Messenger RNA encoding constitutively active Toll-like receptor 4 enhances effector functions of human T cells.

Adoptive T cell therapy of cancer employs a large number of ex-vivo-propagated T cells which recognize their targets either by virtue of their endogenous T cell receptor (TCR) or via genetic reprogramming. However, both cell-extrinsic and intrinsic mechanisms often diminish the in-vivo potency of these therapeutic T cells, limiting their clinical efficacy and broader use. Direct activation of human T cells by Toll-like receptor (TLR) ligands induces T cell survival and proliferation, boosts the production of proinflammatory cytokines and augments resistance to regulatory T cell (Treg) suppression. Removal of the TLR ligand-binding region results in constitutive signalling triggered by the remaining cytosolic Toll/interleukin-1 receptor (TIR) domain. The use of such TIR domains therefore offers an ideal means for equipping anti-tumour T cells with the arsenal of functional attributes required for improving current clinical protocols. Here we show that constitutively active (ca)TLR-4 can be expressed efficiently in human T cells using mRNA electroporation. The mere expression of caTLR-4 mRNA in polyclonal CD8 and CD4 T cells induced the production of interferon (IFN)-γ, triggered the surface expression of CD25, CD69 and 4-1BB and up-regulated a panel of cytokines and chemokines. In tumour-infiltrating lymphocytes prepared from melanoma patients, caTLR-4 induced robust IFN-γ secretion in all samples tested. Furthermore, caTLR-4 enhanced the anti-melanoma cytolytic activity of tumour-infiltrating lymphocytes and augmented the secretion of IFN-γ, tumour necrosis factor (TNF)-α and granulocyte-macrophage colony-stimulating factor (GM-CSF) for at least 4 days post-transfection. Our results demonstrate that caTLR-4 is capable of exerting multiple T cell-enhancing effects and can potentially be used as a genetic adjuvant in adoptive cell therapy.

Endogenous hepoxilin A3, produced under short duration of high shear-stress, inhibits thrombin-induced aggregation in human platelets.

The effect of short duration of shear-stress (350 dyne/cm2, 20 ms) on platelet-aggregation has been assessed. This treatment inhibits thrombin-induced but not ADP- or collagen-induced aggregation. The inhibitory effect is mediated by endogenous hepoxilin A3. This conclusion is based on the following observations: (a) The shear-stress effect is abolished by lipoxygenase inhibitors. (b) Hepoxilin A3 mimics the shear-stress effect.

Human platelets exposed to mechanical stresses express a potent lipoxygenase product.

Human platelets exposed to hypotonicity undergo regulatory volume decrease (RVD), controlled by a potent, yet labile, lipoxygenase product (LP). LP is synthesized and excreted during RVD affecting selectively K+ permeability. LP is assayed by its capacity to reconstitute RVD when lipoxygenase is blocked. Centrifugation for preparing washed platelets (1,550 x g, 10 min) is sufficient to express LP activity, with declining potency in repeated centrifugations, indicating that it is not readily replenishable. When platelet suspension flows in a vinyl tubing (1 mm i.d.), at physiological velocity, controlled at 90-254 cm/s, LP formation increases as a function of velocity but declines as result of increasing the tubing length. Stirring the platelets in an aggregometer cuvette for 30 s, yields no LP unless the stirring is intermittent. No associated platelet lysis or aggregation are observed following the mechanical stress applications. These results demonstrate that although mechanical stresses result in LP production, the mode of its application plays a major role. These results may indicate that LP is synthesized under pathological conditions and could be of relevance to platelets behavior related to arterial stenosis.

Altered arachidonic acid metabolism in urate crystal induced inflammation.

Gout is an acute rheumatic disorder that occurs in connection with the deposition of monosodium urate (MSU) crystals in the joints. This disease is characterized by intermittent episodes of severe pain and inflammatory joint swelling which are seemingly driven by prostaglandins. In this study we investigated the effect of MSU crystals on arachidonic acid (AA) metabolism in the mouse. We have demonstrated that prostaglandins and other AA metabolites were transiently formed after MSU crystal injection with peak levels occurring after 10 min. In contrast, free AA levels remained high for 2-4 hours after MSU crystal injection. By contrast, when exogenous AA was administered instead of MSU crystals, both the eicosanoids and AA diminished at the same high rates. The metabolism of exogenously administered AA to eicosanoids was inhibited by pretreatment with MSU crystals. No inhibition of AA metabolism was observed when mice were pretreated with AA itself, Ca2+ ionophore (A23187), or zymosan. We conclude that the MSU crystal treatment of mice results in a transient eicosanoid production which is followed by attenuated AA metabolism. It could be that MSU crystals similarly inhibit AA metabolism in gout and thereby limit the duration of gout attacks.

