Staphylococcal footpad infection in mice.

Local footpad infection in mouse was investigated with 55 clinically isolated strains of Staphylococcus aureus. When 10(7) viable cells were inoculated into the footpad, local swelling and bacterial growth resulted after 24 hr. With a dose of 10(6) cells, moderate swelling was observed after a few hours but the reaction had almost disappeared after 24 hr. About 75% of the staphylococcal strains tested caused footpad edema in mice at doses of 10(7) cells. A statistical comparison of the virulence of the organisms on intravenous and intraperitoneal injection with that in inducing footpad swelling is also reported.     

Genetic Control of Immune Responses to HIV-1 env DNA Vaccine

We investigated the genetic control of immunoglobulin production and the delayed-type hypersensitivity (DTH) response produced by an HIV-specific DNA vaccine using several strains of mice. Murine antigen-specific immunoglobulin production was determined by ELISA. The DTH response was assessed in terms of the footpad swelling reaction. All strains of mice, except for B10.RIII and B10.T(6R), exhibited strong immunoglobulin production and footpad swelling in response to the DNA vaccine. In vitro treatment of lymphoid cells with monoclonal antibodies showed that the footpad swelling response was mediated by CD4⁺8^? and Ia— T cells. However, CD8⁺ T cells did not suppress footpad swelling. There was no difference in the induction of HIV-specific immunoglobulin production or DTH response induced by the DNA vaccine among the strains, suggesting that HIV-specific DNA vaccine is useful for immunizing various populations against HIV-1.     

Infectivity and Early Antibody Response to Borrelia burgdorferi Sensu Lato Isolated in Japan in Outbred Mice

Borrelia burgdorferi sensu lato isolated from Ixodes ovatus (B. japonica), I. persulcatus and patients with erythema migrans (EM) in Japan were determined on infectivity and arthritis induction-activity in outbred mice. Infectivity of B. japonica was weak and did not induce the development of footpad swelling by subcutaneous (s.c.) inoculation into the footpad. Challenged strain, NO129-M of B. japonica, to ddY mice were reinoculated to the mice at various cell numbers (1 × 10-1 × 10⁶ cells/mouse). The strain isolated from the mouse did not reinfect ddY mice and did not induce the production of specific antibody to the homologous strain. On the other hand, strains from I. persulcatus and patients with EM in Japan infected the mice and induced a serious inflammatory response in Borrelia-inoculated footpad as well as strains belonging to the three genospecies, B. burgdorferi sensu stricto, B. garinii, and B. afzelii, related to Lyme disease, from North America and Europe. The mice were infected with 10 cells of strain HP1 isolated from I. persulcatus in Hokkaido and of strain 297 isolated from a patient in the U.S.A. by subcutaneous inoculation into the hind footpad, or by intradermal inoculation into the back. Antigens of ca. 20, 23–24 (Osp C), 29, 39, 41 (flagellin) and 45 kDa reacted with the pooled sera from mice inoculated with strains HP1 and 297, but Osp A and Osp B did not.     

T Cell Response to Borrelia garinii,Borrelia afzelii,and Borrelia japonica in Various Congenic Mouse Strains

The infectivity and T cell response to Borrelia garinii SIKA2, Borrelia afzelii BFOX, and Borrelia japonica 0612, the organisms that cause Lyme disease in Japan, were examined in various inbred and congenic strains of mice. Infectivity differed among the species: B. garinii SIKA2 and B. afzelii BFOX were each able to infect 90% to 100% of C3H/He mice; B. japonica 0612 was able to infect only 20% of C3H/He mice. The pattern of infectivity to various inbred and congenic strains of mice may influence the pathogenicity of the organism and the clinical signs of Lyme disease. Cross-reactivity between Borrelia antigens was observed, but there was no cross-reactivity between Borrelia antigens and Leptospira antigens. We evaluated the genetic control of the delayed-type hypersensitivity (DTH) reaction in the form of footpad swelling produced by Borrelia antigens using viable or sonicated bacteria as sensitization. Differences in strains of mice infected by viable antigen were observed. However, all strains of mice showed a strong DTH reaction using sonicated antigens without genetic background. A DTH reaction in the form of footpad swelling did not appear to be associated with genetic background. The footpad reaction was mediated by CD4⁺8^? and Ia^? T cells, as revealed by in vitro monoclonal antibody treatment. However, CD8⁺ T cells did not suppress footpad swelling. These results indicate that many antigenic epitopes of the Borrelia spirochete can stimulate the DTH reaction.     

