Virus-like particle-based countermeasures against Rift Valley fever virus.

Rift Valley fever virus (RVFV) is an arbovirus that causes significant morbidity and mortality in both humans and livestock. With increased world travel and the threat of bioterrorism, there is a real risk of RVFV spreading to naïve geographical areas (Trans. R. Soc. Trop. Med. Hyg., 73, 1979, 618; MMWR Morb. Mortal. Wkly Rep., 49, 2000, 905). The introduction of RVFV would cause critical public health, agricultural and economic damage. Despite the clear need for an efficacious vaccine, there are no United States (US) Food and Drug Administration or US Department of Agriculture approved vaccines against RVFV. To address this need, a virus-like particle (VLP)-based vaccine candidate was developed. First, a non-replicating chimeric RVF VLP vaccine candidate was generated that protected mice and rats against a lethal RVFV challenge. This was followed by the development and optimization of conditions for production of RVF VLPs in insect and mammalian cells. Immunological studies demonstrated that VLP-based vaccine candidates elicit both humoral and cellular immune responses. Subsequent challenge studies using a lethal wild-type RVFV strain under high-containment conditions showed that RVF VLP vaccine candidates can completely protect mice and rats.

Mild fumarase deficiency and a trial of low protein diet.

We report clinical findings in a 12-year-old girl with a mild case of fumarase deficiency who continues to make progress. She has two novel mutations of the fumarase gene [c.521C>G (p.P174R) and c.908T>C (p.L303S)]. A trial of low protein diet did not reduce fumaric aciduria.

Argininosuccinate lyase deficiency: longterm outcome of 13 patients detected by newborn screening.

Argininosuccinate lyase deficiency is a urea cycle disorder which can present in the neonatal period with hyperammonemic encephalopathy, or later in childhood with episodic vomiting, growth and developmental delay. Abnormal hair, hepatomegaly, and hepatic fibrosis are unique features of this disorder. Twelve patients with argininosuccinate lyase deficiency were ascertained between 4 and 6 weeks of age by urine amino acid screening. One infant in a previously identified family was diagnosed shortly after birth. Diagnosis was confirmed by enzyme assay in red blood cells and/or skin fibroblasts. At the time of last follow-up, patients had been followed for 13-33 years. All patients were asymptomatic at detection, 7 had slightly increased blood ammonia, and all were initially treated with low-protein diet. Utilization of (14)C-citrulline by intact skin fibroblasts measured by (14)C incorporation into macromolecules was 74-135% of the control mean for 7 of the 8 patients studied. Nine patients had normal development, 4 had learning disability, 6 had EEG abnormalities, 3 had seizure disorder. None had any episodes of hyperammonemic coma. None had hepatomegaly. Patients detected by screening had higher enzyme activity measured by the (14)C-citrulline incorporation assay than comparison groups of patients with neonatal-onset and with late-onset detected by clinical disease. The ability to utilize (14)C-citrulline by intact fibroblasts seems to correlate with clinical outcome and may have prognostic value. It is likely that early diagnosis and treatment contributed to the relatively mild clinical course of the study group.

The αGal HyperAcute(®) Technology: enhancing immunogenicity of antiviral vaccines by exploiting the natural αGal-mediated zoonotic blockade.

The αGal HyperAcute(®) Technology exploits a robust zoonotic blockade to enhance potency of antiviral vaccines. Naturally acquired immunity against the common αGal epitope [galactose-alpha(1,3)-galactose-beta(1,4)N-acetylglucosamine-R (Gal-α(1,3)-Gal-ß(1,4)-GlcNAc-R)] is facilitated by the loss of a key enzyme in the epitope's biosynthetic pathway. As human cells are devoid of this epitope, chronic stimulus from gut flora leads to high levels of circulating anti-αGal antibodies and the development of a robust immune pathway. As the αGal epitope is immediately recognized as foreign, the naturally acquired αGal immune pathway in humans serves as a strong barrier to zoonotic infection. The αGal HyperAcute(®) Technology takes advantage of this natural process to facilitate the rapid presentation of modified antigens to antigen-presenting cells, leading to a strong immune response. The evolutionary immunity to αGal ensures that the presence of αGal epitopes on antigens will lead to a robust immune response involving cross-activation of T(H)1 immunity, characterized by cytokine secretion and increased phagocytic activity, and T(H)2 immunity characterized by high antibody titres. αGal epitopes can be applied to antiviral vaccines by biological, enzymatic or chemical means. Several detection methods that directly and indirectly verify αGal addition are discussed. Enhanced immunogenicity (humoral and cellular) of αGal-modified vaccines is shown for several antiviral vaccine candidates. αGal modification of antiviral vaccine components leads to enhanced immunogenicity. The existing body of literature describing the utility of αGal epitopes as a safe and robust immunostimulatory and -modulatory agent in humans supports the basis for applying the αGal HyperAcute(®) Technology to the improvement of antiviral vaccines, both new and currently approved.

