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Biomedical subjects

D Kobiler

Publications and source records attributed to D Kobiler.

At least 19 recordsLinked to original sources
PubMed2003

Bone marrow-derived, endothelial progenitor-like cells as angiogenesis-selective gene-targeting vectors.

Human and mouse proliferation-competent, bone marrow or peripheral circulation derived endothelial progenitor-like cells (EPCs) were isolated, expanded and genetically engineered ex vivo to express the beta galactosidase (beta-gal), green fluorescence protein or thymidine kinase (TK) genes using retrovirus-mediated gene transfer. Genetically labeled EPCs were transplanted into sublethally irradiated tumor-bearing mice and were found to migrate to and incorporate into the angiogenic vasculature of growing tumors while maintaining transgene expression. Ganciclovir treatment resulted in significant tumor necrosis in animals previously administered TK-expressing EPCs with no systemic toxicity. These results demonstrate the feasibility of using genetically modified EPCs as angiogenesis-selective gene-targeting vectors and the potential of this approach to mediate non-toxic and systemic antitumor responses.

Animals↗
PubMed2001

Viral neuroinvasion as a marker for BBB integrity following exposure to cholinesterase inhibitors.

Exposure to the nerve agent soman, an irreversible cholinesterase (ChE) inhibitor, results in changes in blood-brain barrier permeability attributed to its seizure-induced activity. However, smaller BBB changes may be independent of convulsions. Such minor injury may escape detection. A nonneuroinvasive neurovirulent Sindbis virus strain (SVN) was used as a marker for BBB permeability. Peripheral inoculation of mice with 2 x 10(3) plaque forming units (PFU) caused up to 10(5) PFU/ml viremia after 24 hours with no signs of central nervous system (CNS) infection and with no virus detected in brain tissue. Intra-cerebral injection of as low as 1-5 PFU of the same virus caused CNS infection, exhibited 5-7 days later as hind limb paralysis and death. Soman (0.1-0.7 of the LD50) was administered at peak viremia (1 day following peripheral inoculation). Sublethal soman exposure at as low as 0.1 LD50 resulted in CNS infection 6-8 days following inoculation in 30-40% of the mice. High virus titer were recorded in brain tissue of sick mice while no virus was detected in healthy mice subjected to the same treatment. No changes in the level of viremia or changes in viral traits were observed in the infected mice. The reversible anticholinesterases physostigmine (0.2 mg/kg, s.c.) and pyridostigmine (0.4 mg/kg, i.m.) injected at a dose equal to 0.1 LD50, induced similar results. Thus, both central and peripheral anticholinesterases (anti-ChEs) induce changes in BBB permeability sufficient to allow, at least in some of the mice, the invasion of this otherwise noninvasive but highly neurovirulent virus. This BBB change is probably due to the presence of cholinesterases in the capillary wall. SVN brain invasion served here as a highly sensitive and reliable marker for BBB integrity.

Alphavirus Infections↗
PubMed2001

Detection of specific Bacillus anthracis spore biomarkers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied for the characterization of Bacillus anthracis spore biomarkers. B. anthracis spores were extracted under a simple procedure, followed by linear mode analysis, using sinapinic acid as the matrix. Several markers with a mass range of 4-7 kDa were detected in three B. anthracis strains: Vollum, Sterne and V770-NP1-R. Similar spectra were also obtained for spore extracts of two members of the B. cereus group: B. thuringiensis and B. cereus, but not for B. mycoides, B. subtilis or B. licheniformis, suggesting that these markers are specific to closely related members of the B. cereus group. When alpha-cyano-4-hydroxycinnamic acid was used as the matrix, at least four additional new markers within a mass range of 2-4 kDa could be detected in all B. anthracis spore extracts. These markers, corresponding to a molecular weight of 2528.3, 2792.4, 3077.4, and 3590.7 Da, have not been observed in extracts of the three closely related Bacillus species - B. cereus, B. thuringiensis and B. mycoides. These unique B. anthracis biomarkers, which were isotopically resolved and reproducibly detected in the highly accurate MALDI-TOFMS reflectron mode, may be useful as a basis for rapid and specific identification of B. anthracis strains.

