Beta amino acid-modified and fluorescently labelled kisspeptin analogues with potent KISS1R activity.

Kisspeptin analogues with improved metabolic stability may represent important ligands in the study of the kisspeptin/KISS1R system and have therapeutic potential. In this paper we assess the activity of known and novel kisspeptin analogues utilising a dual luciferase reporter assay in KISS1R-transfected HEK293T cells. In general terms the results reflect the outcomes of other assay formats and a number of potent agonists were identified among the analogues, including ß(2) -hTyr-modified and fluorescently labelled forms. We also showed, by assaying kisspeptin in the presence of protease inhibitors, that proteolysis of kisspeptin activity within the reporter assay itself may diminish the agonist outputs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Prospective study of enhanced recovery after surgery protocol in children undergoing reconstructive operations.

BACKGROUND: Enhanced recovery after surgery (ERAS) protocol is a set of peri-operative strategies to increase speed of recovery. ERAS is well established in adults but has not been well studied in children. OBJECTIVE: The purpose of the current study was to establish the safety and efficacy of an ERAS protocol in pediatric urology patients undergoing reconstructive operations. It was hypothesized that ERAS would reduce length of stay and decrease complications when compared with historical controls. STUDY DESIGN: Institutional Review Board approval was obtained to prospectively enroll patients aged <18 years if they had undergone urologic reconstruction that included a bowel anastomosis. ERAS included: no bowel preparation, administration of pre-operative oral carbohydrate liquid, avoidance of opioids, regional anesthesia, laparoscopy when feasible, no postoperative nasogastric tube, early feeding, and early removal of intravenous fluids (IVF). Recent (2009-2014) historical controls were propensity matched in a 2:1 ratio on age, sex, ventriculoperitoneal shunt status and whether the patient was undergoing bladder augmentation. Outcomes were protocol adherence, length of stay (LOS), emergency department (ED) visits, re-admission within 30 days, re-operations and adverse events occurring within 90 days of surgery. RESULTS: A total of 26 historical and 13 ERAS patients were included. Median ages were 10.4 (IQR 8.0-12.4) and 9.9 years (IQR 9.1-11), respectively (P = 0.94) (see Summary Table). There were no significant between-group differences in prior abdominal surgery (38% vs 62%), rate of augmentation (88% vs 92%) or primary diagnosis of spina bifida (both 62%). ERAS significantly improved use of pre-operative liquid load (P < 0.001), avoidance of opioids (P = 0.046), early discontinuation of IVF (P < 0.001), and early feeding (P < 0.001). Protocol adherence improved from 8/16 (IQR 4-9) historically to 12/16 (IQR 11-12) after implementation of ERAS. LOS decreased from 8 days to 5.7 days (P = 0.520). Complications of any grade per patient decreased from 2.1 to 1.3 (OR 0.71, 95% CI 0.51-0.97). There were fewer complications per patient across all grades with ERAS. No differences were seen in emergency department (ED) visits, re-admissions and re-operations. DISCUSSION: Implementation improved consistency of care delivered. Tenets of ERAS that appeared to drive improvements included maintenance of euvolemia through avoidance of excess fluids, multimodal analgesia, and early feeding. CONCLUSION: ERAS decreased length of stay and 90-day complications after pediatric reconstructive surgery without increased re-admissions, re-operations or ED visits. A multicenter study will be required to confirm the potential benefits of adopting ERAS.

Mycophenolate mofetil therapy for refractory inflammatory bowel disease.

BACKGROUND: Mycophenolate mofetil (MMF) is an immunomodulatory drug, and its use in inflammatory bowel disease has previously been reported. The aim of this study was to review the Leeds Colitis Clinic experience of the safety and efficacy of MMF in treating patients with refractory Crohn's disease (CD) and ulcerative colitis (UC). This is an extension of a previously published study from our center with a longer follow-up period and approximately twice the number of patients. METHODS: A retrospective analysis was performed of the records of all patients treated with MMF for inflammatory bowel disease over a 5-year period. RESULTS: Of 70 patients identified, 67 had previously been treated with azathioprine unsuccessfully. Seventeen of the 70 patients had been successfully maintained in remission with MMF for an average duration of 33 months. Treatment with MMF was discontinued for 53 patients, 17 because of side effects and 36 because they had not responded to the treatment. CONCLUSIONS: In our series, 17 patients (24.3%) had a sustained steroid-free remission with MMF therapy. Nineteen patients (27%) experienced side effects, of which 17 (24.3% of the total group) had to discontinue therapy. An additional 36 (51.4%) required an escalation in medical therapy or surgery because of failure of the MMF therapy. MMF may have a role in the treatment of refractory inflammatory bowel disease, especially in patients who have previously failed standard therapies such as azathioprine.

