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JANEWAY'S IMMUNOBIOLOGY, 9TH EDITION

CHAPTER 2: INNATE IMMUNITY: THE FIRST LINES OF DEFENSE

Anatomic barriers and initial chemical defenses

2-1 Infectious diseases are caused by diverse living agents that replicate in their hosts

2.1 Multiple choice: Antibodies, complement proteins, and phagocytic cells provide
effective protection against all of the following types of infections in Figure Q2.1, except:

Figure Q2.1

2.2 Multiple choice: Pathogenic infections induce damage to the host by a variety of
mechanisms. While many mechanisms are direct effects of the pathogen, some
damaging mechanisms result from the immune response to the infection, as illustrated in
Figure Q2.2. Examples of damage caused by the host immune response are:

Page 1 of 19
 Figure Q2.2

2.3 True/False: Mucosal surfaces and external epithelia are major routes of pathogenic
infection. Mucosal surfaces are found in tissues such as the gastrointestinal tract, the
reproductive tract and the mouth and respiratory tract. While the mouth and respiratory
tract are routes of virus but not bacterial infections, the gastrointestinal tract is the route
for bacterial but not virus infections.

2-2 Epithelial surfaces of the body provide the first barrier against infection

2.4 Multiple choice: Epithelial surfaces provide the first line of defense against infection by
the use of several types of mechanisms. One of the chemical mechanisms used by
epithelia is:
A. Joining of epithelial cells by tight junctions
B. Secretion of antimicrobial peptides by epithelial cells

Page 2 of 19
 C. Production of mucus, tears, or saliva in the nose, eyes, and oral cavity
D. Movement of mucus by cilia
E. Peristalsis in the gastrointestinal tract

2.5 Multiple choice: Women with urinary tract infections caused by E. coli are generally
treated with a course of antibiotics. A common complication of the antibiotic treatment is
the occurrence of a vaginal yeast infection caused by Candida albicans, an organism
that is normally present in very low numbers in the human vaginal tract. This
complication occurs because:
A. The E. coli infection damages the reproductive epithelium, causing a breach in
the tight junctions and allowing invasion by the Candida albicans.
B. The E. coli infection induces adhesion molecule expression on the reproductive
epithelium, allowing attachment of the yeast.
C. The antibiotic treatment kills all strains of fungi present in the reproductive tract,
except the Candida albicans.
D. The E. coli infection causes gastrointestinal distress leading to diarrhea.
E. The antibiotics kill many of the commensal organisms in the reproductive tract,
allowing overgrowth of the fungus.

2-3 Infectious agents must overcome innate host defenses to establish a focus of
infection

2.6 Short answer: Our environment contains masses of microorganisms, many of which
reside as commensal organisms on our body’s mucosal and epithelial surfaces without
causing disease. What two features distinguish a pathogenic microbe from these
commensal microbes?

2-4 Epithelial cells and phagocytes produce several kinds of antimicrobial proteins

2.7 Multiple choice: Streptococcus pneumoniae is a Gram-positive bacterium that

colonizes the mucosal surface of the upper respiratory tract in humans. The presence of
this bacterium in the nose and throat is widespread in the population, and in most
people, colonization with Strep. pneumoniae is asymptomatic. Figure Q2.7 shows a
comparison of in vitro growth curves of the wild-type strain of Strep. pneumoniae, as well
as a Strep. pneumoniae mutant strain with a defect in one bacterial gene. The graph on
the right shows the growth curve following addition of lysozyme during the logarithmic
phase of bacterial growth.

Page 3 of 19
 Figure Q2.7
Which statement could account for the data in these graphs?
A. Strain B is wild-type Strep. pneumoniae, and strain A is a mutant that cannot
modify its peptidoglycan to be lysozyme-resistant.
B. Strain B is wild-type Strep. pneumoniae, and strain A is a mutant that that
expresses increased levels of LPS.
C. Strain A is wild-type Strep. pneumoniae, and strain B is a mutant that cannot
modify its peptidoglycan to be lysozyme-resistant.
D. Strain A is wild-type Strep. pneumoniae, and strain B is a mutant that secretes an
enzyme that inactivates lysozyme.
E. Strain A is wild-type Strep. pneumoniae, and strain B is a mutant that cannot
grow well in vitro.

2.8 Multiple choice: The production of antimicrobial peptides is one of the most
evolutionarily ancient mechanisms of defense for multicellular organisms, and most
eukaryotic species make many different forms of these proteins. For instance, human
paneth cells in the gastrointestinal epithelium make 21 different defensins. The reason
for this diversity of antimicrobial peptides is:
A. Epithelial cells make different forms than those made by neutrophils.
B. Neutrophils make many different defensins and store them as inactive proteins in
their secretory granules.
C. Most of them are produced only in response to infection.
D. The production of different peptides is induced following a bacterial infection
versus a fungal infection.
E. Each one has distinct activities against Gram-negative bacteria, Gram-positive
bacteria, or fungi.

2.9 True/False: Neutrophils regulate the production of active cathelicidins (a class of

antimicrobial peptides) by segregating the inactive propeptide from the processing

Page 4 of 19
 enzyme that cleaves and activates it in two different types of cytoplasmic granules.
These two types of granules are induced to fuse with phagosomes after ingestion of
microbes, bringing the processing enzyme and the propeptide together.

The complement system and innate immunity

2-5 The complement system recognizes features of microbial surfaces and marks them
for destruction by coating them with C3b

2.10 Multiple choice: Although the complement cascade can be initiated by antibodies
bound to the surface of a pathogen, complement activation is generally considered to be
an innate immune response. This is because:
A. Two of the three pathways for complement activation are initiated by
constitutively produced recognition molecules that directly interact with microbial
surfaces.
B. When the complement cascade leads to the formation of a membrane-attack
complex, the pathogen is killed.
C. Several of the soluble products generated by complement activation lead
promote the inflammatory response.
D. Complement proteins bound to the pathogen promote uptake and destruction by
phagocytic cells.
E. The C3 convertase is only produced when complement activation is initiated by
antibody binding to a pathogen.

2.11 Multiple choice: The formation of the C3 convertase is a key step in complement
activation that occurs in all three complement pathways. This enzyme cleaves C3 in
blood plasma, leading to a conformational change in the C3b fragment that exposes its
reactive thioester group. The activated C3b is potentially harmful to the host, if it
becomes covalently attached to a host cell, rather than to the surface of a pathogen.
This deleterious outcome is largely avoided by:
A. The inability of active C3b to diffuse away in the blood plasma.
B. The inability of active C3b to covalently attach to the membranes of eukaryotic
cells.
C. The rapid hydrolysis of active C3b in solution, rendering it inactive.
D. The tight binding of active C3b to the C3 convertase.
E. The ability of active C3b to recruit phagocytic cells.

2-6 The lectin pathway uses soluble receptors that recognize microbial surfaces to
activate the complement cascade

2.12 Short answer: Infants and young children with deficiencies in specific complement
components present with recurrent respiratory infections caused by extracellular
bacteria. The peak age of susceptibility is between 6 and 12 months after birth. At this
time, as shown in Figure Q2.12, maternal antibodies acquired by the child during fetal
gestation are nearly gone, but the child is not yet generating robust antibody responses
to new infections, as indicated by the low circulating levels of IgG and IgA. As children
with this immunodeficiency get older, they outgrow this disease and show no further
evidence of these recurrent infections. Based on this information, name one likely gene
deficiency (in the complement system) that could cause this primary immunodeficiency,
and the specific complement pathway likely to be affected. Explain your answer.

