Cytokine release assays: current practices and future directions.

As a result of the CD28 superagonist biotherapeutic monoclonal antibody (TGN 1412) "cytokine storm" incident, cytokine release assays (CRA) have become hazard identification and prospective risk assessment tools for screening novel biotherapeutics directed against targets having a potential risk for eliciting adverse pro-inflammatory clinical infusion reactions. Different laboratories may have different strategies, assay formats, and approaches to the reporting, interpretation, and use of data for either decision making or risk assessment. Additionally, many independent contract research organizations (CROs), academic and government laboratories are involved in some aspect of CRA work. As a result, while some pharmaceutical companies are providing CRA data as part of the regulatory submissions when necessary, technical and regulatory practices are still evolving to provide data predictive of cytokine release in humans and that are relevant to safety. This manuscript provides an overview of different approaches employed by the pharmaceutical industry and CROs, for the use and application of CRA based upon a survey and post survey follow up conducted by ILSI-Health and Environmental Sciences Institute (HESI) Immunotoxicology Committee CRA Working Group. Also discussed is ongoing research in the academic sector, the regulatory environment, current limitations of the assays, and future directions and recommendations for cytokine release assays.

Defective inflammatory response in interleukin 6-deficient mice.

Systemic and localized inflammation elicit a number of host responses which include fever, cachexia, hypoglycemia, and major changes in the concentration of liver plasma proteins. Interleukin 6 (IL-6) is considered an important mediator of the inflammatory response, together with IL-1 and tumor necrosis factor alpha (TNF-alpha). The purpose of this study was to unequivocally determine the role of IL-6 in these phenomena making use of IL-6-deficient mice that we have recently generated by gene targeting. We report here that in the absence of IL-6, mice are unable to mount a normal inflammatory response to localized tissue damage generated by turpentine injection. The induction of acute phase proteins is dramatically reduced, mice do not lose body weight and only suffer from mild anorexia and hypoglycemia. In contrast, when systemic inflammation is elicited through the injection of bacterial lipopolysaccharide (LPS), these parameters are altered to the same extent both in wild-type and IL-6-deficient mice, demonstrating that under these conditions IL-6 function is dispensable. Moreover, we show that LPS-treated IL-6-deficient mice produce three times more TNF-alpha than wild-type controls, suggesting that increased TNF-alpha production might be one of the compensatory mechanisms through which a normal response to LPS is achieved in the absence of IL-6. We also show that corticosterone is normally induced in IL-6-deficient mice, demonstrating that IL-6 is not required for the activation of the hypothalamic-pituitary-adrenal axis. Our results reinforce the idea that different patterns of cytokines are involved in systemic and localized tissue damage, and identify IL-6 as an essential mediator of the inflammatory response to localized inflammation.

Molecular mapping and detoxification of the lipid A binding site by synthetic peptides.

Endotoxin [lipopolysaccharide (LPS)], the major antigen of the outer membrane of Gram-negative bacteria, consists of a variable-size carbohydrate chain that is covalently linked to N,O-acylated beta-1,6-D-glucosamine disaccharide 1,4'-bisphosphate (lipid A). The toxic activity of LPS resides in the lipid A structure. The structural features of synthetic peptides that bind to lipid A with high affinity, detoxify LPS in vitro, and prevent LPS-induced cytokine release and lethality in vivo were defined. The binding thermodynamics were comparable to that of an antigen-antibody reaction. Such synthetic peptides may provide a strategy for prophylaxis and treatment of LPS-mediated diseases.

Upregulation of uncoupling protein 2 mRNA in genetic obesity: lack of an essential role for leptin, hyperphagia, increased tissue lipid content, and TNF-alpha.

