Immunological Probes Research Articles

BI-26 * HOW MUCH INSIGHT CAN A STEREOTACTIC BIOPSY DELIVER?

INTRODUCTION: Besides standard evaluation of histology, immunology and molecular markers as MGNT, IDH etc. an additional screening for biomarkers, genetic alterations and epigenetic features deriving from tissue from primary or recurrent brain tumors may harbor potential as prognostic and predictive information. With this pilot study we intended to examine in how far tissue obtained by stereotactic biopsy can be utilized for extended evaluation. METHODS: Stereotactically guided serial tissue samples (1mm diameter) from 16 patients were analyzed. Besides clinically obligatory routine histo-pathological, immunological and molecular evaluation (e.g. GFAP, S100, Vimentin, MAP2, EGFR, IDH1, p53 and MIB-1) probes were also utilized for analysis for further immunohistochemistry, for genomic DNA extraction, for RNA and protein extraction: The biochemical analysis was done by Western blot using various antibodies (e.g. ALDH1, AHR, GFAP, beta-actin). Furthermore, molecular techniques were applied including the methylation status of the MGMT promoter by bi-sulfit conversion of the DNA and following PCR and the IDH1/2 mutation status verification by pyro-sequencing. RESULTS: In most of the 16 patients the clinical routine diagnostic could be evaluated including MGMT promotor- and IDH1/2 mutation status (two diffuse astrocytomas, two anaplastic astrocytomas, nine glioblastomas, three other tumors). Genomic DNA could be extracted from stereotactic probes. RNA extraction was also possible, but the amount of cDNA obtained from the RNA from stereotactic samples was significant lower in 14/16 patient samples than the amount of cDNA normally used for quantitative RT-PCR from open resection. Besides, there was no biopsy-related morbidity. CONCLUSION: Tissue provided by stereotactically guided serial biopsies in patients with primary and recurrent brain tumors is safe and feasible. Besides clinical routine diagnostic the tissue can also sufficiently be used for of a variety of additional immunohistochemical, biochemical, and molecular analyses. This information may provide valuable and potential treatment relevant factors.

Duality of the Immune Response in Cancer: Lessons Learned from Skin

The skin not only acts as a physical barrier to pathogens and toxins, but also functions as an immunological barrier constantly responding to environmental insults (e.g., UV radiation, chemical carcinogens, and oncogenic viruses). Resident and recruited immune cells respond to these types of insults by directly or indirectly inducing secretion of damage response molecules (e.g., proinflammatory cytokines, chemokines, matrix remodeling enzymes, reactive oxygen mediators, and so on) in an attempt to clear damaged cells and pathogens such that tissue homeostasis can be reinstated. Instead, when damage is chronic and/or results in somatic alterations leading to altered proliferative or apoptotic programming of epithelial cells, inflammation that was initially an acute response instead becomes chronic. In this scenario, chronic inflammation acts as a promoting force that fosters early neoplastic progression and underscores data revealing that chronic exposure to environmental toxins and pathogens is a risk factor for cancer development (Coussens and Werb, 2002). How does activation of what should be an acute response instead foster neoplasia? The series of events discussed above is initiated by tissue-resident innate immune cells (dendritic cells, mast cells, macrophages, and γδ T cells) responding to damage response proteins, including damage-associated molecular patterns, pathogen-associated molecular patterns, Toll-like receptor ligands, colony stimulating factors, cytokines (tumor necrosis factor-α), and chemokines released from “damaged” epithelial cells (Balkwill et al., 2005; Balkwill, 2009). Upon engagement of these damage signals, resident immune cells are activated, and they respond by degranulation or secretion of a diversity of mediators that in turn results in activation of resident mesenchymal support cells (fibroblasts, adipocytes, mural, and endothelial cells), recruitment of peripheral blood leukocytes into “damaged” tissue, as well as engagement of cells of the adaptive immune system, both locally and peripherally (Balkwill et al., 2005). Dendritic cells, and to a lesser extent macrophages, are antigen-presenting cells that activate B and T cells to mount an adaptive immune response. Upon antigen recognition, B cells, as well as CD4+ and CD8+ T cells, undergo clonal expansion and mount responses specific to presented antigens. Although all of these tissue responses are otherwise entirely “normal,” during early neoplasia, they fail to resolve (Dvorak, 1986). Thus, chronic inflammation underlies the earliest stages of cancer development (Balkwill and Mantovani, 2001; Coussens and Werb, 2001, 2002). As such, chronic inflammation is now accepted as a hallmark of cancer development (Hanahan and Weinberg, 2011), where both innate and adaptive immune cells exert either pro- or anti-tumor activities dependent on their activation state and the microenvironment in which they reside (Balkwill et al., 2005; de Visser et al., 2006; Hanahan and Coussens, 2012). Although early studies of skin focused on the suppressive effects of leukocytes on carcinogenesis, we now recognize that proliferation and survival of epithelial cells harboring genomic alterations are sustained by chronic inflammatory pathways; understanding the nuances of these support mechanisms has yielded a diversity of new anticancer targets currently being utilized in the clinic.

