Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.

A deficiency in microsomal triglyceride transfer protein (MTP) causes the human lipoprotein deficiency syndrome abetalipoproteinemia. However, the role of MTP in the assembly and secretion of VLDL in the liver is not precisely understood. It is not clear, for instance, whether MTP is required to move the bulk of triglycerides into the lumen of the endoplasmic reticulum (ER) during the assembly of VLDL particles. To define MTP's role in hepatic lipoprotein assembly, we recently knocked out the mouse MTP gene (Mttp). Unfortunately, achieving our objective was thwarted by a lethal embryonic phenotype. In this study, we produced mice harboring a "floxed" Mttp allele and then used Cre-mediated recombination to generate liver-specific Mttp knockout mice. Inactivating the Mttp gene in the liver caused a striking reduction in VLDL triglycerides and large reductions in both VLDL/LDL and HDL cholesterol levels. The Mttp inactivation lowered apo B-100 levels in the plasma by >95% but reduced plasma apo B-48 levels by only approximately 20%. Histologic studies in liver-specific knockout mice revealed moderate hepatic steatosis. Ultrastructural studies of wild-type mouse livers revealed numerous VLDL-sized lipid-staining particles within membrane-bound compartments of the secretory pathway (ER and Golgi apparatus) and few cytosolic lipid droplets. In contrast, VLDL-sized lipid-staining particles were not observed in MTP-deficient hepatocytes, either in the ER or in the Golgi apparatus, and there were numerous cytosolic fat droplets. We conclude that MTP is essential for transferring the bulk of triglycerides into the lumen of the ER for VLDL assembly and is required for the secretion of apo B-100 from the liver.

Defining the atherogenicity of large and small lipoproteins containing apolipoprotein B100.

Apo-E-deficient apo-B100-only mice (APOE:(-/-)APOB:(100/100)) and LDL receptor-deficient apo-B100-only mice (LDLR:(-/-)APOB:(100/100)) have similar total plasma cholesterol levels, but nearly all of the plasma cholesterol in the former animals is packaged in VLDL particles, whereas, in the latter, plasma cholesterol is found in smaller LDL particles. We compared the apo-B100-containing lipoprotein populations in these mice to determine their relation to susceptibility to atherosclerosis. The median size of the apo-B100-containing lipoprotein particles in APOE:(-/-)APOB:(100/100) plasma was 53.4 nm versus only 22.1 nm in LDLR:(-/-)APOB:(100/100) plasma. The plasma levels of apo-B100 were three- to fourfold higher in LDLR:(-/-)APOB:(100/100) mice than in APOE:(-/-)APOB:(100/100) mice. After 40 weeks on a chow diet, the LDLR:(-/-)APOB:(100/100) mice had more extensive atherosclerotic lesions than APOE:(-/-)APOB:(100/100) mice. The aortic DNA synthesis rate and the aortic free and esterified cholesterol contents were also higher in the LDLR:(-/-)APOB:(100/100) mice. These findings challenge the notion that all non-HDL lipoproteins are equally atherogenic and suggest that at a given cholesterol level, large numbers of small apo-B100-containing lipoproteins are more atherogenic than lower numbers of large apo-B100-containing lipoproteins.

Effects of ibudilast on oxycodone-induced analgesia and subjective effects in opioid-dependent volunteers.

