Retroviral-mediated gene transfer into human myeloma cells

SUMMARY. SW-480 cells, derived from a primary human colon adenocarcinoma, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma. SW-480 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin (5 U/ml) but unaffected by apyrase (10 U/ml). This TCIPA was also unaffected by cysteine proteinase inhibition with E-64 (10 μM) but was limited by cell pretreatment with phospholipase A₂. SW-480 cell suspension caused marked dose-dependent decreases in plasma recalcification times using normal, factor VIII-deficient and factor IX-deficient human plasma. This effect was potentiated with cell lysates but inhibited in intact cells pretreated with sphingosine. SW-480 cell suspension did not affect the recalcification time of factor VII-deficient plasma. Moreover, monoclonal antibody against human tissue factor completely abolished SW-480 TCIPA. Taken together, these data suggest that SW-480 TCIPA arises from SW-480 tissue factor activity expression. Trigramin and rhodostomin, RGD-containing snake venom peptides, which antagonize the binding of fibrinogen to platelet membrane glycogen IIb/IIIa, prevented SW-480 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against platelet membrane glycoprotien IIb/IIIa and Ib prevent SW-480 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC₅₀ 0.09 μM) and rhodostomin (IC₅₀ 0.03 μM) were about 6000 and 18000 times, respectively, more potent than GRGDS (IC₅₀ 0.56mM).     

Effects of different surface treatments on shear bond strength in two different ceramic systems

The purpose of this study was to evaluate the influence of different surface treatments (sandblasting, acid etching, and laser irradiation) on the shear bond strength of lithium disilicate-based core (IPS Empress 2) and feldspathic ceramics (VITA VM 9). One hundred ceramic discs were divided into two groups of 50 discs each for two ceramic systems: IPS Empress 2 (group I) and VITA VM 9 (group II). Each of the two groups was further divided into five surface treatment groups (ten each) as follows: group SB, sandblasting with alumina particles (50 μm); group HF, 5 % hydrofluoric acid etching; group L, Er:YAG laser irradiation (distance, 1 mm; 500 mJ; 20 Hz; 10 W; manually, noncontact R14 handpiece); group SB–L, sandblasting + Er:YAG laser; and group HF–L, 5 % hydrofluoric acid + Er:YAG laser. Luting cement (Panavia 2.0) was bonded to the ceramic specimens using Teflon tubes. After 24 h of water storage, a shear bond strength test was performed using a universal testing machine at a crosshead speed of 0.5 mm/min. The data were analyzed with a two-way analysis of variance (ANOVA) and Tukey's honestly significant difference tests (α?=?0.05). The two-way ANOVA indicated that the shear bond strength was significantly affected by the surface treatment methods (p?<?0.05), but there was no significant interaction between the ceramic systems. Group SB–L had the highest mean values for each ceramic system. Sandblasting, followed by Er:YAG laser irradiation, enhanced the bond strength, indicating its potential use as an alternative method. The atomic force microscopic evaluation revealed that group SB had the most distinct sharp peaks among the groups.     

Congenital lobar emphysema: evaluation and long-term follow-up of thirty cases at a single center

