduce) into primary cultures of human mesangial cells. METHODS: Primary cultures of human mesangial cells were transduced with an adenoviral vector (rAv beta-gal) containing a CMVllacZ promoter-reporter expression cassette coding for beta-galactosidase (beta-gal). We assessed soluble and histologic beta-gal activity, morphology, and phenotypic expression of mesangial cell transductants, durability of transduced mesangial cells by measuring transgene expression following trypsinization or after prolonged periods in culture and metabolic stability following transduction (as assessed by fibronectin biosynthesis). RESULTS: We showed that rAv beta-gal efficiently transduced mesangial cells in a dose-dependent fashion at a multiplicity of infectious units (MOI) ranging from 1 to 400 plaque forming units/cell (pfu/cell). One hundred percent of mesangial cells were transduced at an MOI of 100 pfu/cell. By electron microscopic evaluation, viral particles of approximately 85-90 nm were demonstrated in the cytoplasm of transduced cells. Following transduction, legal levels rose rapidly and were 10-fold greater than baseline levels after 2 hours. Beta-gal levels continued to rise for 7 days following transduction. Transduction with rAv beta-gal was well tolerated; mesangial cell transductants maintained normal morphology and phenotype, tolerated 3 cycles of trypsinization and maintained normal constitutive production of fibronectin. CONCLUSIONS: Gene transfer with adenovirus is an effective, well tolerated approach for introducing DNA into primary cultures of human mesangial cells.

Recombinant adeno-associated viral vectors mediate long-term transgene expression in muscle.

Gene transfer to muscle holds overt promise for the treatment of inherited myopathies, lysosomal storage disorders, and serum protein deficiencies. In addition, muscle could provide a reservoir for delivery of therapeutic molecules like blood clotting factors, erythropoietin, or insulin. To date, successful gene transfer to muscle has been limited by the inefficiency of the vector delivery systems and the transient nature of gene expression. In this paper, we show that a vector based on recombinant adeno-associated virus (rAAV) can efficiently transduce adult mouse skeletal muscle. Transduced myofibers escape immune elimination and transgene expression is robust beyond 5 months. Importantly, input vector DNA appears to undergo conversion from single-stranded genomes to high-molecular-weight concatameric forms. These data suggest that rAAV might have a significant advantage over many other viral and nonviral gene delivery methods, and holds significant promise as a vector for gene transfer to mature muscle.

Gene transfer to the intestinal tract: a new approach using selective injection of the superior mesenteric artery.

Gene transfer to the intestinal tract has many potential applications, including complementation of single gene disorders, genetic immunization, and ectopic production of therapeutic molecules. Because the intralumenal approach to vector administration has not been highly successful, we tested whether the circulation can be used as a route to transfer genes to intestinal cells. The superior mesenteric artery (SMA) and vein (SMV) of adult Lewis rats were isolated and an adenoviral vector expressing the Escherichia coli LacZ gene was injected into the SMA. In one set of experiments, both vessels remained patent throughout the entire procedure. In a second group of animals, both vessels were occluded by clamping the SMA 1 cm distal to the injection site and the SMV proximal to the portal vein. In the absence of vascular clamps, gene transfer was evident throughout the small bowel, localized near the serosal surface within the muscularis propria. Occlusion of the SMA and SMV limited gene delivery to a short segment of bowel and shifted beta-galactosidase activity toward the mucosal surface. At the level of microscopy, most of the transduction events were in the lamina propria; transduced mucosal epithelial cells were occasionally observed. These data demonstrate that intestinal gene transfer can be accomplished through the circulation, and that targeting specific regions is feasible.

Gastrointestinal gas formation and infantile colic.

Gastrointestinal gas causes distress in many patients and their parents. Most often, patients do not have an actual increase in gastrointestinal gas volume, but rather their complaints derive from a misunderstanding of normal physiology, a misinterpretation of symptoms (colic), or an increase in intestinal sensitivity (irritable bowel syndrome). Symptoms from actual increases in intestinal gas volume are seen most frequently in children who swallow excessive amounts of air, have a dysmotility syndrome, or consume foods containing poorly absorbed carbohydrates. Although many therapies are used in the treatment of gas-related symptoms, under close scrutiny, the commonly recommended agents (e.g. simethicone) do not have proven efficacy. An understanding of the physiology of gas production and disposal is of practical use to pediatricians in determining the appropriate method of intervention for patients with these complaints.

