Site-specific immunophenotyping of keloid disease demonstrates immune upregulation and the presence of lymphoid aggregates.

BACKGROUND: Keloid disease (KD) is a common fibroproliferative disorder of unknown aetiology. T cells and macrophages are increased in KD and are thought to contribute to its pathogenesis. However, while a link between inflammation and fibrotic disorders is well known for other disorders, it remains undetermined in KD. OBJECTIVES: Systematically to immunophenotype the inflammatory infiltrate of KD in situ in a site-specific manner, and to compare this with normal skin and scar tissue. METHODS: Sixty-eight keloid cases were screened for the presence of all three (intralesional, perilesional and extralesional) keloid-associated specific tissue sites. Subsequently, a complete set of 25 keloid biopsies (from different patients) was compared with normal skin (n = 11) and normal scar (n = 11) samples and subjected to systematic, site-specific quantitative immunohistomorphometry and histochemistry, using a range of immunological markers of B cells, T cells, macrophages, mast cells (MCs) and Langerhans cells. RESULTS: T cells, B cells, degranulated and mature MCs (coexpressing OX40 ligand) and alternative macrophages (M2) were all significantly increased in intralesional and perilesional KD sites compared with normal skin and scar tissue (P < 0·05). Additionally, 10 of 68 KD cases (15%) showed the presence of distinctive lymphoid aggregates, which resembled mucosa-associated lymphoid tissue (MALT). CONCLUSIONS: The increased number and activity of MCs and M2 may implicate inflammation in the fibrotic process in KD. The distinct KD-associated lymphoid aggregate resembles MALT, for which we propose the term 'keloid-associated lymphoid tissue' (KALT). It may perpetuate inflammatory stimuli that promote KD growth. KALT, MCs and M2 are promising novel targets for future KD therapy.

Poly(A) RT-PCR measurement of diagnostic genes in pancreatic juice in pancreatic cancer.

BACKGROUND: The last decade has seen significant progress in understanding the molecular biology of pancreatic adenocarcinoma. There is now an urgent need to translate these molecular techniques to clinical practice in order to improve diagnosis and prediction of response to treatment. The objectives of this study are to utilise poly(A) RT-PCR to measure expression levels of diagnostic Indicator genes, selected from microarray studies, of RNA from intraoperatively sampled pancreatic ductal juice and to correlate these expression levels with those in matched pancreatic tissue resection samples. METHODS: Intraoperative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreaticoduodenectomy for suspected tumour. RNA was isolated and poly(A) PCR and real-time PCR used to measure expression levels of 30 genes. Spearman's rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. RESULTS: Of the 30 Indicator genes measured, just one, ANXA1, showed a significant correlation of expression level between pancreatic juice and tissue samples, whereas three genes, IGFBP3 (P0.035), PSCA (P0.001) and SPINK1 (P0.05), showed significantly different expression between cancerous and benign pancreatic tissue samples. CONCLUSIONS: These results demonstrate that RNA analysis of pancreatic juice is feasible using the poly(A) cDNA technique, that correlation of gene expression exists between pancreatic juice and tissue for very few genes and that gene expression profiling can distinguish between benign and malignant pancreatic tissue. This indicates possible use of the technique for measurement of Indicator genes in pancreatic tissue for diagnosis of pancreatic cancer from very small tissue samples.

Quantum dots light up pathology.

Quantum dots (QDs) are novel nanocrystal fluorophores with extremely high fluorescence efficiency and minimal photobleaching. They also possess a constant excitation wavelength together with sharp and symmetrical tunable emission spectra. These unique optical properties make them near-perfect fluorescent markers and there has recently been rapid development of their use for bioimaging. QDs can be conjugated to a wide range of biological targets, including proteins, antibodies, and nucleic acid probes, rendering them of particular interest to pathology researchers. They have been used in multiplex immunohistochemistry and in situ hybridization, which when combined with multispectral imaging, has enabled quantitative measurement of gene expression in situ. QDs have also been used for live in vivo animal imaging and are now being applied to an ever-increasing range of biological problems. These are detailed in this review, which also acts to outline the important advances that have been made in their range of applications. The relative novelty of QDs can present problems in their practical use and guidelines for their application are given.

