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K Lazaridis

Publications and source records attributed to K Lazaridis.

8 recordsLinked to original sources
PubMed2004

Characterisation of TGF-beta2 signalling and function in a human lens cell line.

There is increasing evidence implicating Transforming growth factor beta (TGF-beta) in pathological states of the lens. However, the underlying signalling mechanisms in human cells have not been fully examined. We have therefore investigated in a human lens cell line, FHL 124, the signalling characteristics of TGF-beta and Smad proteins. Moreover, we have tested the effectiveness of a fully human monoclonal anti-TGF-beta2 antibody, CAT-152, in suppressing TGF-beta2 induced changes in a number of conditions. FHL 124 cells were routinely cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% FCS. Characterisation of the cell line was determined using Affymetrix gene microarrays and compared to native human lens epithelium. Cells were serum starved for 24 hr prior to exposure to TGF-beta2 in the presence and absence of CAT-152. Non-stimulated cells served as controls. Smad 4 localisation was observed by immunocytochemistry. To study Smad-dependent transcriptional activity, cells were transfected with SBE4-luc, an artificial smad-specific reporter, using Fugene-6. Transcriptional activity was determined by luciferase activity. Gene expression was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR). Proliferation was determined by 3H-thymidine DNA incorporation. Growth and contraction were assessed using a scratch and patch assay. Affymettrix gene microarrays identified 99.5% homology between FHL 124 cells and the native lens epithelium with respect to expression pattern of the 22,270 genes on the chip. Moreover, FHL 124 cells expressed phenotypic markers, alphaA-crystallin and pax6 along with lens epithelial cell specific marker FoxE3. Immunocytochemical studies revealed the presence of Smad 4 which following TGF-beta2 exposure accumulated in the cell nucleus. Furthermore, Smad-dependent transcriptional activity was also stimulated. TGF-beta2 enhanced the expression of mRNA levels of alpha smooth muscle actin (alphaSMA) and connective tissue growth factor (CTGF). Exposure to TGF-beta2 resulted in a relatively small inhibition of 3H-thymidine incorporation of FHL 124 cells. However, a more marked contractile effect was also observed. In serum-supplemented medium, growth rates and TGF-beta induced contraction were enhanced. Treatment with 0.1-10 microg ml(-1) CAT-152 dose-dependently inhibited 10 ng ml(-1) TGF-beta2 induced effects in the presence and absence of serum. Exposure of FHL 124 cells to TGF-beta therefore induces Smad translocation, transcription, expression of transdifferentiation markers and induces marked contraction. Treatment with CAT-152 can effectively inhibit these responses. TGF-beta2 induced changes can also persist long after the period of exposure and when in the presence of serum TGF-beta induced contraction is enhanced. The work presented therefore demonstrates a platform technology to study TGF-beta2 signalling in human lens epithelial cells and provides evidence to show TGF-beta2 can be a potent factor in the development of posterior capsule opacification following cataract surgery.

Actins↗
PubMed2001

The 5' untranslated region of Rhopalosiphum padi virus contains an internal ribosome entry site which functions efficiently in mammalian, plant, and insect translation systems.

Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5' UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5' UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, and Drosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5' IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.

5' Untranslated Regions↗
PubMed1995

Analysis of transforming growth factor beta and other cytokines in autoimmune exocrinopathy (Sjögren's syndrome).

Cytokines play a major role in tissue destruction caused by autoimmune dysregulation. In Sjögren's syndrome (SS) patients, salivary glands are the target organs for autoimmune tissue damage. In the present study, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to look for cytokine mRNA expressed in SS salivary glands. Focus score was used to determine the severity of the lesions. Cytokine production in supernatants of the salivary gland cell culture was measured by enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining was used to identify the local presence of transforming growth factor beta (TGF-beta). Interleukin (IL)-2, IL-6, and IL-10 mRNA were expressed in moderate to severe SS salivary gland lesions. TGF-beta mRNA was constitutively expressed in normal and SS salivary glands. In SS salivary gland cell cultures, IL-6 and IL-10 proteins were produced. TGF-beta production was reduced in high focus score SS glands. Normal and minimally involved SS salivary gland ductal epithelium and acinar cells were found to produce TGF-beta by immunostaining. In conclusion, an excess production of IL-2, IL-6, and IL-10 and a reduced production of the immunosuppressive cytokine, TGF-beta, may be responsible for the progression of the salivary gland lesion in SS. Specific immunotherapy can now be designed based on mechanisms to correct this cytokine imbalance and benefit patients with autoimmune diseases, such as SS.

