Formation of the arthrodial membrane in the blue crab, Callinectes sapidus.

In this study the pattern of arthrodial membrane deposition in Callinectes sapidus was determined by histological and ultrastructural examination of tissues from the carpus joint of the cheliped collected during premolt, ecdysis, postmolt, and intermolt. Apolysis in the arthrodial membrane occurs at stage D(0) and is synchronous with apolysis of the calcified cuticle. Epicuticle formation begins at early stage D(1) and is completed in late stage D(1). Procuticle deposition starts at D(2) and continues until ecdysis. Numerous cytoplasmic extensions occur throughout the lamellae. Component fibers of the arthrodial membrane are intimately associated with dense plaques on the apical membrane of the underlying hypodermal cells, suggesting a site for fiber polymerization. Deposition of the arthrodial membrane continues after ecdysis, with most of the cuticle thickening occurring during stage C. When stained with PAS and counterstained with hematoxylin, a difference can be discerned between preecdysial and postecdysial procuticle of the arthrodial membrane, a distinction not made in previous studies. The boundary between the arthrodial membrane and calcified cuticle is thicker than either of the two layers and the layers overlap rather than butting up against one another. This pattern suggests that underlying hypodermal cells have to produce multiple types of cuticle over the molt cycle. A summary of the various molting patterns in C. sapidus suggests that the control of these diverse events may prove to be complex.

Cathepsin S regulates the expression of cathepsin L and the turnover of gamma-interferon-inducible lysosomal thiol reductase in B lymphocytes.

Loading of antigenic peptide fragments on major histocompatibility complex class II molecules is essential for generation of CD4(+) T cell responses and occurs after cathepsin-mediated degradation of the invariant chain chaperone molecule. Cathepsins are expressed differentially in antigen presenting cells, and mice deficient in cathepsin S or cathepsin L exhibit severely impaired antigen presentation in peripheral lymphoid organs and the thymus, respectively. To determine whether these defects are due solely to the block in invariant chain cleavage, we used cathepsin-deficient B cells to examine the role of cathepsins S and B in the degradation of other molecules important in the class II presentation pathway. Our data indicate that neither cathepsin S nor B is critical for H-2M degradation or processing of precursor gamma-interferon-inducible lysosomal thiol reductase (GILT) to a mature thiol reductase, but suggest a role for cathepsin S in the turnover of mature GILT and in regulating levels of mature cathepsin L protein in B cells. Despite the presence of mature cathepsin L protein, no enzyme activity could be detected in B cells or dendritic cells. These experiments suggest a novel mechanism by which these functionally important enzymes may be regulated.

Impaired invariant chain degradation and antigen presentation and diminished collagen-induced arthritis in cathepsin S null mice.

Cathepsins have been implicated in the degradation of proteins destined for the MHC class II processing pathway and in the proteolytic removal of invariant chain (Ii), a critical regulator of MHC class II function. Mice lacking the lysosomal cysteine proteinase cathepsin S (catS) demonstrated a profound inhibition of Ii degradation in professional APC in vivo. A marked variation in the generation of MHC class II-bound Ii fragments and presentation of exogenous proteins was observed between B cells, dendritic cells, and macrophages lacking catS. CatS-deficient mice showed diminished susceptibility to collagen-induced arthritis, suggesting a potential therapeutic target for regulation of immune responsiveness.

Formation of the inner branchiostegal cuticle of the blue crab, Callinectes sapidus.

