Innate antimicrobial peptide protects the skin from invasive bacterial infection.

In mammals, several gene families encode peptides with antibacterial activity, such as the beta-defensins and cathelicidins. These peptides are expressed on epithelial surfaces and in neutrophils, and have been proposed to provide a first line of defence against infection by acting as 'natural antibiotics'. The protective effect of antimicrobial peptides is brought into question by observations that several of these peptides are easily inactivated and have diverse cellular effects that are distinct from antimicrobial activity demonstrated in vitro. To investigate the function of a specific antimicrobial peptide in a mouse model of cutaneous infection, we applied a combined mammalian and bacterial genetic approach to the cathelicidin antimicrobial gene family. The mature human (LL-37) and mouse (CRAMP) peptides are encoded by similar genes (CAMP and Cnlp, respectively), and have similar alpha-helical structures, spectra of antimicrobial activity and tissue distribution. Here we show that cathelicidins are an important native component of innate host defence in mice and provide protection against necrotic skin infection caused by Group A Streptococcus (GAS).

Stability of dapsone in two oral liquid dosage forms.

OBJECTIVE: Dapsone use in pediatric patients is increasing; however, the currently available tablet dosage form cannot be used in young children. The objective of our study was to determine the stability of dapsone in two oral suspensions stored at two temperatures. METHODS: Commercially available dapsone tablets (25 mg) were used to prepare the suspensions: the first in simple syrup and water with citric acid, the second in 1:1 Ora Sweet:Ora Plus to yield a concentration of 2.0 mg/mL. The dosage forms were stored in 10 amber plastic prescription bottles. Five were stored at 25 degrees C and five at 4 degrees C. Three samples were taken from each of five bottles at 0, 7, 14, 28, 42, 56, 70, and 91 days (n = 15). Dapsone concentrations in each sample were measured in duplicate by a validated and stability-indicating HPLC method; the pH of each sample was also determined. The drug was considered stable if the mean concentration > or =90% of the original concentration. RESULTS: The mean concentrations of dapsone were >95% of the initial concentrations for 91 days at both 4 degrees C and 25 degrees C in each suspension. There was a slight darkening of the samples stored at 25 degrees C. CONCLUSIONS: Dapsone was stable in two suspensions prepared from commercially available tablets for at least three months at 4 degrees C and 25 degrees C.

Stability of propylthiouracil in extemporaneously prepared oral suspensions at 4 and 25 degrees C.

The stability of propylthiouracil in two extemporaneously prepared suspensions at 4 and 25 degrees C was studied. Commercially available 50-mg propylthiouracil tablets were used to prepare suspensions in 1:1 Ora-Sweet:Ora-Plus and in 1:1 1% methylcellulose:Simple Syrup, NF, to yield a propylthiouracil concentration of 5 mg/mL. Each suspension was stored in 10 amber plastic prescription bottles, 5 of them at 4 degrees C and the other 5 at 25 degrees C. Three samples were taken from each bottle at 0, 7, 14, 28, 42, 56, 70, and 91 days for assay by stability-indicating high-performance liquid chromatography. More than 90% of the initial propylthiouracil concentration was retained in both suspensions for 70 days at 25 degrees C and for 91 days at 4 degrees C. There were no changes in physical appearance, color, or odor, and the pH remained essentially unchanged. Propylthiouracil 5 mg/mL in two extemporaneously prepared oral suspensions was stable for at least 70 days at 25 degrees C and for at least 91 days at 4 degrees C.

Candida lambica polyarthritis in a patient with chronic alcoholism.

We describe a patient with an indolent polyarthritis over a period of several years caused by Candida lambica probably acquired from a contaminated wound. C. lambica has not been previously reported to cause infectious arthritis. Hematogenous spread was manifest by 4 separate sites of involvement. Chronic alcoholism was the only apparent risk factor for dissemination. The infection seems to be environmentally acquired.

Potentiation of human estrogen receptor alpha transcriptional activation through phosphorylation of serines 104 and 106 by the cyclin A-CDK2 complex.

Both estradiol binding and phosphorylation regulate transcriptional activation by the human estrogen receptor alpha (ER). We have previously shown that activation of the cyclin A-CDK2 complex by overexpression of cyclin A leads to enhanced ER-dependent transcriptional activation and that the cyclin A-CDK2 complex phosphorylates the ER N-terminal activation function-1 (AF-1) between residues 82 and 121. Within ER AF-1, serines 104, 106, and 118 represent potential CDK phosphorylation sites, and in this current study, we ascertain their importance in mediating cyclin A-CDK2-dependent enhancement of ER transcriptional activity. Cyclin A overexpression does not enhance transcriptional activation by an ER derivative bearing serine-to-alanine changes at residues 104, 106, and 118. Likewise, the cyclin A-CDK2 complex does not phosphorylate this triple-mutated derivative in vitro. Individual serine-to-alanine mutations at residues 104 and 106, but not 118, decrease ER-dependent transcriptional enhancement in response to cyclin A. The same relationship holds for ER phosphorylation by cyclin A-CDK2 in vitro. Finally, enhancement of ER transcriptional activation by cyclin A is evident in the absence and presence of estradiol, as well as in the presence of tamoxifen, suggesting that the effect of the cyclin A-CDK2 on ER transcriptional activation is AF-2-independent. These results indicate that the enhancement of ER transcriptional activation by the cyclin A-CDK2 complex is mediated via the AF-1 domain by phosphorylation of serines 104 and 106. We propose that these residues control ER AF-1 activity in response to signals that affect cyclin A-CDK2 function.

Regulation of estrogen receptor transcriptional enhancement by the cyclin A/Cdk2 complex.

We have found that ectopic expression of cyclin A increases hormone-dependent and hormone-independent transcriptional activation by the estrogen receptor in vivo in a number of cell lines, including HeLa cells, U-2 OS osteosarcoma cells and Hs 578Bst breast epithelial cells. This effect can be further enhanced in HeLa cells by the concurrent expression of the cyclin-dependent kinase activator, cyclin H, and cdk7, and abolished by expression of the cdk inhibitor, p27(KIP1), or by the expression of a dominant negative catalytically inactive cdk2 mutant. ER is phosphorylated between amino acids 82 and 121 in vitro by the cyclin A/cdk2 complex and incorporation of phosphate into ER is stimulated by ectopic expression of cyclin A in vivo. Together, these results strongly suggest a direct role for the cyclin A/cdk2 complex in phosphorylating ER and regulating its transcriptional activity.

Glucocorticoid receptor-mediated cell cycle arrest is achieved through distinct cell-specific transcriptional regulatory mechanisms.

Glucocorticoids inhibit proliferation of many cell types, but the events leading from the activated glucocorticoid receptor (GR) to growth arrest are not understood. Ectopic expression and activation of GR in human osteosarcoma cell lines U2OS and SAOS2, which lack endogenous receptors, result in a G1 cell cycle arrest. GR activation in U2OS cells represses expression of the cyclin-dependent kinases (CDKs) CDK4 and CDK6 as well as their regulatory partner, cyclin D3, leading to hypophosphorylation of the retinoblastoma protein (Rb). We also demonstrate a ligand-dependent reduction in the expression of E2F-1 and c-Myc, transcription factors involved in the G1-to-S-phase transition. Mitogen-activated protein kinase, CDK2, cyclin E, and the CDK inhibitors (CDIs) p27 and p21 are unaffected by receptor activation in U2OS cells. The receptor's N-terminal transcriptional activation domain is not required for growth arrest in U2OS cells. In Rb-deficient SAOS2 cells, however, the expression of p27 and p21 is induced upon receptor activation. Remarkably, in SAOS2 cells that express a GR deletion derivative lacking the N-terminal transcriptional activation domain, induction of CDI expression is abolished and the cells fail to undergo ligand-dependent cell cycle arrest. Similarly, murine S49 lymphoma cells, which, like SAOS2 cells, lack Rb, require the N-terminal activation domain for growth arrest and induce CDI expression upon GR activation. These cell-type-specific differences in receptor domains and cellular targets linking GR activation to cell cycle machinery suggest two distinct regulatory mechanisms of GR-mediated cell cycle arrest: one involving transcriptional repression of G1 cyclins and CDKs and the other involving enhanced transcription of CDIs by the activated receptor.

