Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C.

The limb girdle and congenital muscular dystrophies (LGMD and CMD) are characterized by skeletal muscle weakness and dystrophic muscle changes. The onset of symptoms in CMD is within the first few months of life, whereas in LGMD they can occur in late childhood, adolescence or adult life. We have recently demonstrated that the fukutin-related protein gene (FKRP) is mutated in a severe form of CMD (MDC1C), characterized by the inability to walk, leg muscle hypertrophy and a secondary deficiency of laminin alpha2 and alpha-dystroglycan. Both MDC1C and LGMD2I map to an identical region on chromosome 19q13.3. To investigate whether these are allelic disorders, we undertook mutation analysis of FKRP in 25 potential LGMD2I families, including some with a severe and early onset phenotype. Mutations were identified in individuals from 17 families. A variable reduction of alpha-dystroglycan expression was observed in the skeletal muscle biopsy of all individuals studied. In addition, several cases showed a deficiency of laminin alpha2 either by immunocytochemistry or western blotting. Unexpectedly, affected individuals from 15 families had an identical C826A (Leu276Ileu) mutation, including five that were homozygous for this change. Linkage analysis identified at least two possible haplotypes in linkage disequilibrium with this mutation. Patients with the C826A change had the clinically less severe LGMD2I phenotype, suggesting that this is a less disruptive FKRP mutation than those found in MDC1C. The spectrum of LGMD2I phenotypes ranged from infants with an early presentation and a Duchenne-like disease course including cardiomyopathy, to milder phenotypes compatible with a favourable long-term outcome.

Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan.

The congenital muscular dystrophies (CMD) are a heterogeneous group of autosomal recessive disorders presenting in infancy with muscle weakness, contractures, and dystrophic changes on skeletal-muscle biopsy. Structural brain defects, with or without mental retardation, are additional features of several CMD syndromes. Approximately 40% of patients with CMD have a primary deficiency (MDC1A) of the laminin alpha2 chain of merosin (laminin-2) due to mutations in the LAMA2 gene. In addition, a secondary deficiency of laminin alpha2 is apparent in some CMD syndromes, including MDC1B, which is mapped to chromosome 1q42, and both muscle-eye-brain disease (MEB) and Fukuyama CMD (FCMD), two forms with severe brain involvement. The FCMD gene encodes a protein of unknown function, fukutin, though sequence analysis predicts it to be a phosphoryl-ligand transferase. Here we identify the gene for a new member of the fukutin protein family (fukutin related protein [FKRP]), mapping to human chromosome 19q13.3. We report the genomic organization of the FKRP gene and its pattern of tissue expression. Mutations in the FKRP gene have been identified in seven families with CMD characterized by disease onset in the first weeks of life and a severe phenotype with inability to walk, muscle hypertrophy, marked elevation of serum creatine kinase, and normal brain structure and function. Affected individuals had a secondary deficiency of laminin alpha2 expression. In addition, they had both a marked decrease in immunostaining of muscle alpha-dystroglycan and a reduction in its molecular weight on western blot analysis. We suggest these abnormalities of alpha-dystroglycan are caused by its defective glycosylation and are integral to the pathology seen in MDC1C.

Dysbindin, a novel coiled-coil-containing protein that interacts with the dystrobrevins in muscle and brain.

The dystrophin-associated protein complex (DPC) is required for the maintenance of muscle integrity during the mechanical stresses of contraction and relaxation. In addition to providing a membrane scaffold, members of the DPC such as the alpha-dystrobrevin protein family are thought to play an important role in intracellular signal transduction. To gain additional insights into the function of the DPC, we performed a yeast two-hybrid screen for dystrobrevin-interacting proteins. Here we describe the identification of a dysbindin, a novel dystrobrevin-binding protein. Dysbindin is an evolutionary conserved 40-kDa coiled-coil-containing protein that binds to alpha- and beta-dystrobrevin in muscle and brain. Dystrophin and alpha-dystrobrevin are co-immunoprecipitated with dysbindin, indicating that dysbindin is DPC-associated in muscle. Dysbindin co-localizes with alpha-dystrobrevin at the sarcolemma and is up-regulated in dystrophin-deficient muscle. In the brain, dysbindin is found primarily in axon bundles and especially in certain axon terminals, notably mossy fiber synaptic terminals in the cerebellum and hippocampus. These findings have implications for the molecular pathology of Duchenne muscular dystrophy and may provide an alternative route for anchoring dystrobrevin and the DPC to the muscle membrane.

