Effect of proteolytic activity on virulence of Candida albicans in burned mice.

Total circulating proteolytic activity (PA) was determined by measuring the acid-soluble 125I-protein fragments generated per 100 microliters of serum incubated with 125I-protein at 37 degrees C for 15 min. Normal mice had low circulating PA (1.3 +/- 0.2 micrograms/100 microliters), and burned mice had a higher average PA; the actual value depended on the time of measurement postburn. We measured the effect on mortality and on circulating PA of challenging normal and burned mice with high-virulence strain Candida albicans MY 1044 and its less virulent mutant MY 1049. Burned and normal mice challenged with a high dose (10(5)) of MY 1044 had high mortality (greater than 90%) and high circulating PA (greater than 33 micrograms generated per 100 microliters). Burned mice challenged with a lower dose (10(4] of MY 1044 had moderate mortality (63%) and lower PA (27.2 +/- 4.2 micrograms/100 microliters). All other groups of mice, including burned mice challenged with 10(5) MY 1049, had low mortality (less than 10%), and PAs were less than 22 micrograms/100 microliters. Augmentation of burned mice challenged with 10(5) MY 1049 with proteinase significantly increased mortality; with treatment of burned mice challenged with 10(5) MY 1044 with proteinase inhibitor significantly decreased mortality. We conclude that mortality correlated with total circulating PA; that the contribution to this net PA was the background PA level in the normal mice, the PA associated with the burn, and the PA caused by infection with a C. albicans strain with a particular virulence; that most deaths caused by C. albicans occurred past a PA threshold of 25 micrograms/100 microliters in the host; and that the number of burned and infected mice that died of candidiasis could be modulated by the addition of proteinases or proteinase inhibitors to the host. This last finding may lead to some novel treatments for candidiasis in burned hosts.     

Assays for total and antigen-specific polymeric IgA in serum based on binding to secretory component

Binding assays with secretory component (SC) were used to detect polymeric IgA antibody to E. coli lipopolysaccharide and to estimate total polymeric IgA in sera from 14 patients with alcoholic liver disease and eight normal controls. Radioiodinated human SC was shown to bind to polymeric IgA and IgM but not to monomeric IgA, secretory IgA or IgG. Serum aliquots (0.5 ml) were totally depleted of IgM using 2 ml anti-IgM affinity columns and the effluent sera were titrated in microtitre plates coated with lipopolysaccharide, the binding of polymeric IgA being detected by adding 10 ng radiolabelled SC. Total polymeric IgA was measured via its capacity to inhibit the binding of 5 ng labelled SC to IgM coated wells, quantitation being achieved by comparison with the inhibition produced by purified polymeric IgA. Total lipopolysaccharide-specific IgA antibody was detected by ELISA in sera from both patients and controls, 1185 +/- 793 and 56 +/- 19 U/100 microliters (mean +/- SD), respectively; but polymeric IgA antibody was detected only in patients' sera (131 +/- 214 U/100 microliters). The concentration of total polymeric IgA was higher in patients' sera than in control sera (488 +/- 333 and less than 120 micrograms/ml respectively).     

Interaction of hyaluronic acid with mucin, evaluated by gel permeation chromatography

Hyaluronic acid (HA) is known to increase the ocular bioavailability of ophthalmic drugs not only for its viscous properties but also for its specific affinity for ocular mucins. This phenomenon, called bio- or mucoadhesion, can be evaluated in vitro by mechanical tests which, however, require considerable amounts of mucin (M) that are difficult to obtain from ocular surfaces. Thus, we developed an alternative method, based on gel permeation liquid chromatography, to examine the interaction of HA with microgram quantities of mucin. HA (from human umbilical cord or rooster comb) were fractionated using a Sepharose CL-4B column, before and after incubation with porcine gastric mucin (PGM), and the fractions were analyzed by a specific assay based on the histological dye Stains-all. PGM interacted with high molecular weight (M.W). HA, causing the displacement of low M.W., non-covalently bound, HA fragments, which were eluted under a distinct chromatographic peak. By quantitating the relative area of this peak, an evaluation of the mucoadhesion of HA could be obtained. This method could be useful to study the interaction between HA and microgram quantities of ocular M (mucin), obtained from individual patients or normal subjects.     

