Impact of CMV Reactivation, Treatment Approaches, and Immune Reconstitution in a Nonmyeloablative Tolerance Induction Protocol in Cynomolgus Macaques.

BACKGROUND: Cytomegalovirus (CMV) infection is a serious complication in immunosuppressed patients, specifically transplant recipients. Here, we describe the development and use of an assay to monitor the incidence and treatment of CMV viremia in a Cynomolgus macaque model of bone marrow transplantation (BMT) for tolerance induction. We address the correlation between the course of viremia and immune reconstitution. METHODS: Twenty-one animals received a nonmyeloablative conditioning regimen. Seven received cyclosporine A for 28 days and 14 received rapamycin. A CMV polymerase chain reaction assay was developed and run twice per week to monitor viremia. Nineteen recipients were CMV seropositive before BMT. Immune reconstitution was monitored through flow cytometry and CMV viremia was tracked via quantitative polymerase chain reaction. RESULTS: Recipients developed CMV viremia during the first month post-BMT. Two animals developed uncontrollable CMV disease. CMV reactivation occurred earlier in cyclosporine A-treated animals compared with those receiving rapamycin. Post-BMT, T-cell counts remained significantly lower compared with pretransplant levels until CMV reactivation, at which point they increased during the viremic phase and approached pretransplant levels 3 months post-BMT. Management of CMV required treatment before viremia reached 10 000 copies/mL; otherwise clinical symptoms were observed. High doses of ganciclovir resolved the viremia, which could subsequently be controlled with valganciclovir. CONCLUSIONS: We developed an assay to monitor CMV in Cynomolgus macaques. CMV reactivation occurred in 100% of seropositive animals in this model. Rapamycin delayed CMV reactivation and ganciclovir treatment was effective at high doses. As in humans, CD8 T cells proliferated during CMV viremia.

Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity.

Although the signaling events that induce different forms of programmed cell death are well defined, the subsequent immune responses to dying cells in the context of cancer remain relatively unexplored. Necroptosis occurs downstream of the receptor-interacting protein kinases RIPK1 and RIPK3, whose activation leads to lytic cell death accompanied by de novo production of proinflammatory mediators. Here, we show that ectopic introduction of necroptotic cells to the tumor microenvironment promotes BATF3+ cDC1- and CD8+ leukocyte-dependent antitumor immunity accompanied by increased tumor antigen loading by tumor-associated antigen-presenting cells. Furthermore, we report the development of constitutively active forms of the necroptosis-inducing enzyme RIPK3 and show that delivery of a gene encoding this enzyme to tumor cells using adeno-associated viruses induces tumor cell necroptosis, which synergizes with immune checkpoint blockade to promote durable tumor clearance. These findings support a role for RIPK1/RIPK3 activation as a beneficial proximal target in the initiation of tumor immunity. Considering that successful tumor immunotherapy regimens will require the rational application of multiple treatment modalities, we propose that maximizing the immunogenicity of dying cells within the tumor microenvironment through specific activation of the necroptotic pathway represents a beneficial treatment approach that may warrant further clinical development.

Characterization, biology, and expansion of regulatory T cells in the Cynomolgus macaque for preclinical studies.

Reliable in vitro expansion protocols of regulatory T cells (Tregs) are needed for clinical use. We studied the biology of Mauritian Cynomolgus macaque (MCM) Tregs and developed four in vitro Treg expansion protocols for translational studies. Tregs expanded 3000-fold when artificial antigen presenting cells (aAPCs) expressing human CD80, CD58 and CD32 were used throughout the culture. When donor peripheral blood mononuclear cells (PBMCs) were used as the single source of APCs followed by aAPCs, Tregs expanded 2000-fold. Tregs from all protocols suppressed the proliferation of anti-CD2CD3CD28 bead-stimulated autologous PBMCs albeit with different potencies, varying from 1:2-1:4 Treg:PBMC ratios, up to >1:32. Reculture of cryopreserved Tregs permitted reexpansion with improved suppressive activity. Occasionally, CD8 contamination was observed and resolved by resorting. Specificity studies showed greater suppression of stimulation by anti-CD2CD3CD28 beads of PBMCs from the same donor used for stimulation during the Treg cultures and of autologous cells than of third-party PBMC responders. Similar to humans, the Treg-specific demethylated region (TSDR) within the Foxp3 locus correlated with suppressive activity and expression of Foxp3. Contrary to humans, FoxP3 expression did not correlate with CD45RA or CD127 expression. In summary, we have characterized MCM Tregs and developed four Treg expansion protocols that can be used for preclinical applications.

