Gas6 and the Tyro 3 receptor tyrosine kinase subfamily regulate the phagocytic function of Sertoli cells

The apoptotic spermatogenic cells and residual bodies are phagocytosed and degraded by Sertoli cells during spermatogenesis. The mechanisms of this process are largely unknown. Here, we demonstrate that Gas6 and its receptors, the Tyro 3 subfamily of receptor tyrosine kinases (RTKs; Tyro 3, Axl, and Mer), regulate the phagocytic function of Sertoli cells. The phagocytic ability of Sertoli cells increased by five times in the presence of Gas6 in serum-free medium when compared with controls. The Sertoli cells lacking Mer showed a 35% reduction in phagocytosis of apoptotic spermatogenic cells when compared with wild-type (WT) controls, whereas the Sertoli cells lacking Tyro 3 or Axl exhibited phagocytic activity comparable with the controls. Notably, the Sertoli cells lacking all three members of the Tyro 3 RTK subfamily showed a dramatic decrease in phagocytic ability of 7.6-fold when compared with WT Sertoli cells. The deficiency in phagocytosis by the triple-mutant Sertoli cells was due to the deficit in binding of the Sertoli cells to apoptotic germ cells. These findings suggest that Mer is responsible for triggering phagocytosis of apoptotic spermatogenic cells by Sertoli cells and that Tyro 3, Axl, and Mer participate in recognizing and binding apoptotic germ cells by Sertoli cells in a redundant manner. Gas6 is a functional ligand of the Tyro 3 RTK subfamily in mediating phagocytic ability of Sertoli cells.     

Arrested apoptosis without nuclear fragmentation produced by efferent duct ligation in round spermatids and multinucleated giant cells of rat testis

The apoptotic process evoked by efferent duct ligation in the testes of adult rats was followed for 10 days by differential staining for haematoxylin-eosin, periodic acid-Schiff and a modified trichrome technique in optical microscopy and by ultrastructural localization of acid phosphatase. Round spermatids showed the first effects of efferent duct ligation. At day 3 after ligation, annular clumps of chromatin with typical apoptotic characteristics appeared against the nuclear membrane of these cells. Afterwards, membranous structures and a wide separation between the two layers of the nuclear membrane were observed but nuclear fragmentation did not occur and apoptotic granules were not seen. Cytoplasmic components were also altered, and severely damaged organoids and empty vacuoles lacking acid phosphatase reaction were frequently seen. On day 2 after efferent duct ligation, multinucleated giant cells appeared, which displayed similar characteristics as spermatids and showed no acid phosphatase reaction. Although abnormal spermatids and multinucleated giant cells were surrounded by the cytoplasm of Sertoli cells, neither lysosomal acid phosphatase nor phagocytic activity was detected. It is concluded that efferent duct ligation specifically affects round immature spermatids eliciting a partial nuclear apoptotic response that is not accompanied by autophagic or heterophagic activity and without lysosomal participation in Sertoli cells.     

Enhanced phagocytosis of Aggregatibacter actinomycetemcomitans cells by macrophages activated by a probiotic Lactobacillus strain

The activation of phagocytosis is one important approach to clearing pathogenic cells in a host. This study evaluated the ability of probiotic lactobacilli to induce phagocytic activity as well as the clearance of a periodontal pathogen, Aggregatibacter actinomycetemcomitans. First, the activation of phagocytosis was found by using lyophilized dead cells. Probiotic Lactobacillus strains significantly enhanced the phagocytic activity of macrophage cells, indicating that the probiotic lactobacilli have a remarkable ability to stimulate the macrophages. Essentially, 3 Lactobacillus strains tested did not have any critical toxic effect on the murine macrophage, and Lactobacillus johnsonii NBRC 13952 showed the least cytotoxic effect on the RAW264.7 macrophages. The expression of classically activated macrophage markers, IL-1β, and cluster of differentiation 80 increased by L. johnsonii NBRC 13952; however, there was no significant difference for IL-18. The highest phagocytic activity by macrophages was found in a condition in which the macrophage activated by L. johnsonii NBRC 13952 functions to kill the cells of A. actinomycetemcomitans. Correlating with the result, a high amount of hypodiploid DNA (SubG1) was detected from the macrophage cells stimulated by L. johnsonii NBRC 13952. Taken together, the results suggest that macrophages activated by the Lactobacillus strain can facilitate the phagocytosis of A. actinomycetemcomitans cells by linking with enhanced apoptotic activities. In conclusion, L. johnsonii NBRC 13952 has a certain role in activating the RAW264.7 macrophages, thereby counteracting the infection of A. actinomycetemcomitans.     

