Molecular basis of cross-species reactivities of human versus porcine CTLA-4

The binding motif of human CTLA-4 is well known to be MYPPPY and for porcine CTLA-4 the binding motif is LYPPPY. Is this single amino acid difference of methionine (M) versus leucine (L) critical for the CTLA-4 binding? Recently, we have reported that the recombinant soluble porcine CTLA-4 was incapable of binding to human CD80. In this study we mutated L to M in the binding motif of the soluble porcine CTLA-4 and mutated M to L in the binding motif of the soluble human CTLA-4. We then analyzed how these mutations affected the binding affinity of the mutants to both porcine and human CD80⁺ cells. The soluble porcine CTLA-4-L97M mutant partially lost its binding affinity to porcine CD80 compared to the wild-type and conferred very weak binding ability to human CD80. These results indicate that the L in the binding motif of porcine CTLA-4 is important for determining its binding ability to porcine CD80. Wild-type soluble human CTLA-4 binds to both human and porcine CD80 with comparable affinity, however, the soluble human CTLA-4-M97L mutant almost lost its binding ability to human CD80 and increased its binding ability to porcine CD80. These results indicate that M in the human CTLA-4 binding motif is extremely critical for its binding to human CD80. Those data suggest that the human CTLA-4 based recombinant protein drugs such as human CTLA-4-Ig can be used and/or tested in a porcine model. Conversely, the use of porcine CTLA-4 based recombinant protein drugs such as porcine CTLA-4-Ig is restricted to swine models. The difference in binding specificity of CTLA-4 observed in this study may be useful for studies such as pig to nonhuman primate xeno-transplantation. Porcine CTLA-4- and human CTLA-4-M97L mutant-based recombinant protein drugs can be used to specifically block the direct presentation by donor antigen presenting cells in pig to nonhuman primate xeno-transplantation. Human CTLA-4-M97L mutant-based recombinant protein drugs will be more ideal as it is without immunogenicity to human being.     

Broad-spectrum analgesic efficacy of IBNtxA is mediated by exon 11-associated splice variants of the mu-opioid receptor gene

μ-Opioids remain vastly important for the treatment of pain, and would represent ideal analgesics if their analgesic effects could be separated from their many side effects. A recently synthesized compound, iodobenzoylnaltrexamide (IBNtxA), acting at 6-transmembrane (6-TM) splice variants of the μ-opioid receptor gene, was shown to have potent analgesic actions against acute, thermal pain accompanied by a vastly improved side-effect profile compared to 7-TM-acting drugs such as morphine. Whether such analgesia can be seen in longer-lasting and nonthermal algesiometric assays is not known. The current study demonstrates potent and efficacious IBNtxA inhibition of a wide variety of assays, including inflammatory and neuropathic hypersensitivity and spontaneous pain. We further demonstrate the dependence of such analgesia on 6-TM μ-opioid receptor variants using isobolographic analysis and the testing of Oprm1 (the μ-opioid receptor gene) exon 11 null mutant mice. Finally, the effect of nerve damage (spared nerve injury) and inflammatory injury (complete Freund’s adjuvant) on expression of μ-opioid receptor variant genes in pain-relevant central nervous system loci was examined, revealing a downregulation of the mMOR-1D splice variant in the dorsal root ganglion after spared nerve injury. These findings are supportive of the potential value of 6-TM-acting drugs as novel analgesics.     

