Characterization of specific cDNA background synthesis introduced by reverse transcription in RT-PCR assays.

To block expression of NMDA receptor NR1 subunit, we injected into rat hippocampus a Herpes Simplex Virus type 1 derived vector bearing a sequence for NR1 antisense. RT-PCR assays with RNA from hippocampus of animals injected either with NR1 antisense vector, control vector or vehicle, showed an amplification signal compatible with NR1 antisense which could be attributed either to an endogenous NR1 antisense or to an artifact. RT-PCR was performed either with different primers or without primers in the RT, using RNA from different tissues. RNAse protection assay was carried out to characterize the amplified signal nature. Our results show that the template for the unexpected amplified fragment was NR1 mRNA currently expressed in nervous tissue. We considered this basal amplification of a mRNA in a RT-PCR assay as "background amplification". After background subtraction, a significant signal only remained when samples from NR1 antisense vector injected animals were used, demonstrating that this was the only source for NR1 antisense. Background amplification at RT in the absence of primers, can constitute a troubling factor in quantitative nucleic acid determination, leading to major interference, particularly when both sense and antisense sequences are present in the sample. Since RT introduced a significant background signal for every gene analyzed, we propose that RT must be always performed also without primers. Then, this signal should be identified, quantified and subtracted from the specific reaction amplification signal.

Progress and prospects: biological properties and technological advances of herpes simplex virus type 1-based amplicon vectors.

The last two years have seen significant advances in our understanding of the cellular innate responses elicited or activated by the entry of amplicon particles, which may, in part, explain the transient nature of transgene expression often observed in cells infected with helper-free amplicon stocks. At the technological level, the most consistent progress has been in strategies to enhance the stability of transgene cassettes, either through integration into host chromosomes or through the conversion of the amplicon genome into a replication-competent extrachromosomal element.

Cytokine, chemokine, and co-stimulatory fusion proteins for the immunotherapy of solid tumors.

This chapter describes the generation of novel reagents for the treatment of cancer using fusion proteins constructed with natural ligands of the immune system. Immunotherapy is a powerful therapeutic modality that has not been fully harnessed for the treatment of cancer. We and others have hypothesized that if the proper immunoregulatory ligands can be targeted to the tumor, an effective immune response can be mounted to treat both established primary tumors and distant metastatic lesions. Though it is generally believed that immunotherapy has the potential to treat only residual disease, we offer evidence that this approach can, by itself, destroy large tumor masses and produce lasting remissions of experimental solid tumors. From these studies, three major classes of immune activators, namely, cytokines, chemokines, and costimulatory molecules, have been shown to generate antitumor responses in animal models. In addition, the reversal of immune tolerance by the deletion of T regulatory (Treg) cells has been shown to be equally important for effective immunotherapy. In an attempt to identify reagents that can provide an enhanced immune stimulation and treatment of cancer, our laboratory has developed a novel monoclonal antibody targeting approach, designated Tumor Necrosis Therapy (TNT), which utilizes stable intracellular antigens present in all cell types but which are only accessible in dead and/or dying cells. Since tumors contain necrotic and degenerating regions that account for 30-80% of the tumor mass, this targeting approach can be used to deliver therapeutic reagents to the core of tumors, a site abundant in tumor antigens. In our first set of reagents, a panel of cytokine fusion proteins was genetically engineered using monoclonal antibody chimeric TNT-3 (chTNT-3) directed against necrotic regions of tumors (single-stranded DNA) fused with IL-2, or GM-CSF, or TNFalphaa, or IFNgamma. Tested against different solid tumors, these reagents were found to mount an effective although transient immune response to tumor especially when used in combination. To improve upon these results, additional chTNT-3 fusion proteins using the liver-expression chemokine (LEC) and the costimulatory molecule B7.1 were constructed. Both of these reagents were found to work significantly better than the above cytokine fusion proteins due to their ability to stimulate multiple arms of the immune system deemed useful for cancer immunotherapy. Finally, the Tumor Necrosis Factor Superfamily (TNFSF) gene DC137L was used to generate chTNT-3 antibody (targeted) and soluble Fc (untargeted) fusion proteins. When used alone, both forms of costimulatory fusion proteins were found to produce in a s dose-dependent manner, complete regression of murine solid tumors. Evidence is presented to show that Treg cells play an important role in suppressing antitumor immunity since the deletion of these cells, when used in combination with LEC or costimulatory fusion proteins, produced profound and effective treatment with sustained memory. It is hoped that these data will further the preclinical development of soluble Fc and antibody based fusion proteins fro the immunotherapy of cancer.

