Biomedical subjects

C W Roberts

Publications and source records attributed to C W Roberts.

At least 55 records · Page 3Linked to original sources

PubMed1995

Antibody responses to Toxoplasma gondii antigen in human peripheral blood lymphocyte-reconstituted severe-combined immunodeficient mice reproduce the immunological status of the lymphocyte donor.

These studies describe the production of specific antibodies in human peripheral blood lymphocyte-reconstituted severe-combined immunodeficient (PBL-SCID) mice following vaccination with antigen from the protozoan parasite Toxoplasma gondii. To determine the effect of previous exposure of the lymphocyte donor to antigen, human-PBL-SCID animals were created by transferring peripheral blood lymphocytes from either a single T. gondii-seronegative or a single seropositive donor. These reconstituted animals were subsequently inoculated with T. gondii soluble tachyzoite antigen (STAg) entrapped within non-ionic surfactant vesicles as an immunological adjuvant. Animals were bled at pre-determined time points post-vaccination and the expression of human anti-STAg antibodies in the plasma determined by enzyme-linked immunosorbent assay. Human antibodies specific for STAg were readily inducible in both groups of reconstituted animals, although the pattern of isotype production differed markedly between groups. The response in animals reconstituted with lymphocytes from the T. gondii-seronegative donor consisted primarily of IgM and subsequently of IgG (predominantly IgG1). In animals reconstituted with lymphocytes from the seropositive donor, no parasite-specific IgM could be demonstrated. The detectable response to STAg consisted entirely of human antibodies of the IgG isotype (IgG1), indicative of a memory-type response. These results mimicked exactly the antibody responses that would be expected had the lymphocyte donors been directly challenged with either the antigen or the live infectious agent, demonstrating that the immune system within these animals is functional and reproducible with regard to both the primary and secondary responses of the human donors.

Animals↗

PubMed1995

Accurate determination of adjuvant-associated protein or peptide by ninhydrin assay.

Modern peptide and subunit vaccines are increasingly having to rely on the use of immunological adjuvants to achieve effective immunity. However, the only adjuvant currently approved for use in humans is aluminium hydroxide, although many adjuvants are currently under preclinical development. Determining immunogen concentration in the presence of adjuvants such as aluminium hydroxide gel, liposomes or NISV has proved to be problematic. One approach has been to use radiolabelled antigens to extrapolate concentration to a preparation using native immunogen. However, the use of a colorimetric assay would allow greater flexibility in terms of immunogen used and would reduce costs and remove safety problems. Of the colorimetric methods we have examined thus far, only the manual ninhydrin assay has produced consistent results with detection of microgram quantities of protein or peptide in the presence of NISV or Alhydrogel, but not liposomes. As the assay relies on the detection of free amino groups after protein hydrolysis, peptides as well as proteins may be effectively determined irrespective of amino acid composition, a considerable advantage over other colorimetric assay systems.

Adjuvants, Immunologic↗

PubMed1995

Sex-determined resistance to Toxoplasma gondii is associated with temporal differences in cytokine production.

Examination of a wide range of inbred mice of diverse genetic backgrounds and major histocompatibility complex haplotypes revealed a dramatic difference in the susceptibilities of males and females to Toxoplasma gondii infection. Female mice were found to be more susceptible to acute infection, as determined by higher mortality levels, than male mice, while those female mice surviving to have chronic infections harbored more cysts in their brains than did surviving males. This phenomenon was therefore investigated in greater depth immunologically in the BALB/K mouse, a strain showing moderate susceptibility to infection with T. gondii. Plasma tumor necrosis factor alpha (TNF-alpha) levels were elevated in both male and female BALB/K mice on days 8 and 10 postinfection, but not thereafter, with males producing significantly higher levels than females. However, it was not until day 12 postinfection that the first deaths occurred, and these were among female mice, indicating that TNF-alpha production was not responsible for mortality. In vitro examination of T. gondii-specific T-cell proliferative responses from day 15 postinfection onwards revealed significantly higher stimulation indices in male mice than in their female counterparts. This difference was most apparent in splenocyte cultures initiated at day 15 postinfection, where complete suppression of proliferation was noted in the splenocytes from female mice but not from male mice. Analysis of tissue culture supernatants from these cultures revealed distinct differences in the kinetics of production as well as the quantities of gamma interferon (IFN-gamma) and interleukin 10 (IL-10) produced. Spleen cells from male mice produced higher levels of IFN-gamma in the early stages of infection than those from female mice. IFN-gamma levels were highest in the supernatants from male splenocyte cultures initiated at day 15 postinfection. Similar levels of IFN-gamma were not obtained from the supernatants of female splenocyte cultures until day 22 postinfection. IL-10 production, on the other hand, peaked at maximal levels in the cell cultures from both sexes initiated at day 22 postinfection. These results suggest that, in male mice, a rapid response to infection with high levels of TNF-alpha and IFN-gamma helps to control parasite multiplication, after which IL-10 production may be important in down regulating these potentially harmful inflammatory mediators. The failure of female mice to respond quickly in terms of T-cell proliferation and IFN-gamma production compared with their male counterparts may account for their poor survival rates and higher cyst burdens.

