Elevated soluble MUC1 levels and decreased anti-MUC1 antibody levels in patients with multiple myeloma

Soluble MUC1 (sMUC1) levels are elevated in many MUC1(+) cancers. We and others have shown that MUC1 is expressed on multiple myeloma (MM) plasma cells and B cells. In this study, we measured sMUC1 levels in bone marrow (BM) plasma from 71 MM patients and 21 healthy donors (HDs), and in peripheral blood (PB) plasma from 42 MM patients and 13 HDs using an immunoassay that detects the CA27.29 epitope of MUC1. sMUC1 levels were found to be significantly greater (mean 31.76 U/mL, range 5.69 to 142.48 U/mL) in MM patient BM plasma versus HD BM plasma (mean 9.68 U/mL, range 0.65 to 39.83 U/mL) (P <. 001). Importantly, BM plasma sMUC1 levels were related to tumor burden because sMUC1 levels were significantly higher for MM patients with active disease (34.62 U/mL, range 5.69 to 142.48 U/mL) versus MM patients with minimal residual disease (16.16 U/mL, range 5.7 to 56.68 U/mL) (P =.0026). sMUC1 levels were also elevated in the PB plasma of MM patients (32.79 U/mL, range 4.15 to 148.84 U/mL) versus HDs (18.47 U/mL, range 8.84 to 42.49) (P =.0052). Lastly, circulating immunglobulin M (IgM) and IgG antibodies to MUC1 were measured in 114 MM patients and 31 HDs, because natural antibodies to MUC1 have been detected in patients with other MUC1-bearing malignancies. These studies demonstrated lower levels of circulating IgM (P <.001) and IgG (P =.078) antibodies to MUC1 in MM patients compared with HDs. Our data therefore show that in MM patients, sMUC1 levels are elevated and correlate with disease burden, whereas anti-MUC1 antibody levels are decreased.     

Annual risk of tuberculous infection in four defined zones of India: a comparative picture.

SETTING: Twenty-six selected districts in India. OBJECTIVES: To estimate the average annual risk of tuberculous infection (ARTI) in four defined zones in the country. STUDY DESIGN: A tuberculin survey was conducted in selected clusters of 26 districts in four defined zones of India. Children 1-9 years of age were subjected to tuberculin testing with ITU PPD RT23 with Tween 80, and the maximum transverse diameter of induration was measured 72 h later. Prevalence of infection was estimated using the cut-off point method (Method I) and the mirror-image technique (Method II) among children without bacille Calmette-Guérin scar. Results from individual zones have been reported earlier, and the results from all four zones are presented here as a consolidated summary. RESULTS: The ARTI computed from estimated prevalence was found to be lowest in the southern zone (Method I: 1.1%, Method II: 1.0%). It was higher in the eastern zone (1.3% by both methods) and highest in the western (Method I: 1.8%, Method II: 1.6%) and northern zones (1.9% by both methods). The proportion of infected children was found to be significantly higher in urban than in rural areas in all zones. CONCLUSION: The intensified tuberculosis control efforts need to be sustained for many years.     

Improved tuberculosis case detection through public-private partnership and laboratory-based surveillance, Kannur District, Kerala, India, 2001-2002.

BACKGROUND: Efforts to intensify global tuberculosis (TB) control are limited by difficulties in coordinating with private doctors. More than half of Indian TB patients may initially consult a private provider, but many are neither diagnosed accurately nor treated effectively. We established and evaluated a public-private partnership based on surveillance of TB detected in private laboratories and use of standardised directly observed treatment regimens. METHODS: In one district, the governmental TB control programme offered training in microscopy to all large private sector laboratories, and educated private physicians on the importance of microscopy for TB diagnosis. We reviewed records from participating private laboratories and all publicly diagnosed patients. RESULTS: Of 2328 pulmonary TB patients registered from July 2001 to December 2002, 404 (17%) were detected in the private sector. The annual new AFB-positive case notification rate increased by 21%, from 27.8/100,000 in 2000 to 33.5/100,000 in 2002. Surveillance at private laboratories found an additional 260 nonregistered AFB-positive patients. CONCLUSIONS: This public-private partnership substantially increased TB case detection and established a sustainable framework for private sector involvement in TB control. In the setting of a strong public sector programme, the combination of active surveillance of private laboratories along with physician sensitisation is a promising approach to improve TB case detection.     

