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PubMed1997

Antibody levels to diphtheria, tetanus, and rubella in infants vaccinated while on PD: a Study of the Pediatric Peritoneal Dialysis Study Consortium.

To determine whether infants who receive routine childhood immunizations while on chronic peritoneal dialysis (CPD) develop protective antibody levels/titers, we measured antibody levels/titers in infants vaccinated with diphtheria/tetanus/pertussis (DTP) and measles/mumps/rubella (MMR) while on CPD. Eight CPD patients (median age 19 months, range 9-39 months) had measurement of antibody to diphtheria and tetanus toxoids. Seven of the 8 (88%) had protective levels of IgG antibody to both toxoids. The single patient who did not have protective antibody to either diphtheria or tetanus had a low total serum IgG. However, 3 other patients who had low IgG had protective antibody levels. Serial measurements of antibody to tetanus and diphtheria were obtained in 3 of the 8 patients. All maintained protective levels to both diphtheria and tetanus toxoids for as long as 24 months postvaccination. Antibody to rubella was also measured in 5 CPD patients (median 29 months, range 19-39 months), and all had protective antibody titers despite the fact that 3 had low total serum levels. In conclusion, most but not all infants immunized while on CPD have protective antibody levels/titers to diphtherial, tetanus, rubella. Alteration of the routine schedule for immunizations does not appear to be necessary. However, periodic measurements of antibody may be indicated, particularly to live vial vaccines, prior to transplantation.

Antibodies, Bacterial↗
PubMed1976

Acute clinical hypocalcemic myocardial depression during rapid blood transfusion and postoperative hemodialysis: a preventable complication.

Despite experimental evidence that myocardial depression resulting from rapid transfusion of ACD blood (citrate binds ionic calcium) is avoidable by simultaneous calcium administration, most hypovolemic patients receive calcium either after transfusion or not at all. Similar iatrogenic hypocalcemic myocardial depression occurs in normovolemic patients with known myocardial damage who are dialyzed for acute uremia when ACD blood prime is used at high initial flow rates (350 c.c. per minute) and when dialysis is performed against low calcium dialysate (2.5 mEq. per liter or less). This study tests the hypotheses that (1) rapid transfusion of as little as one unit of CPD blood causes a significant reduction in ionized calcium, (2) the depressive effect of CPD blood is significant and similar to that of ACD blood, (3) rapid blood transfusion (ACD or CPD) is safe if calcium is given simultaneously, (4) addition of calcium to the extracorporeal heparinized blood prime used in dialysis prevents initial depression, and (5) hemodynamic instability during dialysis is prevented when the dialysate is normocalcemic. From the results of our study, we made the following conclusions: (1) Ionized calcium is reduced significantly by rapid transfusion of CPD blood; (2) acute myocardial depression noted with CPD blood is similar to that previously observed with ACD blood and is prevented during transfusion of either type of blood by simultaneous calcium administration; and (3) hemodialysis in patients who have had cardiac surgery is safe if calcium is added to blood prime and dialysate is made normocalcemic.

Blood Preservation↗
PubMed1998

Assessment of the effects of various UV sources on inactivation and photoproduct induction in phage T7 dosimeter.

The correlation between the biologically effective dose (BED) of a phage T7 biological dosimeter and the induction of cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts ((6-4)PD) in the phage DNA was determined using seven various UV sources. The BED is the inactivation rate of phage T7 expressed in HT7 units. The CPD and (6-4)PD were determined by lesion-specific monoclonal antibodies in an immunodot-blot assay. The various lamps induced these lesions at different rates; the relative induction ratios of CPD to (6-4)PD increased with increasing effective wavelength of irradiation source. The amount of total adducts per phage was compared to the BED of phage T7 dosimeter, representing the average number of UV lesions in phage. For UVC (200-280 nm radiation) and unfiltered TL01 the number of total adducts approximates the reading; however, UV sources having longer effective wavelengths produced fewer CPD and (6-4)PD. A possible explanation is that although the most relevant lesions by UVC are the CPD and (6-4)PD, at longer wavelengths other photoproducts can contribute to the lethal damage of phages. The results emphasize the need to study the biological effects of solar radiation because the lesions responsible for the lethal effect may be different from those produced by various UV sources.

