A novel ELISpot method for adherent cells

The purpose of this study was to develop an enzyme-linked immunospot assay (ELISpot assay) that can be used with human adherent cells. While standard enzyme-linked immunosorbent assays (ELISAs) are available and widely used and ELISpot assays are used for nonadherent lymphocytes, no ELISpot assay has been developed for adherent cells. We used primary human fibroblasts from four different tissues (myometrium, lung, gingiva, and orbit), either unstimulated or interleukin (IL)-1beta-activated, to evaluate an ELISpot assay. Antibody pairs for IL-6 and IL-8 were used and results were compared to a standard ELISA. We found that we could reliably detect IL-6 and IL-8 spots with as few as 10 fibroblasts. Optimal cell numbers were 50 cells per well incubated for 8 h, although spots appeared as early as 2 h after incubation. Spots were absent when cells, primary, or secondary anti-cytokine antibodies were omitted from the protocol. Spot number and size can be ascertained using current automated ELISpot reader technology. The frequency of IL-6 and IL-8-producing human fibroblasts could also be determined. For example, 60% of the lung fibroblasts express IL-6, but IL-8 can be detected from only 40% of the cells. Approximately 80% of the human orbital fibroblasts make IL-6, whereas approximately 50% generate IL-8 following IL-1beta stimulation. These new findings show that fibroblasts from different human tissues display different frequencies of cytokine production and this further supports the concept of fibroblast diversity. The sensitivity of this new ELISpot assay is adequate for cytokine detection in just a few cells, unlike the standard ELISA. It should permit ascertaining the frequency of fibroblasts and other adherent cells that produce cytokines and, if desired, can be used in tandem with a standard ELISA to determine total cytokine produced. Moreover, the assay is suitable for normal human adherent cells that are often short-lived and difficult to cultivate.     

Bilateral Skin Conductance and Skin Potential in Schizophrenic and Normal Subjects: The Identification of the Fast Habituator Group of Schizophrenics

Chronic schizophrenics from three different hospitals were compared to normal subjects on skin conductance parameters. In addition to “Responders” and “Nonresponders” as reported by Gruzelier and Venables (1972), a group of “Fast Habituator” schizophrenics was found. These subjects produce one or at most two responses before habituation in an orienting series. The SC waveform of fast habituator subjects shows long latency, slow risetime and long recovery, although the amplitude of response is within normal limits.     

Lipophosphoglycan expression and virulence in Ricin-resistant variants of Leishmania major

Lipophosphoglycan (LPG) of Leishmania is a polymorphic molecule comprising an alkylglycerol anchor, a conserved oligosaccharide core and a species-specific polymer of oligosaccharide repeats joined by phosphodiester bonds. This molecule, together with the membrane polypeptide gp63, has been implicated as a parasite receptor for host macrophages. To examine the role of LPG in parasite infectivity glycosylation variants of Leishmania major were generated by chemical mutagenesis of a virulent cloned line V121 and variants with modified LPG selected using the galactose-specific lectin Ricinus communis II (RCA II). Twenty RCA II-resistant primary clones were generated. Analysis of LPG profile by immunoblotting using LPG-specific monoclonal and polyclonal antibodies revealed that some of the clones were LPG-deficient. Three clones that did not bind any LPG-specific antibodies but expressed normal levels of the M_r 63 000 glycoprotein (gp63), a second parasite receptor for host, were chosen for detailed studies.All three clones expressed, at least to some extent, a surface molecule which could be labeled by mild periodate oxidation and sodium borotritide and behaved like LPG by hydrophobic interaction chromatography. All clones also bound a well-characterized monoclonal antibody L157 directed to the core oligosaccharide of LPG, but did not bind another monoclonal antibody, CA7AE, to an epitope on a repeating unit shared by Leishmania donovani and L. major LPG. A third monoclonal antibody, 5E6, recognizing LPG on the surface of wild-type V121 promastigotes bound only to RCA II-resistant clone 3A2-C3 and was restricted to an internal structure. The LPG molecule that this clone expressed was a form of LPG by its chromatographic behavior and by its monosaccharide and alkylglycerol composition. Clone 3A2-C3 was the only one to infect mice in vivo and survive in macrophages in vitro, albeit at a much reduced rate compared to wild-type V121 promastigotes. The data suggest that some form of LPG may be necessary to ensure parasite infectivity.     