Embryo cell wall properties in relation to development and desiccation in the recalcitrant-seeded Encephalartos natalensis (Zamiaceae) Dyer and Verdoorn.

Plant cell walls are dynamic entities that may change with development, differ between plant species and tissue type and play an important role in responses to various stresses. In this regard, the present investigation employed immunocytochemistry to determine wall composition and possible changes during development of immature and mature embryos of the recalcitrant-seeded cycad Encephalartos natalensis. Fluorescent and gold markers, together with cryo-scanning and transmission electron microscopy (TEM) were also used to analyse potential changes in the cell walls of mature embryos upon desiccation. Immature cell walls were characterised by low- and high methyl-esterified epitopes of pectin, rhamnogalacturonan-associated arabinan, and the hemicellulose xyloglucan. Arabinogalactan protein recognised by the LM2 antibody, along with rhamnogalacturonan-associated galactan and the hemicellulose xylan, were not positively localised using immunological probes, suggesting that the cell walls of the embryo of E. natalensis do not possess these epitopes. Interestingly, mature embryos appeared to be identical to immature ones with respect to the cell wall components investigated, implying that these may not change during the protracted post-shedding embryogenesis of this species. Drying appeared to induce some degree of cell wall folding in mature embryos, although this was limited by the abundant amyloplasts, which filled the cytomatrical space. Folding, however, was correlated with relatively high levels of wall plasticisers typified by arabinose polymers. From the results of this study, it is proposed that the embryo cell walls of E. natalensis are constitutively prepared for the flexibility required during cell growth and expansion, which may also facilitate the moderate cell wall folding observed in mature embryos upon drying. This, together with the abundant occurrence of amyloplasts in the cytomatrix, may provide sufficient mechanical stabilisation if water is lost, even though the seeds of this species are highly desiccation-sensitive.

Quantification of eggs and sperm in the Black-lip pearl oyster Pinctada margaritifera using an enzyme-linked immunosorbent assay (ELISA)

We have developed immunological probes to quantify eggs and sperm of the Black-lip pearl (BLP) oyster Pinctada margaritifera. The western blot assay revealed that the polyclonal antibodies developed in this study specifically recognized only egg and sperm proteins. These polyclonal antibodies also showed high sensitivities to the antigens, detecting 0.31–10 μg/ml of the egg or sperm proteins in an indirect enzyme-linked immunosorbent assay (ELISA). Accordingly, we used an indirect ELISA to quantify the eggs and sperm in BLP oysters collected in December 2009 from Weno Island in Micronesia and in May 2010 from Tahiti. The gonad somatic index (GSI), a ratio of gonad weight to somatic tissue weight, of the females collected from Weno Island ranged from 3.6 to 18.4 %, while the GSI of the females collected from Tahiti ranged from 5.6 to 12.6 %. Similarly, the GSI of the male BLP oysters from Weno Island ranged from 0.8 to 8.5 %, while the GSI of the males from Tahiti ranged from 4.8 to 7.5 %. These results lead to the conclusion that the immunological probes developed in this study can be successfully applied to quantify BLP oyster gonadal tissues and that these probes can be used in studies of BLP oyster reproductive ecology based on their sensitivity, rapidity, and affordability.

Abstract 4796: Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody.