Phagocytosis as a potential mechanism for microbial defense of mouse placental trophoblast cells

Trophoblast giant cells are active phagocytes during implantation and post-implantation. Phagocytosis decreases during placental maturation as the phagocytic function of nutrition is gradually replaced by the direct uptake of nutrients by the labyrinth zone trophoblast. We hypothesize that, after placental maturation, trophoblast cells maintain phagocytic functions for purposes other than nutrition. This study employs histological techniques to examine the ability of trophoblast cells to phagocytose microorganisms (yeast or bacteria)--in vivo in females receiving thioglycolate to activate macrophages and in vitro in the presence of phagocytic promoters such as interferon-gamma and complement component C3. Placental trophoblast cells from the second half of gestation show basal phagocytosis that can be dramatically up-regulated by these promoters when microorganisms are inoculated into pregnant animals or introduced into culture systems. Stimulated trophoblast cells phagocytosed organisms more rapidly and in greater numbers than non-stimulated trophoblast exposed to the same numbers of organisms. Taken together, our results indicate that trophoblast cells do not lose their ability to phagocytose during the placentation process, which may imply that trophoblast cells participate in embryonic and fetal innate immune defense through elimination of microorganisms present at the maternal-fetal interface.     

Influence of involution on intramammary phagocytic defense mechanisms.

Mammary secretions (n = 34 cows) and mammary phagocytes (n = 18 cows) were collected throughout the nonlactating (dry) period to determine changes in intramammary phagocytic defense mechanisms. Mammary secretions were evaluated for their ability to support phagocytosis of Staphylococcus aureus by neutrophils from donor cows and mammary phagocytes for phagocytic and chemiluminescence activity. Ability of secretions to support phagocytosis decreased with advancing length of the dry period. This effect was more pronounced when dry cow secretions constituted 50% of the phagocytic mixture. Phagocytic activity of mammary phagocytes decreased with advancing dry period when autologous secretion was used in the incubation mixture. With homologous secretion, the percentage of phagocytosis increased 5 to 6 d after drying off compared with before drying off and then gradually decreased throughout the remainder of the dry period. Chemiluminescence activity (log10 counts per minute) of mammary phagocytes was lower during the dry period and decreased with advancing dry period. Results indicated diminishing ability of secretions to support phagocytosis and diminished phagocytic and bactericidal mechanisms during the dry period.     

Beta-carotene and vitamin A effects on bovine phagocyte function in vitro during the peripartum period

The effects of in vitro supplementation of beta-carotene, retinol, and retinoic acid on phagocyte function during the peripartum period were assessed. Blood was collected at wk -4, -1, 0 (calving), 1, and 4; mammary secretions were collected at wk -1, 0, 1, and 4 from 14 Holstein cows for the isolation of phagocytic cells. Blood polymorphonuclear leukocytes and mammary macrophages and polymorphonuclear leukocytes (phagocytic cells) were assayed for phagocytic and intracellular kill abilities of Staphylococcus aureus in the presence of beta-carotene and retinol at 10(-8) and 10(-7) M and retinoic acid at 10(-9) and 10(-8) M. Phagocytosis by blood or milk phagocytic cells was not influenced by beta-carotene. However, beta-carotene enhanced kill by blood and milk phagocytic cells during certain prepartum and post-partum periods. In contrast to beta-carotene, retinol and retinoic acid either had no effect or suppressed phagocytosis and kill. These results are interpreted to suggest a mechanism by which beta-carotene affords the mammary gland protection against infection, i.e., through enhanced intracellular kill by phagocytes.     

