14.4: Clinical Considerations - Biology

14.4: Clinical Considerations - Biology

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Learning Objectives

  • Explain the differences between modes of action of drugs that target fungi, protozoa, helminths, and viruses

Because fungi, protozoa, and helminths are eukaryotic, their cells are very similar to human cells, making it more difficult to develop drugs with selective toxicity. Additionally, viruses replicate within human host cells, making it difficult to develop drugs that are selectively toxic to viruses or virus-infected cells. Despite these challenges, there are antimicrobial drugs that target fungi, protozoa, helminths, and viruses, and some even target more than one type of microbe. Table (PageIndex{1}), Table (PageIndex{2}), Table (PageIndex{3}), and Table (PageIndex{4}) provide examples for antimicrobial drugs in these various classes.

Antifungal Drugs

The most common mode of action for antifungal drugs is the disruption of the cell membrane. Antifungals take advantage of small differences between fungi and humans in the biochemical pathways that synthesize sterols. The sterols are important in maintaining proper membrane fluidity and, hence, proper function of the cell membrane. For most fungi, the predominant membrane sterol is ergosterol. Because human cell membranes use cholesterol, instead of ergosterol, antifungal drugs that target ergosterol synthesis are selectively toxic (Figure (PageIndex{1})).

The imidazoles are synthetic fungicides that disrupt ergosterol biosynthesis; they are commonly used in medical applications and also in agriculture to keep seeds and harvested crops from molding. Examples include miconazole, ketoconazole, and clotrimazole, which are used to treat fungal skin infections such as ringworm, specifically tinea pedis (athlete’s foot), tinea cruris (jock itch), and tinea corporis. These infections are commonly caused by dermatophytes of the genera Trichophyton, Epidermophyton, and Microsporum. Miconazole is also used predominantly for the treatment of vaginal yeast infections caused by the fungus Candida, and ketoconazole is used for the treatment of tinea versicolor and dandruff, which both can be caused by the fungus Malassezia.

The triazole drugs, including fluconazole, also inhibit ergosterol biosynthesis. However, they can be administered orally or intravenously for the treatment of several types of systemic yeast infections, including oral thrush and cryptococcal meningitis, both of which are prevalent in patients with AIDS. The triazoles also exhibit more selective toxicity, compared with the imidazoles, and are associated with fewer side effects.

The allylamines, a structurally different class of synthetic antifungal drugs, inhibit an earlier step in ergosterol biosynthesis. The most commonly used allylamine is terbinafine (marketed under the brand name Lamisil), which is used topically for the treatment of dermatophytic skin infections like athlete’s foot, ringworm, and jock itch. Oral treatment with terbinafine is also used for the treatment of fingernail and toenail fungus, but it can be associated with the rare side effect of hepatotoxicity.

The polyenes are a class of antifungal agents naturally produced by certain actinomycete soil bacteria and are structurally related to macrolides. These large, lipophilic molecules bind to ergosterol in fungal cytoplasmic membranes, thus creating pores. Common examples include nystatin and amphotericin B. Nystatin is typically used as a topical treatment for yeast infections of the skin, mouth, and vagina, but may also be used for intestinal fungal infections. The drug amphotericin B is used for systemic fungal infections like aspergillosis, cryptococcal meningitis, histoplasmosis, blastomycosis, and candidiasis. Amphotericin B was the only antifungal drug available for several decades, but its use is associated with some serious side effects, including nephrotoxicity (kidney toxicity).

Amphotericin B is often used in combination with flucytosine, a fluorinated pyrimidine analog that is converted by a fungal-specific enzyme into a toxic product that interferes with both DNA replication and protein synthesis in fungi. Flucytosine is also associated with hepatotoxicity (liver toxicity) and bone marrow depression.

Beyond targeting ergosterol in fungal cell membranes, there are a few antifungal drugs that target other fungal structures (Figure (PageIndex{2})). The echinocandins, including caspofungin, are a group of naturally produced antifungal compounds that block the synthesis of β(1→3) glucan found in fungal cell walls but not found in human cells. This drug class has the nickname “penicillin for fungi.” Caspofungin is used for the treatment of aspergillosis as well as systemic yeast infections.

Although chitin is only a minor constituent of fungal cell walls, it is also absent in human cells, making it a selective target. The polyoxins and nikkomycins are naturally produced antifungals that target chitin synthesis. Polyoxins are used to control fungi for agricultural purposes, and nikkomycin Z is currently under development for use in humans to treat yeast infections and Valley fever (coccidioidomycosis), a fungal disease prevalent in the southwestern US.1

The naturally produced antifungal griseofulvin is thought to specifically disrupt fungal cell division by interfering with microtubules involved in spindle formation during mitosis. It was one of the first antifungals, but its use is associated with hepatotoxicity. It is typically administered orally to treat various types of dermatophytic skin infections when other topical antifungal treatments are ineffective.

There are a few drugs that act as antimetabolites against fungal processes. For example, atovaquone, a representative of the naphthoquinone drug class, is a semisynthetic antimetabolite for fungal and protozoal versions of a mitochondrial cytochrome important in electron transport. Structurally, it is an analog of coenzyme Q, with which it competes for electron binding. It is particularly useful for the treatment of Pneumocystis pneumonia caused by Pneumocystis jirovecii. The antibacterial sulfamethoxazole-trimethoprim combination also acts as an antimetabolite against P. jirovecii.

Table (PageIndex{1}) shows the various therapeutic classes of antifungal drugs, categorized by mode of action, with examples of each.

Table (PageIndex{1}): Common Antifungal Drugs
Mechanism of ActionDrug ClassSpecific DrugsClinical Uses
Inhibit ergosterol synthesisImidazolesMiconazole, ketoconazole, clotrimazoleFungal skin infections and vaginal yeast infections
TriazolesFluconazoleSystemic yeast infections, oral thrush, and cryptococcal meningitis
AllylaminesTerbinafineDermatophytic skin infections (athlete’s foot, ring worm, jock itch), and infections of fingernails and toenails
Bind ergosterol in the cell membrane and create pores that disrupt the membranePolyenesNystatinUsed topically for yeast infections of skin, mouth, and vagina; also used for fungal infections of the intestine
Amphotericin BVariety systemic fungal infections
Inhibit cell wall synthesisEchinocandinsCaspofunginAspergillosis and systemic yeast infections
Not applicableNikkomycin ZCoccidioidomycosis (Valley fever) and yeast infections
Inhibit microtubules and cell divisionNot applicableGriseofulvinDermatophytic skin infections

Exercise (PageIndex{1})

How is disruption of ergosterol biosynthesis an effective mode of action for antifungals?


Jack, a 48-year-old engineer, is HIV positive but generally healthy thanks to antiretroviral therapy (ART). However, after a particularly intense week at work, he developed a fever and a dry cough. He assumed that he just had a cold or mild flu due to overexertion and didn’t think much of it. However, after about a week, he began to experience fatigue, weight loss, and shortness of breath. He decided to visit his physician, who found that Jack had a low level of blood oxygenation. The physician ordered blood testing, a chest X-ray, and the collection of an induced sputum sample for analysis. His X-ray showed a fine cloudiness and several pneumatoceles (thin-walled pockets of air), which indicated Pneumocystis pneumonia (PCP), a type of pneumonia caused by the fungus Pneumocystis jirovecii. Jack’s physician admitted him to the hospital and prescribed Bactrim, a combination of sulfamethoxazole and trimethoprim, to be administered intravenously.

P. jirovecii is a yeast-like fungus with a life cycle similar to that of protozoans. As such, it was classified as a protozoan until the 1980s. It lives only in the lung tissue of infected persons and is transmitted from person to person, with many people exposed as children. Typically, P. jirovecii only causes pneumonia in immunocompromised individuals. Healthy people may carry the fungus in their lungs with no symptoms of disease. PCP is particularly problematic among HIV patients with compromised immune systems.

PCP is usually treated with oral or intravenous Bactrim, but atovaquone or pentamidine(another antiparasitic drug) are alternatives. If not treated, PCP can progress, leading to a collapsed lung and nearly 100% mortality. Even with antimicrobial drug therapy, PCP still is responsible for 10% of HIV-related deaths.

The cytological examination, using direct immunofluorescence assay (DFA), of a smear from Jack’s sputum sample confirmed the presence of P. jirovecii (Figure (PageIndex{3})). Additionally, the results of Jack’s blood tests revealed that his white blood cell count had dipped, making him more susceptible to the fungus. His physician reviewed his ART regimen and made adjustments. After a few days of hospitalization, Jack was released to continue his antimicrobial therapy at home. With the adjustments to his ART therapy, Jack’s CD4 counts began to increase and he was able to go back to work.