Syncoilin, a novel member of the intermediate filament superfamily that interacts with alpha-dystrobrevin in skeletal muscle.

Dystrophin coordinates the assembly of a complex of structural and signaling proteins that are required for normal muscle function. A key component of the dystrophin protein complex is alpha-dystrobrevin, a dystrophin-associated protein whose absence results in neuromuscular junction defects and muscular dystrophy. To gain further insights into the role of alpha-dystrobrevin in skeletal muscle, we used the yeast two-hybrid system to identify a novel alpha-dystrobrevin-binding partner called syncoilin. Syncoilin is a new member of the intermediate filament superfamily and is highly expressed in skeletal and cardiac muscle. In normal skeletal muscle, syncoilin is concentrated at the neuromuscular junction, where it colocalizes and coimmunoprecipitates with alpha-dystrobrevin-1. Expression studies in mammalian cells demonstrate that, while alpha-dystrobrevin and syncoilin associate directly, overexpression of syncoilin does not result in the self-assembly of intermediate filaments. Finally, unlike many components of the dystrophin protein complex, we show that syncoilin expression is up-regulated in dystrophin-deficient muscle. These data suggest that alpha-dystrobrevin provides a link between the dystrophin protein complex and the intermediate filament network at the neuromuscular junction, which may be important for the maintenance and maturation of the synapse.

Alternative splicing of dystrobrevin regulates the stoichiometry of syntrophin binding to the dystrophin protein complex.

Dystrophin coordinates the assembly of a complex of structural and signalling proteins that is required for normal muscle function. A key component of the dystrophin-associated protein complex (DPC) is alpha-dystrobrevin, a dystrophin-related and -associated protein whose absence results in muscular dystrophy and neuromuscular junction defects [1,2]. The current model of the DPC predicts that dystrophin and dystrobrevin each bind a single syntrophin molecule [3]. The syntrophins are PDZ-domain-containing proteins that facilitate the recruitment of signalling proteins such as nNOS (neuronal nitric oxide synthase) to the DPC [4]. Here we show, using yeast two-hybrid analysis and biochemical binding studies, that alpha-dystrobrevin in fact contains two independent syntrophin-binding sites in tandem. The previously undescribed binding site is situated within an alternatively spliced exon of alpha-dystrobrevin, termed the variable region-3 (vr3) sequence, which is specifically expressed in skeletal and cardiac muscle [5,6]. Analysis of the syntrophin-binding region of dystrobrevin reveals a tandem pair of predicted alpha helices with significant sequence similarity. These alpha helices, each termed a syntrophin-binding motif, are also highly conserved in dystrophin and utrophin. Together these data show that there are four potential syntrophin-binding sites per dystrophin complex in skeletal muscle: two on dystrobrevin and two on dystrophin or utrophin. Furthermore, alternative splicing of dystrobrevin provides a mechanism for regulating the stoichiometry of syntrophin association with the DPC. This is likely to have important consequences for the recruitment of specific signalling molecules to the DPC and ultimately for its function.

Different dystrophin-like complexes are expressed in neurons and glia.

Duchenne muscular dystrophy is a fatal muscle disease that is often associated with cognitive impairment. Accordingly, dystrophin is found at the muscle sarcolemma and at postsynaptic sites in neurons. In muscle, dystrophin forms part of a membrane-spanning complex, the dystrophin-associated protein complex (DPC). Whereas the composition of the DPC in muscle is well documented, the existence of a similar complex in brain remains largely unknown. To determine the composition of DPC-like complexes in brain, we have examined the molecular associations and distribution of the dystrobrevins, a widely expressed family of dystrophin-associated proteins, some of which are components of the muscle DPC. beta-Dystrobrevin is found in neurons and is highly enriched in postsynaptic densities (PSDs). Furthermore, beta-dystrobrevin forms a specific complex with dystrophin and syntrophin. By contrast, alpha-dystrobrevin-1 is found in perivascular astrocytes and Bergmann glia, and is not PSD-enriched. alpha-Dystrobrevin-1 is associated with Dp71, utrophin, and syntrophin. In the brains of mice that lack dystrophin and Dp71, the dystrobrevin-syntrophin complexes are still formed, whereas in dystrophin-deficient muscle, the assembly of the DPC is disrupted. Thus, despite the similarity in primary sequence, alpha- and beta-dystrobrevin are differentially distributed in the brain where they form separate DPC-like complexes.

