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Objective: To assess whether it is possible to measure changes in cord cross-sectional area during a 1-year period in patients with MS reliably., Background: Involvement of the spinal cord in MS is extremely common and an important element in the development of disability. Although little relation has been shown between the cord lesion load and disability, a strong correlation between spinal cord atrophy and the expanded disability status scale (EDSS) has been demonstrated in cross-sectional studies., Method: A highly reproducible semiautomated technique that measures the cross-sectional area of the cord at the C2 level was applied to 13 healthy control subjects and 28 patients serially., Results: This study confirms that patients have significantly smaller cords than control subjects at baseline (control subjects: mean 80.95 mm2, patients: mean 71.25 mm2, p = 0.01) and demonstrates that patients have a significant loss in cord cross-sectional area during 12 months, which was not seen in control subjects (p < 0.001). This reduction in cord size was most marked in the primary progressive patients who had a mean cord cross-sectional area loss of 3.52 mm2 (5.2%) and least in the secondary progressive (-0.26 mm2, 0.7%) and benign patients (-0.41 mm2, 0.8%). The baseline cord cross-sectional area correlated strongly with the EDSS (r = -0.52, p = 0.005) and with disease duration (r = -0.75, p < 0.001); however, there was no significant difference in cord area (p = 0.69) or change in cord area (p = 0.51) between those patients with a definite increase in EDSS and those without., Conclusion: This study demonstrates, for the first time, that it is possible to measure changes in cord cross-sectional area over time. The serial measurement of spinal cord atrophy may thus make an important contribution to the evaluation of therapeutic efficacy, especially in primary progressive disease.

B-cell heterogeneity studies have historically focused upon BALB/c mice and their derivatives. In contrast, the B cells of DBA/2J mice, a prototype strain for the study of the endogenous minor lymphocyte stimulatory (Mls) viral superantigen Mls-1a, have not been extensively investigated. DBA/2J B cells, by functioning as Mls-1a antigen-presenting cells, influence their own differentiation and diversity by inducing the proliferation and differentiation of specific CD4 T-cell subsets. In this report, the B cells of DBA/2J and BALB/c mice were compared for their ability to restore B-cell function in severe combined immunodeficient (SCID) recipients. Although spleen and bone marrow cells from these strains exhibited similar restoration of serum IgM production, the transfer of DBA/2J B cells into SCID mice led to greater IgG1 production. The peritoneal cells of DBA/2J mice consisted of a lower percentage of B-1 B cells and were less capable of restoring B-cell function after transfer into SCID recipients. These differences are discussed with respect to the possible role of viral superantigens in influencing B-lymphocyte diversity.

Mitochondria are responsible for the generation of ATP to fuel muscle contraction. Hypermetabolic stresses imposed upon muscles can lead to mitochondrial proliferation, but the resulting mitochondria greatly resemble their progenitors. During the mitochondrial biogenesis that accompanies phenotypic adaptation, the stoichiometric relationships between functional elements are preserved through shared sensitivities of respiratory genes to specific transcription factors. Although the properties of muscle mitochondria are generally thought to be highly conserved across species, there are many examples of mitochondrial differences between muscle types, species and developmental states and even within single cells. In this review, we discuss (1) the nature and regulation of gene families that allow coordinated expression of genes for mitochondrial products and (2) the regulatory mechanisms by which mitochondrial differences can arise over physiological and evolutionary time.

An isolated, perfused rainbow trout liver preparation was developed to investigate the action of nonthyroidal hormones on hepatic thyroid hormone metabolism. Several assessments were made of the stability and viability of the preparations under a range of conditions, including measures of lactate dehydrogenase flux and tissue ATP and glycogen content, all of which indicated that the perfused liver was stable for the 60-min perfusion period. Moreover, the liver preparations were responsive to an epinephrine challenge and, throughout the series of experiments, sustained hepatic glucose release. Triiodo-L-thyronine (T3) flux from the liver preparation was significantly increased by the provision of thyroxine (T4) substrate. Epinephrine and bovine thyroid stimulating hormone (TSH) were perfused alone and in combination with T4 to evaluate the effect of these hormones on T3 flux from the liver. Both epinephrine and TSH significantly enhanced hepatic T3 flux in the absence of T3 substrate, but neither had an additional effect on T3 flux when perfused in combination with T4. The results of the study suggest that a relationship exists between the circulating levels of nonthyroid hormones and peripheral thyroid hormone metabolism that may be receptor-mediated.

