An Accidental Nutritionist.

My career as an accidental nutritionist began with my immersion in cholera control, a cyclone disaster, a smallpox epidemic, and formal training in ophthalmology and epidemiology. Interest in blindness prevention inexplicably led me to (re)pioneer the effects, treatment, and prevention of vitamin A deficiency, while faced with intense criticism by many leading scientists in the nutrition community. The resulting efforts by the World Health Organization and UNICEF in support of programs for the global control of vitamin A deficiency still face vocal opposition by some senior scientists, despite having been estimated to have saved tens of millions of children from unnecessary death and blindness. This entire journey was largely an accident!

Anti-Inflammatory and Anti-Apoptotic Effects of Acer Palmatum Thumb. Extract, KIOM-2015EW, in a Hyperosmolar-Stress-Induced In Vitro Dry Eye Model.

The aim of this study was to assess the anti-inflammatory and anti-apoptotic effects of KIOM-2015EW, the hot-water extract of maple leaves in hyperosmolar stress (HOS)-induced human corneal epithelial cells (HCECs). HCECs were exposed to hyperosmolar medium and exposed to KIOM-2015EW with or without the hyperosmolar media. Tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 production and apoptosis were observed, and the activation of mitogen-activated protein kinases (MAPKs) including extracellular signal regulated kinase (ERK), p38 and c-JUN N-terminal kinase (JNK) signaling and nuclear factor (NF)-κB was confirmed. Compared to isomolar medium, the induction of cell cytotoxicity significantly increased in HCECs exposed to hyperosmolar medium in a time-dependent manner. KIOM-2015EW-treatment significantly reduced the mRNA and protein expression of pro-inflammatory mediators and apoptosis. KIOM-2015EW-treatment inhibited HOS-induced MAPK signaling activation. Additionally, the HOS-induced increase in NF-κB phosphorylation was attenuated by KIOM-2015EW. The results demonstrated that KIOM-2015EW protects the ocular surface by suppressing inflammation in dry eye disease, and suggest that KIOM-2015EW may be used to treat several ocular surface diseases where inflammation plays a key role.

A Comparison of the Effects of Benzalkonium Chloride on Ocular Surfaces between C57BL/6 and BALB/c Mice.

Models of benzalkonium chloride (BAC)-induced ocular disruption have been created and are widely used in various animals. This study aimed to compare the effects of BAC on the ocular surfaces of C57BL/6 and BALB/c mice. C57BL/6 and BALB/c mice were treated separately with BAC eye-drops at different concentrations. Eyes were evaluated by scoring epithelial disruption, corneal opacity and neovascularization in vivo, and by histological assays with hematoxylin/eosin (H/E) and periodic acid-Schiff stainings and by determining the expression of inflammatory factors in vitro on Days 7 and 14. The in vivo corneal epithelial disruption, corneal edema/opacity and neovascularization, which were in accordance with the results of the H/E staining and peaked at Day 7, were observed in a dose-dependent manner in the BAC-treated mice, with more severe signs in the C57BL/6 mice than the BALB/c mice. The loss of conjunctival goblet cells in the conjunctivas and the increasing expression of monocyte chemoattractant protein 1 (MCP-1), growth-regulated protein alpha (GROa) and macrophage inflammatory protein-1 alpha (MIP-1a) in the corneas were found in a dose-dependent manner in both strains of mice. Topical application of BAC can dramatically disrupt the ocular surfaces of C57BL/6 and BALB/c mice, and the disruptions were much more severe in the C57BL/6 mice that received high doses of BAC.

Topical Application of Apricot Kernel Extract Improves Dry Eye Symptoms in a Unilateral Exorbital Lacrimal Gland Excision Mouse.

The purpose of this study was to investigate the therapeutic effects of topical application of apricot kernel extract (AKE) in a unilateral exorbital lacrimal gland excision mouse model of experimental dry eye. Dry eye was induced by surgical removal of the lacrimal gland. Eye drops containing 0.5 or 1 mg/mL AKE were administered twice a day from day 3 to day 7 after surgery. Tear fluid volume and corneal irregularity scores were determined. In addition, we examined the immunohistochemical expression level of Muc4. The topical administration of AKE dose-dependently improved all clinical dry eye symptoms by promoting the secretion of tear fluid and mucin. Thus, the results of this study indicate that AKE may be an efficacious topical agent for treating dry eye disease.

