Rat retinal pigment epithelial cells express an inducible form of nitric oxide synthase and produce nitric oxide in response to inflammatory cytokines and activated T cells.

C4-deficient (C4D) and Albany strains of guinea-pigs transplacentally and neonatally infected with Treponema pallidum showed distinctive patterns of humoral immune responses. Congenitally infected progeny of both strains originated from dams intradermally (i.d.) infected at mid-pregnancy with virulent T. pallidum. In the neonatal groups families of C4D and Albany strains consisting of 1-3-day-old offspring and their mothers were i.d. infected with a similar dose of T. pallidum. Regardless of the strain, asymptomatic congenitally infected guinea-pigs (n = 16) responded from the first day of life with high levels of IgM [T. pallidum (TP) ELISA] antitreponemal antibodies and up to 85% presented with IgM CIC (circulating immune complexes) and IgM RF (rheumatoid factor). Although relatively high levels of IgM antitreponemal antibodies persisted in these animals throughout the 4-month experimental period, significant levels of host IgG antitreponemal antibodies were detectable after 2-3 months of age. Neonatally infected guinea-pigs of both strains (n = 27) responded similar to the infected sow but with relatively lower levels of IgM and IgG antitreponemal antibodies at 1 and 4 weeks, respectively, both of which increased with the time of infection. Antibodies were also detected in these animals by fluorescent treponemal antibody adsorption test (FTA-ABS). Unlike congenital syphilis, neonatally infected animals developed IgG-CIC after 2-3 months of infection and none of them showed any RF. In neonatal syphilis, FTA-ABS antibody levels were closely associated with the onset of lesions, whereas those of TP ELISA were not. The distinctive immune responses observed in these experimental models have the potential to differentiate between congenitally and neonatally infected human infants, even though the current clinical management is the same.     

Treponema pallidum subsp. pertenue displays pathogenic properties different from those of T. pallidum subsp. pallidum

The present study described the susceptibility of C4D guinea pigs to cutaneous infection with Treponema pallidum subsp. pertenue Haiti B strain. The general manifestations of the disease in adults and neonates differ, to a certain degree, from those induced by T. pallidum subsp. pallidum Nichols strain. Noticeable differences between the infections were reflected in the character of the skin lesions, their onset and persistence, and the kinetics of the humoral response. The incidence and dissemination of cutaneous yaws lesions in very young guinea pigs were remarkably different from the low frequency observed in a similar age group of syphilis infection, 100 versus 17%, respectively. Moreover, as opposed to T. pallidum subsp. pallidum, T. pallidum subsp. pertenue does not cross the placenta. Offspring born to yaws-infected mothers did not produce immunoglobulin M antibodies and their organs, examined by PCR and rabbit infectivity test (RIT), were all negative. Examination of a large number of tissues and organs in adult, neonate, and maternal yaws by PCR and RIT clearly demonstrated that, unlike syphilis, there was a low incidence and short persistence of the yaws pathogen in internal organs. These findings stress the dermotropic rather than the organotropic character of yaws and provide further evidence of distinctive biological and pathological differences between yaws and venereal syphilis.     

Experimental congenital syphilis: guinea pig model.

Neonates born to female guinea pigs of either a highly susceptible (C4D) or a resistant (Albany) strain, infected prior to or during pregnancy with a single dose of Treponema pallidum, showed in their sera from the first day of life immunoglobulin M (IgM) antibodies to T. pallidum, circulating immune complexes consisting of IgM antibodies and treponemal antigens, and IgM rheumatoid factor. Although the animals were asymptomatic for a 6-month observation period, several lines of evidence indicated that they were infected in utero. Molecular analysis of whole sera, purified serum IgM fraction, or dissociated immune complexes demonstrated IgM reactivity against one (47 kDa) or more of several T. pallidum peptides (15, 17, 37, 42, 45, and 87 kDa) recognized as integral membrane components. Sequential analysis of the neonates' sera by immunoblot and enzyme-linked immunosorbent assay, using alcohol-treated T. pallidum, T. phagedenis biotype Reiter, and T. vincentii, demonstrated early IgM antibodies followed 3 to 4 months later by IgG2- and IgG1-specific antibodies to T. pallidum. Moreover, an infectivity test done in five rabbits with pooled tissue extracts prepared from liveborn or stillborn animals evoked a seroconversion in two rabbits (reactive Venereal Disease Research Laboratory and fluorescent treponemal antibody tests), suggesting the presence of T. pallidum in the organs. Sera from neonates born to either T. phagedenis biotype Reiter-injected mothers or three normal pregnant females were all serologically negative. The model offers new possibilities for exploration of factors responsible for asymptomatic infection often observed in human congenital syphilis.     

