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Live imaging of the gastrointestinal tract with two-photon microscopy (TPM) has proven to be a useful tool for mucosal immunologists. It provides deep penetration of live tissues with reduced phototoxicity and photobleaching and thus excels in deciphering dynamic immunological processes that require cell motility and last minutes through hours. The few studies that employed this technique in the gut have uncovered new aspects of mucosal immunity. They focused mainly on adaptive immunity in the small intestine and exposed the details of important interactions among several epithelial and hematopoietic cell types. TPM can be employed either on explanted tissue or intravitally, as has been practiced in our lab. Intravital TPM preserves physiological conditions more faithfully, but it is a demanding technique that requires dedicated personnel. To achieve success, the peristaltic motility of the intestine must be curbed, surgical and photonic damage must be minimized, and tissue degradation must be delayed and controlled for. Here we briefly review published studies that employed intravital TPM in the gut, describe our own technique for imaging the intestinal Peyer's patches (PPs) and villi, and present some observations we made using this technique., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Palmatier and Rovner (2015) attempt to establish the construct validity of the Comparison Question Test (CQT) by citing extensive research ranging from modern neuroscience to memory and psychophysiology. In this comment we argue that merely citing studies on the preliminary process theory (PPT) of the orienting response (OR) or neuroimaging research on deception without a clear specification of their connection to the CQT is insufficient for construct validity. Moreover, PPT cannot account for observed differential heart rate responses found in both CQT and Concealed Information Test (CIT) research. Furthermore, Palmatier and Rovner ignore the many other deficiencies of the CQT, such as lack of proper control and standardization, which cannot be resolved by any psychological or psychophysiological theory. In sum, we show that Palmatier and Rovner failed in their mission to establish construct validity of the CQT, and their article provides no solution to the many other deficiencies of this test., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Kindlin-3 is an integrin-binding focal adhesion adaptor absent in patients with leukocyte and platelet adhesion deficiency syndrome and is critical for firm integrin-dependent leukocyte adhesion. The role of this adaptor in leukocyte diapedesis has never been investigated. In the present study, the functions of Kindlin-3 in this process were investigated in effector T lymphocytes trafficking to various lymphoid and nonlymphoid tissues. In vitro, Kindlin-3-deficient T cells displayed severely impaired lymphocyte function antigen-1-dependent lymphocyte adhesion but partially conserved very late antigen-4 adhesiveness. In vivo, the number of adoptively transferred Kindlin-3-deficient T effectors was dramatically elevated in the circulating pool compared with normal effectors, and the Kindlin-3 mutant effectors failed to enter inflamed skin lesions. The frequency of Kindlin-3-deficient T effectors arrested on vessel walls within inflamed skin-draining lymph nodes was also reduced. Strikingly, however, Kindlin-3-deficient effector T cells accumulated inside these vessels at significantly higher numbers than their wild-type lymphocyte counterparts and successfully extravasated into inflamed lymph nodes. Nevertheless, on entering these organs, the interstitial motility of these lymphocytes was impaired. This is the first in vivo demonstration that Kindlin-3-stabilized integrin adhesions, although essential for lymphocyte arrest on blood vessels and interstitial motility, are not obligatory for leukocyte diapedesis.

The bone marrow (BM) hosts memory lymphocytes and supports secondary immune responses against blood-borne antigens, but it is unsettled whether primary responses occur there and which cells present the antigen. We used 2-photon microscopy in the BM of live mice to study these questions. Naïve CD8(+) T cells crawled rapidly at steady state but arrested immediately upon sensing antigenic peptides. Following infusion of soluble protein, various cell types were imaged ingesting the antigen, while antigen-specific T cells decelerated, clustered, upregulated CD69, and were observed dividing in situ to yield effector cells. Unlike in the spleen, T-cell responses persisted when BM-resident dendritic cells (DCs) were ablated but failed when all phagocytic cells were depleted. Potential antigen-presenting cells included monocytes and macrophages but not B cells. Collectively, our results suggest that the BM supports crosspresentation of blood-borne antigens similar to the spleen; uniquely, alongside DCs, other myeloid cells participate in crosspresentation.

Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders and can mediate immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. Here, we address this barrier using donorderived central memory CD8(+) T cells (Tcms), directed against third-party antigens. Our results show that fully allogeneic or (hostXdonor)F1-Tcm, support donor chimerism (> 6 months) in sublethally irradiated (5.5Gy) mice, without GVHD symptoms. Chimerism under yet lower irradiation (4.5Gy) was achieved by combining Tcm with short-term administration of low-dose Rapamycin. Importantly, this chimerism resulted in successful donor skin acceptance, whereas third-party skin was rejected. Tracking of host anti-donor T cells (HADTCs), that mediate TDBMT rejection, in a novel bioluminescence-imaging model revealed that Tcms both induce accumulation and eradicate HADTCs in the LNs,concomitant with their elimination from other organs, including the BM. Further analysis with 2-photon microcopy revealed that Tcms form conjugates with HADTCs, resulting in decelerated and confined movement of HADTCs within the LNs in an antigen-specific manner. Thus, anti-third-party Tcms support TDBMT engraftment under reduced-conditioning through lymph-node sequestration and deletion of HADTCs, offering a novel and potentially safe approach for attaining stable hematopoietic chimerism.

The CX3C chemokine family is composed of only one member, CX3CL1, also known as fractalkine, which in mice is the sole ligand of the G protein-coupled, 7-transmembrane receptor CX3CR1. Unlike classic small peptide chemokines, CX3CL1 is synthesized as a membrane-anchored protein that can promote integrin-independent adhesion. Subsequent cleavage by metalloproteases, either constitutive or induced, can generate shed CX3CL1 entities that potentially have chemoattractive activity. To study the CX3C interface in tissues of live animals, we generated transgenic mice (CX3CL1cherry:CX3CR1gfp), which express red and green fluorescent reporter genes under the respective control of the CX3CL1 and CX3CR1 promoters. Furthermore, we performed a structure/function analysis to differentiate the in vivo functions of membrane-tethered versus shed CX3CL1 moieties by comparing their respective ability to correct established defects in macrophage function and leukocyte survival in CX3CL1-deficient mice. Specifically, expression of CX3CL1(105Δ), an obligatory soluble CX3CL1 isoform, reconstituted the formation of transepithelial dendrites by intestinal macrophages but did not rescue circulating Ly6Clo CX3CR1hi blood monocytes in CX3CR1gfp/gfp mice. Instead, monocyte survival required the full-length membrane-anchored CX3CL1, suggesting differential activities of tethered and shed CX3CL1 entities.

Perioperative suppression of NK activity has been suggested to compromise host resistance to tumor progression. Here, we sought to develop a clinically applicable preoperative regimen to prevent immunosuppression and promotion of metastasis by stress or surgery. The synthetic ds-RNA, poly I-C, was used in vivo in F344 rats, based on its alleged in vitro ability to protect immunocytes from suppression by cAMP elevating agents. Different regimens of poly I-C were studied in controls and in rats subjected to a pharmacological stressor, swim stress, or surgical stress. Resistance to lung experimental metastasis of the syngeneic non-immunogenic MADB106 mammary adenocarcinoma was assessed. Numbers of circulating and marginating-pulmonary NK cells and their cytotoxicity against the MADB106 and YAC-1 target lines were also studied. Our findings established a regimen of repeated low-dose poly I-C administration with minimal side effects (0.2mg/kg i.p. 5, 3, and 1day before tumor inoculation). This regimen, while hardly affecting resistance levels in non-stressed animals, prevented all stressors from promoting metastases. These beneficial effects occurred in the presence of a primary tumor and in both sexes. Poly I-C increased the numbers of NK cells, and, on a per NK cell basis, while not increasing cytotoxicity, profoundly protected marginating-pulmonary NK cells from suppression by surgery. This study suggests a non-toxic clinically translatable prophylactic use of poly I-C to target the critical perioperative period. By increasing the number of marginating-pulmonary NK cells, and by transforming them into a mode of resistance to immunosuppression, this approach may reduce postoperative metastasis in cancer patients.

An attempt was made to assess the efficiency of the finger pulse waveform length (FPWL), in detection of concealed information. For this purpose, two mock-theft experiments were designed. In the first, 40 guilty participants were examined while electrodermal, respiration and finger pulse volume were recorded. Results showed that detection accuracy with the FPWL was at least as good as the accuracy obtained with the other two measures (respiration changes and skin conductance responses). Detection efficiency was further improved when a combination of FPWL with the other two measures was used. In the second experiment, 39 guilty and 23 innocent participants were instructed to deny knowledge while the transducers were not attached to them. Then, the same questions were repeated while electrodermal, respiration and finger pulse volume were recorded. Results showed reduced rates of identification compared to the first experiment, which were explained by habituation. However, finger pulse was less affected by habituation than both respiration and skin conductance. Results suggested that the FPWL might be a useful addition to the existing measures in the detection of concealed information.