Bacteriophage T7↗
PubMed2005

The new paradigm of continuing education in surgery.

Concerns regarding the shortcomings of traditional continuing medical education (CME) have led to widespread acceptance of the concept of continuous professional development (CPD). Continuous professional development focuses on the individual learning needs of physicians across the continuum of their professional careers and encompasses a broad range of educational activities. Continuous professional development activities of practicing surgeons should be integrated with the core competency of practice-based learning and improvement (PBLI), which involves a cycle of 4 steps-identifying areas for improvement, engaging in learning, applying new knowledge and skills to practice, and checking for improvement. The effectiveness of CPD and PBLI should be objectively evaluated by assessing their impact on surgeons' learning and performance and on patient care outcomes. A portfolio may be used to document CPD and PBLI activities and can serve as a valuable learning and assessment tool. It should contain information on the personal learning projects pursued by the surgeon. Continuous professional development of the entire surgical team and education of patients and their families need to be considered within the context of the surgeon's CPD efforts to positively affect patient care. Thus, a concept of global 360 degrees education should be embraced. Continuous professional development integrated with PBLI can help surgeons address their specific learning needs and play a pivotal role in surgeons' providing the best care to patients.

Education, Medical, Continuing↗
PubMed1978

Morphological changes in isolated lymphocytes during preparation for SEM: freeze drying versus critical-point drying.

A quantitative comparison of isolated lymphocytes prepared for SEM by the critical-point drying (CPD) and freeze drying (FD) methods revealed that the mean cellular diameter dropped from 7.9 micron after fixation to a final diameter 6.9 micron after FD, and to 5.7 micron after CPD. In addition to their larger sizes, FD lymphocytes were immediately distinguishable by their more complex surfaces, featuring wider microvilli which often emanated from ridges on the cell surface and branched extensively near their bases. The mean width of microvilli was 0.22 micron after FD and 0.12 micron after CPD. The number of microvilli per cell was essentially the same by the two methods. In view of these findings, a critical comparison of the CPD and FD methods using the particular cells or tissue to be investigated is an essential prelude to a rigorous SEM study.

Freeze Drying↗
PubMed2001

Improved method for measuring the ensemble average of strand breaks in genomic DNA.

The cis-syn cyclobutane pyrimidine dimer (CPD) is the major photoproduct induced in DNA by low wavelength ultraviolet radiation. An improved method was developed to detect CPD formation and removal in genomic DNA that avoids the problems encountered with the standard method of endonuclease detection of these photoproducts. Since CPD-specific endonucleases make single-strand cuts at CPD sites, quantification of the frequency of CPDs in DNA is usually done by denaturing gel electrophoresis. The standard method of ethidium bromide staining and gel photography requires more than 10 microg of DNA per gel lane, and correction of the photographic signal for the nonlinear film response. To simplify this procedure, a standard Southern blot protocol, coupled with phosphorimage analysis, was developed. This method uses random hybridization probes to detect genomic sequences with minimal sequence bias. Because of the vast linearity range of phosphorimage detection, scans of the signal profiles for the heterogeneous population of DNA fragments can be integrated directly to determine the number-average size of the population.

Animals↗
PubMed2002

EGFR-independent activation of ERK1/2 mediates growth inhibition by a PTPase antagonizing K-vitamin analog.

