Design and methods for the Ranger Resilience and Improved Performance on Phospholipid bound Omega-3's (RRIPP-3 study).

Intake of nutrients fundamental for optimal neuronal function is of increasing interest. The potential importance of omega-3 highly unsaturated fatty acids (HUFAs) for optimizing emotional states, cognitive function, and mental health has been demonstrated in observational studies and randomized controlled trials. Omega-3 (HUFAs), specifically EPA (eicosapentaenoic acid) and docosahexaenoic acid (DHA), are concentrated in neural tissues and are essential for neural function, normative neurodevelopment, neurotransmitter, and neural immune functions. Omega-3 HUFAs must be obtained from the diet, predominantly from marine sources such as fish and other seafood. HUFAs also can be found in a variety of dietary supplements (omega-3 fatty acid esters, fish oil and krill oil). As dietary supplements, omega-3 HUFAs (fatty acid esters, fish and krill oils) differ substantially in their physicochemical properties and nutrient content. Here we present the design and methods for the Ranger Resilience and Improved Performance on Phospholipid bound Omega-3's (RRIPP-3) study. RRIPP-3 was a double blind, randomized, controlled trial among individuals in the United States (US) Army Infantry Basic Officer Leaders Course (IBOLC) and following US Ranger School training (RC) at Fort Benning, GA of omega-3 HUFA on krill oil versus placebo supplementation. The RRIPP-3 study sought to determine if krill oil supplementation with omega-3 HUFAs supports aspects of cognitive functioning critical to battlefield success when measured immediately after an intense combat simulation. Sub-analyses addressed basic improvements in IBOLC performance. We also describe additional outcome measures critical for interpretation of the study results, such as diet and other dietary supplement use.

The Use of Comfort Kits to Optimize Adult Cancer Pain Management.

BACKGROUND: Pain is one of the most feared of all symptoms for the cancer patient. Some studies estimate that up to 90% of all cancer patients experience pain. Advances in pharmaceuticals and expert provider knowledge have improved pain management overall for the patient with cancer; however, complementary therapies can synergize medications to provide optimal pain relief while decreasing the side effect profile. Despite this, nurses may have limited access to such resources. Many therapies can be administered directly by the bedside/chairside nurse with minimal training and the nurse can then teach the patient and family how to use the selected complementary therapy after leaving the hospital or clinic. OBJECTIVES: The oncology nurse will be able to identify several easy-to-implement complementary therapies that can supplement pharmacologic pain management for cancer patients. METHODS: As a quality project, comfort kits, containing such items as handheld massagers, guided imagery audiotapes, and aromatherapy essential oils, were distributed for use with patients through unit-based pain resource nurses. ANALYSIS: More than 500 comfort kit items were tracked by the pain clinical nurse specialist during the comfort kit trial, both by medical record review and by follow-up phone calls to patients. During the comfort kit trial, average pain intensity decreased by 2.25 points on a 0-10 scale in the 24-hour period after use of the item from the comfort kit. Patients also had an overall decrease in the use of pharmacologic pain interventions and an increase in ambulation in the 24-hour period after implementation. CONCLUSIONS: Comfort kits allow nurses easy access to inexpensive tools to supplement pharmaceutical pain management. Optimizing nonpharmacologic pain management can increase patient and nurse satisfaction, improve overall pain management, and decrease untoward side effects.

Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant.

A variable region fusion strategy was used to generate an immunosuppressive antibody based on a novel "stalk-knob" structural motif in the ultralong complementary-determining region (CDR) of a bovine antibody. The potent Kv1.3 channel inhibitory peptides Moka1-toxin and Vm24-toxin were grafted into different CDRs of the humanized antibodies BVK and Synagis (Syn) using both ß-sheet and coiled-coil linkers. Structure-activity relationship efforts led to generation of the fusion protein Syn-Vm24-CDR3L, which demonstrated excellent selectivity and potency against effector human memory T cells (subnanomolar to picomolar EC50 values). This fusion antibody also had significantly improved plasma half-life and serum stability in rodents compared with the parent Vm24 peptide. Finally, this fusion protein showed potent in vivo efficacy in the delayed type hypersensitivity in rats. These results illustrate the utility of antibody CDR fusions as a general and effective strategy to generate long-acting functional antibodies, and may lead to a selective immunosuppressive antibody for the treatment of autoimmune diseases.

