Evidence that ADP hydrolysis by human cells is related to thrombogenic potential1

Washed monolayer cultures of human cells provoke the platelet release reaction to an extent comparable to irreversible aggregation with the following frequencies: endothelial, 0.0; smooth muscle; 0.67; fibroblast, 0.31. The ability of each cell type to hydrolyze exogenous adenine nucleotides was also compared. Endothelial cells and fibroblasts readily hydrolyze ATP and ADP, but smooth muscle cells have very little ectonucleotidase activity. The kinetic data suggest that the ability of endothelial cells to degrade ADP may contribute to their non-thrombogenic nature.These observations suggest that vascular-type cells may retain some of their non-thrombogenic characteristics in culture and that this $\text{in vitro}$ system may serve as a model for studying contributions of each portion of the vessel wall to hemostasis and thrombosis.     

Characterization of human monocyte-derived microglia-like cells

Microglial cells are central to brain immunity and intervene in many human neurological diseases. The aim of this study was to develop a convenient cellular model for human microglial cells, suitable for HIV studies. Microglia derive from the hematogenous myelomonocytic lineage, possibly as a distinct subpopulation but in any case able to invade the CNS, proliferate, and differentiate into ameboid and then ramified microglia in the adult life. We thus attempted to derive microglia-like cells from human monocytes. When cultured with astrocyte-conditioned medium (ACM), monocytes acquired a ramified morphology, typical of microglia. They overexpressed substance P and the calcium binding protein Iba-1 and dimly expressed class II MHC, three characteristics of microglial cells. Moreover, they also expressed a potassium inward rectifier current, another microglia-specific feature. These monocyte-derived microglia-like cells (MDMi) were CD4(+)/CD14(+), evocative of an activated microglia phenotype. When treated with lipopolysaccharide (LPS), MDMi lost their overexpression of substance P, which returned to untreated monocyte-derived macrophage (MDM) level. Compared with MDM, MDMi expressed higher CD4 but lower CCR5 levels; they could be infected by HIV-1(BaL), but produced less virus progeny than MDM did. This model of human microglia may be an interesting alternative to primary microglia for large scale in vitro HIV studies and may help to better understand HIV-associated microgliosis and chronic inflammation in the brain.     

Synthesis and release of neurotoxic kynurenine metabolites by human monocyte-derived macrophages

We studied the regulation of the kynurenine pathway of tryptophan metabolism in human monocyte-derived macrophages (MDM) with the aim of evaluating macrophage involvement in inflammatory neurological disorders.

Cultured MDM metabolized tryptophan and released kynurenine metabolites, including the excitotoxin quinolinic acid (QUIN). Lipopolysaccharides (LPS) or the pro-inflammatory cytokines INFγ and TNF increased, while IL 4 or IL 10 inhibited the rate of tryptophan metabolism and the release of QUIN.

The incubation media of INFγ-exposed MDM caused neuronal death in primary cultures of mixed cortical cells. Glutamate receptor antagonists or poly(ADP-ribose) polymerase inhibitors significantly reduced this death, thus suggesting new possibilities for the treatment of neuronal damage in neuroinflammatory disorders.     

Interactions between HIV-infected monocyte-derived macrophages and human brain microvascular endothelial cells result in increased expression of CC chemokines

The presence of perivascular monocytic infiltration is a major hallmark of HIV-1-associated dementia. Since CC chemokines are chemoattractant cytokines that are able to attract T cells and monocytes/macrophages to sites of inflammation, and since infiltrating monocytes/macrophages remain in close contact with the brain endothelium, we investigated whether interactions between HIV-1-infected macrophages and brain endothelium result in an altered chemokine production. We found an increased mRNA expression of monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, and RANTES by macrophages after HIV-1 infection. Interactions between HIV-infected macrophages and brain microvascular endothelial cells resulted in an additional upregulation of chemokine mRNA expression, during cell-cell contact as well as in a trans-well system. Since IL-1 beta can function as a modulator of chemokine expression we investigated if interleukin-1 beta could be involved in the regulation of chemokine induction. Coculturing of HIV-infected macrophages and endothelial cells resulted in immune-activation as indicated by increased mRNA expression of IL-1 beta. Subsequently, addition of a neutralizing antibody against IL-1 beta resulted in altered chemokine expression by macrophages, but not by endothelial cells. Thus, IL-1 beta appears to play a major role in the regulation of chemokines during cellular interactions in HIV-associated dementia, but other factors may also be involved.     

