Use of FTA gene guard filter paper for the storage and transportation of tumor cells for molecular testing.

CONTEXT: Efficient methods of storing tumor specimens for molecular testing are needed in the modern surgical pathology laboratory. The FTA Gene Guard system is a novel method for the collection and room temperature storage of blood samples for DNA testing. The method uses index card-sized filter papers that provide an ideal medium on which to store tumor specimens for DNA testing. OBJECTIVE: To determine whether FTA filter paper can be used in the surgical pathology laboratory to store tumor cells for DNA testing. DESIGN: Cell suspensions were prepared from 60 surgical specimens, and DNA was extracted either immediately or after storage on FTA paper. The DNA extracted by each method was tested by polymerase chain reaction (PCR) for the beta-globin and interferon gamma genes, and the results were compared. Fifteen lymph node specimens stored on FTA paper were then tested for immunoglobulin heavy chain (IgH) gene rearrangement by PCR, and these results were compared with those obtained for immediately extracted DNA.SETTING: University medical center. RESULTS: The DNA extracted from cells stored on FTA paper performed as well in the PCR as the freshly extracted DNA in nearly all cases (>95%). The results of tests for IgH gene rearrangements showed 100% concordance between the 2 methods of DNA extraction.Conclusion.-Cells from surgical specimens can be stored on FTA paper for extended lengths of time, and DNA can be extracted from these cells for PCR-based testing. FTA filter paper is a reliable medium for the storage and/or transport of tumor cells for PCR-based DNA analysis.     

Gastric myeloid metaplasia: a case report and review of the literature

We report a case of gastric myeloid metaplasia in an 89- year-old woman with agnogenic myeloid metaplasia. The lesions were fortuitously discovered on upper endoscopy. The antral mucosa was thickened and polypoid, and on histologic examination contained immature granulocytes, megakaryocytes, and a few erythroblasts without desmoplastic stromal reaction. The granulocytes were positive for CD15, CD68, and myeloperoxidase on immunohistochemistry, and the megakaryocytes showed positive reactivity for factor VIII. Gastric myeloid metaplasia is a very rare event, and to our knowledge only 6 cases have been reported in the literature to date. It usually occurs in patients with advanced myeloproliferative syndrome. Gastric myeloid metaplasia often has a pseudotumoral appearance, leading to digestive symptoms. Histologic diagnosis is straightforward when trilinear hematopoietic elements are identified in gastric biopsies. Immunohistochemistry with anti-factor VIII antibody can be useful to confirm the presence of megakaryocytes.     

Pseudomonas aeruginosa degrades pulmonary surfactant and increases conversion in vitro

Although it is known that surfactant lipids and proteins are altered in patients with Pseudomonas aeruginosa infections, the mechanisms and implications of these alterations are not clear. In this study, the effects of P. aeruginosa on the surfactant large aggregate fraction were examined using an in vitro surface area cycling model. Large aggregates were isolated from porcine bronchoalveolar lavage fluid and incubated with supernatants from P. aeruginosa cultures (PAO1, parent strain; PAO1-A1, lasA-negative mutant; PAO1-B1, elastase-negative mutant) or purified elastase. Amounts of surfactant protein (SP)-A and SP-B, phospholipid content, and large aggregate conversion were assessed. In addition, lipid degradation was assessed by incubating a mixture of radiolabeled phospholipids with P. aeruginosa supernatants. The results demonstrated that SP-A was degraded by PAO1 and PAO1-A1 supernatants, and by purified elastase. SP-B was degraded by PAO1 and PAO1-B1 supernatants, but not by elastase. P. aeruginosa supernatants degraded phospholipids, a process inhibited by ZnCl(2). P. aeruginosa supernatants and elastase increased conversion. The data suggest that protein degradation facilitates increased conversion, and that phospholipid degradation and conversion enhance degradation of surfactant proteins. In conclusion, P. aeruginosa secretes multiple virulence factors that cooperate to result in degradation of surfactant components and alteration of large aggregate conversion.     

Screening for antibacterial activity in 72 species of wood-colonizing fungi by the Vibrio fisheri bioluminescence method

Resistance of pathogenic bacteria to antibiotics leads scientists to discover new antibacterial drugs. Ninety samples of wood-colonizing fungi were cultivated on agar plates, and their extracts tested for antibacterial activity using the Vibrio fischeri bioluminescence test. Two fungi species, Serpula lacrymans and Nectria vilior, were found to be a potential new source of thermostable antibiotics. Vibrio fischeri bioluminescence test was found to be a useful method for antibacterial activity screening from the samples of natural origin.     

IL-4 induces IL-6 and signs of allergic-type inflammation in the nasal airways of nonallergic individuals

In addition to its more widely recognized role in promoting IgE synthesis, we speculate that interleukin-4 (IL-4) may modulate both allergic- and nonallergic-type inflammatory processes in the airway mucosa. We examined in vivo the effect of IL-4 on granulocyte and cytokine homeostasis in the nasal airways of nonallergic volunteers. Ten (N = 10) healthy subjects received nasal IL-4 (10 microg) or saline (0.9%) challenges on separate occasions. Nasal lavage was obtained before and 24 h after nasal challenges. We report that IL-4 induced a significant increase in IL-6 and produced elevated levels of eosinophils and neutrophils compared to saline. These data demonstrate that IL-4 can modulate both allergic- and nonallergic-type inflammatory responses in the nasal airways of nonallergic individuals.     

