Screening for Comorbidity of Sleep Disorders in Career Firefighters.

BACKGROUND: The purpose of this study was to assess the prevalence and sequelae of insomnia, obstructive sleep apnea (OSA), and comorbid OSA and insomnia (COMISA). METHOD: In the morning, after a shift end, Midwest career firefighters ( N = 89) in a midsized city completed an electronic battery of questionnaire to screen for OSA, daytime sleepiness, insomnia, presleep arousal, nightmares, mental and physical health symptoms, and a one-night sleep diary. RESULTS: Prevalence of firefighters exceeding screening thresholds: OSA: 54%; insomnia: 30%; COMISA: 17%; four or more nightmares per month: 15%. Firefighters who met criteria for COMISA had shorter total sleep time, less restful and worse sleep quality, higher depression and anxiety symptoms, and presleep arousal symptoms than firefighters without self-reported sleep problems. CONCLUSIONS: Many firefighters are at elevated risk of individual behavioral sleep disorders, COMISA, and daytime dysfunction.

Phenobarbital and symptom-triggered lorazepam versus lorazepam alone for severe alcohol withdrawal in the intensive care unit.

A symptom-triggered lorazepam regimen is the standard for treating alcohol withdrawal syndrome (AWS) in an inpatient setting. However, in severe AWS, lorazepam requirements can reach significant amounts and lead to risk of delirium and propylene glycol toxicity. Phenobarbital has been shown to be an effective adjunctive therapy for AWS, reducing benzodiazepine use, in the emergency department. The purpose of this study is to determine the efficacy and safety of phenobarbital in adjunct to symptom-triggered lorazepam for severe AWS vs. lorazepam alone in the intensive care unit (ICU). A retrospective cohort was conducted at Cleveland Clinic hospitals from 2013 to 2018 of ICU patients with AWS receiving either phenobarbital adjunct to symptom-triggered lorazepam or lorazepam alone. The primary outcome was the total duration of treatment. Secondary outcomes include ICU length of stay, change in CIWA-Ar score at 24 h, incidence of hypotension, mechanical ventilation, and serum osmolar gap. A total of 72 ICU patients were included with 36 patients in each arm. The median duration of treatment in the phenobarbital adjunct arm was 2.7 days (IQR = 1.7-6.4), compared to 3.1 days (IQR = 1.6-4.8) in the lorazepam arm (p = 0.578). The median ICU length of stay was similar between both arms [4.1 days (IQR = 2.4-8.4) vs. 4.5 days (IQR = 2.8-6.1), p = 0.727]. The average change in CIWA-Ar from baseline at 24 h was significantly lower for those who received phenobarbital (1.8 ± 9.0 vs. 6.5 ± 8.5, p = 0.028). Three patients in the phenobarbital-adjunct group received mechanical ventilation after starting phenobarbital treatment. There were no new incidences of hypotension or increased osmol gap >10 mmol/L after starting treatment in both groups. In conclusion, phenobarbital is an effective adjunct to symptom-triggered lorazepam in severe alcohol withdrawal in the ICU with no significant difference in adverse events.

Epigenetic silencing of a multifunctional plant stress regulator.

The central regulator of the ethylene (ET) signaling pathway, which controls a plethora of developmental programs and responses to environmental cues in plants, is ETHYLENE-INSENSITIVE2 (EIN2). Here we identify a chromatin-dependent regulatory mechanism at EIN2 requiring two genes: ETHYLENE-INSENSITIVE6 (EIN6), which is a H3K27me3 demethylase also known as RELATIVE OF EARLY FLOWERING6 (REF6), and EIN6 ENHANCER (EEN), the Arabidopsis homolog of the yeast INO80 chromatin remodeling complex subunit IES6 (INO EIGHTY SUBUNIT). Strikingly, EIN6 (REF6) and the INO80 complex redundantly control the level and the localization of the repressive histone modification H3K27me3 and the histone variant H2A.Z at the 5' untranslated region (5'UTR) intron of EIN2. Concomitant loss of EIN6 (REF6) and the INO80 complex shifts the chromatin landscape at EIN2 to a repressive state causing a dramatic reduction of EIN2 expression. These results uncover a unique type of chromatin regulation which safeguards the expression of an essential multifunctional plant stress regulator.

