Application of newly synthesized detergents in the side chain degradation of plant sterols by Mycobacterium fortuitum.

Newly synthesized detergents of polyoxyethylene polyoxypropylene co-polymers (Co-EOPO), polyoxyethylene polyoxypropylene adducts of the diethylene triamine (DETA-EOPO), and steroidal detergents (SDD) stimulate significantly side chain degradation of plant sterols referring to both the sterol degradation and the formation of the product, 9 alpha-hydroxy-androsta-4-ene-3,17-dione (9 OH-AD), by Mycobacterium fortuitum NRRL-B-8119. Highest stimulative effects were observed with derivatives of the DETA-EOPO group. Using compounds of the Co-EOPO group optimal sterol transformation rates were found for polymers with a polyoxypropylene domain of 1400-2000 in molecular weight and with a polyoxyethylene content of 20-50%. Mostly efficient steroidal detergents tested were the 4-cholesten-3-(O-carboxymethyl)-oxime and the amino acid adducts of sterol-3 beta-chlorocarbonates. Detergents were favourably applied as pre-formed sterol-detergent complexes (9:1/w:w) in submers fermentation procedures. By treating harvested resting cells of M. fortuitum with derivatives of Co-EOPO or DETA-EOPO a considerable activation of the cells takes place concerning the sterol degradation and the formation of 9 OH-AD at a high yield in subsequent transformation process on buffer. In solubilization experiments we revealed that there is no correlation between the detergent mediated solubilization of the hydrophobic sterol substrate in the aqueous medium and the transformation activity of the bacterial cells. The detergents are assumed to interact with a multicomponent mesophase (FMCM) which is placed between cells and sterol surface and mediates the transport.     

Interferon-γ-activated human granulocytes kill ingested Mycobacterium fortuitum more efficiently than normal granulocytes

Although shortly after the onset of a mycobacterial infection granulocytes are present at the site of inflammation, the role of granulocytes in the elimination of mycobacteria is not well understood. In vitro studies with, for example Mycobacterium tuberculosis or M. bovis, are hampered by the slow proliferation and clumping of the bacteria. To avoid these disadvantages, we developed a model using the atypical mycobacterium M. fortuitum. The present study concerned two questions: whether human granulocytes are able to phagocytose and intracellularly kill opsonized M. fortuitum and whether intracellular killing of these bacteria can be enhanced by treatment of the granulocytes with recombinant human interferon-γ (rIFN-γ). The results showed that normal granulocytes phagocytosed opsonized M. fortuitum rapidly, but did not kill these bacteria effectively. The intracellular killing of M. fortuitum was significantly enhanced by incubation of the granulocytes with rIFN-γ for 18 h before the start of the killing assay. Since these rIFN-γ-pretreated granulocytes did not release more O₂^? and H₂O₂ upon stimulation with phorbol 12-myristate 13-acetate or opsonized M. fortuitum than control granulocytes, non-oxidative killing mechanisms are probably involved in the enhanced killing of M. fortuitum.     

A novel nucleotide-containing antigen for human blood γδ T lymphocytes

The stimulation of human γδ T cells by mycobacteria occurs through recognition of four distinct nonpeptide phosphorylated antigens termed TUBag1–4. Among these latter, TUBag4 has already been biochemically characterized as a γ-X derivative of 5′-deoxythymidine triphosphate (Constant, P., Davodeau, F., Peyrat, M. A., Poquet, Y., Puzo, G., Bonneville, M. and Fournié, J.-J., Science 1994. 264: 267). However, despite chemical synthesis of weakly stimulatory nucleotide-containing analogs, these mycobacterial compounds remained the sole nucleotide-containing antigens actually isolated from natural sources. Here, we present the complete isolation of the TUBag3 antigen from Mycobacterium fortuitum and demonstrate that this nonpeptide molecule contains a 5′-UTP nucleotide moiety. On selected Vγ9/Vδ2 clones, T cell responses can be triggered with nanomolar concentrations of TUBag3. Like crude mycobacterial extracts, this purified nucleotide conjugate elicits a strong polyclonal response of γδ PBL from healthy donors. Furthermore, we present evidence that this compound is distinct from the recently synthesized γ-isopentenyl 5′-UTP, a nucleotide conjugate of isopentenyl pyrophosphate that was found to be stimulatory for human γδ T cells (Tanaka, Y., Morita, C. T., Tanaka, Y., Nieves, E., Brenner, M. B. and Bloom, B. R., Nature 1995. 375: 155). Since it appears that both mycobacterial nucleotide antigens are molecules structurally related to peculiar precursors of nucleic acid synthesis, we propose that TUBag-reactive T cells might be specifically devoted to surveillance of proliferating cells.     

