Assessment of caffeine exposure: caffeine content of beverages, caffeine intake, and plasma concentrations of methylxanthines

The caffeine content of all tea or coffee beverages consumed by 17 healthy adults over 24 hours was measured. Plasma caffeine, theophylline, theobromine, and paraxanthine concentrations were determined over the same 24 hours. The average caffeine content per drink was 60.4 +/- 21.8 mg for instant coffee (14-fold range), 80.1 +/- 19.2 mg for brewed coffee (2.8-fold range), and 28.8 +/- 13.7 mg for tea (5.5-fold range). The number of drinks of coffee and tea consumed was a poor index of actual caffeine intake (r2 = 0.42). Caffeine intake correlated poorly with the 24-hour average caffeine concentration (r2 = 0.41), but there was a very good correlation between a single plasma caffeine concentration measured at 5 PM and the 24-hour average concentration (r2 = 0.94). The same was true for paraxanthine (r2 = 0.86). Paraxanthine accounted for 67.3% of the total dimethylxanthines in plasma, while theobromine and theophylline accounted for 24.4% and 8.3%, respectively. Mean caffeine clearance was 1.2 +/- 0.3 ml/min/kg. Plasma caffeine concentration before the first drink in the morning correlated very poorly with caffeine clearance (r2 = 0.07), even when adjusted for caffeine intake (r2 = 0.21).     

Art in medical education: Especially plastic surgery

The importance of art studies in the training of plastic surgeons has not been well recognized. Presently, very few medical schools offer courses on art or include it in the humanities. Because the study of art is a great experience that helps to develop the trained eye, the inclusion of art in medical education is recommended. For plastic and aesthetic surgeons, art knowledge can greatly add to the development of surgical skill. Courses in drawing, modeling, and casting are recommended along with lectures or seminars on art appreciation.Delivered as the Nojarova Lecture at the New York Academy of Medicine, the New York Regional Society of Plastic and Reconstructive Surgery, March 3, 1990. Also given at the Italian-American Conference of Plastic Surgeons, Venice, Italy, September 23, 1990     

Isolation of encephalomyocarditis virus among stillborn and post-weaning pigs in Quebec

Summary Encephalomyocarditis (EMC) virus was isolated from aborted fetuses and lungs of suckling pigs from three Quebec pig farms that experienced outbreaks of reproductive failure in sows and respiratory problems in suckling and post-weaning piglets. Multifocal interstitial pneumonia and mild non-suppurative myocarditis and meningoencephalitis were the more significant histopathological lesions observed in piglets. Vero cells were found to be more sensitive than BHK-21 cells and pig cell lines for primary isolation of EMC virus. The Quebec EMC virus isolates were highly virulent for mice and were antigenically related to reference strain of EMC virus as demonstrated by indirect immunofluorescence, seroneutralization and Western immunoblotting. Specific virus neutralization antibody titers up to 1:12,800 were detected in samples of thoracic or abdominal fluids of the aborted fetuses.     

Two vasopressin-like peptides in the pig testis?

Vasopressin (VP)-like immunoreactivity (IR) has been located in the testes of several species of mammal. There is evidence that most of this IR in the rat does not represent authentic arginine vasopressin (AVP) and that a second AVP-like peptide may exist. We have studied testis samples from the pig, which produces lysine vasopressin (LVP) in its pituitary, and have found both LVP- and AVP-like IR. High-performance liquid chromatography (HPLC) of testis extracts showed two peaks of VP-IR. The first peak co-eluted with authentic LVP and was recognized only by antisera which cross-reacted with LVP. The second peak co-eluted with authentic AVP and was recognized by antisera raised against AVP. Both VP-like peptides bound to a neurophysin affinity column and the HPLC elution profiles of the bound peptides were similar to those of the authentic hormones. When the LVP-like material was oxidized with performic acid, a peak of IR running in the same position as oxidized authentic LVP on HPLC was produced. Similarly, the performic acid-oxidized AVP-like material co-eluted with oxidized authentic AVP. The presence of both LVP- and AVP-like peptides in the pig testis may mean that more than one gene is involved. A second VP-like gene could also explain the anomalies of VP-IR in other species.     

