CD20-targeted measles virus shows high oncolytic specificity in clinical samples from lymphoma patients independent of prior rituximab therapy.

New therapeutic modalities for B-cell non-Hodgkin's lymphomas (B-NHL) are needed, especially for relapsing and aggressive subtypes. Toward this end, we previously generated a fully CD20-targeted and armed measles virus, and tested its efficacy in a xenograft model of mantle cell lymphoma (MCL). Here, we quantify its spread in peripheral blood mononuclear cells and/or tissue of patients with different histological subtypes of B-NHL, including splenic marginal zone lymphoma (SMZL). CD20-targeted MV efficiently infects lymphoma cells from SMZL and MCL while sparing most cells in the CD20-negative population, in contrast to the parental vaccine-lineage MV, which infects CD20-positive and CD20-negative cells equally. Rituximab therapy (4-8 months before relapse) did not interfere with the infectivity and specificity of MV(green)H(blind)antiCD20 in patient lymphoma samples. Thus, CD20-targeted oncolytic virotherapy is likely to be effective after previous antiCD20 therapy.

Mantle cell lymphoma salvage regimen: synergy between a reprogrammed oncolytic virus and two chemotherapeutics.

Measles virus (MV)-PNP H(blind)antiCD20 is a CD20-targeted and prodrug convertase-armed MV that temporarily controls growth of lymphoma xenografts in severe combined immunodeficiency (SCID) mice in combination with fludarabine phosphate (fludarabine). Herein, we examine the replication of this targeted virus and of a vaccine-lineage MV in disease bulks and circulating cells from mantle cell lymphoma (MCL) patients, and show that only the targeted virus is specific for CD20-expressing cells. We then assessed the efficacy of different regimens of administration of this virus in combination with fludarabine and cyclophosphamide (CPA) in an MCL xenograft model. We show that CPA administration before the beginning of virus treatment enhances oncolytic efficacy, likely through temporary immunosuppression. An interval of 1 week between intravenous virus administration and fludarabine treatment further enhanced oncolysis, by synchronizing maximum prodrug convertase expression with fludarabine availability. Finally, three 23-day courses of triple sequential treatment with CPA, virus and fludarabine treatment resulted in complete regression of the xenografts. Secondary disease symptoms interfered with survival, but average survival times increased from 22 to 77 days. These studies document a reprogrammed oncolytic virus, consolidating the effects of two chemotherapeutics, a concept well suited for a phase I clinical trial for MCL patients for whom conventional therapies have failed.

C5a is important in the tubulointerstitial component of experimental immune complex glomerulonephritis.

Interstitial injury is the hallmark of glomerulonephritis which is progressing to end-stage renal disease (ESRD). In humans and experimental animals, we have shown that interstitial disease is accompanied by up-regulation of complement components in tubular epithelial cells. Glomerulonephritis was induced in mice by the intraperitoneal injection of horse spleen apoferritin (HSA) and lipopolysaccharide (LPS). In addition to wild-type C57/B6 mice, animals in which the C5a receptor had been deleted (C5aR KO) were used. Animals were killed after 3 or 6 weeks, and kidneys harvested. At three weeks, both groups had evidence of mild mesangial matrix expansion and increased cellularity; there were no crescents, sclerotic lesions, or interstitial disease. At six weeks, glomerular lesions were advanced, but identical in the two groups. Both groups had evidence of an identical pattern of C3 gene expression in the tubular epithelium by in situ hybridization. There was a marked difference, however, in the extent of interstitial injury. Wild-type animals had significantly greater numbers of infiltrating interstitial cells, greater expansion of the peritubular space, more tubular atrophy, and more apoptotic tubular cells than did C5aR KOs. The anaphylotoxic fragment of C5, C5a, is not likely to be important in the glomerular component of this model of progressive glomerulonephritis. On the other hand, the interstitial component is markedly attenuated in knockout animals. These data support a role for complement in the interstitial component of this glomerulonephritis model. They are consistent with our hypotheses of a role for complement in the progression of some forms of glomerulonephritis to ESRD.

Evidence of a role for C4 in modulating interstitial inflammation in experimental glomerulonephritis.

