Ischemia disrupts myosin I beta in renal tubules.

In these studies we have examined rat kidneys biochemically and microscopically to determine where myosin I beta is located before, during, and after an acute ischemic injury. Myosin I beta is present in multiple tubule segments including the brush border (BB) of the proximal tubule cell (PTC). Its distribution is severely altered by a 15-min renal artery clamp. Myosin I beta is present in the urine during reflow and is found in the numerous cellular blebs arising from the damaged PTC and other tubules. Two hours of reflow result in a decrease in BB myosin I beta staining and an increase in its cytoplasmic staining. Interestingly, the return of the F-actin in the BB precedes the return of the myosin I beta, suggesting that this myosin I isoform may not play a role in rebuilding the microvilli after an ischemic injury. A nonstructural role for this myosin, such as transport or channel regulation, is supported by its presence in many tubule segments, all of which have transport and channel requirements but do not all contain microvilli.

Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection.

BACKGROUND: Pneumocystis carinii causes pneumonia in immunocompromised patients with a high morbidity and mortality rate, but the interaction between this organism and the host cell is not well understood. The purpose of this research was to study the response of host cells to P. carinii infection on a molecular level. RESULTS: The technique of mRNA differential display was used to detect genes whose expression may be affected by P. carinii infection. The nucleotide sequence of one differentially displayed DNA fragment was found to be identical to that of the rat mitochondrial ATPase 6 gene, which is a subunit of the F0F1-ATP synthase complex. A four-fold increase in expression of this gene was verified by Northern blot analysis of total RNA extracted from P. carinii-infected rat lung versus that from mock-infected rat lung. Localization of the cells containing ATPase 6 mRNA was accomplished by in situ hybridization. In sections of non-infected rat lung, these cells were found lining the distal parts of the respiratory tree and in apical areas of the alveoli. Histological location of these cells suggested that they were Clara cells and type II pneumocytes. This hypothesis was confirmed by co-localizing the mRNAs for ATPase 6 and surfactant protein B (SP-B) to the same cells by two-color fluorescent in situ hybridization. CONCLUSIONS: The ATPase 6 gene is over expressed during P. carinii infection, and type II pneumocytes and Clara cells are the cell types responsible for this over-expression.

Haemophilus ducreyi associates with phagocytes, collagen, and fibrin and remains extracellular throughout infection of human volunteers.

In a previous study, Haemophilus ducreyi was found in the pustule and dermis of samples obtained at the clinical end point in the human model of infection. To understand the kinetics of localization, we examined infected sites at 0, 24, and 48 h after inoculation and at the clinical end point. Immediately after inoculation, bacteria were found predominantly in the dermis but also in the epidermis. Few bacteria were detectable at 24 h; however, by 48 h, bacteria were readily seen in the pustule and dermis. H. ducreyi was associated with polymorphonuclear leukocytes and macrophages in the pustule and at its base, but was not associated with T cells, Langerhans' cells, or fibroblasts. H. ducreyi colocalized with collagen and fibrin but not laminin or fibronectin. Association with phagocytes, collagen, and fibrin was seen as early as 48 h and persisted at the pustular stage of disease. Optical sectioning by confocal microscopy and transmission electron microscopy both failed to demonstrate intracellular H. ducreyi. These data identify collagen and fibrin as potentially important targets of adherence in vivo and strongly suggest that H. ducreyi remains extracellular throughout infection and survives by resisting phagocytic killing in vivo.

Characterization of a multicopy family of genes encoding a surface-expressed serine endoprotease in rat Pneumocystis carinii.

A unique family of genes encoding serine endoproteases related to the Saccharomyces cerevisiae serine endoprotease kexin was identified in Pneumocystis carinii. Unlike previously described serine endoprotease genes that are single copies, multiple copies of the P. carinii serine endoprotease are distributed throughout the genome. The proteins predicted by these variant genes demonstrate sequence variability, but they retain the conserved active sites associated with endoprotease activity. The serine endoprotease was localized to the organism surface by immunohistochemical and immunofluorescence studies and to the electron lucent layer of the cyst wall by immunoelectron microscopy. The findings of multiple copies of the serine endoprotease gene in the P. carinii genome, and its localization to the cell surface, suggest that this protease plays an important role in the biology of P. carinii and that antigenic variation of the surface-expressed serine endoprotease may be a strategy for immune evasion. P. carinii serine endoprotease provides a novel target for chemotherapeutic and immune-based approaches to the treatment of P. carinii pneumonia.

Airborne particulates in the OR environment.

