Intracellular reservoir of Streptococcus pyogenes in vivo: a possible explanation for recurrent pharyngotonsillitis.

Numerous theories have been presented that attempt to explain the frequent recurrences of pharyngotonsillitis caused by Streptococcus pyogenes; these recurrences occur after seemingly adequate antibiotic treatment. We previously have demonstrated that Spyogenes can survive for up to 7 days intracellularly in immortalized human respiratory epithelial cells grown in an antibiotic supplemented medium. Viable S pyogenes were externalized and established an extracellular infection, whenever the extracellular antibiotic was removed. We have investigated the presence of intracellular S pyogenes in two in vivo studies using respiratory epithelial cells collected from patients with tonsillitis and the tonsils of asymptomatic carriers. Electron microscopy and immunohistochemistry demonstrated intracellular S pyogenes in pharyngeal epithelial cells in 13 of 14 patients with tonsillitis (93%). Furthermore, intracellular S pyogenes were found in macrophage-like cells in eight (73%) and in epithelial cells in four (36%) tonsils from 11 asymptomatic S pyogenes carriers. These in vivo data strongly support the hypothesis that intracellular S pyogenes can constitute a reservoir of bacteria with the potential to cause reinfections.

Sarcocystis buffalonis n.sp. (Protozoa: Sarcocystidae) from the water buffalo (Bubalus bubalis) in Vietnam.

Sarcocystis buffalonis n. sp. is proposed for a species forming thick-walled, macroscopic sarcocysts in skeletal muscles and the esophagus of the water buffalo (Bubalus bubalis). Sarcocysts of S. buffalonis were found in 68 (10.5%) of 647 buffalo carcasses examined grossly at slaughter in Ho Chi Minh City in southern Vietnam. Sarcocystis buffalonis sarcocysts were 1-8 mm long and 0.1-0.5 mm wide. The cyst wall was 3-7.7 microns thick and had palisadelike villar protrusions that were constricted at the base, expanded laterally in the mid-region, and tapered distally. The villar protrusions contained microfilaments and electron-dense granules. Sarcocysts of Sarcocystis fusiformis, the other well-known macroscopic species occurring in water buffalo, were also found in 60 of the 68 animals infected with S. buffalonis. Sarcocysts of S. fusiformis were thin walled and had characteristic cauliflowerlike villar protrusions. Two of 7 cats fed isolated S. buffalonis sarcocysts were found to have 12 x 8 microns sporocysts in their intestine or feces 10 days after inoculation.

Occurrence of multinucleated giant cells in the appendix of clinically healthy rabbits.

The spontaneous formation of multinucleated giant cells was observed in the appendix of clinically healthy adult rabbits that were free of infection with intestinal viruses, pathogenic bacteria, fungi and parasites. Giant cells occurred singly and in aggregates. They were of the foreign body and of the Langhans' type, but intermediate forms were also noticed. Ultrastructurally, the hallmark of these appendiceal polykaryons were large phagolysomal fields harbouring amorphous debris and remains of cytoplasmic organelles and bacteria. The bacteria in the appendiceal tissues were neither of a special type nor acid-fast. The aetiology and significance of appendiceal giant cells remains to be clarified.

A bovine viral diarrhoea/mucosal disease-like syndrome in moose (Alces alces): investigations on the central nervous system.

A disease of unknown aetiology has been observed in moose. The animals showed signs of a bovine viral diarrhoea/mucosal disease-like syndrome, and central nervous disturbances. Brains from adult female moose were investigated by means of histology, immunohistochemistry, electron microscopy, virology, and bacteriology. The results indicate that the nervous signs were not associated with a spongiform encephalopathy. The lesions suggest a viral aetiology, although all the virological investigations have so far proved negative.

Equine herpes virus 1 (EHV-1) in liver, spleen, and lung as demonstrated by immunohistology and electron microscopy.

