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15.1: Primary Immunodeficiency - Biology

15.1: Primary Immunodeficiency - Biology


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Learning Objectives

  1. Define primary immunodeficiency.
  2. Compare and contrast conventional and novel primary immunodeficiencies.
  3. Name four categories of conventional immunodeficiencies and give an example of each.

A primary immunodeficiency is usually an immunodeficiency that one is born with. Until recently, primary immunodeficiencies were defined as a rare recessive genetic defect in the immune responses that involved the development of B-lymphocytes, T-lymphocytes, or both and resulted in multiple, recurrent infections during infancy. Depending on the disorder, the lymphocytes in question were either completely absent, present in very low levels, or present but not functioning normally. These disorders represent the conventional immunodeficiencies.

However, based on our increased understanding of the human genome and immune responses it now appears that there are a multitude of common, less severe primary immunodeficiencies involving just one or more of the huge number of genes involved in the immune responses. These so called novel primary immunodeficiencies involve the decreased ability to combat just a single type of infection or a narrow range of infections. The conventional primary immunodeficiencies were grouped as follows:

Conventional: B-lymphocyte Disorders

In the case of B-lymphocyte disorders, there may be may be greatly decreased humoral immunity but cell-mediated immunity , mediated by T-lymphocytes, remains normal.

1. Agammaglobulinemias: Few if any antibodies are produced and there are reduced B-lymphocyte numbers. The person is very susceptible to recurrent infections by common pyogenic bacteria such as Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitidis, and Hemophilus influenzae. These bacteria have antiphagocytic capsules that are normally eliminated by antibodies through opsonization. Examples include X-linked agammaglobulinemia and Autosomal recessive agammaglobulinemia.

2. Hypogammaglobulinemias /Isotype Defects: Decreased general antibody production or decrease production of a single isotype of antibody. Examples include:

  • IgG2 subclass deficiency: A person is unable to produce the subclass of IgG called IgG2 but can produce other classes of antibodies. There is increased susceptibility to bacterial infections.
  • Selective IgA deficiency: A person is unable to make IgA but can produce other classes of antibodies. There is increased susceptibility to bacterial infections and certain protozoan infections.
  • Combined Variable Immunodeficiency (CVID): Hypogammaglobulinemia with normal or decreased numbers of B-lymphocytes.

More severe forms such as agammaglobulinemia are treated with artificially-acquired passive immunization - periodic injections of large amounts of immune globulin (IG or IVIG).

Conventional: T-lymphocyte Disorders

In the case of T-lymphocyte disorders, there is little or no cell-mediated immunity if the disorder involves T8-lymphocytes and/or T4-lymphocytes. There may also be decreased humoral immunity if there is a disorder involves T4-lymphocytes.

1. MHC Expression Defects

  • MHC-I deficiency. Decreased levels of MHC-I production and reduced T8-lymphocyte numbers.
  • Bare lymphocyte syndrome. Decreased levels of MHC-II, decreased numbers of T4-lymphocytes, and decreased T4-dependent antibody production by B-lymphocytes.

2. T-Lymphocyte Signaling Defects

  • Wiskott-Aldrich syndrome. Defective T-lymphocyte activation and defective leukocyte mobility.
  • Proximal TCR signaling defects. Defective cell-mediated immunity and defective T4-dependent antibody production by B-lymphocytes.

3. Familial Hemophagocytic Lymphohistiocytosis

  • Perforin deficiencies. Defective CTL and NK cell function; uncontrolled activation of macrophages and CTLs.
  • Granule fusion defects. Defective CTL and NK cell function; uncontrolled activation of macrophages and CTLs.
  • X-linked lymphoproliferative syndrome. Defective CTL and NK cell function; uncontrolled activation of macrophages and CTLs. Uncontrolled Epstein-Barr virus - induced B-lymphocyte proliferation.

Conventional: Combined B- and T-lymphocyte Disorders (Severe Combined Immunodeficiency Disease or SCID)

Severe combined immunodeficiency disease or SCID affects both humoral immunity and cell-mediated immunity . There is a defect in both B-lymphocytes and T-lymphocytes, or just T-lymphocytes in which case the humoral deficiency is due to the lack of T4-helper lymphocytes.

1. Cytokine-Signaling Defects

  • Autosomal recessive SCID. Shows a marked decrease in T-lymphocytes but normal to increased levels of B-lymphocytes. There is reduced antibody levels due to the lack of T4-helper lymphocytes.
  • X-linked recessive SCID. There is reduced antibody levels due to the lack of T4-helper lymphocytes.

2. Defects in Nucleotide Salvage Pathways

  • PNP deficiency. Shows a progressive decrease in both T-lymphocytes, B-lymphocytes, and NK cells, as well as reduced antibody levels.
  • ADA deficiency. Shows a progressive decrease in both T-lymphocytes, B-lymphocytes, and NK cells, as well as reduced antibody levels.

3. Defects in V(D)J Recombination (Combinatorial Diversity)

  • RAG1 or RAG2 deficiency. Shows an absence or deficiency of both T-lymphocytes and B-lymphocytes, as well as reduced antibody levels.
  • ARTEMIS defects. Shows an absence or deficiency of both T-lymphocytes and B-lymphocytes, as well as reduced antibody levels.

4. Defective Thymus Development

The thymus is needed for the development of T-lymphocytes from stem cells.

  • DiGeorge syndrome. Shows decreased levels of T-lymphocytes, normal levels of B-lymphocytes, and reduced antibody levels.
  • Defective pre-TCR checkpoint. Shows decreased levels of T-lymphocytes, normal or reduced levels of B-lymphocytes, and reduced antibody levels.

Conventional: Innate Immunity Disorders

  • Chronic granulomatous disease. No oxygen-dependant killing pathway in phagocytes. Recurrent intracellular bacterial and fungal infections.
  • Leukocyte adhesion deficiencies. Defective leukocyte adhesion, diapedesis , and migration. Recurrent bacterial and fungal infections.
  • Chediak-Higashi syndrome. Defective vesicle fusion and lysosomal function in neutrophils, dendritic cells, macrophages and other cells. Recurrent infections by pyogenic bacteria.

