Loratadine Combats Methicillin-Resistant Staphylococcus aureus by Modulating Virulence, Antibiotic Resistance, and Biofilm Genes.

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved to become resistant to multiple classes of antibiotics. New antibiotics are costly to develop and deploy, and they have a limited effective lifespan. Antibiotic adjuvants are molecules that potentiate existing antibiotics through nontoxic mechanisms. We previously reported that loratadine, the active ingredient in Claritin, potentiates multiple cell-wall active antibiotics in vitro and disrupts biofilm formation through a hypothesized inhibition of the master regulatory kinase Stk1. Loratadine and oxacillin combined repressed the expression of key antibiotic resistance genes in the bla and mec operons. We hypothesized that additional genes involved in antibiotic resistance, biofilm formation, and other cellular pathways would be modulated when looking transcriptome-wide. To test this, we used RNA-seq to quantify transcript levels and found significant effects in gene expression, including genes controlling virulence, antibiotic resistance, metabolism, transcription (core RNA polymerase subunits and sigma factors), and translation (a plethora of genes encoding ribosomal proteins and elongation factor Tu). We further demonstrated the impacts of these transcriptional effects by investigating loratadine treatment on intracellular ATP levels, persister formation, and biofilm formation and morphology. Loratadine minimized biofilm formation in vitro and enhanced the survival of infected Caenorhabditis elegans. These pleiotropic effects and their demonstrated outcomes on MRSA virulence and survival phenotypes position loratadine as an attractive anti-infective against MRSA.

Preparedness planning for pandemic influenza among large US maternity hospitals.

The objective of this investigation was to determine the state of pandemic influenza preparedness and to delineate commonly reported challenges among a sample of larger US national maternity hospitals. This was done given the recent emphasis on hospital disaster planning and the disproportionate morbidity and mortality that pregnant women have suffered in previous influenza pandemics. An internet-based survey was sent to all 12 members of the Council of Women's and Infants' Specialty Hospitals. Questions addressed hospital demographics and overall pandemic preparedness planning, including presence of a pandemic planning committee and the existence of written plans addressing communications, surge capacity, degradation of services, and advance supply planning. Nine of 12 (75%) hospitals responded. All had active pandemic planning committees with identified leadership. The majority (78%) had written formal plans regarding back-up communications, surge/overflow capacity, and degradation of services. However, fewer (44%) reported having written plans in place regarding supply-line/stockpiling of resources. The most common challenges noted were staff and supply coordination, ethical distribution of limited medical resources, and coordination with government agencies. In conclusion, the majority of the Council of Women's and Infants' Specialty Hospitals maternity hospitals have preliminary infrastructure for pandemic influenza planning, but many challenges exist to optimize maternal and fetal outcomes during the next influenza pandemic.

Fc gamma R expression on NK cells influences disease severity in rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with autoantibodies, the best known of which is rheumatoid factor (RF). RF/IgG complexes interact with FcgammaR on the surface of neutrophils, NK cells and monocyte/macrophages. We have analyzed the expression pattern and allelic polymorphisms of three FcgammaR genes (FcgammaRIIA, FcgammaRIIC and FcgammaRIIIA) in a large sample of RA patients and normal donors. We have found that the level of FcgammaR (CD16 and CD32) expression on NK cells is lower in RA patients than in normal individuals. Genotypic analysis demonstrated that the CD32 isoform expressed by the majority of RA patients was not the activating FcgammaRIIc1 isoform, commonly seen in normal individuals, but rather the inhibitory FcgammaRIIb isoform. The combination of the FcgammaRIIIA-176F allele with a lack of CD32 expression in NK cells appeared to be characteristic of RA subjects with aggressive disease. Since FcgammaRII and FcgammaRIIIA are predominantly expressed by NK cells, these data further suggest that FcgammaR-mediated activation of NK cells could be a disease-determining factor in RA patients.

Identification of the CD32/FcgammaRIIc-Q13/STP13 polymorphism using an allele-specific restriction enzyme digestion assay.

We have recently reported that in addition to FcgammaRIIIa (CD16), approximately 45% of normal individuals also express FcgammaRIIc (CD32) on their natural killer (NK) cells. We found this expression to be regulated by an allelic polymorphism localized in the first extracellular exon (EC1) of the FcgammaRIIC gene, corresponding to aa 13. This is determined by a single nucleotide substitution, which results in either a functional open reading frame (glutamine-Q) or a premature stop codon (STP). Identification of this polymorphism provided a good explanation for the lack of CD32 expression previously observed with NK cells in some normal individuals. Here, we describe a new method for detection of FcgammaRIIc allelism based on RT-PCR amplification followed by an allele-specific restriction enzyme digestion. This method is rapid, reliable and time saving, as compared to the currently available allele-specific oligo-nucleotide probe-based Southern Blotting.

