The impact of frailty on intra-hospital survival in older patients with COVID-19 infection: the importance of early identification. SEMI-COVID National Registry.

BACKGROUND: Emerging evidence suggests that frailty may be a significant predictor of poor outcomes in older individuals hospitalized due to COVID-19. This study aims to determine the prognostic value of frailty on intrahospital patient survival. METHODS: This observational, multicenter, nationwide study included patients aged 70 years and older who were hospitalized due to COVID-19 in Spain between March 1 and December 31, 2020. Patient data were obtained from the SEMI-COVID-19 Registry of the Spanish Society of Internal Medicine. Frailty was assessed using the Clinical Frailty Scale. The primary outcome was hospital survival. Cox proportional hazards models were used to assess predictors of survival. RESULTS: A total of 1,878 participants (52% men and 48% women) were included, with 1,351 (71.9%) survivors and 527 (28.1%) non-survivors. The non-survivor group had higher mean age (83.5 vs. 81 years), comorbidities (6.3 vs. 5.3 points on the Charlson index), degree of dependency (26.8% vs. 12.4% severely dependent patients), and frailty (34.5% vs. 14.7% severely frail patients) compared to survivors. However, there were no differences in terms of sex. Our results demonstrate that a moderate-severe degree of frailty is the primary factor independently associated with shorter survival [HR 2.344 (1.437-3.823; p<0.001) for CFS 5-6 and 3.694 (2.155-6.330; p<0.001) for CFS 7-9]. CONCLUSION: Frailty is the main predictor of adverse outcomes in older patients with COVID-19. The utilization of tools such as the Clinical Frailty Scale is crucial for early detection in this population.

Clinical characteristics of patients hospitalized with COVID-19 in Spain: Results from the SEMI-COVID-19 Registry. / Características clínicas de los pacientes hospitalizados con COVID-19 en España: resultados del Registro SEMI-COVID-19.

BACKGROUND: Spain has been one of the countries most affected by the COVID-19 pandemic. OBJECTIVE: To create a registry of patients with COVID-19 hospitalized in Spain, in order to improve our knowledge of the clinical, diagnostic, therapeutic, and prognostic aspects of this disease. METHODS: A multicentre retrospective cohort study, including consecutive patients hospitalized with confirmed COVID-19 throughout Spain. Epidemiological and clinical data, additional tests at admission and at seven days, treatments administered, and progress at 30 days of hospitalization were collected from electronic medical records. RESULTS: Up to June 30th 2020, 15,111 patients from 150 hospitals were included. Their median age was 69.4 years (range: 18-102 years) and 57.2% were male. Prevalences of hypertension, dyslipidemia, and diabetes mellitus were 50.9%, 39.7%, and 19.4%, respectively. The most frequent symptoms were fever (84.2%) and cough (73.5%). High values of ferritin (73.5%), lactate dehydrogenase (73.9%), and D-dimer (63.8%), as well as lymphopenia (52.8%), were frequent. The most used antiviral drugs were hydroxychloroquine (85.6%) and lopinavir/ritonavir (61.4%); 33.1% developed respiratory distress. Overall mortality rate was 21.0%, with a marked increase with age (50-59 years: 4.7%, 60-69 years: 10.5%, 70-79 years: 26.9%, ≥80 years: 46.0%). CONCLUSIONS: The SEMI-COVID-19 Network provides data on the clinical characteristics of patients with COVID-19 hospitalized in Spain. Patients with COVID-19 hospitalized in Spain are mostly severe cases, as one in three patients developed respiratory distress and one in five patients died. These findings confirm a close relationship between advanced age and mortality.

[Clinical characteristics of patients hospitalized with COVID-19 in Spain: Results from the SEMI-COVID-19 Registry]. / Características clínicas de los pacientes hospitalizados con COVID-19 en España: resultados del Registro SEMI-COVID-19.

