Adverse obstetric history and ectopic pregnancy

OBJECTIVE: To determine whether women with an adverse obstetric history are at greater risk of ectopic pregnancy. STUDY DESIGN: A retrospective, case-control study was conducted on ectopic (case) and intrauterine (control) pregnancies at New York Methodist Hospital between January 2000 and April 2004. There were 67 patients in each group, matched by age and gravidity. Number of pregnancies (G) was stratified by parity (P1-4). Interruption of pregnancy prior to 20 weeks (P3) was further stratified into spontaneous and voluntary abortion and prior ectopic pregnancy. RESULTS: There were 285 ectopic and 286 control pregnancies. There were more term births (P1: 239 vs. 100) and more live children (P4: 241 vs. 103) in the control group, (p < 0.001), but the number of interrupted pregnancies (P3) was higher (182 vs. 43, p < 0.001) in the ectopic group. The ectopic group had more voluntary (144) and spontaneous interruptions (38) than the control group (18 and 25) (p < 0.001). The ectopic group had more surgeries (57), with 34 dilation and curettages; the control group had 30 surgeries and 11 dilation and curettages (p < 0.001). CONCLUSION: Women with an adverse obstetric history that included interrupted pregnancies were more likely to have ectopic pregnancies.     

Consensus recommendations of pediatric transfusion medicine objectives for clinical pathology residency training programs

BACKGROUND: Pediatric transfusion medicine (PTM) is a subspecialty of transfusion medicine with no formal training program and few specialists. The Pediatric Transfusion Medicine Academic Awardees (PedsTMAA) group surveyed PTM content experts to identify relevant objectives for the first formal PTM curriculum. STUDY DESIGN AND METHODS: Eight North American PTM experts were invited to participate in a two‐step consensus process. PTM‐related objectives compiled from a review of existing training documents were organized into a survey. Experts were asked to rate each objective for relevancy for a clinical pathology trainee. Content validity indexes (CVIs) and asymmetric confidence intervals (ACIs) of expert ratings and analysis of respondents' comments were used to identify relevant objectives. RESULTS: Six experts participated and reviewed 117 objectives. Based on content validity criteria (CVI ≥ 0.83 and lower‐limit 95% ACI ≥ 3), a total of 65 objectives were considered relevant. Twenty‐three objectives were rated “very relevant” by all the experts while some proposed objectives were determined to be not relevant, out of date, or inappropriate for a resident trainee level. CONCLUSIONS: The PedsTMAA group identified 65 objectives for a PTM curriculum. Twenty‐three represent a clear core set of objectives and should be considered for clinical pathology training. The next step is to consider the teaching strategies and evaluation methods that will be employed to best deliver this content addressing competency in medical knowledge.     

Procedure‐specific preoperative red blood cell preparation and utilization management in pediatric surgical patients

BACKGROUND: Data‐driven practices in preoperative red blood cell (RBC) preparation for pediatric surgical procedures are not well established. Adaptation of established adult preparation guidance methods to pediatric populations may improve perioperative RBC utilization. STUDY DESIGN AND METHODS: A retrospective audit of preoperative RBC preparation volumes (Vp) and intraoperative RBC transfusion volumes (Vt) for pediatric surgical procedures was undertaken at a large children's hospital from January to June 2006. RBC preparation‐to‐transfusion volume (mL/kg) ratios (P:T) were calculated for all surgeries, subspecialties, and select procedures. P:T equals Vp divided by Vt. Resulting P:Ts were compared to a target P:T of 2:1. A model for maximum procedure‐specific Vp (Vp‐max) defined Vp‐max as the RBC transfusion volume able to meet the needs of 80% of patients undergoing an individual surgical procedure. Vp‐max values were applied to the study data set to predict the impact on P:Ts and Vp. RESULTS: RBCs were prepared for 332 surgical procedures and transfused during 113 procedures. P:T was 3.5:1 for total surgical procedures (subspecialty range, 2.7:1‐46:0), exceeding the 2:1 target. Vp‐max modeling for spinal fusion, craniotomy for neoplasia, craniotomy for seizure, and craniosynostectomy yielded P:T ratios of 1.5:1, 1.5:1, 1.7:1, and 1.0:1, respectively, predicting a 30% decrease in Vp for these four surgical procedures. CONCLUSIONS: P:Ts for pediatric surgical procedures at this institution indicate potentially excessive preoperative RBC preparations. Determination of data‐driven procedure‐specific Vp may increase the efficiency of preoperative RBC preparation practices.     

