Autoinflammatory disorders of the innate immune system present with recurrent episodes of inflammation often beginning in early childhood. While there are now more than 30 genetically-defined hereditary fever disorders, many patients lack a clear diagnosis. Many pediatric patients are often grouped with patients with periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome despite failing to meet diagnostic criteria. Here, we categorize these patients as syndrome of undifferentiated recurrent fever (SURF), and identify the unique features which distinguish them from the PFAPA syndrome. SURF patients were more likely to report gastrointestinal symptoms of nausea, vomiting and abdominal pain, and experienced inconsistent responses to on-demand steroid therapy compared to PFAPA patients. For this previously undefined cohort, an optimal course of therapy remains uncertain, with medical and surgical therapies largely driven by parental preference. A subset of patients with SURF underwent tonsillectomy with complete resolution. Flow cytometric evaluation demonstrates leukocytic populations distinct from PFAPA patients, with reduced CD3+ T cell numbers. SURF patient tonsils were predominantly characterized by an IL-1 signature compared to PFAPA, even during the afebrile period. Peripheral blood signatures were similar between groups suggesting that PFAPA and SURF patient tonsils have localized, persistent inflammation, without clinical symptoms. These data suggest that SURF is a heterogenous syndrome on the autoinflammatory disease spectrum.

Periodic Fever, Aphthous stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome is an inflammatory disorder of childhood classically characterized by recurrent fevers, pharyngitis, stomatitis, cervical adenitis, and leukocytosis. While the mechanism is unclear, previous studies have shown that tonsillectomy can be a therapeutic option with improvement in quality of life in many patients with PFAPA, but the mechanisms behind surgical success remain unknown. In addition, long-term clinical follow-up is lacking. In our tertiary care center cohort, 62 patients with PFAPA syndrome had complete resolution of symptoms after surgery (95.3%). Flow cytometric evaluation demonstrates an inflammatory cell population, distinct from patients with infectious pharyngitis, with increased numbers of CD8+ T cells (5.9% vs. 3.8%, p < 0.01), CD19+ B cells (51% vs. 35%, p < 0.05), and CD19+CD20+CD27+CD38-memory B cells (14% vs. 7.7%, p < 0.01). Cells are primed at baseline with increased percentage of IL-1β positive cells compared to control tonsil-derived cells, which require exogenous LPS stimulation. Gene expression analysis demonstrates a fivefold upregulation in IL1RN and TNF expression in whole tonsil compared to control tonsils, with persistent activation of the NF-κB signaling pathway, and differential microbial signatures, even in the afebrile period. Our data indicates that PFAPA patient tonsils have localized, persistent inflammation, in the absence of clinical symptoms, which may explain the success of tonsillectomy as an effective surgical treatment option. The differential expression of several genes and microbial signatures suggests the potential for a diagnostic biomarker for PFAPA syndrome.

One Sentence Summary Recurrent tonsillitis is a multifactorial disease associated with an aberrant tonsillar germinal center response to Group A Streptococcus. ABSTRACT Recurrent Group A Streptococcus (GAS) tonsillitis (RT) is a common indication for pediatric tonsillectomy. ‘Strep throat’ is highly prevalent among children; yet, it is unknown why some children develop RT. To gain insights into this classic childhood disease, we performed phenotypic, genotypic, and functional studies on pediatric GAS RT and non-RT tonsils. We observed significantly smaller germinal centers in GAS RT tonsils, and underrepresentation of GAS-specific germinal center follicular helper (GC Tfh) CD4+ T cells. RT children exhibited reduced antibody responses to GAS virulence factor SpeA. Risk and protective HLA Class II alleles for RT were identified. Finally, SpeA induced granzyme B+ GC Tfh cells in RT tonsils that had capacity to kill B cells. Together, these observations suggest that RT susceptibility can occur due to genetic differences that can result in aberrant GC Tfh cells and poor antibody responses to GAS SpeA.