Abstract NADPH oxidase (Nox) family enzymes (Nox1-5 and Duox1-2) generate reactive oxygen species (ROS) and modulate cellular pathways that regulate tumorigenesis. Recent studies have proposed that Nox5 plays a role in the proliferative capacity of Hairy cell leukemia, Barrett's esophageal adenocarcinoma, and in prostate cancer cells. Although implicated in several malignancies, the role of Nox5 in tumor cell biology is still unclear. This impediment is due to a lack of reliable immunological probes as well as the fact that little is known regarding Nox5 expression in human tumors. In this study, we report for the first time the generation and characterization of a mouse monoclonal antibody against a recombinant Nox5 protein (600-746), and the expression profile of Nox5 in human tumors by tissue microarray analysis. We also report the detection of endogenous Nox5 protein in the UACC-257 human melanoma cell line using our novel Nox5 antibody. Specificity of the antibody for Nox5 was confirmed by generating stable Nox5 over-expressing UACC-257 cell clones, and by transiently silencing both endogenous Nox5 and stably overexpressed Nox5. Additionally, no cross-reactivity of the antibody with other Nox homologs was detected. Sub-cellular fractionation studies revealed that Nox5 was predominantly expressed in the membranes of UACC-257 cells and its Nox5 overexpressing clones. Immunofluorescence, confocal microscopy, and immunohistochemistry demonstrated that Nox5 localizes throughout parental UACC-257 cells and Nox5 overexpressing clones, with perinuclear enhancement. To evaluate the clinical significance of Nox5 in human cancer, we determined the expression of Nox5 protein in a representative set (382 samples) of human tumors. Tissue microarray analysis revealed substantial Nox5 overexpression in several human tumors of epithelial origin including breast, colon, lung, prostate, and ovarian cancers, as well as non-epithelial malignancies (glioblastoma multiforme, melanoma, and lymphoma). Expression was markedly increased in tumors compared to normal tissues of the same organ. Collectively, our study provides the first demonstration of Nox5 overexpression in human tumors by tissue microarray, and reports on the first mouse monoclonal antibody against Nox5. The elevated expression of Nox5 in tumors provides impetus to explore the functional significance of Nox5 in the context of tumor development and maintenance. Citation Format: Smitha Antony, Yongzhong Wu, Stephen M. Hewitt, Miriam R. Anver, Guojian Jiang, Jennifer L. Meitzler, Han Liu, Agnes Juhasz, Jiamo Lu, Krishnendu K. Roy, James H. Doroshow. Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4796. doi:10.1158/1538-7445.AM2013-4796

A Novel Subcellular Machine Contributes to Basal Junction Remodeling in the Seminiferous Epithelium1

Tubulobulbar complexes are cytoskeleton-related membrane structures that develop at sites of intercellular attachment in mammalian seminiferous epithelium. At apical junctions between Sertoli cells and spermatids, the structures internalize adhesion junctions and are a component of the sperm release mechanism. Here we explore the possibility that tubulobulbar complexes that form at the blood-testis barrier are subcellular machines that internalize basal junction complexes. Using electron microscopy, we confirmed that morphologically identifiable tight and gap junctions are present in basal tubulobulbar complexes in rats. In addition, immunological probes for claudin-11 (CLDN11), connexin-43 (GJA1), and nectin-2 (PVRL2) react with linear structures at the light level that we interpret as tubulobulbar complexes, and probes for early endosome antigen 1 (EEA1) and Rab5 (RAB5A) react in similar locations. Significantly, fluorescence patterns for actin and claudin-11 indicate that the amount of junction present is dramatically reduced over the time period that tubulobulbar complexes are known to be most prevalent during spermatogenesis. We also demonstrated, using electron microscopy and fluorescence microscopy, that tubulobulbar complexes develop at basal junctions in primary cultures of Sertoli cells and that like their in vivo counterparts, the structures contain junction proteins. We use this culture system together with transfection techniques to show that junction proteins from one transfected cell occur in structures that project into adjacent nontransfected cells as predicted by the junction internalization hypothesis. On the basis of our findings, we present a new model for basal junction remodeling as it relates to spermatocyte translocation in the seminiferous epithelium.

