Altered parvalbumin basket cell inputs in the dorsolateral prefrontal cortex of schizophrenia subjects.

Cortical circuitry dysfunction in schizophrenia has been studied at many different levels of resolution, but not at the most basic unit of network organization--synaptic inputs. Multi-label electron or confocal light microscopy is required to examine specific types of synaptic inputs, and application of these methods to quantitatively study disease-related changes in human postmortem tissue has not been feasible for technical reasons. We recently developed a multi-label confocal light microscopic approach that makes possible the systematic identification and quantification of synaptic inputs, and of the relative levels of proteins localized to these inputs, in human postmortem tissue. We applied this approach to quantify parvalbumin basket cell (PVBC) inputs in area 9 of the dorsolateral prefrontal cortex from schizophrenia and matched comparison subjects. Tissue sections were triple-labeled for the 65 kD isoform of glutamic acid decarboxylase (GAD65), PV and the GABA(A) receptor α1 subunit. PVBC axonal boutons were defined as PV/GAD65 dual-labeled puncta, and PVBC inputs were defined as a PVBC bouton that overlapped a GABA(A) receptor α1 subunit punctum. The density of PVBC inputs was unchanged in subjects with schizophrenia, but levels of PV protein were lower in PVBC boutons. In concert with prior reports, these findings indicate that PVBC dysfunction in schizophrenia reflects molecular and not structural alterations in these cells and their axon terminals.

Relationship of cannabinoid CB1 receptor and cholecystokinin immunoreactivity in monkey dorsolateral prefrontal cortex.

Exposure to cannabis impairs cognitive functions reliant on the circuitry of the dorsolateral prefrontal cortex (DLPFC) and increases the risk of schizophrenia. The actions of cannabis are mediated via the brain cannabinoid 1 receptor (CB1R), which in rodents is heavily localized to the axon terminals of cortical GABA basket neurons that contain cholecystokinin (CCK). Differences in the laminar distribution of CB1R-immunoreactive (IR) axons have been reported between rodent and monkey neocortex, suggesting that the cell type(s) containing CB1Rs, and the synaptic targets of CB1R-IR axon terminals, may differ across species; however, neither the relationship of CB1Rs to CCK-containing interneurons, nor the postsynaptic targets of CB1R and CCK axon terminals, have been examined in primate DLPFC. Consequently, we compared the distribution patterns of CB1R- and CCK-IR structures, determined the proportions of CB1R and CCK neurons that were dual-labeled, and identified the synaptic types and postsynaptic targets of CB1R- and CCK-IR axon terminals in macaque monkey DLPFC. By light microscopy, CB1R- and CCK-IR axons exhibited a similar laminar distribution, with their greatest densities in layer 4. Dual-label fluorescence experiments demonstrated that 91% of CB1R-IR neurons were immunopositive for CCK, whereas only 51% of CCK-IR neurons were immunopositive for CB1R. By electron microscopy, all synapses formed by CB1R-IR axon terminals were symmetric, whereas CCK-IR axon terminals formed both symmetric (88%) and asymmetric (12%) synapses. The primary postsynaptic target of both CB1R- and CCK-IR axon terminals forming symmetric synapses was dendritic shafts (81-88%), with the remainder targeting cell bodies or dendritic spines. Thus, despite species differences in laminar distribution, CB1Rs are principally localized to CCK basket neuron axons in both rodent neocortex and monkey DLPFC. These axons target the perisomatic region of pyramidal neurons, providing a potential anatomical substrate for the impaired function of the DLPFC associated with cannabis use and schizophrenia.

Reactivation of latent human cytomegalovirus in CD14(+) monocytes is differentiation dependent.