BACKGROUND: Spain has been one of the countries most affected by the COVID-19 pandemic. OBJECTIVE: To create a registry of patients with COVID-19 hospitalized in Spain, in order to improve our knowledge of the clinical, diagnostic, therapeutic, and prognostic aspects of this disease. METHODS: A multicentre retrospective cohort study, including consecutive patients hospitalized with confirmed COVID-19 throughout Spain. Epidemiological and clinical data, additional tests at admission and at seven days, treatments administered, and progress at 30 days of hospitalization were collected from electronic medical records. RESULTS: Up to June 30th 2020, 15,111 patients from 150 hospitals were included. Their median age was 69.4 years (range: 18-102 years) and 57.2% were male. Prevalences of hypertension, dyslipidemia, and diabetes mellitus were 50.9%, 39.7%, and 19.4%, respectively. The most frequent symptoms were fever (84.2%) and cough (73.5%). High values of ferritin (73.5%), lactate dehydrogenase (73.9%), and D-dimer (63.8%), as well as lymphopenia (52.8%), were frequent. The most used antiviral drugs were hydroxychloroquine (85.6%) and lopinavir/ritonavir (61.4%); 33.1% developed respiratory distress. Overall mortality rate was 21.0%, with a marked increase with age (50-59 years: 4.7%, 60-69 years: 10.5%, 70-79 years: 26.9%, ≥ 80 years: 46.0%). CONCLUSIONS: The SEMI-COVID-19 Network provides data on the clinical characteristics of patients with COVID-19 hospitalized in Spain. Patients with COVID-19 hospitalized in Spain are mostly severe cases, as one in three patients developed respiratory distress and one in five patients died. These findings confirm a close relationship between advanced age and mortality.

Hepatic insulin resistance both in prediabetic and diabetic patients determines postprandial lipoprotein metabolism: from the CORDIOPREV study.

BACKGROUND/AIMS: Previous evidences have shown the presence of a prolonged and exaggerated postprandial response in type 2 diabetes mellitus (T2DM) and its relation with an increase of cardiovascular risk. However, the response in prediabetes population has not been established. The objective was to analyze the degree of postprandial lipemia response in the CORDIOPREV clinical trial (NCT00924937) according to the diabetic status. METHODS: 1002 patients were submitted to an oral fat load test meal (OFTT) with 0.7 g fat/kg body weight [12 % saturated fatty acids (SFA), 10 % polyunsaturated fatty acids (PUFA), 43 % monounsaturated fatty acids (MUFA), 10 % protein and 25 % carbohydrates]. Serial blood test analyzing lipid fractions were drawn at 0, 1, 2, 3 and 4 h during postprandial state. Postprandial triglycerides (TG) concentration at any point >2.5 mmol/L (220 mg/dL) has been established as undesirable response. We explored the dynamic response in 57 non-diabetic, 364 prediabetic and 581 type 2 diabetic patients. Additionally, the postprandial response was evaluated according to basal insulin resistance subgroups in patients non-diabetic and diabetic without pharmacological treatment (N = 642). RESULTS: Prevalence of undesirable postprandial TG was 35 % in non-diabetic, 48 % in prediabetic and 59 % in diabetic subgroup, respectively (p < 0.001). Interestingly, prediabetic patients displayed higher plasma TG and large triacylglycerol-rich lipoproteins (TRLs-TG) postprandial response compared with those non-diabetic patients (p < 0.001 and p = 0.003 respectively). Moreover, the area under the curve (AUC) of TG and AUC of TRLs-TG was greater in the prediabetic group compared with non-diabetic patients (p < 0.001 and p < 0.005 respectively). Patients with liver insulin resistance (liver-IR) showed higher postprandial response of TG compared with those patients with muscle-IR or without any insulin-resistance respectively (p < 0.001). CONCLUSIONS: Our findings demonstrate that prediabetic patients show a lower phenotypic flexibility after external aggression, such as OFTT compared with nondiabetic patients. The postprandial response increases progressively according to non-diabetic, prediabetic and type 2 diabetic state and it is higher in patients with liver insulin-resistance. To identify this subgroup of patients is important to treat more intensively in order to avoid future cardiometabolic complications.