Animals↗

PubMed1995

Development expression of Hox11 and specification of splenic cell fate.

Hox11 is the first member of a novel class of orphan homeobox genes. We report that Hox11 is expressed in a discrete temporal and spatially segmented pattern during embryonic development and appears critical for the specification of splenic cell fate. Expression is first observed in the developing muscle plates of branchial arches 1, 2, 3 and 4/6, and subsequently within motor neurons of cranial nerves V, VII, IX, and X, which innervate these muscles. Hox11 serves as a molecular maker distinguishing branchial from somatic motor nuclei. Additionally, Hox11 is expressed in the surface ectoderm of the first branchial arch in the region destined to become the tongue and teeth and then in ganglia innervating this area. However, Hox11-deficient mice have no apparent morphological of functional defects within these structures. Notably the closely related homeobox genes, Hox11L.1 and Hox1L1.2, were not expressed in a redundant pattern. Neither Hox11L1 nor Hox11L2 was expressed in the branchial arches or their motor nuclei within wild-type or Hox11-/- mice. Beginning at E11.5, Hox11 is normally expressed at a single site in the abdomen within splanchnic mesoderm destined to form the spleen, and Hox11-/- mice have no spleen. We noted no increase in cell death within the dorsal mesogastrium of Hox11-deficient mice. Instead the dorsal mesogastrium fails to separate from the stomach. Hox11-/- mice display a larger stomach and possibly pancreas, suggesting that these mesodermal cells now contribute to other organs.

Animals↗

PubMed1994

Hox11 controls the genesis of the spleen.

Many homeobox genes are clustered in a linear array along a chromosome, reflecting their ordered expression along the anterior-posterior axis of the embryo. Expression patterns as well as grafting, ectopic expression and loss-of-function experiments suggest that the Hox genes encode a combinatorial system of positional specification along that axis. In contrast, the function of orphan homeobox genes located at sites outside the four mammalian Hox clusters is less well understood. To assess the functional role of the orphan homeobox gene Hox11, we have generated Hox11-deficient mice through gene targeting. Hox11-/- mice have no spleen, but otherwise appear normal. Hox11 is normally expressed in the splenic anlage arising from the splanchnic mesoderm. Hox11-/- embryos have no cellular organization at the site of splenic development but all other splanchnic derivatives develop normally. Hox11 controls the genesis of a single organ, providing new insight into the genetic regulation of morphogenesis.

Animals↗

PubMed1994

Congenital toxoplasmosis in the Balb/c mouse: prevention of vertical disease transmission and fetal death by vaccination.

