Heterogeneity in mantle carbon content from CO2-undersaturated basalts.

The amount of carbon present in Earth's mantle affects the dynamics of melting, volcanic eruption style and the evolution of Earth's atmosphere via planetary outgassing. Mantle carbon concentrations are difficult to quantify because most magmas are strongly degassed upon eruption. Here we report undegassed carbon concentrations from a new set of olivine-hosted melt inclusions from the Mid-Atlantic Ridge. We use the correlations of CO2 with trace elements to define an average carbon abundance for the upper mantle. Our results indicate that the upper mantle carbon content is highly heterogeneous, varying by almost two orders of magnitude globally, with the potential to produce large geographic variations in melt fraction below the volatile-free solidus. Such heterogeneity will manifest as variations in the depths at which melt becomes interconnected and detectable, the CO2 fluxes at mid-ocean ridges, the depth of the lithosphere-asthenosphere boundary, and mantle conductivity.

Analysis of gastrointestinal physiology using a novel intestinal transit assay in zebrafish.

Gastrointestinal function depends upon coordinated contractions to mix and propel food through the gut. Deregulation of these contractions leads to alterations in the speed of material transit through the gut, with potentially significant consequences. We have developed a method for visualizing intestinal transit, the physiological result of peristaltic contractions, in larval zebrafish. This method allows direct, non-invasive observation of luminal content as it traverses the gut. Using this method, we characterized gastrointestinal transit in zebrafish larvae at 7 days postfertilization. In addition, we used this transit assay to assess the physiological consequences of reduced or absent enteric neurones on intestinal transit in larval zebrafish. This may facilitate the use of the zebrafish for investigating the effect of compounds and candidate genes on gastrointestinal motility.

Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo.

Adeno-associated virus 2 (AAV) vectors are currently in use in Phase I/II clinical trials for gene therapy of cystic fibrosis and hemophilia B. Although 100% of murine hepatocytes can be targeted by AAV vectors, the transgene expression is limited to approximately 5% of hepatocytes. Since the viral genome is a single-stranded DNA, and single strands of both polarities are encapsidated with equal frequency, it has been suggested that failure to undergo DNA strand-annealing accounts for the lack of efficient transgene expression. We and others, on the other hand, have proposed that failure to undergo viral second-strand DNA synthesis attributes to the observed low efficiency of transgene expression. We have previously documented that a cellular protein, designated FKBP52, when present in phosphorylated forms, inhibits the viral second-strand DNA synthesis, and consequently, limits transgene expression in nonhepatic cells, whereas unphosphorylated forms of FKBP52 have no effect. To further evaluate whether phosphorylated FKBP52 is also involved in regulating AAV-mediated transgene expression in murine hepatocytes, we generated transgenic mice overexpressing the cellular T-cell protein tyrosine phosphatase (TC-PTP) protein, known to catalyze dephosphorylation of FKBP52, as well as mice deficient in FKBP52. We demonstrate here that dephosphorylation of FKBP52 in TC-PTP transgenic (TC-PTP-TG) mice, and removal of FKBP52 in FKBP52-knockout (FKBP52-KO) mice results in efficient transduction of murine hepatocytes following tail-vein injection of recombinant AAV vectors. We also document efficient viral second-strand DNA synthesis in hepatocytes from both TC-PTP-TG and FKBP52-KO mice. Thus, our data strongly support the contention that the viral second-strand DNA synthesis, rather than DNA strand-annealing, is the rate-limiting step in the efficient transduction of hepatocytes, which should have implications in the optimal use of recombinant AAV vectors in human gene therapy.

Testing the role of the cell-surface molecule Thy-1 in regeneration and plasticity of connectivity in the CNS.

