Swine Dialyzable Spleen Extract as Antiviral Prophylaxis

JOURNAL OF MEDICINAL FOOD J Med Food 00 (0) 2015, 1–8 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2014.0176

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Swine Dialyzable Spleen Extract as Antiviral Prophylaxis Giovanna Merchand-Reyes,1,2 Lenin Pavo´n,2 Gilberto Pe´rez-Sa´nchez,1,2 Said Va´zquez-Leyva,1 Nohemı´ Salinas-Jazmı´n,1 Marco Velasco-Vela´zquez,3 Emilio Medina-Rivero,1 and Sonia Mayra Pe´rez-Tapia1,4 1

Unit of R&D in Bioprocesses (UDIBI), National School of Biological Sciences, National Polytechnic Institute, Mexico City, Mexico. 2 Department of Psychoimmunology, National Institute of Psychiatry, ‘‘Ramo´n de la Fuente,’’ Mexico City, Mexico. 3 School of Medicine, National Autonomous University of Mexico, Mexico City, Mexico. 4 Unit of Research, Development and Innovation in Medicine and Biotechnology/ Transfer Factor Project (UDIMEB/PFT), National School of Biological Sciences, National Polytechnic Institute, Mexico City, Mexico. ABSTRACT Worldwide, the most highly consumed meat is of porcine origin. The production and distribution of swine meat are affected by diverse health matters, such as influenza and diarrhea, which cause head losses and require the use of antibiotics and other drugs in hog farms. To stimulate newborn piglet immune responses and increase resistance to infections, we developed a spray-drying technique to produce dried swine dialyzable spleen extract (sDSE), an immunomodulator. Based on the size-exclusion ultra performance liquid chromatography quantitative analysis, it was possible to recover up to 58% of the product after the drying process. The biological activity of orally administered dried sDSE increased mouse survival and induced cytokine production in a herpes infection model.

KEY WORDS:  immunoregulation  piglet food supplement  process development  ultra performance liquid chromatograph

the United States, infections with the porcine reproductive and respiratory syndrome (PRRS) virus decrease reproduction and fattening, and in farms throughout the world, influenza negatively impacts the development of weaning pigs.6,7 Swine production in Mexico is of no exception. The prevalence of PRRS has been studied in hog farms in the state of Nuevo Leon, Mexico, where the percentage of seropositive pigs varies between 14% and 56% depending on the stage of production.8 In April 2014, Mexican pork production suffered an impact due to another virus, porcine diarrhea virus, which affected 30% of the pig population.9 Therefore, preventing infections in weaning pigs is crucial to improve pork meat production and avoid economic losses. It is possible that young swine are more susceptible to infections due to decreased levels of cytokines, such as IL-1b and TNF-a, and diminished interferon gamma (IFN-c) production in CD4 + lymphocytes compared with adult pigs.10,11 To improve immune responses in weaning pigs and reduce the risk of infection, the use of immunomodulators has been proposed.11 The dialyzable leukocyte extract (DLE) is a mixture of low-molecular-weight peptides obtained from leukocytes and can be obtained from healthy human donors or swine spleens.12,13 DLE was first described

INTRODUCTION

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wine is the most highly consumed source of meat worldwide and principally produced in the European Union, China, and the United States of America.1 According to data from the Foreign Agriculture Service, Mexico is a major exporter of swine meat to the United States of America (20% of the total exports) and the eighth major producer of pork in the world.2,3 In Mexico, pork meat production has increased by 4.25% from 1992 to 2008; furthermore, the production increased from 15.2 to an estimate of 17.1 million of heads from 2008 to 2014.1,4 Infectious diseases within hog farms are a major problem affecting the cost, quantity, and quality of pork meat production. However, the use of antibiotics, as prophylaxis or treatment, is believed to negatively impact meat quality.5 In

Manuscript received 8 November 2014. Revision accepted 7 March 2015. Address correspondence to: Dr. Lenin Pavo´n, Department of Psychoimmunology, National Institute of Psychiatry ‘‘Ramo´n de la Fuente Mun˜iz’’ Calzada MexicoXochimilco, 101 Colonia San Lorenzo Huipulco, Mexico DF 14370, Mexico, E-mail: [email protected] or Dr. Sonia Mayra Pe´rez-Tapia, Unit Bioprocess Research and Development (UDIBI), National School of Biological Sciences, National Polytechnic Institute, Plan de Ayala s/n Colonia Santo Tomas, Miguel Hidalgo, Mexico DF 01134, Mexico, E-mail: [email protected]

