Raymond B. Baggs

Potentiated and preferential effects of combined paraquat and maneb on nigrostriatal dopamine systems: environmental risk factors for Parkinson's disease?

The absence of any compelling basis for a heritable basis of idiopathic Parkinsons disease (PD) has focused attention on environmental exposures as causative agents. While the herbicide paraquat has repeatedly been implicated, its impact on dopamine systems following systemic exposures is equivocal. The restricted focus on paraquat also ignores the extensive geographical overlap of its use with other agrichemicals known to adversely impact dopamine systems, including ethylenebisdithiocarbamate fungicides such as maneb. The present study sought to determine whether combined exposures to paraquat and maneb would produce additive effects and support a multiple-hit environmental contribution to PD. C57BL/6 mice were exposed to either paraquat (5-10 mg/kg) or maneb (15-30 mg/kg) i.p. alone or in combination once a week for 4 weeks. Sustained decreases in motor activity immediately following injections were consistently observed only with combined exposures, with activity levels returning to control values 24 h later. Concurrently, levels of dopamine and metabolites and dopamine turnover were increased immediately post-injection only by combined exposures, and returned to control levels or below within 48 h. Reductions in tyrosine hydroxylase immunoreactivity, measured 3 days after the last injection, resulted only from combined exposure and were detected in dorsal striatum, but not in the nucleus accumbens. The fact that combined exposures resulted in potentiated effects that appear to target nigrostriatal dopamine systems suggests that these combinations may be important environmental risk factors for Parkinsonism. These findings also raise questions about the adequacy of current risk assessment guidelines for these chemicals which are based on effect levels derived from exposures to single agents.

Age-related irreversible progressive nigrostriatal dopaminergic neurotoxicity in the paraquat and maneb model of the Parkinson's disease phenotype

While advancing age is the only unequivocally accepted risk factor for idiopathic Parkinsons disease, it has been postulated that exposure to environmental neurotoxicants combined with ageing could increase the risk for developing Parkinsons disease. The current study tested this hypothesis by exposing C57BL/6 mice that were 6 weeks, 5 months or 18 months old to the herbicide paraquat, the fungicide maneb or paraquat + maneb, a combination that produces a Parkinsons disease phenotype in young adult mice. Paraquat + maneb‐induced reductions in locomotor activity and motor coordination were age dependent, with 18‐month‐old mice most affected and exhibiting failure to recover 24 h post‐treatment. Three months post‐treatment, reductions in locomotor activity and deficits in motor coordination were sustained in 5‐month‐old and further reduced in 18‐month‐old paraquat + maneb groups. Progressive reductions in dopamine metabolites and dopamine turnover were greatest in 18‐month‐old paraquat + maneb and paraquat groups 3 months post‐treatment. Increased tyrosine hydroxylase enzyme activity compensated for striatal tyrosine hydroxylase protein and/or dopamine loss following treatment in 6‐week‐old and 5‐month‐old, but not 18‐month‐old paraquat and paraquat + maneb mice. Numbers of nigrostriatal dopaminergic neurons were reduced in all age groups following paraquat alone and paraquat + maneb exposure, but these losses, along with decreases in striatal tyrosine hydroxylase protein levels, were progressive in 18‐month‐old paraquat and paraquat + maneb groups between 2 weeks and 3 months post‐exposure. Collectively, these data demonstrate enhanced sensitivity of the ageing nigrostriatal dopamine pathway to these pesticides, particularly paraquat + maneb, resulting in irreversible and progressive neurotoxicity.

Early alterations in extracellular matrix and transforming growth factor β gene expression in mouse lung indicative of late radiation fibrosis

