Analysis of HER2 expression changes from breast primary to brain metastases and the impact of HER2-low expression on overall survival.

BACKGROUND: There are limited data regarding HER2-low expression dynamics between matched primary tumors and brain metastases (BrMs) in breast cancer. HER2-low expression has emerged as a new therapeutic biomarker for highly active antibody-drug conjugates with emerging intracranial activity. METHODS: Patients with metastatic breast cancer (MBC) and BrMs seen at an NCI-designated center between 2003-2023 were identified. HER2 expression was defined as HER2-positive (3+,2+/ISH amplified), HER2-low (1+,2+/ISH negative), or HER2-0 by ASCO-CAP guidelines. Estrogen receptor (ER) status was defined as ER≥1%. Multivariate survival analyses by Cox proportional hazard models were determined from time of BrM resection to death or last follow-up between the 3 subtypes, controlling for ER and age. RESULTS: Among 197 matched primary and resected BrMs, 81% exhibited HER2 expression in the brain:61% HER2-positive, 20% HER2-low, and 19% HER2-0. Concordance was high in HER2-positive primary tumors with 100% retaining HER2 expression (97% retained HER2+ expression and 2.7% switched to HER2-low). HER2-0 primaries frequently showed HER2 gain in BrMs to HER2-low (35%) or HER2-positive (5.4%) status. Among 48 HER2-low primary tumors, 52% were discordant for HER2 status in the brain with 21% testing HER2-positive and 31% testing HER2-0. In adjusted analyses, patients with HER2-positive BrMs had significantly lower death risk than patients with HER2-low BrMs (HR=0.41, P=0.0006); no difference was observed between HER2-0 and HER2-low. CONCLUSIONS: In this retrospective analysis, HER2 expression is common in breast cancer BrMs, emphasizing the need for improved, non-invasive diagnostics. Patients with HER2-low and HER2-0 BrMs face inferior survival, presenting an unmet clinical need.

Multi-wavelength multi-distance diffuse correlation spectroscopy system for assessment of premature infants' cerebral hemodynamics.

Infants born at an extremely low gestational age (ELGA, < 29 weeks) are at an increased risk of intraventricular hemorrhage (IVH), and there is a need for standalone, safe, easy-to-use tools for monitoring cerebral hemodynamics. We have built a multi-wavelength multi-distance diffuse correlation spectroscopy device (MW-MD-DCS), which utilizes time-multiplexed, long-coherence lasers at 785, 808, and 853 nm, to simultaneously quantify the index of cerebral blood flow (CBFi) and the hemoglobin oxygen saturation (SO2). We show characterization data on liquid phantoms and demonstrate the system performance on the forearm of healthy adults, as well as clinical data obtained on two preterm infants.

Enhancing diffuse correlation spectroscopy pulsatile cerebral blood flow signal with near-infrared spectroscopy photoplethysmography.

Significance: Combining near-infrared spectroscopy (NIRS) and diffuse correlation spectroscopy (DCS) allows for quantifying cerebral blood volume, flow, and oxygenation changes continuously and non-invasively. As recently shown, the DCS pulsatile cerebral blood flow index (pCBFi) can be used to quantify critical closing pressure (CrCP) and cerebrovascular resistance (CVRi). Aim: Although current DCS technology allows for reliable monitoring of the slow hemodynamic changes, resolving pulsatile blood flow at large source-detector separations, which is needed to ensure cerebral sensitivity, is challenging because of its low signal-to-noise ratio (SNR). Cardiac-gated averaging of several arterial pulse cycles is required to obtain a meaningful waveform. Approach: Taking advantage of the high SNR of NIRS, we demonstrate a method that uses the NIRS photoplethysmography (NIRS-PPG) pulsatile signal to model DCS pCBFi, reducing the coefficient of variation of the recovered pulsatile waveform (pCBFi-fit) and allowing for an unprecedented temporal resolution (266 Hz) at a large source-detector separation (>3 cm). Results: In 10 healthy subjects, we verified the quality of the NIRS-PPG pCBFi-fit during common tasks, showing high fidelity against pCBFi (R2 0.98±0.01). We recovered CrCP and CVRi at 0.25 Hz, >10 times faster than previously achieved with DCS. Conclusions: NIRS-PPG improves DCS pCBFi SNR, reducing the number of gate-averaged heartbeats required to recover CrCP and CVRi.

