Burden of illness and costs among paediatric haemophilia patients with and without central venous access devices treated in US hospitals.

INTRODUCTION: Central venous access devices (CVADs) facilitate repeated or urgent treatments for paediatric haemophilia patients, but are associated with complications. This study examined the burden of illness, healthcare utilization and costs for CVADs in a real-world hospital setting. MATERIALS AND METHODS: This study included haemophilia patients ages ≤18 years with discharges during 2006-2014 in the US Premier Healthcare Database. Haemophilia was identified using ICD-9 diagnosis codes and CVAD exposure using billing information. After matching haemophilia patients with and without CVADs on demographic and clinical characteristics, we compared infection, thrombosis, length of stay (LOS), inflation-adjusted hospital cost (2014 $USD) and readmission outcomes using generalized estimating equation models adjusted for hospital teaching status. RESULTS: Among 4793 paediatric haemophilia patients treated at one of 548 hospitals, a total of 197 patients were identified with CVAD exposure. The matched sample included 310 haemophilia patients (155 CVAD and 155 non-CVAD). CVAD cases had greater frequencies of all-cause infections (29% vs 17%, P = .01) and thrombosis (6% vs 1%, P = .06), longer adjusted mean LOS (9.5 vs 4.7 days, P = .002), higher adjusted mean inpatient total hospitalization costs ($47200 vs $25389, P = .02) as well as more inpatient and outpatient visits at 30-, 60- and 90-days (P < .05 for all differences) compared with non-CVAD patients. CONCLUSION: Paediatric haemophilia patients with CVADs experienced greater infection rates, healthcare utilization and higher hospitalization costs compared with non-CVAD patients. The results of this study may inform further research efforts to understand the costs and benefits of novel treatment alternatives for young haemophilia patients requiring CVADs.

Early menarche and blood pressure in adulthood: systematic review and meta-analysis.

Background: It has been reported that early menarche is associated with high blood pressure and hypertension. However, some studies have failed to observe such association. We carried out a systematic review and meta-analysis on the association of early menarche with hypertension and high blood pressure in adulthood. Methods: PUBMED, SciELO, Scopus and LILACS databases were searched. Studies that evaluated the association of early menarche with hypertension or high blood pressure, among women aged 20 years or more were included. Random effects models were used to pool the estimates. Meta-regression was used to evaluate the contribution of different co-variables to heterogeneity. Results: We identified 17 studies with 18 estimates on the association of early menarche with hypertension and high blood pressure. The odds of hypertension/high blood pressure was higher among women with early menarche [pooled (OR):1.25; 95% confidence interval (CI): 1.17-1.34; P < 0.001]. In the meta-regression analysis, studies evaluating 1500 subjects or more had a higher pooled OR [1.27; 95%CI (1.19;1.36)] than those with less participants. Although funnel plots showed some asymmetry, Egger tests were not statistically significant. Therefore, it is unlikely that the observed association was to publication bias. Conclusions: Early menarche is associated with hypertension among adult woman.

Immediate postoperative extubation in bilateral lung transplantation: predictive factors and outcomes.

BACKGROUND: We reviewed our experience with tracheal extubation in the operating room (E-OR) among cystic fibrosis patients requiring bilateral lung transplantation to evaluate safety and determine predictive factors of E-OR. METHODS: The charts of 89 recipients (from May 2007 to June 2013) were analysed. Patients were divided into E-OR and E-ICU (intensive care unit extubation) groups. Data are expressed as numbers (percentages) or medians [25th-75th percentiles]. RESULTS: There were 41 patients in the E-OR group (46%). Donor and recipient characteristics were similar between groups. Intraoperative complications occurred less frequently in the E-OR group, and fluid and transfusion requirements were lower. Postoperative courses were different in the E-OR group, including a lower rate of grade 3 primary graft dysfunction (0 compared with 19 patients, P<0.0001) and shorter ICU (5.0 [3.7-7.2] compared with 11.5 [7.0-15.5] days) and hospital stays (22.0 [18.0-25.5] compared with 33.0 [25.0-56.5] days, respectively; P<0.0001 for both). The 1 yr survival rates were similar: 95% in the E-OR group and 98% in the E-ICU group. A statistical model built on a development cohort of 60 randomly selected patients predicted 95% of E-OR instances in this cohort and 82% of E-OR instances in the validation cohort (28 patients). Predictive factors were complications during single-lung ventilation (second graft implantation), complications during bipulmonary ventilation (end of surgery), and the ratio of arterial partial pressure of oxygen to fractional inspired oxygen (end of surgery). CONCLUSIONS: Our protocol allowed for extubation of 46% of bilateral lung transplant patients without increased postoperative risks.