We have previously demonstrated reactivation of latent human cytomegalovirus (HCMV) in myeloid lineage cells obtained from healthy donors. Virus was obtained from allogenically stimulated monocyte-derived macrophages (Allo-MDM), but not from macrophages differentiated by mitogenic stimulation (ConA-MDM). In the present study, the cellular and cytokine components essential for HCMV replication and reactivation were examined in Allo-MDM. The importance of both CD4(+) and CD8(+) T cells in the generation of HCMV-permissive Allo-MDM was demonstrated by negative selection or blocking experiments using antibodies directed against both HLA class I and HLA class II molecules. Interestingly, contact of monocytes with CD4 or CD8 T cells was not essential for reactivation of HCMV, since virus was observed in macrophages derived from CD14(+) monocytes stimulated by supernatants produced by allogeneic stimulation of peripheral blood mononuclear cells. Examination of the cytokines produced in Allo-MDM and ConA-MDM cultures indicated a significant difference in the kinetics of production and quantity of these factors. Further examination of the cytokines essential for the generation of HCMV-permissive Allo-MDM identified gamma interferon (IFN-gamma) but not interleukin-1 or -2, tumor necrosis factor alpha, or granulocyte-macrophage colony-stimulating factor as critical components in the generation of these macrophages. In addition, although IFN-gamma was crucial for reactivation of latent HCMV, addition of IFN-gamma to unstimulated macrophage cultures was insufficient to reactivate virus. Thus, this study characterizes two distinct monocyte-derived cell types which can be distinguished by their ability to reactivate and support HCMV replication and identifies the critical importance of IFN-gamma in the reactivation of HCMV.

The Sar1 GTPase coordinates biosynthetic cargo selection with endoplasmic reticulum export site assembly.

Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro. We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection. These Sar1-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro. By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures. Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites.

Evidence that dynamin-2 functions as a signal-transducing GTPase.

The role of dynamin GTPases in the regulation of receptor-mediated endocytosis is well established. Here, we present new evidence that the ubiquitously expressed isoform dynamin-2 (dyn2) can also function in a signal transduction pathway(s). A 200-fold overexpression of dyn1, the 70% identical neuronal isoform, has no effect. Our data suggest that dyn2 can act as a signal transducing GTPase affecting transcriptional regulation.

Traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway.

The pathway of transport of the cystic fibrosis transmembrane regulator (CFTR) through the early exocytic pathway has not been examined. In contrast to most membrane proteins that are concentrated during export from the ER and therefore readily detectable at elevated levels in pre-Golgi intermediates and Golgi compartments, wild-type CFTR could not be detected in these compartments using deconvolution immunofluorescence microscopy. To determine the basis for this unusual feature, we analyzed CFTR localization using quantitative immunoelectron microscopy (IEM). We found that wild-type CFTR is present in pre-Golgi compartments and peripheral tubular elements associated with the cis and trans faces of the Golgi stack, albeit at a concentration 2-fold lower than that found in the endoplasmic reticulum (ER). delta F508 CFTR, a mutant form that is not efficiently delivered to the cell surface and the most common mutation in cystic fibrosis, could also be detected at a reduced concentration in pre-Golgi intermediates and peripheral cis Golgi elements, but not in post-Golgi compartments. Our results suggest that the low level of wild-type CFTR in the Golgi region reflects a limiting step in selective recruitment by the ER export machinery, an event that is largely deficient in delta F508. We raise the possibility that novel modes of selective anterograde and retrograde traffic between the ER and the Golgi may serve to regulate CFTR function in the early secretory compartments.

Long-term infection and transformation of dermal microvascular endothelial cells by human herpesvirus 8.

Human herpesvirus 8 (HHV8) infects Kaposi's sarcoma (KS) spindle cells in situ, as well as the lesional endothelial cells considered to be spindle cell precursors. The HHV8 genome contains several oncogenes, suggesting that infection of endothelial and spindle cells could induce cellular transformation and tumorigenesis and promote the formation of KS lesions. To investigate the potential of HHV8 infection of endothelial cells to contribute to the development of KS, we have developed an in vitro model utilizing dermal microvascular endothelial cells that support significant HHV8 infection. In contrast to existing in vitro systems used to study HHV8 pathogenesis, the majority of dermal endothelial cells are infected with HHV8 and the viral genome is maintained indefinitely. Infection is predominantly latent, with a small percentage of cells supporting lytic replication, and latency is responsive to lytic induction stimuli. Infected endothelial cells develop a spindle shape resembling that of KS lesional cells and show characteristics of a transformed phenotype, including loss of contact inhibition and acquisition of anchorage-independent growth. These results describe a relevant model system in which to study virus-host interactions in vitro and demonstrate the ability of HHV8 to induce phenotypic changes in infected endothelial cells that resemble characteristics of KS spindle cells in vivo. Thus, our results are consistent with a direct role for HHV8 in the pathogenesis of KS.