The noncalcified inner branchiostegal cuticle, which lines the branchial chamber, was examined histologically and ultrastructurally over the molt cycle in the blue crab, Callinectes sapidus. In intermolt crabs (stage C4 ) the epithelium underlying the inner cuticle is cuboidal and has abundant intercellular spaces and a prominent basement membrane. Apolysis occurs at stage D0 and dissolution of the cuticle is accompanied by the formation of numerous lysosomes in the epithelium. During stage D1 , cells increase in height, apical mitochondria become more abundant, and the cuticle continues to be resorbed. An epicuticle is formed in early D2 , arising from a fusion of small subunits apparently attached to short apical microvilli. Cuticle deposition continues through D2 and is complete by stage D3 . By the time cuticle deposition is complete, the epithelium has become extremely columnar and cells are filled with bundles of microtubules. In stage D4 , an amorphous electron-dense core appears in the microtubule-filled cells, which are attached to the cuticle at their apical end and anchored to their basement membrane at the basal surface. These microtubule-filled cells persist through ecdysis, stage E, but during stage A1 the cores disappear and some organelles begin to reappear in the cytoplasm. By stage A2 , the cells return to the cuboidal morphology seen in intermolt and remain so throughout the remainder of the molt cycle. This new pattern of cuticle deposition resembles that observed in the gills of crustaceans in that the cuticle is uncalcified and there is no postecdysial cuticle formation. However, instead of apolysis being delayed until just before ecdysis, the inner cuticle is formed during the first half of premolt, allowing the epithelial cells time to differentiate into a morphology that provides tensile strength for the stress of ecdysis. These new observations demonstrate that cuticle formation can follow very diverse structural and temporal patterns. In order to integrate and coordinate these diverse patterns, it is suggested that a suite of feedback mechanisms must be present. J. Morphol. 240:267-281, 1999. © 1999 Wiley-Liss, Inc.

Immune tolerance mediated by recombinant proteolipid protein prevents experimental autoimmune encephalomyelitis.

Proteolipid protein (PLP), a transmembrane protein expressed only in the central nervous system (CNS), is a candidate target autoantigen for autoimmune-mediated demyelination. We have evaluated the effect of a recombinant form of the PLP protein, delta PLP4, in a murine model of experimental autoimmune encephalomyelitis (EAE). PLP-specific T-cell responses were observed following immunization of SJL/J, PL/J and SWR mice with delta PLP4, demonstrating processing of the protein to several distinct antigenic epitopes. Clinical EAE associated with inflammation and demyelination in the CNS also developed after sensitization of mice with delta PLP4 in adjuvant. Conversely, tolerance to delta PLP4 in adult mice and prevention of PLP peptide 139-151-induced EAE was induced by intravenous injection of soluble delta PLP4. The prevention of disease onset was paralleled by a significant reduction in demyelination and CNS inflammatory cell infiltration and diminished PLP139-151-specific T-cell proliferative responses. These results are consistent with the establishment of peripheral T-cell tolerance and reinforce the notion that recombinant myelin antigens and intravenous tolerance induction may prove useful in the modulation of the human demyelinating disease, multiple sclerosis (MS).

Humanized porcine VCAM-specific monoclonal antibodies with chimeric IgG2/G4 constant regions block human leukocyte binding to porcine endothelial cells.

Discordant xenografts surviving the initial hyperacute rejection phase may be subject to cellular rejection processes mediated by infiltrating leukocytes including T cells, NK cells and monocytes. The stable adhesion of these cell types to endothelial cells is due to the molecular interaction of the integrins VLA-4 and LFA-1 with their ligands vascular cell adhesion molecule (VCAM) and ICAM-1 present on the endothelial cells. Human VLA-4 binds to porcine VCAM, and blocking mAbs specific for porcine VCAM have been developed. We have localized the epitope of the anti-porcine VCAM blocking mAbs 2A2 and 3F4 to domains 1 and 2, respectively. Humanized antibodies (IgG4 isotype) were constructed from these anti-porcine VCAM antibodies and demonstrated to inhibit adhesion of Ramos, Jurkat and YT cells, as well as purified resting and activated human T cells, to porcine aortic endothelial cells (PAEC). These cell types express both LFA-1 as well as VLA-4, suggesting blockade of human VLA-4 interaction with porcine VCAM may alone be sufficient to significantly impair adhesion of human leukocytes to porcine endothelial cells. The chimeric anti-porcine VCAM (pVCAM) HuG4 antibodies promoted increased adhesion of Fc receptor (FcR) positive cells such as U937 monocytic cells to PAEC. In contrast, chimeric anti-porcine VCAM antibodies created using the CH1 and hinge region from human IgG2 and the CH2 and CH3 regions from human IgG4 (HuG2/G4 antibodies) inhibited binding of FcR positive cells to PAEC. These chimeric anti-pVCAM antibodies should allow delineation of the in vivo role of VLA-4/VCAM interaction in porcine-to-primate xenotransplants. Further, the design of the HuG2/G4 antibodies should render them efficacious in multiple settings requiring elimination of FcR binding.

