Molecular and functional expression of voltage-operated calcium channels during osteogenic differentiation of human mesenchymal stem cells.

We used the patch-clamp technique and RT-PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L-type Ca2+ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L-type Ca2+ channel function. INTRODUCTION: During osteogenic differentiation, mesenchymal stem cells from human bone marrow (hMSCs) must adopt the calcium handling of terminally differentiated osteoblasts. There is evidence that voltage-operated calcium channels (VOCCs), including L-type calcium channels, are involved in regulation of osteoblast function. We therefore studied whether VOCCs play a critical role during osteogenic differentiation of hMSCs. MATERIALS AND METHODS: Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, beta-glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca2+ channel alpha1 subunits was shown by semiquantitative or single cell RT-PCR. Voltage-activated calcium currents of hMSCs were measured with the whole cell voltage-clamp technique. RESULTS: mRNA for the pore-forming alpha1C and alpha1G subunits of the L-type and T-type Ca2+ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L-type Ca2+ currents to a subpopulation of hMSCs was confirmed by single cell RT-PCR, where mRNA for the alpha1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine-sensitive L-type Ca2+ currents were found in 13% of cells cultured in MM and in 12% of the cells cultured in ODM. Under MM and ODM culture conditions, the cells positive for L-type Ca2+ currents were significantly larger than cells without Ca2+ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L-type Ca2+ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization. CONCLUSION: These results suggest that, in the majority of hMSCs, Ca2+ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L-type Ca2+ channels does not affect early osteogenic differentiation of hMSCs.     

Multilocular cystic nephroma: MR imaging appearance with current techniques,including gadolinium enhancement

The purpose of the study was to define the MRI appearance of multilocular cystic nephroma (MLCN), using current MR techniques, including gadolinium (Gd)-enhanced sequences. Seven patients with MLCN underwent MR imaging with the following sequences: T1-weighted spin echo with fat suppression (TIFS, five patients), T1-weighted spoiled gradient echo (SGE, seven patients), T2-weighted fast spin echo (two patients), and Gd-enhanced TIFS (seven patients) and SGE (seven patients). MLCN was histologically proven by resection of the mass in six patients and by observation of typical imaging features with stability in appearance over a 6-month period in one patient. Lesion morphology and signal intensity (SI) features were retrospectively evaluated. MRI features of MLCN included a solitary cystic lesion with thin internal septations in six patients and a cluster of closely grouped cysts similar in size in one patient. Individual cystic spaces demonstrated SI, varying from low to high on T1-weighted images in three patients and demonstrated low-to-intermediate SI in four patients. Herniation of the lesions into the renal collecting system and thin enhancing septa were demonstrated in all patients. A complex cystic renal lesion with enhancing septa and herniation into the renal collecting system are the characteristic MR findings of MLCN. The direct multiplanar capability of MR may optimally show the relationship of MLCN to the renal pelvis and, thus, facilitate correct diagnosis.     

Selective use of intraoperative sentinel lymph node pathological evaluation in breast cancer

Background

In breast cancer staging, the need for intraoperative sentinel lymph (SLN) evaluation is not well established. This study compares intraoperative use of touch preparation (TP), frozen section (FS), and factors that may influence the selective use of intraoperative SLN analysis.

Methods

Breast cancer patients (1998-2007) undergoing SLN evaluation were retrospectively reviewed.

Results

Of 205 SLN procedures, 157 cases underwent intraoperative evaluation, 43% (FS) and 57% (TP) with positive pathology in 21% and 20%, respectively. The false negative case rate was 16% for TP versus 12% for FS. Of T1, low-grade tumors, 9% were intraoperatively positive, versus 43% of T2-3, moderate- to high-grade tumors (P = .006). Additional positive axillary nodes were found in 43% of the higher risk patients versus 0% in the lower risk groups.

Conclusions

Both TP and FS are accurate for intraoperative SLN evaluation and can be selectively applied to breast cancer staging in low- and high-risk groups.     

Histological verification of 11C-choline-positron emission/computed tomography-positive lymph nodes in patients with biochemical failure after treatment for localized prostate cancer

OBJECTIVES

To evaluate the potential of ¹¹C‐choline‐positron emission tomography (PET)/computed tomography (CT) for planning surgery in patients with prostate cancer and prostate‐specific antigen (PSA) relapse after treatment with curative intent.

PATIENTS AND METHODS

We retrospectively reviewed the charts of 10 patients with PSA recurrence after either external beam radiation (two) or radical retropubic prostatectomy (eight) for prostate cancer, and who had a laparoscopic lymphadenectomy for suspicious lymph nodes detected on ¹¹C‐choline‐PET/CT. The histological results and PET/CT findings were compared.

