Pleural fluid residue as a diagnostic tool for cytology-negative malignant pleural effusion: A proof-of-concept study.

Background: Pleural fluid residue, or macroscopic tissue, circulating freely in the pleural fluid obtained through direct filtration, may carry diagnostic histopathological information. We aimed to determine the histopathological concordance of pleural fluid residue in diagnosing TPE and MPE, compared with conventional pleural biopsy. This was a prospective cohort study of consecutive inpatients with cytology-negative exudative effusion who underwent pleuroscopy and had their initial suctioned pleural fluid filtered for residue samples. Pleural fluid residue demonstrated malignant cells in four out of seven cases of pleural biopsy-confirmed malignancy. Pleural fluid residue has comparable cytomorphology but reduced cellularity compared with pleural biopsy. No tuberculous histological features were present in the pleural fluid residue samples. In this preliminary study pleural fluid residue provided histopathological information for malignant pleural effusion, but no incremental diagnostic information for tuberculous effusion. However larger and more definitive studies are required to clarify these findings, and to explore the utility and suitability of pleural fluid residue for mutational analysis. What the study adds: This study demonstrates the potential of pleural fluid residue as a non-invasive diagnostic method for confirming malignancy in cytology-negative exudative effusion. What are the implications of the findings: In resource-limited settings or patients contraindicated for pleural biopsy, pleural fluid residue may provide a viable diagnostic alternative; however, this observation needs further validation.

An ectoprotein kinase of group C streptococci binds hyaluronan and regulates capsule formation.

A 56-kDa protein had been isolated and cloned from protoplast membranes of group C streptococci that had erroneously been identified as hyaluronan synthase. The function of this protein was reexamined. When streptococcal membranes were separated on an SDS-polyacrylamide gel and renatured, a 56-kDa protein was detected that had kinase activity for a casein substrate. When this recombinant protein was expressed in Escherichia coli and incubated in the presence of [32P]ATP, it was responsible for phosphorylation of two proteins with 30 and 56 kDa that were not present in the control lysate. The 56-kDa protein was specifically phosphorylated in an immunoprecipitate of a detergent extract of the recombinant E. coli lysate with antibodies against the 56-kDa protein, indicating that it was autophosphorylated. The E. coli lysate containing the recombinant protein could bind hyaluronan, and hyaluronan binding was abolished by the addition of ATP. Kinetic analysis of hyaluronan synthesis and release from isolated protoplast membranes indicated that phosphorylation by ATP stimulated hyaluronan release and synthesis. Incubation of membranes with antibodies to the 56-kDa protein increased hyaluronan release. The addition of [32P]ATP to intact streptococci led to rapid phosphorylation of two proteins, 56 and 75 kDa each at threonine residues. This phosphorylation was neither observed with [32P]phosphate nor in the presence of trypsin, indicating that the kinase was localized extracellularly. The addition of ATP to growing group C streptococci led to increased hyaluronan synthesis and release. However marked differences were found between group A and group C streptococci. Antibodies against the 56-kDa protein from group C streptococci did not recognize proteins from group A strains, and a homologous DNA sequence could not be detected by polymerase chain reaction or Southern blotting. In addition, Group A streptococci did not retain a large hyaluronan capsule like group C strains. These results indicated that the 56-kDa protein is an ectoprotein kinase specific for group C streptococci that regulates hyaluronan capsule shedding by phosphorylation.

The results of vitreous surgery for chronic macular holes.

PURPOSE: To evaluate the visual and anatomic results of macular hole surgery in eyes that have had symptoms of a macular hole for 2 years or longer. METHODS: Fifty-one eyes with chronic macular holes (> or = 2 years' duration) were treated in a retrospective analysis of the results of vitrectomy, 16% perfluoropropane gas tamponade, and one of three adjunctive agents (bovine transforming growth factor beta-2, recombinant transforming growth factor beta-2, or autologous platelet extract). Of 51 eyes, 45 (88.2%) were examined 3 months after surgery. Visual acuity of these 45 eyes was measured preoperatively and 3 months postoperatively using the Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart. Twenty-eight eyes (62.2%) had no prior vitrectomy and 17 eyes (37.8%) had a prior vitrectomy for the macular hole that failed. RESULTS: The macular holes had a mean duration of 3.7 years and were successfully closed in 32 of 51 total eyes (62.7%) and 32 of 45 eyes (71.1%) that were examined 3 months after surgery. The mean preoperative visual acuity was 20/100 -2 and the mean visual acuity at 3 months was 20/80 for a mean gain of 6.6 ETDRS letters (+ 1.3 lines). Of 45 eyes, 17 (37.8%) were 20/63 or better 3 months after surgery; 21 (46.7%) gained 2 or more ETDRS lines. There was no statistically significant difference in macular hole closure (P = 0.311) or visual acuity change (P = 0.095) in eyes with or without prior vitreous surgery. Eyes with macular holes between 2-2.99 years experienced a somewhat better anatomic and functional result overall than eyes with macular hole from 3-14 years, duration, but the visual acuity change was not statistically significant (P = 0.187). There was substantial variability in visual improvement among eyes with successful closure of the chronic macular hole. CONCLUSIONS: Macular holes of > or = 2 years' duration may be more difficult to close successfully than are more recent macular holes, and the visual improvement appears to be less favorable. Many eyes with chronic macular holes in our study gained substantial visual acuity, so vitreous surgery can be considered in selected eyes with chronic macular holes based on visual needs.

