Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Neuroprotection of medical IOP-lowering therapy

Intraocular pressure (IOP)-lowering therapy has been shown to arrest or retard the progression of optic neuropathy typical for glaucoma and can, thus, be described as neuroprotective. At present, six classes of medical therapy are employed, namely parasympathomimetics, alpha/beta-sympathomimetics, β-blockers, carbonic anhydrase inhibitors, α₂-adrenergic receptor agonists and prostaglandin analogues. For several of these substances, some experimental evidence exists of a possible neuroprotective mechanism, beyond their IOP-lowering activity. β-Blockers are involved in the up-regulation of brain-derived neurotrophic factor (BDNF) and can decrease glutamate-mediated NMDA receptor activation. Not only systemic but also topical carbonic anhydrase inhibitors are able to increase retinal blood flow. α₂-Adrenergic receptor agonists can up-regulate the formation of BDNF and anti-apoptotic factors. Prostaglandin analogues increase blood flow to the eye, possibly including the retina. To date, evidence for a neuroprotective effect independent of IOP regulation in human glaucoma is scarce and has only been shown to be likely for the α₂-adrenergic receptor agonist, brimonidine.     

Largest vertebrate vomeronasal type 1 receptor gene repertoire in the semiaquatic platypus

Vertebrate vomeronasal chemoreception plays important roles in many aspects of an organism's daily life, such as mating, territoriality, and foraging. Vomeronasal type 1 receptors (V1Rs) and vomeronasal type 2 receptors (V2Rs), 2 large families of G protein-coupled receptors, serve as vomeronasal receptors to bind to various pheromones and odorants. Contrary to the previous observations of reduced olfaction in aquatic and semiaquatic mammals, we here report the surprising finding that the platypus, a semiaquatic monotreme, has the largest V1R repertoire and nearly largest combined repertoire of V1Rs and V2Rs of all vertebrates surveyed, with 270 intact genes and 579 pseudogenes in the V1R family and 15 intact genes, 55 potentially intact genes, and 57 pseudogenes in the V2R family. Phylogenetic analysis shows a remarkable expansion of the V1R repertoire and a moderate expansion of the V2R repertoire in platypus since the separation of monotremes from placentals and marsupials. Our results challenge the view that olfaction is unimportant to aquatic mammals and call for further study into the role of vomeronasal reception in platypus physiology and behavior.     

GE-25-like immunoreactivity in the rat eye

This study aimed to investigate the presence and distribution of the chromogranin A-derived peptide GE-25 in the rat eye. The molecular form detected by the GE-25 antiserum was evaluated in the rat trigeminal ganglion, retina and remaining tissues of the rat eye by means of Western blots and the distribution pattern of GE-25-like immunoreactivity was studied in the rat eye and rat trigeminal ganglion by immunofluorescence. One single band of approximately 70kDa was stained in the trigeminal ganglion and retina which represents the uncleaved intact chromogranin A indicating that the proteolytic processing of chromogranin A to GE-25 is limited in these tissues. Sparse GE-25-like immunoreactive nerve fibers were visualized in the corneal stroma, at the limbus around blood vessels, in the sphincter and dilator muscle and stroma of the iris, in the stroma of the ciliary body and ciliary processes and in the stroma and around blood vessels in the choroid. This distribution pattern is characteristic for neuropeptides whereas the presence of immunoreactivity in the corneal endothelium and in Müller glia in the retina is atypical. GE-25-like immunoreactivity was found in small to medium-sized ganglion cells in the rat trigeminal ganglion clearly indicating that the nerve fibers in the rat eye are of sensory origin. The colocalization of GE-25-immunoreactivity with SP-immunoreactivity in the rat ciliary body is in agreement with the presumption of the sensory nature of the innervation of the anterior segment of the eye by GE-25.     

Longitudinal analysis of taurine induced effects on the tear proteome of contact lens wearers and dry eye patients using a RP-RP-Capillary-HPLC-MALDI TOF/TOF MS approach

Tear proteomic studies revealed distinct similarities between contact lens wearers and dry eye patients. AMO Complete® multipurpose contact lens cleaning solutions containing taurine seem to have a beneficial effect regarding contact lens induced dry eye. To illuminate the effect of taurine on the tear proteome of contact lens wearers and sicca patients we developed a gel-based RP-RP capillary HPLC–MALDI TOF/TOF MS strategy. Two contact lens wearer groups, one using eye drops containing 0.05% taurine; the other for control physiological NaCl solution were monitored. Also, a third group of sicca patients using taurine solution was studied (N = 4 individuals/group). Tear pools of each group at six time points over 5 weeks were analyzed. In summary 267 tear proteins were identified. We found a protein subset showing a linear taurine response with R² values ≥ 0.5. Taurine effects were detected predominantly in the contact lens group demonstrated by distinct level decreases. Most protein candidates were related to inflammation. Since levels of these proteins differentiate from those of a healthy non-contact lens wearer reference they are supposed to be involved in contact lens induced dry eye and should be focused on in further studies.     

Retinal ganglion cell loss is accompanied by antibody depositions and increased levels of microglia after immunization with retinal antigens

Background

Antibodies against retinal and optic nerve antigens are detectable in glaucoma patients. Recent studies using a model of experimental autoimmune glaucoma demonstrated that immunization with certain ocular antigens causes an immun-mediated retinal ganglion cell loss in rats.

