A simple and rapid model for hair-follicle regeneration in the nude mouse.

BACKGROUND: Methods for hair-follicle regeneration are important tools for investigating signalling and cytokines during hair-follicle morphogenesis and cycling. Several animal models for hair reconstitution have been established; however, these models have several shortcomings. AIM: To develop a simple and rapid model for hair induction in nude mouse. METHODS: We designed an improved flap model (IFM) for hair regeneration based on the existing flap assay. Histological sections and scanning electron microscopy were used to evaluate the regenerated hair. The fates of grafted cells were traced by fluorescence. The time required for hair induction was analysed and compared. RESULTS: IFM produced a large number of normal hairs, and the time required for hair induction using IFM was 20.67 ± 0.67 days, compared with 29.33 ± 0.67 days for the traditional flap assay. CONCLUSIONS: The time required for hair regeneration is considerably shortened with IFM. We speculate that this is due to increased blood supply at the transplantation sites.

Higher mortality in patients with right hemispheric intracerebral haemorrhage: INTERACT1 and 2.

BACKGROUND AND PURPOSE: Controversy exists over the prognostic significance of the affected hemisphere in stroke. We aimed to determine the relationship between laterality of acute intracerebral haemorrhage (ICH) and poor clinical outcomes. METHODS: A subsidiary analysis of the INTERACT Pilot and INTERACT2 studies--randomised controlled trials of patients with spontaneous acute ICH with elevated systolic blood pressure (BP), randomly assigned to intensive (target systolic BP <140 mm Hg) or guideline-based (<180 mm Hg) BP management. Outcomes were the combined and separate end points of death and major disability (modified Rankin scale (mRS) scores of 3-6, 6 and 3-5, respectively) at 90 days. RESULTS: A total of 2708 patients had supratentorial/hemispheric ICH and information on mRS at 90 days. Patients with right hemispheric ICH (1327, 49%) had a higher risk of death at 90 days compared to those with left hemispheric ICH after adjustment for potential confounding variables (OR, 1.77 (95% CI 1.33 to 2.37)). There were no differences between patients with right and left hemispheric ICH regarding the combined end point of death or major disability or major disability in the multivariable-adjusted models (1.07 (0.89 to 1.29) and 0.85 (0.72 to 1.01), respectively). CONCLUSIONS: Right hemispheric lesion was associated with increased risk of death in patients with acute ICH. The laterality of the ICH does not appear to affect the level of disability in survivors. TRIAL REGISTRATION NUMBER: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00226096 and NCT00716079.

A study of microemboli monitoring of atherosclerotic thrombotic cerebral infarction and artery stenosis.

This study aimed to assess the relationship between the recurrence and prognosis of patients with acute middle cerebral artery infarction, atherosclerotic brain infarction, and the existence of microemboli. We continuously enrolled patients with acute atherosclerotic thrombotic cerebral infarction artery stenosis. We performed transcranial Doppler color ultrasound micro emboli monitoring, color Doppler ultrasound carotid artery tests, intracranial and carotid artery magnetic resonance angiography, impairment evaluation of nerve function, and registration of stroke recurrence and stroke mortality. Of the 49 patients enrolled in the study, 123 main arteries presented atherosclerotic stenosis or formed plaques, and 33 patients had symptomatic stenosis. Patients with symptomatic stenosis have a higher incidence of microemboli than patients with asymptomatic stenosis (P = 0.009). The microembolus-positive rate increased in patients with unstable plaques (P = 0.001). Patients who were microembolus-negative were more likely to show a neural function deficient NIHSS (National Institutes of Stroke Scale) score improvement than patients who were microembolus-positive at one week (P = 0.026). However, we found no significant difference between mRS (modified rankin scale) score (P = 0.319), relapse, and death (P = 0.179). The rate of microembolus-positivity increased in patients with atherosclerotic thrombotic cerebral infarction and unstable plaques. Patients who were microembolus-negative were more likely to show an improvement of neural function deficiency than patients with microembolus-positivity at one week (P = 0.026).

Cardiac right-to-left shunt subtypes in Chinese patients with cryptogenic strokes: a multicenter case-control study.

