Sverre-Henning Brorson

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole.

The aim of this study was to examine the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole. Because B. burgdorferi is a microaerobic bacterium like Helicobacter pylori, metronidazole (MZ) was chosen in the susceptibility test. For both microaerobic and aerobic incubation the normal mobile spirochetes were resistant to this antibiotic with an MBC ≥512 μg/ml. Conversion of mobile spirochetes to cystic forms was not observed when they were incubated with MZ. When they were incubated under microaerobic conditions, the biologically active cystic forms had an MBC ≥4 μg/ml, but the MBC was ≥32 μg/ml with aerobic incubation at 37°C. Staining with acridine orange (AO), dark field microscopy (DFM), and transmission electron microscopy (TEM) revealed that the contents of the cysts were degraded when the concentration of MZ was ≥MBC. Some cysts were also ruptured. When incubated with a sufficient concentration of MZ, core structures did not develop inside the cysts, and AO revealed less RNA in the cysts. Our observations may help efforts to treat resistant infections caused by B. burgdorferi with a combination of MZ and other antibiotics in order to eradicate both cystic and mobile forms of B. burgdorferi.

Destruction of spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic Tigecycline

Persistence of tissue spirochetes of Borrelia burgdorferi as helices and round bodies (RBs) explains many erythema-Lyme disease symptoms. Spirochete RBs (reproductive propagules also called coccoid bodies, globular bodies, spherical bodies, granules, cysts, L-forms, sphaeroplasts, or vesicles) are induced by environmental conditions unfavorable for growth. Viable, they grow, move and reversibly convert into motile helices. Reversible pleiomorphy was recorded in at least six spirochete genera (>12 species). Penicillin solution is one unfavorable condition that induces RBs. This antibiotic that inhibits bacterial cell wall synthesis cures neither the second “Great Imitator” (Lyme borreliosis) nor the first: syphilis. Molecular-microscopic techniques, in principle, can detect in animals (insects, ticks, and mammals, including patients) helices and RBs of live spirochetes. Genome sequences of B. burgdorferi and Treponema pallidum spirochetes show absence of >75% of genes in comparison with their free-living relatives. Irreversible integration of spirochetes at behavioral, metabolic, gene product and genetic levels into animal tissue has been documented. Irreversible integration of spirochetes may severely impair immunological response such that they persist undetected in tissue. We report in vitro inhibition and destruction of B. burgdorferi (helices, RBs = “cysts”) by the antibiotic Tigecycline (TG; Wyeth), a glycylcycline protein-synthesis inhibitor (of both 30S and 70S ribosome subunits). Studies of the pleiomorphic life history stages in response to TG of both B. burgdorferi and Treponema pallidum in vivo and in vitro are strongly encouraged.

A rapid method for generating cystic forms of Borrelia burgdorferi, and their reversal to mobile spirochetes.

Mobile Borrelia burgdorferi were transferred to distilled water (106 per ml). The cultures were observed by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). 95% of the spirochetes were converted to cysts after 1 min, and after 4 h no normal mobile borreliae were observed. When transferred to growth medium (BSK‐H), the cysts became smaller and more irregular, and were filled with organic substances. After 1 day, 1–5 thin structures sprouted from the cysts. They continued to grow in both length and thickness until they attained a normal spirochetal structure. Finally, these new‐born spirochetes detached from the cysts, by which time their mobility had become normal. The present method for producing large amounts of cystic forms of B. burgdorferi is well suited for further studies of this unique microbe.

Antibody penetration into LR-White sections

The purpose of this investigation is to study the ability of antibodies to penetrate sections of LR-WHITE resin. The methods used in this study were the following: (1) Reembedding of sections labeled with immunogold (1 nm) and peroxidase/DAB/gold chloride, (2) tilting of ultrathin sections treated with immunogold (1 nm), (3) immunolabeling of cylindrical structures embedded in LR-WHITE, (4) application of primary and secondary antibodies on opposite sides of ultrathin sections. Fibrin and human pituitary tissue was embedded in LR-WHITE and treated with anti-fibrinogen or anti-ACTH respectively (ACTH = Adrenocorticotropic hormone). No indication of antibody penetration into the section were found with either of the methods, contrary to findings in earlier publications. The significance of this result is that antigens cannot be demonstrated in the interior of LR-WHITE sections with post-embedding techniques. Furthermore, LR-WHITE resin may be used for quantitative immunoelectron microscopy, and the resin may be used for double immunogold labeling since the application of immunoreagents on opposite sides of the sections is completely safe.

