Slow Delivery Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via Modulation of Immunodominance.

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes.

Vaccine-Induced Protection from Homologous Tier 2 SHIV Challenge in Nonhuman Primates Depends on Serum-Neutralizing Antibody Titers.

Passive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection after vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers. Repeat intrarectal challenge with homologous tier 2 SHIVBG505 led to rapid infection in unimmunized and low-titer animals. High-titer animals, however, demonstrated protection that was gradually lost as nAb titers waned over time. An autologous serum ID50 nAb titer of ∼1:500 afforded more than 90% protection from medium-dose SHIV infection. In contrast, antibody-dependent cellular cytotoxicity and T cell activity did not correlate with protection. Therefore, Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained.

Velopharyngeal Characteristics in Aarskog-Scott Syndrome: A Case Report.

Aarskog-Scott syndrome (AAS), also known as facio-digito-genital syndrome, is a rare heterogenous syndrome characterized by facial dysmorphism, brachydactyly, and genetic abnormalities. Although severe craniofacial abnormalities have been reported in AAS, little is known about speech and resonance issues in AAS. Specifically, published data to date have only indicated reports of hypernasality associated with a cleft palate in AAS. This case report provides clinical and anatomic information surrounding hypernasal speech in the absence of an overt cleft palate in a patient with AAS.

Outpatient clinical pharmacy practice in the face of COVID-19 at a cancer center in New York City.

PURPOSE: With the rapid spread of COVID-19 in New York City since early March 2020, innovative measures were needed for clinical pharmacy specialists to provide direct clinical care safely to cancer patients. Allocating the workforce was necessary to meet the surging needs of the inpatient services due to the COVID-19 outbreak, which had the potential to compromise outpatient services. We present here our approach of restructuring clinical pharmacy services and providing direct patient care in outpatient clinics during the pandemic. DATA SOURCES: We conducted a retrospective review of electronic clinical documentation involving clinical pharmacy specialist patient encounters in 9 outpatient clinics from March 1, 2020 to May 31, 2020. The analysis of the clinical pharmacy specialist interventions and the impact of the interventions was descriptive. DATA SUMMARY: As hospital services were modified to handle the surge due to COVID-19, select clinical pharmacy specialists were redeployed from the outpatient clinics or research blocks to COVID-19 inpatient teams. During these 3 months, clinical pharmacy specialists were involved in 2535 patient visits from 9 outpatient clinics and contributed a total of 4022 interventions, the majority of which utilized telemedicine. The interventions provided critical clinical pharmacy care during the pandemic and omitted 199 in-person visits for medical care. CONCLUSION: The swift transition to telemedicine allowed the provision of direct clinical pharmacy services to patients with cancer during the COVID-19 pandemic.

Evaluation of toxicity of carmustine with or without bevacizumab in patients with recurrent or progressive high grade gliomas.

PURPOSE: An increased incidence in hematologic toxicity has been reported with the addition of bevacizumab to lomustine for patients with recurrent or progressive high grade gliomas (HGG). Data regarding incidence of toxicity with combination bevacizumab and carmustine is limited. The purpose of this study is to compare toxicity of single agent carmustine and carmustine plus bevacizumab for patients with HGGs. METHODS: This single center retrospective study at Memorial Sloan Kettering Cancer Center included pathologically confirmed HGG with age ≥ 18 years who received carmustine between January 2003 and May 2017. RESULTS: Sixty-five patients with HGGs collectively received 110 doses of BCNU during the specified time period. Sixteen patients received single agent BCNU (30 doses); 49 patients received combination bevacizumab with BCNU (80 doses). There was no significant difference in incidence or grade of toxicity between single agent and combination therapy with respect to hepatotoxicity, leukopenia, lymphopenia, neutropenia, anemia, and thrombocytopenia. Rates of grade 3 and 4 neutropenia (20% vs 13.8%, p = 0.55) and thrombocytopenia (23.3% vs 23.8%, p = 1) did not differ between single agent BCNU versus combination therapy. When stratified based on dose ( < 150 mg/m2, 150 mg/m2, > 150 mg/m2), there was no statistically significant difference between the two groups with respect to grade 3 and 4 neutropenia or thrombocytopenia. CONCLUSIONS: This is the first study to report the toxicity of carmustine with or without bevacizumab for the treatment of recurrent and refractory HGG. The addition of bevacizumab to carmustine did not increase incidence or grade of hematologic toxicity when compared to single agent carmustine.

