Rare mutations in a domain crucial for V3-loop structure prevail in replicating HIV from long-term non-progressors.

OBJECTIVE: To evaluate the role of the selective forces exerted by the host on the HIV-1 structures involved in viral entry. DESIGN AND METHODS: The V3 region of the env gene was analysed in cell-free HIV-1 RNA from 17 infected subjects: 11 long-term non-progressors (LTNP) and six symptomless, typical progressor patients. To evaluate the potential biological significance of one of the rare variants detected in the LTNP, it was reproduced by recombinant PCR into a HIV-1 molecular clone. RESULTS: The intrapatient divergence of the V3-loop sequences averaged 8.62% in LTNP and 5.29% in progressors, although LTNP displayed lower divergence from the clade B consensus than progressors (16.65 and 19.76%, respectively). The analysis of non-synonymous and synonymous substitutions indicated that selective pressure was exerted in this region in both LTNP and progressors. Individual peculiarities (unique and rare V3-loop variants) emerged, however, in most sequences from LTNP, and variants bearing mutations in a domain crucial for the V3-loop structure were more prevalent in LTNP (P = 0.0012). The pNL4-3-derived mutant reproducing a V3-loop variant detected in a LTNP was efficiently expressed upon transfection, but the mutant virus was nearly completely unable to infect CD4+ cell lines, activated primary peripheral blood lymphocytes, or monocyte-derived macrophages, suggesting that a defect impaired the entry phase of the replication cycle. CONCLUSIONS: The results indicate that host factors impose selective constraints on the evolution of the HIV-1 structures involved in viral entry. In LTNP, these factors are likely to force the virus into attenuated variants.

Pleomorphic xanthoastrocytoma of the retina.

We report two cases of pleomorphic xanthoastrocytomas of the retina. The immunoreactivity to CD68, Fe (Pearls' technique), astrocytes (argentic technique), and antibodies to glial fibrillary acidic protein was tested in tumor cells. Both neoplasms contained cells immunoreactive for glial fibrillary acidic protein and CD68. These tumors, observed in the CNS, first reported by Kepes et al. were found in the retina as well-circumscribed masses with a cystic component. Many large pleomorphic cells contained vesicular and enlarged nuclei with a homogeneous eosinophilic cytoplasm, prominent nucleoli, and calcium deposits. Some cells had a markedly swollen lipidized cytoplasm, whereas others were spindle-shaped and contained multiple nuclei. These findings suggest that our two specimens have an astroglial lineage and are similar to pleomorphic xanthoastrocytomas of the CNS brain and spinal cord. Both reported cases were in female patients who were in their 20s and had glaucoma. After 10 years both are free of disease.

Scanning electron microscopy of the trabecular meshwork in normal and glucomatous eyes.

The ultrastructure of trabeculectomy specimens obtained from enucleated eyes was compared to that of 23 similar specimens extracted from glaucomatous eyes. It was thus possible to study the trabecular meshwork from Schwalbe's line to the iris root, the corneal endothelium, the canal of Schlemm, its inner and outer walls, and the collectors. In the glaucomatous specimens it was possible to verify, for the first time, two aspects which show clinical correlation. The first aspect is pseudoexfoliation: we find in the trabecular meshwork two types of deposits in great quantity. One is formed by a dense irregular fibrillar net, with a diameter oscillating betwwwn 600 and 830 A, surrounding the trabeculae; the others are pigment granules. Both fill the intertrabecular spaces. The first type is also observed on the corneal endothelium lining Schwalbe's ring, stressing the visibility of the intercellular spaces. The second aspect is the pectinate ligament (mesodermic remnant) in late-onset congenital glaucoma. Its pattern is characteristically different from that of the corneoscleral meshwork.

Comparable biological and molecular determinants in HIV type 1-infected long-term nonprogressors and recently infected individuals.

Human immunodeficiency virus type 1 (HIV-1) isolability, rate of replication, phenotype, plasma viremia, and specific intracellular transcripts were cross-sectionally analyzed in 61 HIV-1-seropositive individuals to evaluate the correlations between the virological and molecular correlates of protection and progression in different clinical subsets: recently infected subjects (RIs), long-term nonprogressors (LTNPs), late progressors (LPs), and typical progressors (TPs). Comparison of the major virological and molecular features of HIV-1 infection has defined distinct profiles for different subsets of patients. LTNPs or RIs, as well as LPs or TPs, exhibited similar titers of coculture p24 antigen; the differences between the former and the latter were statistically significant at all the time points tested (p = 0.0001; 0.0003 and 0.0001). Whereas LTNPs and RIs revealed comparable low levels of indexes of viral replication, LPs and TPs showed higher genome and mRNA copy numbers (p = 0.0004 and p = 0.0008, respectively). We demonstrated close biological and molecular similarities between RIs and LTNPs on the one hand, and LPs and TPs on the other. In LTNPs both viral biological properties and viral load are important determinants of the course of the disease.

