Biomedical subjects

S R Watson

Publications and source records attributed to S R Watson.

At least 19 recordsLinked to original sources

PubMed2001

The immune system and gene expression microarrays--new answers to old questions.

The recent increase in availability of gene expression technologies has the potential to dramatically expand our understanding of cellular immunology in molecular detail. Expression levels of tens of thousands of genes can be measured in dozens of samples in only a few days, and this data can be integrated with sequence informatics to tentatively assign some (limited) functional information to a majority of these genes. In this review we discuss some initial applications of these new tools to the fields of lymphocyte and monocyte differentiation pathways, the tolerance or immunity decision process, and B cell transformation. These examples illustrate the power of unbiased, 'wide-net', approaches both to drive immunological research in previously unexpected directions and to confirm classic tenets of immunology.

Animals↗

PubMed2000

Vestibular hypersensitivity to sound (Tullio phenomenon): structural and functional assessment.

OBJECTIVES: To establish the role of high-resolution CT imaging and tests of vestibulocollic reflexes in diagnosing and understanding the pathogenesis of the Tullio phenomenon. BACKGROUND: The Tullio phenomenon is a syndrome in which acoustic stimulation produces symptoms and signs of vestibular activation. It has previously been associated with an abnormally low threshold for click-evoked vestibulocollic responses and also with dehiscence of the roof of the anterior (superior) semicircular canal on high-resolution CT scans of the temporal bones. METHODS: High-resolution CT scans of the temporal bones and vestibulocollic responses in sternocleidomastoid to both clicks and transmastoid galvanic stimulation (3 mA/2 msec) were studied in four patients with the Tullio phenomenon (one bilateral). RESULTS: Click-evoked thresholds were low for all affected ears (four at 65 dB nHL, one at 55 dB nHL) and normal (>70 dB nHL) for the three unaffected ears. In contrast, galvanic-evoked vestibulocollic responses were symmetric and of normal size in all patients. The bony roof of the anterior (superior) semicircular canal was thin, possibly absent, on CT of all affected ears and also in two out of three unaffected ears. CONCLUSIONS: The normal galvanic vestibulocollic responses indicate that sound sensitivity in patients with the Tullio phenomenon is likely to occur distal to the vestibular nerve, probably at the level of the receptors. Both click hypersensitivity and dehiscence of the anterior (superior) semicircular canal are associated with the Tullio phenomenon but as the CT scan abnormality can occur in clinically unaffected ears, click testing is important for specific diagnosis. Abnormal sound sensitivity, as demonstrated by click responses, confirms that the radiologic abnormality is function significant.

Adult↗

PubMed2000

Anti-L-selectin aptamers: binding characteristics, pharmacokinetic parameters, and activity against an intravascular target in vivo.

Therapeutic and diagnostic applications have been envisioned for aptamers, a class of oligonucleotide ligands that bind their target molecules with high affinity and specificity (Gold, J. Biol. Chem. 270, 13581-13584, 1995). To identify parameters that are important for the in vivo activity of aptamers acting on intravascular targets, we have studied binding characteristics in vitro, pharmacokinetic parameters in Sprague-Dawley rats, and inhibitory activity in a SCID mouse/human lymphocyte model of lymphocyte trafficking for both 2'F pyrimidine 2'OH purine RNA and ssDNA anti-human L-selectin aptamers. The data indicate that aptamers with low nanomolar affinity are suitable candidates for use as in vivo reagents and that nonspecific binding to vascular cells is not an issue for efficacy. As is often observed for other reagents, plasma clearance is biphasic. Both the distribution phase and the clearance rate strongly affect in vivo activity. Pharmacokinetic parameters and in vivo activity are significantly improved by conjugating aptamers to a carrier molecule, such as polyethylene glycol (PEG). Most active in vivo is 1d40, a 2'F pyrimidine 2'OH purine aptamer conjugated to 40 kDa PEG. At a dose of 5.4 nmol/kg body weight, its duration of effect (time to 50% inhibition) is 11.2 hours, and at 1 mg or 90 nmol/kg, its plasma clearance rate (CL) is 0.4 ml/min/kg. Its ED50 is estimated to be 80 pmol/kg in preinjection dose-response experiments, compared with 4 pmol/kg for the dimeric anti-L-selectin antibody DREG56. Further improvement of in vivo activity is expected from nucleotide modifications that increase resistance to nuclease digestion for aptamers where mass is not rate limiting for clearance. Because the relationship of clearance to conjugate molecular weight (MW) is not the same for all aptamers, it is advisable to determine the relationship at the outset of in vivo studies. In summary, the data suggest that properly formulated aptamers have the capacity to be effective therapeutic agents against intravascular targets.

