Roquin--a multifunctional regulator of immune homeostasis.

Roquin-1 (Rc3h1) is an E3 ubiquitin ligase originally discovered in a mutational screen for genetic factors contributory to systemic lupus erythematosus-like symptoms in mice. A single base-pair mutation in the Rc3h1 gene resulted in the manifestation of autoantibody production and sustained immunological inflammation characterized by excessive T follicular helper cell activation and formation of germinal centers. Subsequent studies have uncovered a multifactorial process by which Roquin-1 contributes to the maintenance of immune homeostasis. Through its interactions with partner proteins, Roquin-1 targets mRNAs for decay with inducible costimulator being a primary target. In this review, we discuss newly discovered functions of Roquin-1 in the immune system and inflammation, and in disease manifestation, and discuss avenues of further research. A model is presented for the role of Roquin in health and disease.

Hearts from DCD donors display acceptable biventricular function after heart transplantation in pigs.

Cardiac transplantation is in decline, in contrast to other solid organs where the number of solid organ transplants from donors after circulatory death (DCD) is increasing. Hearts from DCD donors are not currently utilized due to concerns that they may suffer irreversible cardiac injury with resultant poor graft function. Using a large animal model, we tested the hypothesis that hearts from DCD donors would be suitable for transplantation. Donor pigs were subjected to hypoxic cardiac arrest (DCD) followed by 15 min of warm ischemia and resuscitation on cardiopulmonary bypass, or brainstem death (BSD) via intracerebral balloon inflation. Cardiac function was assessed through load-independent measures and magnetic resonance imaging and spectroscopy. After resuscitation, DCD hearts had near normal contractility, although stroke volume was reduced, comparable to BSD hearts. DCD hearts had a significant decline in phosphocreatine and increase in inorganic phosphate during the hypoxic period, with a return to baseline levels after reperfusion. After transplantation, cardiac function was comparable between BSD and DCD groups. Therefore, in a large animal model, the DCD heart maintains viability and recovers function similar to that of the BSD heart and may be suitable for clinical transplantation. Further study is warranted on optimal reperfusion strategies.

Bone marrow cells produce a novel TSHbeta splice variant that is upregulated in the thyroid following systemic virus infection.

Although cells of the immune system can produce thyroid-stimulating hormone (TSH), the significance of that remains unclear. Using 5' rapid amplification of cDNA ends (RACE), we show that mouse bone marrow (BM) cells produce a novel in-frame TSHbeta splice variant generated from a portion of intron 4 with all of the coding region of exon 5, but none of exon 4. The TSHbeta splice variant gene was expressed at low levels in the pituitary, but at high levels in the BM and the thyroid, and the protein was secreted from transfected Chinese hamster ovary (CHO) cells. Immunoprecipitation identified an 8 kDa product in lysates of CHO cells transfected with the novel TSHbeta construct, and a 17 kDa product in lysates of CHO cells transfected with the native TSHbeta construct. The splice variant TSHbeta protein elicited a cAMP response from FRTL-5 thyroid follicular cells and a mouse alveolar macrophage (AM) cell line. Expression of the TSHbeta splice variant, but not the native form of TSHbeta, was significantly upregulated in the thyroid during systemic virus infection. These studies characterize the first functional splice variant of TSHbeta, which may contribute to the metabolic regulation during immunological stress, and may offer a new perspective for understanding autoimmune thyroiditis.

