A STD/HIV prevention trial among adolescents in managed care

OBJECTIVE: To determine if sexually transmitted diseases (STDs), including human immunodeficiency virus (HIV) infection, risk assessment, and education tools provided as part of office-based primary care reduce adolescent risky sexual behaviors. DESIGN: A randomized intervention trial with 3- and 9-month follow-up. SETTING: Five staff-model managed care sites in Washington, DC (n = 19 pediatricians). PATIENTS: Consecutive 12- to 15-year-olds receiving a general health examination; 81% minority. Participation rate = 215/432 (50%). Nine-month follow-up rate = 197/215 (92%). INTERVENTION: Audiotaped STD risk assessment and education about staying safe (safer = condoms, safest = abstinence). MAIN OUTCOME MEASURES: Adolescent-reported sexual intercourse and condom use. RESULTS: More intervention adolescents reported pediatrician discussion on 11/13 sexual topics. Although more vaginal intercourse (odds ratio [OR] = 2.46, 95% confidence interval [CI] = 1.04-5.84) was reported in the intervention group at 3 months, this was not true of overall sexual intercourse (OR = 1.55, 95% CI =.73-3.32). More sexually active adolescents reported condom use in the intervention group at 3 months (OR = 18.05, 95% CI = 1.27-256.03). At 9 months, there were no group differences in sexual behaviors; however, more signs of STD were reported by the control (7/103) than the intervention group (0/94). CONCLUSIONS: STD risk assessment and education tools administered in a single office visit facilitated STD/HIV prevention education. Any impact on sexual activity and condom use was short-lived. Further research is needed to develop brief, office-based sexual risk reduction for young adolescents.     

Spinal distribution and metabolism of 2'-O-(2-methoxyethyl)-modified oligonucleotides after intrathecal administration in rats

Intrathecal (IT) delivery of antisense oligodeoxynucleotides (ASO) has been used to study the function of specific gene products in spinal nociception. However, a lack of systematic studies on the spinal distribution and kinetics of IT ASO is a major hurdle to the utilization of this technique. In the present study, we injected rats IT with 2'-O-(2-methoxyethyl) modified phosphorothioate ASO (2'-O-MOE ASO) and examined anatomical and cellular location of the ASO in the spinal cord and dorsal root ganglia (DRG) by immunocytochemistry. At 0.5 h after a single IT injection, immunostaining for ISIS 13920 (a 2'-O-MOE ASO targeting h-ras) localized superficially in the lumbar spinal cord, while at 24 h the immunostaining was distributed throughout the spinal cord and was predominantly intracellular. Double staining with cell type specific antibodies indicated that the ASO was taken up by both glia and neurons. ASO immunoreactivity was also observed in DRG after IT ISIS 13920. Capillary gel electrophoresis analysis showed that ISIS 22703, a 2'-O-MOE ASO targeting the alpha isozyme of protein kinase C (PKC), remained intact in spinal cord tissue and cerebrospinal fluid up to 24 h after the injection and no metabolites were detected. In contrast, after IT ISIS 11300, an unmodified phosphorothioate ASO with the same sequence as ISIS 22703, no full-length compound was detectable at 24 h, and metabolites were seen as early as 0.5 h. IT treatment with ISIS 22703 at doses that effectively down-regulated PKCalpha mRNA in spinal cord did not affect the mRNA expression in DRG. In summary, 2'-O-MOE ASO displayed high stability in spinal tissue after IT delivery, efficiently distributed to spinal cord, and internalized into both neuronal and non-neuronal cells. ASO are able to reach DRG after IT delivery; however, higher doses may be required to reduce target gene in DRG as compared with spinal cord.     

Chaperonin-mediated assembly of wild-type and mutant subunits of human propionyl-CoA carboxylase expressed in Escherichia coli

We developed a bacterial expression system for the human alpha and beta cDNAs of propionyl-CoA carboxylase (PCC). These cDNAs (less the putative mitochondrial matrix targeting presequences) were co-expressed in Escherichia coli on one plasmid vector with each cDNA having its own IPTG-inducible promoter. Only negligible amounts of active PCC were measured despite the presence of both alpha and beta subunits as indicated by Western blot analysis and the almost complete biotinylation of the alpha subunit. Co-expression of this plasmid with a second plasmid vector over-expressing the E. coli chaperonin proteins, groES and groEL, resulted in a several hundred-fold increase in PCC specific activity, to a level comparable with that found in crude human liver extracts. PCC was partially purified on monomeric avidin affinity resin and the presence of both alpha and beta subunits was demonstrated, thereby confirming the assembly of both subunits into an active enzyme. Deficiency of either alpha PCC or beta PCC results in propionic acidemia, an autosomal recessive disorder. We used this expression system to characterize one missense mutation previously described in five Japanese alleles, namely C1283T (Thr428lle) in beta PCC. This mutation, when expressed in E.coli under the same conditions as that of wild-type PCC, had null activity, despite the presence of assembled alpha PCC and beta PCC subunits. This bacterial expression system can be useful for analysis of either alpha PCC or beta PCC mutations. Our findings indicated that the groES and groEL chaperonin proteins were essential for folding and assembly of the human PCC heteromeric subunits.      

Spinal antinociceptive action of loperamide is mediated by opioid receptors in the formalin test in rats

Opioids like morphine produce antinociception after intrathecal administration. Being hydrophilic in nature, morphine also spreads rostrally which leads to respiratory depression. Loperamide has been reported to produce antinociception after both intracisternal and intrathecal administration. It is also hydrophobic, which could restrict its diffusion in the spinal canal. However, the mechanism of its antinociceptive action after intrathecal administration is not definitely known. In the present study, the antinociceptive effect of loperamide was evaluated by the formalin test. It significantly inhibited Phase II flinching behavior. This antinociceptive effect was reversed by pre-administration of naloxone indicating that it was predominantly due to activation of opioid receptors.     

Spinal N-methyl-D-aspartate receptors and nociception-evoked release of primary afferent substance P

Dorsal horn N-methyl-D-aspartate (NMDA) receptors contribute significantly to spinal nociceptive processing through an effect postsynaptic to non-primary glutamatergic axons, and perhaps presynaptic to the primary afferent terminals. The present study sought to examine the regulatory effects of NMDA receptors on primary afferent release of substance P (SP), as measured by neurokinin 1 receptor (NK1r) internalization in the spinal dorsal horn of rats. The effects of intrathecal NMDA alone or in combination with D-serine (a glycine site agonist) were initially examined on basal levels of NK1r internalization. NMDA alone or when co-administered with D-serine failed to induce NK1r internalization, whereas activation of spinal TRPV1 receptors by capsaicin resulted in a notable NK1r internalization. To determine whether NMDA receptor activation could potentiate NK1r internalization or pain behavior induced by a peripheral noxious stimulus, intrathecal NMDA was given prior to an intraplantar injection of formalin. NMDA did not alter the formalin-induced NK1r internalization nor did it enhance the formalin paw flinching behavior. To further characterize the effects of presynaptic NMDA receptors, the NMDA antagonists DL-2-amino-5-phosphonopentanoic acid (AP-5) and MK-801 were intrathecally administered to assess their regulatory effects on formalin-induced NK1r internalization and pain behavior. AP-5 had no effect on formalin-induced NK1r internalization, whereas MK-801 produced only a modest reduction. Both antagonists, however, reduced the formalin paw flinching behavior. In subsequent in vitro experiments, perfusion of NMDA in spinal cord slice preparations did not evoke basal release of SP or calcitonin gene-related peptide (CGRP). Likewise, perfusion of NMDA did not enhance capsaicin-evoked release of the two peptides. These results suggest that presynaptic NMDA receptors in the spinal cord play little if any role on the primary afferent release of SP.     

Overview and update on molecular testing in non-small cell lung carcinoma utilizing endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) samples

Lung carcinoma remains one of the most frequent and aggressive human neoplasms. Fortunately, in the last decades, the increasing knowledge of the molecular mechanisms leading to cancer development has allowed the use of targeted therapies with improvement of prognosis in many patients. Clinical management has also changed after the introduction of endobronchialultrasonographic bronchoscopy that allows a conservative staging of lung tumors, avoiding the need of mediastinoscopy for lymph node staging. Lung pathologists and cytopathologists are facing the challenge of giving the more comprehensive prognostic and predictive information with ever smaller tissue or cytological samples. The aim of this review is to summarize the molecular testing for non-small cell lung carcinoma and how pathologists can contribute to the patient's outcome with a conscious management of biological samples.     

Synthesis and biological activities of cyclic lanthionine enkephalin analogues: delta-opioid receptor selective ligands

The synthesis and biological test results of a series of enkephalin analogues incorporating the lanthionine modification are presented. The syntheses of four monosulfide-bridged analogues of enkephalins, Tyr-c[D-Ala(L)-Gly-Phe-D-Ala(L)]-OH (1a), Tyr-c[D-Val(L)-Gly-Phe-D-Ala(L)]-OH (1b), Tyr-c[D-Ala(L)-Gly-Phe-Ala(L)]-OH (1c), and Tyr-c[D-Val(L)-Gly-Phe-Ala(L)]-OH (1d), where Ala(L) and Val(L) denote the lanthionine amino acid ends linked by a monosulfide bridge to form the lanthionine structure, were successfully carried out via preparation of the linear peptide on solid support and cyclization in solution. In vitro binding assays against mu-, delta-, and kappa-opioid receptors and in vitro tests using GPI and MVD assays revealed that the dimethyl lanthionine analogues 1b and 1d, denoted as D-Val(L) in position 2, show substantial selectivity toward the delta-opioid receptor, while the unsubstituted analogues 1a and 1c, denoted as D-Ala(L) in position 2, bind to both mu- and delta-opioid receptors. The in vivo thermal escape assay by intrathecal administration showed that the analogues 1b and 1d are among the most potent ligands at producing antinociception through the delta-opioid receptor. The picomolar potencies of analogues 1a and 1c in the intrathecal (it.) assay strongly indicate that mu- and delta-opioid receptors interact synergistically to modulate the antinociceptive responses.