Ultrastructure of Brucella abortus L-forms induced by penicillin in a liquid and in a semisolid medium

Brucella abortus L-forms were induced by 5.0 or 10.0 mug of penicillin/ml in a broth medium containing 0.3 m sucrose, and in a semisolid medium containing 10% calf serum and 20.0, 40.0, or 60.0 mug of penicillin/ml. After 96 hr of incubation, L-forms of various sizes and shapes were observed. Basic structures of the L-forms were similar whether induced in liquid or semisolid medium. L-forms had two "unit" membranes, each consisting of two outer dense layers separated by a lucent layer. A few large, irregularly shaped organisms in penicillin-treated broth cultures had additional surface material and were referred to as "transitional" forms. In contrast with L-forms, the bacterial cells were fairly uniform in size and shape, were smaller, and had a more complex cell wall structure. Small bodies limited by a "unit" membrane were present within and around numerous L-forms from liquid and semisolid medium cultures. Other internal membranous structures were also seen in some L-forms. Most Brucella L-forms described in this paper reverted to bacteria in the absence of penicillin and were structurally characteristic of unstable L-forms.     

L-Form Induction, Morphology, and Development in Two Related Strains of Erysipelothrix rhusiopathiae

Two related strains of Erysipelothrix rhusiopathiae, one the parent and the other an L-form revertant, were studied for their propensity or ability to produce L-forms under the influence of penicillin. The parent strain produced L-forms in nutrient solid media in an osmolarity range between 0.85 and 5.0% NaCl concentration whereas the revertant strain did so between 0.5 and 3.0% NaCl concentration. When various hyperosmolar media were tried without penicillin, recovery of L-forms from the revertant strain was optimal at a salt concentration of 2.0%, whereas the parent strain occasionally produced a few L-forms on 3.0% salt medium only. The process of penicillin-induced transformation from bacteria to L-form followed an unusual morphological sequence, beginning with beading of the bacterial body, followed by disintegration into granules from which the L-form colony derived. No large bodies were seen during the initial process of L-form induction, but they evolved later from the original granules and had the potential to reproduce L-type growth. The spontaneous development of L-forms in hyperosmolar media had a different morphological sequence starting with elongation of the bacteria into filaments which later developed polar and central dilatations from which granules and L-type growth developed. The differences in biological behavior between these related bacterial strains suggest that the revertant strain developed new properties, probably of genetic origin. Consequently, the assumption that L-forms revert to the "parent" bacteria may not always be justified. It can be made only after the biological properties of the parent and the revertant organisms have been properly identified.     

Treatment and prevention of experimental pneumonias, caused by L forms of bacteria]

On a model of experimental pneumonia in mice caused by the L-forms of bacteria against the background of diminished immunity a study was made of the therapeutic efficacy of penicillin, lincomycin, lysozyme and gamma-globulin. Lincomycin, particularly in combination with biologically-active preparations, proved to be expedience for the treatment and prophylaxis of pneumonia caused by the mentioned bacteria; a rapid arrest of pneumonic process occurred and more animals survived. In the greater percentage of cases the use of penicillin was accompanied by generalization of the process.     

Nyctotheroides puytoraci: stimulation of encystment in toads, Bufo regularis, injected with rheumatoid arthritis patients' urine.

Nyctotheroides puytoraci, a ciliated protozoan parasite first described by Essawy (M.Sc. thesis, Alexandria University, Alexandria, Egypt, 1978), reacted by encystation in toad hosts Bufo regularis that had been injected subcutaneously with urine of patients with rheumatoid arthritis. It is speculated that carcinogenic tryptophan metabolites present in the injected urine reach parasites in the recta of treated host animals and are the inducers of encystment. Increased encystment was obtained when hosts were injected with the urine of rheumatoid arthritis patients who had been given 2 g l-tryptophan orally. On the other hand, injection of B. regularis with the urine of rheumatoid arthritis patients who had been given 100 mg of pyridoxine HCl did not induce increased cyst formation in the parasite. The abnormality of tryptophan metabolism in rheumatoid arthritis patients was readily corrected by the administration of pyridoxine.     