Page 5 of 19
 Figure Q2.12

2.13 Multiple choice: Mannose binding lectins (MBL) and ficolins are the two classes of
proteins that can initiate the lectin pathway of complement activation. These proteins are
selective for activating complement on the surfaces of microbial pathogens rather than
host cells because:
A. Their higher-order oligomeric structure can be assembled only after the
monomers first bind to pathogen membranes.
B. They only recruit MASP (MBL-associated serine proteases) proteins when bound
to pathogen surfaces and not when bound to host cells.
C. They only undergo the conformational change needed to activate MASP proteins
when bound to a pathogen and not when bound to a host cell.
D. They only bind to carbohydrate side chains and oligosaccharide modifications
found on pathogen surfaces but not on host cell membranes.
E. The activated MASP proteins are rapidly inactivated by hydrolysis when present
on the surface of a host cell.

2-7 The classical pathway is initiated by activation of the C1 complex and is homologous
to the lectin pathway

2.14 Multiple choice: The classical complement pathway is initiated by C1q binding to the
surface of a pathogen. In some cases, C1q can directly bind the pathogen, for instance
by recognizing proteins of bacterial cell walls, but in most cases C1q binds to IgM
antibodies that are bound to the pathogen surface. How does this IgM-binding feature of
C1q contribute to rapid, innate immune responses rather than to slow, adaptive
responses?
A. C1q induces B lymphocytes to begin secreting antibody within hours of pathogen
exposure.
B. Natural antibody that binds to many microbial pathogens is produced prior to
pathogen exposure.
C. C1q binds to C-reactive protein which then binds to IgM on the pathogen surface.
D. C1q directly induces inflammation, recruiting phagocytes and antibodies from the
blood into the infected tissue.
E. C1q binds to dendritic cells in the infected tissue, inducing them to secrete
inflammatory cytokines.

2.15 True/False: The classical and lectin pathways of complement activation converge at the
step of C3 activation. However, the initiating steps of each pathway use protein
components and enzymatic mechanisms that share no similarity with each other.

Page 6 of 19
2-8 Complement activation is largely confined to the surface on which it is initiated

2.16 Multiple choice: Opsonization of pathogens by both antibodies and complement

proteins (C3b) leads to uptake and destruction of the pathogen by phagocytic cells that
express both Fc receptors and complement receptors. Which of the following in Figure
Q2.16 is the most efficient form of dual opsonization of the pathogen by antibody and
C3b to maximize phagocytosis?

Figure Q2.16

2-9 The alternative pathway is an amplification loop for C3b formation that is accelerated
by properdin in the presence of pathogens

2.17 Multiple choice: The alternative pathway of complement activation has an important
role in innate immunity, due to its ability to greatly amplify the amount of C3b deposited
onto the pathogen surface. This amplification occurs because:
A. The C3 convertase of the alternative pathway is much more active than those of
the classical and lectin pathways.
B. The C3 convertase of the alternative pathway works as a soluble enzyme in the
plasma.
C. The C3 convertase of the alternative pathway cannot be inactivated by
complement regulatory factors in the host.
D. The C3 convertase of the alternative pathway is more efficiently recruited to
pathogen surfaces than the C3 convertases of the classical and lectin pathways.
E. The C3 convertase of the alternative pathway contains C3b, and can generate
more of itself.

2.18 True/False: The C3 convertase of the alternative complement pathway amplifies the
overall magnitude of complement activation regardless of which of the three pathways
initiated the complement activation initially.

2.19 Multiple choice: Patients with recurrent infections of Neisseria meningitidis, an

extracellular bacterial pathogen that causes meningitis, were examined to determine the
underlying cause of their immunodeficiency. A subset of these patients were found to
have defects in complement activation on the bacterial surface, a process that for this
bacterium is dominated by alternative complement activation leading to C3b deposition
on the pathogen surface. When neutrophils from these patients were examined in vitro,
the results in Figure Q2.19 were obtained.

Page 7 of 19
 Figure Q2.19
Based on these data, the identity of the green neutrophil mediator in Figure Q2.19 is
likely to be:
A. Complement factor B
B. The C3 convertase
C. Factor P (properdin)
D. C3b
E. Mannose-binding lectin (MBL)

2-10 Membrane and plasma proteins that regulate the formation and stability of C3
convertases determine the extent of complement activation

2.20 Multiple choice: One form of anemia results when individuals have a deficiency in the
enzyme phosphatidylinositol glycan A (PIGA). This enzyme is required for the
membrane attachment of proteins anchored by glycolipids to the plasma membrane,
using what is called a ‘GPI-linkage.’ Included in the group of GPI-linked cell surface
proteins is DAF/CD55. These individuals become anemic because:
A. DAF/CD55 prevents the lysis of red blood cells by infecting pathogens.
B. DAF/CD55 normally prevents the spleen from clearing healthy red blood cells
from the circulation.
C. In the absence of PIGA, the red blood cell membrane is bare of proteins allowing
increased access of complement activating proteins to attach to the cell
membrane.
D. DAF/CD55 is a complement inhibitory protein that inactivates any C3 convertase
that may form on host cell surfaces.
E. In the absence of PIGA, red blood cells are unable to synthesize high levels of
hemoglobin.

2-11 Complement developed early in the evolution of multicellular organisms

Page 8 of 19
2.21 Short answer: In vertebrates, complement activation generally involves a pathogen
recognition step followed by a proteolytic cascade that produces the effector proteins
that function in opsonization, membrane attack, and inflammation.
a) Which of these is likely to be the most evolutionarily primitive aspect of the
complement system?
b) Which pathway of complement initiation is likely to be the one that most recently
evolved?

2-12 Surface-bound C3 convertase deposits large numbers of C3b fragments on

pathogen surfaces and generates C5 convertase activity

2.22 True/False: The C3 convertase amplifies the process of complement activation by

generating large amounts of C3b and cleaving large numbers of C5 molecules.

2-13 Ingestion of complement-tagged pathogens by phagocytes is mediated by

receptors for the bound complement proteins

2.23 Multiple choice: Even when the complement cascade fails to proceed beyond
generating the C3 convertase, complement activation is effective at inducing pathogen
uptake and destruction. This process of immune protection is mediated by:
A. Activation of complement inhibitory receptors on phagocytes that promote
pathogen uptake
B. Activation of soluble proteases in the serum that disrupt pathogen membranes
C. Engagement of complement receptors on phagocytes by C3b and its cleavage
products which promotes phagocytosis
D. Engagement of complement receptors on B cells that promotes antibody
production
E. Stimulation of antimicrobial peptide secretion by phagocytes

2.24 Multiple choice: B cells express a complement receptor that binds to C3b cleavage
products, such as iC3b and C3dg. When a B cell with an antigen receptor that
specifically recognizes that pathogen also has its complement receptor stimulated
because the pathogen is opsonized with these C3 fragments, B cell activation is greatly
enhanced. Due to this mechanism, B cells can be activated by much lower
concentrations of antigen (in this case, the pathogen) than if the antigen is devoid of
complement components. This mechanism functions to:
A. Ensure that pathogens are readily detected by the adaptive immune system
before they replicate to high levels in the host
B. Prevent B cells from being activated in response to antigens that are not
pathogens
C. Allow B cells to phagocytose the pathogen and help destroy it
D. Induce increased rounds of B cell replication to make more pathogen-specific B
cells
E. Allow the B cell to block pathogen replication by interfering with multiple
pathogen surface functions

2-14 The small fragments of some complement proteins initiate a local inflammatory
response

2.25 Short answer: Recent studies using mouse models of pulmonary inflammation (a model

Page 9 of 19
 for human asthma) have found that mice deficient in the C3a receptor have greatly
reduced disease symptoms when challenged with inhaled preparations containing
extracts of the fungal pathogen Aspergillus fumigatus. Specifically, the C3a receptor-
deficient mice showed reduced influx of granulocytes and lymphocytes into the lung and
reduced fluid in the lung after challenge. What is the explanation for these findings?