Examination of attachment and phagocytic uptake of Listeria species by mammalian intestinal cells

Strains of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria welshimeri and Listeria seeligeri isolated from diverse source were examined in an attempt to determine if their potential for attachment to intestinal epithelial cells and their behavior towards gut phagocytes was similar. Blood phagocytes were included for a comparison of their phagocytic activity with gut macrophages. Guinea pig small intestine was the source of intestinal epithelial cells and gut macrophages for the assays. Attachment of the bacteria to whole tissue was performed by incubation at 37°C of Listeria with small pieces of bowel, followed by staining and examination under light microscopy. Phagocytic cells were isolated from the gut by the use of EDTA, collagenase and DNAse, and peripheral blood cells were separated by differential centrifugation with Ficoll-Hypaque. Phagocytic activity was allowed to take place for 15 min at 37°C and the phagocytic index (PI) was determined from the number of ingested bacteria. Assays with gentamicin were included in order to assess the fate of ingested Listeria with respect to viability. Attachment of Listeria stains to the gut epithelial cells varied and could not be correlated either with the strain or with the source of the bacteria. The most significant differences in phagocytic activity were observed at the gut cell level. Overall, the PI was higher for L. monocytogenes than for L. innocua. A great variability occurred in the results and was due mostly to animal variation.     

Initiation of testicular tubulogenesis is controlled by neurotrophic tyrosine receptor kinases in a three-dimensional Sertoli cell aggregation assay

The first morphological sign of testicular differentiation is the formation of testis cords. Prior to cord formation, newly specified Sertoli cells establish adhesive junctions, and condensation of somatic cells along the surface epithelium of the genital ridge occurs. Here, we show that Sertoli cell aggregation is necessary for subsequent testis cord formation, and that neurotrophic tyrosine kinase receptors (NTRKs) regulate this process. In a three-dimensional cell culture assay, immature rat Sertoli cells aggregate to form large spherical aggregates (81.36+/-7.34 microm in diameter) in a highly organized, hexagonal arrangement (376.95+/-21.93 microm average distance between spherical aggregates). Exposure to NTRK inhibitors K252a and AG879 significantly disrupted Sertoli cell aggregation in a dose-dependent manner. Sertoli cells were prevented from establishing cell-cell contacts and from forming spherical aggregates. In vitro-derived spherical aggregates were xenografted into immunodeficient nude mice to investigate their developmental potential. In controls, seminiferous tubule-like structures showing polarized single-layered Sertoli cell epithelia, basement membranes, peritubular myoid cells surrounding the tubules, and lumen were observed in histological sections. By contrast, grafts from treatment groups were devoid of tubules and only few single Sertoli cells were present in xenografts after 4 weeks. Furthermore, the grafts were significantly smaller when Sertoli cell aggregation was disrupted by K252a in vitro (20.87 vs 6.63 mg; P<0.05). We conclude from these results that NTRK-regulated Sertoli-Sertoli cell contact is essential to the period of extensive growth and remodeling that occurs during testicular tubulogenesis, and our data indicate its potential function in fetal and prepubertal testis differentiation.     

山莴苣苦素改善FFA诱导的HepG2细胞脂肪变性及氧化应激的研究

目的:研究山莴苣苦素对游离脂肪酸(FFA)诱导的HepG2细胞脂肪变性的改善作用,并初步探讨其可能作用机制.方法:观察山莴苣苦素对HepG2细胞活力的影响.采用FFA诱导培养HepG2细胞,构建脂肪变性模型(NAFLD体外模型),并用山莴苣苦素干预72h后,检测各组细胞内脂滴数量、甘油三酯(TG)和总胆固醇(TC)含量...     

Dynamics of intracellular calcium induced by lactate and glucose in rat pachytene spermatocytes and round spermatids