Mycobacterium fortuitum infection after anterior cruciate ligament reconstruction using a polylactic acid bioabsorbable screw: Case report

We report a case of pretibial sinus and abscess after anterior cruciate ligament reconstruction using a polylactic acid tricalcium phosphate bioabsorbable screw for tibial fixation. Mycobacterium fortuitum was identified as the pathogen after specific mycobacterial cultures were obtained from operative specimens. M. fortuitum is a known but rare cause of periprosthetic infection. Diagnosis is often delayed as routine microbiological cultures do not utilise specific culture requirements for mycobacterial growth. There have been several reports in the literature of sterile abscesses associated with bioabsorbable screws. To our knowledge, this is the first case report of a non-tuberculous mycobacterial infection associated with a bioabsorbable implant. This case illustrates that post-operative Mycobacterium infection can occur as a complication of ACL reconstruction with bioabsorbable screw fixation and should be considered in the differential diagnosis of post-operative periprosthetic infection.     

Identification of mycobacteria to the species level by automated restriction enzyme fragment length polymorphism analysis

An automated method for the restriction fragment length polymorphism (RFLP) analysis for the differentiation of mycobacteria to the species level is described. After polymerase chain reaction (PCR) amplification of a sequence of the gene encoding the 65-kDa surface antigen common to all mycobacteria the product was investigated by RFLP analysis. For accurate determination of fragment sizes the asymmetrically fluorescein-labelled PCR product was partially digested with restriction site enzymes BstEII and HaeIII. The fragments obtained were analysed electrophoretically using an automated laser fluorescence DNA sequencer. Determination of fragment sizes revealed a deviation of ±1 base pair (bp; 0.6%) when compared to expected sizes. The validity of this approach was confirmed by analysing mycobacterial DNA obtained from pure cultures of Mycobacterium (M.) tuberculosis and alcohol-fixed smears as well as paraffin-embedded sputa of patients with culture-proven tuberculosis. Additionally a diagnostic algorithm was established by investigation of cultured M. bovis, M. bovis bacille Calmette-Guérin, M. avium, M. intracellulare and M. fortuitum. The method allows the identification of restriction enzyme sites which are only 40 bp apart. Partial restriction enzyme digestion of asymmetrically fluorescence-labelled PCR products will presumably lead to the discovery of new restriction enzyme sites.     

Antigen 85C on Mycobacterium bovis, BCG and M. tuberculosis promotes monocyte-CR3-mediated uptake of microbeads coated with mycobacterial products.

The uptake in monocytes of monodispersed latex microbeads precoated with whole bacillus Calmette-Guérin (BCG) cells, Mycobacterium tuberculosis sonicate or culture fluid, or antigen (Ag) from the culture fluid was examined by microscopy. There was a significantly higher cell association of beads coated with whole BCG cells, the secreted 85C component of the Ag 85 complex or M. tuberculosis sonicate than of phosphate-buffered saline (PBS)-treated control beads. Antibodies (Ab) to Ag 85 inhibited the uptake of BCG- and Ag 85C-treated beads. A monoclonal antibody (mAb) to complement receptor type 3 (CR3), but not mAb to CR1, inhibited the uptake of Ag 85C-coated beads, indicating that the mycobacterial Ag-dependent uptake of particles was mediated via CR3 on the monocytes. This points to the existence of a ligand on Ag 85C which may promote monocyte uptake of M. bovis, BCG and M. tuberculosis.     

