T and B cell clonal expansion in Ras-associated lymphoproliferative disease (RALD) as revealed by next-generation sequencing.

Ras-associated lymphoproliferative disease (RALD) is an autoimmune lymphoproliferative syndrome (ALPS)-like disease caused by mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) or neuroblastoma RAS viral (V-Ras) oncogene homologue (NRAS). The immunological phenotype and pathogenesis of RALD have yet to be studied extensively. Here we report a thorough immunological investigation of a RALD patient with a somatic KRAS mutation. Patient lymphocytes were analysed for phenotype, immunoglobulin levels and T cell proliferation capacity. T and B cell receptor excision circles (TREC and KREC, respectively), markers of naive T and B cell production, were measured serially for 3 years. T and B cell receptor repertoires were studied using both traditional assays as well as next-generation sequencing (NGS). TREC and KREC declined dramatically with time, as did T cell receptor diversity. NGS analysis demonstrated T and B clonal expansions and marked restriction of T and B cell receptor repertoires compared to healthy controls. Our results demonstrate, at least for our reported RALD patient, how peripheral T and B clonal expansions reciprocally limit lymphocyte production and restrict the lymphocyte receptor repertoire in this disease. Decreased naive lymphocyte production correlated with a clinical deterioration in our patient's immune status, suggesting that TREC and KREC may be used as an aid in monitoring disease progression. Both the methodologies used here and the conclusions regarding immune homeostasis may be applicable to the research of ALPS and other immune dysregulation syndromes.

In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma.

BACKGROUND: Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects. METHODS: We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ(9)-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells. RESULTS: Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts. CONCLUSIONS: Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl.

Hyperbaric oxygen can induce neuroplasticity and improve cognitive functions of patients suffering from anoxic brain damage.

PURPOSE: Cognitive impairment may occur in 42-50% of cardiac arrest survivors. Hyperbaric oxygen therapy (HBO2) has recently been shown to have neurotherapeutic effects in patients suffering from chronic cognitive impairments (CCI) consequent to stroke and mild traumatic brain injury.The objective of this study was to assess the neurotherapeutic effect of HBO2 in patients suffering from CCI due to cardiac arrest. METHODS: Retrospective analysis of patients with CCI caused by cardiac arrest, treated with 60 daily sessions of HBO2. Evaluation included objective computerized cognitive tests (NeuroTrax), Activity of Daily Living (ADL) and Quality of life questionnaires. The results of these tests were compared with changes in brain activity as assessed by single photon emission computed tomography (SPECT) brain imaging. RESULTS: The study included 11 cases of CCI patients. Patients were treated with HBO2, 0.5-7.5 years (mean 2.6 ± 0.6 years) after the cardiac arrest. HBO2 was found to induce modest, but statistically significant improvement in memory, attention and executive function (mean scores) of 12% , 20% and 24% respectively. The clinical improvements were found to be well correlated with increased brain activity in relevant brain areas as assessed by computerized analysis of the SPECT imaging. CONCLUSIONS: Although further research is needed, the results demonstrate the beneficial effects of HBO2 on CCI in patients after cardiac arrest, even months to years after the acute event.

Increased susceptibility to mild neonatal stress in MTHFR deficient mice.

Early life stress is shown to have a life-span outcome on human and animal behavior, increasing the risk for psychopathology. The gene methylenetetrahydrofolate reductase (MTHFR), which encodes for a key enzyme in one carbon metabolism, shows a high prevalence of polymorphism in patients with developmental disorders. Here we examined the hypothesis that MTHFR deficiency results in an increased susceptibility of the developing brain to mild neonatal stress (NS). Mild NS failed to alter corticosterone levels in young and adult Wt mice. However, an elevated level of corticosterone was found in the MTHFR deficient-NS female, exemplifying enhanced sensitivity to NS. Behavioral phenotyping of Wt and MTHFR deficient mice provides evidence that the effect of mild NS may be amplified by the MTHFR deficient genotype. Distinct behavioral characteristics were altered in male and female mice. In general, three patterns of influence on mice behavior were observed: (1) an additive suppressive effect of NS and MTHFR deficiency on exploration and activity was evident in females; (2) stress related parameters were significantly sensitive to genotype in females, presenting an interaction between genotype and sex; (3) various aspects of behavior in a social setting were modified preferably in males by genotype, NS and the interaction between the two, while females exhibited a smaller effect that was restricted to NS with no genotype effect. Overall, our results support an interaction between mild NS, the MTHFR genotype and sex. We suggest using this animal model to study the molecular mechanism linking these two risk factors and their involvement in neurodevelopmental disorders such as schizophrenia and autism.

