Dia Kamel

Mechanisms of ANCA-mediated leukocyte-endothelial cell interactions in vivo.

Anti-myeloperoxidase (anti-MPO) antibodies have been implicated in the pathogenesis of small-vessel vasculitis, but the molecular mechanisms by which these antibodies contribute to disease are unknown. For determination of how anti-MPO antibodies affect inflammatory cell recruitment in small-vessel vasculitis, intravital microscopy was used to monitor leukocyte behavior in the accessible cremasteric microvessels under various experimental conditions. After local pretreatment of the cremaster muscle with cytokines (TNF-alpha, IL-1beta, or keratinocyte-derived chemokine), administration of anti-MPO IgG to wild-type mice reduced leukocyte rolling in favor of augmented adhesion to and transmigration across the endothelium. This led to a decrease in the number of systemic circulating leukocytes and, similar to the early events in the development of vasculitic lesions, an increase in leukocyte recruitment to renal and pulmonary tissue. TNF-alpha led to the greatest recruitment of inflammatory cells, and IL-1beta led to the least. When anti-CD18 was co-administered, anti-MPO IgG did not affect leukocyte rolling, adhesion, or transmigration; similarly, anti-MPO IgG did not produce these effects in Fc receptor gamma chain-/- mice. This study provides direct in vivo evidence of enhanced leukocyte-endothelial cell interactions in the presence of anti-MPO IgG and highlights the critical roles of Fcgamma receptors and beta2 integrins in mediating these interactions. In addition, it suggests that neutrophils primed by cytokines in the presence of anti-MPO IgG can have systemic effects and target specific vascular beds.

Lowp53 protein expression in salivary gland tumours compared with lung carcinomas

Fifty-one salivary gland tumours (23 pleomorphic adenomas, 5 Warthins tumours, 12 mucoepidermoid carcinomas, 7 adenoid cystic carcinomas, 3 undifferentiated carcinomas and 1 acinic cell tumour) and 27 lung carcinomas (18 squamous cell carcinomas, 6 adenocarcinomas and 3 small cell carcinomas) were analysed immunohistochemically for the expression ofp53 nuclear phosphoprotein. Eight out of 51 (16%) salivary gland tumours werep53 positive. Three of these were benign and 5 malignant. All 3 benign salivary gland tumours were pleomorphic adenomas and expressed only occasional nuclear positivity with less than 1% of tumour cells positive. Of the 5p53-positive malignant tumours, 3 were mucoepidermoid carcinomas and 2 undifferentiated carcinomas. The malignant salivary gland tumours expressed more than 1% of positive nuclei in every case. Seventeen lung carcinomas werep53 positive (63%). Thirteen of these were squamous cell carcinomas, 3 were adenocarcinomas and 1 small cell lung carcinoma. The results show that mutations of thep53 gene may be infrequent in salivary gland tumours when compared with lung carcinomas. The relatively indolent course of some histological types of malignant salivary gland tumours could be associated with the preservation of the non-mutatedp53 gene in most of these tumours. The presence ofp53 positivity in some pleomorphic adenomas might, on one hand, suggest thatp53 gene alterations are also present in these tumours; on the other hand, the accumulation of thep53 protein in these tumours might also be due to some unknown mechanism, not necessarily related top53 gene mutation.

