Pomalidomide in combination with dexamethasone results in synergistic anti‐tumour responses in pre‐clinical models of lenalidomide‐resistant multiple myeloma

Pomalidomide is an IMiD^® immunomodulatory agent, which has shown clinically significant benefits in relapsed and/or refractory multiple myeloma (rrMM) patients when combined with dexamethasone, regardless of refractory status to lenalidomide or bortezomib. (Schey et al, 2004 ; San Miguel et al, 2013; Richardson et al, 2014; Scott, 2014 ) In this work, we present preclinical data showing that the combination of pomalidomide with dexamethasone (PomDex) demonstrates potent anti‐proliferative and pro‐apoptotic activity in both lenalidomide‐sensitive and lenalidomide‐resistant MM cell lines. PomDex also synergistically inhibited tumour growth compared with single‐agent treatment in xenografts of lenalidomide‐resistant H929 R10‐1 cells. Typical hallmarks of IMiD compound activity, including IKZF3 (Aiolos) degradation, and the downregulation of interferon regulatory factor (IRF) 4 and MYC, seen in lenalidomide‐sensitive H929 MM cell lines, were also observed in PomDex‐treated lenalidomide‐resistant H929 MM cells. Remarkably, this resulted in strong, synergistic effects on the induction of apoptosis in both lenalidomide‐sensitive and resistant MM cells. Furthermore, gene expression profiling revealed a unique differential gene expression pattern in PomDex‐treated samples, highlighted by the modulation of pro‐apoptotic pathways in lenalidomide‐resistant cells. These results provide key insights into molecular mechanisms of PomDex in the lenalidomide‐resistant setting.     

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.The germinal center (GC) reaction is a critical immunological process that occurs in draining lymph nodes after immunization (1). The GC response consists of antigen-specific B cells undergoing affinity maturation through a process of somatic hypermutation (SHM) of the B-cell receptor. SHM is necessary for producing high-affinity Ab responses after immunizations and infections. Influenza neutralizing Abs have substantial SHM. Particularly high levels of SHM, 15–30% amino acid mutation (2, 3), are present and necessary for broad Ab neutralization of diverse HIV strains (4, 5). Therefore, as candidate influenza and HIV vaccines are evaluated for the ability to induce broadly neutralizing antibodies (bnAbs), the quantitation and functional characterization of GC responses will be a key parameter for study. Serological analysis of vaccine-specific Ab titers provides important information, but those data are limited. Serological outcomes are measured at time points long after initial immunizations. Neutralizing Ab responses are commonly only measurable after multiple boosts. Those outcomes likely depend on GC activity and affinity maturation at much earlier time points. Several state of the art HIV vaccine strategies rely on long, multistage immunization protocols (6, 7). With bnAb responses as the goal, means of early analysis of the immune response will be essential to understand and improve on vaccination schemes that may end in failure or only partial success. One critical parameter to assess will be the ability of each immunization to generate GC responses.Central to the GC reaction and SHM is the interaction of GC B cells with germinal center T follicular helper (GC Tfh) cells (8). GC Tfh cells are both required and limiting for the GC reaction (9, 10). GC Tfh cells control the number of GC B-cell divisions and therefore, the amount of SHM by individual GC B-cell clones (11). Currently, the preferred means of quantifying the GC response is the cellular enumeration and analysis of GC Tfh and GC B cells (8). However, in human and nonhuman primate (NHP) vaccination studies, direct analysis of draining lymph node tissues is impractical or undesirable for fear of removing the primary site of the ongoing immune response. Therefore, identification of a plasma biomarker for GC activity would be of great value in immunization studies as well as have utility in a number of other biomedically relevant contexts.The CXCL13 [chemokine (C-X-C motif) ligand 13]–CXCR5 [chemokine (C-X-C motif) receptor 5] chemokine axis plays a major role in organizing both B-cell follicles and GCs (12, 13). CXCL13 is expressed by both follicular dendritic cells (14) and GC Tfh cells (15, 16) in the B-cell follicles. B- and Tfh-cell expressions of CXCR5, the receptor for CXCL13, are necessary for migration to the B-cell follicle. Although CXCL13 acts locally, it can also be detected in human plasma in the steady state, and perturbations in plasma CXCL13 have been associated with immune activity (17–21). Because GC Tfh cells regulate the size of the GC response and can be major producers of CXCL13, we explored whether plasma CXCL13 changes may reflect lymphoid tissue GC activity.     

