Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot.

Background: The inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The ELISpot assay is a functional bioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis on whether the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity. Methods: Mice were made septic using sublethal cecal ligation and puncture (CLP). Blood and spleens were harvested serially and ex vivo IFN-γ and TNF-α production were compared by ELISpot and ELISA. The capability of ELISpot to detect changes in innate and adaptive immunity due to in vivo immune therapy with dexamethasone, IL-7, and arginine was also evaluated. Results: ELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example dexamethasone, arginine, and IL-7 in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and ELISA results tended to parallel one another although some differences were noted. Conclusion: ELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow the in vivo effects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.

Diagnosis and Treatment of Posteromedial Elbow Impingement in the Throwing Athlete.

PURPOSE OF REVIEW: Posteromedial elbow impingement is a common pathological entity in the throwing athlete. The posteromedial articulation of the elbow is a significant stabilizer to medial elbow forces and valgus stress noted during repetitive throwing. This current review investigates recent literature regarding the relevant anatomy, diagnosis, and treatment of posteromedial impingement in the thrower. RECENT FINDINGS: Improvements in advanced imaging have provided accurate and detailed diagnostic capability for the assessment of the throwers' elbow. After failure of conservative measures, arthroscopic treatment of posteromedial elbow impingement with posteromedial osteophyte removal has demonstrated excellent outcomes with a reliable return to play in the competitive thrower. In addition to a thorough history and physical examination, MRI, MR arthrogram, CT, and dynamic ultrasound imaging modalities are useful in the assessment of a presumed diagnosis of posteromedial impingement. Arthroscopic decompression with posteromedial osteophyte removal provides effective clinical results and return to play. The surgeon should be careful to avoid creating medial ulnar collateral instability by means of over-resection of the posteromedial olecranon.

Molecular characterization of the translocation breakpoints in the Down syndrome mouse model Ts65Dn.

Ts65Dn is a mouse model of Down syndrome: a syndrome that results from chromosome (Chr) 21 trisomy and is associated with congenital defects, cognitive impairment, and ultimately Alzheimer's disease. Ts65Dn mice have segmental trisomy for distal mouse Chr 16, a region sharing conserved synteny with human Chr 21. As a result, this strain harbors three copies of over half of the human Chr 21 orthologs. The trisomic segment of Chr 16 is present as a translocation chromosome (Mmu17(16)), with breakpoints that have not been defined previously. To molecularly characterize the Chrs 16 and 17 breakpoints on the translocation chromosome in Ts65Dn mice, we used a selective enrichment and high-throughput paired-end sequencing approach. Analysis of paired-end reads flanking the Chr 16, Chr 17 junction on Mmu17(16) and de novo assembly of the reads directly spanning the junction provided the precise locations of the Chrs 16 and 17 breakpoints at 84,351,351 and 9,426,822 bp, respectively. These data provide the basis for low-cost, highly efficient genotyping of Ts65Dn mice. More importantly, these data provide, for the first time, complete characterization of gene dosage in Ts65Dn mice.

Tetrahydrohyperforin prevents cognitive deficit, Aß deposition, tau phosphorylation and synaptotoxicity in the APPswe/PSEN1ΔE9 model of Alzheimer's disease: a possible effect on APP processing.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration of cognitive abilities, amyloid-ß peptide (Aß) accumulation and synaptic alterations. Previous studies indicated that hyperforin, a component of the St John's Wort, prevents Aß neurotoxicity and some behavioral impairments in a rat model of AD. In this study we examined the ability of tetrahydrohyperforin (IDN5607), a stable hyperforin derivative, to prevent the cognitive deficit and synaptic impairment in an in vivo model of AD. In double transgenic APPswe/PSEN1ΔE9 mice, IDN5706 improves memory and prevents the impairment of synaptic plasticity in a dose-dependent manner, inducing a recovery of long-term potentiation. In agreement with these findings, IDN5706 prevented the decrease in synaptic proteins in hippocampus and cortex. In addition, decreased levels of tau hyperphosphorylation, astrogliosis, and total fibrillar and oligomeric forms of Aß were determined in double transgenic mice treated with IDN5706. In cultured cells, IDN5706 decreased the proteolytic processing of the amyloid precursor protein that leads to Aß peptide generation. These findings indicate that IDN5706 ameliorates AD neuropathology and could be considered of therapeutic relevance in AD treatment.

Neurobiological effects of Hyperforin and its potential in Alzheimer's disease therapy.