Bacillus anthracis↗
PubMed2001

vFLIP protects PC-12 cells from apoptosis induced by Sindbis virus: implications for the role of TNF-alpha.

Sindbis virus (SV) is an alphavirus used as a model for studying the pathogenesis of viral encephalitis. In this study we examined the effects and the mechanisms involved in the apoptosis induced by SV in PC-12 cells, and the role of a vFLIP in this process. Infection of PC-12 cells with a neurovirulent strain of SV, SVNI, induced cell apoptosis. Overexpression of vFLIP encoded by the HHV-8 or treatment with a caspase-8 inhibitor inhibited cell apoptosis. SVNI induced an increase in the expression of tumor necrosis factor alpha (TNF-alpha), and pre-treatment of the cells with an anti-TNF-alpha blocking antibody or with soluble TNF-alpha receptor abrogated the apoptotic effect of SVNI. Moreover, TNF-alpha R1 knockout mice were more resistant to the cytopathic effects of the virus as compared to control animals. Our results indicate that the apoptosis induced by SVNI is mediated by activation of caspase-8, and that TNF-alpha plays an important role in the apoptotic response.

Animals↗
PubMed2001

Search for correlates of protective immunity conferred by anthrax vaccine.

Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found (r(2) = 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing-antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD(50)) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.

Animals↗
PubMed2000

Differential regulation of Bcl-2 and Bax expression in cells infected with virulent and nonvirulent strains of sindbis virus.

Sindbis virus is an alphavirus that infects cells in either lytic or persistent infection. In this study we examined the effects of Sindbis virus on cell apoptosis and on the expression of Bcl-2 and Bax. Of the two strains studied, SVA and SVNI, only the neurovirulent strain, SVNI, induced apoptosis of astrocytes and PC-12 cells. SVA, which infects cells in a persistent manner, induced up-regulation of bcl-2 mRNA and Bcl-2 protein, whereas SVNI induced an increase in Bax levels. Our results indicate a differential regulation of Bcl2 and Bax expression by SVA and SVNI, which may be associated with the apoptotic potential of the viruses.

Animals↗
PubMed2000

A live attenuated West Nile virus strain as a potential veterinary vaccine.

This article reviews the development of two attenuated West Nile virus (WNV) variants, WNI-25 and WNI-25A. These variants have lost the neuroinvasion trait of the parental virus. Attenuation was achieved through serial passages in mosquito cells and neutralization escape from WNV-specific monoclonal antibody. Genetic analysis reveals amino acid changes between the parental and each of the variants. The attenuated variants preserve the ability to replicate in mice and geese and to induce a protective immune response. WNI-25A was found to be a genetically stable virus. This variant was successfully used as a live vaccine to protect geese against a wild-type virulent WNV field isolate that closely resembles the WNV isolated during the 1999 New York epidemic.

Animals↗
PubMed2000

CNS penetration by noninvasive viruses following inhalational anesthetics.

The effects of inhalational anesthetics on brain penetration by the neurovirulent noninvasive West Nile virus (WN-25) were studied in mice. WN-25 injected intracerebrally causes encephalitis and kills adult mice, but when injected intraperitoneally (i.p.) it is unable to invade the brain and kill. Under stress conditions, this strain causes encephalitis and death even after i.p. inoculation. In the study described in this paper, we used two inhalational anesthetics, a single short-term exposure to 2% halothane for 10 min in oxygen, or 70% nitrous oxide (N2O) for 30 min in air. Both inhalational anesthetics induced WN-25 encephalitis and death in 33% and 20% of the tested mice, respectively. Exposure of inoculated mice to halothane for prolonged periods or for repeated exposures (two or three times) markedly increased the mortality rate (up to 75%). Exposure to 30% CO2, a known modulator of blood-brain barrier (BBB) activity, was used as a positive control (80% mortality). No death was observed in the control non-exposed injected mice. Virus levels were found to be more than 10(7) plaque-forming units (PFU)/brain in all moribund mice. Additional parameter demonstrating the "stressor-like" nature of inhalation anesthetics was the induction of a significant decrease in weight of the lymphoid organs of inoculated mice. We suggest that inhalational anesthetics induces BBB breaching with subsequent entrance of the noninvasive WN-25 virus into the brain, causing encephalitis and death.

