Virulence of protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement.

The gene coding for protein A (spa) of Staphylococcus aureus 8325-4 has been inactivated by substituting part of the spa coding sequence for a DNA fragment specifying resistance to ethidium bromide. The in vitro-constructed spa::EtBrr substitution mutation was introduced into the S. aureus chromosome by recombinational allele replacement. Southern blot hybridization showed that the in vitro-constructed mutation was present in the chromosomal spa locus. We have previously reported the inactivation of the alpha-toxin gene (hly) by allele replacement with an in vitro-constructed hly::Emr (erythromycin resistance) mutation (M. O'Reilly, J.C.S. de Azavedo, S. Kennedy, and T.J. Foster, Microb. Pathogen. 1:125-138, 1986). A double Spa- Hly- mutant was constructed by transduction. The virulence of Spa- and Hly- mutants was tested by experimental infection of mice. When subcutaneous injections were given, Hly- mutants formed a flat, darkened lesion, whereas Hly+ strains caused a raised, cream lesion. Alpha-toxin was shown to be a major factor in forming subcutaneous lesions and in causing the death of mice injected intraperitoneally. Spa- mutants were slightly less virulent than their Spa+ counterparts, which suggests that protein A is also a virulence factor of S. aureus.     

Correlates of body image dissatisfaction among overweight women seeking weight loss

Predictors of body image dissatisfaction (BID) among obese people are poorly understood. In 79 obese women seeking weight reduction, associations with BID of self-esteem, youth teasing, adult teasing, and internalization of sociocultural appearance standards (ISAS) were studied. Analyses revealed that only self-esteem, adult teasing, and ISAS predicted BID. Results highlight the importance of adulthood self-esteem and interpersonal-cultural context--rather than childhood experiences--in predicting adulthood BID.     

Interleukin-21 is a T-helper cytokine that regulates humoral immunity and cell-mediated anti-tumour responses

Cytokines and their receptors represent key targets for therapeutic intervention. Ligands are being used to supplement cell numbers that become depleted as a result of disease (organ failure, infection) or subsequent disease treatments (i.e. chemotherapy). Conversely, the inhibition of target cell binding by cytokines is an established strategy for abrogating pathologic cellular activities common to many immunological diseases. Considerable effort in biomedical research is being focused on the cytokine families that play a dominant role in regulating immunity and then prioritizing each member for its therapeutic potential. Currently, the interleukin-2 (IL-2) family of cytokines is widely recognized for its central involvement in controlling lymphocyte function and is the most explored for medical utility. Collectively, these proteins (or their antagonists) are either marketed drugs or have received advanced testing for an impressive array of indications including cancer, infectious disease, transplantation, inflammation and allergic asthma. Here we review the current understanding of IL-21, the most recent member of this cytokine family to be discovered. As will be discussed, IL-21 shares many of the same attributes as its relatives in that it has broad immunoregulatory activity and can modulate both humoral and cell-mediated responses. Its ability to stimulate durable anti-tumour responses in mice defines one therapeutic indication that merits clinical development.     

Dielectric properties of low-water-content tissues

The dielectric properties of two low-water-content tissues, bone marrow and adipose tissue, were measured from 1 kHz to 1 GHz. From 1 kHz to 13 MHz, the measurements were performed using a parallel-plate capacitor method. From 10 MHz to 1 GHz, a reflection coefficient technique using an open-ended coaxial transmission line was employed. The tissue water contents ranged from 1 to almost 70% by weight. The dielectric properties correlate well with the values predicted by mixture theory. Comparison with previous results from high-water-content tissues suggests that bone marrow and adipose tissues contain less motionally altered water per unit dry volume than do the previously studied tissues with lower lipid fractions. The high degree of structural heterogeneity of these tissues was reflected in the large scatter of the data, a source of uncertainty that should be considered in practical applications of the present data.     

The effect of elevated intracranial pressure on the vibrational response of the ovine head

Although potentially fatal increases in intracranial pressure (ICP) can occur in a number of pathological conditions, there is no reliable and noninvasive procedure to detect ICP elevation and quantitatively monitor changes over time. In this experimental study, the relationships between ICP elevation and the vibrational response of the head were determined. An ovine animal model was employed in which incremental increases in ICP were elicited and directly measured through intraventricular cannulae. At each ICP increment, a vibration source elicited a flexural response of the animal's head that was measured at four locations on the skull using accelerometers. Spectral analysis of the responses showed changes in proportion to ICP change up to roughly 20 cm H₂O (15 mm Hg) above normal; a clinically significant range. Both magnitude and phase changes at frequencies between 4 and 7 kHz correlated well (γ>0.92) with ICP across the study group. These findings suggest that the vibrational response of the head can be used to monitor changes in ICP noninvasively.     

