Macular pigment and lutein supplementation in retinitis pigmentosa and Usher syndrome.

PURPOSE: To determine macular pigment (MP) in patients with inherited retinal degeneration and the response of MP and vision to supplementation of lutein. METHODS: Patients with retinitis pigmentosa (RP) or Usher syndrome and normal subjects had MP optical density profiles measured with heterochromatic flicker photometry. Serum carotenoids, visual acuity, foveal sensitivity, and retinal thickness (by optical coherence tomography [OCT]) were quantified. The effects on MP and central vision of 6 months of lutein supplementation at 20 mg/d were determined. RESULTS: MP density in the patients as a group did not differ from normal. Among patients with lower MP, there was a higher percentage of females, smokers, and light-colored irides. Disease expression tended to be more severe in patients with lower MP. Inner retinal thickness by OCT correlated positively with MP density in the patients. After supplementation, all participants showed an increase in serum lutein. Only approximately half the patients showed a statistically significant increase in MP. Retinal nonresponders had slightly greater disease severity but were otherwise not distinguishable from responders. Central vision was unchanged after supplementation. CONCLUSIONS: Factors previously associated with lower or higher MP density in normal subjects showed similar associations in RP and Usher syndrome. In addition, MP in patients may be affected by stage of retinal disease, especially that leading to abnormal foveal architecture. MP could be augmented by supplemental lutein in many but not all patients. There was no change in central vision after 6 months of lutein supplementation, but long-term influences on the natural history of these retinal degenerations require further study.

Susceptibility of B-cell lymphoma to human antibodies encoded by the V4-34 gene.

Our previous studies have shown that mAbs derived from the human V4-34 gene bind and kill human B-lymphocytes via membrane disruption. This study demonstrates the cytotoxicity of two V4-34 encoded mAbs, 216 and Z2D2, towards human B-cell lymphoma. In vitro, 216 and Z2D2 are cytotoxic to a variety of B-cell lymphomas obtained from patient biopsies. In vivo, increased survival was observed with both mAbs in a lymphoma model developed in scid mice with human B-cell line Nalm-6. Studies in mice show that these mAbs are well tolerated with minimum side effects. Since 216 and Z2D2 show increased toxicity towards cycling cells, V4-34 mAb-based therapy can be additive with drugs that block cell-cycle progression. Stem cells that are V4-34 mAb ligand negative would not be depleted. Together, these studies recommend an evaluation of the two completely human mAbs in a phase I trial for B-cell lymphoma.

Senescence of foveal and parafoveal cone sensitivities and their relations to macular pigment density.

Foveal and parafoveal increment thresholds were measured for 50 observers (12-88 years of age) under conditions that isolated retinal mechanisms dominated by short- (S-), middle- (M-), or long- (L-) wave-sensitive cones. Thresholds were obtained on the plateau of the threshold-versus-intensity function of each isolated mechanism and were referred to the retina by using individual measurements of ocular media and macular pigment density. Age-related increases in foveal thresholds, specified at the retina, were found for all three cone mechanisms. Parallel sensitivity losses for each cone mechanism were also observed at 4 degrees and 8 degrees in the temporal retina. A significant positive correlation was found between foveal macular pigment density and the S-cone, but not the M- and L-cone, log sensitivity difference (0 degrees-8 degrees) specified at the retina. This relation is expected from the hypothesis that the macular pigment protects the photoreceptors from senescent losses in sensitivity. However, because this result is independent of age, it is interpreted as being due to local gain changes resulting from differential filtering of incident light by the macular pigment between the fovea and the parafovea.

Recognition of auto- and exoantigens by V4-34 gene encoded antibodies.

The antigenic specificities of 24 V4-34-encoded monoclonal antibodies were compared with the amino acid sequence. The specificities were divided into three categories, red blood cells, B lymphocytes and auto/exoantigens. Six anti-I monoclonal antibodies, with multiple substitutions in their VH region, did not bind B lymphocytes or auto/exoantigens. Reactivity to these two antigens segregated with the 16 anti-i monoclonal antibodies, which were derived from the near germline V4-34 gene. All anti-i monoclonal antibodies bound B lymphocytes, albeit with varying intensities. B-cell binding correlated with basic amino acids in the VH-CDR3. Reactivity to auto/exoantigens was demonstrated only by a subset anti-i monoclonal antibodies and did not correlate with B-lymphocyte or i-antigen binding. These anti-ssDNA reactive monoclonal antibodies had basic amino acids in the VH-CDR3, strongly supporting the suggested role of arginine in DNA binding. However, an arginine-rich CDR3 was not enough to ensure DNA reactivity, since six other anti-i monoclonal antibodies that fulfilled this criteria did not bind ssDNA. Thus it is possible that the anti-DNA reactivity of V4-34-encoded monoclonal antibodies is mediated by the classic antigen-binding groove generated by the CDRs of the heavy/light chains. In contrast, anti-B-cell/i-antigen reactivity is mediated, unconventionally, by the V4-34 protein with a dominant influence of the VH-CDR3.

