Amifostine (WR-2721), a cytoprotective agent during high-dose cyclophosphamide treatment of non-Hodgkin's lymphomas: a phase II study

Clinical trials indicate that amifostine may confer protection on various normal tissues without attenuating anti-tumor response. When administered prior to chemotherapy or radiotherapy, it may provide a broad spectrum of cytoprotection including against alkylating drugs. The mechanism of protection resides in the metabolism at normal tissue site by membrane-bound alkaline phosphatase. Toxicity of this drug is moderate with hypotension, nausea and vomiting, and hypocalcemia being observed. We report a phase II study using amifostine as a protective drug against high-dose cyclophosphamide (HDCY) (7 g/m2), used to mobilize peripheral blood progenitor cells (PBPC) and to reduce tumor burden. We enrolled 29 patients, 22 (75.9%) affected by aggressive and 7 (24.1%) by indolent non-Hodgkin's lymphoma (NHL), who were submitted to 58 infusions of amifostine and compared them with a historical group (33 patients) affected by aggressive NHL and treated with VACOP-B followed by HDCY. The most important results in favor of amifostine were the reduction of intensity of cardiac, pulmonary and hepatic toxicity, and a significant reduction of frequency and severity of mucositis (P = 0.04). None of the 29 patients died in the protected group, while in the historical group 2/33 patients died because of cardiac or pulmonary toxicity and 2 patients stopped therapy due to toxicity. Amifostine did not prevent the aplastic phase following HDCY. PBPC collection and hematological recovery were adequate in both groups. The number of CFU-GM (colony-forming units-granulocyte/macrophage) colonies and mononuclear cells in the apheresis products was significantly higher in the amifostine group (P = 0.02 and 0.01, respectively). Side effects were mild and easily controlled. We conclude that amifostine protection should be useful in HDCY to protect normal tissues, with acceptable side effects.

Clinical results of the use of mitotane for adrenocortical carcinoma

Mitotane (o,p'-DDD) acts mainly as an inhibitor of intramitochondrial pregnenolone and cortisol synthesis. Its adrenolytic effect depends on metabolic activation due to conversion to o,p'-DDA and o,p'-DDE. The drug has been used for 40 years in the treatment of adrenocortical carcinoma, mainly its regional and metastatic stage, as an adjuvant to surgical resection of the tumor. In the medical literature there are controversial opinions about its efficacy for the treatment of adrenocortical carcinoma. In our experience, mitotane administered immediately after surgery appeared to be much more efficient than when administered later. We have administered this drug in all cases of microscopically confirmed adrenocortical carcinoma, irrespectively of stage at the time of surgery, for fear of a false too optimistic classification. In our series of 82 patients with adrenocortical carcinoma, 59 patients have been treated with mitotane, 32 of them immediately after surgery, and 27 with a delay of 2 to 24 months. Today there are 18 survivors in the group of patients treated with mitotane soon after the operation and only 6 survivors in the group receiving mitotane with a delay. All patients were simultaneously given replacement therapy. Undesired effects of mitotane administration included increased aminotransferase and alkaline phosphatase activity, decreased white cell, platelet or red cell number, and myasthenia. Furthermore, we used mitotane with good results in Cushing's syndrome of non-malignant origin as pre-treatment before surgery or in long-term treatment for patients with poor tolerance of other adrenal inhibitors.