Opioid-induced glial activation is hypothesized to contribute to the development of tolerance to opioid-induced analgesia. This inpatient, double-blind, placebo-controlled, within-subject and between-groups pilot study investigated the dose-dependent effects of ibudilast, a glial cell modulator, on oxycodone-induced analgesia. Opioid-dependent volunteers were maintained on morphine (30mg, PO, QID) for two weeks and received placebo ibudilast (0mg, PO, BID) during the 1st week (days 1-7). On day 8, participants (N=10/group) were randomized to receive ibudilast (20 or 40mg, PO, BID) or placebo for the remainder of the study. On days 4 (week 1) and 11 (week 2), the analgesic, subjective, and physiological effects of oxycodone (0, 25, 50mg/70kg, PO) were determined. Analgesia was measured using the cold pressor test; participants immersed their hand in cold water (4°C) and pain threshold and pain tolerability were recorded. Oxycodone decreased pain threshold and tolerability in all groups during week 1. During week 2, the placebo group exhibited a blunted analgesic response to oxycodone for pain threshold and subjective pain ratings, whereas the 40mg BID ibudilast group exhibited greater analgesia as measured by subjective pain ratings (p≤0.05). Oxycodone also increased subjective drug effect ratings associated with abuse liability in all groups during week 1 (p≤0.05); ibudilast did not consistently affect these ratings. These findings suggest that ibudilast may enhance opioid-induced analgesia. Investigating higher ibudilast doses may establish the utility of pharmacological modulation of glial activity to maximize the clinical use of opioids.

Dose escalation in non-small-cell lung cancer using three-dimensional conformal radiation therapy: update of a phase I trial.

PURPOSE: High-dose radiation may improve outcomes in non-small-cell lung cancer (NSCLC). By using three-dimensional conformal radiation therapy and limiting the target volume, we hypothesized that the dose could be safely escalated. MATERIALS AND METHODS: A standard phase I design was used. Five bins were created based on the volume of normal lung irradiated, and dose levels within bins were chosen based on the estimated risk of radiation pneumonitis. Starting doses ranged from 63 to 84 Gy given in 2.1-Gy fractions. Target volumes included the primary tumor and any nodes >or= 1 cm on computed tomography. Clinically uninvolved nodal regions were not included purposely. More recently, selected patients received neoadjuvant cisplatin and vinorelbine. RESULTS: At the time of this writing, 104 patients had been enrolled. Twenty-four had stage I, four had stage II, 43 had stage IIIA, 26 had stage IIIB, and seven had locally recurrent disease. Twenty-five received chemotherapy, and 63 were assessable for escalation. All bins were escalated at least twice. Although grade 2 radiation pneumonitis occurred in five patients, grade 3 radiation pneumonitis occurred in only two. The maximum-tolerated dose was only established for the largest bin, at 65.1 Gy. Dose levels for the four remaining bins were 102.9, 102.9, 84 and 75.6 Gy. The majority of patients failed distantly, though a significant proportion also failed in the target volume. There were no isolated failures in clinically uninvolved nodal regions. CONCLUSION: Dose escalation in NSCLC has been accomplished safely in most patients using three-dimensional conformal radiation therapy, limiting target volumes, and segregating patients by the volume of normal lung irradiated.

Abnormal gastrocolic response in patients with intestinal pseudo-obstruction.

Gastrointestinal motility disturbances after endogenous stimuli have been demonstrated only in the esophagus and small intestine in patients with chronic idiopathic pseudo-obstruction. The 1,000-calorie meal stimulated a significant increase in spike and contractile activity in normal subjects. No increase in colonic spike or contractile activity occurred in patients with chronic idiopathic pseudo-obstruction even though colonic motility increased normally following the administration of neostigmine methylsulfate. These findings suggest that the normal response to eating is absent in patients with chronic idiopathic intestinal pseudo-obstruction even though their smooth muscle has been shown to be responsive to cholinergic stimulation. This loss of a gastrocolic response is further evidence for a disorder of the neurohumoral control mechanisms in patients with chronic idiopathic intestinal pseudo-obstruction.

83-kilodalton heat shock proteins of trypanosomes are potent peptide-stimulated ATPases.

A Crithidia fasciculata 83-kDa protein purified during a separate study of C. fasciculata trypanothione synthetase was shown to have ATPase activity and to belong to the hsp90 family of stress proteins. Because no ATPase activity has previously been reported for the hsp90 class, ATP utilization by C. fasciculata hsp83 was characterized: this hsp83 has an ATPase kcat of 150 min-1 and a Km of 60 microM, whereas the homologous mammalian hsp90 binds ATP but has no ATPase activity. Crithidia fasciculata hsp83 undergoes autophosphorylation on serine and threonine at a rate constant of 3.3 x 10(-3) min-1. Similar analysis was performed on recombinant Trypanosoma cruzi hsp83, and comparable ATPase parameters were obtained (kcat = 100 min-1, Km = 80 microM, kautophosphorylation = 6.3 x 10(-3) min-1). The phosphoenzyme is neither on the ATPase hydrolytic pathway nor does it affect ATPase catalytic efficiency. Both C. fasciculata and T. cruzi hsp83 show up to fivefold stimulation of ATPase activity by peptides of 6-24 amino acids.

Cloning and expression in Escherichia coli of a hypoxanthine-guanine phosphoribosyltransferase-encoding cDNA from Plasmodium falciparum.

The enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of Plasmodium falciparum plays a key role in the salvage of preformed purine nucleotides from parasite-infected erythrocytes. Since P. falciparum cannot synthesize purines de novo, development of inhibitors specific for the parasite HGPRT should be an effective method of chemotherapy. To provide sufficient amounts of HGPRT for biochemical and crystallographic analysis, we have isolated the P. falciparum HPRT cDNA sequence and expressed it in an Escherichia coli strain deficient for both de novo purine synthesis and guanine utilization (strain GP120). GP120 cells containing the P. falciparum HPRT plasmid vector (pRD500), when grown in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) which induces the tac promoter of the expression vector, produce a novel protein of 26 kDa, which is in agreement with the predicted Mr deduced from the HPRT cDNA open reading frame. In addition, we have demonstrated significant HGPRT activity in cell-free extracts of GP120[pRD500] cultures grown in minimal medium containing xanthine, as the sole source of purines, and IPTG.

New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments.

Two new shuttle vectors have been constructed by fusing the Escherichia coli plasmid pUC9 with the Staphylococcus aureus plasmids pU110 and pC194. The resulting hybrids replicate in both E. coli and Bacillus subtilis and contain seven restriction sites within a part of the lacZ gene. Insertion of foreign DNA into those sites can be easily detected in E. coli and hybrid plasmids can subsequently be transformed into B. subtilis.

A randomized, placebo-controlled dose-comparison trial of haloperidol for psychosis and disruptive behaviors in Alzheimer's disease.

OBJECTIVE: The goal of this study was to compare the efficacy and side effects of two doses of haloperidol and placebo in the treatment of psychosis and disruptive behaviors in patients with Alzheimer's disease. METHOD: In a 6-week random-assignment, double-blind, placebo-controlled trial (phase A), haloperidol, 2-3 mg/day (standard dose), and haloperidol, 0.50-0.75 mg/day (low dose), were compared in 71 outpatients with Alzheimer's disease. For the subsequent 6-week double-blind crossover phase (phase B), patients taking standard- or low-dose haloperidol were switched to placebo, and patients taking placebo were randomly assigned to standard- or low-dose haloperidol. RESULTS: For the 60 patients who completed phase A, standard-dose haloperidol was efficacious and superior to both low-dose haloperidol and placebo for scores on the Brief Psychiatric Rating Scale psychosis factor and on psychomotor agitation. Response rates according to three sets of criteria were greater with the standard dose (55%-60%) than the low dose (25%-35%) and placebo (25%-30%). The advantage of standard dose over low dose was replicated in phase B. In phase A, extrapyramidal signs tended to be greater with the standard dose than in the other two conditions, primarily because of a subgroup (20%) who developed moderate to severe signs. Low-dose haloperidol did not differ from placebo on any measure of efficacy or side effects. CONCLUSIONS: The results indicated a favorable therapeutic profile for haloperidol in doses of 2-3 mg/day, although a subgroup developed moderate to severe extrapyramidal signs. A starting dose of 1 mg/day with gradual, upward dose titration is recommended. The narrow therapeutic window observed with haloperidol may also apply to other neuroleptics used in Alzheimer's disease patients with psychosis and disruptive behaviors.

The dexamethasone suppression test: identification of subtypes of depression.

