Assessment of the targeting specificity of a fluorescent albumin conceived as a preclinical agent of the liver function.

In the context of increasing liver diseases, no contrast agent is currently available in Europe and the United States to directly assess the liver function. Only neolactosylated human serum albumin is being clinically used in Asia. In order to perform preclinical studies in the context of liver diseases, we conceived a fluorescent lactosylated albumin for the quantification of liver functional cells (l-Cyal). Precise characterization was achieved in order to determine the amounts of lactose and Cyanine 5 (Cy5) coupled to the albumin. In addition, potential aggregation was characterized by asymmetrical flow field-flow fractionation hyphenated to multi-angle light scattering (AF4-MALS). The optimal functionalized albumin exhibited a mass greater than 87 kDa which corresponds to the addition of 34 lactose moieties per protein and 1-2 Cy5 labels. Also, no significant formation of aggregates could be identified due to the modification of the native albumin. In healthy mice, the accumulation of l-Cyal in the liver and its selectivity for hepatocyte cells were shown by optical imaging and flow cytometry. Administration of l-Cyal to mice bearing liver metastases showed a reduced signal in the liver related to a decrease in the number of hepatocytes. The l-Cyal bioimaging contrast agent could be particularly useful for assessing the state of liver related diseases.

Radiation therapy facility risk analysis in Brazil with SEVRRA software.

Risk assessment deals with processes, accident-initiating events, barriers and risk ratings to unveil the fragility and weakness of some processes; within this study, specifically related to radiation therapy facilities. Barriers are technical or organizational safety measures put in place to avoid, prevent, detect, control, reduce or mitigate the consequences of an accident once an initiating event has occurred. In this work, radiological risk analysis was performed for a set of 20 Brazilian radiotherapy facilities making use of the freeware sevrra risk-management software. The objective of this study was to define parameters that could be useful in creating an overall risk profile. This profile would be helpful for establishing priorities for decision making and support a risk-informed regulatory process. The most relevant missing barriers in facilities were identified according to three parameters: the 'importance index', 'impacted facilities index' and the 'barrier-effectiveness index'. Barriers such as 'in vivo dosimetry in the first treatment session', 'weekly in vivo dosimetry to detect errors in the dose delivering process', 'annual external audit for the control of reference dose rate' and 'independent verification of calibration by various medical physicists with a different dosimetry equipment' were found to be the most effective in reducing the risk level of the facilities. The present investigation reinforces the need to strengthen the mechanisms that guarantee the effectiveness of such barriers in radiation therapy procedures.

Cost effectiveness of collagen crosslinking for progressive keratoconus in the UK NHS.

BACKGROUND: Keratoconus is a progressive degenerative corneal disorder of children and young adults that is traditionally managed by refractive error correction, with corneal transplantation reserved for the most severe cases. UVA collagen crosslinking is a novel procedure that aims to prevent disease progression, currently being considered for use in the UK NHS. We assess whether it might be a cost-effective alternative to standard management for patients with progressive keratoconus. METHODS: We constructed a Markov model in which we estimated disease progression from prospective follow-up studies, derived costs derived from the NHS National Tariff, and calculated utilities from linear regression models of visual acuity in the better-seeing eye. We performed deterministic and probabilistic sensitivity analyses to assess the impact of possible variations in the model parameters. RESULTS: Collagen crosslinking is cost effective compared with standard management at an incremental cost of £ 3174 per QALY in the base case. Deterministic sensitivity analysis shows that this could rise above £ 33,263 per QALY if the duration of treatment efficacy is limited to 5 years. Other model parameters are not decision significant. Collagen crosslinking is cost effective in 85% of simulations at a willingness-to-pay threshold of £ 30,000 per QALY. CONCLUSION: UVA collagen crosslinking is very likely to be cost effective, compared with standard management, for the treatment of progressive keratoconus. However, further research to explore its efficacy beyond 5 years is desirable.

Induction of porcine regulatory cells by mycophenolic Acid-treated dendritic cells.