The minimally important difference in the Rhinoconjunctivitis Total Symptom Score in grass-pollen-induced allergic rhinoconjunctivitis.

BACKGROUND: The minimally important difference (MID) has been defined as the smallest improvement considered worthwhile by a patient. The MID has not been estimated for the Rhinoconjunctivitis Total Symptom Score (RTSS). METHODS: In a prospective multicentre study, patients consulting for grass-pollen-induced allergic rhinitis (AR) recorded a 15-point global rating of change scale (GRCS) score and the Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ) score on a weekly basis and the individual symptom scores comprising the RTSS on a daily basis over two consecutive weeks. The MID in the RTSS was determined with anchor-based methods (using the GRCS and the RQLQ) and a distribution-based method [based on the RTSS' standard deviation (SD)]. RESULTS: The study population comprised 806 patients (253 children, 250 adolescents and 303 adults). During the first week of the study, the mean ± SD RTSSs for these age groups were 6.5 ± 3.3, 6.8 ± 3.4 and 7.0 ± 3.4, respectively. For an improvement of 2 points in the GRCS or 0.5 points in the RQLQ score, the regression analysis yielded MIDs in the RTSS of 1.24 ± 0.17 and 1.12 ± 0.14 in children, 1.33 ± 0.14 and 1.20 ± 0.13 in adolescents and 1.13 ± 0.14 and 0.89 ± 0.12 in adults, respectively. When applying distribution-based methods, the MID ranged from 1.09 to 1.13 (based on 0.33 SDs of the first-week RTSS) and from 1.22 to 1.40 (based on 0.5 SDs of the difference in RTSSs between the first and second weeks). CONCLUSION: The MID in the RTSS was consistently estimated as 1.1-1.3 (and could conceivably be rounded to 1) in patients with grass-pollen-induced AR.

Age at menarche and risk of type 2 diabetes among African-American and white women in the Atherosclerosis Risk in Communities (ARIC) study.

AIMS/HYPOTHESIS: We examined race differences in the association between age at menarche and type 2 diabetes before and after adjustment for adiposity. METHODS: We analysed baseline and 9-year follow-up data from 8,491 women (n = 2,505 African-American, mean age 53.3 years; n = 5,986 white, mean age 54.0 years) in the Atherosclerosis Risk in Communities (ARIC) study. Stratifying by race, we used logistic regression to estimate the OR for prevalent diabetes at baseline, and Cox proportional hazard models to estimate the HR for incident diabetes over follow-up according to age at menarche category (8-11, 12, 13, 14 and 15-18 years). RESULTS: Adjusting for age and centre, we found that early age at menarche (8-11 vs 13 years) was associated with diabetes for white, but not African-American women in both the prevalent (white OR 1.72, 95% CI 1.32, 2.25; African-American OR 1.13, 95% CI 0.84, 1.51; interaction p = 0.043) and incident models (white HR 1.43, 95% CI 1.08, 1.89; African-American HR 1.20, 95% CI 0.87, 1.67; interaction p = 0.527). Adjustment for adiposity and lifestyle confounders attenuated associations for prevalent (white OR 1.41, 95% CI 1.05, 1.89; African-American OR 0.94, 95% CI 0.68, 1.30; interaction p = 0.093) and incident diabetes (white HR 1.22, 95% CI 0.92, 1.63; African-American HR 1.11, 95% CI 0.80, 1.56; interaction p = 0.554). CONCLUSIONS/INTERPRETATION: Early menarche was associated with type 2 diabetes in white women, and adulthood adiposity attenuated the relationship. We did not find a similar association in African-American women. Our findings suggest that there may be race/ethnic differences in the influence of developmental factors in the aetiology of type 2 diabetes, which merit further investigation.

