Increased mortality in systemic inflammatory response syndrome patients with high levels of coagulation factor VIIa.

BACKGROUND: The tissue factor (TF)- Factor VIIa (FVIIa) complex has a pivotal role in inflammatory and coagulation responses in patients with systemic inflammatory response syndrome (SIRS) and sepsis. Because zymogen FVII (FVII) and FVIIa compete for binding to TF, their plasma levels determine if a catalytically active TF-FVIIa complex will be formed. OBJECTIVE: To study mortality in SIRS patients as a function of FVIIa and FVII levels in plasma. METHODS: This was a cohort study of 275 patients presenting with SIRS, aged 18 years or older and with an anticipated Intensive Care Unit (ICU) stay of at least 24 h. FVIIa was measured using a novel, quantitative assay that recognizes FVIIa, but not FVII. All-cause hospital mortality was followed over a period of 60 days. RESULTS: The percentage of FVII measured as FVIIa was higher in non-survivors than survivors (2.8%, IQR = 1-5.5% vs. 1.5%, IQR = 0.6-3.3%; P = 0.034). High levels of FVIIa were associated with decreased 60-day cumulative survival (62% vs. 81%, P = 0.030); the opposite was observed for FVII (84% vs. 76%, P = 0.039). Patients with high-FVIIa and low-FVII levels had a three-fold increased hazard ratio (HR) compared with the patients that had low-FVIIa and high-FVII levels (HR = 3.24, 95% confidence interval [CI] = 1.41-7.36). This association persisted after adjusting for the APACHE IV score (adjusted HR = 2.75, 95% CI = 1.2-6.27). CONCLUSIONS: SIRS patients with high-FVIIa and low-FVII on admission have an increased mortality risk, an association that is independent from the parameters included in the APACHE IV score.

Cardiac biomarkers as indicators of hemodynamic adaptation during postasphyxial hypothermia treatment.

BACKGROUND: Little is known about the effects of hypothermia on the cardiovascular system in term newborns with neonatal encephalopathy. OBJECTIVES: To evaluate whether mild hypothermia for neonatal encephalopathy is cardioprotective as indicated by the cardiac biomarkers cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP). METHODS: This was an observational cohort study of infants treated for perinatal asphyxia. In infants, mild total body hypothermia treatment of 33.5°C during 72 h was initiated (n = 20). Samples of cTnI and BNP were collected before the start of hypothermia, at 24 and 48 h after birth, and after rewarming (84 h). BNP and cTnI values were then compared with BNP and cTnI values of asphyxiated infants not treated with hypothermia (n = 28). RESULTS: No differences were found between the groups in clinical patient characteristics or inotropic support. The hypothermia-treated patients seemed to be clinically more affected (5-min Apgar score, p < 0.05; umbilical artery pH, p = 0.08), but showed similar encephalopathy scores. Significantly lower values for BNP were found in hypothermia- compared to nonhypothermia-treated infants at 48 h and at normothermia after rewarming [144 pmol/l (95-286) vs. 75 pmol/l (45-143), 182 pmol/l (73-341) vs. 43 pmol/l (24-163)]. No differences were found for cTnI concentrations between both groups. CONCLUSIONS: The raised, but similar, cTnI values between hypothermia- and nonhypothermia-treated infants indicate similar myocardial damage in both groups. The lower BNP levels during hypothermia treatment suggest that hypothermia after perinatal asphyxia exerts a beneficial effect on cardiac function.

Left ventricular assist device: a functional comparison with heart transplantation.

