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.

Recruitment of new sites of synaptic transmission during the cAMP-dependent late phase of LTP at CA3-CA1 synapses in the hippocampus.

Long-term potentiation at CA3-CA1 hippocampal synapses exhibits an early phase and a late phase, which can be distinguished by their underlying molecular mechanisms. Unlike the early phase, the late phase is dependent on both cAMP and protein synthesis. Quantal analysis of unitary synaptic transmission between a single presynaptic CA3 neuron and a single postsynaptic CA1 neuron suggests that, under certain conditions, the early phase of LTP involves an increase in the probability of release of a single quantum of transmitter from a single presynaptic release site, with no change in the number of quanta that are released or in postsynaptic sensitivity to transmitter. Here, we show that the cAMP-induced late phase of LTP involves an increase in the number of quanta released in response to a single presynaptic action potential, possibly due to an increase in the number of sites of synaptic transmission between a single CA3 and a single CA1 neuron.

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.

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.

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.

Pressure exposure unmasks differences in release properties between high and low yield excitatory synapses of a single crustacean axon.

The cellular mechanisms underlying the effect of high pressure on synaptic transmission at two types of synapses were studied in the opener muscle of the lobster walking leg. Excitatory postsynaptic currents (EPSCs) were recorded using a loose macropatch clamp technique at normal pressure and 3.5, 6.9 MPa helium pressure. Responses of the single excitatory axon could be grouped into two types: low yield (L) synapse exhibiting a small mean EPSC with a considerable number of failures, and high yield (H) synapse having a larger mean EPSC with very few failures. The change in several synaptic transmission parameters indicated that high pressure similarly reduced presynaptic evoked release in both L and H synapses. However, some differences in the kinetics and probability of release could be detected. A major difference was the spontaneous miniature EPSCs (mEPSCs) activity. Many of the mEPSC, observed only in L synapses, were 'giant' (size of 2-5 q). High pressure selectively increased the frequency of the giant mEPSCs in the L synapse but had little effect on their amplitude histogram. High pressure depressed evoked synaptic transmission in both synapses by modulating the presynaptic quantal release parameters, but concomitantly enhanced spontaneous quantal release in L synapses by an unknown mechanism.

Regional differences in technetium-99m-ECD clearance on brain SPECT in healthy subjects.

UNLABELLED: The aim of this study was to evaluate the in vivo stability of ECD brain SPECT. METHODS: Twenty normal volunteers (35.4 +/- 9.1 yr) each had six ECD scans at 30, 60, 120, 240, 360 and 480 min postinjection. Each scan was acquired for 24 min using a triple-head SPECT system. Average counts per pixel were measured from frontal, temporal, parietal, occipital, cerebellum, basal ganglia, thalamus and white matter regions. ECD clearance rates were calculated by fitting regional time activity data to a monoexponential equation. Regional gray-to-white matter (G/W) and gray-to-cerebellum (G/C) ratios were calculated for each scan. Analysis of variance was used to compare regional ECD clearance and ratio measurements. RESULTS: The average ECD clearance was 4.3%/hr. There was a significant regional variation in the ECD clearance, being higher for occipital (6.34%/hr) but lower for both white matter (2.39%/hr) and thalamus (2.45%/hr). Both G/W and G/C ratios showed a significant regional variation with time. The overall G/W ratio was 2.13 at 30 min and became progressively lower after 2 hr, reaching 1.78 at 8 hr. All regional G/W ratios declined with time except for thalamus where it remained constant at 2.15. The overall G/C ratio was 0.984 at 30 min but it declined after 4 hr, reaching 0.955 at 8 hr. All regional G/C ratios declined with time except for thalamus where it increased progressively from 0.955 to 1.120 at 8 hr. CONCLUSION: ECD clears from normal brain slowly and shows a significant regional variation. As a result, G/W contrast begins to decrease after 2 hr and the gray-matter activity pattern becomes significantly different after 4 hr. Therefore, the optimal imaging time may be between 30-120 min. However, images obtained up to 4 hr still maintain the initial gray-matter activity pattern.

Noninvasive quantification of dopamine D2 receptors with iodine-123-IBF SPECT.

UNLABELLED: Iodine-123-iodobenzofuran (IBF) is a potent dopamine D2 receptor ligand suited for quantitative receptor studies. The purpose of this study was to evaluate three noninvasive methods of estimating the receptor parameter k3/k4 in humans with IBF-SPECT. METHODS: Scans were acquired every 5 min for 180 min using a triple-headed SPECT system following a bolus injection of IBF (296 +/- 37 MBq) in 14 normal volunteers. k3/k4 was estimated by the peak equilibrium ratio (RPE) method and two proposed methods: a variation of the graphic method that derives the ratio of ligand distribution volumes (RV) and area ratio (RA) method, in which the ratio is calculated from the areas under the specific binding and nondisplaceable activity curves. RESULTS: The mean RPE, RV and RA were 2.74 +/- 0.40, 3.06 +/- 0.42 and 2.26 +/- 0.28, respectively. Both RPE and RA underestimated RV. The relationship between RPE or RA and RV was linear (p < or = 10(-5), RA showed higher correlation (r = 0.94) with RV than did RPE (r = 0.90). Simulations based on a tracer kinetic model showed that RV, unlike RPE or RA, is affected by neither regional cerebral blood flow (rCBF) nor peripheral clearance rate (CR) of IBF. All three measures showed a significant decline with increasing age (r = 0.54-0.58, p < 0.05). CONCLUSION: RV is preferred because it provides a theoretically valid estimate of k3/k4, independently of rCBF or CR. Alternatively, RA might be preferred to RPE because the former is simpler than the latter to implement yet the former provides a measure that equally well correlates with k3/k4.

