Intracellular Ca2+ and pacemaking within the rabbit sinoatrial node: heterogeneity of role and control

Recent studies have proposed that release of calcium from the sarcoplasmic reticulum (SR) modulates the spontaneous activity of the sinoatrial node (SAN). Previously we have shown that several calcium regulatory proteins are expressed at a lower level in the centre of the SAN compared with the periphery. Such differences may produce heterogeneity of intracellular calcium handling and pacemaker activity across the SAN. Selective isolations showed that the centre of the SAN is composed of smaller cells than the periphery. Measurements of cytosolic calcium in spontaneously beating cells showed that diastolic calcium, systolic calcium, the calcium transient amplitude and spontaneous rate were greater in larger (likely to be peripheral) cells compared with smaller (likely to be central) SAN cells. The SR calcium content was greater in larger cells, although SR recruitment was more efficient in smaller cells. The sodium–calcium exchanger and sarcolemmal calcium ATPase had a lower activity and the exchanger was responsible for a larger proportion of sarcolemmal calcium extrusion in smaller cells compared with larger cells. Ryanodine had a greater effect on the spontaneous calcium transient in larger cells compared with smaller cells, and slowed pacemaker activity in larger cells but not smaller cells, thus abolishing the difference in cycle length. This study shows heterogeneity of intracellular calcium regulation within the SAN and this contributes to differences in pacemaker activity between cells from across the SAN. The smallest central cells of the leading pacemaker region of the SAN do not require SR calcium for spontaneous activity nor does disruption of the SR alter pacemaking in these primary pacemaker cells.     

Molecular characterization of 22q11 deletion in a three-generation family with maternal transmission

Haploinsufficiency of chromosome 22q11.2 is a well-established cause of both the DiGeorge anomaly and the velocardiofacial syndrome. This condition shows a continuous spectrum of phenotypic manifestations with a considerable inter- and intrafamilial variability. We report on a three-generation family with four members sharing the same 3 Mb long deletion but showing different phenotypic expression. In the first generation, the deleted patient has hypernasal speech and suffers from recurrent psychotic episodes. Two of her offspring inherited the deletion. One of these, a male, has hypernasal speech, low-set ears, hypocalcemia, severe development delay, and tetralogy of Fallot. The other, a female, has hypernasal speech, minor facial anomalies, and very mild mental retardation. Her daughter has tetralogy of Fallot, velopharyngeal insufficiency, and mild facial anomalies. This family is an example of the widely variable phenotypic expressivity of the 22q11.2 deletion. There is no correlation between the size of the deletion and the phenotypic manifestations. Genetic background and/or environmental factors could explain the different phenotypes observed in the affected members of the family.     

Cytotoxicity of glutaraldehyde crosslinked collagen/poly(vinyl alcohol) films is by the mechanism of apoptosis

Collagen has been investigated as a potential natural biomaterial, because of its occurrence in the extracellular matrix. Collagen requires crosslinking in this context, by reagents that are often cytotoxic. Glutaraldehyde is one such agent that is potentially cytotoxic. The aim of this study was to determine the cause of poor cell attachment and growth on collagen/poly(vinyl alcohol) bioartificial composite films, when crosslinked with glutaraldehyde. Dehydrothermal crosslinking was used as a comparison. Human osteoblasts were observed to undergo apoptosis on glutaraldehyde crosslinked films dependent on concentration of collagen present. Higher collagen content resulted in higher levels of apoptosis with poor cell attachment and spreading of remaining cells. Post-treatment of films with 8% L-glutamic acid prevented the apoptotic response of osteoblasts and allowed attachment and spreading. The addition of 100 nM insulin-like growth factor-1 to the culture medium also prevented apoptosis. Glutaraldehyde toxicity of crosslinked collagen has been demonstrated in this study, the mechanism of which is apoptosis. This study indicates that poor biocompatibility and induction of apoptosis on collagen/poly(vinyl alcohol) films crosslinked by glutaraldehyde are attributed to glutaraldehyde components on the surface of the films (not residual glutaraldehyde), whose effects can be quenched by glutamic acid, and prevented by insulin-like growth factor-1.     

