The kinetics, function, and regulation of P-selectin expressed by human brain microvessel endothelial cells in primary culture.

P-selectin is an endothelial cell adhesion glycoprotein expressed on the cell surface early in inflammation where it binds to blood leukocytes. This study examines the expression, function, and regulation of P-selectin in primary cultures of human brain microvessel endothelial cells (HBMEC). Surface expression of P-selectin was minimal in unstimulated HBMEC; however, it was significantly augmented upon stimulation with histamine (10(-7)-10(-3) M) and thrombin (0.01-1 U/ml). Expression increased rapidly at 10 min and remained elevated at 60 min. Immunogold electron microscopy showed that histamine (10(-7) M) increased surface expression preferentially on the apical surface of subconfluent monolayers. A cell binding assay showed that the adhesion of polymorphonuclear leukocytes (PMNs) to confluent monolayers was augmented after histamine treatment. Histamine-induced surface expression of P-selectin was blocked by the histamine H2 receptor antagonist cimetidine. The H1 receptor antagonist mepyramine had no effect. Expression was reduced by the extracellular calcium chelator EDTA and blocked by the cyclic AMP phosphodiesterase inhibitor rolipram. Thus histamine and thrombin both increase P-selectin expression in HBMEC. Histamine mediates expression through the H2, but not the H1, receptor and calcium, whereas expression is reduced by cyclic AMP. The histamine-induced expression increases PMN binding to the HBMEC. These data suggest that P-selectin plays a role in the recruitment of acute inflammatory cells to the CNS.

Expression and function of the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) in an in vitro model of the human blood--brain barrier.

The interaction of B7 molecules with their ligand provides important accessory signals for optimal T cell activation and proliferation. In this study the in vitro expression of B7-1 and B7-2 by human brain microvessel endothelial cells (HBMEC) was investigated by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. In addition, the contribution of B7 molecules to T cell proliferation on cerebral endothelial cells was studied by coincubating purified CD4+ T cells with resting or cytokine activated HBMEC. Untreated cultures constitutively expressed B7-2 RNA and surface protein, but lacked B7-1 expression. Treatment with TNF-alpha and IFN-gamma upregulated B7-2 and induced de novo expression of B7-1. Monoclonal blocking antibodies to B7-1 or B7-2 and human CTLA-4Ig chimeric protein significantly reduced the ability of HBMEC to support alpha-CD3-induced proliferation of CD4+ T lymphocytes. Expression of B7 glycoproteins and the ability to provide secondary signals for T cell proliferation suggest a potential role of the human cerebral endothelium in T cell activation during the early stages of central nervous system inflammation.

Expression of the beta-chemokines RANTES and MIP-1 beta by human brain microvessel endothelial cells in primary culture.

The mechanisms that regulate inflammatory cell recruitment across the blood-brain barrier (BBB) during CNS inflammation have not been fully characterized. Likely players in this process include the chemokines, small secondary messengers of inflammation capable of subset-specific leukocyte activation and chemoattraction. Primary cultures of human brain microvessel endothelial cells (HBMEC) were examined for their in vitro expression of the beta chemokines RANTES and MIP-1beta. Untreated HBMEC expressed low levels of RANTES and MIP-1beta RNA that were significantly upregulated following cytokine treatment. Parallel studies performed on human umbilical vein endothelial cells (HUVEC) showed induction of RANTES but not MIP-1beta RNA. Following stimulation with LPS, TNF-alpha, IFN-gamma, and IL-1beta alone or in combination, HBMEC released significant amounts of RANTES and MIP-1beta into the culture supernatants. RANTES secretion by HUVEC could be induced only with TNF-alpha/IFN-gamma. Both RANTES and MIP-1beta were detected by immunocytochemistry on the apical and basal surfaces of HBMEC, as well as bound to basal lamina-like material under the basal cell surface. Cytokine stimulation induced significant increase of RANTES and MIP-1beta molecules associated with the EC surface and subendothelial matrix. The expression of RANTES and MIP-1beta by HBMEC suggests that these chemokines may play an important role in mediating inflammatory responses and leukocyte trafficking across the BBB.