In this study mean 4 p.m. cortisol levels were significantly higher in patients with major depression than in control subjects or in patients with bipolar depression or dysthymic-related disorders. Moreover, the distribution of values differed significantly among groups. Eighteen of 45 patients with major depression had cortisol levels of 10 micrograms/dl or more, compared with 2 of 20 bipolar depressed patients and 0 of 31 controls. Patients with very high cortisol levels (15 micrograms/dl or more) tended to fulfill criteria for major depression with mood-congruent psychosis. The distribution of values in the major depression group also suggested the existence of three major subgroups. The authors discuss the implications of these data.

Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens.

A combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to identify phage clones capable of binding to the surface of Candida albicans blastoconidia. Single chain antibody variable fragments (scFv) derived from three clones detected C. albicans antigens by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blotting. The antigens detected were conserved among different strains of C. albicans and several other Candida species. Two scFv clones detected antigens specifically expressed by C. albicans blastoconidia; the third detected antigens in both blastoconidia and filamentous forms of C. albicans. The antigens containing the epitopes recognized by all three scFv could be extracted from blastoconidia by dithiothreitol, suggesting attachment to the cell wall via sulfhydryl bonds. Epitope detection by the scFv was sensitive to treatment of C. albicans blastoconidia with sodium periodate, but not proteinase K, indicating the cognate epitopes were composed of carbohydrate. Antigenic determinants for each of the three scFv were detected by immunohistochemical staining of skin sections from a model of cutaneous candidiasis, demonstrating expression in vivo. Through selection for the ability to bind intact organisms, the phage display system provides a means to rapidly identify monoclonal binding ligands to Candida surface antigens. Being entirely human, mature antibodies generated from the scFv have potential utility in the treatment of candidiasis.

Overall survival after prostate-specific-antigen-detected recurrence following conformal radiation therapy.

PURPOSE: To study the significance, in terms of overall and cause-specific survival, of biochemical failure after conformal external-beam radiation therapy (RT) for prostate cancer. METHODS AND MATERIALS: Of the 1844 patients in the Radiation Oncology prostate cancer database, 718 were deemed eligible. Patients excluded were those with N1 or M1 disease, those treated after radical prostatectomy, those who received hormone therapy before radiation therapy, and those who died, failed clinically, or had no PSA response in the first 6 months after RT. Patients included were required to have a minimum of 2 post-RT PSAs separated by at least 1 week. Biochemical relapse was defined as 3 consecutive PSA rises. This resulted in 154 patients with biochemical failure. Survival was calculated from the third PSA elevation. The rate of rise of PSA was calculated by fitting a regression line to the four rising PSAs on a ln PSA vs. time plot. RESULTS: There were 41 deaths among the 154 patients with failure in 23 of the 41 due to prostate cancer. The overall survival after failure was 58% at 5 years, while the cause-specific failure was 73% at 5 years. Among the 154 failures, several factors were evaluated for an association with overall survival: age at failure, pre-RT PSA, PSA at second rise, PSA nadir, time from RT to failure, time to nadir, Gleason score, T-stage, and rate of rise, both from the nadir and from the beginning of the rise. None of these factors were significantly associated with an increased risk of death. As expected, the group of patients with biochemical failure have significantly worse prognostic factors than those without biochemical failure: median pre-RT PSA 15.9 vs. 9.0 (p < 0.001), and Gleason score of 7 or greater for 48% of subjects vs. 40% (p = 0.1). Relative PSA rise and slope of ln PSA vs. time were associated with cause-specific mortality (p < 0.001 and p = 0.007, respectively). CONCLUSION: Overall survival after conformal radiotherapy for prostate cancer remains high 5 years after biochemical failure. This high survival rate occurs even though the group of patients with biochemical failure has worse than average adverse preradiation prognostic factors. Thus, although biochemical failure can identify patients who have recurrent disease after RT, the ultimate relationship between this endpoint and death remains to be better defined.

Expression and localization of Trypanosoma cruzi hsp60.