CXCL13 is a plasma biomarker of germinal center activity.

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

A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood.

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

Cytokine-Independent Detection of Antigen-Specific Germinal Center T Follicular Helper Cells in Immunized Nonhuman Primates Using a Live Cell Activation-Induced Marker Technique.

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

Hypoalbuminemia is significantly associated with increased clearance time of high dose methotrexate in patients being treated for lymphoma or leukemia.

As a weak acid, methotrexate (MTX) is bound to serum albumin and has variable protein binding. The purpose of this study was to assess serum albumin's relationship with MTX pharmacokinetics by comparing MTX clearance and toxicities between patients with normal serum albumin to those with hypoalbuminemia. This single-center retrospective study included adult patients with leukemia or lymphoma who received their first MTX at a dose ≥1 g/m2. Hypoalbuminemia was defined as serum albumin ≤3.4 g/dL. MTX clearance was defined as the first documented time the MTX level ≤0.05 µM. Fisher's exact tests and Wilcoxon rank sum tests were used to examine differences in toxicities, and Cox proportional hazard regression was used to assess relationship with time to clearance. Of 523 patients identified, 167 patients were evaluable. One hundred thirty-five patients had normal serum albumin and 32 had hypoalbuminemia. Hypoalbuminemia was associated with a higher proportion of patients experiencing edema, ascites or pleural effusions (34 vs. 12 %, p = 0.006), and the concomitant use of nephrotoxic agents (41 vs. 20 %, p = 0.021). Hypoalbuminemia was associated with a significantly longer time to MTX clearance (median 96 vs. 72 h, p = 0.004). In addition, patients with hypoalbuminemia had a higher proportion of hyperbilirubinemia and significantly longer hospitalization (median 14 vs. 5 days, p < 0.001). In conclusion, hypoalbuminemia was associated with increased time to MTX clearance and increased length of hospitalization. High dose MTX is safe to administer in patients with low albumin levels, with appropriate leucovorin rescue, and good supportive care.

Grade V Liver Injury Presented With Peritonitis Treated With Stapler-Assisted Hepatic Segmentectomy: A Case Report.

The liver is one of the most commonly injured solid organs in blunt abdominal trauma. In patients who are hemodynamically normal, most cases of blunt liver injuries are managed conservatively. At present, nonoperative management (NOM) is the standard of care for both minor and severe liver injuries. Usually, patients with severe liver injuries, i.e., grades IV and V, are treated with surgical intervention versus angioembolization depending if patients are hemodynamically stable or not. We present a hemodynamically stable 53-year-old male patient with a grade V blunt liver injury with complete avulsion of the left lobe of the liver after a motor vehicle collision (MVC). Very few cases of complete hepatic avulsions have been published in the literature. We discuss surgical management with stapler-assisted hepatectomy in emergency trauma laparotomy for bleeding control.

Esophageal Replacement by Colonic Interposition for the Surgical Management of Acute Necrotic Gastric Volvulus: A Case Report.

Acute gastric volvulus, a condition where the stomach rotates around itself, is a rare clinical entity that requires prompt identification and immediate intervention to prevent life-threatening complications. Upon diagnosis, an emergent exploratory laparotomy is the procedure of choice, especially if complications, such as obstruction, ischemia, or perforation, are present. Management techniques and surgical corrections vary depending on the degree of obstruction, the consequent damage to surrounding structures, and the functional reservoir. We present a case of acute gastric volvulus with extensive esophageal and gastric necrosis requiring total gastrectomy and partial esophagectomy. We discuss the patient's operative management requiring colonic interposition with esophagocolonic anastomosis to reconnect this patient's gastrointestinal tract.

