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India is a densely populated country of 0.4 billion children. However, it is alarming to know that parents and caregivers are oblivious to the prevention of, maintenance of, and treatment of oral hygiene and dental care of children. A government-proposed system, or one endorsed by paediatric dentistry associations, must be implemented with a military mindset. Without it, the paediatric dental workforce will be overburdened with the responsibility of treating these kids to help them maintain their dental and oral well-being in another ten or so years, when refined sugars become more and more prevalent in their diets. This will prevent them from making room for a healthy general well-being. Pediatric dentistry is a specialty of great importance that is largely neglected by the public with the mind that offsets the importance of primary dentition. This large-scale negligence and lack of knowledge among the public and even some specialty professionals definitely need attention and awareness. It, therefore, becomes our duty as pediatric dental professionals to inform and educate and help the concerned create large-scale know-how of what the profession is, what it stands for, what it can do to the children and adolescents of this nation, and what impact good orodental health can have on the country's overall growth. [ABSTRACT FROM AUTHOR]

Pediatric dentists' role in radiographic exposure of children plays a vital role in their normal growth and development. Radiographs act as a diagnostic tool in identifying the physical condition of the patients. With the technological advancements, there has been a reduction of radiation exposure to patients and clinicians. However, the use of dental radiography should be carefully managed since it can cause some side effects toward normal cells and tissues especially in growing child. A self-administered questionnaire consisting of around twenty questions with both "yes," "no," and multiple-choice pattern will be prepared to obtain information about knowledge, attitude, and practice on radiation exposure protection from pediatric dentists. Dental radiography is a must have tool in our practice, but the judicious use of it is recommended and to be self-mandated. In this study, we will be assessing the knowledge, awareness, and practice of radiography among pediatric dentists which will be analyzed and inferred and will serve as a wake-up call to our professionals to safeguard the future generation. [ABSTRACT FROM AUTHOR]

Background: The periodontal diseases comprise of a group of diseases involving gingiva, periodontal ligament, cementum, and alveolar bone. Although multifactorial, the main etiological factor in causing periodontitis is the presence of microorganisms. Animal model studies have shown a positive significant correlation between the presence of dental plaque and occurrence of periodontal pathologies. Hence, we aim to isolate and evaluate the microbial flora in patients with chronic generalize periodontitis. Materials and Methods: A total of 100 patients reporting with the problem of chronic periodontitis were included in the study. Samples were collected twice. Initially, before the commencement of any treatment and second, following scaling and root planning treatment along with antibiotic therapy after 1 week. Specimens were sent to the microbiology laboratory for evaluation and assessment. McConkey agar, 5% sheep blood agar and agar plates containing hematin and Vitamin K were used for assessment of growth of colonies of microorganisms. Results: Average age of the patients was between 45 and 65 years. 76 of the isolates from the patients were polymicrobial in nature. 34 polymicrobial specimens were combination of 2 isolates while 42 samples were a combination of 3 or more isolates. 89% of the total isolates were strictly anaerobes while remaining were aerobic in nature. Fusobacterium species were the most common among the anaerobes. Conclusion: Periodontitis patients comprises of a wide morphological diverse microbial flora which should be considered while planning treatment. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to determine whether interleukin-1 receptor antagonist and/or interleukin-1beta gene polymorphisms influence multifetal pregnancy outcome.Study Design: Maternal and neonatal buccal swabs from 51 multifetal gestations were analyzed for interleukin-1 receptor antagonist and interleukin-1beta alleles. Outcome data were obtained subsequently.Results: Fetal carriage of interleukin-1 receptor antagonist allele 1 was more than twice as prevalent as the carriage of allele 2. Preterm premature rupture of membranes was observed in 12 of 24 pregnancies (50.0%) in which 2 fetuses tested positive for interleukin-1 receptor antagonist allele 2, as opposed to only 3 of 27 pregnancies (11.1%) in which 1 or neither fetus tested positive for interleukin-1 receptor antagonist allele 2 (P=.005). Similarly, 20 of 26 neonates (76.9%) with documented morbidity tested positive for interleukin-1 receptor antagonist allele 2, as compared with 36 of 78 neonates (46.2%) without morbidity (P=.007). Fetal or maternal interleukin-1beta polymorphisms or maternal interleukin-1 receptor antagonist polymorphisms were unrelated to pregnancy outcome.Conclusion: Fetal carriage of interleukin-1 receptor antagonist allele 2 was associated with both preterm premature rupture of membranes and neonatal morbidity in women with multifetal pregnancies. [ABSTRACT FROM AUTHOR]

Background: Lymph node metastasis is a critical prognostic factor for patients with gastric carcinoma (GC). Sentinel lymph node (SLN) mapping has the potential to identify the initial site of draining lymph node metastasis and reduce the extent of surgical lymphadenectomy. This study aimed to evaluate the diagnostic accuracy of SLN mapping in GC., Methods: The study enrolled 129 GC patients undergoing total or partial gastrectomy with D2 lymphadenectomy and indocyanine green fluorescence-guided SLN mapping. The primary outcomes were the negative predictive value (NPV) and sensitivity of SLN mapping. The secondary outcomes were clinicopathologic factors associated with SLN mapping accuracy and successful SLN mapping., Results: The SLN detection rate in this study was 86.8 %. The study had an overall NPV of 83.1 % and an overall sensitivity of 65.8 %. The NPV was found to be significantly higher in the patients with no lymphovascular invasion (LVI) than in those with LVI (96.0 % vs 59.3 %; p < 0.001) and in the patients whose pathologic T (pT) stage lower than 3 than in those whose T stage was 3 or higher (92.0 % vs 66.7 %; p = 0.009). The sensitivity of SLN mapping was 50 % in the patients with no LVI and 33 % in the patients with a pT stage lower than 3., Conclusion: The study results showed that for patients with early-stage GC with no LVI, negative SLN findings may represent a potential additive predictor indicating the absence of regional LN metastasis. However, given the low sensitivity rates noted, further research is needed to identify specific patient populations that may benefit from SLN mapping in GC., (© 2024. Society of Surgical Oncology.)

Background: The incidence of diffuse-type gastric cancer is increasing steadily in the United States, Europe, and Asia. This subtype is known for aggressive clinical characteristics and transmural invasion. However, T1a diffuse-type cancers have been observed to have a better 5-year, disease-specific mortality than stage-matched intestinal tumors, supporting a clinical difference in these early-stage cancers., Methods: Data on all living patients with T1a gastric adenocarcinoma with a finding of signet ring cell morphology on pathology and ≥1 year of follow-up from 2013 to 2023 at Memorial Sloan Kettering Cancer Center (MSK) was collected from a prospectively maintained database. Patients with known CDH1 or CTNNA1 mutations were excluded., Results: In 7 of 30 patients, sporadic pathologically confirmed T1a signet ring cell (diffuse) cancer identified on initial biopsy was no longer detectable upon subsequent biopsy or resection with mean follow-up of 50 months., Conclusions: These cases allude to the distinct pathways of carcinogenesis in T1a signet ring cell cancers. Potential factors that may underlie the spontaneous regression of these T1a cancers include complete removal at initial biopsy, immune clearance, and lack of survival advantage conferred by signet ring cell genetic alterations in these cases. Given their more indolent behavior at an earlier stage, we suggest that these lesions can be closely followed by endoscopy in select circumstances with thorough disease assessment and an experienced care team., (© 2024. Society of Surgical Oncology.)

