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Erythrocytosis is associated with increased red blood cell mass and can be either congenital or acquired. Congenital secondary causes are rare and include germline variants increasing haemoglobin (Hb)-oxygen affinity (e.g., Hb or bisphosphoglycerate mutase (BPGM) variants) or affecting oxygen-sensing pathway proteins. Here, we describe five adults from three kindreds with erythrocytosis associated with heterozygosity for BPGM variants, including one novel. Functional analyses showed partial BPGM deficiency, reduced 2,3-bisphosphoglycerate levels and/or increased Hb-oxygen affinity. We also review currently known BPGM variants. This study contributes to raising awareness of BPGM variants, and in particular that heterozygosity for BPGM deficiency may already manifest clinically., (© 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Background: Chemotherapy (CT)-induced neutropenia and febrile neutropenia (FN) can lead to changes in the treatment plan, potentially worsening the cancer outcome. This study evaluated the effect of the glycopegylated granulocyte-colony stimulating factor lipegfilgrastim, used as primary (PP) or secondary prophylaxis (SP), on treatment modifications in adult patients receiving cytotoxic CT with or without biological/targeted therapy (BT) for solid and haematological tumours., Methods: This phase 4, prospective, observational study was conducted in eight centres in the Netherlands, in 2015-2017. Other study objectives were to characterise the population of cancer patients receiving lipegfilgrastim, to evaluate the incidence of CT-induced neutropenic events, and to assess safety., Results: Of 142 patients, 73.94% had breast cancer and 55.63% received CT in the adjuvant setting. Most patients received lipegfilgrastim as PP (74.65%) and were at low (34.51%) or high risk (39.44%) of FN. CT dose delays were recorded for 22.64% and 36.11% of patients receiving lipegfilgrastim for PP and SP, respectively. CT dose reductions were recorded for 2.11% of patients; no CT dose omissions and one BT dose omission occurred. FN and grade III/IV neutropenia were reported for 5.63% and 9.86% of patients, respectively; associated hospitalisations were rare. The most frequently lipegfilgrastimrelated adverse events (AE) were myalgia, bone pain, and back pain. Serious AEs (55) were reported for 30 (21.13%) patients. There were two deaths, unrelated to lipegfilgrastim administration., Conclusion: Administration of lipegfilgrastim in routine clinical practice in the Netherlands results in limited CT/BT dose modifications and low incidence of neutropenic events, with no new safety concerns.

Background: This study evaluated the effectiveness of a screening and stepped care program (the TES program) in reducing psychological distress compared with care as usual (CAU) in patients with metastatic colorectal cancer starting with first-line systemic palliative treatment., Patients and Methods: In this cluster randomized trial, 16 hospitals were assigned to the TES program or CAU. Patients in the TES arm were screened for psychological distress with the Hospital Anxiety and Depression Scale and the Distress Thermometer/Problem List (at baseline and 10 and 18 weeks). Stepped care was offered to patients with distress or expressed needs, and it consisted of watchful waiting, guided self-help, face-to-face problem-solving therapy, or referral to specialized mental healthcare. The primary outcome was change in psychological distress over time, and secondary outcomes were quality of life, satisfaction with care, and recognition and referral of distressed patients by clinicians. Linear mixed models and effect sizes were used to evaluate differences., Results: A total of 349 patients were randomized; 184 received the TES program and 165 received CAU. In the TES arm, 60.3% of the patients screened positive for psychological distress, 26.1% of which entered the stepped care program (14.7% used only watchful waiting and 11.4% used at least one of the other treatment steps). The observed low use of the TES program led us to pursue a futility analysis, which showed a small conditional power and therefore resulted in halted recruitment for this study. No difference was seen in change in psychological distress over time between the 2 groups (effect size, -0.16; 95% CI, -0.35 to 0.03; P>.05). The TES group reported higher satisfaction with the received treatment and better cognitive quality of life (all P<.05)., Conclusions: As a result of the low use of stepped care, a combined screening and treatment program targeting psychological distress in patients with metastatic colorectal cancer did not improve psychological distress. Our results suggest that enhanced evaluation of psychosocial concerns may improve aspects of patient well-being.