Focal cerebral ischaemia induces corticotropin releasing factor (CRF) vascular immunoreactivity in rat occluded hemisphere.

Corticotropin-releasing factor (CRF) induces the dilatation of cerebral blood vessels and increases cerebral blood flow (CBF). CRF receptor antagonists reduce ischaemic damage in the rat. In the present study, the expression of CRF around cerebral vessels has been investigated in the rat. No CRF immunoreactivity was identified around pial or intracerebral vessels in the absence of cerebral ischaemia. Four hours after middle cerebral artery occlusion (MCAo), intensely CRF-positive blood vessels were evident on the ischaemic cortical surface and in the peri-infarct and infarct zone. Increased CRF immunoreactivity was also detected in swollen axons in subcortical white matter, caudate nucleus and lateral olfactory tract of the ipsilateral hemisphere, consistent with the failure of axonal transport. These data provide morphologic support for a role of CRF in the pathophysiology of cerebral ischaemia.

Case-control study of risk factors for fatal and non-fatal injury in crashes of rotary-wing aircraft.

INTRODUCTION: There have been few studies of the risk factors for fatal injury in air crashes of rotary-wing aircraft, and none of risk factors for all serious injury (fatal and non-fatal) in these aircraft. The aim of the study was to identify the potentially modifiable risk factors for injury in civil rotary-wing aircraft crashes in New Zealand. METHOD: We analyzed records from all reported civil rotary-wing aircraft crashes in New Zealand between 1988 and 1994. Air crash data from the official databases were merged with nationwide injury records and information obtained from Coroner's files. Crashes where the pilot-in-command was fatally injured were compared with crashes where the pilot-in-command was not fatally injured on 50 variables, covering pilot, aircraft, environmental, and operational characteristics. A second analysis compared crashes where the pilot-in-command was seriously injured (either fatally or non-fatally) with crashes where the pilot-in-command was not hospitalized with an injury. A series of multivariate logistic regression analyses were conducted to estimate the odds associated with each of the factors identified by the univariate analyses. RESULTS: The most significant risk factors for all serious injury were: (a) not obtaining a weather briefing, (b) off-airport location of the crash site, (c) flights carried out for air transport purposes, and (d) non-solo flights. Other risk factors, significant for fatal injury only, included post-crash fire and the nature of the crash terrain. Factors within the control of the pilot, environmental, and flight characteristics are the key determinants of the injury outcome of civil rotary-wing aircraft crashes.

Inter-rater reliability of the APD, SFU and UTD grading systems in fetal sonography and MRI.

INTRODUCTION: Antenatal hydronephrosis (ANH) is frequently detected on screening obstetric ultrasonography. Common ANH grading systems include the anterior-posterior diameter (APD) and the Society for Fetal Urology (SFU) grading system. Recent developments in the management of ANH include the use of fetal magnetic resonance imaging (MRI), and a new grading system - Urinary Tract Dilation (UTD). This study reviewed patients who underwent fetal MRI and ultrasound, and compared the grading systems across these imaging modalities. MATERIALS AND METHODS: Patients who underwent paired fetal MRI and ultrasound studies between January 2012 and January 2014 were included. Two pediatric urologists and a pediatric radiologist reviewed the studies. Data collected included APD, SFU grade, and UTD grade. Fleiss' kappa statistic determined the inter-rater reliability (IRR) of the SFU and UTD grading within each imaging modality. Intra-class correlation assessed the consistency of the APD measurements. RESULTS: Forty-seven patients and 88 renal units were evaluated. Median gestational age was 22 weeks. Kappa values of the SFU grading system indicated fair IRR for ultrasound imaging and moderate IRR for MRI imaging, while the UTD grading system reached moderate IRR for both. The IRR of the SFU grading system was improved with the use of MRI, while the UTD grading system was no different. The APD intraclass correlation coefficient improved significantly when measured by MRI. As the ultrasound SFU grade increased, the odds of the MRI SFU grade being scored higher increased by a factor of 3.7. There was no difference between ultrasound and MRI when using the UTD grading system. DISCUSSION: This study was the first to assess the UTD system in a cohort of patients who underwent paired ultrasound and MRI studies. The results suggested that the UTD system might improve IRR, compared with the SFU system. The use of fetal MRI may improve the IRR of the SFU grading system. It also found that the proportion of SFU grades was affected by the imaging modality, raising the possibility that MRI 'overcalls' the SFU grade, compared with ultrasound. This difference was not observed using the UTD grading system. The most important limitation was the selection bias favoring complex pathology with severe ANH diagnosed at an early gestational age. CONCLUSIONS: In this unique cohort, the UTD system improved IRR when compared to the SFU grading system. Fetal MRI improved the IRR of the SFU grading system, and improved the APD intraclass correlation. The SFU grading was likely to be higher when assessed by MRI vs ultrasound, but the UTD grade was not affected by the imaging modality.