Methodology/Principal Findings

Rats immunized with a retinal ganglion cell layer homogenate (RGA) had a reduced retinal ganglion cell density on retinal flatmounts (p = 0.007) and a lower number of Brn3⁺retinal ganglion cells (p = 0.0001) after six weeks. The autoreactive antibody development against retina and optic nerve was examined throughout the study. The levels of autoreactive antibodies continuously increased up to 6 weeks (retina: p = 0.004; optic nerve: p = 0.000003). Additionally, antibody deposits were detected in the retina (p = 0.02). After 6 weeks a reactive gliosis (GFAP density: RGA: 174.7±41.9; CO: 137.6±36.8, p = 0.0006; %GFAP⁺ area: RGA: 8.5±3.4; CO: 5.9±3.6, p = 0.006) as well as elevated level of Iba1⁺ microglia cells (p = 0.003) was observed in retinas of RGA animals.

Conclusions/Significance

Our findings suggest that these antibodies play a substantial role in mechanisms leading to retinal ganglion cell death. This seems to lead to glia cell activation as well as the invasion of microglia, which might be associated with debris clearance.     

Serum and Antibodies of Glaucoma Patients Lead to Changes in the Proteome,Especially Cell Regulatory Proteins,in Retinal Cells

Purpose

Previous studies show significantly specifically changed autoantibody reactions against retinal antigens in the serum of glaucoma and ocular hypertension (OHT) patients in comparison to healthy people. As pathogenesis of glaucoma still is unknown the aim of this study was to analyze if the serum and antibodies of glaucoma patients interact with neuroretinal cells.

Methods

R28 cells were incubated with serum of patients suffering from primary open angle glaucoma (POAG), normal tension glaucoma (NTG) or OHT, POAG serum after antibody removal and serum from healthy people for 48 h under a normal or an elevated pressure of 15000 Pa (112 mmHg). RGC5 cells were additionally incubated with POAG antibodies under a normal pressure. Protein profiles of the R28 cells were measured with Seldi-Tof-MS, protein identification was performed with Maldi-TofTof-MS. Protein analysis of the RGC5 cells was performed with ESI-Orbitrap MS. Statistical analysis including multivariate statistics, variance component analysis as well as calculating Mahalanobis distances was performed.

Results

Highly significant changes of the complex protein profiles after incubation with glaucoma and OHT serum in comparison to healthy serum were detected, showing specific changes in the cells (e.g. Protein at 9192 Da (p<0.001)). The variance component analysis showed an effect of the serum of 59% on the cells. The pressure had an effect of 11% on the cells. Antibody removal led to significantly changed cell reactions (p<0.03). Furthermore, the incubation with POAG serum and its antibodies led to pro-apoptotic changes of proteins in the cells.

Conclusions

These studies show that the serum and the antibodies of glaucoma patients significantly change protein expressions involved in cell regulatory processes in neuroretinal cells. These could lead to a higher vulnerability of retinal cells towards stress factors such as an elevated IOP and eventually could lead to an increased apoptosis of the cells as in glaucoma.     

Soulamarin Isolated from Calophyllum brasiliense (Clusiaceae) Induces Plasma Membrane Permeabilization of Trypanosoma cruzi and Mytochondrial Dysfunction

Chagas disease is caused by the parasitic protozoan Trypanosoma cruzi. It has high mortality as well as morbidity rates and usually affects the poorer sections of the population. The development of new, less harmful and more effective drugs is a promising research target, since current standard treatments are highly toxic and administered for long periods. Fractioning of methanol (MeOH) extract of the stem bark of Calophyllum brasiliense (Clusiaceae) resulted in the isolation of the coumarin soulamarin, which was characterized by one- and two-dimensional ¹H- and ¹³C NMR spectroscopy as well as ESI mass spectrometry. All data obtained were consistent with a structure of 6-hydroxy-4-propyl-5-(3-hydroxy-2-methyl-1-oxobutyl)-6″,6″-dimethylpyrane-[2″,3″:8,7]-benzopyran-2-one for soulamarin. Colorimetric MTT assays showed that soulamarin induces trypanocidal effects, and is also active against trypomastigotes. Hemolytic activity tests showed that soulamarin is unable to induce any observable damage to erythrocytes (c_max. = 1,300 µM). The lethal action of soulamarin against T. cruzi was investigated by using amino(4-(6-(amino(iminio)methyl)-1H-indol-2-yl)phenyl)methaniminium chloride (SYTOX Green and 1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[4-(chloromethyl)phenyl]-2,3,6,7,12,13,16,17-octahydro-chloride (MitoTracker Red) as fluorimetric probes. With the former, soulamarin showed dose-dependent permeability of the plasma membrane, relative to fully permeable Triton X-100-treated parasites. Spectrofluorimetric and fluorescence microscopy with the latter revealed that soulamarin also induced a strong depolarization (ca. 97%) of the mitochondrial membrane potential. These data demonstrate that the lethal action of soulamarin towards T. cruzi involves damages to the plasma membrane of the parasite and mitochondrial dysfunction without the additional generation of reactive oxygen species, which may have also contributed to the death of the parasites. Considering the unique mitochondrion of T. cruzi, secondary metabolites of plants affecting the bioenergetic system as soulamarin may contribute as scaffolds for the design of novel and selective drug candidates for neglected diseases, mainly Chagas disease.