Methods for detecting apoptosis in thyroid diseases.

Over the last few years, the importance of apoptosis in determining the fate of thyrocytes in autoimmune thyroid disease has been the topic of intense investigation. It is now clear that thyrocytes from patients with Hashimoto's thyroiditis are destroyed as a result of an apoptotic process. However, there is no general consensus on whether the intrathyroidal lymphocytes or the thyrocytes themselves are responsible for their death. The use of a wide range of techniques has contributed to the assessment of this process both in situ on thyroid sections and in vitro on thyroid cell preparations. The apoptosis field of research is rapidly evolving and as the pathways to cell death become unravelled, novel methods will emerge. As each technique offers some advantage, it is critical to know the most suitable method for a specific study. Equally, each method also has intrinsic limitations. Thus, to achieve reliable results, it is necessary to use more than one technique per study. In addition, techniques related to the measurement of the expression of pro-apoptotic and anti-apoptotic genes have been contributing to the study of the susceptibility of the cells to apoptosis and/or to their ability to kill themselves or neighbouring cells. In this review we will focus on the most relevant techniques.

Secondary neoplasms of the breast: a survey of the 20th Century.

BACKGROUND: A 90-year archive of surgical and postmortem material was reviewed to establish the incidence, presentation, and pathology of tumors secondary to the breast. METHODS: A search was performed on all cases contained within the files of the Pathology Department of the Royal London Hospital from 1907 to 1999. RESULTS: Sixty patients were identified with unequivocal, secondary, nonmammary neoplasms involving the breast. Hematologic tumors predominated, particularly among the more recent cases, with carcinoma less frequent overall but more numerous in the earlier part of the 20th century. There were several surprising and sometimes unique findings, with occasional metastases from primary tumors of the esophagus, retina, pancreas, thyroid, and skin. Other primary tumor sites included the stomach, lungs, and kidney. In line with expectations, the majority of tumors occurred in women. CONCLUSIONS: Secondary tumors to the breast are rare. In the current series, these tumors comprised 3% of the breast tumors in the files under review. The majority of these were metastases from the contralateral breast. However, the existence of metastatic nonmammary tumors to the breast should be appreciated so that secondary tumors from unusual sites are not overlooked, particularly with the widespread use of fine-needle aspiration cytology and needle core biopsies for preoperative diagnosis. In fact, 0.43% of the breast malignancies in the current report originated from sites outside the breast. Of these one-third represented spread from an occult primary. Furthermore, the current data suggest a move from carcinomatous metastases to hematologic malignancies over the last century, possibly reflecting earlier diagnosis of the former and an increase in the prevalence of the latter.

Thyrocyte targets and effectors of autoimmunity: a role for death receptors?

In Hashimoto's thyroiditis, thyrocytes die by apoptosis. Whether this is the result of impaired antiapoptotic gene expression or hyperexpression of proapoptotic signals or other mechanisms is not fully established. Following the suggestion that thyrocytes from Hashimoto's glands die by a fratricidal killing mediated by Fas/Fas ligand, we have investigated whether thyroid cells from different clinical conditions are able to kill Fas-expressing target cells. We have studied whether this effector ability was mediated by Fas/Fas ligand, perforin or other death receptors/ligands, i.e., tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/tumor necrosis factor-related apoptosis-inducing ligand receptor (TRAIL-R). We have confirmed that thyroid preparations can kill Fas-expressing HUT78 targets through apoptosis. Cell death was only partially dependent on Fas/Fas ligand but it was trypsin-sensitive. Blocking perforin did not affect Fas-expressing target killing while caspase inhibitors had a consistent although limited effect. Thyroid cells were not sensitive to TRAIL/TRAIL-R. We have also found that both thyrocytes and lymphocytes from Graves' disease thyroids were effective at killing autologous and heterologous Fas-expressing targets. Conversely, killing of these targets could be shown only with lymphocytes (but not with thyrocytes) from Hashimoto's glands. In Hashimoto's thyroiditis, thyrocytes were poorly functional while lymphocytes were able to operate as effectors. It is envisaged that thyrocyte death in Hashimoto's would result from autologous thyrocyte killing perpetrated by lymphocytes. Death receptors/ligands would appear to play a role. However, a caspase-independent mechanism may also coexist and contribute to cell death in Hashimoto's thyroiditis.

