Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes.

Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCß showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules.

Naturalez neuronal de los neuroblastomas de la clasificación de la Río Hortega del sistema nervioso central: estudio con técnicas argénticas e inmunohistoquímicas / Neuroblastomas neuronal nature of the classification of the Hortega River of the central nervous system. Argénticas study and immunohistochemical techniques

El objetivo fue demostrar que los llamados meduloblastomas del Sistema Nervioso Central son neuroblastomas, tumores de origen neural con identidad propia. Siguiendo a Del Río Hortega y Polak se estudiaron 33 meduloblastomas cerebelares clínica y anatomopatológicamente demostrados. Se utilizaron técnicas de anilinas, impregnación argéntica y de inmunohistoquímica. Se analizó extensamente la bibliografía haciendo énfasis en el origen e histogénesis de las neuronas. Se tuvo en cuenta un principio básico que para reconocerlas se debe teñir el núcleo, el citoplasma y sus prolongaciones. Con las técnicas habituales de hematoxilina- eosina se observa sólo el núcleo y citoplasma, con la inmunohistoquímica se caracteriza la estirpe y con las técnicas argénticas las prolongaciones, con las que Cajal, Del Río Hortega, Golgi y otros estudiaron y descubrieron las células del SNC normal y patológico, cuya teoría de la neurona de Cajal no fue superada hasta la actualidad.

SUMMARY: The purpose of this study was to demonstrate that the called meduloblastoma of the central nervous system, are neuroblastomas, tumors of neural origin with its own identity .Following the published studies from Del Rio Hortega and Pollak, we studied 33 cerebellar medulloblastoma, clinically and pathologically demonstrated We used silver, aniline stains and immunohistochemestry. A comprehensive literature review was conducted, with emphasis on the origin and histogenesis of neurons. A basic principle was considered to recognize a neuron, the nucleus, the cytoplasm and the neuronal extensions must be stained. With standard hematoxilin and eosin techniques we only observed the nucleus and the cytoplasm. The immunohistochemestry showed the cell line origin and the silver stain make evident the neuronal extensions. Del Rio Hortega, Cajal, Golgi and others discovered and studied the normal and pathological central nervous system cell. The Cajal’s neuron theory was unsurpassed even today.

Treatment of high-risk neuroblastoma with anti-GD2 antibodies.

Anticancer monoclonal antibodies (mAbs) targeting specific antigens on the tumour surface are increasingly being applied in cancer treatment. Potential advantages include long half-life, low toxicity, high affinity and specificity. In order to develop novel immune therapies for high-risk cancers, finding tumour targets that are not widely shared by normal cells is a goal. GD2-disialoganglioside is one of them. It is expressed on the surface of a variety of tumours with no curative therapies for patients with advanced disease. In childhood, neuroblastoma is the most common GD2-expressing tumour. Because of this tumour-selective expression, it is an attractive target for tumour-specific therapies such as antibody therapy. Over the last two decades, several anti-GD2 antibodies have been developed. To reduce both toxicity and development of human anti-mouse antibodies (HAMA), research efforts have primarily focused on exploring anti-GD2 antibodies that substitute mouse components by human ones. This review will examine antibodies currently undergoing clinical testing as well as the most recent advances to improve antibody therapy for patients with high-risk neuroblastoma.

Morphometric evaluation of NB84, synaptophysin and AgNOR is useful for the histological diagnosis and prognosis in peripheral neuroblastic tumors (pNTs).

OBJECTIVE: To study the importance of NB84, synaptophysin and AgNOR and explore the quantitative association of these factors with diagnosis and outcome as well as the association between NB84 and AgNOR and other tumor and stromal factors in twenty-eight peripheral neuroblastic tumors. METHODS: We assessed AgNORs, NB84, synaptophysin and several other markers in tumor tissues from 28 patients with primary neuroblastic tumors. The treatment included: surgery for stage 1, chemotherapy and bone marrow transplantation for most of stages 3 and 4. Histochemistry, immunohistochemistry and morphometry were used to evaluate the amount of tumor staining for AgNOR, NB84 and synaptophysin; the outcome for our study was survival time until death due to recurrent neuroblastic tumors. RESULTS: Only stage (p<0.01), AgNOR (p<0.01), NB84 (p<0.01) and synaptophysin (p=0.01) reached statistical significance as prognostic indicators. CONCLUSIONS: Determination of NB84 and synaptophysin are useful tools for the diagnosis of peripheral neuroblastic tumors The association of the evaluation of AgNOR expression by the tumor cells may provide an important contribution to the prognostic evaluation and management approach of the patients.

