Production of the Shwartzman Reaction with Microbial L Forms

Study of potential pathogenicity of microbial L forms was done by the localized Shwartzman reaction. Stable L forms of Proteus mirabilis served as skin preparation in rabbits for induction of Shwartzman reaction by subsequent intravenous injection of either P. mirabilis L forms or Escherichia coli endotoxin. The intensity of the reaction was positively correlated to numbers of L forms in the skin. L forms also served as the intravenous challenge. In vivo multiplication of L forms was not a prerequisite for the reaction, as it could be produced with nonviable, osmotically lysed L forms. The reaction produced with L forms in the skin was more intense than that produced with the parent bacterial form. These latter observations, coupled with the demonstration that L forms disappeared from the skin (lysed?) after 4 hr, in contrast to bacteria which were recoverable for 72 hr (duration of study), suggest release of endotoxin by L forms as a pathogenic mechanism.     

Listeria monocytogenes L Forms I. Induction, Maintenance, and Biological Characteristics

L forms were induced from 15 of 16 strains of Listeria monocytogenes on penicillin gradient plates incubated under aerobic conditions. The culture medium for maintenance of these L forms must contain an electrolyte in a concentration of 1% or sucrose in a concentration of 10%. The electrolytes NaCl, KCl, or MgSO(4) were used in both induction and maintenance media. Induction of L forms occurred more rapidly on media containing KCl. Listeria L forms had the same fermentation reactions as the parent bacterium. The L-form growth in liquid medium was slow, not extensive, and appeared as clumps on the bottom of culture tubes. The morphology of Listeria L forms was similar to that reported for other bacterial L forms. The L forms derived from strain 10403, serotype 1, were stable after two or more passages on penicillin media. They did not revert to the bacterial form after 40 subcultures on penicillin-free media. Some L-form colonies derived from strain 10403 did revert to the bacterial form when transferred directly from induction plates to penicillin-free media. Studies of the growth characteristics for L forms derived from strain 10403 gave the following results: an optimal temperature of 30 C, high electrolyte or sucrose concentration necessary for induction and maintenance, and no requirement for serum.     

Hemocytoblastoid reaction of thymic subcapsular cells to diphtheria toxoid

Subcapsular cells lining the thymic stroma vary from low to high forms, while others have a hemocytoblastoid aspect. The purpose of the present study was to establish whether the transformation of the low forms into hemocytoblastoid subcapsular cells can be induced by an antigen. Rats given 10 Lf of diphtheria toxoid intramediastinally were killed at periods ranging from 3 to 24 hours later. Other rats were injected with ³H-thymidine at various intervals after the toxoid injection, and were killed one hour later. The observations revealed a rapid hemocytoblastoid transformation of subcapsular cells following administration of the toxoid. The transformation is detectable as early as three hours after the injection and can be completed after nine hours. Radioautography revealed that DNA duplication is initiated rapidly in the transforming subcapsular cells, since it can be completed 9 to 12 hours after the toxoid injection. Other observations suggested the transformation of reactive perithymic fibroblasts into subcapsular cells as well as the transformation of hemocytoblastoid fibroblasts and subcapsular cells into free hemocytoblastoid cells.     

Antitumor effect of heat-killed Lactobacillus plantarum L-137 through restoration of impaired interleukin-12 production in tumor-bearing mice

 We have previously reported that heat-killed Lactobacillus plantarum L-137 is a potent inducer of interleukin-12 (IL-12) in vivo as well as in vitro in mice. In order to develop effective usage of L. plantarum L-137 for tumor immunotherapy, we examined its antitumor effect against DBA/2 mice inoculated with syngenic P388D1 tumor cells in different treatment schedules. Daily injection of L. plantarum L-137 from the day of tumor inoculation induced a steep increase in plasma IL-12 only after the first injection but not after subsequent injections, and had no effect on tumor growth and survival time. In contrast, daily injection of L. plantarum L-137 from the 7th day after tumor inoculation exerted a marked antitumor effect but such an effect was not evident in mice treated with L. plantarum L-137 twice a week from the 7th day. IL-12 production was considerably impaired at the first injection but steeply increased after the third injection in the mice injected daily with L. plantarum L-137 from the 7th day. Our results suggest that daily administration of L. plantarum L-137 is required to exert an antitumor effect at the late stages of tumor development when IL-12 production is considerably impaired. Received: 15 July 1999 / Accepted: 28 January 2000     

