Vitamin D supplementation and bone turnover in advanced heart failure: the EVITA trial.

Low vitamin D status is common in patients with heart failure and may influence bone health. A daily vitamin D dose of 4000 IU (moderately high dose) for 3 years had however no effect on parameters of bone metabolism, even in patients with very low vitamin D status. INTRODUCTION: Low vitamin D status is common in patients with heart failure (HF) and has been related to disturbed bone turnover. The present study investigated the effect of a daily vitamin D3 dose of 4000 IU on bone turnover markers (BTMs) in patients with advanced HF and 25-hydroxyvitamin D (25OHD) concentrations < 75 nmol/L. METHODS: In this pre-specified secondary analysis of a randomized controlled trial, we assessed in 158 male HF patients (vitamin D group: n = 80; placebo group: n = 78) between-group differences in calciotropic hormones (25OHD, 1,25-dihydroxyvitamin D [1,25(OH)2D], intact parathyroid hormone [iPTH]), and BTMs (cross-linked C-telopeptide of type I collagen, bone-specific alkaline phosphatase, undercarboxylated osteocalcin). Comparisons were performed at the end of a 3-year vitamin D supplementation period with adjustments for baseline values. RESULTS: Compared with placebo, vitamin D increased 25OHD on average by 54.3 nmol/L. At study termination, 25OHD and 1,25(OH)2D were significantly higher (P < 0.001 and P = 0.007, respectively), whereas iPTH tended to be lower in the vitamin D group than in the placebo group (P = 0.083). BTMs were initially within their reference ranges and did not differ significantly between groups at study termination, neither in the entire study cohort nor when data analysis was restricted to the subgroup of patients with initial 25OHD concentrations < 30 nmol/L (n = 54) or to patients with initial hyperparathyroidism (n = 65) (all P values > 0.05). CONCLUSIONS: A daily vitamin D3 dose of 4000 IU did not influence BTMs. Data indicate that vitamin D supplementation will not lower bone turnover in male patients with heart failure.

Randomized supplementation of 4000 IU vitamin D3 daily vs placebo on the prevalence of anemia in advanced heart failure: the EVITA trial.

BACKGROUND: Low 25-hydroxyvitamin D (25OHD) levels (< 75 nmol/l) are inversely associated with anemia prevalence. Since anemia and low 25OHD levels are common in patients with heart failure (HF), we aimed to investigate whether vitamin D supplementation can reduce anemia prevalence in advanced HF. METHODS: EVITA (Effect of Vitamin D on Mortality in Heart Failure) is a randomized, placebo-controlled clinical trial in patients with initial 25OHD levels < 75 nmol/l. Participants received either 4000 IU vitamin D3 daily or a matching placebo for 36 months. A total of 172 patients (vitamin D group: n = 85; placebo group: n = 87) were investigated in this pre-specified secondary data analysis. Hemoglobin (Hb) and other hematological parameters were measured at baseline and study termination. Assessment of between-group differences in anemia prevalence and Hb concentrations was performed at study termination, while adjusting for baseline differences. RESULTS: In the vitamin D and placebo group, baseline proportions of patients with anemia (Hb < 12.0 g/dL in females and < 13.0 g/dL in males) were 17.2% and 10.6%, respectively (P = 0.19). At study termination, the proportion of patients with anemia in the vitamin D and placebo groups was 32.2% and 31.8%, respectively (P > 0.99). There was no between-group difference in change in the Hb concentrations (- 0.04 g/dL [95%CI:-0.53 to 0.45 g/dL]; P = 0.87). Results regarding anemia risk and Hb concentrations were similar in the subgroup of patients with chronic kidney disease (vitamin D group: n = 26; placebo group: n = 23). Moreover, results did not differ substantially when data analysis was restricted to patients with deficient baseline 25OHD levels. CONCLUSIONS: A daily vitamin D supplement of 4000 IU did not reduce anemia prevalence in patients with advanced HF. Data challenge the clinical relevance of vitamin D supplementation to increase Hb levels. TRIAL REGISTRATION: The study was registered at EudraCT (No. 2010-020793-42) and clinicaltrials.gov ( NCT01326650 ).

[Heart and combined heart-lung transplantation. Indications, chances and risks]. / Herz- und Herz-Lungen-Transplantation. Indikationen, Chancen, Risiken.