Intrathecal somatostatin, somatostatin analogs, substance P analog and dynorphin A cause comparable neurotoxicity in rats.

Rats chronically implanted with intrathecal catheters received intrathecal injections (10 microliters followed by 10 microliters saline flush) of either saline (n = 5), somatostatin (100 micrograms, n = 10), the somatostatin analog BIM 23003 (100 micrograms, n = 5), the somatostatin analog SMS 201-995 (100 micrograms, n = 5), the substance P analog [D-Pro2, D-Trp7,9] SP (10 micrograms, n = 10), or dynorphin A (1-17) (20 nmol, n = 8). These doses (somatostatin, substance P and dynorphin A) were selected based on previous studies in which they caused significant motor deficits. Effects on thermal cutaneous nociception, behavior, motor function and spinal cord histopathology were evaluated. All peptides caused severe neurotoxicity, evidenced by flaccid hind leg paralysis and lumbar spinal neuronal degeneration, which was accompanied by an inflammatory reaction in meninges and spinal gray matter. Histopathological changes had developed within 24 h after injection of somatostatin, substance P analog and dynorphin A, showing mild to severe neuronal degeneration and mild inflammatory responses in spinal cord and meninges. Significant antinociceptive effects, due to severe neurotoxic effects, were only observed following intrathecal injection of SMS 201-995 and the substance P analog. Potential neurotoxic mechanisms of the different peptides are discussed.     

Enhancement of tonic and phasic events of rapid eye movement sleep following bilateral ibotenic acid injections into centralis lateralis thalamic nucleus of cats.

The excitotoxin ibotenic acid (1.2-2.6 microliters of 50 micrograms/microliters) was injected bilaterally into the thalamic centralis lateralis nucleus of chronically implanted cats in order to study the effects of tonic excitation followed by destruction of perikarya on the sleep-waking cycle and its electrographic correlates. Ibotenate injections were performed under mild ketamine anaesthesia. Immediately afterwards, the animals showed behavioural arousal accompanied first by ocular nystagmiform movements and then by pontogeniculooccipital waves. By 6-10 h post-injection, the numbers of rapid eye movement sleep episodes, but not their duration, increased compared to the preinjection control period. The injection sites were histologically confirmed using conventional Thionin stains. Additional control was provided by retrograde transport of wheat-germ agglutinin conjugated with horseradish peroxidase. The present results suggest that a population of neurons important for ocular saccades, pontogeniculooccipital waves, and the state of desynchronized sleep is present in the internal medullary lamina, in particular in the centralis lateralis nuclei.     

Ocular anterior segment pathologies and tear film changes in patients with psoriasis vulgaris

Ocular manifestations in patients with psoriasis vulgaris have been investigated in only a small number of studies. Our purpose was to identify tear film function and ocular pathologies associated with psoriasis vulgaris in patients who had received neither oral retinoids nor phototherapy. We examined 62 eyes of 31 patients with psoriasis and 60 eyes of 30 age-and-sex matched healthy volunteers. In addition to complete ocular and dermatological examination, tear film function (i.e., tear secretion and tear film stability) were assessed by the Schirmer-I test, as well as by tear film break-up time. None of the controls had any ocular abnormalities, whereas 67.74% of patients with psoriasis had various anterior segment pathologies (P<0.00009). The most prevalent finding was chronic blepharoconjunctivitis (64.5%), as the only pathology (n=9) or in association with other findings, including nonspecific corneal opacities (n=4), cataract (n=3), both corneal opacities and cataract (n=2), and corneal pigment dispersion (n=2). The Schirmer-I test results revealed comparable mean values in the patient group (9.8+-4.2 mm) and in the controls (11.2+-3.7 mm; P=0.078). However, mean tear film break-up time was significantly shorter in the patients (7.2+-2.5 sec) than in the healthy persons (11.7+-3.1 sec; P=0.001). In agreement with some previous reports, our findings clearly demonstrated that early ocular involvement occurs in patients with psoriasis vulgaris, irrespective of the history of previous therapeutic modalities (e.g., retinoid therapy and phototherapy). Thus, the present findings are suggestive of the contributory role of primary etiologic factors of psoriasis in the pathogenesis of ocular changes in patients with psoriasis vulgaris.     