[HSV1-derived vectors: a powerful and flexible system for genes transfers]. / Les vecteurs amplicons dérivés du virus HSV1 : un système souple et puissant pour le transfert de gènes.

Amplicons are non-integrative defective herpes simplex type 1 (HSV1) derived vectors. Their genomes are entirely free of viral genes, making these vectors non toxic for infected cells and non pathogenic for inoculated animals. In addition, amplicon vectors possess the unique property of delivering up to 150 kbp of foreign DNA. These characteristics make amplicon vectors one of the most powerful and promising viral vectors for gene transfer. This review illustrates several interesting applications using amplicon vectors, as well as problems that need to be resolved in order to obtain stable and physiological transgene expression.

Knocking-down the NMDAR1 subunit in a limited amount of neurons in the rat hippocampus impairs learning.

Amplicon vectors derived from herpes simplex virus type 1 were built to modify NMDA receptors by expressing antisense RNA for the essential NR1 subunit. Their ability to modify endogenous levels of NR1 was tested in cultures of rat embryo neocortical neurons. We studied behaviour and tested for expression in adult rats injected with those vectors into the dorsal hippocampus to find out which cells and how many appear involved in memory formation. Rats injected with vectors expressing NR1 antisense performed significantly worse than control rats in an inhibitory avoidance task. Immunohistochemistry was performed in brain slices from the same animals. The transduced cells represented 6-7% of pyramidal neurons in CA1, showing that a single gene knockdown of NR1 in a small number of neurons significantly impaired memory formation. Perhaps neurons undergoing synaptic plasticity are more susceptible to NR1 knockdown, and hence NMDAR are particularly required in those neurons undergoing synaptic plasticity during learning, or perhaps, and more likely, there is not a high level of redundancy in the hippocampal circuits involved, leading to the idea that a certain level of NR1 expression/availability appears necessary for memory formation in most of CA1 pyramidal neurons.

Hippocampal infection with HSV-1-derived vectors expressing an NMDAR1 antisense modifies behavior.

Herpes simplex virus-derived amplicon vectors simultaneously expressing the open reading frame encoding NR1 subunit of the NMDA receptor, either in sense or antisense orientation, as well as the open reading frame encoding the green fluorescent protein (GFP), as distinct transcription units, were constructed. Vector expression in cells was demonstrated by GFP-fluorescence, immunofluorescence, Western blots and RT-PCR. The vectors were inoculated into the dorsal hippocampus of adult male rats, which were then trained for habituation to an open field and for inhibitory avoidance to a foot-shock. Those animals injected with vectors expressing NR1 protein showed habituation to a new environment, and achieved the criteria for a step-down inhibitory avoidance to a foot-shock. In contrast, animals injected with vectors carrying the NR1 open reading frame in antisense position, showed neither habituation nor appropriate performance in the inhibitory avoidance task. There was no evidence for motor impairment or motivational disturbance, since all the animals exhibit similar behavior and performance in the training sessions. Hence, the impaired performance might be due to either amnesia or disability to record events. Transgene expression in brain, as revealed by GFP fluorescence, was mainly observed in pyramidal cells of CA1, but also in CA3. Therefore, our results strongly support the participation of hippocampal NR1 subunit in habituation to a new environment, but also in recording events for the inhibitory avoidance task. Hence, amplicon vectors appear to be useful tools to modify endogenous gene expression at a defined period, in restricted brain regions, and should allow investigating in vivo functions of genes.

Gene transfer of NMDAR1 subunit sequences to the rat CNS using herpes simplex virus vectors interfered with habituation.