Induction of Apoptosis by PQ1, a Gap Junction Enhancer that Upregulates Connexin 43 and Activates the MAPK Signaling Pathway in Mammary Carcinoma Cells.

The mechanism of gap junction enhancer (PQ1) induced cytotoxicity is thought to be attributed to the change in connexin 43 (Cx43) expression; therefore, the effects of Cx43 modulation in cell survival were investigated in mammary carcinoma cells (FMC2u) derived from a malignant neoplasm of a female FVB/N-Tg(MMTV-PyVT)634Mul/J (PyVT) transgenic mouse. PQ1 was determined to have an IC50 of 6.5 µM in FMC2u cells, while inducing an upregulation in Cx43 expression. The effects of Cx43 modulation in FMC2u cell survival was determined through transfection experiments with Cx43 cDNA, which induced an elevated level of protein expression similar to that seen with PQ1 exposure, or siRNA to silence Cx43 protein expression. Overexpression or silencing of Cx43 led to a reduction or an increase in cell viability, respectively. The mitogen-activated protein kinase (MAPK) family has been implicated in the regulation of cell survival and cell death; therefore, the gap junctional intercellular communication (GJIC)-independent function of PQ1 and Cx43 in the Raf/Mitogen-activated protein kinase/ERK kinase/extracellular-signal-regulated kinase (Raf-MEK-ERK) cascade of cellular survival and p38 MAPK-dependent pathway of apoptosis were explored. PQ1 treatment activated p44/42 MAPK, while the overexpression of Cx43 resulted in a reduced expression. This suggests that PQ1 affects the Raf-MEK-ERK cascade independent of Cx43 upregulation. Both overexpression of Cx43 and PQ1 treatment stimulated an increase in the phosphorylated form of p38-MAPK, reduced levels of the anti-apoptotic protein Bcl-2, and increased the cleavage of pro-caspase-3. Silencing of Cx43 protein expression led to a reduction in the phosphorylation of p38-MAPK and an increase in Bcl-2 expression. The mechanism behind PQ1-induced cytotoxicity in FMC2u mammary carcinoma cells is thought to be attributed to the change in Cx43 expression. Furthermore, PQ1-induced apoptosis through the upregulation of Cx43 may depend on p38 MAPK, highlighting that the effect of PQ1 on gap junctions as well as cellular survival via a MAPK-dependent pathway.

Glyceollin I Reverses Epithelial to Mesenchymal Transition in Letrozole Resistant Breast Cancer through ZEB1.

Although aromatase inhibitors are standard endocrine therapy for postmenopausal women with early-stage metastatic estrogen-dependent breast cancer, they are limited by the development of drug resistance. A better understanding of this process is critical towards designing novel strategies for disease management. Previously, we demonstrated a global proteomic signature of letrozole-resistance associated with hormone-independence, enhanced cell motility and implications of epithelial mesenchymal transition (EMT). Letrozole-resistant breast cancer cells (LTLT-Ca) were treated with a novel phytoalexin, glyceollin I, and exhibited morphological characteristics synonymous with an epithelial phenotype and decreased proliferation. Letrozole-resistance increased Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression (4.51-fold), while glyceollin I treatment caused a -3.39-fold reduction. Immunofluorescence analyses resulted of glyceollin I-induced increase and decrease in E-cadherin and ZEB1, respectively. In vivo studies performed in ovariectomized, female nude mice indicated that glyceollin treated tumors stained weakly for ZEB1 and N-cadherin and strongly for E-cadherin. Compared to letrozole-sensitive cells, LTLT-Ca cells displayed enhanced motility, however in the presence of glyceollin I, exhibited a 68% and 83% decrease in invasion and migration, respectively. These effects of glyceollin I were mediated in part by inhibition of ZEB1, thus indicating therapeutic potential of glyceollin I in targeting EMT in letrozole resistant breast cancer.