Use of plasma iodine assay for diagnosing thyroid disorders.

AIMS--To examine the advantage of systematic plasma iodine assays in establishing the thyroid function of patients with thyroid disorders. METHODS--Iodine was determined by inductively coupled plasma mass spectrometry (ICPMS) in the plasma of 799 patients consulting for possible thyroid disorders, indicated by FT4 and TSH assays. RESULTS--Plasma iodine was below 40 micrograms/l in 57 (7%) patients, most of whom had hypothyroidism; 40-80 micrograms/l in 439 (55%) patients, most of whom had normal thyroid hormone function; 80-250 micrograms/l in 240 (30%) patients, most of whom had hyperthyroidism; and above 250 micrograms/l in 63 (8%) patients, almost all of whom had iodine overload caused by iodinated drugs, particularly amiodarone, resulting in euthyroidism (24%), hyperthyroidism (36%), and hypothyroidism (16%). Sixty five (7%) had been treated with amiodarone and 27 (3%) with other iodinated drugs. More than 10% of patients with thyroid disorders therefore had an iodine overload. CONCLUSIONS--The determination of total plasma iodine using the simple, accurate ICPMS technique, should be carried out in patients consulting for thyroid disorders, particularly for the detection of an iodine overload.     

Rabbit trachealis tension responses to receptor-mediated agonists are diminished by elastase.

The present study examined the effects of elastase, in concentrations present in respiratory secretions, on airway smooth muscle contractile responses in vitro and the magnitude of the airway epithelial inhibition of smooth muscle tension. Experiments were performed on 126 full-thickness tracheal strips from 25 rabbits. Isometric tension responses to acetylcholine (10(-8) to 10(-4) M) and potassium chloride (10 to 110 mM) were examined before and after a 5-min exposure to either porcine pancreatic elastase (PPE) or human neutrophil elastase (HNE). PPE (5 to 40 micrograms/100 microliters) reduced the tension response to acetylcholine but had no effect on the tension response to potassium chloride. PPE and HNE (20 micrograms/100 microliters) produced similar effects. Mechanical removal of the epithelium per se significantly (P less than 0.005) decreased the ED50 response to acetylcholine but did not affect maximal tension. However, the airway epithelial inhibitory effect on the acetylcholine tension response was similar in the presence and absence of PPE (20 micrograms/100 microliters). These data suggest that the diminution of tracheal smooth muscle tension responses to receptor-mediated agonists induced by elastase is a direct effect on the muscle and is not mediated by an effect of elastase on the respiratory epithelium.     

4种国产和3种进口第4代HIV抗原抗体检测试剂的质量评价

目的 对4种国产和3种进口第4代HIV诊断试剂的质量进行评价.方法 利用HIV抗体阴性样品库和核酸阳性样品库、BBI阳转血清盘样品等,对4种国产和3种进口第4代试剂的敏感性和特异性、检测HIV-1早期感染的能力进行分析.结果 7种第4代试剂的敏感性均为100％ (95％ CI:99.86％～100％),且1份HIV-1感染窗口期样品均检测为阳性,其中1种进口试剂“8+”值较大(1.0892),其余6种试剂的“δ+”值均比较小(0.0836 ～0.3003).对阴性样品,7种试剂均存在不同程度的假阳性(特异性为97.80％ ～ 99.60％,“δ-”值为-1.3803 -0.4778).对BBI阳转血清盘样品,国产试剂的阳转血清相对敏感性系数为-0.500～0,2种进口试剂则为-0.600和-0.700.结论 7种试剂均具有较高的敏感性和特异性,第4代HIV试剂用于血液筛查可发现HIV感染窗口期样本,对减少HIV传播的风险有一定的意义.但进口第4代试剂检测HIV-1早期感染的能力强于国产第4代试剂.     

