Detection of pulsation in unruptured cerebral aneurysms by ECG-gated 3D-CT angiography (4D-CTA) with 320-row area detector CT (ADCT) and follow-up evaluation results: assessment based on heart rate at the time of scanning.

PURPOSE: Many epidemiological studies on unruptured cerebral aneurysms have reported that the larger the aneurysm, the higher the risk of rupture. However, many ruptured aneurysms are not large. Electrocardiography (ECG)-gated 3D-computed tomography angiography (4D-CTA) was used to detect pulsation in unruptured cerebral aneurysms. The differences in the clinical course of patients in whom pulsation was or was not detected were then evaluated. METHODS: Forty-two patients with 62 unruptured cystiform cerebral aneurysms who underwent 4D-CTA and follow-up 3D-CTA more than 120 days later were studied. The tube voltage, tube current, and rotation speed were 120 kV, 270 mA, and 0.35 s/rot., respectively. ECG-gated reconstruction was performed, with the cardiac cycle divided into 20 phases. Patients with heart rates higher than 80 bpm were excluded, so 37 patients with 56 aneurysms were analyzed. RESULTS: Pulsation was detected in 20 of the 56 unruptured aneurysms. Of these 20 aneurysms, 6 showed a change in shape at the time of follow-up. Of the 36 aneurysms in which pulsation was not detected, 2 showed a change in shape at follow-up. There was no significant difference in the follow-up interval between the two groups. The aneurysms in which pulsation was detected were significantly more likely to show a change in shape (P = 0.04), with a higher odds ratio of 7.286. CONCLUSION: Unruptured aneurysms in which pulsation was detected by 4D-CTA were more likely to show a change in shape at follow-up, suggesting that 4D-CTA may be useful for identifying aneurysms with a higher risk of rupture.

Subcutaneous hematoma associated with manual cervical massage during carotid artery stenting. A case report.

We describe a patient with subcutaneous hematoma associated with manual cervical massage during carotid artery stenting.A 73-year-old man with left cervical carotid artery stenosis presented with left amaurosis fugax. We performed carotid artery stenting using distal embolic protection with balloon occlusion. Dual antiplatelet therapy was maintained in the periprocedural period and an anticoagulant agent was administered during the procedure. Because the aspiration catheter became entrapped by the stent, it did not reach the distal side of the stenotic lesion, and manual compression of the cervical region was therefore performed. Immediately afterwards, a subcutaneous hemorrhage occurred in the cervical region. There was no postoperative dyspnea due to enlargement of the hematoma, which was absorbed spontaneously.Cervical subcutaneous hematoma can occur in the cervical region due to cervical massage in patients who are receiving adjuvant antiplatelet therapy and anticoagulation therapy.

Actinohivin, a novel anti-HIV protein from an actinomycete that inhibits syncytium formation: isolation, characterization, and biological activities.

Blocking human immunodeficiency virus (HIV) entry into target cells is an important goal of HIV and acquired immune deficiency syndrome (AIDS) therapies. We have searched for anti-HIV substances from microorganisms using a syncytium formation assay system constructed with HeLa/CD4/Lac-Z and HeLa/T-env/Tat cells. We discovered a novel anti-HIV protein that inhibits syncytium formation, designated as actinohivin, from a cultured broth of a soil isolate, actinomycete strain K97-0003. ESI mass spectrometry of actinohivin isolated from the culture filtrate showed an ion with molecular mass of 12,520.3 Da. The amino acid sequence was determined by N-terminal Edman degradation of the intact protein and peptide fragments formed by endoproteinase digestions. Actinohivin consists of a 114-amino-acid chain that exhibits internal sequence triplication. Actinohivin inhibited both T-cell and macrophage tropic syncytium formation, with IC(50) values of 60 and 700 nM, respectively, and the cytopathic effect of HIV-1(IIIB) in MT-4 cells, with IC(50) value of 230 nM.

Comparative study of the effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on generation and mobilization of neutrophils in cyclophosphamide-treated neutropenic mice.

