Publisher Summary This chapter discusses preparation of isolated rat liver cells The early mechanical and chemical methods for liver-cell preparation were relatively successful in converting liver tissue to a suspension of isolated cells The successful preparation of intact liver cells by perfusion with collagenase is technically quite difficult The major method for preparation of nonparenchymal liver cells is based on the selective sensitivity of parenchymal cells toward proteases By incubation of rat liver minces with pronase, most of the liver parenchyma is digested, while nonparenchymal cells remain intact and can be recovered from the incubate Similar results have been reported with trypsin digestion of collagenase-dispersed liver minces, but pronase appears to be more effective The most common procedure is to perfuse the liver briefly with pronase before it is minced and incubated with the enzyme Such direct pronase methods have been used by several investigators with yields of nonparenchymal liver cells reported to be in the range 2–15 × 10 6 cells/gm liver

Preparation of isolated rat liver cells.

The general view that only adaptive immunity can build immunological memory has recently been challenged. In organisms lacking adaptive immunity, as well as in mammals, the innate immune system can mount resistance to reinfection, a phenomenon termed "trained immunity" or "innate immune memory." Trained immunity is orchestrated by epigenetic reprogramming, broadly defined as sustained changes in gene expression and cell physiology that do not involve permanent genetic changes such as mutations and recombination, which are essential for adaptive immunity. The discovery of trained immunity may open the door for novel vaccine approaches, new therapeutic strategies for the treatment of immune deficiency states, and modulation of exaggerated inflammation in autoinflammatory diseases.

http://science.sciencemag.org/content/sci/352/6284/aaf1098.full.pdf

Trained immunity: A program of innate immune memory in health and disease

Immune memory is a defining feature of the acquired immune system, but activation of the innate immune system can also result in enhanced responsiveness to subsequent triggers. This process has been termed 'trained immunity', a de facto innate immune memory. Research in the past decade has pointed to the broad benefits of trained immunity for host defence but has also suggested potentially detrimental outcomes in immune-mediated and chronic inflammatory diseases. Here we define 'trained immunity' as a biological process and discuss the innate stimuli and the epigenetic and metabolic reprogramming events that shape the induction of trained immunity.

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Defining trained immunity and its role in health and disease

(1→3)-β-d-Glucans as biological response modifiers: a review of structure-functional activity relationships

The present review is focused on the main achievements realized in the lysozyme research field since the meeting held in 1972 to commemorate the fiftieth anniversary of the discovery of this enzyme. Despite of extensive structural, physico-chemical, crystallographic, genetic, immunological and evolutionary studies devoted to lysozymes, their biological role is still not exactly known.

What's new in lysozyme research? Always a model system, today as yesterday.

A soluble fraction of particulate glucan was prepared and evaluated for its anti-tumor and anti-bacterial activity. Thin-layer chromatographic analysis indicated that the soluble preparation was composed of a variety of polyglucoses. Intravenous administration of soluble or particulate glucan resulted in significant reductions in the growth of a syngeneic anaplastic mammary carcinoma and melanoma B16. Survival data demonstrated that intravenous administration of soluble or particulate glucan prolonged survival of A/J and C57BL/6J mice with subcutaneous tumor implants. As regards to bacterial infections, soluble and particulate glucan decreased renal necrosis in S. aureus challenged mice as compared to control mice. Although the exact nature of the active soluble fraction(s) of glucan remains to be delineated, these studies demonstrate that a soluble glucan preparation exhibits significant anti-tumor and anti-staphylococcal activity. The active soluble fraction of particulate glucan may be preferable to particulate glucan in view of the inherent ease of parenteral administration.