Microdeletions and Microduplications in Patients with Congenital Heart Disease and Multiple Congenital Anomalies

Objective. Multiple genetic syndromes are caused by recurrent chromosomal microdeletions or microduplications. The increasing use of high‐resolution microarrays in clinical analysis has allowed the identification of previously undetectable submicroscopic copy number variants (CNVs) associated with genetic disorders. We hypothesized that patients with congenital heart disease and additional dysmorphic features or other anomalies would be likely to harbor previously undetected CNVs, which might identify new disease loci or disease‐related genes for various cardiac defects. Design. Copy number analysis with single nucleotide polymorphism‐based, oligonucleotide microarrays was performed on 58 patients with congenital heart disease and other dysmorphic features and/or other anomalies. The observed CNVs were validated using independent techniques and validated CNVs were further analyzed using computational algorithms and comparison with available control CNV datasets in order to assess their pathogenic potential. Results. Potentially pathogenic CNVs were detected in twelve of 58 patients (20.7%), ranging in size from 240 Kb to 9.6 Mb. These CNVs contained between 1 and 55 genes, including NRP1, NTRK3, MESP1, ADAM19, and HAND1, all of which are known to participate in cardiac development. Conclusions. Genome‐wide analysis in patients with congenital heart disease and additional phenotypes has identified potentially pathogenic CNVs affecting genes involved in cardiac development. The identified variant loci and the genes within them warrant further evaluation in similarly syndromic and nonsyndromic cardiac cohorts.     

Ankylosing spondylitis diagnosis in US patients with back pain: identifying providers involved and factors associated with rheumatology referral delay

This study aimed to identify providers involved in diagnosing ankylosing spondylitis (AS) following back pain diagnosis in the USA and to identify factors leading to the delay in rheumatology referrals. The Truven Health MarketScan® US Commercial Database was searched for patients aged 18–64 years with back pain diagnosis in a non-rheumatology setting followed by AS diagnosis in any setting during January 2000–December 2012. Patients with a rheumatologist visit on or before AS diagnosis were considered referred. Cox regression was used to determine factors associated with referral time after adjusting for age, sex, comorbidities, physician specialty, drug therapy, and imaging procedures. Of 3336 patients included, 1244 (37 %) were referred to and diagnosed by rheumatologists; the others were diagnosed in primary care (25.7 %), chiropractic/physical therapy (7 %), orthopedic surgery (3.8 %), pain clinic (3.6 %), acute care (3.4 %), and other (19.2 %) settings. Median time from back pain diagnosis to rheumatology referral was 307 days and from first rheumatologist visit to AS diagnosis was 28 days. Referred patients were more likely to be younger (hazard ratio [HR]?=?0.986; p?<?0.0001), male (HR?=?1.15; p?=?0.0163), diagnosed with uveitis (HR?=?1.49; p?=?0.0050), referred by primary care physicians (HR?=?1.96; p?<?0.0001), prescribed non-steroidal anti-inflammatory drugs (HR?=?1.55; p?<?0.0001), disease-modifying antirheumatic drugs (HR?=?1.33; p?<?0.0001), and tumor necrosis factor inhibitors (HR?=?1.40; p?=?0.0036), and to have had spinal/pelvic X-ray prior to referral (HR?=?1.28; p?=?0.0003). During 2000–2012, most patients with AS were diagnosed outside of rheumatology practices. The delay before referral to rheumatology was 10 months; AS diagnosis generally followed within a month. Earlier referral of patients with AS signs and symptoms may lead to more timely diagnosis and appropriate treatment.     

Branched chain and aromatic amino acids change acutely following two medical therapies for type 2 diabetes mellitus

ObjectiveElevated circulating levels of branched chain and aromatic amino acids (BCAA/AAAs) are associated with insulin resistance and incident type 2 diabetes (T2D). BCAA/AAAs decrease acutely during an oral glucose tolerance test (OGTT), a diagnostic test for T2D. It is unknown whether changes in BCAA/AAAs also signal an early response to commonly used medical therapies for T2D.Materials and MethodsA liquid chromatography–mass spectrometry approach was used to measure BCAA/AAAs in 30 insulin sensitive (IS) and 30 insulin resistant (IR) subjects before and after: 1) one dose of a sulfonylurea medication, glipizide, 5 mg orally; 2) two days of twice daily metformin 500 mg orally; and 3) a 75-g OGTT. Percent change in BCAA/AAAs was determined after each intervention.ResultsFollowing glipizide, which increased insulin and decreased glucose in both subject groups, BCAA/AAAs decreased in the IS subjects only (all P < 0.05). Following metformin, which decreased glucose and insulin in only the IR subjects, 4 BCAA/AAAs increased in the IR subjects at or below P = 0.05, and none changed in the IS subjects. Following OGTT, which increased glucose and insulin in all subjects, BCAA/AAAs decreased in all subjects (P < 0.05).ConclusionsBCAA/AAAs changed acutely during glipizide and metformin administration, and the magnitude and direction of change differed by the insulin resistance status of the individual and the intervention. These results indicate that BCAA/AAAs may be useful biomarkers for monitoring the early response to therapeutic interventions for T2D.     

