肾包膜内注射骨髓间充质干细胞在链脲佐菌素糖尿病模型大鼠体内的定位及功能测评

目的:观察骨髓间充质干细胞在体外不诱导条件下,移植于链脲佐菌素所致糖尿病大鼠后在胰腺的定位及胰岛素分泌情况。方法:实验于2004-08/2006-03在2级实验室唐山工人医院中心实验室和具有国家认证的屏障动物环境实验室华北煤炭医学院动物中心完成。①实验材料:青年清洁级SD大鼠购自华北煤炭医学院动物中心(SCXK2002-0003),4周龄,体质量180～250g,动物级别:SPF/VAF。②实验方法:体外培养雄性SD大鼠骨髓间充质干细胞,并采用流式细胞仪鉴定。选取雌性SD大鼠,按65mg/kg剂量腹腔注射链脲佐菌素建立糖尿病大鼠模型。给药后二三天,大鼠血糖升高,当血糖≥16.7mmol/L且稳定2d,认为糖尿病模型建立成功。将建模成功的40只大鼠完全随机分为对照组、实验组,每组20只。对照组大鼠右侧肾包囊注射磷酸缓冲液0.2mL,实验组大鼠右侧肾包囊下注射骨髓间充质干细胞0.2mL,细胞数为4×105L-1。于移植前及移植后第3,5,7天采大鼠尾静脉血检测血糖。③实验评估:于移植前及注射后7d采大鼠心脏血检测C-肽值,移植后7d取大鼠胰腺组织作石蜡切片,应用原位杂交技术检测Y染色体细胞。结果:40只大鼠全进入结果分析。①骨髓间充质干细胞鉴定:骨髓间充质干细胞呈梭形、放射状,形成数个细胞克隆时排列整齐,原代培养7～10d即可长满,按1∶3传代,生长四五天达融合。流式细胞术表面标志鉴定显示,培养的原代骨髓间充质干细胞表面标志CD90阳性,基质细胞表面标志CD44阳性,造血细胞表面标志CD45及内皮细胞表面标志CD31阴性。②大鼠尾静脉血血糖值、心脏血C-肽值分析:血糖结果显示,对照组治疗后第3,5,7天血糖值及C-肽值与治疗前相比,无明显变化(P>0.05);实验组治疗后第3,5,7天血糖值较治疗前明显下降,治疗后第7天C-肽值明显上升,与对照组对应时间比较,差异均显著[实验组治疗前:(21.75±2.44)mmol/L,(0.21±0.01)μg/L;实验组治疗后:(12.23±1.95)mmol/L,(14.00±2.85)mmol/L,(12.75±2.86)mmol/L,(0.26±0.01)μg/L;对照组治疗后:(21.63±1.34)mmol/L,(23.03±1.11)mmol/L,(22.48±1.92)mmol/L,(0.21±0.01)μg/L,P均<0.01]。③原位杂交技术检测Y染色体细胞:对照组大鼠胰腺组织切片中未见黄绿荧光Y染色体,实验组大鼠胰腺组织切片中可见黄绿荧光Y染色体,存在于胰腺腺泡间隙,或胰腺组织中。结论:雄性大鼠骨髓间充质干细胞在肾包囊注射下可到达雌性糖尿病大鼠胰腺组织,使糖尿病大鼠血糖下降,C-肽值升高,具有分化为胰岛素分泌细胞的可能性。     

Enhanced proteolysis of plasma von Willebrand factor in thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome

To examine whether enhanced in vivo proteolysis of von Willebrand factor (vWF) would account for the reported loss of larger multimers in acute thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), we studied eight patients with acute TTP/HUS whose blood samples were collected into an anticoagulant containing a cocktail of protease inhibitors to impede in vitro proteolysis. In all, enhanced proteolytic degradation of vWF was expressed as a relative decrease in the intact 225-Kd subunit of vWF and a relative increase in the 176-Kd fragment. However, instead of the loss of larger forms of normal multimers reported by other investigators, the plasma of all but one of our patients (whether they had TTP or HUS) contained a set of larger than normal (supranormal) multimers. Hence, although proteolytic fragmentation of vWF was enhanced during acute TTP/HUS, this phenomenon was not associated with the loss of larger multimers. In the five patients who survived the acute disease and underwent plasma exchange (three with HUS and two with chronic relapsing TTP), subunits and fragments returned to normal values, and supranormal multimers were no longer detected in plasma. In conclusion, even though vWF proteolysis is enhanced in acute TTP/HUS, it does not lead to loss of larger multimers.     

