FluoroSpot Analysis of TLR-Activated Monocytes Reveals Several Distinct Cytokine-Secreting Subpopulations.

Monocytes have long been considered a heterogeneous group of cells both in terms of morphology and function. In humans, three distinct subsets have been described based on their differential expression of the cell surface markers CD14 and CD16. However, the relationship between these subsets and the production of cytokines has for the most part been based on ELISA measurements, making it difficult to draw conclusions as to their functional profile on the cellular level. In this study, we have investigated lipoteichoic acid (LTA)- and lipopolysaccharide (LPS)-induced cytokine secretion by monocytes using the FluoroSpot technique. This method measures the number of cytokine-secreting cells on the single-cell level and uses fluorescent detection, allowing for the simultaneous analysis of two cytokines from the same population of isolated cells. By this approach, human monocytes from healthy volunteers could be divided into several subgroups as IL-1ß, IL-6, TNF-α and MIP-1ß were secreted by larger populations of responding cells (25.9-39.2%) compared with the smaller populations of GM-CSF (9.1%), IL-10 (1.3%) and IL-12p40 (1.2%). Furthermore, when studying co-secretion in FluoroSpot, an intricate relationship between the monocytes secreting IL-1ß and/or IL-6 and those secreting TNF-α, MIP-1ß, GM-CSF, IL-10 and IL-12p40 was revealed. In this way, dissecting the secretion pattern of the monocytes in response to TLR-2 or TLR-4 stimulation, several subpopulations with distinct cytokine-secreting profiles could be identified.

Effects of the dual endothelin receptor antagonist tezosentan and hypertonic saline/dextran on porcine endotoxin shock.

AIM: To evaluate the haemodynamic effects of the dual endothelin receptor antagonist tezosentan, both alone and combined with hypertonic saline/dextran (HSD), on porcine endotoxin shock, with focus on cardiopulmonary circulation. The effects on gas exchange and short-term survival were also studied. METHODS: A prospective, randomized experimental study was carried out. Thirty-two anaesthetized pigs underwent pulmonary and carotid artery catheterization. Following haemodynamic stabilization and baseline measurements, endotoxaemia was induced by an Escherichia coli-endotoxin infusion over 180 min and the animals observed another 120 min. After 60 min of endotoxaemia, directly before intervention, animals were randomized into four groups: a tezosentan group, an HSD group, a combined tezosentan/HSD group and a control group. The consequent haemodynamic effects and blood gas results were recorded. RESULTS: The endotoxin infusion reduced mean arterial blood pressure from 111 +/- 14 (mean +/- standard deviation) to 77 +/- 27 mmHg and cardiac index from 126.9 +/- 27.2 to 109.3 +/- 22.6 mL min(-1) kg(-1) within 90 min in the control group. In addition, endotoxin simultaneously increased mean pulmonary artery pressure from 24 +/- 17 to 38 +/- 19 mmHg and reduced arterial oxygenation from 18.9 +/- 2.0 to 12.2 +/- 5.3 kPa. Tezosentan, alone and combined with HSD, reversed the pulmonary hypertension and prevented the reduction in cardiac index and arterial oxygenation, resulting in reduced metabolic acidosis. Additionally, in the tezosentan group, the mean arterial blood pressure was reduced to the same level as in controls, an effect not prevented by the addition of HSD. It was found that all three interventions improved survival rates. CONCLUSION: Tezosentan, alone and in combination with HSD, improved cardiac index and arterial oxygenation. The addition of HSD to tezosentan treatment did not improve the endotoxin-induced hypotension, but beneficial effects on microcirculation and systemic oxygenation were seen despite low perfusion pressure, as indicated by increased SvO(2) and reduced metabolic acidosis.

Beneficial effects of hypertonic saline/dextran on early survival in porcine endotoxin shock.