"Mitotic drive" of expanded CTG repeats in myotonic dystrophy type 1 (DM1)

In myotonic dystrophy type 1 (DM1), an expanded CTG repeat shows repeat size instability in somatic and germ line tissues with a strong bias toward further expansion. To investigate the mechanism of this expansion bias, 29 DM1 and six normal lymphoblastoid cell lines (LBCLs) were single-cell cloned from blood cells of 18 DM1 patients and six normal subjects. In all 29 cell lines, the expanded CTG repeat alleles gradually shifted toward further expansion by "step-wise" mutations. Of these 29 cell lines, eight yielded a rapidly proliferating mutant with a gain of large repeat size that became the major allele population, eventually replacing the progenitor allele population. By mixing cell lines with different repeat expansions, we found that cells with larger CTG repeat expansion had a growth advantage over those with smaller expansions in culture. This growth advantage was attributable to increased cell proliferation mediated by Erk1,2 activation, which is negatively regulated by p21(WAF1). This phenomenon, which we designated "mitotic drive" , is a novel mechanism which can explain the expansion bias of DM1 CTG repeat instability at the tissue level, on a basis independent of the DNA-based expansion models. The lifespans of the DM1 LBCLs were significantly shorter than normal cell lines. Thus, we propose a hypothesis that DM1 LBCLs drive themselves to extinction through a process related to increased proliferation.     

Quadriceps-hamstring EMG activity during functional, closed kinetic chain exercise to fatigue

It has been hypothesized that the ability of the neuromuscular system to co-contract muscles for joint stabilization may be impaired during the development of fatigue. The purpose of this study was to examine muscle activation of the quadriceps and hamstring muscles during a prolonged closed kinetic chain exercise, the forward lunge. Eight males and two females [mean (SD) age 26.0 (2.3) years, height 177.2 (13.6) cm, body mass 82.8 (17.1) kg] with no prior knee pathology volunteered for this study. Subjects performed repeated forward lunges onto their dominant leg at the cadence of one full lunge cycle every 2 s, until the point of volitional failure. Digital switches were positioned to record foot-strike and knee-strike of the lunge leg at the midpoint of the lunge, as well as heel-strike upon return to stance. During the lunge performance, surface electromyographic (EMG) signals of the vastus lateralis (VL), vastus medialis (VM), biceps femoris (BF), and semitendinosus (ST) muscles of the supporting leg were measured. Heart rate was also monitored every 30 s during the performance. All EMG data were full-wave rectified, partitioned into up and down phases, and integrated over the entire exercise period. The results demonstrated a significant increase in activation of the VL, VM, and BF during performance of the forward lunge to volitional failure (P < 0.05). No significant increase was shown for the ST. Heart rate increased significantly over the course of the lunge. These findings suggest that activation of the VL, VM, and BF muscles occurs as a unit during performance of the forward lunge during both concentric and eccentric lunge phases. Accepted: 25 October 1999     

Targeting of CD45 protein tyrosine phosphatase activity to lipid microdomains on the T cell surface inhibits TCR signaling

CD45, a transmembrane protein tyrosine phosphatase (PTP), can either positively or negatively regulate Src-family protein tyrosine kinase (PTK) activity in vivo. It is proposed that TCR-initiated signaling requires the segregation of PTP activities from the engaged TCR, based upon the differential membrane compartmentalization on the T cell surface. To test the importance of CD45 exclusion from lipid microdomains for proper TCR signaling, a chimeric molecule was generated by fusing the CD45 cytoplasmic region, which contains the PTP domains, to the amino-terminal 12 amino acids of Lck, which target Lck to lipid microdomains. Using 3A9 T lymphocyte hybridoma (3A9H) cells whose TCR recognizes hen egg-white lysozyme (HEL), Lck-CD45 expression resulted in its targeting to lipid microdomains. The 3A9H cells expressing Lck-CD45 were reduced in their responses to HEL or co-cross-linking of CD3 and CD4, as assessed by IL-2 production and Ca(2+) mobilization. Src-family PTK activity associated with lipid microdomains was also decreased. These results suggest that the segregation of CD45 from proximal TCR signaling components is necessary for TCR signaling and that the targeting of CD45 PTP activity to lipid microdomains on the T cell surface results in decreased sensitivity of TCR-mediated signaling.     