Expression of complement messenger RNAs by human endothelial cells.

This study evaluated complement mRNA expression in human brain microvessel endothelial cells (HBMEC), human umbilical vein endothelial cells (HUVEC), and cells of the human derived ECV304 line. Cerebral endothelial cells and HUVEC expressed detectable levels of complement gene mRNAs for the C1q B-chain, C1r, C1s, C2, C3, C4, C5, C7, C8 gamma-subunit and C9. In addition to C6 mRNA, C1q and C9 were not detected in ECV304 cells. These results indicate that endothelial cells may be a source of complement proteins in brain and other organs of the body.

A1 functions at the mitochondria to delay endothelial apoptosis in response to tumor necrosis factor.

Tumor necrosis factor (TNF) does not cause endothelial apoptosis unless the expression of cytoprotective genes is blocked. We have previously demonstrated that one of the TNF-inducible cytoprotective genes is the Bcl-2 family member, A1. A1 is induced by the action of the transcription factor, NFkappaB, in response to inflammatory mediators. In this report we demonstrate that, as with other cell types, inhibition of NFkappaB initiates microvascular endothelial apoptosis in response to TNF. A1 is able to inhibit this apoptosis over 24 h. We demonstrate that A1 is localized to and functions at the mitochondria. Whereas A1 is able to inhibit mitochondrial depolarization, loss of cytochrome c, cleavage of caspase 9, BID, and poly(ADP-ribose) polymerase, it does not block caspase 8 or caspase 3 cleavage. In contrast, A1 is not able to prevent endothelial apoptosis by TNF over 72 h, when NFkappaB signaling is blocked. On the other hand, the caspase inhibitor, benzyloxycarbonyl-VAD-formylmethyl ketone, completely blocks TNF-induced endothelial apoptosis over 72 h. Our findings indicate that A1 is able to maintain temporary survival of endothelial cells in response to TNF by maintaining mitochondrial viability and function. However, a mitochondria-independent caspase pathway eventually results in endothelial death despite mitochondrial protection by A1.

Characterization of ABCB9, an ATP binding cassette protein associated with lysosomes.

We have cloned full-length human and mouse cDNAs of ABCB9, which encodes a predicted multiple-spanning transmembrane domain and a nucleotide-binding domain with Walker motifs. It is therefore designated as a "half" ATP binding cassette (ABC) transporter. Northern analysis shows that the ABCB9 mRNA is expressed at a high level in testes and moderate levels in brain and spinal cord. A splice variant mRNA deleted in the last pair of predicted transmembrane segments was shown to be expressed in human tissues. Phylogenetic analysis indicates that ABCB9 is closely related to TAP1 and TAP2, two "half" ABC proteins found in endoplasmic reticulum. ABCB9 protein colocalized with the lysosomal markers, LAMP1 and LAMP2, in transfected cells. ABCB9 protein appears to be most highly expressed in the Sertoli cells of the seminiferous tubules in mouse and rat testes. These cells have high levels of phagocytosis and secretory activities. These findings pave the way for further investigation into the potential novel function of ABCB9 in lysosomes.

Blood mononuclear cells in patients with HTLV-I-associated myelopathy: lymphocytes are highly activated and adhesion to endothelial cells is increased.

