Humanization and simultaneous optimization of monoclonal antibody.

Antibody humanization is an essential technology for reducing the potential risk of immunogenicity associated with animal-derived antibodies and has been applied to a majority of the therapeutic antibodies on the market. For developing an antibody molecule as a pharmaceutical at the current biotechnology level, however, other properties also have to be considered in parallel with humanization in antibody generation and optimization. This section describes the critical properties of therapeutic antibodies that should be sufficiently qualified, including immunogenicity, binding affinity, physiochemical stability, expression in host cells and pharmacokinetics, and the basic methodologies of antibody engineering involved. By simultaneously optimizing the antibody molecule in the light of these properties, it should prove possible to shorten the research and development period necessary to identify a highly qualified clinical candidate and consequently accelerate the start of the clinical trial.

Engineered antibody Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa(R131) and FcγRIIa(H131).

Engaging inhibitory FcγRIIb by Fc region has been recently reported to be an attractive approach for improving the efficacy of antibody therapeutics. However, the previously reported S267E/L328F variant with enhanced binding affinity to FcγRIIb, also enhances binding affinity to FcγRIIa(R131) allotype to a similar degree because FcγRIIb and FcγRIIa(R131) are structurally similar. In this study, we applied comprehensive mutagenesis and structure-guided design based on the crystal structure of the Fc/FcγRIIb complex to identify a novel Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa(R131) and FcγRIIa(H131). This novel variant has more than 200-fold stronger binding affinity to FcγRIIb than wild-type IgG1, while binding affinity to FcγRIIa(R131) and FcγRIIa(H131) is comparable with or lower than wild-type IgG1. This selectivity was achieved by conformational change of the C(H)2 domain by mutating Pro to Asp at position 238. Fc variant with increased binding to both FcγRIIb and FcγRIIa induced platelet aggregation and activation in an immune complex form in vitro while our novel variant did not. When applied to agonistic anti-CD137 IgG1 antibody, our variant greatly enhanced the agonistic activity. Thus, the selective enhancement of FcγRIIb binding achieved by our Fc variant provides a novel tool for improving the efficacy of antibody therapeutics.

Phylogenetic identification of Sparganum proliferum as a pseudophyllidean cestode by the sequence analyses on mitochondrial COI and nuclear sdhB genes.

Sparganum proliferum is a larval cestode for which the adult stage is unknown. It is characterized by the continuous branching and budding when parasitized to humans, and causes fatal human sparganosis. However, the biological features of S. proliferum, including its taxonomic status, still remain obscure. Our previous investigation suggested that S. proliferum might be phylogenetically distinct from Spirometra erinaceieuropaei, by the analysis on mitochondrial NADH dehydrogenase subunit 3 (ND3) gene. However, mitochondrial DNA sequence in Platyhelminth is known to have heteroplasmy within a species. Therefore, in the present study, we have investigated the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene and the partial nucleotide sequences of nuclear coded succinate dehydrogenase iron-sulfur protein subunit gene (sdhB). The results clearly demonstrated that S. proliferum is a distinct species from S. erinaceieuropaei, and that S. proliferum belongs to the order Pseudophyllidea.

Three epiphyseal fractures (distal radius and ulna and proximal radius) and a diaphyseal ulnar fracture in a seven-year-old child's forearm.

SUMMARY: The authors report a rare case of fracture separations at both ends of the radius combined with an epiphyseal and diaphyseal fracture of the ipsilateral ulna. A seven-year-old girl fell one story and sustained a closed injury of her forearm. A closed reduction was unsuccessful, and an open reduction was performed with three of the four fractures being secured with Kirschner wires. These wires were removed one month later, and range-of-motion exercises were started. Thirty months after surgery, both forearms were equal in length, although the proximal radial epiphyseal line appeared partially closed. Joint motions, including forearm rotation, were normal. Radiologically, the ulnar diaphysis and the radial neck were posteriorly convex 20 degrees and 18 degrees, respectively.

Lack of B cell leakiness in BALB/cA-nu, scid double mutant mice.

