Identification of a major recombination hotspot in patients with short stature and SHOX deficiency

Human growth is influenced not only by environmental and internal factors but also by a large number of different genes. One of these genes, SHOX, is believed to play a major role in growth, since defects in this homeobox-containing gene on the sex chromosomes lead to syndromal short stature (Leri-Weill dyschondrosteosis, Langer mesomelic dysplasia, and Turner syndrome) as well as to idiopathic short stature. We have analyzed 118 unrelated patients with Leri-Weill dyschondrosteosis and >1,500 patients with idiopathic short stature for deletions encompassing SHOX. Deletions were detected in 34% of the patients with Leri-Weill dyschondrosteosis and in 2% of the patients with idiopathic short stature. For 27 patients with Leri-Weill dyschondrosteosis and for 6 with idiopathic short stature, detailed deletion mapping was performed. Analysis was performed by polymerase chain reaction with the use of pseudoautosomal polymorphic markers and by fluorescence in situ hybridization with the use of cosmid clones. Here, we show that, although the identified deletions vary in size, the vast majority (73%) of patients tested share a distinct proximal deletion breakpoint. We propose that the sequence present within this proximal deletion breakpoint "hotspot" region predisposes to recurrent breaks.     

Expansion and functional relevance of high-avidity myelin-specific CD4+ T cells in multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease in which myelin-specific T cells are believed to play a crucial pathogenic role. Nevertheless, so far it has been extremely difficult to demonstrate differences in T cell reactivity to myelin Ag between MS patients and controls. We believe that by using unphysiologically high Ag concentrations previous studies have missed a highly relevant aspect of autoimmune responses, i.e., T cells recognizing Ag with high functional avidity. Therefore, we focused on the characterization of high-avidity myelin-specific CD4+ T cells in a large cohort of MS patients and controls that was matched demographically and with respect to expression of MHC class II alleles. We demonstrated that their frequency is significantly higher in MS patients while the numbers of control T cells specific for influenza hemagglutinin are virtually identical between the two cohorts; that high-avidity T cells are enriched for previously in vivo-activated cells and are significantly skewed toward a proinflammatory phenotype. Moreover, the immunodominant epitopes that were most discriminatory between MS patients and controls differed from those described previously and were clearly biased toward epitopes with lower predicted binding affinities to HLA-DR molecules, pointing at the importance of thymic selection for the generation of the autoimmune T cell repertoire. Correlations between selected immunological parameters and magnetic resonance imaging markers indicate that the specificity and function of these cells influences phenotypic disease expression. These data have important implications for autoimmunity research and should be considered in the development of Ag-specific therapies in MS.     

Reservoirs of antibiotic resistance genes

A potential concern about the use of antibiotics in animal husbundary is that, as antibiotic resistant bacteria move from the farm into the human diet, they may pass antibiotic resistance genes to bacteria that normally reside in a the human intestinal tract and from there to bacteria that cause human disease (reservoir hypothesis). In this article various approaches to evaluating the risk of agricultural use of antibiotics are assessed critically. In addition, the potential benefits of applying new technology and using new insights from the field of microbial ecology are explained.     

The Full-Length Isoform of Human Papillomavirus 16 E6 and Its Splice Variant E6* Bind to Different Sites on the Procaspase 8 Death Effector Domain

