Once focused mainly on the characterization of neoplasms, immunohistochemistry (IHC) today is used in the investigation of a broad range of disease processes with applications in diagnosis, prognostication, therapeutic decisions to tailor treatment to an individual patient, and investigations into the pathogenesis of disease. This review addresses the technical aspects of immunohistochemistry (and, to a lesser extent, immunocytochemistry) with attention to the antigen-antibody reaction, optimal fixation techniques, tissue processing considerations, antigen retrieval methods, detection systems, selection and use of an autostainer, standardization and validation of IHC tests, preparation of proper tissue and reagent controls, tissue microarrays and other high-throughput systems, quality assurance/quality control measures, interpretation of the IHC reaction, and reporting of results. It is now more important than ever, with these sophisticated applications, to standardize the entire IHC process from tissue collection through interpretation and reporting to minimize variability among laboratories and to facilitate quantification and interlaboratory comparison of IHC results.

https://journals.sagepub.com/doi/pdf/10.1177/0300985813505879

When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry--the red, brown, and blue technique.

Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles

Marsh PD, Devine DA. How is the development of dental biofilms influenced by the host? J Clin Periodontol 2011; 38 (Suppl. 11): 28–35. doi: 10.1111/j.1600-051X.2010.01673.x.

Abstract
Background: The host provides environmental conditions that support diverse communities of microorganisms on all environmentally-exposed surfaces of the body.

Materials and Methods: To review the literature to determine which properties of the host substantially influence the development of dental biofilms.

Results: The mouth facilitates the growth of a characteristic resident microbiota. The composition of the oral microbiota is influenced by temperature, pH, and atmosphere, as well as by the host defences and host genetics. In addition, the host supplies endogenous nutrients and a variety of surfaces for biofilm formation. In health, the resident oral microbiota forms a symbiotic relationship with the host, regulated by active host–microbe cross talk. This resident microbiota is sensitive to perturbations in the host environment, especially to changes in nutrient supply and pH, so that previously minor components of the microbiota can become more competitive (and vice versa), resulting in reorganization of biofilm community structure.

Conclusion: The host environment dictates the composition and gene expression of the resident microbiota. Changes in oral environmental conditions can disrupt the normal symbiotic relationship between the host and its resident microbes, and increase the risk of disease.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1600-051X.2010.01673.x

How is the development of dental biofilms influenced by the host

Porphyromonas gingivalis, one of the causative agents of adult periodontitis, attaches and forms biofilms on substrata of Streptococcus gordonii. Coadhesion and biofilm development between these organisms requires the interaction of the short fimbriae of P. gingivalis with the SspB streptococcal surface polypeptide. In this study we investigated the structure and binding activities of the short fimbriae of P. gingivalis. Electron microscopy showed that isolated short fimbriae have an average length of 103 nm and exhibit a helical structure with a pitch of ca. 27 nm. Mfa1, the major protein subunit of the short fimbriae, bound to SspB protein, and this reaction was inhibited by purified recombinant Mfa1 and monospecifc anti-Mfa1 serum in a dose-dependent manner. Complementation of a polar Mfa1 mutant with the mfa1 gene restored the coadhesion phenotype of P. gingivalis. Hence, the Mfa1 structural fimbrial subunit does not require accessory proteins for binding to SspB. Furthermore, the interaction of Mfa1 with SspB is necessary for optimal coadhesion between P. gingivalis and S. gordonii.

https://iai.asm.org/content/iai/73/7/3983.full.pdf

Short Fimbriae of Porphyromonas gingivalis and Their Role in Coadhesion with Streptococcus gordonii

Establishment of a community is considered to be essential for microbial growth and survival in the human oral cavity. Biofilm communities have increased resilience to physical forces, antimicrobial agents and nutritional variations. Specific cell-to-cell adherence processes, mediated by adhesin-receptor pairings on respective microbial surfaces, are able to direct community development. These interactions co-localize species in mutually beneficial relationships, such as streptococci, veillonellae, Porphyromonas gingivalis and Candida albicans. In transition from the planktonic mode of growth to a biofilm community, microorganisms undergo major transcriptional and proteomic changes. These occur in response to sensing of diffusible signals, such as autoinducer molecules, and to contact with host tissues or other microbial cells. Underpinning many of these processes are intracellular phosphorylation events that regulate a large number of microbial interactions relevant to community formation and development.

/pdf/microbial-interactions-in-building-of-communities-2cpesvqswm.pdf

Microbial interactions in building of communities

Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components.

Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2.

Porphyromonas gingivalis is implicated in the occurrence of adult periodontitis. We have previously identified major outer membrane proteins from P. gingivalis, which include representative virulence factors such as gingipains, a 75 kDa major protein, RagA, RagB, and putative porin. Fimbriae, another important virulence factor, exist on the cell surface. In this study, we identified major supernatant proteins. They were fimbrilin, the 75 kDa major protein, gingipains and their adhesin domains. Microscopic examination showed that supernatant proteins formed vesicle-like and fimbrial structures. To learn more about the character of this bacterium, we examined effects of growth temperature on localization and expression of these virulence factors. In general, localization of major virulence factors did not change at the various growth temperatures used. Most of the 75 kDa major protein, RagA, RagB, and putative porin were found in the envelope fraction, not in cell-free culture supernatant. Gingipains were found in both the envelope fraction and supernatant. More than 80% of fimbriae were associated with cells, less than 20% migrated to the supernatant. Most fimbriae existed in the whole cell lysate, although there was a small amount in the envelope fraction. When the growth temperature was increased, expression of fimbriae, gingipains, the 75 kDa major protein, RagA, and RagB decreased. However, temperature had almost no effect on expression of putative porin. The tendency for expression of major virulence factors to decrease at higher temperatures may enable P. gingivalis to survive under hostile conditions.

Analysis of major virulence factors in Porphyromonas gingivalis under various culture temperatures using specific antibodies

Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3'-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.

https://informahealthcare.com/doi/pdf/10.3109/10520295.2012.687117

Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride

Tannerella forsythensis (Bacteroides forsythus), an anaerobic gram-negative potential periodontal pathogens in the progression of periodontitis. T. forsythensis has unique bacterial protein profiles containing major proteins with apparent molecular weight of more than 200-kDa shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. It is also known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane revealed by electron microscopy. On the other hand, electron microscopy showed that the best preservation of structure was obtained when cells were postfixed with OsO4, but this resulted in very low levels of gold particles labeling. Therefore, cells were applied to pieces of filter paper and freeze-substituted by plung-freezing in Liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR-White resin. We also examined the relation between high molecular weight proteins and S-layer in energy-filtering transmission electron microscopy (EF-TEM) to visualize 3,3’diaminobenzidene, tetrahydrochloride (DAB) reaction. The three-window method in electron spectroscopic images (ESI) of nitrogen (N) element, reflecting the presence of DAB moieties by the DAB reaction solution, horseradish peroxidase (HRP)conjugated secondary antibodies instead of immunogold particles obtained by the EF-TEM. The mapping patterns of net N element were restricted to the outermost cell surface.

Immunocytochemical approach for surface layer proteins of freeze-substituted Tannerella forsythensis by energy-filtering transmission electron microscopy.

Jun Iwami

Papers

Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2.

Analysis of major virulence factors in Porphyromonas gingivalis under various culture temperatures using specific antibodies

Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride

Immunocytochemical approach for surface layer proteins of freeze-substituted Tannerella forsythensis by energy-filtering transmission electron microscopy.