Activation of Src kinase Lyn by the Kaposi sarcoma-associated herpesvirus K1 protein: implications for lymphomagenesis

The K1 gene of Kaposi sarcoma-associated herpesvirus (KSHV) encodes a transmembrane glycoprotein bearing a functional immunoreceptor tyrosine-based activation motif (ITAM). Previously, we reported that the K1 protein induced plasmablastic lymphomas in K1 transgenic mice, and that these lymphomas showed enhanced Lyn kinase activity. Here, we report that systemic administration of the nuclear factor kappa B (NF-kappaB) inhibitor Bay 11-7085 or an anti-vascular endothelial growth factor (VEGF) antibody significantly reduced K1 lymphoma growth in nude mice. Furthermore, in KVL-1 cells, a cell line derived from a K1 lymphoma, inhibition of Lyn kinase activity by the Src kinase inhibitor PP2 decreased VEGF induction, NF-kappaB activity, and the cell proliferation index by 50% to 75%. In contrast, human B-cell lymphoma BJAB cells expressing K1, but not the ITAM sequence-deleted mutant K1, showed a marked increase in Lyn kinase activity with concomitant VEGF induction and NF-kappaB activation, indicating that ITAM sequences were required for the Lyn kinase-mediated activation of these factors. Our results suggested that K1-mediated constitutive Lyn kinase activation in K1 lymphoma cells is crucial for the production of VEGF and NF-kappaB activation, both strongly implicated in the development of KSHV-induced lymphoproliferative disorders.     

The role of endoscopic retrograde cholangiopancreatography in acute and chronic pancreatitis

Endoscopic retrograde cholangiopancreatography (ERCP) plays a pivotal role in the management of patients with acute and chronic pancreatitis. Whereas endoscopic observation during ERCP permits recognition of abnormalities involving the major and minor duodenal papillae such as papillary tumors or choledochocele, radiographic evaluation enables the detection of structural abnormalities of pancreaticobiliary ducts like strictures or calculi. Sphincter of Oddi manometry, a technical advance of ERCP, is essential for the diagnosis of sphincter of Oddi dysfunction, which may present clinically as recurrent pancreatitis. Because structural alterations of the pancreatic duct forms the hallmark of chronic pancreatitis, ERCP is highly sensitive and specific in diagnosing chronic pancreatitis. Apart from its diagnostic role, ERCP offers a variety of possibilities for therapeutic interventions in selected problems associated with pancreatitis. Endoscopic papillectomy and mucosal resection for tumors of the papilla, unroofing of a choledochocele, and sphincterotomy for sphincter ablation in sphincter of Oddi dysfunction are some of the therapeutic interventions possible during ERCP. Pancreatic ductal hypertension, which is considered to be the major pathophysiologic mechanism for disabling abdominal pain in chronic pancreatitis, also can be managed by ERCP-directed treatments. Pancreatic sphincterotomy, dilation of strictures, lithotripsy, extraction of calculi, and deployment of endoprosthesis constitute the commonly used therapeutic techniques in this situation. Besides offering a noninvasive alternative, these treatments are associated with a favorable clinical outcome comparable with that of operative treatments. Nevertheless, complications such as acute pancreatitis, bleeding, perforation, or sepsis may occur in 5% to 10% of patients undergoing these procedures. Therefore, careful selection of patients, appropriate preoperative care, and a team approach, including surgeon, interventional radiologist, and endoscopist, are important.     

Infrequency of colonization with Oxalobacter formigenes in inflammatory bowel disease: possible role in renal stone formation

