Clonal character of F1 hybrid lymphocyte subset recognition of parental cells in one-way mixed lymphocyte cultures.

Proliferation of F(1) hybrid lymphocytes in mixed lymphocyte cultures is stimulated by mitomycin-blocked parental cells. The demonstration of this phenomenon using F(1) hybrids derived from congenic lines of mice establishes that the stimulation is controlled by genes in or closely linked to the major histocompatibility locus chromosome region. In agreement with the finding that tumor-bearing mice have an increased capacity for primary alloantigen recognition, it was observed that the F(1) hybrid response to parent was also augmented by tumor bearing. Chromosomal analysis of dividing cells in one-way mixed cultures confirms that F(1) cells, and not the blocked parental cells, enter mitosis. Stimulation of F(1) cells by a soluble mediator liberated by the parental cells was not observed and mitomycin blocking of parental cells seems to be a completely effective blocking agent ensuring that parental cells can not enter DNA synthesis. The specificity and clonal nature of F(1) recognition of parent was demonstrated using a 5-bromodeoxyuridine-suicide procedure. Distinct clones of lymphocytes in F(1) spleen cell populations seem to recognize one or the other parent, but not both, in such experiments. These observations and others in tumor systems suggest that most or all heterozygous organisms may possess potentially self-reactive clones of lymphocytes.

Evidence from cultured leucocytes of blood cell chimerism in ex-parabiotic frogs.

Postmetamorphic diploid and triploid frogs that had earlier been joined in parabiotic union from embryonic life until metamorphosis were each found to be chimeric with respect to their blood cells, as revealed in chromosome preparations of cultured leucocytes. Blood cells precursorsmost likely were interchanged when the ex-parabionts shared a common circulation in embryonic life,and the exchanged precursor cells apparently homed in the hematopoietic tissues of the hosts. The tolerance which exparabiotic pairs of frogs exhibit toward grafts of each other's skin is attributable to the blood cell chimerism.

Can a herpes simplex virus type 1 neuroinvasive score be correlated to other risk factors in Alzheimer's disease?

Herpes simplex virus type 1 (HSV-1) is latent in the nervous system of most humans. Ball [Can J Neurol Sci 9 (1982) 303] first suggested the hypothesis that HSV-1 could be involved in the pathogenesis of Alzheimer's Disease (AD) by noting that regions of the brain particularly and earliest affected in AD were the same as those most damaged during HSV encephalitis. Data from Itzhaki's research suggests that HSV-1 in the brain and the carriage of an apolipoprotein E allele 4 (ApoE e4) together confer risk for AD [J Pathol 97 (2002) 395], [Mol Chem Neuropathol 28 (1996) 135], [Alzheimer's Rep 1 (1998) 173], [Biochem Soc Trans 26 (1998) 273]. Of the two other studies based on Itzhaki's findings, one showed similar results [Lancet 349 (1997) 1102], and the other showed a similar trend [Lancet 351 (1998) 1330], [Lancet 352 (1998) 1312]. To further examine the role of HSV-1 in the etiology of AD, we have formulated a Neuroinvasive Score that quantifies the presence and viral load of HSV-1 in eight brain regions. These regions are: entorhinal cortex, hippocampus, pons, cerebellum, and neocortex (temporal, parietal, occipital, and frontal). We hypothesize that the Neuroinvasive Score that encompasses the presence, amount, and extent of HSV-1 spreading (neuroinvasiveness), will correlate with the genetic risk factor, ApoE e4, in the assessment of autopsy samples from AD patients. If the neuroinvasive score can be directly correlated to the different stages of AD (mild, moderate, severe), this will strengthen the hypothesis that HSV-1 is involved in AD and that ApoE e4 also confers risk for the development and progression of AD.

Mitogen-stimulated lymphocytes release biologically active corticotropin.

