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The intense accumulation of prostate-specific membrane antigen (PSMA) radioligands in salivary glands is still not well understood. It is of concern for therapeutic applications of PSMA radioligands, because therapeutic radiation will damage these glands. A better understanding of the uptake mechanism is, therefore, crucial to find solutions to reduce toxicity. The aim of this study was to investigate whether the accumulation of PSMA-targeting radioligands in submandibular glands (SMGs) can be explained with PSMA expression levels using autoradiography (ARG) and immunohistochemistry (IHC). Methods: All patients gave written informed consent for further utility of the biologic material. The SMG of 9 patients, pancreatic tissue of 4 patients, and prostate cancer (PCA) lesions of 9 patients were analyzed. Tissue specimens were analyzed by means of PSMA-IHC (using an anti-PSMA-antibody and an immunoreactivity score system [IRS]) and ARG using 177 Lu-PSMA-617 (with quantification of the relative signal intensity compared with a PSMA-positive standard). The SUV max in salivary glands, pancreas, and PCA tissues were quantified in 60 clinical 68 Ga-PSMA-11 PET scans for recurrent disease as well as the 9 primary tumors selected for ARG and IHC. Results: PCA tissue samples revealed a wide range of PSMA staining intensity on IHC (IRS = 70-300) as well as in ARG (1.3%-22% of standard). This variability on PCA tissue could also be observed in 68 Ga-PSMA-11 PET (SUV max , 4.4-16) with a significant correlation between ARG and SUV max ( P < 0.001, R 2 = 0.897). On IHC, ARG, and 68 of 3.1 ± 1.1). The SMG tissue displayed only focal expression of PSMA limited to the intercalated ducts on IHC (IRS = 10-15) and a minimal signal on ARG (1.3% ± 0.9%). In contrast, all SMG showed a high max of 3.1 ± 1.1). The SMG tissue displayed only focal expression of PSMA limited to the intercalated ducts on IHC (IRS = 10-15) and a minimal signal on ARG (1.3% ± 0.9%). In contrast, all SMG showed a high 68 Ga-PSMA-11 accumulation on PET scans (SUV max Our results indicate that the high accumulation of PSMA radioligands in salivary glands does not correspond to high PSMA expression levels determined using ARG and IHC. These findings provide evidence, that the significant accumulation of PSMA radioligands in SMG is not primarily a result of PSMA-mediated uptake.Conclusion: Our results indicate that the high accumulation of PSMA radioligands in salivary glands does not correspond to high PSMA expression levels determined using ARG and IHC. These findings provide evidence, that the significant accumulation of PSMA radioligands in SMG is not primarily a result of PSMA-mediated uptake., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

Background: Soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) belongs to the immunoglobulin superfamily and is involved in amplification of the inflammatory response to bacterial infection. This cross-sectional study aims to investigate the levels of sTREM-1 in gingival crevicular fluid (GCF) of individuals without periodontitis and with chronic periodontitis (CP) or generalized aggressive periodontitis (GAgP) and their association with the levels of key periodontal pathogens in subgingival plaque., Methods: GCF and subgingival plaque samples were obtained from healthy sites of participants without periodontitis (n = 20) and periodontitis sites of patients with CP (n = 22) and GAgP (n = 20). sTREM-1 levels in GCF were measured by enzyme-linked immunosorbent assay. Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans levels in subgingival plaque were analyzed by quantitative real-time polymerase chain reaction., Results: sTREM-1 levels in GCF were higher in CP and GAgP than healthy sites by 3.6- and 4.4-fold, respectively, with no significant differences between the two forms of periodontitis. Moreover, sTREM-1 levels in GCF were positively correlated with site-specific clinical periodontal parameters and levels of P. gingivalis, T. denticola, and T. forsythia, but not A. actinomycetemcomitans, in subgingival plaque., Conclusion: Increased GCF levels of sTREM-1 at diseased sites and their positive correlation with clinical and microbiologic parameters strengthen the association of this inflammatory marker with periodontitis.

The Triggering Receptor Expressed on Myeloid cells 1 (TREM-1) is a cell-surface receptor of the immunoglobulin superfamily, involved in the propagation of the inflammatory response to bacterial challenge. Soluble (s)TREM-1 is released from the cell surface during the course of infection and is a useful inflammatory biomarker in the early diagnosis of systemic sepsis. The hypothesis of this study was that oral and systemic levels of sTREM-1 are elevated in periodontitis. Therefore, the aim was to investigate, by ELISA, the sTREM-1 concentrations in saliva and serum of individuals without periodontitis (control) and persons with chronic or generalized aggressive periodontitis. In saliva, sTREM-1 concentrations were higher in chronic and aggressive periodontitis than in the control group, by 3.3-fold and 5.6-fold, respectively. In serum, these differences were 1.7-fold and 2-fold, respectively. However, there were no significant differences between the two forms of periodontitis, neither in saliva nor in serum. Salivary and serum sTREM-1 levels positively correlated with full-mouth clinical periodontal parameters. In conclusion, the increased oral and systemic levels of sTREM-1 in periodontitis denote a value for this molecule as a biomarker for the disease and may also have implications in the association between periodontal infections and systemic inflammatory response.

