Implementation of an Italian multicentric care network for early HIV infection diagnosis: 1999-2001 report.

BACKGROUND: Diagnosis of a new HIV infection during the primary phase (PHI) is sometimes misleading in a primary care setting. Since 1999 the Italian network for the study of acute HIV infection (ISAI) has been operative. At the time of PHI diagnosis the case is reported to the coordinating centre and enrolled in the National Register which records all epidemiological, demographic and clinical information. PATIENTS AND METHODS: From 1999 to September 2001, 51 symptomatic or asymptomatic patients with diagnosis of primary HIV infection were signalled to the coordinating centre. At screening, assessments were: interview to collect demographic and epidemiological data, clinical history (regarding PHI signs and symptoms) and, if available, relevant index case information; physical examination; routine hematology and chemistry; lymphocyte count; plasma HIV-RNA. In a subset of patients PBMC HIV-DNA, HIV-RNA, resistance genotyping and HIV subtype characterization were assessed. RESULTS: 74.5% of patients were males and all but four were Italian. Hetero and homosexual contacts were the prevalent route of HIV transmission. Forty-five patients (89%) were symptomatic and the most frequent signs and symptoms were: fever, lymphadenopathy, malaise and pharyngodinia. Baseline reverse-transcriptase (RT) and protease (PR) genotyping analysis was available for 29 patients. Only one of 29 patients harbored a virus with a resistance-associated mutation in the RT region (215Y); NNRTI mutations were identified in 3 of 29 patients. In the remaining 20 (69%) patients no mutations were found in the RT region. Sequence data from PR region were successfully obtained in 21 patients. Only one of these had a high-level resistance mutation (46L); in an additional 10 cases 1 or more secondary mutations were identified. The remaining 10 patients harbored a PR region wild type virus. One patient presenting two secondary mutations in the PR region, even if highly adherent and tolerant to drug regimen, showed a slow viral load decrease. CONCLUSIONS: Our cohort confirms the uptrend of new infections through unsafe sexual contacts involving both homosexual and heterosexual couples. Genotype sequencing for antiretroviral resistant viral variants describes a low prevalence of RT resistance-associated mutations, as well as primary mutations in the PR region. On the contrary, a higher prevalence of PR gene polymorphisms and mutations is not known with any certainty to confer resistance to NRTI and NNRTI. The identification of antiretroviral drug resistant HIV strains is strategic for clinical and therapeutical intervention, even though from a public health point of view cost-efficacy must be considered.

An immunogenetic map of Lombardy (Northern Italy).

For this study we consulted the Bone Marrow Donors' Registry of Lombardy (Italy) and analyzed 43937 HLA-A,B phenotypes and 13922 HLA-A,B,DR phenotypes. We estimated the HLA-A,B and HLA-A,B,DR haplotype frequencies via the maximum-likelihood method. We analyzed the genetic structure of the 11 provinces of Lombardy by means of Principal Component Analysis and Correspondence Analysis, and estimated the variety of the different haplotypes at provincial level and the percentage of unique phenotypes at village level. We found 11189 different HLA-A,B phenotypes, 661 different HLA-A,B haplotypes and more than 4000 different HLA-A,B,DR haplotypes. We identified 20 villages, in Western Lombardy, very rich in unique/rare phenotypes. Here we report a formula which allows the identification of a putative donor matched for two haplotypes with a recipient. This result may be of great importance for the genetic study of the population of Lombardy and, even more, for bone marrow transplantation programs.

Claudin 5 is transiently expressed during the development of the retinal pigment epithelium.