BALB/cA mice homozygous for both nu and scid mutations (BALB/cA-nu/nu, scid/scid) were developed by mating between BALB/cA-scid and BALB/cA-nu. These mice have greater longevity than C.B-17-scid because no thymic lymphoma occurs in them unlike in the latter. C.B-17-scid is known to show the leaky phenomenon in which a few clones of functional T and B cells develop in aged C.B-17-scid. Unexpectedly, the leaky B cells and T cells were absent or suppressed in BALB/cA-nu, scid mice when cytokine expressions were determined by RT-PCR, lymphocyte phenotypes by flow cytometry and serum immunoglobulin levels by ELISA. These results indicate that B cell leakiness may be induced by leaked T cells. BALB/cA-nu, scid mice may be useful as a recipient in allo- and xeno-transplantation experiments because of the absence of both thymic lymphomas and leakiness, in addition to lack of hair.

Prophylactic effect of FK463, a novel antifungal lipopeptide, against Pneumocystis carinii infection in mice.

The prophylactic effect of FK463, a new water-soluble echinocandin-like lipopeptide with inhibitory activity against 1, 3-beta-D-glucan synthase, against Pneumocystis carinii infection was investigated with the severe combined immunodeficient (SCID) mouse model. Treatment with FK463, pentamidine, and saline only was performed for 6 weeks from the day after the SCID mice were inoculated intranasally with infected lung homogenates. FK463 at 0.2 or 1.0 mg/kg of body weight, pentamidine at 4 mg/kg, or saline was subcutaneously administered daily into the backs of the SCID mice. The effects of the drugs were evaluated by detection of P. carinii cysts in mouse lung homogenates by toluidine blue O staining, lung histology, and PCR amplification of a P. carinii-specific DNA fragment from the lungs. P. carinii cysts were detected in the lungs of all mice administered saline. In contrast, no cysts were detected in mice administered both doses of FK463 and pentamidine. A specific DNA fragment was amplified from all mice administered saline and at least half or more of the mice administered FK463 and pentamidine. These results indicate that FK463 acts on cyst wall formation but not on trophozoite proliferation and is extremely effective in preventing P. carinii-associated pneumonia. These results suggest that FK463 is potentially useful as a prophylactic agent against P. carinii infection.

CD4(+) cells are indispensable for ulcer development in murine cutaneous leishmaniasis.

One of the most characteristic clinical features in cutaneous leishmaniasis is the development of nodules followed by ulcerations at the site of infection. Leishmania amazonensis-infected mice show similar ulcerative lesions. Leishmania-infected severe combined immunodeficiency (SCID) mice, however, have been shown to develop nonulcerative nodules. In the present study, the roles of T cells in ulceration were examined using SCID mice in cell reconstitution experiments. After development of nonulcerative nodules, SCID mice were inoculated with splenocytes from either Leishmania-infected or naive immunocompetent mice, resulting in ulceration in all mice. When naive splenocytes were depleted of CD4(+), CD8(+), or B220(+) cell populations and the remaining cells were injected into Leishmania-infected SCID mice after the development of nodules, only SCID mice inoculated with splenocytes depleted of CD4(+) cells did not show ulceration. The evidence obtained in this study clearly shows that the CD4(+) cell population is indispensable for ulceration in leishmaniasis lesions of SCID mice.

Mutant presenilin 2 transgenic mice. A large increase in the levels of Abeta 42 is presumably associated with the low density membrane domain that contains decreased levels of glycerophospholipids and sphingomyelin.

The N141I mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease in the Volga German families. We previously reported that mouse brains harboring mutant PS2 contained increased levels of amyloid beta protein (Abeta) 42 in the Tris-saline-soluble fraction (Oyama, F., Sawamura, N., Kobayashi, K., Morishima-Kawashima, M., Kuramochi, T., Ito, M., Tomita, T., Maruyama, K., Saido, T. C., Iwatsubo, T., Capell, A., Walter, J., Grünberg, J., Ueyama, Y., Haass, C. and Ihara, Y. (1998) J. Neurochem. 71, 313-322). Here, using a new extraction protocol, we quantitated the Abeta40 and Abeta42 levels in the Tris-saline-insoluble fraction. The insoluble Abeta levels were found to be higher than the soluble Abeta levels, and the insoluble Abeta42 levels were markedly increased in mutant PS2 transgenic mice. To investigate the origin of the insoluble Abeta42, we prepared the detergent-insoluble, low density membrane fraction. This fraction from two independent lines of mutant PS2 transgenic mice contained remarkably increased levels of Abeta42 and significantly low levels of glycerophospholipids and sphingomyelin. This unexpected finding suggests that a large increase in the levels of Abeta42 in mutant PS2 mice is presumably induced through alterations of the lipid composition in the low density membrane domain in the brain.