Porphyromonas gingivalis lipopolysaccharide induces tumor necrosis factor-alpha and interleukin-6 secretion, and CCL25 gene expression, in mouse primary gingival cell lines: interleukin-6-driven activation of CCL2.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis infection is strongly associated with periodontitis. Although P. gingivalis is known to elicit a strong inflammatory response, details of that remain fragmentary. To understand the local response to P. gingivalis, primary cell lines derived from mouse gingival tissues were exposed to P. gingivalis or Escherichia coli lipopolysaccharide, and the production of interleukin-6 and tumor necrosis factor-alpha was measured. CCL25 gene expression was measured by real-time polymerase chain reaction. Cells stimulated with combinations of interleukin-6, soluble interleukin-6 receptor and/or soluble gp130 were assayed for CCL2 and tumor necrosis factor-alpha secretion. MATERIAL AND METHODS: Primary cell lines were generated from mouse gingival tissues. Enzyme-linked immunosorbent assays were used to determine cytokine levels, and real-time polymerase chain reaction was used to quantify CCL25 gene expression. RESULTS: Exposure to P. gingivalis lipopolysaccharide but not to E. coli lipopolysaccharide resulted in significantly elevated levels of both interleukin-6 and tumor necrosis factor-alpha, and stimulation with P. gingivalis lipopolysaccharide also upregulated CCL25 gene expression. In one of three experiments, interleukin-6 induced CCL2 secretion, whereas interleukin-6 plus soluble interleukin-6 receptor induced CCL2 secretion in all three experiments, suggesting that both direct interleukin-6 signaling and interleukin-6 trans-signaling may be involved. However, because soluble gp130 did not inhibit trans-signaling, and because direct stimulation of gingival cells with soluble gp130 resulted in CCL2 secretion, the possibility exists that soluble gp130 forms binary complexes with soluble interleukin-6 receptor that promote direct interleukin-6 stimulation. CONCLUSION: These findings define a pathway in which exposure of gingival cells to P. gingivalis induces the release of interleukin-6 and tumor necrosis factor-alpha; interleukin-6, in turn, induces CCL2 secretion.

Food-grade delivery system for controlled gene expression in Lactococcus lactis.

A food-grade system for the delivery of desired genes to Lactococcus lactis, their inducible expression, and their transfer to related strains was established. Based on the thermosensitive pG(+)host replicon, two types of plasmid vectors were constructed which contained sections of either the chromosomal leu operon of L. lactis or the tel operon from the lactococcal sex factor. Genes cloned into the leu or tel sequences of these vectors were delivered to the homologous regions of the chromosome or the sex factor through two single crossovers, leading to integration of the recombinant plasmids and subsequent excision of the vector portions. Inducible transcription of integrated genes was achieved by using the nisin-controlled expression (NICE) system. To establish the signal transduction genes nisRK in L. lactis, the vectors pLNG1363 (targeted to the chromosome) and pUK500 (targeted to the sex factor) were constructed. Fusions of six different peptidase genes (pep) from Lactobacillus delbrueckii with the nisin-inducible promoter P(nisA) were delivered to the sex factor with derivatives of the vector pUK300. Food-grade recombinants of L. lactis were constructed which had the nisRK genes and individual P(nisA)::pep fusions integrated either separately into the chromosome and the sex factor or simultaneously into the sex factor. With both types of recombinants, expression of P(nisA)::pep fusions after induction with nisin was demonstrated. Depending on the loci used for integration of nisRK, variable induction rates were observed. Furthermore, an engineered sex factor carrying a P(nisA)::pepI fusion was transfered by conjugation between two strains of L. lactis at a frequency of 4 x 10(-4).

Multiple levels of activation of murine CD8(+) intraepithelial lymphocytes defined by OX40 (CD134) expression: effects on cell-mediated cytotoxicity, IFN-gamma, and IL-10 regulation.

The involvement of OX40 (CD134) in the activation of CD8(+) intestinal intraepithelial lymphocytes (IELs) has been studied using freshly isolated IELs and in vitro CD3-stimulated IELs. Although freshly isolated CD8(+) IELs exhibited properties of activated T cells (CD69 expression and ex vivo cytotoxicity), virtually all CD8(+) IELs from normal mice were devoid of other activation-associated properties, including a lack of expression of OX40 and the ligand for OX40 (OX40L) and an absence of intracellular IFN-gamma staining. However, OX40 and OX40L expression were rapidly up-regulated on CD8 IELs following CD3 stimulation, indicating that both markers on IELs reflect activation-dependent events. Unlike IELs, activated lymph node T cells did not express OX40L, thus indicating that OX40-OX40L communication in the intestinal epithelium is part of a novel CD8 network. Functionally, OX40 expression was exclusively associated with IELs with active intracellular IFN-gamma synthesis and markedly enhanced cell-mediated cytotoxicity. However, OX40 costimulation during CD3-mediated activation significantly suppressed IL-10 synthesis by IELs, whereas blockade of OX40-OX40L by anti-OX40L mAb markedly increased IL-10 production. These findings indicate that: 1) resident CD69(+)OX40(-) IELs constitute a population of partially activated T cells poised for rapid delivery of effector activity, 2) OX40 and OX40L expression defines IELs that have undergone recent immune activation, 3) OX40(+) IELs are significantly more efficient CTL than are OX40(-) IELs, and 4) the local OX40/OX40L system plays a critical role in regulating the magnitude of cytokine responses in the gut epithelium.

