Increased resistance to Salmonella infection of hypoferremic mice fed a low-protein diet

BALB/c and CBA/CA mice fed a protein-deficient diet developed a plasma hypoferremia corresponding to a 30 percent lowering of serum iron concentration. This hypoferremia persisted as long as the diet was maintained. Hypoferremic CBA/CA mice had increased resistance to Salmonella typhimurium C5 infection, as shown by the reduced lethal activity and the decreased growth of the bacteria in the spleen and in the peritoneal exudate of the deficient animals. This induced resistance was abolished after injection of iron or Desferal into the restricted animals. Such resistance was not observed with BALB/c mice fed a protein-deficient diet, in spite of the plasma hypoferremia. The growth of S. typhimurium C5 in the spleen and in the peritoneal exudate of these animals did not differ from the growth observed in control animals fed a protein-sufficient diet. This study suggests that hypoferremia induced by a protein-deficient diet is probably involved in the enhancement of resistance of CBA/CA mice to Salmonella infection, and that the phenomenon is host-strain dependent.     

The pathological study of enterosiderosis in guinea pigs

Enterosiderosis in both SPF Hartley guinea pigs and vitamin C-deficient animals of the same strain were studied by light and electron microscopy. Enterosiderosis was detected in all animals in the present study. Macrophages, inclosing yellowish-brown pigments and erythrocytes, appeared in the lamina propria of the intestinal mucosa, mainly in the cecum. These pigments in the macrophages were positive for Prussian blue, PAS and the Nile blue reaction. Residual bodies containing highly electron-dense ferritin-like particles, lipofuscin granules and debris of phagocytized erythrocytes were found by electron microscopy in the macrophages. In vitamin C-deficient guinea pigs, the number of macrophages, including the same above pigments, appeared in the lamina propria of the intestinal mucosa, and there was severe enterosiderosis. In the absorptive cells of the intestinal mucous membrane, granules positive for the Prussian blue reaction appeared only in the duodenum. These findings strongly suggest that the pigments in the macrophages in enterosiderosis of the guinea pigs were mixtures of iron and lipofuscin granules and that the iron is derived from erythrocytes phagocytized by macrophages in the lamina propria, but not from iron absorbed by epithelial cells.     

Bronchoalveolar eosinophilia in guinea pigs harboring inapparent infections of Paraspidodera uncinata

During the course of experiments examining the changes in cell populations in bronchoalveolar lavage (BAL) fluid in 3-11-wk-old guinea pigs, a marked increase in the numbers of eosinophils was observed in BAL fluid in untreated control animals from historical levels (observations made over the previous 2 yr) of 8.8 +/- 1.5% to levels greater than 16% and up to 44%. Repeated occurrence of this phenomenon in several different groups of guinea pigs that appeared clinically normal and the impact on our experimental studies led us to attempt to identify the cause of increased inflammatory cell numbers in these guinea pigs. Examination in 2 groups of animals of whole blood and lung tissue for the presence of bacteria or fungi revealed minor bacterial infections in one group but not the other, whereas both exhibited elevated eosinophil numbers. At necropsy, 41.7% and 60% of the animals in the 2 groups harbored the nematode Paraspidodera uncinata. Guinea pigs exhibiting eosinophil numbers in BAL fluid comparable to our historical levels were then inoculated with approximately 100 embryonated eggs of P. uncinata and developed elevated eosinophilia in BAL fluid compared to sham-inoculated animals (significant at 2 of the 3 examination times postinoculation). These findings suggest that P. uncinata is capable of causing changes in inflammatory cell populations in the lungs of guinea pigs and illustrate the importance of subclinical or inapparent infections in experimental design and interpretation.     

An evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs

Sodium ampicillin was administered subcutaneously to 350-550 g male Dunkin Hartley guinea pigs at doses of 6, 8 and 10 mg/kg tid for 5 days. Over a period of 12 days, the lowest ampicillin dose appeared to be tolerated well. However, significant body weight reduction and mortality occurred with the two higher dosage regimens. Cecal cultures of dead animals confirmed the presence of Clostridium difficile, an organism associated with antibiotic-induced enterotoxemia. Assay of serum collected from ampicillin-treated animals revealed ampicillin concentrations of approximately 10 micrograms/ml at 5 minutes post-dosing which fell precipitously to less than 0.2 micrograms/ml at 60 minutes. Determination of biliary ampicillin levels during the 60 minutes after administration of a single 10 mg/kg SQ dose revealed concentrations ranging from 18 micrograms/ml to 90 micrograms/ml. Estimates of total urinary ampicillin content after a single 10 mg/kg SQ dose were less than 500 micrograms/animal at 7.5 minutes, but increased to greater than 2000 micrograms/animal at 60 minutes after dosing. Results of this study indicated that due to its short serum half-life, sodium ampicillin probably has little systemic therapeutic efficacy in guinea pigs. Because high concentrations of ampicillin accumulated in the urine and bile, the antibiotic probably would have therapeutic efficacy for urinary and intestinal infections. However, its associated toxicity at large doses probably precludes its use. In view of the rapid clearance of ampicillin in guinea pigs in comparison to other species, the pharmacokinetics of other antibiotics, especially those reported to be less toxic for guinea pigs, should be considered.     

