The median palmar cutaneous nerve in normal subjects and CTS.

OBJECTIVE: The neurophysiological confirmation of carpal tunnel syndrome (CTS) relies on detecting abnormal median nerve transcarpal conduction in the presence of unaffected comparator nerves. We compare the palmar cutaneous median branch (PCBm) with the ulnar sensory nerve conduction to digit 5 (US(5)) as comparator nerves for diagnosing CTS. METHODS: In a prospective case control study of patients with clinically defined carpal tunnel syndrome and normal subjects, we determined and compared the PCBm and US(5) conduction velocity. RESULTS: We examined 57 hands with clinically defined CTS and 59 control hands. Comparison showed highly significantly slowed PCBm conduction (p<0.0001) but not for US(5) conduction (p=0.488). Using a 3 percentile cut-off for abnormality derived from controls, PCBm conduction velocity was abnormal in 46% of CTS hands. CONCLUSIONS: The high frequency of PCBm nerve conduction abnormality in CTS suggests that this nerve should not be used as a comparator nerve for the neurophysiological diagnosis of CTS. This finding may help explain some of the extension of sensory symptoms outside the median nerve distribution in CTS. SIGNIFICANCE: In CTS frequent abnormality of PCBm conduction makes this a poor comparator nerve and may explain extension of sensory symptoms beyond the median nerve.     

Does host complement kill Borrelia burgdorferi within ticks?

The Lyme disease spirochete, Borrelia burgdorferi, inhabits the gut lumen of the tick vector. At this location the spirochete is exposed to host blood when a tick feeds. We report here on studies that were done with normal and complement-deficient (C3-knockout) mice to determine if the host complement system killed spirochetes within the vector. We found that spirochete numbers within feeding nymphs were not influenced by complement, most likely because host complement was inactivated within the vector. The Lyme disease outer surface protein A (OspA) vaccine is a transmission-blocking vaccine that targets spirochetes in the vector. In experiments with mice hyperimmunized with OspA, complement was not required to kill spirochetes within nymphs and to block transmission from nymphs to the vaccinated host. However, host complement did enhance the ability of OspA antibody to block larvae from acquiring spirochetes. Thus, the effects of OspA antibody on nymphal transmission and larval acquisition appear to be based on different mechanisms.     

The Human MitoChip: a high-throughput sequencing microarray for mitochondrial mutation detection

Somatic mitochondrial mutations are common in human cancers, and can be used as a tool for early detection of cancer. We have developed a mitochondrial Custom Reseq microarray as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The MitoChip contains oligonucleotide probes synthesized using standard photolithography and solid-phase synthesis, and is able to sequence >29 kb of double-stranded DNA in a single assay. Both strands of the entire human mitochondrial coding sequence (15,451 bp) are arrayed on the MitoChip; both strands of an additional 12,935 bp (84% of coding DNA) are arrayed in duplicate. We used 300 ng of genomic DNA to amplify the mitochondrial coding sequence in three overlapping long PCR fragments. We then sequenced >2 million base pairs of mitochondrial DNA, and successfully assigned base calls at 96.0% of nucleotide positions. Replicate experiments demonstrated >99.99% reproducibility. In matched fluid samples (urine and pancreatic juice, respectively) obtained from five patients with bladder cancer and four with pancreatic cancer, the MitoChip detected at least one cancer-associated mitochondrial mutation in six (66%) of nine samples. The MitoChip is a high-throughput sequencing tool for the reliable identification of mitochondrial DNA mutations from primary tumors in clinical samples.     

On the probability that a novel variant is a disease-causing mutation

When a novel variant is found in a patient and not in a group of controls, it becomes a candidate for the disease-causing mutation in that patient. At present, no sampling theory exists for assessing the probability that the novel SNP might actually be a neutral variant. We have developed a population genetics-based method for calculating a P-value for a mutation-detection effort. Our method can be applied to a heterozygous patient, a homozygous patient, with or without inbreeding, or to a patient who is a compound heterozygote. Additionally, the method can be used to calculate the probability of finding a neutral variant at frequencies that differ between a group of patients and a group of controls, given some length of sequence examined. This method accounts for the multiple testing that is inherent in identification of variants through sequencing, to be used in subsequent case-control analyses. We show, for example, that for complete resequencing of 10 kb, the probability of finding a neutral variant in a patient and not in 50 controls is about 15%. Thus, discovery of a variant in a patient and not in a group of controls is, on its own, very weak evidence of involvement with disease.     

