Effects of alpha-tocopherol on gingival expression of inducible nitric oxide synthase in the rats with experimental periodontitis and diabetes.

BACKGROUND: The aim of this study was to investigate effects of α-tocopherol and/or insulin on the number of gingival inducible nitric oxide synthase (iNOS) positive cells in rats with experimental periodontitis with or without streptozotocin (STZ)-induced diabetes. MATERIALS AND METHODS: A total of 60 Sprague-Dawley rats were divided into three groups: Group I: The group without diabetes; Group II: The group with STZ-induced diabetes; Group III: The group with STZ-induced diabetes receiving insulin therapy. All animals received anesthesia, and 3/0 silk suture was inserted around the mandibular molar teeth. All groups were divided into subgroups receiving saline (Groups IA, IIA, IIIA) and α-tocopherol injection (Groups IB, IIB, IIIB). After a period of 3 weeks, all rats were sacrificed, and the number of gingival iNOS positive cells was analyzed using image analysis software. RESULTS: Applying α-tocopherol suppressed the number of gingival iNOS positive cells in Groups IB, IIB, and IIIB compared to application of saline (Groups IA, IIA, and IIIA) (P < 0.05). Numbers of gingival iNOS positive cells were found to be similar in the Groups I and III (P > 0.05). CONCLUSIONS: Within limitations of the current study, this is the first study one may suggest that α-tocopherol may reduce oxidative damage in the gingiva of the rats with periodontitis with or without STZ-induced diabetes and increase effects of insulin.

Effects of iloprost and piracetam in spinal cord ischemia-reperfusion injury in the rabbit.

STUDY DESIGN: Experimental Study. OBJECTIVES: The aim of this study was to investigate the neuroprotective effects of iloprost and piracetam on spinal cord ischemia/reperfusion (I/R) injury in the rabbit. SETTINGS: The Experimental Research Center of Selcuk University, Konya, Turkey. METHODS: A total of 24 rabbits were divided into four groups of six rabbits each, as follows: group 1 (n = 6) sham, laparotomy only; group 2 (n = 6) I/R; group 3 (n = 6) I/R+iloprost; and group 4 (n = 6) I/R+piracetam. I/R was established in groups 2, 3 and 4. Subsequently, they were followed up neurologically for 24 h until the rabbits were killed; biochemical and histopathological examinations of samples from the spinal cord were carried out. RESULTS: Neurological examination results were significantly better in the iloprost and piracetam groups compared with the I/R group (P < 0.05). Neuroprotection was achieved with iloprost and piracetam by suppressing malondialdehyde (P < 0.05), increasing glutathione peroxidase activity (P < 0.05) and decreasing the xanthine oxidase level. In histopathological assessment, iloprost and piracetam groups were statistically different from the I/R group in terms of the number of apoptotic neurons in gray matter and white matter, as well as in terms of degenerated neurons and glial cells (P < 0.05). No statistical difference was determined between the four groups in the number of degenerated glial cells (P > 0.05). CONCLUSION: This study has shown that iloprost and piracetam have neuroprotective effects in I/R injury both neurologically and histopathologically because of inhibition of lipid peroxidation.

The effects of chronic administration of sumatriptan and dipyrone on serotonergic system in the rat brain: an immunohistochemical study.

OBJECTIVE: To investigate the effects of chronic high dose sumatriptan and dipyrone treatment on central serotonergic system in rats. MATERIALS AND METHODS: Male Sprague-Dawley rats (seven per group) were daily injected with sumatriptan (3 mg/kg), dipyrone (400 mg/kg) or saline for 30 days. The brains of animals were surgically removed and immunohistochemically stained for serotonin. Serotonin-positive stained cells were counted automatically by using a computerized image analysis program. Statistical analysis carried out using one-way ANOVA followed by post hoc Tukey test. RESULTS: A significant decrease in serotonin-positive cells in the brainstem was observed after chronic sumatriptan administration while chronic use of dipyrone induced a significant increase in serotonin-positive cells both in the cortex and midbrain. CONCLUSION: Our data suggest that central serotonergic system might be modified by chronic use of sumatriptan and dipyrone.

The effects of resveratrol administration on lipid oxidation in experimental renal ischemia-reperfusion injury in rats.