A comparison of videotape and audiotape assessment of patient-centredness in family physicians' consultations.

The aim of this study was to compare videotape and audiotape assessments of doctors' patient-centred behaviour in primary care consultations. The patient-centredness scale of Henbest and Stewart was used to measure the quality of 856 doctor-patient interactions in 258 consultations performed by 47 primary care physicians recorded on videotape. Assessments were performed once using only the sound track and a second time using both the sound and video-tracks. On a nominal quality scale of 0-3, the average score for all consultations was 1.94 (S.D. 0.63) by audio assessment, and 1.94 (S.D. 0.59) by video assessment (P<0.8). There was excellent agreement between audio and video scores over a wide range of scores. Little information (<5%) was lost when using audiotape compared to videotape. It was technically easier to assess the video recordings. Audio recording is equivalent to video recording for the assessment of patient-centredness using the scale of Henbest and Stewart in primary care consultations.

Cholesteatoma: an epidemiologic study among members of kibbutzim in northern Israel.

In this study 3,056 members (10 years of age and older) of the kibbutzim in the northern part of Israel were examined in order to determine the prevalence of chronic otitis media and cholesteatoma. In addition to demographic factors, the influence of altitude was investigated, but the differences were not found to be significant. The prevalence of chronic otitis media was found to be 0.95% and of cholesteatoma, 0.4%. Of the patients who had chronic otitis media, 41% suffered from cholesteatoma as well. Chronic otitis media was more prevalent in males than females (0.02 greater than p greater than 0.01) in the Ashkenazic than Sephardic populations (0.05 greater than p greater than 0.02), and in industrial and agricultural workers (0.05 greater than p greater than 0.02), as compared to others. Among cholesteatoma patients a family history was found in 64%. In 71% of patients, illness began in infancy. Fifteen percent of the patients did not know of their disease until they were examined in this study. These results suggest a high rate of cholesteatoma among chronic otitis media patients and emphasize the need for performing more extensive studies to determine the rate of cholesteatoma in the general population. The necessity for preventive ear examinations, earlier diagnosis, and treatment of cholesteatoma is emphasized.

Development of a chemiluminescent optical fiber immunosensor to detect Streptococcus pneumoniae antipolysaccharide antibodies.

A chemiluminescent-based optical fiber immunosensor was developed for the detection of antipneumococcal antibodies. This was accomplished by developing a different chemical procedure utilizing 3-aminopropyl trimethoxysilane and cyanuric chloride to conjugate pneumococcal cell wall polysaccharides to the optical fiber tips, and by improving the sensitivity of the photodetection system. The lowest titer of antipneumococcal antibodies detected by the optical fiber was at a 1:819,200 dilution. The lowest corresponding value by standard enzyme-linked immunosorbent assay was at a 1:98,415 dilution. It was concluded that the optical immunosensor system is an accurate and sensitive method to detect antipneumococcal antibodies and may be an adequate tool to monitor antibodies in specimens such as saliva and urine.

[Short course for primary physicians care].

This department of family medicine has been challenged with helping a group of Russian immigrant physicians find places in primary care clinics, quickly and at minimal expense. A 3-month course was set up based on the Family Practice Residency Syllabus and the SFATAM approach, led by teachers and tutors from our department. 30 newly immigrated Russian physicians participated. The course included: lectures and exercises in treatment and communication with patients with a variety of common medical problems in the primary care setting; improvement of fluency in Hebrew relevant to the work setting; and information on the function of primary care and professional clinics. Before-and-after questionnaires evaluating optimal use of a 10- minute meeting with a client presenting with headache were administered. The data showed that the physicians had learned to use more psychosocial diagnostic question and more psychosocial interventions. There was a cleared trend toward greater awareness of the patient's environment, his family, social connections and work. There was no change in biomedical inquiry and interventions but a clear trend to a decrease in recommendations for tests and in referrals. The authors recommend the following didactic tools: adopting a biopsychosocial attitude, active participation of students in the learning situation, working in small groups, use of simulations and video clips, and acquiring basic communication experience.

