Looking Beyond Respiratory Cultures: Microbiome-Cytokine Signatures of Bacterial Pneumonia and Tracheobronchitis in Lung Transplant Recipients.

Bacterial pneumonia and tracheobronchitis are diagnosed frequently following lung transplantation. The diseases share clinical signs of inflammation and are often difficult to differentiate based on culture results. Microbiome and host immune-response signatures that distinguish between pneumonia and tracheobronchitis are undefined. Using a retrospective study design, we selected 49 bronchoalveolar lavage fluid samples from 16 lung transplant recipients associated with pneumonia (n = 8), tracheobronchitis (n = 12) or colonization without respiratory infection (n = 29). We ensured an even distribution of Pseudomonas aeruginosa or Staphylococcus aureus culture-positive samples across the groups. Bayesian regression analysis identified non-culture-based signatures comprising 16S ribosomal RNA microbiome profiles, cytokine levels and clinical variables that characterized the three diagnoses. Relative to samples associated with colonization, those from pneumonia had significantly lower microbial diversity, decreased levels of several bacterial genera and prominent multifunctional cytokine responses. In contrast, tracheobronchitis was characterized by high microbial diversity and multifunctional cytokine responses that differed from those of pneumonia-colonization comparisons. The dissimilar microbiomes and cytokine responses underlying bacterial pneumonia and tracheobronchitis following lung transplantation suggest that the diseases result from different pathogenic processes. Microbiomes and cytokine responses had complementary features, suggesting that they are closely interconnected in the pathogenesis of both diseases.

Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense.

Bacterial pneumonia is an increasing complication of HIV infection and inversely correlates with the CD4(+) lymphocyte count. Interleukin (IL)-17 is a cytokine produced principally by CD4(+) T cells, which induces granulopoiesis via granulocyte colony-stimulating factor (G-CSF) production and induces CXC chemokines. We hypothesized that IL-17 receptor (IL-17R) signaling is critical for G-CSF and CXC chemokine production and lung host defenses. To test this, we used a model of Klebsiella pneumoniae lung infection in mice genetically deficient in IL-17R or in mice overexpressing a soluble IL-17R. IL-17R-deficient mice were exquisitely sensitive to intranasal K. pneumoniae with 100% mortality after 48 h compared with only 40% mortality in controls. IL-17R knockout (KO) mice displayed a significant delay in neutrophil recruitment into the alveolar space, and had greater dissemination of K. pneumoniae compared with control mice. This defect was associated with a significant reduction in steady-state levels of G-CSF and macrophage inflammatory protein (MIP)-2 mRNA and protein in the lung in response to the K. pneumoniae challenge in IL-17R KO mice. Thus, IL-17R signaling is critical for optimal production of G-CSF and MIP-2 and local control of pulmonary K. pneumoniae infection. These data support impaired IL-17R signaling as a potential mechanism by which deficiency of CD4 lymphocytes predisposes to bacterial pneumonia.

Targeting the IL-22/IL-22BP axis enhances tight junctions and reduces inflammation during influenza infection.

The seasonal burden of influenza coupled with the pandemic outbreaks of more pathogenic strains underscore a critical need to understand the pathophysiology of influenza injury in the lung. Interleukin-22 (IL-22) is a promising cytokine that is critical in protecting the lung during infection. This cytokine is strongly regulated by the soluble receptor IL-22-binding protein (IL-22BP), which is constitutively expressed in the lungs where it inhibits IL-22 activity. The IL-22/IL-22BP axis is thought to prevent chronic exposure of epithelial cells to IL-22. However, the importance of this axis is not understood during an infection such as influenza. Here we demonstrate through the use of IL-22BP-knockout mice (il-22ra2-/-) that a pro-IL-22 environment reduces pulmonary inflammation during H1N1 (PR8/34 H1N1) infection and protects the lung by promoting tight junction formation. We confirmed these results in normal human bronchial epithelial cells in vitro demonstrating improved membrane resistance and induction of the tight junction proteins Cldn4, Tjp1, and Tjp2. Importantly, we show that administering recombinant IL-22 in vivo reduces inflammation and fluid leak into the lung. Taken together, our results demonstrate the IL-22/IL-22BP axis is a potential targetable pathway for reducing influenza-induced pneumonia.

SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery.

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic resulting from zoonotic transmission of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Severe symptoms include viral pneumonia secondary to infection and inflammation of the lower respiratory tract, in some cases causing death. We developed primary human lung epithelial infection models to understand responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface cultures of proximal airway epithelium and 3D organoid cultures of alveolar epithelium were readily infected by SARS-CoV-2 leading to an epithelial cell-autonomous proinflammatory response. We validated the efficacy of selected candidate COVID-19 drugs confirming that Remdesivir strongly suppressed viral infection/replication. We provide a relevant platform for studying COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and future emergent respiratory pathogens. ONE SENTENCE SUMMARY: A novel infection model of the adult human lung epithelium serves as a platform for COVID-19 studies and drug discovery.

Lung epithelial cells: therapeutically inducible effectors of antimicrobial defense.

Lung epithelial cells are increasingly recognized to be active effectors of microbial defense, contributing to both innate and adaptive immune function in the lower respiratory tract. As immune sentinels, lung epithelial cells detect diverse pathogens through an ample repertoire of membrane-bound, endosomal, and cytosolic pattern-recognition receptors (PRRs). The highly plastic epithelial barrier responds to detected threats via modulation of paracellular flux, intercellular communications, mucin production, and periciliary fluid composition. Epithelial PRR stimulation also induces production of cytokines that recruit and sculpt leukocyte-mediated responses, and promotes epithelial generation of antimicrobial effector molecules that are directly microbicidal. The epithelium can alternately enhance tolerance to pathogens, preventing tissue damage through PRR-induced inhibitory signals, opsonization of pathogen-associated molecular patterns, and attenuation of injurious leukocyte responses. The inducibility of these protective responses has prompted attempts to therapeutically harness epithelial defense mechanisms to protect against pneumonias. Recent reports describe successful strategies for manipulation of epithelial defenses to protect against a wide range of respiratory pathogens. The lung epithelium is capable of both significant antimicrobial responses that reduce pathogen burdens and tolerance mechanisms that attenuate immunopathology. This manuscript reviews inducible lung epithelial defense mechanisms that offer opportunities for therapeutic manipulation to protect vulnerable populations against pneumonia.

Exogenous administration of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury.

Heme oxygenase-1 (HO-1) confers protection against a variety of oxidant-induced cell and tissue injury. In this study, we examined whether exogenous administration of HO-1 by gene transfer could also confer protection. We first demonstrated the feasibility of overexpressing HO-1 in the lung by gene transfer. A fragment of the rat HO-1 cDNA clone containing the entire coding region was cloned into plasmid pAC-CMVpLpA, and recombinant adenoviruses containing the rat HO-1 cDNA fragment Ad5-HO-1 were generated by homologous recombination. Intratracheal administration of Ad5-HO-1 resulted in a time-dependent increase in expression of HO-1 mRNA and protein in the rat lungs. Increased HO-1 protein expression was detected diffusely in the bronchiolar epithelium of rats receiving Ad5-HO-1, as assessed by immunohistochemical studies. We then examined whether ectopic expression of HO-1 could confer protection against hyperoxia-induced lung injury. Rats receiving Ad5-HO-1, but not AdV-betaGal, a recombinant adenovirus expressing Escherichia coli beta-galactosidase, before exposure to hyperoxia (>99% O2) exhibited marked reduction in lung injury, as assessed by volume of pleural effusion and histological analyses (significant reduction of edema, hemorrhage, and inflammation). In addition, rats receiving Ad5-HO-1 also exhibited increased survivability against hyperoxic stress when compared with rats receiving AdV-betaGal. Expression of the antioxidant enzymes manganese superoxide dismutase (Mn-SOD) and copper-zinc superoxide dismutase (CuZn-SOD) and of L-ferritin and H-ferritin was not affected by Ad5-HO-1 administration. Furthermore, rats treated with Ad5-HO-1 exhibited attenuation of hyperoxia-induced neutrophil inflammation and apoptosis. Taken together, these data suggest the feasibility of high-level HO-1 expression in the rat lung by gene delivery. To our knowledge, we have demonstrated for the first time that HO-1 can provide protection against hyperoxia-induced lung injury in vivo by modulation of neutrophil inflammation and lung apoptosis.