During the development of chick retinal pigment epithelium (RPE), the permeability and selectivity of the epithelium's tight junctions are continuously modulated. Overall paracellular permeability decreases, but selectivity increases. Because the claudin family of transmembrane proteins appears to provide the structural basis for selectivity, we examined the expression of claudins as a function of development in chick RPE. Degenerate primers were used with the reverse transcriptase-polymerase chain reaction (RT-PCR) to obtain complete sequences of chick claudins 3 and 5. Northern blotting and semi-quantitative RT-PCR demonstrated that claudin 5 was expressed in RPE, but claudin 3 was expressed only in the choroid layer of the eye. Northern blotting, semiquantitative RT-PCR and immunoblotting demonstrated that the expression of claudin 5 was transient, with peak levels of expression between embryonic days 10 and 14. Primary cultures were used to demonstrate that factors secreted by the neural retina induced the expression of claudin 5 nearly 3-fold if RPE was isolated from embryonic day 7 embryos. There was little effect if RPE was isolated from embryonic day 14. The upregulation of claudin 5 correlates with permeability changes that occur during the intermediate stage of RPE development. Interestingly, claudin 5 must be replaced during the late stage of development when the number and complexity of tight junctional strands increases. This would imply more changes in selectivity.

Defects in the MITF(mi/mi) apical surface are associated with a failure of outer segment elongation.

The loss of MITF function in the MITF(mi/mi)mouse affects not only RPE differentiation, but also the development of rod photoreceptor outer segments. Our data indicate that opsin immunoreactivity is detected in the cell membrane and along the ONL/RPE border of developing MITF(mi/mi)rod photoreceptors and that rod outer segment morphogenesis is initiated. Although molecules associated with the outer segment continued to be expressed, outer segments did not elongate and develop stacked organized discs perpendicular to the RPE. The MITF(mi/mi)RPE also failed to form apical microvilli and lacked the apical network of the phosphoprotein ezrin seen in wild type tissue. The MITF(mi/mi)RPE basal surface was loosely organized and retained ezrin labelling which indicated some degree of differentiation. The correlation seen in our data suggest that there may be a link between the failure of the RPE apical domain to form and lack of rod outer segment elongation.

Differential regulation of tight junction permeability during development of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) is an epithelial region of the blood-brain barrier. During embryogenesis, permeability of the barrier gradually decreases. A culture model of RPE development revealed differences in how tight junctions regulate the paracellular diffusion of ionic and nonionic solutes (Ban Y and Rizzolo LJ. Mol Vis 3: 18, 1997). To examine these differences, the permeation of ionic and nonionic monosaccharides was compared with mannitol, and the permeation of the alkali metals was compared with sodium. The order of permeation was 3-O-methlyglucose = glucosamine = mannitol > N-acetylneuraminic acid. The ratio of N-acetylneuraminic acid to mannitol permeability decreased with embryonic age of the RPE or exposure to retinal-conditioned medium. Neither the ratio nor the permeability was affected by inhibiting transcytosis. The ratio increased if tight junctions were disrupted in low-calcium medium. The permeation of cations followed the sequence cesium > rubidium > potassium = sodium > lithium and was unaffected by embryonic age or retinal-conditioned medium. These results are considered in terms of a model in which the size distribution, charge, or number of open junctional pores could be modulated. It suggests that different subpopulations of pores can be regulated independently during development.

Regulation of glucose transporters during development of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) separates the outer retina from its blood supply. To satisfy the retina's large requirement for glucose, the RPE expresses high levels of glucose transporters. In most rat cells, the transporter GLUT3 provides a basal level of transport, but the expression of GLUT1 can be regulated. The opposite is true in chicken (P. Wagstaff, H.Y. Kang, D. Mylott, P.J. Robbins, M.K. White, Characterization of the avian GLUT1 glucose transporter: differential regulation of GLUT1 and GLUT3 in chicken embryo fibroblasts, Mol. Biol. Cell 6 (1995) 1575-1589). We examined chick RPE to determine which isoform is regulated during development, and if the neural retina regulates GLUT expression. By RT-PCR, RPE expressed GLUT1 and GLUT3, but not GLUT2. Only the level of GLUT1 increased between E5 and E18. A corresponding increase in GLUT1 protein was observed by immunoblotting. Most of the increase occurred between E14 and E18, which corresponds to the late stage of tight junction development. A culture model of development was used to examine the intermediate phase, which extends from E7 to E14. While medium conditioned by the neural retina decreased paracellular diffusion across the tight junctions, it increased diffusion through the glucose transporters. Unlike mammals, chick upregulates different isoforms in quiescent RPE and proliferating fibroblasts. Further, the upregulation of glucose transport is coordinated with the development of tight junctions in the blood-retinal barrier.