Frequency-encoding Thr17 phospholamban phosphorylation is independent of Ser16 phosphorylation in cardiac myocytes.

Both Ser(16) and Thr(17) of phospholamban (PLB) are phosphorylated, respectively, by cAMP-dependent protein kinase (PKA) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). PLB phosphorylation relieves cardiac sarcoplasmic reticulum Ca(2+) pump from inhibition by PLB. Previous studies have suggested that phosphorylation of Ser(16) by PKA is a prerequisite for Thr(17) phosphorylation by CaMKII and is essential to the relaxant effect of beta-adrenergic stimulation. To determine the role of Thr(17) PLB phosphorylation, we investigated the dual-site phosphorylation of PLB in isolated adult rat cardiac myocytes in response to beta(1)-adrenergic stimulation or electrical field stimulation (0. 1-3 Hz) or both. A beta(1)-adrenergic agonist, norepinephrine (10(-9)-10(-6) m), in the presence of an alpha(1)-adrenergic antagonist, prazosin (10(-6) m), selectively increases the PKA-dependent phosphorylation of PLB at Ser(16) in quiescent myocytes. In contrast, electrical pacing induces an opposite phosphorylation pattern, selectively enhancing the CaMKII-mediated Thr(17) PLB phosphorylation in a frequency-dependent manner. When combined, electric stimulation (2 Hz) and beta(1)-adrenergic stimulation lead to dual phosphorylation of PLB and exert a synergistic effect on phosphorylation of Thr(17) but not Ser(16). Frequency-dependent Thr(17) phosphorylation is closely correlated with a decrease in 50% relaxation time (t(50)) of cell contraction, which is independent of, but additive to, the relaxant effect of Ser(16) phosphorylation, resulting in hastened contractile relaxation at high stimulation frequencies. Thus, we conclude that in intact cardiac myocytes, phosphorylation of PLB at Thr(17) occurs in the absence of prior Ser(16) phosphorylation, and that frequencydependent Thr(17) PLB phosphorylation may provide an intrinsic mechanism for cardiac myocytes to adapt to a sudden change of heart rate.

Human acute myeloblastic leukemia-ascites model using the human GM-CSF- and IL-3-releasing transgenic SCID mice.

To generate an appropriate model for human acute myeloblastic leukemia (AML), we have successfully established a human hematopoietic growth factor-dependent AML cell line (TF-1 and UT-7/GM)-ascites model using human granulocyte-macrophage colony-stimulating factor (hGM-CSF)- and human interleukin 3 (hIL-3)-releasing transgenic (Tg)-SCID mice. When 1 x 10(7) cells of TF-1, a human erythroleukemia cell line, were transplanted into the peritoneum of irradiated Tg-SCID mice (TF-1 ip/Tg-SCID mice), TF-1 cells grew in both the single cell suspension form (asTF-1) and solid form in ascites and invaded various tissues: lungs, liver, pancreas, and genitals, 3-6 weeks following transplantation. Subsequently, 0.5-1 x 10(7) cells of UT-7/GM, a subline of the UT-7 human megakaryoblastic leukemia cell line, grown in the back of hGM-CSF Tg-SCID mice after subcutaneous inoculation, were transplanted into the peritoneum of other irradiated hGM-CSF Tg-SCID mice. After 4 weeks, UT-7/GM cells (asUT-7/GM) also grew in the same manner as TF-1 cells in hGM-CSF Tg-SCID mice. Analysis of the cells from the peritoneum and tissues by PCR amplifying ALU and human GM-CSF receptor beta sequences and by immunohistochemical staining using anti-human CD45 revealed that they possessed the original characteristics of the parental cells. To confirm the usefulness of this human AML-ascites model, experimental treatment of AML cells grown in these mice was carried out with a differentiation inducer, delta-aminolevulinic acid (deltaALA), which induces hemoglobin synthesis for TF-1 in vitro and is thus regarded as an anti-leukemia drug candidate. Unexpectedly, growth promotion of TF-1 cells was observed in the treated TF-1 ip/hIL-3 Tg-SCID mice without differentiation to erythroid cells after treatment with delta-ALA (5 mM) for 7 days. These results indicate that Tg-SCID mice can support the growth of human hematopoietic growth factor-dependent AML cell lines which are usually rejected by SCID mice, without modification of the parental cell characteristics. In addition, this Tg-SCID leukemia-ascites model may become a useful preclinical tool for estimation of drug efficacy in vivo, since the drug candidate which was promising in vitro did not act in the same manner in vivo.