BOVINE SERUM ALBUMIN (BSA) AS A REAGENT AGAINST NON-SPECIFIC IMMUNOGOLD LABELING ON LR-WHITE AND EPOXY RESIN

The purpose of this study was to examine how different incubation times with different concentrations of bovine serum albumin (BSA) affect the amount of non-specific immunogold labeling on epoxy sections and LR-White sections. Immunogold labeling was performed on epoxy sections and LR-White sections of renal tissue with IgG-deposits and fibrin clots, and the antibodies used were anti-IgG and anti-fibrinogen, respectively. The sections were incubated with different concentrations of BSA prior to application of primary antibodies, and the length of this pre-incubation step varied between 0 and 4 h. During the incubation with primary antibodies, BSA was added in the same concentration as in the pre-incubation step. The results showed that the non-specific labeling on the resin decreased significantly when the concentration of BSA or the length of the preincubation step was increased. The non-specific labeling was usually higher on the epoxy resin than on the LR-White resin when using the same conditions with respect to BSA. But, when the preincubation step with BSA lasted 4 h, the non-specific labeling was somewhat lower on epoxy resin than on the acrylic LR-White resin, without respect to the concentration of BSA. The specific labeling for both fibrinogen and IgG decreased slightly when the concentration of BSA and incubation time increased, probably due to the steric hindrance performed by BSA molecules on the section. Blocking procedures with at least 1 h incubation time for the blocking step with at least 5% BSA are recommended for both epoxy and LR-White sections.

Mechanism for antigen detection on deplasticized epoxy sections

The purpose of this investigation was to explain why deplasticizing of epoxy sections gives higher immunogold labeling than non-deplasticizing. The methods used were the following: (1) Comparison of the ratio of immunogold labeling of deplasticized and non-deplasticized sections with gold particles of different sizes and comparison of this ratio with respect to sections of different thickness, (2) the tilt method (Brorson et al., 1994). Human kidney tissue with amyloid A depositions, human fibrin, and human pituitary tissue were embedded, sections were deplasticized on grids, treated with anti-Aa, anti-fibrinogen or anti-ACTH (ACTH = adrenocorticotropic hormone), and reembedded on grids. Indications of significant antibody penetration were found only at the periphery of structures (ACTH-vesicles). This penetration was about 30 nm. The ratios of immunogold labeling of deplasticized and non-deplasticized sections were approximately 2, 5 and 1 for amyloid, fibrin and ACTH, respectively, and were independent of the gold particle size. No significant differences of gold labeling were found between thicker and thinner deplasticized epoxy sections regardless the gold particle size. No significant differences of gold labeling between deplasticized epoxy sections and LR-White sections were found on interior areas of ACTH-vesicles or amyloid A plaques. The increased labeling of deplasticized epoxy sections compared to normal epoxy sections seemed to be mainly a surface phenomenon. The practical significance of this observation is that deplasticizing of epoxy sections may be a better method for localizing antigens at the periphery of structures than the use of other resin embedding media. Deplasticizing of epoxy sections may be a method of choice in a pathological laboratory to detect antigens in routinely embedded tissues.

The combination of high-accelerator epoxy resin and antigen retrieval to obtain more intense immunolabeling on epoxy sections than on LR-white sections for large proteins

The purpose of this study was to examine how antigen retrieval affected the yield of immunogold labeling on epoxy sections based on embedding with different amounts of accelerator. The concentration of accelerator DMP-30 (tri(dimethyl amino methyl) phenol) was varied in the range of 0-8% in the processing of the tissue for epoxy embedding. Immunogold labeling was performed on epoxy sections and LR-White sections of fibrin clots and renal tissue with IgG-deposits, and the antibodies used were anti-fibrinogen anti-IgG and, respectively. For some of the sections antigen retrieval was performed by heating the sections in citrate buffer. In all cases, the yield of immunogold labeling increased following antigen retrieval. The increase (%) in the yield of immunogold labeling as a result of antigen retrieval was larger for epoxy sections than for LR-White sections. The immunolabeling on high-accelerator epoxy sections exposed to antigen retrieval was about 20% more intense than on untreated LR-White sections both for IgG and fibrinogen. In addition to breaking fixations bonds introduced by the chemical fixation, we believe that the antigen retrieval also breaks bonds between the epoxy resin and the embedded tissue. The combination of increased amount of accelerator during tissue processing for epoxy embedding and antigen retrieval by heating in citrate buffer is a potent method for increasing specific immunolabeling on epoxy sections.