Vertical disease transmission only occurs in Balb/c mice infected with Toxoplasma gondii for the first time during pregnancy. This is similar to the situation in humans, where a previous infection with T. gondii tends to give life-long immunity against reinfection and fetal disease transmission. The Balb/c mouse therefore provides a suitable model to study the effectiveness of T. gondii vaccine candidates. A soluble tachyzoite antigen (STAg) preparation was used to vaccinate female Balb/c mice. STAg was inoculated subcutaneously into Balb/c mice in phosphate-buffered saline (PBS), emulsified in Freund's complete adjuvant (FCA), or entrapped within non-ionic surfactant vesicles (NISV). While all inocula induced cellular immunity as measured by parasite-specific spleen cell proliferation in vitro, the highest mean proliferative values were observed in spleens from mice where NISV had been used as the adjuvant and the lowest values were observed where FCA had been used. More importantly, cultures from the NISV/STAg vaccinated mice produced significantly more gamma-interferon than the other experimental groups. This vaccine formulation was therefore identified as that most likely to induce protective immunity against toxoplasmosis. Mice were inoculated subcutaneously with either NISV/STAg or STAg in PBS 4 and 2 weeks before mating and infected orally with 20 tissue cysts of T. gondii on day 12 of pregnancy. The incidence of fetal infection and death in these mice and non-vaccinated infected dams was compared. Of 84 pups born to 14 non-vaccinated dams 45 were viable, of which 18 were found to be infected on reaching maturity.(ABSTRACT TRUNCATED AT 250 WORDS)

Abortion, Veterinary↗

PubMed1994

Influence of macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) on Toxoplasma gondii infection in mice.

Functional studies have shown that the murine macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) regulates macrophage priming/activation for antimicrobial activity via the tumour necrosis factor-alpha (TNF-alpha)-dependent production of reactive nitrogen intermediates. Since Toxoplasma gondii also parasitizes macrophages, is a stimulator of endogenous TNF-alpha release, and is sensitive to nitric oxide-mediated killing in activated macrophages, studies were carried out using chromosome 1 congenic mouse strains to determine whether Lsh influences T. gondii infection. Two interesting observations were made: (i) contrary to expectation, mice carrying the Lsh-resistant allele died earlier over the acute phase of infection than Lsh-susceptible mice; and (ii) Lsh-resistant mice which survived this acute phase of infection showed lower brain cyst numbers than the Lsh-susceptible mice. Whilst the latter occurred independently of route of inoculation (oral, intraperitoneal, or subcutaneous), the former was influenced both by the route of inoculation and the genetic background on which the Lsh-resistant allele had been isolated. Hence, following oral administration of 20 brain cysts of the RRA strain of T. gondii, mice carrying the Lsh-resistant allele on a B10 genetic background showed a significantly enhanced rate of mortality over the acute (first 8-12 days) phase of infection than B10 Lsh-susceptible mice. Although this acute phase of infection in B10 background mice was accompanied by an increase in serum TNF-alpha levels in both Lsh-resistant and -susceptible mouse strains, early mortality preceded the TNF-alpha peak, and administration of neutralizing rabbit anti-TNF-alpha did not significantly enhance survival. Hence, inflammatory mediators other than TNF-alpha appear to be responsible for the increased rate of acute mortality observed in resistant mice. Infection intraperitoneally led to delayed mortality in B10 mice, with the mean time to 50% mortality now being significantly longer in Lsh-resistant than in Lsh-susceptible mice. On a BALB genetic background, it was the i.p. route of infection which led to acute mortality and more rapid death in the Lsh-resistant strain. When a less virulent inoculum was used and mortality delayed, Lsh-susceptible mice died more rapidly, and i.p. administration of rabbit anti-TNF-alpha led to 100% mortality between days 8 and 10 of infection in both susceptible and resistant mouse strains, consistent with a crucial protective role for TNF-alpha during this phase of infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Temporal differences in the expression of mRNA for IL-10 and IFN-gamma in the brains and spleens of C57BL/10 mice infected with Toxoplasma gondii.

C57BL/10 Sc Sn (B10) mice infected orally with Toxoplasma gondii tissue cysts were killed at regular intervals up to day 116 post infection (p.i.) and their brains excised. These were used either to count the total number of cysts in the brain, for RNA purification or histopathological studies. Mortality levels in a parallel group of T. gondii infected B10 mice were also monitored and regular plasma samples taken to measure specific antibody production. Seventy per cent of mice died within the first 35 days of infection. Thereafter deaths were infrequent. Inflammation in the brain was apparent from day 10 onwards and by day 25 there was widespread astrocyte activation, perivascular cuffing, meningitis and extensive encephalitis. Total cyst numbers increased rapidly from day 15 to day 35 when they peaked. By day 60, however, cyst numbers had dropped dramatically and this decrease continued through to day 116. Using the polymerase chain reaction mRNA transcripts for IFN-gamma were detected from the first time point sampled, day 25 p.i., until the end of the study. Transcripts for IL-10, an inhibitor of IFN-gamma production, release and activity, were not detected until day 70. The predominant antibody detected against T. gondii was IgG2a but not IgG1. Significantly transcripts for IFN-gamma were found in the spleens of infected but not non-infected animals. Our results suggest that an inflammatory response associated with IFN-gamma production in B10 mice eventually controls T. gondii infection. After the cyst burden has dropped dramatically transcripts for IL-10 are detected in the brain, perhaps to suppress inflammation, and limit pathology.