Portable, high speed blood flow measurements enabled by long wavelength, interferometric diffuse correlation spectroscopy (LW-iDCS).

Diffuse correlation spectroscopy (DCS) is an optical technique that can be used to characterize blood flow in tissue. The measurement of cerebral hemodynamics has arisen as a promising use case for DCS, though traditional implementations of DCS exhibit suboptimal signal-to-noise ratio (SNR) and cerebral sensitivity to make robust measurements of cerebral blood flow in adults. In this work, we present long wavelength, interferometric DCS (LW-iDCS), which combines the use of a longer illumination wavelength (1064 nm), multi-speckle, and interferometric detection, to improve both cerebral sensitivity and SNR. Through direct comparison with long wavelength DCS based on superconducting nanowire single photon detectors, we demonstrate an approximate 5× improvement in SNR over a single channel of LW-DCS in the measured blood flow signals in human subjects. We show equivalence of extracted blood flow between LW-DCS and LW-iDCS, and demonstrate the feasibility of LW-iDCS measured at 100 Hz at a source-detector separation of 3.5 cm. This improvement in performance has the potential to enable robust measurement of cerebral hemodynamics and unlock novel use cases for diffuse correlation spectroscopy.

Functional Time Domain Diffuse Correlation Spectroscopy.

Time-domain diffuse correlation spectroscopy (TD-DCS) offers a novel approach to high-spatial resolution functional brain imaging based on the direct quantification of cerebral blood flow (CBF) changes in response to neural activity. However, the signal-to-noise ratio (SNR) offered by previous TD-DCS instruments remains a challenge to achieving the high temporal resolution needed to resolve perfusion changes during functional measurements. Here we present a next-generation optimized functional TD-DCS system that combines a custom 1,064 nm pulse-shaped, quasi transform-limited, amplified laser source with a high-resolution time-tagging system and superconducting nanowire single-photon detectors (SNSPDs). System characterization and optimization was conducted on homogenous and two-layer intralipid phantoms before performing functional CBF measurements in six human subjects. By acquiring CBF signals at over 5 Hz for a late gate start time of the temporal point spread function (TPSF) at 15 mm source-detector separation, we demonstrate for the first time the measurement of blood flow responses to breath-holding and functional tasks using TD-DCS.

Diffuse correlation spectroscopy blood flow monitoring for intraventricular hemorrhage vulnerability in extremely low gestational age newborns.

In premature infants with an extremely low gestational age (ELGA, < 29 weeks GA), dysregulated changes in cerebral blood flow (CBF) are among the major pathogenic factors leading to germinal matrix/intraventricular hemorrhage (GM/IVH). Continuous monitoring of CBF can guide interventions to minimize the risk of brain injury, but there are no clinically standard techniques or tools for its measurement. We report the feasibility of the continuous monitoring of CBF, including measures of autoregulation, via diffuse correlation spectroscopy (DCS) in ELGA infants using CBF variability and correlation with scalp blood flow (SBF, served as a surrogate measure of systemic perturbations). In nineteen ELGA infants (with 9 cases of GM/IVH) monitored for 6-24 h between days 2-5 of life, we found a strong correlation between CBF and SBF in severe IVH (Grade III or IV) and IVH diagnosed within 72 h of life, while CBF variability alone was not associated with IVH. The proposed method is potentially useful at the bedside for the prompt assessment of cerebral autoregulation and early identification of infants vulnerable to GM/IVH.

Open-source FlexNIRS: A low-cost, wireless and wearable cerebral health tracker.

