Reduction in obesity-related comorbidities: is gastric bypass better than sleeve gastrectomy?

BACKGROUND: Bariatric surgery is currently the most effective treatment for morbid obesity. It provides not only substantial weight loss, but also resolution of obesity-related comorbidities. Laparoscopic sleeve gastrectomy (LSG) has rapidly been gaining in popularity. However, there are limited data on the reduction of obesity-related comorbidities for LSG compared to laparoscopic Roux-en-Y gastric bypass (LRYGB). The aim of this study was to assess the effectiveness of laparoscopic LSG versus LRYGB for the treatment of obesity-related comorbidities. METHODS: A total of 558 patients who underwent either LSG or LRYGB for morbid obesity at the Westchester Medical Center between April 2008 and September 2010 were included. Data were collected prospectively into a computerized database and reviewed for this study. Fisher's exact test analyses compared 30-day, 6-month, and 1-year outcomes of obesity-related comorbidities. RESULTS: A total of 558 patients were included in the analysis of obesity-related comorbidity resolution; 200 underwent LSG and 358 underwent LRYGB. After 1 year, 86.2 % of the LSG patients had one or more comorbidities in remission compared to 83.1 % LRYGB patients (P = 0.688). With the exception of GERD (-0.09 vs. 50 %; P < 0.001), similar comorbidity remission rates were observed between LSG and LRYGB for sleep apnea (91.2 vs. 82.8 %; P = 0.338), hyperlipidemia (63 vs. 55.8 %; P = 0.633), hypertension (38.8 vs. 52.9 %; P = 0.062), diabetes (58.6 vs. 65.5 %; P = 0.638), and musculoskeletal disease (66.7 vs. 79.4 %; P = 0.472). CONCLUSIONS: Laparoscopic sleeve gastrectomy markedly improves most obesity-related comorbidities. Compared to LRYGB, LSG may have equal in reducing sleep apnea, hyperlipidemia, hypertension, diabetes, and musculoskeletal disease. LRYGB appears to be more effective at GERD resolution than LSG.

Analytical assay validation for acute myeloid leukemia measurable residual disease assessment by multiparametric flow cytometry.

BACKGROUND: Measurable residual disease (MRD) assessed by multiparametric flow cytometry (MFC) has gained importance in clinical decision-making for acute myeloid leukemia (AML) patients. However, complying with the recent In Vitro Diagnostic Regulations (IVDR) in Europe and Food and Drug Administration (FDA) guidance in the United States requires rigorous validation prior to their use in investigational clinical trials and diagnostics. Validating AML MRD-MFC assays poses challenges due to the unique underlying disease biology and paucity of patient specimens. In this study, we describe an experimental framework for validation that meets regulatory expectations. METHODS: Our validation efforts focused on evaluating assay accuracy, analytical specificity, analytical and functional sensitivity (limit of blank (LoB), detection (LLoD) and quantitation (LLoQ)), precision, linearity, sample/reagent stability and establishing the assay background frequencies. RESULTS: Correlation between different MFC methods was highly significant (r = 0.99 for %blasts and r = 0.93 for %LAIPs). The analysis of LAIP specificity accurately discriminated from negative control cells. The assay demonstrated a LoB of 0.03, LLoD of 0.04, and LLoQ of 0.1%. Precision experiments yielded highly reproducible results (Coefficient of Variation <20%). Stability experiments demonstrated reliable measurement of samples up to 96 h from collection. Furthermore, the reference range of LAIP frequencies in non-AML patients was below 0.1%, ranging from 0.0% to 0.04%. CONCLUSION: In this manuscript, we present the validation of an AML MFC-MRD assay using BM/PB patient specimens, adhering to best practices. Our approach is expected to assist other laboratories in expediting their validation activities to fulfill recent health authority guidelines.

Identification of accessory spleens during laparoscopic splenectomy is superior to preoperative computed tomography for detection of accessory spleens.