Uncoupling protein 2 (UCP2) has been proposed to play a prominent role in the regulation of energy balance. UCP2 mRNA expression is upregulated in white adipose tissue (WAT) and liver, but is not altered in skeletal muscle in genetically obese ob/ob mice. The mechanisms involved in the upregulation of UCP2 in obesity have not been investigated. We have now examined the potential role of leptin, hyperphagia, increased tissue lipid content, and overexpression of tumor necrosis factor (TNF)-alpha in the upregulation of UCP2 mRNA expression in the liver and WAT in ob/ob mice. Treatment of ob/ob mice with leptin for 3 days significantly reduced their food intake but had no effect on the upregulation of UCP2 mRNA levels in the liver or WAT. To investigate the effect of feeding and higher tissue lipid content on the upregulation of UCP2 in liver and WAT, we compared UCP2 mRNA levels in ad-libitum fed and 72-h fasted control and ob/ob mice. In controls, fasting had no effect on UCP2 mRNA levels in liver, but increased UCP2 mRNA in WAT suggesting that the effects of fasting on UCP2 mRNA levels are tissue-specific. In ob/ob mice, fasting did not lower UCP2 mRNA levels in liver or WAT suggesting that the upregulation of UCP2 in ob/ob mice is not merely a direct consequence of increased food intake. 72-h fasting lowered hepatic total lipid content by 34% and 36% in control and ob/ob mice, respectively, without any corresponding decrease in hepatic UCP2 mRNA levels, suggesting that the enhanced UCP2 expression in the liver of ob/ob mice is not secondary to lipid accumulation in their livers. Although TNF-alpha has been shown to acutely increase UCP2 mRNA levels in liver and WAT, and is overexpressed in adipose tissue in obesity, deletion of the genes for both TNF receptors in ob/ob mice produces a further increase in UCP2 mRNA expression in liver and adipose tissue indicating a paradoxical inhibitory role. Taken together, these results suggest that the upregulation of UCP2 mRNA levels in the liver and WAT of ob/ob mice is not due to the lack of leptin, hyperphagia, increased tissue lipid content, or over-expression of TNF-alpha.

Leptin regulation of the immune response and the immunodeficiency of malnutrition.

Leptin is a 16 kDa protein mainly produced by adipose tissue in proportion to adipose tissue mass. Originally thought to be a satiety factor, leptin is a pleiotropic molecule. In addition to playing a role in energy regulation, leptin also regulates endocrine and immune functions. Both the structure of leptin and that of its receptor suggest that leptin might be classified as a cytokine. The secondary structure of leptin has similarities to the long-chain helical cytokines family, which includes interleukin 6 (IL-6), IL-11, CNTF, and LIF, and the leptin receptor is homologous to the gp-130 signal-transducing subunit of the IL-6-type cytokine receptors. Leptin plays a role in innate and acquired immunity. Leptin levels increase acutely during infection and inflammation, and may represent a protective component of the host response to inflammation. More important, leptin deficiency increases susceptibility to infectious and inflammatory stimuli and is associated with dysregulation of cytokine production. Leptin deficiency also causes a defect in hematopoiesis. Leptin regulates T cells responses, polarizing Th cells toward a Th1 phenotype. Low leptin levels occurring during starvation mediate the neuroendocrine and immune dysfunction of starvation.

Leptin in the regulation of immunity, inflammation, and hematopoiesis.

Leptin, the product of the ob gene, is a pleiotropic molecule that regulates food intake as well as metabolic and endocrine functions. Leptin also plays a regulatory role in immunity, inflammation, and hematopoiesis. Alterations in immune and inflammatory responses are present in leptin- or leptin-receptor-deficient animals, as well as during starvation and malnutrition, two conditions characterized by low levels of circulating leptin. Both leptin and its receptor share structural and functional similarities with the interleukin-6 family of cytokines. Leptin exerts proliferative and antiapoptotic activities in a variety of cell types, including T lymphocytes, leukemia cells, and hematopoietic progenitors. Leptin also affects cytokine production, the activation of monocytes/macrophages, wound healing, angiogenesis, and hematopoiesis. Moreover, leptin production is acutely increased during infection and inflammation. This review focuses on the role of leptin in the modulation of the innate immune response, inflammation, and hematopoiesis.

Delineation of a 1-cM region on distal 5q containing the locus for corneal dystrophies Groenouw type I and lattice type I and exclusion of the candidate genes SPARC and LOX.

Granular Groenouw type I (CDGG1) and lattice type 1 (CDL1) corneal dystrophies are two distinct potentially blinding conditions. These two entities were recently mapped to a region on chromosome 5q. We have investigated 2 families of Swiss origin with CDGG1 and CDL1 by linkage analysis. Our data show a maximum lod score of 5.38 at theta = 0.00 for marker D5S393 in CDL1 and 4.17 at theta = 0.00 for D5S658 in CDGG1. When combined, these families show a maximum low score of 9.22 for D5S393 at theta = 0.00. This confirms previous reports. Furthermore, we describe a recombination centromeric to D5S399 in a member of the CDL1 family. Haplotype analysis in the 4 branches of the CDGG1 family demonstrated a common chromosomal region including D5S393 and D5S399 in all the affected members. By combining our data with previously reported mapping information and assuming that CDGG1 and CDL1 are allelic manifestations of the same gene, we can refine the location of the CDGG1/CDL1 gene to a 1-cM region on chromosome 5q. Using candidate genes in the 5q22-q32 interval, we investigated the possibility that mutations in the SPARC or LOX genes cause these corneal diseases. Several recombinations occurred between these two genes and CDGG1/CDL1 in our 2 families, thus excluding this hypothesis.

