Maintenance of normal platelet mass in anemic Belgrade rats and their response to iron.

The Belgrade (b/b) rat has severe hypochromic, microcytic anemia accompanied by mild thrombocytopenia and a 49% reduction in megakaryocytes (MKs). The platelet counts are decreased only 34%, but relative platelet size measured by two independent methods averages 50% greater than controls. Thus, the platelet mass of the b/b rat is within the normal range. The marrow MK progenitors (MK colony-forming units, CFU-MK) respond linearly to increased colony-stimulating activity in vitro, but they are reduced 68% and form smaller colonies than normal. Flow cytometric analysis of MK ploidy indicates that significantly more MKs are distributed into the low and high ploidy classes compared with the normal, and the mean ploidy is similar. The b/b rat maintains effective thrombocytopoiesis in spite of a severe reduction in MK progenitors, primarily by an increased rate of maturation of the endomitotic compartment. Iron treatment partially arrests the b/b anemia and is associated with a significant increase in CFU-MK, a normalized MK ploidy distribution, and a significant decrease in platelet size. The favorable response to iron therapy suggests that the megakaryocytopenia is secondary to the severe anemia and results from stem cell commitment to the erythroid lineage.

Alga associated with diarrhea in patients with acquired immunodeficiency syndrome and in travelers.

Spherical bodies resembling coccidian oocysts and measuring 8.0 to 9.0 microns in diameter were seen in the stools of eight persons with explosive, watery diarrhea. Seven had recently traveled to tropical countries, mostly in the Caribbean, and four had acquired immunodeficiency syndrome. The structures were easily discernible in wet mounts by light microscopy and contained variable numbers of granular inclusions, but were refractory to, or stained partially with, 12 commonly used laboratory stains. Electron microscopy revealed an outer fibrillar coat, a thin cell wall, granules, and organelles which were not surrounded by membranes. One type of organelle was similar to the thylakoid photosynthesizing organelles of blue-green algae (cyanobacteria). These findings indicate that the bodies may be a species of blue-green algae.

Human serologic response to subcellular antigens of Trichomonas vaginalis.

Trichomonas vaginalis organisms were processed by differential centrifugation to yield defined subcellular fractions. The various fractions were processed by methods to enhance solubility of membranes and other components. The soluble materials were analyzed for immunogenicity by a quantitative kinetic enzyme-linked immunosorbent assay (k-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) technique using human patient serum. The principal antibodies in the human serum were directed toward cytosol components, not against membrane components, although all fractions contained multiple antigenic moieties.

In vitro infection of human monocytes with human T lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV).

We explored the possibility that normal human monocytes can be infected with the retrovirus human T lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV). The T4 antigen, believed to be the receptor for HTLV-III/LAV binding to CD4 cells, is found on monocytes at low levels. Anti-T4A, which recognizes an epitope on the T4 molecule, inhibits viral binding to monocytes, and virus inhibits anti-T4A binding, although inhibition in both cases is not total. Virus particles were detected in HTLV-III/LAV-pulsed monocytes by electron microscopy as early as 10 min and for up to 3 days after inoculation, although budding virus was not observed. Monocytes were exposed to virus, were washed, and were cultured. Monocyte cultures were monitored by conventional assays for virus replication: immunofluorescence detection of cytoplasmic virus, supernatant reverse transcriptase activity, and supernatant virus antigen. These assays were either negative or at the lower limits of positivity. However, the amount of infectious virus was shown to increase over time in monocyte cultures by harvesting monocytes or their culture supernatants and titrating them into assay cultures containing stimulated T cells. Virus recovery from monocytes and virus recovery from T cells differed both quantitatively and qualitatively. Recovery from T cells and T cell supernatants peaked at 3 to 6 days and declined thereafter. Recovery from monocytes and monocyte supernatants increased over time in culture and never attained the levels of T cell cultures. Taken together, these studies indicate that HTLV-III/LAV binds to monocytes via the T4 molecule and enters the cells. Infectious virus is retained and increases with time in infected monocyte cultures. Both viral binding and infection are at low levels compared with levels in T cells. Unlike the usual infection of T cells characterized by high level virus replication with cell depletion, the infection appears to be persistent in monocytes.

Comparison of Legionella pneumophila, L. micdadei, L. bozemanii, and L. dumoffii by transmission electron microscopy.