CD4+ T cell-independent vaccination against Pneumocystis carinii in mice.

Host defenses are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes. Moreover, deficient CD4+ T lymphocytes impair vaccination approaches to prevent opportunistic infection. Therefore, we investigated a CD4+ T cell-independent vaccine approach to a prototypic AIDS-defining infection, Pneumocystis carinii (PC) pneumonia. Here, we demonstrate that bone marrow-derived dendritic cells (DCs) expressing the murine CD40 ligand, when pulsed ex vivo by PC antigen, elicited significant titers of anti-PC IgG in CD4-deficient mice. Vaccinated animals demonstrated significant protection from PC infection, and this protection was the result of an effective humoral response, since adoptive transfer of CD4-depleted splenocytes or serum conferred this protection to CD4-deficient mice. Western blot analysis of PC antigen revealed that DC-vaccinated, CD4-deficient mice predominantly reacted to a 55-kDa PC antigen. These studies show promise for advances in CD4-independent vaccination against HIV-related pathogens.

Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury.

We examined the effects of upregulation of heme oxygenase-1 (HO-1) in steatotic rat liver models of ex vivo cold ischemia/reperfusion (I/R) injury. In the model of ischemia/isolated perfusion, treatment of genetically obese Zucker rats with the HO-1 inducer cobalt protoporphyrin (CoPP) or with adenoviral HO-1 (Ad-HO-1) significantly improved portal venous blood flow, increased bile production, and decreased hepatocyte injury. Unlike in untreated rats or those pretreated with the HO-1 inhibitor zinc protoporphyrin (ZnPP), upregulation of HO-1 by Western blots correlated with amelioration of histologic features of I/R injury. Adjunctive infusion of ZnPP abrogated the beneficial effects of Ad-HO-1 gene transfer, documenting the direct involvement of HO-1 in protection against I/R injury. Following cold ischemia/isotransplantation, HO-1 overexpression extended animal survival from 40% in untreated controls to about 80% after CoPP or Ad-HO-1 therapy. This effect correlated with preserved hepatic architecture, improved liver function, and depressed infiltration by T cells and macrophages. Hence, CoPP- or gene therapy-induced HO-1 prevented I/R injury in steatotic rat livers. These findings provide the rationale for refined new treatments that should increase the supply of usable donor livers and ultimately improve the overall success of liver transplantation.

Novel role for IL-22 in protection during chronic Mycobacterium tuberculosis HN878 infection.

Approximately 2 billion people are infected with Mycobacterium tuberculosis (Mtb), resulting in 1.4 million deaths every year. Among Mtb-infected individuals, clinical isolates belonging to the W-Beijing lineage are increasingly prevalent, associated with drug resistance, and cause severe disease immunopathology in animal models. Therefore, it is exceedingly important to identify the immune mechanisms that mediate protection against rapidly emerging Mtb strains, such as W-Beijing lineage. IL-22 is a member of the IL-10 family of cytokines with both protective and pathological functions at mucosal surfaces. Thus far, collective data show that IL-22 deficient mice are not more susceptible to aerosolized infection with less virulent Mtb strains. Thus, in this study we addressed the functional role for the IL-22 pathway in immunity to emerging Mtb isolates, using W-Beijing lineage member, Mtb HN878 as a prototype. We show that Mtb HN878 stimulates IL-22 production in TLR2 dependent manner and IL-22 mediates protective immunity during chronic stages of Mtb HN878 infection in mice. Interestingly, IL-22-dependent pathways in both epithelial cells and macrophages mediate protective mechanisms for Mtb HN878 control. Thus, our results project a new protective role for IL-22 in emerging Mtb infections.