1. The aim is to study some roles of the hippocampal NMDA receptor, by modifying the expression of the essential NR1 subunit, with temporal and spatial restrictions in the central nervous system (CNS) of the rat. 2. Due to their neurotropism and the size of inserts they can accomodate, herpes simplex virus type-1 (HSV-1) derived amplicon vectors were used to transfer sequences, either in sense (+) or antisense (-) orientations, of the NR1 subunit gene, or of the green fluorescent protein (GFP) gene, into the CNS. 3. Vector expression in cell lines was followed by GFP autofluorescence, immunofluorescence and western blot. 4. The vectors were inoculated into the dorsal hippocampus of adult male Wistar rats, which were evaluated for habituation to an open field, and then, for expression of the transgenes, by autofluorescence and western blot; the expression mainly happened in pyramidal cells of CA1. 5. The animals injected with vectors carrying the NR1(+) transgene showed habituation to the new environment, as also happened with rats injected with vectors carrying only the GFP transgene. 6. In contrast, animals injected with vectors carrying NR1(-) sequence, did not show habituation. This might be retrograde amnesia or disability to record the trace, suggesting that the NR1 subunit in the dorsal hippocampus, is involved in habituation to a new environment. 7. HSV-1 derived amplicon vectors appear to be useful tools to modify endogenous gene expression, at a defined period, in restricted regions of the CNS.

Therapeutic efficacy in experimental polyarthritis of viral-driven enkephalin overproduction in sensory neurons.

Rheumatoid arthritis is characterized by erosive inflammation of the joints, new bone proliferation, and ankylosis, leading to severely reduced locomotion and intense chronic pain. In a model of this disease, adjuvant-induced polyarthritis in the rat, neurons involved in pain transmission and control undergo plastic changes, especially at the spinal level. These changes affect notably neurons that contain opioids, such as enkephalins deriving from preproenkephalin A (PA) precursor protein. Using recombinant herpes simplex virus containing rat PA cDNA, we enhanced enkephalin synthesis in sensory neurons of polyarthritic rats. This treatment markedly improved locomotion and reduced hyperalgesia. Furthermore, the progression of bone destruction slowed down, which is the most difficult target to reach in the treatment of patients suffering from arthritis. These data demonstrate the therapeutic efficacy of enkephalin overproduction in a model of systemic inflammatory and painful chronic disorder.

Chimaeric Lym-1 monoclonal antibody-mediated cytolysis by neutrophils from G-CSF-treated patients: stimulation by GM-CSF and role of Fc gamma -receptors.

Chimaeric Lym-1 (chLym-1) is a monoclonal antibody generated by fusing the variable region genes of murine Lym-1 to human gamma1 and kappa constant regions. Owing to its selectivity and avidity for human malignant B cells, it is an attractive candidate for developing immune-interventions in B-lymphomas. In the attempt to identify rational bases for optimizing potential chLym-1 related therapeutic approaches, we studied the ability of this ch-mAb to trigger neutrophil-mediated Raji cell cytolysis in cooperation with two neutrophil-related cytokines, G-CSF and GM-CSF. ChLym-1 triggered low levels of cytolysis by normal neutrophils but induced consistent cytolysis in neutrophils from individuals treated with G-CSF. When exposed to GM-CSF, neutrophils from subjects treated with G-CSF became potent effectors, also leading to 75% lysis. By using mAbs specific for distinct FcgammaRs, normal neutrophils were inhibited by mAb IV.3, suggesting the intervention of FcgammaRII, constitutively expressed on the cells. On the other hand, neutrophils from patients treated with G-CSF were inhibited by mAb IV.3 plus mAb 197, a finding consistent with a cooperative intervention of FCgammaRII and G-CSF-induced FcgammaRI. The anti-FcgammaRIII mAb 3G8 promoted significant enhancement of the neutrophil cytolytic efficiency. Therefore, neutrophil FcgammaRIII behaves as a down-regulator of the cytolytic potential. The present findings suggest new attempts to develop mAb-based and G-CSF/GM-CSF combined immune-interventions in B lymphomas.

