Intermediate-dose cytarabine plus mitoxantrone versus standard-dose cytarabine plus daunorubicin for acute myeloid leukemia in elderly patients.

Background: The combination of intermediate-dose cytarabine plus mitoxantrone (IMA) can induce high complete remission rates with acceptable toxicity in elderly patients with acute myeloid leukemia (AML). We present the final results of a randomized-controlled trial comparing IMA with the standard 7 + 3 induction regimen consisting of continuous infusion cytarabine plus daunorubicin (DA). Patients and methods: Patients with newly diagnosed AML >60 years were randomized to receive either intermediate-dose cytarabine (1000 mg/m2 twice daily on days 1, 3, 5, 7) plus mitoxantrone (10 mg/m2 days 1-3) (IMA) or standard induction therapy with cytarabine (100 mg/m2 continuously days 1-7) plus daunorubicin (45 mg/m2 days 3-5) (DA). Patients in complete remission after DA received intermediate-dose cytarabine plus amsacrine as consolidation treatment, whereas patients after IMA were consolidated with standard-dose cytarabine plus mitoxantrone. Results: Between February 2005 and October 2009, 485 patients were randomized; 241 for treatment arm DA and 244 for IMA; 76% of patients were >65 years. The complete response rate after DA was 39% [95% confidence interval (95% CI): 33-45] versus 55% (95% CI: 49-61) after IMA (odds ratio 1.89, P = 0.001). The 6-week early-death rate was 14% in both arms. Relapse-free survival curves were superimposable in the first year, but separated afterwards, resulting in 3-year relapse-free survival rates of 29% versus 14% in the DA versus IMA arms, respectively (P = 0.042). The median overall survival was 10 months in both arms (P = 0.513). Conclusion: The dose escalation of cytarabine in induction therapy lead to improved remission rates in the elderly AML patients. This did not translate into a survival advantage, most likely due to differences in consolidation treatment. Thus, effective consolidation strategies need to be further explored. In combination with an effective consolidation strategy, the use of intermediate-dose cytarabine in induction may improve curative treatment for elderly AML patients.

A papain-induced disc degeneration model for the assessment of thermo-reversible hydrogel-cells therapeutic approach.

Nucleus pulposus (NP) regeneration by the application of injectable cell-embedded hydrogels is an appealing approach for tissue engineering. We investigated a thermo-reversible hydrogel (TR-HG), based on a modified polysaccharide with a thermo-reversible polyamide [poly(N-isopropylacrylamide), pNIPAM], which is made to behave as a liquid at room temperature and hardens at > 32 °C. In order to test the hydrogel, a papain-induced bovine caudal disc degeneration model (PDDM), creating a cavity in the NP, was employed. Human mesenchymal stem cells (hMSCs) or autologous bovine NP cells (bNPCs) were seeded in TR-HG; hMSCs were additionally preconditioned with rhGDF-5 for 7 days. Then, TR-HG was reversed to a fluid and the cell suspension injected into the PDDM and kept under static loading for 7 days. Experimental design was: (D1) fresh disc control + PBS injection; (D2) PDDM + PBS injection; (D3) PDDM + TR-HG (material control); (D4) PDDM + TR-HG + bNPCs; (D5) PDDM + TR-HG + hMSCs. Magnetic resonance imaging performed before and after loading, on days 9 and 16, allowed imaging of the hydrogel-filled PDDM and assessment of disc height and volume changes. In gel-injected discs the NP region showed a major drop in volume and disc height during culture under static load. The RT-PCR results of injected hMSCs showed significant upregulation of ACAN, COL2A1, VCAN and SOX9 during culture in the disc cavity, whereas the gene expression profile of NP cells remained unchanged. The cell viability of injected cells (NPCs or hMSCs) was maintained at over 86% in 3D culture and dropped to ~72% after organ culture. Our results underline the need for load-bearing hydrogels that are also cyto-compatible.

Role of hypoxia and growth and differentiation factor-5 on differentiation of human mesenchymal stem cells towards intervertebral nucleus pulposus-like cells.