Glycolytic metabolism in meiotic and post-meiotic spermatogenic cells shows differentiation-related changes. The developmental and physiological significance of these metabolic changes is not known. The aim of the present study was to test the hypothesis that glucose and lactate metabolism can modulate intracellular calcium [Ca2+](i) in spermatogenic cells in an opposing and dynamic manner. Fluorescent probes were used to measure [Ca2+](i) and pH(i), and HPLC was used to measure intracellular adenine nucleotides and mitochondrial sensing of ATP turnover. [Ca2+](i) in pachytene spermatocytes and round spermatids was modulated by changes in lactate and glucose concentrations in the media. The kinetics and magnitude of the [Ca2+](i) changes induced by lactate and glucose were different in meiotic and post-meiotic spermatogenic cells. The presence of glucose in the medium induced a decrease in pH(i) in spermatogenic cells. This glucose-induced pH(i) decrease occurred later than the changes in [Ca2+](i), which were also observed when the pH(i) decrease was inhibited, indicating that the glucose-induced [Ca2+](i) increase was not a consequence of pH(i) changes. Hexose phosphorylation in glycolysis was part of the mechanism by which glucose metabolism induced a [Ca2+](i) increase in spermatogenic cells. The sensitivity of [Ca2+](i) to carbohydrate metabolism was higher in round spermatids than in pachytene spermatocytes. Thus, differentiation-related changes in carbohydrate metabolism in spermatogenic cells determine a dynamic and differential modulation of their [Ca2+](i) by glucose and lactate, two substrates secreted by the Sertoli cells.     

Duplicated insertion mutation in the microtubule-associated protein Spag5 (astrin/MAP126) and defective proliferation of immature Sertoli cells in rat hypogonadic (hgn/hgn) testes

Male rats with hypogonadism (hgn/hgn) experience sterility from testicular dysplasia, which is controlled by a single recessive gene, hgn. The postnatal growth of the seminiferous tubules was severely affected. In this study, we localized the hgn locus to a 320 kb region on rat chromosome 10 and detected the insertion of a 25 bp duplication into the sixth exon of the sperm-associated antigen 5 (Spag5/astrin/MAP126) gene, which codes for a microtubule-associated protein. This mutation results in a truncated Spag5 protein lacking the primary spindle-targeting domain at the C terminus. Immunological staining with antibodies to markers for Sertoli and germ cells during the early postnatal period indicated that the abnormal mitosis with dispersed chromosomes in hgn/hgn testes occurs in proliferating Sertoli cells. Therefore, apoptotic Sertoli cell death would result from the disorganization of the spindle apparatus caused by defective Spag5. These findings suggested that the Spag5 is essential for testis development in rats and that the hgn/hgn rat is a unique animal model for studying the function of Spag5.     

Claudin-11 expression and localisation is regulated by androgens in rat Sertoli cells in vitro

Claudin-11 and occludin are protein components in tight junctions (TJs) between Sertoli cells which are important for the maintenance of the blood-testis barrier. Barrier formation occurs during puberty, with evidence suggesting hormonal regulation of both claudin-11 and occludin. This study aimed to investigate the regulation of claudin-11 and occludin mRNA expression by testosterone (T) and FSH and their immunolocalisation at rat Sertoli cell TJs in vitro, and to correlate any steroid regulation with the functional capacity of TJs. Sertoli cells formed functional TJs within 3 days as assessed by transepithelial electrical resistance (TER). Both T and dihydrotestosterone significantly (P < 0.01) increased TER twofold and claudin-11 mRNA two- to threefold within 3 days. FSH partially stimulated TER and claudin-11 mRNA, but estradiol had no effect. T also promoted claudin-11 localisation into extensive intercellular contacts. In contrast to claudin-11, Tand FSH did not change occludin mRNA expression, however, T promoted localisation of occludin at cell contacts in a similar manner to claudin-11. Addition of flutamide to T-stimulated cells caused a twofold decrease in both TER and claudin-11 mRNA expression, and resulted in the loss of both proteins from cell contacts. This effect was reversible following flutamide removal. It is concluded that androgens i) co-regulate claudin-11 mRNA expression and TER, implicating claudin-11 in TJ formation and ii) promote the localisation of claudin-11 and occludin at Sertoli cell contacts. Hence, the ability of androgens to maintain spermatogenesis in vivo is partly via their effects on TJ proteins and regulation of the blood-testis barrier.     