Two secreted retinal factors regulate different stages of development of the outer blood-retinal barrier.

The retinal pigment epithelium (RPE) lies at the interface between the neural retina and the choriocapillaries where it forms a blood-retinal barrier. Like endothelial regions of the blood-brain barrier, the development of the RPE barrier is a gradual, multistep process. A culture model of chick RPE was used to study this development. The permeability of the tight junctions that limit diffusion between neighboring RPE cells was measured as the transepithelial electrical resistance (TER). Embryonic day 14 (E14) retinas were used to make a conditioned medium that lowered the permeability of cultured RPE. The TER of cultures prepared from E14 RPE was twice that of E7 RPE. In each culture, retinal conditioned medium increases the TER 2-2.5 fold. The active factors of conditioned medium that affected each culture had different physical properties. The factor that affected E7 was protease-resistant with a Mr<10 kDa, but the factor that affected E14 appeared to be a protein of approximately 49 kDa. Unlike the effect of astrocyte conditioned medium on endothelia, retinal conditioned medium did not act synergistically with cAMP. These data indicate that the chick retina, which lacks astrocytes, uses different diffusible factors to regulate different stages of tight junction development.

Polarization of the Na+, K(+)-ATPase in epithelia derived from the neuroepithelium.

The neuroepithelium generates a fascinating group of epithelia. One of their intriguing properties is how they polarize the distribution of the Na+, K(+)-ATPase. Typically, this ion pump is concentrated in the basolateral membrane, but it is concentrated in the apical membranes of the retinal pigment epithelium and the epithelium of the choroid plexus. A comparison of their development with that of systemic epithelia yields insights into how cells polarize the distribution of this and other membrane proteins. The polarization of the Na+, K(+)-ATPase depends upon the interplay between different sorting signals and different types of polarity mechanisms. These include intracellular targeting signals that direct the delivery of newly synthesized proteins, and maintenance signals that stabilize proteins in the proper membrane domain. Conflicting signals appear to be arranged in a hierarchy that can be rearranged as cells respond to certain environmental stimuli. Part of this response is mediated by changes in the distribution and composition of the cortical cytoskeleton.

Protein-binding domains of the tight junction protein, ZO-2, are highly conserved between avian and mammalian species.

The tight junction is composed of many proteins and includes three members of the MAGUK (membrane-associated, guanylate kinase-like) protein family: ZO-1, ZO-2, and ZO-3. ZO-2 was cloned and sequenced from embryonic chicken retina. Antibodies against a short ZO-2 peptide immunolabeled the outer limiting membrane (an adherens junction of the neural retina) and the apical junctional complexes of the retinal pigment epithelium. Each ZO family member contains a homologous series of protein-binding domains: three distinct PDZ domains and src homology 3 (SH3), guanylate kinase-like (GuK), and acidic domains. Compared with human and canine ZO-2s, the PDZ and SH3 domains are the most conserved (90-95% amino acid sequence identity). These domains are only 50-71% identical with the homologous domains of ZO-1 and ZO-3. Although the sequence is less conserved for regions that link the protein-binding domains, the length of those regions is conserved in ZO-2s. The postacidic (C-terminal) region is the least conserved. The evolutionary pressure to maintain the sequence of the protein-binding domains suggests that homologous domains have different functions in ZO-1, ZO-2, and ZO-3.

Polarity and the development of the outer blood-retinal barrier.