Atypical behaviour and survival of Streptococcus pyogenes L forms during intraperitoneal infection in rats

Groups of rats were injected intraperitoneally with cell wall-deficient (L) forms of Streptococcus pyogenes, with their parental (S) forms, as well as with a combined inoculum of both forms (S+L). Peritoneal exudate samples were harvested on days 1, 3, 7, 15 and 30 after challenge and were investigated by microbiological, electron microscopic, cytometric and biochemical methods. Parental S forms were isolated from peritoneal exudate samples up to day 15 post infection, while L form cultures were isolated until the end of the examined interval. Electron microscopic examination revealed continuous adhesion of L forms on the macrophage surface as well as intracellular persistence inside them. It was demonstrated that the intraperitoneal inflammatory response to L form infection was higher than to the other infections and the monocyte-macrophage populations were predominant. The established atypical behaviour and long survival of S. pyogenes L forms in the rat's peritoneum could explain some of the mechanisms of the pathogens' persistence as well as the reasons for chronic streptococcal infections.     

Experimental Infection with Parent and L-Phase Variants of Neisseria meningitidis

Thirty-six strains of Neisseria meningitidis, including groups A, B, and C, produced L forms in vitro in the presence of an osmotic stabilizer and high concentrations of horse serum using penicillin as the transforming agent. Transformation to L growth occurred most readily among strains recently isolated from patients, and an unusually high rate of transformation was observed in 7 of the 36 strains. Revertant L strains developed diplococcal colonies on blood-agar and L colonies on sucrose-serum-penicillin-agar-always in a ratio of approximately 10 to 100 diplococcal colonies to 1 L colony. Using mucin as a host depressant, comparison was made between parent and revertant L strains of their initial pathogenicity and development of virulence by serial mouse passage. In general, revertant L strains showed the same pathogenic characteristics as the parent. Heart blood cultures from mice dying of infection with revertant L strains retained their ability to grow as L forms on penicillin media. Three stable L strains were completely avirulent for mice, although persistence of L forms could be demonstrated in peritoneal exudate for 6 days after inoculation.     

Biochemical and genetic evidence for a macromolecular -glucuronidase complex in microsomal membranes

In the mouse β-glucuronidase is present in both microsomes and lysosomes and the enzyme at both sites is coded by the same structural gene. Electrophoresis on polyacrylamide gels showed that liver, kidney and lung from normal strains contained five enzyme forms designated L, M1, M2, M3 and M4 in order of decreasing mobility toward the anode. Band L is found primarily in lysosomes and is a tetramer of 260,000 molecular weight. Bands M1 to M4 are found exclusively in microsomes and range in molecular weight from 310,000 to 470,000. The increase in molecular weight is due to sequential addition of an accessory protein chain. When glucuronidase is highly induced in kidneys of female mice by injection of dihydrotestosterone, a sixth electrophoretic form of glucuronidase, designated X, appears. Form X appears early in induction, is localized in microsomes, and has a molecular weight (260,000) equal to that of the tetramer form L.Mice homozygous for the eg ° mutation, and thus deficient in microsomal glucuronidase, completely lack the microsomal forms M1 to M4. They do contain form X, and this increases after testosterone induction in kidney. The form X present in eg ° mice is indistinguishable from the form X seen in normal induced kidney.It appears that mice synthesize two different tetrameric forms of glucuronidase from the same structural gene. One, form L, is lysosomal; the other, form X, gives rise to microsomal enzyme forms M1 to M4 by the successive addition of up to four accessory protein chains. The eg ° mutant is blocked in the conversion of X to M1.     