Acute hepatitis due to fluoxetine therapy.

Fluoxetine-induced hepatotoxicity is generally considered of minimal clinical importance and is not well recognized. Asymptomatic increases in liver enzyme values have been observed in 0.5% of patients who take long-term fluoxetine therapy. This report details 2 cases of acute hepatitis believed to be caused by fluoxetine. Three cases of acute hepatitis caused by fluoxetine have been reported previously. The mechanism of fluoxetine-induced hepatotoxicity is unknown. Although routine monitoring of liver function may not be cost-effective, physicians should be alert to the possibility of fluoxetine-associated hepatitis and consider early discontinuation of the drug if this condition is suspected.

Staging renal carcinoma. What is sufficient?

We retrospectively reviewed the roentgenographic and pathologic staging of 64 patients with renal cell carcinoma to assess the role of the various staging modalities (ie, angiography, venacavography, bone scanning, ultrasound, computed tomography [CT], and magnetic resonance imaging). Specific attention was directed at detecting vena cava thrombus and metastatic bone disease, factors with a significant impact on the therapeutic approach. The findings support the role of CT as the principle tool for overall staging and the observation that venacavography is not indicated if CT has excluded caval thrombus. Similarly, routine bone scans are not warranted in the absence of an elevated alkaline phosphatase level or bone pain. The key to the more efficient utilization of imaging resources is understanding the capabilities of the technology available.

An in vitro model for clonal anergy in continuously growing antigen-specific B-cell lines.

Two continuously growing nonmalignant B-cell lines specific for the hapten DNP have been used to study tolerance in developing B cells. These cell lines have previously been shown to consist of small cells without sIgM but with cytoplasmic mu chains, and mature sIgM- and sIgD-bearing cells. When the sIgM-negative cells are placed in culture, mature DNP-specific B cells begin to appear. The studies reported here have shown that when these cell lines were propagated in the presence of either 200 micrograms/ml or 1 mg/ml of the tolerogen DNP-MGG there was no inhibition of cell line growth as measured by thymidine incorporation, and no inhibition of receptor expression by maturing B cells. The cell line lymphocytes propagated in the presence of 200 micrograms/ml DNP-MGG for 7, 30, 45, or 60 days became tolerant and the tolerance persisted for at least 6 days after removal of DNP-MGG. However, tolerance was lost between 6 and 10 days after removal of DNP-MGG. Propagation of the cell lines for 30 days in either DNP-KLH or DNP-Ficoll produced the same results. Limiting dilution cultures of cell line lymphocytes made tolerant by growing them for 30 days in the presence of DNP-MGG demonstrated that there was a marked decrease in precursor frequency compared to controls. However, cell line lymphocytes made tolerant by a 48-hr incubation with DNP-MGG did not have a significant decrease in precursor frequency. These data suggest that tolerance induced by growing these cell lines in the presence of DNP-MGG is a valid in vitro model of tolerance in developing antigen-specific B cells. Tolerance induced in this model is consistent with the clonal anergy hypothesis, but requires the continued presence of DNP-MGG to maintain unresponsiveness. This suggests that clonal anergy can occur in B cells but may not be the sole mechanism of self tolerance for those antigens which are sequestered from the immune system.

Tolerance and immunity in antigen-specific B-lymphocyte lines: early receptor binding of either antigen or tolerogen initiates an immune response.

