Ex vivo restimulation of human PBMC expands a CD3+CD4-CD8- γδ+ T cell population that can confound the evaluation of CD4 and CD8 T cell responses to vaccination.

The measurement of vaccine-induced humoral and CD4(+) and CD8(+) cellular immune responses represents an important correlate of vaccine efficacy. Accurate and reliable assays evaluating such responses are therefore critical during the clinical development phase of vaccines. T cells play a pivotal role both in coordinating the adaptive and innate immune responses and as effectors. During the assessment of cell-mediated immunity (CMI) in subjects participating in a large-scale influenza vaccine trial, we identified the expansion of an IFN-γ-producing CD3(+)CD4(-)CD8(-) γδ (+) T cell population in the peripheral blood of 90/610 (15%) healthy subjects. The appearance of CD3(+)CD4(-)CD8(-) γδ (+) T cells in the blood of subjects was transient and found to be independent of the study cohort, vaccine group, subject gender and ethnicity, and ex vivo restimulation conditions. Although the function of this population and relevance to vaccination are unclear, their inclusion in the total vaccine-specific T-cell response has the potential to confound data interpretation. It is thus recommended that when evaluating the induction of IFN-γ-producing CD4(+) and CD8(+) immune responses following vaccination, the CD3(+)CD4(-)CD8(-) γδ (+) T cells are either excluded or separately enumerated from the overall frequency determination.

Expression of interleukin-6, leukemia inhibitory factor and their receptors by colonic epithelium and pericryptal fibroblasts.

BACKGROUND AND AIM: The cellular configuration of the human colon suggests a predetermined organization that creates specific microenvironments. The role of pericryptal fibroblasts in this microenvironment has been the subject of considerable speculation. This study examined the expression of growth factors and their receptors by colonic crypt epithelium and pericryptal fibroblasts. METHODS AND RESULTS: Pericryptal fibroblast cells were isolated and cultured from decrypted human colonic mucosa. The pericryptal fibroblast cells expressed messenger RNA (mRNA) for interleukin-6 (IL-6), leukemia inhibitory factor (LIF), LIF receptor alpha, and the common coreceptor glycoprotein 130 (GP130), but not the IL-6 receptor alpha. Interleukin-6 protein expression was confirmed by the analysis of conditioned medium and immunohistochemistry. In comparison, normal colonic epithelial cells express mRNA for LIF but not IL-6 as well as the receptors for GP-130, IL-6 receptor alpha but not LIF receptor alpha. As cultures of normal human colonic epithelial cells were not available, the conditioned medium was assayed from established colon carcinoma cell lines and demonstrated a secretion of LIF but not IL-6 protein. CONCLUSION: The expression of reciprocal cytokine and receptor expression suggest that there is a paracrine relationship between pericryptal fibroblasts and colonic epithelium.

The phosphatidylinositol 3'-kinase p85alpha gene is an oncogene in human ovarian and colon tumors.

Phosphatidylinositol 3'-kinases (PI3ks) are a family of lipid kinases that play a crucial role in a wide range of important cellular processes associated with malignant behavior including cell growth, migration, and survival. We have used single-strand conformational polymorphism/heteroduplex analysis to demonstrate the presence of somatic mutations in the gene for the p85alpha regulatory subunit of PI3k (PIK3R1) in primary human colon and ovarian tumors and cancer cell lines. All of the mutations lead to deletions in the inter-SH2 region of the molecule proximal to the serine608 autoregulatory site. Expression of a mutant protein with a 23 amino acid deletion leads to constitutive activation of PI3k providing the first direct evidence that p85alpha is a new oncogene involved in human tumorigenesis.

Id2 is a target of the beta-catenin/T cell factor pathway in colon carcinoma.

