Tumor necrosis factor induces acute phase proteins in rats

Inoculation of WAG rats with recombinant mouse tumor necrosis factor results in a rapid and marked increase in several acute phase proteins in the serum (haptoglobin, alpha 1 acid glycoprotein, alpha 2 macroglobulin) and in the plasma (fibrinogen). We conclude that TNF may play an important role in the inflammatory response in vivo and possibly in the pathogenesis of inflammatory disorders.     

Measurement of serum amyloid A protein concentrations as test of renal allograft rejection in patients with initially non-functioning grafts.

Serum amyloid A protein concentrations were monitored in 10 renal transplant recipients who required dialysis after transplantation because of an initially non-functioning graft. Fifteen rejection episodes were identified by repeated fine needle aspiration biopsies of the grafts. All rejections were characterised by pronounced increases in serum amyloid A concentrations, the mean peak value being 363 (SE 57) mg/1 as compared with a mean preoperative concentration of 14 (5) mg/1. The rise in concentrations preceded the start of anti-rejection treatment by an average of 2.5 days in eight of the rejection episodes, in five episodes it occurred the same day, and in two episodes it occurred the next day. With exclusion of the predictable surgery induced rise in values, which peaked on the second postoperative day, there were 17 increases in amyloid A concentrations peaking at greater than or equal to 100 mg/1; in two cases they were not related to documented rejection. These findings show that measurements of serum amyloid A concentration provide a valuable non-invasive aid in identifying acute renal allograft rejection, including that in patients whose graft does not function initially.     

Correlation of persistently high serum amyloid A protein and C-reactive protein concentrations with rapid progression of secondary amyloidosis.

The importance of serum amyloid A protein in the progression of renal failure was studied over three years in 28 patients with secondary (amyloid A type) amyloidosis predominantly due to rheumatoid arthritis. Creatinine clearance, the amount of protein in the urine, and serum amyloid A and C-reactive protein concentrations were determined regularly. Linear regression analysis showed a close correlation between the change in creatinine clearance each year and both serum amyloid A concentrations (20 patients: r= -0.83, p less than 0.001) and C-reactive protein concentrations (28 patients: r= -0.80, p less than 0.001). The correlation between serum amyloid A and C-reactive protein concentrations was also significant (317 parallel measurements: r=0.81, p less than 0.001). These findings suggest that monitoring serum amyloid A or C-reactive protein concentrations is valuable in assessing the prognosis in secondary amyloidosis and that therapeutic measures that lower serum amyloid A concentrations may reduce the formation of amyloid.     

Adoptive chemoimmunotherapy of a syngeneic murine lymphoma with long-term lymphoid cell lines expanded in T cell growth factor

Summary Recently techniques have been developed for the long-term growth of cytotoxic T-lymphoid cells in vitro with T cell growth factor (TCGF). We have investigated the use of these in vitro-expanded T cells for the immunotherapy of a disseminated syngeneic murine FBL-3 lymphoma. In this model, mice with disseminated tumor were treated on day 5 with 180 mg cytoxan/kg and then 5 h later were given lymphoid cells IP. In vivo-immunized lymphocytes resulted in significantly improved survival in three of three experiments, curing 52% of 38 animals, compared with treatment with cytoxan alone (0 of 31 cured) or cytoxan plus unimmunized cells (0 of 40 cured) (P<0.0005). In vivo-immunized lymphocytes were re-exposed to FBL-3 tumor in vitro for 5 days in complete medium (CM) or lectin-free TCGF (LF-TCGF). Both groups showed significantly improved survival in six of six experiments. Cytoxan cured 17% of 66 animals, while cytoxan plus normal lymphocytes after IVS cured 6% of 47 animals. In vivo-immunized cells resensitized in vitro to FBL-3 in CM or LF-TCGF cured 82% of 50 animals (P<0.001) and 72% of 61 animals (P<0.001), respectively. Cells from in vivo- and in vitro-sensitized lymphocytes exhibited no cytotoxicity in our in vitro ⁵¹Cr-release assay; expansion of these cells resulted in significant specific lysis of fresh FBL-3 targets. Adoptive transfer of immune lymphocytes resensitized to FBL-3 tumor in vitro and expanded in LF-TCGF conferred a significant survival benefit (P<0.001, curing 7 of 27 animals) compared with all controls. These expanded cells were then continuously grown in LF-TCGF for 2 1/2 months. Again, in vivo-immunized lymphocytes resensitized to FBL-3 tumor and expanded in LF-TCGF for 2 1/2 months cured 56% of the animals with disseminated tumor, significantly prolonging survival over that recorded in any control group (P<0.0002). Irradiation of these same cells totally abolished their efficacy. Clones were generated from IVS and continuously grown in LF-TCGF. Two of these clones were very cytotoxic for fresh FBL-3 (>4,000 lytic units/10⁶ cells). When adoptively transferred to mice in this chemoimmunotherapy model these cytotoxic clones significantly enhanced survival over that recorded following treatment with cytoxan alone (P<0.00001), though prolongation of survival was small. Implications of these results for application of these techniques to other less antigenic tumors and human cancers are discussed.     