Rapid and transient reduction in circulating thyroid hormones following systemic antigen priming: implications for functional collaboration between dendritic cells and thyroid.

The thyroid hormones T(3) (tri-iodothyronine) and T(4) (thyroxine) are disseminated throughout the body via the circulation and are maintained across a range of physiological concentrations under the control of thyroid-stimulating hormone (TSH). T(3) (and T(4) after conversion to T(3)) influences many biological activities, including gene expression and protein synthesis, though little is known about the nature of pituitary-thyroid immune interactions. In the present study we show that serum T(3) and T(4) levels are sharply but transiently reduced during the first 24 h of systemic antigen exposure and that this is followed by suppressed levels of free T(4), after which there is rapid recovery to normal levels. Splenic dendritic cells, depending upon the stage of maturation/activation, were found to be a rich source of TSH, and CD11c(+) cells with dendritic cell morphology were present in the thyroid 1-3 days after antigen exposure. Moreover, antigen priming of hypophysectomized mice that are unable to make pituitary-derived TSH resulted in significant increases in circulating T(4), implying that compensation in the drop in thyroid hormones can be regulated from extrapituitary sources. These findings thus identify a novel set of immune-endocrine interactions that transpire during the early phase of antigen exposure, and they suggest that under appropriate conditions the immune system directly participates in the process of maintaining physiological homeostasis by contributing to the regulatory control of thyroid hormone activity.

Analgesia for circumcision in a paediatric population: comparison of caudal bupivacaine alone with bupivacaine plus two doses of clonidine.

BACKGROUND: Clonidine is often used to improve the duration and quality of analgesia produced by caudal epidural blockade, although the optimum dose of clonidine with bupivacaine remains uncertain. METHODS: We compared the effect of clonidine, 1 and 2 microg x kg(-1), added to bupivacaine (1.25 mg x kg(-1)) with that of bupivacaine alone in 75 male children undergoing elective circumcision. RESULTS: There was a trend towards increasing duration of analgesia with increasing dose of clonidine [group B (bupivacaine) 280.7 (171.6) min, C1 (bupivacaine + clonidine 1 microg x kg(-1)) 327.8 (188.3) min and C2 (bupivacaine + clonidine 2 microg x kg(-1)) 382.0 (200.6) min], although this difference was not statistically significant. Mean time to arousal from anaesthesia was significantly prolonged with clonidine 2 microg kg(-1) (group C2 21.3 (13-36) min, group C1 14.0 (6-25) min and group B 14.4 (2-32) min. Supplementary analgesic requirements and incidence of adverse effects were low, with no differences between the groups. CONCLUSIONS: For paediatric circumcision, under general anaesthesia, the addition of clonidine 2 microg x kg(-1) to low volume (0.5 ml x kg(-1)) caudal anaesthetics has a limited clinical benefit for children undergoing circumcision.

Immune-endocrine interactions of the hypothalamus-pituitary-thyroid axis: integration, communication and homeostasis.

The immune and neuroendocrine systems are two essential physiological components of mammalian organisms. Although each is primarily committed to a set of tasks involved, on the one hand, in the protection from infection and disease, and on the other hand, in the regulation of metabolism and other physiological activities, there is also evidence indicating that active and dynamic collaborations exist between those systems in the execution of their designated functions. These interactions occur at many stages of embryonic and neonatal development, and they are a continual part of the normal homeostatic balance needed to maintain health. The present review discusses various historical and contemporary perspectives of immune-endocrine interactions involving the hypothalamus-pituitary-thyroid axis, and offers a hypothesis of how this aspect of the neuroendocrine system participates directly in the immune response to antigenic challenge, infection and disease.

CD43 potentiates CD3-induced proliferation of murine intestinal intraepithelial lymphocytes.

