Isolation of multiple mRNAs from a few eukaryotic cells: a fast method to obtain templates for RT-PCR.
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Antigens, Neoplasm↗
Biomedical subjects
C Schaefer
Publications and source records attributed to C Schaefer.
Cystic fibrosis heterozygote screening in 5,161 pregnant women.
A screening program for cystic fibrosis (CF) heterozygotes was conducted in a large HMO prenatal population, to evaluate the level of interest among eligible patients, the effectiveness of prescreening education, attitudes toward the screening process, psychological effects, and utilization of prenatal diagnosis and its outcomes. The heterozygote identification rate and frequency of specific CFTR mutations were also assessed. Identified carriers were offered genetic counseling and testing of male partners. Prenatal diagnosis was offered if both parents were identified as carriers. A total of 5,161 women underwent carrier testing; 947 others completed survey instruments only. The acceptance rate of screening was high (78%), and pretest education by videotape was generally effective. Adverse psychological effects were not reported. Participants generally found screening to be desirable and useful. Screening identified 142 female heterozygotes, 109 couples in which the male partner was not a carrier, and 7 high-risk couples. The incidence of R117H mutations was much higher than expected. The number of identified carriers was much lower in Hispanics than in Caucasians. We conclude that large-scale prenatal screening for CF heterozygotes in the absence of a family history of CF is an acceptable method for identifying couples at risk for affected fetuses. Sufficient pretest education can be accomplished efficiently, test insensitivity is well accepted, adverse psychological events are not observed, and general patient satisfaction is high.
Adult↗
Regional cerebral blood flow changes associated with risperidone treatment in elderly schizophrenia patients: a pilot study.
Although there is evidence that some schizophrenia patients may have altered regional cerebral blood flow patterns, few studies have addressed the relationship between cortical activity and changes in psychiatric symptoms following treatment, particularly in the elderly. We took advantage of an existing safety and tolerance study of risperidone in the elderly and examined the possible relationship between changes in psychiatric symptoms following risperidone and changes in relative cortical perfusion in a group of 6 elderly schizophrenia patients. All subjects were at least 65 years old and diagnosed with schizophrenia according to DSM-III-R criteria. The patients were assessed using the Positive and Negative Syndrome Scale for Schizophrenia (PANSS) and Mini-Mental Status Examination (MMSE) and had single photon emission computed tomography (SPECT) studies before and at least 3 weeks after change of their previous neuroleptic to risperidone. The frontal/total cortex and temporal/total cortex counts in the slice ratios, and 99mTechnitium-hexamethylpropylene amine oxime (99mTc-HMPAO) percentage uptake in the whole cortical area in the slice were used for data analysis. With risperidone, patients (age 66-81) scored better on the PANSS, particularly in the positive symptom subtests. The changes in positive symptom scores correlated directly with those in frontal and temporal relative activity and 99mTc-HMPAO percentage uptake in the whole cortical area in the slice. Our findings suggest that the improvement in psychotic symptoms after risperidone is associated with a decrease in frontal and temporal activity and a reduction in 99mTc-HMPAO percentage uptake in the entire cortical area in the slice and agree with data from younger populations. Comparative studies assessing the therapeutic impact of neuroleptics on cortical activity in different age groups could be helpful in examining both the mechanisms of action of various drugs and the links between symptoms and specific brain areas.
Aged↗
Psychologic stress in the workplace and spontaneous abortion.
The relation of work-related psychologic stress to spontaneous abortion was examined in a prospective study of 3,953 pregnant, employed members of a prepaid health plan in California, recruited between 1990 and 1991. Information on occupation, psychologic stress-related factors at work, social support at work, physical exertion at work, life events, pregnancy worries, and potential confounders was obtained by a telephone interview. Psychologic job stress and social support at work were assessed using an abbreviated version of the instrument developed by Karasek and colleagues (Los Angeles: University of Southern California, 1986). Results from the multivariate model indicated that, overall, stressful work was not associated with an increased risk of spontaneous abortion. However, interactions were observed between stressful work and maternal age over 32 years (p = 0.04), cigarette smoking (p = 0.02), and primigravidity (p = 0.06). Relative to the odds ratio for stressful work in young, nonsmoking, multigravid women without a history of two or more spontaneous abortions, the odds ratios for spontaneous abortion given stressful work were higher by 2.45 (95% confidence interval (CI) 1.03-5.81) for older women, 2.96 (95% CI 1.16-7.52) for smokers, and 2.27 (95% CI 0.97-5.27) for primigravid women.
