An evaluation of the long-term effectiveness of Gatekeeper™ intersphincteric implants for passive faecal incontinence.

BACKGROUND: Implantation of Gatekeeper™ prostheses presents an option for the treatment of passive faecal incontinence (FI). Whilst preliminary results are encouraging, long-term data regarding its sustained benefit are limited. The aim of this study was to assess and evaluate the long-term clinical function and quality of life of patients with passive faecal incontinence who were treated with Gatekeeper™ prostheses. METHODS: This was a single centre, single surgeon retrospective study of prospectively collected clinical data in patients with FI treated between June 2012 and May 2019. Patients with passive FI with symptoms refractory to conservative treatment and endoanal ultrasonography showing intact or disrupted internal anal sphincter were included. Formal clinical and quality of life assessments were carried out using the St. Mark's Incontinence Score (SMIS) and Faecal Incontinence Quality of Life (FIQoL) questionnaires at baseline, 3 months, 6 months, 12 months and then annually. Endoanal ultrasonography was performed both before and after surgery. RESULTS: Forty patients (14 males, 26 females) with a median age of 62.5 (range 33-80) years were treated with the Gatekeeper™ implant. The majority of patients (87.5%) received six implants. There were no peri or post-operative complications. Prosthesis migration was observed in 12.5% patients. The median follow-up duration was 5 years (interquartile range (IQR) 3.25-6.00 years). A sustained improvement in median SMIS and FIQoL scores from baseline to follow-up was noted. Significant differences were observed between the median baseline SMIS score and last follow-up score of 16.00 (IQR 15.00-16.75) to 7.00 (IQR 5.00-8.00) respectively (p < 0.001), a 56.25% decrease. The overall median FIQoL score showed a significant improvement from 7.95 (IQR 7.13-9.48) to 13.15 (IQR 12.00-13.98) (p < 0.001) a 65.40% increase. CONCLUSIONS: Gatekeeper™ implantation is a safe approach to treating passive FI and is minimally invasive, reproducible and has minimal complications. Long-term sustained clinical improvement is achievable beyond 5 years. Careful patient selection is paramount, as is consistency of technique and follow-up protocol.

Lack of clonal BCRA2 gene deletion on chromosome 13 in chronic lymphocytic leukaemia.

Chromosome 13q deletion is among the most common cytogenetic abnormalities in chronic lymphocytic leukaemia (CLL). We investigated the 13q14.3 deletion in 44 CLL patients by Southern blotting following purification of clonal B CLL cells to >90%. Two sets of probes were used to investigate the site of clonal deletion, the D13S25 and D13S319 markers (at 13q14.3) and probes for exons 11 and 26-27 of the BRCA2 gene (at 13q12). Homozygous and heterozygous deletion at the 13q14.3 region was found in five and 17 patients, respectively. Despite the recent report of the BRCA2 gene involvement in >80% of CLL patients, we failed to detect a single case of homozygous or heterozygous deletion involving the 13q12 region. Our data support previous findings that the 13q14.3, and not the 13q12 region, is the major site of candidate tumour suppressor gene(s) in CLL.

Deletion of chromosome 13 (band q14) but not trisomy 12 is a clonal event in B-chronic lymphocytic leukaemia (CLL).

Chromosomal abnormalities are detected by conventional cytogenetic or FISH analysis in 50% of chronic lymphocytic leukaemias (CLL). Trisomy 12 and del 13q14 account for 70% of these abnormalities. The incidence of these two abnormalities was studied in CLL patients by Southern blot analysis using a highly purified B-cell malignant population (CD5 > 95%, CD3 < 5%). Probes for the D13S25 marker on chromosome 13 band q14 and for the RBTN3 gene on chromosome 12 band p12-13, were used. Deletion of the D13S25 was detected in 17/42 patients (43%) in a homozygous (9.5%) or heterozygous (30%) configuration. Deletion of the D13S25 marker appears to be a clonal and early event in CLL development since it is detected in > 95% of the malignant clonal population. Conversely, trisomy 12 is rarely a clonal event (5/33 patients, 15%) and a varying proportion of cells carrying this abnormality can be demonstrated in 30% of CLL patients (10/33 patients).