Animals↗

PubMed1993

Intraocular miotics and postoperative inflammation.

Sixty patients scheduled for planned extracapsular cataract extraction with intraocular lens (IOL) implantation were randomly assigned to receive acetylcholine chloride, carbachol, or balanced salt solution (BSS) immediately after IOL placement and at the end of the surgical case. As part of the postoperative evaluation, patients were examined at the first and eighth postoperative day with a Kowa FC-1000 laser flare and cell meter to assess objectively the amount of postoperative inflammation. At day one and day eight, the amount of cell and flare in the carbachol group was significantly greater than in the acetylcholine group or in the BSS control group. The acetylcholine group had significantly less flare than the BSS group at day one; yet there was no difference between the two groups at day eight, nor was there a significant difference in the amount of cells between day one and day eight. It is presumed that the prolonged miosis seen in the carbachol group delays the re-establishment of the blood-aqueous barrier after surgery, causing the inflammatory response.

Acetylcholine↗

PubMed1993

Influence of genes within the MHC on mortality and brain cyst development in mice infected with Toxoplasma gondii: kinetics of immune regulation in BALB H-2 congenic mice.

Previous work has shown that genes within the major histocompatibility complex (MHC) of the mouse influence resistance and susceptibility to Toxoplasma gondii infection. Initial studies presented here using B10 H-2 congenic and recombinant haplotype mice inoculated via the oral route with the low virulence Beverley strain of T. gondii confirm the D region localization of MHC-linked control of brain cyst number. All B10 mice were, however, exquisitely sensitive to minor changes in virulence of the parasite inoculum resulting in high mortality during the early acute phase of infection. Further experiments examining mortality and brain cyst number in BALB MHC congenic mice inoculated via different routes indicated that the BALB background would provide a more favourable genetic environment in which to analyse kinetics of MHC controlled immune regulation following infection via the natural (oral) route. In studies comparing d and k haplotype mice a dramatic inverse relationship between splenic CD4:CD8 T cell ratios and brain cyst number was observed, particularly in the strain (BALB/K; H-2k) most susceptible to high brain cyst numbers and subsequent toxoplasmic encephalitis. Of particular interest was the observation that splenomegaly and the relative increase in the splenic CD8 T cell population preceded and accompanied the very dramatic and rapid increase in brain cyst formation. The results suggest that the too rapid development of a potent anti-parasite response in the viscera may drive the parasite to encyst in the brain.

Animals↗

PubMed1993

Optical zone diameters for photorefractive corneal surgery.

PURPOSE: To examine the physiological optics of photorefractive corneal surgery and to study the effect on glare production of the optical zone diameter. METHODS: An optical analysis computer program was used to generate rays that define the edge of the optical zone for any given pupil size and glare-free field. RESULTS: The optical zone diameter must be based on the postoperative corneal curvature because the determines magnification of the pupil. The minimal optical zone diameter of uniform optical power was determined both for myopic and hyperopic surgery and for two values of anterior chamber depth. CONCLUSIONS: Optical zone diameters must be at least as large as the entrance pupil diameter to preclude glare at the fovea, and larger than the entrance pupil to preclude parafoveal glare.

Computer Simulation↗

PubMed1993

A comparison of topical non-steroidal anti-inflammatory drugs to steroids for control of post cataract inflammation.

