Immunofluorescent localization of C-erbB-2 oncoprotein in breast cancer: a preliminary study

Monoclonal antibody CIBCgp185 of IgG2a isotype has been generated against C-erbB-2 oncoprotein using BT474 breast carcinoma cell line as immunogen. Earlier studies have revealed that this MAb has potential application as diagnostic tool in the detection of breast cancers overexpressing C-erbB-2. In the present study, the reactivity pattern of this MAb was studied on frozen sections of 28 malignant and 15 normal breast tissues and on cultured mammary tumor cell lines by indirect immunofluorescent staining. Results indicated that gp185C-erbB-2 was overexpressed in 6 malignant specimens, indicating correlation with immunohistochemical analysis studied previously. These results indicate that immunofluorescence might also be used to study C-erbB-2 overexpression in breast cancers and could serve as a confirmatory test for IHC. However, further studies with large number of cases are needed to confirm these results.     

Recent advances in targeting the autotaxin-lysophosphatidate-lipid phosphate phosphatase axis in vivo

Extracellular lysophosphatidate(LPA) is a potent bioactive lipid that signals through six G-protein-coupled receptors.This signaling is required for embryogenesis,tissue repair and remodeling processes.LPA is produced from circulating lysophosphatidylcholine by autotaxin(ATX),and is degraded outside cells by a family of three enzymes called the lipid phosphate phosphatases(LPPs).In many pathological conditions,particularly in cancers,LPA concentrations are increased due to high ATX expression and low LPP activity.In cancers,LPA signaling drives tumor growth,angiogenesis,metastasis,resistance to chemotherapy and decreased efficacy of radiotherapy.Hence,targeting the ATX-LPA-LPP axis is an attractive strategy for introducing novel adjuvant therapeutic options.In this review,we will summarize current progress in targeting the ATX-LPA-LPP axis with inhibitors of autotaxin activity,LPA receptor antagonists,LPA monoclonal antibodies,and increasing low LPP expression.Some of these agents are already in clinical trials and have applications beyond cancer,including chronic inflammatory diseases.     

CD44 expression is a feature of prostatic small cell carcinoma and distinguishes it from its mimickers

Small cell neuroendocrine carcinoma of the prostate is a rare variant of prostatic cancer that shares morphologic similarity with prostatic adenocarcinoma of Gleason 5 pattern. It has also been considered morphologically and immunohistochemically indistinguishable from small cell neuroendocrine carcinomas of other origins. CD44 is a cell-surface molecule proposed to identify cancer stem/progenitor cells in prostate cancer. We performed immunohistochemical study for CD44 expression in 11 cases of prostatic small cell neuroendocrine carcinoma and compared its patterns of expression with 73 cases of prostatic adenocarcinoma and 47 cases of small cell neuroendocrine carcinomas of other organs. Strong and diffuse membrane staining for CD44 was observed in 100% of the prostatic small cell neuroendocrine carcinomas. In conventional adenocarcinomas of the prostate, positive staining was only seen in rare, scattered tumor cells; and CD44 staining was negative in most of the small cell neuroendocrine carcinomas of nonprostate origin. The difference in CD44 expression between small cell neuroendocrine carcinomas of the prostate and those of other organs are statistically significant (P < .001). Our study demonstrates the utility of immunohistochemical staining for CD44 in distinguishing prostatic small cell neuroendocrine carcinoma from its mimickers including prostatic adenocarcinoma of Gleason 5 pattern and small cell neuroendocrine carcinomas of other organs. CD44 is the first marker that shows a high degree of tissue/organ specificity for small cell neuroendocrine carcinomas. Because CD44 is a putative marker of prostate cancer stem cells, the strong and diffuse expression of CD44 and the lack of expression of prostate luminal differentiation markers androgen receptor and prostatic specific antigen in prostatic small cell neuroendocrine carcinomas suggest that the tumor cells may retain cancer stem cell features.     

An analysis of B-cell epitope discontinuity

Although it is widely acknowledged that most B-cell epitopes are discontinuous, the degree of discontinuity is poorly understood. For example, given that an antigen having a single epitope that has been chopped into peptides of a specific length, what is the likelihood that one of the peptides will span all the residues belonging to that epitope? Or, alternatively, what is the largest proportion of the epitope's residues that any peptide is likely to contain? These and similar questions are of direct relevance both to computational methods that aim to predict the location of epitopes from sequence (linear B-cell epitope prediction methods) and window-based experimental methods that aim to locate epitopes by assessing the strength of antibody binding to synthetic peptides on a chip. In this paper we present an analysis of the degree of B-cell epitope discontinuity, both in terms of the structural epitopes defined by a set of antigen-antibody complexes in the Protein Data Bank, and with respect to the distribution of key residues that form functional epitopes. We show that, taking a strict definition of discontinuity, all the epitopes in our data set are discontinuous. More significantly, we provide explicit guidance about the choice of peptide length when using window-based B-cell epitope prediction and mapping techniques based on a detailed analysis of the likely effectiveness of different lengths.     

