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Purpose: Rituximab [chimeric anti-CD20 monoclonal antibody], alone or combined with chemotherapy, is used in the treatment of non–Hodgkin's lymphoma (NHL). Rituximab binds to CD20 and inhibits intracellular survival/growth pathways leading to chemo/immunosensitization of tumor cells in vitro. The contribution of rituximab Fc-FcR interaction in signaling is not known. This study examined the role of Fc-FcR interactions in rituximab-induced signaling using rituximab (Fab')2 fragments as well as rituximab devoid of the CH2 Fc-binding domain (CH2−).Experimental Design: Rituximab (CH2−) and rituximab (Fab')2 were tested for their activity on B-NHL cell lines. Cell signaling and sensitization to chemotherapy and immunotherapy were examined. The in vitro studies were validated in mice bearing tumor xenografts.Results: Although the modified antibodies were defective in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity functions, they retained all other biological activities such as inhibition of cell proliferation, induction of cell aggregation, and apoptosis induction. In addition, similar to rituximab, the modified antibodies inhibited the activity of cell survival/growth pathways and their associated transcription factors (e.g., NF-κB, YY1, SP-1), and signal transducers and activators of transcription 3 (STAT-3), and downregulated the expression of antiapoptotic gene products, such as Bcl-2/Bclxl, which regulate drug resistance. The modified antibodies, similar to rituximab, sensitized resistant B-NHL cells to both CDDP and Fas ligand–induced apoptosis. Furthermore, treatment of nude mice bearing Raji tumor cell xenografts with the combination of rituximab (Fab')2 or rituximab and CDDP resulted in similar and significant inhibition of tumor growth.Conclusion: These findings reveal that rituximab-mediated inhibition of intracellular signaling pathways and leading to chemo/immuno-sensitization of resistant B-NHL is Fc independent. (Clin Cancer Res 2009;15(21):6582–94)

Supplementary Data from Rituximab-Mediated Cell Signaling and Chemo/Immuno-sensitization of Drug-Resistant B-NHL Is Independent of Its Fc Functions

Tuberculosis remains the most pervasive infectious disease and the recent emergence of multiple or even fully drug-resistant strains increases the risk and emphasizes the need for more efficient and better drug treatments. A key feature of mycobacteria pathogenesis is the metabolic switch during infection and expression of virulence genes is often adapted to specific infection conditions. This study aims to identify genes that are involved in the establishment and maintenance of the infection. To answer these questions, we have applied Transposon Sequencing (Tn-Seq) inM. marinum, an unbiased genome-wide strategy that combines saturation insertional mutagenesis and high throughput sequencing. This approach allowed us to precisely identify the localization and relative abundance of insertions in pools of Tn mutants. The essentiality and fitness cost, in terms of growth advantage and disadvantage of over 105mutants were quantitatively compared betweenin vitroand different stages of infection in two evolutionary distinct hosts,D. discoideumand BV2 microglial cells. We found that 57% of TA sites in theM. marinumgenome were disrupted and that 568 genes (10.2%) are essential forM. marinum, which is comparable to previous Tn-Seq studies onM. tuberculosis. The major pathways involved in the survival ofM. marinumduring infection ofD. discoideumwere related to vitamin metabolism, theesx-1operon, as well as the mce1 operon.

This study was aimed at validating the diagnostic accuracy of ultrasound (US) scans in pre-operative evaluation of flexor tendon injuries in the hand and to determine its value in the management of such injuries and in the prediction of patient outcome. This descriptive cross-sectional prospective study included 35 patients with penetrating trauma to the volar aspect of the hand or wrist with questionable clinical findings. They had 50 injured tendons and were candidates for exploratory surgery versus physiotherapy. They underwent pre-operative US to guide their management. Ultrasound results were compared with the operative findings as the gold standard test. Patients were followed up postoperatively, and functional outcome was assessed and correlated with pre-operative sonographic findings. Our results indicate that sonographic examination of hand tendon injury has high accuracy in diagnosing complete or partial flexor injuries, with 100% accuracy, sensitivity and specificity in diagnosing full-thickness tears as well as tenosynovitis of hand flexor tendons. Zonal II injury was the most frequent among our study population and correlated with poorest functional outcome after rehabilitation at 3 mo follow-up. US of the hand is a fast, inexpensive and potentially indispensable dynamic tool for accurate assessment of flexor tendon injuries. It provides data on the extent of injury that effectively helps both set up an appropriate operative plan and predict the patient's functional outcome post-operatively, which in turn will have a direct impact on the patient's rehabilitation plan and lifestyle. Thus, it should be a fundamental part of the management of patients with tendon injuries.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manipulates the host phagosome maturation pathway to replicate intracellularly. Mycobacterium marinum, a closely-related species, and Dictyostelium discoideum, a social amoeba and alternative phagocytic host, have been used as models to study host-pathogen interactions occurring during mycobacterial infections. Vacuolins, functional homologues of the mammalian flotillins, organize membrane microdomains and play a role in vesicular trafficking. Various pathogens have been reported to manipulate their membrane association and function. During infection of D. discoideum with M. marinum, Vacuolin C was specifically and highly induced and all three vacuolin isoforms were enriched at the mycobacteria-containing-vacuole (MCV). In addition, absence of vacuolins reduced escape from the MCV and conferred resistance to M. marinum infection. Moreover, ESAT-6, the membrane-disrupting virulence factor of M. marinum, was less associated with membranes when vacuolins were absent. Together, these results suggest that vacuolins are important host factors that are manipulated by mycobacteria to inflict membrane damage and escape from their compartment.

The Dictyostelium discoideum-Mycobacterium marinum host-pathogen system is a well-established and powerful alternative model system to study mycobacterial infections. In this chapter, we will describe three microscopy methods that allow the precise identification and quantification of very diverse phenotypes arising during infection of D. discoideum with M. marinum. First, at the lowest end of the scale, we use the InfectChip, a microfluidic device that enables the long-term monitoring of the integrated history of the infection course at the single-cell level. We use single-cell analysis to precisely map and quantitate the various fates of the host and the pathogen during infection. Second, a high-content microscopy setup was established to study the infection dynamics with high-throughput imaging of a large number of cells at the different critical stages of infection. The large datasets are then fed into a deep image analysis pipeline allowing the development of complex phenotypic analyses. Finally, as part of its life cycle, single D. discoideum amoebae aggregate by chemotaxis to form multicellular structures, which represent ordered assemblies of hundreds of thousands of cells. This transition represents a challenge for the monitoring of infection at multiple scales, from single cells to a true multicellular organism. In order to visualize and quantitate the fates of host cells and bacteria during the developmental cycle in a controlled manner, we can adjust the proportion of infected cells using live FAC-sorting. Then, cells are plated in defined humidity conditions on optical glass plates in order to image large fields, using tile scans, with the help of a spinning disc confocal microscope.

The Dictyostelium discoideum-Mycobacterium marinum host-pathogen system is a well-established and powerful alternative model system to study mycobacterial infections. In this chapter, we will describe three microscopy methods that allow the precise identification and quantification of very diverse phenotypes arising during infection of D. discoideum with M. marinum. First, at the lowest end of the scale, we use the InfectChip, a microfluidic device that enables the long-term monitoring of the integrated history of the infection course at the single-cell level. We use single-cell analysis to precisely map and quantitate the various fates of the host and the pathogen during infection. Second, a high-content microscopy setup was established to study the infection dynamics with high-throughput imaging of a large number of cells at the different critical stages of infection. The large datasets are then fed into a deep image analysis pipeline allowing the development of complex phenotypic analyses. Finally, as part of its life cycle, single D. discoideum amoebae aggregate by chemotaxis to form multicellular structures, which represent ordered assemblies of hundreds of thousands of cells. This transition represents a challenge for the monitoring of infection at multiple scales, from single cells to a true multicellular organism. In order to visualize and quantitate the fates of host cells and bacteria during the developmental cycle in a controlled manner, we can adjust the proportion of infected cells using live FAC-sorting. Then, cells are plated in defined humidity conditions on optical glass plates in order to image large fields, using tile scans, with the help of a spinning disc confocal microscope.