Pulmonary hypertension in type 1 Gaucher's disease: genetic and epigenetic determinants of phenotype and response to therapy

Type 1 Gaucher's disease (GD) is recognized for striking but unexplained phenotypic diversity. Rarely, severe pulmonary hypertension (PH) may occur in GD but its clinical spectrum, determinants or its response to enzyme replacement therapy (ERT)+/-vasodilators is not known. One hundred and thirty-four consecutive patients with Type 1 GD were screened to estimate right ventricular systolic pressure (RVSP) by Doppler echocardiography. Ninety-four patients were on ERT and 40 were untreated. Eight additional GD patients were studied that represented consecutive tertiary referrals with severe PH. Angiotensin converting enzyme (ACE) gene polymorphisms and acid beta-glucosidase gene (GBA) mutations were determined by DNA analysis. Mild, asymptomatic PH (RVSP>35<50 mmHg) was prevalent in Type 1 GD: 30% in untreated patients and 7.4% among patients receiving ERT (P<0.001). Splenectomy was strongly associated with severe, life-threatening PH: all patients with severe PH (RVSP 50-130 mmHg) were asplenic compared to only 31% of patients with RVSP<50 mmHg (Odds ratio [OR] 28.8, 95% CI 1.6-531.6, P<0.001). Other characteristics of patients presenting with severe PH were poor compliance to ERT (4/9 patients) or no ERT (5/9 patients), a family history of a sib with GD and PH (2/2 patients), an excess of ACE I allele (OR 2.3, 95% CI 1.1-4.9, P=0.034) and an excess of non-N370S GBA mutation (OR 6.0, 95% CI 1.1-33, P=0.003). Severe PH was ameliorated by ERT+/-vasodilators during 4.6+/-4.0 yr (range 1-12 yr) follow-up; three patients were initially considered for lung transplantation but improved such that they are no longer active transplant candidates. Our study reveals a remarkable predisposition for PH in type 1 GD. Progression to severe, life-threatening PH occurs in the presence of additional genetic factors (non-N370S GBA mutation, positive family history, and ACE I gene polymorphism) and epigenetic modifiers (i.e., asplenia and female sex). Splenectomy should be avoided and in high-risk patients, ERT+/-vasodilators/coumadin should be initiated.     

Epstein-Barr viral load as a marker of lymphoma in AIDS patients

Epstein-Barr virus (EBV) is implicated in the pathogenesis of acquired immunodeficiency syndrome (AIDS) lymphoma, and viral DNA is present within the malignant cells in about half of affected patients. We examined the extent to which EBV viral load is elevated in the plasma of AIDS lymphoma patients compared to AIDS patients with opportunistic infections. Sixty-one AIDS patients were studied including 35 with lymphoma (24 non-Hodgkin, six Hodgkin, and five brain lymphoma) and 26 with various opportunistic infections. In situ hybridization revealed EBV encoded RNA (EBER) expression in the malignant cells of 17/28 AIDS lymphomas (61%). In 232 serial plasma samples from 35 lymphoma patients and in 128 samples from AIDS controls, EBV viral load was assayed by quantitative-polymerase chain reaction (Q-PCR) using a TaqMan probe targeting the BamH1W sequence. EBV was detected in plasma from all 17 EBER-positive AIDS lymphoma patients, with viral loads ranging from 34 to 1,500,000 copies per ml (median 3,210). Viral load usually fell rapidly upon initiation of lymphoma therapy and remained undetectable except in two patients with persistent tumor. In 11 AIDS patients, whose lymphoma lacked EBER expression, and in 26 control patients without lymphoma, levels of EBV in plasma were usually low or undetectable (range 0-1,995 and 0-2,409, median 0 and 0, respectively). There was no association between EBV viral load and human immunodeficiency virus (HIV) load or CD4 count. In conclusion, EBV viral load shows promise as a tool to assist in diagnosis and management of EBV-related lymphoma patients.     

Progenitor egress from the bone marrow after allergen challenge: role of stromal cell-derived factor 1alpha and eotaxin

BACKGROUND: CCR3 expression on CD34+ cells mediates migration to eotaxin in vitro. CXCR4 and stromal cell-derived factor (SDF)-1alpha are important for stem cell homing to hemopoietic compartments. OBJECTIVE: To study chemokine-mediated progenitor cell traffic in allergic inflammation. METHODS: Bone marrow (BM) aspirates were obtained at baseline from normal subjects; atopic subjects without asthma; and subjects with asthma before, 5 hours after, and 24 hours after allergen inhalation (dual and early responders). Changes in chemokine receptor expression and migration were assessed. RESULTS: Expression of CXCR4, but not CCR3, on BM CD34+ cells was greater in normal subjects compared with atopic subjects with asthma. Likewise, SDF-1alpha, but not eotaxin, stimulated a greater migrational response by BM CD34+ cells from normal subjects compared with subjects with asthma. For all subjects, a positive correlation was found between intensity of CXCR4 expression and magnitude of CD34+ cell response to SDF-1alpha. Allergen inhalation attenuated both intensity of CXCR4 expression and SDF-1alpha levels in marrow from dual compared with early responders 24 hours postallergen. In contrast, the intensity of CCR3 expression on BM CD34+ cells increased in dual compared with early responders at 24 hours postallergen. In addition, an increase in migrational responsiveness of BM CD34+ cells to eotaxin and a decrease to SDF-1alpha 24 hours postallergen was found in dual responder subjects with asthma. CONCLUSION: After allergen inhalation in subjects with asthma, a downregulation in CXCR4 intensity on BM CD34+ cells and a reduction in BM SDF-1alpha levels may reduce progenitor retention to marrow stroma promoting peripheral egress, possibly mediated by the CCR3/eotaxin axis.     

