Studies on sperm antigenicity. I. Delayed hypersensitivity to spermatozoa

Delayed hypersensitivity to human and guinea pig sperm was demonstrated in guinea pigs of the Rockefeller strain by immunization with H₃₇Ra adjuvant. The reaction in vivo was demonstrated by skin testing the animals and in vitro by the capillary method. It was found that the sensitivity is not only directed towards the sensitizing antigen, but also shows cross-reactivity. Thus, peritoneal exudate cells derived from guinea pigs sensitized to human sperm were inhibited by guinea pig sperm. This cross-reactivity revealed the possibility of a tissue specific antigen. In addition, supernatants obtained after incubation of the sensitized lymph node cells with the specific antigen were found to be spermatotoxic.     

The guinea pig model of diisocyanate sensitization. I. Immunologic studies

Two strains of guinea pigs were parenterally immunized with well-characterized diisocyanate-protein conjugates. Hapten-specific IgE antibodies were detected in the sera of English short-hair strain guinea pigs immunized with either toluene diisocyanate-human serum albumin (TDI-HSA) or hexamethylene diisocyanate-HSA (HDI-HSA) when these sera were analyzed by the 168 hr passive cutaneous anaphylaxis (PCA) technique followed by intravenous challenges with conjugates of respective ligands coupled to an unrelated carrier protein, transferrin. IgG1 antibodies and precipitating antibodies were demonstrated in Hartley strain guinea pigs immunized with TDI/HDI-HSA conjugates. The hapten specificity of these antibodies was proved by PCA inhibition experiments and antibody absorption experiments. In the precipitating antibody system, this was further confirmed by immunoelectrophoretic analysis. Cross-reactivity between HDI and TDI was not observed in the PCA experiments. However, apparent cross-reactivity in the double gel diffusion experiments was due to new antigenic determinants formed by isocyanates after conjugation with proteins. It was therefore apparent that immune responses of guinea pigs immunized with protein conjugates of bifunctional isocyanates were heterogeneous and involved multiple specificities for hapten, carrier protein, and new antigenic determinants. It was postulated that the complex nature of the immune response generated by diisocyanate compounds in the guinea pig may also serve as a more appropriate model of isocyanate-induced human sensitivity reactions, which are known to involve diverse immunologic and nonimmunologic mechanisms.     

Radioimmunoassay for human type II collagen.

Human articular cartilage type II collagen (h coll.II) was purified and used to develop a radioimmunoassay. The sequential saturation procedure allowed a sensitivity of 3 ng/tube. The intra and between assay coefficients of variation were less than 10 and 20% respectively in the linear part of the curve. The assay was highly specific for native human articular type II collagen. There was no cross-reactivity with other constituents of cartilage: human proteoglycans, fibronectin, laminin and hyaluronic acid did not interfere with the assay. No cross-reactivity existed with bovine collagen types I, III, IV. However, native collagens from human placenta (I, III, IV, V, VI), rat and calf skin type I collagens and bovine type II collagen produced a weak cross-reaction only at high doses. Concerning the latter, inhibition curves were not parallel. Parallelism of inhibition curves were observed for dilution of type II collagen, produced by human chondrocytes in three-dimensional culture. All of these characteristics indicate that radioimmunoassy of type II collagen is a very sensitive and specific method available for the study and quantification of type II collagen in in vitro experimental conditions.     

In vivo and in vitro immunological cross-reactions between basic encephalitogen and synthetic basic polypeptides capable of suppressing experimental allergic encephalomyelitis