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at 1940s and has been used as an immunoregulator to treat human infections and other inflammatory diseases.14–18 The majority of research on the application of DLE has focused on clinical use in the treatment of human disease, with very little research on potential veterinary applications. Wang et al. used swine dialyzable spleen extract (sDSE) as an adjuvant in a porcine parvovirus vaccine. In mice, the sDSE adjuvant improved immunological vaccine responses by increasing the number of CD4 + lymphocytes and IFN-c production.19 However, a wider industrial use of sDSE has not yet been fully explored. In this study, we describe a scalable process to produce sDSE to meet the market demand. This process achieves greater production in a solid form that can be easily distributed without disrupting the physicochemical characteristics or biological activity of the product. The pharmaceutical formulation described here may facilitate the veterinary use of sDSE. MATERIALS AND METHODS Swine dialyzable spleen extracts Dried sDSE was produced by the Unidad de Desarrollo e Investigacio´n en Bioprocesos (UDIBI) at the Escuela Nacional de Ciencias Biolo´gicas, Instituto Polite´cnico Nacional, Mexico. Spleens from adult swine were provided by the slaughterhouse Health Inspection Type (TIF) number 240, ‘‘Rastro Mun˜ora,’’ which follows the Mexican standards (NOM-009-ZOO-1994). Spleens were transported with refrigeration to the production site, then cleaned, and sanitized with 70% ethanol. Cells contained in spleen extract Spleens were pressed to obtain a biological cell extract and followed up to osmotic shock with purified water containing 0.15% potassium sorbate (Sigma Aldrich). The spleen extract was clarified with four different Sartopure filter MidiCaps (20, 8, 1.2, and 0.65 lm) and a peristaltic pump (Sartorius). Following clarification, the extract was passed through a Sartocon slice Hydrosart with 10 kDa cutoff cartridges (Sartorius). The extract was dried as described below. Two batches were produced to test smallscale and large-scale production. Drying of sDSEs We normalized our drying condition using a standardized batch of DLE Transferon, which is a human-DLE composed by an heterogeneous mixture of low-molecularweight peptides (below 12 kDa) that modulate the immune response (batch number 12H16; UDIMEB).12 Five hundred milliliters of Transferon, containing 947 mg of humanDLE, was formulated with maltodextrin (30 dextrose equivalents; CP Ingredients) in two different proportions: 0.3 or 0.5 g/mL. The formulations were dried using a Niro Mobile Minor 2000 (GEA) with a nozzle parallel fluid and the following conditions: entrance temperature, 170C; exit temperature, 76C; air pressure, 1 bar. The dried

products were solubilized at 50 or 100 mg/mL and analyzed by size-exclusion ultra performance liquid chromatography (SE-UPLC). The best drying condition was chosen based on UPLC profiles and the percentage recovered. For the production of dried sDSE, maltodextrin was added as an excipient at 0.3 g/mL in the DLE solution, because it was more efficient and allowed for an accurate quality analysis. sDSE or Transferon recovery was calculated by comparing the total quantity of dried product against the total quantity of liquid product. sDSE characterization by SE-UPLC For the characterization of dried sDSE, we used SEUPLC as previously described for the characterization of DLE.12 Briefly, 100 mg of dried sDSE was weighed and dissolved in 1 mL of MilliQ water (Millipore); 3 lL of the solution at 30C was injected in an SE-UPLC H class Acquity system with an Acquity BEH 125 size exclusion column 1.7 lm (4.6 · 150 mm; Waters). The chromatograms were generated with a tunable ultraviolet detector (Waters) at 280 nm. The workflow was isocratic at 0.4 mL/min of 50 mM monobasic/dibasic sodium phosphate-buffered solution pH 6.8 (Mallinckdrodt Baker), with 150 mM sodium chloride (Mallinckdrodt Baker) and ultrapure MilliQ water (Millipore); running time was 15 min. To estimate the molecular weight of the product, we used a gel filtration standard (Biorad). The equipment was controlled with Empower software (Waters). sDSE protein concentration To estimate protein concentration in dried sDSE, we used standard curve analysis with SE-UPLC because maltodextrin interfered with conventional methods of calculating protein concentration, such as BCA, Bradford, or UV absorption (data not shown). Before drying, aliquots of sDSE were collected, and protein concentration was quantified with a BCA kit according to the manufacturer’s specifications.20 Once the liquid SDE protein concentration was determined, different volumes were injected into the H class Acquity UPLC (Waters): 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 lL. As a background control, 5.0 lL of potassium sorbate 1.5 mg/mL solution was injected into the UPLC and the signal was subtracted from experimental results. The total area for each chromatogram was quantified and a standard curve was constructed against protein standards. For dried sDSE, 50 mg of powder was dissolved in 1 mL of water and 5 or 10 lL was injected; the total area of the chromatogram was compared with the standard curve. Each sample was measured thrice to evaluate reproducibility. Biological activity of sDSE: survival test We used a previously reported herpes simplex mouse model to evaluate the biologic activity of the dried sDSE.21 Briefly, 5-week-old male BALB/c mice were separated in groups (n = 10): healthy control, infected, maltodextrin treated, sDSE batches 1 and 2 treated, and Transferon control.