Ocular echometry in the diagnosis of congenital glaucoma.

Thirty-three eyes of 18 normal infants and children and 36 eyes of infants in whom congenital glaucoma had been diagnosed were measured by echometry. The anterior chamber depth, vitreous length, and axial length were notably greater in glaucomatous eyes, while the lens thickness was smaller in glaucomatous eyes. The axial length of normal eyes was found to increase with age. The confidence interval of the normal growth curve allows the prediction of the maximum normal axial length for any given age. In congenital glaucoma cases in which the visual fields and daily pressure curve cannot be evaluated, clinical echometry is proposed as a valuable diagnostic and follow-up procedure.

Prof. Marc Amsler and the Cantonal Hospital Eye Clinic, Zurich.

Prof. Marc Amsler was an outstanding teacher of ophthalmology and a highly creative clinician, investigator and designer of instruments and surgical procedures. Well known for his design of the Amsler grid, he also conducted investigations into anterior chamber puncture and the aqueous humor in uveitis. In this biographical sketch the author recalls his experience working with Prof. Amsler in Zurich in 1955, during which time the author made important discoveries regarding the increased permeability of the blood-aqueous barrier in exfoliation syndrome.

Molecular monitoring of human immunodeficiency virus type 1 infection.

Over the past few years, considerable technical effort has been directed to developing molecular methods that would allow an effective approach to the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection and its monitoring. Indeed, quantitative molecular techniques have opened the way for a new type of direct study of untreated and treated HIV-1 infected subjects. The understanding of the immunopathogenesis of HIV-1 infection has increased significantly with the introduction of advanced virological and molecular methods for accurate quantitative analysis of HIV-1 activity; powerful methodologies answer (directly and in real time) most questions generated by pathogenic research and by the novel anti-viral strategies introduced in clinical practice. The data from pilot diagnostic applications of quantitative techniques have clarified important features of the natural history of HIV-1 infection. Moreover, an increasing amount of data indicate the need for second-level laboratory facilities for the clinical management of infected patients; virological aspects and some genetic features of the hosts concerning HIV-1 co-receptors (all the co-receptors so far identified are members of, or related to, the transmembrane, chemokine-receptor family) need to be elucidated for the complete diagnostic evaluation of HIV-1-infected subjects.

[Retinal detachment and pigment dispersion syndrome]. / Amotio retinae und Pigmentdispersionssyndrom.

PATIENTS: The frequency of retinal detachment has been studied in 107 consecutive patients suffering from pigmentary glaucoma. The frequency of retinal detachment in pigmentary glaucoma is 12%, while in the normal population is only 0.001%. Pigmentary glaucoma is a disease which may manifest itself for the first time either as a retinal detachment or with symptoms of pigmentary glaucoma. CONCLUSION: Therapy with miotics affecting accommodation is completely contraindicated.

The chamber angle in exfoliation syndrome. Clinical and pathological findings.

The clinical picture of the chamber angle in capsular glaucoma is described. One or more waves of pigment overpass Schwalbe's line and accumulate on the posterior face of the cornea. These waves are found in the chamber angle between 4 and 8 o'clock across 6 o'clock. The pathological anatomy of the chamber angle in the exfoliation is studied with light microscopy, scanning electronmicroscopy and transmission electronmicroscopy in relation with the material of pseudoexfoliation and pigment granules and the difference with the pigmentary glaucoma is shown.

Large optic nerve heads: megalopapilla or megalodiscs.