Animals↗

PubMed1999

Memory CD4 cells do not migrate into peripheral lymphnodes in the absence of antigen.

Memory T cells are thought to protect against previously encountered pathogens in part by preferentially recirculating through the lymphoid tissues where they were primed and where challenge with antigen (Ag) is likely to occur. In this study, we examined the distribution of memory CD4 cells after priming, and analyzed their capacity to localize in lymph nodes after transfer to normal and Ag-primed recipients. Immunization induced a high frequency of Ag-specific CD4 cells in the primary response in draining lymph nodes and spleen. Thereafter, the numbers in lymph nodes declined dramatically whereas frequencies in the spleen were unchanged, suggesting that memory CD4 cells primarily reside in or recirculate through the spleen. Indeed, memory CD4 cells, unlike naive CD4 cells, failed to home to lymph nodes after adoptive transfer to normal recipients and were detected predominantly in the spleen for extended periods, suggesting that recirculation through lymph nodes was limited. Memory cells also did not home to lymph nodes recipients in response to specific Ag, but subsequently, recruitment that could be blocked with monoclonal antibodies to CD44 and LFA-1 and was independent of naive cells did occur. The data indicate that memory and naive CD4 cells can be distinguished on the basis of their patterns of circulation.

Animals↗

PubMed1998

Vestibulocollic reflexes evoked by short-duration galvanic stimulation in man.

1. Vestibular-dependent responses in leg muscles following transmastoid galvanic stimulation have been well characterized. Here we describe the properties of vestibulocollic responses evoked by transmastoid galvanic stimulation. 2. In twelve healthy human subjects we examined the averaged responses in unrectified sternocleidomastoid (SCM) EMG evoked by transmastoid stimulation using current pulses of 4 mA intensity and 2 ms duration. In ten subjects we also examined the effects of unilateral vestibular stimulation with the indifferent electrode at the vertex. In further experiments we studied the effects of different levels of background muscle activation, head position, current intensity and current duration. We compared these responses with click-evoked vestibulocollic responses in SCM. 3. A clearly defined biphasic response, beginning with a surface positivity, was recorded in the SCM ipsilateral to the side of cathode placement in all subjects. We refer to this as the p13/n23 [g] (galvanic) response, given the close similarity, in terms of waveform and latencies, to the previously described click-evoked p13/n23 vestibulocollic response. The amplitude of this response was linearly related to background muscle activation, current intensity and current duration, but independent of head position. Unilateral galvanic stimulation revealed the p13/n23 [g] response to be solely generated by the cathode. 4. A biphasic response beginning with a surface negativity (n12/p20 [g]) contralateral to the cathode was seen in all subjects and was generated by both the cathode contralaterally and the anode ipsilaterally. 5. Both the p13/n23 [g] and n12/p20 [g] potentials were abolished by selective vestibular nerve section and unaffected by severe sensorineural deafness. 6. We conclude that galvanic stimulation evokes short-latency vestibulocollic reflexes. These vestibulocollic reflexes have properties that are distinct from those described for galvanic-evoked vestibular reflexes in leg muscles, and which may be related to their differing physiological roles.