Response and fertility of dairy heifers following injection with prostaglandin F(2alpha) during early, middle or late diestrus

After an observed estrus, 250 dairy heifers were injected once with 25 mg of PGF(2alpha) either on cycle days 5 through 7 (E), 8 through 11 (M) or 12 through 15 (L). For five days after the PGF(2alpha) injection, heifers were inseminated at about 12 h after estrus was first observed. Observed estrual response rates were 43.0%, 83.6% and 100% for E, M and L, respectively. Average time from PGF(2alpha) to observation of estrus for E, M and L was 59, 70 and 72 h. Conception rates for heifers responding to PGF(2alpha) were 56.8%, 62.1% and 78.3% for E, M and L, respectively. Based on blood samples drawn at the time of PGF(2alpha) injection, progesterone concentration was significantly correlated with response rate but not with conception rate. When compared with M and L, E had a significantly lower response rate and conception rate as well as a shorter period between injection of PGF(2alpha) and observation of estrus.     

Induction of L Forms of Haemophilus influenzae in Culture and Their Demonstration in Human Bronchial Secretions

Transitional forms and round bodies of Haemophilus influenzae were identified in sputa from patients with chronic bronchitis who were receiving penicillin therapy for H. influenzae infections. In vitro growth of L forms of this organism was induced by penicillin and glycine and was studied for comparison with development in vivo. Variant forms demonstrated in sputum were similar to variant forms observed in penicillin-induced L colonies. Recurrence of infection after cessation of therapy was related to reversion of persisting L forms to bacillary forms. That these forms were derived from H. influenzae was established by direct staining with fluoresceinlabeled specific antibody. This demonstration that transitional forms and round bodies of H. influenzae occurred in vivo suggests that L forms of bacteria may be significant in chronic or recurrent infections.     

Mycobacteriophage L5 infection of Mycobacterium bovis BCG: implications for phage genetics in the slow-growing mycobacteria

Mycobacteriophage L5 is a well-characterized temperate phage that forms stable lysogens in Mycobacterium smegmatis . The host range of L5 is, however, unclear because previous reports suggested that it does not infect slow-growing mycobacteria such as Mycobacterium tuberculosis and bacille Calmette-Guérin (BCG). Moreover, luciferase reporter phage derivatives of L5 failed to produce light from BCG, suggesting that infection is blocked at or before the stage of DNA injection. In this study, we demonstrate that L5 infection of slow growing mycobacteria specifically requires a high concentration of Ca²⁺, conditions that differs from those required for infection of M. smegmatis by L5 and for infection of BCG by the closely related phage D29. In addition, we show that there are specific genetic determinants of L5 that confer the ability to infect slow growing mycobacteria, without altering infection of M. smegmatis . These observations extend the use of phage L5 for the diagnosis and analysis of tuberculosis and other mycobacterial diseases.     

Retrograde Axonal Transport of Locally Synthesized Phosphoinositides in the Rat Sciatic Nerve

Although autoradiography has demonstrated local incorporation of [3H]inositol into axonal phospholipids after intraneural injection, retrograde axonal transport of phosphatidylinositol has only been demonstrated after injection of lipid precursor into the cell body regions (L4 and L5 dorsal root ganglia) of the sciatic nerve. We now report the retrograde axonal transport of inositol phospholipids synthesized locally in the axons. Following microinjection of myo-[3H]inositol into the rat sciatic nerve (50-55 mm distal to L4 and L5 dorsal root ganglia), a time-dependent accumulation of 3H label occurred in the dorsal root ganglia ipsilateral to the injection site. The ratio of dpm present in the ipsilateral dorsal root ganglia to that in the contralateral dorsal root ganglia was not significantly different from unity between 2 and 8 h following isotope injection but increased to 10-12-fold between 24 and 72 h following precursor injection. By 24 h following precursor injection, the ipsilateral/contralateral ratio of the water-soluble label in the dorsal root ganglia still remained approximately 1.0, whereas the corresponding ratio in the chloroform/methanol-soluble fraction was approximately 20. The time course of appearance of labeled lipids in the ipsilateral dorsal root ganglia after injection of precursor into the nerve at various distances from the dorsal root ganglia indicated a transport rate of at least 5 mm/h. Accumulation of label in the dorsal root ganglia could be prevented by intraneural injection of colchicine or ligation of the sciatic nerve between the dorsal root ganglia and the isotope injection site. These results demonstrate that inositol phospholipids synthesized locally in the sciatic nerve are retrogradely transported back to the nerve cell bodies located in the dorsal root ganglia.     