Impedance-gradient electrode reduces skin irritation induced by transthoracic defibrillation

A new type of disposable external defibrillation electrode has been developed to reduce the skin irritation commonly associated with defibrillation and synchronised cardioversion. This design employs an impedance gradient to reduce the proportion of current delivered to the electrode periphery. The temperature distribution under the new electrode was compared with that of four other types of commercially available electrodes after repeated high-energy biphasic defibrillation discharges to domestic swine. Skin temperature distributions were acquired using non-invasive thermography. Measurements of the maximum temperature rise at each electrode site, taken 3.6s after the fifth defibrillation discharge, demonstrated that the new impedance-gradient electrode produced 50–60% less skin heating than two of the three uniform-impedance electrode designs. Histological examination of erythematous sites excised 24h after defibrillation quantified the associated skin damage using a scoring protocol developed for this study. In contrast to previous studies, histological examinations demonstrated second-degree skin burns following defibrillation. The new electrode design, however, induced 44–46% less skin damage than two of the traditional uniform-impedance electrodes.     

A novel ELISpot method for adherent cells

The purpose of this study was to develop an enzyme-linked immunospot assay (ELISpot assay) that can be used with human adherent cells. While standard enzyme-linked immunosorbent assays (ELISAs) are available and widely used and ELISpot assays are used for nonadherent lymphocytes, no ELISpot assay has been developed for adherent cells. We used primary human fibroblasts from four different tissues (myometrium, lung, gingiva, and orbit), either unstimulated or interleukin (IL)-1beta-activated, to evaluate an ELISpot assay. Antibody pairs for IL-6 and IL-8 were used and results were compared to a standard ELISA. We found that we could reliably detect IL-6 and IL-8 spots with as few as 10 fibroblasts. Optimal cell numbers were 50 cells per well incubated for 8 h, although spots appeared as early as 2 h after incubation. Spots were absent when cells, primary, or secondary anti-cytokine antibodies were omitted from the protocol. Spot number and size can be ascertained using current automated ELISpot reader technology. The frequency of IL-6 and IL-8-producing human fibroblasts could also be determined. For example, 60% of the lung fibroblasts express IL-6, but IL-8 can be detected from only 40% of the cells. Approximately 80% of the human orbital fibroblasts make IL-6, whereas approximately 50% generate IL-8 following IL-1beta stimulation. These new findings show that fibroblasts from different human tissues display different frequencies of cytokine production and this further supports the concept of fibroblast diversity. The sensitivity of this new ELISpot assay is adequate for cytokine detection in just a few cells, unlike the standard ELISA. It should permit ascertaining the frequency of fibroblasts and other adherent cells that produce cytokines and, if desired, can be used in tandem with a standard ELISA to determine total cytokine produced. Moreover, the assay is suitable for normal human adherent cells that are often short-lived and difficult to cultivate.     

Identification of an antigenic subtype of eastern equine encephalitis virus isolated from a human.

Eastern equine encephalitis (EEE) virus was isolated from the cerebrospinal fluid of a 6-year-old male who had clinically diagnosed aseptic meningitis and subsequently died. Several standard serologic tests that use polyclonal antibody and indirect immunofluorescence and hemagglutination inhibition tests that use monoclonal antibody provided evidence that the isolate was an antigenic subtype of prototype North American EEE virus. We believe that this is the first evidence of an antigenic subtype of EEE virus.     

Analysis of the targeting of the hypermutational machinery and the impact of subsequent selection on the distribution of nucleotide changes in human V rearrangements

Summary: B cells are unique in that they generate and tolerate a high rate of mutations in their antigen receptor genes and employ these mutations as a basis of avidity maturation. The precise role of the mutational machinery versus subsequent selection in determining the frequency and distribution of mutations has not been fully analyzed. To address these issues, the influence of the intrinsic mutational machinery and subsequent selection on the frequency and distribution of mutations in the expressed human immunoglobulin repertoire was analyzed. Analysis of non-productively rearranged v_H genes from individual human B cells provided an opportunity to examine the immediate impact of somatic hypermtitation without superimposed selective influences. Comparison with the frequency and distribution of mutations in the productively rearranged human V_H genes permitted an estimate of the influences of subsequent selection.