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R H Jacobson

Publications and source records attributed to R H Jacobson.

At least 37 records · Page 2Linked to original sources
PubMed1996

Lyme disease: laboratory diagnosis of infected and vaccinated symptomatic dogs.

Serological assays for detection of canine antibodies to the Lyme agent generally have been difficult to validate because an acceptable standard of comparison such as unequivocal proof of infection status has not been available. For practical and logistical reasons, it has not been possible to use culture of organism from infected animals, seroconversion in a large number of field dogs, or clinical criteria as the standard of comparison for validation of assays. Therefore, estimates of diagnostic sensitivity and specificity based on an appropriate gold standard have not been available. When it was discovered how to infect laboratory dogs via ticks infected with Borrelia burgdorferi, it was possible to define the kinetics and magnitude of the antibody response that might be expected in nature. ELISA and Western immunoblot data from experimental dogs were then compared and correlated with results of the same tests on dogs from endemic and nonendomic areas. Coupled with studies on cross-reactive antibodies elicited from other infectious agents or autoimmune phenomena, it was possible to account for interfering antibodies and to establish estimates of diagnostic sensitivity and specificity for the ELISA based on objective criteria. Such validated assays can predict, with a relatively high degree of proficiency, the infection and/or vaccinal status of animals. These assays have shown that some dogs, vaccinated with the commercially available whole-cell Lyme bacterins develop typical signs of Lyme disease but have no evidence of an underlying infection; antibody elicited only by the vaccine and not by infection is detectable in these animals. Western immunoblot can also confirm infection in animals of equivocal ELISA status if their bands have been evaluated for specificity of antibodies to B burgdorferi. Serology can be a very useful aid in the diagnosis of Lyme disease, but it requires that the assays used have been subjected to rigorous validation criteria. When that is not performed, an unacceptable level of false-positive and false-negative test results is virtually assured.

Animals↗
PubMed1995

Recombinant OspA protects dogs against infection and disease caused by Borrelia burgdorferi.

Twenty-two specific-pathogen-free beagles were vaccinated with recombinant OspA (ospA gene derived from Borrelia burgdorferi B31) alone or with adjuvant (QuilA, Montanide ISA25, or aluminum hydroxide) at 6 weeks of age. Thirteen dogs were used as nonvaccinated controls with or without adjuvant. Three dogs were kept as contact controls and received neither vaccine nor challenge. Six weeks or 6 months after the first vaccination, the vaccinated (20 of 22) and nonvaccinated dogs (13) were challenged by exposure to adult ticks (Ixodes scapularis) naturally that were infected with B. burgdorferi (tick infection rate, > or = 60%) and that were collected from Westchester County, N.Y. Protection from infection was evaluated by culture for B. burgdorferi from skin biopsies taken near the sites of tick bites. Skin biopsies were taken at monthly intervals for 3 months. B. burgdorferi was not isolated from any of the vaccinated dogs. In contrast, 12 of 13 control dogs challenged by exposure to the ticks were culture positive. The histopathology of the joint capsules 3 months after the challenge was used to evaluate protection from arthritis. Eight of 13 control dogs showed synovitis in single or multiple joints, while only 1 of the 22 vaccinated dogs had a single focus of mild inflammation in a single joint. At the time of the challenge, the vaccinated dogs had antibody to B. burgdorferi, which was demonstrable by kinetic enzyme-linked immunosorbent assay, Western blotting (immunoblotting), and a serum growth inhibition assay. The vaccinal antibody declined gradually after the challenge, especially in dogs vaccinated with OspA without adjuvants. Antibodies in the challenge control dogs were only detectable by 4 to 6 weeks after the challenge and remained at high levels until the termination of the study. Contact control dogs showed no antibody responses or histopathologic lesions and were culture negative. By Western blot analysis, antibodies to OspA first appeared in the sera of vaccinated dogs 3 weeks after the first vaccination. The absence of additional bands after the challenge suggests that infection in vaccinated dogs was blocked. Results from this study show that vaccination with recombinant OspA protected dogs against infection and disease after an experimental challenge with B. burgdorferi by exposure to ticks.

Animals↗
PubMed1995

Sera from OspA-vaccinated dogs, but not those from tick-infected dogs, inhibit in vitro growth of Borrelia burgdorferi.