Antiprotozoan Drugs

There are a few mechanisms by which antiprotozoan drugs target infectious protozoans (Table (PageIndex{3})). Some are antimetabolites, such as atovaquone, proguanil, and artemisinins. Atovaquone, in addition to being antifungal, blocks electron transport in protozoans and is used for the treatment of protozoan infections including malaria, babesiosis, and toxoplasmosis. Proguanil is another synthetic antimetabolite that is processed in parasitic cells into its active form, which inhibits protozoan folic acid synthesis. It is often used in combination with atovaquone, and the combination is marketed as Malarone for both malaria treatment and prevention.

Artemisinin, a plant-derived antifungal first discovered by Chinese scientists in the 1970s, is quite effective against malaria. Semisynthetic derivatives of artemisinin are more water soluble than the natural version, which makes them more bioavailable. Although the exact mechanism of action is unclear, artemisinins appear to act as prodrugs that are metabolized by target cells to produce reactive oxygen species (ROS) that damage target cells. Due to the rise in resistance to antimalarial drugs, artemisinins are also commonly used in combination with other antimalarial compounds in artemisinin-based combination therapy (ACT).

Several antimetabolites are used for the treatment of toxoplasmosis caused by the parasite Toxoplasma gondii. The synthetic sulfa drug sulfadiazine competitively inhibits an enzyme in folic acid production in parasites and can be used to treat malaria and toxoplasmosis. Pyrimethamine is a synthetic drug that inhibits a different enzyme in the folic acid production pathway and is often used in combination with sulfadoxine (another sulfa drug) for the treatment of malariaor in combination with sulfadiazine for the treatment of toxoplasmosis. Side effects of pyrimethamine include decreased bone marrow activity that may cause increased bruising and low red blood cell counts. When toxicity is a concern, spiramycin, a macrolide protein synthesis inhibitor, is typically administered for the treatment of toxoplasmosis.

Two classes of antiprotozoan drugs interfere with nucleic acid synthesis: nitroimidazoles and quinolines. Nitroimidazoles, including semisynthetic metronidazole, which was discussed previously as an antibacterial drug, and synthetic tinidazole, are useful in combating a wide variety of protozoan pathogens, such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis. Upon introduction into these cells in low-oxygen environments, nitroimidazoles become activated and introduce DNA strand breakage, interfering with DNA replication in target cells. Unfortunately, metronidazole is associated with carcinogenesis (the development of cancer) in humans.

Another type of synthetic antiprotozoan drug that has long been thought to specifically interfere with DNA replication in certain pathogens is pentamidine. It has historically been used for the treatment of African sleeping sickness (caused by the protozoan Trypanosoma brucei) and leishmaniasis (caused by protozoa of the genus Leishmania), but it is also an alternative treatment for the fungus Pneumocystis. Some studies indicate that it specifically binds to the DNA found within kinetoplasts (kDNA; long mitochondrion-like structures unique to trypanosomes), leading to the cleavage of kDNA. However, nuclear DNA of both the parasite and host remain unaffected. It also appears to bind to tRNA, inhibiting the addition of amino acids to tRNA, thus preventing protein synthesis. Possible side effects of pentamidine use include pancreatic dysfunction and liver damage.

The quinolines are a class of synthetic compounds related to quinine, which has a long history of use against malaria. Quinolines are thought to interfere with heme detoxification, which is necessary for the parasite’s effective breakdown of hemoglobin into amino acids inside red blood cells. The synthetic derivatives chloroquine, quinacrine (also called mepacrine), and mefloquine are commonly used as antimalarials, and chloroquine is also used to treat amebiasis typically caused by Entamoeba histolytica. Long-term prophylactic use of chloroquine or mefloquine may result in serious side effects, including hallucinations or cardiac issues. Patients with glucose-6-phosphate dehydrogenase deficiency experience severe anemia when treated with chloroquine.

Table (PageIndex{2}): Common Antiprotozoan Drugs
Mechanism of ActionDrug ClassSpecific DrugsClinical Uses
Inhibit electron transport in mitochondriaNaphthoquinoneAtovaquoneMalaria, babesiosis, and toxoplasmosis
Inhibit folic acid synthesisNot applicableProquanilCombination therapy with atovaquone for malaria treatment and prevention
SulfonamideSulfadiazineMalaria and toxoplasmosis
Not applicablePyrimethamineCombination therapy with sulfadoxine (sulfa drug) for malaria
Produces damaging reactive oxygen speciesNot applicableArtemisininCombination therapy to treat malaria
Inhibit DNA synthesisNitroimidazolesMetronidazole, tinidazoleInfections caused by Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis
Not applicablePentamidineAfrican sleeping sickness and leishmaniasis
Inhibit heme detoxificationQuinolinesChloroquineMalaria and infections with E. histolytica
Mepacrine, mefloquineMalaria

Exercise (PageIndex{2})

List two modes of action for antiprotozoan drugs.

Antihelminthic Drugs

Because helminths are multicellular eukaryotes like humans, developing drugs with selective toxicity against them is extremely challenging. Despite this, several effective classes have been developed (Table (PageIndex{3})). Synthetic benzimidazoles, like mebendazole and albendazole, bind to helminthic β-tubulin, preventing microtubule formation. Microtubules in the intestinal cells of the worms seem to be particularly affected, leading to a reduction in glucose uptake. Besides their activity against a broad range of helminths, benzimidazoles are also active against many protozoans, fungi, and viruses, and their use for inhibiting mitosis and cell cycle progression in cancer cells is under study.2 Possible side effects of their use include liver damage and bone marrow suppression.

The avermectins are members of the macrolide family that were first discovered from a Japanese soil isolate, Streptomyces avermectinius. A more potent semisynthetic derivative of avermectin is ivermectin, which binds to glutamate-gated chloride channels specific to invertebrates including helminths, blocking neuronal transmission and causing starvation, paralysis, and death of the worms. Ivermectin is used to treat roundworm diseases, including onchocerciasis (also called river blindness, caused by the worm Onchocerca volvulus) and strongyloidiasis (caused by the worm Strongyloides stercoralis or S. fuelleborni). Ivermectin also can also treat parasitic insects like mites, lice, and bed bugs, and is nontoxic to humans.

Niclosamide is a synthetic drug that has been used for over 50 years to treat tapeworm infections. Although its mode of action is not entirely clear, niclosamide appears to inhibit ATP formation under anaerobic conditions and inhibit oxidative phosphorylation in the mitochondria of its target pathogens. Niclosamide is not absorbed from the gastrointestinal tract, thus it can achieve high localized intestinal concentrations in patients. Recently, it has been shown to also have antibacterial, antiviral, and antitumor activities.345

Another synthetic antihelminthic drug is praziquantel, which used for the treatment of parasitic tapeworms and liver flukes, and is particularly useful for the treatment of schistosomiasis (caused by blood flukes from three genera of Schistosoma). Its mode of action remains unclear, but it appears to cause the influx of calcium into the worm, resulting in intense spasm and paralysis of the worm. It is often used as a preferred alternative to niclosamide in the treatment of tapeworms when gastrointestinal discomfort limits niclosamide use.

The thioxanthenones, another class of synthetic drugs structurally related to quinine, exhibit antischistosomal activity by inhibiting RNA synthesis. The thioxanthenone lucanthone and its metabolite hycanthone were the first used clinically, but serious neurological, gastrointestinal, cardiovascular, and hepatic side effects led to their discontinuation. Oxamniquine, a less toxic derivative of hycanthone, is only effective against S. mansoni, one of the three species known to cause schistosomiasis in humans. Praziquantel was developed to target the other two schistosome species, but concerns about increasing resistance have renewed interest in developing additional derivatives of oxamniquine to target all three clinically important schistosome species.

Table (PageIndex{3}): Common Antihelminthic Drugs
Mechanism of ActionDrug ClassSpecific DrugsClinical Uses
Inhibit microtubule formation, reducing glucose uptakeBenzimidazolesMebendazole, albendazoleVariety of helminth infections
Block neuronal transmission, causing paralysis and starvationAvermectinsIvermectinRoundworm diseases, including river blindness and strongyloidiasis, and treatment of parasitic insects
Inhibit ATP productionNot applicableNiclosamideIntestinal tapeworm infections
Induce calcium influxNot applicablePraziquantelSchistosomiasis (blood flukes)
Inhibit RNA synthesisThioxanthenonesLucanthone, hycanthone, oxamniquineSchistosomiasis (blood flukes)

Exercise (PageIndex{3})

Why are antihelminthic drugs difficult to develop?

Antiviral Drugs

Unlike the complex structure of fungi, protozoa, and helminths, viral structure is simple, consisting of nucleic acid, a protein coat, viral enzymes, and, sometimes, a lipid envelope. Furthermore, viruses are obligate intracellular pathogens that use the host’s cellular machinery to replicate. These characteristics make it difficult to develop drugs with selective toxicity against viruses.