Death of the autoimmune thyrocyte: is it pushed or does it jump?

Programmed cell death or apoptosis is central both in physiology during development and in disease. The mechanism of apoptosis is under the control of antiapoptotic survival genes of the Bcl-2 family and proapoptotic death receptors of the TNF superfamily (Fas, TNFR, TRAILR). Following death signal, the death receptor binds to its own receptor and initiates, through binding of adaptors, a cascade of events mediated by the autoproteolytic activation of specific enzymes called caspases. This enzyme activation is ultimately responsible for the dissembly of basic nuclear and cytoplasmic cell structures leading to cell death. In certain cell systems, antiapoptotic genes of the Bcl-2 family prevent the proapoptotic pathway. One of their roles is to maintain mitochondrial function integrity. In autoimmune destructive thyroiditis high levels of apoptosis have been demonstrated particularly within the destructed follicles near the infiltrated areas in comparison to Graves' disease and non autoimmune glands. In Hashimoto's thyroiditis Fas expression has been found increased on thyrocytes and in vitro can be modulated by proinflammatory cytokines. FasL expression on thyrocytes remains controversial. Thyroid cells from Graves' disease and multinodular glands are known to kill Fas expressing target cells although Hashimoto's thyrocytes are not efficient effector cells. Intrathyroidal lymphocytes from Hashimoto's thyroids maintain functional killer activity. These findings would suggest that intrathyroidal lymphocytes could be responsible for thyrocyte death in vivo. Whether this mechanism is Fas/FasL, TRAIL/TRAILR dependent can not be confirmed as specific blocking reagents were not able to inhibit cell induced death. In Hashimoto's thyroiditis an impairment of Bcl-2 and Bcl-X anitapoptotic genes on thyrocytes has also been detected. Bcl-X expression can be down-regulated in vitro by incubation with cytokines. These findings suggest that thyrocyte death may not exclusively be the result of specific interactions between death receptor and their ligands but it may involve simultaneous impairment of protective genes of the Bcl-2 family. Whether the impairment of the Bcl-2 family is a direct consequence of environmental stimuli or is the result of an intrinsic thyrocyte (mitochondrial?) alteration is as yet not known.

Future of thyroid surgery and training surgeons to meet the expectations of 2000 and beyond.

What is the future of thyroid surgery in the new millennium? How can surgeons keep abreast of advances in thyroid endocrinology, genetics, surgical therapy, and other aspects of thyroid disease management? How should surgeons be trained to become highly competent in thyroid disease and to perform safe, effective thyroid operative procedures? Nine internationally recognized endocrine surgeons were asked to express their views on these and related subjects. They noted that advances in molecular biology, pathology, and genetics of thyroid disease should allow more tailored surgical approaches during the twenty-first century. Current training of general surgical residents in thyroid and other types of endocrine surgery is highly variable, which may contribute to increased complication rates and number of second operations. The leadership for addressing these deficiencies and promoting a more organized approach to thyroid disease management should come from national endocrine surgery associations and their leaders. It is incumbent upon endocrine surgeons to maintain their central role in the management of many aspects of thyroid disease. Organizing teams of specialists into thyroid centers (centers of excellence) can (1) increase efficiency; (2) increase quality of care; (3) decrease costs; (4) encourage a more individualized approach to surgery; (5) lower complication rates; and (6) foster innovation in technology and disease management. Two years of additional fellowship training in thyroid and endocrine surgery is now being advocated by increasing numbers of national endocrine surgical associations as the best way to prepare surgeons for society's needs for highly skilled, competent thyroid surgeons of the future.

Integrin beta1 upregulation in MCF-7 breast cancer cells by angiotensin II.