To examine the differential in vivo effects of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the recovery from neutropenia caused by chemotherapy, we directly compared the actions of these CSFs on neutrophil count in the bone marrow, spleen, and peripheral blood of mice treated with an anticancer drug, cyclophosphamide (CPA). Both G-CSF and GM-CSF stimulated the generation of neutrophils in bone marrow, but GM-CSF is only about 1/20 as effective as G-CSF. Both CSFs also stimulated the release of the generated neutrophils therefrom. G-CSF mobilized the neutrophils into both the circulation pool and the marginal pool, and these neutrophils were able to migrate into the inflammatory site when stimulated by casein. On the other hand, GM-CSF mobilize the neutrophils into the marginal pool but not the circulation pool, and they were not able to migrate into the inflammatory site. These findings indicate that G-CSF physiologically plays an important role as a factor which stimulates neutrophil mobilization from bone marrow into circulation as well as neutrophil generation in the bone marrow.

Characterization, molecular cloning and expression of megakaryocyte potentiating factor.

We examined whether the conditioned media of 64 kinds of cell lines, which have been maintained by a protein-free culture system, could produce megakaryocyte potentiating (Meg-POT) activity. In these cell lines, HPC-Y5, established from human pancreatic cancer, was shown to have the highest level of activity. The megakaryocyte potentiating factor (MPF) was purified from its conditioned medium by a combination of ion-exchange chromatography, gel filtration and reversed-phase HPLC. The purified MPF showed Meg-POT activity almost equal to human (Hu) interleukin 6 (IL-6) in the presence of murine IL-3 in a colony-forming assay with mouse bone marrow cells. The molecular weight of MPF was estimated to be 33 kDa by SDS-PAGE. Glycopeptidase F digestion and amino sugar analysis of the factor demonstrated that MPF is a glycoprotein carrying at least one N-linked sugar chain. The N-terminal amino acid sequence of MPF was determined to be Leu-Ala-Gly-Glu-Thr-Gly-Gln-Glu-Ala-Ala-Pro-Leu-Asp-Gly-Val-Leu-Ala-Asn. The same or homologous amino acid sequence has not been found in known proteins, demonstrating that MPF may be a novel cytokine which has Meg-POT activity. Then, we isolated HuMPF cDNA from an HPC-Y5 cDNA library using polymerase chain reaction and plaque hybridization methods. The HuMPF cDNA encodes a polypeptide consisting of 622 amino acids, including a signal peptide of 33 amino acids, and with a deduced molecular weight of 68 kDa, although HPC-Y5 cells secrete a 33 kDa form of HuMPF. HuMPF cDNA does not show any significant homology with other known sequences. The cDNA was expressed in COS-7 and Chinese hamster ovary (CHO) cells, and Meg-POT activity was detected in their culture supernatant. The COS-7 cells secreted only a 33 kDa recombinant (r)HuMPF, however, an additional 30 kDa form was detected in the culture medium of CHO cells. The 33 kDa rHuMPF from CHO cells showed Meg-POT activity, but not the purified 30 kDa rHuMPF. The difference in structure and activity between the 33 and 30 kDa forms of HuMPF was ascribed to the existence in the 33 kDa form of the C-terminal 25 amino acid residues. The expression of MPF mRNA was examined by Northern blot analysis using labeled MPF cDNA as a probe. MPF mRNA was detected in HPC-Y5 cells, with an approximate molecular size of 2.4 kb. We also examined the expression of the MPF gene in various human tissues, and the 2.4 kb band was detected only in lung. Then, the immunohistocytochemical analysis and in situ hybridization revealed that MPF-producing cells were identified as lung macrophages. MPF may exhibit other biological activities such as regeneration of the lung tissues.

Quantitative in vivo assay of human granulocyte colony-stimulating factor using cyclophosphamide-induced neutropenic mice.

