Lynda Charters

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Carotenoids are pigments that occur naturally and, as well as providing the red-to-orange colours of many fruits and vegetables, have important functions in the eye. One such carotenoid, beta-carotene, is an essential nutrient and the major precursor of vitamin A.

Beta-carotene is converted in the human body to two metabolites: 11-cis-retinal and all-transretinoic acid. Through those molecules, it affects major physiological functions (vision, cellular differentiation, metabolic control, reproduction and embryonic development and organogenesis), according to Dr Johannes von Lintig, a professorin the Department of Pharmacology at Case Western Reserve University, Cleveland, Ohio, United States.

In addition to (pro) vitamin A, other carotenoids are absorbed and accumulated in the central retina (the macula lutea) of the human eyes, namely lutein, zeaxanthin and meso-zeaxanthin. All of these substances reduce chromatic aberrations, increase visual acuity and prevent oxidative stress to photoreceptors.

The most important research finding regarding carotenoid and vitamin A metabolism is the association with numerous diseases, such as xerophthalmia; age-related macular degeneration; micro-ophthalmic syndromes; retinitis pigmentosa; and iris, retinal and choroidal coloboma.

Although a greatdeal is known about vitamin A metabolism in the retina, Dr von Lintigwas interested in the mechanism of carotenoid accumulation in the macula. His laboratory identified a carotenoid scavenger gene,beta-carotene-oxygenase 2 (BCO2), which encodes a mitochondrial enzyme expressed in the inner membrane of the mitochondria that breaks down carotenoids, such as zeaxanthin, into smaller entities.

The enzyme is important in producing the colours seen in nature. As Dr von Lintig and colleagues from Columbia University, New York, US,recently demonstrated, it is responsible for, among other things, the colours in betta fish. In addition, investigators showed that genetic variation in the BCO2gene influences the colours of feathers and skin in several bird species.

These genetic investigations have also been conducted in human eyes, where carotenoids accumulate in distinct patterns. BCO2 expressionis high in the peripheral retina,where there are no carotenoids, and low in the central fovea,where the carotenoids accumulate. This is reflected in single-cell sequencing.

The research into cone photoreceptors showed some surprising findings. The photoreceptors are rich in mitochondria, which is surprising because they rely on glycolysis rather than respiration mediated by these powerhouses of the cell. 

“Based on our research, we propose that photoreceptors are rich in mitochondria because, in the peripheral retina, BCO2-dependent carotenoid breakdown takes place in these organelles. In the central retina, the carotenoids are amassed in the mitochondria because of the absence of BCO2,” Dr von Lintig said.

However, an unresolved question is how the carotenoids enter the mitochondria. Dr von Lintig pointed out that carotenoids are lipids and cannot simply diffuse in an aqueous environment as sugars or amino acids do. They need specific transport proteins to move from one membrane to the other.

Dr von Lintig looked for candidate transport proteins that are expressed in the photoreceptors and found that the one with the highest expression in photoreceptors is Aster-B. “Aster-B was recently characterised in the context of cholesterol metabolism and transports cholesterol to mitochondria for steroid hormone production in the adrenal gland,” Dr von Lintig said. The next question addressed was whether Aster-B was really expressed in the retina.

To answer this question, the investigators analysed eye samples to determine whether Aster-B helped the carotenoids access the mitochondria. Interestingly, opposite expression patterns of BCO2 and Aster-B were found in the retina of human donor eyes. Immunohistochemistry showed Aster-B expression in the inner and outer segments of the photoreceptors.

Dr von Lintig also reported that Aster-B proteins, which constitute a novel family of lipid transfer proteins, have a domain-like structure. The proteins have a transmembrane domain in the endoplasmic reticulum, a lipid-binding domain and a phosphatidylserine-binding domain that connects them to another membrane. Aster-B proteins resemble bridges that catalyse the movement of lipids, such as cholesterol, from one membrane tothe other.

To determine whether the Aster proteins perform the same task with carotenoids, Dr von Lintig and colleagues invented a novel test system for which they bioengineered a new strain of bacteria that produced carotenoids. They expected that recombinant Aster proteins should extract the pigments from membranes when expressed in this bacterial strain. 

“That is exactly what they did,” he said. “Molecular modelling showed that the bulky carotenoid molecules fit perfectly into the binding cavity of Aster proteins.”

The investigators concluded that BCO2, which metabolises carotenoids and expresses in the peripheral retina, and Aster-B, which transports proteins, are important players in macula pigment metabolism and are responsible for the distinct pattern of accumulation of the pigments in the human retina.

