STAT1 Is Required for Decreasing Accumulation of Granulocytic Cells via IL-17 during Initial Steps of Colitis-Associated Cancer.

Signal transducer and activator of transcription 1 (STAT1) acts as a tumor suppressor molecule in colitis-associated colorectal cancer (CAC), particularly during the very early stages, modulating immune responses and controlling mechanisms such as apoptosis and cell proliferation. Previously, using an experimental model of CAC, we reported increased intestinal cell proliferation and faster tumor development, which were consistent with more signs of disease and damage, and reduced survival in STAT1-/- mice, compared with WT counterparts. However, the mechanisms through which STAT1 might prevent colorectal cancer progression preceded by chronic inflammation are still unclear. Here, we demonstrate that increased tumorigenicity related to STAT1 deficiency could be suppressed by IL-17 neutralization. The blockade of IL-17 in STAT1-/- mice reduced the accumulation of CD11b+Ly6ClowLy6G+ cells resembling granulocytic myeloid-derived suppressor cells (MDSCs) in both spleen and circulation. Additionally, IL-17 blockade reduced the recruitment of neutrophils into intestinal tissue, the expression and production of inflammatory cytokines, and the expression of intestinal STAT3. In addition, the anti-IL-17 treatment also reduced the expression of Arginase-1 and inducible nitric oxide synthase (iNOS) in the colon, both associated with the main suppressive activity of MDSCs. Thus, a lack of STAT1 signaling induces a significant change in the colonic microenvironment that supports inflammation and tumor formation. Anti-IL-17 treatment throughout the initial stages of CAC related to STAT1 deficiency abrogates the tumor formation possibly caused by myeloid cells.

Antibody-Based Inhibition of Pathogenic New World Hemorrhagic Fever Mammarenaviruses by Steric Occlusion of the Human Transferrin Receptor 1 Apical Domain.

Pathogenic clade B New World mammarenaviruses (NWM) can cause Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers. Sequence variability among NWM glycoproteins (GP) poses a challenge to the development of broadly neutralizing therapeutics against the entire clade of viruses. However, blockade of their shared binding site on the apical domain of human transferrin receptor 1 (hTfR1/CD71) presents an opportunity for the development of effective and broadly neutralizing therapeutics. Here, we demonstrate that the murine monoclonal antibody OKT9, which targets the apical domain of hTfR1, can sterically block cellular entry by viral particles presenting clade B NWM glycoproteins (GP1-GP2). OKT9 blockade is also effective against viral particles pseudotyped with glycoproteins of a recently identified pathogenic Sabia-like virus. With nanomolar affinity for hTfR1, the OKT9 antigen binding fragment (OKT9-Fab) sterically blocks clade B NWM-GP1s and reduces infectivity of an attenuated strain of Junin virus. Binding of OKT9 to the hTfR1 ectodomain in its soluble, dimeric state produces stable assemblies that are observable by negative-stain electron microscopy. A model of the OKT9-sTfR1 complex, informed by the known crystallographic structure of sTfR1 and a newly determined structure of the OKT9 antigen binding fragment (Fab), suggests that OKT9 and the Machupo virus GP1 share a binding site on the hTfR1 apical domain. The structural basis for this interaction presents a framework for the design and development of high-affinity, broadly acting agents targeting clade B NWMs. IMPORTANCE Pathogenic clade B NWMs cause grave infectious diseases, the South American hemorrhagic fevers. Their etiological agents are Junin (JUNV), Guanarito (GTOV), Sabiá (SABV), Machupo (MACV), Chapare (CHAV), and a new Sabiá-like (SABV-L) virus recently identified in Brazil. These are priority A pathogens due to their high infectivity and mortality, their potential for person-to-person transmission, and the limited availability of effective therapeutics and vaccines to curb their effects. While low homology between surface glycoproteins of NWMs foils efforts to develop broadly neutralizing therapies targeting NWMs, this work provides structural evidence that OKT9, a monoclonal antibody targeting a single NWM glycoprotein binding site on hTfR1, can efficiently prevent their entry into cells.

