Plasma VEGF - a candidate biomarker for response to treatment with bevacizumab in HHT patients.

BACKGROUND: Recurrent epistaxis is the principal symptom of hereditary hemorrhagic telangiectasia (HHT). Currently, there is no standard therapy for this condition. Bevacizumab (anti-VEGF) treatment has been under intense investigation but treatment effects vary greatly between individuals. There are currently no markers to predict anti-VEGF therapeutic response in HHT patients. METHODS: We evaluated plasma VEGF levels in 13 HHT patients and correlated values with i) degree of epistaxis, measured by visual analog scale (VAS), epistaxis severity score (ESS), and patient bleeding diaries ii) the prevalence of extranasal manifestations, iii) the HHT subtype and iv) the treatment response to intranasal submucosal bevacizumab. RESULTS: Plasma VEGF was elevated in all 13 HHT patients compared to reference levels and showed a moderate correlation with VAS and duration of moderate bleeding events. In patients treated with intranasal submucosal bevacizumab plasma VEGF levels showed a strong correlation with the degree of reduction of mild bleeding events and VAS. CONCLUSIONS: The role of plasma VEGF as a potential predictive biomarker for therapeutic response to bevacizumab treatment warrants further investigation in larger prospective studies.

Erythropoietin reduces experimental autoimmune encephalomyelitis severity via neuroprotective mechanisms.

BACKGROUND: Treatment with erythropoietin (Epo) in experimental autoimmune encephalomyelitis (EAE), the rodent model of multiple sclerosis (MS), has consistently been shown to ameliorate disease progression and improve overall outcome. The effect has been attributed to modulation of the immune response and/or preservation of the central nervous system (CNS) tissue integrity. It remains unclear, however, if (a) Epo acts primarily in the CNS or the periphery and if (b) Epo's beneficial effect in EAE is mainly due to maintaining CNS tissue integrity or to modulation of the immune response. If Epo acts primarily by modulating the immune system, where is this modulation required? In the periphery, the CNS or both? METHODS: To address these questions, we used two well-characterized transgenic mouse strains that constitutively overexpress recombinant human Epo (rhEpo) either systemically (tg6) or in CNS only (tg21) in a MOG-induced EAE model. We assessed clinical severity, disease progression, immunomodulation, and CNS tissue integrity, including neuronal survival. RESULTS: Although disease onset remained unaffected, EAE progression was alleviated in transgenic animals compared to controls with both lines performing equally well showing that expression of Epo in the periphery is not required; Epo expression in the CNS is sufficient. Immunomodulation was observed in both strains but surprisingly the profile of modulation differed substantially between strains. Modulation in the tg21 strain was limited to a reduction in macrophages in the CNS, with no peripheral immunomodulatory effects observed. In contrast, in the tg6 strain, macrophages were upregulated in the CNS, and, in the periphery of this strain, T cells and macrophages were downregulated. The lack of a consistent immunomodulatory profile across both transgenic species suggests that immunomodulation by Epo is unlikely to be the primary mechanism driving amelioration of EAE. Finally, CNS tissue integrity was affected in all strains. Although myelin appeared equally damaged in all strains, neuronal survival was significantly improved in the spinal cord of tg21 mice, indicating that Epo may ameliorate EAE predominantly by protecting neurons. CONCLUSIONS: Our data suggests that moderate elevated brain Epo levels provide clinically significant neuroprotection in EAE without modulation of the immune response making a significant contribution.

Expression of nogo-a is decreased with increasing gestational age in the human fetal brain.

