Chronic Heart Failure and Comorbid Renal Dysfunction - A Focus on Type 2 Cardiorenal Syndrome.

The most important advancements in the Cardiorenal syndrome (CRS) are its definition and subsequent classifications. When the predominant pathology and pathophysiology is the heart, i.e. chronic heart failure (CHF), and where any renal impairment (RI) subsequent to this is secondary, the classification is type 2 CRS. There are unique differences in the pathophysiology and progression of individual subclasses. It is important to understand the evolution of CHF and consequences of subsequent RI as they are becoming increasingly prevalent, aggravate morbidity and mortality and limit many therapeutic options. In this paper we discuss the significance of the type 2 CRS patients in the context of the thematic series.

Direct real-time quantitative PCR for measurement of host-cell residual DNA in therapeutic proteins.

Real-time quantitative PCR (qPCR) is important for quantification of residual host cell DNA (resDNA) in therapeutic protein preparations. Typical qPCR protocols involve DNA extraction steps complicating sample handling. Here, we describe a "direct qPCR" approach without DNA extraction. To avoid interferences of DNA polymerase with a therapeutic protein, proteins in the samples were digested with proteinase K (PK) in the presence of sodium dodecyl sulfate (SDS). Tween 20 and NaCl were included to minimize precipitation of therapeutic proteins in the PK/SDS mix. After PK treatment, the solution was applied directly for qPCR. Inhibition of DNA polymerase by SDS was prevented by adding 2% (v/v) of Tween 20 to the final qPCR mix. The direct qPCR approach was evaluated for quantification of resDNA in therapeutic proteins manufactured in Chinese hamster ovary (CHO) host cells. First, direct qPCR was compared with qPCR applied on purified DNA ("extraction qPCR"). For both qPCRs, the same CHO-specific primers and probes were used. Comparable residual DNA levels were detected with both PCR approaches in purified and highly concentrated drug proteins as well as in in-process-control samples. Finally, the CHO-specific direct qPCR protocol was validated according to ICH guidelines and applied for 25 different therapeutic proteins. The specific limits of quantification were 0.1-0.8ppb for 24 proteins, and 2.0ppb for one protein. General applicability of the direct qPCR was demonstrated by applying the sample preparation protocol for quantification of resDNA in therapeutic proteins manufactured in other hosts such as Escherichia coli and mouse cells.

Role of interferons in LPS hypersensitivity.

The innate immune response to Gram-negative bacteria depends mainly on the ability of the host to respond to the LPS component. Consequently, the state of LPS sensitivity at the time of infection and the numbers of invading bacteria (i.e. the amounts of LPS) are primary factors determining the innate responses provoked by Gram-negative pathogens. LPS sensitivity increases following treatment of mice with live or killed micro-organisms. Two types of sensitization have been recognized, strong, IFN-gamma-dependent and moderate IFN-gamma-independent. IL-12 and IL-18 are intimately involved in the induction of IFN-gamma by bacteria. We showed that Gram-negative bacteria induce IFN-gamma in mice also by an IFN-beta-dependent pathway that requires IL-18 and is independent of IL-12 signaling. This pathway is STAT4 dependent, the activation of which is directly linked to IFN-beta. Further, IFN-beta can be replaced by IFN-alpha. While different components of Gram-negative bacteria induce IL-12 and IL-18, LPS seems to be the only component in these bacteria capable of inducing IFN-beta. Therefore, the IFN-beta pathway of IFN-gamma induction, unlike the IL-12 pathway, proceeds only in LPS responder mice. The IFN-alpha/beta-dependent pathway is expected to play a role whenever IFN-alpha or IFN-beta, and IL-18 are produced concomitantly during infection.

Cutting edge: a murine, IL-12-independent pathway of IFN-gamma induction by gram-negative bacteria based on STAT4 activation by Type I IFN and IL-18 signaling.

IFN-alphabeta is a potent immunoregulatory cytokine involved in the defense against viral and bacterial infections. In this study, we describe an as yet undefined IFN-alphabeta-dependent pathway of IFN-gamma induction in mice. This pathway is based on a synergism of IFN-alphabeta and IL-18, and is independent of IL-12 signaling yet dependent on STAT4. In contradiction to current dogma, we show further that IFN-alphabeta alone induces tyrosine phosphorylation of STAT4 in murine splenocytes of different mouse strains. This pathway participates in the induction of IFN-gamma by Gram-negative bacteria and is therefore expected to play a role whenever IFN-alpha or IFN-beta and IL-18 are produced concomitantly during bacterial, viral, or other infections.

Expression and role of CD14 in mice sensitized to lipopolysaccharide by Propionibacterium acnes.

Propionibacterium acnes-primed mice develop an IFN-gamma-dependent hypersensitivity towards LPS. Since CD14 plays a key role in LPS-induced cell activation the regulation and function of CD14 in this sensitization process were studied in IFN-gamma R-/- and the respective wild-type (wt) mice. In unprimed mice, CD14 (mRNA and protein) was either absent (liver) or only weakly expressed in organs (spleen, lung) and in plasma. Priming with P. acnes led to a moderate, mainly IFN-gamma-dependent up-regulation of CD14. LPS challenge of unprimed mice induced an IFN-gamma-independent increase in CD14 mRNA and CD14 protein. LPS challenge of P. acnes-primed mice induced a strong CD14 overexpression. This response was completely absent in IFN-gamma R-/- mice and is therefore strictly IFN-gamma-dependent. The requirement for CD14 in LPS hyper-responsiveness was assessed by comparing CD14-/- and the respective wt mice with respect to their ability to produce TNF and IFN-gamma, two recognized indices of LPS activity. LPS challenge without priming led to a weaker cytokine reaction in CD14-/- than in wt mice. However, priming with P. acnes enhanced the cytokine response to LPS in both wt and CD14-/- mice, although in the latter absolute levels of cytokines were lower. Therefore, hyperreactivity to LPS is characterized by an up-regulation of CD14, but the sensitization by P. acnes is not CD14 dependent.