Target organs of infection in guinea pigs with acquired congenital syphilis.

The target organs of infection in guinea pigs with asymptomatic acquired or congenital syphilis were identified by PCR and in some cases by rabbit infectivity test (RIT). The prevalence of Treponema pallidum DNA was examined in the following seven organs: the inguinal and mesenteric lymph nodes, spleen, liver, kidney, heart, and brain. Test samples consisted of 95 organs from two genetically different strains of female guinea pigs (C4-deficient and Albany) with different susceptibilities to cutaneous infection by T. pallidum and 195 organs from their asymptomatic offspring. Twenty organs from dams of both strains injected with heat-killed T. pallidum and 19 organs from their progeny served as negative controls. The infections of mothers and neonates were documented by PCR, RIT, and serology. Though any of the organs tested could be infected, there was a spirochetal predilection for some anatomical locations, such as the lymph nodes, heart, and brain, regardless of the strain, route of maternal infection, and age. None of the 49 organs collected from control animals were positive by PCR. In infected C4-deficient dams, one to four organs were positive by PCR, whereas the organs of 7 of their 27 (25%) asymptomatic offspring were treponemal DNA negative, despite evidence of immunoglobulin M treponemal antibodies. Comparative analysis done by both PCR and RIT on a limited number of samples showed 90% agreement between results. An examination of multiple samples obtained from single organs demonstrated that even within 24 h of spirochetemia, when most organs appeared to be infected, not all samples from an individual organ were positive by PCR. A specific immunological response in guinea pigs with congenital syphilis was a more consistent parameter of vertical transmission than was an analysis of T. pallidum DNA.     

Adoptive transfer of immunity to Treponema pallidum Nichols infection in inbred strain 2 and C4D guinea pigs.

T lymphocytes purified from lymph nodes and spleens of chancre-immune, inbred strain 2 guinea pigs, when infused into syngeneic guinea pigs, conferred protection against challenge with Treponema pallidum subsp. pallidum Nichols. No protection was conferred by similar injections of cell suspensions from normal guinea pigs or guinea pigs immunized with T. phagedenis biotype Reiter or T. pallidum-free testis supernatants from infected rabbits. Similar results were obtained with homozygous C4D guinea pigs. After several months of infection, 2 of 11 strain 2 and 1 of 8 strain C4D recipients of T. pallidum-immune cells developed an erythematous reaction of short duration at the injection site; 2 of these recipients were positive for T. pallidum. Throughout the experimental period the humoral response to treponemal antigens was substantially lower in the adoptively immune guinea pigs than in various unprotected control groups. Passive immunity to infection with T. pallidum, however, seems to be dose related, since asymptomatic infection persisted for as long as 3 months after challenge in strain 2 guinea pigs transfused with 10(8) T. pallidum-immune lymphocytes, but not in C4D recipients of twice as many immune cells.     