Local Sustained Delivery of Anti-IL-17A Antibodies Limits Inflammatory Bone Loss in Murine Experimental Periodontitis.

Periodontal disease (PD) is a chronic destructive inflammatory disease of the tooth-supporting structures that leads to tooth loss at its advanced stages. Although the disease is initiated by a complex organization of oral microorganisms in the form of a plaque biofilm, it is the uncontrolled immune response to periodontal pathogens that fuels periodontal tissue destruction. IL-17A has been identified as a key cytokine in the pathogenesis of PD. Despite its well documented role in host defense against invading pathogens at oral barrier sites, IL-17A-mediated signaling can also lead to a detrimental inflammatory response, causing periodontal bone destruction. In this study, we developed a local sustained delivery system that restrains IL-17A hyperactivity in periodontal tissues by incorporating neutralizing anti-IL-17A Abs in poly(lactic-coglycolic) acid microparticles (MP). This formulation allowed for controlled release of anti-IL-17A in the periodontium of mice with ligature-induced PD. Local delivery of anti-IL-17A MP after murine PD induction inhibited alveolar bone loss and osteoclastic activity. The anti-IL-17A MP formulation also decreased expression of IL-6, an IL-17A target gene known to induce bone resorption in periodontal tissues. This study demonstrates proof of concept that local and sustained release of IL-17A Abs constitutes a promising therapeutic strategy for PD and may be applicable to other osteolytic bone diseases mediated by IL-17A-driven inflammation.

Novel preventive and therapeutic strategies against HIV infection.

Since its first isolation in 1983, over 77 million people became infected with the human immunodeficiency virus (HIV), and only one case has been reported in which the virus was completely removed from the body. A recent second case was reported that remains to be confirmed. Antiretroviral therapy (ART) manages to control blood viral replication and, consequently, to restore -at least partially- the functions of the immune system, with a notable positive impact on the morbidity and mortality associated with the virus. However, given the difficulty in eliminating the virus from the body, treatment should be given for life. This long-term exposure to antiretroviral drugs implies the risk of generating intolerance, toxic effects, gaps in adherence and the potential selection of resistance mutations. Another limitation is the high cost of treating 37 million persons living with HIV, most of whom are living in resource-limited countries and relying on international aid initiatives. Having these challenges in mind, there is general agreement that new approaches for preventing and treating HIV infection are needed to control the epidemic, while vaccine development efforts continue. In this regard, new generation broadly neutralising monoclonal antibodies (bnMAbs) against the HIV viral envelope protein can prevent virus acquisition, reduce viremia, enhance immunity, and induce the killing of infected cells in animal models of HIV infection. Most importantly, some clinical trials have shown that bnMAbs could effectively d ecrease viremia and delay viral rebound in people chronically infected with HIV.

Desde su primer aislamiento en 1983, el virus de la inmunodeficiencia humana (HIV) ha infectado a más de 77 millones de personas y solo se ha documentado un caso en el cual el virus fue removido completamente del organismo; aún resta confirmar un segundo caso informado recientemente. El tratamiento antirretroviral logra controlar la replicación viral en el plasma y en consecuencia recuperar (al menos parcialmente) la actividad del sistema inmune, con una notable reducción de la morbilidad y la mortalidad asociadas a la infección por HIV. Sin embargo, ante la dificultad para eliminar completamente el virus del organismo, es necesario continuar el tratamiento de por vida. Esto implica la exposició n a largo plazo a drogas antirretrovirales con riesgo de generar intolerancia, efectos tóxicos, brechas en la adherencia y selección de mutantes resistentes. Otro aspecto a considerar es la carga económica que implica tratar a 37 millones de personas infectadas con HIV, la mayoría de ellas en países que solo pueden afrontar esos costos con ayuda internacional. Por ello, hasta tanto se disponga de una vacuna capaz de prevenir la infección de todas las formas circulantes del HIV, es necesario desarrollar nuevas herramientas terapéuticas capaces de complementar y potenciar los efectos del tratamiento antirretroviral. Diversos ensayos preclínicos sugieren que la administración pasiva de anticuerpos monoclonales dirigidos contra la glicoproteína de envoltura viral podría prevenir la infección, reducir la carga viral, estimular la respuesta inmune y favorecer la eliminación de células infectadas con HIV.

