Pediatric Nonclinical Drug Testing (Pediatric Nonclinical Drug Testing: Principles, Requirements, and Practices) || Juvenile Immunodevelopment in Minipigs

12Juvenile

Immunodevelopmentin Minipigs

Andre H. Penninks, Geertje J.D. van Mierlo, Frieke Kuper, Cor J. Snel,Niels-Christian Ganderup, and Andre P.M. Wolterbeek

12.1 INTRODUCTION: WHY JUVENILE DEVELOPMENT STUDIESAND WHY IN MINIPIGS?

Change in the legislative landscape governing approval of new medicinal productsin Europe and North America is the main driving force behind the recent focusof the pharmaceutical industries on juvenile toxicity testing. Pediatric regulationhas been in place in the United States since 1997 [1,2], and more recently, since2006 [3], in the European Union also. Before that time, drugs, which had beenevaluated for safety and efficacy in adult subjects, were most often prescribed offlabel, with doses being adjusted to the lower body weights of children. It hassince become clear that, as the aforementioned regulations testify, children are not“small adults.” Anatomical and functional development, as well as metabolic andphysiological processes, may differ considerably between children and adults. Even“children” as used here, is a broad expression that spans ages from extreme pretermneonates to children in their late teens. The differences mentioned above are by nomeans exhaustive; age may also impact underlying mechanisms of disease, diseaseprogression, and the pharmacokinetic and pharmacodynamic response to drugs. In

Pediatric Nonclinical Drug Testing: Principles, Requirements, and Practices,First Edition. Edited by Alan M. Hoberman and Elise M. Lewis.© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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232 JUVENILE IMMUNODEVELOPMENT IN MINIPIGS

the European Union, a Marketing Authorization Application (MAA) must containa pediatric investigation plan (PIP), waiver, or deferral in order to be filed, whichis not the case in the United States. In the European Union, the PIP is discussedearlier in the regulatory process, compared to the United States.

The rat is the species of choice for juvenile toxicity studies, even though rats arenot easily compared to human adults, a caveat that can be overcome to some extentby the considerable volume of background data available on various developmentalaspects of the rat.

In some instances, such as for logistic and/or scientific reasons, a nonrodentspecies may be required as a second species for juvenile toxicity studies. Numerouspractical challenges, such as small litter size, long gestation period, age of sexualmaturity, and housing conditions, are considered when choosing nonrodent species.With regard to most of these issues, the minipig looks favorable when compared todog, rabbit, or nonhuman primate [4]. Some of the biological characteristics of theminipig are favorable compared to dogs and nonhuman primates; particularly, thelitter size is relatively large (five newborns per primaparus sow) and the birth weightof 400–500 g makes manipulation for dosing and examination straightforwardand surgical intervention possible (e.g., catheterization or implantation of vascularaccess port, telemetric equipment, etc.). Moreover, the size at birth allows forsampling of larger blood volumes if needed, the piglets are born fairly autonomous(they walk around within minutes after being born) and are fairly robust, they reachsexual maturity at an early age (three to four months for males and four to fivemonths for females), they grow fast (about 2 kg/mo during the first year), and thereis often less emotive stress on staff when working with piglets than, for example,with puppies. This latter aspect is merely a reflection of the societal view of pigsas consumption (food) animals and has nothing to do with the minipig’s capacityto experience pain and suffering.

Within the past 10 years, the minipig is being increasingly used in toxicologicaland pharmacological studies, and to a lesser extent, also in juvenile studies. For usein juvenile toxicity studies, the developmental characteristics of main organ systemsof the minipig still require some improvement. Therefore, the aim of our study isto gain more knowledge about physical and sensory development of the minipig(developmental landmarks); hematological, clinical chemistry, and immunologicalparameters; as well as the histopathology of main organs (including lymphoidorgans/tissues) during early development.

The immune system of the pig has been studied extensively, particularly in rela-tion to different infectious diseases in pigs and xenotransplantation. The knowledgeof the pig immune system is, therefore, more extensive than that of other nonrodentspecies. Along with similarities to other mammals, some differences also exist inthe basic structure of the pig immune system. For instance, the lymph nodes have aninverted morphology when compared to that of other nonrodents and humans; how-ever, this does not affect lymph node function but results in different lymphocytetrafficking in the lymph node [5]. Moreover, the lymphocyte subset compositiondiffers, and unlike rodents and humans, pigs have quite a lot of γδ-T cells [6], highnumbers of CD4+CD8+ double-positive T cells in their periphery like monkeys

ANIMALS 233

[7], and quite high numbers of natural killer (NK) cells (up to 60%) [8]. Thesedifferences do not result in differences in the overall functioning of the immunesystem.