Influence of mouse liver stored vitamin A on the induction of mutations (Ames tests) and SCE of bone marrow cells by aflatoxin B1, benzo(a)pyrene, or cyclophosphamide

Male mice (C57BL/6J) at 2 weeks of age were divided into two groups and maintained on a vitamin A-deficient or vitamin A-(retinyl acetate) supplemented diet. After 8 weeks, the average liver vitamin A concentration of mice fed on vitamin A-deficient or -supplemented diet was 36 +/- 7 micrograms/g vs 287 +/- 22 micrograms/g, respectively. Uninduced liver S9 fractions were prepared from both groups of mice and used to activate (with cofactors) the precarcinogens aflatoxin B1 (AFB), cyclophosphamide (CPP), dimethylbenz(a)anthracene (DMBA), and benzo(a)pyrene (BP) in the Salmonella mutagenicity assay. S9 fraction prepared from both groups of mice failed to activate CPP to metabolites mutagenic in tester strains TA100 and TA1535 or to activate DMBA to metabolites mutagenic in TA100, but effectively activated AFB and BP to metabolites mutagenic in TA98. Comparison of activation activities of S9 prepared from liver of mice fed a high or low level of vitamin A was made with T98 treated with AFB or BP using three doses of S9 (50, 100, and 200 microliters/plate). S9 fractions from mice with a high liver vitamin A level were consistently less potent than S9 fractions from mice with a low liver vitamin A level in activating AFB to its mutagenic metabolites. This effect was not observed in BP-treated plates. Administration of AFB to groups of mice with a high liver vitamin A level induced significantly less SCE in bone marrow cells than did administration of AFB to mice with a low liver vitamin A level. This differential sensitivity was not observed when the two groups of mice were treated with either BP or CPP. The possible relationship between vitamin A levels in vivo and mutagenesis or carcinogenesis are discussed briefly.     

Hypertonic KCI, NaCl and capsaicin intracamerally causes release of substance P-like immunoreactive material into the aqueous humor in rabbits

We have investigated release of substance P-like immunoreactivity (SPLI) into the anterior chamber of the rabbit eye evoked by stimuli which cause non-cholinergic miosis. In a recent study such miosis was reported to be blocked by the substance P analogue (D-Arg1, D-Pro2, D-Trp7,9, Leu11)-SP. Mechanical intracranial antidromic trigeminal nerve stimulation caused marked SPLI release presumably from primary sensory nerve endings in the anterior part of the eye. Intermittent stimulation for 20 min was not more effective than stimulation for 10 min. Intracameral injection of either 20 microliters 4.65 M KCI, 20 microliters 4.65 M NaCl or 100 micrograms capsaicin also caused SPLI release. Intracameral injection of 70 microliters 150 mM KCI, 28 micrograms prostaglandin E1 or 200 micrograms of compound 48/80 did not cause detectable SPLI release.     