Effects of chondrocyte-derived extracellular matrix in a dry eye mouse model.

PURPOSE: The occurrence of repetitive dry eye is accompanied by inflammation. This study investigated the anti-inflammatory effects of chondrocyte-derived extracellular matrix (CDECM) on the cornea and conjunctiva in a dry eye mouse model. METHODS: Dry eyes were experimentally induced in 12- to 16-week-old NOD.B10.H2(b) mice (Control) via subcutaneous injections of scopolamine (muscarinic receptor blocker) and exposure to an air draft for 10 days (desiccation stress [DS] 10D group). Tear volume and corneal smoothness were measured at 3, 5, 7, and 10 days after the instillation of PBS (PBS group) or CDECM (CDECM group). The corneas and conjunctivas were sectioned and stained with hematoxylin and eosin (H&E) and periodic acid Schiff (PAS). The expression of inflammatory markers (i.e., tumor necrosis factor-α [TNF-α], matrix metalloproteinase-2 [MMP-2], MMP-9, intercellular adhesion molecule-1 [ICAM-1], and vascular cell adhesion molecule-1 [VCAM-1]) was detected by quantitative real-time (qRT)-PCR and western blotting. All data were statistically processed using SPSS version 18.0. RESULTS: The instillation of CDECM after the removal of the DS increased tear production by up to 3.0-fold, and corneal smoothness improved to 80% compared to the PBS group (p<0.05). In the CDECM group, the detachment of the corneal epithelial cells was reduced by 73.3% compared to the PBS group, and the conjunctival goblet cell density was significantly recovered to the control levels (p<0.05). The expression of inflammatory factors was decreased in the cornea and conjunctiva of the CDECM group compared to the PBS group. CONCLUSIONS: These observations suggest that CDECM induced effective anti-inflammatory improvements in the cornea and conjunctiva in this experimental model of dry eye.

Expression of SIRT1 and oxidative stress in diabetic dry eye.

OBJECTIVE: To explore the expression of SIRT1 with oxidative stress and observe physiological and pathological changes in the corneas as well as the association between SIRT1 and oxidative stress of diabetic dry eyes in mice. METHOD: Forty-eight C57BL/6Jdb/db mice at eight weeks of age were divided randomly into two groups: the diabetic dry eye group and the diabetic group. An additional forty-eight C57BL/6J mice at eight weeks of age were divided randomly into two groups: the dry eye group and the control group. Every mouse in the dry eye groups (diabetic and normal) was injected with scopolamine hydrobromide three times daily, combined with low humidity to establish a dry eye model. After the intervention, phenol red cotton string tests and corneal fluorescein staining were performed. In addition, HE staining and immunofluorescence were done. Expression of SIRT1 in the cornea was examined by real-time PCR and Western Blot and expression of FOXO3 and MnSOD proteins was detected by Western Blot. RESULTS: At one, four, and eight weeks post intervention, all of the groups except the controls showed significant decreases in tear production and increases in the corneal fluorescein stain (P<0.05 vs control). Between the experimental groups, the diabetic dry eye group had the least tear production and the highest corneal fluorescein stain score (P<0.05). As the disease progressed, all of the experimental groups showed obviously pathological changes in HE staining, particularly the diabetic dry eye group. In the 1(st) and 4(th) week, the expression of SIRT1, FOXO3, and MnSOD were significantly higher in the diabetic DE and DM groups but lower in the DE group compared to the controls (P<0.05). In the 8(th) week, the expression of SIRT1, FOXO3, and MnSOD was significantly down-regulated in the diabetic DE group and the DM group (P<0.05). Immunofluorescence showed similar results. CONCLUSION: In the condition of diabetic dry eye, tear production declined markedly coupled with seriously wounded corneal epithelium. Oxidative stress in the cornea was enhanced significantly and the expression of SIRT1 was decreased.

