Evaluation of a lymph node proliferation assay for its ability to detect pharmaceuticals with potential to cause immune-mediated drug reactions

Hypersensitivity reactions to systemically administered drugs cannot be predicted using available preclinical models. This research is a collaborative project to evaluate the ability of the Lymph Node Proliferation Assay (LNPA) to predict systemic hypersensitivity caused by pharmaceuticals. The assay design is a modification of the Local Lymph Node Assay with the major modification being injection of the test substance subcutaneously to achieve a known systemic exposure to the drug. Fourteen compounds were evaluated in the LNPA. These were two clinically negative drugs (Metformin, phenobarbital), an assay positive control (streptozotocin), eight human hypersensitivity positive drugs (sulfamethoxazole, procainamide, clonidine, ofloxacin, nevirapine, abacavir, lamotrigine, zomepirac), and 3 investigational drugs (CM40874, CM40954 and CM40420), one of which caused hypersensitivity in primates. Hypersensitivity-positive drugs were classified as such based on at least two of three independent data sources: U.S. FDA postmarketing database, drug labeling information, and clinical trial data. All drugs were tested in multiple laboratories for a total of 2-12 evaluations per compound. The pure drug substance was used for testing if it could be obtained commercially, otherwise the marketed drug formulation was used. Neither of the negative control drugs showed a positive reaction in the test system. Four of the eight hypersensitivity positive drugs showed a mixed or positive reaction. Two of the three investigational compounds gave a positive response. A smaller number of LNPAs were run concurrently using footpad injection and evaluation of the popliteal lymph node and gave generally comparable results. Additional development may increase the reproducibility of the assay and facilitate detection of drugs that require metabolic activation to become allergenic, or drugs for which there is dose-limiting toxicity. The data suggest that this method might be useful as a first-line screen to identify candidate drugs that are more likely to cause a high prevalence of human drug hypersensitivity.     

Human endometrial perfusion after tubal occlusion

We examined variations in human endometrial microvascular perfusion across one menstrual cycle in women who had undergone tubal ligation and did not report unusual menstruation. Endometrial red blood cell flux was monitored by laser Doppler fluxmetry via a fibreoptic probe atraumatically inserted transvaginally into the uterus of each of 13 conscious volunteers. The observations obtained have been compared with those previously reported from a matched control group of women [B.J.Gannon et al., Hum. Reprod., 12, 132-139 (1997)]. Women who had undergone tubal occlusion for sterilization exhibited greater endometrial perfusion during menstruation (cycle days 0-5), at the time of ovulation (cycle days 13-16) and in the late secretory phase (cycle days 23-28) than occurred in controls. In addition, vasomotion in the study group was lower than that in controls in the early and late secretory phase (cycle days 17-22 and 23-28). Tubal occlusion appeared to alter endometrial perfusion. It is possible that the reported menstrual changes in women following tubal ligation are a consequence of altered endometrial perfusion; a possible causative relationship is discussed.      

Transfected Plasmodium knowlesi produces bioactive host gamma interferon: a new perspective for modulating immune responses to malaria parasites

Transgenic pathogenic microorganisms expressing host cytokines such as gamma interferon (IFN-gamma) have been shown to manipulate host-pathogen interaction, leading to immunomodulation and enhanced protection. Expression of host cytokines in malaria parasites offers the opportunity to investigate the potential of an immunomodulatory approach by generating immunopotentiated parasites. Using the primate malaria parasite Plasmodium knowlesi, we explored the conditions for expressing host cytokines in malaria parasites. P. knowlesi parasites transfected with DNA constructs for expressing rhesus monkey (Macaca mulatta) IFN-gamma under the control of the heterologous P. berghei apical membrane antigen 1 promoter, produced bioactive IFN-gamma in a developmentally regulated manner. IFN-gamma expression had no marked effect on in vitro parasite development. Bioactivity of the parasite-produced IFN-gamma was shown through inhibition of virus cytopathic effect and confirmed by using M. mulatta peripheral blood cells in vitro. These data indicate for the first time that it is feasible to generate malaria parasites expressing bioactive host immunomodulatory cytokines. Furthermore, cytokine-expressing malaria parasites offer the opportunity to analyze cytokine-mediated modulation of malaria during the blood and liver stages of the infection.     

Intensity generalization with positively reinforcing intracranial stimulation.

Rats were implanted with electrodes in positively reinforcing areas of the septal region or medial forebrain bundle. Rewarding brain stimulation, at an intermediate intensity, was established as a conditioned stimulus for a shuttle box avoidance response. When the avoidance response was learned, test trials were run using stimulation intensities 10 and 20 μa above or below the original CS intensity. Later, self-stimulation rates were collected at each current level employed in the generalization tests. The degree of stimulus intensity generalization correlated highly with changes in “reinforcement strength” demonstrating that the latter can be a salient perceptual dimension.     