BACKGROUND: Hypertonic saline/dextran (HSD) has been shown to have beneficial effects in haemorrhagic shock. These effects, with improved haemodynamics and organ perfusion, would in theory also be of benefit in septic shock. However, this is less studied. We have therefore further evaluated the effect of additional treatment with HSD in a porcine endotoxin shock model. METHODS: Sixteen anaesthetized pigs were used. A continuous infusion of endotoxin (LPS EC) was increased stepwise during 30 min to a rate of 5 microg/kg/h. The infusion was discontinued after 3 h and the animals were observed for another 2 h. The animals received continuous basal fluid resuscitation with isotonic Ringer's glucose 2.5% at a rate of 20 ml/kg/h throughout the experiment. After 1 h of endotoxin infusion, the animals were randomized to additional treatment with HSD, 4 ml/kg over 5 min, or the same volume of isotonic saline. Every 30 min, haemodynamics and mixed venous saturation (SvO2) were measured via a pulmonary artery catheter. Regional blood flow rates were measured continuously by perivascular ultrasonic flow probes. The metabolic response was measured by arterial blood gas analysis. RESULTS: The endotoxin put all animals into a progressive hypodynamic circulatory shock during the experiment. Treatment with HSD improved survival rate to 8/8 compared with controls 3/8. There was a transient circulatory recovery with improved central and regional haemodynamics, accompanied by stabilized metabolic response. CONCLUSION: Treatment with additional HSD improves survival in an early phase of endotoxin shock. Generally improved haemodynamics and oxygenation of peripheral tissues are suggested as possible mechanisms.

Randomized trial of adjuvant tamoxifen in node negative postmenopausal breast cancer. Stockholm Breast Cancer Study Group.

The paper presents long-term results of a randomized trial of adjuvant tamoxifen (40 mg daily for 2 or 5 years) versus surgery alone including 1,347 postmenopausal patients with histologically negative axillary nodes and a tumour diameter less than or equal to 30 mm. Data on the estrogen receptor status of the primary tumour were available in 1,136 patients (84%). At a median follow-up of 7 years (range 1.7-13.0 years) there was a significant prolongation of the recurrence-free survival among those allocated to tamoxifen (p less than 0.01), significantly fewer deaths due to breast cancer (p = 0.02) and a trend towards improved overall survival (p = 0.11). The treatment benefit was restricted to patients with ER-positive tumours. There was no significant reduction of breast cancer recurrences in the tamoxifen group among patients whose tumours were classified as ER-negative. The results support and extend previous studies in showing a long-term benefit of tamoxifen in postmenopausal breast cancer patients with node-negative, estrogen receptor positive disease.

Adjuvant tamoxifen in early-stage breast cancer: effects on intercurrent morbidity and mortality.

Intercurrent mortality and the pattern of inpatient hospital care was studied among 1,846 postmenopausal patients included in the Stockholm randomized trial of adjuvant tamoxifen (40 mg daily for 2 years) versus no adjuvant endocrine therapy. The median follow-up time was 54 months (range, 2 to 123 months). The patients were matched to the Swedish National Registry of Causes of Death and a computerized register covering about 95% of all hospital admissions in Stockholm County. There was no significant difference in the pattern of intercurrent mortality among the tamoxifen and control patients. The total number of hospital admissions was similar in both groups, but the tamoxifen patients were admitted significantly less frequently because of immunologic diseases (relative risk [RR] = 0.4; 95% confidence interval [CI], 0.2 to 0.9). Admissions because of thrombotic diseases were slightly, but not significantly, more frequent among the tamoxifen patients (RR = 1.2; 95% [CI], 0.6 to 2.3). The risk of hospital stay for benign gynecologic diseases other than prolapse or uterine bleeding was increased in the tamoxifen group (RR = 3.2; 95% CI, 1.2 to 8.6). No significant differences were found for diseases related to arteriosclerosis or osteoporosis. The study confirms and extends previous reports, which have shown that tamoxifen has few and usually mild side effects. However, the current results should be judged cautiously because of the relatively short median follow-up time (4.5 years) and the limitation of data in detecting morbidity that does not necessarily result in hospitalization.

Contralateral primary tumors in breast cancer patients in a randomized trial of adjuvant tamoxifen therapy.