Administration of human granulocyte colony-stimulating factor (hG-CSF) to mice with cyclophosphamide (CPA)-induced neutropenia for 4 consecutive days from the day after the CPA dosing (100 mg/kg) resulted in a dose-dependent increase in the peripheral blood neutrophil count 6 hours after the final hG-CSF injection. Within the hG-CSF dose range of 0.1 to 10 micrograms per mouse per day, there was a strong linear relationship (r greater than .9) between the logarithm of the dose and the peripheral blood neutrophil count in the treated mice. Using the same hG-CSF preparation, 38 experiments indicated that the regression lines are highly reproducible. Such an association never occurred with intact mice, and 100 mg/kg of CPA induced the highest response to hG-CSF. This linear relationship between the two variables allows us to determine the biologic potency of a test hG-CSF preparation relative to a reference standard using a parallel line assay, with a coefficient of precision of around .2. When assayed by this bioassay procedure, which we have termed CPA-mouse assay, natural hG-CSF and recombinant hG-CSF (produced by Chinese hamster ovary cells) were nearly equipotent in specific biologic activity. These results confirm the CPA-mouse assay as an especially useful assay method for quantifying the in vivo activity of hG-CSF.

Structures of the sugar chains of recombinant human granulocyte-colony-stimulating factor produced by Chinese hamster ovary cells.

Recombinant human granulocyte-colony-stimulating factor (G-CSF) was purified from Chinese hamster ovary cells transfected with human G-CSF cDNA. The recombinant human G-CSF was treated with alkaline borohydride and the oligosaccharide-alditols liberated were fractioned by gel filtration on a Bio-Gel P-4 column, followed by high-performance liquid chromatography by use of a strong anion exchanger. Two oligosaccharide-alditols were obtained and their structures were identified by component analysis and 500-MHz 1H-NMR spectroscopy. The structures of the sugar chains were NeuAc alpha 2-3Gal beta 1-3GalNAcol and NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAcol.

Induction of neutrophilic granulocytosis in mice by administration of purified human native granulocyte colony-stimulating factor (G-CSF).

Mice were subcutaneously (sc) injected once a day for up to 15 days with a purified human native G-CSF sample at a dose of 2.5 micrograms/injection or with control samples with or without added endotoxin. In the G-CSF-treated mice, blood neutrophil counts began to rise as early as 2 hours after the first injection, reached a level 8 times above the preinjection level after 15 days of injections with marked elevation of all progenitor cell levels in spleen, and returned to normal within 48 hours after cessation of the injections. Such neutrophilia was observed even when endotoxin-resistant C3H/HeJ mice were used, but not in control mice. It is possible that repeated G-CSF injections after administration of cyclophosphamide (CY) in mice could accelerate recovery of granulopoiesis with a rather transient rise in blood neutrophil counts.

Purification and characterization of human granulocyte colony-stimulating factor (G-CSF).

A colony-stimulating factor (CSF) has been purified to homogeneity from the serum-free medium conditioned by one of the human CSF-producing tumor cell lines, CHU-2. The molecule was a hydrophobic glycoprotein (mol. wt 19,000, pI = 6.1 as asialo form) with possible O-linked glycosides. Amino acid sequence determination of the molecule gave a single NH2-terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological activity was apparently specific for a neutrophilic granulocyte-lineage of both human and mouse bone marrow cells with a specific activity of 2.7 X 10(8) colonies/10(5) non-adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G-CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis.

The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor.

Two different cDNAs for human granulocyte colony-stimulating factor (G-CSF) were isolated from a cDNA library constructed with mRNA prepared from human squamous carcinoma cells, which produce G-CSF constitutively. The nucleotide sequence analysis of both cDNAs indicated that two polypeptides coded by these cDNAs are different at one position where three amino acids are deleted/inserted. When the two cDNAs were introduced into monkey COS cells under the SV40 early promoter, both of them produced proteins having authentic G-CSF activity and some difference in the specific activity was suggested. A human gene library was then screened with the G-CSF cDNA and the DNA fragment containing the G-CSF chromosomal gene was characterized by the nucleotide sequence analysis. The human G-CSF gene is interrupted by four introns and a comparison of the structures of the two G-CSF cDNAs with that of the chromosomal gene indicated that the two mRNAs are generated by alternative use of two 5' splice donor sequences in the second intron of the G-CSF gene. When the G-CSF chromosomal gene was expressed in monkey COS cells by using the SV40 enhancer two mRNAs were detected by S1 mapping analysis.

Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor.

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation, and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently, Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively, and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein, and by using oligonucleotides as probes, have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore, Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells.