Germline-enforced enrichment for charged amino acids in TCR beta chain (TCRß) complementarity determining region 3 (CDR-B3) alters T cell development, repertoire content, and antigen recognition.

T cell receptor beta chain (TCRß) diversity (Dß) gene segments are highly conserved across evolution, with trout Dß1 sequence identical to human and mouse Dß1. A key conserved feature is enrichment for glycine in all three Dß reading frames (RFs). Previously, we found that replacement of mouse Dß1 with a typical immunoglobulin DH sequence, which unlike Dß is enriched for tyrosine, leads to an increase in the use of tyrosine in TCRß complementarity determining region 3 (CDR-B3) after thymic selection, altering T cell numbers, CDR-B3 diversity, and T cell function. To test whether the incorporation of charged amino acids into the Dß sequence in place of glycine would also influence T cell biology, we targeted the TCRß locus with a novel glycine-deficient DßDKRQ allele that replaces Dß1 coding sequence with charged amino acids in all three reading frames. Developing T cells using DßDKRQ expressed TCR CDR-B3s depleted of tyrosine and glycine and enriched for germline-encoded lysine, arginine, and glutamine. Total thymocytes declined in number during the process of ß selection that occurs during the transition from the DN3bc to DN4 stage. Conventional thymocyte and T cell numbers remained reduced at all subsequent thymic stages and in the spleen. By contrast, regulatory T cell numbers were increased in Peyer's patches and the large intestine. In terms of functional consequences, T cell reactivity to an ovalbumin immunodominant epitope was reduced. These findings buttress the view that natural selection of Dß sequence is used to shape the pre-immune TCRß repertoire, affecting both conventional and regulatory T cell development and influencing epitope recognition.

Replacement of TCR Dß With Immunoglobulin DH DSP2.3 Imposes a Tyrosine-Enriched TCR Repertoire and Adversely Affects T Cell Development.

Enrichment for tyrosine in immunoglobulin CDR-H3 is due in large part to natural selection of germline immunoglobulin DH sequence. We have previously shown that when DH sequence is modified to reduce the contribution of tyrosine codons, epitope recognition is altered and B cell development, antibody production, autoantibody production, and morbidity and mortality following pathogen challenge are adversely affected. TCRß diversity (Dß) gene segment sequences are even more highly conserved than DH, with trout Dß1 identical to human and mouse Dß1. We hypothesized that natural selection of Dß sequence also shapes CDR-B3 diversity and influences T cell development and T cell function. To test this, we used a mouse strain that lacked Dß2 and contained a novel Dß1 allele (DßYTL) that replaces Dß1 with an immunoglobulin DH, DSP2.3. Unlike Dß1, wherein glycine predominates in all three reading frames (RFs), in DSP2.3 there is enrichment for tyrosine in RF1, threonine in RF2, and leucine in RF3. Mature T cells using DßYTL expressed TCRs enriched at particular CDR-B3 positions for tyrosine but depleted of leucine. Changing Dß sequence altered thymocyte and peripheral T cell numbers and the T cell response to an ovalbumin immunodominant epitope. The differences in tyrosine content might explain, at least in part, why TCRs are more polyspecific and of lower affinity for their cognate antigens than their immunoglobulin counterparts.

Preimmune Control of the Variance of TCR CDR-B3: Insights Gained From Germline Replacement of a TCR Dß Gene Segment With an Ig D H Gene Segment.

We have previously shown that the sequence of the immunoglobulin diversity gene segment (D H ) helps dictate the structure and composition of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3). In order to test the role of germline D sequence on the diversity of the preimmune TCRß repertoire of T cells, we generated a mouse with a mutant TCRß DJC locus wherein the Dß2-Jß2 gene segment cluster was deleted and the remaining diversity gene segment, Dß1 (IMGT:TRDB1), was replaced with DSP2.3 (IMGT:IGHD2-02), a commonly used B cell immunoglobulin D H gene segment. Crystallographic studies have shown that the length and thus structure of TCR CDR-B3 places amino acids at the tip of CDR-B3 in a position to directly interact with peptide bound to an MHC molecule. The length distribution of complementarity determining region 3 of the T cell receptor beta chain (CDR-B3) has been proposed to be restricted largely by MHC-specific selection, disfavoring CDR-B3 that are too long or too short. Here we show that the mechanism of control of CDR-B3 length depends on the Dß sequence, which in turn dictates exonucleolytic nibbling. By contrast, the extent of N addition and the variance of created CDR3 lengths are regulated by the cell of origin, the thymocyte. We found that the sequence of the D and control of N addition collaborate to bias the distribution of CDR-B3 lengths in the pre-immune TCR repertoire and to focus the diversity provided by N addition and the sequence of the D on that portion of CDR-B3 that is most likely to interact with the peptide that is bound to the presenting MHC.

HIV-1 gp140 epitope recognition is influenced by immunoglobulin DH gene segment sequence.

Complementarity Determining Region 3 of the immunoglobulin (Ig) H chain (CDR-H3) lies at the center of the antigen-binding site where it often plays a decisive role in antigen recognition and binding. Amino acids encoded by the diversity (DH) gene segment are the main component of CDR-H3. Each DH has the potential to rearrange into one of six DH reading frames (RFs), each of which exhibits a characteristic amino acid hydrophobicity signature that has been conserved among jawed vertebrates by natural selection. A preference for use of RF1 promotes the incorporation of tyrosine into CDR-H3 while suppressing the inclusion of hydrophobic or charged amino acids. To test the hypothesis that these evolutionary constraints on DH sequence influence epitope recognition, we used mice with a single DH that has been altered to preferentially use RF2 or inverted RF1. B cells in these mice produce a CDR-H3 repertoire that is enriched for valine or arginine in place of tyrosine. We serially immunized this panel of mice with gp140 from HIV-1 JR-FL isolate and then used enzyme-linked immunosorbent assay (ELISA) or peptide microarray to assess antibody binding to key or overlapping HIV-1 envelope epitopes. By ELISA, serum reactivity to key epitopes varied by DH sequence. By microarray, sera with Ig CDR-H3s enriched for arginine bound to linear peptides with a greater range of hydrophobicity but had a lower intensity of binding than sera containing Ig CDR-H3s enriched for tyrosine or valine. We conclude that patterns of epitope recognition and binding can be heavily influenced by DH germ line sequence. This may help explain why antibodies in HIV-infected patients must undergo extensive somatic mutation in order to bind to specific viral epitopes and achieve neutralization.

Synovial Sarcoma-AV Malformation Collision in the Anterior Mediastinum.

Arteriovenous malformation (AVM) and synovial sarcomas are both rare lesions in the mediastinum. Rarer still is a collision tumor in that region. Herein we present a case of a collision tumor comprised of AVM and synovial sarcoma in a 76-year-old man, presenting with pneumonia. Imaging showed a vascular lesion that spontaneously ruptured, causing enlargement of the mass and hemothorax. The resected specimen revealed the malignant second component. This report is a discussion of the never-before reported lesion.

Inactivated HSV-2 in MPL/alum adjuvant provides nearly complete protection against genital infection and shedding following long term challenge and rechallenge.

Herpes Simplex Virus Type 2 (HSV-2) infection can result in life-long recurrent genital disease, asymptomatic virus shedding, and transmission. No vaccine to date has shown significant protection clinically. Here, we used a mouse model of genital HSV-2 infection to test the efficacy of a vaccine consisting of whole, formalin-inactivated HSV-2 (FI-HSV2) formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Vaccine components were administered alone or as a prime-boost immunization together with DNA vaccines encoding a truncated glycoprotein D2 (gD2t) and two conserved HSV-2 genes necessary for virus replication, UL5 (DNA helicase) and UL30 (DNA polymerase). Our results show: (1) compared with mock immunized controls, mice immunized with FI-HSV2 plus MPL/alum consistently showed protection against disease burden and total viral shedding while the mice immunized with gD2t protein with MPL/alum did not; (2) protection against genital disease and viral replication correlated with the type of boost in a prime-boost immunization with little advantage afforded by a DNA prime; (3) intramuscular (i.m.) immunization with FI-HSV2 in MPL/Alhydrogel adjuvant provided nearly complete protection against vaginal HSV-2 shedding after a lethal intravaginal (i.vag.) short-term challenge and long-term rechallenge; (4) single formulation immunization with DNA vaccines, FI-HSV2, and MPL in an aluminum phosphate (Adju-Phos) adjuvant did not increase protection relative to FI-HSV2/MPL/Adju-Phos alone; and (5) addition of MPL/alum to the FI-HSV2 was required for optimal protection against disease, viral replication, and latent virus load in the dorsal root ganglia (DRG). Most notably, an optimized vaccine formulation of FI-HSV2 MPL/Alhydrogel given i.m. completely protected against detectable vaginal HSV-2 shedding in the majority of animals and HSV-2 latent DNA in the DRG of all animals.

Seeking safe harbor. How to navigate antikickback and Stark barriers to clinical integration.

