Baseline characteristics of children in the early glasses study.

PURPOSE: The relationship between refractive error at age 1 and the risk of developing amblyopia or accommodative esotropia, and the protection offered by early glasses, is unknown. These are determined in the Early Glasses Study, a prospective, population-based, longitudinal, randomized controlled study. We report baseline findings. METHODS: Healthy children aged 12-18 months were recruited at Children's Healthcare Centres (CHCs) and received an entry orthoptic examination followed by cycloplegic retinoscopy. Children with amblyopia, strabismus, ophthalmic disease or very high refractive error were excluded. Those exceeding the AAPOS 2003 Criteria (> + 3.5D spherical equivalent (SE), > 1.5D astigmatism, > 1.5D anisometropia) were randomized into wearing glasses or not, and are followed-up by research orthoptists. Other children are followed-up by regular vision screening at CHCs and visual acuity is measured in all children at age 4. RESULTS: Parents of 865 children were called, 123 were excluded. Of 742 children enrolled, 601 underwent the entry orthoptic examination at age 14.5 ± 1.7 months. Mean SE was + 1.73 ± 1.18D, astigmatism -0.70 ± 0.44D, anisometropia 0.21D (IQR: 0-0.25). Of 62 (10.3%) children exceeding the Criteria, 52 were randomized into wearing glasses or not. Of 539 other children, 522 are followed up at CHCs. In total, 31 were excluded: 2 had strabismus and amblyopia, 7 strabismus, 2 amblyopia suspect, 1 strabismus suspect, 1 squinting during sinusitis, 4 excessive refractive error, 9 myopia, 2 ptosis, 1 oculomotor apraxia, 1 Duane syndrome, 1 congenital nystagmus. CONCLUSION: Prevalence of strabismus (10/601) was as expected, but prevalence of amblyopia (2/601) was low, suggesting that common amblyopia develops later than generally thought. KEY MESSAGES: What is known High refractive errors cause amblyopia, but no study has determined the exact relationship between the kind and size of refractive error at age 1 and the risk to develop amblyopia, and assessed the protective effect of glasses in a controlled, population-based, longitudinal study. What is new At baseline, 601 children received a full orthoptic examination followed by retinoscopy in cycloplegia at the age of 14.5 ± 1.7 months; 10.3% had high refractive error exceeding spherical equivalent > + 3.5D, > 1.5D astigmatism, > 1D oblique astigmatism or > 1.5D anisometropia. The prevalence of amblyopia was lower (0.3%) than expected, suggesting that most amblyopia develops after the first year of life. The prevalence of anisometropia, associated with amblyopia in older children, was low (0.8%).

Modulation of systemic cytokine levels by implantation of alginate encapsulated cells.

The availability of cell lines that are transfected with IL-4, IL-5 and IFN-gamma cytokine genes permits the prolonged in vivo delivery of functional cytokines in relatively large doses for the modulation of specific immune responses. Often the transfected cells are xenogeneic or allogeneic to the experimental animal and have to be encapsulated in such a way that no cellular response by the host will be induced. Alginate has proven to be a simple matrix for encapsulating cells under mild conditions suitable for in vivo implantation. Encapsulated cells express the transfected IL-4 gene for at least 14 days after in vivo implantation and were shown to be functional during that period by modulating ongoing IgE responses. The application of adherent growing transfected cells permits dose-response titrations and provides an easy method for local and systemic cytokine delivery. Alternatively, hybridoma cells can be encapsulated and the secreted antibody monitored in the serum. It was found that no host immune response was triggered by alginate encapsulated cells. The efficiency of treatment by encapsulated hybridoma cells was shown to be equivalent to that of injecting purified antibodies.

Physical complaints and emotional stress related to routine diagnostic procedures of the fertility investigation.