The K-vitamin analog Cpd 5 or [2-(2-mercaptoethanol)-3-methyl-1,4-napthoquinone] is a potent cell growth inhibitor in vitro and in vivo, likely due to arylation of enzymes containing a catalytic cysteine. This results in inhibition of protein tyrosine phosphatase (PTPase) activity with resultant hyperphosphorylation of EGF receptors (EGFR) and ERK1/2 protein kinases, which are downstream to EGFR in the MAPK pathway. We used NR6 fibroblast cells, which lack endogenous EGFR and its variant cells transfected with different EGFR mutants to assess the contribution of the EGFR-mediated signaling pathway to Cpd 5-mediated ERK activation and cell growth inhibition. Cpd 5 treatment resulted in enhanced phosphorylation of EGFR at carboxyl-terminal tyrosines. This phosphorylation and activation of EGFR were found to be necessary neither for growth inhibition nor for the activation of the downstream kinases ERK1/2, since both occurred in EGFR-devoid mutant cells. U0126 and PD 098059, specific inhibitors of MEK1/2, the ERK1/2 kinases, antagonized both cell growth inhibition and ERK1/2 phosphorylation mediated by Cpd5. Cpd 5 was also found to inhibit ERK1/2 phosphatase(s) activity in lysates from all the cells tested, irrespective of their EGFR status. These results show that EGFR-independent ERK1/2 phosphorylation was involved in the mechanism of Cpd5 mediated growth inhibition. This is likely due to the observed antagonism of ERK phosphatase activity. A candidate PTPase was found to be Cdc25A, a recently identified ERK phosphatase.

Cell Division↗
PubMed1983

Autocrine and paracrine growth stimulation of cells derived from human skin.

A sensitive serum-free culture system was used to demonstrate that cells derived from normal human skin release soluble mediators which can modulate keratinocyte, melanocyte, and fibroblast growth in vitro. In M199 supplemented with epidermal growth factor, hydrocortisone, insulin, transferrin, triidothyronine, bovine serum albumin, and an extract of bovine hypothalamus, keratinocytes underwent approximately three to five cumulative population doublings (CPD) over a 7-day period. Addition of 20% keratinocyte-conditioned medium more than doubled keratinocyte growth and increased fibroblast growth 25-40% above controls. Dermal fibroblasts maintained in the same serum-free hormone-supplemented medium (SFHSM) alone underwent approximately 4.5-5.5 CPD over a 7-day period; 20% fibroblast-conditioned medium increased fibroblast growth 50-80% and increased keratinocyte growth 30-50%. Melanocytes maintained in the same SFHSM underwent approximately 1 CPD over a 14-day period and 2 CPD if the medium was supplemented with 2% fetal bovine serum (FBS). Addition of 20% melanocyte-conditioned medium more than doubled melanocyte growth in either SFHSM or in medium containing 2% FBS, but decreased both keratinocyte and fibroblast growth up to 30-60%. None of the conditioned media altered cellular morphology. These data provide the first demonstration of mutual growth modulation by cell types normally contiguous in vivo and expand existing evidence for autocrine and paracrine growth regulation by normal human cells.

Adult↗
PubMed1992

Preservation and visualization of molecular structure in detergent-extracted whole mounts of cultured cells.

Today's electron microscopes have a resolution sufficient to resolve supramolecular structures. However, the methods used to prepare biological samples for electron microscopy often limit our ability to achieve the resolution that is theoretically possible. We use whole mounts of detergent-extracted cells grown on Formvar-coated gold grids as a model system to evaluate various steps in the preparation of biological samples for high resolution scanning electron microscopy (SEM). Factors that are important in determining the structure and composition of detergent-extracted cells include the nature of the detergent and the composition of the extraction vehicle. Chelation of calcium is extremely important to stabilize and preserve the cytoskeletal filaments. We have also demonstrated both morphologically and by gel electrophoresis that treatment of cells with bifunctional protein crosslinkers before or during extraction with detergent can significantly enhance the preservation of both proteins and supramolecular structures. The methods used to dry samples are a major determinant of the quality of structural preservation. For cytoskeletons freeze-drying (FD) is superior to critical point-drying (CPD), one reason being that CPD samples have to be dehydrated, thereby causing more shrinkage as compared to FD samples. The high pressures to which samples are exposed during CPD may also cause increased shrinkage, and water contamination during CPD causes severe structural damage. We have obtained the best structural preservation of detergent-extracted and fixed cells by manually plunging them into liquid propane and drying over night in a freeze-dryer. The factor that most limits achievement of high resolution in SEM is the metal coat, which has to be very thin, uniform, and free of grain in order not to hide structures or to create artifactual ones. We have found that sputter-coating with 1-3 nm of tungsten (W) or niobium (Nb) gives extremely fine-grained films as well as satisfactory emission of secondary electrons. These samples can also be examined at high resolution by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). The best preservation and visualization of supramolecular structures have been obtained using cryosputtering, in which the samples are freeze-dried and then sputter-coated within the freeze-dryer while still frozen.

