Integration and differentiation of human embryonic stem cells transplanted to the chick embryo.

Human embryonic stem (ES) cells are pluripotent cells that can differentiate into a large array of cell types and, thus, hold promise for advancing our understanding of human embryology and for contributing to transplantation medicine. In this study, differentiation of human ES cells was examined in vivo by in ovo transplantation to organogenesis-stage embryos. Colonies of human ES cells were grafted into or in place of epithelial-stage somites of chick embryos of 1.5 to 2 days of development. The grafted human ES cells survived in the chick host and were identified by vital staining with carboxyfluorescein diacetate or use of a green fluorescent protein-expressing cells. Histologic analysis showed that human ES cells are easily distinguished from host cells by their larger, more intensely staining nuclei. Some grafted cells differentiated en masse into epithelia, whereas others migrated and mingled with host tissues, including the dorsal root ganglion. Colonies grafted directly adjacent to the host neural tube produced primarily structures with the morphology and molecular characteristics of neural rosettes. These structures contain differentiated neurons as shown by beta-3-tubulin and neurofilament expression in axons and cell bodies. Axons derived from the grafted cells penetrate the host nervous system, and host axons enter the structures derived from the graft. Our results show that human ES cells transplanted in ovo survive, divide, differentiate, and integrate with host tissues and that the host embryonic environment may modulate their differentiation. The chick embryo, therefore, may serve as an accessible and unique experimental system for the study of in vivo development of human ES cells.     

Modeling for Lesch-Nyhan disease by gene targeting in human embryonic stem cells

Human embryonic stem (ES) cells are pluripotent cells derived from blastocyst-stage embryos. It has been suggested that these cells should play a major role in transplantation medicine and be able to advance our knowledge in human embryology. We propose that these cells should also play a vital role in the creation of models of human disorders. This aspect would be most valuable where animal models failed to faithfully recapitulate the human phenotype. Lesch-Nyhan disease is caused by a mutation in the HPRT1 gene that triggers an overproduction of uric acid, causing gout-like symptoms and urinary stones, in addition to neurological disorders. Due to biochemical differences between humans and rodents, a mouse lacking the HPRT expression will fail to accumulate uric acid. In this research we demonstrate a model for Lesch-Nyhan disease by mutating the HPRT1 gene in human ES cells using homologous recombination. We have verified the mutation in the HPRT1 allele at the DNA and RNA levels. By using selection media, we show that HPRT1 activity is abolished in the mutant cells, and the HPRT1-cells show a higher rate of uric acid accumulation than the wild-type cells. Therefore, these cells recapitulate to some extent the characteristics of Lesch-Nyhan syndrome and can help researchers further investigate this genetic disease and analyze drugs that will prevent the onset of its symptoms. We therefore suggest that human diseases may be modeled using human ES cells.     