From the Cover: Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity

The four dengue virus (DENV) serotypes, DENV-1, -2, -3, and -4, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million acute infections annually. Infection confers long-term protective immunity against the infecting serotype, but secondary infection with a different serotype carries a greater risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. The single most effective measure to control this threat to global health is a tetravalent DENV vaccine. To date, attempts to develop a protective vaccine have progressed slowly, partly because the targets of type-specific human neutralizing antibodies (NAbs), which are critical for long-term protection, remain poorly defined, impeding our understanding of natural immunity and hindering effective vaccine development. Here, we show that the envelope glycoprotein domain I/II hinge of DENV-3 and DENV-4 is the primary target of the long-term type-specific NAb response in humans. Transplantation of a DENV-4 hinge into a recombinant DENV-3 virus showed that the hinge determines the serotype-specific neutralizing potency of primary human and nonhuman primate DENV immune sera and that the hinge region both induces NAbs and is targeted by protective NAbs in rhesus macaques. These results suggest that the success of live dengue vaccines may depend on their ability to stimulate NAbs that target the envelope glycoprotein domain I/II hinge region. More broadly, this study shows that complex conformational antibody epitopes can be transplanted between live viruses, opening up similar possibilities for improving the breadth and specificity of vaccines for influenza, HIV, hepatitis C virus, and other clinically important viral pathogens.The four dengue virus serotypes (DENV-1, -2, -3, and -4), transmitted by Aedes spp. mosquitoes, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million new infections annually (1). Primary infection with one serotype confers long-term immunity against that serotype, but repeat infection with a different serotype has an increased risk of potentially fatal severe dengue disease (2), including dengue hemorrhagic fever and dengue shock syndrome. This risk has been attributed, at least in part, to the ability of some cross-reactive antibodies to enhance infection of Fc-receptor bearing cells. The consensus is that, to be safe and effective, any dengue vaccine must simultaneously induce neutralizing antibodies (NAbs) to all four serotypes. However, DENV vaccine development has progressed slowly, highlighted by the disappointing results of the live-attenuated Sanofi Pasteur tetravalent DENV vaccine trial in Thailand (3). Progress is hindered, in part, because the epitopes targeted by the type-specific human NAbs critical for long-term protection (4, 5) remain poorly defined, limiting our understanding of natural DENV immunity and slowing effective vaccine development.The DENV envelope glycoprotein (E) (Fig. 1A) is the major surface-exposed DENV antigen and the principle target of NAbs. The E structure consists of three distinct domains: I, II, and III (EDI, EDII, and EDIII) (6, 7); EDIII is a continuous peptide extending from domain I and forming an Ig-like fold, whereas EDI and EDII are discontinuous and connect by four peptide linkers that form the EDI/EDII hinge. We and others have recently described potent human DENV NAbs that bind to epitopes around the EDI/EDII hinge (8, 9). To more fully explore the significance of this antigenic region, we used reverse genetics and synthetic biology to transplant the EDI/EDII antigenic region from DENV-4 into a DENV-3 background and showed by both gain- and loss-of-function assays that the EDI/EDII hinge region is the primary target of the long-lived DENV serotype-specific NAb response.Open in a separate windowFig. 1.(A) Cartoon representation of the DENV-3 E dimer with EDI (red), EDII (yellow), and EDIII (blue). The EDI/EDII hinge region is circled. Location of the critical residues associated with escape mutations and mutagenesis-mapped 5J7 residues are shown. Residues critical for 5J7 binding identified by shotgun mutagenesis loss of binding of E glycoprotein expressed in HEK-293T cells are shown in magenta. Mutations associated with both viral escape from 5J7 and loss of binding in HEK-293T cells (L53 and K128) are shown in orange, and the single residue identified by escape mutation alone (Q269K270_insK) is shown in cyan. All critical residues were individually identified but are shown on a single E dimer for simplicity. (B) Cartoon representation of the DENV E dimer with the locations of variable EDI/EDII hinge residues transplanted between rDENV-3 and rDENV-4 to make rDENV-3/4 shown in green. (C) Primary sequence alignment and secondary structure of rDENV-3 E, rDENV-3/4 E, and rDENV-4 E. Secondary structure is indicated above the primary sequence and color-coded to the corresponding domains on the tertiary structure (A and B). Arrows indicate β-sheets, cylinders indicate helices, and lines indicate spanning loops and strands. Binding, escape, and hinge residues shown in A and B are indicated by corresponding colors in the rDENV-3 sequence. Amino acid residues transplanted between rDENV-3/4 and rDENV-4 are indicated in green for both sequences.     

Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions

Dengue is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 50 countries. Even though infected individuals develop potent and long-lasting serotype-specific neutralizing antibodies (Abs), the epitopes engaged by human neutralizing Abs have not been identified. Here, we demonstrate that the dengue virus (DENV)-specific serum Ab response in humans consists of a large fraction of cross-reactive, poorly neutralizing Abs and a small fraction of serotype-specific, potently inhibitory Abs. Although many mouse-generated, strongly neutralizing monoclonal antibodies (mAbs) recognize epitopes that are present on recombinant DENV envelope (E) proteins, unexpectedly, the majority of neutralizing Abs in human immune sera bound to intact virions but not to the ectodomain of purified soluble E proteins. These conclusions with polyclonal Abs were confirmed with newly generated human mAbs derived from DENV-immune individuals. Two of three strongly neutralizing human mAbs bound to E protein epitopes that were preserved on the virion but not on recombinant E (rE) protein. We propose that humans produce Abs that neutralize DENV infection by binding a complex, quaternary structure epitope that is expressed only when E proteins are assembled on a virus particle. Mapping studies indicate that this epitope has a footprint that spans adjacent E protein dimers and includes residues at the hinge between domains I and II of E protein. These results have significant implications for the DENV Ab and vaccine field.     

Infection of mice with lyme disease spirochetes constitutively producing outer surface proteins a and B

Outer surface protein A (OspA) of the Lyme disease spirochete is primarily produced in the tick vector. OspA, which is a receptor for attaching spirochetes to the tick gut, is down regulated as the spirochetes leave the tick and enter the mammalian host. Although OspA is not a major antigen produced in the mammal, the protein appears to be produced under some conditions and production has been linked to more severe disease. A Lyme disease vaccine based on recombinant OspA has been approved for human use. However, the vaccine is no longer available, in part because of fears that OspA causes arthritis in people. To further understand the consequences of OspA production in the host, we created a Borrelia burgdorferi mutant that was unable to down regulate OspA. C3H/HeN mice infected with this mutant developed a specific anti-OspA immune response, and the spirochetes were unable to persist in these mice. In contrast, immunodeficient SCID mice were persistently infected with the mutant. We conclude that spirochetes producing OspA and B from the flaB promoter in immunocompetent mice stimulate an immune response that clear the bacteria without any signs of disease development in the mice.     

SDPR functions as a metastasis suppressor in breast cancer by promoting apoptosis

