Two randomised phase II trials of subcutaneous interleukin-2 and histamine dihydrochloride in patients with metastatic renal cell carcinoma

Histamine inhibits formation and release of phagocyte-derived reactive oxygen species, and thereby protects natural killer and T cells against oxidative damage. Thus, the addition of histamine may potentially improve the efficacy of interleukin-2 (IL-2). Two randomised phase II trials of IL-2 with or without histamine dihydrochloride (HDC) in patients with metastatic renal cell carcinoma (mRCC) were run in parallel. A total of 41 patients were included in Manchester, UK and 63 in Aarhus, Denmark. The self-administered, outpatient regimen included IL-2 as a fixed dose, 18 MIU s.c. once daily, 5 days per week for 3 weeks followed by 2 weeks rest. Histamine dihydrochloride was added twice daily, 1.0 mg s.c., concomitantly with IL-2. A maximum of four cycles were given. The Danish study showed a statistically significant 1-year survival benefit (76 vs 47%, P=0.03), a trend towards benefit in both median survival (18.3 vs 11.4 months, P=0.07), time to PD (4.5 vs 2.2 months, P=0.13) and clinical benefit (CR+PR+SD) (58 vs 37%, P=0.10) in favour of IL-2/HDC, whereas the UK study was negative for all end points. Only three patients had grade 4 toxicity; however, two were fatal. A randomised phase III trial is warranted to clarify the potential role of adding histamine to IL-2 in mRCC

Accumulation of natural killer cells after hepatic artery embolisation in the midgut carcinoid syndrome.

Eleven patients with disseminated midgut carcinoid tumour disease were subjected to hepatic artery embolisation. In six patients, lymphocytosis with a predominance of NK cells occurred and the cytotoxic activity of isolated lymphocytes increased. A relation between NK cell accumulation and subsequent radiological and biochemical response was observed, and it is suggested that anti-tumour mechanisms other than ischaemia may contribute to the therapeutic response in these patients

Impact of killer-immunoglobulin-like receptor and human leukocyte antigen genotypes on the efficacy of immunotherapy in acute myeloid leukemia

Interactions between killer-immunoglobulin-like receptors (KIRs) and their HLA class I ligands are instrumental in natural killer (NK) cell regulation and protect normal tissue from NK cell attack. Human KIR haplotypes comprise genes encoding mainly inhibitory receptors (KIR A) or activating and inhibitory receptors (KIR B). A substantial fraction of humans lack ligands for inhibitory KIRs (iKIRs), that is, a 'missing ligand' genotype. KIR B/x and missing ligand genotypes may thus give rise to potentially autoreactive, unlicensed NK cells. Little is known regarding the impact of such genotypes in untransplanted acute myeloid leukemia (AML). For this study, NK cell phenotypes and KIR/HLA genotypes were determined in 81 AML patients who received immunotherapy with histamine dihydrochloride and low-dose IL-2 for relapse prevention (NCT01347996). We observed that presence of unlicensed NK cells impacted favorably on clinical outcome, in particular among patients harboring functional NK cells reflected by high expression of the natural cytotoxicity receptor (NCR) NKp46. Genotype analyses suggested that the clinical benefit of high NCR expression was restricted to patients with a missing ligand genotype and/or a KIR B/x genotype. These data imply that functional NK cells are significant anti-leukemic effector cells in patients with KIR/HLA genotypes that favor NK cell autoreactivity

Two novel human cytomegalovirus NK cell evasion functions target MICA for lysosomal degradation

NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αβ and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1–6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12–US21; a genetic arrangement, which is suggestive of an ‘accordion’ expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family

Cimetidine increases survival of colorectal cancer patients with high levels of sialyl Lewis-X and sialyl Lewis-A epitope expression on tumour cells

Cimetidine has been shown to have beneficial effects in colorectal cancer patients. In this study, a total of 64 colorectal cancer patients who received curative operation were examined for the effects of cimetidine treatment on survival and recurrence. The cimetidine group was given 800 mg day−1 of cimetidine orally together with 200 mg day−1 of 5-fluorouracil, while the control group received 5-fluorouracil alone. The treatment was initiated 2 weeks after the operation and terminated after 1 year. Robust beneficial effects of cimetidine were noted: the 10-year survival rate of the cimetidine group was 84.6% whereas that of control group was 49.8% (P<0.0001). According to our previous observations that cimetidine blocked the expression of E-selectin on vascular endothelium and inhibited the adhesion of cancer cells to the endothelium, we have further stratified the patients according to the expression levels of sialyl Lewis antigens X (sLx) and A (sLa). We found that cimetidine treatment was particularly effective in patients whose tumour had higher sLx and sLa antigen levels. For example, the 10-year cumulative survival rate of the cimetidine group with higher CSLEX staining, recognizing sLx, of tumours was 95.5%, whereas that of control group was 35.1% (P=0.0001). In contrast, in the group of patients with no or low levels CSLEX staining, cimetidine did not show significant beneficial effect (the 10-year survival rate of the cimetidine group was 70.0% and that of control group was 85.7% (P=n.s.)). These results clearly indicate that cimetidine treatment dramatically improved survival in colorectal cancer patients with tumour cells expressing high levels of sLx and sLa