BACKGROUND AND PURPOSE: Data on the possible association between cardiac right-to-left shunt (RLS) and cryptogenic stroke are lacking in Asians. RLS and its subtypes in Chinese cryptogenic stroke patients were investigated. METHODS: Patients (n = 153, mean age 42 ± 10 years, 81 male) with cryptogenic stroke from four medical centers in China and 135 healthy volunteers (mean age 34 ± 8 years, 54 male) were recruited. Contrast transcranial Doppler was used to assess the prevalence of RLS. A three-level RLS categorization was applied as follows: none, 0 microbubbles (MBs); small, 1-25 MBs; and large, >25 MBs. RLS was considered latent if it occurred only after the Valsalva maneuver or permanent when it occurred also during normal respiration. RESULTS: Overall, RLS (P = 0.02), large RLS (P < 0.001) and permanent RLS (P = 0.02) were more frequently detected in patients with cryptogenic stroke than in healthy volunteers. The prevalences of small RLS and latent RLS in the two groups were similar (22% vs. 21% and 11% vs. 10%, respectively). The proportion of large RLSs amongst the subjects with RLS was much higher in the patient group than in healthy volunteers (45% vs. 18%, P < 0.001), whilst the proportion of permanent RLS was similar (72% vs. 64%, P = 0.11). Most large RLSs in the patient group (22/27, 81%) were permanent RLSs. CONCLUSIONS: Cardiac RLS is associated with cryptogenic stroke in Chinese. However, the higher prevalence of overall RLS in the patient group was mainly due to the increased proportion of large RLSs. The results only support large RLSs as a pathological condition.

Effects of predistention with normal saline containing adrenaline on blood-vessel injury during epidural catheter placement.

This study investigated the effects of predistention with normal saline containing adrenaline on vascular plexus injury during epidural catheter placement. Three hundred parturients undergoing caesarean sections were randomly divided into three groups. Group I (n = 102) received an epidural injection with 5 ml normal saline; group II (n = 93) received 5 ml normal saline containing adrenaline (5 µg/ml); group III (n = 100) received direct epidural catheter placement. Five women were excluded from the analysis for technical reasons. The incidence of bloody fluid in the epidural needle was significantly lower in groups I and II compared with group III (eight [7.8%] and seven [7.5%] versus 17 [17.0%], respectively). There were no significant differences in the incidence of bloody fluid in the epidural catheter or in the incidence of intravascular epidural catheter placement between the three groups. Predistention with 5 ml normal saline before catheter insertion reduced the incidence of blood-vessel injury during epidural catheter placement, but adrenaline provided no additional protective effects.

Significance of perihematomal edema in acute intracerebral hemorrhage: the INTERACT trial.

BACKGROUND: Uncertainty surrounds the effects of cerebral edema on outcomes in intracerebral hemorrhage (ICH). METHODS: We used data from the INTERACT trial to determine the predictors and prognostic significance of "perihematomal" edema over 72 hours after ICH. INTERACT included 404 patients with CT-confirmed ICH and elevated systolic blood pressure (BP) (150-220 mm Hg) who had the capacity to commence BP lowering treatment within 6 hours of ICH. Baseline and repeat CT (24 and 72 hours) were performed using standardized techniques, with digital images analyzed centrally. Predictors of growth in edema were determined using generalized estimating equations, and its effects on clinical outcomes were estimated using a logistic regression model. RESULTS: Overall, 270 patients had 3 sequential CT scans available for analyses. At baseline, there was a highly significant correlation between hematoma and perihematomal edema volumes (r(2) = 0.45). Lower systolic BP and baseline hematoma volume were independently associated with absolute increase in perihematomal edema volume. History of hypertension, baseline hematoma volume, and earlier time from onset to CT were independently associated with relative increase in edema volume. Both absolute and relative increases in perihematomal edema growth were significantly associated with death or dependency at 90 days after adjustment for age, gender, and randomized treatment, but not when additionally adjusted for baseline hematoma volume. CONCLUSIONS: The degree of, and growth in, perihematomal edema are strongly related to the size of the underlying hematoma of acute intracerebral hemorrhage, and do not appear to have a major independent effect in determining the outcome from this condition.

Unique induction of p21(WAF1/CIP1)expression by vinorelbine in androgen-independent prostate cancer cells.