SWINE DIALYZABLE SPLEEN EXTRACT AS ANTIVIRAL PROPHYLAXIS

Mice were anesthetized and inoculated with 5 · 106 PFU/mL Herpes Simplex Virus Type 1 (ATCC) by cutaneous scarification on 1 cm2 of plucked dorsal skin. Oral treatment with dried Transferon or sDSE equivalent to 0.5 lg/dose started on day 2 postinfection and was given every other day through day 10. Control treatment consisted of the vehicle (maltodextrin) equivalent of 0.5 lg of sDSE solubilized in 200 lL of water. To evaluate survival, mice were monitored over 20 days in a 12-h dark/12-h light cycle with food and water provided ad libitum. When infection-associated symptoms became severe (total loss of mobility), animals were euthanized and counted as dead for survival analysis. Experimental procedures were approved by the Ethics Committee of the Unidad de Desarrollo, Investigacio´n e Innovacio´n Me´dica y Biotecnolo´gica at the Escuela Nacional de Ciencias Biolo´gicas, Instituto Polite´cnico Nacional (protocol TU/IB/012/

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010/PRO). All animal experiments were performed in accordance with the Mexican Guidelines for the Production, Care, and Use of Laboratory Animals (NOM-062-ZOO1999) and the International Guide for the Care and Use of Laboratory Animals. In vivo cytokine production To determine the in vivo immunologic response to DSE treatment, we measured the levels of IFN-c, IL-6, and TNF-a as previously described in an independent experiment.21 Briefly, blood samples were collected 7 days from mice following infection with herpes simplex 1 and the serum was obtained according to the literature.21 Groups of mice were randomly selected for the following conditions (n = 3): uninfected, infected, vehicle control (maltodextrin), dried sDSE

FIG. 1. A description of the dried swine dialyzable spleen extract (sDSE) production process is shown. Color images available online at www.liebertpub.com/jmf

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FIG. 2. Transferon maintains its physicochemical profile after drying. Transferon, a standardized human dialyzable leukocyte extract (DLE), was spray dried with maltodextrin at 0.3 g/mL. After drying, 300 mg of powder was diluted in 1 mL of water and analyzed by size-exclusion ultra performance liquid chromatography (SE-UPLC). Profiles of DLE analyses performed before and after drying are shown.

0.5 lg/dose, and liquid Transferon 0.75 lg/dose (previous studies have shown similar results by using Transferon at 0.5 or 0.75 lg/dose).21 Collected serum samples were stored at - 20C before use. The concentrations of cytokines in each sample were quantified using the Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Biosciences) and a FACS Aria flow cytometer according to the manufacturer’s instructions. Analysis was performed with CBA FCAP Array Software V.3.0 (BD Biosciences); after obtaining cytokine concentration, every value was divided by the uninfected control to normalize the results. Statistical analysis

was used as an internal control to standardize the process. Formulation with maltodextrin allowed us to evaluate the chromatographic profile of the product. Based on our analysis, Transferon maintains its original characteristics once dried (Fig. 2), with 46.7% recovery of the total vehicle weight. Characterization of dried sDSE An analysis of both the percent recovery and product concentration after drying sDSE was performed (Table 1). Table 1. Results of Dialyzable Extract Powder Production