A calcium antagonist protects against doxorubicin-induced impairment of calcium handling in neonatal rat cardiac myocytes.

The effects of doxorubicin (DOX) on intracellular calcium transients and the cardioprotective effects of a calcium antagonist on DOX-induced impairment of calcium handling were examined in neonatal rat cultured cardiac myocytes. Cultured cardiac myocytes isolated from neonatal Wistar-Kyoto rats were treated with DOX for 24 h. Field-stimulated calcium transients in single myocytes were measured in the presence or absence of isoproterenol using fura-2/AM. Calcium transients were also measured after the addition of DOX to myocytes pretreated with a calcium antagonist, benidipine. DOX reduced the amplitude, maximum velocity of increase and decrease of calcium transients and prolonged the time course of calcium transients and impaired the beta-adrenoceptor responsiveness of calcium transients in a concentration-dependent manner. The DOX-induced impairment of calcium transients and beta-adrenoceptor responsiveness was improved by 10(-8) mol/L of benidipine. However, these improvements decreased with increasing concentrations of benidipine. DOX impaired both the mobilization and removal of intracellular calcium ions in contraction-relaxation cycles and the response of calcium transients to beta-adrenoceptor stimulation. Appropriate concentration of benidipine ameliorated DOX-induced impairment of calcium dynamics, suggesting that benidipine, a long-acting calcium antagonist, has potential clinical usefulness on DOX-induced abnormal calcium handling.

Non-ulcerative cutaneous lesion in immunodeficient mice with Leishmania amazonensis infection.

Cutaneous leishmaniasis begins as papules or nodules at the site of promastigote inoculation. The next key pathogenic event in this disease is the formation of an ulcer at this site. Leishmania infection in immunodeficient mice, however, showed non-ulcerative cutaneous lesions suggesting the involvement of the immune system in ulcer formation. Severe combined immunodeficient (SCID), recombination-activating gene 2 knockout (RAG-2-/-), and immunocompetent mice were inoculated subcutaneously with cultured L. amazonensis promastigotes. Macroscopic nodules appeared at the inoculation site within 2 weeks of infection in all the mice and gradually extended to the surrounding skin tissue. Although nodules of immunocompetent mice ulcerated within 6 weeks, immunodeficient mice did not form ulcers even after 25 weeks of inoculation. These results strongly suggest the importance of functional T and B cells in ulcer formation of cutaneous leishmaniasis and are consistent with clinical features of non-ulcerative cutaneous leishmaniasis in some AIDS patients. The present study also indicates that the L. amazonensis-infected immunodeficient mouse model might be suitable for studying the mechanisms of ulcer formation in cutaneous leishmaniasis.

Developmental changes in enzyme activities and in structural features of rat masticatory muscle mitochondria.

The functional ability of a muscle is closely related to the activities of the mitochondria, which are energy-producing organelles in muscle cells. The development of the mammalian masticatory muscle progresses dramatically when feeding behavior changes from suckling to mastication, but it is unclear how the energy-producing systems of the mitochondria change. In this paper, the development of rat masticatory muscle mitochondria was investigated in terms of enzyme activities of the mitochondrial respiratory chain and the structural and numerical development of mitochondria, especially regarding the change in feeding behavior from suckling to mastication. Using isolated mitochondria from the masticatory muscle, we measured succinate dehydrogenase, NADH dehydrogenase, succinate-O2 oxidoreductase, and NADH-O2 oxidoreductase. These were found to be increased in the 15-day postnatal rat compared with the 0- to 10-day postnatal rat. The structural development of mitochondria was gradual in the 0- to 15-day postnatal rat. However, a notable increase was found in the cross-sectional area of mitochondria between 10 and 15 days postnatally. The number of mitochondria per muscle fiber was apparently constant during the same period. We demonstrated that the change in feeding behavior was well-correlated with an increase in mitochondrial enzyme activity, also supported by the early structural development of mitochondria.