Distribution of Transglutaminase in Pear Pollen Tubes in Relation to Cytoskeleton and Membrane Dynamics

Transglutaminases (TGases) are ubiquitous enzymes that take part in a variety of cellular functions. In the pollen tube, cytoplasmic TGases are likely to be involved in the incorporation of primary amines at selected peptide-bound glutamine residues of cytosolic proteins (including actin and tubulin), while cell wall-associated TGases are believed to regulate pollen tube growth. Using immunological probes, we identified TGases associated with different subcellular compartments (cytosol, membranes, and cell walls). Binding of cytosolic TGase to actin filaments was shown to be Ca(2+) dependent. The membrane TGase is likely associated with both Golgi-derived structures and the plasma membrane, suggesting a Golgi-based exocytotic delivery of TGase. Association of TGase with the plasma membrane was also confirmed by immunogold transmission electron microscopy. Immunolocalization of TGase indicated that the enzyme was present in the growing region of pollen tubes and that the enzyme colocalizes with cell wall markers. Bidimensional electrophoresis indicated that different TGase isoforms were present in distinct subcellular compartments, suggesting either different roles or different regulatory mechanisms of enzyme activity. The application of specific inhibitors showed that the distribution of TGase in different subcellular compartments was regulated by both membrane dynamics and cytoskeleton integrity, suggesting that delivery of TGase to the cell wall requires the transport of membranes along cytoskeleton filaments. Taken together, these data indicate that a cytoplasmic TGase interacts with the cytoskeleton, while a different TGase isoform, probably delivered via a membrane/cytoskeleton-based transport system, is secreted in the cell wall of pear (Pyrus communis) pollen tubes, where it might play a role in the regulation of apical growth.

Phenotypic characterization of mononuclear inflammatory cells following equine hydroxyapatite/collagen block grafting in rats

To measure the inflammatory changes associated with the implantation of an equine hydroxyapatite and collagen-containing block graft (eHAC block) in a rodent model system, an eHAC block graft was implanted subcutaneously in rats. Control groups included saline, turpentine oil, and human mineralized particulate allograft (hMPA). Animals were sacrificed and tissue samples obtained after three days, as well as after 1, 2, 4 and 8 weeks. A panel of immunologic probes was used to identify circulatory monocytic cells (ED1), resident mononuclear phagocytes (ED2), mononuclear phagocytes of lymphoid origin (ED3), expression of Ia antigen (OX6), T-cells (OX19), and B-cells (OX33). Immunocytochemical localization was performed and mononuclear cells localized with each immunologic probe counted. Rat sera obtained after eight weeks were used for nitrocellulose dot-blotting to assess circulating anti-equine immunoglobulins. Statistical analysis was performed using two-way analysis of variance, in conjunction with the Bonferroni correction to account for multiple comparisons. A transient increase in monocytes at 3 days and 1 week was observed in all groups, but was significantly higher in the turpentine control (P < 0.0001). A significant increase in the numbers of mononuclear cells detected with clones ED2 and ED3 was observed in specimens from the turpentine group, in contrast to the other groups in the 3 day to 4 week interval (P < 0.0001), as well as within all time periods (P < 0.0001). A statistically significant difference in numbers of ED3-positive cells was observed in the hMPA group compared to the saline and the eHAC block groups after one week (P < 0.0001). Significantly more OX6-positive cells were observed in the turpentine group, compared to other groups (3 days to 1 week; P < 0.0001). T-lymphocytes were essentially absent except for rats given turpentine (after 1 week). No B-lymphocyte response was found and none of the rats developed systemic anti-equine antibodies. These data indicate that a cellular immune response is not elicited following implantation with the eHAC block graft, which might serve as an alternative material for regenerative therapy.

Cytosolic subunits of ATP synthase are localized to the cortical endoplasmic reticulum‐rich domain of the ascidian egg myoplasm

Previously, we revealed that p58, one of the ascidian maternal factors, is identical to the alpha-subunit of F1-ATP synthase (ATPα), a protein complex of the inner mitochondrial membrane. In the current study, we used immunological probes for ascidian mitochondria components to show that the ascidian ATPα is ectopically localized to the cytosol. Virtually all mitochondrial components were localized to the mitochondria-rich myoplasm. However, in detail, ATP synthase subunits and the matrix proteins showed different localization patterns. At least at the crescent stage, transmission electron microscopy (TEM) distinguished the mitochondria-less, endoplasmic reticulum (ER)-rich cortical region and the mitochondria-rich internal region. ATPα was enriched in the cortical region and MnSOD was limited to the internal region. Using subcellular fractionation, although all of the mitochondria components were highly enriched in the mitochondria-enriched fraction, a considerable amount of ATPα and F1-ATP synthase beta-subunit (ATPβ) were recovered in the insoluble cytoplasmic fraction. Even under these conditions, F1-ATP synthase gamma-subunit (ATPγ) and F0-ATP synthase subunit b (ATPb) were not recovered in the insoluble cytoplasmic fraction. This result strongly supports the exomitochondrial localization of both ATPα and ATPβ. In addition, the detergent extraction of eggs supports the idea that these cytosolic ATP synthase subunits are associated with the egg cytoskeleton. These results suggest that the subunits of ATP synthase might play dual roles at different subcellular compartments during early development.

