Biomedical subjects

M Gad

Publications and source records attributed to M Gad.

At least 19 recordsLinked to original sources

PubMed2006

Characterization of monocyte-derived dendritic cells maturated with IFN-alpha.

Dendritic cells (DC) are promising candidates for cancer immunotherapy. These cells can be generated from peripheral blood monocytes cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). In order to obtain full functional capacity, maturation is required, but the most potent reagents such as LPS or polyriboinosinic polyribocytidylic acid (Poly I:C) are not approved for clinical use. We tested the ability of type I interferon (IFN) to induce such maturation. We found that 24-h IFN-alpha co-culture of day 7 monocyte-derived DC generated with GM-CSF and IL-4 induces increased numbers of DC positive for CD54 and CD40 together with the co-stimulatory molecule CD80 but not the activation marker CD83. Also, IFN-alpha maturation leads to an increase in IP-10 and MCP-1 chemokine secretion, but only a minor increase in IL-12p40 secretion. In line with this, maturation with IFN-alpha has only a small effect on induction of autologous T-cell stimulatory capacity of the DC. However, an increase in DC allogeneic T-cell stimulatory capacity was observed. These data suggest that IFN-alpha has a potential as a maturation agent used in DC-based cancer vaccine trials, but not as a single reagent.

Apoptosis↗

PubMed2005

Regulatory T cells in experimental colitis.

Induction and maintenance of peripheral tolerance are important mechanisms to maintain the balance of the immune system. Growing evidence indicates that dysregulation of mucosal T cell responses may lead to loss of tolerance to commensal flora and to the development of inflammatory bowel diseases (IBD). Many studies suggest that active suppression of enteroantigen reactive cells mediated by regulatory T cells contributes to the maintenance of natural intestinal immune homeostasis. The use of the multiple animal models has not only improved our understanding of IBD, but also contributed to new suggestions of treatment strategies involving the use of regulatory T cells. The present review summarizes our current knowledge of regulatory T cells and their involvement in experimental IBD. The well-characterized SCID T cell transfer model and the naturally occurring regulatory CD4+CD25+ T cells are highlighted.

Animals↗

PubMed2005

Phenotypic and functional characterization of clinical grade dendritic cells generated from patients with advanced breast cancer for therapeutic vaccination.

Dendritic cells (DC) are promising candidates for cancer immunotherapy. However, it is not known whether in vitro-generated monocyte-derived DC from cancer patients are altered compared with DC from healthy donors. In a clinical phase I/II study, monocyte-derived DC were generated in vitro utilizing granulocyte macrophage colony-stimulating factor and rh-interleukin-4 (IL-4) and used for cancer immunotherapy. In this study, we tested the effect of various maturation cocktails and performed a comparative evaluation of the DC phenotype and functional characteristics. Polyriboinosinic polyribocytidylic acid (Poly I:C) + tumour necrosis factor-alpha (TNF-alpha) induced significant IL-12 p70 secretion, which was increased after addition of a decoy IL-10 receptor. The lymph node homing chemokine receptor CCR-7 expression was induced by TNF-alpha + IL-1beta + IL-6 + prostaglandin E2 but was not induced by Poly I:C + TNF-alpha. In general, DC from patients had an intermediate maturity phenotype with a significantly higher expression of CD40 and CD54 compared with healthy donors. In vitro analyses showed an unimpaired capacity of the patient-derived DC for antigen-specific (cytomegalovirus, tetanus and keyhole limpet haemocyanin) T-cell stimulation, whereas the allostimulatory capacity of patient-derived DC was significantly decreased. These data suggest that patient-derived DC are more differentiated but are less sensitive to maturation-inducing agents than DC obtained from healthy individuals.

Breast Neoplasms↗

PubMed2003

CD4+ T regulatory cells from the colonic lamina propria of normal mice inhibit proliferation of enterobacteria-reactive, disease-inducing Th1-cells from scid mice with colitis.

Adoptive transfer of CD4+ T cells into scid mice leads to a chronic colitis in the recipients. The transferred CD4+ T cells accumulate in the intestinal lamina propria (LP), express an activated Th1 phenotype and proliferate vigorously when exposed ex vivo to enteric bacterial antigens. As LP CD4+ T cells from normal BALB/c mice do not respond to enteric bacterial antigens, we have investigated whether colonic LP-derived CD4+ T cells from normal mice suppress the antibacterial response of CD4+ T cells from scid mice with colitis. LP-derived CD4+ T cells cocultured with bone marrow-derived dendritic cells effectively suppress the antibacterial proliferative response of CD4+ T cells from scid mice with colitis. The majority of these LP T-reg cells display a nonactivated phenotype and suppression is independent of antigen exposure, is partly mediated by soluble factor(s) different from IL-10 and TGF-beta, and is not prevented by the addition of high doses of IL-2 to the assay culture. Functionally and phenotypically the T-reg cells of the present study differ from previously described subsets of T-reg cells. The presence of T cells with a regulatory potential in the normal colonic mucosa suggests a role for these cells in the maintenance of local immune homeostasis of the gut.