Pigmentation in intrinsically aged skin of A1 guinea pigs

It is known that skin often shows irregular pigmentation during aging which is frequently associated with hyperpigmentation. Many studies have utilized brownish A1 guinea pigs to investigate the pathogenesis of ultraviolet (UV)-induced skin pigmentation, however, changes associated with intrinsic aging in A1 guinea pig skin have not been documented. To characterize such changes, skin from the dorsal and neck areas of 20-week, 1-, 2-, 3- and 5-yr-old guinea pigs was examined. Skin color was measured using a colorimeter, and biopsy specimens were stained with Masson-Fontana, L-3,4-dihydroxyphenylalanine (DOPA), and antibodies against KIT (ACK-45), gp100 (HMB-45) and S-100 proteins. The L* value of skin color decreased with aging and melanin deposits increased in the epidermis. Further, DOPA+, gp100+ and S-100+ melanocytes increased, indicating that the number of melanocytes had increased with age, whereas KIT+ melanocytes did not increase in dorsal skin and actually decreased in neck skin with aging. Further, rippled pigmented areas appeared in the neck skin of the 3-yr-old animals, and in the dorsal and neck skin of 5-yr-old guinea pigs in the absence of UV irradiation. Melanocytes were distributed uniformly in younger skin, whereas they were clustered in older skin. UV irradiation caused an increase in the number of melanocytes, although they were not clustered. These results are the first to provide evidence that pigmentation is induced in the skin of intrinsically aged A1 guinea pigs in the absence of UV irradiation, a process that differs from that elicited by UV irradiation.     

Host-parasite relationships in experimental airborne tuberculosis. I. Preliminary studies in BCG-vaccinated and nonvaccinated animals

Smith, D. W. (University of Wisconsin, Madison), E. Wiegeshaus, R. Navalkar, and A. A. Grover. Host-parasite relationships in experimental airborne tuberculosis. I. Preliminary studies in BCG-vaccinated and nonvaccinated animals. J. Bacteriol. 91:718-724. 1966.-Previous studies from this laboratory on immunogenicity and allergenicity of defatted mycobacterial vaccines involved subcutaneous challenge of guinea pigs and killing of the animals 6 weeks later to evaluate the amount of disease. This type of experiment has discontinued in this laboratory in favor of an airborne challenge type of experiment, with the advantages that animals can be challenged with small numbers of bacilli by a natural route, and the number of primary lesions, the rate of spread from those lesions, and the rate of bacillary multiplication can be used to evaluate protection. Experiments to determine uniformity of infection showed that a fair degree of uniformity resulted when seven guinea pigs were exposed simultaneously, and were studied 3 weeks later to determine numbers of primary lesions and bacilli in the tissues. A less satisfactory degree of uniformity was obtained when more animals were exposed at one time. BCG-vaccinated and nonvaccinated animals were studied to determine the earliest time and the optimal time for killing the animals to detect the effects of vaccination. In guinea pigs, the degree of protection assessed by lesion counts is time-dependent, but the degree of protection assessed by viable counts of bacilli in the tissues was relatively constant 3 to 12 weeks after infection. Mice vaccinated subcutaneously with BCG were not protected against infection at any interval between 2 and 19 weeks. Guinea pigs vaccinated subcutaneously with the same lot of vaccine were protected as judged by counts of viable bacilli in the tissues 3 weeks after infection.     