Animals↗
PubMed1997

C-terminal incorporation of fluorogenic and affinity labels using wild-type and mutagenized carboxypeptidase Y.

The ability to carry out specific C-terminal modification or labeling of peptides and proteins has a broad range of applications. It is well established that this may be achieved by protease-catalyzed transacylation reactions and that carboxypeptidase Y (CPD-Y) is suitable for this due to its broad specificity and stability in the presence of denaturants. Furthermore, CPD-Y is characterized by a S'1 binding site that is open to solvent and, thus, capable of catalyzing a transpeptidation reaction with nucleophiles that extend beyond the perimeter of the active site. However, one major drawback with CPD-Y is that the yield of the reaction is highly dependent on the nature of the leaving group; e.g., with large apolar leaving groups the yield of the reaction does not exceed 15%. In the present publication it is demonstrated that mutants of CPD-Y, designed for low leaving group dependence, efficiently incorporate biocytin amide as well as a new fluorescent nucleophile, N'-Abz-Lysine amide (ablysin amide), into peptides and proteins.

Affinity Labels↗
PubMed1996

Peptide aldehyde complexes with wheat serine carboxypeptidase II: implications for the catalytic mechanism and substrate specificity.

The structures of two ternary complexes of wheat serine carboxypeptidase II (CPD-WII), with a tetrapeptide aldehyde and a reaction product arginine, have been determined by X-ray crystallography at room temperature and -170 degrees. The peptide aldehydes, antipain and chymostatin, form covalent adducts with the active-site serine 146. The CPD-WII antipain arginine model has a standard crystallographic R-factor of 0.162, with good geometry at 2.5 A resolution for data collected at room temperature. The -170 degrees C model of the chymostatin arginine complex has an R-factor of 0.174, with good geometry using data to 2.1 A resolution. The structures suggest binding subsites N-terminal to the scissile bond. All four residues of chymostatin are well-localized in the putative S1 through S4 sites, while density is apparent only in S1 and S2 for antipain. In the S1 site, Val340 and 341, Phe215 and Leu216 form a hydrophobic binding surface, not a pocket, for the P1 phenylalanyl side-chain of chymostatin. The P1 arginyl of antipain also binds at this site, but the positive charge appears to be stabilized by additional solvent molecules. Thus, the hybrid nature of the S1 site accounts for the ability of CPD-WII to accept both hydrophobic and basic residues at P1. Hydrogen bonds to the peptide substrate backbone are few and are made primarily with side-chains on the enzyme. Thus, substrate recognition by CPD-WII appears to have nothing in common with that of the other families of serine proteinases. The hemiacetal linkages to the essential Ser146 are of a single stereoisomer with tetrahedral geometry, with an oxygen atom occupying the "oxyanion hole" region of the enzyme. This atom accepts three hydrogen bonds, two from the polypeptide backbone and one from the positively-charged amino group of bound arginine, and must be negatively charged. Thus, the combination of ligands forms an excellent approximation to the oxyanion intermediate formed during peptide hydrolysis. Surprisingly, the (R) stereochemistry at the hemiacetal linkage is opposite to that expected by comparison to previously determined structures of peptide aldehydes complexed with Streptomyces griseus proteinase A. This is shown to be a consequence of the approximate mirror symmetry of the arrangement of catalytic groups in the two families of serine proteases and suggests that the stereochemical course of the two enzymatic reactions differ in handedness.

Aldehydes↗
PubMed2001

Early (N70m) neuromagnetic signal topography and striate and extrastriate generators following pattern onset quadrant stimulation.