Quantitative multiplexed quantum dot immunohistochemistry.

Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.

Gene expression analysis of myeloid and lymphoid lineage markers during mouse haematopoiesis.

Expression profiling of haematopoietic cells is hampered by the heterogeneous nature of haematopoietic tissues and the absolute rarity of early unrestricted progenitors. To overcome this, the expression profile of lymphoid and myeloid-associated genes (LEF1, EBF, CD19, Sox-4, B29, CD45, C-fms, lysozyme, PU.1 and CD5) were investigated in 40 mouse myeloid haematopoietic precursors covering the entire haematopoietic hierarchy from multipotential to committed single lineages. The lineage-specific expression seen in single-cell studies was confirmed by examining fractionated bone marrow, whole tissues and differentiation of the multipotent cell line FDCP (Factor Dependent Cell Paterson) mix. Analysis of the 40 single myeloid precursors failed to detect expression of lymphoid-associated genes, LEF1, EBF, CD19 and CD5, despite detection in lymphoid cell controls. Surprisingly, the lymphoid-associated genes, Sox-4 and B29 were detected in the single myeloid precursors, which was confirmed in bone marrow and a multipotential myeloid cell line. The pattern of Sox-4 and B29, is consistent with a potential role in the commitment of bipotential granulocytic/macrophage precursors towards the granulocyte or macrophage lineage. In addition to providing baseline values for myeloid and lymphoid lineage markers during mouse haematopoiesis, these results highlight the importance of single-cell analysis in the study of complex tissues.

Comparison of gene-expression profiles in parallel bone marrow and peripheral blood samples in acute myeloid leukaemia by real-time polymerase chain reaction.

BACKGROUND: Gene signatures (Indicator genes) in bone marrow that provide more precise prognostication in haematological malignancy have been identified by microarray expression studies. It would be beneficial to measure these diagnostic signatures in peripheral blood. AIMS: To determine the degree of correspondence of gene expression for a set of Indicator genes between bone marrow and peripheral blood in acute myeloid leukaemia (AML). METHODS: Parallel bone marrow aspirate and peripheral blood samples were obtained from 19 patients diagnosed with AML and mononuclear cells isolated from both sample types. mRNA was globally amplified by polyadenylated real-time polymerase chain reaction (polyA RT-PCR); the expression of 15 AML Indicator genes, identified from previous microarray studies, was measured by RT-PCR. All values were normalised to the mean expression of three housekeeping genes (IF2-beta, GAP and RbS9) and were statistically compared using SPSS software. RESULTS: No significant difference in expression between bone marrow and peripheral blood was observed for 10 of the genes (leptin receptor, CD33, adipsin, proteoglycan 1, MB-1, cyclin D3, hSNF2b, proteasome iota, HkrT-1 and E2A), indicating its possible use in monitoring disease activity in peripheral blood samples, whereas c-myb, HOXA9, LYN, cystatin c and LTC4s showed significantly different expression between bone marrow and peripheral blood samples. CONCLUSION: These results indicate a possible use for the method in monitoring AML in peripheral blood by RT-PCR measurement of Indicator genes. In addition, the initial use of polyA PCR facilitates translation to very small clinical samples, including fractionated cell populations, of particular importance for monitoring haematological malignancy.

Chronic herpes simplex virus type-2 infection and HIV viral load.

By December 2003, the estimated adult HIV/AIDS prevalence rate in sub-Saharan Africa was 7.5-8.5%, and rates of herpes simplex virus type-2 (HSV-2) infection among adults aged >30 years ranged from 60% to 82%. However, little is known about the natural history of HIV/HSV-2 co-infection in this population. We evaluated HIV viral load and CD4+ cell counts among persons with and without chronic HSV-2 co-infection in a cross-sectional study of HIV-infected persons not receiving antiretroviral therapy. HSV-2 and HIV co-infection was associated with a 0.3 log copies/mL higher HIV viral load compared with persons without HSV-2 infection (P=0.014). Chronic HSV-2 infection may have a negative effect on the clinical course of persons with HIV.