We investigated how resveratrol affects lipid oxidation during experimental renal ischemia-reperfusion injury in rats. We used 48 adult male rats assigned to five groups: group 1, control; group 2, renal ischemia; group 3, renal ischemia + reperfusion; group 4, resveratrol + renal ischemia; group 5, resveratrol + renal ischemia + reperfusion. Plasma and renal tissue malondialdehyde (MDA), and erythrocyte and renal tissue glutathione (GSH) levels were measured and histologic changes in the renal tissue were examined. Ischemia-reperfusion affected the MDA-GSH balance adversely and caused histopathological changes in the renal tissue of the ischemia and ischemia + reperfusion groups. Resveratrol treatment normalized MDA and GSH levels as well as the histopathology that occurred in the renal tissue of the ischemia and ischemia + reperfusion groups.

Melatonin treatment for prevention of oxidative stress: involving histopathological changes.

This study was undertaken to test the effect of irradiation on the histopathology of the hypothalamus and cerebral cortex. In addition, the probable effects of radiotherapy on the activities of antioxidant enzymes and levels of nitric oxide (NO) in the plasma were investigated as well. The effects of melatonin treatment on radiotherapy-based central nervous system (CNS) damage were also studied. For this purpose, the rats were randomized into four groups. The first group was the control group (sham-exposed group), the second group received only melatonin, the third group was irradiated and the fourth group received both melatonin and irradiation. Plasma samples of rats were collected for measuring the activities of superoxide dismutase (SOD), catalase (CAT) and the levels of NO. 24 h after the interventions, tissue samples were obtained from the hypothalamus and the cerebral cortex for the light microscopic investigations. These tissues were mostly affected by radiation. The results indicated that the application of radiation significantly enhanced the levels of plasma SOD and NO. On the other hand, melatonin pretreatment prevented the decrease in plasma CAT activity induced by irradiation. It was found that the application of melatonin could significantly prevent the irradiation-induced damages. Light microscopic results revealed that the damage of the CNS by radiation was prevented by the application of melatonin.

The effect of human amniotic fluid on mandibular distraction osteogenesis.

The aim of this study was to evaluate the effects of local administration of human amniotic fluid (HAF) on newly formed bone obtained by mandibular distraction osteogenesis (DO) with histomorphometry. A unilateral mandibular osteotomy at the left corpus was performed in 32 adult male rabbits. After a 5-day latency period, the left mandibles were lengthened by mandibular DO over 5 days, at a rate of 1mm/day, via a custom-made distractor. After the distraction, the rabbits were divided randomly into four groups: 0.3 ml HAF was injected into the distraction gap followed by 21 (group 1) or 45 (group 2) days of consolidation; or 0.3 ml normal saline (NS) was administered followed by 21 (group 3) or 45 (group 4) days of consolidation. Mandibles were removed at the end of the consolidation period and investigated histomorphometrically. The newly formed bone area (NFBA) and number of fibroblasts increased significantly in the HAF groups compared to the NS groups (NFBA: group 1 vs. group 3, P<0.05; group 2 vs. group 4, P<0.01; fibroblasts: group 1 vs. group 3, and group 2 vs. group 4, P<0.05), and also in both 45-day consolidation groups compared to the 21-day consolidation groups (NFBA: group 1 vs. group 2, and group 3 vs. group 4, P<0.001; fibroblasts: group 1 vs. group 2, and group 3 vs. group 4, P<0.01). Additionally, the numbers of osteoblasts and capillaries were increased significantly at 45 days of consolidation compared to 21 days in both the HAF and NS groups (osteoblasts: group 1 vs. group 2, P<0.01; group 3 vs. group 4, P<0.05; capillaries: group 1 vs. group 2, and group 3 vs. group 4, P<0.01). Histomorphometric analysis demonstrated that local HAF administration effectively accelerated bone formation. Thus, a HAF injection procedure could improve new bone formation around the bone in maxillofacial operations such as DO.

Prevention of lens damage associated with cigarette smoke exposure in rats by alpha-tocopherol (vitamin E) treatment.