The extensive volume of published literature pertaining to the pig immunesystem and its functioning has also opened novel opportunities for the potentialuse of the minipig in assessing responses of the immune system to biopharma-ceuticals (or small molecular entities) for safety and/or efficacy purposes. Someimmune responses in pigs following exposure to some classical immunosuppressivedrugs have been reported [9,10]. More recently, several quantitative and qualita-tive immune end points were evaluated in a subacute immunotoxicity study inGottingen Minipigs® following exposure to the classical immunosuppressive com-pounds cyclosporin A and dexamethasone [11,12].

To evaluate the potential utility of the minipig as a test model for the safety eval-uation of new biopharmaceuticals (e.g., proteins, monoclonal antibodies), it is ofutmost importance that the biopharmaceutical is biologically active in the minipig,for example, the homologous molecular targets in minipig tissues should react withthe human biopharmaceutical in development, resulting also in pharmacologicalactivity.

This chapter reports on the developmental aspects of lymphoid organs (organweights and microscopy), selected hematology parameters (white blood cell (WBC)differentiation, lymphocyte subset analysis), and clinical chemistry parameters (totalprotein, albumin and albumin/globulin (A/G) ratio) in minipig piglets from neonatalstage to six months of age. The development in time of the other main organs/tissues(∼20) will be published separately by Wolterbeek and colleagues.

12.2 STUDY DESIGN

Physical and sensory development of the minipig (developmental landmarks),hematological and clinical chemistry parameters, immunological parameters, andhistopathology of main organs during early development were recorded in thisstudy. Seven groups of Gottingen Minipigs were killed at approximately (postnataldays) (PNDs) 2, 7, 14, and 28 and at about 8 weeks, 3 months, and 6 months ofage. All groups contained four males and four females, except for the group killedon day 7, which consisted of six males and one female. As the sex ratio of thepiglets delivered was 58%, the piglets killed on day 7 had this uneven distributionof males:females (6:1).

12.3 ANIMALS

12.3.1 Methods

The study was conducted with male and female Gottingen Minipigs that wereobtained from Ellegaard Gottingen Minipigs®, Denmark. Five pregnant minipig


sows delivered the piglets for the different groups up to week 4. Minipigs olderthan four weeks at necropsy were shipped directly from the breeder. After arrival,the animals were acclimatized to the laboratory conditions. The animals were keptin indoor pens, according to the European guidelines for housing of laboratoryminipigs. Pregnant animals were housed individually and kept together with theirpiglets after delivery. Other animals were housed in groups of four animals persex in indoor pens. The animals were housed under conventional conditions in oneroom, which was ventilated with ∼10 air changes per hour and maintained at atemperature of 20–24◦C with a relative humidity of at least 40% and not exceeding70% other than during room cleaning and meteorological circumstances. Lightingwas artificial with a 12/12-h light/dark cycle.

Animals were socialized to staff by frequent handling (e.g., by rubbing the backand udders of the sows and by hand feeding apples). Furthermore, the number ofbiotechnicians caring for the farrowing sows was limited.

After birth, the sows were given 0.5 mL oxytocin by intramuscular (IM) injec-tion in the neck muscle. The piglets were individually identified by ink marks ontheir backs. One day after birth, they were given an IM injection of ferridextran(1 mL/kg) in the neck muscle to prevent anemia.

The sows were fed a commercial diet (SMP (E) SQC) and received a measuredamount of food once or twice each day (600 g/d). Each batch of diet was analyzedby the supplier (SDS Special Diets Services, Whitham, England) for nutrients andcontaminants.

Piglets were offered a solid piglet diet, 30 g per piglet twice daily, from PND14 until weaning (piglet diet was delivered by Ellegaard Gottingen Minipigs). The6-week old piglets were fed a combination of piglet diet and SDS minipig diet.Feed and apples in limited amounts were also used to socialize the animals. Theanimals were provided with domestic tap water suitable for human consumption(quality guidelines according to Dutch legislation based on EC Council Directive98/83/EC).

