This simple, universally applicable staging system stratifies patients with both ATTRwt and ATTRv amyloid cardiomyopathy into prognostic categories. It will be of value in the design of forthcoming clinical trials of novel amyloid-specific therapies.

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Antibody-drug conjugates (ADCs) utilizing cysteine-directed linker chemistry have cytotoxic drugs covalently bound to native heavy-heavy and heavy-light interchain disulfide bonds. The manufacture of these ADCs involves a reduction step followed by a conjugation step. When tris(2-carboxyethyl)phosphine (TCEP) is used as the reductant, the reaction stoichiometry predicts that for each molecule of TCEP added, one interchain disulfide should be reduced, generating two free thiols for drug linkage. In practice, the amount of TCEP required to achieve the desired drug-to-antibody ratio often exceeds the predicted, and is variable for different lots of monoclonal antibody starting material. We have identified the cause of this variability to be inconsistent levels of interchain trisulfide bonds in the monoclonal antibody. We propose that TCEP reacts with each trisulfide bond to form a thiophosphine and a disulfide bond, yielding no net antibody free thiols for conjugation. Antibodies with higher levels of trisulfide bonds require a greater TCEP:antibody molar ratio to achieve the targeted drug-to-antibody ratio.

During production of therapeutic monoclonal antibodies (mAb), it is highly desirable to remove and control antibody aggregates in the manufacturing process to minimize the potential risk of immunogenicity to patients. During process development for the production of a recombinant IgG in a CHO cell line, we observed atypical high variability from 1 to 20% mAb aggregates formed during cell culture that negatively impacted antibody purification. Analytical characterization revealed the IgG aggregates were mediated by hydrophobic interactions likely caused by misfolded antibody during intracellular processing. Strikingly, data analysis showed an inverse correlation of lower cell culture temperature producing higher aggregate levels. All cultures at 37°C exhibited ≤ 5% aggregates at harvest. Aggregate levels increased 4-12-fold in 33°C cultures when compared to 37°C, with a corresponding 2-4-fold increase in heavy chain (HC) and light chain (LC) mRNA. Additionally, 37°C cases showed a greater excess of LC to HC mRNA levels. Endoplasmic reticulum (ER) chaperone expression and ER size also increased 25-75% at 33°C versus 37°C but to a lesser extent than LC and HC mRNA, consistent with a potential limiting ER folding capacity at 33°C for this cell line. Finally, we identified a 2-5-fold increase in mAb aggregate formation at 33°C compared to 37°C cultures for three additional CHO cell lines. Taken together, our observations indicate that low culture temperature can increase antibody aggregate formation in CHO cells by increasing LC and HC transcripts coupled with limited ER machinery.

Debate continues regarding the influence of dietary fats and sugars on the risk of developing metabolic diseases, including insulin resistance and nonalcoholic fatty liver disease (NAFLD). We investigated the effect of two eucaloric diets, one enriched with saturated fat (SFA) and the other enriched with free sugars (SUGAR), on intrahepatic triacylglycerol (IHTAG) content, hepatic de novo lipogenesis (DNL), and whole-body postprandial metabolism in overweight males. RESEARCH DESIGN AND METHODSSixteen overweight males were randomized to consume the SFA or SUGAR diet for 4 weeks before consuming the alternate diet after a 7-week washout period. The metabolic effects of the respective diets on IHTAG content, hepatic DNL, and wholebody metabolism were investigated using imaging techniques and metabolic substrates labeled with stable-isotope tracers. RESULTSConsumption of the SFA diet significantly increased IHTAG by mean 6 SEM 39.0 6 10.0%, while after the SUGAR diet IHTAG was virtually unchanged. Consumption of the SFA diet induced an exaggerated postprandial glucose and insulin response to a standardized test meal compared with SUGAR. Although whole-body fat oxidation, lipolysis, and DNL were similar following the two diets, consumption of the SUGAR diet resulted in significant (P < 0.05) decreases in plasma total, HDL, and non-HDL cholesterol and fasting b-hydroxybutyrate plasma concentrations. CONCLUSIONSConsumption of an SFA diet had a potent effect, increasing IHTAG together with exaggerating postprandial glycemia. The SUGAR diet did not influence IHTAG and induced minor metabolic changes. Our findings indicate that a diet enriched in SFA is more harmful to metabolic health than a diet enriched in free sugars.Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver-related conditions, ranging from steatosis (characterized by an accumulation of intrahepatic triacylglycerol [IHTAG]) to nonalcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma, and is the most prevalent liver disease worldwide (1). There appears to exist a bidirectional relationship between NAFLD and metabolic disease; the presence

Haemophagocytic lymphohisticytosis (HLH) is a syndrome of uncontrolled, severe systemic inflammation (hyperinflammation) arising either from a genetic immune system defect [primary (pHLH)] or triggered as a complication of malignancy, infection, or rheumatologic disease [secondary (sHLH)]. Patients with HLH often have non-specific symptoms and become progressively and critically unwell, with fever, cytopenia and multi-organ failure. Untreated, HLH is almost universally fatal, but even when treated, mortality is high, particularly when HLH complicates malignancy. HLH is managed with immunosuppression, and this can seem difficult to justify in such unwell patients. This review aims to examine the diagnostic and treatment challenges posed by sHLH and to improve recognition among rheumatologists who, being expert in the management of multisystem diseases and in the use of immunosuppression, are ideally placed to deliver care and build an evidence base for better disease characterization and treatment.