Comparison of the Cellient(™) automated cell block system and agar cell block method.

OBJECTIVE: To compare the Cellient(TM) automated cell block system with the agar cell block method in terms of quantity and quality of diagnostic material and morphological, histochemical and immunocytochemical features. MATERIALS AND METHODS: Cell blocks were prepared from 100 effusion samples using the agar method and Cellient system, and routinely sectioned and stained for haematoxylin and eosin and periodic acid-Schiff with diastase (PASD). A preliminary immunocytochemical study was performed on selected cases (27/100 cases). Sections were evaluated using a three-point grading system to compare a set of morphological parameters. Statistical analysis was performed using Fisher's exact test. RESULTS: Parameters assessing cellularity, presence of single cells and definition of nuclear membrane, nucleoli, chromatin and cytoplasm showed a statistically significant improvement on Cellient cell blocks compared with agar cell blocks (P < 0.05). No significant difference was seen for definition of cell groups, PASD staining or the intensity or clarity of immunocytochemical staining. A discrepant immunocytochemistry (ICC) result was seen in 21% (13/63) of immunostains. CONCLUSION: The Cellient technique is comparable with the agar method, with statistically significant results achieved for important morphological features. It demonstrates potential as an alternative cell block preparation method which is relevant for the rapid processing of fine needle aspiration samples, malignant effusions and low-cellularity specimens, where optimal cell morphology and architecture are essential. Further investigation is required to optimize immunocytochemical staining using the Cellient method.

Infection control in the outpatient setting.

This article discusses aspects of ambulatory care that increase the difficulty of practicing infection control in this setting or that require infection control staff to use different methods than they would use in the inpatient setting. The article reviews basic infection control precautions that apply to the outpatient setting in general and specific precautions that apply to dialysis centers and physical therapy programs. The article also describes outbreaks that have occurred in the outpatient setting, defines the deficiencies in infection control practice that caused the outbreaks, and discusses methods to prevent transmission of pathogens in the outpatient setting.

Infection control issues in construction and renovation.

Construction or renovation projects in hospitals pose special challenges. Infection control personnel should be involved in all phases of these projects to ensure that patients, visitors, and staff are protected from unnecessary exposure to infectious agents. Infection control personnel must identify the infection risks posed by each project and must plan ways to minimize the risk. Infection control personnel also must ensure that municipal, county, state, and federal infection control guidelines and regulations are met. This article will discuss basic infection control issues encountered during construction and renovation, offer practical suggestions for addressing these issues, discuss common questions that infection control personnel must address, and describe outbreaks related to construction and renovation.

Exposure workups.

Exposure workups are an important responsibility for infection control personnel. A well-designed plan for investigating exposures, which includes appropriate algorithms, will enable infection control personnel to evaluate exposures rapidly and consistently so that nosocomial transmission is minimized. Infection control personnel should use their own data to develop policies and procedures that suit the needs of their facility. After they have implemented the plan, infection control personnel should continue to collect data on exposures so they can continuously improve their performance.

A prolonged outbreak of methicillin-resistant Staphylococcus aureus in the burn unit of a tertiary medical center.

OBJECTIVE: To report an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in our burn unit and the steps we used to eradicate the organism. DESIGN AND SETTING: Outbreak investigation in the burn unit of a 900-bed tertiary-care medical center. OUTBREAK: Between March and June 1993, MRSA was isolated from 10 patients in our burn unit. All isolates had identical antibiograms and chromosomal DNA patterns. CONTROL MEASURES: Infection control personnel encouraged healthcare workers to wash their hands after each patients contact. The unit cohorted all infected or colonized patients, placed each affected patient in isolation, and, if possible, transferred the patient to another unit. Despite these measures, new cases occurred. Infection control personnel obtained nares cultures from 56 healthcare workers, 3 of whom carried the epidemic MRSA strain. One healthcare worker cared for six affected patients, and one cared for five patients. We treated the three healthcare workers with mupirocin. Subsequently, no additional patients became colonized or infected with the epidemic MRSA strain. CONCLUSIONS: The outbreak ended after we treated healthcare workers who carried the epidemic strain with mupirocin. This approach is not appropriate in all settings. However, we felt it was justified in this case because of a persistent problem after less intrusive measures.