The time-dependent clearance of virulent Treponema pallidum in susceptible and resistant strains of guinea pigs is significantly different

The kinetics of clearance of Treponema pallidum spp. pallidum Nichols from skin and testes of susceptible C4-deficient (C4D) and -resistant Albany (Alb) strains of guinea pigs (gps) was evaluated using the polymerase chain reaction (PCR) and the rabbit infectivity test (RIT). For each strain there were two groups of animals, one infected with virulent T. pallidum (TP) and one control injected with heat-killed treponemes (HKTP). The kinetic studies and their statistical analysis showed that in the C4D strain the microbial clearance in both tissues was significantly slower (p < 0.005) and still incomplete at 3 months after infection. In the Alb strain the clearance was faster and apparently completed within a month. A greater permissiveness in bacterial growth in C4D compared to Alb appears to be one critical factor determining the different rate of local elimination after primary infection. In both strains there was some correlation between the severity and duration of cutaneous lesions and the local persistence of viable organisms. This correlation was not observed in testes. These studies suggest a genetic basis for the strain-specific susceptibility and resistance phenotypes in the pathogenesis of syphilis.     

Immunization of guinea pigs with recombinant TmpB antigen induces protection against challenge infection with Treponema pallidum Nichols.

Treponema pallidum-susceptible guinea pigs of strain C4D were immunized with recombinant T. pallidum antigens TmpA, TmpB, TmpC, and TmpA plus TmpB plus TmpC; with Escherichia coli membranes; or with adjuvant alone. Animals in groups of five received six immunizing injections, each of 100 micrograms of antigen incorporated in RIBI adjuvant. After the sixth immunization, all experimental and nonimmunized controls were intradermally challenged with 3 x 10(6) T. pallidum Nichols freshly extracted from infected rabbit testes. Although high titers of antitreponemal antibodies in the fluorescent-treponemal-antibody test or an enzyme-linked immunosorbent assay were evoked in all animals immunized with recombinant antigens, only guinea pigs receiving TmpB antigen demonstrated protection expressed by the development of significantly (P less than 0.01) smaller, atypical lesions of significantly (P less than 0.01) shorter duration and devoid of or containing fewer T. pallidum organisms than lesions in the remaining immunized and control animals.     

Studies on the pathogenesis of experimental autoimmune renal tubulointerstitial disease in guinea pigs. VI. Induction of renal lesions by active or passive immunization of strain 2 guinea pigs.

It has been reported that strain 2 guinea pigs do not develop experimental autoimmune renal tubulointerstitial disease (RTD) by active or passive immunization. Under our experimental conditions strain 2 guinea pigs are susceptible to the induction of RTD. Typical moderate to severe renal lesions were seen 22 days after immunization with rabbit tubular basement membrane in complete Freund's adjuvant. Most Albany strain guinea pigs had severe RTD. Susceptibility to induction of RTD by passive transfer of antitubular basement membrane autoantibodies was studied in Albany (A) and strain 2 (2) guinea pigs, as well as in A leads to A, A leads to 2, and 2 leads to A radiation chimeras. Only some strain 2 guinea pigs had mild to severe renal lesions by day 9, but by day 19 all had typical histologic renal abnormalities. Albany guinea pigs already had moderate to severe RTD by days 9-10. Strain 2 differed from Albany recipients by a noticeable delay in the onset of lesions. Bone marrow transplants between Albany and strain 2 did not affect these susceptibility differences. A leads to 2 recipients responded like strain 2, and 2 leads to A like Albany. This indicates that the delay of onset of RTD in strain 2 is not a defect in the bone marrow-derived inflammatory cells.     

Strain- and age-associated differences in lymphocyte phenotypes and immune responsiveness in C4-deficient and Albany strains of guinea-pigs.

Spleen lymphocytes from C4-deficient (C4D) and Albany strains of guinea-pigs, 1-7 days, 3-6 and 12-16 months old, genetically related to inbred strains 13 and 2 respectively, were analysed in terms of their expression of cell surface markers, allogenic and T- and B-cell mitogenic responses, and interleukin-1 (IL-1) and IL-2 production. There were strain- and age-associated differences in phenotypic expression and immune responsiveness levels. In both strains a significant shift in immunocompetence apparently occurs postnatally before 3-6 months of age, with no further significant changes noticed in animals 12-16 months old. Phenotypic changes in cell surface markers did not always correlate with functional capability of lymphoid cells. H159+ (pan T) and H155+ (CD4) lymphocyte number and levels of T-cell responsiveness (mitogenic and allogenic responses, and IL-2 production) were higher in C4D neonates compared with age-matched Albany guinea-pigs or with young animals of the same strain. On the other hand, 31D2+ (B) lymphocytes in a significantly higher proportion in Albany neonates compared with similarly aged C4D, did not correlate at this age or at any other time with their proliferative response to lipopolysaccharide (LPS) or dextran sulphate (DS), two B-cell-specific mitogens.     