Mycobacterium marinum is a model organism for pathogenic Mycobacterium species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. These pathogens enter phagocytes and replicate within the Mycobacterium-containing vacuole, possibly followed by vacuole exit and growth in the host cell cytosol. Mycobacteria release siderophores called mycobactins to scavenge iron, an essential yet poorly soluble and available micronutrient. To investigate the role of M. marinum mycobactins, we purified by organic solvent extraction and identified by mass spectrometry the lipid-bound mycobactin (MBT) and the water-soluble variant carboxymycobactin (cMBT). Moreover, we generated by specialised phage transduction a defined M. marinum ΔmbtB deletion mutant predicted to be defective for mycobactin production. The M. marinum ΔmbtB mutant strain showed a severe growth defect in broth and phagocytes, which was partially complemented by supplying the mbtB gene on a plasmid. Furthermore, purified Fe-MBT or Fe-cMBT improved the growth of wild type as well as ΔmbtB mutant bacteria on minimal plates, but only Fe-cMBT promoted the growth of wild-type M. marinum during phagocyte infection. Finally, the intracellular growth of M. marinum ΔmbtB in Acanthamoeba castellanii amoebae was restored by coinfection with wild-type bacteria. Our study identifies and characterises the M. marinum MBT and cMBT siderophores and reveals the requirement of mycobactins for extra- and intracellular growth of the pathogen.

Flotillins are lipid raft residents involved in membrane trafficking and recycling of plasma membrane proteins. Dictyostelium discoideum uses phagocytosis to kill, digest and feed on bacteria. It possesses three flotillin-like vacuolins that are strongly associated with membranes and that gradually accumulate on maturing phagosomes. Absence of vacuolins reduced adhesion and particle recognition resulting in a drastic reduction in the uptake of various types of particles. This was caused by a block in the recycling of plasma membrane components and the absence of their specific cortex-associated proteins. In addition, absence of vacuolins also impaired phagolysosome biogenesis, without significantly impacting killing and digestion of a range of bacteria. Strikingly, both absence and overexpression of vacuolins induced a strong downregulation of myosin VII (also known as MyoI) expression, as well as its binding partner talin A. Episomal expression of myosin VII fully rescued defects in uptake and adhesion but not in phagosome maturation. These results suggest a dual role for vacuolins: a novel mechanism involving membrane microdomains and myosin VII–talin A in clustering phagosomal receptors and adhesion molecules at the plasma membrane, and a role in phagolysosomal biogenesis.

Flotillins are lipid raft residents involved in membrane trafficking and recycling of plasma membrane proteins.

The metazoan flotillins are lipid rafts residents involved in membrane trafficking and recycling of plasma membrane proteins.Dictyostelium discoideum, a social soil amoeba, uses phagocytosis to digest, kill and feed on bacteria.D. discoideumpossesses three flotillin-like proteins, termed VacA, VacB and the recently identified VacC. All three vacuolins gradually accumulate on postlysosomes and, like flotillins, are strongly associated with membranes and partly with lipid rafts. Vacuolins are absolutely required for uptake of various particles. Their absence impairs particle recognition possibly because of defective recycling of plasma membrane or cortex-associated proteins. In addition, vacuolins are involved in phagolysosome biogenesis, although this does not impact digestion and killing of a wide range of bacteria. Furthermore, vacuolin knockout affects early recruitment of the WASH complex on phagosomes, suggesting that vacuolins may be involved in the WASH-dependent plasma membrane recycling. Altogether, these results indicate that vacuolins act as the functional homologues of flotillins inD. discoideum.

Tuberculosis remains the most pervasive infectious disease and the recent emergence of multiple drug-resistant strains emphasizes the need for more efficient drug treatments. The experimentally versatile Dictyostelium discoideum – Mycobacterium marinum infection model provides a powerful system to study mycobacteria pathogenicity and host response. In this study, a time-resolved transcriptomic analysis of the amoeba D. discoideum was performed to decipher the different host pathways impacted during infection. We investigated how D. discoideum fine-tunes its gene expression in response to M. marinum infection by assessing the transcriptomic profile covering the critical stages of entry, establishment of a permissive niche, proliferation and dissemination (1, 3, 6, 12, 24 and 48 hours post infection). Differential gene expression provided a fingerprint of the transcriptome of the host cell in the presence of mycobacteria, and helped identify specific markers and molecular signatures of infection. Enrichment pathway analysis showed that most of the Biological Processes (BP) of upregulated genes at early time point of infection hinted towards damage response and cellular defence, especially in specific pathways involved in membrane repair (ESCRT) and bacteria elimination (autophagy). Whereas at late time points of infection, BP related to starvation were upregulated. Some other signatures were more unexpected, such as cell cycle (downregulation of cytosolic large & small ribosomal subunits) and upregulation of metabolic adaptations (lipids transport).

Legionella pneumophila and tubercular Mycobacteria are the causative agents of potentially fatal diseases due to their pathogenesis but also to the emergence of antibiotic resistance that limits treatment strategies. The aim of our study is to explore the antimicrobial activity of a small ligand-based chemical library of 1,255 structurally diverse compounds. These compounds were screened in a combination of three assays, two monitoring the intracellular growth of the pathogenic bacteria, Mycobacterium marinum and L. pneumophila, and an additional anti-virulence plaque assay for M. marinum. We set up these assays using two amoeba strains, the genetically tractable Dictyostelium discoideum and the free-living amoeba Acanthamoeba castellanii. In summary, sixty-four compounds showed anti-infective/anti-virulence activity in at least one of the 3 assays. The intracellular assays hit rate varied between 1.7% (n=22) for M. marinum and 2.8% (n=35) for L pneumophila with 7 compounds in common between both pathogens. In parallel, 1.2 % (n= 15) of the tested compounds were able to restore D. discoideum growth in presence of M. marinum spiked in a lawn of Klebsiella pneumoniae. We also validated the generality of the hit compounds identified using the A. castellanii-M. marinum anti-infective screen in the powerful D. discoideum-M. marinum host-pathogen model. The characterization of anti-infective and antibacterial hits in the latter infection model revealed compounds able to reduce intracellular growth more than 50% at 30 μM. Our studies underline the relevance of using a combination of low-cost and low-complexity assays with full 3R compliance associated with a rationalized focused library of compounds to help identifying new chemical scaffolds and dissect some of their properties prior to run further compounds development steps.

