Partial correction of an inherited biochemical defect of skeletal muscle by grafts of normal muscle precursor cells.

We have attempted to use allografts of normal muscle precursor cells (mpc) to insert donor nuclei, containing a normal genome, into growing or regenerating skeletal muscle fibres of mice with an inherited deficiency of the enzyme phosphorylase kinase (PhK). Analysis of the glucose-6-phosphate isomerase (GPI) isoenzymes of treated muscles showed that myonuclei of donor origin became incorporated into host muscle fibres in 8 of 9 regenerating autografts, but PhK activity was found only in the 3 grafts into which the largest numbers (1-3 x 10(6)) of mpc had been implanted. Following injection of normal mpc into growing PhK-deficient skeletal muscle, mosaic fibres containing myonuclei of donor origin were detected in only 11 of 192 muscles examined from 64 mice, but, of these 11 muscles, 5 contained PhK activity detectable by two separate assays in a further 4 muscles activity was detected by one or other assay.

The mdx mouse skeletal muscle myopathy: I. A histological, morphometric and biochemical investigation.

Skeletal muscle has been examined in a colony of the mdx strain of myopathic mice. Sixty-five mice from 22 to 303 days of age, showed extensive and recurrent areas of necrosis and regeneration of muscle fibres, often accompanied by active cellular infiltration. Morphometry of the soleus muscle revealed an abnormal proportion of small and large muscle fibres; over half of the muscle fibres contained 'central' (non-peripheral) nuclei. No histochemical muscle fibre-type grouping was detected. Serum activities of muscle-derived enzymes were greatly elevated in all animals and probably reflect enzyme leakage from damaged muscle fibres. Histological evidence of a cardiomyopathy was found in 13 mice. The mdx myopathy thus shows features seen in Duchenne muscular dystrophy. Mdx differs from Duchenne dystrophy principally in that it exhibits a greater degree of compensatory muscle regeneration and an absence of fibro-fatty replacement of muscle fibres.

Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse.

Normal muscle precursor cells, prepared by the enzymatic disaggregation of neonatal mouse muscle, were implanted into an area of regenerating muscle in a genetically different inbred strain. This was done in an attempt to determine, first, whether donor muscle precursor cells prepared in this way would fuse with the developing muscle fibres of the host; and second, whether in the "mosaic" muscle fibres thus formed donor as well as host genes were expressed. As markers of the host and donor genes we used the allelic isoenzyme variants of glucose-6-phosphate isomerase (GPI). In 43 out of 60 grafts we detected the presence of a "hybrid" isoenzyme intermediate between host and donor types. This "hybrid" indicated that donor muscle precursor cells had fused with regenerating host muscle cells, and had expressed their GPI genes within the resulting mosaic muscle fibres. We have developed this technique with a view to inserting normal genes into genetically abnormal myopathic muscle.

Severity of glomerulonephritis induced in different strains of suckling mice by infection with lymphocytic choriomeningitis virus: correlation with amounts of endogenous interferon and circulating immune complexes.

Renal lesions due to neonatal infection with lymphocytic choriomeningitis virus were studied in three different strains of mice known to produce different amounts of viral interferon. Very severe ultrastructural lesions similar to those induced by exogenous interferon were found as early as day 8 in C3H mice which produced the highest amount of interferon. Further studies could not be performed in these mice since all died by day 14. Balb/c mice produced the lowest amount of interferon and had very mild ultrastructural lesions. An intermediate pattern was found in Swiss mice. After 30 days of infection, severe immune complex type glomerulonephritis detectable by light microscopy and immunofluorescence was observed in Swiss mice whereas mild lesions only were found in Balb/c mice. Circulating immune complexes were present in both strains but in greater amounts of Swiss than Balb/c mice. These results suggest that two factors at least are important in the development of glomerulonephritis: interferon produced early in life and the load of circulating immune complexes.

Interferon as a cause of endoplasmic reticulum abnormalities within hepatocytes in newborn mice.

An ultrastructural examination of livers from newborn mice, injected with potent partially purified or highly purified mouse interferon or with lymphocytic choriomeningitis (LCM) virus, has revealed the presence of tubular aggregates associated with the granular endoplasmic reticulum in the cytoplasm of hepatocytes after either treatment. Thus the lesion was observed in A2G and Swiss mice after interferon injections. It was also seen in C3H mice after LCM infection, the liver being examined at a time when the interferonaemia in the injected mice was known to be at its peak. The aggregate resembles the tubular systems associated with the endoplasmic reticulum described in various tissues in both human and animal diseases. These observations raise the possibility that in some of the cases previously described the lesion has been interferon induced.

Cyclosporin A as a means of preventing rejection of skeletal muscle allografts in mice.

