We report results of a study utilizing a recently developed tissue diagnostic method, based on label-free spectral techniques, for the classification of lung cancer histopathological samples from a tissue microarray. The spectral diagnostic method allows reproducible and objective diagnosis of unstained tissue sections. This is accomplished by acquiring infrared hyperspectral data sets containing thousands of spectra, each collected from tissue pixels about 6 mm on edge; these pixel spectra contain an encoded snapshot of the entire biochemical composition of the pixel area. The hyperspectral data sets are subsequently decoded by methods of multivariate analysis, which reveal changes in the biochemical composition between tissue types, and between various stages and states of disease. In this study, a detailed comparison between classical and spectral histopathology (SHP) is presented, which suggests SHP can achieve levels of diagnostic accuracy that is comparable to that of multi-panel immunohistochemistry. KEYWORDS: artificial neural network analysis; histopathology; immunohistochemistry; lung cancer; spectral histopathology This paper reports a large-scale study of a new technology to classify four common forms of lung cancers, and distinguish them from normal tissues. The new methodology introduced here utilizes optical measurements on unstained tissue 1 for spectral data acquisition, and does not utilize any immunohistochemical or other stains or labels for classification. As the diagnostic procedure is instrument based and utilizes trained computer algorithms for classification, this method offers reproducibility, complete objectivity and improved accuracy over present methodology.Optical methods have been used in histology and pathology ever since these methods were first described. After all, staining tissues or cells by hematoxylin/eosin (H&E), followed by (visual) microscopic examination is a form of spectral analysis: different compartments of the cell respond differently to basophilic and eosinophilic stains and thus, allow a 'spectral analysis' using the eye as a detector. This method can reveal an amazing amount of information but is inherently subjective. More recent optical methods have used image capture at a few selected wavelengths, and computer analysis of the image planes, for tissue analysis. 2 Immunohistochemistry (IHC), to date the most advanced optical method to detect the presence of certain cancer signatures or markers 3,4 uses detection of specific antibodies labeled with easily observable stains.The new approach reported here is based on the observation of inherent spectral signatures (as opposed to any external stains or labels used to treat the sample) of cellular components to aid classical cytopathology and histopathology. 5 The paradigm for the spectral approach is that the transition from normal tissue or cells to diseased states is accompanied by changes in the overall biochemical composition of the tissue, along with well-known changes in cellular morphology and tissue archite...

Development of a vaccine to protect against cryptococcosis is a priority given the enormous global burden of disease in at-risk individuals. Using glucan particles (GPs) as a delivery system, we previously demonstrated that mice vaccinated with crude Cryptococcus-derived alkaline extracts were protected against lethal challenge with Cryptococcus neoformans and Cryptococcus gattii. The goal of the present study was to identify protective protein antigens that could be used in a subunit vaccine. Using biased and unbiased approaches, six candidate antigens (Cda1, Cda2, Cda3, Fpd1, MP88, and Sod1) were selected, recombinantly expressed in Escherichia coli, purified, and loaded into GPs. Three mouse strains (C57BL/6, BALB/c, and DR4) were then vaccinated with the antigen-laden GPs, following which they received a pulmonary challenge with virulent C. neoformans and C. gattii strains. Four candidate vaccines (GP-Cda1, GP-Cda2, GP-Cda3, and GP-Sod1) afforded a significant survival advantage in at least one mouse model; some vaccine combinations provided added protection over that seen with either antigen alone. Vaccine-mediated protection against C. neoformans did not necessarily predict protection against C. gattii. Vaccinated mice developed pulmonary inflammatory responses that effectively contained the infection; many surviving mice developed sterilizing immunity. Predicted T helper cell epitopes differed between mouse strains and in the degree to which they matched epitopes predicted in humans. Thus, we have discovered cryptococcal proteins that make promising candidate vaccine antigens. Protection varied depending on the mouse strain and cryptococcal species, suggesting that a successful human subunit vaccine will need to contain multiple antigens, including ones that are species specific.

Keratins that are overexpressed selectively in human carcinomas may offer diagnostic and prognostic utility. In this study, we show that high expression of keratin-17 (K17) predicts poor outcome in patients with cervical cancer, at early or late stages of disease, surpassing in accuracy either tumor staging or loss of p27 KIP1 as a negative prognostic marker in this setting. We investigated the mechanistic basis for the biologic impact of K17 through loss-and gain-of-function experiments in human cervix, breast, and pancreatic cancer cells. Specifically, we determined that K17 functions as an oncoprotein by regulating the subcellular localization and degradation of p27 KIP1 . We found that K17 was released from intermediate filaments and translocated into the nucleus via a nuclear localization signal (NLS), specific among keratins, where it bound p27 KIP1 during G 1 phase of the cell cycle. p27 KIP1 lacks a nuclear export signal (NES) and requires an adaptor for CRM1 binding for nuclear export. In K17, we defined and validated a leucine-rich NES that mediated CRM1 binding for export. Cervical cancer cells expressing K17 mutations in its NLS or NES signals exhibited an increase in levels of nuclear p27 KIP1 , whereas cells expressing wild-type K17 exhibited a depletion in total endogenous p27 KIP1 . In clinical specimens of cervical cancer, we confirmed that the expressions of K17 and p27 KIP1 were inversely correlated, both across tumors and within individual tumors. Overall, our findings establish that K17 functions specially among keratins as an oncoprotein by controlling the ability of p27 KIP1 to influence cervical cancer pathogenesis. Cancer Res; 75(17); 3650-62. Ó2015 AACR.

Aspergillus fumigatus causes invasive pulmonary disease in immunocompromised hosts and allergic asthma in atopic individuals. We studied the contribution of lung eosinophils to these fungal diseases. By in vivo intracellular cytokine staining and confocal microscopy, we observed that eosinophils act as local sources of IL-23 and IL-17. Remarkably, mice lacking eosinophils had a >95% reduction in the percentage of lung IL-23p19+ cells as well as markedly reduced IL-23 heterodimer in lung lavage fluid. Eosinophils killed A. fumigatus conidia in vivo. Eosinopenic mice had higher mortality rates, decreased recruitment of inflammatory monocytes, and decreased expansion of lung macrophages after challenge with conidia. All of these functions underscore a potential protective role for eosinophils in acute aspergillosis. Given the postulated role for IL-17 in asthma pathogenesis, we assessed whether eosinophils could act as sources of IL-23 and IL-17 in models where mice were sensitized to either A. fumigatus antigens or ovalbumin (OVA). We found IL-23p19+ IL-17AF+ eosinophils in both allergic models. Moreover, close to 95% of IL-23p19+ cells and >90% of IL-17AF+ cells were identified as eosinophils. These data establish a new paradigm in acute and allergic aspergillosis whereby eosinophils act not only as effector cells but also as immunomodulatory cells driving the IL-23/IL-17 axis and contributing to inflammatory cell recruitment.