Brucella suis, facultative intracellular bacterial pathogen of mammals, and Agrobacterium tumefaciens, a plant pathogen, both use a VirB type IV secretion system (T4SS) to translocate effector molecules into host cells. HspL, an α-crystalline-type small heat-shock protein, acts as a chaperone for the Agrobacterium VirB8 protein, an essential component of the VirB system. An Agrobacterium mutant lacking hspL is attenuated due to a misfunctional T4SS. We have investigated whether IbpA (BRA0051), the Brucella HspL homologue, plays a similar role. Unlike HspL, IbpA does not interact with VirB8, and an IbpA mutant shows full virulence and no defect in VirB expression. These data show that the Brucella α-crystalline-type small heat-shock protein IbpA is not required for Brucella virulence. Significance and Impact of Study Many bacteria use type IV secretion systems (T4SS), multi-protein machines, to translocate DNA and protein substrates across their envelope. Understanding how T4SS function is important as they play major roles in the spread of plasmids carrying antibiotic resistance and in pathogenicity. In the plant pathogen Agrobacterium tumefaciens, HspL, an α-crystalline-type small heat-shock protein, acts as a chaperone for the essential type IV secretion system component VirB8. Here, we show that this is not the case for all T4SS; in the zoonotic pathogen Brucella suis, IbpA, the protein most related to HspL, does not play this role.

The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication-permissive compartment termed the Mycobacterium-containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid-borne genes. Fluorescence microscopy of M. marinum-infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol-3-phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V-ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape.

Symptoms of gastroenteritis compatible with a food-borne outbreak started appearing among employees at the central headquarters of a “Bank” located in Beirut, Lebanon, on the evening of Monday 17 May 2004. Earlier on that day, many employees had had lunch at the bank cafeteria. All of those with signs and symptoms had eaten the main dish of chicken noodles au gratin served at the cafeteria on that day. The chicken noodle dish had been brought from the kitchen of a local catering company in 2 large serving containers. This catering company has an excellent reputation in Beirut and had been serving the Bank for more than a year with no previous problems reported. Public health officers alerted to the outbreak went to the caterer on May 18 in line with legal regulations. Based on the mandate of an obsolete law they were supposed to “destroy” the remains of the “poisoned” food items. This kind of policy usually renders all evidence-based epidemiological investigations impossible. Fortunately, a portion of leftovers of the chicken noodles had been hidden by the caterers from the public health food inspectors, in anticipation of potential court action. Those leftovers were sent on 18 May to the Central Laboratory, the government reference laboratory in Beirut. On 19 May, testing of the remains of the incriminated dish demonstrated the presence of S. enterica Serovar Enteritidis, thus confirming the contamination of the dish.

The primary limitation of IgG antibodies for radioimmunotherapy of solid tumors is their prolonged serum half-life, leading to dose-limiting bone marrow toxicity at doses providing inadequate radiation to the tumor. A humanized C(H)2 domain-deleted variant of the anti-TAG-72 antibody CC49 (HuCC49DeltaC(H)2) has faster blood clearance, compared to the IgG, while retaining tumor targeting. We compared the pharmacokinetics and tumor uptake of (111)In-HuCC49DeltaC(H)2 in BALB/c mice and a colon carcinoma (LS-174T) mouse xenograft with that of (111)In-labeled chimeric CC49 (cCC49), an antibody with pharmacokinetics similar to the humanized CC49 parent. Immuno-conjugates of HuCC49DeltaC(H)2 and cCC49 prepared with the (111)In chelator Mx-DTPA (1-isothiocyantobenzyl-3-methyldiethylenetriaminepentaacetic acid) retained low nM affinity and radiolabeling protocols provided greater than 95% radio-incorporation with (111)In while retaining greater than 80% immunoreactivity. Blood clearance of (111)In-HuCC49DeltaC(H)2 in BALB/c mice was monoexponential (t(1/2) 5.4 hours) and faster than (111)In-cCC49 (biexponential clearance; t1/2Delta 1.5 hours; t1/2beta 162 hours). The (111)In-HuCC49DeltaC(H)2 also cleared more rapidly from the blood in the murine xenograft. At 1 hour postinjection, blood concentrations for (111)In-HuCC49DeltaC(H)2 and (111)In-cCC49 were comparable (25.5 injected dose per g [%ID/g] and 21.3 %ID/g, respectively); tumor uptake for (111)In- HuCC49DeltaC(H)2 was 7.9 %ID/g, compared to 7.5 %ID/g for (111)In-cCC49. However, at 24 hours, blood concentration for (111)In-HuCC49DeltaC(H)2 was less than (111)In-cCC49 (0.9 %ID/g versus 5.2 %ID/g, respectively) with comparable tumor retention (14.4 %ID/g versus 19.0 %ID/g, respectively). Faster blood clearance of (111)In-HuCC49DeltaC(H)2 and tumor localization comparable to that of (111)In-cCC49 provided a fourfold improved tumor-to-blood ratio for (111)In-HuCC49DeltaC(H)2 at 24 hours postinjection.

The current excitement about molecular targeted therapies has driven much of the recent dialog in cancer diagnosis and treatment. Particularly in the biologic therapy of cancer, identifiable antigenic T-cell targets restricted by MHC molecules and the related novel stress molecules such as MICA/B and Letal allow a degree of precision previously unknown in cancer therapy. We have previously held workshops on immunologic monitoring and angiogenesis monitoring. This workshop was designed to discuss the state of the art in identification of biomarkers and surrogates of tumor in patients with cancer, with particular emphasis on assays within the blood and tumor. We distinguish this from immunologic monitoring in the sense that it is primarily a measure of the tumor burden as opposed to the immune response to it. Recommendations for intensive investigation and targeted funding to enable such strategies were developed in seven areas: genomic analysis; detection of molecular markers in peripheral blood and lymph node by tumor capture and RT-PCR; serum, plasma, and tumor proteomics; immune polymorphisms; high content screening using flow and imaging cytometry; immunohistochemistry and tissue microarrays; and assessment of immune infiltrate and necrosis in tumors. Concrete recommendations for current application and enabling further development in cancer biometrics are summarized. This will allow a more informed, rapid, and accurate assessment of novel cancer therapies.

Keliximab, a Primatized IgG1 CD4 mAb, was reconfigured to an IgG4 antibody. The gamma4 constant region was further modified by substituting glutamic acid for serine at position 235 in the CH2 domain (IgG4-E), to remove residual binding to Fcgamma receptors, and substitution of serine with proline at position 228 in the hinge region (IgG4-PE) for greater stability. Pharmacokinetic analysis in rats gave a t(1/2) of approximately 4 days for IgG4-E and 9 days for IgG4-PE, consistent with a greater stability of the IgG4-PE molecule. The effects on T cell subsets were assessed in chimpanzees given escalating doses of IgG4-PE: 0.05 mg/kg on Day 16, 1.5 mg/kg dose on Day 43, and 15 mg/kg on Day 85. Receptor modulation was observed at the two highest doses, but no depletion of T cells at any dose. The in vitro and in vivo results demonstrate the potential of this IgG4-PE mAb for use in human trials.

A major challenge facing the development of subunit vaccines comprised of well-defined recombinant antigens is their weak immunogenicity and inability to induce effective cytotoxic T cell (CTL) responses. Adjuvants aimed at increasing the immunogenicity of recombinant antigens remain a focus in vaccine development. The potency of an adjuvant is linked to specific stimulation of T cell responses, involving TH1 and TH2 subsets of CD4(+) T helper cells and CD8(+) CTL and B cell-mediated antibody responses. As a result of the existence of two distinct intra-cellular pathways for antigen processing, immunization with exogenous antigens often shows a greater propensity for T helper and antibody responses, but not CD8(+) CTL responses. However, existing experimental evidence suggests that CD8(+) CTLs, which are critical in the elimination of viral-infected and neoplastic cells, can be elicited with soluble antigens when delivered in appropriate formulations or adjuvants. This review focuses on the properties of PROVAX adjuvant in inducing antigen-specific CTL responses, antibody responses and tumor regression in experimental models and its potential application for the development of recombinant cancer vaccines.