PURPOSE: To evaluate megalopapillas in order to determine whether they constitute a different population, according to their specific features. MATERIAL: Evaluation of the optic nerve head of 405 eyes divided into 172 normal eyes, 168 pre-perimetric glaucomas (phase 3 or 4), 30 primary congenital glaucomas (children with pure congenital glaucomas operated more than once and diagnosed within the first year of age), and 35 megalopapillas. METHOD: The examinations were performed with the HRT (Heidelberg Retina Tomograph), using a wavelength of 680 nm and the new standard reference plane. Each examination results from the mean of 3 image acquisitions with a SD lower than n = 20. Each optic nerve head was studied in 360 degrees (segments), as well as in quadrants and octants separately (predefined segments). The visual fields were assessed with either the Octopus 1-2-3 or the Octopus 101, programs G2 and G2x (three complete phases). The intraocular pressure was measured by means of daily pressure curves including 7 measurements with applanation tonometry at the office and the first one at 6/7 a.m. with the patient still in bed. Gonioscopic examinations were also performed for classification purposes. RESULTS: By comparing all the stereometric parameters with the Total Area (disc area) and the Rim Volume it has been concluded that the megalopapilla group is different from both the normal control group and the glaucoma group. The disc area of the congenital glaucomas was not statistically different from the megalopapillas, but it was in terms of rim volume and other parameters. CONCLUSION: megalopapilla is an entity characterized by a large optic nerve head which may appear abnormal, with an increased cup, but associated with a normal rim volume, normal visual field and normal IOP. Special attention should be given to its differentiation from pseudoglaucomatous diseases. DISCUSSION: The frequency of megalopapillas seems to be quite higher than in congenital optic nerve head anomalies, but is has been virtually ignored by the literature worldwide so far.

Malignant melanoma of the conjunctiva. Report of a case.

Malignant melanomas of the conjunctiva are extremely rare in children. The authors report an 11-year-old boy who had a nodule at the limbus in a pigmented area since early childhood. The lesion was excised and the diagnosis of atypical compound nevus was made. Five months later, the patient returned with multiple nodules in the bulbar and palpebral conjunctiva. One of the lesions was excised, and the diagnosis was malignant melanoma. He was lost for follow-up for 6 months; then he presented a large pigmented, vascularized mass protruding through the palpebral aperture. Results of systemic workup for metastatic disease were negative, and an exenteration was performed. A few months later, cervical lymphadenopathy developed followed by extensive metastatic disease.

Antiglaucomatous drugs effects on optic nerve head flow: design, baseline and preliminary report.

PURPOSE: To determine the effect of the most frequent used antiglaucomatous drugs on the optic nerve head flow. To see the response of the autoregulation system in hypertensive, preperimetric and advanced perimetric glaucomas. MATERIALS: In this preliminary report, optic nerve head parameters and retinal perfusion values were measured in 25 healthy controls as well as in 72 glaucomatous optic nerve heads. The glaucomatous sample was divided into three groups: 24 eyes treated with Betaxolol, 24 with Brinzolamide and 24 treated with Brimonidine (each of these groups was conformed by 8 hypertensive glaucomas, 8 preperimetric glaucomas, and 8 perimetric glaucomas). METHODS: Patients were examined with scanning laser Doppler flowmetry (wave length 638 nm), and flow, volume and velocity indices were assessed in each report. Perfusion maps were analyzed with the new SLDF software, version 3.2 (automatic full field perfusion image analizer). Examinations were also performed with the Heidelberg Retina Tomograph (wave length 638 nm), using the new standard reference plane. Visual fields were performed with the Octopus 123 or 101 perimeters, programs Glx and G2, with 3 phases completed. Diurnal pressure curve with applanation tonometry and gonioscopy (for classification) were also performed. RESULTS: In this preliminary report, it was found that eyes in Hypertensive or preperimetric stages of glaucoma, seem to use their regulation systems in order to preserve their optic nerve head circulation, while advanced perimetric glaucomas seem not be able to preserve their optic nerve head flow. And that antiglaucomatous drugs were not able to increase optic nerve head flow neither. DISCUSSION: Optic nerve head blood supplies is regulated by an autoregulation system. This phenomenon may act in normals, hypertensive and preperimetric glaucomas, but it seems not be able to preserve blood flow in advanced glaucomas. Optic nerve head flow returns to normal values with treatment in glaucoma first stages, while it seems that it could not be restored in advanced glaucomas.

Optic nerve head behavior in Posner-Schlossman syndrome.