St. John's Wort (SJW) has been used medicinally for over 5,000 years. Relatively recently, one of its phloroglucinol derivatives, hyperforin, has emerged as a compound of interest. Hyperforin first gained attention as the constituent of SJW responsible for its antidepressant effects. Since then, several of its neurobiological effects have been described, including neurotransmitter re-uptake inhibition, the ability to increase intracellular sodium and calcium levels, canonical transient receptor potential 6 (TRPC6) activation, N-methyl-D-aspartic acid (NMDA) receptor antagonism as well as antioxidant and anti-inflammatory properties. Until recently, its pharmacological actions outside of depression had not been investigated. However, hyperforin has been shown to have cognitive enhancing and memory facilitating properties. Importantly, it has been shown to have neuroprotective effects against Alzheimer's disease (AD) neuropathology, including the ability to disassemble amyloid-beta (Abeta) aggregates in vitro, decrease astrogliosis and microglia activation, as well as improve spatial memory in vivo. This review will examine some of the early studies involving hyperforin and its effects in the central nervous system (CNS), with an emphasis on its potential use in AD therapy. With further investigation, hyperforin could emerge to be a likely therapeutical candidate in the treatment of this disease.

DcR2 (TRAIL-R4) siRNA and adenovirus delivery of TRAIL (Ad5hTRAIL) break down in vitro tumorigenic potential of prostate carcinoma cells.

High levels of decoy receptor 2 (DcR2; TRAIL-R4) expression are correlated with TRAIL resistance in prostate cancer cells. In addition, upregulation of TRAIL death receptor (DR4 and DR5) expression, either by ionizing radiation or chemotherapy, can sensitize cancer cells to TRAIL. Considering more than half of human cancers are TRAIL resistant, modulation of surface TRAIL receptor expression appears to be an attractive treatment modality to counteract TRAIL resistance. In this study, three siRNA duplexes targeting DcR2 receptor were tested. Ad5hTRAIL infections were performed to overexpress human full-length TRAIL to induce cell death, and the in vitro tumorigenic potential of prostate cancer cells was assessed using colony-forming assays on soft agar. The DU145 and LNCaP prostate cancer cell lines, which express high levels of DcR2, were resistant to Ad5hTRAIL-induced death. Downregulation of surface DcR2 expression by siRNA sensitized these prostate cancer cell lines to Ad5hTRAIL. In addition, DcR2 siRNA-mediated knockdown of DcR2, followed by Ad5hTRAIL infection, dramatically reduced the in vitro tumorigenic potential of prostate cancer cells. Collectively, our results suggest the potential for combining receptor-specific siRNA with TRAIL in the treatment of certain cancers.

Dynamics of a three-variable nonlinear model of vasomotion: comparison of theory and experiment.

The effects of pharmacological interventions that modulate Ca(2+) homeodynamics and membrane potential in rat isolated cerebral vessels during vasomotion (i.e., rhythmic fluctuations in arterial diameter) were simulated by a third-order system of nonlinear differential equations. Independent control variables employed in the model were [Ca(2+)] in the cytosol, [Ca(2+)] in intracellular stores, and smooth muscle membrane potential. Interactions between ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca(2+) stores and transmembrane ion fluxes via K(+) channels, Cl(-) channels, and voltage-operated Ca(2+) channels were studied by comparing simulations of oscillatory behavior with experimental measurements of membrane potential, intracellular free [Ca(2+)] and vessel diameter during a range of pharmacological interventions. The main conclusion of the study is that a general model of vasomotion that predicts experimental data can be constructed by a low-order system that incorporates nonlinear interactions between dynamical control variables.

Enhancement of Ad5-TRAIL cytotoxicity against renal cell carcinoma with histone deacetylase inhibitors.