Many antiviral drugs are nucleoside analogs and function by inhibiting nucleic acid biosynthesis. For example, acyclovir(marketed as Zovirax) is a synthetic analog of the nucleoside guanosine (Figure (PageIndex{4})). It is activated by the herpes simplex viral enzyme thymidine kinase and, when added to a growing DNA strand during replication, causes chain termination. Its specificity for virus-infected cells comes from both the need for a viral enzyme to activate it and the increased affinity of the activated form for viral DNA polymerase compared to host cell DNA polymerase. Acyclovir and its derivatives are frequently used for the treatment of herpes virus infections, including genital herpes, chickenpox, shingles, Epstein-Barr virus infections, and cytomegalovirus infections. Acyclovir can be administered either topically or systemically, depending on the infection. One possible side effect of its use includes nephrotoxicity. The drug adenine-arabinoside, marketed as vidarabine, is a synthetic analog to deoxyadenosine that has a mechanism of action similar to that of acyclovir. It is also effective for the treatment of various human herpes viruses. However, because of possible side effects involving low white blood cell counts and neurotoxicity, treatment with acyclovir is now preferred.

Ribavirin, another synthetic guanosine analog, works by a mechanism of action that is not entirely clear. It appears to interfere with both DNA and RNA synthesis, perhaps by reducing intracellular pools of guanosine triphosphate (GTP). Ribavarin also appears to inhibit the RNA polymerase of hepatitis C virus. It is primarily used for the treatment of the RNA viruses like hepatitis C (in combination therapy with interferon) and respiratory syncytial virus. Possible side effects of ribavirin use include anemia and developmental effects on unborn children in pregnant patients. In recent years, another nucleotide analog, sofosbuvir (Solvaldi), has also been developed for the treatment of hepatitis C. Sofosbuvir is a uridine analog that interferes with viral polymerase activity. It is commonly coadministered with ribavirin, with and without interferon.

Inhibition of nucleic acid synthesis is not the only target of synthetic antivirals. Although the mode of action of amantadine and its relative rimantadine are not entirely clear, these drugs appear to bind to a transmembrane protein that is involved in the escape of the influenza virus from endosomes. Blocking escape of the virus also prevents viral RNA release into host cells and subsequent viral replication. Increasing resistance has limited the use of amantadine and rimantadine in the treatment of influenza A. Use of amantadine can result in neurological side effects, but the side effects of rimantadine seem less severe. Interestingly, because of their effects on brain chemicals such as dopamine and NMDA (N-methyl D-aspartate), amantadine and rimantadine are also used for the treatment of Parkinson’s disease.

Neuraminidase inhibitors, including olsetamivir (Tamiflu), zanamivir (Relenza), and peramivir (Rapivab), specifically target influenza viruses by blocking the activity of influenza virus neuraminidase, preventing the release of the virus from infected cells. These three antivirals can decrease flu symptoms and shorten the duration of illness, but they differ in their modes of administration: olsetamivir is administered orally, zanamivir is inhaled, and peramivir is administered intravenously. Resistance to these neuraminidase inhibitors still seems to be minimal.

Pleconaril is a synthetic antiviral under development that showed promise for the treatment of picornaviruses. Use of pleconaril for the treatment of the common cold caused by rhinoviruses was not approved by the FDA in 2002 because of lack of proven effectiveness, lack of stability, and association with irregular menstruation. Its further development for this purpose was halted in 2007. However, pleconaril is still being investigated for use in the treatment of life-threatening complications of enteroviruses, such as meningitis and sepsis. It is also being investigated for use in the global eradication of a specific enterovirus, polio.6 Pleconaril seems to work by binding to the viral capsid and preventing the uncoating of viral particles inside host cells during viral infection.

Viruses with complex life cycles, such as HIV, can be more difficult to treat. First, HIV targets CD4-positive white blood cells, which are necessary for a normal immune response to infection. Second, HIV is a retrovirus, meaning that it converts its RNA genome into a DNA copy that integrates into the host cell’s genome, thus hiding within host cell DNA. Third, the HIV reverse transcriptase lacks proofreading activity and introduces mutations that allow for rapid development of antiviral drug resistance. To help prevent the emergence of resistance, a combination of specific synthetic antiviral drugs is typically used in ART for HIV (Figure (PageIndex{5})).

The reverse transcriptase inhibitors block the early step of converting viral RNA genome into DNA, and can include competitive nucleoside analog inhibitors (e.g., azidothymidine/zidovudine, or AZT) and non-nucleoside noncompetitive inhibitors (e.g., etravirine) that bind reverse transcriptase and cause an inactivating conformational change. Drugs called protease inhibitors (e.g., ritonavir) block the processing of viral proteins and prevent viral maturation. Protease inhibitors are also being developed for the treatment of other viral types.7 For example, simeprevir (Olysio) has been approved for the treatment of hepatitis C and is administered with ribavirin and interferon in combination therapy. The integrase inhibitors (e.g., raltegravir), block the activity of the HIV integrase responsible for the recombination of a DNA copy of the viral genome into the host cell chromosome. Additional drug classes for HIV treatment include the CCR5 antagonists and the fusion inhibitors (e.g., enfuviritide), which prevent the binding of HIV to the host cell coreceptor (chemokine receptor type 5 [CCR5]) and the merging of the viral envelope with the host cell membrane, respectively. Table (PageIndex{4}) shows the various therapeutic classes of antiviral drugs, categorized by mode of action, with examples of each.

Table (PageIndex{4}): Common Antiviral Drugs
Mechanism of ActionDrugClinical Uses
Nucleoside analog inhibition of nucleic acid synthesisAcyclovirHerpes virus infections
Azidothymidine/zidovudine (AZT)HIV infections
RibavirinHepatitis C virus and respiratory syncytial virus infections
VidarabineHerpes virus infections
SofosbuvirHepatitis C virus infections
Non-nucleoside noncompetitive inhibitionEtravirineHIV infections
Inhibit escape of virus from endosomesAmantadine, rimantadineInfections with influenza virus
Inhibit neuraminadaseOlsetamivir, zanamivir, peramivirInfections with influenza virus
Inhibit viral uncoatingPleconarilSerious enterovirus infections
Inhibition of proteaseRitonavirHIV infections
SimeprevirHepatitis C virus infections
Inhibition of integraseRaltegravirHIV infections
Inhibition of membrane fusionEnfuviritideHIV infections

Exercise (PageIndex{4})

Why is HIV difficult to treat with antivirals?

To learn more about the various classes of antiretroviral drugs used in the ART of HIV infection, explore each of the drugs in the HIV drug classes provided by US Department of Health and Human Services at this website.

Key Concepts and Summary

  • Because fungi, protozoans, and helminths are eukaryotic organisms like human cells, it is more challenging to develop antimicrobial drugs that specifically target them. Similarly, it is hard to target viruses because human viruses replicate inside of human cells.
  • Antifungal drugs interfere with ergosterol synthesis, bind to ergosterol to disrupt fungal cell membrane integrity, or target cell wall-specific components or other cellular proteins.
  • Antiprotozoan drugs increase cellular levels of reactive oxygen species, interfere with protozoal DNA replication (nuclear versus kDNA, respectively), and disrupt heme detoxification.
  • Antihelminthic drugs disrupt helminthic and protozoan microtubule formation; block neuronal transmissions; inhibit anaerobic ATP formation and/or oxidative phosphorylation; induce a calcium influx in tapeworms, leading to spasms and paralysis; and interfere with RNA synthesis in schistosomes.
  • Antiviral drugs inhibit viral entry, inhibit viral uncoating, inhibit nucleic acid biosynthesis, prevent viral escape from endosomes in host cells, and prevent viral release from infected cells.
  • Because it can easily mutate to become drug resistant, HIV is typically treated with a combination of several antiretroviral drugs, which may include reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, and drugs that interfere with viral binding and fusion to initiate infection.


  1. 1 Centers for Disease Control and Prevention. “Valley Fever: Awareness Is Key.” Accessed June 1, 2016.
  2. 2 B. Chu et al. “A Benzimidazole Derivative Exhibiting Antitumor Activity Blocks EGFR and HER2 Activity and Upregulates DR5 in Breast Cancer Cells.” Cell Death and Disease 6 (2015):e1686
  3. 3 J.-X. Pan et al. “Niclosamide, An Old Antihelminthic Agent, Demonstrates Antitumor Activity by Blocking Multiple Signaling Pathways of Cancer Stem Cells.” Chinese Journal of Cancer 31 no. 4 (2012):178–184.
  4. 4 F. Imperi et al. “New Life for an Old Drug: The Anthelmintic Drug Niclosamide Inhibits Pseudomonas aeruginosa Quorum Sensing.” Antimicrobial Agents and Chemotherapy 57 no. 2 (2013):996-1005.
  5. 5 A. Jurgeit et al. “Niclosamide Is a Proton Carrier and Targets Acidic Endosomes with Broad Antiviral Effects.” PLoS Pathogens 8 no. 10 (2012):e1002976.
  6. 6 M.J. Abzug. “The Enteroviruses: Problems in Need of Treatments.” Journal of Infection 68 no. S1 (2014):108–14.
  7. 7 B.L. Pearlman. “Protease Inhibitors for the Treatment of Chronic Hepatitis C Genotype-1 Infection: The New Standard of Care.” Lancet Infectious Diseases 12 no. 9 (2012):717–728.


  • Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University) with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free at


Norvasc ® is indicated for the treatment of hypertension, to lower blood pressure. Lowering blood pressure reduces the risk of fatal and nonfatal cardiovascular events, primarily strokes and myocardial infarctions. These benefits have been seen in controlled trials of antihypertensive drugs from a wide variety of pharmacologic classes including Norvasc.

Control of high blood pressure should be part of comprehensive cardiovascular risk management, including, as appropriate, lipid control, diabetes management, antithrombotic therapy, smoking cessation, exercise, and limited sodium intake. Many patients will require more than one drug to achieve blood pressure goals. For specific advice on goals and management, see published guidelines, such as those of the National High Blood Pressure Education Program's Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC).

Numerous antihypertensive drugs, from a variety of pharmacologic classes and with different mechanisms of action, have been shown in randomized controlled trials to reduce cardiovascular morbidity and mortality, and it can be concluded that it is blood pressure reduction, and not some other pharmacologic property of the drugs, that is largely responsible for those benefits. The largest and most consistent cardiovascular outcome benefit has been a reduction in the risk of stroke, but reductions in myocardial infarction and cardiovascular mortality also have been seen regularly.

Elevated systolic or diastolic pressure causes increased cardiovascular risk, and the absolute risk increase per mmHg is greater at higher blood pressures, so that even modest reductions of severe hypertension can provide substantial benefit. Relative risk reduction from blood pressure reduction is similar across populations with varying absolute risk, so the absolute benefit is greater in patients who are at higher risk independent of their hypertension (for example, patients with diabetes or hyperlipidemia), and such patients would be expected to benefit from more aggressive treatment to a lower blood pressure goal.

Some antihypertensive drugs have smaller blood pressure effects (as monotherapy) in black patients, and many antihypertensive drugs have additional approved indications and effects (e.g., on angina, heart failure, or diabetic kidney disease). These considerations may guide selection of therapy.

Norvasc may be used alone or in combination with other antihypertensive agents.

Coronary Artery Disease (CAD)


Expression profiling studies in human tumors have enabled new insights into the genes and pathways that contribute to tumorigenesis and spurred the development of gene expression signatures prognostic of patient outcomes. Genes comprising prognostic signatures often provide clues to the pathobiological mechanisms that drive cancer progression. With the aim of discovering genes with statistical associations with breast cancer recurrence, we and others have identified a number of genes with roles in cellular proliferation [1–6], including multi-gene proliferation signatures that directly reflect tumor proliferative capacity [1, 4–7]. These signatures are highly significantly associated with poor patient outcomes, consistent with the view that uncontrolled cell proliferation is a central feature of neoplastic disease and, ultimately, a contributing factor in metastatic progression [8, 9]. Indeed, proliferation-associated genes are common components of many previously reported prognostic gene signatures, including Genomic Health's 21-gene Oncotype Dx test [10, 11] (Genomic Health, Inc., Redwood City, CA, USA), and frequently account for the majority of the prognostic power driving the performance of these signatures [12–14]. Thus, a clear biological understanding of how prognostic genes relate to different aspects of tumor pathobiology is imperative to both the optimal construction of prognostic models and the elucidation of key regulators of cancer behavior.

In recent years, we and others have observed that elevated expression levels of many genes involved in immune response pathways are associated with reduced risk of breast cancer recurrence [15–19]. These observations support the view that cancer-leukocyte interactions in the microenvironment of established tumors may function to limit the growth and metastatic progression of breast cancer [20–22]. However, the extent to which these genes reflect different effector cell populations, or contribute to patient prognosis in the presence of other predictive biomarkers such as proliferation, remains unclear.

In this report, we investigate the biological origins of coordinately expressed genes in breast cancer that exhibit statistical associations with patient distant metastasis-free survival (DMFS). We identify gene clusters indicative of tumor-immune cell interactions that organize into three distinct immunity-related gene signatures, or metagenes, and shed light on their prognostic implications for tumors of differing proliferative capacity with an emphasis on highly proliferative breast cancers and the most aggressive intrinsic molecular subtypes in particular.

2. Classification and Diagnosis of Diabetes


Diabetes can be classified into the following general categories:

1. Type 1 diabetes (due to autoimmune β-cell destruction, usually leading to absolute insulin deficiency, including latent autoimmune diabetes of adulthood)

2. Type 2 diabetes (due to a progressive loss of β-cell insulin secretion frequently on the background of insulin resistance)

3. Specific types of diabetes due to other causes, e.g., monogenic diabetes syndromes (such as neonatal diabetes and maturity-onset diabetes of the young), diseases of the exocrine pancreas (such as cystic fibrosis and pancreatitis), and drug- or chemical-induced diabetes (such as with glucocorticoid use, in the treatment of HIV/AIDS, or after organ transplantation)

4. Gestational diabetes mellitus (GDM diabetes diagnosed in the second or third trimester of pregnancy that was not clearly overt diabetes prior to gestation)

It is important for providers to realize that classification of diabetes type is not always straightforward at presentation, and misdiagnosis may occur. ​ Children with type 1 diabetes typically present with polyuria/polydipsia, and approximately one-third present with diabetic ketoacidosis (DKA). Adults with type 1 diabetes may not present with classic symptoms and may have a temporary remission from the need for insulin. The diagnosis may become more obvious over time and should be reevaluated if there is concern.

Screening and Diagnostic Tests for Prediabetes and Type 2 Diabetes

The diagnostic criteria for diabetes and prediabetes are shown in Table 2.2/2.5. Screening criteria are listed in Table 2.3.

Criteria for the Screening and Diagnosis of Prediabetes and Diabetes

Criteria for Testing for Diabetes or Prediabetes in Asymptomatic Adults


2.6 Screening for prediabetes and type 2 diabetes with an informal assessment of risk factors or validated tools should be considered in asymptomatic adults. B

2.7 Testing for prediabetes and/or type 2 diabetes in asymptomatic people should be considered in adults of any age with overweight (BMI 25–29.9 kg/m 2 or 23–27.4 kg/m 2 in Asian Americans) or obesity (BMI ≥30 kg/m 2 or ≥27.5 kg/m 2 in Asian Americans) and who have one or more additional risk factors for diabetes (Table 2.3). B

2.8 Testing for prediabetes and/or type 2 diabetes should be considered in women with overweight or obesity planning pregnancy and/or who have one or more additional risk factors for diabetes (Table 2.3). C

2.9 For all people, testing should begin at age 45 years. B

2.10 If tests are normal, repeat testing carried out at a minimum of 3-year intervals is reasonable, sooner with symptoms. C

2.13 Risk-based screening for prediabetes and/or type 2 diabetes should be considered after the onset of puberty or after 10 years of age, whichever occurs earlier, in children and adolescents with overweight (BMI ≥85th percentile) or obesity (BMI ≥95th percentile) and who have additional risk factors for diabetes. (See Table 2.4 for evidence grading of risk factors.) B

Risk-Based Screening for Type 2 Diabetes or Prediabetes in Asymptomatic Children and Adolescents in a Clinical Setting

An assessment tool such as the ADA risk test ( is recommended to guide providers on whether performing a diagnostic test for prediabetes or previously undiagnosed type 2 diabetes is appropriate.

Marked discrepancies between measured A1C and plasma glucose levels should prompt consideration that the A1C assay may not be reliable for that individual, and one should consider using an A1C assay without interference or plasma blood glucose criteria for diagnosis. (An updated list of A1C assays with interferences is available at Unless there is a clear clinical diagnosis based on overt signs of hyperglycemia, diagnosis requires two abnormal test results from the same sample or in two separate test samples. If using two separate test samples, it is recommended that the second test, which may either be a repeat of the initial test or a different test, be performed without delay. If the patient has a test result near the margins of the diagnostic threshold, the provider should follow the patient closely and repeat the test in 3–6 months.

Certain medications, such as glucocorticoids, thiazide diuretics, some HIV medications, and atypical antipsychotics, are known to increase the risk of diabetes and should be considered when deciding whether to screen.

14.4: Clinical Considerations - Biology

Key Dates
Release Date: June 9, 2015

Issued by
National Institutes of Health (NIH)

The National Institutes of Health (NIH) is committed to improving the health outcomes of men and women through support of rigorous science that advances fundamental knowledge about the nature and behavior of living systems. Sex and gender play a role in how health and disease processes differ across individuals1, and consideration of these factors in research studies informs the development and testing of preventive and therapeutic interventions in both sexes. This notice focuses on NIH's expectation that scientists will account for the possible role of sex as a biological variable in vertebrate animal and human studies. Clarification of these expectations is reflected in plans by NIH's Office of Extramural Research (OER) to update application instructions and review questions once approved by the Office of Management and Budget (OMB), these updates will take effect for applications submitted for the January 25, 2016, due date and thereafter. Please refer to NOT-OD-15-103 for further consideration of NIH expectations about enhancing reproducibility through rigor and transparency.