Dogs were challenged with Borrelia burgdorferi by exposure to ticks, with or without prior protection from infection by recombinant OspA (rOspA) vaccination. Sera from these dogs were tested for their capability to inhibit the growth of B. burgdorferi in vitro. Bacterial growth was detected by a color change in the culture medium, and the optical density was measured with a spectrophotometer in microtiter plates. By growth inhibition, which was complement dependent, the color change was lacking after 5 days of incubation. Over a 1-year study, nonvaccinated dogs infected by exposure to ticks showed high antibody titers to B. burgdorferi by kinetic enzyme-linked immunosorbent assay (KELA). The same sera did not inhibit spirochetal growth or did so only at a low dilution. These results corresponded to the lack of OspA and OspB antibodies seen in Western blots (immunoblots), and these dogs were not protected from infection or disease. In contrast, dogs immunized with rOspA prior to challenge with infected ticks produced high antibody titers, as determined by KELA, but their sera also had high growth-inhibiting antibody titers. Western blot analysis showed a strong band in the 32-kDa region when the sera of these dogs were tested. When adjuvant was administered with rOspA, antibody titers by both KELA and growth inhibition were higher and persisted longer in the immunized dogs. All dogs immunized with rOspA were protected from infection and disease.

Animals↗
PubMed1994

Three-dimensional structure of beta-galactosidase from E. coli.

The beta-galactosidase from Escherichia coli was instrumental in the development of the operon model, and today is one of the most commonly used enzymes in molecular biology. Here we report the structure of this protein and show that it is a tetramer with 222-point symmetry. The 1,023-amino-acid polypeptide chain folds into five sequential domains, with an extended segment at the amino terminus. The participation of this amino-terminal segment in a subunit interface, coupled with the observation that each active site is made up of elements from two different subunits, provides a structural rationale for the phenomenon of alpha-complementation. The structure represents the longest polypeptide chain for which an atomic structure has been determined. Our results show that it is possible successfully to study non-viral protein crystals with unit cell dimensions in excess of 500 A and with relative molecular masses in the region of 2,000K per asymmetric unit. Non-crystallographic symmetry averaging proved to be a very powerful tool in the structure determination, as has been shown in other contexts.

Amino Acid Sequence↗
PubMed1994

BALB/c mice infected with Brucella abortus express protracted polyclonal responses of both IgG2a and IgG3 isotypes.

A polyclonal IgG2a response dependent on the secretion of endogenous IFN-gamma has been demonstrated in BALB/c mice injected with killed whole cells of Brucella abortus [1]. Here we report intense and protracted polyclonal responses of IgG2a and also of IgG3 isotypes in BALB/c mice undergoing primary infections with B. abortus attenuated vaccine strain 19 or virulent strain 2308. Ratios of total serum Ig levels between infected mice and age matched controls were greater than 38 for IgG3 and greater than 12 for IgG2a between weeks 4 and 8 post-infection. Polyclonal increases of IgM and IgG1 that were proportionally much lower (ratios < 2 and < or = 3, respectively) also occurred in infected mice during this time. It is hypothesized that both IgG3 and IgG2a polyclonal responses required IFN-gamma, which was induced by B. abortus primarily in a T cell-independent fashion during the first weeks of infection, and from T cells thereafter.

Animals↗
PubMed1994

Antibody-mediated protection against Brucella abortus in BALB/c mice at successive periods after infection: variation between virulent strain 2308 and attenuated vaccine strain 19.

In BALB/c mice antibodies specific for the O polysaccharide (OPS) as well as T lymphocytes mediate protective immunity to Brucella abortus. We performed quantitative analyses of isotypes of OPS antibodies generated during primary infections, and tested the protective qualities of antisera at successive stages of infection against B. abortus strain 2308, representative of the wild type, and attenuated vaccine strain 19. IgM antibodies predominated during the first 3-4 weeks of infection. IgG3 antibodies increased slowly for the first 3 weeks but then rose rapidly and persisted at high levels (> 300 micrograms/ml). IgG1, IgG2a and IgG2b antibodies had increased slightly by week 4 and then remained at low to moderate levels (< 70 micrograms/ml). Week 2 serum pools (IgM high, IgG3 low or undetectable) transferred substantial protection against 2308 (> or = 1 log unit) which increased relatively little (to 1.2-1.5 log units) with later sera that were high in IgG antibodies. In contrast, week 2 sera conferred low levels of protection against 19 (< 0.6 log units), but protection was dramatically increased (to > or = 2.3 log units) with sera obtained 1 week later that had slightly increased IgG antibodies. Monoclonal IgM antibodies also provided better protection against 2308 than 19, while monoclonal IgG3 antibodies protected much better against 19. Strain 19 opsonized with antibodies taken at any stage of infection was killed within normal macrophages, whereas comparably opsonized 2308 underwent intracellular replication. Phagocytosis of 2308 was better than of 19 when brucellae were opsonized with either polyclonal IgM or IgG3 antibodies, and the difference between strains was more extreme following IgM opsonization. The data suggest an explanation for differences in the growth curves of 2308 and 19 in spleens of BALB/c mice. Higher numbers achieved by 19 at week 2 could result from extracellular replication owing to ineffectual opsonization by IgM antibodies, while the precipitous decline of 19 beginning at week 3 could be caused by the increase in more effective IgG3 opsonins that facilitate its rapid intracellular destruction.

