Evidence of malignant hyperthermia in patients administered triggering agents before malignant hyperthermia susceptibility identified: missed opportunities prior to diagnosis.

Malignant hyperthermia (MH) is a hypermetabolic disorder of skeletal muscle triggered almost exclusively by potent inhalational agents and suxamethonium. Signs of an MH reaction are non-specific and may be confused with the presentation of other problems such as sepsis and overheating of a patient. A high index of suspicion is needed to be aware of an early presentation of MH. Nine patients are presented who showed abnormal signs with an earlier anaesthetic where the possible diagnosis of an MH reaction was missed. These patients either presented later with an MH reaction, confirmed by DNA analysis and in some cases in vitro contracture testing, or were diagnosed by the identification of a causative mutation confirming MH susceptibility. The MH clinical grading scale is helpful in determining the likelihood that clinical indicators indicate a possible MH reaction. Masseter muscle rigidity is a known sign of MH, confirmed in this report by positive in vitro contracture testing and DNA analysis. Several uncommon muscle disorders have a high association with MH, and postoperative myalgia unrelated to suxamethonium can be a sign which is associated with MH. These reports emphasise the importance of a thorough family history (as the MH status was known by the family in four patients), a high index of suspicion for MH, and documentation of the possibility of MH susceptibility in the anaesthesia record.

High altitude hypoxia, a mask and a Street. Donation of an aviation BLB oxygen mask apparatus from World War 2.

The history of hypoxia prevention is closely inter-related with high altitude mountain and aviation physiology. One pioneering attempt to overcome low inspired oxygen partial pressures in aviation was the BLB mask-named after the three designers-Walter M Boothby, W Randolph Lovelace II and Arthur H Bulbulian. This mask and its variations originated just prior to World War 2 when aircraft were able to fly higher than 10,000 feet and pilot hypoxia affecting performance was an increasing problem. We give a brief description of the mask and its designers and discuss the donation of a model used by the British War Office in October 1940 and donated to the Harry Daly Museum at the Australian Society of Anaesthetists by the family of Dr Fred Street. Dr Street was a pioneering paediatric surgeon in Australia and served as a doctor in the Middle East and New Guinea in World War 2. He received the Military Cross.

Cancer dormancy. VII. A regulatory role for CD8+ T cells and IFN-gamma in establishing and maintaining the tumor-dormant state.

Dormant tumor cells resistant to ablative cancer therapy represent a significant clinical obstacle due to later relapse. Experimentally, the murine B cell lymphoma (BCL1) is used as a model of tumor dormancy in mice vaccinated with the BCL1 Ig. Here, we used this model to explore the cellular mechanisms underlying dormancy. Our previous studies have demonstrated that T cell-mediated immunity is an important component in the regulation of tumor dormancy because Id-immune T cells adoptively transferred into passively immunized SCID mice challenged with BCL1 cells significantly increased the incidence and duration of the dormant state. We have extended these observations and demonstrate that CD8+, but not CD4+, T cells are required for the maintenance of dormancy in BCL1 Ig-immunized BALB/c mice. In parallel studies, the transfer of Id-immune CD8+ cells, but not Id-immune CD4+ cells, conferred significant protection to SCID mice passively immunized with nonprotective levels of polyclonal anti-Id and then challenged with BCL1 cells. Furthermore, the ability of CD8+ T cells to induce a state of dormancy in passively immunized SCID mice was completely abrogated by treatment with neutralizing alpha-IFN-gamma mAbs in vivo. In vitro studies demonstrated that IFN-gamma alone or in combination with reagents to cross-link the surface Ig induced both cell cycle arrest and apoptosis in a BCL1 cell line. Collectively, these data demonstrate a role for CD8+ T cells via endogenous production of IFN-gamma in collaboration with humoral immunity to both induce and maintain a state of tumor dormancy.