Pharmacokinetics and tolerability of a novel long-acting glucagon-like peptide-1 analog, CJC-1131, in healthy and diabetic subjects.

OBJECTIVE: The safety, tolerability, pharmacokinetics and preliminary pharmacodynamics of single rising doses of a novel GLP-1 analog, CJC-1131, was evaluated. METHODS: CJC-1131 was subcutaneously injected in 8 groups (1.5 - 20.5 microg/kg) of healthy subjects (each group of six subjects included 1 placebo per dose level). CJC-1131 was also injected subcutaneously in 6 groups (1.5 - 12 microg/kg) of Type 2 diabetic patients after a 9-day washout period from their own anti-diabetic medication. Each group of 8 patients included 2 placebo-treated patients. Seven blood glucose measurements were taken daily, and meal tolerance tests were performed on the day before dosing and on Day 3. RESULTS: CJC-1131 was quickly absorbed from the subcutaneous space, and a less than dose-proportional increase was found in Cmax. The half-life of CJC-1131 varied from 8.9 - 14.7 days in healthy subjects and from 9.1 - 13.8 days in patients. The maximum tolerated dose in healthy subjects was established at 12 microg/kg with nausea and vomiting being the dose-limiting events. These events occurred generally in the morning after dosing. Blood glucose levels in patients decreased on Day 1 in proportion with dose, with a maximum average decrease of 4.1 mmol/l in the highest dose group. Higher doses appeared to be related to a slight weight loss in patients. CONCLUSIONS: Conjugation to albumin led to a major prolongation of the half-life of GLP-1. The tolerability of this potential antidiabetic drug seems to be limited only by gastrointestinal complaints.

Predictors of mitral annulus enlargement? A real-time three-dimensional transesophageal study.

BACKGROUND: Mitral annulus (MA) enlargement can be observed in various cardiac conditions but respective influence of left atrial (LA) and left ventricle (LV) size remained unclear. METHODS: In 120 patients who underwent a clinically indicated 3D-transesophageal-echocardiography, 30 atrial fibrillation (AF), 30 secondary mitral regurgitation (SMR), 30 primary myxomatous mitral regurgitation (PMR) and 30 mitral stenosis (MS), we evaluated the association between MA area (MA-area) and LA volume (LAvol) measured using the biplane area-length method, end-diastolic (LVEDV) and end-systolic (LVESV) volumes measured using the biplane Simpson method. MA-area was measured based on 3D datasets using QLab10. RESULTS: MA-area was correlated to LVEDV (r = 0.42, p < 0.0001), LVESV (r = 0.29, p = 0.001) but more markedly to LAvol (r = 0.62, p < 0.0001). Correlation between MA-area and LAvol was sustained in all subsets whereas MA-area was not correlated to LVEDV and LVESV in patients with SMR and with PMR (all p > 0.10). In multivariate analysis main predictors of MA-area were LAvol (p < 0.0001) and myxomatous etiology of MR (p = 0.0003) followed by LVEDV (p = 0.006) and LVESV (p = 0.02). CONCLUSION: In a population of patients with a wide range of LA/LV size related to various conditions, LA volume and myxomatous MR etiology appeared as main predictors of MA size whereas LV size had a more modest influence.

Molecular mechanisms of McArdle's disease (muscle glycogen phosphorylase deficiency). RNA and DNA analysis.