A Kaplan–Meier plot of animal survival was generated and analyzed for statistical significance with Mantel–Cox tests (a = 0.05) using the software Prism 5 Project V 5.0 (GraphPad). Cytokine production in serum was analyzed using the same software to perform post-tests following one-way ANOVA with the Bonferroni method to compare between groups. RESULTS A standardized method for drying sDSE This alternative drying process allows sDSE to be preserved and transported without special conditions. The process, depicted in Figure 1, consists of four parts: disaggregation, clarification, ultrafiltration, and spray drying. A batch of Transferon, a commercially available human-DLE,

Initial spleen weight (g) Total dialyzable extract used/obtained (mg) Maltodextrin added (g) Amount of powder recovered (g) Dialyzable extract concentration in powder (lg/mg) Dialyzable extract recovered after drying (mg) Dialyzable extract recovered after drying (%)

Transferon (control)

Batch 1

Batch 2

— 947.00

538.84 784.38

2285.30 6112.00

150.00 69.95

306.00 209.01

2400.00 2022.00

3.53

2.27

1.77

246.92

474.45

3578.94

26.07

60.5

58.6

SWINE DIALYZABLE SPLEEN EXTRACT AS ANTIVIRAL PROPHYLAXIS

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FIG. 3. Large-scale and small-scale production of dried sDSE results in similar physicochemical profiles. (A) After drying, sDSE was dissolved in water and analyzed by SE-UPLC. (B) sDSE chromatogram (solid line) was compared to a molecular weight marker (dashed line), which demonstrates that DSE is below a molecular weight of 17 kDa.

To characterize the dried sDSE product, we used SEUPLC. Two different batches of dried sDSE had similar characterization profiles; therefore, we believe that the process is reproducible (Fig. 3A). Each profile is composed of five main peaks, with the major peak corresponding to the preserving agent (data not shown). The molecular weight of dried sDSE was between 0.2 and 17 kDa, which was expected due to the ultrafiltration process (Fig. 3B). Biological activity and cytokine production in vivo To verify that dried sDSE effectively protects against infections, similar to Transferon, we use a previously established herpes infection mouse model.21 Dried Transferon was protective against herpes infection (Fig. 4), supporting previous reports that treating mice with the product is 30% more effective than providing no treatment at all.21 Similar results were observed with our two dried batches of sDSE,

FIG. 4. Dried sDSE treatment protects mice from herpes simplex infection. Mice were infected dermally with herpes simplex virus and treated with dried sDSE or Transferon. Data were analyzed by the Mantel–Cox test (a = 0.05).

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FIG. 5. Dried sDSE induces cytokine production in vivo. Murine serum levels of interferon gamma (IFN-c), TNF-a, and IL-6 were measured 7 days after herpes simplex infection (*P < .05 by one-way ANOVA).

which protected 70% and 60% of mice, respectively, compared with controls. This suggests that dried sDSE can protect against herpes simplex infection. During the infection, the levels of IFN-c, TNF-a, and IL-6 were measured at day 7 (Fig. 5). There was a significant increase in IFN-c in sDSE-treated mice compared with untreated infected mice and uninfected mice. However, TNF-a levels remained unchanged, and there was a slight decrease in IL-6 production when mice were administered sDSE. DISCUSSION Dialyzable extracts are a complex mixture of lowmolecular-weight peptides from different sources that have been widely studied due to their immunoregulatory properties in different animal models of human diseases. sDSE, an extract obtained through disrupted spleen cells from pigs, has shown to induce cytokine production in vitro and in vivo, indicating an immunomodulatory activity that may be useful for veterinary use.13,19 In this study, we describe a process to dry sDSE without altering its main attributes: physicochemical characteristics and biological activity. A biotherapeutic human-DLE distributed in Mexico, Transferon is the only one product to the best of our knowledge that had the category of a drug worldwide and can be used as standard to analyze dried sDSE properties.12 Previous studies in Transferon have shown that its chromatographic profile is constant in relation with its biologic activity.12,21 Also, the chromatographic profile of Transferon did not show changes after drying with the excipient. To characterize dried sDSE, we decided to apply the same test before search properties like peptide composition. In addition, other quality control tests were performed to assure the safety of the product, like endotoxin levels and microbiological assays (data not shown). This product may be included in a balanced hog diet on farms as prophylaxis against infection. Production of soluble sDSE has been previously described.22 However, veterinarian use of the soluble form would require refrigerated transportation (to assure stability of the product over prolonged periods) and complex diet mixing. The described sDSE drying process not only improves production