VH4-34 encoded antibodies in systemic lupus erythematosus: a specific diagnostic marker that correlates with clinical disease characteristics.

OBJECTIVE: To determine the clinical significance of elevated serum levels of VH4-34 encoded antibodies (VH4-34 Ab) with respect to the diagnosis and clinical characteristics of systemic lupus erythematosus (SLE). METHODS: Ninety-five patients with SLE and 344 controls were studied. The controls included 34 healthy individuals, 282 patients with nonautoimmune diseases, and 28 patients with autoimmune diseases other than SLE. VH4-34 Ab levels were measured by inhibition ELISA using anti-idiotope monoclonal antibody (9G4). SLE disease activity, severity, and damage were assessed by visual analog scales, Systemic Lupus Activity Measure, Lupus Severity of Disease Index, and Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index. RESULTS: Fifty-two of 95 patients with SLE had elevated levels of VH4-34 Ab compared to 18 of 344 controls (5%), giving a sensitivity of 55% and a specificity of 95% for elevated VH4-34 Ab as a serologic test for SLE. The positive predictive value of elevated VH4-34 under these conditions was 74-85%. In this study, anti-dsDNA was not VH4-34 encoded. Significant correlations between VH4-34 and disease activity and severity indices were observed (r = 0.29-0.50). The relative risk for severe disease in SLE patients with VH4-34 antibody level in the highest tertile compared to the lowest tertile was 5.25. Twenty-five of 29 patients with lupus nephritis and 6 of 6 patients with central nervous system (CNS) lupus had elevated VH4-34 Ab. CONCLUSION: With a specificity of 94-95%, the VH4-34 antibody assay may prove valuable as a confirmatory diagnostic test for SLE. In patients with known SLE, serum VH4-34 Ab levels correlate with overall disease severity and activity, but not damage, and with nephritis and CNS lupus.

Effects of known variations in photopigments on L/M cone ratios estimated from luminous efficiency functions.

The extent to which known variations in photopigment lambda max and optical density may affect cone ratios estimated from the spectral luminous efficiency function (LEF) was examined. LEFs were generated using L- and M-cone fundamentals, one of which had been shifted in lambda max (+/- 1, 2, 4 or 6 nm) or varied in peak optical density (increased or decreased by 10, 25 or 50%). A curve-fitting program was then used to estimate the L/M cone ratios for the generated LEFs assuming standard L- and M-cone fundamentals. These modeling exercises indicate that L/M cone ratios estimated from LEFs are highly correlated with long-wave sensitivity and with known variations in L-cone lambda max. Variations in M-cone lambda max and photopigment optical density for both cone types are also correlated with L/M cone ratios, but have much less impact on the estimated ratios.

M- and L-cones in early infancy: I. VEP responses to receptor-isolating stimuli at 4- and 8-weeks of age.

A silent-substitution technique combined with measures of the visually-evoked potential (VEP) was used to determine whether M- and L-cones are functional in early infancy. Data were successfully collected from twenty six infants in response to three receptor-isolation conditions (rod, M- and L-cone isolation) and a luminance-modulation condition. The efficacy of the receptor-isolation conditions was first verified by measuring VEP responses from both dichromatic and color-normal adults to each of the receptor-isolation conditions. Both 4- and 8-week-old infants demonstrated VEP responses to the M- and L-cone isolating stimuli, though the amplitude of the the responses at 4-weeks were reduced compared to those at 8-weeks. These data suggest that the functioning of M- and L-cones can be differentiated as early as 4-weeks of age.

M- and L-cones in early infancy: II. Action spectra at 8 weeks of age.

