Transfection of drug-specific T-cell receptors into hybridoma cells: tools to monitor drug interaction with T-cell receptors and evaluate cross-reactivity to related compounds

In the context of drug hypersensitivity, our group has recently proposed a new model based on the structural features of drugs (pharmacological interaction with immune receptors; p-i concept) to explain their recognition by T cells. According to this concept, even chemically inert drugs can stimulate T cells because certain drugs interact in a direct way with T-cell receptors (TCR) and possibly major histocompatibility complex molecules without the need for metabolism and covalent binding to a carrier. In this study, we investigated whether mouse T-cell hybridomas transfected with drug-specific human TCR can be used as an alternative to drug-specific T-cell clones (TCC). Indeed, they behaved like TCC and, in accordance with the p-i concept, the TCR recognize their specific drugs in a direct, processing-independent, and dose-dependent way. The presence of antigen-presenting cells was a prerequisite for interleukin-2 production by the TCR-transfected cells. The analysis of cross-reactivity confirmed the fine specificity of the TCR and also showed that TCR transfectants might provide a tool to evaluate the potential of new drugs to cause hypersensitivity due to cross-reactivity. Recombining the alpha- and beta-chains of sulfanilamide- and quinolone-specific TCR abrogated drug reactivity, suggesting that both original alpha- and beta-chains were involved in drug binding. The TCR-transfected hybridoma system showed that the recognition of two important classes of drugs (sulfanilamides and quinolones) by TCR occurred according to the p-i concept and provides an interesting tool to study drug-TCR interactions and their biological consequences and to evaluate the cross-reactivity potential of new drugs of the same class.

Production of monoclonal antibodies specific for native equine IgE and their application to monitor total serum IgE responses in Icelandic and non-Icelandic horses with insect bite dermal hypersensitivity

Immunoglobulin E forms a minor component of serum antibody in mammals. In tissues IgE is bound by FcvarepsilonRI receptors on the surface of mast cells and mediates their release of inflammatory substances in response to antigen. IgE and mast cells have a central role in immunity to parasites and the pathogenesis of allergic diseases in horses and other mammals. This paper describes the production of several novel monoclonal antibodies that detect native equine IgE in immunohistology, ELISA and Western blotting. An antigen capture ELISA to quantify equine IgE in serum has been developed using two of these antibodies. The mean serum IgE concentration of a group of 122 adult horses was 23,523ng/ml with a range of 425-82,610ng/ml. Total serum IgE of healthy horses was compared with that of horses with insect bite dermal hypersensitivity (IBDH) an allergic reaction to the bites of blood feeding insects of Culicoides or Simulium spp. IBDH does not occur in Iceland where Culicoides spp. are absent, but following importation into mainland Europe native Icelandic horses have an exceptionally high incidence of this condition. In the present study Icelandic horses with IBDH had significantly higher total IgE than healthy Icelandic horse controls (P<0.05). By contrast in horses of other breeds the difference in total serum IgE between those affected with IBDH and healthy controls was not statistically significant. Total serum IgE was also monitored in a cohort of Icelandic horses prior to import into Switzerland and for a period of 3 years thereafter. High levels of serum IgE were present in all horses at the start of the study but dropped in the first year after import. Thereafter the total serum IgE remained low in Icelandic horses that remained healthy but rose significantly (P<0.05) in those that developed IBDH. These results support the conclusion that IBDH is a type I hypersensitivity response to insect allergens but indicate that IBDH in Icelandic horses may have a different pathogenesis from the same condition in other breeds.

Intrapatient reproducibility of the BISxp monitor

BACKGROUND: The Bispectral Index (BIS) reportedly reflects anesthetic depth. It is recommended that anesthetic agents should be titrated to maintain the BIS between 40 and 60 arbitrary BIS units during anesthesia. For anesthesia providers to follow this recommendation, the monitor should be predictably affected by different anesthetic agents and have good interpatient and intrapatient reproducibility. The authors hypothesized that when two BISxp devices (Aspect Medical Systems, Newton, MA) are placed concurrently on the same patient, their readings are concordant throughout the anesthetic period. METHODS: Simultaneous BIS recordings from two BISxp monitors were obtained during anesthesia at 5-s intervals from 12 participants. RESULTS: In total 22,860 concurrent paired BIS readings were obtained. For 10.7% of the time, there were sustained periods of 30 s or greater where the readings suggested a different depth of anesthesia. For 6% of the time, there were sustained periods of 30 s or greater where the readings differed by 10 or more arbitrary BIS units. The regression coefficient (R) for the two devices was 0.65 (range, 0.35-0.92). There was zero bias between the devices, and the 95% limits of agreement ranged between -18 and +17. CONCLUSION: A conflicting anesthetic management was suggested by the simultaneous BIS readings 10.7% of the time. These results suggest that BISxp does not always provide a reproducible single number. Anesthesia providers should not rely exclusively on the BIS reading when assessing depth of anesthesia.

