Novel approach for efficient resonance tracking in photoacoustic gas sensor systems based on a light-induced wall signal.

Photoacoustic gas sensing is a method suited for the detection of radiation absorbing molecular species in the gas phase. Due to the backgroand-free detection, it has considerable benefits in the measurement of very low concentrations down to the parts-per-trillion range. Yet in resonant systems, the resonance frequency depends on several parameters like temperature or gas composition and therefore must be continuously determined. In the present work, we propose a new method of tracking the resonance frequency using a photoacoustic signal generated at the walls of the resonant cell. The method has been evaluated with two different photoacoustic setups intended for the detection of NO2. We further propose an algorithm for finding the resonance frequency and evaluated the performance thereof. With this method, it is possible to detect the resonance frequency of a cylindrical and a dumbbell-shaped cell in less than two seconds and with an accuracy < 0.06% and < 0.2%, respectively.

Fit-for-Discharge Criteria after Esophagectomy: An International Expert Delphi Consensus.

There are no internationally recognized criteria available to determine preparedness for hospital discharge after esophagectomy. This study aims to achieve international consensus using Delphi methodology. The expert panel consisted of 40 esophageal surgeons spanning 16 countries and 4 continents. During a 3-round, web-based Delphi process, experts voted for discharge criteria using 5-point Likert scales. Data were analyzed using descriptive statistics. Consensus was reached if agreement was ≥75% in round 3. Consensus was achieved for the following basic criteria: nutritional requirements are met by oral intake of at least liquids with optional supplementary nutrition via jejunal feeding tube. The patient should have passed flatus and does not require oxygen during mobilization or at rest. Central venous catheters should be removed. Adequate analgesia at rest and during mobilization is achieved using both oral opioid and non-opioid analgesics. All vital signs should be normal unless abnormal preoperatively. Inflammatory parameters should be trending down and close to normal (leucocyte count ≤12G/l and C-reactive protein ≤80 mg/dl). This multinational Delphi survey represents the first expert-led process for consensus criteria to determine 'fit-for-discharge' status after esophagectomy. Results of this Delphi survey may be applied to clinical outcomes research as an objective measure of short-term recovery. Furthermore, standardized endpoints identified through this process may be used in clinical practice to guide decisions regarding patient discharge and may help to reduce the risk of premature discharge or prolonged admission.

Spleen preserving management of a non-parasitic splenic cyst in pregnancy.

Non-parasitic splenic cysts are rare entities. In pregnancy, they are rarer still, with as few as seven cases reported in the literature. There is little consensus regarding the optimal management of this condition in pregnancy. Although small, the theoretical risk of intrapartum splenic rupture is associated with a fetal mortality rate as high as 70%. The authors of at least three case reports advocate total splenectomy as first-line management of splenic cyst in pregnancy. Paradoxically, spleen conserving surgery is the recognised gold standard treatment for symptomatic splenic cysts in non-pregnant patients. We present a case of a large maternal splenic cyst that was treated successfully with a laparoscopic cystectomy.

Reliability of health-related quality-of-life indicators in cancer survivors from a population-based sample, 2005, BRFSS.

OBJECTIVE: The current emphasis in cancer survivorship research, which includes health-related quality of life (HRQoL), drives the need to monitor the nation's cancer burden. Routine, ongoing public health surveillance tools, such as the Behavioral Risk Factor Surveillance System (BRFSS), may be relevant for this purpose. STUDY DESIGN: A subsample of the 2005 Missouri BRFSS was used to estimate test-retest reliability of HRQoL questions among persons who did and did not report a personal cancer history. METHODS: Retest interviews were conducted by telephone 14-21 days after the initial data collection (n=540, 67% response rate). Reliability was estimated overall and by cancer history using intraclass correlation coefficients (ICCs) and kappa statistics. RESULTS: The majority of retest respondents were White, female and married, with 13% reporting a history of cancer. Overall, point estimates of the reliability coefficients ranged from moderate to excellent (kappa=0.57-0.75). There were no statistically significant differences in test-retest reliability between persons with and without a history of cancer, except for self-reported pain (ICC=0.59 and ICC=0.78, respectively). CONCLUSIONS: In general, BRFSS questions appear to have adequate reliability for monitoring HRQoL in this community-dwelling population, regardless of cancer history.

Can the Expanded Disability Status Scale be assessed by telephone?