PURPOSE Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The expression of late radiation injury can be found immediately after irradiation by measuring messenger RNA (mRNA) abundance. METHODS AND MATERIALS To determine if extracellular matrix mRNA and transforming growth factor beta abundance was affected acutely after irradiation, we measured mRNA levels of collagen I (CI), collagen III (CIII), collagen IV (CIV), fibronectin (FN), and transforming growth factor beta (TGF beta 1,2&3) in mouse lungs on day 1 and day 14 after graded doses of radiation. C57BL/6 female mice were irradiated with a single dose to the thorax of 5 or 12.5 Gy. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabelled cDNA probes for CI, CIII, CIV, FN, TGF beta 1,2&3 and a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Autoradiographic data were quantified by video densitometry and results normalized to GAPDH. RESULTS Changes in the expression of CI, CIII, CIV, FN and TGF beta 1,2&3 were observed as early as 1 day after exposure. Through 14 days, changes in mRNA up to 5-fold were seen for any one dose. Dose related changes as high as 10-fold were also evident. The CI:CIII ratio increased gradually for the 5 Gy dose at 14 days postirradiation while the CI:CII ratio for the 12.5 Gy dose decreased by approximately 4-fold as compared to the control. CONCLUSION These studies suggest that alterations in expression of extracellular matrix and TGF beta mRNA occur very early after radiation injury even at low doses and may play a role in the development of chronic fibrosis.

Developmental Exposure to the Pesticides Paraquat and Maneb and the Parkinson’s Disease Phenotype

Idiopathic Parkinsons disease (PD) is associated with advanced age, but it is still unclear whether dopaminergic neuronal death results from events initiated during development, adulthood, or represents a cumulative effect across the span of life. This study hypothesized that paraquat (PQ) and maneb (MB) exposure during critical periods of development could permanently change the nigrostriatal dopamine (DA) system and enhance its vulnerability to subsequent neurotoxicant challenges. C57BL/6 mice were treated daily with saline, 0.3 mg/kg PQ, 1 mg/kg MB or PQ + MB from post-natal (PN) days 5 to 19. At 6 weeks, a 20% decrease in activity was evident only in the PQ + MB group, with a further decline (40%) observed at 6 months. A subset of mice were re-challenged as adults with saline, 10 mg/kg PQ, 30 mg/kg MB, or PQ + MB 2 x a week for 3 weeks. Mice exposed developmentally to PQ + MB and rechallenged as adults were the most affected, showing a 70% reduction in motor activity 2 weeks following the last rechallenge dose. Striatal DA levels were reduced by 37% following developmental exposure to PQ + MB only, butfollowing adult re-challenge levels were reduced by 62%. A similar pattern of nigral dopaminergic cell loss was observed, with the PQ + MB treated group exhibiting the greatest reduction, with this loss being amplified by adult re-challenge. Developmental exposure to PQ or MB alone produced minimal changes. However, following adult re-challenge, significant decreases in DA and nigral cell counts were observed, suggesting that exposure to either neurotoxicant alone produced a state of silent toxicity that was unmasked following adult re-exposure. Taken together, these findings indicate that exposure to pesticides during the PN period can produce permanent and progressive lesions of the nigrostriatal DA system, and enhanced adult susceptibility to these pesticides, suggesting that developmental exposure to neurotoxicants may be involved in the induction of neurodegenerative disorders and/or alter the normal aging process.

Pathogenesis of vertebral metastasis and epidural spinal cord compression.

The authors have studied the sequential events in the process of vertebral metastasis that result in spinal cord compression. Different tumor cell lines were injected into the systemic arterial circulation of syngeneic or nude mice, and they were killed at timed intervals after injection or when they became paraplegic. The following observations were made. The tumor cells lodged and grew in the hematopoietic bone marrow of the vertebrae. Cancer cells in the vertebral marrow cavity invaded into the spinal canal through the foramina of the vertebral veins rather than destroying the cortical bone. Tumor cell lines that grew in an infiltrative fashion migrated toward a posterior location in the spinal canal, and compressed the spinal cord from a posterior direction. Tumor cell lines that grew as compact tumors formed a tumor mass at the same location from which the cells emerged from the vertebra, and compressed the cord predominantly from an anterior direction. Radiographic evidence of vertebral metastasis was a late event, and commonly associated with significant compression of the cord and extraosseous tumor. These experimental findings may help to establish better diagnostic and treatment strategies for patients with metastatic disease of the spine.