Nutritional status of hemodialysis patients with secondary hyperparathyroidism

The repercussions of secondary hyperparathyroidism on the nutritional status of chronic renal failure patients have not been well established. Therefore, the aim of this study was to compare the nutritional indices of hemodialysis patients with and without secondary hyperparathyroidism. Sixteen hemodialysis patients with serum parathyroid hormone (PTH) levels higher than 420 pg/ml (hyperparathyroidism group) were matched for gender, age and length of dialysis treatment to 16 patients with serum PTH between 64 and 290 pg/ml (control group). The following parameters were assessed: anthropometric indices (body mass index, skinfold thickness, midarm muscle circumference and body fat), 4-day food diaries, protein catabolic rate, biochemical indices (blood urea nitrogen, serum creatinine, albumin, ionized calcium, inorganic phosphorus, serum alkaline phosphatase, PTH, pH and HCO3) and dialysis efficiency. We did not observe differences in the anthropometric indices between the two groups. Only calcium intake was significantly different between groups (307.9 mg/day for the hyperparathyroidism group vs 475.8 mg/day for the control group). Protein catabolic rate tended to be higher in the hyperparathyroidism group compared to the control group (1.3 vs 0.9 g kg-1 day-1; P = 0.08). Except for blood urea nitrogen (86.4 vs 75.7 mg/dl), alkaline phosphatase (175 vs 65 U/l) and PTH (898 vs 155 pg/ml), no other differences were found between groups in the biochemical indices studied. PTH was directly correlated with protein catabolic rate (r = 0.61; P<0.05) and length of dialysis (r = 0.53; P<0.05) only in the hyperparathyroidism group. Considering the indices used, we could not demonstrate the deleterious effect of high PTH levels on the nutritional status of hemodialysis patients. Indirect evidence, however, suggests an action of PTH on protein metabolism.

In vitro cytotoxicity of the LDE: daunorubicin complex in acute myelogenous leukemia blast cells

Acute myelogenous leukemia (AML) blast cells show high-affinity degradation of low-density lipoprotein (LDL), suggesting an increased expression of cellular LDL receptors. LDE is a lipid microemulsion easily synthesized in vitro which is known to mimic the metabolic pathway of LDL. We used LDE as a carrier for daunorubicin and assayed the cytotoxicity of the complex using AML blast cells since RT-PCR analysis showed that AML cells express LDL receptor mRNA. The LDE:daunorubicin complex killed 46.7% of blast cells and 20.2% of normal bone marrow cells (P<0.001; Student t-test). Moreover, this complex destroyed AML blast cells as efficiently as free daunorubicin. Thus, LDE might be a suitable carrier of chemotherapeutic agents targeting these drugs to neoplastic cells and protecting normal tissues.

Regenerated rat skeletal muscle after periodic contusions

In the present study we evaluated the morphological aspect and changes in the area and incidence of muscle fiber types of long-term regenerated rat tibialis anterior (TA) muscle previously submitted to periodic contusions. Animals received eight consecutive traumas: one trauma per week, for eight weeks, and were evaluated one (N = 8) and four (N = 9) months after the last contusion. Serial cross-sections were evaluated by toluidine blue staining, acid phosphatase and myosin ATPase reactions. The weight of injured muscles was decreased compared to the contralateral intact one (one month: 0.77 ± 0.15 vs 0.91 ± 0.09 g, P = 0.03; four months: 0.79 ± 0.14 vs 1.02 ± 0.07 g, P = 0.0007, respectively) and showed abundant presence of split fibers and fibers with centralized nuclei, mainly in the deep portion. Damaged muscles presented a higher incidence of undifferentiated fibers when compared to the intact one (one month: 3.4 ± 2.1 vs 0.5 ± 0.3%, P = 0.006; four months: 2.3 ± 1.6 vs 0.3 ± 0.3%, P = 0.007, respectively). Injured TA evaluated one month later showed a decreased area of muscle fibers when compared to the intact one (P = 0.003). Thus, we conclude that: a) muscle fibers were damaged mainly in the deep portion, probably because they were compressed against the tibia; b) periodic contusions in the TA muscle did not change the percentage of type I and II muscle fibers; c) periodically injured TA muscles took four months to reach a muscle fiber area similar to that of the intact muscle.

