Assessment of an anti-alpha-toxin monoclonal antibody for prevention and treatment of Staphylococcus aureus-induced pneumonia.

Alpha-toxin (AT) is a major virulence factor in the disease pathogenesis of Staphylococcus aureus. We previously identified a monoclonal antibody (MAb) against AT that reduced disease severity in a mouse dermonecrosis model. Here, we evaluate the activity of an affinity-optimized variant, LC10, in a mouse model of S. aureus pneumonia. Passive immunization with LC10 increased survival and reduced bacterial numbers in the lungs and kidneys of infected mice and showed protection against diverse S. aureus clinical isolates. The lungs of S. aureus-infected mice exhibited bacterial pneumonia, including widespread inflammation, whereas the lungs of mice that received LC10 exhibited minimal inflammation and retained healthy architecture. Consistent with reduced immune cell infiltration, LC10-treated animals had significantly lower (P < 0.05) proinflammatory cytokine and chemokine levels in the bronchoalveolar lavage fluid than did those of the control animals. This reduction in inflammation and damage to the LC10-treated animals resulted in reduced vascular protein leakage and CO2 levels in the blood. LC10 was also assessed for its therapeutic activity in combination with vancomycin or linezolid. Treatment with a combination of LC10 and vancomycin or linezolid resulted in a significant increase (P < 0.05) in survival relative to the monotherapies and was deemed additive to synergistic by isobologram analysis. Consistent with improved survival, the lungs of animals treated with antibiotic plus LC10 exhibited less inflammatory tissue damage than those that received monotherapy. These data provide insight into the mechanisms of protection provided by AT inhibition and support AT as a promising target for immunoprophylaxis or adjunctive therapy against S. aureus pneumonia.

Full-length dystrophin gene transfer to the mdx mouse in utero.

In utero gene therapy for genetic diseases, such as muscular dystrophies, offers potential advantages over postnatal treatment including vector delivery at the earliest point in the disease and treatment prior to full maturation of the immune system. This study examines in utero gene delivery of full-length murine dystrophin to the murine mdx model for Duchenne muscular dystrophy using a high-capacity adenoviral vector. We examined dystrophin expression, spread of vector, morphology and specific force production of the tibialis anterior muscle 9 weeks after intramuscular in utero injection. Recombinant dystrophin was expressed in the hindlimb muscles, with the majority of animals having expression in two muscles of the injected hindlimb. The dystrophin-glycoprotein complex was restored in those muscle fibers expressing recombinant dystrophin. Analysis of the percentage of dystrophin-expressing muscle fibers with centrally placed nuclei revealed effective protection from cycles of degeneration and regeneration normally seen in muscle fibers lacking dystrophin. However, due to low levels of muscle gene transfer, further advances in the efficiency of adenoviral vector-mediated gene delivery would be required for clinical applications of in utero gene therapy for primary myopathies such as Duchenne muscular dystrophy.

Effect of undernutrition on contractile and fatigue properties of rat diaphragm during development.

The present study was designed to assess the effects of combined pre- and postnatal undernutrition on the in vitro contractile and fatigue properties of the rat diaphragm during development. In vitro direct stimulation of costal diaphragm from control (CTL) and undernourished (UN) rats was done on postnatal days 1, 4, 14, 21, 30, 40, 50, and 60. Combined pre- and postnatal undernutrition resulted in stunted animal growth but did not alter the diaphragm-to-total body weight ratio. Twitch contraction time, half-relaxation time, and force-frequency relationships were not consistently affected by undernutrition. Specific twitch force and specific tetanic force were also unchanged in the UN group. Fatigue resistance was high and comparable in UN and CTL groups at days 1 and 4. At day 14 and thereafter, fatigue resistance declined but was consistently higher in the UN than in the CTL group. We conclude that combined pre- and postnatal undernutrition results in a significant increase in fatigue resistance of the diaphragm compared with CTL, whereas diaphragm muscle contractile properties are not appreciably affected by prolonged undernutrition.

Contractile properties of the rat external abdominal oblique and diaphragm muscles during development.