To study the mechanisms of the development of hormone refractory prostate cancer, we established an androgen-independent (AI) prostate cancer cell line derived from hormone-dependent (AD) LNCaP cells. Our previous studies have demonstrated that AI cells are deficient in expression of p21(WAFl/CIP1) (p21) due to overexpressed AR and are resistant to apoptosis. In this study, the induction of p53 and p21 expression by vinorelbine (Navelbine) was compared between AD and AI cells in an attempt to understand the difference(s) in apoptotic signalling pathways in these cells. Using a series of deletion of p21 reporter constructs, we found that vinorelbine mediated p21 induction in a p53-dependent manner in AD cells. In contrast, p21 expression restored by vinorelbine in AI cells was found to be through both p53-dependent and-independent pathways. In the absence of two p53 binding sites, Spl-3 and Spl-4 sites, in the promoter of human p21 gene, were found to be required for vinorelbine-mediated p21 activation. No p21 induction was observed by paclitaxel in AI cells. Exposure of AI cells to paciltaxel followed by vinorelbine produced synergism. Our data, thus, provide a basis for the synergistic combination of vinorelbine and paclitaxel for the treatment of advanced prostate cancer.

High anticancer efficacy of L-proline-m-bis (2-chloroethyl) amino-L-phenylalanyl-L-norvaline ethyl ester hydrochloride (MF13) in vivo.

The anticancer efficacy of the new anticancer tripeptide, L-proline-m-bis (2-chloroethyl) amino-L-phenylalanyl-L-norvaline ethyl ester hydrochloride (MF13), was investigated in mice. MF13 showed a therapeutic effect in liquid tumors and induced complete remission even in late stage malignancies. MF13 also inhibited human colon cancer growth in nude mice by more than 85% (volume, p<0.001). It acted in a dose-dependent manner and induced a complete regression of tumor in 20% of the mice when the initial dose was high (15 mg/kg, i.p.). Human melanoma exhibited a response to MF13 similar to colon cancer. Activity of MF13 in murine hepatoma in vivo was stronger than its precursor m-sarcolysin (p<0.001). Tumor cells in peritoneal cavities of the MF13 treated (s.c.) mice underwent an irreversible apoptosis. Side effects of MF13 were the transient depression of hemopoiesis and loss of body weight, which vanished within 9-10 days. LD50 of MF13 of a single i.p. injection was 27 mg/kg (94 mg/m2), 11 times higher than the therapeutic dose of a single injection.

Antibody responses to HIV-1 antigens are higher in HIV-1(+) intravenous drug users than in HIV-1(+) homosexuals.

Immune responses to HIV-1 infection of 42 HIV-1-positive asymptomatic intravenous drug users (IVDUs) were compared with those of 135 HIV-1-infected asymptomatic homosexual men in the present study. Twenty-five HIV-1(-) individuals served as normal controls. The comparison included antibody responses to five computer-predicted epitopes of HIV-1 p17, and viral proteins gp120 and p24 as well as p17. Major immunophenotypes were also investigated. Results showed that antibody responses to the five epitopes were significantly higher in the IVDUs. A larger proportion of the IVDUs, with respect to that of homosexuals, showed positive antibody responses to p24 and p17, respectively. However, the antibody response to gp120 was similar between the two cohorts. Immunophenotyping showed that HIV-1(+) homosexuals had higher profiles in most of the major subsets than did the IVDUs, especially in the total count of lymphocytes, absolute numbers of CD3+ cells and CD8+ cells. It appeared that the HIV-1(+) IVDU cohort had higher antibody responses to most of the viral antigens, but had lower levels of lymphocyte subsets in comparison with HIV(+) homosexuals.

[Experimental studies of the protective effects of basic fibroblast growth factor and Radix Salviae Miltiorrhizae on brain injury in rats caused by repeated exposures to +Gz].