Abstract: Although significant progress has been made in understanding the genetic basis of primary hemophagocytic lymphohistiocytosis (HLH), the pathogenesis of secondary HLH, the more prevalent form, remains unclear. Among the various conditions giving rise to secondary HLH, HLH in patients with lymphoma (HLH-L) accounts for a substantial proportion. In this study, we investigated the role of somatic mutations in the pathogenesis of HLH-L in a cohort of patients with T- and/or natural killer-cell lymphoma. We identified a 3-time higher frequency of mutations in FAS pathway in patients with HLH-L. Patients harboring these mutations had a 5-time increased HLH-L risk. These mutations were independently associated with inferior outcome. Hence, our study demonstrates the association between somatic mutations in FAS pathway and HLH-L. Further studies are warranted on the mechanistic role of these mutations in HLH-L., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Objective: Here, we characterize differences in the genetic and microbial profiles of GC in patients of African (AFR), European, and Asian ancestry., Background: Gastric cancer (GC) is a heterogeneous disease with clinicopathologic variations due to a complex interplay of environmental and biological factors, which may affect disparities in oncologic outcomes.., Methods: We identified 1042 patients with GC with next-generation sequencing data from an institutional Integrated Mutation Profiling of Actionable Cancer Targets assay and the Cancer Genomic Atlas group. Genetic ancestry was inferred from markers captured by the Integrated Mutation Profiling of Actionable Cancer Targets and the Cancer Genomic Atlas whole exome sequencing panels. Tumor microbial profiles were inferred from sequencing data using a validated microbiome bioinformatics pipeline. Genomic alterations and microbial profiles were compared among patients with GC of different ancestries., Results: We assessed 8023 genomic alterations. The most frequently altered genes were TP53 , ARID1A , KRAS , ERBB2 , and CDH1 . Patients of AFR ancestry had a significantly higher rate of CCNE1 alterations and a lower rate of KRAS alterations ( P < 0.05), and patients of East Asian ancestry had a significantly lower rate of PI3K pathway alterations ( P < 0.05) compared with other ancestries. Microbial diversity and enrichment did not differ significantly across ancestry groups ( P > 0.05)., Conclusions: Distinct patterns of genomic alterations and variations in microbial profiles were identified in patients with GC of AFR, European, and Asian ancestry. Our findings of variation in the prevalence of clinically actionable tumor alterations among ancestry groups suggest that precision medicine can mitigate oncologic disparities., Competing Interests: C.M.V. is an uncompensated consultant and shareholder in Paige AI. S.V. is an advisor for Immunai and has been a consultant for Koch Disruptive Technologies. M.F. Berger has provided services to AstraZeneca, Eli Lilly and Company, and PetDx, Inc. M.F. B. has ownership in Kazia Therapeutics, Ltd. and a fiduciary role in the de Beaumont Foundation. N.D.S. has provided services to Cambridge Innovation Institute, Harvard T.H. Chan School of Public Health, Innovation in Cancer Informatics, and Seoul National University. V.E.S. has received speaking honoraria from Merck Pharmaceuticals. The remaining authors report no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: We sought to define criteria associated with low lymph node metastasis risk in patients with submucosal (pT1b) gastric cancer from 3 Western and 3 Eastern countries., Summary Background Data: Accurate prediction of lymph node metastasis risk is essential when determining the need for gastrectomy with lymph node dissection following endoscopic resection. Under present guidelines, endoscopic resection is considered definitive treatment if submucosal invasion is only superficial, but this is not routinely assessed., Methods: Lymph node metastasis rates were determined for patient groups defined according to tumor pathological characteristics. Clinicopathological predictors of lymph node metastasis were determined by multivariable logistic regression and used to develop a nomogram in a randomly selected subset that was validated in the remainder. Overall survival was compared between Eastern and Western countries., Results: Lymph node metastasis was found in 701 of 3166 (22.1%) Eastern and 153 of 560 (27.3%) Western patients. Independent predictors of lymph node metastasis were female sex, tumor size, distal stomach location, lymphovascular invasion, and moderate or poor differentiation. Patients fulfilling the National Comprehensive Cancer Network guideline criteria, excluding the requirement that invasion not extend beyond the superficial submucosa, had a lymph node metastasis rate of 8.9% (53/594). Excluding moderately differentiated tumors lowered the rate to 3.4% (10/296). The nomogram's area under the curve was 0.690. Regardless of lymph node status, overall survival was better in Eastern patients., Conclusions: The lymph node metastasis rate was lowest in patients with well differentiated tumors that were ≤3 cm and lacked lymphovascular invasion. These criteria may be useful in decisions regarding endoscopic resection as definitive treatment for pT1b gastric cancer., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Immunocompromised individuals and particularly those with hematologic malignancies are at increased risk for SARS-CoV-2-associated morbidity and mortality due to immunologic deficits that limit prevention, treatment, and clearance of the virus. Understanding the natural history of viral infections in people with impaired immunity due to underlying conditions, immunosuppressive therapy, or a combination thereof has emerged as a critical area of investigation during the COVID-19 pandemic. Studies focused on these individuals have provided key insights into aspects of innate and adaptive immunity underlying both the antiviral immune response and excess inflammation in the setting of COVID-19. This review presents what is known about distinct states of immunologic vulnerability to SARS-CoV-2 and how this information can be harnessed to improve prevention and treatment strategies for immunologically high-risk populations., Competing Interests: Declaration of interests M.-A.P. reports honoraria from Abbvie, Astellas, Bristol-Myers Squibb, Celgene, Equilium, Incyte, Karyopharm, Kite/Gilead, Merck, Miltenyi Biotec, MorphoSys, Novartis, Nektar Therapeutics, Omeros, OrcaBio, Takeda, and VectivBio AG, Vor Biopharma. He serves on DSMBs for Cidara Therapeutics, Medigene, Sellas Life Sciences, and Servier, and the scientific advisory board of NexImmune. He has ownership interests in NexImmune and Omeros. He has received research support for clinical trials from Incyte, Kite/Gilead, Miltenyi Biotec, and Novartis. He serves in a volunteer capacity as a member of the Board of Directors of the American Society for Transplantation and Cellular Therapy (ASTCT) and Be The Match (National Marrow Donor Program, NMDP), as well as on the CIBMTR Cellular Immunotherapy Data Resource (CIDR) Executive Committee. M.V.D.B. has received research support and stock options from Seres Therapeutics and stock options from Notch Therapeutics and Pluto Therapeutics; he is a Scientific Co-founder and consultant for Thymofox, Inc; he has received royalties from Wolters Kluwer; has consulted, received honorarium from, or participated in advisory boards for Seres Therapeutics, WindMIL Therapeutics, Rheos Medicines, Merck & Co, Inc., Magenta Therapeutics, Frazier Healthcare Partners, Nektar Therapeutics, Notch Therapeutics, Forty Seven Inc., Ceramedix, Lygenesis, Pluto Therapeutics, GlaxoSmithKline, Da Volterra, Vor Biopharma, Novartis (Spouse), Synthekine (Spouse), and Beigene (Spouse); he has IP Licensing with Seres Therapeutics and Juno Therapeutics and holds a fiduciary role on the Foundation Board of DKMS (a nonprofit organization). S.V. is an advisor for Immunai and has provided consulting services for Koch Disruptive Technologies., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: The microbiome is hypothesized to have a significant impact on cancer development. In gastric cancer (GC), Helicobacter pylori is an established class I carcinogen. However, additional organisms in the intratumoral microbiome play an important role in GC pathogenesis and progression. In this study, we characterize the full spectrum of the microbes present within GC and identify distinctions among molecular subtypes., Methods: A microbiome bioinformatics pipeline that is generalizable across multiple next-generation sequencing platforms was developed. Microbial profiles for alpha diversity and enrichment were generated for 2 large, demographically distinct cohorts: (1) internal Memorial Sloan Kettering Cancer Center (MSKCC) and (2) The Cancer Genome Atlas (TCGA) cohorts. A total of 520 GC samples were compared with select tumor-adjacent nonmalignant samples. Microbiome differences among the GC molecular subtypes were identified., Results: Compared with nonmalignant samples, GC had significantly decreased microbial diversity in both MSKCC and TCGA cohorts ( P <0.05). Helicobacter , Lactobacillus , Streptococcus , Prevotella , and Bacteroides were significantly more enriched in GC samples when compared with nonmalignant tissue ( P <0.05). Microsatellite instability-high GC had distinct microbial enrichment compared with other GC molecular subtypes., Conclusion: Distinct patterns of microbial diversity and species enrichment were identified in patients with GC. Given the varied spectrum of disease progression and treatment response of GC, understanding unique microbial signatures will provide the landscape to explore key microbial targets for therapy., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Durable T cell responses to SARS-CoV-2 antigens after infection or vaccination improve immune-mediated viral clearance. To date, population-based surveys of COVID-19 adaptive immunity have focused on testing for IgG antibodies that bind spike protein and/or neutralize the virus. Deployment of existing methods for measuring T cell immunity could provide a more complete profile of immune status, informing public health policies and interventions.

