There is a need for biomarkers to predict efficacy of adjuvant chemotherapy in resected non-small cell lung cancer (NSCLC). Presented is a combined cisplatin and vinorelbine marker from a previously validated model system [1] tested in two cohorts. The profiles consist of correlated in vitro cytotoxicity of cisplatin and vinorelbine and mRNA expressions. Then each profile is correlated to mRNA expression of 3500 tumors. The cohorts are 1) a publically available dataset with 133 completely resected stage Ib-II NSCLC patients, 71 of whom received adjuvant cisplatin and vinorelbine (ACT) and 62 patients who had no adjuvant treatment (OBS) [2] and 2) 95 stage Ib-IIIb completely resected NSCLC patients who all received adjuvant cisplatin and vinorelbine [3]. Endpoint is cancer specific survival. The combined cisplatin and vinorelbine profiles scored as a continuous covariate showed 1) a Hazard Ratio (HR) of 0.265 ((95% CI:0.079-0.889), p = 0.032) in the ACT cohort (sensitive versus resistant), and no significant discrimination in the OBS cohort (HR = 1.328 (95% CI:0.46-3.835), p = 0.60). A multivariate model adjusted for stage demonstrated significance for ACT (HR = 0.284 (95% CI:0.086-0.944), p = 0.040) but not for OBS (HR = 1.702 (95% CI: 0.575-5.036), p = 0.34). The combined profiles resulted in 2) a significant prediction for up to 3 years from surgery (HR = 0.143 (95% CI:0.038-0.542), p = 0.004, scored as a continuous covariate). A multivariate model adjusting for stage showed that the predictor remained significant (HR = 0.123 (95% CI:0.030-0.512), p = 0.004). A pooled analysis of the two treated cohorts resulted in a significant prediction (HR = 0.187, (95% CI:0.069-0.508), p = 0.001) up to 3 years from surgery using a random effects model. The combined profiles demonstrate that NSCLC patients who benefit from cisplatin and vinorelbine can be identified. The profiles did not discriminate patients in the untreated arm. This holds promise for a predictive effect of the profiles and it is currently being validated in a prospective study [4]. [1] PLoS ONE 2016, 11(2): e0148070. [2] Zhu et al JCO 2010;28:4417-4424. [3] ASCO 2016 abstract e20007. [4] AACR 2016 Abstract CT154.

7013 Background: In two Phase III trials (INTACT 1 & 2), gefitinib (‘Iressa’, ZD1839) in combination with first-line platinum-based chemotherapy regimens failed to show benefit in terms of survival when compared with chemotherapy alone in patients (pts) with NSCLC. The primary objective of this analysis was to evaluate if EGFR staining was predictive of survival. Methods: Pretreatment tumor biopsies were assessed by immunohistochemistry for EGFR using the DakoCytomation pharmDx assay™. The percentage of tumor cells was assessed using 4 levels of intensity: no staining, 0; weak, 1+; moderate, 2+; strong, 3+; as well as the presence of complete membrane staining. 516 pts (INTACT 1/2, 219/297) with tumor samples fully evaluable for EGFR were analyzed in the per protocol population of the combined trials. The analysis was stratified by trial and performed independently for pts randomized to placebo or gefitinib. A restricted backwards elimination Cox regression analysis was conducted to identify independent EGFR factors. Variables found to be significant (p<0.1) were also tested for treatment interaction to determine if they served as predictive factors. Results: The demographics of the analysis population were representative of the overall population in each trial as were common disease characteristics. The analysis showed that two EGFR-based variables are independent strong prognostic factors for both placebo- and gefitinib-treated patients. However, no interactions with treatment (ie no value for predicting benefit of gefitinib treatment) were indicated for either of these two variables (p=0.8688 and 0.4967) or for any other EGFR-based variables. Conclusion: Given the overall trial results for INTACT 1 & 2 showing no survival benefit between gefitinib and placebo, it was highly unlikely that EGFR variables would identify a subset of pts who receive survival benefit. The data presented support this hypothesis. 'Iressa' is a trademark of the AstraZeneca group of companies 'PharmDx assay' is a trademark of DakoCytomation Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca Pharmaceuticals; DakoCytomation AstraZeneca; DakoCytomation; Genentech AstraZeneca AstraZeneca AstraZeneca Pharmaceuticals