Epithelioid angiomyolipoma of kidney with atypical nuclear features and intranuclear inclusions on cytology

Described herein are the cytological findings of epithelioid angiomyolipoma (EAML) of the kidney with atypical nuclear features mistaken for renal cell carcinoma (RCC) in a 61‐year‐old male patient. Aspirates from this large renal mass were cellular and showed epithelioid cell clusters with focally crowded nuclei showing moderate anisonucleosis, small nucleoli, and prominent eosinophilic intranuclear inclusions. Failure to recognize the scanty adipose tissue component and preponderance of epithelioid cells with nuclear pleomorphism lead to a diagnosis of RCC on cytology. On histology, the tumor was essentially composed of epithelioid and spindle cells that showed the typical immunoprofile of an angiomyolipoma and only occasional foci of typical AML were seen. The hilar lymph node was involved in contiguity. However, in view of lack of obvious features of malignancy, the tumor was labeled as EAML with atypical features. Immunocytochemistry on the destained cytology aspirates revealed strong smooth muscle actin staining of all cells. To conclude, EAML can mimic a RCC. In such instances, lack of arborizing vasculature, absence of cytoplasmic fatty vacoulation, crowded nuclei with intranuclear inclusions, and lack of prominent nucleoli along with typical immunophenotype of EAML may assist in the cytology diagnosis. Diagn. Cytopathol. 2011;39:278–282. © 2010 Wiley‐Liss, Inc.     

Inhibiting N-glycan processing increases the antibody binding affinity and effector function of human natural killer cells

Novel approaches are required to improve the efficacy of immunotherapies and increase the proportion of patients who experience a benefit. Antibody-dependent cell-mediated cytotoxicity (ADCC) contributes to the efficacy of many monoclonal antibodies therapies. Natural killer (NK) cells mediate ADCC, though responses are highly variable and depend on prior treatment as well as other factors. Thus, strategies to increase NK cell activity are expected to improve multiple therapies. Both cytokine treatment and NK cell receptor engineering are being explored to increase ADCC. Post-translational modifications, including glycosylation, are widely recognized as mediators of cellular processes but minimally explored as an alternative strategy to increase ADCC. We evaluated the impact of treatment with kifunensine, an inhibitor of asparagine-linked (N-)glycan processing, on ADCC using primary and cultured human NK cells. We also probed affinity using binding assays and CD16a structure with nuclear magnetic resonance spectroscopy. Treating primary human NK cells and cultured YTS-CD16a cells with kifunensine doubled ADCC in a CD16a-dependent manner. Kifunensine treatment also increased the antibody-binding affinity of CD16a on the NK cell surface. Structural interrogation identified a single CD16a region, proximal to the N162 glycan and the antibody-binding interface, perturbed by the N-glycan composition. The observed increase in NK cell activity following kifunensine treatment synergized with afucosylated antibodies, further increasing ADCC by an additional 33%. These results demonstrate native N-glycan processing is an important factor that limits NK cell ADCC. Furthermore, optimal antibody and CD16a glycoforms are defined that provide the greatest ADCC activity.     

Fabrication and statistical optimization of surface engineered PLGA nanoparticles for naso-brain delivery of ropinirole hydrochloride: in-vitro–ex-vivo studies

Ropinirole hydrochloride (RPN), a nonergot dopamine D₂-agonist used in the management of Parkinson’s disease, has poor oral bioavailability (52%) due to extensive hepatic metabolism. The intent of present research work was aimed at design and statistical optimization of RPN-loaded poly (lactic-co-glycolic acid) (PLGA)-based biodegradable nanoparticles (NPs) surface modified using natural emulsifier, vitamin E (d-α-tocopheryl polyethylene glycol 1000 succinate [TPGS]) for direct nose-to-brain delivery in order to avoid hepatic first-pass metabolism, and improve therapeutic efficacy with sustained drug release. RPN-NPs were prepared by modified nanoprecipitation technique and optimized using 2³ factorial design of experiment. The effect of polymer and emulsifier concentration was evaluated on particle size and entrapment efficiency (EE%). Formulation PL₆ was considered as desirable with highest EE% (72.3?±?6.1%), PS (279.4?±?1.8?nm), zeta potential (?29.4?±?2.6?mV), and cumulative drug diffusion of 96.43?±?3.1% in 24?h. The ANOVA results for the dependent variables demonstrated that the model was significant (p value?<?0.05) for response variables. Histopathological study of optimized batch (PL₆) demonstrated good retention of NPs with no severe signs of damage on the integrity of nasal mucosa. Differential scanning calorimetry revealed the absence of any chemical interaction between RPN, PLGA, and TPGS while SEM study confirmed spherical shape of optimized NPs. Accelerated stability studies of freeze-dried optimized batch demonstrated negligible change in the average PS and EE% after storage at 25?±?2?°C/60?±?5% (relative humidity (RH) for the period of three months. The promising results of optimized batch suggested practicability of investigated system for enhancement of bioavailability and brain targeting of CNS acting drugs like RPN.     

Reconstruction of 3D dynamic contrast-enhanced magnetic resonance imaging using nonlocal means

Purpose

To develop and test a nonlocal means‐based reconstruction algorithm for undersampled 3D dynamic contrast‐enhanced (DCE) magnetic resonance imaging (MRI) of tumors.

Materials and Methods

We propose a reconstruction technique that is based on the recently proposed nonlocal means (NLM) filter which can relax trade‐offs in spatial and temporal resolutions in dynamic imaging. Unlike the original application of NLM for image denoising, the MR reconstruction framework here can offer high‐quality images from undersampled k‐space data. The method is based on enforcing similarity constraints in terms of neighborhoods of pixels rather than individual pixels. The method was applied on undersampled 3D DCE imaging of breast and brain tumor datasets and the results were compared to sliding window reconstructions and to a compressed sensing method using total variation constraints on the images.

Results

Undersampling factors of up to five were obtained with the proposed approach while preserving the spatial and temporal characteristics. The NLM reconstruction method offered improved performance over the sliding window and the total variation constrained reconstruction techniques.

Conclusion

The reconstruction framework here can give high‐quality images from undersampled DCE MRI data and has the potential to improve the quality of DCE tumor imaging. J. Magn. Reson. Imaging 2010;32:1217–1227. © 2010 Wiley‐Liss, Inc.