Stimulation of uterine cell cytokine production by ovarian hormones.

PROBLEM: Although leukocytes do not possess significant numbers of ovarian steroid hormone receptors, their numbers in the endometrium vary consistently, relative to the menstrual cycle. The possibility that cell types within the endometrium express leukocyte-attracting genes in response to ovarian hormones was investigated. METHOD OF STUDY: Endometrial biopsies were collected 10 days post-leutinizing hormone surge; the cell types were separated and cultured individually for 5 days in the presence of increasing amounts of estrogen or progesterone. Following culture, RNA was collected from cells and reverse-transcription-polymerase chain reaction was used to determine relative levels of gene expression of monocyte chemotactic proteins (MCP)-1, -2, and -3, and interleukin (IL)-12 p35 and p40. RESULTS: Although both endometrial stroma and glands were able to make MCP mRNA, steady-state levels of gene expression did not vary significantly relative to hormone treatment. The same was found for the p35 molecule of the IL-12 gene; however, differences were observed for the p40 subunit. CONCLUSIONS: Within the human endometrium, chemokines other than MCP and IL-12 are most likely responsible for cycle-related leukocyte recruitment.

Evidence for superantigen involvement in preeclampsia.

PROBLEM: Preeclampsia is the leading cause of maternal morbidity and premature fetal delivery in the United States, most likely involving the immune system in disease genesis. In this report, we tested the hypothesis that a superantigen phenomenon is an important factor in the pathogenesis of the disease. METHOD OF STUDY: A semi-quantitative polymerase chain reaction (PCR) was used to assess T-cell receptor (TCR) beta chain variable (Vbeta) regions as an indicator of T-cell expansion in both peripheral blood and basal plate of preeclamptic patients. All the subjects were also molecularly typed to identify their HLA-class II alleles. RESULTS: In peripheral blood of the majority of the patients, there was a high abundance of the Vbeta4 gene family, which was not observed in the control group. Polyclonality of this Vbeta gene family was confirmed by analysis of the Valpha chain and the complementary determining region 3 (CDR3). The majority of patients carried the Human Leukocyte Antigens (HLA)-DRB1*13 allele. CONCLUSION: We present evidence for the existence of a superantigen-like effect in at least a subset of patients with preeclampsia.

Ovarian responses in women to recombinant follicle-stimulating hormone and luteinizing hormone (LH): a role for LH in the final stages of follicular maturation.

During the follicular phase of the menstrual cycle, FSH stimulates follicular growth, granulosa cell aromatase activity, induction of LH receptors on the granulosa cell membrane, and estradiol secretion. As a result of negative feedback of estradiol on the pituitary, serum FSH concentrations decline. Despite the fall in FSH concentrations, the maturing follicle continues to develop to the preovulatory stage. In a prospective randomized trial, we tested the hypothesis that a key mechanism by which the dominant follicle continues to develop in the face of decreasing concentration of FSH is by acquiring LH responsiveness. In 24 women, pituitary gonadotropin secretion was down-regulated with a GnRH agonist. Follicular growth was then stimulated with recombinant human FSH (r-hFSH) until a 14-mm follicle was identified by ultrasound. The women were then randomized to 1 of 4 groups for a 2-day period: continued r-hFSH treatment, substitution of r-hFSH with saline, low dose r-hLH (150 IU, twice daily), or high dose r-hLH (375 IU, twice daily). Serum estradiol concentrations in the women receiving saline declined by the end of the 2-day randomization period. In contrast, serum estradiol concentrations continued to rise in women receiving either r-hFSH or r-hLH compared with those in the saline-treated group (P < 0.05). Pregnancies occurred in each of the gonadotropin treatment groups. These findings indicate that once FSH initiates follicular growth, either FSH or LH is capable of sustaining follicular estradiol production. Extrapolating these findings to the normal menstrual cycle suggests that the maturing follicle may continue to develop in the presence of diminishing FSH concentrations by acquiring the capacity to respond to LH.

Methotrexate effects on trophoblast and the corpus luteum in early pregnancy.