Doxorubicin cardiotoxicity: diastolic cardiac myocyte dysfunction as a result of impaired calcium handling in isolated cardiac myocytes.

We examined intracellular calcium transients of isolated single cardiac myocytes from rats with doxorubicin (DOX)-induced cardiomyopathy with simultaneous measurement of cell motion. DOX was administered i.p. to Sprague-Dawley rats at 2.5 mg/kg once a week for 10 weeks. Field-stimulated calcium transients and simultaneous cell motion in single myocytes were measured in the presence or absence of isoproterenol using fura-2/AM. Histopathologic examination revealed slight changes. The time courses of both calcium transients and cell motion were significantly prolonged by DOX. There was a slight but not significant reduction in parameters of contractility in both calcium transients and cell motion. The beta-adrenoceptor responsiveness of both calcium transients and cell motion was not significantly impaired compared with the controls. Our data indicated that, despite the slight histologic changes in the heart in DOX-induced cardiomyopathy, impaired sequestration of intracellular free calcium ions in individual myocytes may be one factor leading to diastolic dysfunction. Monitoring of diastolic function is important to detect early cardiotoxicity caused by DOX.

Cytokine dependent growth of human TF-1 leukemic cell line in human GM-CSF and IL-3 producing transgenic SCID mice.

Although severe combined immunodeficient (SCID) mice are considered useful as an animal model for human hematopoietic diseases, the complete reconstruction of human hematopoietic cells can not be established even in these mice. This appears to be because human cytokines, adhesion molecules and extracellular matrices which support differentiation and growth of human hematopoietic cells differ from those in animals. To improve this animal model, we attempted to produce transgenic (Tg) mice producing human interleukin 3 (hIL-3) and human granulocyte macrophage colony stimulating factor (hGM-CSF) with the homozygote of the scid gene. We established two Tg mouse lines, one releasing both 0.5-1 ng/ml of hIL-3 and 0.05-0.2 ng/ml of hGM-CSF in their sera and another releasing only high (2-10 ng/ml) levels of hGM-CSF. When human cytokine-dependent myeloid cell line, TF-1, was subcutaneously transplanted into these two Tg-SCID mouse lines, TF-1 could be successfully engrafted and grew in all lines of Tg-SCID mice but not in control mice. We also observed that TF-1 grows in GM-CSF Tg-SCID mice in a dose dependent manner in vivo and IL-3 shows an additive effect on its growth. These results indicated that these Tg-SCID mice were an useful in vivo model for investigating human leukemogenesis, especially the role of IL-3 and GM-CSF in leukemogenesis.

Mutant presenilin 2 transgenic mouse: effect on an age-dependent increase of amyloid beta-protein 42 in the brain.

The N141I missense mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease (AD) in the Volga German families. We have generated transgenic mouse lines overexpressing human wild-type or mutant PS2 under transcriptional control of the chicken beta-actin promoter. In the brains of transgenic mice, the levels of human PS2 mRNA were found to be five- to 15-fold higher than that of endogenous mouse PS2 mRNA. The amyloid beta-protein (Abeta) 42 levels in the brains of mutant PS2 transgenic mice were higher than those in wild-type PS2 transgenic mice at the age of 2, 5, or 8 months. In addition, the Abeta42 levels appeared to increase steadily in the mutant PS2 transgenic mouse brains from 2 to 8 months of age, whereas there was only a small increase in wild-type transgenic mice between the ages of 5 and 8 months. There was no definite difference in the levels of N-terminal and C-terminal fragments between wild-type and mutant PS2 transgenic mice at the age of 2, 5, or 8 months. These data show a definite effect of the PS2 mutation on an age-dependent increase of Abeta42 content in the brain.

An angiotensin-converting enzyme inhibitor protects against doxorubicin-induced impairment of calcium handling in neonatal rat cardiac myocytes.