but also eliminates the issues mentioned above for use in veterinary applications. The sDSE drying process requires four main steps: tissue disruption, clarification, ultrafiltration, and spray drying. The first three steps are based on the production process of human-DLE, such as Transferon.12 The use of water for tissue disruption and cell lysis allows the release of intracellular peptides, the main component of sDSE. Clarification and ultrafiltration select components with a molecular weight less than 10 kDa. We used SE-UPLC analysis of Transferon to assess the best drying conditions that limit the loss of physicochemical properties. Two maltodextrin solution concentrations were tested for drying: 0.3 or 0.5 g/mL. Although the excipient generates a chromatographic background, it is easy to eliminate it for DLE profile analysis (Fig. 2). The peptide composition of sDSE was distributed between 0.2 and 17 kDa, as described previously for Transferon.12 Furthermore, this demonstrates that the spray-drying technique suitably preserves the physicochemical properties of the product. To determine if the established drying conditions are reproducible at greater scales, we produced two batches of sDSE: 1 or 10 L of product generated. For batches 1 and 2 of 149.06 g and 762.82 g of tissue extracted, we obtained 0.53% and 0.80% of sDSE (calculated against total spleen weight), respectively, after ultrafiltration. Furthermore, when the product was dried, we obtained 474.45 and 3578.94 mg per batch, reaching a recovery of 60.5% and 58.6% of sDSE in the dried preparation (Table 1). This indicates that the drying process is reproducible between the two scales. Regarding the peptide content of the product, batches 1 and 2 contained 2.27 and 1.77 mg/g of product. Importantly, this difference is influenced by the volume dried and the initial amount of spleen obtained per litter. We found that batches 1 and 2 had similar SE-UPLC profiles and, similar to Transferon, are composed of peptides within 0.2 and 17 kDa (Fig. 3A and B).12 The sDSE profiles have 4–5 peaks. The main peak corresponds to the preservative potassium sorbate (data not shown), whereas the others correspond to sDSE peptides. Although dried sDSE was designed as a supplement to prevent swine diseases in hog farms, the study scope is to

SWINE DIALYZABLE SPLEEN EXTRACT AS ANTIVIRAL PROPHYLAXIS

test the sDSE product in small species before our species of interest, to demonstrate the safety and efficacy. Using a swine model requires large facilities that allow us to handle farm animals, controlled environmental conditions, and high costs. Dialyzable extracts, specifically spleen DLE, have previously been tested by other authors in interspecies experiments; hence, we tested our product in a standardized herpes simplex 1 infection mice model used to evaluate human dialyzable extracts.21,23 Human-DLE is an effective treatment for herpetic patients.24 Similarly, sDSE is an effective treatment for herpes simplex 1 infection. Groups treated with water or maltodextrin had a 30% survival rate at day 13, whereas groups treated with dried sDSE reached a survival rate of 60–70%. This survival rate mimicked that reached by the Transferon-positive control. Therefore, maltodextrin does not interfere with DLE or sDSE biologic activity. Furthermore, sDSE has the same effectiveness as in Transferon in the protection against herpes infection. We confirmed in previous reports that Transferon induces IFN-c production in vitro and in vivo.12,25 For many years, this effect has been attributed to the activity of DLE.21 To determine if the effect of sDSE is related to IFN-c induction, we measured serum concentrations from mice seven days after infection.21 We used a standardized batch to evaluate cytokine production due to similarities in physicochemical properties and mice survival between the two batches. Although the literature established that there were no changes in the survival effect by using Transferon at doses of 0.5 and 0.75 lg, future studies must consider using the same dose of the reference to compare it with products from different sources.21 As seen in Figure 5, sDSE treatment significantly exacerbates IFN-c production in mice compared with untreated controls. A hog farm study demonstrated that treating 21-day-old pigs with 77 lg/kg of DLE for 30 days significantly increased IFN-c production compared to age-matched controls; this result is consistent with our study as sDSE induced cytokine production.26 The production of IFN-c may also protect against candidiasis infection based on murine DLE treatment in a different mouse model.27 TNF-a is a second inflammatory cytokine related to DLE treatment. In this study, we did not observe changes in TNF-a production following treatment with dried sDSE or Transferon, although previous data indicate that Transferon causes a decrease in this cytokine.21 Finally, we observed a change in the levels of serum IL-6 following sDSE treatment. In another study with a similar response to human-DLE treatment shown here and previously reported, IL-6 levels slightly decreased in infected mice treated with dried sDSE.21 It is important to consider that dried sDSE and Transferon are made from different sources (swine spleen and human leukocytes, respectively), and thus the production processes carry some differences.28 Although we can see similitudes in mice survival, it is possible that slight peptide differences between both species lead to a different mechanism of action and different cytokine production modulation. In conclusion, we developed an alternative method for the production of dried sDSE that preserves its physicochemical