2-15 The terminal complement proteins polymerize to form pores in membranes that can
kill certain pathogens

2.26 Multiple choice: The terminal components of the complement pathway assemble to
form a membrane attack complex that can induce pathogen lysis and death. Yet,
evidence indicates that this feature of complement is less important than the earlier
steps that promote pathogen opsonization and induce inflammation. This conclusion is
based on:
A. In vitro experiments showing that very few species of bacteria are susceptible to
lysis by the membrane attack complex
B. Experiments indicating that only bacteria, but not viruses or fungi, are susceptible
to lysis by the membrane attack complex
C. The very low levels of terminal complement components in the serum
D. The fact that other mammalian species lack the terminal components of the
complement pathway needed to form the membrane attack complex
E. The limited susceptibility to infections of patients with deficiencies in terminal
complement components

2-16 Complement control proteins regulate all three pathways of complement activation
and protect the host from their destructive effects

2.27 Multiple choice: Multiple pathways for regulating complement activation limit the
potential damage caused by complement deposition on host cells or caused by the
spontaneous activation of complement proteins in the plasma. Genetic deficiencies in
these mechanisms often lead to chronic inflammatory diseases, but in some cases can
paradoxically lead to increased susceptibility to bacterial infections. This latter outcome
may occur because:
A. Complement regulatory proteins have dual functions in inhibiting and promoting
complement activation.
B. Uncontrolled complement activation leads to the depletion of serum complement
proteins.
C. The inhibition of the membrane attack complex by complement regulatory
proteins normally leads to enhanced activation of the early steps of the
complement pathway.
D. Complement regulatory proteins normally cause the rapid depletion of plasma
complement factors.
E. Uncontrolled complement activation recruits the majority of phagocytic cells,
leaving few remaining to fight infections in the tissues.

2.28 Short answer: Although homozygous deficiencies in complement regulatory proteins

cause serious diseases, more subtle alterations in the balance of complement activation
versus inhibition have been found to contribute to disease susceptibility. Describe the
genetic evidence linking subtle alterations in complement regulatory proteins to disease
susceptibility.

Page 10 of 19
2-17 Pathogens produce several types of proteins that can inhibit complement activation

2.29 Multiple choice: The importance of complement activation as an innate immune

defense against infections is illustrated by:
A. The evolution of complement avoidance strategies by many pathogens
B. The large number of proteins involved in the complement pathway
C. The large number of complement regulatory pathways expressed by the host
D. The existence of three different mechanisms for initiating complement activation
E. The ability of the membrane attack complex to lyse some pathogens

2.30 True/False: Several pathogens produce proteins, either membrane-bound or secreted,

that inactivate C3b that might be deposited on the pathogen surface. C3b is specifically
targeted due to its central position in all three complement pathways.

2.31 Synthesis question: Four different clinical isolates of the Gram-positive bacterium,
Staphylococcus aureus, are tested for their abilities to resist innate immune defense
mechanisms. For these experiments, each bacterial strain is first grown in culture to
achieve log-phase replication, and then cultures are supplemented with dilutions of
human serum containing normal serum proteins as well as antibodies capable of binding
to S. aureus. One hour later, the cultures are analyzed and the numbers of live bacteria
are quantitated. The data from this experiment are shown in Figure Q2.31A.

Figure Q2.31A

a) From these data, what general conclusions can be reached about the four strains of
S. aureus?

To identify the bactericidal mechanisms killing each strain of S. aureus, the serum is
depleted of complement C3 by running it over an anti-complement C3 antibody affinity
column. The experiment above is then repeated and the data are shown in Figure
Q2.31B.

Page 11 of 19
 Figure Q2.31B

b) What is the most likely mechanism accounting for the killing of strain D in this
experiment?

To determine whether strains A and C are susceptible to the same microbicidal pathway,
the serum is depleted of antibody by running over an anti-human immunoglobulin affinity
column. Following this treatment, it is found that strain A, but not strain C is still killed by
incubation with the serum.

c) From these data, what is the most likely mechanism killing S. aureus strain A? What
about strain C?

2.32 Synthesis question: Pseudomonas aeruginosa is a Gram-negative bacterium that

causes severe, and often life-threatening infections in immunocompromised individuals.
In susceptible individuals, P. aeruginosa can establish infections in a wide range of
tissues, including the lung, the GI tract, the eye, the ear, the urinary tract, the skin, and
the blood. This bacterium is common in the environment, and is found on the skin of
approximately 5% of healthy individuals. It is often found on hospital equipment, such as
ventilators and catheters, and as a consequence, P. aeruginosa accounts for ~10% of
hospital-acquired infections. To study the role of complement in the early innate immune
response to P. aeruginosa, the following studies in mice were performed. Mice deficient
in complement C3 or C5 (C3-/- or C5-/-, respectively) were infected by intratracheal
inoculation with 105 colony forming units (CFU) of P. aeruginosa, and survival was
monitored over the first 72 hrs post-infection. The data from these studies are shown in
Figure Q2.32. Genetic data from human population studies also indicate that Individuals
with genetic deficiencies in one of the collectins or ficolins show increased susceptibility
to P. aeruginosa infections.

Page 12 of 19
 Figure Q2.32

a) Based on these data, evaluate the importance of complement in protection against P.

aeruginosa infection, and describe the most likely complement pathway(s) involved in
pathogen recognition and in pathogen destruction.

Another group of individuals that are highly susceptible to P. aeruginosa infections are
patients with the disease cystic fibrosis. These individuals suffer from the production of a
thick mucus secretion in their lungs, which clogs the bronchial tubes. A similar increase
in viscosity of bodily secretions is seen in these patients’ sweat, digestive fluid, and
gastrointestinal mucus. In these patients, the most common form of lung infection is that
of P. aeruginosa.

b) What is the most likely explanation for the increased susceptibility of cystic fibrosis
patients to P. aeruginosa and other infections?

Page 13 of 19
ANSWERS

2.1: B.
All extracellular forms of pathogens are targets for antibodies, complement, phagocytic cells and
antibody-dependent immune clearance mechanisms. Once a pathogen, such as a virus or
intracellular protozoan, invades a cell and begins replicating in the cell, these mechanisms are
no longer able to clear the infection. These intracellular stages of pathogenic infection require
cellular responses, such as those mediated by T cells or NK cells.

2.2: C.
Pathogens cause direct tissue damage by the production of exotoxins or endotoxins, as well as
by direct cytopathic effects. Tissue damage caused by the host immune response include
damage caused by cell-mediated immunity and by the accumulation of immune complexes.

2.3: False.
Both bacterial and virus infections can use both the mouth and respiratory tract and the
gastrointestinal tract. There is no route of infection that is specific for a single category of
pathogen.

2.4: B.
Anti-microbial peptides are produced by epithelia at all mucosal and epidermal surfaces. These
chemicals are important in immune protection against microbial pathogens. All other choices are
mechanical mechanisms by which surface epithelia protect against infections, not chemical
mechanisms.