ABERRANT ACCUMULATION OF P53 ASSOCIATES WITH KI67 AND MITOTIC COUNT IN BENIGN SKIN LESIONS

Sixty‐two skin samples from patients with a variety of benign disorders (20 cases of psoriasis, 14 cases of chronic dermatitis, 11 seborrhoeic keratoses, 11 cases of lichen planus), and seven normal skin samples, were stained immunohistochemically with a polyclonal antibody (CM‐1) to p53, and a monoclonal antibody to Ki67, using the avidin‐biotin complex method, p53‐positive keratinocytes could be found in most of these lesions. The percentage of p53‐positive cells was, however, far lower than usually seen in p53‐positive malignant tumours. No p53 reactivity was observed in the normal skin samples. Variable Ki67 reactivity was observed in all skin samples. Overall, the number of Ki67‐positive cells was higher in skin samples in which the proportion of p53‐positive cells was high (>0.5% of total epidermal cell population) (P=0.004). This also applied separately to psoriatic and non‐psoriatic lesions (P=0.028 and P=0.033, respectively). In cases with >10% of Ki67‐positive cells, there were significantly more mitoses (P<0.001). This association applied to both psoriasis and the other lesions studied (P=0.024 and P <0.001, respectively). The results show that immunohistochemically detectable accumulation of p53 is a frequent finding in non‐neoplastic skin lesions. As p53 positivity was associated with the proliferation marker Ki67, the accumulation of p53 is possibly a response to an increased proliferation rate of the keratinocytes in these skin diseases, or alternatively it may be associated with apoptosis.

Presence of human papillomavirus DNA and abnormal p53 protein accumulation in lung carcinoma.

BACKGROUND: In some carcinomas inactivation of the tumour suppressor gene product p53, either by point mutation or indirectly by the human papillomavirus (HPV), has been suggested as two alternative routes to malignant transformation. To test this hypothesis in lung tumours, 43 lung carcinomas were analysed by in situ hybridisation and polymerase chain reaction (PCR) for the presence of HPV DNA, and the results were compared with p53 protein immunohistochemical analysis. METHODS: The presence of HPV DNA in lung carcinoma was detected by nucleic acid in situ hybridisation for HPV types 6, 11, 16, 18, 31, and 33 using nonradioactively labelled DNA probes. Polymerase chain reaction (PCR) analysis was performed on all cases showing positive HPV DNA labelling by in situ hybridisation and in an additional 13 negative cases. Abnormal nuclear accumulation of the p53 protein was revealed by immunohistochemistry using the avidin-biotin-peroxidase complex method and a CM-1 polyclonal anti-human p53 antibody and a monoclonal mutation-specific Pab 240 p53 antibody. RESULTS: HPV DNA was found by in situ hybridisation in 13 lung carcinomas (30%). In all these cases subtype-specific HPV DNA could also be detected by PCR. Abnormal p53 protein accumulation was seen in 21 of the 43 carcinomas (49%), of which 18 were HPV negative. Twelve (57%) of the CM-1 positive cases were also positive for the mutation-specific antibody Pab 240. There was an obvious inverse relationship between the presence of papilloma viral DNA and abnormal p53 protein accumulation. CONCLUSIONS: p53 plays an important part in the development of lung carcinomas and, in some cases, HPV may contribute to it by binding and inactivating the p53 protein.

Comparative analysis ofp53 protein immunoreactivity in prostatic, lung and breast carcinomas

In this study we analysed the expression ofp53 protein in a total of 143 carcinomas immunohistochemically. These consisted of 34 prostatic adenocarcinomas, 59 lung and 50 breast carcinomas. In 28 cases, an average of 2–3 additional sections from different tumour areas were analysed. Forty-nine of the 143 carcinomas (34%) showed typical nuclear immunoreactivity by immunohistochemical staining with thep53 antibody CM-1. Two of the 34 prostatic carcinomas (6%) werep53 positive while 25 of the 59 lung carcinomas (43%) and 22 of the 50 breast carcinomas (44%) showed positivity forp53. By grade: 49% of grade III tumours, 36% of grade II and 5% of grade I tumours werep53 positive. There were significantly morep53-positive cases in grade II–III tumours than in grade I tumours (P= 0.001) when all tumours were taken into account. Further, there were significantly morep53-positive cases in grade III than in grade I–II tumours (P=0.001). In lung tumours there were significantly morep53-positive cases in grade II–III tumours than in grade I tumours (P=0.018). Similarly, there were significantly morep53-positive tumours in grade III breast tumours than in grade I–II tumours (P=0.003). The low incidence ofp53 positivity in prostate carcinomas suggests that mutations of thep53 gene are not as frequent in the neoplastic transformation of these tumours as in lung or breast carcinomas. The association ofp53 positivity with tumours of higher grade suggests thatp53 mutations lead to tumours of a more aggressive type. The analysis of tumours by multiple sections indicates thatp53 positivity is not evenly distributed in tumour tissue. Therefore, analysis of additional tumour areas may reveal positivity some cases, which is not evident if only one section is studied.