The polarization response of a ferroelectric ceramic displays nonlinear behavior at high applied fields due to saturation effects. Weak-field dielectric aging imposes additional nonlinearity on the hysteresis loops of these materials. Harmonic analysis using a discrete Fourier transform (DFT) permitted an observation of the change in the polarization frequency spectrum as Pb(Mg(1/3)Nb(2/3))O (3)-based relaxor ceramics aged. It also facilitated the calculation of ac current, power, and "internal bias field". The results show that particular harmonics in the polarization signal are sensitive indications of aging behavior. The average power dissipated at weak ac fields was found to decrease with aging time; and the power dissipated at strong fields tended to increase.

Yersinia pestis, the aetiological agent of plague, has in the past caused social devastation on a scale unmatched by other infectious diseases. There is still a public health problem from plague, with at least 2000 cases reported annually. Most of these cases are of the bubonic form. Occasionally bubonic plague develops into pneumonic plague, and this form of the disease can spread rapidly between susceptible individuals. The recent outbreak of plague in India highlighted the potential for plague to explosively re-appear, and modern mass transport systems mean that there is the potential for the rapid spread of disease. Against this background, there is a need to ensure that vaccines and antibiotics are available to prevent and treat the disease. Progress has been made in devising a sub-unit vaccine, effective against bubonic and pneumonic plague. Antibiotics must be given in the early stages of disease to effect a cure.

V antigen of Yersinia pestis is a multifunctional protein that has been implicated as a protective antigen, a virulence factor, and a regulatory protein. A series of V-antigen truncates expressed as glutathione S-transferase (GST) fusion proteins (GST-V truncates) have been cloned and purified to support immunogenicity and functionality studies of V antigen. Immunization studies with GST-V truncates have identified two regions of V antigen that confer protection against Y. pestis 9B (a fully virulent human pneumonic plague isolate) in a mouse model for plague. A minor protective region is located from amino acids 2 to 135 (region I), and a major protective region is found between amino acids 135 and 275 (region II). In addition, analysis of IgG titers following immunization suggested that the major antigenic region of V antigen is located between amino acids 135 and 245. A panel of monoclonal antibodies raised against recombinant V antigen was characterized by Western blotting against GST-V truncates, and epitopes of most of the monoclonal antibodies were mapped to region I or II. Monoclonal antibody 7.3, which recognizes an epitope in region II, passively protected mice against challenge with 12 median lethal doses of Y. pestis GB, indicating that region II encodes a protective epitope. This is the first report of a V-antigen-specific monoclonal antibody that will protect mice against a fully virulent strain of Y. pestis. The combined approach of passive and active immunization has therefore confirmed the importance of the central region of the protein for protection and also identified a previously unknown protective region at the N terminus of V antigen.

A novel approach to making fusions of F1 and V antigens, which may be incorporated into a live recombinant vaccine for plague, was developed. The nucleotide sequences encoding Yersinia pestis V antigen (lcrV) and the mature form of F1 antigen (caf1) were amplified by PCR with primers which included tails. At the 3' end of caf1 and the 5' end of lcrV, the tails encoded one of three six- or eight-amino acid linkers or their complementary sequences. The DNA overlap in each linker region was used to prime a second PCR to generate three F1/V fusions, which were cloned into pUC18. The resulting plasmids expressed fusion proteins consisting of F1 and V antigens, separated by the linkers Gly-Ser-Ile-Glu-Gly-Arg, Ser-Ala-Pro-Gly-Thr-Pro or Ser-Ala-Pro-Gly-Thr-Pro-Ser-Arg. As shown by Western blotting of bacterial cell lysates with anti-V and anti-F1 sera, the level of expression and degree of degradation of the three fusion proteins was similar. To investigate the immunogenicity of F1/V, one of the plasmids, placFV6 which encoded the Gly-Ser-Ile-Glu-Gly-Arg linker, was electroporated into the attenuated Salmonella typhimurium strain SL3261 (aroA). Mice receiving two intravenous doses of 5 x 10(6) cfu SL3261/placFV6 developed serum anti-V and anti-F1 IgG titres, with similar IgG1:IgG2a isotype ratios, and T cell responses specific for V and F1 antigens. Six weeks after vaccination, mice were challenged subcutaneously with 7.4 x 10(2) or 7.4 x 10(4) LD50s of Y. pestis strain GB, and a significant degree of protection was demonstrated. These results demonstrate the potential of co-expressing Y. pestis antigens as fusion proteins to develop a live recombinant vaccine against plague.