Tolerance induction in murine allogeneic transplantation is relatively easy, often by induction of regulatory T cells (Treg). Unfortunately, the implementation of these models in clinical situations has not yielded reliable protocols of tolerance induction in humans. Our project sought to create a preclinical model of tolerance induction in large animals. Our current efforts seek to induce and characterize porcine Treg, obtaining dendritic cells (DC) able to preferentially stimulate them. DCs were differentiated from blood monocytes with porcine recombinant interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for 6 days. These DCs were then stimulated by human CD40 ligand-transfected L cells with or without mycophenolic acid (MPA) for 48 hours. We analyzed surface marker expression, cytokine synthesis, and ability to stimulate allogeneic peripheral blood mononuclear cells (PBMC). The porcine lymphocytes underwent 4 rounds of 1-week stimulation with allogeneic DC treated or not with MPA. At the end of this coculture we analyzed their capacity to suppress allogeneic PBMC proliferation induced by mature DC. Our results showed that porcine DCs pretreated with MPA display a low expression of B7 costimulatory molecules, produce low levels of IL-12, and induce weak proliferation of allogeneic lymphocytes. Moreover, after 4 rounds of stimulation with MPA-treated DCs, PBMCs were able to inhibit an alloreactive response. These preliminary results suggested induction of a regulatory T-cell population that we are currently seeking to characterize.

Effects of dietary scFOS on immunoglobulins in colostrums and milk of bitches.

Fructo-oligosaccharides (scFOS) are prebiotic ingredients that improve protection against pathogens probably through promoting the growth of gastrointestinal bacteria-like Bifidobacteria and Lactobacilli: this stimulation may lead to a better development of immune repertoire and/or stimulation of the local immune response. According to the existence of the immune entero-mammary link, we were wondering if the dietary supplementation with scFOS could enhance the mucosal immunoglobulin level in mammary secretions. Results in this study show that bitches supplemented with scFOS exhibit higher colostrum and milk IgM content without concomitant effect on IgG1, IgG2 and IgA. In addition, intranasally immunized puppies exhibited a trend to higher Bordetella bronchiseptica-specific IgM immune response. The dietary supplementation with scFOS increased the IgM level in colostrums and milk of bitches by mechanisms which remain to be elucidated.

Evaluations of the renal cell carcinoma model Caki-1 using a silicon based microvascular casting technique.

The vascular development of orthotopically grown human renal cell carcinoma (Caki-1) was examined using a silicon based casting technique in nude mice. Images taken of these vascular casts showed Caki-1 tumors to be highly vascularized and invasive. The spread of the tumor within the cortex of the kidney revealed that Caki-1 recruits the kidney's own vasculature, destroying the functional glomeruli in the process. The loss of these glomeruli was further highlighted by the presence of enlarged glomeruli resulting from the development of super nephrons. Vessel size and density measurements were then made in this model. This was done using both computer-based and manual measurement methods. In the vessel size studies the computer-based method tended to overestimate the number of larger diameter vessels whereas the vessels density assessment showed good agreement between the two techniques. Nevertheless, both methods showed that Caki-1 tumors possessed a higher proportion of larger diameter vessels and a lower vessel density than normal kidney cortex. In summary, silicon based vascular casting proved to be a simple and effective tool for the study of tumor vasculature. In particular this technique could readily be used to examine the invasion of tumor into normal tissue. The computer-based technique for evaluating vessel number and vascular density was found to have merit in both normal and tumor tissues, particularly in the vascular density studies. However, in both settings this technique did tend to overestimate the number of larger diameter vessels.

T and IgA B lymphocytes of the pharyngeal and palatine tonsils: differential expression of adhesion molecules and chemokines.

The pharyngeal (Ph) and palatine (Pa) tonsils, although located in different regions of the upper aero-digestive tract (UADT), are thought to protect the respiratory tract similarly against infections by inducing and disseminating T and surface IgA(+) (sIgA(+)) B cells. We investigated the factors controlling the migratory properties of T and sIgA(+) B lymphocytes in the UADT of pigs by comparing the expression of vascular addressins, homing receptors and chemokine transcripts in Ph/Pa tonsils, Peyer's patches (PP) and their draining lymph nodes (LN). The vascular addressin PNAd was detected on high endothelial venules in both tonsils, whereas mucosal addressin cell adhesion molecule-1, otherwise present in PP and mesenteric LN, was not detected. More importantly, the vascular cell adhesion molecule-1 (VCAM-1) addressin was present in Ph tonsil and LN but neither in Pa tonsil nor in PP vascular cells, whereas both T and sIgA(+) B lymphocytes displayed similar levels of alpha4beta1(high) integrin, the ligand of VCAM-1. Analysis of transcript levels for several lymphoid (CCL19, CXCL12 and CCL21) and epithelial chemokines also demonstrated opposite chemokine mRNA ratios for Ph tonsil (CCL28 > CCL25) and PP, with Pa tonsil expressing very low levels of CCL28. Collectively, these data indicate that the differential compartmentalization of sIgA(+) lymphocytes between Pa and Ph tonsils may partly result from the differential expression of VCAM-1 and CCL28. They also suggest that tonsillar addressins and epithelial chemokines, rather than the cells intravasating it, control the regionalization of sIgA(+) lymphocytes in the UADT.