Ornithine aminotransferase deficiency: diagnostic difficulties in neonatal presentation.

We describe two unrelated cases of ornithine aminotransferase (OAT) deficiency with rare neonatal presentation of hyperammonaemia. The diagnosis in the neonatal presentation of OAT deficiency is hampered as hyperornithinaemia is absent. Enzyme and mutation studies confirmed the diagnosis. OAT deficiency should be included in differential diagnosis of neonatal hyperammonaemia.

Inflammation, homocysteine, and vitamin B6 status after ischemic stroke.

BACKGROUND AND PURPOSE: Epidemiological studies have described an association between low vitamin B6 (measured as pyridoxal 5'-phosphate [PLP]) and ischemic stroke, independent of homocysteine (tHcy). We investigated B6 status, tHcy, and inflammation (measured by C-reactive protein [CRP]) in patients with stroke and controls. METHODS: Consecutive cases with new ischemic stroke were compared with matched controls. Fasting tHcy, PLP, and CRP were measured. RESULTS: The adjusted odds ratio of low PLP in the highest compared with the lowest CRP quartile was 16.6 (2, 139.9, P=0.01). Age, CRP, supplemental vitamin use, and albumin were independent predictors of PLP (P<0.05 for all). No relationship was observed between CRP and tHcy. CONCLUSIONS: The relationship between inflammation and low B6 status may partially explain the findings of previous epidemiological studies.

Stroke in young patients with hyperhomocysteinemia due to cystathionine beta-synthase deficiency.

BACKGROUND: Although hyperhomocyst(e)inemia (Hyper-Hcy) may predispose to atherosclerosis and venous thrombosis, the mechanisms of stroke associated with Hyper-Hcy are not defined. METHODS: Clinical and biochemical phenotypes and genetic features of three unrelated patients with premature stroke and severe Hyper-Hcy due to cystathionine beta-synthase (CBS) deficiency are described. Plasma Hcy and amino acids were measured by fluorescence polarization immune assay and ion exchange chromatography. Analysis of the CBS and methylenetetrahydrofolate reductase genes was performed by restriction enzyme digestion and sequence analysis. RESULTS: Two of the three index cases had no known diagnosis of homocystinuria and initially presented with embolic cerebral and retinal infarction in mid-adulthood. Mechanisms of cerebrovascular disease were carotid intraluminal thrombosis, arterial dissection, and possible cardiac embolism. Family screening revealed additional members with clinically silent homocystinuria and severe Hyper-Hcy. Excluding tall stature in two individuals, all had mild phenotypes, without classic findings of CBS deficiency. Plasma total and free Hcy, methionine, and urine Hcy were elevated. Genotyping revealed heterozygous CBS mutations (I278T, D444N, G307S) in affected individuals. CONCLUSION: Artery-to-artery embolism and dissection may cause stroke in young adults with homocystinuria. The results also support a rationale for screening for Hyper-Hcy in young adults with stroke without a phenotype suggestive of classic homocystinuria.

Measurement of post-lens tear thickness.

