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.     

Suppression of hyperglycemia in NOD mice after inoculation with recombinant vaccinia viruses

In autoimmune (type 1) diabetes, autoreactive lymphocytes destroy pancreatic β-cells responsible for insulin synthesis. To assess the feasibility of gene therapy for type 1 diabetes, recombinant vaccinia virus (rVV) vectors were constructed expressing pancreatic islet autoantigens proinsulin (INS) and a 55-kDa immunogenic peptide from glutamic acid decarboxylase (GAD), and the immunomodulatory cytokine interleukin (IL)-10. To augment the beneficial effects of recombinant virus therapy, the INS and GAD genes were fused to the C terminus of the cholera toxin B subunit (CTB). Five-week-old non-obese diabetic (NOD) mice were injected once with rVV. Humoral antibody immune responses and hyperglycemia in the infected mice were analyzed. Only 20% of the mice inoculated with rVV expressing the CTB::INS fusion protein developed hyperglycemia, in comparison to 70% of the mice in the uninoculated animal group. Islets from pancreatic tissues isolated from euglycemic mice from this animal group showed no sign of inflammatory lymphocyte invasion. Inoculation with rVV producing CTB::GAD or IL-10 was somewhat less effective in reducing diabetes. Humoral antibody isotypes of hyperglycemic and euglycemic mice from all treated groups possessed similar IgG1/IgG2c antibody titer ratios from 19 to 32 wk after virus inoculation. In comparison with uninoculated mice, 11-wk-old NOD mice injected with virus expressing CTB::INS were delayed in diabetes onset by more than 4 wk. The experimental results demonstrate the feasibility of using rVV expressing CTB::INS fusion protein to generate significant protection and therapy against type 1 diabetes onset and progression.     

Mitochondrial DNA repair of oxidative damage in mammalian cells

Nuclear and mitochondrial DNA are constantly being exposed to damaging agents, from endogenous and exogenous sources. In particular, reactive oxygen species (ROS) are formed at high levels as by-products of the normal metabolism. Upon oxidative attack of DNA many DNA lesions are formed and oxidized bases are generated with high frequency. Mitochondrial DNA has been shown to accumulate high levels of 8-hydroxy-2'-deoxyguanosine, the product of hydroxylation of guanine at carbon 8, which is a mutagenic lesion. Most of these small base modifications are repaired by the base excision repair (BER) pathway. Despite the initial concept that mitochondria lack DNA repair, experimental evidences now show that mitochondria are very proficient in BER of oxidative DNA damage, and proteins necessary for this pathway have been isolated from mammalian mitochondria. Here, we examine the BER pathway with an emphasis on mtDNA repair. The molecular mechanisms involved in the formation and removal of oxidative damage from mitochondria are discussed. The pivotal role of the OGG1 glycosylase in removal of oxidized guanines from mtDNA will also be examined. Lastly, changes in mtDNA repair during the aging process and possible biological implications are discussed.     

Tick-borne nyamanini virus replicates in the nucleus and exhibits unusual genome and matrix protein properties

Tick-borne Nyamanini virus (NYMV) is the prototypic member of a recently discovered genus in the order Mononegavirales, designated Nyavirus. The NYMV genome codes for six distinct genes. Sequence similarity and structural properties suggest that genes 1, 5, and 6 encode the nucleoprotein (N), the glycoprotein (G), and the viral polymerase (L), respectively. The function of the other viral genes has been unknown to date. We found that the third NYMV gene codes for a protein which, when coexpressed with N and L, can reconstitute viral polymerase activity, suggesting that it represents a polymerase cofactor. The second viral gene codes for a small protein that inhibits viral polymerase activity and further strongly enhances the formation of virus-like particles when coexpressed with gene 4 and the viral glycoprotein G. This suggests that two distinct proteins serve a matrix protein function in NYMV as previously described for members of the family Filoviridae. We further found that NYMV replicates in the nucleus of infected cells like members of the family Bornaviridae. NYMV is a poor inducer of beta interferon, presumably because the viral genome is 5' monophosphorylated and has a protruding 3' terminus as observed for bornaviruses. Taken together, our results demonstrate that NYMV possesses biological properties previously regarded as typical for filoviruses and bornaviruses, respectively.