BACKGROUND: Missed accessory spleen (AcS) can cause recurrence of hematologic disease after splenectomy. The objective of the study was to determine whether detection of AcS is more accurate with preoperative computed tomography (CT) scan or with exploration during laparoscopic splenectomy. METHODS: A retrospective chart review was performed for 75 adult patients who underwent laparoscopic splenectomy for various hematologic disorders from 1999 to 2009. Preoperative CT scans were performed in all patients. Patients were followed for recurrence of disease, and a scintigraphy scan was performed in those with suspected missed AcS. RESULTS: The most common diagnosis was idiopathic thrombocytopenic purpura in 29 patients (39%), followed by non-Hodgkin's lymphoma in 22 patients (29%). Sixteen AcSs were found during surgery in 15 patients (20%), and preoperative CT scan identified 2 of these. Twelve AcSs were located at the splenic hilum (75%). Nine patients experienced recurrence of their disease, and none had a missed AcS on subsequent scintigraphy. Sensitivity of exploratory laparoscopy for detection of AcS was 100%, and for preoperative CT scan was 12.5% (P = .005). CONCLUSION: Exploratory laparoscopy during splenectomy is more accurate than preoperative imaging with CT scan for detection of AcS. Preoperative CT scan misses AcS frequently and should not be obtained for the purpose of its identification.

Multimarker scores of Th1 and Th2 immune cellular profiles in peripheral blood predict response and immune related toxicity with CTLA4 blockade and IFNα in melanoma.

Neoadjuvant therapy with ipilimumab in combination with high dose IFNα was evaluated in patients with locally/regionally advanced melanoma in a previously reported clinical trial [NCT01608594]. In this study, peripheral immune cell profiling was performed in order to investigate the underlying mechanisms of tumor immune susceptibility and resistance. Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 28) were collected at baseline and then at 6-weeks, 3-months and 12-months. High complexity (14-color) flow cytometry, designed to detect key immunological biomarkers was used to evaluate the frequencies of immune cell subsets. Statistical significance was determined using R-package employing Kruskal's test. We found that higher levels of Th1 cells at baseline (defined as CD45RA- CCR6- CXCR3+ CCR4-) correlated with the preoperative radiological response (p = 0.007) while higher Th2 cells (defined as CD45RA- CCR6- CXCR3- CCR4+) were associated with progressive disease (p = 0.009). A multimarker score consisting of higher levels of Th1 cells and CD8+ central memory T-cells was associated with pathologic complete response (pCR) (p = 0.041) at surgical resection. On the other hand, high TIM3 expression on T-cells correlated with gross viable tumor (p = 0.047). With regard to immune related toxicity, higher levels of phenotypically naive (defined as CCR7+CD45RA+) and effector memory (defined as CCR7-CD45RO+) CD8+ T-cells (p = 0.014) or lower levels of Th2 cells were associated with lower toxicity (p = 0.024). Furthermore, a multimarker score consisting of higher CD19+ and CD8+ cells was associated with lower toxicity (p = 0.0014). In conclusion, our study yielded mechanistic insights related to the immune impact of CTLA4 blockade and IFNα and potential biomarkers of immune response and toxicity.

Monitoring CAR-T cell kinetics in clinical trials by multiparametric flow cytometry: Benefits and challenges.

Exceptional clinical responses produced by the first chimeric antigen receptor T [CAR-T] cell therapies, and their entry into commercial markets prompted a logarithmic increase in the number of next generation CAR-T clinical trials. As a result, there is a growing interest in understanding the analytical approaches utilized for reliable monitoring of these "living" drugs, and the challenges encountered during their clinical development. Multiparametric flow cytometry (MFC) assays have played a crucial role in understanding the phenotype and function of first approved CAR-T therapies. Herein, three main areas for monitoring CAR-T therapies in clinical trials are discussed: (1) analytical considerations critical for development of MFC assays for the reliable enumeration of CAR-T levels, (2) operational challenges associated with clinical trial sampling and transportation, and (3) differential cellular kinetics observed by MFC and qPCR analyses and their relationship with efficacy (measurable residual disease levels). Initial experiences described here may enable design of fit-for-purpose tools and help to more rapidly advance the development of next generation CAR-T therapies.

Best practices for the development, analytical validation and clinical implementation of flow cytometric methods for chimeric antigen receptor T cell analyses.