Morphometric evaluation of NB84, synaptophysin and AgNOR is useful for the histological diagnosis and prognosis in peripheral neuroblastic tumors (pNTs)

OBJECTIVE: To study the importance of NB84, synaptophysin and AgNOR and explore the quantitative association of these factors with diagnosis and outcome as well as the association between NB84 and AgNOR and other tumor and stromal factors in twenty-eight peripheral neuroblastic tumors. METHODS: We assessed AgNORs, NB84, synaptophysin and several other markers in tumor tissues from 28 patients with primary neuroblastic tumors. The treatment included: surgery for stage 1, chemotherapy and bone marrow transplantation for most of stages 3 and 4. Histochemistry, immunohistochemistry and morphometry were used to evaluate the amount of tumor staining for AgNOR, NB84 and synaptophysin; the outcome for our study was survival time until death due to recurrent neuroblastic tumors. RESULTS: Only stage (p<0.01), AgNOR (p<0.01), NB84 (p<0.01) and synaptophysin (p=0.01) reached statistical significance as prognostic indicators. CONCLUSIONS: Determination of NB84 and synaptophysin are useful tools for the diagnosis of peripheral neuroblastic tumors The association of the evaluation of AgNOR expression by the tumor cells may provide an important contribution to the prognostic evaluation and management approach of the patients.

OBJETIVO: Estudar a importância dos marcadores NB84 e AgNOR e explorar as relações quantitativas entre esses marcadores com o diagnóstico e prognóstico assim como as relações entre NB84 e AgNOR e outros marcadores tumorais e estromais em 28 tumores neuroblásticos periféricos. MÉTODOS: Examinamos AgNOR, NB84 e sinaptofisina e vários outros marcadores em tecidos tumorais de vinte e oito pacientes com tumors neuroblásticos primários. Tratamento dos pacientes incluiu: cirurgia para o estágio 1, quimioterapia e transplante de medula óssea para a maioria dos pacientes nos estágios 3 e 4. Utilizamos histoquímica, imunohistoquímica e morfometria para avaliar a intensidade e extensão de expressão do AgNOR, NB84 e sinaptofisina, tendo o prognóstico dos pacientes incluído o tempo de sobrevida até a morte por recurrência dos tumores neuroblásticos. RESULTADOS: Estadiamento (p<0.01), AgNOR (p<0.01), NB84 (p<0.01) e sinaptofisina (p=0.01) foram marcadores independents de sobrevida. CONCLUSÕES: A determinação dos marcadores NB84 e sinaptofisina mostrou-se como uma ferramenta útil no diagnóstico dos tumors neuroblásticos periféricos; a associação desses marcadores à expressão de AgNOR pelas células tumorais contribuiu à determinação do prognóstico e estabelecimento do protocolo terapêutico para os pacientes.

Humoral and cellular responses raised against the human HER2 oncoprotein are cross-reactive with the homologous product of the new proto-oncogene, but do not protect rats against B104 tumors expressing mutated neu.

The neu proto-oncogene encodes a plasma membrane protein belonging to the epidermal growth factor receptor family. The cell line B104, derived from BDIX rat neuroblastoma, carries a point mutation in neu, and forms a tumor when injected into these rats. The human homologue of the neu oncogene (here called HER2) is overexpressed in certain types of cancer. Rats were immunized with HER2 protein (HER2) to investigate a possible cross-reaction between the homologous proteins which could protect them against subsequent inoculation with B104. Specific antibody in the serum was measured by cell-based enzyme-linked immunoabsorbent assay and fluorescence immunocytochemistry, and delayed-type hypersensitivity by an ear assay. Sera from animals immunized with the HER2 extracellular domain (HER2-ECD) reacted with both HER2- and neu-expressing cells. In the ear assay, a significant cellular response to both HER-ECD (P < 0.05) and neu protein (P < 0.001) was observed in HER2-ECD-immunized rats. However, the growth of B104 tumors in rats was not affected by preimmunization with HER2-ECD. The results indicate that an autoreactive immune response to neu was induced by immunization with HER2-ECD, but was too weak to affect the growth of the neu-bearing tumor.