Human papillomavirus 16 is a causative agent of most cases of cervical cancer and has also been implicated in the development of some head and neck cancers. The early viral E6 gene codes for two alternatively spliced isoforms, E6_large and E6*. We have previously demonstrated the differential effects of E6_large and E6* binding on the expression and stability of procaspase 8, a key mediator of the apoptotic pathway. Additionally, we have reported that E6 binds to the FADD death effector domain (DED) at a novel E6 binding domain. Sequence similarities between the FADD and procaspase 8 DEDs suggested a specific region for E6_large/procaspase 8 binding, which was subsequently confirmed by mutational analysis as well as by the ability of peptides capable of blocking E6/FADD binding to also block E6_large/caspase 8 binding. However, the binding of the smaller isoform, E6*, to procaspase 8 occurs at a different region, as deletion and point mutations that disrupt E6_large/caspase 8 DED binding do not disrupt E6*/caspase 8 DED binding. In addition, peptide inhibitors that can block E6_large/procaspase 8 binding do not affect the binding of E6* to procaspase 8. These results demonstrate that the residues that mediate E6*/procaspase 8 DED binding localize to a different region on the protein and employ a separate binding motif. This provides a molecular explanation for our initial findings that the two E6 isoforms affect procaspase 8 stability in an opposing manner.The relationship between viruses and cancers is reflected in the observation that viral infections account for approximately 10 to 15% of the cancer burden worldwide (6, 60). This makes viral infections one of the preventable risk factors of cancer. Viruses are associated with several human malignancies, including hepatitis B and C virus-associated hepatocellular carcinomas (48), Epstein-Barr virus-associated nasopharyngeal carcinomas and lymphomas (36), and human T-cell leukemia virus-associated adult T-cell leukemia (8, 28). Although there is a correlation between infection and the onset of cancer, the frequency of infection supersedes the incidence of cancer inception, suggesting that the presence of the virus alone is not sufficient to trigger carcinogenesis. Progression from viral infection to tumor development therefore requires additional environmental and cellular factors in addition to the expression and activity of virus-encoded proteins (40).High-risk strains of human papillomavirus (HPV) (high-risk HPV [HR-HPV]) such as HPV16 and HPV18 have been implicated in most cases of cervical cancer and also in a subset of head and neck cancers (24, 26, 39). Infection with oncogenic strains of HPV represents up to 75% of all infections. Furthermore, 1/10 of all deaths among women worldwide can be attributed to HR-HPV-related cancers (44, 45). The key players in promoting cell transformation and immortalization following HPV infection are the viral early proteins E6 and E7. These proteins are well known for their ability to interact with the tumor suppressor p53 or members of the retinoblastoma family of proteins including pRb, p107, and p130, respectively (3, 17, 41). In addition to p53, HR-HPV E6 (HR-E6) binds to a number of cellular proteins involved in various aspects of cell proliferation and virus survival (reviewed in references 34 and 53). Our laboratory has reported that E6 binds to key mediators of the apoptotic pathway including tumor necrosis factor (TNF) R1 (22), the FADD death effector domain (DED) (21), and the procaspase 8 DED (20) and, in doing so, impedes apoptosis from taking place.As noted above, HR-E6 binds to TNF R1, blocking the adaptor molecule TRADD from binding to the membrane receptor. Similarly, the binding of HR-E6 to the FADD DED, a molecule common to the TNF-, Fas L-, and TRAIL-mediated extrinsic pathways of apoptosis, leads to the accelerated degradation of FADD and thereby inhibits the binding of additional downstream molecules necessary for programmed cell death. Additionally, we have reported that HR-E6 binds to procaspase 8, another molecule common to all three receptor-mediated pathways. The importance of procaspase 8 can be demonstrated by the many proteins produced by viruses to either inactivate or inhibit this apoptotic mediator in order to evade clearance by the host immune response. Such proteins include the herpes simplex virus R1 subunit that interferes with caspase 8 activation (31); the molluscum contagiosum virus MC159 protein that binds to the DEDs of both FADD and procaspase 8, thereby inhibiting their interaction (25); the human herpesvirus 8 FLICE protein that obstructs procaspase 8 cleavage and prevents its activation (4); and the cowpox virus serpin CrmA, which, along with the human cytomegalovirus UL136 proteins, inhibits caspase 8 activation (50, 56). In a like manner, HR-HPV16 produces the early protein E6 that binds to procaspase 8. Interestingly, however, we have found that the two splice products of the E6 gene, E6_large, a protein of about 16 kDa, and E6*, a protein less than half the size of E6_large, bind to and affect procaspase 8 stability differentially. While the large isoform accelerates the degradation of procaspase 8, leading to its destabilization, the short isoform leads to the stabilization of protein expression and an increase in activity. These observations suggest that the bindings of these two E6 isoforms have different functional consequences and may well localize to different regions on procaspase 8.We have previously identified a novel E6 binding site on the FADD DED (54). Based on sequence comparisons between the DEDs of FADD and procaspase 8, we proposed that the binding motif that mediates oncoprotein binding to both proteins would be similar. To test this possibility, we performed a series of mutational and peptide competitor-based experiments and discovered that the motifs on caspase 8 and on FADD that mediate binding between E6 and its cellular partner are indeed similar. Interestingly, however, the motif by which E6* binds to procaspase 8 is located in another region of the protein. These findings provide a molecular explanation for our previously reported observations concerning the differential effects of the binding of each isoform to the procaspase 8 DED. These findings also demonstrate the ability of peptide inhibitors to successfully impair E6 binding to its cellular targets and contribute to the discovery of therapeutic agents that are effective against cervical cancer.     

