Early signs of gait deviation in Duchenne muscular dystrophy.

BACKGROUND: Most analytical studies found in literature only focus on specific aspects of Duchenne muscular dystrophy (DMD) gait and posture (joint range of motion, standing balance, variations of gait spatial-temporal parameters). Some of them analyze single cases and do not provide a comprehensive evaluation of locomotion. There are few studies about DMD gait patterns, most of them concerning small groups of patients, sometimes not homogeneous, in which the clinical manifestations of the next stages of DMD were present. AIM: The goal of our study was to analyze the characteristics of gait patterns in early stage patients, when clinical and functional evaluation do not allow to quantify initial walking worsening or to identify the changes adopted to compensate for muscle weakness. SETTING: Gait Analysis Laboratory by using a six-camera motion capture system (Vicon, Oxford Metrics, UK), set at a sampling rate of 60 Hz. Subjects were asked to walk barefoot at their usual cadence, along a 10-m walkway, where one force platform (Kistler, Switzerland), embedded in the middle portion of the pathway, measured the foot-ground reaction forces. Retroreflective markers were placed on the subjects according to the protocol described in Davis et al. POPULATION: A group of 15 patients aging from 5 to 6.8 years was compared with a similar age control group composed of 9 healthy children. RESULTS: Spatial and temporal parameters showed significant differences between the two groups: cadence was increased and step length was decreased significantly in the DMD group. We found a significant increase in the range of anterior-posterior pelvic tilt and in pelvic rotation. In the frontal plane there was a tendency for an increased pelvic obliquity. Dynamic range of motion in sagittal plane showed a significant difference at the ankle, with an increased plantarflexion in swing in the dystrophic patients. Maximum dorsiflexion was reduced in the DMD group. Kinetic analysis showed significant differences in power generation and absorption at the hip joint and at the ankle joint. At knee there was a reduced flexor moment in mid-stance. Ankle showed a reduced dorsiflexor moment in terminal stance and pre-swing with a consequent reduction in the peak-to-peak excursion. CONCLUSION AND CLINICAL REHABILITATION IMPACT: It was shown that instrumented gait analysis, being more sensitive than other clinical and functional assessment methods, allowed to quantify the very early modifications characterizing locomotion worsening in the first stage of the DMD.

Functional changes in Duchenne muscular dystrophy: a 12-month longitudinal cohort study.

OBJECTIVE: The aim of the study was to assess different outcome measures in a cohort of ambulant boys with Duchenne muscular dystrophy (DMD) over 12 months in order to establish the spectrum of possible changes in relation to age and steroid treatment. METHODS: The study is a longitudinal multicentric cohort study. A total of 106 ambulant patients with DMD were assessed using the 6-minute walk test (6MWT) and North Star Ambulatory Assessment (NSAA) at baseline and 12 months. Clinical data including age and steroid treatment were collected. RESULTS: During the 12 months of the study, we observed a mean decline of 25.8 meters in the 6MWT with a SD of 74.3 meters. On NSAA, the mean decline was 2.2 points with a SD of 3.7. Not all the boys with DMD in our cohort showed a decline over the 12 months, with young boys showing some improvement in their 6MWT and NSAA scores up to the age of 7. NSAA and the 6MWT had the highest correlation (r = 0.52, p < 0.001). CONCLUSIONS: This study provides longitudinal data of NSAA and 6MWT over a 12-month period. These data can be useful when designing a clinical trial.

Reliability of the North Star Ambulatory Assessment in a multicentric setting.

The aim of this study was to investigate the suitability of the North Star Ambulatory Assessment as a possible outcome measure in multicentric clinical trials. More specifically we wished to investigate the level of training needed for achieving a good interobserver reliability in a multicentric setting. The scale was specifically designed for ambulant children with Duchenne Muscular Dystrophy and includes 17 items that are relevant for this cohort. Thirteen Italian centers participated in the study. In the first phase of the study we provided two training videos and an example of the scale performed on a child. After the first session of training, all the 13 examiners were asked to send a video with an assessment performed in their centre and to score all the videos collected. There were no difficulties in performing the items and in obtaining adequate videos with a hand held camera but the results showed a poor interobserver reliability (<.5). After a second training session with review and discussion of the videos previously scored, the same examiners were asked to score three new videos. The results of this session had an excellent interobserver reliability (.995). The level of agreement was maintained even when the same videos were rescored after a month, showing a significant intra-observer reliability (.95). Our results suggest that the NSAA is a test that can be easily performed, completed in 10 min and can be used in a multicentric setting, providing that adequate training is administered.

