Regeneration of cochlear efferent nerve terminals after gentamycin damage.

Chickens recover auditory function after hair cell loss caused by ototoxic drug damage or acoustic overstimulation, indicating that mechanisms exist to reestablish appropriate neuronal connections to regenerated hair cells. However, despite similar hair cell regeneration times, hearing recovery takes substantially longer after aminoglycoside than after sound damage. We have therefore begun examining damage and regeneration of efferent nerve terminals by immunolabeling whole-mount cochleae for differentially localized synaptic proteins and by visualizing the distribution of label with confocal microscopy. In undamaged cochleae, the synaptic proteins synapsin and syntaxin show similar distribution patterns corresponding to the large cup-like terminals on short hair cells. After gentamycin administration, these terminals are disrupted as hair cells are lost, leaving smaller, more numerous synapsin-reactive structures in the sensory epithelium. Syntaxin reactivity remains associated with the extruded hair cells, indicating that the presynaptic membrane is still attached to the postsynaptic site. In contrast, after sound damage, both synapsin and syntaxin reactivity are lost from the epithelium with extruded hair cells. As regenerated hair cells differentiate after gentamycin treatment, the synapsin labeling associated with cup-like efferent endings reappears but is not completely restored even after 60 d of recovery. Thus, efferent terminals are reestablished much more slowly than after sound damage (), consistent with the prolonged loss of hearing function. This in vivo model system allows comparison of axonal reconnection after either complete loss (sound damage) or partial disruption (gentamycin treatment) of axon terminals. Elucidating the differences in recovery between these injuries can provide insights into reinnervation mechanisms.

Sound damage and gentamicin treatment produce different patterns of damage to the efferent innervation of the chick cochlea.

Both sound exposure and gentamicin treatment cause damage to sensory hair cells in the peripheral chick auditory organ, the basilar papilla. This induces a regeneration response which replaces hair cells and restores auditory function. Since functional recovery requires the re-establishment of connections between regenerated hair cells and the central nervous system, we have investigated the effects of sound damage and gentamicin treatment on the neuronal elements within the cochlea. Whole-mount preparations of basilar papillae were labeled with phalloidin to label the actin cytoskeleton and antibodies to neurofilaments, choline acetyltransferase, and synapsin to label neurons; and examined by confocal laser scanning microscopy. When chicks are treated with gentamicin or exposed to acoustic overstimulation, the transverse nerve fibers show no changes from normal cochleae assayed in parallel. Efferent nerve terminals, however, disappear from areas depleted of hair cells following acoustic trauma. In contrast, efferent nerve endings are still present in the areas of hair cell loss following gentamicin treatment, although their morphological appearance is greatly altered. These differences in the response of efferent nerve terminals to sound exposure versus gentamicin treatment may account, at least in part, for the discrepancies reported in the time of recovery of auditory function.

Expression of a quail bHLH transcription factor is associated with adrenergic development in trunk neural crest cultures.

1. Expression of chick type 1 basic helix-loop-helix transcription factor GbHLH1.4 persists in several embryonic regions, including some where neural crest cells differentiate (Helms, J. A., et al., Mech. Dev. 48:93-108, 1994.) We have cloned portions of the quail homologue (designated QbHLH) in order to investigate its expression and possible function in quail neural crest cultures. Three sets of polymerase chain reaction primers were used to amplify cDNA sequences encompassing much of the coding region outside the bHLH domain. Two of the primer sets amplified a single band from all quail and chick tissues tested. The third set of primers produced two bands, differing by a 72-base pair insertion, both of which which were present in all tissues assayed. 2. The quail sequences showed greater than 97% nucleotide identity with GbHLH1.4. In situ hybridization of cultured quail neural crest cells showed expression in some, but not all, cells throughout the first 2 weeks in culture. 3. Tyrosine hydroxylase immunoreactivity correlated particularly well with QbHLH expression, although substantial subpopulations of cells with other phenotypes also express QbHLH. 4. In some cells, only limited regions of the cytoplasm showed hybridization with QbHLH probes, indicating possible mRNA localization. 5. The expression of QbHLH in neural crest cultures is consistent with its role as a relatively widely expressed helix-loop-helix dimerization partner and suggests that it may function by interacting with cell type-specific partners to regulate expression of genes involved in the development and maintenance of several phenotypes.

Persistent correlation between expression of a sulfated carbohydrate antigen and adrenergic differentiation in cultures of quail trunk neural crest cells.