The purified recombinant V antigen from Yersinia pestis, expressed in Escherichia coli and adsorbed to aluminum hydroxide, an adjuvant approved for human use, was used to immunize outbred Hsd:ND4 mice subcutaneously. Immunization protected mice from lethal bubonic and pneumonic plague caused by CO92, a wild-type F1+ strain, or by the isogenic F1- strain C12. This work demonstrates that a subunit plague vaccine formulated for human use provides significant protection against bubonic plague caused by an F1- strain (C12) or against substantial aerosol challenges from either F1+ (CO92) or F1-(C12) Y. pestis.

Direct effects of 3,5,3'-triiodothyronine (T3) and 3,5-diiodothyronine (T2) on the metabolism of the goldfish (Carassius auratus) were assessed using mitochondria isolated from liver and red muscle. Following a 5-min incubation with either T3 or T2, the oxidation rates of substrates involved in amino acid and carbohydrate metabolism and lipid catabolism were measured. State 3 oxidation of pyruvate was significantly higher for liver mitochondria treated with T2 and for red muscle mitochondria incubated with T3 when compared to control mitochondria. Rapid elevation of state 3 rates of substrate oxidation by thyroid hormones may be important in mediating diurnal changes in mitochondrial metabolism. Significant increases in liver and red muscle mitochondrial state 4 rates were also observed for pyruvate in T2- and T3-treated mitochondria and for glutamate in T3-treated mitochondria.

In this study, we have determined the limit of protection achievable by immunisation with sub-units of Yersinia pestis against the development of plague in an experimental animal model. Co-immunisation with the purified culture-derived F1 and the recombinant V sub-units afforded a greater level of protection than with either sub-unit alone. The protection given by the combined sub-units was several orders of magnitude greater than that afforded by the whole cell killed (Cutter USP) vaccine and was equivalent to that achieved by vaccination with EV76, the live attenuated Y. pestis vaccine strain. However, the combined sub-unit vaccine has clear advantages over the live vaccine in terms of safety of use and absence of side-effects.

The gene encoding V antigen from Yersinia pestis was cloned into the plasmid expression vector pGEX-5X-2. When electroporated into Escherichia coli JM109, the recombinant expressed V antigen as a stable fusion protein with glutathione S-transferase. The glutathione S-transferase-V fusion protein was isolated from recombinant E. coli and cleaved with factor Xa to yield purified V antigen as a stable product. Recombinant V antigen was inoculated intraperitoneally into mice and shown to induce a protective immune response against a subcutaneous challenge with 3.74 x 10(6) CFU of virulent Y. pestis. Protection correlated with the induction of a high titer of serum antibodies and a T-cell response specific for recombinant V antigen. These results indicate that V antigen should be a major component of an improved vaccine for plague.

An attenuated Salmonella typhimurium strain which expressed the F1 capsular antigen of Yersinia pestis was constructed by transformation of S. typhimurium SL3261 with plasmid pFGAL2a, a derivative of pUC18 which contained the caf1 gene without the leader sequence. The recombinant was used to vaccinate mice intragastrically and intravenously. The immunity induced was able to protect mice against challenge with a virulent strain of plague. Protection correlated with the induction of high titers of immunoglobulin G in serum samples and a specific T-cell response.

RNA-based amplification systems that have been recently described are dependent upon the presence of more than one enzyme. In an attempt to minimize the number of polymerases required for efficient amplification, we have studied the template specificity of bacteriophage T3 RNA polymerase. A synthetic bacteriophage T3 promoter was covalently attached to an RNA template. The T3 promoter-RNA complex was found to be selective for its native polymerase, and dependent upon the presence of all four ribonucleoside precursors. The product of the RNA-directed transcription is complementary to the initial template.

To test whether exposure to dichlorvos vapors for treatment of mouse ectoparasites resulted in temporary cessation of breeding, we exposed harem breeding groups of mice to varying concentrations of dichlorvos vapors and examined the effects of exposure on litter frequency and litter size. All exposure levels resulted in decreased plasma cholinesterase concentrations in treated mice for up to 10 days following the completion of exposure. Litter frequency and size were unaffected by dichlorvos exposure, and gestation times were not prolonged. Therefore, treatment with dichlorvos vapors during breeding did not affect reproduction in exposed mice.

