Mutation analysis of PVRL1 in patients with non-syndromic cleft of the lip and/or palate in Guangdong.

Non-syndromic cleft of the lip and/or palate (NSCLP) is a very common birth defect; the poliovirus receptor-like 1 gene (PVRL1) has been identified as a genetic risk factor for NSCLP in patients from Norway, the Philippines, and South America. Given the considerable variation in allele frequencies across these geographical regions, this study explored the relationship between NSCLP and mutations of PVRL1 in patients from Guangdong, China. We recruited 171 NSCLP patients and 100 volunteers, and divided our samples into 2 groups: a sequencing group and a mass spectrometry group. In the sequencing group, we screened for mutations in exons 2 and 5 of PVRL1 by polymerase chain reaction and direct sequencing in 71 NSCLP patients and 100 volunteers. In the mass spectrometry group, we screened for amino acid mutations in α-spliced transcript codons 112, 131, and 395, and in the ß-spliced transcript codon 1082 using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis in 100 NSCLP patients and 100 volunteers. No mutations were detected in either PVRL1 exons 2 or 5 in the 71 NSCLP patients and 100 volunteers, nor did we find mutations of α-spliced transcript codons 112, 131, 395 and the ß-spliced transcript codon 1082 in any of the 100 NSCLP patients and 100 volunteers. Thus, mutations in exons 2 and 5 of PVRL1, and T334A, A391T, G1183A in the α-spliced transcript, and G1082T in the ß-spliced transcript do not participate in the development of NSCLP in patients from Guangdong.

A new area in the human brain associated with learning and memory: immunohistochemical and functional MRI analysis.

Previous studies identified a new brain area, the marginal division (MrD), at the caudomedial border of the neostriatum in the brain of the rat, cat and monkey. The MrD was distinguishable from the rest of the striatum by the presence of spindle-shaped neurons, specific connections, and dense immunoreactivity for neuropeptides and monoamines in fibers, terminals and neuronal somata. Behavioral testing demonstrated that the MrD contributes to learning and memory in the rat. In the present study, the structure and the function of the MrD were investigated in the human brain. The presence of spindle-shaped neurons and the distribution of neurotransmitters in the MrD were evaluated by immunocytochemical methods. The function of the MrD was identified with functional magnetic resonance imaging (fMRI) of healthy volunteers tested with an auditory digital working memory task. Highly active areas were observed in the prefrontal cortex and MrD with left sided predominance during performance of the task, but other parts of the neostriatum were not excited and the MrD was not activated in a control test of non-working memory. The results of the present investigation therefore indicate the existence of a new area associated with learning and memory function in the human brain. The MrD probably plays an important role in the execution of digital working memory and appears to link the limbic system and the basal nucleus of Meynert. The MrD may also be involved in the mechanism of Alzheimer's disease.

A new subdivision, marginal division, in the neostriatum of the monkey brain.

A new subdivision, the "marginal division" (MrD), was discovered at the caudal border of the striatum and surrounds the rostral edge of the globus pallidus in the rat brain in our previous studies. The neuronal somata of the MrD are mostly fusiform in shape with their long axes lining dorsoventrally. The MrD is more densely filled with substance P (SP)-, Leucine-enkephalin (L-Enk)-, dynorphin B-, neurotensin-, somatostatin- and cholecystokinin (CCK)-immunoreactive fibers and terminal-like structures than the rest of the striatum. The MrD was confirmed in the cat neostriatum as well. The present study intended to explore whether the MrD exists in the monkey neostriatum (putamen) with Nissl, histochemical and immunohistochemical methods. A band of fusiform neurons were obviously identified at the caudomedial edge of the putamen. These neurons lie outside the lateral medullary lamina and indirectly surround the rostrolateral border of the globus pallidus. The abundance of SP-, L-Enk-, neuropeptide Y-, CCK-, dopamine- and serotonin-positive fibers and terminal-like structures with a few positive fusiform neurons accumulating at the caudomedial border of the putamen obviously distinguishes this zone from the rest of neostriatum and globus pallidus. The acetylcholinesterase (AChE) positive and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) containing fusiform neurons are distinctly visualized in the same zone. The morphological figure and the location of these neurons, and the histochemical and immunohistochemical characteristics of this area coincide well with those of the MrD in the rat and cat striatum. This study thus convincingly identifies the existence of the MrD in the monkey neostriatum. It is fairly asserted that the MrD is a universal structure in the mammalian brain.