'Popeye, the Sailor' cartoons taught children that eating spinach boosts strength and helps defend against bullies. Lötscher and colleagues report that dietary deficiency of magnesium ions (Mg 2+ ), against which eating spinach is an excellent antidote, impairs the activity of a key adhesion molecule, LFA-1, and hinders the ability of CD8 + T cells to grapple with assorted bullies, such as tumors and bacteria., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Signaling through JAK1 and/or JAK2 is common among tumor and nontumor cells within peripheral T-cell lymphoma (PTCL). No oral therapies are approved for PTCL, and better treatments for relapsed/refractory disease are urgently needed. We conducted a phase 2 study of the JAK1/2 inhibitor ruxolitinib for patients with relapsed/refractory PTCL (n = 45) or mycosis fungoides (MF) (n = 7). Patients enrolled onto 1 of 3 biomarker-defined cohorts: (1) activating JAK and/or STAT mutations, (2) ≥30% pSTAT3 expression among tumor cells by immunohistochemistry, or (3) neither or insufficient tissue to assess. Patients received ruxolitinib 20 mg PO twice daily until progression and were assessed for response after cycles 2 and 5 and every 3 cycles thereafter. The primary endpoint was clinical benefit rate (CBR), defined as the combination of complete response, partial response (PR), and stable disease lasting at least 6 months. Only 1 of 7 patients with MF had CBR (ongoing PR > 18 months). CBR among the PTCL cases (n = 45) in cohorts 1, 2, and 3 were 53%, 45%, and 13% (cohorts 1 & 2 vs 3, P = .02), respectively. Eight patients had CBR > 12 months (5 ongoing), including 4 of 5 patients with T-cell large granular lymphocytic leukemia. In an exploratory analysis using multiplex immunofluorescence, expression of phosphorylated S6, a marker of PI3 kinase or mitogen-activated protein kinase activation, in <25% of tumor cells was associated with response to ruxolitinib (P = .05). Our findings indicate that ruxolitinib is active across various PTCL subtypes and support a precision therapy approach to JAK/STAT inhibition in patients with PTCL. This trial was registered at www.clincialtrials.gov as #NCT02974647., (© 2021 by The American Society of Hematology.)

