TNF-alpha inhibits insulin action in liver and adipose tissue: A model of metabolic syndrome.

Several studies suggest that TNF-alpha contributes to the development of insulin resistance (IR). We compared transcriptional profiles of rat H-411E liver cells exposed to insulin in the absence or presence of TNF-alpha. We identified 33 genes whose expression was altered by insulin, and then reversed by TNF-alpha. Twenty-six of these 33 genes created a single network centered around: insulin, TNF-alpha, p38-MAPK, TGFb1; SMAD and STAT1; and enzymes and cytokines involved in apoptosis (CASP3, GADD45B, IL2, TNF-alpha, etc.). We analyzed our data together with other data of gene expression in adipocytes and found a number of processes common to both, for example, cell death and inflammation; intercellular signaling and metabolism; G-Protein, IL-10 and PTEN signaling. Moreover, the two datasets combined generated a single molecular network that further identified PTEN (a phosphatase) as a unique new link between insulin signaling, IR, and apoptosis reflecting the pathophysiology of "metabolic syndrome".

Multimaterial Dual Gradient Three-Dimensional Printing for Osteogenic Differentiation and Spatial Segregation.

In this study of three-dimensional (3D) printed composite ß-tricalcium phosphate (ß-TCP)-/hydroxyapatite/poly(ɛ-caprolactone)-based constructs, the effects of vertical compositional ceramic gradients and architectural porosity gradients on the osteogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (MSCs) were investigated. Specifically, three different concentrations of ß-TCP (0, 10, and 20 wt%) and three different porosities (33% ± 4%, 50% ± 4%, and 65% ± 3%) were examined to elucidate the contributions of chemical and physical gradients on the biochemical behavior of MSCs and the mineralized matrix production within a 3D culture system. By delaminating the constructs at the gradient transition point, the spatial separation of cellular phenotypes could be specifically evaluated for each construct section. Results indicated that increased concentrations of ß-TCP resulted in upregulation of osteogenic markers, including alkaline phosphatase activity and mineralized matrix development. Furthermore, MSCs located within regions of higher porosity displayed a more mature osteogenic phenotype compared to MSCs in lower porosity regions. These results demonstrate that 3D printing can be leveraged to create multiphasic gradient constructs to precisely direct the development and function of MSCs, leading to a phenotypic gradient. Impact Statement In this study, three-dimensional (3D) printed ceramic/polymeric constructs containing discrete vertical gradients of both composition and porosity were fabricated to precisely control the osteogenic differentiation of mesenchymal stem cells. By making simple alterations in construct architecture and composition, constructs containing heterogenous populations of cells were generated, where gradients in scaffold design led to corresponding gradients in cellular phenotype. The study demonstrates that 3D printed multiphasic composite constructs can be leveraged to create complex heterogeneous tissues and interfaces.

Fabrication and mechanical characterization of 3D printed vertical uniform and gradient scaffolds for bone and osteochondral tissue engineering.