The involvement of CD43 in cell proliferation of murine intestinal intraepithelial lymphocytes (IEL) has been studied in in vitro CD3-stimulated cell cultures. In the presence of either IL-2 or IL-15, CD3 stimulation of IEL resulted in low levels of proliferation as measured by thymidine incorporation, whereas no proliferation occurred upon CD3 stimulation in the absence of cytokines. The combination of both cytokines to IEL cultures synergistically enhanced CD3-induced proliferation by approximately threefold that of cultures supplemented with either cytokine alone. Most importantly, however, proliferation of IEL was significantly greater when CD3 stimulation occurred in conjunction with CD43 triggering, indicating that CD43 functions as a coactivational signal for murine IEL. These findings indicate that a spectrum of potential proliferative responses exist among murine IEL depending on the types and combinations of signals received, and that because under normal conditions murine IEL are largely devoid of CD28 expression, a classical T-cell coactivational molecule, the capacity for high-level IEL proliferation may reside with CD43.

Salmonella pathogenicity island 2-encoded type III secretion system mediates exclusion of NADPH oxidase assembly from the phagosomal membrane.

Salmonella typhimurium requires a type III secretion system encoded by pathogenicity island (SPI)-2 to survive and proliferate within macrophages. This survival implies that S. typhimurium avoids or withstands bactericidal events targeted to the microbe-containing vacuole, which include intraphagosomal production of reactive oxygen species (ROS), phagosomal acidification, and delivery of hydrolytic enzymes to the phagosome via fusion with lysosomes. Recent evidence suggests that S. typhimurium alters ROS production by murine macrophages in an SPI-2-dependent manner. To gain insights into the mechanism by which S. typhimurium inhibits intraphagosomal ROS production, we analyzed the subcellular distribution of NADPH oxidase components during infection of human monocyte-derived macrophages by wild-type (WT) or several SPI-2 mutant strains of S. typhimurium. We found that the membrane component of the NADPH oxidase, flavocytochrome b(558), was actively excluded or rapidly removed from the phagosomal membrane of WT-infected monocyte-derived macrophages, thereby preventing assembly of the NADPH oxidase complex and intraphagosomal production of superoxide anion. In contrast, the NADPH oxidase assembled on and generated ROS in phagosomes containing SPI-2 mutant S. typhimurium. Subversion of NADPH oxidase assembly by S. typhimurium was accompanied by increased bacterial replication relative to that of SPI-2 mutant strains, suggesting that the ability of WT S. typhimurium to prevent NADPH oxidase assembly at the phagosomal membrane represents an important virulence factor influencing its intracellular survival.

Salmonella pathogenicity island 2-encoded proteins SseC and SseD are essential for virulence and are substrates of the type III secretion system.

Survival of Salmonella enterica serovar Typhimurium within host phagocytic cells is a critical step in establishing systemic infection in mice. Genes within Salmonella pathogenicity island 2 (SPI-2) encode a type III secretion system that is required for establishment of systemic infection. Several proteins encoded by SPI-2 have homology to type III secreted proteins from enteropathogenic Escherichia coli and Yersinia and, based on that homology, are predicted to be secreted through the SPI-2 type III secretion system. We have investigated the roles of two of these proteins, SseC and SseD. We demonstrate here that the SseD protein is required for systemic Salmonella infection of the mouse, and we confirmed the virulence requirement for the SseC protein. Experiments were performed, using cellular fractionation and immunoblotting, to identify the subcellular location of the SseC and SseD proteins. Both proteins were found to localize predominantly to the bacterial cell membrane. In addition, our work revealed that SseC and SseD are exposed to the extracellular environment and are loosely associated with the bacterial membrane. Furthermore, localization of SseC and SseD to the bacterial membrane was found to require a functional SPI-2 type III secretion system. Collectively, these results indicate that the SseC and SseD proteins are secreted by the SPI-2 type III secretion system to the bacterial membrane in order to perform their virulence functions.

Immune function of thyroid stimulating hormone and receptor.

Thyroid stimulating hormone (TSH) is a central component of the hypothalamus-pituitary-thyroid axis. Although TSH is known for its important biological effects as a neuroendocrine used to regulate thyroid hormone activity and subsequent metabolic functions, TSH also has been shown to be produced and used by cells ofthe mammalian immune system. Moreover, recent findings have linked the use of TSH by cells of the immune system in humans and mice to a group of monocytic cells and lymphocytes--primarily dendritic cells, macrophages, and subset of naïve peripheral T cells. Other studies have demonstrated the capacity of dendritic cells and monocytes to produce biologically active TSH, thereby pointing to a process of paracrine or autocrine TSH-mediated communication during the earliest stages of an immune response to antigen. In this article, these and other features of TSH immune-endocrine interactions are discussed in the context of an intrinsic TSH immunological pathway. Additionally, a hypothesis is proposed in which TSH produced by cells of the immune system during acute antigen exposure plays a dual role, consisting on the one hand of TSH communication during antigen-driven immune activation while concomitantly serving to regulate physiological homeostasis by modulating and adjusting thyroid hormone activity.