Evaluation of vertical transmission of Toxoplasma gondii in Calomys callosus model after reinfection with heterologous and virulent strain.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes a variety of clinical syndromes, but the infection is severe in immunocompromised individuals and during pregnancy due to the possibility of transplacental transmission of the parasite causing congenital toxoplasmosis. Vertical transmission of the parasite usually occurs when females are primarily infected during pregnancy. Calomys callosus is resistant to T. gondii ME49 strain, which presents a moderate virulence and congenital disease occurs only during the acute phase of infection. The aim of this study was to determine whether vertical transmission occurs when females of C. callosus chronically infected with ME49 strain of T. gondii are reinfected with a highly virulent strain (RH, type I). Females were infected with cysts of the ME49 strain. On the 1st day of pregnancy, animals were reinfected with tachyzoites of the RH strain. In the 19th day of pregnancy, placentas and embryos were processed for morphological analysis, immunohistochemistry and for detection of the parasite by PCR and mouse bioassay. Morphological and immunohistochemical analyses revealed the presence of parasites only in placental tissues. Mouse bioassay results showed seroconversion only in mice that were inoculated with placental tissues. Also, T. gondii DNA was detected only in placental samples. Congenital toxoplasmosis does not occur in C. callosus females chronically infected with the moderately virulent ME49 strain of T. gondii and reinfected with the highly virulent RH strain, thus indicating that primary T. gondii infection before pregnancy leads to an effective long-term immunity preventing transplacental transmission to the fetus.

Azithromycin inhibits vertical transmission of Toxoplasma gondii in Calomys callosus (Rodentia: Cricetidae).

Toxoplasma gondii infection during pregnancy may cause severe consequences to the embryo. Current toxoplasmosis treatment for pregnant women is based on the administration of spiramycin or a drug combination as sulphadiazine-pyrimethamine-folinic acid (SPFA) in cases of confirmed fetal infection. However, these drugs are few tolerated and present many disadvantages due to their toxic effects to the host. The aim of this study was to evaluate the effectiveness of different treatments on the vertical transmission of T. gondii, including azithromycin, Artemisia annua infusion, spiramycin and SPFA in Calomys callosus as model of congenital toxoplasmosis. C. callosus females were perorally infected with 20 cysts of T. gondii ME49 strain at the day that a vaginal plug was observed (1st day of pregnancy - dop). Treatment with azithromycin, A. annua infusion, and spiramycin started at the 4th dop, while the treatment with SPFA started at the 14th dop. Placenta and embryonic tissues were collected for morphological and immunohistochemical analyses, mouse bioassay and PCR from the 15th to 20th dop. No morphological changes were seen in the placenta and embryonic tissues from females treated with azithromycin, spiramycin and SPFA, but embryonic atrophy was observed in animals treated with A. annua infusion. Parasites were found in the placenta and fetal (brain and liver) tissues of animals treated with SPFA, A. annua infusion and spiramycin, although the number of parasites was lower than in non-treated animals. Parasites were also observed in the placenta of animals treated with azithromycin, but not in their embryos. Bioassay and PCR results confirmed the immunohistochemical data. Also, bradyzoite immunostaining was observed only in placental and fetal tissues of animals treated with SPFA. In conclusion, the treatment with azithromycin showed to be more effective, since it was capable to inhibit the vertical transmission of T. gondii in this model of congenital toxoplasmosis.

Susceptibility to vertical transmission of Toxoplasma gondii is temporally dependent on the preconceptional infection in Calomys callosus.

