Randomized, controlled, dose-range study of Ro 25-8315 given before and after a high-dose combination chemotherapy regimen in patients with metastatic or recurrent breast cancer patients.

PURPOSE: To evaluate the safety, pharmacokinetics, and efficacy of three different dose levels of pegylated granulocyte colony-stimulating factor (Ro 25-8315) on progenitor cell mobilization and hematologic recovery in cancer patients. PATIENTS AND METHODS: Breast cancer patients (n = 36) were randomly assigned to receive before (part I) and after (part II) chemotherapy either a single-dose injection of Ro 25-8315 (20 microg/kg, n = 9; 60 microg/kg, n = 9; 100 microg/kg, n = 10) or a standard daily dose of filgrastim (part I, 10 microg/kg/d; part II, 5 microg/kg/d) (control group, n = 8). RESULTS: Overall, Ro 25-8315 was well tolerated. In part I, more progenitor cell mobilization was observed with Ro 25-8315 100 microg/kg. The peak of circulating CD34(+) cells was obtained at day +5 in the four groups, and the absolute neutrophil count (ANC) returned to less than 20 x 10(9)/L by day +15. In part II, high levels of circulating CD34(+) cells (> 20 cells/microL) were obtained in all four groups. The chemotherapy-induced neutropenia (< 1 x 10(9)/L) was similar in the four groups. Ro 25-8315 100 microg/kg was more effective than filgrastim in reducing the number of patients with an ANC less than 0.5 x 10(9)/L on day +12 after chemotherapy. CONCLUSION: A single injection of Ro 25-8315 100 microg/kg might be the optimal dose for steady-state peripheral-blood progenitor cell mobilization. A single injection of 20, 60, or 100 microg/kg could be as efficient as daily administration of filgrastim to correct chemotherapy-induced cytopenia. The optimal dose of Ro 25-8315 should be determined according to the planned chemotherapy regimen.

Pharmacodynamics and pharmacokinetics of single doses of subcutaneous pegylated human G-CSF mutant (Ro 25-8315) in healthy volunteers: comparison with single and multiple daily doses of filgrastim.

Ro 25-8315 is produced by conjugation of rhG-CSF mutant with polyethylene glycol (PEG). The purpose of this study was to examine the pharmacodynamics and pharmacokinetics of Ro 25-8315 in comparison with Filgrastim (rhG-CSF). Subjects received single subcutaneous doses of Ro 25-8315 ranging from 10 to 150 microg/kg using a double-blind, randomized, placebo-controlled design. Filgrastim was administered as a single dose (5 or 10 microg/kg) and, following a 14-day washout period, daily for 7 days. Ro 25-8315 increased absolute neutrophil count (ANC) by 6- to 8-fold and CD34+ cell count more than 30-fold at the highest doses tested. Single doses (60-150 microg/kg) of Ro 25-8315 and multiple doses of Filgrastim had similar effects on ANC and CD34+, although Ro 25-8315 had a greater effect on CFU-GM. The pharmacokinetics of Ro 25-8315 were dose-dependent, with peak concentrations and area under the serum concentration-time curve (AUC) increasing 100-fold over the range of doses studied. Time to reach peak concentration (T(max)) and half-life of Ro 25-8315 averaged 20-30 hr at all doses, approximately three times longer than with Filgrastim. Adverse events were not serious and occurred with similar frequency with both products. Pegylation of rhG-CSF mutant results in more desirable pharmacokinetic properties and a longer duration of action with effective increases in ANC and measures of peripheral blood progenitor cell mobilization for at least 1 week.

Characterization of Otostrongylus circumlitus from Pacific harbor and northern elephant seals.