Comparative tumor‐inhibitory and anti‐bacterial activity of soluble and particulate glucan

Glucan is a potent reticuloendothelial stimulant whose immunobiological activity is mediated, in part, by an increase in the number and function of macrophages. In studying the role of glucan as a mediator of antibacterial activity, we attempted to ascertain the ability of glucan to modify the mortality of mice with experimentally induced Gram-positive bacteremia, and to enhance antibacterial defenses in rats as denoted by serum lysozyme and phagocytic activity. After intravenous administration of glucan, serum lysozyme concentrations were increased approximately sevenfold over control concentrations. The increase in serum lysozyme appeared to parallel the glucan-induced increase in phagocytosis and induced hyperplasia of macrophages. Prior treatment of mice with glucan significantly enhanced their survival when they were challenged systemically with Staphylococcus aureus. These studies indicate that glucan confers an enhanced state of host defense against bacterial infections.

https://science.sciencemag.org/content/sci/199/4335/1340.full.pdf

Increased resistance to Staphylococcus aureus infection and enhancement in serum lysozyme activity by glucan

We conducted studies with mice, rats, and monkeys which demonstrated the ability of glucan to induce either nonspecific or specific enhancement of host resistance to infectious diseases. Intravenous pretreatment of mice with glucan significantly enhanced the survival of mice challenged with either Venezuelan equine encephalomyelitis (VEE) virus or Rift Valley fever virus. Pretreatment was beneficial when initiated 3 days before challenge with VEE virus and 7 days before challenge with Rift Valley fever virus. Treatment of mice after VEE challenge did not increase their survival compared with controls. Glucan pretreatment of rats provided increased resistance to both intraperitoneal and low-dose aerosol challenges with virulent Francisella tularensis when the glucan was given intravenously, but not when it was administered intranasally. In contrast, intranasal glucan pretreatment enhanced the survival of mice when they were challenged by aerosol with Pseudomonas pseudomallei, whereas intravenous glucan pretreatment did not increase survival. mice given glucan combined with a marginally immunogenic dose of VEE vaccine were more resistant to homologous virus challenge than were mice given either Freund complete adjuvant plus vaccine or vaccine alone. Similarly, both primary and secondary VEE antibody titers in cynomolgus monkeys given glucan with VEE vaccine were significantly greater than titers in vaccine controls.

Glucan-induced enhancement of host resistance to selected infectious diseases.

The reticuloendothelial stimulant glucan, a beta-1,3-polyglucose component of the cell wall of Saccharomyces cerevisiae, was evaluated for its ability to modify Staphylococcus aureus-induced lethality in normal and leukemic mice. In normal mice the intravenous injection of glucan (0.45 mg per mouse) 7 and 4 days prior to intravenous challenge with S. aureus (1.0 x 10(9)) resulted in a significantly increased survival. Histological examination of the kidneys revealed that glucan significantly inhibited renal necrosis associated with systemic staphylococcal diseases. Further studies indicated that glucan administration not only enhanced survival of leukemic mice, but also increased survival of leukemic mice with experimentally induced staphylococcal speticemia. These data denote that glucan enhances nonspecific resistance to S. aureus sepsis, promotes survival during leukemic episodes, and increases survival time of leukemic mice with experimentally induced staphylococcal infection.

Protective effect of glucan against systemic staphylococcus aureus septicemia in normal and leukemic mice.

Glucan, a macrophage stimulant, was evaluated for its ability to alter survival and phagocytic dysfunction in mice challenged with mouse hepatitis virus strain MHV-A59. Administration of glucan before the mice were challenged with the virus significantly prolonged median survival time but did not modify overall mortality compared with control mice given dextrose. Maximal effectiveness was achieved when glucan was administered both before and after the viral challenge. In contrast to the marked hepatic parenchymal cell necrosis observed in the control mice, glucan-treated mice exhibited reduced pathology. Intraperitoneal administration of MHV-A59 resulted in a significant depression of phagocytic activity compared with controls that were not exposed to the virus. The enhancement in phagocytic function in glucan-treated control mice was unaltered in virus-challenged, glucan-treated mice. Thus glucan is capable of increasing survival, inhibiting hepatic necrosis, and maintaining an activated state of phagocytic activity in mice challenged with MHV-A59. Macrophage stimulants may have a significant role in the modification of virally induced hepatic lesions.

N. R. Di Luzio

Papers

Comparative tumor‐inhibitory and anti‐bacterial activity of soluble and particulate glucan

Increased resistance to Staphylococcus aureus infection and enhancement in serum lysozyme activity by glucan

Glucan-induced enhancement of host resistance to selected infectious diseases.

Protective effect of glucan against systemic staphylococcus aureus septicemia in normal and leukemic mice.

Glucan-induced modification of murine viral hepatitis.