Orthotopic heart transplantation (HTX) is nowadays the worldwide accepted gold standard for the treatment of terminal heart failure. The main indications for HTX are non-ischemic dilatative (54%) and ischemic (37%) heart failure. In the acute phase after HTX the survival rate is approximately 90%. Good short and long-term results with survival rates ranging from 81% after 1 year to more than 50% after 11 years demonstrate that there is currently no real treatment alternative to HTX for treatment of end-stage heart failure. In the case of irreversible pulmonary hypertension in combination with end-stage heart failure or complex congenital heart syndromes, a combined heart and lung transplantation (HLTX) is necessary. Compared with HTX the short-term survival of HLTX is reduced, mostly for technical reasons. Improved long-term results after HTX and HLTX are a result of highly specialized transplantation units and effective immunosuppression. However, a major problem is the shortage of organ donors in Germany and the resulting long waiting times for patients with frequently occurring blood groups of up to 10 months for transplantation. The consequence of the latter is the ever increasing number of implanted cardiac assist devices in patients not only as a bridge to transplant but also as destination therapy.

Bosentan effects on hemodynamics and clinical outcome in heart failure patients with pulmonary hypertension awaiting cardiac transplantation.

BACKGROUND: In heart failure (HF) patients, pulmonary hypertension (PH) is associated with a poor prognosis. We assessed whether low dose treatment with the dual endothelin-1 receptor antagonist bosentan is associated with improved hemodynamics and clinical outcome in these patients. METHODS: We performed a retrospective data analysis in 82 end-stage heart failure patients on the waiting list for cardiac transplantation since January 2006. All patients had pulmonary arterial pressure >35 mmHg, pulmonary vascular resistance >240 dyn × s × cm-5, and/or a transpulmonary gradient (TPG) >15 mmHg. Fifty-four patients received a median dose of 125 mg bid bosentan (BOS group), and 28 patients received standard medical treatment (CON group). Data were assessed until June 2009. RESULTS: Hemodynamic parameters improved significantly in the BOS group but remained unchanged in the CON group. The percentage of patients who fell below the thresholds of PAP, PVR, and TPG for cardiac transplantation increased significantly by 20.3%, 34.5%, and 20.8%, respectively (p = 0.007-0.013) in the BOS group, but did not change significantly in the CON group. One-year survival on the waiting list was approximately 20% higher in the BOS group than in the CON group (p = 0.020). Bosentan treatment remained an independent predictor of reduced mortality risk on the waiting list after propensity score adjustment (relative risk = 0.107; 95% CI: 0.013-0.869; p = 0.036). CONCLUSION: Treatment with the endothelin-1 antagonist bosentan is associated with improvements in hemodynamics and clinical outcome in end-stage heart failure patients with PH. If these results can be confirmed by randomized controlled trials, bosentan may represent a treatment option in these patients.

Vitamin D supplementation of 4000 IU daily and cardiac function in patients with advanced heart failure: The EVITA trial.

BACKGROUND: Data regarding the effects of vitamin D on cardiac function are inconclusive. METHODS: In a post-hoc analysis of the EVITA (Effect of vitamin D on mortality in heart failure) trial, we investigated whether a daily vitamin D3 supplement of 4000 IU for three years affects echocardiography parameters like left ventricular end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), and LV ejection fraction (LVEF) in patients with advanced heart failure (HF) and 25­hydroxyvitamin D levels <75 nmol/L. Of 400 patients enrolled, 199 were assigned to vitamin D and 201 to placebo. We assessed time × treatment interaction effects using linear mixed models and analyzed in subgroups vitamin D effects at 12 and 36 months post-randomization using analysis of covariance with adjustments for baseline values. RESULTS: At baseline, values of LVEDD, LVESD, and LVEF were 67.5 ±â€¯10.5 mm, 58.9 ±â€¯12.0 mm, and 30.47 ±â€¯10.2%, respectively. There were no time × treatment interaction effects on LV echocardiographic parameters in the entire study cohort, neither at 12 months nor at 36 months post-randomization (P-values > 0.05). However, in the subgroup of patients aged ≥50 years, vitamin D treatment was associated with an increase in LVEF of 2.73% (95%CI: 0.14 to 5.31%) at 12 months post-randomization (n = 311). The increase was slightly attenuated to 2.60% (95%CI: -2.47 to 7.67%) at 36 months post-randomization (n = 242). CONCLUSION: Our data indicate that vitamin D supplementation does not significantly improve cardiac function in all patients with advanced HF. However, vitamin D probably improves LV function in HF patients aged ≥50 years.