Alterations of the L-forms of a sporebearing bacillus

A large pleomorphic gram-negative bacillus developed as a contaminant on blood-agar. Spores were formed in one culture. L-forms were produced with penicillin on blood-agar with 2.5% NaCl; they grew well when transplanted to agar with 0.5% NaCl. After several transplants and long incubation of the L-forms without penicillin, in three transplants small gram-negative pleomorphic bacilli grew, but no L-forms. This occurred once on blood-agar and twice on 30% gelatin. The growth obtained from these small bacilli was similar in morphology and in the physical properties of the organisms to the altered L-forms of Proteus and Salmonella. Multiplication of the pleomorphic organisms and development of branching filaments from the round forms was apparent. The original large gram-negative bacillus was regularly recovered from the L-forms, and was recovered several times from the descendants of the small bacilli. These observations are essentially similar to those made with L-forms of Proteus and with an L-form studied in 1952, indicating alterations in L-forms of bacteria which do not produce B type L-forms.     

Induction of Bacillus brevis L-forms

L-forms of Bacillus brevis were induced and maintained in L-phase medium supplemented with inactivated horse serum with a combination of penicillin (80 u/ml) and cephalosporin (5 μg/ml) in liquid medium and penicillin (200 u/ml) in diphasic culture. These L-forms failed to grow on solid media.     

The clinical spectrum and treatment of Lyme disease

Lyme disease was recognized as a separate entity because of close geographic clustering of affected children in Lyme, Connecticut, with what was thought to be juvenile rheumatoid arthritis. It then became apparent that Lyme disease is a complex, multisystem disorder. The illness usually begins in summer with erythema chronicum migrans and associated symptoms (stage 1). Weeks to months later, some patients develop neurologic or cardiac abnormalities (stage 2), and weeks to years later, many patients develop intermittent attacks of arthritis (stage 3), which may become chronic, with erosion of cartilage and bone. Patients with severe and prolonged illness have an increased frequency of the B-cell alloantigen, DR2. For patients with early Lyme disease, tetracycline appears to be the most effective drug, then penicillin, and finally erythromycin. High-dose intravenous penicillin is effective for the later stages of the disease.     

Microtubular structures in group D streptococcal L-forms

Summary Microtubular structures, apparently continuous with the plasmalemma, have been observed in thin sections of two strains of group D streptococcal L-forms. The tubules had an external diameter of about 250 Å and a hollow core 100–150 Å in diameter. The tubules were found protruding either into or out of the L-form cells and were only found in cultures growing in the presence of penicillin.     

Synovial fluid leukocyte apoptosis is inhibited in patients with very early rheumatoid arthritis

Synovial leukocyte apoptosis is inhibited in established rheumatoid arthritis (RA). In contrast, high levels of leukocyte apoptosis are seen in self-limiting crystal arthritis. The phase in the development of RA at which the inhibition of leukocyte apoptosis is first apparent, and the relationship between leukocyte apoptosis in early RA and other early arthritides, has not been defined. We measured synovial fluid leukocyte apoptosis in very early arthritis and related this to clinical outcome. Synovial fluid was obtained at presentation from 81 patients with synovitis of < or = 3 months duration. The percentages of apoptotic neutrophils and lymphocytes were assessed on cytospin preparations. Patients were assigned to diagnostic groups after 18 months follow-up. The relationship between leukocyte apoptosis and patient outcome was assessed. Patients with early RA had significantly lower levels of neutrophil apoptosis than patients who developed non-RA persistent arthritis and those with a resolving disease course. Similarly, lymphocyte apoptosis was absent in patients with early RA whereas it was seen in patients with other early arthritides. The inhibition of synovial fluid leukocyte apoptosis in the earliest clinically apparent phase of RA distinguishes this from other early arthritides. The mechanisms for this inhibition may relate to the high levels of anti-apoptotic cytokines found in the early rheumatoid joint (e.g. IL-2, IL-4, IL-15 GMCSF, GCSF). It is likely that this process contributes to an accumulation of leukocytes in the early rheumatoid lesion and is involved in the development of the microenvironment required for persistent RA.     

