Nitric oxide (NO) conveys a variety of messages between cells, including signals for vasorelaxation, neurotransmission, and cytotoxicity. In some endothelial cells and neurons, a constitutive NO synthase is activated transiently by agonists that elevate intracellular calcium concentrations and promote the binding of calmodulin. In contrast, in macrophages, NO synthase activity appears slowly after exposure of the cells to cytokines and bacterial products, is sustained, and functions independently of calcium and calmodulin. A monospecific antibody was used to clone complementary DNA that encoded two isoforms of NO synthase from immunologically activated mouse macrophages. Liquid chromatography-mass spectrometry was used to confirm most of the amino acid sequence. Macrophage NO synthase differs extensively from cerebellar NO synthase. The macrophage enzyme is immunologically induced at the transcriptional level and closely resembles the enzyme in cytokine-treated tumor cells and inflammatory neutrophils.

A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon y and bacterial lipopolysaccharide by sequential anion-exchange, affinity, and gel filtration chromatography. SDS/PAGE of the purified NO synthase gave three closely spaced silver-staining protein bands between 125 and 135 kDa. When assayed in the presence of L-arginine, NADPH, tetrahydrobiopterin, FAD, and reduced thiol, purified NO synthase had a specific activity of 1313 nmol of NOj plus NOj per min per mg. The apparent Km of the enzyme for L-arginine and NADPH was 2.8 and 0.3 FM, respectively. Addition of calcium ions with or without calmodulin did not increase the activity of the purified enzyme, and NO synthesis was not altered by calmodulin inhibitors. Gel filtration chromatography indicated that the induced NO synthase was catalytically competent as a dimer of -250 kDa but could be dissociated into inactive monomers of -130 kDa in the absence of L-arginine, FAD, and tetrahydrobiopterin. Upon heat denaturation, NO synthase released 1.1 mol of FAD and 0.55 mol of FMN per mol of 130-kDa subunit. Thus, inducible macrophage NO synthase differs in several respects from constitutive NO synthases and is one of very few eukaryotic enzymes containing both FAD and FMN.The free radical nitric oxide (NO) or a NO-releasing product is synthesized within mammalian immune, cardiovascular, and neural systems, where it functions as a signaling or cytotoxic molecule (for reviews, see refs. 1-3). The mammalian enzymes that generate NO are not completely characterized. Current evidence suggests that there are at least two forms. One is constitutively expressed, requires calcium ions and a calcium-binding protein such as calmodulin for its activation, and participates in signal transduction by generating NO in response to increased intracellular calcium levels, leading to activation of soluble guanylyl cyclase by NO (1, 4-7). The other form is expressed in cells only after several hours of exposure to cytokines like interferon y (IFN-y) and/or microbial products such as bacterial lipopolysaccharide (LPS) (8)(9)(10). Immunologically induced NO synthase participates in the destruction of microbial pathogens and tumor cells and contributes to shock associated with sepsis (2, 3, 11, 12). The inducible NO synthase appears to be antigenically distinct from constitutive NO synthase (13).Despite their differences in regulation and function, evidence suggests that the constitutive and induced NO synthases are catalytically similar. Both types utilize NADPH and, where it was tested, tetrahydrobiopterin as redox cofactors (4-7, 14, 15) and convert L-arginine to NO and L-citrulline with one atom of molecular oxygen being incorporated into L-citrulline (16). In cases where it was tested, both constitutive and inducible activities were enhanced by exogenous FAD (7, 17) and inhibited irreversibly by the flavoprotein inhibitor diphenyleneiodonium (18), suggesting that NO synthases may be flavoproteins. Howev...

Autoantibodies against factor VIII (FVIII) are rare but can cause life-threatening bleeding requiring costly factor replacement and prolonged immunosuppression. We report 4 consecutively treated patients whose acquired FVIII inhibitors responded rapidly to immunosuppressive regimens that included rituximab, a monoclonal antibody against CD20 ؉ B cells. Three patients had spontaneously occurring inhibitors. The fourth, a patient with mild hemophilia A, developed both an autoantibody and an alloantibody following recombinant FVIII treatment. Pretreatment FVIII activities ranged from less than 1% to 4% and inhibitor titers from 5 to 60 Bethesda units (BU). One patient with polymyalgia rheumatica who developed the inhibitor while receiving prednisone responded to single agent rituximab. The hemophilia patient had rapid resolution of the autoantibody, whereas the alloantibody persisted for months. Responses continue off treatment from more than 7 to more than 12 months. This report adds to the growing evidence that rituximab has efficacy in immune disorders resulting from autoantibody formation. (Blood. 2002;100: 3426-3428)

