Clinical testing of three novel transient receptor potential cation channel subfamily V member 1 antagonists in a pharmacodynamic intradermal capsaicin model.

BACKGROUND: The transient receptor potential cation channel subfamily V 1 (TRPV1) is involved in nociception and has thus been of interest for drug developers, as a target for novel analgesics. However, several oral TRPV1 antagonists have failed in development, and novel approaches to target TRPV1 with innovative chemistry are needed. METHOD: This work describes an intradermal microdosing approach in humans for pharmacodynamic deductions and pharmacological profiling of compounds. First, a human capsaicin model was developed, to generate pharmacodynamic translational data (Study Part A, n = 24). Then, three small molecule TRPV1 antagonists (AZ11760788, AZ12048189 and AZ12099548) were investigated in healthy volunteers (Study Part B, n = 36), applying the established model. Pain and flare were assessed by Visual Analogue Score and laser Doppler, respectively. RESULTS: The developed model proved useful for pharmacologic deductions; all compounds caused a dose-dependent inhibition of capsaicin-induced pain and flare responses, with a rank order potency of AZ11760788 > AZ12048189 â‰« AZ12099548. In addition, the dose-response data showed that the minimal antagonist concentrations needed to inhibit TRPV1 was ≥6-7 times the equilibrium dissociation constant for each compound. CONCLUSION: With careful design of a pharmacodynamic translational human pain model, it was possible to rank order TRPV1 efficacy among three investigational TRPV1 antagonists, and to estimate human efficacious concentrations. SIGNIFICANCE: This fast and cost-effective translational approach allows for generation of human target engagement information early in drug development. This could be of value for other development programmes where pharmacological targets are expressed in peripheral sensory nerves.

Preclinical characterization of three transient receptor potential vanilloid receptor 1 antagonists for early use in human intradermal microdose analgesic studies.

BACKGROUND: The transient receptor potential vanilloid receptor 1 (TRPV1) is a nonselective cation channel involved in the mediation of peripheral pain to the central nervous system. As such, the TRPV1 is an accessible molecular target that lends itself well to the understanding of nociceptive signalling. This study encompasses preclinical investigations of three molecules with the prospect to establish them as suitable analgesic model compounds in human intradermal pain relief studies. METHODS: The inhibitory effectiveness was evaluated by means of in vitro assays, TRPV1 expressing Chinese hamster ovary cells (CHO-K1) and rat dorsal root ganglion cultures in fluorescent imaging plate reader and whole cell patch clamp systems, as well as in vivo by capsaicin-evoked pain-related behavioural response studies in rat. Secondary pharmacology, pharmacokinetics and preclinical safety were also assessed. RESULTS: In vitro, all three compounds were effective at inhibiting capsaicin-activated TRPV1. The concentration producing 50% inhibition (IC50 ) determined was in the range of 3-32 nmol/L and 10-501 nmol/L using CHO-K1 and dorsal root ganglion cultures, respectively. In vivo, all compounds showed dose-dependent reduction in capsaicin-evoked pain-related behavioural responses in rat. None of the three compounds displayed any significant activity on any of the secondary targets tested. The compounds were also shown to be safe from a toxicological, drug metabolism and pharmacokinetic perspective, for usage in microgram doses in the human skin. CONCLUSION: The investigated model compounds displayed ideal compound characteristics as pharmacological and translational tools to address efficacy on the human native TRPV1 target in human skin in situ. SIGNIFICANCE: This work details the pharmaceutical work-up of three TRPV1-active investigational compounds, to obtain regulatory approval, for subsequent use in humans. This fast and cost-effective preclinical development path may impact research beyond the pain management area, as it allows human target engagement information gathering early in drug development.

IDH1 and IDH2 mutations in pediatric acute leukemia.