The carbohydrate antigen recognized by monoclonal antibodies such as HNK-1 (first characterized as recognizing human natural killer cells) and NC-1 (raised against quail neural-crest-derived cells) is found on a number of molecules associated with cell differentiation in vertebrates [42]. Previous work has shown that subpopulations of cultured quail trunk neural crest cells can be separated by fluorescence-activated cell sorting (FACS) on the basis of expression of this carbohydrate antigen. When neural crest cells are separated after 2 days in culture, adrenergic cells develop preferentially within the HNK-1-reactive subpopulation [27]. We wished to investigate whether the capacity for adrenergic differentiation remained associated with the HNK-1-positive cell population at later times in vitro, when the percentage of HNK-1-reactive cells has declined. The present study found that neural crest cells separated according to HNK-1-reactivity after 4 days in culture also showed preferential development of adrenergic cells in HNK-1-positive-enriched cultures, indicating that the HNK-1 epitope is persistently expressed in vitro on cells with adrenergic potential after 4 days of culture. To investigate the possible function of this epitope in development of the adrenergic phenotype, HNK-1 was added to unsorted neural crest cell cultures. The presence of antibody resulted in a decrease in the percentage of HNK-1-reactive cells during the initial 24 h after replating, but had no effect on the number of catecholamine-positive cells which developed after 7 days. We conclude that the epitope recognized by the HNK-1 antibody does not appear to function in the induction of the adrenergic phenotype. However, this antigenic determinant is useful as a predictive early marker which defines a subset of neural crest cells that includes those with the ability to undergo adrenergic differentiation.

Large chondroitin sulfate proteoglycans of developing chick CNS are expressed in cerebral hemisphere neuronal cultures.

Chondroitin sulfate proteoglycans (CSPG) of the extracellular matrix may play regulatory roles in central nervous system (CNS) development. We have examined the expression of two large CSPGs of the embryonic chick brain, which can be differentiated using the monoclonal antibodies HNK-1 and S103L, in cultures of embryonic day 6 chick cerebral hemisphere neurons. Western blot analysis following immunoprecipitation and endoglycosidase treatment revealed that these cultures produce S103L- and HNK-1-reactive proteoglycans which are biochemically indistinguishable from the CSPGs (previously) identified in homogenized chick embryo brain extracts. The HNK-1-reactive CSPG accumulated in the medium throughout the course of cultures. In contrast, the S103L-reactive CSPG was found in a neuron-associated form during the period of aggregate establishment in culture, as well as in a soluble form secreted into the medium. Immunocytochemical staining of cultures with the S103L antibody localized reactivity to most neurons during the period of aggregate formation, while neuronal processes and the few flat cells present (presumably neuroblasts and early glia) were negative. Cell selection experiments confirmed that neurofilament-positive cells were the source of the S103L-reactive CSPG. The use of differential fixation techniques suggested that the cell-associated S103L reactivity may be intracellular. Because of this pattern of expression and localization, we propose that the developmentally regulated S103L-reactive CSPG may play a role in neuronal migration arrest and organization of neurons into functional aggregates.

Two immunologically and developmentally distinct chondroitin sulfate proteolglycans in embryonic chick brain.

Two different chondroitin sulfate proteoglycans (CSPG) in embryonic chick brain were distinguished by immunoreactivity either with S103L, a rat monoclonal antibody which reacts specifically with an 11-amino-acid region in the chondroitin sulfate domain of the core protein of chick cartilage CSPG (Krueger, R. C., Jr., Fields, T. A., Mensch, J. R., and Schwartz, N. B. (1990) J. Biol. Chem. 265, 12088-12097), or with HNK-1, a mouse monoclonal antibody which reacts with a 3-sulfoglucuronic acid residue on neural glycolipids and glycoproteins (Chou, D. K. H., Ilyas, A., Evans, J. E. Costello, C., Quarles, R. H., and Jungawala, F. B. (1986) J. Biol. Chem. 261, 11717-11725) but not with both antibodies. This specific immunoreactivity was used to separate the two CSPGs for further characterization. The S103L reactive brain proteoglycan had a core protein of similar size to cartilage CSPG (370 kDa) but exhibited a smaller hydrodynamic size (K(av) of 0.308). It was substituted predominantly with chondroitin sulfate chains and virtually no keratan sulfate chains. The HNK-1 reactive CSPG had a smaller core protein (340 kDa), an even smaller hydrodynamic size (K(av) of 0.564), and was substituted with both chondroitin sulfate and keratan sulfate chains. Glycosidase digestion patterns with endo-beta-galactosidase, N-glycosidase F, neuraminidase, and O-glycosidase, and reactivity with an antibody to the hyaluronate binding region also showed significant differences between the two brain CSPGs. Expression of the S103L reactive brain CSPG was developmentally regulated from embryonic day 7 through 19 with a peak in core protein on day 13, and in mRNA expression at day 10. In contrast the HNK-1 reactive brain CSPG was constitutively present from day 7 through hatching. These data suggest that these two distinct core proteins are immunologically and biochemically unique translation products of two different CSPG genes.

