Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Two silicone nontoxic fouling release coatings: Hydrosilation cured PDMS and CaCO3 filled,ethoxysiloxane cured RTV11

Two silicone coatings have been evaluated for barnacle adhesion. One coating is an unfilled hydrosilation cured polydimethylsiloxane (PDMS) network, while the other is a room temperature vulcanized (RTV), filled, ethoxysiloxane cured PDMS elastomer, RTV11^?. The adhesion strength of one species of barnacle, Balanus eburneus, to the hydrosilation coatings is in the range of 0.37–0.60 kg cm^‐2 while the corresponding range for RTV11 is 0.64–0.90 kg cm^‐2. The easier release of B. eburneus from the hydrosilation cured network compared to RTV11 is discussed in relationship to differences in bulk and surface properties. Preliminary results suggest bulk modulus may be the most important parameter in determining barnacle adhesion strength. In light or mechanical property analysis, a re‐evaluation of surface properties and chemical stability is presented.     

Assessment of Blood Collection from the Lateral Saphenous Vein for Microfilaria Counts in Mongolian Gerbils (Meriones unguiculatus) Infected with Brugia pahangi

The NIH guidelines for survival bleeding of mice and rats note that using the retroorbital plexus has a greater potential for complications than do other methods of blood collection and that this procedure should be performed on anesthetized animals. Lateral saphenous vein puncture has a low potential for complications and can be performed without anesthesia. Mongolian gerbils (Meriones unguiculatus) are the preferred rodent model for filarial parasite research. To monitor microfilaria counts in the blood, blood sampling from the orbital plexus has been the standard. Our goal was to refine the blood collection technique. To determine whether blood collection from the lateral saphenous vein was a feasible alternative to retroorbital sampling, we compared microfilaria counts in blood samples collected by both methods from 21 gerbils infected with the filarial parasitic worm Brugia pahangi. Lateral saphenous vein counts were equivalent to retroorbital counts at relatively high counts (greater than 50 microfilariae per 20 µL) but were significantly lower than retroorbital counts when microfilarial concentrations were lower. Our results indicate that although retroorbital collection may be preferable when low concentrations of microfilariae need to be enumerated, the lateral saphenous vein is a suitable alternative site for blood sampling to determine microfilaremia and is a feasible refinement that can benefit the wellbeing of gerbils.Abbreviations: FR3, Filariasis Research Reagent Resource CenterLymphatic filariasis a major threat to human health worldwide. More than one billion people in more than 90 countries around the globe are at risk from lymphatic filariasis, and between 120 and 150 million people are infected.^9,11,25 Infection with the filarioid parasitic worms Brugia malayi and Wuchereria bancrofti can result in severe sequelae, including elephantiasis and hydrocoele formation.^3,11,15,25 In addition to the clinical manifestations of filariasis are the potential associated psychologic, social, and cultural effects in persons exhibiting visible signs of infection.^9,23,34The life cycle of filarioid nematodes requires an arthropod intermediate host and a definitive vertebrate host. Within the definitive host, dioecious adult filarial nematodes reproduce sexually. Inseminated adult female worms then release live, sheathed microfilariae into the lymph that circulate in the peripheral blood.²¹ In the case of B. malayi and W. bancrofti, the intermediate host is the mosquito.²¹ When an uninfected mosquito ingests a blood meal from an infected human, ingested microfilariae unsheathe to penetrate the midgut of the mosquito to reach the thoracic muscles and molt twice, to become the infectious third-stage larvae. The third-stage larvae then migrate to the mosquito''s proboscis and can infect another human when the mosquito takes a blood meal.^10,11 The third-stage larvae enter the new host''s lymphatic system which is their final location, where they undergo 2 molts into adults.Because of the complexity of filarioid life cycles, research involving these parasites can be logistically challenging. Although mice can be infected with W. bancrofti, they do not maintain the infection.³⁵ Furthermore, there is no suitable nonhuman host that can maintain a patent infection, with the exception of the silvered leaf monkey (Trachypithecus cristatus).⁹ Because the closely related parasites B. malayi and B. pahangi have more extensive host ranges than does W. bancrofti, they are easier to maintain in a research setting. Domestic cats (Felis catus) can be experimentally infected with B. malayi and develop a patent infection, and both domestic cats and dogs (Canis familiaris) can be experimentally infected with B. pahangi^13,29,37 and are suitable for obtaining microfilaremic blood for experimental feeding of mosquitoes. The Mongolian gerbil can be infected with B. pahangi. Because replacing a phylogenetically higher species with a lower species is preferable³⁶ and because performing experiments involving dogs and cats can be logistically difficult and cost-prohibitive, many researchers prefer a rodent model, specifically gerbils.The Filariasis Research Reagent Resource Center (FR3) is an NIH center whose mandate is to support filariasis research worldwide. The FR3 provides parasitic and molecular resources, as well as training in animal procedures, to researchers from many nations. The FR3 maintains both B. malayi and B. pahangi, and researchers occasionally require gerbils with patent infections. Because the required level of microfilaria counts varies among investigators, an accurate microfilaria count must be obtained prior to the shipment of gerbils. For example, some experiments require that live mosquitoes feed directly on infected gerbils, and when the microfilaria level is too low, the mosquitoes do not become infected. Conversely when the level is too high, the migration of microfilariae and the later larval stages can kill the mosquitoes. Historically, the FR3 has used retroorbital sampling under general anesthesia to obtain the blood for microfilaria counts.²⁸ Although this method has been fairly successful, the FR3 has encountered occasional complications secondary to the procedure, including exophthalmia and, rarely, death under anesthesia. The NIH guidelines for survival bleeding of mice and rats notes that compared with other blood collection methods, retroorbital sampling has a greater potential for complications. The guidelines recommend a 10- to 14-d period between retroorbital blood collections and state that the procedure is “…best conducted under general anesthesia.”³¹ By comparison, collecting blood from the lateral saphenous vein is considered to have a low potential for complications or tissue damage, can be performed without general anesthesia,^12,18,31 and can be performed repeatedly, even daily.³¹In the current study, we proposed to refine the blood collection method being used by FR3 by developing sampling from the lateral saphenous vein as the new standard blood-collection method for monitoring microfilaremia. Our goal was to assess blood collection from the lateral saphenous vein as a feasible refinement technique to potentially replace retroorbital sampling by determining whether the microfilaria counts in blood collected from the lateral saphenous vein without anesthesia were sufficiently similar to those from retroorbital blood sampling with anesthesia to provide adequate information about the microfilaremia level.     