Colonic epithelial apoptosis during conventional and endoluminal aortic surgery.

BACKGROUND: This study characterized the initial modes of colonic mucosal injury during aneurysm surgery and correlated these with proinflammatory cytokine release into the colonic and systemic circulations. METHODS: Twenty-four patients undergoing conventional open aortic aneurysm repair and ten who had endovascular aneurysm repair (EVAR) were recruited. Mucosal biopsies were taken from the sigmoid colon immediately before and after surgery, for histological examination. Inferior mesenteric vein (IMV) and peripheral blood from patients who had conventional surgery was assayed for interleukin (IL) 1 beta, IL-6 and tumour necrosis factor (TNF) alpha. Only peripheral blood from patients who had EVAR was assayed. RESULTS: Conventional aneurysm repair resulted in a threefold increase in columnar epithelial apoptosis. There was a 26-fold increase in IL-6 in IMV blood within 5 min of reperfusion, with an equivalent rise in peripheral blood after 30 min. A 20-fold rise in peripheral blood TNF-alpha was observed after surgery. Splanchnic IL-6 correlated positively with cross-clamp time and increased apoptosis. No histological changes were seen after EVAR. There were no intraoperative cytokine changes during EVAR, although a postoperative increase in IL-6 and TNF-alpha was observed. CONCLUSION: The lack of columnar epithelial apoptosis following EVAR reflects the relatively minor ischaemic injury incurred during this procedure.

Adipocytic proportion of bone marrow is inversely related to bone formation in osteoporosis.

AIMS: Preliminary studies have suggested that there is an increase in adipocytic tissue in osteoporotic (OP) bone, supporting in vitro evidence for a switch in differentiation of stromal cells from the osteoblastic to the adipocytic lineage. To investigate this the variation of the ratio of adipose tissue to haemopoietic/stromal tissue in OP bone was measured. METHODS: The ratio of adipocytic to haemopoietic/stromal tissue (A/H) was measured by semi-automated image analysis in iliac crest biopsies from 127 patients with osteoporosis (84 female patients, 48 male patients; mean age, 55 years; range, 5-80). Fourteen patients with normal histomorphometric data (nine women; five men; mean age, 48 years; range 21-70) acted as controls. RESULTS: The ratio of A/H was higher in OP bone than in the normal controls (OP mean 43.06% v normal mean 22.4%; p < 0.001). Multiple regression analysis showed that 98.5% of the variability in the A/H ratio was the result of age and several measures of bone formation, including cancellous wall thickness, osteoid volume, cancellous thickness, cortical wall thickness, cancellous apposition rate, and bone formation rate, together with cancellous separation (each significant at p < 0.001). Those with the greatest effect on the A/H ratio (in decreasing order) were cancellous apposition rate, osteoid volume, and age. CONCLUSIONS: Cancellous apposition rate, osteoid volume, and age were associated with the increase in the proportion of adipose tissue present in OP bone. Of these, cancellous apposition rate reflects osteoblast activity, indicating that the increase in the volume of adipose tissue in osteoporosis is associated with reduced bone formation, supporting the postulated switch in differentiation of stromal cells from the osteoblastic to the adipocytic pathway in osteoporosis.

Intra-tumoural microvessel density in human solid tumours.

