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ABSTRACTMacroautophagy/autophagy proteins have been linked with the development of immune-mediated diseases including lupus, but the mechanisms for this are unclear due to the complex roles of these proteins in multiple immune cell types. We have previously shown that a form of noncanonical autophagy induced by ITGAV/alpha(v) integrins regulates B cell activation by viral and self-antigens, in mice. Here, we investigate the involvement of this pathway in B cells from human tissues. Our data reveal that autophagy is specifically induced in the germinal center and memory B cell subpopulations of human tonsils and spleens. Transcriptomic analysis show that the induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of ITGAV/alpha(v) integrin-dependent autophagy in human B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells, we found that ITGAV/alpha(v)-dependent autophagy limits activation of specific pathways related to B cell responses, while promoting others. These data provide new mechanistic links for autophagy and B-cell-mediated immune dysregulation in diseases such as lupus.

Acute pancreatitis (AP), resulting from inflammation of the pancreas, accounts for more than 300,000 US hospital discharges per year. Although glucose intolerance has been known as a complication of severe AP, this effect was thought to be transient. Recently, cohort studies and meta-analysis of 24 published studies of 1100 patients who survived one or more episodes of AP revealed that 30% to 40% of patients developed diabetes or impaired glucose tolerance within 3 to 4 years of even a single episode of AP. The National Institute of Diabetes and Digestive and Kidney Diseases funded the Type 1 Diabetes in Acute Pancreatitis Consortium (T1DAPC) to undertake a prospective observational study of the occurrence of diabetes during an AP episode or subsequently, with emphasis on type 1 diabetes. Key factors for funding T1DAPC are the increasing incidence and prevalence of AP, its association with the development of type 1 diabetes and other forms of diabetes after AP, its complications, and associated health care cost. The T1DAPC structure, governance, and research objectives are described in this article. The DREAM (Diabetes RElated to Acute pancreatitis and its Mechanisms) studies to be undertaken by the T1DAPC are described in other articles in this journal's issue.

The association between acute pancreatitis (AP) and diabetes mellitus (DM) has long been established, with the initial descriptions of AP patients presenting with DM after a bout of AP published in the 1940s and 50s. However, the potential mechanisms involved, particularly those components related to the immune system, have not been well defined. The Diabetes RElated to Acute pancreatitis and its Mechanisms (DREAM) study is a multicenter clinical study designed to understand the frequency and phenotype of DM developing after AP. This article describes one objective of the DREAM study: to determine the immunologic mechanisms of DM after AP, including the contribution of β-cell autoimmunity. This component of the study will assess the presence of islet autoimmunity, as well as the magnitude and kinetics of the innate and adaptive immune response at enrollment and during longitudinal follow-up after 1 or more episodes of AP. Finally, DREAM will evaluate the relationship between immune features, DM development, and pancreatitis etiology and severity.

Intratracheal instillation of apoptotic cells enhances resolution of experimental lung inflammation by incompletely understood mechanisms. We report that this intervention induces functional regulatory T lymphocytes (Tregs) in mouse lung experimentally inflamed by intratracheal administration of lipopolysaccharide. Selective depletion demonstrated that Tregs were necessary for maximal apoptotic cell–directed enhancement of resolution, and adoptive transfer of additional Tregs was sufficient to promote resolution without administering apoptotic cells. After intratracheal instillation, labeled apoptotic cells were observed in most CD11c+CD103+myeloid dendritic cells migrating to mediastinal draining lymph nodes and bearing migratory and immunoregulatory markers, including increased CCR7 and β8 integrin (ITGB8) expression. In mice deleted for αv integrin in the myeloid line to reduce phagocytosis of dying cells by CD103+dendritic cells, exogenous apoptotic cells failed to induce transforming growth factor-β1 expression or Treg accumulation and failed to enhance resolution of lipopolysaccharide-induced lung inflammation. We conclude that in murine lung, myeloid phagocytes encountering apoptotic cells can deploy αv integrin–mediated mechanisms to induce Tregs and enhance resolution of acute inflammation.