Congenital lobar emphysema (CLE) is characterized by overdistension and air-trapping in the affected lobe, and is one of the causes of infantile respiratory distress. In this report, we review our 27 years of experience with 30 CLE patients. Patients' medical records were evaluated with regard to age, clinical presentation, diagnostic methods, associated diseases, treatment, histopathologic findings, and final clinical and laboratory findings at the end of a long-term period. The mean age of 30 patients (18 male) at diagnosis was 4.9 +/- 6.7 months (range, 2 days-2.5 years). Tachypnea, dyspnea, cough, cyanosis, wheezing, hoarseness, and decreased breath sounds on the affected side were the main symptoms and clinical findings. On chest X-rays, emphysema was seen in all patients; shift/herniation to the opposite lung, atelectasis, and pneumothorax were observed in 16, 5, and 2 cases, respectively. Computerized tomography of the thorax was performed in 16 cases and revealed emphysema at affected lobe/lobes in all, a shift/herniation to the opposite side in 12 cases, and atelectasis of neighbor lobe/lobes in 7 cases. All 8 patients who had perfusion scintigraphy showed reduced perfusion in the affected lobe. Narrowed and flaccid bronchi were detected in one patient by using flexible bronchoscopy. Blood gas analysis was performed in 11 patients, and hypoxia and hypercarbia were revealed in 9 and 7 of these patients, respectively. The most common affected lobe was the left upper lobe (57%), followed by the right upper lobe (30%) and right middle lobe (27%). Two lobes were involved in 4 patients. Associated abnormalities were observed in 5 patients. Twenty-one patients underwent lobectomy; 9 were followed conservatively. Ages at diagnosis were significantly younger in surgically treated patients. Emphysema was detected in all pathological specimens, with an additional bronchial cartilage deficiency in 2 patients. In the surgically treated group, 2 patients died and 2 patients were lost to follow-up. In the conservatively treated group, one patient was lost to follow-up. Mean follow-up duration of all patients was 63.2 +/- 56.2 months (range, 1-209 months). At follow-up visits, all patients were doing well. In surgically treated patients, chest X-rays were normal (9 cases), or showed hyperlucency on the operated side (6 cases) or chronic changes in the operation area (2 cases). Hyperexpansion in the affected lobe was found to be reduced in all cases in the conservatively treated group.     

HMGB‐1, TLR4, IL‐1R1, TNF‐α, and IL‐1β: novel epilepsy markers?

Aim: The purpose of this study was to compare HMGB‐1, TLR4, IL‐1β, IL‐1R1, and TNF‐α levels in patients with mild and severe epilepsy with those in a healthy control group. Methods: Children aged 4–17 years, diagnosed with epilepsy for at least three years and with no progressive neurological disease, metabolic disease or infection, were selected for the study. The severe epilepsy group consisted of 28 children with at least one episode a week despite receiving three or more antiepileptic drugs. The mild epilepsy group consisted of 29 children with no seizures in the previous year, receiving only one antiepileptic drug, while 27 healthy children were selected as the control group. HMGB‐1, TLR4, IL‐1R1, TNF‐α and IL‐1β levels were investigated in these three groups. The MRI findings and clinical characteristics of the patients in the epilepsy group were also compared with these markers. Results: HMGB‐1, TLR4, TNF‐α, and IL‐1β levels in the severe epilepsy group were higher than in the control group and the mild epilepsy group (p<0.05), and were higher in the mild epilepsy group than in the control group (p<0.05). IL‐1R1 was also higher in the severe epilepsy group than in the control group (p<0.05). Conclusion: In this first report to identity a possible correlation between HMGB‐1, TLR4, IL‐1β, IL‐1R1, and TNF‐α levels and severity of epilepsy, our data demonstrates that the serum level of these cytokines is higher in cases of drug‐refractory epilepsy.     

Reduced bone mineral density in childhood chronic idiopathic thrombocytopenic purpura treated with high-dose methylprednisolone