A 60-kDa heat shock protein (hsp60) is involved in mitochondrial protein folding and assembly of oligomeric protein complexes in the mitochondrial matrix. Here we report the isolation of Trypanosoma cruzi hsp60 cDNAs, the determination of the organization and chromosomal location of the genes, and the assessment of the heat-regulated expression and subcellular location of the protein. T. cruzi hsp60 is encoded by a multigene family organized in two allelic direct tandem arrays on a chromosome of 1.6 Mb. The regulation of hsp60 expression by heat is complex. While the hsp60 mRNA level is 6-fold higher at 37 degrees C than at either 26 degrees C, the hsp60 protein level remains essentially constant across all temperatures examined. Further analysis of the protein by two-dimensional immunoblotting revealed the existence of multiple isoforms that, with increasing temperature, shift in relative abundance from the more basic to the more acidic. A combination of immunofluorescence microscopy and cell fractionation was used to show that hsp60 is distributed throughout the matrix of the mitochondrion--a location distinct from that of the 70-kDa mitochondrial hsp, mtp70, which is associated with the kinetoplast.

Increasing the protection of newborn infants in cars.

Recently the American Academy of Pediatrics instituted a major campaign ("The First Ride--A Safe Ride") in order to encourage all parents to use an infant restraint seat for their newborn's first ride in an automobile--the ride home from the hospital. In the present study the effect of the behavior of the hospital staff on parents' use of infant restraint seats was examined. This study involved 30 mother-infant pairs who were selected sequentially from an obstetrics unit and randomly assigned to two groups. A control group was discharged from the obstetrics unit with no particular emphasis on car safety and no loaner restraint seat available. An experimental group was offered a loaner restraint seat at the time of discharge, with a staff person demonstrating how to put the infant into the restraint seat, how to carry the infant in the seat out to the car, and how to fasten the restraint seat in the automobile with the auto lap belt. Correct use of the loaner restraint seat on the first ride home was observed in 67% of the experimental mothers and in none (0%) of the control mothers. Although this difference was no longer significant at four- to six-week follow-up this study points out the short-term impact that hospital staff can have on the parents' use of restraint seats. Additional techniques are needed to maintain parents' use of restraint seats throughout childhood.

Advances in non-nicotine pharmacotherapy for smoking cessation.

Progress in understanding the pharmacological nature of tobacco addiction, along with the modest success rates achieved by the nicotine replacement therapies, has provided the major impetus for the development of non-nicotine drugs as smoking cessation aids. This article reviews evidence from controlled trials of several non-nicotine medications for the treatment of nicotine dependence. Clonidine was the first non-nicotine medication to show efficacy for smoking cessation in multiple studies, but its effect was found to be limited at best. Positive results across several trials have been consistently demonstrated for amfebutamone (bupropion). Encouraging results have also been observed for nortriptyline and moclobemide. Studies of combined treatments using non-nicotine medications (amfebutamone, mecamylamine, oral dextrose) with nicotine replacement therapy suggest increased efficacy relative to treatments using one or the other treatment strategy alone. Thus, available evidence indicates that non-nicotine drug treatments offer a promising panoply of therapeutic strategies for the addicted smoker.

Nutritional therapy in Crohn's disease.

The value of nutritional support in the prevention and treatment of malnutrition in Crohn's disease is undisputed but its role in primary therapy continues to be debated. Controlled trials have demonstrated that enteral nutrition induces remission rates comparable to that of corticosteroid therapy in Crohn's disease and remains the treatment of choice for specific subgroups such as children with signs of growth impairment and patients with intolerable steroid-induced side effects. The mechanism by which an enteral diet induces remission in Crohn's disease is unclear. Bowel rest, reduced antigenic load, nutritional effects, the provision of trophic amino acids, modification of gut flora, intestinal permeability, or fecal pH have been proposed. Equally, the fat profile of the feed may reduce pro-inflammatory ecosanoid synthesis and thus modify disease activity. Maintaining long-term remission remains a challenge in the management of this disease. Cyclic administration of enteral diets, maintenance drug therapy, fat manipulated formulas, or fish oil therapy may be strategies to prolong diet-induced remission. In the future, nutrient derivatives that play a role in the protective processes of the intestinal mucosa may have application in nutritional therapy in Crohn's disease.