We have investigated lymphocyte subpopulations and blood mononuclear cell (MNC) adhesion to activated endothelial monolayers in patients with human T lymphotropic virus type I (HTLV-I) associated myelopathy (HAM), in HTLV-I asymptomatic carriers (carriers), and in seronegative controls. HAM patients and carriers had higher levels of CD4(+)CD29(+) "memory cells" than controls (P < 0.05). The percentage of CD3(+)CD27(-) "primed T cell" was elevated in patients with HAM (P < 0.05), but not in carriers. HAM patients had higher levels of CD8(+)CD57(+) "cytotoxic cells" (P < 0.05) than controls and carriers. The percentages of CD4(+) cells coexpressing activation markers HLA-DR and CD25, and of CD8(+) cells expressing HLA-DR, were significantly higher in HAM patients and carriers than in controls. Functional experiments indicated that MNC from HAM patients adhered more to activated endothelial monolayers than MNC from carriers or controls. Blocking studies demonstrated that the adhesion molecules VLA-4 and ICAM-1 and also L-selectin all contributed to increased binding. Analysis of expression of molecules involved in adhesion indicated that in HAM patients, L-selectin (CD62L) expression on CD4(+) and CD8(+) subsets was lower than in controls. Interestingly, HAM patients had a lower percentage of CD4(+) subsets expressing L-selectin than carriers (P < 0.05). In contrast, the percentage of CD4(+) and CD8(+) cells expressing VLA-4 (CD49d) was found to be higher in both HAM patients and carriers compared with controls. After 2 days in culture without mitogen, the percentage of T cells expressing ICAM-1 (CD54) increased in culture in carriers and more profoundly in HAM, but not in controls (P < 0. 05). After culture, T cells expressing the early activation antigen CD69 were also increased in HAM and carriers (P < 0.05) but not in controls. Interestingly, the levels of CD8(+) cells coexpressing activation antigen HLA-DR and CD38 were higher in HAM patients compared with both carriers and controls (P < 0.05) after culture. These findings are consistent with the observations that HTLV-I produces chronic lymphocyte activation with increased adhesion. This may be sufficient to initiate events leading to central nervous system inflammation and ultimately to HAM.

Agonist-stimulated calcium entry in primary cultures of human cerebral microvascular endothelial cells.

Primary cultures of human cerebral microvascular endothelial cells (HCMEC) were loaded with fura-2. The intracellular free Ca2+ concentration ([Ca2+]i) was measured by digital imaging microscopy. Agonists ATP (100 micro), thrombin (10 units/ml), and histamine (25 microM) induced a transient [Ca2+]i increase. Histamine (100 microM) induced a biphasic [Ca2+]i increase with an initial [Ca2+]i peak followed by a [Ca2+]i plateau. The [Ca2+]i plateau was blocked by the receptor-operated Ca2+ channel (ROC) blockers SK&F 96365 and NCDC, indicating a contribution by Ca2+ influx through ROC to the [Ca2+]i plateau. However, this [Ca2+]i plateau was not blocked by the voltage-gated Ca2+ channel (VGC) blocker diltiazem (DTZ). Depolarization with 80K+ or application of the VGC agonist BAY K 8644 did not alter the resting [Ca2+]i; but 80K+ reduced the histamine (100 microM) induced [Ca2+]i plateau. These results show that HCMEC are devoid of functional VGC. Thus the membrane potential (Em) regulates Ca2+ entry mainly by enhancing the electrochemical Ca2+ gradient, such that hyperpolarization increases while depolarization decreases [Ca2+]i. Blockade of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) by CPA increased [Ca2+]i. This effect was dependent on extracellular Ca2+ and reduced by iberiotoxin (IBTX) blockade of Ca2+-activated K+ channels (Kca), suggesting a role for Kca in regulating Ca2+ influx. Ca2+ is the principal activator of endothelial nitric oxide synthase (eNOS), which stimulates cyclic GMP production. The final result that the eNOS inhibitor L-NAME enhanced the histamine (100 microM) induced [Ca2+]i plateau suggests a negative feedback loop (via cGMP) of endothelial NO on its own synthesis in the regulation of endothelial [Ca2+]i signal.

Expression and function of lymphocyte function associated antigen-3 (LFA-3) at the blood-brain barrier.