PURPOSE: To evaluate whether repeated courses of high-dose methylprednisolone (HDMP) affect the lumbar spine bone mineral density (BMD) in children with chronic idiopathic thrombocytopenic purpura (ITP). MATERIALS AND METHODS: This study included 24 patients with chronic ITP and 149 healthy controls. The patients were allocated into three groups according to the number of HDMP courses (30 mg/kg per day as a single dose for 7 days); group 1 (10 patients), group 2 (9 patients), and group 3 (5 patients) had received less than 5, 6-10, and more than 10 courses, respectively. Lumbar spine BMD and body composition were measured using dual energy X-ray absorptiometry of lumbar spine (L2-L4), and volumetric bone mineral density (vBMD) values were calculated and compared with the controls. The z score of the vBMD was also calculated and compared in the patients of each other groups. Serum markers of the bone turnover were measured to exclude other factors that could effect BMD. RESULTS: The vBMD values of the patients, corrected BMDs for age, were significantly lower than the values of controls (P = 0.018). It was significantly lower in group 3 than groups 1 and 2 (P = 0.005 and P = 0.006, respectively), but there was no statistically significant difference between groups 1 and 2 (P = 0.87). The vBMD z scores were significantly lower in group 3 than in groups 1 and 2 (P = 0.003 and P = 0.004, respectively), and also in group 2 than in group 1 (P = 0.034). There were a weak negative correlation between the cumulative dose of HDMP and vBMD (r = -0.39, P = 0.054), and strong negative correlation between the cumulative dose of HDMP and vBMD z score (r = -0.63, P = 0.001). CONCLUSION: Children with chronic ITP are at risk for decreased BMD because of the repeated courses of HDMP; especially more than 2100 mg of cumulative dose. We therefore recommend that BMD should be monitored in patients with chronic ITP who received repeated courses of HDMP.     

Permanent transfemoral pacemaker implantation in a child with Maroteaux Lamy syndrome

Permanent transfemoral pacing has been described as an alternative route in patients in whom the superior venous approach is not feasible. This report describes the use of the femoral venous approach to insert a permanent pacemaker in a child with Maroteaux Lamy syndrome who has complete atrioventricular block and abnormal subclavian venous anatomy. Transfemoral pacing may be a suitable alternative in children with short stature.     

Anti-myeloma activity of endogenous and adoptively transferred activated natural killer cells in experimental multiple myeloma model

OBJECTIVE: Despite advances in autologous stem cell transplantation and chemotherapy, multiple myeloma (MM) remains an incurable disease. Due to the role of natural killer (NK) cells in host resistance against several tumors, it is of interest to explore the anti-MM activity of NK cells. For this reason, we aimed to determine if NK cells provide anti-MM activity following interleukin-2 (IL-2) administration, and if ex vivo activated and intravenously administered NK cells prolong survival in MM-bearing C57BL/KaLwRij mice. METHODS: The anti-MM effect of IL-2 was tested by intraperitoneal injection into the 5T33MM-inoculated mice. Subsequently, in vivo effector cell depletions were performed by administration of anti-NK1.1 or anti-CD8 monoclonal antibodies. Finally, magnetically separated and activated NK cells from splenocytes of C57BL/KaLwRij mice were adoptively transferred to tumor-bearing mice in conjunction with IL-2 treatment. RESULTS: IL-2 administration into MM-bearing mice significantly prolonged their survival. This effect was diminished by in vivo depletion of NK cells. Adoptive transfer of activated NK cells showed a significant in vivo anti-MM effect that was dependent on cell dose. Biodistribution of the marked adoptively transferred NK cells correlated with MM cells' homing sites. CONCLUSION: These data suggest that activated NK cells have a promising potential in adoptive immunotherapy for MM.     

Enhanced ouabain resistance gene as a eukaryotic selection marker

Current selection markers allow selection by antibiotics or fluorescent/magnetic sorting by green fluorescent protein or membrane antigens. Antibiotic selection proceeds on a time scale of weeks, and flourescence-activated cell sorting requires complex equipment and may generate false-positive results when selection is performed too early after transduction with membrane markers. We have characterized an endogenous eukaryotic selection marker, the ouabain resistance gene (Oua(r)), which has the potential for quick and efficient in vitro selection of target cells. The Oua(r) used by us is derived from the rat alpha(1) isoform of Na(+),K(+)-ATPase, where leucine at position 799 is substituted for cysteine by targeted mutagenesis. This mutation confers resistance to more than 1 mM ouabain in vitro. We show that cells transfected with plasmid or transduced with a retrovirus vector encoding Oua(r) can be selected efficiently with ouabain in 48 hr and that a pure population of cells can be obtained. The ouabain resistance gene may be useful as a selection marker in general molecular biology, preclinical, and clinical applications because of its short selection time and also because of the safety of ouabain for human use.