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Recent and historical evidence is consistent with the view that atherosclerosis is an infectious disease or microbial toxicosis impacted by genetics and behavior. Because small bacterial-like particles, also known as nanobacteria have been detected in kidney stones, kidney and liver cyst fluids, and can form a calcium apatite coat we posited that this agent is present in calcified human atherosclerotic plaques. Carotid and aortic atherosclerotic plaques and blood samples collected at autopsy were examined for nanobacteria- like structures by light microscopy (hematoxylin-eosin and a calcium-specific von Kossa staining), immuno-gold labeling for transmission electron microscopy (TEM) for specific nanobacterial antigens, and propagation from homogenized, filtered specimens in culture medium. Nanobacterial antigens were identified in situby immuno-TEM in 9 of 14 plaque specimens, but none of the normal carotid or aortic tissue (5 specimens). Nanobacteria-like particles were propagated from 26 of 42 sclerotic aorta and carotid samples and were confirmed by dot immunoblot, light microscopy and TEM. [3H]L-aspartic acid was incorporated into high molecular weight compounds of demineralized particles. PCR amplification of 16S rDNA sequences from the particles was unsuccessful by traditional protocols. Identification of nanobacteria-like particles at the lesion supports, but does not by itself prove the hypothesis that these agents contribute to the pathogenesis of atherosclerosis, especially vascular calcifications.

Recent and historical evidence is consistent with the view that atherosclerosis is an infectious disease or microbial toxicosis impacted by genetics and behavior. Because small bacterial-like particles, also known as nanobacteria have been detected in kidney stones, kidney and liver cyst fluids, and can form a calcium apatite coat we posited that this agent is present in calcified human atherosclerotic plaques. Carotid and aortic atherosclerotic plaques and blood samples collected at autopsy were examined for nanobacteria-like structures by light microscopy (hematoxylin-eosin and a calcium-specific von Kossa staining), immuno-gold labeling for transmission electron microscopy (TEM) for specific nanobacterial antigens, and propagation from homogenized, filtered specimens in culture medium. Nanobacterial antigens were identified in situby immuno-TEM in 9 of 14 plaque specimens, but none of the normal carotid or aortic tissue (5 specimens). Nanobacteria-like particles were propagated from 26 of 42 sclerotic aorta and carotid samples and were confirmed by dot immunoblot, light microscopy and TEM. [3H]L-aspartic acid was incorporated into high molecular weight compounds of demineralized particles. PCR amplification of 16S rDNA sequences from the particles was unsuccessful by traditional protocols. Identification of nanobacteria-like particles at the lesion supports, but does not by itself prove the hypothesis that these agents contribute to the pathogenesis of atherosclerosis, especially vascular calcifications.

We investigated the expression of gap junction connexins26, -32, and -43 in normal, reactive, and diseased human lymphoid tissue with single and double immunolabeling and confocal laser scanning microscopy. In all tissues, connexin43 positivity was detected in follicular dendritic cells positive for CD21 and CD35 antigens, around lymphoendothelial cells moderately positive for Factor VIII, CD31 and cathepsin-D antigens; and somewhat in vascular endothelia including high endothelial venules strongly positive for Factor VIII and CD31 antigens. The ultrastructural hallmark of gap junctions, pentalaminar structures with appropriate spacing, was found in follicular dendritic cell processes. Connexin43 was also detected between smooth muscle and stromal cells of the gut, in capsular fibroblasts, and in tonsil epithelium. Neither connexin32 nor -26 was revealed, except for connexin26 in the tonsil epithelium. In follicular dendritic cells, connexin43 co-localized closely with the desmosomal proteins desmoplakin and desmoglein, suggesting that cell adherence has a role in gap junction formation. Most connexin43 was observed in sinus lining cells of lymph nodes involved in malignancies and in follicular dendritic cells in the light zone of germinal centers where maturing but still proliferating lymphocytes are situated. In the light of their distribution, gap junctions may play a part in regulating the growth of germinal centers and in integrating activating or controlling signals in follicular dendritic and sinus lining cell networks. Because connexin43 is the connexin of stromal cells, finding it in follicular dendritic cells in consistent with the proposal that these cells originate from resident stromal cells.

A novel one-step double immunolabeling method was elaborated on the basis of the simultaneous application of preformed molecular complexes of two primary antibodies with their specific secondary antibodies labeled with different enzymes. Treatment with a rat monoclonal antibody (MAb), M1-8, pre-coupled with horseradish peroxidase-linked sheep anti-rat immunoglobulins, and enzyme reaction revealed by the 3-amino-9-ethylcarbazole/hydrogen peroxide reaction, resulted in red-brown intracytoplasmic staining of interdigitating reticular cells in the lymph nodes of Balb/c mice. Another molecular complex, made of mouse anti-Ia MAb with alkaline phosphatase-linked rabbit anti-mouse immunoglobulins, applied at the same time and then developed with naphthol AS-BI-phosphate/fast blue BB as substrate, yielded blue surface staining of this cell type in addition to labeling of B-lymphocytes. The method described provides the possibility of relatively rapid double antigen detection where the binding sites of the secondary antibodies are saturated by the specific primary immunoglobulins. This approach seems to avoid nonspecific binding of primary antibodies to Fc receptors, and the unwanted binding of secondary antibodies with cell surface immunoglobulins on B-lymphocytes or with crossreactive primary antibodies used in the other sequence, if the primary antibodies and the tissue are the same or crossreactive animal species.