Health-related quality of life in survivors of Wilms' tumor and advanced neuroblastoma: aA cross-sectional study.

PURPOSE: In pediatric oncology, Wilms' tumor and advanced neuroblastoma represent opposite ends of the spectra of survival probability and therapeutic intensity. Consequently, it was envisaged that survivors of Wilms' tumor would enjoy better health status and health-related quality of life (HRQL) than survivors of advanced neuroblastoma. PATIENTS AND METHODS: Health status questionnaires were sent to the parents of all eligible children and to the children themselves if they were > or = 8 years of age. Responses were received from 84% of 93 eligible families. Responses were converted by established algorithms into levels of two multiattribute health status classification systems known as Health Utilities Index Mark 2 and Mark 3. These systems are linked to measures of preference, in the form of multiattribute utility functions, which provide scores of morbidity for single-attribute levels and of global HRQL for comprehensive health states. RESULTS: A greater burden of morbidity was identified in the survivors of advanced neuroblastoma than in survivors of Wilms' tumor based on the assessments of the parents of these children. In particular, survivors of advanced neuroblastoma exhibited deficits in hearing and speech. It is possible that this morbidity burden reflects the prevalent use of platinum compounds (causing ototoxicity) in this group. Within parent-child dyads there was a high level of percentage agreement on responses in all attributes except cognition. CONCLUSION: Extension of this study to a larger sample size of patients will provide clarification of these observations.

Corticotrophin-releasing factor receptors: from molecular biology to drug design.

Corticotrophin-releasing factor (CRF) acts within both the brain and the periphery to coordinate the overall response of the body to stress. The involvement of the CRF systems in a variety of both CNS and peripheral disease states has stimulated great interest in this peptide as a potential site of therapeutic intervention. The recent cloning of multiple CRF receptor subtypes has precipitated a new era in CRF research that has allowed precise molecular, pharmacological and anatomical examination of mammalian CRF receptors. In this article, Derek Chalmers and colleagues highlight the major differences between the two classes of CRF receptors, CRF1 and CRF2, and a functionally related CRF-binding protein, and discuss the relevance of these sites to the ongoing development of CRF-based therapeutics.

Comparative analysis of the nonA region in Drosophila identifies a highly diverged 5' gene that may constrain nonA promoter evolution.

A genomic fragment from Drosophila virilis that contained all the no-on-transientA (nonA) coding information, plus several kilobases of upstream material, was identified. Comparisons of nonA sequences and the gene nonA-like in D. melanogaster, a processed duplication of nonA, suggest that it arose before the split between D. melanogaster and D. virilis. In both species, another gene that lies <350 bp upstream from the nonA transcription starts, and that probably corresponds to the lethal gene l(1)i19, was identified. This gene encodes a protein that shows similarities to GPI1, which is required for the biosynthesis of glycosylphosphatidylinositol (GPI), a component for anchoring eukaryotic proteins to membranes, and so we have named it dGpi1. The molecular evolution of nonA and dGpi1 sequences show remarkable differences, with the latter revealing a level of amino acid divergence that is as high as that of transformer and with extremely low levels of codon bias. Nevertheless, in D. melanogaster hosts, the D. virilis fragment rescues the lethality associated with a mutation of l(1)i19e, as well as the viability and visual defects produced by deletion of nonA(-). The presence of dGpi1 sequences so close to nonA appears to have constrained the evolution of the nonA promoter.