There is evidence that mesenchymal stem cells (MSCs) can differentiate towards an intervertebral disc (IVD)-like phenotype. We compared the standard chondrogenic protocol using transforming growth factor beta-1 (TGFß) to the effects of hypoxia, growth and differentiation factor-5 (GDF5), and coculture with bovine nucleus pulposus cells (bNPC). The efficacy of molecules recently discovered as possible nucleus pulposus (NP) markers to differentiate between chondrogenic and IVD-like differentiation was evaluated. MSCs were isolated from human bone marrow and encapsulated in alginate beads. Beads were cultured in DMEM (control) supplemented with TGFß or GDF5 or under indirect coculture with bNPC. All groups were incubated at low (2 %) or normal (20 %) oxygen tension for 28 days. Hypoxia increased aggrecan and collagen II gene expression in all groups. The hypoxic GDF5 and TGFß groups demonstrated most increased aggrecan and collagen II mRNA levels and glycosaminoglycan accumulation. Collagen I and X were most up-regulated in the TGFß groups. From the NP markers, cytokeratin-19 was expressed to highest extent in the hypoxic GDF5 groups; lowest expression was observed in the TGFß group. Levels of forkhead box F1 were down-regulated by TGFß and up-regulated by coculture with bNPC. Carbonic anhydrase 12 was also down-regulated in the TGFß group and showed highest expression in the GDF5 group cocultured with bNPC under hypoxia. Trends in gene expression regulation were confirmed on the protein level using immunohistochemistry. We conclude that hypoxia and GDF5 may be suitable for directing MSCs towards the IVD-like phenotype.

Molecular mechanisms in clathrin-mediated membrane budding revealed through subcellular proteomics.

Subcellular proteomics is a powerful new approach that combines subcellular fractionation and MS (mass spectrometry) to identify the protein complement of cellular compartments. The approach has been applied to isolated organelles and major suborganellar structures and each study has identified known proteins not previously understood to associate with the compartment and novel proteins that had been described only as predicted open-reading frames from genome sequencing data. We have utilized subcellular proteomics to analyse the protein components of CCVs (clathrin-coated vesicles) isolated from adult brain. Accounting for identified fragmented peptides allows for a quantitative assessment of protein complexes associated with CCVs, and the identification of many of the known components of post-fusion synaptic vesicles demonstrates that a main function for brain CCVs is to recycle synaptic vesicles. In addition, we have identified a number of novel proteins that participate in CCV formation and function at the trans-Golgi network and the plasma membrane. Characterization of two of these proteins, NECAP1 and NECAP2, has led to the identification of a new consensus motif that mediates protein interactions with the clathrin adaptor protein 2. These studies highlight the ability of proteomics to reveal new insights into the mechanisms and functional roles of subcellular compartments.

Carotenoids are decreased in biopsies from colorectal adenomas.

UNLABELLED: A lower intake of carotenoids is associated with an increased risk of colorectal cancer. In order to take advantage of the chemopreventive properties of carotenoids, it is necessary to determine carotenoid concentration at the target tissue. As early stages in the adenoma-carcinoma sequence of colorectal cancer might be susceptible to chemoprevention, we sought to determine carotenoid concentrations in biopsies from colorectal adenomas. METHODS: Biopsies from colorectal adenomas and non-involved mucosa were taken from seven patients. For controls, biopsies were obtained from the ascending and descending colon of patients without polyps (n = 5). Concentration of carotenoids (alpha-, beta-carotene, lutein, lycopene, zeaxanthin, beta-cryptoxanthin) were determined by optimizing gradient HPLC-analysis. Results are expressed as pmol/microg DNA. RESULTS: Except for alpha-carotene, all carotenoids could reliably be detected in all specimens. In control patients carotenoid concentrations were highest in the ascending colon, being followed by the descending colon and non-involved mucosa from polyp-carriers. In colorectal adenomas all carotenoids were significantly reduced as compared to-non-involved mucosa (beta-carotene: 0.37 vs 0.19, P<0.03; lycopene: 0.34 vs 0.21, P<0.06, beta-cryptoxanthin: 0.14 vs 0.09, P<0.03, zeaxanthin: 0.18 vs 0.09, P<0.02; lutein: 0.18 vs 0.13,P <0.02). CONCLUSION: All carotenoids investigated are reduced in colorectal adenomas, suggesting that mucosal carotenoids could serve as biomarkers for predisposition to colorectal cancer. Moreover, anti-tumor activity exerted by carotenoids is limited due to mucosal depletion. We speculate that supplementation of a larger array of carotenoids might be beneficial for patients with colorectal adenoma.

The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism.