Activation of beta-catenin/T cell factor (TCF) transcription as a result of mutations in the adenomatous polyposis coli (APC) and/or beta-catenin genes occurs in the majority of colon tumors. An increasing number of genes, including c-myc and cyclin D1, have been implicated as targets of this pathway. We now report that the dominant negative helix-loop-helix regulator Id2 is also a target of the beta-catenin/TCF transcription pathway in colon adenocarcinoma. Investigation of the mechanism for the overexpression of Id2 in colon carcinoma cells demonstrated that the Id2 promoter is activated, and the Id2 protein is up-regulated by beta-catenin. Conversely, reducing free beta-catenin blocked this induction of promoter activity. We have also used an electrophoretic mobility shift assay and supershift to identify a motif in the Id2 promoter that binds to TCF4 protein. Site-directed mutagenesis of this motif abolished promoter reporter activity. Both transfection of Id2 into SW480 cells and induction of Id2 in HT29 colon cells was found to increase anchorage-independent survival of these cells. Growing evidence associates disruption to Id2 expression with tumorigenesis, and our findings suggest that this dysregulation of Id2 expression is due to the activation of the beta-catenin/TCF pathway.

Functional abnormalities in protein tyrosine phosphatase epsilon-deficient macrophages.

Protein tyrosine phosphatase epsilon (PTP epsilon)-deficient mice were generated by targeted deletion of exons 3, 4, and 5 of the Ptpre gene. Mice homozygous for this deletion (Ptpre(Delta3-5)) were fertile, bred and developed normally and exhibited no overt phenotype. However, closer examination of the function of macrophages from these mice revealed a defect in the regulation of the respiratory burst. While bacterial lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNFalpha) were able to prime bone marrow-derived macrophages (BMM) from wild type (Ptpre(+)) macrophages for an enhanced respiratory burst, they were unable to do so in macrophages from PTP epsilon-deficient mice. PTP epsilon-deficient BMM also had abnormalities in cytokine production with a reduced ability to produce TNFalpha and enhanced IL-10 production in response to challenge with LPS. These findings suggest an important role for PTP epsilon in the control of macrophage function.

Methylation of exon 2 of p16 is associated with late stage oesophageal cancer.

Methylation of the p16 gene was studied in 16 oesophageal tumours. Five (31%) of the tumours were found to be methylated in exon 1 and eight (50%) were methylated in exon 2. The loss of p16 protein correlated with methylation of exon 1 (P = 0.005). However, methylation of exon 2, but not exon 1, was found to be associated with late stage tumours (P = 0.01). We conclude that the methylation of exon 2 of p16 may have effects on the progression of oesophageal tumours that are independent of the expression of the p16 protein.

Microsatellite instability in gastrointestinal tract tumours.

Although a number of studies have documented microsatellite instability (MSI) in gastrointestinal tumours, the clinical significance is uncertain. In this study the MSI status and clinicopathological features were examined in gastric and colorectal tumours. Eighty-four gastrointestinal tumours were examined for MSI. Normal and tumour DNA isolated from the same patients were analysed at five different microsatellite loci. Clinical features of these patients were also collated and compared with MSI status. High level MSI (MSI-H) (as defined by instability in 2 or more microsatellites) was detected in 6 out of 47 (13%) colon tumours and 6 of 37 (16%) gastric tumours. The frequency of MSI-H between these groups was not statistically significant (P = 0.36). There was no significant correlation with patient age or gender, UICC stage, or degree of differentiation of the tumour. This was true both when analysed as a group, as well as when divided into colon and gastric sites. Our results confirm that a proportion of sporadic tumours from the colon and stomach exhibit an MSI-H phenotype. However, there was no significant relationship between the presence of MSI and any of the clinicopathological characteristics studied.

Clonogenic growth of epithelial cells from normal colonic mucosa from both mice and humans.