Tumor necrosis factor-alpha induces hepatic insulin resistance in obese Zucker (fa/fa) rats via interaction of leukocyte antigen-related tyrosine phosphatase with focal adhesion kinase.

The molecular mechanism whereby tumor necrosis factor-alpha (TNF-alpha) induces insulin resistance in obesity is not well understood. Previously, we have shown that inhibition of TNF-alpha improved hepatic insulin sensitivity in obese Zucker rats without altering the tyrosine phosphorylation of liver insulin receptors (IRs), which indicates that the TNF-alpha and insulin-signaling cascades interact distally to the IR. To assess the effects of TNF-alpha on signaling molecules downstream from the IR, we analyzed the tyrosine phosphorylation patterns of liver homogenate proteins from TNF-alpha-neutralized fa/fa rats and showed that focal adhesion kinase (FAK) was consistently hyperphosphorylated (4.5-fold). Moreover, intravenous insulin increased hepatic FAK phosphorylation in a time-dependent manner in Sprague-Dawley rats, which suggests that TNF-alpha may induce hepatic insulin resistance by preventing FAK phosphorylation in response to insulin treatment. To explore the cellular mechanism whereby TNF-alpha regulates phosphorylation of FAK in the liver, we measured c-Src kinase activity and the abundance of 3 major protein tyrosine phosphatases (PTPs) (PTP-1B, leukocyte antigen-related tyrosine phosphatase [LAR], and src homology 2 domain-containing protein-tyrosine phosphatase [SHPTP-2]) in liver homogenates from obese Zucker rats after TNF-alpha blockade. Hepatic c-Src kinase activity was unaltered, but LAR protein was reduced by 75%. In addition, TNF-alpha blockade reduced hepatic PTP activity toward tyrosine phosphorylated FAK by 70%, and this was accounted for by immunodepletion of LAR. Incubation of HepG2 cells with TNF-alpha increased LAR protein levels in a dose-dependent manner. Additionally, pretreatment with TNF-alpha abolished insulin-stimulated tyrosine phosphorylation of FAK in HepG2 cells but had no effect on IR tyrosine phosphorylation or expression. These data suggest that TNF-alpha promotes LAR expression and thus prevents insulin-mediated tyrosine phosphorylation of FAK. This probably represents the interface between TNF-alpha and insulin signaling in the liver.

Inhibition of TNF-alpha processing and TACE-mediated ectodomain shedding by ethanol.

Alcohol (EtOH) is a well-documented immunosuppressant. Acute EtOH-induced immunosuppression is partially due to suppression of tumor necrosis factor alpha (TNF-alpha) secretion. We investigated the mechanism of acute EtOH-induced TNF-alpha suppression in two monocytic cell lines, Mono Mac 6 and DRM. EtOH inhibited TNF-alpha secretion in a dose-dependent manner. However, TNF-alpha transcription was not affected by EtOH. Enzyme-linked immunosorbent assay and confocal microscopy showed that EtOH treatment increased cell-associated TNF-alpha. Ectodomain shedding of TNF-alpha from the cell surface is mediated by TNF-alpha converting enzyme (TACE). In contrast with TNF-alpha, EtOH did not inhibit interleukin-8 (IL-8) secretion, which does not require shedding. Furthermore, TNF p75 receptor shedding, a biomarker for TACE activity, was inhibited by EtOH in both cell lines. EtOH also inhibited TNF p75 receptor shedding in TACE-reconstituted fibroblasts, suggesting that EtOH inhibits the shedding process. These data show that acute EtOH exposure can posttranscriptionally suppress TNF-alpha production, resulting in specific defects in immune defense.