Phosphoprotein enriched in astrocytes-15 is expressed in mouse testis and protects spermatocytes from apoptosis

Phosphoprotein enriched in astrocytes (PEA-15) is a 15 kDa acidic serine-phosphorylated protein expressed in different cell types, especially in the CN. We initially detected the expression of PEA-15 in primary cultures of Sertoli cells. To assess the presence and localization of PEA-15 in the mouse testis, we studied the expression pattern of the PEA-15 protein by immunohistochemistry and mRNA by in situ hybridization. Both the protein and the mRNA of PEA-15 were localized in the cytoplasm of Sertoli cells, all types of spermatogonia, and spermatocytes up till zygotene phase of the meiotic prophase. Subsequently, with ongoing development of the spermatocytes, the expression decreased and was very low in the cytoplasm of diplotene spermatocytes. To analyze the possible role of PEA-15 in the developing testis, null mutants for PEA-15 were examined. As the PEA-15 C terminus contains residues for ERK binding, we studied possible differences between the localization of the ERK2 protein in wild type (WT) and PEA-15(-/-)mice. In the WT testis, ERK2 was localized in the cytoplasm of Sertoli cells, B spermatogonia, preleptotene, leptotene, and zygotene spermatocytes, whereas in the KO testis, ERK2 was primarily localized in the nuclei of these cells and only little staining remained in the cytoplasm. Moreover, in PEA-15-deficient mice, significantly increased numbers of apoptotic spermatocytes were found, indicating an anti-apoptotic role of PEA-15 during the meiotic prophase. The increased numbers of apoptotic spermatocytes were not found at a specific step in the meiotic prophase.     

Stage and season effects on cell cycle and apoptotic activities of germ cells and Sertoli cells during spermatogenesis in the spiny dogfish (Squalus acanthias)

To understand the processes involved in the spatial and temporal maturation of testicular cells in Squalus acanthias, we used standard morphometry, proliferating-cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) immunohistochemistry. Except for immature spermatocysts (germinal zone, GZ; early-stage pre-meiotic, E-PrM), the number of cysts in all subsequent stages and the total number of cysts in the spermatogenic progression varied seasonally. The spermatogenic cycle spans about 2 years and is interrupted by germcell clone deletion via apoptosis at the mitosis-meiosis transition in April/May, manifesting as a zone of degeneration (ZD). Rate of displacement of the ZD across the testis diameter indicates that late-stage premeiotic (L-PrM) generations 12-13 require 9-10 months to reach the mature-spermatid stage. Also, the number of cysts completing spermatogenesis is approximately 4-5-fold less than the number that entered spermatogenesis proper 2 years earlier. Pronounced gonocytogenesis in the germinal ridge was coincident with ZD formation in April/May, but it was absent in the fall when mature spermatogonial and meiotic activities had resumed. Whereas strong Sertoli cell PCNA immunoreactivity dominated the GZ cyst cell-cycle activities throughout the year, except during the spring/summer months, the spermatogonial- and Sertoli-cell PCNA indices in E-PrM cysts were inversely related. PCNA immunoreactivity in spermatocytes was seasonal and dependent on the stage of meiosis. TUNEL labelling was limited to spermatogonia and increased stage-dependently in the PrM region (L-PrM = mid-stage PrM >E-PrM >GZ), correlating with ZD formation, in a season-dependent manner. Results imply that effects of normal regulatory factors in Squalus are stage- and process-specific.     

Developmental ability of cloned embryos from neural stem cells

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei, suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs, cumulus cells, Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However, the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%), but lower than that obtained by cloning mice from other cell nuclei (2.2-3.5%). Although the in vitro developmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g., 85% in Sertoli cells and 75% in cumulus cells), the developmental rate to the morula-blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g., 50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point for in vitro development of cloned embryos depends on the donor cell type.     