Stark and antikickback rules complicate clinical integration.

Immunization with herpes simplex virus 2 (HSV-2) genes plus inactivated HSV-2 is highly protective against acute and recurrent HSV-2 disease.

To date, no vaccine that is safe and effective against herpes simplex virus 2 (HSV-2) disease has been licensed. In this study, we evaluated a DNA prime-formalin-inactivated-HSV-2 (FI-HSV2) boost vaccine approach in the guinea pig model of acute and recurrent HSV-2 genital disease. Five groups of guinea pigs were immunized and intravaginally challenged with HSV-2. Two groups were primed with plasmid DNAs encoding the secreted form of glycoprotein D2 (gD2t) together with two genes required for viral replication, either the helicase (UL5) and DNA polymerase (UL30) genes or the single-stranded DNA binding protein (UL29) and primase (UL52) genes. Both DNA-primed groups were boosted with FI-HSV2 formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Two additional groups were primed with the empty backbone plasmid DNA (pVAX). These two groups were boosted with MPL and alum (MPL-alum) together with either formalin-inactivated mock HSV-2 (FI-Mock) or with FI-HSV2. The final group was immunized with gD2t protein in MPL-alum. After challenge, 0/9 animals in the group primed with UL5, UL30, and gD2t DNAs and all 10 animals in the mock-immunized control group (pVAX-FI-Mock) developed primary lesions. All mock controls developed recurrent lesions through day 100 postchallenge. Only 1 guinea pig in the group primed with pVAX DNA and boosted with FI-HSV2 (pVAX-FI-HSV2 group) and 2 guinea pigs in the group primed with UL5, UL30, and gD2t DNAs and boosted with FI-HSV2 (UL5, UL30, gD2t DNA-FI-HSV2 group) developed recurrent lesions. Strikingly, the UL5, UL30, gD2t DNA-FI-HSV2 group showed a 97% reduction in recurrent lesion days compared with the mock controls, had the highest reduction in days with recurrent disease, and contained the lowest mean HSV-2 DNA load in the dorsal root ganglia.

Anterior cruciate ligament reconstruction using achilles tendon allograft: an assessment of outcome for patients age 30 years and older.

Achilles allografts have become popular for anterior cruciate ligament (ACL) reconstructions in older patients. Primary ACL reconstructions using Achilles tendon allografts in patients age 30 years and older are successful in restoring the knee to "normal" or "near normal." During a three-year period, the two senior authors performed 65 primary ACL reconstructions using Achilles tendon allografts in patients aged 30 years and older. Our exclusion criteria were periarticular fracture, ipsilateral/contralateral knee ligament injury, and previous or concomitant osteotomy or cartilage restoration procedure. Each patient was evaluated via physical examination, functional and arthrometric testing, and radiographic and subjective outcome. Knees were considered normal, near normal, or abnormal based on the International Knee Documentation Committee (IKDC) system. Forty-three patients were examined at an average of 33 months (minimum, 24 months) postoperatively. At the time of ACL reconstruction, 35% had normal articular cartilage in all three compartments and 70% had meniscal tears. No re-ruptures occurred. While 24% had mean maximal translation differences less than or equal to 3 mm, none had side-to-side differences greater than 5 mm. Postoperative IKDC, Activities of Daily Living, and Activity Rating Scale scores averaged 88, 94, and 7.7, respectively. Despite the overall favorable outcomes, 29% had worsened radiographic grades at follow-up. Using an Achilles allograft for ACL reconstruction in patients older than 30 years, we restored over 90% of knees to normal or near normal while limiting postoperative complications. Poor subjective results may be related less to instability and more to pain, which may result from progressive arthritis.

Are one or two dangerous? Quinine and quinidine exposure in toddlers.

Quinine and quinidine have been cited as drugs that may cause significant morbidity and mortality in toddlers who ingest one or two pills. The use of both of these drugs has declined in the United States since the 1980s. A review of the literature and Poison Control data reveals that large quinine and quinidine ingestions, although rare in this country, may lead to severe toxicity and death related to cardiovascular and neurological effects in both children and adults. Although the majority of cases of quinine and quinidine toxicity in toddlers occur after ingestions of more than two pills, a single report each of severe toxicity after the equivalent of an ingestion of two pills or less by a toddler exists for both quinine and quinidine. Although the risk to the toddler exposed to one or two tablets seems to be small, triage to an Emergency Department is warranted after quinidine ingestion of any amount and after quinine ingestion that exceeds the age-appropriate therapeutic dose.