In vivo and in vitro gene transfer and expression in rat intestinal epithelial cells by E1-deleted adenoviral vector.

The intestine is proposed to be an attractive target site for somatic gene therapy due to a large mass of proliferating tissue and stem cells in the crypts. Previous studies using a retroviral vector have shown that a reporter gene, bacterial beta-galactosidase (beta-Gal), can be transferred and expressed in the small intestinal epithelial cell. However, transduction efficiency is relatively low in rat and mice intestines. In the present study, we employed an E1-deleted adenoviral vector (which encodes the beta-Gal gene) to investigate the feasibility of gene transfer into rat small intestinal epithelial cell lines and small intestines in male Sprague-Dawley rats. In in vitro studies, expression of AdCMV beta gal was quantitatively measured in IEC-6 and IEC-18 cell cultures using X-Gal histochemistry and chemiluminescent reporter gene assays. The results indicate that AdCMV beta gal can be efficiently transferred into intestinal epithelial cell lines and transgene expression is virus concentration dependent. In in vivo studies, a 5F intestinal feeding tube was used to deliver the vector to the duodenal segment of the rat. Expression of AdCMV beta gal was primarily localized to the epithelium of the intestinal tract. Transduction efficiency of the transgene was seen in the duodenum, jejunum, ileum, and, to a lesser extent, the colon. Moreover, following a single or secondary administration of recombinant adenovirus, efficient expression of AdCMV beta gal in the intestinal tract peaked at 3 days and decreased by 7 and 14 days. No antiadenoviral antibody response was detected in the serum after a single or secondary challenge with this virus. These findings demonstrate that an E1-deleted adenoviral vector, when administered through an oral-duodenal tube, transfers genetic material more successfully in the intestinal epithelium in the small intestine when compared to the large intestine. A single or secondary challenge with adenoviral vector does not cause enhanced host immune responses to this virus. It suggests that successful gene transduction by the repeat administration of the adenoviral vector makes it an alternative candidate for gene therapy applications in intestinal diseases and metabolic deficiencies.

Use of transient CD4 lymphocyte depletion to prolong transgene expression of E1-deleted adenoviral vectors.

E1-deleted adenoviral vectors are increasingly being utilized for in vivo gene transfer. The potential use of these vectors is limited by transient expression of the transgene and a markedly reduced rate of transduction following readministration, presumably due to a host immune response to the vector. We hypothesized that CD4+ lymphocytes are necessary to generate an immune response to these vectors and that administration of a depleting anti-CD4 antibody (GK1.5) might prolong transgene expression in vivo. We found that pretreatment of mice with a single injection (transient depletion) or weekly injections of GK1.5 (persistent depletion), markedly prolonged expression of an adenovirus-encoded tumor necrosis factor (TNF) inhibitor or luciferase gene compared to controls. Moreover, mice treated with GK1.5 showed no antiadenoviral antibody response to repeat administration of the vector and a second adenoviral transgene could be expressed in these animals. However, control mice developed a significant neutralizing antibody response that prevented transgene expression with administration of a second adenovirus. These findings demonstrate that manipulation of the host immune response may expand potential applications of gene transfer utilizing adenoviral vectors.

Nondepleting anti-CD4 antibody treatment prolongs lung-directed E1-deleted adenovirus-mediated gene expression in rats.

E1-deleted adenoviral vectors are efficient vectors for somatic cell gene therapy, but transgene expression is limited in part by a cytotoxic T cell response directed against virally transduced cells. Moreover, the development of a neutralizing antibody response limits secondary gene transfer with these vectors. Therapy with a depleting anti-CD4 antibody permits prolonged transgene expression in the lung and liver of mice. Furthermore, transient depletion of CD4+ lymphocytes blocks neutralizing antibody production and therefore allows repeat administration and expression of E1-deleted recombinant adenovirus. In this study, we investigated the efficacy of a novel nondepleting anti-CD4 antibody (RIB 5/2) in a model of lung-directed gene therapy in outbred rats. Treatment with RIB 5/2 permitted prolonged reporter gene expression and reduced adenovirus-induced peribronchial and alveolar inflammation in the lung. Moreover administration of RIB 5/2 blocked the development of an anti-adenoviral neutralizing antibody response in the lung and permitted secondary administration and expression of a recombinant adenovirus. These data support the role of immunomodulation in prolonging in vivo transgene expression by recombinant adenovirus.