Information from patients who are unable to continue their visits to a study centre may be of major importance for the interpretation of results in multiple sclerosis (MS) clinical trials. To validate a questionnaire based on the Expanded Disability Status Scale (EDSS), patients in five different European centres were assessed independently by pairs of trained EDSS raters, first by telephone interview and a few days later by standardized neurological examination. Seventy women and 40 men with an average age of 43.7 years (range 19-74 years) were included in the study. Mean EDSS score at the last visit was 4.5 (0-9). EDSS assessment by telephone was highly correlated with the EDSS determined by physical examination (Pearson's correlation coefficient = 0.95). An intraclass correlation coefficient (ICC) of 94.8% was found for the total sample; 77.6% and 86%, respectively, for patients with EDSS < 4.5 (n = 46) and > 4.5 (n = 64). Kappa values for full agreement were 0.48; for variation by +0.5 steps and +1.0 steps, 0.79 and 0.90, respectively. Best agreement could be found in higher EDSS scores, where assessment by telephone interview might be needed most. The telephone questionnaire is a valid tool to assess EDSS score in cases where the patient is unable to continue visiting a study centre or in long-term follow-up of trial participants.

gammadelta T cells are critical for the induction of anterior chamber-associated immune deviation.

Anterior chamber-associated immune deviation (ACAID) is a systemic form of tolerance that is elicited by introducing antigens into the anterior chamber of the eye. ACAID is characterized by deficiencies in delayed-type hypersensitivity and complement-fixing antibodies upon subsequent challenge with antigen. The mechanisms responsible for the generation of this form of tolerance are not yet completely clear. Here we asked whether gammadelta T cells, which are critical in the induction of oral tolerance and nasal tolerance, play a role in ACAID. The percentage of splenic gammadelta T cells was higher in mice that received antigen via the anterior chamber compared to untreated mice. In addition, CD44 was up-regulated on some splenic gammadelta and alphabeta T cells after the intraocular injection of antigen. Moreover, administration of antigen into the anterior chamber did not induce ACAID in the C57BL/6 mice pretreated with anti-mouse delta-chain monoclonal antibody or in the gammadelta T-cell-receptor-deficient (delta-/-) mice. gammadelta T cells from wild-type mice reconstituted ACAID when transferred into the delta-/- mice before injection of antigen, verifying that the deficiency in delta-/- mice results from the lack of gammadelta T cells rather than from an inadvertent change caused by deletion of the delta-chain. These findings indicate that gammadelta T cells play a very important role in ocular tolerance.

Highly abundant genes in the transcriptosome of human and baboon CD34 antigen-positive bone marrow cells.

Nonhuman primates are useful large animal model systems for the in vivo study of hematopoietic stem cell biology. To better understand the degree of similarity of the hematopoietic systems between humans and baboons, and to explore the relevance of such studies in nonhuman primates to humans, this study was designed to compare the global gene expression profile of bone marrow CD34(+) cells isolated from these 2 species. Human complementary DNA (cDNA) filter arrays containing 25 920 human cDNAs were surveyed for this purpose. The expression pattern and relative gene abundance of the 2 RNA sources were similar, with a correlation coefficient of 0.87. A total of 15 970 of these cDNAs were expressed in human CD34(+) cells, of which the majority (96%) varied less than 3-fold in their relative level of expression between human and baboon. Reverse transcriptase-polymerase chain reaction analysis of selected genes confirmed that expression was comparable between the 2 species. No species-restricted transcripts have been identified, further reinforcing the high degree of similarity between the 2 populations. A subset of 1554 cDNAs, which are expressed at levels 100-fold and greater than background, is described, which includes 959 expressed sequence tags and uncharacterized cDNAs, and 595 named genes, including many that are clearly involved in hematopoiesis. The cDNAs reported here represent a selection of some of the most highly abundant genes in hematopoietic cells and provide a starting point to develop a profile of the transcriptosome of CD34(+) cells.

Herpes simplex virus-induced keratitis: evaluation of the role of molecular mimicry in lesion pathogenesis.