The retinal pigment epithelium (RPE) is a monolayer that separates the outer surface of the neural retina from the choriocapillaris. Because the choriocapillaris is fenestrated, it is the RPE that forms the outer blood-retinal barrier and regulates the environment of the outer retina. Like all epithelia and endothelia, the ability of RPE to regulate transepithelial transport depends upon two properties: apical tight junctions to retard diffusion through the paracellular spaces of the monolayer, and an asymmetric distribution of proteins to regulate vectorial transport across the monolayer. During development, these properties form gradually. Initially, the tight junctions are leaky, and the RPE exhibits only partial polarity. As the neural retina and choriocapillaris develop, there are progressive changes in the composition of the apical junctional complexes, the expression of cell adhesion proteins, and the distribution of membrane and cytoskeletal proteins. Development can be used to dissect the multiple mechanisms that establish and maintain polarity and barrier function. These mechanisms are regulated by the interactions that develop between the RPE and its neighboring tissues. This review discusses the remodeling of the apical, lateral and basal plasma membranes of RPE that occurs during normal development, and establishes a framework to integrate the data obtained from multiple species. It examines the progress in understanding how environmental interactions regulate this development.

Differentiation and transdifferentiation of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies between the retina and the choroid of the eye and plays a vital role in ocular metabolism. The RPE develops from the same sheet of neuroepithelium as the retina and the two derivatives become distinguished by different expression patterns of a number of transcription factors during embryonic development. As the RPE layer differentiates it expresses a set of unique molecules, many of which are restricted to certain regions of the cell. PRE cells undergo both a loss of polarity and a loss of expression of many of these cell type-specific molecules when placed in monolayer culture. The RPE of many species, including mammals, can be induced to transdifferentiate by growth factors such as basic fibroblast growth factor. Under the influence of such factors the RPE is triggered to alter expression of a wide array of molecules and to take on a retinal epithelium fate, from which differentiated retinal cell types including rod photoreceptors can be produced.

A culture model of development reveals multiple properties of RPE tight junctions.

PURPOSE: A culture model was used to examine the development of tight junctions in the retinal pigment epithelium (RPE). METHODS: Chick RPE was isolated on embryonic day 7 (E7), E10 or E14 and cultured on laminin-coated filters. Barrier properties were stimulated with E14 retinal conditioned medium. Morphology was characterized by confocal microscopy. Permeability was determined by measuring the flux of horseradish peroxidase (HRP), radiolabeled inulin and mannitol, and the transepithelial electrical resistance (TER). Changes in the expression of ZO-1 and a related protein, ZO-1LP, were determined by immunoblotting. RESULTS: RPE from each age formed epithelial monolayers of similar height, but the density of the cultures varied in parallel with density changes in vivo. The cultures appeared to regulate the permeability to ions and nonionic solutes independently. With embryonic age, there was a progressive decrease in permeability that first affected larger and then smaller tracers. Despite a small decrease in the permeability to mannitol, there was a large decrease in the permeability to ions. This suggests that in E14 cultures tight junctions discriminated by charge, as well as size. Although E14 retinal conditioned medium reduced the permeability to all solutes, it appeared to regulate size discrimination more than charge discrimination. Despite large effects on permeability, conditioned medium had no effect on the expression of ZO-1 or ZO-1LP. CONCLUSIONS: The ability of tight junctions to discriminate on the basis of charge and size is regulated independently during development. The permeability of tight junctions cannot be predicted by the level of ZO-1 expression.

Remodeling of junctional complexes during the development of the outer blood-retinal barrier.