Adoptive Transfer↗

PubMed2003

Immunobiological effects of glucosamine in vitro.

Glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) were assayed in vitro for their effects on proliferation, cytotoxicity and cytokine secretion in primary and secondary mixed lymphocyte cultures (MLCs). In addition, we studied the effect of GlcN and GlcNAc on the proliferation of purified CD4+ T cells exposed to immobilized anti-CD3 antibody. The present data show that GlcN, but not GlcNAc, inhibits CD4+ T-cell proliferation, the generation of alloreactive cytotoxic T lymphocytes (CTLs) and the secretion of interferon-gamma (IFN-gamma) and interleukin-5 (IL-5) in primary MLC. In secondary T helper-2 (Th2)-polarized MLC, GlcN, but not GlcNAc, inhibits IL-4 and IL-5 secretion, whereas no effect was found on IFN-gamma secretion in Th1-polarized MLC. Dendritic cells treated with GlcN showed a 75-80% decreased capacity for antigen cross-presentation and allostimulation. In cellular bioassays, GlcN was shown to inhibit the stimulatory activity of IL-4 and IL-2, as well as the cytotoxic activity of tumour necrosis factor-alpha (TNF-alpha). In conclusion, GlcN suppresses unprimed T-cell responses by interfering with antigen-presenting cell functions and by a direct inhibitory effect on T-cell proliferation. In addition, GlcN inhibits the secretion of cytokines in antigen-stimulated unprimed T cells and primed Th2-polarized cells.

Adjuvants, Immunologic↗

PubMed2003

Method for patterning stretched DNA molecules on mica surfaces by soft lithography.

Lambda DNA was stretched and patterned on mica surface using soft lithography. A highly diluted solution of amino propyl trimethoxy silane in hexane was deposited on a line patterned polydimethylsiloxane (PDMS) stamp. The functionalized stamp was then used to pick up DNA by molecular combing while the line patterns are parallel to the liquid surface. The stamp was then microcontact printed on freshly cleaved mica. We successfully obtained stretched DNA pattern on mica surface. DNA was found to be stretched in patterns perpendicular to those carved on the stamp. The stretched DNA population was large enough to be used for molecular biology mapping studies. Furthermore, the possibility of locating stretched DNA molecules in the desired position by stamping makes this method a good candidate for assembling non-semiconductor molecular devices.

Aluminum Silicates↗

PubMed2002

Patterning DNA on microm scale on mica.

Double-stranded DNA molecules were patterned by selective adsorption to aminosilane patterns on mica surfaces. Line patterns with 10 microm spacing were made by photolithography and transferred to a polymer stamp. The stamp was then used for applying aminosilane molecules by microcontact-printing technique on mica substrates. We applied DNA in Tris-EDTA (TE) buffer solution on the patterned substrate, and incubated it for 5 min at room temperature. The sample was then rinsed with pure water, and dried with nitrogen gas. Tapping mode force microscopy showed that DNA was adsorbed selectively on the aminosilanized parts of the mica substrate. We also tried to bridge two aluminum electrodes with DNA using AC electrophoresis.

Adsorption↗

PubMed2001

Accumulation of plant galactolipid affects cell morphology of Escherichia coli.

Monogalactosyldiacylglycerol (MGDG) is a major constituent of thylakoid membrane in chloroplasts. Therefore, it is considered to have an important role in the maintenance of the complicated structure of the thylakoid membrane. We have succeeded in cloning the enzyme for MGDG synthesis and overexpressed it in Escherichia coli. In this study we analyzed the morphology of the E. coli harboring the gene. The fatty acid composition of its membrane lipids did not differ between the wild type and transformant, except for the appearance of MGDG. However, transformant cells appeared to be elongated. DAPI staining revealed the entire intracellular region of filamentous cells to be stained; therefore, the elongation of the cells is probably due to a defect in cell division. Atomic force microscopy revealed that the transformant had a smooth but scratched surface. It was concluded that the excessive accumulation of a non-bilayer lipid, MGDG, interfered with the translocation of proteins across the plasma membrane, including those for cell division.

Escherichia coli↗

PubMed2001

Radically altered T cell receptor signaling in glycopeptide-specific T cell hybridoma induced by antigen with minimal differences in the glycan group.