Interleukin-23 (IL-23) is required for inflammatory Th17 cell function in experimental autoimmune encephalomyelitis (EAE), and IL-23 blockade reduces the number of effector Th17 cells in the CNS. We report that pro-inflammatory Th17 cells express high integrin β3that is IL-23 dependent. Integrin β3was not upregulated on all activated T cells; rather, integrin β3was upregulated along with its functional partner integrin αvon effector Th17 cells and “ex-Th17” cells, and αvβ3hiRORγt+cells expanded during EAE. Integrin αvβ3inhibitors ameliorated clinical signs of EAE, and integrin β3deficiency on CD4+T cells alone was sufficient to block EAE induction. Furthermore, integrin-β3-deficient Th17 cells, but not Th1 cells, were impaired in their ability to induce EAE. Integrin β3−/−T cells induced smaller demyelinated lesions and showed reduced spread and accumulation within the CNS, corresponding with impaired extracellular-matrix-mediated migration. Hence, integrin β3is required for Th17 cell-mediated autoimmune CNS inflammation.

SmartSAMM is the New Zealand dairy industry's new mastitis extension (advisory) programme, which helps farmers capture the benefits of improved udder health on the farm. SmartSAMM builds on the success of the SAMM Plan (the Seasonal Approach to Managing Mastitis Plan), first released in the early 1990s. the SAMM plan relied on a "one-size-fits-all", technical approach to mastitis extension. SmartSAMM aims to help farmers develop customised solutions for their herd. the first innovation, "Healthy udder" was released to farmers in October 2011, followed by the SmartSAMM website in June 2012. Coupled with the launch of proactive management initiatives by Fonterra, the major milk processor, in December 2011, and favourable weather, the average milk SCC for Fonterra dropped by 12%, from 212,000 cells per ml in the 2010/11 season to 187,000 cells per ml for the 2011/12 season. [ABSTRACT FROM AUTHOR]

Integrin-mediated cell adhesion and signaling events are essential for the proper development and homeostasis of most epithelial tissues. Dysregulation of integrin expression and function can cause abnormal epithelial cell proliferation and/or differentiation, contributing to the pathogenesis of malignant epithelial cancers. Here we report on the use of a conditional knockout strategy exploiting the Cre/Lox technology to study the in vivofunctions of αv integrins during epithelial cell proliferation and differentiation. We show that genetic ablation of αv integrin expression in basal epithelial cells of the eyelid skin and conjunctiva causes the formation of tumors that are strikingly similar to the malignant epithelial cancer, squamous cell carcinoma. These data suggest a mechanism whereby αv integrins normally suppress epithelial cell proliferation, likely via adhesion to ECM ligands, as well as by the modulation of intracellular signaling cascades. We propose that αv gene deletion eliminates normal integrin-mediated growth suppression, ultimately leading to cellular transformation and tumorigenesis. Hence, these studies reveal a novel tumor suppressor-like function of αv integrins and provide a genetically tractable mouse model for studying the pathogenesis of squamous cell carcinoma and related cancers of epithelial origin, as well as to test and develop novel therapeutic compounds to treat or prevent squamous cell carcinoma of the skin.