CaM kinase II-alpha activity,levels and Ca/calmodulin dependent phosphorylation of substrate proteins in mice brain during fatal murine cerebral malaria

The activity and levels of CaM kinase II-alpha was investigated in the cytosolic and membrane fraction of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). In parallel, Ca(2+)/Calmodulin dependent phosphorylation of target substrate proteins was studied using syntide-2 as substrate. Pathology of FMCM resulted in decreased CaM kinase-II activity in both cortex and cerebellum though western analysis revealed no appreciable changes in the levels of CaM kinase-II alpha in cytosol and membrane fractions from control and cerebral malaria infected brain. Given the abundant expression of Cam kinase-II in neuronal tissue, its significance in neurotransmitter release and synthesis and signal transduction during apoptosis, decreased levels of enzyme activity and altered phosphorylation of substrate proteins by CaM kinase II may serve as important cues in understanding the CaM kinase signal transduction events central to neurological disorders during FMCM.     

Preparation of Fluorescent Carboxyl and Amino Functionalized Polystyrene Particles by Miniemulsion Polymerization as Markers for Cells

Summary: A series of fluorescent polystyrene latex particles with carboxyl and amino functionalities on their surface were synthesized by the miniemulsion technique. The fluorescent dye N‐(2,6‐diisopropylphenyl)perylene‐3,4‐dicarboximide (PMI) was incorporated into the copolymer nanoparticles formulated from styrene and acrylic acid or styrene and aminoethyl methacrylate hydrochloride. The resulting latexes were stable and showed a monodisperse size distribution. The particle size depended on the amount and nature of the functional comonomer and was in the range 100–175 nm. All latexes were characterized by transmission electron microscopy (TEM), dynamic light scattering, UV‐Vis spectroscopy and zeta potential measurements. The amount of surface functional groups was determined by electrolyte titration. Furthermore, the functionalized fluorescent particles were utilized as markers for HeLa cells and cell uptake was visualized using fluorescence microscopy. The correlation of the uptake of nanoparticles with the surface charge was determined by FACS measurements.

Confocal fluorescent microscopy of HeLa cells after the uptake of amino functionalized particles (green).     

Tumor necrosis factor-alpha expression in the brain during fatal murine cerebral malaria: evidence for production by microglia and astrocytes.

Fatal murine cerebral malaria (FMCM) is an immunopathological process. The depletion of CD4+ T cells, or the administration of antioxidants or antibodies against certain cytokines, protect the mice against cerebral complications. We previously have shown that astrocytes, microglia, and monocytes play a role in the development of FMCM, suggesting that an active immune response occurs locally within the central nervous system. We now have investigated the functional involvement of glia and monocytes in FMCM by assessing 1) the production, 2) the temporal appearance, and 3) the cellular source of cytokine mRNA and protein in the brain. Brain sections from uninfected and FMCM mice were analyzed for the presence of cytokine mRNA and protein by in situ hybridization and immunohistochemistry. Tumor necrosis factor (TNF)-alpha mRNA and protein were associated with microglia and astrocytes, monocytes, and the cerebral vascular endothelium in FMCM mice but not uninfected animals. TNF-alpha mRNA was first detected several days before the animals showed cerebral symptoms and died. Interleukin (IL)-1 beta mRNA was found in the brains of both uninfected and FMCM mice. However, IL-1 beta protein was associated only with monocytes, the meningeal vascular endothelium, and neurons in the fronto-parietal cortex in the FMCM brains. No IL-4 or IL-6 mRNAs were detected in either group. These results provide the strongest evidence to date that cytokines, in particular TNF-alpha, produced locally in the central nervous system play a role in the pathogenesis of FMCM.     