RESULTS

In all, 22 suspicious lymph nodes were found on PET/CT, and 14 on conventional CT or magnetic resonance imaging. Comparing the conventional imaging showed concordance in 13 lymph nodes. Three of the 10 patients had no metastatic lymph node disease on definitive histology. The mean (sd ) PSA level for these patients was 1.0 (0.4) ng/mL, whereas that in patients with lymph node metastases was 15.1 (9.2) ng/mL (statistically significant difference, P < 0.05). The positive predictive value was seven of 10. All of the patients initially regressed, with PSA increases after lymphadenectomy. Two of the patients are being managed by watchful waiting, two had radiotherapy of the prostate fossa and two had chemotherapy with docetaxel. Four patients were treated by hormone‐deprivation therapy. After a mean (sd ) follow up of 11 (7) months, one patient died, one has PSA progression, but none of those with negative histology has clinical signs of local recurrence.

CONCLUSIONS

¹¹C‐choline‐PET is a valuable tool for detecting recurrent prostate cancer, but the limited positive predictive value should lead to a critical interpretation of the results.     

Cardioprotective effects of the serine protease inhibitor aprotinin after regional ischemia and reperfusion on the beating heart

OBJECTIVE: Early coronary reperfusion of the ischemic myocardium is a desired therapeutic goal to preserve myocardium. However, reperfusion itself contributes to an additional myocardial injury (ie, reperfusion injury), which has been attributed to neutrophil infiltration with subsequent release of proteases and oxygen-derived radicals. We studied the effects of the serine protease inhibitor aprotinin (Trasylol) on myocardial ischemia and reperfusion in a rat model. METHODS: The effects of aprotinin (5000 and 20,000 U/kg) were examined in vivo in a rat model of regional myocardial ischemia (20 minutes) and long-term reperfusion (24 hours). Cardioprotecive effects were determined by means of measurement of creatine kinase and myeloperoxidase activity within the myocardium, as well as histochemical analysis. RESULTS: Aprotinin (20,000 U/kg) administrated 2 minutes before reperfusion significantly attenuated myocardial injury expressed as creatine kinase washout compared with that seen in vehicle-treated rats (65 +/- 25 vs 585 +/- 98 creatine kinase difference in units per 100 mg, P <.01). Administration of 5000 U/kg of the protease inhibitor resulted in partial inhibition of myocardial reperfusion injury. Moreover, cardiac myeloperoxidase activity in the ischemic myocardium, a marker of neutrophil accumulation, was significantly reduced after aprotinin treatment. Histologic analysis of the reperfused myocardium demonstrated reduced polymorphonuclear leukocyte infiltration and reduced tissue injury. Furthermore, aprotinin treatment resulted in decreased induction of cardiac myocyte apoptosis compared with that seen in vehicle-treated rats. CONCLUSIONS: Inhibition of serine proteases with aprotinin appears to be an effective means of preserving ischemic myocardium from reperfusion injury, even after 24 hours of reperfusion. Aprotinin might exert cardioprotection through inhibition of polymorphonuclear leukocyte-induced myocardial injury and inhibition of reperfusion-induced apoptosis of cardiac myocytes.     

Follicular penetration and targeting

In the past, intercellular penetration was assumed to be the most important penetration pathway of topically applied substances. First hints that follicular penetration needs to be taken into consideration were confirmed by recent investigations, presented during the workshop "Follicular Penetration and Targeting" at the 4th Intercontinental Meeting of Hair Research Societies", in Berlin 2004. Hair follicles represent an efficient reservoir for the penetration of topically applied substances with subsequent targeting of distinct cell populations, e.g., nestin-expressing follicular bulge cells. The volume of this reservoir can be determined by differential stripping technology. The follicular penetration processes are significantly influenced by the state of the follicular infundibulum; recent experimental investigations could demonstrate that it is essential to distinguish between open and closed hair follicles. Topically applied substances can only penetrate into open hair follicle. Knowledge of follicular penetration is of high clinical relevance for functional targeting of distinct follicular regions. Human hair follicles show a hair-cycle-dependent variation of the dense neuronal and vascular network. Moreover, during hair follicle cycling with initiation of anagen, newly formed vessels occur. Thus, the potential of nestin-expressing hair follicle stem cells to form neurons and blood vessels was investigated.     

Noncycloplegic photorefractive screening in pre-school children with the "PowerRefractor" in a pediatric practice.

