Case 46

Published on 18/02/2015 by admin

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CASE 46

Anne is 20 months of age and has been hospitalized with a fever, irritability, and moderate dehydration. Blood work suggests a bacterial infection, with elevated neutrophils (see Appendix for reference value), although chest radiograph and urinalysis are normal. A lumbar puncture taken yesterday showed an elevated white blood cell count, and preliminary culture results today suggest growth of Neisseria meningitidis. She has been receiving intravenous antibiotics for 30 hours, and there is some improvement in her condition. Physical examination indicated that Anne was below the 50th percentile for weight, had recurrent diarrhea, and had seborrheic dermatitis, more commonly referred to as “cradle cap.” Detailed question of Anne’s clinical history revealed that she had had recurrent infections, but none had ever been this bad. Detailed family history reveals that a distant male cousin and an aunt both died at an early age (<2 years) of meningitis. What further tests might you order? What is the significance of the history and findings?

QUESTIONS FOR GROUP DISCUSSION

1. Bacterial infections characterize defects in B cells, complement, or phagocytes. Explain why a major B cell defect (e.g., agammaglobulinemia) is unlikely in this case. Discuss how you would test for defects in phagocytes. To test for complement defects you would measure overall complement activity using the CH50 test. Assuming that the results of this test show a complement defect, how would you proceed? What single test would allow you to determine if the defect is in the terminal pathway or the classical pathway?

2. Because preliminary culture results suggested growth of Neisseria meningitidis, Anne was diagnosed with systemic inflammatory response syndrome (SIRS), formerly referred to simply as “sepsis” during infection. Review the processes that are activated during inflammation (see Case 23), with emphasis on the role of interleukin (IL)-1 and tumor necrosis factor (TNF). Note that the current dogma states that SIRS leads to the excessive release of cytokines that produce fever, shock, and often death.

3. Infections with Gram-negative bacteria lead to their lysis, release of lipopolysaccharide (LPS/endotoxin) from the cell wall outer membrane, and subsequent secretion of TNF by monocytes and macrophages. Describe how these events are connected. Include in your discussion each of the following terms: lipopolysaccharide binding protein (LBP), soluble CD14, MD-2, and TLR-4. How would you explain the fact that in animal models of endotoxin-induced peritonitis and in human SIRS trials blocking both TNF and TNF antagonists increased mortality?

4. With respect to question 4, how would you explain the following: (a) in humans, mortality associated with meningococcemia correlates with high TNF levels; (b) in SIRS patients (infected with Gram-negative bacteria), antagonists of IL-1 receptor, TNF, and endotoxin do not improve outcome; and (c) mice that have mutations in the TLR-4 gene have increased mortality to SIRS induced by infection with Gram-negative bacteria.

5. Review the differentiation of Thp cells to Th1 or Th2 cells secreting type 1 and type 2 cytokines, respectively. List these cytokines. What factors influence the polarization of a response to either a Th1 or a Th2 cell? An increased level of IL-10 in SIRS patients is a predictor of mortality. What is the role of IL-10 in the regulation of Thp cell activation? What is the consequence of increased IL-10 on the production of type 1 cytokines by Th1 cells?

6. Antigen presentation by macrophages after phagocytosis of necrotic cells polarizes the Thp differentiation to that of a Th1 phenotype, whereas phagocytosis of apoptosed cells polarizes the response to that of a Th2 phenotype. Patients who died of SIRS have evidence for apoptosis-induced loss of immune cells. What cytokines would you expect to be increased in these patients? What effect would this have on the production of type 1 cytokines? Based on this information, why would apoptosis-induced death of immune cells be a predictor of mortality?

7. Explain why neutrophils are regarded as “double-edged swords” in SIRS. What is the rationale for the administration of the cytokine granulocyte colony-stimulating factor (G-CSF)? Explain the role of activated protein C in the extrinsic coagulation system. How do you reconcile the use of recombinant protein C with studies in which G-CSF increased survival rates in patients with SIRS? How would you rationalize the use of this therapeutic agent with studies that showed that antigen presentation by macrophages that have ingested apoptosed cells leads to the induction of Th2 cells secreting type II cytokines (e.g., interleukin [IL]-10) and the fact that increased IL-10 is an independent predictor of mortality?

