Biomedical subjects
H R Smith
Publications and source records attributed to H R Smith.
Evidence for thymic regulation of autoimmunity in BXSB mice: acceleration of disease by neonatal thymectomy.
BXSB mice spontaneously develop an autoimmune syndrome characterized by autoantibody production, immune complex renal disease, and lymphadenopathy containing B cells. The male BSXB mouse has a rapid onset of disease that is determined by defective stem cells. Although the B cell hyperactivity has been traced to the stem cell, previous studies did not evaluate the T cells of BXSB mice with regard to a role in modifying their natural history. The present study was carried out in an attempt to determine whether or not thymic regulation played a role in modulating the stem-B cell abnormalities in BXSB mice. It was found that neonatal thymectomy led to a marked increase in autoantibody production (anti-RBC, anti-DNA), a dramatic increase in lymphadenopathy, and worsening of renal disease. Flow cytometric analysis of the lymph node cells from intact and thymectomized mice demonstrated a loss of Lyt-2+ cells from the thymectomized mice and an increased proportion of Ly-1+, Thy-1.2- cells. These results indicate that although the B cell hyperactivity of BXSB mice is determined by a marrow stem cell defect, thymic regulation normally serves to dampen that B cell hyperactivity; neonatal thymectomy reduces the thymic regulation of autoimmunity in the BXSB males and leads to excessive B cell numbers and function.
Induction of autoimmunity in normal mice by thymectomy and administration of polyclonal B cell activators: association with contrasuppressor function.
In order to gain insight into the role of polyclonal B cell activation in the development of autoimmunity, non-autoimmune mice were given chronic injections of polyclonal B cell activators (PBA). In addition, to assess the contribution of T cell regulation of such PBA-induced B cell hyperactivity, the additional effect of postnatal thymectomy was studied. Mice that were postnatally thymectomized and given PBA (LPS +/- poly rI X rC) thrice weekly were found to have elevated levels of IgG and significantly increased serum concentrations of anti-ssDNA. This anti-DNA production was greater than that observed with PBA alone or with thymectomy alone. The entire experiment was repeated with different non-autoimmune mice with the same result. Numbers of proliferating cells in the spleens of the mice in the various groups were analysed by flow cytometry. The number of cells in S+G2+M phases of the cell cycle was significantly increased by PBA or thymectomy alone as well as by the combination. As a result, B cell proliferation was not sufficient to result in maximal anti-ssDNA; an additional T cell defect was required. This was further studied in an in vitro assay for suppressor and contrasuppressor activity. In three separate experiments, mice which were clinically autoimmune were found to have defective suppressor function and the presence of abnormal contrasuppressor activity, whereas non-autoimmune controls had normal suppressor function and no contrasuppressor function. These results indicate that the combination of PBA and thymectomy can most easily induce autoimmunity. The autoimmune state so induced in non-autoimmune strains was associated with a failure of normal suppressor function and the abnormal presence of contrasuppressor function. These results have important implications for spontaneously occurring autoimmune diseases.
The effect of the X-linked immune deficiency gene (xid) upon the Y chromosome-related disease of BXSB mice.
BXSB mice develop a lupus-like disease characterized by B cell hyperplasia, hypergammaglobulinemia, autoantibodies, nephritis and coronary artery disease. To determine the subset of B cells responsible for disease in these mice, we bred a congenic BXSB.xid strain (greater than 99.2% inbred) with the xid gene that deletes a subset of splenic B cells. Because BXSB disease is associated with the Y chromosome, BXSB males and the autoimmune cross (NZB X BXSB)F1 males were studied. BXSB.xid males had profoundly reduced lymphoid hyperplasia, hypergammaglobulinemia, autoantibodies, renal disease, cardiac disease, and markedly prolonged survival. (NZB.xid/+ X BXSB)F1 males also demonstrated a marked protection associated with the xid gene. These studies suggest that the autoimmune disease of BXSB males is dependent upon the B cell subset deleted by xid.
Genetic and molecular studies of plasmids coding for colonization factor antigen I and heat-stable enterotoxin in several escherichia coli serotypes.