The aim of this study was to map out the extent of the physical complaints and emotional stress due to diagnostic routines of the infertility work-up. To this end a questionnaire was sent to 96 consecutive couples visiting an infertility clinic of a university hospital. The results indicate that women often have physical complaints as a result of the hysterosalpingography (59%) and the diagnostic laparoscopy (47%) and mostly experience these diagnostic procedures as very stressful. Both the postcoital test and the semen analysis caused a moderate amount of stress. The other diagnostic procedures, including physical examination of both sexes, recording of the basal temperature and taking blood for hormonal determinations, were accompanied by fewer complaints and much less stress. It is concluded that the role of the hysterosalpingography and the diagnostic laparoscopy in the routine infertility work-up needs to be reconsidered in view of the burden they pose to the women involved.

Role of IL-4 in persistent IgE formation.

Antigen-specific immunoglobulin E (IgE) responses against T-cell-dependent antigens, like allergens, can only be generated by cognate interaction between B-cells and T-helper (Th2) cells. This interaction is a prerequisite, donating the two signals that are essential for IgE production: CD40 ligation with its ligand gp39 and exposure to interleukin (IL)-4. Cytokine-mediated immunotherapy geared at intervention in allergic diseases, therefore aims at inhibiting the production or action of IL-4. In our view, based on two findings, this approach is simplistic. The first is that anti-IL-4 based approach (by complex formation between IL-4 and soluble IL-4 receptors or serum binding proteins) may actually increase the persistence of IL-4 and its effects instead of inhibiting them. Secondly, we have good evidence in mouse model systems that a period of exposure to IL-4 will result in an increased population of gamma 1,epsilon-double positive B-cells allowing an increased serum IgE level to persist for extensive periods of time. These B-cells turn out to be partially independent of IL-4 for their IgE formation. Moreover, these B-cells are partially independent of a cognate interaction with T-cells for their subsequent IgE synthesis. Therefore, we hypothesize that an approach geared solely at inhibiting IL-4 is not sufficient for decreasing persistent IgE levels in allergic patients.

Secondary IgE responses in vivo are predominantly generated via gamma 1 epsilon-double positive B cells.

We have recently developed a model in which mice were treated with IL-4 after primary immunization, resulting in elevated total serum IgG1 and IgE levels, but decreased antigen-specific levels and memory formation for these isotypes. In this report, we describe that these effects of IL-4 are mediated at the B cell and not the T-cell level. Major changes occurred in the gamma 1 epsilon-double positive B-cell population which is increased as a result of IL-4 treatment. Moreover, it is shown that gamma 1 epsilon-double positive B cells can develop in vitro out of gamma 1-positive primed B cells and that these double positive cells can differentiate into IgG1- and IgE-secreting cells. The existence of gamma 1 epsilon-double positive memory B cells can explain the differences in cytokine dependence of TNP-specific memory IgG1 and IgE responses found after adoptively transferring primed spleen cells into irradiated naive recipients. Whereas the IL-4 independent TNP-specific memory IgG1 responses could be blocked efficiently by neutralizing IL-5 and IL-6, TNP-specific memory IgE responses were virtually not susceptible to such treatment. These IgE responses were also not susceptible to IFN-gamma, used in doses that could inhibit the primary IgE response. Inhibition of the TNP-specific memory IgG1 response by neutralizing IL-5 and IL-6 is accompanied by a 10-fold increase of the IL-4 independent TNP-specific IgE memory response. These data indicate that secondary IgE responses primarily result from B cells that are either switched to IgG1, or are double positive for IgG1 and IgE, thereby suggesting a minor role for epsilon-single positive B cells in secondary IgE responses.

Prolonged in vivo IL-4 treatment inhibits antigen-specific IgG1 and IgE formation.

