Severe Dengue Epidemic, Sri Lanka, 2017.

In 2017, a dengue epidemic of unexpected magnitude occurred in Sri Lanka. A total of 186,101 suspected cases and 440 dengue-related deaths occurred. We conducted a comprehensive analysis of this epidemic by comparing national surveillance data for 2017 with data from the preceding 5 years. In all Sri Lanka districts, dengue incidence in 2017 increased significantly over incidence during the previous 5 years. Older schoolchildren and young adults were more clinically symptomatic than those at extremes of age. Limited virologic surveillance showed the dominant circulating variant was dengue virus type 2 cosmopolitan genotype in the most affected district. One quarter of total annual cases were reported 5 weeks after the southwest monsoon started. Changes in vector abundance were not predictive of the increased incidence. Direct government expenditures on dengue control activities in 2017 were US $12.7 million. The lessons learned from this outbreak are useful for other tropical nations facing increasing dengue incidence.

Hormone sensitive lipase mRNA in both monocyte and macrophage forms of the human THP-1 cell line.

The identity of the neutral cholesteryl ester hydrolase (CEH) in human monocyte/macrophages is uncertain. Prior studies indicate that hormone sensitive lipase (HSL) is a major CEH in mouse macrophages, and that HSL mRNA is present in human THP-1 monocytes. In the present study, HSL mRNA expression was examined in THP-1 cells as a function of differentiation status and cholesterol enrichment. By RT-PCR with primer pairs that span exon boundaries, HSL mRNA was demonstrated in THP-1 monocytes and phorbol-ester differentiated THP-1 macrophages. cDNA identities were confirmed by sequencing. By Northern blotting, with HSL cDNA as probe, THP-1 monocytes were found to contain HSL mRNA of approximately 3 and 3.9 kb. In THP-1 macrophages, the 3 kb mRNA was greatly diminished, while the level of the 3.9 kb mRNA was maintained. mRNA of approximately 3 and 3.9 kb are those expected of the 86-kDa (adipocyte) and 117-kDa (testicular) HSL isoforms, respectively. The presence of the testicular isoform mRNA was confirmed in THP-1 cells by amplification and sequencing of an isoform-specific cDNA. Additionally, Northern-blot comparisons showed that the 3 and 3.9 kb mRNA in THP-1 comigrated with the HSL mRNA in 3T3-L1 adipocytes and rat testis, respectively. The level of the 3.9 kb mRNA did not vary greatly with cholesterol enrichment. Thus, the HSL gene is transcribed in THP-1 cells both before and after differentiation into macrophages; after differentiation, the predominant mRNA is that for the 117-kDa isoform. This isoform is a CEH, and may mediate some CE turnover in THP-1 cells.

FGF receptor availability regulates skeletal myogenesis.

Fibroblast growth factors (FGFs) and their receptors are critical participants in embryonic development, including the genesis of skeletal, cardiac, and smooth muscle. FGF signaling is mediated through interactions between multiple FGF ligands and transmembrane tyrosine kinase receptors, resulting in activation of a number of signal transduction pathways. Skeletal myocytes express FGF ligands and receptors in a coordinated fashion, suggesting that these molecules participate in autocrine signaling in the myocyte. Endogenously produced FGF has been shown to inhibit myogenesis, but the role of FGF receptor availability in directing myocyte proliferation and differentiation has not been established. To determine the contribution of receptor availability to the regulation of myogenesis, receptor availability was either increased by expressing a full-length FGF receptor-1 or decreased by expressing a truncated FGF receptor-1 in cultured skeletal myocytes. Constitutive expression of a full-length FGF receptor-1 increased myocyte proliferation and delayed differentiation. Conversely, a reduction in functional FGF receptor signaling by expression of a truncated FGF receptor-1 decreased proliferation and enhanced differentiation of myocytes. These data demonstrate that FGF receptor availability plays a critical regulatory role in skeletal myogenesis.

Is oral albuterol effective for acute cough in non-asthmatic children?

Fifty-nine non-asthmatic children with acute cough were randomized to receive oral albuterol or placebo for 7 d. There was a similar, rapid rate of resolution of acute cough for the two groups, but more shaking or trembling in those treated with albuterol (5/30 vs 0/29; p = 0.05). In ambulatory children with acute cough who have no history of asthma and a normal chest examination, oral albuterol does not reduce the frequency or duration of cough.