IL-5 is required for eosinophil recruitment, crystal deposition, and mononuclear cell recruitment during a pulmonary Cryptococcus neoformans infection in genetically susceptible mice (C57BL/6).

CBA/J (highly resistant), BALB/c (moderately resistant), and C57BL/6 (susceptible) mice displayed three resistance patterns following intratracheal inoculation of Cryptococcus neoformans 52. The inability to clear the infection correlated with the duration of the eosinophil infiltrate in the lungs. The role of IL-5 in promoting the pulmonary eosinophilia and subsequent inflammatory damage in susceptible C57BL/6 mice was investigated. C57BL/6 mice developed a chronic alveolar, peribronchiolar, and perivascular eosinophilia following C. neoformans infection. This resulted in the accumulation of intracellular Charcot-Leyden-like crystals in alveolar macrophages by wk 4 and the extracellular deposition of these crystals in the bronchioles with associated destruction of airway epithelium by wk 6. IL-5 mRNA was expressed in the lungs, and injections of anti-IL-5 mAb prevented eosinophil recruitment and crystal deposition but did not alter cryptococcal clearance. Depletion of CD4+ T cells (but not CD8+) ablated IL-5 production by lung leukocytes in vitro and eosinophil recruitment in vivo. Neutralization of IL-5 also inhibited the recruitment of macrophages, CD8+ T lymphocytes, and B lymphocytes by 47 to 57%. Anti-IL-5 mAb inhibited CD4+ T lymphocyte recruitment by 30% but did not affect neutrophil recruitment. Thus, the development of a chronic eosinophil infiltrate in the lungs of C. neoformans-infected C57BL/6 mice is a nonprotective immune response that causes significant lung pathology. Furthermore, IL-5 promotes the recruitment and activation of eosinophils, resulting in the recruitment of additional macrophages and lymphocytes into the lungs.

IL-12 and IFN-gamma are required for initiating the protective Th1 response to pulmonary cryptococcosis in resistant C.B-17 mice.

A murine model was used to assess the role of cytokines in initiating protective T-cell-mediated immunity in the lung. A pulmonary infection was initiated by intratracheal inoculation of Cryptococcus neoformans (Cne). Previously, we had established that Cne lung clearance was mouse-strain-specific: C.B-17 mice were resistant and developed a Th1-like response, whereas C57BL/6 mice were susceptible and did not develop a Th1 response. In the present study we showed that monoclonal anti-interferon-gamma (IFN-gamma) and anti-interleukin-12 (IL-12) antibody administration prior to infection of resistant C.B-17 mice inhibited lung clearance of Cne. Cytokine profiles of lung and lung-associated lymph nodes (LALN) from monoclonal antibody (mAb)-treated C.B-17 mice were switched from Th1-like to Th2-like, and mAb-treated C.B-17 mice exhibited lung eosinophilia, which was absent in control C.B-17 mice. Additionally, C.B-17 mice treated with anti-IFN-gamma and anti-IL-12 mAb demonstrated a significantly lower percentage of lung macrophages expressing inducible nitric oxide synthase (iNOS) than did control mice. These studies clearly demonstrate that both IFN-gamma and IL-12 are required for initiation of a Th1 response in resistant C.B-17 mice.

Tumor dormancy and cell signaling. V. Regrowth of the BCL1 tumor after dormancy is established.

The majority of BALB/c mice immunized with the BCL1 lymphoma-derived idiotype (Id+) IgM and subsequently challenged with BCL1 tumor cells develop a state of tumor dormancy. The vast majority of dormant lymphoma cells are in cell cycle arrest, but there are also residual replicating cells. In the present studies, we attempted to define features of both the dormant lymphoma cells and the host that lead to escape from dormancy. Escape from dormancy occurs at a steady rate over a 2-year period, suggesting that it is a stochastic process. We found that, in the majority of mice, escape was due to the emergence of genetic variants that were no longer susceptible to the anti-Id-mediated induction of dormancy. Ten percent of these variants were Id-; the remainder were Id+ but could grow in the presence of anti-Id antibodies, suggesting that there were mutations in molecules involved in one or more mIg-mediated negative-signaling pathways. In two of five such escapees, alterations in either Syk, HS1, and/or Lyn were observed. In a small percentage of mice, a low titer of circulating anti-Id antibody before tumor challenge correlated with a subsequent, more rapid loss of dormancy.