To investigate the frequency of isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations in pediatric acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), we sequenced these genes in diagnostic samples from 515 patients (227 AMLs and 288 ALLs). Somatic IDH1/IDH2 mutations were rare in ALL (N=1), but were more common in AML, occurring in 3.5% (IDH1 N=3 and IDH2 N=5), with the frequency higher in AMLs with a normal karyotype (9.8%). The identified IDH1 mutations occurred in codon 132 resulting in replacement of arginine with either cysteine (N=3) or histidine (N=1). By contrast, mutations in IDH2 did not affect the homologous residue but instead altered codon 140, resulting in replacement of arginine with either glutamine (N=4) or tryptophan (N=1). Structural modeling of IDH2 suggested that codon 140 mutations disrupt the enzyme's ability to bind its substrate isocitrate. Accordingly, recombinant IDH2 R140Q/W were unable to carry out the decarboxylation of isocitrate to α-ketoglutarate (α-KG), but instead gained the neomorphic activity to reduce α-KG to R(-)-2-hydroxyglutarete (2-HG). Analysis of primary leukemic blasts confirmed high levels of 2-HG in AMLs with IDH1/IDH2 mutations. Interestingly, 3/5 AMLs with IDH2 mutations had FLT3-activating mutations, raising the possibility that these mutations cooperate in leukemogenesis.

Neutralizing IL-21 and IL-15 inhibits pro-inflammatory cytokine production in rheumatoid arthritis.

Interleukin IL-21 and IL-15 belong to the common gamma-chain receptor family. IL-15 represents a novel therapeutic target in rheumatoid arthritis (RA), whereas less is known about the role of IL-21 in human inflammatory diseases. We have analysed the effects of blocking IL-21 and IL-15 on spontaneous production of pro-inflammatory cytokines in RA synovial cell cultures. RA synovial membrane cells were cultured in the presence of an IL-21R-Fc chimera or a neutralizing IL-15 antibody and production of tumour necrosis factor (TNF)alpha, IL-6 and IL-1beta was measured by enzyme-linked immunosorbent assay (ELISA). Expression of IL-21 and IL-15 in RA synovium was measured by RT-PCR and ELISA. mRNA for IL-21 and IL-21R was detected in the culture cell lysates. Protein for IL-15 was found at detectable levels in the cell lysates. Both the IL-21R-Fc chimera and anti-IL-15 antibody inhibited cytokine release, although substantially more IL-21R-Fc was needed. IL-21R-Fc at the highest dose (100 microg/ml) significantly reduced TNFalpha production by 50%, IL-6 by 57% and IL-1beta by 81%. Anti-IL-15 antibody (5 microg/ml) significantly inhibited TNFalpha release by 51%, IL-6 by 37% and IL-1beta by 82% in line with previous published observations. The data confirm that IL-15 plays a role in RA and suggests that IL-21 is also involved in driving the pro-inflammatory cytokine response in RA.

Nitric oxide contributes to cytokine-induced apoptosis in pancreatic beta cells via potentiation of JNK activity and inhibition of Akt.

AIMS/HYPOTHESIS: Pro-inflammatory cytokines cause beta cell secretory dysfunction and apoptosis--a process implicated in the pathogenesis of type 1 diabetes. Cytokines induce the expression of inducible nitric oxide (NO) synthase (iNOS) leading to NO production. NO contributes to cytokine-induced apoptosis, but the underlying mechanisms are unclear. The aim of this study was to investigate whether NO modulates signalling via mitogen-activated protein kinases (MAPKs) and Akt. MATERIALS AND METHODS: MAPK activities in INS-1 cells and isolated islets were determined by immunoblotting and in vitro kinase assay. Apoptosis was determined by ELISA measurement of histone-DNA complexes present in cytoplasm. RESULTS: Apoptosis in INS-1 cells induced by IL-1beta plus IFNgamma was dependent on NO production as demonstrated by the use of the NOS blocker NG-methyl-L-arginine. Accordingly, an NO donor (S-nitroso-N-acetyl-D, L-penicillamine, SNAP) dose-dependently caused apoptosis in INS-1 cells. SNAP activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but suppressed the activity of extracellular signal-regulated kinase MAPK. In rat islets, NOS inhibition decreased JNK and p38 activities induced by a 6-h exposure to IL-1beta. Likewise, IL-1beta-induced JNK and p38 activities were lower in iNOS(-/-) mouse islets than in wild-type islets. In human islets, SNAP potentiated IL-1beta-induced JNK activation. The constitutive level of active, Ser473-phosphorylated Akt in INS-1 cells was suppressed by SNAP. IGF-I activated Akt and protected against SNAP-induced apoptosis. The anti-apoptotic effect of IGF-I was not associated with reduced JNK activation. CONCLUSIONS/INTERPRETATION: We suggest that NO contributes to cytokine-induced apoptosis via potentiation of JNK activity and suppression of Akt.