Hepcidin, interleukin-6 and hematological iron markers in males before and after heart surgery

Anemia of inflammation in patients with acute or chronic acute-phase activation is a common clinical problem. Hepcidin is a peptide shown to be the principal regulator of the absorption and systemic distribution of iron. Main inducers of hepcidin are iron overload, hypoxia and inflammation, where the latter has been linked to hepcidin via increased interleukin-6 (IL-6). This article addresses the impact and time course of postoperative acute-phase reaction in humans following heart surgery on prohepcidin, hepcidin, hematological markers and IL-6 concentrations. Serum concentrations of prohepcidin, hepcidin, IL-6 and hematological iron parameters were studied in five male patients without infection before and after heart surgery. This study, which is the first to report the impact on serum hepcidin and serum prohepcidin concentrations in patients following surgery, clearly demonstrates the induction of hypoferremia due to the postoperative acute-phase reaction. Significant changes were seen for serum iron concentration, transferrin saturation, total iron binding capacity and hemoglobin concentration. A significant increase in ferritin concentration was seen 96-144 h postoperatively. Additionally, there were significant alterations in both serum hepcidin after 96-144 h and serum prohepcidin after 48 h compared with preoperative values. Serum prohepcidin decreased, whereas serum hepcidin increased. In conclusion, changes in serum prohepcidin were followed by an increase in serum hepcidin. This speaks in favor of a chain of action where proteolytic trimming of serum prohepcidin results in increased serum hepcidin. However, hypoferremia appeared prior to the changes in serum prohepcidin and serum hepcidin.     

Local immunity in the parenteral immunization of guinea pigs with a ribosomal dysentery vaccine

After immunization of guinea pigs with Shigella sonnei ribosomal vaccine O-antibodies appeared not only in the blood serum of the animals, but also in their lacrimal fluid. Since no correlation between the levels of serum and secretory antibodies was detected and since the time course of changes in these antibody levels was quite different (serum antibodies reached their peak on day 7 while secretory antibodies, on day 14 after vaccination), antibodies in lacrimal fluid were supposed to reflect local immune response induced by parenteral administration of ribosomal vaccine, irrespective of systemic immune response. The peak of secretory O-antibodies coincided in time with the period of the highest protection of guinea pigs from Shigella keratoconjunctivitis. The animals with a high level of secretory antibodies were better protected from Shigella infection than those with a low level of secretory antibodies. These data suggest that locally produced O-antibodies play an important role in protective immunity induced by parenteral administration of the ribosomal vaccine.     

Transfer Agent of Immunity

An immune ribonucleic acid (RNA) preparation was extracted with phenol from the spleens of guinea pigs immunized with diphtheria toxoid. Antibody-carrying cells were detected by immunocyte adhesion as rosette-forming cells. When germ-free rats, conventional guinea pigs or mice were injected intraperitoneally with this preparation, the rosette-formers were detected in either peritoneal exudate cells or spleen cells, whereas serum antibodies were unable to be detected thus far in such animals. Two injections with this preparation did not cause any remarkable increase in the number of rosette-formers, and serum antibody was also not detectable. By contrast, a high titer of serum antibody was demonstrated and the number of rosette-formers increased shortly after an injection of a small amount of diphtheria toxoid into guinea pigs which had previously received an injection with immune RNA. This reaction indicates a secondary response of antibody formation. However, secondary responses were not induced by injections of immune RNA preparations in guinea pigs primed with either diphtheria toxoid or immune RNA preparation. These facts suggest that immune RNA preparations did not contain antigens or fragments thereof and the immune response induced by RNA preparation is not the same as that induced by stimulation by the antigen itself. These results moreover can be accounted for by the notion that the immune RNA preparation is able to induce “memory” cells capable of responding to a secondary stimulus with an antigen and producing a high titer of serum antibody.     

Study of anaphylectic hypersensitivity manifestations to blood protein in guinea pigs with using the neutrophil damage test in vitro]

The paper is devoted to the study of hypersensitivity of anaphylactic type to horse serum in guinea pigs by means of neutrophil injury index in vitro. Simultaneously, for control, the same method was applied to the study of delayed hypersensitivity manifestations; for this purpose allergy to hemolytic staphylococcus was reproduced in these animals. The state of hypersensitivity of "anaphylactic type" against the foreign serum in guinea pigs was not accompanied by any enhanced blood neutrophil reactions, whereas high values of the test under study were characteristic of the process of guinea pigs sensitization with staphylococcus culture.     

Virulence of Trichophyton mentagrophytes infecting steroid-treated guinea pigs

The skin of steroid-treated and normal guinea pigs was inoculated with Trichophyton mentagrophytes and the clinical infection was observed. Organisms isolated from these two groups of animals were cultured on Mycosel® agar and inoculated into untreated guinea pigs to obtain a new infection. In vitro growth inhibition studies were also performed on these organisms using a tolnaftate disc. The character and duration of infection, colonial growth, new infection and tolnaftate inhibition were essentially the same for both the steroid-treated and control groups of animals.     