Normal aging of offspring mice of mothers with induced inflammation during pregnancy.

Intrauterine inflammation is a major risk for offspring neurodevelopmental brain damage and may result in cognitive limitations and poor cognitive and perceptual outcomes. In the present study we tested the possibility that prenatal exposure to a high level of inflammatory factors may increase the risk for neurodegeneration in aging. The effect of systemic maternal inflammation (MI), induced by lipopolysaccharide (LPS) on offspring brain aging, was examined in 8 month old (adult) and 20 month old (aged) offspring mice. A significant effect of age was found in the distance and velocity of exploration in the open field in both groups. In addition, MI aged offspring covered longer distances and enter frequently to the center of the field compared with the aged control group. Although only little difference was found in the aged MI offspring compared with the control offspring, the overall profile of behavior of these mice differs from that of the control group, as detected by clustering analysis. The expression of the death-associated protein FAS-ligand and the amount of apoptotic cell death were examined in the brains of aged offspring. Similar levels of FAS-ligand expression and parallel density of apoptotic cells were detected in the brains of aged mice of control and MI groups. Altogether, moderate systemic MI was not found to increase the risk for cell death in the aged offspring; limited effect was found in mice profile of behavior.

Remodeling of hippocampal GABAergic system in adult offspring after maternal hypoxia and magnesium sulfate load: immunohistochemical study.

A strong relationship between hypoxia and fetal brain damage has been described. Specific susceptibility of the GABAergic neurons to these conditions may be crucial to the damage induced. We have previously shown, in a mouse model, that maternal pretreatment with magnesium sulfate (Mg) partially prevented the behavioral consequences of maternal hypoxia in the adult offspring. Here, we tested the effect of maternal hypoxia and maternal Mg load on the GABAergic system of 8-month-old offspring. The immunoreactivity (IR) of several proteins expressed in GABAergic neurons and inhibitory synapses was analyzed in the following regions of the adult offspring brain: hippocampus, cortical M1, caudate putamen, and lateral globus pallidus. Maternal hypoxia reduced the density of parvalbumin (PV)-IR neurons in the hippocampus. The density of PV-IR and calbindin (CB)-IR neurons was also reduced in the deep and superficial layers of the M1. Maternal pretreatment with Mg had a prophylactic action in the superficial, but not the deep, layers of M1. Also, in offspring from the maternal hypoxia group, the vesicular GABA transporter (VGAT)-IR was enhanced in the hippocampal CA1 and hilus regions. No effect of maternal hypoxia on VGAT-IR was observed in the M1. However, maternal pretreatment with Mg enhanced VGAT-IR and glutamate decarboxylase-IR in the deep layers of the M1. In the globus pallidus, maternal hypoxia enhanced CB-IR, which was prevented by maternal pretreatment with Mg. In conclusion, maternal hypoxia induced a loss of PV-IR and CB-IR neurons; maternal pretreatment with Mg partially protected these neuron populations. An increase in proteins of inhibitory synapses, observed under hypoxic conditions in several brain regions, may be a result of some compensatory mechanism.

Specific neurodevelopmental damage in mice offspring following maternal inflammation during pregnancy.

Intrauterine inflammation is a major risk for offspring neurodevelopmental brain damage and may result in cognitive limitations and poor cognitive and perceptual outcomes. Pro-inflammatory cytokines, stimulated during inflammatory response, have a pleotrophic effect on neurons and glia cells. They act in a dose-dependent manner, activate cell-death pathways and also act as trophic factors. In the present study, we have examined in mice the effect of short, systemic maternal inflammation on fetal brain development. Maternal inflammation, induced by lipopolysaccharide (LPS) at gestation day 17, did not affect morphogenic parameters and reflex development during the first month of life. However, maternal inflammation specifically increased the number of pyramidal and granular cells in the hippocampus, as well as the shrinkage of pyramidal cells, but not of the granular cells. No additional major morphological differences were observed in the cerebral cortex or cerebellum. In accordance with the morphological effects, maternal inflammation specifically impaired distinct forms of learning and memory, but not motor function or exploration in the adult offspring. The specific deficiency observed, following maternal inflammation, may suggest particular sensitivity of the hippocampus and other associated brain regions to inflammatory factors during late embryonic development.

