Immunochemical and morphometric features of astrocyte reactivity vs. plaque location in Alzheimers disease

The quantitative relationship between glial fibrillary acidic protein (GFAP) hyper-reactivity and -amyloid protein (AP) deposition was investigated by double immunoperoxidase labeling of hippocampal and entorhinal cortex sections from five Alzheimer s disease (AD) cases and five age-matched controls. AP plaques, which were absent in controls, were found in all AD samples, without significant differences in number or perimeter according to their location among the regions studied. In contrast, the mean number of GFAP (+) cells was significantly greater in the hippocampus than in the entorhinal cortex from AD cases (49 vs.39). Although at lower values (30 vs. 20), predominance of astrocyte hyperplasia in hippocampus as compared with entorhinal cortex was also found in control samples. Concomitant astrocyte hypertrophy, as defined by surface density (Sv) values of GFAP-immunoreactive material exceeding those of control means, affected a similar proportion of cells in the hippocampus (73%) and the entorhinal cortex (74%) from AD cases. Since an increased number of GFAP (+) cells in the hippocampus was not accompanied by an increased number and/or perimeter of neighbouring plaques, such differential hyper-reactivity in samples from AD patients, as well as in those with normal aging, seems to depend partially on the regional location of the involved astrocyte. (AU)

Immunochemical and morphometric features of astrocyte reactivity vs. plaque location in Alzheimer's disease

The quantitative relationship between glial fibrillary acidic protein (GFAP) hyper-reactivity and -amyloid protein (AP) deposition was investigated by double immunoperoxidase labeling of hippocampal and entorhinal cortex sections from five Alzheimer´s disease (AD) cases and five age-matched controls. AP plaques, which were absent in controls, were found in all AD samples, without significant differences in number or perimeter according to their location among the regions studied. In contrast, the mean number of GFAP (+) cells was significantly greater in the hippocampus than in the entorhinal cortex from AD cases (49 vs.39). Although at lower values (30 vs. 20), predominance of astrocyte hyperplasia in hippocampus as compared with entorhinal cortex was also found in control samples. Concomitant astrocyte hypertrophy, as defined by surface density (Sv) values of GFAP-immunoreactive material exceeding those of control means, affected a similar proportion of cells in the hippocampus (73%) and the entorhinal cortex (74%) from AD cases. Since an increased number of GFAP (+) cells in the hippocampus was not accompanied by an increased number and/or perimeter of neighbouring plaques, such differential hyper-reactivity in samples from AD patients, as well as in those with normal aging, seems to depend partially on the regional location of the involved astrocyte.

Human serum antibodies against shared antigens of different stages of Trichinella spiralis: relevance of glycan and protein epitopes.

Taking into account that antibodies against the surface antigens of newborn larvae (anti-NBL Abs) present in sera from individuals with chronic trichinellosis recognize antigenic determinants of the excretory-secretory muscle larva products (ML-ESP), and that these products are mainly glycoproteins, the aim of this work was to assess the frequency of anti-NBL Abs in sera from individuals with acute and chronic trichinellosis, to analyse the relevance of glycan and protein epitopes of the ML-ESP in the cross-reactivity phenomenon, and its correlation with the host's serum response towards these products. Anti-NBL surface Abs were determined in sera by indirect immunofluorescence. The degree of recognition by serum and purified anti-NBL Abs was evaluated comparatively before and after chemical deglycosylation of ML-ESP by immunoelectrotransfer blot assay. Results showed that 64% of the sera from individuals with acute trichinellosis and 35% of those belonging to the chronic phase had anti-NBL Abs, and also that the protein epitopes are the major ones responsible for the cross-reactivity phenomenon involving the ML-ESP and the NBL surface during both phases of the infection, while glycan epitopes are immunodominant in the stimulation of the host's immune system. A modulatory phenomenon in the immune response generated towards Trichinella spiralis NBL driven by the ML-ESP is postulated.

Long-lasting astrocyte reaction to persistent Junin virus infection of rat cortical neurons.