Cholesteryl ester hydrolysis in J774 macrophages occurs in the cytoplasm and lysosomes.

The relationship of cholesteryl ester hydrolysis to the physical state of the cholesteryl ester in J774 murine macrophages was explored in cells induced to store cholesteryl esters either in anisotropic (ordered) inclusions or isotropic (liquid) inclusions. In contrast to other cell systems, the rate of cholesteryl ester hydrolysis was faster in cells containing anisotropic inclusions than in cells containing isotropic inclusions. Two contributing factors were identified. Kinetic analyses of the rates of hydrolysis are consistent with a substrate competition by co-deposited triglyceride in cells with isotropic inclusions. In addition, hydrolysis of cholesteryl esters in cells with anisotropic droplets is mediated by both cytoplasmic and lysosomal lipolytic enzymes, as shown by using the lysosomotropic agent, chloroquine, and an inhibitor of neutral cholesteryl ester hydrolase, umbelliferyl diethylphosphate. In cells containing anisotropic inclusions, hydrolysis was partially inhibited by incubation in media containing either chloroquine or umbelliferyl diethylphosphate. Together, chloroquine and umbelliferyl diethylphosphate completely inhibited hydrolysis. However, when cells containing isotropic inclusions were incubated with umbelliferyl diethylphosphate, cholesteryl ester hydrolysis was completely inhibited, but chloroquine had no effect. Transmission electron microscopy demonstrated a primarily lysosomal location for lipid droplets in cells with anisotropic droplets and both non-lysosomal and lysosomal populations of lipid droplets in cells with isotropic droplets. These results support the conclusion that there is a lysosomal component to the hydrolysis of stored cholesteryl esters in foam cells.

Monitoring heparin therapy: APTT results from partial- vs full-draw tubes.

Results of the activated partial thromboplastin time (APTT) test can be influenced by biologic, preanalytic, and analytic variables. When a patient is being treated with unfractionated heparin, the correct interpretation of the APTT test result is essential. Laboratories should evaluate all variables influencing this result, particularly when determining the "therapeutic" range for heparin treatment. This study compared the APTT results assayed on specimens drawn into 2 different types of evacuated blood collection tubes. A statistically significant difference was seen in the results when the APTT was outside the reference interval, including results in the therapeutic range for unfractionated heparin. When the therapeutic range is determined by the laboratory, the evacuated blood collection tube system must be standardized.

Nighttime dosing assures postdistribution sampling for therapeutic drug monitoring of digoxin.

To study the appropriateness of phlebotomy for digoxin therapeutic drug monitoring (TDM) in outpatients, we conducted a retrospective chart review, a computer search of all previous TDM testing, and a questionnaire of all outpatients (n = 86) who had serum digoxin determinations between April 10 and April 28, 1992 (585 tests). In patients who took digoxin at the same time daily (40 patients, 300 tests), 52% of tests were performed on inappropriate samples drawn within 6 h of the last dose. No patient who took digoxin after 1700 had inappropriate tests. Phlebotomy for serum digoxin determinations before distribution of digoxin is complete is a common problem in outpatients, leading to clinically uninterpretable test results. Postdistribution sampling can be assured by nighttime dosing, and this recommendation has been implemented at our hospital.

cAMP stimulates cholesteryl ester clearance to high density lipoproteins in J7774 macrophages.

The regulation by cAMP of cholesteryl ester hydrolysis and net depletion of cellular cholesteryl ester (cholesteryl ester clearance) in J774 murine macrophages was explored. Using Sandoz 58035 to selectively inhibit acyl CoA:cholesterol acyltransferase, we showed that the absolute rate of cholesteryl ester hydrolysis was stimulated 2-fold in J774 cells by the cAMP analogues 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate and dibutyryl-cAMP. The rate of hydrolysis was also stimulated by prostaglandin E1, by cholera toxin, and by a mixture of forskolin and isobutylmethylxanthine, but was not affected by epinephrine or dibutyryl-cGMP. These data demonstrate that cholesteryl ester hydrolysis in J774 cells can be stimulated by cAMP-dependent protein kinase. Cholesteryl ester clearance from J774 cells was achieved upon incubation with high density lipoproteins (HDL) plus CPT-cAMP but not with HDL alone. HDL-mediated cholesteryl ester clearance was dependent on the concentration of both HDL and CPT-cAMP. The data suggest that the defect responsible for the lack of HDL-mediated cholesteryl ester clearance in J774 cells involves a failure to modulate cAMP levels.