BACKGROUND: A growing number of patients with end-stage heart failure undergo implantation of ventricular assist devices as a bridge to heart transplantation. OBJECTIVES: In this study we investigated whether functional and haemodynamic recovery after implantation is sufficient to warrant the use of them as long-term alternative to heart transplantation. METHODS: We compared peak VO(2) of a group of patients three months after implantation of a ventricular assist device and three months after heart transplantation. Furthermore, we analysed the degree of haemodynamic recovery, by comparing plasma levels of BNP and creatinine before and after implantation of the device. RESULTS: After implantation of a ventricular assist device, exercise capacity improved considerably; three months after implantation peak VO(2) was 20.0+/-4.9 ml/kg/min (52% of predicted for age and gender). After heart transplantation exercise capacity improved even further; 24.0+/-3.9 ml/ kg/min (62% of predicted for age and gender) (p<0.001). In the three months after implantation, BNP plasma levels decreased from 570+/-307 pmol/l to 31+/-25 pmol/l and creatinine levels decreased from 191+/-82 mumol/l to 82+/-25 mumol/l, indicating significant unloading of the ventricles and haemodynamic recovery. CONCLUSION: With regard to functional and haemodynamic recovery, the effect of implantation of a ventricular assist device is sufficient to justify its use as an alternative to heart transplantation. (Neth Heart J 2008;16:41-6.).

Introduction of preoperative radiotherapy in the treatment of operable rectal cancer in the Southwest region of the Netherlands.

INTRODUCTION: After publication of the results of the Dutch TME-trial preoperative radiotherapy followed by TME-surgery was introduced in July 2001 in the region of the comprehensive cancer centre Rotterdam as standard treatment for rectal cancer. The aim of this study is to identify the compliance to a new standardized treatment protocol i.e. the introduction of preoperative radiotherapy and to analyze the results of rectal cancer treatment in the Cancer Centre Rotterdam Region. PATIENTS AND METHODS: A total of 521 patients with adenocarcinoma of the rectum were included in the period from 2001 to 2003. All patients were treated with curative intent. RESULTS: There was a significant increase of preoperative radiotherapy for patients with a tumour in the lower two-third of the rectum (21% versus 69%, p<0.001). Peri-operative mortality rate was 2.7% and overall anastomotic leakage rate was 10.3%. There was a significant increase in the occurrence of anastomotic leakage in end-to-end anastomoses (p<0.0001). Most anastomotic leakages occurred when patients were operated in between 4 and 8 days after the end of radiotherapy. Several aspects such as continence for urine and faeces and sexual functions were poorly registered. The total number of lymph nodes registered in pathology reports was low. The rate of reported circumferential margins increased from 37% to 70% after feedback to the regional pathology working group. CONCLUSION: The regional quality of rectal cancer surgery is conform preset quality-demands. There was a significant increase in the percentage preoperative radiotherapy, but still about 25% of patients who qualified for radiotherapy did not receive radiation. Pathology reports improved during registration, which illustrates the importance of registration to assess and improve quality of rectal cancer treatment.

Precision, accuracy and linearity of radiometer EML 105 whole blood metabolite biosensors.

The analytical performance of a new, whole blood glucose and lactate electrode system (EML 105 analyser. Radiometer Medical A/S. Copenhagen, Denmark) was evaluated. Between-day coefficients of variation were < or = 1.9% and < or = 3.1% for glucose and lactate, respectively. Recoveries of glucose were 100 +/- 10% using either aqueous or protein-based standards. Recoveries of lactate depended on the matrix, being underestimated in aqueous standards (approximately -10%) and 95-100% in standards containing 40 g/L albumin at lactate concentrations of 15 and 30 mmol/L. However, recoveries were high (up to 180%) at low lactate concentrations in protein-based standards. Carry-over, investigated according to National Clinical Chemistry Laboratory Standards EP10-T2, was negligible (alpha = 0.01). Glucose and lactate biosensors equipped with new membranes were linear up to 60 and 30 mmol/L, respectively. However, linearity fell upon daily use with increasing membrane lifetime. We conclude that the Radiometer metabolite biosensor results are reproducible and do not suffer from specimen-related carry-over. However, lactate recovery depends on the protein content and the lactate concentration.

alphaV Integrins on HT-29 colon carcinoma cells: adhesion to fibronectin is mediated solely by small amounts of alphaVbeta6, and alphaVbeta5 is codistributed with actin fibers.