Rheumatology outcomes: the patient's perspective. A multicentre focus group interview study of Swedish rheumatoid arthritis patients.

OBJECTIVES: Patients with rheumatoid arthritis (RA) and clinicians have different views about benefits from treatments. More knowledge is needed about how patients assess outcomes in order to update current measurements. METHODS: Focus group interviews were performed at four Swedish rheumatology clinics. A total of 25 patients with RA were included, representing a wide range of ages and disease duration. Predetermined topics relating to important outcomes from and satisfaction/dissatisfaction with RA treatments were discussed. RESULTS: The participants' initial outcome assessments included physical and psychosocial items, which comprised overall treatment goals such as impairment in social roles, fatigue, daily activities and self-confidence. The identified themes were 'Normal life', 'Physical capacity', 'Independence' and 'Well-being'. Satisfaction with treatment was associated with the quality of communication between staff and the patient. The participants assumed this as a prerequisite for a treatment to work. Patients wanted to be accepted as experts on their own bodies, and expected all clinicians to be experts on RA. This made it possible for patients to 'take charge' of their life situation. Good resources for and access to rheumatology care were desired. CONCLUSIONS: Suggesting a holistic approach to rheumatology care, the study results indicate that the illness and outcomes have to be evaluated within an individual RA patient's total life situation, described in the identified themes: 'Normal life', 'Physical capacity', 'Independence' and 'Well-being'. Development and validation of measurements covering these issues is suggested. More research is needed about communication and how patients experience their roles in the rheumatology clinic.

Human glutathione transferase A1-1 demonstrates both half-of-the-sites and all-of-the-sites reactivity.

A study of the kinetics of a heterodimeric variant of glutathione transferase (GST) A1-1 has led to the conclusion that, although the wild-type enzyme displays all-of-the-sites reactivity in nucleophilic aromatic substitution reactions, it demonstrates half-of-the-sites reactivity in addition reactions. The heterodimer, designed to be essentially catalytically inactive in one subunit due to a single point mutation (D101K), and the two parental homodimers were analyzed with seven different substrates, exemplifying three types of reactions catalyzed by glutathione transferases (nucleophilic aromatic substitution, addition, and double-bond isomerization reactions). Stopped-flow kinetic results suggested that the wild-type GST A1-1 behaved with half-of-the-sites reactivity in a nucleophilic aromatic substitution reaction, but steady-state kinetic analyses of the GST A1-D101K heterodimer revealed that this was presumably due to changes to the extinction coefficient of the enzyme-bound product. In contrast, steady-state kinetic analysis of the heterodimer with three different substrates of addition reactions provided evidence that the wild-type enzyme displayed half-of-the-sites reactivity in association with these reactions. The half-of-the-sites reactivity was shown not to be dependent on substrate size, the level of saturation of the enzyme with glutathione, or relative catalytic rate.

Melatonin protects against streptozotocin, but not interleukin-1beta-induced damage of rodent pancreatic beta-cells.

In the present study, we examined whether melatonin can protect rodent pancreatic islets against streptozotocin (STZ) and interleukin-1beta (IL-1beta)-induced suppression of beta-cell function. Formation of free radicals, DNA damage and extensive DNA repair leading to depletion of intracellular nicotinamide adenine dinucleotide (NAD) may mediate STZ toxicity. Activation of inducible nitric oxide synthase and nitric oxide (NO) formation may cause IL-1beta -induced beta-cell impairment. We also studied the effect of melatonin against STZ-induced hyperglycemia in C57BL/Ks mice. For in vitro studies, cultured rat islets were exposed to melatonin (100 microM-1 mM) 30 min prior to STZ (0.5 mM) or IL-1beta (25 U/mL) addition. After an additional 30 min incubation with STZ, islet function and NAD content were analyzed either acutely or after 18 hr of recovery in fresh culture medium. For IL-1beta experiments, islets were incubated for 48 hr with the cytokine before evaluation of islet function. We found that melatonin counteracted STZ-induced inhibition of glucose metabolism and insulin release in cultured rat islets after 18 hr of recovery. Moreover, NAD levels were higher in the melatonin-treated group at this time point. Melatonin had no effect on IL-1beta-induced islet inhibition of glucose oxidation or NO formation. Diabetes induced by STZ (140 mg/kg body weight; i.v.) was effectively prevented by administration of melatonin (100 mg/kg body weight; i.p.) 30 min before STZ injection. We conclude that the protective effects of melatonin against beta-cell damage may be related to interference with DNA damage and poly(ADP-ribose) polymerase (PARP) activation rather than through effects on NO generation pathways.

