Amniotic fluid alpha-fetoprotein determination at the time of genetic amniocentesis: has it outlived its usefulness?

The purpose of this retrospective study was to evaluate the utility of routine measurement of amniotic fluid alpha-fetoprotein levels at the time of second trimester genetic amniocentesis (mean gestational age, 17.3 weeks +/- 2.5 weeks standard deviation; median, 16.8 weeks; range, 15 to 22 weeks). During the study period 7174 patients underwent second trimester genetic amniocentesis. Outcome data were available in all cases. In 79 (1.1%) cases the amniotic fluid alpha-fetoprotein level was > or = 2.0 multiples of the median. Thirty-three of the 79 (42%) patients had normal ultrasonograms, and in 31 of 33 (94%) the amniotic fluid alpha-fetoprotein level was between 2.0 and 3.0 multiples of the median. Forty-six of the 79 (58%) patients had abnormal ultrasonographic findings, and of these, 82% were neural tube defects, abdominal wall defects, or cystic hygromas. Acetylcholinesterase was positive in 37 cases, all of which had abnormal ultrasonographic findings. None of the fetuses with negative findings on sonographic screening had detectable abnormalities at birth. In this study, with over 7000 patients, amniotic fluid alpha-fetoprotein and acetylcholinesterase levels did not increase the detection of fetal abnormalities. On the basis of these results, routine measurement of amniotic fluid alpha-fetoprotein level at the time of routine genetic amniocentesis (15 to 22 weeks) does not appear justified.     

Cytopathological analysis of sputum in patients with airflow obstruction and significant smoking histories

Advances in the understanding of lung cancer biology have led to observations that specific genetic changes occur in premalignant dysplasia. These observations have occurred predominantly in molecular studies of resected lung tumors and consequently, they may not be fully representative of those biological abnormalities characterizing premalignant lesions in individuals without overt lung cancer. Studies of premalignant epithelial cell biology and chemoprevention are needed in this patient subgroup. Such an initiative is now underway through the lung cancer Specialized Program of Research Excellence (SPORE) grant awarded to the University of Colorado Cancer Center (and affiliated institutions) by the National Cancer Institute. To identify participants for the early detection and chemoprevention trials of the Colorado SPORE, we initiated a sputum cytology screening program targeting persons with chronic obstructive pulmonary disease and smoking histories of 40 or more pack-years. During the first 26 months after activation of the screening program, sputum samples from 632 participants were evaluated. Of these, 533 (84%) of the subjects submitted specimens deemed adequate for cytopathological interpretation; 99 (16%) provided sputum samples unsuitable for cytodiagnosis. Of those participants who submitted adequate samples, 48% had cytodiagnoses of mild dysplasia, 26 % had moderate to severe dysplasia, and 2% presented with carcinoma in situ or invasive carcinoma. Logistic regression modeling was pursued to determine whether selected demographic and/or clinical status variables could be identified as statistically significant predictors of the specific cytological outcome to be expected (mild dysplasia, moderate dysplasia, and so forth). The only apparent associations found from both univariate and multivariate analyses were that the total number of pack-years of smoking history decreased with severity of cytodiagnosis and that those individuals with mild or moderate dysplasia were more likely to be ex-smokers than those with grades of regular metaplasia or lower. Based on the initial results of the Colorado SPORE sputum cytology screening program, we conclude that persons with chronic obstructive pulmonary disease and 40 or more pack-years of smoking history have a high prevalence of premalignant dysplasia detectable through sputum cytology and should be targeted for research programs focusing on lung cancer prevention, early detection, and exploratory biomarker studies.     

Genetic determinants of p53-induced apoptosis and growth arrest

Previous studies have suggested that expression of p53 in cancer cells can result in either growth arrest or apoptosis. Accordingly, expression of p53 in a series of colorectal cancer cell lines yielded growth arrest in some lines (A-lines) and apoptosis in others (D-lines). To investigate the basis of this difference, we evaluated the role of p21WAF1/Cip1, a known mediator of p53-induced growth arrest. Inactivation of p21 by homologous recombination converted an A-line to a D-line, suggesting that p21 could protect cells from apoptosis. However, examination of p53-induced p21 expression in naturally occurring D-lines and A-lines demonstrated that the induction of p21 could not account for the differential response to p53. Moreover, when a D-line was fused to an A-line, the resulting hybrid cells underwent apoptosis in response to p53, indicating that the apoptosis pathway was dominant over the growth arrest pathway. Therefore, the apoptotic response to p53 in colorectal cancer cells is modulated by at least two factors: p21-mediated growth arrest that can protect cells from apoptosis in A-cells, and trans-acting factors in D-cells that can overcome this protection, resulting in cell death.     