Immunoperoxidase labeling was performed in histological sections from rat brain harvested during acute (10-30 days), clinically inapparent (90-270 days) and late (450-540 days) stages of Junin virus-induced neurological disease. In frontoparietal cortex, count of viral antigen (+) neurons peaked during the acute period (27.7+/-6.8), dropped within the intermediate (4.8+/-4.0 to 1.4+/-1.1) and increased (7.6+/-4.3) at the onset of the late neurological syndrome. In infected vs. control rats, the number of GFAP (+) astrocytes maximized during the acute stage (19+/-4 vs. 11+/-5), and from the end of the intermediate (27+/-5 vs. 21+/-5) up to the late (37+/-7 vs. 26+/-6) periods. In turn, surface density of GFAP (+) material in infected samples peaked at 0.196+/-0.066, while it failed to exceed 0.090+/-0.043 in controls. Both astrocyte hypertrophy relapsing into chronicity, as depicted by surface density, and astrocyte hyperplasia preceding the onset of the late neurological syndrome, support their pathogenic contribution to disease expression.

Combined measurement of diabetes mellitus immunological markers: an assessment of its benefits in adult-onset patients.

The convenience of combining the measurement of antibodies to glutamic acid decarboxylase (GADA), protein tyrosine phosphatase (IA-2A), and autoantibodies to insulin (IAA) in diabetic patients was assessed. We analysed 71 type 1 and 115 adult-onset diabetic patients. The latter were grouped into three categories according to the time of evolution to insulin dependence. The main findings were as follows: (i) in type 1 diabetes, the combined analysis of GADA and IA-2A showed a sensitivity of 87.4% and was not appreciably improved by adding IAA; (ii) out of 31 adults who required insulin immediately or within the first two years of diagnosis, 41.9, 29.0, and 6.5% were positive for at least one, two or all three, and all three markers, respectively; GADA was the most prevalent (35.5%) and IA-2A the least represented (16.1%); (iii) 34 adult patients with slow evolution to insulin dependence showed a completely different profile: 5.9% were GADA positive and 23.5% were IAA positive and no double or triple positivity was observed as all patients were IA-2A negative; and (iv) 50 type 2 patients who had not required insulin treatment showed a low incidence of GADA (4%) as the only marker present. We conclude that a combined double-antigen test for GADA and IA-2A is a useful strategy for prospective screening of type 1 diabetes. However, in adults, the profile of individual markers discloses the course to insulin dependence. Therefore, it seems advisable to measure the markers separately, to allow a better classification of these patients, and help define their treatment.

Immunologic and genetic markers in insulin-dependent diabetes mellitus (type 1) in an Argentine population.

The objective was to evaluate the prevalence and association of several markers (islet cell antibodies: ICA, insulin autoantibodies: IAA, glutamic acid decarboxylase antibodies: GADA and ICA512 antibodies: ICA512A) along with HLA DQB1 genotype in type 1 diabetes mellitus of recent onset, including siblings and individuals without any history of this disease, in an Argentine population. A total of 79 children with type 1 diabetes mellitus of recent onset were studied, as well as 79 control children, and 68 healthy siblings of type 1 diabetic cases. IAA, ICA, GADA, ICA512A and HLA DQB1 alleles were determined. Sensitivity was 67.1% for ICA, 36.7% for IAA, 74.6% for GADA and 63.4% for ICA512A. None of the control subjects was positive for the immunological markers. Combined sensitivity of ICA-IAA-GADA was 89.8%, similar to the ICA512A-GADA (87.3%) or ICA512A-GADA-IAA combination (91.1%). GADA correlated positively with ICA, but no such correlation was found between IAA, ICA512A and ICA. IAA correlated negatively and GADA positively with age. IAA was associated to DQB1*0201, whereas ICA and ICA512A associated to DQB1*0302. Among siblings, 3/68 (4.4%) were positive for IAA and a single case (1.5%) was positive for GADA and one for ICA512A. Our findings show that the combination of multiple tests increases the sensitivity for prediction, with the ICA512A-GADA combination proving highly sensitive and equivalent to other proposed combinations, such as ICA-IAA-GADA.

Immunologic and genetic markers in insulin-dependent diabetes mellitus (type 1) in an Argentine population.

The objective was to evaluate the prevalence and association of several markers (islet cell antibodies: ICA, insulin autoantibodies: IAA, glutamic acid decarboxylase antibodies: GADA and ICA512 antibodies: ICA512A) along with HLA DQB1 genotype in type 1 diabetes mellitus of recent onset, including siblings and individuals without any history of this disease, in an Argentine population. A total of 79 children with type 1 diabetes mellitus of recent onset were studied, as well as 79 control children, and 68 healthy siblings of type 1 diabetic cases. IAA, ICA, GADA, ICA512A and HLA DQB1 alleles were determined. Sensitivity was 67.1

for ICA, 36.7

for IAA, 74.6

for GADA and 63.4

for ICA512A. None of the control subjects was positive for the immunological markers. Combined sensitivity of ICA-IAA-GADA was 89.8