The triggering receptor expressed on myeloid cells 1 (TREM-1) is a cell surface receptor of the immunoglobulin superfamily, with the capacity to amplify pro-inflammatory cytokine production. Porphyromonas gingivalis is a Gram-negative anaerobic species highly implicated in inflammatory periodontal disease, with potential involvement in systemic inflammation. Porphyromonas gingivalis positively regulates TREM-1 expression and production in monocytic cells. Subantimicrobial doses of doxycycline (SDD) are used as an adjunct treatment in periodontal therapy, because of their anti-inflammatory properties. The aim of this study was to investigate the effect of SDD on P. gingivalis-induced TREM-1 expression and secretion by the myelomonocytic cell line MonoMac-6. After 24 h of challenge, P. gingivalis enhanced TREM-1 gene expression by the cells, with a concomitant increase in soluble TREM-1 release. Nevertheless, SDD concentrations between 2 and 10 μg mL(-1) abolished TREM-1 expression and release, already after 4 h of administration. Moreover, SDD reduced P. gingivalis-induced interleukin-8 secretion, confirming its anti-inflammatory effects. In conclusion, SDD inhibits bacterially induced TREM-1, and this effect may partly account for its generalized anti-inflammatory properties. This could partly explain the clinical efficacy of SDD as an adjunctive treatment for periodontal disease, but may also indicate that SDD could serve as a suitable modulator of systemic inflammatory responses., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are able to lyse suitable target cells. CTLs recognize a specific peptide epitope presented by major histocompatibility complex (MHC) class I molecules on the target cells, whereas NK cells lyse targets that express no or low MHC class I molecules. Using perforin-deficient mice, we provide evidence that both NK cells and CTLs exclusively use perforin-dependent cytolysis as an effector mechanism in vitro, as well as in vivo. This review summarizes the most important role of perforin-dependent cytolysis in a wide variety of bacterial and viral infections, in tumour surveillance, in immunopathology and in autoimmunity.

In rat kidney, megalin, a member of the low density lipoprotein receptor gene family, is the sole glycoprotein which carries oligo/poly alpha2,8 deaminoneuraminic acid (KDN) as a posttranslational modification. We have investigated immunoprecipitated megalin from rat brain, lung and placenta, mouse yolk sac carcinoma and megalin synthesizing carcinoma cell lines, for presence of this unique glycan structure. Our immunoblot analysis revealed the presence of oligo/poly alpha2,8 KDN on megalin in all the studied normal tissues and carcinoma cells. Furthermore, it is demonstrated to be part of oligosaccharides O-glycosidically linked to megalin.

Recently, poly alpha2,8 deaminoneuraminic acid (poly alpha2,8 KDN) was demonstrated in various embryonic and adult mammalian tissues. This study reports the purification and characterization of the single poly alpha2,8 KDN-bearing glycoprotein from rat kidney. Amino acid sequences of proteolytic fragments shared homology with megalin, a member of the LDL receptor family. Immunochemical analysis supported this finding, since immunoprecipitated poly alpha2,8 KDN-bearing glycoprotein was immunoreactive with anti-megalin antibodies in Western blotting and conversely immunoprecipitated megalin was immunoreactive with the monoclonal anti-poly alpha2,8 KDN antibody. Furthermore, receptor-associated protein affinity-purified megalin reacted with the anti-poly alpha2,8 KDN antibody. By immunoelectron microscopy, labeling for both poly alpha2,8 KDN and megalin coincided in the brush border, endocytic invaginations and vesicles, and apical dense tubules of proximal convoluted tubules. Immunoreactivity for poly alpha2,8 KDN on purified megalin was abolished by beta-elimination reaction but not by N-glycosidase F treatment. These data identified megalin as the sole glycoprotein of rat kidney, which contains poly alpha2,8 KDN present on O-glycosidically linked oligosaccharides. Furthermore, this study shows that megalin carries N-glycosidically linked hybrid and complex-type oligosaccharides terminating with sialic acid. Both poly alpha2,8 KDN and sialic acids on megalin may contribute to the binding of Ca2+ and cationic ligands.

Aldehyde fixation of tissues often adversely affects the reactivity of cellular proteins with antibodies. A most commonly used retrieval technique in immunohistochemistry is high-temperature microwave heating of sections from formaldehyde-fixed and paraffin-embedded tissues. Here we report that pretreatment of paraffin and ultrathin cryosections with N-glycanase F to remove N-glycosidically linked oligosaccharides can result in a dramatic increase in specificity and intensity of immunogold labeling for sugar moieties present on O-glycosidically linked oligosaccharides. This is demonstrated in the immunolocalization of poly alpha2,8 KDN (KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) of megalin in rat kidney. The mechanism of this retrieval procedure is most probably based on the elimination of sterical hindrance by large N-glycosidically linked oligosaccharides. Furthermore, we demonstrate that exposure of ultrathin cryosections to acidic conditions (pH 5.5) at ambient temperature prior to immunogold labeling can result in an increased labeling intensity. This effect was observed for megalin immunoreactive sites in proximal tubular epithelia of rat kidney. It is proposed that the mechanism of this retrieval procedure is based on the depolymerization of methylen and polymethylen bridges introduced by formaldehyde in the acidic milieu.

