Idiotype-anti-idiotype network. II. Activation of silent clones by treatment at birth with idiotypes is associated with the expansion of idiotype-specific helper T cells.

BALB/c mice immunized with bacterial levan (BL) produce a vigorous antibody response that fails to include antibodies expressing the idiotype of the beta 2 leads to 6 fructosan-binding myeloma protein ABPC48 (A48). Treatment of newborn BALB/c mice at 1 d of age with 0.1-10 microgram of either the A48 myeloma protein or monoclonal proteins that share idiotopes with the A48 family, followed by immunization with BL 2-4 wk later, produces an anti-BL response that is dominated by the A48Id. Various degrees of activation of the A48Id BL response were observed by injecting mice with A48 monoclonal protein only up until 3 wk of age. Activation of the A48Id clones by treating with A48 monoclonal protein was ineffective in mice who were older than 4 wk. Elicitation of an A48Id BL response required specific antigenic stimulation with either beta 2 leads to 6 or beta 2 leads to 1 fructosan epitopes, because it does not occur after injection with TNP-Ficoll in spite of the A48 treatment. The expansion of A48Id clones in mice treated at birth with A48 monoclonal protein is associated with an increase in A48Id-specific helper T cells. The binding specificity of these cells was demonstrated by infusing them into nu/nu BALB/c mice and observing that they rendered help that enalbed the animal to mount an anti-TNP response after immunization only with A48-TNP, but not with MOPC384-TNP conjugates. The helper activity of these cells is sensitive to the effects of treatment with anti-Lyt-1.2 antibodies plus complement. A predominantly A48Id BL-specific response can be transferred into lethally irradiated mice by infusing them with purified T and B cells from A48-treated mice. The transfer of this response can be ablated by treating the T cells with anti-Lyt-1.2 antibodies plus complement. These results indicate that A48Id-specific helper cells possess the ability to select the A48Id-bearing B cell precursors for expression, thus exerting a fine-tuning effect on the idiotypic expression of the anti-BL repertoire. We propose that this idiotype-induced idiotype response, which can be, in principal, induced by idiotypes provided by the mother, plays an important role in the expansion of precursors of antibody-forming cells during embryonic as well as postnatal life.

Idiotype-antiidiotype regulation. V. The requirement for immunization with antigen or monoclonal antiidiotypic antibodies for the activation of beta 2 leads to 6 and beta 2 leads to 1 polyfructosan-reactive clones in BALB/c mice treated at birth with minute amounts of anti-A48 idiotype antibodies.

The anti-beta 2 leads to 6 fructosan antibodies sharing the idiotypes (Id) of ABPC48 (A48) monoclonal protein represent a silent fraction of the anti-beta 2 leads to 6 fructosan repertoire, since these antibodies cannot be detected during a conventional immune response elicited by bacterial levan (BL). However, the administration at birth of minute amounts of anti-A48 Id antibodies causes a long-lasting activation of A48 Id+-bearing clones. This activation is related to direct interaction of anti-A48 Id antibodies with precursors bearing the A48 Id+ immunoglobulin receptor, since an A48 Id+ response can be transferred with highly purified B cells in lethally irradiated mice. The maturation of these precursors into A48 Id+ anti-beta 2 leads to 6 fructosan antibody-secreting cells requires challenge by the antigen. Isoelectric focusing (IEF) data showed that in 1-mo-old mice an UPC10 (U10)-like spectrotype was observed, whereas in 3-mo-old mice, a new spectrotype binding BL rather than inulin (In) was identified. This spectrotype was observed only in CXBJ mice, the single strain in which an A48 Id+ response was observed. The antigenic challenge can be replaced by a monoclonal anti-A48 Id antibody (i.e., 17-38). Interestingly, in 1-mo-old BALB/c mice treated with anti-A48 Id antibodies, the challenge with 17-38 monoclonal antibody led to the activation of A48 Id- anti-beta 2 leads to 6 fructosan-reactive clones with BALB/c type IEF spectrotypes, whereas in 3-mo-old BALB/c mice treated with anti-A48 Id antibodies, the challenge with 17-38 monoclonal antibody led to the activation of W3082 IdX+ anti-beta 2 leads to 6 and beta 2 leads to 1 fructosan-reactive clones. In these animals, inhibition of A48 Id+ anti-beta 2 leads to 6 fructosan clones was observed. This antibody probably represents a homobody carrying the internal image of the antigen, which through its paratope suppresses the A48 Id+ response and through its Id activates an A48 Id- anti-beta 2 leads to 6 fructosan response in 1-mo-old mice and in 3-mo-old mice leads to an anti-beta 2 leads to 6 and beta 2 leads to 1 fructosan response dominated by the W3082 IdX.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of myeloma growth in mice by antigen and regulatory idiotopes.