Restrictive clonal allocation in the chimeric mouse brain.

Whether, and to what extent, lineage restriction contributes to the organization of the mammalian brain remains unclear. Here we address this issue by examining the distribution of clonally related cells in chimeric mice generated by injecting genetically tagged embryonic stem (ES) cells into blastocyst embryos. Our examination of postnatal chimeric brains revealed that the vast majority of labeled ES cell descendents were confined within a different subset of brain regions in each animal. Moreover, the deployment of labeled cells in different brain regions was distinctive. The pattern of ordered and binomial colonization suggested that early diversified founder cells may constrain the fates of their descendants through a restriction of dispersion. In addition, the symmetrical distribution of ES cell descendants suggests that bilaterally corresponding structures may arise from a common set of progenitor cells. Finally, clones of cells formed a continuous band within the deep strata of the neocortex. This later finding in conjunction with the radial distribution of clones in remaining layers observed in previous studies indicates that the cerebral neocortex may derive from two groups of founder cells, which is consistent with the hypothesis of dual phylogenetic origins of the mammalian cerebral cortex.

Inhibition of complement activity by humanized anti-C5 antibody and single-chain Fv.

Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5G1.1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9. Humanized 5G1.1 antibody, Fab and scFv molecules have been produced by grafting the complementarity determining regions of 5G1.1 on to human framework regions. Competitive ELISA analysis indicated that no framework changes were required in the humanized variable regions for retention of high affinity binding to C5, even at framework positions predicted by computer modeling to influence CDR canonical structure. The humanized Fab and scFv molecules blocked complement-mediated lysis of chicken erythrocytes and porcine aortic endothelial cells in a dose-dependent fashion, with complete complement inhibition occurring at a three-fold molar excess, relative to the human C5 concentration. In contrast to a previously characterized anti-C5 scFv molecule, the humanized h5G1.1 scFv also effectively blocked C5a generation. Finally, an intact humanized h5G1.1 antibody blocked human complement lytic activity at concentrations identical to the original murine monoclonal antibody. These results demonstrate that humanized h5G1.1 and its recombinant derivatives retain both the affinity and blocking functions of the murine 5G1.1 antibody, and suggest that these molecules may serve as potent inhibitors of complement-mediated pathology in human inflammatory diseases.

Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein.

It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.

Complement inhibition with an anti-C5 monoclonal antibody prevents acute cardiac tissue injury in an ex vivo model of pig-to-human xenotransplantation.

Prevention of hyperacute xenograft rejection in the pig-to-primate combination has been accomplished by removal of natural antibodies, complement depletion with cobra venom factor, or prevention of C3 activation with the soluble complement inhibitor sCR1. Although these strategies effectively prevent hyperacute rejection, they do not address the relative contribution of early (C3a, C3b) versus late (C5a, C5b-9) activated complement components to xenogeneic organ damage. To better understand the role of the terminal complement components (C5a, C5b-9) in hyperacute rejection, an anti-human C5 mAb was developed and tested in an ex vivo model of cardiac xenograft rejection. In vitro studies demonstrated that the anti-C5 mAb effectively blocked C5 cleavage in a dose-dependent manner that resulted in complete inhibition of both C5a and C5b-9 generation. Addition of anti-C5 mAb to human blood used to perfuse a porcine heart prolonged normal sinus cardiac rhythm from a mean time of 25.2 min in hearts perfused with unmodified blood to 79,296, or > 360 min when anti-C5 mAb was added to the blood at 50 micrograms/ml, 100 micrograms/ml, or 200 micrograms/ml, respectively. In these experiments, activation of the classical complement pathway was completely inhibited. Hearts perfused with blood containing the highest concentration of anti-C5 mAb had no histologic evidence of hyperacute rejection and no deposition of C5b-9. These experiments suggest that the activated terminal complement components C5a and C5b-9, but not C3a or C3b, play a major role in tissue damage in this porcine-to-human model of hyperacute rejection. They also suggest that targeted inhibition of terminal complement activation by anti-C5 mAbs may be useful in clinical xenotransplantation.