Purpose: We conducted a phase II study evaluating pembrolizumab plus gemcitabine, vinorelbine, and liposomal doxorubicin (pembro-GVD) as second-line therapy for relapsed or refractory (rel/ref) classical Hodgkin lymphoma (cHL) (ClinicalTrials.gov identifier: NCT03618550)., Methods: Transplant eligible patients with rel/ref cHL following first-line therapy were treated with two to four cycles of pembrolizumab (200 mg intravenous [IV], day 1), gemcitabine (1,000 mg/m 2 IV, days 1 and 8), vinorelbine (20 mg/m 2 IV, days 1 and 8), and liposomal doxorubicin (15 mg/m 2 , days 1 and 8), given on 21-day cycles. The primary end point was complete response (CR) following up to four cycles of pembro-GVD. Patients who achieved CR by labeled fluorodeoxyglucose-positron emission tomography (Deauville ≤ 3) after two or four cycles proceeded to high-dose therapy and autologous hematopoietic cell transplantation (HDT/AHCT). HDT/AHCT was carried out according to institutional standards, and brentuximab vedotin maintenance was allowed following HDT/AHCT., Results: Of 39 patients enrolled, 41% had primary ref disease and 38% relapsed within 1 year of frontline treatment. 31 patients received two cycles of pembro-GVD, and eight received four cycles. Most adverse events were grade 1 or two, whereas few were grade 3 and included transaminitis (n = 4), neutropenia (n = 4), mucositis (n = 2), thyroiditis (n = 1), and rash (n = 1). Of 38 evaluable patients, overall and CR rates after pembro-GVD were 100% and 95%, respectively. Thirty-six (95%) patients proceeded to HDT/AHCT, two received pre-HDT/AHCT involved site radiation, and 13 (33%) received post-HDT/AHCT brentuximab vedotin maintenance. All 36 transplanted patients are in remission at a median post-transplant follow-up of 13.5 months (range: 2.66-27.06 months)., Conclusion: Second-line therapy with pembro-GVD is a highly effective and well-tolerated regimen that can efficiently bridge patients with rel/ref cHL to HDT/AHCT., Competing Interests: Alison J. MoskowitzHonoraria: Seattle GeneticsConsulting or Advisory Role: Seattle Genetics, Takeda, Imbrium Therapeutics, Merck, Janpix, Kyowa Kirin International, miRagen, ADC Therapeutics, Bristol Myers SquibbResearch Funding: Incyte, Seattle Genetics, Merck, Bristol Myers Squibb, miRagen, ADC Therapeutics, BeiGene Gunjan ShahResearch Funding: Janssen, Amgen Anita KumarStock and Other Ownership Interests: BridgebioConsulting or Advisory Role: Celgene, Kite, a Gilead company, AstraZeneca/MedImmuneResearch Funding: AbbVie/Genentech, Adaptive Biotechnologies, Celgene, Seattle Genetics, AstraZeneca/MedImmune, Pharmacyclics Oscar LahoudConsulting or Advisory Role: MorphoSysTravel, Accommodations, Expenses: MorphoSysOpen Payments Link: https://openpaymentsdata.cms.gov/physician/225358/summary Connie L. BatleviStock and Other Ownership Interests: Moderna Therapeutics, Novavax, Pfizer, Bristol Myers Squibb, Regeneron, ViatrisHonoraria: DAVAOncologyConsulting or Advisory Role: Lifesci Capital, GLG, Juno Therapeutics, Celgene, Seattle Genetics, Kite, a Gilead company, TG Therapeutics, Karyopharm TherapeuticsResearch Funding: Janssen Biotech, Novartis, Epizyme, Xynomic Pharma, Bayer, Roche, AutolusOpen Payments Link: https://openpaymentsdata.cms.gov/physician/2778694 Paul HamlinConsulting or Advisory Role: Juno Therapeutics, Karyopharm Therapeutics, Celgene, Sandoz, AstraZeneca/MerckResearch Funding: Spectrum Pharmaceuticals, Seattle Genetics, Janssen, Portola Pharmaceuticals, GlaxoSmithKline, Molecular Templates, Incyte David J. StrausConsulting or Advisory Role: Takeda, Seagen, EpizymeResearch Funding: Takeda Ildefonso Rodriguez-RiveraStock and Other Ownership Interests: Pfizer Philip CaronStock and Other Ownership Interests: AstraZeneca, Bristol Myers Squibb, GlaxoSmithKline, Johnson & Johnson/Janssen, Novartis, Pfizer, Teva Steven M. HorwitzConsulting or Advisory Role: Celgene, Millennium, Kyowa Hakko Kirin, Seattle Genetics, ADC Therapeutics, Verastem, Takeda, Astex Pharmaceuticals, Kura Oncology, Acrotech Biopharma, C4 Therapeutics, Janssen Oncology, Trillium Therapeutics, Vividion Therapeutics, Myeloid Therapeutics, Ono Pharmaceutical, Secura Bio, Shoreline Biosciences Inc, TubulisResearch Funding: Celgene, Seattle Genetics, Takeda, Kyowa Hakko Kirin, Aileron Therapeutics, ADC Therapeutics, Verastem, 47, Trillium Therapeutics, Daiichi Sankyo, Affimed Therapeutics, Secura Bio Lorenzo FalchiConsulting or Advisory Role: GenmabResearch Funding: Roche, Genmab Erel JoffeConsulting or Advisory Role: AstraZeneca, Epizyme Christina LeeHonoraria: WebMDConsulting or Advisory Role: Intellisphere LLC M. Lia PalombaStock and Other Ownership Interests: Seres TherapeuticsHonoraria: Flagship Biosciences, Evelo Therapeutics, Jazz Pharmaceuticals, Therakos, Amgen, Merck, Seres TherapeuticsConsulting or Advisory Role: Flagship Biosciences, Novartis, Evelo Therapeutics, Jazz Pharmaceuticals, Therakos, Amgen, Merck, Seres Therapeutics, Kite, a Gilead company, Novartis, BeiGeneResearch Funding: Seres TherapeuticsPatents, Royalties, Other Intellectual Property: Intellectual Property Rights, Juno intellectual property rights Ariela NoyConsulting or Advisory Role: MorphoSys, Janssen Biotech, Prime OncologyResearch Funding: Pharmacyclics, Rafael PharmaceuticalsTravel, Accommodations, Expenses: Pharmacyclics, Janssen Oncology Matthew J. MatasarStock and Other Ownership Interests: MerckHonoraria: Genentech, Roche, Bayer, Pharmacyclics, Seattle Genetics, Takeda, Immunovaccine, ADC Therapeutics, Karyopharm TherapeuticsConsulting or Advisory Role: Genentech, Bayer, Merck, Juno Therapeutics, Roche, Teva, Rocket Medical, Seattle Genetics, Daiichi Sankyo, TakedaResearch Funding: Genentech, Roche, GlaxoSmithKline, Bayer, Pharmacyclics, Janssen, Rocket Medical, Seattle Genetics, Immunovaccine, IGM BiosciencesTravel, Accommodations, Expenses: Genentech, Roche, Seattle Genetics, Bayer Georgios PongasHonoraria: Atara Biotherapeutics, Curio Science, OncLive/MJH Life SciencesTravel, Accommodations, Expenses: Atara Biotherapeutics Gilles SallesHonoraria: Roche/Genentech, Janssen, Celgene, Gilead Sciences, Novartis, AbbVie, MorphoSysConsulting or Advisory Role: Roche/Genentech, Gilead Sciences, Janssen, Celgene, Novartis, MorphoSys, Epizyme, Alimera Sciences, Genmab, Debiopharm Group, Velosbio, BMS, BeiGene, Incyte, Miltenyi Biotec, Ipsen Santosha VardhanaHonoraria: AgiosConsulting or Advisory Role: Immunai, ADC Therapeutics Gottfried von KeudelllHonoraria: PharmacyclicsConsulting or Advisory Role: Pharmacyclics, Merck, Incyte Ahmet DoganConsulting or Advisory Role: Seattle Genetics, Takeda, EUSA Pharma, AbbVie, Peerview, Physicans' Education ResourceResearch Funding: Roche/Genentech Andrew D. ZelenetzHonoraria: NCCN, Clinical Care Options, Oncology Information Group, PER, PlexusConsulting or Advisory Role: Gilead Sciences, Amgen, Genentech/Roche, Celgene, AstraZeneca, DAVAOncology, MorphoSys, BeiGene, MEI Pharma, Vaniam Group, Verastem, Pharmacyclics, Karyopharm Therapeutics, Debiopharm Group, Seattle Genetics, Quant Health Ltd, Kite, a Gilead company, Curis, Coherus Biosciences, Juno/Celgene/Bristol Myers Squibb, Curio ScienceResearch Funding: Genentech/Roche, Gilead Sciences, MEI Pharma, BeiGene Craig H. MoskowitzConsulting or Advisory Role: Merck Sharp & Dohme, Molecular Templates, Takeda, AstraZeneca, IncyteResearch Funding: AstraZeneca, Merck Sharp & DohmeNo other potential conflicts of interest were reported.