A T cell hybridoma raised against the synthetic glycopeptide T(72)(Tn) was used to study whether the initial TCR signaling events are markedly different when the hybridoma is stimulated with glycopeptides closely related to the cognate glycopeptide antigen. T(72)(Tn) has an alpha-D-GalNAc group O-linked to the central threonine in the decapeptide VITAFTEGLK, and the hybridoma is known to be highly specific for this carbohydrate group. T(72)(Tn)-pulsed APC induced tyrosine phosphorylation of the TCR-zeta 21- and 23-kDa proteins and the downstream p42/44 MAP kinase and strong IL-2 secretion. APC pulsed with T(72)(alpha-D-GlcNAc), which differs from T(72)(Tn) solely by the orientation of a hydroxy group in the carbohydrate structure, completely failed to induce detectable tyrosine phosphorylation and IL-2 secretion. APC pulsed with S(72)(Tn), which differs from T(72)(Tn) by not having a methyl group in the serine amino acid side chain to which the glycan is attached, induced partial tyrosine phosphorylation of the TCR-zeta 21-kDa protein, no tyrosine phosphorylation of the MAP kinases and no IL-2 production. Molecular modeling of the MHC/glycopeptide complex revealed that the dramatic difference between the stimulatory power of T(72)(Tn) and T(72)(alpha-D-GlcNAc) is mainly due to very small differences in the TCR exposed carbohydrate structure.

Cell Line↗

PubMed2001

Method for orienting DNA molecules on mica surfaces in one direction for atomic force microscopy imaging.

An efficient method was developed to stretch DNA molecules on an atomically flat surface for AFM imaging. This method involves anchoring DNA molecules from their 5' ends to amino silanized mica surfaces. N-Succinimidyl6-[3'-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP), a heterobifunctional cross-linker with a flexible spacer arm was used for this purpose. Immobilization was carried out by introducing a thiol group to the 5' end of DNA by PCR. Thiolated molecules were then reacted with the cross linker to conjugate with its 2-pyridyl disulphide group via sulfhydryl exchange. The resulting complex was deposited on amino silanized mica where NHS-ester moiety of the cross linker reacted with the primary amino group on the surface. Samples were washed by a current of water and dried by an air jet in one direction parallel to the surface. DNA molecules were fully stretched in one direction on imaging them by AFM.

Aluminum Silicates↗

PubMed2000

Method for stretching DNA molecules on mica surface in one direction for AFM imaging.

An efficient method was developed to stretch DNA molecules on an atomically flat surface for AFM imaging. This method involves anchoring DNA molecules from their 5' ends to amino silanized mica surfaces. N-Succinimidyl6-[3'-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP), a heterobifunctional cross-linker with a flexible spacer arm was used for this purpose. The immobilization process was carried out by introducing a thiol group to the 5' end of DNA by PCR. Thiolated molecules were then reacted with the cross linker to conjugate with its 2-pyridyl disulphide group via sulfhydryl exchange. The resulting complex was deposited on amino silanized mica where NHS-ester moiety of the cross linker reacted with the primary amino group on the surface. Samples were washed by a current of water and dried by an air jet in one direction parallel to the surface. DNA molecules were shown to be fully stretched in one direction on imaging them by AFM.

Aluminum Silicates↗

PubMed1997

Mapping cell wall polysaccharides of living microbial cells using atomic force microscopy.

Functionalized atomic force microscope tips were used to sense specific forces of interaction between ligand-receptor pairs and to map the positions of polysaccharides on a living microbial cell surface. Gold-coated tips were functionalized with concanavalin A using a cross-linker with a spacer arm of 15.6 A. It was possible to measure the binding force between concanavalin A and mannan polymers on the yeast (Saccharomyces cerevisiae) cell surface. This force ranged from 75 to 200 pN. The shape of the force curve indicated that the polymers were pulled away from the cell surface for a fairly long distance that sometimes reached several hundred nanometres. The distribution of mannan on the cell surface was mapped by carrying out the force measurement in the force volume mode of atomic force microscopy (AFM). During the measurement, the maximum cantilever deflection after contact between the tip and the sample was kept constant at 10 nm using trigger mode to keep the pressing force on the sample surface as gently as possible at a force of 180 pN. This regime was used to minimize the non-specific adhesion between the tip and the cell surface. Specific molecular recognition events took place on specific areas of the cell surface that could be interpreted as reflecting a non-uniform distribution of mannan on the cell surface.

Cell Membrane↗

PubMed1995

Method for immobilizing microbial cells on gel surface for dynamic AFM studies.

The processes of cell growth and budding of the yeast cells Saccharomyces cerevisiae, which were gently immobilized on 3% agar and submerged in culture medium, were successfully imaged with an atomic force microscope for 6-7 h. Similar experiments on chemically fixed cells did not detect any appreciable change in their appearance except in a few scannings at the very beginning, indicating that the dissolution of agar and/or scraping of its surface by the scanning tip, if any, did not significantly interfere with the images taken thereafter. The increment in the height of many of the untreated cells, accompanied by their lateral enlargement, was taken as an indication of successful imaging of the growth process of yeast cells, together with an image of a growing daughter cell attached to its mother cell.

