A blocking antibody against canine CSF-1R maturated by limited CDR mutagenesis

CSF-1R is a receptor mostly associated with the mononuclear phagocytic system. However, its expression within tumors has been linked with poor prognosis in both humans and dogs. Accordingly, several reports have demonstrated the beneficial effects of blocking CSF-1R in model systems of cancer. In this study, we generated a monoclonal antibody that could block CSF-1R in dogs as the first step to develop an anticancer drug for this species. Initially, an antibody was raised by the hybridoma methodology against the fragment responsible for receptor dimerization. mAb3.1, one of the resulting hybridoma clones, was able to bind macrophages in fixed tissues and was shown to inhibit cells of the mononuclear phagocytic line. Nevertheless, mAb 3.1 could not bind to some glycoforms of the receptor in its native form, while also demonstrating cross-reactivity with other proteins. To enhance binding properties of the mAb, five amino acids of the complementarity-determining region 2 of the variable heavy chain of mAb3.1 were mutated by PCR, and the variant scFv clones were screened by phage display. The selected scFv clones demonstrated improved binding to the native receptor as well as increased anti-macrophage activity. The resulting scFv antibody fragment presented here has the potential for use in cancer patients and in inflammatory diseases. Furthermore, this work provides insights into the use of such restricted mutations in antibody engineering.</p

Assessment of MMP-9, TIMP-1, and COX-2 in normal tissue and in advanced symptomatic and asymptomatic carotid plaques

<p>Abstract</p> <p>Background</p> <p>Mature carotid plaques are complex structures, and their histological classification is challenging. The carotid plaques of asymptomatic and symptomatic patients could exhibit identical histological components.</p> <p>Objectives</p> <p>To investigate whether matrix metalloproteinase 9 (MMP-9), tissue inhibitor of MMP (TIMP), and cyclooxygenase-2 (COX-2) have different expression levels in advanced symptomatic carotid plaques, asymptomatic carotid plaques, and normal tissue.</p> <p>Methods</p> <p>Thirty patients admitted for carotid endarterectomy were selected. Each patient was assigned preoperatively to one of two groups: group I consisted of symptomatic patients (n = 16, 12 males, mean age 66.7 ± 6.8 years), and group II consisted of asymptomatic patients (n = 14, 8 males, mean age 67.6 ± 6.81 years). Nine normal carotid arteries were used as control. Tissue specimens were analyzed for fibromuscular, lipid and calcium contents. The expressions of MMP-9, TIMP-1 and COX-2 in each plaque were quantified.</p> <p>Results</p> <p>Fifty-eight percent of all carotid plaques were classified as Type VI according to the American Heart Association Committee on Vascular Lesions. The control carotid arteries all were classified as Type III. The median percentage of fibromuscular tissue was significantly greater in group II compared to group I (<it>p </it>< 0.05). The median percentage of lipid tissue had a tendency to be greater in group I than in group II (<it>p </it>= 0.057). The percentages of calcification were similar among the two groups. MMP-9 protein expression levels were significantly higher in group II and in the control group when compared with group I (p < 0.001). TIMP-1 expression levels were significantly higher in the control group and in group II when compared to group I, with statistical difference between control group and group I (p = 0.010). COX-2 expression levels did not differ among groups. There was no statistical correlation between MMP-9, COX-2, and TIMP-1 levels and fibrous tissue.</p> <p>Conclusions</p> <p>MMP-9 and TIMP-1 are present in all stages of atherosclerotic plaque progression, from normal tissue to advanced lesions. When sections of a plaque are analyzed without preselection, MMP-9 concentration is higher in normal tissues and asymptomatic surgical specimens than in symptomatic specimens, and TIMP-1 concentration is higher in normal tissue than in symptomatic specimens.</p