Novel Immunodeficiencies

While the rare conventional primary immunodeficiencies mentioned above are still very important, based on our increased understanding of the human genome and immune responses it now appears that there are a multitude of common, less severe primary immunodeficiencies. These so called novel primary immunodeficiencies relate to an individual’s own unique genetics and can involve one or more of many immunity genes, ranging from any of the huge number of genes conferring protective immunity in general, to individual genes conferring specific immunity to a single pathogen.

It is now thought that almost every person suffers from one form of primary immunodeficiency or another. Unlike the classical primary immunodeficiencies, however, these primary Examples include:

  • Disorders of the interleukin-12/interferon-gamma pathway appear to make individuals more susceptible to Mycobacterium and Salmonella infections.
  • Disorders of the TLR-3 pathway makes individuals more susceptible to herpes simplex virus encephalitis.
  • Disorders of the toll-interleukin 1 receptor/nuclear factor kappa B pathway makes individuals more susceptible to staphylococcal and pneumococcal infections.
  • Disorders of properdin and terminal components of the complement pathways make individuals more susceptible to Neisseria infections.
  • People with chronic sinusitis that does not respond well to treatment have decreased activity of TLR-9 and produce reduced levels of human beta-defensin 2, as well as mannan-binding lectin needed to initiate the lectin complement pathway.

Summary

  1. Immunodeficiency results in an inability to combat certain diseases.
  2. A primary immunodeficiency is usually an immunodeficiency that one is born with.
  3. Conventional primary immunodeficiencies are rare recessive genetic defect in the immune responses that involved the development of B-lymphocytes, T-lymphocytes, or both and resulted in multiple, recurrent infections during infancy. Depending on the disorder, the lymphocytes in question were either completely absent, present in very low levels, or present but not functioning normally.
  4. Conventional primary immunodeficiencies include B-lymphocyte disorders, T-lymphocyte disorders, Severe combined immunodeficiency disease or SCID,and innate immunity disorders.
  5. B-lymphocyte disorders may result in greatly decreased humoral immunity but cell-mediated immunity, mediated by T-lymphocytes, remains normal.
  6. T-lymphocyte disorders may result in little or no cell-mediated immunity if the disorder involves T8-lymphocytes and/or T4-helper lymphocytes. There may also be decreased humoral immunity if there is a disorder involves T4-helper lymphocytes.
  7. Severe combined immunodeficiency disease deficiencies affect both humoral immunity and cell-mediated immunity may result in a defect in both B-lymphocytes and T-lymphocytes, or just T-lymphocytes in which case the humoral deficiency is due to the lack of T4-helper lymphocytes.
  8. Innate immunity disorders are due to defects in genes that play a role in innate immune responses.
  9. Novel primary immunodeficiencies include a multitude of common, less severe primary immunodeficiencies involving just one or more of the huge number of genes involved in the immune responses resulting in the decreased ability to combat just a single type of infection or a narrow range of infections.

A single-center pilot study in Malaysia on the clinical utility of whole-exome sequencing for inborn errors of immunity

Primary immunodeficiency diseases refer to inborn errors of immunity (IEI) that affect the normal development and function of the immune system. The phenotypic and genetic heterogeneity of IEI have made their diagnosis challenging. Hence, whole-exome sequencing (WES) was employed in this pilot study to identify the genetic etiology of 30 pediatric patients clinically diagnosed with IEI. The potential causative variants identified by WES were validated using Sanger sequencing. Genetic diagnosis was attained in 46.7% (14/30) of the patients and categorized into autoinflammatory disorders (n=3), diseases of immune dysregulation (n=3), defects in intrinsic and innate immunity (n=3), predominantly antibody deficiencies (n=2), combined immunodeficiencies with associated and syndromic features (n=2), and immunodeficiencies affecting cellular and humoral immunity (n=1). Of the 15 genetic variants identified, two were novel variants. Genetic findings differed from the provisional clinical diagnoses in seven cases (50.0%). This study showed that WES enhances the capacity to diagnose IEI, allowing more patients to receive appropriate therapy and disease management.

Keywords: bioinformatics analysis genetic diagnosis genetic variant inborn errors of immunity whole-exome sequencing.


15.1 The Genetic Code

In this section, you will explore the following questions:

  • What is the “Central Dogma” of protein synthesis?
  • What is the genetic code, and how does nucleotide sequence prescribe the amino acid and polypeptide sequence?

Connection for AP ® Courses

Since the rediscovery of Mendel’s work in the 1900s, scientists have learned much about how the genetic blueprints stored in DNA are capable of replication, expression, and mutation. Just as the 26 letters of the English alphabet can be arranged into what seems to be a limitless number of words, with new ones added to the dictionary every year, the four nucleotides of DNA—A, T, C, and G—can generate sequences of DNA called genes that specify tens of thousands of polymers of amino acids. In turn, these sequences can be transcribed into mRNA and translated into proteins which orchestrate nearly every function of the cell. The genetic code refers to the DNA alphabet (A, T, C, G), the RNA alphabet (A, U, C, G), and the polypeptide alphabet (20 amino acids). But how do genes located on a chromosome ultimately produce a polypeptide that can result in a physical phenotype such as hair or eye color—or a disease like cystic fibrosis or hemophilia?

The Central Dogma describes the normal flow of genetic information from DNA to mRNA to protein: DNA in genes specify sequences of mRNA which, in turn, specify amino acid sequences in proteins. The process requires two steps, transcription and translation. During transcription, genes are used to make messenger RNA (mRNA). In turn, the mRNA is used to direct the synthesis of proteins during the process of translation. Translation also requires two other types of RNA: transfer RNA (tRNA) and ribosomal RNA (rRNA). The genetic code is a triplet code, with each RNA codon consisting of three consecutive nucleotides that specify one amino acid or the release of the newly formed polypeptide chain for example, the mRNA codon CAU specifies the amino acid histidine. The code is degenerate that is, some amino acids are specified by more than one codon, like synonyms you study in your English class (different word, same meaning). For example, CCU, CCC, CCA, and CCG are all codons for proline. It is important to remember the same genetic code is universal to almost all organisms on Earth. Small variations in codon assignment exist in mitochondria and some microorganisms.

Deviations from the simple scheme of the central dogma are discovered as researchers explore gene expression with new technology. For example the human immunodeficiency virus (HIV) is a retrovirus which stores its genetic information in single stranded RNA molecules. Upon infection of a host cell, RNA is used as a template by the virally encoded enzyme, reverse transcriptase, to synthesize DNA. The viral DNA is later transcribed into mRNA and translated into proteins. Some RNA viruses such as the influenza virus never go through a DNA step. The RNA genome is replicated by an RNA dependent RNA polymerase which is virally encoded.