Quantitative electrochemical detection of cathepsin B activity in breast cancer cell lysates using carbon nanofiber nanoelectrode arrays toward identification of cancer formation.

The proteolytic activity of cathepsin B in complex breast cell lysates has been measured with alternating current voltammetry (ACV) using ferrocene (Fc)-labeled-tetrapeptides immobilized on nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). Four types of breast cells have been tested, including normal breast cells (HMEC), transformed breast cells (MCF-10A), breast cancer cells (T47D), and metastatic breast cancer cells (MDA-MB-231). The detected protease activity was found increased in cancer cells, with the MDA-MB-231 metastatic cancer cell lysate showing the highest cathepsin B activity. The equivalent cathepsin B concentration in MDA-MB-231 cancer cell lysate was quantitatively determined by spiking recombinant cathepsin B into the immunoprecipitated MDA-MB-231 lysate and the HMEC whole cell lysate. The results illustrated the potential of this technique as a portable multiplex electronic device for cancer diagnosis and treatment monitoring through rapid profiling the activity of specific cancer-relevant proteases. FROM THE CLINICAL EDITOR: Breast cancer is the most common cancer in women. In this report, the authors applied the technique of nanoelectrode arrays to try to detect and compare cathepsin B activities in normal and breast cancer cells. It was found that protease activity correlated positively with the degree of malignancy cancer cells. Taking this further, this technique may be useful for rapid diagnosis of cancer in the future.

Increase of gap junction activities in SW480 human colorectal cancer cells.

BACKGROUND: Colorectal cancer is one of the most common cancers in the United States with an early detection rate of only 39%. Colorectal cancer cells along with other cancer cells exhibit many deficiencies in cell-to-cell communication, particularly gap junctional intercellular communication (GJIC). GJIC has been reported to diminish as cancer cells progress. Gap junctions are intercellular channels composed of connexin proteins, which mediate the direct passage of small molecules from one cell to the next. They are involved in the regulation of the cell cycle, cell differentiation, and cell signaling. Since the regulation of gap junctions is lost in colorectal cancer cells, the goal of this study is to determine the effect of GJIC restoration in colorectal cancer cells. METHODS: Gap Junction Activity Assay and protein analysis were performed to evaluate the effects of overexpression of connexin 43 (Cx43) and treatment of PQ1, a small molecule, on GJIC. RESULTS: Overexpression of Cx43 in SW480 colorectal cancer cells causes a 6-fold increase of gap junction activity compared to control. This suggests that overexpressing Cx43 can restore GJIC. Furthermore, small molecule like PQ1 directly targeting gap junction channel was used to increase GJIC. Gap junction enhancers, PQ1, at 200 nM showed a 4-fold increase of gap junction activity in SW480 cells. A shift from the P0 to the P2 isoform of Cx43 was seen after 1 hour treatment with 200 nM PQ1. CONCLUSION: Overexpression of Cx43 and treatment of PQ1 can directly increase gap junction activity. The findings provide an important implication in which restoration of gap junction activity can be targeted for drug development.

Quantitative electrochemical detection of cathepsin B activity in complex tissue lysates using enhanced AC voltammetry at carbon nanofiber nanoelectrode arrays.

