Monitoring and impact of fluconazole serum and cerebrospinal fluid concentration in HIV‐associated cryptococcal meningitis‐infected patients

Objectives The aim of the present study was to assess fluconazole pharmacokinetic measures in serum and cerebrospinal fluid (CSF); and the correlation of these measures with clinical outcomes of invasive fungal infections. Methods A randomized trial was conducted in HIV‐infected patients receiving three different regimens of fluconazole plus amphotericin B (AmB) for the treatment of cryptococcal meningitis. Regimens included fluconazole 400 mg/day+AmB (AmB+Fluc400) or fluconazole 800 mg/day+AmB (AmB+Fluc800) (14 days followed by fluconazole alone at the randomized dose for 56 days); or AmB alone for 14 days followed by fluconazole 400 mg/day for 56 days. Serum (at 24 h after dosing) and CSF samples were taken at baseline and days 14 and 70 (serum only) for fluconazole measurement, using gas–liquid chromatography. Results Sixty‐four treated patients had fluconazole measurements: 11 in the AmB group, 12 in the AmB+Fluc400 group and 41 in the AmB+Fluc800 group. Day 14 serum concentration geometric means were 24.7 mg/L for AmB+Fluc400 and 37.0 mg/L for AmB+Fluc800. Correspondingly, CSF concentration geometric means were 25.1 mg/L and 32.7 mg/L. Day 14 Serum and CSF concentrations were highly correlated with AmB+Fluc800 (P<0.001, r=0.873) and AmB+Fluc400 (P=0.005, r=0.943). Increased serum area under the curve (AUC) appears to be associated with decreased mortality at day 70 (P=0.061, odds ratio=2.19) as well as with increased study composite endpoint success at days 42 and 70 (P=0.081, odds ratio=2.25 and 0.058, 2.89, respectively). Conclusion High fluconazole dosage (800 mg/day) for the treatment of HIV‐associated cryptococcal meningitis was associated with high serum and CSF fluconazole concentration. Overall, high serum and CSF concentration appear to be associated with increased survival and primary composite endpoint success.     

Altered adhesion molecules expression on peripheral blood mononuclear cells from patients with systemic sclerosis and clinical correlations

The aim of the study was to evaluate the expressions of adhesion molecules (AM) on peripheral blood mononuclear cells (PBMNC) from systemic sclerosis (SSc) patients. Thirty-one SSc patients (ACR) and 20 normal subjects were selected for the study. PBMNC were analyzed for LFA-1α, LFA-1β, ICAM-3, ICAM-1, and l-selectin expressions. ICAM-3 expression was decreased while ICAM-1 was increased on SSc PBMNC, compared to controls (p = 0.04 and 0.003, respectively). A positive association was found between LFA-1α (r = 0.37, p = 0.03), LFA-1β (r = 0.38, p = 0.002), ICAM-3 (r = 0.42, p = 0.01), and l-selectin (r = 0.38, p = 0.03) expressions and greater number of immunosuppressive drugs taken by SSc patients. Also, anti-centromeric positive SSc patients had lower expressions of LFA-1α, LFA-1β, ICAM-3, and l-selectin. Lower expression of ICAM-3 and higher expression of ICAM-1 suggest that AMs may be involved in the pathogenesis of scleroderma.     

Intrinsic electronic conductivity of individual atomically resolved amyloid crystals reveals micrometer-long hole hopping via tyrosines