2.5: E.
Commensal organisms associated with all epithelial surfaces provide protection against
colonization by pathogenic microbes. One mechanism is by competition for nutrients as well as
for attachment sites on epithelial surfaces. Another mechanism is by producing metabolites that
are toxic to other organisms. When these commensal microorganisms are eliminated by
antibiotic treatment, pathogenic microbes are able to step into the void and establish an
infection.

2.6: The first important feature of a pathogenic microbe is that it must establish a replicating
colony of organisms in our body. This can occur by the pathogen crossing an epithelial barrier
and replicating in the tissue, or by attaching to the epithelial surface and establishing a colony
there. The second feature is that the pathogen needs to have special mechanisms to evade the
innate immune response.

2.7: C.
Strep. pneumoniae is a Gram-positive bacterium that readily colonizes the human nose and
mouth, due to the resistance of its peptidoglycan to degradation by lysozyme, an enzyme that is
abundant in tears, saliva, and mucus. Wild-type Strep. pneumoniae is naturally lysozyme-
resistant because a substantial proportion of GlcNAc residues in its peptidoglycan are
deacetylated, and no longer substrates for lysozyme-mediated hydrolysis. This accounts for the
prevalence of Strep. pneumoniae in the upper respiratory tract of healthy humans, where
lysozyme is present at high concentrations. When one of these enzymes is missing, as in strain
B, the bacteria become lysozyme-sensitive, and are killed by the lysozyme.
fungi.

Page 14 of 19
2.8: E.
The diversity of antimicrobial peptides is a reflection of the diversity of microbial pathogens that
they attack. Some antimicrobial peptides are active against Gram-negative bacteria, while
others are only active against Gram-positive bacteria. Other antimicrobial peptides are only
active against fungal pathogens, and some are able to disrupt the membrane envelopes of
some viruses.

2.9: True.
All antimicrobial peptides, including cathelicidins, are produced as inactive propeptides. The
active forms of the peptides are generated following proteolytic cleavage of the propeptides.
Neutrophils constitutively produce cathelicidins, which are synthesized as inactive propeptides.
The inactive cathelicidin propeptides are stored in secondary granules, whereas the cleavage
enzyme, neutrophil elastase, is stored in primary granules. These two types of granules are
induced to fuse with phagocytic vesicles, called phagosomes, after the neutrophil has engulfed
a pathogen. This fusion brings the cleavage enzyme together with the cathelicidin propeptide,
leading to cathelicidin activation.

2.10: A.
There are three pathways for initiating complement activation. One of them, known as the
classical pathway, occurs when the pathogen has antibodies bound to its surface, leading to
recruitment of C1q. The other two pathways, the lectin pathway and the alternative pathway, are
initiated by mechanisms that do not require antibodies directed against the pathogen surface.
These latter two pathways are dependent on constitutively produced, and therefore ‘innate’
recognition molecules that directly bind to pathogen surfaces, initiating complement activation.

2.11: C.
Active C3b is highly labile, and is rapidly inactivated by hydrolysis. This prevents the C3b from
remaining active should it diffuse away from the pathogen surface where it was activated by the
C3 convertase.

2.12: MBL or MASP.

Infants and small children with defects in MBL or MASP show recurrent upper respiratory
infections by extracellular bacteria. This is due to a defect in the lectin pathway of complement
activation. When maternal antibodies wane and the child is not yet generating robust antibody
responses on its own, complement activation cannot proceed by the classical pathway. During
this time, protection against upper respiratory bacterial infections is highly dependent on the
lectin pathway, initiated by MBL or collectin binding to the pathogen. The information provided
rule out the alternative pathway, which is initiated by spontaneous C3 cleavage. If there was a
defect in C3, or a downstream component of the complement cascade shared by all three
pathways, the recurrent infections would not disappear as children age.

2.13: D.
MBL and ficolins have binding specificity for carbohydrate side chains and oligosaccharide
modifications that are unique to microbial pathogens, and not found on host cells. MBL binds to
mannose, fucose, and GlcNac residues, which are common on microbial glycans; in contrast,
MBL does not bind to sialic residues, which terminate vertebrate glycans. Ficolins have
specificity for binding to oligosaccharides containing acetylated sugars, a structure also only
found on pathogen surfaces, not on host cells.

2.14: B.
Natural antibody, which is primarily of the IgM class, is produced in the body prior to pathogen

Page 15 of 19
exposure. These antibodies are widely reactive with many microbial pathogens, although they
generally have low affinity for the pathogen. However, since IgM is a pentamer of IgM
monomers, each IgM pentamer has 10 binding sites for antigen, allowing even low affinity
antibodies to bind, due to the increased avidity of multiple binding sites. This natural antibody
will then recruit C1q, leading to complement activation. Since the natural antibody pre-exists
prior to pathogen exposure, this response is rapid and is considered part of the innate immune
response.

2.15: False.
The initiating steps of the classical and lectin pathways of complement activation are remarkably
conserved in their mechanisms. The pathogen recognition component of the classical pathway,
C1q, has structural similarity to MBL and the ficolins. The C1r and C1s components of the
classical pathway, that are activated to form the serine protease, are closely related to the
MASP proteins of the lectin pathway.

2.16: A.
The most efficient form of opsonization by antibody plus C3b is when the complement protein is
covalently linked to the antibody molecule. This leads to efficient engagement of both Fc
receptors and complement receptors on phagocytic cells.

2.17: E.
The C3 convertase (C3bBb) of the alternative pathway contains C3b, allowing it to generate
more of itself and amplify the overall level of C3b formed. Once additional molecules of C3b are
made by C3bBb, these can recruit additional molecules of factor B and the plasma protease
factor D. Factor D cleaves factor B, and one of the products, Bb, remains associated with C3b,
forming more active C3 convertase.

2.18: True.
The C3 convertase of the alternative pathway contains C3b, allowing it to generate more of itself
and amplify the overall level of C3b formed. Since C3b is a common intermediate for all three
pathways of complement activation, once the initial C3b is generated by any of the pathways,
the recruitment of factor B, and cleavage by factor D can proceed. By this mechanism, the initial
C3b generated forms an amplification loop leading to more C3b, regardless of how the initial
C3b was made.

2.19: C.
Factor P (properdin) is made by neutrophils and stored in their granules. When neutrophils are
activated by the presence of pathogens, factor P is released. Factor P binds to and stabilizes
the reactive form of C3 (C3-H20) and the C3 convertase C3bBb. In the absence of factor P, the
alternative complement pathway is inefficient, due to the rapid spontaneous inactivation of C3-
H20 and C3bBb. This pathway is particularly important in protection against Neisseria
meningitidis, and patients that are deficient in producing factor P are highly susceptible to
infections with this pathogen.

2.20: D.
Host cells express several complement-regulatory proteins on their surface. These proteins
function to rapidly inactivate any C3bBb (active C3 convertase) that may form on the host cell
membrane. Several of these complement regulatory proteins use GPI-linkages to attach to the
host cell membrane. Included in this group is DAF/CD55, which competes with factor B for
binding to C3b on the cell surface, and displaces Bb from any active C3 convertase that has
already formed. The absence of DAF/CD55 makes host cells susceptible to complement-

Page 16 of 19
mediated lysis. For reasons that are not entirely clear, red blood cells are particularly
susceptible to complement-mediated lysis and the absence of the GPI-linked subset of
complement regulatory proteins is sufficient to cause red blood cell lysis leading to anemia.