New insights into the neuroanatomical distribution and phylogeny of opioids and POMC-derived peptides in fish

This review re-evaluates the use of immunological probes to map enkephalinergic, dynorphinergic, and endorphinergic circuits in the CNS of lobe-finned fishes, ray-finned fishes, and cartilaginous fishes in light of the characterization of proenkephalin, prodynorphin, and POMC sequences from representatives of these groups of fish over the past 20years. The use of α-MSH specific antisera is a reliable method for detecting POMC immunopositive cell bodies and fibers. Since α-MSH and β-endorphin are co-localized in the same neurons, these studies also reveal the distribution of endorphinergic networks. Met-enkephalin specific antisera can be used to detect enkephalinergic circuits in the CNS of gnathostomes because of the ubiquitous presence of this pentapeptide in the proenkephalin sequences of gnathostomes. However, the use of leu-enkephalin specific antisera to detect enkephalinergic networks is more problematic. While this immunological probe is appropriate for analyzing enkephalinergic networks in mammals and perhaps teleosts, for the lungfishes and cartilaginous fishes this probe is more likely able to detect dynorphinergic circuits. In this regard, there is a need to re-examine dynorphinergic networks in non-mammalian gnathostomes by using species specific antisera directed against dynorphin end-products.

Immunological toolbox available for in situ exploration of pectic homogalacturonan and its modifying enzymes in fruits and vegetables and their derived food products

Abstract Pectin and its modifications during growth, ripening, processing and storage play a crucial role in determining the textural properties of fruits and vegetables and their derived food products. Insight into the (enzyme-catalysed) modifications of homogalacturonan, the major structural domain of this polysaccharide which strongly influences the pectin structure–function properties, can be obtained by in situ research tools. Such in situ knowledge is of common interest to both plant and food scientists and requires a good view on available in situ probes and their possible applications, both in a plant physiological and a food-related context. Therefore, the aim of this review is to briefly discuss the available probes towards pectic homogalacturonan and its modifying enzymes and to overview the obtained results using pectin probes, pectin methylesterase probes and pectin methylesterase inhibitor probes in both domains. The presented overview of the available data on in situ research of pectic homogalacturonan and its modifying enzymes emphasises the possibilities of more integrated approaches. Industrial relevance Pectin is a highly important component of plant-based food products. This polysaccharide, naturally present in plant cell walls, is prone to modifications imposed by enzyme activity and/or processing conditions. As the properties of pectin influence product quality attributes such as texture, this macromolecule has been widely investigated in the processing of plant-based foods. An emerging technology for the in situ investigation of food components is using immunological probes. Within this context, the available immunological toolbox for pectic homogalacturonan and its modifying enzymes is summarised in this review article.

Internalization of adhesion junction proteins and their association with recycling endosome marker proteins in rat seminiferous epithelium

Tubulobulbar complexes (TBCs) are elaborate cytoskeleton-related structures that are formed in association with intercellular junctions in the seminiferous epithelium. They consist of a cylindrical double-membrane core composed of the plasma membranes of the two attached cells, cuffed by a dendritic network of actin filaments. TBCs are proposed to be subcellular machines that internalize intercellular junctions during the extensive junction remodeling that occurs during spermatogenesis. At the apical sites of attachment between Sertoli cells and spermatids, junction disassembly is part of the sperm release mechanism. In this study, we used immunological probes to explore junction internalization and recycling at apical TBCs in the rat seminiferous epithelium. We demonstrate that β1-integrin and nectin 2 were concentrated at the ends of TBCs and for the first time show that the early endosome marker RAB5A was also distinctly localized at the ends of TBCs that appear to be the 'bulbar' regions of the complexes. Significantly, we also demonstrate that the 'long-loop' recycling endosome marker RAB11A was co-distributed with nectin 2 at junctions with early spermatids deeper in the epithelium. Our results are consistent with the hypothesis that TBCs associated with late spermatids internalize adhesion junctions and also indicate that some of the internalized junction proteins may be recycled to form junctions with the next generation of spermatids.