Hepatocellular carcinoma (HCC) is the most common primary hepatic cancer. As it often presents at an advanced stage, it generally has a poor prognosis. Spontaneous regression is a rare finding in HCC, and is often associated with tumour ischemia or a systemic inflammatory response. We herein present the case of a Caucasian patient with spontaneous regression of advanced HCC, not associated with either mechanism. Our patient was a 74-year old Caucasian male who presented with advanced biopsy-proven HCC, with α-fetoprotein levels of >16,600 kU/l. On computed tomography examination, multiple metastases were identified. Due to the poor performance status and as the patient declined treatment, he was referred to a general practitioner for supportive palliative care. Six months later, the patient was alive and feeling well. He had suffered a cerebrovascular accident with ensuing partial hemiparesis, and had been initiated on enalapril, furosemide and curcumin. On re-evaluation, the lung lesions had disappeared, whereas both the liver and peritoneal lesions had decreased in size. Following a review of the literature, the conclusion was that this case of spontaneous regression of advanced HCC was not caused by ischemia, inflammation, or any other previously reported mechanism. Further research is required to elucidate the mechanisms underlying spontaneous regression of HCC.

Background: Carcinoembryonic antigen is the commonly used tumor marker in patients with colorectal cancer, and CA 19-9 might be an additional marker. The aim of this retrospective study was to investigate whether CA 19-9 levels can be used to monitor the disease process in patients with colorectal cancer who had no elevated CEA levels. The secondary aim was to determine if preoperative increased levels of CEA and CA 19-9 were associated with mortality., Materials and Methods: Two sets of data from patients with histologically confirmed colorectal cancer, were included in a single-center study. First, patients with a minimum of 3 serial measurements of CA 19-9 and CEA tumor markers were related to the clinical course of their disease. Second, patients with preoperative levels of CEA and CA 19-9 were related to survival., Results: In patients with colorectal cancer and 3 serial measurements of tumor markers, 7.3% had only increased CA 19-9 levels without increased CEA levels, and 55.4% of the patients had an increase of CA 19-9 and CEA levels. In the patients with available preoperative markers, patients with only an increase of CA 19-9 had a significantly decreased 5-year survival compared with patients with an increase of only CEA (P = .013)., Conclusion: CA 19-9 can be used as additional marker to follow the disease process in patients with colorectal cancer without an increase in CEA level. Patients with preoperative increased CA 19-9 level had a poorer 5-year survival than patients with preoperative increased CEA levels., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Patients with von Willebrand disease (VWD), the most common inherited bleeding disorder, display large variation in bleeding tendency, which is not completely related to VWF levels. The cause of variability in clinical expression is largely unknown. The effect of plasma fibrinolytic capacity on bleeding tendency in VWD patients has not been investigated. We hypothesized that enhanced fibrinolysis may result in a more severe bleeding phenotype. Therefore, we measured the fibrinolytic potential in patients with moderate or severe VWD to investigate the contribution of fibrinolysis to the bleeding tendency. Fibrinolytic potential was measured as plasma clot lysis time (CLT) with and without addition of potato carboxypeptidase inhibitor (PCI) in 638 patients with moderate or severe VWD who participated in a nationwide multicentre cross-sectional study. Bleeding severity was measured using the Bleeding Score (BS).The CLTs were significantly longer, indicative of hypofibrinolysis, in men compared to women with VWD [106.2 (IQR 95.7-118.1) vs. 101.9 (IQR 92.8-114.0) min]. The CLTs prolonged with increasing age. No association was found between VWF or FVIII levels and CLT, or between VWF or FVIII levels and CLT(+PCI) . No association was observed for BS in a model with 10log-transformed CLT, adjusted for age, gender, VWF:Act and FVIII [b = 6.5 (95%CI -0.3 to 13.4)]. Our study showed that the plasma fibrinolytic potential does not influence bleeding tendency in VWD patients and therefore does not explain the variability in bleeding phenotype in VWD., (© 2011 Blackwell Publishing Ltd.)