Effects of storage duration and volume on the quality of leukoreduced apheresis‐derived platelets: implications for pediatric transfusion medicine

BACKGROUND: Platelet (PLT) storage adversely affects PLT structure and function in vitro and is associated with decreased PLT recovery and function in vivo. In pediatric transfusion medicine, it is not uncommon for small residual volumes to remain in parent units after aliquot preparation of leukoreduced apheresis‐derived PLTs (LR‐ADP). However, limited data exist regarding the impact of storage on residual small‐volume LR‐ADP. STUDY DESIGN AND METHODS: Standard metabolic testing was performed on residual volumes of LR‐ADP after aliquot removal and PLT aggregometry using a dual agonist of ADP and collagen was performed on stored, small‐volume aliquots (10‐80 mL) created from an in vitro model of PLT storage. RESULTS: Seventy‐seven LR‐ADP underwent metabolic (n = 67) or metabolic and aggregation (n = 10) studies. All products maintained a pH value of more than 6.89 throughout storage. Lactate and pCO₂ increased proportionally with longer storage time. Regardless of acceptable metabolism during storage, aggregation in 10‐ to 20‐mL aliquots was impaired by Day 4 and aliquots less than 40 mL demonstrated the most dramatic decrease in aggregation from baseline. CONCLUSIONS: Despite maintenance of acceptable metabolic conditions, residual volumes of LR‐ADP develop impaired aggregation in vitro that may adversely affect PLT survival and function in vivo. At volumes below 40 mL, LR‐ADP revealed reduced aggregation. As a result, it is recommended to monitor and record volumes of LR‐ADP used for pediatric transfusion. Moreover, once LR‐ADP attain a volume of 50 mL or less on Day 4 or Day 5 of storage, consider discarding these products until their in vivo efficacy can be studied.     