Chemiluminescence immunoassay based on dual signal amplification strategy of Au/mesoporous silica and multienzyme functionalized mesoporous silica

► The increased amount of monoclonal antibody in Au/SiO 2 led to a wider linear range. ► Due to the increased HRP tags in HRP–Ab 2 /SiO 2 , signal amplification achieved. ► A simple dual amplification immunoassay achieved with flow injection analysis. A chemiluminescent dual signal amplification strategy for the determination of α-fetoprotein (AFP) was proposed based on a sandwich immunoassay format. Monoclonal antibody of AFP immobilized on the gold nanoparticles doped mesoporous SiO 2 (Au/SiO 2 ) were prepared and used as a primary antibody. Horseradish peroxidase (HRP) and HRP-labeled secondary antibody (Ab 2 ) co-immobilized into the mesoporous SiO 2 nanoparticles (HRP–Ab 2 /SiO 2 ) were used as the labeled immunological probe. Due to the high ratio surface areas and pore volumes of the mesoporous SiO 2 , not only the amount of AFP monoclonal antibody but also the amount of the modified HRP and Ab 2 in HRP–Ab 2 /SiO 2 were largely increased. Thus the chemiluminescent signal was amplified by using the system of luminol and H 2 O 2 under the catalysis of HRP. Under the optimal conditions, two linear ranges for AFP were obtained from 0.01 to 0.5 ng mL −1 and 0.5 to 100 ng mL −1 with a detection limit of 0.005 ng mL −1 (3 σ ). The fabricated signal amplification strategy showed an excellent promise for sensitive detection of AFP and other tumor markers.

Investigation of lignan accumulation in developing Linum usitatissimum seeds by immunolocalization and HPLC

Lignans are widely distributed plant metabolites associated with a large range of biological activities. An immunological probe was previously developed to immunolocalize lignans in mature flaxseed ( Linum usitatissimum) and allowed to show that they are mainly localized in the secondary wall of the sclerite cells layer of the outer integument of the seed. In order to precise the temporal regulation of these secondary metabolites, L. usitatissimum plants were cultivated and their developing seeds were investigated according to their SDG (secoisolariciresinol diglucoside) content (main lignan in seeds) by immunolocalization and HPLC techniques. The HPLC results showed that SDG content increased as a function of the developmental stage to reach a maximum of about 9 mg/g at the end of seed maturation. In addition, the seed dissection during seed maturation allowed to observe a similar pattern of evolution of SDG content in the outer integument whereas this content decreased in the inner integument and the endosperm. These results were confirmed by immunolocalization experiments.

In vivo 6-thioguanine-resistant T cells from melanoma patients have public TCR and share TCR beta amino acid sequences with melanoma-reactive T cells

In vivo hypoxanthine–guanine phosphoribosyltransferase (HPRT)-deficient T cells (MT) from melanoma patients are enriched for T cells with in vivo clonal amplifications that traffic between blood and tumor tissues. Melanoma is thus a model cancer to test the hypothesis that in vivo MT from cancer patients can be used as immunological probes for immunogenic tumor antigens. MT were obtained by 6-thioguanine (TG) selection of lymphocytes from peripheral blood and tumor tissues, and wild-type T cells (WT) were obtained analogously without TG selection. cDNA sequences of the T cell receptor beta chains (TRB) were used as unambiguous biomarkers of in vivo clonality and as indicators of T cell specificity. Public TRB were identified in MT from the blood and tumor of different melanoma patients. Such public TRB were not found in normal control MT or WT. As an indicator of T cell specificity for melanoma, the >2600 MT and WT TRB, including the public TRB from melanoma patients, were compared to a literature-derived empirical database of >1270 TRB from melanoma-reactive T cells. Various degrees of similarity, ranging from 100% conservation to 3-amino acid motifs (3-mer), were found between both melanoma patient MT and WT TRBs and the empirical database. The frequency of 3-mer and 4-mer TRB matching to the empirical database was significantly higher in MT compared with WT in the tumor (p=0.0285 and p=0.006, respectively). In summary, in vivo MT from melanoma patients contain public TRB as well as T cells with specificity for characterized melanoma antigens. We conclude that in vivo MT merit study as novel probes for uncharacterized immunogenic antigens in melanoma and other malignancies.

