Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes.

Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson's correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis.

Gene expression and distribution of key bone turnover markers in the callus of estrogen-deficient, vitamin D-depleted rats.

An experimental rat model was used to test the hypothesis that in osteoporosis (OP) the molecular composition of the extracellular matrix in the fracture callus is disturbed. OP was induced at 10 weeks of age by ovariectomy and a vitamin D(3)-deficient diet, and sham-operated animals fed normal diet served as controls. Three months later a closed tibial fracture was made and stabilized with an intramedullary nail. After 3 and 6 weeks of healing, the animals were killed and the fracture calluses examined with global gene expression, in situ mRNA expression, and ultrastructural protein distribution of four bone turnover markers: osteopontin, bone sialoprotein, tartrate-resistant acid phosphatase, and cathepsin K. Global gene expression showed a relatively small number of differently regulated genes, mostly upregulated and at 3 weeks. The four chosen markers were not differently regulated, and only minor differences in the in situ mRNA expression and ultrastructural protein distribution were detected. Gene expression and composition of fracture calluses are not generally disturbed in experimental OP.

An in vitro study of the activity of telithromycin against mobile and cystic forms of Borrelia afzelii.

In this study the new ketolide telithromycin was tested in vitro against motile and cystic forms of Borrelia afzelii, one of the species of Borrelia burgdorferi sensu lato. Acridine orange staining, dark field microscopy, and transmission electron microscopy were the techniques used to study the influence of telithromycin on the bacteria. The activity was unexpectedly high, 0.0003 microg/ml < MBC < or = 0.0006 microg/ml for the mobile forms after 7 days of incubation at 34 degrees C. MIC was < 0.00015 microg/ml. It is likely that the agent works bacteriostatically and kills in a time-dependent and concentration-independent way, by binding tightly to the ribosomes. The agent was not able to prevent cyst formation, and the cysts were not affect ed at an in vivo achievable concentration. Electron microscopy also supports the hypothesis of telithromycin being an effective agent against the mobile bacteria.

[Lupus nephritis with crystal structures in glomerulopathy ]. / Lupusnephritis mit Kristallstrukturen in glomerulären Ablagerungen.

Lupus nephritis is a common phenomenon in Systemic Lupus Erythematosus (SLE). We analyzed a renal biopsy of a 30-year-old woman with SLE. The clinical history showed a typical SLE with generalized symptoms without demonstrable lupus coagulant, positive for anti-nuclear antibodies and anti-ds-DNA antibodies but negative for rheumatoid factor, cryoglobulins and antiphospholipid antibodies. A paraproteinemia for IgA, IgG and IgM was not detectable. Using light, electron and immunoelectron microscopy electron-dense deposits were noted in subepithelial, subendothelial and mesangial position. Most remarkably, the electron-dense deposits and mesangial areas in the vicinity of deposits contained an electron-dense crystalline material. The crystalline structures were composed of IgG and kappa light chains, while they were negative for IgM, IgA and lambda light chains, as demonstrated by immunoelectron microscopy. As far as we know, this is the first case of lupus nephritis with crystalline structures. Since we could not detect cryoglobulinemia or paraproteinemia, other mechanisms possibly favor organization of macromolecular structures.

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

In this work the susceptibility of mobile and cystic forms of Borrelia burgdorferi to hydroxychloroquine (HCQ) was studied. The minimal bactericidal concentration (MBC) of HCQ against the mobile spirochetes was > 32 microg/ml at 37 degrees C, and > 128 microg/ml at 30 degrees C. Incubation with HCQ significantly reduced the conversion of mobile spirochetes to cystic forms. When incubated at 37 degrees C, the MBC for young biologically active cysts (1-day old) was > 8 microg/ml, but it was > 32 microg/ml for old cysts (1-week old). Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that the contents of the cysts were partly degraded when the concentration of HCQ was > or = MBC. At high concentrations of HCQ (256 microg/ml) about 95% of the cysts were ruptured. When the concentration of HCQ was > or = MBC, core structures did not develop inside the cysts, and the amount of RNA in these cysts decreased significantly. Spirochetal structures inside the cysts dissolved in the presence of high concentrations of HCQ. When the concentration of HCQ was > or = MBC, the core structures inside the cysts were eliminated. These observations may be valuable in the treatment of resistant infections caused by B. burgdorferi, and suggest that a combination of HCQ and a macrolide antibiotic could eradicate both cystic and mobile forms of B. burgdorferi.

pH-dependent effect of heat-induced antigen retrieval of epoxy section for electron microscopy.