Field sensitivities were measured under conditions of M- and L-cone isolation for seven infants (8-12 weeks-old) and two adults, using silent-substitution and the visually evoked potential (VEP). The efficacy of the receptor-isolation conditions were first verified by measuring psychophysical and VEP-derived action spectra from two color-normal adults under conditions of M- and L-cone isolation. M- and L-cone action spectra obtained from the two methods were found to be similar to the Smith and Pokorny M- and L-cone fundamentals, respectively. The VEP-derived action spectra obtained from infants and adults were well fit by the Smith and Pokorny M- and L-cone fundamentals. These data, in conjunction with our previous study, confirm that M- and L-cones are operating by 8 weeks and possibly as early as 4 weeks of age.

The area of complete scotopic spatial summation enlarges with age.

The maximal area of complete scotopic spatial summation (Ricco's area) was determined for 50 subjects ranging in age from 19 to 87 yr. Increment thresholds were measured for 10-ms, 520-nm circular test lights of varying diameters that were superimposed and concentric with a 10 degrees, 640-nm circular background. The test lights were imaged in Maxwellian view along the horizontal meridian, 6 degrees nasal from a foveal fixation point. The results demonstrate a statistically significant enlargement of Ricco's area with age. The average angular subtenses of Ricco's areas for the ten youngest (mean = 26 yr) and ten oldest (mean = 75 yr) observers were approximately 48 and 69 arc min, respectively. Model simulations based on a series of optical transfer functions of the eye and varying degrees of intraocular light scatter for younger and older observers show that preneural factors cannot account for these results. Therefore changes in neural mechanisms must be invoked to explain the enlargement in the size of Ricco's area under scotopic conditions.

M- and L-cones in early infancy: III. Comparison of genotypic and phenotypic markers of color vision in infants and adults.

Genetic analyses were performed on five male children (approximately 3 years), two suspect color-normals and three suspects for congenital color vision deficiencies. These classifications were based on visually-evoked potential (VEP) responses to M- and L-cone-isolating stimuli obtained in a previous study when each subject was either 4- or 8-weeks old. The present analyses were performed in a blind study to characterize the genotypes of these subjects. Four male adults with various color vision phenotypes were also tested as a control. DNA was isolated using a non-invasive technique followed by polymerase chain reaction (PCR) amplification and restriction enzyme analysis to examine the genomic DNA of each subject. The genetic analyses confirmed the VEP identification of two color defective infants, and were consistent with the diagnosis of two other infants as color normal. A third infant was predicted by VEP analysis to have a protan defect, but he did not have a gene array typically found in protan observers.

Cytotoxicity of murine B lymphocytes induced by human VH4-34 (VH4.21) gene-encoded monoclonal antibodies.

We have previously described specific binding and cytotoxicity of human B lymphocytes by VH4-34 gene-derived anti-i cold agglutinin (CA) mAbs. Here we demonstrate that the carbohydrate ligand recognized by human VH4-34 anti-i CA mAbs is also expressed on murine B lymphocytes. Similar to human B cells, binding of murine B lymphocytes by VH4-34-derived anti-i CA mAbs leads to rapid cytotoxicity of target cells as tested both in vitro and in vivo. Moreover, the mechanism leading to murine B cell death is also similar to human B cells, since morphologically identical membrane pores were detected within 15 min of mAb exposure by scanning electron microscopy. The conservation of the carbohydrate ligand across species provides an ideal system to study the function of human VH4-34 gene derived Abs in immune regulation.

Sensitizing properties of spectral lights in 4-month-old human infants.

Previous studies of infant attention, learning, and memory have revealed that certain stimulus properties may increase an infant's arousal or excitation level, thereby increasing responsiveness and facilitating the encoding and processing of information. In a series of experiments aimed at identifying stimulus determinants of sensitization, we examined visual responses from 4-month-old infants to spectral lights. Habituation data were obtained from 92 full-term infants separated into one of five groups. Each group viewed either a broadband white light (correlated color temperature approximately 2800 K) or one of four different spectral lights (lambda d = 470, 510, 570, or 650 nm) approximately corresponding to the elemental hues blue, green, yellow and red, respectively, for adults with normal trichromatic vision. Stimuli were equated in luminous efficiency for a standard infant observer. Stimulus fixation was recorded for twelve 10-s presentations, each separated by 10-s interstimulus intervals (ISIs). The results show that mean fixation times to the red and green lights were significantly greater than those for the blue and white light. Mean fixation time for the yellow light was also reduced (significantly) compared to the red but not the green light. These results suggest that the chromatic properties of red and green spectral lights may be more sensitizing to infants than those of the blue, yellow, or white lights.

Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies, II.