Immunomodulation and adenoviral gene transfer to the lungs of nonhuman primates.

Previous data from our laboratory and others have demonstrated a critical role for the CD4+ T lymphocyte in in vivo immune responses to recombinant adenoviral vectors. In rodent models, this subset of T cells is required for T cell proliferation, subsequent cytotoxic T cell generation, and production of anti-adenoviral antibodies by B cells. Both depleting and nondepleting anti-CD4 antibodies can attenuate these immune responses to recombinant adenovirus. On the basis of these data, we hypothesized that a nondepleting CDR-engrafted anti-human CD4 antibody (OKT4A) with cross-reactivity to rhesus macaques would attenuate both T and B cell responses to intrapulmonary administration of recombinant adenovirus and permit prolonged reporter gene expression and permit secondary gene transfer. Juvenile rhesus macaques were treated with PBS or OKT4A antibody (10 mg/kg) daily beginning 1 day prior to and up to 11 days after gene transfer. OKT4A resulted in significant attenuation of lymphocyte recruitment into the lung, lymphocyte-proliferative responses to both adenovirus capsid proteins and transgene protein, and adenovirus-induced interferon-gamma elaboration in whole blood and hilar lymph nodes. However, OKT4A was ineffective in attenuating adenovirus-induced IL-4 production in whole blood or hilar lymph nodes, generating neutralizing anti-adenoviral antibodies, or permitting secondary gene transfer. As all the monkeys in this protocol had baseline-detectable anti-adenoviral antibodies by ELISA that were nonneutralizing, analogous to most patients with cystic fibrosis, we postulate that anti-CD4 did not block the proliferation of memory B cells. Moreover, these data suggest that for transient immunomodulation to be successful, strategies need to focus specifically on B cell activation independent of CD4+ T cell help.

The treatment of malignant mesothelioma with a gene modified cancer cell line: a phase I study.

Malignant mesothelioma is a tumor of the pleura for which there is no satisfactory treatment. It is almost universally fatal, regardless of the stage of the tumor at the time of diagnosis. Current treatment modalities include surgery, chemotherapy, and radiation therapy, although in some series none of these modalities is superior to no treatment at all. Because of the dismal prognosis for patients with malignant mesothelioma, a new mode of treatment is desperately needed. A promising area of research into the treatment of various malignancies is gene therapy. Recent studies have demonstrated the utility of exposing tumor cells to cells transduced to express the Herpes simplex virus gene for thymidine kinase (HSV-tk). By virtue of their expression of HSV-tk, the transduced cells are rendered susceptible to the antiviral drug, ganciclovir (GCV). and nearby tumor cells are killed by a phenomenon termed the bystander effect. In this protocol we propose a Phase I trial to study the safety and determine the maximal tolerated dose of an HSV-tk-transduced ovarian cancer cell line (PA1-STK cells) infused into the pleural cavities of patients with malignant pleural mesothelioma, followed by systemic administration of ganciclovir. The hope is that administration of ganciclovir will result in killing of the HSV-tk transduced ovarian cancer cells as well as the nearby malignant mesothelioma cells. This is a standard dose-escalation protocol.