PURPOSE: To provide a framework for typical refractive development, as measured without cycloplegia with a commercial infrared photorefractor. To evaluate the usefulness of the screening for refractive errors, we retrospectively analyzed the data of a large number of unselected children of different ages in a pediatric practice in Tuebingen, Germany. METHODS: During the standard regular preventive examinations that are performed in 80% to 90% of the young children in Germany by a pediatrician (the German "U1 to U9" system), 736 children were also measured with the first generation PowerRefractor (made by MCS, Reutlingen, Germany, but no longer available in this version). Of those, 172 were also measured with +3 D spectacles to find out whether this helps detect hyperopia. Children with more than +2 D of hyperopia or astigmatism, more than 1.5 D of anisometropia, or more than 1 D of myopia in the second year of life were referred to an eye care specialist. The actions taken by the eye care specialist were used to evaluate the merits of the screening. RESULTS: The average noncycloplegic spherical refractive errors in the right eyes declined linearly from +0.93 to +0.62 D over the first 6 years (p < 0.001)-between 1.5 and 0.5 D less hyperopic than in published studies with cycloplegic retinoscopy. As expected, +3 D spectacle lenses moved the refractions into the myopic direction, but this shift was not smaller in hyperopic children. The average negative cylinder magnitudes declined from -0.89 to 0.48 D (linear regression: p < 0.001). The J0 components displayed high correlations in both eyes (p < 0.001) but the J45 components did not. The average absolute anisometropias (difference of spheres) declined from 0.37 to 0.23 (linear regression: p < 0.001). Of the 736 children, 85 (11.5%) were referred to an eye care specialist. Of these, 52 received spectacles (61.2%), 14 (16.4%) were identified as "at risk" and remained under observation, and 18 (21.2%) were considered "false-positive." CONCLUSIONS: Non cycloplegic photorefraction provides considerably less hyperopic readings than retinoscopy under cycloplegia. Additional refractions performed through binocular +3-D lenses did not facilitate detection of hyperopia. With the referral criteria above, 11% of the children were referred to an eye care specialist, but with a 20% false-positive rate. The screening had some power to identify children at risk but the number of false-negatives remained uncertain.     

Diagnostic odyssey of a cutaneous mycobacteriosis rare in central Europe

Cutaneous infection with Mycobacterium chelonae is an uncommon disease, although this atypical mycobacterium is an acid-fast bacillus ubiquitous in the environment. It is often misdiagnosed and treated as a fungal or common bacterial infection. We report a case of disseminated atypical mycobacterial skin infection of a 72-year-old woman who was treated with different topical and systemic antimycotic and antibiotic drugs over a period of 5 months without remarkable improvement. Eventually, repeated tissue cultures on special medium and performance of PCR led to the diagnosis of M. chelonae infection. The patient was treated successfully with oral clarithromycin within 8 weeks. In case of abscessing cutaneous infection, M. chelonae should be considered in the differential diagnosis of prolonged disease when common antibiotics are not effective after 2-4 weeks of treatment.     

Low-dose total body irradiation (TBI) and fludarabine followed by hematopoietic cell transplantation (HCT) from HLA-matched or mismatched unrelated donors and postgrafting immunosuppression with cyclosporine and mycophenolate mofetil (MMF) can induce durable complete chimerism and sustained remissions in patients with hematological diseases

Toxicities of high-dose conditioning regimens have limited the use of conventional unrelated donor hematopoietic cell transplantation (HCT) to younger, medically fit patients. Based on preclinical studies, an HCT approach has been developed for elderly or medically infirm patients with HLA-matched or mismatched unrelated donors. In this study, 52 patients with hematological diseases were included. Most (88%) had preceding unsuccessful conventional HCT or refractory/advanced disease. Patients were treated with fludarabine 30 mg/m(2)/d from days -4 to -2, 2 Gy total body irradiation on day 0, cyclosporine at 6.25 mg/kg twice daily from day -3, and mycophenolate mofetil at 15 mg/kg twice daily from day 0. Durable donor chimerism was attained in 88% of the patients. By day 28, a median of 100% of CD56(+) cells were of donor origin. Granulocyte and T-cell donor chimerism increased to medians of 100% on day 56 and day 180 (range, 55%-100%), respectively. Acute GVHD, grade II, was seen in 42% (CI, 29%-56%); grade III in 8% (CI, 0%-15%); and grade IV in 13% (CI, 4%-23%) of patients; it was fatal in 9%. The 100-day transplantation-related mortality was 11%. Complete remissions, including molecular remissions, were seen in 45% of patients with measurable disease before transplantation. Mortality from disease progression was 27% at one year. With a median follow-up of 19 months, 18 of the 52 patients (35%) were alive and 25% were in remission. HCT from HLA-matched or mismatched unrelated donors can be performed with a reduced intensity conditioning regimen in patients ineligible for conventional HCT.     

Liver dysfunction in Pennsylvania's multitransfused hemophiliacs

Transaminase values [alanine amino transferase (ALT) and aspartate amino transferase (AST)] and markers for hepatitis B were serially determined in 558 hemophiliacs exposed to blood products. Hepatitis B surface antigen (HB_sAg) persistent for over 12 months was present in 6% of the patients. Antibody to hepatitis B surface antigen (anti-HBs) was noted in 90% of the 259 patients treated with factor VIII or IX concentrates but in only 49% of the 43 patients treated with fresh frozen plasma (FFP) or cryoprecipitate. Persistently abnormal transaminase values were noted in 31% of the patients treated with commercial concentrates but in only one (2%) of the patients exposed to cryoprecipitate or FFP. This difference continued even when the two groups of patients were matched for the amount of blood products, up to 50, 000 units, which they had received in the study period. In the concentrate-treated patients, no correlation could be found between transaminase values and the number of units of factor VIII or IX they had received during the six years of the study (1973–1978).Supported in part by the Pennsylvania Hemophilia Centers and the Pennsylvania State-Wide Hemophilia Program.