Women now account for roughly half of all participants in NIH-supported clinical research, which is subject to NIH's Policy on the Inclusion of Women in Clinical Research.2 However, more often than not, basic and preclinical biomedical research has focused on male animals and cells.3 An over-reliance on male animals and cells may obscure understanding of key sex influences on health processes and outcomes.

Accounting for sex as a biological variable begins with the development of research questions and study design. It also includes data collection and analysis of results, as well as reporting of findings. Consideration of sex may be critical to the interpretation, validation, and generalizability of research findings. Adequate consideration of both sexes in experiments and disaggregation of data by sex allows for sex-based comparisons and may inform clinical interventions. Appropriate analysis and transparent reporting of data by sex may therefore enhance the rigor and applicability of preclinical biomedical research.4


Background and aims

High prevalence of diabetes makes it an important comorbidity in patients with COVID-19. We sought to review and analyze the data regarding the association between diabetes and COVID-19, pathophysiology of the disease in diabetes and management of patients with diabetes who develop COVID-19 infection.


PubMed database and Google Scholar were searched using the key terms ‘COVID-19’, ‘SARS-CoV-2’, ‘diabetes’, ‘antidiabetic therapy’ up to April 2, 2020. Full texts of the retrieved articles were accessed.


There is evidence of increased incidence and severity of COVID-19 in patients with diabetes. COVID-19 could have effect on the pathophysiology of diabetes. Blood glucose control is important not only for patients who are infected with COVID-19, but also for those without the disease. Innovations like telemedicine are useful to treat patients with diabetes in today’s times.


This study was supported by the Irish Health Research Board (HRB) PhD Scholars Programme in Health Services Research, run jointly by Royal College of Surgeons in Ireland (RCSI), Trinity College Dublin (TCD) and University College Cork (UCC). The authors would like to thank Dr Siobhán Jennings, Health Services Executive, and Dr Brendan McAdam, Beaumont Hospital, for their guidance and contribution to the specific research project that highlighted these ethical challenges.

Telomeres and age-related disease: how telomere biology informs clinical paradigms

Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, and McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Address correspondence to: Mary Armanios, Department of Oncology, Johns Hopkins University School of Medicine, 1650 Orleans St., Cancer Research Building I Room 186, Baltimore, Maryland 21287, USA. Phone: 410.502.3817 Fax: 410.955.0125 E-mail: [email protected]

Find articles by Armanios, M. in: JCI | PubMed | Google Scholar

Telomere length shortens with age and predicts the onset of replicative senescence. Recently, short telomeres have been linked to the etiology of degenerative diseases such as idiopathic pulmonary fibrosis, bone marrow failure, and cryptogenic liver cirrhosis. These disorders have recognizable clinical manifestations, and the telomere defect explains their genetics and informs the approach to their treatment. Here, I review how telomere biology has become intimately connected to clinical paradigms both for understanding pathophysiology and for individualizing therapy decisions. I also critically examine nuances of interpreting telomere length measurement in clinical studies.

Understanding basic biological mechanisms holds the potential to advance clinical paradigms. The emerging impact of telomerase and telomere biology in medicine provides a clear example of this promise. Research in this area was initially sparked by fundamental questions about how genomes are protected at chromosome ends, and focused on curiosity-driven questions in maize, yeast, and protozoa ( 1 ). These highly conserved molecular mechanisms have now led to unforeseen benefits for understanding idiopathic disease and have opened a new area of translational research. Here I review the trajectory of the evolving role of telomere biology in clinical paradigms and highlight how it has become central to understanding the pathophysiology of age-dependent disorders as well as for informing new approaches to their treatment.

Telomeres define the ends of linear chromosomes. They are made up of repetitive DNA sequences that are bound by specialized proteins. The human telomeric DNA sequence is a tandem repeat of TTAGGG that extends several kilobases (a mean of 10 kilobases in umbilical cord blood) ( 2 – 4 ). The telomere-binding complex of proteins, known as shelterin, together with telomere DNA, functions as a dynamic unit that protects chromosome ends from being recognized as broken DNA, thus preventing their degradation and participation in fusion events ( 5 ). Telomeres are therefore essential for the maintenance of genomic integrity.

Telomerase is the specialized polymerase that synthesizes new telomere repeats ( 6 , 7 ). It offsets the shortening that normally occurs with cell division since the replication machinery does not copy fully to the ends. Telomerase has two essential core components, the telomerase reverse transcriptase (TERT) and the telomerase RNA (TR), the latter of which provides the template for telomere repeat addition ( 8 – 10 ). In human cells, telomerase is the primary mechanism by which telomeric DNA is synthesized de novo. As will be discussed, mutations in the TERT and TR genes are considered the most common cause of inherited human telomere-mediated disease ( 11 ). Even with mild perturbations in telomerase activity, telomere length homeostasis is disturbed and manifests in what has become recognized as a discrete syndrome complex, which recapitulates age-dependent disease processes ( 12 , 13 ). As such, these mutations and their clinical consequences are the primary focus of this Review.

Telomeres have long been linked to processes of cellular aging. Since the 1990s it has been known that telomere length predicts the onset of replicative senescence ( 14 , 15 ), a permanent state of cell cycle arrest that primary cells reach after they undergo a finite number of cell divisions ( 16 ). The fact that telomeres also shorten in vivo in humans with advancing age made a further compelling case for the idea that telomeres play a role in age-related processes. The evidence reviewed here shows that telomere shortening is sufficient to provoke age-related pathology. Several factors ensure that telomere shortening is a default state in somatic cells. Although telomerase offsets the end-replication problem, its levels are tightly regulated and only a few telomeres are elongated in each cell cycle ( 17 ). Therefore, even cells that may be relatively enriched for telomerase activity, such as hematopoietic stem cells, undergo telomere shortening with age ( 4 ). The incremental elongation of telomeres by telomerase can also be seen across generations ( 18 ). For both humans and mice, the telomere length of parentes determines the telomere length of their offspring ( 19 – 21 ). These observations have further established telomere length as a unique genotype (at times referred to as “the telotype”) and as a source of genetic variation across human populations ( 22 ).

When telomeres become critically short, they become dysfunctional and activate a DNA damage response that resembles double-strand breaks ( 23 ). The resulting signaling cascade provokes apoptosis and/or a permanent cell cycle arrest that, until recently, has been considered the primary functional consequence of senescence. Cell type–dependent factors determine whether apoptosis, senescence, or a combined phenotype predominates in response to dysfunctional telomeres ( 14 , 24 , 25 ), although the molecular effectors that discriminate between these pathways are not entirely understood.

Recently, a more complex understanding of the senescence phenotype has been emerging and suggests a closer link to disease mechanisms than was previously appreciated. For example, although senescent cells are quiescent in the cell cycle, for reasons that are not entirely clear, their gene expression profile is altered ( 26 ). One consequence of this altered gene expression is that senescent cells secrete a predictable profile of cytokines, chemokines, and proteases into culture media, a phenotype known as the senescence-associated secretory phenotype (SASP) ( 27 , 28 ). In vivo, the SASP has been hypothesized to play a role in the clearance of damaged cells ( 29 ). Telomere dysfunction is furthermore associated with a state of decreased cellular metabolic activity ( 25 , 30 ). In mice with short telomeres, defective cellular metabolism in the setting of senescence manifests as mitochondrial dysfunction and aberrant Ca 2+ signaling that cause insulin secretory defects by pancreatic β cells ( 25 ). These defects disturb glucose homeostasis in vivo. The fact that cellular senescence is associated with defective signaling and metabolism provides new contexts for understanding mechanisms of degenerative disease with age, particularly because these defects might occur in the absence of overt histopathology ( 25 ).

The most compelling evidence that telomeres contribute to aging comes from the fact that mutant telomerase and telomere genes cause telomere shortening that manifests in age-related phenotypes (see Telomere syndrome manifestations that overlap with human age-related phenotypes). Because telomere shortening is acquired universally with age, these disorders have a particular relevance for understanding mechanisms of age-related disease. Telomere-mediated disorders show two hallmarks of age-related disease: degenerative organ failure and a cancer-prone state ( 31 ). Age-related disease is additionally marked by atherosclerosis however, premature vascular disease has not been reported and does not, in our experience, seem to be accelerated in individuals with telomere disorders.