Effects of Lactobacillus casei on Pseudomonas aeruginosa infection in normal and dexamethasone-treated mice

A single intraperitoneal injection of Lactobacillus casei YIT 0003 into normal or dexamethasone-treated mice led to nonspecific resistance against intraperitoneal challenge with lethal doses of Pseudomonas aeruginosa PAO 3047. The enhanced resistance was retained for 14 days (P less than 0.05) after injection with living L. casei. In contrast, the statistically significant duration of the enhanced resistance in mice treated intraperitoneally with living L. acidophilus YIT 0075 was only 5 days. The in vivo killing activity of peritoneal exudate cells (PECs) against P. aeruginosa 5 and 7 days after intraperitoneal injection of living L. casei was significantly higher than in the case of PECs elicited by L. acidophilus. In the case of intravenous injection of heat-killed L. casei before intraperitoneal challenge with P. aeruginosa, there were no survivors in the late period after administration of L. casei. A high correlation existed between the patterns of in vivo killing of P. aeruginosa by PECs and survival rate of mice injected intravenously with heat-killed L. casei. The reduced in vivo killing activity of PECs from dexamethasone-treated mice against P. aeruginosa infection was also augmented by the intraperitoneal injection of heat-killed L. casei. These results indicate that L. casei possesses a resistance-enhancing capacity against P. aeruginosa infection in vivo. Differences in the duration of the enhanced resistance caused by L. casei and by L. acidophilus may be due to differences in chemical composition and/or physicochemical properties of the cell walls of the two kinds of bacteria.     

Study of dinitrosyl-iron complexes pharmacokinetics and accumulation in depot in rat organs

Protein-bound dinitrosyl-iron complexes (DNIC) in rat whole blood and organs were studied after intravenous injection of this substance with glutathione ligand (DNIC-GH). The effect of DNIC-GH injection on NO level (including NO physiological forms) in hydrophobic areas of rat tissues was also studied in normal physiological blood circulation condition. It has been shown, that after DNIC-GH injection the concentration of protein-bound DNICs in rat whole blood and organs rapidly reached maximum values, and then gradually decreased, that pointed to decomposition of DNIC molecules, coupled with NO release. At the beginning of the experiment the rates of DNIC decay in rat heart and lung were substantially higher, as compared with those in liver and kidney. By spin trappping it has been demonstrated that DNIC-GH, as a source of NO physiological forms (including S-nitrosothiols), in normal physiological blood circulation influence heart more selectively, as compared with the other organs.     

Protective Capacity of L-Form Salmonella typhimurium against Murine Typhoid in C3H/HeJ Mice

L forms of Salmonella typhimurium LT2 conferred strong protection to a lethal challenge with its parental bacterium on innately hypersusceptible C3H/HeJ mice, and its minimal protective dose was approximately 150 L-forming units. Although L-form S. typhimurium was avirulent for C3H/HeJ mice, it multiplied slowly in both the liver and spleen with the maximal growth 2–3 weeks after immunization and thereafter it persisted in the liver until 24 weeks. Protective immunity began to work between 4 and 6 weeks after immunization, and it remained active as long as the L forms colonized the liver (until 24 weeks after immunization). Vaccination with the L form induced a population of T cells responding to L-form whole-cell lysate (WCL), while delayed-type hypersensitivity (DTH) to the extract of S. typhimurium was induced after the establishment of solid immunity. Moreover, neither T-cell responses nor DTH to heat-killed S. typhimurium was generated. In addition, antibody responses were elicited to WCL but not to heat-killed S. typhimurium. These results indicate that protection conferred by the L forms is attributable to the persistent colonization of the L forms rather than the presence of DTH, and also that Salmonella cytoplasmic antigens are involved in induction of immunological responses by vaccination with the L forms.     

