Identification of human neutrophil defensins (HNPs1-3) in biopsies of squamous cell carcinomas of the human tongue

It has previously been reported that the a-defensins, found in the granules of polymorphonuclear leukocytes (neutrophils/ PMNs), are cytolytic for human tumour cells in vitro. Objective: To identify and quantify the a- defensins, HNP-1, HNP-2 and HNP-3 in healthy and tumour tissue from patients with oral squamous cell carcinoma using HPLC, mass spectrometry and amino acid sequencing. Methods: All patients (n=5) were diagnosed with oral squamous cell carcinoma of the tongue.Biopsy tissue from the site of the tumour (n=5) and a non-affected region of the tongue (n=5) was snap frozen and subsequently stored at -70 ºC until analysed. Peptides were extracted from the 10 tissue biopsies using acidified ethanol. Peptide extracts were separated by reverse-phase HPLC . All tumour and control tissue samples were individually analysed under identical conditions with a flow rate of l ml/min, ambient column temperature and absorbance detection at 214 and 280 nm. Fractions (1ml) were collected automatically. HPLC fractions were analysed by MALDI-MS using a linear time-of-flight Voyager DE-mass spectrometer (PerSeptive Biosystems, UK). Using this system the detection limit was 10 fmol. Peptides with molecular masses corresponding to those reported for the a-defensins were deemed of interest and were further subject to complete structural analysis by automated Edman degradation using an Applied Biosystems 491 Procise microsequencer. Results: MALDI-MS revealed a triad of peptides of molecular masses 3442 Da, 3371 Da and 3486 Da in both healthy and tumour tissue. Full length sequence data were obtained for the three a-defensins, unequivocally identifying their presence in both tumour and healthy tissue. Analysis of the MALDI-MS and sequence data indicated that the a-defensins were overexpressed (up to 12 fold) in tumour tissue. Conclusion: This study demonstrates the feasibility of screening tumour tissue for novel peptides/proteins using HPLC and MALDI-MS.The role of a-defensins in oral squamous cell carcinoma of the tongue requires further investigation.

“Unmixing” Tissue Gene Expression Signatures from Tumor Biopsies

Emergent molecular measurement methods, such as DNA microarray, qRTPCR, and many others, offer tremendous promise for the personalized treatment of cancer. These technologies measure the amount of specific proteins, RNA, DNA or other molecular targets from tumor specimens with the goal of “fingerprinting” individual cancers. Tumor specimens are heterogeneous; an individual specimen typically contains unknown amounts of multiple tissues types. Thus, the measured molecular concentrations result from an unknown mixture of tissue types, and must be normalized to account for the composition of the mixture. For example, a breast tumor biopsy may contain normal, dysplastic and cancerous epithelial cells, as well as stromal components (fatty and connective tissue) and blood and lymphatic vessels. Our diagnostic interest focuses solely on the dysplastic and cancerous epithelial cells. The remaining tissue components serve to “contaminate” the signal of interest. The proportion of each of the tissue components changes as a function of patient characteristics (e.g., age), and varies spatially across the tumor region. Because each of the tissue components produces a different molecular signature, and the amount of each tissue type is specimen dependent, we must estimate the tissue composition of the specimen, and adjust the molecular signal for this composition. Using the idea of a chemical mass balance, we consider the total measured concentrations to be a weighted sum of the individual tissue signatures, where weights are determined by the relative amounts of the different tissue types. We develop a compositional source apportionment model to estimate the relative amounts of tissue components in a tumor specimen. We then use these estimates to infer the tissuespecific concentrations of key molecular targets for sub-typing individual tumors. We anticipate these specific measurements will greatly improve our ability to discriminate between different classes of tumors, and allow more precise matching of each patient to the appropriate treatment

Intra-individual variation of serum prostate specific antigen levels in men with benign prostate biopsies

To determine the intra-individual (physiological) variation of prostate-specific antigen (PSA) measurements in men after a benign prostatic biopsy. Sixty-four men were prospectively assessed, all of whom had a benign prostatic biopsy within the preceding 13 months. The degree of intra-individual variability was established by calculating the coefficient of variation on four PSA levels obtained from each patient weekly over a month. Six patients were subsequently diagnosed with prostate cancer and their data are presented separately. In the remaining 58 patients the median (range) individual mean PSA value was 6.3 (0.5-34.1) ng/mL. The median (range) coefficient of variation within the group was 9.5 (2.4-76.1)%. There was a clear linear relationship between mean PSA level and the standard deviation. In 48 of the 63 patients analysed, the coefficient of variation for serum PSA values in the group as a whole was greater than the variation claimed for the assay technique. The significance of the linear relationship between PSA and the standard deviation is discussed, with particular reference to those men who had a benign prostate biopsy.