Lack of muscle glycogen phosphorylase activity leads to McArdle's disease, a rare metabolic myopathy. To investigate its molecular basis at the nucleic acid level, we isolated muscle phosphorylase cDNA clones from a human cDNA library in Escherichia coli plasmid pBR 322. Subcloning of one insertion of M13 bacteriophage permitted its definite identification by sequencing. Northern blot experiments revealed one specific messenger RNA of 3.4 kilobases found uniquely in tissues expressing muscle phosphorylase. We show that McArdle's disease exhibits a molecular heterogeneity at the messenger RNA level. In eight unrelated cases of McArdle's disease in which no inactive proteins had been detected, we assayed muscle biopsies for phosphorylase mRNA by Northern blotting. In five cases, no muscle phosphorylase mRNA could be detected, while in three other cases, normal length mRNA was present in lower amounts. Moreover, Southern blot analysis of DNA isolated from white blood cells in four McArdle patients revealed no major deletion or rearrangements of the phosphorylase gene as compared with controls.

Dietary and hormonal regulation of aldolase B gene expression.

Aldolase B is an enzyme of the glycolytic pathway whose activity and mRNA levels in the liver fluctuate according to dietary status. Both the enzyme activity and the mRNA concentration decline during fasting and increase four- to eightfold upon refeeding of a carbohydrate-rich diet. The mechanism, however, of the mRNA induction remains unknown. To elucidate the mechanisms that regulate this induction responsive to dietary stimuli, we have studied the roles of hormones and glycolytic substrates on aldolase B gene expression in three tissues that synthesize the enzyme. Using a cDNA probe complementary to rat aldolase B mRNA, we determined the amount of cytoplasmic RNAs in the liver, kidney, and small intestine of normal, adrenalectomized, thyroidectomized, diabetic, and glucagon- or cAMP-treated animals refed either a fructose-rich or a maltose-rich diet. The in vivo hormonal control of gene expression was found to be very different in the three organs tested. In the liver, cortisone and thyroid hormones were required for the induction of the specific mRNA by carbohydrates, while in the kidney none of the hormonal modifications tested altered the level of mRNA induction. In the liver, but not in the kidney, diabetes and glucagon administration abolished the induction of aldolase B mRNAs in animals refed the maltose-rich diets. In the small intestine, only diabetes and thyroidectomy affected the gene expression. Finally, no induction occurred when normal fasted rats were given any of the hormones. Thus, the in vivo hormonal control of liver aldolase B gene expression differs significantly from that of kidney and small intestine. In the liver, the mRNA induction requires the presence of dietary carbohydrates, of permissive hormones, and the cessation of glucagon release, while in the kidney, the induction of the mRNAs by fructose occurs regardless of the hormonal status of the animals. The hormonal control of aldolase B mRNA levels in the small intestine is intermediate.

Homozygous PPT1 Splice Donor Mutation in a Cane Corso Dog With Neuronal Ceroid Lipofuscinosis.

A 10-month-old spayed female Cane Corso dog was evaluated after a 2-month history of progressive blindness, ataxia, and lethargy. Neurologic examination abnormalities indicated a multifocal lesion with primarily cerebral and cerebellar signs. Clinical worsening resulted in humane euthanasia. On necropsy, there was marked astrogliosis throughout white matter tracts of the cerebrum, most prominently in the corpus callosum. In the cerebral cortex and midbrain, most neurons contained large amounts of autofluorescent storage material in the perinuclear area of the cells. Cerebellar storage material was present in the Purkinje cells, granular cell layer, and perinuclear regions of neurons in the deep nuclei. Neuronal ceroid lipofuscinosis (NCL) was diagnosed. Whole genome sequencing identified a PPT1c.124 + 1G>A splice donor mutation. This nonreference assembly allele was homozygous in the affected dog, has not previously been reported in dbSNP, and was absent from the whole genome sequences of 45 control dogs and 31 unaffected Cane Corsos. Our findings indicate a novel mutation causing the CLN1 form of NCL in a previously unreported dog breed. A canine model for CLN1 disease could provide an opportunity for therapeutic advancement, benefiting both humans and dogs with this disorder.

