A histological survey of green fluorescent protein expression in 'green' mice: implications for stem cell research.

AIMS: The transgenic enhanced green fluorescent protein (EGFP) expressing 'green' mouse (C57BL/6-TgN(ACTbEGFP)1Osb) is a widely used tool in stem cell research, where the ubiquitous nature of EGFP expression is critical to track the fate of single or small groups of transplanted haematopoietic stem cells (HSC). Our aim was to investigate this assumed ubiquitous expression by performing a detailed histological survey of EGFP expression in these mice. METHODS: Fluorescent microscopy of frozen tissue sections was used to perform a detailed histological survey of the pattern of EGFP expression in these mice. Flow cytometry was also used to determine the expression pattern in blood and bone marrow. RESULTS: Three patterns of EGFP expression were noted. In most tissues there was an apparently stochastic variegation of the transgene, with individual cell types demonstrating highly variable rates of EGFP expression. Certain specific cell types such as pancreatic ductal epithelium, cerebral cortical neurones and glial cells and glomerular mesangial cells consistently lacked EGFP expression, while others, including pancreatic islet cells, expressed EGFP only at extremely low levels, barely distinguishable from background. Lastly, in the colon and stomach the pattern of EGFP expression was suggestive of clonal inactivation. Only cardiac and skeletal muscle showed near ubiquitous expression. CONCLUSIONS: These findings raise questions regarding the 'ubiquitous' expression of EGFP in these transgenic mice and suggest caution in relying overly on EGFP alone as an infallible marker of donor cell origin.

DPC4/Smad4 expression and outcome in pancreatic ductal adenocarcinoma.

PURPOSE: Prognostic indicators in pancreatic cancer (PC) are poorly defined and difficult to quantify preoperatively, hence they may lead to inappropriate patient selection for treatment. We examined the protein expression of key cell-cycle regulatory and cell-signaling molecules that occur at high frequency in PC and assessed their relationship to clinicopathologic parameters, response to operative resection, and outcome. PATIENTS AND METHODS: We identified 348 patients with pancreatic ductal adenocarcinoma and assessed the influence of reported clinicopathologic prognostic factors and the expression of the cell-cycle regulatory genes p21(WAF1/CIP1) (CDKN1A), cyclin D1 (CCND1), p53, and p16(INK4A) (CDKN2) and the cell-signaling molecule DPC4/Smad4 (MADH4) using immunohistochemistry in a subgroup of 129 patients. RESULTS: Independent prognostic factors in resected patients were tumor size greater than 45 mm (P =.0015), involvement of surgical margins (P <.0001), and perineural invasion (P =.014). Loss of DPC4/Smad4 expression cosegregated with resectability (P <.0001) and was associated with improved survival after resection (P <.0001), whereas resection did not improve survival in patients whose tumor expressed DPC4/Smad4 (P =.5). Aberrant expression of p21(WAF1/CIP1), cyclin D1, p53, or p16(INK4A) was not associated with a difference in survival. CONCLUSION: Tumor size (> 45 mm), resection margin involvement, and perineural invasion were independent prognostic factors. Preoperative assessment of DPC4/Smad4 expression has potential as a prognostic indicator in patients with PC since resection did not benefit those patients whose cancers expressed DPC4/Smad4 and accurate assessment of DPC4/Smad4 expression, unlike tumor size, margin status, and perineural invasion, does not require resection.

Target-seeking behavior of plasma glucose with exercise in type 1 diabetes.