U19/Eaf2 knockout causes lung adenocarcinoma, B-cell lymphoma, hepatocellular carcinoma and prostatic intraepithelial neoplasia.

Upregulated gene 19 (U19)/ELL-associated factor 2 (Eaf2) is a potential human tumor suppressor that exhibits frequent allelic loss and downregulation in high-grade prostate cancer. U19/Eaf2, along with its homolog Eaf1, has been reported to regulate transcriptional elongation via interaction with the eleven-nineteen lysine-rich leukemia (ELL) family of proteins. To further explore the tumor-suppressive effects of U19/Eaf2, we constructed and characterized a murine U19/Eaf2-knockout model. Homozygous or heterozygous deletion of U19/Eaf2 resulted in high rates of lung adenocarcinoma, B-cell lymphoma, hepatocellular carcinoma and prostate intraepithelial neoplasia. Within the mouse prostate, U19/Eaf2 deficiency enhanced cell proliferation and increased epithelial cell size. The knockout mice also exhibited cardiac cell hypertrophy. These data indicate a role for U19/Eaf2 in growth suppression and cell size control as well as argue for U19/Eaf2 as a novel tumor suppressor in multiple mouse tissues. The U19/Eaf2 knockout mouse also provides a unique animal model for three important cancers: lung adenocarcinoma, B-cell lymphoma and hepatocellular carcinoma.

Noninvasive ventilation with positive airway pressure in paediatric intensive care.

AIM: The aim of the present study is to retrospectively evaluate the effectiveness of noninvasive pressure ventilation in the 24-bed Pediatric Intensive Care Unit (PICU) of the G. Gaslini Institute during a 24-month period. METHODS: A retrospective analysis of the characteristics (pH, CO2, SpO2, respiratory rate, oxygen requirement) of patients treated with noninvasive mechanical ventilation for different acute pathologies has been performed. RESULTS: Twenty patients (mean age 7.4+/-0.28 years) with acute respiratory failure due to different pathologies were treated with noninvasive mechanical ventilation. They were divided into 2 groups: the hypoxic group, suffering from pulmonary diseases, and the hypercapnic group, presenting a failure of the mechanical strength or increased dead space. Modalities of ventilation were pressure assisted/controlled or pressure support, delivered through nasal or facial masks. Fifteen out of 20 patients presented a marked improvement of oxygenation and ventilation. Mean times of treatment were 69 and 200 h in the hypoxic and hypercapnic groups, respectively. Five patients required intubation. Two patients presented reversible skin lesions over the nasal bridge. CONCLUSIONS: Noninvasive ventilation can be used in PICU. Major advantages regard immunocompromised children and patients with exacerbations from chronic respiratory diseases, whereas the exact role of noninvasive positive pressure ventilation in patients affected by acute respiratory distress syndrome is still controversial.

GLI1 localization in the germinal epithelial cells alternates between cytoplasm and nucleus: upregulation in transgenic mice blocks spermatogenesis in pachytene.

The zinc finger transcription factor GLI1 is the mediator of signaling by members of the Hedgehog (Hh) family. Male mice in which Desert hedgehog (Dhh), an Hh homologue expressed in Sertoli cells of the testis, was knocked out are sterile, suggesting that the Dhh/GLI1 pathway plays a role in spermatogenesis. Using an antiserum raised against human GLI1, we found that during the first round of spermatogenesis, GLI1 expression is initially cytoplasmic, then shifts to the nuclei of Sertoli and germ cells, and finally shifts back to the cytoplasm. In the adult mouse testis, GLI1 expression localized to the nuclei of germ cells, beginning with pachytene cells and persisting through round spermatids. Localization of GLI1 in elongating spermatids shifted from the nucleus to the cytoplasm and became associated with microtubules. We also examined a line of transgenic mice that overexpressed human GLI1. Male mice in this line were sterile. Spermatogenesis was blocked at the pachytene stage, and a subset of the morphologically indistinguishable pachytene cells underwent apoptosis. Patched-2, which is a Dhh receptor, and Fused, another component of the signal transduction pathway, are expressed in Leydig cells and in primary and secondary spermatocytes. Expression of GLI1 in the same cell types as Patched-2 and Fused and the disruption of spermatogenesis by GLI1 overexpression suggest that GLI1 is the mediator of the Dhh signal in the testis, and that it may be a regulator of spermatogenesis.