Desiccating stress-induced chemokine expression in the epithelium is dependent on upregulation of NKG2D/RAE-1 and release of IFN-γ in experimental dry eye.

The Th1-associated chemokines CXCL9, CXCL10, and CXCL11 coordinate migration of CXCR3(+) Th1 cells. The objective of this study was to evaluate the role of the innate immune system in stimulating chemokine expression in an experimental model of dry eye and bridge the gap between innate and adaptive immunity. Desiccating stress (DS) induced very early (6 h) expression and production of Th1-associated chemokines in cornea and conjunctiva of C57BL/6 and RAG1 knockout (KO) mice, demonstrating that chemokine expression does not require innate T cells. We then demonstrated that activating the innate immune system prior to adoptive transfer of T cells to RAG1KO mice increased disease severity. Interestingly, lack of induction of chemokines CXCL9, CXCL10, and CXCL11 in IFN-γKO mice provided evidence that their expression requires IFN-γ for induction. Treatment of RAG1KO mice with anti-NK1.1 prevented the increase of CXCL9, CXCL10, and CXCL11 in response to DS, compared with isotype controls. Additionally, DS increased the expression of NKG2D in the conjunctiva. The expression of the NKG2D ligand, retinoic acid early inducible gene 1, also increased at the ocular surface at both the protein and gene levels. Neutralization of NKG2D at the ocular surface decreased the expression of CXCL9, CXCL10, CXCL11, and IFN-γ. In summary, upregulation of CXCL9, CXCL10, and CXCL11 expression in experimental dry eye is T cell-independent, requiring IFN-γ-producing NKG2D(+) NK cells that are activated in response to DS-induced stress signals. This study provides insight into the events that trigger the initial immune response in dry eye pathology.

Chemokine receptors CCR6 and CXCR3 are necessary for CD4(+) T cell mediated ocular surface disease in experimental dry eye disease.

CD4(+) T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4(+) T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4(+) T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4(+) T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease.

Surfactant protein D contributes to ocular defense against Pseudomonas aeruginosa in a murine model of dry eye disease.

Dry eye disease can cause ocular surface inflammation that disrupts the corneal epithelial barrier. While dry eye patients are known to have an increased risk of corneal infection, it is not known whether there is a direct causal relationship between these two conditions. Here, we tested the hypothesis that experimentally-induced dry eye (EDE) increases susceptibility to corneal infection using a mouse model. In doing so, we also examined the role of surfactant protein D (SP-D), which we have previously shown is involved in corneal defense against infection. Scopolamine injections and fan-driven air were used to cause EDE in C57BL/6 or Black Swiss mice (wild-type and SP-D gene-knockout). Controls received PBS injections and were housed normally. After 5 or 10 days, otherwise uninjured corneas were inoculated with 10(9) cfu of Pseudomonas aeruginosa strain PAO1. Anesthesia was maintained for 3 h post-inoculation. Viable bacteria were quantified in ocular surface washes and corneal homogenates 6 h post-inoculation. SP-D was measured by Western immunoblot, and corneal pathology assessed from 6 h to 4 days. EDE mice showed reduced tear volumes after 5 and 10 days (each by ∼75%, p<0.001) and showed fluorescein staining (i.e. epithelial disruption). Surprisingly, there was no significant difference in corneal pathology between EDE mice and controls (∼10-14% incidence). Before bacterial inoculation, EDE mice showed elevated SP-D in ocular washes. After inoculation, fewer bacteria were recovered from ocular washes of EDE mice (<2% of controls, p = 0.0004). Furthermore, SP-D knockout mice showed a significant increase in P. aeruginosa corneal colonization under EDE conditions. Taken together, these data suggest that SP-D contributes to corneal defense against P. aeruginosa colonization and infection in EDE despite the loss of barrier function to fluorescein.

Preoperative evaluation for periorbital surgery.