The inhibition of CTGF/CCN2 activity improves muscle and locomotor function in a murine ALS model.

Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset progressive neurodegenerative disease characterized by upper and lower motoneuron degeneration. A total of 20% of familial ALS (fALS) cases are explained by mutations in the superoxide dismutase 1 (SOD1) enzyme. Although more than 20 years have passed since the generation of the first ALS mouse model, the precise molecular mechanisms of ALS pathogenesis remain unknown. CTGF/CCN2 is a matricellular protein with associated fibrotic activity that is up-regulated in several chronic diseases. The inhibition of CTGF/CCN2 with the monoclonal neutralizing antibody FG-3019 reduces fibrosis in several chronic disorders including the mdx mice, a murine model for Duchenne muscular dystrophy (DMD). In this work, we show that there are increased levels of CTGF/CCN2 in skeletal muscle and spinal cord of hSOD1G93A mice. In this scenario, we show evidence that FG-3019 not only reduces fibrosis in skeletal muscle of hSOD1G93A mice, but also improves muscle and locomotor performance. We demonstrate that treatment with FG-3019 reduces muscle atrophy in hSOD1G93A mice. We also found improvement of neuromuscular junction (NMJ) innervation together with a reduction in myelin degeneration in the sciatic nerve, suggesting that alterations in nerve-muscle communication are partially improved in FG-3019-treated hSOD1G93A mice. Moreover, we also found that CTGF/CCN2 is expressed in astrocytes and neurons, predominantly in dorsal areas of spinal cord from symptomatic hSOD1G93A mice. Together, these results reveal that CTGF/CCN2 might be a novel therapeutic target to ameliorate symptoms and improve the quality of life of ALS patients.

Development and characterization of novel chimeric monoclonal antibodies for broad spectrum neutralization of rabies virus.

Current post-exposure prophylaxis for rabies virus infection has several limitations in terms of supply, cost, safety, and efficacy. Attempts to replace human or equine rabies immune globulins (HRIG or ERIG) have been made by several companies and institutes. We developed potent monoclonal antibodies to neutralize a broad spectrum of rabies viruses by screening hybridomas received from the U.S. Centers for Disease Control and Prevention (CDC). Two kinds of chimeric human antibodies (chimeric #7 and #17) were constructed by cloning the variable regions from selected hybridomas and the constant region of a human antibody. Two antibodies were bound to antigenic site III and I/IV, respectively, and were able to neutralize 51 field isolates of rabies virus that were isolated at different times and places such as Asia, Africa, North America, South America, and Australia. These two antibodies neutralize rabies viruses with high efficacy in an in vivo test using Syrian hamster and mouse models and show low risk for adverse immunogenicity.

Does native Trypanosoma cruzi calreticulin mediate growth inhibition of a mammary tumor during infection?

