Ex vivo characterization and isolation of rare memory B cells with antigen tetramers.

Studying human antigen-specific memory B cells has been challenging because of low frequencies in peripheral blood, slow proliferation, and lack of antibody secretion. Therefore, most studies have relied on conversion of memory B cells into antibody-secreting cells by in vitro culture. To facilitate direct ex vivo isolation, we generated fluorescent antigen tetramers for characterization of memory B cells by using tetanus toxoid as a model antigen. Brightly labeled memory B cells were identified even 4 years after last immunization, despite low frequencies ranging from 0.01% to 0.11% of class-switched memory B cells. A direct comparison of monomeric to tetrameric antigen labeling demonstrated that a substantial fraction of the B-cell repertoire can be missed when monomeric antigens are used. The specificity of the method was confirmed by antibody reconstruction from single-cell sorted tetramer(+) B cells with single-cell RT-PCR of the B-cell receptor. All antibodies bound to tetanus antigen with high affinity, ranging from 0.23 to 2.2 nM. Furthermore, sequence analysis identified related memory B cell and plasmablast clones isolated more than a year apart. Therefore, antigen tetramers enable specific and sensitive ex vivo characterization of rare memory B cells as well as the production of fully human antibodies.

Does the dual use of toluidine blue and hematoxylin and eosin staining improve basal cell carcinoma detection by Mohs surgery trainees?

BACKGROUND: Although a number of Mohs surgeons currently use Toluidine blue stain, alone or in combination with hematoxylin and eosin (H&E), the effects on the trainees' histologic accuracy of adding toluidine blue to their H&E training was unknown. OBJECTIVE: To assess a trainees' histological accuracy when trained in a unit that routinely employs the dual staining technique and to determine whether the addition of toluidine blue improves, or impairs, the training process. METHODS: A fellow examined slides from 403 consecutive Mohs cases over 3 months, from the start of his training period. H&E slides for each case were examined first, followed by the toluidine blue slides, with recordings made of the diagnosis based on each. The fellows' findings were then checked against those of the senior Mohs surgeons and a consultant histopathologist. RESULTS: According to H&E alone, the fellow completely excised 96.3% of 352 basal cell carcinomas; this increased to 99.7% by adding toluidine blue. False-positive rates were 1.5% for H&E alone and 1.7% when using both stains. CONCLUSION: The addition of toluidine blue increased the diagnostic accuracy of the trainee, and we encourage the use and teaching of this stain in Mohs surgery.

Acute generalised exanthematous pustulosis following intravitreal Ranibizumab.

Acute generalised exanthematous pustulosis, or AGEP, is a well documented cutaneous drug reaction. It typically occurs within 48 hours of oral antibiotics, but can be caused by other medications and, occasionally, after viral infections. We present a case of AGEP following intravitreal injection of Ranibizumab, a monoclonal antibody vascular endothelial growth factor inhibitor.

Loss of abutment teeth for double crown-retained removable partial dentures compared to clasp-retained removable partial dentures: a systematic review and meta-analysis.

Objective: To analyze the loss of abutment teeth for double crown-retained removable partial dentures (DC-RPDs) compared to clasp-retained removable partial dentures (C-RPDs). Method and materials: A search was conducted in the Ovid MEDLINE, Embase, Web of Science databases, and a manual search. The search was conducted based on the PICO framework with inclusion and exclusion criteria. After extracting the data of selected studies, a meta-analysis was performed to estimate abutment loss with 95% confidence interval (CI). The statistical significance was defined as P < .05, and the heterogeneity of the data was assessed based on the chi-squared test and I2 statistics. Risk of bias assessment was conducted using Cochrane Risk of Bias tool and Newcastle-Ottawa Scale. Results: A total of 4,692 records were identified from electronic databases and 38 studies were included for quantitative synthesis of 3,393 subjects with 13,552 abutment teeth. A total of 1,226 abutment teeth were lost with > 4,016 RPDs. Seven studies were compounded for > 668 C-RPDs (mean follow-up time ≤ 5 years) and six studies for 893 C-RPDs (mean follow-up time < 5 years), where the estimates of abutment loss were 5% (95% CI 2% to 8%) and 8% (95% CI 5% to 13%), respectively. The data were not significantly different (P = .1), and were heterogenous between the studies (τ2 ≥ 0.34, I2 ≥ 87.38%). Thirteen studies were compounded for 1,223 DC-RPDs (mean follow-up time ≤ 5 years) and eight studies for 1,033 DC-RPDs (mean follow-up time > 5 years), where the estimates of abutment loss were 6% (95% CI 5% to 8%) and 12% (95% CI 8% to 18%), respectively. The data were heterogenous (τ2 ≥ 0.17, I2 ≥ 75.86%), and were significantly different between the studies (P = .005). Overall, C-RPDs were not significantly different from DC-RPDs in abutment loss (P ≥ .3). A significant predictor for abutment loss was follow-up time with DC-RPDs (P = .005), where the risk of abutment loss per year was 18% (P = .0001). In contrast, follow-up time was not a significant factor for C-RPDs (P = .1). None of the included studies were at high risk of bias. Conclusion: Within the limitations of the current systematic review and meta-analysis, abutment loss was not significantly different between C-RPDs and DC-RPDs. A significant predictor was follow-up time for DC-RPDs, whereas this factor was not significant for C-RPDs. Further research is needed to investigate critical factors for abutment loss with RPDs.

