Effects of intense exercise in chronic spinal cord injury.

BACKGROUND: Exercise has beneficial effects on muscle and motor function after spinal cord injury (SCI). Little is known regarding effects of prolonged intense exercise (IE) in humans with chronic SCI. DESIGN: Prospective, non-randomized, controlled observational study. The intervention was either a multimodal IE program (n=21) or a control (CTL) intervention consisting of self-regulated exercise (n=8). OBJECTIVE: Measure sensorimotor function over 6 months in relation to an IE program. SETTING: Single outpatient center. SUBJECTS: Subjects with chronic SCI (n=29 total), mainly ASIA Impairment Scale A and B, injury levels C4-T11. RESULTS: Baseline neurological assessments (for example, ASIA motor score, 39+/-3 vs 42+/-5, IE vs CTL, P>0.5, mean+/-s.e.m.) did not differ between the two groups. During the 6 months, IE subjects averaged 7.3+/-0.7 h per week exercise, not significantly different from CTL subjects (5.2+/-1.3 h per week, P>0.1). However, after 6 months, IE subjects showed significantly greater motor gains than CTL subjects in the main outcome measure, ASIA motor score (change of 4.8+/-1.0 vs -0.1+/-0.5 points, P=0.0001). The main outcome measure was calculated by ASIA motor score. These IE subject ASIA motor gains correlated with number of exercise hours per week (r=0.53, P<0.02), and with type of specific IE components, particularly load bearing. CONCLUSIONS: Multimodal IE can significantly improve motor function in subjects with chronic SCI. An organized program may provide greater motor benefits than a self-regulated program; load bearing might be of particular value. IE might have therapeutic value in chronic SCI, and as an adjunct to other restorative therapies.

MUC1 isoform specific monoclonal antibody 6E6/2 detects preferential expression of the novel MUC1/Y protein in breast and ovarian cancer.

The products of the MUC1 gene are known to be highly expressed in human breast cancer cells. The best characterized MUC1 protein is a polymorphic, type 1 transmembrane molecule containing a large extracellular domain composed primarily of a variable number of 20 amino acid tandem repeats. We have recently identified a novel protein product of the MUC1 gene, the MUC1/Y protein, that is also a transmembrane protein but is devoid of the tandem repeat array and its immediate flanking sequences. To analyze its expression in tumor cells we generated monoclonal antibodies directed against the MUC1/Y extracellular domain (anti-MUC1/Yex MAbs). Epitope mapping identified the MAb, 6E6, which recognized the MUC1/Y isoform with exquisite specificity- the repeat-array-containing MUC1 isoform could not compete out this immunoreactivity. A 30mer peptide which is unique for MUC1/Y and corresponds to the "join" region generated by the MUC1/Y specific splice, abrogated all 6E6 MAb immunoreactivity towards MUC1/Y. Immunoprecipitation of the MUC1/Y protein with 6E6 MAbs revealed that, in contrast with the proteolytic cleavage of the tandem-repeat-array-containing MUC1 isoform, MUC1/Y is not cleaved. Flow cytometry analyses using the 6E6 MAbs demonstrated that the MUC1/Y isoform is expressed on the cell surface of both MCF-7 breast cancer cells and malignant epithelial cells present in effusions obtained from breast and ovarian cancer patients. Our results unequivocally establish that the MUC1/Y protein is expressed on the surface of breast cancer cells and cells of other epithelial malignancies. The anti-MUC1/Y MAbs described here can target MUC1/Y expressing tumor cells in vivo and are likely to be important reagents both for epithelial tumor diagnosis and immunotherapy.

Inhibition of the hydrolytic and transpeptidase activities of rat kidney gamma-glutamyl transpeptidase by specific monoclonal antibodies.

Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long-term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.

Detection of a secreted MUC1/SEC protein by MUC1 isoform specific monoclonal antibodies.

Although MUC1 proteins are known to be secreted by breast cancer cells, the mechanism of their release from the cell is still obscure. Our previously reported MUC1 cDNA sequences suggested the existence of a secreted MUC1 isoform, MUC1/SEC, that includes a sequence of intron 2, and terminates prematurely at a stop codon within this intron. It is thus devoid of a transmembrane domain. As no formal evidence for MUC1/SEC expression at the protein level had been provided, we generated monoclonal antibodies (mAbs) against a peptide sequence (sec peptide) that is unique for the MUC1/SEC protein. Two anti-sec peptide mAbs were obtained which reacted strongly with (a) the immunizing peptide, (b) recombinant MUC1/SEC protein, and (c) MUC1 proteins secreted from breast cancer cells. The immunoreactivity of the anti-sec peptide mAbs with MUC1 proteins secreted by breast cancer cells was specifically inhibited by the sec peptide-it was completely unaffected by a peptide sequence that represents a MUC1 repeat motif. Significantly, the anti-sec peptide mAbs also detected MUC1/SEC protein in sera of breast cancer patients. We have established here that these mAbs recognize the MUC1/SEC isoform via a peptide sequence which is unique for the MUC1/SEC protein. Our studies thus demonstrate that the MUC1/SEC protein is a bona-fide MUC1 isoform and that its expression may contribute to the secretion of MUC1 proteins by secretory epithelial cells in general and breast cancer cells in particular.