Alpha-mannosidase in human red cells.

1. A search for lysosomal hydrolases and related enzymes has been made in hemolysates from human and rabbit red cells. Apart from acid phosphatases, significant activities were found only for alpha-mannosidase, neutral alpha-glucosidase and beta-hexosaminidase. 2. alpha-Mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) activity per cell in human red blood cells was 200-times lower than in white cells. The optimal pH was 5.5--6.0. Electrophoresis on cellulose acetate showed three bands. Hemolysates from four patients with mannosidosis were not deficient in alpha-mannosidase. pH activity curves and elctrophoretic pattern were similar to those of controls. From its biochemical and genetic properties, it is concluded that red cell mannosidase differs from the lysosomal acid mannosidase.

Hexosaminidase C in Tay-Sachs and Sandhoff disease.

1. Hexosaminidase C has been purified from human placenta. Complete separation from hexosaminidases A and B was achieved. 2. The following properties of hexosaminidase C differ from those of the A and B isozymes. Presence in the supernatant rather than the lysosomes, neutral pH optimum, higher molecular weight, lack of activity on beta-N-acetylgalactosamine derivatives, and lack of immunological relationship. 3. Hexosaminidase C is active in patients deficient in hexosaminidases A and B and can be recognized by its characteristic electrophoretic mobility. It is concluded that the genetic origin of hexosaminidase C is probably different from that of hexosaminidases A and B.

Molecular heterogeneity of rabbit heart phosphorylase kinase.

Phosphorylase kinase (ATP: phosphorylase-b phosphotransferase, EC 2.7.1.38) from rabbit heart, when submitted to electrophoresis on Pevikon, separates into two discrete peaks A and B. The two peaks have been analyzed using reelectrophoresis, chromatography on DEAE-cellulose, thermal stability, inactivation by EGTA (ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) and reaction with an anti-muscle phosphorylase kinase antiserum. It can be concluded that rabbit heart extracts contain two isozymes of phosphorylase kinase. The more negatively charged isozyme seems to be identical with the muscle enzyme. The other isozyme resembles the liver enzyme but differs from the major fraction of the latter by its charge. It is likely that there exist at least three molecular types of phosphorylase kinase.

Studies on the nature of different molecular forms of glucose-6-phosphate dehydrogenase purified from human leukocytes.

Several molecular forms of human glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) corresponding to different stages of post-synthetic modifications have been purified from human leukocytes. The various enzyme forms were different in their specific activity, their kinetic properties and their isoelectrofusing pattern. The molecular weight of the subunits of the different forms was not modified. The changes in the electrofocusing pattern were not due to modifications of the N-terminal ends, the oxidation of thiol groups or the non-covalent fixation of an acid molecule upon the enzyme. Carboxypeptidase B cleaved a C-terminal lysine from the different enzyme forms and shifted the isoelectric point of the different enzyme active bands towards the acid pH. The different enzyme forms studied here seemed to result from the action upon 'native glucose-6-phosphate dehydrogenase' of 'modifying factors' especially abundant in some leukemic granulocytes. The modifying factors did not seem to be consumed during the 'modification' of glucose-6-phosphate dehydrogenase. Moreover, the storage for one year of unmodified enzyme resulted in changes in its electrofocusing pattern similar to those quickly induced by the 'modifying factors'. Consequently it appears that the modifying factors are catalysts of the modification of special residues of glucose-6-phosphate dehydrogenase. The hypothesis that this modification involves the deamination of asparagine or glutamine residues is put forward.

Hyperanodic forms of human glucose-6-phosphate dehydrogenase.