Plasmids coding for colonization factor antigen I (CFA/I) and heat-stable enterotoxin (ST) were identified in 10 strains of human enterotoxigenic Escherichia coli. The strains, which belonged to serogroups O63, O114, O128, and O153, were isolated in Bangladesh, Latin America, Spain, and South Africa. Two strains produced heat-labile enterotoxin in addition to ST. CFA/I-ST plasmids were mobilized from two O128 strains into E. coli K-12 with the R factor R1-19K-. Like the prototype CFA/I-ST plasmid NTP113, mobilized previously from an E. coli O78 strain into K-12, these two plasmids were non-autotransferring. All 10 CFA/I-ST plasmids were incompatible with NTP113 and had molecular weights ranging from 59 X 10(6) to 72 X 10(6). The molecular properties of seven of these plasmids were compared with those of six CFA/I-ST plasmids previously mobilized from O78 strains from Ethiopia, South Africa, and Bangladesh and with those of one plasmid coding for CFA/I, ST and heat-labile enterotoxin from a South African strain of serogroup O63. Digestion with the restriction endonuclease HindIII showed that several plasmids had very similar fragment patterns and two were identical. Generally, a larger proportion of HindIII fragments were of common size in digests of plasmids identified in strains from related geographical areas, regardless of serogroup. However, all except one plasmid shared five or six HindIII fragments of the same size, one of which had been shown previously to be involved in CFA/I production. There was at least 90% DNA homology between CFA/I-ST plasmids with a molecular weight of about 58 X 10(6) from O78 strains from different sources. Most of the DNA sequences of these plasmids were present in a larger CFA/I-ST plasmid (72 X 10(6) from an O128 strain. The results of genetic and molecular studies suggest that CFA/I and ST production is determined by very similar plasmids in different serogroups of human enterotoxigenic E. coli from several sources.
Mapping of a plasmid, coding for colonization, factor antigen I and heat-stable enterotoxin production, isolated from an enterotoxigenic strain of Escherichia coli.
The non-autotransferring plasmid NTP113 codes for production of colonization factor antigen I and heat-stable enterotoxin, NTP113, which has a molecular weight of 58 X 10(6), was digested with BamHI, EcoRI, and HindIII and combinations of these restriction endonucleases, and the products of these digestions were analyzed by agarose gel electrophoresis. The results were used to construct a partial restriction map of NTP113. Transposons coding for resistance to ampicillin, kanamycin, and tetracycline were inserted into NTP113, and we obtained a series of deletion mutants, as determined by the loss of tetracycline or kanamycin resistance from strains carrying the insertion mutants. A number of plasmid mutants obtained by insertion or deletion did not code for colonization factor antigen I, but most of these mutants still coded for heat-stable enterotoxin production. The position of the inserted transposons and of the deletions were determined on the restriction map. Two regions of NTP113 were required for the expression of colonization factor antigen I, and the two sites were separated by a length of DNA corresponding to a molecular weight of about 25 X10(6).
Bullous eruption of systemic lupus erythematosus. Dramatic response to dapsone therapy.
Four patients with systemic lupus erythematosus developed a nonpruritic vesiculobullous eruption that was unresponsive to high-dose systemic corticosteroid therapy. In three patients the eruption was not associated with a flare of systemic disease. Biopsy results showed neutrophilic microabscesses at the dermal papillary tips and perivascular lymphohistiocytic infiltrates. Direct immunofluorescence of normal appearing skin not exposed to the sun was positive in all four patients. Due to the unresponsiveness to corticosteroid therapy and the striking histologic resemblance to dermatitis herpetiformis, each of the patients was treated with dapsone. Within 24 hours each patient had prompt cessation of the appearance of new lesions. Improvement of the eruption did not correlate with improvement of the systemic manifestations of their lupus erythematosus. The rapid response to dapsone therapy suggests that dapsone is useful in treating bullous lesions of systemic lupus erythematosus.
Studies of the induction of anti-DNA in normal mice.
Injection of PBA has previously been demonstrated to induce anti-DNA. In the present study, we found that the combination of neonatal thymectomy and chronic administration of PBA (LPS + poly rI . rC) led to significantly higher anti-DNA levels than either PBA or thymectomy separately. These results suggested that a thymic regulatory process normally serves to suppress anti-DNA after chronic PBA exposure. Indeed, antigen-nonspecific suppressor function was found to be deficient in such thymectomy + PBA-treated mice. In addition, the cells of such mice in vitro interfered with the development of normal suppressor function by control cells.
Studies of immune abnormalities in systemic lupus erythematosus.
Systemic lupus erythematosus (SLE) in humans and in mice appears to be a syndrome with different cellular bases. In individuals, the illness is influenced by a variety of factors, including genetic, hormonal, immune, and environmental. In mice, SLE can be induced with polyclonal B-cell activators and thymectomy. Retardation of disease occurs with the gene xid on an NZB background. Tolerance studies indicate that tolerance depends upon a normal thymus. In addition, females can be nontolerant with a single immune defect, whereas males can become tolerant with only one defect; they become nontolerant with two immune defects. These studied may help to explain the prevalence of SLE in females and the protective effects of androgens. Human SLE is characterized by excessive B cell activity and impaired T cell activity, especially in active disease. A scheme by which the disease becomes activated is put forth. The details of cell-cell dialogue are becoming clearer with study of the autologous mixed lymphocyte reaction. T 4+ cells provide helper signals for T 8+ cells. Macrophages and T cells combine to regulate the AMLR. The AMLR itself gives rise to a variety of functional cells and serves as an amplification system. This system is defective in active SLE. Preliminary attempts have been made to separate human SLE into subgroups on the basis of the ratio of helper to suppressor cells (RT). A low RT is associated with renal disease, whereas a high RT characterizes patients with a multisystem illness with less important kidney involvement.