The Nucleotide Sensor ZBP1 and Kinase RIPK3 Induce the Enzyme IRG1 to Promote an Antiviral Metabolic State in Neurons.

As long-lived post-mitotic cells, neurons employ unique strategies to resist pathogen infection while preserving cellular function. Here, using a murine model of Zika virus (ZIKV) infection, we identified an innate immune pathway that restricts ZIKV replication in neurons and is required for survival upon ZIKV infection of the central nervous system (CNS). We found that neuronal ZIKV infection activated the nucleotide sensor ZBP1 and the kinases RIPK1 and RIPK3, core components of virus-induced necroptotic cell death signaling. However, activation of this pathway in ZIKV-infected neurons did not induce cell death. Rather, RIPK signaling restricted viral replication by altering cellular metabolism via upregulation of the enzyme IRG1 and production of the metabolite itaconate. Itaconate inhibited the activity of succinate dehydrogenase, generating a metabolic state in neurons that suppresses replication of viral genomes. These findings demonstrate an immunometabolic mechanism of viral restriction during neuroinvasive infection.

Endothelial Wnt/ß-catenin signaling reduces immune cell infiltration in multiple sclerosis.

Disruption of the blood-brain barrier (BBB) is a defining and early feature of multiple sclerosis (MS) that directly damages the central nervous system (CNS), promotes immune cell infiltration, and influences clinical outcomes. There is an urgent need for new therapies to protect and restore BBB function, either by strengthening endothelial tight junctions or suppressing endothelial vesicular transcytosis. Although wingless integrated MMTV (Wnt)/ß-catenin signaling plays an essential role in BBB formation and maintenance in healthy CNS, its role in BBB repair in neurologic diseases such as MS remains unclear. Using a Wnt/ß-catenin reporter mouse and several downstream targets, we demonstrate that the Wnt/ß-catenin pathway is up-regulated in CNS endothelial cells in both human MS and the mouse model experimental autoimmune encephalomyelitis (EAE). Increased Wnt/ß-catenin activity in CNS blood vessels during EAE progression correlates with up-regulation of neuronal Wnt3 expression, as well as breakdown of endothelial cell junctions. Genetic inhibition of the Wnt/ß-catenin pathway in CNS endothelium before disease onset exacerbates the clinical presentation of EAE, CD4+ T-cell infiltration into the CNS, and demyelination by increasing expression of vascular cell adhesion molecule-1 and the transcytosis protein Caveolin-1 and promoting endothelial transcytosis. However, Wnt signaling attenuation does not affect the progressive degradation of tight junction proteins or paracellular BBB leakage. These results suggest that reactivation of Wnt/ß-catenin signaling in CNS vessels during EAE/MS partially restores functional BBB integrity and limits immune cell infiltration into the CNS.

Response to dual HER2 blockade in a patient with HER3-mutant metastatic breast cancer.

BACKGROUND: HER3 activating mutations have been shown in preclinical models to be oncogenic and ligand-independent, but to depend on kinase-active HER2. PATIENTS AND METHODS: Whole-exome sequencing of the primary HER2-negative breast cancer and its HER2-negative synchronous liver metastasis from a 46-year-old female revealed the presence of an activating and clonal HER3 G284R mutation. RESULTS: HER2 dual blockade with trastuzumab and lapatinib as third-line therapy led to complete metabolic response in 2 weeks and confirmed radiological partial response after 8 weeks. Following the resection of the liver metastasis, the patient remains disease-free 40 weeks after initiation of the HER2 dual blockade therapy. Immunohistochemical analysis demonstrated a substantial reduction of phospho-rpS6 and phospho-AKT in the post-therapy biopsy of the liver metastasis. DISCUSSION: This is the first-in-man evidence that anti-HER2 therapies are likely effective in breast cancers harboring HER3 activating mutations.

[Stoutness and prognosis of female non-metastatic breast cancer: results from a French observational cohort study]. / Corpulence et pronostic du cancer du sein non métastatique de la femme: résultats d'une étude de cohorte observationnelle française.