Measles virus (MV) and vesicular stomatitis virus (VSV) are both members of the Mononegavirales but are only distantly related. We generated two genetically stable chimeric viruses. In MGV, the reading frames of the MV envelope glycoproteins H and F were substituted by a single reading frame encoding the VSV G glycoprotein; MG/FV is similar but encodes a G/F hybrid in which the VSV G cytoplasmic tail was replaced by that of MV F. In contrast to MG/FV, MGV virions do not contain the MV matrix (M) protein. This demonstrates that virus assembly is possible in the absence of M; conversely, the cytoplasmic domain of F allows incorporation of M and enhances assembly. The formation of chimeric viruses was substantially delayed and the titers obtained were reduced about 50-fold in comparison to standard MV. In the novel chimeras, transcription and replication are mediated by the MV ribonucleoproteins but the envelope glycoproteins dictate the host range. Mice immunized with the chimeric viruses were protected against lethal doses of wild-type VSV. These findings suggest that it is feasible to construct MV variants bearing a variety of different envelopes for use as vaccines or for gene therapeutic purposes.

The expression of the neural cell adhesion molecule L1 is altered by neuronal activity and promotes neurite outgrowth in vitro. To study the effects of L1 on learning and synaptic plasticity, transgenic mice have been created which express L1 ectopically in glial fibrillary acidic protein (GFAP) expressing astrocytes. Ninety mice, including GFAP-L1-transgenic mice from two genetic backgrounds and their littermates, were tested for swimming navigation learning in the Morris water maze according to a standardized protocol. While learning the position of an invisible target platform and also relearning its position after relocation, GFAP-L1-transgenic mice spent a greater fraction of their swim time in the target quadrant. Moreover, they showed a more rapid improvement of escape performance during the first day of training. Factor analysis revealed that this difference in swimming pattern could not be explained by non-cognitive factors. Factor analysis also revealed that, during a probe trial, the GFAP-L1-transgenic mice spent comparatively less time in the old target quadrant than predicted by the increased searching they had shown during acquisition learning. Hence, ectopic expression of L1 by astrocytes in mice appears to be linked to a factor which increases behavioural flexibility and selectivity while learning and relearning, but concomitantly may lead to a relative reduction of spatial retention.

Neutralizing antiviral B cell responses differ in various aspects from the many usually measured B cell responses specific for protein in adjuvants. In particular, such neutralizing antiviral B cell responses are more rapidly induced, reach higher titers, are longer lived, and are efficiently generated without adjuvants. Evidence is summarized here that the repetitiveness of many viral antigens is a key factor responsible for the efficiency of these B cell responses, amplifying B cells early and rapidly for potent IgM responses and also for efficient switching to IgG. The data reviewed indicate that B cells discriminate antigen patterns via the degree of surface Ig-cross-linking and use antigen repetitiveness as a self/nonself discriminator.

Immune surveillance against tumors usually depends on T cell recognition of tumor antigens presented by major histocompatibility complex (MHC) molecules, whereas MHC class I- tumors may be controlled by natural killer (NK) cells. Perforin-dependent cytotoxicity is a major effector function of CD8+ MHC class I-restricted T cells and of NK cells. Here, we used perforin-deficient C57BL/6 (PKO) mice to study involvement of perforin and Fas ligand in tumor surveillance in vivo. We induced tumors in PKO and normal C57BL/6 mice by (a) injection of different syngeneic tumor cell lines of different tissue origin in naive and primed mice; (b) administration of the chemical carcinogens methylcholanthrene (MCA) or 12-O-tetradecanoylphorbol-13-acetate (TPA) plus 7,12-dimethylbenzanthracene (DMBA), or (c) by injection of acutely oncogenic Moloney sarcoma virus. The first set of models analyzes the defense against a tumor load given at once, whereas the last two sets give information on immune defense against tumors at the very moment of their generation. Most of the tumor cell lines tested were eliminated 10-100-fold better by C57BL/6 mice in an unprimed situation; after priming, the differences were more pronounced. Lymphoma cells transfected with Fas were controlled 10-fold better by PKO and C57BL/6 mice when compared to untransfected control cells, indicating some role for FasL in tumor control. MCA-induced tumors arose more rapidly and with a higher incidence in PKO mice compared to C57BL/6 or CD8-deficient mice. DMBA+TPA-induced skin papillomas arose with similar high incidence and comparable kinetics in both mouse strains. C57BL/6 and PKO mice have a similar incidence of Moloney murine sarcoma and leukemia virus-induced sarcomas, but tumors are larger and regression is retarded in PKO mice. Thus, perforin-dependent cytotoxicity is not only a crucial mechanism of both cytotoxic T lymphocyte- and NK-dependent resistance to injected tumor cell lines, but also operates during viral and chemical carcinogenesis in vivo. Experiments addressing the role of Fas-dependent cytotoxicity by studying resistance to tumor cell lines that were stably transfected with Fas neither provided evidence for a major role of Fas nor excluded a minor contribution of Fas in tumor surveillance.

