Search PubMed⌕ Search

Biomedical subjects

R Alam

Publications and source records attributed to R Alam.

At least 37 records · Page 2Linked to original sources
PubMed1999

The mapping of the Lyn kinase binding site of the common beta subunit of IL-3/granulocyte-macrophage colony-stimulating factor/IL-5 receptor.

It has been shown that a membrane-proximal region within common beta (betac) receptor of IL-3/granulocyte-macrophage CSF/IL-5 (amino acids 450-517) is important for Lyn binding. We have shown previously that Lyn kinase is physically associated with the IL-5R betac subunit in unstimulated cells. The result suggests that this association involves binding modules that are not activation or phosphorylation dependent. The objective of this study was to map the exact Lyn binding site on betac. Using overlapping and/or sequential peptides derived from betac 450-517, we narrowed down the Lyn binding site to nine amino acid residues, betac 457-465. The P-->A mutation in this region abrogated the binding to Lyn, indicating a critical role of proline residues. We created a cell-permeable Lyn-binding peptide by N-stearation. This cell-permeable peptide blocked the association of Lyn, but not Jak2 with betac in situ. We also investigated the betac binding site of Lyn kinase. Our results suggest that the N-terminal unique domain of Lyn kinase is important for binding to betac receptor. To our knowledge, this is the first molecular identification of the Lyn binding site of betac receptor. This finding may help develop specific inhibitors of Lyn-coupled signaling pathways.

Amino Acid Sequence↗
PubMed1999

Regulation of eosinophil apoptosis: transduction of survival and death signals.

Since eosinophils are prominent in allergic inflammation, investigators became interested in how these cells accumulate in tissues and their role within the inflammatory cascade. There is increasing evidence from several laboratories that eosinophil numbers are regulated in vivo, not only by eosinophil production in the bone marrow, but also by the amount of eosinophil apoptosis. Moreover, it has been directly demonstrated that eosinophil apoptosis is delayed in allergic inflammatory sites, and that this mechanism contributes to the expansion of these cells in tissue. In this article, we review recent studies that shed light on the intracellular pathways that control eosinophil apoptosis.

Animals↗
PubMed1998

Lyn, Jak2, and Raf-1 kinases are critical for the antiapoptotic effect of interleukin 5, whereas only Raf-1 kinase is essential for eosinophil activation and degranulation.

Interleukin (IL)-5 has been shown to activate many signaling molecules in eosinophils, but their functional relevance remains unknown. We have examined the functional relevance of Lyn, Jak2, and Raf-1 kinases in eosinophil survival, upregulation of adhesion molecules and degranulation. To this goal we used Lyn and Raf-1 antisense (AS) oligodeoxynucleotides (ODN) to inhibit the expression of these proteins and tyrphostin AG490 to specifically block the activation of Jak2. We have demonstrated that all three kinases are important for IL-5- induced suppression of eosinophil apoptosis. However, Lyn and Jak2 tyrosine kinases are not important for the upregulation of CD11b and the secretion of eosinophil cationic protein. In contrast, Raf-1 kinase is critical for both these functions. This is the first identification of specific signaling molecules responsible for three important functions of eosinophils. We have established a central role for Raf-1 kinase in regulating eosinophil survival, expression of beta2 integrins and degranulation. Further, there appears to be a dissociation between two receptor-associated tyrosine kinases, i.e., Lyn and Jak2, and the activation of Raf-1 kinase. The delineation of the functional relevance of signaling molecules will help design therapeutic approaches targeting specific eosinophil function.

Apoptosis↗
PubMed1998

Diisocyanate antigen-enhanced production of monocyte chemoattractant protein-1, IL-8, and tumor necrosis factor-alpha by peripheral mononuclear cells of workers with occupational asthma.