This trial determined the safety and efficacy of the combination regimen clarithromycin (Biaxin), lenalidomide (Revlimid), and dexamethasone (BiRD) as first-line therapy for multiple myeloma. Patients received BiRD in 28-day cycles. Dexamethasone (40 mg) was given orally once weekly, clarithromycin (500 mg) was given orally twice daily, and lenalidomide (25 mg) was given orally daily on days 1 to 21. Objective response was defined by standard criteria (ie, decrease in serum monoclonal protein [M-protein] by at least 50%, and a decrease in urine Mprotein by at least 90%). Of the 72 patients enrolled, 65 had an objective response (90.3%). A combined stringent and conventional complete response rate of 38.9% was achieved, and 73.6% of the patients achieved at least a 90% decrease in Mprotein levels. This regimen did not interfere with hematopoietic stem-cell harvest. Fifty-two patients who did not go on to receive transplants received continued therapy (complete response, 37%; very good partial response, 33%). The major adverse events were thromboembolic events, corticosteroid-related morbidity, and cytopenias. BiRD is an effective regimen with manageable side effects in the treatment of symptomatic, newly diagnosed multiple myeloma. This trial was registered at www.clinicaltrials.gov as

• PomCyDex results in a higher overall response rate than pomalidomide and dexamethasone.• PomCyDex is an effective, all oral regimen for refractory myeloma patients.Pomalidomide and low-dose dexamethasone (PomDex) is standard treatment of lenalidomide refractory myeloma patients who have received >2 prior therapies. We aimed to assess the safety and efficacy of the addition of oral weekly cyclophosphamide to standard PomDex. We first performed a dose escalation phase 1 study to determine the recommended phase 2 dose of cyclophosphamide in combination with PomDex (arm A). A randomized, multicenter phase 2 study followed, enrolling patients with lenalidomide refractory myeloma. Patients were randomized (1:1) to receive pomalidomide 4 mg on days 1 to 21 of a 28-day cycle in combination with weekly dexamethasone (arm B) or pomalidomide, dexamethasone, and cyclophosphamide (PomCyDex) 400 mg orally on days 1, 8, and 15 (arm C). The primary end point was overall response rate (ORR). Eighty patients were enrolled (10 in phase 1 and 70 randomized in phase 2: 36 to arm B and 34 to arm C). The ORR was 38.9% (95% confidence interval [CI], 23-54.8%) and 64.7% (95% CI, 48.6-80.8%) for arms B and C, respectively (P 5 .035). As of June 2015, 62 of the 70 randomized patients had progressed. The median progression-free survival (PFS) was 4.4 (95% CI, 2.3-5.7) and 9.5 months (95% CI, 4.6-14) for arms B and C, respectively (P 5 .106). Toxicity was predominantly hematologic in nature but was not statistically higher in arm C. The combination of PomCyDex results in a superior ORR and PFS compared with PomDex in patients with lenalidomide refractory multiple myeloma.

No abstract

Multiple myeloma, the second most common hematopoietic cancer, ultimately becomes refractory to treatment when selfrenewing multiple myeloma cells begin unrestrained proliferation by unknown mechanisms. Here, we show that one, but not more than one, of the three early G 1 D cyclins is elevated in each case of multiple myeloma. Cyclin D1 or D3 expression does not vary in the clinical course, but that alone is insufficient to promote cell cycle progression unless cyclindependent kinase 4 (cdk4) is also elevated, in the absence of cdk6, to phosphorylate the retinoblastoma protein (Rb). By contrast, cyclin D2 and cdk6 are coordinately increased, thereby overriding the inhibition by cdk inhibitors p18 INK4c and p27 Kip1 and phosphorylating Rb in conjunction with the existing cdk4. Thus, cyclin D1 pairs exclusively with cdk4 and cdk6 pairs only with cyclin D2, although cyclin D2 can also pair with cdk4 in multiple myeloma cells. The basis for this novel and specific cdk/D cyclin pairing lies in differential transcriptional activation. In addition, cyclin D1-or cyclin D3-expressing multiple myeloma cells are uniformly distributed in the bone marrow, whereas cdk6-specific phosphorylation of Rb occurs in discrete foci of bone marrow multiple myeloma cells before proliferation early in the clinical course and is then heightened with proliferation and disease progression. Mutually exclusive cdk4/cyclin D1 and cdk6/ cyclin D2 pairing, therefore, is likely to be a critical determinant for cell cycle reentry and progression and may play a pivotal role in the expansion of self-renewing multiple myeloma cells. (Cancer Res 2005; 65(24): 11345-53)

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH4) for dimerization and NO production. Mutation The radical nitric oxide (NO) has emerged as an important signaling and cytotoxic molecule in metazoan physiology (1)(2)(3)(4). NO is synthesized from L-Arg, oxygen, and NADPH by variably regulated isoforms of NO synthase (NOS). Products of the three known mammalian NOS genes (4) include two (NOS1 and NOS3) that are constitutively expressed and activated by binding calmodulin in response to elevated Ca2+ and one [inducible NOS (iNOS); NOS2] that is activated transcriptionally and binds calmodulin without an elevation of