Language and cultural barriers can impede communication between patients and clinicians, exacerbating health inequity. Additional complications can arise when family members, intending to protect their loved ones, ask clinicians to lie or not disclose to patients their diagnoses, prognoses, or intervention options. Clinicians must express respect for patients' and families' cultural, religious, and social norms regarding health care decision making, but they might also be ethically troubled by some decisions' effects on patients' health outcomes. This article suggests strategies for clinicians trying to overcome linguistic and cultural barriers to equitable patient care., (© 2021 American Medical Association. All Rights Reserved.)

BACKGROUNDUnderstanding outcomes and immunologic characteristics of cellular therapy recipients with SARS-CoV-2 is critical to performing these potentially life-saving therapies in the COVID-19 era. In this study of recipients of allogeneic (Allo) and autologous (Auto) hematopoietic cell transplant and CD19-directed chimeric antigen receptor T cell (CAR T) therapy at Memorial Sloan Kettering Cancer Center, we aimed to identify clinical variables associated with COVID-19 severity and assess lymphocyte populations.METHODSWe retrospectively investigated patients diagnosed between March 15, 2020, and May 7, 2020. In a subset of patients, lymphocyte immunophenotyping, quantitative real-time PCR from nasopharyngeal swabs, and SARS-CoV-2 antibody status were available.RESULTSWe identified 77 patients with SARS-CoV-2 who were recipients of cellular therapy (Allo, 35; Auto, 37; CAR T, 5; median time from cellular therapy, 782 days; IQR, 354-1611 days). Overall survival at 30 days was 78%. Clinical variables significantly associated with the composite endpoint of nonrebreather or higher oxygen requirement and death (n events = 25 of 77) included number of comorbidities (HR 5.41, P = 0.004), infiltrates (HR 3.08, P = 0.032), and neutropenia (HR 1.15, P = 0.04). Worsening graft-versus-host disease was not identified among Allo recipients. Immune profiling revealed reductions and rapid recovery in lymphocyte populations across lymphocyte subsets. Antibody responses were seen in a subset of patients.CONCLUSIONIn this series of Allo, Auto, and CAR T recipients, we report overall favorable clinical outcomes for patients with COVID-19 without active malignancy and provide preliminary insights into the lymphocyte populations that are key for the antiviral response and immune reconstitution.FUNDINGNIH grant P01 CA23766 and NIH/National Cancer Institute grant P30 CA008748.

Tumor cells have devised unique metabolic strategies to garner enough nutrients to sustain continuous growth and cell division. Oncogenic mutations may alter metabolic pathways to unlock new sources of energy, and cells take the advantage of various scavenging pathways to ingest material from their environment. These changes in metabolism result in a metabolic profile that, in addition to providing the building blocks for macromolecules, can also influence cell signaling pathways to promote tumor initiation and progression. Understanding what pathways tumor cells use to synthesize the materials necessary to support metabolic growth can pave the way for new cancer therapeutics. Potential strategies include depriving tumors of the materials needed to grow or targeting pathways involved in dependencies that arise by virtue of their altered metabolis., (© 2019 New York Academy of Sciences.)

The programmed death-1 (PD-1) receptor checkpoint inhibitors nivolumab and pembrolizumab represent an important therapeutic advance in the treatment of relapsed or refractory classical Hodgkin lymphoma (cHL). Clinical trials have shown substantial therapeutic activity and an acceptable safety profile in heavily pretreated patients, resulting in U.S. Food and Drug Administration approval of nivolumab for the treatment of cHL that has relapsed or progressed after either autologous hematopoietic cell transplantation (auto-HCT) and brentuximab vedotin treatment or three or more lines of systemic therapy (including auto-HCT), and of pembrolizumab for adult or pediatric patients with refractory cHL or cHL that has relapsed after three or more prior therapies. Mechanistically, anti-PD-1 therapy prevents inhibitory signaling through PD-1 receptors on T cells, thereby releasing a 'block' to antitumor T-cell responses. However, this disinhibition can also lead to inappropriate T-cell activation and responses against healthy tissues, resulting in immune-mediated adverse events (IMAEs) that affect a number of organ systems. The skin, gastrointestinal, hepatic, and endocrine systems are most commonly involved, typically resulting in rash, colitis, abnormal liver enzyme levels, and thyroiditis, respectively. Notably, pneumonitis is a potentially fatal complication of checkpoint inhibitor immunotherapy. Hematologic oncologists who treat cHL with PD-1 immune checkpoint inhibitors should monitor patients for IMAEs, as early recognition and treatment can rapidly reduce morbidity and mortality. This review focuses on IMAEs during the treatment of relapsed or refractory cHL with nivolumab and pembrolizumab. IMPLICATIONS FOR PRACTICE: This article highlights the importance of monitoring for immune-mediated adverse events (IMAEs) in patients with Hodgkin lymphoma (HL) who receive anti-programmed death-1 (anti-PD-1) therapy, with particular attention given to the recognition and management of such events. The risk of individual IMAEs differs between patients with HL and those with solid tumors, as prior treatments may predispose certain organ systems to specific IMAEs. Accurate and prompt diagnosis of IMAEs is essential for optimal management, allowing PD-1 inhibitor therapy to be restarted in order to maintain disease control. Potential difficulties, such as distinguishing disease progression from pneumonitis, or colitis from diarrhea, are highlighted to raise clinical awareness., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2018.)

Primary mediastinal (thymic) large B cell lymphoma is a subtype of diffuse large B cell lymphoma with distinct clinical, molecular, and genetic features, many of which overlap with Hodgkin lymphoma. Increasingly, initial therapy for these patients has used dose-dense chemotherapy with or without radiation with excellent results. In patients with relapsed and primary refractory disease, outcomes of second-line therapy followed by consolidation with high-dose therapy and autologous stem cell transplantation remains largely undefined. We reviewed the outcomes of 60 transplant-eligible patients with relapsed or refractory primary mediastinal (thymic) large B cell lymphoma enrolled on sequential protocols with uniform second-line therapy with intent to consolidate with autologous stem cell transplant. The estimated 3-year overall and event-free survivals for all patients were 61% and 57%, respectively, and 68% and 65%, respectively, for patients proceeding to stem cell transplant. Multivariable analysis of risk factors before transplant revealed that an incomplete response to initial therapy, advanced Ann Arbor stage at disease progression, and failure to achieve a partial remission or better to second-line therapy to be independently associated with inferior event-free and overall survival. A risk score based on these variables was able to identify patients who are unlikely to respond to conventional second-line strategies. These results suggest that salvage chemoradiotherapy with intent of subsequent high-dose therapy and autologous stem cell transplant is successful in most patients with relapsed and refractory primary mediastinal (thymic) large B cell lymphoma. Alternative strategies are warranted for a significant subset of patients with high-risk disease who are unlikely to be cured with this strategy., (Copyright © 2018. Published by Elsevier Inc.)