Pen-side observations, to detect signs of ill health, reaction to treatment, as wellas moribund or dead animals were conducted and recorded at least once daily.Piglets were weighed on PNDs 1, 4, 7, 14, 21, and 28 as well as at schedulednecropsy in order to calculate the correct organ-to-body-weight ratios.

For necropsy, the animals were sedated by Domitor (medetomidine HCl,1 mg/mL) 0.5 mL/10 kg body weight IM + Dormicum (midazolam, 5 mg/mL)0.5 mL/10 kg body weight IM. Thereafter, the piglets killed on PNDs 1 and 7were anesthetized with Na-pentobarbital (60 mg/mL, ∼1 mL/kg body weight,intracardially) until deep anesthesia, and subsequently, blood was collected fromthe aorta. From all other animals, blood was collected from the heart whileunder sedation; subsequently, the animals were anesthetized with Na-pentobarbital(60 mg/mL, about 1 mL/kg body weight, intracardially) until deep anesthesia.Finally, the animals were euthanized by exsanguination from the axillary arteryand examined for external changes and grossly for pathological changes.

HEMATOLOGY AND CLINICAL CHEMISTRY 235

Piglet body weight

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Figure 12.1 Mean body weight of male (upper line) and female (lower line) piglets up to PND28. N = 5/sex at day 1 and reduced to N = 3/sex at day 25 because of scheduled killing of thepiglets. N = 1/sex at day 28.

12.3.2 Results

12.3.2.1 Body WeightsPiglet mean body weight at PND 1 was 543 g for males and 422 g for females andincreased to 3225 and 2180 g, respectively, at PND 28 (Fig. 12.1). Weight gainswere approximately 91 g/d for males and 65 g/d for females during this period.Before being killed, terminal body weights of the offspring of the sows on PNDs1–4, 7, 14, and 28 and of the minipigs of the second shipment at two, three, andsix months of age were measured (Fig. 12.2). At two months of age, the bodyweights for males and females were equal again. At three and six months of age,the females were slightly heavier than the males; however, these differences werenot significant.

12.4 HEMATOLOGY AND CLINICAL CHEMISTRY

12.4.1 Methods

Blood was collected in tubes with K3-EDTA as anticoagulant (for hematology deter-minations), in tubes containing citrate (for prothrombin time determination), andin heparinized tubes (for clinical chemistry). The parameters determined for hema-tology were hemoglobin (Hb), packed cell volume (PCV), red blood cells (RBCs),reticulocytes, thrombocytes, WBCs, differential WBC (absolute and relative), pro-thrombin time, and calculated values for mean corpuscular volume (MCV), meancorpuscular hemoglobin (MCH), and MCH concentration (MCHC). Of the clinicalchemistry parameters measured, only results for total protein, albumin, and A/Gratio were reported.


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Figure 12.2 Mean terminal body weight of male and female juvenile and young pigs up to sixmonths of age. N = 4/sex except at day 7, when N = 6 for males and N = 1 for females.

12.4.2 Results

There were no remarkable differences in the hematology results between male andfemale piglets. RBC numbers were stable during the first four weeks of life, afterwhich they gradually increased from ∼5–6 × 1012 to ∼8.3 × 1012 cells in malesand from 5.3 × 1012 to 7.3 × 1012 cells in females at six months of age (Fig. 12.3).Hb and PCV reached stable values from week 8 of age, like RBC numbers, withhighest values at six months of age for both sexes (data not shown). The calculatedvalues for MCV, MCH, and MCHC showed minor fluctuations over time. From lowlevels (∼1.5%) at PNDs 1–4, the number of reticulocytes remarkably increased (upto 10–15%) on PNDs 7 and 14, and declined again to stable lower levels (∼0.5%)from three months of age in both males and females. The number of thrombocytesand the prothrombin time were almost constant in time.

During the first 28 days, the number of WBCs was around 4–6 × 109 cells/Lfor both male and female piglets and the percentage of lymphocytes was around50%–60% of the WBCs (Fig. 12.3). On PNDs 1–4, WBC differentiation was notpossible because of the high number of normoblasts. Probably because of partlyclotted blood samples, the WBC value (and SD) of males on PND 28 was alsosomewhat higher with a high SD. The results presented for females on PND 7 arefrom one female only. At two months of age, both male and female piglets hadthe highest number of WBCs, which slightly declined to values of approximately9 × 109 cells/L at six months of age. The percentage of neutrophils is rather highin minipigs, and in this study, it reached values of ∼45% and 25% of the WBCnumbers for males and females at six months of age, respectively.

