Is there a Role for Frozen Section Evaluation of Parotid Masses After Preoperative Cytology or Biopsy Diagnosis?

Fine-needle aspiration (FNA) biopsy reliably diagnoses parotid gland lesions preoperatively, whereas intraoperative frozen section (FS) has the additional benefit of assessing surgical margins and refining diagnoses; however, the role of FS in the setting of prior FNA diagnosis is not well established. Our aim was to determine whether FS should still be performed after a prior FNA/ CNB diagnosis. Parotid gland resections from January 2009 to January 2020 were identified; however, only patients who had both FNA and FS constituted our study population. For the purpose of statistical analysis, FNA diagnoses were classified into non-diagnostic (ND), non-neoplastic (NN), benign neoplasm (BN), indeterminate, and malignant. FS diagnoses were classified into benign, indeterminate, or malignant. Resections were dichotomized into benign and malignant and regarded as the gold standard to subsequently calculate diagnostic accuracy of FNA and FS. A total of 167 parotid gland resections were identified, but only 76 patients (45.5%) had both FNA and FS. In 35 cases deemed as benign preoperatively, three (8.6%) were reclassified as malignant on FS. Out of 18 lesions reported as malignant on FNA, four (22.2%) were interpreted as benign on FS, with three of these benign lesions confirmed on permanent slides. In addition, in patients with both FNA and FS, compared to FNA, FS was able to provide a definitive diagnosis in all five ND cases and in 61.1% (11/18) of indeterminate tumors. Intraoperative assessment provided a relative increase of 33.3% in specificity and 38.5% in positive predictive value when compared to preoperative FNA. The addition of FS to FNA was helpful to further refine the diagnoses of parotid gland lesions, which may provide better guidance for surgical intervention.

Adhesion molecules involved in hepoxilin A3-mediated neutrophil transepithelial migration.

A common feature underlying active states of inflammation is the migration of neutrophils (PMNs) from the circulation and across a number of tissue barriers in response to chemoattractant stimuli. Although our group has recently established a discreet role for the PMN chemoattractant, hepoxilin A3 (HXA3) in the process of PMN recruitment, very little is known regarding the interaction of HXA3 with PMNs. To characterize further the event of HXA3-induced PMN transepithelial migration, we sought to determine the adhesion molecules required for migration across different epithelial surfaces (T84 intestinal and A549 airway cells) relative to two well-studied PMN chemoattractants, formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4). Our findings reveal that the adhesion interaction profile of PMN transepithelial migration in response to HXA3 differs from the adhesion interaction profile exhibited by the structurally related eicosanoid LTB4. Furthermore, unique to PMN transepithelial migration induced by gradients of HXA3 was the critical dependency of all four major surface adhesion molecules examined (i.e. CD18, CD47, CD44 and CD55). Our results suggest that the particular chemoattractant gradient imposed, as well as the type of epithelial cell monolayer, each plays a role in determining the adhesion molecules involved in transepithelial migration. Given the complexities of these interactions, our findings are important to consider with respect to adhesion molecules that may be targeted for potential drug development.

Anaesthesia equipment for resource-poor environments.

The design of anaesthesia equipment for use in hospitals in the developing world must take into account the local conditions, particularly whether reliable supplies of compressed oxygen and electricity are available. Designs should ensure that maintenance is feasible locally. International standards should encourage the design of suitable equipment to ensure safe anaesthesia for patients worldwide.

Anaesthesia services in developing countries: defining the problems.

We describe the use of a questionnaire to define the difficulties in providing anaesthesia in Uganda. The results show that 23% of anaesthetists have the facilities to deliver safe anaesthesia to an adult, 13% to deliver safe anaesthesia to a child and 6% to deliver safe anaesthesia for a Caesarean section. The questionnaire identified shortages of personnel, drugs, equipment and training that have not been quantified or accurately described before. The method used provides an easy and effective way to gain essential data for any country or national anaesthesia society wishing to investigate anaesthesia services in its hospitals. Solutions require improvements in local management, finance and logistics, and action to ensure that the importance of anaesthesia within acute sector healthcare is fully recognised. Major investment in terms of personnel and equipment is required to modernise and improve the safety of anaesthesia for patients in Uganda.

Life-threatening pulmonary hypertension and right ventricular failure complicating calcium and phosphate replacement in the intensive care unit.