Outpatient parenteral antibiotic therapy. Management of serious infections. Part II: Amenable infections and models for delivery. Pneumonia and chronic lung disease.

The need for medical support is the determining factor when selecting patients with acute pneumonia for outpatient therapy. Patients are often too old or too sick for early discharge, but a large subgroup can continue parenteral therapy as outpatients. Other pneumonia patients, as well as patients with infectious flares of chronic lung disease, can be treated with outpatient therapy alone.

What's new in the management of infectious diseases.

The 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) was held late last year in Anaheim, California. Sponsored by the American Society for Microbiology, the conference is designed to encourage the exchange of new information among microbiologists, clinicians, pharmacologists, pathologists, and members of related disciplines. The following highlights include those that may be of particular clinical interest to physicians in the oncology specialties.

Differential cerebrovascular responsiveness in spontaneously hypertensive rats following antihypertensive treatment with clonidine and verapamil.

Numerous studies have been reported examining the effects of antihypertensive treatment on peripheral vascular responsiveness in spontaneously hypertensive rats (SHR). This study was conducted to determine the effects of chronic treatment with 2 antihypertensive agents on cerebrovascular responsiveness in male SHR and Wistar-Kyoto (WKY) rats. SHR and WKY (3-4 weeks old) received either placebo, clonidine (CLON, 10 mg pellet) or verapamil (VER, 5 mg pellet). Vascular reactivity studies on the basilar artery, using standard smooth muscle bath techniques, were conducted following 6 weeks of treatment. Both CLON and VER significantly attenuated the rise in blood pressure in SHR. Basilar artery responsiveness to KCl, serotonin (5-HT), and calcium were significantly increased whereas responses to acetylcholine (ACH), isoproterenol (ISO) and sodium nitroprusside (SNP) were significantly reduced in SHR compared to WKY. CLON had no effect on basilar artery responsiveness to either the contractile or relaxation agents in SHR. However, although responses to KCl, 5-HT and calcium were not affected by VER in SHR, VER significantly increased the responses to ACH, ISO and SNP. Neither CLON nor VER treatment affected basilar artery responsiveness to any of the agents in WKY. These data demonstrate that, even though CLON and VER have similar antihypertensive effects, differential effects of the 2 agents on cerebrovascular responsiveness in the SHR are apparent. This would suggest that the vascular effects of VER and CLON are dependent upon the mechanism of action of the agents and not simply due to prevention of the elevation in blood pressure.

Selective assisted hatching of human embryos.

Overall results of assisted hatching by zona drilling using acidic Tyrode's solution performed during three randomised trials in 330 in vitro fertilisation (IVF) patients are presented. It was demonstrated retrospectively and prospectively that assisted hatching by zona drilling was effective in embryos with thick zonae (> 15 microns). This procedure is called selective assisted hatching. In order to investigate whether the success rate of embryos with thin zonae (< 13 microns) can be improved further, a fourth trial was executed in 40 consenting patients. Embryos with thin zonae were left intact in one group (control), while the outside of zonae of similar embryos were thinned with acidic Tyrode's solution. Results thus far indicate that embryos with thin zonae do not benefit from this technique. Embryonic implantation (fetal heartbeat per embryo) was high (26%-27%) in both arms of the trial, probably as a result of selective zona drilling of low prognosis embryos with thick zonae. A method is presented for quantifying zona hardening in human embryos. The exposure to acidic Tyrode's solution of each embryo was expressed as a function of the duration to pierce the zona and the diameter of the needle. Preliminary findings suggested that embryonic viability is correlated with zona hardening. In order to test the hypothesis that extracellular fragments may affect embryonic viability, small amounts of fragments were removed from embryos during assisted hatching. The pregnancy rate in 36 patients with extracted fragments was relatively high (41%) considering the poor morphology of the embryos involved.

Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis.

Assisted hatching by zona drilling using acidic Tyrode's solution was performed during three randomized trials in 330 in-vitro fertilization patients. The trials were designed in order to study the overall effect of the procedure and whether characteristic patient [i.e. maternal age and basal levels of follicle stimulating hormone (FSH)] and embryonic features (i.e. zona pellucida thickness) are important for the decision to perform assisted hatching routinely. Couples (n = 137; Trial 1) in whom the female partner had normal basal FSH levels were randomized in a control group (without micromanipulation) and a zona drilling group (all embryos micromanipulated). The incidence of implantation (67/239; 28%) of zona-drilled embryos compared favourably with that of control embryos (49/229; 21%), but the difference was not significant. Retrospective analysis revealed that those embryos whose zonae were thicker than 15 microns were rescued. In order to test the validity of this finding, selective assisted hatching was performed on embryos with a poor prognosis in 163 other patients (Trial 2). The couples were randomized into a control group and a group in which embryos were selectively zona-drilled, based on zona thickness and other embryonic features. The rate of embryonic implantation in the selectively zona-drilled group was 25% (70/278), significantly (P less than 0.05) higher than that of control embryos (51/285; 18%). Although it was demonstrated retrospectively and prospectively that assisted hatching by zona drilling is effective in embryos with thick zonae (greater than or equal to 15 microns), patients whose embryos have thin (less than 13 microns) zonae may be jeopardized by the procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin-induced elevation of 1,2-diacylglycerol and stimulation of growth of FRTL-5 cells are not dependent on inositol lipid hydrolysis.

We reported that TSH, which stimulates cAMP accumulation and proliferation of FRTL-5 thyroid cells, chronically increases the 1,2-diacylglycerol (1,2-DG) content of FRTL-5 cells. Because activation of inositol lipid hydrolysis by a phospholipase-C enzyme would generate 1,2-DG, we compared the effects of TSH on inositol lipid metabolism to TSH-induced increases in 1,2-DG content and stimulation of cAMP accumulation and cell growth. Acute stimulation of inositol lipid hydrolysis did not occur with doses of 1000 microU/ml or lower, but did occur with TSH doses of 3000 microU/ml and higher, with rates between 1-4%/h. More importantly, in cells chronically exposed to TSH, the rate of inositol lipid hydrolysis was increased only at TSH doses of 10,000 microU/ml or greater, and the maximum rate was 4-5%/h. When cells were growth arrested by TSH deprivation, there was no change in the content of inositol phosphates or polyphosphoinositides. In contrast to the high doses of TSH required to stimulate inositol lipid hydrolysis, TSH-induced elevation of 1,2-DG content and stimulation of cAMP accumulation and growth occurred at physiological TSH concentrations, with minimal effective doses in the range of 1-10 microU TSH/ml, and half-maximally effective doses between 50-200 microU TSH/ml. These data suggest that inositol lipid hydrolysis does not mediate the proliferative response to TSH in FRTL-5 cells and is not the mechanism by which increases in 1,2-DG content occur at physiological TSH concentrations.

Coronary thrombosis and platelet/fibrin microemboli in death associated with acute myocardial infarction.