Acoustic Stimulation↗

PubMed1998

Vestibular-evoked electromyographic responses in soleus: a comparison between click and galvanic stimulation.

The aim of this study was to demonstrate, if possible, vestibulospinal reflex responses in soleus using a stimulus known to be capable of exciting vestibular afferents, namely 100-dB (NHL) clicks. We were able to show short-latency electromyographic (EMG) responses after clicks in five of eight normal subjects, and then we compared these responses with those after transmastoid galvanic stimulation (12 normal subjects). Stimulation of the side towards which the head was rotated (i.e. the side facing backwards) with either clicks or the cathode (anode applied to the opposite side) gave an initial excitatory response in soleus, while click or cathodal stimulation of the opposite side (i.e. the side facing forwards) gave an initial inhibitory response. Onset latencies and modulation with changes in postural task were identical for both click- and galvanic-evoked responses. In addition, there was a significant correlation between the amplitudes of the responses in soleus after click and galvanic stimulation (R2=0.72). These similarities suggest that the earliest reflex responses in soleus after clicks and galvanic stimulation may be mediated by a common central pathway. In contrast, there was no correlation between the amplitudes of responses evoked by 100-dB clicks in soleus and those evoked by the same stimulus in the sternocleidomastoid. We conclude that vestibular activation by clicks can evoke reflex responses in lower-limb muscles and these responses have similar characteristics to the earliest responses evoked by galvanic vestibular stimulation.

Acoustic Stimulation↗

PubMed1998

Maintained ocular torsion produced by bilateral and unilateral galvanic (DC) vestibular stimulation in humans.

This study was designed to measure ocular movements evoked by galvanic (DC) stimulation using computerised video-oculography. Long duration (>30 s) galvanic vestibular stimulation at currents of up to 5 mA through large-area surface electrodes over the mastoid processes causes maintained changes in the ocular torsional position of both eyes in healthy human subjects. With the subject seated and the head held firmly, torsion was measured by a computer-based image-processing system (VTM). Torsion was recorded in darkness, with or without a single fixation point. With bilateral stimulation, the upper poles of both eyes always torted away from the side of cathode placement and toward the anode. For unilateral stimulation, torsion was directed away from the cathode or toward the anode. The magnitude of ocular torsion was dependent on current strength: with bilateral stimulation the peak torsion was on average 2.88 degrees for 5-mA current intensity compared with 1.58 degrees for 3 mA. A smaller amplitude of torsion was obtained for unilateral stimulation. The average peak torsion was the same for both eyes for all forms of stimulation. Our findings indicate that low-intensity galvanic stimulation evokes ocular torsion in normal subjects, an effect which is consistent with an action on otolith afferents.

Adult↗

PubMed1998

Galvanic stimulation evokes short-latency EMG responses in sternocleidomastoid which are abolished by selective vestibular nerve section.

OBJECTIVE: To describe vestibulocollic responses in sternocleidomastoid (SCM) evoked by transmastoid galvanic (DC) stimulation. METHODS: We studied the averaged responses in the unrectified EMG of SCM to transmastoid galvanic stimulation (5 mA/2 ms) and also to 100 dB clicks. Two patients with Meniere's disease were studied both before and after unilateral selective vestibular nerve section. RESULTS: Transmastoid galvanic stimulation produced a positive-negative biphasic EMG response at short latency in the SCM ipsilateral to the side of cathode placement, which resembled that which followed vestibular activation by loud clicks (p13/n23). Selective unilateral vestibular nerve section abolished this galvanic-evoked response. CONCLUSIONS: Galvanic-evoked vestibulocollic responses can be recorded in SCM. This is a new method of studying vestibular reflex function which may have application in the clinical assessment of vestibular disorders.

Adult↗

PubMed1998

Blockade of both L-selectin and alpha4 integrins abrogates naive CD4 cell trafficking and responses in gut-associated lymphoid organs.