Overexpression, one-step purification and characterization of UDP-glucose dehydrogenase and UDP-N-acetylglucosamine pyrophosphorylase.

Two enzymes of the Leloir pathway, UDP-GlcNAc pyrophosphorylase and UDP-Glc dehydrogenase, which are involved in the synthesis of activated sugar nucleotides have been cloned, overexpressed in Escherichia coli, and purified to homogeneity in only one step by chelation-affinity chromatography. The gene KfaC of E. coli K5 was thus demonstrated to encode UDP-Glc DH. Some properties of the cloned enzymes, such as stability, pH dependence, and substrate kinetics, were studied in order to facilitate the use of these enzymes in carbohydrate synthesis, especially in the synthesis of hyaluronic acid.

Molecular mechanisms and genetics of hyaluronan biosynthesis.

Hyaluronan is an extremely important polysaccharide from both the biological and commercial points of view. This review summarizes the present state of the art concerning the polymer and our understanding of the molecular mechanisms of its synthesis with emphasis on the implications of this understanding for polysaccharide engineering of hyaluronan.

Heparin prevents antigen-induced airway hyperresponsiveness: interference with IP3-mediated mast cell degranulation?

We hypothesized that heparin, because of its antiallergic and/or anti-inflammatory properties, modifies airway hyperresponsiveness (AHR). We studied the effects of inhaled heparin on AHR induced by specific antigen or by platelet-activating factor (PAF), a proinflammatory mediator. Specific lung resistance (sRL) was measured in 17 allergic sheep before, immediately after, and serially for up to 2 h after airway challenge with either specific antigen or PAF. Airway responsiveness was expressed as the cumulative provocative dose of carbachol that increased sRL to 4 cmH2O/s [PD4, in breath units (BU; 1 BU = 1 breath of 1% carbachol solution)]. PD4 was determined on a baseline day and on various experimental days 2 h after airway challenge with antigen or PAF, without or after pretreatment with inhaled heparin (1,000 U/kg). Pretreatment with inhaled heparin prevented antigen-induced bronchoconstriction and postantigen AHR. PD4 was 26 +/- 2.6 (SE), 12 +/- 1.7, and 22 +/- 2.8 BU on baseline, antigen control, and postheparin days, respectively. Heparin given immediately after the antigen challenge failed to modify the magnitude and/or duration of antigen-induced bronchoconstrictor response or postantigen AHR. Heparin also failed to prevent PAF-induced changes in sRL and AHR. In vitro heparin inhibited anti-immunoglobin E- and 1,4,5-inositol triphosphate-mediated degranulation of rat peritoneal mast cells without attenuating the effects of the Ca2+ ionophore A-23187. These data suggest that in "acute responders" heparin prevents antigen-induced bronchoconstriction and AHR, possibly by inhibiting 1,4,5-inositol triphosphate-dependent mast cell mediator release and not by its anti-inflammatory action.

The effect of pars plana vitrectomy and transforming growth factor-beta 2 without epiretinal membrane peeling on full-thickness macular holes.

PURPOSE: Surgical techniques for the treatment of macular holes generally include removal of the overlying cortical vitreous and/or epiretinal membranes. The authors demonstrate that by using vitrectomy, posterior hyaloid removal, fluid-gas exchange, and transforming growth factor-beta 2 (TGF-beta 2), a growth factor that modulates the wound healing process, epiretinal membrane peeling can be avoided and the surgical procedure thereby simplified without compromising results. METHODS: A total of 24 eyes of 24 patients with stage 2, 3, or 4 full-thickness macular holes were treated. Of 24 patients, 1 was lost to follow-up after suffering a stroke; the remaining 23 (17 females and 6 males) (age range, 11-81 years; mean, 64 years) were followed for 5 to 16 months (mean, 12 months). Preoperative best-corrected visual acuity ranged from 20/50 to 20/400 (mean, 20/125). A standardized vitrectomy was performed with posterior hyaloid removal and, after a near-complete fluid-air exchange, 0.1 ml of a solution containing 1330 ng of TGF-beta 2 was instilled over the macular hole. No attempts were made to peel epiretinal membranes or drain fluid from the macular hole. RESULTS: Of 23 eyes, 22 (96%) had resolution of the surrounding subretinal fluid and flattening of the macular hole (1 patient required a second procedure, in which visual improvement of 20/30 was achieved); 11 (48%) had visual acuities of 20/40 or better, 19 (85%) had visual acuities of 20/60 or better, and 19 (85%) showed an improvement in visual acuity of at least two lines (mean, 3.8 lines). The authors saw no retinal pigment epithelial mottling. CONCLUSION: The authors' results demonstrate that treatment of macular holes using vitrectomy, fluid-gas exchange, and the instillation of a solution containing TGF-beta 2, without epiretinal membrane peeling, maintains efficacy while simplifying surgery.