The effects of HER upon early and late stages of BBN-induced bladder cancer in rats were examined. Female Fischer 344 rats were administered HER in the diet either before and during or continuously after BBN administration and were monitored periodically for up to 2 years. The total dose of BBN was 600 mg administered over a 6-week period. In a separate experiment, the effects of HER administration to syngeneic recipients of a transplanted primary bladder cancer were examined. No effects on neoplastic development were observed as the result of HER treatment before and during carcinogen administration. However, at the 1-year sacrifice, there was a significant increase in bladder tumor incidence in the animals receiving BBN followed by continuous retinoid treatment versus animals receiving BBN only. At the 2-year sacrifice, there was a significant increase in tumor progression in the continuous retinoid group versus the animals receiving BBN alone, based upon grading and staging of tumors, although tumor incidences were not significantly different. In the transplantation experiment, more recipients (9/20 versus 2/20) receiving continuous HER had large, anaplastic tumors following 9 months of observation than did control animals. This study supports the view that retinoids should not be considered as only inhibitors of carcinogenesis, but rather as modifiers which vary in their effects depending upon factors yet to be understood.

Gastric foreign bodies were induced in laboratory rabbits by orogastric infusion of a liquid latex containing radiopaque dye which polymerizes in the acid environment within the stomach to form a solid mass. The rabbits were monitored clinically and radiographically for 6 months at which time gastrotomies were performed to remove the masses, followed by a 4-week observation period. None of the 14 rabbits became ill or anorectic during the 6-month period of clinical monitoring, and all gained weight. At gastrotomy, eight rabbits had both latex bezoars and trichobezoars . Though two rabbits died post-operatively from respiratory complications, the remaining 12 rabbits recovered without complications and regained their pre-operative body weight within 4 weeks. Five of 10 rabbits necropsied 1 month after gastrotomy had gastric trichobezoars . This led to a survey of the stomachs of 208 healthy slaughter-rabbits which revealed that 48 (23.1%) had trichobezoars weighing 1 to 24 g. The data suggested that gastric trichobezoars were common in rabbits and that few animals with these foreign bodies developed chronic anorexia. In addition, there appeared to be a high likelihood that trichobezoars recur in a high percentage of rabbits within several weeks postoperatively.

Baboons with long term, indwelling, intravascular catheters developed clinical signs of renal and hepatic impairment. These included proteinuria and hypoalbuminemia without edema, and albumin to globulin ratios were reversed. Serum IgM, IgG, rheumatoid factor, and liver enzyme concentrations were above normal. Immunofluorescent staining of renal glomerular capillary loops was positive for IgG, IgM, B1c, and C4. Major microscopic lesions were membranoproliferative glomerulonephritis, chronic active hepatitis, degenerative arthritis, and chronic sialoadenitis. Electron microscopy of renal glomeruli demonstrated dense deposits in a variety of locations, mesangial cell interpositioning, and foot process fusion. These alterations, found in conjunction with the isolation of Staphylococcus aureus from the blood of affected baboons as well as the intravascular catheters, suggested that chronic bacterial infection was important in the pathogenesis of this disease.

A laboratory animal technician experienced undulating fever, chills, and myalgia 3 days after he was bitten by a laboratory rat. The clinical symptoms subsided with antibiotic therapy, but recurrent fever, malaise, and joint pain occurred when therapy was discontinued. Streptobacillus moniliformis was cultured from the patient's blood.

Shope papillomavirus induced benign and malignant tumors from both wild and domestic rabbits were analyzed by the Northern blotting technique for the presence of viral-specific RNAs. Virus-producing benign tumors of wild cottontail rabbits are shown to contain two major RNA species approximately 5000 and 3000 bases in length that originate in the early region and include the majority of the L1 and L2 late open reading frames. Non-productive tumors including wild-rabbit carcinomas, and benign warts and primary and metastatic carcinomas of domestic rabbits uniformly are shown to contain two major viral-specific RNA species, 2400 and 1400 bases in length. These transcripts map entirely within the early region of the viral genome. In addition, Southern blot analysis of metastatic tumors indicates that the viral DNA remains exclusively extrachromosomal and apparently unrearranged supporting previous findings with epithelial malignancies.

The lipopolysaccharides (LPSs) of five isolates of Pasteurella multocida from rabbits were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblots, and enzyme-linked immunosorbent assay. Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of purified unaggregated LPSs resembled those of semirough strains of gram-negative enterobacteria and consisted of one or two bands that migrated within an interval just ahead or slightly behind the migration of the Ra chemotype of "Salmonella minnesota," which has a molecular size of 4.3 kilodaltons. Polyclonal rabbit antisera to P. multocida whole cells used in Western blots and enzyme-linked immunosorbent assays of unabsorbed and LPS-absorbed antisera revealed that the LPS of these isolates of P. multocida contained at least two types of antigens: a nonserospecific antigen and a serospecific antigen. The LPSs of four isolates each had a different serospecific antigen. The nonserospecific antigen was expressed in two isolates and was the only demonstrable LPS antigen in one other isolate.