Identification of two distinct MYC breakpoint clusters and their association with various IGH breakpoint regions in the t(8;14) translocations in sporadic Burkitt-lymphoma.

The chromosomal translocation t(8;14) is the hallmark of Burkitt's-lymphoma (BL) and fuses the proto-oncogene c-MYC to the IGH locus. We analyzed the genomic structure of MYC/IGH fusions derived from a large series of 78 patients with t(8;14) and asked (i) whether distinct breakpoint clusters exist within the MYC gene and (ii) whether any pairwise association between particular IGH and MYC breakpoints exist. Identification of such associations will help elucidate the etiology of the breaks on the MYC locus. Scan statistic analyses revealed two distinct, but large clusters within c-MYC containing 60/78 (77%) of the breakpoints. Clusters 1 and 2 were 560 and 779 bp in length within a 4555 bp breakpoint cluster region. Breaks within IGH switch mu and joining region did not differ with respect to their corresponding MYC breakpoints. However, there was a highly significant correlation between breakpoints 5' of MYC cluster 1 and fusions to IGH switch gamma region and breakpoints downstream of MYC cluster 2 and fusions to IGH switch alpha region (chi(2)-test: P<0.005). Chromatin changes governing choice of IGH-Fc region recombination may parallel changes in the MYC gene 5' region chromatin leading to some degree of coordinated ontological specificity in breakpoint location.

Unusual case of an 18-year-old heart transplant recipient with endocardial fibroelastosis.

An 18-year-old female Japanese patient who suffered from heart failure and severe pulmonary hypertension was referred to our clinic. The etiology of her cardiomyopathy was unclear. Inhaled prostacyclin therapy resulted in an improvement of pulmonary arterial pressure and allowed us to avoid lung transplantation. Heart transplantation resulted in a complete remission of her respiratory function. Autopsies of the explanted heart revealed massive endomyocardial fibroelastosis. We concluded that endomyocardial fibroelastosis has to be considered a cause of heart failure in young adults with unclear cardiomyopathy.

Unusual pacemaker implantation through a left sided superior vena cava via anonymous vein after heart transplantion.

We report the case of a 56-year-old male heart transplant recipient, who underwent postoperative pacemaker implantation through a left sided superior vena cava (LSVC) via anonymous vein. We describe our successful management of this case. We suggest that the specific anatomic conditions should be considered in all heart transplant recipients with LSVC if pacemaker implantation is necessary postoperatively.

Papillary fibroelastoma of the ascending aorta presenting with cardiogenic shock.

We describe an uncommon case of a 58-year-old woman who presented with cardiogenic shock. The echocardiography examination revealed a papillary fibroadenoma located in the ascending aorta which was subsequently surgically treated.

Frequent detection of hepatitis B core antibodies in heart transplant recipients without preceding hepatitis B infection.

It is unclear whether heart donors positive for hepatitis B core antibodies (anti-HBc) can transfer hepatitis B virus (HBV) infection to immunosuppressed heart recipients, or whether passive transfer of anti-HBc simulates a hepatitis B infection. Therefore, we performed a case-controlled study in 46 heart recipients who all tested negative for hepatitis B antigen (HbsAg), antiHBc, and hepatitis B surface antibodies before heart transplantation. Twenty-three patients (group 1) received hearts from anti-HBc-positive donors, while 23 other patients (group 2) received hearts from anti-HBc-negative donors. After heart transplantation, anti-HBc were present in 65.0% of blood samples among group 1 and 47.8% of the blood samples among group 2 (P > .05). HbsAg was undetectable in blood samples of all patients of both study groups. The immunoglobulin preparation that we regularly use for immune suppression immediately after heart transplantation contained a relatively high concentration of anti-Hbc antibodies. The nearly identical presence of anti-HBc in both study groups indicated that passive transfer via immunoglobulin preparations rather than HBV infection is the cause for the anti-HBc detected in heart recipients. Since only a small volume of blood is transferred with the donor heart, it seems to be rather unlikely that the donor heart might be the source of anti-HBc. In summary, we observed no evidence for HBV infection in those heart recipients who received organs from anti-HBc-positive donors. Moreover, our data demonstrated that the presence of anti-HBc in heart recipients frequently occurs but does not necessarily indicate a preceding HBV infection.