Toxoplasma gondii is an obligate intracellular parasite that causes a variety of clinical syndromes, but the infection is more severe in immunocompromised individuals and in cases of congenital toxoplasmosis. This study aimed to verify if the susceptibility to vertical transmission of Toxoplasma gondii is temporally dependent on the preconceptional infection in Calomys callosus. Twelve C. callosus females were infected with 20 cysts of T. gondii ME49 strain and divided into three groups of four animals that were mated after approximately 10 days (group 1), 30 days (group 2), and 50 days (group 3) of infection. The animals were sacrificed from the 17th to 20th day of pregnancy, when placentas and embryos were collected for morphological and immunohistochemical studies, mouse bioassay for evaluating seroconversion and PCR for detecting parasite DNA. Serum samples from C. callosus females and mice used in bioassay were analysed for the detection of IgG antibodies to T. gondii by ELISA. Detection of T. gondii was observed by mouse bioassay and PCR in placentas and embryos from C. callosus females infected around 10 days pre-conception. However, only placentas, but not embryos, from females infected around 30 and 50 days pre-conception showed positivity for parasite DNA and seroconversion by mouse bioassay. In conclusion, this study model shows that vertical transmission of T. gondii may take place when maternal infection occurs within one month before conception, thus demonstrating the time of preconceptional seroconversion that rule out a risk of congenital toxoplasmosis.

Increased elastin expression in astrocytes of the lamina cribrosa in response to elevated intraocular pressure.

PURPOSE: To determine whether abnormal elastin synthesis in the glaucomatous optic nerve head and lamina cribrosa is due to elevated intraocular pressure (IOP) or secondary to axonal injury, monkeys with elevated IOP and with optic nerve transection were compared. METHODS: Unilateral, chronic elevated IOP was induced in 11 rhesus monkeys by laser scarification of the trabecular meshwork. IOP was monitored weekly and maintained within 25 to 45 mm Hg for 7 to 36 weeks. In 6 monkeys, unilateral, optic nerve transection was performed, and monkeys were killed after 4 weeks. Optic nerve damage was assessed by stereoscopic slit-lamp biomicroscopy and fundus photography and by confocal scanning laser ophthalmoscopy. The eyes were enucleated and processed for immunohistochemistry and in situ hybridization and for electron microscopic immunogold detection of elastin. Axonal loss was evaluated in cross sections of the optic nerve stained with phenylenediamine. RESULTS: Compared with normal contralateral controls, the lamina cribrosa of eyes with elevated IOP exhibited markedly increased elastin and the presence of elastotic aggregates in the extracellular matrix and upregulation of elastin mRNA in the astrocytes. In transected eyes, elastin appeared as fine fibers in the lamina cribrosa, without elastotic aggregates, and without new synthesis or abnormal deposition of elastin. At the transected site, new synthesis of elastin was present in the pia mater but not in astrocytes in the glial scar. CONCLUSIONS: This study demonstrates that abnormal elastin synthesis in experimental glaucomatous optic neuropathy in the monkey is specific to elevated IOP and not secondary to axonal loss. The mechanisms by which elevated IOP induces enhanced elastin synthesis in laminar astrocytes are unknown but differ from those involved in acute axonal injury such as transection, where inflammation and breakdown of the blood-nerve barrier occur.

Selective expression of neural cell adhesion molecule (NCAM)-180 in optic nerve head astrocytes exposed to elevated hydrostatic pressure in vitro.