Delayed-type hypersensitivity to allogeneic mouse epidermal cell antigens

Footpad swelling response was used to measure the alloantigenicity of epidermal cells (ECs) in delayed-type hypersensitivity (DTH). Strong footpad swelling was oberserved 3 h after the challenge, and it continued for 48 h after the challenge. Genetical incompatibility between the recipients and the ECs was required to induce significant footpad swelling. H-2 or non-H-2 incompatibility between mice and ECs in the sensitization phase sufficed to develop significant footpad swelling. Incompatibility caused by point mutation in the A region induced strong responses when B6. C-H-2 ^bm12 mice were immunized with B6/J ECs, but the disparity in immuno-globulin h (Igh) allotype genes was insufficient. H -Y antigen on ECs could also elicit the DTH response. Semiallogeneic F₁-derived ECs sensitized the parental recipients. The responses were successfully transferred by immune lymph node cells, but not by immune sera. Treatment of these immune lymph node cells with monoclonal antibodies plus complement revealed that the cells responsible for DTH transfer were Lyt-1⁺2^–, Ia^– T cells.Abbreviations used in this paper DNFB 2,4-dinitro-1-fluorobenzene - DTH delayed-type hypersensitivity - ECs epidermal cells - HBSS Hanks' balanced salt solution - MHC major histocompatibility complex - PBS phosphate-buffered saline     

Experimental Induction of Lyme Arthritis in Outbred Mice

Outbred ddY mice inoculated with live cells of Borrelia burgdorferi strain 297 into hind footpad displayed swelling of the footpad at days 7 to 11 after inoculation. Marked neutrophilic infiltration was observed in the subcutaneous tissue and the part of bone tissue which was partially destroyed, and synovial layer of articular capsule was thickened and protruded into the joint space in the histopathological examination of footpad inoculated with live Borrelia cells. The inflammation peaked at day 7 and B. burgdorferi was cultured from bladder and heart of the mice at day 14 after inoculation. The mice inoculated with heat-inactivated cells at 56 C for 30 min did not show any significant histopathological change. In this mice model, nontreated littermates were not infected in contact with infected littermates for 14 days of experimental period. The outbred ddY mice model is useful for evaluating the effectiveness of vaccination against Lyme disease.     

Effects of light-dark cycles on the circadian conidiation rhythm inNeurospora crassa

The effects of 24 hr light-dark cycles on the circadian conidiation rhythm inNeurospora crassa were compared among will-typefrq ⁺ and clock mutantsfrq ⁺,frq ³,frq ⁷,frq ⁹ andfrq ¹¹. The minimum length of the light period necessary for complete entrainment to the light-dark cycles was almost 2 hr infrq ⁺,frq ³ andfrq ⁷ strains. The minimum duration of the dark period necessary for the appearance of circadian conidiation was almost 4 hr in all of the strains except thefrq ¹¹ strain. The phase of the conidiation rhythm was dependent on the light to dark transition in thefrq ¹ strain in all light-dark cycles examined and in thefrq ⁺ andfrq ³ strains when the light period was shorter than 16 hr. In contrast, the phase of thefrq ⁷ strain was dependent on the light to dark transition when the light period was shorter than 10 hr.     

Fonsecaea pedrosoi Conidia Induces Activation of Dendritic Cells and Increases CD11c+ Cells in Regional Lymph Nodes During Experimental Chromoblastomycosis

The chromoblastomycosis is a subcutaneous mycosis with a high morbidity rate, Fonsecaea pedrosoi being the largest etiologic agent of this mycosis, usually confined to the skin and subcutaneous tissues. Rarely people get the cure, because the therapies shown to be deficient and few studies report the host–parasite relationship. Dendritic cells (DCs) are specialized in presenting antigens to naïve T lymphocytes inducing primary immune responses. Therefore, we propose to study the migratory capacity of DCs after infection with conidia of F. pedrosoi. The phenotype of DCs was evaluated using cells obtained from footpad and lymph nodes of BALB/c mice after 12, 24 and 72 h of infection. After 24 and 72 h of infection, we found a significant decrease in DCs in footpad and a significant increase in the lymph nodes after 72 h. The expression of surface markers and co-stimulatory molecules were reduced in cells obtained from footpad. To better assess the migratory capacity of DCs migration from footpad, CFSE-stained conidia were injected subcutaneously. We found that after 12 and 72 h, CD11c⁺ cells were increased in regional lymph nodes, leading us to believe that DCs (CD11c⁺) were able to phagocytic conidia present in footpad and migrated to regional lymph nodes.