We studied the in vitro contractile and fatigue properties of the rat external abdominal oblique (EAO) and costal diaphragm (DIA) muscles during postnatal development. Isometric twitch contraction (CT) and half-relaxation (RT1/2) times were longer in both the EAO and DIA muscles during the early postnatal period and decreased with age. In the first postnatal week, the CT and RT1/2 were longer in the EAO than the DIA muscle. At 14 days of age and thereafter, the CT and RT1/2 were shorter in the EAO than in the DIA muscle. Force-frequency relationships of the EAO and DIA muscles changed during postnatal development such that the relative force (percent maximum) generated at lower frequencies (less than 15 pulses/s) decreased with age. Moreover the relative force generated by the EAO muscle at lower frequencies was greater than that of the DIA muscle during the early postnatal period but less than that of the DIA muscle in adults. The specific force of both the EAO and DIA muscles increased progressively with age. There were no differences in specific force between the EAO and DIA muscles at any age. The fatigability of the EAO and DIA muscles was comparable during the early postnatal period and increased in both muscles with postnatal development. In adults the EAO muscle was more fatigable than the DIA muscle. We conclude that the contractile and fatigue properties of the EAO and DIA muscles undergo significantly different postnatal transitions, which may reflect their functional involvement in sustaining ventilation.

Control of genioglossal muscle activity in the anesthetized piglet: the role of vagal afferents.

We examined genioglossal muscle electromyogram activity during room air breathing and hyperoxic hypercapnia in 10 anesthetized (halothane) newborn piglets before and after bilateral midcervical vagotomy. With vagal afferents intact, genioglossal activity was absent during room air breathing in 10/10 study animals and was recruited in only 4/10 piglets during carbon dioxide breathing. After vagotomy, genioglossal activity remained absent in 9/10 study animals during room air breathing but was recruited in 10/10 piglets during the hypercapnic gas exposure at arterial CO2 tensions comparable to prevagotomy levels. We conclude that vagal afferent feedback modulates genioglossal activity in anesthetized newborn piglets and exerts an inhibitory influence on the activity of this muscle during hyperpnea induced by carbon dioxide breathing.

Diaphragmatic electromyogram power-spectral analysis as a function of reduced end-expiratory lung volume.

We examined the centroid frequency (Fc) of the electromyogram power-frequency spectra from the costal (EMGco) and crural (EMGcr) diaphragms at functional residual capacity and at reduced end-expiratory lung volume (EELV) (induced by abdominal banding) in six anesthetized newborn piglets. EMGco and EMGcr were recorded from bipolar electrodes embedded in the costal and crural diaphragms respectively. A fast Fourier transformation of ECG free EMGco and EMGcr was used to compute the power-frequency spectra and calculate the Fc of EMGco and EMGcr. The nitrogen washout technique was used to measure EELV. Abdominal banding induced a reduction in EELV of 30.6% functional residual capacity (range 22-39%). The mean Fc of EMGcr was not significantly altered by the reduction in EELV, whereas the mean Fc of the EMGco fell in every animal at reduced EELV by 13 +/- 8% of baseline Fc (p less than 0.05). We conclude that alterations in lung volume alone can determine changes in the EMGco power spectrum and Fc. Investigators performing EMGco power spectral analysis should consider EELV status when interpreting their findings.

Practical anterior segment fluorescein angiography.

Anterior segment fluorescein angiography is a useful investigative technique for diagnosing and assessing anterior segment disease. However, a number of technical problems are inherent in current methodology, such as confounding reflexes, off-axis illumination, and shallow depth of field. We developed a simple technique using readily available equipment, polarized light, and an easy-to-construct auxiliary bracket to provide an inexpensive and optimal photographic system for anterior segment fluorescein angiography.

Expiratory abdominal muscle activity during ventilatory chemostimulation in piglets.

We examined abdominal muscle minute electromyographic (EMG) activity (peak moving time average EMG x respiratory rate) during eupnea, hyperoxic hypercapnia (8% CO2-40% O2-balance N2), and hypoxia (13% O2) in 12 anesthetized (0.5% halothane) newborn piglets. In addition, we assessed the role of vagal afferent pathways in the abdominal muscles' response to ventilatory chemostimulation by examining abdominal EMG activity (EMGab) before and after bilateral cervical vagotomy in five animals. Phasic expiratory EMGab was observed in 11 of 12 piglets during eupnea. Hypercapnia was associated with a sustained augmentation of minute EMGab (444 +/- 208% control). In contrast, hypoxia consistently augmented (1 min, 193 +/- 33% control) then diminished (5 min, 126 +/- 39% control) minute EMGab. Vagotomy resulted in a decline in peak moving time average EMGab by approximately one-half (48 +/- 18% control); the abdominal muscles' response to ventilatory chemostimulation, however, was qualitatively unchanged. We conclude that 1) expiration during eupnea in anesthetized newborn piglets is associated with phasic EMGab; 2) both hypercapnia and hypoxia augment minute EMGab; however, only hypercapnia is associated with sustained augmentation; and 3) although vagal afferents have a role in modulating the base-line level of EMGab, other extravagal mechanisms appear to determine the pattern of EMGab in response to ventilatory chemostimulation.

