Fc receptor-like 4 and 5 define human atypical memory B cells.

Atypical memory B cells accumulate in chronic infections and autoimmune conditions, and commonly express FCRL4 and FCRL5, respective IgA and IgG receptors. We characterized memory cells from tonsils on the basis of both FCRL4 and FCRL5 expression, defining three subsets with distinct surface proteins and gene expression. Atypical FCRL4+FCRL5+ memory cells had the most discrete surface protein expression and were enriched in cell adhesion pathways, consistent with functioning as tissue-resident cells. Atypical FCRL4-FCRL5+ memory cells expressed transcription factors and immunoglobulin genes that suggest poised differentiation into plasma cells. Accordingly, the FCRL4-FCRL5+ memory subset was enriched in pathways responding to endoplasmic reticulum stress and IFN-γ. We reconstructed ongoing B-cell responses as lineage trees, providing crucial in vivo developmental context. Each memory subset typically maintained its lineage, denoting mechanisms enforcing their phenotypes. Classical FCRL4-FCRL5- memory cells were infrequently detected in lineage trees, suggesting the majority were in a quiescent state. FCRL4-FCRL5+ cells were the most represented memory subset in lineage trees, indicating robust participation in ongoing responses. Together, these differences suggest FCRL4 and FCRL5 are unlikely to be passive markers but rather active drivers of human memory B-cell development and function.

Multiple factors influence the contribution of individual immunoglobulin light chain genes to the naïve antibody repertoire.

BACKGROUND: The naïve antibody repertoire is initially dependent upon the number of germline V(D)J genes and the ability of recombined heavy and light chains to pair. Individual VH and VL genes are not equally represented in naïve mature B cells, suggesting that positive and negative selection also shape the antibody repertoire. Among the three member murine Vκ10 L chain family, the Vκ10C gene is under-represented in the antibody repertoire. Although it is structurally functional and accessible to both transcriptional and recombination machinery, the Vκ10C promoter is inefficient in pre-B cell lines and productive Vκ10C rearrangements are lost as development progresses from pre-B cells through mature B cells. This study examined VH/Vκ10 pairing, promoter mutations, Vκ10 transcript levels and receptor editing as possible factors that are responsible for loss of productive Vκ10C rearrangements in developing B cells. RESULTS: We demonstrate that the loss of Vκ10C expression is not due to an inability to pair with H chains, but is likely due to a combination of other factors. Levels of mRNA are low in sorted pre-B cells and undetectable in B cells. Mutation of a single base in the three prime region of the Vκ10C promoter increases Vκ10C promoter function in pre-B cell lines. Pre-B and B cells harbor disproportionate levels of receptor-edited productive Vκ10C rearrangements. CONCLUSIONS: Our findings suggest that the weak Vκ10C promoter initially limits the amount of available Vκ10C L chain for pairing with H chains, resulting in sub-threshold levels of cell surface B cell receptors, insufficient tonic signaling and subsequent receptor editing to limit the numbers of Vκ10C-expressing B cells emigrating from the bone marrow to the periphery.

Diversity of the antibody response to tetanus toxoid: comparison of hybridoma library to phage display library.

Monoclonal antibodies are important tools in research and since the 1990s have been an important therapeutic class targeting a wide variety of diseases. Earlier methods of mAb production relied exclusively on the lengthy process of making hybridomas. The advent of phage display technology introduced an alternative approach for mAb production. A potential concern with this approach is its complete dependence on an in vitro selection process, which may result in selection of V(H)-V(L) pairs normally eliminated during the in vivo selection process. The diversity of V(H)-V(L) pairs selected from phage display libraries relative to an endogenous response is unknown. To address these questions, we constructed a panel of hybridomas and a phage display library using the spleen of a single tetanus toxoid-immunized mouse and compared the diversity of the immune response generated using each technique. Surprisingly, the tetanus toxoid-specific antibodies produced by the hybridoma library exhibited a higher degree of V(H)-V(L) genetic diversity than their phage display-derived counterparts. Furthermore, the overlap among the V-genes from each library was very limited. Consistent with the notion that accumulation of many small DNA changes lead to increased antigen specificity and affinity, the phage clones displayed substantial micro-heterogeneity. Contrary to previous reports, we found that antigen specificity against tetanus toxoid is encoded by both V(κ) and V(H) genes. Finally, the phage-derived tetanus-specific clones had a lower binding affinity than the hybridomas, a phenomenon thought to be the result of random pairing of the V-genes.