Synergistic convergence of microbiota-specific systemic IgG and secretory IgA

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Blunting airway eosinophilic inflammation results in a decreased airway neutrophil response to inhaled LPS in patients with atopic asthma: a role for CD14

Recent data demonstrate that atopic inflammation might enhance airway responses to inhaled LPS in individuals with atopic asthma by increasing CD14 expression on airway macrophages. We sought to determine whether blunting airway eosinophilic inflammation decreases CD14 expression and the subsequent airway polymorphonuclear neutrophil (PMN) response to inhaled LPS in subjects with atopic asthma. Twelve such subjects underwent a 2-week, placebo-controlled trial of inhaled steroid (440 microg fluticasone propionate [FP] twice per day); this was followed 48 hours later by an inhaled LPS (5 microg) challenge. A comparison of LPS-induced inflammatory cells in sputum, CD14 expression, and methacholine responsiveness with FP or placebo was conducted. Flow cytometry was used to analyze membrane-bound CD14 expression (mean fluorescence intensity) on sputum macrophages. We report that 48 hours before inhaled LPS challenge (baseline), FP significantly blunted airway eosinophils (cells per milligram; P =.04) and mCD14 expression (mean fluorescence intensity; P =.03) but did not decrease the number of PMNs (cells per milligram). Six hours after LPS challenge, airway PMNs and mCD14 expression were significantly decreased for FP in comparison with placebo (P =.04). Our data suggest that decreasing airway allergic inflammation with corticosteroids results in both decreased expression of CD14 on airway monocytic cells and a decreased PMN response to inhaled LPS.     

Cytokine induction of heat shock protein in human granulosa-luteal cells

The infiltration of leukocytes is a characteristic feature ofluteolysis in humans. Leukocytes are known to generate physiologicalinducers of cell stress such as cytokines which have been implicatedas mediators of functional luteal regression. In cells exposedto stress, a response characterized by an increase in heat shockprotein (HSP) synthesis occurs. Recently, the induction of HSP-70in rat luteal cells has been shown to inhibit luteinizing hormone(LH) and cAMP-sensitive progesterone production, possibly byinterfering with the translocation of cholesterol to the mitochondrialcytochrome P450_SCC. We therefore investigated whether HSP-70is induced in human granulosa-luteal cells and its relationshipto steroidogenesis. [³⁵S]Methionine labelling showed an increasein a 70 kDa protein after heat treatment which was demonstratedto be HSP-70 by Western analysis using monoclonal antibodiesagainst the constitutive and inducible forms of HSP-70. Inductionof HSP-70 in human granulosa-luteal cells was also seen withinterferon (IFN) (10 ng/ml), tumour necrosis factor (TNF)-     

CD14-dependent airway neutrophil response to inhaled LPS: role of atopy

BACKGROUND: Inhaled endotoxin (LPS) is associated with airway neutrophilic (PMN) inflammation in both asthmatic and control subjects, with asthmatic subjects demonstrating possibly higher sensitivity. CD14 is the principal receptor mediating LPS responses in vivo. It is unknown whether constitutive CD14 can predict the magnitude of the PMN response after LPS inhalation and whether atopy plays a role in this response. OBJECTIVE: We sought to examine associations between constitutive airway CD14 expression and LPS-induced PMNs after 5 microg of LPS inhalation and to examine associations between markers of atopy (eosinophils and eosinophil cationic protein) and CD14 expression and LPS-induced PMNs. METHODS: Ten atopic asthmatic subjects and 8 healthy control subjects inhaled 0.9% saline and LPS (Escherichia coli 026:B6, 5 microg) separated by 3 weeks. Induced sputum was collected at 24 hours before and 6 hours after inhalation. Induced sputum was analyzed for total and differential cell counts and soluble markers (soluble [s]CD14, eosinophil cationic protein, IL8, and total protein). Flow cytometry was used to analyze membrane-bound CD14 expression. RESULTS: Significant associations were found between the LPS-induced PMN response (PMNs per milligram of sputum) and both constitutive sCD14 (R = 0.7, P =.005) and membrane-bound CD14 (R = 0.9, P =.01). Asthmatic subjects demonstrated significantly higher levels of constitutive sCD14 compared with control subjects, and baseline eosinophils were significantly associated with baseline sCD14 (R = 0.7, P =.01) and LPS-induced PMNs (R = 0.6, P =.03). CONCLUSION: Constitutive airway CD14 expression can predict the magnitude of the PMN response after inhaled LPS. Atopy appears to play a role in the level of CD14 expression and may contribute to LPS sensitivity in asthmatic subjects.