Proteins are commonly known to transfer electrons over distances limited to a few nanometers. However, many biological processes require electron transport over far longer distances. For example, soil and sediment bacteria transport electrons, over hundreds of micrometers to even centimeters, via putative filamentous proteins rich in aromatic residues. However, measurements of true protein conductivity have been hampered by artifacts due to large contact resistances between proteins and electrodes. Using individual amyloid protein crystals with atomic-resolution structures as a model system, we perform contact-free measurements of intrinsic electronic conductivity using a four-electrode approach. We find hole transport through micrometer-long stacked tyrosines at physiologically relevant potentials. Notably, the transport rate through tyrosines (10⁵ s⁻¹) is comparable to cytochromes. Our studies therefore show that amyloid proteins can efficiently transport charges, under ordinary thermal conditions, without any need for redox-active metal cofactors, large driving force, or photosensitizers to generate a high oxidation state for charge injection. By measuring conductivity as a function of molecular length, voltage, and temperature, while eliminating the dominant contribution of contact resistances, we show that a multistep hopping mechanism (composed of multiple tunneling steps), not single-step tunneling, explains the measured conductivity. Combined experimental and computational studies reveal that proton-coupled electron transfer confers conductivity; both the energetics of the proton acceptor, a neighboring glutamine, and its proximity to tyrosine influence the hole transport rate through a proton rocking mechanism. Surprisingly, conductivity increases 200-fold upon cooling due to higher availability of the proton acceptor by increased hydrogen bonding.