Schizophrenia has been associated with altered immunity and reduced occurrence of autoimmune diseases and malignancies. A few studies in schizophrenic patients have assessed natural killer cell activity (NKA), but no consistent findings have emerged. However, NKA was assessed using standard procedures and in the absence of autologous serum and the various cytokines that modulate NKA and appear to be abnormal in schizophrenic patients. In the current study, therefore, the number of NK cells and the activity of the individual NK cell were assessed in whole blood shortly after blood withdrawal, in both the presence and the absence of autologous serum. Twenty-nine schizophrenic patients (11 nonmedicated), 8 nonschizophrenic control patients (bipolar and personality disorders), and 31 age-matched healthy controls were studied. Schizophrenic patients showed higher NKA per NK cell than controls and nonschizophrenic patients. This difference remained significant even when the nonmedicated schizophrenics, who showed the highest levels of NKA, were excluded. However, the increase in NKA was more pronounced in the presence of serum and was reduced to an insignificant level when serum was removed from the same samples. In both schizophrenic patients and controls, smokers and women showed lower NKA. Numbers of NK cells did not differ among groups, although medication affected blood concentration of other leukocytes. These findings indicate that the effects of serum factors, psychiatric medication, gender, and smoking should be considered when assessing NKA in schizophrenic patients. The observed higher NKA may help explain the surprising reports of low incidence of lung cancer and other malignancies in schizophrenic patients, despite their higher rate of smoking., (Copyright 2000 Academic Press.)

Background: The objective of this study was to evaluate the acquisition, generalization, and extinction of conditioned physiologic responses to aversive stimuli in posttraumatic stress disorder (PTSD)., Methods: Thirty-six PTSD patients, 20 individuals with past trauma and no current PTSD, and 30 mentally healthy individuals without exposure to major trauma underwent a differential aversive conditioning experiment. Bursts of 105 dB white noise were used as unconditioned stimuli (UCSs), and 35x24 mm slides of different colors served as either CS+ (paired) or CS- (unpaired) stimuli. Heart rate (HR) and nondominant palm skin conductance (SC) were measured at rest and between 1 and 4 sec following each CS presentation., Results: The PTSD group showed higher levels of resting SC and resting HR, larger SC responses to the initial presentation of unpaired CSs, larger HR responses following paired CS+ stimuli, larger SC responses to unpaired CS- during acquisition and extinction, and larger SC and HR responses to CS+ during extinction. The group differences in responses to CS+ during extinction remained statistically significant after controlling for age, resting physiologic levels, and initial responsivity., Conclusions: PTSD is associated with elevated autonomic responses to both innocuous and aversive stimuli, with larger responses to unpaired cues and with reduced extinction of conditioned responses.

In the differentiation-of-deception paradigm (DDP), the experimental and control conditions, respectively, consist of questions answered deceptively (D) and honestly (H). Previous DDP studies with the electrodermal SCR as the dependent variable have yielded the basic increase in responding to D relative to H questions (D > H), and have indicated that this effect is probably not due to cognitive factors such as differential retrieval difficulty, and is also relatively unaffected by motivational factors. To test the notion that the D > H effect does not represent genuine deception because of the elimination of the element of choice in the DDP, the present study varied, between two groups of 16 subjects, the degree to which subjects could choose which questions they would answer deceptively. If choice were necessary, or even important, for the differentiation-of-deception phenomenon, the D > H effect should have been greater in the free-choice condition, but the (nonsignificant) trend was in a direction opposite to this prediction. Another orthogonally-varied, between-subject manipulation, was the relative frequency of D and H items. The basic electrodermal D > H phenomenon, including the curious lack of response habituation during the session, has now been duplicated over a variety of conditions, but the mechanisms underlying the phenomenon are far from being well understood.

This study focused on the effects of mental countermeasures on psychophysiological detection in a guilty knowledge paradigm. Two experiments which utilized a 3 x 2 between subjects factorial design were conducted. Two types of mental countermeasures (specific dissociations from the relevant stimulus and continuous dissociation throughout the entire test, as well as a control-no countermeasure condition were used). Each group was further subdivided into two conditions--a high attention condition created by motivational instructions and a deceptive verbal response to the relevant question; and a low attention condition in which no motivational instructions were provided and no verbal response was required. The results of both experiments (one was conducted in a field set up, and the other utilized more standard experimental equipment and measurement procedures) revealed the following pattern: the item-specific countermeasures tended to increase psychophysiological detection, whereas the continuous dissociations tended to decrease detection efficiencies. The pattern was consistent across attention conditions and experiments but it was stronger in the field experiment where overall detection efficiency was relatively high. In other respects the present results replicated previous findings and revealed a significant effect for the attention factor.