Recurrent Group A Streptcoccus (GAS) tonsillopharyngitis or “strep throat” is a common pediatric disease. Tonsils are the nidus of GAS infection. Tonsils contain germinal centers (GC), consisting of T follicular helper cells (Tfh) and germinal center B cells. GC Tfh cells are CD4+T cells that instruct GC B cells, allowing for the generation of high quality humoral immune responses. Why some children are susceptible to recurrent GAS tonsillitis remains a mystery. We have established a cohort of pediatric patients who have undergone tonsillectomy for either (1) recurrent tonsillitis (RT, N = 48) or (2) sleep apnea (non-RT, N = 64). We performed phenotypic, histologic, and genotypic analyses on the cohort. To address GAS-specific responses, we developed the activation-induced marker (AIM) assay to quantify GAS-specific GC Tfh responses. Tonsils from age-matched children with RT have significantly fewer GC Tfh cells (P < 0.0001) and GC B cells (P = 0.011) than non-RT tonsils. Histologically, RT tonsils have significantly smaller germinal center areas (P = 0.0002), but not fewer germinal centers. Moreover, we have observed HLA class II associations in the cohort with the identification of “At Risk” and “Protective” alleles for RT. Overall, these data suggests that children with RT may have an immunosusceptibility to recurrent GAS infection. As GC Tfh cells instruct nearby GC B cells through close contact and not through robust cytokine production, it is very challenging to quantify human antigen-specific GC Tfh cells (Dan et al, Journal of Immunology 2016). To address if RT tonsils have a GAS GC deficit, we utilized the AIM assay. We observed GAS-specific GC Tfh cell responses well above the background negative control bacterium (P < 0.0001). More importantly, we observed a significant impairment in the abundance of GAS-specific GC Tfh among GAS-specific antigen-experienced CD4+T cells in RT tonsils compared with non-RT tonsils (P = 0.025). Reduced GAS-specific GC Tfh cells suggest that children with recurrent tonsillitis have defective GAS-specific Tfh responses, likely resulting in fewer GAS-specific opsonizing antibodies and a predisposition toward recurrent infection. We are continuing to explore these germinal center deficits. All authors: No reported disclosures.

Soft tissue fillers are continuing to increase in popularity throughout North America and worldwide as a means of wrinkle and fold reduction, facial volume restoration, and contour enhancement [1]. A 3% increase in soft tissue fillers was noted in one year from 2013 to 2014 in the American Society of Plastic Surgery report 2014 [2]. Yet fillers are implants and essentially foreign bodies that can potentially trigger an inflammatory response in some individuals [3].

Significantly higher levels of plasma CXCL13 [chemokine (C-X-Cmotif) ligand 13] were associated with the generation of broadly neutralizing antibodies (bnAbs) against HIV in a large longitudinal cohort of HIV-infected individuals. Germinal centers (GCs) perform the remarkable task of optimizing B-cell Ab responses. GCs are required for almost all B-cell receptor affinity maturation and will be a critical parameter to monitor if HIV bnAbs are to be induced by vaccination. However, lymphoid tissue is rarely available from immunized humans, making the monitoring of GC activity by direct assessment of GC B cells and germinal center CD4(+) T follicular helper (GC Tfh) cells problematic. The CXCL13-CXCR5 [chemokine (C-X-C motif) receptor 5] chemokine axis plays a central role in organizing both B-cell follicles and GCs. Because GC Tfh cells can produce CXCL13, we explored the potential use of CXCL13 as a blood biomarker to indicate GC activity. In a series of studies, we found that plasma CXCL13 levels correlated with GC activity in draining lymph nodes of immunized mice, immunized macaques, and HIV-infected humans. Furthermore, plasma CXCL13 levels in immunized humans correlated with the magnitude of Ab responses and the frequency of ICOS+ (inducible T-cell costimulator) Tfh-like cells in blood. Together, these findings support the potential use of CXCL13 as a plasma biomarker of GC activity in human vaccine trials and other clinical settings.