Defects in signal transduction pathways in chronic B lymphocytic leukemia cells.

B chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL) cells are refractory to many of the signals which activate normal B cells but are stimulated to proliferate by tumor necrosis factor (TNF). Cell signalling by TNF is mediated in part by the induction of the transcription factor families AP-1 and NF-kappa B. In some cellular contexts, these factors play a role in regulating cell cycle transit. AP-1 binds DNA as dimers of jun and fos family proteins and is regulated by a cascade of protein kinases which eventually activate a mitogen-activated protein kinase (MAP kinase) and also by protein kinase C. Three pathways have been implicated in the activation of NF-kappa B by extracellular ligands. 1, the activation of protein kinase C by diacylglycerol generated by ligand-mediated activation of phosphatidylcholine hydrolysis, 2, stimulation of specific protein kinases by ceramide generated following activation of a sphingomyelinase by diacylglycerol and 3, a novel pathway involving ligand-induced generation of free radical species. In B-CLL and HCL cells, the generation of nuclear-localized c-jun and c-fos proteins (components of AP-1) in response to TNF or PMA appears to be blocked. Whereas PMA failed to induce NF-kappa B in these cells, this factor was readily induced by TNF. TNF induction of NF-kappa B was abolished by antioxidants, suggesting involvement of the free radical pathway. The data discussed here suggest defects in coupling of some protein kinase C-dependent pathways in B-CLL and HCL cells and that TNF is able to bypass these blocks by the activation of NF-kappa B via a free radical-dependent pathway which is independent of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of calcium ion-regulating inositol polyphosphate second messengers is impaired in subpopulations of human leukemia cells.

Inositol 1,4,5-triphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) are calcium-regulating second messenger molecules generated following the binding of a wide range of hormones and growth factors to their receptors. The actions of these messengers, which play important roles in the regulation of cell proliferation as well as in other signaling pathways, are terminated by the action of a 5-phosphomonoesterase (5-PME) enzyme. We have assayed this enzyme in normal and malignant hemopoietic cells. Extracts from normal bone marrow cells and peripheral blood mononuclear cells (PBMNC) degraded [3H]IP3 at rates of 74.5 (+/- 3.4) and 84.5 (+/- 7.9) pmol/min/micrograms protein, respectively. PME activity in 10/13 (77%) acute lymphoblastic leukemia samples were significantly below the normal range and the enzyme was completely undetectable in three (23%) of these. Enzyme activity in 8/9 (89%) chronic lymphocytic leukemia samples were below the normal range, being undetectable in three of these (33%). Nine of 24 (38%) acute myeloid leukemia samples contained low 5-PME levels, which was undetectable in one sample. Reduced 5-PME activity was detected in 2/7 (28%) of chronic granulocytic leukemia samples. The data here are consistent with the hypothesis that a reduced rate of degradation of IP3 and IP4 in some leukemia cells may result in the aberrant operation of signaling pathways, possibly including those involved in the control of cell proliferation.

Redox reagents and staurosporine inhibit stimulation of the transcription regulator NF-kappa B following tumour necrosis factor treatment of chronic B-leukaemia cells.