The MEG signal generated by sinusoidal grating pattern onset at 1 and 3 cpd, presented randomly to the four quadrants, was analyzed in terms of gross signal properties and current dipole modeling and for a subset of subjects with magnetic field tomography (MFT). In all subjects a prominent wave was identified with a peak latency around 70 ms (N70m), modulated by spatial frequency and varying systematically with the stimulation quadrant. Sensors over occipital areas recorded stronger responses with lower field quadrants, while the signal for sensors a few centimeters more superior was stronger with upper quadrant stimuli. A strong signal in inferior occipitotemporal areas was less sensitive to upper and lower field stimulation and was stronger in the left hemisphere with contralateral (right) visual field stimulation. For lower visual field stimulation a good fit to the average data was obtained with a single dipole for 3 cpd, but was less consistent across run repetitions for 1 cpd. Neither the single-dipole model nor the two-dipole model produced a good fit across runs with the upper field stimuli. MFT solutions identified overlapping activity in striate and extrastriate areas in all conditions. The MFT solutions in the V1/V2 at the N70m were highly reproducible across run repetitions for 1 and 3 cpd, and consistent with the cruciform model, even though they were often weaker than simultaneously activated extrastriate generators. Extrastriate generators in V5 and the human homologue of V6, which were variable across run repetitions at N70m, settled to highly reproducible activations between 100 and 200 ms.

Adult↗
PubMed1988

Juvenile Parkinson's disease with widespread Lewy bodies in the brain.

A clinico-pathological case report on a case of juvenile Parkinson's disease (JPD) with widespread Lewy bodies (LBs) in the brain is presented with comparative morphological studies on two demented cases of "classical" Parkinson's disease (CPD) with disease onset at an older age. The clinical and histological pictures of this JPD case were typical of Parkinson's disease, excepting numerous Lewy bodies in the cerebrum. There were no neurofibrillary change nor senile plaques throughout the CNS. The distribution and histochemical and ultrastructural characters of the histological lesions (i.e., LBs) in the JPD and the two CPD cases were investigated and compared. The comparison showed no qualitative but only quantitative differences between the two types of Parkinson's disease. The present study also revealed that in CPD cases significant numbers of LBs could be present in the cerebral cortex, amygdaloid and claustrum. These lesions can be in part responsible for dementia in CPD.

Adult↗
PubMed1993

Defective antibody response to Hemophilus influenzae type b immunization in children receiving peritoneal dialysis.

Pediatric end-stage renal disease patients, maintained on chronic peritoneal dialysis (CPD), may have a variety of immunological abnormalities, including hypogammaglobulinemia and poor responses to vaccines. We measured antibody levels to Hemophilus influenzae type b (Hib) in 24 CPD patients. Eight children received primary Hib immunization while undergoing CPD. Of these, 1 of 8 (12%) lacked protective levels of antibody. In another child, who had an initial protective response, antibody levels were undetectable 12 months after immunization. Sixteen of the patients had not been immunized with Hib vaccine because they were more than 5 years old when the vaccine was licensed. In this group, 5 of 16 (31%) lacked protective levels of anti-Hib antibody. Of those available for follow-up, 3 responded normally to Hib vaccine. It is not sufficient to provide childhood immunizations to CPD patients with the assumption that those immunizations will lead to long-lived immunity. Antibody levels should be measured within a month of immunization and at regular intervals thereafter to document immunity.

Adolescent↗
PubMed1992

Chronic peritoneal dialysis in paediatrics: experience of a national registry.

The results of the first 3 years' collaboration of the Italian Registry of Paediatric Chronic Peritoneal Dialysis (CPD) (1986-1988) are presented. This Registry acquired data on the majority of the paediatric patients treated with CPD in Italy, thus providing a national picture in a field where few nationwide surveys are available. Patients of less than 15 years of age at the start of dialysis were enrolled and clinical data collected until the age of 19 years. The number of nephrological paediatric centres participating in the Registry increased from 7 in 1986 to 11 in 1988. The total number of patients on CPD was 70 and the percentage of dialysed children treated with CPD ranged from 40.2% to 43.6%. Data on 89 peritoneal catheters were collected: during 1417 dialysis-months 70 catheter-related complications were observed (1:20.8 dialysis-months); actuarial catheter survival was 92.7% at 6 months, 84.8% at 1 year and 68.8% at 2 years. the incidence of peritonitis changed from 1 episode every 10.9 patient-months in 1986 to 1 every 19.8 in 1988. Abdominal hernias were the other main clinical complication observed. The survival of patients was 92.5% at 3 years, while the technique survival at the same time was 84%.

