Synthesis and biological assessment of 3,7-dihydroxytropolones.

3,7-Dihydroxytropolones (3,7-dHTs) are highly oxygenated troponoids that have been identified as lead compounds for several human diseases. To date, structure-function studies on these molecules have been limited due to a scarcity of synthetic methods for their preparation. New synthetic strategies towards structurally novel 3,7-dHTs would be valuable in further studying their therapeutic potential. Here we describe the successful adaptation of a [5 + 2] oxidopyrilium cycloaddition/ring-opening for 3,7-dHT synthesis, which we apply in the synthesis of a plausible biosynthetic intermediate to the natural products puberulic and puberulonic acid. We have also tested these new compounds in several biological assays related to human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV) in order to gain insight into structure-functional analysis related to antiviral troponoid development.

Expression of specific cytolytic activity by H-2I region-restricted, influenza virus-specific T lymphocyte clones.

Among murine class II major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) clones specific for type A influenza virus, we have identified both noncytolytic clones and clones exhibiting H-2 I region-restricted cytolytic activity. After appropriate antigenic stimulation, both cytolytic and noncytolytic clones proliferated in the absence of exogenous interleukin 2. All of the clones possess the Thy-1.2+, Lyt-1+2-, L3T4+ phenotype. The class II MHC restriction of viral recognition by the CTL clones was mapped by proliferation using recombinant mouse strains and by inhibition of cytotoxic activity with monoclonal antibodies directed to class II MHC products and L3T4a. The restriction specificity of two CTL clones was unambiguously assigned to the E beta d chain by using L cell transfectant lines expressing E alpha kE beta d or E alpha kE beta k gene products. Analysis of the viral specificity of the cloned lines revealed subtype-specific and crossreactive patterns of viral antigen recognition; the pattern of viral antigen specificity exhibited by each clone in proliferation and cell-mediated cytotoxicity was identical. Each CTL clone also demonstrated antigen-dependent release of helper factor(s) that promoted in vitro primary anti-SRBC responses. Finally, the cytotoxic effector function of the class II MHC-restricted CTL clones was mediated by direct lysis of virus-infected cells, and not by secretion of a cytolytic lymphokine.

Differences in antigen presentation to MHC class I-and class II-restricted influenza virus-specific cytolytic T lymphocyte clones.

We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA-specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II-restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets.

Increased number of white matter lesions in patients with familial cerebral cavernous malformations.

BACKGROUND AND PURPOSE: Familial cerebral cavernous malformations, an autosomal dominant disorder, result in excess morbidity and mortality in affected patients. The disorder is most prevalent in the Southwest United States, where the affected families are most often carriers of the CCM1-KRIT1 Common Hispanic Mutation. The brain and spinal cord parenchyma in these individuals is usually affected by multiple cavernous malformations. Previous studies have shown abnormalities of endothelial cell junctions and the blood-brain barrier in cerebral cavernous malformations. Endothelial cell abnormalities have also been described in pathologic studies of white matter hyperintensities. We compared the prevalence of white matter hyperintensities in a population with known familial cerebral cavernous malformations. MATERIALS AND METHODS: We examined 191 subjects with familial cerebral cavernous malformations who were enrolled into an institutional review board-approved study. All carry the same Common Hispanic Mutation in the CCM1 gene. Each subject underwent 3T MR imaging, including gradient recalled-echo, SWI, and FLAIR sequences. The number of cavernous malformations and the number of nonhemorrhagic white matter hyperintensities were counted. Subjects older than 60 years of age were excluded due to the high prevalence of white matter lesions in this population, and children younger than 6 were excluded due to potential sedation requirements. Logistic regression analysis was performed to determine the prevalence of abnormal white matter hyperintensities in those with familial cerebral cavernous malformations compared with healthy controls or those with sporadic cerebral cavernous malformation within the familial cerebral cavernous malformations group; it was also performed to evaluate the associations between abnormal white matter hyperintensities and age, sex, headaches, thyroid disease, diabetes, hypertension, hyperlipidemia, seizure history, or modified Rankin Scale score. RESULTS: Familial CCM1 carriers have a higher prevalence of abnormal white matter hyperintensities (15.4%) compared with both control populations (2.1% and 2.5%, respectively) (P < .05). Logistic regression showed no statistical association with sex, headaches, hyperlipidemia, hypertension, thyroid disease, seizure history, number of cerebral cavernous malformations, or modified Rankin Scale score among those with familial cerebral cavernous malformation. An expected correlation with age was shown. CONCLUSIONS: Familial CCM1 carriers have not only an increased number of cerebral cavernous malformations but also an increased number of white matter T2 hyperintensities, spatially distinct from cerebral cavernous malformations, which exceeded that of a healthy population. Clinical findings did not explain the association with abnormal white matter hyperintensities in the familial cerebral cavernous malformation population. To our knowledge, these relationships have not been previously reported. This finding suggests an additional manifestation of endothelial abnormalities in this population.