Median infective dose of Treponema pallidum determined in a highly susceptible guinea pig strain.

The median infective dose of Treponema pallidum subsp. pallidum and the production of immunity to reinfection in C4D guinea pigs have been determined with 10(1) to 10(6) organisms per infective dose. The mean infective dose is 10(2) organisms, and immunity--in those animals that demonstrated lesions--developed after 4.5 months postinfection.     

Humoral response in Treponema pallidum-infected guinea pigs: I. Antibody specificity.

Young male inbred strain 2 guinea pigs were infected intradermally with 8 X 10(7) Treponema pallidum extracted from a rabbit orchitis, and 5 months later reinfected with 10(7) T. pallidum. Ninety percent of the animals developed symptomatic lesions after initial infection but none on challenge. Immunoblotting of sera obtained at intervals after infection or reinfection showed antibodies against T. pallidum antigen (TP), nonpathogenic treponemes--T. phagedenis biotype Reiter (TR), T. refringens strain Noguchi (TN), and T. vincentii (TV)--as well as normal rabbit serum (NRS) and normal rabbit testes extract (NRT). Antibodies reacting with TP were detected as early as 17 days (five polypeptides) and steadily rose (at 3 months 17 polypeptides were seen). Cross-reacting antibodies to TR, TN, TV, or rabbit proteins decreased within 3 to 5 months. After reinfection, the antibodies to NRS increased more sharply than the anti-treponemal antibodies. Adsorption with TR and NRS of sera obtained after infection or reinfection produced a reduction of antibodies to TP by 75-87%.     

Infection of guinea pigs with vesicular stomatitis New Jersey virus Transmitted by Culicoides sonorensis (Diptera: Ceratopogonidae)

Intrathoracically inoculated Culicoides sonorensis Wirth & Jones were capable of transmitting vesicular stomatitis New Jersey virus (family Rhabdoviridae, genus Vesiculovirus, VSNJV) during blood feeding on the abdomen of six guinea pigs. None of the guinea pigs infected in this manner developed clinical signs of vesicular stomatitis despite seroconversion for VSNJV. Guinea pigs infected by intradermal inoculations of VSNJV in the abdomen also failed to develop clinical signs of vesicular stomatitis. Three guinea pigs given intradermal inoculations of VSNJV in the foot pad developed lesions typical of vesicular stomatitis. Transmission by the bite of C. sonorensis may have facilitated guinea pig infection with VSNJV because a single infected C. sonorensis caused seroconversion and all guinea pigs infected by insect bite seroconverted compared with 50% of the guinea pigs infected by intradermal inoculation with a higher titer VSNJV inoculum. The role of C. sonorensis in the transmission of VSNJV is discussed.     

Immunogenicity of three recombinant Treponema pallidum antigens examined in guinea pigs

The immunogenicity of recombinant treponemal antigens TmpA, TmpB and TmpC incorporated in RIBI adjuvant and injected into inbred strain 2 guinea pigs has been examined. The immune status of these animals has been challenged by infection with Treponema pallidum, Nichols. The immune response evaluated by the fluorescent-antibody test, microhemagglutination test and ELISA demonstrated high titers of antibodies to the T. pallidum antigens. The immunoblot analysis proved that the antibodies were directed to the 43-(Tmp A) 34- (Tmp B) and 35-kdalton (Tmp C) polypeptides. Antibodies cross-reacting with Treponema phagedenis biotype Reiter were, however, also detected. In spite of high titers of antibodies the animals were not protected against challenging infection with 10(8) organisms of T. pallidum.     