Proteomic fingerprinting of HIV-1-infected human monocyte-derived macrophages: a preliminary report

Mononuclear phagocytes (MP; blood monocytes, alveolar, lymph node, and brain macrophages and microglia) are vehicles for dissemination and principle target cells for human immunodeficiency virus type 1 (HIV-1) infection. Notably, viral persistence in macrophages occurs despite ongoing phagocytic, intracellular killing, innate and adaptive immune responses. To assess potential pathways for how HIV-1 may bypass antiviral MP responses, we used proteomic tests to evaluate protein fingerprints of HIV-1-infected human monocyte-derived macrophages 7 days after viral infection. By using weak cation exchange chips, 58 proteins were found up- or down-regulated after HIV-1(ADA) infection. Several of these proteins were identified by microsequencing. It is probable that cellular proteins identified by proteomic fingerprinting could assist in unraveling how persistent viral infection occurs in MP lineage cells. Moreover, this evolving technology can be utilized to unravel changes in immune activities initiated by interactions between virus, environmental cues and drugs of abuse.     

Differences in kinetics of human cytomegalovirus cell-free viral release after in vitro infection of human microglial cells, astrocytes and monocyte-derived macrophages.

Microglial cells and astrocytes isolated from human embryonic proencephalon were compared to monocyte-derived macrophages (MDM) for their ability to replicate human cytomegalovirus (HCMV) in vitro. A specific cytopathic effect was observed in microglial cells and astrocytes, but not in MDM. A high percentage of glial cells but a low percentage of MDM expressed immediate-early and late viral antigens. The ability of HCMV-infected microglial cells and astrocytes to release viral particles in their supernatants was significantly higher than that of infected MDM. Human microglial cells and astrocytes at an early stage of development are highly susceptible to HCMV infection.     

The nature of macrophages (foam cells) in neurinomas. Tissue culture study

Summary Fourteen cases of neurinomas of variable location are studied by tissue culture technic in an attempt to typify the foam cells as primary or superimposed elements in the tumor population. Our results demonstrate that the in vitro behavior of the neurinomas is constant and characteristic and that three cell types are found in them: fusiform, star-shaped cells and macrophages. There appears that the macrophage is an evolutive aspect of the star-shaped cells and probably of the fusiform one. On this basis, macrophage and foam cells of neurinomas must be considered as primary.     

HIV-1 infected monocyte-derived macrophages affect the human brain microvascular endothelial cell proteome: new insights into blood-brain barrier dysfunction for HIV-1-associated dementia

Blood-brain barrier (BBB) compromise and transendothelial migration of HIV-infected leukocytes into the central nervous system (CNS) underlies the neuropathogenesis of HIV-1 infection. How this occurs is incompletely understood. We used a proteomic platform integrating difference gel electrophoresis and tandem mass spectrometry peptide sequencing to determine the effects that HIV-1-infected macrophages have on human brain microvascular endothelial cell (HBMEC) protein profiles. HIV-1 infected monocyte-derived macrophages (MDM) induced the upregulation of over 200 HBMEC proteins. These included metabolic, voltage-gated ion channels, heat shock, transport, cytoskeletal, regulatory, and calcium binding proteins. Results were validated by Western blot analysis. We conclude that HIV-1-infected MDM affect the HBMEC proteome and, in this way, affect BBB dysfunction and the development of HIV-1 CNS disease.     

Proteomic fingerprinting of human immunodeficiency virus type 1-associated dementia from patient monocyte-derived macrophages: A case study

The emergence of a subset of circulating monocytes during human immunodeficiency virus type 1 (HIV-1) disease has been shown to correlate with cognitive impairment. Thus, it is hypothesized that diagnostic protein profiles may be obtained from these cells from patients with or at risk for HIV-1-associated dementia (HAD). To address this possibility, we used ProteinChip assays to define a unique monocyte-derived macrophage (MDM) protein fingerprint during HAD and whether it is affected by highly active antiretroviral therapy (HAART). The study included five Hispanic women, one with HAD, two HIV-1-infected without cognitive impairment, and two seronegative controls. All patients were matched by age and immune status. Monocytes were recovered from the peripheral blood leukocytes by Percoll gradient centrifugation and allowed to differentiate in vitro for 7 days. Cell lysates and supernatants were collected from the MDM and analyzed by surface enhanced laser desorption/ionization-time of flight ProteinChip assays. Seven unique protein peaks between 3.0 and 20.0 kDa were found in the HAD MDM sample. Each of these proteins were abrogated after HAART. Additional studies extending this one time point determination would serve to confirm the general utility of MDM protein profiling for the diagnosis and monitoring of HAD.     