Activation of C4 releases into the fluid phase a fragment of the alpha chain, C4a. Unlike the analogous fragments of C3 and C2, there is no evidence for an anaphylatoxic effect of C4a. There is actually some in vitro evidence that it could have a modulating effect on inflammation by inhibiting monocyte chemotaxis. We induced an immune complex glomerulonephritis in wild-type (WT) and C4 knock out (C4KO) mice. Although the glomerular component of the disease did not differ in the two groups of animals, there were marked differences in the accompanying tubulo-interstitial injury. Compared to WT animals, the C4KO mice had significantly more infiltrating interstitial cells (1910 vs 2720/mm(2)), foci of tubular atrophy (6.3 vs 14.8/section), and interstitial space (22 vs 30% of cortex). C4 is expressed constitutively by renal tubular epithelial cells. These data support a role for such local C4 in modulating interstitial inflammation, consistent with in vitro experiments.

Glomerulonephritis associated with deficiencies and polymorphisms of complement components encoded in the class III region of the MHC.

An association between the complement system and immune complex glomerular disease in humans has long been recognized. In fact, much of our early understanding of the immunochemistry of complement activation developed with the study of acute and chronic glomerulonephritis (1). This manuscript will examine associations between glomerulonephritis and the three complement components encoded within the major histocompatibility complex: C4, C2, and factor B (B). The mechanisms by which deficiencies or polymorphisms in these components can mediate disease will be examined.

The C5a receptor is expressed by human renal proximal tubular epithelial cells.

The C5a receptor is expressed by a variety of cell types. These studies demonstrate by immunohistochemistry that the receptor is present on the surface of proximal and distal tubular epithelial cells from normal kidney. In addition, the receptor was detected on transitional epithelial cells of the ureter and bladder. Primary proximal tubular cultures and a proximal tubular cell line both also expressed the C5a receptor, as demonstrated by immunofluorescence and by FACS analysis. The presence of mRNA encoding the receptor was confirmed by reverse transcriptase-polymerase chain reaction analysis. As opposed to its effect on glomerular mesangial cells, the receptor did not mediate a proliferative response by the proximal tubular cells. C5a also did not enhance the synthesis/secretion of transforming growth factor-beta 1, monocyte chemoattractant protein-1, platelet-derived growth factor-AB or tumour necrosis factor-alpha by cultured proximal tubular cells. Therefore, although the C5a receptor clearly is expressed by proximal tubular cells, clarification of its functional relevance on this cell type awaits further studies.

Evidence of a role for local complement expression in a murine model of progressive glomerulonephritis.

C57/B6 mice received intraperitoneal horse spleen apoferritin (4 mg) with lipopolysaccharide (0.05 mg); control mice received 0.15 M NaCl. Control and treated animals were killed weekly for 6 wk; blood and urine specimens were obtained, and tissue samples were secured. Treated animals showed evidence of significant chronic disease, with proteinuria, hematuria, and uremia. A mild glomerulonephritis was present at 2 wk, with significant proliferative glomerulonephritis at 4 wk, progressing to chronic disease with tubulointerstitial changes at 6 wk. Changes at each time period were uniform between animals. C3 mRNA was first detected by in situ hybridization at 3 wk. Message was restricted to proximal tubular and periglomerular epithelial cells. Presence of C3 message preceded the development of interstitial inflammation and fibrosis by 1-2 wk, and its location and intensity paralleled the evolving interstitial disease. Although extensive mesangial C3 protein deposits appeared early, there was never C3 message in glomeruli or infiltrating cells. Before C3 message became apparent, two cytokines known to up-regulate C3 transcription in vitro, IL-1 and IL-6, were detected by immunohistochemistry. The temporal sequence in this model is consistent with our hypothesis that local synthesis and activation of C3 in tubular epithelium is important to the interstitial component of chronic glomerulonephritis. The process is independent of the deposition of circulating complement in the glomerulus, but may be triggered by glomerular cytokines.

The phenotype of SLE associated with complete deficiency of complement isotype C4A.