Regulation of the Rev1–pol ζ complex during bypass of a DNA interstrand cross‐link

DNA interstrand cross‐links (ICLs) are repaired in S phase by a complex, multistep mechanism involving translesion DNA polymerases. After replication forks collide with an ICL, the leading strand approaches to within one nucleotide of the ICL (“approach”), a nucleotide is inserted across from the unhooked lesion (“insertion”), and the leading strand is extended beyond the lesion (“extension”). How DNA polymerases bypass the ICL is incompletely understood. Here, we use repair of a site‐specific ICL in Xenopus egg extracts to study the mechanism of lesion bypass. Deep sequencing of ICL repair products showed that the approach and extension steps are largely error‐free. However, a short mutagenic tract is introduced in the vicinity of the lesion, with a maximum mutation frequency of ~1%. Our data further suggest that approach is performed by a replicative polymerase, while extension involves a complex of Rev1 and DNA polymerase ζ. Rev1–pol ζ recruitment requires the Fanconi anemia core complex but not FancI–FancD2. Our results begin to illuminate how lesion bypass is integrated with chromosomal DNA replication to limit ICL repair‐associated mutagenesis.     

Unassisted blood collection from unanesthetized rats and gerbils

Blood collection is a common procedure in animal research. This column describes humane methods of collecting blood from the rat and the gerbil that can be carried out by a single person on an awake animal.     

Targeting tumour necrosis factor alleviates signs and symptoms of inflammatory osteoarthritis of the knee

Introduction

Inflammation associated with synovial expression of TNFα is a recognised feature of osteoarthritis (OA), although no studies have yet reported beneficial effects of anti-TNFα therapy on clinical manifestations of inflammation in OA.

Methods

We conducted an open-label evaluation of adalimumab over 12 weeks in 20 patients with OA of the knee and evidence of effusion clinically. Inclusion criteria included daily knee pain for the month preceding study enrolment and a summed pain score of 125 to 400 mm visual analogue scale on the Western Ontario and McMaster University Osteoarthritis Index (WOMAC) pain subscale. The primary outcome was the Osteoarthritis Research Society International/Outcome Measures in Rheumatology Clinical Trials (OARSI/OMERACT) response criterion at week 12. Secondary outcomes included the WOMAC pain score 20% and 50% improvement, WOMAC stiffness and function scores, patient and physician global visual analogue scale, as well as target joint swelling.