A Primatized anti-CD4 monoclonal antibody (MAb), CE9.1, with V-domain from cynomolgus macaque (showing 92% homology with human consensus sequence V-domains), and a human IgG1 constant region, was characterized in vitro and in vivo in chimpanzees. This MAb binds human CD4 with Kd of 1.0 nM and was also able to bind to human IgG Fc receptors (Fc gamma R). However, despite being of the IgG1 subclass, CE9.1 did not bind to complement component C1q, nor did it mediate complement-dependent cytotoxicity. Examination of T cells from a number of species showed restricted reactivity for CE9.1, recognizing only human and chimpanzee CD4. In both human and chimpanzee MLRs, it had an IC50 of about 10.0 ng/mL. Therefore, a chimpanzee in vivo model was used to characterize CE9.1, CE9.1 caused transient decrease in the number of lymphocytes bearing the CD4 receptor starting at doses of 0.3 mg/kg in an in vivo dose ranging study in one chimpanzee. This effect was reversed within approximately 7 days. In a multiple high-dose study in which 10.0 mg/kg of CE9.1 was administered at intervals of 1-3 months, there was a dramatic loss of CD4 marker with a reciprocal increase in the number of CD3+ CD8- CD4- cells. The CD4 receptor was totally undetectable on these lymphocytes for 1-2 weeks, with a gradual, but complete, reversal within 4 weeks. We interpret these observations as receptor modulation because, although there was apparent loss of CD4+ lymphocytes, an equivalent number of CD3+CD8- T lymphocytes were present in circulation in all four chimpanzees treated with 10.0 mg/kg CE9.1. Even at this high dose, only limited reduction of CD4+ T lymphocytes was observed in these animals. These observations are in sharp contrast to what has been reported in rodents or in human clinical studies using other IgG1 mAbs to human CD4. CD8 counts, although variable, remained unaffected by CE9.1 treatment. No adverse events were observed following administration of CE9.1 to chimpanzees, and there was no detectable host immune responses to the Primatized MAb.

BACKGROUND: Genome analysis indicated that the new species Brucella microti possesses a potentially functional glutamate decarboxylase (GAD) system involved in extreme acid resistance in several foodborne bacteria. The contribution of this system in adaptation of B. microti to an acidic environment, including the intracellular vacuole and stomach, was investigated. RESULTS: B. microti was GAD positive and able to export its product, γ-aminobutyrate, to the extracellular medium. The resistance of B. microti to acid stress (pH 2.5) was glutamate dependent. Mutants affected in the GAD system lost this resistance, demonstrating its direct involvement in survival under these conditions. The reciprocal heterologous complementation of mutants with the GAD systems of Escherichia coli or B. microti confirmed conserved functions in both bacterial species. A gad mutant was not attenuated during infection of macrophages, where Brucella resides in an acidified vacuole at a pH of 4-4.5 during the early phase of macrophage infection, but GAD contributed to the survival of B. microti in a murine model following oral infection. CONCLUSIONS: This work provides first evidence that the GAD system might play an essential role in the resistance of an environment-borne, pathogenic Brucella species to extreme acid shock and during passage through the host stomach following oral infection.

Murine monoclonal antibody 2B8 specifically recognizes the CD20 phosphoprotein expressed on the surface of normal B lymphocytes and B- cell lymphomas. The light- and heavy-chain variable regions of 2B8 were cloned, after amplification by the polymerase chain reaction, into a cDNA expression vector that contained human IgG1 heavy chain and human kappa-light chain constant regions. High-level expression of chimeric- 2B8 antibody (C2B8) was obtained in Chinese hamster ovary cells. Purified C2B8 exhibited antigen binding affinity and human-tissue reactivity similar to the native murine antibody. In vitro studies showed the ability of C2B8 to bind human C1q, mediate complement- dependent cell lysis of human B-lymphoid cell lines, and lyse human target cells through antibody-dependent cellular cytotoxicity. Infusion of macaque cynomolgus monkeys with doses ranging from 1.6 mg/kg to 6.4 mg/kg resulted in greater than 98% depletion of peripheral blood (PB) B cells and 40% to 70% depletion of lymph node B cells. Recovery of PB B cells usually started at 2 weeks after treatment and required 60 to greater than 90 days to reach normal levels. As much as 95% depletion of B cells in peripheral lymph nodes and bone marrow was observed following weekly injections of 16.8 mg/kg antibody. No toxicity was observed in any of the animals. These results offer the possibility of using an “immunologically active” chimeric anti-CD20 antibody as an alternative approach in the treatment of B-cell lymphoma.

International audience; In murine infections, Brucella microti exhibits an atypical and highly pathogenic behavior resulting in a mortality of 82%. In this study, the possible involvement of the virB type IV secretion system, a key virulence factor of Brucella sp., in this lethal phenotype was investigated. As previously described for B. suis, expression of the virB operon of B. microti was induced in acid minimal medium, partially mimicking intracellular environment. Early neutralization of cellular compartments abolished intracellular replication of B. microti, showing that acidity of the Brucella-containing vacuole is an essential trigger. A ΔvirB mutant of B. microti exhibited strong attenuation in murine and human macrophages in vitro. Interestingly, infection with this mutant was not lethal in Balb/c mice and lacked the typical intrasplenic peak at 3 days post-infection, hence demonstrating that lethality of B. microti in murine infection absolutely requires a functional virB operon.

Spirogermanium (1; 8,8-diethyl-N,N-dimethyl-2-aza-8- germaspiro[4.5]decane-2-propanamine dihydrochloride) is a potent cytotoxic agent in vitro which has demonstrated limited activity in experimental animal tumor models. Subsequently, it has been reported that spirogermanium has antiarthritic and suppressor cell-inducing activity. We have synthesized a series of substituted 8-hetero-2-azaspiro[4.5]decane and 9-hetero-3-azaspiro[5.5]undecane analogues of spirogermanium to identify the heteroatom requirements for in vivo antiarthritic and suppressor cell-inducing activity. This structure-activity relationship study has identified that appropriately substituted silicon and carbon analogues of spirogermanium retain both antiarthritic and immunosuppressive activity, with the 8,8-dipropyl (carbon) analogue being among the most active. Following the identification of N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine++ + dihydrochloride (9) as a more active analogue than spirogermanium, a series of 8,8-dipropyl analogues with various amine substituents were synthesized. A number of these analogues had activity similar to that of 9. A correlation between activity in the adjuvant arthritic rat and the ability to induce suppressor cells (r = 0.894, p less than 0.001) suggests an association between the two pharmacologic effects. While the precise biochemical mechanism(s) for the pharmacological activity is unclear, these data suggest that compounds within this series, e.g., N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine++ + dihydrochloride, may provide effective therapy in diseases of autoimmune origin and/or the prevention of rejection in tissue transplantation.

Immunoglobulin variable region genes from non-human primates, cynomolgus macaques, were shown to have 85%-98% homology with human immunoglobulin sequences and yet macaques are phylogenetically distant enough to respond against conserved human antigens. Immunoglobulin genes were isolated from monkeys immunized with human CD4 antigen and a human/monkey chimeric anti-CD4 antibody with 91-92% homology to human immunoglobulin framework regions was cloned and expressed. The antibody has an apparent affinity of 3.2 x 10(-11) M and exhibits potent immunosuppressive properties in vitro.

