Differential gene expression during terminal erythroid differentiation.

Terminal erythroid differentiation in mammals is the process whereby nucleated precursor cells accumulate erythroid-specific proteins such as hemoglobin, undergo extensive cellular and nuclear remodeling, and ultimately shed their nuclei to form reticulocytes, which then become mature erythrocytes in the circulation. Little is known about the mechanisms that enable erythroblasts to undergo such a transformation. We hypothesized that genes involved in these mechanisms were likely expressed at restricted times during the differentiation process and used differential display reverse transcriptase polymerase chain reaction as a first step in identifying such genes. We identified three differentially expressed cDNAs that we termed late erythroblast (LEB) 1-3. None of these cDNAs were previously identified as being expressed in erythroblasts and their patterns of expression indicated they are likely to be involved in the differentiation process. LEB-1 cDNA was derived from the gene A330102K04Rik (approved gene symbol Apoll1), and shares homology with members of the apolipoprotein L family in humans. LEB-3 cDNA was derived from the novel gene D930015E06Rik, that has no known function. LEB-2 cDNA was derived from the gene ranBP16 (approved gene symbol Xpo7), a nuclear exportin. D930015E06Rik mRNA is also strongly expressed in the testis and was localized to a region of the seminiferous tubule where secondary spermatocytes and early spermatids are found, suggesting a role for D930015E06Rik in spermatogenesis as well as terminal erythroid differentiation. We have thus identified three genes not previously described as being expressed in erythroblasts that could be relevant in elucidating mechanisms involved in terminal erythroid differentiation.

Localization and quantitation of eotaxin mRNA in human nasal polyps.

Nasal Polyps (NPs) are the most common mass lesions found in the nose. NPs cause airway obstruction, prevent normal sinus function, and can lead to infection of the eye, facial bones and central nervous system. The predominant cell type inhabiting NPs is the eosinophil, and the chemokine eotaxin is believed to play an important role in NP eosinophilia. The objective of this study was to localize and quantitate expression of eotaxin mRNA in human NPs. Total RNA was isolated from NPs that were collected from 5 patients who had undergone polypectomy. Portions of these polyps were also fixed in formalin, embedded in paraffin, and sectioned onto slides for use in in situ hybridization. Total RNA from one patient was used in a reverse transcriptase polymerase chain reaction using eotaxin specific primers to generate a human eotaxin cDNA. The eotaxin cDNA was cloned and used to generate probes for Northern blot analyses and for use in in situ hybridization (ISH). Eotaxin mRNA was detected by Northern analyses in all patient samples, though the relative expression level in each patient varied. ISH localized the expression of eotaxin mRNA specifically in eosinophils in 2 of the 3 patients in the study for whom the embedded polyp tissue appeared sufficiently well preserved for mRNA localization. Our findings suggest that eosinophilia in NPs is likely a self-amplification process whereby increasing numbers of eosinophils are recruited to enter the polyp as a result of production of eotaxin by eosinophils already within the polyp.

Expression of AhR and ARNT mRNA in cultured human endometrial explants exposed to TCDD.

