A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.

INTRODUCTION: Discrepancies have been previously reported for one-stage clotting and chromogenic assays for FVIII activity analysis. Inter-laboratory variations in instruments, method of clot detection, assay set-up, reference standard calibration, reagent source and reagent composition all contribute to assay variability. AIM: To characterise multilaboratory assay variability in measuring ADYNOVATE, OBIZUR and ADVATE FVIII activity in human plasma and survey multinational FVIII activity assay preferences. METHODS: As samples from patients treated with either of the FVIII products are not available in the quantities required for a systematic collaborative study, haemophilia A plasma was spiked in vitro with either ADYNOVATE (PEGylated rFVIII), OBIZUR [Porcine Sequence Antihaemophilic Factor (Recombinant)] or ADVATE at high (0.80 IU or U mL-1 ), medium (0.20 IU or U mL-1 ) and low (0.05 IU or U mL-1 ) FVIII concentrations, based on labelled potencies. Clinical laboratories used their routine FVIII activity assay to determine FVIII activity of each product. Thirty-five data sets using one-stage clotting assay and 11 sets using chromogenic assay were obtained. RESULTS: A vast majority of laboratories (98%) prefer and rely on the one-stage clotting assay. Mean recoveries across all concentrations were 113%, 120% and 127% for ADYNOVATE, OBIZUR and ADVATE respectively. Assay variation was comparable between ADVATE, ADYNOVATE and OBIZUR with inter-laboratory percent coefficients of variation (%CV) ranging from 11 to 22%. Mean chromogenic assay results were 116%, 51% and 113% for ADYNOVATE, OBIZUR and ADVATE respectively. Inter-laboratory CV's were similar for ADYNOVATE, OBIZUR and ADVATE. CONCLUSIONS: One-stage clotting assays can and will be used with sufficient accuracy and precision for the measurement of ADYNOVATE, OBIZUR and ADVATE in plasma samples from subjects with haemophilia A. Chromogenic assay underestimates OBIZUR potency, particularly at lower concentrations.

A straightforward method for determination of the sevoflurane metabolite hexafluoroisopropanol in urinary occupational medical samples by headspace-gas chromatography mass spectrometry.

Biological monitoring of the unmodified sevoflurane and its metabolite hexafluoroisopropanol (HFIP) in urine samples was proposed to determine the individual exposure levels of the medical staff. In this study, a method for simultaneous determination of both compounds in urine using static headspace-gas chromatography-mass spectrometry (HS-GC-MS) was developed. The method is linear over a broad concentration range from 1 to 1000 µg/L (r2 > 0.999) and shows high precision. Limits of quantification (LOQ) are 0.6 µg/L for sevoflurane and 3 µg/L for HFIP, representing an excellent sensitivity without the necessity of analyte enrichment. The method was successfully applied in a German pilot-study to monitor both compounds in samples from medical personnel working in operating theatres. Urinary concentrations of HFIP ranged between < LOQ and 145 µg/L, while sevoflurane was below the LOD in all samples.

Suberoylanilide hydroxamic acid: a potential epigenetic therapeutic agent for lung fibrosis?

Pulmonary fibrosis represents a fatal stage of interstitial lung diseases of known and idiopathic aetiology. No effective therapy is currently available. Based on an indication-discovery approach we present novel in vitro evidence that the histone deacetylases inhibitor suberoylanilide hydroxamic acid (SAHA), an FDA approved anti-cancer drug, has antifibrotic and anti-inflammatory potential. Human lung fibroblasts (fetal, adult and idiopathic adult pulmonary fibrosis) were treated with transforming growth factor (TGF)-beta 1 with or without SAHA. Collagen deposition, alpha-smooth muscle actin (alpha-SMA) expression, matrix metalloproteinase (MMP)1 activity, tissue inhibitor of MMP (TIMP)1 production, apoptosis and cell proliferation were assessed. Pro-inflammatory cytokines relevant to pulmonary fibrosis were assayed in SAHA-treated human peripheral blood mononuclear cells (PBMC) and its subpopulations. SAHA abrogated TGF-beta 1 effects on all the fibroblast lines by preventing their transdifferentiation into alpha-SMA positive myofibroblasts and increased collagen deposition without inducing apoptosis. However, MMP1 activity and TIMP1 production was modulated without a clear fibrolytic effect. SAHA also inhibited serum-induced proliferation of the fibroblast lines and caused hyperacetylation of alpha-tubulin and histone. Cytokine secretion was inhibited from PBMC and lymphocytes at nonapoptotic concentrations. Taken together, these data demonstrate combined antifibrotic and anti-inflammatory properties of SAHA, suggesting its therapeutic potential for pulmonary fibrosis.