External intercostal muscle activity during acute hypoxia in the kitten.

The effects of acute hypoxia on the recruitment of external intercostal muscle activity were determined in 12 kittens, aged 14 to 36 days. The animals were anesthetized with 1.23 +/- 0.23% halothane and bipolar electrodes were placed in the costal and crural diaphragm and in dorsal external intercostal muscles. Acute hypoxia was induced by the animals breathing 13% oxygen; arterial gases were sampled during baseline conditions and at 1 and 5 min after induction of hypoxia. Peak-moving average (PA) and minute electromyogram (EMG) activity (PA x f) were recorded during baseline conditions and at 1 and 5 min after onset of acute hypoxia. At 1 min of acute hypoxia, PA and PA x f of the costal diaphragm, crural diaphragm, and external intercostal muscles were significantly increased above baseline values (P less than 0.01). After 5 min of acute hypoxia, PA of all three muscles remained elevated above baseline values (P less than 0.05) but PA x f returned toward baseline levels. Respiratory frequency remained unchanged during the hypoxic stimulus. These data document that the newborn is capable of increasing inspiratory external intercostal muscle EMG activity during acute hypoxia. We speculate that this phasic recruitment could be of physiologic benefit to the newborn by stabilizing the complaint chest wall and by increasing the contribution of rib cage expansion to tidal breathing.

Use of cross-polarized light in anterior segment photography.

A photographic technique improved the quality of anterior segment photography by using one polarizer in the illumination path and another polarizer that was perpendicularly oriented to the first in front of the camera film back. A significant improvement was noted by impartial masked observers who assessed standard photographs of the iris, conjunctiva, and sclera. A crosslike artifact, observed in photographs using cross-polarizers, was present and originated from corneal birefringence. The significance of the artifact and its possible diagnostic applications were determined.

Genioglossal recruitment during acute hypoxia and hypercapnia in kittens.

We examined genioglossal and diaphragmatic EMG activities in one- and two-month-old anesthetized kittens during acute exposures to hypoxic (13% or 10% O2) and hyperoxic hypercapnic (8% CO2/50% O2/balanced N2) gas mixtures. Phasic genioglossal EMG activity, frequently characterized by a combined inspiratory-expiratory discharge pattern, was observed in 3 of 8 one-month-old vs. 7 of 7 two-month-old kittens during hypercapnia (Chi-square P less than 0.05). The percentage of kittens recruiting genioglossal activity during hypoxic exposures was similar at both ages (1 month, 75%; 2 month, 83%). Analysis of the breath-by-breath response during trials of hypoxia in which genioglossal recruitment was observed, however, revealed that the one-month-old kittens exhibited phasic genioglossal activity in only 40 +/- 27% of the stimulated breaths, compared to 63 +/- 26% for the two-month-old ones (P less than 0.05) at each level of hypoxia. In this regard, the genioglossal response to hypoxia in one-month-old kittens was frequently characterized by early and only transient recruitment (when diaphragmatic activity was at its peak), while genioglossal recruitment was more sustained in two-month-old animals. These data indicate that genioglossal activity in kittens is often recruited during exposures to hypercapnia and hypoxia, and suggest that such recruitment is more frequent with increasing postnatal age.

Costal and crural diaphragm, and intercostal and genioglossal electromyogram activities during spontaneous augmented breaths (sighs) in kittens.

Spontaneously occurring augmented breaths (sighs) are common in infants. The pattern of electrical activity of the inspiratory muscles of the thorax and upper airway during augmented breaths, however, has not been fully characterized in this less than fully mature age group. We therefore examined costal and crural diaphragm and external intercostal and genioglossal EMG activities during spontaneous augmented breaths (n = 46) in 10 anesthetized (1.35% halothane) 1-month-old kittens breathing room air. EMG responses were assessed by comparing the spontaneous augmented breaths (AB) to the five immediately preceding breaths (control). The peak moving time average EMG activity observed during the AB was 240 +/- 32% (mean +/- SD) of control for the costal diaphragm, 279 +/- 66% of control for the crural diaphragm, and 274 +/- 68% of control for the external intercostal muscle. The mean increase in EMG activity during the AB was not significantly different among these three muscle groups (P greater than 0.25). Genioglossal EMG activity during AB was observed in only 1 of 10 study animals. These results document that during AB in anesthetized kittens, activity of the thoracic inspiratory muscles (costal/crural diaphragm and external intercostal muscles) increase in parallel, suggesting that they are modulated in a uniform manner. The infrequent observance of genioglossal activity during AB suggests that either 1) halothane anesthesia depresses genioglossal activity more than diaphragmatic and intercostal activity during AB or 2) that genioglossal recruitment is not necessary to maintain upper airway patency during this period of heightened respiratory drive.