A significant extent of immunological cross-reactivity has been demonstrated between the basic encephalitogenic protein of bovine origin and several synthetic amino acid copolymers which have suppressive effect on experimental allergic encephalomyelitis (EAE). This cross-reactivity has been conclusively established on the cellular level, both in vivo by means of delayed hypersensitivity skin tests and in vitro using transformation of sensitized lymphocytes, as measured by incorporation of radioactive thymidine. The in vitro experiments have been conducted with lymph node cells from guinea pigs of both random bred and inbred strains, and on spleen and lymph node cells from rabbits. Definite cross-reactivity was observed between the basic encephalitogen and all the synthetic copolymers which were previously shown effective in suppression of EAE, whereas ineffective copolymers or unrelated proteins did not show any cross-reactivity. In the case of strain 2 guinea pigs and rabbit lymph node cells the cross-reactivity in vitro was manifested by direct cross-stimulation of the lymphocytes, whereas in random-bred or strain 13 guinea pigs and rabbit spleen cells, the cross-reaction was detected only by means of specific inhibition of the homologous stimulation by the heterologous antigen. A limited extent of cross-reactivity was observed on the humoral level as well; antibodies provoked in guinea pigs against the synthetic copolymer Cop 1 cross-reacted in the passive cutaneous anaphylaxis assay with the bovine basic encephalitogen.     

Response of the vagina of the spayed guinea pig to low or high doses of estrogen

Spayed ginea pigs injected daily for 6 to 10 days with 10, 50, 150 μg of estradiol cyclopentylpropionate (ECP) developed a mucified vaginal epithelium, similar to that observed during pregnancy or following treatment of ovariectomized animals with 5 mg progesterone and 1 μg of ECP. The epithelium did not mucify in spayed guinea pigs given 0.01 to 1.0 μg ECP daily for 10 or more days. Vaginal cornification developed only in animals treated with 1 μg ECP but the reaction was transitory and the keratinized cells were soon replaced by a stratified squamous epithelium. Spayed and adrenalectomized guinea pigs injected daily with 150 μg ECP developed a stratified squamous epithelium rather than a mucified type. However, the vaginal epithelium was mucified in ovariectomized-adrenalectomized guinea pigs receiving 150 μg ECP and corticoids or progesterone. This suggests that after treatment with large doses of estrogen the adrenal of the spayed guinea pig produces progestin-like hormones. These progestins interacting with exogenous estrogen are responsible for vaginal mucification. On the other hand, spayed rats maintained on 1 or 150 μg of ECP showed continuous vaginal cornification, which indicates that there are species differences in the ability of large doses of estrogen to influence the adrenal.     

Development of rheumatoid factors and anti-F(ab')2 antibodies in guinea pigs immunized with type II bovine collagen

The purpose of this report is to present results demonstrating for the first time the development of rheumatoid factors to rabbit IgG-Fc as well as antirabbit F(ab')2 antibodies in guinea pigs after chronic sensitization with purified native type II bovine collagen. The sensitized animals also developed antibodies to guinea pig Ig. Antibodies to rabbit Ig arose as early as 3 weeks after bovine type II collagen injection and persisted for as long as 80 weeks when the experiment was terminated. The anti-Ig antibodies did not cross-react with the type II bovine collagen. Despite development and persistence of higher titers of RF and anti-F(ab')2 antibodies in the immunized animals, the animals failed to show clinical evidence of inflammatory polyarthritis. These results indicate that rheumatoid factors as well as antibodies to F(ab')2 arise independently of the clinical expression of disease.     

Activation of lordosis in ovariectomized guinea pigs by free and esterified forms of estrone, estradiol-17 beta and estriol

The threshold doses of estradiol-17β, estrone, estriol, estradiol-17β-3-benzoate and estrone-3-benzoate required to activate lordosis in ovariectomized adult guinea pigs were determined by injecting these steroids in combination with progesterone. The doses of the 3 free steroids which activated lordosis in about 50% of the animals ranged from 20 to 50 μg/animal. In contrast, threshold doses of estradiol benzoate and estrone benzoate were only 0.4 and 1.7 μg/animal, respectively. The data indicate that (1) conversion to estradiol-17β is not an absolute requirement for activation of lordosis and (2) esterified forms of estradiol and estrone are far more potent in inducing lordosis than the corresponding free forms of these steroids. The increased potency of the esterified estrogens may be attributable to their prolonged action. Testosterone propionate also induced lordosis in 6/18 ovariectomized guinea pigs when given in a dose of 500 μg/animal for 7 days.     