Many biological processes require electron transport over far longer distances than the 25 Å allowed by single-step tunneling (1). The aromatic amino acids tyrosine and tryptophan, strategically placed between cofactors in proteins, can provide the critical stepping-stones for multistep hopping by acting as intermediaries along the electron transfer route as in Photosystem II and ribonucleotide reductase (1, 2). In addition, chains of aromatic residues have been proposed to move hole carriers from enzyme active sites to the exterior of proteins to avoid oxidative damage caused by redox cofactors, but direct experimental evidence of charge transport beyond the nanometer scale is lacking (3, 4). The ubiquity of such chains observed in proteins, and the established role of aromatic amino acids in the above biological processes, motivates mechanistic studies of how electron transport via aromatic residues can scale over mesoscopic (hundreds of nanometers) to microscopic distances (1, 2).A remarkable example of biological long-distance electron transport is by soil (5, 6) and sediment bacteria (7, 8) that carry electrons to remote acceptors hundreds of micrometers (5, 9) to centimeters away (7–10), 10,000 times the size of the cell. This long-distance transport enables bacteria to get rid of electrons derived from metabolism and survive in harsh environments that lack soluble, membrane-ingestible electron acceptors such as oxygen. Polymerized cytochromes produced by soil bacteria function as “microbial nanowires” by transporting electrons through seamlessly stacked hemes (5, 6). In addition, filamentous proteins produced by both soil (11) and sediment bacteria (8) have been shown to be conductive, even in the absence of cytochromes (8). However, the underlying conductivity mechanism is unclear. Aromatic residues present in these filamentous proteins of soil (11) and sediment bacteria (10) have been proposed to carry electrons over long distances. However, this hypothesis is open to question because previous studies of DNA conductivity have shown that charge transport through aromatic bases is not possible beyond nanometer distances (12, 13), even using a very large driving force (>5 V) and synthetic bases (13).As the structures and composition of the abovementioned filamentous proteins produced by soil and sediment bacteria are unknown, here we use amyloid proteins with stacked tyrosine residues as a model system to evaluate the possibility and mechanism of long-distance electron transport via aromatic residues (Figs. 1 and ​and2A).2A). Many soluble proteins aggregate into an insoluble amyloid state to form elongated, unbranched fibrils that are associated with several fatal diseases, such as Alzheimer’s, type II diabetes, and some types of cancers (14). Amyloids are attractive model systems to evaluate protein conductivity mechanisms due to their unique ability to form several distinct, highly ordered, self-replicating, and stable biomimetic structures (Fig. 1). Short peptide-based amyloids are particularly attractive because of their computationally guided, autonomous, and high-precision synthesis as well as their diverse chemical properties and biocompatibility.Open in a separate windowFig. 1.Strategy to evaluate protein conductivity mechanism using short peptides as building blocks for model systems. (A) Amino acid sequences of the short peptides used with tyrosine (yellow) and zinc (gray) highlighted. (B) Light microscopy image of microcrystals. (Scale bar, 50 µm.) (C–F) Atomic structures of microcrystals with gray arrows indicating the direction of the fibril axis. (C) X1, Zn²⁺NNQQNY (PDB ID 5K2E); (D) X2, GNNQQNY (PDB ID 5K2G); (E) X3, Zn²⁺GGVLVN (PDB ID 3PPD). (F) X4, KVQIINKKL (PDB ID 6NK4). Tyrosine edge-to-edge distances are 3.5 Å. Chemical elements are shown as follows: green, carbon; blue, nitrogen; red, oxygen; and gray, zinc.Open in a separate windowFig. 2.Intrinsic conductivity measurements of amyloids reveal micrometer-long charge transport through stacked tyrosines. (A) A schematic for measuring the intrinsic conductivity of a protein crystal (black) using four probes. (Inset) Atomic structure of X1 showing stacked tyrosines. (B, Left) AFM image of X1 crystal spanning multiple electrodes and (Right) corresponding height profile at the red dotted line. (Scale bar: B, 1 µm.) (C) Representative current–voltage (I–V) curves taken by two-probe electrical measurements. The dashed lines are linear fits to the data shown. (Inset) Zoomed I–V curve for low-conductive microcrystals. (D) Comparisons of the four-probe (black) and two-probe (gray) conductivity of the microcrystals. (Inset) Zoomed values (below X2 two-probe dashed red line) on a log scale. (E) Average resistance values of microcrystals measured using two-probe, contact, AC impedance spectroscopy (IS), and four-probe methods. Bars represent mean + SEM of multiple replicates [n (two-probe) = 19, 8, 9, 4, and n (four-probe) = 8, 9, 6 for X1, X2, X3, and X4, respectively. n (IS) = 11]. (F) Comparison of current–voltage profile for X1 measured using (Left) four- and (Right) two-probe method.In addition to their biomedical importance, amyloids are attractive biomaterials due to their high stability and capacity to reproduce themselves by seeding, enabling the development of self-repairing and replicating biomimetic materials (14). However, two major bottlenecks remain in the use of amyloids as a model system to evaluate conduction mechanisms and for the development of protein-based multifunctional biomaterials. First, like most proteins, amyloids lack electronic or optical functionality (15, 16), and in prior studies of protein conductivity, high contact resistance—either between proteins or at the protein–electrode interface—has been shown to mask the intrinsic electronic properties (17). Second, in contrast to over 100,000 structures of globular proteins, only a few dozen amyloid fibril structures are available (14), and conformational changes induced by experimental conditions hinder the elucidation of structure–function correlations.To address both these bottlenecks, here we employ a strategy to measure intrinsic conductivity of pristine amyloid microcrystals, with atomic resolution structures (18, 19), aiming to correlate structure with function by avoiding modification or tagged molecules that can induce large conformational changes (20–22). We use individual microcrystals with defined geometry to perform measurements of charge transport through stacked tyrosines, or along the fibril axis (Fig. 1). Notably, we employ a four-electrode system with ionically blocking electrodes and measure steady-state electronic current (9, 17) to avoid artifacts due to contact resistance (23, 24), polarization resistance (25), and ionic currents (23), which are known to mask the intrinsic conductivity of the protein (23, 26). Our studies show that amyloid proteins can efficiently transport charges over micrometer distances under ordinary thermal conditions without any need for redox-active metal cofactors. In contrast to single-step tunneling commonly observed in proteins, we find evidence for a hopping mechanism, previously observed experimentally only in synthetic conjugated molecular wires (27, 28), to account for long-distance conductivity.     

