Cutting edge: a TLR9 cytoplasmic tyrosine motif is selectively required for proinflammatory cytokine production.

Compartmentalization of nucleic acid sensing TLR9 has been implicated as a mechanism to prevent recognition of self nucleic acid structures. Furthermore, recognition of CpG DNA in different endosomal compartments leads to the production of the proinflammatory cytokine TNF-α, or type I IFN. We previously characterized a tyrosine-based motif at aa 888-891 in the cytoplasmic tail of TLR9 important for appropriate intracellular localization. In this article, we show that this motif is selectively required for the production of TNF, but not IFN. In response to CpG DNA stimulation, the proteolytically processed 80-kDa fragment is tyrosine phosphorylated. Although Y888 is not itself phosphorylated, the structure of this motif is necessary for both TLR9 phosphorylation and TNF-α production in response to CpG DNA. We conclude that bifurcation in TLR9 signaling is regulated by a critical tyrosine motif in the cytoplasmic tail.

Negative regulation of signaling by a soluble form of toll-like receptor 9.

Nucleic acid structures are highly conserved through evolution and when self nucleic acids are aberrantly detected by toll-like receptors (TLRs) they contribute to autoimmune disease. For this reason, multiple regulatory mechanisms exist to prevent immune responses to self nucleic acids. TLR9 is a nucleic acid-sensing TLR that is regulated at multiple levels including association with accessory proteins, intracellular localization and proteolytic processing. In the endolysosomal compartment TLR9 is proteolytically processed to an 80 kDa form (p80) and this processing is a prerequisite for activation. Here, we identified a soluble form of TLR9 (sTLR9) generated by a novel proteolytic event that cleaved TLR9 between amino acids 724-735. Similar to p80, sTLR9 was generated in endosomes. However, generation of sTLR9 was independent of the cysteine protease cathepsin B, active at acidic pH, but partially dependent on cathepsin S, a protease active at neutral pH. Most importantly, sTLR9 inhibited TLR9-dependent signaling. Altogether, these data support a model where an intrinsic proteolytic processing mechanism negatively regulates TLR9 signaling. A proper balance between the independent proteolytic events probabably contributes to regulation of TLR9-mediated innate immunity and prevention of autoimmune disease.

An expert commentary on essential equipment, supplies and culture media in the assisted reproductive technology laboratory.

The assisted reproductive technology (ART) laboratory is a complex system designed to sustain the fertilization, survival, and culture of the preimplantation embryo to the blastocyst stage. ART outcomes depend on numerous factors, among which are the equipment, supplies and culture media used. The number and type of incubators also may affect ART results. While large incubators may be more suitable for media equilibration, bench-top incubators may provide better embryo culture conditions in separate or smaller chambers and may be coupled with time-lapse systems that allow continuous embryo monitoring. Microscopes are essential for observation, assessment, and micromanipulation. Workstations provide a controlled environment for gamete and embryo handling and their quantity should be adjusted according to the number of ART cycles treated in order to provide a steady and efficient workflow. Continuous maintenance, quality control and monitoring of equipment are essential and quality control devices such as the thermometer, and pH-meter are necessary to maintain optimal culture conditions. Tracking, appropriate delivery and storage conditions, and quality control of all consumables are recommended so that adequate quantity and quality are available for use. Embryo culture media have evolved: preimplantation embryos are cultured either by sequential media or single-step media that can be used for interrupted or uninterrupted culture. There is currently no sufficient evidence that any individual commercially-available culture system is better than others in terms of embryo viability. In this review, we aim to analyze the various parameters that should be taken into account when choosing the essential equipment, consumables and culture media systems that will create optimal culture conditions and provide the most effective patient treatment.

Optimizing embryological aspects of oocyte retrieval, oocyte denudation, and embryo loading for transfer.