Stabilization of mRNAs contributes to the strong and rapid induction of genes in the inflammatory response. The signaling mechanisms involved were investigated using a tetracycline-controlled expression system to determine the half-lives of interleukin (IL)-6 and IL-8 mRNAs. Transcript stability was low in untreated HeLa cells, but increased in cells expressing a constitutively active form of the MAP kinase kinase kinase MEKK1. Destabilization and signal-induced stabilization was transferred to the stable beta-globin mRNA by a 161-nucleotide fragment of IL-8 mRNA which contains an AU-rich region, as well as by defined AU-rich elements (AREs) of the c-fos and GM-CSF mRNAs. Of the different MEKK1-activated signaling pathways, no significant effects on mRNA degradation were observed for the SAPK/JNK, extracellular regulated kinase and NF-kappaB pathways. Selective activation of the p38 MAP kinase (=SAPK2) pathway by MAP kinase kinase 6 induced mRNA stabilization. A dominant-negative mutant of p38 MAP kinase interfered with MEKK1 and also IL-1-induced stabilization. Furthermore, an active form of the p38 MAP kinase-activated protein kinase (MAPKAP K2 or MK2) induced mRNA stabilization, whereas a negative interfering MK2 mutant interfered with MAP kinase kinase 6-induced stabilization. These findings indicate that the p38 MAP kinase pathway contributes to cytokine/stress-induced gene expression by stabilizing mRNAs through an MK2-dependent, ARE-targeted mechanism.

Morphometric evaluation of bronchial glands of beagle dogs.

Using lung tissues of beagle dogs, we developed a method for objective estimation of bronchial glands. Defined generations (5th, 10th, 15th and 20th) of bronchi were collected according to the microdissection method by Plopper et al. (1983) [1]. Transverse sections were prepared for both light microscopic and electron microscopic studies. The point counting using prints made by a video copy processor was performed for estimation of the volume density of bronchial glands. Bronchial glands were observed throughout the canine bronchial tree. Moreover, a distinct difference was found in the volume density of bronchial glands depending on the lobe examined and the generation number. Recently, we performed a long-term inhalation study using beagle dogs in order to elucidate toxic effects of sulfur(IV) and acidic sulfate aerosols (Heyder et al., 1994) [2]. In addition to the quantitative analysis of surface epithelia, the volume density of submucosal glands in the bronchial wall was measured according to the method described above. As preliminary result, there was a tendency to an increased volume density of glands in the exposure group. Detailed quantitative evaluation of bronchial glands may enable detection of finite changes induced by low level concentrations of air pollutants.

Iron deficiency and iron overload in Swedish male adolescents.

OBJECTIVES: The present study was undertaken to confirm or reject recent findings indicating a high prevalence of iron deficiency in Swedish male adolescents; a second aim was to study the prevalence of genetic iron overload. DESIGN: The diagnostic criteria were: anaemia: Hb < 130 g L-1 (a): iron deficiency: serum ferritin (SF) < 12 micrograms L-1 + transferrin saturation (TS) < 16% (b): iron deficiency anaemia a + b. Iron overload: SF (90th percentile) + TS (90th percentile) in repeat tests. SETTING: Central Sweden. SUBJECTS: A total 3975 men aged 18 years studied on enrollment into military service. RESULTS: Serum ferritin averaged 36.8 micrograms L-1. Anaemia was present in 0.5%, iron deficiency anaemia in 0.17% and iron deficiency in 0.4%. If iron deficiency is defined as SF < 16 micrograms L-1, as was recently suggested, the prevalence would be 2.8%. Such a cut-off value would include 73% normal people (false positives). Iron overload had the same prevalence as iron deficiency, 0.4%. CONCLUSIONS: Iron stores, as measured by serum ferritin, are small in young men studied at the end of their growth spurt. However, iron deficiency is rare. Therefore, the present study has not been able to confirm the high prevalence of iron deficiency recently reported. A prevalence of genetic haemochromatosis of 0.4%, confirms earlier findings and would mean that 12.6% of the population are heterozygotic carriers of the iron-loading genes. These findings give no support for a proposed, more effective iron-enrichment of food. It is not needed and can be harmful.

Therapy of vulvar carcinoma.

