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.

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.

IL-18-binding protein protects against lipopolysaccharide- induced lethality and prevents the development of Fas/Fas ligand-mediated models of liver disease in mice.

IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (K(D) 0.3-5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-gamma production by KG1 cells (EC(50) = 0.3 microg/ml). In mice challenged with an LD(90) of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-gamma production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-gamma production induced by LPS (5 mg/kg) with ED(50) of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-gamma production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-gamma and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1alpha and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-gamma and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.

WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection.

WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors. The physiological role of WSX-1, which is expressed mainly in T cells, was investigated in gene-targeted WSX-1-deficient mice. IFN-gamma production was reduced in isolated WSX-1(-/-) T cells subjected to primary stimulation in vitro to induce Th1 differentiation but was normal in fully differentiated and activated WSX-1(-/-) Th1 cells that had received secondary stimulation. WSX-1(-/-) mice were remarkably susceptible to Leishmania major infection, showing impaired IFN-gamma production early in the infection. However, IFN-gamma production during the later phases of the infection was not impaired in the knockout. WSX-1(-/-) mice also showed poorly differentiated granulomas with dispersed accumulations of mononuclear cells when infected with bacillus Calmette-Guerin (BCG). Thus, WSX-1 is essential for the initial mounting of Th1 responses but dispensable for their maintenance.

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.

Reduced leptin levels in starvation increase susceptibility to endotoxic shock.

Malnutrition compromises immune function, reducing resistance to infection. We examine whether the decrease in leptin induced by starvation increases susceptibility to lipopolysaccharide (LPS)- and tumor necrosis factor (TNF)-induced lethality. In mice, fasting for 48 hours enhances sensitivity to LPS. Decreasing the fasting-induced fall in leptin by leptin administration markedly reduced sensitivity to LPS. Although fasting decreases basal leptin levels, LPS treatment increased leptin to the same extent as in fed animals. Fasting increased basal serum corticosterone; leptin treatment blunted this increase. Fasting decreased the ability of LPS to increase corticosterone; leptin restored the corticosterone response to LPS. Serum glucose levels were decreased in fasted mice and LPS induced a further decrease. Leptin treatment affected neither basal glucose nor that after LPS. LPS induced a fivefold greater increase in serum TNF in fasted mice, which was blunted by leptin replacement. In contrast, LPS induced lower levels of interferon-gamma and no differences in interleukin-1beta in fasted compared to fed animals; leptin had no effect on those cytokines. Furthermore, fasting increased sensitivity to the lethal effect of TNF itself, which was also reversed by leptin treatment. Thus, leptin seems to be protective by both inhibiting TNF induction by LPS and by reducing TNF toxicity.

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-deficient (ob/ob) mice are protected from T cell-mediated hepatotoxicity: role of tumor necrosis factor alpha and IL-18.

The role of leptin was investigated in two models of T cell-mediated hepatitis: the administration of Con A or of Pseudomonas aeruginosa exotoxin A (PEA). In both models, leptin-deficient (ob/ob) mice were protected from liver damage and showed lower induction of tumor necrosis factor (TNF) alpha and IL-18 compared with their lean littermates. Neutralization of TNF-alpha reduced induction of IL-18 by either Con A (70% reduction) or PEA (40% reduction). Pretreatment of lean mice with either soluble TNF receptors or with an anti-IL-18 antiserum significantly reduced Con A- and PEA-induced liver damage. The simultaneous neutralization of TNF-alpha and IL-18 fully protected the mice against liver toxicity. However, neutralization of either IL-18 or TNF-alpha did not inhibit Con A-induced production of IFN-gamma. Thymus atrophy and alterations in the number of circulating lymphocytes and monocytes were observed in ob/ob mice. Exogenous leptin replacement restored the responsiveness of ob/ob mice to Con A and normalized their lymphocyte and monocyte populations. These results demonstrate that leptin deficiency leads to reduced production of TNF-alpha and IL-18 associated with reduced T cell-mediated hepatotoxicity. In addition, both TNF-alpha and IL-18 appear to be essential mediators of T cell-mediated liver injury.

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.

Leptin deficiency enhances sensitivity to endotoxin-induced lethality.