Evaluation of diagnostic tests with multiple diagnostic categories

The evaluation of diagnostic tests attempts to obtain one or more statistical parameters which can indicate the intrinsic diagnostic utility of a test. Sensitivity, specificity and predictive value are not appropriate for this use. The likelihood ratio has been proposed as a useful measure when using a test to diagnose one of two disease states (e.g. disease present or absent). In this paper, we generalize the likelihood ratio concept to a situation in which the goal is to diagnose one of several non-overlapping disease states. A formula is derived to determine the post-test probability of a specific disease state. The post-test odds are shown to be related to the pre-test odds of a disease and to the usual likelihood ratios derived from considering the diagnosis between the target diagnosis and each alternate in turn. Hence, likelihood ratios derived from comparing pairs of diseases can be used to determine test utility in a multiple disease diagnostic situation.     

Eucaryotic Expression of the Nucleocapsid Protein Gene of Porcine Circovirus Type 2 and Use of the Protein in an Indirect Immunofluorescence Assay for Serological Diagnosis of Postweaning Multisystemic Wasting Syndrome in Pigs

The purpose of this study was to develop a sensitive, rapid, and inexpensive immunofluorescence assay (IFA) using a recombinant porcine circovirus type 2 (PCV2) nucleocapsid protein for the serological detection of PCV2-specific antibodies in pig sera. The viral nucleocapsid protein encoded by the PCV2 ORF2 gene has recently been identified as the most immunoreactive viral protein that carries type-specific antigenic determinants. The ORF2 sequence of the IAF-2897 strain of PCV2 has been cloned into a pCEP5 eucaryotic expression vector under the control of the cytomegalovirus promoter, downstream of a polyhistidine sequence tag. The recombinant plasmid was used in transfection experiments with human epithelial kidney 293 cells that were further tested, and positive expression of the viral nucleocapsid protein was confirmed by IFA and Western blotting. Strong, specific fluorescence was observed in the nuclei of transfected cells. Test specificity to PCV2 was verified with several related infectious agents. Sensitivity was compared to that of standard IFA using PCV2-infected cells by evaluating the reactivities of 44 field serum samples from pigs on farms with a porcine population suffering from postweaning multisystemic wasting syndrome. The recombinant nucleocapsid-based test was able to detect 15 more positive-testing pigs than the PCV2-based IFA. Therefore, the relative sensitivity of the latter test was estimated at only 57.1% compared to that of the recombinant nucleocapsid-based test. The recombinant fusion protein has been purified by affinity chromatography and is being used to develop further sensitive serological tests.     

Multiplex PCR for detection and typing of porcine circoviruses

Sets of oligonucleotide primers were designed according to the sequences of the open reading frames (ORFs) ORF1 and ORF2 of the prototype nonpathogenic PK-15 strain of porcine circovirus (PCV) type 1 (PCV-1). By the PCR performed with the various primer sets, genomic DNA or RNA from other bacterial or viral pathogens of the respiratory tracts of pigs could not be amplified. A positive amplification reaction could be visualized with DNA extracted from a viral suspension containing as few as 10 viral particles per ml. No DNA fragment could be amplified from lysates of continuous porcine cell lines (PT, ST, and PFT cells) known to be negative for PCV. When tested with clinical samples from pigs, the results of the single PCR method showed nearly 93% (13 of 14 samples) correlation with histopathological and immunohistochemical findings. Interestingly, subclinical PCV infections could be detected by single PCR with clinical samples that have been submitted from animals with irrelevant cases of respiratory and/or enteric problems. On the basis of the nucleotide sequences of PCV strains (PCV-2) recently associated with outbreaks of postweaning multisystemic wasting syndrome (PWMS) in Quebec, Canada, pig farms, other primers were designed from the PCV-1 genome, and these primers failed to amplify genomic fragments specific to the ORF1 or ORF2 genes of clinical isolates associated with PWMS but amplified DNA from the PCV-1 strain. Two rapid multiplex PCR (mPCR) methods have been developed to distinguish between both genotypes of PCV. By those two mPCR methods, (i) species-specific primer pairs were used to amplify a DNA fragment of 488 bp specific for the ORF2 genes of both genotypes, whereas a 375-bp fragment was amplified from the ORF1 gene of the PCV-1 strain only, or (ii) species-specific primer pairs were used to amplify a DNA fragment of 646 bp specific for the ORF1 genes of both genotypes, whereas a 425-bp fragment was amplified from the ORF2 gene of the PCV-1 strain only. By both mPCR methods, a PCV-2 infection was demonstrated in tissues of 94.2% (33 of 35) of the sick pigs tested, in agreement with previous findings showing the close association of this new genotype of PCV with outbreaks of PMWS in Europe and North America. On the other hand, a PCV-1 infection was confirmed in only 5.7% (2 of 35) of the pigs, and confirmation of a mixed infection with PCV-2 was obtained by a single PCR with PCV-2-specific primers.     