BACKGROUND: Previous studies have shown a significant association between confirmed diisocyanate-induced asthma (DOA) and in vitro production of diisocyanate antigen-stimulated histamine-releasing factors by PBMCs. Chemokines found in PBMC supernatants are known to express histamine-releasing factor activity. OBJECTIVE: PBMCs of diisocyanate-exposed workers were tested in vitro for diisocyanate antigen-specific enhancement of monocyte chemoattractant protein-1 (MCP-1), monocyte chemoattractant protein-3 (MCP-3), macrophage inflammatory protein-1alpha, RANTES, IL-8, and T-cell cytokines that could play a regulatory role in chemokine synthesis (IL-4, IL-5, IFN-gamma, and TNF-alpha. METHODS: Secretion of chemokines and cytokines was determined by quantitative immunochemical assays of PBMC supernatants. Synthesis of mRNA for beta-chemokines was determined by reverse transcription-polymerase chain reaction. RESULTS: PBMCs of workers with DOA showed significantly enhanced secretion for MCP-1 compared with diisocyanate-exposed asymptomatic workers (P < .05). In vitro induction of antigen-stimulated MCP-1 mRNA synthesis in cultured PBMCs was demonstrated by reverse-transcription polymerase chain reaction. Quantitation of cytokines in supernatants showed increased mean production of IL-8 and TNF-alpha. IFN-gamma, IL-4, and IL-5 were not enhanced in subjects with DOA. CONCLUSION: Antigen stimulation of MCP-1 and TNF-alpha suggest that diisocyanate-specific cellular immune reactions result in activation of macrophages, which may be important in the pathogenesis of DOA.

Adult↗
PubMed1998

A brief review of the immune system.

This article provides a brief review of the immune system and describes the features of innate and adaptive immunity and their similarities and differences. The mechanism of antigen presentation and major histocompatibility complex restriction is discussed as well as the structure and function of T cells and B cells. Three tables present a concise description of cytokines, interleukins, and chemokines.

Antigen Presentation↗
PubMed1998

Cell-specific expression of RANTES, MCP-1, and MIP-1alpha by lower airway epithelial cells and eosinophils infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the major cause of acute bronchiolitis in infancy, a syndrome characterized by wheezing, respiratory distress, and the pathologic findings of peribronchial mononuclear cell infiltration and release of inflammatory mediators by basophil and eosinophil leukocytes. Composition and activation of this cellular response are thought to rely on the discrete target cell selectivity of C-C chemokines. We demonstrate that infection in vitro of human epithelial cells of the lower respiratory tract by RSV induced dose- and time-dependent increases in mRNA and protein secretion for RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha). Production of MCP-1 and MIP-1alpha was selectively localized only in epithelial cells of the small airways and lung. Exposure of epithelial cells to gamma interferon (IFN-gamma), in combination with RSV infection, induced a significant increase in RANTES production that was synergistic with respect to that obtained by RSV infection or IFN-gamma treatment alone. Epithelial cell-derived chemokines exhibited a strong chemotactic activity for normal human blood eosinophils. Furthermore, eosinophils were susceptible to RSV and released RANTES and MIP-1alpha as a result of infection. Therefore, the inflammatory process in RSV-induced bronchiolitis appears to be triggered by the infection of epithelial cells and further amplified via mechanisms driven by IFN-gamma and by the secretion of eosinophil chemokines.

Bronchi↗
PubMed1998

The mechanism of IL-5 signal transduction.

Cytokines are important regulators of hematopoiesis. They exert their actions by binding to specific receptors on the cell surface. Interleukin-5 (IL-5) is a critical cytokine that regulates the growth, activation, and survival of eosinophils. Because eosinophils play a seminal role in the pathogenesis of asthma and allergic diseases, an understanding of the signal transduction mechanism of IL-5 is of paramount importance. The IL-5 receptor is a heterodimer of alpha- and beta-subunits. The alpha-subunit is specific, whereas the beta-subunit is common to IL-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF) receptors and is crucial for signal transduction. It has been shown that there are two major signaling pathways of IL-5 in eosinophils. IL-5 activates Lyn, Syk, and JAK2 and propagates signals through the Ras-MAPK and JAK-STAT pathways. Studies suggest that Lyn, Syk, and JAK2 tyrosine kinases and SHP-2 tyrosine phosphatase are important for eosinophil survival. In contrast to their survival-promoting activity, Lyn and JAK2 appear to have no role in eosinophil degranulation or expression of surface adhesion molecules. Raf-1 kinase, on the other hand, is critical for eosinophil degranulation and adhesion molecule expression. Btk is involved in IL-5 stimulation of B cell function. However, it does not appear to be important for eosinophil function. Thus a clear segregation of signaling molecules based on their functional importance is emerging. This review describes the signal transduction mechanism of the IL-3/GM-CSF/IL-5 receptor system and compares and contrasts IL-5 signaling between eosinophils and B cells.