Anesthetics, Inhalation↗
PubMed2000

Attenuated nontoxinogenic and nonencapsulated recombinant Bacillus anthracis spore vaccines protect against anthrax.

Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 x 10(7) spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (>/=100 microgram/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.

Animals↗
PubMed1999

The role of host immunocompetence in neuroinvasion of Sindbis virus.

Viral infections of the central nervous system (CNS) following peripheral inoculation of Sindbis viruses were studied. The use of viral strains, which vary in their neuroinvasive and neurovirulent properties, and various strains of mice, which differ in immunocompetence, revealed several pathways of viral neuroinvasion in adult mice. A genetic-trait dependent mechanism was exhibited by the neuroinvasive viruses, showing a similar pattern in all mice strains tested. A second mechanism, dependent on a prolonged high viral load, was exhibited by a noninvasive variant in Severe Combined ImmunoDeficient (SCID) mice. The absence of antiviral antibodies in SCID mice allowed the maintenance of a long-term high viremia, leading to a random entry to the CNS and proliferation in brain tissue. An additional pathway for neuroinvasion was induced upon disruption of the blood-brain barrier activity by exogenous reagents and was demonstrated in cases of short lived high viremia of noninvasive viruses.

Alphavirus Infections↗
PubMed1999

A single nucleotide change in the 5' noncoding region of Sindbis virus confers neurovirulence in rats.

Two pairs of Sindbis virus (SV) variants that differ in their neuroinvasive and neurovirulent traits in mice have been isolated. Recently, we mapped the genetic determinants responsible for neuroinvasiveness in weanling mice. Here, we extend this study to newborn and adult rats and to rat neuronal cultures. Remarkably, certain aspects of the pathogenesis of these strains in rats were found to be quite distinct from the mouse model. Suckling rats were susceptible to all four isolates, and replication in the brain was observed after both intraperitoneal and intracranial (i.c.) inoculation. None of the isolates was neuroinvasive in adult rats, although all replicated after i.c. inoculation. For the isolate pair that was highly neurovirulent in mice, SVN and SVNI, only SVNI caused death after i.c. inoculation of adult rats. Similarly, only SVNI was cytotoxic for primary cultures of mature neurons. The genetic determinants responsible for the pathogenic properties of SVNI were mapped to the E2 glycoprotein and the 5' noncoding region (5'NCR). Substitution of two amino acids in SVN E2 with the corresponding residues of SVNI (Met-190 and Lys-260) led to paralysis in 3- and 5-week-old rats. More dramatically, a single substitution in the 5'NCR of SVN (G at position 8) transformed the virus into a lethal pathogen for 3-week-old rats like SVNI. In 5-week-old rats, however, this recombinant was attenuated relative to SVNI by 2 orders of magnitude. Combination of the E2 and 5'NCR determinants resulted in a recombinant with virulence properties indistinguishable from those of SVNI. These data indicate that the 5'NCR and E2 play an instrumental role in determining the age-dependent pathogenic properties of SV in rats.

5' Untranslated Regions↗
PubMed1997

Astrocyte activation by Sindbis virus: expression of GFAP, cytokines, and adhesion molecules.