The stability of mutagenic chemicals stored in solution

Using the Salmonella/microsome assay, we have evaluated the stability of mutagenic responses of chemicals stored frozen over a period of 18 months. Each of the standard mutagens was prepared in January 1982, and aliquots were stored at -20 degrees C and at -80 degrees C. Sodium azide (NaN3) was dissolved in water; 4-nitro-o-phenylenediamine (4NOP), 4-nitroquinoline-N-oxide (4NQO), benzo(a)pyrene (B[a]P), and 2-aminoanthracene (2AA) were dissolved in DMSO, all at 100 micrograms/ml. At various times, aliquots were removed, thawed, and tested in parallel with freshly prepared mutagen samples using strain TA100 in a standard plate test and freshly prepared Aroclor 1254-induced rat liver S-9 mix where needed. 4NOP (2-10 micrograms/plate), 4NQO (0.01-0.10 micrograms/plate), B(a)P (0.5-2.5 micrograms/plate), and 2AA (0.25-2.0 micrograms/plate) showed no significant differences between the freshly prepared solutions and the solutions stored at -20 degrees C and -80 degrees C. NaN3 (0.1-0.8 micrograms/plate) did show a statistically significant difference, with the fresh samples giving the lowest mean responses (over all doses) and the -80 degrees C treatment giving the highest. The freezing of mutagen solutions is adaptable to routine use and provides the advantage of reducing the time required to prepare positive control chemicals and reducing the exposure of laboratory personnel to known mutagens.     

Quantitation of components of the alternative pathway of complement (APC) by enzyme-linked immunosorbent assays

Sensitive enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies have been developed to specifically detect components of the alternative pathway of complement in human blood plasma. Normal values of the factor B split products Ba (1.01 +/- 0.30 micrograms/ml, mean +/- SD), Bb (0.65 +/- 0.23 micrograms/ml), of the C3-fragments C3b/iC3b/C3dg (17.9 +/- 5.7 micrograms/ml), native factor B (238 +/- 48 micrograms/ml), factor D (1.05 +/- 0.27 micrograms/ml), and factor H (702 +/- 292 micrograms/ml) were determined in the EDTA-plasma of healthy probands (n = 55). The simultaneous quantitation of the main cleavage products and of control proteins in the plasma samples permits precise analysis of the activation of the alternative pathway of complement in various disease states. In addition, we describe a method for the specific depletion of factor B prior to fragment-specific assays utilizing monoclonal antibodies conjugated to paramagnetic beads. The latter should permit the quantitation of other complement split products.     

Prostacyclin release and cytotoxicity of peritoneal cells are inversely related in pregnant and non-pregnant mice infected with herpes simplex virus

Cytotoxicity of peritoneal cells in a HSV-infected murine model is attenuated in late pregnancy. Prostacyclin (PGI2) is elevated at this time in reproductive tissues and has been implicated in the regulation of the immune response. The purpose of this study was to estimate PGI2 in the peritoneal wash or culture supernatants of peritoneal cells obtained from uninfected and HSV-infected pregnant and virgin mice using a radioimmunoassay for 6-keto-prostaglandin F1 alpha. The peritoneal wash of uninfected pregnant and virgin mice contained high levels of 6-keto-PGF1 alpha, 505 +/- 51 pg/100 microliters, (mean +/- S.E., n = 15), and 200 +/- 19 pg/100 microliters, (n = 30), ad did peritoneal effector and target cell cultures (1,159 +/- 118 pg/100 microliters, n = 6, and 1,057 +/- 207 pg/100 microliters, n = 7), respectively. HSV-infection induced in vitro cytotoxicity and suppressed the release of 6-keto-PGF 1 alpha (r = -0.897, P less than 0.05, n = 18). Its concentration was significantly higher (14-fold, P less than .05) in the peritoneal wash, but not in the cell culture, of pregnant (212 +/- 29 pg/100 microliters, n = 19) as compared to virgin mice (18.5 +/- 3.4 pg/100 microliters, n = 27). The levels of 6-keto-PGF1 alpha were inversely correlated (P less than .05) with the combined effects of HSV-infection and cytotoxicity.     