BACKGROUND: For several decades now an antagonism between Trypanosoma cruzi infection and tumor development has been detected. The molecular basis of this phenomenon remained basically unknown until our proposal that T. cruzi Calreticulin (TcCRT), an endoplasmic reticulum-resident chaperone, translocated-externalized by the parasite, may mediate at least an important part of this effect. Thus, recombinant TcCRT (rTcCRT) has important in vivo antiangiogenic and antitumor activities. However, the relevant question whether the in vivo antitumor effect of T. cruzi infection is indeed mediated by the native chaperone (nTcCRT), remains open. Herein, by using specific modified anti-rTcCRT antibodies (Abs), we have neutralized the antitumor activity of T. cruzi infection and extracts thereof, thus identifying nTcCRT as a valid mediator of this effect. METHODS: Polyclonal anti-rTcCRT F(ab')2 Ab fragments were used to reverse the capacity of rTcCRT to inhibit EAhy926 endothelial cell (EC) proliferation, as detected by BrdU uptake. Using these F(ab')2 fragments, we also challenged the capacity of nTcCRT, during T. cruzi infection, to inhibit the growth of an aggressive mammary adenocarcinoma cell line (TA3-MTXR) in mice. Moreover, we determined the capacity of anti-rTcCRT Abs to reverse the antitumor effect of an epimastigote extract (EE). Finally, the effects of these treatments on tumor histology were evaluated. RESULTS: The rTcCRT capacity to inhibit ECs proliferation was reversed by anti-rTcCRT F(ab')2 Ab fragments, thus defining them as valid probes to interfere in vivo with this important TcCRT function. Consequently, during infection, these Ab fragments also reversed the in vivo experimental mammary tumor growth. Moreover, anti-rTcCRT Abs also neutralized the antitumor effect of an EE, again identifying the chaperone protein as an important mediator of this anti mammary tumor effect. Finally, as determined by conventional histological parameters, in infected animals and in those treated with EE, less invasive tumors were observed while, as expected, treatment with F(ab')2 Ab fragments increased malignancy. CONCLUSION: We have identified translocated/externalized nTcCRT as responsible for at least an important part of the anti mammary tumor effect of the chaperone observed during experimental infections with T. cruzi.

Glycoprotein-Specific Antibodies Produced by DNA Vaccination Protect Guinea Pigs from Lethal Argentine and Venezuelan Hemorrhagic Fever.

UNLABELLED: Several members of the Arenaviridae can cause acute febrile diseases in humans, often resulting in lethality. The use of convalescent-phase human plasma is an effective treatment in humans infected with arenaviruses, particularly species found in South America. Despite this, little work has focused on developing potent and defined immunotherapeutics against arenaviruses. In the present study, we produced arenavirus neutralizing antibodies by DNA vaccination of rabbits with plasmids encoding the full-length glycoprotein precursors of Junín virus (JUNV), Machupo virus (MACV), and Guanarito virus (GTOV). Geometric mean neutralizing antibody titers, as measured by the 50% plaque reduction neutralization test (PRNT(50)), exceeded 5,000 against homologous viruses. Antisera against each targeted virus exhibited limited cross-species binding and, to a lesser extent, cross-neutralization. Anti-JUNV glycoprotein rabbit antiserum protected Hartley guinea pigs from lethal intraperitoneal infection with JUNV strain Romero when the antiserum was administered 2 days after challenge and provided some protection (∼30%) when administered 4 days after challenge. Treatment starting on day 6 did not protect animals. We further formulated an IgG antibody cocktail by combining anti-JUNV, -MACV, and -GTOV antibodies produced in DNA-vaccinated rabbits. This cocktail protected 100% of guinea pigs against JUNV and GTOV lethal disease. We then expanded on this cocktail approach by simultaneously vaccinating rabbits with a combination of plasmids encoding glycoproteins from JUNV, MACV, GTOV, and Sabia virus (SABV). Sera collected from rabbits vaccinated with the combination vaccine neutralized all four targets. These findings support the concept of using a DNA vaccine approach to generate a potent pan-arenavirus immunotherapeutic. IMPORTANCE: Arenaviruses are an important family of emerging viruses. In infected humans, convalescent-phase plasma containing neutralizing antibodies can mitigate the severity of disease caused by arenaviruses, particularly species found in South America. Because of variations in potency of the human-derived product, limited availability, and safety concerns, this treatment option has essentially been abandoned. Accordingly, despite this approach being an effective postinfection treatment option, research on novel approaches to produce potent polyclonal antibody-based therapies have been deficient. Here we show that DNA-based vaccine technology can be used to make potently neutralizing antibodies in rabbits that exclusively target the glycoproteins of several human-pathogenic arenaviruses found in South America, including JUNV, MACV, GTOV, and SABV. These antibodies protected guinea pigs from lethal disease when given post-virus challenge. We also generated a purified antibody cocktail with antibodies targeting three arenaviruses and demonstrated protective efficacy against all three targets. Our findings demonstrate that use of the DNA vaccine technology could be used to produce candidate antiarenavirus neutralizing antibody-based products.