The use of pregame hyperhydration with intravenous fluids in National Collegiate Athletic Association Football Bowl Subdivision teams.

OBJECTIVE: To determine the frequency of pregame intravenous fluid hyperhydration (PIVFH) usage, administration protocols, indications, complications, and perceived efficacy by National Collegiate Athletic Association Football Bowl Subdivision (NCAA-FBS) teams. DESIGN: Cross-sectional survey study. SETTING: National Collegiate Athletic Association Football Bowl Subdivision. PARTICIPANTS: Head athletic trainers from NCAA-FBS institutions. INTERVENTION: Voluntary, anonymous 15-item validated online survey instrument. MAIN OUTCOME MEASURES: Number of teams reporting use of PIVFH. RESULTS: The survey response rate was 64% (77 of 120). Thirty percent of respondents reported the utilization of PIVFH, administered to an average of 2 to 3 players. The most common reasons cited for using PIVFH were to prevent muscle cramps (95%), heat illness (79%), and dehydration (68%). Additionally, 47% of programs used PIVFH to improve player exercise tolerance and 47% per player request. Twenty-four percent of programs that used PIVFH reported the occurrence of associated complications. Only 15% of respondents believed that PIVFH improved their teams' overall performance. CONCLUSIONS: PIVFH is a common practice among NCAA-FBS teams. PIVFH is used most often to prevent muscle cramps, dehydration, and heat illness. The relatively few numbers of players per team who receive PIVFH suggest that higher risk individuals were targeted for administration.

Seventy-five percent of National Football League teams use pregame hyperhydration with intravenous fluid.

OBJECTIVE: To determine the number of National Football League (NFL) teams using pregame hyperhydration with intravenous fluid (IVF), the most common protocols for using the IVF, the complications from this routine, and the perceived efficacy of this treatment. DESIGN: Cross-sectional survey study. SETTING: National Football League. PARTICIPANTS: The head athletic trainer from all 32 NFL teams received and completed the survey. INTERVENTION: A survey designed to answer the aforementioned topics. MAIN OUTCOME MEASURES: Number of teams using IVF to hyperhydrate euvolemic players before NFL games. RESULTS: All teams (100%, 32 of 32) responded. Seventy-five percent (24 of 32) of NFL teams used pregame hyperhydration with IVF. On average, 5 to 7 players per team per game received IVF. Players received an average of 1.5 L of fluid, an average of 2.5 hours before the game. When the athletic trainers were asked to mark all the reasons why they use pregame hyperhydration with IVF, the most commonly cited reason was muscle cramps (23 of 24). However, player request was the number one answer (10 of 24) when the athletic trainers were asked to mark only the primary reason for giving IVF. Of the 27 head athletic trainers who had used pregame hyperhydration with IVF in the past, 13 (48%) reported complications. CONCLUSIONS: Pregame hyperhydration is a common practice in the NFL. Because pregame hyperhydration with IVF is often a player- driven routine and has potential complications, more scientific studies are needed to determine its true efficacy.

Giant cell tumor at tibial screw site after anterior cruciate ligament reconstruction.

We report a case of giant cell tumor that occurred in the proximal tibia of a 52-year-old man 13 years after bone-patella-bone anterior cruciate ligament reconstruction. The tumor was at the site of the metal interference screw. We discuss the differential diagnosis of proximal tibia lesions that occur after anterior cruciate ligament reconstruction and the importance of recognizing potentially life-threatening sports tumors.

How to treat the stiff total knee arthroplasty?: a systematic review.