Sindbis virus (SV) is a member of the alphaviruses which has served as a model system for studying viral encephalitis. Although astrocytes are reported to be involved in the pathogenesis of various virus-related diseases, the effects of SV on astrocyte function have not been reported. In this study we compared the effects of two strains of SV, SVA, and SVNI, which differ in their neurovirulent properties, on astrocytes with the use of cultured mouse astrocytes and the rat C6 glial cell line. We found that although both strains can similarly infect and replicate in astrocytes, they induced different changes in the function of these cells. The neurovirulent strain, SVNI, induced a decrease in cell number and a marked increase in the expression of GFAP, whereas SVA did not alter these parameters. In addition, SVNI induced the secretion of the cytokines TNF-alpha and IL-6, the expression of adhesion molecules, and the production of the neurotrophic factor NGF. In contrast, SVA induced smaller increases in the secretion of IL-6 and NGF but did not alter the secretion of TNF-alpha and the expression of the adhesion molecules. Neither virus induced the secretion of IL-2, IL-4, IL-10 and IFN-gamma or the expression of iNOS in the cells. These results indicate that astrocytes, similar to neurons, can serve as target cells to SV infection in the CNS. Moreover, the infection of astrocytes by SVNI leads to changes characteristic of reactive astrogliosis which may contribute to the pathogenesis of SV-induced encephalitis by enhancing the local immune response in the CNS.

Animals↗
PubMed1996

West Nile virus neuroinvasion and encephalitis induced by macrophage depletion in mice.

The encephalitic West Nile virus and its nonneuroinvasive variant, WN-25, were used to study the effect of macrophage depletion on viral invasion of the central nervous system. The in vivo elimination of macrophages was achieved by use of liposome-encapsulated drug dichloromethylene diphosphonate. Depletion of macrophages had an exacerbating effect on the course of the viral infection, exhibited by higher and extended viremia and accelerated development of encephalitis and death. Using a low dose of West Nile virus (5 PFU/mouse), an increase in mortality (from 50% to 100%) due to macrophage depletion was demonstrated. Furthermore, the attenuated noninvasive variant WN-25 showed high and prolonged viremia in the macrophage depleted mice (approximately 5 log 10 PFU/ml versus 2 in control mice), that allowed the penetration of the virus into the central nervous system. The mortality rate caused by the attenuated virus in the macrophage-depleted mice was 70-75%, as compared to complete survival in the control inoculated mice. These results indicate a significant role of macrophages in the non-specific immediate defence system of the organism in case of viral infection.

Aedes↗
PubMed1996

Cold stress-induced neuroinvasiveness of attenuated arboviruses is not solely mediated by corticosterone.

In previous studies we have shown that various stress paradigms can induce the penetration of noninvasive, attenuated viruses into the central nervous system (CNS). Since glucocorticoids levels are elevated during stress, we compared the effect of cold stress and corticosterone (CS) injection on neuroinvasiveness of a non-invasive encephalitic virus, WN-25 (West Nile). Exposure of inoculated mice to cold stress or CS resulted in high viremia and a marked increase in mortality when compared to control untreated mice. Exposure of WN-25 inoculated mice to cold treatment or CS injection led to high blood virus levels as compared to nontreated mice (3.2 and 3.1 vs > 1 log 10 PFU/ml). Cold stress or CS (5000 ng/mouse) treatment caused a mortality rate of 70% and 50% of the WN-25 inoculated mice respectively. No mortality was recorded in control inoculated groups (p < 0.05). Passive transfer serum from uninfected cold stressed mice to WN-25 inoculated nonstressed mice, resulted in similar mortality. The levels of CS in passive transferred serum from cold stressed animals was 500 ng/ml, only 2% (100 vs. 5000 ng) of the CS dose required to obtain a similar effect on viral penetration and mortality when CS was injected directly. Therefore, we concluded that CS was not the sole factor responsible for the cold stress effect on the viral infection outcome.

Animals↗
PubMed1994

Loss of active neuroinvasiveness in attenuated strains of West Nile virus: pathogenicity in immunocompetent and SCID mice.