B-chronic lymphocytic leukaemia (B-CLL) and hairy cell leukaemia cells (HCL) are refractory to stimulation by several cytokines which activate normal B-cells. However, tumour necrosis factor (TNF) promotes the proliferation of these cells. TNF regulates some of its cellular responses via the transcription factor NF-kappa B. Using an electrophoretic mobility shift assay, we demonstrate that TNF treatment of B-CLL and HCL cells in vitro resulted in the augmentation of NF-kappa B levels. In haemopoietic cell lines, TNF induction of NF-kappa B is mediated via the generation of reactive oxygen intermediates and by the activation of protein kinase C (PKC). We have used activators and inhibitors of these pathways to unravel TNF signalling in the cells of ten patients with B-CLL and two with HCL, using the increase in NF-kappa B levels following TNF treatment as an end point. Raising glutathione levels with N-acetyl cysteine substantially reduced NF-kappa B induction by TNF in two of four samples tested. These data suggest that redox mechanisms are involved in TNF signalling in these cells. Treatment with the PKC activator phorbol myristate acetate failed to activate NF-kappa B suggesting that this enzyme does not mediate the induction of NF-kappa B in these cells. However, the protein kinase inhibitor staurosporine inhibited TNF induction of NF-kappa B in four of five samples, suggesting that staurosporine-sensitive protein kinases (other than PKC) are involved in the signalling pathway. Our results suggest that PKC-independent pathways, including pathways sensitive to redox reagents, mediate the induction of NF-kappa B by TNF in chronic B-leukaemia cells. Additionally, these data suggest that defects in PKC-mediated pathways may contribute to the general reluctance of B-CLL and HCL cells to respond to mitogenic signals.

Regulation of transcription factors NF kappa B and AP-1 following tumour necrosis factor-alpha treatment of cells from chronic B cell leukaemia patients.

Malignant B lymphocytes from patients with B-chronic lymphocytic leukaemia (B-CLL) or hairy cell leukemia (HCL) are refractory in vitro to mitogenic stimulation by several agents which trigger proliferation of normal B cells. Tumour necrosis factor (TNF) is a growth factor for these malignant cells, although the proliferative response is usually small. TNF regulates some of its cellular responses via induction of the transcription factors NF kappa B and AP-1 (jun/fos). The induction of NF kappa B by TNF is mediated via a novel signalling pathway involving the generation of reactive oxidative intermediates. Induction of jun and fos proteins (polypeptide components of AP-1) are mediated via pathways involving protein kinase C and the protein kinase encoded by the raf proto-oncogene. Here we have used an electrophoretic mobility shift assay to show that TNF induced NF kappa B in malignant cells isolated from 3/3 HCL and 15/15 B-CLL patients. By contrast, phorbol myristate acetate (PMA), a direct activator of protein kinase C, failed to activate this transcription factor in 1/1 HCL and 5/5 B-CLL isolates. The induction of jun and fos proteins (as detected by Western blot analysis) showed greater heterogeneity. Nuclear jun was induced by TNF in 5/12 chronic B cell leukaemia isolates. PMA induced this protein in 4/5 samples. Nuclear fos was induced by TNF in only 2/12 isolates and by PMA in 2/5. The data suggest that the pathways for the activation of jun and fos by TNF are defective in some B-CLL and HCL cells and that these defects may be heterogeneous. The induction of AP-1 is crucial in securing the mitogenic response to TNF. It is therefore plausible that these lesions may contribute to the refractory nature of B-CLL and HCL cells to proliferative stimuli in vitro.

Alpha-interferon (alpha-IFN) protects B-chronic lymphocytic leukaemia cells from apoptotic cell death in vitro.

B-chronic lymphocytic leukaemia cells (CLL) are prone to apoptotic cell death when cultured in vitro. Apoptosis and loss of the bcl-2 protein is prevented in CLL cells cultured in the presence of interleukin-4. In this study we analysed the effects of alpha-IFN on the DNA fragmentation, bcl-2 protein levels and cell survival in purified B-cells from 16 CLL patients. Alpha-IFN (10(3) U/ml) reduced the degree of spontaneous DNA fragmentation of CLL cells after a 30 h culture period (from a mean of 22.2% in control cultures to 10.5%, P < 0.01). This inhibition was accompanied by preservation of bcl-2 protein and an increased survival of CLL cells compared to control cultures. In parallel, alpha-IFN inhibited hydrocortisone induced DNA fragmentation in CLL cells. The effects of alpha-IFN on DNA synthesis of CLL cells were variable (in two patients a decrease and in seven an increase in 3H-thymidine uptake) and did not correlate with the effect on DNA fragmentation. In conclusion, our data suggest that alpha-IFN, like IL-4 and gamma-IFN, inhibits apoptosis of CLL cells. These in vitro data indicate that the clinical responses of some CLL patients to alpha-IFN cannot be explained by a direct cytotoxic effect of alpha-IFN on circulating CLL cells. Alternatively, alpha-IFN may inhibit the proliferation of the small fraction of clonogenic CLL progenitors, or interfere with cellular interactions necessary for the survival and growth of CLL cells.