HT-29 colon carcinoma cells form liver metastases upon intrasplenic injection, and adhesion to fibronectin under the liver microvascular liver endothelium is likely to be important for metastasis formation. We have therefore studied the integrins involved in fibronectin adhesion. This was not affected by blocking antibodies against the beta1, alpha3, and alpha5 integrin subunits, but it was blocked by an RGD-containing peptide, indicating involvement of RGD-dependent non-beta1 alphaV integrins. Both alphaVbeta5 and alphaVbeta6 were detected on HT-29 cells. Blocking mAb against alphaV, but not against alphaVbeta5, abolished adhesion. From a HT-29 cell lysate, only alphaVbeta6 bound to a fibronectin-Sepharose column. Thus, alphaVbeta6 is the main fibronectin receptor on HT-29 cells, despite the very low levels of alphaVbeta6 and the much higher levels of alphaVbeta5. The HT29 cells did not spread on fibronectin in the absence of serum, not even after a three- to fourfold increase in alphaVbeta6 levels, induced by interleukin 4. The cells did spread on vitronectin. Using immunofluorescence we observed that both on vitronectin and on fibronectin alphaVbeta5 was arranged in a striped pattern, aligned with actin fibers, and not in focal adhesions. On fibronectin, but not on vitronectin, alphaVbeta6 was concentrated in a punctate pattern at the periphery of cell islands.

Adhesion of lymphoma cells to fibronectin: differential use of alpha 4 beta 1 and alpha 5 beta 1 integrins and stimulation by the 9EG7 mAb against the murine beta 1 integrin subunit.

Murine ESb and MDAY-D2 lymphoma cells are highly metastatic, in particular to the liver, and are highly invasive in hepatocyte cultures. This may involve adhesion to hepatocyte surface-associated fibronectin (Kemperman et al., 1994, Cell Adh. and Communic. 2:45). Both ESb and MDAY-D2 cells express the fibronectin receptor alpha 4 beta 1, and MDAY-D2 cells in addition also alpha 5 beta 1. Yet, adhesion of ESb cells to fibronectin was low, and MDAY-D2 cells did not adhere at all, but adhesion of both cells was stimulated by phorbol myristate acetate (PMA) and Mn2+. In ESb cells, this adhesion was mediated by alpha 4 beta 1. In MDAY-D2 cells, however, only alpha 5 beta 1 was involved, despite alpha 4 beta 1 levels similar to ESb cells. The alpha 4 beta 1 integrin was functional since it mediated adhesion of MDAY-D2 cells to VCAM-1. An alpha 5 beta 1-negative variant of MDAY-D2 adhered to fibronectin and this was mediated by alpha 4 beta 1. These results indicate that alpha 4 beta 1 function in these cells is suppressed in the presence of alpha 5 beta 1. Adhesion of ESb cells to hepatocytes was inhibited by anti-alpha 4 antibody, but only by 30%, and fibronectin adhesion was found to have no role in the interaction of MDAY-D2 cells with hepatocytes. This suggests that alpha 4 beta 1 and alpha 5 beta 1 function is not activated during this interaction. The 9EG7 antibody against mouse beta 1 integrin was described to inhibit beta 1 integrins (Lenter et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 9051). In contrast, we observed that 9EG7 stimulated beta 1-integrin function: Adhesion of ESb and MDAY-D2 cells not only to fibronectin, but also to laminin was induced or enhanced.

Adhesion mechanisms in liver metastasis formation.