Mapping of murine IgE epitopes involved in IgE-Fc epsilon receptor interactions

The generation of anti-IgE monoclonal antibodies has permitted the identification of various serological epitopes on the IgE molecule. The relationship of the sites on IgE recognized by such antibodies to the Fc epsilon receptor (Fc epsilon R) interaction site has been determined using cross-inhibition studies. However, interpretation of this type of experiment is limited by problems of steric hindrance. Thus, to accomplish precise mapping on the IgE molecule of the Fc epsilon R interaction site and the binding sites of various anti-IgE mAb, we employed site-directed mutagenesis of the IgE heavy chain gene. To this end we have constructed and expressed a recombinant murine constant epsilon heavy chain (C epsilon) gene bearing a (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH region. Several site-specific mutants in the C epsilon 3 and C epsilon 4 domains of this recombinant C epsilon gene were prepared and expressed by transfection into the light chain-producing J558L myeloma cell line. The resulting IgE antibodies were tested for binding to mast cells and to various anti-IgE mAb. The mutants produced include a proline to histidine point mutant at amino acid residue 404 in the C epsilon 3 domain, a mutant with a truncated C epsilon 4 domain, a mutant with a 45 amino acid deletion in the carboxy end of C epsilon 3, and a chimeric human C epsilon in which the human C epsilon 3 was replaced by the homologous mouse C epsilon 3 domain. These mutants have permitted the localization, to the C epsilon 3 domain, of the epitopes recognized by the 84.1C and 95.3 anti-IgE mAb. The 84.1C mAb recognizes a site on IgE which is identical or very close to the Fc epsilon R binding site, and 95.3 recognizes a site on IgE which is related, but not identical to the Fc epsilon R binding site. The antigenic determinant recognized by the 51.3 mAb, which is inefficient at blocking the IgE-Fc epsilon R interaction, has been mapped to the C epsilon 4 domain. When tested for binding to the Fc epsilon R on RBL-2H3 cells, the point mutant bound to the Fc epsilon R with twofold reduced affinity, while the C epsilon 3 deletion mutant and the mutant truncated in C epsilon 4 lost all receptor binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antibody fragments selected by phage display against the nuclear localization signal of the HIV-1 Vpr protein inhibit nuclear import in permeabilized and intact cultured cells

The HIV-1 Vpr protein harbors a nuclear localization signal in its N-terminal domain. A peptide bearing this domain and which is designated VprN has been used as a target to screen a phage display single chain Fv (scFv) library. Here we report the isolation of anti-VprN scFv fragments from this library. The purified scFv fragments were able to bind the VprN peptide in an ELISA-based system and to inhibit VprN-mediated nuclear import in permeabilized as well as in intact microinjected cells. Furthermore, the anti-VprN scFv fragments recognized the full-length recombinant Vpr protein and inhibited its nuclear import. The same scFv fragments did not inhibit nuclear import mediated by the nuclear localization signal of the SV40 large T-antigen demonstrating a specific effect. The use of the described inhibitory anti-VprN scFv fragments to study nuclear import of viral karyophilic proteins and their therapeutic potential is discussed.     

Haptoglobin-related protein as a serum marker in malignant lymphoma

A novel serum 21 kDa haptoglobin-related protein (Hpr) was investigated in patients with malignant lymphoma, to evaluate its correlation with clinical and histologic features at presentation and its possible role as a tumor marker for patient outcome. One hundred fifty eight serum samples were taken from 88 patients with non-Hodgkin’s lymphoma (n=58) and Hodgkin’s disease (n=30) at presentation and in the course of follow-up. Sera from 61 healthy volunteers served as normal controls. Serum Hpr levels in the lymphoma patients (median 430xl0³ u/ml, range 0-4000xl0³) were significantly higher than in the control group (median 68xl0³ u/ml, range 0-180xl0³) (p=0.0001). Higher median Hpr values were detected in patients with advanced disease (p=0.013), “B” symptoms (p=0.029) and in males (p=0.053). There was also a significant correlation between Hpr and erythrocyte sedimentation rate (p=0.028). Serial determinations showed a significant decrease of the initial Hpr values obtained after treatment in 41 patients, 38 of whom achieved complete remission. In the follow-up period additional Hpr measurements were taken from 17 patients. Three of them eventually relapsed, and showed increased Hpr levels at the time of relapse. Hpr levels remained low in 11 of 14 patients who maintained complete remission, and increased in three. In conclusion, serum Hpr is a new serum tumor marker of potential use in the clinical setting of lymphoma. This work is dedicated to the memory of Dr. Arie H. Bartal, a dedicated oncologist and friend. This work was supported by Chemotech Thechnologies Ltd., by grant no. 3676 from the Chief Scientist’s Office of the Ministry of Health, Israel, and by the Fund for Promotion of Research in the Technion.     