Recently identified peptides involved in the regulation of body weight.

The application of molecular and genetic techniques to the study of body weight regulation have produced exciting new insights into the physiological systems governing energy expenditure, appetite, and metabolic signaling. A number of new peptides have been identified that play important roles in these regulatory systems. These include the hormone leptin, the short and long forms of the leptin receptor, uncoupling proteins, agouti protein, melanocortin receptor isoforms, melanin-concentrating hormone, and the proteins responsible for tub and fat, two monogenic mouse models of obesity. This article reviews some of the new insights gained from studies of these peptides. Although much of this new knowledge has come from studies of obesity, there may be implications for the clinical syndromes associated with weight loss. As more is learned about these systems, potential new targets for therapeutic intervention will likely become evident. These interventions may develop first as obesity treatments, but investigators and clinicians involved in the care of cachectic patients should follow these scientific developments as well.

Mechanisms of interleukin-2-induced depression of hepatic cytochrome P-450 in mice.

Interleukin-2 (15 micrograms/mouse, i.p. twice daily for 4 days and once on the 5th day) significantly lowered cytochrome P-450 and heme content and increased heme oxygenase mRNA accumulation; the activities of 7-ethoxycoumarin O-deethylase, ethoxy- and pentoxyphenoxazone O-dealkylases were decreased. The activity of the type O form of hepatic xanthine oxidase increased, but there was no increase in lipid peroxide, expressed in terms of microsomal malondialdehyde. In vivo inactivation of xanthine oxidase activity by feeding mice with tungstate did not substantially change the degree of interleukin-2-induced cytochrome P-450 depression, suggesting that the two processes are not causally linked. Induction of tolerance to endotoxin by a 4-day pretreatment with lipopolysaccharide resulted in 50% protection against this depression despite inhibition of the interleukin-2 induced formation of tumor necrosis factor. This suggests that the release of tumor necrosis factor per se does not fully account for the depression of cytochrome P-450. Dexamethasone, already used in patients to reduce the toxicity of interleukin-2 therapy, provided full protection against the cytochrome P-450 depression.

Urinary TNF-binding protein (TNF soluble receptor) protects mice against the lethal effect of TNF and endotoxic shock.

We tested the effect of urinary TNF-binding protein (uTBP) on the toxic effect of TNF (0.5 micrograms/mouse, i.v.) in adrenalectomized mice sensitized with IL-1 to increase susceptibility to TNF. In this experimental model, mortality was 67%, but decreased to 13% when uTBP (250 micrograms/mouse, i.v.) was administered simultaneously with TNF. The protective effect of uTBP was dose-dependent, and time course experiments indicated a protective effect when uTBP was administered before or up to one hour after TNF. Some protection was also obtained when uTBP was given three hours after TNF. Urinary TBP improved the survival of mice after a lethal dose of LPS (1.2 mg/mouse, i.p.), suggesting its possible efficacy in the therapy of septic shock or other TNF-mediated pathologies.

Ultrasound biomicroscopy of eyes undergoing deep sclerectomy with collagen implant.

AIMS: To assess the intraocular pressure (IOP) lowering mechanism of deep sclerectomy with collagen implant (DSCI), a non-penetrating glaucoma surgery. METHODS: Nine eyes of nine patients with medically uncontrolled open angle glaucoma underwent DSCI. Ultrasound biomicroscopy (UBM) of the sclerectomy site was performed 1 month after surgery. The following factors were assessed: length and height of collagen implant, and thickness of the residual trabeculocorneal membrane. RESULTS: Postoperative IOP decreased significantly in all nine eyes from a preoperative mean value of 25.8 (SD 4.8) mm Hg to a postoperative (1 month) mean value of 11.3 (6.3) mm Hg (p = 0.001). In all nine eyes, UBM at 1 month after surgery showed a subconjunctival filtration through the thin trabeculocorneal membrane and through the scleral flap around the collagen implant. In four cases, a hypoechoic area in the suprachoroidal space was observed and might represent ciliary body detachment or be due to suprachoroidal drainage of aqueous humour through the thin deep scleral wall. At 1 month after surgery the mean trabeculocorneal membrane thickness was 110.1 (16.8) microns, and the mean length and height of the collagen implant were 2.3 (0.1) mm and 1.1 (0.1) mm respectively. CONCLUSION: DSCI lowered IOP by allowing aqueous filtration through a thin trabeculocorneal membrane to the subconjunctival space and, eventually, to the suprachoroidal space.