Cytotoxic T lymphocytes (CTL) are induced specifically against viral and tumor antigens presented by major histocompatibility complex class I molecules on the surface of infected or transformed cells. Intracellular synthesized antigens are processed and associated with class I antigens within cells before presentation on the cell surface. Because of this special requirement for CTL induction, exogenous soluble antigens do not, in general, induce specific CTL responses. To overcome this problem, various laboratories have resorted to the use of vaccinia virus and other replicating expression vectors for intracellular antigen delivery leading to the stimulation of humoral and cell-mediated immunity to specific proteins. However, for human use it is safer to use purified and defined antigens for inducing immune responses. Using soluble ovalbumin and human immunodeficiency virus glycoprotein gp120, we have explored the possibility of using an antigen formulation consisting of squalane and Tween 80 to elicit antigen-specific CTL responses in mice. We have demonstrated that this antigen formulation is a potent inducer of CD8+, class I-restricted, antigen-specific CTLs. The CTL priming induced by soluble antigen in squalane/Tween 80 resembles the reported response to the vaccinia recombinant containing human immunodeficiency virus envelope protein and by splenocytes cytoplasmically loaded with soluble ovalbumin. The ramifications of these findings for vaccine development are discussed.

Purpose: Rituximab [chimeric anti-CD20 monoclonal antibody], alone or combined with chemotherapy, is used in the treatment of non–Hodgkin's lymphoma (NHL). Rituximab binds to CD20 and inhibits intracellular survival/growth pathways leading to chemo/immunosensitization of tumor cells in vitro. The contribution of rituximab Fc-FcR interaction in signaling is not known. This study examined the role of Fc-FcR interactions in rituximab-induced signaling using rituximab (Fab')2 fragments as well as rituximab devoid of the CH2 Fc-binding domain (CH2−).Experimental Design: Rituximab (CH2−) and rituximab (Fab')2 were tested for their activity on B-NHL cell lines. Cell signaling and sensitization to chemotherapy and immunotherapy were examined. The in vitro studies were validated in mice bearing tumor xenografts.Results: Although the modified antibodies were defective in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity functions, they retained all other biological activities such as inhibition of cell proliferation, induction of cell aggregation, and apoptosis induction. In addition, similar to rituximab, the modified antibodies inhibited the activity of cell survival/growth pathways and their associated transcription factors (e.g., NF-κB, YY1, SP-1), and signal transducers and activators of transcription 3 (STAT-3), and downregulated the expression of antiapoptotic gene products, such as Bcl-2/Bclxl, which regulate drug resistance. The modified antibodies, similar to rituximab, sensitized resistant B-NHL cells to both CDDP and Fas ligand–induced apoptosis. Furthermore, treatment of nude mice bearing Raji tumor cell xenografts with the combination of rituximab (Fab')2 or rituximab and CDDP resulted in similar and significant inhibition of tumor growth.Conclusion: These findings reveal that rituximab-mediated inhibition of intracellular signaling pathways and leading to chemo/immuno-sensitization of resistant B-NHL is Fc independent. (Clin Cancer Res 2009;15(21):6582–94)

SK&F 105809 {2-(4- methylsulfinylphenyl )-3-(4- pyridyl )-6,7- dihydro -[5H]- pyrrolo [l,2-a] imidazole} was determined to be a prodrug for the sulfide metabolite SK&F 105561 {2-(4- methyltniophenyl )-3-(4- pyridyl )-6,7- dihydro -[5H]- pyrrolo [1,2-a] imidazole} which inhibited interleukin-1 (IL-1) production in vitro and both 5-lipoxygenase (5-LO) and prostaglandin H (PGH) synthase activities in vitro and ex vivo . SK&F 105561 inhibited partially purified 5-LO with a half-maximal concentration ( ic 50 ) of 3 μM. This inhibition was reversible, independent of preincubation time, and dependent on the concentration of the substrate arachidonic acid. SK&F 105561 also inhibited purified PGH synthase with the potency dependent on the level of peroxidase activity. The ic 50 was 100 μM in the absence of peroxidase activity, whereas an ic 50 of 3 μM was observed in the presence of peroxidase activity. Using human monocytes, SK&F 105561 inhibited A23187-stimulated prostaglandin E 2 (PGE 2 ) and leukotriene B 4 (LTB 4 ) production with ic 50 values of 0.1 and 2 μM, respectively. In addition, IL-1 production by lipopolysaccharide-stimulated human monocytes was also inhibited ( ic 50 2 μM). Oral administration of SK&F 105809 to rats resulted in a dose-related generation of SK&F 105561 and in the inhibition of thromboxane B 2 and LTB 4 production ex vivo with a half-maximal dose ( ed 50 ) of 15 and 60 mg/kg, respectively. SK&F 105561 showed weak inhibitory activity on 12-lipoxygenase with an ic 50 of greater than 200 μM. Neither SK&F 105561 nor SK&F 105809 inhibited the stimulated-turnover of arachidonic acid-containing phospholipids in human monocytes or the activity of cell-free phospholipases A 2 and C. Moreover, neither SK&F 105561 nor SK&F 105809 antagonized the binding of LTB 4 or leukotriene D 4 to membrane receptors. From these results, SK&F 105561, the active principle of SK&F 105809, acts as an inhibitor of both inflammatory cytokine and eicosanoid production.

SK&F 105809 [2-(4-methylsulfinylphenyl)-3-(4-pyridyl)- 6,7-dihydro-[5H]-pyrrolo[1,2,a] imidazole] demonstrated unique antiinflammatory activities in murine models that are resistant to selective cyclooxygenase (CO) inhibitors. Both edema and inflammatory cell infiltration induced by the topical application of arachidonic acid to the mouse ear were decreased by SK&F 105809 (ED50 values of 44 mg/kg, p.o.). Polymorphonuclear leukocyte (PMN) infiltration following the intraperitoneal injection of either monosodium urate crystal or carrageenan was inhibited with ED50 values of 64 and 72 mg/kg, p.o., respectively. These inflammatory responses were unaffected by the selective cyclooxygenase inhibitor naproxen. SK&F 105809 also inhibited leukotriene B4 (LTB4) and prostaglandin E2 production in vivo in arachidonic acid-induced inflammatory exudates (ED50 values of 41 and 15 mg/kg, p.o., respectively). The inhibition of LTB4 production preceded the inhibition of PMN infiltration. The impact of inhibition of both 5-lipoxygenase (5-LO) and CO was seen with platelet-activating factor-induced vascular permeability which was inhibited markedly by SK&F 105809. However, the 5-LO inhibitor, phenidone, only strongly inhibited when coadministered with the selective CO inhibitor, indomethacin. In spite of a short half-life (14-18 min) for both SK&F 105809 and the active metabolite SK&F 105561 [2-(4- methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole], the pharmacological activity lasted at least 1.5 hr. The biochemical evidence of inhibition of interleukin-1 (IL-1) production and 5-LO and CO activity, in vitro, by the metabolite (SK&F 105561) seen in the companion paper (Marshall PJ, Griswold DE, Breton J. Webb EF, Hillegass LM, Sarau HM, Newton J Jr, Lee JC, Bender PE and Hanna N, Pharmacology of the pyrroloimidazole, SK&F 105809--I. Inhibition of inflammatory cytokine production and of 5-lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid. Biochem Pharmacol 42: 813-824, 1991) and inhibition of the fluid and cellular phases of the inflammatory response, in vivo, by SK&F 105809 suggest that this compound possesses a unique profile of activity.