Nogo is a member of the reticulon family. Our understanding of the physiological functions of the Nogo-A protein has grown over the last few years, and this molecule is now recognized as one of the most important axonal regrowth inhibitors present in central nervous system (CNS) myelin. Nogo-A plays other important roles in nervous system development, epilepsy, vascular physiology, muscle pathology, stroke, inflammation, and CNS tumors. Since the exact role of Nogo-A protein in human brain development is still poorly understood, we studied its cellular and regional distribution by immunohistochemistry in the frontal lobe of 30 human fetal brains. Nogo-A was expressed in the following cortical zones: ependyma, ventricular zone, subventricular zone, intermediate zone, subplate, cortical plate, and marginal zone. The number of positive cells decreased significantly with increasing gestational age in the subplate and marginal zone. Using different antibodies, changes in isoform expression and dimerization states could be shown between various cortical zones. The results demonstrate a significant change in the expression of Nogo-A during the development of the human brain. The effects of its time- and region-specific regulation have to be further studied in detail.

Listeria meningitis: identification of a cerebrospinal fluid inhibitor of macrophage listericidal function as interleukin 10.

The killing of bacteria gaining access to the central nervous system is insufficient and requires bactericidal antibiotics for treatment. The inefficient host response in cerebrospinal fluid (CSF) is thought to be due to impaired phagocytosis in CSF, and low local concentration of antibody and complement. In addition, the CSF may contain inhibitors, disabling phagocytes to eliminate bacteria. We have assessed the bactericidal activity of macrophages in the presence of CSF from mice infected intracerebrally with Listeria monocytogenes (LM). Pretreatment of J774A.1 macrophages with interferon gamma (IFN-gamma) resulted in high levels of nitric oxide-dependent intracellular killing of LM. CSF taken from mice 24 h after infection (CSF-LM 24) contained IFN-gamma and induced killing of LM by macrophages. However, pulsing J774A.1 cells with IFN-gamma in the presence of CSF obtained from mice at later time points (48 h) rendered macrophages partly permissive for intracellular Listeria growth. The inhibitor detected in CSF-LM 48 was identified as IL-10 since: (a) IL-10 dose dependently impaired the listericidal activity of IFN-gamma-activated macrophages; (b) anti-IL-10 antibodies abrogated the bacterial growth permissive effect of CSF-LM 48; and (c) IL-10 was detected in CSF-LM 48 but not in CSF-LM 24 or CSF of mock-injected animals (CSF-Co). Likewise, IL-10 was found in the CSF of 95% of patients with bacterial meningitis.

Tumor necrosis factor alpha in cerebrospinal fluid during bacterial, but not viral, meningitis. Evaluation in murine model infections and in patients.

To evaluate the potential role of cachectin/TNF-alpha in the pathogenesis of bacterial and viral meningitis, concentrations and kinetics of TNF-alpha were determined in cerebrospinal fluid (CSF). After intracerebral, but not systemic, infection with Listeria monocytogenes in mice, TNF-alpha was detected as early as 3 h after infection reaching maximum titers after 24 h. However, TNF-alpha was not found in serum during the course of Listeria infection. In contrast to bacterial meningitis, no TNF-alpha was detected at any time in CSF of mice suffering from severe lymphocytic choriomeningitis induced by intracerebral infection with lymphocytic choriomeningitis virus. This difference is striking since both model infections led to a massive infiltration of polymorphonuclear and mononuclear leukocytes into the meninges and CSF. The results found for the two model infections were paralleled by findings in humans; CSF from three out of three patients with bacterial meningitis examined during the first day of hospitalization showed significant levels of TNF-alpha; none of the CSF obtained later than 3 d after hospitalization was positive. In addition, similarly to what was found in mice with viral meningitis, zero out of seven patients with viral meningitis had detectable TNF-alpha in CSF.

Transforming growth factor beta 2 inhibits cerebrovascular changes and brain edema formation in the tumor necrosis factor alpha-independent early phase of experimental pneumococcal meningitis.