Oocyte retrieval, oocyte denudation, and embryo transfer are crucial processes during assisted reproduction technology (ART). Air quality in the ART laboratory, temperature, pH of the media used and the time interval between oocyte retrieval and insemination are all critical factors. Anesthesia is required for oocyte retrieval, however, evidence regarding the potential impact of different methods (general anesthesia, conscious sedation, and local anesthesia) on the clinical outcomes is unclear. The optimal timing of oocyte denudation following retrieval has not been established. Regarding the mechanical denudation process, there is a lack of evidence to demonstrate the safest minimum inner diameter of denuding pipettes used to complete the removal of granulosa cells surrounding the oocytes. During embryo transfer, many clinics worldwide flush the catheter before embryo loading, in an attempt to potentially rinse off any toxic agents; however, there is insufficient evidence to show that flushing the embryo transfer catheter before loading increases the success of ART outcome. Considering the serious gaps in knowledge in ART practice, the aim of this review is to provide an updated overview of the current knowledge regarding the various steps and techniques involved in oocyte retrieval, oocyte denudation, and embryo loading for transfer.

Early response of mucosal epithelial cells during Toxoplasma gondii infection.

The innate immune response of mucosal epithelial cells during pathogen invasion plays a central role in immune regulation in the gut. Toxoplasma gondii is a protozoan intracellular parasite that is usually transmitted through oral infection. Although much of the information on immunity to T. gondii has come from i.p. infection models, more recent studies have revealed the importance of studying immunity following infection through the natural peroral route. Oral infection studies have identified many of the key players in the intestinal response; however, they have relied on responses detected days to weeks following infection. Much less is known about how the gut epithelial layer senses and reacts during initial contact with the pathogen. Given the importance of epithelial cells during pathogen invasion, this study uses an in vitro approach to isolate the key players and examine the early response of intestinal epithelial cells during infection by T. gondii. We show that human intestinal epithelial cells infected with T. gondii elicit rapid MAPK phosphorylation, NF-kappaB nuclear translocation, and secretion of IL-8. Both ERK1/2 activation and IL-8 secretion responses were shown to be MyD88 dependent and TLR2 was identified to be involved in the recognition of the parasite regardless of the parasite genotype. Furthermore, we were able to identify additional T. gondii-regulated genes in the infected cells using a pathway-focused array. Together, our findings suggest that intestinal epithelial cells were able to recognize T. gondii during infection, and the outcome is important for modulating intestinal immune responses.

A Web-Based Global Educational Model for Training in Semen Analysis during the COVID-19 Pandemic.

PURPOSE: In response to the COVID-19 pandemic, the American Center for Reproductive Medicine (ACRM) transitioned its annual training in assisted reproductive technology (ART) from a hands-on, laboratory-based training course to a fully online training endorsed by the American College of Embryology. Here we describe our experience and assess the quality of an online training format based on participant outcomes for the first three modules of a planned series of online ART training. MATERIALS AND METHODS: These modules included manual semen analysis, sperm morphology and ancillary semen tests (testing for leukocytospermia, sperm vitality, and anti-sperm antibody screening). The virtual format consisted of lecture presentations featuring laboratory protocols with corresponding video demonstrations of routine techniques and best practices. Practical scenarios, troubleshooting, and clinical interpretation of laboratory results were also discussed. At the end of each module, an optional multiple choice question test was held as a prerequisite to obtain certification on the topics presented. Course quality was assessed using participant responses collected via online surveys. RESULTS: The digital delivery methods used were found to have largely or completely met the participants' expectations for all questions (>85%). The majority (>87%) of the participants either strongly agreed or agreed that the course content was well-structured with appropriate depth, and that their overall expectations of the course had been met. CONCLUSIONS: This training format appears to be a realistic teaching option to freely share highly specialized expertise and technical knowledge with participants from anywhere in the world with varying levels of competency or experience.

A peptide derived from human bactericidal/permeability-increasing protein (BPI) exerts bactericidal activity against Gram-negative bacterial isolates obtained from clinical cases of bovine mastitis.