2.21:
a) The most primitive form of a complement system is one that resembles our alternative
complement pathway, with ancestral homologs of C3 and factor B that make a C3
convertase. This provides a mechanism for opsonizing infecting bacteria and increasing
their phagocytosis by phagocytic cells. These ancestral homologs of C3 and factor B
have been found in echinoderms, and may even have existed in even more primitive
organisms such as corals and sea anemones.
b) The latest evolutionary development in the complement system is the classical pathway,
which makes use of antibody binding to initiate complement activation. The adaptive
immune system, including the production of antibodies, is only found in vertebrates.

2.22: False.
The C3 convertase does generate large numbers of C3b molecules which become attached to
the pathogen surface in the vicinity of the convertase. This enzyme can only cleave C5 when
bound to a molecule of C3b, generating the C5 convertase. The generation of the C5
convertase occurs at a much lower level than the C3 convertase, and many fewer molecules of
C5 than C3 are cleaved.

2.23: C.
Phagocytes have a variety of receptors that recognize C3b and fragments of C3b, such as iC3b.
Engagement of these complement receptors stimulates phagocytosis of the C3b-coated
pathogen, leading to pathogen destruction.

2.24: A.
The complement receptor on B cells, CD21, is often referred to as the B cell co-receptor. When
this receptor is engaged together with the B cell antigen receptor, the B cell can be activated by
much lower concentrations of antigen compared to antigen lacking ligands for CD21.
Experiments have indicated that CD21 stimulation can reduce the concentration of antigen
needed to activate the B cell by 100–1000-fold. This allows B cells to detect small numbers of
infecting pathogens, to initiate an adaptive response prior to the occurrence of a high pathogen
load in the host.

2.25: When complement is activated in the lung in response to the inhaled preparations of the
fungus, the C3 convertase generates C3a. C3a induces a local inflammatory response in the
lung, by acting on the vascular endothelial cells. This response includes increased vascular
permeability, leading to an increase of fluid in the lung, and also acts to up-regulate adhesion
molecules on the local vascular endothelium. As a result, there is increased recruitment of
granulocytes, monocytes, and lymphocytes into the lung.

2.26: E.
Patients with genetic deficiencies in terminal complement components show only a limited
increase in susceptibility to infection. These individuals are more susceptible to infection by
Neisseria species that cause gonorrhea or meningitis. Otherwise, these individuals show no
other increased susceptibility to infection, indicating that formation of the membrane attack
complex is a less important aspect of complement activation compared to the earlier steps that
lead to opsonization of the pathogen as well as inducing inflammation.

Page 17 of 19
2.27: B.
Individuals with a genetic defect in factor I are subject to recurrent infections with pyogenic (pus-
forming) extracellular bacterial infections. This occurs because, in the absence of factor I,
uncontrolled complement activation ends up depleting the complement proteins from the
plasma. This leads to impaired complement activation on these bacteria, and therefore, to
diminished clearance of these infections.

2.28: Two types of genetic alterations in complement regulatory proteins have been linked to
disease susceptibility. First, individuals heterozygous for mutations in one of several
complement regulatory proteins (MCP, factor I, factor H). These individuals are predisposed to
develop a hemolytic disease that leads to damage to platelets and red blood cells, and to kidney
inflammation. Second, individuals with particular single-nucleotide polymorphisms in the gene
for factor H are predisposed to macular degeneration, an age-related disease that causes
blindness. Furthermore, polymorphisms in other complement genes have been found to
contribute to the susceptibility to age-related macular degeneration.

2.29: A.
One of the best indicators of the importance of an immune protective mechanism is the
development by pathogens of strategies to evade that mechanism. In the case of the
complement pathway, many pathogens have evolved strategies to avoid complement activation
on their surface. These include the expression of proteins that attract complement regulatory
proteins to their surface, in an effort to mimic host cell surfaces that can inactivate complement.
An additional strategy is to secrete proteins that directly inhibit components of the complement
pathway.

2.30: True.
All three pathways of complement activation converge on the assembly of a C3 convertase that
produces C3b bound to the pathogen surface. Therefore, pathogens that can inactivate bound
C3b can interfere with complement activation that might be initiated by any of the three
pathways. This makes C3b an ideal target for an immune evasions strategy.

2.31:
a) Strain A and strain C are equally sensitive to a serum component (or set of components)
that can kill S. aureus. Strain B is resistant to all possible serum components that can kill
S. aureus. Strain D is also sensitive to a serum component(s) that can kill S. aureus, but
is killed by a different mechanism than the one(s) killing strains A and C. This latter
conclusion is based on the observation that the factor(s) killing strain D are no longer
active when the serum is diluted 1:16, whereas the activities that kill strains A and C are
still active at this dilution.b) Strains A and C are no longer killed when complement C3
is depleted from the serum. This indicates that these two strains are susceptible to
complement-mediated lysis. The killing of strain D is not affected by depletion of C3,
indicating a distinct mechanism. Strain D is most likely being killed by defensins, or
another antimicrobial peptide. It is unlikely that strain D is being killed by lysozyme in
these experiments, as lysozyme is predominantly found in tears and saliva, rather than
in serum.
c) Since strain A is still killed following depletion of antibody, but not following depletion of
C3, it is likely that strain A is susceptible to the lectin pathway of complement activation,
initiated by binding of ficolin (or collectin) to the bacterial surface. Ficolins are the most
likely initiators of complement activation in this case, as they are at high concentrations
in the serum. Although it is theoretically possible that strain A is killed by the alternative
pathway of complement activation, this possibility is unlikely. The spontaneous

Page 18 of 19
 hydrolysis of C3 produces a fluid-phase C3 convertase that is very short-lived. This fluid-
phase C3 convertase is stabilized by properdin (factor P), which is only produced by
neutrophils when they are activated by pathogens. Therefore, there is unlikely to be
factor P in the serum used in this study, and therefore, the alternative pathway of
complement activation would be very inefficient.

Strain C is no longer killed following depletion of antibody. Therefore, strain C is likely

susceptible to the classical pathway of complement activation.

2.32:
a) Complement is essential for early protection against this dose of P. aeruginosa in mice.
In the absence of either C3 or C5, all of the infected mice succumb to the infection by 2
days post-infection. Based on the human population studies indicating that individuals
deficient in collectins or ficolins are also more susceptible to this bacterial infection, it is
likely that the lectin pathway of complement activation is the primary mechanism for
initiating the complement cascade. Since C5 is required, this points to an important role
for the membrane attack complex in pathogen destruction. However, it is also possible
that the requirement for C5 is due to the role of C5a in promoting inflammation, leading
to the recruitment of phagocytic cells to the site of infection. In this latter case, pathogen
destruction would be due to uptake by phagocytic cells.
b) Due to their defect in regulating the viscosity of bodily secretions, the mechanical and
chemical mechanisms of immune resistance at the body’s epithelial barriers do not
function normally. Instead of serving as a fluid that flushes away potential pathogens at
these barriers, the mucus of cystic fibrosis patients functions as a stagnant reservoir
ideal for bacterial colonization. Furthermore, the thick mucus prevents the normal
diffusion of antimicrobial enzymes and peptides that are secreted into the mucus.

Page 19 of 19
Another random document with
no related content on Scribd:
 I have a distinct recollection of so much. Then I think I
must have lain stunned for half-a-minute. My first clear
thought was of thankfulness at having escaped the black
deep water, so awfully close.

"Not yet death!" flashed through my mind; and I said

aloud, "How foolish I have been!"