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properties and biological activity. This product enhanced survival and regulated cytokine production in our murine herpes infection model. These results provide a basis to test sDSE in our species of interest for potential large-scale industrial purposes.

ACKNOWLEDGMENT This research received financial support from the National Council of Science and Technology (CONACyT) under Project number CONACYT-INFR-2014-01-225313. AUTHOR DISCLOSURE STATEMENT S.V.-L., G.P.-S., N.S.-J., and S.M.P.-T. are employees or have been compensated for their work by Unidad de Desarrollo, Investigacio´n e Innovacio´n Me´dica y Biotechnolo´gica (UDIMEB), the producer of Transferon. All other authors declare no competing financial interests. REFERENCES 1. Bobadilla Soto E, Espinoza Ortega A, Martı´nez Castan˜eda FE: Dina´mica de la produccio´n porcina en Me´xico de 1980 a 2008. Rev Mex Cienc Pecu 2010;1:251–268. 2. Orr DEJ, Shen YR: World pig production, opportunity or threat? Swine Nutrition Conference Proceedings 2006;2006:3–8. 3. Meyer S, Steiner L: Daily Livestock Report. 2010;8. 4. Garcı´a Arias KI: Panorama del porcino. Mexico: Financiera Nacional de Desarrollo Agropecuario, Rural, Forestal y Pesquera, 2014. 5. Blaha T: Animal health, animal welfare, pre-harvest food safety and protecting the environment as key elements of animal production. Proceedings of The International Society for Animal Hygiene (ISAH) 2005. 6. Meng XJ: Emerging and re-emerging swine viruses. Transbound Emerg Dis 2012;59:85–102. 7. Janke BH: Clinicopathological features of swine influenza. In: Swine Influenza (Richt JA, Webby RJ, eds.). Springer Berlin, Heidelberg, Germany, 2013, pp. 69–83. ´ valos 8. Salinas Mele´ndez JA, Lara Arias J, Flores Andrade H, A Ramı´rez R, Za´rate Ramos JJ, Riojas Valde´s V, et al: Presencia de animales seropositivos al sı´ndrome reproductivo y respiratorio porcino en Nuevo Leo´n. Vet Me´x 2008;39:215–221. 9. Me´xico reporta mortal virus porcino en 17 estados. El Universal 2014 May 22. 10. Lewis DB, Yu CC, Meyer J, English BK, Kahn SJ, Wilson CB: Cellular and molecular mechanisms for reduced interleukin 4 and interferon-gamma production by neonatal T cells. J Clin Invest 1991;87:194–202. 11. Zelnickova P, Leva L, Stepanova H, Kovaru F, Faldyna M: Agedependent changes of proinflammatory cytokine production by porcine peripheral blood phagocytes. Vet Immunol Immunopathol 2008;124:367–378. 12. Medina-Rivero E, Merchand-Reyes G, Pavo´n L, Va´zquez-Leyva S, Pe´rez-Sa´nchez G, Salinas-Jazmı´n N, et al: Batch-to-batch reproducibility of Transferon. J Pharm Biomed Anal 2014;88: 289–294. 13. Hofer M, Vacek A, Lojek A, Hola J, Streitova D: Ultrafiltered pig leukocyte extract (IMUNOR) decreases nitric oxide formation and hematopoiesis-stimulating cytokine production in

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