Ethidium bromide-induced demyelination of the sciatic nerve of adult Wistar rats

Peripheral nerve ultrastructure was assessed after single or multiple local injections of the intercalating dye ethidium bromide. Thirty-four adult Wistar rats of both sexes were divided into five groups and maintained in a controlled environment with rat chow and water ad libitum throughout the experiment. The experimental animals were injected with 1 µl of 0.1% ethidium bromide in 0.9% saline into the central third of the left sciatic nerve 1 (group 1), 2 (group 2), 4 (group 3), 6 (group 4) or 8 (group 5) times. In groups 2 to 5 the injections were made at 28-day intervals. Control animals received the same amount of 0.9% saline. The animals were killed at different times after injection: group 1 at 7 days (2 rats) and 15 days (2 rats); for groups 2, 3, 4 and 5, all rats were killed 10 days after the last injection and the lesions were investigated by light and transmission electron microscopy. In the acute lesions, intoxicated Schwann cells showed a vacuolated cytoplasm and separation of the sheaths from the axon. Myelin sheaths underwent progressive vesiculation and subsequent segmental demyelination. Myelin debris were withdrawn by macrophages and remyelination by Schwann cells was prominent. With the increase in the number of injections collagen fibers also increased in number and progressively enveloped smaller numbers of remyelinated axons composing new fascicles. Wallerian degeneration of fibers apparently not affected by ethidium bromide was more intense in the nerves from groups 4 and 5. The peripheral nerve repairs itself after demyelinating challenges with a profusion of collagen fibers and new fasciculations. This experimental model is valid to mimic recurrent demyelinating neuropathies.

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Stimulatory effects of adenosine on prolactin secretion in the pituitary gland of the rat

We investigated the effects of adenosine on prolactin (PRL) secretion from rat anterior pituitaries incubated in vitro. The administration of 5-N-methylcarboxamidoadenosine (MECA), an analog agonist that preferentially activates A2 receptors, induced a dose-dependent (1 nM to 1 µM) increase in the levels of PRL released, an effect abolished by 1,3-dipropyl-7-methylxanthine, an antagonist of A2 adenosine receptors. In addition, the basal levels of PRL secretion were decreased by the blockade of cyclooxygenase or lipoxygenase pathways, with indomethacin and nordihydroguaiaretic acid (NDGA), respectively. The stimulatory effects of MECA on PRL secretion persisted even after the addition of indomethacin, but not of NDGA, to the medium. MECA was unable to stimulate PRL secretion in the presence of dopamine, the strongest inhibitor of PRL release that works by inducing a decrease in adenylyl cyclase activity. Furthermore, the addition of adenosine (10 nM) mimicked the effects of MECA on PRL secretion, an effect that persisted regardless of the presence of LiCl (5 mM). The basal secretion of PRL was significatively reduced by LiCl, and restored by the concomitant addition of both LiCl and myo-inositol. These results indicate that PRL secretion is under a multifactorial regulatory mechanism, with the participation of different enzymes, including adenylyl cyclase, inositol-1-phosphatase, cyclooxygenase, and lipoxygenase. However, the increase in PRL secretion observed in the lactotroph in response to A2 adenosine receptor activation probably was mediated by mechanisms involving regulation of adenylyl cyclase, independent of membrane phosphoinositide synthesis or cyclooxygenase activity and partially dependent on lipoxygenase arachidonic acid-derived substances.

Insulin impairs the maturation of chondrocytes in vitro

The precise nature of hormones and growth factors directly responsible for cartilage maturation is still largely unclear. Since longitudinal bone growth occurs through endochondral bone formation, excess or deficiency of most hormones and growth factors strongly influences final adult height. The structure and composition of the cartilaginous extracellular matrix have a critical role in regulating the behavior of growth plate chondrocytes. Therefore, the maintenance of the three-dimensional cell-matrix interaction is necessary to study the influence of individual signaling molecules on chondrogenesis, cartilage maturation and calcification. To investigate the effects of insulin on both proliferation and induction of hypertrophy in chondrocytes in vitro we used high-density micromass cultures of chick embryonic limb mesenchymal cells. Culture medium was supplemented with 1% FCS + 60 ng/ml (0.01 µM) insulin and cultures were harvested at regular time points for later analysis. Proliferating cell nuclear antigen immunoreactivity was widely detected in insulin-treated cultures and persisted until day 21 and [³H]-thymidine uptake was highest on day 14. While apoptosis increased in control cultures as a function of culture time, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-labeled cells were markedly reduced in the presence of insulin. Type II collagen production, alkaline phosphatase activity and cell size were also lower in insulin-treated cultures. Our results indicate that under the influence of 60 ng/ml insulin, chick chondrocytes maintain their proliferative potential but do not become hypertrophic, suggesting that insulin can affect the regulation of chondrocyte maturation and hypertrophy, possibly through an antiapoptotic effect.