Studies of the effect of tolerance-inducing compounds on B lymphocytes have been complicated by the fact that it is technically difficult to completely isolate the antigen-specific B cell from the effects of T cells or T-cell factors. We have used our cell lines of nonmalignant dinitrophenyl (DNP)-specific B lymphocytes derived from normal mice, which have no contaminating T cells, to study the effect of DNP-murine IgG2a (DNP-MGG), a tolerogen which is not normally immunogenic, on antigen-specific B lymphocytes. Preincubation with DNP-MGG for 48 hr, both in the presence and absence of T-cell factors from EL-4 supernatant prior to adding the antigen DNP-Ficoll, can induce tolerance in cell line B lymphocytes. The suppression is antigen-specific since preincubation with fluorescein-MGG or unconjugated MGG does not suppress the anti-DNP response. At least a 36-hr incubation is required for tolerance induction in B lymphocytes, but a 6-hr preincubation with DNP-MGG augments the immune response to DNP-Ficoll. Lymphocytes incubated for 6 or 24 hr with DNP-MGG prior to adding EL-4 supernatant and filler cells without DNP-Ficoll exhibited an immune response equal to that elicited by DNP-Ficoll and T-cell factors. A 6-hr pulse with a DNP-conjugated polymer of D-glutamic acid and D-lysine (DNP-dGL), a B-cell tolerogen which does not bind to Fc receptors, elicited the same immune response as seen with a 6-hr pulse of DNP-MGG but a 48-hr preincubation with DNP-dGL induced tolerance. Thus, it is likely that the initial binding of the tolerogen to the immunoglobulin receptor on the mature B cell elicits an activation signal similar to that seen with the antigen. The suppressive effect of the tolerogen itself appears to occur at a later stage of the process of the B-cell activation, proliferation, and differentiation.

Macrophage-derived soluble factors mediate suppression induced by 2,4-dinitrophenyl-conjugated mouse IgG in hybridoma cells.

Our laboratory has established that 2,4-dinitrophenyl-conjugated mouse IgG (DNP-MGG) can specifically suppress proliferation, antibody synthesis, and secretion in vivo in two anti-DNP secreting cell lines: hybridoma 35-12 and myeloma MOPC-315. In the present study an in vitro system was used to further analyze the mechanism of suppression of hybridoma 35-12 cells (HC) by DNP-MGG. It was found that DNP-MGG-induced suppression of HC requires macrophages (M phi) and occurs only in eclipsed HC which are mainly small, nonsecreting cells. The M phi-mediated suppression is DNP specific, requires no M phi-HC cell contact, and does not involve killing of eclipsed HC. M phi culture supernatant alone cannot mediate suppression, but supernatants obtained by culturing M phi with either HC or supernatant from HC culture can mediate suppression of eclipsed HC in the presence of DNP-MGG. DNP-MGG is not required for the generation of effective M phi factors, but it is required for suppression of HC in the presence of M phi factors. Indomethacin cannot reverse M phi-mediated suppression, suggesting prostaglandins may not be the M phi factors. These data suggest that M phi-derived factors which are not prostaglandins in nature may play a role in B-cell regulation and in B-cell suppression induced by tolerogenic forms of antigen.

Central suppression of monoclonal B cells: DNP-MGG suppresses proliferation and immunoglobulin synthesis in anti-DNP-secreting hybridoma and myeloma.

Our laboratory has established that 2,4-dinitrophenyl-conjugated mouse IgG (DNP-MGG) can specifically suppress the anti-DNP secretion in hybridoma 35-12 and plasmacytoma MOPC-315 cells. To further study the mechanism of this suppression, the effect of DNP-MGG on anti-DNP synthesis and cell proliferation was investigated in these cell lines. Cultured tumor cells (1 X 10(6] were injected ip into syngeneic mice. These mice were then given either 1 mg MGG or 1 mg DNP-MGG. At different days after injection, tumor cells obtained from these mice were assayed for anti-DNP secretion, anti-DNP synthesis, cell proliferation, and tumor cell size. When the anti-DNP secretion was suppressed by DNP-MGG, the intracellular synthesis of anti-DNP, demonstrated by [3H]leucine incorporation into DNP-binding activity, was also suppressed. Simultaneous assays of [3H]thymidine incorporation demonstrated that proliferation was also suppressed. Tumor cells injected ip into mice normally become small nonsecreting cells and later return to preinjection size and secrete antibody. Those cells whose antibody synthesis and proliferation were suppressed by DNP-MGG remained smaller.

A prospective analysis of the arthritis syndrome and immune function in jejunoileal bypass patients.