Differential expression of the beta2-adrenergic receptor by Th1 and Th2 clones: implications for cytokine production and B cell help.

An important function of the sympathetic nervous system is to maintain homeostasis by modulating the level of cellular activity in many diverse organ systems. The sympathetic neurotransmitter norepinephrine modulates the level of T and B lymphocyte activity by binding to the beta2-adrenergic receptor (beta2AR). The present study was designed to elucidate the mechanism by which stimulation of the beta2AR affects both Th1/Th2 cell cytokine production and Th1/Th2 cell-dependent Ab production. Clones of murine Th1/Th2 cells were exposed to the beta2AR agonist terbutaline before activation by Ag-presenting B cells. Terbutaline exposure of Th1 cells before activation inhibited IFN-gamma production by Th1 cells and subsequent IgG2a production by B cells. IgG2a inhibition was prevented by addition of the betaAR antagonist nadolol or exogenous IFN-gamma. In contrast to Th1 cells, terbutaline did not affect either IL-4 production by Th2 cells or subsequent IgG1 production by B cells. Although baseline levels of intracellular cAMP were similar in both subsets, terbutaline induced an increase in cAMP levels in Th1 cells only. Radioligand binding studies showed that a detectable number of beta2AR binding sites were present on Th1 cells, but not on Th2 cells. Immunofluorescence analyses showed that Th1 cells expressed a higher level of the beta2AR cytoplasmic carboxyl terminus than did Th2 cells. These results show that expression of the beta2AR binding site by Th1 cells, but not by Th2 cells, establishes a physiologic mechanism for selective modulation of Th1 cell IFN-gamma production and IFN-gamma-dependent IgG2a production, provided that beta2AR stimulation occurs before cell activation by a B cell.

The production of soluble interleukin 4 receptors is preferentially regulated by the murine Th2 cell subset.

In order to understand how the endogenous production of soluble IL-4 receptors (sIL-4r) is regulated, the authors tested prototypic clones of Th1 and Th2 murine CD4+ T cell subsets for their ability to regulate their expression of sIL-4r. Results showed that although both types of clones produced low levels of sIL-4r under resting conditions, only the Th2 clones upregulated sIL-4r expression following antigenic stimulation. Inhibition of endogenous IL-4 with a neutralizing anti-IL-4 mAb had only a minor (approximately 20%) inhibitory effect on sIL-4r production by the Th2 cells, and addition of rIL-4 to Th1 cells resulted only in a modest two-fold increase in sIL-4r levels, suggesting that IL-4 is not the only factor that regulates sIL-4r production and that the ability of Th2 clones to upregulate sIL-4r expression can be relatively independent of IL-4. Indeed, the production of sIL-4r by Th2 cells was found to be regulated by cell contact and/or IL-1 mediated signals. Transcripts for both sIL-4r and mIL-4r were detected by RT-PCR on both resting and activated Th1 and Th2 cells, with the relative levels of expression being moderately higher in the Th2 clones. Moreover, the expression of sIL-4r-specific transcripts appeared to increase to a greater extent than those of mIL-4r after activation of Th2 cells with APCs, both in the presence and absence of antigen. Taken together, these results predict that increased sIL-4r production in vivo might be preferentially associated with Th2-type responses and indicate that even though the production of IL-4 and sIL-4r is mediated by the same cells (i.e. Th2 cells), the synthesis of sIL-4r can be regulated independently from that of IL-4 through alternative signals such as cell contact and/or IL-1. These properties may allow for changing ratios of sIL-4r to IL-4 and sIL-4r to mIL-4r during different phases of an immune response and are consistent with a regulatory role for sIL-4r on IL-4 activity in vivo.