Glaucomatous optic neuropathy is usually associated with elevated intraocular pressure. Optic nerve head astrocytes may respond to intraocular pressure by stimulation of pressure-sensitive mechanoreceptors on the cell surface. Neural cell adhesion molecule (NCAM) a transmembrane protein, mediates cell adhesion and migration. The NCAM 180 isoform increases in astrocytes of glaucomatous optic nerve head. We characterized the relative expression of NCAM isoforms in human optic nerve head astrocytes grown under elevated hydrostatic pressure. Astrocytes cultured from normal human optic nerve heads were exposed to either atmospheric or continuous hydrostatic pressure of 60 mm Hg, and analyzed at 6-48 h. Changes in cell shape, immunoreactivity, and distribution of GFAP, actin and NCAM were observed in pressure-treated cultures. Newly synthesized (35)S-labeled NCAM protein immunoprecipitated from cell lysates was increased 2-fold within 24 h after exposure to elevated pressure compared to control. The increase in NCAM synthesis was primarily due to the NCAM 180 isoform. A significant increase in NCAM 180 mRNA levels was detected by RT-PCR and Northern blots in cultured optic nerve head astrocytes within 6 h after exposure to elevated pressure. NCAM 180 mRNA and protein synthesis decreased after 24 h and returned to control levels by 48 h. Our data indicate that NCAM 180 transcription and synthesis in astrocytes is stimulated by elevated hydrostatic pressure. Because NCAM 180 interacts with the cytoskeleton through an extended cytoplasmic tail, a selective and transient increase in NCAM 180 in optic nerve head astrocytes exposed to elevated pressure may be relevant to the migration and interactions of reactive astrocytes in glaucoma.

Hydrostatic pressure stimulates synthesis of elastin in cultured optic nerve head astrocytes.

Elastin is a major component of the extracellular matrix (ECM) of the lamina cribrosa in the optic nerve head in humans and nonhuman primates. The lamina cribrosa appears to be the site of damage to the retinal ganglion cell axons in glaucomatous optic neuropathy, characterized in many patients by elevated intraocular pressure (IOP). Type 1B astrocytes are the major cell type in the lamina, synthesize elastic fibers during development, express increased elastin mRNA, and synthesize abnormal elastin in glaucoma. In this study, we determined the effect of elevated hydrostatic pressure on the synthesis of elastin by type 1B astrocytes in culture. Type 1B astrocytes were exposed to gradients of hydrostatic pressure and tested for proliferation, morphology, synthesis, and deposition of elastin. Trichloroacetic acid (TCA) and immunoprecipitation of radiolabeled protein determined total new protein and elastin synthesis. Proteins from the conditioned media were analyzed by Western blot. Levels of elastin mRNA were determined by in situ hybridization. Cell proliferation increased approximately 2-fold after exposure to pressure for one day, approximately 5-fold after 3 and 5 days of exposure to pressure. Confocal and electron microscopic cytochemistry showed a marked increase in intracellular elastin in astrocytes exposed to pressure, as compared with controls. Intracellular elastin was associated with the RER-Golgi region and with the cytoskeleton. Total protein and elastin synthesis increased significantly (P < 0.05) at 3- and 5-day exposure to pressure, as well as the level of elastin mRNA. Elastin protein in the media increased with the level of pressure. These results indicate that hydrostatic pressure stimulates type 1B astrocytes to synthesize and secrete soluble elastin into the media. In glaucoma, type 1B astrocytes may respond to IOP-related stress with increased expression of elastin and formation of elastotic fibers leading to loss of elasticity and tissue remodeling.

Synthesis of elastic microfibrillar components fibrillin-1 and fibrillin-2 by human optic nerve head astrocytes in situ and in vitro.