7024 Background: Two double-blind placebo-controlled randomized multicenter trials (INTACT 1 & 2) in NSCLC failed to show survival benefit for gefitinib (‘Iressa’, ZD1839) in combination with first-line platinum-based chemotherapy. However, these large studies provide an opportunity to evaluate if EGFR staining is prognostic for survival. Methods: Tumor biopsies (n=516) taken between diagnosis and start of therapy were assessed by immunohistochemistry for EGFR using the DakoCytomation EGFR pharmDx assay™. The analysis was stratified by trial and performed independently for patients randomized to placebo and gefitinib. A restricted backwards elimination Cox regression analysis was conducted to identify independent EGFR factors that were statistically significant (p<0.10) and these were also tested for treatment interaction to assess if they served as predictive factors. Results: The analysis found 2 statistically significant EGFR-based prognostic factors representing the growth pattern and percent membrane staining in patients treated with gefitinib (p=0.0023, df=2) and placebo (p=0.0128, df=2). A further analysis including interaction terms of the EGFR variables with treatment confirmed these as highly significant (p<0.0001, df=2) but not the interaction. The EGFR factors remained independently prognostic when conditioning for previously found factors (PS2; stage IV; bone, brain, liver metastasis; sex; histology; weight loss) and were comparably significant (p=0.0011 and p=0.0158). Results will also be presented for secondary endpoints (time to progression, objective response). Conclusion: While the majority of previous studies indicate higher EGFR expression correlates with poor survival, the analysis from 2 large Phase III trials provides statistically significant evidence that the combination of EGFR expression and growth pattern is a strong prognostic indicator for improved survival within this setting. ‘Iressa’ is a trademark of the AstraZeneca group of companies ‘PharmDx assay’ is a trademark of DakoCytomation Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca Pharmaceuticals; DakoCytomation DakoCytomation; Genentech AstraZeneca AstraZeneca

Accelerated repopulation in head-and-neck carcinomas might be related to the expression of proliferative factors such as epidermal growth factor receptor (EGFr). The present study focuses on the prognostic value of EGFr for T-site control and the relation to tumor cell differentiation and overall treatment time. We studied 336 patients treated with primary radiotherapy using 66-68 Gy, 2 Gy per fraction and overall treatment times of 912, 612, or 512 weeks. Pretreatment biopsies were stained for EGFr.Thirty-five percent of the carcinomas had less than 50% of the area stained for EGFr. Small T-size and well-differentiated tumors was associated with a high degree of staining (p = 0.001 and p = 0.002, respectively). EGFr was of poor prognostic influence regarding local control in patients treated with 9 weeks split-course, whereas the opposite was found for patients given accelerated treatment in 5 weeks. A similar relationship between outcome, overall treatment time, and differentiation has previously been shown. The two parameters were analyzed together by separating the tumors with low EGFr and/or poor differentiation from tumors with well/moderate differentiation and high EGFr, resulting in odds ratios for T-site failure of 12 (1.43-104), 0.91 (0.51-1.65), and 0.43 (0.17-1.08), for treatment times of 912, 612, and 512 weeks, respectively. The tumor response to variations in fractionation is heterogeneous, and the prognostic impact of EGFr and differentiation might be relative and dependent on the overall treatment time of radiotherapy.

Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in response to viral infection. The aim of this study was to explore the function and mechanism of MAPK signaling pathway in enterovirus 71 (EV71) infection of human rhabdomyosarcoma (RD) cells.Apoptosis of RD cells was observed using annexin V-FITC/PI binding assay under a fluorescence microscope. Cellular RNA was extracted and transcribed to cDNA. The expressions of 56 genes of MAPK signaling pathway in EV71-infected RD cells at 8 h and 20 h after infection were analyzed by PCR array. The levels of IL-2, IL-4, IL-10, and TNF-α in the supernatant of RD cells infected with EV71 at different time points were measured by ELISA.The viability of RD cells decreased obviously within 48 h after EV71 infection. Compared with the control group, EV71 infection resulted in the significantly enhanced releases of IL-2, IL-4, IL-10 and TNF-α from infected RD cells (p < 0.05). At 8 h after infection, the expressions of c-Jun, c-Fos, IFN-β, MEKK1, MLK3 and NIK genes in EV71-infected RD cells were up-regulated by 2.08–6.12-fold, whereas other 19 genes (e.g. AKT1, AKT2, E2F1, IKK and NF-κB1) exhibited down-regulation. However, at 20 h after infection, those MAPK signaling molecules including MEKK1, ASK1, MLK2, MLK3, NIK, MEK1, MEK2, MEK4, MEK7, ERK1, JNK1 and JNK2 were up-regulated. In addition, the expressions of AKT2, ELK1, c-Jun, c-Fos, NF-κB p65, PI3K and STAT1 were also increased.EV71 infection induces the differential gene expressions of MAPK signaling pathway such as ERK, JNK and PI3K/AKT in RD cells, which may be associated with the secretions of inflammatory cytokines and host cell apoptosis.