Oxygen radicals contribute to antigen-induced airway hyperresponsiveness in conscious sheep.

We previously showed that oxygen radicals can induce airway hyperresponsiveness (AHR) in allergic sheep. The purpose of this study was to determine whether antigen challenge results in the generation of free oxygen radicals and if these radicals contribute to antigen-induced AHR. We first determined baseline airway responsiveness in seven Ascaris suum-sensitive sheep by calculating the cumulative provocative concentration of carbachol in breath units (BU; one BU defined as one breath of a 1% wt/vol carbachol solution) that increased specific lung resistance (SRL) 400% over baseline (PC400). On a different day, the sheep underwent inhalation challenge with A. suum antigen, SRL was measured before and immediately after challenge and then hourly for 2 h, at which time SRL had returned to baseline. The postchallenge PC400 was then measured. This procedure was repeated on separate occasions, each at least 14 days apart, except that the sheep were treated with an aerosol of catalase (CAT; 38 mg in 3 ml deionized water), the enzyme that catalyzes the decomposition of hydrogen peroxide (H2O2), at three different times: Trial 1, before antigen and then every 30 min after antigen challenge for 2 h; Trial II, 1 and 2 h after antigen challenge; and Trial III, only at 2 h after antigen challenge. In the control trial, antigen challenge caused a transient (mean +/- SEM) 303 +/- 48% increase in SRL over baseline (p < 0.05), and 2 h later, PC400 was reduced to 11.0 +/- 1.7 BU from a prechallenge value of 24.8 +/- 1.9 BU (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Hyaluronate synthase: cloning and sequencing of the gene from Streptococcus sp.

The complete nucleotide sequence of hyaluronate synthase from Streptococcus sp. and its flanking regions is presented. The gene locus was designated has. Southern-blotting results suggested that the gene was conserved in hyaluronate-producing streptococci. A putative translation-initiation codon was identified and the open reading frame consists of 1566 bp, specifying a protein of 56 kDa. Sequences resembling the promoter and ribosome-binding site of Gram-positive organisms are found upstream of the synthase. The predicted amino-acid sequence reveals the presence of a 35-residue signal peptide. The sequence has some similarity to bacterial peptide-binding proteins.

Lipid mediators contribute to oxygen-radical-induced airway responses in sheep.

The purpose of this study was to determine if the bronchoconstriction and airway hyperresponsiveness (AHR) resulting from aerosolized xanthine (x; 0.1%)-xanthine oxidase (xo; 4.1 U) and the subsequent production of oxygen radicals is mediated by the secondary generation of lipid mediators. In seven conscious sheep, specific lung resistance (SRL) was measured before and after x-xo challenge; approximately 30 min later when SRL had returned to baseline, airway responsiveness to carbachol was determined from dose-response curves by calculating the cumulative provocating dose of carbachol in breath units (BU, defined as one breath of a 1% wt/vol carbachol solution) that increased SRL 400% over baseline (PD400). Inhaled x-xo caused in immediate increase in SRL of 162 +/- 36% (mean +/- SE; p less than 0.05) over baseline and decreased PD400 from a baseline value of 32.5 +/- 5.0 to 16.6 +/- 1.7 BU (p less than 0.05). Pretreatment with the H2O2 scavenger, catalase (CAT,; 38 mg aerosol), methylprednisolone succinate (MS; 1 mg/kg given intravenously), the cyclooxygenase inhibitor, indomethacin (IND; 2 mg/kg given intravenously), and the PAF antagonist, WEB-2086 (3 mg/kg given intravenously) all attenuated the x-xo-induced increase in SRL (p less than 0.05); the leukotriene D4 antagonist, MK-571 (5 mg by aerosol) had no effect. All agents inhibited the x-xo-induced decrease in PD400: mean BUs were 27 after CAT, 32 after WEB-2086, 34 after IND, 31 after MS, and 25 after MK-571 (all p less than 0.05 versus x-xo alone).(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of fibrosis and transforming growth factor-beta type 2 levels in the eye.