Cytokine-induced inhibition of insulin release from mouse pancreatic beta-cells deficient in inducible nitric oxide synthase.

Cytokines may participate in islet destruction during the development of type 1 diabetes. Expression of inducible nitric oxide synthase (iNOS) and subsequent NO formation induced by IL-1 beta or (IL-1 beta + IFN-gamma) may impair islet function in rodent islets. Inhibition of iNOS or a deletion of the iNOS gene (iNOS -/- mice) protects against cytokine-induced beta-cell suppression, although cytokines might also induce NO-independent impairment. Presently, we exposed wild-type (wt, C57BL/6 x 129SvEv) and iNOS -/- islets to IL-1 beta (25 U/ml) and (IL-1 beta (25 U/ml) + IFN-gamma (1000 U/ml)) for 48 h. IL-1 beta and (IL-1 beta + IFN-gamma) induced a significant increase in NO formation in wt but not in iNOS -/- islets. Both IL-1 beta and (IL-1 beta + IFN-gamma) impaired glucose-stimulated insulin release and reduced the insulin content of wt islets, while (IL-1 beta + IFN-gamma) reduced glucose oxidation rates and cell viability. IL-1 beta exposure to iNOS -/- islets impaired glucose-stimulated insulin release, increased insulin accumulation and reduced the insulin content, without any increase in cell death. Exposure to (IL-1 beta + IFN-gamma) had no effect on iNOS -/- islets except reducing the insulin content. Our data suggest that IL-1 beta may inhibit glucose-stimulated insulin release by pathways that are not NO-dependent and not related to glucose metabolism or cell death.

Novel experimental strategies to prevent the development of type 1 diabetes mellitus.

Type 1 diabetes is an autoimmune disease leading to extensive destruction of the pancreatic beta-cells. Our research focusses on the role of beta-cells during the course of the disease, aiming at finding novel strategies to enhance beta-cell resistance against the cytotoxic damage inflicted by the immune system. Special attention has been paid to the possibility that cytokines released by the immune cells infiltrating the pancreatic islets can directly suppress and kill beta-cells. Certain cytokines (interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma) either alone or in combination, are able to activate signal transduction pathways in beta-cells leading to transcription factor activation and de novo gene expression. In this context, it has been found that induction of inducible nitric oxide synthase mediates an elevated production of nitric oxide, which impairs mitochondrial function and causes DNA damage eventually leading to apoptosis and necrosis. However, other induced proteins SUCH AS heat shock protein 70 and superoxide dismutase may reflect a defense reaction elicited in the beta-cells by the cytokines. Our strategy is to further seek for proteins involved in both destruction and protection of beta-cells. Based on this knowledge, we plan to apply gene therapeutic approaches to increase expression of protective genes in beta-cells. If this is feasible we will then evaluate the function and survival of such modified beta-cells in animal models of type 1 diabetes such as the NOD mouse. The long-term goal for this research line is to find novel approaches to influence beta-cell resistance in humans at risk of developing type 1 diabetes.

Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis.

Osteopetrosis includes a group of inherited diseases in which inadequate bone resorption is caused by osteoclast dysfunction. Although molecular defects have been described for many animal models of osteopetrosis, the gene responsible for most cases of the severe human form of the disease (infantile malignant osteopetrosis) is unknown. Infantile malignant autosomal recessive osteopetrosis (MIM 259700) is a severe bone disease with a fatal outcome, generally within the first decade of life. Osteoclasts are present in normal or elevated numbers in individuals affected by autosomal recessive osteopetrosis, suggesting that the defect is not in osteoclast differentiation, but in a gene involved in the functional capacity of mature osteoclasts. Some of the mouse mutants have a decreased number of osteoclasts, which suggests that the defect directly interferes with osteoclast differentiation. In other mutants, it is the function of the osteoclast that seems to be affected, as they show normal or elevated numbers of non-functioning osteoclasts. Here we show that TCIRG1, encoding the osteoclast-specific 116-kD subunit of the vacuolar proton pump, is mutated in five of nine patients with a diagnosis of infantile malignant osteopetrosis. Our data indicate that mutations in TCIRG1 are a frequent cause of autosomal recessive osteopetrosis in humans.