Cross-modal generality of the gating deficit

Auditory P50/M50 paired-click studies have established an association between schizophrenia and impaired sensory gating in the auditory modality. However, the presumed cross-modal generality of the gating deficit has received little study. The present study examined gating in area 3b of primary somatosensory cortex to evaluate patients' somatosensory gating at this first stage of cortical processing. One hundred twenty-two channels of magnetoencephalography (MEG) data were collected from 27 subjects with chronic schizophrenia and 21 controls during a somatosensory paired-pulse paradigm with a 75- or 500-ms interstimulus interval. M20 somatosensory responses were localized using magnetic source imaging, and a gating ratio was calculated. In a subset of these subjects, MEG was also done for the standard auditory paradigm to assess M50 gating. Patients showed abnormal auditory M50 gating but normal somatosensory M20 gating. Results argue against a cross-modal gating deficit in primary somatosensory cortex.     

BDNF-induced facilitation of afferent-evoked responses in lamina II neurons is reduced after neonatal spinal cord contusion injury

We previously reported that brain-derived neurotrophic factor (BDNF), a pronociceptive neurotransmitter, induces synaptic facilitation of excitatory postsynaptic current (EPSC) in lamina II neurons of neonatal rats up to P14 in a N-methyl-d-aspartate (NMDA) receptor-dependent manner. Here we used the patch-clamp technique to study synaptic and NMDA-evoked responses in transverse spinal slices in the lumbar enlargement as well as the ability of BDNF to modify these responses from 1 day to 6 wk after neonatal contusion. In older uninjured animals (>P14), BDNF continued to evoke synaptic facilitation although superfusion of NMDA (in TTX) induced inward current of significantly smaller amplitude than that observed in younger rats. After contusion injury, BDNF was unable to facilitate dorsal root-evoked EPSCs in lamina II neurons despite the finding that NMDA-evoked currents were only slightly smaller than those observed in age-matched uninjured animals. These findings suggest that although BDNF-induced facilitation of the AMPA/kainate receptor-mediated response to dorsal root stimulation is maintained in the mature dorsal horn from intact rats, BDNF may no longer elicit these pronociceptive actions after neonatal contusion injury. The lack of change in NMDA-evoked currents in contused cords suggests that diminished NMDA receptor function is not the major cause of the decline in BDNF action after contusion. It seems more likely that diminished trkB expression and enhanced expression of truncated trkB receptors in the contused cord play a significant role in determining the reduced effect of BDNF under these conditions.     

Molecular analysis as a tool in the differential diagnosis of VHL disease-related tumors.

Von Hippel-Lindau (VHL) disease is an autosomal dominant tumor syndrome, in which hemangioblastomas (HBs), clear cell renal cell carcinomas (RCCs), and pheochromocytomas are the most frequently encountered tumors. The differential diagnosis of dedifferentiated tumors in general can be difficult, as standard histologic and immunohistochemical investigations do not always allow a definitive diagnosis. We used molecular genetic analysis to resolve the differential diagnosis of sarcomatoid RCC versus pheochromocytoma of a (peri)renal tumor in a VHL patient. Germline mutation analysis identified the C407T mutation, which has been related to a VHL phenotype in which pheochromocytomas are rare. Chromosomal imbalances detected in the tumor by CGH showed a pattern typical for RCCs and not for pheochromocytomas. CGH analysis of the multiple tumors of this VHL patient revealed a comparable karyotype in the metastatic tumors and the (peri)renal tumor. Concordantly, although the germline mutation was detected in all analyzed tumors, LOH 3p was only detected in the (peri)renal mass and most metastases. Overall, based on all genetic data, this tumor corroborated a diagnosis of metastatic sarcomatoid RCC. In line with these observations is the immunopositivity for the RCC-specific RC38 detected in the (peri)renal mass and the metastases that was not detected in pheochromocytomas. The RCC specific marker G250 was uninformative as it stains positive in all types of VHL tumors. This case report illustrates the promising role of genetic analysis in the differential diagnosis of histologically dedifferentiated tumors.