Characterization of monoclonal antibodies to the hemagglutinin-esterase glycoprotein of a bovine coronavirus associated with winter dysentery and cross-reactivity to field isolates.

Seven hybridoma cell lines producing monoclonal antibodies (MAbs) to the hemagglutinin-esterase (HE) glycoprotein of bovine coronavirus (BCV) were obtained from BALB/c mice that were immunized with an enriched peplomeric fraction of the winter dysentery (WD)-associated strain BCQ.2590. The specificities of these MAbs to either the dimeric (140-kDa) or the monomeric (65-kDa) form of the HE glycoprotein were determined by Western immunoblotting experiments with purified virus and immunoprecipitation tests with [35S]methionine-labelled infected cell extracts. Four of these anti-HE MAbs inhibited the hemagglutinating activity of the virus and three weakly neutralized its infectivity in vitro. In addition, competition binding assays allowed for the definition of two independent antigenic domains (domains A and D) and two overlapping antigenic domains (domains B and C) for the HE glycoprotein of the WD-associated strain; epitopes located within antigenic domain A were not associated with hemagglutination inhibition (HAI) and virus neutralization activities. In HAI tests, the four anti-HA MAbs defined two distinct antigenic subgroups among 24 BCV field isolates that have been associated with either typical outbreaks of WD or neonatal calf diarrhea (NCD) in Quebec dairy herds from 1986 to 1996. The Quebec WD-associated strains of BCV, as well as some of the NCD-associated strains isolated since 1991, fell within a subgroup distinct from that of the prototype Mebus strain.     

The cholesterol-lowering drug probucol increases apolipoprotein E production in the hippocampus of aged rats: implications for Alzheimer's disease

Several recent epidemiological studies have proposed that cholesterol-lowering drug Statin may provide protection against Alzheimer's disease (AD). Probucol is a non-Statin cholesterol-lowering drug and a potent inducer of apolipoprotein E (apoE) production in peripheral circulation. A recent clinical study using Probucol in elderly AD subjects revealed a concomitant stabilisation of cognitive symptoms and significant increases in apoE levels in the cerebral spinal fluid in these patients. To gain insight into the mechanisms underlying these effects, we treated a cohort of aged male rats (26-month-old) with oral dose of Probucol for 30 days. Specifically, we examined the effects of Probucol on apoE production and its receptors (low density lipoprotein receptor [LDLr] and low density lipoprotein receptor-related protein [LRP]), astroglial marker of cell damage (glial fibrillary acidic protein [GFAP]), markers of neuronal synaptic plasticity and integrity (synaptosomal associated protein of 25 kDa [SNAP-25] and synaptophysin) as well as cholesterol biosynthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase [HMGCoAr]) in the hippocampus. We report that Probucol induces the production of apoE and one of its main receptors, LRP, increases HMGCoAr (rate-limiting enzyme in cholesterol synthesis), substantially attenuates age-related increases in glial activation, and induces production of synaptic marker SNAP-25, a molecule commonly associated with synaptogenesis and dendritic remodeling.These findings suggest that Probucol could promote neural and synaptic plasticity to counteract the synaptic deterioration associated with brain aging through an apoE/LRP-mediated system. Consistent with the beneficial effects of other cholesterol-lowering drugs such as the Statin, Probucol could also offers additional benefits based on apoE neurobiology.