Susceptibility of diverse primary HIV isolates with varying co-receptor specificity's to CXCR4 antagonistic compounds

The chemokine receptors CCR5 and CXCR4 are an obvious target for HIV therapies. Two compounds, T-22 and AMD-3100, have been shown to inhibit infection of CXCR4-using HIV-1 isolates. The specificity of T-22 and AMD-3100 was further confirmed by their ability to block entry of HIV-1 in GHOST-CXCR4 transfected cells with no effect on viral entry in the GHOST-CCR5 cells. The ability of T-22 to block replication of diverse HIV-1 isolates (group M, subtypes A, B, D, E, and F as well as group O) and HIV-2 primary isolates with varying coreceptor specificities ranging from exclusive CCR5 usage to multiple coreceptor usage was examined in detail. T-22 was found to be highly effective (>90%) at blocking infection of diverse HIV-1 (subtypes A-F, and group O) and HIV-2 isolates that use multiple coreceptors in human PBMCs homozygous for a 32-bp deletion in CCR5 (CCR5-/-), but less effective in CCR5 +/+ PBMCs. Additionally, sequential primary HIV-1 isolates obtained from a longitudinal cohort who had switched from single coreceptor usage to a broad range of multiple receptors could be blocked effectively by both T-22 and AMD-3100 in CCR5-/- PBMCs. Our data suggest that CXCR4 antagonistic compounds are highly effective in blocking the entry of X4-tropic HIV-1, and that these compounds could be a useful additive to current anti-retroviral therapy for clinical management of HIV disease.     

Pemphigus vulgaris: the role of IL-1 and IL-1 receptor antagonist in pathogenesis and effects of intravenous immunoglobulin on their production

Intravenous immunoglobulin (IVIG) is increasingly being used for the treatment of autoimmune diseases. In the present report, the role of IVIG on in vivo and in vitro production of IL-1 and IL-1 receptor antagonist (Ra) was studied in patients with pemphigus vulgaris (PV). Serum samples from 20 untreated patients with active PV prior to initiation of systemic therapy, 20 patients receiving IVIG treatment, 20 patients in clinical remission after conventional therapy, and 20 normal human controls were studied to determine the serum levels of IL-1alpha, IL-1beta, and IL-1Ra. The in vitro production of these cytokines was measured in the culture supernatant of peripheral blood mononuclear cells (PBMC) from 10 PV patients immediately before and after IVIG therapy and from age and sex-matched 10 healthy donors simultaneously. Elevated levels of IL-1alpha and IL-1beta were detected (i) in the serum of untreated PV patients with active disease prior to systemic therapy and (ii) before IVIG infusions in patients receiving IVIG therapy. These increased levels are statistically significant when compared to the levels in healthy controls (P < 0.01). A marked reduction of IL-1alpha and IL-1beta was detected (i) in the serum of patients in prolonged clinical remission and (ii) immediately after IVIG infusion in those patients on IVIG therapy. Increased level of IL-1Ra was detected in PV patients in prolonged clinical remission and after IVIG infusion in those receiving IVIG therapy. These differences were statistically significant when compared to the levels in normal controls and to the levels in the sera of patients with active disease (P < 0.01) or just before the beginning of IVIG infusion (P < 0.01). Similar differences in the levels of IL-1alpha, IL-1beta, and IL-1Ra were found in the culture supernatant of PBMC isolated from the PV patients pre and post IVIG therapy. These observations suggests that, compared to normal controls, patients with active PV have reversed levels of IL-1alpha, IL-1beta, and IL-1Ra. IVIG therapy may down-regulate production of IL-1alpha and IL-1beta and enhance production of IL-1Ra, in vivo and in vitro. This might be one of the important mechanisms by which IVIG produces its early therapeutic effects in pemphigus vulgaris.     