Use of circularly polarized light in fundus and optic disc photography.

Circularly polarized light was used in fundus and optic disc photography to reduce reflected glare and photographic artifacts originating from the ocular media and the lenses of the fundus camera. A standard fundus camera was used, with circular polarizers placed in the illumination and observation paths. The quality of fundus photography was improved significantly in eyes with intraocular lenses, cataract, vitreous haze, or high myopia as well as in normal eyes, in fundus photography of the retinal periphery, and in optic disc photography.

Recruitment of intercostal muscle activity during hypercapnia in kittens.

Little is known about the respiratory behavior of the intercostal muscles within a neonatal and developmental context. We, therefore, examined intercostal muscle electromyographic (EMG) activity in kittens (1 month old, n = 8; 2 months old, n = 7) during eupnea and heightened respiratory drive induced by hypercapnia. The kittens were anesthetized with halothane (1.25-1.50%) at comparable minimum alveolar concentrations and were studied in the prone position during an acute exposure to hyperoxic hypercapnia (8% CO2, 50% O2, balance N2) for 7 min. We recorded EMG activities from bipolar electrodes embedded in the intercostal (dorsolateral thorax, 4th-5th interspace) (EMGic) and costal diaphragm (EMGdi) muscles. Peak moving time average EMG measurements served as our index of muscle activity. Phasic inspiratory EMGic activity was present during eupnea in all animals. EMGic and EMGdi increased significantly above baseline levels during hypercapnic exposure with the increase in EMGic (133 +/- 27%) being comparable to that observed in EMGdi (141 +/- 28%) (P = 0.40). No differences in EMGic (P = 0.64) or EMGdi (P = 0.88) recruitment were noted between age groups. These data indicate that hypercapnia augments intercostal muscle EMG activity in kittens and suggest that such activity parallels costal diaphragmatic EMG recruitment. We conclude that EMGic recruitment is a frequent respiratory phenomenon associated with hypercapnically stimulated diaphragmatic muscle activity in kittens. We speculate that intercostal muscle recruitment stabilizes the compliant chest wall of the newborn and helps sustain inspiratory force generation during stimulated breathing.

Use of a modified procedure for treating small amounts of red blood cells with 2-aminoethylisothiouronium bromide.

Kell null red blood cell samples can be prepared using 2-aminoethylisothiouronium bromide (AET). This article describes a modification whereby only three drops of a 5% red blood cell suspension may be AET treated. This procedure has been used routinely in the authors' laboratory for more than a year. One patient's serum contained anti-Kpb plus anti-C and anti-D. Tests with panel cells pretreated with AET made it possible to identify underlying Rh antibodies without using a panel of genetic Kpb negative red blood cells. Of 24 red blood cell eluates from patient sample with warm autoantibodies, one had specificity within the Kell blood group system. This small volume AET-treatment method is a quick screen for the differentiation of a Kell-related specificity from a non-Kell specificity of both warm autoantibodies and alloantibodies to high-incidence antigens.

Autoantibodies mimicking anti-Jkb plus anti-Jk3 associated with autoimmune hemolytic anemia in a primipara who delivered an unaffected infant.

A serum sample from a nontransfused 25-year-old Caucasian primipara contained weak anti-Jkb plus anti-Jk3. The direct antiglobulin test on the patient's red cells was strongly positive. Anti-Jkb was recovered in a heat eluate. An ether eluate contained anti-Jk3. Her red cells types as Jk(a-b+)Jk:3. The anti-Jkb and anti-Jk3 reactivity was completely absorbed both by red cell samples lacking and red cells possessing the corresponding antigens, indicating these antibody specificities to be of the mimicking variety. At 41 weeks gestation she delivered a normal, healthy infant with no detectable serum or cell bound antibody.