Elwyn Loh

Monocyte-chemoattractant protein 1 gene expression in intestinal epithelial cells and inflammatory bowel disease mucosa

BACKGROUND Monocyte-chemoattractant protein 1 (MCP-1) activates macrophages and increases the migration of monocytes into tissue during inflammation. It was hypothesized that MCP-1 expression is involved in intestinal inflammation. METHODS MCP-1 protein was detected by immunohistochemistry and immunoprecipitation. Biological activity of MCP-1 was assessed using a chemotactic assay. MCP-1 messenger RNA (mRNA) levels were measured by quantitative reverse-transcription polymerase chain reaction. RESULTS In normal mucosa, MCP-1 was predominantly present in surface epithelium. In contrast, inflamed mucosa from patients with ulcerative colitis or Crohns disease contained multiple cells immunoreactive for MCP-1, including spindle cells, mononuclear cells, and endothelial cells. Furthermore, MCP-1 mRNA expression was markedly increased in inflamed intestinal biopsy specimens from patients with inflammatory bowel disease. MCP-1 was detected in isolated intestinal epithelial cells and in conditioned media from Caco-2 cells. Caco-2 cell-conditioned media stimulated monocyte chemotaxis activity that was inhibited by anti-MCP-1 antibodies. Constituitive MCP-1 mRNA levels in Caco-2 cells were up-regulated by interleukin 1 beta and down-regulated by dexamethasone. CONCLUSIONS In addition to lamina propria macrophages, endothelial cells, and spindle cells, intestinal epithelial cells are able to produce MCP-1. MCP-1 is expressed constitutively in the intestinal colonic mucosa and is up-regulated during inflammation.

Rituximab (anti-CD20 monoclonal antibody) therapy for progressive intermediate-grade non-Hodgkin's lymphoma after high-dose therapy and autologous peripheral stem cell transplantation.

We evaluated the response and toxicity of rituximab in the setting of progressive intermediate grade non-Hodgkin’s lymphoma (NHL) after autologous peripheral stem cell transplantation (PSCT). Seven patients with a median age of 59 years (45–62), ECOG performance status 0–1, and CD20-positive diffuse large cell lymphoma with progression after PSCT were treated. All patients initially received 4-weekly infusions of rituximab (375 mg/m2). The maximum response was three CR and four PR. Median progression-free survival was 197 days (range 60–282). With a median follow-up of 204 (115–299) days, the patients’ disease status is classified as two CR, one PR, and four PD. Four of five patients with ECOG performance status of 1 prior to treatment showed improvement to status 0 after treatment with rituximab. While follow-up is short, these results suggest that rituximab has significant activity in intermediate-grade non-Hodgkin’s lymphoma that has relapsed after PSCT.

Effect of Cytoplasmic Domain Mutations on the Agonist-stimulated Ligand Binding Activity of the Platelet Integrin αIIbβ3

Function of the platelet integrin αIIbβ3 is regulated by agonist-generated signals interacting with its cytoplasmic tails. When αIIbβ3 is expressed in Epstein-Barr virus-transformed B lymphocytes, stimulation of the cells with phorbol 12-myristate 13-acetate results in αIIbβ3-mediated lymphocyte adherence to immobilized fibrinogen, as well as soluble fibrinogen binding to αIIbβ3, indicating that agonists increase the affinity of αIIbβ3 for fibrinogen in these cells. To address the contribution of the αIIb and β3 cytoplasmic tails to this process, we mutated each tail and expressed the mutants in B lymphocytes. Truncation of the αIIb tail did not impair unstimulated or stimulated lymphocyte adherence to fibrinogen, regardless whether the truncation was proximal or distal to the conserved GFFKR sequence. However, deleting GFFKR or replacing it with alanines markedly reduced αIIbβ3 expression due to impaired intracellular assembly of αIIbβ3 heterodimers, probably due to a mutation-induced change in the conformation of αIIb. Introducing β3 mutations known to impair αIIbβ3 function in platelets into the cytoplasmic tail of β3 in lymphocytes also impaired αIIbβ3 function in these cells. These studies demonstrate that the cytoplasmic tail of αIIb is not required for αIIbβ3 function in lymphocytes, although the presence of GFFKR in the αIIb tail is required for αIIb to interact with β3. Additionally, they indicate that signals interacting with the β3 cytoplasmic tail are responsible for the ability of agonists to stimulate αIIbβ3 function.