OBJECTIVE: Our purpose was to determine whether methotrexate affects the trophoblast or corpus luteum when administered for abortion. STUDY DESIGN: A randomized controlled trial was performed in women requesting an abortion up to 49 days' gestation. Twenty patients were treated with intramuscular methotrexate 50 mg/m2 (10 women) or 60 mg/m2 (10 women). Serum beta-human chorionic gonadotropin, progesterone, and 17-hydroxyprogesterone levels were determined at baseline and then serially after methotrexate administration for the first 24 hours, then every 24 hours for 7 days. On the seventh day misoprostol 800 microg was administered vaginally. RESULTS: Serum beta-human chorionic gonadotropin increased at a lower rate than occurs in normal pregnancy. Progesterone levels averaged 56.9 +/- 19.8 nmol/L at baseline and 45.5 +/- 20.5 nmol/L (P = .01) 1 week after methotrexate. Progesterone decreased in 16 women over the 7 days and increased in the other 4; these latter women all aborted after a single dose of misoprostol. Levels of 17-hydroxyprogesterone plateaued during the first day after methotrexate administration; both progesterone and 17-hydroxyprogesterone declined simultaneously between the third and fourth day after methotrexate. CONCLUSIONS: Methotrexate most likely primarily affects trophoblast production of human chorionic gonadotropin, as evidenced by a blunting of the expected increase in serum beta-human chorionic gonadotropin resulting in less support for the production of progesterone by the corpus luteum. However, changes in progesterone levels after methotrexate administration were inconsistent and are unlikely to represent the ultimate effect of methotrexate in abortion. The less-than-normal increase in serum beta-human chorionic gonadotropin levels after methotrexate administration is most likely a result of disruption of cytotrophoblast syncytialization. This disruption may be the true effect of methotrexate in destabilizing the implantation site of an early pregnancy.

Effects of methotrexate on trophoblast proliferation and local immune responses.

Methotrexate is a folic acid analogue that has been used successfully for the treatment of ectopic pregnancy and, in conjunction with misoprostol, for medical abortions of early intrauterine pregnancies. To administer the most efficacious treatment requires knowledge of the mechanism underlying the induction of methotrexate-induced abortion. This study was designed to ascertain trophoblast integrity, proliferation and differentiation following administration of methotrexate. In addition, to determine if methotrexate affects the local uterine immune response, we ascertained the numbers and identities of decidual leukocytes following treatment. Ten women with undesired intrauterine pregnancies of 42-49 days gestation were recruited to receive methotrexte 50 mg/m2 i.m. A suction aspiration was performed 7 days later. Tissues from gestational age-matched elective surgical abortions were used as controls. Additionally, specimens from women who received methotrexate and misoprostol for abortion in a clinical trial of oral methotrexate in combination with misoprostol, who had a suction abortion because of continued embryonic cardiac activity 14 days after the methotrexate, were evaluated. Immunoreactivity to proliferating cell nuclear antigen and cyclin D3 antibodies was used to demonstrate a marked reduction in the proliferation index of cytotrophoblasts from methotrexate-treated abortions. Methotrexate treatment failures and non-treated pregnancies had a much higher proliferation index. There was no direct destruction of the syncytiotrophoblast, as indicated by the continued presence of human placental lactogen and beta-human chorionic gonadotrophin proteins. A decrease in the total number of leukocyte cells was observed in the decidua of methotrexate-treated samples, with the large granular lymphocyte (LGL) cells showing the greatest decline in numbers. Our conclusions from this study are that methotrexate acts primarily to derail the normal developmental programme of the trophoblast stem cell population, as well as to decrease LGL cell numbers in the decidua.

Endometrial leukocytes are altered numerically and functionally in women with implantation defects.

PROBLEM: A significant cohort of women with autoimmune thyroid disease (ATD) also suffer from reduced fertility. The finding that neither exogenous hormones nor donor eggs correct the infertility suggests that the problem involves an inherent endometrial defect. METHOD OF STUDY: Endometrial leukocyte populations in women with ATD were quantitated by immunohistochemistry. A semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique was used to examine the expression of transforming growth factor (TGF)-beta 1, interleukin (IL)-4, IL-10, and interferon (IFN)-gamma in the endometrial samples. RESULTS: A significant increase in the endometrial T-cell population was observed in women with ATD compared to controls. The relative abundance of IL-4 and IL-10 was decreased in women with ATD compared to controls, whereas IFN-gamma was increased. No difference was noted in the abundance of TGF-beta 1. The source of cytokine production for IL-4, IL-10, and IFN-gamma was the endometrial leukocytes. CONCLUSIONS: Both the leukocyte numbers and cytokine expression profile were altered significantly in a well-defined group of women with implantation defects.

Decidual leukocyte populations in ectopic pregnancies.

OBJECTIVE: To determine whether the recruitment of decidual leukocytes during pregnancy is the same in intrauterine pregnancies (IUPs) versus ectopic pregnancies (EPs). DESIGN: Intrauterine decidual samples from both EPs and IUPs were obtained for the evaluation of leukocyte populations. SETTING: In vitro experiment. PATIENT(S): Women with EPs and women with IUPs. MAIN OUTCOME MEASURE(S): Immunohistochemical identification of decidual leukocyte populations. RESULT(S): We have analyzed the decidual leukocyte populations from three women with EPs by immunohistochemical analysis. The data demonstrate a leukocyte infiltration similar to that found in decidua from normal pregnancies. CONCLUSIONS(S): These data support the hypothesis that decidual leukocyte recruitment and/or increases during pregnancy is primarily hormonally regulated.