Viruses are suspected but usually unproven triggering factors in autoimmunity. One favored mechanism to explain the role of viruses in the genesis of autoimmunity is molecular mimicry. An immunoinflammatory blinding lesion called herpetic stromal keratitis (HSK) that follows ocular infection with herpes simplex virus (HSV) is suggested to result from a CD4(+) T-cell response to a UL6 peptide of HSV that cross-reacts with a corneal autopeptide shared with the immunoglobulin G2a(b) (IgG2a(b)) isotype. The present report reevaluates the molecular mimicry hypothesis to explain HSK pathogenesis. Our results failed to reveal cross-reactivity between the UL6 and IgG2a(b) peptides or between peptide reactive T cells and HSV antigens. More importantly, animals infected with HSV failed to develop responses that reacted with either peptide, and infection with a recombinant vaccinia UL6 vector failed to cause HSK, in spite of generating UL6 reactivity. Other lines of evidence also failed to support the molecular mimicry hypothesis, such as the failure to affect HSK severity upon tolerization of susceptible BALB/c and B-cell-deficient mice with IgG2a(b) or UL6 peptides. An additional study system revealed that HSK could be induced in mouse strains, such as the OT2 x RAG1(-/-) mice (T cell receptor transgenic recognizing OVA(323-339)) that were unable to produce CD4(+) T-cell responses to any detectable HSV antigens. Our results cast doubt on the molecular mimicry hypothesis as an explanation for the pathogenesis of HSK and indicate that if autoimmunity is involved its likely proceeds via a bystander activation mechanism.

Peptide affinity for MHC influences the phenotype of CD8(+) T cells primed in vivo.

Priming C57BL/6 mice with dominant antigenic peptides of ovalbumin (OVA) or bovine insulin (INS) in complete Freund's adjuvant generates antigen-specific, H-2K(b)-restricted, CD8(+) CTL. OVA-CTL produced type 1 cytokines IFN-gamma and TNF-alpha, whereas INS-CTL produced IL-5 and IL-10 with low levels of IL-4 and IFN-gamma. Here, we investigate whether differential binding affinities of the OVA and INS peptides to H-2K(b) influence the phenotype of the CD8(+) CTL. OVA(257-264) was found to have significantly higher binding affinity than the INS A-chain(12-21) toward K(b). Exchanging the MHC anchor residues between the OVA and INS peptides reversed the K(b) binding capacity of the altered peptides. The lower affinity, altered OVA peptides induced CTL that produced IL-5 and IL-10 in addition to IFN-gamma, whereas high binding affinity, altered INS peptides induced CTL that produced IFN-gamma but not IL-5 or IL-10. These data suggest that MHC binding affinity of peptides can regulate the phenotype of the resulting CD8(+) T cells.

Noscapine inhibits tumor growth with little toxicity to normal tissues or inhibition of immune responses.

Noscapine, a phthalideisoquinoline alkaloid derived from opium, has been used as an oral anti-tussive agent and has shown very few toxic effects in animals or humans. Recently, we reported that noscapine binds stoichiometrically to tubulin and promotes microtubule polymerization. Noscapine causes growth arrest of tumor cells in mitosis and induces apoptosis of tumor cells in vitro. Previous experiments also showed that noscapine has potent antitumor activity in mice when administered parenterally or by gastric lavage. Here, we report that the anti-mitotic effect was specific to noscapine since closely related compounds did not inhibit the growth of a lymphoma cell line. In addition, noscapine was shown to be effective in reducing the growth of the lymphoma and increasing the survival of tumor-bearing mice when administered in the drinking water. It is noteworthy that, noscapine showed little or no toxicity to kidney, liver, heart, bone marrow, spleen or small intestine at tumor-suppressive doses. Furthermore, oral noscapine did not inhibit primary immune responses, which are critically dependent upon proliferation of lymphoid cells. Thus, our results indicate that noscapine has the potential to be an effective chemotherapeutic agent for the treatment of human cancer.

Antigenic epitopes regulate the phenotype of CD8+ CTL primed by exogenous antigens.

We previously reported that insulin-specific, MHC class I-restricted CTL precursors can be primed by injecting C57BL/6 mice with bovine insulin in CFA. These bovine insulin-primed CTL displayed a type 0 CTL phenotype, producing IL-4, IL-5, IL-10, low levels of IFN-gamma, but no TNF-alpha. By contrast, CTL generated from C57BL/6 mice primed with OVA in CFA produced IFN-gamma and TNF-alpha but no IL-4, IL-5, or IL-10 and therefore were classified as type 1 CTL. Although CD4+ T cell subsets have been compared extensively in the literature, CTL subsets are less well characterized. Here, the phenotype, function, and requirements for the in vivo activation of type 1 and type 0 CTL cells were studied. Although both types of CTL express many of the same cell-surface Ags, OVA-specific CTL but not bovine insulin-primed CTL expressed CT-1, a carbohydrate epitope of CD45, and bovine insulin-primed CTL but not OVA-specific CTL expressed Fas constitutively. Priming of CTL was abrogated by depletion of phagocytic cells but not CD4+ T cells, whereas depletion of CD4+ T cells but not phagocytic cells inhibited Ab responses in the same mice. Neither endogenous IL-4 nor the dose of priming Ag altered the CTL phenotypes, but the antigenic peptides of OVA and bovine insulin were key to determining the differentiation of either type 1 or type 0 CTL. To our knowledge, this is the first time that antigenic epitopes have been demonstrated to influence the phenotype of Ag-specific CTL responses. These results may be relevant to the development of peptide vaccines in which a particular type of CTL response is desired.