Porcine vascular cell adhesion molecule (VCAM) mediates endothelial cell adhesion to human T cells. Development of blocking antibodies specific for porcine VCAM.

Vascular cell adhesion molecule (VCAM) is expressed on activated endothelial cells and binds to the alpha 4 beta 1 integrin receptor, very late antigen-4 (VLA-4), expressed on human lymphoid cells. Anti-VCAM mAbs have been shown to prolong allograft survival. To explore the role of porcine VCAM (pVCAM) in xenotransplantation, a recombinant secreted form of pVCAM (spVCAM) was expressed in 293-EBNA cells and purified by metal affinity chromatography. A human lymphoid cell line bound to spVCAM in a VLA-4-dependent manner. Using spVCAM as an immunogen, we developed three anti-pVCAM mAbs that reacted with cell surface pVCAM on porcine aortic endothelial cells (PAEC) but not to human VCAM on human umbilical vein endothelial cells. Pairwise interaction analysis indicated that these mAbs recognized distinct epitopes on pVCAM. Two anti-pVCAM mAbs, 2A2 and 3F4, inhibited the binding of Ramos cells to spVCAM, while the third, 5D11, did not. Similarly, mAbs 2A2 and 3F4 inhibited binding of Ramos cells or human peripheral blood T cells to activated PAEC. The extent of inhibition with mAbs 2A2 and 3F4 was comparable to the inhibition obtained with a blocking mAb to human VLA-4. These anti-pVCAM mAbs will provide a means to specifically block pVCAM in a xenograft setting and allow the determination of the role of pVCAM in a primary xenogeneic immune response.

Reconstitution of invariant chain function in transgenic mice in vivo by individual p31 and p41 isoforms.

MHC class II molecules associate with invariant chain (li) during biosynthesis. If facilitates folding of class II molecules, interferes with their association with peptides, and is involved in their transport. The murine Ii gene encodes two chains, p31 and p41. The role of these isoforms has been studied in vitro only in inappropriate antigen-presenting cells. To circumvent this problem, we have generated invariant chain-deficient mice (delta Ii), which express exclusively the p31 and p41 isoforms. Low level expression of p31 or p41 is not sufficient for rescuing high levels of cell surface class II expression. However, low levels of the typical compact dimer conformation indicative of tight peptide binding are observed. Thus, both isoforms participate in class II folding and assembly. Furthermore, p31 and p41 retrieve the CD4+ T cell population, which is reduced in the (delta Ii) mice. Moreover, the immune response to protein antigen is restored by both isoforms.

Male mice defective in the DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis.

Using gene targeting in embryonic stem cells, we have derived mice with a null mutation in a DNA mismatch repair gene homolog, PMS2. We observed microsatellite instability in the male germline, in tail, and in tumor DNA of PMS2-deficient animals. We therefore conclude that PMS2 is involved in DNA mismatch repair in a variety of tissues. PMS2-deficient animals appear prone to sarcomas and lymphomas. PMS2-deficient males are infertile, producing only abnormal spermatozoa. Analysis of axial element and synaptonemal complex formation during prophase of meiosis I indicates abnormalities in chromosome synapsis. These observations suggest links among mismatch repair, genetic recombination, and chromosome synapsis in meiosis.