L-Phase variants ofAgromyces ramosus

Agromyces ramosus, which is a numerically prevalent bacterium in soil, was easily induced into the L-phase by growing it on agar media containing low levels of penicillin or glycine. The L-forms were stable after initial contact with the inducing agent and could not be reverted to the bacterial form by any of the procedures tried. These results are discussed in relation to a possible natural occurrence of L-forms of this bacterium in soil.     

The DING protein: an autocrine growth-stimulatory protein related to the human synovial stimulatory protein

A synovial stimulating protein (SSP) has previously been isolated from rheumatoid arthritis synovial fluid and from the culture fluid of rheumatoid arthritis synovial fibroblasts. We have previously isolated, from skin fibroblast cultures, a 40 kDa hirudin-binding protein, which had amino acid sequence homology with the SSP. We sought to clarify the relationship, if any, between the SSP and the hirudin-binding protein. We show that the hirudin-binding protein is immunologically cross-reactive with a protein identical with, or very similar to, the SSP. This hirudin-binding protein is produced by normal and rheumatoid arthritis fibroblasts in culture, and also by cervical carcinoma cells. Traces of an SSP-like protein, and of proteins intermediate in size between the SSP and the hirudin-binding protein, suggest that the hirudin-binding protein may be proteolytically derived from the SSP. An SSP-like protein of about 200 kDa is present in all synovial fluid samples, arthritic and normal, indicating that its presence is not a primary cause of rheumatoid arthritis. There is no evidence for the existence of smaller fragments of the SSP-like protein in synovial fluid. A cDNA sequence, coding for part of the 40 kDa protein, has been obtained. The derived amino acid sequence indicates that a domain, previously identified in the dishevelled gene from Drosophila melanogaster, is present in this protein. Peptides predicted from the cDNA sequence were used to raise antisera, which recognise both the 40 kDa protein and the SSP-like protein. One of the antibody preparations is a good inhibitor of fibroblast proliferation, which confirms the autocrine growth-stimulatory role originally proposed for these proteins.     

Flow cytometric detection of type 1 (IL-2, IFN-gamma) and type 2 (IL-4, IL-5) cytokines in T-helper and T-suppressor/cytotoxic cells in rheumatoid arthritis, allergic asthma and atopic dermatitis.

Type 1 cytokines (a.o. IL-2 and IFN-gamma) play an important role in the pathogenesis of rheumatoid arthritis. On the other hand, IgE-mediated diseases such as allergic asthma and atopic dermatitis show a type 2 cytokine (amongst others IL-4 and IL-5) profile.This study examined simultaneously the intracellular production of IL-2, IFN-gamma, IL-4 and IL-5 in T-lymphocytes of patients with rheumatoid arthritis during treatment with methotrexate or salazopyrin, patients with allergic asthma or atopic dermatitis under stable treatment, compared to healthy controls.A three-colour flow cytometric analysis was used for cytokine detection in T-helper cells and T-suppressor/cytotoxic cells.Compared to controls, patients with symptomatic atopic dermatitis showed an increased number of IL-4-producing T-helper lymphocytes in basal circumstances (P=0.001), in contrast to asymptomatic allergic asthma patients. Compared to controls, rheumatoid arthritis patients, treated with salazopyrin, showed an increased number of IL-2-producing T-helper and T-suppressor/cytotoxic lymphocytes after in vitro stimulation with PMA and ionomycin (P=0.01). In contrast, rheumatoid arthritis patients, treated with methotrexate, a more potent disease modifying drug, did not show this type 1 cytokine profile. A positive correlation was found between the number of IFN-gamma producing T-helper cells and disease activity (Ritchie Index and number of swollen joints) in both rheumatoid arthritis patient groups.Active atopic dermatitis patients showed a type 2 cytokine profile, whereas stable asthma patients with lower disease activity did not show a predominance of type 2 cytokines. Rheumatoid arthritis patients under treatment with salazopyrin had a type 1 cytokine profile, which could not be demonstrated in patients treated with methotrexate. This imbalance between type 1 and type 2 cytokines in different immune mediated disorders can be related with treatment and the grade of disease activity. These results stress the need for further investigation of the influence of therapy on cytokine profiles.     