     

Effect of pH and Oxygen Tension on Staphylococcal Growth and Enterotoxin Formation in Fermented Sausage

Commercial fermented 0sausages that contained significant numbers of viable coagulase-positive staphylococci were found to have the growth localized in the outermost areas of the sausage where oxygen tension was highest. Staphylococci were found to be more acid-tolerant aerobically than anaerobically. With chemical acidulation of sausage, growth could be controlled both aerobically and anaerobically with approximately 1.5% glucono delta lactone. Biological acidulation with a high inoculum of Pediococcus cerevisiae inhibited anaerobic staphylococcal growth but failed to suppress aerobic growth completely. A staphylococcal count of approximately 4 × 10⁷ cells/g of sausage appeared to be necessary to produce detectable enterotoxin A within 24 hr in sausage. A minor difference existed in the relative rates of production of the different types of enterotoxin. Detectable enterotoxin A was produced in 24 hr in sausage held in atmospheres containing 10, 15, and 20% oxygen. In an atmosphere containing 5% oxygen, toxin was detected after 48 hr of incubation. No toxin was detected after 120 hr under anaerobic conditions. Most staphylococcal strains tested initiated growth and produced detectable enterotoxin aerobically at a pH of 5.1 in broth media. Anaerobically, however, most strains failed to produce detectable enterotoxin below pH 5.7.     

PHYSICOCHEMICAL EFFECTS OF ALDEHYDES ON THE HUMAN ERYTHROCYTE

The effects of formaldehyde, acetaldehyde, and glutaraldehyde on human red blood cells were investigated. It was found that (a) The surface negative charge of the erythrocytes at pH 7 was increased 10% by glutaraldehyde, but not by the other two aldehydes. (b) The effect of incomplete fixation of the red blood cells was demonstrated by hemoglobin leakage studies The leakage of hemoglobin subsequent to formaldehyde treatment was especially pronounced Acetaldehyde-fixed cells showed some leakage of hemoglobin after an hour of exposure to the fixative, whereas glutaraldehyde-fixed cells showed no hemoglobin leakage. (c) All three aldehydes caused K⁺ leakage during fixation. The concentrations of K⁺ in the fixing solutions all reached the same level, but whereas the leakage with glutaraldehyde was immediate, that with formaldehyde was more gradual and that with acetaldehyde reached a steady state only after 24 hr. (d) The effects of the aldehydes on red cell deformability and swelling revealed that glutaraldehyde hardened the cells within 15 min, formaldehyde within 5 hr, while acetaldehyde required at least 24 hr to produce appreciable fixation. (e) The hematocrit changes accompanying the fixation process depended upon cell volume changes and loss of deformability.     

Effects of pertussis toxin on delayed-type hypersensitivity responses and on the activity of suppressor T cells on the responses

Experiments were performed on mice to investigate the effects of pertussis toxin (PT) on delayed-type hypersensitivity (DTH) to ovalbumin (OA) and on the activity of suppressor T cells on the DTH (DTH-Ts). Mice immunized with alum-precipitated ovalbumin showed a transient DTH, which was determined as footpad swelling which disappeared 2 weeks after immunization. Maximal footpad swelling was observed 24 hr after DTH elicitation. On the other hand, when mice received PT (2 micrograms/mouse) at the time of immunization, the transient DTH became an enhanced and persistent DTH, which persisted for at least 4 weeks. In addition, the time of maximum footpad swelling was delayed from 24 to 48 hr after DTH elicitation. The immune spleen T cells from PT-treated mice showed a persistently high ability to transfer DTH into syngenic naive mice. DTH-Ts was induced in spleens of mice injected iv with OA-coupled syngeneic spleen cells. However, when these mice received PT at the time of suppressor induction, their spleen cells revealed considerably reduced suppressor activity. The activity of DTH-Ts was also reduced when DTH-Ts were either treated in vitro with PT or transferred into PT-injected recipient mice. From these results, interference with the suppressor function of DTH-Ts from PT was considered to be, at least in part, as an enhancing mechanism of DTH.     