Bovine and human insulin activate CD8+-autoreactive CTL expressing both type 1 and type 2 cytokines in C57BL/6 mice.

CD8+ T cells down-regulate a variety of immune responses. For example, porcine and human insulin do not stimulate Abs in C57BL/6 mice because CD8+ T cells inhibit CD4+ helper T cells. By contrast, bovine insulin induces Ab in C57BL/6 mice, and removal of CD8+ T cells does not alter this response. This raises the question of whether porcine, but not bovine, insulin activates CD8+ T cells or whether both insulins activate CD8+ T cells but CD4+ helper T cells are differentially inhibited by them. In this study, we show that insulin-specific CD8+ CTL can be cultured from C57BL/6 mice primed with either bovine or human insulin in CFA. Thus, exogenous Ags, besides OVA, induce CD8+ CTL when administered in an adjuvant, suggesting this is a typical response. These CTL are H-2Kb restricted and produce IL-5, IL-10, IFN-gamma, and small amounts of IL-4, which is distinct from IFN-gamma and TNF-alpha that are typically secreted by virus-specific CTL. Moreover, the CTL primed with either bovine or human insulin recognize an A-chain peptide that is identical to the mouse insulin sequence. That foreign proteins, which are closely related to self-proteins, activated autoreactive, CD8+ T cells in vivo is a novel finding. It raises the possibility that self-reactive CTL may be activated by cross-reacting Ags and once activated they might participate in autoimmunity. These results also suggest that down-regulation of insulin-specific responses by autoreactive CD8+ T cells is most likely due to the differential sensitivity of bovine and human insulin-specific CD4+ T cells.

Bovine insulin and porcine or human insulin prime distinct CD4(+) T cell subsets in C57BL/6 mice.

H-2(b) mice produce insulin-specific antibody when injected with bovine but not porcine or human insulin. Nevertheless, CD4(+) T cells have been cloned from C57BL/6 mice primed with porcine, human, and bovine insulin. Here we tested the hypothesis that CD4(+) T cells from C57BL/6 mice primed with porcine or human insulin are functionally distinct from those primed with bovine insulin. Our results show that variants of insulin that stimulate antibody responses induced Th2 clones, whereas variants of insulin that fail to stimulate antibody induced Th0 clones. Th0 clones triggered delayed-type hypersensitivity (DTH) in adoptive recipients, whereas Th2 clones did not. Insulin variants that primed Th0 clones also directly primed for DTH responses, while variants that activated Th2 clones did not. Thus, induction of Th2 clones correlated with the ability of mice to make antibody responses to insulin while development of Th0 clones correlated with DTH responses and the failure to produce antibody.

Evaluation of graft-host response for various tissue sources and animal models.

The efficacy of pancreatic islet transplants in correcting hyperglycemia and slowing the progression of complications in diabetics has been confirmed by many experimental and clinical studies. Unfortunately, the availability of human islets is extremely limited and, therefore, treatment of large numbers of human diabetic patients will almost certainly require either the use of islets harvested from animals (xenografts) or the use of insulin-secreting genetically modified cells of either human or animal origin. There is currently no effective regimen which will allow long-term survival of xenogeneic islets from widely unrelated donor-recipient combinations, such as pig-to-rodent, pig-to-dog, or pig-to-primate. There is considerable interest in the development of immunoisolation techniques for protection of donor islets. However, most materials used in immunoisolation devices are relatively bio-incompatible. Poly-L-lysine-alginate microcapsules are biocompatible and provide an optimal geometry for transmembrane diffusion of insulin and nutrients. Microcapsules allow long-term survival of xenogeneic islets in diabetic rodents or dogs with induced diabetes. However, mice and rats with spontaneous diabetes destroy encapsulated islet grafts within 2 to 3 weeks. Biopsies reveal large numbers of macrophages, immunoglobulins and limited numbers of helper and cytotoxic T-cells in the peri-microcapsule environment of the peritoneal cavity. Cytokines have been identified in peritoneal fluid from mice with islet grafts and may play a role in encapsulated islet destruction. Targeted immunomodulation by treatment of recipients with either anti-helper T-cell antibodies, or fusion proteins which block costimulatory interactions between antigen presenting cells and host T-cells have demonstrated synergy in significant prolongation of encapsulated islet xenograft survival in NOD mice with spontaneous diabetes. Technical improvements in microcapsule design also have contributed to prolonged graft survival. "Double-wall" microencapsulation provides a more durable microcapsule and islet pretreatment prior to encapsulation reduces the frequency of defective capsules with islets entrapped in the membrane. Long-term durability of encapsulated islet grafts remains a concern and further improvements in microcapsule design are a prerequisite to clinical trials.