Converting modified health assessment questionnaire (HAQ), multidimensional HAQ,and HAQII scores into original HAQ scores using models developed with a large cohort of rheumatoid arthritis patients

Objective

The Stanford Health Assessment Questionnaire Disability Index (HAQ) is the gold standard functional status questionnaire in rheumatology, but it is lengthy. Three shorter versions, the modified HAQ (MHAQ), the Multidimensional HAQ (MDHAQ), and the HAQII are often used in outcomes research as HAQ substitutes. We developed conversion formulas between these modified versions and the original HAQ.

Methods

Analysis was limited to the comparison of rheumatoid arthritis (RA) patients at a random observation when the HAQ was recorded in conjunction with the MHAQ (n = 29,596), the MDHAQ (n = 13,665), or the HAQII (n = 15,823). Development models were randomly limited to 80% of the data (development sample) and the remaining 20% was used for model validation.

Results

Two conversion formulas were developed for each of the MHAQ, the MDHAQ, and the HAQII: a short model and a long model inclusive of questions common to both the modified measures and the original HAQ. Short models explained 81–83%, and long models 82–86%, of the variance. Predicted HAQ values of zero were assigned to all cases with an MDHAQ or HAQII score of zero, with remaining cases used for model estimation. Bland‐Altman plots demonstrated good concordance between actual and predicted values for each measure. The validation sample closely approximated the results from the development sample (0.005 ≤ ΔR² ≤ 0.009) for each measure.

Conclusion

We have developed and validated highly accurate conversion formulas from the MHAQ, MDHAQ, and HAQII to the original HAQ in a large sample of RA patients. The developed models are useful for conversion of measures in the research setting. Because of substantial variability at the individual patient level, application of the formulas to individual patients is inadvisable.     

Multiprofessional survey of protocol use in the intensive care unit

Purpose

To date, there has been no large multicenter, multiprofessional evaluation of protocol and guideline use in the intensive care unit (ICU). The primary purpose of this study was to describe national availability, development, implementation, and assessment of protocols in ICUs. A secondary objective was to compare perceived utility by ease of use, patient safety, cost containment, and compliance of protocols between nurses, physicians, and pharmacists.

Materials and Methods

The survey was developed and tested for validity by 15 clinicians who identified additional domains of interest. An additional 15 clinicians of the 3 different professions evaluated the survey for relevancy and appropriateness of responses. Three survey experts evaluated survey construction. The survey was uploaded to a Web survey tool and pilot tested for clarity and ease of completion.

Results

The overall response rate for the survey was 18.1% (n = 614). Popular methods of education for protocol implementation included staff meetings (85.3%) and unit-specific in-services (77.7%). Protocols were most often updated when new information was available (40.8%) or every 12 months (17.9%). The most common limitation to development and implementation was limited personnel resources (24.5%) and physicians not wanting to use them (21.3%), respectively. Clinicians indicated that protocols made their job easier and improved cost containment some or most of the time. Sepsis protocols were identified as most useful in promoting patient outcomes by all 3 professions.

Conclusions

The types of protocols available appear to be those assisting with management of high-alert medications. Overcoming the perceived barriers of protocol use within ICUs requires personnel for development and physician support. A better protocol review process may be necessary to assure optimal content, desired outcomes, and consistency with Institute for Safe Medication Practices guidelines.