Metastatic dissemination of breast cancer cells represents a significant clinical obstacle to curative therapy. The loss of function of metastasis suppressor genes is a major rate-limiting step in breast cancer progression that prevents the formation of new colonies at distal sites. However, the discovery of new metastasis suppressor genes in breast cancer using genomic efforts has been slow, potentially due to their primary regulation by epigenetic mechanisms. Here, we report the use of model cell lines with the same genetic lineage for the identification of a novel metastasis suppressor gene, serum deprivation response (SDPR), localized to 2q32-33, a region reported to be associated with significant loss of heterozygosity in breast cancer. In silico metaanalysis of publicly available gene expression datasets suggests that the loss of expression of SDPR correlates with significantly reduced distant-metastasis–free and relapse-free survival of breast cancer patients who underwent therapy. Furthermore, we found that stable SDPR overexpression in highly metastatic breast cancer model cell lines inhibited prosurvival pathways, shifted the balance of Bcl-2 family proteins in favor of apoptosis, and decreased migration and intravasation/extravasation potential, with a corresponding drastic suppression of metastatic nodule formation in the lungs of NOD/SCID mice. Moreover, SDPR expression is silenced by promoter DNA methylation, and as such it exemplifies epigenetic regulation of metastatic breast cancer progression. These observations highlight SDPR as a potential prognostic biomarker and a target for future therapeutic applications.The metastatic progression of breast cancer accounts for the majority of disease-related mortality. A major rate-limiting step in metastasis is the loss of function of the metastasis suppressor genes, which block a cascade of crucial steps including the loss of adhesion of primary tumor cells, intravasation into the blood and lymphatics with subsequent extravasation at distant sites, and the formation of new colonies. Despite the identification of the first metastasis suppressor gene, nonmetastatic 23 (NM23), nearly two decades ago (1), only a handful of new metastasis suppressors have been identified in recent years using candidate gene approaches (2, 3). It is likely that the current catalog of metastasis suppressor genes remains incomplete despite the vast sequencing efforts due to the possibility that a subset of genes regulated by epigenetic mechanisms may have eluded traditional discovery procedures (4–6). To identify these elusive metastasis suppressor genes, which are functionally compromised in late-stage disease (7–9), we took advantage of a well-established breast cancer progression cell line model system sharing the same genetic linage (Fig. 1A) (10). This model system consists of five cell lines that represent the various stages of breast cancer progression based on the MCF10A cell line: MCF10AneoT (NeoT), MCF10AT1Kcl2 (MII), MCF10CA1h (MIII), and MCF10CA1a (MIV). NeoT cells were generated by overexpression of HRAS in MCF10A cells and rarely exhibit growth following injection into nude mice. MII cells were generated by single xenograft passaging of NeoT cells. When injected subcutaneously (s.c.) into nude mice, MII cells generally form benign tumors that progress to carcinoma one out of four times; hence they mimic the early stage, carcinoma in situ. MIII and MIV cells were isolated from tumors formed by MII cells. MIII cells represent carcinoma, as in general they metastasize at a very low frequency, which requires a prolonged incubation period. On the other hand, MIV cells have the potential to readily seed lung metastases and represent the final stages of a breast cancer, metastatic carcinoma. We compared the gene expression profiles of these latter three model cell lines and leveraged large amounts of publically available breast tumor gene expression profiling data (11–13) by applying multiple bioinformatics filters to identify candidate metastasis suppressor genes.Open in a separate windowFig. 1.Identification of SDPR as a candidate metastasis suppressor gene. (A) Schematic depiction of the generation of breast cancer progression cell line model system. The model consists of five cell lines representing different stages of breast cancer progression. MI, normal breast epithelial cells; NeoT and MII, carcinoma in situ; MIII, carcinoma; and MIV, metastatic carcinoma. (B) Hierarchical clustering of gene expression profiles from MII, MIII, and MIV cells for the genes whose expression differ at least twofold between each cell line. Two clusters, cluster 6 and 7, are magnified because expressions of the genes in these two clusters are significantly suppressed in metastatic MIV cells compared with nonmetastatic MII and MIII. (C) The schematic summary of our strategy for the selection of SDPR as the top candidate metastasis suppressor.Here, we report the discovery of the phosphatidylserine-interacting protein, serum deprivation response (SDPR) (also known as cavin-2), as a bona fide metastasis suppressor. Thus far, studies on SDPR function have been limited to its role as a regulator of caveolae formation (14), and its potential direct involvement in cancer has not been previously described. However, it has been reported that SDPR expression is down-regulated significantly in not only breast cancer but also in cancers of kidney and prostate (15). Moreover, SDPR protein down-regulation was observed in serum from patients with malignant kidney tumors, and hence it was suggested as a possible diagnostic marker to discriminate malignant tumors from benign formations (16). Interestingly, SDPR is localized to 2q32-33, a region with a significant level of loss of heterozygosity that is associated with a high degree of recurrence in breast cancer (17, 18). Our results indicate that SDPR is capable of specifically inhibiting the metastatic growth of breast cancer cells.     

MODELING THE EFFECTS OF IMMUNIZATIONS TIMING ON CHILD HEALTH OUTCOMES IN INDIA

Timely vaccinations of children in developing countries are important for reducing morbidity and mortality, which are Millennium Development Goals. However, a majority of children do not possess vaccination cards compiling information on timing. We investigated the benefits of vaccination cards for the uptake of immunizations against diphtheria, pertussis and tetanus (DPT), polio, tuberculosis (BCG), and measles using data on over 200,000 Indian children from the District Level Health and Facility Survey 3. Methodological issues such as whether parents of children with higher morbidity levels may have them vaccinated were investigated. The results from the models for DPT, polio, measles, and BCG vaccinations showed significant beneficial effects of maternal education, household possessions, and access to health care facilities. Moreover, models for children's ages at the time of vaccination showed significant interactions between maternal education and access to and availability of health care facilities. Finally, models for child morbidity due to diarrhea, cough, and fever showed that timely vaccinations against DPT, access to piped water, and cooking with electricity or natural gas were associated with lower morbidity. Overall, issuing paper or electronic vaccination cards to children is likely to enhance timely uptake of various immunizations thereby reducing child morbidity. Copyright © 2013 John Wiley & Sons, Ltd.