Currently, there is great interest in making neuroimaging widely accessible and thus expanding the sampling population for better understanding and preventing diseases. The use of wearable health devices has skyrocketed in recent years, allowing continuous assessment of physiological parameters in patients and research cohorts. While most health wearables monitor the heart, lungs and skeletal muscles, devices targeting the brain are currently lacking. To promote brain health in the general population, we developed a novel, low-cost wireless cerebral oximeter called FlexNIRS. The device has 4 LEDs and 3 photodiode detectors arranged in a symmetric geometry, which allows for a self-calibrated multi-distance method to recover cerebral hemoglobin oxygenation (SO2) at a rate of 100 Hz. The device is powered by a rechargeable battery and uses Bluetooth Low Energy (BLE) for wireless communication. We developed an Android application for portable data collection and real-time analysis and display. Characterization tests in phantoms and human participants show very low noise (noise-equivalent power <70 fW/√Hz) and robustness of SO2 quantification in vivo. The estimated cost is on the order of $50/unit for 1000 units, and our goal is to share the device with the research community following an open-source model. The low cost, ease-of-use, smart-phone readiness, accurate SO2 quantification, real time data quality feedback, and long battery life make prolonged monitoring feasible in low resource settings, including typically medically underserved communities, and enable new community and telehealth applications.

A Contact-Sensitive Probe for Biomedical Optics.

Capacitive proximity sensing is widespread in our everyday life, but no sensor for biomedical optics takes advantage of this technology to monitor the probe attachment to the subject's skin. In particular, when using optical monitoring devices, the capability to quantitatively measure the probe contact can significantly improve data quality and ensure the subject's safety. We present a custom novel optical probe based on a flexible printed circuit board which integrates a capacitive contact sensor, 3D-printed optic fiber holders and an accelerometer sensor. The device can be effectively adopted during continuous monitoring optical measurements to detect contact quality, motion artifacts, probe detachment and ensure optimal signal quality.

Similar Frequency and Inducibility of Intact Human Immunodeficiency Virus-1 Proviruses in Blood and Lymph Nodes.

BACKGROUND: The human immunodeficiency virus (HIV)-1 latent reservoir (LR) in resting CD4+ T cells is a barrier to cure. LR measurements are commonly performed on blood samples and therefore may miss latently infected cells residing in tissues, including lymph nodes. METHODS: We determined the frequency of intact HIV-1 proviruses and proviral inducibility in matched peripheral blood (PB) and lymph node (LN) samples from 10 HIV-1-infected patients on antiretroviral therapy (ART) using the intact proviral DNA assay and a novel quantitative viral induction assay. Prominent viral sequences from induced viral RNA were characterized using a next-generation sequencing assay. RESULTS: The frequencies of CD4+ T cells with intact proviruses were not significantly different in PB versus LN (61/106 vs 104/106 CD4+ cells), and they were substantially lower than frequencies of CD4+ T cells with defective proviruses. The frequencies of CD4+ T cells induced to produce high levels of viral RNA were not significantly different in PB versus LN (4.3/106 vs 7.9/106), but they were 14-fold lower than the frequencies of cells with intact proviruses. Sequencing of HIV-1 RNA from induced proviruses revealed comparable sequences in paired PB and LN samples. CONCLUSIONS: These results further support the use of PB as an appropriate proxy for the HIV-1 LR in secondary lymphoid organs.

The association of α4ß7 expression with HIV acquisition and disease progression in people who inject drugs and men who have sex with men: Case control studies.