BACKGROUND AND AIM: Calcium oxalate renal stones (RS) and hyperoxaluria are common in patients with inflammatory bowel disease (IBD). The absence of intestinal oxalate degrading bacteria, Oxalobacter formigenes, may cause hyperoxaluria in IBD. The aim of the present study was to examine: (i) the colonization of O. formigenes in patients with IBD and controls and to correlate its presence with urinary oxalate excretion; and (ii) urinary analytes contributing to RS in IBD. METHODS: Stool samples were studied for O. formigenes using polymerase chain reaction and Southern blotting in patients with IBD (n = 48: ulcerative colitis, 37; Crohn's disease, 11), RS (n = 87) and healthy subjects that were used as controls (n = 48). Levels of urinary oxalate, citrate, calcium, magnesium, creatinine and uric acid were estimated spectrophotometrically in each patient and in 13 controls for 24 h. RESULTS: Five of the 48 (10.4%) patients with IBD had RS. Five of the 48 (10.4%) patients with IBD, 25 of the 87 (29%) with RS and 27 of the 48 (56%) controls were colonized with O. formigenes (P < 0.001 for RS vs controls and P = 0.01 for RS vs IBD). Patients without O. formigenes had higher urinary oxalate than those with it (IBD, median 0.48 [range 0.11-2.09]vs 0.43 [range 0.16-1.10] mmol/24 h, P = NS; RS, median 0.59 mmol/24 h, range 0.14-1.90 vs 0.44 mmol/24 h, range 0.23-0.97; P = 0.008, Mann-Whitney U-test). Median excretion of oxalate was higher in IBD and RS than in controls (0.47 [0.11-2.09], 0.56 [0.14-1.9] and 0.41 [0.21-0.62] mmol/24 h; P < 0.01), respectively. Median calcium was also higher in IBD and RS than in controls (6.50 [1.38-21.00], 6.78 [1.55-20.30] and 4.99 [1.47-9.60] mmol/24 h; P < 0.05, Kruskal-Wallis H-test), respectively. Median urinary magnesium was higher in IBD than in RS and controls (4.57 [1.50-12.30], 3.60 [0.90-6.35] and 2.49 [0.74-4.80]; P < 0.001, Kruskal-Wallis H-test), respectively. Urinary citrate excretion was comparable in IBD, RS and controls. CONCLUSIONS: Patients with IBD and RS rarely have O. formigenes in their stools as compared with controls; this may contribute to hyperoxaluria in IBD. Hyperoxaluria and hypercalciuria may contribute to RS in patients with IBD. Hypermagnesuria in patients with IBD may protect them from RS.     

Parent decision making for life support for extremely premature infants: from the prenatal through end-of-life period

Most deaths of extremely premature infants occur in the perinatal period. Yet, little is known about how parents make life support decisions in such a short period of time. In the paper, how parents make life support decisions for extremely premature infants from the prenatal period through death from the perspectives of parents, nurses, and physicians is described. Five cases, comprised of five mothers, four neonatologists, three nurses, and one neonatal nurse practitioner, are drawn from a larger collective case study. Prenatal, postnatal and end-of-life interviews were conducted, and medical record data were obtained. In an analysis by two research team members, mothers were found to exhibit these characteristics: desire for and actual involvement in life support decisions, weighing pain, suffering and hope in decision making, and wanting everything done for their infants. All mothers received decision making help and support from partners and family, but relationships with providers were also important. Finally, external resources impacted parental decision making in several of the cases. By understanding what factors contribute to parents' decision making, providers may be better equipped to prepare and assist parents when making life support decisions for their extremely premature infants.     

Type of Continuing Bonds Expression and Its Comforting Versus Distressing Nature: Implications for Adjustment Among Bereaved Mothers

This study investigated type of continuing bonds (CB) expression and its comforting versus distressing nature in relation to psychosocial adjustment among bereaved mothers. Twenty-eight mothers whose child had died within the previous five years participated in a CB interview in which they rated the extent they used each of 11 different types of CB expression during the past month and the degree to which they experienced each of the CB expressions as comforting and distressing. CB expressions involving illusions and hallucinations of the deceased child were predictive of greater distress whereas those involving belief that the deceased child was aware of the mother or communicating with her through dreams were not associated with symptoms, but instead linked to greater spirituality. Furthermore, mothers who reported CB as more comforting than distressing had lower symptom ratings. The implications of the findings for the attachment theory perspective on unresolved loss are discussed.     