Endometriosis is a debilitating disease found in 10-15% of reproductive-age women and is characterized by the presence of endometrial tissue outside of the uterus. The present study characterizes the expression of AhR and ARNT mRNA in a human endometrial explant culture model in the absence and presence of TCDD exposure. In a parallel, companion study using this model, TCDD exposure was shown to induce CYP1A1 mRNA, CYP1B1 mRNA, EROD (7-ethoxyresorufin-O-deethylase) activity, and CYP1B1 protein in human endometrial explants. Explants were prepared from specimens obtained at laparoscopy or laparotomy from women undergoing surgery for tubal ligation, endometriosis, or pelvic pain unrelated to endometriosis. These specimens were a subset of the specimens used in the parallel study. The explants were cultured in medium containing 10 nM estradiol (E(2)) or 1 nM estradiol plus 500 nM progesterone (E(2) + P(4)) with or without TCDD (first 24 h). After culture, AhR and ARNT mRNA expression were quantified by RT-PCR. TCDD treatment significantly increased the expression of AhR mRNA, but not ARNT mRNA. The expression of both genes was similar for all individual explants and the ratio of AhR:ARNT mRNA expression across all samples was 1.7 to 1.8. Constitutive AhR mRNA expression was donor age dependent (increasing with age), while ARNT mRNA expression was donor age and tissue phase dependent (increased in older and proliferative phase specimens). Similar to results in the parallel study on expression of CYP1A1 mRNA, CYP1B1 mRNA, EROD activity, and CYP1B1 protein, the presence of endometriosis did not affect the expression of AhR or ARNT mRNA, either constitutively or following TCDD exposure. However, the detection of disease-specific change was limited by small sample size and variability in tissue cycle phase. The human endometrial explant culture model will be useful for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase, hormonal exposure, and donor age.

Effect of TCDD exposure on CYP1A1 and CYP1B1 expression in explant cultures of human endometrium.

Endometriosis is a debilitating disease estimated to affect 10% of reproductive-age women and characterized by the growth of endometrial tissue outside of the uterus. The present study characterizes a human endometrial explant culture model for studying the direct effects of TCDD exposure by assessing the expression of CYP1A1 and CYP1B1 mRNA (Northern blotting), protein (Western blotting), and activity (7-ethoxyresorufin-O-deethylase; EROD) in explants cultured with and without TCDD. Explants were obtained at laparoscopy or laparotomy from women undergoing surgery for tubal ligation, endometriosis, or pelvic pain unrelated to endometriosis. The explants were cultured with 10 nM estradiol (E(2)) or 1 nM E(2) plus 500 nM progesterone (P(4)) with or without TCDD (first 24 h). The expression of CYP1A1 and CYP1B1 mRNA was greatest with 10 nM TCDD and increased up to 72 h after initial exposure. EROD activity increased up to 120 h. Explants from a secretory phase biopsy became reorganized in culture and formed a new epithelial membrane, while maintaining basic endometrial morphology and viability for up to 120 h. At 24 h, TCDD significantly increased CYP1A1 and CYP1B1 mRNA, and at 72 h, TCDD significantly increased EROD activity and CYP1B1 protein compared to explants cultured without TCDD for similar times. CYP1B1 protein also exhibited substantial constitutive expression that was similar in uncultured biopsies, where CYP1B1 protein was immunolocalized in the cytoplasm of epithelial glands, with only occasional patches of protein in the surface epithelial membrane. In explants cultured with and without TCDD exposure, CYP1B1 protein was localized in the cytoplasm of the new surface epithelial membrane and glands closest to the surface. CYP1A1 protein was not detected in uncultured biopsies or explants. Both younger age (age 30 and under) and proliferative phase were associated with higher TCDD-induced EROD activity in specimens treated with E(2):P(4). No significant endometriosis-related differences were observed for any of the biomarkers, but the detection of disease-specific change was limited by small sample size and variability in tissue-cycle phase. The human endometrial explant culture model will be useful for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase and hormonal exposure.

Focal neurological signs in hepatic encephalopathy in cirrhotic patients: an underestimated entity?