A 43-year-old man developed septic shock and acute lung injury after surgery to drain an ischiorectal abscess. In the intensive care unit he initially improved but developed severe hypoxaemia, right ventricular failure and pulmonary hypertension 90 min after receiving intravenous calcium gluconate and potassium phosphate, best explained by the formation of a calcium-phosphate precipitant that resulted in aggregate anaphylaxis. His rapid deterioration and lack of response to conventional therapies necessitated support with extracorporeal membrane oxygenation that was life saving. This adverse event has altered local practice regarding calcium and phosphate replacement and has implications for all intensive care units.

Regulation of Salmonella-induced neutrophil transmigration by epithelial ADP-ribosylation factor 6.

Salmonella typhimurium elicits an acute inflammatory response in the host intestinal epithelium, characterized by the movement of polymorphonuclear leukocytes (PMN) across the epithelial monolayer to the intestinal lumen. It was recently shown that SipA, a protein secreted by S. typhimurium, is necessary and sufficient to drive PMN transmigration across model intestinal epithelia (Lee, C. A., Silva, M., Siber, A. M., Kelly, A. J., Galyov, E., and McCormick, B. A. (2000) Proc. Natl. Acad Sci. USA 97, 12283-12288). However, the epithelial factors responsible for this process have not been identified. Here, for the first time, we demonstrate that S. typhimurium-induced PMN transmigration across Madin-Darby canine kidney-polarized monolayers is regulated by the GTPase ARF6. Apically added S. typhimurium promoted the translocation of ARF6 and its exchange factor ARNO to the apical surface. Overexpression of a dominant-negative mutant of ARF6 inhibited Salmonella-induced PMN transmigration, which was due to a reduction in apical release of the PMN chemoattractant PEEC (pathogen-elicited epithelial chemoattractant), without affecting bacterial internalization. Furthermore, ARF6 and its effector phospholipase D (PLD) were both required for bacteria-induced translocation of protein kinase C (PKC) to membranes. These results describe a novel signal transduction pathway, in which Salmonella initiates an ARF6- and PLD-dependent lipid signaling cascade that, in turn, directs activation of PKC, release of PEEC, and subsequent transepithelial PMN movement.

Cadaverine prevents the escape of Shigella flexneri from the phagolysosome: a connection between bacterial dissemination and neutrophil transepithelial signaling.

Shigella flexneri causes bacillary dysentery in humans by invading epithelial cells of the colon, which is characterized by an acute polymorphonuclear leukocyte (PMNL)-rich inflammation. Our recent studies demonstrated that cadaverine, a polyamine, specifically acts to abrogate transepithelial signaling to PMNL induced by S. flexneri. Here, insight is provided into the cellular mechanisms by which cadaverine attenuates the ability of Shigella species to induce PMNL signaling. It was found that cadaverine retards the lysis of the Shigella species-containing vacuole, suggesting that a blockade is established, in which the pathogen is prevented from adequately interacting with the cytoskeleton. Furthermore, an IcsA mutant of S. flexneri that cannot interact with the cytoskeleton and spreads intercellularly fails to induce transmigration of PMNL. Results indicate that cadaverine-induced compartmentalization of Shigella species to the phagolysosome might be a protective response of the host that directly contributes to the diminished ability of PMNL to transmigrate across model intestinal epithelia.

The GTPase Rac1 selectively regulates Salmonella invasion at the apical plasma membrane of polarized epithelial cells.

The bacterial pathogen Salmonella typhimurium colonizes its animal hosts by inducing its internalization into intestinal epithelial cells. This process requires reorganization of the actin cytoskeleton of the apical plasma membrane into elaborate membrane ruffles that engulf the bacteria. Members of the Rho family of small GTPases are critical regulators of actin structure, and in nonpolarized cells, the GTPase Cdc42 has been shown to modulate Salmonella entry. Because the actin architecture of epithelial cells is organized differently from that of nonpolarized cells, we examined the role of two Rho family GTPases, Cdc42 and Rac1, in invasion of polarized monolayers of MDCK cells by S. typhimurium. Surprisingly, we found that endogenous Rac1, but not Cdc42, was activated during bacterial entry at the apical pole, and that this activation required the bacterial effector protein SopE. Furthermore, expression of dominant inhibitory Rac1 but not Cdc42 significantly inhibited apical internalization of Salmonella, indicating that Rac1 activation is integral to the bacterial entry process. In contrast, during basolateral internalization, both Cdc42 and Rac1 were activated; however, neither GTPase was required for entry. These findings, which differ significantly from previous observations in nonpolarized cells, indicate that the host cell signaling pathways activated by bacterial pathogens may vary with cell type, and in epithelial tissues may further differ between plasma membrane domains.