HER2 is an erbB/HER type I tyrosine kinase receptor that is frequently over-expressed in malignant epithelial tumours, Herceptin, a humanised mouse monoclonal antibody to HER2, is proven therapeutically in the management of metastatic breast cancer, significantly prolonging survival when combined with cytotoxic chemotherapeutic agents, Immunohistochemical studies suggest that non-small-cell lung cancer (NSCLC) tumours may over-express HER2, Our aim was to evaluate HER2 gene amplification and semi-quantitative immuno-expression in NSCLC. A total of 344 NSCLC cases were immunostained far HER2 expression in 2 centres using the HercepTest. Fluorescence in situ hybridisation (FISH) analysis for HER2 gene amplification was performed on most positive cases and a subset of negative cases. Fifteen cases (4.3%) demonstrated 2+ or 3+ membranous HER2 immuno-expression. There was no correlation between immuno-expression and tumour histology or grade. Tumours from higher-stage disease were more often HercepTest-positive (p < 0.001). All 4 HercepTest 3(+) cases demonstrated gene amplification, One of the 5 2+ cases tested for gene amplification showed areas of borderline amplification and areas of polyploidy. None of the 19 HercepTest-negative cases demonstrated gene amplification or polyploidy (p < 0.001). Gene amplification was demonstrated in all HercepTest 3+ scoring NSCLC cases. Unlike breast cancer, gene amplification and HER2 protein over-expression assessed by the HercepTest appeared to be uncommon in NSCLC. Herceptin may therefore target only a small proportion of NSCLC tumours and be of limited clinical value in this disease, particularly in the adjuvant setting, (C) 2001 Wiley-Liss, Inc.

Tumor angiogenesis, a major requirement for tumor outgrowth and metastasis formation, is regulated by pro- and anti-angiogenic factors. We have studied the expression of a panel of angiogenic factors, and of the angiogenesis inhibitor angiostatin, in a panel of human melanoma cell lines giving rise to xenografts with different vascular densities. Angiogenic-factor expression was analyzed in vitro (cell lines) and in vivo (xenografts), both at mRNA (RT-PCR and Northern blot) and at protein level (ELISA and Western blot). In vitro angiostatin generation was assessed by Western-blot analysis. Expression of bFGF and VEGF was clearly correlated with a high degree of vascularization, confirming the importance of these factors for tumor angiogenesis. In addition, there was exclusive or elevated in vitro expression of angiogenic factors IL-8, PDGF-AB, and, to a lesser extent, midkine in cell lines that formed highly vascularized tumors. A similar angiogenic-factor-expression pattern was found in the corresponding xenografts, with the exception of VEGF. in most cell lines, this factor had low expression in vitro which was strongly enhanced in vivo. Although all 8 melanoma cell lines were able to excise the angiostatin fragment from the plasminogen parent molecule in vitro, cell lines BLM and M14 showed the most potent angiostatin generation. In vitro angiostatin generation by cell lysates prepared from melanoma xenografts was comparable in all xenograft types. Thus, in our model system we found no correlation between angiostatin generation and vascular density. Our study has limited the number of pro-angiogenic factors that may be involved in melanoma angiogenesis, and provides evidence for the notion that regulation of tumor angiogenesis is dependent on multiple factors. Inhibition of angiogenesis for therapeutic purposes, therefore, should preferably not concentrate on a single factor. (C) 2000 Wiley-Liss, Inc.