Histopathological studies on the local reactions induced by complete Freund's adjuvant (CFA), bacterial lipopolysaccharide (LPS), and synthetic lipopeptide (P3C) conjugates

The inflammatory reactions following subcutaneous application of adjuvants revealed characteristic pathological patterns. The injection of complete Freund's adjuvant (CFA) resulted in the formation of large lipid deposits encircled by an inflammatory reaction and concentrically arranged collagen bundles. Bacterial lipopolysaccharide (LPS) caused granulomatous aggregations of mononuclear cells with thrombotic vessel occlusions. Inoculation of the lipopeptide adjuvants induced accumulation of mononuclear cells with only minimal fibrotic changes which were resolved after day 28. Lipopeptide conjugates based on the head group tripalmitoyl-S-glyceryl-cysteinyl-serin (P3CS) can thus be used as effective immunogens and adjuvants without long-term tissue damage.     

The effect of cytostatic drugs on collagen metabolism in guinea pigs

Azathiprine (0.25 mg/100 g body wt.) and cyclophosphamide (0.2 mg/100 g body wt.) were administered to guinea pigs, orally every day during 4 weeks. Azathioprine caused a decrease in total collagen content in skin, liver and bone and decreased hydroxyproline and hydroxylysine concentration in blood serum, and urinary excretion of collagen catabolites. No effect of cyclophosphamide on total collagen in skin and liver was observed. An increase in collagen content in bone in cyclophosphamide-treated guinea pigs was found. A decrease of hydroxyproline and hydroxylysine in blood serum and urine was observed after cyclophosphamide treatment. The results support the view that cytostatic drugs inhibit collagen metabolism, and azathioprine significantly affects collagen biosynthesis, and cyclophosphamide influences inhibition of catabolic processes of collagen.     

Identification of antibody epitopes in the CB-11 peptide of bovine type II collagen recognized by sera from arthritis-susceptible and -resistant rhesus monkeys.

Sera from eight rhesus monkeys that had been immunized with native bovine type II collagen were tested for antibodies to cyanogen bromide peptides (CB peptides) of type II collagen by Western blotting. The monkeys produced IgG antibodies to a number of different CB peptides, with five out of eight animals producing antibodies to the CB-11 peptide (four arthritic, one non-arthritic). Antibody epitopes on the CB-11 peptide of bovine type II collagen recognized by these sera were investigated by epitope mapping. Peptides (8-mers overlapping by seven amino acids) representing the CB-11 region were synthesised and the sera screened for binding to these peptides to determine areas of high IgG antibody binding to this region of type II collagen. The profiles obtained were not identical, though there were some epitopes that were commonly recognized. Antibodies to one epitope, also present in human type II collagen, were found only in the sera of two animals with the severest arthritis. The technique of epitope mapping has successfully identified a number of epitopes within the CB-11 peptide of type II collagen recognized by antibodies from bovine type II collagen-immunized monkeys. Studies on the relevance of responses to the identified epitopes can now be undertaken.     

Delayed-type hypersensitivity responses in infected mice elicited by cytoplasmic fractions of Cryptococcus neoformans.

Four subcellular fractions of Cryptococcus neoformans prepared by differential centrifugation of disrupted whole yeast and a 3-day culture filtrate were examined for their ability to elicit delayed-type hypersensitivity in sensitized animals. The methods used to detect sensitization were (i) the footpad swelling test and inhibition of peritoneal macrophage migration in mice and (ii) skin testing in guinea pigs. Two entities, the post-mitochondrial supernatant and the culture filtrate, showed considerable activity in the footpad test, with 26- and 30-microliter 24-h swellings, respectively, at 6 weeks after infection. With the latter there was interference from a strong antibody-mediated 4-h skin reaction. The post-mitochondrial supernatant produced strong delayed-type hypersensitivity in guinea pigs at a dose of 69 microgram, and there was no demonstrable cross-reactivity in animals sensitized with heterologous fungi. The footpad swelling in mice correlated well with the macrophage migration inhibition test, with 71% inhibition in mice infected subcutaneously with C. neoformans at 6 weeks. However, mice infected intravenously developed poorer cell-mediated immunity than the subcutaneously infected mice. The post-mitochondrial supernatant was found to contain detectable amounts of cryptococcal capsular polysaccharide.     