Vaccinia virus DNA replication occurs in endoplasmic reticulum-enclosed cytoplasmic mini-nuclei.

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ~45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [(3)H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.

Sertoli cell-specific rescue of fertility, but not testicular pathology, in Dax1 (Ahch)-deficient male mice.

DAX1 is an orphan member of the nuclear hormone receptor superfamily of transcription factors. Our recent characterization of Dax1 (Ahch)-deficient male mice revealed a primary testicular defect resulting in hypogonadism and sterility. The progressive degeneration of the germinal epithelium, independent of abnormal gonadotropin and testosterone production, suggested an intrinsic loss of Dax1 function in the Sertoli cells. To test this hypothesis, we assessed the effect of Sertoli cell-specific expression of a human DAX1 (AHC) transgene driven using the promoter of the Müllerian inhibiting substance (MIS) gene. The MIS-DAX1 transgene partially rescued the mutant phenotype of the Dax1-deficient male mice. Although testicular morphology remained abnormal, fertility was restored to levels matching that of wild-type littermates. Examination of several markers of sperm fertilizing capability revealed significant improvements in MIS-DAX1-rescued mice. Epididymal sperm count and sperm motility were greater in 12-week-old rescued mice than in age-matched Dax1-deficient mice. The ability of sperm to undergo an immediate acrosome reaction was impaired in Dax1-deficient animals, and sperm from Dax1-deficient mice fertilized only 8.2 +/- 6.8% of eggs in vitro, significantly less than rescue (67.8 +/- 19.1%) and wild-type (88.9 +/- 3.9%) sperm. These results indicate that Dax1 expression in Sertoli cells is adequate to overcome crucial thresholds related to sperm production and function. However, the failure to completely rescue the testicular pathology of Dax1-deficient mice suggests that Dax1 expression in other somatic cells is essential for normal testicular development.

Absence of proximal duct apoptosis in the ventral prostate of transgenic mice carrying the C3(1)-TGF-beta type II dominant negative receptor.

BACKGROUND: Prostatic epithelial cells are sensitive to the inhibitory effects of TGF-beta. However, TGF-beta signaling in the prostate is dependent on androgenic status. Under the in vivo conditions, it is difficult to dissociate the effect of TGF-beta from that of androgen on the prostate. METHODS: The objective of the present study was to create and verify a transgenic mouse system in which epithelial cells of the ventral prostate are insensitive to the actions of TGF-beta. By using a modified prostate-specific promoter, C3(1), the TGF-beta dominant negative receptor is only expressed in the epithelial cells of the ventral prostate, and these cells are resistant to TGF-beta. Morphology of transgenic animal prostates was compared to wild-type animal prostates by immunohistochemistry and microscopy. RESULTS: The prostate of transgenic mice exhibited an abnormal morphology with multiple layers of epithelial cells lining the proximal ducts, in contrast to the simple cuboidal monolayer of cells seen in the normal prostate. This observation was accompanied by a loss of apoptosis in this region, as seen by TUNEL assay. There was no significant difference in serum levels of testosterone between the wild-type and transgenic animals. CONCLUSIONS: These results demonstrated that a loss of sensitivity to TGF-beta results in the accumulation of multiple layers of epithelial cells in the proximal region of the ventral prostate. This abnormal growth illustrates that TGF-beta plays an important role in regulating prostate growth. The current transgenic system can be used as an experimental model to study the functional role of TGF-beta in prostatic growth and function.

Transgenic mice demonstrate a testis-specific promoter for lactate dehydrogenase, LDHC.

The mammalian genome encodes a family of lactate dehydrogenase (LDH) isozymes. Two of these, ldha and ldhb, are expressed ubiquitously. The ldhc gene is active only in the germinal epithelium during spermatogenesis. In our analysis of ldhc gene regulation, we found that a 60-base pair promoter sequence was sufficient for testis-specific expression in an in vitro transcription assay. To confirm these findings, a genomic fragment containing 100 base pairs overlapping the transcription start site was isolated and linked to the Escherichia coli lacZ gene. We report that this genomic fragment drives testis-specific expression in transgenic mice. We conclude that transcription of the transgene and possibly of the endogenous ldhc gene is restricted to leptotene/pachytene primary spermatocytes.

A cis-acting element that directs the activity of the murine methylation modifier locus Ssm1.