Detection of Ag-specific CD4+ T cells is central to the study of many human infectious diseases, vaccines, and autoimmune diseases. However, such cells are generally rare and heterogeneous in their cytokine profiles. Identification of Ag-specific germinal center (GC) T follicular helper (Tfh) cells by cytokine production has been particularly problematic. The function of a GC Tfh cell is to selectively help adjacent GC B cells via cognate interaction; thus, GC Tfh cells may be stingy cytokine producers, fundamentally different from Th1 or Th17 cells in the quantities of cytokines produced. Conventional identification of Ag-specific cells by intracellular cytokine staining relies on the ability of the CD4+ T cell to generate substantial amounts of cytokine. To address this problem, we have developed a cytokine-independent activation-induced marker (AIM) methodology to identify Ag-specific GC Tfh cells in human lymphoid tissue. Whereas Group A Streptococcus–specific GC Tfh cells produced minimal detectable cytokines by intracellular cytokine staining, the AIM method identified 85-fold more Ag-specific GC Tfh cells. Intriguingly, these GC Tfh cells consistently expressed programmed death ligand 1 upon activation. AIM also detected non-Tfh cells in lymphoid tissue. As such, we applied AIM for identification of rare Ag-specific CD4+ T cells in human peripheral blood. Dengue, tuberculosis, and pertussis vaccine–specific CD4+ T cells were readily detectable by AIM. In summary, cytokine assays missed 98% of Ag-specific human GC Tfh cells, reflecting the biology of these cells, which could instead be sensitively identified by coexpression of TCR-dependent activation markers.

A range of current candidate AIDS vaccine regimens are focused on generating protective HIV-neutralizing Ab responses. Many of these efforts rely on the rhesus macaque animal model. Understanding how protective Ab responses develop and how to increase their efficacy are both major knowledge gaps. Germinal centers (GCs) are the engines of Ab affinity maturation. GC T follicular helper (Tfh) CD4 T cells are required for GCs. Studying vaccine-specific GC Tfh cells after protein immunizations has been challenging, as Ag-specific GC Tfh cells are difficult to identify by conventional intracellular cytokine staining. Cytokine production by GC Tfh cells may be intrinsically limited in comparison with other Th effector cells, as the biological role of a GC Tfh cell is to provide help to individual B cells within the GC, rather than secreting large amounts of cytokines bathing a tissue. To test this idea, we developed a cytokine-independent method to identify Ag-specific GC Tfh cells. RNA sequencing was performed using TCR-stimulated GC Tfh cells to identify candidate markers. Validation experiments determined CD25 (IL-2Rα) and OX40 to be highly upregulated activation-induced markers (AIM) on the surface of GC Tfh cells after stimulation. In comparison with intracellular cytokine staining, the AIM assay identified >10-fold more Ag-specific GC Tfh cells in HIV Env protein-immunized macaques (BG505 SOSIP). CD4 T cells in blood were also studied. In summary, AIM demonstrates that Ag-specific GC Tfh cells are intrinsically stingy producers of cytokines, which is likely an essential part of their biological function.

Follicular helper T cells (Tfh cells) are required for T cell help to B cells, and BCL6 is the defining transcription factor of Tfh cells. However, the functions of BCL6 in Tfh cells have largely remained unclear. Here we defined the BCL6 cistrome in primary human germinal center Tfh cells to assess mechanisms of BCL6 regulation of CD4 T cells, comparing and contrasting BCL6 function in T and B cells. BCL6 primarily acts as a repressor in Tfh cells, and BCL6 binding was associated with control of Tfh cell migration and repression of alternative cell fates. Interestingly, although some BCL6-bound genes possessed BCL6 DNA-binding motifs, many BCL6-bound loci were instead characterized by the presence of DNA motifs for AP1 or STAT. AP1 complexes are key positive downstream mediators of TCR signaling and external stimuli. We show that BCL6 can directly bind AP1, and BCL6 depends on AP1 for recruitment to BCL6-binding sites with AP1 motifs, suggesting that BCL6 subverts AP1 activity. These findings reveal that BCL6 has broad and multifaceted effects on Tfh biology and provide insight into how this master regulator mediates distinct cell context-dependent phenotypes.

Periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA) syndrome is an inflammatory disorder of childhood classically characterized by recurrent fevers, pharyngitis, stomatitis, cervical adenitis and leukocytosis. Little is known about the true incidence, natural course, pathogenesis, and appropriate therapy in this recently described syndrome. While the mechanism is unclear, previous studies have shown that tonsillectomy can be a therapeutic option with improvement in quality of life in many patients with PFAPA, but long-term clinical follow up is lacking.