The content presented in this section supports the Learning Objectives outlined in Big Idea 1 and Big Idea 3 of the AP ® Biology Curriculum Framework. The Learning Objectives merge Essential Knowledge content with one or more of the seven Science Practices. These Learning Objectives provide a transparent foundation for the AP ® Biology course, along with inquiry-based laboratory experiences, instructional activities, and AP ® Exam questions.

Big Idea 1 The process of evolution drives the diversity and unity of life.
Enduring Understanding 1.B Organisms are linked by lines of descent from common ancestry.
Essential Knowledge 1.B.1 Organisms share many conserved core processes and features that evolved and are widely distributed among organisms today.
Science Practice 3.1 The student can pose scientific questions.
Science Practice 7.2 The student can connect concepts in and across domain(s) to generalize or extrapolate in and/or across enduring understandings and/or big ideas.
Learning Objective 1.15 The student is able to describe specific examples of conserved core biological processes and features shared by all domains or within one domain of life, and how these shared, conserved core processes and features support the concept of common ancestry for all organisms.
Big Idea 3 Living systems store, retrieve, transmit and respond to information essential to life processes.
Enduring Understanding 3.A Heritable information provides for continuity of life.
Essential Knowledge 3.A.1 DNA, and in some cases RNA, is the primary source of heritable information.
Science Practice 6.5 The student can evaluate alternative scientific explanations.
Learning Objective 3.1 The student is able to construct scientific explanations that use the structure and functions of DNA and RNA to support the claim that DNA and, in some cases, that RNA are the primary sources of heritable information.

Teacher Support

The Central Dogma has been validated by many experiments. The flow of information from DNA to mRNA to polypeptide is the common scheme in all cells, both prokaryotic and eukaryotic. The information in DNA is contained in the sequence of nitrogenous bases. Next question is, How is the sequence of the nitrogenous bases translated into amino acids? A combination of two out of the four letters gives 16 possible amino acids (4 2 = 16) for example, AA, or AC but, there 20 amino acids. A combination of three bases gives 64 possible sets (4 3 = 64) for example, AAA or AAC. A combination of three bases in a row is a codon or “triplets.” This gives rise to more than enough combinations for the 20 common acids. Some amino acids are specified by a single codon, for example, methionine and tryptophan others are encoded by up to six independent codons, for example, leucine.

Although protein synthesis follows the same general scheme in prokaryotes and eukaryotes, the detailed mechanism of each can be quite different. The presence of the nuclear membrane adds a layer of complexity to the process. In prokaryotes, transcription and translation are tightly coupled. As soon as the 5'-end of a mRNA has been transcribed from the template strand of DNA, ribosomes can latch onto it and polypeptide synthesis begins. Eukaryotic cells use a more complex series of steps. The enzyme RNA polymerase forms the transcription initiation complex with many proteins called transcription factors. The product of transcription, mRNA undergoes several modifications that change its stability and facilitate export from the nucleus. These extra steps allow greater control over gene expression. Although prokaryotic mRNA is not generally modified, eukaryotic mRNA strands undergo the addition of a methyl-guanosine cap at the 5'-end and a poly-adenosine tail at the 3'- end, without which they may not exit the nucleus. The mRNA also undergoes splicing to remove introns, the non–protein–coding regions of the gene. Protein translation depends on the presence of ribosomes, mRNA, a full complement of tRNA molecules, many enzymes, and many protein factors. As the polypeptide is synthesized, it starts folding into its three-dimensional structure. Further modifications will ensure that the protein is fully functional and shipped to its destination.

Ask the students what a dogma is. It will serve as an introduction to deviations from the Central Dogma. Viruses show numerous variations. The Human Immunodeficiency Virus (HIV) is a retrovirus. Its genome is encoded in RNA molecules which serve as a template for the synthesis of DNA by a virally encoded enzyme called reverse transcriptase. Point out that this enzyme, which is not found in humans, is the target of many anti-HIV medications. The flu virus carries non-coding strands of RNA molecules which are replicated in the host cell by a RNA-dependent RNA polymerase, an enzyme encoded in the viral genome. In the case of the flu virus, there is no DNA stage at all. The flow of information is RNA to RNA to proteins. Closer to “home,” the telomeres, the ends of the linear chromosomes in eukaryotes, are replicated by a special enzyme, a telomerase, which synthesizes DNA from an RNA template.

Just as we transfer information using letters and numbers, the cell transfers information using molecules. Emphasize the similarities between writing and the genetic code. Tell the students that much of the vocabulary of molecular genetics is borrowed from editing: transcription, translation, proofreading, missense, nonsense, etc.

Although the chapter does not use the term “open reading frame,” tie it to Figure 15.4. An open reading frame is a DNA sequence that follows a start codon and ends with a stop codon. A long open reading frame is likely to be a gene.

Teacher Support

Students confuse the vocabulary used to describe the Central Dogma. Copying information from DNA to RNA is transcription because the language is the same. Both are constructed using nucleotides. When a polypeptide is synthesized, the building blocks or “letters” have switched to amino acids. It is a translation. Although not quite identical, show students an example similar to the following:

Dog to Dog (transcription) to Canis (translation)

The first two words represent transcription. The letters are just copied. The last word has the same meaning, “dog” in Latin, but now the language is different.

Consider using the word “redundant” to help explain the meaning of the word “degenerate” in this context. Students confuse the fact that the code is degenerate—several codons can encode the same amino acid—with the fact that the genetic code is universal, which means that the same codon, AUG as an example, is translated as methionine in all cells. The confusion arises from students learning the two concepts at the same time. Give examples of changes in the codons which result in the same amino acids. Although the gene sequence is different, the polypeptide is the same. Remind students that each codon specifies one amino acid, but the reverse is not true. Depending on the amino acid, more than one codon will translate to the same amino acid.

Explain that many proteins of interest are synthesized in bacteria and yeast by inserting the genes for the proteins in the host expression systems. This is possible because the code is universal. If a gene coding for human insulin is inserted in the chromosomes of E. coli, the bacteria will synthesize human insulin.