Over the last decade assessment of angiogenesis has emerged as a potentially useful biological prognostic and predictive factor in human solid tumours. With the development of highly specific endothelial markers that can be assessed in histological archival specimens, several quantitative studies have been performed in various solid tumours. The majority of published studies have shown a positive correlation between intra-tumoural microvessel density, a measure of tumour angiogenesis, and prognosis in solid tumours. A minority of studies have not demonstrated an association and this may be attributed to significant differences in the methodologies employed for sample selection, immunostaining techniques, vessel counting and statistical analysis, although a number of biological differences may account for the discrepancy. In this review we evaluate the quantification of angiogenesis by immunohistochemistry, the relationship between tumour vascularity and metastasis, and the clinicopathological studies correlating intra-tumoral microvessel density with prognosis and response to anti-cancer therapy. In view of the extensive nature of this retrospective body of data, comparative studies are needed to identify the optimum technique and endothelial antigens (activated or pan-endothelial antigens) but subsequently prospective studies that allocate treatment on the basis of microvessel density are required.

A study of four cases of extra-orbital giant cell angiofibroma with documentation of some unusual features.

AIMS: To document the clinical, light microscopic, immunohistochemical and ultrastructural features of four cases of extra-orbital giant cell angiofibromas. METHODS AND RESULTS: Sections of formalin-fixed paraffin-embedded specimens were studied by haematoxylin and eosin, reticulin and immunohistochemical stains. Electron microscopy was carried out in two cases on tissue fixed in formalin. The age of the patients ranged from 30 to 41 years. Two patients presented with a soft tissue swelling in the left groin, one patient had a left axillary soft tissue lump and one patient presented with a parotid lump. All lesions were well circumscribed and contained variably cellular and vascularized tissue composed of round to spindle cells with a patternless arrangement, scattered multinucleate giant cells and pseudovascular spaces conforming to the description of giant cell angiofibroma. Mononuclear and multinucleate tumour cells were both positive for vimentin and CD34; one tumour exhibited focal S100 protein and GFAP positivity. Both of the tumours examined by electron microscopy showed fibroblastic features, but in addition one contained cells having Schwannian features. All four patients were well without recurrent disease on follow-up (average 25 months). CONCLUSION: Giant cell angiofibroma shares many features with solitary fibrous tumour and giant cell fibroblastoma and shows a wider distribution than initially recognized. Rarely, Schwannian differentiation may be observed in these tumours.

Dual colour FISH in paraffin wax embedded bone trephines for identification of numerical and structural chromosomal abnormalities in acute myeloid leukaemia and myelodysplasia.

AIMS/BACKGROUND: The advent of new treatments for haematological malignancies has led to the need for a correlation between cytogenetic and morphological abnormalities. This study aimed to achieve this by the application of interphase cytogenetics to marrow trephine sections, a technique not previously reported for formalin fixed, paraffin wax embedded trephine biopsies. METHODS: Dual colour fluorescence in situ hybridisation (FISH) was used to detect numerical and structural abnormalities in routinely processed paraffin wax embedded trephine biopsies. Three cases with t(8;21) and three with t(15;17) were analysed, together with a case of trisomy 8. Chromosome specific probes were hybridised with sections and disclosed by fluorescein isothiocyanate and rhodamine/Texas red labelled antidigoxigenin and antibiotin amplification; translocations were identified by colocalisation of probes using a double wavelength bypass filter. RESULTS: A translocation signal was present in 12% and 11.5% of the cells counted in the t(8;21) and t(15;17) cases, respectively, but in none of the normal controls (p < 0.001). In the case of trisomy 8, 9% of the cells counted contained three hybridisation signals for chromosome 8, whereas no cell contained more than two in the normal control (p < 0.001). CONCLUSIONS: This technique is useful for archived routinely processed material, enabling it to be used as a research tool but also, and perhaps more importantly, in clinical practice.

Can positron emission tomography improve the quality of care for head-and-neck cancer patients?