Clinical isolates of Legionella pneumophila, L. micdadei, L. bozemanii, and L. dumoffii were grown on charcoal-yeast extract agar from a living-medium inoculum and prepared for transmission electron microscopy by three different methods. Cells of all four Legionella species possessed cytoplasmic vacuoles, a gram-negative type of cell envelope with a dense peptidoglycan-like layer, a ruthenium red-positive polysaccharide capsule, and a single subpolar flagellum. The dense polysaccharide capsule seen on cells of L. micdadei was separated from the outer membrane by an extra layer of electron-lucent material that was not present on cells of the other species examined.

Cultivation of Neisseria gonorrhoeae under low-oxygen conditions.

Sixteen Neisseria gonorrhoeae strains were examined for their abilities to grow under reduced oxygen tension. Low oxygen tensions were developed by evacuation-replacement procedures in which anaerobes and oxidation-reduction indicators were used as controls. All strains survived 96 h in a medium reduced to below -125 mV without visible growth. Detectable growth occurred at 0.05% oxygen, and 33% of normal colony size under air (21% oxygen) was obtained at 0.15% oxygen. Population selection did not determine survival and growth, but carbon dioxide was required. Characteristic colony morphologies were not evident at the lower oxygen concentrations. Colonial variation was not influenced during survival under anaerobic conditions or growth under low oxygen levels (0.15%). Medium differences were not significant affectors. We concluded that N. gonorrhoeae will grow under tensions suitable for anaerobes, and will demonstrate certain modifications of behavior under these conditions.

Flagella on Legionnaires' disease bacteria: ultastructural observations.

Scanning and transmission electron microscopic studies were done on five strains of flagellated Legionella pneumophila cultured for 1 to 3 days on charcoal yeast extract agar or yeast extract broth. Ultrastructurally, each strain consisted of pleomorphic, flagellated bacilli, many of which showed the typical pinching type of division as seen with other gram-negative bacteria. Most of the flagellated organisms, regardless of the strain, had a single, relatively straight or undulant polar flagellum, about 25 nm in diameter. In some instances, retraction of the bacterial cell membranes made the flagellar basal bodies (bulbs) visible when observed by transmission electron microscopy. The bulbous nature of these bodies appears to be different from the basal structures described for Escherichia coli and other gram-negative bacteria. Long "streamers," previously observed with the light microscope, appear to be fimbriae (or pili) that have a diameter approximatley half that of the flagella and are somewhat coiled and bent at irregular intervals.

Intracellular multiplication of Legionella pneumophila in cultured human embryonic lung fibroblasts.

Cell cultures of normal human embryonic lung fibroblasts were inoculated with two strains of Legionella pneumophila, the etiological agent of Legionnaires disease. Large numbers of intact organisms, including many dividing forms, were seen within fibroblasts by light and electron microscopy. Intracellular multiplication of organisms was demonstrated by the progressive increase in bacterial colony counts from plated extracts of infected fibroblast cultures in which extracellular multiplication had been eliminated by bactericidal concentrations of antibiotics. It is evident that L. pneumophila can thrive in an intracellular environment, and this property may be significant in the pathogenesis of Legionnaires disease.

Fatal pneumonia caused by Legionella pneumophila, serogroup 3: demonstration of the bacilli in extrathoracic organs.

An immunosuppressed patient with malignant lymphoma died of acute pneumonia caused by Legionella pneumophila. Bacilli of serogroup 3 were detected in areas of pnemonia, in a mediastinal lymph node, and in the liver and spleen by direct immunofluorescence done on tissue obtained at autopsy. That the extrathoracic fluorescent material represented intact bacteria rather than antigenic fragments or antigen-antibody complexes was confirmed by finding intracellular bacilli in the liver by electron microscopy. To our knowledge, this case represents the first example of fatal disease attributed to serogroup 3 L. pneumophila and the first case in which L. pneumophila has been demonstrated in extrathoracic organs.

Ultrastructure of the Legionnaires' disease bacterium. A study using transmission electron microscopy.