beta-Glucuronidase mRNA was purified from androgen-induced mouse kidney by immunoadsorption of polysomes to protein A-Sepharose. Cell-free translation of mRNA isolated from the protein A bound RNA followed by immunoprecipitation revealed that beta-glucuronidase mRNA represented approximately 2% of the purified mRNA fraction. This mRNA preparation was used to produce complementary DNA clones by recombination with pBR322. Clones containing sequences that were enriched during the purification procedure were selected by differential colony hybridization. These were further screened for homology with beta-glucuronidase mRNA by hybrid-selected translation. A beta-glucuronidase cDNA clone, designated pGUS7, was identified by these criteria. With this plasmid, the abundance of beta-glucuronidase mRNA in total poly(A) mRNA from androgen-induced mouse kidney was estimated to be less than 0.04%. The beta-glucuronidase cDNA plasmid hybridized to a mRNA of 2.6 kb in length, which was induced in an androgen receptor dependent fashion over a time course of 21 days. Treatment of female mice with a single dose of testosterone (10 mg) revealed that beta-glucuronidase mRNA concentration begins to increase between 12 and 24 h after hormone administration.

The Gus-s alpha allele of the mouse beta-glucuronidase gene exhibits a high degree of inducibility by androgens due to its linkage with the Gus-r alpha regulatory locus. We isolated Gus-s alpha on a 28-kilobase pair fragment of mouse chromosome 5 and found that it contains 12 exons and 11 intervening sequences spanning 14 kilobase pairs of this genomic segment. The mRNA cap site was identified by ribonuclease protection and primer extension analyses which revealed an unusually short 5' noncoding sequence of 12 nucleotides. Proximal regulatory sequences in the 5'-flanking DNA and the complete sequence of the Gus-s alpha mRNA transcript were also determined. Comparison of the amino acid sequence determined from the Gus-s alpha nucleotide sequence with that of human beta-glucuronidase indicated that the two human mRNA species differ due to alternate splicing of an exon homologous to exon 6 of the mouse gene.

A method is described for the high-level transcription of any DNA segment using bacteriophage RNA polymerases (RNAPs). A synthetic mobile promoter with a template-complementary 3' extension is ligated to the target sequence of interest. Transcription with an appropriate RNAP results in an amplification of approx. 70-fold. In the presence of heterologous DNA, bacteriophage RNAPs are shown to be specific for their cognate mobile promoters.

A dot-immunobinding assay was developed to detect serum IgG specific for lipopolysaccharide of rabbit isolates of P. multocida. The assay detected serum IgG as early as 1 week after experimental subclinical nasal infection, whereas 8 weeks were required to detect antibody by a gel diffusion precipitin test. The assay was more reliable than nasal cultures, in that up to 46% of 16 weekly nasal washings of some infected rabbits failed to yield P. multocida. The bacterial antigen (proteinase k digested cell lysate) used in the assay reacted with IgG that did not cross-react with lipopolysaccharide antigens of B. bronchiseptica, P. pneumotropica or P. hemolytica. The assay is sensitive and specific, easily performed, cost effective, requires no special laboratory instruments and provides a permanent easily stored record.

Adjuvant arthritis (AA) in rats is susceptible to cell-mediated passive transfer. Collagen-induced arthritis (CIA) in rats is susceptible to passive transfer with antibody to type II collagen. We report here the development of strikingly severe arthritis in Lewis rats as the result of synergy between passively transferred antibody to type II collagen from rats with CIA and concanavalin A (Con A)-stimulated lymph node or spleen cells from syngeneic rats with AA. Similar synergy was seen in rats with AA given anticollagen antibody, in rats with CIA given Con A-stimulated adjuvant spleen cells, and in rats actively immunized with CII and complete Freund's adjuvant. The synergistic process caused a very severe polyarthritis, characterized by marked swelling and erythema in all the joints of the distal extremities, with histologic and radiographic evidence of early, extensive erosion of articular cartilage. Synergy was apparent if the lymphoid cells from AA rats were given up to 1 mo after a single injection of anticollagen antibody. No synergy was seen when normal rat immunoglobulin or anti-ovalbumin antibody was substituted for anticollagen antibody, when Con A-stimulated lymphoid cells from normal rats or donors with CIA were used, or when Con A-stimulated AA lymphoid cells were irradiated before transfer. Synergy between separate immune effector mechanisms may represent a general phenomenon in the pathogenesis of inflammatory joint disease.