Is microscopic assessment of macroscopically normal hysterectomy specimens necessary?

AIM: To determine whether microscopic examination of macroscopically normal hysterectomy specimens yields findings that could alter subsequent clinical management. METHODS: All pathology reports on hysterectomy specimens submitted to the department of histopathology at the Northern General Hospital from January 1997 to December 1998 were reviewed. Cases were included for further assessment if the hysterectomy specimen was regarded as macroscopically normal by a consultant pathologist and if the patient had no history of, or suspicion of, neoplastic disease. The subsequent microscopic findings from these cases were assessed to determine whether any lesions of clinical importance were identified. RESULTS: Eight hundred and fifty four specimens were reviewed, of which 139 were suitable for inclusion. Only one of the 139 cases harboured a microscopic abnormality that necessitated specific clinical follow up; this was a focus of cervical intraepithelial neoplasia 2 (CIN 2). On follow up of that patient, no further neoplastic disease was identified. CONCLUSION: Microscopic assessment of macroscopically normal hysterectomy specimens does not contribute to patient management and is unnecessary in an era of manpower shortage and cost containment.

Overview of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with "w". As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).

Summary of the first round analyses of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The reactivity of 155 monoclonal antibodies submitted to the Third International Workshop on Swine Leukocyte Differentiation Antigens, together with 41 internal standards, was analysed by flow cytometry on 29 different pig cell targets as well as two human cell targets as a means of establishing suitable panels of monoclonal antibodies for more detailed clustering analyses by the various subsections of the workshop. Results were collected either without further gating, with gating based on FS/SS characteristics or with gating based on the co-expression of a reference antibody in two-colour flow cytometry. The CD or SWC reactivity of the internal standards had been established in previous workshops. Data sets were subsequently analysed by statistical clustering using the Leucocyte Typing Database IV software. The resulting 18 cluster groups were allocated to the appropriate second round sections of the workshop, after reviewing the overall cellular reactivity of each cluster as well as the specificity of known standards which clustered in a group.

Behavioural, growth and immune consequences of early weaning in one-week-old large-white piglets.

Genetic improvement in sows' prolificity is limited by their milk capacities, which do not allow all piglets to survive or grow normally. This experiment compared the behaviour, growth and immune responses of piglets that were weaned early at 6 days of age (EW) vs. control Large White piglets' (C) suckled by their mothers. Behaviour of 9 litters of 5 to 8 piglets in each group were observed from d5 to d20. All piglets were weighed from birth to d74. Three piglets from each group were slaughtered on d36 for immunological analysis. Until they began to eat dry food, EW piglets walked and vocalised more than C piglets. After that time, when resting, they were less often lying down and more frequently in contact with littermates under the heater. Aggressive behaviour and belly-nosing were more frequent. They displayed a more marked growth check after weaning than did C piglets until 28 days of age. In EW piglets, at 36 days of age, there was a higher density of T- and B-lymphocytes in the gut epithelium and lamina propria, fespectively, in relation to the size of lymphoid follicles of Peyer's patches. The results indicate great behavioural adaptation capacities of very early-weaned piglets, together with earlier maturation of their gut immune system.

Fetal target organs of graft-versus-host reaction induced in utero by injection of maternal cells in a pig model.