Prophylactic treatment with the anti-estrogen tamoxifen may reduce the risk of breast cancer because estrogens are thought to act as promoters in the pathogenesis of the disease. This article presents results on the incidence of contralateral new primary tumors among 1846 postmenopausal breast cancer patients included in a randomized trial of adjuvant tamoxifen therapy for 2 or 5 years after surgery versus no adjuvant endocrine therapy. The median follow-up was 7 years (range, 3-13 years). There was a significant reduction of contralateral breast cancer in the 931 patients in the tamoxifen group versus that in the 915 control patients (29 versus 47 cases, respectively; P = .03). The cumulative incidence at 10 years in the tamoxifen group and the control group was 5% and 8%, respectively. Analysis of the relative hazard of contralateral tumor over time showed that the benefit with tamoxifen therapy was greatest during the first 1-2 years, but there was a continued risk reduction during the entire follow-up period, i.e., more than 10 years after cessation of treatment. There was no significant difference in the number of contralateral cancers in the patients randomly assigned to 2 or 5 years of treatment, but the 95% confidence interval of the relative hazard was wide. The proportion of estrogen receptor-negative contralateral breast cancers was higher in the tamoxifen group than in the control group. There was no difference, however, between the two groups in recurrence-free survival time from the diagnosis of the contralateral cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Randomized study of mammography screening--preliminary report on mortality in the Stockholm trial.

In March 1981, 40,318 women in Stockholm, aged 40-64, entered a randomized trial of breast cancer screening by single-view mammography alone versus no intervention in a control group of 20,000 women. The attendance rate during the first screening round was 81 per cent and the cancer detection rate was 4.0 per 1000 women. The detection the rate fell to 3.1 per 1000 in the second round, which was completed in October 1985. During 1986 the controlled design of the study was broken and the control women were invited once to screening which was completed the same year. A total of 428 cases of breast cancer were thus diagnosed in the study group and 439 in the adjusted control group. After a mean follow-up of 7.4 years the number of breast cancer deaths in the study and control groups was 39 and 30 respectively. The relative risk of breast cancer death (screening versus control) was 0.71 (95 per cent confidence interval: 0.4-1.2). Among women older than 50 years at entry the relative risk was 0.57 (95 percent confidence interval: 0.3-1.1). Cancer deaths among women under 50 were few and perhaps because of this no mortality reduction was seen in this age group. The estimate of mortality reduction lies between the results from two earlier Swedish randomized controlled trials.

Randomized trial of adjuvant tamoxifen combined with postoperative radiation therapy or adjuvant chemotherapy in postmenopausal breast cancer.

From 1976 to 1984, 427 postmenopausal women with high-risk breast cancer (pN + or pT greater than 30 mm) were randomized between postoperative radiation therapy (RT), radiation therapy plus tamoxifen (RT-TAM), adjuvant chemotherapy (CT), or chemotherapy plus tamoxifen (CT-TAM). Surgery was a modified radical mastectomy in all cases. The radiation therapy was given with high-voltage techniques and included the chest wall and regional nodes. The dose was 4600 cGy/4 1/2 weeks. Tamoxifen was given at a dose of 40 mg daily for 2 years. The adjuvant chemotherapy consisted of 12 cycles of cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) (or chlorambucil, methotrexate, and 5-fluorouracil [LMF] for patients entered before 1978). At a median follow-up time of 6 1/2 years the recurrence-free survival was significantly better for patients allocated to radiation therapy compared to chemotherapy and for patients allocated to tamoxifen compared to no adjuvant endocrine treatment (P less than 0.01). At 10 years the recurrence-free survival for patients in the RT-TAM, RT, CT-TAM, and CT groups was 63%, 57%, 47%, and 31%, respectively. A significant reduction of treatment failures with tamoxifen was only observed among patients with estrogen receptor-positive tumors. The overall survival difference in favor of patients allocated to radiation therapy or tamoxifen was not significant: the respective survival percentage at 10 years in the RT-TAM, RT, CT-TAM, and CT group was 65%, 62%, 52%, and 50%. The results indicate that postoperative radiation therapy continues to play an important role in the primary management of postmenopausal women with high-risk breast cancer and that the addition of tamoxifen may further improve the results among ER-positive patients.