AIM: Our objective was to study the prognostic value of stoutness in non-metastatic breast cancer, from a population of French women. METHODS: We constituted a large observational cohort of patients followed since a first unilateral breast cancer without distant dissemination. Stoutness was assessed using the body mass index (BMI, in kg/m(2)) at the time of diagnosis. Patients were classified into the four main categories of BMI, defined according to the World Health Organization recommendations. The risk of prognosis events was analyzed according to the BMI categories. To this end, survival analyses were achieved. RESULTS: The patients having a BMI value of at least 25 kg/m(2) presented significantly higher risks of death and metastasis recurrences when they were compared to the patients having a normal value of BMI. The multivariate analyses found a modest increase of risk, about 10 to 20%, depending on the degree of fatness. It reached about 20 to 50% according to the univariate analyses. The obese patients (BMI >or= 30 kg/m(2)) had an increase of 50% of the risk of second primary cancers, comparatively to the patients having a normal value of BMI. Regarding contralateral, nodal and local recurrences, the survival analyses did not achieve any significant relationship with stoutness. CONCLUSION: A poorer prognosis is observed when breast cancer patients have a value of BMI that matches at least with overweight. Contrary to the results of few recent surveys, underweight patients do not present a poorer prognosis than normal weight patients. Excess of weight represents a modifiable factor in order to improve female breast cancer prognosis.

Prostate cancer is highly predictable: a prognostic equation based on all morphological variables in radical prostatectomy specimens.

PURPOSE: We determine whether biochemical prostate specific antigen (PSA) failure can be accurately predicted from preoperative serum PSA combined with 6 morphological variables from radical retropubic prostatectomy specimens in men with peripheral zone cancers. The unexpected limitation imposed by preoperative serum PSA on biochemical failure led us to compare peripheral zone to transition zone cancers. MATERIALS AND METHODS: A total of 326 peripheral zone and 46 transition zone cancers treated only with radical retropubic prostatectomy were followed for a minimum of 3 years (mean and median greater than 5). All prostates were sectioned at 3 mm. intervals and morphological variables were quantitated using the Stanford technique. Biochemical failure was defined as serum PSA 0.07 ng./ml. or greater and increasing. Multivariate logistic regression was used to identify variables with the most independent influence on biochemical failure and derive a clinical equation to predict failure in peripheral zone cancers. The validity of the predictive equation was assessed by out of sample validation and cross validation techniques. The 46 transition zone cancers were compared to the 326 peripheral zone cancers by Student's t and Wilcoxon tests. RESULTS: Of the peripheral zone failures 60% occurred in the first year after radical retropubic prostatectomy and 95% had occurred by the end of year 4. The highest preoperative serum PSA was 23 ng./ml. among the 181 men biochemically free of disease. Only 15.8% of 57 men with PSA greater than 15 ng./ml. were biochemically disease-free. For the 48 transition zone cancers cure rates were independent of serum PSA with 6 men having PSA greater than 50 ng./ml. Biochemical disease-free status was noted in 80% of transition zone compared to 56% of peripheral zone cancers (p = 0.0009). The most important variables predicting biochemical disease-free status for peripheral zone cancers were percent Gleason grade 4/5, cancer volume, serum PSA and prostate weight. Foci of vascular invasion, intraductal cancer and lymph nodes were less significant variables, and capsular penetration, positive surgical margins and seminal vesical invasion were insignificant. The multivariate logistic equation for predicting failure in peripheral zone cancers was highly accurate and requires only 2 to 3 minutes with a simple calculator. CONCLUSIONS: Failure of radical retropubic prostatectomy to cure peripheral zone prostate cancer is highly predictable based on 6 morphological variables from the prostatectomy specimen and serum PSA. The level of serum PSA profoundly limits biochemical cure rates in peripheral zone cancers. Transition zone cancers have a high cure rate, despite high serum PSA and adverse morphological variables. Men with serum PSA greater than 15 and perhaps even greater than 10 ng./ml. have such a low cure rate for peripheral zone cancer that re-biopsy attempts appear indicated to prove a transition zone location or else therapy other than radical retropubic prostatectomy should be sought. Pathologists should indicate whether the primary (largest) cancer is in the peripheral or transition zone to prevent overoptimistic reports of cure with radical prostatectomy procedures, as 85% of all tumors are in the peripheral zone.

Biological determinants of cancer progression in men with prostate cancer.

CONTEXT: The recent increase in ability to diagnose prostatic adenocarcinoma has created a dilemma for treatment decisions. OBJECTIVE: To determine whether prostate cancer progression is associated with a modified version of the Gleason grading system together with selected morphologic and clinical variables. DESIGN: Retrospective analysis of a cohort of patients with peripheral zone prostate cancers who underwent surgery between August 1983 and July 1992. SETTING: University hospital. PATIENTS: Radical prostatectomy specimens from 379 men treated only by surgical excision were prospectively studied for 8 morphologic variables using previously standardized techniques. Variables were percentage of each cancer occupied by Gleason grade 4/5 (% Gleason grade 4/5, the Stanford modified Gleason scale), cancer volume, vascular invasion, lymph node involvement, seminal vesicle invasion, capsular penetration, positive surgical margin, prostate weight, and preoperative prostate-specific antigen (PSA) level. MAIN OUTCOME MEASURE: Biochemical progression of prostate cancer as indicated by serum PSA level of 0.07 ng/mL and increasing. RESULTS: Cancer grade expressed as % Gleason grade 4/5 and cancer volume were highly predictive of disease progression. In a Cox proportional hazards model that included % Gleason grade 4/5, the traditional Gleason score was not an independent predictor of treatment failure. Positive lymph node findings and intraprostatic vascular invasion were the only other variables that remained significant at the .01 level. CONCLUSION: The % Gleason grade 4/5, cancer volume, positive lymph node findings, and intraprostatic vascular invasion were independently associated with prostate cancer progression, defined by an increasing PSA level. Techniques to accurately measure cancer volume and % Gleason grade 4/5 are needed to better predict which patient will experience cancer progression. The commonly accepted predictors of progression-capsular penetration and positive surgical margins-were not independently predictive of failure after radical prostatectomy.