A vesicular stomatitis virus glycoprotein-specific, class II restricted, CD4+ T-cell clone was obtained and the unidentified T-cell receptor alpha chain cloned in order to establish a T-cell receptor (TCR) alpha chain transgenic mouse line. Polymerase chain reaction (PCR) strategies have been developed to clone TCR chain genes starting from T-cell cDNA. There remain difficulties, however, in cloning the functional TCR alpha chain due to the complexity of the protocols applied if the variable (V) alpha region rearranged is not known. The strategy described here allows the identification and cloning of alpha chains that are not recognized by any of the anti-V alpha monoclonal antibodies available: three 5' consensus V alpha primers designed from all known V alpha gene sequences and a primer specific for the constant (C) alpha region were used and the PCR product sequenced. The T-cell clone displayed two alpha gene rearrangements, only one of which giving rise to a functional protein. The alpha chain used by the T-cell clone contained a V alpha 3.1 and a J alpha region which has been described only at the genomic level, but never in a functional TCR. The complete alpha chain gene was cloned by enriching the alpha chain-encoding cDNA by ligation-anchored PCR and using an alpha specific primer pair. The use of the present method, even if the sequence of the 5' untranslated (UT) region of the alpha chain is not known, is also discussed.

CD40-CD40 ligand (CD40L) interaction is required for the generation of antibody responses to T-dependent antigens as well as for the development of germinal centers and memory B cells. The role of the CD40-CD40L interaction in the induction of antigen-specific. Th cells and in mediating Th cell effector functions other than cognate help for B cells is less well understood. Using CD40- and CD40L-deficient mice together with lymphocytic choriomeningitis virus and vesicular stomatitis virus as viral model antigens, this study corroborates earlier findings that no lg isotype switching of virus-specific antibodies was measurable upon infection of CD40- or CD40L-deficient mice. In contrast, in vivo induction of virus-specific CD4+ T cells measured by proliferation and cytokine secretion of primed virus-specific Th cells in vitro was not crucially dependent on the CD40-CD40L interaction. In addition, virus-specific Th cells primed in a CD40-deficient environment, adoptively transferred into CD40-competent recipients, were able to mediate lg isotype switch. Th-mediated effector functions distinct from and in addition to T-B collaboration were analyzed in CD40- and CD40L-deficient and normal mice: (a) local inflammatory reactions upon LCMV infection mediated by LCMV-specific Th cells were not dependent on a functional CD40-CD40L interaction, (b) cytokine-mediated protection by CD4+ T cells primed by vesicular stomatitis virus against a challenge infection with recombinant vaccinia virus expressing the glycoprotein of vesicular stomatitis virus was found to be equivalent in CD40L-deficient and normal mice. Thus, CD40-CD40L interaction plays a crucial role in T-B interactions for Th-dependent activation of B cells but not, or to a much lesser extent, in T cell activation, antigen-specific Th cell responses in vitro, and for interleukin-mediated Th cell effector functions in vivo.

It is widely accepted that T cells play an important role in the destruction of beta cells leading to autoimmune type I diabetes, but the involved effector mechanisms have remained unclear. We addressed this issue by testing the role of perforin-dependent cytotoxicity in a disease model involving transgenic mice expressing glycoprotein of lymphocytic choriomeningitis virus (LCMV-GP) in the beta cells of the endocrine pancreas. In such mice, LCMV infection leads to a potent LCMV-GP-specific T cell response resulting in rapid development of diabetes. We report here that in perforin-deficient LCMV-GP transgenic mice, LCMV infection failed to induce diabetes despite the activation of LCMV-GP-specific T cells. Deletion of nu beta 6+ T cells in Mls-1a perforin-deficient mice and the activation of LCMV-GP-specific T cells in perforin-deficient LCMV-GP transgenic mice, however, indicated that thymic tolerance induction by negative selection was not affected by the disruption of the perforin gene and that there is no fundamental difference between the T cell repertoires of normal control and perforin-deficient mice. In addition, adoptive transfer of LCMV-GP-specific TCR transgenic perforin-deficient T cells activated by LCMV-GP recombinant vaccinia virus led to marked insulitis with infiltration of CD4+ and CD8+ T cells without the development of diabetes. These findings indicate that perforin-dependent cytotoxicity is not required for the initiation of insulitis but is crucial for the destruction of beta cells in the later phase of the disease process. Other mechanisms or soluble factors present in the inflammatory islet infiltrate apparently lack the ability to efficiently induce diabetogenic beta cell damage.