Immunoelectron microscopy on epoxy sections without deplasticizing to detect glomerular immunoglobulin and complement deposits in renal diseases

Twenty renal biopsies were studied by immunoelectron microscopy (IEM) after embedding in epoxy resin. Immunogold labeling for immunoglobulins and complement C3 was performed on the epoxy sections, which were not subjected to any kind of etching or deplasticizing prior to the immunolabeling. The concentration of accelerator, DMP‐30 (Tri (Dimethyl Amino Methyl) Phenol), was increased in the infiltration and embedding steps far beyond the values normally used to make immunolabeling of these antigens possible on epoxy sections. The sections were stained with tannic acid accompanied by uranyl acetate and lead citrate. Immunofluorescence (IF) for light microscopy was carried out on frozen sections of parallel tissue samples. Some cases with IgA‐nephritis demonstrated a higher sensitivity for IEM than IF, in the sense that smaller amounts of antigen were detectable with IEM. Ultrastructural preservation with this method was approximately the same as that usually seen on epoxy‐embedded material. By combining excellent immunolabeling with nearly optimal ultrastructural morphology in one procedure, this method is useful particularly in situations where the material available is limited, such as in studies of renal biopsies. As far as we know, this is the first time that immunoglobulins have been satisfactorily immunolabeled on epoxy sections without etching or deplasticizing.

The use of post‐embedding immunoelectron microscopy in the diagnosis of glomerular diseases

Fifty renal biopsies were studied by immunoelectron microscopy after embedding in a partly hydrophilic polyacrylic resin (LR White). Immunofluorescence studies were carried out on frozen sections of parallel tissue samples. Polyacrylic embedding gave good preservation of the renal ultrastructure and precise localization of immunoglobulin and C3c antibodies within glomerular electron‐dense deposits. Non‐specific staining of plasma proteins within vascular lumina could easily be detected. There was good correlation between immunoelectron and immunofluorescence microscopy. Immunoelectron microscopy is a very sensitive method, which can detect small amounts of antigen. More cases were, however, positive by immunofluorescence than by immunoelectron microscopy. This discrepancy may be explained by difference in sample size, and by difference in resolution of morphological details (electron microscopy versus fluorescence microscopy).

Comparison of the Immunogold Labeling of Single Light Chains and Whole Immunoglobulins with Anti-κ on LR-White and Epoxy Sections

The purpose of this study was to examine the intensity of the immunogold labeling of kappa light chains as single molecules and as parts of whole immunoglobulin molecules in LR-White sections and epoxy sections both practically and theoretically. Human renal tissues including deposits of kappa light chains and immune complex deposits of IgA were embedded in both LR-White resin and epoxy resin. Immunogold labeling was performed on unetched thin sections of both resins with anti-kappa or anti-IgA. In all relevant cases the immunolabeling was intense on the LR-White sections. Single kappa light chains were intensely labeled also on the epoxy sections, although not as intensely as on LR-White sections. In contrast, the immunogold labeling of whole immunoglobulins with anti-kapp and anti-IgA was weak and hardly positive on the epoxy sections. Consequently, the quotient labelinglr-white/labelingepoxy for anti-kappa was significantly lower for labeling of single light chains (3.6) than for labeling of whole immunoglobulins (15.9). The corresponding quotient for labeling of whole immunoglobulins with anti-IgA was 17.0. Supported by theoretical considerations, it is believed that the copolymerization between the epoxy resin and the antigens allows the knife to cleave the large whole immunoglobulins more easily than the smaller single kappa chains. This prevents satisfactory immunolabeling of whole immunoglobulins on epoxy sections whether anti-kappa or anti-IgA is used as antibodies, while single kappa chains are easily immunolabeled.