An in vivo model for elucidation of the mechanism of tumor necrosis factor-alpha (TNF-alpha)-induced insulin resistance: evidence for differential regulation of insulin signaling by TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce insulin resistance in cultured cells as well as in animal models. The aim of this study was to map the in vivo mechanism whereby TNF-alpha contributes to the pathogenesis of impaired insulin signaling, using obese and lean Zucker rats in which TNF-alpha activity was inhibited through adenovirus-mediated gene transfer. We employed a replication-incompetent adenovirus-5 (Ad5) vector to endogenously express a TNF inhibitor (TNFi) gene, which encodes a chimeric protein consisting of the extracellular domain of the human 55-kDa TNF receptor joined to a mouse IgG heavy chain. Control animals consisted of rats infected with the same titer of adenovirus carrying the lac-z complementary DNA, encoding for beta-galactosidase. There was a significant reduction in plasma insulin and free fatty acid levels in TNFi obese rats 2 days following Ad5 administration. The peripheral insulin sensitivity index was 50% greater, whereas hepatic glucose output was completely suppressed during hyperinsulinemic glucose clamps in TNFi obese animals, with no differences observed between the two lean groups. The improvement in peripheral and hepatic sensitivity to insulin seen in the obese animals was independent of insulin receptor (IR) number and insulin binding affinity for IR. However, TNF-alpha neutralization led to a 2.5-fold increase in tyrosine phosphorylation of IR in skeletal muscle, whereas this was unchanged in liver. There was also a 4-fold increase in particulate protein tyrosine phosphatase activity of skeletal muscle in TNFi obese animals vs. beta-galactosidase controls, whereas protein tyrosine phosphatase activity in liver was unchanged. These results suggest that TNF-alpha is a mediator of insulin resistance in obesity and may modulate IR signaling in skeletal muscle and liver through different pathways. TNF-alpha may affect insulin action in the liver either at sites distal to the IR or indirectly, possibly because of increased provision of gluconeogenic substrates or altered counterregulation. In addition, the Ad5-mediated gene delivery system employed here provides an in vivo model that is efficient and economical for exploring mechanisms involved in TNF-alpha-induced insulin resistance in various genetic models of obesity-linked diabetes.

Cytokine therapeutics for infectious diseases.

Cytokines are potent molecules, which function as growth factors and orchestrate both innate and adaptive immune responses. Over the last two decades the number of molecules in this class have greatly expanded, and as the biology of these factors is better understood, several of these factors have entered the clinical arena to support or augment components of the immune response. Recently the use of cytokines/growth factors has been studied in patients without a defective immune system but either have significant infection or infection with drug resistant organisms. The use of cytokines as adjuvants in the treatment of infectious diseases is reviewed both in the context of protein and gene-based therapies.

Chimerism analysis in sex-mismatched murine transplantation using quantitative real-time PCR.

Marine experimental stem cell transplantations require the accurate discrimination and quantification of donor cells from host cells. A Y-chromosome-specific, quantitative real-time PCR (kinetic PCR) protocol for blood-derived DNA was developed. The assay sensitivity was extremely high with accurate detection of only 10 pg (six copies of Y target DNA) in a variable background of female DNA background ranging from 2.5 to 50 ng. The dynamic range of the assay provided accurate results ranging from 2.2 x 10(-2)% to 100% of male DNA in female background. The kinetic PCR assay can be used in all mouse strains, and a sample size as low as 2.5 ng total DNA is sufficient for analysis. Therefore, kinetic PCR allows engraftment kinetic studies on repeated blood draws of individual animals with no need for sacrifice. Compared to conventional PCR, the assay is much simplified, as neither the accurate adjustment of sample DNA concentration nor a post-reaction analysis procedure is required. The procedure is simple, free of radioactivity, and permits a throughput of 500-600 reactions per day.

Intralobar pulmonary sequestration presenting as congestive heart failure in a neonate.

We recently evaluated a premature infant with intralobar pulmonary sequestration which presented with signs of CHF. The infant initially underwent ligation of a PDA but subsequently developed tachycardia, tachypnea, and a continuous murmur which radiated to the back. The diagnosis of a pulmonary sequestration was suggested by Doppler echocardiography and confirmed by aortography. The sequestration was successfully treated by surgical removal of the involved lobe.