Complete deficiency of the complement C4A isotype is a known genetic risk factor for systemic lupus erythematosus (SLE). The disease phenotype of C4A-deficient patients has never been defined. Among 200 patients with SLE from five centers, 18 (9%) with C4A deficiency were identified. These individuals were compared to those who were C4A replete with regard to a series of clinical and serologic features. The only significant differences between the two groups were in the presence of renal disease (C4A deficient, 11%; C4A replete, 46%; P < 0.006) and a decrease in the serum concentrations of C3 (C4A deficient, 11%; C4A replete, 35%; P < 0.04). There was also a trend for the C4A-deficient individuals to have milder disease. In light of the tendency for C4A-deficient individuals to have lower serum concentrations of C4, it is important that such patients not be subjected to overly aggressive efforts to "normalize" their C4 levels.

Regulated expression of complement factor B in the human kidney.

We have previously demonstrated regulated expression of C3 in the proximal renal tubular epithelial cells of humans. To test the hypothesis that local alternative pathway complement activation could contribute to the tubulointerstitial component of chronic renal disease, we examined factor B gene expression in human kidneys. 35S riboprobes were generated from a human factor B cDNA. By in situ hybridization, proximal tubular factor B message was seen in 17 kidneys with various nephropathies. The expression was most intense in organs with evidence of interstitial inflammation, and its localization paralleled the inflammation. As was the case with C3 and C4, there was never any evidence of glomerular factor B message, nor was any seen in infiltrating inflammatory cells. In eight normal kidney tissues, factor B expression was either absent or restricted to rare foci of interstitial infiltration. The proximal renal tubular epithelium of humans appears to express the genes for both components of the alternative pathway convertase, C3 and factor B. These locally produced components may be important mediators of the interstitial inflammation that is common to all progressive nephritides.

Annexin VI overexpression targeted to heart alters cardiomyocyte function in transgenic mice.

Annexin VI is a member of a family of Ca(2+)-dependent phospholipid-binding proteins that is expressed in many tissues, including the heart. It is a regulator of membrane-associated events, including the skeletal muscle ryanodine-sensitive Ca2+ release channel and the cardiac Na+/Ca2+ exchanger. The potential roles of annexin VI in Ca2+ signaling in cardiac myocytes were evaluated by targeting its overexpression to the hearts of transgenic mice. Expression of full-length human annexin VI cDNA was targeted to the heart using the alpha-myosin heavy chain gene promoter (Subramaniam, A., W. K. Jones, J. Gulick, S. Wert, J. Neumann, and J. Robbins. J. Biol. Chem. 266: 24613-24620, 1991). Five transgenic lines exhibited at least 10-fold overexpression of annexin VI protein in both atria and ventricles. Pathological evaluation indicated mice overexpressing annexin VI had enlarged dilated hearts, acute diffuse myocarditis, lymphocytic infiltration, moderate to severe fibrosis throughout the heart, and mild fibrosis around the pulmonary veins of the lungs. Contractile mechanics of cardiomyocytes isolated from hearts of transgenic animals showed frequency-dependent reduced percent shortening and decreased rates of contraction and relaxation compared with control animals. Cardiomyocytes isolated from transgenic animals had lower basal levels of intracellular free Ca2+ and a reduced rise in free Ca2+ following depolarization. After stimulation, intracellular free Ca2+ returned to basal levels faster in transgenic cells than in cells from control animals. These data demonstrate that the overexpression of annexin VI in the heart disrupts normal Ca2+ homeostasis and suggests that this dysfunction may be due to annexin VI regulation of pumps and/or exchangers in the membranes of cardiomyocytes.

Plasma interleukin-1 alpha and beta, tumor necrosis factor-alpha, and lipopolysaccharide concentrations during pulmonary exacerbations of cystic fibrosis.

Earlier studies have reported the presence of interleukin-1 (IL-1) and tumor necrosis factor (TNF) in the plasma of patients with cystic fibrosis (CF), but the results have been inconsistent. To investigate the relationships among plasma IL-1 alpha, IL-1 beta, TNF, lipopolysaccharide (LPS), and clinical status, measurements were made before and after 14 days of intravenous antibiotic therapy in 13 patients with CF. In addition, whole blood cytokine production rates were measured in 18 hr cultures stimulated with 10 micrograms/mL LPS or sterile saline (control). On admission, patients with CF had significantly greater plasma levels of LPS and IL-1 alpha compared with 20 healthy adult controls. In response to antibiotic therapy, the patients had statistically significant increases in weight, oxygen saturation, chest radiograph score, and forced expiratory volume in 1 second. They had significant decreases in pulse rate, residual volume/total lung capacity ratio, white blood count, neutrophil count, LPS concentration, and resting energy expenditure per kg body weight. There were no significant changes in the plasma concentrations of IL-1 alpha, IL-1 beta, or TNF and no significant changes in the basal or stimulated whole blood production rates of IL-1 alpha, IL-1 beta, or TNF. The immunological variables did not correlate significantly with clinical measurements of severity or the presence of fever. It is likely that in CF local pulmonary effects of cytokines are of more pathophysiologic significance than systemic effects.