Isografts and allografts of skeletal muscle inserted into the limbs of mice initially degenerate. After some 5 to 8 days newly formed myotubes appear in the graft which develop into mature muscle fibers. In nontolerant hosts allografts are rejected between the 10th and 12th days. In mice treated with cyclosporin A, this effect persists for some 12 days after the end of treatment. Isoenzyme marker studies indicate that the regenerated graft is composed of both host and donor tissue. Donor isoenzyme does not persist when grafts are rejected.

The contribution of exogenous cells to regenerating skeletal muscle: an isoenzyme study of muscle allografts in mice.

A sequential study of 180 allografts of minced skeletal muscle has been made in mice, in 124 of which tolerance was induced. The host/donor composition of grafts was assessed in terms of their content of isoenzymes of glucose-6-phosphate isomerase characteristic of host and donor strains. From 0-5 days implanted muscle fibre fragments uniformly underwent degeneration. New myotubes appeared at day 5. Both host and donor isoenzyme were found during this period. In "non-tolerant" hosts, grafts were rejected at 8-12 days, after which only host isoenzyme was found and the graft site usually lacked muscle, consisting of fibrofatty connective tissue. In the few instances where muscle was found in such grafts, this was necessarily formed from host precursor cells which had migrated into the graft site. In "tolerant" hosts, grafts contained up to 80 per cent. of the muscle and usually yielded both host and donor isoenzyme. Where "hybrid" isoenzyme was found, it was probable that host muscle precursor-cells had entered grafts and fused with donor muscle.

Histogenesis of the supraoptic and paraventricular neurosecretory cells of the mouse hypothalamus.

Using tritiated thymidine in mice it was found that the precursor cells of neurons in the supraoptic and paraventricular nuclei cease proliferating by the 13th embryonic day. The significance of the early origin of these neurons is discussed.

Interferon-induced disease in mice and rats.

Treatment of newborn mice with potent mouse interferon preparations resulted in an acute "early" syndrome characterized by inhibition of growth, delay in maturation of several organs, diffuse liver cell necrosis and death. When interferon treatment was discontinued at 1 week of life, mice appeared to recover, but subsequently developed a progressive glomerulonephritis ("late syndrome"). Treatment of newborn rats with potent rat interferon preparations also resulted in inhibition of growth, delay in maturation, and the subsequent development of glomerulonephritis. After infection at birth with lymphocyte choriomeningitis (LCM) virus, most strains of mice developed a similar acute early syndrome and surviving mice subsequently developed glomerulonephritis. We postulated that the endogenous interferon induced by LCM virus early in life was partially responsible for these syndromes. Administration of a potent anti-mouse interferon serum to LCM virus-infected mice neutralized the circulating endogenous interferon and inhibited the development of both the early and late syndromes. Our results suggest that large amounts of exogenous or endogenous interferon at a crucial stage of rapid growth or development of mice and rats can induce lesions in several different organs. Some lesions (i.e. the kidney) only become apparent weeks or even months after exposure to interferon.

Post meningococcal acute glomerular nephritis.

A case of meningococcal meningitis is described in which 10 days later there developed the histological lesions of acute exsudative proliferative glomerular nephritis without proteinuria, hematuria, hypertension or salt and water retention. The relationship between structural and functional changes in the kidney in glomerular nephritis is discussed in the light of these findings.

Microtubular number in the tractus hypophyseus of newborn normal rats and newborn rats with congenital diabetes insipidus.

Evidence has already been adduced suggesting that an increase in microtubular number occurs in the tractus hypophyseus of rats stressed by the administration of hypertonic saline, and of rats with congenital diabetes insipidus (CDI). Since the tractus hypophyseus in these animals shows high secretory activity, it seems likely that the microtubular increase reflects the participation of microtubules in axoplasmic transport. To exclude, however, a congenital microtubular abnormality in CDI, affected newborn rats were examined. In these, the microtubular number was normal, thus suggesting that the increase in microtubular number seen in adult animals was not a congenital morphological abnormality. However, by 4 days of age there was a slight but statistically significant increase in microtubular number in affected rats, a change probably attributable to increased secretory activity.

Isolated glomerulonephritis with mesangial IgA deposits.

Mesangial deposits of IgA, occurring in the absence of systemic disease known to be associated with nephritis, were detected by immunofluorescence microscopy in renal biopsy specimens from 25 patients (4% of 630 specimens studied). Associated deposits of C3 were always present, usually with IgG, but IgM deposits were less common and C1q was never seen. On light microscopy most of the biopsy specimens showed mesangial of focal nuclear proliferation though some were normal. Fifteen of the 25 patients presented with macroscopic haematuria, which was usually recurrent and preceded by a sore throat, whereas the remaining, and usually older, patients presented with persistent proteinuria and were more likely to have impaired renal function. This incidence of "mesangial IgA disease" is less than that reported by French workers. There was a significantly high incidence of familial renal disease among these patients. No abnormalities of serum complement or IgA concentration were found.