Effects of crystalloid solutions on circulating lactate concentrations: Part 1. Implications for the proper handling of blood specimens obtained from critically ill patients.

OBJECTIVES: a) To test the hypothesis that circulating lactate concentrations are the same in simultaneously collected arterial and central venous blood specimens; b) to test the hypothesis that even small amounts of crystalloid solutions, which are inadequately "cleared" from these indwelling arterial and venous catheters, can lead to clinically important and misleading changes in the measured lactate values. DESIGN: A prospective, multiexperiment study. SETTING: A critical care research laboratory and a 20-bed intensive care unit (ICU). PATIENTS: Three hundred fifty-five patients. INTERVENTIONS: Blood samples were collected. MEASUREMENTS AND MAIN RESULTS: Experiment 1: Simultaneously collected arterial and central venous blood specimens were obtained on 148 occasions from 48 medical ICU patients receiving no lactated Ringer's solution (RL). Arterial and central venous lactate values were nearly identical in these patients. The correlation between the arterial and central venous lactate concentrations was excellent (r2 = .85; p < .0001) and the agreement between the arterial and central venous lactate concentrations was also excellent (bias and precision = 0.04 mmol/L and +/- 0.38 mmol/L, respectively). Experiment 2: Arterial and mixed venous blood samples were obtained from 100 percutaneous transluminal coronary angioplasty (PTCA) and 75 cardiac surgical patients immediately before the performance of these cardiac procedures. We found the central venous lactate concentrations to be higher than arterial lactate values in the cardiac surgical group, and there was a very poor correlation (r2 = .07) between arterial and central venous lactate values in the cardiac surgical group. The correlation between central venous and arterial lactate concentrations in the PTCA patients was excellent (r2 = .84) and similar to the findings of experiment 1. Since the cardiac surgical patients received RL and the PTCA patients received no RL, we speculated that the intravenous infusion of RL in the cardiac surgical group accounted for these discordant findings. To test this speculation, we performed experiments 3 and 4. Experiment 3: In a large bench study, blood specimens were divided into multiple 1-mL aliquot portions, to which 0.01, 0.05, 0.10, 0.50, or 1.0 mL of various crystalloid solutions, containing or not containing RL, were added. In a volume-dependent and linear manner, solutions containing RL increased the circulating lactate concentration from 10% to > 400% of the baseline lactate value. In a volume-dependent and linear fashion, the non-RL crystalloid solutions decreased the lactate concentration by 0 to 66% of the baseline nondiluted lactate concentration. Experiment 4: In 30 different cardiac surgical patients, we simultaneously obtained central venous and arterial blood specimens. Patients this time received no RL, and catheter lines were adequately cleared (removal > 5 mL) of crystalloid solutions. We found a correlation (r2 = .82; p < .0001) that was virtually identical to the findings of experiment 1 and to the findings in the PTCA group of experiment 2. CONCLUSIONS: a) Arterial and central venous lactate concentrations are similar in hemodynamically stable critically ill patients, b) Even small amounts of RL-containing solutions in catheters used for blood sampling may cause false increases in the circulating lactate concentration. c) Even small amounts of non-RL crystalloid solutions in catheters used for blood sampling may falsely decrease circulating lactate values. d) When blood specimens are drawn from indwelling catheters, all crystalloid solutions must be cleared from the line.

Use of the glycerin test in the diagnosis of post-traumatic perilymphatic fistulas.

A new application of the glycerin test in the diagnosis of post-traumatic perilymphatic fistulas is described. Temporary disappearance of abnormal responses to the fistula and Quix tests and improvement in the hearing occurred. The glycerin test was useful in confirming the diagnosis of post-traumatic perilymphatic fistula in 13 patients in whom fistulas were found at middle ear exploration.