OBJECTIVES: Focal neurological signs have been poorly documented in the course of hepatic encephalopathy in cirrhotic patients because they are not mentioned in any textbooks of liver diseases. Having the opportunity to observe such cases, we underwent a prospective study to determine incidence, characteristics, associated factors, prognostic significance, and outcome of this rare form of hepatic encephalopathy. METHODS: Over a 12-month period, all cirrhotic patients hospitalized in the intensive care unit of our department for hepatic encephalopathy were prospectively studied. Patients with clinical and electroencephalogram evidences of hepatic encephalopathy were examined by a senior physician and, in cases of focal neurological signs, underwent examination by a neurologist, CT scan, lumbar punction, and cerebral magnetic resonance imaging and echo Doppler examination of neck and head vessels. Clinical and biological parameters were compared in patients during episodes with and without focal neurological signs, and outcome was noted. RESULTS: Thirty-four cirrhotic patients were hospitalized for 48 episodes of hepatic encephalopathy; two of these patients with cerebral hematoma were excluded. Twenty-four patients exhibited 38 hepatic encephalopathy episodes without focal neurological signs (82.6%), and eight patients exhibited eight hepatic encephalopathy episodes with focal neurological signs (17.4%) that were hemiplegia and hemiparesia in six patients (75%). In all patients, cerebral CT scan and cerebrospinal fluid examination disclosed no abnormalities, as neither did cerebral magnetic resonance imaging (n = 5) and echo Doppler examination of neck and head vessels (n = 5). Except for female sex, which was more often encountered in patients with focal neurological signs (p < 0.05), there were no differences between episodes with and without focal neurological signs for any of the parameters studied. In surviving patients who recovered from hepatic encephalopathy (7/8), focal neurological signs disappeared without recurrences after follow up of 6 months (3-12). CONCLUSIONS: Hepatic encephalopathy with focal neurological signs when carefully searched is not uncommon. It could be more frequent in cirrhotic females, is reversible, and has no prognostic significance.

Recognition and management of Tillaux fractures in adolescents.

Tillaux fractures are relatively uncommon Salter Harris III fractures of the tibia. The importance of recognizing this fracture is that a residual deformity in the joint surface can lead to premature degenerative arthritis. For this reason, it is important that accurate imaging to assess the congruity of the joint, as well as adequate reduction, is obtained. These fractures can occur in adolescents in the 18-month period during which the distal tibial epiphysis is closing. These injuries occur either by lateral rotation of the foot or by medial rotation of the leg on the fixed foot. Closed reduction is sufficient in most cases; however, if a gap of > or = 2 mm of the articular surface remains, open reduction is usually required to adequately reduce the articular surface. Orthopedic injuries are one of the most common reasons children are brought to the emergency department (ED). Most of these injuries are easily managed by splinting, with outpatient orthopedic follow-up. However, certain fractures need closer evaluation and immediate consultation with an orthopedic surgeon. One relatively uncommon fracture that needs special attention is the Tillaux fracture. Paul Jules Tillaux first described this particular fracture in 1892. He performed experiments on cadavers and found that stress to the anterior inferior tibiofibular ligament could lead to this type of avulsion fracture, which today is termed the Tillaux fracture. The distal tibial epiphysis is involved, and the mechanism usually is forced external rotation of the foot in a 12- to 14-year-old adolescent. This fracture only occurs during a certain time of adolescence, owing to the differential growth rate of the epiphysis, and only under certain circumstances. The fracture is of great importance because it involves a major weight-bearing articular surface. A residual deformity of the joint surface can lead to premature degenerative arthritis. We present a patient with a Tillaux fracture to elaborate on the mechanism of injury and to summarize the importance of its recognition and imaging and treatment options.

Air vs ground transport and outcome in trauma patients requiring urgent operative interventions.

OBJECTIVE: To study trauma patients requiring urgent operative interventions to determine whether transport mode was associated with outcome difference. METHODS: Eligible patients were injured adults and children who presented over a 57-month period to the emergency department (ED) at the study hospital (annual ED census 36,000) after air or ground transport from trauma scenes or referring hospitals. Patients included were those whose ED lengths of stay were <60 minutes prior to transfer to an operating room. Data collected included injury severity score (ISS), ED and hospital lengths of stay, and mortality. Continuous data, which were not distributed normally, were analyzed using Wilcoxon nonparametric analysis. Categorical variables were analyzed using chi-square testing. Multivariate logistic regression was used to account for confounding variables and isolate the effects of transport mode on mortality. Alpha for all tests was set at 0.05. RESULTS: 272 patients were eligible for study; 168 air medical and 104 ground transports. No between-group differences were found for ED length of stay, ISS, or mortality. A significantly longer hospital stay was found for air-transported patients. Subgroup analysis of patients with penetrating trauma and ISS of > or =25 revealed mortalities of 28% and 45% for air- and ground-transported patients, respectively; this difference was not statistically significant (p = 0.24), but the study had a power of only 22% to detect a difference at this magnitude. CONCLUSION: This study failed to identify, but had insufficient power to rule out, outcome benefit to air medical transport in a subset of trauma patients requiring urgent operative interventions.