Tool parameters to minimize temperature changes in bone drilling.

BACKGROUND: Drilling is a common technique used in orthopedic surgery procedures but causes increases in temperature that can lead to cell damage and death. The extent of this depends largely on the magnitude of the increase in temperature. The commonly accepted limit to prevent osteonecrosis is less than 47 °C for 60 s. There is controversy when it comes to the optimal drilling parameters that limit temperature increases and cell death. In addition to this, less research has been done on the drilling effects in the osteochondral area of joints. Osteochondral tissue damage can interfere with the daily lives of patients and if severe enough will need to be treated. We hypothesize that increasing tool speed and drill bit size will increase temperature that could be above the osteonecrosis limit. METHODS: Ex-vivo experiments were conducted on porcine shoulder joints that tested the thermal effects of different tool speeds and drill bit sizes. A thermal camera was used to record and measure real time temperature changes while drilling. Three drill bit sizes and five tool speeds were used. Statistical analyses includes Welch's ANOVA with Games-Howell Post Hoc analyses, multivariate linear regression, and surface response regression were used to explore the association of tool speeds and drill bit size on temperature. RESULTS AND CONCLUSIONS: All the tool speed and drill bit size combinations lead to an increase in temperature that were under the commonly accepted limit. The highest temperature reached was 44 °C with a tool speed of 1150 RPM and 3070 RPM and drill bit size 5.159 mm. It was found that increasing the tool speed increased the temperature change and increasing the drill bit size increased the temperature change.

Circadian rhythm shifts and alcohol access in adolescence synergistically increase alcohol preference and intake in adulthood in male C57BL/6 mice.

BACKGROUND: Adolescents have a natural tendency to be night owls, maintaining delayed circadian rhythms, and this rhythm is in direct conflict with the early wake times required during the school year. This leads to 'social jetlag', chronic circadian stress or desynchrony (CD) in which the rhythm of the intrinsic body clock is out of sync with behavior. CD increases alcohol intake in adolescents and adults, yet it is unknown whether adolescent CD also increases long-term addiction risk. The goal of this study was to determine whether adolescent alcohol intake in CD would increase adult alcohol preference and intake in male C57BL/6 J mice. METHODS: We measured free access alcohol intake, water intake, and wheel-running activity during a normal 12 h (h) baseline photoperiod and then during shifting lighting schedules (Experiment 1) or a shortened circadian day (Experiment 2). RESULTS: In Experiment 1, altered lighting produced a persistent increase in adolescent alcohol intake and in binge-like drinking (drinking at least 5 licks per minute, with no more than a 1 min break in drinking) in adulthood, but only a transient increase in total alcohol intake for the first week after alcohol was reintroduced in adulthood. In Experiment 2, the circadian shift produced a significant increase in alcohol intake in both adolescence and adulthood. Molecular analysis demonstrated changes in plasma corticosterone and neuronal markers of stress and addiction at the conclusion of these experiments in the CD and alcohol-exposed groups. CONCLUSIONS: Thus, we conclude that circadian stress during adolescence is sufficient to produce a long-lasting susceptibility to alcohol use.

Is more better? Increased doses of high dose methotrexate and addition of rituximab is associated with improved outcomes in a large primary CNS lymphoma cohort.