8. Intensive insulin therapy has been shown to result in lower morbidity and mortality in patients with SIRS. Although the mechanism of action is unknown, insulin prevents apoptotic death of cells. What effect would this have on IL-10 levels? Discuss the studies that could be used to rationalize the use of insulin therapy.

9. Newer therapies for SIRS that are under consideration include interferon gamma (IFNγ), IL-12, and C5a. Provide a rationale for each of these as potential therapeutic agents.

10. Ischemic preconditioning is a term used to describe a phenomenon in which a transient episode of myocardial ischemia subsequently protects the individual from severe myocardial ischemia. Extrapolate this concept to SIRS patients who survive SIRS sepsis.

RECOMMENDED APPROACH

Implications/Analysis of Family History

Detailed family history revealed that a distant male cousin and an aunt both died at an early age (<2 years) of meningitis. The fact that this disease susceptibility is so rare in the family suggests that we should be considering disorders with an autosomal recessive mode of inheritance.

Implications/Analysis of Clinical History

Anne’s immediate problem is the Neisseria meningitidis infection, which is the cause of the overwhelming sepsis, now referred to as systemic inflammatory response syndrome (SIRS). Although deficiencies in B cells, phagocytes, and complement are considerations in patients with severe bacterial infections, the fact that this is a Neisseria infection suggests that we should be focusing our attention on possible deficiencies in the terminal pathway of complement. Patients with deficiencies in the proteins that compose the membrane attack complex are particularly susceptible to infections with Neisseria species (Fig. 46-1).

FIGURE 46-1 Neisseria gonorrhoeae. Gram stain of urethral pus from a patient with gonorrhea. Note that the diplococci occur mostly intracellularly. (×1000.)

(From Hart CA, Shears P: Color Atlas of Medical Microbiology, 2nd ed. St. Louis, Mosby, 2004.)

Note, however, that in addition to Anne’s immediate problem of SIRS, she has a triad of symptoms—recurrent diarrhea, seborrheic dermatitis (which often manifests as cradle cap), and failure to thrive. Therefore, we should be attempting to determine if there is a defect that will account for both SIRS and the symptoms described.

Implications/Analysis of Laboratory Investigation

Results of blood work and culture were consistent with a strong inflammatory response to systemic infection with Neisseria meningitidis. Although the immediate problem facing Anne and her physician is SIRS, it is important to find the underlying problem for the uncontrolled spread of this infection. The most widely used test to screen for deficiencies in the classical or terminal pathway is the CH50 test. Low levels of CH50 indicate that there is a deficiency in either the classical or the terminal pathway. If the defect is in the terminal pathway, an AH50 test will indicate a deficiency as well.

In Anne’s case both the AH50 and CH50 were low, indicating that the defect was in one of the proteins that make up the terminal pathway. Further laboratory tests would be required to identify the particular component that is defective. (Note: the AH50 test is only available in specialized laboratories.)

Additional Laboratory Tests

Although one could request a measure of each of the terminal pathway components, Anne’s clinical history and results of the physical examination suggest that she may have a deficiency in C5. Patients with deficiencies in C5 present with Leiner’s disease, which is characterized by recurrent diarrhea, seborrheic dermatitis (which often manifests as cradle cap), and failure to thrive. Anne’s serum sample was sent to a specialized laboratory to test for a defect in C5. (C5 is only measured in specialized laboratories.)

DIAGNOSIS

Anne was diagnosed with a deficiency of C5, also referred to as Leiner’s disease. A deficiency in C5 is a risk factor for infections with Neisseria species and SIRS. Neisseria meningitidis, a Gram-negative bacterium, is surrounded by a polysaccharide capsule whose composition defines the serogroup of the pathogen. This capsule prevents phagocytosis of this pathogen in the absence of opsonins. Five serogroups (A, B, C, Y, and W-135) cause most of the infections in North America. (Note: C5b, which is a proteolytic fragment of C5, is a component of the membrane attack complex.)