Fifty patients undergoing phacoemulsification with posterior chamber intraocular lens implantation were randomly assigned to receive either diclofenac sodium 0.1% eye drops (Voltaren Ophthalmic, CibaVision Ophthalmics, Duluth, GA) or prednisolone acetate 1.0% eye drops (Pred Forte, Allergan Pharmaceuticals, Irvine, CA) as their postoperative anti-inflammatory medication. The patients were examined one day, one week, and one month after surgery, and their postoperative inflammation was evaluated both by slit lamp assessment of cell and flare, and by objective measurement of cell and flare with the Kowa FC-1000 laser cell and flare meter. At each visit, the level of postoperative inflammation was the same for the two study groups. Thus diclofenac sodium was as effective an anti-inflammatory agent for postoperative inflammation as prednisolone acetate.

Cataract Extraction↗

PubMed1992

Kinetics of cytokine mRNA production in the brains of mice with progressive toxoplasmic encephalitis.

C57BL/10 ScSn (B10) mice infected orally with Toxoplasma gondii were killed on days 5, 10, 15, 20 and 30 post infection and their brains excised. These were either used to count total tissue cyst numbers or divided for RNA purification and histopathological studies. The first signs of inflammation were on day 10 post infection, before the appearance of cysts in the brain, and correlating with the appearance of activated astrocytes. These mice had a mild meningitis with areas of encephalitis. Small numbers of cyst stages were first observed in the brain on day 15 and by day 20 the cyst numbers had increased dramatically but were not always associated with inflammation. After this time point, total cyst numbers did not increase significantly though there developed a marked variation in tissue cyst size with larger cysts becoming more numerous. The use of the polymerase chain reaction to assist in the amplification of brain RNA allowed the characterization of the kinetics of cytokine production within the brains of these animals. Only IL-1 alpha was found to be expressed constitutively in control mice. Transcripts for other cytokines associated with activated monocytes, microglial cells and astrocytes [tumor necrosis factor (TNF)-alpha and interleukin (IL)-6] were present on day 10, (IL-6) and day 15 (TNF-alpha) post infection. Thereafter, these cytokines were present in all infected animals. Of the T cell-associated cytokines, IL-4, a characteristic product of the T helper (Th)2 cell subset, was detected on days 10 and 15, while granulocyte macrophage colony stimulating factor (GM-CSF) which can be produced not only by this cell type but also by Th1 cells and CD8+ T cells, was also present on day 15 but not thereafter. Transcripts for interferon (IFN)-gamma, present from day 15 post infection, were probably produced by CD8+ T cells, as IL-2 which would indicate Th1 cell involvement was only detected 30 days after infection. The continual presence of IFN-gamma and TNF-alpha, cytokines with reported anti-toxoplasmic activity, in the CNS of B10 mice throughout the latter half of the experimental period did not diminish the severity of infection. These results indicate that the CD4+ Th2 subset may allow a rapid rise in cyst numbers and so be important in determining susceptibility to toxoplasmic encephalitis.

Animals↗

PubMed1992

Studies on a murine model of congenital toxoplasmosis: vertical disease transmission only occurs in BALB/c mice infected for the first time during pregnancy.

The incidence of congenital toxoplasmosis was determined by an ELISA in the litters of BALB/c mice which had been infected 8 weeks before mating, on day 12 of pregnancy, or on both these occasions. Of those mice given the infection for the first time on day 12 of pregnancy, 5 out of 6 gave birth to infected litters with approximately 50% of the individuals in each litter being infected. BALB/c mice which had been infected 8 weeks before mating did not give birth to infected litters, even if they were reinfected on day 12 of pregnancy. Following infection BALB/c mice were found to harbour significantly fewer tissue cysts than the congenic H-2 derivative BALB/K strain. However, chronically infected BALB/K mice also failed to produce infected litters, indicating that tissue cyst burden in the dam did not influence congenital infection at least on the BALB background. This study demonstrates that BALB/c dams chronically infected with Toxoplasma gondii, have immunity capable of protecting their embryos from congenital infection, even if the dams are reinfected during pregnancy. Our results demonstrate that the BALB/c mouse can be used as a model of human or ovine congenital T. gondii infection suitable for testing putative vaccines.

Animals↗

PubMed1992

Detection of cytokine mRNA in the brains of mice with toxoplasmic encephalitis.