Eight genes have been implicated in monogenic telomere disorders (reviewed in ref. 22 ). The most prevalent are heterozygous mutations in TERT and TR, which cause autosomal dominant disease. The dominant mode of inheritance occurs as a result of the sensitivity of telomere maintenance to telomerase levels, even when only one allele is perturbed ( 12 , 18 , 32 – 35 ). Mutations in TERT and TR usually cause significant morbidity after the reproductive age is reached, and a greater number of offspring are affected as a result of their dominant mode of inheritance. They are thus estimated to be the most prevalent cause of inherited telomere disorders, comprising at least 90% of cases ( 11 ). Mutations in genes encoding the X-linked telomerase accessory component, DKC1, which is essential for human TR stability, and the autosomal shelterin gene, TINF2, explain a significant subset of pediatric telomere syndrome cases, especially in the setting of dyskeratosis congenita, which was the first genetic disorder to be linked to telomere biology ( 36 – 38 ). Biallelic mutations in the conserved telomere component 1 gene, CTC1, which plays a putative role in telomere lagging strand synthesis, have also been recently implicated in rare autosomal recessive cases that also have predominantly pediatric presentations ( 39 – 41 ). There remains a subset of cases with inherited telomere phenotypes for which the mutant genes are unknown their identification is the focus of ongoing research.

Telomere-mediated disease has diverse presentations that span the age spectrum. Their type, age of onset, and severity depend on the extent of the telomere length defect. In infancy, severe telomere shortening manifests as developmental delay, cerebellar hypoplasia, and immunodeficiency, features that are recognized in the rare Hoyeraal-Hreidarsson syndrome ( 42 ). In children and young adults, telomere-mediated disease causes bone marrow failure and at times may be recognized in the mucocutaneous syndrome dyskeratosis congenita, which is defined by a triad of mucocutaneous features — skin hyperpigmentation, dystrophic nails, and oral leukoplakia ( 33 , 43 , 44 ). Telomere-mediated disease manifests in adults as isolated or syndromic clustering of idiopathic pulmonary fibrosis (IPF), liver cirrhosis, and bone marrow failure ( 31 ). Mutant TERT and TR genes account for 8%–15% of familial and 1%–3% of sporadic pulmonary fibrosis cases ( 45 – 47 ). Because IPF affects at least 100,000 individuals in the United States, it is considered the most prevalent manifestation of the telomere disorders ( 11 ). An individual who carries a telomerase mutation will therefore most frequently be clinically recognized as an adult with familial pulmonary fibrosis. Adult-onset telomere disease may rarely also manifest as sporadic or familial myelodysplastic syndrome or acute myeloid leukemia ( 48 – 50 ). The co-occurrence of IPF and bone marrow failure within a single family is highly predictive for the presence of a germline telomerase defect ( 51 ).

Although the manifestations of telomere-mediated disease occur in multiple organs and may appear clinically different, it has been proposed that their shared short telomere length defect unifies them under the umbrella of a single syndrome continuum ( 12 , 22 , 31 , 45 , 46 , 51 ). This molecular classification is significant because the telomere defect is present in the germline of these patients and thus, even when a single presentation predominates, complications that are relevant to managing symptoms and averting complications may arise in other organs. The regrouping of what have historically been considered unrelated disorders provides new clinical insights as these conditions significantly overlap. The consideration of the telomere syndromes as a single spectrum exemplifies how a molecular classification of disease may help explain previously mysterious complications of treatment and refine clinical approaches.

The clinical manifestations of telomere shortening can be divided into two broad categories: those affecting high-turnover tissues and those affecting low-turnover tissues. This distinction is important for understanding disease patterns because the high-turnover phenotypes tend to appear first in pediatric populations and represent more severe disease (ref. 51 and Figure 1). For example, telomere syndromes in infancy manifest as severe immunodeficiency, which affects B cells, T cells, and NK cells, coincident with the extraordinary replicative demands on the adaptive immune system during this period of development ( 42 , 52 , 53 ). Bone marrow phenotypes tend to appear later in children and young adults as isolated cytopenias or aplastic anemia ( 43 , 45 , 51 , 54 ). The hematopoietic defects have been studied in animal models and represent a stem cell failure state whereby short telomere length limits both stem cell number and function ( 33 , 51 , 55 – 57 ). The telomere-mediated bone marrow failure phenotype is stem cell autonomous because allogeneic stem cell transplantation can reverse this state. The gastrointestinal epithelium, another high-turnover compartment, is also affected in a subset of patients who develop an enteropathy marked by villous blunting that resembles celiac disease ( 53 ). These intestinal phenotypes are similarly thought to be caused by stem cell failure that appears as villous atrophy in mice with short telomeres ( 18 , 58 ).

Clinical manifestations of telomere disorders and their onset relative to tissue turnover rate. Shown are representative images of diagnostic histopathology and radiographic studies in patients with telomere-mediated disease (AD) and 5-ethynyl-2′-deoxyuridine (EdU) incorporation detected in corresponding mouse tissues (EH). The estimated turnover rate of more than 90% of cells is indicated for each pair of images. (A) Photomicrograph of a bone marrow biopsy showing an acellular marrow replaced by adipose tissue with only remnants of hematopoiesis, taken from an individual with aplastic anemia. Image reproduced with permission from Annual Reviews of Genomics and Human Genetics ( 31 ). (B) Histopathology of a duodenal biopsy from a patient with telomere-mediated enteropathy shows profound villous atrophy. Image reproduced with permission from Aging Cell ( 53 ). (C) Abdominal CT scan image from a patient with liver cirrhosis, as evidenced by the nodular liver surface, the caudate lobe hypertrophy, and splenomegaly. (D) Lung windows of a chest CT scan from a carrier of the telomerase mutation show classic basilar honeycombing changes pathognomonic for IPF. (E) Flow cytometry plot of EdU incorporation in the bone marrow after a short (2-hour) pulse, showing that nearly one-third of the cells have undergone division. (F) Immunohistochemistry of intestinal section after a EdU pulse (5 days) shows that nearly all enteric epithelial cells are positively labeled (brown). (G) Brown staining shows EdU-labeled hepatocytes after EdU labeling (14 days). (H) Image of terminal bronchiole shows EdU-positive lung epithelial cells (red) identified by the Clara cell antigen (green) after 14 day label.

More commonly, telomere-mediated disease manifests in slow-turnover tissues, such as the lung and the liver (Figure 1). These phenotypes frequently appear as de novo adult-onset disease, in contrast to the pediatric presentations of dyskeratosis congenita and related disorders. IPF presents at a mean age between 50 and 60 years (range 31–87) ( 35 , 45 , 47 , 51 , 59 , 60 ), and telomere-related cryptogenic liver fibrosis, based on reported cases, presents at a mean of 37 years (range 20–57) ( 12 , 59 , 61 ). The mechanisms of these adult-onset disorders can also be distinguished in animal models. In contrast to the high-turnover phenotypes that are readily evident in the telomerase knockout mouse, telomere dysfunction in slow-turnover organs serves as the first of multiple acquired “hits” that contribute ultimately to organ failure (Figure 2). For example, mice with short telomeres do not develop de novo lung phenotypes, but acquire them only after chronic injury such as with cigarette smoke ( 62 ). Similarly, liver damage is only detected when mice with short telomeres are challenged with carbon tetrachloride ( 63 ). In the endocrine pancreas, telomere dysfunction cooperates with genetically induced endoplasmic reticulum stress to cause β cell apoptosis and manifest in worsening diabetes severity ( 25 ). Therefore, in tissues in which adult cell turnover is minimal, telomere dysfunction disturbs organ homeostasis because of cumulative hits in long-lived cells and eventually culminates in what appears as irreversible adult-onset disease (Figure 2). The cell types responsible for the telomere-induced fibrotic disorders are not known, but it has been hypothesized that these disorders, similar to the telomere-dependent bone marrow and intestinal defects, represent stem cell failure states ( 45 ). This framework has important implications for treatment strategies, as discussed below.

Model for understanding the mechanisms of telomere-mediated disease in high- and low-turnover tissues. In high-turnover tissues (left), cell replication is the primary determinant of disease onset. In contrast, in low-turnover tissues (right), other genetic and acquired hits contribute to disease onset. In both cases, telomere dysfunction induces apoptosis and/or senescence. The senescence phenotype may be associated with gene expression changes, mitochondrial dysfunction, aberrant Ca 2+ signaling, and the SASP.

Telomere length is the primary determinant of disease onset and predominant presentation in telomere disorders. This observation is supported by the fact that in families that carry mutant telomerase genes and display autosomal dominant inheritance, the disease worsens and appears earlier with each successive generation as the telomere length shortens ( 12 , 64 ). Genetic anticipation due to telomere shortening was first recognized in telomerase-null mice, which develop worsening phenotypes with successive breeding ( 18 , 58 , 65 ). In very late generations, mice die at pre-reproductive ages, which eventually limits the genetic lineage ( 19 ). The severity of the genetic anticipation in human families correlates in part with the extent of telomerase loss of function — families with functionally null telomerase alleles show more evident changes in onset across consecutive generations, in contrast to families that carry hypomorphic mutations ( 12 , 35 ). Telomere phenotypes also evolve in autosomal dominant telomere syndromes. In older generations, slow-turnover disease tends to predominate, with IPF being the primary first complication. In later generations, a bone marrow failure–predominant phenotype often comes to attention first ( 51 ). Therefore, a single telomerase gene mutation can have heterogeneous manifestations within a given family ( 51 ). This evolving pattern is unique to these Mendelian disorders and distinguishes the telomere syndromes from other conditions that show genetic anticipation, such as the trinucleotide repeat expansion syndromes ( 66 ). Clinically, this pattern of inheritance poses particular challenges to genetic counseling discussions with at-risk individuals, as the type and onset of disease may be heterogeneous and difficult to predict.