In a vaccine model, selected substitution of a highly stimulatory T cell epitope of hen's egg lysozyme into a Salmonella flagellin does not result in a homologous, specific, cellular immune response and may alter the way in which the total antigen is processed.

A 13 amino acid peptide corresponding to a potent BALB/c mouse T cell epitope of hen's egg lysozyme (HEL) was substituted singly at five sites in the d flagellin of Salmonella muenchen. The resulting chimeric proteins were unable to expand T cells capable of being stimulated by the HEL epitope and induced T cell populations which either failed to respond or responded at a low level to a normally highly stimulatory flagellin T cell epitope that was present in all chimeras. The results suggested that substitution of a T cell epitope in flagellin may alter the processing of the resulting immunogen.

Memory T cell development in the absence of specific antigen priming.

Numerous studies have shown that memory T cell development is Ag dependent and specific. In the present study, we show that memory responses can be made against an Ag to which there has been no prior exposure. In unimmunized DO11.10 mice, which carry alpha and beta transgenes that encode a TCR specific for OVA, CD45RB(low) memory cells express the transgenic TCR. These cells can be stimulated by OVA to proliferate and perform typical memory functions, such as secrete diverse lymphokines and provide cognate help to B cells, despite the fact that the mice were never exposed to OVA. Thus, memory cells can be generated in the absence of specific Ag. The data also demonstrate that the transgenic TCR-bearing memory T cells possess endogenous TCR alpha-chains, which permit the expression of a second TCR. In DO11.10/RAG(-/-) mice, the endogenous alpha-chains are eliminated, and the T cells can only express the transgenic TCR. In these mice, no memory cells were observed. Thus, it is the additional TCR that appears to drive memory cell generation. Once induced, memory function may be triggered through the transgenic receptor. Since dual TCR-bearing cells have been shown to exist in nontransgenic mice and humans, our results provide evidence that one mechanism for the maintenance of memory responses to a specific Ag is through stimulation of the second TCR by another Ag. Further, these findings have important implications for understanding aberrant immune responses, such as those that occur in autoimmunity.

Cytokine-mediated communication between dendritic epidermal T cells and Langerhans cells. In vitro studies using cell lines.

Murine epidermis contains two leukocyte populations: Langerhans cells (LC), which are APC of dendritic cell (DC) lineage, and dendritic epidermal T cells (DETC), which are members of the tissue-type gamma delta T cell family. Despite close physical approximation in vivo, the extent to which LC and DETC affect each other's function has remained unknown. We addressed this question using the long term DC line XS52 and the gamma delta T cell line 7-17, both of which were established from mouse epidermis, and both of which retain important features of the resident populations from which they were derived. XS52 DC proliferated maximally when cocultured with gamma-irradiated 7-17 DETC. They also proliferated in response to culture supernatants collected from anti-CD3- or Con A-activated 7-17 DETC, but not from nonstimulated DETC. In both systems, DETC-induced XS52 DC growth was inhibited partially (up to 70%) by Abs against granulocyte/macrophage CSF (GM-CSF) or CD115 (CSF-1 receptor) and nearly completely (up to 90%) by both together. Among 28 tested cytokines, only GM-CSF, CSF-1, IL-4, and IL-13 promoted XS52 DC growth significantly. Anti-IL-4 failed to inhibit DETC-induced XS52 cell growth, and IL-4 was not detectable in DETC supernatants. Thus, we conclude that GM-CSF and CSF-1 (and perhaps IL-13) account for the DC growth-promoting activity secreted by DETC. These results suggest that during coculture, XS52 DC activate 7-17 DETC to secrete both GM-CSF and CSF-1. In fact, when cultured with XS52 DC, 7-17 DETC also elevated their expression of the gamma c receptor and acquired proliferative responsiveness to their own growth factor IL-15. We propose that LC and DETC in situ may interact with each other in a similar manner, thereby regulating their residence and function.