Nonafact(®), an ultrapure, monoclonal antibody-purified factor IX concentrate (FIX) was developed to minimize risk of thrombotic complications and viral transmission. To investigate the pharmacokinetics, efficacy and safety, phase III/IV studies were performed in the Netherlands and Poland from 1996 to 2007. The mean half-life, in vivo response and recovery of Nonafact(®) were 18.7 (SD 2.0) h, 1.1 (SD 0.2) IU dL(-1) per IU kg(-1) b.w. of FIX infused and 49% (SD 10%), respectively. Eleven surgical procedures were performed in eight patients. During two surgeries, both high-risk, blood loss was observed. No postoperative bleeding occurred. The in vivo recovery of FIX was higher than expected. In the phase III follow-up study, 26 previously treated patients (PTP) were included with a median follow-up of 1130 days. From the 1617 minor bleedings, 80.5% was stopped after a single infusion. In the phase IV study thirteen patients were treated for a median study period of 737 days. In the two follow-up studies the investigators rated the effect of Nonafact(®) as excellent/good in 95% of major bleedings. Surgeries for which Nonafact(®) was given prophylactically were without bleeding problems. In total more than 10 million units of Nonafact(®) were used during almost 120 person-years. Only one minor adverse event was reported. No inhibitors, viral transmissions and thrombogenic events occurred. In conclusion, Nonafact(®) is safe and provides excellent haemostasis in haemophilia B patients treated for spontaneous bleeding or undergoing surgical procedures. Due to the excellent in vivo recovery characteristic, treatment with Nonafact(®) is cost saving compared to other FIX products., (© 2011 Blackwell Publishing Ltd.)

Although hypercalcaemia is often encountered during the course of malignant disease, hypocalcaemia appears to be rather rare. We describe a 37-year-old patient with metastatic carcinoma of the breast, who developed extreme hypocalcaemia (as low as 0.75 mmol calcium per litre) after chemotherapy. This is caused by a combination of hungry-bone syndrome and an insufficient parathyroid response. The latter may be the result of a direct toxic effect of chemotherapy on parathyroid hormone (PTH) synthesis possibly in combination with microscopic tumour infiltration in the parathyroid glands. Correction of the extreme hypocalcaemia over a period of 100 days by oral and intravenous calcium supplementation, corresponding to a total of 352 gram elemental calcium (1/3 of the total body calcium), resulted in gradual symptomatic relief. The possible mechanisms for these findings are discussed and the literature is briefly reviewed.

We have prospectively monitored treatment of haemophilia patients with inhibitors by recombinant factor VIIa (rFVIIa) administered by continuous infusion to obtain more insight in the underlying factors of the clinical efficacy of this administration method. At present, 43 treatment episodes of 14 different Dutch haemophilia inhibitor patients are included in the database. Analysis of the data showed a discrepancy between the efficacy of rFVIIa continuous infusion treatment of acute and surgical bleeds in the oral cavity [one (14%) effective, two (29%) partially effective, four (57%) not effective] and other parts of the body [29 (80%) effective, four (11%) partially effective, two (6%) not effective, one (3%) impossible to classify]. Patients who had acute or surgical oral cavity bleeds, uncontrolled by rFVIIa continuous infusion, reacted favourably to rFVIIa continuous infusion in other locations of the body. Acute bleeding episodes in the oral cavity, which could not be controlled by rFVIIa continuous infusion, stopped when the treatment regimen was switched to rFVIIa bolus injections. Finally, haemostatic control during dental extractions was excellent after the initial rFVIIa bolus injection preceding the continuous infusion, but rebleeds occurred in all patients within 48 h under rFVIIa continuous infusion coverage. These observations suggest that the efficacy of rFVIIa continuous infusion depends, at least in part, on the location of the body in which the bleeding occurs and that rFVIIa bolus injections are more effective than rFVIIa continuous infusion in the oral cavity. We hypothesize that the inability of rFVIIa continuous infusion treatment to sufficiently inhibit fibrinolysis is the underlying cause of the decreased efficacy of rFVIIa continuous infusion treatment in the oral cavity.