BACKGROUND & AIMS: The factors controlling the proliferation and differentiation of the colonic mucosa are unknown and have proved difficult to identify mainly because of a lack of in vitro methods for studying the proliferative cells of the mucosa. METHODS: We have developed a novel method of preparing a viable single-cell suspension from isolated crypts and cloning these single cells. RESULTS: We have obtained clonogenic growth from this single-cell suspension with an average of 1 colony per 10(5) cells in control cultures. Addition of conditioned medium from the LIM1863 colon carcinoma cell line increased the mean colony number to 11 +/- 3 per 10(5) cells. The cells forming the colonies are still viable after 4 weeks in culture. The epithelial nature of the cells was confirmed by ultrastructural and immunohistochemical methods with staining for keratin 8 and 18 and anti-human epithelial membrane-specific antigen and a positive result on polymerase chain reaction for keratin 19. CONCLUSIONS: We have successfully cloned single cells from disaggregated colonic crypts from both human and murine colonic mucosa. We have also demonstrated the presence of an active clonogenic factor in the conditioned medium of a colon carcinoma cell line. Assays show that the clonogenic activity in the conditioned medium is not caused by the presence of any of the epidermal growth factor family of growth factors. This is the first report of a clonogenic assay for epithelial cells of normal colonic mucosa.

Determination of clonality in patients who present with diagnostic dilemmas: a laboratory experience and review of the literature.

Identification of rearrangements in the immunoglobulin heavy chain (IgH), T cell receptor (TCR) and other genes assists in the classification of hemopoietic disorders. This study reviews a 5 year experience of genotyping as an assessment of clonality in a central referral laboratory. Patients were referred for assessment if abnormal hemopoietic populations were identified and where standard morphology, cytochemistry or immunophenotyping techniques were equivocal and unable to establish the diagnosis. Analysis of the antigen receptor genes (IgH gene and TCR gamma locus) was performed in 230 patients by either Southern blotting or the polymerase chain reaction (PCR). Clonal rearrangements of either loci could be demonstrated in 91/230 patients: 56/161 patients analyzed by Southern blotting and 48/125 analyzed by polymerase chain reaction. A subgroup of patients (n = 88) was analyzed for rearrangement of the immunoglobulin heavy chain gene by both techniques. Discordant results were observed in 18 of these patients (20%). Analysis of the TCR gamma locus in a separate group demonstrated discordant results in eight of 40 patients examined (20%). Clinical outcome could be available in 61 patients (median time: 42 months, range 1-68 months): for those in whom a rearrangement was detected approximately 80% went on to develop a lymphoproliferative disorder although this diagnosis was not able to be made at the time the sample was taken. In a subset of patients (n = 14) who presented with lymphocytosis after bone marrow transplantation (seven) or solid organ transplant (seven), clonal lymphoid populations were demonstrated in approximately 50% of cases. The majority of these cases demonstrated the presence of Epstein-Barr virus (EBV) by PCR. The clonality of EBV infection was assessed by Southern blotting and the significance of these findings are discussed. This study explores the differences between Southern blotting and PCR as applied to the study of antigen receptor rearrangement studies. We conclude that detecting a clonal population is valuable in patients where standard diagnostic techniques are equivocal. However, the inability to detect a clonal population should be treated with caution and interpreted in light of other investigations.

Deregulated c-myc expression overrides IFN gamma-induced macrophage growth arrest.

Induction of c-myc gene expression is an essential response to growth promoting agents, including colony-stimulating factor 1 (CSF-1). Down regulation of c-myc expression occurs in response to a variety of negative growth regulators in many cell types. However, for many of these systems the causal link between c-myc down regulation and growth arrest remains to be established. Here we show for CSF-1-dependent BAC1.2F5 mouse macrophages that interferon-gamma (IFN gamma) results in a midlate G1 phase decrease of CSF-1-dependent c-myc mRNA and subsequent cell cycle arrest. Introduction of a deregulated c-myc gene into these cells, which prevents the IFN gamma-mediated decrease in c-myc expression, overrides the cell cycle arrest and restores CSF-1-dependent growth in the presence of the cytokine. This result contrasts with the macrophage growth arrest induced by cAMP elevation, which also suppresses c-myc expression, but is not overcome by a deregulated c-myc gene. These results show that inhibition of c-myc expression is an essential component in IFN gamma-mediated cell cycle arrest and demonstrates that distinct mechanisms contribute to IFN gamma- and cAMP-mediated growth arrest in macrophages.

Endoglin: a 180-kD endothelial cell and macrophage restricted differentiation molecule.