The effect of antigen, activation of normal A48Id+ B-cell clones, and monoclonal antibodies sharing A48-UPC10 regulatory idiotopes on the in vivo growth of spleen adapted ABPC48 myeloma cells has been examined. Immunogenic doses of bacterial levan have no detectable effects, whereas tolerogenic doses substantially delay the growth of the myeloma. The activation of normal A48Id+ B cell clones also has no apparent effect on the growth of the ABPC48 myeloma cells. Among a panel of 15 monoclonal antibodies expressing A48-UPC10 regulatory idiotopes and expressing VH genes derived from the VH441-4 germ line gene family, 5 were able to provide a long lasting but not definitive idiotype specific protection against the ABPC48 myeloma cells.

Histogenetic and morphological in vivo and in vitro data concerning androgen-estrogen-induced scent gland tumor in the Syrian hamster.

The scent gland tumor of the Syrian hamster is induced by exogenous androgen and estrogen. Microscopic nodules are induced normally in old males by endogenous androgen. The histogenesis of the scent gland tumor is complex and not completely understood. In this study microscopic preneoplastic nodules and macroscopic tumors were studied by light and electron microscopy, and the macroscopic tumors were grown in tissue culture on collagen-coated coverslips and on sponge foam matrices by the organ culture method. The cultures were fed with an unfiltered fetal calf serum-bovine serum ultrafiltrate medium, which contained endogenous androgen-estrogen, 110-100 pg and could maintain growth without additional androgen-estrogen. Exogenous androgen-estrogen was also added to some cultures. Scent gland tumors grown in organ culture contained cells of two shapes, spindle and ovoid arranged in cords. Cultures on coverslips showed radiating outgrowths of spindle cells suggesting either mesenchymal or Schwann cells. By electron microscopy, both in vivo and in vitro preneoplastic and tumor samples contained cells with segments of basal lamina, micropinocytotic vesicles, and junctional complexes. These features were similar to those of poorly differentiated experimental malignant rat schwannomas maintained in similar in vitro systems. Tumors grown in vivo and in vitro were associated with collagen fibrils with a periodicity ranging from 400 to 1075 A. The evidence reported in this paper suggests that one component of the scent gland tumor is an androgen-estrogen-induced poorly differentiated schwannoma.

Immunoradiometric and immunohistochemical demonstration of neuron-specific enolase in experimental rat gliomas.

A number of neural and nonneural tumor cell lines of rat and human origin were assayed for neuron-specific enolase (NSE) by radioimmunoassay. Most neural tumor cell lines had appreciably higher levels of NSE than did the nonneural tumor cell lines, the highest levels being found in two anaplastic rat glioma lines ( F98 and T24). These two lines contained more than twice the amount of NSE found in a rat pheochromocytoma line (PC12) and in neuroblastoma lines derived from rats ( B35 and B50 ) or humans (IMR-32 and SHSY - 5Y ). Several of the rat glioma and schwannoma lines were inoculated intracerebrally into syngeneic rats. In the resulting tumors, NSE was demonstrable by immunohistochemistry only in those from the F98 and T24 cell lines. A number of ethylnitrosourea-induced rat tumors were also examined immunohistochemically for NSE: NSE was demonstrated in three anaplastic gliomas; three astrocytomas; and two mixed gliomas. Reactive astrocytes were also positive. Fibroadenomas of apocrine and mammary glands in rats were weakly positive, but other extraneural tumors tested were negative. Since normal neuronal elements, axonal swellings, and amine precursor uptake and decarboxylation cells are strongly positive for NSE, whereas glia and most other normal cells are negative, we hypothesize that the elevated metabolic demands imposed on neoplastic and reactive glial cells and on some extraneural tumors necessitate the opening up of metabolic pathways that are normally operative only in neurons and neuroendocrine cells, therefore resulting in the synthesis of the more stable neuron-specific form of enolase.