Objective: Our purpose was to resolve the apparent contradiction between the endothelium-dependent and endothelium-independent vasodilator effects of 17 beta-estradiol reported in different studies., Study Design: The inner diameters of isolated pressurized spontaneously constricted muscle arterioles (diameter = 63 microm) from Wistar rats (n = 21) were measured during exposure to 17 beta-estradiol, and the role of the endothelium and the influence of sex were assessed., Results: A dose-dependent dilatation was observed during exposure to 17 beta-estradiol concentrations from 10(-10) to 10(-4) mol/L. Arterioles of female rats displayed significantly more dilatation than vessels from male rats. The dilatation was significantly less in endothelium-denuded arterioles or after pretreatment with and in the presence of a nitric oxide synthase inhibitor., Conclusions: These results provide strong evidence that, in addition to an endothelium-independent effect, 17 beta-estradiol has a dose-dependent, endothelium-mediated, rapid vasodilatory effect on muscle arterioles from the rat, which is stronger in female rats than in male rats.

Idiopathic thrombocytopenic purpura (ITP) can follow a viral infection. We describe severe thrombocytopenia in two adult patients preceded by varicella zoster and Epstein-Barr virus infection, respectively. The differences between acute and chronic ITP are discussed, as well as therapeutic options.

Haemophilia A is usually a genetic deficiency of coagulation factor VIII (F VIII). The development of antibodies against F VIII is a well known and frequent complication in the treatment of haemophilia A. Rarely, a F VIII inhibitor arises spontaneously, causing a condition which is known as acquired haemophilia A. We describe a patient with acquired haemophilia A and pemphigus, who presented with spontaneous haematomas of the extremities. Laboratory tests showed an activated partial thromboplastin time (aPTT) of 71 s (normal: 26-36 s), a F VIII concentration of 9% (normal: 60-140%), and a F VIII inhibitor-activity of 7.5 Bethesda Units/ml (B.U./ml, normal: 0). The haematomas disappeared within a few days and the laboratory tests normalized within 6 weeks, after administration of a booster of oral corticosteroids. One and a half years after the corticosteroids were stopped, both the clinical and the laboratory course of the patient has been uneventful. As far as we know, the combination of acquired haemophilia A and pemphigus has been reported in the literature only three times before. The diagnosis acquired haemophilia A should be considered in a patient presenting with a newly arisen haemorrhagic diathesis.

Background: Venom immunotherapy is effective in preventing systemic reactions in patients with a history of an anaphylactic reaction to Hymenoptera stings. It is uncertain how long venom immunotherapy should be continued., Objective: We evaluated whether the duration of venom immunotherapy given to yellow jacket-sensitive patients related to the risk of an anaphylactic reaction to a later sting., Methods: Seventy-five yellow jacket-sensitive patients (29 male and 46 female) received a median number of three in-hospital sting challenges from a live insect in 3 subsequent years after discontinuation of venom immunotherapy. An anaphylactic reaction to one or more of the sting challenges was considered a relapse. We analyzed whether patients with and patients without a relapse differed in terms of gender, age, preimmunotherapy skin test data, preimmunotherapy level of venom-specific IgE, severity of the field-sting reaction that preceded immunotherapy, severity of the reaction to the sting challenge that preceded immunotherapy, adverse reactions to immunotherapy, changes in IgE and IgG4 levels during immunotherapy, duration of immunotherapy, and presence of venom-specific IgE after cessation of therapy., Results: Venom immunotherapy was given for a median duration of 40 months (range, 7 to 120 months). Relapses were observed in six patients. In two of them, a rather severe anaphylactic reaction was observed after the second sting challenge. No relation was found between duration of venom immunotherapy and relapse risk. The relapse rate was higher among patients with high levels of specific IgE before and after immunotherapy. During therapy, the mean level of specific IgE decreased. This decline persisted in the 3 following years. No relapses of sting reactions were observed among patients without detectable specific IgE., Conclusion: Discontinuation of venom immunotherapy appears safe for patients with pretreatment IgE antibodies if these antibodies can no longer be detected during immunotherapy. For the remaining patients, a treatment period of 3 years may suffice. After discontinuation of immunotherapy, a clinical sting challenge can be considered to estimate the patient's current grade of hypersensitivity.