Otostrongylus circumlitus (Railliet, 1899) from Pacific harbor seals (Phoca vitulina richardsi) and northern elephant seals (Mirounga angustirostris) were examined using morphological and molecular methods to determine whether northern elephant seals along the central California coast are infected by the same species of Otostrongylus as are Pacific harbor seals in the same area. Fixed nematodes were examined and measured using light microscopy. The polymerase chain reaction (PCR) was used to amplify and sequence the second internal transcribed spacer (ITS-2) and D3 expansion (26S) regions of ribosomal DNA of O. circumlitus from Pacific harbor and northern elephant seals. The ITS-2 region was also amplified from Parafilaroides sp. from the Pacific harbor seal, northern elephant seal, and California sea lion (Zalophus californianus) and used for restriction fragment length polymorphism (RFLP) analysis. Morphologically, it was not possible to distinguish O. circumlitus from Pacific harbor and northern elephant seals, and over a consensus length of 443 base pairs (bp) for ITS-2 and 321 bp for D3 the sequences of O. circumlitus from both hosts were identical. With the PCR-RFLP assay, it was possible to distinguish O. circumlitus from Parafilaroides sp. The results suggest that O. circumlitus is the same species in Pacific harbor and northern elephant seals, and molecular methods make it possible to distinguish this nematode from related nematodes.

Venereal worms: sexually transmitted nematodes in the decorated cricket.

The nematode, Mehdinema alii, occurs in the alimentary canal of the decorated cricket Gryllodes sigillatus. Adult nematodes occur primarily in the hindgut of mature male crickets, whereas juvenile nematodes are found in the genital chambers of mature male and female crickets. Here, we present experimental evidence for the venereal transmission of M. alii in G. sigillatus. Infectivity experiments were conducted to test for transmission via oral-fecal contamination, same-sex contact, and copulation. The infective dauers of the nematode are transferred from male to female crickets during copulation. Adult female crickets harboring infective dauers subsequently transfer the nematode to their next mates. Thus, M. alii is transmitted sexually during copulation.

Molecular systematics of Mesocestoides sPP (cestoda: mesocestoididae) from domestic dogs (Canis familiaris) and coyotes (Canis latrans).

The genus Mesocestoides Vaillant, 1863 includes tapeworms of uncertain phylogenetic affinities and with poorly defined life histories. We previously documented 11 cases of peritoneal cestodiasis in dogs (Canis familiaris L.) in western North America caused by metacestodes of Mesocestoides spp. In the current study, DNA sequences were obtained from metacestodes collected from these dogs (n = 10), as well as proglottids from dogs (n = 3) and coyotes (Canis latrans Say, 1823 [n = 2]), and tetrathyridia representing laboratory isolates of M. corti (n = 3), and these data were analyzed phylogenetically. Two nuclear genetic markers, 18S ribosomal DNA and the second internal-transcribed spacer (ITS 2), were sequenced. Phylogenetic analysis of the 18S rDNA data recovered a monophyletic group composed of all samples of Mesocestoides spp., distinct from closely related outgroup taxa (Amurotaenia Akhmerov, 1941 and Tetrabothrius Rudolphi, 1819). Initial analysis of the ITS 2 data resolved 3 clades within Mesocestoides. Two proglottids from dogs formed a basal clade, a second clade was represented by tetrathyridial isolates, and a third clade included all other samples. Interpretation of these data from an apomorphy-based perspective identified 6 evolutionary lineages. We also assessed whether metacestodes from dogs (n = 4) are capable of asexual proliferation in laboratory mice. One tetrathyridial and 2 acephalic isolates from dogs proliferated asexually. Further investigation is warranted to determine which of the lineages represent distinct species and to determine the life history strategies of Mesocestoides spp.

Arginine kinase and phosphoarginine, a functional phosphagen, in the rhabditoid nematode steinernema carpocapsae.

Moderate activity of arginine kinase was found in Steinernema carpocapsae, an entomopathogenic nematode. In the forward reaction, 4.60 and 3.12 micromol ATP/min/mg protein was produced in infectious third-stage juveniles (J3s) and adult nematodes, respectively. For the reverse reaction, 3.20 and 2.27 micromol phosphoarginine/min/mg protein was produced by J3s and adults, respectively. The K(m)s for phosphoarginine and ADP were 0.73 and 0.42 mM, respectively, in the forward reaction, whereas in the reverse reaction, the K(m)s were 0.37 and 2.35 mM for arginine and ATP, respectively, for the enzyme from J3s. The pH optimum for the forward reaction was 7.2 and 7.3 in J3s and adults, respectively. The pH optimum was elevated for the reverse reaction, 7.8 and 7.9-8.5 in J3s and adults, respectively. In the J3s, the in vitro optima for arginine kinase activity was correlated with the in vivo tissue pH in hypoxic (6.9) and aerobic (7.5) J3s estimated by in vivo flow 31P-NMR.