, similar to the ICA512A-GADA (87.3

) or ICA512A-GADA-IAA combination (91.1

). GADA correlated positively with ICA, but no such correlation was found between IAA, ICA512A and ICA. IAA correlated negatively and GADA positively with age. IAA was associated to DQB1*0201, whereas ICA and ICA512A associated to DQB1*0302. Among siblings, 3/68 (4.4

) were positive for IAA and a single case (1.5

) was positive for GADA and one for ICA512A. Our findings show that the combination of multiple tests increases the sensitivity for prediction, with the ICA512A-GADA combination proving highly sensitive and equivalent to other proposed combinations, such as ICA-IAA-GADA.

A quantitative approach to correlate astrocyte differentiation and phagocytic activity.

A triple staining procedure (PAP labeling for GFAP, PAS reaction for added yeast cells and hematoxylin for nuclear staining of the whole cell monolayer) had disclosed that Junin virus infection enhanced phagocytic activity by inducing greater astrocyte differentiation. Here, we resorted to a mathematical approach for simultaneous evaluation of astrocyte differentiation and potential phagocytosis. At light microscopy level, the total number of: a) PAS-stained yeast cells, b) PAS-stained yeast cells associated to GFAP-positive astrocytes, c) GFAP-positive astrocytes, and d) total number of GFAP-labeled and non-labeled astrocytes, were counted within the monolayer area delimited by a grid with a total area of 0.01 mm2. As the percentage of PAS-stained yeast cells associated to GFAP-positive astrocytes correlated significantly with the percentage of GFAP-positive astrocytes for the three yeast cell incubation times (24, 48 and 72 h), a mathematical approach involving a so-called beta parameter representing the percentage of differentiated astrocytes capable of taking up 50% of added yeast cells, was developed. Since beta value dropped along yeast cell incubation time, and more markedly in Junin-virus infected samples, a numerical value was thus available to assess enhanced phagocytic activity in astrocytes undergoing differentiation. Therefore, the application of a mathematical approach to cell monolayers subjected to current staining techniques, allows more objective analysis of data provided by cursory visualization at light microscopy level.

A quantitative approach to correlate astrocyte differentiation and phagocytic activity.

A triple staining procedure (PAP labeling for GFAP, PAS reaction for added yeast cells and hematoxylin for nuclear staining of the whole cell monolayer) had disclosed that Junin virus infection enhanced phagocytic activity by inducing greater astrocyte differentiation. Here, we resorted to a mathematical approach for simultaneous evaluation of astrocyte differentiation and potential phagocytosis. At light microscopy level, the total number of: a) PAS-stained yeast cells, b) PAS-stained yeast cells associated to GFAP-positive astrocytes, c) GFAP-positive astrocytes, and d) total number of GFAP-labeled and non-labeled astrocytes, were counted within the monolayer area delimited by a grid with a total area of 0.01 mm2. As the percentage of PAS-stained yeast cells associated to GFAP-positive astrocytes correlated significantly with the percentage of GFAP-positive astrocytes for the three yeast cell incubation times (24, 48 and 72 h), a mathematical approach involving a so-called beta parameter representing the percentage of differentiated astrocytes capable of taking up 50

of added yeast cells, was developed. Since beta value dropped along yeast cell incubation time, and more markedly in Junin-virus infected samples, a numerical value was thus available to assess enhanced phagocytic activity in astrocytes undergoing differentiation. Therefore, the application of a mathematical approach to cell monolayers subjected to current staining techniques, allows more objective analysis of data provided by cursory visualization at light microscopy level.

Cell differentiation increases astrocyte phagocytic activity. A quantitative analysis of both GFAP labeling and PAS-stained yeast cells.