Thy-1 is a cell-surface signaling molecule of the Ig superfamily implicated in the regulation of neurite outgrowth, synaptic function and plasticity. There is, however, no consensus as to its precise function in the nervous system, and it remains unclear or untested as to what its role is in the development, maintenance and plasticity of neuronal connectivity in the intact brain and whether it is essential for any of the purported functions which have been attributed to it based largely on in vitro bioassays. Here, we have engineered transgenic mice with a targeted deletion of the Thy-1 gene and, after characterizing the development of their corticospinal and thalamocortical pathways, subjected them at adulthood to paradigms of axonal regeneration and plasticity which can be readily induced during development. Quantitative analyses of the brains and spinal cords of adult null mutants showed normal cellular organization, normal anatomical features of the corticospinal and thalamocortical pathways, and basic neurophysiological properties of thalamocortical synaptic transmission which were quantitatively indistinguishable from wild-type mice. Despite the absence of Thy-1, corticospinal axons in adult mutants failed to exhibit overt regeneration following spinal cord lesion; likewise, the terminal arbors of ventrobasal thalamocortical axons also failed to reorganize in adult barrel cortex in response to whisker cautery, although they did so during a developmental critical period identical to that displayed by wild-type mice.Taken together, these results suggest that Thy-1 is not essential for the normal development and maintenance of major axon pathways and functional synaptic connections, nor would it appear to be critically important for inhibiting or promoting axonal growth, regeneration and plasticity in the developing and mature CNS.

Adeno-associated virus type 2-mediated gene transfer: role of cellular FKBP52 protein in transgene expression.

Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful vector for human gene therapy, the transduction efficiencies of AAV vectors vary greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays a crucial role in AAV-mediated transgene expression (K. Y. Qing, X.-S. Wang, D. M. Kube, S. Ponnazhagan, A. Bajpai, and A. Srivastava, Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997). We have documented a strong correlation between the phosphorylation state of ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing, B. Khuntrirat, C. Mah, D. M. Kube, X.-S. Wang, S. Ponnazhagan, S. Z. Zhou, V. J. Dwarki, M. C. Yoder, and A. Srivastava, J. Virol. 72:1593-1599, 1998). We have also established that the ssD-BP is phosphorylated by epidermal growth factor receptor protein tyrosine kinase and that the tyrosine-phosphorylated form, but not the dephosphorylated form, of ssD-BP prevents AAV second-strand DNA synthesis and, consequently, results in a significant inhibition of AAV-mediated transgene expression (C. Mah, K. Y. Qing, B. Khuntrirat, S. Ponnazhagan, X.-S. Wang, D. M. Kube, M. C. Yoder, and A. Srivastava, J. Virol. 72:9835-9841, 1998). Here, we report that a partial amino acid sequence of ssD-BP purified from HeLa cells is identical to a portion of a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein 52 (FKBP52). FKBP52 was purified by using a prokaryotic expression plasmid containing the human cDNA. The purified protein could be phosphorylated at both tyrosine and serine or threonine residues, and only the phosphorylated forms of FKBP52 were shown to interact with the AAV single-stranded D-sequence probe. Furthermore, in in vitro DNA replication assays, tyrosine-phosphorylated FKBP52 inhibited AAV second-strand DNA synthesis by greater than 90%. Serine- or threonine-phosphorylated FKBP52 caused approximately 40% inhibition, whereas dephosphorylated FKBP52 had no effect on AAV second-strand DNA synthesis. Deliberate overexpression of FKBP52 effectively reduced the extent of tyrosine phosphorylation of the protein, resulting in a significant increase in AAV-mediated transgene expression in human and murine cell lines. These studies corroborate the idea that the phosphorylation status of the cellular FKBP52 protein correlates strongly with AAV transduction efficiency, which may have important implications for the optimal use of AAV vectors in human gene therapy.

Recombinant human parvovirus B19 vectors: erythrocyte P antigen is necessary but not sufficient for successful transduction of human hematopoietic cells.