Animals↗
PubMed1993

Expression and secretion of outer surface protein (OSP-A) of Borrelia burgdorferi from Escherichia coli.

Outer surface protein A (OspA) is encoded by the ospA gene from Borrelia burgdorferi. This protein induces immunity against infection in mice. The cloning and expression of OspA in Escherichia coli have been previously described, but the secretion of OspA into culture media in E. coli has not yet been reported. In this report we demonstrate that a chimeric OspA protein was secreted into culture media by E. coli when it also harbors the hemolysin secretion genes hlyBD. The OspA fusion protein was also overexpressed from a T7 promoter and purified by immobilized metal ion chromatography. This was possible because the fusion protein contains six histidyl residues in its N-terminus.

Antigens, Surface↗
PubMed1993

Trichinella spiralis: the effect of specific antibody on muscle larvae in the small intestines of weaned rats.

Weaned rats were passively immunized with rat IgG2c monoclonal antibodies previously shown to protect infant rats against challenge with Trichinella spiralis. Although the antibodies did not protect rats against infection, the course of larval establishment in the intestine was altered in a way that has not been described previously. Specifically, larvae that invaded the epithelium were inhibited from migrating from intestinal tissue into saline during the standard recovery procedure. The inhibition was reversed at a time coincident with the first moult. Whole serum from infected rats had a similar effect. These results show that specific antibodies influence T. spiralis larvae in the intestinal epithelium, yet fail to effect their expulsion from adult rats.

Animals↗
PubMed1993

Babesia bovis: in vitro phagocytosis promoted by immune serum and by antibodies produced against protective antigens.

The in vitro phagocytosis of both Babesia bovis-infected red cells and of parasites exposed by lysis of infected red blood cells is demonstrated in a phagocytic mouse model. Twenty-four B. bovis immune sera were tested alone or as a pool as were antibodies (DS antibodies) raised against a B. bovis protective fraction, prepared by dextran sulfate precipitation. All the immune sera failed to promote significant levels of phagocytosis, whereas the other antibodies (DS antibodies) consistently induced phagocytosis of infected cells in all the experiments carried out. This study shows that antibody specificity is critical to the opsonization of infected red cells and parasites during in vitro phagocytosis and suggests that phagocytosis is one of the mechanisms in the in vivo immune response against Babesia species.

Animals↗
PubMed1993

Cross-reactivity between B. burgdorferi and other spirochetes affects specificity of serotests for detection of antibodies to the Lyme disease agent in dogs.

Western immunoblots, the kinetics-based enzyme-linked immunosorbent assay (KELA), and the microagglutination test were used to evaluate cross-reactivity among antibodies to serovars of Leptospira interrogans (leptospiral serovars), and B. burgdorferi from naturally infected dogs, and to Serpulina (Treponema) hyodysenteriae from vaccinated rabbits. Whole-cell lysates from Borrelia spp., leptospiral serovars, and Serpulina spp. were used for SDS-PAGE, western blots, and KELA. Crossreactivity occurred between the antibodies to B. burgdorferi and leptospiral serovars when tested on the heterologous antigens. Antibodies to leptospiral serovars tended to cross-react more strongly with antigens of B. burgdorferi spp. than did antibodies to B. burgdorferi when tested against antigens of leptospiral serovars. The antibodies against B. burgdorferi showed a lesser degree of cross-reactivity to the antigens of S. hyodysenteriae and S. innocens than they did to leptospiral serovars. We conclude that cross-reactivity occurs between B. burgdorferi and leptospiral serovars. Validation and interpretation of ELISA tests for detection of antibody activity to whole cell lysates of the Lyme agent must take this cross-reactivity into consideration. Conversely, dogs infected with the Lyme agent do not show significant cross-reactivity in the microagglutination test for antibody to the leptospiral serovars.

Agglutination Tests↗
PubMed1993

Experimental Lyme disease in dogs produces arthritis and persistent infection.