Reduced oxygenation of a variety of cells results in transcriptional upregulation of several genes, including the hematopoietic hormone erythropoietin, the angiogenic vascular endothelial growth factor (VEGF), and glycolytic enzymes such as aldolase. Recently, the heme protein cytochrome b558 of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex has been proposed as a key component of the oxygen-sensing mechanism. Cytochrome b558 consists of the p22phox and gp91phox subunits and is essential for superoxide generation in phagocytes and B lymphocytes. Mutations in these subunits result in cytochrome b558-negative chronic granulomatous disease (cytb- CGD), an inherited disorder in humans characterized by reduced microbicidal activity due to deficient superoxide generation. To test whether NADPH oxidase is involved in oxygen sensing, we exposed wild-type B-cell lines as well as cytb- CGD-derived B cell lines, deficient in either p22phox or gp91phox, to hypoxia (1% oxygen) or CoCl2 (100 mumol/L) and compared the mRNA levels of VEGF and aldolase with the untreated controls. Northern blot analysis revealed unimpaired basal and inducible expression of VEGF and aldolase mRNA in all four cytb- CGD-derived B-cell lines compared with wild-type cells. Furthermore, reconstitution of cytochrome b558 expression in cytb- CGD-derived B cells by transfection with p22phox or gp91phox expression vectors did not modify VEGF and aldolase mRNA expression. Thus, cytochrome b558 of the NADPH oxidase complex appears not to be essential for hypoxia-activated gene expression and can be excluded as a candidate for the putative universal oxygen sensor.

The neutralizing antibody response against vesicular stomatitis virus (VSV) was studied to analyze conditions necessary for induction of mature B cells. The glycoprotein of VSV (VSV-G) is expressed as repetitive epitopes that are in a rigid densely packed paracristalline form in the virus envelope; in contrast, VSV-G is present in a mobile randomly ordered form on infected cells and in micelles. We found that the rigid, paracristalline form of VSV-G spaced about 5-10 mm on VSV virons induced potent primary and secondary neutralizing B cell responses which were independent of T helper cells, whereas the more randomly distributed, mobile forms of VSV-G induced primary and secondary B cell responses that were more tightly controlled by T helper cells. These data suggest that (i) cross-linking is a critical signal for B cell activation and antibody production, and that this signal alone does not necessarily anergize or delete mature B cells, (ii) the more regularly and rigidly ordered in a distance of 5-10 nm repetitive identical antigenic determinants are, the less are primary and secondary B cell responses controlled by T cells. We therefore propose that B cells take antigen organization as a marker for foreigness.

Adaptive immune surveillance by T cells against infections and tumors depends on the presence of antigenic peptides presented by major histocompatibility complex (MHC) molecules. If antigenic tumor-specific peptides or MHC class I molecules are absent, the adaptive T cell immune response fails. Natural killer (NK) cells seem to complement the specific T cells by recognizing target cells lacking MHC class I (e.g. RMA-S). The role of perforin, which is crucially involved in T cell and NK cell-mediated target cell lysis, was evaluated in mice lacking perforin with respect to their capacity to eliminate a syngeneic lymphoid tumor. Here, we show that growth of MHC class I RMA-S tumor cells in unprimed mice was controlled by NK cells through perforin-dependent cytotoxicity.

The pregnant uterus of humans and rodents contains a population of granulated lymphoid cells, which, in the mouse, are called granular metrial gland (GMG) cells and have been described to express high levels of perforin. Since there is evidence for cytolytic activity of these cells and since perforin is a crucial effector molecule for the lytic action of cytotoxic T cells and natural killer cells, we evaluated the function of perforin in the pregnant uterus by using perforin-deficient mice. Perforin-deficient female mice were found to reproduce as efficiently as normal control females when bred either with syngeneic or allogeneic males. However, perforin-deficient mice differed from normal mice in that the frequency of GMG cells was significantly higher within maternal blood spaces and within several compartments of the feto-maternal interface. Proliferating GMG cells, identified by [3H] thymidine incorporation, were observed during more advanced stages of pregnancy when compared to normal controls. In contrast to normal mice, perforin-deficient mice did not display GMG cells attached to degenerating trophoblasts; instead perforin-deficient GMG cells were often observed in association with small maternal lymphocytes. In addition, the lack of transmission of lymphocytic choriomeningitis virus from infected pregnant perforin-deficient mice to the fetuses argued against a role of perforin expression by GMG cells in prevention of virus transmission from the mother to the fetus. Our data indicate that functional perforin is not necessary for successful pregnancies. The morphological changes in the pregnant uterus of perforin-deficient mice might, however, point to a certain, as-yet undefined function of perforin in the uterus of pregnant normal mice, which is functionally compensated in perforin-deficient mice.