The time development of direct hemolytic plaques: implications for the binding of IgM to cell surface haptens.

We studied the time development of direct hemolytic plaques in thin layers containing either sheep red blood cells (SRBC) directly haptenated (DH) with trinitrophenyl, or SRBC indirectly haptenated (IH) with dinitrophenyl coupled to human serum albumin. The DH-SRBC tend to be sparsely haptenated while the IH-SRBC tend to have very high local hapten densities. We observed marked differences in the growth of plaques for the two differently haptenated SRBC. Plots of the plaque radius squared vs time show that the slope of those curves that developed in a lawn of DH-SRBC tended to be constant while the slope of those curves that developed in a lawn of IH-SRBC tended to decrease with time. These results are what is predicted from theory if: IgM binds to DH-SRBC through attachments that rapidly dissociate, if IgM binds to IH-SRBC through attachments that very slowly dissociate, and if (3) both types of bound IgM can fix and activate complement.

Non-specificity of anti-carbonic anhydrase C antibody as a marker in human neurooncology.

Because the presence of carbonic anhydrase C (CA C) has been demonstrated in the oligodendrocytes of the mouse, rat and man, anti-CA C serum has been considered to be a possible specific marker for these cells. In order to determine its value in human neurooncology, specimens from 110 human tumors from the central and peripheral nervous systems as well as from five cases of cerebral infarction and two of multiple sclerosis were tested immunohistochemically by the peroxidase-antiperoxidase method with anti-CA C serum. Reactive astrocytes, oligodendrocytes in the neural parenchyma surrounding tumors, and neurons included in areas of neoplasia showed CA C immunopositivity. In 92% of the astrocytomas and 56% of the glioblastomas variable numbers of tumor cells were positive. Some tumor cells positive for glial fibrillary acidic protein in ependymomas and astroblastomas were also CA C-positive. Schwannomas (86%), neurofibromas (100%) and meningiomas (86%) showed CA C positivity of the tumor cells, as did choroid plexus papillomas and gangliogliomas. However, all the medulloblastomas, neuroblastomas, central neurocytomas or melanomas tested in this study were entirely CA C-negative. In some examples of squamous cell carcinoma, leiomyoma, leiomyosarcoma and fibrous histiocytoma, CA C-positive neoplastic cells were also demonstrated. Our findings indicate that since various types of neoplastic and reactive cells express CA C positivity, the anti-CA C serum cannot be used as a specific marker for any tumor in human neurooncology.

Mixed capillary hemangioblastoma and glioma. A redefinition of the "angioglioma".

The histopathologic features of four cases of mixed capillary hemangioblastoma and glioma are described. In three cases, two of which arose in the cerebellum and one in the spinal cord, the hemangioblastic component may have originated from a neoplastic proliferation of the exuberant vascular stroma in a glial tumor. In a fourth case, a cerebellar hemangioblastoma was surrounded by a peripheral rim of atypical neoplastic-looking astrocytes ("reactive glioma"). The controversial concept of the "angioglioma" is reviewed, and it is proposed that the term be used to designate only true mixed tumors of glial and vascular tissue origin whose histologic features conform to the examples described in this report.

GFA protein reactivity in nerve sheath tumors: a polyvalent and monoclonal antibody study.