Our results strongly indicate that integrin mediated adhesion between metastasizing tumour cells and hepatocytes has a decisive role in the formation of liver metastases. However, it is also clear that tumour cells may use different adhesion pathways and furthermore that the adhesion may be modulated by several factors. The role of adhesion has been demonstrated most clearly for LFA1 on T cell hybridomas, which interacts with ICAM1 present in the liver. Alternative pathways must exist, however, given the high invasive capacity of ESb cells, which is apparently LFA1 independent. A possible alternative is adhesion to fibronectin, which is present in abundance on the hepatocyte surface, both in vivo and in vitro. As there is no basement membrane under the endothelium of liver microvessels, so that tumour cells cannot adhere to laminin and collagen type IV as in other organs, adhesion to this fibronectin may be particularly important for metastasis to the liver. Many tumour types can use this pathway, and many different fibronectin receptors may be involved, including VLA-4, VLA-5 and several alpha V integrins. For carcinomas there is another possibility: Fibronectin receptor deficient cells may still adhere using the integrin alpha 6 beta 4, which binds to an unknown ligand present on the hepatocyte surface. Modulation of adhesion can occur in several ways. One example is steric hindrance by mucins that may strongly affect and even abrogate adhesion, despite high levels of appropriate integrins. Another is the activation required for integrins on lymphoma cells, the best known example of which is the activation of LFA1 on T cell hybridomas. It will be evident, therefore, that the role of adhesion in the formation of liver metastases can only be fully understood if the complete set of adhesion molecules on the tumour cells is known as well as their functional status and the possible effects of both cellular and extracellular modulating factors.

Prognostic factors for survival after breast conserving therapy for stage I and II breast cancer. The role of local recurrence.

Risk factors for local recurrence (LR) in a univariate analysis had a significant correlation with survival. Local and distant failure could not be regarded as independent events. We undertook a multivariate survival analysis to study the relation between LR and survival. In a retrospective study of 1026 patients treated with tumorectomy, axillary dissection and radiotherapy, factors associated with disease-specific survival (DSS) were analysed. Actuarial estimates for DSS are 91% at 5 years and 86% at 10 years. The multivariate analysis revealed five factors: clinical stage, number of affected axillary nodes, histological grade, degree of tubule formation and left-sided primary tumour. Controlling for these factors, LR appeared to be significantly correlated with DSS. The hazard rate of DSS was estimated to increase by a factor 8.8 (95% confidence interval 4.6-16.8) upon occurrence of a LR. Local recurrence per se, apart from the identified prognostic factors, is a risk factor for DSS. The exact mechanism by which LR has an influence on survival cannot be clarified from these data.

Adhesion of carcinoma cells to rat hepatocytes and rat fibronectin is inhibited by the OPAR monoclonal antibody, which is directed against a rat liver-specific carbohydrate epitope.

The OPAR mouse monoclonal antibody (mAb) directed against rat hepatocytes was previously shown to inhibit adhesion of TA3/Ha mammary carcinoma cells to hepatocytes. The antigen is abundantly present at the surface of hepatocytes beneath the endothelium of liver capillaries where we have observed invasion of carcinoma cells to occur. The OPAR mAb reacted with three major bands on a Western blot of liver plasma membrane proteins. The same proteins were also seen upon immunoprecipitation from iodinated liver plasma membrane proteins. We have isolated OPAR antigens by lectin wheat germ agglutinin (WGA) and OPAR affinity chromatography. Amino acid sequence analysis revealed that two of the bands were alpha 1-macroglobulin and C4-binding protein, which are serum components produced by hepatocytes. The presence of the epitope on distinct proteins and our previous observation that it can be detected in the Golgi apparatus but not in the endoplasmic reticulum, suggested that OPAR reacts with a liver-specific glycoconjugate. Loss of OPAR reactivity after neuraminidase and N-glycosidase F treatment showed that the epitope contains sialic acid residues on N-linked sugar moieties. OPAR also reacted with rat fibronectin, and inhibited adhesion of TA3/St cells to fibronectin. This explains the inhibition by the OPAR mAb of TA3/St cell adhesion to hepatocytes, which we have shown to be due mainly to interaction with hepatocyte surface-associated fibronectin. However, adhesion of the related TA3/Ha cells to hepatocytes, which is mediated by the alpha 6 beta 4 integrin, and does not involve binding to fibronectin, is also inhibited. This suggests that alpha 6 beta 4 on liver-metastasizing carcinoma cells binds to an OPAR epitope-carrying glycoprotein produced by hepatocytes.

