Extraocular muscle (EOM) is spared in Duchenne muscular dystrophy. Here, we tested putative EOM sparing mechanisms predicted from existing dystrophinopathy models. Data show that mdx mouse EOM contains dystrophin-glycoprotein complex (DGC)-competent and DGC-deficient myofibers distributed in a fiber type-specific pattern. Up-regulation of a dystrophin homologue, utrophin, mediates selective DGC retention. Counter to the DGC mechanical hypothesis, an intact DGC is not a precondition for EOM sarcolemmal integrity, and active adaptation at the level of calcium homeostasis is not mechanistic in protection. A partial, fiber type-specific retention of antiischemic nitric oxide to vascular smooth muscle signaling is not a factor in EOM sparing, because mice deficient in dystrophin and alpha-syntrophin, which localizes neuronal nitric oxide synthase to the sarcolemma, have normal EOMs. Moreover, an alternative transmembrane protein, alpha7beta1 integrin, does not appear to substitute for the DGC in EOM. Finally, genomewide expression profiling showed that EOM does not actively adapt to dystrophinopathy but identified candidate genes for the constitutive protection of mdx EOM. Taken together, data emphasize the conditional nature of dystrophinopathy and the potential importance of nonmechanical DGC roles and support the hypothesis that broad, constitutive structural cell signaling, and/or biochemical differences between EOM and other skeletal muscles are determinants of differential disease responsiveness.

Extraocular muscle is uniquely spared from damage in merosin-deficient congenital muscular dystrophy. Using a murine model, we have tested the hypothesis that the maintenance of calcium homeostasis is mechanistic in extraocular muscle protection. Atomic absorption spectroscopy has demonstrated a strong correlation between the perturbation of calcium homeostasis in hindlimb muscle that is severely damaged and the absence of changes in calcium in extraocular muscle. If, as in other skeletal muscles, extraocular muscle fibers are destabilized by merosin deficiency, we would expect an increase in total muscle calcium coupled with an adaptive response in the high capacity/speed of the sarcoplasmic reticulum of the eye muscle. However, we have not observed the expected increases in total muscle calcium content, Ca2+-ATPase activity, Na+/Ca2+ exchanger content, or smooth ER Ca2+-ATPase content that are predicted by this model. Instead, these results indicate that the increased membrane permeability that characterizes, and is potentially mechanistic in, myofiber degeneration in muscular dystrophy does not occur in merosin-deficient extraocular muscle. Thus, the high-capacity calcium-scavenging systems are not primarily responsible for extraocular muscle protection in muscular dystrophy.

A solid-phase enzyme-linked immunosorbent assay (ELISA) with monoclonal secondary antibodies was used to detect matrix protein and nucleoprotein of influenza A. The sensitivity of the ELISA for highly purified A/Brazil nucleoprotein and matrix protein was 0.05 and 1.0 ng, respectively. Nasal washes from 10 of 20 adult subjects with culture-proven, naturally acquired infection caused by A/Brazil/11/78-like influenza virus were positive in the test, and 2 of 13 subjects with rhinovirus infection were falsely positive. To determine if ELISA results could be improved, nasal washes were obtained from 21 adult volunteers who had been inoculated intranasally with wild-type A/Korea/1/82 (five subjects) or A/Korea recombinants with matrix protein or RNA-2 protein of A/Ann Arbor/6/60 (16 subjects), and the nasal washes were processed by a variety of methods. Prompt addition of sodium azide to the nasal washes to limit bacterial growth, avoidance of freezing, and the use of an antiproteolytic agent all failed to improve ELISA results noticeably. Under the best conditions, ELISA was positive in only 12 of the 21 experimentally infected subjects and in 1 of 15 uninfected controls. Positive ELISA results in experimentally infected subjects correlated significantly with the titer of live virus in the nasal washes (r = +0.506; P less than 0.001). Detection of gradient-purified whole influenza virus or isolated core antigen in ELISA was inhibited by prior incubation with nasal washes, and the inhibitory activity was only partly decreased by heat treatment of the secretions. At present, the use of ELISA for detection of influenza antigens in respiratory secretions is not sufficiently sensitive or specific for routine laboratory diagnosis of influenza.

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Upregulation of tropoelastin (TE) gene expression in rat lung interstitial fibroblasts normally occurs during alveolar septation. TE message increases at the end of the first week of life, peaks on days 9-11, and returns to barely detectable levels over the next 7-10 days. Our previous in situ hybridization studies indicated that exposure of pups to > 95% oxygen from 3 to 13 days of age interfered with the increased in TE gene expression in interstitial fibroblasts normally seen during septation. However, when the pups were returned to room air, lung fibroblast TE message levels increased, exceeding levels seen in control lungs during the exposure. In addition, TE message levels remained elevated for a week after levels in control lungs had returned to background. A possible interpretation of these results was that the developmentally regulated increase in TE messenger RNA (mRNA) was downregulated by the hyperoxic exposure but resumed when the pups were returned to a normoxic environment. We report herein the results of a subsequent study conducted to determine whether continued hyperoxic exposure beyond day 13 would further delay the peak in TE mRNA. Rat pups were exposed to 95% O2 from 5 to 17 days of age. TE and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) message levels in lung interstitial fibroblasts were assessed by in situ hybridization. As observed in pups exposed from 3 to 13 days, hyperoxic exposure from days 5 to 17 also extended the period during which TE mRNA levels were elevated. After exposure, TE message levels were 99%, 262%, and 223% of controls on days 19, 21, and 23 respectively. In addition, delaying the exposure 2 days until the pups were 5 days old resulted in an upregulation of TE message, relative to control values, during the hyperoxic exposure. In hyperoxic pups, values for TE message expression were 105%, 152%, 168%, and 144% of control pups on days 9, 11, 13, and 16 respectively. The influence on peak TE message expression of postnatal age at the time of exposure was further explored to verify the results of the 3-13 and 5-17 day exposures. When pups were exposed continuously from 2, 3, 4, 5, or 6 days until 11 days of age, the results of both in situ hybridization and Northern blot analysis confirmed our previous observations, demonstrating that the postnatal age at which hyperoxic exposure is initiated influences TE message expression in the developing lung.