Since efficiency of phagocytic potential in activated astrocytes is still a subject of controversy, an attempt was made to quantify simultaneously phagocytic activity and astrocyte differentiation. Resorting to Junin virus, known to induce astrocyte activation, infected vs control samples of cultured rat astroglial cells were serially harvested up to day 12 post-inoculation (pi), and subjected to a triple staining procedure consisting in immunoperoxidase labeling of GFAP, periodic acid-Schiff (PAS) reaction in added baker's yeast cells and hematoxylin for nuclear staining of the whole cell monolayer. Adopting GFAP labeling as a specific marker of astrocyte differentiation, the immunoprecipitate development over time was measured. Direct calculation of the initial reaction rate was feasible given its linear behavior during the first 10 min, so that GFAP amount was regarded proportional to peroxidase activity. As determined by digital image analysis, mean optical density (MOD) values of GFAP in infected samples increased from 0.618 +/- 0.082 at day 1 pi to 0.825 +/- 0.125 at day 3, leveling off at 1.010 +/- 0.101 as from day 9, while control uninfected samples remained unchanged at roughly 0.6 during the entire observation period. In turn, phagocytosis was quantified by PAS staining densitometry, whose intensity varied according to wall degradation of yeast cells. MOD levels of PAS-stained phagocytized yeast cells were significantly lower (p < 0.05) in infected vs control cultures at 48 and 72 h following their addition to the astroglial monolayer. According to simultaneous quantification of two components of astrocyte response to viral infection, it is concluded that phagocytic activity increases with astrocyte differentiation.

Cell differentiation increases astrocyte phagocytic activity. A quantitative analysis of both GFAP labeling and PAS-stained yeast cells.

Since efficiency of phagocytic potential in activated astrocytes is still a subject of controversy, an attempt was made to quantify simultaneously phagocytic activity and astrocyte differentiation. Resorting to Junin virus, known to induce astrocyte activation, infected vs control samples of cultured rat astroglial cells were serially harvested up to day 12 post-inoculation (pi), and subjected to a triple staining procedure consisting in immunoperoxidase labeling of GFAP, periodic acid-Schiff (PAS) reaction in added bakers yeast cells and hematoxylin for nuclear staining of the whole cell monolayer. Adopting GFAP labeling as a specific marker of astrocyte differentiation, the immunoprecipitate development over time was measured. Direct calculation of the initial reaction rate was feasible given its linear behavior during the first 10 min, so that GFAP amount was regarded proportional to peroxidase activity. As determined by digital image analysis, mean optical density (MOD) values of GFAP in infected samples increased from 0.618 +/- 0.082 at day 1 pi to 0.825 +/- 0.125 at day 3, leveling off at 1.010 +/- 0.101 as from day 9, while control uninfected samples remained unchanged at roughly 0.6 during the entire observation period. In turn, phagocytosis was quantified by PAS staining densitometry, whose intensity varied according to wall degradation of yeast cells. MOD levels of PAS-stained phagocytized yeast cells were significantly lower (p < 0.05) in infected vs control cultures at 48 and 72 h following their addition to the astroglial monolayer. According to simultaneous quantification of two components of astrocyte response to viral infection, it is concluded that phagocytic activity increases with astrocyte differentiation.

Glial fibrillary acidic protein (GFAP) immunochemical profile after junin virus infection of rat cultured astrocytes.

Cultured astrocytes derived from newborn rat brain were inoculated with Junin virus (JV) to characterize their response to infection by means of their glial fibrillary acidic protein (GFAP) immunochemical profile. Samples from 1 to 11 days post-inoculation (pi), as well as matched controls, were serially harvested for GFAP labeling by peroxidase-antiperoxidase (PAP) method. It was only at day 3 that significantly greater values of GFAP staining (P < 0.05) were disclosed by three complementary approaches: image analysis, ELISA and immunoblot densitometry. Since such increase was abolished by Triton X-100 treatment, soluble GFAP fraction appeared responsible for the early though transient enhancement of GFAP immunoreactivity that followed viral inoculation.

Growth inhibition of herpes simplex virus-type 1 in calphostin C-treated astrocytes.

The aim was to evaluate the effects of calphostin C (CC), a protein kinase C inhibitor, on lytic herpes simplex virus-type 1 infection of cultured rat astrocytes. At 24 h postinjection, the cell culture receiving CC treatment at 50 nM concentration showed decreased cell detachment and retraction versus untreated infected controls; likewise, the infective virus yield was significantly lower in a dose-dependent manner. In contrast, image analysis failed to disclose differences in viral antigen immunolabeling at low drug concentrations thus suggesting that CC-induced inhibition of cytopathic effects and infectivity taken place through mechanisms not involving viral protein synthesis. Given the low dose required and the apparent lack of cytotoxic effects, present findings encourage additional studies on CC antiviral potential in the whole organism.

Histometric demonstration of periastrocytic fibronectin loss during herpes simplex virus infection.