The proteolytic activity of a cancer-related enzyme cathepsin B is measured with alternating current voltammetry (ACV) using ferrocene (Fc) labeled tetrapeptides attached to nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). This combination enables the use of high AC frequencies (~1kHz) with enhanced electrochemical signals. The specific proteolysis of the Fc-peptide by cathepsin B produces decay in the ACV peak current versus the reaction time. The exponential component of the raw data can be extracted and defined as the "extracted proteolytic signal" which allows consistent quantitative analyses using a heterogeneous Michaelis-Menten model. A "specificity constant" kcat/KM = (3.68 ± 0.50) × 10(4)M(-1)s(-1) for purified cathepsin B was obtained. The detections of cathepsin B activity in different concentrations of whole lysate of human breast tissue, tissue lysate spiked with varied concentrations of cathepsin B, and the tissue lysate after immunoprecipitation showed that there is ~13.4 nM higher cathepsin B concentration in 29.1 µg mL(-1) of whole tissue lysate than the immunoprecipitated sample. The well-defined regular VACNF NEAs by e-beam lithography show a much faster kinetics for cathepsin B proteolysis with kcat/KM = 9.2 × 10(4)M(-1)s(-1). These results illustrate the potential of this technique as a portable multiplex electronic system for cancer diagnosis by rapid protease profiling of serum or blood samples.

The effect of antineoplastic drugs in a male spontaneous mammary tumor model.

Male breast cancer is a rare disease. The limited number of clinical cases has led to the primary treatments for men being derived from female breast cancer studies. Here the transgenic strain FVB/N-Tg(MMTV-PyVT)634Mul/J (also known as PyVT) was used as a model system for measuring tumor burden and drug sensitivity of the antineoplastic drugs tamoxifen, cisplatin, and paclitaxel on tumorigenesis at an early stage of mammary carcinoma development in a male mouse model. Cisplatin treatment significantly reduced tumor volume, while paclitaxel and tamoxifen did not attenuate tumor growth. Cisplatin treatment was shown to induce apoptosis, grossly observed by reduced tumor formation, through reduced Bcl-2 and survivin protein expression levels with an increase in caspase 3 expression compared to control tumors. Tamoxifen treatment significantly altered the hormone receptor expression levels of the tumor, while additionally upregulating Bcl-2 and Cyclin D1. This suggests an importance in hormonal signaling in male breast cancer pathogenesis. The results of this study provide valuable information toward the better understanding of male breast cancer and may help guide treatment decisions.

Electrochemical Protease Biosensor Based on Enhanced AC Voltammetry Using Carbon Nanofiber Nanoelectrode Arrays.

We report an electrochemical method for measuring the activity of proteases using nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). The VACNFs of ~150 nm in diameter and 3 to 5 µm in length were grown on conductive substrates and encapsulated in SiO2 matrix. After polishing and plasma etching, controlled VACNF tips are exposed to form an embedded VACNF NEA. Two types of tetrapeptides specific to cancer-mediated proteases legumain and cathepsin B are covalently attached to the exposed VACNF tip, with a ferrocene (Fc) moiety linked at the distal end. The redox signal of Fc can be measured with AC voltammetry (ACV) at ~1 kHz frequency on VACNF NEAs, showing distinct properties from macroscopic glassy carbon electrodes due to VACNF's unique interior structure. The enhanced ACV properties enable the kinetic measurements of proteolytic cleavage of the surface-attached tetrapeptides by proteases, further validated with a fluorescence assay. The data can be analyzed with a heterogeneous Michaelis-Menten model, giving "specificity constant" kcat /Km as (4.3 ± 0.8) × 104 M-1s-1 for cathepsin B and (1.13 ± 0.38) × 104 M-1s-1 for legumain. This method could be developed as portable multiplex electronic techniques for rapid cancer diagnosis and treatment monitoring.

Peptide hydrogelation and cell encapsulation for 3D culture of MCF-7 breast cancer cells.

Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing.

Gap junction enhancer increases efficacy of cisplatin to attenuate mammary tumor growth.

