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Point-of-care antibiotic susceptibility test for urinary tract infections.

The HIV reservoir is largely composed of latently infected memory CD4 + T cells that persist during antiretroviral therapy and constitute a major barrier toward HIV eradication. In particular, CD4 + T follicular helper cells have been demonstrated as key targets for viral replication and persistence under ART.

HIV associated immune activation (IA) is associated with increased morbidity in people living with HIV (PLWH) on antiretroviral therapy, and remains a barrier for strategies aimed at reducing the HIV reservoir. The underlying mechanisms of IA have not been definitively elucidated, however, persistent production of Type I IFNs and expression of ISGs is considered to be one of the primary factors. Plasmacytoid DCs (pDCs) are a major producer of Type I IFN during viral infections, and are highly immunomodulatory in acute HIV and SIV infection, however their role in chronic HIV/SIV infection has not been firmly established. Here, we performed a detailed transcriptomic characterization of pDCs in chronic SIV infection in rhesus macaques, and in sooty mangabeys, a natural host non-human primate (NHP) species that undergoes non-pathogenic SIV infection. We also investigated the immunostimulatory capacity of lymph node homing pDCs in chronic SIV infection by contrasting gene expression of pDCs isolated from lymph nodes with those from blood. We observed that pDCs in LNs, but not blood, produced high levels of IFNα transcripts, and upregulated gene expression programs consistent with T cell activation and exhaustion. We apply a novel strategy to catalogue uncharacterized surface molecules on pDCs, and identified the lymphoid exhaustion markers TIGIT and LAIR1 as highly expressed in SIV infection. pDCs from SIV-infected sooty mangabeys lacked the activation profile of ISG signatures observed in infected macaques. These data demonstrate that pDCs are a primary producer of Type I IFN in chronic SIV infection. Further, this study demonstrated that pDCs trafficking to LNs persist in a highly activated state well into chronic infection. Collectively, these data identify pDCs as a highly immunomodulatory cell population in chronic SIV infection, and a putative therapeutic target to reduce immune activation., Author summary For people living with HIV (PLWH), persistent immune activation is an obstacle to optimal health. In this study, we investigate the immunostimulatory potential of plasmacytoid dendritic cells in chronic SIV infection using comparative RNA-Seq. We observed that pDCs from SIV-infected rhesus macaques have highly activated profiles relative to uninfected animals; in contrast, pDCs from SIV-infected natural host sooty mangabeys had expression profiles similar to cells from uninfected animals. In chronically infected RMs, pDCs from lymph nodes maintained activation profiles elevated at levels even higher than those in the blood. Further, transcripts for the immunostimulatory cytokine family IFNA were readily detected in LN homing pDCs, but not those from blood. These data confirm pDCs as a major producer of Type I IFN in chronic SIV infection, and identify them as a target for immunotherapy.

Lymph nodes (LN) and their resident T follicular helper CD4+ T cells (Tfh) are a critical site for HIV replication and persistence. Therefore, optimizing antiviral activity in lymphoid tissues will be needed to reduce or eliminate the HIV reservoir. In this study, we retained effector immune cells in LN of cART-suppressed, SIV-infected rhesus macaques by treatment with the lysophospholipid sphingosine-1 phosphate receptor modulator FTY720 (fingolimod). FTY720 was remarkably effective in reducing circulating CD4+ and CD8+ T cells, including those with cytolytic potential, and in increasing the number of these T cells retained in LN, as determined directly in situ by histocytometry and immunohistochemistry. The FTY720-induced inhibition of T cell egress from LN resulted in a measurable decrease of SIV-DNA content in blood as well as in LN Tfh cells in most treated animals. In conclusion, FTY720 administration has the potential to limit viral persistence, including in the critical Tfh cellular reservoir. These findings provide rationale for strategies designed to retain antiviral T cells in lymphoid tissues to target HIV remission., Author summary FTY720 (fingolimod), a drug approved by the FDA for treatment of multiple sclerosis, blocks the egress of lymphocytes from the lymph node (LN). To determine whether FTY720 retention activity could improve cytolytic responses in the LN and affect SIV persistence, we studied for the first time tolerability and biological activity of two doses of FTY720 in cART-suppressed, SIV-infected rhesus macaques. FTY720 was remarkably effective in reducing circulating CD4+ and CD8+ T cells, including those with cytolytic potential, and in increasing the number of cytolytic T cells in LN. FTY720 administration reduced SIV-DNA content in blood as well as in LN Tfh cells in most of the animals. These results suggest that FTY720 limits viral persistence, including Tfh cellular reservoir, by increasing the number of cytolytic cells in the LN, critical site for HIV/SIV replication and persistence.