A novel approach for herpes simplex virus type 1 amplicon vector production, using the Cre-loxP recombination system to remove helper virus.

Helper-dependent HSV vectors (commonly known as HSV amplicons) are able to transfer genes into both dividing and quiescent cells, and thus have the potential to be widely used as vectors in physiological studies and gene therapy. Historically, these vectors were produced by superinfection with a helper virus that furnished all the trans-acting functions required for amplification and packaging of vector genomes into HSV-1 particles. In these systems, however, large amounts of potentially harmful helper virus are present in the vector stocks, thus restricting the use of these vectors. New helper virus-free packaging systems have been developed that utilize transfection of helper functions rather than infection and thus produce safer vector stocks. The vector titers as well as the amounts of particles obtained with these systems are, however, limited by the impossibility to reamplify the vector stocks. In this article, we present a novel system for producing large amounts of high-titer amplicon vector with low contamination by helper viruses. This system is based on the use of the Cre-loxP recombination system, which allows efficient deletion of the packaging signal of an HSV-1 recombinant helper virus (HSV-1-LaL) on Cre-expressing cells (TE-CRE30).

Characterization of a phage display-derived human monoclonal antibody (NHS76) counterpart to chimeric TNT-1 directed against necrotic regions of solid tumors.

To eliminate the human anti-mouse antibody (HAMA) response seen in patients treated with murine and chimeric antibodies, fully human monoclonal antibodies (MAbs) are now being developed. Tumor Necrosis Therapy (TNT) is an approach to tumor targeting that utilizes MAbs directed against common intracellular antigens such as nucleic acids, accessible only in necrotic areas of solid tumors. By binding to the necrotic core of tumors, these TNT MAbs can circumvent many of the limitations of MAbs directed against tumor cell surface antigens. Chimeric TNT-1 (chTNT-1) was first developed from the parent murine antibody by genetically engineering the murine variable regions to the human IgG(1) and kappa constant regions. Although the chimeric antibody's behavior was similar to that of the murine version, the 35% murine homology it shares allows for the potential of a HAMA response. A human antibody derived from a phage display library, designated NHS76, has been developed with similar binding characteristics to the TNT-1. To demonstrate that this genetically engineered human counterpart to chTNT-1 has similar pharmacokinetic characteristics, in vivo behavior, and targeting abilities, both antibodies were rigorously tested in parallel. For these studies, biodistribution analysis in LS174T human colon tumor-bearing nude mice was performed to compare the uptake levels in tumor and normal organs. In addition, mouse imaging and autoradiographic studies were conducted to demonstrate positive uptake in necrotic regions of tumor and negative uptake in viable tissues and organs. The results of these studies confirm the comparable nature of both antibodies and provide the necessary preclinical data to show the suitability of NHS76 as an improved product for the therapy of solid tumors in man.

Monoclonal Lym-1 antibody-targeted lysis of B lymphoma cells by neutrophils. Evidence for two mechanisms of FcgammaRII-dependent cytolysis.

Human neutrophils incubated with the anti-HLA-DR mAb Lym-1, plus PMA, induced significant cytolysis of B lymphoma cells compared with Lym-1 and PMA alone. The effect of PMA was independent of the ability of the compound to stimulate neutrophil-respiratory burst. In fact, first, neutrophils from a patient with chronic granulomatous disease were cytolytically effective in spite of their inability to produce oxidants. Second, various kinase inhibitors exerted different effects on the PMA-stimulated cytolytic system and neutrophil-oxidative burst. Previous studies have shown the involvement of the FcgammaRII, CD11b-CD18 integrins, and CD66b glycoproteins in the Lym-1 mAb-dependent cytolysis by GM-CSF-stimulated neutrophils. The present PMA-stimulated system was inhibited by the anti-FcgammaRII mAb IV.3, the anti-CD18 mAb MEM 48, and the anti-CD11b mAb 2LPM19c but not by the anti-CD66b mAb 80H3 and N-acetyl-D-glucosamine. Furthermore, the PMA- and GM-CSF-stimulated cytolysis was insensitive and sensitive to inhibition by pertussis toxin, respectively. Thus, the use of PMA and GMCSF as neutrophil stimulants uncovers the existence of distinct mechanisms of Lym-1 mAb-mediated cytolysis.