Interleukin-4 inhibits apoptotic cell death and loss of the bcl-2 protein in B-chronic lymphocytic leukaemia cells in vitro.

When monoclonal B cells from B-chronic lymphocytic leukaemia (B-CLL) patients are cultured in vitro, they die by apoptosis. Apoptotic cell death occurred in the B cells from 20/24 B-CLL patients after 26-30 h in in vitro culture, with 14.3-59.0% (mean 33.6%) of their DNA being fragmented in approximately 180 base pair multimers. After 8-10 d culture, 90-100% of the B-CLL cells were dead. Cell death and DNA fragmentation were inhibited in the presence of 0.5-5 ng/ml human recombinant interleukin-4 (IL-4) and viable monoclonal B cells could be maintained in culture up to 3 weeks. At 5 ng/ml, IL-4 reduced DNA fragmentation after a 26-30 h culture to 2.2-33.3% (mean 14.9%). IL-4 inhibited apoptosis without stimulating cell proliferation. In four patients the cells were resistant to apoptosis in vitro and they could be maintained for up to 4 weeks in culture medium alone. DNA fragmentation in all patients was increased in the presence of the RNA synthesis inhibitor actinomycin-D. Western blot analysis of cell lysates showed expression of the bcl-2 protein in all 11 B-CLL patients studied. However, during culture, bcl-2 protein levels were preserved only in patients resistant to apoptosis and were reduced in those susceptible to apoptosis. Reduction of bcl-2 protein levels was inhibited in cells cultured in the presence of IL-4. These data offer an explanation for the difference between the long life in vivo and rapid death in vitro of B-CLL cells and indicate that IL-4 may participate in the extended survival of these non-dividing cells in vivo.

The use of clonogenic assays in assessing the response of human lung cancer cell lines to alpha and gamma interferons alone or in combination with adriamycin.

The antiproliferative and cytotoxic effects of purified IFN-alpha and recombinant IFN-gamma were investigated using both direct cell counting and a clonogenic assay on a panel of 5 established human lung cancer cell lines and for 2 of them also on their multidrug-resistant counterparts. There was considerable heterogeneity in the response of the cell lines to the IFNs in terms of growth inhibition. Clonogenic assay of IFN-treated cells indicated that, where a cell line had responded markedly to an IFN, only a small fraction of the cells remaining after IFN treatment were clonogenically viable. When cells were placed into the clonogenic assay in the presence of IFNs, the time course of colony formation was different from that seen in the control cultures for most of the cell lines. The measured "surviving fraction" was greatly dependent upon the time of colony counting. When the effects of IFNs in combination with ADM were studied, conclusions regarding the interaction of the effects of the agents also depended upon the time at which colonies were counted.

The MTT assay underestimates the growth inhibitory effects of interferons.

The growth inhibitory effects of interferons, IFN-alpha and IFN-gamma on human lung cancer cell lines were studied using both a tetrazolium (MTT) colorimetric assay and direct cell counting. Significant discrepancies between the two assays were observed, the MTT assay consistently underestimating the growth inhibitory effects of the IFNs. There was no direct chemical effect of the IFNs on the tetrazolium reduction process. IFN treated cells showed increased cell size compared with control cells, although there was little or no change in cell cycle distribution. Mitochondrial activity was 30-50% greater in IFN-gamma treated cells (COR-L23) than the controls. Reduced formazan production per cell was observed in medium which had supported cell growth for several days. Differential 'medium conditioning' led to a difference in formazan production per cell between IFN and control cells and this was the major basis of the observed discrepancy. This discrepancy was not due to the differences in the glucose concentrations between these media. However, differences in pH between the media proved to be the major contributory factor of the discrepancy.