Macrophages and granulocytes seem to play a key role in the pathogenesis of bacterial meningitis. Transforming growth factor beta (TGF-beta) leads to macrophage deactivation, as well as to inhibition of cytokine production and of endothelial granulocyte adhesion. We have investigated the influence of TGF-beta on regional cerebral blood flow (rCBF), intracranial pressure (ICP), and brain edema formation during the early phase of experimental meningitis. Rats which were inoculated intracisternally with live pneumococci or with pneumococcal cell wall hydrolyzed by the M1 muramidase (PCW-M) developed an increase of rCBF and ICP within 4 h postintracisternal challenge. A single intraperitoneal injection of TGF-beta 2 but not of TGF-beta 2 vehicle-control prevented the changes of rCBF. Furthermore, TGF-beta 2 significantly reduced the increase of ICP in rats inoculated with PCW-M. Likewise, the elevation of brain water content after intracisternal injection of pneumococci or PCW-M was blocked by pretreatment of rats with TGF-beta 2. TGF-beta 1 exhibited similar inhibitory effects in PCW-M-injected rats. The beneficial effects of TGF-beta 2 on the initial phase after pneumococcal inoculation seem to be tumor necrosis factor alpha- (TNF-alpha) independent since (a) intracisternal or intraperitoneal injection of neutralizing anti-TNF-alpha antibodies did not significantly influence rCBF, ICP, and brain water content in PCW-M-induced meningitis; and (b) TNF-alpha was only occasionally detected at low levels in cerebrospinal fluid at 4 h after PCW-M application.

Tumor necrosis factor alpha and lymphotoxin alpha are not required for induction of acute experimental autoimmune encephalomyelitis.

Immunization of mice with myelin components results in experimental autoimmune encephalomyelitis (EAE), which is mediated by myelin-specific CD4(+) T cells and anti-myelin antibodies. Tumor necrosis factor alpha (TNF-alpha) and lymphotoxin alpha (LT-alpha) are thought to be involved in the events leading to inflammatory demyelination in the central nervous system. To ascertain this hypothesis 129 x C57BL/6 mice with an inactivation of the tnf and lta genes (129 x C57BL/6(-/-)) and SJL/J mice derived from backcrosses of the above mentioned mutant mice (SJL-/-) were immunized with mouse spinal cord homogenate (MSCH) or proteolipid protein. Both 129 x C57BL/6(-/-) mice and SJL-/- mice developed EAE. In SJL-/- mice immunized with MSCH, a very severe form of EAE with weight loss, paralysis of all four limbs, and lethal outcome was observed. The histologic hallmark was an intense perivascular and parenchymal infiltration with predominantly CD4(+) T cells and some CD8(+) T cells associated with demyelination in both brain and spinal cord. These results indicate that TNF-alpha and LT-alpha are not essential for the development of EAE.

Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

Interleukin 6 production in experimental cerebral malaria: modulation by anticytokine antibodies and possible role in hypergammaglobulinemia.

The production of Interleukin 6 (IL-6) was studied during experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA (PbA) infection. IL-6 is present in the serum of mice with ECM, the highest concentrations being observed in mice with full-blown neurological syndrome. High IL-6 levels were also observed, however, in the absence of pathology in nonlethal malaria infection. These data suggest that IL-6 is produced in large amounts during malaria infection, but does not play a major role in the pathogenesis of ECM. A modulation of IL-6 production in ECM was achieved by in vivo treatment with other anticytokine antibodies: antibodies to interferon (IFN-gamma) or to tumor necrosis factor (TNF) abolished the rise of IL-6, while anti-IL-3 and anti-granulocyte/macrophage colony-stimulating factor antibodies only partially prevented this rise, suggesting that the two cytokines IFN-gamma and TNF are important intermediates in IL-6 production. Passive immunization against IL-6 did not prevent ECM, but significantly reduced serum IgG levels in malaria-infected mice. Thus, by its effects on B cells, IL-6 may be involved in hypergammaglobulinemia and immune-complex diseases, e.g., glomerulonephritis observed during malaria infection.

Production of B cell stimulatory factor-2 and interferon gamma in the central nervous system during viral meningitis and encephalitis. Evaluation in a murine model infection and in patients.