Targeting tumor-associated antigens to the MHC class I presentation pathway.

There is little doubt that cytotoxic T lymphocytes (CTLs) can kill tumor cells in-vivo. However, most CTL-inducing immunization protocols examined so far in cancer patients have yielded only limited clinical benefits, underscoring the urge to improve current approaches for the effective induction of tumor-reactive CTLs. The tumor side of the immunological frontline is armed with large masses, high mutability and an arsenal of immune evasion and suppression mechanisms. Accordingly, the confronting CTLs should come in large numbers, recognize an assortment of MHC class I (MHC-I) bound tumor-associated peptides and be brought into action under effective immunostimulatory conditions. Naïve CTLs are activated to become effector cells in secondary lymphoid organs, following their productive encounter with MHC-I-bound peptides at the surface of dendritic cells (DCs). Therefore, many cancer vaccines under development focus on the optimization of peptide presentation by DCs at this critical stage. The elucidation of discrete steps and the subsequent identification of inherent bottlenecks in the MHC-I antigen presentation pathway have fueled elaborate efforts to enhance vaccine efficacy by the rational targeting of proteins or peptides, formulated into these vaccines, to this pathway. Protein- and gene-based strategies are accordingly devised to deliver tumor-associated peptides to selected cellular compartments, which are essential for the generation of functional CTL ligands. Many of these strategies target the conventional, endogenous route, while others harness the unique pathways that enable DCs to present exogenous antigens, known as cross-presentation. Here we dissect the intricate machinery that produces CTL ligands and examine how knowledge-based cancer vaccines can target the sequence of workstations, biochemical utensils and molecular intermediates comprising this production line.

Original article: low regulatory volume decrease rate in platelets from ischemic patients: a possible role for hepoxilin a(3) in thrombogenicity.

Hepoxilin-A(3) (Hx-A(3)) is produced by platelets in response to shear-stress. It has an antithrombotic effect on platelets. A low Hx-A(3) level may contribute to the high thrombogenic state that exists in patients with acute coronary syndromes. Since we have previously demonstrated that the regulatory volume decrease (RVD) of human platelets exposed to hypotonic solutions is controlled by Hx-A(3) it is possible that the RVD rate reflects Hx-A(3) activity. In this study, the RVD rate of platelets taken from a healthy control group (n=21) was compared to that of patients with chronic ischemic heart disease (n=23), acute ischemic heart disease (n = 24) and acute myocardial infarction (MI, n = 29). The RVD rate of the control group was significantly higher than the other three groups (P < 0.001). The addition of 100 nM of Hx-A, to the platelets of eight patients with MI increased their RVD rate to that of the controls. Patients with diabetes mellitus or hypertension have the lowest RVD rates. Medications such as aspirin, heparin, and streptokinase did not affect the Hx-A(3) activity of platelets obtained from patients with ischemic heart disease. The results of the present study indicate that patients with acute ischemia may have a low level of platelet Hx-A(3) activity. This possible low level of Hx-A, activity may be associated with a failure to develop an antithrombotic reaction to the shear-stress forces generated during acute ischemia.

Lipoxygenase product controls the regulatory volume decrease of human platelets.

SUMMARY. Blood platelets exposed to hypotonic medium are known to undergo a regulatory volume decrease (RVD), mediated by increased conductive permeability of K(+) and Cl(-). It is presently shown that RVD in platelets is controlled by a lipoxygenase product, apparently by a selective regulation of K(+) permeability. This conclusion is supported by the following observations: (a) lipoxygenase inhibitors, nordihydroguaiaretic acid (NDGA), N-(3-phenoxycinnamyl)-acetohydroxamic acid (BW A4C), methyl 2-[(3,4-dihydro-3,4-dioxo-naphthalenyl)amino]-benzoate (CGS 8515), and 5,8,11,14-eicosatetraynoic acid, inhibit RVD with IC(50) values of 3,6,10, and 5 µM, respectively. In contrast, aspirin and indomethacin, known cycloxygenase inhibitors, are innocuous; (b) an eluate from platelets undergoing RVD restores RVD in NDGA-treated platelets; (c) the eluate is unstable (t(1/2) = 8 s and 2 min in the presence and absence of platelets, respectively); furthermore, albumin promotes platelet RVD; (d) known lipoxygenase products, 12-hydroperoxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid and leukotriene D(4), restore RVD in NDGA-treated platelets at 0.1-1.0 µM; (e) the extended hypotonic swelling of gramicidin-treated platelets, expressing Cl(-) permeability, is insensitive to NDGA. It is hypothesized that the lipoxygenase product selectively opens K(+) channels in platelets, in analogy with the effect of lipoxygenase products in cardiac atrial cells and Aplysia sensory neurons.

