Peptides modulating conformational changes in secreted chaperones: From in silico design to preclinical proof of concept

Blocking conformational changes in biologically active proteins holds therapeutic promise. Inspired by the susceptibility of viral entry to inhibition by synthetic peptides that block the formation of helix–helix interactions in viral envelope proteins, we developed a computational approach for predicting interacting helices. Using this approach, which combines correlated mutations analysis and Fourier transform, we designed peptides that target gp96 and clusterin, 2 secreted chaperones known to shift between inactive and active conformations. In human blood mononuclear cells, the gp96-derived peptide inhibited the production of TNFα, IL-1β, IL-6, and IL-8 induced by endotoxin by >80%. When injected into mice, the peptide reduced circulating levels of endotoxin-induced TNFα, IL-6, and IFNγ by >50%. The clusterin-derived peptide arrested proliferation of several neoplastic cell lines, and significantly enhanced the cytostatic activity of taxol in vitro and in a xenograft model of lung cancer. Also, the predicted mode of action of the active peptides was experimentally verified. Both peptides bound to their parent proteins, and their biological activity was abolished in the presence of the peptides corresponding to the counterpart helices. These data demonstrate a previously uncharacterized method for rational design of protein antagonists.     

Androgens, estrogens, and hydroxysteroid dehydrogenases

The task of regulating both production and activity of potent androgens and estrogens in human physiology is largely relegated to the hydroxysteroid dehydrogenases (HSDs). Although over two dozen enzymes with HSD activities have been described, we only understand the physiology of a small number, and for only one enzyme has the function been unequivocally determined by the study of human mutations. The physiology of the HSDs derive from their enzymatic activities, which in turn derive from their respective structures. In general, pairs of enzymes that drive steroid flux in opposite directions are found, and we have been studying the biochemical principles which enable dichotomous enzymes to perform their specific functions. In general, these directional preferences in intact cells are governed by relative affinities for nicotinamide adenine dinucleotide (phosphate) cofactors [NAD(P)(H)] and concentration gradients of these cofactors in subcellular compartments. For the reductive HSDs human 17betaHSD type 1 and rat AKR1C9, we can attenuate or reverse the directional preference in intact cells by site-directed mutagenesis in the cofactor-binding domain or by glucose deprivation, but the magnitude of such changes vary with the different enzymes.     

The clinical significance of isolation of two different organisms from the urine of patients with an indwelling catheter

Background

We evaluated the clinical significance of urine cultures from patients with an indwelling urinary catheter (UC) from which 2 different pathogens were isolated.

Methods

Urine cultures from patients with a UC from which 2 different organisms were isolated were randomly divided into a control group (culture results were reported as usual) and a study group (culture results were reported as “mixed growth”). Endpoints included change in antibiotic treatment, use of broad spectrum agents, time for clinical improvement, and duration of admission.

Results

A total of 81 cultures met the inclusion criteria. Antibiotic treatment was changed after 72–96 h in 19 (48%) study patients and in 25 (61%) controls (NS). There was no difference regarding narrowing or broadening of antibiotic spectrum, and duration of hospitalization was similar. In each group, 15 (36%) patients died.

Conclusion

Our findings imply that laboratory work-up of 2 pathogens from patients with an indwelling catheter may be discarded.     

Safety and Immunogenicity of Multimeric-001—a Novel Universal Influenza Vaccine

Objective

A new vaccine, Multimeric-001, containing conserved linear epitopes from the HA, NP, and M1 proteins of influenza type A and type B strains was designed to protect against seasonal and pandemic influenza virus strains, regardless of mutations. We assessed its safety and tolerability and characterized humoral and cellular immune responses elicited by its administration.

Methods

Sixty healthy volunteers received either 250 or 500 μg injections, with or without adjuvant (Montanide ISA 51VG), or matching placebo. Two intramuscular injections were administered, 21 days apart.

Results

Treatment was well tolerated and no significant adverse events were noted. Forty-two days after first injection, there was a 50-fold and 37-fold increase in IgG titers against Multimeric-001 protein, following the adjuvanted 500 and 250 μg doses, respectively. Sera from immunized subjects lysed MDCK cells infected with strains of influenza representing the major strains that infect humans: H1N1, H3N2, and influenza B. Proliferation of peripheral blood mononuclear cells as well as increase in IL-2 and IFN-gamma secretion occurred following incubation with the vaccine.

Conclusion

This vaccine model differs fundamentally from the current influenza virus vaccines, as it does not contain the variable regions of the virus hemagglutinin and hence does not induce hemagglutination inhibition antibodies that serve as surrogate markers for protection. In order to demonstrate the potential efficacy of the Multimeric-001, an alternative assay was employed, in which the lysis of MDCK cells infected with different virus strains was shown, with the involvement of the complement mechanism. The humoral and cellular responses suggest a cross-immunity of the vaccine toward influenza virus strains regardless of mutations. These results corroborate the protective effect of the vaccine, previously shown in animals. Larger-scale studies are under way to further substantiate the safety and efficacy of the vaccine candidate.     

