The stress interview as a tool for examining physiological reactivity

Most studies of cardiovascular reactivity utilize tasks that are highly standardized and controlled. However, there is some concern whether the information obtained from these tasks is relevant to understanding the cardiovascular responses to behavioral stimuli encountered in real life. Historically, various forms of a stress interview have been used to examine physiological concomitants of emotional arousal. However, these tools fell into disuse because of ethical concerns, their intensive time requirements, and the perception that the interviews could not be standardized. We have developed a short (16-minute) interview that is not aversive or threatening. In studies on 24 normotensives and 19 hypertensives, the interview elicited greater blood pressure elevations than those elicited by mathematical calculations or cold pressor (p less than 0.0001). The interview increased systolic and diastolic pressures by 38/32 mm Hg in hypertensives and 31/24 mm Hg in normotensives. The variance in BP elicited by this interview is not appreciably greater than that found in highly structured tasks such as mathematical calculations. The test-retest stability of the interview is comparable to that of resting baseline blood pressure and is superior to that of mathematical calculations or cold pressor. Techniques such as the interview may be at least as useful as highly standardized tasks and possibly more valid as models for examining cardiovascular reactivity.     

Epidemiological studies of Streptococcus pneumoniae in infants: methods of isolating pneumococci.

A prospective study of the natural history of pneumococcal infection, which involves serial culture studies in healthy infants from 6 weeks of age onward, is in progress in our laboratory. This report describes results of a comparison of several methods for the isolation and identification of Streptococcus pneumoniae from the nasopharynges and throats of these infants. Sheep blood agar, sheep blood agar with gentamicin sulfate (gentamicin agar), and mouse inoculation with 4-h broth cultures were used. Gentamicin agar proved superior to plain sheep blood agar as a solid culture medium, especially in enhancing the recovery of pneumococci from throat cultures. With gentamicin agar, similar carrier rates were found for both culture sites (nasopharynx and throat). In addition, gentamicin agar proved superior to mouse inoculation for the recovery of carrier strains from 131 nasopharyngeal culture samples processed by both methods. Sixty of 131 samples were positive for pneumococci, 25% of which would have been missed had mouse inoculation alone been used. In only three instances did we recover a strain by mouse inoculation that failed to grow on gentamicin agar; conversely, 15 strains were isolated on gentamicin agar but could not be recovered from mice. The latter observation might be explained by the fact that certain carrier strains may be relatively mouse avirulent. The use of blood agar containing gentamicin appears to offer a simple and inexpensive method for the recovery of S. pneumoniae and, in our opinion, provides an ideal method for the identification of pneumococcal carriers as well as for the recovery of these strains from clinical material such as sputum or ear exudates, where other and less fastidious organisms may also be present.     

Effects of digoxin in infants with congested circulatory state due to a ventricular septal defect

Digoxin alone was used to treat a congested circulatory state in 21 infants (mean age, 2.7 months; mean weight, 3.8 kg) with a ventricular septal defect. The dose was adjusted on the basis of pharmacokinetics to achieve a mean steady-state concentration of 1.6 +/- 0.3 ng of digoxin per milliliter of serum. The mean red-cell level of sodium-potassium ATPase fell from 23.1 +/- 7.0 to 12.6 +/- 5.2 nmol per milligram per minute with treatment. Only 6 of the 21 patients had an inotropic response, as reflected by echocardiographic measurements, but the drug was of clinical benefit to 12 infants (including these 6). These results show that not all infants with a congested circulatory state due to a ventricular septal defect benefit from digoxin therapy. Furthermore, in some subjects clinical improvement occurs in the absence of a measurable inotropic response.     

Isolation of a new clathrin heavy chain gene with muscle-specific expression from the region commonly deleted in velo-cardio-facial syndrome

Velo-cardio-facial syndrome (VCFS) and DiGeorge syndrome (DGS) are developmental disorders characterized by a spectrum of phenotypes including velopharyngeal insufficiency, conotruncal heart defects and facial dysmorphology among others. Eighty to eighty-five percent of VCFS/DGS patients are hemizygous for a portion of chromosome 22. It is likely that the genes encoded by this region play a role in the etiology of the phenotypes associated with the disorders. Using a cDNA selection protocol, we isolated a novel clathrin heavy chain cDNA (CLTD) from the VCFS/DGS minimally deleted interval. The cDNA encodes a protein of 1638 amino acids. CLTD shares significant homology, but is not identical to the ubiquitously expressed clathrin heavy chain gene. The CLTD gene also shows a unique pattern of expression, having its maximal level of expression in skeletal muscle. Velopharyngeal insufficiency and muscle weakness are common features of VCFS patients. Based on the location and expression pattern of CLTD, we suggest hemizygosity at this locus may play a role in the etiology of one of the VCFS-associated phenotypes.      