Fifty-two patients undergoing jejunoileal bypass surgery were prospectively evaluated to determine: 1) the incidence of the associated arthritic syndrome; 2) whether we could identify patients at risk for arthritis prior to surgery; and 3) changes in immune function. The incidence of arthritis was 28% and was frequently associated with dermatitis. No preoperative clinical or laboratory parameters identified those patients at risk to develop rheumatic problems. Circulating immune complexes were found in both arthritis and non-arthritis patients after surgery. Mean serum levels of IgA rose significantly after surgery only in patients who developed arthritis, but remained within the normal range. No other immunologic abnormalities were noted.

Specific suppression of hybridoma immunoglobulin secretion by hapten-conjugated mouse IgG: a model of B-cell tolerance.

In an effort to establish a model system for studying B-cell tolerance, the effects of hapten-conjugated isologous mouse IgG on the secretion of antibody by a mouse hybridoma cell line were studied. The hybridoma cell line 35-12 (HC) secretes IgM antibody to the hapten dinitrophenyl (DNP). After HC cells are injected intraperitoneally into BALB/c mice, the cells initially undergo a marked reduction in the proportion of PFC/10(6) followed by a return of PFC to pretransfer levels by 7-10 days. Hapten-conjugated mouse IgG (DNP-MGG), which induces tolerance to DNP in normal mouse B cells, also induces suppression of HC PFC when administered within the first 2 days after the transfer. Administration of tolerogen either 2 days before injection of HC or after the PFC response has returned to preinjection levels fails to give suppression. Suppression is dose dependent and hapten specific since immunogenic hapten-carrier conjugates (e.g., DNP-KLH, DNP-Ficoll) and fluorescein-MGG are not suppressive. T cells may not be required for suppression since hybridoma cells inoculated into nude mice are also suppressed by DNP-MGG. These results suggest that hybridoma cells undergo a change from nonsecreting to secreting cells during in vivo growth and that administration of tolerogen during the nonsecreting stage inhibits antibody secretion.

SAA suppression of immune response in vitro: evidence for an effect on T cell-macrophage interaction.

SAA has been shown previously to inhibit the in vitro antibody response of normal murine spleen cells to SRBC. This effect is nonspecific but does not directly suppress B cells. To evaluate the mechanisms of this suppression, the in vitro antibody response to SRBC was studied using cells from cytoxan-treated mice. No evidence for preformed suppressor cells was found. Also, preincubation of normal cells with SAA failed to reveal evidence for the generation of suppressor cells in vitro. The addition of 2-mercaptoethanol to cultures completely reversed the suppression caused by SAA. Addition of either normal T cells or normal macrophages to cultures partially reversed suppression caused by SAA, but only a combination of T cells and macrophages could completely reverse the suppression. These data are consistent with an effect of SAA on macrophage-T cell interaction in the generation of the immune response to T-dependent antigen. No evidence for the participation of suppressor cells as has been demonstrated for alpha-fetoprotein was found.

The effect of cyclic nucleotides on tolerance induction by dinitrophenyl-isologous immunoglobulin G.

When tolerance to a hapten is induced by injecting the hapten conjugated to isologous immunoglobulin (Ig) G (dinitrophenyl (DNP)-IgG), the hapten remains on the surface of the antigen binding cell (ABC) for the duration of the tolerant state. Although this has been interpreted as a receptor blockade of ABC, it does not necessarily indicate that physical blocking of antigen receptors is the only mechanism of tolerance. Data are presented which suggest that cyclic nucleotides play a role in the in vitro induction of tolerance by DNP-IgG. Agents that elevate intracellular cyclic adenosine 3',5' monophosphate (cAMP) levels were added to lymphocytes during tolerance induction in vitro by DNP-IgG. Dibutyryl cAMP, theophylline, aminophylline, and isobutyl alpha-methylxanthine were able to inhibit the induction of tolerance to DNP. On the other hand, addition of dibutyryl cyclic guanosine 3',5'-monophosphate (cGMP) to lymphocytes incubated with doses of DNP-IgG that ordinarily are too low to induce tolerance facilitated tolerance induction. The possible role of these intracellular second messengers in the induction to tolerance is discussed.