Ecto-ATPase: an activation marker necessary for effector cell function.

Ecto-ATPase, a transmembrane enzyme that catalyzes the hydrolysis of extracellular ATP (ATPe) to ADP and inorganic phosphate, is expressed upon cell activation. Ecto-ATPase is inhibited by non-hydrolyzable ATP analogues, which are competitive inhibitors of the catalytic reaction, and the ATP analogue affinity label. 5'-p-(fluorosulfonyl)benzoyl adenosine (5'-FSBA), which irreversibly inhibits the catalytic activity. These nucleotide antagonists do not cross the cell membrane and are specific for ecto-ATPase in T cells, B cells and NK cells. Inhibition of ecto-ATPase by both reversible and irreversible nucleotide antagonists results in the inhibition of antigen-induced cytokine secretion and cytolytic activity of T cells. Likewise, granule release and cytolytic activity of NK cells as well as antibody secretion and spontaneous proliferation by B-cell hybridomas are inhibited. Inhibition of ecto-ATPase does not influence effector cell-target cell conjugate formation, but acts, in part, by regulating the influx of extracellular calcium that is necessary to maintain cellular activation. Thus, further elucidation of ecto-ATPase regulation and expression and its interaction with intracellular signal transduction events will provide a basis for understanding the role of the hydrolysis of ATPe by ecto-ATPase in lymphocyte effector function.

Opium alkaloid noscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells.

An alkaloid from opium, noscapine, is used as an antitussive drug and has low toxicity in humans and mice. We show that noscapine binds stoichiometrically to tubulin, alters its conformation, affects microtubule assembly, and arrests mammalian cells in mitosis. Furthermore, noscapine causes apoptosis in many cell types and has potent antitumor activity against solid murine lymphoid tumors (even when the drug was administered orally) and against human breast and bladder tumors implanted in nude mice. Because noscapine is water-soluble and absorbed after oral administration, its chemotherapeutic potential in human cancer merits thorough evaluation.

Antigen is required for the activation of effector activities, whereas interleukin 2 Is required for the maintenance of memory in ovalbumin-specific, CD8+ cytotoxic T lymphocytes.

The mechanisms that maintain memory in T cells are not completely understood. We have investigated the role of antigen and interleukin (IL)-2 in the growth and maintenance of CD8+ T cells using a cytolytic T cell line specific for ovalbumin (OVA)257-264 presented by H-2Kb. This line does not secrete IL-4 or IL-2; hence, stimulation with the OVA-transfected EL4 line (E.G7-OVA) does not induce proliferation without addition of exogenous growth factors. Furthermore, this line can be maintained continuously by weekly addition of irradiated, splenic filler cells and IL-2, with or without E.G7-OVA. Although IL-2 induced proliferation of these cytotoxic T lymphocytes (CTLs), production of interferon gamma and tumor necrosis factor alpha required stimulation of the CTL with E. G7-OVA. The kinetics of lymphokine secretion after stimulation by E. G7-OVA were the same whether the CTL had been maintained with or without antigen (Ag). In addition, both CTL lines killed E.G7-OVA target cells within 4 h. Thus, the effector functions of these CTLs were rapidly induced by T cell receptor (TCR) occupancy. CTLs cultured with or without Ag also served as memory T cells when parked for 100 d in unirradiated, syngeneic recipients without OVA. In the absence of OVA, the precursor frequency was identical in spleens of normal and beta2-microglobulin knockout recipients, but significantly less in IL-2 knockout mice. The decline of memory in the absence of IL-2 supports data from other investigators, suggesting that cell cycling is important to the maintenance of CD8+ T cell memory. These data also suggest that stimulation of OVA-specific CTLs by lymphokines seems to be more important to maintaining memory than stimulation of TCRs by cross-reactive peptides complexed to class I molecules.