Spatial and temporal patterns of transneuronal labeling in CNS neurons after injection of pseudorabies virus into the sciatic nerve of adult rats.

The distribution of labeled neurons in the brain and spinal cord was studied after injecting the Bartha strain of pseudorabies virus (PRV) into the sciatic nerve to provide a baseline for studying neural circuitry after spinal cord injury (SCI) and regeneration. Following a single injection of viral particles into the left sciatic nerve, PRV labeling was found in the spinal cord at 2 days post-injection (p.i.). Increasing complexity in viral labeling from the spinal cord to supraspinal regions became apparent with increasing survival time. In brain regions, several neuronal groups that regulate sympathetic outflow, such as the rostroventrolateral medulla, the lateral paragigantocellular nuclei, and the A5 cells, were densely labeled. However, relatively sparse labeling was noticed in the lateral vestibular nuclei, the red nucleus and the motor cortex whose spinal projections regulate somatic motor function, although those areas were abundantly labeled with Fast blue (FB) in a double-labeling experiment in which FB was co-injected into the lumbar cord. The pattern of viral labeling became more complex beyond 5 days p.i. when increased numbers of cell groups were labeled with PRV but not FB. In addition, some infected neurons started to lyse, as evidenced by a decrease in viral labeling at 7 days p.i. Thus, the 5th day post-viral injection would appear to be an appropriate survival time to obtain maximal labeling with acceptable specificity. We suggest that transneuronal labeling using PRV should be appropriate for studying multi-neural circuitry after SCI and regeneration.

Expectancy and outcome in prescriptive vs. exploratory psychotherapy.

Client ratings of the credibility of prescriptive and exploratory therapies were compared, and correlated with clinical improvement. Both expectancies and outcomes favoured prescriptive therapy in the first, but not the second, period of a cross-over design, and modest correlations were obtained between credibility and improvement. Regression analysis suggested that the impact of expectancy on outcome was secondary to the treatment effect rather than its cause.

Familial versus sporadic cavernous malformations: differences in developmental venous anomaly association and lesion phenotype.

BACKGROUND AND PURPOSE: CCMs are commonly associated with DVAs, but the incidence of association in familial CCM is unknown. The presence of a DVA significantly complicates surgical management of a CCM because of the risk of compromised venous drainage. In this investigation, we compared the incidence of a DVA in the presence of a CCM in sporadic and familial CCM cases comprising predominantly familial CCM with the Southwestern US common Hispanic mutation (or Q455X mutation) of CCM1. MATERIALS AND METHODS: Retrospective review was performed of 112 patients identified with CCM. MR imaging review included the presence or absence of a DVA and number, location, size, and signal-intensity characteristics of CCMs. Record review included patient and family history and documented genetic mutations. Statistical analysis was performed by using the Fisher exact and 2-sample t tests. RESULTS: Eighty-one cases were familial, 18 were sporadic, and 13 were indeterminate. There were a total of 2212 CCMs: 2176, 21, and 15 in the familial, sporadic, and indeterminate groups, respectively. There was a close association of CCM and DVA (an apparent combined vascular lesion) in 8 of 18 (44%) sporadic cases and only 1 possible such association in the familial cases. The difference was highly statistically significant (P < .0001). CONCLUSIONS: Familial CCMs are unlikely to be associated with DVAs, and sporadic CCMs have a high rate of association with DVA. This difference in imaging features of familial and sporadic CCMs suggests the possibility of a different developmental mechanism.

