Iw McDicken

Detection of interleukin-8 mRNA and protein in human colorectal carcinoma cells

Interleukin-8 (IL-8) is a member of the chemokine family of pro-inflammatory chemotactic cytokines and is secreted by some human colorectal carcinoma cell lines. We have used in situ hybridisation and immunohistochemistry to determine whether IL-8 mRNA and protein, respectively, are produced by human colorectal carcinoma cells in vivo. IL-8 mRNA was detected within the cytoplasm of tumour cells in all nine samples tested, including that of a tumour which had metastasised to a lymph node. Non-involved colonic mucosa within the same tissue blocks showed much weaker labelling. IL-8 protein was detected in 74% (23/31) of tumour samples and was mainly localised to the tumour cell cytoplasm. In 30% of cases, staining was heterogeneous, with between 1 and 30% of cells being positive. In some tumour cells, IL-8 showed a perinuclear distribution resembling that found by in situ hybridisation. Some infiltrating leucocytes, endothelial cells and fibroblast-like cells within the tumour sections were also positive for IL-8 mRNA and protein. The possibilities that colorectal tumours produce IL-8 to aid invasion and/or metastasis or as a tumour growth factor are discussed.

p75 nerve growth factor receptor in the vesical urothelium of patients with neuropathic bladder: an immunohistochemical study.

Introduction: Nerve growth factor (NGF), apart from its role as a growth factor, appears to be involved in neuroimmune interactions and in tissue inflammation. Low-affinity nerve growth factor receptor (p75 NGFR), if demonstrated in the urothelium, could provide the means for (1) NGF-mediated modulation of the urothelial response to inflammation; (2) NGF-mediated autocrine/paracrine regulation of urothelial proliferation; and (3) p75 NGFR-mediated induction of apoptosis. Objectives: To investigate the presence of p75 NGFR in the vesical urothelium of patients with neuropathic bladder by immunohistochemical methods. Setting: A hospital-based study of consecutive, unselected, adult patients of either sex with neuropathic bladder, undergoing procedure on the urinary tract in a Regional Spinal Injuries Centre located in the north-west of England. Intervention: Cold cup biopsies were taken from the trigone of the neuropathic urinary bladder of 26 patients with neuropathic bladder. Immunohistochemical studies were performed using antiNGF-receptor human monoclonal antibody which reacts with the low affinity receptor (p75 NGFR). Results: Both neural and epithelial structures showed positive immunostaining for p75 NGFR. The basal layer of the transitional epithelium showed strongly positive immunostaining for p75 NGFR in all the 26 cases. The luminal layer of transitional epithelium showed varying degree of positive immunostaining in 12 patients. The nerve fibres showed positive immunostaining for p75 NGFR. In many cases, the positively-stained nerve fibres were coursing very close to the basal layer of the urothelium almost entering the urothelium; however, no NGFR-positive intra-epithelial terminals could be seen. The positively-stained single nerve fibres and positively-stained thicker nerve bundles were seen in abundance in the submucosa but they were present in a sparse manner in the muscularis layer. Conclusion: The presence of p75 NGFR was demonstrated in the urothelium of neuropathic bladder of all the 26 patients with neuropathic bladder. This observation may have potential therapeutic implications.

Evidence for keratinocyte immortalization in high-grade squamous intraepithelial lesions of the cervix infected with high-risk human papillomaviruses.

In this study, we demonstrate that expression of cyclin B protein is up-regulated and persists into the upper epithelial layers in parallel with cyclin A expression in high-grade squamous intraepithelial lesions (SIL) infected with human papillomaviruses 16, 31, 33, 51, 58, 66, and 67 (n = 33). In contrast, low-grade SIL infected with human papillomaviruses 16, 18, 31, 33, 39, 51, 52, 56, 58, and 66 (n = 27) show weaker cyclin B expression confined to basal and parabasal cells despite extension of cyclin A and Ki67 expression into superficial cells. Moreover, aneusomy is present in 20% of the high-grade lesions but in none of the low-grade lesions. The persistent expression of cyclin B in high-grade SIL, and the restriction of aneusomy to high-grade SIL suggest that there is cell cycle progression. In combination with in vitro studies, this provides evidence that high-grade SIL lesions have undergone immortalization.