BACKGROUND: The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier by separating the neural retina from fenestrated capillaries in the choroid. The barrier depends upon tight junctions within the apical junctional complexes that bind neighboring cells. During development, permeability decreases as the apical junctional complex gradually matures. To investigate this process, the composition of the apical junctional complex was monitored during RPE development in chicken embryos. METHODS: Permeability was monitored by incubating freshly isolated RPE/choroid in medium containing horseradish peroxidase followed by histochemical staining and electron microscopy. The expression of the tight junction proteins, ZO-1 and occludin, was determined by immunofluorescence and immunoblotting. Development of the RPE apical junctional complex was to compared to the homologous complex that forms the outer limiting membrane of the neural retina. RESULTS: The apical junctional complex of the RPE was permeable to horseradish peroxidase until embryonic day 10-12. Two putative forms of ZO-1 had approximately the same molecular mass as mammalian ZO-1 and were present in the apical junctional complexes at different stages of development. We identified one form as ZO-1, because it was present in mature RPE and shared an epitope with the rodent isoforms, ZO-1 alpha+ and ZO-1 alpha-. The second form lacked this epitope but was identified by a polyclonal antibody to ZO-1. It was designated the ZO-1-like protein (ZO-1LP). On embryonic day 3, occludin and ZO-1LP were observed along the apical surface of the neuroepithelium that gave rise to the RPE and the neural retina. In the neural retina, occludin expression decreased just before inner segments were formed, but ZO-1LP expression continued in the outer limiting membrane throughout development. During RPE development, occludin expression was constant or increased slightly. By contrast, ZO-1LP was gradually replaced by ZO-1 and total ZO-1 immunoreactive proteins decreased more than 10x. CONCLUSIONS: A gradual change in the composition of the apical junctional complexes accompanied the period of barrier formation. In RPE, ZO-1 gradually replaced ZO-1LP, but the decrease in ZO-1 expression suggests its functions during junction formation are not directly related to junction permeability. By contrast, occludin was lost and ZO-1LP retained where an adherens junction forms the permeable, outer limiting membrane.

The distribution of Na+,K(+)-ATPase and 5A11 antigen in apical microvilli of the retinal pigment epithelium is unrelated to alpha-spectrin.

The retinal pigment epithelium was used to study the relationship between the cortical cytoskeleton and two plasma membrane proteins that associate with it. These proteins were the Na+,K(+)-ATPase, an ion pump, and the 5A11 antigen, a member of the immunoglobulin superfamily of receptor proteins. The cytoskeleton was marked by two of its constituents, alpha-spectrin and ankyrin. Ankyrin links the Na+,K(+)-ATPase to spectrin in many cells. The RPE is of interest, because unlike most epithelia it distributes the Na+,K(+)-ATPase to the apical membrane. The development of polarity was studied during chick embryogenesis. On embryonic day 6 (E6), each of these proteins was observed in the apical and lateral plasma membranes. As development proceeded, only the Na+,K(+)-ATPase was removed from the lateral membranes. Beginning on E12, ankyrin, spectrin and 5A11 appeared together in patches along the basal plasma membrane. By E16, these patches coalesced into a uniform distribution along the basal membrane. At the apical pole, alpha-spectrin appeared near the base of the microvilli, but was undetected in the microvilli themselves. This distribution resembled the distribution of alpha-spectrin in the intestine and proximal kidney tubule. By contrast, a pool of ankyrin and 5A11 and nearly all the Na+,K(+)-ATPase appeared in the microvilli. Despite its segregation from alpha-spectrin, the Na+,K(+)-ATPase appeared to associate with a macromolecular complex, as judged by extraction with Triton X-100. Changes in spectrin distribution could not be related to changes in isoform expression, as only one isoform of beta-spectrin was detected by co-immunoprecipitation with alpha-spectrin. By contrast, multiple ankyrin-like peptides could be identified by immunoblotting. These data illustrate some of the unique properties of RPE microvilli. These properties prevent the Na+,K(+)-ATPase from complexing with the alpha-spectrin-based cytoskeleton by sequestering the enzyme into the compartment where its activity is required.

The neural retina maintains integrins in the apical membrane of the RPE early in development.