SFAT-AM: short family therapy in ambulatory medicine. Treatment approach in 10-15 minute encounters.

The question of the possibilities latent within an appointment lasting on average only 10-15 minutes, under the pressures of a heavy workload in a public clinic, has occupied the authors for three years. SFAT-AM: Short Family Therapy in Ambulatory Medicine, has been developed with aim of offering solutions to this question. The theoretical background is taken from family medicine with a biopsychosocial (b.p.s.) systems approach. The authors formulated series of basic doctor-patient encounters which last on average 10-15 minutes. The basic encounter is a kind of didactic checklist model which presents to the doctor possibilities latent within. Emphasis is placed on the art of treatment and the meeting itself as therapeutic. During the meeting, patient's satisfaction, psychosocial information and the doctor's inner voice help the participants adjust to each other and move from stage to stage. The approach also relates to the possibility of joint work together with colleagues or with members of different teams in the clinic. The doctor makes use of communication techniques and reaches a b.p.s. diagnosis which will be the basis for suggesting a treatment. The presented model was tried by the authors and by trainees as part of a specialization course in family medicine. The trainees received between 20 and 70 academic hours of instruction. From the doctors' report it can be seen that SFAT-AM can be used in a primary clinic. Future research should give more specific answers to questions about the model's desirability, cost-efficiency, and job satisfaction. (Doctors will be referred to in the male gender throughout the article).

Initiation of RVD response in human platelets: mechanical-biochemical transduction involves pertussis-toxin-sensitive G protein and phospholipase A2.

Platelets revert hypotonic-induced swelling by the process of regulatory volume decrease (RVD). We have recently shown that this process is under the control of endogenous hepoxilin A3. In this work, we investigated the mechanical-biochemical transduction that leads to hepoxilin A3 formation. We demonstrate that this process is mediated by pertussis-toxin-sensitive G protein, which activates Ca(2+)-insensitive phospholipase A2, and the sequential release of arachidonic acid. This conclusion is supported by the following observations: (i) RVD response is blocked selectively by the phospholipase A2 inhibitors manoalide and bromophenacyl-bromide (0.2 and 5 microM, respectively) but not by phospholipase C inhibitors. The addition of arachidonic acid overcame this inhibition; (ii) extracellular Ca2+ depletion by EGTA (up to 10 mM) does not affect RVD; (iii) intracellular Ca2+ depletion by BAPTA-AM (100 microM) inhibits RVD but not hepoxilin A3 formation, as tested by the RVD reconstitution assay; (iv) RVD is inhibited by the G-protein inhibitors, GDP beta S (1 microM) and pertussis toxin (1 ng/ml). This inhibition is overcome by addition of arachidonic acid or hypotonic cell-free eluate that contains hepoxilin A3; (v) NaF, 1 mM, induces hepoxilin A3 formation, tested by the RVD reconstitution assay; and (vii) GDP beta S inhibits hepoxilin A3 formation associated with flow. Therefore, it seems that G proteins are involved in the initial step of the mechanical-biochemical transduction leading to hepoxilin A3 formation in human platelets.

Rapid quantitation of a large scope of eicosanoids in two models of inflammation: development of an electrospray and tandem mass spectrometry method and application to biological studies.