We studied glial fibrillary acidic (GFA) protein immunoreactivity in 30 schwannomas, including two intracerebral examples, 26 neurofibromas and 12 neuromas using the immunoperoxidase method with a polyvalent antiserum (PVAS) and three well-characterized monoclonal antibody (MAb) preparations. Twelve of the schwannomas, including both intracerebral tumors, two of the neurofibromas and none of the neuromas immunostained with PVAS. Except for one schwannoma, all the PVAS-positive tumors were positive with two of the MAb preparations. While both of the intracerebral schwannomas were positive with the third MAb, none of the extracerebral tumors were. Our results suggest that: 1) human nerve sheath tumors contain cells having polypeptides that share epitopes with GFA protein, but 2) these polypeptides differ from astrocytic GFA protein by at least one epitope, and 3) the location of the tumors in relation to the central nervous system may influence GFA protein immunoreactivity.

Patterns of epithelial metaplasia in malignant gliomas. I. Papillary formations mimicking medulloepithelioma.

Adenoid-like formations resembling ducts and glands or forming a cribriform pattern have previously been described in malignant gliomas, resulting in some cases in a confusion with metastatic adenocarcinoma. The interpretation of these structures as being composed of anaplastic glial cells rests partly on the presence of transitions to more differentiated neoplastic astrocytes and partly on the positivity of some of these cells for glial fibrillary acidic protein. In this report two cases are presented in which the adenoid pattern was associated with papillary formations mimicking the arrangement of a medulloepithelioma. These structures represent a form of aberrant neoplastic differentiation in a malignant glioma rather than the expression of an embryonal neuroepithelial neoplasm.

Leukoencephalopathy in Waldenström's macroglobulinemia. Immunohistochemical and electron microscopic observations.

The clinical and pathologic features of a case of Waldenström's macroglobulinemia with leukoencephalopathy are reported. Multiple cerebral foci of demyelination, accompanied to a lesser extent by axonal degeneration, were associated with perivascular infiltrates of plasmacytoid lymphocytes and with permeation of the white matter by macroglobulins. Immunohistochemical studies demonstrated a predominance of IgM kappa within the blood plasma, in cerebral blood vessel walls, in the foci of demyelination, and within perivascular histiocytes. Electron microscopy disclosed the presence, in macrophages and pericytes, of membrane-bound cytoplasmic inclusions consisting of tubular arrays, suggestive of cryoglobulin deposits. We hypothesize that the high serum levels of macroglobulins accompanied by lymphoplasmocytic infiltrates may, either by way of viscosity-related ischemia, or by a direct toxic effect, have caused abnormal vascular permeability, infiltration of the cerebral parenchyma by paraproteins, and, ultimately, focal degeneration of the white matter.

Patterns of epithelial metaplasia in malignant gliomas. II. Squamous differentiation of epithelial-like formations in gliosarcomas and glioblastomas.

Six cases are reported (four gliosarcomas and two glioblastomas) in which the epithelial-like areas of glial anaplasia showed focal squamous cell differentiation, characterized by the development of epithelial whorls, keratin pearls and immunopositivity for cytokeratin. The expression of glial fibrillary acidic protein and the development of squamous metaplasia usually were mutually exclusive. Autopsy findings in two patients and clinical work-up in five failed to disclose a primary extraneural malignancy. It is suggested that squamous differentiation may represent an extreme form of epithelial metaplasia in a malignant glioma. This possibility should be kept in mind in the diagnostic evaluation of such cases, especially in view of the current emphasis on the immunomorphologic demonstration of intermediate filament tumor markers.

A simple method for coating native polysaccharides onto nitrocellulose.

A method for coating native, non-derivatized, polysaccharide (PS) onto nitrocellulose (NC) for identifying PS-specific antibodies has been developed. The new feature of this method is that PS molecules are vacuum filtered onto NC in their native state by devices that can accommodate NC of different sizes and shapes. PS-coated NC disks were used to localize antibody secreting hybridoma cells cultured on filter paper disks. These were analyzed by blotting with size-matched PS-coated NC disks and specific antibodies secreted by individual colonies were detected by enzyme-linked immunoblot. In another application of this method, immune sera were separated by isoelectric focusing and the gels were blotted with PS-coated NC sheets. The spectrotype and isotype of antibodies that bound to the NC were examined using isotype specific enzyme-linked antibody. These immunoblots showed high resolution and specificity. The advantages of this method are that the PS used for coating does not need to be derivatized in order to bind the NC, and that smaller quantities of PS may be utilized by this coating method when compared to other techniques. This provides a useful tool to ask many questions regarding the immune response to PS.