Teacher Support

Give students examples of codons and ask them to find the matching amino acid. Bring to their attention that typographical errors are a great source of mutations. They should proofread their sequences carefully.

The Science Practice Challenge Questions contain additional test questions for this section that will help you prepare for the AP exam. These questions address the following standards:
[APLO 3.4][APLO 3.25]

The cellular process of transcription generates messenger RNA (mRNA), a mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the mRNA template converts nucleotide-based genetic information into a protein product. Protein sequences consist of 20 commonly occurring amino acids therefore, it can be said that the protein alphabet consists of 20 letters (Figure 15.2). Each amino acid is defined by a three-nucleotide sequence called the triplet codon. Different amino acids have different chemistries (such as acidic versus basic, or polar and nonpolar) and different structural constraints. Variation in amino acid sequence gives rise to enormous variation in protein structure and function.

The Central Dogma: DNA Encodes RNA RNA Encodes Protein

The flow of genetic information in cells from DNA to mRNA to protein is described by the Central Dogma (Figure 15.3), which states that genes specify the sequence of mRNAs, which in turn specify the sequence of proteins. The decoding of one molecule to another is performed by specific proteins and RNAs. Because the information stored in DNA is so central to cellular function, it makes intuitive sense that the cell would make mRNA copies of this information for protein synthesis, while keeping the DNA itself intact and protected. The copying of DNA to RNA is relatively straightforward, with one nucleotide being added to the mRNA strand for every nucleotide read in the DNA strand. The translation to protein is a bit more complex because three mRNA nucleotides correspond to one amino acid in the polypeptide sequence. However, the translation to protein is still systematic and colinear , such that nucleotides 1 to 3 correspond to amino acid 1, nucleotides 4 to 6 correspond to amino acid 2, and so on.

The Genetic Code Is Degenerate and Universal

Given the different numbers of “letters” in the mRNA and protein “alphabets,” scientists theorized that combinations of nucleotides corresponded to single amino acids. Nucleotide doublets would not be sufficient to specify every amino acid because there are only 16 possible two-nucleotide combinations (4 2 ). In contrast, there are 64 possible nucleotide triplets (4 3 ), which is far more than the number of amino acids. Scientists theorized that amino acids were encoded by nucleotide triplets and that the genetic code was degenerate . In other words, a given amino acid could be encoded by more than one nucleotide triplet. This was later confirmed experimentally Francis Crick and Sydney Brenner used the chemical mutagen proflavin to insert one, two, or three nucleotides into the gene of a virus. When one or two nucleotides were inserted, protein synthesis was completely abolished. When three nucleotides were inserted, the protein was synthesized and functional. This demonstrated that three nucleotides specify each amino acid. These nucleotide triplets are called codons . The insertion of one or two nucleotides completely changed the triplet reading frame , thereby altering the message for every subsequent amino acid (Figure 15.4). Though insertion of three nucleotides caused an extra amino acid to be inserted during translation, the integrity of the rest of the protein was maintained.

Scientists painstakingly solved the genetic code by translating synthetic mRNAs in vitro and sequencing the proteins they specified (Figure 15.5).

In addition to instructing the addition of a specific amino acid to a polypeptide chain, three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called nonsense codons , or stop codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also serves as the start codon to initiate translation. The reading frame for translation is set by the AUG start codon near the 5' end of the mRNA.

The genetic code is universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis. Conservation of codons means that a purified mRNA encoding the globin protein in horses could be transferred to a tulip cell, and the tulip would synthesize horse globin. That there is only one genetic code is powerful evidence that all of life on Earth shares a common origin, especially considering that there are about 10 84 possible combinations of 20 amino acids and 64 triplet codons.

Link to Learning

Transcribe a gene and translate it to protein using complementary pairing and the genetic code at this site.

  1. If there is an error in translation, the correct lipids will not be made for signaling, storage of energy or to perform vital functions. This can cause hereditary and age-related diseases.
  2. Translation is the process in which a particular segment of DNA is copied into RNA (mRNA) by the enzyme RNA polymerase. Error in such copying can lead to various hereditary and age-related diseases.
  3. Translation is the process used by ribosomes to synthesize proteins from amino acids. If there is an error in this process, the correct proteins will not be made to build important body tissue or perform vital functions thus leading to hereditary and age-related diseases.
  4. Translation is the process Golgi bodies use to synthesize proteins from amino acids. If there is an error in this process, the correct proteins will not be made to build important body tissue or perform vital functions.

Science Practice Connection for AP® Courses

Think About It

  • A strand of DNA has the nucleotide sequence 3'……GCT GTC AAA TTC GAT……5'. What is the sequence of mRNA that is complementary to this DNA sequence? Using the chart of codons in the text, determine the sequence of amino acids which can be generated from this strand of DNA.
  • How does degeneracy of the genetic code make cells less vulnerable to mutations? What is an advantage of degeneracy with respect to the negative impact of random mutations on natural selection and evolution?

Teacher Support

The first question is an application of Learning Objective 3.1 and Science Practice 6.5 because students are explaining how the language of DNA can be transcribed and translated into a sequence of amino acids.

The second set of questions are an application of Learning Objective 1.15 and Science Practice 3.1 because students are asked to raise questions about the universal genetic code and the impact of its degeneracy on mutations.

Answer

  • 3'…GCT GTC AAA TTC GAT…5'
  • mRNA 5'……CGA CAG UUU AAG CUA……3'
  • peptide…Arg Gln Phe Lys Leu……

Degeneracy is believed to be a cellular mechanism to reduce the negative impact of random mutations. Codons that specify the same amino acid typically only differ by one nucleotide. In addition, amino acids with chemically similar side chains are encoded by similar codons. This nuance of the genetic code ensures that a single-nucleotide substitution mutation might either specify the same amino acid but have no effect or specify a similar amino acid, preventing the protein from being rendered completely nonfunctional.

Scientific Method Connection

Which Has More DNA: A Kiwi or a Strawberry?

Question: Would a kiwifruit and strawberry that are approximately the same size (Figure 15.6) also have approximately the same amount of DNA?