PURPOSE: A method to measure the tear film beneath a soft contact lens, referred to as post-lens tear thickness (PLTT), would have many applications to contact lens research. In this study a noninvasive technique for measuring the PLTT is presented. METHODS: The feasibility of measuring the tear layer by optical pachometry was first assessed using a model eye. The baseline corneal thickness (B) of both eyes of 21 subjects was measured, etafilcon-A ionic disposable soft contact lenses (58% water) were inserted, and the total thickness (T) of the cornea, contact lens, and PLTT were measured. After the pachometry readings the lenses were removed and their center thickness (C) determined. The PLTT was calculated using the equation: PLTT = T-(B+C). Two sets of measurements of T were performed at 15 and 25 minutes after lens insertion. The entire procedure was repeated at a second visit. RESULTS: The pachometry measurements of the small aqueous reservoir between the model eye and the lens closely matched those obtained by direct microscopic measurement. For human PLTT, the mean values (and 95% confidence intervals) for right eyes on visits 1 and 2 were 11 (8, 13) and 12 (10, 15) microm, respectively, and for left eyes were 12 (10, 15) and 11 microm (8, 14) microm, respectively. CONCLUSIONS: It is possible to measure the post-lens tear thickness using optical pachometry. The variability between repeated measurements suggests that with careful sample size planning, the technique is sufficiently precise to be useful in group assessments of PLTT.

Cystathionine beta-synthase mutations in homocystinuria.

The major cause of homocystinuria is mutation of the gene encoding the enzyme cystathionine beta-synthase (CBS). Deficiency of CBS activity results in elevated levels of homocysteine as well as methionine in plasma and urine and decreased levels of cystathionine and cysteine. Ninety-two different disease-associated mutations have been identified in the CBS gene in 310 examined homocystinuric alleles in more than a dozen laboratories around the world. Most of these mutations are missense, and the vast majority of these are private mutations. The two most frequently encountered of these mutations are the pyridoxine-responsive I278T and the pyridoxine-nonresponsive G307S. Mutations due to deaminations of methylcytosines represent 53% of all point substitutions in the coding region of the CBS gene.

Radioisotope concentrator gene therapy using the sodium/iodide symporter gene.

We demonstrate a novel method of concentrating radiation for tumor imaging or killing. The rat sodium/iodide symporter gene (rNIS) was cloned into a retroviral vector for transfer into cancer cells to mimic the iodide uptake of thyroid follicular cells. In vitro iodide transport shows that the symporter functions similarly in rNIS-transduced tumor cells and rat thyroid follicular cells. rNIS-transduced and control nontransduced (NV) human A375 melanoma xenografts established in vivo in athymic nude mice were imaged using a gamma camera after i.p. injections of 123I. The rNIS-transduced human A375 melanoma tumors are visually distinguishable from and accumulate significantly more radionuclides than NV tumors. In vitro clonogenic assays confirm efficacy and clearly show that rNIS-transduced A375 human melanoma, BNL.1 ME murine transformed liver, CT26 murine colon carcinoma, and IGROV human ovarian carcinoma can be selectively killed by the induced accumulation of 131I. Thus, NIS-based gene therapy may have both diagnostic and therapeutic applications for cancer.

Contact lens base curve prediction from videokeratography.

PURPOSE: To assess whether the contact lens to cornea-bearing relationship, as determined from the fluorescein pattern, can be predicted from videokeratography. METHODS: Nineteen non-rigid gas permeable (RGP) lens wearers were each tested for fluorescein patterns with a series of seven RGP contact lenses of different base curves, and compared to a theoretical estimate of the fitting relationship from videokeratography. The experimentally determined alignment lens was then compared to the theoretical alignment (TA) value as determined from the central curvature and eccentricity. RESULTS: The mean difference in lens choice between the TA and experimental alignment (EA) values was -0.01 +/- 0.04 mm and between the simulated keratometric (KA) readings and the EA choice was 0.11 +/- 0.05 mm. CONCLUSION: A knowledge of the eccentricity value from videokeratography allowed a better prediction of the base curve to cornea relationship than was provided by only a central corneal measurement.

Molecular analysis and prenatal diagnosis of human fumarase deficiency.

Fumarase deficiency is a rare autosomal recessive disorder of the citric acid cycle causing severe neurological impairment. The cDNA for both the rat and human enzymes has been cloned previously and shown to encode a coding region of 1.46 kb. To scan for mutations in fumarase-deficient patients we amplified the coding region of fumarase from fibroblast/lymphoblast cDNA employing the oligonucleotide primers designed from the published human and rat cDNA sequence. We then directly sequenced the polymerase chain reaction product. In seven unrelated patients, we detected four missense mutations (A265T, D383V, F269C, K187R), a nonsense mutation (W458X), a 3-bp AAA insertion that introduces an additional lysine residue at codon 435, and a spontaneous new mutation resulting in a 74-bp deletion (66del74). Seven at-risk pregnancies were monitored with one prenatal diagnosis of fumarase deficiency by molecular analysis and favorable outcome of the other pregnancies as predicted by enzyme assay of cultured fetal cells or molecular analysis.