Ten aborted foals, diagnosed as infected with Equine Herpes Virus 1 (EHV-1) on histopathological criteria, were examined for the presence of EHV-1 using immunohistology as the investigative instrument. The primary reagent was an antiserum specific for viral envelope glycoproteins. Immunohistology localised EHV-1 to areas of liver necrosis and to the cytoplasm of infected Kupffer cells and hepatocytes. Cytoplasmic immunolabelling was also prominent in reticular cells of the red pulp of the spleen and in intact and degenerated bronchiolar epithelium. Cytoplasmic immunolabelling was seen in morphologically unchanged cells and in cells containing intranuclear inclusion bodies. Three aborted foetuses with no histological signs of EHV-1 infection were negative when immunostained for EHV-1. Detection by electron microscopy of EHV-1 virions confirmed the EHV-1 specificity of the immunolabelling procedure.

Mechanisms of absorption enhancement by medium chain fatty acids in intestinal epithelial Caco-2 cell monolayers.

Sodium salts of medium chain fatty acids (MCFAs) enhance the absorption of hydrophilic drugs across the intestinal mucosa, but the mechanism behind the effect is largely unknown. In this study, the dose-dependent effects of the sodium salts of four MCFAs, C6 (caproate), C8 (caprylate), C10 (caprate) and C12 (laurate), on the permeability of the hydrophilic marker molecule [14C]mannitol were studied in monolayers of the human intestinal epithelial cell line, Caco-2, grown on permeable supports. C8, C10 and C12, but not C6, enhanced the permeability of [14C]mannitol in a dose-dependent manner. Comparison of the cellular effects of the MCFAs at concentrations that gave comparable (8.1- to 8.5-fold) absorption enhancement showed that: 1) C8 was active as absorption enhancer only when the tonicity of the medium was increased; 2) absorption enhancement mediated by C10 was related to a redistribution of the cytoskeleton and structural dilatations in the tight junctions; and 3) C12 was without effect on the cytoskeleton and cellular morphology. Studies on C10 under anisotonic conditions showed that deviations from isotonicity enhanced its effect. These results suggest that structurally similar MCFAs display dramatic differences in their mechanism of action. In addition, the effects of osmolality provide an explanation for the previously reported variability in the efficacy of MCFAs as absorption enhancers.

Sarcocystis species in skeletal muscle of otter (Lutra lutra).

Cysts of a Sarcocystis species were found in large numbers in skeletal muscle of an otter (Lutra lutra) which was raised in Norway and died in captivity in Sweden. This is the first report of Sarcocystis infection in the otter. The sarcocysts were 0.3-2.3 mm long and 0.06-0.25 mm wide. As judged by light microscopy the sarcocyst walls were thin (< 3 microns) with a serrated surface but without visible projections. By transmission electron microscopy, the sarcocyst wall measured 0.6-1.8 microns and had minute undulations covering the entire sarcocyst surface giving the wall a wavy appearance. Septa were indistinct. The sarcocysts contained few metrocytes and numerous bradyzoites. Sarcocysts were not found in 69 other otters subjected to necropsy in Sweden.

An ultrastructural study of spontaneous chronic lung lesions in asymptomatic rabbits.

An electron microscopical study of chronic lung lesions in 12 clinically healthy, purpose bred laboratory rabbits (eight of which were free from infections with known respiratory pathogens but 4 of them carried a natural B. bronchiseptica infection) revealed focal chronic interstitial pneumonia, vascular changes and focal chronic bronchiolitis. In addition, severe endothelial changes and intravascular deposition of collagen were observed in septal capillaries. In type I pneumocytes and septal capillary endothelium we noticed numerous rounded structures, 70-90 nm in diameter, which consisted of a limiting two layer membrane enclosing an irregularly rounded electron-dense centre surrounded by a more electron-lucent halo. These structures appeared free in the cytoplasm, or they were attached to or apparently budding from membranes other than the plasmalemma. Particles located extracellularly were not found. Whether the structures described were involved in the genesis of the lesions found remains to be elucidated.

The cell surface phenotype of human natural interferon-alpha producing cells as determined by flow cytometry.