Anchored PCR: Amplification with single-sided specificity

PCR (polymerase chain reaction) usually requires two specific primers to amplify the DNA bracketed by the two primers. In many circumstances one wishes to characterize unknown sequences contiguous to a known segment of DNA and thus two specific primers are not available. Strategies to overcome this limitation have been developed by adding an anchor to the unknown gene segments and amplifying with the anchor and a specific primer. This paper describes two such techniques, the first in which an anchor is added to cDNA by terminal deoxynucleotidyl transferase, and the second in which an anchor is ligated onto double-stranded DNA.

A consensus primer to amplify both α and β chains of the human T cell receptor

Abstract The use of reverse transcriptase in conjunction with the polymerase chain reaction (RT-PCR) has proven invaluable in the analysis of the T cell receptor (TCR) repertoire of different populations of T cells. However, the presence of a variable region in the T cell receptor has hindered the design of primers for the 5′ end of the TCR cDNA. We describe the design and use of a degenerate consensus primer that allows amplification of both the α and β chains of the human TCR. We have used this primer in the analysis of the TCR distribution of T cell clones, peripheral blood lymphocytes and lymphocytes residing in tissue. In addition, the primer has allowed the identification of an alternative splice site in the β chain constant region which cannot translate into a functional constant region. We have found the primer to be easy to use, sensitive and specific.

Regulation of alphaIIb beta3 function in human B lymphocytes.

We studied the function of the platelet integrin αIIbβ3 using a B lymphocyte model in which αIIbβ3 can be induced to interact with fibrinogen using phorbol myristate acetate (PMA). To determine whether a G protein-coupled receptor could also activate αIIbβ3 in lymphocytes, we coexpressed the human formyl peptide receptor (fPR) and αIIbβ3, finding that the fPR agonist formyl Met-Leu-Phe (fMLP)-stimulated lymphocyte adherence to immobilized fibrinogen and binding of soluble fibrinogen to the lymphocyte surface. The response to fMLP, but not PMA, was abrogated by pertussis toxin, indicating that the fPR was coupled to the G-protein Gαi, whereas the protein kinase C inhibitor bisindolylmaleimide I inhibited the response to both fMLP and PMA, indicating that signaling from the fPR included protein kinase C. On the other hand, the tyrosine kinase inhibitor genistein, the Syk inhibitor piceatannol, and the RhoA inhibitor C3 exoenzyme had no effect, implying that neither tyrosine phosphorylation nor the GTPase RhoA were involved. Furthermore, whereas micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of αIIbβ3 with fibrinogen, nanomolar concentrations actually induced fibrinogen binding to unstimulated cells. Our studies demonstrate that αIIbβ3 expressed in B lymphocytes can be activated by a physiologic agonist and outline an activating pathway that includes Gαi, protein kinase C, and the actin cytoskeleton.

Autologous stem-cell transplant after conventional dose adjuvant chemotherapy for high-risk breast cancer: Impact on the delivery of local-regional radiation therapy

BACKGROUND High-dose chemotherapy with autologous stem-cell transplantation is used increasingly in the treatment of poor-prognosis primary breast cancer. Because these patients may be cured with standard multimodality therapy, it is important to address both the efficacy of transplantation, and its effect on the delivery of standard treatments including local radiation therapy. PATIENTS AND METHODS Patients with high risk primary breast cancer were treated with high-dose cyclophosphamide and thiotepa and stem-cell transplant following surgery and conventional-dose adjuvant chemotherapy. Outcome, including sites of failure and delivery of local radiation therapy, was assessed for 103 patients. RESULTS Overall and disease-free survival rates at 18 months were 83% (+/- 4%) and 77% (+/- 4%) respectively. Twenty patients (19.4%) received radiation therapy prior to transplant. Of the remaining 83, 77 received radiation therapy after transplant. Overall, 5 (19.2%) of 26 first sites of recurrence were local alone. For patients receiving radiation prior to transplant, 3 of 7 (43%, 95% CI: 6%-80%) sites of first recurrence were local, while 2 of 19 (10.5%, 95% CI: 0%-24.5%) sites of first recurrence were local alone in patients receiving post-transplant radiation or no radiation. CONCLUSION Transplantation does not appear to significantly compromise the delivery or outcome of local radiation therapy for primary breast cancer.