Intolerance of Uncertainty Moderates the Association Between Potentially Morally Injurious Events and Suicide Ideation and Behavior Among Combat Veterans

Exposure to potentially morally injurious events (PMIEs) among combat veterans has been acknowledged as significant stressful combat events that may lead to mental health problems, including self‐injurious thoughts and behavior (SITB). However, few studies have examined the risk and protective factors that can explain the conditions in which PMIEs may contribute to the development and maintenance of SITB. In the current study, we aimed to examine the association between PMIEs and SITB among combat veterans and explore the moderating roles of intolerance of uncertainty (IU) in this association. A volunteer sample of 191 Israeli combat veterans was recruited during 2017. Participants completed validated self‐report questionnaires in a cross‐sectional study. Results indicated that two separate measures of PMIEs, the Perceived Perpetration by Oneself and Others subscale of the Moral Injury Events Scale (MIES) and the Causes subscale of the Moral Injury Questionnaire (MIQ–Causes), were positively associated with higher levels of SITB. Moreover, beyond the contributions of reserve duty, posttraumatic stress symptoms, and depressive symptoms, MIQ–Causes scores significantly predicted current SITB. Importantly, under low and average levels of inhibitory IU, significant positive effects were revealed for the MIQ–Causes on current SITB, R² = .34. Although veterans exposed to PMIEs are more prone to SITB, even years after their release from military service, their IU may temper the link between experiences of PMIEs and SITB.     

Immunohistochemical staining for cyclin D1 and Ki-67 aids in the stratification of atypical ductal hyperplasia diagnosed on breast core biopsy

A diagnosis of atypical ductal hyperplasia (ADH) after breast core biopsy usually is followed by an excisional biopsy to exclude the presence of a more significant lesion. To determine whether the immunohistochemical expression of cyclin D1 (CyD1) and Ki-67 can aid in case stratification for the likelihood of finding ductal carcinoma in situ (DCIS) on subsequent excision, we immunohistochemically stained 21 consecutive ADH cases diagnosed by core biopsy, and proliferation indices (PIs) were calculated for each case. Fluorescence in situ hybridization to detect CCND1 amplification was performed in 10 cases. In 5 cases, DCIS (with or without invasive carcinoma) was identified in the subsequent excision. The mean PICyD1 and PIKi-67 for these cases were significantly higher than in the remainder (P = .03 and P = .05, respectively). The sensitivities of PICyD1 and PIKi-67 for the presence of DCIS on subsequent excision were 100%, and the specificities were 75% and 69%, respectively. The specificity of the 2 markers combined was 88%. The number of cells with CCND1 amplification was higher in cases with DCIS or ADH on subsequent excision. Immunostaining for CyD1 and Ki-67 might help stratify cases of ADH on core biopsy and identify patients unlikely to have DCIS found on excision.     

Major underestimation and overestimation of visual blood loss during cesarean deliveries: can they be predicted?

Purpose

The surgeons’ visual estimation is the most widely used method for estimating blood loss (BL) while performing cesarean deliveries (CDs). Major BL underestimation may adversely influence obstetric decision making, and result in delaying interventions. Major BL overestimation may result in unnecessary costly interventions. Therefore, we aimed to identify independent predictors for major BL underestimation and overestimation during CDs.

Methods

All CDs performed between 11/2008 and 6/2016, in a university-affiliated hospital, were reviewed for demographic and surgical data, including the surgeons’ reported estimated BL (EBL). Calculated BL (CBL) was calculated by multiplying the calculated maternal blood volume by the percent of hematocrit decrease. Multivariate logistic regressions were performed to identify independent risk factors for major BL underestimation (CBL?EBL ≥ 400 ml) and overestimation (EBL?CBL ≥ 400 ml).

Results

During the study period, 3655 CDs were analyzed, of which 420 met the criterion for major BL underestimation and 1214 for major BL overestimation. Urgent surgery (aOR = 2.83; 95% CI 2.06–3.89), general anesthesia (aOR = 2.39; 95% CI 1.71–3.33), and higher surgeon experience (aOR = 1.03; 95% CI 1.01–1.06) were found to be independent risk factors for major BL underestimation, while any previous CD (aOR = 0.47; 95% CI 0.33–0.67) decreased the risk of underestimation. Any previous CD (aOR = 1.29; 95% CI 1.05–1.58) and intra-abdominal adhesions (aOR = 1.37; 95% CI 1.11–1.70) were found to be independent risk factors for major BL overestimation, while urgent CD (aOR = 0.50; 95% CI 0.41–0.60) decreased the risk of overestimation.

Conclusion

Various factors can predict major underestimation and overestimation of BL during CDs. Recognizing these factors can assist in the interpretation of visual EBL and improve obstetric decision making.