The aim was to examine how the pH in the antigen retrieval medium (citrate) affects the yield of immunogold labeling of epoxy sections. Renal swine tissue with glomerular immune complex deposits with reactivity against IgG was embedded in epoxy resin. Prior to immunogold labeling with anti-IgG, ultrathin sections from these blocks were exposed to antigen retrieval by heating in citrate solution (pH 6, 9 or 12) at 95 degrees C in a PCR-machine or at 121 or 135 degrees C min in an autoclave. The level of immunogold labeling was significantly higher for pH 12 than for pH 6 when heated at 95 degrees C (50% more intense), but at the cost of the ultrastructural preservation of the tissue. At pH 12 and temperature 135 degrees C the epoxy sections were completely destroyed. The sections which had been heated at 135 degrees C, pH 6 appeared significantly better both with respect to intensity of immunogold labeling (85% more intense) and to ultrastructural preservation than those which were heated at 95 degrees C, pH 12. Therefore, our results indicate that relatively low pH (pH 6) and high temperature is the method of choice, but low temperature and high pH can be used when an autoclave is not available.

Heat-induced antigen retrieval of epoxy sections for electron microscopy.

The purpose of this manuscript is to review the literature on the use of heat-induced antigen retrieval methods to enhance the immunolabeling of epoxy sections at the electron microscopical level. The history of the development of antigen retrieval by heating formaldehyde fixed paraffin sections in a buffer solution is given in short, and how this technique has been extended to resin sections and in particular epoxy sections is explained. Theories for the mechanism of enhancement of the immunolabeling of epoxy sections by the heat-retrieval method are discussed, and it is finally speculated whether most of the mechanisms for antigen retrieval on epoxy sections in heated buffer solution are essentially the same as for conventional immunoenhancing by deplastizing and etching. The more accelerator used in the processing of the tissue the more intense the immunolabeling of the heated epoxy sections becomes. The intensity of immunolabeling of the epoxy sections increases with the temperature in the heated buffer solution, and the intensity is significantly higher at high autoclave temperatures than at 95 degrees C, Heat-induced antigen retrieval is also compared with other, conventional techniques for enhancing the immunolabeling of epoxy sections.

Increased level of immunogold labeling of epoxy sections by rising the temperature significantly beyond 100 degrees C in the antigen retrieval medium.

The purpose of this study was to compare the level of immunogold labeling of epoxy sections when the sections were subjected to antigen retrieval at different temperatures. Renal swine tissue with glomerular immune complex deposits with reactivity against IgG and C3 was embedded in epoxy resin. Sections from these blocks were exposed to antigen retrieval by heating in citrate solution at temperatures in the range of 25-135 degrees C. Immunogold labeling with anti-IgG and anti-C3 was performed on the heated sections. The level of immunogold labeling increased significantly in the direction of increased heat. Interestingly, the level of immunogold labeling was significantly higher when exposed to heating in the autoclave (121 and 135 degrees C) than at temperatures just below the normal boiling point. Sections stained with anti-C3 turned from almost negative labeling when heated at 95 degrees C to strong positive labeling when heated at 135 degrees C (11 times increased). The intensity of the immunogold labeling with anti-IgG increased almost three times when raising the temperature in the retrieval medium from 95 to 135 degrees C. The practical significance of these results is that antigen retrieval of epoxy sections should be performed by heating in aqueous solutions at 135 degrees C or higher to obtain maximum immunolabeling.

Deplasticizing or etching of epoxy sections with different concentrations of sodium ethoxide to enhance the immunogold labeling.

The study's purpose was to obtain improved "deplasticizing" of epoxy sections for immunoelectron microscopy. Epoxy-embedded renal swine tissue with immune complex deposits was used. Ultrathin sections were mounted on uncoated grids or on carbon-stabilized formvar grids. The sections were exposed to different concentrations of sodium ethoxide, and they were subjected to immunogold labeling with anti-IgG. Etching with > or =8% of saturated solution gave completely deplasticized sections. Sections etched with 2-4% solution were only partly deplasticized, but these sections were detached if mounted on uncoated grids, and the yields of immunolabeling were significantly decreased compared with the deplasticized ones. Sections exposed to < or =1% solution were not detached from the uncoated grids. Double-sided labeling of uncoated sections etched with 1% solution yielded approximately the same immunolabeling as for the completely deplasticized formvar-supported sections, and they gave better ultrastructural preservation of the tissue. We have established that etching epoxy sections on non-supported grids with a diluted solution of sodium ethoxide may be preferable for immunoelectron microscopy.

A comparative study of the immunogold labeling on H(2)O(2)-treated and heated epoxy sections.