Silencing of chromosomal domains has been described in diverse systems such as position effect variegation in insects, silencing near yeast telomeres, and mammalian X chromosome inactivation. In mammals, silencing is associated with methylation at CpG dinucleotides, but little is known about how methylation patterns are established or altered during development. We previously described a strain-specific modifier locus, Ssm1, that controls the methylation of a complex transgene. In this study we address the questions of the nature of Ssm1's targets and whether its effect extends into adjacent sequences. By examining the inheritance of methylation patterns in a series of mice harboring deletion derivatives of the original transgene, we have identified a discrete segment, derived from the gpt gene of Escherichia coli, that is a major determinant for Ssm1-mediated methylation. Methylation analysis of sequences adjacent to a transgenic target indicates that the influence of this modifier extends into the surrounding chromosome in a strain-dependent fashion. Implications for the mechanism of Ssm1 action are discussed.

Expression of lambda and kappa genes can occur in all B cells and is initiated around the same pre-B-cell developmental stage.

Transgenic mice that carry a lambda 2 transgene under the control of the V lambda 2 promoter and the E lambda 2-4 enhancer (lambda 2E lambda mice) are described. A high proportion of B cells in the spleen and the bone marrow express the lambda transgene on the cell membrane. lambda 2 protein is synthesized by all lambda 2E lambda-derived spleen B-cell hybridomas that have retained the transgene, suggesting that all B cells have the ability to express lambda genes. Feedback inhibition of endogenous kappa-gene rearrangement is significant, but not complete. The results are similar to those with transgenic mice expressing the same lambda 2 transgene under the control of the heavy-chain enhancer (lambda 2EH mice). Although the lambda 2EH transgene is expressed before the lambda 2E lambda transgene, feedback inhibition seems to occur at about the same stage of B-cell development, regardless of the timing of expression of the lambda transgenes. Apparently, feedback is not necessarily coincident with the assembly of a heavy-chain/light-chain complex in pre-B cells. Expression of lambda in the normal fetal liver coincides with the expression of kappa; thus, it appears that lambda-gene transcription is not delayed. The differential rearrangement of kappa and lambda genes is discussed in the light of these findings.

Immunoglobulin gamma 2b transgenes inhibit heavy chain gene rearrangement, but cannot promote B cell development.

Transgenic mice with a gamma 2b transgene were produced to investigate whether gamma 2b can replace mu in the development of B lymphocytes. Transgenic gamma 2b is present on the surface of B cells. Young transgenic mice have a dramatic decrease in B cell numbers, however, older mice have almost normal B cell numbers. Strikingly, all gamma 2b-expressing B cells in the spleen also express mu. The same is true for mice with a hybrid transgene in which the mu transmembrane and intracytoplasmic sequences replace those of gamma 2b (gamma 2b-mumem). The B cell defect is not due to toxicity of gamma 2b since crosses between gamma 2b transgenic and mu transgenic mice have normal numbers of B cells. Presence of the gamma 2b transgene strongly enhances the feedback inhibition of endogenous heavy chain gene rearrangement. Light chain genes are expressed normally, and the early expression of transgenic light chains does not improve B cell maturation. When the endogenous mu locus is inactivated, B cells do not develop at all in gamma 2b transgenic mice. The data suggest that gamma 2b cannot replace mu in promoting the developmental maturation of B cells, but that it can cause feedback inhibition of heavy chain gene rearrangement. Thus, the signals for heavy chain feedback and B cell maturation appear to be different.

Rearrangement and expression of immunoglobulin genes in transgenic mice.

Transgenic mice are discussed which carry a rearrangement test transgene. The methylation status of the transgene varies, depending on the background mouse strain. When the transgene is bred into the C57BL/6 strain, it is completely methylated and not rearranged in lymphoid organs. After several generations of crossing into DBA/2 or SJL the transgene becomes unmethylated and rearranges at high frequency. A strain specific modifier of DNA methylation (Ssm-1) was mapped close to the Friend virus susceptibility locus (Fv-1) on mouse chromosome 4. Rearranged transgenes from spleen, bone marrow and thymus of adult mice or fetal liver were cloned and sequenced. A great variety of joints was found, with about 1/3 being in the correct reading frame. Small deletions into the V- and J-coding ends as well as N region additions contributed to the variability. The fetal joints showed no N regions. Since no functional immunoglobulin (Ig) gene can be created from this artificial test gene, the data indicate that the rearrangement mechanism of the fetus differs from that of the adult.