We have previously described complement-independent killing of human B lymphocytes by two IgM MoAbs derived from the VH4-34 (VH4.21) gene. Analysis of 17 independently derived VH4-34-encoded MoAbs shows that B cell toxicity is not limited to the two described MoAbs, but is a general property shared by a subset of MoAbs derived from the VH4-34 gene. As observed by two independent microscopy techniques, giant membrane pores were formed on target B cells within 10-15 min of exposure to cytotoxic VH4-34-derived MoAbs. Toxicity by individual MoAb correlated directly to its B cell binding intensity measured by FACS, i.e. stronger the binding greater the killing. Sequence analysis showed that V(H) region in germ-line or in near germ-line configuration was necessary but not sufficient for B cell binding. In addition, a particular sequence motif enriched in basic amino acids in the CDR3 may be required to supplement the reactivity mediated by the V(H) region of the MoAb molecule. VH4-34-encoded antibodies that fulfil the above sequence requirements have cold agglutinin activity towards the i antigen of cord erythrocytes. In vivo, such anti-i/anti-B cell antibodies are rarely detected in healthy adults, but serum levels are dramatically elevated in selective pathological conditions, such as systemic lupus erythematosus and infectious mononucleosis. This strict regulation may be related to the novel and rapid mechanism of human B cell toxicity demonstrated by antibodies encoded by a single human V(H) gene.

Heavy chain variable gene usage by human B-1 lymphocytes and polyreactive autoantibodies.

To evaluate the role of B-1 cells and polyreactive autoantibodies in the development of adult immune repertoire, it is necessary to assess their immunoglobulin heavy chain variable-gene usage. We thus screened 28 independently derived human polyreactive MAbs from fetal and adult splenic B lymphocytes for their VH-region usage. We demonstrate that the polyreactivity of the IgM antibodies secreted by B-1 cells is not the result of the expression of particular variable region gene families. All six VH families are represented roughly in proportion to their estimated family size. Furthermore, the representation of the six families appears similar in polyreactive MAbs derived from fetal or adult lymphocytes.

VH4-34 (VH4.21) gene expression in the chronic arthritides of childhood: studies of associations with anti-lipid A antibodies, HLA antigens, and clinical features.

OBJECTIVE: To determine if the germ line gene VH4-34 (VH4.21) encodes the antimonophosphoryl lipid A (MPL) polyspecific antibodies found in oligoarticular arthritis of childhood. METHODS: Sera from a range of rheumatic diseases of childhood were assayed for VH4-34 derived antibodies by ELISA using the antiidiotype monoclonal antibody 9G4. Results were compared to assays for anti-MPL antibodies, C4d, and Bb, and for HLA type, joint count, and sedimentation rate. RESULTS: VH4-34 derived antibodies were elevated in all diseases studied except rheumatoid factor positive polyarticular disease. In oligoarticular arthritis, VH4-34 gene expression correlated with C4d concentration, and VH4-34 encoded globulins were more concentrated in synovial fluid than in blood. No association was found with HLA type. An association between VH4-34 expression and IgG anti-MPL was found in sera from patients from Cincinnati but not from Stanford. No other evidence supported a direct association between VH4-34 derived and anti-MPL antibodies in these children. CONCLUSION: The expression of VH4-34 is increased in several rheumatic diseases of childhood, but, as in adults, not in rheumatoid arthritis. VH4-34 expression is not associated with HLA type. The polyspecific autoantibody nature of some VH4-34 derived antibodies may explain the wide range of the unusual antibodies found in oligoarticular arthritis.

Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies.

We have previously described two human cold agglutinin MoAbs 216 and A6(H4C5), that are derived from the VH4-34 (VH4.21) gene that bind specifically to a cell surface ligand on human B lymphocytes. In this study, we report that binding of 216 and A6(H4C5) leads to rapid killing of target B cells. This complement-independent cytotoxicity was measured by three independent assays, cell viability dye uptake on FACS, 3H-thymidine uptake, and the 3(4,5)-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxicity was specific for CD20+ mononuclear cells in human spleen and peripheral blood. The MoAbs were also cytotoxic to human B cell lines Nalm-6, OCI-LY8, Arent and SUP-B8, but not to T cell lines HuT 78 and PEER. As observed by scanning electron microscopy, membrane pores were formed within 15 min of exposure to the MoAbs. Cytotoxic activity was dependent on MoAb concentration and temperature of exposure. Killing with greater at 4 degrees C than 37 degrees C. Sodium azide and EDTA did not block the cytotoxic activity. No DNA fragmentation typical of apoptosis was observed. This rapid cytotoxic activity, independent of physiologic cellular process and independent of complement, suggests a novel mechanism of all death via membrane perturbations.