Adhesion of osteoblast-like cells on nanostructured hydroxyapatite

The response of osteoblast-like cells seeded on hydroxyapatite (HAp) substrates consisting of nanosized crystals was investigated. Various types of HAp nanocrystals, such as nanofibers, nanoneedles and nanosheets, were selectively prepared as substrate through the hydrolysis of a solid precursor crystal of CaHPO₄ in alkaline solutions by varying the pH and ion concentrations. Although all the substrates were macroscopically flat and smooth, the nanoscale topography influenced cell activity, including the adhesion, proliferation, elongation and formation of actin stress fibers. The presence of fine nanoneedles and nanofibers on the surface restricted the cellular activities, while the cells steadily proliferated on a nanoscopically smooth surface of large grains and on a substrate consisting of wide nanosheets. These results suggest that the adhesion and subsequent responses of osteoblast-like cells were affected by the contact domain size between the cell and the substrate. Isolated small domains of the nanostructured HAp limited focal adhesion formation in the cells associated with the formation of stress fibers. Stable adhesion with contact domains larger than 100 nm in width was suggested to be required for cell survival. On the other hand, insufficient adhesion on the fine nanoneedles was found to lead to apoptosis.     

Construction and growth properties of a yeast strain defective in sterol 14-reductase

Summary We have transformed Saccharomyces cerevisiae with a genomic library contained in the replicative vector pFL44. The resulting transformants were screened for resistance to fenpropidin, a specific inhibitor of sterol 14-reductase. A plasmid was isolated that transformed yeast both to resistance to fenpropidin and to an increased specific activity of sterol 14-reductase. Sterol analysis of transformed cells grown in the presence of increasing concentrations of the inhibitor confirmed that resistance was a consequence of over-production of sterol 14-reductase. By chromosomal gene disruption, we have, for the first time, constructed yeast strains defective in sterol 14-reductase. As expected, since yeast in unable to take up sterols in aerobiosis, the disrupted strains do not grow in the presence of oxygen, even if exogenous sterols are supplied. However, disrupted cells grow in anaerobiosis with exogenous oleic acid and ergosterol supplemens. They also grow in aerobiosis if they bear an additional mutation allowing sterol uptake. In this last growth condition the cells require a sparking ergosterol supplementation (25nM) and accumulate ignosterol (ergosta-8, 14-dienol) as the end-product of the sterol pathway. These results reveal that ignosterol is not obviously toxic to yeast membranes and strongly suggest that the molecular basis of the antifungal-activity morpholine and piperidine is directly related to the specific inhibition of ergosterol formation.     

Generation of Multinucleated Giant Cells In Vitro by Culture of Human Monocytes with Mycobacterium bovis BCG in Combination with Cytokine-Containing Supernatants

Multinucleated giant cells (MGC), a characteristic feature of tuberculous granulomas, form by fusion of monocytes or macrophages, but little is known about the mechanism of the fusion process itself. Several studies report an indirect effect of mycobacteria, i.e., induction of a soluble lymphocyte-derived fusion factor following stimulation by mycobacteria or mycobacterial products. The aim of our study was to determine whether contact with mycobacteria can induce MGC formation from human monocytes in vitro. Stimulation of monocytes with Mycobacterium bovis bacillus Calmette-Guérin (BCG) in combination with cytokine-containing supernatants of herpesvirus saimiri-transformed human T-cell clones (T-SN) led to MGC formation with fusion rates of about 27%. In contrast, stimulation with one component alone induced only low fusion rates of up to 10%. Heat-killed BCG in combination with T-SN induced monocyte fusion to the same extent as live mycobacteria. BCG culture supernatant, BCG lysate, or inert particles in combination with T-SN did not induce MGC formation. Experiments using transwell plates containing a semipermeable membrane revealed that induction of the fusion process is dependent on direct contact of monocytes and mycobacteria. MGC formation induced by BCG plus T-SN could be inhibited by addition of monoclonal antibodies to gamma interferon (but not tumor necrosis factor alpha) as well as to the β chain (CD18) of β2-integrins. These results demonstrate that contact with mycobacteria in combination with cytokine-containing supernatants is able to induce human monocytes to form MGC and that membrane-bound molecules of mycobacteria and monocytes are involved in the fusion process.     