The clock gene period of the housefly, Musca domestica, rescues behavioral rhythmicity in Drosophila melanogaster. Evidence for intermolecular coevolution?

In Drosophila, the clock gene period (per), is an integral component of the circadian clock and acts via a negative autoregulatory feedback loop. Comparative analyses of per genes in insects and mammals have revealed that they may function in similar ways. However in the giant silkmoth, Antheraea pernyi, per expression and that of the partner gene, tim, is not consistent with the negative feedback role. As an initial step in developing an alternative dipteran model to Drosophila, we have identified the per orthologue in the housefly, Musca domestica. The Musca per sequence highlights a pattern of conservation and divergence similar to other insect per genes. The PAS dimerization domain shows an unexpected phylogenetic relationship in comparison with the corresponding region of other Drosophila species, and this appears to correlate with a functional assay of the Musca per transgene in Drosophila melanogaster per-mutant hosts. A simple hypothesis based on the coevolution of the PERIOD and TIMELESS proteins with respect to the PER PAS domain can explain the behavioral data gathered from transformants.

An evaluation of mouthguard requirements and dental injuries in New Zealand rugby union.

OBJECTIVES: To document the effects of compulsory mouthguard wearing on rugby related dental injury claims made to ACC, the administrator of New Zealand's accident compensation scheme. METHODS: An ecological study was conducted. Estimates of mouthguard wearing rates were available from prospective studies conducted in 1993, 2002, and 2003. Rugby related dental injury claims were available for the period 1995-2003. Player numbers were available from 1998. Mouthguard wearing was made compulsory during match play for rugby players at under 19 level and below at the beginning of the 1997 season, and for all grades of domestic rugby at the beginning of the 1998 season. Greater powers of enforcement were provided to referees at the beginning of the 2003 season. RESULTS: The self reported rate of mouthguard use was 67% of player-weeks in 1993 and 93% in 2003. A total of 2644 claims was reported in 1995. There was a 43% (90% confidence interval 39% to 46%) reduction in dental claims from 1995 to 2003. On the reasonable assumption that the number of players and player-matches remained constant throughout the study period, the relative rate of injury claims for non-wearers versus wearers was 4.6 (90% confidence interval 3.8 to 5.6). The cumulative savings in claim costs compared with the cost per year if claim numbers had remained constant from 1995 is 1.87 million NZD. CONCLUSION: Although ecological studies have acknowledged weaknesses, the findings provide evidence that mouthguard use is a simple and effective injury prevention strategy for rugby players. The use of mouthguards for all players in both matches and contact practice situations is strongly recommended.

Playing conditions, player preparation and rugby injury: a case-control study.

This paper investigates the effect of player preparation, ground conditions and weather conditions upon the injury risk for Rugby Union players. A population-based case-control study was performed using a sample (n= 1043) of New Zealand Rugby Union players aged 16 y and above. Details concerning game preparation (warm-up and usual position), and ground and weather conditions (precipitation, wind and temperature) were obtained from the players. If players were injured during the season (n= 624) they were asked to provide details about the game in which they were last injured. Uninjured players (n= 419) provided details about the last game in which they played. Injuries were more likely to occur when games were played on hard grounds or in calm or warm conditions. Playing out of position and the duration of warming up did not significantly alter the risk of injury. When player preparation, ground and weather conditions, grade, age, playing position and rugby experience were simultaneously controlled for, hard ground and the absence of wind were associated with increased risk. The influence of these factors may be indirect, through adaptation to the conditions in which a game is played.

Distinct hematopoietic support by two human stromal cell lines.

OBJECTIVE: The hematopoietic microenvironment is complex, and the role of myofibroblast in its function is crucial. In order to obtain a stable model reflecting this particular cell type, we have previously established human bone marrow cell lines from primary myofibroblastic Stro1(+) population (pStro1(+)). We placed HPV16 E6 and E7 expression under the control of different promoters. Here, we have characterized and studied the hematopoietic support for two cell lines corresponding to the promoters alpha-SM (alphaSM-56 line) and SV40 (SV40-56 line). MATERIALS AND METHODS: The expression profile was analyzed at the RNA level by gene array and at the protein level by Western blot, flow cytometry, and ELISA. Hematopoietic support determined using colony-forming unit (CFU) and stroma-adherent colony-forming cell (SA-CFC) assays. RESULTS: The phenotype of cell lines was not significantly modified compared with primary myofibroblastic cells. They secreted a broad spectrum of hematopoietic cytokines and nonspecific mediators. The two lines allowed the growth of hematopoietic precursors and had different support capabilities. CONCLUSIONS: We have extensively characterized two novel human bone marrow stromal cell lines. They retained a myofibroblastic phenotype and have substantial but different hematopoietic support capabilities. These lines provided a basis for determining stromal factors involved in stem-cell regulation.