Interleukin-6 secretion by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro.

Interleukin-6 (IL-6) is a pluripotent cytokine that may play a role in pulmonary defense against bacterial pathogens. We have quantitated the response of bovine alveolar macrophages (bAM) to bacterial lipopolysaccharide (LPS; E. coli 055: B5) in vitro using the IL-6 sensitive 7TD1 cell line. Bacteria LPS in the absence of serum induced IL-6 secretion from bAM (1 x 10(6) ml-1) over a range of LPS concentrations from 10 ng ml-1 to 10 micrograms ml-1. This resulted in IL-6 levels ranging from approximately 5 to over 200 U ml-1.IL-6 secretion by from approximately 5 to over 200 U ml-1.IL-6 secretion by LPS-stimulated bAM was increased by 24 h poststimulation, and continued to increase up to 72 h after stimulation. Fetal bovine serum (FBS, 1% vol/vol; 320 micrograms ml-1) enhanced IL-6 secretion from macrophages in the presence of LPS by approximately 10-fold compared with LPS alone. A bovine serum fraction (1 microgram ml-1 protein) prepared using ion-exchange chromatography also markedly enhanced IL-6 secretion versus LPS alone. The stimulatory effect of IL-6-like activity in the bAM supernatants was neutralized by an anti-human IL-6 polyclonal antibody. Northern blot analysis revealed increased IL-6 mRNA at 2 h poststimulation with LPS + FBS, peak levels at 4 h, and levels were decreased by 6 h poststimulation. Results suggest that IL-6 is secreted by bovine alveolar macrophages, and that bacterial LPS and serum components synergize to produce this response.

CD14 and tissue factor expression by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro.

The membrane-associated CD14 receptor (mCD14) is a monocyte/macrophage differentiation antigen, and it has been demonstrated to serve as a receptor for bacterial lipopolysaccharide (LPS; endotoxin). Binding of LPS to mCD14 has been shown to be associated with LPS-induced macrophage, monocyte, and neutrophil activation in humans. In this report, we describe the presence and function of an mCD14-like receptor on bovine alveolar macrophages (bAM). An immunofluorescence technique and flow cytometric analysis indicated binding of anti-human CD14 monoclonal antibodies (MAb) My4, 3C10, and 60bd to bAM. Binding of anti-CD14 MAb (3C10 and MY4) was reduced over 20% by pretreatment of bAM with phosphatidylinositol-specific phospholipase C (0.5 to 1.0 U/ml), indicating that bovine mCD14 is a glycosyl phosphatidylinositol-anchored protein. In addition, pretreatment of bAM with anti-CD14 MAb decreased binding of 125I-labeled LPS to macrophages, suggesting that bovine mCD14 serves as a receptor for LPS. A cDNA probe based on the human sequence for CD14 was used in Northern (RNA) blot analysis, and hybridization to human monocyte CD14 yielded the expected 1.5-kb band. Hybridization to bovine mRNA yielded a 1.5-kb band plus an unexpected 3.1-kb band. Constitutive expression of bovine CD14 mRNA was observed, and the expression level was modestly elevated in bAM stimulated for 24 h with LPS (1 ng/ml) in the presence of bovine serum. The function and activation of bAM were assessed by quantitation of tissue factor (TF) expression on the cells using an activated factor X-related chromogenic assay and S-2222 substrate. LPS (1 ng/ml)-mediated upregulation of TF expression on bAM was dependent on the presence of bovine serum components, and TF expression was inhibited by anti-CD14 MAb. In addition, TF mRNA levels in LPS-stimulated bAM were decreased by pretreatment of cells with anti-CD14 MAb (MAb 60bd, 10 micrograms/ml).

Signal transduction pathways of bacterial lipopolysaccharide-stimulated bovine vascular endothelial cells.

