M. A. Forbes

Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor with a key role in several pathological processes, including tumour vascularization. Our preliminary observations indicated higher VEGF concentrations in serum samples than in matched plasma samples. We have now demonstrated that this difference is due to the presence of VEGF within platelets and its release upon their activation during coagulation. In eight healthy volunteers, serum VEGF concentrations ranged from 76 to 854 pg ml(-1) and were significantly higher (P < 0.01) than the matched citrated plasma VEGF concentrations, which ranged from < 9 to 42 pg ml(-1). Using platelet-rich plasma, mean (s.d.) platelet VEGF contents of 0.56 (0.36) pg of VEGF 10(-6) platelets were found. Immunocytochemistry demonstrated the cytoplasmic presence of VEGF within megakaryocytes and other cell types within the bone marrow. From examination of the effects of blood sample processing on circulating VEGF concentrations, it is apparent that for accurate measurements, citrated plasma processed within 1 h of venepuncture should be used. Serum is completely unsuitable. The presence of VEGF within platelets has implications for processes involving platelet and endothelial cell interactions. e.g. wound healing, and in tumour metastasis, when platelets adhering to circulating tumour cells may release VEGF at points of adhesion to endothelium, leading to hyperpermeability and extravasation of cells.

The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis — Preliminary findings

Proteomics‐based studies offer a powerful complementary approach to DNA/RNA‐based investigations and are now being applied to investigate aspects of many diseases including cancer. However, the heterogeneous nature of tissue samples often makes interpretation difficult. We have undertaken a study into the potential use of a novel laser capture microdissection (LCM) system to isolate cells of interest for subsequent proteomic analysis. Retrieval of selected cells is achieved by activation of a transfer film placed in contact with a tissue section, by a laser beam (30 or 60 μm diameter) which is focused on a selected area of tissue using an inverted microscope. The precise area of film targeted by the laser bonds to the tissue beneath it and these cells are then lifted free of surrounding tissue. Although the technique has been shown to be readily compatible with subsequent analysis of nucleic acids, little information is yet available regarding the application of protein‐based analyses to the captured tissue. We report here preliminary data regarding the potential use of the LCM system in combination with two‐dimensional electrophoresis to examine protein profiles of selected tissue areas. Electrophoretic profiles of proteins from normal and malignant renal tissue samples showed little change following LCM, nine selected proteins showed identical mass spectrometric sequencing profiles, and two selected proteins retained antigenicity. Dissection of epithelial tissue from a sample of normal human cervix resulted in enrichment of some proteins compared with analysis of the whole tissue. LCM will be a valuable adjunct to proteomic studies although further detailed validation is necessary.

The acute phase protein response in patients receiving subcutaneous IL‐6

IL‐6, tumour necrosis factor‐α (TNE‐α) and IL‐1 are thought to be the key mediators of the acute phase response although much of the evidence is based on in vitro studies. It is not clear to what extent each of the acute phase proteins are regulated in vivo by each of these cytokines. The aim of this study was to examine the effects of IL‐6 treatment in eight patients with cancer on the concentrations of an extensive range of positive and negative acute phase proteins. It was part of a larger investigation to assess the value of IL‐6 in the management of chemotherapy‐induced thrombocytopenia. IL‐6 was administered by a daily subcutaneous injection for 7 days at a dose level of 1, 3. or 10 μg/kg/day. Increases in the positive acute phase proteins, serum amyloid A. C‐reactive protein. α1‐acid glycoprotein, α1‐antichymotrypsin, haptoglobin, α1‐antitrypsin, fibrinogen, complement component C3, and caeruloptasmin, were observed, with the greatest incremental changes and fastest responses being seen for C‐reactive protein and serum amyloid A protein. The negative acute phase proteins transferrin, transthyretin and retinol binding protein all fell to a nadir within 48‐96 h after the first IL‐6 injection. Increases in complement component C4 were only found in two patients, which may be related to the increase in circulating TNF‐α concentrations found only in these patients. This study has therefore shown that IL‐6 is capable of causing changes in the majority of acute phase proteins in vivo. Although secondary induction of TNF‐α was not observed in the majority of patients examined, it is still possible however that other cytokines involved in regulation of the acute phase response, such as IL‐1, may have been induced and contributed to the overall response.

Serum concentrations of soluble adhesion molecules in patients with colorectal cancer.