Lymphocyte function associated antigen-3 (LFA-3) is a cell surface glycoprotein involved in antigen independent T-cell activation and proliferation. The expression and function of LFA-3 at the blood-brain barrier were studied in an in vitro model consisting of primary cultures of human brain microvessel endothelial cells (HBMEC). Surface expression of LFA-3 was detected by immunogold silver staining and the presence of RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Unstimulated HBMEC in primary culture constitutively express LFA-3 on their surface. Expression is only marginally upregulated following stimulation with tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma). Similarly, LFA-3 RNA is present constitutively in unstimulated HBMEC with minimal increase after co-incubation with TNF-alpha and IFN-gamma. The function of LFA-3 as a costimulatory molecule on HBMEC was investigated by incubating purified CD4+ T-lymphocytes with resting or IFN-gamma treated HBMEC monolayers. Proliferation of alpha-CD3 activated CD4+ T-cells was significantly increased upon incubation with resting or activated endothelial cells. Monoclonal antibodies to LFA-3 consistently blocked the proliferative response by 64-76%. The ability of the cerebral endothelium to express LFA-3 and provide secondary signals for T-cell proliferation suggests that cerebral EC may be important in the initiation of inflammatory responses in the human central nervous system.

In vitro adhesion and migration of T lymphocytes across monolayers of human brain microvessel endothelial cells: regulation by ICAM-1, VCAM-1, E-selectin and PECAM-1.

Increased lymphocyte traffic across an altered blood-brain barrier (BBB) is a prominent and early event in inflammatory and immune-mediated CNS diseases. The factors that control the entry of lymphocytes into the brain have not been fully elucidated. In this study, primary cultures of human brain microvessel endothelial cells (HBMEC) were used to investigate the role of endothelial cell (EC) adhesion molecules in the adhesion and migration of peripheral blood T lymphocytes across TNF-alpha treated and untreated monolayers. Adhesion of T cells to unstimulated HBMEC was minimal and few of the adherent cells migrated across the monolayers. Treatment of HBMEC with TNF-alpha augmented adhesion by 5-fold. The binding to activated EC was significantly, but not completely, inhibited by monoclonal antibodies (mAbs) to ICAM-1 and VCAM-1, whereas adhesion to unstimulated EC was blocked by mAb to ICAM-1 but not VCAM-1. Transendothelial migration of lymphocytes increased by up to 30-fold following treatment of HBMEC with TNF-alpha. Migration across activated monolayers, but not across untreated EC, was almost completely blocked by Ab to ICAM-1 and significantly inhibited by Abs to PECAM-1 and E-selectin. VCAM-1 was not utilized during transendothelial migration. Ultrastructurally, pseudopodia from lymphocytes contacted finger-like cytoplasmic projections on EC and eventually penetrated the EC cytoplasm at focal points along the apical surface. Migrating lymphocytes moved either through the EC cytoplasm or between adjacent EC across intercellular contacts. The overlying monolayers showed no evidence of disruption and intercellular junctions appeared intact over the migrated T cells. These studies indicate that adhesion and migration of T lymphocytes across the cerebral endothelial barrier are distinct processes that depend upon the activation state of EC and are controlled by diverse receptor-ligand interactions.

Glomeruloid vascular structures in glioblastoma multiforme: an immunohistochemical and ultrastructural study.

Microvascular proliferation and glomeruloid vascular structures are important histopathological features of glioblastoma multiforme (GBM). The nature of cells participating in the formation of these structures remains unclear and is the subject of this study. To define these cells better, immunohistochemical markers directed against Factor VIII-related antigen (FVIIIR:Ag), alpha smooth-muscle actin (alpha-SMA), and the lectin Ulex europaeus agglutinin type I (UEA-I) were used. Cells lining the vascular channels and a large number of proliferating abluminal cells participating in glomeruloid vascular structure formation showed positive cytoplasmic staining for FVIIIR:Ag and UEA-I. Abluminal and luminal cells were variably labeled for alpha-SMA. Ultrastructurally, complex aggregates of focally anastomosing capillaries with narrow lumina composed the glomeruloid vascular structure. Endothelial cells were hyperplastic, varied in size and shape, overlapped focally, and contained numerous cytoplasmic filaments. Tight junctions bound together adjacent and overlapping endothelial cells. Weibel-Palade bodies, usually absent from brain microvessels, were present in increased numbers in the newly formed capillaries. Each capillary loop was surrounded by basal lamina encompassing a discontinuous layer of pericytes. This study indicates that glomeruloid vascular structures in GBM are complex aggregates of newly formed microchannels lined with hyperplastic endothelial cells that have an altered morphological phenotype and that these microchannels are supported by basal lamina and pericytes and are devoid of astrocytic end-feet.