A new solid-phase immunoassay for terminal deoxynucleotidyl transferase: analysis of TdT antigen in cells, plasma, and serum

A solid-phase immunoassay for terminal deoxynucleotidyl transferase has been developed using a primary antibody-coated polystyrene bead and secondary antibody conjugated with horseradish peroxidase. The immunoassay was compared with assays for enzyme activity and detection of antigen with immunofluorescence using cells from peripheral blood and bone marrow from patients with leukemia or lymphoma. In each instance, the solid-phase immunoassay correlated correctly with cellular samples judged to be positive by other tests. However, the level of detection of terminal transferase antigen in plasma or serum of patients with leukemia did not reflect accurately the level of terminal transferase in neoplastic cells. The solid-phase immunoassay was greater than 100-fold more sensitive than conventional assays for enzyme activity, rendering it potentially useful for quantitatively monitoring terminal transferase in patients with leukemia.     

An unusual case of dental infection by Pseudomonas aeruginosa causing a brain abscess: case report

A brain abscess may result when dental infection reaches the brain through contiguous anatomic cavities such as the maxillary sinus, the ethmoid sinus and the orbital cavity. It is an important complication and should be treated aggressively. Such treatment would include the excision of the etiological factor, drainage and adjuvant antibiotic therapy. The present case concerns a 23 year old woman who presented at the General Hospital of Nova Iguaçu with complaints of pain in the right side of the face and was diagnosed with acute sinusitis. Antibiotics and analgesics were prescribed to treat the disease. However, after 10 days, she returned to the emergency room, presenting with proptosis of the right eyeball, subconjunctival haemorrhage, ophthalmoplegia and intense pain in the right orbit, in addition to headaches. After computed tomography was performed, she was diagnosed with a brain abscess in the frontal lobe with the involvement of the maxillary right first molar, the maxillary sinus, the ethmoid sinus and the orbital cavity. With culturing of the secretion, the growth of Pseudomonas aeruginosa colonies was evident. Treatment consisted of a craniotomy to drain the brain abscess, a Caldwell–Luc procedure to drain the right maxillary sinus, dental extraction and aggressive antibiotic therapy. After 6 weeks, the patient was discharged with no neurological sequelae.     

The incidence,spectrum and outcome of paediatric trauma managed by the Pietermaritzburg Metropolitan Trauma Service

Introduction

The Pietermaritzburg Metropolitan Trauma Service (PMTS) has run a systematic quality improvement programme since 2006. A key component included the development and implementation of an effective surveillance system in the form of an electronic surgical registry (ESR). This study used data from the ESR to review the incidence, spectrum and outcome of paediatric trauma in Pietermaritzburg, South Africa.

Methods

The ESR was reviewed, and all cases of paediatric trauma managed between 1 January 2012 and 30 July 2014 were retrieved for analysis.

Results

During the study period, 1,041 paediatric trauma patients (724 male, 69.5%) were managed by the PMTS, averaging a monthly admission of 36. The mean age was 10.9 years (standard deviation: 5.4 years). The mechanism of injury (MOI) was blunt trauma in 753 patients (72.3%) and penetrating trauma in 170 (16.3%). Pedestrian vehicle collisions accounted for 21% of cases and motor vehicle collisions for a further 11%. Intentional trauma accounted for 282 patients (27.1%) and self-inflicted trauma for 14 cases (1.3%). Ninety patients admitted to the intensive care unit and fifty-one required high dependency unit admission. There were 17 deaths, equating to an in-hospital mortality rate of 1.7%. A total of 172 children died on the scene of an incident. There were 35 road traffic related deaths, 26 suicides by hanging, 27 deaths from blunt assault and 23 deaths from penetrating assault. The overall mortality rate for paediatric trauma was 18.2%.

Conclusions

The ESR has proved to be an effective surveillance system and has enabled the accurate quantification of the burden of paediatric trauma in Pietermaritzburg. This has improved our understanding of the mechanisms and patterns of injury, and has identified a high incidence of intentional and penetrating trauma as well as road traffic collisions. These data can be used to guide strategies to reduce the burden of paediatric trauma in our environment.     