Adult↗
PubMed1993

Induction of lupus-associated autoantibodies by immunization with native and recombinant Ig polypeptides expressing a cross-reactive idiotype 4B4.

A human mAb designated 4B4 with anti-Sm activity was derived from a patient with systemic lupus erythematosus. This antibody expressed a lupus-associated cross-reactive Id, partially related to the monoclonal murine anti-Sm (Y2) from MRL/lpr mice. Studies were performed to investigate the ability of 4B4 to induce lupus in nonautoimmune-prone mice. BALB/c mice immunized with 4B4 produced antibodies to dsDNA, ssDNA, Sm ribonucleoprotein, and mouse Fc fragment. There was no antibody activity against SSA/Ro, SSB/La, and hen egg lysozyme. Ag inhibition studies show that the autoantibodies were not polyreactive. Mice were also immunized with r4B4 polypeptides representing the H/L heterodimer, H chain and L chain. Autoantibodies were induced in mice immunized against the H/L and H polypeptides. No autoantibodies were induced in mice immunized with recombinant L chain. Furthermore, from 20 to 68% of antibody activity to Sm or dsDNA could be inhibited with anti-Id antiserum (either anti-4B4 or Y2). The autoantibody was initially IgM and then underwent an isotype switch to IgG. These results show that lupus-associated autoantibodies can be induced by immunization with 4B4 and that the 4B4 VH region is important in this induction process. The finding of murine IgG autoantibody expressing a cross-reactive Id similar to the immunizing 4B4 suggests a role for anti-idiotypic Th cells in this autoimmune response.

Animals↗
PubMed1993

Cloning of a human IgM autoantibody bearing a cross-reactive idiotype in a lambda expression vector: a new approach to studying autoantibodies.

The monoclonal antibody 4B4 is a human IgM,kappa which expresses a cross-reactive lupus-associated idiotype and has anti-Sm binding activity. We find that the VL nucleotide sequence of 4B4, like the 4B4 VH region, is encoded by unmutated germline genes. The 4B4 VH and VL were cloned into the ImmunoZap lambda expression vector to produce three recombinant immunoglobulin polypeptides. These recombinant polypeptides expressed, respectively, either the 4B4 VH or VL alone or a VH/VL heterodimer. ELISA showed that the VH/VL heterodimer retained anti-Sm antibody activity. The 4B4 idiotype was found predominantly on the VH. This report describes: (i) a method for producing recombinant immunoglobulin molecules from an IgM-secreting B cell line and (ii) the ability of recombinant antibody fragments expressed in Escherichia coli to retain the structural and antigenic properties of the native molecule.

Amino Acid Sequence↗
PubMed1993

Human kappa chain expression in a lambda phage vector: methods of isolating amplified cDNA affects cloning efficiency.

Three common methods of isolating amplified DNA were evaluated for their effects on cloning efficiency in a phage vector designed to express human kappa chain. The "glass milk" technique gave higher cloning efficiency and protein expression than phenol-chloroform extraction or microfiltration. This shows that the quality of amplified cDNA should be considered when studying the human antibody repertoire using this vector system.

Bacteriophage lambda↗
PubMed1991

Early exercise test in acute myocardial infarction treated with intravenous streptokinase.

The aim of this study was to assess the value of the early exercise test (ET) in patients with acute myocardial infarction (AMI) treated with IV streptokinase (SK). The authors studied 70 patients with first AMI; 31 were treated with SK and 39 were not. Before discharge everyone was given early exercise up to 5-6 METs and catheterized within 22.9 +/- 7.2 days. There was no significant difference in the number of positive ETs between the two groups (11/31 and 14/39 respectively). There was significant difference in favor of: (1) the recanalization of the infarct-related artery in the SK group, (2) the negative ET in patients with recanalized vessels in both groups, (3) the positive ET in patients with multi-vessel coronary disease. It is concluded that the results of early ET in patients with AMI are related to the recanalization of the infarct-related artery and the coexistence of multi-vessel coronary artery disease, regardless of SK treatment. Patients with successful thrombolysis have negative ET more frequently.

Cardiac Catheterization↗