AIMS: Integrins are a major family of cell adhesion molecules whose function is perturbed in tumour invasion and metastasis. Angiotensin II (A II) is well-known in the systemic control of water and electrolyte homeostasis and haemodynamics, but recent evidence points to an additional local renin-angiotensin system (RAS) with possible long-term trophic effects including carcinogenesis. METHODS: The effect of angiotensin II on MCF-7 human breast cancer cell line integrin expression was evaluated with immunocytochemistry (ICC) and immunoprecipitation (IP). RESULTS: The experiments demonstrated a 1.40 +/- 0.14-fold increase in beta, integrin expression on MCF-7 cells following treatment with A II. CONCLUSIONS: These findings report the first evidence of an association between integrins and the RAS in human breast cancer cells and suggest a novel research avenue for future anti-metastatic strategies, through the manipulation of cell adhesion mechanics, in the management of invasive human breast cancer.

The magnetization transfer characteristics of human breast tissues: an in vitro NMR study.

A series of freshly excised human breast tissues was analysed using a nuclear magnetic resonance spectrometer and then subjected to routine histopathology examination. Tissues comprised normal parenchymal, adipose, fibrocystic, fibroadenoma and malignant types. An inversion-recovery sequence performed both with and without magnetization transfer allowed T1, T1s, M0 and Ms values to be obtained. From this information, the magnetization transfer rate constant, K, was calculated for each tissue sample. These data show that T1s provided greater discrimination between neoplasic and normal tissues than did T1. However, neither T1s nor K values provided a means of discriminating between benign and malignant disease.

A potential diagnostic application of magnetization transfer contrast: an in vitro NMR study of excised human thyroid tissues.

A series of freshly excised thyroid tissues was analysed using a nuclear magnetic resonance spectrometer and then subjected to routine histo-pathology examination. Whilst simple T1 values for normal tissue and goitre are not significantly different, the degree of intra-subject T1 and T1s variability is shown to be an indicator of benign thyroid disease. Using data collected from an inversion-recovery sequence performed with and without magnetization transfer, a magnetization transfer rate constant was calculated for each tissue sample. These data suggest that this parameter may provide in vivo discrimination between follicular cancer and follicular adenoma.

Transcription of the prorenin gene in normal and diseased breast.

The angiotensin II type 1 (AT1) receptor is present in a wide variety of human and animal tissues, and is particularly abundant in epithelial cells. Because of this, and because it is known that tissue renin angiotensin systems (RASs) exist that have specific local functions, we investigated the expression and localisation of components of the RAS in normal and diseased breast tissue. Using a monoclonal antibody to the AT1 receptor, immunocytochemistry confirmed that the AT1 receptor was characteristically distributed in ductal epithelial cells in both normal and malignant tissue, and in most, although not all, cells in invasive tumours. Transcription of prorenin mRNA was studied by in situ hybridisation, using a DIG-ddUTP tail-labelled probe specific for the human prorenin gene. In normal tissue, and in cases of ductal carcinoma in situ, prorenin mRNA was distributed in myoepithelial cells and in a band of connective tissue cells completely surrounding the AT1-containing ductal epithelial cells. This prorenin transcribing tissue was disrupted and attenuated in invasive tumours, and in some of these, prorenin mRNA transcription could not be detected at all. Functions ascribed to the tissue RASs include regulation of mitosis and tissue modelling, as well as fluid and electrolyte transport. The results presented here strongly suggest the possibility that a tissue RAS may also be present in the breast, closely coupled to the provision of angiotensin II to the AT1 receptors in ductal epithelial cells. This mechanism is disrupted in cancer.

Short-term restorative nutrition in malnourished patients: pro's and con's of intravenous and enteral alimentation using compositionally matched nutrients.