Ibrutinib has previously been shown to inhibit Bruton's tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK), which mediate B-cell and T-cell receptor signaling, respectively. BTK inhibition with ibrutinib has demonstrated impressive clinical responses in a variety of B-cell malignancies. Whether ibrutinib inhibition of ITK can lead to clinical response in T-cell malignancies is unknown. We hypothesized that ibrutinib-mediated ITK inhibition in T-cell lymphoma would result in decreased signaling through the T-cell receptor pathway and promote antitumor immune response by driving selective cytotoxic Th1 CD4 effector T-cell differentiation. This pilot clinical trial evaluated 2 dose levels of ibrutinib: 560 and 840 mg orally daily. Fourteen patients with relapsed, refractory peripheral T-cell lymphoma and cutaneous T-cell lymphoma were enrolled. Both dose levels were safe and well tolerated, and no dose-limiting toxicities were observed. One patient achieved a partial response (overall response rate, 8% [1/13]). ITK occupancy studies demonstrated a mean occupancy of 50% (range, 15%-80%). Higher ITK occupancy of more than 50% correlated with higher serum levels of tumor necrosis factor-α and interferon-γ and favored a Th1 phenotype. Our data suggest that ibrutinib inhibition of ITK has limited clinical activity in T-cell lymphoma. This study is registered at www.clinicaltrials.gov as #NCT02309580., (© 2018 by The American Society of Hematology.)

Few studies have evaluated the role of antibacterial prophylaxis during neutropenia in patients with multiple myeloma undergoing autologous hematopoietic stem cell transplantation (HSCT). At our center, levofloxacin prophylaxis was initiated in June 2006 in patients with myeloma who were undergoing autologous HSCT. We compared the incidence of bloodstream infection (BSI) and fever and neutropenia (FN) within 30 days of transplantation before (January 2003 to May 2006) and after (June 2006 to April 2010) the initiation of levofloxacin prophylaxis in patients undergoing autologous HSCT for myeloma. We also compared rates of BSI and FN during the same time periods in autologous HSCT recipients with lymphoma who did not receive antibacterial prophylaxis during either time period. After the initiation of levofloxacin prophylaxis, the BSI rate decreased from 41.2% (49 of 119) to 14.7% (23 of 156) and the rate of FN decreased from 91.6% to 60.9% in patients with myeloma (P < .001, for each). In contrast, rates of BSI (43.1% versus 47.3%; P = .50) and FN (98.8% versus 97.1%; P = .63) did not change in patients with lymphoma. Levofloxacin prophylaxis was independently associated with decreased odds of BSI (odds ratio, .27; 95% confidence interval, .14 to .51; P < .001) and FN (odds ratio, .18; 95% confidence interval, .09 to .36; P < .001) in multivariate analysis. Patients with myeloma had a nonsignificant increase in the risk of BSI due to levofloxacin-resistant Enterobacteriaceae (5% versus 1%, P = .08) and Clostridium difficile infection (7% versus 3%, P = .12) after the initiation of levofloxacin prophylaxis but did not have higher rates of BSI due to other resistant bacteria. Levofloxacin prophylaxis is associated with decreased risk of BSI and FN in patients with myeloma undergoing autologous HSCT., (Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca(2+)]i) elevation. TCR activation triggers increased [Ca(2+)]i and can arrest T-cell motility in vitro. However, the requirement for [Ca(2+)]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca(2+) release-activated Ca(2+) (CRAC) channel pathway required for [Ca(2+)]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant-negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naïve CD4(+) T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca(2+)]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen-specific ORAI1-DN T cells had a twofold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest., (© 2013 The Authors. European Journal of Immunology published by Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim.)

The role of non-muscle myosin IIA (heavy chain encoded by the non-muscle myosin heavy chain 9 gene, Myh9) in immunological synapse formation is controversial. We have addressed the role of myosin IIA heavy chain protein (MYH9) in mouse T cells responding to MHC-peptide complexes and ICAM-1 in supported planar bilayers - a model for immunological synapse maturation. We found that reduction of MYH9 expression levels using Myh9 siRNA in proliferating mouse CD4(+) AND T cell receptor (TCR) transgenic T cells resulted in increased spreading area, failure to assemble the central and peripheral supramolecular activation clusters (cSMAC and pSMAC), and increased motility. Surprisingly, TCR microcluster speed was reduced marginally, however TCR microclusters dissipated prior to forming a cSMAC. TCR microclusters formed in the Myh9 siRNA-treated T cells showed reduced phosphorylation of the Src family kinase (SFK) activation loop and displayed reduced cytoplasmic calcium ion (Ca(2+)) elevation. In addition, Myh9 siRNA-treated cells displayed reduced phosphorylation of the Cas-L substrate domain - a force-dependent SFK substrate - which was observed in control siRNA-treated cells in foci throughout the immunological synapse except the cSMAC. Cas-L exhibited TCR ligation-dependent induction of phosphorylation. These results provide further evidence that T cell activation is modulated by intrinsic force-generating systems and can be viewed as a mechanically responsive process influenced by MYH9.

Recognition of self-peptide-MHC (pMHC) complexes by CD4 T cells plays an important role in the pathogenesis of many autoimmune diseases. We analyzed formation of immunological synapses (IS) in self-reactive T cell clones from patients with multiple sclerosis and type 1 diabetes. All self-reactive T cells contained a large number of phosphorylated T cell receptor (TCR) microclusters, indicative of active TCR signaling. However, they showed little or no visible pMHC accumulation or transport of TCR-pMHC complexes into a central supramolecular activation cluster (cSMAC). In contrast, influenza-specific T cells accumulated large quantities of pMHC complexes in microclusters and a cSMAC, even when presented with 100-fold lower pMHC densities. The self-reactive T cells also maintained a high degree of motility, again in sharp contrast to virus-specific T cells. 2D affinity measurements of three of these self-reactive T cell clones demonstrated a normal off-rate but a slow on-rate of TCR binding to pMHC. These unusual IS features may facilitate escape from negative selection by self-reactive T cells encountering very small amounts of self-antigen in the thymus. However, these same features may enable acquisition of effector functions by self-reactive T cells encountering large amounts of self-antigen in the target organ of the autoimmune disease.