仿刺参雌性生殖腺皂苷的免疫增强活性研究

目的：以仿刺参雌性生殖腺皂苷(gonad saponins from female of Apostichopus japonicus,AGS)为研究对象,探究其免疫增强活性。方法：通过对巨噬细胞吞噬能力和细胞因子的测定,研究AGS的体外免疫调节活性;通过对免疫低下小鼠胸腺、脾脏指数,碳廓清指数与吞噬指数,腹腔巨噬细胞增殖活性,脾细胞增殖活性,自然杀伤细胞(natural killer cell,NK cell)活力,T淋巴细胞亚群水平及小鼠血清细胞因子的测定,分析AGS在小鼠体内的免疫调节活性。结果：体外实验表明,AGS能提高小鼠体外巨噬细胞吞噬能力,促进TNF-α、IL-6、IFN-γ的分泌,且各组与正常组相比差异显著(P<0.05);体内实验表明,与模型组相比,中剂量组和高剂量组AGS均能显著提高小鼠胸腺、脾脏指数、碳廓清指数和吞噬指数(P<0.05),显著增强腹腔巨噬细胞和脾细胞增殖活性(P<0.05),显著增强NK细胞的活性(P<0.05),显著提高CD4⁺/CD8⁺水平(P<0.05),显著促进TNF...     

Expression of Col1a1, Col1a2 and procollagen I in germ cells of immature and adult mouse testis

The objective of this study was to compare the expression of Col1a1, Col1a2, and procollagen I in the seminiferous tubules of immature and adult mice and to characterize the cellular expression pattern of procollagen I in germ cells during spermatogenesis in order to provide necessary groundwork for further functional studies in the process of spermatogenesis. Microarray analysis demonstrated that Col1a1 and Col1a2 were abundantly expressed in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice, and the expression levels of Col1a1 and Col1a2 mRNA were validated using a semi-quantitative RT-PCR assay. Western blot analysis further confirmed that procollagen I was expressed at a higher level in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice. Immunohistochemical analysis revealed that type A spermatogonia were positive for procollagen I in the testis of 6-day-old mice, whereas Sertoli cells were negative for this protein. The in vivo procollagen I staining in type A spermatogonia was corroborated in spermatogonia exhibiting a high potential for proliferation and the ability to form germ cell colonies in in vitro culture. Moreover, procollagen I was also detected in type A spermatogonia, intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes in the adult mouse testes, but positive staining disappeared in more differentiated germ cell lineages detaching from the basement membrane, including leptotene spermatocytes, pachytene spermatocytes, round spermatids and elongated spermatids. These data suggest that Col1a1, Col1a2 and procollagen I are associated with type A spermatogonia and play a potential role in mediating the detachment and migration of germ cells during spermatogenesis.     

不同极性酸浆提取液对体外诱导的非酒精性脂肪肝细胞的影响

探讨不同极性酸浆提取液对体外诱导的非酒精性脂肪肝(NAFLD)细胞的影响及其可能的作用机制。用不同极性有机溶剂对酸浆进行萃取,采用MTT法筛选医用脂肪乳注射液使用浓度、酸浆提取液作用浓度,光学显微镜观察油红O染色情况,全自动生化仪检测甘油三酯(TG)、谷丙转氨酶(ALT)、谷草转氨酶(AST)和谷氨酰转肽酶(GGT)水平。与正常肝细胞株HL7702相比,模型组油红O染色后细胞浆内有大量被染成橘红色的脂滴,细胞内TG、ALT、AST和GGT水平显著升高(p0.01)。与模型组相比,不同酸浆提取物治疗12 h组细胞浆内橘红色的脂滴数量明显减少,TG、ALT、AST和GGT水平显著降低(p0.01)。在体外成功建立非酒精性脂肪肝的细胞模型,不同极性酸浆提取液处理能减轻NAFLD细胞中TG蓄积和ALT、AST和GGT的泄漏量。     

Susceptibility of Escherichia coli isolated from intramammary infections to phagocytosis by bovine neutrophils.

Thirteen Escherichia coli isolated from naturally occurring IMI were tested for susceptibility to phagocytosis by bovine blood neutrophils. Isolates were opsonized in pooled serum collected from nine healthy lactating cows. Bacteria isolated from IMI first diagnosed within 3 d after calving were more resistant to phagocytosis than were isolates from IMI originating during either the first half of the dry period or later during lactation. Duration of the IMI was negatively correlated with both phagocytic index and percentage of neutrophils phagocytizing within bacterial isolates from IMI originating at calving and during lactation. Phagocytosis was independent of duration of IMI within isolates from IMI originating during the first half of the dry period. Susceptibility to in vitro phagocytosis by neutrophils was not related to O antigen serotype, encapsulation, or growth in dry cow secretion.