Morphological, molecular and biological characterization of Mehdinema alii (Nematoda: Diplogasterida) from the decorated cricket (Gryllodes sigillatus).

The nematode Mehdinema alii was recovered from the decorated cricket Gryllodes sigillatus (Walker). Morphometric comparisons are presented from 3 populations. The nematode is characterized by dense arrays of spines on the cuticle of the anterior half of the body and a highly elongate, tubular stoma with a dorsal denticle in the glottoid region. Females have a protruding vulva. Young females are amphidelphic, but the anterior ovary disappears in older females bearing multiple developing juveniles. The male is monorchic with asymmetrically placed genital papillae, distally fused spicules, and a highly complex gubernaculum bearing 2 cuticularized thorns that protrude through a separate, postcloacal opening. Adult nematodes are located primarily in the hindgut, whereas juveniles or dauers occur mainly in the genital chamber of both male and female crickets. Male crickets are significantly more likely to be infected than females. This male-biased infection may be linked to the venereal transmission mechanism of the dauers. Although morphologically unusual in many respects, placement of M. alii in Diplogasterida is supported by both the morphology of the anterior digestive tract as well as analysis of its 18S rDNA sequence. These sequence data suggest that M. alii groups most closely with members of the Cylindrocorporidae.

Diagnostic procedures and treatment of eleven dogs with peritoneal infections caused by Mesocestoides spp.

An 8-year-old spayed Schnauzer with a distended abdomen was examined because of straining to urinate and suspected urinary tract infection. Abdominal radiography revealed a ground-glass appearance, and ultrasonography revealed numerous cystic structures in the peritoneal cavity. Examination of an aspirate of abdominal fluid revealed tissues consistent with metacestodes. Tissues were definitively identified as Mesocestoides spp on the basis of polymerase chain reaction amplification of restriction fragment length polymorphisms. The dog required several courses of treatment with fenbendazole to eliminate the infection. This was 1 of 11 dogs infected with Mesocestoides metacestodes. Treatment involving the use of praziquantel and albendazole were ineffective, but fenbendazole successfully cleared Mesocestoides infections in 5 of 6 dogs.

[Effects of temperature on the viability and infectivity of preparasitic larvae of Romanomermis yuanenesis].

Romanomermis yuanenesis (Mermithidae: Nematoda) was found in Henan, China (Song and Peng, 1987), which has a broad host range in Culicinae mosquito and has been used successfully in field test for control of culex tritaeniorhynchus, culex fatigans and Aedes albopictus in Sichuan, Yunnan, Guangxi and Henan Provinces. This study was attempted to determine the viability and infectivity of preparasitic larvae in various temperatures. The cultures containing R. yuanenesis eggs were flooded 2h with distilled water, filtered and blocked with 1% agarose. Put the filter paper into water, then the motile preparasites separated from the unhatched eggs and got through the agarose membrane into water. About 200ml water containing preparasites free from eggs were held at 26 degrees C-28 degrees C, 16 degrees C-18 degrees C and -2 degrees C to 2 degrees C for test. The motility or lack of motility was used as the criterion to distinguish the living and dead nematodes. The rate of infection of mosquitoes and the rate of parasitism of nematodes were used to show the infectivity of the preserved preparasites. The results showed that at -2 degrees C to 2 degrees C, more than 90% of preparasitic larvae of R. yuanenesis survived for 8 days and the rate of mosquito infection was 87.5% to 100%, but at 26 degrees C-28 degrees C and 16 degrees C-18 degrees C the survival times of 90% preparasites were only 24 hours and 48 hours respectively. It indicates the low temperature preservation may prolong the survival time and keep the infectivity of these preparasitic larvae.