Agar↗

PubMed1994

Vector competence of autogenous and anautogenous Culex pipiens mosquitoes for Wuchereria bancrofti.

Vector competence of autogenous and anautogenous Culex pipiens; derived from North Sinai Governorate, Egypt, for the human filaria parasite Wuchereria bancrofti was studied. After feeding on the same microfilaremic volunteers both biotypes were readily infected with the parasite (infection rates > 80%) and supported its development to the infective stage within 11 - 12 days. Infective rates of both autogenous and anautogenous mosquitoes were similar (> 95%). However, autogenous Cx. pipiens developed significantly less number of infective stage larvae (4.7 +/- 1.4 L3 / female) than did anautogenous siblings (6.7 +/- 3.6 L3 / female) (P < 0.05). Moreover, autogenous females were observed to contain twice the number of L3 larvae in the thoracic muscles and less larvae in the head region compared to autogenous counterparts. Vector competence characteristics of Cx. pipiens derived from a filariasis endemic area in Qalubiya Governorate were similar to those of anautogenous mosquitoes of North Sinai. These findings indicate that autogenous Cx. pipiens may be less efficient vector of W. bancrofti in endemic areas of Egypt.

Animals↗

PubMed1991

Percutaneous ethanol injection for the treatment of symptomatic cystic metastases from ovarian carcinoma. Work in progress.

Two patients with symptomatic cystic metastases from ovarian epithelial carcinoma underwent ultrasound (US)-guided percutaneous aspiration and temporary injection of 99% ethanol into the cyst. In the first case, the patient initially underwent surgical resection of the mass and received systemic chemotherapy, but the cyst recurred 2 months later. Percutaneous aspiration and ethanol sclerotherapy were performed twice in the second case; fluid reaccumulated 2 months after the initial procedure. No side effects occurred. During the follow-up period (8 months in the first case and 4 months in the second), no clinical recurrence of the initial symptoms was noted. At the end of that period, a recurrent but asymptomatic cystic lesion was revealed at US examination in the first case. In the second case, a minimal asymptomatic residual collection was depicted with computed tomography. The results indicate that this technique should be considered in patients with symptomatic cystic metastases from ovarian carcinoma and may have potential benefit in the palliative treatment of such lesions.

Administration, Cutaneous↗

PubMed1991

Preoperative segmental localization of hepatic metastases: utility of three-dimensional CT during arterial portography.

To evaluate the accuracy of a fast three-dimensional (3D) reconstruction technique in determining the segmental location of hepatic metastases, 14 patients (40 metastases) were prospectively investigated before surgery with dynamic two-dimensional (2D) computed tomography during sequential arterial portography (CTAP). All patients underwent subsequent hepatic tumor resection within 4 days. After computer-generated mapping of hepatic venous structures with high attenuation and metastases with low attenuation, 3D reconstruction was performed. Thirty-six of the 40 lesions (90%) were detected with 2D and 3D CTAP. The accuracy in determining the segmental location of hepatic metastases was 78% (28 of the 36 metastases) for 2D CTAP and 94% (34 of the 36 metastases) for 3D CTAP. The difference in localization rates between 2D CTAP and 3D CTAP was statistically significant. The 3D CTAP technique provides vital data unattainable with other imaging modalities that improve the preoperative assessment of the resectability of hepatic metastases and allows planning a safer surgical approach.

Adult↗

PubMed1991

Primary carcinoid tumor of the seminal vesicles: CT and MR findings.

A case of primary carcinoid tumor of the seminal vesicles is presented. To our knowledge, this case documented by CT and MRI is unique in that it is the first described in this location. The tumor appears homogeneous and intensely enhanced after IV on CT, and hypointense on MRI on both T1- and T2-weighted images. On T2-weighted images, the tumor is heterogeneous.

Carcinoid Tumor↗

PubMed1984

Evidence that the cytosolic activity of 3-hydroxybutyrate dehydrogenase in chicken liver is L-3-hydroxyacid dehydrogenase.

Classical fractionation studies showed that chicken liver contains two enzymes which can oxidize DL-3-hydroxybutyrate. The cytosolic enzyme is specific for the L-(+) isomer and accounts for 60% of the total activity. The mitochondrial activity is specific for the D-(-) isomer and accounts for 40% of the total activity. Kinetic studies showed that L-gulonic acid is a competitive inhibitor of the enzyme. We conclude that the cytosolic enzyme is the previously described L-3-hydroxyacid dehydrogenase.

3-Hydroxybutyric Acid↗