PURPOSE: Fluoro-2-deoxy-d-glucose-positron emission tomography (FDG-PET) is a functional imaging modality that measures the relative uptake of 18FDG with PET. The purpose of this review is to assess the potential contribution of FDG-PET scans to the treatment of head-and-neck cancer patients. METHODS AND MATERIALS: Data were assessed from the literature with attention to what additional information may be gained from the use of FDG-PET in four clinical settings: (1) detection of occult metastatic disease in the neck, (2) detection of occult primaries in patients with neck metastases, (3) detection of synchronous primaries or metastatic disease in the chest, and (4) detection of residual/recurrent locoregional disease. RESULTS: Although the data are somewhat conflicting, FDG-PET appears to add little additional value to the physical examination and conventional imaging studies (supplemented by biopsy when appropriate) for the detection of subclinical nodal metastases, unknown primaries, or disease in the chest. However, FDG-PET scans are quite useful in differentiating residual/recurrent disease from treatment-induced normal tissue changes. A positive FDG-PET scan at 1 month after radiotherapy is highly indicative of the presence of residual disease, and a negative scan at 4 months after treatment is highly predictive of tumor eradication. CONCLUSIONS: Large-scale studies using newer generation equipment and more defined methods are needed to more rigorously assess the potential of FDG-PET in the detection of subclinical primary or simultaneous secondary tumors and of nodal or systemic spread. Currently, however, FDG-PET can contribute to the detection of residual/early recurrent tumors, leading to the timely institution of salvage therapy or the prevention of unnecessary biopsies of irradiated tissues, which may aggravate injury.

Tomato Ve disease resistance genes encode cell surface-like receptors.

In tomato, Ve is implicated in race-specific resistance to infection by Verticillium species causing crop disease. Characterization of the Ve locus involved positional cloning and isolation of two closely linked inverted genes. Expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing 28 or 35 potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXLphi or YXXphi endocytosis signals (phi is an amino acid with a hydrophobic side chain). A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. These structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences.

Disappearance of well-differentiated carcinoma of the prostate: effect of transurethral resection of the prostate, prostate-specific antigen, and prostate biopsy.

OBJECTIVES: To characterize the effect of prostate-specific antigen (PSA) and transurethral resection of the prostate (TURP) on the rate of diagnosis of well-differentiated (WD) prostate cancer (PCa) and PCa mortality. METHODS: All cases of PCa and rates of TURP at both Wilford Hall and Brooke Army Medical Centers between 1984 and 1995 were reviewed. Tumor grade was compared between prostate needle biopsy and TURP. The pattern of diagnosis was analyzed annually and for two time periods: pre-PSA (1984 to 1988) and post-PSA (1989 to 1995). RESULTS: The number of WD tumors fell by 50% over the period of study and was caused by a fall in number of TURPs as well as in WD tumors detected by TURP. PSA for early detection of PCa began in 1988, and within 5 years a more than 50% fall in the rate of metastatic disease was witnessed. These two events (PSA screening and fall in TURPs) led to an increase from 57% to 92% of tumors that were both clinically significant and potentially curable. CONCLUSIONS: These data help explain the fall in the rate of diagnosis of WD PCa. The resultant increase in the diagnosis of moderately and poorly differentiated PCa, coupled with the dramatic fall in the rate of diagnosis of metastatic PCa, may explain the reports of a fall in PCa mortality. If this observation is replicated in other populations, it may provide further impetus for a stronger recommendation for early detection with PSA and digital rectal examination.

Osteoporosis in men: a cellular endocrine perspective of an increasingly common clinical problem.