Use of albumin in intensive care units in the United kingdom.

OBJECTIVE: To review the use of intravenous albumin solutions in intensive care units in the United Kingdom. METHODS: A postal questionnaire was sent to the clinical directors of all intensive care units in the United Kingdom (n = 292) asking about their use and indications for intravenous albumin solutions. RESULTS: Responses were received from 261 (89.4%) intensive care units (ICUs). The units were classified as general ICUs (n = 198), paediatric ICUs (n = 22) and combined intensive care/coronary care units (ICU/CCUs) (n = 41). Of the 261 units that replied, 181 (69.3%) reported using intravenous albumin, although the indications varied between units particularly in paediatric intensive care units. The alternatives to albumin also varied between the units. The general ICUs favoured hydroxyethyl starch (n = 129, 65.2%) and/or gelatin solutions (n = 87, 43.9%), as did the combined ICU/CCUs (n = 28, 68.3% and/or n = 23, 56.1% respectively). However, of the paediatric ICUs that used an alternative to albumin solutions (n = 21/22), 12 favoured crystalloid solutions (54.5%) and 9 favoured gelatin solutions (40.9%). We also assessed the impact of the recent review by the Cochrane Injuries Group Reviewers on the use of albumin and found that the respondents of 131 units (50.2%) reported that this study influenced their use of intravenous albumin. Of the 80 units that did not use albumin solutions, 33 units reported that they had ceased using intravenous albumin following the review from the Cochrane Injuries Group Reviewers. CONCLUSIONS: Approximately two-thirds of intensive care units in the United kingdom reported using intravenous albumin, although the indications varied between units. In many of these units the use of intravenous albumin had been influenced by the recent review by the Cochrane Injuries Group Reviewers on the use of albumin.

A secreted Salmonella protein induces a proinflammatory response in epithelial cells, which promotes neutrophil migration.

In response to Salmonella typhimurium, the intestinal epithelium generates an intense inflammatory response consisting largely of polymorphonuclear leukocytes (neutrophils, PMN) migrating toward and ultimately across the epithelial monolayer into the intestinal lumen. It has been shown that bacterial-epithelial cell interactions elicit the production of inflammatory regulators that promote transepithelial PMN migration. Although S. typhimurium can enter intestinal epithelial cells, bacterial internalization is not required for the signaling mechanisms that induce PMN movement. Here, we sought to determine which S. typhimurium factors and intestinal epithelial signaling pathways elicit the production of PMN chemoattractants by enterocytes. Our results suggest that S. typhimurium activates a protein kinase C-dependent signal transduction pathway that orchestrates transepithelial PMN movement. We show that the type III effector protein, SipA, is not only necessary but is sufficient to induce this proinflammatory response in epithelial cells. Our results force us to reconsider the long-held view that Salmonella effector proteins must be directly delivered into host cells from bacterial cells.

Salmonella enterica serovar typhimurium-dependent regulation of inducible nitric oxide synthase expression in macrophages by invasins SipB, SipC, and SipD and effector SopE2.

When Salmonella enterica invades mammalian cells, it activates signals leading to increased expression of inflammatory mediators. One such mediator is nitric oxide (NO), which is produced under control of the enzyme inducible NO synthase (iNOS). Induction of iNOS in response to Salmonella infection has been demonstrated, but the bacterial effector molecules that regulate expression of the enzyme have not been identified. In the study reported here, an analysis of Salmonella-dependent iNOS expression in macrophages was carried out. Wild-type Salmonella strains increased the levels of both iNOS protein and mRNA in murine macrophage cell lines in an invasion-independent fashion. Mutant strains lacking a functional pathogenicity island 1-encoded type III secretion system, as well as strains lacking the invasins SipB, SipC, and SipD, were impaired in iNOS induction. Complementation experiments indicated that all three of the invasins were required for induction of iNOS expression. These results suggested that an effector protein, translocated into macrophages via the type III secretion system in a SipB-, SipC-, and SipD-dependent manner, might be the ultimate mediator of iNOS induction. In keeping with this idea, a mutant strain deficient in SopE2, a recently described homolog of SopE, was found to be impaired in the induction of iNOS expression. These observations suggest that iNOS expression is regulated by signals activated by SopE2 (and possibly SopE) and that the role of SipB, SipC, and SipD in this process is to facilitate translocation of the relevant effector.