The mucin epiglycanin on TA3/Ha carcinoma cells prevents alpha 6 beta 4-mediated adhesion to laminin and kalinin and E-cadherin-mediated cell-cell interaction.

TA3/Ha murine mammary carcinoma cells grow in suspension, do not adhere to extracellular matrix molecules, but do adhere to hepatocytes and form liver metastases upon intraportal injection. Recently we showed that the integrin alpha 6 beta 4 on the TA3/Ha cells is involved in adhesion to hepatocytes. However, despite high cell surface levels of alpha 6 beta 4, TA3/Ha cells do not adhere to the alpha 6 beta 4 ligands laminin and kalinin. Here we show that this is due to the mucin epiglycanin that is highly expressed on TA3/Ha cells. Some monoclonal antibodies generated against epiglycanin induced capping of most of the epiglycanin molecules. TA3/Ha cells treated with these mAb did adhere to laminin and kalinin, and an epithelial monolayer was formed on kalinin, with alpha 6 beta 4 localized in HD1-containing hemidesmosome-like structures and E-cadherin at the cell-cell contact sites. Similar results were obtained after treatment of TA3/Ha cells with O-sialoglycoprotein endopeptidase which removes all epiglycanin. In addition, the enzyme induced E-cadherin-mediated cell-cell aggregation. Both treatments also enhanced the adhesion to hepatocytes, but given the potent antiadhesive effect of epiglycanin it is remarkable that nontreated TA3/Ha cells adhere to hepatocytes at all. We found that during this interaction, epiglycanin was redistributed. We conclude that epiglycanin can completely prevent both intercellular and matrix adhesion, but that this effect can be overcome in certain intercellular interactions because of the induced redistribution of the mucin.

Dose and volume effects on fibrosis after breast conservation therapy.

PURPOSE: To analyze factors involved in the development of fibrosis in the boost area after breast conservation therapy (BCT) in patients treated with continuous low dose rate iridium implants following 50 Gy whole breast irradiation. METHODS AND MATERIALS: Fibrosis was estimated by palpation in 404 patients by four physicians. The median follow-up (FUP) duration was 70 months (range 30-133 months). Original implant data were used for reconstruction and dose-volume calculations. The total dose of the external whole breast irradiation and iridium implants was expressed in Normalized Total Dose (NTD): the total dose given in fractions of 2 Gy, which is biologically equivalent to the actual dose given according to the linear-quadratic model, using an alpha/beta value of 2 Gy, and 1.5 h for the recovery half-life of sublethal damage repair. To identify predictors of fibrosis we used a proportional odds model in a polychotomous logistic regression analysis. RESULTS: Seven independent factors were identified that were related to the severity of fibrosis: age, duration of FUP, clinical T-size, photon beam energy, NTD level, implant volume, and adjuvant chemotherapy. From the proportional odds model, a volume exponent could be estimated (0.16 +/- 0.04) that enabled us to determine dose-effect relations for different volumes. A 10-fold higher risk of fibrosis was seen when the total dose was above 79 Gy as compared with doses lower than 70 Gy. A fourfold increase in risk of fibrosis was seen for each 100 cm3 increase in irradiated boost volume. The use of adjuvant chemotherapy resulted in a twofold increase in the risk of fibrosis (dose modifying factor approximately 1.08). The application of Co-60 beams had a similar effect. The relative odds for the other factors were smaller (1.4 for each 10 years of older age, and 1.2 for clinical T-size over 20 mm). The FUP-period had a nonlinear effect: relative odds 2.2 at 6 years, 3.6 at 7-8 years, and 2.8 at 9-11 years. The dose rate (mean 0.57, range 0.26-0.89 Gy/h) had no influence on the development of fibrosis and there was no correlation between dose rate and irradiated volume. CONCLUSIONS: To optimize cosmetic results after BCT, both the total dose and the irradiated volume should be kept as low as possible. Minimum effective dose levels still have to be established. The boost volume can be minimized by more conformal brachytherapy techniques and optimal localization. It may be worthwhile to take adjuvant chemotherapy into account in decisions on boost dose levels.