Testing of maternal sera in pregnancies at risk for neonatal alloimmune thrombocytopenia.

The utility of prenatal testing of maternal serum for platelet-reactive antibody was assessed in 25 women at risk of delivering infants with neonatal alloimmune thrombocytopenia (NAT). Seventeen women were incompatible with their husbands for the PlA1 antigen and three for Baka; in five families, no demonstrable platelet-specific antigen incompatibility was found. Analysis of the clinical outcome demonstrated that women with platelet-specific antibody detectable in any of the assays at any time during gestation were at risk of delivering thrombocytopenic infants (neonatal platelet count 31,250/microliters if mother did have antibody, as compared with 138,750/microliters if she did not; p less than 0.005). When only PlA1-incompatible pregnancies were examined, this association remained significant (mean neonatal platelet count in infants exposed to anti-PlA1, 34,285/microliters; that in infants not so exposed, 243,000/microliters; p less than 0.001). Changes in antibody strength throughout pregnancy did not correlate with the severity of NAT. The combination of the antigen-capture enzyme-linked immunosorbent assay and the indirect immunofluorescence test appeared to be most sensitive in detecting relevant platelet-specific alloantibodies. It is concluded that the detection of platelet-specific alloantibody in maternal serum in pregnancies at risk for NAT predicts moderate to severe NAT. However, the failure to detect such antibody does not always predict a normal neonatal platelet count.

Evaluation of factor VIII-rich cryoprecipitate and the plasma fibronectin-rich, heparin-precipitable fraction prepared from single-donor plasma units.

A plasma fibronectin-rich component was prepared by heparin-induced 4 degrees C precipitation of fresh or stored (21 days at 4 degrees C), single-donor plasma. The recovery of plasma fibronectin was 45 percent at a concentration of 0.05 mg heparin per ml (7.5 units/ml) and 75 percent at 0.1 mg per ml (15 units/ml). The biologic activity of plasma fibronectin, as assessed by the spreading of Chinese hamster ovary cells or attachment of monocytes to gelatin-coated surfaces, was similar to that of plasma fibronectin concentrates made from fresh or stored plasma. Only 20 to 30 percent of the factor VIII activity in fresh plasma was recovered in cryoprecipitate produced after the heparin-induced precipitate containing fibronectin was removed. Cryoprecipitate prepared from the supernatant plasma that remains after heparin-induced cold precipitation in the presence of CaCl2 (5 mM) contained approximately 50 percent less factor VIII. The relatively low recovery of factor VIII in cryoprecipitate prepared from fibronectin-depleted plasma makes cryoprecipitation an unsuitable method of producing fibronectin-rich and factor VIII-rich components effectively from a single unit of fresh plasma. However, heparin-induced cold precipitation provides an efficient method for preparing plasma fibronectin concentrates from small plasma pools or single units of stored or fresh plasma.

Use of PGE1 for preparation of platelet concentrates.

Platelets stored as platelet concentrates (PCs) undergo biochemical, functional, and morphological changes that may inhibit their hemostatic effectiveness. Prostaglandin E1 (PGE1) (5.9 X 10(-8) M) added to platelet-rich plasma (PRP) immediately before PC preparation reduced platelet activation during PC preparation and storage as determined by inhibition of beta-thromboglobulin release and thromboxane B2 production. However, PCs prepared with PGE1 contained less glycogen after storage than PCs prepared by standard methods. All PC platelets lost their ability to aggregate in response to adenosine diphosphate (ADP) during storage, but sensitivity to dual stimulation with epinephrine and ADP was retained. These findings suggest that addition of PGE1 to PRP may permit production of platelets that are hemostatically superior to conventional PCs. In vivo studies to evaluate this possibility are indicated.