In a prospective controlled clinical study 30 patients with moderate degree of malnutrition, normal liver and kidneys, and a functioning gastrointestinal tract were randomized to receive a free amino acid and small peptide enteral diet (15 patients) or an isonitrogenous isocaloric parenteral support for at least 10 days (total energy: 2900 kcal, nitrogen: 14.5 g, carbohydrates: 380 g, fat: 112 g, N/non protein calories: 1/175). The parenteral and enteral diets had the same protein/lipid/carbohydrate composition. The data indicated that both routes led to positive nitrogen balance. Nitrogen equilibrium was achieved by day 3 in the TPN group and by day 5 in the enteral group. There were no significant changes in serum albumin within either group. Serum level of transferrin reached a significant increase in both groups (p = 0.003). Thyroxine-binding prealbumin rose significantly in both groups as well (p = 0.019 and 0.004 respectively). Statistically significant rises in lymphocyte counts (p = 0.003 and 0.001 respectively), in levels of C3 (p = 0.009 and 0.001 respectively), IgA (p = 0.002), IgG (p = 0.004 and 0.003 respectively) and IgM (p = 0.004) occurred in either treatment group. There was a high incidence of negative skin tests at the start of the study in the enteral group (73.3%) and the TPN group (60%). By the end of the study the incidence of negative results for this test was 40.0% and 26.6% respectively. Despite maintenance of similar glucose levels in both groups, TPN led to significantly (p = 0.000) higher serum insulin levels. The serum insulin increased almost linearly over the study period, and eventually prevented fat mobilization and lipolysis, so that free fatty acid levels had fallen significantly (p = 0.000). A significant elevation of the liver enzymes over the study period occurred in the TPN group, but not in the enterally fed patients. The present findings provide no evidence that semi-elemental diets are in any way inferior to isonitrogenous isocaloric regimes parenterally given for a short period of time.

Analysis of apoptosis in relation to tissue destruction associated with Hashimoto's autoimmune thyroiditis.

The level of apoptosis has been investigated in thyroid tissue from eight patients with Graves's disease, one with Hashitoxicosis, three with Hashimoto's thyroiditis, and five patients with multinodular goitre, using flow cytometry and an in situ immunofluorescence technique. Cryostat sections have also been studied for Bcl-2 and APO-1/Fas expression in the thyrocytes and infiltrating lymphocytes, to determine their susceptibility to apoptosis. An increased level of apoptosis was detected in Hashimoto's glands. This was associated with decreased Bcl-2 staining and a patchy APO-1/Fas reactivity on thyrocytes. In addition, APO-1/Fas expression was noted within the germinal centres of lymphoid follicles. It is suggested that the dysregulation of apoptosis-related genes could be an important factor in the progression of destructive thyroid autoimmune disease.

Angiotensin II type 1 receptor expression in human breast tissues.

We demonstrate the expression of angiotensin II type 1 (AT1) receptors in normal and diseased human breast tissues. Using monoclonal antibody 6313/G2, directed against a specific sequence in the extracellular domain of the AT1 receptor, immunocytochemical analysis revealed positive immunoreactivity in membrane and cytoplasm of specific cell types. Immunoblotting of solubilized proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) from benign and malignant tumours identified a single immunoreactive species with a molecular mass of approximately 60 kDa, consistent with that of the mature glycosylated receptor. In studies of [125I]angiotensin II binding using breast membrane preparations, concentrations of specific angiotensin II binding sites were found to range from 1.8 to 100 fmol mg(-1) protein, with a K(d) of approximately 60 nM. Most of the specifically bound [125I]angiotensin II was displaced by losartan, a specific angiotensin II type 1 receptor antagonist, while less was displaced by the AT2 receptor type antagonist, CGP42112A, thus confirming the prevalence of AT1 receptors in this tissue type. These data suggest that the renin-angiotensin system may be involved in normal and abnormal breast tissue function.

Beta-2 microglobulinuria and long-term outcome following curative surgery for primary hyperparathyroidism.

Twentyseven patients admitted for surgery for primary hyperparathyroidism were studied preoperatively. Fifteen were normotensive and 12 were either hypertensive, diastolic blood pressure > 95 mmHg or had a raised serum creatinine measuring 109.7 (55-171) mumol/l. The beta 2 microglobulin (beta 2M) urinary excretion was measured on all patients. The beta 2M levels were raised in all patients preoperatively even in those patients who had normal values for conventional renal function tests. Following curative surgery the values are returned to the normal range. The patients were followed up for a mean of 4.2 (2.8-5.6) years. Six patients who were initially normotensive subsequently developed hypertension and the initial beta 2M ratio was significantly higher 386 (122-680) micrograms/l compared to those who remained normotensive 186 (95-340) micrograms/l (p < 0.05). Even higher preoperative beta 2M excretion was found in the initial hypertensive group 505 (87-1160) micrograms/l compared to those who later developed hypertension 386 (122-680) micrograms/l, p < 0.001. This preliminary study suggests that preoperative beta 2M urinary excretion may be of value in identifying those patients who will subsequently develop hypertension, an identified long term cause of death in patients operated on for primary hyperparathyroidism. Further studies are indicated.