Development of antibodies against secoisolariciresinol – Application to the immunolocalization of lignans in Linum usitatissimum seeds

Lignans are widely distributed plant metabolites associated with a large range of biological activities. In order to gain insight into their biosynthesis and their spatio-temporal accumulation an immunological probe was developed. Secondary metabolites generally have too small molecular weight to be antigenic and have to be associated with a carrier protein. Secoisolariciresinol was chosen as the hapten and was linked to bovine serum albumin via a spacer arm, the p-aminohippuric acid. The artificial antigen was injected to New Zealand rabbits. The successful production of polyclonal antibodies against secoisolariciresinol was assessed with indirect enzyme immunosorbent assay (ELISA) by comparison with pre-immune serum and by competitive assays using dilutions of secoisolariciresinol standards. The antibodies had an IC 50 value of 94 μg/ml and showed moderate cross-reactivities with structurally related compounds. They were thus used to immunolocalize lignans in flaxseed ( Linum usitatissimum), one of the richest sources of lignans. The immunohistochemical labeling allowed us to localize for the first time lignans in planta. They are mainly localized in the secondary wall of the sclerite cells of the outer integument of the seed. A very light labeling is also observed in cytoplasmic inclusions of the endosperm. The results were correlated with HPLC analytical results which enabled to evaluate the relative lignan quantities: in flaxseed about 90% of the metabolites are localized in the outer integument.

Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques

Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a protozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decomposing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti- P. olseni antibody developed in this study were peptides with molecular masses of 55.9, 24.0, and 19.2 kDa. Immunofluorescent assay indicated that the rabbit anti- P. olseni IgG was specific to all life stages, including the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells were successfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, where P. olseni–associated clam mortality has recurred for the past decade. Purified cells were positively stained with the rabbit anti- P. olseni antibody in an immunofluorescence assay, confirming for the first time the presence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia were observed in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from the feces and pseudofeces of infected clams and confirmed by PCR. The clams released 1–2 prezoosporangia per day through feces. The data suggested that the fecal discharge and decomposition of the infected clam tissue could be the two major P. olseni transmission routes.

Immunocytochemical Localization of a 58kDa Glycoprotein of Epididymal Fluid of Rhesus Monkey (Macaca mulatta)

Jamal, F. and Srivastav, A. 2010. Immunocytochemical localization of a 58kDa glycoprotein of epididymal fluid of rhesus monkey (Macaca mulatta). J. Appl. Anim. Res., 37: 177–183. A 58kDa glycoprotein of epididymal fluid of rhesus monkey, using lectins Lens culinaris agglutinin (LCA) and Ricinus communis agglutinin (RCA), was studied raising polyclonal antisera against this protein to study cross-reactivity with the epididymal fluids of mice, rat, hamster, guinea pig, rabbit and Triton X-100 human sperm extract. The present study aimed at delineating the distribution of this 58kDa antigen in testis, epididymis, vas deferens, seminal vesicle and prostate tissues of rhesus monkey using immunological probes, depicting their ontogeny and its association with maturing spermatozoa. The epitopes cross-reacting with 58kDa antisera were predominantly distributed in the stroma region and luminal spermatozoa throughout the epididymis, vas deferens and prostate tissues. The epitopes against this 58kDa antisera have been conserved and could be explored for immuno-contraception in humans.

Immunolocalization of β-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica

Compression wood (CW) contains higher quantities of beta-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of beta-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of beta-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of beta-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S(1) layer. At this stage, CW was strongly labeled in the S(1) layer, whereas no label was observed in the S(1) layer of NW and OW. Immunogold labeling showed that beta-1-4-galactan in the S(1) layer of CW tracheids significantly decreased during the formation of the S(2) layer. Most beta-1-4-galactan labeling was present in the outer S(2) region in mature CW tracheids, and was absent in the inner S(2) layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of beta-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask beta-1-4-galactan epitopes. The study clearly showed that beta-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica.

A developmental study of enteric neuron migration in the grasshopper using immunological probes

Pioneering migratory enteric neurons on the midgut of a locust embryo at 63 to 65% E. The neurites are labelled immunofluorescently by an antibody against grasshopper fasciclin I (red). The merged DIC image illustrates the overall morphology. From Knipp and Bicker, Developmental Dynamics 238:2837–2849, 2009.