Vascular supply of the central nervous system of the land snail Megalobulimus oblongus (Gastropoda, Pulmonata)

The vascularization of the central nervous system of the snail Megalobulimus oblongus was studied by injection of carmine-gelatin solution into the arterial system and using a histochemical technique for the detection of alkaline phosphatase. The central nervous system of M. oblongus is irrigated by the anterior aorta, from which a series of small branches emerge that supply the subesophageal nervous ganglia. In turn, these branches give rise to a series of smaller vessels that irrigate the buccal bulb, the anterior portion of the foot, the cerebral ganglia, the dorsal body gland, and the anterior portion of the reproductive system. No hemolymph vessels were detected within nervous tissue although such vessels were found in the periganglionic connective sheath. This connective sheath contains vascular loops and had a series of overlaps and projections that follow the contour of the nervous ganglia. This arrangement permits a larger area of interaction between the surface of the nervous tissue and the hemolymph and reduces the distance between the deepest portion of a given ganglion and the hemolymph vessels.

Noninvasive classification of liver disease in asymptomatic and oligosymptomatic male alcoholics

The predominant type of liver alteration in asymptomatic or oligosymptomatic chronic male alcoholics (N = 169) admitted to a psychiatric hospital for detoxification was classified by two independent methods: liver palpation and multiple quadratic discriminant analysis (QDA), the latter applied to two parameters reported by the patient (duration of alcoholism and daily amount ingested) and to the data obtained from eight biochemical blood determinations (total bilirubin, alkaline phosphatase, glycemia, potassium, aspartate aminotransferase, albumin, globulin, and sodium). All 11 soft and sensitive, and 13 firm and sensitive livers formed fully concordant groups as determined by QDA. Among the 22 soft and not sensitive livers, 95% were concordant by QDA grouping. Concordance rates were low (55%) in the 73 firm and not sensitive livers, and intermediate (76%) in the 50 not palpable livers. Prediction of the liver palpation characteristics by QDA was 95% correct for the firm and not sensitive livers and moderate for the other groups. On a preliminary basis, the variables considered to be most informative by QDA were the two anamnestic data and bilirubin levels, followed by alkaline phosphatase, glycemia and potassium, and then by aspartate aminotransferase and albumin. We conclude that, when biopsies would be too costly or potentially injurious to the patients to varying extents, clinical data could be considered valid to guide patient care, at least in the three groups (soft, not sensitive; soft, sensitive; firm, sensitive livers) in which the two noninvasive procedures were highly concordant in the present study.

Effect of bullfrog (Rana catesbeiana) oil administered by gavage on the fatty acid composition and oxidative stress of mouse liver

The aim of the present study was to investigate the effects of daily intragastric administration of bullfrog oil (oleic, linoleic and palmitoleic acid-rich oil), corresponding to 0.4% of body weight for four weeks, on fatty acid composition and oxidative stress (lipid peroxidation and catalase activity) in mouse liver. The activities of aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT), biomarkers of tissue injury, were determined in liver homogenates and serum. The proportions of 18:2n-6, 20:4n-6, 20:5n-3, and 22:6n-3 (polyunsaturated fatty acids, from 37 to 60%) in the total fatty acid content were increased in the liver of the bullfrog oil-treated group (P < 0.05) compared to control. At the same time, a significant decrease in the relative abundance of 14:0, 16:0, and 18:0 (saturated fatty acids, from 49 to 25%) was observed. The hepatic content of thiobarbituric acid reactive substances (TBARS) was increased from 2.3 ± 0.2 to 12.3 ± 0.3 nmol TBA-MDA/mg protein and catalase activity was increased from 840 ± 32 to 1110 ± 45 µmol reduced H2O2 min-1 mg protein-1 in the treated group. Bullfrog oil administration increased AST and ALP activities in the liver (from 234.10 ± 0.12 to 342.84 ± 0.13 and 9.38 ± 0.60 to 20.06 ± 0.27 U/g, respectively) and in serum (from 95.41 ± 6.13 to 120.32 ± 3.15 and 234.75 ± 11.5 to 254.41 ± 2.73 U/l, respectively), suggesting that this treatment induced tissue damage. ALT activity was increased from 287.28 ± 0.29 to 315.98 ± 0.34 U/g in the liver but remained unchanged in serum, whereas the GGT activity was not affected by bullfrog oil treatment. Therefore, despite the interesting modulation of fatty acids by bullfrog oil, a possible therapeutic use requires care since some adverse effects were observed in liver.