Telomere-mediated organ failure typically has a protracted course, especially in adults who may have subclinical disease for many years before becoming symptomatic ( 67 ). In some cases, for example with an offending insult such as an infection or an exposure to drug toxicities, acute declines can be sustained. In the past, this progressive course has led to a view of telomere disorders (such as aplastic anemia and IPF) as autoimmune processes, and to their empiric treatment with immunosuppression ( 33 ). With clear causal links to telomere defects, and with a growing appreciation for the full spectrum of telomere phenotypes, it is now possible to identify affected patients and thus to refine the treatment approach. Patients with telomere-related syndromes are known to have a higher incidence of adverse events with cytotoxic therapies ( 44 ) which makes the diagnostic considerations particularly important. The current treatment for telomere-mediated organ failure is primarily supportive, and its complete reversal is feasible only with organ transplantation. Below I highlight some examples in which telomere biology has affected clinical paradigms.

Patients with telomere syndromes may have subtle cosmetic features of aging (e.g., premature hair graying), but dysmorphic features are not sensitive and, in our experience, not sufficiently robust to make the diagnosis, even with training. In the setting of bone marrow transplantation, such diagnostic decisions are particularly imperative because patients with telomere syndrome have historically had poor outcomes with conventional bone marrow transplantation (reviewed in ref. 44 ). Morbidity and mortality occur primarily because of pulmonary and liver toxicity related to chemotherapy used in standard conditioning transplant regimens. With appreciation for the broad telomere-related clinical spectrum, and with the availability of DNA sequencing and telomere length measurement, improved selection has allowed for the testing of reduced-intensity regimens in dedicated studies for patients with telomere disorders. This approach has shown promising short-term outcomes ( 68 ).

IPF treatment is another evolving area in which telomere biology challenges current treatment approaches. IPF is a progressive disorder with a mean survival of 3 years from diagnosis ( 69 ). No approved treatments for IPF are currently available, and lung transplantation is accessible to only a small subset of patients who develop end-stage lung disease (less than 5%) ( 70 , 71 ). Although telomerase mutations are the most commonly identifiable genetic cause for familial pulmonary fibrosis, short telomere length in pathological ranges is a common feature even in IPF patients without mutations ( 11 ). The telomere length defect is likely in the germline, as it is concurrently seen in multiple leukocyte subsets as well as lung epithelial cells ( 46 ). This observation has led to the idea that short telomere length may be a risk factor for this disease ( 46 ). In support of the idea that telomere length might play a role in driving apparently sporadic IPF is the observation that a subset of IPF patients concomitantly develops cryptogenic liver cirrhosis, another telomere-mediated phenotype ( 46 ). In the past two decades, the idea that IPF may be an immune-mediated disease has led to the use of immunosuppressive therapy outside and within clinical trials (ref. 72 and references therein). A recent phase III trial that randomized patients with IPF to the immunosuppressive regimen of N-acetyl cysteine alone, a combination of N-acetyl cysteine, prednisone, and azathioprine, or placebo alone was stopped early because the mortality rate in the group receiving combination treatment was 8-fold higher than that in the placebo group ( 73 ). The majority of deaths were reported as respiratory in nature, but it remains unclear whether they were indirectly related to systemic toxicity. Although the role of telomere defects in sporadic forms of IPF is not yet fully understood, the lack of efficacy combined with the increased toxicity seen in recent immunosuppression trials suggests that future clinical approaches to IPF treatment should account for the fact that patients with this form of idiopathic interstitial pneumonia may be exquisitely sensitive to cytotoxic drugs. IPF patients also fare poorly with cancer treatment, an observation that is not commonly noted in patients with other lung disorders ( 74 ). It has been suggested that the apparently irreversible scarring pattern of IPF may represent a stem cell failure state that will not amenable to reversal with immunosuppression similar to telomere-mediated aplastic anemia ( 45 ). Ultimately, fundamental research in lung biology following the telomere genetic clues has the potential to open paths to new treatment paradigms for age-dependent fibrotic lung disease.

One important breakthrough that has emerged from the study of human monogenic disorders is the delineation of clinically meaningful thresholds for telomere shortening. Through the use of the telomere length method of flow cytometry and fluorescence in situ hybridization ( 75 ), the lymphocyte telomere length in patients with telomere syndrome can be stratified relative to age-matched controls in the population. Early studies that have examined this tool in the monogenic telomere disorders suggest that a threshold below the tenth percentile is sensitive, and below the first percentile is fairly specific, for distinguishing individuals who carry mutant telomere genes from their relatives who are noncarriers ( 45 , 46 , 76 ). These ranges have allowed for the use of this validated method for testing telomere length in the diagnostic work-up of suspected telomere disorders.

The fact that certain age-adjusted thresholds of telomere length have predictive value in clinical settings is significant because short leukocyte telomere length has been associated with numerous disease states and environmental factors, including chronic inflammatory states such as cancer (reviewed in ref. 77 ), cardiovascular disease (reviewed in ref. 20 ), and acquired states such as emotional stress, poor socioeconomic status, and education levels (reviewed in refs. 78 , 79 ). Although some of these variables have shown statistically significant telomere shortening consistently across studies, the biological consequences of this relative shortening cannot be equated with the severe telomere length defects seen in the monogenic telomere disorders (Figure 3). While the differences may be statistically significant, the absolute telomere length change in some cases may be small and might therefore reflect acquired replicative stress states rather than telomere-driven degenerative changes such as with the monogenic telomere syndromes. This important caveat should be considered in the interpretation of telomere epidemiology studies.

Telomere syndromes have defined pathological ranges of telomere shortening. Although short telomere length (TL) has been associated with numerous conditions, in some cases, the shortening reflects acquired replicative stress states rather than telomere-driven degenerative changes. (A) Putative dataset showing large effect size and short telomere length outside of the normal age-adjusted range. (B) Small and statistically significant change in telomere length in hypothetical dataset is less likely to reflect a telomere-mediated process.

Clinical observations in patients with telomere syndromes also shed light on the role of telomeres in cancer, which until recently had been primarily studied in cell culture and animal models. Like other DNA repair disorders, telomere disorders are cancer prone however, the overall incidence is relatively low ( 80 ). The cancer-related mortality in patients with telomere syndrome is not known, but it has been estimated that 10% of patients with dyskeratosis congenita are diagnosed with cancer ( 54 , 80 ). However, that estimate likely includes skin squamous cell cancers, which are prevalent in this group of patients and are usually not lethal ( 54 ). Cancers in dyskeratosis congenita have a predilection for high-turnover tissues, with squamous cell carcinomas of the skin and upper aerodigestive tract, myelodysplasia, and acute myeloid leukemia being the most common ( 80 ). In a cohort of adults with IPF with TERT mutations, 10% self-reported a history of cancer, although this rate was not adjusted for age or other exposures ( 60 ). These clinical observations make it clear that although telomere syndrome patients are at significantly increased risk for developing cancer, degenerative disease accounts for the majority of the morbidity and mortality in at least 90% of cases.

The relatively low overall incidence of cancers in patients with telomere disorders underscores the fact that in the presence of an intact DNA damage response, short telomere length predominantly causes cell loss in humans. These observations are in line with the long-hypothesized role of telomere shortening as a powerful tumor-suppressive mechanism ( 81 ). Studies in animal models have shown that short telomeres suppress tumorigenesis by mediating p53-dependent apoptosis and senescence ( 82 ). In mice with short telomeres that also lack p53, genomic instability fuels carcinogenesis ( 83 – 85 ). Whether short telomere length in human cancers may contribute to genomic instability at a low level remains a question of ongoing study. Other explanations have been hypothesized to underlie the tumor-prone nature of telomere syndromes, such as compromised immunosurveillance due to the associated immunodeficiency phenotype ( 22 ). The stem cell exhaustion state itself has also been proposed to contribute to tumorigenesis, and this would explain the tumor-prone nature of stem cell failure states, such as occurs with non–telomere-mediated aplastic anemia. The clinical study of disease driven by telomere defects provides a unique opportunity to refine current ideas about the role of telomere dysfunction in human cancer development and progression.