An effective preoperative evaluation is critical to the success of aesthetic surgery for the periorbital region. This review emphasizes important history questions and examination techniques, including advice for the nonophthalmologist surgeon, to help avoid ocular complications. Aesthetic surgery of the periocular area is a rewarding endeavor for both the patient and surgeon. Proper preoperative evaluation helps to avoid some of the pitfalls of operating in this delicate area and increases the likelihood of a positive outcome.

Accumulation of secretory vesicles in the lacrimal gland epithelia is related to non-Sjögren's type dry eye in visual display terminal users.

Previous observations in a rat model of a non-Sjögren's syndrome (non-SS) type of dry eye seen in users of visual display terminals (VDT) indicated that secretory vesicle (SV) accumulation in the lacrimal gland epithelia contributes to the condition. Here, to examine this possibility in humans, we compared the lacrimal gland histology and percent SV area in the cytoplasm of acinar epithelial cells using light microscopy and transmission electron microscopy, in patients with VDT work-related non-SS dry-eye (VDT group), SS-induced dry-eye, and autopsied normal controls. In addition, the VAMP8 (vesicle-associated membrane protein 8, an exocrine-pathway molecule) and Rab3D (mature vesicle marker) were histochemically examined in lacrimal gland tissue sections. The lacrimal gland acini were larger in the VDT group than in the SS group, and the percent SV area was significantly higher in the VDT group than in the normal controls (P = 0.021) or SS group (P = 0.004). Immunostaining revealed abnormal distributions of VAMP8 in the VDT and SS groups. Rab3D was more strongly expressed in the cytoplasm of acinar epithelial cells in the VDT group than in that of normal controls. The duration of VDT use was significantly longer in the VDT group than in the other groups. These findings suggest that excessive SV accumulation in the acinar epithelia may contribute to the reduced tear secretion in VDT users.

Hyperosmolarity potentiates toxic effects of benzalkonium chloride on conjunctival epithelial cells in vitro.

PURPOSE: Benzalkonium chloride (BAK), the most commonly used preservative in eye drops, is known to induce ocular irritation symptoms and dry eye in long-term treated patients and animal models. As tear film hyperosmolarity is diagnostic of some types of dry eye disease, we determined in vitro on conjunctival epithelial cells the cytoxicity of BAK in hyperosmolar conditions through cell viability, apoptosis, and oxidative stress assays. METHODS: The Wong Kilbourne derivative of Chang conjunctival epithelial cells were cultured for 24 h or 48 h either in NaCl-induced hyperosmolar conditions (400-425-500 mOsM), in low concentrations of BAK (10(-4)%, 3.10(-4)%, and 5.10(-4)%), or in combination of both. We investigated cell viability through lysosomal integrity evaluation, cell death (cell membrane permeability and chromatin condensation), and oxidative stress (reactive oxygen species, superoxide anion) using spectrofluorimetry. Immunohistochemistry was performed for cytoskeleton shrinkage (phalloidin staining), mitochondrial permeability transition pore (cytochrome c release), the apoptosis effector active caspase-3, and the caspase-independent apoptosis factor AIF. We also observed early effects induced by the experimental conditions on the conjunctival cell layers using phase contrast imaging of live cells. RESULTS: As compared to standard culture solutions, hyperosmolar stress potentiated BAK cytotoxicity on conjunctival cells through the induction of oxidative stress; reduction of cell viability; cell membrane permeability increase; cell shrinkage with cell blebbing, as shown in phase contrast imaging of live cells; and chromatin condensation. Like BAK, but to a much lesser extent, hyperosmolarity increased cell death in a concentration-dependent manner through a caspase-dependent apoptosis characterized by a release of cytochrome c in the cytoplasm from mitochondria and the activation of caspase-3. Moreover, the caspase-independent apoptosis factor AIF was found translocated from mitochondria to the nucleus in both conditions. CONCLUSIONS: This study showed increased cytotoxic effects of BAK in hyperosmotic conditions, with characteristic cell death processes, namely caspase-dependent and independent apoptosis and oxidative stress. As BAK is known to disrupt tear film, which could promote evaporative dry eye and tear hyperosmolarity, BAK could promote the conditions enhancing its own cytotoxicity. This in vitro hyperosmolarity model thus highlights the risk of inducing a vicious cycle and the importance of avoiding BAK in patients with dry eye conditions.