Plasmodium falciparum: involvement of additional receptors in the cytoadherence of infected erythrocytes to microvascular endothelial cells.

The involvement of additional ligands in the cytoadhesion of PRBC to endothelial cells was studied by the use of human microvascular endothelial cells (HMEC-1), brain microvascular endothelial cells (HBEC-51), umbilical vein endothelial (HUVEC), and C32 melanoma cells as well as soluble CD36, ICAM-1, and thrombospondin in the adhesion assays. Immunostaining showed that ICAM-1 and thrombospondin were expressed by all cell lines, whereas CD36 and VCAM-1 were expressed constitutively only by C32 melanoma cells and HBEC-51, respectively; none of these cells had basal expression of E-selectin. Bindings of the parental HB3 parasite strain to HMEC-1 and HUVEC were higher than that to HBEC-51 and C32 melanoma cells. Selections by panning the parental HB3 through HMEC-1 (HB3EC-6 line) or C32 melanoma cells (HB3C32-6 to HMEC-1 was higher than that to C32 melanoma cells. Antibody or peptide blockade against CD36, ICAM-1, and thrombospondin or preincubation of target cells with TNF-alpha and IFN-gamma did not significantly alter the binding intensity of HB3EC-6 to HMEC-1 and HB3C32-6 to C32 melanoma cells. Preincubation of HMEC-1 with IL-4, however, reduced its binding with HB3EC-6. In vitro selection did not enhance the binding of PRBC to plate-bound CD36 or thrombospondin; binding to ICAM-1 was negligible. The binding of both selected lines was inhibited by dextran sulfate and sulfatides, but not by chondroitin sulfate A. These results suggested that in addition to CD36 and thrombospondin, sulfated glycoconjugates were probably concurrently utilized by these PRBC as receptors. Experiments with freshly isolated Kenyan parasites indicated that they also exhibited a similar mechanism of binding to endothelial cells.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1) expression by human brain microvessel endothelial cells in primary culture.

PECAM-1 expression was investigated in primary cultures of human brain microvessel endothelial cells (HBMEC). HBMEC constitutively express PECAM-1 along their apical cell surface, advancing processes and on the basal surface at points of contact with the extracellular matrix. Surface expression is not altered by cytokine or lipopolysaccharide treatment. This distribution may mediate cell-cell contract and migration during angiogenesis and HBMEC-leukocyte interactions in CNS inflammation.

Regualtion by cytokines and lipopolysaccharide of E-selectin expression by human brain microvessel endothelial cells in primary culture.

E-selectin is an adhesion molecule expressed on endothelial cells after treatment with inflammatory agents in vitro and in inflammatory diseases in vivo. Interactions between leukocytes and endothelial cells are mediated partly through this adhesion molecule. In this study, the kinetic expression of E-selectin by human cerebral endothelium was studied under standard conditions and following treatment of primary cultures with bacterial lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interferon-gamma (IFN-gamma). Surface expression of E-selectin was detected by immunocytochemistry, ELISA and immunoelectron microscopy. Untreated human cerebral endothelial cells constitutively expressed low levels of E-selectin. Treatment with LPS, TNF-alpha and IL-beta increased the mean level of E-selectin expression per cell and the percentage of cells expressing E-selectin, in a time and concentration-dependent manner. E-selectin expression was maximal by 4 h post-stimulation and returned unstimulated levels by 48 h. LPS and TNF-alpha were most effective followed by IL-1beta, while the IFN-gamma had no effect on E-selectin expression. Immunoelectron microscopy demonstrated that E-selectin was preferentially expressed on the apical surface of unstimulated and TNF-alpha treated cells. Cytokine stimulation induced a several-fold increase of E-selectin expression on the apical and to a lesser extent on the basal cell surface. Modulation of E-selectin expression on cerebral endothelium by inflammatory cytokines suggests a potentially important role of this adhesion molecule in the recruitment of leukocytes in central nervous (CNS) inflammation.