No remarkable time-related changes were observed for the clinical chemistryparameters, total protein (g/L), albumin (g/L), and A/G ratio, except for the PNDs1–4 and PND 7 values for albumin and A/G ratio (Table 12.1). The total protein

HEMATOLOGY AND CLINICAL CHEMISTRY 237

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Figure 12.3 Absolute WBC, lymphocyte, and RBC counts in PBMCs of male and femaleminipigs up to six months of age. N = 4/sex, except at day 7, when N = 6 for males and N = 1for females.

levels were almost constant for both sexes up to three months of age, with valuesbetween 50 and 60 g/L. At six months of age, the total protein content was ∼64 g/Lfor both sexes. Albumin values were ∼10 g/L at PNDs 1–4, rose to ∼33 g/L onPND 14, remained stable at ∼32 g/L until three months of age, and reached finallevels of 40 g/L at six months of age. Consequently, the A/G ratio also startedsomewhat low on PNDs 1–4 (0.24), rose to ∼1.30 on PND 14, remained constant


TABLE 12.1 Mean Clinical Chemistry Values for Total Protein, Albumin, andAlbumin/Globulin Ratio up to Six Months of Age

Total Protein (g/L) Albumin (g/L) Albumin/GlobulinRatio

Age Males Females Males Females Males Females

Day 1–4 56 ± 12 54 ± 7 10 ± 1 10 ± 2 0.24 ± 0.05 0.23 ± 0.03Day 7 51 ± 6 53 23 ± 4 21 0.78 ± 0.70 0.66Day 14 59 ± 2 56 ± 4 33 ± 3 31 ± 3 1.25 ± 0.13 1.29 ± 0.21Day 28 58 ± 3 58 ± 1 35 ± 4 36 ± 2 1.60 ± 0.39 1.68 ± 0.102 mo 52 ± 3 51 ± 5 31 ± 1 30 ± 3 1.46 ± 0.13 1.39 ± 0.033 mo 54 ± 2 57 ± 12 31 ± 2 33 ± 2 1.33 ± 0.07 1.39 ± 0.186 mo 63 ± 2 64 ± 5 38 ± 3 40 ± 3 1.54 ± 0.16 1.68 ± 0.22

N = 4/sex except at day 7, where N = 6 for males and N = 1 for females.

around 1.4 up to three months of age, and was finally ∼1.6 at six months of agefor both sexes.

12.5 LYMPHOCYTE SUBSET ANALYSIS

12.5.1 Methods

The blood, collected in heparinized tubes just before necropsy, was processed toobtain a single-cell suspension used for lymphocyte subset analysis. Blood sam-ples were diluted in PBS, after which PBMC were isolated using Ficoll-Paque.After separation, cells were washed twice in PBS, after which cell suspensionswere prepared in an enriched RPMI medium. The number of cells was determinedusing an automated hematology analyzer (K-800, Sysmex Toa, Kobe, Japan). Asuspension with a final cell concentration of 10–25 × 106 cells/mL was preparedin an enriched RPMI medium. PBMC suspensions were frozen in 10% DMSO/40%FCS at −80◦C for at least one day and stored in liquid nitrogen for future analysis.Immunophenotyping of WBCs by multiparameter flow cytometric analysis wereconducted using these stored single-cell suspensions.

Monoclonal antibodies conjugated with either fluorescein isothiocyanate (FITC),or phycoerythrin (PE) were used. In all cases, specific antibodies against pig anti-gens were used, except the B-cell-FITC, which was directed against a broad rangeof species. The following antibody combinations were used to label each single-cellsuspension:

1. CD3-FITC (1:100; Pharmingen, #559582)/CD4-PE (1:50; Abcam,#AB25354) for staining helper T cells;

2. CD3-FITC/CD8-PE (1:250; Abcam, #AB25414) for staining cytotoxic Tcells;

3. CD4-PE/CD8-FITC for staining double-positive cells;

4. B-cell-FITC (CD79a; 1:5; Abcam, #AB74693) for staining of B cells;

LYMPHOCYTE SUBSET ANALYSIS 239

5. CD2-PE (two-step stain) (CD2 pur (1:2000; Serotec, MCA 1156) and Goatantirat IgM FITC (1:400; Serotec, #302009)/CD3-FITC for staining CD2+ Tcells;

6. Isotype control antibodies

After thawing and washing with PBMC, for each labeling, 100–150 μL of theblood cell suspension was put in a well of a 96-well plate. After washing withPBS/1% BSA, the supernatants were discarded and the cell pellets were resus-pended in 50 μL of the appropriate diluted antibody mixture and subsequentlyincubated for 20–30 min at 4◦C in the dark. After washing with PBS/1% BSA toremove unbound antibodies, the cell pellet was resuspended in 4% paraformalde-hyde. Analyses were done using a BD FACSCanto™ II flow cytometer within oneweek after staining.