Chimeric Antigen Receptor (CAR) T cells are recognized as efficacious therapies with demonstrated ability to produce durable responses in blood cancer patients. Regulatory approvals and acceptance of these unique therapies by patients and reimbursement agencies have led to a significant increase in the number of next generation CAR T clinical trials. Flow cytometry is a powerful tool for comprehensive profiling of individual CAR T cells at multiple stages of clinical development, from product characterization during manufacturing to longitudinal evaluation of the infused product in patients. There are unique challenges with regard to the development and validation of flow cytometric methods for CAR T cells; moreover, the assay requirements for manufacturing and clinical monitoring differ. Based on the collective experience of the authors, this recommendation paper aims to review these challenges and present approaches to address them. The discussion focuses on describing key considerations for the design, optimization, validation and implementation of flow cytometric methods during the clinical development of CAR T cell therapies.

Neoadjuvant Pembrolizumab and High-Dose IFNα-2b in Resectable Regionally Advanced Melanoma.

PURPOSE: Neoadjuvant immunotherapy may improve the clinical outcome of regionally advanced operable melanoma and allows for rapid clinical and pathologic assessment of response. We examined neoadjuvant pembrolizumab and high-dose IFNα-2b (HDI) therapy in patients with resectable advanced melanoma. PATIENTS AND METHODS: Patients with resectable stage III/IV melanoma were treated with concurrent pembrolizumab 200 mg i.v. every 3 weeks and HDI 20 MU/m2/day i.v., 5 days per week for 4 weeks, then 10 MU/m2/day subcutaneously 3 days per week for 2 weeks. Definitive surgery followed, as did adjuvant combination immunotherapy, completing a year of treatment. Primary endpoint was safety of the combination. Secondary endpoints included overall response rate (ORR), pathologic complete response (pCR), recurrence-free survival (RFS), and overall survival (OS). Blood samples for correlative studies were collected throughout. Tumor tissue was assessed by IHC and flow cytometry at baseline and at surgery. RESULTS: A total of 31 patients were enrolled, and 30 were evaluable. At data cutoff (October 2, 2019), median follow-up for OS was 37.87 months (range, 33.2-43.47). Median OS and RFS were not reached. Radiographic ORR was 73.3% [95% confidence interval (CI): 55.5-85.8], with a 43% (95% CI: 27.3-60.1) pCR rate. None of the patients with a pCR have had a recurrence. HDI and pembrolizumab were discontinued in 73% and 43% of patients, respectively. Correlative analyses suggested that intratumoral PD-1/PD-L1 interaction and HLA-DR expression are associated with pCR (P = 0.002 and P = 0.008, respectively). CONCLUSIONS: Neoadjuvant concurrent HDI and pembrolizumab demonstrated promising clinical activity despite high rates of treatment discontinuation. pCR is a prognostic indicator.See related commentary by Menzies et al., p. 4133.

Central pancreatectomy without anastomosis.

BACKGROUND: Central pancreatectomy has a unique application for lesions in the neck of the pancreas. It preserves the distal pancreas and its endocrine functions. It also preserves the spleen. METHODS: This is a retrospective review of 10 patients who underwent central pancreatectomy without pancreatico-enteric anastomosis between October 2005 and May 2009. The surgical indications, operative outcomes, and pathologic findings were analyzed. RESULTS: All 10 lesions were in the neck of the pancreas and included: 2 branch intraductal papillary mucinous neoplasms (IPMNs), a mucinous cyst, a lymphoid cyst, 5 neuroendocrine tumors, and a clear cell adenoma. CONCLUSION: Central pancreatectomy without pancreatico-enteric anastomosis for lesions in the neck and proximal pancreas is a safe and effective procedure. Morbidity is low because there is no anastomosis. Long term endocrine and exocrine function has been maintained.

Immunological characterization and therapeutic activity of an altered-peptide ligand, NBI-6024, based on the immunodominant type 1 diabetes autoantigen insulin B-chain (9-23) peptide.