Examination of unplanned 30-day readmissions to a comprehensive cancer hospital.

PURPOSE: The Centers for Medicare and Medicaid Services (CMS), under the Hospitals Readmissions Reductions Program, may withhold regular reimbursements for excessive 30-day readmissions for select diagnoses. Such penalties imply that some readmissions reflect poor clinical decision making or care during the initial hospitalization. We examined factors related to potentially preventable readmissions in CMS patients at a tertiary cancer hospital. METHODS: The medical records of all CMS patients with unplanned readmissions within 30 days of index admission were reviewed over 6 months (October 15, 2011-April 15, 2012). Each readmission was classified as not preventable or potentially preventable. Factors associated with potentially preventable readmissions were sought. RESULTS: Of 2,531 inpatient admissions in CMS patients over 6 months, 185 patients experienced at least one readmission for 282 total readmissions (11%). Median time to readmission was 9 days (range, 0 to 30 days). The most common causes for first readmission were new diagnoses not present at first admission (n = 43, 23%), new or worsening symptoms due to cancer progression (n = 40, 21%) and complications of procedures (n = 25, 13%). There were 38 (21%) initial readmissions classified as potentially preventable. Use of total parenteral nutrition at the time of discharge was associated with potentially preventable readmission (P = .028). CONCLUSION: Most unplanned readmissions to a tertiary cancer hospital are related to progression of disease, new diagnoses, and procedure complications. Minimizing readmissions in complex cancer patients is challenging. Larger multi-institutional datasets are needed to determine a reasonable standard for expected readmission rates.

Novel mouse model of autosomal semidominant adult hypophosphatasia has a splice site mutation in the tissue nonspecific alkaline phosphatase gene Akp2.

UNLABELLED: Deactivating mutations in the TNSALP gene cause HPP. Akp2(-/-) mice model severe infantile HPP, but there is no model for the relatively mild adult form. Here we report on mice with an induced mutation in Akp2 that affects splicing. The phenotype of homozygotes mirror aspects of the adult form of HPP. INTRODUCTION: Hypophosphatasia (HPP) is a clinically varied skeletal disorder resulting from deficiency of tissue nonspecific alkaline phosphatase (TNSALP). Mice lacking Akp2 model infantile HPP characterized by skeletal hypomineralization, impaired growth, seizures, and perinatal mortality. No animal model exists to study the less severe forms of the disease that typically present in later life. MATERIALS AND METHODS: N-ethyl-N-nitrosourea (ENU) mutagenesis was used to generate mouse models of human disease. A mouse with low plasma alkaline phosphatase (ALP) activity was identified by our clinical chemistry screen. Its offspring were used for inheritance studies and subjected to biochemical, histological, and radiological phenotyping. DNA was extracted for mapping and osteoblasts harvested for functional studies. RESULTS: We showed semidominant inheritance of the low ALP phenotype and mapped the underlying point mutation to Akp2. Affected offspring bear the splice site mutation 862 + 5G>A-a hypomorphic allele named Akp2(Hpp). The same mutation has been reported in a patient. Akp2(Hpp/+) mice have approximately 50% of normal plasma ALP but display no other biochemical or skeletal abnormalities. Unlike Akp2(-/-) mice, Akp2(Hpp/Hpp) mice have normal initial skeletal development and growth, a normal lifespan and do not have seizures. TNSALP is low but detectable in Akp2(Hpp/Hpp) plasma. Osteoblasts display approximately 10% of normal ALP activity and reduced intracellular inorganic phosphate levels, yet are capable of normal mineralization in vitro. TNSALP substrates are significantly elevated in urine (inorganic pyrophosphate and phosphoethanolamine) and plasma (pyridoxal 5'-phosphate), whereas plasma inorganic pyrophosphate levels are normal. Akp2(Hpp/Hpp) mice develop late-onset skeletal disease, notably defective endochondral ossification and bone mineralization that leads to arthropathies of knees and shoulders. CONCLUSIONS: Akp2(Hpp/Hpp) mice mirror a number of clinical features of the human adult form of HPP. These mice provide for the first time an animal model of late onset HPP that will be valuable in future mechanistic studies and for the evaluation of therapies such as those aimed at HPP.