Intracellular Cre-mediated deletion of the unique packaging signal carried by a herpes simplex virus type 1 recombinant and its relationship to the cleavage-packaging process.

To gain further insight on the function of the herpes simplex virus type 1 (HSV-1) packaging signal (a sequence), we constructed a recombinant virus containing a unique a sequence, which was flanked by two loxP sites in parallel orientation. The phenotype of this recombinant, named HSV-1 LaL, was studied in cell lines which either express or do not express Cre recombinase. Although LaL virus multiplication was only slightly reduced in standard cell lines, its growth was strongly inhibited in Cre-expressing cells. In these cells, a sequences were detected mostly in low-molecular-weight DNA circles, indicating that they had been excised from virus DNA by site-specific recombination. Deletion of the a sequences from the viral genome resulted in the accumulation of uncleaved replication intermediates, as observed by pulsed-field gel electrophoresis. B-type capsids also accumulated in these cells, as shown both by electron microscopy and by sucrose gradient sedimentation. Further examination of the status of a sequences in Cre-expressing cells indicated that high-level amplification of this sequence can occur in the absence of the cleavage-packaging process. Moreover, the amplified a signals in small circular DNA molecules remained uncleaved, indicating that these molecules were not able to efficiently interact with the cleavage-packaging machinery. The cleavage-packaging machinery and the structural proteins required to assemble virions were, however, functional in HSV-1 LaL-infected Cre-expressing cells, since this system could be used to package plasmid DNA harboring an origin of virus replication and one normal a signal. This is the first study in which accumulation both of uncleaved replication intermediates and of B capsids has been obtained in the presence of the full set of proteins required to package virus DNA.

Molecular cloning and functional expression of contortrostatin, a homodimeric disintegrin from southern copperhead snake venom.

Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Through antagonism of integrins alphaIIbbeta3, alpha5beta1, alphavbeta3, and alphavbeta5, contortrostatin inhibits platelet aggregation and disrupts cancer cell adhesion and invasion. We cloned cDNA from a library made from the venom gland cells of Agkistrodon contortrix contortrix using polymerase chain reaction. We found that the contortrostatin gene is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. The precursor cDNA is 2027 bp with a 1449-bp open reading frame. The disintegrin domain is 195 bp encoding 65 amino acids. Like other members of the disintegrin family, each subunit of contortrostatin has an RGD site, and the cysteine alignment is conserved. The disintegrin domain of the cDNA has been expressed in a eukaryotic expression system as a homodimeric fusion protein with an immunoglobulin. The recombinant protein is recognized by an antiserum against native contortrostatin in Western blot. Both the native and recombinant proteins bind to integrins alphavbeta3 and alphavbeta5. Like native contortrostatin, the recombinant fusion protein inhibits platelet aggregation, blocks cancer cell adhesion to fibronectin and vitronectin, and prevents invasion of cancer cells through a Matrigel barrier. The success of functional expression not only validates the cDNA cloning of this disintegrin, but also provides adequate material for functional studies of contortrostatin.

Single amino acid substitution in the Fc region of chimeric TNT-3 antibody accelerates clearance and improves immunoscintigraphy of solid tumors.

