Small Granular Deposits Research Articles

The gut microbiota induces melanin deposits that act as substrates for fimA-mediated aggregation of Salmonella Typhimurium and enhance infection of the German cockroach vector.

When Salmonella Typhimurium is ingested by German cockroaches, the bacteria replicate in the gut and persist for at least 7 d, enabling transmission in the feces. However, the mechanisms that facilitate survival and persistence in the cockroach gut remain poorly detailed. We previously reported the formation of biofilm-like aggregate populations of S. Typhimurium in the gut of cockroaches upon ingestion. We also reported that deletion of the type-1 fimbrial subunit of S. Typhimurium, fimA, leads to a reduced bacterial load in the cockroach gut. Here, we link these observations and provide further insight into the mechanism and function of S. Typhimurium aggregation in the gut of the cockroach. We show that S. Typhimurium but not Escherichia coli forms aggregated populations in the cockroach gut, and that aggregate formation requires fimA but not the biofilm formation-related genes csgA and csgD. Furthermore, we show that S. Typhimurium aggregates are formed using small granular deposits present in the cockroach gut, which exhibit properties consistent with melanin, as substrates. These melanin deposits are prevalent in the guts of both immature and adult cockroaches from laboratory colonies and are correlated with increased gut bacterial density while being entirely absent in gnotobiotic cockroaches reared without exposure to environmental bacteria, indicating they are induced as a response to the gut microbiota. When cockroaches lacking melanin deposits in the gut are fed S. Typhimurium, they exhibit lower rates of infection than those harboring melanin deposits, demonstrating that microbiota-induced melanin deposits enhance infection of the gut of the vector. IMPORTANCE Cockroaches, including the German cockroach (Blattella germanica), can be both mechanical and biological vectors of pathogenic bacteria. Together, our data reveal a novel mechanism by which S. Typhimurium interacts with the cockroach gut and its microbiota that promotes infection of the vector. These findings exemplify the emerging but underappreciated complexity of the relationship between cockroaches and S. Typhimurium.

Two mutations in the transforming growth factor beta-induced gene associated with familial Lattice corneal dystrophy.

To report a phenotypic variant pedigree of lattice corneal dystrophy (LCD) associated with two mutations, R124C and A546D, in the transforming growth factor beta-induced gene (TGFBI). A detailed ocular examination was taken for all participants of a LCD family. Peripheral blood leukocytes from each participant were extracted to obtain the DNA. Polymerase chain reaction (PCR) of all seventeen exons of TGFBI gene was performed. The products were sequenced and analyzed. Histological examination was carried out after a penetrating keratoplasty from the right eye of proband. Genetic analysis showed that the proband and all 6 affected individuals harbored both a heterozygous CGC to TGC mutation at codon 124 and a heterozygous GCC to GAC mutation at codon 546 of TGFBI. None of the 100 control subjects and unaffected family members was positive for these two mutations. Ocular examination displayed multiple refractile lattice-like opacities in anterior stroma of the central cornea and small granular deposits in the peripheral cornea. The deposits were stained positively with Congo red indicating be amyloid in nature and situated mainly in the anterior and middle stroma. We observed a novel LCD family which carried two pathogenic mutations (R124C and A546D) in the TGFBI gene. The phenotypic features were apparently different from those associated with corresponding single mutations. The result reveals that although the definite mutation is the most important genetic cause of the disease, some different modifier alleles may influence the phenotype.

Combined IgG4κ and IgG1λ deposition in the glomerular and tubular basement membrane accompanied by autoimmune neutropenia (AIN) and immune thrombocytopenia (ITP).

A 48-year-old male was admitted to our hospital with nephrotic syndrome. Light-microscopic examination of a renal biopsy specimenshowed almost normal glomerular appearance, however, immunofluorescence examination revealed linear and granular IgG deposits on the glomerular basement membrane (GBM), accompanied by slight IgG deposition in the tubular basement membrane (TBM). Further investigation of the IgG subclass and light chain staining revealed that the glomerular deposits were composed of IgG1 and IgG4, with both κ and λ light chains, while the tubular deposits were composed of only IgG4 and κ light chains. The electron-microscopic findings of small granular deposits in the GBM and TBM closely resembled those of light and heavy chain deposition disease (LHCDD). Immunoelectron microscopy confirmed the presence of κ and λ chains in the GBM and TBM, however, only significant κ chain deposition was found in the TBM. There was no evidence of monoclonal gammopathy. Clinically, the patient subsequently developed neutropenia and thrombocytopenia associated with the presence of anti-neutrophil antibody and anti-GPIIb/IIIa antibody-producing B cells in the blood. Oral steroid administration was initiated, which led to amelioration of the neutropenia, thrombocytopenia and proteinuria. This may be a very rare case of combined IgG4κ and IgG1λ deposition disease accompanied by autoimmune neutropenia (AIN) and immune thrombocytopenia (ITP) suggestive of biclonal immunoglobulin deposition disease (BIDD). Investigation of the IgG subclass and of the light chains was useful for recognizing the clonality of the immunoglobulin deposits in the kidney.