Antigenic evidence for host origin of exudative fluids in lesions of Treponema pallidum-infected rabbits.

Mucoid fluid accumulating within syphilitic lesions has been considered to be of Treponema pallidum origin. To test this assumption, we examined testicular exudative fluids from T. pallidum-infected rabbits for the presence of T. pallidum antigens by various sensitive immunochemical methods, including Western blot analysis. Antigenic analysis of these fluids revealed host components but not treponemal antigens. Prolonged immunization of rabbits, guinea pigs, and a goat with this material in complete Freund adjuvant elicited low titers (fluorescent-treponemal-antibody test titer, less than or equal to 10) of antitreponemal antibodies in the rabbits and guinea pigs but not in the goat. The data suggest that these mucoid fluids are of host origin. The presence of mucopolysaccharides in these fluids may be related to the infective process. The possible mechanism by which mucopolysaccharides protect T. pallidum from immune mechanisms and its potential relationship to the pathogenesis of the disease are discussed.     

Enhanced primary resistance to Treponema pallidum infection and increased susceptibility to toxoplasmosis in T-cell-depleted guinea pigs.

Strain 2 guinea pigs made T-cell deficient by thymectomy and irradiation and protected with syngeneic bone-marrow cells (TXB guinea pigs) have a surprisingly high level of resistance to cutaneous syphilis and to the dissemination of treponemes to the draining lymph node. Compared with normal euthymic controls infected with Treponema pallidum Nichols, syphilitic TXB guinea pigs developed fewer and less severe skin lesions and their lymph nodes contained lower numbers of treponemes. Associated with this evidence for enhanced innate resistance was the ability of the TXB host to produce, during each test interval of a primary infection, more antitreponemal antibodies than that of their euthymic counterparts. Similar levels of partial protection against cutaneous and disseminated syphilitic infection and elevated antibody levels occurred in challenged normal guinea pigs passively immunized with lymphocytes from T. pallidum-infected TXB donors. In contrast, the capacity of the TXB host to be protected against a lethal infection with the unrelated intracellular protozoan parasite Toxoplasma gondii was greatly impaired unless it received an intravenous infusion of normal syngeneic thymocytes. These seemingly paradoxical results are explained primarily in terms of a residual T-helper-cell population in the TXB guinea pig which is large and competent enough to generate antisyphilis, but not anti-Toxoplasma, immunity.     

Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen.

A cloned Treponema pallidum antigen, designated 4D, was purified from Escherichia coli predominantly as a 190-kilodalton (kd) polypeptide, although higher oligomeric forms exist. Extensive proteolysis of 4D created a limit digestion product of 90 kd which retained antigenicity with sera from patients with primary, secondary, early latent, late latent, and tertiary syphilis. A molecule indistinguishable from 90-kd 4D in size, isoelectric point, and antigenicity was isolated from T. pallidum after proteolysis. The 190- and 90-kd forms of 4D were stable at 68 degrees C but converted to 19- and 14-kd species, respectively, after boiling in sodium dodecyl sulfate. The low-molecular-weight species did not react with syphilitic sera. Rabbits immunized with the purified 4D antigen developed antibodies which immobilized virulent T. pallidum in a complement-dependent assay system, suggesting that the antigen has a native surface location.     

Pathogenicity of wild-type and temperature-sensitive mutants of herpes simplex virus type 2 in guinea pigs.