A strain-specific modifier on mouse chromosome 4 controls the methylation of independent transgene loci.

A transgene, pHRD, is highly methylated in 12 independent mouse lines when in a C57BL/6 strain background, but becomes progressively less methylated when bred into a DBA/2 background. Transgenes inherited from the mother are generally more methylated; however, this parental effect disappears following continued breeding into the nonmethylating strain. Mapping experiments using BXD recombinant inbred mice as well as other inbred strains indicate that a single strain-specific modifier (Ssm-1) linked to, but distinct from, Fv-1 is responsible for the strain effect. In addition to the methylated and unmethylated transgenic phenotypes, certain mice exhibit a partial methylation pattern that is a consequence of an unusual cellular mosaicism. The pHRD transgene, containing target sequences for the V(D)J recombinase, undergoes site-specific recombination only in lymphoid tissues. This V-J joining is restricted primarily to unmethylated transgene copies.

Inhibition of immunoglobulin gene rearrangement by the expression of a lambda 2 transgene.

The rearrangement of Ig genes is known to be regulated by the production of H and kappa L chains. To determine whether lambda L chains have a similar effect, transgenic mice were produced with a lambda 2 gene. It was necessary to include the H chain enhancer, since a lambda gene without the added enhancer did not result in transgene expression. The lambda 2 transgene with the H enhancer was expressed in lymphoid cells only. The majority of the B cells of newborn transgenic mice produced lambda, whereas kappa + cells were reduced. Concomitantly, serum levels of kappa and kappa mRNA were diminished. By 2 wk after birth the proportion of kappa-expressing cells was dramatically increased. Adults had reduced proportions of B cells that produced lambda only, but the levels of lambda were still higher than in normal littermates. Also, kappa + cells were still lower than in normal mice. Analysis of hybridomas revealed that reduction of kappa gene rearrangement was the basis for the decreased frequency of kappa + cells. Furthermore, many cells also contained an unrearranged H chain allele. It was concluded that feedback inhibition by the lambda 2 together with endogenous H protein may have inhibited recombinase activity in early pre-B cells, leading to inhibition of both H chain and kappa gene rearrangement. Thus, lambda 2 can replace kappa in a feedback complex. The levels of serum lambda 1 and, to a lesser degree, of spleen lambda 1 mRNA were reduced in the lambda 2 transgenic mice. However, the proportion of hybridomas with endogenous lambda gene rearrangement was at least as high as in normal mice. It was therefore concluded that the suppression of functional lambda 1 may be a consequence of decreased selection of endogenous lambda-producing cells because of the excess of transgenic lambda. The escape of kappa-producing cells from feedback inhibition may be the result of several mechanisms that operate to varying degrees, among them: (a) kappa rearrangement during a period in which the recombinase is still active after appearance of a lambda 2/mu stop signal; (b) a B cell lineage that is not feedback inhibited at the pre-B cell stage; (c) subthreshold levels of transgenic lambda 2 in some pre-B cells; and (d) loss of the lambda 2 transgenes in rare pre-B cells.

Analysis of human C8 with monoclonal antibodies. Characterization of a monoclonal antibody that recognizes free C8 alpha-gamma subunit.

The eighth component of human C is essential for the formation of the membranolytic C attack complex. C8 has a unique structure in that two covalently linked chains, C8 alpha and C8 gamma, are associated non-covalently with the third chain, C8 beta. In order to study the structure and assembly of the C8 molecule, a panel of mAb has been produced against the C component C8. Eight of these mAb had reactivity to the C8 alpha-gamma subunit, whereas four reacted with C8 beta. One of the C8 alpha-gamma mAb, C8A2, had specificity for an epitope on the C8 alpha-chain and exhibited no cross-reactivity to any of the other terminal C components, including C8 beta. C8A2 inhibited the hemolytic activity of the C8 alpha-gamma subunit but had no effect on the activity of fluid phase whole C8 or C8 within membrane-bound C5b-8. Functional experiments suggest that C8A2 inhibits C8 alpha-gamma activity by interfering with its interaction with the C8 beta-chain. In an enzyme immunoassay using the C8A2 mAb, free C8 alpha-gamma subunit could be detected in both homozygous and heterozygous C8 beta-deficient serum. However, only low level binding was observed when homozygous C5- and C7-deficient sera were tested. Thus the mAb, C8A2, recognizes an epitope expressed on the C8 alpha-gamma subunit but not on intact C8 and can detect free C8 alpha-gamma in the presence of native C8.