BACKGROUND: α4ß7 is a gut-homing integrin heterodimer that can act as a non-essential binding molecule for HIV. A previous study in heterosexual African women found that individuals with higher proportions of α4ß7 expressing CD4+ T cells were more likely to become infected with HIV, as well as present with faster disease progression. It is unknown if this phenomenon is also observed in men who have sex with men (MSM) or people who inject drugs (PWID). METHODS: MSM and transgender women who seroconverted as part of the HVTN 505 HIV vaccine trial and PWID who seroconverted during the ALIVE cohort study were selected as cases and matched to HIV-uninfected controls from the same studies (1:1 and 1:3, respectively). Pre-seroconversion PBMC samples from cases and controls in both studies were examined by flow cytometry to measure levels of α4ß7 expression on CD4+ T cells. Multivariable conditional logistic regression was used to compare α4ß7 expression levels between cases and controls. A Kaplan-Meier curve was used to examine the association of α4ß7 expression pre-seroconversion with HIV disease progression. FINDINGS: In MSM and transgender women (n = 103 cases, 103 controls), there was no statistically significant difference in the levels of α4ß7 expression on CD4+ T cells between cases and controls (adjusted odds ratio [adjOR] =1.10, 95% confidence interval [CI]=0.94,1.29; p = 0.246). Interestingly, in PWID (n = 49 cases, 143 controls), cases had significantly lower levels of α4ß7 expression compared to their matched controls (adjOR = 0.80, 95% CI = 0.68, 0.93; p = 0.004). Among HIV-positive PWID (n = 47), there was no significant association in HIV disease progression in individuals above or below the median level of α4ß7 expression (log-rank p = 0.84). INTERPRETATION: In contrast to findings in heterosexual women, higher α4ß7 expression does not predict HIV acquisition or disease progression in PWID or MSM. FUNDING: This study was supported in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health. The study was also supported by extramural grants from NIAID T32AI102623 (E.U.P.), and UM1AI069470.

Racial differences in α4ß7 expression on CD4+ T cells of HIV-negative men and women who inject drugs.

INTRODUCTION: We performed a cross-sectional study of HIV-uninfected men and women who inject drugs from the ALIVE cohort to examine if black men and women who inject drugs have higher levels of CD4+ T cells expressing the integrin heterodimer α4ß7 compared to white men and women. MATERIALS AND METHODS: Flow cytometry was used to examine expression of α4ß7 and other markers associated with different functional CD4+ T cell subsets in both men and women who inject drugs. RESULTS: Higher levels of α4ß7, CCR5, and CCR6 were observed on CD4+ T cells from black participants compared with white participants. In a multivariable model, α4ß7 expression differed by race, but not sex, age, or other factors. DISCUSSION: Black men and women express higher percentages of α4ß7 expressing CD4+ T cells, which may play a role in HIV disease.

Characterization of Elite Suppressors Cell-Associated HIV-1 mRNA at Baseline and with T Cell Activation

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Objective: Elite Controllers or Suppressors (ES) are patients who control HIV replication without antiretroviral therapy. In this study, we compared baseline and inducible HIV-1 mRNA levels in CD4+ T cells from ES and chronic progressors (CPs) receiving suppressive antiretroviral therapy. Methods: We quantified basal levels of cell associated HIV-1 mRNA in CD4+ T cells isolated from CPs and ES. Additionally, we measured the fold upregulation of intracellular HIV-mRNA after stimulation of CD4+ T cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and quantified the amount of HIV-mRNA levels released into culture supernatant. Results: ES have significantly less cell associated HIV-mRNA per 5x106 cells (p = 0.003); 8 of 10 CPs had quantifiable HIV-1 mRNA at baseline, whereas this was present in only 2 of 10 ES. Upon stimulation with PMA and ionomycin, 4 of 5 CPs and 7 of 9 ES showed increased cell associated HIV-mRNA. Interestingly, released HIV-1 mRNA could be detected in supernatants of CD4+ T cells stimulated with PMA/ionomycin from 5 of 8 ES. Conclusion: Our results demonstrate that while the baseline levels of cell associated HIV-1 mRNA are significantly lower in ES compared to CPs, stimulation of CD4+ T cells results in a comparable relative upregulation of viral transcription.

Defective HIV-1 Proviruses Are Expressed and Can Be Recognized by Cytotoxic T Lymphocytes, which Shape the Proviral Landscape.