Control of the production of soluble interleukin-4 receptors: implications in immunoregulation.

Soluble cytokine receptors (sCR) are generated in vivo through proteolytic cleavage of the membrane-bound receptors or by direct translation of mRNAs specifically encoding the soluble forms. Despite their widespread presence in biological fluids, the physiological role of endogenous sCR as immunoregulatory molecules is not yet well understood. In vivo, exogenous soluble interleukin-4 receptors (sIL-4R) have been shown to have both agonistic and antagonistic effects on IL-4 responses, depending on the relative concentration ratios of sIL-4R to IL-4. In an effort to elucidate the potential role of endogenous sIL-4R in the regulation of IL-4 responses, the mechanisms controlling the production of sIL-4R have been investigated. Although many cell types are able to constitutively produce low levels, production of sIL-4R is significantly up-regulated in vitro by T cell activation and IL-4. The ability of splenic cells to produce sIL-4R and the serum levels of sIL-4R have consistently been found to be increased during immune responses characterized by T cell activation and IL-4 secretion (Th2 responses). In agreement, clones of Th2, but not Th1, cells were found to significantly up-regulate sIL-4R production following antigenic stimulation. However, the production of sIL-4R by Th2 cells appears to be independent from that of IL-4 and can also be induced by cell contact and/or IL-1-dependent pathways. Taken together, these observations suggest that the production of sIL-4R in vivo is closely associated with the secretion of IL-4, and are consistent with the notion that endogenous sIL-4R are involved in the regulation of IL-4 activity during immune responses.

Early cytokine production in pulmonary Cryptococcus neoformans infections distinguishes susceptible and resistant mice.

A murine pulmonary infection model utilizing intratracheal inoculation of Cryptococcus neoformans was used to analyze cytokines produced in response to opportunistic pathogens acquired via the respiratory tract. The specific question asked was whether early cytokine secretion in lung-associated lymph nodes (LALN) would predict whether this organism would be cleared from the lung. Lung colony-forming units (CFU) were analyzed in two strains of mice over 12 wk, and lung clearance was found to be strain dependent. C.B-17 mice reduced their lung CFU burden between day 7 and day 14 of infection, had significantly higher in lung CFU than C.B-17 mice. The capacity of cells from lungs and LALN to secrete cytokines was significantly different between the strains when assessed at day 7 and day 14 after inoculation. When compared with sensitive C57BL/6 mice 7 days after infection, resistant C.B-17 mice demonstrated (1) increased interferon-gamma secretion by LALN cells in vitro in response to media alone, heat-killed cryptococci, and the T cell mitogen concanavalin A and (2) increased interleukin (IL)-2 secretion by both LALN and lung cells in response to concanavalin A. IL-4 and IL-10 were comparable or undetectable in both mouse strains, whereas IL-5 was significantly higher in all lung cell cultures of C57BL/6 mice. Thus, an early regional Th1 immune response in C.B-17 mice correlated with resistance to the organism, whereas the absence of this response in C57BL/6 mice correlated with susceptibility.

Gastro-oesophageal reflux and adverse respiratory events in children under anaesthesia.

The incidence of gastro-oesophageal reflux in children undergoing general anaesthesia has not previously been studied. One-hundred-and-twenty children (ASA Class 1-2) were studied intraoperatively using continuous oesophageal pH monitoring. The incidence of reflux was 2.5% (3 of 120). None of these three patients had an adverse respiratory event. There was no correlation between reflux and adverse respiratory events. Thirteen patients had minor respiratory events without evidence of acid reflux. Gastro-oesophageal reflux does occur in healthy paediatric patients having minor surgery, but was not a significant cause of the adverse respiratory events that occurred in our study.