DNA-level diversity and relatedness of Helicobacter pylori strains in shantytown families in Peru and transmission in a developing-country setting.

The efficiency of transmission of a pathogen within families compared with that between unrelated persons can affect both the strategies needed to control or eradicate infection and how the pathogen evolves. In industrialized countries, most cases of transmission of the gastric pathogen Helicobacter pylori seems to be from mother to child. An alternative model, potentially applicable among the very poor in developing countries, where infection is more common and the sanitary infrastructure is often deficient, invokes frequent transmission among unrelated persons, often via environmental sources. In the present study, we compared the genotypes of H. pylori from members of shantytown households in Peru to better understand the transmission of H. pylori in developing-country settings. H. pylori cultures and/or DNAs were obtained with informed consent by the string test (a minimally invasive alternative to endoscopy) from at least one child and one parent from each of 62 families. The random amplified polymorphic DNA fingerprints of 57 of 81 (70%) child-mother strain pairs did not match, nor did the diagnostic gene sequences (>1% DNA sequence difference), independent of the child's age (range, 1 to 39 years). Most strains from siblings or other paired family members were also unrelated. These results suggest that H. pylori infections are often community acquired in the society studied. Transmission between unrelated persons should facilitate the formation of novel recombinant genotypes by interstrain DNA transfer and selection for genotypes that are well suited for individual hosts. It also implies that the effective prevention of H. pylori infection and associated gastroduodenal disease will require anti-H. pylori measures to be applied communitywide.

Partially formed native tertiary interactions in the A-state of cytochrome c.

Considerable insight into protein structure, stability, and folding has been obtained from studies of non-native states. We have studied the extent of native tertiary contacts in one such molecule, the A-state of yeast iso-1-ferricytochrome c. Previously, we showed that the interface between the N and C-terminal helices is completely formed in the A-state. Here, we focus on interactions essential for forming the heme pocket of eukaryotic cytochromes c. To determine the extent of these interactions, we used saturation mutagenesis at the evolutionarily invariant residue leucine 68, and measured the free energy of denaturation for the native states and the A-states of functional variants. We show that, unlike the interaction between the terminal helices, the native interactions between the 60s helix and the rest of the protein are not completely formed in the A-state.

Basal forebrain lesions impair tactile discrimination and working memory.

Rats received bilateral injections of the excitotoxin, N-methyl-D,L aspartate, which resulted in degeneration of basal forebrain cholinergic (BFC) neurons in the nucleus basalis magnocellularis. Most tests of general neurological function revealed no differences between control rats and those with BFC lesions and where differences were found they appeared to be due to hyperemotionality. Rats with BFC lesions demonstrated significant deficits in working memory, as evaluated in an 8-arm radial maze. In addition, these rats showed a severe impairment in tactile discrimination learning, an effect of BFC lesions not previously demonstrated. We propose that cholinergic deafferentation of the somatosensory cortex with consequent disruption in somatosensory information processing might account at least in part for this effect.

A happy state of mind: a history of mild elation, denial of disability, optimism, and laughing in multiple sclerosis.