60 Hertz magnetic field exposure assessment for an investigation of leukemia in telephone lineworkers.

The purpose of this paper is to present the assessment of magnetic field exposure conducted as a part of a nested case-control investigation of leukemia mortality in telephone lineworkers. For the purposes of exposure classification, telephone company jobs were initially divided into two classes: those with potential for working in an electric environment, referred to as linework jobs, and those not working in an electric environment, referred to as nonlinework jobs. Linework jobs were further divided into the following four categories: outside plant technicians (OPT), installation/maintenance/repair (IMR) technicians, central office technicians (COT), and cable splicing technicians (CST). These job groupings were based on similarity of work tasks and exposure environments. Emdex data-logging dosimeters were used to measure personal exposures to ELF magnetic fields for 204 telephone company workers. Three general classes of exposure indices were calculated for each exposure record: measures of central tendency, measures of peak or maximum exposure, and measures of exposure variability. CSTs had the highest full-shift mean and median exposure, 4.3 and 3.2 mG, respectively. CSTs also ranked the highest, with average peak, average 95th percentile, and average time above background equal to 99.2 mG, 11.1 mG, and 156 min, respectively. In addition, the results suggest the OPT and IMR technicians have exposures similar to nonlineworkers. Exposure classifications, therefore, which misclassify all lineworkers into one "telephone lineworker" job grouping are not appropriate and future studies should concentrate on cable splicing technicians.

Transgenic mice expressing constitutive levels of IL-2 in islet beta cells develop diabetes.

IL-2 plays an important role in the clonal expansion of T cells during an immune response and it has been implicated in autoimmune disease. To examine the role of IL-2 in the regulation of peripheral tolerance we produced transgenic mice in which the expression of murine IL-2 was directed by the rat insulin II promoter. The IL-2 transgene was expressed specifically in the pancreas. Islets from transgenic mice synthesized biologically active IL-2. Expression of IL-2 in the pancreas resulted in a massive inflammatory response directed at the beta cells of the pancreas. The infiltrate consisted primarily of B cells and CD4+ and CD8+ T cells. The infiltrate resulted in destruction of the insulin-producing beta cells and diabetes, but there was no evidence for antigen specificity. The results suggest that local IL-2 production elicits the recruitment and activation of cells capable of destroying beta cells by non-antigen-specific mechanisms.

Mice deficient for the CD40 ligand.

To study the potential roles of CD40L in immune responses, we generated CD40L-deficient mice by gene targeting. Similar to the effects of CD40L mutations in humans (hyper-IgM syndrome), CD40L-deficient mice have a decreased IgM response to thymus-dependent antigens, fail altogether to produce an antigen-specific IgG1 response following immunization, yet respond normally to a T-independent antigen, TNP-Ficoll. Moreover, these mice do not develop germinal centers in response to thymus-dependent antigens, suggesting an inability to develop memory B cell responses. Although CD40L-deficient mice have low levels of most circulating immunoglobulin isotypes, they do not exhibit the spontaneous hyper-IgM syndrome seen in humans, at least up to 12 weeks of age. In summary, our study confirms the important role of CD40-CD40L interactions in thymus-dependent humoral immune responses and germinal center formation.

Evidence that activation of human T cells by porcine endothelium involves direct recognition of porcine SLA and costimulation by porcine ligands for LFA-1 and CD2.