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC₅₀ (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC₅₀ (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC₅₀ of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC₅₀ of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the newly emerged, highly transmissible coronavirus responsible for the ongoing COVID-19 pandemic, which is associated with 136 million cases and almost 3 million deaths worldwide as of April 12, 2021 (https://coronavirus.jhu.edu/map.html). While three vaccines have recently been approved by the FDA, there are still no clinically approved small-molecule drugs available for the treatment of this disease except Remdesivir, and the effectiveness of the vaccines against immune escape variants might be reduced. Multiple therapeutic strategies have been proposed (1−2), including both viral and host proteins, but none have yet been fully validated for clinical application. One class of protein targets which have shown promising results are proteolytic enzymes including the viral proteases (1, 3–5), Papain-Like Protease (PLpro) and the 3C-like or “Main Protease” (3CL or MPro), and several host proteases involved in viral entry, replication, and effects on the immune system creating the life-threatening symptoms of COVID-19 infection (4–6). The latter include various members of the cathepsin family of cysteine proteases, including cathepsin L, furin, and the serine proteases factor Xa, plasmin, elastase, tryptase, TMPRSS2, and TMPRSS4.Coronavirus (SARS CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus [MERS]) entry is mediated by the viral spike protein, which must be cleaved by host proteases in order to trigger membrane fusion and entry into the host cell after binding to the host cell receptor Angiotensin Converting Enzyme-2 (ACE2) (7–10). This is mediated by initial cleavage at the S1/S2 junction of spike, which is thought to occur during processing in the producer cell, followed by cleavage at the S2′ site either by serine proteases at the cell surface or by cathepsin proteases in the late endosome or endolysosome (9, 10). Whether serine or cathepsin proteases are used for S2′ cleavage is cell type dependent. While entry into Calu-3 (human lung epithelial) or HAE (primarily human airway epithelial) cells is cathepsin independent, entry into Vero cells (African green monkey kidney epithelial), which do not express the required serine proteases, depends exclusively on cathepsins (7, 9–11).TMPRSS2 (12) is a type II transmembrane serine proteases (TTSP) (13) that has been shown to be crucial for host cell viral entry and spread of SARS-CoV-2 (7, 8, 14–16), as well as SARS-CoV (17, 18), MERS-CoV (19), and influenza A viruses (20–27). The Spike protein requires proteolytic processing/priming by TMPRSS2 to mediate entry into lung cells; thus, small-molecule inhibitors of this target offer much promise as new therapeutics for COVID-19 and other coronavirus diseases (7, 8). TMPRSS2 expression levels dictate the entry route used by SARS-CoV-2 to enter cells, as reported recently (28). In cells that express little or no TMPRSS2, cell entry occurs via the endosomal pathway, and cleavage of spike protein is performed by cathepsin L. It has been demonstrated that the TMPRSS2-expressing lung epithelial Calu-3 cells are highly permissive to SARS-CoV-2 infection. The irreversible serine protease inhibitors Camostat (7) and Nafamostat (29) are effective at preventing host cell entry and replication of SARS-CoV-2 in Calu-3 cells through a TMPRSS2-dependent mechanism (14, 15).Herein, we report on the discovery of a class of substrate-based ketobenzothiazole (kbt) inhibitors of TMPRSS2 with potent antiviral activity against SARS-CoV-2 which are significantly improved over Camostat and Nafamostat. Several compounds were found to be strong inhibitors of viral entry and replication, with EC₅₀ (half-maximal effective concentration) values exceeding the potency of Camostat and Nafamostat and without cytotoxicity. Newly developed compound MM3122 (4) has excellent pharmacokinetics (PK) and safety in mice and is thus a promising lead candidate drug for COVID-19 treatment.     

Transfusion management of sickle cell patients during bone marrow transplantation with matched sibling donor

BACKGROUND: Sickle cell disease (SCD) patients have unique transfusion considerations during bone marrow transplantation (BMT), including prophylaxis against stroke and alloimmunization. Characterization of transfusion requirements is important for blood bank and clinician patient management.
STUDY DESIGN AND METHODS: A retrospective analysis of red blood cell (RBC) and platelet (PLT) transfusion of SCD patients during myeloablative matched sibling donor (MSD) BMT at one institution from 1993 to 2007 was performed. Patient characteristics (RBC blood group antibodies, ABO-incompatible donor, BMT-related morbidity) and transfusion practices (RBC phenotype matching, transfusion threshold, and blood age) were assessed for effect on total RBC transfusion volumes.
RESULTS: Twenty-seven patients received MSD BMT with 96% survival and 0% rejection. Six alloimmunized patients received RBCs with extended phenotype matching (C, c, E, e, K, Fy^a, Jk^b), 14 nonalloimmunized received limited matching (C, c, E, e, K), and 7 did not have phenotype matching. Among 26 survivors, a median seven RBC transfusions (range, 3-15) and 13.5 PLT transfusions (range, 4-48) per patient were administered, equivalent to 64 mL/kg RBCs (range, 22-122 mL/kg) and 106 mL/kg PLTs (range, 26-343 mL/kg). BMT-related morbidity predicted increased RBC transfusions (p = 0.006). Venoocclusive disease was associated with greater RBC (p = 0.016) and PLT transfusion volumes (p = 0.016). Greater phenotype matching was associated with decreased RBC transfusions (p = 0.0247).
CONCLUSIONS: SCD patients have high transfusion support during MSD BMT. Communication of BMT complications to the blood bank is essential for transfusion inventory management. Phenotype matching decreased RBC transfusions in this cohort and warrants further investigation in SCD transfusion therapy.     