Increased procoagulant activity of vascular endothelial cells may be an important component in the pathogenesis of intravascular coagulation associated with gram-negative bacterial diseases. Two bovine endothelial cell (BEC) lines isolated from pulmonary arteries (ENS-2 and ENT-18) were used in this study to investigate procoagulant signal transduction pathways of endotoxin (lipopolysaccharide, LPS)--stimulated BECs. The endothelial cell line ENS-2 was sensitive to LPS as demonstrated by tissue factor (TF) expression, but in contrast, the ENT-18 endothelial cell line was unusually resistant to the effects of LPS. No remarkable quantitative difference in binding of radiolabeled LPS was detected between the two endothelial cell lines. A protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate, PMA) failed to induce TF expression in either cell line at concentrations ranging from 0.05 to 1.00 microM when used as a sole stimulus for the endothelial cells. However, when PMA was used in combination with LPS, PMA enhanced the stimulatory effect of LPS on the endothelial cells. In parallel experiments, PKC inhibitors (H-7 and GF 109203X) interfered with the stimulatory effect of LPS on the cells by decreasing tissue factor expression. We also found that an activator of adenylate cyclase, forskolin, similarly inhibited LPS-induced tissue factor activity. In contrast, protein tyrosine kinase inhibitors (genistein, lavendustin A) had no inhibitory effect on LPS-induced endothelial cell tissue factor expression. Our results collectively suggest that activation of PKC is an important step in stimulation of endothelial cells by LPS, and that LPS and phorbol esters may synergize to produce an enhanced stimulatory effect. Our results also suggest participation of cAMP in controlling LPS-mediated stimulation of endothelial cells, but fail to demonstrate a role for protein tyrosine kinase activity.

Serum components enhance bacterial lipopolysaccharide-induced tissue factor expression and tumor necrosis factor-alpha secretion by bovine alveolar macrophages in vitro.

We have compared the effect of bacterial lipopolysaccharide (LPS) in combination with normal adult bovine serum (NBS), fetal bovine serum (FBS), or a bovine serum fraction on tissue factor expression and tumor necrosis factor alpha (TNF-alpha) secretion by bovine alveolar macrophages. At a concentration of 1 ng/ml, bacterial LPS alone failed to induce measurable tissue factor expression by the macrophages, but the presence of FBS, NBS, or a fraction of normal pooled bovine serum isolated by ion-exchange chromatography (fraction 2) markedly potentiated the effect of LPS. A protein concentration of 64 micrograms/ml NBS, 192 micrograms/ml FBS, and only 640 ng/ml fraction 2 was required to induce maximal tissue factor expression on the macrophages in combination with 1 ng/ml LPS. Comparison of quantities of added serum protein required to induce maximal potentiating effects indicated that fraction 2 was 100 times more potent than whole NBS and 300 times more potent than whole FBS. We similarly found that TNF-alpha secretion by macrophages exposed to LPS was responsive to serum and was highly responsive to fraction 2. LPS alone (1 ng/ml) induced a relatively low level of TNF-alpha secretion by the macrophages, and the presence of FBS, NBS, or fraction 2 potentiated the effect of LPS. A concentration of 64.0 micrograms/ml NBS, 320.0 micrograms/ml FBS, and 3.2 micrograms/ml fraction 2 serum protein induced near-maximal TNF-alpha secretion by the macrophages. Comparison of the concentration of serum protein required to induce these potentiating effects indicated that fraction 2 was approximately 20 times more potent than whole NBS and 100 times more potent than whole FBS. The stimulatory effect of LPS plus fraction 2 serum proteins was dependent on the CD14 receptor, as monoclonal antibodies directed against CD14 (My4, 60bd; 10 micrograms/ml) inhibited tissue factor expression and TNF-alpha secretion by the macrophages.

Simultaneous measurements of NO, OH, and the major species in turbulent flames.

We present a method for obtaining time-resolved point measurements of the pollutant nitric oxide (NO), hydroxyl (OH), the temperature, and the major species-using a combination of laser-induced fluorescence and Rayleigh/Raman scattering-in turbulent flames. Using single-shot measurements of temperature and the major species concentrations, we calculate the mixture fraction, which characterizes the state of mixing in the gas, and the corrections for the NO and OH fluorescence. For NO the time-resolved detection limit is ~ 10 parts in 10(6). We demonstrate this facility with measurements of NO versus the mixture fraction in a turbulent nonpremixed H(2) jet flame.