Adolescent↗
PubMed1997

Contrast sensitivity in diabetic pregnancy.

PURPOSE: To evaluate the correlation between the changes in contrast sensitivity and retinopathy throughout pregnancy in diabetic women with mild background diabetic retinopathy. METHODS: Contrast sensitivity (Vistech 6500 Contrast Test System) was measured in 22 type I diabetic women with mild background retinopathy [0-16 microaneurysms (MAs)/eye and occasional small intraretinal hemorrhages] and 10 healthy pregnant women at the 12th and 32nd weeks of pregnancy and 3 months after delivery. Red-free fundus photography was also performed at all visits. RESULTS: Throughout pregnancy contrast sensitivity was lower in the diabetics than in the controls at 1.5, 3.0, and 6.0 cycles per degree (cpd). During the course of pregnancy, contrast sensitivity decreased at 1.5 and 6.0 cpd in patients with more than five new MAs compared to those with fewer than five new MAs during pregnancy. Likewise, contrast sensitivity decreased at 6 cpd in patients with an increase in MA count during pregnancy and at 6 and 12 cpd in patients with a baseline MA count of two or more MAs. CONCLUSIONS: Contrast sensitivity, especially at low to mid-range spatial frequencies, is decreased in diabetic women with mild retinopathy. Even small changes in retinopathy are reflected in a decrease in contrast sensitivity during and after pregnancy. Thus, contrast sensitivity seems to be a sensitive indicator of changes in minimal background retinopathy.

Adult↗
PubMed1995

Cytokine production by peripheral blood mononuclear cells from pediatric chronic peritoneal dialysis patients.

Previous reports have documented impaired cytokine production by peritoneal macrophages in chronic peritoneal dialysis (CPD) patients. To determine if this observed defect was a reflection of systemic mononuclear cell dysfunction, the function of peripheral blood mononuclear cells obtained from pediatric patients on CPD was assessed after stimulation with lipopolysaccharide (LPS). Levels of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) mRNA and protein were measured before and after stimulation with LPS. There was no significant difference in the response of mononuclear cells from CPD patients and normal controls in terms of increase in TNF-alpha mRNA [median stimulation index (SI) = 6.6 vs. 3.7, P = 0.35] or IL-1 beta mRNA (median SI = 6.2 vs. 6.5, P = 1.0). There was also no significant difference between the median increase in TNF-alpha protein secretion (median 372 pg/ml vs. 373 pg/ml, P = 0.60). These results suggest that systemic mononuclear cell function may be intact in CPD patients, and therefore this does not account for the dysfunction of peritoneal macrophages that has been previously reported.

Adolescent↗
PubMed2000

Models to assess nitrogen losses in pediatric patients on chronic peritoneal dialysis.

To develop models to estimate nitrogen (N) losses of children on chronic peritoneal dialysis (CPD) from easily measurable indexes and laboratory tests, we measured the N content and all nitrogenous compounds in dialysate (D), urine (U), and feces over 3 days in 19 pediatric patients on CPD. Total measured N losses (TNm) were 5.56+/-2.26 g/day (69.9+/-11.1% in dialysate, 16.3+/-10.6% in urine, and 13.6+/-4.6% in feces). Correlation coefficients between measured dialysate and urinary N losses and the single nitrogenous compounds indicated values of over 0.9 only for urea in dialysate and urine; fecal N losses correlated well with body surface area (BSA). Taking into account these correlations, we developed a univariate additive model and three multivariate models to predict total estimated N losses (TNe). The best prediction of TNm was obtained with model 3, which considered not only urea output in dialysate and urine but also dialysate protein loss and BSA: TNe (g/day)=0.03+/-1.138 UN urea+0.99 DN urea+1.18 BSA+0.965 DN protein. A confirmatory analysis performed on a second group of 23 pediatric patients on CPD, using all four models, showed a higher percentage of studies with a relative difference between TNm and TNe less than 10% for model 3 than for the other models. Thus, N losses of pediatric patients on CPD can be estimated from measured urea and protein losses in dialysate and urea loss in urine, together with BSA.

Adolescent↗