Serum factors in the progression of human immunodeficiency virus type 1 infection to AIDS.

We studied the prevalence of four serum factors in individuals at different stages of human immunodeficiency virus-1 (HIV-1) infection. Soluble interleukin-2 receptors (sIL-2R) were elevated in all antibody-positive groups compared with high-risk, antibody-negative controls. Paraproteins, usually of the IgG-kappa isotype, were found in the sera of a significant number of HIV-1-infected individuals as were antibodies to lymphocytes (ALAs). Serum factors that inhibit proliferation of peripheral blood mononuclear cells from healthy donors appear late in the course of infection and were associated with increasing clinical severity. Measurement of these factors may prove to be useful in defining the stages of infection and in predicting the appearance or exacerbation of symptoms. They may also play a role in the development of the HIV-1-induced immune defects that lead to the expression of clinical acquired immunodeficiency syndrome.

Lysis of fresh murine mammary tumor cells by syngeneic natural killer cells and lymphokine-activated killer cells.

We have compared the ability of natural killer (NK) cells from two substrains of C3H mice that differ with respect to their susceptibility to the development of mammary adenocarcinomas to lyse fresh syngeneic mammary tumor cells. Single cell suspensions of mammary tumors from retired breeder females were used as targets in 22-h 51Cr-release cytotoxicity assays with syngeneic NK cells. Tumor cell suspensions were prepared by enzymatic digestion of finely minced tissue followed by centrifugation through a discontinuous Percoll gradient. Effector cells were prepared by passing spleen cells over nylon wool followed by centrifugation through Percoll fraction 7. Syngeneic NK cells had significant levels of lysis against 5/8 tumors studied. NK cells from low risk animals (C3Heb/FeJ) consistently demonstrated greater cytotoxicity against tumor cell preparations than did effectors from the high tumor substrain (C3H/OuJ). Study of cytocentrifuge preparations stained with Wright-Giemsa revealed that the two substrains were identical with respect to the number of azurophilic granules present in the cytoplasm of their NK cells. We have also shown that lymphokine-activated killer (LAK) cells can be generated from splenocytes in C3H mice. While LAK cells from both substrains were capable of lysing fresh syngeneic mammary tumor cells in vitro, LAK cells from the animals at high risk for the formation of mammary adenocarcinomas had greater cytotoxicity against tumor cell suspensions than LAK cells from the low tumor substrain.

Cellular targets of antilymphocyte antibodies in AIDS and LAS.

Many patients with acquired immune deficiency syndrome (AIDS) and lymphadenopathy syndrome (LAS) have serum antilymphocyte antibodies. The targets of these antibodies are neither sex nor HLA directed. There is disagreement about the cell subset at risk. We examined the cellular specificity of antilymphocyte antibodies from AIDS and LAS patients by microcytotoxicity on positively selected lymphocytes, by using double-labeling immunofluorescence, and by using well-characterized continuous cell lines as targets. We find that most AIDS/LAS patients have antibodies to T and B cells, that immunofluorescence is somewhat more sensitive than microcytotoxicity, and that the antibodies are directed to both T4+ and T4- lymphoid cells.

Inhibition of in vitro lymphocyte proliferation by serum from acquired immune deficiency syndrome patients depends on the ratio of cells to serum in culture.

The effect of serum from patients with acquired immune deficiency syndrome (AIDS) on cultures of normal human peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA) has been studied. It has been found that AIDS sera are inhibitory when compared with control sera from healthy individuals. The inhibitory activity in the AIDS patients' sera is dilutable with normal serum, is dependent on the number of cells present in culture and the amount of serum added, and cannot be attributed to a deficiency of nutrients in these sera. Inhibition of proliferation occurs even when AIDS serum is added to cultures of normal cells several hours after stimulation with PHA. In one patient who was being treated with plasmapheresis, decreases in serum inhibitory activity were found after pheresis procedures and were coincident with increases in the number of circulating T4-positive lymphocytes.