Growth of human cytomegalovirus in primary macrophages.

Human cytomegalovirus (HCMV) is a major human pathogen that causes considerable disease among immunocompromised individuals. A primary infection results in life-long persistence of the virus in a latent form. HCMV is known to be transferred by blood products, bone marrow, and solid organs, but the cell type that carries the latent infection has been difficult to identify. We have recently demonstrated reactivation of latent HCMV in allogeneically stimulated monocyte-derived macrophages (Allo-MDM). Reactivation occurred only in macrophages produced by allogeneic but not mitogenic stimulation. The presence of dendritic cell markers on some Allo-MDM cells suggested that these macrophages were related to dendritic cells. However, dendritic cells obtained by stimulation of monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) were not permissive for HCMV infection. The cellular and cytokine components which are essential for HCMV replication and reactivation of virus were also examined in Allo-MDM. The importance of both CD4- or CD8-positive T cells in the generation of HCMV permissive Allo-MDM was demonstrated by negative selection or blocking experiments using antibodies directed against both HLA class I and HLA class II molecules. Examination of the cytokines essential for the generation of HCMV permissive Allo-MDM identified gamma-interferon (IFN-gamma, but not IL-1, IL-2, tumor necrosis factor alpha, or GM-CSF as critical components in the generation of these macrophages. However, addition of IFN-gamma to unstimulated macrophage cultures was insufficient to reactivate virus. These results indicate the importance of a specific moncyte stimulus in the generation of a unique HCMV permissive macrophage phenotype as well as why virus is commonly reactivated in transplant patients.

Steady-state plasma membrane expression of human cytomegalovirus gB is determined by the phosphorylation state of Ser900.

Human cytomegalovirus (HCMV) infection of an astrocytoma cell line (U373) or human fibroblast (HF) cells results in a differential cell distribution of the major envelope glycoprotein gB (UL55). This 906-amino-acid type I glycoprotein contains an extracellular domain with a signal sequence, a transmembrane domain, and a 135-amino-acid cytoplasmic tail with a consensus casein kinase II (CKII) site located at Ser900. Since phosphorylation of proteins in the secretory pathway is an important determinant of intracellular trafficking, the state of gB phosphorylation in U373 and HF cells was examined. Analysis of cells expressing wild-type gB and gB with site-specific mutations indicated that the glycoprotein was equally phosphorylated at a single site, Ser900, in both U373 and HF cells. To assess the effect of charge on gB surface expression in U373 cells, Ser900 was replaced with an aspartate (Asp) or alanine (Ala) residue to mimic the phosphorylated and nonphosphorylated states, respectively. Expression of the Asp but not the Ala gB mutation resulted in an increase in the steady-state expression of gB at the plasma membrane (PM) in U373 cells. In addition, treatment of U373 cells with the phosphatase inhibitor tautomycin resulted in the accumulation of gB at the PM. Interestingly, the addition of a charge at Ser900 trapped gB in a low-level cycling pathway at the PM, preventing trafficking of the protein to the trans-Golgi network or other intracellular compartments. Therefore, these results suggest that a tautomycin-sensitive phosphatase regulates cell-specific PM retrieval of gB to intracellular compartments.

Human cytomegalovirus persistently infects aortic endothelial cells.