C57Bl/10 ScSn mice infected with Toxoplasma gondii developed a meningoencephalitis, characterized by areas of tissue destruction and cellular infiltration including foci of neutrophils. Large numbers of cyst stages were found throughout the brain but were not always associated with inflammation. The use of immunocytochemistry to detect glial fibrillary acidic protein, an astrocyte specific marker, showed a widespread astrocyte activation. This was particularly prominent in areas of intense inflammation but cysts were negative for glial fibrillary acidic protein, indicating that astrocytes were not host cells for the bradyzoites. The use of the polymerase chain reaction to assist in the amplification of total brain RNA allowed the characterization of the cytokines being produced locally within the brains of infected animals. beta-actin transcripts were detected in all of the uninfected and infected mice. In only one of the seven uninfected control mice were other transcripts found. Transcripts for tumour necrosis factor-alpha, interleukin-1 alpha and beta, interleukin-6, macrophage inflammatory protein-1 and interferon-gamma as well as the CD4 marker were detected in all of the infected mice. However, transcripts for IL-2 and IL-4 were not present. Several of the cytokines present are capable of initiating meningeal inflammation and may play a role in the immunopathogenesis of toxoplasmic encephalitis.

Animals↗

PubMed1991

Deregulation of a homeobox gene, HOX11, by the t(10;14) in T cell leukemia.

Molecular cloning of the t(10;14)(q24;q11) recurrent breakpoint of T cell acute lymphoblastic leukemia has demonstrated a transcript for the candidate gene TCL3. Characterization of this gene from chromosome segment 10q24 revealed it to be a new homeobox, HOX11. The HOX11 homeodomain is most similar to that of the murine gene Hlx and possesses a markedly glycine-rich variable region and an acidic carboxyl terminus. HOX11, while expressed in liver, was not detected in normal thymus or T cells. This lineage-restricted homeobox gene is deregulated upon translocation into the T cell receptor locus where it may act as an oncogene.

Amino Acid Sequence↗

PubMed1991

Cataract surgery in anticoagulated patients.

A prospective study was performed on 31 patients having planned extracapsular cataract extraction with posterior chamber intraocular lens implantation. The patients were considered to be anticoagulated because of the medications they were taking. The patients were instructed to continue their usual medications throughout the perioperative period including the day of surgery. All patients had routine narcoleptic sedation and retrobulbar anesthesia. The surgical technique was altered to use an inferior corneal traction suture and a single planed clear corneal incision. No intraoperative or postoperative anterior chamber bleeding was seen. The observed complications were increased awareness of corneal sutures, increased endothelial cell loss, delayed visual rehabilitation from with-the-rule astigmatism, and transient corneal edema. All patients achieved 20/40 or better visual acuity without corneal edema by three months post-surgery.

Aged↗

PubMed1991

CD8+ T cells are the major lymphocyte subpopulation involved in the protective immune response to Toxoplasma gondii in mice.

The ability of the major T cell subsets to adoptively transfer resistance to T. gondii infection was studied. Spleen cells harvested from mice with a 3-month T. gondii infection and cells from uninfected mice were enriched for T cells by nylon/wool purification. Adoptive transfer of these cells from both groups of donor mice led to a significant increase in the survival of syngeneic recipient mice infected intraperitoneally with 20 T. gondii cysts. Increased survival was mediated particularly by CD4-depleted but also, to a lesser extent, CD8-depleted subpopulations. These results were confirmed in T cell reconstituted athymic nude mice. Unfractionated T cells from chronically infected donors produced a significant inhibition of cyst formation in the brains of recipient mice measured 10 weeks after infection compared with control mice. The inhibition of cyst formation was ablated by pretreating T cells with anti-CD8 antibody and complement, but not anti-CD4 antibody and complement. Mice receiving cells from infected donors produced an early increase in their IgG1 and IgG2a antibody titres compared with mice given cells from uninfected animals. The depletion of either CD8+ or CD4+ immune cells appeared to have little effect on the antibody responses in recipient mice and there was no correlation between antibody levels and immunity. The results indicate that CD8+ T lymphocytes from convalescent T. gondii-infected BALB/c mice are the principal mediators of resistance to T. gondii, although CD4+ T cells appear to be involved during the acute phase of infection.

Animals↗