Next I had a sense that I was very much hurt

somewhere; but I thought I would get up; and when I tried
to move an inch, the pain in my knee was so fearful, that I
was obliged to desist at once.

I do not fancy I made any sound, for screaming is not

at all in my line; but I did feel dismayed. The position was
not an enviable one. I hoped that the pain might lessen
soon; but it did not.

Then I recollected that I must try to make known where

I was: and I called repeatedly—"Maggie!" "Denham!"
"Help!" But there was no response. Indeed, I scarcely
expected any. Even if the rest of my party had not already
gone home without me,—and I began to feel sure that this
must be the true state of the case,—they would content
themselves easily with the conjecture that I might have
started first alone, and would not search far. The woman in
the cottage had very likely retired with her family for the
night. Unless a passer-by came near the edge of
Gurglepool, my voice from the depth would be unheard;
and stray passers-by, on such a spot and at such an hour,
were in the highest degree improbable.

I tried again to rise: but in vain. I tried to drag myself,

crawling, to the path, only a yard or two off, but I could not.
The least motion gave intolerable agony.
 Darkness seemed to be coming all at once, in a rush.
Outside Gurglepool, no doubt, it was pleasant twilight still;
but I lay in black shadow; the straight rocky sides rising
steeply for sixty feet or more, all around, in a circle broken
only by the path. Small bushes sprouted out here and there
from some tiny ledge; and overhead was a circular grey
sky. That was all I could see. Dim light above, under the
grey sky-roof; no light below. I could just make out the
surface of the still water, near to my side. No sound or stir
of life was to be heard.

It was strangely solemn to be there, all alone; fir from

any human being; clear and calm in mind; but unable to
stir.

While I kept absolutely still, the pain was so far

bearable, that I could think. But the more I thought, the
more I saw that I could do nothing, except endure passively
until help should come. To climb the path was physically
impossible.

Help of course would come in time:—but when?

 It was strangely solemn to be there all alone.

That was the question. If the rest of the party had

started without me, they would not expect me to arrive till
perhaps an hour after themselves: and then they would
wait before doing anything practical. I knew how indignant
and grieved Thyrza would be, at the mere thought of my
having been left behind alone. Perhaps she would see Mr.
Stockmoor: or send some one to meet me. By that time,
however, I scarcely saw what she or anybody could do. The
walk over the hills in darkness would be no easy matter:
and how could they guess where I might be found?

I saw all this very plainly, and it did seem that I should
almost certainly have to remain where I was until the
morning. The marvel to me now is that I could view the
prospect so quietly. I do not think it was stupefaction. I only
felt that Christ my Master was with me—absolutely and
actually present—whatever might happen: that He would
never forsake His own. And four little simple lines kept
running in my head:

"His Arm is beneath me,

His Eye is above:
His Spirit within me
Says—'Rest in My love.'"

It seemed at last a certainty that the trick, of which I

had not liked to suspect the others, had really been
planned. Otherwise I must surely have heard their voices
calling my name, when they returned from the search.

"Poor children! How silly of them!" I thought. For I knew

that in punishing me, they would—as is so often the case—
have punished themselves. And then I reverted to Thyrza,
and I did grieve to picture her trouble.

Suppose she went to the Farm, and told Mr. Stockmoor!

And suppose—suppose Arthur Lenox were there still! Would
he come in search of the missing governess? I felt that
anything I might have to endure would be worth such a
consummation.

The sound of a slow drop—drop—drop on the pool-

surface was followed by big splashes upon my face. Rain
came fast; no soft shower, but a pelting sheet of water,
hissing down the muddy pathway. Ascent would be worse
than ever after this. I should have been thankful for my
waterproof cloak, lying far above on the edge. In five
minutes my clothes were soaked.
 Blacker and blacker grew the sky, heavier and heavier
the rain. It was one of Nature's shower-baths. I was soon
thoroughly chilled and shivering, less able to bear up. I
remember the thought occurring, "Even if I live till daylight,
this may mean fatal illness,—may mean the worst!" And
then the question, "Would it be 'the worst' to me?" And a
murmured, "Even so, my Father,—if so it should seem good
in Thy sight."

How long a time passed thus I cannot tell, for I could

not see the face of my watch. Every two or three minutes I
still called forlornly for aid, though I felt the effort to be
almost useless.

After what must have been a considerable while, I tried

to change my position. In so doing, I put out one hand,
perhaps a foot off,—not on the bank, as I expected, for it
splashed into water.

Then—the pool was rising!

At once I understood. The woman had explained to me

how these waters did rise in heavy rain, slowly mounting up
and up, towards the mouth of the hole, curdling fiercely
round like water in a saucepan vehemently stirred, and
finally "boiling" over on the grass outside.

I think I must have been getting at last a little stupefied

with pain and cold; for I kept picturing this to myself, in a
dreamy fashion, wondering if the waters would carry me up
as they rose, and would whirl me round in eddying circles,
till finally I was cast out upon the grassy slope.

Or I might instead be sucked downwards, drawn into

the quiet river below, carried through dark underground
passages, and perhaps, a mile or two farther on, be washed
out through holes into light of day, just where the hidden
river bubbles up once more upon its stony bed, as I had
seen it when driving past in the dog-cart.

Maggie would be the one to be pitied,—poor Maggie! I

felt such intense compassion for her. I thought of Eustace,
and of Keith's death. It did seem strange, if something akin
to that were to happen again in the family. Not the same,
yet so far alike that Maggie would certainly be blamed for
my death. People would say, "How terrible for Maggie! Such
a result from one little bout of girlish temper and silliness!"
But would that be true? Was it not rather the end of a long
downsliding on Maggie's part: a persistent yielding to ill
temper and perversity?

I think I wanted to live most of all for Maggie's sake. It

seemed to me that my death just then would throw such a
shadow over her life.

Of Miss Millington I thought little, and this now seems to

me singular. Maggie's face haunted me. I kept seeing the
rounded peach-bloom cheeks, and the sweet half-shy grey
eyes, just as I had seen them when she stepped forward to
speak to Arthur Lenox. And, strange to say, the face grew
more dear, just because he had looked upon it admiringly.

Until those lonely hours in Gurglepool hollow, I never

dreamt how I loved Maggie, despite all her coldness. I can
recall saying, with quite a gleam of joy, "If I get through
this, I shall be able now to write to my friend as she wishes,
about her darling."

The downpour continued, and the pool still rose. I could

feel the water creeping, creeping, like a snake of ice about
my feet.

I found myself wondering what the process of drowning

would be like. Should I just fade away into a peaceful
unconsciousness, or would there be struggling and
oppression? Two or three descriptions which I had read
came to me, written by some who had gone through the
actual experience, so far as all loss of sense. "Not worse in
any case than what many have to bear in their own beds," I
thought.

And—"When thou passest through the waters I will be

with thee!" was as if whispered to my mind.

"Why, I am passing through them now," I said aloud.

Yet how far I realised danger, I do not know. For in the

midst of all this, I tried to reckon how many hours must
pass before I could hope to be rescued. Then I wondered
again whether—perhaps—Arthur Lenox might come. And I
seemed to see him and Maggie wandering together, out of
my reach.

Consciousness must have been a little vague at times.

Somehow it did not occur to me to try again to move. I had
quite ceased to call for help, and the very wish to be saved
faded gradually away. I hardly even observed that by-and-
bye the rain came to an end, and the pool was no longer
rising. All this must have taken much time: how much I
cannot tell.

