Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.     

骨髓干细胞移植对心肌梗死大鼠血流动力学指标和心脏功能的影响

目的:心肌梗死所致的细胞缺失和瘢痕形成是心力衰竭乃至死亡的病理基础,目前药物治疗、介入治疗和外科手术均不能替代坏死心肌和彻底改善心脏功能。观察骨髓干细胞移植对心肌梗死大鼠血流动力学指标和心功能的影响。方法:实验于2005-03/2006-10在哈尔滨医科大学附属第一医院细胞移植中心完成。①实验动物:SD雄性大鼠60只作为细胞移植的受体,随机数字表法分成假手术组、心肌梗死组、细胞移植组,20只/组。另取SD雄性幼鼠10只作为骨髓干细胞的供体。实验过程中对动物的处置符合动物伦理学标准。②实验方法:取幼鼠股骨骨髓,Percoll分离后收取细胞层,加入含体积分数为0.1的胎牛血清、100IU/mL青霉素、100g/mL链霉素的DMEM营养液,差速贴壁法分离骨髓干细胞,达80%～90%融合时采用胰酶 乙二胺四乙酸消化传代。向含有第3代骨髓干细胞的培养液中加入5-氮杂胞嘧啶核苷进行诱导,3周后行BrdU标记,离心后配制成1×1012L-1的细胞悬液用于移植。心肌梗死组、细胞移植组大鼠建立心肌梗死模型,假手术组未结扎冠状动脉。细胞移植组吸取0.2mL骨髓干细胞悬液注射到瘢痕组织中,心肌梗死组注入等量干细胞培养液基质,假手术组不予任何移植处理。③实验评估:术后4周,利用导管和心动超声技术检测各组大鼠左室舒张末期内压、左室收缩末期内压、左室压力最大变化值、左室压力最小变化值、等容时间常数和心率。结果:术后4周,与假手术组比较,心肌梗死组左室收缩末期内压、左室压力最大变化值、左室压力最小变化值均明显降低(P<0.01),左室舒张末期内压、等容时间常数均明显增高(P<0.01);与心肌梗死组比较,细胞移植组以上各项指标均明显好转(P<0.01)。结论:骨髓干细胞移植到瘢痕心肌组织中,能改善心肌梗死后大鼠的血流动力学参数和心脏功能。     

Stroke unit Nurse Managers' views of individual and organizational factors liable to influence evidence‐based practice: A survey

The uptake of evidence into practice may be impeded or facilitated by individual and organizational factors within the local context. This study investigated Nurse Managers of New South Wales, Australia, stroke units (n = 19) in their views on: leadership ability (measured by the Leadership Practices Inventory), organizational learning (measured by the Organizational Learning Survey), attitudes and beliefs towards evidence‐based practice (EBP) and readiness for change. Overall Nurse Managers reported high‐level leadership skills and a culture of learning. Nurse Managers' attitude towards EBP was positive, although nursing colleague's attitudes were perceived as less positive. Nurse Managers agreed that implementing evidence in practice places additional demands on staff; and almost half (n = 9, 47%) reported that resources were not available for evidence implementation. The findings indicate that key persons responsible for evidence implementation are not allocated sufficient time to coordinate and implement guidelines into practice. The findings suggest that barriers to evidence uptake, including insufficient resources and time constraints, identified by Nurse Managers in this study are not likely to be unique to stroke units. Furthermore, Nurse Managers may be unable to address these organizational barriers (i.e. lack of resources) and thus provide all the components necessary to implement EBP.     

Impact of Balance Confidence on Daily Living Activities of Older People with Knee Osteoarthritis with Regard to Balance,Physical Function,Pain, and Quality of Life – A Preliminary Report

Objectives: The objective of this study was to explore the impact of balance confidence on different activities of daily living (ADL) in older people with knee osteoarthritis (OA).

Methods: Forty-seven consecutive participants with knee OA were included in this cross-sectional study. They were divided according to the results of the Activities-specific Balance Confidence (ABC) Scale into a group with a low level of confidence in physical functioning (ABC < 50, n = 22) and a group with moderate and high levels of confidence (ABC ≥ 50, n = 25).

Results: In the ABC < 50 group, the effect of pain on ADL, the physician’s global assessment of the disease, and the Western Ontario and McMaster Universities Osteoarthritis Index scores were significantly higher, while quality of life (Short form-36) was lower compared to the ABC ≥ 50 group. No significant difference was found between the two groups regarding the static and dynamic balance measurements.

Conclusions: Older people with knee OA who were less confident in their daily physical activities had more physical difficulties and a greater effect of pain on ADL, lower quality of life, and a higher physician’s global assessment, but no differences were obtained in balance tests.

Clinical Implications: In people with knee OA, decreased balance confidence is associated with more physical difficulties, an increased effect of pain on ADL, and lower quality of life. An improved awareness of decreased balance confidence may lead to more effective management of older people with knee OA by improving their mobility and QOL through rehabilitation. Furthermore, future research in that direction is warranted.     