Brevinin-2-related peptide (BR-II), a novel antimicrobial peptide isolated from the skin of frog, Rana septentrionalis, shows a broad spectrum of antimicrobial activity with low haemolytic activity. It has also been shown to have antiviral activity, specifically to protect cells from infection by HIV-1. To understand the active conformation of the BR-II peptide in membranes, we have investigated the interaction of BR-II with the prokaryotic and eukaryotic membrane-mimetic micelles such as sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N-terminus tryptophan residue of BR-II interacts with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane-mimetic micelles induce an α-helix conformation in BR-II. We have also determined the solution structures of BR-II in DPC and SDS micelles using NMR spectroscopy. The structural comparison of BR-II in the presence of SDS and DPC micelles showed significant conformational changes in the residues connecting the N-terminus and C-terminus helices. The ability of BR-II to bind DNA was elucidated by agarose gel retardation and fluorescence experiments. The structural differences of BR-II in zwitterionic versus anionic membrane mimics and the DNA binding ability of BR-II collectively contribute to the general understanding of the pharmacological specificity of this peptide towards prokaryotic and eukaryotic membranes and provide insights into its overall antimicrobial mechanism.

Emerging evidence indicates a central role for the microbiome in immunity. However, causal evidence in humans is sparse. Here, we administered broad-spectrum antibiotics to healthy adults prior and subsequent to seasonal influenza vaccination. Despite a 10,000-fold reduction in gut bacterial load and long-lasting diminution in bacterial diversity, antibody responses were not significantly affected. However, in a second trial of subjects with low pre-existing antibody titers, there was significant impairment in H1N1-specific neutralization and binding IgG1 and IgA responses. In addition, in both studies antibiotics treatment resulted in (1) enhanced inflammatory signatures (including AP-1/NR4A expression), observed previously in the elderly, and increased dendritic cell activation; (2) divergent metabolic trajectories, with a 1,000-fold reduction in serum secondary bile acids, which was highly correlated with AP-1/NR4A signaling and inflammasome activation. Multi-omics integration revealed significant associations between bacterial species and metabolic phenotypes, highlighting a key role for the microbiome in modulating human immunity.

Objective The sudden closure of 30 out of 54 acute psychiatric beds in Cornwall presented a stressful challenge to staff but also a natural experiment on how a service dealt with this situation. We aimed to evaluate the outcomes of patients needing to leave the closed ward, how bed occupancy rates were affected and the impact on admission rates. Design A service evaluation of the impact of the ward closure. Setting A comprehensive secondary NHS mental health service in Cornwall serving 550,000 population. Main outcome measures The destination of the patients needing to leave the acute unit, the effect of the closure on bed occupancy, admission rates and serious untoward incidents. Results Of 26 patients needing to be moved from the acute ward, only 10 needed an acute psychiatric bed. None of the seven patients who had been on the ward longer than nine weeks needed an acute unit. Admission rates fell over the subsequent three months. There was no increase in serious incidents due to the closure. Conclusions This naturalistic event suggests that many patients on acute units could be cared for elsewhere, especially recovery/rehabilitation care environments, if political and financial urgency is present. Admission rates are responsive to the pressure on beds.

The objective of this work is two-fold. First, this work focuses on detection of alive Homo sapiens. Second, it focuses on obstacle detection and collision avoidance. The main aim of this work is to maintain both needs simultaneously. To define the path more precisely, fuzzy set theory is used which tackle with the intermediate values of direction. A Sugeno fuzzy controller algorithm is used which takes three inputs labelled as obstacle position obtained through ultrasonic sensor, obstacle distance through IR sensor and CO2 sensor for finding presence of carbon-dioxide. The work is evaluated on different parameters. As compare to other rescue robots, the number of passes used by our proposed rescue robot is comparatively lesser. The other parameter is wheel computation which is used for measuring the motion. The proposed rescue robot is more flexible while determining its optimising path.