Modelling 3D control of upright stance using an optimal control strategy

A 3D balance control model of quiet upright stance is presented, based on an optimal control strategy, and evaluated in terms of its ability to simulate postural sway in both the anterior–posterior and medial–lateral directions. The human body was represented as a two-segment inverted pendulum. Several assumptions were made to linearise body dynamics, for example, that there was no transverse rotation during upright stance. The neural controller was presumed to be an optimal controller that generates ankle control torque and hip control torque according to certain performance criteria. An optimisation procedure was used to determine the values of unspecified model parameters including random disturbance gains and sensory delay times. This model was used to simulate postural sway behaviours characterised by centre-of-pressure (COP)-based measures. Confidence intervals for all normalised COP-based measures contained unity, indicating no significant differences between any of the simulated COP-based measures and corresponding experimental references. In addition, mean normalised errors for the traditional measures were < 8%, and those for most statistical mechanics measures were ～3–66%. On the basis these results, the proposed 3D balance control model appears to have the ability to accurately simulate 3D postural sway behaviours.     

Role of the Phosphatidylserine Receptor TIM-1 in Enveloped-Virus Entry

The cell surface receptor T cell immunoglobulin mucin domain 1 (TIM-1) dramatically enhances filovirus infection of epithelial cells. Here, we showed that key phosphatidylserine (PtdSer) binding residues of the TIM-1 IgV domain are critical for Ebola virus (EBOV) entry through direct interaction with PtdSer on the viral envelope. PtdSer liposomes but not phosphatidylcholine liposomes competed with TIM-1 for EBOV pseudovirion binding and transduction. Further, annexin V (AnxV) substituted for the TIM-1 IgV domain, supporting a PtdSer-dependent mechanism. Our findings suggest that TIM-1-dependent uptake of EBOV occurs by apoptotic mimicry. Additionally, TIM-1 enhanced infection of a wide range of enveloped viruses, including alphaviruses and a baculovirus. As further evidence of the critical role of enveloped-virion-associated PtdSer in TIM-1-mediated uptake, TIM-1 enhanced internalization of pseudovirions and virus-like proteins (VLPs) lacking a glycoprotein, providing evidence that TIM-1 and PtdSer-binding receptors can mediate virus uptake independent of a glycoprotein. These results provide evidence for a broad role of TIM-1 as a PtdSer-binding receptor that mediates enveloped-virus uptake. Utilization of PtdSer-binding receptors may explain the wide tropism of many of these viruses and provide new avenues for controlling their virulence.     

Glucose‐based microbial production of the hormone melatonin in yeast Saccharomyces cerevisiae

Melatonin is a natural mammalian hormone that plays an important role in regulating the circadian cycle in humans. It is a clinically effective drug exhibiting positive effects as a sleep aid and a powerful antioxidant used as a dietary supplement. Commercial melatonin production is predominantly performed by complex chemical synthesis. In this study, we demonstrate microbial production of melatonin and related compounds, such as serotonin and N‐acetylserotonin. We generated Saccharomyces cerevisiae strains that comprise heterologous genes encoding one or more variants of an L‐tryptophan hydroxylase, a 5‐hydroxy‐L‐tryptophan decarboxylase, a serotonin acetyltransferase, an acetylserotonin O‐methyltransferase, and means for providing the cofactor tetrahydrobiopterin via heterologous biosynthesis and recycling pathways. We thereby achieved de novo melatonin biosynthesis from glucose. We furthermore accomplished increased product titers by altering expression levels of selected pathway enzymes and boosting co‐factor supply. The final yeast strain produced melatonin at a titer of 14.50 ± 0.57 mg L^?1 in a 76h fermentation using simulated fed‐batch medium with glucose as sole carbon source. Our study lays the basis for further developing a yeast cell factory for biological production of melatonin.     