Practice parameter: pharmacologic treatment of spasticity in children and adolescents with cerebral palsy (an evidence-based review): report of the Quality Standards Subcommittee of the American Academy of Neurology and the Practice Committee of the Child Neurology Society.

OBJECTIVE: To evaluate published evidence of efficacy and safety of pharmacologic treatments for childhood spasticity due to cerebral palsy. METHODS: A multidisciplinary panel systematically reviewed relevant literature from 1966 to July 2008. RESULTS: For localized/segmental spasticity, botulinum toxin type A is established as an effective treatment to reduce spasticity in the upper and lower extremities. There is conflicting evidence regarding functional improvement. Botulinum toxin type A was found to be generally safe in children with cerebral palsy; however, the Food and Drug Administration is presently investigating isolated cases of generalized weakness resulting in poor outcomes. No studies that met criteria are available on the use of phenol, alcohol, or botulinum toxin type B injections. For generalized spasticity, diazepam is probably effective in reducing spasticity, but there are insufficient data on its effect on motor function and its side-effect profile. Tizanidine is possibly effective, but there are insufficient data on its effect on function and its side-effect profile. There were insufficient data on the use of dantrolene, oral baclofen, and intrathecal baclofen, and toxicity was frequently reported. RECOMMENDATIONS: For localized/segmental spasticity that warrants treatment, botulinum toxin type A should be offered as an effective and generally safe treatment (Level A). There are insufficient data to support or refute the use of phenol, alcohol, or botulinum toxin type B (Level U). For generalized spasticity that warrants treatment, diazepam should be considered for short-term treatment, with caution regarding toxicity (Level B), and tizanidine may be considered (Level C). There are insufficient data to support or refute use of dantrolene, oral baclofen, or continuous intrathecal baclofen (Level U).

Contributions of antibody and T cell subsets to protection elicited by immunization with a replication-defective mutant of herpes simplex virus type 1.

Replication-defective mutants of herpes simplex virus 1 (HSV-1) elicit immune responses in mice that reduce acute and latent infection after corneal challenge and are protective against development of disease. To understand the basis for the protective immunity induced by this new form of immunization, we investigated the contribution of various components of the immune response to protection against corneal infection and disease. Passive transfer of sera from mice immunized with the replication-defective mutant virus, d301, its parental HSV-1 strain, or uninfected cell lysate was used to examine the role of antibody. Despite posttransfer neutralizing antibody titers equivalent to those in control mice directly immunized with mutant virus, recipients of immune serum showed no reductions in primary replication in the eye, keratitis, or latent infection of the nervous system. However, immune serum protected mice from encephalitis and death. To examine the contribution of T cell subsets to protection, mice were immunized once with mutant virus and then were depleted in vivo of CD4+ or CD8+ T cells prior to corneal challenge. CD4 depletion resulted in higher titers of challenge virus in the eye at 3 to 4 days after challenge compared to control mice. Latent infection of the nervous system was increased by depletion of CD4+ T cells but not by depletion of CD8+ T cells keratitis developed only in a portion of the CD8+ T cell-depleted mice, suggesting that an immunopathologic potential of CD4+ T cells is held in check when immune CD8+ T cells are also present. Taken together, these data support a role for antibody induced by immunization with a replication-defective virus principally in protecting the central nervous system from disease, roles for CD4+ T cells in reducing primary replication in the eye and protecting against latent infection of the nervous system, and a role for CD8+ T cells in regulating the immunopathologic activity of CD4+ T cells.

Mechanisms of immunization with a replication-defective mutant of herpes simplex virus 1.

