Screening for gene expression fluctuations reveals latency-promoting agents of HIV.

Upon treatment removal, spontaneous reactivation of latently infected T cells remains a major barrier toward curing HIV. Therapies that reactivate and clear the latent reservoir are only partially effective, while latency-promoting agents (LPAs) used to suppress reactivation and stabilize latency are understudied and lack diversity in their mechanisms of action. Here, we identify additional LPAs using a screen for gene-expression fluctuations (or "noise") that drive cell-fate specification and control HIV reactivation from latency. Single-cell protein dynamics of a minimal HIV gene circuit were monitored with time-lapse fluorescence microscopy. We screened 1,806 drugs, out of which 279 modulate noise magnitude or half autocorrelation time. Next, we tested the strongest noise modulators in a Jurkat T cell latency model and discovered three LPAs that would be overlooked by quantifying their mean expression levels alone. The LPAs reduced reactivation of latency in both Jurkat and primary cell models when challenged by synergistic and potent combinations of HIV activators. The two strongest LPAs, NSC 401005 and NSC 400938, are structurally and functionally related to inhibitors of thioredoxin reductase, a protein involved in maintaining redox balance in host cells. Experiments with multiple functional analogs revealed two additional LPAs, PX12 and tiopronin, and suggest a potential LPA family, within which some are commercially available and Food and Drug Administration-approved. The LPAs presented here may provide new strategies to complement antiretroviral treatments. Screening for gene expression noise holds the potential for drug discovery in other diseases.

Approach to the Patient with Unknown Primary Squamous Cell Carcinoma of the Head and Neck.

OPINION STATEMENT: Head and neck cancer of unknown primary (HNCUP) is increasingly encountered in both community otolaryngology practice and the academic head and neck cancer program. A stepwise diagnostic evaluation will identify many primary sites. However, true HNCUP remains common in high-volume practices after appropriate examination, imaging, and biopsies. The prognosis for the majority of the patients is good, owing to the common association with high-risk HPV, and putative oropharyngeal primary origin. With high oncologic control rates, judicious treatment selection is essential to optimize functional outcomes.

Immunotherapy for Head and Neck Squamous Cell Carcinoma.

PURPOSE OF REVIEW: Discussion of current strategies targeting the immune system related to solid tumors with emphasis on head and neck squamous cell carcinoma (HNSCC).This review will outline the current challenges with immunotherapy and future goals for treatment using these agents. RECENT FINDINGS: Agents targeting immune checkpoint receptors (IR) such as program death 1 (PD1) have been used in the clinical realm for melanoma and non-small cell lung cancer (NSCLC), and the use of these agents for these malignancies has provided crucial information about how and why patients respond or not to inhibitory checkpoint receptor blockade therapy (ICR). The anti PD1 agent, nivolumab, was recently approved by the FDA as a standard of care regimen for patients with platinum refractory recurrent/metastatic (R/M) HNSCC. Molecular pathways leading to resistance are starting to be identified, and work is underway to understand the most optimal treatment regimen with incorporation of immunotherapy. ICR has renewed interest in the immunology of cancer, but resistance is not uncommon, and thus understanding of these mechanisms will allow the clinician to appropriately select patients that will benefit from this therapy.

Immunotherapy for Head and Neck Squamous Cell Carcinoma: A Review of Current and Emerging Therapeutic Options.

Advances in the field of cancer immunotherapy have occurred rapidly over the past decade. Exciting results from clinical trials have led to new treatment options and improved survival for patients with a myriad of solid tumor pathologies. However, questions remain unanswered regarding duration and timing of therapy, combination regimens, appropriate biomarkers of disease, and optimal monitoring of therapeutic response. This article reviews emerging immunotherapeutic agents and significant clinical trials that have led to advancements in the field of immuno-oncology for patients with head and neck squamous cell carcinoma. IMPLICATIONS FOR PRACTICE: This review article summarizes recently developed agents that harness the immune system to fight head and neck squamous cell carcinoma. A brief review of the immune system and its role in cancer development is included. Recently completed and emerging therapeutic trials centering on the immune system and head and neck cancer are reviewed.

Processing of vestibular inputs by the medullary lateral tegmental field of conscious cats: implications for generation of motion sickness.