A novel method of studying fascicle architecture in relaxed and contracted muscles

A muscle’s architecture, described by geometric variables such as fascicle pennation angles or lengths, plays a crucial role in its functionality. Usually, single parameters are used to estimate force vectors or lengthening rates, thereby assuming that they represent the architecture properly and are constant during contraction. To describe muscle architecture in more detail and compare relaxed and contracted states, we developed and validated a new approach. The m. soleus of the laboratory rat was shock-frozen while relaxed and under isometric contraction, reconstructed three-dimensionally from histological sections, and fascicle lengths, curvatures and pennation angles, as well as the shape of the aponeuroses were analysed. Remarkable differences in volume distribution and the shapes of the aponeuroses as well as locally varying changes in the fascicle architecture were observed. While the mean pennation angle increased by only 2° due to contraction, local changes of up to 4° were observed. Fascicle curvature increased in the distal but remained unchanged in the proximal parts. Our approach may help to identify functional subunits within the muscle, i.e., regions with homogeneous architectural properties. Our results are discussed regarding the input parameters essential for realistic muscle modelling and challenge maximum isometric force estimations that are based on the physiological cross-sectional area or the Hill-model.     

Postnatal allometry of the skeleton in Tupaia glis (Scandentia: Tupaiidae) and Galea musteloides (Rodentia: Caviidae)--a test of the three-segment limb hypothesis

During the evolution of therian mammals, the two-segmented, sprawled tetrapod limbs were transformed into three-segmented limbs in parasagittal zig-zag configuration (three-segment limb hypothesis). As a consequence, the functional correspondence of limb segments has changed (now: scapula to thigh, upper arm to shank, fore arm plus hand to foot). Therefore, the scapula was taken into account in the current study of the postnatal growth of the postcranial skeleton in two small mammalian species (Tupaia glis, Galea musteloides). Comparisons were made between the functionally equivalent elements and not in the traditional way between serially homologous segments. This study presents a test of the three-segment limb hypothesis which predicts a greater ontogenetic congruence in the functionally equivalent elements in fore and hind limbs than in the serially homologous elements. A growth sequence, with decreasing regression coefficients from proximal to distal, was observed in both species under study. This proximo-distal growth sequence is assumed to be ancestral in the ontogeny of eutherian mammals. Different reproductive modes have evolved within eutherian mammals. To test the influence of different life histories on ontogenetic scaling during postnatal growth, one species with altricial juveniles (Tupaia glis) assumed to be the ancestral mode of development for eutherians and one species with derived, precocial young (Galea musteloides) were selected. The growth series covered postnatal development from the first successive steps with a lifted belly to the adult locomotory pattern; thus, functionally equivalent developmental stages were compared. The higher number of allometrically positive or isometrically growing segments in the altricial mammalian species was interpreted as a remnant of the fast growth period in the nest without great locomotor demands, and the clearly negative allometry in nearly all segments in the precocial young was interpreted as a response to the demand on early locomotor activity. Different life histories seem to have a strong influence on postnatal ontogenetic scaling; the effects of the developmental differences are still observable when comparing adults of the two species.     

TRPV1+ sensory neurons control beta cell stress and islet inflammation in autoimmune diabetes

In type 1 diabetes, T cell-mediated death of pancreatic beta cells produces insulin deficiency. However, what attracts or restricts broadly autoreactive lymphocyte pools to the pancreas remains unclear. We report that TRPV1(+) pancreatic sensory neurons control islet inflammation and insulin resistance. Eliminating these neurons in diabetes-prone NOD mice prevents insulitis and diabetes, despite systemic persistence of pathogenic T cell pools. Insulin resistance and beta cell stress of prediabetic NOD mice are prevented when TRPV1(+) neurons are eliminated. TRPV1(NOD), localized to the Idd4.1 diabetes-risk locus, is a hypofunctional mutant, mediating depressed neurogenic inflammation. Delivering the neuropeptide substance P by intra-arterial injection into the NOD pancreas reverses abnormal insulin resistance, insulitis, and diabetes for weeks. Concordantly, insulin sensitivity is enhanced in trpv1(-/-) mice, whereas insulitis/diabetes-resistant NODxB6Idd4-congenic mice, carrying wild-type TRPV1, show restored TRPV1 function and insulin sensitivity. Our data uncover a fundamental role for insulin-responsive TRPV1(+) sensory neurons in beta cell function and diabetes pathoetiology.