Epstein–Barr virus (EBV) is a human herpes virus with oncogenic potential, associated with several malignancies. The EBV-encoded latent membrane protein 1 (LMP1) is one of nine proteins regularly expressed in virally infected and immortalised B lymphocytes. We now document the consistent immunoreactivity for LMP1 in 90% of 65 nevi and melanomas, using the monoclonal antibody cocktail CS1–4. The immunocytochemical findings, however, were not confirmed using reverse-transcription polymerase chain reaction (RT-PCR) experiments, which failed to demonstrate any actual expression of LMP1 mRNA. In situ hybridisation for EBV-encoded RNAs (EBERs 1 and 2) and PCR amplification of EBV genomic sequences also failed to document any viral infection. Several normal and neoplastic human tissues have also been immunostained for LMP1, without any positive staining, with the exception of a minor percentage of skin melanocytes and of normal blasts of the myeloid and erythroid lineages. We conclude that the vast majority of nevi and melanomas express a still uncharacterised molecule, cross-reacting with anti-LMP1 (CS1–4) antibodies, which may be considered a consistent marker of melanocytic proliferations. The immunoreactivity of normal and neoplastic human tissues for the anti-LMP1 reagent should not be taken as evidence of EBV infection.

Angiostatin is a tumor-derived angiogenesis inhibitor consisting of an internal fragment of plasminogen. Little is known about the production of angiostatin by human tumors. In this study, we examined the in vitro angiostatin-generating capacities of a panel of human tumor cell lines (total n = 75) and the proteolytic molecule(s) involved. Angiostatin formation was determined by assessing the level of plasminogen digestion in conditioned medium by Western-blot analysis. We found that the capacity to produce angiostatin is a common feature of many cell lines, depending on the tumor type. All 6 bladder-carcinoma and 6 out of 7 prostate-carcinoma cell lines showed intermediate to potent angiostatin-generating activity. In contrast, only 2 out of 7 colon-carcinoma and 2 out of 9 renal-cell carcinoma cell lines were able to generate angiostatin at intermediate levels. Out of 25 melanoma cell lines, only one line failed to generate angiostatin. In the other cell-line groups (cervix, breast and ovary), angiostatin formation varied. Remarkably, angiostatin bands were not of equal size in all plasminogen digests. Since reported data have indicated that plasminogen activators (uPA and tPA) were able to excise the angiostatin fragment from the plasminogen parent molecule via plasmin generation, we determined levels of uPA and tPA and PAI-1 antigen in the conditioned media, and correlated the results with angiostatin-generating capacity. Whereas prostate- and bladder-carcinoma lines capable of generating high levels of angiostatin showed high uPA levels, angiostatin generation in melanoma cell lines was correlated with tPA levels. Generally, angiostatin non-producers did not express uPA or tPA. In 6 out of 75 cell lines, however, we found angiostatin generation combined with low or absent levels of plasminogen activator, suggesting the involvement of alternative proteolytic pathways in the generation of angiostatin.

AIMS--To prepare a rabbit antiserum equivalent to MIB 1 to permit the simultaneous assessment of cell proliferation and other markers of interest using double labelling studies. METHODS--Rabbits were immunised with a synthetic peptide deduced from the cDNA sequence coding for the Ki-67 antigen. Serum samples were tested for immunoreactivity using different immunobiochemical methods. RESULTS--A polyclonal antiserum was derived which detects the native as well as recombinant parts of the Ki-67 antigen in different test systems. Furthermore, the antiserum stains the Ki-67 antigen in routinely processed, paraffin wax embedded material. CONCLUSIONS--After antigen unmasking by microwave treatment the antiserum described here represents a powerful tool for the determination of growth fractions even in archival material. It is especially suitable for double staining experiments in combination with monoclonal antibodies.

Immunohistochemical identification of neuroendocrine tumour markers in paraffin embedded material from 22 tumours (5 small cell carcinomas of the lung (SCCL), 12 carcinoids, 2 medullary thyroid carcinomas, 2 pheochromocytomas and one paraganglioma) with electron microscopically verified dense-core granules revealed neuron-specific enolase in all but one tumour, synaptophsin in 15/22 (2 SCCL and 6 carcinoids negative), chromogranin in 16/22 (all SCCL and one carcinoid negative), and endocrine granule constituent in 18/22 (4 SCCL negative). The Grimelius silver methods stained 13/22 (all cases of SCCL and 4 carcinoids negative).