There were cries at last,—shouts,—and I saw lanterns

above, gleaming through the darkness. I tried to call, but
could not, for my voice seemed gone; and I thought, "It
does not matter; they will find my cloak;" which indeed
came to pass.

Then I knew that somebody was descending the path,

followed by somebody else. I have been told since that I
was lying half in water, and my remembrance of the
exclamations around confirms this.
 Some one drew me back gently,—so gently, that I
believed it must be Arthur. I did not say his name, but I
managed to look up, and I saw—not Captain Lenox, but Sir
Keith Denham.

For a moment, I could hardly believe that it was Sir

Keith,—his face was so stern and grieved and pale. I felt no
surprise at seeing him. There was one sharp stab of
disappointment; and then all other thoughts were lost in the
pain of being moved.

I shall never forget the ascent of that path; though

indeed it was managed beautifully. Two other men helped
Sir Keith and Mr. Stockmoor; and sometimes one or another
slipped. They could not help it; but the least jar was terrible
to me; and I did not lose sense for a moment.

Then followed the long long drive in the waggonette,

with its ceaseless jolting. Thyrza was there, and she held
me in her dear arms all the while, tears often running down
her cheeks. I cannot remember my first sight of Thyrza.
They say that she was on the edge of Gurglepool, and that
almost the only words I spoke were just these, "I am so
sorry for poor Maggie." The remark would be natural
enough; but I can remember little of anything, beyond the
pain, and Thyrza's distress, and Sir Keith's stern gentleness.

We reached home at last, and faces and voices came

round. The sound of Maggie's sobbing went to my heart,
and I believe I burst into tears then for the first time. They
kept her away from me.

In the early morning, a doctor from Beckbergh arrived.

I had thought the pain in my knee all night as much as I
could possibly endure: but I had to bear worse from his
hands. It was not a case of broken bones, but of severe
dislocation, with terrible bruises and swelling. At first he
feared permanent injury to the bone. That fear, I am
thankful to say, is now going off. He told me everything
depended on absolute rest and stillness for the limb; and
indeed I have done my best to be quiet, though it was not
easy.

For three weeks, only Lady Denham and Thyrza and

Rouse were allowed in my room. During some days I had a
sharp touch of rheumatic fever, from lying so long in wet
clothes. Things are much better now, and I have permission
to amuse myself by writing a little at times: so when alone
with Thyrza, I ask for my journal. The knee has still to be
kept motionless. But my doctor speaks of the improvement
in it as astonishingly rapid.

"Thanks, partly, to your being so good a patient," he

says.

It was strange that Lady Denham and Sir Keith should

have unexpectedly arrived at the Farm that very afternoon.
Captain Lenox had left only one hour earlier, walking off
with his carpet-bag, and telling nobody where he meant to
go. Sometimes I do long to know what passed between him
and Miss Millington,—but of course I shall never hear.

Friday. September 18.—Having written the above,

piecemeal, up to this day, I hope to resume my more
regular journalising.

It is now over four weeks since the accident. Maggie

and the twins come in daily to see me: but they are all
three more or less constrained and uncomfortable. Nona
chatters. Elfie looks pinched and forlorn. Maggie seems at a
loss what to say or do. I have seen none of them alone, and
scarcely an allusion has been made to the real cause of my
illness. I think it best to wait; not to try to force any
expressions of regret. There is unhappily an adverse
influence.

Miss Millington has not been near me yet. I am told that

she says, "It is kinder not to crowd the room."

CHAPTER XXVI.
AUTHORSHIP—WHETHER? AND HOW?

FROM MISS GRAHAM TO MAGGIE.

Tuesday. September 15.

DEAR MISS ROMILLY,—I am sorry that I could

not write sooner about your MS., but work has
been pressing.

I think I warned you in my last letter that if

you would have an opinion from me as to your
powers, it must be an honest opinion. That does
not at all mean that what I say must finally
settle the question for you. I may take a
different view of the matter from somebody
else; and I may be mistaken. But what I think I
must say. It would be no kindness to lure you
on with false promises, contrary to my real
expectation.

You have sent me a good deal more than the

few pages for which I asked. I have waited till I
could look carefully through the whole: though
twenty pages would have been enough.

The first question is respecting this particular

MS., and I can unhesitatingly advise you not to
offer it to any publisher: for no publisher will
undertake to bring it out. There is a want of
plot, a want of style, a want of care and finish,
a want of force and interest, from beginning to
end, which must tell fatally against it.

It is astonishing how few young people—or

people of any age—have any clear idea of what
is required in writing for the press. They have a
vague impression that the best writers can
"dash off" a thing effectively in a hurry, when
required; therefore, they suppose, all that a
young and unpractised hand has to do is to sit
down when the fancy seizes him or her, scribble
recklessly whatever comes into his or her head,
and be sublimely sure that "anything will do" for
a much-enduring public.

I do not deny that many experienced writers

can "dash off" a thing well, or that the most
rapid writing is often the best. But the rush of
sudden power is generally the outcome of hard
thinking, often of hard struggling up to it. I am
not certain whether you will understand what I
mean; and if not, further words will scarcely
 y
make my meaning clear. Of course there have
been instances of hasty and brilliant hits from
unpractised hands. These, however, are so rare
that ordinary mortals—perhaps I should say
ordinary would-be authors—have no business to
count on any such possibility. In ninety-nine
cases out of a hundred, to say the least,
success presupposes hard work.

I have noted in pencil on your MS. a few of

the more egregious errors in style and
grammar. Some of them might be corrected by
careful re-writing, if the story were worth
further attention: which it is not!

Now we come to the second question,—as to

your future. Is it, or is it not, worth while for
you to set the vocation of literature before your
mind as a distinct aim?

I am more reluctant here to give a decisive

opinion. You are young still. You may have
certain latent powers which might be worth
developing. Carelessly as your MS. is scribbled,
I detect a certain ease of expression, rather
beyond that of the ordinary run of girls. The
plot is no plot: and the characters are feeble:
but about the little boy there is an occasional
touch of reality, which deserves commendation.

You will not count this too encouraging, yet it

is all I can honestly say: There are no such
signs of marked talent, still less of any spark of
genius, that I may venture to say, "Go on, and
prosper."
 It is for you to decide whether you will give
up literary efforts, and be content to live a
simple womanly life,—that may be busy and
beautiful enough, if you will,—or whether you
will prepare to enter the lists.

If you decide on the last,—mark my words!—

it will not mean ease, or laziness, or self-
indulgence. A successful literary career is no
idle career. And the sooner you begin—not to
publish, but to prepare for future publishing,—
the better.

Though you cannot write yet for the press,

you must write and re-write, for practice. You
must read much and steadily. You must study
life and human nature. You must go through the
best authors, with careful noting of the style of
each. You must bind yourself to habits of
regular work, and not allow your plans to be
lightly broken. Authorship is business, not play:
and it must be treated as business.

It may be that your literary bent is strong

enough not to be checked by all this: that you
have in your heart a conviction of future
success, which will nerve you to meet toil and
failure undaunted until you do succeed: that
you feel or believe yourself so distinctly called
of God to this career, as to render it a duty for
you to go straight forward.

If so, I would not deter you. Strive your

utmost: and in time you will learn whether or
no you really are called to it; whether or no,
any measure of the gift is really yours.
 But if you merely think it would be nice to
write because a great many people write in
these days; or because you want to make a
little money, and authorship seems the easiest
fashion of doing so,—then you had better give
up the notion at once. That does not mean
success.