The blood group P antigen, known to be abundantly expressed on erythroid cells, has been reported to be the cellular receptor for parvovirus B19. We have described the development of recombinant parvovirus B19 vectors with which high-efficiency, erythroid lineage-restricted transduction can be achieved (S. Ponnazhagan, K. A. Weigel, S. P. Raikwar, P. Mukherjee, M. C. Yoder, and A. Srivastava, J. Virol. 72:5224-5230, 1998). However, since a low-level transduction of nonerythroid cells could also be detected and since P antigen is expressed in nonerythroid cells, we reevaluated the role of P antigen in the viral binding and entry into cells. Cell surface expression analyses revealed that approximately 75% of primary human bone marrow mononuclear erythroid cells and approximately 31% of cells in the nonerythroid population were positive for P antigen. Two human erythroleukemia cell lines, HEL and K562, and a human promyelocytic leukemia cell line, HL-60, were also examined for P antigen expression and binding and entry of the vector. HEL and K562 cells showed intermediate levels, whereas HL-60 cells demonstrated high levels of expression of P antigen. However, the efficiency of vector binding to these cells did not correlate with P antigen expression. Moreover, despite P antigen positivity and efficient viral binding, HEL, K562, and HL-60 cells could not be transduced with the vector. Low levels of P antigen expression could also be detected in two primary cell types, human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblasts (NHLF). In addition, vector binding occurred in both cell types and was inhibited by globoside, indicating the involvement of P antigen in virus binding to these cells. These primary cells could be efficiently transduced with the recombinant vector. These data suggest that (i) P antigen is expressed on a variety of cell types and is involved in binding of parvovirus B19 to human cells, (ii) the level of P antigen expression does not correlate with the efficiency of viral binding, (iii) P antigen is necessary but not sufficient for parvovirus B19 entry into cells, and (iv) parvovirus B19 vectors can be used to transduce HUVEC and NHLF. These studies further suggest the existence of a putative cellular coreceptor for efficient entry of parvovirus B19 into human cells.

Aerobic exercise and resting blood pressure: a meta-analytic review of randomized, controlled trials.

In this study the authors used the meta-analytic approach to examine the effects of aerobic exercise on resting systolic and diastolic blood pressure in adults. Forty-seven clinical trials representing a total of 72 effect sizes in 2543 subjects (1653 exercise, 890 control) met the criteria for inclusion. Statistically significant exercise-minus-control decreases were found for changes in resting systolic and diastolic blood pressure in both hypertensive (systolic, -6 mm Hg, 95% CI, -8 to -3; diastolic, -5 mm Hg, 95% CI, -7 to -3) and normotensive (systolic, -2 mm Hg, 95% CI, -3 to -1; diastolic, -1 mm Hg, 95% CI, -2 to -1) groups. The differences between groups were statistically significant (systolic, p=0.008; diastolic, p=0.000). Relative decreases were approximately 4% (systolic) and 5% (diastolic) in hypertensives, and 2% (systolic) and 1% (diastolic) in normotensives. It was concluded that aerobic exercise reduces resting systolic and diastolic blood pressure in adults. (c) 2001 by CHF, Inc.

The evolution of lipophilin genes from invertebrates to tetrapods: DM-20 cannot replace proteolipid protein in CNS myelin.

The proteolipid protein (PLP) gene encodes two myelin-specific protein isoforms, DM-20 and PLP, which are members of the highly conserved lipophilin family of transmembrane proteins. While the functions of this family are poorly understood, the fact that null mutations of the PLP gene cause leukodystrophy in man is testament to the importance of DM-20 and PLP in normal CNS function. PLP differs from DM-20 by the presence of a 35 amino acid domain exposed to the cytoplasm, which is not encoded by other lipophilin genes and appears to have arisen in amphibians approximately 300 million years before present. However, the lipophilin gene family can be traced back at least 550 million years and is represented in Drosophila and silkworms. Thus, from an evolutionary perspective PLP can reasonably be anticipated to perform functions in CNS myelin that cannot be accomplished by other lipophilins. Herein we use a novel knock-in strategy to generate mice expressing wild-type levels of a Plp gene that has been modified to encode only DM-20. Although DM-20 is incorporated into functional compact myelin sheaths in young animals, our data show that the 35 amino acid PLP-specific peptide is required to engender the normal myelin period and to confer long-term stability on this multilamellar membrane.