Permanent alterations of the L-forms of proteus and salmonella under various conditions

L-forms obtained from three strains of Proteus and from one strain of Salmonella have been kept for 15 to 20 years by weekly or monthly transfers on agar plates containing penicillin. The morphology and growth requirements of these strains have changed. They now grow abundantly on the surface of agar and in broth. The cultures consist of large bodies, small granules, and transitional forms. These organisms are more resistant to distortion and stain more deeply than organisms of the usual L-forms. In broth and to a lesser extent on agar, branching filaments develop, on the ends of which both the large, round organisms and small organisms are produced. The filaments are a transitional stage in the development of the cultures. Usual bacillary forms were not present in the culture and did not appear in successive transfers in the absence of penicillin. Bacilli reappeared on exposure of the L cultures to the influence of a spore-bearing bacillus. A similar transformation of L-forms has also been observed developing within a short time in recently isolated A and B type L cultures of one Proteus strain during the process of reversion to the bacterial form. The altered cultures are fixed in a stage of transition between the B type L-form and the regular bacteria.     

Seroprevalence of parvovirus B19 IgG in children affected by juvenile idiopathic arthritis

Parvovirus (PV) B19 is the causative agent of the childhood disease erythema infectiosum. An association of PV B19 with chronic arthropathies, sometimes resembling rheumatoid arthritis or juvenile idiopathic arthritis (JIA), has repeatedly been described. Other studies, however, have failed to identify any such relationship. In order to study further whether there is a link between PV B19 and JIA, we determined the prevalence of PV B19 specific IgG antibodies in serum samples from children with rheumatoid diseases and compared it with the prevalence in unaffected children We reasoned that if there is an association between PV B19 and JIA, then the prevalence of PV B19 IgG in the children with JIA should be higher than in the control group. PV B19 IgG status was tested in 406 children with JIA and related diseases, and in 146 children constituting a control group. The percentage of PV B19 IgG positive children was not significantly elevated in the disease subgroups compared with age-matched control groups. In conclusion, our findings do not support the hypothesis that human parvovirus B19 is involved in the pathogenesis of JIA.     

Neutrophil responsiveness to chemoattractant tripeptide in rheumatoid arthritis

Neutrophils isolated from medication-free rheumatoid arthritis (RA) patients were assayed for responsiveness to the bacterial chemoattractant tripeptide formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). Rheumatoid arthritis neutrophil preparations contained significantly lower percentages of rapidly migrating cells. This relative hyporesponsiveness of RA neutrophils was related to impaired sensing of chemotactic gradients. Rheumatoid neutrophil abnormalities in sensing of and responding to chemotactic gradients were not associated with resting or f-Met-Leu-Phe-induced changes in arachidonic acid metabolism.     

Comparison between penicillamine and sulphasalazine in rheumatoid arthritis: Leeds-Birmingham trial.