The relationship between the so-called natural interferon-alpha (IFN-alpha) producing cell (IPC), stimulated to produce IFN-alpha by herpes simplex virus type 1 (HSV), and of dendritic cells (DC) in peripheral blood leucocytes was investigated. The simultaneous expression of cell surface antigens and intracellular IFN-alpha in the HSV-stimulated IPC (HSV-IPC) was examined by flow cytometry (FCM). The HSV-IPC were infrequent, < 0.3% of the mononuclear leucocytes, and with homogeneous light scatter characteristics. The HSV-IPC were confirmed to lack leucocyte lineage specific markers, and to express CD4, CD36 and HLA-DR. Furthermore, they expressed high levels of CD44, CD45RA and CD45RB, and lower levels of CD40, CD45R0, CD72 and CD83. The HSV-IPC expression of CD13, CD33 and Fc epsilon RI were weak but significant, while no CD5, CD11b, CD16, CD64, CD80 or CD86 were detected. Sorted pure HSV-IPC had irregular shaped nuclei, many mitochondria and vesicles, and rugged cell membranes without veils. Sorted HSV-IPC stimulated proliferation of autologous T cells from HSV immune donors. Thus, the HSV-IPC in many respects resemble immature DC, but clearly differ from typical mature DC. However, they may also represent a specialized population of efficient IFN-alpha producing cells.

Internalization of iscom-borne antigens and presentation under MHC class I or class II restriction.

Exogenous nonreplicating antigens (Ag) incorporated into immunostimulating complexes (iscoms) induce CTL responses under MHC class I restriction. A requirement for inducing CTL responses is that the Ag is delivered to the cytosol of antigen-presenting cells (APC), a route restricted to endogenously produced Ag. To investigate the mechanisms by which iscoms elicit MHC class I-restricted responses, the intracellular distribution of influenza virus envelope proteins incorporated in iscoms (flu-iscoms) or in micelles (flumicelles) was studied in vitro using murine peritoneal cells (PEC). Ultrathin sections of cells pulsed with biotinylated flu-iscoms or flu-micelles were analyzed by electron microscopy after detection of the biotin label by reaction with streptavidin-gold. PEC pulsed with flu-iscoms showed a pattern of scattered gold particles distributed in clear and dense vesicles as well as in the intracellular space but not associated with organelles. In cells pulsed with flu-micelles, Ag was also detected in most cellular compartments but at a considerably lower concentration. The intracellular distribution of particulate Ag in iscom or micelle form was confirmed by lysis and differential centrifugation of Ag-pulsed APC. Furthermore, P815 cells pulsed with flu-iscoms were lysed by specific immune effectors showing that the iscom-Ag was processed and presented by class I-expressing APC. Flu-iscoms were internalized about 50-fold more efficiently than ovalbumin iscoms (ovaiscoms) suggesting that the nature of the protein and/or the presence of cellular receptors are important factors influencing the capacity of APC to take up iscom-borne proteins. PEC accounted for the most active internalization of iscom-borne Ag, although splenic dendritic cells and B cells also took up fluiscoms with remarkable efficiency.

Phylogenetic placement and characterization of a new alpha-2 proteobacterium isolated from a patient with sepsis.

An alpha-2 proteobacterium, previously unknown as determined by its phylogenetic characteristics and the DNA sequence of its 16S rRNA gene, was isolated from a patient who presented an unusual clinical picture, including high remitting fever and multiorgan involvement. The bacterium was detected in multiple plasma samples, obtained during the acute phase of the disease, after cocultivation in cell culture media. Electron microscopy of the organism showed a three-layer laminar cell wall and electron-dense granules within the cytoplasm, as well as a polar flagellum. By means of PCR followed by sequencing of amplified 16S ribosomal DNA fragments, the bacterium was found to differ from all species for which ribosomal sequence information is available. It is here provisionally named the Rasbo bacterium. At a subsequent relapse, the bacterium was identified in pericardial fluid both by PCR/sequencing and by direct electron microscopy. At a second relapse, it was again cultured from plasma. After in vitro adaptation to solid media, the MICs of various antibiotics could be determined. A transient immunoglobulin M (IgM) but no IgG response to the bacterium was found by an indirect immunofluorescence test, as well as by an immobilization test during the acute phase of the disease.