Potentiation of excitotoxicity in transgenic mice overexpressing neuronal cyclooxygenase-2.

In this study we describe the generation of a transgenic mouse model with neuronal overexpression of the human cyclooxygenase-2, h(COX)-2, to explore its role in excitotoxicity. We report that overexpression of neuronal hCOX-2 potentiates the intensity and lethality of kainic acid excitotoxicity in coincidence with potentiation of expression of the immediate early genes c-fos and zif-268. In vitro studies extended the in vivo findings and revealed that glutamate excitotoxicity is potentiated in primary cortico-hippocampal neurons derived from hCOX-2 transgenic mice, possibly through potentiation of mitochondrial impairment. This study is the first to demonstrate a cause-effect relationship between neuronal COX-2 expression and excitotoxicity. This model system will allow the systematic examination of the role of COX-2 in mechanisms of neurodegeneration that involve excitatory amino acid pathways.

Targeted deletion of the Vgf gene indicates that the encoded secretory peptide precursor plays a novel role in the regulation of energy balance.

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

Paradoxical locomotor behavior of dopamine D1 receptor transgenic mice.

The behavioral effects of augmenting dopamine D1 receptor expression in the brain were investigated in mice incorporating additional copies of the mouse D1 receptor gene. Two transgenic lines showed increases in brain D1 receptor binding sites, which were greatest in extrastriatal regions. The full D1 agonist SKF 81297, when administered systemically to control animals, stimulated a dose-dependent increase in locomotor activity. In contrast, in D1 receptor overexpressing transgenic mice, this drug caused a marked suppression of locomotion due to a decrease in the frequency of movement initiation. Amphetamine and cocaine induced comparable locomotor activation in both transgenic animals and their control littermates. In the transgenic animals, D1 agonist-induced rearing and climbing behaviors were suppressed. However, on rotarod testing, the agonist-treated transgenic and control mice performed comparably, indicating that sensorimotor coordination was unaffected. These studies demonstrate that altering the levels of D1 receptor expression reverses the effects of D1 agonism on locomotor initiation and rearing.

Role of the glucagon receptor COOH-terminal domain in glucagon-mediated signaling and receptor internalization.

The binding of glucagon to its hepatic receptor is known to result in a number of effects, including the intracellular accumulation of cAMP, the mobilization of intracellular Ca2+, and the endocytosis of glucagon and its receptor into intracellular vesicles. In this study, we begin to define the functional role of the COOH-terminal tail of the human glucagon receptor in glucagon-stimulated signal transduction and receptor internalization. We have created and expressed in Chinese hamster ovary (CHO) cells five truncation mutants in which the COOH-terminal 24, 56, 62, 67, and 73 amino acids have been removed. Cells expressing relevant truncated receptors were assayed for cell surface expression by immunofluorescence, for ligand-binding properties, for cAMP and Ca2+-mediated signal transduction properties, and for receptor endocytosis. In addition, a mutant receptor containing seven serine-to-alanine mutations in the COOH-terminal tail was studied. Our results reveal the following: 1) a region of the COOH-terminal tail that is required for proper cell surface expression, 2) the COOH-terminal 62 amino acids, which comprise the majority of the tail, are not required for ligand binding, cAMP accumulation, or Ca2+ mobilization, and 3) phosphorylation of the COOH-terminal tail is crucial for glucagon-stimulated receptor endocytosis.

Contrast of survey results between state and a cohort of nonstate mycobacteriology laboratories: changes in laboratory practices.