Maternal hypoxia during pregnancy induces fetal neurodevelopmental brain damage: partial protection by magnesium sulfate.

Fetal low brain oxygenation may be an outcome of maternal complications during pregnancy and is associated with increased risk of cerebral palsy and periventricular leukomalacia in newborns. One treatment used for prevention of fetal brain damage is maternal treatment with MgSO(4). Although this treatment is indicated to reduce the risk of cerebral palsy in newborns, its use remains controversial. We have shown previously that pretreatment with MgSO(4) in a mouse model of maternal hypoxia prevented a delay in the development of motor reflexes induced by hypoxia. We demonstrate here that pretreatment with MgSO(4) reduces hypoxia-induced motor disabilities in adult offspring. This effect is associated with histologic protection of the Purkinje cells in the cerebellum and stabilization of brain-derived neurotrophic factor (BDNF) levels in the cerebellum. MgSO(4) did not prevent the reduction in cerebral cortex cell density and cell size induced by maternal hypoxia, however, nor did it interfere with the modulation of BDNF and nerve growth factor (NGF) expression in the cerebral cortex. MgSO(4) pretreatment also prevented the impairment of short-term memory (30 min, P < 0.05) but not long-term memory (7 days). Nevertheless, maternal pretreatment with MgSO(4) reduced CA1 cell layer width and induced alterations in both NGF and BDNF in the hippocampus. These results support the prophylactic effect of MgSO(4) against motor disabilities; however, they may also indicate possible harmful effects on the cerebral cortex and hippocampus.

Multilineage hematopoietic engraftment after allogeneic peripheral blood stem cell transplantation without conditioning in SCID patients.

Successful stem cell transplantation for patients with severe combined immunodeficiency (SCID) from matched family donors without conditioning results in engraftment of T lymphocytes. B lymphocytes engraft in only 50% of the cases, while myelopoiesis and erythropoiesis remain of host origin. Full hematopoietic engraftment was reported in one case after bone marrow transplantation without conditioning for a SCID patient. We studied three SCID patients who were transplanted with unmodified mobilized peripheral blood from HLA-identical family sex-mismatched members. They received megadoses of stem cells (18-23 x 10(6)CD34/kg). In contrast to the expected mixed chimerism that usually occurs in the absence of conditioning, we found in our patients 100% donor cell engraftment based on fluorescence in situ hybridization (FISH) and microsatellite techniques. Subset analysis of the engrafted cells using a multiparametric system enabling a combined analysis of morphology, immunophenotyping and FISH showed that both T and B lymphocytes and myeloid cells were of donor origin in two patients, while T lymphocytes and myeloid cells were of donor origin in the third. In the two cases with ABO incompatibility, erythroid engraftment was evidenced by blood group conversion from recipient to donor type. Multilineage donor engraftment is possible in SCID patients even without conditioning.

Perinatal exposure to GABA-transaminase inhibitor impaired psychomotor function in the developing and adult mouse.

Antiepileptic drugs acting through the potentiation of GABA-ergic pathways have harmful effects on brain development. Increased risk of impaired intellectual development was reported in children born to women treated for epilepsy during pregnancy. Here we examined the vulnerability of the developing brain to treatment with one of the new antiepileptic drugs--vigabatrin--during two time periods in newborn mice (postnatal days 1-7 and 4-14) which parallel the third trimester of human embryo brain development. Delayed development of sensory and motor reflexes, reduced mobility in the open field, impaired object recognition and deficient spatial learning and memory were observed independently of the treatment period. On the contrary, specific susceptibility to the age of exposure was detected in various motor functions. A number of morphological correlates may explain these behavioral alterations; a transient increase in CA1 pyramidal cell layer (P < 0.001) and decrease in granular cell layer (P < 0.05) in hippocampus were detected at postnatal day 7. In addition, a significantly lower cell density was observed in the adult mouse brain in all layers of the M2 cerebral cortex of mice treated during days 4-14, compared to the controls (P < 0.05). Our findings demonstrated short- and long-term deleterious effects of vigabatrin treatment and suggest a specific vulnerability of the developing motor system to GABA enhancement during the first postnatal week.