A synthetic standard DNA construct for use in quantification of murine cytokine mRNA molecules.

A synthetic DNA construct has been developed as a standard molecule whereby murine cytokine mRNA molecules can be quantified by the reverse transcription-polymerase chain reaction (RT-PCR). The construct, designated Cytoquant 1, allows the quantification of murine IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha, TGF-beta, GM-CSF, CD4, CD8, HPRT and beta-actin mRNA levels. This technique is based on the amplification of a transcribed RNA molecule from Cytoquant 1 as an internal standard control in both the RT and PCR reactions. The quantification data from these analyses are expressed in absolute values, i.e. molecules/cell, which allows the data derived from separate experiments to be compared. In this study, mRNAs encoding beta-actin, IL-10, IFN-gamma and GM-CSF have been quantitated in both Th1 and Th2 cell clones with, and without, stimulation. The quantitative analysis data are highly reproducible and cytokine mRNA concentrations are reflective of restricted cytokine secretion patterns. Furthermore, constitutive cytokine mRNA levels are detectable in resting cells, eliminating the need for exogenous stimulation. The high degree of sensitivity and accuracy make this methodology uniquely suited for the study of T-cell subset cytokine expression in both in vivo and in vitro biological models.

Tumor dormancy and cell signaling. II. Antibody as an agonist in inducing dormancy of a B cell lymphoma in SCID mice.

Tumor dormancy can be induced in a murine B cell lymphoma (BCL1) by immunizing BALB/c mice with the tumor immunoglobulin (Ig) before tumor cell challenge. In this report, we have investigated the immunological and cellular mechanisms underlying the induction of dormancy. BCL1 tumor cells were injected into SCID mice passively immunized with antibody against different epitopes on IgM or IgD with or without idiotype (Id)-immune T lymphocytes. Results indicate that antibody to IgM is sufficient to induce a state of dormancy. Antibodies against other cell surface molecules including IgD and CD44 (Pgp1) had no effect on tumor growth. Id-immune T cells by themselves also had no effect on tumor growth in SCID mice. However, simultaneous transfer of anti-Id and Id-immune T cells enhanced both the induction and duration of the dormant state. In vitro studies indicated that antibody to IgM induced apoptosis within several hours and cell cycle arrest by 24 h. Hyper cross-linking increased apoptosis. The Fc gamma RII receptor played little or no role in the negative signaling. Antibodies that did not negatively signal in vitro did not induce dormancy in vivo. The results suggest that anti-IgM plays a decisive role in inducing tumor dormancy to BCL1 by acting as an agonist of IgM-mediated signal transduction pathways.

Lyn tyrosine kinase signals cell cycle arrest but not apoptosis in B-lineage lymphoma cells.

Signal transduction initiated by binding of antibodies to cell surface molecules can have an important impact on the growth of tumor cells. The malignant behavior of the murine lymphoma BCL1 can be suppressed and the neoplastic cells can be induced to enter a dormant state by in vivo ligation of membrane immunoglobulin. Anti-CD19 antibodies can prolong the survival of SCID mice challenged with the human Burkitt lymphoma cell line, Daudi. Here, we show that cross-linking of membrane immunoglobulin on both murine BCL1 and human Daudi cells initiates a cascade of signals leading to the induction of both apoptosis and cell cycle arrest in vitro. Using antisense oligonucleotides, we demonstrate that the immunoglobulin-associated Lyn tyrosine kinase is required for anti-immunoglobulin-mediated cell cycle arrest but is not required for the signal leading to apoptosis. These results define a branch point in the cytosolic signaling pathways mediating cell cycle arrest and apoptosis. In Daudi cells, Lyn is also critical for cell cycle arrest induced by anti-CD19 signaling. Thus, the Lyn tyrosine kinase may be an important mediator of cell cycle arrest in neoplastic B lymphocytes and, perhaps, other cell types.