Hypothesized molecular mechanisms for aging in modern biology have abounded. These have included stem cell failure, mitochondrial dysfunction, genotoxic stress, and epigenetic changes. Recent cumulative evidence points to telomere shortening as sufficient to provoke all these mechanisms. The manifestations of telomere-mediated disease, especially in adults, can be subtle and are often indistinguishable from the slow, gradual functional decline that is a hallmark of aging. The compelling clinical evidence therefore points to telomere shortening itself as being sufficient, or perhaps more broadly representing forms of genotoxic stress that contribute to age-related changes.

In the past decade, telomere biology has provided a molecular rationale for unifying a group of historically considered unrelated disorders under the umbrella of telomere syndromes. The rich, context-dependent clinical presentations of these single-gene disorders and their now appreciated overlap highlight how a molecularly based understanding of disease can refine clinical care at the bedside. This new understanding underscores how the interpretation of increasingly available genetic information might require clinical contextualization before it can be readily applied. Beyond these conceptual considerations, telomere biology has of late brought new tools for diagnosis as well as for understanding disease mechanisms in areas that have long been perplexing to clinicians. Such novel paradigms are particularly needed when it comes to approaching difficult problems such as IPF. The coming years will undoubtedly point to new examples of how the biology of these DNA ends may advance clinical care.

I am particularly indebted to Jonathan Alder for helpful discussions and for assistance with the figures, and to Carol Greider for critical comments on the manuscript. I acknowledge funding support from the NIH (grants R21 HL104345 and RO1 CA160433), the Maryland Stem Cell and Commonwealth Foundations, and the Flight Attendants Medical Research Institute.

Conflict of interest: The author has declared that no conflict of interest exists.

Reference information: J Clin Invest. 2013123(3):996–1002. doi:10.1172/JCI66370.

Host—fungus interaction

Key factors involved in fungal infections seem to be shared among distantly related fungi pathogenic to different hosts [24, 33].

The respiratory tract is the main entrance for fungi, among them Zygomycetes. Inhaled aerosolized spores are removed by the movements of ciliated epithelial cells. If some manage to overcome this barrier the alveolar macrophages phagocytose and destroy most spores. Healthy gastrointestinal tract and skin are good barriers to Zygomycetes. It is not surprising that most skin infections are a consequence of direct inoculation due to severe trauma. Gastrointestinal infections are most common in neonates, who have not yet developed proper immune mechanisms.

The description of host pathogen interactions at a molecular level shows differences in the mechanisms of aspergillosis and mucormycosis, including host immune response and susceptibility to macrophage-induced hyphal damage [34]. Furthermore, biological features of the causing agent determine the location and progress of the infection, leading to a more localized infection in entomophthoromycosis and a progressive and potentially disseminated infection in mucormycosis. Entomophthorales specialized in cutin degradation exert keratinolytic enzymes as a consequence entomophthoromycoses are usually superficial and gastrointestinal infections. In contrast to Entomophthorales, human-infecting Mucorales are usually thermo-tolerant, which makes them able to invade internal body parts and form a chronic infection.

The interplay between pathogens and hosts varies among fungal agents and the host condition. However, some general trends can be drawn. Most of the mechanisms are known from animal data the latter should be considered with caution as the inflammatory reaction is different in rodents and apes. The role of the immune system in preventing and eliminating fungal infections involves an inflammatory reaction. Inflammation triggered by Zygomycota is usually less visible than in aspergillosis. Ascomycota are recognized by both TRL2 and TRL4 receptors, whereas Zygomycota are recognized solely by TLR2 receptors [34]. There is another major difference compared with aspergillosis regarding the involvement of T lymphocytes. This is because innate immunity is indispensable in fighting a Zygomycota infection and the acquired immunity involvement is not that pronounced. However, Mucorales-specific T cells and NK cells can be found both in patients and in healthy individuals. These findings pave the way for potential diagnostic tests and adoptive immunotherapy [35].

One of the most characteristic features of zygomycoses is their occurrence in diabetic patients. Some aspects of the interplay between the fungus and diabetic patients has been revealed at a molecular level. During the infection R. oryzae binds to GRP78 receptors [36, 37]. The expression of GRP78 receptor coding genes is affected by acidosis and by iron and glucose levels, which are observed in diabetic patients (high blood sugar levels and available iron). In such a scenario R. oryzae become more resistant to neutrophils, which are susceptible to low pH conditions. One taxon may produce hyphae and spores of variable size. Phagocyte-induced damage is related to target mass bigger hyphae are harder to phagocytose. Schmidt and colleagues [38] demonstrated that both unstimulated and IL-2-prestimulated human NK cells damage Rhizopus oryzae hyphae, but do not affect resting conidia. They concluded that the damage of the fungus is mediated, at least in part, by perforin. R. oryzae hyphae decrease the secretion of immunoregulatory molecules by NK cells, such as IFN-γ and RANTES (regulated on activation, normal T-cell expressed and secreted), indicating an immunosuppressive effect of the fungus.

A study on the activity of human polymorphonuclear leukocytes (PMNLs) against R. oryzae, R. microsporus, and L. corymbifera revealed that interferon (IFN)-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) augment the hyphal damage of all three zygomycetes. Additionally, this effect was more pronounced against L. corymbifera than against both Rhizopus species [39].

Genus-specific patterns could be drawn from pulled data from multiple infections. It is currently known that Cunninghammella is more aggressive than Lichtheimia in diabetes patients [34]. It induces low TNFα levels and is resistant to hyphae damage by macrophages. In rare cases it can invade an immunocompetent host [40], which suggests some of the specific properties of Cunninghammella compared with other Mucorales. Cunninghammella infections have higher mortality rates than other, more common infections [8].

The New Standard for Single-Cell Functional Biomarkers

Explore our award-winning IsoLight System.

Unprecedented Ability

The IsoLight is the only technology able to detect highly potent single-cell functional subsets and measure the true cytokines secretion of every cell. Our proprietary & patented “Proteomic Barcoded” IsoCode Chip, named the Scientist’s #1 innovation, detects 30+ cytokines per cell. You can run 1,000+ cells on a single chip, giving you the unprecedented ability to reveal the functional phenotype differences of each immune cell.

Fully Automated

Load up to 8 IsoCode Chips onto the IsoLight system for a completely automated and hands-off end-to-end workflow. All cellular analysis and washing steps associated with ELISA platforms are automated, minimizing the risk of human error. With advanced fluidics and precision imaging, live single cells are automatically detected within an onboard incubator, helping you get to answers in days, not weeks.

Actionable Insights

The intuitive IsoSpeak software push-button user interface enables researchers to automatically visualize, target, and utilize data from direct, functional cytokine profiling of single cells. Make sense of complex single-cell data with meaningful visualizations automatically generated for you. Connect the dots and get answers faster, with detailed informatics & statistics on cellular potency and durability, all at your fingertips.

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How Our Unique Biology Works

Our functional biology can accurately detect what individual T-cells are secreting for the first time, rather than estimations. These unique cellular subsets are depicted as the blue highly polyfunctional cells (cells that secrete two or more cytokines) in the top right of the graph.

Samples with multiple subsets of these cells have high polyfunctional strength, which has correlated with outcome.

32-Plex Single-Cell Adaptive Immune Panel

The ability to capture the range of relevant cytokines from each immune cell represents a unique secreted protein multiplexing capability. The mechanism for both clinical effects and cytotoxicity can be heavily mediated by cytokines (functional proteins through which immune cells send and receive signals).

Stratify Donor/Patient Response by Cellular Cytokine Signature

Polyfunctional Activation Topology Principal Component Analysis (PAT-PCA) is a high dimensional map showing which cells secrete multiple cytokines per cell and groups them into corresponding functional groups.

The Dominant subgroups will emerge in the PAT-PCA graphs, representing significant multi-functional subsets driving the overall response.

Samples with a higher response dominate the graph. Differences between donors are highlighted through the location of their polyfunctional subsets in the graph.

Group Bioprocessing Correlations by Potent Polyfunctional Cells

A polyfunctional heat map is a visualization comparing the frequency at which various functional and polyfunctional groups are secreted by the sample. Use Heterogeneity Heat Maps to uncover the critical cell subpopulations that exist only in the condition/group of interest.

Discover Cell Product Potency Differences with PSI Cytokine Signatures

The Single Cell Polyfunctional Strength Index (PSI) aggregates all single-cell, multi-dimensional secretions from a sample into a single index. The readout combines the polyfunctionality of a sample (frequency of cells secreting multiple cytokines) with the signal intensities for each single-cell across the secreted cytokines of the sample. The displayed index is color-coded to show the contribution from different categories of cytokines (e.g., effector vs. stimulatory cytokines).

PSI is the most novel and revealing metric for measuring the potency of different immune cell types, helping top researchers accelerate their immunotherapy programs from discovery to predicting response.

Watch the video: Antibody Therapy in Acute Leukemia: Clinical Considerations in Conjunction With HSCT (October 2022).