Numerical abnormalities of chromosomes 1, 11, 17, and X are associated with stromal invasion in serous and mucinous epithelial ovarian tumours

The clinical behaviour of ovarian tumours of low malignant potential (LMP) is unpredictable and it has been suggested that the majority of these lesions have no invasive potential. This study has analysed 92 epithelial ovarian tumours [11 mucinous cystadenomas, 18 mucinous LMP tumours, 15 mucinous carcinomas (9 FIGO stage I), 16 serous cystadenomas, 15 serous LMP tumours, and 17 serous carcinomas (11 FIGO stage I)] for numerical abnormalities of chromosomes 1, 11, 17, and X by interphase cytogenetics. Overall, numerical aberrations were identified in none of the cystadenomas, 15 per cent of serous LMP tumours, 17 per cent of mucinous LMP tumours, 67 per cent of mucinous carcinomas, and 82 per cent of invasive serous carcinomas. In mucinous LMP tumours, chromosome gains were associated with spindled nuclear morphology. Chromosome abnormalities were significantly more frequent in invasive mucinous (overall p< 0·01; stage I p< 0·05) and serous (overall p< 0·001; stage I p< 0·01) carcinomas than in the corresponding LMP tumours. No significant relationship between either stromal invasion or tumour type and the pattern of chromosome loss or gain was identified, although monosomy X was identified almost exclusively in invasive serous carcinomas. These observations are consistent with the concept that LMP tumours are unlikely to be precursors of ovarian carcinoma, but suggest that chromosome instability is important in the development of the invasive phenotype. Copyright

A study of cytokeratin 20 immunostaining in the urothelium of neuropathic bladder of patients with spinal cord injury

BackgroundNormal urothelium is characterised by terminally differentiated superficial cells, which express cytokeratin 20 in the cytoplasm. In contrast, cultured human stratified urothelium, which does not undergo complete terminal differentiation of its superficial cells, does not express cytokeratin 20. If spinal cord injury (SCI) affects urothelial differentiation or induces squamous or other metaplastic change undetected by histological analysis, the superficial urothelial cells of the neuropathic bladder might be expected to show absence of immunostaining for cytokeratin 20.Patients and MethodsWe studied immunostaining for cytokeratin 20 in bladder biopsies taken from 63 consecutive SCI patients. Immunostaining was performed on paraffin-embedded tissue using a mouse monoclonal antibody (clone: Ks20.8).ResultsOf 63 biopsies, the epithelium was scarce in two. Eight biopsies showed squamous metaplasia and immunostaining for cytokeratin 20 was absent in all the eight biopsies. Of the remaining 53 cases, in which the umbrella cell layer of the urothelium was intact, immunostaining for cytokeratin 20 was seen only in ten biopsies.ConclusionSuperficial cells in the transitional epithelium showed immunostaining for cytokeratin 20 in 10 of 53 bladder biopsies taken from SCI patients. The reasons for this could be either that there is an underlying metaplasia or that changes in the neuropathic bladder affect urothelial differentiation. Taken with evidence from other systems, such as loss of cytokeratin 20 expression from static organ cultures of urothelial tissue, this might suggest that other factors, such as impairment of voluntary voiding in SCI patients, could affect expression of markers such as cytokeratin 20.

Localization of C-X-C and C-C chemokines to renal tubular epithelial cells in human kidney transplants is not confined to acute cellular rejection.

Chemokines are important mediators of leucocyte chemoattraction to inflammatory sites. Previous work has shown that the expression of some chemokines is upregulated during renal transplant rejection. The objectives of the present study were to determine whether chemokine expression is increased during renal transplant rejection. Immunohistochemistry was used to localize the C-X-C (alpha) chemokine interleukin-8 (IL-8) and the C-C (beta) chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1beta (MIP-1beta) in 30 needle biopsies of human kidney transplants taken for diagnosis of renal dysfunction. Urine samples from transplant patients taken immediately prior to biopsy were assayed for chemokine content using enzyme-linked immunosorbent assays (ELISAs). Results from groups of patients having different clinicopathological diagnoses were then compared. All three chemokines were detected in most renal transplant biopsies showing acute cellular rejection but, although infiltrating leucocytes were often positive, staining was predominantly localized to renal tubular epithelium. Staining for MCP-1 was generally weaker than for the other chemokines, and collecting tubules were usually stained more strongly than proximal convoluted tubules. Tubular epithelial staining was also found in biopsies from patients without signs of acute cellular rejection. There were significantly higher amounts of IL-8 in the urine of patients with acute cellular rejection, even when patients with urinary tract infections were excluded, but mean titres of urinary MIP-1beta did not differ between patient groups. This was also found when titres were normalized for urine volume and creatinine levels. Production of IL-8, MCP-1 and MIP-1beta is not confined to kidney transplants showing acute cellular rejection, and may be a relatively nonspecific response of tubular epithelial cells to renal damage.

