Endothelial activation and inflammation biomarkers in children and adolescents with sickle cell disease.

Sickle cell disease pathogenesis is a complex interplay of multiple factors associated with vascular endothelial activation, intense oxidative stress, and increased sickle cell adhesion. The aim of this study was to determine and compare three panels of plasma circulating biomarkers at 'steady state' and during veno-occlusive crises (VOC) in a cohort of children and adolescents with SCD and healthy controls. The following biomarkers were assessed: acute phase reactants, endothelial factors, and adhesion molecules. Forty-one SCD pediatric patients and 28 healthy children were enrolled. Patients at 'steady state' presented significantly elevated plasma levels of endothelin-1 (ET-1), soluble-VCAM-1 (sVCAM-1), soluble P-selectin (sP-selectin), and d-dimers compared to the control group. ET-1, sP-selectin, platelet-derived growth factor (PDGF), von Willebrand factor (vWf), d-dimers, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) seems to represent additional, but not independent, prognostic markers of VOC crisis. Elevated plasma levels of sP-selectin, ET-1, and sVCAM-1 were associated with VOC frequency. The present study provides preliminary evidence of a possible association between these biomarkers and the endothelial activation at steady state and VOC in childhood SCD. Further prospective studies are required to confirm the potential independent prognostic value of these markers in different stages of pediatric SCD.

Hearing loss in children with sickle cell disease.

Hearing loss in children with sickle cell disease. Sickle cell anemia (S/S) has been associated with a high incidence of hearing loss mostly of the sensorineural type (SNHL). Twenty-four patients with sickle cell disease (13 female and 11 male; 22 patients belonging to the S/beta(+)-Thal and 2 to the S/S phenotype) with a mean age of 12.5 +/- 3.6 years and hemoglobin (Hb) levels range 6.5-11 g/dl underwent ENT examination, pure tone audiometry, speech audiometry, tympanometry, auditory reflex evaluation, tone decay test and brain auditory evoked potentials (BAEP) in order to evaluate the presence, type and degree of hearing loss. Only one patient (4.6%) who also sustained an infarct of the middle cerebral artery, demonstrated a unilateral SNHL in high tone frequencies exceeding 70 dB, as well as a prolonged III-V interpeak latency at the same side. No abnormalities were detected in the control group. These findings suggest a low incidence of SNHL in Greek SCD patients probably due to different hematological and clinical profile (S/beta(+)-Thal).

Stabilization and reactivation of the p53 tumor suppressor protein in nontumorigenic revertants of HeLa cervical cancer cells.

We demonstrated previously that loss of in vitro transformation and in vivo tumorigenicity in two independent revertant clones of HeLa cells (designated HA and HF) resulted from dominant-acting genetic changes. Analysis of the p53 tumor suppressor gene revealed stabilization and at least partial restoration of wild-type p53 transactivation properties pathways in both revertants of HPV-induced cell transformation. The half-lives of the p53 protein and both of the HA and HF clones were increased approximately 4 fold compared with the parental HeLa cells (16, 17, and 4 min, respectively). The levels of E6 viral protein expression were similar in the three cell lines, whereas the levels of the ubiquitin ligase protein, E6 associated protein (E6-AP), were elevated in the revertants. Western blot analysis of immunoaffinity-purified p53 demonstrated that stabilization of p53 in the revertants was correlated with a reduction in the in vivo formation of complexes involving the E6 oncoprotein and p53. Stabilization of p53 function in the revertants did not result from mutations in either the p53 or E6-AP genes. Despite the observed stabilization and restoration of p53 transactivation function in the revertants, exposure of the revertants to DNA-damaging agents did not result in elevated levels of p21(waf-1) protein and failed to induce growth arrest in the G1 phase of the cell cycle. However, p53-independent induction of p21(waf-1) protein also failed to induce the G1 phase of the cell cycle. Thus, restoration of wild-type p53 transactivation activity in the HA and HF revertants is insufficient to induce G1 arrest and reversion from HPV-induced cell transformation in our model system.

Blunt diaphragmatic rupture.