The CD4 cell surface glycoprotein which is expressed primarily by a subset of T lymphocytes plays a key role in normal immune responses. In the immunopathogenesis of AIDS, it serves as the high-affinity receptor for HIV, facilitating viral attachment and entry into CD4 + cells. As such, the truncated soluble form of this molecule (sT4) has been proposed as a therapeutic drug for the treatment of AIDS whereby it would act as decoy for viral entry into cells or facilitate elimination of soluble viral envelope glycoprotein. In a study designed to look at the effect of sT4 on immune function, sT4 was administrated to experimentally naive primates. In this report, we show that administration of sT4 to cynomolgus macaque monkeys over a period of up to 3 weeks results in antibody responses with specificities for human CD4 molecules. Antisera thus generated bound sT4 and cell surface CD4 expressed on human T lymphocytes but failed to bind to cynomolgus lymphocytes. These antibodies caused no apparent adverse effects on normal immune functions of the cynomolgus macaques. We conclude from these data that the antibody response to soluble CD4 in cynomolgus monkeys is directed at determinants present on human CD4 but absent on monkey CD4. The restricted xenogeneic specificity of the antibody response indicates that soluble CD4 may not be highly immunogenic in syngeneic hosts. The present study also shows that these antibodies can block HIV-induced syncytium formation indicating that the antibodies bind to regions on the CD4 molecule close to the HIV-env gp120 binding site. The gp120 binding site, which resides within the N-terminal V1 domain of CD4, encompasses a region which corresponds to the complementarity determining regions (CDRs) of immunoglobulins. The CDR-like regions of CD4-V1 manifest the greatest species divergence, are tolerant to experimental in vitro mutagenesis, and generate the predominant antibody response in mice immunized with human CD4 indicating that differences in the V1 sequence between human and other nonhuman primates may localize to this regions.

SK&F 101926, a synthetic peptide, is a potent antagonist of vasopressin at both the V2 and the V1 receptors. Following intravenous administration of SK&F 101926 (5 mg/kg), mean arterial pressure (MAP) immediately fell 75 mm Hg. Heart rate increased approximately 50 beats/min. Cutaneous flushing and cyanosis appeared approximately 2 to 5 min after the SK&F 101926 administration. Three of the five rats died within 40 min with no improvement in either color or MAP. The two surviving animals slowly recovered from these symptoms. The hypotension and flushing recorded in these studies resembled the effects during hypotensive shock. SK&F 101926 degranulated rat peritoneal mast cells in vitro as measured by the liberation of histamine. Analogs of SK&F 101926 were identified having reduced activity to release histamine from mast cells in vitro. The activity of these analogs to release histamine in vivo was also tested, as reflected by rat paw edema. A positive correlation was found between the potency to produce edema in vivo and the potency to release mast cell histamine in vitro (r = 0.94, p less than 0.05). In addition, compounds that released mast cell histamine and induced rat paw edema also produced hypotension and death when administered intravenously, while analogs which produced minimal histamine release in vitro produced minimal or no cardiovascular changes or lethality in vivo at the same dosages (5 mg/kg). Finally, cyproheptadine (10 mg/kg), an antagonist at both the serotonin and the histamine receptors, blunted the effects of SK&F 101926 on MAP and blocked the lethality. Pretreatment with a combination of histamine (H1 and H2) antagonists provided little protection against the SK&F 101926-induced toxicity. These data indicate that the cardiovascular toxicity of SK&F 101926 (and related peptides) is mediated via the release of autocoids from mast cells. Serotonin appears to play a major role in mediating the cardiovascular toxicity of SK&F 101926.

More than 50% of patients with aggressive B lymphomas and the majority of patients with low grade lymphomas are not cured by current therapeutic strategies. The lymphomas express the B cell antigen CD20 on the cell surface and this antigen serves as target for antibody-directed therapies. Clinical studies with encouraging results have been underway with the use of a chimeric anti-CD20 antibody (IDEC-C2B8), consisting of human IgG1-6 constant regions and variable regions from the murine monoclonal anti-CD20 antibody IDEC-2B8. This study investigated the potential anti-tumor therapeutic value of combination treatment with anti-C2B8 and cytotoxic drugs. The in vitro study examined the sensitizing effect of C2B8 antibody on the DHL-4 B lymphoma line to various cytotoxic agents. Cytotoxicity was determined by the MTT assay. Surface and cytoplasmic proteins were determined by flow cytometry. Pretreatment of DHL-4 with C2B8 resulted in inhibition of cell proliferation and cell death and a fraction of the cells underwent apoptosis. While the DHL-4 tumor cells were relatively resistant to several cytotoxic drugs, pretreatment with C2B8 rendered the cells sensitive to TNF-alpha, ricin, diphtheria toxin (DTX), adriamycin and cisplatin but not to VP-16. Chemosensitization of DHL-4 tumor cells was not due to downmodulation of either the MDR-1 or bcl-2 gene products. However, treatment of DHL-4 with C2B8 inhibited TNF-alpha secretion. These findings demonstrate that C2B8 antibody potentiates the sensitivity of DHL-4 tumor cells to several cytotoxic agents. Further, the findings suggest that combination treatments with C2B8 antibody and drugs may be of clinical benefit in the treatment of patients with resistant aggressive B lymphomas.

Background : Point mutations in the ras proto-oncogene that activate its oncogenic potential occur in approximately 30% of human cancers. Previous studies have demonstrated that T-cell immunity against some forms of mutant Ras proteins could be elicited, and some effectiveness against tumors expressing activated Ras has been reported. Purpose : The goal of this study was to determine if immunization of mice with two forms of mutant Ras protein can induce high levels of Ras mutation-specific T-cell immunity in vitro and tumor regression in vivo. Methods : Mice (BALB/c or C3H/HeJ) were immunized subcutaneously at 2-week intervals with purified Ras oncoproteins mixed with the immunologic adjuvants Antigen Formulation or QS-21, both of which have been shown to enhance the induction of T-cell-mediated immunity when included as components of soluble protein vaccines. In some experiments, mice were immunized directly with heat-killed Escherichia coli that had been induced to express one of the mutant Ras proteins. Spleen cells plus lymph node cells from Ras-immunized mice were tested in vitro for lysis of syngeneic Ras-expressing tumor cells and proliferation in response to mutant Ras peptides. For some of the cytolytic activity experiments, the spleen cells were grown under T H 1 conditions (growth in presence of interleukin 2, interferon gamma, and an antibody directed against interleukin 4 to stimulate a cell-mediated immune response) or T H 2 conditions (growth in presence of interleukins 2 and 4 to stimulate a humoral immune response). The specificity of immunity was examined in vivo by challenge of Ras-immunized mice with syngeneic tumor cells expressing mutant Ras oncoproteins (HaBalb, i.e., BALB/c mouse cells expressing Ras with arginine substituted at amino acid position 12 [Arg 12 Ras] ; C3HL61, i.e., C3H/HeJ mouse cells expressing Ras with leucine substituted at position 61 [Leu 61 Ras]). Ten mice per group were used in each experiment. Results : Proliferative and cytolytic T-cell responses directed against the Arg 12 Ras protein were generated in BALB/c mice, resulting in protection against challenge with cells expressing Arg 12 Ras and therapeutic benefit in mice bearing established tumors expressing this protein. In C3H/HeJ mice, high levels of cytolytic and proliferative responses were induced against Leu 61 Ras. Immunization with heat-killed E. coli genetically engineered to express Leu 61 Ras also led to the induction of anti-Ras T-cell immunity. T cells grown under T H 1 conditions were cytolytic against Ras-transformed tumor cells, whereas those grown under T H 2 conditions were not. Conclusions : Immunization as described here leads to Ras mutation-specific antitumor immunity in vitro and in vivo, with therapeutic efficacy in an established tumor model.