Differential expression of S100B and S100A6(1) in the human fetal and aged cerebral cortex.

S100B and S100A6 (calcylin) are two members of the S100 Ca(2+)-binding protein family and have been localized in the mammalian nervous system. However, information on their distribution in the human nervous system, especially in the developing human fetal brain, is scarce. In the present study, an immunocytochemical method was used to examine the spatio-temporal protein expression patterns of S100B and S100A6 in normal human fetal hippocampus, entorhinal cortex and occipital cortex. Normal aged adult human brain specimens were also included for comparison. From week 15 onwards, an increase with advancing gestation age in both the number and staining intensity of S100B positive, astrocyte-like cells was found in the pyramidal layer of the hippocampus, while both the molecular and polymorphic layers showed similar S100B immunoreactivities at all stages examined. A decrease in the immunoreactivities was found in the molecular layer of the aged adult hippocampus while other layers exhibited immunoreactivities similar to those of the late fetus. At week 15, the molecular, pyramidal and ganglionic/multiform layers of the entorhinal cortex also showed positive S100B immunoreactivities which were maintained throughout the rest of the gestation and in adult specimens. In the occipital cortex, the numbers of positive cells for all layers were about twofold higher than those found in the hippocampus and entorhinal cortex, and immunoreactivities detected in the granular layer increased from week 21, reaching a plateau at around week 27. S100B positive fibers were also found at week 30 but were not observed in aged adult specimens. S100A6 positive cells were on the whole fewer in number than those of S100B in the brain regions examined. The S100A6 immunoreactivities which were localized in some pyramidal neuron-like and some glial-like cells of the pyramidal and molecular layers of the hippocampus increased by midgestation and became weak in the late fetus and in aged adult specimens. Weakly stained S100A6 positive cells were also observed in the entorhinal cortex throughout the gestation and in aged adult cortex. S100A6 immunoreactivities were weak in the fetal occipital cortex. They were also localized in the glial-like cells of the aged adult occipital cortex. The differential spatio-temporal expression of S100B and S100A6 proteins suggests that the proteins play different roles in different brain regions during development and in adulthood.

Properties of acetylcholine receptor ion channels in the acutely dissociated neurons of the marginal division in the rat striatum.

Cell-attached mode of patch clamp technique was employed to investigate the properties of acetylcholine (ACh)-induced ion channels in acutely dissociated neurons from the marginal division (MrD) of rat striatum. Two types of conductance states (25 pS and 60 pS) were recorded. The 25 pS channel (more than 80%) was the main type in the neurons of MrD and was described here. The amplitudes of inward currents increased with hyperpolorization and the reversing potential was about 0 mV. Both single short opening and long burst openings were observed in MrD neurons. Two-time constants of these two kinds of ion channels are 0.29 ms, 1.84 ms and 1.96 ms, 18.24 ms, respectively. Average close time can be fitted with two exponential functions, the two time constants are 1.7 ms and 54 ms. Probability of channel opening is about 0.012 and no voltage-dependence was found. The properties of reversing potential, voltage-independence and the form of agonist to the ion channels indicated that the recorded channel currents flow through AChR channels. The mAChR is involved in slow synaptic transmission and Ach can not induce the opening of mAChR ion channel. The binding site of ACh to AChR and the nAChR ion channel are the same protein, ACh can only activate nAChR ion channel directly. Therefore, the recorded ion channels in the present study are nAChR ion channels. The results suggest that nAChR ion channels exist in the neurons of MrD and the MrD probably is involved in learning and memory mechanism of the brain.