Based on the recommendations of a 1992 conference on tuberculosis, the Centers for Disease Control and Prevention (CDC) established programs for upgrading mycobacteriology laboratories by providing them with monies and focused training. In 1991, state public health laboratories were surveyed to determine the methods they were using for primary Mycobacterium tuberculosis testing and their turnaround times for reporting testing results. A similar survey of nonstate laboratories participating in the National Laboratory Training Network-sponsored, M. tuberculosis-focused training programs was conducted from May 1992 to June 1993. In 1994, follow-up surveys of both the state- and nonstate-laboratory cohorts were conducted with the questionnaire from the initial survey plus additional questions that asked about interventions and changes occurring in the laboratory since the original survey. Although both cohorts showed increases in the percentages of laboratories meeting the recommended turnaround times for reporting M. tuberculosis testing results and using the recommended rapid methods for testing, generally, the increases made by the state laboratories were greater. By June 1994, all state laboratories were using a rapid method for M. tuberculosis isolate identification compared with 88% of the nonstate laboratories. The percentage of laboratories identifying isolates within the recommended 21 days also increased more in the group of state laboratories than in the group of nonstate laboratories (state laboratories, 22 to 73%; nonstate laboratories, 55 to 59%). Responses from the follow-up survey showed large differences in the percentages of laboratories that received CDC funding (state laboratories, 100%; nonstate laboratories, 6%) and participated in M. tuberculosis training (state laboratories, 98%; nonstate laboratories, 45%). These results indicate that adequate funding and focused training are critical in maintaining state-of-the-art mycobacteriology laboratories.

Expression of Thy-1/lacZ fusion genes in the CNS of transgenic mice.

Thy-1 is a cell surface glycoprotein of unknown function that is found on nerve cells and mature T-lymphocytes. To study the regulation of Thy-1 gene expression, mouse Thy-1.2 genomic sequences were joined to various marker sequences and the resulting chimeric constructs were used to produce nearly three dozen independent lines of transgenic mice. The starting point for our studies was an 8.2 kb EcoRI fragment that begins 1.7 kb 5' to the transcription start site and ends with 1.3 kb of 3' flanking sequences. Addition of a small marker oligonucleotide to the 3' untranslated region of this fragment had little or no effect on gene regulation. All of the lines derived from injection of this construct expressed the transgene in the appropriate tissues. Thus, as expected, the Thy-1.2 genomic fragment contains all of the information necessary for tissue-specific, position-independent expression of the modified transgene. Unexpectedly, Thy-1/lacZ hybrid genes did not mimic this behavior. Using either mRNA or histochemical detection of lacZ protein, these constructs were expressed in patterns that varied dramatically from line to line. This behavior suggests that integration site-specific effects dominate the cis-active Thy-1 regulatory elements leading to wide variability of expression. This is further emphasized by the observation that the bacterial reporter protein was found in a few non-neuronal cell-types, in contrast to the known pattern of native Thy-1 expression. These results suggest that either the Thy-1.2 sequences which are necessary for appropriate brain-specific expression are not contained solely within the proposed CNS enhancer in the first intron, or that fusion of the Thy-1.2 sequences with the lacZ coding region may disrupt normal Thy-1 regulatory signals (or result in the creation of new regulatory elements).

Elevation of intracellular cAMP in human T lymphocytes by an anti-CD44 mAb.