Although multiple sclerosis (MS) is often looked on as a disorder of the sensory and motor systems, it can also be associated with changes in emotion and personality. Many patients with MS seem cheerful, optimistic about the future, and strangely unconcerned about their ongoing physical deterioration. In addition, patients with MS have a tendency to break into uncontrollable laughing, even when they have no reason to be happy. This article looks at how these seemingly upbeat affective changes were viewed by early researchers of MS, including Cruveilhier, Charcot, and Moxon during the 19th century and Cottrell, Wilson, and Ombredane in the 1920s. Frequently cited studies on the emotional correlates of MS from the mid-20th century are also presented, and some trends in the more recent literature are identified.

The 'Kennard effect' before Kennard. The early history of age and brain lesions.

The role of age in recovery of function after brain damage has been of particular interest since the mid-1930s when Kennard described sparing of motor function following brain damage in infant monkeys. In the years since her initial papers, this phenomenon has become known as the "Kennard principle." This article describes a number of observations of the Kennard principle prior to Kennard's first publication. Included are descriptions of both early animal research and neurologic cases.

Gustave Dax and the early history of cerebral dominance.

In 1863, 2 years before Paul Broca published his heralded paper on the special role of the left hemisphere in speech, Gustave Dax sent a paper to the Académie de Médecine in Paris, France. His lengthy submission included an insightful memoir presumably written by his father Marc in 1836 and supportive material that he had collected himself. The present article examines the events leading to Gustave's 1863 submission to the Académie. It also presents an English translation of the negative response that this paper received and a translation of the short article that Gustave published in 1865. These materials help to show how cerebral dominance was first discovered, how it was made public, and how the first advocates of the concept were judged by their contemporaries.

Gilles de la Tourette and the discovery of Tourette syndrome. Includes a translation of his 1884 article.

In 1885, Gilles de la Tourette described 9 patients who suffered from a disorder characterized by involuntary movements, echolalia, echopraxia, coprolalia, and strange, uncontrollable sounds. In his article, Gilles de la Tourette presented some earlier descriptions of this disorder. To appreciate what first led Gilles de la Tourette to Tourette syndrome, however, it is necessary to turn to an article that he published a year earlier. In his 1884 article, Gilles de la Tourette cited several movement disorders that he thought were similar to each other, yet different from true chorea. After describing these disorders, namely, "jumping" of Maine, latah of Malaysia, and miryachit of Siberia, he briefly mentioned a boy in Charcot's ward in Paris, France, who seemed to exhibit the same condition. In an addendum, he then said that other cases were now surfacing in Paris and that he would write an additional article describing these individuals. To achieve a more thorough understanding of the events that led Gilles de la Tourette to his 1885 description of the disorder that now bears his name, we herein present an English-language translation of his 1884 article along with a commentary.

Comparison of behavioral effects of nucleus basalis magnocellularis lesions and somatosensory cortex ablation in the rat.

Cholinergic neurons in the nucleus basalis region of the forebrain project to various portions of the cerebral cortex, including somatosensory cortex. Degeneration of these neurons and their cortical projections is a major feature of the neuropathology of Alzheimer's disease. Injecting an excitotoxin into the basal forebrain to destroy nucleus basalis neurons provides a potentially useful animal model for studying the role of these neurons in Alzheimer's disease. Previously, we demonstrated that rats with nucleus basalis excitotoxin lesions performed poorly on a tactile discrimination task and on a test of working memory. In an effort to clarify further the role of impaired memory versus other types of impairment (e.g. disrupted somatosensory processing due to cholinergic deafferentation of somatosensory cortex), we compared a group of rats with bilateral nucleus basalis excitotoxin lesions and a group with bilateral somatosensory cortical ablations on a variety of behavioral tasks. Rats with nucleus basalis lesions performed as well as controls on a battery of neurological tests but exhibited increased emotionality unlike rats with somatosensory cortical ablations which performed poorly on the battery but were not hyperemotional. The two lesion groups were impaired significantly and to a comparable degree in performing two-choice tactile discriminations in a T-maze. In contrast, only rats with nucleus basalis lesions showed deficits in working memory as tested in an eight-arm radial maze. Both lesion groups performed comparably to sham controls on a test of reference memory involving a black/white discrimination in a T-maze. The findings suggest that rats with nucleus basalis lesions manifest disturbances in several of the same spheres (emotionality, somatosensory information processing, memory) that are disrupted in Alzheimer's disease and further confirm the utility of the excitotoxin lesion approach for studying the pathophysiology of Alzheimer's disease.