Despite antiretroviral therapy, HIV-1 persists in memory CD4+ T cells, creating a barrier to cure. The majority of HIV-1 proviruses are defective and considered clinically irrelevant. Using cells from HIV-1-infected individuals and reconstructed patient-derived defective proviruses, we show that defective proviruses can be transcribed into RNAs that are spliced and translated. Proviruses with defective major splice donors (MSDs) can activate novel splice sites to produce HIV-1 transcripts, and cells with these proviruses can be recognized by HIV-1-specific cytotoxic T lymphocytes (CTLs). Further, cells with proviruses containing lethal mutations upstream of CTL epitopes can also be recognized by CTLs, potentially through aberrant translation. Thus, CTLs may change the landscape of HIV-1 proviruses by preferentially targeting cells with specific types of defective proviruses. Additionally, the expression of defective proviruses will need to be considered in the measurement of HIV-1 latency reversal.

Rapamycin-mediated mTOR inhibition uncouples HIV-1 latency reversal from cytokine-associated toxicity.

Current strategies for HIV-1 eradication require the reactivation of latent HIV-1 in resting CD4+ T cells (rCD4s). Global T cell activation is a well-characterized means of inducing HIV-1 transcription, but is considered too toxic for clinical applications. Here, we have explored a strategy that involves a combination of immune activation and the immunosuppressive mTOR inhibitor rapamycin. In purified rCD4s from HIV-1-infected individuals on antiretroviral therapy, rapamycin treatment downregulated markers of toxicity, including proinflammatory cytokine release and cellular proliferation that were induced after potent T cell activation using αCD3/αCD28 antibodies. Using an ex vivo assay for HIV-1 mRNA, we demonstrated that despite this immunomodulatory effect, rapamycin did not affect HIV-1 gene expression induced by T cell activation in these rCD4s. In contrast, treating activated rCD4s with the immunosuppressant cyclosporin, a calcineurin inhibitor, robustly inhibited HIV-1 reactivation. Importantly, rapamycin treatment did not impair cytotoxic T lymphocyte (CTL) recognition and killing of infected cells. These findings raise the possibility of using rapamycin in conjunction with T cell-activating agents in HIV-1 cure strategies.

The mTOR Complex Controls HIV Latency.

A population of CD4 T lymphocytes harboring latent HIV genomes can persist in patients on antiretroviral therapy, posing a barrier to HIV eradication. To examine cellular complexes controlling HIV latency, we conducted a genome-wide screen with a pooled ultracomplex shRNA library and in vitro system modeling HIV latency and identified the mTOR complex as a modulator of HIV latency. Knockdown of mTOR complex subunits or pharmacological inhibition of mTOR activity suppresses reversal of latency in various HIV-1 latency models and HIV-infected patient cells. mTOR inhibitors suppress HIV transcription both through the viral transactivator Tat and via Tat-independent mechanisms. This inhibition occurs at least in part via blocking the phosphorylation of CDK9, a p-TEFb complex member that serves as a cofactor for Tat-mediated transcription. The control of HIV latency by mTOR signaling identifies a pathway that may have significant therapeutic opportunities.

Progress Toward HIV Eradication: Case Reports, Current Efforts, and the Challenges Associated with Cure.

An estimated 35 million people worldwide are infected with HIV, yet a widely applicable cure strategy remains elusive. Recent case reports have suggested that curing HIV infection is possible, renewing excitement about research efforts. We describe those cases and discuss their relevance to the global HIV epidemic. We also review ongoing cure strategies that are transitioning from the lab to the clinic, and the assays and clinical assessments that can be used to evaluate cure interventions.

Ex vivo analysis identifies effective HIV-1 latency-reversing drug combinations.