Objective. To investigate the preventive and theralseutive (therapeutic) effects of basic fibroblast growth factor (bFGF) and Radix Salviae Miltiorrhizae (RSM) on brain injury caused by repeated +Gz exposures. Method. bFGF and RSM were injected intraperitoneally into SD rats before and after repeated +Gz exposures. The contents of excitatory amino acids (EAAs), nitric oxide (NO) and the number of cell apoptosis in the brain were measured, and were compared to those of the control group and normal saline (NS) group. Result. The contents of EAAs, NO and the number of cell apoptosis were significantly higher in repeated +Gz exposures group than those in control group. The values were markedly lower in bFGF and RSM group than those in repeated +Gz exposures group and NS group. Conclusion. bFGF and RSM showed distinct preventive and therapeutic effect on the brain injury induced by repeated +Gz exposures.

3-m-bromoacetylamino benzoic acid ethyl ester: a new cancericidal agent that activates the apoptotic pathway through caspase-9.

The mechanism underlying the cancericidal activity of 3-m-bromoacetylamino benzoic acid ethyl ester (3-BAABE) was investigated. 3-BAABE exerted a strong cancericidal effect on human leukemia and lymphoma cells (IC(50) < 0.2 microgram/mL) and on cell lines of prostate, colon, ductal, and kidney cancer (IC(50) 0.8 to 0.88 microgram/mL). Multiple drug resistance (MDR) had no effect on the susceptibility of human lymphoma cells to 3-BAABE, since Daudi/MDR(20) and wild-type Daudi cells had a similar susceptibility to the cytotoxic effect of 3-BAABE. The cancericidal effect of 3-BAABE, which was not associated with changes in the cell cycle, was mediated by apoptosis. Thus, cells exposed to 3-BAABE displayed the DNA fragmentation ladder characteristic for apoptosis, associated with a marked increase of the activity of apoptosis effector caspases-3 and -6, which was followed by proteolytic cleavage of DNA fragmentation factor (DFF) and poly(ADP-ribose) polymerase (PARP). Exposure of tumor cells to 3-BAABE increased the activity of apical caspase-9, but had no effect on caspase-8. Complete inhibition of 3-BAABE-induced apoptosis was exerted by LEHD-FMK, a caspase-9 inhibitor. DEVD-FMK, a caspase-3 inhibitor, and VEID-FMK, a caspase-6 inhibitor, partially inhibited 3-BAABE-induced apoptosis, whereas exposure to IETD-FMK, a caspase-8 inhibitor, had no effect. The fragmentation and elevated activity of caspase-9 in 3-BAABE-treated cells and the fact that only an inhibitor of caspase-9 abrogated 3-BAABE-induced apoptosis indicate that 3-BAABE is a distinctive compound that elicits apoptosis through a pathway that is limited specifically to activation of apical caspase-9.

[Changes of mRNA expression of IL-1beta and TNF-alpha in rat brains after repeated exposures to +Gz].

Objective. To study changes of mRNA expression of IL-1beta and TNF-alpha in rat brains after repeated exposures to +Gz. Method. Twenty conscious SD rats served as the subjects. They were randomly divided into 5 groups. Using an animal centrifuge, control rats (n = 4) were exposed to +1 Gz and experimental rats (n = 16) were exposed to +14 Gz three times, each for 45 s with 30 min interval in between. The rat brains were taken 30 min, 6 h, 24 h and 48 h after the last centrifuge run and total RNA was isolated. mRNA expression levels of IL-1beta and TNF-alpha in rat brain were measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Result. mRNA expression levels of IL-1beta and TNF-alpha in rat brains taken 30 min, 6 h and 24 h after repeated +Gz exposures were significantly higher than those in control rats, but returned to normal after 48 h. Conclusion. It suggested that mRNA expressions of IL-1beta and TNF-alpha in rat brains can be stimulated by repeated +Gz exposures and the increased expression of IL-1beta and TNF-alpha may play a role in the pathologic course of brain damage induced by +Gz exposures, but the damage is reversible.

Low frequency of the ccr5delta32 HIV-resistance allele in mainland China: identification of the first case of ccr5delta32 mutation in the Chinese population.