Recent developments in 3D printing (3DP) research have led to a variety of scaffold designs and techniques for osteochondral tissue engineering; however, the simultaneous incorporation of multiple types of gradients within the same construct remains a challenge. Herein, we describe the fabrication and mechanical characterization of porous poly(ε-caprolactone) (PCL) and PCL-hydroxyapatite (HA) scaffolds with incorporated vertical porosity and ceramic content gradients via a multimaterial extrusion 3DP system. Scaffolds of 0 wt% HA (PCL), 15 wt% HA (HA15), or 30 wt% HA (HA30) were fabricated with uniform composition and porosity (using 0.2 mm, 0.5 mm, or 0.9 mm on-center fiber spacing), uniform composition and gradient porosity, and gradient composition (PCL-HA15-HA30) and porosity. Micro-CT imaging and porosity analysis demonstrated the ability to incorporate both vertical porosity and pore size gradients and a ceramic gradient, which collectively recapitulate gradients found in native osteochondral tissues. Uniaxial compression testing demonstrated an inverse relationship between porosity, ϕ, and compressive modulus, E, and yield stress, σy, for uniform porosity scaffolds, however, no differences were observed as a result of ceramic incorporation. All scaffolds demonstrated compressive moduli within the appropriate range for trabecular bone, with average moduli between 86 ±â€¯14-220 ±â€¯26 MPa. Uniform porosity and pore size scaffolds for all ceramic levels had compressive moduli between 205 ±â€¯37-220 ±â€¯26 MPa, 112 ±â€¯13-118 ±â€¯23 MPa, and 86 ±â€¯14-97 ±â€¯8 MPa respectively for porosities ranging between 14 ±â€¯4-20 ±â€¯6%, 36 ±â€¯3-43 ±â€¯4%, and 54 ±â€¯2-57 ±â€¯2%, with the moduli and yield stresses of low porosity scaffolds being significantly greater (p < 0.05) than those of all other groups. Single (porosity) gradient and dual (composition/porosity) gradient scaffolds demonstrated compressive properties similar (p > 0.05) to those of the highest porosity uniform scaffolds (porosity gradient scaffolds 98 ±â€¯23-107 ±â€¯6 MPa, and 102 ±â€¯7 MPa for dual composition/porosity gradient scaffolds), indicating that these properties are more heavily influenced by the weakest section of the gradient. The compression data for uniform scaffolds were also readily modeled, yielding scaling laws of the form E ∼ (1 - ϕ)1.27 and σy ∼ (1 - ϕ)1.37, which demonstrated that the compressive properties evaluated in this study were well-aligned with expectations from previous literature and were readily modeled with good fidelity independent of polymer scaffold geometry and ceramic content. All uniform scaffolds were similarly deformed and recovered despite different porosities, while the large-pore sections of porosity gradient scaffolds were significantly more deformed than all other groups, indicating that porosity may not be an independent factor in determining strain recovery. Moving forward, the technique described here will serve as the template for more complex multimaterial constructs with bioactive cues that better match native tissue physiology and promote tissue regeneration. STATEMENT OF SIGNIFICANCE: This manuscript describes the fabrication and mechanical characterization of "dual" porosity/ceramic content gradient scaffolds produced via a multimaterial extrusion 3D printing system for osteochondral tissue engineering. Such scaffolds are designed to better address the simultaneous gradients in architecture and mineralization found in native osteochondral tissue. The results of this study demonstrate that this technique may serve as a template for future advances in 3D printing technology that may better address the inherent complexity in such heterogeneous tissues.

Late graft failure due to dual bone marrow infection with variants A and B of human herpesvirus-6.

Hematopoietic effects of human Herpesvirus-6 (HHV-6) infection following bone marrow transplantation (BMT) include delayed engraftment and early myelosuppression. Variant A has not been isolated after BMT. A case of graft failure is reported following an HLA-identical BMT for chronic myelogenous leukemia (CML) in chronic phase. Evaluation of bone marrow during the period of graft failure revealed variants A and B of HHV-6 by culture, immunofluorescence, polymerase chain reaction (PCR), and immunohistochemistry. Evidence for other cases of graft failure, including cytomegalovirus (CMV), could not be found. A hypothesis is proposed that late graft failure in this case was due to variant A of HHV-6.

Motoneuron uptake from the circulation of the binding fragment of tetanus toxin.

Tetanus toxin enters the central nervous system from the systemic circulation after it is internalized by motoneuron terminals at the neuromuscular junction. We have demonstrated that the atoxic binding fragment (C-fragment) of tetanus toxin is internalized preferentially by motoneurons. We examined the distribution of C-fragment after intravenous injection in the nervous systems of mice by immunohistochemical methods. All animals remained asymptomatic until killed one to two days after injection. C-fragment was found only within neurons with processes outside the blood-brain barrier. Large motoneurons of the spinal cord showed the greatest accumulation of C-fragment. Motoneurons of brain-stem nuclei (particularly facial and trigeminal), also showed substantial label of C-fragment. Small amounts of C-fragment were detected in dorsal root ganglion cells. Affinity of a systemically distributed substance for synaptic components, as well as an inability to cross the blood-brain barrier, may lead to its preferential localization in motoneurons.

Subacute leukoencephalitis caused by CNS infection with human herpesvirus-6 manifesting as acute multiple sclerosis.

Several recent reports have documented the neuroinvasiveness of human herpesvirus-6 (HHV-6) in infants with primary HHV-6 infections, in children and adults with AIDS, in recipients of bone marrow transplants, and in immunologically intact adults and children. CNS infections with HHV-6 can be subacute and are frequently associated with diffuse or multifocal demyelination. We analyzed the CNS tissues of a young woman who died of a demyelinative disease, which was clinically and histopathologically diagnosed as acute multiple sclerosis, for active HHV-6 infection by immunohistochemical staining. The tissues contained a dense and disseminated active HHV-6 infection that was intimately related to the pathologic changes present.

Chronic myelopathy associated with human herpesvirus-6.

Human herpesvirus-6 (HHV-6) is implicated in a variety of neurologic diseases. We report a previously healthy elderly woman with progressive spastic paraparesis. At autopsy the spinal cord showed widespread demyelination, axonal loss, and chronic inflammation. HHV-6 DNA was amplified, using polymerase chain reaction, from spinal cord tissue, and glial cells were immunoreactive with an HHV-6 antibody. These findings suggest that HHV-6 may cause myelopathy.