Glomangioma of the ankle simulating injury to the flexor hallucis longus: a case report

The glomangioma is one of a group of benign tumors of glomus body origin. Commonly found in the distal extremities, they may present with pain, tenderness to palpation, and sensitivity to temperature. Histologically, varying proportions of glomus cells, as well as vascular and smooth muscle elements, are found. Complete surgical excision is usually curative. We report on a glomangioma of the ankle in a 12-year-old male patient that simulated injury to the flexor hallucis longus tendon.     

Intestinal cell cycle regulations. Interactions of cyclin D1, Cdk4, and p21Cip1

OBJECTIVE: The p21Cip1 protein is a potent stoichiometric inhibitor of cyclin-dependent kinase activity, and p21Cip1 mRNA expression is localized to the nonproliferative compartment of the intestinal villus, suggesting an in vivo growth-inhibitory role in the gut. The authors determined whether nontransformed rat intestinal epithelial cells (IECs) underwent reversible cell cycle arrest by contact inhibition, and determined whether increases in the relative amount of p21 associated with cyclin D/Cdk4 protein complexes were associated with cell growth arrest. METHODS: Density arrest was achieved by prolonged culture IEC-6 in confluent conditions (5 or more days). Release from density arrest was achieved by detaching the cells from the culture plate and reseeding them at a 1:4 ratio. The DNA synthesis was estimated by [3H]-thymidine incorporation and expressed as mean plus or minus standard error of the mean (n = 4). Cyclin D1, Cdk4, and p21 mRNA and protein levels were determined by standard Northern and Western blot analyses, respectively. Cyclin D1, Cdk4, and p21 protein complex formation was analyzed by immunoprecipitating the complexes from cell lysates with an antibody to one of the constituents, followed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the precipitated complexes using antibodies to the other proteins. The kinase activity of the immunoprecipitated Cdk4 was determined using recombinant Rb as substrate. RESULTS: The IEC-6[3H]-thymidine incorporation was decreased 7.5-fold from day 1 confluence to day 7 of confluence. Twenty-four hours after release from density arrest, there was a 43-fold increase in [3H]-thymidine incorporation. Cyclin D1 and Cdk4 mRNA levels remained relatively constant during contact inhibition, whereas immunoblotting showed that the levels of cyclin D1 and Cdk4 proteins decreased by 70.9% and 68.7%, respectively, comparing day 3 with day 9 during density arrest. The levels of cyclin D1 increased 5.8-fold and Cdk4 increased by 4.4-fold by 24 hours after reseeding the day 9 density-arrested cultures, coincident with the increase in DNA synthesis. The amount of p21 associated with the cyclin D1 and Cdk4 complex in the density-arrested cells was 170% of that observed in the reseeded, proliferating cells. More important, the p21::Cdk4 ratio was 6.4-fold higher in the density-arrested (quiescent) cells as compared with rapidly proliferating cells by 24 hours after release from growth arrest. Recovery of Cdk4-dependent kinase activity occurred by 4 hours after release from growth arrest, coincident with decreased binding of p21 to the complex. CONCLUSIONS: Intestinal epithelial cells in culture can undergo density-dependent growth arrest. This process involves downregulation of cyclin D1 and Cdk4 at the level of protein expression, whereas the mRNA levels remain relatively unchanged. Further, during contact inhibition, there is more p21 associated with cyclin D1/Cdk4, which further contributes to the inhibition of the kinase complex. The authors also have shown that the process of contact inhibition is reversible, which may explain partly the ability of the intestinal epithelium to increase proliferative activity in response to injury.     