The purpose of this study was to identify elastic microfibrillar components fibrillin-1 and fibrillin-2 in optic nerve heads of adult normal and glaucomatous subjects, in cultured optic nerve head astrocytes (type 1B astrocytes), as well as fibrillin-1 in fetal optic nerve heads. To characterize synthesis and gene expression of microfibrillar proteins in human optic nerve heads and cultured type 1B astrocytes, light microscopy immunohistochemistry, in situ hybridization, and RT-PCR or Northern blots were performed. Our results demonstrated that fibrillin-1 was associated with blood vessels, astrocytes in the glial columns and cribriform plates, and with astrocyte processes in the nerve bundles in all samples. In glaucomatous optic nerves there was enhanced fibrillin-1 immunoreactivity, especially surrounding blood vessels. Fibrillin-2 was localized primarily to blood vessels in all samples, without qualitative differences between normal and glaucomatous samples. In fetal optic nerve heads fibrillin-1 mRNA was localized to glial cells and to the blood vessel walls. In adult optic nerve heads, there was little fibrillin-1 mRNA as detectable by in situ hybridization and RT-PCR. There was no detectable upregulation of fibrillin-1 mRNA in glaucoma. In cultured type 1B astrocytes, fibrillin-1 staining was mostly pericellular. There was little fibrillin-2 immunoreactivity. In conclusion, astrocytes from the optic nerve head deposit elastic microfibrillar components in situ and in vitro, with a predominance of fibrillin-1. Upregulation of fibrillin-1 mRNA was not observed in glaucoma, suggesting that increased transcription may occur early in the disease process. Cultures of type 1B astrocytes from the optic nerve head provides a useful model to study mechanisms regulating the interactions of elastin and the microfibrils in optic nerve head astrocytes.

Transforming growth factor beta isoforms in human optic nerve heads.

AIM: To determine if the isoforms of transforming growth factor beta (TGF-beta) are present in fetal, normal adult, and glaucomatous optic nerve heads. METHODS: To localise cells synthesising TGF-beta, optic nerve heads were stained using antibodies to TGF-beta 1, TGF-beta 2, and TGF-beta 3. To demonstrate synthesis, human optic nerve heads from fetal, glaucomatous, and normal age matched subjects were explanted, cultured overnight, and the culture supernatant was assayed for the presence of TGF-beta 1 and TGF-beta 2 by bioassay. In addition, semiquantitative RT-PCR was performed to determine the gene expression pattern of TGF-beta 2. RESULTS: Immunohistochemistry of glaucomatous samples revealed the presence of intense staining for TGF-beta 2 primarily in astrocytes, whereas TGF-beta 1 was localised to blood vessels. No TGF-beta 3 immunoreactivity was observed. There was little or no expression of TGF-beta in normal optic nerve heads. Optic nerve heads from glaucomatous eyes released 70-100-fold more TGF-beta 2 than normal age matched optic nerve heads. Fetal optic nerve heads released 90-100-fold more TGF-beta 2 than normal adult optic nerve heads. TGF-beta 1 was undetectable by bioassay in all samples tested. There was no apparent increase in TGF-beta 2 gene expression in glaucomatous and fetal eyes, suggesting post-transcriptional regulatory mechanisms. CONCLUSIONS: These results demonstrate that TGF-beta 2 is produced in high levels in the fetal and glaucomatous optic nerve heads, perhaps by a mechanism of post-transcriptional regulation. TGF-beta may be important during development of the optic nerve head and, in glaucoma, TGF-beta 2 may be a mediator of astrocyte reactivation and extracellular matrix remodelling in the lamina cribrosa.

Enhanced tenascin expression associated with reactive astrocytes in human optic nerve heads with primary open angle glaucoma.

Tenascin is a large extracellular matrix glycoprotein expressed in neural and non-neural tissues. In the central nervous system, tenascin is synthesized by astrocytes during development and wound healing, forming barriers and affecting neurite outgrowth. In this study we examined tenascin expression in optic nerve heads of normal and glaucomatous eyes and found that there is upregulation of tenascin mRNA and protein in reactive astrocytes from human glaucomatous optic nerve heads compared to normal age-matched controls. In the prelaminar region there was a band of tenascin immunoreactivity around the blood vessels of glaucomatous, but not in normal eyes. However, tenascin mRNA was only localized to astrocytes, suggesting that astrocytes are the cellular source of tenascin. In the lamina cribrosa, tenascin immunoreactivity and gene expression were localized to astrocytes in the cribriform plates and inside the nerve bundles. In the post-lamina region, tenascin immunoreactivity and gene expression were localized to astrocytes lining the pial septum immediately adjacent to the lamina cribrosa. In normal optic nerve heads, tenascin expression at the mRNA and protein levels was confined to clusters of astrocytes at the level of Bruch's membrane in the prelaminar optic nerve head. In glaucoma, enhanced expression of tenascin may be protective to the axons of the retinal ganglion cells by providing a barrier for humoral and/or blood-borne factors that may cause further neural damage. However, the precise role of tenascin in glaucomatous optic neuropathy is not yet elucidated.