The pathogenicity of herpes simplex virus type 2 strain 186, the wild-type (WT) strain, and four temperature-sensitive (ts) mutants was studied after genital inoculation of female guinea pigs. Infection with the WT virus was generally severe, with extensive skin lesions in 89% and mortality in 37% of inoculated animals. Guinea pigs inoculated with ts mutants manifest remarkably mild disease, with lesions occurring in only 16% of the guinea pits and a mortality rate of 7%. WT virus was recovered from nerve and non-nerve tissues of all acutely infected animals and from the majority of latently infected animals (71%). Virus was isolated from nerve or genital tissues from only 13% of ts mutant-inoculated animals during acute infection and from 7% during latent infection. Three of the seven isolates from mutant-infected animals appeared to be WT virus. Identification of WT and ts mutant isolates was done by biological characterization in selective cell cultures at permissive (33 degrees C) and nonpermissive (38 degrees C) temperatures. One month after initial infection with WT virus, guinea pigs were challenged with the same virus and were completely resistant to overt clinical disease. Animals inoculated with ts mutants A1b and C2b had mild manifestations of disease after challenge with WT virus; however, the capacity of WT virus to establish latent infection was conserved. Although complement-required neutralizing antibodies were detectable after challenge in animals previously inoculated with mutant virus A1b, C2b, or D6b, there was no significant protection against subsequent infection with WT virus. No complement-required neutralizing antibodies were detected in F3b animals after challenge. The present study of WT and ts mutants of herpes simplex virus type 2 in the guinea pig model provides a means for better understanding the mechanisms of pathogenesis and latency after genital infection.     

Modulation of the chemotactic properties of complement fragments C5a and C3 by the anti-inflammatory agent,SC-41930

Cleavage of the fifth component of complement yields C5a, a potent neutrophil (PMN) and eosinophil chemoattractant, and modulator of microvascular permeability. Similarly, but to a lesser degree, C3 increases vascular permeability and histamine release. SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid), an orally-active antiinflammatory agent was tested in anin vivo model of dermal PMN chemotaxis induced by r-hu-C5a and hu-C3. Intradermal injection of C5a in the guinea pig resulted in a significant dose-dependent influx of PMNs at 4 hours as assessed by the dermal levels of myeloperoxidase (MPO). SC-41930 (20 mg/kg) given orally to guinea pigs with intradermal injections of 1 μg C5a significantly (p<0.001) reduced dermal MPO content. SC-41930 was less potent against C3, requiring 40 mg/kg to significantly reduce dermal MPO levels. Agents such as SC-41930, which nullify complement's proinflammatory properties, may well have therapeutic potential.     

Lymphomatoid papulosis. Characterization of skin infiltrates by monoclonal antibodies

Cryostat sections from fully developed papular lesions of lymphomatoid papulosis (histologic subtype A or B) have been examined by immunoenzymatic staining with 24 monoclonal antibodies against lymphoid cells and their subsets. The lesions demonstrated essentially identical cellular compositions and consisted of T-lymphocytes with a peripheral phenotype (Lyt3+, anti-Leu-4+, OKT6-), macrophages (HLA-DR+, EB11+, OKM1+), and Langerhans cells (HLA-DR+, OKT6+). T-helper/inducer cells (anti-Leu-3+) usually dominated over T-suppressor/cytotoxic cells (anti-Leu-2+). In all cases, proportions of the infiltrating T-cells expressed markers associated with activation (HLA-DR, the OKT1O antigen, interleukin-2 receptor) or proliferation (transferrin receptor, the Ki-67 antigen) of lymphoid cells. Furthermore, the infiltrates contained clusters and/or sheets of large cells reactive with antibodies (Ki-1, Ki-24, Ki-27), which recognize Hodgkin's and Reed-Sternberg cells. These data indicate an origin of the cellular infiltrate from transformed or activated lymphoid cells and suggest an interrelationship of lymphomatoid papulosis to Hodgkin's disease.     

Anterior hypothalamic lesions decrease anaphylactic contractions in guinea pig trachea in vitro by reducing histamine and LTC4 reactivity

Discrete lesions in the anterior hypothalamus (AHA) of the guinea pig brain reduce the anaphylactic contraction of the trachea in vitro after active in vivo sensitization by 40%. This difference in anaphylactic contraction does not correlate with a difference in homocytotropic antibodies but coincides with a decreased smooth muscle response to the anaphylactic mediators histamine and leukotriene C4. No difference in the beta-adrenoceptor function of the tracheal preparations can be found. The results suggest that AHA lesions afford protection against anaphylaxis in actively sensitized guinea pigs at least in part through a reduced smooth muscle response to anaphylactic mediators.