OBJECTIVE: To investigate the reproducibility of the plasma glucose (PG) response to exercise in subjects with type 1 diabetes on a nonintensive insulin regimen. RESEARCH DESIGN AND METHODS: Subjects cycled for 45 min at 50% VO(2max) on two occasions (studies 1 and 2) either 1 h after lunch and usual insulin (protocol A) or after overnight fasting without morning insulin (protocol B). Identical diet, activity, and insulin (twice daily neutral and intermediate) were maintained before and during each study day. A total of 13 type 1 diabetic subjects (6 men and 7 women, BMI 24.0 +/- 0.9 kg/m(2) [means +/- SE], age 42.6 +/- 2.7 years, diabetes duration 14.1 +/- 2.8 years) completed protocol A, and 7 (3 men and 4 women, BMI 25.8 +/- 1.3 kg/m(2), age 39.7 +/- 1.3 years, diabetes duration 14 +/- 4.4 years) completed protocol B. RESULTS: In protocol A (fed), the fall in PG during exercise was 4.5 +/- 1.0 and 5.0 +/- 0.8 mmol/l in studies 1 and 2, respectively, whereas in protocol B (fasted), it was 0.6 +/- 0.8 and 3.4 +/- 1.6 mmol/l. Regression analysis of the change in PG in protocol A in study 1 versus study 2 showed poor reproducibility (r(2) = 0.12, P = 0.25) despite uniform conditions. In protocol B, the fall in PG was more reproducible (r(2) = 0.81, P = 0.006). In fed subjects, there was better (P = 0.01) and clinically useful reproducibility of the PG at exercise completion (r(2) = 0.77, P = 0.0001) compared with preexercise. CONCLUSIONS: These results indicate poor reproducibility of the change in PG during exercise after feeding in type 1 diabetes on nonintensive insulin regimens but reasonable reproducibility when fasting. Exercise apparently decreases the glycemic variability after feeding, so that PG concentrations after exercise seek a reproducible "target." Thus, the absolute PG level after a typical bout of exercise in the fed state should be a good guide to carbohydrate or insulin adjustment on subsequent occasions.

Pancreatic intraepithelial neoplasia in association with intraductal papillary mucinous neoplasms of the pancreas: implications for disease progression and recurrence.

The development of pancreatic cancer (PC) several years after curative resection for noninvasive intraductal papillary mucinous neoplasm (IPMN) and the presence of PC distant from IPMN suggest that PC may develop independently of the IPMN. Here, we identified pancreatic intraepithelial neoplasia (PanIN) lesions, the putative precursors of PC, in the ducts of pancreata resected for IPMN and assessed the frequency of molecular aberrations common to PanIN and PC, within these lesions. The protein expression of p53, p21(WAF1/CIP1), cyclin D1, p16(INK4A) and DPC4/Smad4 were examined by immunohistochemistry in 267 PanIN lesions from a cohort of 23 patients with IPMN. Overexpression of p21(WAF1/CIP1) was present in PanIN-1A and -1B lesions and increased in frequency in PanIN-2 and PanIN-3. Overexpression of p53 and cyclin D1, and loss of p16(INK4A) expression were detected in PanIN-2 and PanIN-3 lesions. Loss of DPC4/Smad4 expression occurred only in the PanIN-3 lesions. PanIN lesions that were more dysplastic than the coincident IPMN were identified in 5 of 12 patients, and 2 of these contained a greater number of aberrations in protein expression than the IPMN. PanIN lesions seen in association with IPMN demonstrate molecular and histologic changes identical to PanIN lesions found in association with PC and, in some cases, are more advanced than the associated IPMN. These data suggest that PanIN lesions found in the ducts of a pancreas with IPMN may be relevant to the development of PC either coincident with IPMN or in the remnant pancreas after curative resection of IPMN.

An illustrated consensus on the classification of pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasms.

Invasive pancreatic ductal adenocarcinoma is an almost uniformly fatal disease. Several distinct noninvasive precursor lesions can give rise to invasive adenocarcinoma of the pancreas, and the prevention, detection, and treatment of these noninvasive lesions offers the potential to cure early pancreatic cancers. Noninvasive precursors of invasive ductal adenocarcinoma of the pancreas include pancreatic intraepithelial neoplasias (PanINs), intraductal papillary mucinous neoplasms (IPMNs), and mucinous cystic neoplasms. Diagnostic criteria, including a distinct ovarian-type stroma, and a consistent nomenclature are well established for mucinous cystic neoplasms. By contrast, consistent nomenclatures and diagnostic criteria have been more difficult to establish for PanINs and IPMNs. Because both PanINs and IPMNs consist of intraductal neoplastic proliferations of columnar, mucin-containing cells with a variable degree of papilla formation, the distinction between these two classes of precursor lesions remains problematic. Thus, considerable ambiguities still exist in the classification of noninvasive neoplasms in the pancreatic ducts. A meeting of international experts on precursor lesions of pancreatic cancer was held at The Johns Hopkins Hospital from August 18 to 19, 2003. The purpose of this meeting was to define an international acceptable set of diagnostic criteria for PanINs and IPMNs and to address a number of ambiguities that exist in the previously reported classification systems for these neoplasms. We present a consensus classification of the precursor lesions in the pancreatic ducts, PanINs and IPMNs.