[Sjögren's syndrome: diagnosis and systemic manifestations]. / Circonstances de découverte et manifestations systémiques du syndrome de Gougerot-Sjögren.

Sjögren's syndrome is an autoimmune exocrinopathy characterized by keratoconjunctivis sicca, xerostomia and immune-inflammatory systemic manifestations. The diagnosis is easy to establish when the patient presents with sicca complex as a main symptom, or recurring attacks of parotitis. However, it is way more complex when the disease begins with extraglandular features, such as non erosive polyarticular arthritis, Raynaud's phenomenon, peripheral or central nervous system involvement, kidney disease or interstitial pneumonary disease, or even vasculitis. In such circumstances, diagnosis is often delayed several years.

Comparison of telomere length and association with progenitor cell markers in lacrimal gland between Sjögren syndrome and non-Sjögren syndrome dry eye patients.

PURPOSE: Indicators of aging such as disruption of telomeric function due to shortening may be more frequent in dysfunctional lacrimal gland. The aims of this study were to 1) determine the viability of quantitative fluorescence in situ hybridization of telomeres (telo-FISH) for the assessment of telomere length in lacrimal gland in Sjögren and non- Sjögren syndrome patients; and 2) investigate the relationship between progenitor cell markers and telomere length in both groups. METHODS: Quantitative fluorescence in situ hybridization with a peptide nucleic acid probe complementary to the telomere repeat sequence was performed on frozen sections from human lacrimal gland tissues. The mean fluorescence intensity of telomere spots was automatically quantified by image analysis as relative telomere length in lacrimal gland epithelial cells. Immunostaining for p63, nucleostemin, ATP-binding cassette, sub-family G, member 2 (ABCG2), and nestin was also performed. RESULTS: Telomere intensity in the Sjögren syndrome group (6,785.0±455) was significantly lower than that in the non-Sjögren syndrome group (7,494.7±477; p=0.02). Among the samples from the non-Sjögren syndrome group, immunostaining revealed that p63 was expressed in 1-3 acinar cells in each acinar unit and continuously in the basal layer of duct cells. In contrast, in the Sjögren syndrome group, p63 and nucleostemin showed a lower level of expression. ABCG2 was expressed in acinar cells in both sjogren and non-Sjogren syndrome. CONCLUSIONS: The results of this study indicate that 1) telo-FISH is a viable method of assessing telomere length in lacrimal gland, and 2) telomere length in Sjögren syndrome is shorter and associated with lower levels of expression of p63 and nucleostemin than in non-Sjögren syndrome.

[Phrynoderma secondary to vitamin A deficiency in a patient with biliopancreatic diversion]. / Frinoderma secundario a déficit de vitamina A en un paciente con derivación biliopancreática.

Biliopancreatic diversion (BPD) is a malabsorptivebariatric procedure can lead to the development of several nutritional complications, including fat-soluble vitamins deficiencies. Routine supplementation with vitamins and trace elements and a close follow-up long-term can prevent these nutritional risks. Vitamin A participates in ocular metabolism, epithelial differentiation, growth, and embryogenesis. Have been described several cases of ophthalmological and fetal complications associated with vitamin A deficiency in patients who have undergone BPD. Few information exists in literature about dermatologic manifestations that may occur in these patients. Phrynoderma is a type of follicular hyperkeratosis located on the extensor surfaces of the extremities whose main cause is vitamin A deficiency. We report an exceptional case of severe cutaneous and ocular complications in a patient who had undergone BPD with poor adherence to treatment and postoperative follow-up. Our patient presented simultaneously the characteristic skin lesions of phrynoderma with nytalopia and xerophthalmia in a setting of low serum levels of vitamin A. Treatment with high doses vitamin A obtained the resolution of both processes. We review and discuss the relationship between phrynoderma, malnutrition and vitamin A deficiency.