Depressive-like behaviour induced by an intracerebroventricular injection of streptozotocin in mice: the protective effect of fluoxetine, antitumour necrosis factor-α and thalidomide therapies.

Information on the effect of an intracerebroventricular (i.c.v.) injection of streptozotocin (STZ) on noncognitive behaviour in rodents such as depression states is scarce. Thus, the aim of this study was to examine the depressive-like effect of STZ injected by the i.c.v. route in mice and the potential protective effect of fluoxetine, antitumour necrosis factor-α (anti-TNF-α) and thalidomide. Our results indicated that a single injection of STZ (0.1 mg/site) promoted depressive-like behaviour in the tail suspension and sucrose preference tests without altering either locomotor activity or plasma glucose levels. We also showed that STZ increased TNF-α levels in the hippocampus of mice. Fluoxetine (32 mg/kg, intraperitoneally. 30 min before STZ injection), and the anti-TNF-α antibody (0.1 pg/site, i.c.v.) and thalidomide (3 mg/kg, subcutaneously), coadministered with STZ, prevented these effects. This is the first study to report depressive-like effects of STZ using the i.c.v. route in mice. We concluded that fluoxetine, anti-TNF-α antibody and thalidomide were effective in preventing depressive-like behaviour and the increase in TNF-α levels in the hippocampus of mice induced by an i.c.v. injection of STZ, reinforcing the involvement of TNF-α in the pathophysiology of depression. This model and the mechanisms studied may contribute towards the development of new antidepressant drugs and enhance the options for studying depression.

An inactivated vaccine from a field strain of bovine herpesvirus-1 (BoHV-1) has high antigenic mass and induces strong efficacy in a rabbit model.

Bovine Herpesvirus-1 (BoHV-1) is a DNA virus belonging to the family Herpesviridae, subfamily Alfaherpesvirinae; it is a worldwide pathogen, causing serious economic losses in livestock. In Colombia there have been multiple isolates of BoHV-1 that have been subjected to molecular characterization, classifying most of the country isolates as BoHV-1.1. In the present study we developed and evaluated an ethyleneimine binary inactivated isolate from the native BoHV-1 strain (Córdoba-2) in a rabbit model of vaccination and infection. The vaccine was evaluated in two phases, one of immunogenicity with vaccination and a booster after 21 days, and an evaluation phase of protection against challenge with a highly virulent reference strain. The results demonstrate optimum serum-conversion, with protective neutralizing antibody titers 28 days post vaccination and optimal protection against challenge with the reference strain with decreased clinical signs of infection, protection against the onset of fever and decrease of virus excretion post challenge. In conclusion, our results show the enormous potential that an immunogenic inactivated vaccine has produced from the native BoHV-1.1 strain, which produces a high antigen mass to the vaccine to induce optimal immunity and protection, and it is a strong candidate for evaluation and possible future use in different cattle populations.

HMGB1 and TLR4 mediate skeletal muscle recovery in a murine model of hindlimb ischemia.