Induction of class I MHC-restricted cytotoxic T lymphocyte (CTL) responses by soluble proteins or peptides requires complex adjuvants or carrier systems which are not licensed for use with human vaccines. The data presented in this report show that vaccination with a highly purified recombinant influenza protein antigen in aluminium hydroxide adjuvant, the only adjuvant currently licensed for clinical use, elicited class I restricted CTL and protection from lethal challenge with H1N1 and H2N2 viruses. The antigen (D protein, SK&F 106160) is produced by expression of H1N1 influenza virus-derived cDNA (strain A/PR/8/34) in Escherichia coli, and is composed of the first 81 N-terminal amino acids (aa) of the non-structural protein 1 (NS1) fused via a nine nucleotide non-viral linker sequence to the 157 C-terminal aa of the haemagglutinin 2 subunit (HA2). Previous work by Kuwano et al demonstrated that in vitro stimulation of spleen cells from influenza virus-primed mice, with a partially purified preparation of the D protein, selected for CD8+ CTL clones which facilitated lung clearance of H1N1 and H2N2 viruses. In the current study, these results were extended by studying the responses of mice actively immunized with highly purified D protein in the presence or absence of adjuvants. Vaccination of CB6F1 (H-2dxb) mice with D protein in aluminum hydroxide or Freund's complete adjuvant generated H1N1 cross-reactive, H-2d-restricted, CD8+ CTL directed against an immunodominant HA2 epitope (aa 189-199). D protein without adjuvant did not elicit CTL, regardless of the route of injection. However, long-lived (greater than 6 months) splenic memory CTL were elicited by boosting mice intraperitoneally (i.p.) with the D protein in the absence of adjuvant. In mice injected subcutaneously with D protein in aluminium hydroxide at weeks 0 and 3, survival was increased relative to controls up to 16 weeks beyond the second vaccination, after which time additional boosting was required for protection. Studies in H-2b and H-2k mice vaccinated with the D protein showed that induction of CD4+ T-cell or antibody responses, in the absence of CD8+ CTL, did not correlate with protection. Passive transfer of immune sera from CB6F1 mice was also not protective. This prototype H1N1 recombinant subunit vaccine in aluminium adjuvant should directly address the feasibility of achieving a protective cell-mediated immune response in human influenza.

SK&F 105685 (N,N-Dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine dihydrochloride) is a novel azaspirane which has therapeutic activity in rat models of autoimmune disease. In this study, we have demonstrated that SK&F 105685 is a potent inducer of non-specific suppressor cells (SC). Oral administration of 15–30 mg/kg/day results in the generation of SC in the spleen, lymph nodes and bone marrow, but not the thymus of Lewis rats. Splenic SC suppress Con-A-induced proliferation in co-culture assays at effector-responder ratios of 1:1 to 1:64. SC are radiation resistant (2000 R), non-T, non-B cells, partially adherent to plastic surfaces and are enriched in a 1.07 g/ml fraction of a Percoll density gradient. Their activity is increased, rather than ablated, by indomethacin. No definitive changes in Ig + , OX-19 + , OX-8 + , W3/25 + or asialo GM1 + cells could be detected in the spleens of treated rats compared to control untreated animals. Elevated levels of both radiation-sensitive and radiation-resistant suppressor cells were found in the bone marrow of treated rats in addition to the radiosensitive SC normally present in this tissue.

Rituximab (chimeric anti-CD20 mAb) is currently being used in the therapy of Non-Hodgkin’s B-cell Lymphoma (NHL). It has also been reported that the combination of rituximab and chemotherapy is effective in patients with NHL. Several proposed mechanisms have been suggested for rituximab-mediated effects in vivo and include ADCC, CDC and apoptosis. In addition, we have reported that rituximab can trigger the tumor cells and modifies several intracellular survival signaling pathways resulting in the sensitization of tumor cells to various chemotherapeutic drugs (Alas et al., Clin. Cancer Res.8:836, 2001; Jazirehi et al., Mol. Cancer Therapy2:1183, 2003; Vega et al., Oncogene23:3530, 2004). Rituximab-mediated signaling might have resulted from directly triggering CD20 alone or in conjunction with triggering FcRs on the tumor cells via the Fc fragment of rituximab. This study was designed to distinguish between these two possibilities. Since the CH2 domain in Fc is responsible for its interaction with the Fc receptor, we have genetically engineered a rituximab mAb in which the CH2 domain was deleted. The CHO cells secreted one monomeric form with properly formed disulfides rituximab (CH2−) and a dimeric form rituximab (CH2−) Dimer. The functions of the two CH2-deleted domain antibodies were examined in several systems and compared with those of the native rituximab. We used Ramos and 2F7 B-NHL cell lines as model systems. As expected, we demonstrate that the CH2-deleted antibodies are defective in their ability to mediate ADCC and CDC. However, their biological functions and their ability to signal the cells were not altered and were similar to those observed with rituximab. Hence, the CH2-deleted antibodies inhibited cell proliferation, induced cell aggregation in culture, and induced apoptosis when cross-linked with a secondary anti-human IgG. In addition, the CH2-deleted antibodies triggered the cells similar to rituximab and inhibited the Src kinase pLyn, the downstream p38 MAP kinase, the transcription factors NF-κB, YY1, SP1, and STAT3, and Bcl-2/Bcl-xL expression. These modifications resulted in the sensitization of B-NHL cell lines to both chemotherapeutic drugs and Fas-induced apoptosis. Altogether, these findings demonstrate that rituximab-mediated signaling and modification of intracellular signaling pathways can take place in the absence of any contribution of the Fc fragment cross-linked to the cell Fc receptors. These studies suggest that chemo and immuno-sensitization by rituximab can take place in vivo in the absence of Fc-FcR contributions and thus are of potential clinical relevance.

We have investigated the relationship between CD3 expression and the suppressor T-cell function. We have isolated stable clonal cell lines of the F12.23 suppressor T-cell hybridoma that are either CD3+ or CD3-. These lines were subjected to functional assays including inhibition of in vivo hapten-specific delayed-type hypersensitivity responses, in vitro hapten-specific interleukin 2 responses, as well as hapten-specific cytotoxic T-lymphocyte assays. In all assays, the functional suppressor phenotype absolutely correlated with CD3 surface expression. Furthermore, we have immunoprecipitated heterodimeric proteins that share molecular features with some receptor polypeptides previously described. CD3 polypeptides found on the surface of suppressor T cells are phosphorylated after phorbol ester stimulation. Collectively these studies unambiguously define the suppressive supernatant function as a product of CD3+ receptor-bearing T cells.

Rat liver ribosomes were dissociated into subunits using EDTA, sodium pyrophosphate, high concentrations of KCl, as well as by incubation with puromycin in presence of 0.5 M KCl. The subunits obtained were analyzed using the density gradient centrifugation technique and their ribosomal proteins were separated by means of two-dimensional polyacrylamide gel electrophoresis. The ribosomal protein patterns of the two subunits isolated using each of the dissociating method were compared to the protein patterns of monosomes prepared by puromycin treatment alone. Our results revealed that the use of chelating agents to dissociate the ribosomes resulted in the loss of some ribosomal proteins from the small subunit. On the other hand, the use of KCl in high concentrations to dissociate the ribsosomes did not appear to cause any major loss of proteins from the ribosomes except for some acidic proteins.