Silencing of disease-related genes by small interfering RNAs.

In recent years a new mechanism of posttranscriptional gene silencing has been discovered and named RNA interference. The interference is based on mRNA degradation mediated by small double-stranded RNA molecules approximately 21 nucleotides in length, the so-called short interfering or siRNAs. These molecules are produced from long dsRNAs by Dicer, a dsRNA-specific endonuclease, and cause specific degradation of their mRNA-targets by Watson-Crick base-pairing within a 300 kD multi-enzyme complex named RISC. RNAi is highly conserved between plants and animals of various phyla including mammals. The high sequence-specificity of RNAi makes it a new, promising tool in gene-function analysis as well as in potential therapeutics. In this review the discovery and molecular background of RNAi are summarized and possible fields of application pointed out.

Donor-Derived Cell-Free DNA Is a Novel Universal Biomarker for Allograft Rejection in Solid Organ Transplantation.

BACKGROUND: In solid organ transplantation, sensitive real-time biomarkers to assess the graft health are desirable to enable early intervention, for example, to avoid full-blown rejections. During rejection, high amounts of graft-derived cell-free DNA (GcfDNA) are shed into the blood stream. The quantification of this GcfDNA in allotransplantation is considered to fulfill this need, because it can be measured with great precision and at reasonable cost. PATIENTS AND METHODS: Patients from 2 ongoing studies in kidney (KTx) and heart (HTx) transplantation were monitored blinded on a scheduled basis, by means of a published universal droplet digital polymerase chain reaction to quantify the GcfDNA. RESULTS: Immediately after engraftment, GcfDNA reaches high values (>5% of total cfDNA), with a rapid decrease to values of <0.5% within 1 week. Living-related KTx recipients show lower initial values, reflecting the absence of preservation injury. Episodes of rejection in KTx and HTx are accompanied by a significant increase of GcfDNA (>5-fold) above values in patients without complications, occurring earlier than clinical or biochemical hints to rejection. One case of rejection, which became clinically suspect after 1 year and was proven with biopsy, showed a significant 10-fold increase 3 months earlier. CONCLUSIONS: The quantification of GcfDNA has the potential to detect rejection episodes at early stages, when other means of diagnosis are not effective. The method's noninvasiveness enables the monitoring recipients at intervals that are desired to catch rejections at early actionable stages to prevent full-blown rejection. This biomarker will be particularly valuable in regimens to minimize immunosuppression.

Ribonucleolytic activities in the Escherichia coli in vitro translation system and in its separate components.

mRNA stability is a limiting parameter for the efficiency of in vitro protein biosynthesis. In order to develop strategies to prolong the mRNA half-life, we investigated the ribonuclease activities in the complete Escherichia coli system, in the separate cell fractions 70S ribosomes and S-100 and in the non-cellular fraction. Our results imply that the amount of ribonucleolytic activities and the insensitivity to placental RNase inhibitor in the complete system are due to the 70S ribosome fraction, whereas the generation of small degradation products is due to the S-100 fraction. Remarkably, the human placental RNase inhibitor is able to reduce mRNA degradation in the bacterial S-100 fraction.

The formin-binding protein 17, FBP17, binds via a TNKS binding motif to tankyrase, a protein involved in telomere maintenance.

In acute myelogenous and lymphoid leukemias, rearrangements involving the MLL (mixed lineage leukemia) gene at chromosome 11q23 are frequent. The truncated MLL protein is fused in-frame to a series of partner proteins. We previously identified the formin-binding protein 17 (FBP17) as such an MLL fusion partner. In this study, we explored in vivo physiological interaction partners of FBP17 using a two-hybrid assay and found tankyrase (TNKS), an ADP-ribose polymerase protein involved in telomere maintenance and mitogen-activated protein kinase signaling. We demonstrate that FBP17 binds via a special TNKS-binding motif to tankyrase. The physiological relevance is indicated by co-immunoprecipitation of endogenous proteins in 293T cells.

Cutaneous leiomyosarcoma.