The target organs of graft-versus-host reaction (GVHR) in adult or neonates are the site of multifocal lymphocytic infiltrates. GVHR can also be acquired in utero by maternal cells crossing the placenta, but the fetal target organs are unknown. The aim of this study was to determine these target fetal organs. The distribution pathway of infused labeled lymphocytes within cryostat sections of fetal organs was analyzed, and fetal target organs of infused lymphocytes were investigated in both isogenic and semiallogenic situations. Isogenic cells were observed in less organs and semiallogenic cells were localized in a more restricted number of organs than isogenic cells. Furthermore, the liver, the thyroid, and the spleen were the fetal target organs in the two studied gestations. GALT, thymus, and kidney were also lymphocyte targets in isogenic gestation. In conclusion, isogenic cells induced GVHR in more fetal organs than semiallogenic cells.

Development of an ex vivo model of pig kidney perfused with human lymphocytes. Analysis of xenogeneic cellular reactions.

BACKGROUND: Because of the explosive nature and the extremely rapid process of hyperacute rejection (HAR), significant infiltration of the xenograft by immunocompetent cells is not observed, and the role and the mechanism of action of cell-mediated rejection in discordant xenografts are therefore still under discussion. METHOD: We developed an experimental approach using pig kidneys perfused with human peripheral blood lymphocytes (PBL) in which the immunologic barrier of hyperacute rejection was excluded and which mimics the in vivo situation. RESULTS: PBL retention in the kidney was evaluated at 20-minute intervals for 3 hours. Retention increased from 30% to 80% with the time of perfusion and was specific because significantly fewer syngeneic lymphocytes were retained. Phenotype analysis of recovered PBL showed a significant decrease in natural killer (NK) cells. Immunohistochemical studies revealed the presence of NK cells and T lymphocytes in the glomerular and interstitial tubular structures of the kidney. Functional studies showed a progressive cessation of diuresis and augmentation of renal vascular resistance when the kidney was perfused with PBL. Electron microscopy examinations of kidney sections perfused with PBL showed swollen endothelial zones, suggesting alterations to and damage of the endothelium. CONCLUSIONS: This system provides a valuable model for the study of early discordant xenogeneic cellular rejection and demonstrates the predominance of xenograft infiltration by NK cells.

Mammary gland immunology and neonate protection in pigs. Homing of lymphocytes into the MG.

Since placenta of pregnant sows are impermeable to immunoglobulin passage, the neonates are born agammaglobulinemic; although immunocompetent, they are unable to develop rapidly an immune response which will protect their systemic and mucosal compartments; thus their survival depend upon the passive acquisition of maternal immunity including at least 3 components: i) a systemic humoral immunity, transmitted through colostrum conveying mainly by IgG; these IgG are transferred from maternal serum via Fc gamma receptors on the epithelial cells of mammary gland (MG). ii) a local humoral immunity, especially secretory IgA (IgAs), transmitted mainly by milk (lactogenic immunity) until weaning. IgAs are secreted by MG recruited plasma cells and are excreted in milk via secretory component of epithelial cells: these IgA exhibit a specificity for the antigens present in the maternal digestive tract, the so-called "entero-mammary link"; this link is due to the migration of lymphocytes from the gut to the mammary gland; they are recruited from the blood via the interaction of their homing receptor (alpha 4 beta 7) with the developmentally regulated mucosal vascular addresin MadCAM-1. In the MG, MadCAM-1 increased in pregnancy (probably under oestrogenic stimulation) but regressed in lactation; its density is closely related to the T cell numbers in MG; in contrast the increase in plasma cell numbers is not related to MadCAM-1 density. Thus IgA precursor cells (alpha 4 beta 7 B cells) seem to be recruited by a milk B cell chemoattractant. On the other hand, presence of T and B lymphocytes in MG (some of them originating from the systemic compartment), sustains the attempts of MG immunization and the results sustain the view of a true local immune response. iii) possibly but not formally proved, a cellular immunity transmitted via maternal immunocompetent cells present in mammary secretions; the exported lymphocytes may represent a selected population of lymphocytes after their passage through the MG epithelium.

Dendritic cells enriched from swine thymus co-express CD1, CD2 and major histocompatibility complex class II and actively stimulate alloreactive T lymphocytes.