Intestinal pseudo-obstruction, type 1 anti-neuronal nuclear antibodies and small-cell carcinoma of the lung.

Intestinal pseudo-obstruction is a rare paraneoplastic manifestation of small-cell carcinoma of the lung. This report documents a further case associated with neuronal degeneration of enteric neural plexuses and a high serum titre of immunoglobulin G (IgG) type 1 anti-neuronal nuclear antibodies (ANNA-1).

Thrombopoietin from human embryonic kidney cells stimulates an increase in megakaryocyte size of sublethally irradiated mice.

Previous work showed that treatment of irradiated mice with a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) decreased the degree and duration of thrombocytopenia in the period after irradiation. In an attempt to elucidate the radio-protective effects of TSF, femoral megakaryocyte sizes and numbers were measured in mice treated with 3.0 Gy of 137Cs gamma rays and TSF. For controls, other irradiated mice were given human serum albumin (HSA), the carrier protein for TSF, rabbit anti-mouse platelet serum (RAMPS), or normal rabbit serum (NRS); megakaryocyte sizes and numbers were studied on Days 7-14. The results showed that irradiated, TSF-treated mice had significantly larger megakaryocytes on all days assessed compared to HSA-treated control mice. Likewise, RAMPS-treated mice had significantly larger megakaryocytes 14 days after irradiation compared to NRS-treated mice. Megakaryocyte numbers were significantly depressed in TSF-treated mice on Days 7-10 and 14 and on Day 10 in RAMPS-treated mice, compared to their respective controls. Therefore, irradiated mice treated with TSF yielded results similar to RAMPS-treated mice. Megakaryocyte sizes and numbers were also determined for mice treated with 40,000 U/mouse of interleukin-6 (IL-6), 227 U/mouse of granulocyte-macrophage colony-stimulating factor (GM-CSF), or a combination of both cytokines; bovine serum albumin (BSA) was used as a control for these cytokine treatments. Unlike TSF treatment, GM-CSF significantly increased megakaryocyte numbers on both Days 10 and 14; the combination of both growth factors also increased megakaryocyte numbers on Day 14 compared to BSA-treated control mice. However, megakaryocyte size was decreased in GM-CSF-treated mice and in mice treated with both growth factors on Day 10. High levels of IL-6 failed to affect megakaryocyte size or number significantly on any day evaluated. The data of the present report, showing that TSF significantly increases megakaryocyte sizes and platelet counts of sublethally irradiated mice, indicate that thrombopoietin will be useful in treating patients undergoing bone marrow transplantation and/or patients with platelet production problems.

Chronic administration of transforming growth factor-beta suppresses erythropoietin-dependent erythropoiesis and induces tumour necrosis factor in vivo.

Transforming growth factor beta is a known inhibitor of the proliferation and differentiation of early haematopoietic progenitors but has no effect on mature erythroid cells in vitro. Mice injected with rhTGF beta 1 exhibited severe and progressive suppression of erythropoiesis manifested by a decline of reticulocyte count, marrow erythroblasts and marrow and spleen CFU-E, which could be prevented by administration of erythropoietin. This suppression of erythropoiesis was associated with the appearance of tumour necrosis factor in the blood, development of pronounced cachexia and depression of serum erythropoietin levels. TGF beta induces TNF in vivo that leads to cachexia, decrease of serum erythropoietin levels and suppression of erythropoietin dependent erythropoiesis.

Inheritance of zingiberene in Lycopersicon.