In vitro, T cell-dependent cytotoxicity is mediated by two distinct mechanisms, one being perforin-, the other Fas-dependent. The contribution of both of these mechanisms to clearance of viral infections was investigated in mice for the non-cytopathic lymphocytic choriomeningitis virus (LCMV) and the cytopathic vaccinia, vesicular stomatitis (VSV) and Semliki forest (SFV) viruses. Clearance of an acute LCMV infection was mediated by the perforin-dependent mechanism without measurable involvement of the Fas-dependent pathway. For the resolution of vaccinia virus infection and for resistance against VSV and SFV, however, neither of the two pathways was required. These data suggest that perforin-dependent cytotoxicity mediated by T cells is crucial for protection against non-cytopathic viruses, whereas infections with cytopathic viruses are controlled by nonlytic T cell-dependent soluble mediators such as cytokines (IFN-gamma against vaccinia virus) and neutralizing antibodies (against VSV and SFV).

The role of innate, alpha/beta interferon (IFN-alpha/beta)-dependent protection versus specific antibody-mediated protection against vesicular stomatitis virus (VSV) was evaluated in IFN-alpha/beta receptor-deficient mice (IFN-alpha/beta R0/0 mice). VSV is a close relative to rabies virus that causes neurological disease in mice. In contrast to normal mice, IFN-alpha/beta R0/0 mice were highly susceptible to infection with VSV because of ubiquitous high viral replication. Adoptive transfer experiments showed that neutralizing antibodies against the glycoprotein of VSV (VSV-G) protected these mice efficiently against systemic infection and against peripheral subcutaneous infection but protected only to a limited degree against intranasal infection with VSV. In contrast, VSV-specific T cells or antibodies specific for the nucleoprotein of VSV (VSV-N) were unable to protect IFN-alpha/beta R0/0 mice against VSV. These results demonstrate that mice are extremely sensitive to VSV if IFN-alpha/beta is not functional and that under these conditions, neutralizing antibody responses mediate efficient protection, but apparently only against extraneuronal infection.

Growth of Listeria monocytogenes is mainly controlled by macrophages, which are activated by specific T cells. A potential role of CD8+ T cells by direct lysis of infected cells was investigated in perforin-deficient mice generated by homologous recombination. The absence of perforin-mediated cytotoxicity resulted in delayed clearance of Listeria from the spleen but not the liver after primary infection, overall susceptibility to Listeria however was not increased. Protection against a secondary infection was drastically impaired in perforin-deficient mice. Adoptive transfer of immune spleen cells to recipients revealed that anti-Listeria protection by CD8+ T cells from perforin-deficient versus normal mice was about 10-fold reduced in livers and about 100-fold reduced in the spleen of recipients. CD4+ T cells from immune control and perforin-deficient mice conferred comparable protection. These results indicate that the protective effect of CD8+ T cells against an intracellular bacterium mainly evident in secondary infection is mediated by a perforin-dependent pathway, presumably cytotoxicity, and less by other direct or indirect effector mechanisms.

Safe vaccines should optimally induce both cell-mediated and humoral immunity. Recently, it has been shown that protective cytotoxic T cells (CTLs) can be induced not only with live vaccines, but also with recombinant viral proteins. This report shows in C57BL/6 (H-2b) mice that the recombinant nucleoprotein (N) of vesicular stomatitis virus (VSV) induced protective CTLs but no neutralizing antibodies in mice, whereas the recombinant glycoprotein (G) of VSV alone induced neutralizing antibodies but no CTLs. If the N and G of VSV were coinjected, both CTLs and a long-lasting neutralizing IgG response was measurable, demonstrating that mixed vaccines can be used to induce protective CTLs and antibodies with an efficiency comparable to live virus. In an attempt to define optimal conditions for CTL priming, the intravenous, intraperitoneal and subcutaneous route of injection were compared. Intravenous injection of recombinant VSV-N induced up to 30 times higher responses than the latter two routes. Finally, we tried to define conditions inducing only CTLs and no antibodies binding to the native protein form, or vice versa, only antibodies and no CTLs. Intravenous injection of boiled VSV-N induced a CTL response but no antibodies specific for the native VSV-N, whereas VSV-N injected subcutaneously in incomplete Freund's adjuvant induced high amounts of anti-VSV-N antibodies but virtually no CTLs. The conditions defined here permit vaccines to be designed which would function along selected and defined immunological effector pathways.

Two molecular mechanisms of T cell-mediated cytotoxicity, one perforin-based, the other Fas-based, have been demonstrated. To determine the extent of their contribution to T cell-mediated cytotoxicity, a range of effector cells from normal control or perforin-deficient mice were tested against a panel of target cells with various levels of Fas expression. All cytotoxicity observed was due to either of these mechanisms, and no third mechanism was detected. Thus, the perforin- and Fas-based mechanisms may account for all T cell-mediated cytotoxicity in short-term in vitro assays.