Growth sensitivity of a recombinant simian virus 5 P/V mutant to type I interferon differs between tumor cell lines and normal primary cells

A paramyxovirus SV5 mutant (rSV5-P/V-CPI-) that encodes 6 naturally-occurring P/V gene substitutions is a potent inducer of type I interferon (IFN) and is restricted for low moi growth, two phenotypes not seen with WT SV5. In this study, we have compared the IFN sensitivity of WT SV5 and the rSV5-P/V-CPI- mutant in tumor cell lines and in cultures of normal primary cells. We have tested the hypothesis that differences in IFN induction elicited by WT rSV5 and rSV5-P/V-CPI- are responsible for differences in low moi growth and spread. In contrast to WT SV5, low moi infection of A549 lung carcinoma cells with rSV5-P/V-CPI- resulted in a plateau of virus production by 24-48 h pi when secreted IFN levels were between approximately 100 and 1000 U/ml. Gene microarray and RT-PCR analyses identified IFN genes and IFN-stimulated genes whose expression were increased by infection of A549 cells with WT and P/V mutant viruses. Restricted low moi growth and spread of rSV5-P/V-CPI- in A549 cells was relieved in the presence of neutralizing antibodies to IFN-beta but not TNF-alpha. When A549 or MDA-MB-435 breast tumor cells were pretreated with IFN, both WT and P/V mutant viruses showed delayed spread and approximately 10-fold reduction in virus yield, but infections were not eliminated. Using normal primary human epithelial cells that have undergone limited passage in culture, WT rSV5 and rSV5-P/V-CPI- displayed high moi growth properties that were similar to that seen in A549 cells. However, IFN pretreatment of these primary cells as well as normal human lung cells eliminated low moi spread of both mutant and WT rSV5 infections. Together, these data demonstrate that SV5 growth in normal primary human cells is highly sensitive to IFN compared to growth in some tumor cell lines, regardless of whether the P/V gene is WT or mutant. These results suggest a model in which spread of WT SV5 in normal human cells is dependent on the ability of the virus to prevent IFN synthesis. The implications of these results for the use of recombinant paramyxoviruses as vectors are discussed.     

Use of multiepitope polyproteins in serodiagnosis of active tuberculosis

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of approximately 98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of approximately 93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.     

Functional characterization of GABA(A) receptors in neonatal hypothalamic brain slice

The hypothalamus influences a number of autonomic functions. The activity of hypothalamic neurons is modulated in part by release of the inhibitory neurotransmitter GABA onto these neurons. GABA(A) receptors are formed from a number of distinct subunits, designated alpha, beta, gamma, delta, epsilon, and theta, many of which have multiple isoforms. Little data exist, however, on the functional characteristics of the GABA(A) receptors present on hypothalamic neurons. To gain insight into which GABA(A) receptor subunits are functionally expressed in the hypothalamus, we used an array of pharmacologic assessments. Whole cell recordings were made from thin hypothalamic slices obtained from 1- to 14-day-old rats. GABA(A) receptor-mediated currents were detected in all neurons tested and had an average EC(50) of 20 +/- 1.6 microM. Hypothalamic GABA(A) receptors were modulated by diazepam (EC(50) = 0.060 microM), zolpidem (EC(50) = 0.19 microM), loreclezole (EC(50) = 4.4 microM), methyl-6,7-dimethoxy-4-ethyl-beta-carboline (EC(50) = 7.7 microM), and 5alpha-pregnan-3alpha-hydroxy-20-one (3alpha-OH-DHP). Conversely, these receptors were inhibited by Zn(2+) (IC(50) = 70.5 microM), dehydroepiandrosterone sulfate (IC(50) = 16.7 microM), and picrotoxin (IC(50) = 2.6 microM). The alpha4/6-selective antagonist furosemide (10-1,000 microM) was ineffective in all hypothalamic neurons tested. The results of our pharmacological analysis suggest that hypothalamic neurons express functional GABA(A) receptor subtypes that incorporate alpha1 and/or alpha2 subunits, beta2 and/or beta3 subunits, and the gamma2 subunit. Our results suggest receptors expressing alpha3-alpha6, beta1, gamma1, and delta, if present, represent a minor component of functional hypothalamic GABA(A) receptors.     

Outcomes following adoption of a standardized protocol for abscess drain management in pediatric appendicitis

Background/purposeThough evidence-based clinical pathways for the diagnosis and treatment of pediatric appendicitis have been established, protocols guiding management of percutaneous abscess drains are lacking. We hypothesized a drain management protocol utilizing drain output and clinical factors instead of fluoroscopic drain studies would reduce interventional radiologic procedures without adversely impacting clinical outcomes.MethodsA standardized protocol was uniformly adopted at a tertiary-care children's hospital in April 2016. A retrospective chart review included all cases of appendicitis requiring abscess drainage by interventional radiology three years pre- and postprotocol implementation.ResultsFifty-eight patients (preprotocol = 39, postprotocol = 19) underwent percutaneous abscess drainage, of whom 52 (preprotocol = 34, postprotocol = 18) required a drain. Baseline demographics and clinical presentation were similar across groups. Following protocol implementation, total number of IR procedures decreased from 2.4 to 1.3 per patient (p = 0.004). There was no significant difference in the number of postprocedure diagnostic imaging studies, readmissions, or inpatient days, and there was a trend towards a decrease in number of drain days (10.7 to 5.7, p = 0.067).ConclusionA standardized protocol for management of abscess drains for complicated appendicitis reduced the number of IR procedures without a negative impact on clinical outcomes or increase in alternative imaging studies. This approach may decrease radiation exposure, anesthetic administration, and resource utilization.Type of studyTreatment study (retrospective comparative study).Level of evidenceLevel III.