Continuous local anesthetic infusion for children with spina bifida undergoing major reconstruction of the lower urinary tract.

OBJECTIVE: While many options for postoperative analgesia are available to the general patient population, choices are limited for individuals with spinal dysraphism. We hypothesized that the use of continuous local anesthetic infusion following major reconstruction of the lower urinary tract in children with spina bifida would significantly decrease need for opiate use, while maintaining adequate pain control. MATERIALS AND METHODS: Children with spina bifida who underwent major reconstruction of the lower urinary tract at Children's Hospital Colorado were identified from January, 2003 through January, 2013 were identified. In addition to enterocycstoplasty, procedures included Mitrofanoff or Monti creation, bladder neck reconstruction, and Malone antegrade continence enema. Patients who had local anesthetic infusion catheters placed in the incision were compared to patients without catheters. Opioid consumption was calculated by conversion of any opiates into IV morphine (mg/kg) on postoperative days (POD) 0-3. Pain was assessed by mean and maximum FLACC scores on POD 0-2. Use of antiemetic medications and wound related complications were recorded as secondary metrics. Patients with other etiologies for neurogenic bladder and bowel were excluded. Patients whose pain was assessed by other assessment scales were excluded. Chi-squared analysis was used for nominal variables, students t-test was used for analysis of continuous variables. P values <0.05 were considered significant. RESULTS: 36 myelomeningocele patients who underwent primary enterocystoplasty met the inclusion criteria. All surgeries were open procedures. 24 patients in the infusion catheter group were compared to 12 patients who received primary analgesia by PCA or IV narcotics. There were no significant differences in age, sex, weight or spinal defect level between the two groups. Opioid use, as defined by IV morphine equivalents, was significantly less in the wound soaker group on all PODs. The total opioid use after POD #0-3 was 0.55 mg/kg in the wound soaker group vs 1.66 mg/kg in the IV/PCA group (p = 0.03). FLACC scores were uniformly lower in the wound soaker group, but were not significantly different. There was a significant decrease in need for postoperative antiemetic use in the wound soaker group (36.5% vs 83.3%, p = 0.014). Complications and hospital stay were similar between both groups. DISCUSSION: The advantage of local anesthesia is the reduction of systemic opioids and their subsequent adverse side effects. Our results suggest that in children with spina bifida undergoing major reconstruction of the lower urinary tract narcotic consumption is approximately 1/3 when continuous local anesthetic catheters are placed into the incision. The need for antiemetic medication is also significantly less. While this technique has been validated in a variety of other settings, it may be most beneficial in patients with myelomeningocele or other spinal dysraphism where epidural placement is generally contraindicated and narcotic use may have a particularly deleterious effect on preexisting neurogenic bowel function. The primary limitation of our study is that it is a retrospective review of a limited number of patients. Patients were not randomized and subject to other management differences that could have influenced our results in unknown ways. CONCLUSIONS: Continuous local anesthetic catheters are a simple, effective alternative strategy to provide postoperative analgesia while reducing systemic opiate use and associated adverse effects.

CRF2 alpha and CRF2 beta receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues.

We have recently described the cloning and characterization of a novel corticotropin-releasing factor receptor subtype (CRF2) from rat brain that exists in two alternatively spliced forms, CRF2 alpha and CRF2 beta. These forms differ in their N-terminal coding sequence which results in the production of two distinct receptors of 411 and 431 amino acids, respectively. To assess whether these two forms might represent distinct targets for CRF action, RNase protection and in situ hybridization studies were performed using specific N-terminal cRNA probes. The results showed a differential distribution of the mRNAs for these two receptor forms in the rat. The mRNA for CRF2 alpha is found almost exclusively in the brain, particularly in the hypothalamus, lateral septum, and olfactory bulb, whereas the mRNA for CRF2 beta appears to be both in the brain and in the periphery, with the greatest abundance in the heart and skeletal muscle. Thus, the data suggest that these alternatively spliced forms of the CRF2 receptor may represent functionally distinct CRF receptors. In addition, it highlights the importance of probe specificity for in situ hybridization studies.