We demonstrate that mitogen-activated lymphocytes release a biologically active form of ACTH that stimulates the in vitro release of corticosterone from cocultured rat adrenal cells. Neither nonstimulated lymphocytes nor the addition of mitogens alone to adrenal cell cultures had an effect. The steroidogenic activity could be neutralized by rabbit anti-ACTH serum, but not by a nonimmune serum. Both Concanavalin-A- and lipopolysaccharide-stimulated lymphocytes secrete an ACTH-like molecule with an antigenic specificity identical to pituitary-derived ACTH. Further, the amount of measurable immunoreactive ACTH was far lower than the amount of exogenously added ACTH required to evoke such a vigorous glucocorticoid response, suggesting that local deposition of the hormone results in a higher effective ACTH concentration. In addition, lymphocytes physically isolated from adrenal cells by a semipermeable membrane could stimulate steroidogenesis by 48 h, which corresponds to the rise in ACTH detected by RIA. These results confirm that activated lymphocytes synthesize as well as release biologically active ACTH, thus providing an in vitro model for a bidirectional communication between the endocrine and immune systems.

Immunization with HSV-1 antigen rapidly protects against HSV-1-induced encephalitis and is IFN-gamma independent.

Herpes simplex virus type 1 (HSV-1) infection of mice frequently culminates in fatal encephalitis. Intraperitoneal administration of heat-inactivated HSV-1 0-5 days before infection (active immunization) protected mice from encephalitis. In addition, active immunization 2-5 days before ocular infection with HSV-1 reduced the frequency of establishment of latent HSV-1 infection in the trigeminal ganglion (TG). However, intraperitoneal administration of heat-inactivated HSV-1 did not induce interferon (IFN) production in the peritoneum or serum, as determined by bioassay and ELISA. Intraperitoneal administration of heat-attenuated HSV-1 elicited IFN-gamma but not type I IFN production in the peritoneum. The production of IFN-gamma correlated with the infiltration of CD4 and CD8 cells in the peritoneum as determined by RT-PCR. In addition, there was a significant increase in interleukin (IL)-12 p40, IL-12p35, IL-6, IL-10, and IFN-gamma mRNA in peritoneal cells, as determined by RT-PCR following immunization with heat-attenuated HSV-1, which was not observed using heat-inactivated HSV-1. The results suggest that resistance to HSV-1 is induced rapidly following immunization with viral antigen but that protection against encephalitis is independent of the cytokines that are generated in the peritoneum.

Evidence for antigenic cross-reactivity between herpesvirus and the acetylcholine receptor.

Herpes simplex virus (HSV) is neurotropic and can pass from neuron to neuron at nerve terminals. During the long evolutionary relationship between HSV and vertebrates, this virus may have evolved surface ligands that mimic nerve cell receptors. The present study was undertaken to determine if herpes simplex virus type 1 (HSV-1) has an antigenic relationship with the acetylcholine receptor (AChR). Mice immunized with HSV-1 antigens or an AChR-expressing cell line were tested for antibodies directed against the AChR. By flow cytometry and ELISA, mouse anti-HSV-1 sera were found to contain antibodies that would bind to an epitope on the plasma membrane of AChR-expressing cells. Mice immunized with the AChR-expressing cells were tested for their resistance to HSV-1 infection. Statistically significantly more of the animals immunized with AChR-expressing cells resisted infection and fatal encephalitis, compared to control animals immunized with a cell line not expressing the AChR. Sera from AChR-immunized mice were tested for anti-HSV antibody by ELISA and were found to contain antibodies cross-reactive with HSV-1 antigens. These sera also neutralized virus in a plaque inhibition assay. The results indicate that there are one or more antigenic epitopes shared by herpesvirus and the AChR. Studies are in progress to define the pathogenetic significance of this molecular mimicry.

Cell-mediated immunity in the cornea.

An alternative model for corneal allografting termed the reverse corneal allograft reaction (RCAR) was developed in this study. Spleen cells from an alloimmunized donor were injected into the corneal stroma of the immunizing donor strain or were restimulated in mixed lymphocyte culture and then injected into the corneal stroma of the immunizing strain. The reaction began as a circular opaque site that spread and became irregularly shaped during the first 5 days after cell injection. The epithelial surface of the cornea became uneven and epithelial cell erosions were noted. Histological examination revealed that corneal stromal keratocytes at the site of inoculation had undergone degeneration and the injected cells had migrated toward the epithelial-stromal boundary, wherein a disruption of the basement membrane and disintegration of the epithelial cells occurred. Purified spleen cell subsets injected separately did not mediate the reaction. A suspension of T lymphocytes and class II antigen-positive macrophagelike cells was required to cause the RCAR. This reaction, which mimics a delayed-type hypersensitivity response, was transient, reaching a peak by day 5 and waning by day 8. This experimental model of the corneal allograft reaction shows promise for the study of cells and mediators of the corneal allograft reaction and can be employed as a reproducible system in which to test drug therapies for the treatment of corneal allograft rejection.