Sequential systemic platelet-activating factor and interleukin 8 primes neutrophils in patients with trauma at risk of multiple organ failure.

Plasma from 33 patients at risk of multiple organ failure (MOF) after major trauma was tested for a priming effect on neutrophils, and for the presence of platelet-activating factor (PAF) activity and interleukin (IL) 8. Plasma sampled at 3, 6, 12 and 24 h after injury significantly primed normal neutrophils to release mean(s.e.m.) 1.26(0.19), 1.33(0.26), 1.04(0.14) and 0.86(0.13) nmol superoxide per min per 1.3 x 10(6) neutrophils respectively (P < 0.05). Priming at 3 h after injury was inhibited by mean(s.e.m.) 63.8(7.0) per cent by the PAF antagonist, WEB 2170 (P < 0.01). Mean(s.e.m.) plasma IL-8 was raised at 6 and 12 h after injury to 785(183) and 836(175) pg/ml (P < 0.01). At 12 h after injury the plasma IL-8 level correlated directly with the number of units of red blood cells transfused (r = 0.64, P < 0.01), and was significantly higher in the group of six patients who developed MOF (P < 0.05). These data suggest that after trauma the mediators PAF and IL-8 appear sequentially in the circulation, are potential mechanisms of circulating neutrophil priming, and that IL-8 may also be an early biochemical marker predicting the onset of MOF.

The use of thallium-201 and technetium-99m subtraction scintigraphy for the localisation of parathyroid adenomas.

Parathyroid adenomas account for approximately 85% of cases of primary hyperparathyroidism. Several preoperative localisation techniques exist to aid the surgeon during neck exploration, with varying degrees of success. We report on the results of a modification of the established technique of thallium and technetium subtraction scintigraphy. The operative findings of 60 patients undergoing neck exploration for parathyroid adenoma were correlated with preoperative thallium and technetium subtraction scintigraphy scans. The radio-isotopes were administered in the reverse order to conventional administration, resulting in enhanced imaging. The adenomatous glands were correctly localised in all cases. The 100% sensitivity of this modified scanning technique supports a strategy of unilateral scan-directed neck exploration.

Cardiac output in asymptomatic primary hyperparathyroidism: a stigma of early cardiovascular dysfunction?

The resting cardiac output pre- and postoperatively in 10 patients with asymptomatic primary hyperparathyroidism has been studied. All patients had normal renal function and arterial blood pressure without a previous history of cardiovascular disease. Ten normotensive patients with a non-toxic goitre awaiting thyroidectomy were studied as controls. The mean cardiac output of the hyperparathyroid patients was 7.2 l/minute (range 5.3-8.9) and of the control group 5.8 l/minute (range 5.2-6.3). Following a successful parathyroidectomy with return of the serum calcium to normal, the mean cardiac output was 6.3 l/minute (range 4.9-7.8) (p < 0.04). In 8 of the 10 patients there was a fall in the cardiac output following surgery; in 2 there was an increase. These results suggest that hyperparathyroid patients often have an elevated cardiac output which may fall following a successful parathyroidectomy.

Correlation between albuminuria and positively charged amino acids in gastrointestinal cancer.