BACKGROUND: Multiple modalities have been used to treat the stiff TKA, including manipulation under anesthesia (MUA), arthroscopy, and open arthrolysis. QUESTIONS/PURPOSES: We reviewed the literature to address three questions: (1) How many degrees of ROM will a stiff TKA gain after MUA, arthroscopy, and open arthrolysis? (2) Does the timing of each procedure influence this gain in ROM? (3) What is the number of clinically important complications for each procedure? METHODS: We performed a PubMed search of English language articles from 1966 to 2008 and identified 20 articles, mostly Level IV studies. RESULTS: For patients who have arthrofibrosis after TKA, the gains in ROM after MUA and arthroscopy (with or without MUA) are similar. Open arthrolysis seems to have inferior gains in ROM. MUA is more successful in increasing ROM when performed early but still may be effective when performed late. Arthroscopy combined with MUA still is useful 1 year after the index TKA. The numbers of clinically important complications after MUA and arthroscopy (with or without MUA) are similar. CONCLUSIONS: Stiffness after TKA is a common problem that can be improved with MUA and/or arthroscopic lysis of adhesions with few complications. The low quality of available literature makes it difficult to develop treatment protocols. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Dynamic changes in accessibility, nuclear positioning, recombination, and transcription at the Ig kappa locus.

The 3-megabase Igkappa locus undergoes differentially controlled nuclear positioning events and chromatin structural changes during the course of B cell development. The temporal association of chromatin structural changes, transcription, and recombination at the Igkappa locus was determined in a murine pre-B cell line that can be induced to recombine at the Igkappa locus and in ex vivo-cultured murine pre-B cells. Additionally, the timing of nuclear positioning relative to the temporal order of chromatin structural changes and recombination and transcription was determined. We demonstrate that before induction, the Igkappa locus was poised for recombination; both alleles were in a contracted state, and the enrichment of histone modifications and germline transcripts of specific Vkappa genes were observed. Histone modifications of the Vkappa genes did not vary upon induction but the levels of modifications correlated with the levels of germline Vkappa gene transcripts and recombination. Upon induction, but before VkappaJkappa recombination, centromeric recruitment of single Igkappa alleles occurred. DNase I sensitivity of the entire locus increased gradually over the course of differentiation while the enrichment of histone modifications downstream of the Vkappa genes was increased in the silencer regions upstream of Jkappa1, within the Igkappa sterile transcript, the kappa constant region, the Ekappai and Ekappa3' enhancers, and the recombining sequence. The ex vivo pre-B cells showed similar patterns of histone modifications across the locus except at the Vkappa genes. In this study, H3 acetylation correlated with levels of germline transcripts while H3 methylation correlated with levels of recombination.

Monitoring and modeling of emissions from concentrated animal feeding operations: overview of methods.

Accurate monitors are required to determine ambient concentration levels of contaminants emanating from concentrated animal feeding operations (CAFOs), and accurate models are required to indicate the spatial variability of concentrations over regions affected by CAFOs. A thorough understanding of the spatial and temporal variability of concentration levels could then be associated with locations of healthy individuals or subjects with respiratory ailments to statistically link the presence of CAFOs to the prevalence of ill health effects in local populations. This workgroup report, which was part of the Conference on Environmental Health Impacts of Concentrated Animal Feeding Operations: Anticipating Hazards-Searching for Solutions, describes instrumentation currently available for assessing contaminant concentration levels in the vicinity of CAFOs and reviews plume dispersion models that may be used to estimate concentration levels spatially. Recommendations for further research with respect to ambient air monitoring include accurately determining long-term average concentrations for a region under the influence of CAFO emissions using a combination of instruments based on accuracy, cost, and sampling duration. In addition, development of instruments capable of accurately quantifying adsorbed gases and volatile organic compounds is needed. Further research with respect to plume dispersion models includes identifying and validating the most applicable model for use in predicting downwind concentrations from CAFOs. Additional data are needed to obtain reliable emission rates from CAFOs.

Reconstruction of the anterior cruciate ligament: meta-analysis of patellar tendon versus hamstring tendon autograft.