Acute-phase responses in transgenic mice with CNS overexpression of IL-1 receptor antagonist.

The interleukin-1 (IL-1) receptor antagonist (IL-1ra) is an endogenous antagonist that blocks the effects of the proinflammatory cytokines IL-1alpha and IL-1beta by occupying the type I IL-1 receptor. Here we describe transgenic mice with astrocyte-directed overexpression of the human secreted IL-1ra (hsIL-1ra) under the control of the murine glial fibrillary acidic protein (GFAP) promoter. Two GFAP-hsIL-1ra strains have been generated and characterized further: GILRA2 and GILRA4. These strains show a brain-specific expression of the hsIL-1ra at the mRNA and protein levels. The hsIL-1ra protein was approximated to approximately 50 ng/brain in cytosolic fractions of whole brain homogenates, with no differences between male and female mice or between the two strains. Furthermore, the protein is secreted, inasmuch as the concentration of hsIL-1ra in the cerebrospinal fluid was 13 (GILRA2) to 28 (GILRA4) times higher in the transgenic mice than in the control animals. To characterize the transgenic phenotype, GILRA mice and nontransgenic controls were injected with recombinant human IL-1beta (central injection) or lipopolysaccharide (LPS, peripheral injection). The febrile response elicited by IL-1beta (50 ng/mouse icv) was abolished in hsIL-1ra-overexpressing animals, suggesting that the central IL-1 receptors were occupied by antagonist. The peripheral LPS injection (25 micrograms/kg ip) triggered a fever in overexpressing and control animals. Moreover, no differences were found in LPS-induced (100 and 1,000 micrograms/kg ip; 1 and 6 h after injection) IL-1beta and IL-6 serum levels between GILRA and wild-type mice. On the basis of these results, we suggest that binding of central IL-1 to central IL-1 receptors is not important in LPS-induced fever or LPS-induced IL-1beta and IL-6 plasma levels.

IL-6 is essential in TNF-alpha-induced fever.

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine that orchestrates an array of local and systemic effects. For instance, acute exposure to a high dose of TNF-alpha results in septic shock and fever. We have used interleukin-1beta (IL-1beta)- and interleukin-6 (IL-6)-deficient mice, along with their wild-type equivalents, to define a role for TNF-alpha in fever. Briefly, the mice produced prostaglandin E2-dependent fevers in response to recombinant murine TNF-alpha (rmTNF-alpha). Furthermore, rmTNF-alpha (12 microgram/mouse ip) triggered a febrile response in IL-1beta-deficient mice as well as in their corresponding wild-type controls. In contrast, the IL-6-deficient mice were resistant to rmTNF-alpha (4.5 microgram/mouse ip), although their wild-type counterparts readily mounted a fever. In the IL-6-deficient mice, moreover, the febrile response to rmTNF-alpha could be restored by a central administration of rat recombinant IL-6 (500 ng/mouse icv). We thus conclude that TNF-alpha can trigger fever independent of IL-1beta but dependent on IL-6. We also suggest that central, rather than peripheral, IL-6 (plasma IL-6 was measured 2 h after pyrogenic challenge) is essential in TNF-alpha-induced fever.

Simultaneous measurement of brain and core temperature in the rat during fever, hyperthermia, hypothermia and sleep.

The neuropathological outcome of metabolic, vascular or mechanical insults to the CNS depends on brain temperature; mild hypothermia is neuroprotective, whereas elevated brain temperature can cause additional neural damage. Studies in both animals and humans have shown that the core and the brain temperature do not always concur with one another. It is therefore important to develop methods for monitoring brain temperature. This paper describes an animal model (the rat) in which we have developed a method to measure, at thermoneutral ambient temperature, the brain and core temperature concomitantly, during different drug treatments. We have used this animal model to study body temperature during fever (induced by human recombinant IL-1 beta, 5 microgram/kg, i.p.), stress-induced hyperthermia (handling of the animal), hypothermia (induced by (+/-)-8-hydroxy-2-dipropylaminotetralin hydrobromide, 0.5 mg/kg, i.p. ) and sleep (non-induced, other than by light and diurnal variation). We show that the thermal curves are similar in the brain and the peritoneum, independent of the thermal state.