Expression of dorsal-ventral genes during early development of Rhynchosciara americana embryos

The establishment of dorsal-ventral polarity in Drosophila is a complex process which involves the action of maternal and zygotically expressed genes. Interspecific differences in the expression pattern of some of these genes have been described in other species. Here we present the expression of dorsal-ventral genes during early embryogenesis in the lower dipteran Rhynchosciara americana. The expression of four genes, the ventralizing genes snail (sna) and twist (twi) and the dorsalizing genes decapentaplegic (dpp) and zerknüllt (zen), was investigated by whole-mount in situ hybridization. Sense and antisense mRNA were transcribed in vitro using UTP-digoxigenin and hybridized at 55°C with dechorionated fixed embryos. Staining was obtained with anti-digoxigenin alkaline phosphatase-conjugated antibody revealed with NBT-BCIP solution. The results showed that, in general, the spatial-temporal expression of R. americana dorsal-ventral genes is similar to that observed in Drosophila, where twi and sna are restricted to the ventral region, while dpp and zen are expressed in the dorsal side. The differences encountered were subtle and probably represent a particular aspect of dorsal-ventral axis determination in R. americana. In this lower dipteran sna is expressed slightly later than twi and dpp expression is expanded over the lateral ectoderm during cellular blastoderm stage. These data suggest that the establishment of dorsal-ventral polarity in R. americana embryos follows a program similar to that observed in Drosophila melanogaster.

Cyclosporin A preferentially attenuates skeletal slow-twitch muscle regeneration

Calcineurin, a Ca2+/calmodulin-dependent phosphatase, is associated with muscle regeneration via NFATc1/GATA2-dependent pathways. However, it is not clear whether calcineurin preferentially affects the regeneration of slow- or fast-twitch muscles. We investigated the effect of a calcineurin inhibitor, cyclosporin A (CsA), on the morphology and fiber diameter of regenerating slow- and fast-twitch muscles. Adult Wistar rats (259.5 ± 9 g) maintained under standard conditions were treated with CsA (20 mg/kg body weight, ip) for 5 days, submitted to cryolesion of soleus and tibialis anterior (TA) muscles on the 6th day, and then treated with CsA for an additional 21 days. The muscles were removed, weighed, frozen, and stored in liquid nitrogen. Cryolesion did not alter the body weight gain of the animals after 21 days of regeneration (P = 0.001) and CsA significantly reduced the body weight gain (15.5%; P = 0.01) during the same period. All treated TA and soleus muscles showed decreased weights (17 and 29%, respectively, P < 0.05). CsA treatment decreased the cross-sectional area of both soleus and TA muscles of cryoinjured animals (TA: 2108 ± 930 vs 792 ± 640 µm²; soleus: 2209 ± 322 vs 764 ± 439 m²; P < 0.001). Histological sections of both muscles stained with Toluidine blue revealed similar regenerative responses after cryolesion. In addition, CsA was able to minimize these responses, i.e., centralized nuclei and split fibers, more efficiently so in TA muscle. These results indicate that calcineurin preferentially plays a role in regeneration of slow-twitch muscle.

Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.