OBJECTIVE: To identify (1) predictors of outcome in blunt diaphragmatic rupture (BDR), and (2) factors contributing to diagnostic delay. METHODS: We reviewed the charts and radiographs of 41 patients with BDR treated in our Hospital from 1988 to 1997. There were 35 male (85%) and six female, aged 17-71 (mean: 41) years. BDR was left-sided in 24 cases (58%), right-sided in 15 (36%) and bilateral in two (5%). RESULTS: Two groups of patients can be identified: group A (n = 36, 88%) with acute BDR, and group B (n = 5, 12%) with post-traumatic diaphragmatic hernia (TDH). In group A, immediate diagnosis was made in 35 cases (97%), but only in 26 (72%) preoperatively. In one case, a right BDR was missed on initial evaluation but became apparent 2 weeks later. Associated injuries were present in 34 patients (94%) involving: spleen (n = 18), rib fractures (n = 17), liver (n = 14), lung (n = 11), bowel (n = 7), kidney (n = 5) and other fractures (n = 21). Injury Severity Score (ISS) ranged from 9 to 66 (mean: 31). BDR repair was accomplished through a laparotomy in 22 cases, thoracotomy in 10 and laparo-thoracotomy in four. The overall mortality rate was 16.6% (6/36). Both patients with bilateral BDR died. The patients who died were older than the survivors (mean age: 54 vs. 39 years, P<0.05), were more severely injured (mean ISS: 46 vs. 28, P<0.05) and were in shock (100 vs. 23%, P<0.05). In group B with TDH, diagnosis was delayed for 7-16 months after injury. Four patients had non-specific clinical signs and one strangulation of hollow viscera. One patient had undergone surgery during acute injury but BDR was overlooked. Location of TDH was on the left in three cases and on the right in two. Delay in BDR diagnosis was 12.5% (3/24) in patients with left-sided and 20% (3/15) in patients with right-sided lesions (P>0.1). Repair of TDH was achieved through thoracotomy in all cases. No mortality or major morbidity were encountered. CONCLUSIONS: (1) Predictors of BDR mortality are: age, ISS and hemodynamic status of the patient. (2) Delay in diagnosis does not influence the outcome and is not influenced by the side of BDR location. (3) BDR can easily be missed in the absence of other indications for prompt surgery, where a thorough examination of both hemidiaphragms is mandatory. A high index of suspicion combined with repeated and selective radiologic evaluation is necessary for early diagnosis.

Activation of tumor suppressor genes in nontumorigenic revertants of the HeLa cervical carcinoma cell line.

Two nontransformed revertants of HeLa cells, designated HA and HF, were isolated using a selection procedure based on prolonged retention of the fluorescent dye rhodamine 123 within the mitochondria of HeLa (ATCC CCL2) cells versus normal epithelial cells. Unlike the parental HeLa cells, the revertants expressed markedly reduced levels of the bone-liver-kidney, placental, and intestinal isoforms of alkaline phosphatase, exhibited a flat nonrefractile morphology, and failed to grow in suspension culture. The revertant clones had > 100-fold reduced cloning efficiencies in semisolid medium relative to HeLa cells and failed to induce s.c. tumors when injected into nude mice. Both revertant clones have retained nontransformed phenotypes after 5 years of continuous culture. Southern blot analyses performed with human papillomavirus 18-specific DNA probes indicated that the integrated viral sequences present in HeLa cells remained intact in the revertants. Furthermore, the level of the polycistronic mRNAs encoding the viral E6 and E7 oncogenes were comparable in the parental HeLa cell line and the revertants. Western blot analyses of immunoprecipitated human papillomavirus 18 E6 and E7 proteins further demonstrated that the levels of these viral oncoproteins were comparable in HeLa cells and revertants. Infection with helper-free, defective retroviruses that express E6, E7 or E6 and E7 oncogenes failed to retransform the revertants, suggesting that their nontransformed phenotype did not result from mutations in these viral oncogenes. Cell fusion experiments indicated that the revertant phenotypes of HA and HF cells resulted from mutations in cellular genes that activate one or more tumor suppressor genes.

Sensorineural hearing loss in children with thalassemia major in Northern Greece.

Eighty eight (88) beta-thalassemic patients undergoing regular transfusion- chelation therapy with desferrioxamine (DFO) were studied for ENT problems from 1988 to 1993, as DFO has been implicated for auditory neurotoxicity. The mean age of the patients was 9.66 +/- 3.1 years, their pre-transfusion haemoglobin level was 9 +/- 2 g/dl, serum ferritin level was 2065 +/- 898 ng/ml and the daily DFO dose was 50.7 +/- 9.5 mg/kg for 5 days/week. The ENT study included, ENT examination, pure tone audiometry, speech audiometry, tympanometry, tone decay test and ABR. During this 6-year study 24/88 (27%) patients developed bilateral or ipsilateral sensorineural hearing loss in high tone frequencies, sometimes exceeding 80 dB, which was attributed to DFO toxicity. Therefore, a reduction or temporary withdrawal of DFO followed. After this intervention 12/24 patients recovered almost completely, 7/24 remained stable and 5/24 presented aggravation of their hearing loss. This study confirms the DFO induced auditory neurotoxicity and the necessity of periodical audiology control of beta-thalassemic patients for prompt diagnosis and management of this complication.