Tenascin-C expression by angiogenic vessels in human astrocytomas and by human brain endothelial cells in vitro.

The expression of the extracellular matrix glycoprotein tenascin-C (TN) is enhanced in human astrocytomas and correlates with angiogenesis. To determine whether vascular cells are able to synthesize TN, we investigated the expression of TN protein and mRNA in nine astrocytomas. Immunogold electron microscopy in two glioblastomas multiforme detected the presence of TN in an extracellular perivascular location and to a lesser extent among tumor cells, confirming light microscopy immunohistochemical findings. In situ hybridization of astrocytomas using a digoxigenin-labeled antisense riboprobe detected strong staining for TN mRNA in vascular cells, especially in hyperplastic vessels, including those at the invasive edge of the tumors but not in vessels of normal brains. We observed weaker staining in tumor cells indicating a higher level of TN mRNA in vascular than in tumor cells. No staining was detected with the sense probe. Moreover, we investigated the ability of human brain microvessel endothelial cells (HBMECs) in primary culture to synthesize TN in vitro. Western blot analysis of the culture supernatants from HBMECs detected large amounts of TN. Immunogold silver staining demonstrated the presence of TN on the surface of HBMECs and in the subendothelial matrix. The distribution of TN mRNA in vascular cells of astrocytomas and the ability of HBMECs to synthesize TN in vitro demonstrate that vascular cells, including endothelial cells, are a major source of TN associated with angiogenesis. Furthermore, our results suggest that TN expression may be associated with endothelial cell activation and may play an important role in angiogenesis.

Interferon-beta downregulates interferon-gamma-induced class II MHC molecule expression and morphological changes in primary cultures of human brain microvessel endothelial cells.

Regulation of class II MHC (Ia) antigen expression by interferons beta and gamma was studied in an in vitro model of the blood-brain barrier. Primary cultures of human brain microvessel endothelial cells were incubated with IFN-beta, gamma or a combination of the two cytokines and surface expression of class II MHC molecules was investigated with the immunogold silver staining technique and enzyme-linked immunosorbent assay. Treatment of monolayers with IFN-beta (100-6000 U/ml) failed to induce Class II MHC molecules. Co-incubation with IFN-gamma (100 U/ml), with or without pretreatment with IFN-beta, significantly inhibited the IFN-gamma-induced de novo expression in a concentration-dependent manner. Downregulation was less significant when incubation with both cytokines was preceded by 2-day treatment with IFN-gamma and was not observed in cultures incubated for an additional 4 days with IFN-gamma. Endothelial cells treated with IFN-gamma exhibited prominent morphological changes and frequent overlapping. These changes were not observed in the presence of either IFN-beta or both cytokines in the media. IFN-beta alone, or in combination with IFN-gamma, significantly inhibited the growth of endothelial cells, while only slight inhibition was observed with IFN-gamma. The results of these studies suggest that IFN-beta may function in modulating IFN-gamma-mediated immune responses in the human central nervous system at the level of the blood-brain barrier and this negative regulatory mechanism may be, at least in part, responsible for the recently reported beneficial effect of IFN-beta in relapsing-remitting multiple sclerosis.

Expression of vascular cell adhesion molecule-1 (VCAM-1) by human brain microvessel endothelial cells in primary culture.

Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial cell membrane glycoprotein that has been implicated in leukocyte/endothelial cell interactions in inflammation. In this study, we report the expression of VCAM-1 in primary cultures of human brain microvessel endothelial cells (HBMEC) under standard conditions and following bacterial lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), or interferon-gamma (IFN-gamma) treatment. Surface expression was detected and quantitated by light and immunogold electron microscopy and ELISA. Unstimulated cerebral endothelial cells (EC) constitutively expressed low levels of VCAM-1. LPS, TNF-alpha, or IL-1 beta increased the overall intensity of surface staining and the percentage of cells expressing VCAM-1 in a time- and concentration-dependent manner. LPS had the most potent effect, followed by TNF-alpha and then IL-1 beta. IFN-gamma did not upregulate VCAM-1. The level of VCAM-1 expression increased by 12-24 hr and returned to unstimulated levels by 48 hr. Immunoelectron microscopy demonstrated that VCAM-1 was preferentially localized on the apical as compared to the basal surface in both unstimulated and cytokine-treated cells. In addition, the intensity of immunostaining was significantly greater in stimulated versus unstimulated EC. The polarization and significant upregulation of VCAM-1 after cytokine treatment suggest a possible role of this adhesion molecule in inflammatory and autoimmune processes within the central nervous system.

In vitro interaction of coronaviruses with primate and human brain microvascular endothelial cells.

Primary human and primate brain microvascular endothelial cells were tested for permissiveness to coronaviruses JHM and 229E. While sub-genomic viral RNAs could be detected up to 72 hours post-infection, primate cells were abortively infected and neither virus caused cytopathology. Human cells were non-permissive for JHM but permissive for 229E replication; peak production of progeny 229E and observable cytopathic effects occurred approximately 22 and 32 hour post-infection, respectively. Using the criterion of cytopathology induction in infected endothelial cells, 229E was compared to other human RNA and DNA viruses. In addition, virus induced modulation of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and HLA I was monitored by immunostaining of infected cells.

Additive effect of allopurinol and deferoxamine in the prevention of spinal cord injury caused by aortic crossclamping.

Fourteen domestic swine were divided into two groups. Group A (n = 7) was the control group, in which no pharmacologic intervention was applied. In group B (n = 7), the ischemic-reperfused spinal cord was treated with the combination of allopurinol (50 mg/kg/day for 3 days before the day of operation) and deferoxamine (Desferal, 50 mg/kg administered intravenously over 3 to 4 hours). The administration of deferoxamine was completed 1 hour before crossclamping. The crossclamp was placed on the descending aorta just distal to the left subclavian artery for 30 minutes. Proximal hypertension was controlled with sodium nitroprusside and volume depletion. Methods of assessment included an evaluation of the neurologic status of the animals by quantitative Tarlov criteria, blood flow by radiolabeled microspheres, and histologic examination of the spinal cord. All animals in the control group, group A, were completely paraplegic with 0% recovery by Tarlov criteria at 24 hours after the removal of the crossclamp. In contrast, all animals in group B, in which the combination of allopurinol and deferoxamine was used, completely recovered (100% recovery by Tarlov criteria), and at 24 hours after the ischemic episode they were able to walk with no difficulty and had intact sensation. Functional parameters of these animals fully correlated with the morphologic findings. Widespread acute neuronal injury and vacuolation of neuropil were observed in the control group of animals. In contrast, animals in group B showed much less pronounced morphologic changes after the same period of ischemia. In summary, the combined use of these agents significantly (p < 0.001) reduced the incidence of paraplegia induced by aortic crossclamping with 82% additivity.

Hippocampal synaptic pathology in patients with temporal lobe epilepsy.

Immunostaining of synaptic terminals was studied in the hippocampus of 26 patients who had surgical resections for intractable temporal lobe epilepsy. Two monoclonal antibodies (EP10 and SP12) reactive with distinct synaptic antigens were used on paraffin-embedded tissues. The results indicated qualitative reductions on synaptic terminals in CA4 and other regions where cell loss is reported. The inner molecular layer of the dentate gyrus was observed to have increased synaptic immunostaining. Synaptic terminal loss in CA4 and redistribution in the molecular layer were most frequent in cases with hippocampal sclerosis. However, both forms of synaptic pathology were also noted in most cases where the pathological findings were classified as indefinite, and in some cases associated with mass lesions of the temporal lobe. These results support the importance of neuronal loss and synaptic reorganization as possible mechanisms of illness in epilepsy. They also indicate that synaptic immunostaining may be a useful adjunct to routine neuropathological diagnostic techniques.