Ventilatory acclimatization in awake guinea pigs raised at high altitude

To determine if laboratory strains of guinea pigs bred at sea level (SL) are "pre-adapted" to high altitude (HA), we raised litter-matched weanling Hartley guinea pigs for 4 months at SL, intermediate altitude (IA, 1250 m) or HA (3800 m) and exposed them acutely to 100, 21 and 12% inspired O2 at their respective altitude of residence. Control animals raised at SL were also exposed acutely to 10 and 8% inspired O2. In awake spontaneously breathing guinea pigs raised at SL, resting minute ventilation and tidal volume increased significantly when inspired O2 tension fell below about 60 mm Hg. In guinea pigs raised at IA or HA, ventilation was higher at any given inspired O2 tension in direct relationship to the altitude of residence. Resting hematocrit was also higher in animals raised at HA than at SL. We conclude that the pattern of ventilatory acclimatization to HA exposure in Hartley guinea pigs is similar to that in laboratory rats and human lowlanders; therefore laboratory guinea pigs are not pre-adapted and are suitable animals for the study of adaptation to high altitude.     

Pathogenesis of Lassa virus infection in guinea pigs.

A rodent model for human Lassa fever was developed which uses inbred (strain 13) and outbred (Hartley) guinea pigs. Strain 13 guinea pigs were uniformly susceptible to lethal infection by 2 or more PFU of Lassa virus strain Josiah. In contrast, no more than 30% of the Hartley guinea pigs died regardless of the virus dose. In lethally infected strain 13 guinea pigs, peak titers of virus (10(7) to 10(8) PFU) occurred in the spleen and lymph nodes at 8 to 9 days, in the salivary glands at 11 days, and in the lung at 14 to 16 days. Virus reached low titers (10(4) PFU) in the plasma and brain and intermediate titers in the liver, adrenal glands, kidney, pancreas, and heart. In moribund animals, the most consistent and severe histological lesion as an interstitial pneumonia. In contrast, the brain was only minimally involved. The immune response of lethally infected strain 13 guinea pigs, as measured by the indirect fluorescent antibody test, was detectable within 10 days of infection and was similar in timing and intensity to the fluorescent antibody test response of both lethally infected and surviving outbred animals. In contrast to the fluorescent antibody response, neutralizing antibody developed late in convalescence and was thus detected only in surviving outbred guinea pigs. The availability of a rodent model for human Lassa fever in uniformly susceptible strain 13 guinea pigs should facilitate detailed pathophysiological studies and efficacy testing of antiviral drugs, candidate vaccines, and immunotherapy regimens to develop control methods for this life-threatening disease in humans.     

Experimental evaluation of the pathogenicity of Lassa virus variants

Pathogenicity for guinea pigs and white mice of various Lassa virus variants: native, having had 1 passage in Vero cell culture and 4 passages in newborn white mouse brain; a virus having gone through 10 passages in Vero cells and 8 mouse brain passages (variant No. 10); a small-plaque clone derived from variant No. 10 by the method of plaque-to-plaque cloning (variant No. 11k), was studied. Both the native virus and variant No. 10 were found to be similarly pathogenic for susceptible laboratory animals, while the small-plaque variant of Lassa virus became non-fatal for guinea pigs and white mice. The decline of pathogenic properties in variant No. 11k was shown not to be associated with the presence of temperature-sensitive mutants or defective interfering particles.     

Cell-mediated immunity in malnourished guinea pigs after Mycobacterium bovis BCG vaccination.