A new subdivision of mammalian neostriatum with functional implications to learning and memory.

A new subdivision with distinctive morphological and functional attributes has been identified at the caudomedial margin of the neostriatum and surrounding the rostrolateral border of the globus pallidus in the brains of the rat, cat, monkey, and human. The subdivision is termed marginal division (MrD) based on its location. It is readily distinguishable from the rest of striatum by consisting of spindle-shaped neurons, special connections, and intensely expressed immunoreactivities of many neuropeptides and some monoamines in the fibers, terminals, and neuronal somata. Three-dimensional reconstruction from Nissl-stained sections of the rat brain revealed that the MrD is a flat, pan-shaped zone between the neostriatum and globus pallidus. Functional neuronal connections were delineated by chemical-induced c-Fos expression between the MrD and hippocampus, amygdala, as well as the basal nucleus of Meynert. In rats with chemical lesions of bilateral MrD, learning and memory functions were severely impaired as demonstrated by double blind Y-maze test. Our results thus suggested that the MrD is a distinct part and a universal structure in the neostriatum of the mammalian brain and might play an important role in the mechanism of learning and memory.

[Experimental study on effect of Brucea javanica oil emulsion on rabbit intracranial pressure].

Using the method of intubation into lateral cerebral ventricle, the effect of Brucea javanica oil emulsion (BJOE) venous emulsion and oral emulsion on rabbits with normal and intracranial hypertension respectively were observed, to study whether BJOE could reduce intracranial pressure or not. The results shown that venous emulsion of BJOE had strong action against the elevation of intracranial pressure produced by SNP (P < 0.01) while oral emulsion had mild action against it, which was similar to the clinical observation exhibiting improvement of clinical manifestations after application of BJOE on intracranial hypertension caused by brain metastasis from lung cancer.

Seroepidemiologic study of Cryptosporidium infection in children from rural communities of Anhui, China and Fortaleza, Brazil.

A cluster-sampling, cross-sectional study was conducted for assessing the prevalence of Cryptosporidium infection in children less than 16 years of age from three villages, Dondian, Linshan, and Fuziyin, in rural Anhui in eastern China. Among 320 apparently healthy children less than 10 years of age from Dondian who had stool specimens collected, cryptosporidial oocysts were found in stools of three children from Dondian, and no positive specimens were found in 239 children studied from Linshan. In addition, a total of 610 serum samples from children in these three villages were tested for specific IgG antibody to Cryptosporidium with an enzyme-linked immunosorbent assay (ELISA) and the prevalence rates were 42.3%, 51.7%, and 57.5%, respectively, in Dondian, Linshan, and Fuziyin. Seroprevalence increased progressively with age. No detectable antibody was found in infants between two and six months of age, and seropositivity steadily increased after one year of age. Among 36 sera from adults 15-60 years of age without diarrheal illness in Huanglu villages of rural Chaohu, 50% (18 of 36) were positive. As expected, a good correlation was found in the specific IgG antibody between the paired serum specimens from 30 matched mother-neonates who showed transplacental transfer of IgG. However, little or no IgM antibody was seen in the neonates even though several mothers had a positive anticryptosporidial IgM enzyme-linked immunoassay result. Forty randomly selected serum samples from children less than four years of age in a similarly impoverished semiurban community in Fortaleza, Brazil, where the majority of households also have pit toilets and shared community water supplies and 172 serum samples from patients one month to 29 years of age admitted to the University of Virginia Hospital without diarrhea were also examined. In Fortaleza, almost all children acquired antibody by their second year of life, demonstrating the high prevalence of this infection. In rural Anhui, only about half the children were infected by 5-7 years of age. The overall prevalence rate (16.9%) of seropositivity among children and young adults in Virginia was much lower than in China and Brazil. These results indicate that cryptosporidial infection is ubiquitous, and is highly endemic in these impoverished communities. The difference between China and Brazil may reflect earlier weaning, hygiene practices, poorer water or sanitation, multiple siblings in family and geographic environment in Brazil.