Fasciola hepatica: attempts to immunise rats and mice with metabolic and somatic antigens derived from juvenile flukes.

Attempts were made to vaccinate rats and mice against Fasciola hepatica using either somatic or metabolic antigens derived from juvenile flukes between 10 and 16 days old. None of the regimes tried induced a good resistance to subsequent infection, though metabolic antigens derived from 13-14-day-old flukes when injected subcutaneously into rats with adjuvant did produce some protection to challenge.

Hypersensitivity in rats infected with Fasciola hepatica: possible role in protection against a challenge infection.

Rats infected with Fasciola hepatica showed an increase in intestinal mast cells which reached a peak between four and six weeks and fell to control levels by week 14. Following a challenge infection sensitised rats showed evidence of a transitory mild intestinal anaphylactic response. The numbers of intestinal eosinophils, already increased as a result of the primary infection, were rapidly supplemented. In previously uninfected rats the majority of flukes penetrated the mid gut region, but in sensitised rats there was a shift towards the caecal end. Resistance to challenge and a pronounced intestinal eosinophil response were evident in previously infected rats irrespective of the presence or absence of detectable serum reaginic antibody. Systemic anaphylaxis, induced by intravenous fluke antigen administration, occurred whether serum reagins could be detected or not.

The heterologous protection of rats against a challenge with Fasciola hepatica by prior infection with the nematode Nippostrongylus brasiliensis.

Previous infection of rats with nippostrongylus brasiliensis was shown to result in protection against an oral challenge with Fasciola hepatica metacercariae but not against an intraperitoneal challenge with newly excysted juvenile (NEJ) flukes. The timing of the challenge was important and a double infection with the nematode gave more consistent results than a single. Resistance appeared to be associated with a prior induction of intestinal eosinophilia. Sera from these resistant rats, however, failed to induce eosinophil adherence to NEJ flukes in vitro.

The different stages of Fasciola hepatica capable of inducing immunity and the susceptibility of various stages to immunological attack in the sensitised rat.

Intraperitoneal sensitisation of female Piebald Virol Glaxo rats by the migratory immature stages (two-hours-, six-days- and 14-days-old) of the liver fluke Fasciola hepatica protected them from intraperitoneal challenge with living adult flukes three weeks later. Sensitisation with adult fluke antigen and Freund's complete adjuvant had no effect. Flukes attenuated by gamma irradiation (3-8 kiloroentgen), and so prevented from developing beyond the eight to 10 day stage, did not protect against adult challenge. When rats were sensitised orally with metacercariae and challenged with 14-day-old flukes intraperitoneally the challenge flukes were not adversely affected. It is suggested that although 14-day-old flukes can immunise rats adequately, the fact that they are not killed when placed in sensitised rats may be due to the protective effect of tegumental replacement in these flukes before gaining access to, and safety of, the liver site.

The selective adherence of rat eosinophils to newly excysted Fasciola hepatica in vitro.

Eosinophils present in a mixed population of peritoneal cells, derived from either normal or fluke infected rats, selectively adhered to the tegument of newly excysted Fasciola hepatica in vitro in the presence of immune serum. Adherence occurred independently of complement, was not affected by the age of the sensitising infection and could not be induced by artificially raised antisera to dead fluke antigens.

Failure to demonstrate resistance in goats, sheep and cattle to Fasciola hepatica after infection with Cysticercus tenuicollis.

Goats, sheep and cattle given 1000--1500 eggs of Taenia hydatigena and challenged orally 12 weeks later with 400 Fasciola hepatica metacercariae, showed no evidence of resistance. The fluke burdens were not significantly different from those in control animals.

Resistance of the rat to reinfection with Fasciola hepatica and the possible involvement of intestinal eosinophil leucocytes.

Rats infected three weeks previously with 30 Fasciola hepatica cysts were shown to be highly resistant to oral reinfection, as measured by the recovery of immature flukes from the peritoneal cavity 48 h after challenge and confirmed by liver recoveries three weeks after challenge. Eosinophils were prevalent in the lamina propria of the small intestine three weeks after primary infection and increased markedly after challenge.

The establishment and duration of Fasciola hepatica infections in two strains of rats and the development of acquired resistance.

Male and female rats of the inbred Piebald Virol Glaxo ( PVG) and Sprague Dawley (SD) strains were infected with 20 metacercariae of Fasciola hepatica. Three months after infection there was a highly significant difference (P LESS THAN 0-001) in the fluke burden of the two strains. The PVG rats (average 9-4 flukes) were more susceptible than the SD strain (average 2-8 flukes). The PVG males (11-6 flukes) were also found to be significantly more susceptible than the PVG females (average 7-2 flukes) whereas the sex of the SD rats did not affect the fluke burdens significantly. Seven to eight months after infection the PVG rats had eliminated their flukes. These 'self cured' PVG rats were significantly resistant to oral challenge with 20 metacercariae. In marked contrast the SD rats had not eliminated their flukes at the termination of the experiment 12 months after infection.