Pure glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) is transformed into 'hyperanodic forms' when incubated at acidic pH and in the presence of NADP+ with excess of glucose-6-phosphate or with some 'NADP+ modifying proteins' purified from the same cells. The enzyme hyperanodic forms exhibit low isoelectric point, altered kinetic properties and high lability to heat, urea, and proteolysis. Differences between hyperanodic and native forms of glucose-6-phosphate dehydrogenase are also noted by microcomplement fixation analysis, ultraviolet absorbance difference spectrum and fluorescence emission spectrum. Drastic denaturation of the enzyme by urea and acid treatment did not suppress the difference of isoelectric point between native and hyperanodic forms of glucose-6-phosphate dehydrogenase. From our data we suggest that the conversion into hyperanodic forms could be due to the covalent binding on the enzyme of a degradation product of the pyridine nucleotide coenzyme. This modification could constitute a physiological transient step toward the definitive degradation of the enzyme.

Calmodulin ligands. The interaction of muscle phosphorylase kinase with phosphodiesterase. Comparison of calmodulin ligands in muscle extracts from normal and phosphorylase kinase-deficient mice.

Interactions between phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38) and calmodulin were studied with pure preparations of muscle phosphorylase kinase, and with crude extracts from muscles of control (C57 Black) and deficient (ICR/IAn) mice, which lack muscle phosphorylase kinase activity. Calmodulin was determined by its ability to stimulate a calmodulin-dependent phosphodiesterase. The amount of calmodulin bound to phosphorylase kinase in muscle extract was estimated to a maximum of 30% of the total amount of calmodulin. In the muscle of the deficient strain a decrease of 35% in the total amount of calmodulin was observed. This correlates with the absence of the calmodulin fraction specifically bound to phosphorylase kinase. From sucrose gradient studies we demonstrated that in the presence of Ca2+ the amount of calmodulin bound to phosphorylase kinase was enhanced, compared to the control in the presence of EGTA. This observation was made both in crude extracts and in pure phosphorylase kinase preparations. Sucrose gradient also showed that muscle phosphorylase kinase can be dissociated to low molecular species when extracts are made in the presence of Ca+; this dissociation was found to be related to a Ca2+-dependent proteolytic effect.

Two different species of messenger RNAs specify synthesis of M1 and M2 pyruvate kinase subunits.

A study was performed to determine whether M1 and M2 pyruvate kinases were synthesized under the direction of one or two messenger RNAs. We compared M1 and M2 pyruvate kinases purified from fresh tissues with those neosynthesized under the direction of messenger RNAs from tissues synthesizing either M1 or M2. RNA was isolated from rat muscle, lung, spleen and kidney by ethanol precipitation in 7 M guanidium chloride, translated in rabbit reticulocyte system and newly-synthesized pyruvate kinase subunits were purified by microimmunoaffinity chromatography. Pyruvate kinase from fresh muscle and spleen was purified in one step by a similar process. Muscle and spleen RNA directed the synthesis of M subunits with molecular weights of approx. 61000 and 62000, respectively, the same as those of the corresponding fresh tissue monomers. In addition, peptide maps obtained by partial digestion of neosynthesized M1 and M2 with V8 protease from Staphylococcus aureus confirmed that these polypeptides were clearly different.

Phenotypic characteristics of early-onset autosomal-dominant type 2 diabetes unlinked to known maturity-onset diabetes of the young (MODY) genes.