PURPOSE: The retinal pigment epithelium (RPE) lines the interface between the neural retina and the choroid. Early in chicken development, the beta 1 family of integrins resides in the apical (facing the neural retina) and basolateral (facing the choroid) membranes of RPE. Later in development, integrins reside only in the basolateral membranes, which is more typical of simple transporting epithelia. The authors examined whether the distribution of integrins is regulated by the neural retina. METHODS: Individual integrins were examined by studying the individual alpha-subunits that form heterodimers with the beta 1 subunit. The expression and distribution of these subunits were determined by immunoblotting and immunocytochemistry. RESULTS: Subunits alpha 3 and alpha 6 exemplified contrasting behaviors. Early and late in development, alpha 3 was found only in the basal membranes. As was beta 1, the distribution of alpha 6 was nonpolarized early in development but was basal later in development. The effect of the immature neural retina was determined by reconstituting the RPE:neural retinal interface in explant culture. Absent the neural retina, alpha 6 and beta 1 were removed from the apical membrane. When present, the immature neural retina maintained both subunits in the apical membrane. The neural retina was effective only if the outer (primordial photoreceptor) surface of the retina apposed the RPE. CONCLUSIONS: These data suggest that matrix or intercellular interactions determine the distribution of individual integrins. Further, the changes in integrin distribution during development reflect the maturation of the primordial interphotoreceptor matrix or photoreceptor cell layer.

Diffusible, retinal factors stimulate the barrier properties of junctional complexes in the retinal pigment epithelium.

The retinal pigment epithelium lies at the interface between the neural retina and the choriocapillaris where it forms a blood-retinal barrier. Barrier function requires a polarized distribution of plasma membrane proteins and 'tight' tight junctions. During chicken embryogenesis, these features develop gradually. Although terminal junctional complexes are established by embryonic day 4, the distribution of the Na+/K(+)-APTase is not polarized in all cells of the epithelium until embryonic day 11. Similarly, the tight junctions of early embryos are leaky, but become tight by hatching (embryonic day 21). We used primary cell culture to examine the molecular basis of this gradual induction of polarized function. Pigment epithelium harvested from embryonic day 7, and cultured on filters, formed monolayers coupled by junctional complexes. The distribution of the Na+/K(+)-ATPase was non-polarized and the tight junctions were leaky with a transepithelial electrical resistance of 20-30 omega cm2. To isolate diffusible factors that stimulate the transepithelial electrical resistance, neural retinas from embryonic day 7, 14 or 16 embryos were incubated at 37 degrees C in base medium for 6 hours. The conditioned medium was added to the apical chamber of freshly cultured pigment epithelium. The distribution of the Na+/K(+)-ATPase became basolateral, and the electrical resistance gradually increased two to three times over 6 days. The increase in electrical resistance corresponded to a decrease in the rate of [3H]inulin diffusion across the monolayer. The effectiveness of the conditioned medium increased steadily with increasing age of the neural retina. Rather than increased production of an active factor, apparently different active factors were produced at different ages.(ABSTRACT TRUNCATED AT 250 WORDS)

Apical orientation of the microtubule organizing center and associated gamma-tubulin during the polarization of the retinal pigment epithelium in vivo.

Simple epithelial cells express a morphological and functional polarity along their apical-to-basal axis. During the development of epithelia, a unique reorganization of microtubule arrays is thought to play a fundamental role in the establishment of cell polarity. To begin to understand this process in vivo, we have determined the distribution of gamma-tubulin within developing chicken retinal pigment epithelium (RPE). gamma-Tubulin is a recently discovered centrosomal protein that plays a role in nucleating microtubule growth from the centrosome. Although the RPE monolayer becomes established during embryonic Day 3, cell polarity gradually develops and matures over the next 10-13 days. Our studies reveal that gamma-tubulin is located in a distinct focus subjacent to the apical membrane by embryonic Day 3, the beginning of the polarization process. Using primary cell cultures, we examined the relationship between the establishment of junctional complexes and the reorganization of microtubule arrays. Despite the recovery of junctional complexes and a transepithelial electrical resistance, cultured cells failed to relocate gamma-tubulin foci to a position subjacent to the apical membrane. Rather, these foci remained in the juxtanuclear region. These data indicate that the rearrangement of unique, epithelial microtubule arrays requires more than cell-cell and cell-basement membrane interactions.