A novel technique for the production of hybrid antibodies.

Chemically linked bifunctional antibodies (heteroconjugates) composed of one antibody specific for the TcR/CD3 complex on cytotoxic T cells and another specific for viral antigens expressed on the surface of infected cells have been shown to redirect CTL to lyse virus-infected cells. Hybrid antibodies are bifunctional antibodies produced by the fusion of two hybridomas. As a result of their native dimeric immunoglobulin structure, hybrid antibodies may be more effective than heteroconjugates in vivo. We have developed a unique method for production of hybrid antibodies by infecting each hybridoma with a different retrovirus vector which confers resistance to either G418 or methotrexate. The hybridomas are fused and selected in medium containing both inhibitors. Using this technique, we have produced hybrid antibodies made up of one antibody combining site which binds to the TcR and a second specific for the hemagglutinin of X-31 influenza virus. We show that this hybrid antibody effectively mediates the lysis of virus-infected cells in the presence of appropriate CTL. Thus hybrid antibodies as well as heteroconjugates can redirect CTL to lyse virus-infected targets.

Electron microscopic localization of neuron-specific enolase in rat and mouse brain.

The cellular distribution and intracellular localization of neuron-specific enolase (NSE) has been studied by electron microscopic immunocytochemistry in the brain of the rat and of the mouse. Although the intensity of staining was less in the mouse, the same structures were positive in both species. In the cerebrum, the neuronal perikarya and dendrites were intensely stained, but staining was almost entirely absent in the presynaptic terminals. The deep neurons of the brain stem were also positive. In the cerebellum, perikarya, axons, and parallel fibers of the granule cell neurons were stained as were the synaptic vesicles and presynaptic membranes of the synapses between the parallel fibers and the Purkinje cell dendrites. Golgi cell dendrites, basket cells and their axons, and mossy fibers were also positive. In contrast, the Purkinje cells including their dendrites, and the climbing fibers that formed synapses with the Purkinje cell dendrites were not stained. The majority of the myelinated axons in both the cerebrum and the cerebellum did not stain, but the fibrillary astrocytic processes between myelinated axons in the white matter did. Oligodendroglia, protoplasmic astrocytes, Bergmann glia, astrocytes investing capillaries, and vascular endothelial cells were negative for reaction product. In the positively staining cells and their processes, the positivity was dispersed throughout the cytoplasm and corresponded most closely to the distribution of ribosomes, the granular endoplasmic reticulum, and microtubules. Nuclei, mitochondria, the cisternae of the Golgi complex, myelin lamellae, and most membranes were not stained.

Cytogenesis and differentiation of pineal neoplasms.

Our knowledge of pineal neoplasms is still fragmentary, but a pattern is beginning to emerge in this highly challenging area of neuro-oncology. In vitro studies and more complete morphologic and biochemical characterization are still needed for these very rare neoplasms. Follow-up data from appropriate clinical series are still almost nonexistent for the evaluation of therapeutic effectiveness. As an illustration of neoplastic development, the demonstration of divergent differentiation in pineal parenchymal tumors represents a most intriguing phenomenon in light of the origin, structure, and function of the pineal gland.

The malformative central nervous system lesions in the central and peripheral forms of neurofibromatosis. A neuropathological study of 22 cases.

The neuropathological features of 22 autopsied cases of NF have been reviewed, with special reference to the malformative and proliferative lesions implicating the intracranial and intraspinal neural structures. Eleven cases represented examples of the central form of the disease, and 11 examples of the peripheral form. The central form is defined by the association and multiplicity of cranial and spinal meningeal, nerve-sheath, and glial neoplasms (astrocytomas and ependymomas). Bilateral acoustic schwannomas are a frequent, but not invariable, component of the disease. Central NF is also characterized by the very frequent incidence (9 out of 11 cases) of distinctive malformative CNS lesions, which included intramedullary and perivascular schwannosis, meningioangiomatosis, discrete ependymal ectopias, atypical glial cell nests in the grey matter, and, less frequently, syringomyelia. Many of these hamartomatous changes were closely associated topographically with florid neoplastic lesions. Five of the 11 cases of peripheral NF showed involvement of the CNS by cellular proliferative changes that included subependymal gliofibrillary nodules in 3 cases (causing aqueduct stenosis in 2, with resulting hydrocephalus in 1); hyperplastic meningioencephalic gliosis involving the pons and the cerebellum in 1 case; and micronodular capillary and arteriolar proliferations typical of the vascular form of NF in 1 case. Whereas some of the glial proliferations are probably hamartomatous in nature, others may represent an abnormal productive neuroglial response to adjacent pathological conditions, such as antecedent cerebral hemorrhage or infarct, known to stimulate a proliferative gliosis. Such a response may exhibit morphological features that are indistinguishable from those of an astrocytoma, including leptomeningeal and perivascular invasion. The incidence of proliferative CNS lesions in both the central and the peripheral form of NF indicates that the spectrum of tissues implicated extends beyond those derived solely from the neural crest.

Diagnostic markers in human neurooncology. A progress report.

In this progress report, some of the most commonly used antibodies are discussed in regard to their immunohistochemical application to human neurooncology. The importance of determining the spectrum of antibody immunoreactivity in a wide panel of normal, reactive, and neoplastic tissues is stressed. in atypical and aberrant cases, immunopositivity needs to be interpreted with caution and in the context of all other available data. The demonstration of a well-characterized, cell type-specific marker in a tumor reflects not so much its cytogenesis as its differentiation potential and its capacity for metaplasia. The relation of an abnormal or aberrant expression of antigenic determinants to the process of neoplasia raises a number of intriguing questions to which research in the next few years will likely provide answers.

Immunohistochemical characterization of human retinoblastomas in situ with multiple markers.

We studied paraffin-embedded specimens from 18 surgically enucleated eyes with retinoblastoma by peroxidase-antiperoxidase immunohistochemistry with antibodies against glial fibrillary acidic protein, S-100 protein, Leu 7 epitopes, neuron-specific enolase, the 200-kilodalton subunit of the neurofilament triplet polypeptide, and retinal S-antigen. We found that (1) glial fibrillary acidic protein, S-100 protein, and Leu 7 epitopes were detected only in well-differentiated glial cells that were interpreted as reactive and not neoplastic, (2) undifferentiated neoplastic cells expressed both neuron-specific enolase and retinal S-antigen immunoreactivity, and (3) differentiated cells forming Flexner-Wintersteiner rosettes were found to express neuron-specific enolase, retinal S-antigen, and, occasionally, neurofilament protein. These results support the view that retinoblastomas are composed of neuron-committed cells and favor the origin of these tumors from photoreceptor progenitor cells. We did not find any morphologic or immunohistochemical evidence of glial differentiation from tumor cells that would support the concept that retinoblastoma arises from a primitive neuroectodermal cell capable of divergent differentiation along neuronal and glial lines.

The angioblastic meningioma: a reappraisal of the nosological problem. Light-, electron-microscopic, tissue, and organ culture observations.

The validity of the concept of the angioblastic meningioma, now in dispute, was reexamined by reviewing 79 meningeal and angioblastic tumors of the central nervous system and by comparing the fine structural characteristics and in vitro evolution of 2 typical meningiomas and 1 intracranial hemangiopericytoma. While most tumors show the consistent features of either hemangiopericytoma or hemangioblastoma, there exist transitional forms between these tumors and typical meningioma. There is also a greater degree of morphological overlap at the electron microscopic level than has been recognized up till now. In view of these findings the concept of the angioblastic meningioma deserves to be retained as a generic term to include craniospinal hemangiopericytomas and transitional forms between hemangiopericytoma, hemangioblastoma and classic meningioma. It is postulated that all these tumors share a common origin from polyblastic mesenchymal cells originating in or derived from the meninges.