The nonobese diabetic (NOD) mouse is a good model for human type 1 diabetes, which is characterized by autoreactive T-cell-mediated destruction of insulin-producing islet beta-cells of the pancreas. The 9-23 amino acid region of the insulin B-chain [B((9-23))] is an immunodominant T-cell target antigen in the NOD mouse that plays a critical role in the disease process. By testing a series of B((9-23)) peptide analogs with single or double alanine substitutions, we identified a set of altered peptide ligands (APLs) capable of inhibiting B((9-23))-induced proliferative responses of NOD pathogenic T-cell clones. These APLs were unable to induce proliferation of these clones. However, vaccinations with the APLs induced strong cellular responses, as measured by in vitro lymphocyte proliferation and Th2 cytokine production (i.e., interleukin [IL]-4 and IL-10, but not gamma-interferon [IFN-gamma]). These responses were cross-reactive with the native antigen, B((9-23)), suggesting that the APL-induced Th2 responses may provide protection by controlling endogenous B((9-23))-specific Th1 (i.e., IFN-gamma-producing) pathogenic responses. One of these APLs that contained alanine substitutions at residues 16 and 19 (16Y-->A, 19C-->A; NBI-6024) was further characterized for its therapeutic activity because it consistently induced T-cell responses (e.g., T-cell lines and clones) that were of the Th2 type and that were cross-reactive with B((9-23)). Subcutaneous injections of NBI-6024 to NOD mice administered either before or after the onset of disease substantially delayed the onset and reduced the incidence of diabetes. This study is the first to report therapeutic activity of an APL derived from an islet beta-cell-specific antigen in type 1 diabetes.

Rational design, synthesis, and structure-activity relationships of aryltriazoles as novel corticotropin-releasing factor-1 receptor antagonists.

Following the discovery of the very high binding affinity of 4-anilinopyrimidines against corticotropin-releasing factor receptor-1 (CRF(1)) (e.g., 1, K(i) = 2 nM), a new series of triazoles bearing different groups has been synthesized and evaluated. The compounds were prepared by cyclizations of N-acyl-S-methylisothioureas with alkylhydrazines or by cyclizations with hydrazine followed by alkylation. While members of this series showed potent binding affinity against CRF(1) receptor, there were important differences between the different regio- (7 and 12) and stereoisomeric aryltriazoles where the R(1) or R(2) side chain in 7 has an asymmetric center. In terms of overall potency, aryltriazole analogues such as 7r bearing an N-(alpha-branched benzyl)-N-propylamino side chain were the most potent, followed by analogues such as 7a, with an N-bis(cyclopropyl)methyl-N-propylamino side chain, and analogues such as 7m, with an N-(alpha-branched aliphatic)-N-propylamino side chain. While the N-propyl group was crucial for high potency, we hypothesized that the terminal methyl mimicked the 5-methyl of pyrazolo[1,5-a]pyrimidines 3 and 4. Correlation of the low-energy conformers of compounds of type 3 and 7 generated by computational analyses was very good. The size and shape of the N-alkyl group dramatically changed the potency of the triazoles, which is in contrast to the SAR seen for bicyclic CRF(1) antagonists. In general, the S-enantiomer was much more potent than the corresponding R-isomer. Furthermore, to a limited extent in the aryltriazole series the substituent on the 5-phenyl ring changed the potency up to 9-fold. (S)-1-Methyl-3-[N-(4-fluorophenylpentyl)-N-propyl]amino-5-(2-methoxy-4-dichlorophenyl)-1H-[1,2,4]triazole [(S)-7r] showed very potent binding affinity (K(i) = 2.7 nM) to CRF(1) receptors with an IC(50) of 49 nM in a cAMP inhibition assay.

A high-throughput chemotaxis assay for pharmacological characterization of chemokine receptors: Utilization of U937 monocytic cells.