We have undertaken a detailed study of the expression of GAP-43 and synaptophysin immunoreactivity in the developing postnatal rat retina. We found that these two 'presynaptic' proteins have quite different expression patterns. GAP-43 was first expressed in the optic nerve and the optic fiber layer of the retina, where it disappeared between the 8th and 16th postnatal day. From the 5th postnatal day on, GAP-43 also appeared in the inner plexiform layer, where it was present in three distinct bands. This expression changed little in postnatal development and persisted in the adult retina. GAP-43 was not detected in the outer plexiform layer of the retina. Synaptophysin was absent from the optic nerve and optic fibers at all postnatal stages. It was first expressed in the developing outer plexiform and, with reduced intensity, in the outer nuclear layer between postnatal days 2 and 5. In the inner plexiform layer, synaptophysin could be first detected between postnatal days 8 and 12. The intensity of staining increased during postnatal development in both plexiform layers. The developmental sequence of synaptophysin expression can be correlated with the maturation of presynaptic terminals of photoreceptors and bipolar cells. The rather complex pattern of GAP-43 expression is not easily compatible with a single model of GAP-43 function, and suggests diverse functions of this molecule in the retina.

The axonal cell adhesion molecule, TAG-1/axonin-1, stimulates axonal growth and supports neurite fasciculation in vitro. Using a polyclonal antiserum raised against chick axonin-1, which shares 75% of its sequence with TAG-1 of the rat, we have mapped the distribution of TAG-1/axonin-1 throughout the developing nervous system of the mouse. Although absent from proliferating neuroepithelia and from non-neuronal cells, immunoreactivity for TAG-1/axonin-1 is expressed by stage-specific subpopulations of differentiating neurons from embryonic day 10 to postnatal day 15. It stains their axons and the surface of their parent somata during the early phases of axogenesis. In agreement with a putative role of TAG-1/axonin-1 as an axon-bound growth substrate, immunoreactivity is found in developing spinal and cranial nerves, in corticothalamic projections, as well as in subsets of fasciculating long projecting tracts of the central nervous system, such as the dorsal funiculi of the spinal cord, the lateral olfactory and optic tracts, the fasciculus retroflexus, and the predorsal bundle. High levels of immunoreactivity characterise the development of the cerebellar molecular layer, the corpus callosum, anterior and hippocampal commissure, and of crossed projections in the spinal cord and at several levels of the brainstem. Intense immunoreactivity in fine collaterals of cutaneous afferents, including their growth cones that are in contact with the embryonic skin, suggests a role of TAG-1/axonin-1 in target recognition. While staining is weak on the somata of radially migrating neurons such as cortical neurons and cerebellar granule cells, strong immunoreactivity is associated with neural somata and processes of the three tangential migrations that form the precerebellar nuclei, indicating a possible involvement of TAG-1/axonin-1 in contacts between these neurons and the processes they migrate upon.

The neutralizing immunoglobulin M (IgM) response to vesicular stomatitis virus (VSV) has been shown to be largely T-cell independent in several T-cell-deficient models of mice. By using different antigen froms of VSV, VSV antigen doses could be graded in vivo (infectious > > UV inactivated > formalin inactivated). The present study reveals a T-cell-dependent component of the neutralizing IgM response in nude mice given intravenous injections of low doses of noninfectious UV-inactivated VSV serotype Indiana (VSV-IND) only if the mice are transfused with VSV-IND-specific helper T cells. Instead, nude mice immunized with infectious VSV, which leads to greater antigen doses in vivo, were able to mount an IgM response in the absence of T cells. These results indicate that the IgM response to low doses of VSV-IND glycoprotein (G) is T-cell dependent. Nude mice immunized with infectious VSV also made a variable but low VSV-IND-neutralizing IgG response. A VSV-IND matrix (M)-specific helper T-cell line rendered this response more consistent, much higher, and longer lasting. Thus (i) VSV-G induces a mostly T-cell-independent but partially T-cell-dependent IgM (the latter can be visualized best at low doses of antigen) and (ii) the antibody response to VSV in nude mice proceeds through steps, i.e., IgM and IgG, that are dose dependent. The results suggest that the predominant role of helper T cells may be to expand and maintain the individual steps of differentiating B cells.

Perforin-deficient mice have been generated by homologous recombination to determine whether the effects of CD8+ cytolytic T cells and natural killer cells are mediated by pore formation involving perforin. These mice are viable and fertile and have normal numbers of CD8+ T cells and natural killer cells which do not lyse virus-infected or allogeneic fibroblasts or natural killer target cells in vitro. The mice fail to clear lymphocytic choriomeningitis virus and they eliminate fibrosarcoma tumour cells with reduced efficiency. Perforin is therefore a key effector molecule for T-cell- and natural killer-cell-mediated cytolysis.

The influence of antigen epitope density and order on B cell induction and antibody production was assessed with the glycoprotein of vesicular stomatitis virus serotype Indiana [VSV-G (IND)]. VSV-G (IND) can be found in a highly repetitive form the envelope of VSV-IND and in a poorly organized form on the surface of infected cells. In VSV-G (IND) transgenic mice, B cells were unresponsive to the poorly organized VSV-G (IND) present as self antigen but responded promptly to the same antigen presented in the highly organized form. Thus, antigen organization influences B cell tolerance.