Background: Genes are carried on chromosomes and are made of DNA. All mammals are diploid, meaning they have two copies of each chromosome. However, not all plants are diploid. The common strawberry is octoploid (8n) and the cultivated kiwi is hexaploid (6n). Research the total number of chromosomes in the cells of each of these fruits and think about how this might correspond to the amount of DNA in these fruits’ cell nuclei. Read about the technique of DNA isolation to understand how each step in the isolation protocol helps liberate and precipitate DNA.

Hypothesis: Hypothesize whether you would be able to detect a difference in DNA quantity from similarly sized strawberries and kiwis. Which fruit do you think would yield more DNA?

Test your hypothesis: Isolate the DNA from a strawberry and a kiwi that are similarly sized. Perform the experiment in at least triplicate for each fruit.

  1. Prepare a bottle of DNA extraction buffer from 900 mL water, 50 mL dish detergent, and two teaspoons of table salt. Mix by inversion (cap it and turn it upside down a few times).
  2. Grind a strawberry and a kiwifruit by hand in a plastic bag, or using a mortar and pestle, or with a metal bowl and the end of a blunt instrument. Grind for at least two minutes per fruit.
  3. Add 10 mL of the DNA extraction buffer to each fruit, and mix well for at least one minute.
  4. Remove cellular debris by filtering each fruit mixture through cheesecloth or porous cloth and into a funnel placed in a test tube or an appropriate container.
  5. Pour ice-cold ethanol or isopropanol (rubbing alcohol) into the test tube. You should observe white, precipitated DNA.
  6. Gather the DNA from each fruit by winding it around separate glass rods.

Record your observations: Because you are not quantitatively measuring DNA volume, you can record for each trial whether the two fruits produced the same or different amounts of DNA as observed by eye. If one or the other fruit produced noticeably more DNA, record this as well. Determine whether your observations are consistent with several pieces of each fruit.

Analyze your data: Did you notice an obvious difference in the amount of DNA produced by each fruit? Were your results reproducible?

Draw a conclusion: Given what you know about the number of chromosomes in each fruit, can you conclude that chromosome number necessarily correlates to DNA amount? Can you identify any drawbacks to this procedure? If you had access to a laboratory, how could you standardize your comparison and make it more quantitative?

Imagine if there were 200 commonly occurring amino acids instead of 20. Given what you know about the genetic code, what would be the shortest possible codon length? Explain.

Discuss how degeneracy of the genetic code makes cells more robust to mutations.


Immune disorders are alterations or dysregulations of components of the immune system, be it in the immune cells or their signaling pathways. These alterations can cause either low- (immune deficiency) or hyper-activity (autoimmunity) of the immune system.

Immune system deficiencies occur when immune responses fail to protect the host against infections.

Primary immune deficiencies (PID), such as severe combined immunodeficiency (SCID), occur when some parts of the immune system are absent or deficient. PIDs are usually congenital, deriving from hereditary genetic defects. 1


Diagnosis of Primary mmunodeficiencies

WARNING SIGNS AND SYMPTOMS

The National Institute of Child Health and Human Development recently initiated an educational program to raise awareness of primary immunodeficiencies. As a part of this program, the Jeffrey Modell Foundation developed a list of warning signs for primary immunodeficiency.2 These warning signs, along with other common presenting signs, are listed in Table 3 .2 , 6 , 16 A general approach to the evaluation of patients with suspected primary immunodeficiency is presented in Figure 1 .

Warning Signs of Primary Immunodeficiency Disorders

Eight or more ear infections in one year

Two or more serious sinus infections inone year

Two or more bouts of pneumonia in oneyear

Two or more deep-seated infections, orinfections in unusual areas

Recurrent deep skin or organabscesses

Need for intravenous antibiotic therapyto clear infection

Infections with unusual or opportunisticorganisms

Family history of primary immunodeficiency

Poor growth, failure to thrive

Absent lymph nodes or tonsils

Skin lesions: telangiectasias, petechiae, dermatomyositis, lupus-like rash

Ataxia (with ataxia-telangiectasia)

Oral thrush after one year of age

Adapted with permission from The 10 warning signs of primary immunodeficiency. The Jeffrey Model Foundation, Copyright 2003. Accessed October 6, 2003, at: http://npi.jmfworld.org/patienttopatient/index.cfm?section=warningsigns&ampCFID=4441749&ampCFTOKEN=89405863 , with additional information from references6 and16 .

Warning Signs of Primary Immunodeficiency Disorders

Eight or more ear infections in one year

Two or more serious sinus infections inone year

Two or more bouts of pneumonia in oneyear

Two or more deep-seated infections, orinfections in unusual areas

Recurrent deep skin or organabscesses

Need for intravenous antibiotic therapyto clear infection

Infections with unusual or opportunisticorganisms

Family history of primary immunodeficiency

Poor growth, failure to thrive

Absent lymph nodes or tonsils

Skin lesions: telangiectasias, petechiae, dermatomyositis, lupus-like rash

Ataxia (with ataxia-telangiectasia)

Oral thrush after one year of age

Adapted with permission from The 10 warning signs of primary immunodeficiency. The Jeffrey Model Foundation, Copyright 2003. Accessed October 6, 2003, at: http://npi.jmfworld.org/patienttopatient/index.cfm?section=warningsigns&ampCFID=4441749&ampCFTOKEN=89405863 , with additional information from references6 and16 .

Evaluation for Suspected Primary Immunodeficiency

Algorithm for evaluation of the patient with suspected primary immunodeficiency. (HIV = human immunodeficiency virus CBC = complete blood cell count CH50 = total hemolytic complement assay)

Evaluation for Suspected Primary Immunodeficiency

Algorithm for evaluation of the patient with suspected primary immunodeficiency. (HIV = human immunodeficiency virus CBC = complete blood cell count CH50 = total hemolytic complement assay)

LABORATORY TESTING

When primary immunodeficiency is suspected, initial laboratory studies include a complete blood cell count (CBC) with manual differential, quantitative immunoglobulin measurements (IgG, IgM, IgA), measurements of functional antibodies against immunized antigens, and delayed-type hypersensitivity skin tests (Table 4) .6 , 16 , 17 The CBC with manual differential can detect deficiencies in immune cells and platelets. In most instances, a normal CBC eliminates the diagnosis of T-cell defects or combined B-cell and T-cell defects.