Renal cell carcinoma (RCC) will cause greater than 12,000 deaths in the United States this year. The lack of effective therapy for disseminated RCC has stimulated the search for novel treatments including immunotherapeutic strategies, but poor therapeutic responses and marked toxicity have limited their use. The tumor necrosis factor (TNF) family member TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2L induces apoptosis in various tumor cell types, while having little cytotoxicity against normal cells. In this study, we investigated the tumoricidal potential of a recombinant adenovirus encoding human TNFSF10 (Ad5-TRAIL), alone and in combination with a panel of histone deacetylase inhibitors (HDACi), against the TRAIL/Apo-2L-resistant RCC line 786-O and normal human renal proximal tubule epithelial cells (RPTEC). Ad5-TRAIL was unable to induce apoptosis in either 786-O or RPTEC alone; however, tumor cell apoptosis occurred when Ad5-TRAIL was combined with HDAC inhibition. Except when combined with trichostatin A, RPTEC were not sensitized to Ad5-TRAIL by HDACi. In 786-O, HDAC inhibition induced CAR expression, permitting increased adenoviral infection and transgene expression. It also induced TRAIL-R2 expression, accelerated the death-inducing signaling complex formation and enhanced caspase-8 activation. Our results demonstrate the utility of combining Ad5-TRAIL with HDACi against RCC, and mechanistically define how this combination modulates RCC sensitivity to TRAIL/Apo-2L and adenoviral infection.

Stress avoidance in a common annual: reproductive timing is important for local adaptation and geographic distribution.

Adaptation to local environments may be an important determinant of species' geographic range. However, little is known about which traits contribute to adaptation or whether their further evolution would facilitate range expansion. In this study, we assessed the adaptive value of stress avoidance traits in the common annual Cocklebur (Xanthium strumarium) by performing a reciprocal transplant across a broad latitudinal gradient extending to the species' northern border. Populations were locally adapted and stress avoidance traits accounted for most fitness differences between populations. At the northern border where growing seasons are cooler and shorter, native populations had evolved to reproduce earlier than native populations in the lower latitude gardens. This clinal pattern in reproductive timing corresponded to a shift in selection from favouring later to earlier reproduction. Thus, earlier reproduction is an important adaptation to northern latitudes and constraint on the further evolution of this trait in marginal populations could potentially limit distribution.

Phosphoramidate derivatives of 2',5'-dideoxyadenosine as potential inhibitors of the EDHF phenomenon.

P-site inhibitors of adenyl cyclase, such as the dideoxynucleosides 2',3'-ddA and 2',5-ddA, have been shown to attenuate EDHF phenomenon in rabbit arteries and veins. In order to present the dideoxynucleosides as pre-activated nucleotides and bypass the kinase, as well as to prevent their metabolism to dideoxyinosine by adenosine deaminase, the aryloxyphosphoramidate approach has been successfully applied, initially on the 2',3'-ddA. In the present work a new series of 2',5'-ddA phosphoramidates has been synthesized, representing the first example of phosphoramidate protide not at the 5'-position.

Deterministic nonlinear characteristics of in vivo blood flow velocity and arteriolar diameter fluctuations.

We have performed a nonlinear analysis of fluctuations in red cell velocity and arteriolar calibre in the mesenteric bed of the anaesthetized rat. Measurements were obtained under control conditions and during local superfusion with NG-nitro-L-arginine (L-NNA, 30 microM) and tetrabutylammonium (TBA, 0.1 mM), which suppress NO synthesis and block Ca2+ activated K+ channels (KCa), respectively. Time series were analysed by calculating correlation dimensions and largest Lyapunov exponents. Both statistics were higher for red cell velocity than diameter fluctuations, thereby potentially differentiating between global and local mechanisms that regulate microvascular flow. Evidence for underlying nonlinear structure was provided by analysis of surrogate time series generated from the experimental data following randomization of Fourier phase. Complexity indices characterizing time series under control conditions were in general higher than those derived from data obtained during superfusion with L-NNA and TBA.

Shil'nikov homoclinic chaos is intimately related to type-III intermittency in isolated rabbit arteries: role of nitric oxide.

We provide experimental evidence for the existence of Shil'nikov homoclinic chaos in the fluctuations in flow which can be observed in isolated perfused rabbit ear arteries, and establish a close association between homoclinicity and type-III Pomeau-Manneville intermittent behavior. The transition between the homoclinic scenario and type-III intermittency is clarified by a mathematical model of the arterial smooth muscle cell. Simulations of the effects of nitric oxide (NO) by the vascular endothelium on these patterns of behavior closely match experimental observations.

Induction of necrotic tumor cell death by TRAIL/Apo-2L.