Association of sporadic chondrocalcinosis with a -4-basepair G-to-A transition in the 5'-untranslated region of ANKH that promotes enhanced expression of ANKH protein and excess generation of extracellular inorganic pyrophosphate.

OBJECTIVE: Certain mutations in ANKH, which encodes a multiple-pass transmembrane protein that regulates inorganic pyrophosphate (PPi) transport, are linked to autosomal-dominant familial chondrocalcinosis. This study investigated the potential for ANKH sequence variants to promote sporadic chondrocalcinosis. METHODS: ANKH variants identified by genomic sequencing were screened for association with chondrocalcinosis in 128 patients with severe sporadic chondrocalcinosis or pseudogout and in ethnically matched healthy controls. The effects of specific variants on expression of common markers were evaluated by in vitro transcription/translation. The function of these variants was studied in transfected human immortalized CH-8 articular chondrocytes. RESULTS: Sporadic chondrocalcinosis was associated with a G-to-A transition in the ANKH 5'-untranslated region (5'-UTR) at 4 bp upstream of the start codon (in homozygotes of the minor allele, genotype relative risk 6.0, P = 0.0006; overall genotype association P = 0.02). This -4-bp transition, as well as 2 mutations previously linked with familial and sporadic chondrocalcinosis (+14 bp C-to-T and C-terminal GAG deletion, respectively), but not the French familial chondrocalcinosis kindred 143-bp T-to-C mutation, increased reticulocyte ANKH transcription/ANKH translation in vitro. Transfection of complementary DNA for both the wild-type ANKH and the -4-bp ANKH protein variant promoted increased extracellular PPi in CH-8 cells, but unexpectedly, these ANKH mutants had divergent effects on the expression of extracellular PPi and the chondrocyte hypertrophy marker, type X collagen. CONCLUSION: A subset of sporadic chondrocalcinosis appears to be heritable via a -4-bp G-to-A ANKH 5'-UTR transition that up-regulates expression of ANKH and extracellular PPi in chondrocyte cells. Distinct ANKH mutations associated with heritable chondrocalcinosis may promote disease by divergent effects on extracellular PPi and chondrocyte hypertrophy, which is likely to mediate differences in the clinical phenotypes and severity of the disease.

External GTP-bound transglutaminase 2 is a molecular switch for chondrocyte hypertrophic differentiation and calcification.

Chondrocyte maturation to hypertrophy, associated with up-regulated transglutaminase 2 (TG2) expression, mediates not only physiologic growth plate mineralization but also pathologic matrix calcification and dys-regulated matrix repair in osteoarthritic articular cartilage. TG2-/- mouse chondrocytes demonstrate markedly inhibited progression to hypertrophic differentiation in response to both retinoic acid and the chemokine CXCL1. Here, our objectives were to test if up-regulated TG2 alone is sufficient to promote chondrocyte hypertrophic differentiation and to identify TG2 molecular determinants and potential downstream signals involved. TG2 activities, regulated by nucleotides and calcium, include cross-linking of cartilage matrix proteins, binding of fibronectin, and hydrolysis of GTP and ATP. Following transfection of TG2 site-directed mutants into chondrocytic cells, we observed that wild type TG2, and TG catalytic site and fibronectin-binding mutants promoted type X collagen expression and matrix calcification consistent with chondrocyte hypertrophic differentiation. In contrast, transfected mutants of TG2 GTP binding (K173L) and externalization (Y274A) sites did not stimulate chondrocyte hypertrophy. Recombinant TG2 treatment of bovine cartilage explants demonstrated that extracellular TG2 induced hypertrophy more robustly in the GTP-bound state, confirming an essential role of TG2 GTP binding. Finally, TG2 treatment induced type X collagen in a beta1 integrin-mediated manner, associated with rapid phosphorylation of both Rac1 and p38 kinases that were inhibited by mutation of the TG2 GTP binding site. In conclusion, externalized GTP-bound TG2 serves as a molecular switch for differentiation of chondrocytes to a hypertrophic, calcifying phenotype in a manner that does not require either TG2 transamidation activity or fibronectin binding.