The immunomorphological characteristics of interstitial macrophages with PAS-positive granules were studied in experimentalEscherichia colipyelonephritis in rats, using an anti-E. coliantibody. Immunohistochemical, immunoelectron microscopical, as well as light- and electron microscopical findings were compared at twelve time points between 2 days and 13 weeks after infection. Macrophages with PAS-positive granules were present in the inflammatory infiltrates from the 7th day. The granules were phagolysosomes, filled pre-dominantly with myelin figures. The myelin figures originated mainly from the constituents of the bacterial wall and reacted with the anti-E. coliantibody even 13 weeks after infection. The storage of bacterial residues with preserved antigenic structures for several weeks after infection indicates disturbed phagolysosomal elimination of the bacterial substances in the PAS-positive macrophages. In the formation of macrophages with PAS-positive granules, lysosomal overloading with large amounts of bacteria and cell debris is assumed, leading to consumption of the lysosomal enzymes, consequent incomplete breakdown and retention of the bacterial residues.

Thirteen surgically removed, epoxy resin (Durcupan ACM or Epon 812)-embedded human brain tumors were examined for glial fibrillary acidic protein (GFAP) content in semithin and ultrathin sections with the immunogold-silver staining method. Mild aldehyde fixation and the hydrophobic resin embedding did not interfere with the antigenicity, since silver intensification of the immunogold marker provided excellent visualization of the reaction on both light microscopic and ultrastructural levels. The GFAP reaction was usually localized on the glial intermediate filament bundles, usually correlating well with the amount of filaments. The unstained filamental regions of two ependymomas might correspond to the vimentin expression revealed by double labeling in semithin sections. Occasional GFAP immunopositivity without filamental appearance was observed in one of the oligodendrogliomas, as patchy electron-dense cytoplasmic corpuscules, in Rosenthal fibers and in some mainly necrobiotic tumor cells, reflecting a possible connection with glial filaments.

Double and triple immunocytochemical detection methods for routine use in histopathology were investigated. For double immunostaining, the combinations of immunogold-silver staining (IGSS, black) with an immunoperoxidase method (3-amino-9-ethylcarbazole, red-brown) or with an immunoalkaline phosphatase method (Fast Red TR, red) proved very useful. These were followed by a Haematoxylin counterstain. An alternative approach using immunoperoxidase (red-brown) and immunoalkaline phosphatase (Fast Blue, BB, blue) methods was also successful, particularly for frozen sections of unfixed tissue and for the establishment of intermediate filament coexpression in tumours. The coexistence of desmin with vimentin in rhabdomyosarcoma, and of glial fibrillary acidic protein with vimentin in ependymoma, could be demonstrated directly by means of non-crossreacting murine and rabbit antibodies in the above combinations. The black (IGSS), red-brown (immunoperoxidase) and blue )immunoalkaline phosphatase) colours gave excellent contrast in triple immunostaining. The side-by-side demonstration of helper and suppressor T lymphocytes during renal allograft rejection, of kappa and lambda light chains in B-immunoblastic lymphoma, and of T and B lymphocyte populations in non-Hodgkin's lymphomas provided immediate information on the topography and cellular organization of the tissues.

Skeletal muscle fibre types were identified by using immunohistochemical detection of sarcoplasmic reticulum Ca2+-ATPase and myolobin content in rat gastrocnemius muscle. The strong Ca2+-ATPase-reactive fibres were identical with the fast-twitch population, while the fibres with weak reactivity represented the slow-twitch type. Strong myoglobin immunoreactivity reflected the fast oxidative glycolytic (FOG) and slow oxidative (SO) types. Slight to moderate myoglobin immunostaining was found in the fast glycolytie (FG) fibres. The staining intensity of the different fibre types differed as follows: for Ca2+-ATPase FG>FOG>SO, and for myoglobin FOG>SO>FG. The immunoreactivity of Ca2+-ATPase and myoglobin were well preserved after fixation of the muscles in Bouin's solution, or in formol/acetic acid fixative, and paraffin embedding. Detection of the primary antibodies was carried out by using the avidin-biotin-peroxidase complex, and the immunogold-silver-staining methods. The latter was found to be more sensitive and suitable for postembedding ultrastructural demonstration of the Ca2+-pump enzyme on Durcupan-embedded muscles. The method, using 5 nm immunogold conjugate with silver enhancement, offered the advantages of high sensitivity and excellent visualization of the reaction product. The postembedding detection of sarcoplasmic reticulum Ca2+-ATPase also proved to be useful in the restrospective identification of the main fibre classes in human muscle biopses.