Effect of 40-kHz Ultrasound on Acute Thrombotic Ischemia in a Rabbit Femoral Artery Thrombosis Model Enhancement of Thrombolysis and Improvement in Capillary Muscle Perfusion

BACKGROUND We have shown previously that 40-kHz ultrasound (US) at low intensity accelerates fibrinolysis in vitro with little heating and good tissue penetration. These studies have now been extended to examine the effects of 40-kHz US on thrombolysis and tissue perfusion in a rabbit model. METHODS AND RESULTS Treatment was administered with either US alone at 0.75 W/cm(2), streptokinase alone, or the combination of US and streptokinase. US or streptokinase resulted in minimal thrombolysis, but reperfusion was nearly complete with the combination after 120 minutes. US also reversed the ischemia in nonperfused muscle in the absence of arterial flow. Tissue perfusion decreased after thrombosis from 13. 7+/-0.2 to 6.6+/-0.8 U and then declined further to 4.5+/-0.4 U after 240 minutes. US improved perfusion to 10.6+/-0.5 and 12.1+/-0. 5 U after 30 and 60 minutes, respectively. This effect was reversible and declined to pretreatment values after US was discontinued. Similarly, tissue pH declined from normal to 7.05+/-0. 02 after thrombosis, but US improved pH to 7.34+/-0.03 after 60 minutes. US-induced improvement in tissue perfusion and pH also occurred after femoral artery ligation, indicating that thrombolysis did not cause these effects. CONCLUSIONS 40-kHz US at low intensity markedly accelerates fibrinolysis and also improves tissue perfusion and reverses acidosis, effects that would be beneficial in treatment of acute thrombosis.

Regression of Pulmonary Lesions Produced by Inhaled Titanium Dioxide in Rats

Inhaled ultrafine particles of TiO2 (TiO2-D, 20 nm particle size) lead to a greater pulmonary inflammatory response than larger pigment-grade particles (TiO2-F, 250 nm). Male Fisher 344 rats were exposed for 6 hours a day, 5 days a week, for 3 months to 1) filtered air (control); 2) TiO2-F, 22.3 mg/m3; 3) TiO2-D, 23.5 mg/m3; or 4) crystalline SiO2, a positive control particle (∼ 800 nm particle size, 1.3 mg/m3). Groups of 3-4 animals were sacrificed at 6 and 12 months following the completion of exposure. Pulmonary effects of exposure were evaluated using standard hematoxylin and eosin–stain sections, histochemical stains for collagen, and immunohistochemical assays for cell turnover. Six months after animals were exposed to SiO2, they had moderate focal interstitial fibrosis and moderately severe focal alveolitis. Animals exposed to TiO2-D had slightly less fibrosis. The least fibrosis was seen in the TiO2-F group. At 1 year after exposure, fibrosis was still present but decreased in the SiO2 group. The amount of interstitial fibrosis in the TiO2-D– and TiO2-F–treated animals had largely returned to untreated control levels, although an increased number of alveolar macrophages persisted, usually with retained particles. There was discordance between bromodeoxyuridine and proliferating cell nuclear antigen indices, most probably due to cytokine elaboration in the areas of inflammation, which may have altered the expression of proliferating cell nuclear antigens. There was no detectable fibroblast labeling at the 6-month observation and only very low levels at 12 months. Thus, although initially irritant, TiO2-induced lesions regressed during a 1-year period following cessation of exposure.

Immunization of cotton rats with the fusion f and large g glycoproteins of respiratory syncytial virus rsv protects against rsv challenge without potentiating rsv disease

A formalin-inactivated respiratory syncytial virus (RSV) vaccine tested 22 years ago failed to protect infant vaccinees against RSV infection or disease. Instead, lower respiratory tract disease was enhanced during subsequent infection by RSV. Enhancement of pulmonary pathology is also observed when cotton rats are immunized with formalin-inactivated RSV and subsequently infected with this virus. A major question that must be addressed for each new paramyxovirus vaccine is whether the immunogen possesses the capacity to potentiate disease. In the present study, we evaluated a newly developed purified F and G glycoprotein vaccine over a wide dosage range for immunogenicity, efficacy and capacity to potentiate pulmonary pathology in cotton rats. In addition, a formalin-inactivated RSV vaccine, which served as a positive control for enhancement of pulmonary pathology, was evaluated simultaneously. The results of these comparisons indicate that the purified F and G glycoprotein vaccine was highly immunogenic and was efficacious even in animals that developed low levels of serum-neutralizing antibodies. Furthermore, the F and G vaccine did not induce potentiation of pulmonary pathology. In contrast, formalin-inactivated RSV potentiated RSV pulmonary histopathology, but there was a sparing of potentiation at high and low doses. Both the formalin-inactivated RSV and purified F and G preparations induced a high level of serum antibodies capable of binding to purified F and G glycoproteins but both sets of antibodies had significantly reduced neutralizing activity. These results are encouraging because they suggest that purified paramyxovirus glycoproteins might be used safely as a vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in correlation of mRNA gene expression in mice sensitive and resistant to radiation-induced pulmonary fibrosis.

Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The purpose of this study was to determine if extracellular matrix protein and transforming growth factor beta mRNA expression are altered late in the course of pulmonary fibrosis after irradiation, and then to determine if these changes differ between two strains of mice which vary in their sensitivity to radiation. Radiation-sensitive (C57BL/6) and radiation-resistant (C3H/HeJ) mice were irradiated with a single dose of 5 or 12.5 Gy to the thorax. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabeled cDNA probes for collagens I, III and IV, fibronectin, and transforming growth factor beta 1 and beta 3. Autoradiographic data were quantified by video densitometry and results normalized to a control probe encoding for glyceraldehyde-3-phosphate dehydrogenase. Alterations in mRNA abundance were observed in the sensitive mice at all times, while levels in the resistant mice were unaffected until 26 weeks after irradiation. The relationship between extracellular matrix protein per se and increased mRNA abundance suggests that late matrix protein accumulation may be a function of gene expression. Differences in levels of transforming growth factor beta mRNA may lead to strain-dependent variation in fibrotic response and may also contribute to the radiation-induced component of pulmonary fibrosis.

Formulation of the purified fusion protein of respiratory syncytial virus with the saponin QS-21 induces protective immune responses in Balb/c mice that are similar to those generated by experimental infection.

The feasibility of employing a vaccine composed of the purified fraction 21 of Quillaja saponaria (QS-21) and the fusion (F) protein of respiratory syncytial virus (RSV) to induce protective immune responses in the lower respiratory tract of Balb/c mice was examined. Our goal was to compare local and systemic immune responses with those induced following immunization with the protein adsorbed to aluminium hydroxide (F/ALOH) adjuvant or by experimental infection. Sera from mice vaccinated with the QS-21 formulation (F/QS-21) contained elevated anti-F protein IgG antibody titres that were dependent on the dose of QS-21 employed. Similar to the immune responses generated by experimental infection, the sera from mice vaccinated with F/QS-21 possessed greater capacity to neutralize virus infectivity that was associated with the generation of heightened complement-fixing IgG2a antibody titres. In contrast, vaccination with F/ALOH elicited systemic immune responses that were characterized by a predominance of protein-specific antibodies of the IgG1 subclass and lower neutralizing antibody titres. The capacity of F/QS-21 to facilitate local pulmonary immune responses was also examined and found to be similar to those induced by experimental infection. After virus challenge, a 90-fold increase in the number of F protein-specific antibody-secreting cells was observed and associated with the clearance of virus from the infected lungs. Moreover, elevated levels of antigen-dependent killer cell activity were detected and appeared to be mediated by class I major histocompatibility complex restricted CD8+ T cells. Additional characterization of the pulmonary immune response was performed on the cellular infiltrates obtained after bronchoalveolar lavage and on formalin-fixed lung tissue. The local protective immune responses induced after challenge of the groups immunized with F/QS-21 or infectious virus were significantly different from those elicited in naive control mice injected with adjuvant alone, or in mice immunized with F/ALOH. The cellularity of the lavage fluids from the former groups was characterized by a significantly greater percentage of lymphocytes and less neutrophils. In similar fashion histological evaluation of the lungs from mice immunized with F/QS-21 or infectious virus revealed significantly elevated local immune responses after challenge. In conclusion, the results suggest that formulation with F/QS-21 alters the qualitative and quantitative nature of the immune response to the F glycoprotein when compared with the traditional aluminium-based adjuvants.