Detection of RAG mutations and prenatal diagnosis in families presenting with either T-B- severe combined immunodeficiency or Omenn's syndrome.

It has been recently shown that mutations in both of the recombination activating genes RAG1 and RAG2 are involved in each of the two different types of severe combined immunodeficiency (SCID) syndromes: T-B- SCID and Omenn's syndrome (OS). The objective of the study was to search for novel mutations in the RAG genes and to offer prenatal diagnosis for families that have been identified as at risk of T-B- SCID or OS. Mutation analyses of polymerase chain reaction products of RAG1/RAG2 genes were performed in 14 cases (T-B- SCID = 6 and OS = 8). Consanguinity was reported in seven (50%) families. Four missense mutations in the RAG2 gene in six of eight OS patients and in four of six T-B- SCID patients were detected. The C1845T transition leading to a Tre215Ile substitution is a novel mutation. All but one of the patients were homozygous for the detected mutations, possibly reflecting the consanguinity in these families and the relative rarity of the disease-causing mutations. In addition, three putative polymorphic sites were found. Prenatal diagnosis was offered to seven families, but three of them declined genetic counseling for religious reasons. In the remaining families, four pregnancies were successfully completed, and in one case, the family chose to have an abortion because of a homozygous mutation. Mutations in RAG1/RAG2 genes were detected in only some of the T-B- SCID or OS patients, and the molecular basis for the remaining cases has yet to be elucidated. Important factors such as religious beliefs need to be considered when offering prenatal diagnosis to certain families.

Involvement of tumor necrosis factor alpha in hippocampal development and function.

Tumor necrosis factor alpha (TNFalpha) is a cytokine produced mainly by cells of the immune system. It is also expressed by brain neurons and glia. The physiological role of TNFalpha in the brain is not yet fully clear. Using TNFalpha-deficient mice, we have examined its role in hippocampal development and function. We report here that TNFalpha is involved in the regulation of morphological development in the hippocampus. TNFalpha-deficient mice exhibited an accelerated maturation of the dentate gyrus region and smaller dendritic trees in CA1 and CA3 regions in young mouse. In addition to its involvement in hippocampal morphogenesis, TNFalpha deficiency specifically improved performance of affected mice in behavioral tasks related to spatial memory. Moreover, lack of TNFalpha increased the expression of nerve growth factor (NGF), but not brain-derived neurotrophic factor (BDNF), following performance of the learning task. Our results suggest that TNFalpha actively influences hippocampal development and function. In adult mice, TNFalpha may interfere with memory consolidation, perhaps by regulating NGF levels.

Cell proliferation in response to GABA in postnatal hippocampal slice culture.

Recent studies have implicated the inhibitory neurotransmitter GABA in the regulation of cell migration and proliferation in the embryonic brain. Herein, we examine the possibility that these effects are maintained postnatally. Using 5 days postnatal hippocampus, we tested GABA effects on growth into an incision made in CA1 and the possibility that this response to GABA is mediated via GABA(A)-receptor. Our data shows that GABA promotes neurite growth, cell proliferation and migration up to day 6 in vitro. GABA(A)-receptor is not involved in the trophic response to either exogenous or endogenous GABA. Moreover, GABA induced a 20% increase in NGF secretion to the growth medium, in a similar time frame to its effect on growth. Elevated NGF secretion was suppressed by the inhibition of cell proliferation. These results suggest that GABA can promote growth in postnatal hippocampal tissue. The effect involves cell proliferation and NGF secretion and does not depend on GABA(A)-receptor activation.

Frequent blood-brain barrier disruption in the human cerebral cortex.

1. The blood-brain barrier (BBB) protects the brain from circulating xenobiotic agents. The pathophysiology, time span, spatial pattern, and pathophysiological consequences of BBB disruptions are not known. 2. Here, we report the quantification of BBB disruption by measuring enhancement levels in computerized tomography brain images. 3. Pathological diffuse enhancement associated with elevated albumin levels in the cerebrospinal fluid (CSF) was observed in the cerebral cortex of 28 out of 43 patients, but not in controls. Four patients displayed weeks-long focal BBB impairment. In 19 other patients, BBB disruption was significantly associated with elevated blood pressure, body temperature, serum cortisol, and stress-associated CSF 'readthrough" acetylcholinesterase. Multielectrode electroencephalography revealed enhanced slow-wave activities in areas of focal BBB disruption. Thus, quantification of BBB disruption using minimally invasive procedures, demonstrated correlations with molecular, clinical, and physiological stress-associated indices. 4. These sequelae accompany a wide range of neurological disorders, suggesting that persistent, detrimental BBB disruption is considerably more frequent than previously assumed.