Proteomic fingerprinting of human immunodeficiency virus type 1-associated dementia from patient monocyte-derived macrophages: A case study

The emergence of a subset of circulating monocytes during human immunodeficiency virus type 1 (HIV-1) disease has been shown to correlate with cognitive impairment. Thus, it is hypothesized that diagnostic protein profiles may be obtained from these cells from patients with or at risk for HIV-1-associated dementia (HAD). To address this possibility, we used ProteinChip assays to define a unique monocyte-derived macrophage (MDM) protein fingerprint during HAD and whether it is affected by highly active antiretroviral therapy (HAART). The study included five Hispanic women, one with HAD, two HIV-1-infected without cognitive impairment, and two seronegative controls. All patients were matched by age and immune status. Monocytes were recovered from the peripheral blood leukocytes by Percoll gradient centrifugation and allowed to differentiate in vitro for 7 days. Cell lysates and supernatants were collected from the MDM and analyzed by surface enhanced laser desorption/ionization-time of flight ProteinChip assays. Seven unique protein peaks between 3.0 and 20.0 kDa were found in the HAD MDM sample. Each of these proteins were abrogated after HAART. Additional studies extending this one time point determination would serve to confirm the general utility of MDM protein profiling for the diagnosis and monitoring of HAD.     

Increased level of oxidized LDL-dependent monocyte-derived microparticles in acute coronary syndrome

We measured and compared the levels of plasma soluble (s) P-selectin, sCD40L, platelet-derived microparticles (PDMP), monocyte-derived microparticles (MDMP), and anti-oxidized LDL antibody, to obtain a better understanding of their potential contribution to vascular complications in acute coronary syndrome (ACS). The concentrations of sP-selectin, sCD40L, PDMP, and MDMP in ACS patients were significantly higher than those in normal controls and patients with stable angina. When levels of these markers were compared with differences in concentration of anti-oxidized LDL antibody, all markers were significantly higher in ACS patients with a high level of anti-oxidized LDL antibody. Next, a monocytic cell line (THP-1) was incubated with high shear stress-induced platelet aggregates and PDMP. After incubation,THP-1 cells generated tissue factor-expressing MDMPs. This finding was particularly significant in the presence of oxidized LDL. These findings suggest that elevated levels of MDMPs may be a sign of atherosclerotic development in ACS patients, particularly those who exhibit anti-oxidized LDL antibodies.     

以泡沫细胞浸润为特点的炎性肌肉病伴随大量巨噬细胞一例

目的 报道1例以泡沫细胞浸润为特点的炎性肌肉病伴随大量巨噬细胞的临床和病理特点.方法 患者男性,44岁,因“双上肢无力13个月,加重伴双下肢无力11个月”于2010年6月就诊于我院门诊,病程后期对免疫抑制治疗效果差,既往有类风湿性关节炎病史.肌酸激酶(CK)轻度升高,多种肌炎相关抗体和副肿瘤综合征抗体均为阴性.肌电图示肌源性损害,静止时有强直发放.先后对该患者进行左、右肱二头肌活体组织检查,标本进行组织学、酶组织化学染色和免疫组织化学染色.结果 第1次肌肉活体组织检查显示个别肌纤维坏死和再生,以及CD8阳性T淋巴细胞浸润肌内衣和非坏死肌纤维,肌纤维膜主要组织相容性复合物(MHC) -Ⅰ阳性.第2次肌肉活体组织检查显示束周肌纤维变性,肌束衣的纤维结缔组织呈碎片状改变,可见大量CD6s阳性的泡沫细胞和Touton多核巨细胞浸润.在个别血管周围可见CD20阳性B淋巴细胞和浆细胞,肌内衣中见到散在CDs阳性的T淋巴细胞,MHC-Ⅰ肌纤维膜阳性表达.结论 炎性肌肉病伴随大量巨噬细胞可以表现为肌内衣大量泡沫细胞的浸润,该病可以伴随类风湿性关节炎并对糖皮质激素抵抗.     

HIV-1 and methamphetamine alter galectins -1, -3, and -9 in human monocyte-derived macrophages

Journal of NeuroVirology - Macrophages are key elements of the innate immune system. Their HIV-1 infection is a complex process that involves multiple interacting factors and various steps and is...     