One year follow-up of IOGEL intraocular lenses with ciliary sulcus fixation.

Fifty-one soft hydrogel posterior chamber intraocular lenses (IOLs) were implanted in the ciliary sulcus after planned extracapsular cataract extractions. In nine cases the IOLs were positioned with one haptic in the capsular bag and the other in the ciliary sulcus. During a one-year follow-up, Nd:YAG capsulotomy was performed on five eyes and cystoid macular edema was present in two cases. No other serious complications were encountered. At one year, visual acuity was 20/30 or better in all eyes that did not have preexisting secondary eye problems. To prevent postoperative rotation, decentration, and deformation of the IOL, unnecessary intraocular manipulation and "in-out" positioning of the IOL should be avoided.

Comparison of deep sclerectomy with collagen implant and trabeculectomy in open-angle glaucoma.

PURPOSE: To assess the efficacy and postoperative complications of deep sclerectomy with collagen implant (DSCI), a nonpenetrating filtration procedure. SETTING: Glaucoma Unit, Department of Ophthalmology, University of Lausanne, Switzerland. METHODS: Forty-four eyes of 44 patients with medically uncontrolled open-angle glaucoma had DSCI and a matched control group of 44 patients, trabeculectomy. A superficial scleral flap was raised and a deep sclerectomy performed in the scleral bed. Schlemm's canal was opened, and the cornea was dissected to Descemet's membrane. At that stage, aqueous filtered through the remaining trabeculo-Descemet's membrane. A collagen implant was sutured radially in the scleral bed; the scleral flap and conjunctiva were then closed. Examinations were performed before surgery and postoperatively at 1 and 7 days and 1, 2, 3, 6, 9, 12, 15, 18, and 24 months. RESULTS: The mean follow-up was 14.4 months +/- 6.3 (SD) (range 3 to 24 months). The mean preoperative intraoperative pressure (IOP) was 26.7 +/- 7.3 mm Hg. The mean postoperative IOP was 6.1 +/- 4.5 mm Hg at 1 day and 11.0 +/- 4.4 mm Hg at 1 week; it remained stable for the next 24 months. The success rate, defined as an IOP lower than 21.0 mm Hg without medication, was 69% in the DSCI group and 57% in the trabeculectomy group at 24 months postoperatively (P = .047). The number of postoperative complications was significantly lower in the DSCI group than in the trabeculectomy group. CONCLUSIONS: The success rate of DSCI may be comparable to that of trabeculectomy, with fewer complications.

[Treatment of normal pressure glaucoma with a serotonin S2 receptor antagonist, naftidrofuryl (praxilen)]. / Le traitement du glaucome à pression normale avec un antagoniste des recepteurs S2 de la sérotonine, le naftidrofuryl (praxilen).

35 normal tension glaucoma patients were followed during 8 to 11 month. 12 patients received 2 x 200 mg/day Naftidrofuryl, a serotonine S2 receptor antagonist. As controles, another 23 patients were followed without treatment. The treated group, compared with the 23 non treated patients showed a significant increase in visual acuity (p = 0.001), a significant increase of the MS (p = 0.03), and a non significant decrease of the MD and CLV on the Octopus visual field (p, NS). The physiology and physiopathology of serotonine and S2 receptor antagonist are discussed.

[Hereditary macular retinoschisis]. / Rétinoschisis maculaire héréditaire.

The authors examined and studied a family of Italian origin presenting with hereditary macular retinoschisis. Two first cousins of the third generation were affected by this disease. The mode of transmission of the pathologic gene is compatible with a recessive sex-linked heredity. The correlation between the severity of the cystic macular lesions and their functional consequences is not necessarily absolute. The lesions, which appear to be present very soon after birth, increase in severity with the years without any noticeable changes in macular function, which remains weak but stable.

[Short and long-term advantages and disadvantages of prolene monofilament sutures in penetrating keratoplasty]. / Avantages et inconvénients à court et long terme des sutures au monofilament de Prolène dans les kératoplasties perforantes.

The authors have studied the short and long-term advantages and inconveniences of Prolene monofilament sutures employed in perforating keratoplastic surgery. Contrary to Nylon, Prolene has the advantage of no structural or tensional modifications, postoperative measurements of corneal astigmatism thus remaining more stable with the time. Accordingly, where very little or no astigmatism is noted, the slight or no change in corneal refraction with time is an obvious advantage ot the patient. However, the use of Prolene in the beginning can cause some problems with continuous suture tension, either excessive or insufficient, which can result in serious complications. With training, these difficulties can be overcome and excellent results obtained.