Background: Patients with a history of a serious anaphylactic reaction after a Hymenoptera sting are usually given venom immunotherapy. Because the natural history of Hymenoptera sting anaphylaxis is often of a declining severity, there is a chance of overtreatment., Objective: Identification of patients at risk for a future anaphylactic reaction may reduce the number of patients who need venom immunotherapy., Methods: We investigated the relation between the grade of hypersensitivity to an in-hospital sting challenge and the reaction to a subsequent accidental field sting. From 1982 through 1992, 348 patients with mild or no symptoms after a sting challenge were not given venom immunotherapy. All patients were asked by letter whether they had experienced a subsequent field sting. In case of a sting, the severity of the reaction was further evaluated., Results: Information could be obtained from 327 patients: 129 had been re-stung, and 110 of them had only had a local reaction. Thirteen patients had experienced mild systemic symptoms, and six patients had experienced serious manifestations. In two of the latter group hypotension was observed., Conclusion: In 95% of patients with a previous anaphylactic reaction, the result of the in-hospital sting challenge provided a good prediction of tolerance to a subsequent Hymenoptera field sting.

Three hundred twenty-four patients with a history of yellow jacket- (n = 272) or honeybee- (n = 52) sting anaphylaxis were prospectively subjected to an in-hospital sting challenge. Plasma levels of specific IgE and IgG4, skin venom tests, severity of previous reaction, sex, age, atopic constitution, histamine skin test results, location and number of previous stings, time interval between previous anaphylactic reaction and sting challenge, and time interval between sting challenge and onset of anaphylaxis were studied in relation to the clinical severity of a reaction after sting challenge. A recurrent anaphylactic reaction after sting challenge was observed in 25% of yellow jacket- and in 52% of honeybee-sensitive persons. The severity of this reaction correlated significantly with age and the time interval between sting challenge and onset of anaphylaxis only: older persons with faster reactions had more severe symptoms after sting challenge. None of the current criteria for insect-sting hypersensitivity (IgE, IgG4, skin test) significantly related on an individual basis or in combinations to the reaction after sting challenge. We conclude that the current criteria to assess insect-venom hypersensitivity do not relate to the occurrence and severity of anaphylactic symptoms after an insect-sting challenge.

Tryptase, a neutral protease, is selectively concentrated in the secretory granules of human mast cells, and its release into the circulation serves as a clinical marker of mast cell activation. The current study describes a new, more sensitive ELISA utilizing a newly developed, mouse monoclonal IgG1 antibody for capture called B12 and capable of detecting tryptase in normal plasma and serum. The greater sensitivity of the new immunoassay results in part from a greater portion of tryptase being detected. Mean levels of tryptase in serum from normal subjects from Richmond, Virginia (4.9 ng/ml; n = 56), Munich, Germany (3.8 ng/ml; n = 19), and Amersfoort, The Netherlands (1.9 ng/ml; n = 8) were as indicated. In 62 subjects with ongoing allergic rhinitis, tryptase levels were no different in serum than for 19 normal controls, indicating that local mast cell activation is not necessarily reflected in the circulation. In 61 subjects sensitive to honey bee or yellow jacket venom by history, the 17 destined to have a severe, hypotensive response to a sting challenge had higher levels of tryptase at baseline than mild reactors, nonreactors, and controls, suggesting that baseline levels of tryptase may predict the severity of the clinical response to allergen in sensitive subjects.