Anti-endotoxin human monoclonal antibody A6H4C5 (HA-1A) utilizes the VH4.21 gene.

Human IgM monoclonal antibody A6H4C5 was manufactured by Centocor (Malvern, PA) and used in clinical trials as HA-1A (Centoxin). In vitro, A6H4C6 binds to lipid A and rough-strain, gram-negative bacteria endotoxin. Further analysis of A6H4C5 has shown that it is a polyreactive, cold agglutinin that utilizes the VH4.21 gene segment in germline configuration. It is also a human antibody that binds to human B cells. We have characterized several other independently derived VH4.21 human monoclonal antibodies with the same characteristics as A6H4C5. This group of antibodies may represent a conserved host immune response.

Spectral efficiency measured by heterochromatic flicker photometry is similar in human infants and adults.

Spectral efficiency functions based on heterochromatic flicker photometry (HFP) were measured for three adults and 42 infants using a rapid visually-evoked potential (VEP) method. A 5 degrees-diameter, broadband standard (0.6 cd/m2) was presented in square-wave counterphase (15 Hz) with one of 13 monochromatic lights (420-660 nm; 20 nm steps). The intensity of the monochromatic light was continuously varied while extracting the phase-locked VEP amplitude of the fundamental component. HFP functions measured psychophysically by the method of adjustment were also obtained for the adults. Adult HFP functions from the two methods were found to be essentially the same. Both of these functions were compared to Vos'-modified 2 degrees V(lambda) function and the 10 degrees CIEV(lambda) function. The mean adult data were slightly better fit to the 2 degrees V(lambda) function than to the 10 degrees CIEV(lambda) function, although there was an elevation in sensitivity at 420 and 440 nm. Infant HFP functions were similar to Vos' modified V(lambda) except for an elevation in efficiency at short wavelengths. The mean infant HFP function agreed better with the 10 degrees CIEV(lambda) function than Vos'-modified V(lambda) function, but infant sensitivity was elevated by 0.4 log units at 420 nm compared to the 10 degrees CIE observer. The elevation found at short wavelengths for both adults and infants is attributed to individual and age-related variation in the density of the ocular media, and to reduced macular pigment screening resulting from use of a 5 degrees field size.(ABSTRACT TRUNCATED AT 250 WORDS)

Human CD5+ B lymphocytes (B-1 cells) decrease in peripheral blood during pregnancy.

Pregnancy is a unique immunologic state where a natural homeostasis exists between antigenically different tissues. Several earlier studies have addressed the fluctuations in the number and/or function of lymphocytes, including B cells during pregnancy, but changes within the subsets of B lymphocytes, conventional (CD5-) and B-1 (CD5+), have not been addressed. Here we demonstrate that the frequency of B-1 cells decreases dramatically during pregnancy, whereas the frequency of conventional B cells remains relatively constant. The missing B-1 cells return to pre-pregnancy levels 8-10 weeks after parturition. The polyreactive autoantibodies secreted by B-1 cells have been implicated in autoimmunity and immune regulation. The possible role of B-1 cells during pregnancy will be discussed in that context.

FACS analysis of peritoneal lymphocytes in ovarian cancer and control patients.

In this study different lymphocyte populations in the malignant ascites of 10 patients with ovarian carcinoma and in the peritoneal fluid of 8 control patients (tubal ligation and benign conditions) were analyzed. A panel of monoclonal antibodies against the CD markers of lymphocytes was used to stain different populations and the cells were analyzed on a FACS II (fluorescence-activated cell sorter). The mean percentage of B lymphocytes in the peritoneal cavity of the OVCA patients was 0.18 +/- 0.5% and in the control patients 0.05 +/- 0.07%. There was no significant difference between the two groups. In the OVCA group and in the controls the percentage of T lymphocytes (CD5+) was 23.5% and 17.1% respectively with no significant difference between the groups. These results indicate that B lymphocytes are not present in the human peritoneal cavity. The small numbers of B cells found in this study could be due to contamination with peripheral blood. The human peritoneal cavity contains a cell population which differs from that present in peripheral blood. Significant numbers of B lymphocytes have been reported in the peritoneal cavity of mice. The difference between the lymphocyte population of the human peritoneal cavity and that of rodents implies that data on characterization and function of B lymphocytes in the mouse peritoneal cavity would not be applicable to humans.