Iatrogenic acute hyponatraemia in a college athlete.

Hyponatraemia is one of the most common electrolyte abnormalities, leading to significant morbidity and mortality. In the most basic sense, hyponatraemia can be due to sodium loss or fluid excess. The extracellular fluid status is used to clinically divide hyponatraemia into three categories to help to determine both the cause and treatment required. Hyponatraemic patients can be categorised on the basis of their fluid status as hypovolaemic, euvolaemic, or hypervolaemic. Another distinction to make in evaluating hyponatraemia is whether the onset was acute or chronic in nature. The case presented here is iatrogenic acute hypervolaemic hyponatraemia in a college athlete. The patient presented in respiratory distress with an altered mental status after the administration of hypotonic fluids for treatment of muscle cramps. Treatment included intubation, water restriction, and furosemide, to which he responded favourably. Hyponatraemia should be in the differential diagnosis for patients presenting after intravenous fluid administration.

Omeprazole and the development of acute hepatitis.

Omeprazole has been used with increasing frequency for the treatment of conditions such as reflux oesophagitis, peptic ulcer disease, and Zollinger-Ellison syndrome. Several sequelae have been documented in the literature, but there has been limited indication of significant hepatotoxicity. We present a unique case of acute hepatitis secondary to the use of omeprazole that was resolved spontaneously with discontinuation of the drug.

Apoptosis of late-stage erythroblasts in megaloblastic anemia: association with DNA damage and macrocyte production.

An in vitro model of folate-deficient erythropoiesis has been developed using proerythroblasts isolated from the spleens of Friend virus-infected mice fed an amino acid-based, folate-free diet. Control proerythroblasts were obtained from Friend virus-infected mice fed the same diet plus 2 mg folic acid/kg diet. Our previous studies showed that, after 20 to 32 hours of culture in folate-deficient medium with 4 U/mL of erythropoietin, the folate-deficient proerythroblasts underwent apoptosis, whereas control erythroblasts survived and differentiated into reticulocytes over a period of 48 hours. The addition of folic acid or thymidine to the folate-deficient medium prevented the apoptosis of the folate-deficient erythroblasts, thereby implicating decreased thymidylate synthesis as the main cause of apoptosis in the folate-deficient erythroblasts. In the study reported here, we examined intracellular folate levels, uracil misincorporation into DNA, p53 and p21 proteins, and reticulocyte formation in erythroblasts cultured in folate-deficient or control medium. In all experiments, the folate-deficient erythroblasts cultured in folate-deficient medium gave results that varied significantly from folate-deficient erythroblasts cultured in control medium or control erythroblasts cultured in either folate-deficient or control media. Folate-deficient erythroblasts cultured in folate-deficient medium had marked decreases in all coenzyme forms of folate that persisted throughout culture, increased uracil misincorporation into DNA, persistent accumulations of p53 and p21, and decreased reticulocyte production but increased size of individual reticulocytes. A model of folate-deficient erythropoiesis based on apoptosis of late stage erythroblasts is presented. This model provides explanations for the clinical findings in megaloblastic anemia.

Erythropoietin production by interstitial cells of hypoxic monkey kidneys.