Intraoperative sampling of airborne particulates is rarely performed in the OR environment because of technical difficulties associated with sampling methodologies and because of the common belief that airborne contamination is infrequently associated with surgical site infections (SSIs). In this study, investigators recovered non-viable (i.e., lint) and viable (i.e., microorganisms) particulates during vascular surgery using a personal cascade impactor sampling device. The predominant nonviable particulates recovered during intraoperative sampling were wood pulp fibers from disposable gowns and drapes. Several potential nosocomial pathogens (e.g., Staphylococcus aureus, Staphylococcus epidermidis) and other drug-resistant isolates frequently were recovered from an area adjacent to the surgical field. The widespread presence of airborne particulates during surgery suggests that further studies are warranted to assess the role these particles may play in the development of SSIs or in dissemination of nosocomial pathogens within the OR and hospital environment.

Growth of molluscum contagiosum virus in a human foreskin xenograft model.

Molluscum contagiosum virus (MCV) is a common pathogen causing a troublesome skin condition in many immunocompetent individuals and a widespread, disfiguring affliction in many patients with AIDS. We have successfully infected human foreskin fragments with a patient-derived isolate of MCV. This was demonstrated by exposing the foreskin pieces to a patient lesion extract and implanting the tissue under the renal capsule of athymic mice. Light and electron microscopic examination of infected implants showed the presence of cytoplasmic inclusions containing typical poxvirus particles within 2-3 weeks of implantation. Replication of MCV was established by demonstrating that viable virus was required to produce the cytopathologic effects, and viral DNA replication was demonstrated by incorporation of bromodeoxyuridine into cytoplasmic inclusions. Four additional patient extracts (representing both described MCV types) were also used to successfully infect foreskin implants. A limited number of attempts to pass virus from one infected implant to another were not successful. This system is the most rapid and reproducible for growing MCV that has been reported to date.

Semisynthetic echinocandins affect cell wall deposition of Pneumocystis carinii in vitro and in vivo.

Cyclic lipodepsipeptide compounds of the echinocandin class exhibit broad-spectrum antifungal activity and have been shown to be effective in the treatment of Pneumocystis carinii pneumonia in laboratory animal models. Previous studies have led investigators to propose that these compounds, active against fungal cell walls, are selectively active against the cyst forms of P. carinii. We demonstrate that a semisynthetic, water-soluble echinocandin analog, LY307853, is effective in reducing the number of all life cycle forms of P. carinii and is more effective in mice immunosuppressed with monoclonal antibody to L3T4+ cells than in mice immunosuppressed with dexamethasone. Treatment of P. carinii isolates with LY307853 in a short-term in vitro culture model resulted in cytoarchitectural alterations suggesting that this echinocandin may interfere with the export of surface glycoprotein and the formation of the tubular elements normally found on the surfaces of trophic forms. The cytoarchitectural changes in trophic forms treated in vitro with LY307853 were also observed in trophic forms in the lung tissue of rats treated with a closely related echinocandin analog, LY303366.

Proliferating thymolipoma: ultrastructural, immunohistochemical, and flowcytometric study.

A superior-anterior mediastinal tumor was excised from a 50-year-old man. The 207-g mass was encapsulated and multilobulated. It contained adipose tissue and abnormal thymic tissue. In some areas the thymic tissue was characterized by cords and nests of epithelial cells lying within either the adipose tissue or a myxoid matrix. Other areas were characterized by cortical thymic tissue with increased numbers of epithelial cells. Foci of normal medullary tissue were present. The prominent epithelial cells were immunoreactive for cytokeratin and nonimmunoreactive for vimentin, S-100, chromogranin, and parathyroid hormone. Flow cytometry showed that the lymphocyte populations were consistent with a late cortical thymic phenotype. The tumor was diploid. By electron microscopy, the prominent epithelial cells had desmosomes and a few tonofilaments. The cytoplasm contained additional organelles including mitochondria, polyribosomes, and occasional lysosomes. Nuclei were oval and had relatively smooth contours, prominent nucleoli, and moderate quantities of heterochromatin. Basal lamina was present around many nests and cords of cells. This is the first such study of a tumor with this histology.

Close association of Pneumocystis carinii from infected rat lung with culture cells as shown by light and electron microscopy.

Studies of the association of rat-origin Pneumocystis carinii with culture cells were performed both to learn more about the role of cells in P. carinii culture and to evaluate additional cell lines in an effort to improve culture methods. Proliferation of trophozoites of P. carinii from rat lung in cultures with six lung cell lines was demonstrated by light microscopic evaluations of both Giemsa-stained and immune-specific-stained culture samples. Scanning electron microscopy and transmission electron microscopy were used to study the organism's interaction with culture cells and demonstrated a close association of P. carinii with cells in cell lines that supported growth. Proliferation with the MVILU line was suboptimal and there was less organism interaction with these cells than with other cell lines that allowed proliferation. Two cell lines evaluated, Chinese Hamster ovary CHOKI and CHOLEKI, did not allow proliferation and had no association of P. carinii with cells. Scanning and transmission electron micrographs demonstrated the close association of organisms with rat fetal lung (RFL), human embryonic lung (HEL), human diploid lung (HFL), and feline embryonic lung (AKD) culture cells. It appears that the association of rat-origin P. carinii with cells is essential for parasite proliferation in short-term culture.