The inheritance of the sesquiterpene zingiberene was analyzed in segregating progeny of interspecific crosses among Lycopersicon hirsutum f. hirsutum Humb. and Bonpl. (hir), L. esculentum Mill. cv 'Nova' (esc), and L. hirsutum f. glabratum C.H. Mull (gla). The presence of zingiberene was inherited as a single dominant gene from hir in F2 and BC progeny of esc x hir, and as a single recessive gene in F2 and BC progeny of gla x hir. The segregation of esc x hir, gla x hir, and esc x gla progeny supported a single locus allelomorphic model in which the presence of zingiberene is controlled at a single locus, Z, where the allele from hir confers presence of zingiberene and is dominant to the allele from esc but recessive to that from gla. The presence of zingiberene in esc x hir progeny was not linked to the ability to set fruit or to several fruit characters, and progeny with zingiberene levels comparable to hir were recovered.

Temperature and Photoperiod Influence Trichome Density and Sesquiterpene Content of Lycopersicon hirsutum f. hirsutum.

Resistance to Colorado potato beetle in a clone of Lycopersicon hirsutum f. hirsutum L. is attributed to the presence of the sesquiterpene zingiberene in the type VI leaf trichomes; however, both day/night temperature regimen and photoperiod affect zingiberene content and trichome density. In short days (SD), zingiberene content per trichome is more than 3-fold greater than in long days. In SD, trichome density per unit leaf surface is 2-fold greater at 25/20 degrees C (day/night) than at either 30/25 degrees C or 20/15 degrees C, thus indirectly influencing zingiberene content per cm(2). In long days, temperature regimen had little effect on either trichome density or zingiberene content, although trichome density was greater than or equal to that in SD.

Thrombopoietin from human embryonic kidney cells causes increased thrombocytopoiesis in sublethally irradiated mice.

Previous work showed that mice treated with platelet-specific antiserum prior to whole-body irradiation did not suffer the degree or duration of thrombocytopenia as did irradiated control mice. We now report that a partially purified preparation of a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) mimics the biological effects of platelet-specific antiserum treatment in hematopoietically suppressed mice. Male C3H mice were exposed to 3.0 or 4.5 Gy of 137Cs gamma radiation and injected with a total dose of 4 units (U) of TSF. Human serum albumin (HSA) and rabbit anti-mouse platelet serum-injected mice, along with unirradiated mice, served as controls. Packed cell volumes (PCV), RBC counts, WBC counts, platelet counts, and percentage 35S incorporation into platelets were measured in mice at various days (7-14) following treatment. The results showed that irradiated mice treated with TSF had increased 35S uptake into platelets and higher platelet counts than HSA-treated controls. Also, PCV, RBC counts, and WBC counts of irradiated mice treated with TSF were significantly higher than values for HSA-treated mice. Additional experiments using 40,000 U/mouse of Interleukin-6 (IL-6), 227 U/mouse of granulocyte macrophage-colony stimulating factor (GM-CSF), or a combination of GM-CSF and IL-6 did not show increased platelet counts or 35S incorporation into platelets on Days 10 and 14 when compared to other mice treated with control substances. These results suggest that the radioprotective effects of platelet antibodies reported previously may be due to the release and action of thrombopoietin. These studies also demonstrate that thrombopoietin therapy will modulate the severe thrombocytopenia that occurs in radiation-induced bone marrow suppression.

Saturated fluorescence measurements of the hydroxyl radical in laminar high-pressure C(2)H(6)/O(2)/N(2) flames.

We demonstrate saturation of a transition of the OH molecule in high-pressure flames by obtaining saturation curves in C(2)H(6)/O(2)/N(2) laminar flames at 1, 6.1, 9.2, and 12.3 atm. In addition we present quantitative fluorescence measurements of OH number density at pressures to 12.3 atm. To assess the efficacy of the balanced cross-rate model for high-pressure flames, we compare laser-saturated fluorescence measurements, which were calibrated in an atmospheric-pressure flame, with absorption measurements at 3.1 and 6.1 atm. At 3.1 atm the absorption and fluorescence measurements compare well. At 6.1 atm, however, the concentrations given by laser-saturated fluorescence are ~25% lower than the absorption values, indicating some depletion of the laser-coupled levels beyond that at atmospheric pressure. By using a reasonable estimate for the finite sensitivity to quenching, we anticipate that fluorescence measurements that are calibrated at 1 atm can be applied to flames at ~10 atm with absolute errors within +/-50%.