Comparison of follitropin-beta administered by a pen device with conventional syringe in an ART programme - a retrospective study

Objectives:  This study compares the efficacy and patient tolerance of follitropin-β (recagon) administered using a pen device with conventional syringe in infertile couples undergoing in vitro fertilization/intracytoplasmic sperm injection treatment.
Methods:  Data for 481 patients were retrieved retrospectively for the analysis. Conventional syringe group constituted 204 patients with 217 cycles and 265 patients with 294 cycles in the pen-device group. Down-regulation was achieved with GnRH agonist.
Results:  Comparison of follitropin-β administered with pen and syringe showed the following data, respectively. A total dose of 1909·38/2100·65 IU ( P  < 0·001), duration of stimulation, 9·70/10·47 days ( P  < 0·05), oestradiol levels on the day of human chorionic gonadotropin, 1488·34/1067·63 pg/ml, number of follicles reaching >16-mm size, 9·75/7·34 ( P  < 0·05), number of oocytes retrieved, 13·84/9·55 ( P  < 0·001) and number of embryos available for freezing, 4·56/1·30 ( P  < 0·05), the above data were observed in pen/conventional syringe groups, respectively. The live birth rates per cycle were 28·85% and 30·95% in the conventional syringe/pen-device groups, respectively. Patient tolerance with respect to pain at injection site was better with the pen device ( P  < 0·025).
Conclusion:  The data show that follitropin-β administered with pen device is well tolerated and more efficacious with respect to ovarian stimulation outcome compared with the conventional syringe.     

Gp120-alum boosting of a Gag-Pol-Env DNA/MVA AIDS vaccine: poorer control of a pathogenic viral challenge

Envelope protein immunogens may improve DNA or live-vectored HIV vaccines by complementing antiviral cellular responses with Env antibodies. We tested this concept by administering two immunizations of alum-adjuvanted HIV-1 89.6 gp120 to macaques being primed at weeks 0 and 8 with SHIV 89.6 Gag-Pol-Env DNA and boosted at week 24 with SHIV-89.6 Gag-Pol-Env recombinant modified vaccinia Ankara (MVA). Three hundred micrograms of gp120 was delivered with the second DNA prime and the MVA booster. Eight months after vaccination, all animals were challenged intrarectally with the related, yet serologically distinct, SHIV-89.6P. The gp120 immunizations raised binding, but not neutralizing antibody for the challenge virus, and allowed testing of whether gp120 vaccines that fail to raise neutralizing antibody can improve protection. Following the second gp120 immunization, the plus-gp120 group showed >10 times higher levels of binding antibody than the minus-gp120 group. These levels fell and were overall similar in both groups at the time of challenge. Following the second challenge, both groups had similar temporal patterns and heights of binding and neutralizing antibodies. However, the plus-gp120 group had less consistent control of viremia and higher levels of plasma viral RNA for the first year postchallenge. Assays for complement-dependent enhancing antibody revealed a trend toward higher levels of activity in the plus-gp120 group. This trend did not reach significance in our animal groups of 8. We conclude that gp120 inoculations that fail to raise neutralizing antibody do not improve the efficacy of Gag-Pol-Env DNA/MVA vaccines.     

Nitrolinoleate,a nitric oxide-derived mediator of cell function: synthesis,characterization, and vasomotor activity

Nitric oxide (*NO) and *NO-derived reactive species rapidly react with lipids during both autocatalytic and enzymatic oxidation reactions to yield nitrated derivatives that serve as cell signaling molecules. Herein we report the synthesis, purification, characterization, and bioactivity of nitrolinoleate (LNO2). Nitroselenylation of linoleic acid yielded LNO2 that was purified by solvent extraction, silicic acid chromatography, and reverse-phase HPLC. Structural characterization was performed by IR spectroscopy, 15N-NMR, LC-negative ion electrospray mass spectroscopy (MS), and chemiluminescent nitrogen analysis. Quantitative MS analysis of cell and vessel LNO2 metabolism, using L[15N]O2 as an internal standard, revealed that LNO2 is rapidly metabolized by rat aortic smooth muscle (RASM) monolayers and rat thoracic aorta, resulting in nitrite production and up to 3-fold increases in cGMP (ED50 = 30 microM for RASM, 50 microM for aorta). LNO2 induced endothelium-independent relaxation of preconstricted rat aortic rings, which was unaffected by L(G)-nitro-l-arginine methyl ester addition and inhibited by the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a]quinoxalin-1-one and the *NO scavenger HbO2. These results reveal that synthetic LNO2, identical to lipid derivatives produced biologically by the reaction of *NO and *NO-derived species with oxidizing unsaturated fatty acids (e.g., linoleate), can transduce vascular signaling actions of *NO.