Sensing of Mycobacterium tuberculosis and consequences to both host and bacillus

Mycobacterium tuberculosis (Mtb), the primary causative agent of human tuberculosis, has killed more people than any other bacterial pathogen in human history and remains one of the most important transmissible diseases worldwide. Because of the long-standing interaction of Mtb with humans, it is no surprise that human mucosal and innate immune cells have evolved multiple mechanisms to detect Mtb during initial contact. To that end, the cell surface of human cells is decorated with numerous pattern recognition receptors for a variety of mycobacterial ligands. Furthermore, once Mtb is ingested into professional phagocytes, other host molecules are engaged to report on the presence of an intracellular pathogen. In this review, we discuss the role of specific mycobacterial products in modulating the host's ability to detect Mtb. In addition, we describe the specific host receptors that mediate the detection of mycobacterial infection and the role of individual receptors in mycobacterial pathogenesis in humans and model organisms.     

Reduced surface area in apoptotic rounding of human chang liver cells from serum deprivation

Background: The early stages of apoptosis (programmed cell death) are said to be characterized by internucleosomal DNA fragmentation and “condensation of the cytoplasm” in which cells round up, detach, and increase in density. We studied the causation of apoptotic rounding. Methods: Human Chang liver cells in normal monolayer culture were compared with apoptotic counterparts derived from serum growth factor deprivation. Cell-by-cell analysis using the Coulter EPICS PROFILE II flow cytometer studied (1) the cell cycle from propidium iodide–DNA bindings, (2) uptake of neutral red (NR) dye, a viable cell marker, and (3) cytosolic pH (pH_i) modulations from 2′,7′-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) fluorescence ratios with NH₄Cl prepulsing and forward scatter bitmapping of cell surface area. Morphometric studies were done in the Quantimet 570 image analyser. Uptake of trypan blue, neutral red, and 2 million mol.wt fluoresceinated dextrans was studied by light microscopy. Cytological profiles were examined in light microscopy and transmission and scanning electron microscopy. Results: Three days of serum growth factor deprivation caused confluent flat substrate-attached cells to retract and round up, tethering tenuously to the substrate via thin microvillus attachments only. Ninety percent of cell surface area was lost with this flat-to-round change. There was high trypan blue staining with total loss of proliferative potential, and the entire genome was just fragmented DNA making up the solitary A_o (apoptotic) peak in cell cycle profiles. However, these rounded apoptotic cells also internalized huge 2 million mol.wt dextran particles and impermeant neutral red which is an established viable cell marker. The rounded apoptotic cells had an intensely acidic (pH 5.6) cytosol and therefore a steep [H⁺]_i/[H⁺]_o gradient promoting proton extrusion. The pH_i upshifted dynamically upon acidification, recovering and even exceeding resting level by a whole pH unit. Surface area reduction occurred concomitantly in real time with pH_i upshifts in these apoptotic cells. Acidification and recovery in apoptotic cells also produced enhanced uptake of neutral red. Cytological profiles showed abundant large endocytic channels and endosomes in the rounded apoptotic cells. Conclusion: Gross surface area reduction with evidence of distinctive endocytic activity including uptake of huge 2 million mol.wt dextran particles suggested large channel endocytic internalization as a causal factor in apoptotic rounding, in common with rounding in M-phase and interphase cells with pH_i upshifting where concomitant surface area reduction and uptake of impermeant particles were similarly demonstrable. The reduction in size of the cell envelope, together with consequential concentration pressures, could account for the observed rise in cell density and shrinkage in cell size. As a symptom of continual pH_i upshifting, apoptotic rounding appears to be a recovery-associated response rather than a direct consequence of the disruptive forces causing its death. © 1994 Wiley-Liss, Inc.     