Characterization of Key Glycolytic and Oxidative Enzymes in Steinernema carpocapsae.

The enzyme activities of isocitrate dehydrogenase (ICDH, NADP-specific), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), phosphoenolpyruvate carboxykinase (PEPCK), phosphofructokinase (PFK), pyruvate kinase (PK), and fructose-l,6-bisphosphatase (FBPase) were studied in the third-stage juveniles of Steinernema carpocapsae. Reaction requirements, pH optima, substrate and cofactor kinetic constants were similar to those reported previously from other parasitic helminths with the exception of LDH, which was unstable and could not be characterized for specific activity and kinetic constants. The respective pH optima were 7.5 for ICDH, 8.8 for MDH, 6.5 for PEPCK, 7.3 for PFK, 7.2 for PK, and 7.5 for FBPase. The specific activities for ICDH, MDH, PEPCK, PFK, PK, and FBPase at pH 7.5 were 4.8, 1,300, 22, 25, 35, and 6.8 (nmoles substrate min(1) mg protein(1)), respectively. In summary, the infective juveniles of S. carpocapsae display the metabolism typical of a facultative aerobe.

High energy phosphate metabolites observed by NMR in infective larvae of Haemonchus contortus.

Phosphorus resonances consistent with phosphoarginine and ATP were observed in the in vivo 31P-nuclear magnetic resonance (NMR) spectra of infective larvae of Haemonchus contortus. The level of phosphoarginine quickly declined when nematode suspensions were purged with nitrogen and was restored upon return to aerobic conditions. Saturation transfer NMR demonstrated forward and reverse exchange of phosphorus between phosphoarginine and ATP.

Randomized study of recombinant human granulocyte colony-stimulating factor after high-dose chemotherapy and autologous bone marrow transplantation for high-risk lymphoid malignancies.

PURPOSE: The aim of this prospective randomized trial was to examine the efficacy and safety of filgrastim after high-dose chemotherapy and autologous bone marrow transplantation (ABMT). PATIENTS AND METHODS: Patients with poor-risk non-Hodgkin's lymphoma or relapsed Hodgkin's disease were treated in a randomized, open-label trial to study the use of filgrastim as an adjunct to high-dose chemotherapy and ABMT. Of 43 assessable patients, 19 were randomized to receive filgrastim by continuous subcutaneous infusion at a dose of 10 micrograms/kg/d, 10 to filgrastim 20 micrograms/kg/d, and 14 to a parallel control group that received no filgrastim after ABMT. RESULTS: For all filgrastim-treated patients analyzed together, the median time to neutrophil recovery > or = 0.5 x 10(9)/L after the day of ABMT was significantly accelerated to 10 days compared with 18 days in control patients (P = .0001). The median number of platelet transfusions was identical in both groups. Clinical parameters, including the median number of days with fever (1 v 4, P = .0418) and neutropenic fever (5 v 13.5, P = .0001) were significantly shorter in the filgrastim than in the control group. The number of days on intravenous antibiotics and duration of hospitalization were also shorter in the treated groups; however, the differences did not reach statistical significance. For patients treated with the two different dose levels of filgrastim, the neutrophil recovery and clinical results were similar. Filgrastim-associated toxicity appeared to be minimal, with five adverse events considered at least possibly related to filgrastim: two in the higher-dose group and three in the lower-dose group. All of these were rated moderate, except one case of severe bone pain that did not preclude continued filgrastim treatment at a lower dose. Survival and relapse-free survival were similar for control and filgrastim-treated patients. CONCLUSION: Taken together, the results of this first randomized study support the role of filgrastim given as an adjunct to ABMT in accelerating neutrophil recovery, as well as in reducing treatment-related morbidity and overall duration of the treatment procedure.

Differential effects of transferrin receptor antibodies on growth and receptor expression of human lymphocytic and myelocytic cell lines.