UNLABELLED: Recent studies in antibody catabolism have identified residues at the CH2-CH3 interface of the IgG heavy chain critical for serum persistence of immunoglobulins. Amino acid substitutions in the Fc region of murine IgG1 were shown to drastically accelerate antibody clearance in mice. Our laboratory has previously described a human-mouse chimeric TNT-3 (chTNT-3) monoclonal antibody directed against a universal nuclear antigen that has potential for the radioimmunotherapy of many solid tumors. In the current study, we engineered a chTNT-3 mutant containing a single amino acid substitution, to determine whether a more rapid clearance profile would make the antibody suitable for diagnostic imaging. METHODS: A single amino acid substitution in the CH2 domain of the human gamma1 constant region was made by polymerase chain reaction mutagenesis. High-level expression was achieved using the Glutamine Synthetase Gene Amplification System, and the chTNT-3 mutant was purified by protein A affinity and ion-exchange chromatography. A radioimmunoassay was performed to examine antigen binding, and in vivo studies were undertaken to evaluate clearance and tumor targeting in human tumor xenograft models. RESULTS: The chTNT-3 mutant retained the high affinity of chTNT-3, with a binding constant of 1.5 x 10(-9) mol/L. The mutant was eliminated rapidly from BALB/c mice, with a beta-phase half-life of 33.8 h, compared to 134.2 h for chTNT-3. Moreover, biodistribution studies in human colon tumor-bearing nude mice reflected this accelerated clearance. Tumor levels of the mutant were, respectively, 65%, 39%, and 36% of the tumor levels achieved with the parental chTNT-3 6, 12, and 24 h postinjection. The rapid clearance of the chTNT-3 mutant from the blood resulted in higher tumor-to-normal organ ratios for many normal tissues. Imaging of tumor-bearing mice with 99mTc-labeled chTNT-3 mutant demonstrated early visualization of tumors in 3 different solid tumor xenograft models. CONCLUSION: The accelerated clearance produced by a single amino acid substitution in the Fc region of chTNT-3 leads to improved imaging in tumor-bearing mice. These studies suggest that a rapidly clearing antibody generated by this approach may be useful for the immunoscintigraphy of human tumors.

Genetic engineering of herpes simplex virus and vector genomes carrying loxP sites in cells expressing Cre recombinase.

The prokaryotic Cre-loxP recombination system is a powerful tool that enables in vitro and in vivo site-specific manipulations of the genome of eukaryotic cells as well as of DNA viruses and their derived vectors. This system, however, has not yet been exploited in the context of herpes simplex virus type 1 (HSV-1) infected cells, perhaps because this virus encodes several functions that induce a strong shutoff of cellular protein synthesis, a fact that could preclude expression of cellular-encoded Cre recombinase. In the present study, we show that efficient site-specific recombination can take place in cell lines expressing Cre, even in the context of HSV-1 infection, as evidenced by the engineering of an HSV-1 recombinant virus and several viral vectors carrying one or two loxP sequences. More precisely, we have used this system to induce an irreversible switch in the expression of a viral complex transcription unit encoding two different open reading frames and allowing consecutive expression of two reporter genes. Furthermore Cre recombinations were also used to induce the decatenation of the genomic concatemers harbored by amplicon particles upon infection of cells under nonreplicative conditions, thus enabling the rescue of many independent plasmids corresponding to the original amplicon plasmid used to generate the vectors. Thus the Cre-loxP recombination system can successfully be used for engineering the genome of HSV-1 or HSV-1-based vectors in cultured cells.

Acute gouty arthritis involving a prosthetic knee joint.

A painful and swollen prosthetic joint, accompanied by fever, is considered to be an intra-articular infection until proven otherwise. Acute gout is one of the rare causes of arthritis in a prosthetic joint, and it may be misdiagnosed as an infection, especially when a high leukocyte count is present in the joint fluid. We report a case of crystal-proven, gouty arthritis with a low synovial fluid leukocyte count presenting with pain and swelling in a prosthetic knee and responding to treatment with colchicine and corticosteroids. Physicians taking care of patients with prosthetic joints should be aware that acute gout can occur in an artificial joint and that aspirated prosthetic joint fluid should be routinely screened for crystals independently from the WBC counts obtained.

Monoclonal Lym-1 antibody-dependent cytolysis by neutrophils exposed to granulocyte-macrophage colony-stimulating factor: intervention of FcgammaRII (CD32), CD11b-CD18 integrins, and CD66b glycoproteins.