Successful treatment of invasive aspergillosis in chronic granulomatous disease by granulocyte transfusions followed by peripheral blood stem cell transplantation.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder characterized by impaired microbial killing and susceptibility to bacterial and fungal infections. Cure of the disease can be achieved by stem cell transplantation when performed early in its course, and before severe infections have developed. Invasive aspergillosis constitutes a very high risk for transplantation. We report a 4-year-old boy with X-linked CGD who underwent successful HLA-identical peripheral blood stem cell (PBSC) transplantation during invasive pulmonary aspergillosis and osteomyelitis of the left fourth rib, which was unresponsive to antifungal treatment. During the 2 months prior to the transplant he received G-CSF-mobilized granulocyte transfusions (GTX) from unrelated donors three times a week in addition to the antifungal treatment. This resulted in clinical improvement in his respiratory status. He also received GTX during the aplastic period after the conditioning regimen, until he had engrafted. Post-transplant superoxide generation test revealed that neutrophil function was within normal range. One year post transplant the CT scan showed almost complete clearance of the pulmonary infiltrates and a marked improvement in the osteomyelitic process. Based on other reports and our own experience, GTX can serve as important treatment in patients with CGD who have failed conventional anti-fungal treatment and for whom stem cell transplantation is the only chance for cure.

[Dermatitis from contact with Agave americana].

Various plants induce dermatitis in man. There have been only a few published cases of contact dermatitis caused by Agave americana (AA). We report intentional exposure to AA in a soldier seeking sick leave, and review our previously reported cases. Treatment with oral antihistamines and topical saline compresses resulted in subsidence of the systemic symptoms within 24 h and regression of cutaneous manifestations in 7-10 days. Physicians should be alert to the possibility of self-inflicted contact dermatitis induced by exposure to plants, especially to A. americana. Systemic signs may accompany the cutaneous lesions.

Epidemiology of vascular catheter-related bloodstream infections in a large university hospital in Israel.

Intravascular catheter-related infection and associated bacteraemia constitute a serious and increasing problem among nosocomial infections. As a part of an ongoing survey of positive blood cultures, all catheter-related bloodstream infections (CR-BSI) were reviewed in the authors' Medical Center in 1996, in order to evaluate the magnitude and seriousness of this problem. The largest group (28%) of hospital-acquired bacteraemia by 1 source of infection during 1996 was CR-BSI, identified in 110 patients with 126 episodes. The vascular line was central in 83 (66%), peripheral in 24 (19%), tunnelled in 18 (14%) and arterial in 1 (1%). Among the 83 central CR-BSI no sign of local inflammation was detected in 65%. Gram-positive and gram-negative bacteria shared equal parts among the 145 blood isolates; Staphylococcus aureus was the most common species (43/145, 30%) followed by Klebsiella pneumoniae (15/145, 10%); 11 (8%) isolates were Candida species. Fungal isolates were more common among tunnelled catheter infections than among others (6/18, 33% vs. 5/108, 5%, p < 0.001). Crude mortality was 35% (38/110), while attributable mortality was 14% (15/110), mostly associated with central line infection. Catheter-associated bacteraemias cause significant morbidity and mortality, and have become the most common source of hospital-acquired bacteraemia. There is a need to implement more effective infection-control measures and more advanced technologies in an effort to reduce this unacceptably high incidence.

GABA withdrawal modifies network activity in cultured hippocampal neurons.

Dissociated hippocampal neurons, grown in culture for 2 to 3 weeks, tended to fire bursts of synaptic currents at fairly regular intervals, representing network activity. A brief exposure of cultured neurons to GABA caused a total suppression of the spontaneous network activity. Following a washout of GABA, the activity was no longer clustered in bursts and instead, the cells fired at a high rate tonic manner. The effect of removing GABA could be seen as long as 1 to 2 days after GABA withdrawal and is expressed as an increase in the number of active cells in a network, as well as in their firing rates. Such striking effects of GABA removal may underlie part of the GABA withdrawal syndrome seen elsewhere.