The neuropathogenicity of West Nile virus (WNV) and two derived attenuated strains WN25 and WN25A, was studied in young adult ICR mice and in severe combined immunodeficient (SCID) mice. Similarity in serology and RNA fingerprints were found between WNV and WN25. The viral envelope proteins of the attenuates differed from WNV in their slower mobility in SDS-PAGE due probably to the presence of N-linked glycan. The three strains were lethal to ICR mice by intracerebral (IC) inoculation, but when inoculated intraperitoneally (IP), WNV caused viremia, invaded the CNS and was lethal, whereas the attenuates showed no viremia or invasion of the CNS. The attenuates elicited antibodies to comparable levels as WNV in IP-infected mice, conferring upon them immunity to IC challenge with the wild type. In IP-inoculated SCID mice the three strains exhibited similar high viremiae that lasted until death of the animals. All strains invaded the CNS and proliferated in the mouse brain to similar high titers, but differed largely in the time of invasion: WNV invaded the CNS of SCID mice (and two other mouse strains) much earlier than the attenuates, which showed large intervals in their time of invasion into individual mouse brains within the group. The data presented for SCID mice indicate that WN25 and WN25A have truly lost the neuroinvasive property, and that this property materialized by a prescribed, active process specific for WNV.

Aedes↗
PubMed1992

Viral neuroinvasion and encephalitis induced by lipopolysaccharide and its mediators.

The present study was designed to test the effect of bacterial endotoxin on penetration of viruses into the central nervous system (CNS). As a model we used two neurovirulent viruses that lack neuroinvasive capacity: West Nile virus-25 (WN-25) and neuroadapted Sindbis virus (SVN). Administration of lipopolysaccharide (LPS, 100 micrograms/mouse) to CD-1 mice, followed by WN-25 inoculation resulted in 83% encephalitis and death, compared with less than 5% in controls. The results in SVN-inoculated CD-1 mice were quite similar. LPS-treated mice suffered 62% mortality compared with 6% in the nontreated group. No changes in viral neuroinvasiveness were demonstrated in viruses isolated from brains of encephalitic mice, suggesting that neuroinvasion is not due to a selection process for an invasive variant, but to direct penetration of the viruses through the blood-brain barrier (BBB). LPS did not induce WN-25 encephalitis in LPS-insensitive C3H/HeJ mice, compared with 100% neuroinvasion in C3H/HeB mice. Induction of neuroinvasion could be transferred to C3H/HeJ mice by transfusion with serum obtained from LPS-treated, LPS-responsive mice. Passive immunization of CD-1 mice with anti-mTNF antibodies before LPS administration did not prevent LPS-induced WN-25 encephalitis. Furthermore, neutralization of tumor necrosis factor activity in the serum of LPS-treated mice did not abolish its activity, and transfusion-associated encephalitis was observed after the administration of the neutralized serum with WN-25. We suggest that LPS can contribute to virus penetration from the blood into the CNS, a process which turns a mild viral infection into a severe lethal encephalitis. This effect is mediated by soluble factors, and is probably achieved by injury to cerebral microvascular endothelium and modulation of BBB permeability.

Animals↗
PubMed1992

Dehydroepiandrosterone protects mice inoculated with West Nile virus and exposed to cold stress.

The protective effect of pretreatment with dehydroepiandrosterone (DHEA) on stress-enhanced viral encephalitis was studied in mice exposed to cold following inoculation with West Nile virus (WNV). Exposure of WNV-inoculated mice to cold water (1 +/- 0.5 degrees C, 5 minutes/day for 8 days) resulted in a mortality rate of 83% as compared to 50% in nonstressed mice (p < 0.05). The effect of cold stress was more pronounced when mice were inoculated with WN-25, a noninvasive neurovirulent variant of WNV. Mice infected with WN-25 showed no mortality, whereas cold stressed mice inoculated with the same virus had a mortality rate of 67% (p < 0.05). The administration of DHEA (serial injections of 10-20 mg/kg with or without a loading dose of 1 gm/kg) resulted in a significant reduction in the mortality rate of stressed mice inoculated with either virus (p < 0.05). Virus levels in the blood and brain of the DHEA-treated mice, were significantly lower than in the control groups. DHEA also prevented the involution of lymphoid organs in stressed mice. The present study provides direct evidence of the protective effects of DHEA as an "anti-stress" agent. Its ability to prevent mortality associated with WNV or WN-25, and involution of lymphoid organs caused by stress-induced immunosuppression, supports the notion that its activity is based on the modulation of the host response.

Analysis of Variance↗