Gram-negative bacteria are responsible for approximately one-third of the clinical cases of bovine mastitis and can elicit a life-threatening, systemic inflammatory response. Lipopolysaccharide (LPS) is a membrane component of Gram-negative bacteria and is largely responsible for evoking the inflammatory response. Antibiotic and anti-inflammatory therapy for treating Gram-negative infections remains suboptimal. Bactericidal/permeability-increasing protein (BPI) is a neutrophil-derived protein with antimicrobial and LPS-neutralizing properties. Select peptide derivatives of BPI are reported to retain these properties. The objective of this study was to evaluate the antimicrobial activity of a human BPI-derived synthetic peptide against clinical bovine mastitis isolates of Gram-negative bacteria. A hybrid peptide was synthesized corresponding to two regions of human BPI (amino acids 90-99 and 148-161), the former of which has bactericidal activity and the latter of which has LPS-neutralizing activity. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of this peptide against various genera of bacteria were determined using a broth microdilution assay. The MIC's were determined to be: 16-64 microg/ml against Escherichia coli; 32-128 microg/ml against Klebsiella pneumoniae and Enterobacter spp.; and 64-256 microg/ml against Pseudomonas aeruginosa. The MBC's were equivalent to or 1-fold greater than corresponding MIC's. The peptide had no growth inhibitory effect on Serratia marcescens. The antimicrobial activity of the peptide was retained in the presence of serum, but severely impaired in milk. Further functional evaluation of the peptide demonstrated its ability to completely neutralize LPS. Together, these data support additional investigations into the therapeutic application of BPI to the treatment of Gram-negative infections in cattle.

Antimicrobial activity of bovine bactericidal permeability-increasing protein-derived peptides against gram-negative bacteria isolated from the milk of cows with clinical mastitis.

OBJECTIVE: To evaluate antimicrobial activity of bovine bactericidal permeability-increasing protein (bBPI)-derived synthetic peptides against mastitis-causing gram-negative bacteria. SAMPLE POPULATION: Bacterial isolates from the milk of cows with clinical mastitis. PROCEDURES: 3 peptides were synthesized with sequences corresponding to amino acids 65 to 99 (bBPI(6,599)) or 142 to 169 (bBPI(142,169)) or the combination of amino acids 90 to 99 and 148 to 161 (bBPI(9,099, 148,161)) of bBPI. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of these peptides against bacterial isolates from cows with mastitis were determined by use of a standardized broth microdilution assay. The ability of these peptides to retain their antimicrobial activity in serum and milk was also evaluated. Finally, bacterial lipopolysaccharide (LPS)-neutralizing activity of these peptides was assayed with the Limulus amebocyte lysate test. RESULTS: Of the 3 peptides tested, bBPI(9,099, 148,161) had the widest spectrum of antimicrobial activity, with MIC and MBC values ranging from 16 to 64 Mg/mL against Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp and from 64 to 128 Mg/mL against Pseudomonas aeruginosa. None of the peptides had any growth-inhibitory effect on Serratia marcescens. The antimicrobial activity of bBPI(9,099, 148,161) was inhibited in milk, but preserved in serum. Finally, bBPI(142,169) and bBPI(9,099, 148,161) completely neutralized LPS. CONCLUSIONS AND CLINICAL RELEVANCE: bBPI(9,099, 148,161) is a potent neutralizer of the highly proinflammatory molecule bacterial LPS and has antimicrobial activity against a variety of gram-negative bacteria. The ability of bBPI(9099,148161) to retain antimicrobial activity in serum suggests a potential therapeutic application for this peptide in the management of gram-negative septicemia.

Staphylococcus aureus intramammary infection elicits increased production of transforming growth factor-alpha, beta1, and beta2.

In contrast to other mastitis pathogens, the host response evoked during Staphylococcus aureus intramammary infection is marked by the absence of the induction of critical cytokines, including IL-8 and TNF-alpha, which have established roles in mediating host innate immunity. The elucidation of changes in the expression of other mediators with the potential to regulate mammary inflammatory responses to S. aureus remains lacking. Transforming growth factor (TGF)-alpha, TGF-beta1, and TGF-beta2 are cytokines that regulate mammary gland development. Because these cytokines also have a demonstrated role in mediating inflammation, the objective of the current study was to determine whether S. aureus intramammary infection influences their expression. Ten cows were challenged with S. aureus and milk samples collected. Increases in milk levels of TGF-alpha were evident within 32h of infection and persisted for 16h. Increases in TGF-beta1 and TGF-beta2 levels were detected within 40h of S. aureus infection and persisted through the end of the study. Thus, in contrast to IL-8 and TNF-alpha, S. aureus elicits host production of TGF-alpha, TGF-beta1, and TGF-beta2. This finding may suggest a role for these cytokines in mediating mammary gland host innate immune responses to S. aureus.