Cisplatin treatment has an overall 19% response rate in animal models with malignant tumors. Increasing gap junction activity in tumor cells provides the targets to enhance antineoplastic therapies. Previously, a new class of substituted quinolines (PQs) acts as gap junction enhancer, ability to increase the gap junctional intercellular communication, in breast cancer cells. We examined the effect of combinational treatment of PQs and antineoplastic drugs in an animal model, showing an increase in efficacy of antineoplastic drugs via the enhancement of gap junctions. Mice were implanted with estradiol-17ß (1.7 mg/pellet) before the injection of 1×107 T47D breast cancer cells subcutaneously into the inguinal region of mammary fat pad. Animals were treated intraperitoneally with DMSO (control), cisplatin (3.5 mg/kg), PQ (25 mg/kg), or a combining treatment of cisplatin and PQ. Cisplatin alone decreased mammary tumor growth by 85% while combinational treatment of cisplatin and PQ1 or PQ7 showed an additional reduction of 77% and 22% of tumor growth after 7 treatments at every 2 days, respectively. Histological results showed a significant increase of gap junction proteins, Cx43 and Cx26, in PQ-treated tissues compared to control or cisplatin. Furthermore, evidence of highly stained caspase 3 in tumors of combinational treatment (PQ and cisplatin) was seen compared to cisplatin alone. We have showed for the first time an increase in the efficacy of antineoplastic drugs through a combinational treatment with PQs, a specific class of gap junction enhancers.

C1B domain peptide of protein kinase Cγ significantly suppresses growth of human colon cancer cells in vitro and in an in vivo mouse xenograft model through induction of cell cycle arrest and apoptosis.

Two peptides derived from the C1B domain of protein kinase Cγ (PKCγ) were shown to associate with classical PKC isozymes and modulate their activities. These C1B peptides are designated C1B1 (amino acid residues 101-112) and C1B5 (residues 141-151). Since PKC enzyme activity is shown to be involved in colon cancer development, the effect of C1B peptides on the growth of various human colon cancer cell lines was examined in vitro and in vivo. Sub-micromolar to micromolar levels of both C1B peptides induced approximately 60-70% growth attenuation in multiple colon cancer cell lines in a soft agar tumor colony assay; however, C1B5 peptide was not cytotoxic to normal colon epithelial cells in two dimensional culture. The effect of C1B5 peptide on colony growth of COLO205 cells was reversed by treatment with the PKCα/ß inhibitor, Ro-32-0432. C1B peptide treatment attenuated COLO205 cells via two mechanisms: 1) cell cycle arrest and 2) stimulation of apoptosis. This is evident in G 2 arrest and increases in levels of cleaved caspase 3 and p53 phosphorylated at serine 20. Intratumoral injection of C1B5 peptide (20 mg/kg/day, every three days) markedly attenuated the growth of subcutaneous xenografts of COLO205 cells in SCID mice by 76% compared with the control. Taken together, these results strongly suggest that C1B peptides have negligible effects on normal tissues but are potentially effective chemotherapeutic agents for colon cancer.

Anti-breast cancer agents, quinolines, targeting gap junction.

Cancer cells exhibit many defects in cell communication that contribute to the loss of tissue homeostasis (excess cell proliferation, invasion, and metastasis). The process of cancer formation causes a disruption in cell homeostasis, affecting the ability to respond to extracellular signals, as well as triggering some intracellular events which alter gap junctional intercellular communication (GJIC). Previous research has shown that the first two generations of substituted quinolines have anti-cancer effects in human breast cancer cells. This report presents the synthesis and bioactivities of third generation substituted quinolines. Scrape load/dye transfer studies showed that 100 nM of PQ15, a third generation substituted quinoline, causes a 4.5-fold increase of gap junction activity in T47D breast cancer cells. Furthermore, a significant decrease of cell proliferation and viability was observed in the presence of 200 nM PQ15 compared to control. The expression of α-survivin was reduced to <40% in the treatment of 200 nM PQ15 compared to solvent alone. Alpha-survivin expression is upregulated in human cancers and associated with resistance to chemotherapy, suggesting that α-survivin prolongs the survival of cancer cells. Thus, it has been shown that substituted quinolines stimulate gap junction activity, decrease alpha survivin expression, and subsequently inhibit cancer cell growth. Our findings demonstrate that PQ15 has a promising role in exerting anti-cancer activity in human breast cancer cells.