A.E. Bennett Research Award. Regulation of serotonin1A, glucocorticoid, and mineralocorticoid receptor in rat and human hippocampus: implications for the neurobiology of depression.

BACKGROUND: Disturbances of the limbic-hypothalamic-pituitary-adrenal axis and the serotonin system are commonly found in depressive illness. Studying the effect of stress on these two neurobiological systems may give us important clues into the pathophysiology of affective illness and help us understand how stress and mood disorders are related. METHODS: We studied the effect of chronic unpredictable stress and antidepressant treatment on serotonin 1A (5-HT1A), glucocorticoid (GR), anti mineralocorticoid (MR) receptor levels in rat hippocampus, using in situ hybridization and receptor autoradiography. We also used in situ hybridization to quantify hippocampal 5-HT1A, GR, and MR messenger (mRNA) levels in a small group of suicide victims with a history of depression, compared to matched controls (n = 6). RESULTS: We found that rats subjected to chronic unpredictable stress showed a significant elevation of basal plasma corticosterone compared to nonstressed rats. Chronic stress also caused a decrease in 5-HT1A mRNA and binding in the hippocampus. In addition, chronic stress produced alterations on the MR/GR mRNA ratio in this same region. The decreases in 5-HT1A mRNA and binding, as well as the MR/GR alterations, were prevented in animals that received imipramine or desipramine antidepressant treatment. Zimelidine was unable to reverse stress-induced increases in corticosterone, and was only partially successful in preventing the stress-induced receptor changes in the hippocampus. Suicide victims with a history of depression showed changes that were very similar to the changes found in chronic stress. CONCLUSIONS: Alterations in hippocampal 5-HT1A levels and in the MR/GR balance may be one of the mechanisms by which stress may trigger and/or maintain depressive episodes.

Parity as a factor in incontinence in multiple sclerosis.

Twelve consecutively selected patients with multiple sclerosis and incontinence had electrophysiologic studies performed of the pudendal and perineal innervations of the anal and urinary sphincter. Single-fiber electromyogram density measurements were obtained in the external anal sphincter. Fecal incontinence was found to be unexpectedly frequent. The results suggest that incontinence in patients with multiple sclerosis is often due to the interaction of several factors, including central lesions, lesions of the conus medullaris and, also, coincidental pelvic nerve lesions associated with childbirth. Thus, incontinence is especially a problem in women with this disease.

Regulation of hippocampal 5-HT1A receptor gene expression by dexamethasone.

The effect of dexamethasone, a selective GR agonist, on hippocampal, 5-HT1A receptor mRNA expression and 5-HT1A binding was examined using in situ hybridization histochemistry and in vitro receptor autoradiography. One week after adrenalectomy, both 5-HT1A receptor mRNA expression and 5-HT1A binding were increased throughout the hippocampus. Administration of dexamethasone at the time of adrenalectomy significantly attenuated the increases in 5-HT1A mRNA expression in all hippocampal subfields (p < .05, Fisher Test), although 5-HT1A mRNA levels remained significantly higher than sham levels in all subfields with the exception of CA1. However, 5-HT1A binding levels were responsive to dexamethasone administration only within particular hippocampal subfields, CA1, and dentate gyrus. We conclude that GR occupation negatively regulates 5-HT1A receptor mRNA expression within the hippocampus and that 5-HT1A receptor sites are most sensitive to modulation in those hippocampal subfields expressing higher levels of GR receptors.

Measure of rheumatoid factor in human sera by passive haemagglutination of human erythrocytes carrying immunoglobulin linked by chromic chloride.

Serum rheumatoid factor in sero-positive patients with rheumatoid arthritis may be measured by passive haemagglutination of trypsin-treated human red cells linked with heat-aggregated human IgG by chromic chloride. The results show excellent correlation with those obtained with the classical Rose-Waaler test. The sera may be tested unheated and do not require preliminary absorption with red cells. By this test procedure it should also be possible to analyse the species, allotypic and conformational specificity of different rheumatoid factors.