The frequency and clinical significance of platelet/fibrin microemboli in the microcirculation were investigated in 24 patients whose deaths (before and during hospital admission) were associated with acute myocardial infarction. An acute coronary thrombus was present in all the hearts. In nine hearts an acute thrombus was found in more than one major epicardial coronary artery. A total of 35 acute thrombi were found in the 24 hearts. Platelet/fibrin microemboli were found in 19 (79%) hearts. Eighteen patients died in hospital. The hearts of 16 of these cases showed microemboli; 16 had important arrhythmias or various forms of heart block; 13 showed acute pathological changes in the conduction system. Fourteen of the deaths in hospital were primarily the result of cardiogenic shock and four were primarily caused by arrhythmia. Six of the deaths that occurred before admission to hospital were regarded as being arrhythmic in origin. Three of these showed microemboli and the other three had acute pathological changes in the conduction system. Microemboli were found in two (24%) of 12 control hearts. Coronary thrombosis was found in most deaths caused by acute myocardial infarction and platelet/fibrin microemboli were present in the majority of such hearts. These may arise from the coronary thrombus in the larger upstream vessel supplying the microcirculation.

Distribution of bombesin-like peptides in the alimentary canal of several vertebrate species.

The objective of this study was to quantitate and characterize the variants of bombesin-like immunoreactivity in the alimentary canal of the rat, rabbit, hawk, owl, dog, monkey and human. Bombesin-like immunoreactivity was found throughout the entire gastrointestinal tract of all species studied. In the rat, the highest concentration of bombesin-like immunoreactivity was found in the colon. Gel chromatography showed that bombesin-like immunoreactivity corresponded to gastrin-releasing peptide (GRP-27) and GRP-10. In the dog, the greatest concentration of bombesin-like immunoreactivity was observed in the mucosal layer of the fundus, whereas the concentration of bombesin-like immunoreactivity in the muscle layer of the dog did not vary significantly from region to region. Gel chromatography showed that bombesin-like immunoreactivity in the dog corresponded to GRP-27, bombesin, GRP-10, and a smaller fragment. In the human, the concentration of bombesin-like immunoreactivity did not vary significantly from region to region in the mucosal and muscular layers. Gel chromatography of human fundal mucosa showed that bombesin-like immunoreactivity peaks occur in the regions of GRP-27, bombesin and GRP-10. These findings substantiate the observation that bombesin-like peptides play a variety of roles in the regulation of gut function.

Effect of bombesin and gastrin-releasing peptide on the release of gastric inhibitory polypeptide and insulin in rats.

The objective of this study was to determine whether bombesin- or gastrin-releasing peptide-induced release of insulin occurs before or after the release of gastric inhibitory polypeptide (GIP) in rats. The present results demonstrate that GIP release occurs before insulin release and suggest that bombesin-like peptides and GIP interact to stimulate insulin secretion.

Radioimmunoassay of pancreatic polypeptide in mammalian and submammalian vertebrates using a carboxyl-terminal hexapeptide antiserum.

Pancreatic polypeptide (PP) immunoreactivity in acid-ethanol extracts of the pancreas of representative species of mammals, birds, reptiles, amphibians, and fish was studied by a radioimmunoassay (RIA) that utilizes an antiserum which cross-reacts exclusively with the COOH-terminal hexapeptide of PP (CTPP). PP immunoreactivity in acid-ethanol extracts of rat nonpancreas tissues (stomach, duodenum, skeletal muscle, brain) was also examined. Significant concentrations of PP immunoreactivity were detected in the pancreatic extracts of all species, except fish. Appreciable quantities of PP immunoreactivity were also found in the stomach and duodenum of rats. In all cases, tissue extracts showed parallelism with reference PP (bovine) in the RIA. Gel chromatography (Sephadex G-50sf) of tissue extracts (rat, turtle) demonstrated a major peak of PP immunoreactivity, which eluted in the region of the reference PP. Salamander PP immunoreactivity eluted after bovine PP. In addition, the CTPP RIA can be applied to measure plasma levels of PP in rats, dogs, and humans. By using this PP RIA, we observed that plasma PP levels increase significantly in dogs (P less than 0.05) after intravenous administration of neurotensin. In rats, administration of intravenous bombesin resulted in a significant elevation of plasma PP.