The recirculation of naive lymphocytes from blood to lymph that is initiated in high endothelial venules (HEV) of secondary lymphoid organs such as lymph nodes and Peyer's patches (PP) is regulated by multiple interactions of adhesion receptor/counter-receptor pairs involving both selectins and integrins. We showed previously that blocking of only L-selectin is sufficient to ablate trafficking of naive CD4 cells and the development of their responses in peripheral lymph nodes but not in PP where alpha4beta7 integrins are thought to primarily regulate entry. However, although antibody to alpha4 integrins partially inhibited homing of naive CD4 cells to PP and not to lymph nodes, there was no effect on the development primary responses in these tissues or spleens. Since previous studies indicate that both alpha4beta7 integrins and L-selectin regulate adhesion of naive cells to PP HEV, we examined the effect a blockade of both adhesion pathways on the recirculation of naive CD4 cells. There was no detectable homing of naive CD4 cells to PP or lymph nodes when interactions with both receptors were inhibited, resulting in a profound depletion of naive CD4 cells and loss of antigen responses in these sites. In contrast, increased numbers of naive CD4 cells and responses of higher magnitude were found in the spleen. The results demonstrate recirculation of naive CD4 cells through tissues where entry is controlled through HEV is essential for the local generation of primary responses.

Animals↗

PubMed1998

The recirculation of naive and memory lymphocytes.

It has been clearly shown that continuous recirculation of lymphocytes is crucial for the development of primary immune responses and that naive CD4 cells are distinguished from memory CD4 cells by differences in expression of several adhesion molecules. These findings suggest that changes in migratory behavior accompany the naive to memory cell transition. This area is first reviewed and then to evaluate this hypothesis, we compare the tissue distributions of highly purified naive and memory CD4 cells after transfer to syngeneic recipients. Naive cells which express high levels of L-selectin, and low levels of alpha 4 and beta 2 integrins, and CD44 localized in secondary lymphoid organs and were detectable in these tissues and in the blood for several weeks after transfer. Memory cells, which have a reciprocal phenotype, showed a markedly different distribution, particularly with respect to tissues where entry is controlled through high endothelial venules.

Animals↗

PubMed1997

Isolation and characterization of 2'-fluoro-, 2'-amino-, and 2'-fluoro-/amino-modified RNA ligands to human IFN-gamma that inhibit receptor binding.

CD4+ Th cells produce cytokines that play a pivotal role in the induction and regulation of cell-mediated and humoral immunity. Th1 cells, characterized by their secretion of IFN-gamma, induce macrophage cytotoxicity, delayed hypersensitivity, and enhanced cellular immunity. Secretion of IFN-gamma may even suppress Th2-enhanced humoral immunity. A counterproductive Th1 response and concomitant secretion of IFN-gamma may result in inflammatory and autoimmune diseases. IFN-gamma regulation of T cell function has potential for therapeutic intervention. To isolate high affinity oligonucleotide inhibitors of IFN-gamma activity, combinatorial libraries of RNA molecules modified at the 2' position of pyrimidine nucleotides with fluoro (F), amino (NH2), or a mixture of F and NH2 (2'-F/NH2) were screened using the SELEX (systematic evolution of ligands by exponential enrichment) combinatorial chemistry process. Each modified library of RNA molecules provides an expanded repertoire of molecules with increased structural diversity and unique binding properties. This added diversity increases the possibility of isolating molecules with the desired functional properties. These RNAs modified at the 2' position have also been shown to be nuclease resistant. High affinity ligands to human IFN-gamma from each modified library were isolated and characterized. The K(d)s of these ligands were determined and their secondary structures were predicted. The specificity of these ligands for IFN-gamma binding was confirmed, and their ability to inhibit binding of IFN-gamma to its receptor on A549 human lung carcinoma cells was determined. A 2'-NH2-modified ligand (2'-NH2-30) is described that binds IFN-gamma with high affinity and inhibits IFN-gamma-induced expression of MHC class I and ICAM-1 by human myeloid leukemia cells.