This study was designed to examine a possible relationship between plasma free positively charged amino acid concentrations and the degree of microalbuminuria in patients with gastrointestinal cancer. In 42 consecutive patients (22 men and 20 women), comprised of 25 with histologically proven colorectal or gastric cancer, 9 controls and 8 weight-losing patients with benign gastrointestinal disease urinary albumin and plasma amino acid analysis was performed. Microalbuminuria was more prevalent in weight-losing cancer patients (65%) compared with their weight-stable counterparts, benign gastrointestinal patients and controls. This difference reached statistical significance at the 5% level. Additionally, a significant positive correlation (rs = 0.8, p < 0.05) was observed between ornithine and urinary albumin loss in this group of patients. This study suggests that plasma free amino acid alterations in weight-losing gastrointestinal cancer patients may have an effect on renal tubular protein reabsorption.

Lipid fuel metabolism after abdominal surgery.

The work investigated fuel interrelationships in surgical patients infused with saline (Group I) or glucose (Group II) (13 patients in each group) on the day of surgery and subsequently maintained solely on saline until the fifth postoperative day. Blood concentrations of non-esterified fatty acids (NEFA) and ketone bodies were markedly increased in response to surgical stress on the day of surgery only in patients who were not administered carbohydrate. Increased concentrations of lactate and glucose were observed on the day of surgery in patients infused with either saline or glucose. As both fatty acid and ketone body concentrations were decreased by glucose infusion, impaired glucose utilization immediately after surgery is not a simple consequence of increased oxidation of lipid fuels. Glucose and lactate concentrations declined after the day of surgery. Despite a progressive fall in plasma non-esterified fatty acid concentrations from the first to fifth post-operative days, blood ketone body concentrations were strikingly elevated in both groups of patients. The findings emphasize the role of the liver in post-operative fatty acid turnover.

Micronutrients in gastrointestinal cancer.

The monitoring of micronutrients and the relationship between dietary intake and micronutrient status prior to and after surgery in patients with histologically proven gastrointestinal adenocarcinoma, both weight-stable and weight-losing (> 7.5% of their pre-illness weight) has been studied and the results compared to controls. Plasma vitamin C and red blood cell thiamine levels were significantly lower in weight-losing cancer patients when compared to their weight-stable counterparts (P < 0.05 and P < 0.02 respectively). Weight-losing patients had a lower vitamin C (P < 0.05) and thiamine (P < 0.002) intake, and a higher elevation in plasma C-reactive protein and a lower prealbumin level (P < 0.02), when compared to both weight-stable cancer patients and controls. Plasma vitamin C, prealbumin and C-reactive protein levels remained unchanged after curative resections of the tumours compared to a preoperative value, and there was a highly significant correlation between plasma vitamin C and dietary intake of vitamin C. This study suggests that the lower vitamin C and thiamine status in weight-losing gastrointestinal cancer patients prior to surgery is due to a lower micronutrient intake and an acute phase response to their illness. Dietary intake of vitamin C appears to be the major factor in determining plasma vitamin C concentration following curative surgical resection.

The nature and significance of multiple isoforms of the oestrogen receptor in breast tumours.

Oestrogen receptors (ERs) in breast tumours are highly heterogeneous. In previous studies we have shown that at least four isoforms may exist. These migrate in isoelectric focusing (IEF) gels to isoelectric points (pI values) 6.1, 6.3, 6.6 and 6.8. Of these the first (pI 6.1) corresponds to the 8S isoform as detected by sucrose gradient fractionation, while the others all sediment at 4S. In a series of 66 breast tumours it was found that those at pI 6.3 and pI 6.8 were significantly correlated with the presence of progesterone receptors. To characterize the isoforms more fully, ER isoforms labelled by [3H]oestradiol binding were fractionated by IEF. The results were compared with those obtained after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the H222 anti-ER monoclonal antibody. In other experiments, tumour ER isoforms were covalently labelled with [ring-3H] tamoxifen aziridine and separated by IEF. The individual isoforms were electroeluted from the IEF gel and further analysed by SDS-PAGE and non-denaturing PAGE. In summary, the evidence shows that the isoforms of pI values 6.3, 6.8 and 6.6 have molecular masses of 50, 65 and 70 kDa respectively. In addition, all three of these isoforms, i.e. the pI 6.3, 6.8 and 6.6 isoforms, could form dimers. We conclude that the three isoforms sedimenting at 4S have the capacity to form dimers and thus may have the potential for binding to oestrogen response elements in the genome.