Detection of early squamous metaplasia in bladder biopsies of spinal cord injury patients by immunostaining for cytokeratin 14

Study design: A prospective, immunohistochemical study of bladder biopsies taken from spinal cord injury (SCI) patients.Objectives: To investigate whether cytokeratin 14 immunostaining may be useful to detect early squamous metaplasia in bladder biopsies from patients with SCI.Setting: Southport, United Kingdom.Methods: Biopsy of bladder mucosa was taken from adults with SCI, while they underwent an elective therapeutic procedure in the urinary tract. A total of, 54 biopsies, which showed transitional epithelium only with no evidence of squamous metaplasia on routine H&E staining, formed the study group. In all, 22 biopsies, which showed squamous metaplasia on routine H&E staining, acted as controls. All biopsies were benign with no evidence of dysplasia or malignancy. Immunohistochemical staining for cytokeratin 14 was performed on all biopsies in a single batch, using a standard avidin-biotin complex method.Results: All control biopsies showed positive immunostaining for cytokeratin 14 in basal and parabasal cells in areas of squamous metaplasia. Of the 54 biopsies, which showed only transitional epithelium on H&E staining, immunohistochemistry for cytokeratin 14 showed no staining in 47 biopsies. The remaining seven biopsies showed positive immunostaining for cytokeratin 14 in the epithelium, in individual cells or clusters of basal cells, revealing unexpected early squamous metaplasia in these biopsies.Conclusion: Immunostaining for cytokeratin 14 identifies an early phenotypic switch from transitional to squamous epithelium in bladder mucosa. Cytokeratin 14 staining is sufficiently sensitive to identify early squamous metaplasia, which is not yet evident on examination of routine H&E stained sections. This early identification may be of use in alerting physicians to change bladder management regimens to prevent predisposition to recurrent urinary infection and progression of squamous metaplasia. A cost/benefit analysis should be performed to assess the feasibility of routine cytokeratin 14 immunostaining of bladder biopsies from SCI patients.

Secretory immunoglobulin A in the vesical urothelium of patients with neuropathic bladder–an immunohistochemical study

Study design: A pilot study was carried out on archival material of bladder biopsies taken during 1994 and 1995 from patients with neuropathic bladder.Objectives: To compare the pattern of immunostaining for sIgA in the urothelium of biopsies taken from neuropathic bladder with the biopsies obtained from patients with non-neuropathic bladders.Setting: Regional Spinal Injuries Centre, Southport and Department of Pathology, Royal Liverpool University Hospital, Liverpool.Methods: Formalin-fixed, paraffin-embedded biopsies of bladder mucosa taken from patients with neuropathic urinary bladder (n=43) during 1994 and 1995 were processed for immunostaining with rabbit polyclonal antibody for secretory component of IgA. Archival specimens of bladder biopsies from non-neuropathic bladder were stained as controls. All sections were stained contemporaneously.Results: In all the control biopsies, strong immunostaining for sIgA was observed in the superficial cells of transitional epithelium. In the biopsies taken from patients with neuropathic bladder, immunostaining in the transitional epithelium was variable: strong in 14 cases; moderate in four; faint in 16; and absent in three. Immunostaining for sIgA was absent in all the five biopsies in which the urothelium had undergone squamous metaplasia. One biopsy showed intestinal metaplasia; immunostaining for sIgA was seen in the basal cells.Conclusion: Strong immunostaining for sIgA was observed in the urothelium of all biopsies taken from non-neuropathic bladder. In contrast to this, only 18 of 37 biopsies obtained from neuropathic bladder showed strong or moderate immunostaining for sIgA in the transitional epithelium.