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.

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.

Successful human umbilical cord blood stem cell transplantation without conditioning in severe combined immune deficiency.

A 2-month-old girl with severe combined immunodeficiency (SCID), presented with mild staphylococcal skin infection, lymphopenia, low T cell number, absence of B cells, high number of NK cells, and a negligible response to mitogens. Since her older brother died as a result of SCID 2 years earlier, cord blood was harvested from a sister born 2 1/2 years earlier, who was normal and fully matched both by serology and molecular typing. In view of her clinical condition and in spite of a high number of NK cells with normal activity, HUCBT without preparative conditioning was performed. No G-CSF was administered. Engraftment with mixed chimerism was evident 3 weeks post transplantation. There were no peritransplantation complications. Eighteen months post transplantation, the girl is in excellent condition, blood counts are normal, T cell engraftment is complete, B cell engraftment is proceeding gradually, and the mitogen stimulation tests are normal. Due to the unique nature of HUCB hematopoietic cells, engraftment without conditioning may be possible in patients with SCID with fully matched donors. This is the first HUCBT performed without conditioning.

High CO2-bicarbonate buffer modifies GABAergic inhibitory effect at the crayfish neuromuscular synapse.

gamma-Aminobutyric acid (GABA) activated channels have a considerable permeability to bicarbonate ions (HCO3-), which might alter the efficacy of chloride-dependent synaptic inhibition. Saturation of the bicarbonate-buffered physiologic solution with 15% CO2/85% O2 increased the depolarizing inhibitory postsynaptic potential (IPSP) amplitude in crayfish muscle by 290% due to a shift of +8.33 mV in its reversal potential. Consequently, the normal inhibition exerted by the IPSP on the excitatory postsynaptic potential is reversed to large excitation.

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.

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.

Pentylenetetrazole-induced kindling is prevented by prior treatment with cysteamine.

We have previously demonstrated that in pentylenetetrazole (PTZ)-kindled rats, cysteamine causes prolonged depression of the kindled state. We now report that administration of cysteamine before or during the kindling process prevents attainment of the kindled state. This effect lasts long after cysteamine administration has ceased, suggesting that depletion or somatostatin may not be the only mechanism underlying cysteamine's effect on kindling. The results also support the likelihood that PTZ kindling primarily effects neocortical rather than limbic structures.

GABA metabolism controls inhibition efficacy in the mammalian CNS.

The effects of changes in gamma-aminobutyric acid (GABA) metabolism or inhibitory processes was studied in the perforant path-dentate gyrus synapses in rat cortico-hippocampal slices, and in the monosynaptic-reflex circuit in isolated newborn, rat spinal cord. GABA metabolism was modulated by pharmacological block of either the anabolic enzyme glutamate decarboxylase (GAD) or the catabolic enzyme GABA transaminase (GABA-T). The results support the notion that GABA concentration determines the efficacy of inhibition in these regions of the central nervous system (CNS).

Usefulness of follow-up regional cerebral blood flow measurements by single-photon emission computed tomography in the differential diagnosis of dementia.

The aim of this study was to evaluate whether follow-up measurements of regional cerebral blood flow (rCBF) by single-photon emission computed tomography (SPECT) provide additional information in the differential diagnosis of dementia. Thirty-six patients (70 +/- 14 yr) with suspected dementia who had two technetium 99m-hexamethylpropyleneamineoxime SPECT scans over 18 +/- 7 months were included in this retrospective study. The patients comprised three groups based on the final clinical diagnosis: (1) neurodegenerative disorder (NDD) including Alzheimer's disease (AD) (n = 13), frontotemporal lobe dementia (n = 2), progressive supranuclear palsy (n = 1), and mixed dementia (AD plus multiinfarct dementia [MID]) (n = 3); (2) MID (n = 8); and (3) psychiatric disorders (depression [n = 7], psychosis [n = 1], and anxiety [n = 1]). Blinded to the clinical diagnosis and using visual analysis, the nuclear medicine physicians compared the second scan with the first scan for each patient to characterize temporal changes in rCBF. SPECT findings were categorized into three patterns of rCBF change: worsened, improved, and unchanged. Of the worsened rCBF group, 17 (85%) belonged to the NDD group whereas 2 (10%) and 1 (5%) belonged to the MID and psychiatric disorders groups, respectively. All 5 (100%) of the improved rCBF patients belonged to the psychiatric disorders group. Thus, worsening of rCBF favors the diagnosis of NDD whereas improvement in rCBF may mitigate against the diagnosis of NDD or MID. Follow-up rCBF measurements by SPECT thus provided additional information on the possible cause of dementia. A prospective study to further evaluate the usefulness of follow-up rCBF measurements by SPECT appears warranted.

Treatment of tongue thrust with hypnosis: two case histories.

Tongue thrust is a relatively infrequent habit which can result in disruptive, permanent oral malocclusion, bone changes, and facial disharmony. The use of hypnotic phenomena can augment myofunctional therapy. Temperature control, glove anesthesia, relaxation, and imagery enhance demonstration of the proper way to swallow. The cornerstone of having the patient actually feel the contraction at the insertion of the masseter muscles provides an inner biofeedback which provides a very positive signal that the improper habit is being corrected. Hypnosis can then be used in the manner described to achieve a good clinical result.