Calcium spikes and calcium currents in neurons from the medial preoptic nucleus of rat.

Ca2+ spikes, their contribution to firing patterns, and the underlying Ca2+ currents in neurons of the medial preoptic nucleus of rat were investigated by tight-seal whole-cell recordings in a slice preparation. Two different types of spikes were recorded: Low-threshold spikes were generated from membrane potentials <-75 mV. High-threshold spikes were recorded when K+ currents were reduced, and were readily evoked from membrane potentials near -40 mV. Both types of spikes were blocked by substitution of Co2+ for Ca2+ in the external medium, but were insensitive to 2.0 microM TTX. Under voltage-clamp conditions, two main types of Ca2+ currents were characterized: low-threshold currents that activated at membrane potentials >-60 mV, and high-threshold currents that activated at potentials >-30 mV. The low-threshold current and the low-threshold spike were more sensitive to block by external Ni2+ than to block by Cd2+, whereas the high-threshold current and the high-threshold spike were more sensitive to block by external Cd2+ than to block by Ni2+. Significant fractions of the high-threshold currents were blocked by 10 microM nifedipine, 1.0 microM omega-conotoxin GVIA, 50 nM omega-agatoxin IVA and 1.0 microM omega-conotoxin MVIIC, suggesting the presence of L-, N-, P- and Q-type Ca2+ channels. There were also a high-threshold current component insensitive to the above mentioned toxins. It is proposed that the low-threshold current serves as a trigger for short bursts of fast spikes from hyperpolarized levels, whereas the high-threshold current is involved in the Cd2+-sensitive burst firing seen in relatively depolarized neurons.

Calpains in lens epithelium from patients with cataract.

Lens epithelium from patients with cataract was obtained during surgery and frozen. The samples were subjected to SDS-electrophoresis and Western blotting. Calpains were quantified using polyclonal antibodies against m- and mu-Calpain could be detected but not the isoenzyme mu-calpain, indicating that m-calpain is the significant most important calpain in human lens epithelium. Quantification of m-calpain showed no relationship to age or gender, but there were significant differences between different types of cataract.

Changes in the soluble protein of the human vitreous in vitreoretinal disease.

Samples of the vitreous were analysed in order to identify changes of soluble proteins in vitreo-retinal disease. The soluble proteins of the vitreous were separated on an anion exchange column (Mono-Q). The degree of neutral proteolytic activity in vitreous body was also measured. The vitreous from cataract cases without vitreoretinal disease was characterized by its low content of soluble proteins equivalent to about 1% of that of serum. Albumin and transferrin were the major identified components and their concentrations were approximately 0.85 and 0.03 g/l, respectively. In cases with vitreoretinal disease the vitreous showed changes of total soluble protein and the appearance of additional protein peaks. In patients with PVR the albumin concentration in the vitreous was found to be three times higher as compared to the control group consisting of patients with cataract. Neutral proteolytic activity in the vitreous was relatively low in both normal and pathological vitreous.

A longitudinal study of a population based sample of astigmatic children. I. Refraction and amblyopia.

The refraction changes in 310 children with astigmatism greater than or equal to 1.0 D in at least one eye at one year of age were followed during a period of 3 years. At the age 4 years amblyopia was found in 23 children (7%). The refraction data of these children were compared to the rest of the sample. We found that an increasing astigmatism during the test period was associated with an increased risk to develop amblyopia. The majority of children (n = 280) showed a decrease of their astigmatism, whereas all cases with a marked amblyopia (V.A. less than 0.5) or binocular amblyopia, except one, had an increasing or unchanged astigmatism during the age period 1 to 4 years. Strabismus and oblique astigmatism at any time during the test period was also strongly related to amblyopia. The incidence of strabismus (1%) was unexpectedly low. The study also showed that independent of age there was no simple relationship between amblyopia and refraction errors measured at a single test session. The main conclusion of this study is that failure of emmetropization may play an important role in visual development.