A great deal of enthusiasm is being generated for TRAIL (TNF-related apoptosis-inducing ligand)/Apo-2L as a tumor therapeutic agent because it is cytotoxic to a variety of tumor cell types but not normal cells. Moreover, it is well documented that TRAIL/Apo-2L-induced tumor cell death is a caspase-dependent apoptotic process. Through the use of a transfected cell line expressing murine TRAIL/Apo-2L and a recombinant adenovirus encoding the murine TRAIL/Apo-2L cDNA (Ad5-mTRAIL) against two murine tumor cell lines [TRAMP-C2 (prostate adenocarcinoma) and Renca (renal adenocarcinoma)], we found that mTRAIL/Apo-2L also can kill tumor cells by inducing necrosis. Specifically, we observed the default method of mTRAIL/Apo-2L-induced death in TRAMP-C2 cells was via a necrotic process, characterized by the complete lack of an annexin V(+)/PI(-) population, SAPK/JNK phosphorylation, caspase activation, Bid cleavage, or cytochrome c release. Moreover, the inclusion of zVAD-fmk, an inhibitor of caspase activation, markedly enhanced mTRAIL/Apo-2L-mediated killing of TRAMP-C2. In contrast, apoptosis was induced in TRAMP-C2 using TNF, as measured by the criteria listed above, as was Renca by mTRAIL/Apo-2L. These results demonstrate the natural occurrence of both TRAIL/Apo-2L-induced apoptotic and necrotic signaling mechanisms within tumor cells.

Fractal dimensions of laser doppler flowmetry time series.

Laser Doppler flowmetry (LDF) provides a non-invasive method of assessing cutaneous perfusion. As the microvasculature under the probe is not defined the measured flux cannot be given absolute units, but the technique has nevertheless proved valuable for assessing relative changes in perfusion in response to physiological stress. LDF signals normally show pronounced temporal variability, both as a consequence of the pulsatile nature of blood flow and local changes in dynamic vasomotor activity. The aim of the present study was to investigate the use of methods of nonlinear analysis in characterizing temporal fluctuations in LDF signals. Data were collected under standardised conditions from the forearm of 16 normal subjects at rest, during exercise and on recovery. Surrogate data was then generated from the original time series by phase randomization. Dispersional analysis demonstrated that the LDF data was fractal with two distinct scaling regions, thus allowing the calculation of a fractal dimension which decreased significantly from 1.23 +/- 0.09 to 1.04 +/- 0.02 during exercise. By contrast, dispersional analysis of the surrogate data showed no scaling region.

Wavelet and receiver operating characteristic analysis of heart rate variability.

Multiresolution wavelet analysis has been used to study the heart rate variability in two classes of patients with different pathological conditions. The scale dependent measure of Thurner et al. was found to be statistically significant in discriminating patients suffering from hypercardiomyopathy from a control set of normal subjects. We have performed Receiver Operating Characteristc (ROC) analysis and found the ROC area to be a useful measure by which to label the significance of the discrimination, as well as to describe the severity of heart dysfunction.

Universal scaling properties of type-I intermittent chaos in isolated resistance arteries are unaffected by endogenous nitric oxide synthesis.

Spontaneous fluctuations in flow in isolated rabbit ear resistance arteries may exhibit almost-periodic behavior interrupted by chaotic bursts that can be classified as type-I Pomeau-Manneville intermittency. This conclusion was supported by the construction of parabolic return maps and identification of the characteristic probability distributions for the number of oscillations per laminar segment (n) associated with the type-I scenario. Pharmacological inhibition of nitric oxide (NO) synthesis by the vascular endothelium modulated the dynamics of the reinjection mechanism, and thus the generic shape of the probability distribution for n. Nevertheless, average laminar length was related to a derived bifurcation parameter epsilon according to power-law scaling of the form approximately epsilon(beta), where the estimated critical exponent beta was close to the theoretical value of -0.5 both in the presence and absence of NO synthesis.

Suppression of tumor growth following intralesional therapy with TRAIL recombinant adenovirus.

TRAIL is a member of the tumor necrosis factor superfamily that induces apoptosis in a variety of tumor cell types both in vitro and in vivo, while demonstrating minimal cytotoxicity toward normal tissues. One disadvantage to previous in vivo protocols was the need for large quantities of TRAIL to suppress tumor growth. Here we engineered a replication-deficient adenovirus to encode human TNFSF10 (Ad5-TRAIL) as an alternative to recombinant, soluble TRAIL protein. The results show that TRAIL-sensitive prostate tumor cell targets infected with Ad5-TRAIL undergo apoptosis through the production and expression of TRAIL protein. This activity was limited to TRAIL-sensitive tumor cells, as normal prostate epithelial cells were not killed by Ad5-TRAIL. Furthermore, in vivo administration of Ad5-TRAIL at the site of tumor implantation suppressed the outgrowth of human prostate tumor xenografts in SCID mice. Histologic examination of prostate tumors treated locally with Ad5-TRAIL revealed areas of apoptosis within 24 hours of injection. These results further define Ad5-TRAIL as a novel anti-tumor therapeutic and demonstrate its potential use as a means for treating prostate tumors, as well as other solid tumors, in vivo.