Since Herpes simplex virus-type 1 (HSV-1) is liable to induce modifications and/or loss of immunoreactive fibronectin (FN) in cultured cells, our present goal was to determine whether such loss is attributable to FN binding impairment rather than cell detachment secondary to viral cytopathic effect. For this purpose, we resorted herein to an histometric approach for statistical evaluation of FN pattern in the course of HSV-1 infection of astroglial cell monolayers. The length of FN positive fibers was calculated by means of their tracing on a digitizer tablet; in the same field, cell nucleus count was performed. Recorded data allowed the calculation of an index as the ratio of the length of FN positive fibers over cell nucleus number. On comparing the indices between infected and control cultures, matricial FN loss was found in the former at 48 h post-inoculation, accompanied by cell fusion and retraction, though without cell detachment. A significant loss of FN was thus demonstrated as an event prior to severe cytopathic effect induced by viral infection.

Neural spread of Junin virus in intraperitoneally inoculated rats.

On the basis of an already demonstrated Junín virus (JV) neural route after peripheral footpad infection of newborn rats, here we attempted to determine the viral pathway following intraperitoneal inoculation. As from the 2nd week post-infection, neurological disease developed reaching 84% mortality at 30 days. Immunoperoxidase labeling of viral antigen, concomitantly with infectivity assays and histological examination, was carried out in serially harvested samples. Whenever infectivity was detected, whether by viral rescue from coculture or by conventional isolation, viral antigen staining was achieved. Infective JV was present at threshold levels in spleen and liver from days 2 to 10, and in blood from days 5 to 15. In neural tissues, viral antigen was initially disclosed at day 5 in thoracic rachideal ganglia and related spinal cord segments. From day 7 thereafter, the entire spinal cord was involved; at this stage, first evidence of viral infection was found in brain stem, with subsequent spread to other encephalon structures at day 10. According to harvested samples, no significant differences were found in labeled cell percentages at thoracic vs cervical or lumbar levels of spinal cord. In contrasts, greater involvement of cerebral cortex versus brain stem, hippocampus or cerebellum was demonstrated shortly before death. Although JV antigen was overwhelmingly predominant in neurons, no morphological changes were apparent in such cells. Since rachideal spinal ganglia and spinal cord infection invariably preceded viral spread to encephalon, concomitantly with viral clearance from lymphoreticular organs and blood, a neural pathway seems warranted.

Neural spread of Junin virus in intraperitoneally inoculated rats

Una vez establecida la ruta neural como la seguida por el virus Junín (VJ) a partir de su inoculación intradérmica en ratas lactantes, resultaba de interes determinar cuál era la vía adoptada luego de inoculado intraperitonealmente. Desde la 2da semana se evidenció una enfermedad neurológica que a los 30 días post-infección alcanzó un 84 por ciento de mortalidad. En curso de ese período, se efectuaron cosechas de tejidos extraneurales y neurales para la marcación por inmunoperoxidasa del antígenos viral y el examen histopatológico, asi como la titulación de infectividad que también se extendió a sangre. En todas las muestras de tejido en que se detectó virus infectivo, sea por cocultivo o por aislamiento convencional, se logró la marcación del antígeno viral. El VJ estuvo presente en valores mínimos en bazo e hígado desde el día 2 al 5, y en sangre del 5 al 15. En tejidos neurales, el antígeno viral fue inicialmente revelado al día 5, tanto en ganglios raquídeos torácicos como en los segmentos medulares relacionados. A partir del día 7, la positividad se extendió a la médula espinal en toda su extensión; a la vez, ya había evidencias de presencia viral en tronco cerebral, con disfusión al resto de estructuras encefálicas desde el día 10. Pese a la presencia masiva del antígeno viral en neuromas, dichas células no mostraban cambios morfológicos aparentes. Dado que la infección de ganglios raquídeos y de médula espinal invariablemente precedió al acesso viral a encéfalo, y ello ocurrió en forma concomitante a la desaparición del virus en órganos linfo-reticulares y en sangre, la vía neural parece ser la adoptada por el VJ desde cavidad peritoneal hasta sistema nervioso central