Alignment effects in videokeratography of keratoconus.

PURPOSE: To determine the effects of corneal apex decentration on videokeratography in subjects with keratoconus. We controlled corneal apex decentration by first locating the cone apex position and then shifting the fixation position by known amounts relative to the videokeratograph axis. METHODS: We evaluated the effect of keratoconus apex position on videokeratography by measuring 28 eyes (17 subjects) at 4 corneal positions: the vertex normal, apex, and 1 mm or 2 mm above the apex. RESULTS: When compared to the apex alignment values, the radius of 1 mm (mean difference: 0.27 mm; SD +/- 0.20 mm) and 2 mm (mean difference: 0.82 mm; SD +/- 0.50 mm). The corneal toricity was greater at apex alignment than at the 1 mm (mean: -0.35 D; SD +/- 1.76 D) and 2 mm (mean: 0.62 D; SD +/- 3.23 D) decentrations. CONCLUSIONS: Decentration of the corneal apex in keratoconus produces significant errors in videokeratography when using the standard alignment rather than alignment with the apex. When there is a decentered cone apex, videokeratograph power errors are greater with the standard (sagittal) power map than with the instantaneous (tangential) power map. The standard power map produces values for the apex position that are about 50% too high and values for the apex power that are from 10 to 40% too low. Most cone shapes have significant toricity that is often not detected with standard videokeratograph alignment.

Asymmetric corneal toricity and pseudokeratoconus in videokeratography.

BACKGROUND: The diagnosis of asymmetric corneal toricity in video- keratography is based on the occurrence of an asymmetric bow tie pattern in the corneal map, which is usually attributed to corneal radial asymmetry but can be an artifact produced by misalignment of the videokeratograph with the corneal apex. METHODS: This study determined whether asymmetric bow tie patterns in 16 subjects (ages 24 to 40 years with corneal toricity of 0.37 to 1.50D) could be converted to symmetric bow tie patterns by changing the alignment of the videokeratograph so that it was directed at the corneal apex. RESULTS: In changing from regular to apex alignment the corneal map became more symmetric in 11 eyes, had no change in 3 eyes and increased in 2 eyes. No radius change could be measured centrally, but there were significant changes of greater than 1.00D in the peripheral corneal radii of 8 eyes. CONCLUSIONS: An asymmetric or angled bow tie appearance of a corneal map suggests a decentered apex rather than corneal asymmetry and may lead to a misdiagnosis of the type or magnitude of corneal toricity, keratoconus or other corneal irregularities.

Use of amniotic fluid amino acids in prenatal testing for argininosuccinic aciduria and citrullinaemia.

Prenatal testing of 12 pregnancies at risk for argininosuccinic aciduria due to argininosuccinate lyase (ASAL) deficiency and three pregnancies at risk for citrullinaemia due to argininosuccinate synthatase (ASAS) deficiency was performed by metabolite detection in amniotic fluid and measurement of enzyme activity in uncultured and cultured chorionic tissue and in cultured amniocytes. From our data and those of previous studies, amniotic fluid argininosuccinate measurement alone is clearly a reliable and rapid diagnostic test for both severe and mild ASAL deficiency if maternal ASAL deficiency can be excluded. For prenatal diagnosis of ASAS deficiency, however, both measurement of the amniotic fluid citrulline level and enzyme assay should be employed.

Location of the major corneal reference points.