Murine monoclonal antibody (MoAb) Lym-1 is an IgG2a able to bind HLA-DR variants on malignant B cells and suitable for serotherapeutic approaches in B-lymphoma patients. We have previously shown that Lym-1 can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to trigger neutrophil cytolysis towards Raji cells used as a model of B-lymphoma targets. Here we provide evidence for the intervention of certain neutrophil receptors or surface molecules in this model of cell-mediated lysis. The lysis was completely inhibited by the anti-FcgammaRII MoAb IV.3 and unaffected by the anti-FcgammaRIII MoAb 3G8. This suggests that neutrophil cytolysis involves FcgammaRII without cooperation of this receptor with FcgammaRIII. Moreover, the lysis was inhibited by an anti-CD18 MoAb (MEM48) and by a MoAb specific for carcinoembryonic antigen (CEA)-like and glycophosphatidyl inositol (GPI)-linked glycoproteins (CD66b). Using an immunofluorescence staining procedure, cross-linking of CD66b induced the redistribution of CD11b on neutrophils with distinct areas of CD11b clustering via a process susceptible of inhibition by D-mannose. This is consistent with the ability of CD11b-CD18 and CD66b to undergo lectin-like physical interactions on the neutrophil surface. Such a type of interaction is presumably instrumental for neutrophil cytolytic activity in that the lysis was inhibited by D-mannose and enhanced by the MoAb VIM-12, which mimics the cooperation between CD11b and GPI-anchored molecules by specifically interacting with CD11b lectin-like sites. Therefore, the present results prove the absolute requirement for FcgammaRII in neutrophil GM-CSF/Lym-1-mediated cytolysis and, on the other hand, define the crucial role of CD66b and CD11b/CD18 in the expression of the cell lytic potential.

Pretreatment with a monoclonal antibody/interleukin-2 fusion protein directed against DNA enhances the delivery of therapeutic molecules to solid tumors.

The efficacy of molecular therapies for human malignancies is limited by inadequate accumulation within solid tumors. Our laboratory has developed a novel approach that uses monoclonal antibodies (MAbs) to direct vasoactive proteins to tumor sites to increase local vascular permeability and, in turn, improve the delivery of therapeutic reagents. Previously, we demonstrated that pretreatment with immunoconjugates containing interleukin-2 (IL-2) enhances specific tumor uptake of radiolabeled MAbs without affecting normal tissues. In the present study, we describe a fusion protein consisting of a chimeric antinuclear antibody and IL-2 (chTNT-3/IL-2) and illustrate its potential for improving the delivery of both MAbs and drugs. The ability of pretreatment with chTNT-3/IL-2 to increase specific tumor uptake of the MAb B72.3 was demonstrated in LS174T colon tumor-bearing mice. Tumor accretion of B72.3 increased nearly 3-fold, with no changes in normal tissues. Abrogation of this effect with N(G)-methyl-1-arginine, a chemical inhibitor of nitric oxide synthase, suggests that rapid generation of nitric oxide in the tumor is responsible for the enhanced uptake. To demonstrate that pretreatment with chTNT-3/IL-2 can improve the uptake of other clinically relevant MAbs in different tumor models, additional studies were performed in both lung and prostate xenograft models. Pretreatment with the fusion protein increased specific tumor uptake of the MAb NR-LU-10 in A427 lung tumor-bearing mice and enhanced tumor uptake of the MAb CYT-351 in LNCaP prostate tumor-bearing mice, 2.1-fold and 1.7-fold, respectively. Finally, tumor uptake of the radiolabeled thymidine analogue 125IUdR also increased approximately 3-fold after pretreatment, indicating that this approach can be extended to small molecules such as chemotherapeutic drugs. Because TNT-3 recognizes a universal nuclear antigen accessible in degenerating and necrotic cells within all solid tumors, this strategy may be applicable to the majority of human cancers.

Improving performance in a contracted physician network.

Health care organizations face significant performance challenges. Achieving desired results requires the highest level of partnership with independent physicians. Tufts Health Plan invited medical directors of its affiliated groups to participate in a leadership development process to improve clinical, service, and business performance. The design included performance review, gap analysis, priority setting, improvement work plans, and defining the optimum practice culture. Medical directors practiced core leadership capabilities, including building a shared context, getting physician buy-in, and managing outliers. The peer learning environment has been sustained in redesigned medical directors' meetings. There has been significant performance improvement in several practices and enhanced relations between the health plan and medical directors.