Regulation of Fas ligand-induced apoptosis by TNF.

Fas ligand (FasL, CD95L) expression helps control inflammatory reactions in immune privileged sites such as the eye. Cellular activation is normally required to render lymphoid cells sensitive to FasL-induced death; however, both activated and freshly isolated Fas(+) lymphoid cells are efficiently killed in the eye. Thus, we examined factors that might regulate cell death in the eye. TNF levels rapidly increased in the eye after the injection of lymphoid cells, and these cells underwent apoptosis within 24 h. Coinjection of anti-TNF Ab with the lymphoid cells blocked this cell death. Furthermore, TNFR2(-/-) T cells did not undergo apoptosis in the eyes of normal mice, while normal and TNFR1(-/-) T cells were killed by apoptosis. In vitro, TNF enhanced the Fas-mediated apoptosis of unactivated T cells through decreased intracellular levels of FLIP and increased production of the pro-apoptotic molecule Bax. This effect was mediated through the TNFR2 receptor. In vivo, intracameral injection of normal or TNFR1(-/-) 2,4,6-trinitrophenyl-coupled T cells into normal mice induced immune deviation, but TNFR2(-/-) 2,4,6-trinitrophenyl-coupled T cells were ineffective. Collectively, our results provide evidence of a role for the p75 TNFR in cell death in that TNF signaling through TNFR2 sensitizes lymphoid cells for Fas-mediated apoptosis. We conclude that there is complicity between apoptosis and elements of the inflammatory response in controlling lymphocyte function in immune privileged sites.

Inhibition of murine prostate tumor growth and activation of immunoregulatory cells with recombinant canarypox viruses.

BACKGROUND: Immunization with modified tumor cells carrying recombinant immunomodulatory genes is being explored as cancer immunotherapy. In this study, we examine whether canarypox ALVAC viruses carrying immunostimulatory cytokine genes (granulocyte-macrophage colony-stimulating factor, interleukin 2, interleukin 12, and tumor necrosis factor-alpha) can induce antitumor immunity (to rechallenge) in the RM-1 model of a highly aggressive, weakly immunogenic murine prostate cancer. METHODS: For antitumor activity studies, RM-1 murine prostate cancer cells were infected with the parental ALVAC virus or one or two recombinant ALVAC-cytokine viruses and then injected into male C57BL/6 mice. For rechallenge studies, other mice were first given an injection subcutaneously with irradiated (nonproliferating) recombinant ALVAC-infected RM-1 cells and then (10 days later) with untreated RM-1 cells. For the determination of which immune cells were required for antitumor activity, mice were immunodepleted of CD4, CD8, or natural killer (NK) NK1.1 cells with the corresponding monoclonal antibodies and were then given an injection of ALVAC-cytokine-infected RM-1 cells. For all experiments, tumor outgrowth and animal survival were monitored. RESULTS: After subcutaneous injection into mice, RM-1 cells infected with one (except ALVAC-interleukin 2) or two ALVAC-cytokine recombinants had statistically significantly greater antitumor activity than RM-1 cells infected with parental ALVAC (P<.001 for all; two-sided test). The antitumor activity of RM-1 cells infected with any two ALVAC-cytokine recombinants was greater than, but not statistically significantly different from, that of RM-1 cells infected with any one ALVAC-cytokine recombinant. NK1.1 cells were necessary for antitumor activity, but tumor-specific CD4(+) regulatory T cells were also induced that inhibited CD8(+) RM-1-specific cytotoxic T cells, resulting in the lack of immunity to a rechallenge by RM-1 cells. DISCUSSION: Canarypox viruses can transfer immunostimulatory cytokine genes into RM-1 prostate cancer cells. When such cells were injected into mice, the cytokines induced an antitumor response against this highly aggressive, weakly immunogenic tumor. This response, however, did not protect the mouse against a rechallenge with RM-1 cells because suppressor CD4(+) T cells were induced that inhibited tumor-specific CD8(+) cytotoxic T cells.

A man with a history of skin lesions and seizures.

A 38-year-old man presented with painful growths around his toenails. Since their first occurrence at age three, several had been surgically removed. He had had similar growths around his fingernails and multiple facial lesions since age seven.