Leptin is induced by lipopolysaccharide (LPS) and cytokines. We investigated the role of leptin in LPS-induced toxicity using leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice. Sensitivity to LPS-induced mortality is significantly greater in ob/ob mice compared with their own lean littermates but not in db/db mice. LPS reduced serum glucose in both ob/ob and db/db mice but induced corticosterone only in db/db mice. Despite the very high basal levels of serum leptin in db/db mice, a twofold increase in serum leptin levels was observed after LPS in both db/db mice and their lean littermates. No differences were detected in LPS-induced serum levels of interleukin (IL)-1beta, tumor necrosis factor, macrophage inflammatory protein-1alpha, and interferon-gamma in ob/ob mice compared with their own littermates. In contrast, a blunted induction of IL-10 and IL-1 receptor antagonist (IL-1Ra) was observed in ob/ob mice compared with their littermates. In vitro, leptin induced IL-1Ra production and upregulated the IL-1Ra induction by LPS in macrophages. Moreover, treatment with leptin reversed the increased sensitivity to LPS-induced lethality found in ob/ob mice. These results suggest that leptin participates in the host response to inflammation by modulating the host immune and cytokine responses after LPS.

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.

Induction of UCP2 gene expression by LPS: a potential mechanism for increased thermogenesis during infection.

UCP2 has been proposed to regulate thermogenesis and energy expenditure. To identify potential mechanisms underlying the increased energy expenditure and heat production during infection, we investigated whether LPS and cytokines might increase UCP2 mRNA levels in mice. LPS (100 micrograms, i.p.) increased the expression of UCP2 mRNA in liver (28-fold) and muscle and white adipose tissue (5-fold). In liver, both IL-1 beta (1 microgram, i.p.) and TNF (5 micrograms, i.p.) increased UCP2 mRNA levels, 4- and 3-fold respectively, whereas in muscle and fat tissue, an increase was detectable after TNF, but not IL-1 beta. Indomethacin (10 mg/kg, i.p.) administered immediately before LPS markedly reduced (70%) the ability of LPS to increase UCP2 mRNA in liver, but not in muscle or adipose tissue. These results suggest a role for UCP2 in the heat production and increased energy expenditure that occurs during infection.

IL-1 beta mediates leptin induction during inflammation.

Interleukins (IL) are key mediators of the host response to infection and inflammation. Leptin is secreted by adipose tissue and plays an important role in the control of food intake. Administration of lipopolysaccharide (LPS), tumor necrosis factor (TNF), or IL-1 acutely increases leptin mRNA and protein levels. To investigate the role of IL-1 beta and IL-6 in leptin expression during inflammation, we used IL-1 beta-deficient (-/-) and IL-6 -/- mice. Mice were injected intraperitoneally with LPS or subcutaneously with turpentine, as models of systemic or local inflammation, respectively. In IL-1 beta +/+ mice, both LPS and turpentine increased leptin mRNA and circulating leptin. In contrast, neither LPS nor turpentine increased leptin levels in IL-1 beta -/- mice. In IL-6 +/+ or IL-6 -/- mice, turpentine increased leptin protein to comparable levels. We conclude that IL-1 beta is essential for leptin induction by both LPS and turpentine in mice, but IL-6 is not.

LPS-induced anorexia in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice.

Administration of endotoxin (lipopolysaccharide, LPS) induces profound anorexia. Injection of leptin decreases food intake in mice. Recently, we reported that LPS and cytokines increase leptin levels in hamsters. To further investigate the role of leptin in the LPS-induced anorexia, we administered LPS to leptin receptor-deficient (db/db) and leptin-deficient (ob/ob) mice. We found that LPS caused anorexia in both db/db and ob/ob mice. As might be predicted if leptin had a role in anorexia, the db/db mice were somewhat resistant to LPS-induced anorexia. However the ob/ob mice were more sensitive to LPS-induced anorexia. No differences between db/db and ob/ob mice and their respective littermate were observed in circulating tumor necrosis factor levels after LPS. These data suggest that leptin per se is not essential for LPS-induced anorexia.

Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment.

IL-6-/- mice showed impaired leukocyte accumulation in subcutaneous air pouches. Defective leukocyte accumulation was not due to a reduced migratory capacity of IL-6-/- leukocytes and was associated with a reduced in situ production of chemokines. These observations led to a reexamination of the interaction of IL-6 with endothelial cells (EC). EC express only the gp130 signal transducing chain and not the subunit-specific IL-6R and are therefore unresponsive to IL-6. However, EC are responsive to a combination of IL-6 and soluble IL-6R as measured by the activation of STAT3, chemokine expression, and augmentation of ICAM-1. Activation by IL-6-IL-6R complexes was inhibited by an IL-6 receptor antagonist and potentiated by a superagonist. Hence, in vivo and in vitro evidence supports the concept that the IL-6 system plays an unexpected positive role in local inflammatory reactions by amplifying leukocyte recruitment.

Carrageenan-induced acute inflammation in the mouse air pouch synovial model. Role of tumour necrosis factor.

