Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

The relationship between the PD proprioceptor,the propodite‐dactylopodite joint and the dactylopodite flexor muscle in the walking legs of Cancer pagurus

The mechanical arrangement of the PD organ of Cancer has been studied and equations derived which relate its length to the articulation angle of the PD joint and to the angle which the receptor strand makes with the dactylopodite flexor tendon. These relationships are discussed in the light of the anatomy of the PD articulation.     

Roles of mouse sperm‐associated alpha‐L‐fucosidases in fertilization

Sperm‐associated α‐L ‐fucosidases have been implicated in fertilization in many species. Previously, we documented the existence of α‐L ‐fucosidase in mouse cauda epididymal contents, and showed that sperm‐associated α‐L ‐fucosidase is cryptically stored within the acrosome and reappears within the sperm equatorial segment after the acrosome reaction. The enrichment of sperm membrane‐associated α‐L ‐fucosidase within the equatorial segment of acrosome‐reacted cells implicates its roles during fertilization. Here, we document the absence of α‐L ‐fucosidase in mouse oocytes and early embryos, and define roles of sperm associated α‐L ‐fucosidase in fertilization using specific inhibitors and competitors. Mouse sperm were pretreated with deoxyfuconojirimycin (DFJ, an inhibitor of α‐L ‐fucosidase) or with anti‐fucosidase antibody; alternatively, mouse oocytes were pretreated with purified human liver α‐L ‐fucosidase. Five‐millimolar DFJ did not inhibit sperm–zona pellucida (ZP) binding, membrane binding, or fusion and penetration, but anti‐fucosidase antibody and purified human liver α‐L ‐fucosidase significantly decreased the frequency of these events. To evaluate sperm‐associated α‐L ‐fucosidase enzyme activity in post‐fusion events, DFJ‐pretreated sperm were microinjected into oocytes, and 2‐pronuclear (2‐PN) embryos were treated with 5 mM DFJ with no significant effects, suggesting that α‐L ‐fucosidase enzyme activity does not play a role in post‐fusion events and/or early embryo development in mice. The recognition and binding of mouse sperm to the ZP and oolemma involves the glycoprotein structure of α‐L ‐fucosidase, but not its catalytic action. These observations suggest that deficits in fucosidase protein and/or the presence of anti‐fucosidase antibody may be responsible for some types of infertility. Mol. Reprod. Dev. 80: 273–285, 2013. © 2013 Wiley Periodicals, Inc.     

Identification of amino acids in human colipase that mediate adsorption to lipid emulsions and mixed micelles

The adsorption of colipase is essential for pancreatic triglyceride lipase activity and efficient dietary fat digestion. Yet, little is known about which specific amino acids in the hydrophobic surface of colipase influence adsorption. In this study, we systematically substituted alanine or tryptophan at residues implicated in adsorption of colipase to an interface. We expressed, purified recombinant colipase mutants and characterized the ability of each alanine mutant to restore activity to lipase in the presence of bile salts. The functions of L16A, Y55A, I79A and F84A colipase were most impaired with activities ranging from 20 to 60% of wild-type colipase. We next characterized the fluorescence properties of the tryptophan mutants in the absence and presence of bile–salt–oleic acid mixed micelles. We performed steady-state emission spectra to determine peak shift and I₃₃₀/I₃₅₀ ratio and acrylamide quenching curves to characterize the environment of the residues. The analysis supports a model of adsorption that includes residues Leu 34 and Leu 36 on the 2nd loop, Tyr 55 and Tyr 59 on the 3rd loop and Ile 75 and Ile 79 on the 4th loop. The analysis confirms that Phe 84 is not part of the adsorption surface and likely stabilizes the conformation of colipase. Contrary to the predictions of computer modeling, the results provide strong support for an essential role of Tyr 55 in colipase adsorption to mixed micelles. The results indicate that the adsorption of colipase to mixed micelles is mediated by specific residues residing in a defined surface of colipase.     

Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This “panel-specific” feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.     

Preeclampsia as a Risk Factor for Diabetes: A Population-Based Cohort Study

Background

Women with preeclampsia (PEC) and gestational hypertension (GH) exhibit insulin resistance during pregnancy, independent of obesity and glucose intolerance. Our aim was to determine whether women with PEC or GH during pregnancy have an increased risk of developing diabetes after pregnancy, and whether the presence of PEC/GH in addition to gestational diabetes (GDM) increases the risk of future (postpartum) diabetes.

Methods and Findings

We performed a population-based, retrospective cohort study for 1,010,068 pregnant women who delivered in Ontario, Canada between April 1994 and March 2008. Women were categorized as having PEC alone (n = 22,933), GH alone (n = 27,605), GDM alone (n = 30,852), GDM+PEC (n = 1,476), GDM+GH (n = 2,100), or none of these conditions (n = 925,102). Our main outcome was a new diagnosis of diabetes postpartum in the following years, up until March 2011, based on new records in the Ontario Diabetes Database. The incidence rate of diabetes per 1,000 person-years was 6.47 for women with PEC and 5.26 for GH compared with 2.81 in women with neither of these conditions. In the multivariable analysis, both PEC alone (hazard ratio [HR] = 2.08; 95% CI 1.97–2.19) and GH alone (HR = 1.95; 95% CI 1.83–2.07) were risk factors for subsequent diabetes. Women with GDM alone were at elevated risk of developing diabetes postpartum (HR = 12.77; 95% CI 12.44–13.10); however, the co–presence of PEC or GH in addition to GDM further elevated this risk (HR = 15.75; 95% CI 14.52–17.07, and HR = 18.49; 95% CI 17.12–19.96, respectively). Data on obesity were not available.

Conclusions

Women with PEC/GH have a 2-fold increased risk of developing diabetes when followed up to 16.5 years after pregnancy, even in the absence of GDM. The presence of PEC/GH in the setting of GDM also raised the risk of diabetes significantly beyond that seen with GDM alone. A history of PEC/GH during pregnancy should alert clinicians to the need for preventative counseling and more vigilant screening for diabetes. Please see later in the article for the Editors'' Summary     

Comparative demography of commercially important species of coral grouper,Plectropomus leopardus and P. laevis,from Australia's great barrier reef and Coral Sea marine parks

Understanding the spatial and environmental variation in demographic processes of fisheries target species, such as coral grouper (Genus: Plectropomus), is important for establishing effective management and conservation strategies. Herein we compare the demography of Plectropomus leopardus and P. laevis between Australia's Great Barrier Reef Marine Park (GBRMP), which has been subject to sustained and extensive fishing pressure, and the oceanic atolls of Australia's Coral Sea Marine Park (CSMP), where there is very limited fishing for reef fishes. Coral grouper length-at-age data from contemporary and historical otolith collections across 9.4 degrees of latitude showed little difference in lifetime growth between GBRMP and CSMP regions. Plectropomus laevis populations in GBRMP reefs had significantly higher rates of total mortality than populations in the CSMP. Mean maximum lengths and mean maximum ages of P. laevis were also smaller in the GBRMP than in the CSMP, even when considering populations sampled within GBRMP no-take marine reserves (NTMRs). Plectropomus leopardus, individuals were on average smaller on fished reefs than NTMRs in the GBRMP, but all other aspects of demography were broadly similar between regions despite the negligible levels of fishing pressure in the CSMP. Similarities between regions in growth profiles and length-at-age comparisons of P. laevis and P. leopardus suggest that the environmental differences between the CSMP and the GBRMP may not have significant impacts on lifetime growth. Our results show that fishing may have influenced the demography of coral grouper on the GBR, particularly for the slower growing and longer lived species, P. laevis.