A method to extract cytokines and matrix metalloproteinases from Schirmer strips and analyze using Luminex.

PURPOSE: The Schirmer's test is commonly used in the clinic for the diagnosis of dry eye disease by measuring tear volume. This report describes a procedure which can be used to recover tears from the Schirmer strip for the measurement of multiple tear cytokines as well as matrix metalloproteinases (MMPs) by Luminex technology. METHODS: Cytokine and MMP recovery was determined by using spiked Schirmer strips presoaked with known cytokines or MMPs prepared in PBS with 1% BSA. In a clinical study, tears were collected from 5 subjects using Schirmer strips. Strips were stored on ice immediately after removal from the subject and stored dry at -20 °C for 16-24 h. Cytokines were extracted from the Schirmer strip in 0.5 M NaCl with 0.5% Tween-20. Concentrations of cytokines and MMPs in collected tear samples were analyzed by Luminex using both a 10-cytokine and a 5-MMP kit. RESULTS: The standard curves for the assay in both the kit assay buffer and extraction buffer were identical for 9 of the 10 cytokines and all 5 MMPs. In the clinical sample all the cytokines (interleukin 1α [IL-1α], IL-1ß, IL-1ra, IL-4, IL-6, IL-8, IL-10, IL-13, monocyte chemotactic protein-1 [MCP-1], and tumor necrosis factor-α [TNF-α]) and 5 MMPs (MMP-1, MMP-2, MMP-7, MMP-9, and MMP-10) tested were detected in at least 50% of the 10 subject samples. Recoveries from extracted Schirmer strips were >60% for 8 of the 10 cytokines and all MMPs. CONCLUSIONS: Numerous cytokines and MMPs were detected in the tear samples collected using the Schirmer strip, including many that have been implicated in ocular surface disease. This procedure may be used to evaluate the cytokine and MMP content in tear samples in clinical studies, especially for the evaluation of dry eye therapeutics. Because the Schirmer test is routine in the assessment of dry eye, this method offers the opportunity to evaluate both the quantity and quality of the tears.

Salivary gland involvement in chronic graft-versus-host disease: prevalence, clinical significance, and recommendations for evaluation.

Although xerostomia is a commonly reported complaint in patients with chronic graft-versus-host disease (cGVHD), criteria for evaluating the prevalence and characteristics of salivary gland involvement have not been well defined in this patient population. Previous studies also have made no distinction between salivary and mucosal oral cGVHD. We systematically evaluated signs and symptoms of sicca in a large cohort of patients with cGVHD (n = 101) using instruments widely used to study Sjogren's syndrome. Xerostomia was reported in 60 (77%) patients reporting ocular and 52 (67%) patients reporting oral complaints [corrected]. The salivary flow rate was < or =0.2 mL/min in 27%, and < or =0.1 mL/min in 16%. Histopathological changes, consisting of mononuclear infiltration and/or fibrosis/atrophy, were present in all patients with salivary dysfunction. Importantly, there was no correlation of salivary and oral mucosal involvement in cGVHD. Patients with cGVHD-associated salivary gland involvement had diminished oral cavity-specific quality of life and lower body mass index. Salivary gland involvement is a common and clinically distinct manifestation of cGVHD. Formal evaluation of salivary function using standardized criteria is needed, and this could be incorporated as an outcome measure in clinical trials of cGVHD.

[Vision jeopardizing xerophthalmia in Denmark]. / Synstruende xeroftalmi i Danmark.

A 54-year-old alcoholic was admitted with keratomalacia in 2001. The right cornea had an ulceration and the left cornea had stromal necrosis and a perforation. Serum retinol and serum betacaroten were extremely low. Treatment consisted of high-dose retinol and left sided cornea transplantation. The right eye healed with little scarring, vision: 0.5. The left eye was stabilized after a second cornea transplantation and covering with conjunctiva, vision: hand movement at a distance of 1 m. This proves the existence of symptomatic vitamin A deficiency among socially exposed subjects in Denmark.