IFN-treated neuroblastoma cell lines remain resistant to T cell-mediated allo-killing, and susceptible to non-MHC-restricted cytotoxicity.

Neuroblastoma cell lines can have very low MHC Ag expression. The cell lines are insensitive to allo-killing by primed CTL, but are sensitive to non-MHC-restricted cytotoxicity. IFN-gamma increased class I expression, but the cells remained insensitive to CTL. Susceptibility to nonrestricted effectors was preserved. Class I+ glioma cell lines behaved similarly. The CTL resistance was localized to the recognition phase. Neuroblastoma lines did not form conjugates with primed T cells, but were lysed if they were coupled to the effectors via lectins. The levels of class I expression, and resistance to CTL, were constant over a range of IFN doses. HLA-A,B,C structure and distribution were studied more intensively on one cell line, CHP-100. HLA-A2 and -A3 were present on greater than or equal to 99% of the cells, in a unimodal distribution. After IFN treatment, the levels were similar to B cell controls. In two-dimensional gel electrophoresis, the molecules co-migrated with those of B cell controls. The defect may thus be in accessory proteins that are necessary for T cell recognition or binding, rather than in the structure or distribution of the HLA-A,B,C proteins.

Immunolocalization of neuroblastoma using radiolabeled monoclonal antibody UJ13A.

The monoclonal antibody UJ13A, raised after immunization of mice with human fetal brain, recognized an antigen expressed on human neuroblastoma cell lines and fresh tumors. Antibody was purified and radiolabeled with iodine isotopes using chloramine-T. In preclinical studies, 125I-labeled UJ13A was injected intravenously into nude mice bearing xenografts of human neuroblastoma. Radiolabeled UJ13A uptake by the tumors was four to 23 times greater than that by blood. In control animals, injected with a similar quantity of a monoclonal antibody known not to bind to neuroblastoma cells in vitro (FD44), there was no selective tumor uptake. Nine patients with histologically confirmed neuroblastoma each received 100 to 300 micrograms UJ13A radiolabeled with 1 to 2.8 mCi 123I or 131I. Sixteen positive sites were visible on gamma scans 1 to 7 days after injection: 15 were primary or secondary tumor sites, and one was a false positive; there were two false negatives. In two of the 15 positive sites, tumor had not been demonstrated by other imaging techniques; these were later confirmed as areas of malignant infiltration. No toxicity was encountered.

Abnormalities of the immune system in children with neuroblastoma related to the neoplasm and chemotherapy.

Children with localized and metastatic neuroblastoma were studies to determine their immune status at the time of diagnosis and while they were receiving intensive intermittent chemotherapy; Investigations included leukocyte and differential counts, delayed hypersensitivity response, quantitative serum immunoglobulins, percentages of T and Fc receptor lymphocytes, PHA-induced mitogenesis, and antibody-and PHA-dependent cellular cytoxicity. Abnormalities related to the neoplasm at diagnosis were limited to depressed leukocyte and lymphocyte counts and increased concentrations of serum IgM in patients with metastases to bone marrow and other sites. No abnormalities were observed in those with localized tumors. Intermittent chemotherapy of metastatic neuroblastoma caused immunosuppression. Effects were most marked during five-day courses of chemotherapy; they included abrogation of DH and decreased leukocyte and lymphocyte counts and percentages of Fc receptor lymphocytes. Recovery of DH with partial recovery of leukocyte and lymphocyte counts was observed three weeks, later, prior to the next course, We conclude that both metastatic tumor and chemotherapy cause abnormalities of the immune system in children with neuroblastoma.