A three-dimensional evaluation of microleakage of class V cavities prepared by the very short pulse mode of the erbium:yttrium–aluminium–garnet laser

The aim of this study was to evaluate microleakage along resin restoration in cavities prepared with an erbium:yttrium–aluminium–garnet (Er:YAG) laser, with and without acid etching, and to compare it with that in diamond-drilled cavities. Thirty intact molars were divided into three equal groups. In the teeth in group I, class V cavities were prepared with a diamond drill. Cavities in groups II and III were prepared with an Er:YAG laser (400 mJ/15 Hz for enamel and 250 mJ/10 Hz for dentine). The cavities in groups I and II were acid-etched and adhesive and flowable composite were applied to all cavities. The specimens were first immersed in dye for 24 h and then in 5% nitric acid for 72 h for softening. The fillings were extracted and photographed through a dissecting microscope. The leakage area was measured with specially designed software. The Kruskal–Wallis test showed that the best ranking was group II [mean range (m.r.) = 27.46], followed by group I (m.r. = 33.48) and, lastly, group III (m.r. = 45.15). The differences between groups I and III (P = 0.023) and between groups II and III were statistically significant (P = 0.080). The least microleakage was found in those cavities prepared by Er:YAG laser and subsequently acid-etched, whereas the most leakage was in the lased cavities that had not been etched; the traditional diamond-drilled acid-etched cavities produced medium leakage.     

Muscle strength and bone density in patients with different rheumatic conditions: cross-sectional study

Aim

To explore the relationship between muscle strength and bone density in patients with different rheumatic diseases and to examine whether inflammatory arthritis was more harmful for muscle strength and bone loss than degenerative joint diseases.

Methods

The study included 361 men and women with a mean ± standard deviation age of 60.5 ± 11.4 years and different rheumatic conditions: regional syndromes, osteoarthritis of the hands, shoulders, knees, and hips, and inflammatory arthritis. Maximum voluntary back strength was measured by isometric dynamometry. Bone mineral density (BMD; g/cm²) of the lumbar spine, femoral neck, and distal radius was measured by dual-energy x-ray absorptiometry. Anthropometry and lifestyle characteristics were also assessed.

Results

Back strength was lowest in patients with hand and shoulder osteoarthritis (20.0 ± 17.9 kg), followed by patients with inflammatory arthritis (24.8 ± 19.2 kg). Patients with inflammatory arthritis had the lowest BMD at the mid-radius (0.650 ± 0.115 g/cm²) and femoral neck (0.873 ± 0.137 g/cm²), while patients with hand and shoulder osteoarthritis had the lowest BMD at the mid-radius (0.660 ± 0.101). In both sexes, muscle strength was significantly lower in patients who had lower BMD (T score<-1.0). Multiple regression analysis identified significant predictors of back strength to be spine BMD (P = 0.024) and body mass index (P = 0.004) in men and femoral neck BMD in women (P = 0.004).

Conclusion

Muscle strength decline may be connected to bone loss in patients with rheumatic conditions, especially those with inflammatory joint diseases.There is a concomitant decline in muscle strength of the upper and lower limbs and bone density after the fifth decade of the life (1,2). Impaired muscle function is a common consequence in patients with rheumatic diseases, especially those with inflammatory joint diseases. Muscle strength may also be significantly reduced around joints affected with osteoarthritis. Several studies showed greatly reduced isokinetic strength in patients with rheumatoid arthritis (3-5) and patients with knee osteoarthritis (6).It is also known that muscle strengthening can yield a bone-building effect (7). Exercises with greater loading and higher impact activities produce the greatest skeletal benefit (8). Increased muscle weakness can also compound the problem of low bone density by increasing the risk of falls and fracture. A positive correlation between muscle strength and bone density has been shown in several studies (9-17). Some of them demonstrated the association only in postmenopausal women (12,17) but not in men (9,13), while other found a site-specific correlation between muscle strength and bone mineral density (BMD) (4,12). However, several studies did not find a correlation between any measures of muscle strength and BMD (18,19). With such contradictory reports, it is difficult to make clinically relevant conclusions about the relationship between muscle strength and bone mass, although this may be one of the key factors that affect the rehabilitation outcome.The aim of the study was to assess the differences in muscle strength and bone density between patients with different rheumatic conditions. Since muscle strength is an important determinant of bone density, we explored whether the age-related decline in bone density and muscle strength was more pronounced in patients with inflammatory arthritis than in those with degenerative joint diseases.     