B cells were stained using an antibody recognizing an intracellular epitope ofCD79a. For this staining, cells were fixed with PBS/4% PFA and stored at 2–10◦Cuntil staining. Intracellular staining procedure for samples from males was per-formed immediately after fixing the cells. Samples from females were stored at2–10◦C for five days in PBS/4% PFA before the staining procedure was started.Staining was performed by incubating the cells for at least 15 mi at 2–10◦C in thedark with Perm/wash (BD Biosciences). After centrifugation and removal of thePerm/wash, antibody was added and incubated for 30 min at 2–10◦C in the dark.After washing, cells were resuspended in PBS and measured on a flow cytometer(BD FACSCanto II).

The relative cell numbers (percentage) of the different cell populations weredetermined for each cell sample during phenotypic analysis using BD FACSDiva™software (v6.1.1). Percentages presented are corrected for isotype control staining.

12.5.2 Results

Results of lymphocyte subset analysis of male and female piglets are presented inFig. 12.4. Both in male and in female animals, the percentage of CD3+ T cellsincreases with age. The percentage of CD3+/CD4+ T helper cells rises betweenweek 4 and week 8, after which it remains constant in males. In female animals, theCD3+/CD4+ T helper cells rise between days 1–4 and week 4, staying constantuntil six months of age. For CD3+/CD8+ cytotoxic T cells, a gradual increase isobserved over time in both male and female animals. CD4+/CD8+ double-positivecells fluctuated over time. A rise in these cells over time was expected as this wasreported by Solano-Aguilar et al. [13], Stepanova et al. [14], and Zuckermann andHusmann [15]. Why this rise in CD4+/CD8+ double-positive cells is not observedin the present study is not clear. It is, however, known that the results of lymphocytesubset analysis are influenced by differences in housing and breeding conditions(SPF, Conventional, etc.) [13].

CD2 is seen on a subset of B cells [16], a subset of T cells [16], and cells withNK cell activity [17]. CD2 is regarded to be a developmental marker on B cells[18]. CD2+/CD8+γδ T cells are absent or rare in fetal and germ-free pigs but rise


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Figure 12.4 Lymphocyte subset analysis in male and female minipigs: total CD3+ = totalT lymphocytes, CD3+/CD4+ = T helper lymphocytes, CD3+/CD8+ = cytotoxic T lympho-cytes, CD4+/CD8+ = suppose to be memory T lymphocytes, CD2+/CD3+ = T lymphocytes,CD79a+ = B lymphocytes.

when animals are kept under conventional conditions [19]. In accordance with thestudy by Sinkora et al. [19], in the present study, we seen a rise in the percentageof CD2+ T cells with time in both male and female animals.

In females, the measurements of week 4 do not seem to fit in the overall pictureof the different time points in most of the calculated lymphocyte subtypes. Asall measurements on female PBMC were performed on one day and in the sameanalysis cycle, we believe these data are reliable. Furthermore, also in the literature,fluctuations in lymphocyte populations are observed over time [14].

PATHOLOGY 241

B-cell percentages in female animals are remarkably higher than in male animals.Whether or not this is a true difference is hard to say, as there was a differencein the procedure of the staining between male and female PBMC. Owing to adefect of the flow cytometer, B-cell staining of the female animals was postponed,leaving the PBMC of the female animals stored in PBS/4% paraformaldehydefor five days. The time course of the B-cell percentages across sample points iscomparable in male and female animals. The percentages of B cells are quite stableduring aging. This is consistent with the finding that B-cell repertoire in neonatepiglets is complete [20].

12.6 PATHOLOGY

12.6.1 Methods

After blood collection at necropsy and exsanguination from the axillary artery, allanimals were examined for external changes and for gross pathological changes.