BACKGROUND: We have previously shown that the danger signal high-mobility group box 1 (HMGB1) promotes angiogenesis when administered to ischemic muscle. HMGB1 signals through Toll-like receptor 4 (TLR4) as well as the receptor for advanced glycation end-products (RAGE). However, the actions of these receptors in ischemic injury and muscle recovery are not known. We hypothesize that TLR4 mediates tissue recovery and angiogenesis in response to ischemia. METHODS: Femoral artery ligation was performed in control, TLR4 competent (C3H/HeOuJ) and incompetent (C3H/HeJ) mice, as well as RAGE knockout mice and their C57B6 control counterparts. In other experiments, control mice were pretreated with anti-HMGB1 neutralizing antibody before femoral artery ligation. After 2 weeks, limb perfusion was evaluated using laser Doppler perfusion imaging and reported as the ratio of blood flow in the ischemic to nonischemic limb. Muscle necrosis, fat replacement, and vascular density in the anterior tibialis muscle were quantified histologically. In vitro, TLR4 and RAGE expression was evaluated in human dermal microvascular endothelial cells in response to hypoxia. Human dermal microvascular endothelial cells treated with HMGB1 alone and in the presence of anti-TLR4 antibody were probed for phosphorylated extracellular signal-regulated kinase (ERK), a signaling molecule critical to endothelial cell (EC) angiogenic behavior. RESULTS: Both anti-HMGB1 antibody as well as defective TLR4 signaling in HeJ mice resulted in prominent muscle necrosis 2 weeks after femoral artery ligation. Control HeOuJ mice had less necrosis than TLR4 incompetent HeJ mice, but a greater amount of fat replacement. In contrast to control C3H mice, control C57B6 mice demonstrated prominent muscle regeneration with very little necrosis. Muscle regeneration was not dependent on RAGE. While vascular density did not differ between strains, mice with intact RAGE and TLR4 signaling had less blood flow in ischemic limbs compared with mutant strains. In vitro, EC TLR4 expression increased in response to hypoxia while TLR4 antagonism decreased HMGB1-induced activation of extracellular signal-regulated kinase. CONCLUSIONS: Both HMGB1 and TLR4 protect against muscle necrosis after hindlimb ischemia. However, muscle regeneration does not appear to be tied to vascular density. HMGB1 likely activates angiogenic behavior in ECs in vitro, and this activation may be modulated by TLR4. The improvement in blood flow seen in mice with absent TLR4 and RAGE signaling may suggest anti-angiogenic roles for both receptors, or vasoconstriction induced by TLR4 and RAGE mediated inflammatory pathways.

Inhibition of hypothalamic inflammation reverses diet-induced insulin resistance in the liver.

Defective liver gluconeogenesis is the main mechanism leading to fasting hyperglycemia in type 2 diabetes, and, in concert with steatosis, it is the hallmark of hepatic insulin resistance. Experimental obesity results, at least in part, from hypothalamic inflammation, which leads to leptin resistance and defective regulation of energy homeostasis. Pharmacological or genetic disruption of hypothalamic inflammation restores leptin sensitivity and reduces adiposity. Here, we evaluate the effect of a hypothalamic anti-inflammatory approach to regulating hepatic responsiveness to insulin. Obese rodents were treated by intracerebroventricular injections, with immunoneutralizing antibodies against Toll-like receptor (TLR)4 or tumor necrosis factor (TNF)α, and insulin signal transduction, hepatic steatosis, and gluconeogenesis were evaluated. The inhibition of either TLR4 or TNFα reduced hypothalamic inflammation, which was accompanied by the reduction of hypothalamic resistance to leptin and improved insulin signal transduction in the liver. This was accompanied by reduced liver steatosis and reduced hepatic expression of markers of steatosis. Furthermore, the inhibition of hypothalamic inflammation restored defective liver glucose production. All these beneficial effects were abrogated by vagotomy. Thus, the inhibition of hypothalamic inflammation in obesity results in improved hepatic insulin signal transduction, leading to reduced steatosis and reduced gluconeogenesis. All these effects are mediated by parasympathetic signals delivered by the vagus nerve.