Loss of nonvital abutment teeth for removable partial dentures: a systematic review and meta-analysis.

OBJECTIVE: To analyze the loss of nonvital abutment teeth compared to vital abutment teeth supporting removable partial dentures (RPDs). METHOD AND MATERIALS: An electronic search was conducted in the Ovid MEDLINE, Embase, and Web of Science databases, and supplemented by a manual search. The search was done to identify clinical studies reporting the loss of nonvital abutment teeth compared to vital abutment teeth for RPDs. The data were extracted from each selected article and meta-analysis was performed using a random effects model to estimate adjusted proportion of abutment loss with RPDs along with 95% confidence interval (CI). Risk of bias assessment was conducted using Cochrane Risk of Bias tools and Newcastle-Ottawa Scale. The statistical significance was set as P value < .05. RESULTS: A total of 3,898 records were identified from electronic databases and 8 studies were included for quantitative synthesis of 834 subjects with 1,036 RPDs. No additional records were identified through manual search. Among 1,152 nonvital abutment teeth, 123 teeth were lost. The estimate of nonvital abutment loss was 13% (95% CI 9-18%]. The data were statistically significant (P < .0001), and were heterogenous between the studies (χ2 [df = 7] = 35.9, P < .0001; τ2 = 0.25, I2 = 76.46%). A total of 2,186 vital abutment teeth were compounded where 114 teeth were lost. The estimate of vital abutment loss was 4% (95% CI 2-7%). The data were statistically significant (P < .0001), and were heterogenous between the studies (χ2 [df = 7] = 23.7, P = .01; τ2 = 0.56, I2 = 86.31%). The risk of abutment loss was approximately three times greater with the loss of vitality (odds ratio = 3.04, 95% CI 1.53-6.05; P = .001). In addition, abutment loss was significantly greater with increasing follow-up time (P = .01). None of the included studies were considered to be at high risk of bias. CONCLUSION: Within the limitations of this systematic review and meta-analysis, the loss of nonvital abutment teeth was significantly greater than that of vital abutment teeth for RPDs. Further research is needed to identify critical factors associated with the loss of nonvital abutment teeth.

Tooth reduction guide using silicone registration material along with vacuum-formed thermoplastic matrix.

Adequate tooth reduction is a prerequisite for function, esthetics, and longevity of fixed restorations. A tooth reduction guide may be useful for establishing the proper angulation of the tooth and maximizing periodontal health and restorative success. This article describes a simple and versatile technique for an accurate evaluation of tooth reduction for fixed restorations by using a color-contrasting positive guide of a silicone occlusal registration material processed inside a vacuum-formed clear thermoplastic matrix.

Case Report: A rapidly growing cyst on the scalp.

Trichilemmal carcinoma is a rare tumour derived from the outer root sheath of hair follicles .  It can be difficult to distinguish both clinically and histologically from other skin lesions, particularly squamous cell carcinoma.  We present the case of a 62-year-old female with a 20-year history of three 1-cm cysts on her scalp.  Over a six-month period, a cyst overlying the occiput had become painful and grown in size.  The general practitioner and subsequently local emergency department suspected infection.  The lesion was incised, and the patient was treated with oral antibiotics.  At the time of surgical excision, the lesion measured 3 x 4 cm. Microscopic examination identified rounded dermal lobules of squamous epithelium with trichilemmal keratinization, in keeping with a pre-existing pilar cyst.  There were areas with nuclear pleomorphism, mitoses and an infiltrative architecture.  A diagnosis of trichilemmal carcinoma arising in a pilar cyst was made.  Trichilemmal carcinomas are considered to be a low-grade tumour, but they have the potential to spread to lymph nodes and to metastasise to distant sites in the body, therefore adequate excision and appropriate follow-up are required.