The bovine innate immune response during experimentally-induced Pseudomonas aeruginosa mastitis.

Almost half of all clinical cases of mastitis are caused by Gram-negative bacteria. Among these bacteria, intramammary infection by Pseudomonas aeruginosa remains one of the most refractory to antibiotic therapy. The ability to recognize potentially harmful pathogens whether previously encountered or not, as well as the induction of an initial pro-inflammatory response to these pathogens, are critical components of host innate immunity. Although the innate immune response to another Gram-negative mastitis-causing pathogen, Escherichia coli, has been well-characterized, little is known about the response to other Gram-negative bacteria, including P. aeruginosa. The objective of the current study was to characterize the systemic and localized bovine innate immune response to intramammary infection with P. aeruginosa. The contralateral quarters of ten mid-lactating Holstein cows were challenged with either saline or P. aeruginosa. Following the establishment of infection, milk samples were collected and assayed for changes in cytokine and growth factor concentrations, complement activation, and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), two accessory molecules involved in host recognition of Gram-negative bacteria. Initial increases in milk somatic cell counts were evident within 12h of experimental challenge and remained elevated for >or=3 weeks. Increased permeability of the mammary gland vasculature, as evidenced by elevated milk levels of BSA, was initially observed 20 h post-infection and persisted for 2 weeks. Within 32 h of challenge, increased levels of IL-8, TNF-alpha, IL-10, and IL-12 were detected, however, the elevated levels of these cytokines were not sustained for longer than a 24h period. In contrast, elevations in IL-1beta, IFN-gamma, TGF-alpha, TGF-beta1, TGF-beta2, sCD14, LBP, and activated complement factor 5 (C5a) were sustained for periods of >48 h. Systemic changes were characterized by elevated body temperature, induction of the acute phase protein synthesis of serum amyloid A and LBP, and a transient decrease in circulating neutrophils and lymphocytes. Together, these data demonstrate the capability of the mammary gland to mount a robust innate immune response to P. aeruginosa that is characterized by the induction of pro-inflammatory cytokines, complement activation, and increased levels of accessory molecules involved in Gram-negative bacterial recognition.

TLR9 traffics through the Golgi complex to localize to endolysosomes and respond to CpG DNA.

Toll-like receptor 9 (TLR9) promiscuously binds self- and microbial DNA, but only microbial DNA elicits an inflammatory response. How TLR9 discriminates between self- and foreign DNA is unclear, but inappropriate localization of TLR9 permits response to self-DNA, suggesting that TLR9 localization and trafficking are critical components. The molecular mechanisms controlling the movement of TLR9 may provide new insight into the recognition of DNA in normal and in pathological conditions such as autoimmune systemic lupus erythematosus. We have shown earlier that TLR9 is retained in the endoplasmic reticulum (ER) and it moves to endolysosomes to recognize CpG DNA. Other studies have suggested that TLR9 bypasses the Golgi complex to access endolysosomes. Here, we show that TLR9 translocates from ER to endolysosomes through the Golgi complex and that Golgi export is required for optimal TLR9 signaling. In all, 6-13% of TLR9 constitutively exits the ER, moves through the Golgi complex and resides in lysosomal-associated membrane protein-1-positive vesicles. TLR9 bound to CpG DNA had glycan modifications indicative of Golgi processing confirming that TLR9 travels through the Golgi complex to access CpG DNA in endolysosomes. Together, these data support a model where TLR9 uses traditional secretory pathways and does not bypass the Golgi complex.