The relationship between hormone receptor content and the effect of adjuvant tamoxifen in operable breast cancer.

The relationship between hormone receptor status and the effect of adjuvant tamoxifen in early breast cancer remains controversial. This article presents the results of a randomized trial of adjuvant tamoxifen (40 mg daily for 2 years) versus no adjuvant endocrine therapy in postmenopausal patients. During 1976 to 1984, 1,407 patients were included in the study. Of these, 427 (30%) had high-risk tumors (pN + or pT greater than 30 mm) and were included in a concurrent randomized comparison of postoperative radiotherapy versus adjuvant polychemotherapy. The mean follow-up time was 61/2 years. Tamoxifen improved the recurrence-free survival (RFS) (P less than .01), but the overall survival difference in favor of the tamoxifen-allocated patients was not significant. Data on estrogen (ER) and progesterone receptor (PgR) content were available in 750 patients. Their mean follow-up time was 41/2 years. The effect of tamoxifen was significantly related to ER level (P less than .01). No benefit with tamoxifen was observed among ER-negative patients. The relation to PgR level was of borderline significance (P = .06). Multivariate analysis indicated that most of the interaction between treatment and receptor content was explained by the interaction with ER (P less than .01). The PgR status appeared to modify the effect of tamoxifen among the ER-positive patients and the greatest effect was observed among patients who were positive for both receptors. However, the additional predictive information provided by the PgR assay did not help to identify an unresponsive subgroup of patients.

Additional value of fine-needle aspiration biopsy in a mammographic screening trial.

The Stockholm breast cancer screening trial used single-view mammography as the sole screening method. A majority (63 per cent) of the mammographic selected cases from the first two screening rounds had uncertain mammograms, coded as 3 on an ordinal scale from 1 to 5, where 1 and 2 are dismissed as normal mammograms and 5 stands for a typical cancer. In this group of uncertain mammograms 30 cases were malignant and 431 were non-malignant. The aim of this study was to examine whether surgical biopsy in this group could be replaced by fine-needle aspiration (FNA) biopsy, combined with the information from the mammogram and clinical examination, and whether this diagnostic strategy could select the malignant cases with a high sensitivity. FNA biopsy selected 25 of the 30 mammary carcinomas as definite malignancy or atypia, with a sensitivity of 83 per cent (95 per cent confidence interval: 69-96 per cent), and combined with the information from the mammogram and clinical examination 29 of the malignancies were selected with a sensitivity of 97 per cent (95 per cent confidence interval: 83-100 per cent). In 398 of 431 non-malignant cases the diagnosis was established with the triple diagnostic approach without needing a surgical biopsy. In a clinical follow-up study, up to 64 months after the first screening round, only one false negative case was found, included in the group of 30 malignancies described above. With this strategy the rate of negative surgical biopsies was reduced by 90 per cent in the group with uncertain mammograms and without considerably impairing the reliability of the results.

Radiotherapy, chemotherapy, and tamoxifen as adjuncts to surgery in early breast cancer: a summary of three randomized trials.

The paper summarizes up-dated results of three randomized adjuvant trials from the Stockholm Breast Cancer Group. The objective of all studies included an evaluation of the role of megavoltage radiation in the primary management of patients with early breast cancer. The first trial was started in 1971 and included 960 pre- and postmenopausal patients with operable disease. The study compared adjuvant radiotherapy with surgery alone. All patients were treated with a modified radical mastectomy. There was a sustained improvement of the recurrence-free survival with radiotherapy (p less than 0.001). Among node positive cases radiation reduced the frequency of both loco-regional recurrence (p less than 0.001) and distant metastasis (p less than 0.01). This observation indicates that distant dissemination in subgroups of patients can originate from uncontrolled local deposits of tumor cells, for instance in the regional lymph nodes. No adverse effect from radiation on long-term survival was observed. The second study was started in 1976 and compared postmastectomy radiation with adjuvant chemotherapy in pre- and postmenopausal high-risk patients. At a mean follow-up of 6 1/2 years there was no significant difference in recurrence-free survival between the two treatments. However, postmenopausal patients fared better with radiotherapy (p less than 0.01). In this subgroup, radiation was more effective than adjuvant chemotherapy in reducing both distant metastases (p less than 0.01) and loco-regional recurrences (p less than 0.001). In the third trial--which only included postmenopausal patients--2 years of adjuvant tamoxifen was compared with no adjuvant endocrine treatment. The number of treatment failures was significantly reduced with tamoxifen (p less than 0.01) but there was no significant overall survival benefit. Subset analysis indicated that tamoxifen improved the recurrence-free survival among patients treated with adjuvant chemotherapy (p less than 0.01) but only to a level close to that achieved with radiotherapy alone. Addition of tamoxifen to radiotherapy failed to further increase the recurrence-free survival.