PURPOSE: No graft tissue has consistently shown superiority over others for reconstruction of the anterior cruciate ligament (ACL). Bone-patellar tendon-bone (BPTB) and doubled hamstring tendon (semitendinosus and gracilis) (HT) are the most commonly used autologous grafts. We performed a meta-analysis to compare the effectiveness of ACL reconstruction using either BPTB or HT grafts. TYPE OF STUDY: Systematic review and meta-analysis. METHODS: We searched the MEDLINE database (1966 to April 2003) for English-language randomized or prospective studies comparing BPTB and 3- or 4-strand HT grafts used for ACL reconstruction. For inclusion, studies were required to follow identical rehabilitation protocols within each study, and provide subjective or objective outcome data after a minimum average 2-year follow-up. Comparison data between BPTB and HT for each identified outcome measure were combined using a random-effects model meta-analysis. RESULTS: Eleven reports fulfilled the criteria for inclusion. Outcomes favoring BPTB were found in the following outcome measures: normal Lachman examination (relative risk [RR], 0.91; 95% confidence interval [CI], 0.83-0.99; P = .025), normal pivot-shift (RR, 0.94; 95% CI, 0.88-1.0; P = .067), KT-1000 manual-maximum side-to-side difference of < or = 3 mm (RR, 0.75; 95% CI, 0.55-1.01; P = .057), and fewer reconstructions resulting in flexion loss > 5 degrees (RR, 1.41; 95% CI, 1.01-1.96; P = .04). Intermediate level laxity was more common with the HT graft, as shown by higher rates of abnormal Lachman > 0 (RR, 1.22; 95% CI, 0.99-1.5; P = .06), pivot-shift > 0 (RR, 1.3; 95% CI, 0.96-1.75; P = .09), and KT-1000 manual-maximum side-to-side differences > 3 mm (RR, 1.64; 95% CI, 1.13-2.39; P = .01). Outcome measures that favored HT were absence of patellofemoral crepitance (RR, 1.08; 95% CI, 1.01-1.15; P = .03), fewer results with extension loss > 5 degrees (RR, 0.56; 95% CI, 0.3-1.03; P = .06), and kneeling pain. The incidence of instability, as defined by Lachman grade 2, pivot-shift grade 2, or KT-1000 manual-maximum side-to-side difference > 5 mm, was not significantly different between the 2 grafts. All other outcome measures were not significantly different. CONCLUSIONS: The data presented in this meta-analysis show that the incidence of instability is not significantly different between the BPTB and HT grafts. However, BPTB was more likely to result in reconstructions with normal Lachman, normal pivot-shift, KT-1000 manual-maximum side-to-side difference < 3 mm, and fewer results with significant flexion loss. In contrast, HT grafts had a reduced incidence of patellofemoral crepitance, kneeling pain, and extension loss. The choice of graft by the patient and surgeon must be individualized, and the results of this meta-analysis can aid in the decision by clarifying the risks and benefits of each surgical approach. LEVEL OF EVIDENCE: Level I.

Evolutionary relationships and polymorphisms among V kappa 10 genes from inbred and wild-derived inbred mice.

The V kappa 10 family in BALB/c mice is composed of three members, two of which are utilized in a variety of immune responses. We previously demonstrated that the product of the third gene, V kappa 10C, has never been detected as part of a functional antibody and productive rearrangements are selectively lost during B-cell development. Here we analyzed germline V kappa 10 genes from inbred and wild-derived mice by RFLP and sequencing in order to determine the origin of the V kappa 10C gene, as well as to examine the evolutionary relationships of V kappa 10 genes. Our results demonstrated that the V kappa 10 family is highly conserved across Mus species and subspecies, but that V kappa 10C is rare, being found in only inbred mice of V kappa 10 allelic group b and two of six M. m. domesticus isolates. It was not found in other M. musculus subspecies or M. spretus. V kappa 10A and V kappa 10B were found in all strains, with the exception of one M. m. domesticus isolate, which had only V kappa 10B genes. Overall, V kappa 10A sequences were more highly conserved than V kappa 10B, indicating that different selective pressures may be operating on these genes. The two V kappa 10C sequences from M. m. domesticus were 100% identical to that found in inbred mice. V kappa 10C is more closely related to V kappa 10B than to V kappa 10A and our data suggest that it is a recent duplication of the V kappa 10B gene.