Laboratory Testing for Primary Immunodeficiency Disorders

Complete blood cell count with manual differential

T-cell, B-cell, and mixed B-cell and T-cell defects

Decreased numbers of T cells, B cells, or platelets

Delayed-type hypersensitivity skin test

Negative result signaling possible impaired T-cell response*

Serum IgG, IgM, and IgA levels

Decrease in any or all of the serum immunoglobulins

Antibody testing to specific antigens after vaccination

Decreased or absent antibody response to vaccination †

Total hemolytic complement assay (CH50)

Decreased or absent proteins if there is a deficiency in the classic pathway

Nitroblue tetrazolium test

*— Delayed-type hypersensitivity skin testing involves intracutaneous injection of a recall antigen such as Candida or tetanus toxoid to a previously sensitized patient a negative result could signal impaired T-cell response or lack of exposure .

†— Normal immunoglobulin levels cannot always exclude a deficiency in antibody production therefore, IgG subclasses and antibodies to specific antigens after vaccination against diphtheria, tetanus, and pneumococcus should be measured if humoral deficiencies are still suspected .

‡— Normal cells change the yellow nitroblue tetrazolium dye to a deep blue color, because of the superoxide generated by the oxidative burst function the neutrophils of patients with chronic granulomatous disease remain colorless .

Information from references 6 , 16, and 17 .

Laboratory Testing for Primary Immunodeficiency Disorders

Complete blood cell count with manual differential

T-cell, B-cell, and mixed B-cell and T-cell defects

Decreased numbers of T cells, B cells, or platelets

Delayed-type hypersensitivity skin test

Negative result signaling possible impaired T-cell response*

Serum IgG, IgM, and IgA levels

Decrease in any or all of the serum immunoglobulins

Antibody testing to specific antigens after vaccination

Decreased or absent antibody response to vaccination †

Total hemolytic complement assay (CH50)

Decreased or absent proteins if there is a deficiency in the classic pathway

Nitroblue tetrazolium test

*— Delayed-type hypersensitivity skin testing involves intracutaneous injection of a recall antigen such as Candida or tetanus toxoid to a previously sensitized patient a negative result could signal impaired T-cell response or lack of exposure .

†— Normal immunoglobulin levels cannot always exclude a deficiency in antibody production therefore, IgG subclasses and antibodies to specific antigens after vaccination against diphtheria, tetanus, and pneumococcus should be measured if humoral deficiencies are still suspected .

‡— Normal cells change the yellow nitroblue tetrazolium dye to a deep blue color, because of the superoxide generated by the oxidative burst function the neutrophils of patients with chronic granulomatous disease remain colorless .

Information from references 6 , 16, and 17 .

Caution should be used when assessing immunologic function in newborns. Because of engrafted maternal immune cells, neonates may have both a falsely elevated lymphocyte count and evidence of graft-versus-host disease.18 If severe combined immunodeficiency is strongly suspected and the lymphocyte count is normal or nearly normal, further investigation is warranted to determine the origin of the immune cells.

When a diagnosis is uncertain, additional tests, such as genetic assays or immunophenotyping, might be performed in consultation with a pediatric immunologist.1


Resistance of primary isolates of human immunodeficiency virus type 1 to neutralization by soluble CD4 is not due to lower affinity with the viral envelope glycoprotein gp120.

Recombinant soluble CD4 (rsCD4) has potent antiviral activity against cell line-adapted isolates of the human immunodeficiency virus type 1 (HIV-1) but low activity toward HIV-1 primary isolates from patients. A simple hypothesis proposed to explain this discrepancy, which questions the therapeutic utility of soluble CD4-based approaches, is that the major envelope glycoprotein, gp120, of patient virus has lower affinity for CD4 than does gp120 from laboratory viruses. To test this hypothesis, we have produced pairs of low- and high-passage HIV-1 isolates which, depending on culture passage history, display dramatically different sensitivities to neutralization by rsCD4. Here, we present evidence that the HIV-1 major envelope glycoprotein cDNAs cloned from one such isolate pair show only minor differences in their deduced gp120 primary structures, and these occur outside regions previously shown to be involved in CD4 interactions. In addition, recombinant gp120 from a low-passage rsCD4-resistant patient virus binds rsCD4 with high affinity, equal to that previously measured for recombinant gp120 from high-passage cell line-adapted virus isolates. These data indicate that differences in CD4-gp120 affinity do not account for rsCD4 resistance in HIV-1 recently isolated from patients.


Minimal Region Sufficient for Genome Dimerization in the Human Immunodeficiency Virus Type 1 Virion and Its Potential Roles in the Early Stages of Viral Replication