A functional cra gene is required for Salmonella enterica serovar typhimurium virulence in BALB/c mice.

A minitransposon mutant of Salmonella enterica serovar Typhimurium SR-11, SR-11 Fad(-), is unable to utilize gluconeogenic substrates as carbon sources and is avirulent and immunogenic when administered perorally to BALB/c mice (M. J. Utley et al., FEMS Microbiol. Lett., 163:129-134, 1998). Here, evidence is presented that the mutation in SR-11 Fad(-) that renders the strain avirulent is in the cra gene, which encodes the Cra protein, a regulator of central carbon metabolism.

The secreted effector protein of Salmonella dublin, SopA, is translocated into eukaryotic cells and influences the induction of enteritis.

Salmonella-induced enteritis is associated with the induction of an acute intestinal inflammatory response and net fluid secretion into the lumen of infected mucosa. Proteins secreted by the Inv/Spa type III secretion system of Salmonella play a key role in the induction of these responses. We have demonstrated recently that the Inv/Spa-secreted SopB and SopD effector proteins are translocated into eukaryotic cells via a Sip-dependent pathway and act in concert to mediate inflammation and fluid secretion in infected ileal mucosa. Mutations of both sopB and sopD significantly reduced, but did not abrogate, the enteropathogenic phenotype. This indicated that other virulence factors are involved in the induction of enteritis. In this work, we characterize SopA, a secreted protein belonging to the family of Sop effectors of Salmonella dublin. We demonstrate that SopA is translocated into eukaryotic cells and provide evidence suggesting that SopA has a role in the induction of enteritis.

Bronchopleural fistula complicating group A beta-haemolytic streptococcal pneumonia. Use of a Fogarty embolectomy catheter for selective bronchial blockade.

A 36-year-old woman developed severe group A Streptococcal pneumonia, complicated by a bronchopleural fistula, ARDS and multi-organ failure. We describe the use of selective middle lobe bronchus blockade, with a Fogarty embolectomy catheter, to localise and control the air leak. This allowed effective mechanical ventilation and oxygenation on intensive care and during right middle lobectomy. The patient made a prolonged, but full recovery.

Orchestration of neutrophil movement by intestinal epithelial cells in response to Salmonella typhimurium can be uncoupled from bacterial internalization.

Intestinal epithelial cells respond to Salmonella typhimurium by internalizing this pathogen and secreting, in a polarized manner, an array of chemokines which direct polymorphonuclear leukocyte (PMN) movement. Notably, interleukin-8 (IL-8) is secreted basolaterally and directs PMN through the lamina propria, whereas pathogen-elicited epithelial chemoattractant (PEEC) is secreted apically and directs PMN migration across the epithelial monolayer to the intestinal lumen. While most studies of S. typhimurium pathogenicity have focused on the mechanism by which this bacterium invades its host, the enteritis characteristically associated with salmonellosis appears to be more directly attributable to the PMN movement that occurs in response to this pathogen. Therefore, we sought to better understand the relationship between S. typhimurium invasion and epithelial promotion of PMN movement. First, we investigated whether S. typhimurium becoming intracellular was necessary or sufficient to induce epithelial promotion of PMN movement. Blocking S. typhimurium invasion by preventing, with cytochalasin D, the epithelial cytoskeletal rearrangements which mediate internalization did not reduce the epithelial promotion of PMN movement. Conversely, bacterial attainment of an intracellular position was not sufficient to induce model epithelia to direct PMN transmigration, since neither basolateral invasion by S. typhimurium nor apical internalization of an invasion-deficient mutant (achieved by inducing membrane ruffling with epidermal growth factor) induced this epithelial cell response. These results indicate that specific interactions between the apical surface of epithelial cells and S. typhimurium, rather than simply bacterial invasion, mediate the epithelial direction of PMN transmigration. To further investigate the means by which S. typhimurium induces epithelia to direct PMN movement, we investigated whether the same signaling pathways regulate secretion of IL-8 and PEEC. IL-8 secretion, but not PEEC secretion, was activated by phorbol myristate acetate and blocked by an inhibitor (mg-132) of the proteosome which mediates NF-kappabeta activation. Further, secretion of IL-8, but not PEEC, was activated by an entry-deficient (HilDelta) S. typhimurium mutant or by basolateral invasion of a wild-type strain. Together, these results indicate that distinct signaling pathways mediate S. typhimurium invasion, induction of IL-8 secretion, and induction of PEEC secretion in model intestinal epithelia.