This article is adapted from Dr von Lintig’s presentation at Cleveland Eye Bank Foundation’s 2022 Virtual Vision Research Symposium. He has no financial interest in this subject matter.

Articles

Recent identification of the BCO2 gene and the Aster-B transport protein gives insight into carotenoid distribution in the retina.  

New insights into macular carotenoid accumulation and transport

Great strides have been made in recognising and treating keratoconus. Recent imaging technologies, such as optical coherence tomography, have identified thinning of corneal tissue over the cone, and wave-front aberrometry shows higher-order aberrations that may facilitate the diagnosis.

Yet the pathway to diagnosing the condition is not always free of roadblocks, according to Dr Sumit Garg, professor of cataract, corneal and refractive surgery at the Gavin Herbert Eye Institute, University of California, Irvine, California, United States. Dr Garg said that keratoconus has two components: genetic and environmental. The latter, he said, includes allergies and eye rubbing (during waking and sleeping hours), which is a huge driver of keratoconus progression. If eye rubbing persists, progression can occur despite cross-linking treatment.

Dr Garg also pointed out that the diagnosis can depend on where a patient lives and their genetic make-up. “The keratoconus prevalence is individualised and [keratoconus] can be prevalent in up to 5% of particular populations as shown in a recent study,” he said.

 With the rapid advancements in this field, the next step is determining the role of genetic testing in this patient population.

Genetic testing in patients at risk of keratoconus is in its infancy. Dr Garg reported that AvaGen75 (Avellino) is a test—the first of its kind— that looks at variants of genes reported to be associated with keratoconus. “The test, which looks at 75 genes and more than 2,000 variants, provides a scale ranging from 0 to 100 that defines the genetic risk of a patient developing keratoconus,” Dr Garg said.

The manufacturer reports that the test provides a risk score from data based on multiple gene clusters that have a high correlation with keratoconus, with the goal of providing an early diagnosis. Dr Garg emphasised that keratoconus is polygenic and not binary (i.e., the presence of genes does not necessarily mean that a patient has clinical evidence of keratoconus). He also cited a study

 that found that when genetic testing is coupled with advanced imaging, the chances of identifying keratoconus early are greater.

In his practice, he is offering genetic testing as an option, especially when there is a suspicion of keratoconus. Those who request a refractive surgery and those with family members who have been diagnosed with keratoconus are also offered testing.

Representative results of genetic testing

Dr Garg reported the case of a woman aged 32 years, who was referred for an ocular evaluation for a possible LASIK procedure. The patient was highly myopic with high cylinder, but her vision had been stable. The best-corrected vision was 20/20 bilaterally; she denied eye rubbing.

The topography maps indicated possible posterior changes, with some steepening in the corneal centre, and changes on the ABCD score. The Kmax values were generally thin overall. The patient had no family history of keratoconus.

Genetic testing showed moderate-to-high risk of keratoconus. The decision was made not to perform the refractive procedure and to perform a follow-up examination in 6 months. If the changes were progressive, corneal cross-linking could be considered. If follow-up showed no progression, an implantable collamer lens could be considered.

A second case was that of a woman aged 31 years, who presented after being evaluated for LASIK in another practice, where a photorefractive keratectomy was recommended. The patient, who was a mild myope with low cylinder, had no recent refractive changes and she denied eye rubbing.

The patient’s best-corrected vision was 20/20. The maps did not show any abnormality and the genetic testing was inconclusive. The patient became pregnant and any refractive considerations were postponed.

A third case was that of a man aged 27 years, who presented for a LASIK evaluation. The patient was a mild myope and previously wore contact lenses. The refraction was stable. His maps showed slight corneal thinning but a good residual stromal bed. His father had a history of keratoconus. Dr Garg said that for this patient, genetic testing is the next step and may help in the treatment plan.

“Genetic testing is new and holds a lot of promise,” Dr Garg concluded. “It helps us to risk-stratify our patients, help with clinical quandaries, and hopefully will facilitate earlier diagnosis and screening for keratoconus. It is important that the disease is not binary but rather a spectrum. Patients must understand that if they undergo genetic testing it will not determine whether they have keratoconus. Genetic testing will provide another data point to help identify keratoconus.”

This article is adapted from Dr Garg’s presentation at the American Society of Cataract and Refractive Surgery’s annual meeting in Washington, D.C. He is a consultant to Avellino and Glaukos.

1. Torres Netto EA, Al-Otaibi WM, Hafezi NL, et al. Prevalence of keratoconus in paediatric patients in Riyadh, Saudi Arabia. 