Autografting with cultured marrow in chronic myeloid leukemia: results of a pilot study [see comments]

Incubation of chronic myeloid leukemia (CML) marrow for 10 days in vitro causes a marked and selective loss of very primitive Philadelphia chromosome (Ph)+ as compared with Ph- progenitors. We have autografted 22 patients with CML (16 in first chronic phase [group 1] and 6 with more advanced disease [group 2]) with marrow treated in this way to facilitate restoration of Ph- hematopoiesis after intensive therapy. Hematologic recovery to greater than 0.5 x 10(9)/L neutrophils occurred in 16 patients, and to greater than 20 x 10(9)/L platelets in 15 of 21 evaluable patients at a median of 29 and 48 days postautograft, respectively. Regenerating marrow cells were 100% Ph- in 13 patients and 75% to 94% Ph- in 3. Between 4 and 36 months (median 12) postautograft, Ph+ cells became detectable in all but 1 (who died in remission) of the 13 patients who achieved complete cytogenetic remission. Four of 7 evaluable patients treated with low-dose interferon alpha were returned to complete cytogenetic remission. Thirteen group 1 patients (81%) are alive 1.0 to 5.7 years (median 2.6) after autografting: 4 in complete cytogenetic remission, 2 in hematologic remission, 6 in chronic phase, and 1 in myeloid blast phase. Three group 2 patients (50%) are alive at 2.6, 3.8, and 4.3 years after autografting: 1 in partial cytogenetic remission, 1 in chronic phase, and 1 in accelerated phase. Thus, autografts of cultured marrow can result in prolonged restoration of Ph- hematopoiesis for some patients with CML.     

Quantitation of adenosine-5'-triphosphate used for phosphoinositide metabolism in human erythrocytes

The human erythrocyte actively phosphorylates and dephosphorylates phosphatidylinositol present in the membrane in an apparent "futile cycle." Recent reports have proposed that this phosphorylation/dephosphorylation cycle is a significant consumer of adenosine-5'-triphosphate (ATP) in the erythrocyte. This study details two independent techniques for quantitating the ATP consumed by this phosphoinositide futile cycle. With the first technique a quasi-steady- state labeling of erythrocyte ATP with 32P-phosphate was obtained, and the rate of synthesis of 32P-phosphoinositides was then monitored. The second technique used a novel labeling strategy that allowed only ATP to be labeled with 32P; the transfer of 32P from ATP to phosphoinositides was then an independent measure of the ATP consumed for phosphoinositide synthesis. These two techniques documented that 0.5% to 1.0% of net ATP produced by the erythrocyte is used for phosphoinositide synthesis.     

Preclinical evaluation of a monoclonal antibody targeting the epidermal growth factor receptor as a radioimmunodiagnostic and radioimmunotherapeutic agent

Background and purpose:

The studies described here are the first to evaluate the in vitro and in vivo properties of ¹¹¹In-CHX-A″-panitumumab for radioimmunotherapy (α- and β^--emitters) and radioimmunoimaging (single photon emission computed tomography and positron emission tomography).

Experimental approach:

Twenty-seven human carcinoma cell lines were analysed for expression of epidermal growth factor receptors by flow cytometry. Panitumumab was conjugated with CHX-A″-DTPA (diethylenetriamine-pentaacetic acid) and radiolabelled with ¹¹¹In. Immunoreactivity of the CHX-A″-DTPA-panitumumab and ¹¹¹In-CHX-A″-DTPA-panitumumab was evaluated by radioimmunoassays. Tumour targeting was determined in vivo by direct quantitation of tumour and normal tissues and by γ-scintigraphy.

Key results:

For 26 of 27 human tumour cell lines, 95% of the cells expressed epidermal growth factor receptors over a range of intensity. Immunoreactivity of panitumumab was retained after modification with CHX-A″-DTPA. Radiolabelling of the immunoconjugate with ¹¹¹In was efficient with a specific activity of 19.5 ± 8.9 mCi·mg⁻¹ obtained. Immunoreactivity and specificity of binding of the ¹¹¹In-panitumumab was shown with A431 cells. Tumour targeting by ¹¹¹In-panitumumab was demonstrated in athymic mice bearing A431, HT-29, LS-174T, SHAW or SKOV-3 s.c. xenografts with little uptake observed in normal tissues. The ¹¹¹In-panitumumab was also evaluated in non-tumour-bearing mice. Pharmacokinetic studies compared the plasma retention time of the ¹¹¹In-panitumumab in both non-tumour-bearing and A431 tumour-bearing mice. Tumour targeting was also visualized by γ-scintigraphy.

Conclusions and implications:

Panitumumab can be efficiently radiolabelled with ¹¹¹In with high labelling yields. Based on the efficiency in tumour targeting and low normal tissue uptake, panitumumab may be an effective targeting component for radioimmunodiagnostic and radioimmunotherapeutic applications.