Synthesis of B cell-stimulating factor-2 (BSF-2) and IFN-gamma was shown in cerebrospinal fluids (CSF) collected from mice with experimental viral meningitis. In the CSF, the level of BSF-2 started to increase 24 h after intracerebral infection with lymphocytic choriomeningitis virus (LCMV) with rapid increase after day 4. IFN-gamma was not detected in the CSF before day 5 or 6 after infection, but increased sharply thereafter. In athymic nude mice, LCMV infection did not result in meningitis, and both BSF-2 and IFN-gamma levels were only slightly and transiently elevated. These findings suggest that activated mature T cells are required for development of disease and production of both BSF-2 and IFN-gamma. As observed in mice, BSF-2 was also detected in 16 out of 19 CSF samples collected from patients with acute viral infections of the central nervous system (CNS). Intrathecal production of BSF-2 and IFN-gamma may be instrumental in local production of antiviral antibodies by B lymphocytes/plasma cells invading the CNS during viral CNS disease.

A homozygous deletion of a normal variation locus in a patient with hearing loss from non-consanguineous parents.

BACKGROUND: International databases with information on copy number variation of the human genome are an important reference for laboratories using high resolution whole genome screening. Genomic deletions or duplications which have been detected in the healthy population and thus marked as normal copy number variants (CNVs) can be filtered out using these databases when searching for pathogenic copy number changes in patients. However, a potential pitfall of this strategy is that reported normal CNVs often do not elicit further investigation, and thus may remain unrecognised when they are present in a (pathogenic) homozygous state. The impact on disease of CNVs in the homozygous state may thus remain undetected and underestimated. METHODS AND RESULTS: In a patient with syndromic hearing loss, array comparative genomic hybridisation (array CGH) and multiple ligation dependent probe amplification (MLPA) revealed a homozygous deletion on 15q15.3 of a CNV, inherited from hemizygous carrier parents. The deletion is about 90 kilobases and contains four genes including the STRC gene, which is involved in autosomal recessive deafness (DFNB16). By screening healthy control individuals and review of publicly available CNV data we estimated the frequency of hemizygous deletion carriers to be about 1.6%. CONCLUSION: We characterised a homozygous deletion of a CNV region causing syndromic hearing loss by a panel of molecular tools. Together with the estimated frequency of the hemizygous deletion, these results emphasise the role of the 15q15.3 locus in patients with (syndromic) hearing impairment. Furthermore, this case illustrates the importance of not automatically eliminating registered CNVs from further analysis.

Shallow retardation of the strontium isotope signal of agricultural liming - implications for isoscapes used in provenance studies.

An intensified debate centers on the use of strontium isotopes in surface water run-off as archive for bioavailable signatures in prehistoric provenance studies. Its use has been challenged by a recent suggestion that modern agricultural liming of farmlands exerts a serious imprint on the strontium isotope compositions of these waters. We here present results from a soil profile beneath agricultural farmland in the glaciogenic outwash plain of central West Jutland, Denmark, which show that strontium and its isotope composition derived from lime products is efficiently retained near the surface. Pore waters and bioavailable strontium from the acidic zone below the surface soil depict strontium isotope signatures that can best be explained by a mixture of silicate-derived and relic natural (not agriculturally added) carbonate-derived strontium. We therefore argue that agricultural liming does not contaminate groundwaters and groundwater-supported surface waters, rendering reference maps based on them relevant for modern and past provenance studies.

Intranuclear anchoring of repetitive DNA sequences: centromeres, telomeres, and ribosomal DNA.

Centromeres, telomeres, and ribosomal gene clusters consist of repetitive DNA sequences. To assess their contributions to the spatial organization of the interphase genome, their interactions with the nucleoskeleton were examined in quiescent and activated human lymphocytes. The nucleoskeletons were prepared using "physiological" conditions. The resulting structures were probed for specific DNA sequences of centromeres, telomeres, and ribosomal genes by in situ hybridization; the electroeluted DNA fractions were examined by blot hybridization. In both nonstimulated and stimulated lymphocytes, centromeric alpha-satellite repeats were almost exclusively found in the eluted fraction, while telomeric sequences remained attached to the nucleoskeleton. Ribosomal genes showed a transcription-dependent attachment pattern: in unstimulated lymphocytes, transcriptionally inactive ribosomal genes located outside the nucleolus were eluted completely. When comparing transcription unit and intergenic spacer, significantly more of the intergenic spacer was removed. In activated lymphocytes, considerable but similar amounts of both rDNA fragments were eluted. The results demonstrate that: (a) the various repetitive DNA sequences differ significantly in their intranuclear anchoring, (b) telomeric rather than centromeric DNA sequences form stable attachments to the nucleoskeleton, and (c) different attachment mechanisms might be responsible for the interaction of ribosomal genes with the nucleoskeleton.