Samples of the following lymphoid tissues and organs of all animals werepreserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde:axillary lymph nodes, cervical lymph nodes, GALT (gut-associated lymphoid tis-sue including Peyer’s patches (PP)), BALT (bronchial-associated lymphoid tissue),mesenteric lymph nodes, spleen, sternum with bone marrow, and thymus. Samplesof these tissues and organs were embedded in paraffin wax, sectioned at 5 μm, andstained with hematoxylin and eosin (H&E) for microscopic examination.

12.6.2 Results

12.6.2.1 Organ WeightsBefore the animals were killed, the terminal body weight was evaluated to calculatethe relative organ weights. At necropsy, the cervical lymph nodes, the mesentericlymph nodes, the spleen, and the thymus were weighed before fixation (Fig. 12.5).At day 1–4, the cervical lymph nodes could not be located, and they are thus notincluded in Table 12.2. The mesenteric lymph nodes at that age were difficult todetect and could not be removed properly from the surrounding fatty tissue, hencetheir weights are not recorded either. Overall, the absolute weight of the organsincreased over time. In both male and female piglets, the relative spleen weightincreased during the first month and then stabilized.

12.6.2.2 MicroscopyThe development of lymphoid organs was microscopically examined in all agegroups, and the results are reported in Table 12.2 and Figs. 12.6–12.10.

Thymus Already at day 3, the thymus was fully developed into a distinct medullaand cortex (Table 12.2). The cortex:medulla ratio (“large relative medulla size”)was relatively low in these young animals, when compared with the thymus of

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PATHOLOGY 245

older animals (Fig. 12.6a,b). H&E-stained sections are not optimal to examine theso-called epithelial-free areas [21]. They were observed from day 10 onward butmay have been present at day 8 as well (Fig. 12.6c).

Spontaneous background histopathology was not observed. Ectopic parathyroidtissue in a 26-week-old female was the only remarkable observation.

Spleen As expected, the size of PALS became larger with age and reached itsoptimal size (size relative to overall spleen size) in some males as early as day 14and in females at 8 weeks of age. The B-cell areas (primary follicles) in the whitepulp could be observed from day 14 onward, and germinal centers (secondary folli-cles) developed from 8 weeks of age. Ellipsoids were present at all ages examined(not scored). The marginal zone was not very prominent at any age, except at week26 (and in a single female at day 28 and week 13).

Extramedullary hematopoiesis in the red pulp was seen from day 3 to day 28(weaning) (Fig. 12.7a,c). From two months onward, the bone marrow showed anincrease in adipose tissue (see section titled “Bone Marrow (Sternum)”). Together,these two observations suggest a decrease in demand for hematopoiesis at aroundtwo months of age (Fig. 12.7b). Interestingly, the red pulp was (over)filled witherythrocytes (“blood-filled red pulp”) in 14- and 28-day-old animals, especially inthose with no or only limited hematopoiesis (Fig. 12.7c).

Lymph nodes (Mesenterial, Superficial Cervical, Cervical/Axillary) Cervi-cal/axillary lymph nodes could not be found at necropsy in three-day-old animals,but the superficial cervical and mesenteric lymph nodes were already discernible atthat age (Fig. 12.8a). Germinal center development started in the mesenteric lymphnodes at day 3, but in a single female only. The germinal centers in the cervicallymph nodes were detected at day 7 (Fig. 12.8b,c); while their development wasmuch later in the superficial cervical lymph nodes (males: day 14, females: day 28).The development was complete at week 8 in all three types of lymph nodes, butgerminal centers were never a prominent feature in the superficial cervical lymphnodes.

The characteristic areas with decreased numbers of lymphocytes, or even devoidof lymphocytes, were especially prominent in the superficial cervical lymph nodes(males and females: from day 14 onward), but much less so in the cervical (day28, males) and mesenteric (days 14 and 28) lymph nodes. Apparently, the cervi-cal/axillary and mesenteric lymph nodes are more continuously stimulated than thesuperficial cervical lymph nodes.

Brown pigment accumulated in the subcapsular sinuses of the cervical and super-ficial cervical lymph nodes from day 7 until day 28. In the mesenteric lymph nodes,it was observed at day 7 in all males and at days 7 and 14 in single females. Thepresence of this pigment could not be explained. Black pigment deposits wereobserved in the superficial cervical lymph nodes at almost all time points, exceptfor week 26. The origin of the deposits is unclear (animals were not tattooed).