We have investigated the mechanisms by which subcutaneous immunization of mice with a replication-defective mutant of herpes simplex virus 1 protects against infection of the eye and latent infection of the trigeminal ganglion following corneal challenge. First, we have shown that immunization reduces the number of trigeminal ganglion neurons in challenged animals that express the latency-associated transcript. This indicates that the reduction in the incidence of latent infection by challenge virus is likely due to immune mechanisms and not saturation of the potential sites of latent infection by the immunizing mutant virus itself. Second, the duration of protective immunity against acute infection, keratitis, and latent infection was similar in mice immunized with replication-defective or -competent virus; thus, the replication-defective mutant virus is able to induce durable immunity apparently without spread in the host. Third, although the mutant virus showed no evidence of replication in vivo, it was present in footpad tissue in an infectious form for several days. This surprising observation raises the possibility that continued infection events by input virus over an extended period of time may have a boosting effect on the developing immune response which could explain, at least in part, the capacity of these replication-defective mutant viruses to elicit a robust and durable immunity despite their inability to spread within the host.

Immunization with replication-defective mutants of herpes simplex virus type 1: sites of immune intervention in pathogenesis of challenge virus infection.

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) were used as a new means to immunize mice against HSV-1-mediated ocular infection and disease. The effects of the induced immune responses on pathogenesis of acute and latent infection by challenge virus were investigated after corneal inoculation of immunized mice with virulent HSV-1. A single subcutaneous injection of replication-defective mutant virus protected mice against development of encephalitis and keratitis. Replication of the challenge virus at the initial site of infection was lower in mice immunized with attenuated, wild-type parental virus (KOS1.1) or replication-defective mutant virus than in mice immunized with uninfected cell extract or UV-inactivated wild-type virus. Significantly, latent infection in the trigeminal ganglia was reduced in mice given one immunization with replication-defective mutant virus and was completely prevented by two immunizations. Acute replication in the trigeminal ganglia was also prevented in mice immunized twice with wild-type or mutant virus. The level of protection against infection and disease generated by immunization with replication-defective mutant viruses was comparable to that of infectious wild-type virus in all cases. In addition, T-cell proliferative and neutralizing antibody responses following immunization and corneal challenge were of similar strength in mice immunized with replication-defective mutant viruses or with wild-type virus. Thus, protein expression by forms of HSV-1 capable of only partially completing the replication cycle can induce an immune response in mice that efficiently decreases primary replication of virulent challenge virus, interferes with acute and latent infection of the nervous system, and inhibits the development of both keratitis and systemic neurologic disease.

Quantities of B6 vitamers in human milk by high-performance liquid chromatography. Influence of maternal vitamin B6 status.

A rapid, sensitive procedure is described for the analysis of the B6 vitamers pyridoxal, pyridoxamine, and pyridoxine in human milk from women taking and not taking supplements containing the vitamin using high-performance liquid chromatography with fluorometric detection. Vitamer values represent the sum of their phosphorylated and unphosphorylated forms. Minimum detectable quantities were 1-3 ng. Excellent recoveries of these vitamers in milk were obtained. Similar B6 vitamer concentrations of milk were obtained using the developed high-performance liquid chromatographic and the accepted microbiological techniques. Pyridoxal, actually consisting of pyridoxal plus pyridoxal phosphate, was the predominant B6 vitamer in human milk. The concentration of B6 vitamers in milk was reflective of the maternal vitamin B6 status.

Expression of H-2I region-restricted cytolytic activity by an Lyt-2+ influenza virus-specific T lymphocyte clone.

The expression of Lyt-2 on T lymphocytes has been postulated to correlate closely with restriction by, or alloreactivity to, class I MHC gene products, whereas I region-restricted or alloreactive populations appear to be associated with Lyt-1 and L3T4 expression. However, exceptions to this axiom among alloreactive T cells have been shown to exist. In this report we describe a clonal population of influenza virus-specific T lymphocytes that bears the Lyt-2+, L3T4- phenotype. Notably, this clone is restricted in influenza virus recognition by class II MHC molecules and is cytolytic for virus-infected target cells expressing the appropriate class II molecules. Antibody directed to the Lyt-2 molecule does not inhibit cytolysis.

Antigen form influences induction and frequency of influenza-specific class I and class II MHC-restricted cytolytic T lymphocytes.