Stomatin-like protein 2 (SLP-2) is a member of the stomatin-prohibitin-flotillin-HflC/K (SPFH) superfamily. Recent evidence indicates that SLP-2 is involved in the organization of cardiolipin-enriched microdomains in mitochondrial membranes and the regulation of mitochondrial biogenesis and function. In T cells, this role translates into enhanced T cell activation. Although the major pool of SLP-2 is associated with mitochondria, we show here that there is an additional pool of SLP-2 associated with the plasma membrane of T cells. Both plasma membrane-associated and mitochondria-associated pools of SLP-2 coalesce at the immunological synapse (IS) upon T cell activation. SLP-2 is not required for formation of IS nor for the re-localization of mitochondria to the IS because SLP-2-deficient T cells showed normal re-localization of these organelles in response to T cell activation. Interestingly, upon T cell activation, we found the surface pool of SLP-2 mostly excluded from the central supramolecular activation complex, and enriched in the peripheral area of the IS where signalling TCR microclusters are located. Based on these results, we propose that SLP-2 facilitates the compartmentalization not only of mitochondrial membranes but also of the plasma membrane into functional microdomains. In this latter location, SLP-2 may facilitate the optimal assembly of TCR signalosome components. Our data also suggest that there may be a net exchange of membrane material between mitochondria and plasma membrane, explaining the presence of some mitochondrial proteins in the plasma membrane.

Agonist MHC-peptide complexes in the immunological synapse (IS) signal through T cell receptor (TCR) microclusters (MCs) that converge into a central supramolecular activation cluster (cSMAC). The determinants and function of the cSMAC remain unknown. We demonstrate an essential role for ubiquitin (Ub) and TSG101, but less so for HRS, in signal processing events at the cSMAC. Using siRNA in primary T cells, we show that Ub recognition by TSG101 is required for cSMAC formation, TCR MC signal termination, TCR downregulation, and segregation of TCR-MHC-peptide from PKC-theta-enriched signaling complexes. Weak agonist MHC-peptide induced CD80-dependent TCR MCs that dissociated in the center of the IS without recruiting TSG101. These results support TSG101-dependent recognition of CD80-independent TCR MCs as a molecular checkpoint for TCR downregulation., (Copyright 2010 Elsevier Inc. All rights reserved.)

T cell activation and function require a structured engagement of antigen-presenting cells. These cell contacts are characterized by two distinct dynamics in vivo: transient contacts resulting from promigratory junctions called immunological kinapses or prolonged contacts from stable junctions called immunological synapses. Kinapses operate in the steady state to allow referencing to self-peptide-MHC (pMHC) and searching for pathogen-derived pMHC. Synapses are induced by T cell receptor (TCR) interactions with agonist pMHC under specific conditions and correlate with robust immune responses that generate effector and memory T cells. High-resolution imaging has revealed that the synapse is highly coordinated, integrating cell adhesion, TCR recognition of pMHC complexes, and an array of activating and inhibitory ligands to promote or prevent T cell signaling. In this review, we examine the molecular components, geometry, and timing underlying kinapses and synapses. We integrate recent molecular and physiological data to provide a synthesis and suggest ways forward.

The Ca(2+)-activated K(+) channel KCa3.1 is required for Ca(2+) influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2beta (PI3K-C2beta) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2beta by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2beta siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2beta in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca(2+) influx, whereas silencing of PI3K-C2beta inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2beta colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation.

We have investigated the function of endogenous galectin-3 in T cells. Galectin-3-deficient (gal3(-/-)) CD4(+) T cells secreted more IFN-gamma and IL-4 than gal3(+/+)CD4(+) T cells after T-cell receptor (TCR) engagement. Galectin-3 was recruited to the cytoplasmic side of the immunological synapse (IS) in activated T cells. In T cells stimulated on supported lipid bilayers, galectin-3 was primarily located at the peripheral supramolecular activation cluster (pSMAC). Gal3(+/+) T cells formed central SMAC on lipid bilayers less effectively and adhered to antigen-presenting cells less firmly than gal3(-/-) T cells, suggesting that galectin-3 destabilizes the IS. Galectin-3 expression was associated with lower levels of early signaling events and phosphotyrosine signals at the pSMAC. Additional data suggest that galectin-3 potentiates down-regulation of TCR in T cells. By yeast two-hybrid screening, we identified as a galectin-3-binding partner, Alix, which is known to be involved in protein transport and regulation of cell surface expression of certain receptors. Co-immunoprecipitation confirmed galectin-3-Alix association and immunofluorescence analysis demonstrated the translocation of Alix to the IS in activated T cells. We conclude that galectin-3 is an inhibitory regulator of T-cell activation and functions intracellularly by promoting TCR down-regulation, possibly through modulating Alix's function at the IS.

The interaction between a T cell and an antigen-presenting cell (APC) can trigger a signaling response that leads to T cell activation. Prior studies have shown that ligation of the T cell receptor (TCR) triggers a signaling cascade that proceeds through the coalescence of TCR and various signaling molecules (e.g., the kinase Lck and adaptor protein LAT [linker for T cell activation]) into microdomains on the plasma membrane. In this study, we investigated another ligand-receptor interaction (CD58-CD2) that facilities T cell activation using a model system consisting of Jurkat T cells interacting with a planar lipid bilayer that mimics an APC. We show that the binding of CD58 to CD2, in the absence of TCR activation, also induces signaling through the actin-dependent coalescence of signaling molecules (including TCR-zeta chain, Lck, and LAT) into microdomains. When simultaneously activated, TCR and CD2 initially colocalize in small microdomains but then partition into separate zones; this spatial segregation may enable the two receptors to enhance signaling synergistically. Our results show that two structurally distinct receptors both induce a rapid spatial reorganization of molecules in the plasma membrane, suggesting a model for how local increases in the concentration of signaling molecules can trigger T cell signaling.

Immunological synapses are initiated by signaling in discrete T cell antigen receptor microclusters and are important for the differentiation and effector functions of T cells. Synapse formation involves the orchestrated movement of microclusters toward the center of the contact area with the antigen-presenting cell. Microcluster movement is associated with centripetal actin flow, but the function of motor proteins is unknown. Here we show that myosin IIA was necessary for complete assembly and movement of T cell antigen receptor microclusters. In the absence of myosin IIA or its ATPase activity, T cell signaling was interrupted 'downstream' of the kinase Lck and the synapse was destabilized. Thus, T cell antigen receptor signaling and the subsequent formation of immunological synapses are active processes dependent on myosin IIA.