Cytotoxic T-lymphocyte (CTL) and T-helper-cell responses in various mouse strains were monitored. Protective CTL responsiveness against three proteins of vesicular stomatitis virus was H-2 linked and inducible only in half of the 15 combinations tested (each of five H-2 haplotypes combined with each of three viral proteins), whereas biologically relevant T-helper-cell responses were inducible in all. This suggests that vesicular stomatitis virus exhibits more T-helper-cell than CTL epitopes.

Previously, we isolated a cDNA clone (rBAT-1) of 2.2 kilobase pairs (kb) from a rabbit kidney cortex cDNA library, encoding a protein involved in sodium-independent transport of L-dibasic amino acids, L-cystine, and some neutral amino acids via a system related to b0,(+)-like activity (Bertran, J., Werner, A., Moore, M. L., Stange, G., Markovich, D., Biber, J., Testar, X., Zorzano, A., Palacin, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5601-5605). In Northern blot hybridization using an rBAT-1 cDNA probe, 2.2- and 3.9-kb mRNA species were observed. Here we describe the isolation of a 3.9-kb cDNA clone (rBAT-2) by expression cloning using Xenopus laevis oocytes corresponding to the 3.9-kb mRNA species. On the basis of sequence analysis, in vitro translation (major protein of approximately 78 kDa), and functional analysis (expression of transport function), we conclude that rBAT-1- and rBAT-2-related proteins are identical: 677 amino acids in length, with most likely only one transmembrane-spanning domain. There are seven differences in the nucleotide composition within a common overlap of 2189 nucleotides, resulting in 2 amino acid replacements. In comparison with rBAT-1, rBAT-2 has 26 additional nucleotides at the 5'-end, an identical location of the first polyadenylation signal, and approximately 1.7 kb of 3'-untranslated sequence (rich in AT(U) motifs) prior to a poly(A) tail of 63 adenines. We conclude that rBAT-1 and rBAT-2 encode the same protein and that the major difference seems to be related to the use of different polyadenylation signals.

A cDNA library from rabbit kidney cortex was screened for expression of Na-dependent transport of phosphate (Pi) using Xenopus laevis oocytes as an expression system. A single clone was eventually isolated (designated NaPi-1) that stimulated expression of Na/Pi cotransport approximately 700-fold compared to total mRNA. The predicted sequence of the Na/Pi cotransporter consists of 465 amino acids (relative molecular mass, 51,797); hydropathy profile predictions suggest six (possibly eight) membrane-spanning segments. In vitro translation of NaPi-1/complementary RNA in the presence of pancreatic microsomes indicated NaPi-1 to be a glycosylated protein; four potential N-glycosylation sites are present in the amino acid sequence. Northern blot analysis demonstrated the presence of NaPi-1/mRNA in kidney cortex and liver; no hybridization signal was obtained with mRNA from other tissues (including small intestine). Kinetic analysis of Na/Pi cotransport expressed by NaPi-1/complementary RNA demonstrated characteristics (sodium interaction) similar to those observed in cortical apical membranes. The alignment of 5 amino acid residues (Gly342/Ala381-Xaa-Xaa-Xaa-Xaa-Leu386-Xaa-Xaa-Xaa-P ro390- Arg391) is consistent with a motif proposed for Na-dependent transport systems. We conclude that we have cloned a cDNA for a Na/Pi cotransport system present in rabbit kidney cortex.

Three differently modified forms of beta-glucuronidase are known to exist: a microsomal enzyme form (M) existing in tissues where egasyn, a second microsomal protein, is present; and an acidic (La; complex-type oligosaccharide) and a basic (Lb; non-complex type oligosaccharide) lysosomal form which occur in all mouse tissues. Lb predominates in tissues containing microsomal beta-glucuronidase, La in those lacking it. In pulse-labelling experiments using mouse strain C57BL/6 liver containing egasyn (Eg+/Eg+) and microsomal enzyme, about half of the newly synthesized beta-glucuronidase was processed to the microsomal enzyme form, which was evidently further processed to Lb, and about half directly to La. In contrast, in liver of the congenic line C57BL/6.YBR Es-1b Eg0 that lacks egasyn (Eg0/Eg0) and microsomal enzyme, most of the labelled beta-glucuronidase was processed to La, and only a minor portion to Lb. Newly synthesized enzyme appeared first in microsomal, then in light and heavy lysosomal fractions of Eg+/Eg+ liver. In Eg0/Eg0 liver, no labelled enzyme was measurable in the microsomes, but it appeared rapidly in both types of lysosomes. Taken together these findings indicate that the microsomal enzyme form serves as a precursor of Lb, and that La is synthesized independently. The apparent half-life of La is only two-thirds that of Lb; this fact accounts for the reduced beta-glucuronidase activity in Eg0/Eg0 liver, which contains La as the predominant form.