Summary Infection of the bovine fetus with non‐cytopathic Bovine Viral Diarrhoea virus (BVDV) during the first trimester of gestation frequently results in the establishment of a persistent infection and the birth of viraemic calves with apparent tolerance to the virus. Recently we demonstrated that the infection occurred in T‐, B‐ and null cells as well as in blood monocytes. In order to further characterize the BVDV infected leukocyte subpopulations, attempts were made to identify the infected cells by immuno electron microscopy (IEM). However, the extreme sensitivity of BVDV antigens to the preparative procedures for EM precluded the use of pre‐ and post‐embedding IEM. We therefore resorted to correlative IEM. Subpopulations of peripheral blood mononuclear leukocytes (PBL) from persistently infected calves were enriched for by various immunological methods and tested. Cells were labelled in the living state for surface antigens by an immuno‐gold protocol and subsequently prepared for EM. BVDV‐like particles (40–60 nm) were detected by EM in small cytoplasmic vesicles of the aforementioned cell types, identifiable by the gold marker. The occurrence correlated with the results of the direct virus isolation from cell lysates and immunocytochemical detection of viral antigens on cytospin preparations as well as with the specific detection of viral polypeptides by immunoblotting. Many cells, most notable monocytes and T‐lymphocytes, looked “activated”. A tentative sequence of virus morphogenesis is presented and discussed. Zusammenfassung Vergleichende immun‐elektronenmikroskopische Untersuchungen über die BVD‐Virusinfektion in mononukleären Zellen des peripheren Blutes von persistentvirämischen Kälbern Eine Infektion des Rinderfötus mit nicht‐zytopathischem “Bovinen Virusdiarrhöe Virus” (BVDV) wahrend des ersten Trimesters der Trächtigkeit führt oft zu einer persistierenden Infektion und zur Geburt von Kälbern mit Virämie sowie einer offensichtlichen Virus‐Toleranz. Vor kurzem konnten wir zeigen, daß die Infektion in T‐, B‐ und Nullzellen sowie in Blutmonozyten stattfindet. Um die mit BVDV infizierten Leukozytensubpopulationen weiter zu charakterisieren, wurde versucht, die infizierten Zellen durch Immunoelektronenmikroskopie (IEM) zu identifizieren. Jedoch die extreme Empfindlichkeit der BVDV‐Antigene bei der Aufbereitung zur Elektronenmikroskopie (EM) schloß die Anwendung einer Vor‐ und Nacheinbettung zur IEM aus. Wir entschlossen uns deshalb zur korrelativen IEM. Die Subpopulationen von peripherem Blut mononukleären Leukozyten (PBL) von persistent infizierten Kälbern wurden mittels verschiedener immunologischer Methoden angereichert und getestet. Lebende Zellen wurden zur Oberflächenantigen‐Bestimmung durch ein Immunogold‐Verfahren markiert und danach zur EM aufbereitet. BVDV‐ähnliche Partikel (40–60 nm) wurden mit der EM in kleinen zytoplasmatischen Vesicula der obengenannten Zelltypen ermittelt, und konnten mit dem Goldmarker identifiziert werden. Das Vorkommen korrelierte mit den Ergebnissen der direkten Virusisolation aus Zellysaten und der immunozytochemischen Bestimmung von viralen Polypeptiden durch Immunoblotting. Viele Zellen, hauptsächlich Monozyten und T‐Lymphozyten, sahen “aktiviert” aus. Eine vorläufige Vorstellung vom Ablauf der Virusmorphogenese wird diskutiert.

With a reassortant from a cross of human rotavirus DS-1 (serotype 2) and OSU (serotype 5) it was determined that the OSU major neutralization glycoprotein antigen (VP7) was encoded by gene segment 9. A full-sized cloned cDNA copy of the OSU gene 9 was produced and sequenced. Hybridization of such labelled cDNA with the corresponding segment of a reassortant DS-1 X OSU virus confirmed the coding assignment. Comparison of the deduced amino acid sequence of the VP7 of OSU with those previously determined for five other rotavirus strains, representing four distinct serotypes, revealed some hydrophilic regions that exhibited significant homology and other hydrophilic domains with greater amino acid divergence. In one of the latter hydrophilic domains each of the five serotypes had a distinct amino acid substitution at residue 146, suggesting that it may be involved in serotype specificity.