Metabolie Aspects in Mice Administered Live Salmonella typhimurium

The influence of intraperitoneal inoculation of live Salmonella typhimurium on carbohydrate and lipid metabolisms in mice was investigated at doses of 9.2 × 10⁷ cells, 1.9 × 10⁸ cells, and 3.8 × 10⁸ cells. The hepatic glycogen content in mice at 18 hr after the inoculation decreased in inverse proportion to an increase in the injection dose. The activities of hepatic phosphorylase and G-6-P_ase increased significantly after 2 hr, but after 18 hr the levels of both enzyme activities, especially G-6-P_ase, declined in inverse porportion to an increase in dose of viable cells administered to the mice. The levels of serum glucose and free fatty acids (FFA) in mice markedly decreased at doses of 1.9 × 10⁸ and 3.8 × 10⁸ cells after a transient rise at early stage (1 hr) after the injection. Marked hypertriglyceridemia was seen in infected mice. However, the activity in serum lipoprotein lipase (LPL) was reduced by an increase in the injection dose. The effect of intraperitoneal administration of viable cells on the serum triglyceride level was prevented in mice immunized with S. typhimurium endotoxin or administered with the anti-endotoxin serum. These results indicate that hypertriglyceridemia mainly results from the action of endotoxin in the pathogen. Serum lactate dehydrogenase (LDH) activity markedly increased at the dose of 3.8 × 10⁸ cells within 8–16 hr, and the infected mice exhibited a leakage of isozymes LDH-3 and 5 in the serum 16 hr post-inoculation.     

Isolation,characterization and colonization of 1-aminocyclopropane-1-carboxylate deaminase-producing bacteria XG32 and DP24

Two 1-aminocyclopropane-1-carboxylate deaminase-producing bacterial strains (DP24 and XG32) were isolated from surface-sterilized tomato roots and rizhospere soil. The strains were identified as Pseudomonas fluorescens biovar. IV (XG2) and Erwinia herbicola (DP24) by physiological and biochemical tests, and 16S rRNA gene analysis. Both strains showed positive plant growth-promoting activity when inoculated into cucumber (Cucumis sativus), tomato (Lycopersicon esculentum), pepper (Capsicum annuum) and rapeseed (Brassica napus L.). Colonization ability and behavior of these two strains were determined by treating mutant strains with rifampicin and fluorescence in situ hybridization (FISH) assay with rRNA targeted probes, respectively. Both strains were endophytic colonizers of pepper plants. The behavior of the two strains was not identical. Strain XG32 only colonized the root and reached the max level of 27.7 × 10⁷ c.f.u./g (fresh weight), after 12 days postinoculation, while strain DP24 was able to colonize the roots, stems and leaves. The max level was reached at 40.87 × 10⁷ c.f.u./g (fresh weight) in the roots, 17 × 10⁷ c.f.u./g in the stems after 7 days postinoculation and 44.84 × 10⁷ c.f.u./g in the leaves after 12 days postinoculation.     

Production by Cultured Spleen Cells of Inflammatory Substances and Other Lymphokines that Mediate Delayed-Type Hypersensitivity in Mice

In vitro cultivation of normal mouse spleen cells with human serum albumin generated effector cells that mediate the delayed-type hypersensitivity (DTH) reaction. The cultured cells, when incubated in a serum-free medium for a further 24 hr, released substances (FPRF) which caused a footpad inflammatory reaction at a maximum of 6 hr after injection into normal syngeneic or allogeneic strains of mice, as well as macrophage migration inhibition factor (MIF) and macrophage activating factor (MAF). The DTH-effector cells in the culture were fractionated in the low density layers by discontinuous bovine serum albumin density gradient centrifugation. The effector cells in the low density layers were further enriched in the Lyt 1 subpopulation of T cells when fractionated on a fluorescence activated cell sorter. Cells capable of producing the inflammatory substances (FPRF), MIF and MAF were also enriched in the same fraction containing DTH-effector cells. These results suggest that low density, Lyt 1-positive T cells mediating the DTH reaction produce FPRF as well as MIF and MAF.     

Genetic Control of Immune Responses to a Synthetic Fimbrial Antigen of Actinobacillus actinomycetemcomitans

The incidence of infection by Actinobacillus actinomycetemcomitans, one of the important pathogens in human periodontal diseases, has been reported to be associated with racial background and genetic factors. We attempted to determine the genetic regulation of immune responses to A. actinomycetemcomitans fimbriae, an attachment factor, using various inbred strains of mice. For this purpose, we synthesized an oligopeptide antigen using the amino acid sequence of the fimbriae and conjugated this antigen to branched lysine polymer resin beads. After immunization with the synthetic A. actinomycetemcomitans fimbrial antigen, serum antibody levels and the delayed-type hypersensitivity (DTH) reaction to the antigen were measured by enzyme-linked immunosorbent assay (ELISA) and footpad swelling responses, respectively. The strains of mice found to be high-IgG responders to the antigen were B10.HTT, B10.RIII, B10A (5R) and B10.S (9R). These results indicate that mice with E_β^s: E_α^k, E_β^r: E_α^r and E_β^b: E_α^k respond strongly to the synthetic peptide. All of the high-IgG responders showed a high DTH response. A cell transfer experiment confirmed that CD4 T cells mediated with a DTH response to the synthetic peptide. Thus, the results of this study demonstrate that the immune responses to A. actinomycetemcomitans fimbriae are genetically controlled.     