The dorsolateral reticular formation of the caudal medulla, the lateral tegmental field (LTF), participates in generating vomiting. LTF neurons exhibited complex responses to vestibular stimulation in decerebrate cats, indicating that they received converging inputs from a variety of labyrinthine receptors. Such a convergence pattern of vestibular inputs is appropriate for a brain region that participates in generating motion sickness. Since responses of brainstem neurons to vestibular stimulation can differ between decerebrate and conscious animals, the current study examined the effects of whole-body rotations in vertical planes on the activity of LTF neurons in conscious felines. Wobble stimuli, fixed-amplitude tilts, the direction of which moves around the animal at a constant speed, were used to determine the response vector orientation, and also to ascertain whether neurons had spatial-temporal convergence (STC) behavior (which is due to the convergence of vestibular inputs with different spatial and temporal properties). The proportion of LTF neurons with STC behavior in conscious animals (25 %) was similar to that in decerebrate cats. Far fewer neurons in other regions of the feline brainstem had STC behavior, confirming findings that many LTF neurons receive converging inputs from a variety of labyrinthine receptors. However, responses to vertical plane vestibular stimulation were considerably different in decerebrate and conscious felines for LTF neurons lacking STC behavior. In decerebrate cats, most LTF neurons had graviceptive responses to rotations, similar to those of otolith organ afferents. However, in conscious animals, the response properties were similar to those of semicircular canal afferents. These differences show that higher centers of the brain that are removed during decerebration regulate the labyrinthine inputs relayed to the LTF, either by gating connections in the brainstem or by conveying vestibular inputs directly to the region.

Responses of neurons in the caudal medullary lateral tegmental field to visceral inputs and vestibular stimulation in vertical planes.

The dorsolateral reticular formation of the caudal medulla, or the lateral tegmental field (LTF), has been classified as the brain's "vomiting center", as well as an important region in regulating sympathetic outflow. We examined the responses of LTF neurons in cats to rotations of the body that activate vestibular receptors, as well as to stimulation of baroreceptors (through mechanical stretch of the carotid sinus) and gastrointestinal receptors (through the intragastric administration of the emetic compound copper sulfate). Approximately half of the LTF neurons exhibited graviceptive responses to vestibular stimulation, similar to primary afferents innervating otolith organs. The other half of the neurons had complex responses, including spatiotemporal convergence behavior, suggesting that they received convergent inputs from a variety of vestibular receptors. Neurons that received gastrointestinal and baroreceptor inputs had similar complex responses to vestibular stimulation; such responses are expected for neurons that contribute to the generation of motion sickness. LTF units with convergent baroreceptor and vestibular inputs may participate in producing the cardiovascular system components of motion sickness, such as the changes in skin blood flow that result in pallor. The administration of copper sulfate often modulated the gain of responses of LTF neurons to vestibular stimulation, particularly for units whose spontaneous firing rate was altered by infusion of drug (median of 459%). The present results raise the prospect that emetic signals from the gastrointestinal tract modify the processing of vestibular inputs by LTF neurons, thereby affecting the probability that vomiting will occur as a consequence of motion sickness.

Soft Tissue Reconstruction of Parotidectomy Defect.

This article provides a review of soft tissue reconstructive options for the parotidectomy defect, including skin incision, primary closure, acellular dermis, autologous fat transfer, local and regional flaps, and free tissue transfer. The authors discuss considerations for volume enhancement, skin coverage, prevention of Frey syndrome, tumor surveillance, and potential complications.

Application of the Spider Limb Positioner to Subscapular System Free Flaps.

OBJECTIVE: To demonstrate the application and surgical time savings of the Spider Limb Positioner for subscapular system free flaps in head and neck reconstructive surgery. METHODS: Single institution retrospective chart review and analysis of patients between 2011 and 2019 that underwent a subscapular system free flap either with or without use of the Spider Limb Positioner. One hundred five patients in total were reviewed with 53 patients in the Spider group. The surgical times were compared between the two groups. Patient-specific information regarding average age, laterality of donor site, recipient site, gender, and flap type were reviewed. RESULTS: Forty-one patients in both groups underwent a latissimus free flap. Twelve of 53 in the Spider group and 11/52 in the control group underwent a scapula free flap. The average age in the Spider group at the time of surgery was 64 years. The recipient sites for the Spider groups were reviewed. The free flap was ipsilateral to the defect in 81% of cases. The mean surgical time for the 105 patients without the Spider was 568 minutes versus 486 minutes with a Spider P-value of .003478. CONCLUSION: Use of the Spider Limb Positioner allows for a simultaneous two-team approach during free flap elevation of the subscapular system, which eliminates both dependence on an assistant to support the arm and time consuming positioning changes during flap elevation. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:525-528, 2021.