Abortion, Spontaneous↗
Peptide-specific CTL in tumor infiltrating lymphocytes from metastatic melanomas expressing MART-1/Melan-A, gp100 and Tyrosinase genes: a study in an unselected group of HLA-A2.1-positive patients.
Peptide specificity of cultured tumor-infiltrating lymphocytes (TIL) was systematically investigated in a group of HLA-A2.1+ metastatic melanoma patients consecutively referred to our department for surgical treatment. Seven samples from 6 patients were studied. All surgical specimens showed evidence of gp 100, MART-1/Melan-A and Tyrosinase gene expression as detectable by reverse PCR (rPCR). Cultured TIL from 2 patients displayed cytotoxic activity against autologous or HLA-matched EBV-transformed cells previously pulsed with MART-1/Melan-A27-35 peptide. In contrast, no CTL activity against gp100(280-288) or tyrosinase1-9 peptides could be observed. TIL were then repeatedly stimulated in vitro with the same peptides. After 6 restimulation courses at weekly intervals, specific recognition of gp100(280-288) and MART-1/Melan-A peptides was detectable in 3 and 5 TIL populations, respectively. In one case Tyrosinase1-9-specific CTL could be demonstrated. Two TIL populations from metastases resected from a melanoma patient at 6 months' distance showed a different peptide specificity pattern, and no specific CTL could be generated from simultaneously sampled peripheral blood mononuclear cells (PBMC). All peptide-specific CTL populations also displayed significant cytotoxic activity against HLA-A2.1 matched melanoma cell lines expressing the antigens under investigation. Our data indicate that CTL specific for MART-Melan-A27-35, gp100(280-288) or Tyrosinase1-9 peptides could be expanded with varying frequency from TIL derived from 4 out of 6 HLA-A2.1+ patients whose tumors expressed the genes encoding these tumor-associated antigens (TAA).
Adult↗
Mortality following conjugal bereavement and the effects of a shared environment.
The effect of bereavement on mortality among surviving spouses was examined in a cohort of 12,522 spouse pairs belonging to a prepaid health care plan in northern California. Both spouses were examined and completed a questionnaire between 1964 and 1973, and they were followed for mortality through 1987. Between 1964 and 1987, 1,453 men (12%) and 3,294 women (26%) were bereaved; 440 bereaved men (30%) and 510 women (15%) died during follow-up. Mortality following bereavement was significantly elevated in both men and women after adjusting for age, education, and other predictors of mortality in proportional hazards analyses. The highest relative risks (RRs) of mortality occurred 7-12 months following bereavement. Among women, the RR was 1.9 (95% confidence interval (CI) 1.35-2.71) 7-12 months after bereavement. Among men, the effect of bereavement interacted with prior health status as follows: In men with few health problems, the RR of mortality was 2.12 (95% CI 1.42-3.17); men with many health problems had a RR of 1.56 (95% CI 0.98-2.55) 7-12 months after bereavement. In both men and women, the RR declined after the first year of bereavement but remained above 1.0 for more than 2 years after bereavement. The addition of terms reflecting the influence of the other spouse's characteristics and behaviors did not alter the RR of mortality, indicating no effect of a shared unfavorable environment on mortality following bereavement.
Adult↗
Identification and intracellular location of MAGE-3 gene product.
The human MAGE-3 gene encodes a melanoma antigenic epitope recognized by specific cytotoxic T lymphocytes, but its gene product has not been identified thus far. We produced a recombinant MAGE-3 gene product by expression cloning of the entire reading frame in the context of a fusion protein characterized by a 10-histidine tail, allowing purification by metal chelation on a nickel Sepharose column. The semipurified product was used to generate MAGE-3-specific monoclonal antibodies. One reagent could identify by immunoblotting the native MAGE-3 gene product as a M(r) 48,000 protein in lysates of cell lines showing evidence of MAGE-3 gene expression. No apparent cross-reactivity with recombinant or native MAGE-1 gene product was observed. Immunohistochemistry shows that, closely resembling the MAGE-1 gene product, MAGE-3 is a cytoplasmic protein.
Animals↗
Effect of alpha adrenergic stimulation and carnitine palmitoyl transferase I inhibition on hypertrophying adult rat cardiomyocytes in culture.