Hemifacial spasm producing tremor-like movements of the head.

Tremor is defined as involuntary rhythmic oscillation and is produced by muscle contractions. Hemifacial spasm is rapid involuntary muscle contractions on one side of the face in the distribution of the VIIth nerve. We present a severe case of hemifacial spasm that produces a head-nodding tremor-like movements.

Anti-Fas/APO-1 antibody-mediated apoptosis of cultured human glioma cells. Induction and modulation of sensitivity by cytokines.

Fas/APO-1 is a transmembrane protein of the nerve growth factor/TNF alpha receptor family which signals apoptotic cell death in susceptible target cells. We have investigated the susceptibility of seven human malignant glioma cell lines to Fas/APO-1-dependent apoptosis. Sensitivity to Fas/APO-1 antibody-mediated cell killing correlated with cell surface expression of Fas/APO-1. Expression of Fas/APO-1 as well as Fas/APO-1-dependent cytotoxicity were augmented by preexposure of human malignant glioma cells to IFN gamma and TNF alpha. Further, pretreatment with TGF beta 2, IL1 and IL8 enhanced Fas/APO-1 antibody-induced glioma cell apoptosis whereas other cytokines including TNF beta, IL6, macrophage colony-stimulating factor, IL10 and IL13 had no such effect. None of the human malignant glioma cell lines was susceptible to TNF alpha-induced cytotoxicity. Fas/APO-1 antibody-sensitive glioma cell lines (n = 5), but not Fas/APO-1 antibody-resistant glioma cell lines (n = 2), became sensitive to TNF alpha when co-treated with inhibitors of RNA and protein synthesis. Resistance of human glioma cells to Fas/APO-1 antibody-mediated apoptosis was mainly related to low level expression of Fas/APO-1 and appeared not to be linked to overexpression of the anti-apoptotic protooncogene, bcl-2. Given the resistance of human malignant glioma to surgery, irradiation, chemotherapy and immunotherapy, we propose that Fas/APO-1 may be a promising target for a novel locoregionary approach to human malignant glioma. This strategy gains support from the demonstration of Fas/APO-1 expression in ex vivo human malignant glioma specimens and from the absence of Fas/APO-1 in normal human brain parenchyma.

Increased secretion of tumor necrosis factors alpha, interleukin 1, and interleukin 6 by human mononuclear cells exposed to beta-galactoside-specific lectin from clinically applied mistletoe extract.

A beta-galactoside-specific lectin from proprietary mistletoe extract, recently reported to exhibit immunomodulatory potency in vivo (T. Hajto, K. Hostanska, and H J. Gabius, Cancer Res. 49: 4803-4808, 1989), induced increased secretion of tumor necrosis factor alpha, interleukin 1, and interleukin 6 in cultures of human peripheral blood mononuclear cells. The enhancement of secretion, determined independently by bioassays and enzyme-linked immunosorbent assay-based quantitation, was caused by selective protein-carbohydrate interaction, as revealed by the strict dependence on the presence of the carbohydrate-binding subunit of the specific lectin-binding sugar as well as anti-lectin antibodies. Increased cytokine levels in serum of patients after injection of optimal lectin doses corroborated the in vitro results. Thus, these data provide an explanation for the increases in cellular parameters of the host defense system in vivo, which presents a further step toward their potentially beneficial clinical exploitation in standardized regimens.

Interleukin-8 is produced in neoplastic and infectious diseases of the human central nervous system.