Cellular interactions in effector cell generation and tumor regression mediated by anti-CD3/interleukin 2-activated tumor-draining lymph node cells.

Previous studies have demonstrated that progressive growth of the weakly immunogenic MCA 106 murine sarcoma stimulated, in the draining lymph nodes, the production of tumor-sensitized but not fully functional preeffector lymphocytes. These lymphocytes could develop into specific immune effector cells after sequential in vitro activation with anti-CD3 monoclonal antibody and interleukin 2 (IL-2). In this study, we analyzed cellular requirements for in vivo sensitization of preeffector cells, for generation of immune effector cells by the method of anti-CD3/IL-2 activation, and for adoptive immunotherapy mediated by activated cells. By selective depletion of T-cell subsets in vivo, we found that tumor regression after systemic adoptive immunotherapy required the collaboration of activated CD4+ and CD8+ cells. It was further demonstrated that CD8+ immune cells alone could mediate antitumor effects if exogenous IL-2 was provided in vivo. These results suggest that CD8+ cells served as immediate effector cells, whereas CD4+ immune cells provided a helper function via the secretion of IL-2. During in vitro anti-CD3/IL-2 activation, generation of effector cells depended on the collaborative interaction between previously sensitized CD4+ and CD8+ preeffector cells. At the stage of in vitro activation, the addition of IL-2 could not substitute the function of CD4+ cells. We next examined whether the sensitization of preeffector cells in the draining lymph nodes required cellular interactions between CD4+ and CD8+ T-cells. By in vivo depletion of T-cell subsets during tumor growth, we found that CD4+ cells were sensitized independently of CD8+ cells. More interestingly, in vivo sensitization of CD8+ preeffector cells also occurred independently in the absence of a CD4+ helper cell response. The lack of T-cell-T-cell interactions in vivo may explain the failure of effector cell generation during progressive tumor growth. Taken together, these results demonstrate that the anti-CD3/IL-2 activation defines an immune response distinct from many previously described mechanisms of antitumor immune responses.

Ultrastructure of the marginal division in the rat striatum.

The discovery of a new subdivision in the striatum of the rat, the marginal division, has recently been reported. The marginal division is located at the caudal extent of the striatum, surrounding the rostrolateral border of the globus pallidus, and has different cellular morphology, immunohistochemistry and an efferent projection pattern from those of the main body of the striatum. In the present study, the ultrastructural organization of the marginal division was investigated. Most neuronal somata in the marginal division were fusiform in shape and had a large pale oval nucleus without in-foldings. There were four types of synapses in the marginal division: axo-somatic, axo-dendritic, axo-spinous and axo-axonic. Both symmetric and asymmetric synapses were observed on the somata, dendrites, or dendritic spines. Most of the symmetric synapses contained pleomorphic vesicles, whereas the asymmetric ones contained mainly round vesicles. Individual axo-axo-spinous synapses, which were first described in the striatum, were also observed in the marginal division. These ultrastructural characteristics distinguish the marginal division from the rest of the striatum.

Ultrastructural characteristics of substance P-immunoreactive terminals in marginal division of rat striatum.

In our previous work using immunocytochemical method combined with tract tracing techniques a new subdivision was described in the striatum of the rat. This "marginal division" is more densely filled with substance P, enkephalin and dynorphin B terminals than the rest of the striatum. In the present study, the synaptic organization of the substance P immunoreactive (SPIR) terminals in the marginal division of the rat striatum was studied using electron microscopy and immunocytochemistry for substance P (SP). Four major types of SPIR synapses were identified in the marginal division: axodendritic, axospinous, axo-axonal, and compound synapses. Axodendritic and axospinous synapses, in which the postsynaptic targets were small or large dendrites or spines, were the most common. A few axo-axonic synapses were observed as were several subtypes of compound synapses with more than two synaptic components. SPIR axon terminals formed the presynaptic components of all these synaptic types, but in one case an unlabeled bouton was observed making a synaptic connection onto a SPIR dendrite. Both symmetric and asymmetric SPIR synapses were observed in the marginal division. The vesicles in the SPIR presynaptic boutons were mostly pleomorphic although a few of them were round. The existence of asymmetric synapses, round synaptic vesicles and small postsynaptic dendrites distinguishes the ultrastructure of the marginal division from that of the other parts of the striatum. The complex characteristics of the synaptic organization in the marginal division implies that the SPIR terminals in the marginal division originate from a different source than those in the rest of the striatum. The complexity of the synaptic organization further suggests that the function of the marginal division is different from that of the rest of the striatum.