Adenovirus infections in immunocompromised patients.

Adenovirus infections have been reported in as many as one-fifth of bone marrow transplant (BMT) recipients and patients with acquired immunodeficiency syndrome (AIDS), and in a lesser, though still prominent, proportion of organ transplant recipients. The relative contributions of primary infections versus reactivations from latency in immunocompromised patients remain unclear. Compared with adult BMT recipients, pediatric BMT recipients appear to be infected by adenovirus more frequently and earlier in the post-transplant period. The diagnosis of adenovirus infection is complicated by the existence of > 40 viral serotypes, although certain subgroups are more likely to be involved in certain patient populations. Adenoviruses are responsible for a broad range of clinical diseases that may be associated with high mortality, including pneumonia, hepatitis, encephalitis, hemorrhagic cystitis, and gastroenteritis. The clinical and histopathologic features of adenovirus disease may resemble those of cytomegalovirus disease, potentially complicating the diagnosis. Risk factors for clinical adenovirus disease include the number of sites from which the virus is cultured and, in BMT recipients, the presence of moderate to severe acute graft-versus-host disease.

Human herpesvirus-6 infection in liver transplant recipients: documentation of pathogenicity.

BACKGROUND: The new herpesvirus, human herpesvirus-6 (HHV-6), is able to cause clinical illness after transplantation; however, the pathogenic potential and the clinical features of HHV-6 have not been defined in liver transplant recipients. METHODS: We report the first cases of invasive and symptomatic infection due to HHV-6 in liver transplant recipients. RESULTS: HHV-6 infection occurred in four liver transplant recipients at a median of 50 days after transplant (range 17-90 days). Severe cytopenia was observed in all patients; leukopenia (with median leukocyte count of 1400/mm3) was the most commonly effected bone marrow lineage. One of the four patients had interstitial pneumonitis due to HHV-6. No other virus (e.g., cytomegalovirus) or another pathogen was detected in the lungs, blood, or bone marrow in any of the above patients. CONCLUSIONS: Our data suggest that HHV-6 can be a pathogen in liver transplant recipients; idiopathic bone marrow suppression is its predominant clinical sequelae. Recognition of HHV-6 infection is clinically pertinent because HHV-6 is potentially treatable with the currently available antiviral agents.

Lymphoid cell infection by measles virus in newborn hamsters. Role for monocytes in virus spread to distant sites.

Following intraperitoneal inoculation of measles (HBS) virus into newborn hamsters widespread but variable productive lymphoid tissue infection was detected by a sensitive viral isolation technique. Peritoneal wash cells and spleen were the most common sites of infection. Virus was frequently isolated from the bone marrow and was commonly found in multiple lymph nodes. Thymic infection was only rarely demonstrated. A mononuclear cell associated viremia was demonstrated by Ficollhypaque fractionation of peripheral blood from animals receiving both high- and low-dose virus. Infection was present in both plastic adherent and non-adherent fractions of the blood mononuclear cells. The infected cell population in the spleen had both nylon wool and plastic adherent characteristics and infection was therefore thought to be macrophage-associated. The possible relevance of such macrophage associated infection is discussed with regard to the pathogenesis of natural measles infection and the immunosuppression observed in measles-infected hosts.

Variant B human herpesvirus-6 associated febrile dermatosis with thrombocytopenia and encephalopathy in a liver transplant recipient.

Human herpesvirus 6 (HHV-6) is a recently discovered virus the pathogenicity of which in solid organ transplant recipients has not been defined. We describe a unique febrile syndrome due to disseminated invasive variant B HHV-6 infection in a liver transplant recipient with evidence of direct tissue invasion by the virus. Acute febrile illness characterized by life-threatening thrombocytopenia, progressive encephalopathy and skin rash developed in association with invasive HHV-6 infection in a liver transplant recipient. HHV-6 was isolated from the patient's peripheral blood in cell culture; variant B HHV-6 DNA was detected in the patient's peripheral blood mononuclear cells (PBMC) at a concentration greater than 1000 virus genomes per 10(6) PBMC. A bone marrow biopsy was also positive for HHV-6, documenting direct tissue invasion. Intravenous ganciclovir for three weeks led to a prompt clinical response. Although larger studies are warranted, our case suggests that HHV-6 should be considered in the diagnostic evaluation of patients with fever, cytopenia, and encephalopathy, particularly since HHV-6 is susceptible to ganciclovir and foscarnet.