THERAPY

Anne was treated with antibiotics, along with symptomatic therapy as required. Additionally, Anne should be immunized, recognizing that immunization will only provide protection if the vaccine contains the same polysaccharide capsule serotype as that of the infecting pathogen.

Immunization: Polysaccharide versus Conjugate Vaccines

There are two types of meningococcal vaccines available: a polysaccharide vaccine and a conjugate vaccine. The polysaccharide vaccine (e.g., Menomune, Mencevax) consists of purified meningococcal polysaccharide from serogroups A, C, Y, and W135 but is only effective when administered to individuals over 2 years of age. Protection is relatively short term: 2 to 3 years. In contrast, the conjugate vaccine (e.g., Neis Vac-C, Menjugate) is effective even when administered to children 6 weeks of age, but it consists solely of meningococcal polysaccharide serotype C capsule chemically linked to tetanus toxoid.

Limitations of Meningococcal Vaccines

There is no effective vaccine for protection against serogroup B polysaccharide capsule, which is weakly immunogenic. Unfortunately, serotype B is the most common cause of meningococcal disease in young children and there is no cross-reacting protection from vaccines for other polysaccharide capsule serotypes.

ETIOLOGY: SIRS

Sepsis represents a leading cause of death/disability in the United States, affecting nearly a million people annually, with approximately one fourth of that number dying. Sepsis itself represents an uncontrolled inflammatory response, as witnessed in the Lewis Thomas notion that “the microorganisms that seem to have it in for us … turn out … to be rather more like bystanders. … It is our response to their presence that makes the disease. Our arsenals for fighting off bacteria are so powerful … that we are more in danger from them than the invaders.” Accordingly, sepsis itself is now often referred to instead as SIRS, the systemic inflammatory response syndrome, which occurs during infection, particularly with Gram-negative bacteria. In essence, the immune system goes into overdrive, releasing a flood of cytokines that produce fever, shock, and often death.

Inflammation

Inflammation occurring in response to trauma or infection is complex, involves a number of cells, adhesion molecules, cytokines, chemokines, complement proteins, and coagulation proteins. Many of the processes occurring in inflammation are triggered following the release of tumor necrosis factor (TNF) and IL-1 from activated macrophages. Infections with Gram-negative bacteria lead to the activation of immunologic processes that lead to lysis of bacteria and release of lipopolysaccharide (LPS/endotoxin) from the cell wall outer membrane and subsequent release of TNF from activated macrophages. Release of TNF from activated macrophages occurs after binding of endotoxin-MD-2 complex with toll-like receptor-4 (TLR-4). Complex formation and signaling via TLR-4 occurs only if endotoxin undergoes interactions with, and transfer from, LPS-binding protein (LBP) and soluble CD14. Whether these sequential bindings and transfer modify endotoxin so that it is able to form a complex with MD-2 and TLR-4 is unknown. Signaling via TLR-4, however, activates biochemical pathways that lead to the synthesis and secretion of TNF. Therefore, it is not surprising that TLR-4 mutations in humans make individuals more susceptible to Gram-negative infection. (See Case 7)

Role of Inflammation in SIRS

An awareness of the role of TNF and IL-1 in inflammation has spawned a variety of trials destined to attempt to control the disorder, all including the use of agents that should block the inflammatory cascade, including, but not limited to, corticosteroids, anti-endotoxin antibodies, TNF antagonists, and IL-1 receptor blockage. In general, however, the results of such trials of anti-inflammatory mediators have been poor, leading clinicians to question whether death in patients with sepsis results from uncontrolled inflammation. However, exhaustive meta-analysis of clinical trials using anti-inflammatory agents in SIRS patients has shown that although high doses of anti-inflammatory agents can be harmful in such patients, there is at least a subgroup of patients (∼10%) showing a clear benefit. It must be noted, however, that clinical trials of treatments for sepsis are difficult to interpret for many reasons, not the least of which is the heterogeneity of patients and the high rates of culture-negative sepsis.