Assessment of eicosanoid levels in biological systems is important for understanding their role in cell function and pathophysiological events. Current methods of eicosanoid quantitation are limited by sensitivity, scope, or throughput. The development of a new method for eicosanoid assessment in biological samples by electrospray and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring mode is described here. In this study, 14 biologically significant eicosanoids were quantitated in a single sample. Complete sample analysis required two repeated injections of 5 microliters with an analysis time of 1.5 min/injection. Limits of detection ranged from 0.5 pg for thromboxane B2 (TxB2) to 10 pg for 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha). The reliability, reproducibility, sensitivity, and cross-detection of the method is also described. The MS/MS method was used to explore eicosanoid production in two inflammation models: lipopolysaccharide (LPS)-stimulated human whole blood and carrageenan-challenged rat air pouch. The most abundant metabolites in LPS-stimulated whole blood were prostaglandin E2 (PGE2), TxB2, and 6-keto PGF1 alpha; prostaglandins E1, D2, and F2 alpha and leukotrienes B4 and C4 were detected in lower amounts. Eicosanoid levels determined by MS/MS were similar to those obtained by immunoassay and GC-MS. The most abundant metabolites detected in carrageenan-challenged rat air pouch were PGE2, 6-keto PGF1 alpha, and TxB2. The method described in this work is accurate and rapid and should greatly aid in evaluating the role of multiple eicosanoids in future biological studies.

Regulation of prostaglandin biosynthesis in vivo by glutathione.

Intraperitoneal administration of urate crystals to mice reduced subsequent macrophage conversion of arachidonic acid (AA) to prostaglandins (PGs) and 12-hydroxyeicosatetraenoic acid for up to 6 h. In contrast, levels of 12-hydroxyheptadecatrienoic acid (12-HHT) were markedly elevated. This metabolic profile was previously observed in vitro when recombinant cyclooxygenase (COX) enzymes were incubated with reduced glutathione (GSH). Analysis of peritoneal GSH levels revealed a fivefold elevation after urate crystal administration. The GSH synthesis inhibitor L-buthionine-[S,R]-sulfoximine partially reversed the urate crystal effect on both GSH elevation and PG synthesis. Moreover, addition of exogenous GSH to isolated peritoneal macrophages shifted AA metabolism from PGs to 12-HHT. Urate crystal administration reduced COX-1, but induced COX-2 expression in peritoneal cells. The reduction of COX-1 may contribute to the attenuation of PG synthesis after 1 and 2 h, but PG synthesis remained inhibited up to 6 h, when COX-2 levels were high. Overall, our results indicate that elevated GSH levels inhibit PG production in this model and provide in vivo evidence for the role of GSH in the regulation of PG biosynthesis.

Sensitivity of exponentially growing populations of Escherichia coli to photo-induced psoralen-DNA interstrand crosslinks.

Experimental survival curves for Escherichia coli K 12 (CR 34) were determined after exposure to 4,5',8-trimethylpsoralen and near ultraviolet light. The lethal action was shown to arise exclusively from interstrand crosslinks, cell vulnerability increasing markedly with the doubling time of the culture. To account for these results, two quite different models are considered. The first assumes that a cell survives as long as at least one copy of its genome remains undamaged; a variant of this permits repair by DNA strand exchange. The second model allows for a limited period of time during which DNA repair can take place. A crosslink in a stretch of DNA due to be replicated within this interval constitutes a fatal lesion. Theoretical survival curves are computed for bacterial populations with defined age distributions and chromosome configurations. While the first model completely fails to provide a satisfactory description of the experimental results, the second model does predict the presence of a shoulder in the survival curves and, in one of its forms, it seems to agree rather well with the measured data over a wide range of crosslink concentrations and doubling times.

Hepoxilin A3 is the endogenous lipid mediator opposing hypotonic swelling of intact human platelets.

When human blood platelets are exposed to hypotonic medium they swell first but, shortly thereafter, revert toward their original volume in a process termed regulatory volume decrease (RVD). RVD is the result of an enhanced efflux of K+ and Cl- ions and associated water. Platelet RVD is controlled by a short-lived lipoxygenase-derived product (LP). By using a combination of high-performance liquid chromatography, gas chromatography-mass spectrometry, and RVD reconstitution bioassay, we show that LP is identical with hepoxilin A3. In addition we demonstrate that authentic hepoxilin A3 possesses the same biological properties on RVD reconstitution as LP and that the activity of both compounds is amplified through epoxide hydrolase inhibition with 3,3,3-trichloropropene-1,2-oxide. Therefore, we report here that volume expansion causes the formation and release of hepoxilin A3 from intact human platelets and that this hepoxilin plays a major role in volume regulation.