Local clinical guidelines: description and evaluation of a participative method for their development and implementation

BACKGROUND: National guidelines are rarely followed by immediate changein clinical behaviour. We present our experience of an activeeducational method for local development and implementationof a guideline. OBJECTIVE: To evaluate the effectiveness of a participative method fordeveloping local clinical guidelines. METHODS: A trial in a district of the effect of guideline developmentincorporating active participation of intended recipients onsubsequent relevant prescribing. It was carried out in WirralFamily Health Services Authority district (the Wirral peninsula)comprising 69 general practices covering a population of 345763. An exemplar guideline on ‘hypertension in the elderly’was developed by the method described. The principal recommendeddrug was bendrofluazide 2.5 mg once daily. The differences inprescribed daily doses (PDD) of bendrofluazide 2.5 mg tabletsper quarter per 1000 prescribing units (age-weighted population)between the intervention district and England as a whole wasmeasured. RESULTS: Comparison of the intervention district with England data demonstratesa median difference of 122.49 PDD before and 206.34 PDD afterguideline production, this change is statistically highly significant(Mann-Whitney two-tailed P < 0.0001; 95% CL = 36.51–104.77).Grouped regression analysis shows no significant difference(0.89) in slope gradients before guideline production (P = 0.35,95% CL = –3.97–5.76), but the difference in slopegradients after (12.95) is statistically highly significant(P < 0.0001; 95% CL = 8.17–17.73). The data suggeststhat the change in clinical behaviour persisted for at leasttwo years. CONCLUSION: Participation of intended recipient general practitioners andlocal specialists in the development of a guideline by an activeeducational method as described was followed by a favourablechange in clinical behaviour which persisted for at least twoyears. Keywords. Clinical guidelines, development, evaluation, implementation, participation.     

Resistance of gonadotropin-releasing hormone neurons to glutamatergic neurotoxicity

Although many studies provide evidence that glutamatergic pathways regulate the secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, it is controversial as to whether they act directly upon GnRH neurons. The aim of the current study was to determine whether GnRH neurons are susceptible to the neurotoxic actions of specific glutamate agonists (N-methyl-D-aspartate [NMDA] and kainic acid), the rationale being that neurotoxic loss of GnRH neurons would provide evidence that the perikarya possess specific classes of glutamate receptor. Unilateral 1-microl injections of NMDA (12-120 mM), kainic acid (0.5-2.5 mM), or vehicle were stereotaxically directed at the preoptic area (mPOA)/diagonal band of Broca (dbB) in the region of the organum vasculosum of the lamina terminalis (OVLT) of male adult hamsters (Phodopus sungorus). The number and appearance of GnRH neurons were determined by immunocytochemistry 3-8 days later. The morphology of GnRH neurons in the vicinity of the injection sites appeared normal after both kainic acid and NMDA treatment, and there was no significant decrease in the numbers of GnRH perikarya identified following these treatments. Both agonists caused massive cellular loss when injected directly into cortical areas and striatum. In the experimental studies, there was little neuronal loss within the mPOA or dbB after either toxin, despite clear neuronal loss in areas adjacent to the injection sites, including ventral striatum and olfactory cortex. In follow-up studies, immunocytochemical and in situ hybridisation analysis of the NMDAR1 and NMDAR2 glutamate receptor subunits confirmed their widespread distribution in regions containing GnRH perikarya, but no colocalization within GnRH neurons was observed. The susceptibility of neural areas to NMDA neurotoxicity did not correlate with any difference in the regional expression of these glutamate receptor subunits. The resistance of GnRH neurons to the neurotoxic actions of two different glutamate agonists and the failure to detect colocalisation of NMDAR1 or NMDAR2 subunits within GnRH perikarya are consistent with the notion that the effects of glutamate upon GnRH secretion are not exerted directly upon GnRH cell bodies.     

Peritoneal and splenic B-1 cells are separable by phenotypic, functional, and transcriptomic characteristics

B-1 cells constitute a distinct B cell population with unique phenotypic and functional characteristics. Although the origin of B-1 cells remains controversial, B-1 cells in different locations are generally considered to be part of the same pool. To determine the validity of this assumption, we examined peritoneal and splenic B-1 cells isolated by flow cytometric cell sorting from normal mice for several features. We found that splenic B-1 cells differ from peritoneal B-1 cells in terms of surface antigen expression, viability ex vivo, immunoglobulin secretion in vitro, stimulated cell cycle progression, and expression of Notch family, Notch-dependent, and Notch-associated genes. These results indicate that splenic and peritoneal B-1 cells are not the same and thus dispute the notion that B-1 cells are uniform, and may suggest that different subpopulations of B-1 cells arise separately, home individually, and/or are heavily influenced by local environmental factors.     

Adeno-associated Virus Vectors Serotype 2 Induce Prolonged Proliferation of Capsid-Specific CD8+ T Cells in Mice

Using adoptive transfer models we determined that an adeno-associated viral vector of serotype 2 (AAV2) induces in mice proliferation of CD8⁺ T cells that recognize an epitope within the viral capsid. Proliferation to an endogenous epitope within viral protein (VP)3 could be observed for at least 3 weeks while a foreign epitope placed at multiple copies within VP2 elicited CD8⁺ T cell expansion for at least 10 weeks. These data show that capsid antigens of AAV2 degrade slowly over a period of weeks and during this period provide targets to CD8⁺ T cells.     

DIAPHRAGMATIC HERNIATION OF THE LARGE BOWEL ASSOCIATED WITH UNUSUAL CLINICAL AND ELECTROCARDIOGRAPHIC PRESENTATION

SUMMARY This is the first case reported of combined hiatus hernia of the stomach and herniation of the splenic flexure of the colon through the oesophageal hiatus. It was associated with unusual clinical and electrocardiographic findings. We postulate that these were the result of an increase in intrathoracic pressure induced by the herniating structures.