Combination therapy with anti-CTL antigen-4 and anti-4-1BB antibodies enhances cancer immunity and reduces autoimmunity.

The majority of cancer antigens identified thus far have limited expression in normal tissues. It has been suggested that autoimmune disease is a necessary price for cancer immunity. This notion is supported by a recent clinical trial involving an anti-CTL antigen-4 (CTLA-4) antibody that showed significant clinical responses but severe autoimmune diseases in melanoma patients. To selectively modulate cancer immunity and autoimmunity, we used anti-CTLA-4 and anti-4-1BB antibodies to treat mice with a preexisting cancer, MC38. The combination of the two antibodies led to CD8 T-cell-mediated rejection of large established MC38 tumors and long-lasting immunity to the same tumor cells, although the same regimen was not effective for B16 melanoma. More importantly, whereas individual antibodies induced inflammation and autoimmune manifestations, combination therapy increased cancer immunity while reducing autoimmunity. The reduction of autoimmune effects correlates with an increased function of regulatory T cells. Our results suggest a novel approach to simultaneously enhance cancer immunity and reduce autoimmunity.

Lymphomatoid papulosis presenting as a rash in a patient with HIV infection.

A 43-year-old Malaysian man with well-controlled HIV infection on combination antiretroviral therapy presented with a six-week history of a widespread rash. The patient was otherwise well but was developing new lesions on a daily basis. Referral to Dermatology instigated punch biopsies, which revealed a diagnosis of lymphomatoid papulosis type A. This case highlights the importance of swift referral, especially in cases of spontaneous regression of symptoms, in order to obtain the correct diagnosis. In most patients, this condition tends to be chronic, with its chronicity and benign clinical course setting it apart from cutaneous anaplastic T-cell lymphoma and Hodgkin's disease, which are major entities in the histological differential diagnosis.

Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity.

MICA and MICB are expressed by many human cancers as a result of cellular stress, and can tag cells for elimination by cytotoxic lymphocytes through natural killer group 2D (NKG2D) receptor activation. However, tumors evade this immune recognition pathway through proteolytic shedding of MICA and MICB proteins. We rationally designed antibodies targeting the MICA α3 domain, the site of proteolytic shedding, and found that these antibodies prevented loss of cell surface MICA and MICB by human cancer cells. These antibodies inhibited tumor growth in multiple fully immunocompetent mouse models and reduced human melanoma metastases in a humanized mouse model. Antitumor immunity was mediated mainly by natural killer (NK) cells through activation of NKG2D and CD16 Fc receptors. This approach prevents the loss of important immunostimulatory ligands by human cancers and reactivates antitumor immunity.

Anti-4-1BB monoclonal antibody enhances rejection of large tumor burden by promoting survival but not clonal expansion of tumor-specific CD8+ T cells.

Anti-4-1BB monoclonal antibody (mAb) has been shown to induce antitumor immunity by a CD4/CD8-dependent mechanism, but its direct effect on tumor-specific CD8+ T cells in tumor rejection is unclear. Here we used transgenic CD8+ T cells against the unmutated tumor rejection antigen P1A to analyze whether this mAb can promote CD8+ T-cell function against large tumors in the absence of CD4+ T-helper cells. RAG-2(-/-) mice were challenged with P1A-expressing plasmacytoma J558. Once tumor size reached a diameter of 0.85-1.75 cm, mice were treated with P1A-specific CD8+ CTL (P1CTL) in conjunction with anti-4-1BB mAb or control IgG. All of the mice showed a partial regression of tumor, but mice treated with anti-4-1BB mAb exhibited markedly enhanced tumor rejection, delayed tumor progression, and prolonged survival. Correspondingly, we observed a substantial increase in the number of P1CTL in anti-4-1BB mAb-treated mice. Surprisingly, anti-4-1BB mAb did not accelerate division of the tumor-specific CD8+ T cells, and the increase in tumor-specific T-cell number was due to reduced activation-induced cell death. These results indicate that anti-4-1BB mAb can promote CD8+ T cell-mediated protection against large tumors in the absence of CD4+ T-cell help by promoting P1CTL survival without increasing initial clonal expansion.