Previous studies which demonstrated that interstitial cells of the peritubular capillary bed of the kidneys are the site of erythropoietin (Epo) production have been performed in non-primate species. In this study, kidneys from adult rhesus monkeys exposed to 18 h hypoxia (0.42 atm) with high serum (5685 mU/ml) and kidney (814 mU/g. includes serum EPO in the kidney) levels of Epo were compared with a kidney from a nonhypoxic normal rhesus monkey. Localization of Epo mRNA by in situ hybridization was carried out with either anti-sense or sense RNA probes generated from a 645 base pair KpnI-BgIII fragment of a monkey Epo cDNA. Epo mRNA was demonstrated only in interstitial cells in the peritubular capillary bed of the hypoxic and normal monkey kidneys utilizing the antisense probe. The finding that the same type of cell that produces EPO in mice, rats and sheep also produces EPO in a higher primate species strongly supports the contention that renal interstitial cells also produce EPO in the human.

Amrinone as an antidote in experimental verapamil overdose.

OBJECTIVE: To evaluate the effect of amrinone as a treatment for the hemodynamic effects of verapamil overdose in a canine model. METHODS: This nonblind interventional study was performed in an established canine model of verapamil toxicity, without concurrent control animals. Pentobarbital-anesthetized and instrumented dogs (n = 8) were maintained and observed for 60 minutes or until death. The animals were overdosed with verapamil, 15 mg/ kg IV, over 30 minutes. Hemodynamic parameters, including cardiac index (CI), heart rate (HR), and mean arterial pressure (MAP), were monitored. Completion of the verapamil infusion represented the defined point of toxicity; at that point, all the animals received an amrinone bolus of 2 mg/kg IV over 2 minutes followed by an amrinone drip at 10 micrograms/kg/min. The hemodynamic values at the defined point of toxicity were compared with those obtained postinitiation of the amrinone infusion. RESULTS: Two animals died before the 60-minute observation period elapsed. Baseline CI was 5.6 L/min/m2. Following verapamil-induced toxicity, mean CI was 2.2 L/min/m2. After administration of amrinone, a significant (p < 0.05) increase in CI was observed at 30 minutes (CI = 3.6 L/min/m2), 45 minutes (CI = 4.2 L/ min/m2), and 60 minutes (CI = 4.2 L/min/m2). There was no statistically significant difference noted for MAP or HR compared with "point of toxicity" values. CONCLUSION: Amrinone appears to reverse the depressed cardiac index associated with verapamil overdose in a canine model while having no significant effect on the hypotension or bradycardia.

Cardiac dysrhythmias in severe verapamil overdose: characterization with a canine model.

Verapamil overdose, because of its frequency and severity, represents a significant problem for the emergency physician. With recent search recommending specific therapies for verapamil toxicity, aids to rapid diagnosis hold promise for decreasing morbidity and mortality from overdose of all calcium channel blockers. At this time, diagnosis of verapamil toxicity depends primarily on patient history and identification of cardiac dysrhythmias. This study attempts to improve the diagnostic armamentarium available for verapamil poisoning by analysing cardiac conduction problems seen in a canine model of verapamil toxicity, with the goal of identifying clinically useful dysrhythmia patterns. In 43 verapamil-toxic animals, junctional rhythm without organized atrial activity was the most frequently identified rhythm (55.6%). The next most commonly seen rhythms were tertiary atrioventricular (AV) block (16.3%) and idioventricular rhythm (11.6%); other animals manifested low grade AV block. Of interest, prominent U waves were noted in 25.6% of animals. While these results are subject to the limitations inherent in the use of an animal model, the data generated provide potentially useful patterns of dysrhythmia which may be encountered in humans with verapamil toxicity.

Glucagon and phenylephrine combination vs glucagon alone in experimental verapamil overdose.

