Novel Chromogens for Immunohistochemistry in Spatial Biology.

Spatial relations between tumor cells and host-infiltrating cells are increasingly important in both basic science and clinical research. In this study, we have tested the feasibility of using standard methods of immunohistochemistry (IHC) in a multiplex staining system using a newly developed set of chromogenic substrates for the peroxidase and alkaline phosphatase enzymes. Using this approach, we have developed a set of chromogens characterized by (1) providing fine cellular detail, (2) non-overlapping spectral profiles, (3) an absence of interactions between chromogens, (4) stability when stored, and (5) compatibility with current standard immunohistochemistry practices. When viewed microscopically under brightfield illumination, the chromogens yielded the following colors: red, black, blue, yellow, brown, and green. By selecting compatible color combinations, we have shown feasibility for four-color multiplex staining. Depending on the particular type of analysis being performed, visual analysis, without the aid of computer-assisted image analysis, was sufficient to differentiate up to four different markers.

Recent Advances in Chromogens for Immunohistochemistry.

Various staining strategies and color combinations have been developed to perform single and double immunohistochemical staining on biological samples. However, until recently, the lack of appropriate chromogen color combinations has severely limited many of these methods. Fortunately, this situation has dramatically improved with the introduction of new chromogens and methods of analysis. This article reviews recent trends in multicolor immunohistochemical staining methods that are finding broad applications in both research and clinical laboratories.

A Look into Obstructive Sleep Apnea among Ethiopians.

BACKGROUND: Obstructive Sleep Apnea (OSA) is prevalent throughout the world. However, there are currently limited data concerning the prevalence of OSA in populations that originate from developing countries; the prevalence of OSA is expected to rise in these countries. OSA is poorly characterized amongst Ethiopians, and our study is the first to describe clinical characteristics of OSA among Ethiopians. METHODS: We conducted a retrospective study of primarily Ethiopian patients at an internal medicine clinic in Rockville, Maryland. All patients (n=24) were evaluated for daytime sleepiness using the Epworth Sleepiness Scale (ESS) and received physical examinations and polysomnograms (PSG) by either portable monitoring (Itamar WatchPAT 200 device) or in-lab. Statistical analyses were performed in R. RESULTS: Linear regression model of Body-Mass Index (BMI) and Apnea-Hypopnea Index (AHI) indicated that for every 1-unit increase in BMI, there was a 0.8657-unit increase in AHI (p<0.05). Pearson's correlation coefficient indicateda positive linear relationship between BMI and AHI (0.47) (p<0.05). Adjusted linear regression model for AHI and oxygen saturation indicated that for every 1-unit increase of AHI, there was a 0.8452-unit decrease in nocturnal oxygen saturation (p<0.05). Pearson's correlation coefficient did not demonstrate significance between AHI and oxygen desaturation (p=0.062). Patients received either continuous positive airway pressure (CPAP) (n=15) or oral appliance therapy (n=3). CONCLUSION: All patients who complied with therapy reported improved sleep quality, snoring resolution, and improved daytime alertness. Practitioners in developing countries should suspect OSA in the right clinical setting and offer diagnostic and therapeutic services when available.

A small-molecule inhibitor of the ribonucleolytic activity of human angiogenin that possesses antitumor activity.

The results of previous preclinical and clinical studies have identified angiogenin (ANG) as a potentially important target for anticancer therapy. Here we report the design and implementation of a high-throughput screening assay to identify small molecules that bind to the ribonucleolytic active site of ANG, which is critically involved in the induction of angiogenesis by this protein. Screening of 18,310 compounds from the National Cancer Institute (NCI) Diversity Set and ChemBridge DIVERSet yielded 15 hits that inhibit the enzymatic activity of ANG with K(i) values <100 microM. One of these, NCI compound 65828 [8-amino-5-(4'-hydroxybiphenyl-4-ylazo)naphthalene-2-sulfonate; K(i) = 81 microM], was selected for more detailed studies. Minor changes in ANG or ligand structure markedly reduced potency, demonstrating that inhibition reflects active-site rather than nonspecific binding; these observations are consistent with a computationally generated model of the ANG.65828 complex. Local treatment with modest doses of 65828 significantly delayed the formation of s.c. tumors from two distinct human cancer cell types in athymic mice. ANG is the likely target involved because (i) a 65828 analogue with much lower potency against the enzymatic activity of ANG failed to exert any antitumor effect, (ii) tumors from 65828-treated mice had fewer interior blood vessels than those from control mice, and (iii) 65828 appears to have no direct effect on the tumor cells. Our findings provide considerable support for the targeting of the enzymatic active site of ANG as a strategy for developing new anticancer drugs.

Inhibition of prostate carcinoma establishment and metastatic growth in mice by an antiangiogenin monoclonal antibody.

A neutralizing monoclonal antibody (MAb) 26-2F to human angiogenin, a potent inducer of neovascularization, has been shown previously to prevent or delay the appearance of angiogenin-secreting human colon, fibrosarcoma and lung tumor cell xenografts implanted subcutaneously (s.c.) into athymic mice. In an analogous model system, we report here that the antibody also prevents the establishment of PC-3 androgen-independent human prostate cancer tumors in, on average, 40% of treated mice (p < 0.0001, survivor analysis). Intriguingly, combining MAb 26-2F together with cisplatin and suramin, 2 therapeutic agents that together showed little antitumor activity in the aforementioned model, resulted in an even greater degree of protection (71% protected, p = 0.009 compared to antibody treatment alone). This protective effect persisted several weeks after cessation of treatment. Additionally, prophylactic systemic administration of MAb 26-2F dramatically reduced by 50% the formation of spontaneous regional metastasis originating from primary growth in the prostate gland of PC-3M cells, highly metastatic variants of PC-3. Protection from metastasis was still significant when treatment with MAb 26-2F was delayed until after the primary tumor was well established. The antibody is not directly cytotoxic to either cell type, both of which secrete angiogenin in vitro and when growing as tumors in vivo, but changes the pattern of vascularity in primary tumors growing orthotopically. These findings, together with the observation that angiogenin protein and mRNA are apparently overexpressed in cancerous vs. normal human prostate tissues, demonstrate that angiogenin antagonism represents a promising new approach for preventing progression and metastasis of clinical prostate cancer.