Fibroblast subsets in the human orbit: Thy-1+ and Thy-1- subpopulations exhibit distinct phenotypes

An emerging concept is that fibroblasts are not homogeneous, but rather consist of subsets, capable of producing regulatory mediators that control regional inflammatory responses. Fibroblasts are key effector cells in Graves' ophthalmopathy, responsible for the connective tissue remodeling, and are a rich source of inflammatory mediators. The purpose of this research was to characterize subsets of the fibroblasts in the human orbit. The strategy used was to define fibroblast subpopulations based on surface expression of the Thy-1 antigen. Fibroblast strains derived from human orbital connective tissue exhibit heterogeneous Thy-1 expression. We show, for the first time, separation of orbital fibroblasts into functionally distinct Thy-1+ and Thy-1- subsets using magnetic beading techniques. Both subsets produced the pro-inflammatory cytokine interleukin-6 (IL-6) after stimulation with IL-1beta or the CD40 pathway, whereas Thy-1+ fibroblasts produced higher levels of prostaglandin endoperoxide H synthase-2 (PGHS-2) and prostaglandin E2 (PGE(2)). Thy-1- fibroblasts produced more IL-8 than Thy-1+ fibroblasts, and when treated with interferon-gamma (IFN-gamma) up-regulated MHC class II expression more robustly. Furthermore, CD40 was expressed in a bimodal distribution within each fibroblast subset. These observations suggest that fibroblast subsets in the human orbit play distinct roles in the regulation of immune and inflammatory responses crucial in the initiation and development of thyroid-associated ophthalmopathy.     

Effects of 2 ankle fatigue models on the duration of postural stability dysfunction

Context: Muscle fatigue is generally categorized in 2 ways: that caused by peripheral weakness (peripheral fatigue) and that caused by a progressive failure of voluntary neural drive (central fatigue). Numerous variables have been studied in conjunction with fatigue protocols, including postural stability, maximum voluntary contraction force, and reaction time. When torque recordings fall below 50% of a maximum voluntary contraction, the muscle is described as fatigued, but whether this value is a good indicator of fatigue has not been studied.Objective: To compare the effects of 2 ankle musculature fatigue protocols (30% and 50%) on the duration of postural stability dysfunction.Design: To assess differences between the 30% and 50% fatigue protocols, we calculated a 1 between-groups factor (subjects) and 2 within-groups factors (fatigue, test) analysis of variance.Setting: E.J. Nutter Athletic Training Facility.Patients or Other Participants: Twenty subjects (10 men, 10 women; age = 21.15 ± 2.23 years; height = 172.97 ± 9.86 cm; mass = 70.62 ± 14.60 kg) volunteered for this study. Subjects had no history of lower extremity injury, vestibular or balance disorders, functional ankle instability, or head injury in the past 6 months.Intervention(s): On separate days, subjects performed isokinetic fatiguing contractions of the plantar flexors and dorsiflexors in a 30% protocol (70% decrease in strength) and a 50% protocol (50% decrease in strength).Main Outcome Measure(s): Baseline and postfatigue postural stability scores were determined before and after the isokinetic fatiguing contractions. Plantar-flexion peak-torque measurements were obtained for the 2 fatiguing protocols. Three prefatigue and 12 postfatigue postural stability trials were recorded. Velocities for testing were 60°/s for plantar flexion and 120°/s for dorsiflexion.Results: Sway velocity was significantly greater when the ankle was fatigued to 30% (1.56°/s) than in the 50% condition (1.36°/s). For the 30% protocol, sway was significantly impaired when the pretest condition (1.19°/s) was compared with posttest trial 1 (2.34°/s), trial 2 (2.37°/s), and trial 3 (1.71°/s). For the 50% protocol, sway was significantly impaired when the pretest condition (1.27°/s) was compared with posttest trial 1 (2.02°/s).Conclusions: The 30% fatigue protocol resulted in significantly longer impairment of postural stability than the 50% protocol. Because the 30% protocol resulted in a greater effect but was relatively short-lived (approximately 75 to 90 s), it is more useful for research purposes.     

The putative role of fibroblasts in the pathogenesis of Graves' disease: evidence for the involvement of the insulin-like growth factor-1 receptor in fibroblast activation

Graves' disease when fully expressed affects the thyroid gland and connective tissues of the orbit and pretibium. While the glandular disease is relatively well-characterized, the pathogenesis of the orbital and dermal components remains enigmatic. In the following article, we review some of the evidence suggesting that fibroblast activation in Graves' disease might play an integral role in the tissue remodeling associated with ophthalmopathy. The thyrotropin receptor (TSHR) is expressed at low levels in several connective tissue depots and by their derivative fibroblasts, including those from the orbit. Little direct evidence currently links extra-thyroidal TSHR expression with Graves' disease. Very recent observations now implicate the insulin-like growth factor-1 receptor (IGF-1R) as a fibroblast activating antigen. When immunoglobulins from patients with the disease, with or without clinical ophthalmopathy, bind IGF-1R on the surface of fibroblasts, the receptor becomes activated and upregulates the expression of two T lymphocyte chemoattractants, IL-16 and RANTES. Thus, IGF-1R may represent a second self-antigen with a pathogenic role in extra-thyroidal Graves' disease.