Bruce I. Goldman

L-Type Ca2+ Channel Density and Regulation Are Altered in Failing Human Ventricular Myocytes and Recover After Support With Mechanical Assist Devices

Abstract— Ca2+ influx through the L-type calcium channel (LTCC) induces Ca2+ release from the sarcoplasmic reticulum (SR) and maintains SR Ca2+ loading. Alterations in LTCC properties, their contribution to the blunted adrenergic responsiveness in failing hearts and their recovery after support with LV assist devices (LVAD) were studied. L-type Ca2+ current (ICa,L) was measured under basal conditions and in the presence of isoproterenol (ISO), dibutyryl-cAMP (db-cAMP), Bay K 8644 (BayK), Okadaic acid (OA, a phosphatase inhibitor), and phosphatase 2A (PP2A) in nonfailing (NF), failing (F), and LVAD-supported human left ventricular myocytes (HVMs). Basal ICa,L density was not different in the 3 groups but ICa,L was activated at more negative voltages in F- and LVAD- versus NF-HVMs (V0.5: −7.18±1.4 and −7.0±0.9 versus 0.46±1.1 mV). Both ISO and db-cAMP increased ICa,L in NF- and LVAD- significantly more than in F-HVMs (NF >LVAD> F: ISO: 90±15% versus 77±19% versus 24±12%; db-cAMP: 235%>172%>90%). ISO caused a significant leftward shift of the ICa,L activation curve in NF- and LVAD- but not in F-HVMs. After ISO and db-cAMP, the ICa,L activation was not significantly different between groups. BayK also increased ICa,L more in NF- (81±30%) and LVAD- (70±15%) than in F- (51±8%) HVMs. OA increased ICa, L by 85.6% in NF-HVMs but had no effect in F-HVMs, while PP2A decreased ICa, L in F-HVMs by 35% but had no effect in NF-HVMs. These results suggest that the density of LTCC is reduced in F-HVMs but basal ICa,L density is maintained by increasing in LTCC phosphorylation.

Beneficial effect of novel proteasome inhibitors in murine lupus via dual inhibition of type I interferon and autoantibody-secreting cells

OBJECTIVE To investigate the hypothesis that proteasome inhibition may have potential in the treatment of SLE, by targeting plasmacytoid dendritic cells (PDCs) and plasma cells, both of which are critical in disease pathogenesis. METHODS Lupus-prone mice were treated with the nonselective proteasome inhibitors carfilzomib and bortezomib, the immunoproteasome inhibitor ONX 0914, or vehicle control. Tissue was harvested and analyzed by flow cytometry using standard markers. Nephritis was monitored by evaluation for proteinuria and by histologic analysis of kidneys. Serum anti-double-stranded DNA (anti-dsDNA) levels were measured by enzyme-linked immunosorbent assay (ELISA), and total IgG and dsDNA antibody-secreting cells (ASCs) by enzyme-linked immunospot assay. Human peripheral blood mononuclear cells or mouse bone marrow cells were incubated with Toll-like receptor (TLR) agonists and proteasome inhibitors, and interferon-α (IFNα) levels were measured by ELISA and flow cytometry. RESULTS Early treatment of lupus-prone mice with the dual-targeting proteasome inhibitors carfilzomib or bortezomib or the immunoproteasome-specific inhibitor ONX 0914 prevented disease progression, and treatment of mice with established disease dramatically abrogated nephritis. Treatment had profound effects on plasma cells, with greater reductions in autoreactive than in total IgG ASCs, an effect that became more pronounced with prolonged treatment and was reflected in decreasing serum autoantibody levels. Notably, proteasome inhibition efficiently suppressed production of IFNα by TLR-activated PDCs in vitro and in vivo, an effect mediated by inhibition of both PDC survival and PDC function. CONCLUSION Inhibition of the immunoproteasome is equally efficacious as dual targeting agents in preventing lupus disease progression by targeting 2 critical pathways in disease pathogenesis, type I IFN activation and autoantibody production by plasma cells.

Popliteal venous aneurysm: Report of two cases and review of the world literature

Two new cases of popliteal venous aneurysm are reported and added to the 22 other cases of popliteal venous aneurysm available for review. Both patients were first seen with acute pulmonary embolism and were treated with thrombolytic therapy followed by anticoagulation. Each had recurrent venous thromboembolism before discovery of the popliteal venous aneurysm. One popliteal venous aneurysm was diagnosed with phlebography and the second with venous duplex imaging, confirmed with phlebography. Both were surgically corrected with tangential aneurysmectomy and lateral venorrhaphy. Twenty-four cases of popliteal venous aneurysm are now available for review. Seventy-one percent (17 of 24) presented with pulmonary embolism, 88% (21 of 24) were saccular, and 96% (23 of 24) were located in the proximal popliteal vein. All but two were diagnosed by ascending phlebography. Three patients received no treatment: in two of these the outcome was not documented and the third had occasional pain. Two patients received anticoagulation without subsequent operative repair and both died of recurrent pulmonary emboli. Operative correction resulted in a 75% patency rate with 21% complications, most of which were related to postoperative anticoagulation. No patient who was operated on had subsequent pulmonary embolism, and there were no operative deaths. We suggest that all patients who have pulmonary embolism have lower-extremity venous duplex imaging. All popliteal venous aneurysms should be surgically repaired, inasmuch as nonoperative therapy results in recurrent thromboembolism and an unacceptably high mortality rate. Tangential aneurysmectomy with lateral venorrhaphy is the recommended procedure.