Physiology of transformed glial cells

Much of our present knowledge of glial cell function stems from studies of glioma cell lines, both rodent (C6, C6 polyploid, and TR33B) and human (1321N1, 138MG, D384, R-111, T67, Tp-301MG, Tp-483MG, Tp-378MG, U-118MG, U-251MG, U-373MG, U-787MG, U-1242MG, and UC-11MG). New methods such as patch clamp and Ca²⁺ imaging have lead to rapid progress the last few years in our knowledge about glial cells, where an unexpected presence and diversity of receptors and ion channels have emerged. Basic mechanisms related to membrane potential and K⁺ transport and the presence of voltage gated ion channels (Na⁺, inwardly rectifying K⁺, Ca²⁺ activated K⁺, Ca²⁺, and Cl^? channels) have been identified. Receptor function and intracellular signaling for glutamate, acetylcholine, histamine, serotonin, cathecolamines, and a large number of neuropeptides (bradykinin, cholecystokinin, endothelin, opioids, and tachykinins) have been characterized. Such studies are facilitated in cell lines which offer a more homogenous material than primary cultures. Although the expression of ion channels and receptors vary considerably between different cell lines and comparative studies are rare, a few differences (compared to astrocytes in primary culture) have been identified which may turn out to be characteristic for glioma cells. Future identification of specific markers for receptors on glial and glioma cells related to cell type and growth properties may have great potential in clinical diagnosis and therapy. © 1995 Wiley-Liss, Inc.     

Differentiation of human neural stem cells into retinal cells

We have previously reported that transplanted human neural stem cells (HNSCs) display extensive migration and positional incorporation into the aged rat brain, which is associated with an improvement in cognitive function. In the current study, to investigate whether HNSCs are capable of differentiating into retinal cells, we treated HNSCs with human transforming growth factor-beta3 (TGF-beta3) under a serum-free differentiation condition. After 5 days of differentiation in vitro we detected opsin-immunopositive cells in the culture treated with TGF-beta3. We also transplanted TGF-beta3-treated HNSCs into the rat vitreous cavity. The donor cells migrated and differentiated into opsin-positive cells in the host retinal cell layer. Here we show for the first time that TGF-beta3-treated HNSCs differentiate into retinal cells.     

Appearance of GFAP-positive cells in adult human brain cultures spontaneously decelerated in growth

Cell cultures were derived from adult human brain biopsies [from cortical gray (cultures 9-HB-G and 33-HB-G) and white (culture 14-HB-W) and stroke-injured white matter (culture 33-HB-IW)]. The morphology and growth rate of cultured cells were examined and correlated with the presence of vimentin and glial fibrillary acidic protein (GFAP). The cultures from various brain matters differed in cell morphology and rate of growth but not in GFAP and vimentin staining. Cells of primary and rapidly proliferating cultures were GFAP-negative and vimentin-positive. Spontaneous growth deceleration occurred in culture 14-HB-W within passages 5 to 10 and in cultures 9-HB-G, 33-HB-G, and 33-HB-W within passages 17 to 20. This deceleration, as well as the successive complete growth arrest, were accompanied by an appearance of GFAP-positive cells and an elevated intensity for vimentin staining. We propose that GFAP-positive astrocytes originate from glial precursor cells that migrate from the explants and differentiate under prolonged subcultivation.     

Proteomic analyses of monocyte-derived macrophages infected with human immunodeficiency virus type 1 primary isolates from Hispanic women with and without cognitive impairment

The signature for human immunodeficiency virus type 1 (HIV-1) neurovirulence remains a subject of intense debate. Macrophage viral tropism is one prerequisite but others, including virus-induced alterations in innate and adaptive immunity, remain under investigation. HIV-1-infected mononuclear phagocytes (MPs; perivascular macrophages and microglia) secrete toxins that affect neurons. The authors hypothesize that neurovirulent HIV-1 variants affect the MP proteome by inducing a signature of neurotoxic proteins and thus affect cognitive function. To test this hypothesis, HIV-1 isolates obtained from peripheral blood of women with normal cognition (NC) were compared to isolates obtained from women with cognitive impairment (CI) and to the laboratory adapted SF162, a spinal fluid R5 isolate from a patient with HIV-1-associated dementia. HIV-1 isolates were used to infect monocyte-derived macrophages (MDMs) and infection monitored by secreted HIV-1 p24 by enzyme-linked immunosorbent assay (ELISA). Cell lysates of uninfected and HIV-1infected MDMs at 14 days post infection were fractionated by cationic exchange chromatography and analyzed by surface enhanced laser desorption ionization time of flight (SELDI-TOF) using generalized estimating equations statistics. Proteins were separated by one-dimensional sodium dodecyl sulfatepolyacrylamide gel electrophoresis (1D SDS-PAGE) and identified by tandem mass spectrometry. Levels of viral replication were similar amongst the HIV-1 isolates, although higher levels were obtained from one viral strain obtained from a patient with CI. Significant differences were found in protein profiles between virus-infected MDMs with NC, CI, and SF162 isolates (adjusted P value after multiple testing corrections, or q value <.10). The authors identified 6 unique proteins in NC, 7 in SF162, and 20 in CI. Three proteins were common to SF162 and CI strains. The MDM proteins linked to infection with CI strains were related to apoptosis, chemotaxis, inflammation, and redox metabolism. These findings support the hypothesis that the macrophage proteome differ when infected with viral isolates of women with and without CI.