Effect of Ochratoxin and Aflatoxin on Serum Proteins, Complement Activity, and Antibody Production to Brucella abortus in Guinea Pigs

The effect of ochratoxin alone and in combination with aflatoxin and Brucella abortus antigen on complement activity, serum proteins, and antibody response in guinea pigs was investigated. Ochratoxin did not affect complement activity or antibody response and there was no interaction between ochratoxin and aflatoxin on any of the responses tested. Ochratoxin significantly lowered the level of beta-globulin in serum of guinea pigs. There was no significant interaction between aflatoxin and antigen on lowering of the serum albumin levels of guinea pigs.     

Lack of correlation between steroid sulfatase activities and lipid content in uterus and liver microsomes of guinea pigs

Lipid content and steroid sulfatase activities were determined in liver and uterus microsomes of non-pregnant guinea pigs. The results were compared with values obtained in pregnant and cortisol-treated animals. Steroid sulfatase activities were always higher in pregnant animals, and we supposed that the increase in circulating cortisol in pregnant guinea pigs before parturition has an influence on the membrane-bound sulfatase activities. Sulfatase activities were identical in cortisol-treated and untreated non-pregnant females, although cortisol induced changes in microsomal lipid composition. These results lead us to three conclusions: in intact female guinea pigs, cortisol induces variations in the lipid content of uterus and liver microsomes, especially in the cholesteryl sulfate to phospholipid ratios; the variations of the lipid composition in pregnant animals do not appear to be cortisol-dependent; membrane-bound steroid sulfatase activities are not directly influenced by the lipid composition of microsomes.     

Tubulointerstitial nephritis induced by Tamm-Horsfall protein sensitization in guinea pigs

Experimental tubulointerstitial nephritis (TIN) was induced in guinea pigs by immunization with homologous Tamm-Horsfall protein (THP). This disease was characterized by focal interstitial mononuclear cell infiltration around the distal nephron segments with degeneration of renal tubular cells. Although concomitant granular immunoglobulin deposition on the tubular basement membrane and a rise of serum anti-THP antibodies were recognized, they were related to the severity of the lesion. Lymphocytes from lymph nodes of animals with TIN showed blast transformation in the presence of THP in vitro. Following the transfer of lymphocytes and spleen cells from guinea pigs with THP-induced TIN to nonimmunized animals, the recipient animals developed TIN 7 days later. These observations suggest that TIN induced in guinea pigs by challenge with homologous THP may, at least in part, be related to a cell-mediated immune response.     

Thymic and splenic changes following injection of living Brucella and BCG in the Guinea pig

Summary The thymic and splenic reactions, following injections of BCG and living Brucella M. in the guinea pigs, were studied. Both microorganisms injected intravenously produced thymic involution, maximal after BCG inoculation, followed by regeneration that was complete by day 10 in Brucella M.-treated guinea pigs, and by day 15, in BCG injected guinea pigs. Increase of mitotic index was more accentuated and more persistent after Brucella M. than after BCG treatment in the thymus. After a short involution period the spleen of injected animals increased in size in both groups. The splenic enlargement was dramatic and occurred at an accelerated rate in animals given BCG. It appeared to be the result of a conspicuous involvement of the red pulp by multiple granulomas. In Brucella M. treated guinea pigs the splenic enlargement was less obvious, but the splenic white pulp was more abundant in BCG-treated guinea pigs. Granulomas were observed only in the periarterial sheaths of the white pulp.These observations provide evidence for the hypothesis that injections of both BCG and Brucella M. provoke a proliferation of B and T lymphocytes, a migration of T lymphocytes from the thymus to the T-dependent area of the spleen which seems, perhaps, more marked after injection of Brucella M., and a strong granulomatous histiocytic reaction which is more conspicuous in animals given BCG.     

Modeling of pemphigus vulgaris in guinea pigs]

In 146 guinea pigs with body weight of 150-200 g pemphigus vulgaris (PV) was induced using screening techniques. The animals were injected with PV patients' IgG (IgGPV) and blister fluid (PVBF) with the PV patient mononuclears. Intraperitoneal administration of IgGPV caused dystrophy in epidermis of experimental animals while intracutaneous injection of PVBF resulted in balloon dystrophy. Clinical manifestations of -PV were obtained after two-day intraperitoneal IgGPV administration in a total dose of 1.5 g (6 mg/g) following a cycle of intracutaneous injections of 2.0 ml PVBF in 10-15 sites of guinea pig back. The presence of acantholysis, intraepidermal localization of blisters, pemphigus antibodies fixation on spinous cell surface, and almost 100% mortality of experimental animals were the major criteria for the developed model of PV in guinea pigs. Experimental PV in guinea pigs was abolished due to treatment of PVBF with dexamethasone, contrykal, heating (56 degrees C, 30 min), preincubation with human skin samples, and PVBF cell-free supernatant application. The conclusion was made that for occurrence of pemphigus in laboratory animals a combined effect of pemphigus antibodies, sensitized mononuclears, complement and endogenous proteinases is required.     