Adjuvant tamoxifen in early breast cancer: occurrence of new primary cancers.

The frequency of new primary cancers was studied in 1846 postmenopausal patients included in a randomised trial of tamoxifen as an adjunct to primary surgery for early breast cancer. The median follow-up was 4.5 years (range 0.5-10.5 years). The number of new cancers in the tamoxifen group (n = 57) did not differ significantly from that in the control group (n = 70). However, in tamoxifen patients second breast cancers occurred less often and endometrial cancer occurred more often than in the controls. The increase in endometrial cancers was probably related to the agonistic oestrogenic effects of tamoxifen and was most pronounced in those treated for over 2 years.

The Stockholm breast cancer screening trial--5-year results and stage at discovery.

In screening programmes it is important to assess a preliminary effectiveness of the screening method as soon as possible in order to forecast survival figures. In March 1981 a controlled single-view mammographic screening trial for breast cancer was started in the south of Stockholm. The population invited for screening mammography consisted of 40,000 women aged 40-64 years, and 20,000 women served as a well-defined control group. The main aim of the trial was to determine whether repeated mammographic screening could reduce the mortality in the study population (SP) compared to the control population (CP). The cumulative number of advanced mammary carcinomas in the screening and the control populations from the first five years of screening have shown a tendency towards more favourable stages in the screened population aged 40-64 years. A breakdown by age suggests an effect in age group 50-59 years, but not yet in age groups 40-49 and 60-64 years. When comparing the rates of stage II+ cancer, an increased number is found in the study group. As the total rate of breast cancer is higher in SP than in CP, there ought to be a concealed group of stage II+ cancers in the CP which makes the comparison biased. A new approach has been designed, where an estimation of the 'hidden' number of stage II+ cancers in CP is added to the clinically detected cases, and in this respect a comparison has shown a decrease in the cumulative number of advanced cancers in the SP in relation to the CP (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The Stockholm trial on adjuvant tamoxifen in early breast cancer. Correlation between estrogen receptor level and treatment effect.

The paper presents interim results of an on-going randomized trial of adjuvant tamoxifen (40 mg daily for 2 years) versus no endocrine adjuvant therapy in postmenopausal women with early breast cancer. A total of 1407 patients were included in the study between November 1976 through June 1984. Estrogen receptor (ER) data were available on 1184 patients (84%). The median follow-up was 53 months. Adjuvant tamoxifen increased the recurrence-free interval (P less than 0.01) but had no significant effect on overall survival. Treatment failures were reduced by 25% (P less than 0.01) and deaths by 7% (P greater than 0.05). Tamoxifen mainly decreased the frequency of loco-regional recurrence whereas distant metastases were less affected. The treatment effect was independent of tumor stage but was significantly related to the estrogen receptor (ER) content of the primary tumor. Tamoxifen appeared ineffective among ER negative patients, and the greatest effect was seen among those with high levels of ER. The results indicate that the main mechanism of action of adjuvant tamoxifen is similar to that suggested in advanced disease, i.e. an interaction with the estrogen receptor.

Relationship between certain radiographic and biologic characteristics in breast cancer.

The relationship between mammographic appearance of microcalcifications and tumor stage, malignancy grade, estrogen receptor (ER) content and nuclear DNA pattern was studied in 92 mammary carcinomas. The results indicate that no complementary prognostic guidance can be obtained from the radiographic appearance of a tumor apart from a high probability of a spiculated cancer being ER-rich. The finding of a relatively high proportion of aneuploid and ER-poor carcinomas among tumors with microcalcifications indicates that this radiographic characteristic might be associated with increased growth potential.