Management of Hazardous Waste and Contaminated Land

Regulation of hazardous waste and cleanup of contaminated sites are two major components of modern public policy for environmental protection. We review the literature on these related areas, with emphasis on empirical analyses. Researchers have identified many behavioral responses to regulation of hazardous waste, including changes in the location of economic activity. However, the drivers behind compliance with these costly regulations remain a puzzle, as most research suggests a limited role for conventional enforcement. Increasingly sophisticated research examines the benefits of cleanup of contaminated sites, yet controversy remains about whether the benefits of cleanup in the U.S. exceed its costs. Finally, research focusing on the imposition of legal liability for damages from hazardous waste finds advantages and disadvantages of the U.S. reliance on legal liability to pay for cleanup, as opposed to the government-financed approaches more common in Europe

Plasma Cysteine/Cystine Reduction Potential Correlates with Plasma Creatinine Levels in Chronic Kidney Disease

&lt;b&gt;&lt;i&gt;Background/Aims:&lt;/i&gt;&lt;/b&gt; Oxidative stress has been considered a nontraditional risk factor for cardiovascular disease in the chronic kidney disease (CKD) population, possibly triggered by uremic toxicity. &lt;b&gt;&lt;i&gt;Methods:&lt;/i&gt;&lt;/b&gt; A chromatographic method with coulometric detection was adapted to directly and simultaneously determine cysteine (Cys) and cystine (Cyss) in plasma samples. Healthy subjects and CKD subjects in different stages were analyzed. The free Cys and free Cyss levels in their plasma were determined, and the reduction potential [E&lt;sub&gt;h(Cyss/2Cys)&lt;/sub&gt;] was calculated with the Nernst equation. &lt;b&gt;&lt;i&gt;Results:&lt;/i&gt;&lt;/b&gt; Healthy plasma presented E&lt;sub&gt;h(Cyss/2Cys)&lt;/sub&gt; of –123 ± 7 mV. Plasma E&lt;sub&gt;h(Cyss/2Cys)&lt;/sub&gt; correlated significantly with creatinine levels (p &lt; 0.0001, r = 0.62). &lt;b&gt;&lt;i&gt;Conclusion:&lt;/i&gt;&lt;/b&gt; Plasma E&lt;sub&gt;h(Cyss/2Cys)&lt;/sub&gt; correlated with increased levels of plasma creatinine, supporting the view that uremia triggers oxidative stress. In addition, it may be used as a quantitative oxidative stress biomarker in uremic conditions.</jats:p

Recombinant human interferon‐α14 for the treatment of canine allergic pruritic disease in eight dogs

Background Allergic pruritic diseases are increasingly common in dogs. This group of conditions hampers life quality as pruritus progressively interferes with normal behaviours. Therefore, new treatment modalities for canine allergic pruritic diseases are necessary. While novel drugs have recently reached the market, there is still the need for other therapeutic approaches. Some dogs are refractory even to the newer compounds, and cost is also an important issue for these. Older therapeutic modalities are only moderately successful or have considerable secondary effects, as is the case with glucocorticoids. Objectives Report on the use of recombinant human interferon-alpha 14 (rhIFN-alpha 14) for the treatment of canine allergic pruritus. Following the experience with a similar compound in the Japanese market, it was expected that rhIFN-alpha 14 could alter the Th1/Th2 disbalance that drives these diseases. Methods Here, we present an uncontrolled trial in which eight dogs with clinical diagnosis of allergic pruritus were treated with rhIFN-alpha 14, either orally or via subcutaneous injections. Skin condition, microbiota and anti-interferon antibody levels were assessed. Results The parenteral use of interferon induced hypersensitivity in two of the three dogs in which it was used. The oral administration was consistently safe and could reduce signs of the allergic condition in three of the five treated animals. Treatment also altered the skin microbiota, as verified by next-generation sequencing. Conclusion The present results indicate that rhIFN-alpha 14 is a viable candidate for the treatment of canine allergic pruritus. Future controlled studies are needed, and the oral route is indicated for further trials