The quantitation of lipoprotein lipase mRNA in biopsies of human adipose tissue, using the polymerase chain reaction, and the effect of increased consumption of n-3 polyunsaturated fatty acids

Objective: To examine the effects of the consumption of fish oils on the gene expression of lipoprotein lipase (LPL, EC 3.1.1.34) in human adipose tissue. In order to measure LPL mRNA in adipose tissue samples obtained by needle biopsy from human volunteers a competitive, reverse transcriptase PCR (RT-PCR) protocol was developed. Design: A randomised controlled, single blind cross over dietary study which compared the effects of a low level n-3 polyunsaturated fatty acids (PUFA) using normal foods enriched with eicosapentaenoic (EPA) and docosahexaenoic (DHA) (test diet), with non-enriched but otherwise identical foods (control). The diets were consumed for a period of 22 d with a wash out period of 5 months between the diets. Setting: Free-living individuals associated with the University of Surrey. Subjects: Six male subjects with a mean (±sd) age of 51.2±3.6 y were recruited. Major Outcome Measures: Pre-and postprandial blood samples were taken for the measurement of triacylglycerol (TAG), postheparin LPL activity and adipose tissue samples for the measurement of LPL mRNA levels. Results: Mean LPL expression values were 4.12´105 molecules of LPL mRNA per ng total RNA on the control diet and 4.60´105 molecules of LPL mRNA per ng total RNA on the n-3 PUFA enriched (test) diet. There was no significant difference between the levels of LPL expression following each diet, consistent with the lack of change in TAG levels in response to increased dietary n-3 PUFA intake. However, the change in LPL expression (Test-Control diet) correlated significantly with the change in fasting TAG levels (P=0.03, R=-0.87 and R2=0.75) and with the total area under the TAG-time response curve (P=0.003, R=-0.96 and R2=0.92) in individuals. Conclusions: These findings, although based on a small number of subjects, suggest that LPL expression may be a determinant of plasma TAG levels. The development of this methodology should allow further elucidation of the effects of dietary manipulation and disease processes on lipid clearance and regulation in human subjects.

Transcriptomics and proteomics show that selenium affects inflammation, ctoskeleton, and cancer pathways in human rectal biopsies

Epidemiologic studies highlight the potential role of dietary selenium (Se) in colorectal cancer prevention. Our goal was to elucidate whether expression of factors crucial for colorectal homoeostasis is affected by physiologic differences in Se status. Using transcriptomics and proteomics followed by pathway analysis, we identified pathways affected by Se status in rectal biopsies from 22 healthy adults, including 11 controls with optimal status (mean plasma Se = 1.43 μM) and 11 subjects with suboptimal status (mean plasma Se = 0.86 μM). We observed that 254 genes and 26 proteins implicated in cancer (80%), immune function and inflammatory response (40%), cell growth and proliferation (70%), cellular movement, and cell death (50%) were differentially expressed between the 2 groups. Expression of 69 genes, including selenoproteins W1 and K, which are genes involved in cytoskeleton remodelling and transcription factor NFκB signaling, correlated significantly with Se status. Integrating proteomics and transcriptomics datasets revealed reduced inflammatory and immune responses and cytoskeleton remodelling in the suboptimal Se status group. This is the first study combining omics technologies to describe the impact of differences in Se status on colorectal expression patterns, revealing that suboptimal Se status could alter inflammatory signaling and cytoskeleton in human rectal mucosa and so influence cancer risk.

The reliability of muscle biopsies taken from vastus lateralis

The purpose of this study was to examine whether a single biopsy sample of vastus lateralis could provide an accurate estimate of capillary density (CD) which is indicative of the entire muscle, or whether capillary density is distributed unevenly and varies with muscle depth. Whole muscle cross sections of vastus lateralis were excised post mortem (n=11) for analysis of capillary density at three muscle depths, (superficial, mid and deep regions). Muscle thickness varied widely (17–79mm) across subjects. The distribution of CD throughout the depth of the muscle was homogeneous in 8 subjects, but in 3 subjects it was heterogeneous (p<0.05). In 3 of these subjects there was a significant (p<0.05) effect of sample depth on CD. These data indicate that tissue from a single biopsy will not adequately represent the CD of the entire vastus lateralis in some individuals. Single biopsies from unspecified muscle depth, have routinely been used to estimate CD and fibre type in vastus lateralis. The present study indicates that a more reliable method of analysis would be to use the tissue from two needle biopsies taken at the superficial and deep portions of the muscle from a group of at least 10 subjects. Sampling theory analysis supported this conclusion.