INTRODUCTION: Higher-throughput chemotaxis assays have had limited use in chemokine receptor pharmacology studies mainly because of the unavailability of optimal assay formats in addition to an incompatibility of chemotactic cell backgrounds with other pharmacological assays. Here, we developed a high-throughput 96-well chemotaxis assay for leukocytic cell lines and identified the human U937 monocytic line as an excellent cell background for both chemotaxis and the high-throughput calcium mobilization Fluorescent Imaging Plate Reader (FLIPR) assay. METHODS: Optimal chemotactic conditions were developed using the Neuroprobe MBA96 nondisposable and the Millipore MultiScreen-MIC disposable apparatuses with responses to CXC chemokine receptor (CXCR)-4 endogenously expressed on the human H9 T lymphoma line, and confirmed with Jurkat T cell and U937 monocytic cell lines. RESULTS: The U937 cell line was chosen for site-directed mutagenesis studies with CC chemokine receptor (CCR)-7 because this cell line did not endogenously express this receptor, it demonstrated a good chemotaxis index, and it showed an exceptional ability to mobilize calcium measured via FLIPR. Using the Millipore MultiScreen-MIC and FLIPR assays, alanine substitutions at K130 and Q227 caused threefold shifts in potency for the CCR7 ligand, CCL19, whereas that at K137 had no effect. DISCUSSION: Because these CCR7 mutations have previously been shown not to affect ligand binding, our results here show that these residues are specifically involved in receptor activation signals critical to chemotaxis and underscore the importance of using the U937 cell background to confirm results of chemotaxis with those of the FLIPR assay.

Conformational states of the corticotropin releasing factor 1 (CRF1) receptor: detection, and pharmacological evaluation by peptide ligands.

Previous corticotropin releasing factor 1 (CRF1) receptor characterization has been performed using radiolabeled agonists, which bind predominantly the receptor-G-protein complex. The pharmacological profile of other receptor states, and their abundance, remain poorly characterized. Here we investigated the affinity states of the CRF1 receptor heterologously expressed in Ltk- cells and endogenously expressed in rat cerebellum. In L-CRF1 cell membranes, three agonist affinity states were detected: a very-high affinity receptor-G-protein complex state (eliminated by GTPgammaS) bound by [125I]sauvagine (43 pM, RG); a high affinity state insensitive to GTPgammaS bound by [125I]sauvagine (1.4 nM, termed RO); and a low affinity G-protein-uncoupled state detected by sauvagine displacement of [125I]astressin, a labeled antagonist (120 nM, R). The relative abundance of RG:RO:R was 18%:16%:66%. All three states were demonstrated in rat cerebellum with similar relative abundance (15%:16%:69%). The R state bound CRF with low affinity (270-330 nM), displayed a novel rank order of ligand affinity, and represented the majority of the receptor population in both receptor preparations. This study provides a framework to identify CRF1 receptor conformational states in various receptor preparations.

Downregulation of TGF-ßRII in T effector cells leads to increased resistance to TGF-ß-mediated suppression of autoimmune responses in type I diabetes.

Tregs play an important role in controlling immune responses, particularly autoimmunity. In NOD mouse model, an excellent model for autoimmune diabetes, transfer of Tregs was shown to prevent diabetes, whereas depletion of Tregs in vivo enhanced disease progression, suggesting that Treg dysfunction contributes to the pathogenesis of diabetes. However, the mechanisms leading to Treg dysfunction and their role in diabetes progression has remained unclear. In this study we assessed quantitative and qualitative changes in Tregs during the development of autoimmune diabetes in NOD. We compared female NOD with males that have similar predisposition to but a lower incidence of diabetes and found that Treg numbers remained unchanged between 6 to 16 weeks of age in both groups. Although female Tregs produced lower TGF-ß compared to male, regulatory function of female Tregs was only marginally inferior to male upon GAD65 autoantigen stimulation. GAD65-reactive female Teffectors were more responsive and progressively became refractory to regulation compared to male effectors, in part due to lower expression of TGF-ß RII, accounting for reduced sensitivity to Tregs. Moreover, we unexpectedly found that TGF-ß suppressed IFN-γ production to GAD65 antigen in male, not in female responders. These data suggest that TGF-ß plays a major role in Teff resistance to regulation and Treg dysfunction, and may account for autoimmune diabetes. Our study implies that development of a successful supplemental Treg therapy for halting autoimmunity may require further understanding of Teff responses to regulation in order to generate highly effective Tregs.

Transforming growth factor beta 1 (TGF-beta1) and rapamycin synergize to effectively suppress human T cell responses via upregulation of FoxP3+ Tregs.