A MoAb RMAC8 was generated by immunizing Balb/c mice with cultured human umbilical vein endothelial cells (HUVE). The molecule recognized had a mol. wt of 180 kD non-reduced, 95 kD after reduction and 66 kD in its reduced and N-deglycosylated form. Sequential immunoprecipitation studies with the MoAb 44G4, which recognizes the O- and N-glycosylated homodimer endoglin, showed that both MoAbs recognize the same molecule on HUVE and phorbol myristate acetate (PMA)-stimulated U937 cells. The distribution of the RMAC8-recognized molecule was the same as that described for endoglin, i.e. arterial and venous endothelium, myelomonocytic and pre-B leukaemia cells and cell lines; however, unlike 44G4, RMAC8 also reacted weakly with monocytes and strongly with in vitro differentiated macrophages as well as peritoneal and alveolar macrophages. This distribution of endoglin was confirmed by Northern blot analysis using a full length endoglin cDNA probe. These studies suggest that endoglin is a differentiation marker on macrophages.

Extracorporeal photochemotherapy in the treatment of cutaneous T-cell lymphoma. Complexity of objective evaluation.

The term cutaneous T-cell lymphoma (CTCL) encompasses the spectrum of diseases characterized by a monoclonal proliferation of malignant T lymphocytes predominantly of the mature T-helper cell type. These include mycosis fungoides, which is usually localized to the skin for many years, and the Sezary syndrome, the leukemic variant in which characteristic, bizarre lymphatic cells with deeply indented or cerebriform nuclei, the Sezary cells, infiltrate lymph glands and internal organs such as the spleen, liver, lungs, heart, and bone marrow. The condition is more common in men after their fourth decade. The treatment of CTCL varies depending on the stage of the disease. The skin of the patient is the primary index of effectiveness of therapy. Options range from topical steroids, topical nitrogen mustard, X-irradiation, electron beam irradiation, and 8-methoxypsoralen (8-MOP) combined with ultraviolet A photochemotherapy (PUVA), to leukapheresis and systemic chemotherapy. More recently, extracorporeal photochemotherapy (ECPC) has been introduced. The safety and efficacy of this modality is further investigated in six patients who fulfilled the criteria of diagnosis of CTCL. The problems encountered in objectively evaluating the clinical responses in the patients are outlined and improvements in the protocol to overcome these are suggested. The proposed mechanism of action is an immunostimulatory one and a procedure that is relatively free from side effects; it offers promise as a potential treatment for a difficult cancer.

Mechanisms of lymphocytotoxicity induced by extracorporeal photochemotherapy for cutaneous T cell lymphoma.

Extracorporeal photochemotherapy is an effective treatment for cutaneous T cell lymphoma but its mode of action is uncertain. The reduction in viability of patients' photoirradiated buffy coat lymphocytes was correlated with a 35% increase in DNA single-strand breaks and marked decreases in cellular ATP and NAD levels (to 58 and 34% of control, respectively) immediately after photoirradiation. Complementary in vitro studies were conducted with normal human peripheral blood lymphocytes using a Therakos ultraviolet A (UVA) light box. UVA light was cytotoxic on its own but was potentiated by 8-methoxysporalen. 3-aminobenzamide, a poly (ADP-ribose) synthetase inhibitor, mitigated the cytotoxic effect of ultraviolet A light in the presence of 8-methoxypsoralen in lymphocytes and reduced the amount of nucleotide depletion they caused. 10 J/cm2 of UVA light in the presence of 300 ng/ml 8-methoxypsoralen increased the poly (ADP-ribose) synthetase activity of peripheral blood lymphocytes. Exposing lymphocytes to deoxycoformycin and deoxyadenosine was found to induce biochemical and physical effects similar to those of photochemotherapy. In summary, we have shown that the lymphocytotoxic effect of extracorporeal photochemotherapy for cutaneous T cell lymphoma is apparently mediated by DNA damage, subsequent poly (ADP-ribosyl)ation and adenine nucleotide depletion. It is not known how the DNA damage and resultant biochemical effects relate to the possible immunological mechanism of extracorporeal photochemotherapy; however, it is possible that its effects can be mimicked by other DNA-damaging agents.