Approximately 1 out of every 10 eyes undergoing surgery for retinal detachment develops excessive intraocular fibrosis that can lead to traction retinal detachment and ultimate blindness. This disease process has been termed proliferative vitreoretinopathy (PVR). The ability to monitor and grade this fibrotic response accurately within the eye as well as the ability to aspirate vitreous cavity fluid bathing the fibrotic tissue makes this an ideal setting in which to investigate the development of fibrosis. Although laboratory studies have recently shown that transforming growth factor-beta (TGF-beta) can enhance fibrosis, little clinical evidence is yet available correlating the level of this or other growth factors with the degree of fibrosis in a clinical setting. We have found that vitreous aspirates from eyes with intraocular fibrosis associated with PVR have more than three times the amount of TGF-beta (1,200 +/- 300 pM [SEM]) found in eyes with uncomplicated retinal detachments without intraocular fibrosis (360 +/- 91 pM [SEM]). Using an in vitro assay, 84-100% of the TGF-beta activity could be blocked with specific antibodies against TGF-beta 2, whereas only 10-21% could be blocked by specific antibodies against TGF-beta 1. TGF-beta 1 was used in an animal model of traction retinal detachment. Since beta 1 and beta 2 have essentially identical biologic effects and only human beta 1 was available in quantities required, beta 1 was chosen for these in vivo studies. The injection of TGF-beta1 plus fibronectin (FN) but not TGF-beta1 alone into the vitreous cavity of rabbits resulted in the increased formation of intraocular fibrosis and traction retinal detachments as compared to control eyes. In previous studies, intravitreal FN levels were also found to be elevated in eyes with intraocular fibrosis.

Transection of subfornical organ neural connections diminishes the pressor response to intravenously infused angiotensin II.

Knife-cut lesions were used to assess the participation of the subfornical organ (SFO) in the central pressor action of intravenously administered angiotensin. Knife-cuts of the ventral stalk of the SFO significantly attenuated pressor responses during infusion of 3 doses of angiotensin, although responses to bolus injections were unaffected. These results are consistent with previous work in implicating the SFO as an important mediator of the central pressor action of circulating angiotensin.

Acute tubular necrosis in hepatorenal syndrome: an electron microscopy study.

This report describes light and transmission electron microscopy (LM and EM, respectively) studies of kidneys from five cases of hepatorenal syndrome. The kidneys were removed and fixed for LM and EM between 30 and 120 min after death. All patients had progressive renal failure after admission to the hospital. All cases were jaundiced, had ascites, and exhibited features of hepatic encephalopathy. LM study revealed severe acute tubular lesions (ATL) or, more conventionally, acute tubular necrosis (ATN). EM study demonstrated necrosis of the proximal tubules characterized by swelling, disorganization of the cristae and appearance of dark bodies in the mitochondria, coalescence, fragmentation or displacement of the microvilli, loss of plasma membranes, rupture of the basement membranes, and separation of the cells from the basement membranes. Rupture of tubular basement membranes (tubulorrhexis) and mitochondrial dark bodies suggest an ATN due to ischemia or induced by vasoconstrictor substance(s). Glomerular lesions were infrequent (one in five) and therefore, do not seem to have contributed to renal failure. All cases terminally had extremely low urinary sodium (11 mEq/liter), high urinary potassium (50 mEq/liter), a remarkably low urinary sodium/potassium ratio (0.26, normal = 4.27), and a low urinary osmolality (less than 400 mOsm/kg). From this study we conclude that an ATN of variable severity may be associated with the hepatorenal syndrome. Since this ATN developed without preceding shock, sepsis, or hypotension it is possible that this ATN like that in ischemic acute renal failure may be due to reduced renal blood flow and intense cortical vasoconstriction which has been reported in hepatorenal syndrome. Finally, our data imply that low urinary sodium is consistent with this pathologic lesion in this clinical setting.

Toxemic renal disease in incomplete molar pregnancy.

This communication describes the light, electron, and immunofluorescence microscopy of renal biopsy specimens from a patient with hydatidiform degeneration of the placenta and coexisting fetus. Symptoms of toxemia appeared in the 18th week of gestation and were accompanied by heavy proteinuria, decreased renal function, and disproportionately elevated serum uric acid concentration. The biopsy findings were consistent with the renal lesions of toxemia of pregnancy, although other renal diseases such as Henoch Schonlein nephritis or lupus proliferative glomerulonephritis cannot be excluded. Normal serological tests and disappearance of proteinuria, along with recovery of renal function to normal promptly after termination of pregnancy, tend to rule out other renal diseases. The molar transformation of the placenta, or the interaction with the kidney of certain humoral factors such as human chorionic gonadotropin which was markedly elevated in this patient, may be related to the pathophysiology of toxemia of pregnancy.