The thyrotropin (thyroid-stimulating hormone) receptor is expressed on murine dendritic cells and on a subset of CD45RBhigh lymph node T cells: functional role for thyroid-stimulating hormone during immune activation.

Thyroid-stimulating hormone (TSH), a central neuroendocrine mediator of the hypothalamus-pituitary-thyroid axis, has been shown to affect various aspects of immunological development and function. To gain a better understanding of TSH involvement within the mammalian immune system, the expression and distribution of the TSH receptor (TSHr) has been studied by immunoprecipitation and by flow cytometric analyses. Using highly enriched populations of B cells, T cells, and dendritic cells, trace amounts of TSHr were precipitated from B cells and T cells, whereas high levels of TSHr were precipitated from the dendritic cell fraction. Flow cytometric analyses of TSHr expression on splenic and lymph node T cells revealed a major difference between those tissues in that only 2-3% of splenic T cells were TSHr+, whereas 10-20% of CD4+8- and CD4-8+ lymph node T cells expressed the TSHr, which was exclusively associated with CD45RB(high) cells and was not expressed during or after activation. The TSHr was not present on cells of the immune system during fetal or neonatal life. However, recombinant TSHbeta was found to significantly enhance the phagocytic activity of dendritic cells from adult animals and to selectively augment IL-1beta and IL-12 cytokine responses of dendritic cells following phagocytic activation. These findings identify a novel immune-endocrine bridge associated with professional APCs and naive T cells.

Transcriptional organization and function of invasion genes within Salmonella enterica serovar Typhimurium pathogenicity island 1, including the prgH, prgI, prgJ, prgK, orgA, orgB, and orgC genes.

Salmonella enterica serovar Typhimurium initiates infection of a host by inducing its own uptake into specialized M cells which reside within the epithelium overlaying Peyer's patches. Entry of Salmonella into intestinal epithelial cells is dependent upon invasion genes that are clustered together in Salmonella pathogenicity island 1 (SPI-1). Upon contact between serovar Typhimurium and epithelial cells targeted for bacterial internalization, bacterial proteins are injected into the host cell through a type III secretion system that leads to internalization of the bacteria. Previous work has established that the prgH, -I, -J, and -K and orgA genes reside in SPI-1, and the products of these genes are predicted to be components of the invasion secretion apparatus. We report that an error in the published orgA DNA sequence has been identified so that this region encodes two small genes rather than a single large open reading frame. These genes have been designated orgA and orgB. Additionally, an opening reading frame downstream of orgB, which we have designated orgC, has been identified and partially characterized. Previously published work has indicated that the prgH, -I, -J, and -K genes are transcribed from a promoter distinct from that used by the gene immediately downstream, orgA. Here, we present experiments indicating that orgA expression is driven by the prgH promoter. In addition, using reverse transcriptase PCR analysis, we have found that this polycistronic message extends downstream of prgH to include a total of 10 genes. To more fully characterize this invasion operon, we demonstrate that the prgH, prgI, prgJ, prgK, orgA, and orgB genes are each required for invasion and secretion, while orgC is not essential for the invasive phenotype.

Infiltration of the abdominal wall with local anaesthetic after total abdominal hysterectomy has no opioid-sparing effect.

We have measured the effect of infiltration of the deep and superficial layers of the abdominal wound on morphine consumption and pain for 48 h after operation, in 40 patients undergoing total abdominal hysterectomy, in a double-blind randomized study. Patients received wound infiltration with 0.9% normal saline 40 ml or 40 ml of 0.25% bupivacaine with epinephrine 1:200,000. There were no significant differences between groups in morphine consumption, linear analogue scores for pain at rest or on movement, nausea or sedation during the first 48 h after operation. We conclude that infiltration of the deep and superficial layers of the wound of a Pfannenstiel incision with local anaesthetic solution did not confer additional analgesia in patients undergoing major gynaecological surgery.