Effects of 8-aminoquinolines on the ultrastructural morphology of Pneumocystis carinii.

Primaquine and other 8-aminoquinolines are effective against Pneumocystis carinii in culture and animal models but the way(s) in which they affect P. carinii are not known. This study used transmission electron microscopy to observe early effects of 8-aminoquinolines on P. carinii grown with human embryonic lung fibroblasts in microcarrier suspension culture. The 8-aminoquinolines evaluated were primaquine and Walter Reed Army Institute for Research (WR) compounds WR6026, WR238605 and WR242511. Samples of P. carinii were taken at 0, 3, 6, 12, 24 and 48 hours from culture flasks containing selected concentrations of the drugs. Time matched samples from a parallel culture without drug served as controls. All the 8-aminoquinolines produced similar morphologic alterations of the internal structure of P. carinii. Initially, dilatation of the nuclear envelopes and membranous arrays arising from the reticular system were observed. Later, more organisms displayed large arrays of smooth membranous material often presenting a concentric membranous pattern. Subsequently, cellular organization was lost resulting in necrosis. At concentrations tested WR242511 appeared to be the most effective, producing alterations in many trophozoites after 6 hours of exposure; WR6026 appeared to be the least effective with some organisms unaffected after 48 hours. The changes observed are consistent with damage to the reticular system of P. carinii, which might be caused by oxidation by the 8-aminoquinolines or their metabolites.

Comparative ultrastructure of needle aspiration biopsy and surgical resection specimens of lung tumors.

Ultrastructural examination affords conclusive evidence for classification of lung tumors. Tissue properly fixed for electron microscopy is not available in many cases, however. Ultrastructural diagnosis of resected specimens obviously follows, rather than directs, the surgical treatment. Fine-needle aspiration (FNA) of lung masses is recommended as a means to obtain lung tumor tissue for electron microscopy. Nevertheless, no comparison has been made between ultrastructural information gained from aspiration specimens and resected specimens. Electron microscopy was performed on transthoracic FNA specimens of 10 lung tumors for which surgical resection was subsequently performed. Glutaraldehyde-fixed specimens from FNA and surgical resection were prepared for electron microscopy according to routine procedures. Fixation of the FNA specimens was equivalent or superior to that of the resected specimens in 9 of the cases. Three of the FNA specimens contained necrotic as well as viable tissue. Features essential for diagnosis such as desmosomes, junctions, neurosecretory granules, intermediate filaments, glycogen, lipid, mucin, and microvilli were identifiable in both FNA and resected specimens. FNA specimens therefore yield a representative sample of the ultrastructural features of lung tumors when adequate cellular material is obtained. Use of a coaxial needle sampling technique with immediate microscopic assessment reduces the likelihood of retrieving only blood or necrotic tissue in the electron microscopy specimens.

Improved intracellular morphology of Pneumocystis carinii from rat lung by postfixation with a mixture of potassium ferrocyanide and osmium tetroxide.

Pneumocystis carinii infected rat lungs were postfixed with a mixture of OsO4 and K4Fe(CN)6. A marked improvement in staining of cell membranes, endoplasmic reticulum, nuclear membranes and glycogen was observed. These improvements were seen in both the trophic and cystic forms of the organisms. The addition of K4Fe(CN)6 did not improve the staining of cell walls, microtubules or ribosomes. Trophozoites were seen attached to both type 1 and type 2 pneumocytes by filopodia and/or intercalation of the cell body of P. carinii with the host lung cells. It is expected that the improvement in ultrastructural detail will allow better understanding of the ultrastructure of P. carinii and provide insights into the modes of action of various antimicrobial compounds on this organism.

The effect of primaquine on the ultrastructural morphology of Pneumocystis carinii.

Ultrastructural examination of Pneumocystis grown on WI-38 human embryonic lung fibroblasts and treated with primaquine indicated progressive deterioration of cellular morphology. Thus, primaquine had a cidal effect on the organisms.

Adherence of mucin and non-mucin-producing staphylococci to preclotted and albumin-coated velour knitted vascular grafts.