J64, a monoclonal antibody against the human transferrin receptor, has been shown to induce interleukin-2 production by HUT78 cells. It also causes growth inhibition of several cell lines and stimulated lymphocytes. These effects were also present using transferrin-free culture conditions. In this paper, we dissect cell membrane and intracellular events after binding of J64 and other transferrin receptor antibodies. Incubation of HUT78 and several other cell lines with J64 resulted in an increased number of receptor molecules expressed on the cell surface in contrast to a downmodulation seen with other monoclonal antibodies to the transferrin receptor. This upregulation after treatment with J64 was not due to an increased concentration of transferrin receptor mRNA in these cells or a higher protein synthesis rate. We therefore suggest that J64 causes a redistribution of transferrin receptor molecules from intracellular pools to the cell surface. Additional experiments investigating signal transduction mechanisms revealed no influence of J64 on intracellular Ca2+ concentrations or translocation of protein kinase C. However, an increase of transferrin receptor phosphorylation was seen in HL60 cells after treatment with phorbolester or J64. This phosphorylation of the transferrin receptor might be a signal transduction pathway involved in activation and growth control.

Rapid miniprep isolation of mitochondrial DNA from metacestodes, and free-living and parasitic nematodes.

A method, based on one to isolate supercoiled plasmid DNA from bacterial cells, has been developed to purify mitochondrial DNA (mtDNA) from cestode and nematode tissue easily and efficiently. Starting with as little as 100 mg of helminth tissue, sufficient mtDNA for electrophoretic analysis was extracted. This DNA was essentially free of nuclear DNA and readily digested by restriction endonucleases. Approximately 20% of the mtDNA in helminth tissue was recovered, which is a significant improvement over previously available techniques.

Involvement of the high-affinity receptor for IgG (Fc gamma RI; CD64) in enhanced tumor cell cytotoxicity of neutrophils during granulocyte colony-stimulating factor therapy.

Three different classes of Fc receptors for IgG (Fc gamma R) are currently distinguished in humans, of which polymorphonuclear phagocytes (PMN) normally express both low-affinity receptor classes--Fc gamma RII (CD32) and Fc gamma RIII (CD16). During therapy with granulocyte colony-stimulating factor (G-CSF), neutrophils from patients with various malignancies and different hematologic disorders were found to additionally express high levels of the receptor with high affinity for IgG (Fc gamma RI; CD64). For these patients, the relative fluorescence intensity (rFI) for Fc gamma RI was 5.3 (range, 1.7 to 10.3; n = 19), compared with 1.0 (range, 1.0 to 1.1; n = 8) for healthy donors. The expression of Fc gamma RI during G-CSF therapy could be confirmed by using a panel of six CD64-specific antibodies, and by showing mRNA for Fc gamma RI. So far, three genes for Fc gamma RI have been identified, encoding four distinct transcription products. By reverse transcriptase-polymerase chain reaction technology, transcripts for both membrane-associated isoforms (hFc gamma RIa and hFc gamma RIb2) could be detected. The functional activity of Fc gamma RI on PMN during G-CSF therapy was shown by measuring binding of monomeric human IgG and antibody-dependent cellular cytotoxicity (ADCC). Thus, Fc gamma RI-positive neutrophils displayed enhanced ADCC activity to glioma (A1207), squamous cell (A431), and ovarian (SK-ov3) carcinoma cell lines. The involvement of Fc gamma RI in this increased cytotoxic activity was shown by blocking Fc gamma receptors with monoclonal antibodies, and by using F(ab')2 x F(ab')2-bispecific antibodies with specificities against tumor-related antigens and Fc gamma RI, resulting in solely Fc gamma RI-mediated cytotoxicity. Therapeutically, this additional Fc receptor on PMN may increase the efficacy of experimental antibody therapy.

Phosphoarginine-adenosine triphosphate exchange detected in vivo in a microscopic nematode parasite by flow 31P FT-NMR spectroscopy.