Neural spread of Junin virus in intraperitoneally inoculated rats

Una vez establecida la ruta neural como la seguida por el virus Junín (VJ) a partir de su inoculación intradérmica en ratas lactantes, resultaba de interes determinar cuál era la vía adoptada luego de inoculado intraperitonealmente. Desde la 2da semana se evidenció una enfermedad neurológica que a los 30 días post-infección alcanzó un 84 por ciento de mortalidad. En curso de ese período, se efectuaron cosechas de tejidos extraneurales y neurales para la marcación por inmunoperoxidasa del antígenos viral y el examen histopatológico, asi como la titulación de infectividad que también se extendió a sangre. En todas las muestras de tejido en que se detectó virus infectivo, sea por cocultivo o por aislamiento convencional, se logró la marcación del antígeno viral. El VJ estuvo presente en valores mínimos en bazo e hígado desde el día 2 al 5, y en sangre del 5 al 15. En tejidos neurales, el antígeno viral fue inicialmente revelado al día 5, tanto en ganglios raquídeos torácicos como en los segmentos medulares relacionados. A partir del día 7, la positividad se extendió a la médula espinal en toda su extensión; a la vez, ya había evidencias de presencia viral en tronco cerebral, con disfusión al resto de estructuras encefálicas desde el día 10. Pese a la presencia masiva del antígeno viral en neuromas, dichas células no mostraban cambios morfológicos aparentes. Dado que la infección de ganglios raquídeos y de médula espinal invariablemente precedió al acesso viral a encéfalo, y ello ocurrió en forma concomitante a la desaparición del virus en órganos linfo-reticulares y en sangre, la vía neural parece ser la adoptada por el VJ desde cavidad peritoneal hasta sistema nervioso central (AU)

Histometric demonstration of periastrocytic fibronectin loss during herpes simplex virus infection.

Since Herpes simplex virus-type 1 (HSV-1) is liable to induce modifications and/or loss of immunoreactive fibronectin (FN) in cultured cells, our present goal was to determine whether such loss is attributable to FN binding impairment rather than cell detachment secondary to viral cytopathic effect. For this purpose, we resorted herein to an histometric approach for statistical evaluation of FN pattern in the course of HSV-1 infection of astroglial cell monolayers. The length of FN positive fibers was calculated by means of their tracing on a digitizer tablet; in the same field, cell nucleus count was performed. Recorded data allowed the calculation of an index as the ratio of the length of FN positive fibers over cell nucleus number. On comparing the indices between infected and control cultures, matricial FN loss was found in the former at 48 h post-inoculation, accompanied by cell fusion and retraction, though without cell detachment. A significant loss of FN was thus demonstrated as an event prior to severe cytopathic effect induced by viral infection.

Neural spread of Junin virus in intraperitoneally inoculated rats.

On the basis of an already demonstrated Junín virus (JV) neural route after peripheral footpad infection of newborn rats, here we attempted to determine the viral pathway following intraperitoneal inoculation. As from the 2nd week post-infection, neurological disease developed reaching 84

mortality at 30 days. Immunoperoxidase labeling of viral antigen, concomitantly with infectivity assays and histological examination, was carried out in serially harvested samples. Whenever infectivity was detected, whether by viral rescue from coculture or by conventional isolation, viral antigen staining was achieved. Infective JV was present at threshold levels in spleen and liver from days 2 to 10, and in blood from days 5 to 15. In neural tissues, viral antigen was initially disclosed at day 5 in thoracic rachideal ganglia and related spinal cord segments. From day 7 thereafter, the entire spinal cord was involved; at this stage, first evidence of viral infection was found in brain stem, with subsequent spread to other encephalon structures at day 10. According to harvested samples, no significant differences were found in labeled cell percentages at thoracic vs cervical or lumbar levels of spinal cord. In contrasts, greater involvement of cerebral cortex versus brain stem, hippocampus or cerebellum was demonstrated shortly before death. Although JV antigen was overwhelmingly predominant in neurons, no morphological changes were apparent in such cells. Since rachideal spinal ganglia and spinal cord infection invariably preceded viral spread to encephalon, concomitantly with viral clearance from lymphoreticular organs and blood, a neural pathway seems warranted.

Histometric demonstration of periastrocytic fibronectin loss during herpes simplex virus infection

Since Herpes simplex virus-type 1 (HSV-1) is liable to induce modifications and/or loss of immunoreactive fibronectin (FN) in cultured cells, our present goal was to determine whether such loss is attributable to FN binding impairment rather than cell detachment secondary to viral cytopathic effect. For this purpose, we resorted herein to an histometric approach for statistical evaluation of FN pattern in the course of HSV-1 infection of astroglial cell monolayers. The length of FN positive fibers was calculated by means of their tracing on a digitizer tablet; in the same field, cell nucleus count was performed. Recorded data allowed the calculation of an index as the ratio of the length of FN positive fibers over cell nucleus number. On comparing the indices between infected and control cultures, matricial FN loss was found in the former at 48 h post-inoculation, accompanied by cell fusion and retraction, though without cell detachment. A significant loss of FN was thus demonstrated as an event prior to severe cytopathic effect induced by viral infection.