Integrin αvβ3 is important for cell survival, signaling and migration, particularly during angiogenesis and tumorigenesis, where it has been proposed as a therapeutic target. αvβ3 is up-regulated following transplantation and β3 polymorphisms are associated with increased acute kidney rejection, suggesting that αvβ3 may also play a role in transplant rejection. Here, using a model of allogeneic heart transplantation, we show that allograft survival is prolonged in β3 integrin-deficient (β3−/-) mice. This is associated with Th2-type immune responses and reduced T-cell infiltration into grafts and T cells from β3−/-mice show impaired adhesion and migration, consistent with a role for αvβ3 in transmigration. These studies provide evidence that targeting β3 integrins impairs recruitment of effector cells and alters cytokine production, so prolonging graft survival. We also show that low doses of blocking antibodies against leukocyte function associated antigen-1 (LFA-1)/αLβ2 and very late antigen-4 (VLA-4)/α4β1, when combined with deletion of β3, lead to long-term survival of allografts with no evidence of chronic rejection. Hence we provide strong mechanistic evidence supporting previous genetic studies, demonstrate the involvement of β3 integrins in both acute and chronic rejection and identify β3 as a new target for immunosuppressive therapy.

We examined the effects of administering recombinant bovine cytokines to non-lactating dairy cows and measured mammary gland leucocytes and the involution process. After the final milking, groups of cows were given an intramammary infusion of cytokine in two quarters. These cytokines were recombinant bovine interleukin-2 (rboIL-2) (2×105 units, n=6), recombinant bovine granulocyte-macrophage colony stimulating factor (rboGM-CSF) (500 μg, n=4) and recombinant bovine interleukin-1β (rboIL-1β) (10 μg, n=10). Each animal also received an infusion of phosphate-buffered saline (PBS) in the other two quarters as controls. The rboIL-2 and rboGM-CSF were produced in a yeast expression system, while rboIL-1β was produced in Escherichia coli. Leucocyte numbers, bactericidal activity of leucocytes, and concentrations of citrate and lactoferrin in quarter secretion samples were monitored after infusion of cytokine or PBS. Infusion of rboIL-2 had minimal effect on leucocyte numbers and concentrations of citrate and lactoferrin. Both rboGM-CSF and rboIL-1β induced a rapid increase in the number of neutrophils and macrophages compared with control PBS quarters. Concentrations of lactoferrin in secretions were increased by rboGM-CSF and rboIL-1β compared with control PBS quarters. In addition, infusion of glands with rboIL-1β lowered the citrate∶lactoferrin molar ratio compared with PBS control quarters. The results indicate that intramammary infusion of either rboGM-CSF or rboIL-1β at cessation of milking immediately increased the number of phagocytic cells in the gland. These cytokines, in particular rboIL-1β, also increased the rate of mammary gland involution during the early dry period.

The discriminatory power of two polymerase chain reaction-based DNA fingerprinting methods, random amplified polymorphic DNA and repetitive extragenic palindrome were compared by subtyping 128 isolates of Streptococcus uberiscultured from cows in six different dairy herds in New Zealand. The typing results demonstrated that the majority of isolates possessed unique fingerprint profiles except on occasions where multiple isolates were obtained from individual cows. On these occasions, individual quarters of the mammary gland were generally, but not exclusively, infected by the same strain of bacteria. Both random amplified polymorphic DNA and repetitive extragenic palindromic typing assays were simple to perform, relatively inexpensive ($11.00 per reaction), and provided reliable and reproducible results. Furthermore, when these assays were used in conjunction with each other, they provided a means of confirmation of the specific DNA fingerprint patterns obtained.

An experiment using three New Zealand herds and a total of 632 cows, examined the effect of localised prophylactic treatments with antibiotic at drying-off on the incidence of new intramammary infection during the dry period and at calving. Antibiotic was infused either into the teat canal (0·22 g of dry-cow formulation) or the teat sinus (3·1 g of lactating-cow formulation) of uninfected quarters to eliminate any bacteria present in these locations at the last milking of lactation. These treatments were compared with a negative control (nil treatment) and a positive antibiotic control (infusion of 3·6 g of dry-cow formulation). All antibiotic formulations used the same active ingredient, sodium cloxacillin. No significant reduction in new dry period clinical mastitis was observed for the two localised treatments whereas the positive control treatment achieved 100% reduction in new clinical mastitis compared with untreated control quarters. A 41% reduction (P<0·05) in new Streptococcus uberis infections at calving was associated with the teat canal antibiotic treatment, compared with an 82% reduction (P<0·001) for the positive antibiotic control. Both localised treatments showed a reduced incidence of new intramammary infection (P<0·001) when pooled across periods and pathogens. Teats receiving either the teat canal antibiotic treatment or a full infusion of long acting dry-cow antibiotic had a lower incidence of open teat canals (P<0·05) at 3 weeks after drying-off.