High-dose etoposide, cyclophosphamide, and total body irradiation with allogeneic bone marrow transplantation for patients with acute myeloid leukemia in untreated first relapse: a study by the North American Marrow Transplant Group

Relapse is a major cause of treatment failure following allogeneic bone marrow transplantation (BMT) for acute myeloid leukemia (AML). To reduce the risk of relapse following BMT for patients with hematologic malignancy, our group developed a novel preparative regimen which combines high-dose etoposide with cyclophosphamide and total body irradiation (VPCyTBI). We now report the outcome of therapy with VPCyTBI followed by allogeneic BMT for 40 patients with AML in untreated first relapse. With the exception of increased stomatitis, the toxicity of this regimen was similar to that reported by others for CyTBI. Forty-four months after transplant the actuarial probabilities of disease-free survival (DFS), persistent or recurrent leukemia, and transplant related mortality were .29, .44, and .47 respectively. DFS was improved (P < .01) and risk of persistent or recurrent leukemia reduced (P = .005) among patients with significant (grade > or = 2) acute GVHD. Patients with 30% or more blasts on pre-BMT bone marrow examination were not at increased risk for persistent or recurrent leukemia. We conclude that VPCyTBI with allogeneic BMT is effective therapy for AML in untreated first relapse and that a randomized trial comparing this regimen with CyTBI is warranted.     

Thrombocytopenia in dogs induced by granulocyte-macrophage colony- stimulating factor: increased destruction of circulating platelets

Administration of recombinant canine granulocyte-macrophage colony- stimulating factor (rcGM-CSF) to normal dogs in previous studies induced an increase in peripheral blood neutrophils and a dose- dependent decrease in platelet counts. In six dogs that received the highest tested dose of rcGM-CSF (50 micrograms/kg/d) for a minimum of 12 days, the mean nadir of the platelet count was 46,000/microL (range, 4,000 to 91,000/microL) on day 9 +/- 1.1 after starting therapy, compared with a mean baseline platelet count of 398,000/microL (range, 240,000 to 555,000/microL). In three dogs, survival of autologous 111In- labeled platelets was reduced from a mean of 4.9 days to 1.3 days during the administration of rcGM-CSF. Biodistribution studies with gamma camera imaging indicated that there was an increase in mean hepatic uptake during the administration of rcGM-CSF, from 15% to 44% of the total injected 111In-labeled platelets at 2 hours, whereas splenic uptake was not significantly changed. In contrast, in two evaluable dogs who were recipients of 111In-labeled platelets from matched allogeneic donors receiving rcGM-CSF, platelet survival was not reduced and no increased hepatic uptake was noted. A third dog became alloimmunized to the matched donor platelets and was not evaluable. Immunohistologic studies of liver and spleen were performed with monoclonal antibodies specific for canine gpIIb/IIIa and P-selectin in dogs treated with rcGM-CSF and compared with untreated controls. On treatment, a marked reduction of platelets in the red pulp of the spleen was evident, and in general, the presence of platelet antigen in the liver was unchanged. Therefore, platelets were not being sequestered, but destroyed in the liver and spleen. The platelet antigens, P-selectin and gpIIb/IIIa, were identified in association with Kupffer cells in the liver, but no difference in the number of distribution of these Kupffer cells was found between controls and rcGM- CSF-treated dogs. In the spleen during rcGM-CSF treatment, most platelet antigens were associated with large mononuclear cells in the marginal zone. During administration of rcGM-CSF, CD1c and CD11c expression was increased on Kupffer cells. Platelet P-selectin expression and binding of leukocytes to circulating platelets were unchanged from baseline studies with rcGM-CSF treatment. In conclusion, during the administration of rcGM-CSF to dogs, a local process in the liver and spleen is induced resulting in thrombocytopenia.(ABSTRACT TRUNCATED AT 400 WORDS)     

A comparison of filtered leukocyte-reduced and cytomegalovirus (CMV) seronegative blood products for the prevention of transfusion- associated CMV infection after marrow transplant [see comments]