Excitotoxic lesioning of the rat basal forebrain with S-AMPA: consequent mineralization and associated glial response

Regional depositions of calcium within the basal ganglia, cortex, cerebellum, and white matter and at perivascular sites have been observed in several pathological conditions. These generally indicate signs of ongoing apoptosis or necrotic processes, whereby the activation of glutamate receptors causes a rise in intracellular calcium levels leading to mineralization of neurons, and ultimately to cell death. The selective degeneration of cholinergic neurons in the basal forebrain is a major neuropathological component of Alzheimer’s disease, and may result in abnormal deposition of calcium. In experimental models, selective lesions of the basal forebrain can be induced by intraparenchymal infusions of excito- or immunotoxins targeting cholinergic neurons. Excitotoxic lesions are often accompanied by calcium deposition within affected areas. In a previous study we also noted the presence of unusual deposition in areas close to the site of injections following unilateral S-AMPA-induced lesions of the basal forebrain (T. Perry, H. Hodges, and J. A. Gray, 2001, Brain Res. Bull. 54, 29–48). In this paper, we have characterized these deposits histologically and evaluated the microglial (CD11b) and astrocytic (GFAP) responses at 8 and 16 weeks following lesioning of the nucleus basalis magnocellularis with S-AMPA. The resulting deposits were heterogeneous in morphology and composed primarily of calcium. Small granular deposits were detected around blood vessels, whereas larger calcospherites were situated within the parenchyma. These deposits were more widely dispersed at 16 weeks postlesioning, affected neighboring nuclei, and displayed a progressive increase in size and frequency of occurrence. However, calcification within these regions was differentially associated with microglial and astrocytic reactivity at the two time points. Both microglial and astrocytic responses were pronounced at 8 weeks, whereas at 16 weeks, astrocytic reactivity prevailed and the microglial response was markedly attenuated. Importantly, the pattern of reactivity for microglia detected at 8 weeks was specifically localized to vulnerable nucleated areas prior to their substantial accumulation of calcium deposits, which was clearly evident by 16 weeks. We suggest that the initial microglial response could be used as a selective predictor of tissue necrosis and subsequent calcification, and that astrocytes, which form a glial scar in the affected tissues, may contribute toward the buildup of calcium deposits. The functional relevance of these findings is discussed.

Immune deposits in the glomerular extracellular matrix detected by the quick-freezing and deep-etching method.

Chronic serum sickness nephritis was induced in rats by sensitization with egg albumin. The glomeruli were then examined by the quick-freezing and deep-etching method with immunohistochemical identification of immune complex deposits on replica membranes. In control rats, the glomerular basement membrane showed a three-layered structure. The middle layer was composed of a compact meshwork of fine fibrils that was connected to adjacent endothelial and epithelial cells by perpendicular fibrils in the inner and outer layers. The mesangial matrix contained a similar but looser meshwork structure. In the nephritic rats, small granular deposits were observed within the meshwork, distorting its fine structure. Larger nodular aggregates extended continuously from the middle layer to the epithelial side. Disruption of the connecting fibrils overlying these larger aggregates was noted. The deposits were stained by antiegg albumin or antirat IgG antibodies. Gold particles immunostaining for the sensitizing antigen were also localized within the deposits. These findings suggest that the deposition of immune complexes occurs in the fibrillary meshworks of the mesangium and lamina densa in this experimental model, with the resultant distortion of their meshwork structures and the formation of nodular aggregates.

Immunohistochemical localization of acid alpha-glucosidase in rat liver.

Acid alpha-glucosidase (E.C. 3.2.1.3) was purified more than 60,000-fold from rat liver. Antibody was obtained by injection of this pure enzyme into rabbits with Freund's complete adjuvant. The resultant anti-acid alpha-glucosidase immunoglobulin (Ig) G was digested with pepsin and then F(ab')2 was treated with 2-mercaptoethanol. Coupling of Fab' to horseradish peroxidase was performed according to the method of Wilson and Nakane. Light microscopic observation of the immunohistochemical localization of this enzyme in rat hepatocytes revealed small granular deposits of diaminobenzidine reaction products. The reaction diffusely observed in the hepatocyte cytoplasm of any area. Under the electron microscope, the reaction precipitates were found to be located on the lysosome membrane, particularly on the inner side of the membrane, as small dots. The small vesicles were strongly positive for this reaction. Occasionally positive reaction were also demonstrated in the lumen of the secondary lysosomes. However, the Golgi and its associated structures did not show a positive reaction.

Purification and immunohistochemistry of fetal acid .ALPHA.-glucosidase.

Fetal rat liver acid α-glucosidase was purified more than 20, 000 fold from the fetal rat liver 2 days before delivery. The disc gel electrophoresis of this preparation showed a single band of protein. The Michaelis constants of this enzyme were 3.3mM with maltose and 3.1mM with 4-methylumbelliferyl α-glucoside as substrates. Molecular weight measured by SDS electrophoresis and TOYOPEARL gel filtration was about 100, 000. The pH optima was 4.0 with 4-methylumbelliferyl α-glucoside as substrate and 3.7 with maltose as substrate.Antibody was obtained by injection of this enzyme into rabbits with Freund's complete adjuvant. The anti-fetal acid α-glucosidase IgG showed the cross-reaction to the adult rat liver acid α-glucosidase. F(ab')2 was separated by the filtration of Sephacryl S-200 column chromatography after digestion of anti acid α-glucosidase IgG pepsin and then F(ab')2was treate d with 2-mercaptoethanol. Coupling of Feb', obtained by use of Sephacryl S-200 column chromatography after treatment of F(ab')2 with 2-mercaptoethanol, to horseradish peroxidase was performed according to the method of Wilson and Nakane (14).Light microscopical observation of the immunohistochemical localization of this enzyme in fetal rat and newborn rat liver showed small granular deposits of DAB reaction products in the cytoplasm.