Specific-pathogen-free guinea pigs were vaccinated with viable Mycobacterium bovis BCG and maintained on purified, isocaloric diets containing either 30% or 7.5% casein, or commercial chow. At intervals of 4, 5, 6, and 8 weeks postvaccination, groups of guinea pigs from each diet treatment were skin tested with purified protein derivative and killed. Protein-deficient animals exhibited progressive reductions in total serum proteins and albumin. Significantly greater numbers of viable M. bovis BCG were recovered from the vaccination site and inguinal lymph nodes of protein-deficient guinea pigs at all intervals. In contrast, the development of delayed hypersensitivity was markedly retarded in the 7.5% casein group and was also reduced somewhat in the 30% casein group as compared to chow control. Peripheral blood lymphocytes from protein-deficient animals did not respond normally in vitro to a polyclonal T cell mitogen, phytohemagglutinin. These results demonstrate that protein-calorie malnutrition in this model impairs the development of cell-mediated immunity as evidenced by skin test anergy, lymphocyte hyporesponsiveness, and failure to control levels of viable M. bovis BCG after vaccination.     

Skin Testing of Guinea Pigs and Footpad Testing of Mice With a New Antigen for Detecting Delayed Hypersensitivity to Cryptococcus neoformans

This study was undertaken to evaluate the potential of a cryptococcal culture filtrate antigen, cryptococcin C184, for detecting delayed hypersensitivity in Cryptococcus neoformans-injected animals. The antigen was tested on guinea pigs which had received saline or C. neoformans and on animals sensitized to Histoplasma capsulatum, Blastomyces dermatitidis, Candida albicans, or Sporothrix schenckii. A delayed-type hypersensitivity response was elicited by cryptococcin C184 in C. neoformans-injected guinea pigs, whereas no indurations or erythemas were seen at 48 h after skin testing of saline controls or heterologously sensitized guinea pigs. Besides being specific for Cryptococcus, the antigen showed a high degree of sensitivity and was reproducible. Footpad tests were conducted with the antigen on mice which had previously received either 10(5) viable C. neoformans cells or saline. Delayed hypersensitivity was indicated in the C. neoformans-injected mice by the increase in thickness of antigen-injected footpads when compared with the saline-injected footpads. In control mice, antigen- and saline-injected footpads were comparable in thickness 24 h after injection. Mice sensitized to B. dermatitidis were footpad tested with C184, and no cross-reactivity was demonstrated.     

Antibody-mediated and delayed-type hypersensitivity reactions to Brucella skin test antigens in guinea pigs.

Cutaneous hypersensitivity responses to brucella antigens of different composition were studied in guinea pigs sensitized by infection with smooth brucella or immunization with killed rough brucella in adjuvant. These animals had circulating antibodies to smooth lipopolysaccharide or protein antigens, respectively. Intradermal skin tests, active cutaneous anaphylaxis, passive cutaneous anaphylaxis, and immunodiffusion tests were performed. Delayed-type hypersensitivity reactions uncomplicated by accompanying antibody-mediated reactions were seen only in infected guinea pigs with protein antigen that was entirely free of lipopolysaccharide. In the adjuvant-immunized animals, the protein antigen evoked overlapping antibody-mediated and delayed-type reactions. Lipopolysaccharide and polysaccharide preparations contained varying amounts of protein components. In infected animals, reactions of these antigens were clearly antibody mediated, but participation of delayed-type hypersensitivity could not be excluded. In adjuvant-immunized animals, the antibody-mediated reaction to the lipopolysaccharide preparation was caused by its protein component.     

Response to Mycobacterium bovis BCG vaccination in protein- and zinc-deficient guinea pigs.