Base Sequence↗

PubMed1997

Bone marrow can function as a lymphoid organ during a primary immune response under conditions of disrupted lymphocyte trafficking.

In this study we sought to better understand lymphocyte trafficking patterns and the function of secondary lymphoid organs, such as the spleen, during the generation of virus-specific T cell precursors. Treatment of mice with the Mel-14 mAb to CD62L, the lymph node homing receptor, limits trafficking of naive T cells into lymph nodes through high endothelial venules. Administering Mel-14 following respiratory infection with influenza virus forced the generation of primary virus-specific CD4+ and CD8+ T cell precursors from the mediastinal lymph nodes to the spleen. However, splenectomy did not seriously impede virus clearance from the lung and, despite a substantial reduction of the total lymphocyte pool, the acute T cell responses in the regional lymph nodes were largely normal. Mel-14 treatment of splenectomized mice did not affect clonal expansion of the virus-specific T cells in the MLN, while the response in the cervical lymph nodes was still greatly inhibited. More surprisingly, virus-specific T cell precursors were now detected from days 5 to 6 after infection in the bone marrow (BM) of the splenectomized, Mel-14-treated mice. This was not due to contamination with circulating T cells or infection of BM cells because the distribution profiles of precursor T cells for PBL and BM diverged and PCR analysis showed no evidence of virus replication in the BM. It appears that, under these conditions of disrupted lymphocyte trafficking, the BM can supplant the secondary lymphoid tissue either as a site of primary immune response or as a cache for excess T cell precursors.

Animals↗

PubMed1997

L-selectin is not essential for naive CD4 cell trafficking or development of primary responses in Peyer's patches.

We showed previously that L-selectin-dependent recirculation of naive CD4 cells is essential for development of primary responses in peripheral lymph nodes. Recent studies suggest that L-selectin is also required for lymphocyte entry into gut mucosal lymphoid tissues that include Peyer's patches and mesenteric lymph nodes. Here we show that anti-L-selectin antibody, MEL-14, inhibited homing of a rigorously purified, homogenous population of naive CD4 cells into both of these tissues as well as peripheral lymph nodes, directly demonstrating a role for this receptor in regulating entry into gut-associated sites. However, in intact animals, treatment with MEL-14 resulted in the loss of naive CD4 cells (CD45RBhi, CD44lo from peripheral lymph nodes but not Peyer's patches, whereas mesenteric lymph nodes were intermediate in this regard. In mice primed by parenteral immunization with keyhole limpet hemocyanin (KLH), primary CD4 responses were readily detected in both. Peyer's patches and mesenteric lymph nodes, and were not affected by exposure to MEL-14. Indeed, similar frequencies of KLH-specific CD4 cells were recovered from both of these tissues irrespective of MEL-14 treatment. The results indicate that interactions with L-selectin can be circumvented to allow entry of naive CD4 cells into Peyer's patches but not peripheral lymph nodes.

Animals↗

PubMed1997

EMG responses in the soleus muscles evoked by unipolar galvanic vestibular stimulation.

This study compared the effects of transmastoid galvanic stimulation with unilateral galvanic stimulation of vestibular afferents. We recorded the effects on soleus EMG occurring at short (SL) and medium (ML) latency, both in normal subjects and in patients with previous unilateral vestibular neurectomy. Unipolar cathodal and anodal stimulation on the same side produced opposite effects for both SL and ML responses. Responses to unilateral cathodal or anodal stimulation were smaller, but otherwise resembled those of transmastoid stimulation with the cathode or the anode placed on the same side, respectively. Unilateral cathodal stimulation resulted in a larger SL response, which occurred at shorter latency than unilateral anodal stimulation. With unipolar stimulation on the side of previous vestibular nerve section, typical SL and ML responses were absent. With stimulation of the intact side, the patients showed smaller SL responses than normal subjects with unilateral stimulation. The larger responses to unilateral cathodal compared to unilateral anodal stimulation are consistent with previous reports that cathodal stimulation produces an increase and anodal a decrease in vestibular nerve firing. The smaller SL responses in the patients may be a consequence of central nervous system reorganization following unilateral vestibular nerve section.