Reversal of HIV-1 latency by small molecules is a potential cure strategy. This approach will likely require effective drug combinations to achieve high levels of latency reversal. Using resting CD4+ T cells (rCD4s) from infected individuals, we developed an experimental and theoretical framework to identify effective latency-reversing agent (LRA) combinations. Utilizing ex vivo assays for intracellular HIV-1 mRNA and virion production, we compared 2-drug combinations of leading candidate LRAs and identified multiple combinations that effectively reverse latency. We showed that protein kinase C agonists in combination with bromodomain inhibitor JQ1 or histone deacetylase inhibitors robustly induce HIV-1 transcription and virus production when directly compared with maximum reactivation by T cell activation. Using the Bliss independence model to quantitate combined drug effects, we demonstrated that these combinations synergize to induce HIV-1 transcription. This robust latency reversal occurred without release of proinflammatory cytokines by rCD4s. To extend the clinical utility of our findings, we applied a mathematical model that estimates in vivo changes in plasma HIV-1 RNA from ex vivo measurements of virus production. Our study reconciles diverse findings from previous studies, establishes a quantitative experimental approach to evaluate combinatorial LRA efficacy, and presents a model to predict in vivo responses to LRAs.

Influence of ZIP14 (slc39A14) on intestinal zinc processing and barrier function.

ZIP14 is a zinc transport protein with high expression in the small intestine and liver. Zip14 is upregulated during endotoxemia and leads to increased liver zinc content and transient hypozinemia. Since body zinc status and inflammation are associated with changes in intestinal permeability, we hypothesized that ZIP14 may influence intestinal permeability. Wild-type (WT) and Zip14 knockout (KO) mice were used to determine ZIP14-associated intestinal zinc metabolism and effects on permeability. Fractionation of plasma membranes revealed that ZIP14 is localized to the basolateral membrane of enterocytes. Studies utilizing (65)Zn administered by subcutaneous injection revealed greater zinc accumulation in the SI of Zip14 KO mice compared with WT mice. Isolation of endosomes confirmed the presence of ZIP14. Quantification of endosomal zinc concentration by FluoZin-3AM fluorescence demonstrated that zinc is trapped in endosomes of Zip14 KO mice. Intestinal permeability assessed both by plasma FITC-dextran following gavage and by serum endotoxin content was greater in Zip14 KO mice. Threonine phosphorylation of the tight junction protein occludin, which is necessary for tight junction assembly, was reduced in KO mice. Claudin 1 and 2, known to have an inverse relationship in regards to tight junction integrity, reflected impaired barrier function in KO jejunum. These data suggest involvement of ZIP14 in providing zinc for a regulatory role needed for maintenance of the intestinal barrier. In conclusion, ZIP14 is a basolaterally localized protein in enterocytes and is involved in endosomal trafficking of zinc and is necessary for proper maintenance of intestinal tight junctions.

Gastric and colonic zinc transporter ZIP11 (Slc39a11) in mice responds to dietary zinc and exhibits nuclear localization.

Zinc transporters have been characterized to further understand the absorption and metabolism of dietary zinc. Our goal was to characterize zinc transporter Slc39a11 (ZIP11) expression and its subcellular localization within cells of the murine gastrointestinal tract of mice and to determine if dietary zinc regulates ZIP11. The greatest ZIP11 expression was in the stomach, cecum, and colon. Both Zip11 mRNA and ZIP11 protein were shown to be downregulated during dietary zinc restriction (<1 mg Zn/kg) in the murine stomach tissue but were unaffected in the colon. Acute repletion with zinc did not restore Zip11 mRNA levels in the stomach. Immunohistochemistry (IHC) revealed high ZIP11 levels in the lower regions of gastric glands and parietal cells of the stomach. IHC analysis of the colon showed a marked ZIP11 abundance within the cytoplasm of the colonic epithelial cells. IHC also showed an increase in ZIP11 expression in the colon during zinc restriction. There is a robust abundance of ZIP11 in the nuclei of cells of both stomach and colon. Our experiments suggest that when dietary zinc intake is compromised, the colon may increase zinc transporter expression to improve the efficiency for absorption via increased expression of specific zinc transporters, including ZIP11 and also zinc transporter Slc39a4. In conclusion, ZIP11 is highly expressed within the murine stomach and colon and appears to be partially regulated by dietary zinc intake within these tissues. ZIP11 may play a specialized role in zinc homeostasis within these tissues, helping to maintain mucosal integrity and function.