Targeting the allergen to oral dendritic cells with mucoadhesive chitosan particles enhances tolerance induction

Background: Sublingual immunotherapy (SLIT) efficacy could be improved by formulations facilitating allergen contact with the oral mucosa and uptake by antigen‐presenting cells (APCs). Methods: Two types of chitosan microparticles, differing in size and surface charge, were tested in vitro for their capacity to improve antigen uptake and presentation by murine bone marrow‐derived dendritic cells (BMDCs) or purified oral APCs. T‐cell priming in cervical lymph nodes (LNs) was assessed by intravenous transfer of carboxyfluorescein diacetate succinimidyl ester‐labelled ovalbumin (OVA)‐specific CD4+ T cells and flow cytometry analysis. Ovalbumin‐sensitized BALB/c mice were treated sublingually with soluble or chitosan‐formulated OVA twice a week for 2 months. Airway hyperresponsiveness (AHR), lung inflammation and T‐cell responses in cervical and mediastinal LNs were assessed by whole‐body plethysmography, lung histology and Cytometric Bead Array technology, respectively. Results: Only a mucoadhesive (i.e. highly positively charged) and microparticulate form of chitosan enhances OVA uptake, processing and presentation by murine BMDCs and oral APCs. Targeting OVA to dendritic cells with this formulation increases specific T‐cell proliferation and IFN‐γ/IL‐10 secretion in vitro, as well as T‐cell priming in cervical LNs in vivo. Sublingual administration of such chitosan‐formulated OVA particles enhances tolerance induction in mice with established asthma, with a dramatic reduction of both AHR, lung inflammation, eosinophil numbers in bronchoalveolar lavages, as well as antigen‐specific Th2 responses in mediastinal LNs. Conclusions: Mucoadhesive chitosan microparticles represent a valid formulation for sublingual allergy vaccines.     

The enzyme responsible for the respiratory burst in elicited guinea pig peritoneal macrophages

The enzymatic basis of the respiratory burst induced by phorbol myristate acetate in elicited peritoneal macrophages of the guinea-pig has been studied. The following evidence suggests that a membrane-bound oxidase that preferentially uses NADPH as substrate is the main enzyme responsible for activation of the oxidative metabolism: (1) The supernatant of postnuclear fractions of resting macrophages oxidises NADH and NADPH with formation of O. The activity with both substrates is very low and does not change in the supernatant obtained from activated cells. (2) The cell-free particles of resting macrophages also oxidise both NADH and NADPH with formation of O. The activity of the cell-free particles from activated macrophages does not change when NADH is the substrate. By contrast, the activity of the cell-free particles from activated cells is markedly increased when NADPH is the substrate. (3) In cell-free particles from activated macrophages the K_m for NADPH is about one order of magnitude lower than that for NADH and the V_max with NADPH is double that with NADH. (4) The NADPH oxidase of cell-free particles is insensitive to azide, cyanide, antimycin A and rotenone and is sensitive to the sulphydryl reagent PCMB. All these drugs have the same effect on the respiratory response of intact macrophages. (5) A direct correlation is found between the degree of activation of the respiratory metabolism of intact macrophages and the extent of activation of the NADPH oxidase. A new approach designed to measure the activity of the oxidase soon after the activation of the enzyme has taken place, shows that the NADPH oxidase can account for the respiratory burst of intact macrophages.     