We report the clinical, histopathologic, immunohistologic, and prognostic findings in 19 patients with cutaneous leiomyosarcoma, eight males and 11 females (mean age, 66 years; age range, 41-93 years). The tumors presented mainly as solitary lesions and were located on the head and neck (eight lesions), trunk (four lesions), upper extremities (three lesions), and lower extremities (four lesions). Histopathologically, two predominant growth patterns were observed: nodular (12 cases) and diffuse (seven cases). Neoplasms with a nodular growth pattern were characterized by high cellularity and prominent nuclear atypia, and they showed conspicuous mitoses, several necrotic cells, and sometimes extensive necrotic areas. By contrast, most cutaneous leiomyosarcomas with a diffuse growth pattern revealed low cellularity, well-differentiated smooth muscle cells, inconspicuous mitotic figures, and few or no necrotic cells. Immunohistologic investigations revealed all cutaneous leiomyosarcomas to express vimentin and smooth muscle actin. Pan-muscle actin (HHF-35) was also expressed in most cases (15 lesions). However, only 12 lesions showed positive staining for desmin. Remarkable was the expression of cytokeratins in five lesions. Clinical follow-up revealed local recurrences in five patients (three cases with nodular pattern and two lesions with a diffuse pattern) after a period ranging from 8 months to 3 years after surgical excision. No distant metastases have been observed in our series. We conclude that cutaneous leiomyosarcoma with a diffuse growth pattern may constitute a pitfall in histopathologic diagnosis because of the presence of only subtle criteria for malignancy. Cutaneous leiomyosarcoma may show different immunophenotypes, thus emphasizing the importance of using a large panel of antibodies (smooth muscle actin, HHF-35, desmin, vimentin, cytokeratins, and S-100 protein) in immunohistologic diagnosis. Cutaneous leiomyosarcoma sometimes reveals local recurrences, but it has negligible potential for distant metastases.

Cytoskeleton in neuroectodermally derived epithelial and muscle cells of the human iris and ciliary body.

The cytoskeleton of epithelial and muscle cells of the human iris and ciliary body was analyzed by immunohistochemistry in three morphologically normal formalin-fixed, paraffin-embedded eyes and in 34 eyes containing a uveal melanoma. Both layers of the iris epithelium reacted with monoclonal antibodies (MAb) V9 and Vim 3B4 to vimentin, whereas the ciliary epithelia additionally reacted with MAb CAM 5.2, CK5, KS-B17.2, and CY-90, recognizing cytokeratins 8 and 18. The same cytokeratin MAb labeled the retinal pigment epithelium, which lacked vimentin. The muscle portion of the anterior iris epithelium, which forms the dilator muscle, as well as the sphincter and ciliary muscles, reacted with MAb DE-U-10 to desmin and 1A4 to alpha-smooth muscle actin. The dilator and ciliary muscles also reacted with V9 and Vim 3B4 to vimentin, and some dilator fibers were weakly immunopositive for cytokeratin 8 and 18 with CY-90 and CAM 5.2. The antigenic profile of iris and ciliary epithelia infiltrated by melanoma cells remained unchanged. The intraocular epithelia, which are developmentally related but differ in function, and the intraocular muscles, which differ in origin but are functionally related, have distinct cytoskeletal profiles and may provide insights into the functional significance of intermediate filament expression.

Cytoskeleton in normal and reactive human retinal pigment epithelial cells.

The cytoskeleton of normal and reactive retinal pigment epithelium (RPE) was analyzed immunohistochemically in five light microscopically normal formalin-fixed, paraffin-embedded human eyes enucleated because of orbital tumor and in 44 eyes with a uveal melanoma. In 26 eyes, the RPE overlying the tumor was morphologically normal or atrophic; in 18 eyes, hyperplastic changes were present. Normal RPE cells lacked vimentin, but it was present in 35 of 44 eyes with uveal melanoma. Antibodies that recognize cytokeratins CK8 and CK18 labeled normal and reactive RPE cells in all specimens. Although CAM 5.2 and CY-90 detected RPE cells strongly and quantitatively, clones CK5, KS-B17.2, and pancytokeratin antibody lu-5 reacted weakly and did not label some specimens. Immunoblotting supported the presence of CK8 and CK18 in human RPE. Normal RPE cells did not express other simple epithelial cytokeratins, but both atrophic and hyperplastic reactive RPE cells were labeled with antibodies to CK7 or CK19 in 24 of the 44 eyes. Hyperplastic proliferating RPE cells that formed subretinal membranes reacted positively for alpha-smooth muscle actin in 13 of 18 eyes. Antibodies to CK8 and CK18 are valuable markers of normal and reactive human RPE cells, but a panel of reagents is necessary to document reactive changes in the cytoskeleton. Acquisition of alpha-smooth muscle actin by human RPE cells may be related to their ability to form periretinal membranes and contribute to intraocular proliferative diseases.

