[Distribution of the genotypes of hepatitis C virus in intravenous drug addicts in Argentina]. / Distribución de los genotipos del virus de la hepatitis C en una población Argentina de drogadictos endovenosos.

Intravenous drug addiction (IVD) is an unfrequent risk factor in Argentina, representing less than 10% of patients (pts) with chronic HCV infection seen in our Unit. In order to study the genotypes (Gt) in IVD and compare them with a non drug addicted control population, 68 pts with a history of IVD were enrolled in this study and compared with 68 non drug addict (NDA) pts with chronic HCV, with similar age and gender distribution. In all pts a liver biopsy was performed. Genotyping was done by INNO LiPA (Innogenetics, Belgium). Mean age in both groups was 35 +/- 7.8 years and 50 were males. No difference was observed between both groups in the prevalence of Gt1a, Gt2a/c and in those with mixed infections. The prevalence of Gt1b in IVD was 19.1% and in NDA 38.2% (p = 0.0228). A highly significant difference was also observed in the prevalence of Gt3a, of 42.6% in IVD and only 11.8% in NDA (p = 0.0001). Gt1a was the second most frequent genotype in IVD pts (26.5%). Simultaneous HIV infection was present in 8 IVD pts (11.8%) and in none of NDA group. Liver biopsies showed a higher prevalence of mild chronic hepatitis in NDA (57.3%) than in IVD (32.4%) (p = 0.0058). Severe chronic hepatitis with advanced fibrosis or cirrhosis was more frequent in the Gt3 of the group with IVD when compared with Gt3 of the NDA group. It can be concluded that in accordance with other geographical areas, Gt3a is far more prevalent in intravenous drugs addicts than in the general population in Argentina where Gt1b is more frequent. Mild forms of chronic hepatitis are less frequent in IVD. In spite of the relatively small group with HCV co-infection with HIV, it seems important to note that 2/8 (25%) showed severe hepatitis C or cirrhosis.

Distribución de los genotipos del virus de la hepatitis C en una población Argentina de drogadictos endovenosos. / [Distribution of the genotypes of hepatitis C virus in intravenous drug addicts in Argentina]

Intravenous drug addiction (IVD) is an unfrequent risk factor in Argentina, representing less than 10

of patients (pts) with chronic HCV infection seen in our Unit. In order to study the genotypes (Gt) in IVD and compare them with a non drug addicted control population, 68 pts with a history of IVD were enrolled in this study and compared with 68 non drug addict (NDA) pts with chronic HCV, with similar age and gender distribution. In all pts a liver biopsy was performed. Genotyping was done by INNO LiPA (Innogenetics, Belgium). Mean age in both groups was 35 +/- 7.8 years and 50 were males. No difference was observed between both groups in the prevalence of Gt1a, Gt2a/c and in those with mixed infections. The prevalence of Gt1b in IVD was 19.1

and in NDA 38.2

(p = 0.0228). A highly significant difference was also observed in the prevalence of Gt3a, of 42.6

in IVD and only 11.8

in NDA (p = 0.0001). Gt1a was the second most frequent genotype in IVD pts (26.5

). Simultaneous HIV infection was present in 8 IVD pts (11.8

) and in none of NDA group. Liver biopsies showed a higher prevalence of mild chronic hepatitis in NDA (57.3

) than in IVD (32.4

) (p = 0.0058). Severe chronic hepatitis with advanced fibrosis or cirrhosis was more frequent in the Gt3 of the group with IVD when compared with Gt3 of the NDA group. It can be concluded that in accordance with other geographical areas, Gt3a is far more prevalent in intravenous drugs addicts than in the general population in Argentina where Gt1b is more frequent. Mild forms of chronic hepatitis are less frequent in IVD. In spite of the relatively small group with HCV co-infection with HIV, it seems important to note that 2/8 (25

) showed severe hepatitis C or cirrhosis.

Use of Aplysia neurons for the study of cellular alterations and the resealing of transected axons in vitro.

The present report describes the experimental advantages offered by the combined use of Aplysia neurons and contemporary techniques to analyze the cellular events associated with nerve injury in the form of axotomy. The experiments were performed by transecting, under visual control, the main axon of identified Aplysia neurons in primary culture while monitoring several related parameters. We found that in cultured Aplysia neurons axotomy leads to the elevation of the [Ca2+]i in both the proximal and distal axonal segments from a resting level of 100 nM up to the millimolar range for a duration of 3-5 min. This increase in [Ca2+]i led to identical alterations in the cytoarchitecture of the proximal and distal segments. The formation of a membrane seal over the transected ends by their constriction and the subsequent fusion of the membrane is a [Ca2+]i-dependent process and is triggered by the elevation of [Ca2+]i to the microM level. Seal formation was followed by down-regulation of the [Ca2+]i to control levels. Following the formation of the membrane seal an increase in membrane retrieval was observed. We hypothesize that the retrieved membrane serves as an immediately available membrane reservoir for growth cone extension.