Influence of pressure on permeability of normal and diseased muscular arteries to horseradish peroxidase. A new catheter approach.

The effects of different distending pressures on permeability of dog and human arteries to horseradish peroxidase (HRP) were studied. A new catheter was employed to achieve the distention of defined vessel segments to the desired pressure. Normal dog brachial arteries were studied both post mortem and in vivo. Mildly to moderately diseased human coronary arteries were studied post mortem. A predictable linear relationship between pressure and penetration of HRP into the dog arterial media was found, using pressures of 0, 150, 300 and 500 mm Hg. Postmortem vessels were consistently less permeable than those studied in vivo. Full penetration of the media by HRP was achieved by application of 300 mm Hg pressure for 45 sec with the new catheter. When human coronary lesions were examined under these same conditions, plaques were readily demonstrated to be permeable to HRP, even to a depth of many hundreds of micrometer. Thus, penetration of arterial wall thickness by HRP (Mr 40,000 dalton) is related to the distending pressure applied. Human coronary plaques also show ready penetrance by HRP. The new catheter described allows the application of these pressures to defined segments of the arterial tree.

Allograft Inflammatory Factor-1 Expression Correlates With Cardiac Rejection and Development of Cardiac Allograft Vasculopathy

Background—Standard morphological features of endomyocardial biopsy specimens do not necessarily correlate with the efficacy of immunotherapy or development of cardiac allograft vasculopathy (CAV). We hypothesized that expression of allograft inflammatory factor-1 (AIF-1), a cytokine-inducible, calcium-binding protein associated with vascular smooth muscle cell proliferation, would be associated with allograft rejection and development of CAV. Methods and Results—A total of 157 endomyocardial biopsy specimens from 26 patients with heart transplants were examined for expression of AIF-1 mRNA by semiquantitative reverse transcription–polymerase chain reaction. A significant relation was found between the International Society for Heart and Lung Transplantation rejection grade and expression of AIF-1 (P <0.001). The calculated odds ratio indicates that a biopsy has 2.5 times the chance of AIF-1 expression per grade of rejection. The relative concentrations of AIF-1 and GAPDH mRNA were calculated and the resulting ratios indicated that the amount of AIF-1 mRNA expression is relative to the rejection grade (P <0.02). In grade 1 biopsy specimens, AIF-1 was localized to infiltrating immune cells. In grade 3 biopsy specimens, AIF-1 was observed in immune cells and myocytes. AIF-1 is expressed in vascular and immune cells in coronary arteries with CAV, and persistent expression of AIF-1 in the allograft correlates with development of CAV (P <0.002). Conclusions—Expression of AIF-1 in cardiac allografts correlates with rejection, and the amount of AIF-1 expressed correlates with the severity of rejection. AIF-1 is expressed in coronary arteries with CAV, and persistent expression of AIF-1 in the cardiac allograft is associated with development of CAV.

Hibernation: An opioid-dependent state?☆

Hibernation reduces substantially the heart rate of hamsters as well as the respiratory rate, the body temperature and the arousal level. The heart rate is reversed dramatically by the injection of low doses of Naloxone and in some cases the hamster arouses prematurely from hibernation. The effect is not due to the pain of the injection because saline injections do not produce such changes. The effect requires a pre-existing state of hibernation because Naloxone has no cardioacceleratory or arousal effect in non-hibernating hamsters that have had their heart rate and body temperature decreased substantially during hypothermia. These results suggest that endogenous opioids may contribute specifically to the state of hibernation. Moreover, a physiological role may exist for an anti-opioid system in the promotion of arousal from hibernation.

Prolonged effects of short‐term anti‐CD20 B cell depletion therapy in murine systemic lupus erythematosus