Novel S-Adenosylmethionine Decarboxylase Inhibitors for the Treatment of Human African Trypanosomiasis

Trypanosomiasis remains a significant disease across the sub-Saharan African continent, with 50,000 to 70,000 individuals infected. The utility of current therapies is limited by issues of toxicity and the need to administer compounds intravenously. We have begun a program to pursue lead optimization around MDL 73811, an irreversible inhibitor of S-adenosylmethionine decarboxylase (AdoMetDC). This compound is potent but in previous studies cleared rapidly from the blood of rats (T. L. Byers, T. L. Bush, P. P. McCann, and A. J. Bitonti, Biochem. J. 274:527-533). One of the analogs synthesized (Genz-644131) was shown to be highly active against Trypanosoma brucei rhodesiense in vitro (50% inhibitory concentration, 400 pg/ml). Enzyme kinetic studies showed Genz-644131 to be approximately fivefold more potent than MDL 73811 against the T. brucei brucei AdoMetDC-prozyme complex. This compound was stable in vitro in rat and human liver microsomal and hepatocyte assays, was stable in rat whole-blood assays, did not significantly inhibit human cytochrome P450 enzymes, had no measurable efflux in CaCo-2 cells, and was only 41% bound by serum proteins. Pharmacokinetic studies of mice following intraperitoneal dosing showed that the half-life of Genz-644131 was threefold greater than that of MDL 73811 (7.4 h versus 2.5 h). Furthermore, brain penetration of Genz-644131 was 4.3-fold higher than that of MDL 73811. Finally, in vivo efficacy studies of T. b. brucei strain STIB 795-infected mice showed that Genz-644131 significantly extended survival (from 6.75 days for controls to >30 days for treated animals) and cured animals infected with T. b. brucei strain LAB 110 EATRO. Taken together, the data strengthen validation of AdoMetDC as an important parasite target, and these studies have shown that analogs of MDL 73811 can be synthesized with improved potency and brain penetration.Sleeping sickness, or human African trypanosomiasis (HAT), afflicts 50,000 to 70,000 people across sub-Saharan Africa, with 17,000 new cases reported in the year 2004 (9) and 10,769 reported in 2007 (31). Untreated, the disease is inevitably fatal. Current treatments include drugs first developed over 50 years ago, and while not without efficacy, some have high toxicity and generally need to be administered by intravenous (i.v.) infusion—hardly a practical solution in locations where this disease is prevalent (17). Despite the obvious need for new, easily administered therapies, the rate of development of new drugs for HAT by the pharmaceutical industry has been negligible. Recognizing this, the Drugs for Neglected Diseases Initiative (DNDi) was formed in 2003 to facilitate the formation of partnerships among industry, academia, and public-sector organizations to develop affordable solutions for this urgent unmet medical need (www.dndi.org). Studies presented here were conducted under one such partnership.Polyamines are small-molecule cationic structures that are critical to the survival of eukaryotic cells, including trypanosomes (2, 4, 19). Difluoromethyl ornithine (DFMO) is an inhibitor of ornithine decarboxylase, a key enzyme in the polyamine biosynthetic pathway. DFMO is an effective and relatively well tolerated agent for the treatment of the central nervous system (CNS) second stage of HAT caused by Trypanosoma brucei gambiense. However, DFMO must be given as an i.v. infusion of up to 30 g per day four times per day for 14 days. The amount required and frequency of dosing demonstrate the limitations of this drug in terms of potency, cost, and logistics of administration in the field (10, 16). Nevertheless, the extraordinary efficacy of DFMO serves to validate the polyamine pathway as an attractive target for new drug discovery for trypanosomiasis. A potent, safe, orally bioavailable inhibitor of polyamine biosynthesis would represent a major breakthrough for the treatment of this disease.A second key enzyme in the polyamine biosynthetic pathway is S-adenosylmethionine decarboxylase (AdoMetDC). AdoMetDC is allosterically activated by a novel mechanism unique to trypanosomatid parasites; the functional form of the enzyme is a