几丁糖对干眼症患者眼表功能的影响及其疗效分析

目的：研究几丁糖(Medical Chitosan)对干眼症患者眼表功能的影响及其疗效。方法：采用随机取样法,选取2013年8月至2014年8月到我院就诊的100例(200眼)干眼症患者,并对患者的病症资料进行整理研究。然后将所选取的干眼症患者随机分为2组,每组50例(100眼)。观察组采用几丁糖联合人工泪液的方法对患者进行治疗,对照组则采用人工泪液的治疗方法。治疗后,对两组患者均进行随访1~6个月,观察并记录两组患者的眼表功能变化以及泪膜破裂时间(break-up time, BUT)、Schirmer I试验(Schirmer I test,SIT)、角膜荧光素染色(fluorescein,FL)和视功能评分等指标,最后将两组患者的治疗效果进行比较分析。结果：治疗一段时间后,两组患者BUT时间都明显增加,且观察组高于对照组,差异具有统计学意义(P<0.05);两组患者Schirmer I试验值都明显升高,且实验组高于对照组,差异具有显著性意义(P<0.05);且两组患者的角膜荧光素染色面积都明显减少,且观察组的染色面积小于对照组,同时两组患者的视功能评分都明显升高,且观察组的视功能评分高于对照组,差异具有显著统计学意义(P<0.05)。结论：利用几丁糖治疗患者干眼症取得了良好的效果,有助于患者眼部疾病的康复,减少了患者病情加重的概率,能快速使患者恢复正常生活,减少了病患家属的心理负担,值得广泛应用。     

Effect of Ramadan fasting on tear proteins

Muslims abstain from eating, drinking and smoking from dawn to sunset during the holy month of Ramadan. Prolonged fasting is thought to be among risk factors for many diseases, e.g., cardiovascular, gastrointestinal and various infectious diseases. It could also play a part in several eye diseases, including dry eye syndrome, glaucoma, and cataract. Toxic and oxidative effects due to increased concentrations of some biochemicals as a result of reduction in tear volume thought to play an important role in damaging ocular tissue. Human tear is an important biological fluid similar to blood in many aspects. Tear film is composed of three basic layers i.e. lipid, aqueous and mucin. The tear film covering the ocular surface presents a mechanical and antimicrobial barrier, and endures an optical refractive surface. The aim of this study was to analyze and compare tear protein of volunteers during fasting. Using two reliable analytical methods, i.e. electrophoresis and high performance liquid chromatography (HPLC), we compared tear protein content of sixty volunteers (35 males and 25 females, 23-27 years old) during fasting in holly month of Ramadan (FAST: n = 62) and one month before Ramadan (CTRL: n = 60). The results showed that some identified tear proteins decreased during fasting. On the other hand, the activity of some enzymes such as lysozyme, lactoferrin and alpha amylase also decreased in fasting samples. Electrophoresis results showed that tear protein patterns in FAST (P < 0.05) were different from those of CTRL. There were a few more protein peaks in the FAST group (P < 0.005) than in CTRL.     

Complement proteins and C3 anaphylatoxin in the tears of patients with conjunctivitis

Previous studies in our laboratory have demonstrated pollen-specific IgG antibodies in the tears of patients with vernal conjunctivitis (VC) and elevated tear IgG levels in patients with contact lens-induced giant papillary conjunctivitis (GPC). Tear secretions were examined for complement (C) proteins to determine the role of this effector system in the pathogenesis of these ocular disorders. The tears of VC (15) and GPC (10) patients with active disease had elevated tear levels of both C3 and factor B. By use of transferrin as a marker for the leakage of plasma proteins into the tears, most C3 was locally produced by the conjunctival tissues. Although immune complexes could not be detected in the tear secretions, increased levels of C3 des Arg were present in the tears that suggested complement activation with the generation of anaphylatoxins. These studies suggest that complement may be important in the inflammatory ocular process of VC and GPC and that the generation of anaphylatoxins (C3a), even by nonimmune mechanisms, may contribute to basophil and mast cell activation with the release of inflammatory mediators into the tear secretions.     