Radial maze performance after hippocampal lesions: beneficial effects of nimodipine.

Rats trained in an 8-arm radial maze were given electrolytic lesions of the dorsal hippocampus or sham operations. Within 24 h of surgery, approximately half of the rats in each groups began 14 daily oral treatments with the central calcium channel blocker, nimodipine. Retention testing began seven days after surgery. The untreated rats that received the larger hippocampal lesions did not relearn the maze within one month (2.5 times the preoperative mean number of days). The untreated rats that received the smaller lesions relearned within the allotted time but still showed clear deficits. Nimodipine improved the maze scores of the animals with the smaller lesions, but not those of the animals with the larger lesions. It was unclear whether nimodipine led to the sparing of more cells in the hippocampal region, or whether spared-but-affected cells were returning to normal modes of functioning more rapidly in the group with the smaller lesions. These findings suggest that spared hippocampus was mediating behavior, and extend previous findings from this laboratory showing that nimodipine can enhance recovery of function on higher cognitive tasks after hippocampal lesions.

Nimodipine-enhanced survival of suboptimal neural grafts.

The present experiment was conducted to see whether nimodipine, a calcium channel blocker with the potential to protect vulnerable neurons, could improve the survival rate of striatal grafts under suboptimal conditions. The drug markedly improved graft viability of rats with near-term (E21) striatal implants. Untreated rats showed no surviving neurons while all treated rats showed surviving grafts rich in neuron. It did not affect the survival of striatal grafts from E14/15 donors into the striatum, in which the high rates of survival are normally obtained.

Factors affecting septal graft amelioration of differential reinforcement of low rates (DRL) and activity deficits after fimbria-fornix lesions.

Wound-derived trophic factors released by the injured brain are thought to reach a peak 1-2 weeks after injury. It has been proposed that such factors can promote the survival, growth and functional capacity of embryonic tissue grafts. To test the generality of this hypothesis, control rats and rats with aspirative fimbria-fornix lesions were compared with 5 groups of rats with lesions and septal grafts implanted either in the same session as the lesion or after delays from 10 days to 9 months. Animals were assessed 3 months post-transplantation on an operant differential reinforcement of low rates (DRL) task and on a test of spontaneous locomotor activity. Lesions produced impairments on all measures of DRL performance. Two graft groups showed amelioration of the DRL deficits, one graft group was unchanged, and the deficits were exacerbated in two others. There was no clear relationship between lesion-graft interval and recovery. An inverse relationship was seen, however, between recovery and the developmental age of the donor tissue. In contrast, significant recovery from lesion-induced hyperactivity was observed in the two graft groups with tissue derived from the oldest embryos. There were no clear relationships between recovery on either test, lesion-graft interval, and AChE-positive reinnervation of the host brain. The results provide further evidence that septal grafts can reverse behavioural deficits induced by fimbria-fornix lesions under some conditions, but suggest that the timing of graft surgery may not be as important a factor as donor age in this model system.

Swimming as a method for assessing motor cortex integrity in the rat.

Rats were tested on 6 measures of swimming behavior 1, 2 and 4 weeks after sham operations or bilateral motor cortex lesions that damaged the forelimb areas and axons controlling the axial musculature. Rats with lesions performed more forelimb kicks and showed larger body angles than control animals. Number of hindlimb kicks and speed were within normal limits. Thus, some measures of swimming can be affected by small motor cortex lesions that spare the hindlimb projections.

Brain damage and neuroplasticity: mechanisms of recovery or development?