Evidence Supporting a Role for TNF in SIRS

The theory that sepsis caused death after an uncontrolled stimulation of the immune system came essentially from animal studies, often using high doses of endotoxin or bacteria that induced levels of circulating cytokines (e.g., TNF) far higher in animals than those seen in patients with SIRS. And, in at least one sepsis syndrome in humans—meningococcemia—circulating TNF levels are high and do indeed correlate with mortality.

Evidence Challenging a Role for TNF in SIRS

There is confounding evidence now that cytokines can have beneficial effects in sepsis: (1) in animal models of peritonitis, blocking TNF worsened survival; (2) in a recent clinical trial, a TNF antagonist increased mortality; (3) a strain of mice, C3H/HEJ, is resistant to endotoxin because of a mutation in their TLR-4 gene, but, interestingly, despite their resistance to endotoxin, these mice have increased mortality from SIRS; and (4) recent evidence suggests that TLR-4 mutations in humans may similarly make individuals more susceptible to infection.

Differentiation of Thp to Th1 or Th2 Cells

CD4+ Thp cell differentiation is initiated when these cells interact with antigen-presenting cells that display major histocompatibility complex (MHC) class II antigen peptide and appropriate co-stimulatory molecules on their surface. Thp cells differentiate to either Th1 cells secreting type 1 cytokines or Th2 cells secreting type 2 cytokines. The factors controlling which type of Th cell is activated are complex, but the polarization in T cell cytokine synthesis is known to be influenced by (1) the type of pathogen, (2) the size of the bacterial inoculum, (3) the site of infection, and (4) the type of macrophage stimulation (see later). Type 1 cytokines, which include TNF, interferon gamma (IFNγ), and IL-2, have inflammatory properties; type 2 cytokines (e.g., IL-4 and IL-10) have anti-inflammatory properties.

Type of Macrophage Stimulation in Polarization to Th1 or Th2 cells

Macrophages, antigen-presenting cells, that ingest necrotic tissue release IL-12, a cytokine that promotes differentiation of Thp cells to Th1 cells. Th1 cells secrete type 1 cytokines, which are proinflammatory. In contrast, ingestion of apoptosed cells leads to the induction of Th2 cells secreting type 2 cytokines, which are anti-inflammatory cytokines. (Apoptosis is genetically programmed cell death in which the cells “commit suicide” after the activation of various proteases.)

Regulation of Type 1 and Type 2 Cytokines

Type 1 and type 2 cytokines reciprocally regulate cytokine secretion. Therefore, high levels of IL-10 would lead to a decrease in type 1 cytokine production (i.e., inflammatory cytokines), supporting studies in which anti-inflammatory agents did not rescue patients with SIRS. Further support for a positive role for proinflammatory cytokines comes from studies in which mononuclear cells from patients with sepsis (secondary to burns or trauma) have reduced type 1 and increased type 2 levels of cytokines and reversal of the dominant cytokine profile using IL-12 reduced mortality. IL-12 activates natural killer cells to secrete IFNγ, which promotes differentiation of Thp to Th1 cells secreting type 1 cytokines that downregulate IL-10 production.

Role of IL-10, a Type 2 Cytokine, in SIRS

Studies have shown that a high level of IL-10 in patients with SIRS is an independent predictor of mortality. Interestingly, autopsy studies in persons who died of SIRS sepsis showed evidence for apoptosis-induced loss of immune cells (B cells, CD4 cells, and follicular dendritic cells). Differentiation of Thp cells to Th2 cells predominates in responses in which macrophages that have ingested apoptosed cells are serving as antigen-presenting cells. Because Th2 cells secrete IL-10, this would be consistent with studies that showed that IL-10 was an independent predictor of mortality. Once again, studies in animals showed that prevention of lymphocyte apoptosis improved the likelihood of survival from SIRS. It is important to keep in mind, however, that decreases in cells that undergo apoptosis are likely to impair antibody production, macrophage activation, and antigen presentation.

Role of Neutrophils in SIRS

Neutrophils have been regarded as potential double-edged swords in SIRS, important both for the eradication of pathogens and for excessive release of neutrophil oxidants and proteases responsible for organ injury. Because of the intrapulmonary sequestration of neutrophils and the frequent complication of the acute respiratory distress syndrome (ARDS) in SIRS patients with sepsis, this link between neutrophil activation and organ injury was thought especially to affect the lungs. However, studies using G-CSF to increase the number of neutrophils and enhance their function showed improved survival among SIRS patients!