Inhibitors of neutral cholesteryl ester hydrolase.

p-Nitrophenyl N-butyl, N-octyl, and N-dodecyl carbamates and a newly synthesized diethyl phosphate compound were studied as potential inhibitors of the cholesteryl ester hydrolases of Fu5AH rat hepatoma cells. Whole homogenates of Fu5AH cells were used as an enzyme source for the assay of cholesteryl ester hydrolase activity. All four compounds led to marked inhibition (70-80%) of neutral cholesteryl ester hydrolase activity (assayed at pH 7) at concentrations where the activity of acid cholesteryl ester hydrolase (assayed at pH 4) was unaffected. Cholesteryl ester hydrolysis was also evaluated in intact cultured cells induced to accumulate cholesteryl esters in cytoplasmic lipid droplets by exposure to cholesterol-rich phospholipid dispersions. Hydrolysis was then assessed during subsequent incubations in the presence of an inhibitor of cholesterol esterification. All compounds caused significant inhibition of cholesterol ester hydrolysis with the diethyl phosphate being the most effective. At a concentration that caused greater than 90% inhibition of the hydrolysis of cytoplasmic cholesteryl esters, the compound had only a minimal effect on lysosomal hydrolysis of cholesteryl esters. These results suggest that diethyl phosphates and N-alkylcarbamates may be of value in future studies on the substrate specificities, regulation, and physiological role(s) of cholesteryl ester hydrolases.

Potential problems in the use of commercial preparations of radiolabeled cholesterol.

Through a series of biological and analytical procedures, we demonstrate that a compound purchased from a commercial supplier as [7-3H]cholesterol was not cholesterol. In mouse peritoneal macrophages, this compound was metabolized differently than other radiolabeled cholesterol preparations and was accumulated in the steryl ester pool. In contrast, Fu5AH rat hepatoma cells did not discriminate this compound from cholesterol. Further analysis of the anomalous [7-3H]cholesterol by TLC after cholesterol oxidase treatment and by HPLC indicated that this radiochemical was less polar than cholesterol standard and other radiolabeled cholesterol preparations tested. Mass spectrometry analysis disclosed that the chemical has a similar fragmentation pattern and the same molecular weight (386) as cholesterol.

Influence of high density lipoprotein on esterified cholesterol stores in macrophages and hepatoma cells.

The ability of high density lipoproteins (HDL) to induce the clearance of cholesteryl esters from cultured cells has been explored. Studies using the J774 mouse macrophage cell line showed that these cells are not stimulated to clear esterified cholesterol upon exposure to HDL. This was observed over a wide range of HDL concentrations (10 to 1000 micrograms/ml HDL protein), and the lack of stimulation was not influenced by a number of factors relating to the preparation of the HDL, such as HDL subfraction, varying extents of lecithin:cholesterol acyltransferase modification, or heparin-Sepharose chromatography to remove particles containing apo E. Neither the method of loading the cells with esterified cholesterol nor the physical state of the lipid droplets affected the inability of HDL to elicit esterified cholesterol clearance. In the presence of the acyl CoA:cholesterol acyltransferase inhibitor, Sandoz 58-035, where a high level of intracellular free cholesterol was generated, efflux of only a small fraction of the excess free cholesterol to HDL was observed. J774 cells were able to clear esterified cholesterol efficiently in the presence of cholesterol-free apolipoprotein HDL/phospholipid particles, indicating that the cells have the capacity to clear esterified cholesterol. Fu5AH hepatoma cells and P388.D1 mouse macrophage cells also failed to clear esterified cholesterol in response to HDL. In contrast, mouse peritoneal macrophages cleared esterified cholesterol efficiently to HDL, indicating that there are fundamental differences between mouse peritoneal macrophages and the other cells types studied in regard to cholesterol metabolism as influenced by HDL.