Background: Primary central nervous system lymphoma (PCNSL) is a rare and aggressive primary brain tumor. While high dose methotrexate (HDMTX) regimens remain standard of care, it remains unclear if optimization of HDMTX doses and the addition of rituximab provide clinical benefit. Over the last 30 years, standard care given at Memorial Sloan Kettering Cancer Center (MSKCC) has evolved, allowing the comparison of patients receiving different numbers of HDMTX doses and those treated with and without rituximab. The purpose of this study was to describe outcomes based on treatment pattern changes. Methods: This single-center, retrospective, IRB-approved study at MSKCC included patients with immunocompetent PCNSL, age ≥18 years and diagnosed between 1/1983-12/2017. Overall survival (OS) was modeled from date of last HDMTX for analyses associating HDMTX and OS. Multivariable Cox regression models estimated hazard ratios (HR) and corresponding 95% confidence intervals (CI). Results: There were 546 patients identified with newly diagnosed PCNSL. Median overall survival (mOS) of the entire population was 4.7 years (95% CI: 3.8-5.7 years); 3.3 years (95% CI: 2.7-3.9 years) in patients diagnosed prior to 2006 and 8.1 years (95% CI: 6.6-11.1 years) in patients diagnosed 2006 onwards. Patients receiving ≥6 doses of HDMTX had improved survival compared to those receiving <6 doses of HDMTX (mOS: 7.8 vs. 4.3 years; P=0.001). Patients receiving induction rituximab had improved OS compared to those who did not receive rituximab (mOS: 10.5 vs. 3.2 years; P<0.0001). Patients receiving ≥6 doses of HDMTX and rituximab had greatest mOS at 13 years, with a 70% reduction in death (HR =0.30; 95% CI: 0.19-0.47) adjusting for treatment era, sex, and recursive partitioning analysis (RPA) classes comprising age and karnofsky performance score (KPS). Conclusions: OS for PCNSL has improved significantly over the last few decades. Patients seem to benefit with ≥6 doses of HDMTX and the addition of rituximab, an effect independent of treatment era, age, and KPS.

Design and Synthesis of Novel Cereblon Binders for Use in Targeted Protein Degradation.

Modulating the chemical composition of cereblon (CRBN) binders is a critical step in the optimization process of protein degraders that seek to hijack the function of this E3 ligase. Small structural changes can have profound impacts on the overall profile of these compounds, including depth of on-target degradation, neosubstrate degradation selectivity, as well as other drug-like properties. Herein, we report the design and synthesis of a series of novel CRBN binding moieties. These CRBN binders were evaluated for CRBN binding and degradation of common neosubstrates Aiolos and GSPT1. A selection of these binders was employed for an exploratory matrix of heterobifunctional molecules, targeting CRBN-mediated degradation of the androgen receptor.

DISC1 and reelin interact to alter cognition, inhibition, and neurogenesis in a novel mouse model of schizophrenia.

The etiology of schizophrenia (SCZ) is multifactorial, and depending on a host of genetic and environmental factors. Two putative SCZ susceptibility genes, Disrupted-in-Schizophrenia-1 (DISC1) and reelin (RELN), interact at a molecular level, suggesting that combined disruption of both may lead to an intensified SCZ phenotype. To examine this gene-gene interaction, we produced a double mutant mouse line. Mice with heterozygous RELN haploinsufficiency were crossed with mice expressing dominant-negative c-terminal truncated human DISC1 to produce offspring with both mutations (HRM/DISC1 mice). We used an array of behavioral tests to generate a behavioral phenotype for these mice, then examined the prefrontal cortex and hippocampus using western blotting and immunohistochemistry to probe for SCZ-relevant molecular and cellular alterations. Compared to wild-type controls, HRM/DISC1 mice demonstrated impaired pre-pulse inhibition, altered cognition, and decreased activity. Diazepam failed to rescue anxiety-like behaviors, paradoxically increasing activity in HRM/DISC1 mice. At a cellular level, we found increased α1-subunit containing GABA receptors in the prefrontal cortex, and a reduction in fast-spiking parvalbumin positive neurons. Maturation of adult-born neurons in the hippocampus was also altered in HRM/DISC1 mice. While there was no difference in the total number proliferating cells, more of these cells were in immature stages of development. Homozygous DISC1 mutation combined with RELN haploinsufficiency produces a complex phenotype with neuropsychiatric characteristics relevant to SCZ and related disorders, expanding our understanding of how multiple genetic susceptibility factors might interact to influence the variable presentation of these disorders.