PURPOSE: To analyze the effect of glaucomatocyclitic crisis on the optic nerve head, as well as its consequent structural and hemodynamic changes. To evaluate the surgical indication for Posner-Schlossman syndrome. MATERIALS AND METHODS: Four Posner-Schlossman syndrome cases were evaluated for an analysis of the phenomena occurring in the optic disc by means of retinal confocal tomography and Scanning Laser Doppler Flowmetry, and their correlation with visual function as measured with automated computerized perimetry. In all cases the images were obtained in the mediate period after the attack, while in case 1, measurements were performed in the immediate and mediate periods before, during and after the attack. RESULTS: Significant differences were found between optic nerve head parameters and retinal flow measurements in the different phases evaluated. No permanent optic nerve head damage was demonstrated in any of the cases studied, except for case 4 (Posner-Schlossman syndrome associated with traumatic glaucoma). All the variables analyzed returned to normal values after remission of the acute rise in intraocular pressure. CONCLUSIONS: During the attacks of ocular hypertension the optic nerve head experiences significant morphologic changes and hemodynamic variations; nevertheless since they are transient, they fail to cause permanent damage. There was only one case, in which there was optic disc damage and visual field loss. It can be concluded that surgery should be restricted to those cases with severe and disabling symptoms (relative surgical indication) or to cases with progressive optic neuropathy with visual field loss, when the syndrome is associated with glaucoma (absolute surgical indication).

Quantitative molecular monitoring of human immunodeficiency virus type 1 activity during therapy with specific antiretroviral compounds.

Methods for the absolute quantitation of nucleic acids present in small amounts in biological samples (competitive PCR and competitive reverse transcription PCR) were applied to the direct monitoring of specific anti-human immunodeficiency virus type 1 (HIV-1) therapy. With these techniques, different parameters of HIV-1 activity (including genomic RNA copy numbers in plasma, proviral and late transcript copy numbers in peripheral blood lymphocytes, and mean transcriptional activity per each HIV-1 provirus) were monitored during therapy with azidothymidine or ddI. In most of these treated patients, a direct response to the antiretroviral compounds employed was detected during the first few weeks of treatment, as documented by a fast decrease of all molecular indexes of HIV-1 activity. However, residual viral replication (albeit at minimal levels) was documented during therapy in all subjects monitored in this study. In a minority of the patients under study (3 of 12), the drug-dependent viral inhibition was maintained throughout the observation time (213 to 791 days), but in 9 patients a rebound in viremia level was detected during therapy with competitive reverse transcription PCR. Sequencing analysis of a portion of the HIV-1 gene pol from cell-free virions showed that circulating viral variants bearing at least two mutations compatible with azidothymidine or ddI resistance were detectable in the patients who exhibited a rebound in cell-free HIV-1 genomic RNA copy numbers in plasma but not in one patient who maintained (for 455 days) lowered levels of viral load during ddI treatment.

Dynamics and modulation of human immunodeficiency virus type 1 transcripts in vitro and in vivo.

The dynamics of human immunodeficiency virus type 1 (HIV-1) transcription was analyzed in vitro and in vivo by using a specific molecular approach which allows accurate quantitation of the different classes of viral mRNAs. Unspliced (US) and multiply spliced (MS) HIV-1 transcripts were assayed by competitive reverse transcription (cRT)-PCR, using a single competitor RNA bearing in tandem internally deleted sequences of both template species. Acute HIV-1 infection of primary peripheral blood mononuclear cells (PBMCs), monocytes/macrophages cells, and the A3.01 T-lymphocyte-derived cell line was studied; both classes of HIV-1 mRNAs increased exponentially (r2 > 0.98) at days 1 to 3 and 1 to 4 postinfection in HIV(IIIB)-infected A3.01 cells and PBMCs, respectively, whereas monocytes/macrophages infected with monocytotropic HIV(BaL) exhibited a linear (r2 = 0.81 to 0.94) accumulation of US and MS transcripts. Following induction of chronically infected ACH-2 cells, MS transcripts increased 2 h postinduction and peaked at 5 h (doubling time, 58 min), while at 24 h, US mRNAs increased 3,053-fold compared with basal time (doubling time, 137 min). To address the biopathological significance of HIV-1 expression pattern during infection progression, pilot cross-sectional and longitudinal analyses were carried out with samples from untreated and treated HIV-1-infected patients. In almost all untreated (recently infected, long-term nonprogressor, and progressor) patients, MS transcript levels followed the general trend of systemic HIV-1 activity. In patients under treatment with powerful antiretroviral compounds, viral MS transcripts rapidly fell to undetectable levels, indicating that in vivo, levels of MS mRNAs in PBMCs are closely associated with the number of newly infected cells and suggesting a new role for the quantitative analysis of HIV-1 transcription in infected patients.