Attention deficit/hyperactivity disorder and substance abuse. Diagnostic and therapeutic considerations.

Patterns of association between attention deficit hyperactivity disorder (ADHD) and substance-use disorders are considered. Recent investigations have found that up to 50% of individuals with continuing ADHD symptoms have a substance-use disorder. ADHD appears to represent an independent risk factor for substance abuse. We review clinical challenges posed by the diagnosis of ADHD in substance-abusing populations. Nicotine dependence is also substantially more common among adults with ADHD (40%) than in the general population (26%). While several classes of substances of abuse may ameliorate various symptoms of ADHD, individuals with ADHD may also be vulnerable to substance use because of poor judgment or impulsive behavior in social settings. Evidence is reviewed from genetic studies examining the role of the dopamine D2 (DRD2) gene in the etiology of ADHD. The presence of ADHD may affect the course of adolescent substance abuse in several ways: predicting earlier age of onset, longer duration of substance-use disorder, and progression of alcohol abuse to another drug-use disorder. Individuals with ADHD have been noted to have a shorter interval between the onsets of drug abuse and drug dependence. Such individuals are also at greater risk for treatment failure, as their disruptive behaviors interfere with treatment access and response. Lastly, we review advances in pharmacotherapeutic agents used for treating ADHD and consider the impact of these interventions on comorbid substance-use disorders. We suggest promising areas of focus for clinical research trials targeting the subpopulation of substance abusers with concurrent ADHD.

Fear of AIDS related to development of obsessive-compulsive disorder in a child.

Fear of acquired immune deficiency syndrome (AIDS) has been associated with a variety of psychiatric disorders in adults and in some adolescents. A case is reported of a child with an unrealistic fear of AIDS who developed an obsessive-compulsive disorder. This case illustrates the importance of providing adequate AIDS information to children.

Detection of dengue virus by in situ hybridization.

A non-radioactive in situ hybridization protocol was developed for the detection of dengue virus RNA in fixed tissues and cells. For this purpose a riboprobe was constructed from a 39 base sequence, from the capsid protein coding region of the genome, which is conserved in the four dengue serotypes. The ability of this probe to specifically detect dengue RNA from each serotype was confirmed on brain sections from infected mice. Dengue viral RNA was also detected in in vitro infected human primary endothelial cells which release infectious virus without showing gross cytopathic effect. With clinical samples dengue viral RNA was detected in some preparations of white blood cells from dengue fever patients and in thymus autopsy sections following suspected death from dengue shock syndrome. For dengue samples of undetermined serotype the sensitivity of the short probe was compared to that of an equimolar mixture of long (260 base) probes from the envelope coding region of the four dengue serotypes, provided by Dr. V. Deubel. In those samples examined, sensitivity of the long probe mixture was greater and higher numbers of infected cells were detected.

Endothelial cell monolayers as a model system to investigate dengue shock syndrome.

Monolayers of the human endothelial cell line ECV304 were compared with those from primary endothelial cells from human umbilical cord veins (HUVEC) for potential use as an assay system to investigate vasoactive mediator levels in dengue viral infections. Permeability increases were induced in ECV304 monolayers which were more easily reproduced than in primary cells. The cell line monolayers were considerably more stable which allowed multiple consecutive assays to be undertaken on the same monolayers. Permeability responsiveness was maximal at 2 and 3 days postseeding and declined over a period of 7 days. The cell line formed monolayers which showed time- and concentration-dependent permeability increases in response to thrombin, tumour necrosis factor-alpha (TNF-alpha) or interleukin-1alpha (IL-1alpha) in a manner similar to primary endothelial cells. Permeability increases induced by TNF-alpha were reversible and increased exposure time required a longer recovery period. The cell line, like primary endothelial cells, supported dengue viral replication. Direct infection of confluent monolayers on polycarbonate membranes was not cytolytic and did not increase the permeability of the monolayers over a 15-day period.