The production of macrophage inflammatory protein-1 alpha by human basophils

Macrophage inflammatory protein-1alpha (MIP-1alpha) has previously been shown to be produced by mononuclear cells, eosinophils, and neutrophils. Its production by basophils has not been investigated. The objective of this study was to investigate the production of MIP-1alpha by basophils. Peripheral blood basophils were separated by Percoll gradient centrifugation, cultured overnight, and processed for double immunocytochemistry using Abs against MIP-1alpha and FcepsilonRIalpha (alpha subunit of IgE receptor type 1). We demonstrated that basophils expressed immunoreactive MIP-1alpha upon stimulation with anti-IgE. Less than 5% of the basophils stained for MIP-1alpha without stimulation. The secretion of MIP-1alpha by basophils was studied by ELISA. In these experiments, basophils were further enriched to 65 to 99% (median, 86%) by a negative selection method. Basophils released MIP-1alpha when stimulated by Abs against IgE and FCepsilonRIalpha as well as IL-3 and the calcium ionophore, A23187. In parallel experiments, PBMC, eosinophils, and neutrophils did not produce MIP-1alpha in response to anti-IgE, but they did so in response to A23187. No MIP-1alpha release was detected in platelet preparations. Preincubation with IL-3 (15 min or 18 h) augmented anti-IgE-included basophil MIP-1alpha production. The secretion of MIP-1alpha by basophils was detectable shortly after stimulation and gradually increased over 24 h. Since MIP-1alpha has potent inflammatory and histamine-releasing activities, its production by basophils may indicate a positive feedback mechanism for allergic inflammation.     

Safety and efficacy of oral calcitriol (1,25-dihydroxyvitamin D3) for the treatment of psoriasis

Plaque-type psoriasis has been successfully treated with oral calcitriol, but there has been no long-term follow-up on the safety and efficacy of this calciotropic hormone for psoriasis. In a single centre study, patients were enrolled in an open trial to evaluate the efficacy and safety of oral calcitriol for psoriasis. Of the 85 patients who received oral calcitriol, 88.0% had some improvement in their disease; 26.5, 36.2 and 25.3%, had complete, moderate and slight improvement in their disease, respectively. The mean baseline psoriasis area severity index score (PASI) of 18.4 +/- 1.0 was reduced to 9.7 +/- 0.8 and 7.8 +/- 1.3 after 6 and 24 months on oral calcitriol therapy. Serum calcium concentrations and 24 h urinary calcium excretion increased by 3.9% and 148.2%, respectively, but were not outside the normal range. Bone mineral density remained unchanged. The clearance of creatinine decreased by 13.4% from baseline during the first 6 months of treatment, and thereafter, remained unchanged after 3 years of follow up. An evaluation of creatinine, inulin and paraaminohypurate (PAH) clearance was performed in eight patients. After 6 months on oral calcitriol, there was a 22.5% decline in creatinine clearance but no significant changes were observed in either inulin or PAH clearance, suggesting that calcitriol alters creatinine metabolism or secretion but does not affect renal function. Oral calcitriol is effective and safe for the treatment of psoriasis.     

Tyrosine phosphorylation of the Kv1.3 potassium channel

Kv1.3, a voltage-dependent potassium channel cloned from mammalian brain and T lymphocytes, contains multiple tyrosine residues that are putative targets for tyrosine kinases. We have examined the tyrosine phosphorylation of Kv1.3, expressed transiently in human embryonic kidney (or HEK) 293 cells, by endogenous and coexpressed tyrosine kinases. Tyrosine phosphorylation is measured by a strategy of immunoprecipitation followed by. Western blot analysis, using antibodies that specifically recognize Kv1.3 and phosphotyrosine. Coexpression of the constitutively active tyrosine kinase v-src, together with Kv1.3, causes a large increase in the tyrosine phosphorylation of the channel protein. This phosphorylation of Kv1.3 can be reversed by treatment with alkaline phosphatase before Western blot analysis. Coexpression with a receptor tyrosine kinase, the human epidermal growth factor receptor, also causes an increase in tyrosine phosphorylation of Kv1.3. The effects of endogenous tyrosine kinases were examined by treating Kv1.3-transfected cells with the specific membrane-permeant tyrosine phosphatase inhibitor pervanadate. Pervanadate treatment causes a time- and concentration-dependent increase in the tyrosine phosphorylation of Kv1.3. This increased tyrosine phosphorylation of Kv1.3 is accompanied by a time-dependent decrease in Kv1.3 current, measured by patch-clamp analysis with cell-attached membrane patches. The pervanadate-induced suppression of current and much of the channel tyrosine phosphorylation are eliminated by mutation of a specific tyrosine residue, at position 449 of Kv1.3, to phenylalanine. Thus, there is a continual phosphorylation and dephosphorylation of Kv1.3 by endogenous kinases and phosphatases, and perturbation of this constitutive phosphorylation/dephosphorylation cycle can profoundly influence channel activity.