Rituximab, Methotrexate, Carmustine, Etoposide, and Prednisone (RMBVP) for the treatment of relapsed/refractory primary central nervous system lymphoma: a retrospective single-center study.

Relapsed/refractory Primary Central Nervous System Lymphoma (R/R PCNSL) has a poor prognosis with no established preferred treatment. We report the efficacy and toxicity of a combination chemotherapy regimen: methotrexate, carmustine, etoposide, and prednisone with or without rituximab (RMBVP). This retrospective study included thirty patients who received a median of two 28-day cycles (0.5-5). The median age was 66 years (23-81); median KPS was 70 (30-90); 14 (46.7%) were women. Patients received a median of 2 prior lines of therapy and all received prior methotrexate. Of 29 evaluable patients, the overall response rate was 73.3% (n = 22). Median progression-free survival (PFS) was 15.6 months. Patients who recurred or progressed <12 months since last chemotherapy had a shorter median PFS (7.6 vs 37.6 months). Toxicity was moderate with 20% rates of severe myelosuppression. RMBVP is a tolerable treatment option for R/R PCNSL, with favorable response rates in those with recurrent disease.

Normal human lymph node T follicular helper cells and germinal center B cells accessed via fine needle aspirations.

Germinal centers (GC) are critically important for maturation of the antibody response and generation of memory B cells, processes that form the basis for long-term protection from pathogens. GCs only occur in lymphoid tissue, such as lymph nodes, and are not present in blood. Therefore, GC B cells and GC T follicular helper (TFH) cells are not well-studied in humans under normal healthy conditions, due to the limited availability of healthy lymph node samples. We used a minimally invasive, routine clinical procedure, lymph node fine needle aspirations (LN FNAs), to obtain LN cells from healthy human subjects. This study of 73 LNs demonstrates that human LN FNAs are a safe and feasible technique for immunological research, and suggests benchmarks for human GC biology under noninflammatory conditions. The findings indicate that assessment of the GC response via LN FNAs will have application to the study of human vaccination, allergy, and autoimmune disease.

Systems Biology Methods Applied to Blood and Tissue for a Comprehensive Analysis of Immune Response to Hepatitis B Vaccine in Adults.

Conventional vaccine design has been based on trial-and-error approaches, which have been generally successful. However, there have been some major failures in vaccine development and we still do not have highly effective licensed vaccines for tuberculosis, HIV, respiratory syncytial virus, and other major infections of global significance. Approaches at rational vaccine design have been limited by our understanding of the immune response to vaccination at the molecular level. Tools now exist to undertake in-depth analysis using systems biology approaches, but to be fully realized, studies are required in humans with intensive blood and tissue sampling. Methods that support this intensive sampling need to be developed and validated as feasible. To this end, we describe here a detailed approach that was applied in a study of 15 healthy adults, who were immunized with hepatitis B vaccine. Sampling included ~350 mL of blood, 12 microbiome samples, and lymph node fine needle aspirates obtained over a ~7-month period, enabling comprehensive analysis of the immune response at the molecular level, including single cell and tissue sample analysis. Samples were collected for analysis of immune phenotyping, whole blood and single cell gene expression, proteomics, lipidomics, epigenetics, whole blood response to key immune stimuli, cytokine responses, in vitro T cell responses, antibody repertoire analysis and the microbiome. Data integration was undertaken using different approaches-NetworkAnalyst and DIABLO. Our results demonstrate that such intensive sampling studies are feasible in healthy adults, and data integration tools exist to analyze the vast amount of data generated from a multi-omics systems biology approach. This will provide the basis for a better understanding of vaccine-induced immunity and accelerate future rational vaccine design.