OBJECTIVE: To investigate whether there are forms of early-onset autosomal-dominant type 2 diabetes that are distinct from typical maturity-onset diabetes of the young (MODY) and to characterize their phenotypic characteristics. RESEARCH DESIGN AND METHODS: The study included 220 affected subjects from 29 families in which early-onset type 2 diabetes occurred in multiple generations and was not linked to known MODY genes (MODY gene-negative families). All individuals underwent an oral glucose tolerance test and other clinical measurements aimed at investigating the underlying metabolic defect and the presence of diabetic complications. For comparison, 79 affected carriers of MODY3 (hepatocyte nuclear factor [HNF]-1 alpha) mutations were similarly examined. RESULTS: Subjects from MODY gene-negative pedigrees were diagnosed with diabetes at an older age (36 +/- 17 vs. 21 +/- 10 years, P = 0.0001) and were more frequently obese (52 vs. 18%, P = 0.0001) than MODY3 individuals. MODY gene-negative patients who were insulin treated required more exogenous insulin than did MODY3 subjects (0.7 +/- 0.4 vs. 0.45 +/- 0.2 U.kg-1.day-1, P = 0.04), despite similar C-peptide levels. Among subjects not treated with insulin, MODY gene-negative subjects had significantly higher serum insulin levels, both fasting (16.5 +/- 15 vs. 6.5 +/- 5 microU/ml, P = 0.027) and 2 h after a glucose load (53 +/- 44 vs. 11 +/- 10, P = 0.002). They also had higher serum triglycerides (P = 0.02), higher cholesterol levels (P = 0.02), more hypertension (P = 0.0001), and more nephropathy (P = 0.001). Differences persisted when families were matched for age at diagnosis. CONCLUSIONS: Our findings indicate the existence of forms of early-onset autosomal-dominant type 2 diabetes that are distinct from MODY and are frequently characterized by insulin resistance, similar to later-onset type 2 diabetes. Because of the Mendelian pattern of inheritance, the goal of identifying the genes involved in these forms of diabetes appears to be particularly feasible.

HB-GAM/pleiotrophin: localization of mRNA and protein in the chicken developing leg.

The heparin-binding growth-associated molecule HB-GAM (also named pleiotrophin) is a developmentally-regulated protein that belongs to a new family of heparin-binding molecules with putative functions during cell growth and differentiation. In order to study the localization of HB-GAM during chicken embryogenesis, we produced specific monoclonal antibodies to this factor. HB-GAM protein is first observed at stage 23 in the developing nervous system and later in the forming cartilage. We present an investigation of the HB-GAM mRNA expression and HB-GAM protein distribution in the developing leg by in situ hybridization and immunocytochemical studies. We focused our attention on the development of the tibia, where the HB-GAM protein appears at stage 27-28, i.e., just after the condensation of the mesodermal precursor cells of the chondrocytes. The protein then progressively accumulates in the central part of the embryonic cartilage (diaphysis). It persists until stage 42-44 in the regions where hypertrophic cartilage is being replaced by bone marrow. In contrast to the protein, the transcript is first detected at stage 26-27 and later expressed essentially in the epiphysis until stage 37. Therefore the localization of the mRNA does not parallel that of the protein and our data suggest a long half-life of the protein in the hypertrophic cartilage. In addition, the layer of stacked cells surrounding the cartilage core (usually considered as the osteoprogenitor cells) clearly expresses the HB-GAM message between stages 30-37 whereas differentiated osteoblasts do not. Furthermore, the distribution of HB-GAM protein in the osteoblast/osteoid layer suggests an involvement of this protein in early steps of osteogenesis. HB-GAM is absent from the newly formed bone.

Subspectacular nematodiasis caused by a novel Serpentirhabdias species in ball pythons (Python regius).

Subspectacular nematodiasis was diagnosed in three captive-bred juvenile ball pythons (Python regius) from two unrelated facilities within a 6-month period. The snakes were presented with similar lesions, including swelling of facial, periocular and oral tissues. Bilaterally, the subspectacular spaces were distended and filled with an opaque fluid, which contained nematodes and eggs. Histopathology showed nematodes throughout the periocular tissue, subspectacular space and subcutaneous tissue of the head. The nematodes from both facilities were morphologically indistinguishable and most closely resembled Serpentirhabdias species. Morphological characterization and genetic sequencing indicate this is a previously undescribed rhabdiasid nematode.