The induction of class I and class II MHC-restricted CTL in response to different forms of A/JAP/57 influenza virus was compared. Splenocytes removed from influenza-immune BALB/c mice and stimulated in vitro with infected syngeneic splenocytes are mainly CD8+ (Lyt-2+) and specifically lyse infected Ia- and Ia+ target cells. To a lesser extent they also lyse non-infectious virus-pulsed Ia+ but not Ia- target cells. In contrast, syngeneic stimulators pulsed with non-infectious virus (exogenous Ag) induce effector T cells that specifically lyse both infected and non-infectious virus-pulsed Ia+ target cells. The cells present in this heterogeneous culture predominantly express the CD4 (L3T4) cell surface marker. Frequency analysis by limiting dilution of splenocytes derived directly from influenza-immune mice revealed a similar pattern of precursor induction: In vitro stimulation with infected splenocytes yielded primarily class I MHC-restricted CTL, whereas stimulation with non-infectious virus reciprocally induced primarily class II MHC-restricted CTL. Thus, the Ag form and consequently the intracellular route of viral Ag presentation profoundly influence the MHC restriction of CTL precursors induced.

Association of the reovirus S1 gene with serotype 3-induced biliary atresia in mice.

A panel of serotype 3 (T3) reovirus strains was screened to determine their relative capacities to cause lethal infection and hepatobiliary disease following peroral inoculation in newborn mice. A wide range of 50% lethal doses (LD50s) was apparent after peroral inoculation of the different virus strains. Two of the strains, T3 Abney and T3 clone 31, caused mice to develop the oily fur syndrome associated with biliary atresia. The capacity to cause biliary atresia was not related to the capacity to cause lethal infection, however, because the LD50s of T3 Abney and T3 clone 31 were grossly disparate. Examination of liver and bile duct tissues revealed histopathologic evidence of biliary atresia and hepatic necrosis in T3 Abney-infected mice but not in mice inoculated with a T3 strain of similar virulence or with the hepatotropic T1 Lang strain. The consistency with which T3 Abney-infected mice developed biliary atresia-associated oily fur syndrome permitted us to determine the viral genetic basis of reovirus-induced biliary atresia. Analysis of reassortant viruses isolated from an in vitro coinfection with T3 Abney and T1 Lang indicated a strong association of the hepatobiliary disease-producing phenotype with the T3 Abney S1 gene, which encodes the viral cell attachment protein, sigma 1. Amino acid residues within the sigma 1 protein that were unique to disease-producing T3 strains were identified by comparative sequence analysis. Specific changes exist within two regions of the protein, one of which is thought to be involved in binding to host cell receptors. We hypothesize that changes within this region of the protein are important in determining the tropism of this virus for bile-ductular epithelium.

Direct spread of reovirus from the intestinal lumen to the central nervous system through vagal autonomic nerve fibers.

A crucial event in the pathogenesis of systemic enteric virus infections is entry of virus into the nervous system. Whether enteric virus spreads from the intestinal tract to the central nervous system through nerves or through the bloodstream was examined using a serotype 3 reovirus strain. After peroral inoculation of newborn mice with reovirus, serial histologic sections of small intestine, brain and spinal cord were prepared and stained by immunoperoxidase to detect viral antigen. Three days after inoculation, viral antigen was observed in mononuclear cells of ileal Peyer's patches and in neurons of the adjacent myenteric plexus. Infection first appeared in the central nervous system 1-2 days later in neurons of the dorsal motor nucleus of the vagus nerve. Endothelial cells, meninges, choroid plexus, hypothalamus, and area postrema were not infected, indicating neural rather than bloodborne spread from the intestine. Staining of neurons in the dorsal motor nucleus of the vagus nerve depended on the route of virus inoculation and was independent of the amount of virus in the bloodstream. These results demonstrate that an enteric virus entering a host from the intestinal lumen can spread to the central nervous system through nerve fiber innervating the intestine.

Vaccine-induced serum immunoglobin contributes to protection from herpes simplex virus type 2 genital infection in the presence of immune T cells.