Bac-to-Bac杆状病毒表达系统表达鼠IL-4受体拮抗体蛋白的研究

目的 利用Bac-to-Bac杆状病毒表达系统表达鼠IL-4受体拮抗体 (mIL-4RA)蛋白.方法 PCR方法扩增小鼠IL-4 C118截断型基因,定向克隆入转移质粒pFastBacHTB中,转化感受态DH10Bac细胞,在DH10Bac细胞内重组pFastBacHTB与杆粒发生转座.筛选阳性克隆,提取重组杆粒,转染sf9昆虫细胞株,获取完整重组杆状病毒.反复感染sf9细胞,扩增病毒同时表达目的 蛋白;用ELISA方法进行蛋白鉴定并初步定量.结果 经核苷酸序列测定及PCR方法,鉴定成功获得含mIL-4RA基因的重组杆粒;通过杆粒转染后sf9细胞所表现出来的细胞病变,推断成功转染并获得重组杆状病毒;最后ELISA方法初步定量sf9细胞培养上清中mIL-4RA蛋白表达量为(1.15±0.12) ng/mL.结论 本研究利用Bac-to-Bac杆状病毒表达系统成功表达mIL-4RA蛋白,为进一步研究其生物学活性及功能奠定了实验基础,同时亦为其他蛋白质的真核表达提供了方法学的参考.     

A lower ongoing pregnancy rate can be expected when GnRH agonist is used for triggering final oocyte maturation instead of HCG in patients undergoing IVF with GnRH antagonists

BACKGROUND: Eliciting an endogenous LH surge by GnRH-agonist for the induction of final oocyte maturation may be more physiological compared with the administration of HCG. However, the efficacy of this intervention in patients treated for IVF with GnRH antagonists remains to be assessed. METHODS: 106 patients were randomized to receive either 10 000 IU urinary HCG or 0.2 mg Triptorelin for triggering final oocyte maturation. Ovarian stimulation for IVF was performed with a fixed dose of 200 IU recombinant FSH and GnRH antagonist was started on stimulation day 6. Luteal phase was supported with micronized vaginal progesterone and oral estradiol. The study was monitored continuously for safety and stopping rules were established. RESULTS: No significant differences were present in the number of cumulus-oocyte complexes retrieved, in the proportion of metaphase II oocytes, in fertilization rates or in the number and quality of the embryos transferred between the two groups. However, a significantly lower probability of ongoing pregnancy in the GnRH agonist arm prompted discontinuation of the trial, according to the stopping rules established (odds ratio 0.11; 95% confidence interval 0.02-0.52). CONCLUSIONS: Lower probability of ongoing pregnancy can be expected when GnRH agonist is used for triggering final oocyte maturation instead of HCG in patients undergoing ovarian stimulation for IVF with GnRH antagonists.     

Changes in electromyographic activity,muscle fibre and force production characteristics during heavy resistance/power strength training in middle‐aged and older men and women

The effects of a 6‐month resistance training (2 day/week) designed to develop both strength and power on neural activation by electromyographic activity (EMG) of the agonist and antagonist knee extensors, muscle fibre proportion and areas of type I, IIa, and IIb of the vastus lateralis (VL) as well as maximal concentric one repetition maximum (1 RM) strength and maximal and explosive isometric strength of the knee extensors were examined. A total of 10 middle‐aged men (M40; 42 ± 2), 11 middle‐aged women (W40; 39 ± 3), 11 elderly men (M70; 72 ± 3) and 10 elderly women (W70; 67 ± 3) served as subjects. Maximal and explosive strength values remained unaltered during a 1‐month control period. After the 6‐month training maximal isometric and 1RM strength values increased in M40 by 28 ± 14 and 27 ± 7% (P < 0.001), in M70 by 27 ± 17 and 21 ± 9% (P < 0.001), in W40 by 27 ± 19 and 35 ± 14% (P < 0.001) and in W70 by 26 ± 14 and 31 ± 14% (P < 0.001), respectively. Explosive strength improved in M40 by 21 ± 41% (P < 0.05), in M70 by 21 ± 24% (P < 0.05), in W40 by 32 ± 45% (NS) and in W70 by 22 ± 28% (P < 0.05). The iEMGs of the VL and vastus medialis (VM) muscles increased during the training in M40 (P < 0.001 and 0.05), in M70 (P < 0.001 and 0.05), in W40 (P < 0.001 and 0.05) and in W70 (P < 0.001 and 0.05). The antagonist biceps femoris (BF) activity during the isometric knee extension remained unaltered in M40, in W40, and in M70 but decreased in W70 (from 42 ± 34 to 32 ± 26%; P < 0.05) during the first 2 months of training. Significant increases occurred during the training in the mean fibre areas of type I in W70 (P < 0.05) and of overall type II along with a specific increase in IIa in both W40 (P < 0.05) and in W70 (P < 0.05), while the changes in the male groups were not statistically significant. The individual percentage values for type II fibres at pretraining correlated with the individual values for 1 RM strength in both W70 (r=0.80; P < 0.05) and M70 (r=0.61; P < 0.05) and also at post‐training for maximal isometric torque in W70 (r=0.77, P < 0.05). The findings support the concept of the important role of neural adaptations in strength and power development in middle‐aged and older men and women. The muscle fibre distribution (percentage type II fibres) seems to be an important contributor on muscle strength in older people, especially older women. Women of both age groups appear to be hypertrophically responsive to the total body strength training protocol performed two times a week including heavier and lower (for fast movements) loads designed for both maximal strength and power development, while such a programme has limited effects on muscle hypertrophy in men.     