Exacerbated potassium-induced paralysis of mouse soleus muscle at 37°C vis-à-vis 25°C: implications for fatigue. K+ -induced paralysis at 37°C

The main aim was to investigate the effects of raised [K⁺]_o on contraction of isolated non-fatigued skeletal muscle at 37°C and 25°C to assess the physiological significance of K⁺ in fatigue. Mouse soleus muscles equilibrated at 25°C had good mechanical stability when temperature was elevated to 37°C. The main findings at 37°C vis-à-vis 25°C were as follows. When [K⁺]_o was raised from 4 to 7?mM, there was greater twitch potentiation, but no significant difference in peak tetanic force. At 10?mM [K⁺]_o there was (1) a faster time course for the decline of peak tetanic force, (2) a greater steady-state depression of twitches and tetani, (3) an increase of peak force over 50?C200?Hz (whereas it decreased at 25°C), (4) significant tetanus restoration when stimulus pulse duration increased from 0.1 to 0.25?ms and (5) greater depolarisation of layer-2 fibres, with no repolarisation of surface fibres. These combined data strengthen the proposal that a large run-down of the K⁺ gradient contributes to severe fatigue at physiological temperatures via depolarisation and impaired sarcolemmal excitability. Moreover, terbutaline, a ??₂-adrenergic agonist, induced a slightly greater and more rapid, but transient, restoration of peak tetanic force at 10?mM [K⁺]_o at 37°C vis-à-vis 25°C. A right shift of the twitch force?Cstimulation strength relationship at 10?mM [K⁺]_o was partially reversed with terbutaline to confer the protective effect. Thus, catecholamines are likely to stimulate the Na⁺?CK⁺ pump more powerfully at 37°C to restore excitability and attenuate, but not prevent, the detrimental effects of K⁺.     

Hypertension in people with diabetes in sub-Saharan Africa: revealing the hidden face of the iceberg

AIM: To evaluate the prevalence, awareness, treatment and control of hypertension in a diabetic population of Cameroon, a sub-Saharan African country. METHODS: Two hundreds and ten diabetic patients were consecutively enrolled over a 6-month period. A questionnaire was administered and physical examination done. The JNC VI and the latest WHO criteria were used to diagnose hypertension and diabetes, respectively, and control of hypertension was assessed against five different targets. RESULTS: Ninety-one percent of the participants had type 2 diabetes. Prevalence and awareness rates for hypertension were 66.7% (n=140) and 87.1% (n=122), respectively. Treatment rate among those aware of their hypertension status was 80.3% (n=98). Patients with hypertension were older, more overweight/obese and had a longer duration of diabetes. ACE inhibitors and diuretics were the two most used blood pressure (BP) lowering drugs. Following the ADA/JNC 7 goal, the control rate of hypertension among treated patients was 10.2% (n=10). CONCLUSION: Diabetic patients in Cameroon exhibit a very high prevalence of hypertension and are about three times more affected than the general population. Awareness and treatment rates are high, but the control rate is very low. Large scale studies with intervention component are urgently required.     

Concurrent CMV and EBV DNAemia is significantly correlated with a delay in the response to HAART in treatment-naive HIV type 1-positive patients

The purpose of this study was to assess the qualitative single and multiple herpes virus DNAemia in the peripheral blood leukocytes (PBLs) of HIV-1-positive patients and its impact on the response to highly active antiretroviral therapy (HAART) and immune reconstitution. All (163) HIV-1-positive patients attending "Syngros AIDS Referral Center" from November 2000 to February 2001 were recruited. CMV, HSV-1, HSV-2, EBV, and HHV-8 DNA were detected in PBLs by polymerase chain reaction (PCR). Patients' follow-up comprised regular measurements of CD4(+) T cell count and HIV-1 viral load (VL) for an average period of 21 months. Immune reconstitution was defined as an increase in the CD4 T cell count by above 200 cells/micro l, while response to HAART was defined as a decrease in HIV-1 VL to undetectable levels. Single and multiple herpetic DNAemia in PBLs was found to be significantly higher in HIV-1-positive patients compared to healthy controls (p < 0.02) for all the viruses detected apart from HSV-2, which was not detected in the PBLs of either population. Concurrent CMV and EBV DNAemia significantly correlates with a delay in the response to HAART (p = 0.033) in treatment-naive patients. Untreated patients with a CD4(+) T cell count <200 cells/micro l, and with either CMV or EBV DNAemia, presented a delayed increase in the CD4 count after initiation of HAART (p = 0.035 and p = 0.037 respectively), while multiple herpetic DNAemia in the above patients was borderline associated with immune reconstitution (p = 0.068). Conclusively, CMV and EBV DNAemia may be poor prognostic factors for the response to HAART in treatment-naive HIV-1 patients.     

Genetics of craniosynostosis

Craniosynostosis is a defect of the skull caused by early fusion of one or more of the cranial sutures and affects 3 to 5 individuals per 10,000 live births. Craniosynostosis can be divided into two main groups: syndromic and nonsyndromic. Nonsyndromic craniosynostosis is typically an isolated finding that is classified according to the suture(s) involved. Syndromic craniosynostosis is associated with various dysmorphisms involving the face, skeleton, nervous system, and other anomalies and is usually accompanied by developmental delay. More than 180 syndromes exist that contain craniosynostosis. Secondary effects of craniosynostosis may include vision problems and increased intracranial pressure, among others. The molecular basis of many types of syndromic craniosynostosis is known, and diagnostic testing strategies will often lead to a specific diagnosis.