PURPOSE: To evaluate the possible protective effect and mechanism of alpha-tocopherol (vitamin E) treatment on lens degeneration associated with in vivo exposure to cigarette smoke and to further clarify the role of iron in cigarette smoke-generated lens damage. METHODS: Twenty-eight male Wistar rats were randomly divided into four equal groups. Rats in groups 3 and 4 were exposed to cigarette smoke for 1 hour each day over 90 consecutive days, and rats in groups 1 and 2 were treated in similar fashion but only exposed to room air. Additionally, vitamin E was given to the rats in groups 2 and 4 via intramuscular route. At the end of the study, both eyes of all the animals were enucleated; one eye was prepared for histopathologic examination, and the fellow eye was used for the measurement of iron and calcium levels. RESULTS: Significantly higher iron and calcium levels were observed in the lenses of group 3 rats than in other groups. Similar comparisons performed between groups 1 and 2, groups 1 and 4, and groups 2 and 4 did not show any significant difference. Distinct histopathologic changes in the anterior lens epithelium, such as hyperplasia, hypertrophy, epithelial multilayering, and the presence of epithelial cells over posterior lens capsule, observed in group 3 rats were not present in other groups. CONCLUSIONS: Cataractogenesis after cigarette smoke exposure was associated with an accumulation of iron and calcium in the rat lens, and vitamin E supplementation protected such accumulations and cataractogenesis.

Cataractous changes in rat lens following cigarette smoke exposure is prevented by parenteral deferoxamine therapy.

OBJECTIVES: To test whether iron accumulation in the lens following cigarette smoke exposure is the principal mechanism in smoke-related cataractogenesis and to assess the possible protective effect of deferoxamine mesylate treatment against lenticular degeneration with in vivo exposure to cigarette smoke. METHODS: Thirty-two male Wistar rats were randomly divided into 4 equal groups. Groups 3 and 4 rats were exposed to cigarette smoke for 1 hour each day for 90 consecutive days, and groups 1 and 2 rats were treated in a similar manner but exposed only to room air. In addition, deferoxamine was given subcutaneously to groups 2 and 4 rats. Both eyes of all the animals were then enucleated and 1 eye prepared for histopathological examination. The fellow eye was used to measure iron, calcium, zinc, and copper levels. RESULTS: Significantly higher iron and calcium and lower zinc levels were observed in the lenses of group 3 rats compared with those in the other groups. Similar comparisons performed between groups 1 and 2, 1 and 4, and 2 and 4 did not show any significant difference. Copper concentrations did not differ between groups. Distinct histopathological changes in the anterior lens epithelium, such as hyperplasia, hypertrophy, and epithelial multilayering, and the presence of swollen epithelial cells overlying the posterior lens capsule, observed in group 3 rats, were not present in the other groups. CONCLUSIONS: Cataractogenesis following cigarette smoke exposure in rats was associated with the accumulation of iron, and concurrent deferoxamine therapy prevented such cataract formation. CLINICAL RELEVANCE: Our results may apply to human cataract formation associated with cigarette smoking, so such pathogenesis may be prevented by concurrent parenteral deferoxamine treatment. Clinical studies are needed, however, to determine the value of this suggestion.

Cadmium and iron accumulation in rat lens after cigarette smoke exposure and the effect of vitamin E (alpha-tocopherol) treatment.

PURPOSE: Cadmium accumulation in the lens has been implicated in cataractogenesis of chronic smokers. This study was planned to evaluate whether or not in vivo cigarette smoke exposure causes cadmium accumulation in rat lens, and possible protective effect and mechanism of alpha-tocopherol (vitamin E) treatment on cataractogenesis. METHODS: 28 male Wistar rats were randomly divided into four equal groups. Group 3 and 4 rats were exposed to cigarette smoke over ninety consecutive days, and Group 1 and 2 rats were treated in a similar fashion but exposed only to room air. Additionally, vitamin E was given to Group 2 and 4 rats. RESULTS: Significantly higher iron levels were observed in the lenses of Group 3 rats compared to other groups. With respect to cadmium, Group 3 and 4 rats had significantly higher levels compared to Group 1 and 2 rats. Although vitamin E treatment prevented iron accumulation in Group 4 rats, it had no effect on cadmium concentrations. Distinct histopathological changes observed in Group 3 rats were not present in Group 4 rats. CONCLUSION: Our study demonstrates that in vivo cigarette smoke exposure causes accumulation of cadmium in rat lens and IM vitamin E treatment does not affect this accumulation. The protective effect of vitamin E treatment on smoke exposed rat lens seems to be mediated by blockage of iron accumulation in the lens.

The effects of dimethyl sulfoxide on liver damage caused by ischemia-reperfusion.