Abstract Generation of long term humoral immunity is a complex process predominantly dependent on germinal centers and CD4 T cell help to B cells. Follicular helper T cells (Tfh) are the specialized CD4 T cells for B cell help. We and others have now resolved many of the stages of Tfh differentiation, and molecules involved. BCL6 is the defining transcription factor of Tfh cells. However, the functions of BCL6 in Tfh have largely remained unclear. We have defined the BCL6 cistrome in primary human germinal center Tfh cells to assess mechanisms of BCL6 regulation of CD4 T cells, comparing and contrasting BCL6 function in T and B cells. BCL6 primarily acts as a repressor in Tfh cells, and BCL6 binding was associated with control of Tfh cell migration, Tfh differentiation, and repression of alternative cell fates. Interestingly, although some BCL6 bound genes possessed BCL6 DNA binding motifs, more BCL6-bound loci were instead characterized by the presence of DNA motifs for AP1 or STAT. AP1 complexes are key positive downstream mediators of TCR signaling and external stimuli. We show that BCL6 can directly bind AP1, and AP1 and BCL6 co-occupied BCL6 binding sites with AP1 motifs, suggesting that BCL6 subverts AP1 activity. These findings reveal that BCL6 has broad and multifaceted effects on Tfh biology, and provide insight into how this master regulator mediates distinct cell-context dependent phenotypes.

Branchial cleft cysts, sinuses, and fistulas are the most common congenital lateral neck lesions in children. They arise as a result of an abnormal development of the branchial arches and their corresponding ectoderm-lined branchial clefts. Of these diverse anomalies, second branchial cleft lesions are the most common, accounting for approximately 95% of all branchial arch pathologies. We describe what is to the best of our knowledge the first reported case of an ectopic tooth in a branchial cleft anomaly. The patient was a young girl who had other congenital abnormalities and syndromic features and who was eventually diagnosed with Townes-Brocks syndrome. We describe the clinical presentation, management, pathologic analysis, and postoperative outcomes of this case, and we present a brief review of Townes-Brocks syndrome.

Periodic Fever, Aphthous stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome is an autoinflammatory disorder of childhood and little is known about the underlying etiology. While mutations involving the IL-1 pathway have been identified in other recurrent fever disorders, including TNF-receptor associated periodic syndrome (TRAPS) and cryopyrin-associated periodic syndrome (CAPS), PFAPA syndrome is not traditionally considered to be a hereditary fever disorder.

Periodic Fever, Aphthous stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome is an autoinflammatory disorder of childhood and little is known about the underlying etiology. While mutations involving the IL-1 pathway have been identified in other recurrent fever disorders, including TNF-receptor associated periodic syndrome (TRAPS) and cryopyrin-associated periodic syndrome (CAPS), there is no clear genetic basis for PFAPA. Patient data and detailed family histories were collected for over 170 children with recurrent fevers including 70 patients with PFAPA to create a prospective cohort over a 4-year period. DNA was isolated from blood or tonsillar tissue from recurrent fever patients, and NLRP3 and TNFRSF1A were sequenced. Quantitative real time PCR was used to evaluate IL-36 transcripts in tonsils. Our cohort reflects the diversity of San Diego, without predilection for any specific ethnic background. Family histories revealed 21% of patients have a first degree relative with recurrent fevers and 12% with tonsillitis in childhood, with only 1.4% reporting a history of recurrent infections. We have identified over 30 families with 2-8 affected members. These patients do not possess mutations commonly seen in other autoinflammatory disorders such as CAPS or TRAPS, suggesting that a novel gene may be involved. Upregulation of IL-36 gene expression in tonsils identifies the IL-1 family member IL-36 as a candidate gene. A substantial portion of our families with PFAPA report childhood histories of recurrent fevers that resolved either spontaneously or with tonsillectomy, indicating a possible dominantly inherited trait that impacts the developing immune system, including the tonsils.