Activation by anti-CD3 of tumor-draining lymph node cells for specific adoptive immunotherapy.

Lymph nodes draining progressive tumors contain tumor-sensitized but not functional preeffector T lymphocytes. These cells can acquire antitumor reactivity after stimulation with tumor cells and interleukin-2 (IL-2). We demonstrated here that, in the absence of tumor cells, preeffector cells could be stimulated and expanded by sequential culture with anti-CD3 monoclonal antibody and IL-2. The adoptive transfer of such activated cells mediated immunologically specific reductions of established pulmonary metastases. The therapeutic effects could be enhanced by the administration of IL-2. This activation represents a secondary immune response because effector cells could be generated only from tumor-draining but not from normal or adjuvant-stimulated lymph nodes. Furthermore, treatment of advanced metastases with these cells resulted in prolongation of survival and cure of the disease. Thus, anti-CD3 may serve as a universal reagent for activating tumor-sensitized T lymphocytes for cancer therapy.

Phenotype analyses and cellular mechanisms of the pre-effector T-lymphocyte response to a progressive syngeneic murine sarcoma.

Lymph nodes draining the progressively growing, weakly immunogenic, MCA 105 sarcoma contained tumor-sensitized but not fully functional pre-effector T-cells. These cells could further differentiate to acquire full antitumor effector function for adoptive therapy in an established in vitro sensitization (IVS) procedure. In this study, we utilized selective depletion with antibodies of lymphocyte subsets bearing the L3T4 (CD4) or Lyt-2 (CD8) antigen and of cells bearing the asialo-GM1 (ASGM-1) glycosphingolipid to identify the phenotype of pre-effector cells elicited during progressive tumor growth. Cells from lymph nodes draining a progressive MCA 105 tumor in the footpad were treated with antibodies plus complement prior to IVS. The antitumor efficacy of resulting IVS cells was assessed in adoptive therapy of 3-day established pulmonary MCA 105 metastases. Depletion of Lyt-2+ cells eliminated in vivo antitumor reactivity with concurrent elimination of in vitro cytotoxic activity against the MCA 105 tumor, whereas depletion of L3T4+ cells did not have an impact on either in vivo or in vitro antitumor reactivities. Treatment with ASGM-1 antiserum plus complement was also found to abrogate therapeutic efficacy. However, the in vitro cytotoxic activity was not affected. These results indicate that the pre-effector cells were Lyt-2+, L3T4-, and ASGM-1+. We next examined whether the sensitization of pre-effector cells in vivo required the participation of L3T4+ helper cells. To approach this, mice were depleted of L3T4+, Lyt-2+, or ASGM-1+ cells by antibody injections before tumor inoculation. Treatment with Lyt-2 monoclonal antibody abrogated the pre-effector cell response in the draining lymph nodes, as evidenced by failure to generate therapeutically effective cells following IVS. On the other hand, neither L3T4 nor ASGM-1 antibody treatment affected the generation of pre-effector cells. Thus, sensitization of Lyt-2+ pre-effector cells in response to progressive tumor occurred in the absence of L3T4+ helper cells.

High density of zinc-containing and dynorphin B- and substance P-immunoreactive terminals in the marginal division of the rat striatum.