Cellular neuroimmunologic responses to ocular herpes simplex virus infection.

Infectious herpes simplex virus type 1 (HSV-1) was cultivated from the trigeminal ganglion between days 3 and 21 after ocular infection. T lymphocytes were first seen 9 days after infection and were present in ganglia collected 21 days after corneal infection. Neuron cell bodies expressing cytoplasmic HSV-1 antigens were present in the ganglion by 6 days after infection and could be found up to 21 days after infection although the frequency was low and decreased with time. Neuron cell bodies containing nuclear viral DNA were seen with the same frequency as cells expressing cytoplasmic viral antigens. T lymphocytes were seen surrounding neuron cell bodies some of which contained either cytoplasmic HSV-1 antigens or nuclear HSV-1 DNA.

The immunogenicity of corneal stromal keratocytes.

The purpose of this investigation was to compare the immunogenicity of corneal stromal keratocytes with that of splenic lymphocytes. The corneal stromal keratocytes of two inbred strains of rats were propagated in tissue culture in sufficient numbers to immunize allogeneic recipients and to determine the numbers of these cells required to elicit an allogenic immune response in the recipient. At least 30 x 10(6) tissue culture-propagated keratocytes injected intraperitoneally were required to elicit an alloantigenic response in the recipient strain. This alloantigenic response was measured by determining both the titer and specificity of serum alloantibody and by assessing the cellular immune response generated in the alloimmunized recipients. The results indicate that corneal stromal keratocytes can elicit both an antibody-mediated and a cell-mediated immune response, as indicated by the presence of serum antibody and cytotoxic lymphocytes in immunized recipients. The alloantibody and cellular immune responses were directed against the class I histocompatibility antigens of the immunizing strain. Class II antigens were not detected on the cultured keratocytes.

Functional role and sequence analysis of a lymphocyte orphan opioid receptor.

Pharmacological evidence indicates that lymphocytes express opioid receptors, but this finding has been questioned. By DNA sequencing of reverse transcription-polymerase chain reaction products, we have found that mouse lymphocytes express mRNA encoding an orphan opioid receptor. These mRNA transcripts were detected in the CD4+, CD8+, and CD4- CD8- lymphocyte subpopulations. Northern blot analysis confirmed that splenic lymphocytes express a 1.5-kb orphan opioid receptor mRNA. Fifteen bases encoding Tyr71-Arg75 in the first intracellular loop are alternatively spliced, suggesting that orphan opioid receptor mRNA encodes two receptor subtypes. Treatment of lipopolysaccharide-stimulated lymphocytes with orphan opioid receptor antisense oligonucleotides suppressed polyclonal IgG and IgM production by 50%. Our results provide direct evidence that lymphocytes express an opioid-like receptor gene, and suggest that this receptor plays a functional role in immunocompetence.

Central alpha-adrenergic involvement in morphine-mediated suppression of splenic natural killer activity.

Alpha 1-adrenergic pathways are involved in morphine-induced suppression of murine splenic NK activity. To investigate the level of involvement following morphine administration, the peripheral acting alpha-adrenoceptor antagonist doxazosin and the broad acting alpha-adrenoceptor antagonist phentolamine were employed. Mice preadministered phentolamine (2.0 mg/kg) exhibited a modest but insignificant suppression of splenic NK activity following morphine administration while mice preadministered doxazosin (1.0 mg/kg) or vehicle showed a significant decrease in splenic NK activity following morphine administration. Morphine was also found to significantly (P < 0.01) increase splenic serotonin levels (14.88 +/- 1.62 ng/mg) relative to saline-treated controls (7.3 +/- 0.9 ng/mg). Both phentolamine and doxazosin pretreatment completely or partially blocked morphine-mediated elevation of splenic serotonin levels, respectively. Morphine administration decreased the ability of NK cells to form conjugates with target (YAC-1 lymphoma) cells and decreased the number of active killer cells within the conjugate population. Collectively, these results implicate central alpha-adrenergic involvement following acute morphine administration in suppressing splenic NK activity indirectly through a reduction in the number of effector-target conjugates and active cytolytic effector cells.