BACKGROUND: The major obstacle faced by patients with type 1 diabetes who undergo islet transplantation is a gradual decline in insulin independence. This decline may reflect alloimmune rejection, autoimmune recurrence and toxicity of drugs such as rapamycin to islet beta cells. Thus, there is a pressing need to refine immunosuppressive protocols in order to reduce toxicity to islet grafts and yet prevent rejection. Recent studies demonstrated that TGF-beta1 is a critical cytokine for the regulation of immune responses. In naive T cells, TGF-beta1 induces FoxP3(+) regulatory T cells and thus could promote transplant tolerance. In this study, in vitro experiments were performed to determine whether TGF-beta1 could synergize with low-dose rapamycin and inhibit T cell activation and production of inflammatory cytokines, as well as enhance FoxP3 expression for potential application in islet transplantation. METHODS: Human peripheral blood mononuclear cells were stimulated with either anti-CD3/CD28 or anti-CD3 during TGF-beta1 and rapamycin treatment. RESULTS: TGF-beta1 inhibited T cell proliferation induced with anti-CD3 stimulation, but not with anti-CD3/CD28 stimulation. The combination of these reagents produced a synergistic inhibition of T cell proliferation induced with both anti-CD3/CD28 and anti-CD3 stimulations. Moreover, TGF-beta1 and rapamycin significantly suppressed cytokine production and induced regulatory T cells by upregulating FoxP3 expression. CONCLUSIONS: These results suggest that the combination of TGF-beta1 and low-dose rapamycin can potently inhibit T cell responses in vivo and would be beneficial in supporting islet graft survival by limiting toxicity and preventing immune rejection.

Pharmacological characterization of CXC chemokine receptor 3 ligands and a small molecule antagonist.

The CXC chemokine receptor 3 (CXCR3) is predominantly expressed on T helper type 1 (Th1) cells that are involved in inflammatory diseases. The three CXCR3 ligands CXCL9, CXCL10, and CXCL11 are produced at sites of inflammation and elicit migration of pathological Th1 cells. Here, we are the first to characterize the pharmacological potencies and specificity of a CXCR3 antagonist, N-1R-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl-N-pyridin-3-ylmethyl-2-(4-fluoro-3-trifluoromethyl-phenyl)-acetamide (NBI-74330), from the T487 small molecule series. NBI-74330 demonstrated potent inhibition of [(125)I]CXCL10 and [(125)I]CXCL11 specific binding (K(i) of 1.5 and 3.2 nM, respectively) and of functional responses mediated by CXCR3, such as ligand-induced guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding, calcium mobilization, and cellular chemotaxis (IC(50) of 7 to 18 nM). NBI-74330 was selective for CXCR3 because it showed no significant inhibition of chemotactic responses to other chemokines and did not inhibit radioligand binding to a panel of nonchemokine G-protein coupled receptors. There was a striking difference in potencies among the three CXCR3 ligands, with CXCL11 >> CXCL10 > CXCL9. A comparison of the rank order of K(i) values with the rank order of monocyte production levels of these three ligands revealed a precise inverse correlation, suggesting that the weaker receptor affinities of CXCL9 and CXCL10 were physiologically compensated for by an elevated expression, perhaps to maintain effectiveness of each ligand under physiological conditions.

Identification of CC chemokine receptor 7 residues important for receptor activation.

The binding pocket of family A GPCRs that bind small biogenic amines is well characterized. In this study we identify residues on CC chemokine receptor 7 (CCR-7) that are involved in agonist-mediated receptor activation but not in high affinity ligand binding. The mutations also affect the ability of the ligands to induce chemotaxis. Two of the residues, Lys3.33(137) and Gln5.42(227), are consistent with the binding pocket described for biogenic amines, while Lys3.26(130) and Asn7.32(305), are found at, or close to, the cell surface. Our observations are in agreement with findings from other peptide and chemokine receptors, which indicate that receptors that bind larger ligands contain contact sites closer to the cell surface in addition to the conventional transmembrane binding pocket. These findings also support the theory that chemokine receptors require different sets of interactions for high affinity ligand binding and receptor activation.