Results

Treatment was well tolerated and completed by 17 patients with withdrawals unrelated to lack of efficacy or adverse events. By intention to treat, an OARSI/OMERACT response was recorded in 14 (70%) patients. WOMAC pain 20% and 50% responses were recorded in 14 (70%) patients and eight (40%) patients, respectively. Significant improvement was observed in mean WOMAC pain, stiffness, function, physician and patient global, as well as target joint swelling at 12 weeks (P < 0.0001 for all). After treatment discontinuation, 16 patients were available for assessment at 22 weeks and OARSI/OMERACT response compared with baseline was still evident in 10 (50%) patients.

Conclusion

Targeting TNFα may be of therapeutic benefit in OA and requires further evaluation in controlled trials.

Trial registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT00686439","term_id":"NCT00686439"}}NCT00686439.     

Can erosions on MRI of the sacroiliac joints be reliably detected in patients with ankylosing spondylitis? - A cross-sectional study

Introduction

Erosions of the sacroiliac joints (SIJ) on pelvic radiographs of patients with ankylosing spondylitis (AS) are an important feature of the modified New York classification criteria. However, radiographic SIJ erosions are often difficult to identify. Recent studies have shown that erosions can be detected also on magnetic resonance imaging (MRI) of the SIJ early in the disease course before they can be seen on radiography. The goals of this study were to assess the reproducibility of erosion and related features, namely, extended erosion (EE) and backfill (BF) of excavated erosion, in the SIJ using a standardized MRI methodology.

Methods

Four readers independently assessed T1-weighted and short tau inversion recovery sequence (STIR) images of the SIJ from 30 AS patients and 30 controls (15 patients with non-specific back pain and 15 healthy volunteers) ≤45 years old. Erosions, EE, and BF were recorded according to standardized definitions. Reproducibility was assessed by percentage concordance among six possible reader pairs, kappa statistics (erosion as binary variable) and intraclass correlation coefficient (ICC) (erosion as sum score) for all readers jointly.

Results

SIJ erosions were detected in all AS patients and six controls by ≥2 readers. The median number of SIJ quadrants affected by erosion recorded by four readers in 30 AS patients was 8.6 in the iliac and 2.1 in the sacral joint portion (P < 0.0001). For all 60 subjects and for all four readers, the kappa value for erosion was 0.72, 0.73 for EE, and 0.63 for BF. ICC for erosion was 0.79, 0.72 for EE, and 0.55 for BF, respectively. For comparison, the kappa and ICC values for bone marrow edema were 0.61 and 0.93, respectively.

Conclusions

Erosions can be detected on MRI to a comparable degree of reliability as bone marrow edema despite the significant heterogeneity of their appearance on MRI.     

Evaluating a Parainfluenza Virus 5-Based Vaccine in a Host with Pre-Existing Immunity against Parainfluenza Virus 5

Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a paramyxovirus often referred to as canine parainfluenza virus (CPI) in the veterinary field. PIV5 is thought to be a contributing factor to kennel cough. Kennel cough vaccines containing live PIV5 have been used in dogs for many decades. PIV5 is not known to cause any diseases in humans or other animals. PIV5 has been used as a vector for vaccine development for humans and animals. One critical question concerning the use of PIV5 as a vector is whether prior exposure to PIV5 would prevent the use of PIV5-based vaccines. In this work, we have examined immunogenicity of a recombinant PIV5 expressing hemagglutinin (HA) of influenza A virus subtype 3 (rPIV5-H3) in dogs that were immunized against PIV5. We found that vaccination of the dogs containing neutralizing antibodies against PIV5 with rPIV5-H3 generated immunity against influenza A virus, indicting that PIV5-based vaccine is immunogenic in dogs with prior exposure. Furthermore, we have examined exposure of PIV5 in human populations. We have detected neutralizing antibody (nAb) against PIV5 in 13 out of 45 human serum samples (about 29 percent). The nAb titers in humans were lower than that in vaccinated dogs, suggesting that nAb in humans is unlikely to prevent PIV5 from being an efficacious vector in humans.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.