The concentrations of the soluble adhesion molecules E-cadherin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were investigated in 48 patients with colorectal cancer before treatment, and their relation to clinical, histological and routine laboratory parameters was examined. Data were collected on tumour stage at presentation, presence and sites of metastatic disease, tumour pathology and results of routine laboratory tests. Serum concentrations of ICAM-1 and VCAM-1 were significantly elevated in the patients with colorectal cancer in comparison with a group of healthy subjects (P < 0.00001). Levels of circulating ICAM-1 and VCAM-1 were increased both in patients with local and those with metastatic disease. Although elevated in some patients soluble E-cadherin and E-selectin concentrations were not significantly elevated compared with the control group (P = 0.71 and P = 0.052 respectively). The levels of circulating ICAM-1 were significantly correlated with those of VCAM-1 and E-selectin. A correlation was also found between the serum concentrations of E-selectin and ICAM-1 and alkaline phosphatase, total white cell count and platelet count. VCAM-1 was positively correlated with age and negatively with degree of tumour differentiation and haemoglobin concentration. The biological implications and possible clinical relevance of these findings are discussed.

Circulating soluble adhesion molecules E-cadherin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in patients with gastric cancer.

The concentrations of the soluble adhesion molecules E-cadherin, E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were investigated in 45 patients with gastric cancer before treatment and their correlation with clinical, histological and routine laboratory parameters was examined. Data were collected on tumour stage at presentation, presence and sites of metastatic disease, tumour pathology, survival and results of routine laboratory tests. Serum concentrations of ICAM-1 and VCAM-1 were significantly elevated in the patients with gastric cancer in comparison with the group of healthy subjects (P < 0.00001 and P < 0.0001 respectively). Increased serum concentrations of VCAM-1 were associated with locally advanced and metastatic disease whereas ICAM-1 was significantly elevated both in local and in advanced/metastatic disease. Soluble E-cadherin and E-selectin concentrations did not show any significant elevation in gastric cancer patients. Concentrations of soluble adhesion molecules showed significant correlation with each other (except E-selectin and VCAM-1) and with alkaline phosphatase. Soluble ICAM-1 and VCAM-1 were significantly associated with an elevated total white cell count. Patients with elevated VCAM-1 had significantly poorer survival in comparison with patients with normal serum levels (P = 0.0361).

Bone Alkaline Phosphatase in Rheumatic Diseases

A double monoclonal immunoradiometric assay specific for bone alkaline phosphatase (BAP) was used to determine whether the raised total alkaline phosphatase (TAP) often found in patients with active rheumatoid arthritis (RA) and ankylosing spondylitis (AS) is derived from bone or liver. Fifty-eight patients with RA were compared to 14 with AS and 14 with non-inflammatory rheumatic diseases (NI). None had clinical liver disease and only one had a slightly elevated aspartate transaminase activity. Elevated BAP concentrations were found in seven patients (5 RA, 1 AS, 1 NI), only two of whom also had abnormal TAP. Abnormal TAP activities were found in only three patients (all RA). BAP did not correlate with disease activity in RA or AS. In contrast, TAP correlated with disease activity (assessed by plasma viscosity) in RA (P < 0·02) and AS (P < 0·002) and γ-glutamyl transferase (GGT) also correlated with plasma viscosity in RA (P < 0·01). Both TAP and BAP were significantly correlated with GGT in RA (P < 0·001 and P < 0·02, respectively). These findings are discussed, together with possible reasons for the conflicting nature of some of the observations.

Clinical and Immunobiological Effects of Subcutaneous Recombinant Human Interleukin 12 in Patients with Metastatic Renal Cancer

We examined the biological effects of subcutaneous (s.c.) recombinant human IL-12 (rHuIL-12) in metastatic renal cancer patients. Clinical and immunobiological effects were examined weekly, and detailed pharmacokinetic/pharmacodynamic analyses were carried out in the first treatment cycle. The t 0.5 of IL-12 ranged from 9 to 28 h and the time taken to reach C max from 6 to 24 h. Increases in circulating IFN- n , IL-10, TNF- f , IL-6 and neopterin were seen, but IL-4 was undetectable. Anaemia, neutropenia and lymphocytopenia were seen together with a large decrease in eosinophil number. Successive IL-12 injections produced a marked attenuation in IL-12 and neopterin concentrations, which was reflected in a diminution of the biological and haematological changes. Th1/Th2 and Tc1/Tc2 ratios were assessed by analysis of circulating T-lymphocyte cytoplasmic cytokines, the first time that this technique has been applied to evaluate cytokine therapy. Increases in NK activity were seen in all patients. None of th...