TA3/St, but not TA3/Ha, mammary carcinoma cell adhesion to hepatocytes is mediated by alpha 5 beta 1 interacting with surface-associated fibronectin.

The cell lines TA3/Ha and TA3/St are derived from the same tumor, grow both in suspension and form liver metastases upon intraportal injection. We have studied the interaction of these cell lines with hepatocytes, which is likely to be relevant for liver metastasis formation. Recently we have shown that the integrin alpha 6 beta 4 is involved in adhesion of TA3/Ha cells to hepatocytes. However, we show here that the alpha 6 beta 4-specific antibodies, that inhibit adhesion of TA3/Ha cells, did not affect adhesion of TA3/St cells to hepatocytes. The beta 4 subunit, present at high levels on TA3/Ha cells, was found to be expressed at a much lower level by TA3/St cells. In contrast, TA3/St cells express much more of the beta 1-integrin subunit than TA3/Ha cells. We assessed whether these differences in integrin expression are responsible for the different adhesion mechanisms used by these cell lines. We show that alpha 5 beta 1, which is expressed by TA3/St cells, and not by TA3/Ha cells, is involved in TA3/St adhesion to fibronectin that is associated with the hepatocyte surface. Since fibronectin is the main extracellular matrix component present on the hepatocyte surface underneath the sinusoidal endothelium, alpha 5 beta 1 may be particularly important for metastasis formation in the liver, as compared to other organs.

Risk factors in breast-conservation therapy.

PURPOSE: To identify clinical and pathologic factors associated with an increased risk of local recurrence following breast-conservation therapy (BCT) to assess the safety of this procedure for all subgroups of patients. PATIENTS AND METHODS: The study population consisted of 1,026 patients with clinical stage I and II breast cancer treated between 1979 and 1988 at the Netherlands Cancer Institute. The BCT regimen consisted of local excision and axillary lymph node dissection (ALND) followed by whole-breast irradiation to a total dose of 50 Gy in 2-Gy fractions and boost irradiation (mostly by iridium implant) of 15 to 25 Gy. RESULTS: With a median follow-up duration of 66 months, the actuarial breast recurrence rate was 4% at 5 years, counting all breast recurrences. Univariate analysis showed seven factors to be associated with an increased risk of local recurrence; age, residual tumor at reexcision, histologic tumor type, presence of any carcinoma-in-situ component, vascular invasion, microscopic margin involvement, and whole-breast radiation dose. Three factors remained independently significant after proportional hazard regression analysis: age, margin involvement, and the presence of vascular invasion. When the analysis was repeated, but counting only those breast recurrences that occurred before regional or distant failures, only young age and vascular invasion were independent predictive factors. In the third analysis, factors predicting the necessity of local salvage treatment were analyzed. In this analysis, the possible bias in the former analysis caused by censoring actuarial methods was avoided. The results were the same as in the second analysis, showing young age and vascular invasion as the only independent predictive factors. Breast recurrence rates were 6% for patients less than 40 years of age and 8% for patients with tumors showing vascular invasion. In the absence of risk factors, the breast recurrence rate is only 1% at 5 years. CONCLUSION: Slightly higher recurrence rates were found in patients less than 40 years of age and in patients with tumors showing vascular invasion. The role of margin involvement is uncertain.

The role of integrins and integrin activation in liver metastasis.