DNA Copy Number Changes in Schistosoma-Associated and Non-Schistosoma-Associated Bladder Cancer

DNA copy number changes were investigated in 69 samples of schistosoma-associated (SA) and non-schistosoma-associated (NSA) squamous cell carcinoma (SCC) and transitional cell carcinoma (TCC) of the bladder by comparative genomic hybridization (CGH). DNA copy number changes were detected in 47 tumors. SA tumors had more changes than NSA tumors (mean, 7 vs. 4), whereas the number of changes in SCC and TCC tumors was similar. SA tumors displayed more gains than losses (1.7:1), whereas NSA tumors showed an equal number of gains and losses. Changes that were observed at similar frequencies in SCC and TCC, irrespective of the schistosomal status, included gains and high-level amplifications at 1q, 8q, and 20q and losses in 9p and 13q. These changes may be involved in a common pathway for bladder tumor development and progression independent of schistosomal status or histological subtype. Losses in 3p and gains at 5p were seen only in SCC (P < 0.01) and losses in 5q were more frequent in SA-SCC than in other tumors (P < 0.05). However, changes that were more frequent in TCC than those in SCC included gains at 17q (P < 0.01) and losses in 4q (P < 0.05) and 6q (P < 0.01). Gains and high-level amplifications at 5p were seen only in SA-SCC (P < 0. 01), whereas gains and high-level amplifications with minimal common overlapping regions at 11q13 were more frequently seen both in SA-SCC and SA-TCC tumors (P < 0.01). In addition to the above mentioned alterations, several other changes were also seen at lower frequencies. The variations in the DNA copy number changes observed in TCC, SCC, SA, and NSA bladder carcinomas suggest that these tumors have different genetic pathways.

Human papillomavirus DNA and abnormal p53 expression in carcinoma of the urinary bladder

In this study we analysed 47 bladder carcinomas for the presence of DNA‐HPV subtypes 6, 11, 16, 18, 31 and 33 by nucleic acid in situ hybridization, and for the abnormal accumulation of p53 protein by immunohistochemistry. HPV DNA was found in 27/47 (57%) bladder carcinomas, with multiple subtypes in 20 cases. In squamous cell carcinoma (SCC), HPV DNA was only detected in the superficial layer of the neoplastic epithelium and was found mainly in the nuclear compartment. In contrast, in transitional cell carcinoma (TCC), HPV DNA was also found in deeper parts of the tumour. In about half the cases it was mainly found in the cytoplasmic compartment. In SCC, the HPV DNA labelling occurred in koilocytic cells, while no such association was found in TCC. Abnormal accumulation of p53 protein was found in 24/47 (51%) carcinomas. p53 positivity was found significantly more often in SCC than in TCC (p=0.05). Concurrent HPV positivity and abnormal p53 protein accumulation was found in 18 cases, 14 showing the presence of HPV subtypes 16 and/or 18 DNA. The results demonstrate that HPV DNA occurs widely in urinary tract tumours. Unlike in some other carcinomas, there was no inverse relationship between HPV positivity and abnormal p53 protein accumulation in bladder carcinomas. Thus HPV infection may play a role in the pathogenesis of bladder carcinomas by some mechanism other than inactivation of the p53 protein.