Migratory patterns of B lymphocytes. I. Fate of cells from central and peripheral lymphoid organs in the rabbit and its selective alteration by anti-immunoglobulin.

Homing properties of ³H-adenosine-labeled bone marrow (BM), appendix (App), and mesenteric (Mes) and popliteal (Popl) lymph node (LN) cells were studied in the rabbit. While BM cells 24–48 hr after transfer produced equivalent radioactivity (cpm/mg) in App, Mes, and Popl LN of recipients, App and Popl LN cells did not, showing a highly significant preference for their organ of origin. “Homing to antigen” was shown if Popl LN cells were taken from rabbits immunized to the same antigen as was injected in one footpad of recipients 2 weeks earlier.Pretreatment of App cells with sheep anti-rabbit Ig abolished their preferential localization in App as judged in recipients killed 5 hr after transfer, but this was a transient effect and no longer demonstrable by 24 hr.Histological localization of labeled cells showed the corona of follicles in the center of dome-shaped areas of the App to represent “B-influx areas” after transfer of all cell types and this localization was blocked temporarily (5 hr) by prior incubation of App cells with anti-Ig. Thymus-dependent interfollicular areas showed labeled cells after LN cell transfer, and less after App cells, but none after BM. Emphasized in the discussion are (i) the possible effect of antigen on homing of B cells and (ii) the implications of the findings with respect to the appendix as a peripheral lymphoid organ.     

CHARACTERIZATION OF BLEACHING IN CHLORELLA INDUCED BY SUBOPTIMAL GROWTH TEMPERATURES1

Incubation of cultures of a high-temperature strain of chlorella at 10 C stopped growth and bleached all chlorophyll in the cells in 24 hr. Optimal conditions of light (3.0 mw/cm²), gas (1% CO₂-in-air), and inorganic medium for maximal growth at 39 C were maintained in the transfer from 39 to 12, 10, or 5 C. The bleaching process at 10 C is characterized by a lag period for the first few hours followed by a linear decrease in chlorophyll content of cultures. The amounts of time required to bleach half of the chlorophyll initially present (effective half-times) at 10 C were 14 hr for chlorophyll a and 17 hr for chlorophyll b. Effective half-times of bleaching for total chlorophyll were 47 hr at 12 C and 6 hr at 5 C. Addition of glucose to inorganic medium delayed but did not prevent bleaching. Use of argon gas instead, of 1% CO₂-in-air prevented cells from bleaching in both inorganic and glucose media, and indicated an oxygen requirement for bleaching. Incubation of 6 additional strains of Chlorella at 10 C resulted in responses ranging from bleaching to no growth to growth.     

Effects of lead on hepatocyte ultrastructure in Carassius carassius (L.) var. auratus

Subcellular modifications in hepatocytes of Carassius carassius var. auratus subjected to 24 hr and 48 hr sublethal acute lead (5mg·1⁻¹) exposure were studied by electron microscopy. Cytological alterations were observed after 24 hr of treatment and became more evident after 48 hr. Lead induced an increase in nuclear heterochromatin and alterations in mitochondria, endoplasmic reticulum and Golgi complex ultrastructure. Glycogen granula decreased, and secondary lysosomes and lipid droplets increased. Furthermore, intracytoplasmic lumina with microvilli-bearing surfaces and numerous autophagic vacuola were observed after 48 hr of exposure.     

A Total Synthesis of (±)-Gibberellin A12 via Methyl (±)-7,16-dioxo-17-norkauran-19-oate

¹⁴C-labelled methionine, xanthosine, and 7-methylxan-thosine were given to excised tea shoots. The methyl group of methionine was incorporated into 7-methylxanthosine (ca. 10%) in the earlier period of incubation after the uptake. About 50% of the radioactivity of xanthosine was rapidly incorporated into caffeine via 7-methylxanthosine, 7-methylxanthine, and theobromine within 24 hr. 7-Methylxanthosine was also converted into caffeine at a high rate. The results suggest that the pathway for caffeine biosynthesis is as follows: xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine.