Henrietta Kulaga

Certain host-donor rat strain combinations do not reject brain allografts after systemic sensitization

To investigate the factors related to allogenic brain graft rejection, rat cortex from either LEW-RT1l or BN-RT1n rats was transplanted into brains of several isogenic and allogenic inbred strains: F344-RT1l, LEW-RT1l, BN-RT1n, AO-RT1u, PVG-RT1c, PVG-RT1u, and PVG-RT1l. Each donor and host combination was subsequently subdivided into two subgroups. One group was systematically sensitized twice with donor skin tissue and another group served as a sham-sensitization control. Four weeks after the second sensitization, host brains were examined histologically, and the percentage volume of each graft that showed increased cellularity was estimated. Expression of major histocompatibility complex (MHC) and T cell antigens was also studied immunocytochemically. Almost all grafts in sham-sensitized animals from all groups were accepted with minimal or no reaction. However, two strains (AO-RT1u and F344-RT1l) showed considerable cell infiltration and expression of MHC antigens even without sensitization. After sensitization, almost all allogenic strain combinations showed greater signs of rejection-related responses. The severity of the tissue reaction, however, varied considerably between groups. All grafts from BN-RT1n donors were rejected severely in all host strains. For LEW-RT1l donors, grafts survived well in some host strains (BN-RT1n, AO-RT1u, PVG-RT1c, and PVG-RT1u), even with MHC + non-MHC disparity. Curiously, F344-RT1l hosts rejected LEW-RT1l grafts even though the two strains have the same MHC loci, but different minor histocompatibility (mH) loci. For both donor strains, experimental autoimmune encephalomyelitis-susceptible hosts tended to show more vigorous rejection responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Human Olfactory Neuroepithelial Cells: Tyrosine Phosphorylation and Process Extension Are Increased by the Combination of IL-1β, IL-6, NGF, and bFGF

Olfactory neuroepithelial cells (ONC) grown from biopsies of human donors are a novel cell culture system that may facilitate studies into normal and disease-related human neurobiology. We further characterized the expression of cell surface markers and intermediate filaments, and responses to neurotrophic factors by ONC. ONC are positive for cell surface markers N-CAM, PSA-N-CAM, neutral endopeptidase, N-aminopeptidase, NGF low-affinity receptor homologue (CD40), and transferrin receptor by flow cytometry for the intermediate filament proteins peripherin, vimentin, and NF-H by immunocytochemistry. Responses to neurotrophic factors measured were process outgrowth, cytoskeletal protein expression, and protein phosphorylation. Process outgrowth was increased by interleukin-beta 164-171 (IL-1beta) or by the combination of IL-1beta, interleukin-6 (IL-6), nerve growth factor (NGF), and basic fibroblast growth factor (bFGF). This combination of IL-1beta, IL-6, NGF, and bFGF (16NF) increased expression of two cytoskeletal proteins, NF-H protein and microtubule-associated protein tau. Application of the individual neurotrophic factors IL-1beta, IL-6, NGF, and bFGF increased protein phosphorylation, while 16NF produced an immediate increase in tyrosine phosphorylation of several proteins (MW of 40-80, 120, 150, and 190 kDa). The 16NF combination appears to act through a tyrosine-kinase-mediated pathway to induce process extension and increase NF-H expression. The ONC culture has the potential to be further explored to examine the relationship among process outgrowth, protein phosphorylation, and synergy between neurotrophin and cytokine receptor systems.

Increased cyclic AMP response to forskolin in Epstein-Barr virus-transformed human B-lymphocytes derived from schizophrenics

Abstract Phorbol 12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, elevated basal cyclic AMP levels and enhanced isoproterenol-, prostaglandin E1- (PGE1), forskolin- and cholera toxin-stimulated cyclic AMP accumulation in Epstein-Barr virus (EBV)-transformed human B-lymphocytes. Staurosporine, a PKC inhibitor, significantly antagonized the increase in cyclic AMP accumulation produced by PMA, whereas the inactive phorbol ester, 4α-phorbol 12,13-didecanoate (4αPDD), had no effect. Basal levels of cyclic AMP and the accumulation of cyclic AMP produced by PMA, isoproterenol, PGE1, cholera toxin and the combination of these compounds with PMA were not significantly different between schizophrenics and controls. The cyclic AMP response to forskolin in the presence and absence of PMA was significantly greater in EBV-transformed human B-lymphocytes from schizophrenics. These results suggest that activation of adenylyl cyclase by forskolin is elevated in EBV-transformed B-lymphocytes derived from schizophrenics and that this elevation is further enhanced through a PKC-dependent phosphorylation mechanism.

Evidence for dual infection of rabbits with the human retroviruses HTLV-I and HIV-1

Rabbits experimentally infected with HTLV-I and HIV-1 produced antibody to various viral proteins, and viral DNA could be detected by gene amplification using the polymerase chain reaction. HTLV-I genes were detected in cell lines derived from infected rabbits, and in some cases, both HIV-1 and HTLV-I DNA sequences were demonstrated in peripheral blood cells taken from rabbits one year after experimental infection. The polymerase chain reaction procedure was used to demonstrate the presence of HTLV-I gag, env and tax genes and HIV-1 gag and env genes. The amplified fragments were identified by size and by hybridization to specific probes. The ability of rabbits to support simultaneous infection with HTLV-I and HIV-1 will allow in vivo studies of the possible synergistic effects of these important human pathogens.

The continued search for evidence of retroviral infection in schizophrenic patients

Retroviral infection has been proposed as an etiologic factor in schizophrenia. In an effort to unmask a latent retrovirus, short term cultures of peripheral lymphocytes from 15 schizophrenic subjects and nine normal controls were exposed to ionizing radiation and co-cultured with indicator cells. Reverse transcriptase activity, a marker of retroviral infection, could not be detected in any of the cultures. Our findings are further evidence against a role for retroviral infection in the etiology of schizophrenia.

Growth properties of neural cell lines immortalized with the tsA58 allele of SV40 large T antigen

In vitro growth properties of three CNS-derived cell lines were compared under a variety of culture conditions. The M213-20 and J30a cell lines were each derived from embryonic CNS culture with the temperature-sensitive (ts) allele of SV40 large T antigen, tsA58, while the A7 cell line was immortalized using wild-type SV40 large T antigen. Cells immortalized with tsA58 SV40 large T proliferate at the permissive temperature, 33 degrees C, while growth is expected to be suppressed at the nonpermissive temperature, 39.5 degrees C. Both the M213-20 and J30a cell lines were capable of proliferating at 39.5 degrees C continuously for up to 6 mo. All three cell lines showed no appreciable differences in growth rates related to temperature over a 7-day period in either serum-containing or defined serum-free media. The percentage of cells in S-phase of the cell cycle did not decrease or was elevated at 39.5 degrees C for all three cell lines. After 3 wk at 39.5 degrees C, the three cell lines also showed positive immunostaining using two monoclonal antibodies reacting with different epitopes of SV40 large T antigen. Double strand DNA sequence analyses of a 300 base pair (bp) fragment of the large T gene from each cell line, which included the ts locus, revealed mutations in both the J30a and M213-20 cell lines. The J30a cell line ts mutation had reverted to wild type, and two additional loci with bp substitutions with predicted amino acid changes were also found. While the ts mutation of the M213-20 cells was retained, an additional bp substitution with a predicted amino acid change was found. The A7 cell line sequence was identical to the reference wild-type sequence. These findings suggest that (a) nucleic acid sequences in the temperature-sensitive region of the tsA58 allele of SV40 large T are not necessarily stable, and (b) temperature sensitivity of cell lines immortalized with tsA58 is not necessarily retained.

Phagocytic rabbit cell lines expressing class II MHC products: establishment of cell lines by viral transformation.

Continuous rabbit macrophage‐like cell lines were established after in vitro infection of spleen cells with either Simian virus 40, lymphotropic papovavirus or herpesvirus sylvilagus. These cell lines are characterized as morphologically similar to mature macrophages, esterase positive, and have unrearranged immunoglobulin genes. They possess macrophage functionality because they are highly phagocytic and are able to mediate an antibody dependent cell mediated cytotoxicity assay on chicken red blood cells. Northern blot analysis of total cellular RNA with a DQα probe indicates that the cell lines constitutively express message for class II gene products. In addition, cell sorter analysis indicates that two of these lines display class II antigen at the cell surface.

A plate-binding elisa for use with hybridoma supernatant fluids

An enzyme-linked immunosorbent assay (ELISA) is described for initial screening of hybridoma supernatants. The protocol is flexible enough to be used with a variety of different antigen types with only a few minor modifications. The method is uncomplicated and does not require any special instnumentation or expertise.

Evaluation of cyclic AMP accumulation in EBV-transformed human B-lymphocytes: Effects of dopamine agonists, isoproterenol, prostaglandin E1, cholera toxin, forskolin, and phorbol 12-myristate-13 acetate

1. Phorbol 12-myristate-13-acetate (PMA), a protein kinase C activator, elevated cyclic AMP accumulation in EBV-transformed human B-lymphocytes, and potentiated isoproterenol-, prostaglandin- (PGE1), cholera toxin-, and forskolin-stimulated cyclic AMP accumulation. 2. The dopamine D1 receptor agonist, SKF38393 (10(-7) to 10(-5) MH, had no effect on cyclic AMP accumulation in transformed human B-lymphocytes. 3. The dopamine D2 receptor agonist, quinpirole (10(-7) to 10(-5) MH did not inhibit cyclic AMP accumulation even when cyclic AMP accumulation was maximized by the addition of PMA and forskolin. 4. These data suggest that dopamine D1- and D2-receptor coupling to a cyclic AMP generating system is not present at detectable levels in transformed human B-lymphocytes.