Can trained volunteers provide psychosocial support to patients undergoing radiotherapy? The perspective of patients and volunteers

PurposeClinic-based psychosocial interventions, including volunteer-based ones, may be a cost-efficient and acceptable means of integrating psychosocial support into cancer care during radiotherapy. The present study evaluated a new psychosocial volunteer support program in a large radiotherapy clinic.Methods and MaterialsPatients were asked to complete a demographic and satisfaction with care questionnaire. Clinic volunteers were asked to report their interactions with patients on shift logs.ResultsOf the 182 participating patients, 93 (51%) recalled meeting a volunteer in the clinic, with the 2 most common support types provided being the following: “listening and caring,” and “information on services.” Analysis of 224 volunteers' shift logs indicated that almost all interactions (94%) were initiated by the volunteers, and almost half (47%) involved the patients' companions in the clinic. The most common support type documented was “information and navigation” (71%), followed by “emotional” (47%), “diversional” (21%), and “physical/practical” (17%) support.ConclusionsTrained volunteers can effectively provide clinic-based psychosocial support and information to a high proportion of radiotherapy patients. These findings demonstrate that volunteer support is a feasible means of meeting the psychosocial needs of patients with cancer attending outpatient radiotherapy clinics, who may not require or want professional psychosocial support.     

Milk derived colloid as a novel drug delivery carrier for breast cancer

Triple negative breast cancer has an extremely poor prognosis when chemotherapy is no longer effective. To overcome drug resistance, novel drug delivery systems based on nanoparticles have had remarkable success. We produced a novel nanoparticle component ‘MDC’ from milk-derived colloid. In order to evaluate the anti-cancer effect of MDC, we conducted in vitro and in vivo experiments on cancer cell lines and a primary tumor derived breast xenograft. Doxorubicin (Dox) conjugated to MDC (MDC-Dox) showed higher cancer cell growth inhibition than MDC alone especially in cell lines with high EGFR expression. In a mouse melanoma model, MDC-Dox significantly suppressed tumor growth when compared with free Dox. Moreover, in a primary tumor derived breast xenograft, one of the mice treated with MDC-Dox showed partial regression, while mice treated with free Dox failed to show any suppression of tumor growth. We have shown that a novel nanoparticle compound made of simple milk-derived colloid has the capability for drug conjugation, and serves as a tumor-specific carrier of anti-cancer drugs. Further research on its safety and ability to carry various anti-cancer drugs into multiple drug-resistant primary breast models is warranted.     

Blockage of the Early Events of Mitogenic Signaling by Interferon-gamma in Macrophages in Response to Colony-Stimulating Factor-1

Inhibition of cell proliferation is an important biologic funcphorbol ester, as measured by early gene expression, DNA tion of interferons (IFNs), which has been exploited in therasynthesis and cell proliferation. Although activation, phospeutic treatment of certain hematologic malignancies. Howphorylation, and turnover of the CSF-1 receptor and CSF-1- ever, the molecular mechanism was not clear. We have induced increase in diacylglycerol production remained norrecently shown that IFNs (/ and ) inhibit protein kinase mal, IFN- blocked CSF-1- stimulated activation of mitogen-C (PKC)-dependent (such as PDGF and phorbol ester) but activated protein kinases, Raf-1 kinase, increase in GTP-not PKC-independent (such as epidermal growth factor) actibound Ras and tyrosine phosphorylation, and activation of vation of Raf-1 and mitogen-activated protein kinases protein kinase C  (PKC-). PKC- was required for CSF-1- (MAPK/ERKs) in fibroblasts (Xu et al, Mol Cell Biol 14:8018, induced mitogenic signaling and a primary target for IFN-- 1994), suggesting a novel mechanism by which IFNs execute induced inhibition. Interestingly, although phorbol myristate their antiproliferative function. Monocytes/macrophages acetate stimulated Ras activation, PKC- did not appear to are primary targets in vivo for IFN-, the major activity of be an upstream activator of Ras. These studies clearly indimacrophage-activating factor. In the present study, mechacated that IFN- specifically inhibits PKC- activation, renism of IFN-- induced antiproliferative action in macrosulting in blockage of the early events of mitogenesis in phages in response to colony-stimulating factor- 1 (CSF-1) macrophages in response to CSF-1.