The physiological effects of intramammary infusions of recombinant bovine cytokines in six lactating dairy cows on the quality and yield of milk and the bactericidal activity of milk neutrophils were investigated. Recombinant bovine interleukin-2 (rboIL-2) and interferon-γ (rboIFN-γ) were produced in the yeast Pichia pastoris. Two animals were given rboIL-2 (2×105 units) in two quarters, two animals were given rboIFN-γ (6·5×105 units) in two quarters, and the other two cows received a dose of rboIL-2 in one quarter and rboIFN-γ in a second quarter. In addition, each animal was given phosphate-buffered saline (PBS) in the other two quarters as a control. Somatic cell counts and conductivity of the fore milk were monitored before and after infusion. Neutrophils were isolated from quarter milk samples 36 h after infusion of cytokine or PBS and their bactericidal activities against Staphylococcus aureus were measured in vitro with a colorimetric assay. Quarters infused with rboIL-2 or rboIFN-γ showed significant but transitory increases in both milk somatic cell counts and conductivity when compared with preinfusion values and with control quarters. There were minimal effects on daily milk yield. Neutrophils isolated from milk from quarters infused with rboIL-2 showed enhanced bactericidal activity against Staph. aureus. The bacterial killing from rboIL-2 treated quarters was significantly greater, with a mean of 63·5% compared with a mean of 5·4% for neutrophils taken from uninfected quarters to which PBS had been administered. The bactericidal activities for quarters treated with rboIFN-γ and infected quarters treated with PBS were 15·0 and 30·0% respectively. The results indicate that intramammary infusions of rboIL-2 and rboIFN-γ to lactating cows are well tolerated, and that rboIL-2 can activate milk neutrophils and augment their bactericidal activity.

Twenty-four monozygous twinsets in late lactation (210 d in milk) were used to examine the effects of feed restriction and milking frequency prior to drying off on milk yield and composition in a pastoral dairying system. Cows were assigned to one of four treatment groups for 26 d and were milked either twice or once daily and given either unrestricted or restricted access to feed. Dry matter intakes averaged 16 or 8kg per cow per day, and diets comprised ryegrass and white clover pasture supplemented with 15% pasture silage.

Electrification in developed countries has progressively increased the mean level of extremely low‐frequency electromagnetic fields (ELF‐EMFs) to which populations are exposed; these humanmade fields are substantially above the naturally occurring ambient electric and magnetic fields of ~10‐3Vm‐1and ~10‐13T, respectively. Several epidemiological studies have concluded that ELF‐EMFs may be linked to an increased risk of cancer, particularly childhood leukemia. These observations have been reinforced by cellular studies reporting EMF‐induced effects on biological systems, most notably on the activity of components of the pathways that regulate cell proliferation. However, the limited number of attempts to directly replicate these experimental findings have been almost uniformly unsuccessful, and no EMF‐induced biological response has yet been replicated in independent laboratories. Many of the most well‐defined effects have come from gene expression studies; several attempts have been made recently to repeat these key findings. This review analyses these studies and summarizes other reports of major cellular responses to EMFs and the published attempts at replication. The opening sections discuss quantitative aspects of exposure to EMFs and the incidence of cancers that have been correlated with such fields. The concluding section considers the problems that confront research in this area and suggests feasible strategies.—Lacy‐hulbert, A., Metcalfe, J. C., Hesketh, R. Biological responses to electromagnetic fields. FASEB J. 12, 395–420 (1998)