MRI for differentiating ganglion and synovitis in the chronic painful wrist

OBJECTIVE: The purpose of our study was to determine if preoperative MRI can differentiate between occult ganglion and synovitis in the chronic painful wrist. CONCLUSION: MRI is accurate in preoperatively distinguishing between ganglion and synovitis in the setting of chronic dorsal wrist pain. Four main criteria were useful: margin, shape, internal structure, and enhancement after administration of contrast material, with shape and internal structure being most helpful.

Actigraphy in agitated patients with dementia : Monitoring treatment outcomes

Especially in pharmacotherapeutic research, a variety of methods to monitor behavioural and psychological symptoms of dementia (BPSD) are currently being discussed. To date, the most frequently used of these are clinical scales, which, however, are subjective and highly dependent on personnel resources. In our study, we tested the usefulness of actigraphy as a more direct and objective way to measure day-night rhythm disturbances and agitated behaviour.After a baseline assessment, 24 patients with probable dementia of the Alzheimer type (NINCDS-ADRDA) and agitated behaviour received either 3 mg melatonin (n=7), 2.5 mg dronabinol (n=7), or placebo (n=10) for two weeks. In addition, 10 young and 10 elderly healthy subjects were examined as a control group. Motor activity levels were assessed using an actigraph worn continuously on the wrist of the non-dominant hand. At the beginning and the end of the study, patients' Neuropsychiatric Inventory (NPI) scores were also assessed.In the verum group, actigraphic nocturnal activity (P=0.001), NPI total score (P=0.043), and NPI agitation subscale score (P=0.032) showed significant reductions compared to baseline. The treatment-baseline ratio of nocturnal activity (P=0.021) and treatment-baseline difference of the nocturnal portion of 24 h activity (P=0.012) were reduced. Patients' baseline activity levels were similar to those seen in healthy elderly subjects. Younger healthy subjects exhibited higher motor activity even at night. There was no correlation between actigraphy and NPI.Both actigraphic measures and the gold standard clinical scale were able to distinguish between the verum and placebo groups. However, because they did not correlate with each other, they clearly represent different aspects of BPSD, each of which reacts differently to therapy. As a result, actigraphy may well come to play an important role in monitoring treatment success in BPSD.

Finite element study on fracture patterns in human scaphoid wrist bone due to free fall

Scaphoid is one of the 8 carpal bones found adjacent to the thumb supported proximally by Radius bone. During the free fall, on outstretched hand, the impact load gets transferred to the scaphoid at its free anterior end. Unique arrangement of other carpal bones in the palm is also one of the reasons for the load to get transferred to scaphoid. About half of the total load acting upon carpal bone gets transferred to scaphoid at its distal pole. There are about 10 to 12 clinically observed fracture pattern in the scaphoid due to free fall. The aim of the study is to determine the orientation of the load, magnitude of the load and the corresponding fracture pattern. This study includes both static and dynamic finite element models validated by experiments. The scaphoid model has been prepared from CT scans of a 27 year old person. The 2D slices of the CT scans have been converted to 3D model by using MIMICS software. There are four cases of loading studied which are considered to occur clinically more frequently. In case (i) the load is applied at the posterior end at distal pole whereas in case (ii), (iii) and (iv), the load is applied at anterior end at different directions. The model is given a fixed boundary condition at the region which is supported by Radius bone during the impact. Same loading and boundary conditions have been used in both static and dynamic explicit finite element analysis. The site of fracture initiation and path of fracture propagation have been identified by using max principal stress / gradient and max principal strain / gradient criterion respectively in static and dynamic explicit finite element analysis. Static and dynamic impact experiments were performed on the polyurethane foam specimens to validate the finite element results. Experimental results such as load at fracture, site of fracture initiation and path of fracture propagation have been compared with the results of finite element analysis. Four different types of fracture patterns observed in clinical studies have been identified in this study.

A new method to monitor visual field defects caused by photoreceptor degeneration by quantitative optical coherence tomography

PURPOSE: To correlate the dimension of the visual field (VF) tested by Goldman kinetic perimetry with the extent of visibility of the highly reflective layer between inner and outer segments of photoreceptors (IOS) seen in optical coherence tomography (OCT) images in patients with retinitis pigmentosa (RP). METHODS: In a retrospectively designed cross-sectional study, 18 eyes of 18 patients with RP were examined with OCT and Goldmann perimetry using test target I4e and compared with 18 eyes of 18 control subjects. A-scans of raw scan data of Stratus OCT images (Carl Zeiss Meditec, AG, Oberkochen, Germany) were quantitatively analyzed for the presence of the signal generated by the highly reflective layer between the IOS in OCT images. Starting in the fovea, the distance to which this signal was detectable was measured. Visual fields were analyzed by measuring the distance from the center point to isopter I4e. OCT and visual field data were analyzed in a clockwise fashion every 30 degrees , and corresponding measures were correlated. RESULTS: In corresponding alignments, the distance from the center point to isopter I4e and the distance to which the highly reflective signal from the IOS can be detected correlate significantly (r = 0.75, P < 0.0001). The greater the distance in VF, the greater the distance measured in OCT. CONCLUSIONS: The authors hypothesize that the retinal structure from which the highly reflective layer between the IOS emanates is of critical importance for visual and photoreceptor function. Further research is warranted to determine whether this may be useful as an objective marker of progression of retinal degeneration in patients with RP.