IL-4 is obligatory for primary IgE responses, whereas primary IgG1 and secondary IgE responses are partially IL-4 independent. To investigate the effect of IL-4 on the antigen-specific memory formation for these isotypes, BALB/c mice were treated after primary TNP-KLH immunization with recombinant IL-4 for a period fo 4 months. This prolonged presence of a high IL-4 level resulted in increased serum levels of total IgG1 and IgE, whereas total IgG2a did not change. The expression of CD23, but not I-Ad, increased on the splenic B cells. IL-4 treatment did not affect the IL-4 production by Con A stimulated spleen cells, whereas it did decrease the IFN-gamma production. In the same mice the TNP-specific IgG1 and IgE serum levels, however, were decreased. Similar results were found when the antigen was continuously present during the IL-4 treatment. Furthermore, it was shown that IL-4 decreased the formation of IgG1 and IgE memory cells. These results point to different effects of IL-4 in regulating antigen-specific and bystander responses.

Suppression of polyclonal and antigen-specific murine IgG1 but not IgE responses by neutralizing interleukin-6 in vivo.

The crucial role of interleukin (IL)-4 in the induction of murine IgG1 and IgE responses, which are coupled through the process of sequential isotype switching, has been well documented. Whereas IL-4 is obligatory for the induction of IgE responses, it enhances IgG1 responses. In this study, using neutralizing antibodies, we provide evidence that, besides IL-4, also IL-6 is required for obtaining peak IgG1 responses. The mRNA levels of these two cytokines are coordinately expressed in the spleen of mice immunized with trinitrophenol-keyhole limpet hemocyanin (TNP-KLH). No IL-6 requirement was observed for peak IgE responses. The IL-6 dependence of IgG1 responses was found for both antigen-specific and polyclonal responses. Moreover, it was noted using TNP-KLH and goat anti-mouse (GAM) IgD as antigen that polyclonal IgG1 responses are more dependent on IL-6 than antigen-specific responses. In vitro experiments revealed that exogenous IL-6 neither enhanced nor inhibited the IgG1 and IgE production by naive B cells, suggesting that IL-6 did not interfere with the IL-4-induced isotype switch potential. Primary and memory IgG1 responses were both similarly dependent on IL-6. These observations point to a role of IL-6 in the terminal differentiation of B cells switched to IgG1. Neutralization of IL-6 did not inhibit either antigen-specific or polyclonal IgE responses. Therefore, it was concluded that IL-6 is not involved in the terminal differentiation of B cells switched to IgE. These findings thus provide a distinct role for IL-6, besides IL-4, in regulating murine IgG1 responses. The formation of IgE, however, is completely dependent on IL-4 alone.

The Effect of IFN-gamma, Alum and Complete Freund Adjuvant on TNP-KLH Induced Ig.G(1), IgE and IgG(2a) Responses in Mice.

Adjuvants are considered to play an important role in directing the isotype and amount of antibodies produced upon immunization by conducting the development of either Th-1 or Th-2 cells upon T-cell stimulation. This is based on the different cytokine production patterns that were observed after in vitro resttmulation of T cells isolated from mice immunized with antigen either adsorbed on alum or emulsified in complete Freund adjuvant (CFA). However, other studies suggest that primarily the type of antigen determines which isotypes are produced and to what extent. In these studies, however, IgE was not determined. Therefore, this study examined whether alum and CFA influenced the amount and/or ratio of IgG(1), IgE and IgG(2a) produced after TNP-KLH immunization. Similar levels of IgG(1), IgE and IgG(2a) antibodies were found upon immunization with TNP-KLH either adsorbed on alum or emulsified in CFA. Moreover, administration of IFN-gamma in combination with TNP-KLH adsorbed on alum did not increase the amount of IgG(2a) produced. IFN-gamma treatment resulted in an increased IL-6 and decreased IFN-gamma production by spleen cells upon Con A stimulation, whereas it did not change the IL-4 production in similar conditions. The presented results suggest that upon immunization with TNP-KLH high IL-4 levels are produced, resulting in an antibody response that is dominated by IgG(1), independent of the adjuvant employed. The IL-4 inducing property of TNP-KLH is substantiated by the finding that repeated immunization of mice with TNP-KI, without adjuvant, increases the serum total IgE level. The presented data suggest that the carrier part of TNP-KLH preferentially results in Th-2 cell activity after which the adjuvant merely enhances the antibody responses generated.