83 vulvar carcinoma patients were originally treated in the period between 1970 and 1990. 82 patients presented with squamous cell carcinoma. 70% of the patients were in Stage I or II. It was originally possible to operate on 74 of the 83 patients. A simple or partial vulvectomy was applied 17 times. A bilateral inguinal lymph node excision additionally took place in 6 cases. 51 patients were subjected to radical vulvectomy with inguinofemoral lymph node excision. In 13 cases, pelvic lymph node extirpation was also performed. A posterior pelvic exenteration was performed in 6 cases presenting extensive carcinoma involvement of the vulva. In the remaining 9 patients, either it was not possible to operate, or a nonradical operation could be performed. The primary morbidity, consisting of wound healing disturbances and infections, amounted to 50% in our group. We observed lymphedema in 47% of the cases, although it was clinically important in only 10%. We did not have any primary surgical mortality. The 5-year survival rate was 82% in our patients without inguinofemoral lymph node involvement and only 40% in lymph node metastatic cases. The absolute 5-year cure rate was 66%, or 69% corrected. To be able to give increased preference to less invasive methods an improved prevention and clarification procedure for physicians and patients is necessary.

The effect of iron fortification of the diet on clinical iron overload in the general population.

The gene coding for idiopathic hemochromatosis is prevalent in Sweden, the country with the highest iron fortification of food (42%) in the world. We wanted to study if this highly iron-fortified diet had negative effects on the iron situation in carriers of the iron-loading genes. Iron stores averaged 6.7 grams in male homozygotes who were mainly identified through laboratory screening. It was 3.4 grams in female homozygotes. By HLA typing of family members of these homozygous probands, 39 additional homozygotes and 172 heterozygotes were detected. Serum ferritin averaged 620 micrograms/l in 20 male and 168 micrograms/l in 19 female homozygotes in the family screening. Storage iron as measured by serum ferritin concentration was slightly but significantly higher in male heterozygotes than controls (117 micrograms/l versus 87 micrograms/l, p less than 0.02). There was no further increase in serum ferritin concentration with age after 40 years. Heterozygotes showed no clinical signs of iron damage. These findings do not indicate that carriers of the iron-loading genes in Sweden have been adversely affected by the highly iron-fortified diet of the country.

Acholeplasma laidlawii cells acutely and chronically infected with group 1 acholeplasmavirus.

Interactions between group 1 acholeplasmaviruses and their host cells were studied. Acutely infected, chronically infected and uninfected cultures of Acholeplasma laidlawii strain JA1 were compared by their growth in broth and on agar, by the sensitivities of the uninfected and chronically infected cells to representatives of each of the three groups of acholeplasmaviruses, and by their SDS-PAGE polypeptide profiles. Acutely infected cells resembled uninfected cells by these criteria, except for the fact that progeny virus was being released. Two types of chronically infected cells were found:rapid growers (the same doubling time as uninfected cells) and slow growers. The latter resembled uninfected cells, except for their slower growth and low-level release of virus, and the former was resistant to group 1 viruses and had a unique polypeptide profile. These biological characterizations help to establish the non-lytic, non-cytocidal cycle of the group 1 acholeplasmaviruses.

Liver affection in iron overload studied with serum ferritin and serum aminotransferases.

Liver dysfunction as measured by S-ALAT activity was present in 72% of patients over 40 years of age with HLA-related iron overload, mainly detected by laboratory screening. Liver dysfunction was correlated to the amount of iron stored (r = 0.54, p less than 0.001). When iron was removed by phlebotomy, liver function returned to normal. S-ALAT activity was closely correlated to serum ferritin concentration (r = 0.73, p less than 0.001). Even a mild iron excess can affect hepatocytes and result in increased levels of ferritin and aminotransferases in serum. Patients with "transaminitis" should be investigated for iron overload.

Screening for iron overload using transferrin saturation.

People with parenchymal iron overload exhibit an elevated serum iron concentration and a raised transferrin (TIBC) saturation early in the course of the disease. They can therefore be detected by simple laboratory tests before organ damage has occurred. In this study running for 2 months, 10512 samples from approximately 8750 patients and blood donors were examined in a county hospital in Central Sweden. Abnormal TIBC saturation (greater than 70%) was found in 1.7% of the samples. This abnormality was caused by physiological fluctuations in serum iron in 44%, liver disease in 22%, blood disorder in 10%, iron therapy in 10.5% and parenchymal iron overload in 11.5%. The diagnosis of iron overload was confirmed by measuring the serum ferritin concentration and by performing the desferrioxamine test, liver biopsy, quantitative phlebotomy and family studies including HLA typing. We found a prevalence of iron overload of 0.24%. This figure is almost certainly too low because some affected patients were probably lost because of TIBC desaturation induced by inflammatory conditions.