Tumoural calcium pyrophosphate dihydrate crystal deposition disease presenting clinically as a malignant soft tissue mass diagnosed on fine needle aspiration biopsy.

Tumoural calcium pyrophosphate dihydrate crystal deposition is a rare manifestation of calcium pyrophosphate deposition disease (CPPD). We present the case of a 75-year-old male with a previously resected rectal adenocarcinoma who developed a 5-cm right-sided mass at the base of his neck. Clinically and radiologically the lesion was suspicious for malignancy, possibly of metastatic origin. A bedside fine needle aspirate was performed and the smears were mildly cellular showing histiocytes with numerous intracellular and extracellular crystals. These colourless crystals were mostly short and rhomboid shaped and demonstrated weakly positive birefringence. A diagnosis of tumoural CPPD was made. This case is only the second in the English literature diagnosed on fine needle aspiration biopsy. Tumoural CPPD is well known to be a clinical, radiological and occasionally pathological mimic of malignancy. Several cases have been reported where unnecessary radical surgery was performed for this condition. Fine needle aspiration biopsy, as in this case, can provide a rapid and accurate diagnosis of CPPD, avoiding the need for invasive procedures. Polarisation microscopy is a vital adjunct to confirm this diagnosis.

Autopsy findings in a series of five cases of fetomaternal haemorrhages.

AIMS: Fetal blood cells enter the maternal circulation in up to 95% of pregnancies, but usually in minute volumes. Haemodynamically significant fetomaternal haemorrhage (FMH) is a much rarer event reported in approximately 1 in 2800 pregnancies. Most of the literature on this phenomenon emphasises the clinical aspects, and there is no comprehensive description of the autopsy findings. We present a series of five fatal FMH. The aim of this series is to highlight some of the autopsy findings that may prompt consideration of a diagnosis of FMH and lead to appropriate confirmatory testing and counselling of the affected couple. METHODS: The five cases were referred to the Children's Hospital at Westmead for full autopsy. A Kleihauer-Betke test was performed on the mother's blood within one week of delivery in each case. RESULTS: The infants ranged in age from 27 to 40 weeks gestation (mean 36.6 weeks) with a mean birth weight of 2793 g. The estimated volumes of fetal blood lost ranged from 443 to 104 mL (mean loss 243 mL). The estimated percentage of fetal blood volume loss was an average of 107% (i.e., greater than the entire blood volume of the fetus). No other causes of hydrops were identified. Pallor was often noted, and in most cases the autopsies were markedly bloodless with large vessels collapsed. Where the brain:liver ratio could be applied, two fetuses showed a mild increase in ratio, while one infant showed moderate growth restriction with a ratio of 6.2:1 (normal ratio 2.8:1 on non-macerated fetuses over 28 weeks gestation). Placental abnormalities included thrombosis of the umbilical vein and intervillous 'haematomas' in two cases. The most striking microscopic feature was the presence of intravascular nucleated RBC within virtually all organs. Placental intervillous (i.e., within the maternal vascular compartment) nucleated red blood cells were also seen in all cases. CONCLUSIONS: The autopsy findings of FMH can be subtle and easily overlooked unless a high index of suspicion is maintained. The most reliable autopsy features are pallor, subcutaneous oedema or serous effusions, and intravascular nucleated red blood cells (RBC) in organs or more specifically in the placental intervillous space. In all cases of unexplained fetal death a Kleihauer-Betke test should be performed.

Molecular pathogenesis of precursor lesions of pancreatic ductal adenocarcinoma.

Precursor lesions are assuming greater importance in the study of pancreatic ductal adenocarcinoma. As pancreatic cancer is almost universally fatal due to late clinical presentation and biological aggressiveness, characterisation of its precursor lesions may create scope for early diagnosis and improved outcome with conventional therapies as well as the development of novel therapeutic and preventative strategies. Pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous tumours (IPMTs) are thought to be precursor lesions of ductal adenocarcinoma of the pancreas. Recent work has focused on the molecular aberrations associated with these lesions leading to the formulation of a progression model for pancreatic cancer. Progressive histopathological changes along the progression model are associated with aberrations of cell cycle regulatory and growth factor signalling molecules that occur in pancreatic cancer at high frequency and are common to many cancers. Characterisation of these molecular aberrations provides scope for the development of novel diagnostic and treatment strategies that will ultimately impact on the outcome for people who develop pancreatic cancer.