Prolonged survival of corneal allografts incubated in alloantibody fragments.

BACKGROUND: In this study, we determined the binding characteristics of F(ab')2 alloantibody fragments to corneal antigens and assessed the capacity of these antibody fragments to protect corneal allografts from immune attack. METHODS: Goat anti-rabbit alloantibodies were pepsin-digested and labeled with 125I, and the time course of association and dissociation of the F(ab')2 fragments was determined. Corneal allografts were incubated in unlabeled F(ab')2 fragments and transplanted into allogeneic recipients, and the graft survival times were recorded. RESULTS: Binding of radiolabeled F(ab')2 fragments to rabbit cornea cells reached a maximum at 12 hr. At 32 degrees C (rabbit corneal temperature), the radiolabel eluted rapidly from the cornea, reaching baseline at 72 hr. At 4 degrees C (corneal graft storage temperature), significant amounts remained associated with the cornea at 96 hr. Mean survival time for grafts incubated in F(ab')2 anti-rabbit fragments was significantly greater than that of grafts incubated in nonimmune F(ab')2 fragments. Three of the corneal allografts incubated in goat F(ab')2 anti-rabbit fragments survived for 100 days, whereas the longest surviving control allograft incubated in goat F(ab')2 nonimmune fragments was rejected on day 24. Preincubation of corneas in unlabeled, immune F(ab')2 fragments followed by incubation in radiolabeled, immune F(ab')2 fragments suggested that antigen masking was not a factor in the prolongation of graft survival. CONCLUSION: Based on the binding and release kinetics and the graft survival times, it appears that the protective effect of immune F(ab')2 fragments extends well beyond the binding interval of the antibody fragments to corneal cell membranes.

Progressive nuclear translocation of somatostatin analogs.

UNLABELLED: Optimal cancer radiotherapy using Auger electron emitters requires selective localization of radionuclides in close proximity to tumor DNA. METHODS: Intracellular trafficking of (125)I-Tyr1-somatostatin-14 somatotropin-release inhibiting factor (SRIF) and 2 of its analogs, (125)I-WOC 4a and (111)In-pentetreotide, was studied in human neuroblastoma cells. RESULTS: After 24-h incubation, SRIF was degraded or recycled, whereas its protease-resistant analogs progressively accumulated in nuclear fractions. (111)In-pentetreotide binding to DNA increased over time in somatostatin receptor-positive cells but not in somatostatin receptor-negative cells. CONCLUSION: These in vitro studies show that prolonged exposure to radiolabeled SRIF analogs significantly increases their cellular internalization, nuclear translocation, and DNA binding. Clinically, infusion of radiolabeled somatostatin analogs may enhance tumor uptake and retention and provide more effective in situ radiotherapy.

The role of class II antigen-expressing cells in corneal allograft immunity.

The goal of this study was to investigate the relationship between the presence of Class II antigen-expressing cells (Class II+) cells in the cornea and the generation of allograft immunity. Wistar/Furth (W/F) rat Class II+ cells were injected in various numbers (0.01, 0.1, 1.0, 5.0, 10.0, and 20.0 X 10(6) into the corneal stroma of Fischer 344 (F344) rats. For comparison, the same numbers of W/F Class II+ cells were injected directly into the peritoneal cavity of F344 rats. Also, W/F cells were injected into the corneas of F344 rats and the corneas were "grafted" intraperitoneally in F344 hosts. The results showed that up to 20 X 10(6) class II+ cells injected in situ into the corneal stroma did not elicit a serum cytotoxic antibody response or a splenic or blood cytotoxic T-cell response against donor Class II antigens. In contrast, systemic immune responses were elicited by both direct intraperitoneal injection of large numbers (10 or 20 X 10(6] of allogeneic Class II+ cells and by intraperitoneal grafting of syngeneic corneas carrying similar numbers of allogeneic Class II+ cells. F344 recipients of a syngeneic cornea containing 10 or 20 x 10(6) W/F Class II+ cells or a suspension of W/F cells exhibited an accelerated rejection of W/F skin allografts (13.3 versus 9.0 days, first- versus second-set rejection). These results indicate that the number of Class II+ cells required to elicit a systemic immune response is larger than the number of Class II+ cells present in the normal cornea.