On the role of unmutated antigens in tumor rejection in mice with unperturbed T-cell repertoires.

We investigate here whether P1A, which was the first CTL-recognized and unmutated tumor antigen to be identified, is a tumor rejection antigen for J558 plasmacytoma in mice with an unperturbed T-cell repertoire. We show that although transgenic mice expressing P1A in the thymus have almost complete deletion of P1A-reactive T cells, they reject the B7-1-transfected J558 at a rate comparable with wild-type mice. Thus, P1A is not a necessary tumor rejection antigen for the J558 tumor cells. On the other hand, if anti-P1A CTL response is sufficient for tumor rejection, tumor cells must lose the antigenic epitope to evade CTL destruction. To test this, we analyze whether tumor cells escaping J558-B7 immune spleen cells harbor mutations in the P1A epitope. We find that although the spleen contained a high proportion of P1A-reactive T cells, the recurrent tumor cells have no mutation in the P1A antigenic epitope and remain susceptible to lysis by P1CTL. Thus, the antigen-bearing tumor cells can evade immune destruction in the presence of a high number of P1A-reactive T cells. Taken together, our results demonstrate that in mice with a normal TCR repertoire, substantial numbers of P1A-reactive T cells are neither necessary nor sufficient for tumor rejection and raise interesting questions regarding the significance of T-cell response against unmutated tumor antigens.

Failed adoptive immunotherapy with tumor-specific T cells: reversal with low-dose interleukin 15 but not low-dose interleukin 2.

Adoptive immunotherapy with tumor-specific T cells has emerged as a valid approach for prevention or treatment of diseases, such as melanoma and EBV-associated lymphoma. As interleukin (IL) 15 promotes survival of CD8(+) memory CTLs, we hypothesized that it could be used to enhance antitumor immunity in vivo through the maintenance of adoptively transferred memory CTL. To test this, we treated mice bearing P1A(+) tumors with adoptively transferred T cells possessing a transgenic Valpha8(+) T-cell receptor specific for the P1A tumor antigen (called P1CTL). Mice were then randomized to receive daily low-dose IL-15 (0.5 microg/day) or PBS. Mice receiving the transgenic P1CTL and IL-15 experienced a significantly delayed tumor relapse or complete tumor regression (P < 0.002 compared with PBS), with a striking persistence of the CD8(+) Valpha8(+) P1CTL compared with mice receiving the CD8(+) Valpha8(+) P1CTL and PBS vehicle (26.3 versus 5.1% P < 10(-5)). Animals exhibiting complete tumor regression had a significant population of CD8(+) Valpha8(+) P1CTL (46%) that persisted with IL-15 treatment until 140 days after adoptive transfer and successfully defended them against tumor rechallenge without IL-15. Low-dose IL-2 afforded no protection over vehicle and resulted in lower percentages of T cells with an activated memory phenotype, lower Bcl-2 expression, and lower ex vivo antitumor cytotoxicity compared with mice treated with IL-15. Collectively, the data support the notion that exogenous low-dose IL-15 therapy can enhance and even reverse the limited efficacy of adoptively transferred tumor-specific T-cell therapy and may do so in a fashion that is superior and distinct from exogenous IL-2 therapy.

Recruiting underrepresented minority and low-income high school students into dentistry while educating dental and dental hygiene students about academic careers.

The objectives of this project were to create a program that would expose underrepresented minority (URM) and low income (LI) high school students to dental professions and provide an opportunity for dental and dental hygiene students from URM/LI groups to be engaged in teaching activities. Data were collected from participants during the school years 2009-10 (high school students: N=23, dental students: N=21, dental hygiene students: N=5) and 2010-11 (N=27, N=11, N=3, respectively). The students participated in fifteen Saturday sessions from October through March each year. The data showed that, from the beginning, mentees and mentors were very interested in participating in the program and getting to know each other. Lectures, general program activities, and patient-related events such as organizing a health fair and shadowing during two outreach clinics were evaluated positively by mentees and mentors. The end of program evaluations showed that the program and the mentee-mentor relationships were rated very positively and that the mentees had an increased interest in oral health-related careers. In conclusion, creating opportunities for URM/LI high school students to explore dental careers and for dental and dental hygiene students to engage in teaching resulted in positive experiences for both groups.