Ménétrier disease of childhood: role of cytomegalovirus and transforming growth factor alpha.

OBJECTIVE: Because the role of cytomegalovirus in Ménétrier disease in children remains unclear and recent studies have implicated transforming growth factor alpha in the pathogenesis of this disease in adults, we investigated the possibilities that (1) cytomegalovirus is etiologic in Ménétrier disease in children and (2) transforming growth factor alpha mediates its development. METHODS: The presence of a cytomegaloviral infection and the pattern of transforming growth factor alpha immunolocalization were determined in the gastric mucosa of four pediatric patients with Ménétrier disease, in control subjects (children with normal gastric mucosa, gastritis, or prostaglandin E1-induced antral hyperplasia), and in adults with Ménétrier disease. RESULTS: Evidence of a cytomegaloviral infection was present only in the four children with Ménétrier disease. The pattern of transforming growth factor alpha immunostaining was identical in the specimens from pediatric and adult patients with Ménétrier disease. This pattern was distinct from that found in the pediatric control specimens. CONCLUSIONS: These data strengthen the possibilities that cytomegalovirus is etiologic in children and that transforming growth factor alpha is involved in the pathogenesis of Ménétrier disease in both children and adults.

Characterization of the cystic fibrosis transmembrane conductance regulator promoter region. Chromatin context and tissue-specificity.

Expression of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) is tightly regulated. Using a panel of cell lines expressing different levels of CFTR mRNA, we investigated the mechanisms mediating control of CFTR transcription. In highly expressing cells, multiple sites of transcription initiation can be identified between positions -95 and +50 of the CFTR gene, and an alternatively initiated splice variant transcript is also present. Nonepithelial cell lines expressing very low levels of CFTR utilize a start site at -32. Promoter sequence elements from -83 to +111 are at least partially responsible for dictating CFTR transcriptional tissue-specificity, while multiple elements located farther 5' augment promoter strength. Analysis of the chromatin structure and methylation status of the CFTR promoter region reveals cell line differences which correlate with expression levels, suggesting that the physical context of the CFTR gene in vivo may contribute significantly to appropriate regulation of CFTR transcription. Taken together, these findings indicate that cellular control of CFTR gene expression is likely to be a complex function of several overlapping regulatory pathways.

The molecular biology of cystic fibrosis.

Cystic fibrosis afflicts many children and, as the life expectancy for patients with this disease steadily increases, many adults as well. The identification and characterization of the gene responsible for this disorder provide a basis for understanding the pathophysiology of cystic fibrosis. Basic science advances are rapidly being integrated into the diagnostic and therapeutic regimens used by physicians in the care of patients with this disease.

Simultaneous recovery of bacterial and viral pathogens from cerebrospinal fluid.

Mixed bacterial infection in meningitis is well-documented, but there have been few previous reports of mixed viral-bacterial meningitis. A retrospective analysis of the bacterial and viral cerebrospinal fluid (CSF) cultures from a 1-year period in a 315-bed children's hospital revealed 5 patients with mixed viral-bacterial meningitis among 276 patients with viral and/or bacterial culture-positive meningitis. These 5 accounted for 2.8% of the patients with positive CSF viral cultures and 4.8% of those with positive CSF bacterial cultures. All of the viruses were identified as enteroviruses, and the bacteria were Group B Streptococcus, Group D Salmonella, Streptococcus pneumoniae, Haemophilus influenzae type b and Staphylococcus aureus. The ages of the patients ranged from 10 days to 22 years. The clinical course of each of the illnesses was typical of bacterial meningitis. This relatively high frequency of mixed viral-bacterial meningitis could affect the utility of rapid viral diagnostic tests for CSF viruses.