Animals↗
PubMed1998

RANTES induces nasal mucosal inflammation rich in eosinophils, basophils, and lymphocytes in vivo.

RANTES is a CC chemokine that causes chemotaxis of eosinophils, basophils, and lymphocytes in vitro. The objective of this study was to investigate the effect of RANTES on the influx of inflammatory cells into the nasal mucosa of 12 allergic patients. In the first phase, each patient was challenged with RANTES or diluent on two subsequent days. RANTES caused a significant (p < 0.05) influx of eosinophils as compared with the diluent. The number of eosinophils were 5,548 +/- 1,532/ml and 462 +/- 206/ml after RANTES and diluent challenge, respectively, at the peak of the response at 2 h. There was also a significant influx of metachromatic cells and lymphocytes, but not monocytes, neutrophils, or epithelial cells after RANTES challenge. In the second phase, the patients were first challenged with an allergen and 24 h later, challenged with RANTES or diluent. In the allergen-primed mucosa RANTES induced a significantly higher influx of eosinophils, basophils, and lymphocytes. Further, RANTES caused migration of monocytes and neutrophils, and shedding of epithelial cells. The influx of the inflammatory cells was associated with symptoms of rhinitis. We conclude that RANTES induces a clinically symptomatic inflammatory response in vivo by causing chemotaxis of eosinophils, basophils, and mononuclear cells.

Adult↗
PubMed1997

Src homology 2 protein tyrosine phosphatase (SHPTP2)/Src homology 2 phosphatase 2 (SHP2) tyrosine phosphatase is a positive regulator of the interleukin 5 receptor signal transduction pathways leading to the prolongation of eosinophil survival.

Interleukin-5 (IL-5) regulates the growth and function of eosinophils. It induces rapid tyrosine phosphorylation of Lyn and Jak2 tyrosine kinases. The role of tyrosine phosphatases in IL-5 signal transduction has not been investigated. In this study, we provide first evidence that SH2 protein tyrosine phosphatase 2 (SHPTP2) phosphotyrosine phosphatase plays a key role in prevention of eosinophil death by IL-5. We found that IL-5 produced a rapid activation and tyrosine phosphorylation of SHPTP2 within 1 min. The tyrosine phosphorylated SHPTP2 was complexed with the adapter protein Grb2 in IL-5-stimulated eosinophils. Furthermore, SHPTP2 appeared to physically associate with beta common (betac) chain of the IL-5 receptor (IL-5betacR). The association of SHPTP2 with IL-5betacR was reconstituted using a synthetic phosphotyrosine-containing peptide, betac 605-624, encompassing tyrosine (Y)612. The binding to the phosphotyrosine-containing peptide increased the phosphatase activity of SHPTP2, whereas the same peptide with the phosphorylated Y612--> F mutation did not activate SHPTP2. Only SHPTP2 antisense oligonucleotides, but not sense SHPTP2, could inhibit tyrosine phosphorylation of microtubule-associated protein kinase, and reverse the eosinophil survival advantage provided by IL-5. Therefore, we conclude that the physical association of SHPTP2 with the phosphorylated betac receptor and Grb2 and its early activation are required for the coupling of the receptor to the Ras signaling pathway and for prevention of eosinophil death by IL-5.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Monocyte chemotactic protein-3 (MCP-3)/fibroblast-induced cytokine (FIC) in eosinophilic inflammation of the airways and the inhibitory effects of an anti-MCP-3/FIC antibody.