Lyme disease was reproduced in specific pathogen-free beagle dogs by exposure to Borrelia burgdorferi-infected ticks (Ixodes dammini). Seroconversion and disease frequency were higher after exposure to infected adult ticks than to infected nymphs. Young pups developed clinical disease more readily than older dogs. The incubation period lasted 2-5 months. Acute recurrent lameness with fibrinopurulent arthritis was the dominant clinical sign. Dogs recovered but developed persistent mild polyarthritis. B. burgdorferi persisted in recovered dogs for at least 1 year. Isolation of B. burgdorferi and detection by polymerase chain reaction was most successful from skin biopsies at the site of the tick bite. Antibody to B. burgdorferi antigens was first detected by ELISA and Western blots by 4-6 weeks after exposure. High serum levels persisted during 17 months of observation. In contrast to infection from ticks, inoculation of dogs with cultured B. burgdorferi resulted in seroconversion with a shorter duration of antibody persistence and no clinical disease.

Age Factors↗
PubMed1993

Identification of antigenic sites mediating antibody-dependent enhancement of feline infectious peritonitis virus infectivity.

We have previously demonstrated antibody-dependent enhancement of feline infectious peritonitis virus (FIPV) infection of macrophages using both virus-specific antisera and monoclonal antibodies (MAbs) to the spike (S) protein of FIPV. To increase our understanding of this phenomenon, six representative MAbs from a previously documented group of 12 enhancing MAbs were used to identify epitopes that mediate antibody-dependent enhancement of FIPV infectivity. Analysis of the results of kinetics-based competitive ELISA (K-cELISA) among these six enhancing MAbs grouped the epitopes into two clusters. Because transmissible gastroenteritis virus (TGEV) and FIPV are so closely related antigenically, we also conducted K-cELISA experiments between the FIPV MAbs and TGEV S protein-specific MAbs for which the epitopes had previously been mapped to specific sites on the TGEV S protein. Results of these assays indicated that the two FIPV epitope clusters are homologues of the previously defined TGEV S protein sites A and E/F. In addition, two TGEV S protein-specific MAbs also induced antibody-dependent enhancement of FIPV infection of macrophages. This functional cross-reactivity provides further support for the close antigenic relationship between FIPV and TGEV. Our results provide a preliminary localization of several enhancing epitopes within the amino acid sequence of the FIPV S protein.

Animals↗
PubMed1993

Reproducibility of a commercial enzyme-linked immunosorbent assay for bovine paratuberculosis among eight laboratories.

Interlaboratory reproducibility of an absorbed enzyme-linked immunosorbent assay (ELISA) kit for detection of bovine serum antibodies to Mycobacterium paratuberculosis was evaluated. A panel of 30 bovine sera (15 positives and 15 negatives) was tested in triplicate microtiter wells on each of 2 days at 8 different laboratories. One laboratory had invalid results because of positive or negative serum control optical density (OD) readings beyond the acceptable range specified by the kit. The coefficient of variation (CV) for mean OD values was influenced by low ODs on test negative sera at 2 laboratories, thus the CVs on positive sera were considered a more representative measure of kit reproducibility. Between-well CVs averaged 6.7% +/- 2.8% (mean +/- standard deviation), and between-day CVs averaged 14.5% +/- 9.8% among the 7 laboratories with valid assays on the 15 positive sera. The OD values were converted to positive or negative classifications for each assay well, and the results were compared. Among 1,392 assays in 7 laboratories, 98.6% were in agreement. Eleven of 18 discrepant results were due to a sample that consistently gave OD values near the cutoff for a positive test. Exclusion of that serum from the analysis resulted in a 99.8% rate of agreement among laboratories. Results indicated that the absorbed ELISA kit provided reproducible results within and between laboratories.

Analysis of Variance↗
PubMed1992

Crystallization of beta-galactosidase from Escherichia coli.

Two crystal forms of beta-galactosidase have been obtained from Escherichia coli. One crystal form is hexagonal space group P6222 or enantiomorph, with cell dimensions a = b = 154 A, c = 750 A. The second form is monoclinic, space group P21, with cell dimensions a = 107.9 A, b = 207.5 A, c = 509.9 A, beta = 94.7 degrees. The monoclinic form seems better suited to detailed structural analysis. The crystals are radiation-sensitive, but by using synchrotron radiation in conjunction with a long (400 mm) crystal-to-film distance it was possible to resolve the individual reflections. On the basis of crystal density measurements, there are four tetramers each of molecular weight 465,000 per asymmetric unit. The Patterson function strongly suggests that two of the tetramers are related to the other two by translation. The data are consistent with the tetramers having 222 point symmetry, but this is not proven.