The effect of tissue dose and site of grafting on immunity to corneal allografts.

The purpose of this investigation was to determine the quantitative relationship between corneal alloantigens and host immunity. In addition, the effect of the site of introduction of the corneal antigens on the host response was determined. Two alloantigenic strains of rats were reciprocally grafted at three different locations in the body with carefully quantitated amounts of corneal tissue: (1) orthotopically in the cornea of the eye; (2) subcutaneously; and (3) intraperitoneally. Corneal tissue placed orthotopically into vascularized graft beds did not elicit a systemic immune response. Subcutaneous grafts elicited a weak systemic alloantigenic response, whereas corneal tissue placed in the peritoneal cavity consistently induced a vigorous cellular and humoral alloantigenic response. Eight or more full thickness corneal allografts grafted subcutaneously were required to elicit a systemic response. On the other hand, as few as four corneal allografts placed intraperitoneally invariably elicited a systemic alloimmune response. The results of this investigation demonstrate that both the amount and route of introduction of alloantigen affect the recipient's response to corneal tissue and that the rejection of a single orthotopic cornea graft is a site-limited response. Immune effector cells were not found in the spleens and alloantibodies were not present in the serum of animals that had rejected corneal allografts.

T lymphocytes in the trigeminal ganglia of rabbits during corneal HSV infection.

The results of this investigation reveal, for the first time, the presence of thymus-derived (T) lymphocytes in the trigeminal ganglia of rabbits undergoing primary corneal infection with herpes simplex virus type 1. Infiltration of T cells into the trigeminal ganglion was evident at 15 days after primary ocular infection but these cells were no longer present by 45 days after infection. Corneas and trigeminal ganglia of rabbits sacrificed at 3, 7, 12, 15, 20, 25, 30, 45, 70 and 90 days after infection were assayed for infectious virus and stained for viral antigen and immunoreactive T cells. Infectious virus and cells expressing viral antigens were present in the corneas and trigeminal ganglia during the acute phase (day 0-day 14) of the infection. T cell infiltration of the trigeminal ganglion was present as a perivascular infiltrate along with a sparse scattering of these cells among the nerve fibers. The perivascular infiltration is characteristic of viral infection of a tissue and was not seen in the sections of trigeminal ganglia obtained earlier than 15 days or in ganglia obtained 45 days or more after primary corneal infection. This investigation demonstrates conclusively that the neural ganglia are not completely shielded from the host immune response, as evidenced by the observation that immunocompetent T lymphocytes infiltrate the ganglia subsequent to the infection of a peripheral tissue such as the cornea of the eye.

Topically applied cyclosporine in azone prolongs corneal allograft survival.

The purpose of this study was to develop and test a topical ocular delivery system for the immunosuppressive drug cyclosporine. To this end, cyclosporine was dissolved in the penetration enhancer, Azone, and applied topically to allografted rabbit eyes. The concentration of cyclosporine in the cornea, the aqueous humor, and blood of the treated rabbits was determined by radioimmunoassay. The effect of the cyclosporine-Azone preparation on the survival of corneal allografts was assessed by clinical evaluation of the grafts and by histopathologic and immunohistologic evaluation of the cellular infiltrate in the grafts. Clinically significant concentrations of cyclosporine were measured in the treated corneas but little or no drug was found in the aqueous humor or blood of the treated animals. Cyclosporine in Azone resulted in suppression in the severity and incidence of graft rejection. The suppression of graft rejection was borne out by the immunohistologic observations. Cyclosporine-treated grafts contained significantly fewer infiltrating T lymphocytes than did the drug/solvent-treated allografts, indicating that the topical application of cyclosporine actively inhibited the entry of T cells into the grafts. This study, for the first time, presents a solvent that is apparently not toxic but is effective in delivering immunologically active concentrations of cyclosporine following topical application to the cornea.