New Approaches to Therapy

Recombinant Human Activated Protein C

Activated protein C has a normal regulatory role in coagulation in that it inactivates factors Va and VIIIa, preventing the generation of thrombin, and thus decreasing inflammation by inhibiting platelet activation, neutrophil recruitment, and mast cell degranulation. Activated protein C also has direct antiinflammatory properties, including blocking of the production of cytokines by monocytes and blocking cell adhesion, and in addition it has anti-apoptotic actions that may contribute to its efficacy.

Recombinant human activated protein C, an anticoagulant, is the first anti-inflammatory agent that has proved effective in the treatment of sepsis, showing, in a recent clinical trial, a 20% reduction in the relative risk of death and an absolute risk reduction of about 6%. Note that a major risk associated with activated protein C is hemorrhage (intracranial hemorrhage, a life-threatening bleeding episode, or a requirement for 3 or more units of blood), and thus at present activated protein C is approved only for use in patients with SIRS who have the most severe organ compromise and the highest likelihood of death.

Insulin Therapy

Intensive insulin therapy (blood glucose level of 5 to 6 mmol/L) was also reported to result in lower morbidity and mortality among critically ill patients than conventional therapy (maintaining blood glucose levels at 10 to 11 mmol/L) and was noted to decrease the risk of death in patients with multiorgan failure, regardless of whether they had a history of diabetes. The mechanism of action of insulin in SIRS is unknown, although the phagocytic function of neutrophils is impaired in patients with hyperglycemia and insulin prevents apoptotic cell death from numerous stimuli by activating distinct biochemical pathways (the phosphatidylinositol 3-kinase/Akt pathway). As noted earlier, ingestion of apoptosed cells by antigen-presenting cells leads to the differentiation of Thp cells to Th2 cells that secrete IL-10, as well as other anti-inflammatory cytokines.

Immunotherapy Regimens

A number of other immunologic therapies are under consideration. IFNγ is a potent macrophage activator and has been reported to improve survival in a subgroup of patients with SIRS, possibly by restoring macrophage human leukocyte antigen (HLA)-DR expression (induction of Th1 activation) and TNF production. Similarly, IL-12, another Th1 inducer, reduced mortality from subsequent sepsis when administered after burn injury. In a different approach, anti–complement-activation product (anti-C5a antibodies) decreased the frequency of bacteremia, prevented apoptosis, and improved survival in SIRS patients. Not unexpectedly, given the previous discussion, strategies that block apoptosis of lymphocytes or gastrointestinal epithelial cells (both profoundly increased in almost all models of SIRS and in patient populations) have improved survival in experimental models of sepsis. This is consistent with evidence that mice with sepsis that are deficient in poly-adenosine diphosphate-ribose polymerase 1 (PARP-1), a key molecule in the proapoptotic pathway, have improved survival and that administration of a PARP-1 inhibitor was beneficial in pig models.

Surviving SIRS: A Consequence of Preconditioning?

Ischemic preconditioning is a term used to describe a phenomenon in which a transient episode of myocardial ischemia subsequently protects the individual from severe myocardial ischemia. Similarly, it has been proposed that organ dysfunction in SIRS patients is best explained by “cell hibernation” or “cell stunning.” That is, SIRS is presumed to activate defense mechanisms that cause cellular processes to be reduced to basic “housekeeping” roles. If we are to extrapolate the concept of ischemic preconditioning to SIRS, patients who survive and have secondary exposure to SIRS would fare well. Alternatively, we could use this concept to explain survival of patients with SIRS, saying that earlier infections preconditioned them so that they were able to survive this major infection.

These hypotheses have arisen as a consequence of intriguing findings from autopsy studies that there is discordance between histologic findings and the degree of organ dysfunction seen in patients who died of sepsis. Histologic findings in patients with sepsis and acute renal failure showed only focal injury with preservation of normal glomeruli and renal tubules, as predicted from evidence that patients who survive sepsis and acute renal failure recover baseline renal function.

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