A humanised murine monoclonal antibody with broad serogroup specificity protects mice from challenge with Venezuelan equine encephalitis virus.

In murine models of Venezuelan equine encephalitis virus (VEEV) infection, the neutralising monoclonal antibody 1A3B-7 has been shown to be effective in passive protection from challenge by the aerosol route with serogroups I, II and Mucambo virus (formally VEE complex subtype IIIA). This antibody is able to bind to all serogroups of the VEEV complex when used in ELISA and therefore is an excellent candidate for protein engineering in order to derive a humanised molecule suitable for therapeutic use in humans. A Complementarity Determining Region (CDR) grafting approach using human germline IgG frameworks was used to produce a panel of humanised variants of 1A3B-7, from which a single candidate molecule with retained binding specificity was identified. Evaluation of humanised 1A3B-7 (Hu1A3B-7) in in vitro studies indicated that Hu1A3B-7 retained both broad specificity and neutralising activity. Furthermore, in vivo experiments showed that Hu1A3B-7 successfully protected mice against lethal subcutaneous and aerosol challenges with VEEV strain TrD (serogroup I). Hu1A3B-7 is therefore a promising candidate for the future development of a broad-spectrum antiviral therapy to treat VEEV disease in humans.

Safety and antiviral activity of motavizumab, a respiratory syncytial virus (RSV)-specific humanized monoclonal antibody, when administered to RSV-infected children.

Previously healthy children hospitalized with respiratory syncytial virus (RSV) received motavizumab (3, 15, or 30 mg/kg intravenously), an RSV-specific monoclonal antibody, or placebo. Safety, tolerability, motavizumab concentrations, and immunogenicity were assessed. Cultivatable RSV in the upper respiratory tract was significantly reduced with motavizumab compared with placebo day 1 post-treatment. No adverse events were considered motavizumab-related by site investigators.

Host soluble factors that regulate the synthesis of the major core protein of the bovine leukemia virus (BLV) in a naturally infected neoplastic B-cell line.

Bovine leukemia virus (BLV) is a B-cell tropic Deltaretrovirus that induces a lifelong infection and causes a fatal lymphosarcoma in less than 10% of the infected cattle. BLV is usually present in its host in a transcriptional repressed state but becomes de-repressed a few hours after the infected lymphocytes are cultured in vitro. In the present study we have examined the effect of soluble host factors and various substances on the synthesis of the major BLV protein (p24) in a permanent culture (cell line NBC-10) of neoplastic B-lymphocytes derived from BLV-infected cattle. Certain batches of fetal calf serum (FCS) and bovine platelet lysates (PLy) induced a rapid and drastic increase of the synthesis of BLVp24 in the NBC-10 cells. Neutralization experiments with specific antibodies demonstrated that the transforming growth factor-beta (TGF-beta) was responsible for the stimulatory activity of FCS and PLy on the synthesis of BLVp24 in the NBC-10 cells. Recombinant TGF-beta also stimulated the synthesis of BLVp24 in cultures of peripheral blood mononuclear cells (PBMCs) obtained from BLV-infected cattle. Mitogens, phorbol-myristate-acetate and prostaglandin E(2), previously shown to stimulate the expression of BLV in cultures of PBMC, did not induce the synthesis of BLVp24 in cultures of NBC-10 cells. Plasma, serum and milk from BLV-negative cattle inhibited the synthesis of BLVp24 induced by FCS, PLy or TGF-beta in the NBC-10 cells. The blocking activity was found in the whey and the beta-casein fractions of bovine milk. The relevance of these findings with regard to the previously reported plasma factor (PBB) with blocking activity on the expression of BLV in short-term PBMC cultures is discussed. Based on the information obtained in the present study we have standardized a reproducible and rapid assay system for the identification of factors that regulate the synthesis of BLVp24 in naturally infected neoplastic B cells.