Differential expression of neural cell adhesion molecule isoforms in normal and glaucomatous human optic nerve heads.

Type 1B astrocytes of the human optic nerve head (ONH) constitutively express neural cell adhesion molecule (NCAM) in vivo and in vitro. Increased synthesis of NCAM has been detected in reactive astrocytes in the glaucomatous ONH of human donor eyes. Several NCAM isoforms are generated through alternate RNA splicing in tissue- and disease-specific patterns. In this study, we analyzed expression of NCAM isoforms in ONH of normal donors at different ages and in glaucoma. Total RNA was extracted from ONH of fetal, normal adult and glaucomatous eyes, and cultured human ONH astrocytes, fetal brain astrocytes and an astrocytoma cell line, for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. To distinguish between NCAM 180 and 140 isoforms, exon-specific primer sets covering exons 13-19 were used. Isoform-specific riboprobes were used for in situ hybridization (ISH) in glaucomatous and in age-matched ONH. By RT-PCR, NCAM 140 was the predominant isoform in adult ONH as well as in all cultured cells. NCAM 180 mRNA was strongly expressed in glaucoma, whereas in normal adult tissues it was not detectable. ISH confirmed expression of NCAM in normal adult ONH and localized NCAM 140 mRNA to astrocytes. ISH demonstrated expression of NCAM 180 mRNA in reactive astrocytes in glaucomatous ONH. Our results demonstrate that the NCAM 180 isoform is induced in glaucoma. NCAM 180 may play a role in astrocyte interaction with extracellular matrix (ECM), vessels, axons and other astrocytes and, through its expanded cytoplasmic domain, serve as a signaling molecule for reactive astrocytes during remodeling of the ONH in glaucoma.

Lhx2, a vertebrate homologue of apterous, regulates vertebrate limb outgrowth.

apterous specifies dorsal cell fate and directs outgrowth of the wing during Drosophila wing development. Here we show that, in vertebrates, these functions appear to be performed by two separate proteins. Lmx-1 is necessary and sufficient to specify dorsal identity and Lhx2 regulates limb outgrowth. Our results suggest that Lhx2 is closer to apterous than Lmx-1, yet, in vertebrates, Lhx2 does not specify dorsal cell fate. This implies that in vertebrates, unlike Drosophila, limb outgrowth can be dissociated from the establishment of the dorsoventral axis.

Elastosis of the lamina cribrosa in glaucomatous optic neuropathy.

The purpose of this study was to determine whether elastotic degeneration of the elastin component of the lamina cribrosa occurs in optic neuropathy associated with different types of glaucoma. Human optic nerve heads with primary open-angle, neovascular, chronic angle closure and pseudoexfoliation glaucoma, and with varying duration of disease were compared with age-matched normal eyes, using electron microscopy and immunogold labeling of elastin. The percent area occupied by immunogold-labeled elastin material was determined using a digital image analysis system. In all eyes with a history of glaucoma, elastosis was found in the lamina cribrosa and there was a significantly greater percentage of area occupied by elastin compared with age-matched control eyes (P<0.0001). Among the glaucomatous eyes, pseudoexfoliation glaucoma had the largest area of elastosis, followed by primary open-angle and secondary glaucoma (neovascular and chronic angle closure). In all glaucoma samples, large, confluent elastin aggregates of irregular and varied shapes (elastosis) were observed in the lamina cribrosa and insertion region. These results demonstrate that glaucomatous optic neuropathy is associated with elastosis of the lamina cribrosa, which may contribute to the changes in compliance of the optic nerve heads of glaucomatous eyes.