Endothelial cells (EC) have been implicated as constituting an important cell type in the pathogenesis of human cytomegalovirus (HCMV). Microvascular and macrovascular EC exhibit different biochemical and functional properties depending on the organ of origin. Phenotypic differences between microvascular and macrovascular EC may alter the ability of these cells to support HCMV replication. In this study, we compared the replication of HCMV in primary macrovascular aortic EC (AEC) with that in brain microvascular EC (BMVEC). An examination of IE72, pp65, and gB viral antigen expression in BMVEC and AEC by immunoflourescence revealed similar frequencies of infected cells. Intracellular production of virus was 3 log units greater in BMVEC than in AEC, while equal quantities of extracellular virus were produced in both cell types. HCMV infection of BMVEC resulted in rapid cellular lysis, while the virus was nonlytic and continuously released from HCMV-infected AEC for the life span of the culture. An examination of infected cells by electron microscopy revealed the formation of abundant nucleocapsids in both AEC and BMVEC. However, significant amounts of mature viral particles were only detected in the cytoplasm of BMVEC. These observations indicate that levels of HCMV replication in EC obtained from different organs are distinct and suggest that persistently infected AEC may serve as a reservoir of virus.

Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors.

Reactivation of human cytomegalovirus (HCMV) results in severe disease in AIDS patients and immunocompromised patients receiving blood transfusions or organ or bone marrow grafts. Although the site of HCMV latency is unknown, blood cells have been implicated as a viral reservoir. In this study, we demonstrate HCMV reactivation in vitro from seven consecutive healthy donors through allogeneic stimulation of peripheral blood mononuclear cells (PBMCs). HCMV replication was detected at 17 days poststimulation, and virus was recovered after long-term culture from a macrophage expressing dendritic cell markers. Thus, these observations demonstrate that PBMCs harbor latent HCMV, which reactivates in a myeloid lineage cell upon allogeneic stimulation.

Interferon-gamma and tumor necrosis factor-alpha specifically induce formation of cytomegalovirus-permissive monocyte-derived macrophages that are refractory to the antiviral activity of these cytokines.

Monocytes/macrophages are key cells in the pathogenesis of human cytomegalovirus (HCMV). Although HCMV infection in monocytes is restricted to early events of gene expression, productive infection has been demonstrated in differentiated macrophages in vitro. We examined the cellular and cytokine components that are essential for HCMV replication in Concanavalin A-stimulated monocyte-derived macrophages (MDM). By negative selection, depletion of CD8+ T lymphocytes, but not CD4+ T lymphocytes, CD19+ B cells, or CD56+ NK cells, resulted in a 60-70% reduction in the number of HCMV-infected MDM, and a 4 log decrease in virus production. Neutralization of IFN-gamma and TNF-alpha, but not IL-1, IL-2, or TGF-beta, decreased production of virus by 4 logs and 2 logs, respectively. Subsequently, addition of recombinant IFN-gamma or TNF-alpha to purified monocyte cultures was sufficient to produce HCMV-permissive MDM. While IFN-gamma and TNF-alpha possess antiviral properties, addition of these cytokines to permissive MDM cultures did not affect production of HCMV. Thus, rather than inhibiting replication of HCMV, IFN-gamma and TNF-alpha specifically induce differentiation of monocytes into HCMV-permissive MDM, which are resistant to the antiviral effects of these cytokines.

Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains.

The human cytomegalovirus (HCMV) early glycoprotein products of the US11 and US2 open reading frames cause increased turnover of major histocompatibility complex (MHC) class I heavy chains. Since US2 is homologous to another HCMV gene (US3), we hypothesized that the US3 gene product also may affect MHC class I expression. In cells constitutively expressing the HCMV US3 gene, MHC class I heavy chains formed a stable complex with beta 2-microglobulin. However, maturation of the N-linked glycan of MHC class I heavy chains was impaired in US3+ cells. The glycoprotein product of US3 (gpUS3) occurs mostly in a high-mannose form and coimmunoprecipitates with beta 2-microglobulin associated class I heavy chains. Mature class I molecules were detected at steady state on the surface of US3+ cells, as in control cells. Substantial perinuclear accumulation of heavy chains was observed in US3+ cells. The data suggest that gpUS3 impairs egress of MHC class I heavy chains from the endoplasmic reticulum.