(a)

(b)

(c)

Figure 12.6 (a) Overview of thymus at PND 14; (b) overview of thymus at six months of age;large C:M ratio compared to thymus aged 14 days (C = cortex, dark areas; M = medulla, lightareas); (c) details of thymus at day 14, cortex, medulla, and epithelial-free area.

PATHOLOGY 247

(a)

(b)

(c)

Figure 12.7 (a) Details of spleen, male, PND 7; extramedullary hematopoiesis and distinctellipsoids in the marginal zone and the red pulp. Ellipsoids are macrophages and reticular fibersthat surround a capillary. (b) Details of spleen, female, three months of age; germinal center,capsule, and trabeculae rich in elastic fibers. (c) Details of spleen, male, day 14. Red pulp isfilled with erythrocytes, widely dispersed ellipsoids.


(a)

(c)

(b)

Figure 12.8 (a) Overview of mesenteric lymph node, male, day 3. The lymph nodes of thepig are reversed, with respect to both the cortex and the medulla (the medulla surrounds thecortex, which is centrally located) and the lymph flow. (b) Overview of mesenteric lymph node,male day 8. (c) Overview of mesenteric lymph node, male, day 91.

PATHOLOGY 249

(a) (b)

Figure 12.9 (a) Bone marrow with high cellularity, male, 2 months. (b) Bone marrow withadipose tissue and low numbers of hematopoietic cells, 3 months.

Bone Marrow (Sternum) Hematopoiesis and the presence of mature WBCs(bone marrow cellularity in the sternum) were high until 8 weeks of age, when themajority of the animals began to show an increase of adipose tissue at the cost ofWBCs and their precursors (“decreased cellularity of bone marrow”) (Fig. 12.9a,b).Males may begin with this process slightly earlier, because one male showed thischange already at day 28 and the incidence at week 8 was higher in males than infemales.

Very young animals (males at days 3 and 7; females at day 3) showed a highnumber of blast cells in the bone marrow, suggesting a high number of immaturecells at that age. Although the various cell lineages were not examined in detail andthe different cell types were not counted, mature granulocytes appeared to becomemore prominent with age.

Mucosa-Associated Lymphoid Tissues Mucosa-associated lymphoid tissue inthe small intestines (PP) was found grossly first in the ileum (day 3). From thattime onward, PP were found in the jejunum as well. PP were at first a numberof small primary follicles with inconspicuous interfollicular areas. With age, thesize of the lymphoid tissue increased, especially the size of the follicles. Germinalcenter development was already detectable at day 3, was prominent at day 14–week26 in males (Fig. 12.10a) and week 14 in females, and became less prominent atweek 26 (both males and females) (Fig. 12.10b).

BALT was observed relatively late in the development, namely at day 28. Fromthat time point onward, it was a consistent finding in the animals (Fig. 12.11)


(a) (b)

Figure 12.10 (a) Peyers’s patches, male, day 14. (b) Peyers’s patches, male, six months old.

Figure 12.11 A few small bronchus-associated lymphoid tissues (BALT) in lung of 28-day-oldmale minipig.

SUMMARY 251

12.7 SUMMARY

Piglets were born after a gestation period of 113 days, which is a normal gestationtime for pigs. The mean litter size of almost seven piglets (five, six, seven, eight, andseven pups from the five sows, respectively) is common in the Gottingen minipigs.Piglet body weight at PND 1 (543 g for males, 422 g for females) is comparablewith body weights reported by others [22] Weight gain was rather constant duringthe first month (65 g/d for females and 91 g/d for males).

The number of reticulocytes was strongly increased at PNDs 7 and 14 in bothmale and females and declined to normal levels from 8 weeks of age, suggestiveof a maturation of the erythropoetic system in the first month. This is in line withthe observed high number of normoblasts in blood in these early periods after birthand also the high numbers of blast cells observed in the bone marrow. The numberof normoblasts was high in the first month and decreased to almost zero in week 8.These high numbers of normoblasts and reticulocytes in that period will probablybe associated with the increase in erythrocyte numbers from 4 weeks of age.

The total WBC count rose in the first 8 weeks by a factor of two to three tomaximal values of 14.5 × 109 or 12 × 109 cells/L (males or females), followed bya slight decline to values of approximately 9 × 109 cells/L at six months of age forboth sexes. The increase in WBC count mainly resulted from a rise in lymphocytesand granulocytes, which are both the major contributors to the total WBC count.The percentage of lymphocytes observed in this study was ∼50% for males andalmost 70% for females with neutrophil numbers of, respectively, 45% and 25%of the WBC numbers at six months of age. These values are in agreement withthose reported by Marshall Bioresearch [22] and Jorgensen et al. [23].