Crystallography↗
PubMed1992

Structure of a stabilizing disulfide bridge mutant that closes the active-site cleft of T4 lysozyme.

The engineered disulfide bridge between residues 21 and 142 of phage T4 lysozyme spans the active-site cleft and can be used as a switch to control the activity of the enzyme (Matsumura, M. & Matthews, B.W., 1989, Science 243, 792-794). In the oxidized form the disulfide increases the melting temperature of the protein by 11 degrees C at pH 2. The crystal structure of this mutant lysozyme has been determined in both the reduced and oxidized forms. In the reduced form, the crystal structure of the mutant is shown to be extremely similar to that of wild type. In the oxidized form, however, the formation of the disulfide bridge causes the alpha-carbons of Cys 21 and Cys 142, on opposite sides of the active-site cleft, to move toward each other by 2.5 A. In association with this movement, the amino-terminal domain of the protein undergoes a rigid-body rotation of 5.1 degrees relative to the carboxy-terminal domain. This rotation occurs about an axis passing through the junction of the amino-terminal and carboxy-terminal domains and is also close to the axis that best fits the apparent thermal motion of the amino-terminal domain seen previously in crystals of wild-type lysozyme. Even though the engineered Cys 21-Cys 142 disulfide links together the amino-terminal and carboxy-terminal domains of T4 lysozyme, it does not reduce the apparent mobility of the one domain relative to the other. The pronounced "hinge-bending" mobility of the amino-terminal domain that is suggested by the crystallographic thermal parameters of wild-type lysozyme persists in the oxidized (and reduced) mutant structures. In the immediate vicinity of the introduced disulfide bridge the mutant structure is more mobile (or disordered) than wild type, so much so that the exact conformation of Cys 21 remains obscure. As with the previously described disulfide bridge between residues 9 and 164 of T4 lysozyme (Pjura, P.E., Matsumura, M., Wozniak, J.A., & Matthews, B.W., 1990, Biochemistry 29, 2592-2598), the engineered cross-link substantially enhances the stability of the protein without making the folded structure more rigid.

Bacteriophage T4↗
PubMed1992

Antibody responses to Toxoplasma gondii antigens in aqueous and cerebrospinal fluids of cats infected with T. gondii and FIV.

Antibodies to antigens of Toxoplasma gondii were measured in the aqueous and cerebrospinal fluid (CSF) of 16 specific-pathogen free kittens experimentally infected with feline immunodeficiency virus (FIV), T. gondii, or both pathogens. The results indicated that all cats infected with T. gondii had antibody responses to antigens of T. gondii in both aqueous fluids and CSF. Co-infection with FIV did not affect antibody levels. Aqueous fluids from eyes of cats with toxoplasmic retinochoroiditis did not necessarily have higher antibody levels than those from eyes without lesions. Antibodies to T. gondii were also detected in the CSF of two cats from whose brains no parasites were isolated by in vivo mouse inoculation. Total IgG did not increase significantly in the aqueous fluids and CSF of cats infected with T. gondii whether or not they were also infected with FIV.

Animals↗
PubMed1992

Immunological changes in cats with concurrent Toxoplasma gondii and feline immunodeficiency virus infections.

To examine the immunological changes in cats concurrently infected with feline immunodeficiency virus (FIV) and Toxoplasma gondii, kittens (four per group) were inoculated with FIV, T. gondii, both agents, or no pathogens. Blood mononuclear cells and plasma were collected weekly for lymphocyte assays and serology. At week 14, spleen and lymph node cells were used for lymphocyte assays; brains and mesenteric lymph nodes were used for isolation of T. gondii. More T. gondii organisms were present in tissues of the dually infected cats than in tissues of cats with toxoplasmosis alone. Two dually infected cats and one cat infected with T. gondii developed chorioretinitis. Spleen, lymph node, and blood mononuclear cells from dually infected cats had the greatest reduction in mitogenic responses. By week 3, cats infected with FIV underwent a decrease in the number of CD4 cells that was not changed by concurrent T. gondii infection; the number of CD8 cells increased only in cats infected with T. gondii alone. For cats infected with T. gondii, the responses of lymphocytes to T. gondii antigen were not affected by FIV infection; the responses to FIV antigen were negligible in all groups. Overall, this study indicates that FIV infection favors T. gondii proliferation. Also, the establishment of toxoplasmosis may enhance FIV-induced immunodeficiency and is likely to cause a more rapid disease progression than that from infection with FIV alone.

Animals↗