Radial Forearm Free Tissue Transfer to Clival Defect.

Introduction Reconstruction of craniocervical junction (CCJ) defects after endoscopic endonasal skull base surgery (ESBS) remains challenging, despite advancements in vascularized intranasal and regional flaps. Microvascular free tissue transfers have revolutionized reconstruction in open skull base surgery but have been utilized rarely in ESBS. We describe the use of a radial forearm free flap (RFFF) for reconstruction of a recalcitrant CCJ defect after resection of a clival chordoma. Case Report A 54-year-old female who underwent ESBS for a clival chordoma complicated by a C1-C2 epidural abscess after proton beam therapy presented with pneumocephalus 4 years after her resection ( Fig. 1 ). At the CCJ, she developed a 1-cm skull-base defect. An occult cerebrospinal fluid (CSF) leak persisted despite an extracranial pericranial flap and a lateral nasal wall flap. Her definite reconstruction was a RFFF inset through a transmaxillary approach. Using a maxillary vestibular incision, anterior, lateral, and medial maxillotomies allowed the introduction of the flap into the nasal cavity and the passage of the RFFF pedicle across the posterior maxillary wall, into the premassateric space and to the facial vessels at the mandible. An endonasal inset supplemented with transoral suturing of the distal end of the flap to the posterior oropharynx halted further CSF egress. Vascularization of the flap was confirmed with intraoperative indocyanine green angiography and postoperative computed tomography (CT) angiography and magnetic resonance imaging (MRI). Conclusion A RFFF inset through a transmaxillary approach to the facial vessels has an adequate reconstructive surface and pedicle to cover the central and posterior fossa skull base after ESBS ( Fig. 2 ). The link to the video can be found at: https://youtu.be/rQ5vJKyD5qg .

PD-L1 Mediates Dysfunction in Activated PD-1+ NK Cells in Head and Neck Cancer Patients.

Inhibitory immune-checkpoint receptors (ICRs), including programmed death 1 (PD-1), have been characterized as exhaustion markers on T cells that infiltrate the tumor microenvironment (TME) of many cancer types, including head and neck cancer (HNC). However, expression and function of ICRs, including PD-1, on natural killer (NK) cells remains less defined. NK cells are innate immune effector cells that lyse epidermal growth factor receptor-overexpressing HNC cells via cetuximab-mediated antibody-dependent cytotoxicity. Cetuximab is clinically effective but only in 10% to 15% of patients. Therefore, it is necessary to investigate how immunomodulation with cetuximab or PD-1 blockade might enhance NK cell responses in the TME and improve monoclonal antibody therapeutic efficacy. We observed that expression of PD-1 on NK cells marks an activated phenotype, which was suppressed only after binding programmed death ligand-1 (PD-L1). HNC patients who exhibit higher circulating PD-1+ NK cells associate with better clinical outcome, and these cells are enriched in the TME. Cetuximab-mediated NK cell activation increased PD-1 expression on NK cells in vitro, which was confirmed in vivo in a prospective neoadjuvant cetuximab trial. In contrast, PD-L1 ligation of PD-1+ NK cells diminished their activation status, whereas PD-1 blockade increased cetuximab-mediated NK cell activation and cytotoxicity, but only against HNC targets with high PD-L1 expression. Therefore, blocking the PD-1-PD-L1 axis may be a useful strategy to reverse immune evasion of HNC tumors with high PD-L1 expression during cetuximab therapy by reversing NK cell dysfunction.

Fine-tuning of noise in gene expression with nucleosome remodeling.