An overview is presented of our studies on the interaction between blood-borne tumor cells and the tissues where metastases are formed, in particular the liver. Using blocking antibodies and tumor cell mutants, we have identified the adhesion molecules involved, which so far are all integrins. Strikingly, tumor cell lines that are quite similar, and invade in a comparable fashion, use distinct integrins. Lymphomas that invade the liver massively and diffusely use LFA-1 or fibronectin receptors to adhere to hepatocytes. We have obtained evidence that LFA-1 is activated during the interaction by factors that act through G-protein-coupled receptors, and preliminary results suggest that the same may be true for the fibronectin receptors. Whereas TA3/Ha murine mammary carcinoma cells adhere to hepatocytes via alpha 6 beta 4, TA3/St variant cells of the same tumor bind via the fibronectin receptor alpha 5 beta 1. Adhesion of the TA3/Ha cells appears to be impaired by the mucin epiglycanin that is abundantly present on the surface of these cells.

The integrin alpha 6 beta 4 on TA3/Ha mammary carcinoma cells is involved in adhesion to hepatocytes.

TA3/Ha mammary carcinoma cells form liver metastases upon intraportal injection. We have studied the interaction between these cells and hepatocytes that is likely to be important for liver metastasis formation. We show that the integrin alpha 6 beta 4, which is highly expressed on TA3 cells, is involved in this interaction. Fab fragments, generated from a polyclonal serum against TA3 cells, inhibited TA3-hepatocyte adhesion. By affinity purification on purified alpha 6 beta 4, we isolated alpha 6 beta 4-specific Fab fragments from this anti-TA3 serum. These antibodies were highly specific for alpha 6 beta 4, as demonstrated by Western blot analysis and immunoprecipitation, and inhibited TA3-hepatocyte adhesion. This shows that alpha 6 beta 4, which thus far has only been implicated in cell-matrix adhesion, can also mediate interactions with cell surfaces. Our results suggest that the high levels of alpha 6 beta 4 often expressed by metastatic carcinoma cells contribute to liver metastasis formation.

Breast-conserving therapy for invasive carcinoma: diagnosis of local recurrence.

The way in which local recurrence after breast conserving treatment for invasive carcinoma became apparent, is reported in 44 patients. All patients were followed by regular physical examination and annual mammography. In 36 patients first suspicion of local recurrence was heralded by clinical signs and symptoms, presented between two scheduled routine visits in 12 patients, and at the time of a routine visit in 14 patients. Routine physical examination by surgeon or radiotherapist revealed local recurrence in 10 patients. Local recurrence was detected only by mammography in eight patients. Mammography confirmed the clinical suspicion in seven patients. The remaining 23 patients with clinical overt recurrence showed no signs of recurrent tumour on the mammographs performed after first clinical suspicion. Fine needle aspiration (FNA) cytology confirmed the clinical suspicion in 35 of the 38 tested patients. In our experience, regular physical examination is the mainstay for the detection of local recurrence after breast conserving therapy. Mammography was of limited value but proved more valuable for the early detection of recurrent tumour outside the primary tumour area. Fine needle aspiration cytology is a helpful tool in confirming the diagnosis of local recurrence.

The influence of muscle-conditioned media on chick embryo brainstem neurons in culture.

Brainstem pieces from the trigeminal region of the metencephalic basal plate of 10-day chick embryos were dissociated and cultured in control conditions or in the presence of muscle-conditioned medium (MCM). The MCM was derived from age-matched target tissue relevant to this neuronal region (jaw musculature), from relevant target tissue of an age at which innervation would initially be taking place (4 days), and from nonrelevant target tissue also of an early stage (4-day limb bud). Neuronal survival and differentiation was assessed daily, for 7 days. Survival and differentiation were significantly enhanced by the 4-day jaw MCM compared to both the controls and the cultures grown with 10-day jaw MCM and 4-day limb MCM. These measures in the presence of 10-day jaw MCM and 4-day limb MCM did not differ, but surpassed that seen in control cultures. The results are compared to the more specific responsiveness seen in earlier (2-day) neural tube cultures, and their relationship to in vivo regenerative nerve fiber outgrowth is considered.