Selective modulation of two human monocyte Fc receptors for IgG by immobilized immune complexes.

Two types of IgG FcR, FcRI and FcRII, are constitutively expressed by human monocytes. FcRI (identified by mAb 32.2) binds human (h) IgG, FcRII (identified by mAb IV.3) has a low affinity for hIgG but interacts strongly with murine (m) IgG1. These receptors can be assayed by using indicator E sensitized by hIgG (EA-hIgG) or mIgG1 (EA-mIgG1), respectively. We further characterized these two FcR by modulation studies by using substrate-immobilized immune complexes containing rabbit IgG, goat IgG, or one of the mouse Ig classes or subclasses. After incubating monocytes in microtiter wells containing such immune complexes, binding of the two types of indicator red cells on the apical surface of the monocytes was quantitated using a photometric assay employing the pseudoperoxidase activity of E. No effect on the binding of sensitized E was observed after incubation of monocytes with immune complexes containing mouse IgE, IgA, or IgM, or F(ab')2 fragments of rabbit IgG. High concentrations of immune complexes containing IgG of mouse, rabbit, or goat, however, were able to induce a decrease in binding of both types of sensitized E, suggestive of modulation of both FcRI and FcRII. At lower concentrations of immune complexes, more selective patterns of modulation emerged. Under these conditions, immune complexes containing mIgG1 or mIgG2b, or, surprisingly, goat IgG induced a selective decrease in the binding of EA-mIgG1 (FcRII modulation), while immune complexes containing mIgG2a or rabbit IgG mainly affected the binding of EA-hIgG (FcRI modulation). By using anti-FcR mAb IV.3, it was confirmed that FcRII was modulated from the apical surface of monocytes after incubation on immune complex coated substrates. Selectivity of FcR-modulation was demonstrated by showing that under these conditions binding of anti-C receptor mAb, and several other anti-monocyte mAb did not decrease.

Proteolysis induces increased binding affinity of the monocyte type II FcR for human IgG.

Human monocytes express two types of IgG FcR, Fc gamma RI and Fc gamma RII. These can be assayed by using indicator E sensitized by human IgG (EA-human IgG) or mouse IgG1, (EA-mouse IgG1), respectively. On mouse macrophages, Fc gamma RI is sensitive to trypsin, whereas Fc gamma RII is trypsin resistant. We studied the effects of the proteolytic enzymes pronase and trypsin on human monocyte Fc gamma R. Neither enzyme caused a decrease in rosetting mediated by monocyte Fc gamma RI. Human Fc gamma RII is polymorphic, and monocytes interact either strongly or weakly with mouse IgG1. The interaction of low responder monocytes with mouse IgG1 was dramatically increased (to the level exhibited by high responder monocytes) by protease treatment. The effects of proteases on Fc gamma RII were investigated in more detail by using monocytes from which Fc gamma RI was selectively modulated by using immobilized immune complexes. Proteolysis of such modulated monocytes induced an increased interaction with EA-human IgG. Fc gamma RII appears to mediate this interaction. This conclusion is supported by the observation that after proteolysis, the Fc gamma RII-mediated binding of EA-mouse IgG1 becomes susceptible to inhibition by (monomeric) human IgG. To quantify the effect of proteolytic enzymes on Fc gamma RII, we performed binding studies with cell line K562, that expresses only Fc gamma RII. A significant increase in Ka of Fc gamma RII for dimeric human IgG complexes was observed when K562 cells were treated with protease. To elucidate the mechanism of this enhancement of Ka by proteolysis, we performed immunoprecipitation studies. Neither m.w., nor IEF pattern of Fc gamma RII were influenced by proteolysis. Moreover, the expression of Fc gamma RII was not affected by proteolysis as evidenced by immunofluorescence studies and Scatchard analysis, and neither were Fc gamma RI or Fc gamma RIII induced. We conclude that proteolysis increases the affinity of Fc gamma RII for human IgG, and speculate that such a proteolysis-induced change may also occur in vivo, e.g., at inflammatory sites.