Comparative cardiovascular toxicity in dogs given inotropic agents by continuous intravenous infusion

A comparative study of the toxicity of the inotropic amines isoproterenol hydrochloride (IP), 1-norepinephrine bitartrate (NE), dopamine hydrochloride (DP), and dobutamine hydrochloride (DB) was conducted in beagle dogs (2/sex/dose group). All drugs were administered at doses that produced maximal contractile tension in dog myocardium. Doses, continuously infused for 96 hr, were 0.625, 1.25, and 2.5 micrograms/kg/min IP, 2.5 and 5 micrograms/kg/min NE, and 25, 50, and 100 micrograms/kg/min DP and DB. Three of 4 dogs that received 5 micrograms/kg/min NE and one of 4 given 100 micrograms/kg/min DP died. Pronounced tachycardia (mean peak rate increases from baseline of 88-104 beats/min) was observed at all doses of IP. DB produced a transient moderate tachycardia (mean peak rate increases from baseline of 25-27 beats/min) at 25 and 50 micrograms/kg/min and pronounced tachycardia (mean peak rate increases from baseline of 74 beats/min) at 100 micrograms/kg/min. Moderate bradycardia occurred at both doses of NE and at 25 and 50 micrograms/kg/min DP (mean peak rate decreases from baseline of 42-46 and 22-38 beats/min, respectively). At high doses the 4 inotropes produced focal to multifocal myocardial necrosis located mainly in left ventricle and segmental medial necrosis of the coronary arteries, mainly in small intramural muscular branches. Segmental medial hemorrhage was also seen following administration of high doses of NE and DP. An additional intramural coronary arterial lesion produced by all of the inotropes consisted of a mild periadventitial cellular infiltrate and fibroplasia. The results indicated that NE and DP produced the most severe cardiovascular lesions, followed by IP which produced lesions of more moderate severity. DB produced only slight lesions in comparison to the other 3 inotropic amines.     

Skin genetically engineered as a bioreactor or a 'metabolic sink'

Genetically manipulated human keratinocytes can produce and secrete medically relevant proteins to the circulation. Genetically modified skin may also function as a 'metabolic sink' detoxifying the body of metabolites which accumulate in certain metabolic diseases. At the National Institutes of Health (NIH), Bethesda, Md., a clinical trial investigating the treatment of an ocular disease using the skin as a 'metabolic sink' for ornithine accumulating in gyrate atrophy patients is being prepared. The trial will involve the transplantation of a small patch of autologous keratinocytes, transduced ex vivo, onto the thighs of patients with gyrate atrophy. We are now investigating other diseases where this technology may be applicable such as in the treatment of hyperphenylalaninemia or hypercholesterolemia.     

Simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow cytometry: allergics vs. non-allergics

Tears play an essential role in maintaining corneal and conjunctival integrity by providing a tightly regulated, optimal extracellular environment critical to its numerous functions, which include anti-microbial defense, wound healing and inflammatory responses such as allergies. Elevated levels of inflammatory cytokines have been reported in tears from various ocular disease states. Characterization of tear cytokines has been limited by the small volume (microliter amounts) attainable. This limitation was addressed with the newly developed Becton Dickinson Cytometric Bead Array (CBA), which combines the principles of the "sandwich" immunoassay with the capability of flow cytometry for simultaneous measurement of the characteristics of multiple particles. This technique allows determination of six human cytokine (IFNgamma, TNFalpha, IL-2, IL-4, IL-5, IL-10) concentrations simultaneously in a single tear sample. Tears were collected from the inferior fornix of non-allergic (n=7) and allergic (n=9) donors. Each tear sample or cytokine standard was incubated with a mixture of capture Ab-bead reagent and detector Ab-phycoerythrin (PE) reagent, and analyzed using flow cytometry. All six cytokines were detectable in both non-allergic and allergic tears. Tears from allergic donors contained significantly less IL-10 (p=0.035), and had significant increases in the ratios of TNFalpha/IFNgamma, IL-5/IFNgamma and IL-5/IL-10 (p=0.0008, 0.0124 and 0.011, respectively). The small volume required (5-10 microl/test) by the Cytometric Bead Array allows measurement of all six cytokines from a single collection of tears. This decreases collection time, minimizing the confounding effect of stimulation on cytokine concentration in tears, as well as allowing calculation of cytokine ratios.