The mechanisms involved in bacterial adherence to vascular grafts are important in understanding prosthetic infections. Albumin-coated Dacron (ACD) is a new development in vascular graft fabrication. However, albumin acts as a receptor for certain gram-positive bacterial adhesions. Five pathogenic, coagulase-negative Staphylococcus epidermidis strains were used to measure the differential microbial adherence to ACD versus untreated velour-knitted Dacron (VKD) vascular prostheses. Specimens of VKD, preclotted VKD, and ACD were inoculated with each of the five strains (10(7) colony-forming units/ml) for 2, 4, 8, 12, and 24 hours. After incubation, graft specimens were washed to remove nonadherent organisms and oscillated ultrasonically to remove adherent organisms. The sonication effluent was plated to trypticase soy agar to quantitate the adherent organisms. Adherence was significantly greater (p less than 0.01) to VKD compared with preclotted VKD and ACD at 2, 4, 8, and 24 hours. Four of the five study strains demonstrated significantly greater adherence to VKD than to either ACD or preclotted VKD. Adherence of S. epidermidis increased with exposure time. Albumin bonded to velour-knitted Dacron does not increase coagulase-negative staphylococcal adherence compared with the noncoated vascular prostheses. Binding albumin to vascular prostheses does not increase the risk of staphylococcal colonization.

Fecal peritonitis: microbial adherence to serosal mesothelium and resistance to peritoneal lavage.

Fecal contamination of the peritoneal cavity is a serious and potentially life-threatening event. While numerous models have been developed to study the pathogenesis of intraabdominal infection, to date, most investigations have failed to focus on the adherence of the contaminants to the serosal mesothelium. In the present investigation, the cecal ligation and puncture technique (CLP) was performed in Sprague-Dawley rats to study the following: (a) the kinetics of microbial adherence to the serosal mesothelium, (b) the stability of the aerobic and anaerobic intraperitoneal/mesothelial populations, following extended saline lavage, and (c) the impact of antimicrobial lavage on the stability of the mesothelial microbial populations. The Enterobacteriaceae rapidly colonized the serosal mesothelium and were the predominant flora up to 4 hours post-CLP. After 8 hours, the Bacteroides fragilis group represented the predominant peritoneal wash and mesothelial-associated microorganisms. Extended saline lavage failed to significantly reduce the mesothelial microbial populations. While antimicrobial lavage produced an immediate decrease in mesothelial microbial recovery, the results were transitory and the microbial populations achieved or exceeded prelavage levels at 24 hours postlavage. Microbial colonization of the peritoneal mesothelial surface is a rapid and stable phenomena following penetrating injury to the distal bowel. The results further suggest that the mesothelial populations are resistant to intraperitoneal lavage.

Post-antibiotic effect in Bacteroides fragilis group.

Post-antibiotic effect (PAE) is the transient suppression of bacterial growth after brief antimicrobial exposure. While numerous reports have described PAE with aerobic and facultative microorganisms, virtually no studies have been conducted with anaerobic isolates. Intraabdominal isolates of the Bacteroides fragilis group were exposed for one hour to antibiotic (cefoxitin, cefotetan, and imipenem) concentrations two to four times the minimal inhibitory concentration (MIC). Post-antibiotic effect was described as the difference between the time required for microbial growth in the test versus the control to increase one Log10 above the quantitation observed immediately after drug removal. Bacteroides fragilis, B. ovatus, B. thetaiotaomicron and B. vulgatus exhibit PAEs for all test compounds. The time intervals for the PAEs were strain variable and ranged from six to 50 hours. No PAE was demonstrated with B. distasonis strains by the broth dilution technique. The results suggest that brief high dose exposure of some members of the B. fragilis group to anaerobe active beta-lactams produces a prolonged suppression in growth. In theory, a prolonged PAE could influence the dosage regimentation of selective antibiotics.

Terminal cytoplasmic maturation of human megakaryocytes in vitro.

Several studies suggest that serum factors (thrombopoietins) regulate thrombopoiesis by altering the number, size, ploidy, and maturation rate of megakaryocytes (MK). Various in vivo systems have been used to quantitate these events. In this study, an in vitro system was developed to monitor terminal cytoplasmic maturation of isolated human MK. MK enriched by elutriation, which eliminated the MK progenitors, were suspended in culture with serum from either normal donors (NABS) or patients with aplastic anemia (AAS). In cultures composed of small platelet glycoprotein-positive mononuclear cells and morphologically immature MK, development was characterized by sequential shifts in MK through morphologically recognizable maturation stages I, II, III, and IV over eight days of incubation (I and II only; then I, II, III; II, III, IV; III and IV; then IV only). Platelet formation coincided with the appearances of stage IV cells. Cultures composed of a mixture of all stages followed a similar maturation sequence, only at an accelerated rate. AAS resulted in the more rapid appearances of the mature cells in either system. This study indicates that human MK can undergo terminal cytoplasmic maturation in vitro, and that altering culture conditions (AAS for NABS) can accelerate the rate of maturation. Three major events occur during megakaryocytopoiesis: proliferation of the progenitor cells, polyploidization, and cytoplasmic maturation. Now it is possible to study the terminal steps of differentiation independent of proliferative events.