Disposition of foscarnet during peritoneal dialysis.

OBJECTIVE: To report the disposition of foscarnet in a patient undergoing peritoneal dialysis. CASE SUMMARY: A 34-year-old man with AIDS received foscarnet for the treatment of esophageal cytomegalovirus. We characterized the clearance of foscarnet in this patient during continuous cyclic peritoneal dialysis (CCPD) and continuous ambulatory peritoneal dialysis (CAPD). DISCUSSION: The foscarnet half-lives during CCPD and CAPD were 41.4 and 45.8 hours, respectively. These values are significantly greater than the half-life of 4.5 hours observed in patients with normal renal function and about half that reported in anuric patients undergoing hemodialysis during the interdialytic period. The CCPD and CAPD clearances of foscarnet were 5.8 and 4.5 mL/min, respectively; the CAPD clearances of creatinine and urea nitrogen were 4.1 and 6.0 mL/min, respectively. The patient's estimated total body clearance values of foscarnet during CCPD and CAPD were 9.8 and 8.8 mL/min, respectively. Thus, CCPD and CAPD augmented the patient's residual clearance of foscarnet by 145% and 105%, respectively. CONCLUSIONS: Since incremental increases in residual clearance of 30% or more generally will result in clinically significant changes in a drug's serum concentration, foscarnet dosage needs to be individualized for patients receiving peritoneal dialysis.

Granulocyte-macrophage colony-stimulating factor augmentation of T-cell receptor-dependent and T-cell receptor-independent thymocyte proliferation.

The effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) are not confined to cells of the myeloid lineage. GM-CSF has been shown to have effects on mature T cells and both mature and immature T-cell lines. We therefore examined the GM-CSF responsiveness of murine thymocytes to investigate whether GM-CSF also affected normal immature T lymphocytes. The studies presented here indicate that GM-CSF augments accessory cell (AC)-dependent T-cell receptor (TCR)-mediated proliferation of unseparated thymocyte populations. To identify the GM-CSF responsive cell type, thymic AC and T cells were examined for GM-CSF responsiveness. We found that GM-CSF augmentation of TCR-induced thymocyte proliferation appears to be mediated via augmentation of AC function, and not via direct effects on mature single-positive (SP) thymocytes. Enriched double-negative (DN) thymocytes were also tested for GM-CSF responsiveness. GM-CSF induced the proliferation of adult and fetal DN thymocytes in an AC-independent and TCR-independent single-cell assay. Thus, in contrast to the SP thymocytes, a DN thymocyte population was directly responsive to GM-CSF. GM-CSF therefore may play a direct role in the expansion of DN thymocytes and an indirect role in the expansion of SP thymocytes.

Effect of granulocyte-macrophage colony-stimulating factor on lymphokine-activated killer cell induction.

The treatment of cancer with lymphokine-activated killer (LAK) cells in conjunction with high-dose interleukin-2 (IL-2) has been limited by the toxicity of IL-2 and the narrow range of tumors that respond to therapy. Cytokines that are capable of augmenting lower doses of IL-2 are, therefore, a major focus of research. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF) can augment low-dose IL-2 LAK induction from murine splenocytes. Anti-tumor necrosis factor alpha (anti-TNF alpha) or anti-interferon gamma (anti-IFN gamma) monoclonal antibodies did not inhibit (IL-2 + GM-CSF)-induced LAK generation, indicating that GM-CSF augmentation does not require TNF alpha or IFN gamma activity. Depletion of natural killer cells before culture did not inhibit low-dose IL-2-induced LAK generation or the ability of GM-CSF to augment LAK generation. In contrast, depletion of both CD4+ and CD8+ T cells before culture inhibited the generation of LAK activity. However, depletion of only CD4+ T cells, or only CD8+ T cells, did not inhibit the generation of IL-2 or (IL-2 + GM-CSF) LAK activity. These results suggest that LAK precursors are present in both the CD4+ and CD8+ T-cell populations and suggest that the addition of GM-CSF to low-dose IL-2 may result in the generation of T-derived LAK cells.

The effect of patient movement on resolution.

The effect of long exposure time on patient movement and resulting radiographic resolution was simulated and then measured by visual-light photographic techniques for four radiographic projections. A mathematic relation was then derived to transform these measurements to the maximum resolutions that could have been obtained with radiographs under similar conditions. The results indicate that radiographs of rigid structures such as bones and teeth can be taken with reasonable resolution at exposure times much longer than normally used in clinical practice. It was also found that radiographs taken with the film rigidly fixed to the object being studied will exhibit remarkably less blurring from patient motion than radiographs taken when the patient and the film are not coupled. In addition it was found that motion artifacts are reduced to a minimum when the plane of the film is perpendicular to the radiation beam.