Effect of inhibitors of cell envelope synthesis on β-sitosterol side chain degradation by Mycobacterium sp. NRRL MB 3683

The role of the lipid bilayer and the peptidoglycan of the mycobacterial cell wall in the permeation of β-sitosterol into the cell and its transformation to androst-1-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) was studied. Specific inhibitors were used at concentrations affecting the biosynthesis of the assumed target structures, but causing only partial cell growth inhibition or exerting no effect on growth. m-Fluorophenylalanine and dl-norleucine which are known to disorganize the biosynthesis of amphipatic components of the outer layer of the lipid bilayer, used at concentrations 250 μg/ml and 400 μg/ml, respectively, increased the formation rate of AD + ADD from 0.3 (control) to 0.7 and 0.8 mg products/g dry weight/h. The disorganization of the underlying mycolyl-arabinogalactan structure by the action of ethambutol at the concentration 40 μg/ml, at which the cell growth was apparently not affected, caused the decrease of the product formation from 135 mg/1 to 70 mg/1. In the presence of isoniazid (350 μg/ml) only trace amounts of AD accumulated during 48 hours of transformation indicating much lower activity than that of the intact cells. The most effective among the tested inhibitors of peptidoglycan synthesis were glycine (15 mg/ml) and vancomycin (150 μg/ml) which enhanced the transformation activity of the treated cells nearly three times. Increased transformation rate was also obtained by the action of colistin at concentrations ranging from 10 μg/ml to 15 μg/ml.     

Bacteremia Does Not Affect Cellular Uptake of Ultrafine Particles in the Lungs of Rats

To assess the effects of intra‐abdominal bacteremia on lung cellular function in vivo, we used electron microscopy to quantify the uptake of 6 nm diameter, albumin‐coated colloidal gold particles (overall diam. 20.8 nm) by cells in the lungs of rats made septic by the introduction of live bacteria (E.coli and B. fragilis) into their abdomens. Gold particles were instilled into the trachea 24 hr after bacteremia induction, and lungs were harvested and prepared for electron microscopy 24 hr later. Because bacteremia produces an increase in metabolism, we hypothesized that this might be associated with increased cellular uptake of these particles and also with increased permeability of the alveolar epithelial barrier to them, as bacteremia is also associated with lung injury. We quantified particle uptake by counting particle densities (particles/μm²) within type I and type II epithelial cells, capillary endothelial cells, erythrocytes and neutrophils in the lungs of five septic rats and five sham‐sepsis controls. We also counted particle densities within organelles of these cells (nuclei, mitochondria, type II cell lamellar bodies) and within the alveolar interstitium. We found particles to be present within all of these compartments, although we found no differences in particle densities between bacteremic rats and sham‐sepsis controls. Our results suggest that these 6 nm particles were able to freely cross cell and organelle membranes, and further suggest that this ability was not altered by bacteremia. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Adjuvant disease induced by mycobacteria,determinants of arthritogenicity

Genetic, endocrine and immunological factors are probably involved in adjuvant polyarthritis. The nature of the vehicle and of the mycobacterial components administered also has a major influence. It was originally assumed that arthritogenicity and adjuvanticity of mycobacterial fractions such as wax D were intimately related. Our previous findings showed that the water soluble adjuvant (WSA) ofM. smegmatis which could substitute for mycobacterial cells in Freund's complete adjuvant and induce delayed hypersensitivity was not arthritogenic in the Wistar rat. We have since observed that auto-immune diseases could be elicited by WSA. Therefore experiments were repeated using the very susceptible Lewis strain. The activity of cord factor and of various mycobacterial preparations suspended in mineral or in peanut oil was also evaluated in mice and in normal or hypophysectomized rats.Our present findings confirm the absence of arthritogenicity of WSA in the Lewis strain. They also indicate that cord factor with WSA does not suffice to induce a generalized adjuvant disease, but that a mycobacterial component which could be susceptible to lysozyme treatment is required also. However, the local inflammation of the injected limb was produced by a preparation of cord factor administered in mineral or even in peanut oil. This was observed in normal or hypophysectomized rats and in Swiss mice which were not susceptible to the generalized disease.