Summary Systems biological analysis of immunity to the trivalent inactivated influenza vaccine (TIV) in humans revealed a correlation between early expression of TLR5 and the magnitude of the antibody response. Vaccination of Trl5 −/− mice resulted in reduced antibody titers and lower frequencies of plasma cells, demonstrating a role for TLR5 in immunity to TIV. This was due to a failure to sense host microbiota. Thus, antibody responses in germ-free or antibiotic-treated mice were impaired, but restored by oral reconstitution with a flagellated, but not aflagellated, strain of E. coli . TLR5-mediated sensing of flagellin promoted plasma cell differentiation directly and by stimulating lymph node macrophages to produce plasma cell growth factors. Finally, TLR5-mediated sensing of the microbiota also impacted antibody responses to the inactivated polio vaccine, but not to adjuvanted vaccines or the live-attenuated yellow fever vaccine. These results reveal an unappreciated role for gut microbiota in promoting immunity to vaccination.

Brevinin-2-related peptide (BR-II), a novel antimicrobial peptide isolated from the skin of frog, Rana septentrionalis, shows a broad spectrum of antimicrobial activity with low haemolytic activity. It has also been shown to have antiviral activity, specifically to protect cells from infection by HIV-1. To understand the active conformation of the BR-II peptide in membranes, we have investigated the interaction of BR-II with the prokaryotic and eukaryotic membrane-mimetic micelles such as sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N-terminus tryptophan residue of BR-II interacts with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane-mimetic micelles induce an α-helix conformation in BR-II. We have also determined the solution structures of BR-II in DPC and SDS micelles using NMR spectroscopy. The structural comparison of BR-II in the presence of SDS and DPC micelles showed significant conformational changes in the residues connecting the N-terminus and C-terminus helices. The ability of BR-II to bind DNA was elucidated by agarose gel retardation and fluorescence experiments. The structural differences of BR-II in zwitterionic versus anionic membrane mimics and the DNA binding ability of BR-II collectively contribute to the general understanding of the pharmacological specificity of this peptide towards prokaryotic and eukaryotic membranes and provide insights into its overall antimicrobial mechanism.

Our previous studies have shown that overexpression of aromatase in mammary glands results in the induction of hyperplastic and dysplastic changes in female transgenic mice. In this study we show that overexpression of aromatase in male transgenic mice results in increased mammary growth and histopathological changes similar to gynecomastia. Increased estrogenic activity also results in an increase in estrogen and progesterone receptor expression in the mammary glands of transgenic males as compared to the nontransgenic males, as well as an increase in the expression of various genes involved in cell cycle and cell proliferation. We have also observed an increase in certain growth factors, such as bFGF and TGFbeta, as a result of aromatase overexpression in the male transgenic mammary glands. In order to obtain a better understanding of the biological significance of gynecomastia, a reliable model is necessary to explain the mechanisms and correlations associated with human cancers. This model, can potentially serve as a predictable and useful tool for studying gynecomastia, hormonal carcinogenesis and action of other carcinogens on hormone induced cancers.

Despite recent advances in diagnosis and treatment of testicular cancer, its causes remain unknown. The most common conditions known to be associated with testicular cancer are cryptorchidism, infertility, and overexposure to pesticides or radiation. Recent studies also indicate hormones may play a crucial role in testicular tumorigenesis. Our studies show that about half of the male transgenic mice overexpressing aromatase in testis were infertile and/or had larger than normal testicles. Gross pathology and histological analysis showed the mice to have Leydig cell tumors, unilaterally or bilaterally. Serum estradiol levels for transgenic mice were at least twice as high as those for nontransgenic mice. Expression of aromatase and estrogen receptor were also very high in testicular tissue of transgenic mice compared to nontransgenic mice. Consistent with increased estrogenic activity in the testicular tissue, we also saw an increase in the levels of genes involved in cell cycle that are regulated by the estrogen. To obtain a better understanding of the biological significance of testicular tumorigenesis, a reliable animal model is necessary to clarify the mechanisms and correlations associated with human cancers. Here we describe such a model, which shows that overexpression of aromatase results in increased estrogen production and a changed hormone milieu, leading to the induction of testicular cancer (Leydig cell tumors). This predictable and useful model is a potential tool for the study of testicular tumorigenesis, hormonal carcinogenesis, synergistic action of other carcinogens on hormone-induced tumors, and tumor dependency on endocrine factors.