Regulation of lymphocyte responses to activation of the CD3/TCR complex by other cell surface proteins expressed on lymphocytes is a well established phenomenon. CD44 is an example of such a cell surface protein. Anti-CD44 mAb have been identified which either stimulate or inhibit lymphocyte function. Certain anti-CD44 mAb augment proliferation and IL-2 production by T cells stimulated through the CD2 or CD3/TCR pathways. An anti-CD44 mAb with opposing properties has also been identified. This mAb inhibits activation of human T cells by preventing the rise in [Ca2+]i stimulated by OKT3. The purpose of experiments reported here was to further characterize this phenomenon. The results show that the anti-CD44 mAb, 212.3, does not inhibit inositol phosphate turnover stimulated by OKT3 but does inhibit elevation of intracellular [Ca2+]i in these same cells. Addition of 212.3 to purified human T cells results in a rapid increase in intracellular levels of cAMP. Elevation of cAMP by 212.3 is time- and concentration-dependent. Activation of adenylate cyclase by forskolin also results in elevation of intracellular cAMP and inhibition of the increase in [Ca2+]i stimulated by OKT3. Taken together, these data suggest that CD44 may be positively coupled to adenylate cyclase and that activation of adenylate cyclase by the anti-CD44 mAb, 212.3, may mediate the inhibition of the OKT3-stimulated elevation of [Ca2+]i.

Intracellular distribution of transgenic bacterial beta-galactosidase in central nervous system neurons and neuroglia.

Bacterial beta-galactosidase is widely used as a marker for gene expression and in cell tracing experiments. In a survey of three transgenic mouse lines expressing beta-galactosidase in the central nervous system (CNS) under the control of different promoters, we find substantial variation in the intracellular distribution of the lacZ protein. In line M beta P5, transgene beta-galactosidase expression is driven by a promoter/enhancer fragment from the oligodendrocyte-specific myelin basic protein gene; however, electron microscopy of histochemically stained preparations reveals transgene expression not only in oligodendrocytes but also in some neurons. Immunofluorescence and immunoperoxidase staining show the beta-galactosidase protein distributed throughout the perikaryal cytoplasm of oligodendrocytes and in processes reaching to myelin sheaths. By contrast, immunoreactive protein appears restricted in neurons to one or a few small perikaryal immunoreactive granules. The granules are visible in the electron microscope as amorphous inclusion bodies of moderate electron density and lack a limiting membrane. Histochemical staining patterns with X-gal and Bluo-gal echoed the protein distribution: diffuse distribution of enzyme protein yielded cells filled with substrate, while punctate enzyme distribution yielded restricted or punctate histochemical staining. Examination of two other lines using different promoter/enhancers to drive expression in the CNS showed both diffuse and punctate beta-galactosidase immunolocalization and histochemical staining. The amount of protein synthesized or other properties, yet unidentified, intrinsic to the target cells may determine the intracellular distribution of beta-galactosidase. In retroviral marking studies, clone members have been identified as those cells filled with X-gal reaction product. This approach may underestimate both clone size and the minimum number of divisions separating the members of each clone.

Cellular signaling responses mediated by a novel nucleotide receptor in rabbit microvessel endothelium.

The adenine nucleotide, ATP, elicits an elevation in intracellular ionized calcium concentration ([Ca2+]i) and phospholipase C-mediated phosphatidylinositol hydrolysis and stimulates the synthesis of the prostaglandins E2 and I2 in cultured endothelial cells derived from rabbit cardiac muscle. Use of various ATP analogues indicated that these events did not fit the classical definition of P1 or P2 purinergic receptors and, furthermore, indicated that the receptor(s) mediating these activities was not specific for purines. The rank order of agonist potency on prostaglandin release, elevations in [Ca2+]i, and inositol phosphate response was UTP > or = ATP > ADP > ADP[beta]S = 2-methylthio ATP > adenosine, suggesting that these three cellular responses are coupled to the same or similar receptors. However, the sensitivity of these three cellular responses to added nucleotides was somewhat different. The half-maximum effective concentration (EC50) for ATP stimulation of prostaglandin release was 100 microM, for inositol phosphate turnover it was 25 microM, and for elevations in [Ca2+]i it was < 1 microM. Similar discrepancies in EC50 UTP values for these three cellular responses were also noted. These observations indicate that purine and pyrimidine nucleotides elicit at least three cellular responses in rabbit cardiac muscle microvessel endothelial cells, all demonstrating similar rank orders of potency. However, the differences in EC50 suggest that if these responses are mediated by a single receptor type, it exhibits divergent coupling to various cellular signaling pathways.