Use of prostacyclin to inhibit activation of platelets during preparation of platelet concentrates.

Increased utilization of platelet concentrates (PCs) has created the possibility that demand for this blood component may exceed supply. It seems possible that this problem could be minimized if platelets could be isolated from whole blood and stored with full retention of hemostatic effectiveness. We prepared PCs by adding prostacyclin (PGI2) during the isolation procedure and compared these to PCs prepared without addition of PGI2. At concentration 10(-7) M, PGI2 inhibited activation of platelets during the process of concentration and resuspension by the criteria of minimal thromboxane production and release of beta thromboglobulin (beta-TG), in contrast to concentrates prepared by standard methods. These differences persisted throughout 3 days of storage at room temperature. Our findings suggest that further studies are indicated to determine whether the quality (hemostatic effectiveness) of concentrated platelets can be improved by pharmacologic means.

Chemiluminescence assay for detection of anti-platelet antibodies.

A chemiluminescence (CL) assay for detection of antiplatelet antibodies was developed. Platelets sensitized with a xenogeneic antiplatelet serum or a potent anti-Pla1 serum stimulated granulocytes to produce CL. Other antibodies detectable by 51chromium release and immunofluorescent assays were not discerned by the CL assays.

Platelet inhibitory effects of CRP preparations are due to a co-isolating low molecular weight factor.

We previously reported that C-reactive protein (CRP), an acute phase reactant, inhibits platelet activation through an effect upon a factor(s) critical to ADP-mediated secondary wave platelet aggregation but independent of a direct effect upon platelet contractile elements. However, a role for an accessory factor in this inhibitory effect became of concern because of an inconsistency in the effects of CRP preparations upon the platelet: inhibition was lost upon storage and CRP preparations differed, on a weight basis, in inhibitory capacity and sensitivity to the presence of the CRP ligand C-polysaccharide (CPS(. The studies presented herein were thus intended to assess whether an accessory factor was involved in the inhibition of platelet activation observed with CRP. We report that the activity of the inhibitory CRP preparations resulted from association with a low molecular weight factor (LMF) with an apparent nominal molecular weight of 8300-12,500 and an A280:A260 ratio of approximately 0.4. Purified CRP did not inhibit platelet responsiveness but CRP with associated LMF (CRP-LMF) did. Moreover, the inhibitory capacity of CRP-LMF but not LMF was substantially reversed in the presence of CPS. These studies indicate that the platelet inhibitory properties of CRP preparations result from and are contingent upon the presence of a co-isolating low molecular weight factor.

Modulation of platelet aggregation by native DNA - initial description of platelet receptor type, number and discrimination for native DNA.

Native DNA (dsDNA) induces the aggregation of isolated human platelets. Using isotopically labeled dsDNA (125I-dsDNA) and Scatchard analysis, a single class of platelet receptor was detected with a KD = 190 pM and numbering approximately 275/platelet. This receptor was discriminatory in that heat denatured dsDNA, poly A, poly C, poly C x I and poly C x poly I failed to substantially inhibit either the platelet binding of, or platelet aggregation induced by, dsDNA; by themselves, these polynucleotides were ineffective as platelet agonists. However, poly G, poly I and poly G x I effectively and competitively inhibited platelet binding of the radioligand, independently activated the platelet and when used at a sub-activating concentration decreased the extent of dsDNA stimulated platelet aggregation. These data depict a receptor on human platelets for dsDNA and perhaps certain additional polynucleotides and relate receptor-ligand interactions to a physiologic platelet function.

Circadian rhythms of plasma corticosterone binding activity in the rat and the mouse.

Corticosterone binding activity (CBA) and corticosteroids were measured by competitive protein binding techniques in plasma samples drawn from rats and mice at different times of day. Circacian rhythms of plasma CBA were found in both rats (onset of 14-h daily photoperiod: 0600) and mice (onset of 14-h daily photoperiod: 0700). The plasma CBA rhythm was bimodal in rats with peaks at 1000 and 1800 and unimodal in mice with peak level from 0100 to 0500. Circadian rhythms of plasma corticosteroid concentration with peaks during the late afternoon were confirmed in both rats and mice. The plasma CBA rhythms appear to be related to the circadian rhythms of both locomotor activity and plasma corticosteroid concentration.