Modified vaccinia virus Ankara (MVA) is a safe, attenuated orthopoxvirus that is being developed as a vaccine vector but has demonstrated limited immunogenicity in several early-phase clinical trials. Our objective was to rationally improve the immunogenicity of MVA-based HIV/AIDS vaccines via the targeted deletion of specific poxvirus immune-modulatory genes. Vaccines expressing codon-optimized HIV subtype C consensus Env and Gag antigens were generated from MVA vector backbones that (i) harbor simultaneous deletions of four viral immune-modulatory genes, encoding an interleukin-18 (IL-18) binding protein, an IL-1β receptor, a dominant negative Toll/IL-1 signaling adapter, and CC-chemokine binding protein (MVAΔ4-HIV); (ii) harbor a deletion of an additional (fifth) viral gene, encoding uracil-DNA glycosylase (MVAΔ5-HIV); or (iii) represent the parental MVA backbone as a control (MVA-HIV). We performed head-to-head comparisons of the cellular and humoral immune responses that were elicited by these vectors during homologous prime-boost immunization regimens utilizing either high-dose (2 × 10 8 PFU) or low-dose (1 × 10 7 PFU) intramuscular immunization of rhesus macaques. At all time points, a majority of the HIV-specific T cell responses, elicited by all vectors, were directed against Env, rather than Gag, determinants, as previously observed with other vector systems. Both modified vectors elicited up to 6-fold-higher frequencies of HIV-specific CD8 and CD4 T cell responses and up to 25-fold-higher titers of Env (gp120)-specific binding (nonneutralizing) antibody responses that were relatively transient in nature. While the correlates of protection against HIV infection remain incompletely defined, our results indicate that the rational deletion of specific genes from MVA vectors can positively alter their cellular and humoral immunogenicity profiles in nonhuman primates.

Although elderly patients have physiologic impairments in wound healing, their wounds should be expected to heal with the same frequency of closure as those in younger populations, albeit at a slower rate. However, compared to the general population, the elderly population has a higher incidence of chronic wounds: diabetic foot ulcers, pressure ulcers, and venous stasis ulcers. Experimental and clinical data indicate physiologically impaired healing is characterized by decreased angiogenesis and synthesis of critical growth factors. Further, compared to younger populations, the elderly have a higher rate of mortality associated with specific morbidities, such as sepsis and acute respiratory distress. As these morbidities may develop directly from the wound, early intervention is mandated. In this report, 40 consecutive elderly patients (65-102 years old) with chronic wounds were analyzed. All patients were provided the same treatment protocol and healing was defined as 100% epithelization and no drainage. Despite the wounds presenting in a nonhealing and/or infected state, 73% of these chronic wounds in elderly patients healed. This suggests that elderly patients with diabetic foot ulcers, pressure ulcers, and venous stasis ulcers close their wounds at a similar frequency as younger patients. Therefore, early intervention and comprehensive treatment that includes safe topical therapies, in addition to growth factors and cellular therapy used for chronic wounds, ensure these patients will be spared the morbidities of pain, amputation, osteomyelitis, and even death. We hypothesize that if all elderly patients with chronic wounds are provided early treatment, morbidities (e.g., amputation, sepsis, pain) and associated costs will decrease.

Diabetic foot and pressure ulcers are chronic wounds by definition. They share similar pathogeneses; i.e., a combination of increased pressure and decreased angiogenic response. Neuropathy, trauma, and deformity also often contribute to development of both types of ulcers. Early intervention and proper treatment should result in complete healing of non-ischemic diabetic foot and pressure ulcers, as defined by 100% epithelialization and no drainage (if no osteomyelitis is present). The authors developed the following paradigm, which has proved to be highly effective for complete healing of these wounds: 1) recognition that all patients with limited mobility are at risk for a sacral, ischial, trochanteric, or heel pressure ulcer; 2) daily self-examination of the sacral, ischium, buttocks, hips, and heels of all bed-bound patients and the feet of patients with diabetes with risk factors (e.g., neuropathy); 3) initiation of a treatment protocol immediately upon recognition of a break in the skin (i.e., emergence of a new wound); 4) objective measurement by planimetry of every wound (at a minimum, weekly) and documentation of its progress; 5) establishment of a moist wound-healing environment; 6) relief of pressure from the wound; 7) debridement of all non-viable tissue in the wound; 8) elimination of all drainage and cellulitis; 9) cellular therapy or growth factors for patients with wounds that do not heal rapidly after initial treatment; and 10) continuous physical and psychosocial support for all patients. If this paradigm is followed, most diabetic foot and pressure ulcers are expected to heal.