Herpes simplex type virus 2 (HSV-2) is a sexually transmitted pathogen that causes genital lesions and spreads to the nervous system to establish acute and latent infections. Systemic but not mucosal cellular and humoral immune responses are elicited by immunization of mice with a replication-defective mutant of HSV-2, yet the mice are protected against disease caused by subsequent challenge of the genital mucosa with virulent HSV-2. In this study, we investigated the role of immune serum antibody generated by immunization with a replication-defective HSV-2 vaccine prototype strain in protection of the genital mucosa and the nervous system from HSV-2 infection. Passive transfer of replication-defective virus-immune serum at physiologic concentrations to SCID or B-cell-deficient mice had no effect on replication of challenge virus in the genital mucosa but did significantly reduce the incidence and severity of genital and neurologic disease. In contrast, B-cell-deficient mice immunized with replication-defective HSV-2 were able to control replication of challenge virus in the genital mucosa, but not until 3 days postchallenge, and were not completely protected against genital and neurologic disease. Passive transfer of physiologic amounts of immune serum to immunized, B-cell-deficient mice completely restored their capacity to limit replication of challenge virus in the genital mucosa and prevented signs of genital and systemic disease. In addition, the numbers of viral genomes in the lumbosacral dorsal root ganglia of immunized, B-cell-deficient mice were dramatically reduced by transfer of immune serum prior to challenge. These results suggest that there is an apparent synergism between immune serum antibody and immune T cells in achieving protection and that serum antibody induced by vaccination with replication-defective virus aids in reducing establishment of latent infection after genital infection with HSV-2.

Prolonged replication in the mouse central nervous system of reoviruses isolated from persistently infected cell cultures.

We examined pathogenic characteristics of plaque-purified reoviruses isolated from persistently infected L-cell cultures (PI viruses) after intracranial inoculation into newborn mice. The PI viruses were isolated from independent cultures initiated with high-passage stocks of the wild-type (wt) strain, type 3 Dearing. The virulence of most PI viruses was equivalent to that of the wt strain. However, replication of PI viruses in the central nervous system of infected mice was prolonged to 25 (but not 50) days postinoculation. Thirty-eight percent (n = 186) of mice inoculated with the PI viruses had residual virus detectable in brain tissue 25 days after inoculation, in contrast to only 16% (n = 57) of mice inoculated with wt virus (P = 0.009). Mean residual brain titers were more than 20-fold higher in mice inoculated with PI viruses compared with wt virus (4.3 x 10(4) versus 2.1 x 10(3); P = 0.006). Tropism of PI virus within the brain resembled that of wt virus, and the distribution of PI virus antigen in the brain did not change over time. The extent of necrosis in the brains of mice harboring PI virus 25 days after inoculation was minimal, despite continued presence of high titers of infectious virus. The latter observation resembles the absence of cytopathicity seen in L-cell cultures persistently infected with reovirus. These observations suggest that the interaction of PI viruses with cells can be altered in vivo as well as in cell culture, but virus is eventually cleared from the infected animal.

Recognition of pre-processed endogenous antigen by class I but not class II MHC-restricted T cells.

Class I and class II MHC-restricted T lymphocytes recognize non-native forms of antigen. The presentation of antigen to these two classes of T lymphocytes can occur through distinct pathways. Several mechanisms, including differences in antigen processing in different intracellular compartments, have been proposed to account for these pathway differences. Here we describe a T-cell epitope located on the influenza virus haemaglutinin, which is recognized by both class I and class II MHC-restricted cytolytic T lymphocytes (CTL). When expressed de novo in target cells, from a synthetic minigene encoding only the epitope, this pre-processed antigenic site is recognized by class I but not class II MHC-restricted T lymphocytes, even though target cells treated with the exogenously introduced peptide can be recognized by both classes of T cells. Because endogenous expression of the pre-processed antigenic fragment results in differential presentation to class I and class II MHC-restricted CTL, differences between the two different pathways of presentation could lie not at the level of processing but at the level of targeting and/or interaction of processed antigen with MHC.