INTRODUCTION: The aim of this study was to investigate the effects of dimethyl sulfoxide on liver damage caused by ischemia-reperfusion after portal vein clamping. MATERIAL AND METHODS: Forty New Zealand rabbits were divided into three groups with the portal veins of all the rabbits except the sham group clamped for 30 minutes: group I, sham procedure; group II, control group; and group III, 500 mg/kg DMSO. The drug was administered IM in the left inguinal region 30 minutes before the operation. Blood samples (5 mL) were taken from the animals at 15, 30, and 45 minutes. At the end of the experiment 1 g of liver tissue samples were obtained. Malondialdhyde (MDA), nitric oxide (NO), AST, ALT, and LDH plasma levels were measured in the blood samples. Liver tissue samples stained with hematoxylin eosin were examined under light microscopy for histopathological changes. FINDING: The liver enzymes in both clamping groups increased significantly compared with the sham group (P < .01). Enzyme levels of the DMSO group decreased significantly compared to the control clamping group (P < .05). Similar to the enzyme changes, MDA and NO levels increased in the portal vein clamping versus the sham group and decreased in the drug-administered group versus the control clamped group (P < .03). The severity of histopathological changes was less in the DMSO group than in the clamped controls. CONCLUSION: DMSO decreased the severity of liver damage after portal vein clamping.

Hyperphosphatemic tumoral calcinosis.

Tumoral calcinosis is a rare syndrome characterized by progressively growing and painless masses of calcium phosphate deposits within periarticular areas. Biochemical findings are normal except for an association with hyperphosphatemia. This report describes hyperphosphatemic tumoral calcinosis in a 22-year-old man who had been operated on five times in 5 years because of painless extremity swellings.

CD4+ T cell/CD8+ T cell ratio in the anterior chamber of the eye after penetrating injury and its comparison with normal aqueous samples.

We have recently reported that significantly more CD8+ T-cell activity is present in the aqueous humor compared to peripheral blood. The aim of the current study is to investigate the effect of the ocular trauma on the number of the T lymphocyte subsets in the aqueous humor. CD4+/CD8+ T-cell ratios in the aqueous and blood samples of 12 patients who suffered from traumatic iridocyclitis because of a corneal perforation were compared to those of patients with senile cataracts. We found a relatively higher CD4+/CD8+ cell ratio in the aqueous samples of traumatized patients than cataractous patients. Meanwhile, no difference was present between the aqueous and blood samples of the traumatized patients with respect to the above-mentioned ratio. We suggest that one of the most important factors in maintaining a lower CD4+/CD8+ cell ratio in normal aqueous humor compared to peripheral blood is an intact blood-aqueous barrier. Blood-aqueous barrier may play a participating role in the pathogenesis of immunosuppressive properties of the anterior chamber of the eye by establishing higher CD8+ T-cell activity in the aqueous humor relative to the peripheral blood.

Flow cytometry tear analysis in patients with chronic allergic conjunctivitis.

Tears from patients with chronic allergic conjunctivitis were analyzed with flow cytometry to determine the function of the T lymphocyte-related immunological reactions in the disease pathogenesis. Twenty-eight patients and 22 normal volunteers were studied; tears were obtained with capillary tubes. T helper/T suppressor ratios and the percentages of HLA DR+, CD23+, and CD3+ cells were significantly higher in patients than in controls. This study provides support for the hypothesis that chronic allergic conjunctivitis results from T lymphocyte-related immunological reactions.

A possible mechanism of X-ray-induced injury in rat lens.

PURPOSE: X-ray and other radiation can cause cataract, but the pathogenic mechanism is largely unknown. The aim of this study was to investigate the accumulation of iron in the x-ray-exposed rat lens and its relationship to lens injury. METHODS: Fifty male Wistar rats were divided randomly into five groups of 10. Groups 2 and 4 rats were sham-exposed, groups 3 and 5 were x-ray-treated, and group 1 served as control. X-ray radiation and sham exposure were performed in a similar manner. After 10 and 30 days of exposure, a lens from each rat in groups 2 and 3, and 3 and 5, respectively, were analyzed by flame atomic absorption technique for the assessment of metal content. RESULTS: Significantly decreased zinc and increased iron and calcium concentrations were detected in the lens samples of groups 3 and 5 compared with groups 2 and 4 and controls. Similar results were obtained comparing groups 5 and 3. CONCLUSIONS: We propose that x-ray exposure may cause toxic cell injury of the rat lens via Fenton metals catalyzed damage. Initial lens membrane damage in the radiolytic phase may permit the access of iron resulting in lens damage.