Effect of an enrichment program on DAT scores of potential dental students from disadvantaged backgrounds.

The objectives of this study were to explore whether Dental Admission Test (DAT) training in an enrichment program for potential dental students increases the participants' Perceptual Achievement Test (PAT) and academic average (AA) scores and whether the length of the program and personal factors such as the number of disadvantages correlate with the DAT scores. Data were collected from 361 students in the summer enrichment program at one dental school between 1994 and 2011. Their baseline, midpoint, and end of program PAT and AA DAT scores were collected. Seventy students self-reported official scores. These students' PAT scores increased from 14.40 at baseline to 17.09 at midpoint to 17.84 at program end (p<0.001), and their AA scores increased from 13.53 to 14.09 to 15.12 (p<0.001). Their official scores were higher than the beginning scores (PAT: 14.42 vs. 16.15; p<0.001; AA: 13.61 vs. 16.23; p<0.001). The longer the program, the more the students improved their official scores (PAT: r=0.35; p=0.003; AA: r=0.24; p=0.044). The more disadvantages the students self-reported, the better their official test scores were (PAT: r=0.40; p<0.001; AA: r=0.43; p<0.001). This study found that the DAT training during summer enrichment programs for students from disadvantaged backgrounds led to significant improvements in their DAT scores. The longer the programs, the more the students improved; and the more disadvantages the students had, the more they benefitted.

Prostate cancer immunotherapy.

The interaction between the immune system and prostate cancer has been an area of research interest for several decades. The recent U.S. Food and Drug Administration approval of 2 first-in-class proof-of-concept immunotherapies (sipuleucel-T and ipilimumab) has stimulated broader interest in manipulating immunity to fight cancer. In the context of prostate cancer, the immunotherapy strategies that have garnered the most interest are the therapeutic vaccination strategies, exemplified by sipuleucel-T and PROSTVAC-VF, and immune checkpoint blockade of CTLA-4 and PD-1. Improved understanding of the immune responses generated by these strategies and development of predictive biomarkers for patient selection will guide rational combinations of these treatments and provide building blocks for future immunotherapies.

Immune competence of cancer-reactive T cells generated de novo in adult tumor-bearing mice.

The impact of timing of antigen introduction into fetus and neonates leads to the suggestion that pre-existing antigens are tolerogenic to immunocompetent cells generated thereafter. This hypothesis predicts that in patients with cancer who are undergoing bone marrow transplantation, newly produced T cells with specificity for pre-existing tumor cells will be inactivated by the tumor antigens in the host. Because the effect of tumor cells on developing cancer-reactive T cells has not been investigated, we set out to systematically analyze the impact of tumor cells in the periphery on the development of tumor-reactive T cells in the thymus and their immunocompetence in the periphery. Our data demonstrate that in the host in which a tumor is established in the periphery, the cancer-reactive T cells develop normally, remain fully immunocompetent, become activated in the periphery, and cause regression of large established tumors. The immunocompetence of T cells generated in an antigen-bearing host is also confirmed in a skin graft transplantation model.

B7-deficient autoreactive T cells are highly susceptible to suppression by CD4(+)CD25(+) regulatory T cells.

CD4(+)CD25(+) regulatory T cells (Tregs) suppress immunity to infections and tumors as well as autoimmunity and graft-vs-host disease. Since Tregs constitutively express CTLA-4 and activated T cells express B7-1 and B7-2, it has been suggested that the interaction between CTLA-4 on Tregs and B7-1/2 on the effector T cells may be required for immune suppression. In this study, we report that autopathogenic T cells from B7-deficient mice cause multiorgan inflammation when adoptively transferred into syngeneic RAG-1-deficient hosts. More importantly, this inflammation is suppressed by adoptive transfer of purified wild-type (WT) CD4(+)CD25(+) T cells. WT Tregs also inhibited lymphoproliferation and acquisition of activation markers by the B7-deficient T cells. An in vitro suppressor assay revealed that WT and B7-deficient T cells are equally susceptible to WT Treg regulation. These results demonstrate that B7-deficient T cells are highly susceptible to immune suppression by WT Tregs and refute the hypothesis that B7-CTLA-4 interaction between effector T cells and Tregs plays an essential role in Treg function.