Immunohistochemical study of parathyroid hormone-related protein in vesical transitional epithelium of patients with spinal cord injury.

Introduction: Parathyroid hormone-related protein (PTHrP), in addition to the well-established role in endochrondral bone development, is believed to be an important mediator of cellular growth and differentiation in a number of non-bony tissues. Objectives: To compare the immunohistochemical staining of vesical transitional epithelium to antibodies raised to synthetic peptides of PTHrP composed of amino acid sequences 43–52 and 127–138 in patients with spinal cord injury (SCI) and neuropathic bladder (n=14), and control patients with intact neuraxis and no history of bladder cancer (n=10). Setting: Male SCI patients registered with Regional Spinal Injuries Centre, Southport, England. Intervention: Endoscopic cold cup biopsy from the trigone of the urinary bladder was taken from patients with SCI while they were undergoing a therapeutic procedure in the urinary bladder. The control samples of bladder biopsies were taken from the archives of the Department of Histopathology, District General Hospital, Southport. Immunohistochemistry was performed using rabbit antibodies raised against synthetic peptides of human PTHrP (43–52) and PTHrP (127–138). The biopsies were examined for immunostaining of transitional epithelium. Results: Of the 14 biopsies of SCI patients, positive immunostaining using antibodies to both the PTHrP peptides was found in four cases; five biopsies showed positive immunostaining only to anti-PTHrP (43–52); and five biopsies showed no immunostaining with either of the PTHrP peptides. In contrast, transitional epithelium in the biopsy specimens of ten control subjects with no history of bladder cancer showed no immunostaining with either of the PTHrP peptides. Conclusion: This study revealed that the transitional epithelium of neuropathic urinary bladder exhibits increased predilection for positive immunohistochemical staining for PTHrP (43–52), and to a lesser extent, to PTHrP (127–138), as compared to the vesical transitional epithelium of able bodied individuals with no history of vesical malignancy. The possible role of PTHrP in the cellular differentiation of urothelium of neuropathic bladder, and thereby, in the pathogenesis of cystitis in SCI patients, needs to be explored.

Parathyroid hormone-related protein (1-34) and urothelial redifferentiation in the neuropathic urinary bladder.

Study design: A comparative study of immunostaining for parathyroid hormone-related protein (1–34) (PTHrP (1–34)) in the vesical epithelium of biopsies obtained from patients with non-neuropathic bladder and those with neuropathic bladder.Objectives: To investigate the immunostaining for PTHrP (1–34) in the control cases and in neuropathic bladders showing (1) normal transitional epithelium, (2) hyperplastic transitional epithelium, and (3) squamous metaplasia.Setting: Regional Spinal Injuries Centre, and Department of Cellular Pathology, Southport & Ormskirk Hospitals NHS Trust, Southport, Department of Pathology, Royal Liverpool University Hospital and the Departments of Clinical Chemistry and Cell Biology, The University of Liverpool, Liverpool, England.Methods: Cold cup biopsies of bladder mucosa were taken from patients suffering from neuropathic urinary bladder when they were undergoing a therapeutic procedure in the urinary tract. Immunohistochemistry was performed on these biopsy specimens using a rabbit polyclonal antibody raised to a synthetic peptide corresponding to human PTHrP (1–34). Control group (n=10) consisted of archival biopsies taken from non-neuropathic bladders.Results: In the control group, the transitional epithelium showed no immunostaining, or at the most, very faint positive staining was seen in the transitional epithelium of non-neuropathic bladder. Positive immunostaining to PTHrP (1–34) was seen in the normal transitional epithelium of neuropathic bladder in nine of 13 cases. Hyperplastic transitional epithelium showed positive immunostaining for PTHrP (1–34) in 11 of 13 biopsies from patients with neuropathic bladder. Immunostaining for PTHrP (1–34) was observed in the metaplastic squamous epithelium in 14 of 17 cases with neuropathic bladder.Conclusion: The transitional epithelium of non-neuropathic bladder showed no immunostaining, or at the most, very faint positive staining for PTHrP (1–34). In contrast to this, positive immunostaining for PTHrP (1–34) was observed more frequently in the vesical epithelium of neuropathic bladder. This observation opens up avenues for innovative therapy with PTHrP or its analogues for possible modulation of urothelial differentiation in the neuropathic bladder.