Objective: To study the rate and severity of anaphylactic reaction in relation to plasma levels of cardiovascular mediators in persons with a history of insect-sting anaphylactic shock who were rechallenged with a sting by the same insect., Design: A cohort study with measurements before and after intentional sting challenge., Setting: Intensive care unit of an 830-bed general hospital, a national center of insect-sting anaphylaxis in The Netherlands., Patients: A total of 138 patients referred after a previous anaphylactic reaction to a Hymenoptera sting; and 8 volunteers., Measurements: Signs of anaphylaxis and plasma levels of catecholamines and angiotensins., Main Results: Only 39 of 138 (28%) of patients with a previous insect-sting anaphylactic reaction developed anaphylactic symptoms after sting challenge. Values of cardiovascular mediators and mean arterial pressure did not differ after the challenge from initial values in the volunteers or in the patients with a mild or no reaction after challenge. In the 17 patients with anaphylactic shock, mean arterial pressure decreased from 97 +/- 11 (mean +/- SD) to 65 +/- 17 mm Hg (P < 0.001), epinephrine levels rose from a median of 0.3 nmol/L (range, 0.2 to 2.3 nmol/L) to 2.5 nmol/L (0.2 to 35.7 nmol/L; P < 0.05), norepinephrine from 1.5 nmol/L (0.5 to 6.7) to 5.9 nmol/L (1.6 to 30.9 nmol/L; P < 0.01), and angiotensin II from 61 pmol/L (7 to 217 pmol/L) to 105 pmol/L (11 to 286 pmol/L; P < 0.01), all within 5 minutes after the onset of anaphylactic symptoms. The rise of these mediators correlated with the drop in blood pressure (P < 0.001). Dopamine and angiotensin I levels did not change in any participants., Conclusions: A recurrent insect-sting anaphylactic reaction occurred in only 28% of patients with a previous reaction. During this recurrent reaction, plasma levels of endogenous epinephrine, norepinephrine, and angiotensin II rose in relation to hypotension.

One hundred thirty-eight patients with a previous anaphylactic reaction to a yellow jacket or a honeybee sting, as well as eight volunteers, were subjected to an in-hospital sting challenge. Plasma levels of histamine, tryptase, and prostaglandin D2 (PGD2) during sting challenge were studied in relation to clinical symptoms. Prechallenge levels (mean +/- SD) of histamine, tryptase, and PGD2 were 2 +/- 1 nmol/L, 0.3 +/- 0.3 U/L, and 320 +/- 223 ng/L, respectively. In the volunteers and in none except for one of the nonreacting patients, these levels did not change significantly after challenge. In contrast, mean increases in the group of 18 patients with a mild reaction were significant for histamine and tryptase at one or more time points after the challenge. (Five patients demonstrated no increase in histamine; nine demonstrated no increase in tryptase.) Except for histamine levels in one patient, these increases were considerably more in all 17 patients with a severe reaction, starting from the first anaphylactic symptoms. Fifteen minutes later, peak values were reached of 1275 +/- 2994 nmol of histamine per liter (range, 3 to 12800 nmol/L; median, 11 nmol/L) and 406 +/- 1062 U of tryptase per liter (range, 1.8 to 4400 U/L; median, 17 U/L). This rise in levels inversely correlated with the mean arterial pressure. Plasma levels of PGD2 in severely reacting patients did not differ significantly from those in patients with a mild or no reaction. In conclusion, only 28% of patients with a history of Hymenoptera anaphylaxis developed an anaphylactic reaction after an in-hospital challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

The generation of the anaphylatoxin C3a was measured after a wasp-sting challenge in eight patients with previous anaphylactic reactions to wasp stings. Whereas there was no change in C3a in one patient who showed no reaction and only a slight rise in three patients with mild reactions, C3a rose substantially in the four patients with severe anaphylactic reactions. This complement activation is the first in-vitro variable which correlates with the severity of wasp-sting anaphylactic reactions. A role for complement activation in the pathophysiology of wasp-sting anaphylaxis is therefore suggested.