Second-generation substituted quinolines as anticancer drugs for breast cancer.

Cancer cells have reduced capacity for gap junctional inter-cellular communication (GJIC). One feasible approach to reduce growth of cancer cells is to enhance GJIC. This report shows that a second-generation substituted quinoline, PQ7, has anti-tumor effect. Scrape load/dye transfer and colony growth assays were performed to measure GJIC and tumor formation of T47D breast cancer cells. PQ7 at 500 nM induced a 16-fold increase in the GJIC in T47D cells. In addition to an increase in GJIC, a 50% decrease of colony growth was observed with 100 nM of PQ7. PQ7-treated nu/nu mice showed a 100% regression of xenograft tumor growth of T47D cells. The results show that PQ7 has a promising role in exerting anti-tumor activity in human breast cancer cells.

Transkingdom RNA interference (tkRNAi): a novel method to induce therapeutic gene silencing.

RNA interference is a phenomenon in which specific, endogenous genes are silenced by mRNA degradation. This technology is highly regarded as a potential therapeutic due to its high efficacy and low toxicity. However, the difficulty of delivering RNAi to target cells has impeded the development of RNAi-based therapies. One method to overcome this barrier is the use of a nonpathogenic bacteria vector, Escherichia coli, to deliver RNAi to target cells with high efficacy. In transkingdom interference RNAi (tkRNAi) delivery, E. coli were engineered to transcribe short RNA (shRNA) from a plasmid (TRIP) containing the invasin gene Inv and the listeriolysin O gene Hly. tkRNAi is successful in eliciting efficient gene silencing in vitro and in vivo.

Synthesis and anti-breast cancer activities of substituted quinolines.

Promising anti-breast cancer agents derived from substituted quinolines were discovered. The quinolines were readily synthesized in a large scale from a sequence of reactions starting from 4-acetamidoanisole. The Michael addition product was isolated as the reaction intermediate in the ring closing reaction of 4-amino-5-nitro-2-(3-trifluoromethylphenyloxy)anisole with methyl vinyl ketone leading to 6-methoxy-4-methyl-8-nitro-5-(3-trifluoromethylphenyloxy)quinoline (14). The amino function of 8-amino-6-methoxy-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, prepared from 14, was connected to various side chains via alkylation with N-(3-iodopropyl)phthalimide, Michael addition with acrylonitrile, and reductive amination with various heterocycle carboxaldehydes, such as imidazole-4-carboxaldehyde, thiophene-2-carboxaldehyde, and 2-furaldehyde. Effects of the substituted quinolines on cell viability of T47D breast cancer cells using trypan blue exclusion assay were examined. The results showed that the IC(50) value of 6-methoxy-8-[(2-furanylmethyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline is 16+/-3nM, the lowest IC(50) out of all the quinolines tested. IC(50) values of three other quinolines are in the nanomolar range, a desirable range for pharmacological testing.

Malaria-associated cytokine changes in the placenta of women with pre-term deliveries in Yaounde, Cameroon.

The prevalence of pre-term deliveries (PTDs) is increased in women who become infected with Plasmodium falciparum during pregnancy. Because prematurity is a risk factor for newborns, it is important to identify conditions that contribute to malaria-associated PTDs. Plasmodium falciparum-infected erythrocytes sequester in the placenta and attract activated mononuclear cells that secrete pro-inflammatory cytokines. Increased inflammatory cytokine levels in other microbial infections are associated with PTDs. To determine if such is the case in women with placental malaria, concentrations of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-4 (IL-4), and IL-10 were measured in placental plasma of 391 malaria-infected and -uninfected Cameroonian women with premature and full-term deliveries. Risk factors for malaria-associated PTDs included peripheral and placental parasitemias greater than 1%, maternal anemia, elevated IL-10 levels, and low TNF-alpha:IL-10 ratios due to over-expression of IL-10. Alterations in cytokine levels may contribute to PTDs through the induction of anemia and/or altering cellular immune responses required for eliminating placental parasites.