Long-term, serum supplemented cultures of rat adult ventriculocytes were utilized to study the tropic effects of the alpha-agonist phenylephrine and of the carnitine palmitoyltransferase I inhibitor etomoxir. Cell protein and the rate of incorporation of phenylalanine were measured, corrected for cellular DNA content and utilized as an index for hypertrophy and of anabolic activity of the cells, respectively. The mRNA level of ANF was utilized as an index for the pathological phenotypic change (i.e., switch to fetal gene program), and that of the Na-channel--a constantly expressed gene in normal and hypertrophic cardiomyocytes--served as an internal control. Both mRNAs were quantified at various stages in culture by competitive reverse transcriptase PCR. The size of control myocytes steadily increased for over 3 weeks. The cells were completely redifferentiated and reached a maximum of anabolic activity 2 weeks after plating. Secretion and mRNA levels of ANF were increased severalfold after 7-8 days. Addition of 10 microM phenylephrine considerably speeded up cell growth. Maximum anabolic activity and complete redifferentiation were reached already after 1 week. Levels of mRNA and of ANF release increased 30-40 fold. Interestingly, induction of ANF gene transcription lagged behind the redifferentiation of the cells. Ten microM etomoxir inhibited the oxidation of palmitic acid and stimulated that of exogenous glucose by adult cardiomyocytes. In spite of its clear effect on fuel utilization, etomoxir had no direct hypertrophic effect on the myocytes in culture and did not inhibit the stimulatory action of alpha-agonists. Reactivation of the fetal gene program, as visualized by ANF production, was not reversed by etomoxir.
Animals↗
Review of 47 laparoscopic appendectomies.
STUDY OBJECTIVE: To assess the indications, technique, complications, and outcome of endoscopic appendectomies. DESIGN: Retrospective study with a follow-up of 2 years. SETTING: A gynecologic surgical center in Hamburg, Germany. PATIENTS: Forty-seven women who underwent laparoscopic appendectomy. INTERVENTIONS: Appendectomies were performed under general anesthesia with bipolar coagulation, loop-ligation, and transection. MEASUREMENTS AND MAIN RESULTS: Forty-seven patients have been followed for 2 years. Thirty laparoscopic appendectomies (LA) were incidental and 17 elective. The majority of LA were performed because of pain symptoms. More than 50% of the patients reported complete pain relief after LA. The average duration of surgery was 47 minutes. The average hospital stay was 66 hours. Use of analgesics was low; 19 women required no analgesia. A febrile reaction was registered in 6%. No wound infection occurred. Two serious complications were noted: one acute peritonitis after 4 days, and one stump abscess after 4 weeks. Both patients recovered uneventfully. CONCLUSIONS: Laparoscopic appendectomy is safe surgery if the described technique is followed. The complication rate is low, conversion rate is zero, and late results underline successful treatment of pain.
Abdominal Pain↗
Identification of new oligodendrocyte- and myelin-specific genes by a differential screening approach.
We have isolated several new genes that are specifically expressed by oligodendrocytes in the CNS. This was achieved by differential screening of a rat spinal cord cDNA library with probes derived from normal and from oligodendrocyte-free spinal cord mRNAs. Four of these genes are exclusively expressed by oligodendrocytes: Three of these are not related to known genes, whereas one encodes the myelin oligodendrocyte glycoprotein (MOG). Four other genes are expressed by oligodendrocytes as well as by Schwann cells. One gene codes for apolipoprotein D, which is thought to be involved in lipid metabolism. A second cDNA sequence codes for the recently identified galactosylceramide-synthesizing enzyme UDP-galactose:ceramide galactosyltransferase. The third gene encodes a small protein with four putative transmembrane domains that is related to a T-lymphocyte-specific membrane protein, MAL. The fourth gene encodes the rat homologue of the stearyl-CoA-desaturase 2 (SCD2) gene, which is specifically expressed in the nervous system and involved in the synthesis and regulation of long-chain unsaturated fatty acids essential for myelination. Finally, we found that a member of the beta-tubulin family is highly expressed in oligodendrocytes as well as neurons. The identification of several new proteins that may play a role in myelin synthesis and sheath formation will lead to new insight into this complex mechanism.
Animals↗
Clinical psychological students' subjective stress ratings during their doctoral training.
The Bandura-Rosenthal Metrics for Assessing Stress was used to measure perceived stress among 25 first and 28 second- and third-year clinical Ph.D. students. Analysis gave no significant differences in reported stress across time.
Adaptation, Psychological↗
Prognostic value of Doppler echocardiographic assessment of left ventricular filling in idiopathic dilated cardiomyopathy.
The relation of left ventricular (LV) diastolic filling with the clinical outcome in patients with idiopathic dilated cardiomyopathy (IDC) was examined. LV diastolic filling was assessed by Doppler echocardiography in 57 patients with IDC at the time that the diagnosis was established by angiocardiography. Patients were followed for 29 +/- 16 months. Fifteen patients died: 12 due to progressive congestive heart failure and 3 suddenly. Four other patients underwent cardiac transplantation because of progressive heart failure (1-year survival 86%). Patients who died of congestive heart failure or underwent cardiac transplantation had a steep increase and decrease in the early filling phase as compared with survivors; the peak early Doppler velocity was higher (0.84 +/- 0.16 vs 0.65 +/- 0.21 m/s; p < 0.005), and the deceleration time of the early velocity peak was shorter (117 +/- 26 vs 188 +/- 62 ms; p < 0.001) than in survivors. Surviving patients and those who died suddenly showed similar patterns of LV filling. Deceleration time and peak early Doppler velocity were the strongest predictors of survival as compared with systolic function and clinical status in a Cox proportional-hazards analysis. Patients with a shortened deceleration time (< or = 140 ms) had a significantly reduced 2-year survival rate of 52% (confidence interval 34 to 71%) as compared with those with a longer deceleration time (94%; confidence interval 89 to 98%) (p < 0.001). Evidence was presented for a relation between LV filling and survival in patients with IDC.
Actuarial Analysis↗
Intracellular acidosis in murine fibrosarcomas coincides with ATP depletion, hypoxia, and high levels of lactate and total Pi.
Bioenergetic and metabolic status of murine FSaII tumours were evaluated using 31P MRS, acid extracts ('global' techniques) and quantitative bioluminescence ('microregional' assay). Data obtained from s.c. tumours of varying sizes (44-600 mm3) have been correlated with the oxygenation status evaluated using O2-sensitive needle electrodes. beta-NTP/Pi and phosphocreatine (PCr)/Pi ratios derived from 31P MRS were positively correlated to the median tissue pO2 values. pH declined during growth with intracellular acidosis being evident in tumours > 350 mm3. Whereas lactic acid formation greatly contributed to this decline in small- and medium-sized tumours, ATP hydrolysis and slowing down of the activities of pumps involved in pHi regulation seem to be major factors responsible for intracellular acidification in bulky tumours. PCr levels decreased at an early growth stage, whilst ATP concentrations dropped in bulky malignancies only, coinciding with a decrease in adenylate energy charge and a substantial rise in the levels of total Pi. MRS observable (mobile) Pi was consistently lower than [Pi] measured in acid extracts. On average, median pO2 values of ca 10 mmHg represent a critical threshold for energy metabolism. At higher median O2 tensions, levels of ATP, phosphomonoester and total Pi were relatively constant. This coincided with intracellular alkalosis or neutrality and stable adenylate ratios. On average, median pO2 values < 10 mmHg coincided with intracellular acidosis, ATP depletion, a drop in energy charge and rising Pi levels.
Acidosis↗
[Accuracy of CT angiography in the diagnosis of renal artery stenosis].
Spiral CT in the form of CT angiography (CTA) provides a new method for the investigation of the large vessels. 52 patients with suspected renovascular hypertension were examined by CTA and arterial DSA. The computer tomographic and angiographic findings in 124 renal arteries were compared. Sensitivity of CTA for demonstrating renal artery stenosis was 95%, and for relevant stenoses it actually reached 100%. Specificity was 92%. Positive/negative prediction value was 85% and 100%. CTA proved outstandingly good for demonstrating or excluding renal artery stenosis and was superior to other imaging methods in several respects. CTA requires high contrast values, optimal timing and careful evaluation. Amongst various forms of image reconstruction, interactive viewing of the primary axial images and multiplanar reconstruction in a cine mode are essential and MIP projections are valuable.
Adult↗
Microregional distributions of glucose, lactate, ATP and tissue pH in experimental tumours upon local hyperthermia and/or hyperglycaemia.