Cimetidine modulates the antigen presenting capacity of dendritic cells from colorectal cancer patients

Cimetidine, a H2 receptor antagonist, has been reported to improve survival in gastrointestinal cancer patients. These effects have largely been attributed to the enhancing effects of cimetidine on the host's antitumour cell-mediated immune response, such as inhibition of suppressor T lymphocyte activity, stimulation of natural killer cell activity and increase of interleukin-2 production from helper T lymphocytes. We conducted an in vitro study on the effects of cimetidine on differentiation and antigen presenting capacity of monocyte-derived dendritic cells from advanced colorectal cancer patients and normal controls. As a result, an investigation of expression of surface molecules associated with dendritic cells by flow cytometric analyses showed that cimetidine had no enhancing effect on differentiation of dendritic cells from cancer patients and normal controls. An investigation of [3H]thymidine incorporation by allogeneic mixed lymphocyte reactions revealed that cimetidine increased the antigen presenting capacity of dendritic cells from both materials. Moreover, a higher antigen presenting capacity was observed in advanced cancer patients compared to normal controls. These effects might be mediated via specific action of cimetidine and not via H2 receptors because famotidine did not show similar effects. Our results suggest that cimetidine may enhance the host's antitumour cell-mediated immunity by improving the suppressed dendritic cells function of advanced cancer patients

Isolation and individual electrical stimulation of single smooth-muscle cells from the urinary bladder of the pig

In contrast to striated muscle, measurements on strips of smooth muscle cannot be uniquely interpreted in terms of an array of contractile units. Therefore scaling down to the single-cell level is necessary to gain detailed understanding of the contractile process in this type of muscle. The present study describes the development of a method for isolating contractile single smooth muscle cells from pig urinary bladders. Contractile responses evoked by individual electrical stimulation were used as a measure of cell quality during development of the method. Responses were evaluated by measuring latency, contraction and relaxation times, as indicated by visible length changes, and stored on-line in a computer. Initial length, relative shortening and shortening speed were determined by measuring cell lengths in previously timed still video frames using a computer-controlled crosshair device. Increase of stimulus pulse duration resulted in improved responses, indicating that the observed shortening represented a physiological contractile response. Ultimately this method of evaluation was applied to two sets of cell preparations obtained by two different methods, one using only collagenase digestion, the other using mechanical manipulation as well. Both sets showed two main patterns of response to electrical stimulation: a pattern of contraction upon stimulation followed by enhanced contraction when stimulation was switched off (CK), and a pattern of contraction upon stimulation followed by relaxation when the stimulus was switched off (CR). The set of preparations containing the highest percentage of CR cells was found to be superior (i.e. greater initial length, shorter latency and contraction times, increased shortening and higher shortening speed). The method of isolation used for this set gives a high yield of contractile cells available for experimental use over a long span of time

Effects of Transport Inhibitors on the Cellular Uptake of Carboxylated Polystyrene Nanoparticles in Different Cell Lines

Nanotechnology is expected to play a vital role in the rapidly developing field of nanomedicine, creating innovative solutions and therapies for currently untreatable diseases, and providing new tools for various biomedical applications, such as drug delivery and gene therapy. In order to optimize the efficacy of nanoparticle (NP) delivery to cells, it is necessary to understand the mechanisms by which NPs are internalized by cells, as this will likely determine their ultimate sub-cellular fate and localisation. Here we have used pharmacological inhibitors of some of the major endocytic pathways to investigate nanoparticle uptake mechanisms in a range of representative human cell lines, including HeLa (cervical cancer), A549 (lung carcinoma) and 1321N1 (brain astrocytoma). Chlorpromazine and genistein were used to inhibit clathrin and caveolin mediated endocytosis, respectively. Cytochalasin A and nocodazole were used to inhibit, respectively, the polymerisation of actin and microtubule cytoskeleton. Uptake experiments were performed systematically across the different cell lines, using carboxylated polystyrene NPs of 40 nm and 200 nm diameters, as model NPs of sizes comparable to typical endocytic cargoes. The results clearly indicated that, in all cases and cell types, NPs entered cells via active energy dependent processes. NP uptake in HeLa and 1321N1 cells was strongly affected by actin depolymerisation, while A549 cells showed a stronger inhibition of NP uptake (in comparison to the other cell types) after microtubule disruption and treatment with genistein. A strong reduction of NP uptake was observed after chlorpromazine treatment only in the case of 1321N1 cells. These outcomes suggested that the same NP might exploit different uptake mechanisms to enter different cell types