A microfluidic system for testing the responses of head and neck squamous cell carcinoma tissue biopsies to treatment with chemotherapy drugs

Tumors are heterogeneous masses of cells characterized pathologically by their size and spread. Their chaotic biology makes treatment of malignancies hard to generalize. We present a robust and reproducible glass microfluidic system, for the maintenance and “interrogation” of head and neck squamous cell carcinoma (HNSCC) tumor biopsies, which enables continuous media perfusion and waste removal, recreating in vivo laminar flow and diffusion-driven conditions. Primary HNSCC or metastatic lymph samples were subsequently treated with 5-fluorouracil and cisplatin, alone and in combination, and were monitored for viability and apoptotic biomarker release ‘off-chip’ over 7 days. The concentration of lactate dehydrogenase was initially high but rapidly dropped to minimally detectable levels in all tumor samples; conversely, effluent concentration of WST-1 (cell proliferation) increased over 7 days: both factors demonstrating cell viability. Addition of cell lysis reagent resulted in increased cell death and reduction in cell proliferation. An apoptotic biomarker, cytochrome c, was analyzed and all the treated samples showed higher levels than the control, with the combination therapy showing the greatest effect. Hematoxylin- and Eosin-stained sections from the biopsy, before and after maintenance, demonstrated the preservation of tissue architecture. This device offers a novel method of studying the tumor environment, and offers a pre-clinical model for creating personalized treatment regimens.

Complications and risk factors in transrectal ultrasound-guided prostate biopsies

CONTEXTO E OBJETIVO: A biópsia da próstata não é um procedimento isento de riscos. Existe preocupação com respeito às complicações e quais seriam os melhores antibióticos usados antes do procedimento. O objetivo foi determinar a taxa de complicações e os possíveis fatores de risco para complicação na biópsia da próstata. TIPO DE ESTUDO E LOCAL: Estudo prospectivo clínico, realizado no Hospital das Clínicas de Botucatu. MÉTODOS: Foram realizadas biópsias em 174 pacientes que apresentavam anormalidade ao exame digital da próstata ou antígeno prostático específico maior que 4 ng/ml ou ambos. Todos os pacientes realizaram enema e antibioticoprofilaxia previamente ao exame. As complicações foram anotadas após o término do procedimento e em consultas posteriores. Algumas condições foram investigadas como possíveis fatores de risco para biópsias de próstata: idade, câncer da próstata, diabetes melito, hipertensão arterial sistêmica, antecedentes de prostatite, uso de ácido acetilsalicílico, volume prostático, número de biópsias e uso de sonda vesical. RESULTADOS: As complicações hemorrágicas foram mais comuns (75,3%) enquanto que as infecciosas ocorreram em 19% dos casos. O tipo mais freqüente foi a hematúria, ocorrendo em 56% dos pacientes. A infecção do trato urinário ocorreu em 16 pacientes (9,2%). Sepse foi observada em três pacientes (1,7%). Não houve óbitos. em 20% dos pacientes não foram observadas complicações após o exame. A presença da sonda vesical foi fator de risco para complicações infecciosas (p < 0,05). O número maior de amostras nas biópsias foi relacionado à hematúria, sangramento retal e complicações infecciosas (p < 0,05). As demais condições investigadas não se relacionaram com complicações pós-biópsia da próstata. CONCLUSÕES: As complicações pós-biópsia da próstata foram em sua maioria autolimitadas. A taxa de complicações graves foi baixa, sendo a biópsia de próstata guiada pelo ultra-som segura e eficaz. A retirada de um maior número de fragmentos na biópsia relaciona-se com hematúria, sangramento retal e complicações infecciosas. A sonda vesical foi um fator de risco para complicações infecciosas.

Esophageal and gastric biopsies in symptomatic children and adolescents. Should both be performed?

This study investigated the relationship among the histological diagnosis of esophagitis and gastritis in children and adolescents with gastroesophageal reflux disease (GERD) and/or dyspepsia. Records of 366 patients submitted to endoscopic biopsies were reviewed. Two groups were analyzed: G1 n=258 with esophageal and gastric biopsies, G2 n=108 with gastric biopsies only. For total subjects median age (range) was 8.5y (2mo-19.9y). Helicobacter pylori infection was detected in 30.6 %, median age 12.5y for H pylori-infected and 5.5y for uninfected children. Histological esophagitis was found in 216/258 (83.7 %) and gastritis in 95/258 (36.8 %) of G1. Both biopsies were normal for 13.6 % cases. Normal gastric biopsies were associated with esophagitis in 128/ 163 (78.5 %) of G1, but gastritis was associated with normal esophageal biopsies in only 7/95 (7.4 %) (0<.001). Histological gastritis was found in 80/108 (74.1 %) of G2 patients. Therefore, for symptomatic children both biopsies are indicated.