A distinct subdivision of the striatum has recently been described which is located at the caudomedial margin of the striatum, surrounding the rostrolateral edge of the globus pallidus. This "marginal division" has an internal organization and an efferent distribution which is distinct from the rest of the striatum. The striatum contains moderately high levels of zinc and the neuropeptides enkephalin, dynorphin and substance P. In the present study we have examined the distribution of histologically detectable zinc and of dynorphin B- and substance P-immunoreactivity in the marginal division of the striatum. Each of these substances was more dense within the confines of the marginal division than in the rest of the striatum. These data provide further evidence that the marginal division is a structurally distinct subdivision of the striatum.

Lymphocytes generated by in vivo priming and in vitro sensitization demonstrate therapeutic efficacy against a murine tumor that lacks apparent immunogenicity.

The adoptive transfer of sensitized lymphocytes is an effective means to mediate the regression of established tumors. However, successful therapy can only be demonstrated in animal models where tumors are intrinsically immunogenic, capable of eliciting systemic immunity. To explore the potential of this therapeutic approach to tumors of less immunogenicity, we have selected and used a murine tumor, MCA 102, for the current study because all attempts to immunize syngeneic mice failed. We report here that inoculation of mice with a mixture of tumor cells and a bacterial adjuvant, Corynebacterium parvum led to the production of sensitized, but not fully functional, lymphocytes in the draining lymph nodes (LN). These cells, termed pre-effector cells, could nevertheless further differentiate to acquire full immunologic function by an established in vitro sensitization culture method. In adoptive immunotherapy experiments, transfer of as few as 1.5 X 10(7) in vitro sensitized cells not only reduced established pulmonary MCA 102 metastases but also prolonged survival and cured tumors in a majority of the treated animals. In order to elicit pre-effector cells in vivo, inoculation with both tumor cells and C. parvum was essential. Although a broad range of numbers of MCA 102 tumor cells appeared to be effective, generation of pre-effector cells was dependent on the dose of C. parvum. We have found that a C. parvum dose of 25 micrograms was optimal, whereas higher doses of the adjuvant had suppressive effects. Analysis of the kinetics of their appearance revealed that the generation of pre-effector cells was transient. They were detectable 7 days after in vivo priming followed by a rapid decline. Furthermore, pre-effector cells were detected only in the regional draining LN. No reactivity was demonstrable in the spleen, mesenteric LN, PBL, or bone marrow. Taken together, these results expand the scope of immunotherapy by demonstrating the feasibility of manipulating a limited and obscure immune response to the MCA 102 tumor for therapeutic efficacy.

Effects of Tripterygium hypoglaucum (Lévl.) Hutch on male fertility.

Tripterygium hypoglaucum (Lévl.) Hutch (TH) is a perennial used in Chinese traditional medicine for the treatment of rheumatoid arthritis and various skin disorders. One study showed that daily oral doses of TH significantly reduced the fertility of male rats without apparent toxicity. The effects of daily oral doses of TH on the fertility of men taking it for the treatment of rheumatoid arthritis were evaluated. Sperm concentration, motility and motility grade all were significantly reduced in the 13 men taking TH compared to 11 untreated controls. TH therapy did not affect testosterone, FSH, LH levels, and its antifertility effects appeared to be reversible.

Anterograde and retrograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L) from the globus pallidus to the striatum of the rat.

Iontophoretic administration of PHA-L into the globus pallidus of rats resulted in the labeling of neuronal perikarya in the striatum as well as axons and terminals in the striatum, entopeduncular nucleus, subthalamus and substantia nigra. The labeled striatal perikarya were densely stained in Golgi fashion with virtually complete filling of the dendrites and spines. It is concluded that the striatal cells were filled by the retrograde transport of PHA-L and represent either striatopallidal cells, or striatonigral cells whose axons were interrupted as they passed through the injection site. The anterogradely labeled axon terminals in the striatum were observed in close apposition to the dendrites of the retrogradely labeled neurons suggesting the existence of synaptic contacts between the two groups of cells. This study demonstrates that PHA-L can be transported retrogradely as well as anterogradely following iontophoretic injections.

The 'marginal division': a new subdivision in the neostriatum of the rat.