Expression of neural cell adhesion molecule (NCAM) characterizes a subpopulation of type 1 astrocytes in human optic nerve head.

The human optic nerve contains a heterogeneous population of astrocytes. In situ, a specialized subpopulation of astrocytes was distinguished in the adult optic nerve head by expression of neural cell adhesion molecule (NCAM). To further study the biology of astrocytes, we have developed and characterized cells grown from explanted optic nerve heads and myelinated optic nerves as in vitro model systems. Second or third passage cells were processed for immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP) and cell surface epitopes: CD56/NCAM, HNK-1/NCAM, A2B5, and O4. Synthesis and gene expression of NCAM were characterized by Western blot analysis and RNase protection assay. Cells grown from myelinated optic nerves expressing GFAP, but not NCAM or A2B5, were identified as type 1A astrocytes, and cells expressing GFAP and A2B5, but not NCAM, were identified as type 2 astrocytes. Cells grown from explanted optic nerve head expressing GFAP, NCAM, and O4 were identified as type 1B astrocytes. Expression of NCAM by type 1B astrocytes may provide these cells with adhesion properties that allow them specialized responses in their microenvironment. Astrocytes from the lamina cribrosa may form a functional barrier to prevent myelination of the retina. In glaucoma, these astrocytes may be exposed to stresses due to fluctuation in intraocular pressure and therefore participate in the optic nerve changes associated with glaucomatous optic neuropathy.

Tropoelastin gene expression in optic nerve heads of normal and glaucomatous subjects.

Elastic fibers are a major component of the extracellular matrix in the optic nerve head (ONH) and undergo marked morphological changes during primary open angle glaucoma (POAG). Previous findings indicated that there is reactivation of tropoelastin mRNA synthesis in glaucoma. In this study, we sought to determine the alternative splicing pattern of tropoelastin in the human optic nerve head and in cultured laminar astrocytes. Furthermore, we compared the alternative splicing pattern of normal elastogenesis with that of reactivation of elastin synthesis in patients with primary open angle glaucoma. Our results demonstrate that exons 23 and 32 of tropoelastin are alternatively spliced in the normal ONH as well as in tissue from glaucomatous patients. There are no qualitative differences. We also demonstrated that astrocytes from the ONH synthesize tropoelastin in vitro. In conclusion, we have demonstrated a tropoelastin alternative splicing pattern in the human optic nerve head and laminar astrocytes. Abnormalities in elastic fibers in the ONH of patients with POAG are not due to an aberrant splice variant of tropoelastin. Astrocytes grown from ONH explants may serve as an in vitro model to study extracellular matrix changes in glaucoma.

Influence of androgens and pituitary hormones on the structural profile and secretory activity of the lacrimal gland.

The present study was designed to examine the influence of androgens on the size, morphological appearance, and fluid and protein secretory capacity of the lacrimal gland in a variety of species. For comparison, we also sought to determine whether other hormones might stimulate the growth and secretory ability of this tissue. Lacrimal glands and tears were obtained from intact, surgically-operated and/or hormone-treated rats, mice, guinea pigs and rabbits and the processed for histological and/or image analysis techniques. Our results demonstrated that: 1) androgens modulate the ultrastructural features of rat lacrimal tissue; 2) androgens may influence the weight, morphological aspects, and secretion of the lacrimal gland, however, these effects are not uniform in mice, rats, guinea pigs and rabbits, but instead are dependent upon the species and/or the animal's endocrine status; and 3) treatment with testosterone, prolactin, growth hormone, alpha-melanocyte stimulating hormone, insulin, or thyroxine plus dexamethasone had no impact on the absolute or relative size of the lacrimal gland, or the tear volume, of hypophysectomized male rats. Overall, these findings indicate that androgens may regulate the structure and secretion of the lacrimal gland. However, the nature and extent of this hormone action is species-dependent and may be modified by alterations in the endocrine environment.