Propidium iodide (PI) and diamidinophenylindol (DAPI) were compared with ethidium bromide (EB) in cytotoxicity tests. Double blind studies showed similar results with the 3 dyes in terms of percentages of dead (fluorescing) cells. The dyes were also investigated for their potential combination with the immunofluorescent membrane markers FITC and TRITC. Both EB-FITC and PI-FITC are suitable for conventional HLA-DR typing procedures. Because of its spectral properties, the DAPI-TRITC combination is promising for automated read out of DR-typing tests by image analysis systems.

As part of a study of the antigenic composition of the tegument of mechanically transformed Schistosoma mansoni schistosomula, the phenomenon of coat formation around such schistosomula when incubated in medium containing serum of S. mansoni infected mice was investigated with light and scanning electron microscopy. Coat formation was only observed on freshly transformed schistosomula, while schistosomula that had been incubated overnight at 37 degrees C in medium without serum from infected mice were no longer able to produce a coat. When freshly transformed schistosomula were incubated in medium containing serum from infected mice, it was found that individual spines on the tegument were covered with a coat within 1 h. After 2 h of incubation, the schistosomula were completely covered with a thick coat, starting at the posterior end. This coat was actively shed by the schistosomula after 3 h of incubation. To investigate the mechanism of coat formation, experiments were carried out using serum from uninfected mice, heat-inactivated serum from infected mice, and the IgG fraction of serum from infected mice. From the experiments it was concluded that the coat consisted of antigen-antibody complexes, involving IgG antibodies and complement. A detailed analysis of the easily obtainable coats may lead to a better understanding of the antigenic structure of the cercarial and schistosomular tegumental membrane.

The following fluorochromes were studied as probes for discrimination between living and dead Schistosoma mansoni schistosomula: ethidium bromide (EB), propidium iodide (PI), diamidinophenylindole (DAPI), and carboxyfluoresceine diacetate (C-FDA). While schistosomula stained with EB, PI, or C-FDA showed leakage of fluorochrome into the medium, this was not the case with DAPI. Dead schistosomula, which were stained with DAPI, showed an intense blue fluorescence, while living schistosomula were not stained even after prolonged incubation. In addition, the low DAPI concentration (1 microgram/ml) in the medium proved not to be toxic to the schistosomula, nor did it cause any background fluorescence. These properties make DAPI an ideal probe: the viability of S. mansoni schistosomula in cytotoxicity tests can be continuously monitored in tissue culture trays, using an inverted microscope with simultaneous transmitted light and incident fluorescent light illumination.

Out of 24 primary mammary tumors, arising in rats of the WAG/Rij Wistar strain after low dose irradiation, with or without prolonged treatment with estrogen, a slow-growing, well differentiated adenocarcinoma (MCR-83) was selected. This tumor, induced by radiation alone, is independent of estrogen pellets for growth after transplantation into adult female rats, but nontransplantable into males or ovariectomized females. Measurements of tumor growth and contents of both estrogen and progesterone receptors on three successive passages are not indicative of a rapid progression in growth rate or to hormone independency. Ovariectomy and treatment with tamoxifen give a pronounced inhibition of tumor growth, whereas neither methotrexate nor cyclophosphamide is effective. Growth rate is significantly increased when rats are given 17 beta-estradiol. Flow cytometric DNA analysis as well as in situ S-phase cell detection with anti-bromodeoxyuridine antibodies show a 3-fold increase in S-phase fraction cells within 4 days after the onset of estrogen treatment. No spontaneous metastases have been found so far, but lung nodules develop after i.v. inoculation of tumor cells. From one of these nodules a fast-growing, hormone independent subline (MCR-86) has been derived, showing both lymphatic and hematogenous dissemination upon s.c. transplantation. By showing several features of hormone responsive human disease in its early stage of progression the MCR-83 tumor system may be a clinically relevant model for studies on endocrine regulation of tumor growth and its therapeutic manipulation.