We used the mouse air pouch model of inflammation to study the interaction between cytokines, prostaglandin E(2) (PGE(2)) and cell migration during the various phases of acute local inflammation induced by carrageenan. In serum, the levels of interleukin 1 (IL-1), interleukin 6 (IL-6), tumour necrosis factor (TNF), serum amiloid-A (SAA) and Fe(++) were never different from controls, indicating that no systemic inflammatory changes were induced. Locally the exudate volume and the number of leukocytes recruited into the pouch increased progressively until 7 days after carrageenan. The same was true for PGE(2) production. We could not measure IL-1 but the production of IL-6 and TNF reached a maximum after 5-24 h then quickly decreased. Anti-TNF antibodies inhibited cell migration by 50% 24 h after treatment. Pretreatment with interleukin 10 (IL-10) inhibited TNF production almost completely and cell migration by 60%. Carrageenan-induced inflammation was modulated by anti-inflammatory drugs. Pretreatment with dexamethasone (DEX) or indomethacin (INDO) inhibited cell migration and reduced the concentration of TNF in the exudate. Production of PGE(2) or vascular permeability did not correlate with the number of cells in the pouch. Local TNF seems to play an important role in this model, particularly for leukocyte migration in the first phase of the inflammatory process. In conclusion, the air pouch seems to be a good model for studying the regulation of the early events of local inflammation, particularly the role of cytokines and cell migration.

TNF receptor p55 plays a major role in centrally mediated increases of serum IL-6 and corticosterone after intracerebroventricular injection of TNF.

The aim of this work was to study the relative role of the two TNF receptors (p55 and p75) in the central actions of TNF, studying the elevation of serum corticosterone (CS) and IL-6 levels after injection of recombinant murine (rm)TNF (intracerebroventricularly (i.c.v.)) in normal or p55-deficient (p55 -/-) mice. rmTNF induced high serum IL-6 levels and doubled serum CS in normal mice, whereas no elevation of serum IL-6 or CS was induced in p55 -/- mice. However, a normal CS response was observed in p55 -/- mice after LPS (2.5 microg, i.c.v.). p55 -/- mice also responded, although to a lesser extent than p55 +/+, in terms of LPS-induced IL-6 production. We also injected two agonist Abs specific for the two receptors, alpha p55 and alpha p75. While alpha p55 injected i.c.v. induced a marked elevation in CS and IL-6, alpha p75 induced CS (although less than alpha p55) but no IL-6. rmTNF, which binds both receptors, was more potent in inducing IL-6 and CS than injection of rhTNF, which in mice binds only p55. Finally, we investigated the role of p55 and p75 in IL-6 induction by TNF in a murine brain endothelioma. The results resembled closely those obtained in vivo: rmTNF was more potent than rhTNF and only alpha p55, and not alpha p75, induced IL-6 production. These data indicate that p55 plays a major role in TNF activation of the hypothalamus-pituitary-adrenal axis and in the centrally mediated induction of peripheral IL-6 by TNF, but p75, despite having little IL-6 inductive properties by itself, seems to potentiate p55 induction of IL-6.

Effect of endotoxin in IL-1 beta-deficient mice.

IL-1 plays an important role in the pathophysiologic responses to infection and inflammation, in part by mediating its own production and that of other proinflammatory cytokines. However, the relative contribution of IL-1 alpha and IL-1 beta to the inflammatory response has not been well clarified. Using IL-1 beta-deficient (IL-1 beta -/-) mice, we investigated the specific role of IL-1 beta in the in vivo and in vitro response to LPS. No differences between IL-1 beta +/+ and IL-1 beta -/- mice were observed in circulating levels for IL-1 alpha, IL-6, or TNF-alpha after the systemic administration of either a low (5 micrograms/kg) or high (5 mg/kg) dose of LPS. IL-1 beta -/- mice also had a normal response to LPS in terms of activation of the hypothalamus-pituitary-adrenal axis, hypoglycemia, serum amyloid A production, and anorexia. IL-1 beta -/- mice were normally sensitive to the lethal effect of LPS and were protected against LPS toxicity when pretreated with low-dose LPS. However, in vitro, peritoneal macrophages from IL-1 beta -/- mice stimulated with LPS produced significantly less IL-1 alpha than macrophages from IL-1 beta +/+ mice (p < 0.05). No differences in IL-6 or TNF-alpha synthesis were observed between macrophages from IL-1 beta +/+ and IL-1 beta -/- mice. In summary, our results suggest that either IL-1 beta is not essential for the in vivo systemic response to LPS or that its role can be fulfilled by other cytokines with overlapping activities.

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.

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.