Initial characterization and partial purification of thymic dendritic cells (DC) from miniature swine were carried out with the ultimate goal of using these cells to induce transplantation tolerance in this preclinical animal model. Immunohistochemical analysis of swine thymic tissue sections has shown DC to be large cells located in the medullary and the cortico-medullary regions as evidenced by the presence of surrounding Hassal bodies. These cells exhibit membrane processes and express the CD1, granulocyte/macrophage (G/M), and major histocompatibility complex (MHC) class II surface antigens, as well as the S100 cytosolic and nuclear markers found in humans to be specific for DC. Dendritic cells were purified from thymi following collagenase treatment, Percoll gradient centrifugation, and adhesion steps to plastic. Cells similar in morphology and phenotype to those described in tissue sections were detected in the lighter density layers of the gradient and represented 0.02% of the starting cell number. Removal of plastic nonadherent cells showed enrichment levels similar to those reported for murine and human DC. Two-colour flow cytometric analysis of purified pig DC identified these cells as MHC class IIhi, CD1+, CD2+, and G/M+. The dendritic nature of these cells was confirmed by their potent ability to stimulate alloreactive T lymphocytes. Modification of porcine thymic DC by transfer of allogeneic MHC genes and reinjection into the DC donor should permit testing of the role of this DC subset in the induction of transplantation tolerance.

The mammary gland and neonate mucosal immunity.

The passive mucosal protection of neonate mammals is dependent on the continuous supply until weaning of maternally dimeric IgA (monogastric) and IgG1 (ruminants). This lactogenic (humoral) immunity is linked to the gut, the so-called entero-mammary link, because of the translocation of Ig (IgA and IgG1) or the emigration of IgA lymphoblasts from the gut into the mammary gland (MG); on the other hand, studies on the lymphocyte subsets in the MG of artiodactyls sustained the view of a true local immune response, depending on the MG stage development. Accordingly, the increase of the lactogenic immunity may focus on (1) inductor sites (gut and, possibly, the MG), (2) increase in cell traffic from the gut into the MG, and (3) enhancement at the effector site of the Ig production and excretion in milk. A specific mucosal environment (interleukins and hormones) is responsible for IgM/IgA switch, the induction of mucosal homing receptor onto lymphoblasts and mucosal vascular addressins; very few data are available for the mechanism of lymphoblasts recruitment, either IgA or IgG1, although lactogenic hormones have been implicated in the IgA-blasts homing into the mice MG. After weaning, the neonate is able to mount a gut immune response, but the shortage of the suckling period did not seem to be detrimental for its onset. In soyabean allergy, both piglet and calf exhibited gut villus atrophy, gut accumulation of IgA (swine) and IgG1 (cattle) immunocytes, sustaining the view that a specific environment in ruminant is responsible for both IgA and IgG1 production.

Differential recruitment of T- and IgA B-lymphocytes in the developing mammary gland in relation to homing receptors and vascular addressins.

The mammary gland (MG) develops new vasculature and is colonized by lymphocytes, primarily T-cells, during pregnancy. In contrast, during lactation it is colonized primarily by IgA-containing B-cells (c-IgA cells). To explain this difference, we analyzed the spatiotemporal relationships between lymphocytes that expressed peripheral or mucosal homing receptors (HR) and the location of their vascular counterreceptors using quantitative immunohistochemical techniques. We observed that the density of beta(7+)/CD3(+) T-cells varied with the amount of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1)-stained area. Both increased during pregnancy to peak at delivery, decreased rapidly in early lactation to a steady level in mid- and late lactation, and returned to resting values after weaning. Although 60% of these beta(7+)/CD3(+) T-cells scattered in the epithelium co-expressed alpha(E)beta(7), whereas the remaining 40% in association with blood vessels were alpha(4)beta(7), these results are consistent with a role of MAdCAM-1 in the localization of alpha(4)beta(7+) T-cells. In contrast to T-cells, beta(7+)/c-IgA(+) B plasmablasts (approximately 30% of total c-IgA cells) were located at the alveolar confluence, and their numbers increased in mid- and late lactation when MAdCAM-1 density plateaued. However, both T-and B-cells decreased after weaning. These results show an association between MAdCAM-1 expression level and recruitment of T-cells that does not hold for c-IgA B cells. Furthermore, the recruitment and accumulation of alpha(4)beta(7+) c-IgA cells are reminiscent of locally produced chemoattractants. (J Histochem Cytochem 47:1581-1592, 1999)

The xenotransplantation of goat and human hematopoietic cells to sheep fetuses.