Expression of 5HT1a receptors on activated human T cells. Regulation of cyclic AMP levels and T cell proliferation by 5-hydroxytryptamine.

The neurotransmitter, serotonin (5-hydroxytryptamine, 5HT), has been shown to affect function of cells of the immune system. More recently, specific 5HT receptors have been identified and partially characterized on Jurkat cells. Results presented here characterize the receptor on Jurkat cells as the 5HT1a receptor subtype and show that mitogen-activated but not resting human T cells also express the 5HT1a receptor subtype. Analysis of mRNA in Jurkat cells and activated and resting T cells by PCR or by Northern analysis revealed the presence of 5HT1a receptor. Pharmacologic analysis of this receptor demonstrated that the receptors on Jurkat cells and activated T cells are similar to each other and that they resemble the 5HT1a receptor found in the brain. Analysis of the second messenger pathways activated by the 5HT1a receptor show that ligand-binding to the Jurkat cell 5HT1a receptor results in elevation of intracellular inositol phosphates and Ca2+ and that ligand binding to the 5HT1a receptor on activated T cells modulated intracellular levels of cAMP.

Regulation of the expression of intercellular adhesion molecule 1 in cultured human endothelial cells derived from rheumatoid synovium.

OBJECTIVE: To examine the regulation of intercellular adhesion molecule 1 (ICAM-1) in human synovial microvascular endothelial cells (HSE) and human umbilical vein endothelial cells (HUVE) upon exposure to a variety of agents. METHODS: Cultured endothelial cells were treated with various cytokines alone and in combination. The expression of ICAM-1 was evaluated at several levels, including an investigation of messenger RNA (mRNA) and surface protein expression. RESULTS: Treatment of HSE with interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) resulted in minimal increases in ICAM-1 expression, in contrast to findings with HUVE. Incubation of HUVE or HSE with IL-1 or TNF in combination with interferon-gamma (IFN gamma) greatly potentiated the increase in ICAM-1 surface expression. The synergistic effect of IFN gamma and TNF was confirmed by several methods, including a cell-based enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, immunofluorescence staining, and determination of mRNA levels. IFN gamma also augmented the actions of several other agonists on HSE, i.e., IL-4, lipopolysaccharide, and TNF beta/lymphotoxin. Immunoprecipitation of TNF alpha + IFN gamma-stimulated, 125I-labeled HSE cells with anti-ICAM-1 revealed a single 90-kd band, similar in size to ICAM-1 from HUVE treated in an identical manner. Unexpectedly, IFN gamma alone was a potent stimulus for HSE ICAM-1 mRNA synthesis, but was relatively ineffective in HUVE. CONCLUSION: These studies indicate that IFN gamma plays a critical synergistic role in the regulation of ICAM-1 expression in human synovial endothelial cells.

Superoxide anion production and phagocytosis of crystals by cultured endothelial cells.

OBJECTIVE: To show that cultured human umbilical vein endothelial cells (HUVEC) are capable of phagocytizing inflammation-causing crystals and of generating superoxide anion (SOA) during phagocytosis. METHODS: The superoxide dismutase-inhibitable reduction of nitroblue tetrazolium (NBT) dye was used as a measure of SOA production. Phagocytosis was quantified by light microscopy and confirmed by transmission electron microscopy. Cytochrome C was also studied but was found to undergo spontaneous reduction by monosodium urate (MSU) without cells. RESULTS: Crystals of MSU, calcium oxalate, hydroxyapatite, and calcium pyrophosphate dihydrate (CPPD) were phagocytized and, except for the CPPD crystals, induced NBT reduction. Cholesterol and cholesterol monohydrate were neither phagocytized nor did they induce NBT reduction. CONCLUSIONS: Endothelial cells may be a significant source of oxygen radicals in crystal-associated and other arthritides.