Origins of intestinal intraepithelial lymphocytes: direct evidence for a thymus-derived gamma delta T cell component.

The contribution of T cell precursors from the thymus and the bone marrow to the pool of intestinal intraepithelial lymphocvtes (IELs) has been studied in a system using donor cells from enhanced-green fluorescent protein (EGFP+ ) transgenic mice a doptively transferred into EGFP- recipient mice. Consistent with previous studies, regeneration of gamma delta and alpha beta T cell populations in the intestinal epithelium occurred within 2-3 weeks of bone marrow transfer into irradiatiated EGFP- animals and prior to T cell repopulation of the spleen, of interest, however, although transfer of whole adult EGFP+ thymocytes to non-irradiated EGFP- congenitally-athymic nude mice produced alpha beta T cells in both the spleen and intestine. Gamma delta T cells in significant number were detected only in the intestine of recipient mice. In contrast, transfer of CD3-, CD4-, CD8- immature thymocytes resulted in no detectable T cells in either the intestine or the spleen of nude mice up to twelve weeks post-cell transfer, suggesting that intestinal IELs generated from thymocytes arose from differentiated lineage-committed cells rather than from immature thymocytes. These findings provide direct evidence for both thymus-independent and thymus-dependent sources of intestinal gamma delta T cells, and they suggest that murine IELs consist of diverse groups of T cells with distinct developmental origins.

'Self' tolerance in a parent-->F1 radiation chimera.

The extent to which T cell immune tolerance to self tissue antigens is acquired during intrathymic development, or also may occur elsewhere in the animal, remains unclear. Experiments have been designed to explore this using allogeneic hematopoietic radiation chimeras in which thymectomized CB6F1 (H-2(b/d)) host mice were engrafted with day 16 C57BL/6 (H-2b) fetal thymus tissues, irradiated with 950 rad 3 weeks later, and reconstituted with day 14 C57BL/6 fetal liver cells. Chimeras constructed in this manner had thymus grafts which developed with normal structure and cellularity as determined from histological sections, and had normal proportions of CD4+ 8- and CD4- 8+ peripheral T cells of donor H-2b origin. Mice showed no signs of acute or chronic GVHD when followed for six months and, although T cells from chimeras were non-reactive to donor (H-2b) or host (H-2d) MHC, they responded to third party (H-2k) alloantigens in primary mixed-lymphocyte reactions. To determine whether tolerance might have been induced by radioresistant host hematopoietic cells, mice were treated with anti-I-Ad monoclonal antibody after irradiation and fetal liver cell transfer. The pattern of alloreactivity of T cells from those animals closely resembled that of non-antibody treated mice, suggesting that tolerance to MHC expressed within the host probably was not due to radioresistant class-II-bearing cells in chimeras. These findings imply that immune tolerance to self antigens can be controlled at sites outside the thymus, and they provide further evidence that allogeneic chimeras can be constructed when elimination of mature T cells from the donor hematopoietic pool has been effectively achieved.

Introduction of peptidase genes from Lactobacillus delbrueckii subsp. lactis into Lactococcus lactis and controlled expression.

Peptidases PepI, PepL, PepW, and PepG from Lactobacillus delbrueckii subsp. lactis, which have no counterparts in Lactococcus lactis, and peptidase PepQ were examined to determine their potential to confer new peptidolytic properties to lactococci. Controllable expression of the corresponding genes (pep genes) was achieved by constructing translational fusions with the promoter of the nisA gene (P(nisA)). A suitable host strain, UKLc10, was constructed by chromosomal integration of the genes encoding the NisRK two-component system into the fivefold peptidase-deficient mutant IM16 of L. lactis. Recombinants of this strain were used to analyze growth, peptidase activities, peptide utilization, and intracellular protein cleavage products. After nisin induction of P(nisA)::pep fusions, all of the peptidases were visible as distinct bands in protein gels. Despite the fact that identical transcription and translation signals were used to express the pep genes, the relative amounts of individual peptidases varied considerably. All of the peptidases exhibited activities in extracts of recombinant UKLc10 clones, but only PepL and PepG allowed the clones to utilize specific peptide substrates as sources of essential amino acids. In milk medium, induction of pepG and induction of pepW resulted in growth acceleration. The activities of all five peptidases during growth in milk medium were revealed by high-performance liquid chromatography analyses of intracellular amino acid and peptide pools.