SummaryAimTo demonstrate safety with the use of polymethyl methacrylate (PMMA) microspheres in the infraorbital eyelid area using a deliberate conservative injection in the treatment of rhytids.MethodsA retrospective case series of 289 patients in an outpatient cosmetic dermatology clinic evaluated and treated by one senior provider (NM) of infraorbital rhytids with PMMA from December 2010 to March 2011. Statistical analysis was performed for race, skin type, history of hypertrophic scar, autoimmunity, history of “sensitive skin” and history of prior procedures such as prior facelift, rhinoplasty, and blepharoplasty.ResultsTwo hundred ninety‐one patients underwent at least 1–6 injections of PMMA microspheres into bilateral under eye area. Early complications were edema and ecchymosis. Late complications were identified in 4 of 289 patients who developed small granulomas. All patients who developed granulomas had had a previous lower blepharoplasty (P = 0.00). A history of “sensitive skin” was approaching statistical significance (P = 0.15).ConclusionThis study has shown that PMMA microsphere injection is a safe subdermal technique in the correction of infraorbital rhytids. Safety was demonstrated in 289 patients with only 4 minor complications of small lateral granuloma which all resolved within 4 weeks after intralesion triamcinolone injection. However, this is an off‐label use of a permanent filler not approved for use in the infraorbits and significant caution must be taken with full disclosure to the patient leading to informed consent. Caution in PMMA microsphere injection should be given in the patient with prior blepharoplasty. The advantage of PMMA microsphere is that the result seems to be predictable and natural.

Periodic Fever, Aphthous stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome is an inflammatory disorder of childhood. Little is known about the true incidence, natural course, pathogenesis, and most appropriate therapy. Patient data was collected for over 150 children with recurrent fevers including 70 patients with PFAPA to create a prospective cohort to delineate the natural history of recurrent fever syndromes, with a specific focus on patients with PFAPA, over a 4-year period. In our cohort, the average age of onset is 2.9 years, but diagnosis delayed until 4.9 years. Febrile episodes last 3.3-4.5 days and occur every 23-38 days, with 66% experiencing stomatitis, 84% pharyngitis, and 69% adenitis. The diverse ethnic background of San Diego was reflected in this group, without predilection more common to the hereditary fever syndromes. Family histories revealed 33% of patients with a first degree relative with recurrent fevers or tonsillitis and 50% with a history of tonsillectomy in childhood. For a subset of patients electing to undergo tonsillectomy, symptoms are equivalent to those of entire cohort. Post-operatively, tonsils from patients with PFAPA are notably smaller and grossly friable, although no granulomas or abscesses are noted on histological examination. With an average of 18 months of follow-up, 90% of the patients have not experienced any additional fever episodes. Our cohort demonstrates clinical characteristics consistent with PFAPA. The presence of atopic diseases and family history of fevers, recurrent tonsillitis and tonsillectomy suggests that patients with recurrent fever syndromes including PFAPA may have a general propensity toward immunodysregulation.

Short latency linear vestibular sensory evoked potentials (VsEPs) provide a means to objectively and directly assess the function of gravity receptors in mammals and birds. The importance of this functional measure is illustrated by its use in studies of the genetic basis of vestibular function and disease. Head motion is the stimulus for the VsEP. In the bird, it has been established that neurons mediating the linear VsEP respond collectively to the rate of change in linear acceleration during head movement (i.e. jerk) rather than peak acceleration. The kinematic element of motion responsible for triggering mammalian VsEPs has not been characterized in detail. Here we tested the hypothesis that jerk is the kinematic component of head motion responsible for VsEP characteristics. VsEP amplitudes and latencies changed systematically when peak acceleration level was held constant and jerk level was varied from similar to 0.9-4.6 g/ms. In contrast, responses remained relatively constant when kinematic jerk was held constant and peak acceleration was varied from similar to 0.9 to 5.5 g in mice and similar to 0.44 to 2.75 g in rats. Thus the mammalian VsEP depends on jerk levels and not peak acceleration. We conclude that kinematic jerk is the adequate stimulus for the mammalian VsEP. This sheds light on the behavior of neurons generating the response. The results also provide the basis for standardizing the reporting of stimulus levels, which is key to ensuring that response characteristics reported in the literature by many laboratories can be effectively compared and interpreted. (c) 2011 Elsevier B.V. All rights reserved.