Adult↗

PubMed1996

DNA aptamers block L-selectin function in vivo. Inhibition of human lymphocyte trafficking in SCID mice.

Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics.

Animals↗

PubMed1996

L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes: interactions between the lectin, epidermal growth factor, and consensus repeat domains of the selectins determine ligand binding specificity.

The selectins mediate cellular interactions by binding carbohydrate determinants present on a limited number of glycoprotein ligands. L-selectin binds multiple ligands expressed on endothelial cells, while P-selectin interacts exclusively with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. In this study, L-selectin was shown to bind leukocytes through the P-selectin ligand, PSGL-1, although at lower levels than P-selectin. L-selectin binding to PSGL-1 is specific since it was blocked by Abs to L-selectin or PSGL-1, required appropriate glycosylation of PSGL-1, and was Ca2+ dependent. The contributions of the extracellular domains of the selectins to ligand binding was assessed using a panel of chimeric selectins created by exchange of domains between L-selectin and P- or E-selectin. The lectin and epidermal growth factor domains of L- and P-selectin contributed significantly to binding through similar, if not identical, regions of PSGL-1. The different chimeric selectins revealed that the lectin domain was the dominant determinant for ligand binding, while cooperative interactions between the lectin, epidermal growth factor, and short consensus repeat domains of the selectins also modified ligand binding specificity. L-selectin binding to PSGL-1 expressed by leukocytes may mediate neutrophil rolling on stationary leukocytes bound to cytokine-induced endothelial cells, which was previously reported to be a L-selectin-dependent process.

Amino Acid Sequence↗

PubMed1996

Functional and structural characterization of the eosinophil P-selectin ligand.

Our recent studies have indicated an important role for P-selectin in eosinophil adhesion. We have therefore compared eosinophil and neutrophil binding with nasal polyp endothelium as well as purified P-selectin. We have also compared the structure and expression of the eosinophil and neutrophil P-selectin ligands. Using the frozen section assay, eosinophils bound to 2-fold more blood vessels within the nasal polyp tissue than neutrophils. Up to 10-fold more eosinophils than neutrophils bound per unit length of endothelium. Neutrophil and eosinophil binding was inhibited by a mAb against P-selectin and a P-selectin chimera which binds to the P-selectin ligand. Eosinophils bound with approximately 2-fold greater avidity to purified P-selectin under flow conditions. Using SDS-PAGE we characterized the eosinophil P-selectin ligand as a sialylated, homodimeric glycoprotein consistent with the known structure of PSGL-1. However, expression of PSGL-1 by eosinophils was significantly greater than on neutrophils. The eosinophil ligand had a calculated molecular mass by SDS-PAGE of approximately 10 kDa greater than the neutrophil ligand, which was not due to differences in N-glycosylation. Eosinophils expressed the 15-decapeptide repeat form of PSGL-1 compared with neutrophils that have the 16-decapeptide repeat form. The increased binding of eosinophils, compared with neutrophils, to P-selectin in both an ex-vivo and in vitro assay suggests that P-selectin may have a role directing the specific migration of eosinophils in diseases such as asthma. The increased avidity may be due to increased expression of PSGL-1 by eosinophils, differences in the peptide backbone, or post-translational modifications.

Antibodies, Monoclonal↗

PubMed1996

Lymphocyte migration into tissue: the paradigm derived from CD4 subsets.

The appropriate recirculation and migration of naive, effector and memory T cells into inflamed tissue are precisely controlled by adhesive interactions with vascular endothelium. Analyses of CD4 lymphocytes have indicated that naive and antigen-experienced cells exhibit distinctive patterns of homing and recirculation, and that subsets of cells preferentially localize in different anatomical locations as a consequence of previous antigen exposure and differences in adhesion receptor usage.

Animals↗