The purpose of this study was to compare the intensity of the immunogold labeling of H(2)O(2)-treated and heated epoxy sections. Renal swine tissue with glomerular immune complex deposits with reactivity against IgG was embedded in epoxy resin. Immunogold labeling with anti-IgG was performed on sections from these blocks. Some of these sections were treated by H(2)O(2), others were heated in a citrate solution, while some were not treated at all. Some epoxy sections, which had been exposed to both H(2)O(2) and heat, were also exposed to the same immunolabeling. The heated epoxy sections obtained an yield of specific immunogold labeling, which was twice as large as the labeling of the H(2)O(2)-treated sections. The yield of immunolabeling of the sections that had been exposed to both H(2)O(2) and heat was not significantly different from the sections that were only exposed to heat. The non-treated sections were very weakly labeled with anti-IgG. We believe that both H(2)O(2) and heat have the ability to break some chemical bonds between the epoxy resin and the antigens, but heating in citrate buffer has a larger potential in this respect than H(2)O(2). We interpret the results from the combined treatment with H(2)O(2) and heat in the following way; the bonds that are broken by H(2)O(2) will also be broken by heating in citrate solution. The practical significance of these results is that heating in citrate buffer is a more convenient method for enhancing the immunolabeling of epoxy sections than treatment with H(2)O(2).

Susceptibility of motile and cystic forms of Borrelia burgdorferi to ranitidine bismuth citrate.

Gastrointestinal symptoms accompanying Lyme disease have not been considered in the treatment of Lyme patients yet. Here we examine the effect of ranitidine bismuth citrate (RBC) on motile and cystic forms of Borrelia burgdorferi in vitro, to determine whether it could cure this bacterial infection in the gastrointestinal tract. When motile forms of B. burgdorferi were exposed to RBC for 1 week at 37 degrees C, the minimal bactericidal concentration (MBC) was > 64 mg/ml. At 30 degrees C, the MBC was > 256 mg/ml. When the incubation lasted for 2 weeks at 37 degrees C, the MBC dropped to > 2 mg/ml. Bismuth aggregates were present on the surface of B. burgdorferi when RBC > or = MBC, as shown by transmission electron microscopy (TEM). Cystic forms of B. burgdorferi, exposed to RBC for 2 weeks at 37 degrees C, were examined by cultivation in BSK-H medium (Sigma B3528). They were stained with acridine orange (pH 6.4, pH 7.4) and studied by TEM. The MBC for RBC for young cystic forms (1 day old) and old cysts (8 months old) was estimated to be > 0.125 mg/ml and > 2 mg/ml, respectively. Bismuth aggregates were attached to the cysts and, in some, the pin-shaped aggregates penetrated the cyst wall. The bismuth aggregates also bound strongly to blebs and granules of B. burgdorferi when RBC > or = MBC. When B. burgdorferi is responsible for gastrointestinal symptoms, bismuth compounds may be candidates for eradication of the bacterium from the gastrointestinal tract.

Association between multiple sclerosis and cystic structures in cerebrospinal fluid.

BACKGROUND: The aim of the study was to search for infectious agents in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS). PATIENTS AND METHODS: CSF from ten patients with the diagnosis relapsing remitting MS and from five controls without MS were examined by transmission electron microscopy (TEM), dark field microscopy (DF), interference contrast microscopy (ICM) and UV-microscopic examination of acridine orange staining (AO). All CSF samples from patients and controls were cultured. RESULTS: Cystic structures were observed in CSF of all ten patients by AO and TEM. DF revealed eight cyst-positive patients out of nine. One of five control persons had such structures in the CSF; this person had suffered from erythema migrans. Spirochete or rod-like structures emerged after culturing two of the MS patient CSF samples and these structures could be propagated. CONCLUSION: A significant association of CSF cysts and MS was identified in this small study among residents in a coastal area of southern Norway. The cysts could be of spirochetal origin. Our study may encourage other researchers to study larger patient groups.

Association between apolipoprotein E genotypes and cancer risk in patients with acquired immunodeficiency syndrome.

The apolipoprotein E (apoE) genotype was determined in 197 deceased acquired immunodeficiency syndrome (AIDS) patients treated at Ullevaal Hospital in Oslo, Norway. A full autopsy had been performed on all. Cancer had developed in 71 individuals, mainly lymphomas (46) and Kaposi's sarcomas (18). The apoE genotype distribution was consistent with Hardy-Weinberg equilibrium, and allele frequencies were in the typical Scandinavian range (6.9% apoE2; 75.6% apoE3; and 17.5% apoE4). Cancer cases had a significantly higher frequency of apoE4 alleles than noncancer cases (24.6% and 13.5%, respectively) and a lower frequency of apoE2 alleles (3.5% versus 8.7%). Background factors, such as survival from AIDS diagnosis, could not explain these differences. Our study thus indicates that apoE genotype affects the development of cancers among AIDS patients.