The survival of transected axonal segments of cultured Aplysia neurons is prolonged by contact with intact nerve cells.

Axonal segments transected from their cell body in vivo commonly undergo degeneration within 3-4 days (Wallerian degeneration). In lower vertebrates and invertebrates, however, some transected axonal segments survive for long periods ranging between 30 and 200 days. To circumvent the technical complications of studying the mechanisms underlying long-term survival of transected axons in vivo, we developed an in vitro system. We found previously that isolated axonal segments of cultured Aplysia neurons preserved their morphological integrity for an average duration of 7.6 days (range 2-14 days) and maintain their passive and excitable membrane properties. This survival occurred in the absence of de novo protein synthesis. In the present study we examined the influence of homologous neurons on the survival of transected axonal segments. We found that the average survival time of transected axons was doubled when co-cultured in physical contact with intact homologous neurons (average 15.3 days, range 2-27 days). During this period, the transected axons extended neurites, maintained normal passive and excitable membrane properties, formed electrotonic junctions with the intact neurons and maintained normal free intracellular Ca2+ levels. Consistent with these observations, electron micrographs of the transected axon revealed that the cytoskeletal elements of the axon appeared normal even 20 days after transection. In contrast, the mitochondria and smooth endoplasmic reticulum appeared damaged. As the prolonged survival was conditional on physical contact between the transected axon and the surrounding intact neurons, we suggest that the prolongation of survival time is promoted by the direct transfer of material from the intact neurons to the transected axon. However, co-culture of transected axons with homologous neurons did not fully mimic in vivo conditions, in which transected axons can survive for several months.

Survival of isolated axonal segments in culture: morphological, ultrastructural, and physiological analysis.

Some transected distal axons survive for months in vivo, generate new neurites, and reconnect to proximal segments before degenerating. To determine the factors regulating these phenomena, we studied the behavior of transected axons in cultured metacerebral neurons (MCn) of Aplysia. The neurons were isolated from the ganglia and cultured at 18 degrees C. The morphology, ultrastructure, and electrophysiological properties of the transected axons, as well as their ability to synthesize protein, were examined at different times postaxotomy. Follow-up studies revealed that cultured isolated axonal segments can preserve their morphological integrity for up to 14 days, maintain their passive and active membrane properties for at least 10 days, and extend new neurites and form electrotonic junctions with their proximal segments and intact MCns. De novo protein synthesis is an unlikely mechanism to account for the survival of the isolated axons since they did not incorporate [35S]methionine. We conclude that the viability of transected axons in culture devoid of other cells depends on pools of proteins synthesized prior to the transection and energy stores sufficiently large to maintain neuronal homeostasis.

Resealing of the proximal and distal cut ends of transected axons: electrophysiological and ultrastructural analysis.

The fates of the proximal and distal segments of transected axons differ. Whereas the proximal segment usually recovers from injury and regenerates, the distal segment degenerates. In the present report we studied the kinetics of the recovery processes of both proximal and distal axonal segments following axotomy and its temporal relations to the alterations in the cytoarchitecture of the injured neuron. The experiments were performed on primary cultured metacerebral neurons (MCn) isolated from Aplysia. We transected axons while monitoring the changes in transmembrane potential and input resistance (Rn) by inserting intracellular microelectrodes into the soma and axon. Correlation between the electrophysiological status of the injured axon and its ultrastructure was provided by rapid fixation of the neuron at selected times postaxotomy. Axotomy leads to membrane depolarization from a mean of -55.7 S.D. 12.8 mV to -12.7 S.D. 3.3 mV and decreased Rn from tens of M omega to 1-3 M omega. The transected axons remained depolarized for a period of 10-260 s for as long as the axoplasm was in direct contact with the bathing solution. Rapid repolarization and partial recovery of Rn was associated with the formation of a membrane seal over the cut ends by the constriction and subsequent fusion of the axolema. Prior to the formation of a membraneous barrier, electron-dense deposits aggregate at the tip of the cut axon and appear to form an axoplasmic "plug." Electrophysiological analysis revealed that this "plug" does not provide resistance for current flow and that the axoplasmic resistance is homogenously distributed. The kinetics of injury and recovery processes as well as the ultrastructural changes of the proximal and distal segments are identical suggesting that the different fates of the segments cannot be attributed to differences in the immediate response of the segments to axotomy.