Objective: To evaluate the regulation and expression of CSF-1 and its receptor c-fms in endometrial cells. Design: In vitro study. Setting: Research and teaching institution. Patient(s): None. Intervention(s): In vitro experimental study. Main Outcome Measure(s): The effect of glucocorticoid and oxidative stress on the expression of CSF-1 and c-fms in endometrial cells. Result(s): Cultured nonmalignant EM42 cells not only express CSF-1 and c-fms but are also capable of responding to exogenous CSF-1. We have also seen that glucocorticoids can regulate the expression of CSF-1/ c-fms in endometrial cells. Furthermore, this study shows that oxidative stress plays a significant role in the induction of CSF-1 and its receptor c-fms . Conclusion(s): The results suggest that CSF-1 may promote the growth of nonmalignant endometrial cells in both an autocrine and paracrine manner and that endometrial cells under oxidative stress induce CSF-1 and c-fms .

To directly test the role of aromatase overexpression in tumorigenesis, we have developed a transgenic mouse model and demonstrated for the first time that increased tissue estrogens due to the overexpression of aromatase in mammary glands leads to the induction of various preneoplastic and neoplastic changes that are similar to early breast cancer. Increased estrogenic activity in breast tissue alters the regulation of genes involved in apoptosis, cell cycle and cell proliferation, as well as the levels of various growth factors. Tissue aromatase overexpression is sufficient to induce and maintain early preneoplastic and neoplastic changes in female mice without circulating ovarian estrogen. Overexpression of aromatase in male mammary glands leads to different histopathological changes that are similar to gynecomastia. Increased estrogenic activity in male mice changes the hormonal milieu of testis and results in the induction of Leydig cell tumors. These studies indicate a direct role of aromatase in preneoplastic and neoplastic changes seen in both mammary glands and testis.

To test directly the role of breast-tissue estrogen in initiation of breast cancer, we have developed the aromatase-transgenic mouse model and demonstrated for the first time that increased mammary estrogens resulting from the overexpression of aromatase in mammary glands lead to the induction of various preneoplastic and neoplastic changes that are similar to early breast cancer. Continued overexpression of aromatase that leads to increased breast-tissue estrogen contributes to a number of epigenetic changes in mammary tissue such as alteration in the regulation of genes involved in apoptosis, activation of genes involved in cell cycle and cell proliferation, and activation of a number of growth factors. Our current studies show aromatase overexpression is sufficient to induce and maintain early preneoplastic and neoplastic changes in female mice without circulating ovarian estrogen. Preneoplastic and neoplastic changes induced in mammary glands as a result of aromatase overexpression can be completely abrogated with the administration of the aromatase inhibitor, letrozole. Consistent with complete reduction in hyperplasia, we have also seen downregulation of estrogen receptor and a decrease in cell proliferation markers, suggesting aromatase-induced hyperplasia can be treated with aromatase inhibitors. Our studies demonstrate that aromatase overexpression alone, without circulating estrogen, is responsible for the induction of breast hyperplasia and these changes can be abrogated using aromatase inhibitors.

Objective: To develop and characterize an antiglycodelin antibody using a 15-amino acid synthetic peptide as antigen, derived from the sequence of human glycodelin. Design: We have developed a chicken antiglycodelin-derived peptide antibody and have characterized the antibody with the use of endometrial and ovarian cell lines. The antibody was also tested for its ability to detect glycodelin by ELISA assay, immunocytochemistry, and by Western blot. Setting: Various cell lines, cell culture medium, and amniotic fluid were used in the experiments. Patient(s): Amniotic fluid was collected from pregnant patients in their first trimester of pregnancy. Intervention(s): No intervention. Main Outcome Measure(s): Detection of glycodelin. Result(s): The cell lines RL95-2 (human endometrial carcinoma cells), OVCAR-3 (human ovarian adenocarcinoma cells), HeLa (human cervical epitheloid carcinoma cells), and EM42-D (human endometrial epithelial cells) reacted with the antibody, indicating the presence of glycodelin. A specific 45-kd protein representing glycodelin was detected by Western blot in the amniotic fluid. Conclusion(s): Antipeptide antibodies can be successfully used to detect and quantify the presence of glycodelin in cells and fluids.