Phase I study of safety and pharmacokinetics of a human anticytomegalovirus monoclonal antibody in allogeneic bone marrow transplant recipients.

This study examined the safety and pharmacokinetic profile of a potentially therapeutic and fully human anti-CMV monoclonal antibody (SDZ MSL-109) in a phase I dose escalation trial in patients receiving allogeneic bone marrow transplants. Fifteen adult marrow transplant patients, twelve with chronic myelogenous leukemia and three with acute nonlymphocytic leukemia, in cohorts of five patients each, were administered monoclonal antibody intravenously at doses of 50, 250, and 500 micrograms/kg at approximately three-week intervals for six months. Administration of the monoclonal antibody was associated with minimal side effects and no dose-related toxicity. Antibody elimination curves in all dose groups were consistent with a two-compartment model with an alpha half-life at the low, middle, and high dose groups of 1.03, 0.82, and 0.79 days, and a beta half life of 13.9, 14.0, and 16.5 days, respectively. The volume of distribution decreased with repetitive dosing to approximate the plasma volume in each patient and the pharmacokinetic profile was comparable to that of human IgG. There was no host antiidiotypic or antiallotypic antibody formation, indicating that MSL-109 was not immunogenic. Further studies are warranted to assess the potential efficacy of human monoclonal anti-CMV disease in marrow transplant recipients and other patients with immunodeficiency disorders.

Human herpesvirus-6 in liver transplant recipients: role in pathogenesis of fungal infections, neurologic complications, and outcome.

BACKGROUND: The clinical impact and relevance of human herpesvirus-6 (HHV-6) infection in liver transplant recipients, has not been fully discerned. METHODS: A prospective study of 80 consecutive liver transplant recipients was performed using surveillance cultures for HHV-6 at weeks 2, 3, 4, and 6 after transplantation. Viral isolation was used for the detection of HHV-6. RESULTS: HHV-6 infection occurred in 39% (31 of 80) of the patients. Patients with HHV-6 infection were more likely to have hepatocellular carcinoma as underlying liver disease (P=.09). Mental status changes of unidentifiable etiology were significantly more likely to occur in patients with HHV-6 compared with those without (26%, 9 of 31 vs. 6%, 3 of 49, P=.008). HHV-6 infection was an independent predictor of invasive fungal infections (odds ratio 8.3, 95% confidence interval, 1.2-58.0, P=.03). A significant association between HHV-6 infection and CMV infection after transplantation, CMV recipient and donor serostatus, rejection, or fever of unknown origin, could not be documented. Mortality at last follow-up in patients with HHV-6 infection (29%, 9 of 31) was significantly greater than those without HHV-6 (6%, 3 of 49, P=.008). CONCLUSIONS: Central nervous system complications of unknown etiology after liver transplantation may be related to HHV-6 infection. HHV-6 viremia was an independently significant predictor of invasive fungal infections and was associated with late mortality in liver transplantation recipients.

Human herpesvirus 6 (HHV-6)-associated dysfunction of blood monocytes.

HHV-6 is a recently described member of the herpesvirus family. HHV-6-associated marrow failure and interstitial pneumonitis where macrophages are the primary infected cell type have been described in marrow transplant patients (Carrigan, 1991; Drobyski et al., 1993). In recent studies we have shown that exposure of normal human marrow to HHV-6GS (a type A strain) or several type B strains resulted in suppression of growth factor induced outgrowth of macrophages by > 90% (Burd and Carrigan, 1993). Additional experiments using HHV-6GS to characterize the effects of the virus on peripheral blood monocytes showed that the respiratory burst capacity of these cells as determined by luminol-enhanced chemiluminescence using phorbol myristate acetate as a trigger was decreased by 83% +/- 13% in a series of 5 experiments. The decreased respiratory burst was evident as early as 15 min after exposure to virus. Experiments in which cells were separated on a fluorescence activated cell sorter prior to respiratory burst assay showed that the response was mediated solely by peripheral blood monocytes. The respiratory burst response of virus-exposed cells to opsonized zymosan was not affected, indicating that the virus may selectively interfere with the protein kinase C pathway of cellular activation. Ultracentrifugation of stock material to remove infectious virus showed that the suppressive factor was associated with the supernatant fraction. These findings suggest that HHV-6 infection may be associated with a defect in one of the major monocyte activation pathways, and this could be of importance with respect to persistent infection by HHV-6 in immune compromised patients.

Prevalence of human herpesvirus 6 in lung tissue from children with pneumonitis.