GW654652, the pan-inhibitor of VEGF receptors, blocks the growth and migration of multiple myeloma cells in the bone marrow microenvironment

Previous studies have shown that the multiple myeloma (MM) cell line and MM patient cells express high-affinity vascular endothelial growth factor (VEGF) receptor-1 or Fms-like tyrosine kinase-1 (Flt-1) but not VEGF receptor-2 or Flk-1/kinase insert domain-containing receptor (Flk-1/KDR) and that VEGF triggers MM cell proliferation through a mitogen-activated protein kinase (MAPK)-dependent pathway and migration through a protein kinase C (PKC)-dependent pathway. The present study evaluates the efficacy of the small molecule tyrosine-kinase inhibitor GW654652, which inhibits all 3 VEGF receptors with similar potency. We show that GW654652 acts directly on MM cells and in the bone marrow microenvironment. Specifically, GW654652 (1-10 microg/mL) inhibits, in a dose-dependent fashion, VEGF-triggered migrational activity and cell proliferation of MM cell lines that are sensitive and resistant to conventional therapy. As expected from our previous studies of VEGF-induced signaling and sequelae in MM cells, GW654652 blocked VEGF-induced Flt-1 phosphorylation and downstream activation of AKT-1 and MAPK-signaling cascades. Importantly, GW654652 also inhibits interleukin-6 and VEGF secretion and proliferation of MM cells induced by tumor cell binding to bone marrow (BM) stromal cells. The activity of a pan-VEGF receptor inhibitor against MM cells in the BM milieu, coupled with its lack of major toxicity in preclinical mouse models, provides the framework for clinical trials of this drug class to improve patient outcome in MM.     

Blood pressure control and acceptability of Perindopril and its fixed dose combinations with Amlodipine or Indapamide,in younger patients with hypertension

Objective

Recent hypertension guidelines recommend initiation of treatment with a fixed dose combination of two drugs for more effective and quicker blood pressure control. Few of these have been assessed for efficacy and acceptability. This study examines the short term blood pressure control and acceptability of perindopril, with or without its fixed dose combinations (FDC) with amlodipine and Indapamide in younger patients.

Methods

In a multicentre prospective observational study, patients with stage 1 hypertension were prescribed perindopril 4 mg per day. Those with stage 2 or 3 hypertension were prescribed a single tablet per day of 4 mg perindopril and 5 mg amlodipine (COVERSYL AM), or 4 mg perindopril and 1.25 mg indapamide (COVERSYL PLUS)for 45 days. The primary outcomes were the frequency of patients achieving blood pressure control and the adverse effect of pedal edema.

Results

Of 426 patients, with a mean age of 45 years, distributed throughout India, and an average (SD) baseline systolic/diastolic blood pressure of 157.2 (13.5)/98.6 (7.4), 303 (71.1%) achieved blood pressure control. Mean (SD) SBP/DBP decreased from baseline by 26.9 (12.6), and DBP by 15.4 (7.2) mm Hg. Few patients discontinued treatment, and the frequency of cough that interfered with sleep and ankle edema was low.

Conclusion

In patients requiring combination antihypertensive treatment, the regimen of perindopril alone or its FDC with Indapamide or amlodipine reduces blood pressure effectively, resulting in high rates of blood pressure control over the short term, with a low frequency of side effects including cough and pedal edema.     