Topical negative pressure therapy: Does it accelerate neovascularisation within the dermal regeneration template, Integra? A prospective histological in vivo study

BACKGROUND The use of topical negative pressure (TNP) dressings with dermal regeneration template (DRT), Integra, has improved outcomes and simplified aftercare. Previous clinical studies have suggested accelerated vascularisation; with a reduction in the duration of the 1st stage after the application of Integra, from 2 to 4 weeks to as little as 4 days, but with no histological evidence. However, histological studies, without TNP, have shown that vascularisation occurs between the second and the fourth week. This study set out to examine histologically the rate of DRT neovascularisation when combined with TNP. METHODS Eight patients with nine reconstruction sites were enlisted. Unmeshed Integra and fibrin sealant to promote adherence were used. TNP was applied for the duration between the 1st and the 2nd stages. Patients underwent serial biopsies on days 7, 14, 21 and 28 post-application. The biopsies were stained with H&E and endothelial markers CD31 and CD34. Template vascularisation was assessed as a percentage of the template depth in which patent, canalised vascular channels could be demonstrated. RESULTS The median percentage of the template depth which demonstrated canalised channels was 0%, 20%, 61% and 80% for days, 7, 14, 21 and 28, respectively. CONCLUSION The application of TNP dressings to dermal templates can reduce shearing forces, restrict seroma and haematoma formation, simplify wound care and improve patient tolerance. However, this study could not demonstrate that TNP accelerates neovascularisation as verified by the presence of histologically patent vascular channels.

Proliferating cell nuclear antigen but not p53 or human papillomavirus DNA correlates with advanced clinical stage in renal cell carcinoma

In this study we investigated 56 renal cell carcinomas immunohistochemically for the expression of proliferating cell nuclear antigen (PCNA) and tumour suppressor protein p53. We also analyzed for the presence of human papilloma virus (HPV) DNA subtypes 6, 11, 16, 18, 31 and 33 by in situ hybridization. In carcinomas which showed more than 10% of PCNA positive nuclei there were significantly more cases with invasion (P= 0.032) or metastatic disease (P= 0.047). Nine out of 22 grade III‐IV tumours (40.9%) but only six out of 30 grade I‐II tumours (20%) showed more than 10% of PCNA positive cells (P= 0.097). Patients with 10% or more PCNA positive cells in kidney tumours had more advanced disease at the time of diagnosis than those showing less PCNA positive cells (P= 0.05).

Differential progression of renal scarring and determinants of late renal recovery in sustained dialysis dependent acute kidney injury secondary to myeloma kidney

Background Most cases of dialysis-dependent acute kidney injury due to myeloma cast nephropathy do not recover renal function. Renal biopsy typically shows cast formation, direct tubular injury and interstitial inflammation caused by nephrotoxic monoclonal free light chains (FLC). Established scarring at presentation is rarely severe. There is little data on in situ evolution of renal injury. Aims To conduct a detailed histological study of four patients with cast nephropathy. Methods Cast nephropathy was confirmed by renal biopsy. Treatment consisted of chemotherapy and high cut-off dialysis to maximise extracorporeal removal of FLC and reduce renal toxicity. All four patients remained dialysis dependent at 6 weeks, at which time they underwent a further biopsy. Results Three patients achieved independence from dialysis. Six-week biopsies showed differential changes in chronic damage from no progression, to accelerated progression of scarring from 10% to 42%, despite a rapid and sustained fall in FLC in all patients. In three patients there was a major reduction in intratubular cast numbers; these patients subsequently recovered renal function. In one patient who continued to have high cast formation at 6 weeks there was no subsequent renal recovery. Conclusions Some FLC clones can promote rapid renal scarring. Significant reductions in cast formation on repeat biopsy may identify the potential for late renal recovery. Early diagnosis and treatment may prove crucial in determining renal recovery. Patients who have not recovered renal function after a period of treatment may be usefully reassessed by repeat biopsy for quantitative analysis of chronic damage and cast numbers.