We performed a prospective, randomized trial in CMV seronegative marrow recipients to determine if filtered blood products were as effective as CMV-seronegative blood products for the prevention of transfusion- transmitted CMV infection after marrow transplant. Before transplant, 502 patients were randomized to receive either filtered or seronegative blood products. Patients were monitored for the development of CMV infection and tissue-documented CMV disease between days 21 and 100 after transplant. Infections occurring after day 21 from transplant were considered related to the transfusion of study blood products and, thus, were considered evaluable infections for the purpose of this trial. In the primary analysis of evaluable infections, there were no significant differences between the probability of CMV infection (1.3% v 2.4%, P = 1.00) or disease (0% v 2.4%, P = 1.00) between the seronegative and filtered arms, respectively, or probability of survival (P = .6). In a secondary analysis of all infections occurring from day 0 to 100 post-transplant, although the infection rates were similar, the probability of CMV disease in the filtered arm was greater (2.4% v 0% in the seronegative arm, P = .03). However, the disease rate was still within the prestudy clinically defined acceptable rate of < or = 5%. We conclude that filtration is an effective alternative to the use of seronegative blood products for prevention of transfusion- associated CMV infection in marrow transplant patients.     

Comparison between the SAPIEN S3 and the SAPIEN XT transcatheter heart valves:A single-center experience

AIM To investigate the clinical outcomes of transcatheter aortic valve implantation(TAVI) with the SAPIEN 3 transcatheter heart valve(S3-THV) vs the SAPIEN XT valve(XT-THV).METHODS We retrospectively analyzed 507 patients that underwent TAVI with the XT-THV and 283 patients that received the S3-THV at our institution between March 2010 and December 2015.RESULTS Thirty-day mortality(3.5% vs 8.7%:OR=0.44,P=0.21) and 1-year mortality(25.7% vs 20.1%,P=0.55) were similar in the S3-THV and the XT-THV groups.The rates of both major vascular complication and paravalvular regurgitation(PVR)1 were almost 4 times lower in the S3-THV group than the XT-THV group(major vascular complication: 2.8% vs 9.9%,P0.0001:PVR1: 2.4% vs 9.7%,P0.0001).However,the rate of new pacemaker implantation was almost twice as high in the S3-THV group(17.3% vs 9.8%,P=0.03).In the S3 group,independent predictors of new permanent pacemaker were pre-procedural RBBB(OR=4.9:P=0.001),pre-procedural PR duration(OR=1.14,P=0.05) and device lack of coaxiality(OR=1.13:P=0.05) during deployment.CONCLUSION The S3-THV is associated to lower rates of major vascular complications and PVR but higher rates of new pacemaker compared to the XT-THV.Sub-optimal visualization of the S3-THV in relation to the aortic valvular complex during deployment is a predictor of new permanent pacemaker.     

Frailty under COVID-19 pandemic in Japan: Changes in prevalence of frailty from 2017 to 2021

Background

After the outbreak of the coronavirus disease 2019 (COVID-19), “pandemic-associated-frailty” or profound health deterioration, in older adults has been considered a health concern. In this study, we sought to demonstrate whether pandemic-associated-frailty is occurring in Japan, where the population is aging, by showing the prevalence of frailty before and during the COVID-19 outbreak, using the same method.

Methods

A total of 5222 older adults in Otawara City, Tochigi Prefecture, aged 70 and 75 were surveyed annually using a complete survey, excluding those who were certified as requiring long-term care. Frailty during 2017–2019 before the COVID-19 outbreak and during 2020–2021 during the COVID-19 outbreak was determined using the Kihon Checklist (KCL). Statistical analysis was performed using the χ² test to compare annual frailty status and Kruskal–Wallis test to compare the scores.

Results

The frailty statuses over the five-year period showed a significant decrease in Robust and a significant increase in Pre-frailty and Frailty (p < 0.001). Frailty increased markedly during the second year of the pandemic. Based on the category, the scores deteriorated significantly for activities of daily living (p < 0.001), physical function (p = 0.003), oral function (p < 0.001), outdoor activity (p < 0.001), and depression (p < 0.001). Moreover, there was a significant deterioration in the total score for 25 items (p < 0.001). In addition, a significant deterioration was observed in the total score of 23 items, excluding the social withdrawal affected by self-restraint life (p < 0.001).

Conclusions

The population prevalence of frailty in older adults increased steadily from the pre-pandemic year through the first and second years of the pandemic. Based on the 25 questions of the KCL, two aspects including visiting friends and going out less stood out. This suggests that pandemic-associated-frailty occurred in Japan.