Plasmids coding for colonization factor antigen I and heat-stable enterotoxin production isolated from enterotoxigenic Escherichia coli: comparison of their properties.
We examined seven enterotoxigenic Escherichia coli strains which produced colonization factor antigen I (CFA/I). Four of these strains were from South Africa (three serotype O78:H12 and one serotype O63:H-), one was from Ethiopia (O78:H12), and two were from Bangladesh (O78:H11 and O78:H12). Plasmids coding for CFA/I were mobilized from six of these strains by using resistance or enterotoxin factors. No plasmid was mobilized from the serotype O78:H12 Bangladesh strain. The transconjugants obtained from crosses with the O78 strains also produced heat-stable enterotoxin (ST), and additional investigations showed that CFA/I and ST were coded for by a single non-autotransferring plasmid. These plasmids were fertility inhibition negative, did not restrict any of the coliphages with which they were tested, and were incompatible with each other. Four had molecular weights of approximately 60 X 10(6), and one had a molecular weight of 52 X 10(6). Like the other CFA/I plasmids, the CFA/I plasmid transferred from the O63:H- strain coded for ST, but this plasmid also coded for heat-labile enterotoxin. In most other respects the properties of this plasmid were similar to those of the CFA/I-ST plasmids previously described. The molecular weight of this plasmid was 65 X 10(6). The IncT R-factor Rtsl was marked with a transposon for tetracycline resistance and then transferred into the two Bangladesh wild-type strains. Plasmids which coded for tetracycline resistance, CFA/I, and ST were transferred from these strains. These plasmids were incompatible with Rtsl and with the CFA/I-ST plasmids described above and were recombinants between Rtsl and a CFA/I-ST plasmid. Their properties are also described.
Temperature-sensitive mutants of equine arteritis virus.
Seventeen temperature-sensitive mutants of equine arteritis virus, a nonarthropod-borne togavirus, have been isolated. 5-Fluorouracil, o-methylhydroxylamine and ethyl methanesulphonate were used as mutagens. The mutants were characterized by their ability to synthesize virus RNA and virus proteins at the permissive (35 degrees C) and restrictive temperature (40 degrees C) using autoradiography of cells labelled with 3H-uridine in the presence of actinomycin D and immunofluorescence respectively. Among the mutants, four were unable to synthesize virus RNA and virus proteins at 40 degrees C (RNA-/protein-). The other mutants were RNA-/protein+ (3); RNA +/-/protein- (2); RNA+/protein+ (6) and RNA+/protein- (1).
Plasmids coding for heat-labile enterotoxin production isolated from Escherichia coli O78: comparison of properties.
Nineteen enterotoxigenic Escherichia coli strains of serogroup O78, isolated in different geographical areas from humans with diarrheal diseases, were tested for their ability to transfer enterotoxin production. All of the strains originally produced heat-labile enterotoxin, and 16 also produced heat-stable enterotoxin and colonization factor antigen I. Plasmids coding for the production of heat-labile enterotoxin only were transferred from 13 strains. Some properties of these plasmids were compared. All were fi+, but they could be divided into three groups on the basis of their incompatibility reactions, ability to restrict E. coli K-12 phages, and size. The three heat-labile enterotoxin plasmids isolated from African strains all belonged to one enterotoxin plasmid group. The heat-labile enterotoxin plasmids from the Asian strains were divided into two groups, those from serotype O78.H11 differing from those from serotype O78.H12.
Molecular comparison of plasmids encoding heat-labile enterotoxin isolated from Escherichia coli strains of human origin.
The molecular properties of enterotoxin (Ent) plasmids from 12 Escherichia coli strains of human origin were examined. Ten strains belonged to the O78 serogroup, and the remainder were of serogroup O7 or O159. Eleven plasmids coded for heat-labile enterotoxin (LT), and one coded for heat-stable enterotoxin (ST) and LT. The results of restriction enzyme digests and deoxyribonucleic acid reassociation experiments showed that all of the Ent plasmids were related, and supported the subdivision of the LT plasmids into three groups based on their genetic properties (M. M. McConnell et al., J. Bacteriol. 143: 158-167, 1980). Within group 1, two plasmids from South African strains were indistinguishable but differed in EcoRI and HindIII digests from the LT plasmid that originated from an Ethiopian strain. The three plasmids had >70% homology. The two non-autotransferring group 2 plasmids identified in O78.H11 strains from Bangladesh were indistinguishable. The group 3 plasmids were from strains belonging to serogroups O7 and O78 isolated in Bangladesh, India, and Thailand. They shared >95% homology but showed slight differences in fragment patterns when treated with EcoRI and HindIII. There was 60 to 70% homology between the plasmids of groups 1 and 3, and the group 2 plasmid had 40 to 50% homology with members of these two groups. The autotransferring Ent plasmids had up to 40% homology with R factors of incompatibility groups FI, FII, and FIV.