A novel mechanism for persistence of human cytomegalovirus in macrophages.

Human cytomegalovirus (HCMV) infection of monocyte-derived macrophages (MDM) results in delayed and nonlytic productive viral growth. During late stages of replication, infectious virus remains cell associated in cytoplasmic vacuoles. In order to understand HCMV survival and persistence in MDM, we examined mechanisms involved in the formation and trafficking of HCMV-containing vacuoles in these cells. Utilizing double-label immunofluorescence with antibodies to viral and cellular proteins, HCMV-containing vacuoles were associated with the Golgi apparatus marker mannosidase II but not with markers to early endosomes (transferrin receptor and rab5) or late endosomes and early lysosomes (LAMP-1 and -2). In addition, as late-stage viral infection progressed in MDM, the cells displayed increasing abnormalities in the Golgi apparatus. Analysis of structural features of infected cells revealed the disruption of the microtubule network. These observations suggest a novel mechanism by which HCMV is vacuolized in MDM, avoiding degradation and release from the cell.

Growth kinetics of human cytomegalovirus are altered in monocyte-derived macrophages.

Stimulation of monocytes/macrophages with activated nonadherent cells allows productive nonlytic growth of human cytomegalovirus (HCMV), but the viral replication cycle is delayed relative to replication of HCMV in human fibroblasts. Analysis of infected monocyte-derived macrophage (MDM) mRNA for major immediate-early (MIE 86, 72, and 55) and late (pp65 and gB) gene expression by reverse transcription PCR indicates that transcription peaks at 3 and 7 days postinfection (dpi), respectively. In contrast, in human fibroblast controls, mRNA for MIE and late gene expression peaked at 5 and 48 h postinfection, respectively. Consistent with reverse transcription PCR experiments, double-label antibody experiments first detected MIE antigen expression at 12 h postinfection, peaking at 3 dpi, and late (pp65 or gB) antigen expression at 5 dpi, peaking at 7 dpi. MIE antigen was not detected between 3 and 7 dpi but reappeared and was coexpressed with pp65 in enlarged MDM nuclei at 7 dpi. After 7 dpi, macrophages with numerous vacuoles containing large amounts of pp65 and gB were observed in culture. These vacuoles were frequently seen at cellular contact points, suggesting that cell-to-cell transfer of virus was the major mode of viral transmission. Consistent with this observation, infectious virus was recovered from MDM cellular lysates but not culture supernatant. The delayed growth and compartmentalization of HCMV in macrophages may allow the cell to accommodate the viral replication cycle without cell lysis. In addition, the macrophage may function as a vehicle for cell-to-cell transmission of HCMV.

Cytomegalovirus persistence in macrophages and endothelial cells.

Although human cytomegalovirus (HCMV) rarely causes overt disease in healthy individuals, the virus can be a serious, even life-threatening problem in immunosuppressed or immune-deficient patients and in the setting of maternofetal primary infection. In recent years knowledge about HCMV pathogenesis has increased significantly. Identification of the cell types infected by HCMV in vivo has demonstrated that monocytes/macrophages and endothelial cells are key elements both in latent and acute infection with HCMV. Both cell types can be involved in systemic spread of virus and specific organ disease. While it has been demonstrated that differentiation of monocytes into macrophages renders these cells permissive to productive HCMV infection, there is presently limited knowledge about the pathogenesis of HCMV in endothelial cells (ECs). These cells represent the physiological interface between blood and tissues, display heterologous phenotypes, and are functionally variable, depending on their respective microanatomic environment. Microvascular ECs are the site of monocyte transmigration into organ tissues and therefore are likely to regulate the activation state of infected monocytes. Recently, macrovascular endothelium is receiving increasing attention due to its possible involvement in atherogenesis. In this overview we present recent findings on the role of monocytes/macrophages and endothelial cells in HCMV infection.