In vivo flow 31P NMR spectroscopy of a microscopic nematode, Steinernema carpocapsae, is described. Long-term viability was maintained during analysis by continuous circulation of an oxygenated suspension of the parasite through an NMR spectrometer. Saturation transfer and inversion recovery were employed under flowing conditions to investigate the kinetics of phosphoarginine<-->adenosine triphosphate exchange. The kinetic constants for the forward and reverse reactions were 0.37/s and 1.45/s, respectively. This report is the first to demonstrate a functional phosphagen kinase in the metabolism of a parasitic helminth.

[Susceptibility of thirty-one species of mosquitoes to Romanomermis yunanensis].

A new species of Mermithidae was found parasitizing the larvae of Culex tritaeniorhynchus and Culex fatigans in Henan, China and then named Romanomermis yunanensis. Thirty-one species of Mosquitoes involving six genera have been tested for susceptibility to R. yunanensis and Culicinae mosquito have consistently been highly susceptible. At a 1:5 ratio of mosquito larvae to nematode juveniles, the parasitisms of Aedes aegypti, Ae. albopictus, Culex fatigans, Culex tritaeniorhynchus, Anopheles sinensts, Psorophora columbiae and Culesta inornata were 98.2%, 98.5%, 98.6%, 97.1%, 0%, 98.8%, and 99.0% respectively. R. yunanensis presented a phenomenon of retarted development in Anopheles maculatus, which remained at the parasitic stage both in the larvae and pupa of An. maculatus. The parasitism of An. dirus was 89.7%, but most (88.8%) parasitizing mermithids underwent melanization and died. The comparative susceptibility of some North American mosquitoes to R. yunanesis and R. culicivorax at a 1:5 infective ratio showed that both percentage infection and intensity of infection statistically supported the generalization that, under laboratory conditions, R. yunanesis is a more vigorous and aggressive parasite of Culicinae.

Stable growth transformation of human T lymphocytes by herpesvirus saimiri.

Herpesvirus saimiri induces T-cell lymphomas in various species of New World monkeys and in rabbits, and it is able to immortalize monkey T lymphocytes in vitro. Sequences responsible for these effects have been localized to a region of the genome that varies significantly among the virus subgroups A, B, and C. We now report that infection of human blood lymphocytes and thymocytes with strains of subgroup C, in contrast to viruses of the other subgroups, yields continuously proliferating T-cell lines with the phenotype of mature CD4- or CD8-positive cells. Infection with strains of Herpes-virus saimiri subgroup C can thus be used to generate human T-cell lines for a variety of immunological and developmental studies.

Functional characterization of tumor-infiltrating lymphocytes, lymph-node lymphocytes and peripheral-blood lymphocytes from patients with breast cancer.

Mononuclear cell infiltration is frequently seen within human solid tumors. Effector cells within the tumor site usually fail to exhibit cytotoxic or natural killer activity when freshly isolated; however, they develop potent and sometimes specific cytotoxicity after expansion in IL2. Thus, local tumor environment may influence lymphocyte function. In our study, we disaggregated human breast-cancer and lymph-node tissue to obtain lymphocyte-enriched cell fractions. Besides phenotypic analysis, functional characterization with regard to proliferation and cytokine production of tumor-infiltrating lymphocytes (TIL), peripheral-blood lymphocytes (PBL) and lymph-node lymphocytes (LNL) was the aim of our study. TIL showed an enrichment of CD8+ cells with a corresponding decrease in CD4+ cells in comparison with PBL and LNL. In response to PHA, TIL showed decreased 3H-thymidine uptake, but TIL were significantly stimulated by rhIL2. TIL produced low levels of IL2, TNF and IFN gamma upon mitogen/phorbol ester stimulation, while PBL produce high levels of TNF and IFN gamma but low levels of IL2. Under the same experimental conditions, LNL produce high levels of TNF and IL2 but low levels of IFN gamma. Mitogen-mediated TNF secretion was increased after addition of autologous tumor cells in TIL and LNL, whereas IFN gamma secretion tended to be suppressed. Our results indicate different patterns of activities of TIL, LNL and PBL from breast-cancer patients.