1. The effects of doxorubicin (DOX) on intracellular calcium transients were examined in neonatal rat cultured cardiac myocytes, as were the cardioprotective effects of an angiotensin-converting enzyme (ACE) inhibitor on DOX-induced impairment of calcium handling. 2. Cultured cardiac myocytes isolated from neonatal Wistar-Kyoto rats were treated with DOX for 24 h. Field-stimulated calcium transients in single myocytes were measured in the presence or in the absence of isoproterenol using fura-2/AM. Calcium transients were also measured after the addition of DOX to myocytes pretreated with M-I (an active metabolite of delapril HCL, an ACE inhibitor. 3. Doxorubicin reduced the amplitude and maximum velocity of increase and decrease of calcium transients, prolonged the time-course of calcium transients and impaired the beta-adrenoceptor responsiveness of calcium transients in a dose-dependent manner. The DOX-induced impairment of calcium transients and beta-adrenoceptor responsiveness was improved by M-I. 4. Doxorubicin impaired both the mobilization and sequestration of intracellular calcium ions in contraction-relaxation cycles and the response of calcium transients to beta-adrenoceptor stimulation. The ACE inhibitor ameliorated DOX-induced impairment of calcium dynamics, suggesting ihat M-I, an active metabolite of delapril, protects against DOX-induced abnormal calcium handling leading to cardiac dysfunction.

Pneumocystis carinii cysts are susceptible to inactivation by chemical disinfectants.

The inactivation efficacy of eight disinfectants commonly used in laboratories and animal rooms to inactive Pneumocystis carinii cysts was estimated by experimental infection in C.B-17-scid mice. The disinfectants examined in this study were 70% ethyl alcohol, 10% iodoform, 0.5% hypochlorous acid, two 1% quanternary ammonium salts, 3% hydrogen peroxide, sodium chlorite and 1% cresol soap. The lung homogenates from P. carinii infected C.B-17-scid mice were treated with each disinfectant for 15 min at room temperature, washed with saline, and inoculated into C.B-17-scid mice. Eight weeks after inoculation, lungs from these mice were examined by staining with toluidine blue O to detect P. carinii cysts. PCR amplifying 346 bp of P. carinii specific mitochondrial ribosomal RNA large segments was also performed using DNA extracted from the lungs of the mice. As a result, seven disinfectants, excepting for 0.5% hypochlorous acid, were effective in the inactivation of P. carinii cysts. These results suggest that P. carinii cysts were sensitive to chemical disinfectants even though they have been commonly considered as insensitive.

The roles of preB cell receptor in early B cell development and its signal transduction.

The preB cell receptor is expressed for a short period after mu heavy chain is produced, that is, at the large preB cell stage in B cell development. The severe impairment of B cell differentiation observed in mice deficient for the preB cell receptor clearly demonstrated the importance of the preB cell receptor in B cell development. Analyses of bone marrow precursor B cells in normal and B cell-deficient mutant mice indicated the preB cell receptor transduced signals to drive cell cycle and to induce allelic exclusion. The proliferation of the preB cell receptor-expressing cells leads to the selective expansion of cells which have succeeded in the productive rearrangement of mu heavy chain gene. This process builds up a preB cell pool large enough to generate sufficient numbers of mature B cells. The preB cell receptor appears to induce allelic exclusion by shutting off the expression of recombinase activation gene (RAG). In order to analyse the signal transduction pathway downstream of the preB cell receptor, we have developed a new system in which cross-linking of Ig beta expressed on bone marrow proB cells mimics the signalling through the preB cell receptor to induce differentiation from proB to small preB cells.

Serological studies on porcine Pneumocystis carinii pneumonia: kinetics of the antibody titers in swine herds and the association of porcine reproductive and respiratory syndrome virus infection.

Serological titers to Pneumocystis carinii (Pc) and porcine reproductive and respiratory syndrome virus (PRRSV) were measured on a herd with epidemic Pc pneumonia (case herd) and two comparison herds, by an indirect fluorescent-antibody technique. In the case herd, the geometric mean titer (GMT) for Pc were 1:80 in pigs 1 week old, 1:10 in pigs 5 weeks old, and 1:80 to 1:190 in pigs over 6 weeks old. GMTs for PRRSV were > 1:145 in most of age groups over 7 weeks old. In comparison herds, Pc and PRRSV antibody titers were low in weanling pigs. The results clarified the kinetics of antibodies to Pc and concurrent infection of PRRSV in the case herd.