Increased yield of immunogold labeling of epoxy sections by adding para-phenylendiamine in the tissue processing.

The purpose of this study was to examine if the presence of para-phenylendiamine (PPD) in the tissue processing could increase the yield of immunogold labeling of the epoxy sections. Renal swine tissue with glomerular immune complex deposits with reactivity against IgG was embedded in epoxy resin. PPD was added (1) at the beginning of the dehydration, (2) in the first step with propylene oxide, (3) in the beginning of the dehydration and in all steps with propylene oxide included the infiltration step where propylene oxide and epoxy resin are mixed, or (4) PPD was totally avoided. The tissue was embedded with two different combinations of accelerator. Immunogold labeling with anti-IgG was performed on both non-heated and heated ultrathin sections. The immunogold labeling on the heated sections which were based on processing with PPD in all steps (3) was about 55-65% higher than the corresponding labeling for epoxy sections processed in total absence of PPD (4). The immunolabeling was not significantly increased when the tissue was processed with PPD only in the start of the dehydration (1) or in the first step with propylene oxide (2). We believe that tissue processing with sufficient PPD contributes to reduce the co-polymerization between the antigens and the epoxy polymer in the same way as excess of accelerator does (Brorson and Skjørten, 1996a). The practical significance of this study provides better opportunities for increasing the immunogold labeling of epoxy sections by adding PPD in the tissue processing, and our result may inspire other researchers to develop even more efficient methods for controlling the copolymerization between antigens and epoxy resin.

Comparison of the immunolabeling of heated and deplasticized epoxy sections on the electron microscopical level.

The purpose of this study was to compare the yield of immunogold labeling of heated epoxy sections with the yield of labeling of deplasticized epoxy sections, and to compare the immunolabeling of deplasticized high-accelerator epoxy sections and deplasticized low-accelerator epoxy sections. Renal swine tissue and human thyroid tissue were embedded in both high- and low-accelerator epoxy resin and also in LR-White resin. Immunogold labeling was performed on deplasticized (ethoxide-treated), heated and non-treated ultrathin sections from these specimens. The renal tissue was immunolabeled with anti-IgG, and the thyroid tissue was immunolabeled with anti-thyroglobulin. The ethoxide treatment of the epoxy sections induced complete deplasticizing. The immunogold labeling with anti-IgG on deplasticized epoxy sections of renal tissue demonstrated significantly more intense immunolabeling of immune complex deposits than the corresponding epoxy sections which were exposed to heat in citrate buffer. The results for labeling areas of thyroglobulin substance with anti-thyroglobulin showed no significant differences between deplasticized and heated epoxy sections, probably because the sodium ethoxide partly destroys the antigenicity. Deplasticized high-accelerator epoxy sections showed significantly higher yield of immunolabeling than deplasticized low-accelerator epoxy sections and LR-White sections both for anti-IgG and anti-thyroglobulin. This can be explained by the reduced tendency for the knife to cleave proteins when cutting high-accelerator epoxy sections. High-accelerator epoxy sections which were exposed to heat in citrate buffer were more intensely immunolabeled than similarly treated low-accelerator epoxy sections, in agreement with previous results. The ultrastructural preservation of the tissues of deplasticized epoxy sections was inferior compared with the other sections. This study shows that the choice between deplasticizing technique or heating of epoxy sections has to be considered with respect to the nature of the antigen and to the requirement for ultrastructural preservation.

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 > or = 512 microg/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 > or = 4 microg/ml, but the MBC was > or = 32 microg/ml with aerobic incubation at 37 degrees 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 > or = 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.

Antigen retrieval on epoxy sections based on tissue infiltration with a moderately increased amount of accelerator to detect immune complex deposits in glomerular tissue.

We wanted to examine the effect of antigen retrieval on epoxy sections where the tissue had been infiltrated by resin containing moderately increased amounts of accelerator. The concentration of accelerator DMP-30 (Tri(Dimethyl Amino Methyl) Phenol) was varied in the range of 0% to 4% in the infiltration step of the tissue processing. Some of the epoxy sections were fixed in osmium tetroxide, and for others this fixative was avoided. Immunogold labeling was performed on epoxy sections and LR-White sections of renal tissue with IgG-deposits, and the antibody used was anti-IgG. Antigen retrieval was performed by heating the sections in citrate buffer. The amount of immunogold labeling on retrieved sections increased according to the amount of accelerator the non-osmicated epoxy sections were based on in the infiltration steps. For the osmicated epoxy sections these differences were less pronounced. The immunogold labeling of retrieved epoxy sections was up to 70% of LR-White labeling. In addition to breaking fixation bond 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 in the tissue infiltration and antigen retrieval by heating the sections in citrate buffer is a good method for improving the immunolabeling of epoxy sections.