AIMS/BACKGROUND: Human herpesvirus type 6 (HHV-6) is the aetiological agent of exanthem subitum, and has also been linked with a variety of other diseases. The aim of this study was to investigate the role of HHV-6 in pneumonitis in children. METHODS: Formalin fixed, paraffin wax embedded lung tissue from 33 children (age range two months to 16 years) who died with pneumonitis was subjected to immunohistochemical staining for HHV-6 using an avidin-biotin method. RESULTS: Active HHV-6 infection was demonstrated in four children: a bone marrow transplant recipient with concomitant adenovirus infection, a patient with hepatitis of unknown aetiology, a patient with congenital anomalies, and a patient with congenital immunodeficiency. CONCLUSION: Accurate localisation of HHV-6 is possible in postmortem lung tissue. HHV-6 either alone or in combination with other pathogens may play a role in the development of pneumonitis.

Quantification of measles virus antigens in infected CNS tissue: development of an enzyme linked immunoassay.

An enzyme linked immunoassay procedure has been developed which allows accurate quantification of low levels of measles virus antigens in infected central nervous system (CNS) tissues. In its development a systematic study was made of the effects of a large excess of nonviral proteins on the assay results. It was found that controls using single proteins such as bovine serum albumin were inadequate. Further, results indicate that experimental samples and viral antigen standards must be closely matched for their content of CNS tissue proteins since such proteins exert distinctive quantitative effects, both dramatic and subtle.

Retrograde transneuronal transfer of the C-fragment of tetanus toxin.

The transport of the C-fragment of tetanus toxin after intramuscular injection was studied with immunohistochemical techniques. Brainstems and spinal cords of mice were examined after injections of C-fragment into the tongue or forearm. Labelled motorneurons were observed within 6 h. By two days after injection, the bulk of detectable C-fragment had passed out of motorneurons into the surrounding neuropil. C-fragment, like native tetanus toxin, appears to be transferred from motorneurons to presynaptic structures to an extent not observed with other proteins. It is useful in the study of retrograde transneuronal transfer since it lacks the toxicity of tetanus toxin.

Counselor prompts to increase condom taking during treatment for cocaine dependence.

This study examined whether active prompting would increase the number of free condoms taken from dispensers placed in counselors' offices in a cocaine abuse treatment clinic. Using a combined multiple baseline and reversal design, two teams of counselors were instructed to actively prompt and encourage condom taking during some conditions and to avoid commenting on or encouraging condom use in other conditions. To monitor accuracy of implementing the intervention, counselors completed a checklist for every subject they saw in their office during the day. Overall, the number of condoms taken per visit during prompting conditions was almost six times greater than during baseline conditions. However, implementation declined during the study, and all counselors complained about the intervention. Implications for dispensing free condoms to reduce HIV risk in drug abuse treatment clinics are discussed.

PIP: HIV infection is increasing among noninjecting as well as iv drug users. Of particular concern, are users of cocaine and crack cocaine, who are likely to trade sex for money or drugs and to have multiple sexual partners. This study investigated whether an active counselor intervention could increase the number of free condoms taken from dispensers placed in counselors' offices in a cocaine abuse treatment program in Philadelphia, Pennsylvania (US). A multiple baseline design across 2 teams of counselors with reversals was utilized. During the initial 5-week baseline period, when condoms were displayed on the counselors' desks but no prompts were given, an average of 0.34 and 0.25 condoms were taken per visit per team. During weeks 6-13, when counseling Team 1 utilized prompting and encouraged condom taking, this average increased to 3.17/visit. When Team 2 implemented the intervention (weeks 13-20), an average of 2.78 condoms/visit were taken. When both teams stopped the intervention, the number of condoms taken fell close to baseline levels. Resumption of the intervention increased use, but not to the previous high level. Over the entire 28-week study period, clients took an average of 0.43 condoms/visit during baseline conditions, and 2.45/visit during counselor prompting conditions. The 6-fold increase in condom uptake associated with active encouragement suggests the feasibility of this strategy for cocaine treatment programs. However, the participating counselors voiced irritation with the mandatory, sometimes intrusive intervention. The availability of free condoms in the clinic waiting area or day room represents an alternative strategy documented to increase the taking of condoms.

Effect of dispenser location on taking free condoms in an outpatient cocaine abuse treatment clinic.

Crack cocaine use increases risky sexual behavior and HIV exposure; therefore, safe sexual practices should be encouraged during cocaine addiction treatment. Research indicates that placing condom dispensers in private restrooms increases taking free condoms. We investigated two other dispenser locations (a day room vs. counselors' offices) and found that substantially more condoms were taken when dispensers were in the day room. This is an important issue for public health facilities without private restrooms.