Sulphasalazine was first formulated by Svartz in the early 1940s, specifically for use as a remission inducing drug in rheumatoid arthritis. After the publication of an unfavourable trial, however, the drug was restricted to patients with ulcerative colitis. In the late 1970s sulphasalazine was re-examined in rheumatoid arthritis and favourable results reported in "open" trials. A double blind controlled trial was therefore conducted comparing enteric coated sulphasalazine and D-penicillamine in patients with active rheumatoid arthritis. A total of 63 patients were recruited in two centres; 31 were treated with sulphasalazine and 32 received penicillamine. After 16 weeks'' treatment both drugs had produced significant improvements in clinical score, pain score measured on a visual analogue scale, grip strength, Ritchie articular index, erythrocyte sedimentation rate, and serum C reactive protein concentration. Nausea was the major side effect in the sulphasalazine treated group. No potentially dangerous effects of sulphasalazine were encountered in contrast with those seen in the penicillamine group. The results suggest that sulphasalazine is an effective and safe drug capable of producing remissions in active rheumatoid arthritis. They also lend confidence to the use of preliminary "open" trials as a means of screening for remission inducing drugs in rheumatoid arthritis.     

Membrane structures in stable L-forms of Escherichia coli.

Microtubular and lamellate membrane structures were observed at the log phase of growth in the stable L-forms of Escherichia coli cultured in the absence of penicillin.     

On the pathogenicity of nonstable Brucella L-forms and their revertants.

The pathogenicity of B. abortus 870 L-forms obtained by long-term passaging of virulent culture on media with penicillin and of revertants obtained in vitro and in vivo was studied. L-form cultures stimulated only a mild response of the reticulo-endothelial system of the animal organism, at the same time displaying a certain level of toxicity. In vitro revertants approximated to L-forms, while in vivo revertants stood closer to the initial virulent culture, as regards pathogenicity. This seems to be evidence of a potential danger of brucella L-forms for the human and animal organisms.     

Induction, growth and antibiotic production of Streptomyces viridifaciens L-form bacteria

AIMS: To induce, cultivate and investigate the characteristics of L-form bacteria derived from the filamentous actinomycete Streptomyces viridifaciens. METHODS AND RESULTS: L-forms were induced in a liquid medium supplemented with lysozyme and penicillin. A stable culture which no longer required inducing agents but could still revert, was obtained by the twelfth subculture. The specific growth rate of stable L-forms was faster (0.751) than unstable L-forms (0.361). After the exponential growth phase, the cell diameter continued to increase, as did the percentage of vacuoles. Morphologically, the L-forms appeared as spherical bodies with no signs of differentiation and were sensitive to osmotic stress, indicating removal of the cell wall. The L-forms produced secondary metabolites although much lower levels of antibiotic were assayed in the L-forms compared with the cell walled forms. CONCLUSION: Stable L-form bacteria were induced from S. viridifaciens and their growth characterized. The L-forms produced secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Stable Streptomyces L-forms were induced and have potential as biocontrol agents.     

Fatty Acid Composition of L-Forms of Streptococcus faecalis Cultured at Different Osmolalities

The fatty acid composition of the membranes of three different penicillin-produced L-forms of Streptococcus faecalis was determined: (i) a stable (nonreverting) L-form (T(53)) cultured in brain heart infusion (BHI) with 0.5 M sucrose; (ii) a stable L-form (T(531)) cultured in BHI without sucrose; and (iii) an unstable L-form (T(9)) cultured in BHI with 0.5 M sucrose and 1,000 U of penicillin per ml. L-forms were obtained by centrifugation and lysed by washing in 1 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer. The parent S. faecalis was also cultured in BHI and BHI containing 0.5 M sucrose, and washed with buffer. The fatty acid composition of L-forms of S. faecalis cultured in BHI without sucrose (370 mosmol) had higher C(18:1) and lower C(18) than L-forms cultured in the same media with added 0.5 M sucrose (950 mosmol) in both exponential and stationary cultures. In the stationary phase of growth, C(19) was reduced in the L-forms cultured without sucrose. Similar changes were seen in the parent S. faecalis cultured in the two types of media. These changes in membrane fatty acids may relate to osmo-regulation of the L-forms.