FIG. 1 . The 5′ and 3′ ends of a functional domain of DLS. (A) A schematic image of monomer formation of the E/DLS duplicated mutant (DD-mutant) genome. Genomes of the WT virus form dimers, whereas those of DD-mutant form both dimers and monomers. Solid lines and open circles represent viral genome RNA and E/DLS, respectively. (B) Possible two-dimensional folds of the inserted fragment of each of the constructed mutants. nt., nucleotide. (C) Virion RNA profiles in native agarose gel. Viruses were prepared by transfection of 293T cells with pNLNh (WT) or its derivative mutants. At 48 h posttransfection, culture supernatants were harvested. Virions in the supernatant were collected by ultracentrifugation through a 20% sucrose cushion for isolation of the virion RNA. Open and solid arrowheads denote positions of dimers and monomers, respectively. FIG. 2 . Determination of the necessary and sufficient DLS in virions. (A) Probable two-dimensional folds of the inserted fragment of each of the constructed mutants. (B) Virion RNA profiles in native agarose gel. Virion RNA was isolated, and Northern hybridization was performed as described for Fig. 1. Open and solid arrowheads denote positions of dimers and monomers, respectively. FIG. 3 . Verification of the minimal DLS for its ability to induce RNA-RNA interaction in HIV-1 virions. (A) Virion RNA profiles in native agarose gel. Virion RNA was isolated, and Northern hybridization was performed as described for Fig. 1. Open and solid arrowheads denote positions of dimers and monomers, respectively. Schematic diagrams of mutants are shown above the blots. Solid lines, open circles, gray circles, and gray crosses represent viral genome RNA, authentic E/DLS, Lp4Δ2 fragments, and mutations introduced to knock out E/DLS functions, respectively. (B) A schematic Mfold representative of the verified area. FIG. 4 . Infectivity of mutant viruses. For each graph, the value of the WT was set at 1. Figures show the averages of results of at least two independent experiments. Error bars represent standard errors. (A) Single-round replication assay. M8166/H1Luc cells (1 × 10 6 ) were infected with the same quantity of CA-p24 of WT or mutant viruses pseudotyped with HIV-2 Env. At 24 to 48 h postinfection, cells were lysed and luciferase activity in the cell lysate was measured. (B) CA-p24 production and RNA packaging ability. Quantities of CA-p24 and viral RNA of purified virions were measured with the enzyme-linked immunosorbent assay and the RNase protection assay, respectively. Packaging efficiency was calculated by dividing the quantity of viral RNA by that of CA-p24. (C and D) Viral DNA quantification at early infection steps. A total of 1 × 10 6 MT-4 cells were infected with the same quantity of CA-p24 of WT or mutant viruses pseudotyped with HIV-2 Env. At 20 h postinfection, total cellular DNA was extracted and treated with DpnI overnight to digest methylated plasmid DNA. An equal amount of DNA was subjected to real-time PCR analysis. R/U5, strong-stop DNAs U3, first-strand transferred products Gag, negative-strand late products 2ndTf, second-strand transferred products 2LTR, 2-LTR viral circular DNA Alu, PCR quantification for integrated proviral DNA Infectivity, M8166/H1Luc cell assay as described for Fig. 4A. FIG. 5 . Replication assay of mutants carrying a monomeric genome. (A) Schematic diagrams of replication-competent form mutants. The positions of restriction enzyme sites on the viral genome used for insertion are shown in the upper part of the panel. Diagrams of the mutants are shown in the lower part of the panel. Symbols are the same as those described for Fig. 3. (B) Growth kinetics of viruses. Values are representative of the results of at least three independent experiments. Viruses were prepared by transfection of 293T cells with pNL4-3 (WT) or its derivative mutants (pDDEE+ [DDE], pDDXE+ [DDX], and pDTEXE+ [DTE]). At 48 h posttransfection, culture supernatants of transfected 293T cells were harvested, and equal quantities of CA-p24 were inoculated into MT-4 cells. The supernatants of the cells were harvested every 3 or 4 days. Ten microliters of each cell supernatant was subjected to exogenous RT assay and quantitated by PhosphorImager analysis. PSL, Photostimulierte Lumineszenz. (C) Virion RNA profiles produced from transfected 293T cells and visualized by native Northern blotting analysis. Open and solid arrowheads denote positions of dimers and monomers, respectively. (D) Virion RNA profiles produced from MT-4 cells. Viruses were harvested at their growth kinetic peak point (wild type, 10 days postinfection DDE and DDX, 28 days postinfection). (E) The nature of reversions. The sequences in the vicinity of the fragment-inserted sites are shown. The names of revertant sequences include “rev.” The positions of Lp4Δ2 fragment insertion of DDE and DDX are indicated. The numbers above the sequences represent nucleotide positions of pNL4-3 (WT).

33.4 Disruptions in the Immune System

In this section, you will explore the following questions:

  • What is hypersensitivity?
  • What is autoimmunity, and what is an example of an autoimmune disease?

Connection for AP ® Courses

Much of the information in this section is not within the scope for AP ® . Immune systems can, at times, be defeated by pathogens. For example, some bacteria, including Streptococcus pneumoniae, surround themselves with a capsule that inhibits phagocytes from engulfing them and displaying antigens to the adaptive immune system. Human immunodeficiency virus (HIV), the virus that causes AIDS, infects helper T-cells via their CD4 surface molecules, gradually depleting the number of TH cells in the body this inhibits the adaptive immune system’s capacity to sufficiently respond to infection or tumors that persons with healthy immune systems can defend against. Allergies to pollen or pet dander occur when the immune system attacks the body’s own cells or tissues. Other example of autoimmune diseases include type I diabetes and ALS. In the rejection of transplanted organs, the immune system is responding to unmatched MHC proteins on the cells of the donated (“non-self”) organ. However, the immune system usually responds as it should, defending you against infection and getting you back to your AP ® Biology class as soon as possible.

Information presented and the examples highlighted in the section support concepts outlined in Big Idea 2 of the AP ® Biology Curriculum Framework. The AP ® Learning Objectives listed in the Curriculum Framework provide a transparent foundation for the AP ® Biology course, an inquiry-based laboratory experience, instructional activities, and AP ® exam questions. A learning objective merges required content with one or more of the seven science practices.

Big Idea 2 Biological systems utilize free energy and molecular building blocks to grow, to reproduce, and to maintain dynamic homeostasis.
Enduring Understanding 2.D Growth and dynamic homeostasis of a biological system are influenced by changes in the system’s environment.
Essential Knowledge 2.D.3 Biological systems are affected by disruptions to their dynamic homeostasis.
Science Practice 1.4 The student can use representations and models to analyze situations or solve problems qualitatively and quantitatively.
Learning Objective 2.28 The student is able to use representations or models to analyze quantitatively and qualitatively the effects of disruptions to dynamic homeostasis in biological systems.

Immunodeficiency

Failures, insufficiencies, or delays at any level of the immune response can allow pathogens or tumor cells to gain a foothold and replicate or proliferate to high enough levels that the immune system becomes overwhelmed. Immunodeficiency is the failure, insufficiency, or delay in the response of the immune system, which may be acquired or inherited. Immunodeficiency can be acquired as a result of infection with certain pathogens (such as HIV), chemical exposure (including certain medical treatments), malnutrition, or possibly by extreme stress. For instance, radiation exposure can destroy populations of lymphocytes and elevate an individual’s susceptibility to infections and cancer. Dozens of genetic disorders result in immunodeficiencies, including Severe Combined Immunodeficiency (SCID), Bare lymphocyte syndrome, and MHC II deficiencies. Rarely, primary immunodeficiencies that are present from birth may occur. Neutropenia is one form in which the immune system produces a below-average number of neutrophils, the body’s most abundant phagocytes. As a result, bacterial infections may go unrestricted in the blood, causing serious complications.

Everyday Connection for AP® Courses

This is a white severe combined immunodeficiency (SCID) mouse. SCID mice are used to study the immune system.

Hypersensitivities

Maladaptive immune responses toward harmless foreign substances or self antigens that occur after tissue sensitization are termed hypersensitivities. The types of hypersensitivities include immediate, delayed, and autoimmunity. A large proportion of the population is affected by one or more types of hypersensitivity.