L form of Neisseria gonorrhoeae

Roberts, Richard B. (Walter Reed Army Institute of Research, Washington, D.C.). L form of Neisseria gonorrhoeae. J. Bacteriol. 92:1609-1614. 1966.-L forms were produced by the penicillin gradient plate technique from a recently isolated strain of Neisseria gonorrhoeae. To date, these L forms have had 30 serial passages on medium containing penicillin. Stabilized L forms developed on penicillin-free medium after 10 or more passages in the presence of penicillin. Morphological characteristics of these organisms were identical to L forms of meningococci. Medium and environmental conditions necessary for optimal growth included: Brain Heart Infusion of pH 7.2 to 7.4, 1.1 to 1.3% agar, 10 to 20% sucrose, 10 to 20% horse serum, temperature at 35 to 36 C, and increased CO(2) tension (candle jar). L forms were more resistant than the parent gonococcus to penicillin, ampicillin, methicillin, cycloserine, and cephalothin, whereas both organisms had similar sensitivities to bacitracin, vancomycin, ristocetin, novobiocin, tetracycline, and erythromycin. Revertant gonococci were produced on penicillin-free medium from L forms which had had 1, 5, and 10 serial passages. Morphology and fermentative reactions of revertant strains were identical to those of the parent gonococcus. Revertant strains produced L forms more readily than the parent organism; in fact, L forms from certain revertants did not require penicillin, but only serum and sucrose for their production and propagation on artificial medium.     

Morphology and Reproductive Processes of the L Forms of Bacteria II. Comparative Study of L Forms and Mycoplasma with the Electron Microscope

Representative electron micrographs, from the study of eight strains of L forms and one strain of Mycoplasma, are presented. A- and B-type L forms were derived from two strains of Proteus, two other L forms were derived from a diphtheroid and from a staphylococcus strain, and two strains (designated as LX) were isolated from L forms derived from a group A beta-hemolytic streptococcus and from a staphylococcus. The Mycoplasma strain was isolated from goats. Sections were made of young colonies grown within agar and from parts of surface colonies embedded in the agar. B-type L colonies of Proteus were produced by inoculation of bacteria into media containing penicillin. The large bodies developing from the bacteria and the organisms in B-type L colonies of Proteus, like the parent bacteria, had a cell wall consisting of a plasma membrane and an outer cell wall. The loss of rigidity in the cell wall indicated an alteration in its structure. The A-type L cultures of Proteus consisted of irregular branching masses extending in several directions, of small dense organisms corresponding to the elementary corpuscles present in cultures of Mycoplasma, and of intermediary forms. In contrast to the B-type, all organisms in the A-type colonies were surrounded by a single unit membrane corresponding to the plasma membrane of bacteria. The structures inside the cell membrane, both in the A- and B-type, seemed to correspond to the structure of the parent bacteria, which contained ribosomes and threads of DNA. The elementary corpuscles formed chains and filaments, and, apparently, these corpuscles took part in the multiplication by gradual enlargement. The organisms seen in the cultures of all L forms and Mycoplasma studied, except in the B-type L forms of Proteus, corresponded in size, shape, and structure, as well as in the development of elementary corpuscles, to the organisms in the A-type L form of Proteus. In contrast to the spherical organisms usually seen in broth cultures, the organisms in young cultures of Mycoplasma, which were grown within the agar, were similar in morphology, as well as in the discernible structure of the organisms, to L forms. Significant morphological and structural differences were not apparent between the L forms and Mycoplasma (in cultures grown within agar media) under the conditions of this investigation.     

Enhancement of Host Resistance against Listeria Infection by Lactobacillus casei: Activation of Liver Macrophages and Peritoneal Macrophages by Lactobacillus casei

The functions of liver macrophages and peritoneal macrophages obtained after injection of Lactobacillus casei were examined. Listericidal activity in vivo was enhanced in liver macrophages 13 days after L. casei injection but was somewhat suppressed in the macrophages 2 days after the injection. The listericidal activity in vitro was enhanced in peritoneal macrophages obtained 13 days after L. casei injection but was suppressed in cells obtained 2 days later. The PMA-triggered respiratory burst in the liver macrophages elicited by L. casei was higher than that of resident macrophages. Alkaline phosphodiesterase activity in the liver macrophages was decreased by L. casei injection, as was also the case with peritoneal macrophages. These observations indicate that L. casei augmented cellular functions of both liver and peritoneal macrophages.     