Delayed inflammation decrease is associated with mortality in Tocilizumab-treated critically ill SARS-CoV-2 patients: A retrospective matched-cohort analysis

Little is known about the immuno-inflammatory response to Tocilizumab and its association with outcome in critically-ill SARS-CoV2 pneumonia. In this multicenter retrospective cohort of SARS-CoV-2 patients admitted to three intensive care units between March and April 2020, we matched on gender and SAPS II 21 Tocilizumab-treated patients to 42 non-treated patients. Need for mechanical ventilation was 76% versus 79%. IL-6, C-reactive protein, and fibrinogen had been collected within the first days of admission (T1), 3 d (T2) and 7 d (T3) later. Tocilizumab-treated patients had persistently higher IL-6 plasma levels and persistently lower C-Reactive protein and fibrinogen levels. Among Tocilizumab-treated patients, baseline levels of inflammatory biomarkers were not different according to outcome. Conversely, C-reactive protein and fibrinogen decrease was delayed in non-survivors. C-Reactive protein decreased at T1 in survivors (45 [30–98] vs 170 [69–204] mg/l, P < 0.001) but only at T2 in non-survivors (37 [13–74] vs 277 [235–288], P = 0.03). Fibrinogen decreased at T2 in survivors (4.11 [3.58–4.69] vs 614 [5.61–7.85] g/l, P = 0.005) but not in non-survivors (4.79 [4.12–7.58] vs 7.24 [6.22–9.24] g/l, P = 0.125). Tocilizumab treatment was thus associated with a persistent both increase in plasma IL-6, and decrease in C-reactive protein and fibrinogen. Among Tocilizumab-treated patients, the decrease in inflammatory biomarkers was delayed in non-survivors.     

Low enantioselectivities of human deoxycytidine kinase and human deoxyguanosine kinase with respect to 2'-deoxyadenosine, 2'-deoxyguanosine and their analogs

The antiviral activity of L-nucleoside analogs depends in part on the enantioselectivity of nucleoside kinases which catalyse their monophosphorylation. The substrate properties of human recombinant deoxycytidine kinase (dCK) and human recombinant deoxyguanosine kinase (dGK) with respect to L-adenosine and L-guanosine analogs, in the presence of saturating amounts of ATP and relatively high concentrations of substrates, demonstrated a marked lack of enantioselectivity of both these enzymes. Human dCK catalysed the phosphorylation of D- and L-enantiomers of beta-dA, beta-araA, and beta-dG with enantioselectivities favoring the unnatural enantiomer for the adenosine derivatives and the natural enantiomer for 2'-deoxyguanosine. No other tested L-adenosine or L-guanosine analog was a substrate of dCK. Similarly, D- and L-enantiomers of beta-dA, beta-araA, and beta-dG were substrates of human dGK but with different enantioselectivities compared to dCK, especially concerning beta-dA. The present results indicate that human dCK and dGK have similar properties including substrate properties, relaxed enantioselectivities, and possibly catalytic cycles.     

Unnatural enantiomers of 5-azacytidine analogues: syntheses and enzymatic properties

2'-Deoxy-beta-L-5-azacytidine(L-Decitabine), beta-L-5-azacytidine, and derivatives were stereospecifically prepared starting from L-ribose or L-xylose. D- and L-enantiomers of 2'-deoxy-beta-5-azacytidine were weak substrates of human recombinant deoxycytidine kinase (dCK), whereas both enantiomers of beta-5-azacytidine or the L-xylo-analogues were not substrates of the enzyme. None of the reported derivatives of beta-L-5-azacytidine was a substrate of human recombinant cytidine deaminase (CDA).