Monocyte chemotactic protein-3 (MCP-3)/fibroblast-induced cytokine (FIC), a CC chemokine, is chemotactic for cells that typically infiltrate the late-phase allergic reaction. We developed a mouse model of airway inflammation to study the role of MCP-3/FIC. The immunization of mice with OVA resulted in Ag-specific IgE Ab production and the expression of mRNA for IL-4 in the lung tissue. Two weeks after immunization mice were challenged with the allergen by inhalation. Lungs were lavaged, and the tissue was examined at 2 or 24 h. Allergen challenge resulted in the increased recovery of leukocytes in the lavage fluid, but saline challenge did not. There was a significant increase in eosinophils (29 +/- 8% vs 1.2 +/- 0.2%) and lymphocytes (25 +/- 4% vs 5 +/- 2%) in the bronchoaveolar lavage fluid. Histologic examination of the lung demonstrated intense airway inflammation following OVA challenge. The expression of MCP-3/FIC and other CC chemokines (MCP-1, macrophage inflammatory protein-1alpha, and RANTES) was investigated by reverse transcription-PCR followed by densitometric analyses. The allergen challenge up-regulated the expression of mRNA for MCP-1, MCP-3/FIC, and macrophage inflammatory protein-1alpha at 2 and/or 24 h. Immunocytochemical staining for MCP-3/FIC showed that the allergen challenge induced the expression of MCP-3/FIC predominantly in the airway epithelium. Pretreatment of mice with an anti-MCP-3/FIC Ab significantly inhibited the OVA-induced airway inflammation and the bronchoalveolar lavage eosinophilia (8 +/- 2% vs 46 +/- 11% after control Ab, p < 0.03). We conclude that MCP-3/FIC plays a significant role in the allergen-induced eosinophilic inflammation of the airways.

Animals↗
PubMed1997

Respiratory syncytial virus induces selective production of the chemokine RANTES by upper airway epithelial cells.

The presence of histamine and eosinophil cationic protein in nasopharyngeal secretions of infants with respiratory syncytial virus (RSV)-induced bronchiolitis implies the activation of basophil and eosinophil leukocytes, but the specific mechanism of their recruitment has not been elucidated. Chemokines are potent and selective leukocyte chemotactic molecules that are also expressed by airway epithelial cells. Therefore, the pattern of chemokines produced in response to RSV infection was investigated in primary cultures of human nose- and adenoid-derived epithelial cells. Interleukin-8, growth-related peptide-alpha, and monocyte chemotactic protein-1 were constitutively released by uninfected epithelial cells and were not further enhanced by infection with RSV. RANTES (regulated upon activation, normal T cell-expressed and -secreted), which was present in negligible concentrations in uninfected cultures, was strongly induced by RSV infection, in a dose- and time-dependent manner. Through the release of RANTES, epithelial cells may control the selective concentration and activation of basophils and eosinophils in RSV-infected airway mucosa.

Adenoids↗
PubMed1997

Application of AAPM Radiation Therapy Committee Task Group 23 test package for comparison of two treatment planning systems for photon external beam radiotherapy.

Task Group 23 of the AAPM Radiation Therapy Committee has produced a test package for verification of the accuracy of treatment planning for photon external beam therapy. The package includes measured fundamental beam data for 4 and 18 MV x rays, and 13 test cases with measured dose values at selected points, which serve as the reference for determination of calculated dose accuracy. Test cases include three square fields, two rectangular fields, a wedged field, a blocked field, and an irregular field, as well as cases of an off-center plane, source-to-SOURCE distance (SSD) variation, oblique incidence, and lung and bone inhomogeneities. This package was used to evaluate two treatment planning systems. Although average dose variations in most cases were less than 1%, maximum deviations up to 19% were observed in one planning system and up to 11% in the other. Dose deviations greater than 3% were observed for 51 of 532 points (in the 4 MV case) and for 124 of 586 points (in the 18 MV case). An additional component of the test package is an evaluation of radiological field width at different depths. The largest deviation for width was 17.4 mm for 4 MV and 9.1 mm for 18 MV. In 20% of the widths studied, the deviation observed was greater than 3 mm for the 4 MV case. In the 18 MV case 18% of the widths had deviations greater than 3 mm. Our data indicate that the accuracy of the two treatment planning systems tested here warrants improvement, even for simple treatment geometries involving photon beams, which are generally considered to be well understood. As one of the treatment planning systems is widely used in the US, it is concluded that the accuracy of the currently available commercial treatment planning systems is not adequate for achieving the generally stated goal of +/- 5% accuracy of dose delivery in radiation therapy. The AAPM Task Group 23 test package provides an extremely useful tool for a quantitative analysis of treatment planning systems for photon beam radiation therapy.

Humans↗
PubMed1997

Inhibition of PDGF-mediated proliferation of vascular smooth muscle cells by calcium antagonists.