Protective hinge in insulin opens to enable its receptor engagement

Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated “microreceptors” that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of Phe^B24 to a 60° rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor''s L1–β₂ sheet. Opening of this hinge enables conserved nonpolar side chains (Ile^A2, Val^A3, Val^B12, Phe^B24, and Phe^B25) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.How insulin engages the insulin receptor has inspired speculation ever since the structure of the free hormone was determined by Hodgkin and colleagues in 1969 (1, 2). Over the ensuing decades, anomalies encountered in studies of analogs have suggested that the hormone undergoes a conformational change on receptor binding: in particular, that the C-terminal β-strand of the B chain (residues B24–B30) releases from the helical core to expose otherwise-buried nonpolar surfaces (the detachment model) (3–6). Interest in the B-chain β-strand was further motivated by the discovery of clinical mutations within it associated with diabetes mellitus (DM) (7). Analysis of residue-specific photo–cross-linking provided evidence that both the detached strand and underlying nonpolar surfaces engage the receptor (8).The relevant structural biology is as follows. The insulin receptor is a disulfide-linked (αβ)₂ receptor tyrosine kinase (Fig. 1A), the extracellular α-subunits together binding a single insulin molecule with high affinity (9). Involvement of the two α-subunits is asymmetric: the primary insulin-binding site (site 1^*) comprises the central β-sheet (L1–β₂) of the first leucine-rich repeat domain (L1) of one α-subunit and the partially helical C-terminal segment (αCT) of the other α-subunit (Fig. 1A) (10). Such binding initiates conformational changes leading to transphosphorylation of the β-subunits’ intracellular tyrosine kinase (TK) domains. Structures of wild-type (WT) insulin (or analogs) bound to extracellular receptor fragments were recently described at maximum resolution of 3.9 Å (11), revealing that hormone binding is primarily mediated by αCT (receptor residues 704–719); direct interactions between insulin and L1 were sparse and restricted to certain B-chain residues. On insulin binding, αCT was repositioned on the L1–β₂ surface, and its helix was C-terminally extended to include residues 711–714. None of these structures defined the positions of C-terminal B-chain residues beyond B21. Support for the detachment model was nonetheless provided by entry of αCT into a volume that would otherwise be occupied by B-chain residues B25–B30 (i.e., in classical insulin structures; Fig. 1B) (11).Open in a separate windowFig. 1.Insulin B-chain C-terminal β-strand in the μIR complex. (A) Structure of apo-receptor ectodomain. One monomer is in tube representation (labeled), the second is in surface representation. L1, first leucine-rich repeat domain; CR, cysteine-rich domain; L2, second leucine-rich repeat domain; FnIII-1, -2 and -3; first, second and third fibronectin type III domains, respectively; αCT, α-subunit C-terminal segment; coral disk, plasma membrane. (B) Insulin bound to μIR; the view direction with respect to L1 in the apo-ectodomain is indicated by the arrow in A. Only B-chain residues indicated in black were originally resolved (11). The brown tube indicates classical location of residues B20-B30 in free insulin, occluded in the complex by αCT. (C) Orthogonal views of unmodeled 2F_obs-F_calc difference electron density (SI Appendix), indicating association of map segments with the αCT C-terminal extension (transparent magenta), insulin B-chain C-terminal segment (transparent gray), and Asn^A21 (transparent yellow). Difference density is sharpened (B_sharp = −160 Ų). (D–F) Refined models of respective segments insulin B20–B27, αCT 714–719, and insulin A17-A21 within postrefinement 2F_obs-F_calc difference electron density (B_sharp = −160 Ų). D is in stereo.We describe here the structure and interactions of the detached B-chain C-terminal segment of insulin on its binding to a “microreceptor” (μIR), an L1–CR domain-minimized version of the α-subunit (designated IR310.T) plus exogenous αCT peptide 704–719 (11). Our analysis defines a hinge in the B chain whose opening is coupled to repositioning of αCT between nonpolar surfaces of L1 and the insulin A chain. To understand the role of this hinge in holoreceptor binding and signaling, we designed three insulin analogs containing structural constraints (d-Ala^B20, d-Ala^B23]-insulin, ∆Phe^B25-insulin, and ∆Phe^B24-insulin, where ∆Phe is (α,β)-dehydrophenylalanine (Fig. 2) (12). The latter represents, to our knowledge, the first use of ∆Phe—a rigid “β-breaker” with extended electronic conjugation between its side chain and main chain (SI Appendix, Fig. S1)—as a probe of induced fit in macromolecular recognition. In addition, a fourth analog, active but with anomalous flexibility in the B chain (5, 6) (

Phagocytic cells internalize ZnO particles by FcγII/III-receptor pathway

The present study investigates the process of internalization for bulk ZnO particles in macrophages, and further elucidates the underlying mechanism. Since macrophages are active phagocytes and phagocytosis is a size dependent phenomenon, therefore we hypothesized that bulk ZnO may internalize into macrophages by phagocytic pathways. Interestingly, the phagocytic activity got enhanced in bulk ZnO treated macrophages. Moreover, the bulk ZnO treated macrophages internalized via FcγR-II/III, complement and scavenger–receptor pathways. To confirm the specificity of phagocytic pathway, the uptake was also analyzed in splenocytes where phagocytic (monocytes) and non-phagocytic cells (lymphocytes) are present. It was observed that no significant uptake of bulk ZnO in case of lymphocytes whereas significant uptake in monocytes. Henceforth, our quest for uptake mechanisms also revealed that severe plasma membrane extensions (pseudopodia), FcγR clustering over the surface of macrophages and activation of FcγR signaling were the key players for bulk ZnO uptake; whereas clathrin or caveolae mediated endocytic pathways contributed less. Uptake of these particles was further strengthened by the ZnO-induced activation of the Src-kinase p-Lyn, phospho-tyrosine kinases Syk (spleen tyrosine kinase), p-PLC-γ and PI3K (phosphatidylinositol 3-kinase). Our findings illustrate that the phagocytic nature of macrophages could have led to higher uptake of bulk ZnO.