A 32-bp deletion on the CCR5 gene (ccr5delta32) confers resistance to HIV-1 infection. This deletion is common in Caucasians, but rare in Asians. Since the frequency of the ccr5delta32 allele of Chinese in mainland China has been unknown we investigated the ccr5delta32 mutation in a cohort of 407 Chinese people in this area. A 225-bp fragment of CCR5 encompassing the 32-bp region was analysed by PCR, hybridization and sequencing. Only 1 out of 407 subjects was heterozygous for ccr5delta32 and no homozygotes were detected. The frequency of ccr5delta32 in this cohort is thus 0.00123, i.e. much lower than that of Caucasians. The ccr5delta32 heterozygote is a healthy young man. To our knowledge this is the first ccr5delta32 mutant found in Chinese people. The results indicate that ccr5delta32 does exist in Chinese people, but at very low frequency. This suggests that ccr5delta32 is not a significant factor for the genetic resistance to HIV-1 in Chinese people.

Alterations of T-lymphocyte subsets, soluble IL-2 receptor, and IgE in peripheral blood of children with acute asthma attacks.

BACKGROUND: T-cell activation and alteration of cytokines are involved in the pathogenesis of atopic asthma. However, the profile of circulating T-lymphocyte subsets, related cytokines, and plasma IgE during acute asthma attacks is still unclear. OBJECTIVE: In an attempt to illustrate the dynamics of these parameters in asthma attacks, we investigated the changes of T-cell subsets, lymphocyte activation, soluble IL-2R, and IgE in peripheral blood in children during and after acute asthma attacks. METHODS: This study was carried out in a cohort of Chinese children (n = 59) with acute asthma attacks. Immunoassays were performed when the patients had acute attacks before treatment, and the patients were reexamined in the 4 weeks after the resolution of acute attacks with therapy. Paired t tests were used for the statistical analysis of these patients to compare the data obtained during and after the acute attacks. Twenty healthy, age-matched subjects were used as normal control subjects. Nine children with long-term stable asthma were used as control subjects with stable asthma. RESULTS: CD3+, CD4+, CD8+, and IL-2R+ (CD25+) cells; plasma soluble IL-2 receptor; and IgE were significantly higher in patients with acute attacks than in control subjects. (P <.05, P <.05, P <.001, P <.05, P <.0001, and P <.0001, respectively). Immunoelectron microscopy exhibited an increased expression of IL-2R on lymphocytes in acute attacks as compared to control subjects. The abnormalities returned to normal, with the exception of IgE, when clinical remission was achieved after treatment. Correlation analyses revealed a positive relationship between plasma IgE and soluble IL-2R in asthma attacks (r = 0.83, P =.0001). Plasma IgE and soluble IL-2R of those who were in remission positively correlated with their production in acute attacks (r = 0.58, P =.001 and r = 0.71, P =.0001, respectively). CONCLUSION: This study suggests that (1) the percentage of CD4+, CD8+, or IL-2R+ lymphocytes in peripheral blood was significantly elevated during acute attacks and returned to normal ranges after complete remission was achieved; (2) plasma soluble IL-2R is a sensitive marker for asthma activity; and (3) atopic asthmatic children seem to have a hereditary predisposition of having higher levels of soluble IL-2R in asthma attacks, coinherited with the trait of IgE.

Novel suicide ligands of tubulin arrest cancer cells in S-phase.

It is presently accepted that the mechanism of action for all anti-tumor tubulin ligands involves the perturbation of microtubule dynamics during the G2/M phase of cell division and subsequent entry into apoptosis [1]. In this report, we challenge the established dogma by describing a unique mechanism of action caused by a novel series of tubulin ligands, halogenated derivatives of acetamido benzoyl ethyl ester. We have developed a suicide ligand for tubulin, which covalently attaches to the target and shows potent cancericidal activity in tissue culture assays and in animal tumor models. These compounds target early S-phase at the G1/S transition rather than the G2/M phase and mitotic arrest. Bcl-2 phosphorylation, a marker of mitotic microtubule inhibition by other tubulin ligands was dramatically altered, phosphorylation was rapid and biphasic rather than a slow linear event. The halogenated ethyl ester series of derivatives thus constitute a unique set of tubulin ligands which induce a novel mechanism of apoptosis.

3-(Iodoacetamido)-benzoylurea: a novel cancericidal tubulin ligand that inhibits microtubule polymerization, phosphorylates bcl-2, and induces apoptosis in tumor cells.