The effects of prophylactic zinc and melatonin application on experimental spinal cord ischemia-reperfusion injury in rabbits: experimental study.

STUDY DESIGN: Experimental study. OBJECTIVES: To determine the neuroprotective effects of zinc and melatonin on spinal cord ischemia-reperfusion (I/R) injuries of rabbits. SETTING: The Experimental Research Centre of Selçuk University, Konya, Turkey. METHODS: Twenty-four male rabbits underwent spinal cord ischemia by clamping the thoraco-abdominal aorta for 20 min. Twenty minutes before the aortic clamping, animals received zinc, melatonin or a combination of both. Neurological examination of the animals was performed three times during reperfusion period. The animals were killed 24 h after reperfusion. Spinal cord samples were taken for biochemical and histopathological evaluation. RESULTS: Pre-treated animals with zinc, melatonin or combination displayed better neurological outcomes than the I/R group (P<0.05). Zinc, melatonin and combined treatment prevented spinal cord injury by reducing apoptosis rate (P<0.05) and preserving intact ganglion cell numbers (P<0.05). Zinc pre-treatment protected spinal cord by preventing malondialdehyde (MDA) formation (P=0.002), increasing glutathione peroxidase (GPx) activity (P=0.002) and decreasing xanthine oxidase enzyme activity (P=0.026) at molecular level. Melatonin treatment also resulted with MDA formation (P=0.002), increased GPx activity (P=0.002) and decreased xanthine oxidase activity (P=0.026). CONCLUSION: The results of the study showed that prophylactic zinc and melatonin use in spinal cord I/R not only suppressed lipid peroxidation by activating antioxidant systems but also had significant neuroprotective effects by specifically improving the neurological and histopathological situation.

Analysis of tears in patients with atopic keratoconjunctivitis, using flow cytometry.

Atopic keratoconjunctivitis (AKC) is a chronic allergic eye disease. Although the pathogenesis is not fully understood, some impairment in cell-mediated immunity was suggested by histopathological findings in conjunctival specimens obtained from affected individuals. T-cell infiltration and an enhanced T-helper/T-suppressor cell ratio in conjunctival biopsy specimens were observed previously by immunofluorescence procedures. We analyzed the cells in tears of patients with AKC using flow cytometry (FCM) and compared the results to those of normal subjects to identify the role of T lymphocytes in the pathogenesis of the disease. The tear samples of the patients and normal subjects were collected with capillary tubes, and the surface receptors of cells were detected with FCM. Statistical analyses were performed with Student's t test. The percentages of T cells, activated B cells, and T-helper/T-suppressor cell ratios were found to be higher in the tears of patients with AKC than in controls. We propose that a decreased T-suppressor cell concentration in tears may enhance immunoglobulin-E production of B cells, and the signs and symptoms are provoked by inflammatory mediators liberated from mast cell degranulation.

Mechanisms and comparison of anti-allergic efficacy of topical lodoxamide and cromolyn sodium treatment in vernal keratoconjunctivitis.

PURPOSE: To explore the mechanism of action of topical lodoxamide and cromolyn sodium treatment in vernal keratoconjunctivitis (VKC) and to compare the efficacy of these drugs to each other. DESIGN: Single-investigator, masked, randomized, clinical trial. PARTICIPANTS: Twenty male and 10 female patients between the ages of 6 and 19 years, who were diagnosed as having active VKC, were enrolled in this study. INTERVENTIONS: The patients were randomly divided into two equal groups (groups A and B). Group A patients received topical lodoxamide ophthalmic solution 0.1% (LOS); topical cromolyn sodium ophthalmic solution 4% (CSO) was prescribed to group B patients in a dose of two drops four times daily. MAIN OUTCOME MEASURES: The eye symptom severity scores and clinical signs of the patients were evaluated both in the pre- and post-treatment periods. In addition to the clinical data, conjunctival impression cytologic specimens were obtained from patients both before and after treatment. Impression cytologic specimens were stained using immunohistochemical methods to detect the percentages of CD4(+), CD8(+), CD45RA(+), and CD23(+) cells. Statistical analyses were performed within and between groups. RESULTS: The percentages of CD4(+) and CD23(+) cells in tear samples of patients in groups A and B were significantly higher in the pretreatment stage than post-treatment stage. In the post-treatment stage, group A patients had significantly lower CD4(+) and CD23(+) cell values compared with group B patients. Patient symptom scores and clinical signs were at a significantly lower level after treatment with either LOS or CSO in both groups A and B compared with their pretreatment values. Moreover, group A patients had significantly lower symptom scores and clinical signs than group B patients in the post-treatment stage. CONCLUSIONS: Clinical superiority of LOS over CSO may be linked to its greater effect on the CD4(+) cells, because CD4(+) cells plays a pivotal role in the pathogenesis of VKC.