The presence of interleukin-8 (IL-8), a leukocyte chemotactic factor, was examined in primary and metastatic central nervous system tumors and in nonneoplastic acute meningoencephalitides. In vitro: (a) 11 of 12 glioblastoma cell lines constitutively expressed IL-8 mRNA; (b) 5 of 6 of these cell lines secreted IL-8 protein as detected by enzyme-linked immunosorbent assay and a glucosaminidase release bioassay; and (c) IL-1 beta or tumor necrosis factor was able to augment both IL-8 mRNA steady state levels and protein secretion of all cell lines tested except IN-319. IL-8 was also found in vivo. (a) IL-8 poly A+ mRNA was detected in 2 of 2 low grade astrocytomas, 1 of 2 anaplastic astrocytomas, and 6 of 6 glioblastomas. (b) IL-8 protein was present in the cyst fluid of 1 of 4 low grade astrocytomas, 1 anaplastic astrocytoma, 2 of 2 glioblastomas, 1 oligodendroglioma grade III, and one central nervous system cervical carcinoma metastasis. (c) The cerebrospinal fluid of 3 of 4 metastatic lymphomas, 2 of 16 glioblastomas, 1 of 2 low grade astrocytomas, but none of 3 anaplastic astrocytomas and none of 9 meningiomas contained IL-8. The presence of IL-8 was not restricted to central nervous system tumors as 2 of 2 bacterial meningitis and 5 of 5 acute viral meningitis patients contained considerable IL-8 levels in the cerebrospinal fluid. (d) Immunohistochemical analysis showed IL-8 immunoreactivity in perivascular tumor cells in 11 of 15 glioblastoma sections. These data suggest that IL-8 secretion could be a key factor involved in the determination of the lymphoid infiltrates observed in brain tumors and the development of cerebrospinal fluid pleocytosis in meningoencephalitides.

Posterior sacculation of the uterus in a patient presenting with flank pain at 29 weeks of gestation.

BACKGROUND: Uterine sacculation is a rare complication of pregnancy and may cause substantial peripartal morbidity. CASE: A possible diagnosis of posterior uterine sacculation was raised when a 34-year-old Gravida 1 Para 1 presented with bilateral flank pain at 29 weeks. Sonographic and magnetic resonance imaging findings confirmed the diagnosis and demonstrated bilateral dilated renal pelvises. Bilateral nephrostomas were placed, offering the patient considerable relief. A healthy female newborn was delivered by cesarean at 34 1/7 weeks. Operative findings confirmed the posterior sacculation of the uterus. CONCLUSION: Early diagnosis of sacculation of the uterus is necessary to limit maternal and fetal morbidity and mortality. For a detailed evaluation of the pelvic anatomy, we recommend the use of magnetic resonance imaging in the third trimester.

Treatment of experimental glioma by administration of adenoviral vectors expressing Fas ligand.

Fas ligand (FasL) is a cytokine, produced by activated T cells and NK cells, that triggers apoptosis of Fas-positive target cells including human glioma cells. As shown here, in vitro infection of rat F98 and human LN18 glioma cell lines with recombinant adenovirus (rAd) expressing FasL cDNA under control of the cytomegalovirus promoter (rAd-CMV-FasL) induced striking cytotoxicity in Fas-positive glioma cell lines but not in the Fas-negative F98 glioma subline F98/ZH. The extent of FasL-mediated cytotoxic effects outranged the expectations based on expression of beta-galactosidase (beta-Gal) by F98 cells infected with a control virus expressing the lacZ gene (rAd-CMV-lacZ). The detection of FasL bioactivity in supernatants of infected cells provides evidence of a bystander mechanism involving the cytotoxic action of FasL on uninfected cells. In F98 tumor-bearing rats, infection with rAd-CMV-FasL increased the mean survival time by 50% compared with infection with rAd-CMV-lacZ or untreated controls. These data suggest that viral vector transduction of the FasL gene could be part of a successful glioma gene therapy.