Characterization of in vitro reactivity by BCG-treated guinea pigs to syngeneic line-10 hepatocarcinoma

Summary The purpose of this study was to characterize in vitro the systemic tumor immunity induced by a BCG-intratumoral injection in line-10 hepatocarcinoma established in the skin of inbred guinea pigs (strain 2). Macrophages from BCG-tumor-cured guinea pigs at effector to target cell ratios of 10:1 and 100:1 were cytotoxic in vitro to line-10 tumor cells, and this cytotoxicity was potentiated by autologous serum. Significant cytotoxicity of lymphocytes from BCG-tumor-cured guinea pigs could only be achieved at ratios of 10,000:1, and no effect of autologous serum could be demonstrated. Lymphocytes from both normal and BCG-tumor-cured (line-10 immune) guinea pigs had a significant cytotoxic effect on the highly antigenic line-1 cells at ratios of 1:10,000. Macrophages from both normal and line-10 immune guinea pigs were cytotoxic to line-1 target cells at ratios of 1:100. With respect to specific cytotoxicity (cytotoxicity above and beyond levels achieved with effector cells from normal animals), the only significant difference was demonstrated when line-10 served as target cells and the effector cells were isolated from BCG-tumor-cured (line-10 immune) guinea pigs. Abbreviations used in this paper: BCG, Bacillus Calmette-Guérin; CMEM, complete minimum essential medium; cpm, counts per minute; HBSS, Hanks' balanced salt solution; i.d. intradermally; i.p., intraperitoneally; PEC, peritoneal exudate cells; SDA, superficial distal axillary; ¹²⁵IdUrd, [¹²⁵I]iododeoxyuridine.     

Serum degradation of myelin basic protein with loss of encephalitogenic activity: evidence for an enzymatic process.

Fibrin deposition in parallel with loss of myelin basic protein (MBP), an antigenic constituent of central nervous system (CNS) myelin, within the lesions of animals with experimental allergic encephalomyelitis (EAE) suggested that degradation of MBP by proteolytic activity associated with blood clotting might be an important immunopathologic event in this prototypic autoimmune disease. Following incubation in normal rat serum at 37 °C for more than 4 hr, but not to any comparable degree in plasma, MBP had little or no encephalitogenic activity when bioassayed in guinea pigs or rats. Fragments of increasingly lower molecular weight were demonstrable by polyacrylamide gel electrophoresis after addition of MBP to rat serum; no fragments appeared after incubating the protein in rat plasma. Little or no loss of encephalitogenic activity was observed when MBP was incubated in serum containing protease inhibitors. These findings indicate that the serum-mediated degradation of MBP and concomitant loss of encephalitogenic activity is due to an enzymatic process associated with the coagulation cascade or/and the complement, kallikrein or fibrinolytic pathways. Implications of these findings concerning EAE and the multiple sclerosis process in man are discussed.     

Antibody production by strain 2 and strain 13 guinea pigs: I. Use of a monomeric guinea pig albumin carrier to demonstrate immune response gene control

Homogeneous albumin has been isolated from the serum of strain 13 guinea pigs. The 2,4-dinitrophenyl (DNP) conjugate of this albumin (DNP₉ guinea pig albumin) can be used even at high doses (100 μg) to distinguish easily the nonresponder strain 2 guinea pigs from the responder strain 13 animals. This observation modifies the previous conclusion that clear-cut discrimination of responders and nonresponders requires the use of low doses (i.e., 1 μg) of such a hapten-protein conjugate. Since albumin polymers as well as additional protein contaminants comprise a large proportion of some commercial albumin preparations, these ancillary molecules appear to be responsible for the previous suggestion that protein carriers differed from synthetic peptides in that low immunizing doses were required to distinguish responder from nonresponder animals. That responsiveness in nonresponders can be increased by inclusion of polymeric forms of the antigen in the immunizing mixture raises the possibility that those cells which “process” antigens may play a major role in immune response (Ir) gene control.     

Liver and spleen morphology, ceruloplasmin activity and iron content in serum of guinea pigs exposed to the magnetic field

The chronic exposure of guinea pigs to the magnetic field of induction 0.005 T and 0.3 T cause the morphological changes of spleen and functional disturbances of liver (the histochemical analysis indicate on greater amount of glycogen in hepatocytes). Everyday 1-hour exposure determined the drop in ceruloplasmin activity and an unchanged iron content in the serum of tested animals.