Veneza zonata (Hemiptera: Coreidae)/trypanosomatid relationship: action of hemolymph in vitro and experimental infection

The defense response of Veneza zonata (Hemiptera: Coreidae) against three different trypanosomatid infections was assessed: (1) strain 714TD, a Leptomonas which has V. zonata as vector of a plant trypanosomatid, (2) strain 563TD, a Leptomonas isolated from the digestive tract of Euchistus heros (Hemiptera: Pentatomidae), and (3) Leishmania (L.) amazonensis, a human parasite that cannot infect V. zonata. Experiments with V. zonata hemolymph showed agglutination only of L. (L.) amazonensis culture forms and hemocytic recognition was more intense with this strain. L. (L.) amazonensis also activated the prophenoloxidase system, whereas strains 714TD and 563TD did not activate this system but rather seemed to inhibit phenoloxidase activity. No flagellates were seen in the digestive tract, hemolymph, or salivary glands in insects infected with L. (L.) amazonensis. The digestive tract, the hemolymph, and the salivary glands of insects fed on tomatoes inoculated with 714TD are sequentially invaded by the flagellate, which is inoculated in plants together with saliva. Insects fed on tomatoes inoculated with 563TD exhibited culture forms in the digestive tract (6 days after) and hemocoel (three additional days); however, they died 12 to 14 days after exposure. The salivary glands in insects inoculated in the hemocoel with 714TD strain are rapidly invaded, whereas those with 563TD culture forms died approximately 24 h after infection. Bacterial proliferation in the hemocoel and hemocyte surface blebbing were seen in insects infected only with 563TD strain as the probable pathogenic mechanism of insect death.     

Effects of the chemical forms and valency states of neptunium on its jejunal transfer in the rat

The transfer of various Np(IV) and Np(V) chemical forms across the small intestine of rats was measured in instilled and perfused jejunum. Instillation of Np(V) nitrate together with citrate or DTPA resulted in the same absorption of Np as after instillation of Np(V) nitrate alone (3 per cent per hour). Perfusion of Np(V) nitrate with bicarbonate or DTPA resulted in a similar transfer (2 per cent) but added citrate or ascorbate resulted in reduced transfer (0.8 per cent). Addition of phytate reduced Np transfer in both instilled and perfused jejunum (0.4 per cent). Np(IV) transfer was usually the same as, or less than that of, the corresponding Np(V) forms. Np(IV) transfer was similar in perfused and instilled jejunum, increasing from 0.2 per cent in the presence of citrate and phytate, to 1 per cent with EDTA and DTPA. Except for phytate, all the forms of Np(V) tested behaved like Np(V) nitrate after transfer from the intestine or after intravenous injection. By contrast, the behaviour of Np(IV) varied for all the forms tested and, for a given form, varied as a function of the experimental procedure used, i.e. jejunal instillation, perfusion, or intravenous injection. These findings suggest that the intestinal transfer of Np might occur via the intercellular pathway, and that it is controlled by both the molecular weight of the Np compound and its stability constant.     

Relative larvicidal potentiality of nano-encapsulated Temephos and Imidacloprid against Culex quinquefasciatus

Encapsulation of temephos ranging from 1% to 16% and imidacloprid from 1% to 8% within biodegradable and biocompatible, polyethylene glycol in different ratios was done by using melt-dispersion method. The efficacy of encapsulated forms was evaluated and compared with their non-capsulated forms against larvae of Culex quinquefasciatus. The encapsulated temephos was more toxic than the encapsulated imidacloprid with LC₅₀ values of 0.013, 0.010 and 0.003 mg/L after 24, 48 and 72 h, respectively. No doubt, the non-capsulated temephos and imidacloprid were more effective as compared to their encapsulated forms. However, the same mortality rate was achieved by the slow release of lesser amount of pesticides after encapsulation, e.g., 0.003 mg/L for 8% temephos formulation and 0.019 mg/L for 4% imidacloprid as compared to their non-capsulated form of temephos and imidacloprid (0.004 and 0.021 mL/L) after 72 h of exposure. Thus, encapsulated forms are more economical and eco-friendly due to controlled slow release of their nanoparticles.