Microregional distributions of glucose, lactate and ATP concentrations as well as tissue pH values were determined in subcutaneous rat tumours during normothermia and normoglycaemia, and upon local hyperthermia (HT) and/or hyperglycaemia (HG). Experiments were performed in order to investigate whether, and to what extent, these adjuvant therapeutic measures applied alone or in combination can modify the bioenergetic and metabolic status, parameters that are known to markedly influence the therapeutic response of tumours to heat. Local HT was performed in a saline bath (44 degrees C/2 h) and HG was induced by i.v. infusion of glucose for 2.5 h (blood glucose levels during heating: 35-40 mM). Immediately after treatment, the microregional distributions of glucose, lactate and ATP concentrations were assessed using quantitative bioluminescence and single-photon counting. In corresponding histological sections the fraction of tumour tissue with changes indicating cellular damage was determined. For comparison, global levels of glucose, lactate, ATP, ADP and AMP were measured using enzymatic assays or HPLC. Tumour tissue pH values were recorded immediately after treatment with miniaturised needle glass pH electrodes. Upon HT alone, the microregional glucose distribution remained unchanged. Lactate concentrations significantly increased, resulting in a pH drop of about 0.20 pH units. Mean ATP concentrations decreased without an obvious change in the shape of the distribution curve. The fraction of tumour tissue showing cellular damage increased from 18% (in control tumours) to 27%. Upon HG alone, mean glucose and lactate levels in the tumours increased. Glucose, lactate and pH distributions became broader. Lactate accumulation results in a severe tumour acidosis (mean pH = 6.22). Mean ATP concentrations marginally decreased despite a higher glucose availability, probably because of poorer ATP yield resulting from changes in metabolic channelling (Crabtree effect). The fraction of tumour tissue exhibiting cellular damage was 23%. Following the combined treatment (HT/HG), glucose and lactate levels, and tissue pH were similar to those seen upon HG alone. However, ATP concentrations were lowest under this condition. The variation of tumour ATP concentrations is substantially reduced with only a few tumour areas remaining with ATP levels of at least 0.6 mumol/g. The ATP depletion upon HT/HG is accompanied by a drastic increase in the fraction of tissue areas exhibiting cellular damage to 61%. It may therefore be concluded that only the combined treatment can deplete ATP to such an extent that a pronounced cytotoxic effect is achieved.
Adenosine Triphosphate↗
Doppler echocardiographic assessment of left ventricular filling in idiopathic dilated cardiomyopathy during a one-year follow-up: relation to the clinical course of disease.
In idiopathic dilated cardiomyopathy (IDC), an impaired left ventricular filling as assessed by the Doppler echocardiographic mitral flow pattern is closely related to the severity of congestive heart failure. This study examined the relation of left ventricular filling and the clinical course of the disease in patients with a recent diagnostic procedure and initiation of medical therapy (group 1, n = 15) as compared with patients in a chronic stage of the disease (group 2, n = 24) with the diagnosis established > 1 year before. All patients had to be in sinus rhythm to facilitate the Doppler echocardiographic evaluation of left ventricular filling. The clinical status was assessed by the New York Heart Association classification and a heart failure score at baseline and after a period of 12 +/- 7 months. At baseline the ratio of the peak early/atrial Doppler velocities (VE/VA) was shifted toward the early diastole in group 1 as compared to group 2 (1.84 +/- 1.02 vs 1.12 +/- 0.55; p < 0.05). Symptoms of heart failure were more severe in group 1. During follow-up, VE/VA tended to decrease in group 1 from 1.84 +/- 1.02 to 1.35 +/- 1.03 (p = 0.07) and remained unchanged in group 2 (1.12 +/- 0.55 and 1.34 +/- 1.23; not significant). In a subgroup of 10 patients who underwent repeat right heart catheterization, the decrease of VE/VA coincided with a decrease of the pulmonary capillary wedge pressure.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Three-dimensional vascular imaging--an additional diagnostic tool.
Angiography is still the standard for imaging of vascular structures. However, since the number of projections is limited, complex pathoanatomy may not be sufficiently visible. This study presents two patients in whom 3D reconstruction from Spiral CT data revealed combined vascular lesions undisclosed by angiography. In one case the combination of coarctation and chronic dissection type B, in another the combination of two aneurysms of the celiac trunk in series was disclosed. We conclude that 3D reconstruction can be a valuable asset in the diagnosis of complex vascular pathoanatomy.
Adult↗
[Vascular imaging with spiral-CT. The path to CT-angiography].
Spiral CT is a technique that allows for high-quality two-dimensional angiographic projections and 3D imaging of vascular structures. The authors present the technical and methodological principles of the technique, including scan parameters and parameters of contrast application for various clinical imaging tasks. They present their experience with over 150 clinical cases using spiral CT angiography. Suitable applications of this technique include congenital anomalies, aneurysms, dissections, stenoses, thrombi and vascular tumor involvement. Given a problem-adapted examination technique, pathologic changes in vessels of as little as 2 mm can be visualized. In some cases with complex vascular anatomy, spiral CT angiography can be superior to arterial angiography.
Forecasting↗