Brain damage may be followed by a number of dynamic events including reactive synaptogenesis, rerouting of axons to unusual locations and altered axon retraction processes. In the present theoretical review, the relationship between these morphological changes and behavioral recovery of function is examined from two perspectives. First, an examination of the research literature reveals that the association between these reorganizational events and recovery of function is inconsistent, and it is proposed that in most cases a causal relationship between neural reorganization and behavioral recovery remains speculative at best. It is further noted that aberrant neural circuitry has been associated with neurological dysfunction in many studies. Second, evolutionary considerations suggest that there is little reason to believe that neural reorganizational events emerged to 'heal' damaged brains. Both experimental and evolutionary orientations support the idea that neuronal circuitry changes in response to injury can be better understood as developmental growth processes that are triggered or potentiated in response to cell loss, rather than as recovery or healing processes. The contribution of 'growth' to behavioral recovery of function may be inconsistent because these growth processes are occurring against the backdrop of a damaged brain and may make connections different from those ordinarily seen. Further, they must be considered in conjunction with phenomena such as diaschisis and compensation which may also influence behavioral changes following neural injury.

Effects of one- and two-stage lesions of the posterior hypothalamus on temperature regulation in the rat.

Rats received 1-stage bilateral and sequential unilateral (serial) lesions of the posterior hypothalamus and were tested for the ability to regulate body temperature after a lengthy recovery period. The groups with lesion differed from the sham-operated groups in the cold, although not under ambient or warm conditions. The fact that the serial lesion group performed the same as the 1-stage lesion groups in the cold is significant because earlier tests on these same animals revealed much better recovery after serial lesions in swimming, and a partial serial lesion effect in open field performance.

Behavioral effects of early deprivation of nerve growth factor: some similarities with familial dysautonomia.

Female rats immunized with mouse nerve growth factor develop an antibody (anti-NGF) which reaches offspring through the placenta and via the milk. Pups exposed to maternal anti-NGF have fewer dorsal root and sympathetic neurons. When the offspring are examined on a wide variety of behavioral tests, they exhibit severe deficits in response to stress (ulceration, corticosterone levels), and mild deficits on some sensory and cognitive tasks. Exploratory and motor functions, however, are relatively normal. The pathologic and behavioral profiles of the animals closely mimic the sensory and sympathetic aspects of familial dysautonomia.

Undernutrition and recovery from brain damage: a preliminary investigation.

Rats undernourished early in life did not differ from control animals in acquiring a light-dark discrimination. Posterior cortical lesions impaired retention in both nutritional groups, but the relearning scores of the undernourished animals with lesions were significantly worse than those of the lesion group that had been well fed. Amphetamine was found to enhance recovery, especially in the undernourished group. These data thus show that early nutritional history can be an important factor in accounting for differences in performance following later, focal brain damage, but that pharmacological intervention still can be of great value in these cases.

Behavioral and neurochemical evaluation of a transgenic mouse model of Lesch-Nyhan syndrome.

Two transgenic strains of mutant mice lacking hypoxanthine-guanidine phosphoribosyltransferase (HPRT) activity were examined behaviorally and neurochemically for phenotypic similarity to the human Lesch-Nyhan syndrome. In this syndrome, male children markedly deficient in the enzyme HPRT develop self-mutilation and severe motoric difficulties, and exhibit a pronounced deficiency of dopamine in the basal ganglia. The HPRT-deficient mice showed no evidence of self-mutilation, no detectable motor impairments on tests selected for sensitivity to basal ganglia dysfunction, and no differences in response to apomorphine. Biochemical analyses revealed significantly lower levels of striatal dopamine in the HPRT-deficient mice than in HPRT normal littermates, but the depletion was only of the order of 19%. The results suggest that mice lacking HPRT activity do not phenotypically resemble children born with the same enzymatic deficiency in part because mutant mouse striatal dopamine levels are not as low as those seen in clinical cases with Lesch-Nyhan disease. In contrast to Lesch-Nyhan children, mice may be able to utilize alternative pathways more effectively to maintain purine and neurotransmitter levels within the ranges required for normal brain development and function.