Determinations of some trace and heavy metals in rat lenses after tobacco smoke exposure and their relationships to lens injury.

Cigarette smoking has been implicated in the pathogenesis of cataract, but the pathogenic mechanism by which cigarette smoke causes cataract is yet to be completely understood. There has been suggestion that oxidative damage caused by accumulation of Fenton reagents (iron and copper) in the lens can cause lens damage and possibly cataract. To investigate the accuracy of this theory the study was planned. A number of twenty-four male Wistar rats were divided randomly into experimental and control groups. The experimental group of rats were exposed to cigarette smoke for two hours in each day over sixty consecutive days and the controls were treated in identical fashion but only exposed to room air. At the end of the study period, both eyes of all the animals were enucleated and one eye prepared for histopathological examination and the other used for the measurement of metal levels. The lenses of experimental animals showed significantly decreased zinc and increased iron, and calcium concentration relative to those of sham exposed controls. However, no significant difference was found in the copper contents of the lenses of both groups. Distinct histopathological changes such as hyperplasia, hypertrophia, and multilayering of epithelial cells and elevations of calcium concentration detected in the lenses of experimental group animals suggested that the lens damage was a result of in-vivo exposure to tobacco smoke. We propose that increased metal contents in the lens can cause lens damage by the mechanism of oxidative stress through formation of oxygen radicals via metal catalysed Fenton reaction.

Pharmacological mechanism of topical lodoxamide treatment in vernal keratoconjunctivitis: a flow-cytometric study.

The aim of this study was to investigate the possible effect of lodoxamide 0.1% ophthalmic solution (LOS) on tear T lymphocytes, especially Th2 (T helper 2 subgroup of helper T lymphocytes), in the tear specimens of patients with vernal keratoconjunctivitis (VKC) and to explore the mechanism of the anti-allergic effect of this drug. Twenty-eight active VKC patients were randomly divided into two groups each containing 14 patients. We obtained tear samples from all patients at the beginning of the study. LOS was prescribed to the study group (group A), but the controls (group B) received placebo over the course of 6 weeks. Tear samples were also collected from all patients following a 6-week course of treatment with either LOS or placebo. The percentages of Th1, Th2 and Th cells in the tear samples were measured by using a flow-cytometric technique and the results were compared to each other by using Wilcoxon matched-pairs signed-rank and Mann-Whitney U tests. We found that LOS treatment decreased the percentages of Th2 and Th cells in the tears of group A patients, while placebo had no effect on these values. Th1 cell count in the tears of both groups of patients did not show any significant change between the pre- and posttreatment stages. Our data suggest that LOS exerts, at least, some part of its anti-allergic effect via decreasing the Th2 cell number in the tears of VKC patients.

A flow cytometric study about the immunopathology of vernal keratoconjunctivitis.

BACKGROUND: Vernal keratoconjunctivitis (VKC) is a bilateral seasonal conjunctival inflammation. Exact pathogenesis of the disease is unknown, but some evidences suggest TH lymphocyte-mediated immune reactions. OBJECTIVE: The aim of this study was to investigate the role of the T lymphocyte and its subsets in the pathogenesis of VKC. METHODS: We obtained tear samples from patients with VKC and normal volunteers during active (spring) and quiescent (winter) periods. The patients' records were also obtained for assessment of symptom scores. The percentages of CD4/29+, CD4/45RA+, CD4+, and CD8+ in tear samples were established by using flow cytometry, and the results of all three groups were compared with each other by using the Wilcoxon matched-pair signed-rank test and the Mann-Whitney U test. RESULTS: The percentages of CD4/29+ and CD4+ cells in tears of patients with VKC increased significantly in the active period and decreased to normal levels in the quiescent stage. In contrast, the percentages of CD4/45RA+ and CD8+ cells in tears of patients with VKC did not show any significant change between spring and winter. The patients' symptoms were significantly lower in the quiescent period (winter) compared with the active stage (spring). CONCLUSION: We propose that increased numbers of CD4+ and CD4/29+ cells in tears may be exacerbating the disease during the spring season.