Prevalence of iron overload in central Sweden.

An increase in the iron content of food may be harmful to people with genetic hemochromatosis. We studied the prevalence of this disorder in Sweden, which is the country with the world's highest iron fortification of food. Serum ferritin and transferrin (TIBC) saturation levels were used as initial screening methods. Three (0.5%) of 623 males aged 30-39 years were found to have genetic hemochromatosis. Family studies revealed 10 additional homozygotic family members. A prevalence of 0.5% of homozygotes (q2) implies a gene frequency (q) of 6.9% or a heterozygote frequency (2 x Q) of 13.8%. The high gene frequency may be explained by a possible genetic advantage of heterozygotes in the past. We conclude that idiopathic hemochromatosis is not as rare as previously thought. Affected persons should be detected and treated before irreversible organ damage occurs. This study demonstrates that serum ferritin levels together with TIBC saturation levels are adequate methods for screening populations.

[Improved flow through aortocoronary venous bypasses after anticoagulant therapy. A prospective randomized study]. / Verbesserte Durchgängigkeit aorto-koronarer Venen-Bypasses nach Antikoagulantientherapie. Eine prospektive randomisierte Studie.

To evaluate the effect of oral anticoagulant therapy on graft patency rate during the first 2 months after bypass surgery 174 patients were randomly assigned to treatment with phenprocoumon (89) or to a control group (85) starting on day 7 after bypass surgery. Until day 7 all patients received low dose heparin. There was no significant difference between the two groups with respect to age, sex distribution, number of vessels diseased, left ventricular enddiastolic pressure, preoperative exercise tolerance or number of grafts constructed per patient. All patients underwent angiographic evaluation 8 weeks after bypass surgery. Graft patency rate was 90.4% in the treatment group versus 83.6% in the control group (p less than 0.015). None of the grafts with a flow rate of greater than 90 ml/min was occluded 8 weeks after surgery. Oral anticoagulation improved the patency rate of grafts with a flow of less then 90 ml/min.

Improved graft patency with anticoagulant therapy after aortocoronary bypass surgery: a prospective, randomized study.

We conducted a prospective, randomized study to evaluate the influence of oral anticoagulation on graft patency early after aortocoronary bypass surgery. Eighty-nine patients who received 251 distal venous anastomoses were treated with phenoprocoumon, a vitamin K antagonist, starting on the seventh postoperative day; 84 patients with 238 distal venous anastomoses received no anticoagulation. Both groups were comparable with respect to age, exercise hemodynamics, extent of coronary disease and left ventricular dysfunction. In each group, 2.8 distal anastomoses were constructed per patient. Graft patency after surgery was 90.4% in the treatment group and 84.6% in the control group (p less than 0.015). All anastomoses were patent in 81% and 67% of patients, respectively (p less than 0.02). Flow measurements in 279 grafts suggest that grafts with a flow of less than 90 ml/min benefit from oral anticoagulation. No graft with a flow of more than 90 ml/min was occluded.

Combined radionuclide circulation and static imaging of the brain. Space-occupying lesions.

Combined static and dynamic imaging of the brain was performed in 398 patients with suspected space occupying lesions. The imaging findings were analyzed from two points of view: Firstly, the radionuclide results were compared with pathological anatomical findings and, secondly, the diagnostic yield in patients with various symptoms was evaluated. Thus we obtained the number of false positive and false negative results. The findings of the combined imaging procedure were correctly negative in 85% of the patients with non-focal symptoms and false positive in only 1%. The circulation study alone gave false positive results in 7% and so did the static study alone. The combined imaging procedure was pathologic in 46 of 83 cases of space-occupying lesions (55%). There were only three patients in whom a pathologic circulation study was the only indication of a space-occupying lesion (4%). The static study alone gave correct positive findings in 23 more cases (28%). We conclude that a pathologic blood flow study alone is of limited interest. In the presence of a pathologic static study the circulation procedure gives additional information, especially in meningioma and subdural hematoma. In patients with symptoms indicative of supratentorial space-occupying lesions of the brain combined imaging is recommended.