heterodimer between the active subunit and a paralog (termed prozyme) that arose through gene duplication (32). Gene knockout and small interfering RNA experiments have indicated that suppression of this enzyme by knockdown of either AdoMetDC or its regulatory subunit prozyme is lethal to the parasite (33). Inhibitors of this enzyme have been shown to be highly efficacious in killing trypanosomes in vitro (3, 14) and in curing T. brucei-infected mice (6, 8). However, these agents lack both the potency and, especially, the pharmacokinetic (PK) and tissue (CNS) distribution characteristics (11) that are essential to meet the improved target product profile for a new antitrypanosomal drug.MDL 73811 is an irreversible inhibitor of AdoMetDC and is believed to form a Schiff base with a pyruvate group within the active site of the enzyme (13). The compound displays a high level of selectivity (>100-fold) for killing parasites compared with its toxicity for mammalian cells (24). Reasons for this selectivity are not clear, but it has been postulated that the compound is more readily taken up by the parasites (12). Alternatively, selectivity may result from differences in enzyme turnover (33). The mechanism of killing is believed to be associated with the buildup of S-adenosylmethionine (11). Recent studies utilizing RNA interference silencing, however, suggest that a major method by which AdoMetDC inhibition kills trypanosomes is by depleting reserves of trypanothione (33). Finally, MDL 73811 induces increased expression of the prozyme in T. brucei bloodstream form parasites, providing strong evidence that the primary target of MDL 73811 responsible for parasite death is AdoMetDC inhibition (33). MDL 73811 has been demonstrated to reduce parasitemia within 5 h and to effect cures of acute infections in T. brucei brucei- and T. brucei rhodesiense-infected mice when administered at a dose of 20 mg/kg of body weight twice a day (BID) for 4 days (11, 24). This compound was originally developed in the 1980s by Merrell-Dow as an anticancer therapy.Despite its trypanocidal activity, MDL 73811 is not itself an attractive drug candidate for a number of reasons. This compound is not effective as monotherapy against the CNS stage of infection, although it is curative when given in combination with DFMO (6). It has been hypothesized that DFMO may in some way temporarily alter the blood-brain barrier (BBB) to effect this; however, data to support this contention are currently lacking. Recent studies (28) showed that DFMO by itself penetrated the BBB poorly and that addition of 250 μM DFMO did not improve the uptake of other solutes, although a statistically significant increase in BBB penetration was seen at day 28 and later after the trypanosome infection. Finally, the drug has poor oral bioavailability and was thought to have limited metabolic stability (P. Casara, personal communication). We therefore initiated a program to synthesize analogs of MDL 73811 and determine whether they could overcome these issues. Studies reported here describe initial progress.     

A multicomponent quality improvement intervention to improve blood pressure and reduce racial disparities in rural primary care practices

The Southeastern United States has the highest prevalence of hypertension and African Americans have disproportionately worse blood pressure control. The authors sought to evaluate the effect of a multicomponent practice‐based quality improvement intervention on lowering mean systolic blood pressure (SBP) at 12 and 24 months compared with baseline among 525 patients, and to assess for a differential effect of the intervention by race (African Americans vs white). At 12 months, both African Americans (−5.0 mm Hg) and whites (−7.8 mm Hg) had a significant decrease in mean SBP compared with baseline, with no significant between‐group difference. Similarly, at 24 months, mean SBP decreased in both African Americans (−6.0 mm Hg) and whites (−7.2 mm Hg), with no significant difference between groups. Notably, no significant racial disparity in mean SBP at baseline was shown. The intervention was effective in lowering mean SBP in both African Americans and whites but there was no differential effect of the intervention by race.