OBJECTIVE: To evaluate glucagon and phenylephrine in combination as a treatment for the hemodynamic effects of verapamil overdose. METHODS: Pentobarbital-anesthetized and instrumented dogs were overdosed using a previously developed verapamil overdose model (15 mg/kg IV over 30 minutes). The animals were maintained and observed for 90 minutes or until death. Cardiac output (CO), heart rate (HR), and mean arterial pressure (MAP) were monitored. Following the 30-minute verapamil infusion (toxicity), the control animals received no treatment; the glucagon animals received a 5-mg glucagon bolus and a drip of 5 mg/90 minutes; and the glucagon/phenylephrine animals received the same glucagon therapy plus a phenylephrine drip titrated to 180 micrograms/min over 15 minutes. The groups were compared using analysis of variance: the experimental variables were group and time; the response variables were changes from toxicity for the hemodynamic parameters. Post-hoc comparisons were done with alpha set at 0.05. RESULTS: A significant change in CO was seen in the glucagon group (delta = 2.6 L/min) and the glucagon/phenylephrine group (delta = 1.9 L/min) compared with the control group (delta = 0.8 L/min). The change in CO was significantly larger for the glucagon animals compared with the glucagon/phenylephrine animals. The change in MAP for the glucagon/phenylephrine group (delta = 24 mm Hg) was significant compared with the control group (delta = 14 mm Hg). The MAP change for the glucagon group (delta = 19 mm Hg) was not significantly different from that of either the control or the glucagon/phenylephrine group. The change in glucagon HR (delta = 6 beats/min) was significant compared with the control group (delta = -4 beats/min) and the glucagon/phenylephrine group (delta = -4 beats/min). CONCLUSION: The glucagon/phenylephrine therapy improved MAP compared with the control, but reduced CO and HR compared with glucagon alone. Glucagon/phenylephrine therapy is not as effective as glucagon alone in reversing the hemodynamic effects of experimental verapamil overdose.

Rate of patient workups by non-emergency medicine residents in an academic emergency department.

OBJECTIVE: To quantify the number of patients seen per hour by non-emergency medicine (non-EM) residents in a university hospital ED. METHODS: This retrospective observational study was performed in a university hospital ED and level I trauma center. The facility had no EM residency, but was staffed with 24-hour EM faculty coverage. A computerized tracking system was searched for the number of patients seen by each of 93 non-EM residents for 12 nonconsecutive months. The ED schedule for each month was used to calculate the number of hours worked by each resident. From these figures, the number of patients seen per hour by each resident was calculated. RESULTS: The postgraduate years of training of the residents were as follows: 78 (84%) were PGY1, ten (11%) were PGY2, and five (5%) were PGY3. All the residents combined saw a mean 0.95 +/- 0.20 patients/hour, with a range from 0.58 to 1.75 patients/hour. There was no significant difference between the numbers of patients seen when compared by specialty using the Tukey-Kramer test (alpha = 0.05). CONCLUSION: The rate at which non-EM residents work up patients is consistent with previously reported rates for EM residents.

Reading skills of dietetic interns and readability of dietetics literature.

OBJECTIVE: To assess and compare reading skills of dietetic interns with reading levels of internship references. DESIGN: A standardized reading test, the Nelson-Denny Reading Test, measured reading skills of entering dietetic interns over 7 years. A computerized readability program assessed the readability of references. SETTING: Dietetic internships in university and Veterans Affairs hospitals. SUBJECTS: Of 194 entering interns, 178 (92%) were included and 16 (89%) were omitted. MAIN OUTCOME MEASURES: Nelson-Denny percentile and grade equivalent scores for vocabulary, comprehension, and total. The Fog Index identified reference reading-grade levels. STATISTICAL ANALYSES PERFORMED: Descriptive statistics and analysis of variance. RESULTS: Interns from the two programs did not differ significantly on Nelson-Denny Reading Test scores or in application grade point average. Percentile means and standard deviations were 54.7 +/- 23.8 for vocabulary, 51.2 +/- 25.0 for comprehension, 52.9 +/- 23.9 for total, and 41.6 +/- 24.7 for reading rate. Nearly 20% (33 of 178) of interns read significantly below expected grade level. The fog Index assigned reference grade levels from 6.98 to 21.63 years. CONCLUSIONS: The majority of dietetic interns have strong reading skills and read within the references' reading levels. A minority may experience difficulties reading assignments. Preinternship reading skills assessment could lead to greater success in reading professional literature.