INTRODUCTION: The two principal applications of corneal topography are those pertaining to corneal shape, such as contract lens posterior surface design, and those pertaining to corneal power, such as predicting power changes after refractive surgery. Corneal topography measurements for these applications have advantages when related to two major reference points, the corneal apex for shape and the corneal sighting center for power. METHOD: Instantaneous corneal power, toricity, and axis were measured by videokeratography in 20 right eyes using the standard instrument alignment and alignments at each of the 2 major reference points. RESULTS: In comparing the central corneal measurements by videokeratography using the standard alignment to alignment at the corneal sighting center, there were minimal differences of less than 0.50 D for central power, toricity, and axis. In comparing the results for regular alignment to apex alignment there were significant differences for power (greater than 0.50 D) in 5 eyes, toricity in 3 eyes, and axis shifts of greater than 10 degrees in 5 eyes. In comparing the results for the corneal sighting center to apex alignments there were significant differences for power in 4 eyes, toricity in 2 eyes, and axis in 5 eyes. Changes of greater than 1.00 D occurred in the corneal periphery. CONCLUSIONS: In comparing standard videokeratograph alignment to alignment at the corneal sighting center, there were statistically insignificant differences in videokeratography results for corneal power, toricity, and axis. Significantly larger differences were found in comparing standard alignment and alignment at the corneal apex, especially for measurements in the corneal periphery.

Accuracy of videokeratography for instantaneous radius in keratoconus.

PURPOSE: Present videokeratographs provide corneal radius of curvature measurements in terms of axial (sagittal) radius and, for some instruments, instantaneous (tangential) radius. The EyeSys videokeratograph allows conversion from axial to instantaneous radius based on an algorithm, which assumes a basic corneal shape of elliptical form. METHOD: We tested the accuracy of the EyeSys instantaneous radius algorithm for 29 keratoconic corneas using the criterion that instantaneous radius of the cone apex could be determined accurately by aligning the cone apex with the optic axis of the videokeratoscope. RESULTS: There was a close relation between the instantaneous values obtained from the regular and apex alignments for the 17 eyes with cone apices below 55 D (95% limits of agreement -0.39 to 1.49 D), but not for the 12 eyes above 55 D (95% limits of agreement -4.87 to 6.61 D). The mean distance of the cone position from the center of the map was 1.16 mm for the instantaneous map and 1.59 mm for the axial map. CONCLUSION: Assuming the criterion for accuracy to be the corneal radii found when the videokeratograph was aligned with the cone apex, the instantaneous map for the EyeSys videokeratograph at regular alignment provided reasonably valid measurements for corneas up to 55 D, but large errors existed for some corneas of higher power.

A spline surface algorithm for reconstruction of corneal topography from a videokeratographic reflection pattern.

A videokeratograph quantifies one or more aspects of corneal shape by the computer analysis of a digital image. The image is the reflection formed by illuminating the cornea with a known source pattern. The type of shape information recovered and the accuracy of this information depend upon properties of the computer algorithm used in the analysis. We present a new algorithm that recovers a polynomial spline description of corneal depth.

Shape and refractive powers in corneal topography.

PURPOSE: To compare the relative advantages and disadvantages of four different representations of corneal power--identified as instantaneous, axial, position, and refractive--based on curvature, slope, coordinate position, and focal properties, respectively. METHOD: The four types of corneal power were evaluated by examining their interrelationship for 12 hypothetical corneal shapes chosen to represent the general characteristics of regular, irregular, and surgically altered corneas. RESULTS: There is only a limited association between refractive power and the other three powers, which are based on shape properties. For corneal shapes represented by ellipsoids of low eccentricity (e), refractive power increased as a function of the distance from the reference axis, whereas axial and instantaneous powers were either constant (for e = 0) or decreased, with instantaneous power having the largest decrease. Refractive power decreased for elliptical corneal shapes with eccentricities greater than the reciprocal of the index of refraction, although always decreasing less than the axial or instantaneous power. For various corneal shapes formed by segments of circles (or a polynomial) with large differences in instantaneous power, the refractive power was more closely associated with axial than instantaneous or position powers. CONCLUSIONS: The Purkinje image measurements from videokeratograpy are most closely related to the slope-based axial power because it is slope that determines the direction of reflected rays forming the corneal image. Because of this direct connection, axial power is less sensitive to noise than is refractive, instantaneous, or position power. Present videokeratographs that report axial power provide an approximation of refractive power, but if an exact refractive power is needed it can be calculated easily. Instantaneous power provides the most sensitive measure of local curvature changes, such as those occurring in keratoconus or refractive surgery. There are unique practical applications for each of the four powers.