For the clinical chemistry parameters, total protein, albumin, and A/G ratiopresented, no remarkable time-related changes were observed. Despite that, in pigs,the transfer of immunity (immunoglobulins) from sow to piglets is by the mammarygland (colostrum/milk) and not via the placenta [18]. The total protein levels inpiglets were found to be stable already from PNDs 1–4 after birth. As during thefirst two weeks, the albumin levels rose to a stable level of ∼35 g/L up to sixmonths of age, and the total protein levels were almost constant during the wholeperiod; the A/G ratio also rose only in the first 2 weeks.

Lymphocyte subset analysis revealed that the percentage of CD3+ T cells rosein particular from PNDs 1–4 to 4–8 weeks of age to ∼50% of the PBMCs andremained stable (males) or slightly increased (females) up to six months of age.The percentage of CD3+/CD4+ helper T cells increased still slightly in the firstfour weeks after birth, especially in females but remained almost constant at levelsof about 15%–20% during the whole observation period. For the CD3+/CD8+

cytotoxic T cells, a gradual increase was observed over time to approximately12% of the PBMCs at six months of age, except for the incidental high levelin females at four weeks of age. The percentage of CD4+/CD8+ double-positivecells fluctuated over time between ∼5% and 10% of the PBMCs, although basedon other reports [13–15], an increase was expected. The percentage of CD2+ Tcells was shown to increase over time to ∼30%–35%, which is in agreement with


the observations by Sinkora et al. [19]. The percentage of B cells fluctuated overtime between 8% and 18% for males and 13% and 23% for females. The slightlyhigher levels observed for females might be because of measurement differences.Overall, differences observed in the percentage of lymphocyte subsets in this studyand results of other studies might be influenced by differences in housing andbreeding conditions of the minipigs [13] and differences in the specificities of theantibodies used for the determination of the various subsets.

The absolute organ weights of the thymus increased for females up to six monthsof age (for males up to three months). For spleen, the absolute weights increasedto high levels in both sexes at four weeks followed by a slight decrease at eightweeks, which was followed again by increasing levels up to six months of age.The high absolute spleen weight, especially at four weeks of age, will probablybe related to the histopathologically observed high extramedullary hematopoiesisin the red pulp of the spleen in the first four weeks, which from eight weeksof age decreased again. Together with the observed increase in adipose tissue inthe bone marrow from two months of age, both these observations are indica-tions of a decreased demand for hematopoiesis after about two months of age,which may have resulted in the decreased absolute spleen weight at eight weeksof age compared to the weights at four weeks of age. From eight weeks of age,the absolute spleen weights increased again. Overall, the absolute weights of thecervical and mesenteric lymph nodes increased over time. The relative thymusweight fluctuated in both sexes slightly during the first four weeks, after which itdropped because of the faster body weight growth but remained almost stable upto six months of age. The relative spleen weight increased in both sexes duringthe first four weeks of age which was probably due to the increasing demand forextramedullary hematopoiesis. From eight weeks of age, the relative spleen weightsdropped, probably as a result of the lower demand for extra hematopoiesis, andremained almost constant until six months of age. The relative cervical lymph nodeweights decreased over time for both sexes, whereas the relative mesenteric lymphnodes increased up to four to eight weeks of age, after which they declined becauseof faster body weight growth. The histopathological evaluation of the different lym-phoid organs showed clear development over time, but no obvious differences werenoticed in the lymphoid tissue structures when compared with other species, exceptfor the inverted morphology of the lymph nodes.

REFERENCES

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2. US FDA. Pediatric Research Equity Act. 2003 Dec, Public Law 108–155.

3. Regulation (EC) No. 1901/2006) of the European Parliament and the Council, 12December 2006 on medical products for pediatric use and amending Regulation (EEC)No. 1768/92, Directive 2001/20/EC, Directive 2001/83/EC and Regulation (EC) No.726/2004.

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5. Binns RM, Pabst R. Lymphoid tissue structure and lymphocyte trafficking in the pig.Vet Immunol Immunopathol 1994;43:79–87.

6. Sinkora M, Sinkorova J, Holtmeier W. Development of gamma-delta thymocyte subsetsduring prenatal and postnatal ontogeny. Immunology 2005;115:544–555.

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