2. Chen S, Li XY, Jin JJ, et al. Genetic screening revealed latent keratoconus in asymptomatic individuals. 

According to one study, when genetic testing is coupled with advanced imaging, the chances of identifying keratoconus early are greater.

Genetic testing for keratoconus facilitates earlier recognition of condition

Vernal keratoconjunctivitis (VKC) is a progressive, vision-threatening condition. However, it is rare and may not be in the forefront of clinicians’ minds when evaluating a child with a persistent allergic ocular surface complaint. In fact, 88% of clinicians in one study noted that primary care physicians and paediatricians commonly under-diagnose or misdiagnose the disorder—which then goes untreated or undertreated—largely because the later involvement of the cornea can be misleading.

This finding was reported by Dr Leonard Bielory, a professor of medicine, allergy, immunology, and ophthalmology at Hackensack Meridian School of Medicine in Nutley, New Jersey, United States. The limited awareness of VKC is the focus for Dr Bielory, who emphasised it as a barrier to timely identification and management of this disruptive ocular surface disorder. Frequently, VKC is initially misidentified as an allergy or infection, and referral to a specialist is often delayed until symptoms have become severe.

Some of the corneal and conjunctival findings in vernal conjunctivitis. 

VKC begins in the warmest months worldwide. This characteristic can lead clinicians to confuse it with springtime (or “vernal”) allergic conjunctivitis. The corneal inflammatory component, however, emerges later and is recurring. As Dr Bielory stated, “The condition is thought to mislead clinicians into thinking it is just a seasonal problem [but it] may also have a perennial component that develops with time.”

The key factor, Dr Bielory said, is that VKC should be considered in children who present with a persistent ocular allergic complaint, which may be the more pronounced type of conjunctivitis that can affect the cornea. A clue to diagnosis may emerge when parents report using over-the-counter (OTC) ocular treatments to no avail. “This should be the trigger to additional, more thoughtful involvement by their clinicians, such as referral to allergy specialists and ophthalmologists,” he advised.

The following timeline may be helpful, Dr Bielory explained. During the patient’s first season with an ocular reaction, vernal conjunctivitis and seasonal conjunctivitis may have similar presentations. During the second season of the allergic response, however, the vernal component (associated with warmth and different regional pollinating seasons) begins to progress more than the seasonal component, which remains seasonal.

Then, possibly during the second and third seasons, a great degree of progression may be seen during the year of sensitisation, bearing in mind that there are individual degrees of progression among patients. The patient then moves into the clinical phase.

Dr Bielory explained that a confounding factor in the diagnosis of VKC is how allergies develop (i.e., over time) in infants and young children. Immunoglobulin G antibodies inherited from the mothers protect babies for about 6 months. Immunoglobulin E, the allergy antibody, requires exposure to an offending agent over time, followed by development of the immune response against that agent.

Allergies may begin to manifest in babies 6–9 months after birth. Allergies exhibit nasal and ocular symptoms (rhinoconjunctivitis) in almost equal proportions, as physicians should know.

During the second year for children with developing VKC, symptoms worsen and become more unresponsive to treatment. An allergy work-up is needed, but, Dr Bielory stated, when ocular corticosteroids are prescribed and the patient does not improve, clinicians should suspect something more than seasonal allergic conjunctivitis (i.e., the vernal component).

Minor VKC symptoms include photophobia, tearing, burning and pruritus, with minimal impact on a patient’s quality of life. This scenario is often misdiagnosed as pink eye or allergic conjunctivitis.

Moderate VKC symptoms include photophobia, pruritus and discharge of mucus, with moderate sizes and numbers of cobblestone papillae in the upper tarsal conjunctiva or around the edge of the cornea (limbal form of VKC). Patient quality of life is moderately affected.

Severe symptoms of photophobia, pruritus and discharge of mucus, are accompanied by large cobblestone papillae, punctate epithelial erosions and Horner-Trantas dots. Quality of life is substantially affected, and the disease can potentially threaten vision if left untreated as it starts to affect the visual axis of the cornea.

As mentioned, OTC ocular treatments will not work. Use of oral antihistamines may cause increased ocular dryness over and above the abnormal tear film development in the vernal component.

“With antihistamines, there begins to be an increase in tear film dysfunction that comes with the allergy affecting the ocular surface,” Dr Bielory said. “However, in VKC, over-production of tears in response to corneal irritation usually leads to practitioners increasing the use of antihistamines, compounding the ocular surface issue,” he added.

Nevertheless, within this less-than-clear clinical picture, several treatments can be called into play. Treatment of the ocular surface progresses from lubricants to ophthalmic antihistamines to corticosteroids. Dr Bielory uses the steroid burst treatments over 1–2 weeks to avoid increases in intraocular pressure.

He advised that physicians should be aware of the chronic inflammation that induces the activity of certain cell types that will lead to corneal involvement. At this point, Dr Bielory explained, steroid-sparing treatments should be introduced, such as ciclosporin, which decreases the overall immune response of IL-2 and the cytokines.

Ciclosporin ophthalmic emulsion 0.1% (Verkazia,Santen), a steroid-sparing drug, is the first and only topical immunomodulator approved by the United States Food and Drug Administration for treating adults and children with VKC. In addition, Sinusol Breathe Easy (DRBRX), a non-steroidal OTC product, works by clearing nasal mucus and decongestion, which also benefits the ocular symptoms associated with allergies.

In addition to his role at Hackensack Meridian School of Medicine, Dr Bielory is an adjunct professor at the Center of Aerobiological Research at Rutgers University in New Brunswick, New Jersey, US; and director of the Center of Aerobiological Research at Kean University in Union, New Jersey. Dr Bielory is a consultant to Santen Pharmaceutical and DRBRX.

Ophthalmologists should look past the eyes in patients presenting with a persistent ocular allergic response and be mindful that sight-threatening vernal keratoconjunctivitis is a possibility.

Circumnavigating the bumpy road to vernal keratoconjunctivitis: Time is key

Spanish investigators from the Ophthalmology Service, Hospital Clínico San Carlos, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos, Madrid, led by first author Fernando Ly-Yang, MD, reported the case of a 42-year-old man with monkeypox. The patient had HIV and presented with conjunctivitis.

The patient’s symptoms included lacrimation, pain, and photophobia of the left eye for 6 days. Facial maculopapular lesions developed on his face and foot on the 5th day after symptom onset. The patient had a fever and painful cervical adenopathy and was HIV positive.

The patient had not traveled recently or had contact with monkeypox cases. However, his boyfriend worked in a sex club and had a fever.

Slit-lamp examination of the affected eye showed ulcerations on the eyelid margin, mucoid discharge, and conjunctival whitish, serpinginous, infiltrative lesions with conjunctival thickening. The cornea was unaffected.

Dermatologic evaluation of the skin resulted in a clinical diagnosis with high suspicion for monkeypox.

Polymerase chain reaction testing of the conjunctiva and skin was positive for monkeypox and negative for herpes viruses.

The authors reported that the patient received systemic treatment with 600 mg tecovirimat every 12 hours and intravenous acyclovir 1 gram every 8 hours, and topical zinc sulfate every 8 hours on the skin lesions. Ocular topical treatment included the following drops administered 5 times daily: chlorhexidine 0.2%, ganciclovir 0.15%, moxifloxacin, and povidone iodine 1%.

During the second week of treatment, conjunctival pseudomembranes developed during the second week of treatment. The pseudomembranes were removed and and topical fluorometholone 4 times daily was started.

The skin lesions resolved first and the conjunctival lesions resolved after 4 weeks of treatment.

The authors pointed out that conjunctivitis has been reported in 23% of monkeypox cases, but generally the male patients are younger than 10 years and have other symptoms in contrast to the current patient who was 42 years and did not have systemic signs or symptoms.

“The presence of conjunctival inflammation could occur more often in immunocompromised individuals given that the patient was HIV positive or it could be due to the contiguous spread of the periocular skin lesions,” the investigators wrote.

The investigators also noted that it is unknown if monkeypox is secreted in tears during conjunctivitis. However, the smallpox virus is secreted in tears, and monkeypox is a similar virus. Therefore, conjunctivitis may transmit the disease.

“A case of monkeypox in a patient with HIV with conjunctivitis is documented,” investigators concluded. “Conjunctivitis associated with monkeypox might present before skin lesions and may be a way of transmitting the disease.”

Ly-Yang F, Miranda-Sánchez A, Burgos-Blasco B, et al. Conjunctivitis in an individual with monkeypox. 

. Published Online September 7, 2022. doi:10.1001/jamaophthalmol.2022.3743

Spanish investigators hypothesise that conjunctivitis plays a role in disease transmission.

Author | Lynda Charters

Articles

Genetic testing for keratoconus facilitates earlier recognition of condition

Circumnavigating the bumpy road to vernal keratoconjunctivitis: Time is key

Monkeypox may be linked with conjunctivitis

New insights into macular carotenoid accumulation and transport