Short-lived TCR microclusters and a longer-lived protein kinase Ctheta-focusing central supramolecular activation cluster (cSMAC) have been defined in model immunological synapses (IS). In different model systems, CD28-mediated costimulatory interactions have been detected in microclusters, the cSMAC, or segregated from the TCR forming multiple distinct foci. The relationship between TCR and costimulatory molecules in the physiological IS of T cell-dendritic cell (DC) is obscure. To study the dynamic relationship of CD28-CD80 and TCR interactions in the T cell-DC IS during Ag-specific T cell activation, we generated CD80-eCFP mice using bacterial artificial chromosome transgenic technology. In splenic DCs, endogenous CD80 and CD80-eCFP localized to plasma membrane and Golgi apparatus, and CD80-eCFP was functional in vivo. In the OT-II T cell-DC IS, multiple segregated TCR, CD80, and LFA-1 clusters were detected. In the T cell-DC synapse CD80 clusters were colocalized with CD28 and PKCtheta, a characteristic of the cSMAC. Acute blockade of TCR signaling with anti-MHC Ab resulted in a rapid reduction in Ca(2+) signaling and the number and size of the CD80 clusters, a characteristic of TCR microclusters. Thus, the T cell-DC interface contains dynamic costimulatory foci that share characteristics of microclusters and cSMACs.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added. Then, bilayers are charged with Ni and his-tagged recombinant proteins are added. Finally, lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting, and sites of receptor degradation.

T cell activation through the Ag receptor (TCR) requires sustained signaling from signalosomes within lipid raft microdomains in the plasma membrane. In a proteomic analysis of lipid rafts from human T cells, we identified stomatin-like protein (SLP)-2 as a candidate molecule involved in T cell activation through the Ag receptor. In this study, we show that SLP-2 expression in human primary lymphocytes is up-regulated following in vivo and ex vivo activation. In activated T cells, SLP-2 interacts with components of TCR signalosomes and with polymerized actin. More importantly, up-regulation of SLP-2 expression in human T cell lines and primary peripheral blood T cells increases effector responses, whereas down-regulation of SLP-2 expression correlates with loss of sustained TCR signaling and decreased T cell activation. Our data suggest that SLP-2 is an important player in T cell activation by ensuring sustained TCR signaling, which is required for full effector T cell differentiation, and point to SLP-2 as a potential target for immunomodulation.

Objective: To investigate the association between a tumor necrosis factor-alpha (TNF-alpha) gene polymorphism, vaginal TNF-alpha level, and microbial flora in pregnant women., Methods: Vaginal samples from 203 women at 18-22 weeks' gestation were analyzed for microflora. TNFA-308G>A polymorphism was analyzed by polymerase chain reaction and restriction endonuclease analysis and TNF-alpha concentration was determined by ELISA. Outcome data were subsequently obtained., Results: The vaginal TNF-alpha concentration was elevated in TNFA-308A carriers only in the presence of abnormal vaginal flora. A median TNF-alpha level of 10.94 pg/ml in TNFA-308A carriers with bacterial vaginosis (BV) was significantly higher than that of 1.77 pg/ml in TNFA-308A carriers without BV (P=.02), and 1.72 pg/ml in TNF-308G homozygotes with BV (P=.01)., Conclusion: The TNFA-308G>A polymorphism influences the local TNF-alpha response to altered vaginal microflora. This suggests that the nature of the host response to microbial invasion in the lower female genital is genetically determined.

CD14, the major receptor for bacterial lipopolysaccharide (LPS) as well as other microbial antigens, is a component of the innate immune system. We hypothesized that a single nucleotide C>T polymorphism at position -159 in the CD14 gene that results in elevated CD14 production would influence susceptibility to preterm premature rupture of membranes (PPROM) and spontaneous preterm birth (SPTB) in multi-fetal pregnancies. DNA from 107 mother-twin and three mother-triplet pairs was analyzed. Pregnancy outcomes were obtained after completion of testing. CD14*T homozygosity was present in 39.3% of 28 women whose pregnancies ended with PPROM, as opposed to 18.1% of 72 pregnancies without a SPTB (P=0.03). There was no relation between the fetal CD14 genotype and PPROM. The likelihood ratio (LR) for PPROM was 2.2 for women homozygous for CD14*T. The LR increased to 3.3 and 3.6 if the CD14 polymorphism was present in combination with previously determined maternal polymorphisms in the genes coding for the inducible 70kDa heat shock protein (hsp70-2) and the interleukin-1 receptor antagonist (IL1RN), respectively. Thus, an enhanced maternal pro-inflammatory immune response to LPS may increase susceptibility to PPROM in multi-fetal pregnancies.

Clara cell protein 16 (CC16) is a major immunomodulatory protein produced in the fetal lung. We hypothesized that the mid-trimester amniotic fluid concentration of CC16 would vary according to a +38 CC16 polymorphism in the fetal genome and that increased levels would be an early indicator of subsequent adverse pregnancy outcome. Mid-trimester singleton amniotic fluids from 244 women were assayed for CC16 by ELISA. DNA from fetal cells in 179 amniotic fluids were tested for the A>G polymorphism at position +38 in exon 1 by PCR. Outcome data were obtained from 233 women after completion of laboratory testing. Median CC16 levels were higher in amniotic fluids containing male fetuses than in those with females (p=0.0005). Median amniotic fluid CC16 levels were higher in Hispanics than in Whites and Asians (p<0.05). CC16*G homozygosity was associated with elevated amniotic fluid CC16 concentrations compared to CC16*A homozygotes (p=0.02). Intraamniotic CC16 levels were highest in pregnancies that subsequently resulted in preterm premature rupture of membranes (PPROM) (p=0.01). We conclude that mid-trimester intraamniotic CC16 concentrations vary by gender, ethnicity and fetal CC16 gene polymorphism. Elevated CC16 levels are predictive of subsequent development of PPROM.

Objective: In spite of extensive clinical examinations or autopsies, as many as 15% to 40% of stillbirths remain unexplained. A systemic fetal inflammatory response is an independent risk factor for severe neonatal morbidity, mediated by proinflammatory cytokines. As a major anti-inflammatory cytokine, interleukin-1 receptor antagonist (IL-1ra) plays a crucial role modulating the proinflammatory response. The gene coding for IL-1ra (IL1RN) is polymorphic. We hypothesized that fetal possession of a specific allele, IL-1RN*2, associated with increased proinflammatory responses, may increase susceptibility to intrauterine fetal death., Study Design: Fetal kidney cells were obtained from paraffin blocks of 27 unexplained stillbirths. DNA was isolated and tested for IL-1RN genotypes by polymerase chain reaction. As a control group, DNA from 302 live births was also tested., Results: There was an enhanced rate of IL-1RN*2 homozygocity, 41%, among unexplained stillbirths compared with the control group, 8.6% (P < .001). Histologic analysis of fetal tissues demonstrated a predominant proinflammatory response in IL-1RN*2 homozygote fetuses. Extensive screening (microbiology, maternal serology, placenta histology) did not identify any specific trigger agent., Conclusion: There is an association between unexplained stillbirth and fetal homozygous IL1RN*2 carriage.