Using a combination of anterograde and retrograde (Phaseolus vulgaris leucoagglutinin; PHA-L and wheat germ agglutinin conjugated horseradish peroxidase; WGA-HRP) tract-tracing methods and histochemical techniques, a new subdivision of the neostriatum, the marginal division, has been found in the rat brain. The marginal division is approximately 120 microns wide and is located at the caudal extent of the neostriatum and surrounds the rostral edge of the globus pallidus. The neuronal somata of the marginal division are mostly fusiform in shape, with their long axes running parallel to the border between the striatum and the globus pallidus. Histochemically, the marginal division is lighter in AChE staining, is more densely filled with Met-enkephalin-immunoreactive terminals, and has fewer choline acetyltransferase (ChAT)-immunoreactive neurons than does the rest of the neostriatum. Injections of PHA-L or WGA-HRP demonstrated that the projections of the marginal division differ from those of the main body of the striatum. The striatopallidal projection from the marginal division terminates in the caudal-most part of the globus pallidus which is rich in cholinergic neurons. In contrast, the projection from the main region of the neostriatum terminates in two bands in the globus pallidus, both of which are rostral to the area of termination of the fibres from the marginal division. The striatonigral fibres from the marginal division terminate in the caudal part of the substantia nigra pars reticulata whereas the rest of neostriatum projects to a more rostral region. Based on its cellular morphology, immunohistochemistry and projection pattern, we conclude that the marginal division of the striatum is a distinct subdivision of the neostriatum.

Generation of therapeutic T lymphocytes from tumor-bearing mice by in vitro sensitization. Culture requirements and characterization of immunologic specificity.

We have previously established an in vitro sensitization (IVS) procedure with which lymphocytes from tumor-bearing mice could be expanded and sensitized to acquire antitumor reactivity capable of mediating the regression of established pulmonary metastases from the weakly immunogenic MCA 105 murine sarcoma. Culture conditions required for the optimal generation of therapeutic effector cells were evaluated in the current study. Generation of effector cells by IVS required stimulation by intact tumor cells. Tumor cells killed by heat or disrupted by sonication were ineffective, but the antigenicity of tumor cells was not affected by gamma-irradiation. Long term established tumor cell lines could also serve as antigenic stimulator cells albeit with lower efficiency than fresh tumor cells. IL-2 was essential for cellular proliferation during IVS. The concentration of 1000 U/ml of IL-2 also induced nonspecific lymphokine-activated killer (LAK) activity. However, cytotoxic cells were generated during IVS in response to a broad range of IL-2 concentrations. At low IL-2 concentrations (2 to 10 U/ml), IVS cells were generated which displayed little or no LAK activity, had a greater therapeutic efficacy than those generated with high concentrations of IL-2 (100 to 1000 U/ml). Despite having high LAK activity, IVS cells, from cultures where IL-2 was added 3 or more days after initiation, had no therapeutic effect. Thus, the generation of therapeutic cells occurred independently of LAK cell production. Adoptive immunotherapy with IVS cells from MCA 105 tumor-bearing mice demonstrated cross-reactivity with the immunologically distinct MCA 106 but not the nonimmunogenic MCA 102 tumor. In contrast, IVS cells from MCA 106 tumor-bearing mice exhibited specific in vivo reactivity. In vitro cytotoxicity analyses revealed that IVS cells from MCA 105 and MCA 106 tumor-bearing mice were able to lyse both MCA 105 and MCA 106 target cells, but the reactivity toward inoculating tumors was highest. Considering previous findings that the MCA 105 and MCA 106 sarcomas possessed distinct tumor-specific transplantation Ag, the cross-reactivity observed in this study suggests that the immune response during progressive tumor growth may be different from that elicited in response to active immunization.

The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system.

A combination of the glucose oxidase-diaminobenzidine (DAB) method and the DAB-nickel method can successfully bring out details of immunoreactive structures in immunostained preparations. It is especially beneficial for visualizing fibers and terminals.