Analysis of interval breast carcinomas in a randomized screening trial in Stockholm.

In the interval between screening examinations, some cases of breast cancer are invariably detected clinically in patients whose mammogram was considered to be normal at the earlier screening. During the first interval in the Stockholm study, 60 interval cancers were detected, giving a rate of 1.8 cases/1000 examinations/24 months. About half of these interval cases (31/60) were true interval tumours in that no sign of them could be found on the first mammogram; the other half, non-true, were possible to trace on the first mammogram. It is mainly women under 50 who feature in the interval group, above all in the sub-group of true interval cancers (p less than 0.05). The incidence of interval cancer rises, as expected, with the length of the interval (Fig. 1). In the final six months of the 2-year interval the incidence of interval cancers had risen to 88 per cent of the cancers detected in the control group in the same period of time. The cumulative incidence of interval cancers supports the hypothesis that the distribution of sojourn time in the interval 0-2 years is approximately rectangular. This means that shortening the interval by one-half would halve the number of interval cases. If mammography becomes a wide spread screening method for early detection of breast cancer, the number of non-true interval cancers could be a feed back on the effectiveness of the screening.(ABSTRACT TRUNCATED AT 250 WORDS)

Randomized mammographic screening for breast cancer in Stockholm. Design, first round results and comparisons.

In March 1981 a randomized single-view mammographic screening for breast cancer was started in the south of Stockholm. The screened population in the first round numbered 40,318 women, and 20,000 women served as a well-defined control group. The age groups represented were 40-64 years, and 80.7% of the invited women participated in the study. The first round disclosed 128 breast cancers (113 invasive and 15 noninvasive), or 4.0 per 1,000 women. Mean tumour size was 14.1 mm and axillary lymph node metastases were found in 21.8%. Fifty-five per cent of the tumours were small (less than or equal to 10 mm) or non-invasive, and 71% were stage I. Participation rates are high in all Swedish trials. The present results differ only slightly from other screening programs; the percentages of patients with axillary metastases and stage II tumours are similar in the Stockholm, Malmö and Kopparberg/Ostergötland studies. Comparisons of cancer prevalence in the various Swedish screening trials show that, in comparable age groups, there are some differences, even when the differences in the natural cancer incidence are taken into account. A decreased mortality was found recently in a Swedish trial in ages above 50 years but not below. In the Stockholm study more than one-third of the participants were aged 40-49 years.

Assessment of malignancy potential in so-called interval mammary carcinomas.

Forty-two so-called interval cancers of the breast were studied. The mammograms were reviewed and the tumors were classified as 14 'unrecognized', 9 'observer error', 1 'technical error' and 18 'true' interval cancers. By using a scoring system based upon histologic differentiation, axillary nodal status, estrogen receptor level, and nuclear DNA content, eight ductal carcinomas with high malignancy potential were identified. All of these tumors belonged to the group 'true' interval cancer. The data indicate that the mammographic subgroup 'true' interval cancer identifies the highly malignant tumors. However, even this strictly selected subgroup is heterogeneous since it also includes some tumors with low malignancy potential.

Pancreatic function in patients with hyperparathyroidism. A study with the Lundh test.

To elucidate the assumed relationship between hyperparathyroidism and pancreatitis, the pancreatic function was studied in 20 patients with hyperparathyroidism. In all cases removal of a parathyroid adenoma was followed by normalization of preoperative hypercalcemia. A modified Lundh's test with duodenal aspiration was performed before and after the operation. The volume of the aspirate and its content of electrolytes and pancreatic isoamylase were compared with findings in an age-matched control group. Preoperatively the volume of secretion was significantly less in the patients than in the controls. Carbonate levels were also decreased, but changes in enzymatic activity were slight. Postoperatively there was significant increase in aspirate volume and fall in the level of duodenal calcium. The results suggested an influence of hyperparathyroidism on exocrine pancreatic function, even when no symptoms of pancreatic disease are present.