Although rotavirus has been recognized as the most common etiologic agent of gastroenteritis in infants requiring hospitalization, there are several important gaps in the understanding of rotavirus infection. Obstacles to such an understanding have included difficulties in cultivating the virus from human stools, the lack of a simple animal model to examine the immune response, and differences in the epidemiology of the infection in developed vs. developing countries. There is a strong need for community-based longitudinal studies of rotavirus infections over a period of time, especially in developing countries. This article summarizes current knowledge of the rotavirus structure, genetic variations, antigenic components, epidemiologic features, clinical aspects and treatment, and prevention of rotavirus diarrhea. The typical clinical picture of rotavirus gastroenteritis is indistinguishable from diarrheas of other etiologies; however, dehydration occurs more frequently in children with rotavirus infection. The enzyme-linked immunosorbent assay is generally considered the most efficient and simple method of identifying rotavirus in the stool. Several approaches to rotavirus vaccines are currently being tested, and among the vaccine candidates are strains of rotavirus naturally attenuated for humans, cold-adapted strains, and laboratory-derived reassortants. Also under exploration is the potential of genetic engineering techniques to achieve in vitro production of rotavirus antigen.

A rotavirus-like virus has been isolated from cases of neonatal diarrhoea in piglets. No antigenic relationship with known rotaviruses or with an American strain of a rotavirus-like virus has been demonstrated. Morphologically the virus is similar to known rotaviruses, but it differs in the ability to form syncytia of the enterocytes in the small intestine.

A blocking method of ELISA for the detection and quantification of antibody against porcine rotavirus in serum, colostrum and milk has been compared with a plaque reduction test. The results obtained with the two techniques correlated (Fig. 1). Antibody against rotavirus was demonstrated in 384 serum samples representing 25 swine farms, indicating a widely spread and dense distribution of the infection with porcine rotavirus among Danish swine. The antibody contents in milk samples from 7 gilts and sows from 2 farms with a previously diagnosed problem with rotavirus associated diarrhoe showed a rapid decline during the first few days of the lactation periods (Fig. 1). An increase in the contents of rotavirus specific antibody was observed from day 15 in the milk samples from one of the gilts. The excretion of rotavirus with feces from the 7 litters of piglets were followed through the suckling period. Rotavirus war found in all litters but one and the virus was excreted in periods ranging from 4 to 9 days.

The histology and ultrastructure of the spinal white matter from the dorsolateral funiculus of the third cervical segment was studied in normal control pigs and pigs whose dams were inoculated with the Weybridge congenital tremor strain of swine fever virus in early pregnancy. Only inoculated sows produced abnormal piglets. These showed congenital tremors and ataxia. The severity of clinical signs was related to the degree of spinal myelin deficiency. Morphologically this was quantified by determination of the thickness of myelin investing axons classed according to their diameter. In clinically affected pigs fewer axons were myelinated than normal. Though the myelin sheath thickness increased with increasing axon diameter in all pigs whether clinically normal or not, the increase was less in moderately affected and much less in severely affected pigs. The deficiency of spinal myelin was probably due to delayed or sub-normal myelination accompanied by paranodal myelin abnormalities, myelin degeneration and remyelination.

Of 13 gilts 7 were vaccinated twice at an interval of 3 weeks with an inactivated vaccine against porcine parvovirus (PPV) infection, while the 6 nonvaccinated gilts served as controls. Starting after the 1st vaccination the gilts were bred and, after about 40 days of gestation, challenged intravenously with virulent PPV. The vaccinated gilts produced an antibody respons after the 1st and 2nd vaccination compatible with a primary and a secondary immune response, respectively. The nonvaccinated gilts remained low-titered or PPV antibody negative until after challenge. The gilts were killed after about 90 days of gestation, and their litters were examined. All of 53 fetuses from the vaccinated gilts were alive, and infection with PPV could not be demonstrated. Conversely, 50 of 65 fetuses from the non-vaccinated gilts were infected with PPV, and 43 were dead. In a field study comprising 2 herds, PPV seronegative or lowtitered gilts were vaccinated before mating. There were no obvious signs of reproductive disorders in the 2 herds during the vaccination trials, and the reproductive performance of vaccinated gilts did not differ significantly from that of non-vaccinated gilts.