Although it has been accepted that osteoporosis is common in women, only recently have we become aware that it is also widespread in men; one in twelve men in the UK have osteoporosis. In many cases, there are recognisable causes for their osteoporosis, but a significant proportion (approximately one third) of these men have idiopathic disease. A major problem is that these cases are difficult to treat. An important therapeutic strategy would be to identify men at risk from osteoporosis sufficiently early, so that they can begin preventative measures. Moreover, development of novel means of treating these men would be an important clinical advance. With the emphasis on osteoporosis in women, however, the cellular and molecular basis for male idiopathic osteoporosis (MIO) is still poorly understood. Nevertheless, there are some aspects of skeletal regulation which may be specific for men and which could form the basis for addressing these problems. Thus, the importance of oestrogen in maintaining the adult skeleton in men as well as women implies that bone cells in men can respond to low levels of the hormone. Both oestrogen receptor (ER) alpha and beta are expressed in bone in vivo, which may be important for oestrogen action on bone in men. Furthermore, in osteoporosis generally, there is increasing evidence for defective osteoblast differentiation such that there is a surfeit of adipocytes over osteoblasts. A low peak bone mass is a powerful risk factor for osteoporosis in later life; bone formation and, by implication, osteoblast differentiation, is key to the mechanism by which it is accrued. GH and IGFs are important for regulating osteoblast differentiation. Evidence now suggests that they are associated with bone mineral density, particularly in men. The genes for ERs, GH and IGF-I might be useful candidates with which we can begin to detect men at risk from osteoporosis. Furthermore, the mechanisms by which oestrogen, GH and IGF-I regulate the male skeleton could provide the basis for developing novel means of treating MIO.

A method for classification of HIV exposure category for women without HIV risk information.

An increasing number of cases of human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) among women is reported to state and territorial health departments without exposure risk information (i.e., no documented exposure to HIV through any of the recognized routes of HIV transmission). Because surveillance data are used to plan prevention and other services for HIV-infected persons, developing methods to accurately estimate exposure risk for HIV and AIDS cases initially reported without risk information and assisting states to analyze and interpret trends in the HIV epidemic by exposure risk category is important. In this report, a classification model using discriminant function analysis is described. The purpose of the classification model is to develop a proportionate distribution of exposure risk category for cases among women reported without risk information. The distribution was estimated based on behavioral and demographic data obtained from interviews with HIV-infected women; the interviews were conducted in 12 states during 1993-1996. Variables used in the analysis were alcohol abuse, noninjection-drug use, and crack use; year of HIV/AIDS diagnosis; age; employment; and region. As a result of the classification procedure, nearly all cases among women with no reported risk were classified into an exposure risk category: 81%, heterosexual contact; and 16%, injection-drug use. These proportions are higher than the current redistribution fractions (calculated from risk reclassification patterns and weighted by demographic characteristics) and reflect the increasing proportion of cases among women attributable to heterosexual contact with an infected partner. This report provides one method that could be applied to HIV surveillance data at the national level to estimate the proportion of cases in exposure risk categories. However, because the study in this report is limited in sample size and geographic representativeness, other models are also needed for adjusting risk exposure data at the national, state, and local levels.

Preoperative assessment for and outcomes of mandibular conservation surgery.

INTRODUCTION: The role of marginal mandibulectomy and other conservative resective procedures for patients with early cortical mandibular invasion from squamous carcinoma of the oral cavity remains poorly defined. The purpose of this retrospective study was to evaluate the efficacy of preoperative assessment for bone invasion and the outcomes of different mandibular resective procedures that preserve mandibular continuity. METHODS: The charts of 222 patients treated at the University of Texas M. D. Anderson Cancer Center between 1960 and 1990 were reviewed. All patients had a biopsy-confirmed diagnosis of squamous carcinoma involving either the lower gingiva, floor of mouth, oral tongue, or retromolar trigone. All patients had a surgical resection that involved removing less than a segment of the mandible. Patient data were analyzed to determine the usefulness of preoperative assessment and outcomes of therapy. RESULTS: Clinical evaluation of mandibular bone invasion was more sensitive than radiologic evaluation, whereas radiologic assessment was more specific and had a higher reliability index. The overall local and regional recurrence and distant metastasis rates for all T stages were 14.4%, 18.0%, and 2.7%, respectively. Sixty-nine point eight percent of all patients were without evidence of disease 2 years after treatment. CONCLUSIONS: Mandibular conservation surgery is oncologically safe for patients with early mandibular invasion. Accurate preoperative assessment that combines clinical examination and radiographic evaluation is better than either modality alone, but clinical judgment is still necessary for proper patient selection.