Linked deficiencies in extracellular PP(i) and osteopontin mediate pathologic calcification associated with defective PC-1 and ANK expression.

Osteopontin and PP(i) both suppress hydroxyapatite deposition. Extracellular PP(i) deficiency causes spontaneous hypercalcification, yet unchallenged osteopontin knockout mice have only subtle mineralization abnormalities. We report that extracellular PP(i) deficiency promotes osteopontin deficiency and correction of osteopontin deficiency prevents hypercalcification, suggesting synergistic inhibition of hydroxyapatite deposition. Nucleotide pyrophosphatase phosphodiesterase (NPP) isozymes including PC-1 (NPP1) function partly to generate PP(i), a physiologic calcification inhibitor. PP(i) transport is modulated by the membrane channel protein ANK. Spontaneous articular cartilage calcification, increased vertebral cortical bone formation, and peripheral joint and intervertebral ossific ankylosis are associated with both PC-1 deficiency and expression of truncated ANK in ank/ank mice. To assess how PC-1, ANK, and PP(i) regulate both calcification and cell differentiation, we studied cultured PC-1 -/- and ank/ank mouse calvarial osteoblasts. PC-1 -/- osteoblasts demonstrated approximately 50% depressed NPP activity and markedly lowered extracellular PP(i) associated with hypercalcification. These abnormalities were rescued by transfection of PC-1 but not of the NPP isozyme B10/NPP3. PC-1 -/- and ank/ank cultured osteoblasts demonstrated not only comparable extracellular PP(i) depression and hypercalcification but also marked reduction in expression of osteopontin (OPN), another direct calcification inhibitor. Soluble PC-1 (which corrected extracellular PP(i) and OPN), and OPN itself (> or = 15 pg/ml), corrected hypercalcification by PC-1 -/- and ank/ank osteoblasts. Thus, linked regulatory effects on extracellular PP(i) and OPN expression mediate the ability of PC-1 and ANK to regulate calcification.

One of two chondrocyte-expressed isoforms of cartilage intermediate-layer protein functions as an insulin-like growth factor 1 antagonist.

OBJECTIVE: Aging and osteoarthritic (OA) cartilage commonly demonstrate enhanced expression of the large, transforming growth factor beta (TGFbeta)-inducible glycoprotein cartilage intermediate-layer protein (CILP) as well as enhanced extracellular inorganic pyrophosphate (PPi) that promotes the deposition of calcium pyrophosphate dihydrate crystals. In normal chondrocytes, TGFbeta induces elevated chondrocyte extracellular PPi. Insulin-like growth factor 1 (IGF-1) normally blocks this response and reduces extracellular PPi. However, chondrocyte resistance to IGF-1 is observed in OA and aging. Because CILP was reported to chromatographically fractionate with PPi-generating nucleotide pyrophosphatase phosphodiesterase (NPP) activity, it has been broadly assumed that CILP itself has NPP activity. Our objective was to directly define CILP functions and their relationship to IGF-1 in chondrocytes. METHODS: Using primary cultures of articular chondrocytes from the knee, we defined the function of the previously described CILP (CILP-1) and of a recently described 50.6% identical protein that we designated the CILP-2 isoform. RESULTS: Both CILP isoforms were constitutively expressed by primary cultured articular chondrocytes, but only CILP-1 expression was detectable in cultured knee meniscal cartilage cells. Neither CILP isoform had intrinsic NPP activity. But CILP-1 blocked the ability of IGF-1 to decrease extracellular PPi, an activity specific for the CILP-1 N-terminal domain. The CILP-1 N-terminal domain also suppressed IGF-1-induced (but not TGFbeta-induced) proliferation and sulfated proteoglycan synthesis, and it inhibited ligand-induced IGF-1 receptor autophosphorylation. CONCLUSION: Two CILP isoforms are differentially expressed by chondrocytes. Neither CILP isoform exhibits PPi-generating NPP activity. But, increased expression of CILP-1, via N-terminal domain-mediated inhibitory effects of CILP-1 on chondrocyte IGF-1 responsiveness, could impair chondrocyte growth and matrix repair and indirectly promote PPi supersaturation in aging and OA cartilage.