Allergies

The immune reaction that results from immediate hypersensitivities in which an antibody-mediated immune response occurs within minutes of exposure to a harmless antigen is called an allergy. In the United States, 20 percent of the population exhibits symptoms of allergy or asthma, whereas 55 percent test positive against one or more allergens. Upon initial exposure to a potential allergen, an allergic individual synthesizes antibodies of the IgE class via the typical process of APCs presenting processed antigen to TH cells that stimulate B cells to produce IgE. This class of antibodies also mediates the immune response to parasitic worms. The constant domain of the IgE molecules interact with mast cells embedded in connective tissues. This process primes, or sensitizes, the tissue. Upon subsequent exposure to the same allergen, IgE molecules on mast cells bind the antigen via their variable domains and stimulate the mast cell to release the modified amino acids histamine and serotonin these chemical mediators then recruit eosinophils which mediate allergic responses. Figure 33.27 shows an example of an allergic response to ragweed pollen. The effects of an allergic reaction range from mild symptoms like sneezing and itchy, watery eyes to more severe or even life-threatening reactions involving intensely itchy welts or hives, airway contraction with severe respiratory distress, and plummeting blood pressure. This extreme reaction is known as anaphylactic shock. If not treated with epinephrine to counter the blood pressure and breathing effects, this condition can be fatal.

Delayed hypersensitivity is a cell-mediated immune response that takes approximately one to two days after secondary exposure for a maximal reaction to be observed. This type of hypersensitivity involves the TH1 cytokine-mediated inflammatory response and may manifest as local tissue lesions or contact dermatitis (rash or skin irritation). Delayed hypersensitivity occurs in some individuals in response to contact with certain types of jewelry or cosmetics. Delayed hypersensitivity facilitates the immune response to poison ivy and is also the reason why the skin test for tuberculosis results in a small region of inflammation on individuals who were previously exposed to Mycobacterium tuberculosis. That is also why cortisone is used to treat such responses: it will inhibit cytokine production.

Autoimmunity

Autoimmunity is a type of hypersensitivity to self antigens that affects approximately five percent of the population. Most types of autoimmunity involve the humoral immune response. Antibodies that inappropriately mark self components as foreign are termed autoantibodies. In patients with the autoimmune disease myasthenia gravis, muscle cell receptors that induce contraction in response to acetylcholine are targeted by antibodies. The result is muscle weakness that may include marked difficultly with fine and/or gross motor functions. In systemic lupus erythematosus, a diffuse autoantibody response to the individual’s own DNA and proteins results in various systemic diseases. As illustrated in Figure 33.28, systemic lupus erythematosus may affect the heart, joints, lungs, skin, kidneys, central nervous system, or other tissues, causing tissue damage via antibody binding, complement recruitment, lysis, and inflammation.

Autoimmunity can develop with time, and its causes may be rooted in molecular mimicry. Antibodies and TCRs may bind self antigens that are structurally similar to pathogen antigens, which the immune receptors first raised. As an example, infection with Streptococcus pyogenes (bacterium that causes strep throat) may generate antibodies or T cells that react with heart muscle, which has a similar structure to the surface of S. pyogenes. These antibodies can damage heart muscle with autoimmune attacks, leading to rheumatic fever. Insulin-dependent (Type 1) diabetes mellitus arises from a destructive inflammatory TH1 response against insulin-producing cells of the pancreas. Patients with this autoimmunity must be injected with insulin that originates from other sources.


Primary immunodeficiency diseases: dissectors of the immune system

Summary: The past 50 years have seen enormous progress in this field. An unknown concept until 1952, there are now more than 100 different primary immunodeficiency syndromes in the world's literature. Each novel syndrome has shed new insight into the workings of the immune system, dissecting its multiple parts into unique functioning components. This has been especially true over the past decade, as the molecular bases of approximately 40 of these diseases have been identified in rapid succession. Advances in the treatment of these diseases have also been impressive. Antibody replacement has been improved greatly by the development of human immunoglobulin preparations that can be safely administered by the intravenous route, and cytokine and humanized anticytokine therapies are now possible through recombinant technologies. The ability to achieve life-saving immune reconstitution of patients with lethal severe combined immunodeficiency by administering rigorously T-cell-depleted allogeneic related haploidentical bone marrow stem cells has extended this option to virtually all such infants, if diagnosed before untreatable infections develop. Finally, the past 3 years have witnessed the first truly successful gene therapy. The impressive results in X-linked severe combined immunodeficiency offer hope that this approach can be extended to many more diseases in the future.


Autoimmunity is a type of hypersensitivity to self-antigens that affects approximately five percent of the population. Most types of autoimmunity involve the humoral immune response. An antibody that inappropriately marks self-components as foreign is termed an autoantibody. In patients with myasthenia gravis, an autoimmune disease, muscle-cell receptors that induce contraction in response to acetylcholine are targeted by antibodies. The result is muscle weakness that may include marked difficultly with fine or gross motor functions. In systemic lupus erythematosus, a diffuse autoantibody response to the individual’s own DNA and proteins results in various systemic diseases (Figure 12.23). Systemic lupus erythematosus may affect the heart, joints, lungs, skin, kidneys, central nervous system, or other tissues, causing tissue damage through antibody binding, complement recruitment, lysis, and inflammation.

Figure 12.23 Systemic lupus erythematosus is characterized by autoimmunity to the individual’s own DNA and/or proteins, which leads to varied dysfunction of the organs. (credit: modification of work by Mikael Häggström)

Autoimmunity can develop with time and its causes may be rooted in molecular mimicry, a situation in which one molecule is similar enough in shape to another molecule that it binds the same immune receptors. Antibodies and T-cell receptors may bind self-antigens that are structurally similar to pathogen antigens. As an example, infection with Streptococcus pyogenes (the bacterium that causes strep throat) may generate antibodies or T cells that react with heart muscle, which has a similar structure to the surface of S. pyogenes. These antibodies can damage heart muscle with autoimmune attacks, leading to rheumatic fever. Insulin-dependent (Type 1) diabetes mellitus arises from a destructive inflammatory TH1 response against insulin-producing cells of the pancreas. Patients with this autoimmunity must be treated with regular insulin injections.



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