Design and evaluation of chemically synthesized siRNA targeting the NPM-ALK fusion site in anaplastic large cell lymphoma (ALCL).

The NPM-ALK fusion protein is found in up to 75% of pediatric anaplastic large cell lymphomas (ALCL). The ALK kinase becomes constitutively activated and triggers malignant transformation. Molecular targeting of the tumor-specific NPM-ALK fusion by gene-silencing methods seems to be a promising approach both for the treatment of ALCL and to decipher signaling pathways used by NPM-ALK. We designed and evaluated three chemically synthesized small interfering RNAs (siRNAs) for downregulation of the NPM-ALK fusion mRNA. Compared to HeLa cells transfected with the NPM-ALK expression plasmid only and to an siRNA containing two point mutations, the most potent anti-NPM-ALK siRNA reduced NPM-ALK protein expression in HeLa cells to almost undetectable levels, and the number of cells stained positively for NPM-ALK decreased by 80%. With respect to signaling, expressing of NPM-ALK increased the activity of AKT and ERK in HeLa cells, and this effect could be blocked by the specific siRNA targeting NPM-ALK. Expression of endogenous NPM-ALK mRNA in SR786 ALCL cells decreased by 50%-60% in cells transfected with the NPM-ALK siRNA. However, the amount of NPM-ALK protein was not influenced by a single transfection of the siRNAs against NPM-ALK. Repeated transfections over 8 days led to a significant reduction in NPM-ALK protein but without induction of apoptosis. We believe that the long protein half-life of NPM-ALK, at least 48 hours, limits the application of transiently transfected siRNAs. Nevertheless, RNA interference (RNAi) offers a suitable technique to dissect signaling pathways employed by NPM-ALK and may potentially be used to develop siRNA-based gene therapeutic approaches against NPM-ALK-positive lymphomas.

Tumor cell markers in uveal melanoma.

Monoclonal antibodies to the melanoma-associated antigens HMB-45 and NKI/C3, and for S-100 protein were applied to archival tissue of 43 intraocular melanomas. Tn addition, the expression of the oncoproteins ras-p21 (ras 10) and mutated Ha-ras (E 184) as well as neu/erb-B2 (p185) were immunohistochemically evaluated. Incubation with antibodies to HMB-45 and NKI/C3 revealed consistently moderate to strong staining in all cases. Comparable ras-p21 immunostaining with normal epithelium observed in infiltrating components with a pronounced heterogeneous pattern, was particulary evident in epitheloid tumor cells. In melanomas of the spindle cell type B there was a tendency for patients with neu/erb-B2 positivity to have a worse prognosis. Using the chi-squared test for trend a significant correlation was found between S-100 reactivity, neu/erb-B2 amplification and the clinical outcome.

Lifelong learning of physicians: contributions of different educational phases to practice performance.

This pilot study was designed to find out where and when in their education physicians thought they had acquired the competencies they used in their daily practices five years after completion of their formal training. Specifically, the study sought physicians' views about the relative contributions of seven major phases of medical education (i.e., preclinical, clinical, "practice phases 1 and 2," specialty training, formal continuing education, and independent learning) to their practice performances. In 1991, the authors distributed a questionnaire to 330 physicians participating in a continuing medical education course in Germany, asking them about the relative contributions of these educational phases to their practice performances. A total of 141 (43%) returned questionnaires, of which 114 were suitable for evaluation. Specialty education was believed to have contributed most to the physicians' daily practices, with a median contribution of 20%. Practice-based independent learning, in which 38% participated in groups, contributed, with a median of 18%, and was superior to formal continuing education, with a median of 5%, and to university education, 15%. The frequency distribution of the percentages allotted to the seven phases showed great variation. Thirty-four respondents stated that they had not gained appreciably from preclinical education or formal continuing education but that the other five phases had contributed 50% to 90% of the knowledge and skills they used in their practices.