Inhibition of Shigella flexneri-induced transepithelial migration of polymorphonuclear leucocytes by cadaverine.

Dysentery caused by Shigella species is characterized by infiltration of polymorphonuclear leucocytes (PMNs) into the colonic mucosa. Shigella spp. evolved into pathogens by the acquisition of virulence genes and by the deletion of 'antivirulence' genes detrimental to its pathogenic lifestyle. An example is cadA (encoding lysine decarboxylase), which is uniformly absent in Shigella spp., whereas it is present in nearly all isolates of the closely related non-pathogen Escherichia coli. Here, using monolayers of T84 cells to model the human intestinal epithelium, we determined that the introduction of cadA into S. flexneri and the expression of lysine decarboxylase attenuated the bacteria's ability to induce PMN influx across model intestinal epithelium. Such inhibition was caused by cadaverine generated from the decarboxylation of lysine. Cadaverine treatment of model intestinal epithelia specifically inhibited S. flexneri induction of PMN transepithelial migration, while having no effect on the ability of Salmonella or enteropathogenic E. coli (EPEC) to induce PMN migration. These observations not only provide insight into mechanisms of S. flexneri pathogen evolution and pathogenesis, but also suggest a potential for the use of cadaverine in the treatment of dysentery.

Migration of intestinal intraepithelial lymphocytes into a polarized epithelial monolayer.

Intraepithelial lymphocytes (IEL) are a phenotypically distinct population of lymphocytes that reside in mucosal epithelia, below the intercellular tight junctions. Although adhesive functions of this population have been previously studied, relatively little is known about IEL migration from the microvasculature into the epithelium. We demonstrated that cultured human IEL were capable of migration into polarized epithelial cells in vitro, where they assumed a subjunctional position, identical to that observed in vivo. The migration was rapid and efficient and was directionally polarized, such that IEL migrated into epithelial monolayers from the basolateral, but not the apical, aspect. After a 4-h period of residence, up to one-half of the IEL then exited the monolayer basolaterally. Migration was partially inhibited by pertussis toxin, suggesting a potential mechanism for IEL migration by chemokine receptor-mediated signaling. The conditions and ligand pairs used in IEL migration were different from those for neutrophils, another cell type known to migrate through epithelia. This system may serve as a model for microenvironmental homing of IEL into the epithelium.

Requirement of the Shigella flexneri virulence plasmid in the ability to induce trafficking of neutrophils across polarized monolayers of the intestinal epithelium.

Attachment of an array of enteric pathogens to epithelial surfaces is accompanied by recruitment of polymorphonuclear leukocytes (PMN) across the intestinal epithelium. In this report, we examine how Shigella-intestinal epithelium interactions evoke the mucosal inflammatory response. We modeled these interactions in vitro by using polarized monolayers of the human intestinal epithelial cell line, T84, isolated human PMNs, and Shigella flexneri. We show that Shigella attachment to T84-cell basolateral membranes was a necessary component in the signaling cascade for induction of basolateral-to-apical directed transepithelial PMN migration, the direction of PMN transepithelial migration in vivo. In contrast, attachment of Shigella to the T84-cell apical membrane failed to stimulate a directed PMN transepithelial migration response. Importantly, the ability of Shigella to induce PMN migration across epithelial monolayers was dependent on the presence of the 220-kb virulence plasmid. Moreover, examination of Shigella genes necessary to signal subepithelial neutrophils established the requirement of a functional type III secretion system. Our results indicate that the ability of Shigella to elicit transepithelial signaling to neutrophils from the basolateral membrane of epithelial cells represents a mechanism involved in Shigella-elicited enteritis in humans.