Disease-associated alterations in macrophage functions were assessed by investigating the stages of activation of peritoneal macrophages obtained from adjuvant-induced arthritic rats. The stages of activation were established by defining several functional parameters in macrophages obtained from normal, sterile-irritant injected and Propionibacterium acnes injected animals. Peritoneal macrophages taken from arthritic rats 17 days post adjuvant injection displayed parameters characteristic of activated, but not elicited or resident macrophages. Specifically, an increased number of macrophages was recovered from arthritic rats which spread readily in culture, exhibited enhanced Fc receptor-mediated phagocytosis, increased leucine aminopeptidase ectoenzyme activity, enhanced secretion of prostaglandin E2 and interleukin 1, and ability to lyse tumor cells spontaneously. In addition, these macrophages were impaired in their ability to secrete superoxide anion. These data demonstrate distinct differences in parameters of peritoneal macrophage activation in rats compared to mice and that macrophage activation is associated with disease progression in adjuvant-induced arthritic rats.

The mode of action of the dual inhibitors of eicosanoid metabolism, SK&F 86002 and SK&F 104493 was evaluated on inflammatory cell infiltration induced in mice by carrageenan, monosodium urate crystals, and arachidonic acid. The results were compared to those seen with standard antiinflammatory compounds. Inflammatory cell infiltration was inhibited by SK&F 86002, SK&F 104493, colchicine, and phenidone but not naproxen. In vivo, PMN infiltration induced by LTB4 was inhibited by colchicine but not by SK&F 86002, SK&F 104493, or phenidone treatment. Similarly, in vitro chemotaxis to LTB4 was not inhibited by SK&F 86002. The 5-lipoxygenase inhibitors, SK&F 86002, SK&F 104493, and phenidone inhibited LTB4 production in vivo as well as inflammatory cell infiltration induced by arachidonic acid. The data are consistent with the suggestion that the bicyclic imidazoles inhibit PMN infiltration by virtue of inhibition of LTB4 production.

The effects of SK&F 86002 [5-(4-pyridyl)-6 (4-fluorophenyl)-2,3-dihydroimidazo (2,1-b) thiazole] on the generation of eicosanoids in vitro and on inflammatory responses in vivo are described and compared to other non-steroidal anti-inflammatory drugs. SK&F 86002 inhibited prostaglandin H2 (PGH2) synthase activity (IC50 120 microM) as well as prostanoid production by rat basophilic leukemia (RBL-1) cells (IC50 70 microM) and its sonicate (IC50 100 microM) and human monocytes (IC50 1 microM). In addition, SK&F 86002 inhibited the generation of dihydroxyeicosatetraenoic acid (diHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE) by a high speed supernatant fraction of RBL-1 cells (IC50 10 microM). Cellular production of 5-lipoxygenase products was inhibited by SK&F 86002 as measured by leukotriene B4 (LTB4) generation from human neutrophils (IC50 20 microM), leukotriene C4 (LTC4) generation by human monocytes (IC50 20 microM), and 5-HETE production by RBL-1 cells (IC50 40 microM). The in vivo profile of anti-inflammatory activity of SK&F 86002 supports the dual inhibition of arachidonate metabolism as indicated by its activity in inflammation models that are insensitive to selective cyclooxygenase inhibitors. The responses of arachidonic-acid-induced edema in the mouse ear and rat paw, as well as the cell infiltration induced by carrageenan in the mouse peritoneum and by arachidonic acid in the rat air pouch, were inhibited by SK&F 86002 and phenidone but not by the selective cyclooxygenase inhibitors naproxen and indomethacin.

The dual inhibitors of arachidonic acid metabolism, Smith KlineFrench (SKF) 86002, SKF 104351, and phenidone; the corticosteroid, dexamethasone; and the selective cyclooxygenase inhibitors, ibuprofen, indomethacin, naproxen, and piroxicam were evaluated for their antiarthritic potency in the murine, collagen-induced arthritis model. The ability of these compounds to alter the severity of arthritic lesions and to reduce serum levels of the acute-phase reactant, serum amyloid P component (SAP) were monitored. Serum concentrations of SAP were found to correlate strongly (r = 0.985) with disease severity at day 35 postimmunization. Treatment with SKF 86002, SKF 104351, phenidone, or dexamethasone significantly reduced disease severity, as judged by clinical score (55%, 72%, 41%, and 45% inhibition, respectively) and SAP levels (62%, 94%, 52%, and 94% inhibition, respectively) in arthritic mice. This profile of activity was not shared by the selective cyclooxygenase inhibitors, which did not uniformly inhibit disease activity by both parameters. The results suggest that dual inhibitors of 5-lipoxygenase and cyclooxygenase may prove more effective than selective cyclooxygenase inhibitors as anti-arthritic agents.

There is a close association between levels of natural killer (NK) cell activity and the ability of the host to eliminate circulating tumor cell emboli. Mice that exhibit low levels of NK-cell-mediated cytotoxicity (3-week-old syngeneic mice, 3-week-old allogeneic nude mice, cyclophosphamide- or beta-estradiol-treated mice, and beige mice) also exhibit enhanced survival of tumor cells in the vascular bed of the lung and increased incidence of pulmonary tumor metastasis. Conversely, hosts with high NK cell activity (adult nude mice and syngeneic mice treated with NK-cell-stimulating biological response modifiers (BRM) ) are very resistant to metastasis. Lymphoid adoptive transfer studies have shown that the effector cell responsible for the antimetastatic activity is the NK cell. In these studies, NK cells were highly effective in destroying circulating tumor cells before their extravasation into the organ parenchyma, whereas they exerted only a minimal inhibiting effect on already established micrometastases. The ability to activate NK cells selectively (without subsequently inducing suppressor macrophages) provides a valuable tool for the evaluation of the role of activated NK cells in therapy of tumor metastasis. The validity of this approach is supported by the finding that NK cells activated by BRM are effective in killing, both in vivo and in vitro, solid tumor cells that developed NK-cell-resistance as a result of adaptive growth in vivo or selection during the metastatic process. An understanding of the mechanisms that regulate NK cell activation or suppression as well as elucidation of the circulatory patterns and anatomical compartmentalization of activated NK cells will help achieve a sustained systemic and/or in situ activation of NK cells which may prove effective in the control of cancer metastasis.

The antiinflammatory activity of the structurally novel dual inhibitor of arachidonic acid metabolism, SK&F 86002 was evaluated using arachidonic acid-induced edema and inflammatory cell infiltration. Histological examination demonstrated extensive subcutaneous edema and neutrophil (PMN) accumulation in perivascular and interstitial locations one hour after application of arachidonic acid to the ear. SK&F 86002 and, to a lesser extent, phenidone demonstrated potent inhibition of this inflammatory response following oral and topical administration. Nordihydroguaiaretic acid (NDGA) displayed only topical activity. The selective cyclooxygenase inhibitors ibuprofen and naproxen were either inactive or stimulated ear swelling, Histological evaluation of the lesion in drug-treated animals revealed that SK&F 86002 impaired edema formation and caused a significant reduction in numbers of infiltrating neutrophils. Using arachidonic acid-induced peritoneal exudation, a reduction in the cellular infiltrate was observed after oral treatment with SK&F 86002 or phenidone, but not with naproxen. Taken together, these data illustrate the potent antiinflammatory effects of SK&F 86002 and support the suggestion that 5-lipoxygenase products play a significant role in both the edematous and cellular phases of arachidonic acid-induced inflammation.