Intraperitoneal transmitters in suckling white-footed mice, Peromyscus leucopus.
Intraperitoneal transmitter implantation in suckling white-footed mice, Peromyscus leucopus, was studied. Postnatal morphological development varied. Age ranged from 9 to 16 days, and body weight ranged from 4.4 to 7.3 g. Interference by mothers prevented successful implantation on sucklings which retained their neonatal appearance. Growth was measured in 6 sucklings for 21 days. Observed growth rates based on daily weight gain per 24 h were 0.29 and 0.32 g for implanted and control mice, respectively. Transmitter implantation resulted in a significant short-term weight loss (mean = 0.56 g) which was recouped prior to weaning. There was no evidence of impaired coordination or locomotor disability. This study concluded that intraperitoneal implantation is possible in suckling P. leucopus if surgery is delayed until venter pelage has developed sufficiently and eyes have been open at least 2 days. A few implications of using this technique to radio track small mammals are suggested.
The value of plasmid studies in the epidemiology of infections due to drug-resistant Salmonella wien.
Since 1969 strains of Salmonella wien that are resistant to multiple antibacterial drugs have caused widespread epidemics of enteritis in Europe and North Africa. Of 113 British strains examined between January 1970 and January 1977, 67 were multi-resistant. These strains and 22 strains from six other countries were examined to determine their plasmid content. Two plasmids were found in the vast majority of strains: an FIme factor, conferring resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, spectinomycin, sulfonamides, and tetracyclines (ACKSSpSuT), and a nonautotransferring plasmid of resistance type ASSu. The FIme plasmids have dual incompatibility: they are incompatible with group FI factors and with the MP10 plasmid of Salmonella typhimurium, which belongs to a separate group. Other plasmids found in S. wien included principally a ColIa factor and an autotransferring plasmid that codes for ampicillin resistance and belongs to compatibility group I2. The similarity in plasmid content of strains isolated in widely separated areas suggests that they have a clonal origin.
Application of agarose gel electrophoresis to the characterization of plasmid DNA in drug-resistant enterobacteria.
A simple gel electrophoresis method has been described for the detection of plasmid DNA in bacteria (Meyers et al., 1976). We investigated further the problems encountered in using this method for the analysis of plasmids in wild enterobacterial strains. The migration of open circular and linear plasmid DNA was examined, since these forms sometimes caused difficulty in the interpretation of the plasmid content of uncharacterized strains. Electrophoresis at different agarose concentrations was employed to resolve clearly plasmid DNA from the chromosomal DNA fragments in the crude preparations. Dissociation of some plasmids occurs in Salmonella typhimurium, and this was detected by electrophoresis. The technique was applied to the study of drug-resistant strains of S. typhimurium phage type 208 from several Middle Eastern countries. The cultures carry a drug resistance plasmid of the FIme compatibility group, and at least two other plasmids which were detected and identified by gel electrophoresis. The studies supported and extended the genetic findings and provided information on the distribution of particular plasmids.
Transposition of ampicillin resistance to an enterotoxin plasmid in an Escherichia coli strain of human origin.
We examined a strain of Escherichia coli, serotype O159.H34, of human origin which produced heat-stable and heat-labile enterotoxins, was resistant to ampicillin, and produced colicin. By conjugation and transformation experiments plasmids coding for enterotoxin production (Ent), enterotoxin production and ampicillin resistance (Ap-Ent), ampicillin resistance (Ap), and colicin production were isolated. Both the Ent and Ap-Ent plasmids were autotransferring and belonged to the F-incompatibility complex. However, the Apr Ent+ transconjugants showed differences in their levels of resistance and in their abilities to propagate F-specific phages and to transfer resistance. The results suggested there was transposition from the small Ap plasmid to the Ent plasmid. The Ap-Ent plasmids were larger than the enterotoxin factor and when treated with restriction endonuclease BamHI showed an additional fragment not present in the enterotoxin plasmid. The insertion of ampicillin resistance probably occurred at different sites on the enterotoxin plasmid, resulting in the observed variation in phenotype.
The case history of a continuing medical education film.
The key provisions necessary in the development, implementation, and evaluation of a film designed for Continuing Medical Education are described. A case history approach is utilized with discussion on each of the following points: (1) problem identification, (2) audience, (3) media selection, (4) media production, (5) research design, (6) media distribution, (7) evaluation and (8) results. Attention to each of the above items contributed to this project's success, and reaffirms the important role which the medium of film can have in continuing professional education.