Fate of lyophilized xenogeneic corneal lenticules in intrastromal implantation and epikeratophakia.

The antigenicity of intrastromal and epikeratophakia xenografts of lyophilized corneal tissue was evaluated in nonimmune and immune recipients. Lyophilized feline lenticules were implanted into intrastromal pockets in unsensitized rabbits and rabbits sensitized to the donor cat. In both cases, the grafts remained clear. Sensitized rabbits with clear intrastromal grafts received fresh tissue penetrating keratoplasty grafts from the same donor cat, placed adjacent to the intrastromal grafts. The fresh tissue penetrating keratoplasty grafts were rapidly rejected, while the lyophilized intrastromal grafts remained clear. Cats sensitized to rabbits received lyophilized and rehydrated epikeratophakia grafts shaped from rabbit cornea; these lyophilized grafts also remained clear for the 3-month period of the study. The results indicate that lyophilized and rehydrated corneal stroma, which is devoid of living cells, is not antigenic and is not subjected to immunologic attack, even in cases where the donor and host are of different species and the host has been previously immunized to the donor.

Propranolol suppresses reactivation of herpesvirus.

Herpes simplex virus type 1 (HSV-1) reactivates from the nervous system and causes recurrent disease in end organs such as the eye and the lips. We found that the beta-adrenergic receptor blocker, propranolol, reduces HSV-1 reactivation in an animal model. Mice latent for McKrae strain HSV-1 were injected with propranolol or saline once a day for 3 successive days, and subjected to a brief period of hyperthermia on the second day to induce reactivation. Twenty-four hours after the third injection, swabs of the ocular surface and homogenates of the corneas and trigeminal ganglia were analyzed for the presence of infectious virus and viral DNA. Treatment with propranolol significantly decreased the appearance of infectious virus in the tear film, cornea, and trigeminal ganglia (P < 0.05, chi 2-test). The results suggest a possible new pharmacologic approach to suppressing herpesvirus reactivation in the nervous system and thereby preventing recurrent disease.

9-(4-Hydroxybutyl)-N2-phenylguanine (HBPG), a thymidine kinase inhibitor, suppresses herpes virus reactivation in mice.

In cells of the nervous system, which have little or no cellular thymidine kinase, the pharmacologic inhibition of viral thymidine kinase may prevent the reactivation of herpes virus, which requires phosphorylated thymidine for replication. We tested a newly synthesized inhibitor of viral thymidine kinase, 9-(4-hydroxybutyl)-N2-phenylguanine (HBPG) for its capacity to suppress the reactivation of herpes simplex virus type 1 (HSV-1) in vivo. Mice, latently infected with McKrae strain HSV-1, were treated with intraperitoneal injections of HBPG in a corn oil vehicle (200 mg/kg every 3 h for a total of ten doses), and subjected to hyperthermic stress to stimulate viral reactivation immediately before the third treatment. Three h after the last treatment, the mice were sacrificed, and the presence of infectious virus was determined by culture of ocular surface swabs and trigeminal ganglionic homogenates. Additionally, viral DNA in ganglionic extracts was analyzed by quantitative PCR. Controls included latently infected, stressed animals receiving injections of corn oil vehicle only, and latently infected, drug- and vehicle-treated, unstressed animals. HBPG had a statistically significant inhibitory effect on hyperthermia-induced viral reactivation. Homogenates of trigeminal ganglia and ocular surface swabs from HBPG-treated animals were less likely to contain infectious virus than those of infected, vehicle-treated, stressed controls (P < 0.005, ANOVA). Unstressed controls showed no reactivation. Quantitation of viral DNA in ganglionic extracts demonstrated a 100-fold reduction in the amount of viral DNA in the ganglia of HBPG-treated animals, compared with vehicle-treated controls (P < 0.05, ANOVA). The results indicate that HBPG has an inhibitory effect when given systemically for the suppression of herpes virus reactivation in mice.