BACKGROUND AND PURPOSE: The mechanism by which calcium antagonists (CAs) inhibit proliferation in vascular smooth muscle cells (VSMCs) is not yet fully understood. We investigated the effects of four CAs (clentiazem, verapamil, diltiazem, and nifedipine) on signal transduction pathways activated by platelet-derived growth factor (PDGF). To determine these effects, the levels of inositol phosphates (IPs), protein kinase C (PKC), and the induction of the transcription factor activator protein-1 (AP-1) were measured. METHODS: The mitogenic effect of PDGF on VSMCs was measured by [3H]thymidine incorporated into DNA. IP production was monitored by [3H]myo-inositol incorporation. PKC activation was determined by measurement of myristoylated, alanine-rich C kinase substrate (MARCKS) phosphorylation in digitonin-permeabilized VSMCs. The induction of AP-1 complex was detected by electrophoretic mobility shift assays. RESULTS: Each CA significantly inhibited the [3H]thymidine incorporation into DNA in unstimulated cells. Similar significant decreases in [3H]thymidine incorporation by CAs were observed when cells were stimulated by rPDGF-BB. The phosphorylation of MARCKS mediated by rPDGF-BB was significantly reduced by each CA. Clentiazem and verapamil significantly reduced the expression of AP-I induced by rPDGF-BB (P < .01, P < .05). Clentiazem also significantly reduced the expression of AP-1 induced by rPDGF-AB (P < .05). CONCLUSIONS: PDGF-mediated proliferation of VSMCs correlates with activation of PKC but not with induction of the AP-1 complexes. In addition, our results suggest that CAs block proliferation of VSMCs by inhibiting DNA synthesis, possibly via PKC.

Animals↗
PubMed1996

Inhibition of vascular smooth muscle cell proliferation by the calcium antagonist clentiazem: role of protein kinase C.

The proliferation of vascular smooth muscle cells has been implicated as a causative factor in atherogenesis. Calcium channel blockers have been shown to retard the progression of atherosclerosis. To elucidate the mechanism by which these drugs mediate such actions, we studied the effects of a new calcium antagonist, clentiazem, on the in vitro proliferation of vascular smooth muscle cells. PDGF-induced prolifertion of these cells is markedly inhibited by clentiazem. The probable involvement of protein kinase C (PKC) in this cellular response is suggested. Clentiazem appear to cause inhibition of PKC translocation that is induced by phorbol esters and PDGF-BB and the phosphorylation of the 80 kDa protein substrate of PKC in vascular smooth muscle cells. Moreover, treatment with clentiazem leads to a marked decrease in the number of specific phorbol ester binding sites. Analysis of the membrane bound isoenzymes of protein kinase C revealed that the inhibition was specific to delta enzymes. Arterial cholesterol ester hydrolysis is not significantly altered by clentiazem. Our results suggest that clentiazem may inhibit cell proliferation by regulating cytosolic PKC and preventing its membrane translocation and activation.

Adenosine Monophosphate↗
PubMed1996

The production of macrophage inflammatory protein-1 alpha by human basophils.

Macrophage inflammatory protein-1alpha (MIP-1alpha) has previously been shown to be produced by mononuclear cells, eosinophils, and neutrophils. Its production by basophils has not been investigated. The objective of this study was to investigate the production of MIP-1alpha by basophils. Peripheral blood basophils were separated by Percoll gradient centrifugation, cultured overnight, and processed for double immunocytochemistry using Abs against MIP-1alpha and FcepsilonRIalpha (alpha subunit of IgE receptor type 1). We demonstrated that basophils expressed immunoreactive MIP-1alpha upon stimulation with anti-IgE. Less than 5% of the basophils stained for MIP-1alpha without stimulation. The secretion of MIP-1alpha by basophils was studied by ELISA. In these experiments, basophils were further enriched to 65 to 99% (median, 86%) by a negative selection method. Basophils released MIP-1alpha when stimulated by Abs against IgE and FCepsilonRIalpha as well as IL-3 and the calcium ionophore, A23187. In parallel experiments, PBMC, eosinophils, and neutrophils did not produce MIP-1alpha in response to anti-IgE, but they did so in response to A23187. No MIP-1alpha release was detected in platelet preparations. Preincubation with IL-3 (15 min or 18 h) augmented anti-IgE-included basophil MIP-1alpha production. The secretion of MIP-1alpha by basophils was detectable shortly after stimulation and gradually increased over 24 h. Since MIP-1alpha has potent inflammatory and histamine-releasing activities, its production by basophils may indicate a positive feedback mechanism for allergic inflammation.

Antibodies, Anti-Idiotypic↗