In this study we present a comprehensive evaluation of the molecular interactions between human T cells and porcine aortic endothelial cells (PAEC) that contribute to human T cell activation. Binding assays demonstrated that porcine erythrocytes (E) and PAEC express ligand(s) for the human T cell glycoprotein CD2. Prior incubation of human T cells with a blocking monoclonal antibody directed against CD2 (alpha CD2-BL) completely inhibited T cell/E and T cell/PAEC interaction. Xenogeneic mixed lymphocyte reactions (XMLR) revealed that human PBMC, or highly purified T cells were activated by PAEC in the absence of human antigen-presenting cells (APC). Addition of alpha CD2-BL or alpha LFA-1 to these assays inhibited PAEC-mediated human T cell activation. Furthermore, we demonstrated that highly purified human CD4+ and CD8+ T cells proliferated in response to PAEC and that this response was blocked by monoclonal antibodies directed against LFA-1 and CD2. Addition of alpha SLA class I blocked the proliferation of CD8+ but not CD4+ T cells, indicating direct presentation of SLA class I antigens to human T cells. We have recently shown that expression of the human complement inhibitor (CD59) on PAEC (PAEC-LXSNCD59) rendered these cells resistant to human complement-mediated activation and lysis, suggesting that human CD59 expression on PAEC could be an effective therapy for hyperacute rejection (HAR). However, recent studies have shown that in addition to its role as a complement inhibitor, CD59 binds human T cell CD2 and contributes to T cell activation. We therefore examined whether human CD59 expression on PAEC augmented the human antiporcine T cell response. We demonstrated that human T cells do not display increased binding to or activation by PAEC-LXSNCD59 relative to PAEC controls. Taken together, our data establish that PAEC directly stimulate human T cells in vitro and that interactions between the human accessory molecules CD2, LFA-1 and their PAEC surface ligands contribute to human T cell activation. In addition, the expression of human CD59 on porcine donor organs may confer resistance to human complement-mediated HAR without exacerbating the human antiporcine cellular response.

The invariant chain is required for intracellular transport and function of major histocompatibility complex class II molecules.

The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is thought to act as a chaperone that assists class II during folding, assembly, and transport. To define more precisely the role of Ii chain in regulating class II function, we have investigated in detail the biosynthesis, transport, and intracellular distribution of class II molecules in splenocytes from mice bearing a deletion of the Ii gene. As observed previously, the absence of Ii chain caused significant reduction in both class II-restricted antigen presentation and expression of class II molecules at the cell surface because of the intracellular accumulation of alpha and beta chains. Whereas much of the newly synthesized MHC molecules enter a high molecular weight aggregate characteristic of misfolded proteins, most of the alpha and beta chains form dimers and acquire epitopes characteristic of properly folded complexes. Although the complexes do not bind endogenously processed peptides, class II molecules that reach the surface are competent to bind peptides added to the medium, further demonstrating that at least some of the complexes fold properly. Similar to misfolded proteins, however, the alpha and beta chains are poorly terminally glycosylated, suggesting that they fail to reach the Golgi complex. As demonstrated by double label confocal and electron microscope immunocytochemistry, class II molecules were found in a subcompartment of the endoplasmic reticulum and in a population of small nonlysosomal vesicles possibly corresponding to the intermediate compartment or cis-Golgi network. Thus, although alpha and beta chains can fold and form dimers on their own, the absence of Ii chain causes them to be recognized as "misfolded" and retained in the same compartments as bona fide misfolded proteins.

Leukemia in telephone linemen.

This case-control study examines potential associations between telephone linework and the occurrence of leukemia except chronic lymphocytic leukemia in a primarily retired population of American Telephone and Telegraph Company (AT&T) workers. Cases died between 1975 and 1980. Exposure is defined both by job title and, for workers with complete job histories, by a lifetime exposure score based on industrial hygiene personal monitoring measurements of line and nonline jobs. When the time-weighted average mean for each job is accumulated into a lifetime exposure score, workers with scores above the median for the population show an excess of leukemia 2.5 times higher than workers below the median (95% confidence interval (CI) 0.7-8.6). Those individuals with long duration of employment in jobs with intermittent peak exposures may be at higher risk of leukemia than those with a constant exposure level. Analyses that allow for a latent period suggest the risk is associated with exposures that occurred 10 or more years before death. Workers with peak exposure scores above the median have odds ratios of 2.4 (95% CI 0.7-9.0) and 6.6 (95% CI 0.7-58) for latent periods of 10 and 15 years, respectively. The data suggest an increasing risk with increasing exposure (p for trend = 0.05) when cumulated scores are based on peak exposure scores. Peak exposures tended to occur in cable splicing work and in old telephone switching offices. The numbers in this study are small and observed differences may be due to chance.