OBJECTIVE Although B cells are implicated in the pathogenesis of systemic lupus erythematosus, the role of B cell depletion (BCD) as a treatment is controversial, given the variable benefit in human disease. This study was undertaken to test the effects of BCD therapy in a murine lupus model to better understand the mechanisms, heterogeneity, and effects on disease outcomes. METHODS (NZB x NZW)F(1) female mice with varying degrees of disease severity were treated with an anti-mouse CD20 (anti-mCD20) antibody (IgG2a), BR3-Fc fusion protein (for BAFF blockade), or control anti-human CD20 monoclonal antibody (approximately 10 mg/kg each). Tissue samples were harvested and analyzed by flow cytometry. The development and extent of nephritis were assessed by monitoring proteinuria (using a urine dipstick) and by immunohistochemical analysis of the kidneys. Serum immunoglobulin levels were measured by enzyme-linked immunosorbent assay. RESULTS After a single injection of anti-mCD20, BCD was more efficient in the peripheral blood, lymph nodes, and spleen compared with the bone marrow and peritoneum of normal mice as well as younger mice with lupus. Since depletion of the marginal zone and peritoneal B cells was incomplete and variable, particularly in older mice with established nephritis, a strategy of sequential weekly dosing was subsequently used, which improved the extent of depletion. BAFF blockade further enhanced depletion in the spleen and lymph nodes. Early BCD therapy delayed disease onset, whereas BCD therapy in mice with advanced disease reduced the progression of nephritis. These effects were long-lasting, even after B cell reconstitution occurred, and were associated with a reduction in T cell activation but no significant change in autoantibody production. CONCLUSION The lasting benefit of a short course of BCD therapy in lupus-prone mice with an intact immune system and established disease highlights the validity of this treatment approach.

Glandular cardiac myxomas. Histologic, immunohistochemical, and ultrastructural evidence of epithelial differentiation.

Histologic, histochemical, immunocytochemical, and ultrastructural features of two cardiac myxomas containing glandular elements are reported. Glandular elements in both cases stained positively with both mucicarmine and periodic acid‐Schiff reagent with diastase pretreatment (DPAS). Immunoperoxidase studies demonstrated positivity of the glandular cells for carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), and keratin. Factor VIII‐related antigen (FVIIIAg) was identified only in cells lining vascular spaces. Electron microscopic study of one tumor demonstrated well‐formed glands having basement membranes, junctional complexes, and apical secretory granules. These findings indicate the capacity for true epithelial differentiation of cardiac myxomas and have implications both as regards the histologic diagnosis of these tumors and their histogenesis.

Apoptosis in chronic rejection of human cardiac allografts.

Background. We investigated the role of apoptosis (programed cell death) in the pathogenesis of chronic rejection. Methods. Epicardial coronary arteries from cardiac allografts with chronic rejection were examined for apoptosis by the TUNEL assay. Double labeling was carried out using anti-CD3, anti-CD68, and anti-von Willenbrand factor (vWF) monoclonal antibodies. Additional immunostaining was carried using anti-Fas, anti-Fas-L, and anti-Bcl-2 monoclonal antibodies. Apoptosis-associated oligonucleosomal DNA degradation was assessed by DNA agarose gel electrophoresis. The transcription level of apoptosis-related caspase genes were determined using microarrays. Results. Apoptotic cells (TUNEL+) were detected within the arterial wall and in perivascular areas. Double labeling demonstrated that apoptotic cells included T cells (CD3+), monocyte/macrophages (CD68+), and vascular endothelial cells (VWF+). Numbers and densities of TUNEL+ cells did not correlate with the degree of arterial stenosis. Apoptosis-associated oligonucleosomal DNA degradation was assessed by agarose gel electrophoresis of DNA, which showed DNA fragments of approximately 180 bp and multimers thereof (DNA laddering gel), which are characteristic for DNA fragmentation in apoptotic cells. Microarray analysis demonstrated that the apoptosis related caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, were all transcribed (caspases 8, 9, and 10 were highly up-regulated). These results are consistent with the involvement of apoptosis in chronic rejection. Immunoreactivity for Fas/Fas-L was present at the sites of apoptotic cells. Immunoreactivity for Bcl-2 was present in areas with very few apoptotic cells. Conclusions. Apoptotic cells include T cells, monocyte/macrophages, and endothelial cells. Apoptosis, likely through the Fas/Fas-L system, is involved in the pathogenesis of chronic rejection in cardiac allografts.

Rituximab treatment of fibrillary glomerulonephritis.

Fibrillary glomerulonephritis belongs to a group of disorders characterized by pathogenic deposition of fibrils in glomeruli. This glomerulopathy tends to progress to end-stage kidney disease, and there currently are no treatments of proven benefit, including corticosteroids and cytotoxic agents. Because the glomerular deposits contain an immunoglobulin component, it was postulated that anti-B-cell therapy with rituximab, an anti-CD20 monoclonal antibody, may be effective in the treatment of patients with fibrillary glomerulonephritis. We describe 3 patients with fibrillary glomerulonephritis who were treated with rituximab for nephrotic-range proteinuria. Each patient also received standard antiproteinuria therapy, including blockade of the renin-angiotensin system and strict blood pressure control. All patients showed a decrease in proteinuria to less than 1.5 g/d of protein by 27 months, and kidney function was preserved throughout the duration of therapy and follow-up. No adverse effects were seen with rituximab. These outcomes suggest that treatment with rituximab may be a promising approach to the management of fibrillary glomerulonephritis, an entity previously considered refractory to therapy.