Genetic variation screening and association studies of the adenylate cyclase activating polypeptide 1 (ADCYAP1) gene in patients with type 2 diabetes

Adenylate cyclase activating polypeptide 1 (ADCYAP1) is a pancreatic neuropeptide and modulates glucose-stimulated insulin secretion. The ADCYAP1 gene is located on chromosome 18p11 linked to type 2 diabetes. To test whether it is a candidate gene for type 2 diabetes, screening of the gene in Finnish and Swedish type 2 diabetic patients was done. Two novel SNPs, g.9863G>A (G54D) in exon 3 and g.12712C>G in the 3'-UTR of exon 5 of the ADCYAP1 gene (accession number X60435), were found. PCR-RFLP genotyping was then performed in a total of 253 type 2 diabetic patients and 253 non-diabetic control subjects. Transmission disequilibrium test (TDT) was performed in 132 parent-offspring trios. The G allele frequencies of g.9863G>A (G54D) and g.12712C>G of the ADCYAP1 gene were higher in type 2 diabetic patients than in non-diabetic control subjects (21.0% vs 15.8%, P=0.04; 5.3% vs 3.0%, P=0.045). However, no significant differences in clinical variables was seen between the different genotype carriers, and also no transmission distortion of the G allele of SNP g.9863G>A (G54D) was observed in 132 parent-offspring trios. The present study thus suggest that the variants in the ADCYAP1 gene may not be major influence of the susceptibility to type 2 diabetes in Finnish and Swedish Caucasians.     

Ovarian stimulation by clomiphene citrate and hMG in combination with cetrorelix acetate for ICSI cycles

BACKGROUND: The introduction of GnRH antagonists such as cetrorelix acetate has made possible the simplification of ovarian stimulation. However, the most effective protocol for their administration has not yet been clearly defined. METHODS: Forty women with male-factor infertility undergoing 40 ICSI cycles were included in the study. Clomiphene citrate at 100 mg a day was given from cycle day 3 through day 7. hMG at 150 IU was given on cycle days 4, 6 and 8, and was adjusted from day 9 according to the follicular and hormone responses. Cetrorelix acetate at 2.5 mg was administered when the leading follicle reached 14 mm. The remaining 0.5 mg was divided into two 0.25 mg injections for possible later use. Serum FSH, LH, estradiol and progesterone levels were measured daily from the day of cetrorelix acetate injection until hCG was given. RESULTS: Serum LH level was suppressed effectively for 4 days. Four patients (10%) needed one or two additional injections of 0.25 mg cetrorelix acetate. No premature LH surge was detected in any of the women treated. Sixteen women became pregnant (40%), of which 14 pregnancies (35%) were ongoing at the time of writing. CONCLUSIONS: This study demonstrates that this new protocol is feasible for couples with male-factor infertility undergoing ICSI.     