Groups of specific pathogen-free Hartley strain guinea pigs were vaccinated with viable Mycobacterium bovis BCG and maintained on isocaloric purified diets containing either 30 or 10% protein (ovalbumin) combined with either 50 ppm (microgram/g) or no added zinc. Seven weeks later the guinea pigs were skin tested with purified protein derivative and killed. Both protein and zinc deficiencies had a significant negative impact on growth of the guinea pigs. Both groups consuming the 10% protein diet also demonstrated significant reductions in hematocrit, total serum proteins, and serum albumin, as well as diminished spleen weight. Plasma zinc concentrations were reduced in both low-zinc groups to less than half the value observed in control guinea pigs. Protein deficiency, alone or combined with zinc deficiency, resulted in increased tissue levels of viable M. bovis BCG in the inguinal lymph nodes and subcutaneous vaccination nodule. These same groups exhibited significant impairment in the ability to mount a delayed hypersensitivity reaction. Phytohemagglutinin-driven polyclonal T cell blastogenesis in vitro was significantly diminished in the peripheral lymphocytes of both protein- and protein-zinc-deficient animals at low mitogen doses, but only in the protein-zinc-deficient guinea pigs as the dose of phytohemagglutinin was increased. These results suggest that dietary protein and zinc deficiencies, alone or combined, interfere with immunological responses of the host to vaccination with M. bovis BCG.     

Vaccination and genetic experiments demonstrate that adjuvant-oil-induced arthritis and homologous type II collagen-induced arthritis in the same rat strain are different diseases.

The DA rat is highly susceptible to induction of arthritis after immunization with homologous type II collagen (CII) emulsified in Freund's incomplete adjuvant (FIA), resulting in collagen-induced arthritis (CIA). The DA rat also develops arthritis after injection of FIA alone (oil-induced arthritis (OIA)). This finding allows a direct comparison of two different models for rheumatoid arthritis; one induced with a defined auto-immunogen and one with a pure adjuvant. Both CIA and OIA develop approximately 2 weeks after induction but OIA is a self-limited acute disease whereas CIA induced with homologous CII follows a chronic disease course. Immunization with CII leads to a strong autoantibody response to CII while injection of FIA leads to no or very limited anti-CII antibody response. The Lewis rat develops neither CIA nor OIA while F1 (DA x Lewis) rats develop CIA but not OIA. Olive oil or CII emulsified in olive oil does not induce arthritis in DA rats. Pretreatment with CII in olive oil vaccinates against CIA but not OIA whereas pretreatment with FIA vaccinates against OIA but not CIA. These findings demonstrate that inclusion of CII in the adjuvant leads to a disease distinct from OIA which is characterized by a CII autoimmune response and chronicity of the disease course.     

Immunoreactivities for glutathione <Emphasis Type="Italic">S</Emphasis>-transferases and glutathione peroxidase in the lateral wall of pigmented and albino guinea pig cochlea

Dark-skinned people are known to be more tolerant of ototraumatic noise than are light-skinned people, and pigmented animals are more tolerant of ototraumatic noise and aminoglycoside ototoxicity than are albino animals. Such tolerance may be dependent on the local ability of detoxification and antioxidant enzymes, including glutathione S-transferase (GST) and glutathione peroxidase (GSPx). In the present study, we examined the difference in GST/GSPx expression in the lateral wall of the cochlea between pigmented and albino guinea pigs. Eight-week-old male pigmented and albino guinea pigs were killed by transcardiac perfusion with 2% paraformaldehyde. The cochlear ducts were isolated, further fixed with 4% paraformaldehyde, decalcified, and then embedded in paraffin. Sections prepared at 5-microm thickness were incubated with anti-GST-alpha,-mu,-pi, or anti-GSPx antibody, reacted with Alexa Fluorconjugated secondary antibody, and examined under a Carl Zeiss Axioskop 2 plus fluorescence microscope. The cochlea ducts were also subjected to immunoelectron microscopy for GST-pi by the postembedment method. The stria vascularis of pigmented guinea pigs was strongly immunoreactive for GST-alpha,-mu,-pi, and GSPx, whereas no or only weak immunoreactivities were seen in the stria vascularis of albino guinea pigs. The spiral ligament showed positive but different immunoreactivities for these enzymes between the strains. Double-stained immunofluorescence micrographs for GST-pi and GSPx showed a close resemblance of localization between the two enzymes in both pigmented and albino guinea pigs. At the ultrastructural level, immunoreactivity for GST-pi was localized preferentially in the melanin cells of pigmented guinea pigs. These results suggest that correlation between pigmentation and inner ear susceptibility is, at least partially, attributed to the different distribution of GST/GSPx in the stria vascularis.