One word more. You need not suppose, from

what I have said, that a life of authorship is all
toil or all difficulties. There are grand delights in
it. I can say this from my own experience. I
would not willingly exchange it for any other
life. But there cannot be heights without
valleys: and whether you know anything of the
heights must depend upon whether it is the life
that God has willed for you.

If you decide to pursue your efforts, send me

a short MS. a year or two hence, and I will tell
you how you are getting on.—Believe me, yours
truly—

LETITIA GRAHAM.

FROM MAGGIE TO NELLIE.

Friday. September 18.

DARLING NELLIE,—I promised to send you

the letter from Miss Graham whenever it should
come; so I suppose I must; but you won't like it
any more than I do. I think it's an awfully
stupid letter, and I am sure she can't be at all a
nice sort of person. I wonder if writing books
always makes people get so disagreeable when
they are middle-aged. That is two of them, and
I dare say Gladys will be just the same by-and-
bye, which would be three.

I am sure Gladys hasn't done nearly all that,

—reading and studying and writing everything
over and over again for years and years. Why,
she just began straight off to print books the
moment she wanted to. I don't mean that she
hadn't done any stories before, but not in the
way Miss Graham says; and I have written two
stories. I don't see why I shouldn't begin to
have books printed, when I like, just as Gladys
has. I certainly shan't wait a whole year. And I
don't mean to write to Miss Graham any more.

Miss Con seems getting on all right, only the

doctor won't let her move, except just to be put
on the sofa. I wish she would make haste and
get well: and then Lady Denham could go back
to the Farm and leave us in peace. She is so
unkind to poor dear Millie, and seems to think it
is all Millie's fault and mine that Miss Con fell
down Gurglepool path. And that is so unfair: for
of course we couldn't guess that Miss Con would
choose to tumble in such a place. Millie says it
was very stupid of her,—and so I think. And
Millie is sure Miss Con likes being an invalid,
and having a fuss made. But you mustn't let
Mother see this, because she is fond of Miss
Con.
 I'm so very glad to hear such good accounts
of darling Mother. It does seem almost as if the
being downright ill had made her better. What
does Father mean by saying that perhaps you
will all come home soon? Is there really any
chance of that?

Lady Denham means to have an excursion

one day soon, now Miss Con is well enough to
be left. There's a big cave, miles away, which
we are to see. She and Sir Keith are going, and
she wants to take the twins and Thyrza and me.
I do think she might squeeze poor Millie in too,
but she won't. I've half a mind to stay at home,
if Millie does: only I want to see the cave.—
Believe me, darling, ever your loving sister—

M
AGGIE.

FROM THYRZA TO NELLIE.

PRIVATE September 19.

MY DEAR NELLIE,—I have written very short

letters lately, but nursing has taken up a great
deal of time. And besides—I did not want to say
too much at first. I wanted to leave Maggie to
tell for herself how things have really been. I
think Lady Denham felt the same, from
something she said one day.
 But now all these weeks have gone, and I can
see quite clearly from your letters that Maggie
has not told,—at least that she hasn't said
much. I believe Lady Denham asked her
yesterday how much she had explained things
to you or Father: for I heard her make a
shirking sort of answer. She has learnt that
from Millie. It wasn't Maggie's way—once.

She is writing to-day, but I don't suppose she

will say much: and I think it is time for me to
speak out. You at least ought to understand, for
Miss Con's sake: and you may say just as much
or as little as you like to anybody else.

Isn't it good of Lady Denham to spend all

these weeks in the house, and to look after
everything? You should see the calm way in
which she rides over Millie's fads and tantrums.
I am afraid I do enjoy that. I never liked her or
Sir Keith half so well as I do now.

But about Gurglepool, and the accident,—it

really was the fault of Millie and the girls,—
Millie's most, because she twists Maggie round
her little finger, and Maggie manages the rest.
Only that doesn't set Maggie free from blame.

They were all very much put out, because

Miss Con insisted on going with them to
Gurglepool the first time. She thought it safer.
And they agreed among themselves to leave
her as much as possible alone, while they were
there, as a punishment.
 Then somebody proposed—I can't find out
who, which makes me sure it was Millie,—that
they should slip off, and leave her to walk home
alone. Such a horrid unladylike trick! Nona was
to hide, and they would have a hunt, and Miss
Con was to be frightened and left to watch: and
then they would all slip away, and Nona would
join them outside the valley.

It was done too: and that was how Miss Con

was so hurt. She found Nona's scarf on the
Gurglepool path, and fancied she saw some one
lying below: and in going down, she slipped and
fell. I don't think the scarf was left there on
purpose.

I was at home with Elfie, and Lady Denham

and Sir Keith came in,—quite unexpectedly.
They had only travelled from York that day: and
they seemed very much disappointed to find
Captain Lenox gone.

Well—Millie and the rest came rushing in, all

heated, as if from a race. Millie grew demure in
a moment, when she saw who was there. Of
course, we asked after Miss Con: and Millie
said, "Oh, she's just behind!" which was not
true, though perhaps Millie tried to think it was.
And Maggie grew so red, I felt certain
something was wrong.

Sir Keith took the matter up at once, and

insisted on knowing all: and there was no
getting out of his questions.

Maggie owned at last that it was—"only fun,

but they had started first—just for fun—and of
course Miss Con would find it out directly, and
get home soon."

I never knew till then how severe Sir Keith

can look. One likes him the better for it:
because it wasn't displeasure for himself, but
for somebody else. I detest people to be always
and for ever defending themselves: but
defending others is quite a different thing.

I know I shouldn't like him to look at me as

he looked at Maggie. Lady Denham said
outright, in her quiet way, "I am ashamed of
you, Maggie!" And Sir Keith just turned away
from her, with almost a kind of contempt and I
heard him say to Denham—"You—a gentleman!
—To leave a lady unprotected in such a place
after dusk!"

Then Sir Keith said somebody must go at

once to meet Miss Con. Millie, who was tilting
up her chin in her offended fashion, declared
she couldn't, she was so tired: and Maggie only
looked doleful and said nothing. But Denham
offered at once,—I think he was so ashamed,
he was glad to do anything,—and Nona and I
said we would go too. And then we found that
Sir Keith meant to be with us.

We went a long way, first by the road, and

then over a hill: but of course there were no
signs of Miss Con. And by-and-by Denham was
puzzled about the right path, when it grew
dark. Sir Keith didn't know the short-cut to
Gurglepool, as he had never been that way.
Nona tried to guide us, but she failed too: and
Sir Keith said we must turn back at once, or we
should get lost ourselves, and not be able to
help Miss Con.

To make matters worse, tremendous rain

came on. We were like drowned rats by the
time we reached home. Maggie did look
miserable then, and no wonder. Millie kept
talking, talking perpetually about its being
nobody's fault. The one thing in life that she
does care for, is to shield her precious self from
blame. I suppose I ought not to write so of her,
but I cannot like Millie. She is so untrue.

I can't think what we should have done

without Sir Keith. He ordered out the
waggonette, sent for Mr. Stockmoor, and
arranged for two men to go over the hills with
lanterns, while he and Mr. Stockmoor and I
drove round by the road. It was very good of
Lady Denham to let me go. She made me
change my wet things, and then actually kissed
me, and said, "Don't be frightened, my dear.
Miss Conway has probably found shelter in a
cottage." Of course that did seem likely, only
one could not be sure.

When we reached the valley, the two men

joined us. They had seen nothing of Miss Con,
and I began to be almost in despair, for Mr.
Stockmoor seemed to think she must have
wandered away and been lost on these wild
hills.

We thought it would be best to go first to the