Engineering stochastic fluctuations of gene expression (or "noise") is integral to precisely bias cellular-fate decisions and statistical phenotypes in both single-cell and multi-cellular systems. Epigenetic regulation has been shown to constitute a large source of noise, and thus, engineering stochasticity is deeply intertwined with epigenetics. Here, utilizing chromatin remodeling, we report that Caffeic acid phenethyl ester (CA) and Pyrimethamine (PYR), two inhibitors of BAF250a, a subunit of the Brahma-associated factor (BAF) nucleosome remodeling complex, enable differential and tunable control of noise in transcription and translation from the human immunodeficiency virus long terminal repeat promoter in a dose and time-dependent manner. CA conserves noise levels while increasing mean abundance, resulting in direct tuning of the transcriptional burst size, while PYR strictly increases transcriptional initiation frequency while conserving a constant transcriptional burst size. Time-dependent treatment with CA reveals non-continuous tuning with noise oscillating at a constant mean abundance at early time points and the burst size increasing for treatments after 5 h. Treatments combining CA and Protein Kinase C agonists result in an even larger increase of abundance while conserving noise levels with a highly non-linear increase in variance of up to 63× untreated controls. Finally, drug combinations provide non-antagonistic combinatorial tuning of gene expression noise and map a noise phase space for future applications with viral and synthetic gene vectors. Active remodeling of nucleosomes and BAF-mediated control of gene expression noise expand a toolbox for the future design and engineering of stochasticity in living systems.

Biological mechanisms of immune escape and implications for immunotherapy in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with high morbidity and mortality. Despite advances in cytotoxic therapies and surgical techniques, overall survival (OS) has not improved over the past few decades. This emphasises the need for intense investigation into novel therapies with good tumour control and minimal toxicity. Cancer immunotherapy has led this endeavour, attempting to improve tumour recognition and expand immune responses against tumour cells. While various forms of HNSCC immunotherapy are in preclinical trials, the most promising direction thus far has been with monoclonal antibodies (mAbs), targeting growth factor and immune checkpoint receptors. Preclinical and early phase trials have shown unprecedented efficacy with minimal adverse effects. This article will review biological mechanisms of immune escape and implications for immunotherapy in HNSCC.

PD-1 Status in CD8+ T Cells Associates with Survival and Anti-PD-1 Therapeutic Outcomes in Head and Neck Cancer.

Improved understanding of expression of immune checkpoint receptors (ICR) on tumor-infiltrating lymphocytes (TIL) may facilitate more effective immunotherapy in head and neck cancer (HNC) patients. A higher frequency of PD-1+ TIL has been reported in human papillomavirus (HPV)+ HNC patients, despite the role of PD-1 in T-cell exhaustion. This discordance led us to hypothesize that the extent of PD-1 expression more accurately defines T-cell function and prognostic impact, because PD-1high T cells may be more exhausted than PD-1low T cells and may influence clinical outcome and response to anti-PD-1 immunotherapy. In this study, PD-1 expression was indeed upregulated on HNC patient TIL, and the frequency of these PD-1+ TIL was higher in HPV+ patients (P = 0.006), who nonetheless experienced significantly better clinical outcome. However, PD-1high CD8+ TILs were more frequent in HPV- patients and represented a more dysfunctional subset with compromised IFN-γ secretion. Moreover, HNC patients with higher frequencies of PD-1high CD8+ TIL showed significantly worse disease-free survival and higher hazard ratio for recurrence (P < 0.001), while higher fractions of PD-1low T cells associated with HPV positivity and better outcome. In a murine HPV+ HNC model, anti-PD-1 mAb therapy differentially modulated PD-1high/low populations, and tumor rejection associated with loss of dysfunctional PD-1high CD8+ T cells and a significant increase in PD-1low TIL. Thus, the extent of PD-1 expression on CD8+ TIL provides a potential biomarker for anti-PD-1-based immunotherapy. Cancer Res; 77(22); 6353-64. ©2017 AACR.

Malignant Otitis Externa: Evolving Pathogens and Implications for Diagnosis and Treatment.