Hematopoietic xenografts were carried out in three experiments using goat fetal liver (44-48 days, experiments I and II) or purified human CD 34+ cells (experiment III) as the donor cells. Recipients were sheep fetuses at 41-47 days of gestation. Goat fetal liver cells were either injected without any pretreatment or stimulated by preincubation in a culturing in goat phytohemagglutinin-stimulated lymphocyte supernatant. Human CD 34+ myeloid progenitor cells were purified from bone marrow by minimacs immunomagnetic purification and cultured in medium supplemented with stem cell factor, IL3, and IL6. Goat-sheep chimerism was assessed by flow cytometry analysis (FCA) of peripheral blood and bone marrow cells using a mouse anti-goat CD 45 monoclonal antibody and by karyotype analysis of peripheral blood from goat/sheep chimeras. Human cell engraftment was assessed by polymerase chain reaction amplification of the human DAX1 gene in blood and bone marrow DNA from sheep which had received human cells. In the three experiments, a mean of 76% (26 of 34) of injected fetuses were born alive without any clinical evidence of graft-versus-host disease. Three lambs were found to be goat/sheep chimeric after flow cytometry analysis (peripheral blood and bone marrow) and karyotype (peripheral blood) analysis. Both tissues continued to express goat cells at 6 or 12 months (last assessment) depending on the experiment. No human chimerism was detected using polymerase chain reaction amplification in peripheral blood and bone marrow of any of the six sheep grafted with human cells. These data and those also obtained on other species (human, pig/sheep) show that it is possible to carry out hematopoietic xenografts using the sheep fetus as recipient provided both donor and recipient fetal cells are processed during the period of tolerance to foreign antigens.

Specific cytotoxic lymphocyte response in swine against structural proteins of transmissible gastro-enteritis virus: a study using lymphoblastoid cell line and recombinant vaccinia virus.

To determine the specificity, if any, of cellular cytotoxicity against transmissible gastro-enteritis virus (TGEV) infected cells, we developed a test using B lymphoblasts from a MHC histocompatible (d/d haplotype) cell line (L14), as stimulating and target cells. These cells were previously infected with recombinant vaccinia virus including different TGEV structural genes, either the spike (vS), membrane (vM) or nucleoprotein gene (vN). Lymphocytes from a TGEV immunized (d/d) swine developed a cytotoxic activity after secondary in vitro stimulation in the presence of vS, vM or vN infected L14 cells. The cytotoxic activity was induced and directed against the homologous vS and vM infected cells but no cytotoxic activity occurred at all against vN infected cells. While vM infected cells induced a cytotoxic activity against vM infected cells only, vS infected cells stimulated a cross-reactive cytotoxic activity against vM and vN infected cells in addition to that against vS infected cells. This latter cytotoxicity may be due to an increase in a non-specific background of Natural Killer or lymphocyte activated killer activity, which is seen also after coculture with wild type vaccinia virus (vW) infected cells. Thus these results are of practical importance in two respects. First, lymphoïd B cell lines represent an excellent tool for determining which viral antigens are recognized by cytotoxic lymphocytes and second, they indicate the need to incorporate the M and S genes into a TGEV vaccine to induce cellular immunity against TGEV.

Effect of cryopreservation on chemotaxis of lymphocytes.

Cryopreservation of cells appears to be a potential method of comparing chemotaxis of lymphocytes collected from different anatomical sites at one time in a single assay. Migration of cryopreserved lymphocytes from swine in the absence (spontaneous migration) or presence of N-formyl-methionyl-leucyl-phenylalanine (fMLP, induced migration) was compared to that of fresh lymphocytes, originating from inguinal (ILN) and mesenteric (MLN) lymph nodes, respectively, using a 48-well microchemotaxis chamber. Cryopreservation did not affect the optimal concentration of fMLP for maximal induced migration and did not impair the chemoattractant activity of fMLP as shown by checkerboard assay. However, cryopreservation reduced the extent of fMLP-induced migration by affecting the spontaneous motility of cells, an effect which was greater for MLN than for ILN cells. This reduction was not related to a loss of cell subset and is in keeping with the view that spontaneous and induced migration involve distinct mechanisms. Thus cryopreservation may be of general use to study the migration of lymphocytes by reducing the differences in spontaneous migration of lymphocytes from different sites.