Effect of NMDA receptor antagonist on proliferation of neurospheres from embryonic brain

The N-methyl-D-aspartate (NMDA) receptor, a subtype of ionotropic glutamate receptors, plays an important role in the regulation of neuronal development, learning and memory, and neurodegenerative diseases. NMDA receptor blockade enhances neurogenesis in the hippocampal dentate gyrus in vivo. The effect of NMDA receptor antagonist on proliferation of neural progenitor cells, however, remains to be determined. We investigated changes in the diameter and number of neurospheres derived from the embryonic rat brain after NMDA receptor blockade. Cortical progenitor cells were isolated from gestational day 18 fetal rats according to the Percoll density gradient method. Cultured spheres expressed neural progenitor markers, musashi-1 and nestin. Immunohistochemical analysis demonstrated that cells in Dulbecco's modified Eagle medium/F12 containing 1% fetal bovine serum on day 8 differentiated to MAP-2-positive neurons and GFAP-positive astrocytes. The expression of NR1 and NR2B subunits of the NMDA receptor in neurospheres was detected. Neither brief nor sustained exposure to NMDA altered the diameter and number of neurospheres. Brief exposure to 30 μM MK-801, an NMDA receptor antagonist, decreased the diameter of neurospheres. Sustained exposure to 30 μM MK-801 decreased the diameter and number of neurospheres. Our results provide evidence that MK-801 directly decreased proliferation of neural progenitor cells.     

Ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and the LHRH-agonist buserelin. European Cetrorelix Study Group

In this prospective and randomized study, 188 patients received the luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix, and 85 patients the LHRH agonist buserelin to prevent endogenous luteinizing hormone (LH) surges during ovarian stimulation in in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles. Ultimately, 181 patients (96.3%) in the cetrorelix group, and 77 (90.6%) in the buserelin group, reached the day of the human chorionic gonadotrophin (HCG) injection. The mean number of human menopausal gonadotrophin (HMG) ampoules administered and the mean number of stimulation days with HMG were significantly less in the cetrorelix group than in the buserelin group (P < 0.01). A rise in LH and progesterone concentrations was observed in three of the 188 patients (1.6%) who received cetrorelix. On the day of the HCG administration, more follicles of a small diameter (11-14 mm) were observed in the buserelin group than in the cetrorelix group (P = 0. 02) and the mean serum oestradiol concentration was significantly higher in patients who received buserelin than in those who received cetrorelix (P < 0.01). Similar results were observed in fertilization, cleavage and pregnancy rates in the two groups. In conclusion, the use of the LHRH antagonists might be considered more advantageous because of the short-term application needed to inhibit gonadotrophin secretion, so allowing a reduction in the treatment time in a clinically significant manner.     

Masking,De Lange curves and integration time as revealed by the electroretinogram of a tree shrew (Tupaia chinensis)

In order to elucidate the effects of angiotensin II on renal function, angiotensin II (AII; 1 ng/kg per min) and the AII antagonist 1-sar-8-ala-angiotensin II (AIIA; 200 ng/kg per min) were infused into the renal artery of anesthetized dogs (pentobarbital), on either a high (8 mmol/kg per day for seven days) or a low sodium intake (0.5 mmol/kg). In sodium replete dogs AII produced renal vasoconstriction with decreased RBF (–28%;P<0.001), but with less decrease of GFR (–14%;P<0.001), leading to an increase of FF (+19%;P<0.01),andantidiuresis(–39%;P<0.001); the antinatriuresis (–58%;P<0.001) exceeded the antidiuresis (P<0.001). RBF (–10%;P<0.001) was less pronounced (P<0.001) during AII in sodium deplete dogs, GFR remained unchanged, but FF increased to the same extent (+16%;P<0.05); diuresis and urinary electrolyte excretion were however not affected. AIIA did not affect RBF, GFR, FF, nor diuresis in sodium replete dogs suggesting that endogenous AII has no tonic influence on renal function in these conditions. In sodium deplete animals AIIA produced an 11% (P<0.001) increase of RBF, without changes of GFR; FF decreased by 12% (P<0.01), but diuresis, natriuresis and kaliuresis were not affected.