OBJECTIVE: Malignant otitis externa (MOE) is an invasive infection of the temporal bone that is classically caused by Pseudomonas aeruginosa. Increasingly, however, nonpseudomonal cases are being reported. The goal of this study was to evaluate and compare the clinical presentation and outcomes of cases of MOE caused by Pseudomonas versus non-Pseudomonas organisms. STUDY DESIGN: Retrospective case series with chart review. SETTING: Tertiary care institution. SUBJECTS AND METHODS: Adult patients with diagnoses of MOE between 1995 and 2012 were identified. Charts were reviewed for history, clinical presentation, laboratory data, treatment, and outcomes. RESULTS: Twenty patients diagnosed with and treated for MOE at the University of Pittsburgh Medical Center between 1995 and 2012 were identified. Nine patients (45%) had cultures that grew P aeruginosa. Three patients (15%) had cultures that grew methicillin-resistant Staphylococcus aureus (MRSA). Signs and symptoms at presentation were similar across groups. However, all of the patients with Pseudomonas had diabetes, compared with 33% of MRSA-infected patients (P = .046) and 55% of all non-Pseudomonas-infected patients (P = .04). Patients infected with MRSA were treated for an average total of 4.7 more weeks of antibiotic therapy than Pseudomonas-infected patients (P = .10). Overall, patients with non-Pseudomonas infections were treated for a total of 2.4 more weeks than Pseudomonas-infected patients (P = .25). CONCLUSIONS: A high index of suspicion for nonpseudomonal organisms should be maintained in patients with signs and symptoms of MOE, especially in those without diabetes. MRSA is an increasingly implicated organism in MOE.

Responses of vestibular nucleus neurons to inputs from the hindlimb are enhanced following a bilateral labyrinthectomy.

Vestibular nucleus neurons have been shown to respond to stimulation of afferents innervating the limbs. However, a limitation in the potential translation of these findings is that they were obtained from decerebrate or anesthetized animals. The goal of the present study was to determine whether stimulation of hindlimb nerves similarly affects vestibular nucleus neuronal activity in conscious cats, and whether the responsiveness of neurons to the stimuli is altered following a bilateral labyrinthectomy. In labyrinth-intact animals, the firing rate of 24/59 (41%) of the neurons in the caudal vestibular nucleus complex was affected by hindlimb nerve stimulation. Most responses were excitatory; the median response latency was 20 ms, but some units had response latencies as short as 10 ms. In the first week after a bilateral labyrinthectomy, the proportion of vestibular nucleus neurons that responded to hindlimb nerve stimulation increased slightly (to 24/55 or 44% of units). However, during the subsequent postlabyrinthectomy survival period, the proportion of vestibular nucleus neurons with hindlimb inputs increased significantly (to 30/49 or 61% of units). Stimuli to hindlimb nerves needed to elicit neuronal responses was consistently over three times the threshold for eliciting an afferent volley. These data show that inputs from hindlimb afferents smaller than those innervating muscle spindles and Golgi tendon organs affect the processing of information in the vestibular nuclei, and that these inputs are enhanced following a bilateral labyrinthectomy. These findings have implications for the development of a limb neuroprosthetics device for the management of bilateral vestibular loss.

Thoracic outlet syndrome: pattern of clinical success after operative decompression.

OBJECTIVE: To evaluate the pattern of clinical results in patients with neurogenic thoracic outlet syndrome (N-TOS) after operative decompression and longitudinal follow-up. METHODS: From May 1994 to December 2002, 254 operative sides in 185 patients with N-TOS were treated by the same operative protocol: (1) transaxillary first rib resection and the lower part of scalenectomy for the primary procedure with or without (2) the subsequent upper part of scalenectomy with supraclavicular approach for patients with persistent or recurrent symptoms. This retrospective cohort study included 38 men and 147 women with an age range of 19 to 80 years (mean, 40 years). Evaluated were primary success, defined as uninterrupted success with no procedure performed, and secondary success, defined as success maintained by the secondary operation after the primary failure. Success was defined as > or =50% symptomatic improvement judged by the patient using a 10-point scale, returning to preoperational work status, or both. RESULTS: Follow-up was 2 to 76 months (mean, 25 months). Eighty sides underwent a secondary operation for the primary clinical failure. No technical failures and no deaths occurred < o =30 days after the operations. The complication rate was 4% (13/334) and consisted of 7 pneumothoraxes, 3 subclavian vein injuries, 1 nerve injury, 1 internal mammary artery injury, and 1 suture granuloma. Of 254 operative sides, the primary and secondary success was 46% (118/254) and 64% (163/254). Most the primary failures (90%, 122/136) and the secondary failures (66%, 23/35) occurred < or =18 months after the respective operation. CONCLUSIONS: The long-term results of operations for TOS in this study were much worse than those initially achieved, and most of the primary and secondary failures occurred < or =12 months of the respective operations. A minimum of 18-month follow-up on patients and standardized definition of the outcomes are necessary to determine the true effectiveness and outcome of operative treatment of N-TOS.