3-(Iodoacetamido)-benzoylurea (3-IAABU) is a newly synthesized antitubulin compound with a molecular weight of 347. 3-IAABU exhibited anticancer activity in a variety of tumor cell lines with ID90 in the range of 0.015-0.29 microM for leukemic cells and 0.06-0.92 microM for solid tumors. Higher selectivity against malignant cells was observed with 3-IAABU than that with vinblastine and paclitaxel. It inhibits microtubule assembly in tubulin systems either with or without microtubule-associated proteins (ID50 was 0.1 microM and 1.2 microM, respectively) and microtubule depolymerization was not affected, indicating an inhibition of polymerization by binding of 3-IAABU to the heterodimeric subunit of tubulin. 3-IAABU was shown to inhibit the binding of colchicine, a subunit binding compound, but did not inhibit binding of vinblastine and guanosine 5'-triphosphate/guanosine 5'-diphosphate, indicating that colchicine site corresponds to the site that 3-IAABU locates. Tumor cells treated with 3-IAABU showed scattered chromosomes in metaphase. Normal microtubule architecture or spindle apparatus was absent in these cells; instead, punctuated aggregates of tubulin were found by an immunofluorescent staining. Cell cycle analyses showed an accumulation of tumor cells at M phase after a 4-h treatment with 3-IAABU. The phosphorylated bcl-2 representative of an inactivated form of the oncoprotein was found in the cells 12 h after treatment with 3-IAABU. These cells progressed to apoptosis within 16 h. As a new tubulin ligand, 3-IAABU could be a promising agent in cancer chemotherapy.

Synthesis, cancericidal and antimicrotubule activities of 3-(haloacetamido)-benzoylureas.

The four title compounds (not hitherto reported) were synthesized from 3-aminobenzoic acid through its trifluoroacetic acid-acid chloride derivative, reaction with urea and aminolytic deprotection to yield 3-aminobenzoylurea, followed by unconventional haloacetylation. Three key factors were found essential for antitumor activity: (i) the cytotoxic nature of the halogen: I > Br > Cl > F (ID90 0.014->10 microM); (ii) the position of the halogen: only the 3-position (meta) expressed relevant activity; and (iii) the presence of the urea group (1-position). The selectivity of the bromo and iodo compounds were higher than those of vinblastine and paclitaxel in terms of cytotoxicity (ID50 ratios in nonmalignant myocardial fibroblasts and CEM leukemia cells) and therapeutic indices (P338 leukemia bearing mice). Relevant mechanisms of bioactivity were mitotic arrest and apoptosis. Complete inhibition of microtubule assembly occurred in cell-free systems (at 2.8 versus 2.1 microM for vinblastine); in contrast to paclitaxel, the target compounds did not interfere with microtubule disassembly. The strong cancericidal and antimicrotubular activities of the bromine and iodine compounds justify further exploration of their potential in antineoplastic chemotherapy.

3-Bromoacetylamino benzoylurea (3-BAABU), a new antimicrotubule cancericidal agent applied in cytogenetic analysis in hematology.

3-Bromoacetylamino benzoylurea (3-BAABU) is a newly synthesized antimicrotubule cancericidal compound. In the present study, we investigated the possibility of using 3-BAABU as a mitotic blocking agent for hematologic karyotyping. Treatment with 3-BAABU caused scattering of metaphase chromosomes throughout the cytoplasm both in phytohemagglutinine (PHA)-stimulated human lymphocytes and in human leukemic cells. Kinetic showed a rapid uptake of 3-BAABU by treated cells and irreversibility of its effect. Using 3-BAABU in routine procedure, a karyotype of lymphocytes from a normal male was 46, XY, with normal structure and CEM leukemic line was 85, XX, in a representative spread with abnormalities similar to reports using other blocking agents. Using 3-BAABU in spectral karyotyping, details of translocations in CEM leukemic cells were readily detected in several chromosomes, such as 7 [t(7;11)], 8 [t(8;9)], 9 [t(8;9) & t(9;19)], 11 [t(7;11)], 16 [t(16;18;20)] and 20 [t(1;20)]. 3-BAABU displayed two important characters in cytogenetics, 1) it caused dispersion of chromosomes, avoiding chance of overlap; 2) compared to the conventional mitotic blocking agent, vinblastine sulfate, 3-BAABU exhibited much gentle effect on chromosomes, thus providing more flexibility in time to perform karyotyping.