The Legionnaires' disease (LD) bacterium appeared ultrastructurally identical in human lung, egg yolk membrane, and artificial media, seen as a blunt or tapering rod measuring 0.3 to 0.9 micron in diameter and greater than or equal to 2.0 micron long. Greatly elongated forms were commonly found in cultures and yold sac membranes after 5 to 7 days of growth but were only rarely seen in human lung. The LD bacterium was clearly prokaryotic. Prominent features included electron-lucent nucleoids interspersed among areas of well-defined ribosomes; cleanly circumscribed cytoplasmic vacuoles or granular inclusions; and a double envelope enclosure, each portion consisting of a triple-layered "unit" membrane, approximately 75 A wide. Division always occurred as a pinching, nonseptate process typical of bacteria with a double, gram-negative type of envelope. No definite structure was seen in the periplasmic space that might represent the peptidoglycan layer. These features of the LD bacterium confirm earlier reports of the gram-negative staining reaction of organisms obtained from cultures and preliminary evidence of their gram-negative ultrastructure. We found no unique features that would aid in the ultrastructural differentiation of the LD bacterium from other small gram-negative bacilli.

Ultrastructure of the agent of Legionnaires' disease in the human lung.

This report confirms the gram-negative ultrastructural characteristics of the Legionnaires' disease organism by direct examination of pulmonary tissue from six confirmed cases--two from the original Philadelphia epidemic of 1976 and four from more recent sporadic cases. All microorganisms seen in all six lungs were identical ultrastructurally and were predominantely within intra-alveolar macrophages, as previously observed by light microscopy. They appeared as short, blunt rods that were clearly prokaryotic; i.e., they had diffuse electron-lucent nucleoid areas interspersed among areas of well-defined ribosomes, a pinching nonseptic division, and enclosure within a double envelope consisting of two three-layer "unit" membranes, each approximately 75 A wide. This structure, together with a pinching division, is typical of gram-negative bacteria. The Legionnaires' disease organism multiples both intracellularly and extracellularly in tissue and has no unique ultrastructural features that would aid in its specific identification. These findings are compared with recent reports describing the ultrastructure of what was considered to be the Legionnaires' disease organism in yolk sac and culture medium, and in one human lung.

Differentiation between Prototheca and morphologically similar green algae in tissue.

Evidence that algae are pathogens was provided by the results of electron microscopic studies of tissues from five cattle and sheep suspected of having green algal infections. Chloroplasts were demonstrated in the algae in each case. Prototheca organisms, considered by some to be achloric mutants of green algae, are causative agents of disease in man and animals and may appear morphologically similar to green algae in tissue. However, electron microscopy showed that chloroplasts were absent in these organisms. Light microscopy revealed not only similarities in size, shape, and mode of reproduction, but also a striking difference between the Prototheca organisms and green algae. Unlike Prototheca, the green algae contained abundant cytoplasmic starch granules that were strongly positive by several staining procedures; these granules, which were PAS-negative following diastase digestion, provide a means of differentiating green algae from Prototheca cells in tissue.

Surface ultrastructure of pyruvate kinase-deficient erythrocytes in the Basenji dog.

The scanning electron microscope was used to study the surface morphologic features of erythrocytes from a Basenji dog with hereditary hemolytic anemia due to a pyruvate kinase-deficient erythrocytes (RBC). Cells from this dog were compared with RBC from normal dogs and from dogs with regenerative anemias unrelated to pyruvate kinase deficiency. Demonstration of spherical RBC with uniform spicules on their surface (spheroechinocytes) may provide a morphologic explanation for the shortened erythrocytic life-span previously reported in congenital hemolytic anemia of Basenji dogs. Spherical, spiculated RBC were not found in blood from normal dogs or from anemic dogs with reticulocytoses. The surface of reticulocytes from all dogs with regenerative anemia was roughened, with pronounced folding of the cell membrane.

Human pulmonary adiospiromycosis.

Emmonsia crescens, a fungus that causes granulomatous pulmonary adiospiromycosis, is an uncommon human pathogen. Histopathologic and ultrastructural evaluation of a new case of human adiospiromycosis from Guatemaia showed pulmonary granulomatosis in respose to the inhaled spores of the fungus. The wall of each spore consisted of three zones. A fenestrated layer was clearly demonstrated within the middle zone. Neither budding nor endosporulation was detected. Our findings support the pathogenetic view that E crescens produces local changes by progressive enlargement of the inhaled spores and surrounding granulomas. Characteristically, the fungus does not propagate or disseminate within the lung